Lung fibroblasts participate in the pathogenesis of respiratory diseases, including lung cancer and pulmonary fibrosis. Although fibroblasts are ubiquitous constituents of various organs, their cellular diversity among different organs has been poorly characterized. Here, we aimed to investigate the distinct gene signature of lung fibroblasts that represents its pulmonary origin, and the underlying gene regulatory networks. Promoter-level differential expression analysis by cap analysis of gene expression (CAGE) sequencing revealed distinct gene expression patterns of fibroblasts derived from different anatomical sites and identified 88 coding genes with higher expression in lung fibroblasts relative to other fibroblasts. Multiple key transcription factors important for lung mesenchyme development, including the T-box transcription factors TBX2, TBX4, and TBX5, were enriched in this lung-specific signature and were associated with super-enhancers. TBX4 showed highly specific expression in lung fibroblasts and was required for cell proliferation and collagen gel contraction capacity. Transcriptome analysis revealed that TBX4 could broadly regulate fibroblast-related pathways and partly contribute to super-enhancer-mediated transcriptional programs. Of pathological importance, lung fibroblast-specific genes were globally downregulated in lung cancer-associated fibroblasts (CAFs). Notably, TBX2, TBX4, and TBX5 were downregulated and hypermethylated in lung CAFs, suggesting an association between epigenetic silencing of these factors and phenotypic alteration of lung fibroblasts in cancer. Our study highlights the importance of T-box transcription factors, especially TBX4, and super-enhancers in the roles of lung fibroblasts in pulmonary physiology and pathogenesis.
Chronic Obstructive Pulmonary Disease (COPD) is the third leading cause of morbidity and death and imposes major socioeconomic burdens globally. It is a progressive and disabling condition that severely impairs breathing and lung function. There is a lack of effective treatments for COPD, which is a direct consequence of the poor understanding of the underlying mechanisms involved in driving the pathogenesis of the disease. Toll-like receptor (TLR)2 and TLR4 are implicated in chronic respiratory diseases, including COPD, asthma and pulmonary fibrosis. However, their roles in the pathogenesis of COPD are controversial and conflicting evidence exists. In the current study, we investigated the role of TLR2 and TLR4 using a model of cigarette smoke (CS)-induced experimental COPD that recapitulates the hallmark features of human disease. TLR2, TLR4 and associated co-receptor mRNA expression were increased in the airways in both experimental and human COPD. Compared to WT mice, CS-induced pulmonary inflammation was unaltered in TLR2-deficient (Tlr2-/-), TLR4-deficient (Tlr4-/-) mice. CS-induced airway fibrosis, characterized by increased collagen deposition around small airways, was not altered in Tlr2-/- mice but was attenuated in Tlr4-/- mice compared to CS-exposed WT controls. However, Tlr2-/- mice had increased CS-induced emphysema-like alveolar enlargement, apoptosis and impaired lung function, whilst these features were reduced in Tlr4-/- mice compared to CS-exposed WT controls. Taken together, these data highlight the complex roles of TLRs in the pathogenesis of COPD and suggest that activation of TLR2 and/or inhibition of TLR4 may be novel therapeutic strategies for the treatment of COPD.
Altered sphingolipid metabolism is associated with increased inflammation; however, the impact of inflammatory mediators, including neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. Using a well-characterized mouse model of NE oropharyngeal aspiration, we investigated a potential link between NE-induced airway inflammation and increased synthesis of various classes of sphingolipids, including ceramide species. Sphingolipids in bronchoalveolar lavage fluids (BAL) were identified and quantified using reverse phase high-performance liquid chromatography/electrospray ionization tandem mass spectrometry analysis. BAL total and differential cell counts, CXCL1/keratinocyte chemoattractant (KC) protein levels, and high mobility group box 1 (HMGB1) protein levels were determined. NE exposure increased BAL long chain ceramides, total cell and neutrophil counts, and upregulated KC and HMGB1. The mRNA and protein levels of serine palmitoyltransferase (SPT) long chain subunits 1 and 2, the multimeric enzyme responsible for the first, rate-limiting step of de novo ceramide generation, were determined by Q/RT-PCR and western analyses, respectively. NE increased lung SPT long chain subunit 2 (SPTLC2) protein levels but not SPTLC1, and had no effect on mRNA for either subunit. To assess whether de novo ceramide synthesis was required for NE-induced inflammation, myriocin, a SPT inhibitor, or a vehicle control was administered intraperitoneally 2h prior to NE administration. Myriocin decreased BAL d18:1/22:0 and d18:1/24:1 ceramide, KC and HMGB1 induced by NE exposure. These results support a feed-forward cycle of NE-generated ceramide and ceramide-driven cytokine signaling that may be a potential target for intervention in lung disease typified by chronic neutrophilic inflammation.
Influenza A viruses are highly contagious respiratory pathogens that are responsible for significant morbidity and mortality worldwide on an annual basis. We have shown previously that influenza infection of mice leads to increased ATP and adenosine accumulation in the airway lumen. Moreover, we demonstrated that A1-adenosine receptor activation contributes significantly to influenza-induced acute respiratory distress syndrome (ARDS). However, we found that development of ARDS in influenza-infected mice does not require catabolism of ATP to adenosine by ecto-5'-nucleotidase (CD73). Hence, we hypothesized that increased adenosine generation in response to infection is mediated by tissue non-specific alkaline phosphatase (TNAP), which is a low-affinity, high-capacity enzyme that catabolizes nucleotides in a non-specific manner. In the current study, we found that whole lung and BALF TNAP expression and alkaline phosphatase enzymatic activity increased as early as 2 days post infection (d.p.i.) of C57BL/6 mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1). Treatment at 2 and 4 d.p.i. with a highly-specific quinolinyl-benzenesulfonamide TNAP inhibitor (TNAPi) significantly reduced whole lung alkaline phosphatase activity at 6 d.p.i. but did not alter TNAP gene or protein expression. TNAPi treatment attenuated hypoxemia, lung dysfunction, histopathology, and pulmonary edema at 6 d.p.i. without impacting viral replication. Treatment also improved epithelial barrier function and attenuated cellular and humoral innate immune responses to influenza infection. These data indicate that TNAP inhibition can attenuate influenza-induced ARDS by reducing inflammation and fluid accumulation within the lung. They also further emphasize the importance of adenosine generation for development of ARDS in influenza-infected mice.
Acidic microenvironments commonly occur at sites of inflammation and bacterial infections. In the context of a Pseudomonas aeruginosa infection, we previously demonstrated that acidosis enhances the cellular proinflammatory IL-1β response in vitro. However, how pH alterations affect in vivo IL-1β responses and subsequent IL-1 driven inflammation during infection with P. aeruginosa is unclear. We report herein that acidosis enhances in vivo IL-1β production and downstream IL-1R-dependent responses during infection with P. aeruginosa in models of acute pneumonia and peritonitis. Importantly, we demonstrate that infection with P. aeruginosa within an acidic environment leads to an enhanced production of a subset of proinflammatory cytokines, including CXCL1, IL-6 and CCL2, and increased neutrophil recruitment. Furthermore, with the use of IL-1R1-deficient mice, we identify the contribution of the IL-1 signaling pathway to the acidosis-enhanced inflammatory response and pathology. These data provide insights into the potential benefit of pH regulation during bacterial infections to control disease progression and immunopathology.
In cystic fibrosis (CF) lungs, epithelial Na+ channel (ENaC) hyperactivity causes a reduction in airway surface liquid (ASL) volume, leading to decreased mucocilliary clearance and lung damage. Inhibition of ENaC is an attractive therapeutic option. However, ENaC antagonists have failed clinically due to off-target renal effects. The S18 peptide is a naturally occurring short palate lung and nasal epithelial clone 1 (SPLUNC1)-derived ENaC antagonist that restores ASL height for up to 24 h in CF human bronchial epithelial cultures. However, its efficacy and safety in vivo are unknown. To interrogate the potential clinical efficacy of S18, we assessed its safety and efficacy using airway cultures and animal models. S18-mucus interactions were tested using super resolution microscopy, quartz crystal microbalance with dissipation and confocal microscopy. Human and murine airway cultures were used to measure ASL height. Off-target effects were assessed in conscious mice and anesthetized rats. Morbidity and mortality were assessed in the βENaC-Tg mouse model. Restoration of normal mucus clearance was measured in CFTR(inh)-172 challenged sheep. We found that S18 does not interact with mucus and rapidly penetrated dehydrated CF mucus. Compared to amiloride, an early generation ENaC antagonist, S18 displayed a superior ability to slow ASL absorption, reverse CFTR(inh)-172-induced reduction of mucus transport and reduce morbidity and mortality in the βENaC-Tg mouse, all without inducing any detectable signs of renal toxicity. These data suggest that S18 is the first naturally occurring ENaC antagonist to show improved preclinical efficacy in animal models of CF with no signs of renal toxicity.
Progranulin (PGRN) is a growth factor with multiple biological functions, and has been suggested as an endogenous inhibitor of TNFα-mediated signaling. TNFα is believed as one of the important mediators of the pathogenesis of asthma, including airway hyperresponsiveness (AHR). In the present study, effects of recombinant PGRN on the TNFα-mediated signaling and the antigen-induced hyper-contractility were examined in bronchial smooth muscles (BSMs) both in vitro and in vivo. Cultured human BSM cells (hBSMCs) and male BALB/c mice were used. The mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also received intranasal administrations of recombinant PGRN into the airways 1 hour before each antigen inhalation. In hBSMCs, PGRN inhibited both the degradation of IB-α (an index of NF-B activation) and the up-regulation of RhoA (a contractile machinery-associated protein that contributes to the BSM hyperresponsiveness) induced by TNFα, indicating that PGRN has an ability to inhibit TNFα-mediated signaling also in the BSM cells. In BSMs of the repeatedly antigen-challenged mice, an augmented contractile responsiveness to acetylcholine with an up-regulation of RhoA was observed: both the events were ameliorated by the pretreatments with PGRN intranasally. Interestingly, a significant decrease in the PGRN expression was found in the airways of the repeatedly antigen-challenged mice than those of control animals. In conclusion, exogenously applied PGRN into the airways ameliorated the antigen-induced BSM hyperresponsiveness, probably by blocking TNFα-mediated response. Increasing PGRN levels might be a promising therapeutic for the AHR in allergic asthma.
In vitro and animal studies revealed miRs to be involved in modulation of hypoxia-induced pulmonary hypertension (HPH). However, knowledge on circulating miRs in humans in the context of HPH is very limited. Since symptoms of HPH are non-specific and non-invasive diagnostic parameters do not exist, a disease-specific and hypoxemia-independent biomarker indicating HPH would be of clinical value. To examine whether plasma miR levels correlate with hypoxia-induced increase in pulmonary artery pressures, plasma miRs were assessed in a model of hypoxia-related pulmonary hypertension in humans exposed to extreme altitude. 40 healthy volunteers were repetitively examined during a high altitude expedition up to an altitude of 7050m. Plasma levels of miR-17, -21 and -190 were measured by quantitative Real-time (qRT)-PCR and correlated with systolic pulmonary artery pressure (SPAP), which was assessed by echocardiography. A significant altitude-dependent increase in circulating miR expression was found (all p-values <0.0001). Compared to baseline at 500m, miR-17 changed by 4.72 ± 0.57 fold, miR-21 by 1.91 ± 0.33, and miR-190 by 3.61 ± 0.54 fold at 7050m. MiR-17 and miR-190 were found to be independently correlated with increased SPAP, even after adjusting for hypoxemia. Progressive hypobaric hypoxia significantly affects levels of circulating miR-17, -21, and -190. MiR-17 and -190 significantly correlate with increased SPAP, independently from the extent of hypoxemia. These novel findings provide evidence for an epigenetic modulation of hypoxia-induced increase in pulmonary artery pressures by miR-17 and -190 and suggest a potential value of these miRs as biomarkers for HPH.
The objective of lung development is to generate an organ of gas exchange that provides both a thin gas diffusion barrier and a large gas diffusion surface area, which concomitantly generates a steep gas-diffusion concentration gradient. As such, the lung is perfectly structured to undertake the function of gas exchange: a large number of small alveoli provide extensive surface area within the limited volume of the lung, and a delicate alveolo-capillary barrier brings circulating blood into close proximity to the inspired air. Efficient movement of inspired air and circulating blood through the conducting airways and conducting vessels, respectively, generates steep oxygen and carbon dioxide concentration gradients across the alveolo-capillary barrier, providing ideal conditions for effective diffusion of both gases during breathing. The development of the gas exchange apparatus of the lung occurs during the second phase of lung development - namely, late lung development - which includes the canalicular, saccular and alveolar stages of lung development. It is during these stages of lung development that preterm-born infants are delivered, when the lung is not yet competent for effective gas exchange. These infants may develop bronchopulmonary dysplasia (BPD), a syndrome complicated by disturbances to the development of the alveoli and the pulmonary vasculature. It is the objective of this Perspective article to update the reader about recent developments that further our understanding of the mechanisms of lung alveolarization and vascularization, and the pathogenesis of BPD and other neonatal lung diseases that feature lung hypoplasia.
A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin-remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor, T1R3 is a GPCR, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified however, no studies have focused on T1R3 within the vasculature. We hypothesise that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule, sucralose, attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signalling, through zinc sulfate, T1R3 or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of key signalling molecules, Src, PAK, MLC2, HSP27 and p110αPI3K. Activation of T1R3, by sucralose, protects the pulmonary endothelium from edemagenic-agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin-remodelling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS.
Endothelial-to-mesenchymal transition (EndMT) is a process in which endothelial cells lose polarity and cell-to-cell contacts, and undergo a dramatic remodeling of the cytoskeleton. It has been implicated in initiation and progression of pulmonary arterial hypertension (PAH). However, the characteristics of cells which have undergone EndMT-cells in vivo have not been reported and so remain unclear. To study this, sugen5416 and hypoxia (SuHx)-induced PAH was established in Cdh5-Cre / Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J double-transgenic mice, in which GFP was stably expressed in pan-endothelial cells. After 3 weeks of SuHx, flow cytometry and immunohistochemistry demonstrated CD144-negative and GFP-positive cells (complete EndMT-cells) possessed higher proliferative and migratory activity compared to other mesenchymal cells. While CD144-positive and α-SMA-positive cells (partial EndMT-cells) continued to express endothelial progenitor cell markers, complete EndMT-cells were Sca-1-rich mesenchymal cells with high proliferative and migratory ability. When transferred in fibronectin-coated chamber slides containing smooth muscle media, α-SMA robustly expressed in these cells compared to cEndMT-cells that were grown in maintenance media. Demonstrating additional paracrine effects, conditioned medium from isolated complete EndMT-cells induced enhanced mesenchymal proliferation and migration, and increased angiogenesis as compared to conditioned medium from resident mesenchymal cells. Overall, these findings show that EndMT-cells could contribute to the pathogenesis of PAH both directly by transformation into smooth muscle-like cells with higher proliferative and migratory potency, as well as indirectly, through paracrine effects on vascular intimal and medial proliferation.
The tryptophan metabolite kynurenine is significantly increased in pulmonary arterial hyper-tension (PAH) patients, and it is a potent vasodilator of systemic arteries. Our aim was to in-vestigate the role of kynurenine in the pulmonary circulation. Serum tryptophan, kynurenine and kynurenic-acid levels were measured in 20 idiopathic PAH (IPAH) patients, 20 healthy controls and 20 patients with chronic lung disease or meta-bolic syndrome without-PH. Laser-dissected pulmonary arteries from IPAH and control lungs were tested for the expression of IDO, the rate-limiting enzyme for the conversion from tryptophan to kynurenine. Acute effects of kynurenine were tested in pulmonary vascular preparations, two different models of chronic PH, and in human pulmonary arterial smooth muscle cells (hPASMCs). In IPAH vs. control serum, kynurenine was significantly elevated (3.6±0.2µM vs. 2.6±0.1µM, p<0.0001), and strongly associated with PH (AUC=0.86), but kynurenine levels were not ele-vated in lung disease and metabolic syndrome. Among all investigated tryptophan metabo-lites, kynurenine displayed the strongest correlation with mean pulmonary arterial pressure (mPAP) (:0.770, p<0.0001). Tryptophan was significantly decreased in IPAH lungs, however, IDO expression was not changed. In hPASMCs, kynurenine increased both cAMP and cGMP, in intrapulmonary arteries it relaxed the pre-constriction via NO/cGMP and cAMP pathways, and in two models of established PH it acutely decreased the mPAP. Our data suggest that kynurenine elevation might be specifically associated with mPAP; kynurenine acts on hPASMCs in synergy with NO and exerts acute pulmonary vasodilatation in chronic PH models. Kynurenine might provide both a new biomarker and a new therapeutic option for PH.
Microparticles are a newly recognized class of mediators in the pathophysiology of lung inflammation and injury, but little is known about the factors that regulate their accumulation and clearance. The primary objective of our study was to determine whether alveolar macrophages engulf microparticles and to elucidate the mechanisms by which this occurs. Alveolar microparticles were quantified in bronchoalveolar fluid of mice with lung injury induced by LPS and hydrochloric acid. Microparticle numbers were greatest at the peak of inflammation and declined as inflammation resolved. Isolated, fluorescently labeled particles were placed in culture with macrophages to evaluate ingestion in the presence of endocytosis inhibitors. Ingestion was blocked with cytochalasin D and wortmannin, consistent with a phagocytic process. In separate experiments, mice were treated intratracheally with labeled microparticles and their uptake was assessed though microscopy and flow cytometry. Resident alveolar macrophages, not recruited macrophages, were the primary cell ingesting microparticles in the alveolus during lung injury. In vitro, microparticles promoted inflammatory signaling in LPS primed epithelial cells, signifying the importance of microparticle clearance in resolving lung injury. Microparticles were found to have phosphatidylserine exposed on their surfaces. Accordingly, we measured expression of phosphatidylserine receptors on macrophages and found high expression of MerTK and Axl on the resident macrophage population. Endocytosis of microparticles was markedly reduced in MerTK-deficient macrophages in vitro and in vivo. In conclusion, microparticles are released during acute lung injury and peak in number at the height of inflammation. Resident alveolar macrophages efficiently clear these microparticles through MerTK mediated phagocytosis.
Angiotensin converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-arg9 bradykinin (DABK) in airway epithelial cells based upon a hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/BKB1R axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R (bradykinin receptor B1) axis, release of the proinflammatory chemokines such as CXCL5，MIP2, KC and TNF- MIP2 and TNF-a from airway epithelia, increased neutrophil infiltration and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part due to an impaired ability to inhibit DABK/BKB1R axis mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.
Pneumoconiosis is an occupational disease accompanied by long-term lung impairment for which prediction of prognosis is poorly understood because of the complexity of the inhaled particles. Micro-proton-induced X-ray emission (micro-PIXE) analysis, which is advantageous for high-sensitivity 2D element mapping of lung tissues, was used to investigate element-based predictive factors of prognosis in Chinese patients with welder's and coal miner's pneumoconiosis. Chest radiographs and lung function tests showed that most of the coal miners deteriorated, while symptoms in some welders were alleviated after five years, as determined by comparing percent vital capacity (VC%) and forced expiratory volume in the first second over forced vital capacity (FEV1.0/FVC) to values taken at the initial diagnosis. Micro-PIXE analysis suggested the most abundant particulates in welder's pneumoconiosis were Fe, Mn, and Ti (metallic oxide), accompanied by particulates containing Si, Al, and Ca (aluminum silicate) or only Si (SiO2); the most abundant particulates in coal miner's pneumoconiosis were composed of C, Si, Al, K, and Ti, accompanied by particulates containing Ca or Fe. Particulates containing Al, Si, S, K, Ca, and Ti (orthoclase and anorthite) were correlated with severity of fibrosis. Multivariable linear regression suggested long-term FEV1.0/FVC decrease was independently associated with Si and smoking index, while VC% decrease was associated with Si and Ti. A risk index comprised of these factors was developed to predict the prognosis of pneumoconiosis. Micro-PIXE analysis is feasible for the evaluation of elemental composition and dust exposure, especially for patients whose exposure is mixed or uncertain.
We recently demonstrated that blue light induces vasorelaxation in the systemic mouse circulation, a phenomenon mediated by the non-visual G protein-coupled receptor (GPCR) melanopsin (opsin 4; Opn4). Here we tested the hypothesis that non-visual opsins mediate photorelaxation in the pulmonary circulation. We discovered Opsin 3 (Opn3), Opn4, and G-protein coupled receptor kinase 2 (GRK2) in rat pulmonary arteries (PAs) and in pulmonary arterial smooth muscle cells (PASMCs), where the opsins interact directly with GRK2 as demonstrated with a proximity ligation assay. Light elicited an intensity-dependent relaxation of PAs pre-constricted with phenylephrine (PE), with a maximum response between 400-460 nm (blue light). Wavelength-specific photorelaxation was attenuated in PAs from Opn4-/- mice and further reduced following shRNA-mediated knockdown of Opn3. Inhibition of GRK2 amplified the response and prevented physiologic desensitization to repeated light exposure. Blue light also prevented PE-induced constriction in isolated PAs, decreased basal tone, ablated PE-induced single-cell contraction of PASMCs, and reversed PE-induced depolarization in PASMCs when GRK2 was inhibited. The photorelaxation response was modulated by soluble guanylyl cyclase, but not by protein kinase G or nitric oxide. Most importantly, blue light induced significant vasorelaxation of PAs from rats with chronic pulmonary hypertension and effectively lowered pulmonary arterial pressure (PPA) in isolated intact perfused rat lungs subjected to acute hypoxia. These findings show that functional Opn3 and Opn4 in PAs represent an endogenous "optogenetic system" that mediates photorelaxation in the pulmonary vasculature. Phototherapy in conjunction with GRK2 inhibition could therefore provide an alternative treatment strategy for pulmonary vasoconstrictive disorders.
Our previous study showed that invariant natural killer T (iNKT) cells might act as an adjuvant to promote Th2 inflammatory responses in an OVA-induced mouse model of allergic asthma, but the mechanism remains unknown. To clarify the underlying mechanism through which iNKT cells promote Th2 inflammatory responses, we investigated the modulatory influence of iNKT cells on phenotypic and functional maturation of lung dendritic cells (LDCs) using iNKT cell-knockout mice, specific iNKT cell activation, co-culture experiments, and adoptive transfer of iNKT cells in mouse models of asthma. Our data showed that iNKT cell deficiency could downregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from a mouse model of asthma. However, elevated activation of iNKT cells by α-Galactosylceramide and adoptive transfer of iNKT cells could upregulate surface maturation markers and proinflammatory cytokine secretion of LDCs from mouse models of asthma. Meanwhile, iNKT cells significantly influenced the function of LDCs, markedly enhancing Th2 responses in vivo and in vitro. In addition, iNKT cell can induce LDCs expression of CD206 and RELM-α, reflecting alternative activation of LDCs in a mouse model of asthma. α-Galactosylceramide treatment significantly enhanced expression of CD40L of lung iNKT cells from a mouse model of asthma, and the co-culture experiment of LDCs with iNKT cells showed the blockade of CD40L strongly suppressed surface maturation markers and proinflammatory cytokine production by LDCs. Our data suggest that iNKT cells can promote immunogenic maturation of LDCs to enhance Th2 responses in mouse models of asthma.
Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression using bone marrow derived macrophages (BMDM) from mice lacking specific Toll-like receptors (TLR), TLR adaptor molecules or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [Poly(I:C)] trigger a signaling cascade involving TLR3 or 4, the Trif adaptor, Type I interferons and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (Has1) and syndecan-4 in macrophages. Has1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3 or TLR4 and both MyD88 and Trif. Neither Has1 nor syndecan-4 is dependent on Type 1 interferons. The importance of macrophage-derived versican in lungs was determined using LysM/Vcan-/- mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of Poly(I:C)-treated LysM/Vcan-/- mice, as compared to control mice. IFN-β and IL-10, two important anti-inflammatory molecules, are significantly decreased in both Poly(I:C) treated BMDM from LysM/Vcan-/- mice and BAL fluid from Poly(I:C)-treated LysM/Vcan-/- mice, as compared to control mice. In short, Type I interferon signaling regulates versican expression and versican is necessary for Type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.
During the 1918 influenza pandemic, children experienced substantially lower mortality than adults, a striking but unexplained finding. Whether this was due to enhanced resistance (reduced virus load) or better tolerance (reduced impact of infection) has not been defined. We found that prepubertal mice infected with H1N1 influenza virus also showed greater survival than infected pubertal mice, despite similar virus loads. Transcriptome profiling of infected lungs identified estrogen as a regulator of susceptibility in both sexes, and also linked better survival to late expression of IL-1beta. Blocking puberty with gonadectomy or a GnRH antagonist improved survival. Estrogen or testosterone (which can be converted to estrogen) restored susceptibility of gonadectomized pubertal mice to influenza mortality, but dihydrotestosterone (which cannot be converted to estrogen) did not. Estrogen receptor blockade with fulvestrant in both male and female pubertal mice resulted in improved survival, even when given three days after infection. Moreover, late, but not early, IL-1beta neutralization after infection was also protective. These findings indicate that pubertal increases in estrogen in both sexes are associated with increased mortality during influenza. This helps explain the reduced mortality of children seen with influenza in 1918, and might also be relevant to childhood tolerance to many other infectious diseases.
The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid-subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit ARDS model (repeated airway lavage, overventilation, LPS instillation into airways), we assessed whether the supplementation of surfactant (poractant alfa, S) with inositol-derivatives (inositol-1,2,6-trisphosphate, IP3; phosphatidylinositol-3,5-bisphosphate, PIP2) or phosphatidylglycerol-subfractions (16:0/18:1-POPG; 18:1/18:1-DOPG) would result in improved clinical parameters, and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3/PIP2/POPG), the ventilation efficiency index (S+IP3/POPG), the compliance (S+IP3/POPG) and the resistance (S+POPG) of the respiratory system, and the extra-vascular lung water index (S+IP3/POPG) significantly improved compared to S treatment alone. The inositol-derivatives (mainly S+IP3) exerted their actions by suppressing the acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome NLRP3/ASC/caspase-1 complex, and the pro-fibrotic response represented by the cytokines TGF-β1 and IFN-, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IB-kinase activity was significantly reduced. S+POPG/DOPG treatment inhibited polymorpho-nuclear leukocyte activity (MMP-8, myeloperoxidase), the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol-derivatives and phosphatidylglycerol-subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease, and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.
Idiopathic Pulmonary Fibrosis (IPF) is a progressive fibrotic lung disease, but the mechanisms driving progression remain incompletely defined. We previously reported that the IPF lung harbors fibrogenic mesenchymal progenitor cells (MPCs), which serve as a cell-of-origin for IPF fibroblasts. Proliferating IPF MPCs are located at the periphery of fibroblastic foci in an active cellular front at the interface between the myofibroblast rich focus core and adjacent normal alveolar structures. Among a large set of genes that distinguish IPF MPCs from their control counterparts we identified IL-8 as a candidate mediator of IPF MPC fibrogenicity and driver of fibrotic progression. IPF MPCs and their progeny displayed increased steady state levels of IL-8 and its cognate receptor CXCR1 and secreted more IL-8 than did controls. IL-8 functioned in an autocrine manner promoting IPF MPC self-renewal and the proliferation and motility of IPF MPC progeny. Secreted IL-8 also functioned in a paracrine manner stimulating macrophage migration. Analysis of IPF lung tissue demonstrated co-distribution of IPF MPCs with activated macrophages in the active cellular front of the fibroblastic focus. These findings indicate that IPF MPC derived IL-8 is capable of expanding the mesenchymal cell population and recruiting activated macrophages cells to actively evolving fibrotic lesions.
The mechanisms of aging that are involved in the development of idiopathic pulmonary fibrosis (IPF) are still unclear. Although it has been hypothesized that the proliferation and activation of human lung fibroblasts (hLF) are essential in IPF, no studies have assessed how this process works in an aging lung. Our goal was to elucidate if there were age-related changes on primary hLF isolated from IPF lungs compared to age-matched controls. We investigated several hallmarks of aging in hLF from IPF patients and age-matched controls. IPF hLF have increased cellular senescence with higher expression of β-galactosidase, p21, p16, p53 and cytokines related to the senescence-associated secretory phenotype (SASP) as well as decreased proliferation/apoptosis compared to age-match controls. Additionally, we observed shorter telomeres, mitochondrial dysfunction, and upon TGF-β stimulation, increased markers of ER stress. Our data suggests that IPF hLF develop senescence resulting in a decreased apoptosis and that the development of SASP may be an important contributor to the fibrotic process observed in IPF. These results might change the existing paradigm, which describes fibroblasts as aberrantly activated cells, to a cell with a senescence phenotype.
Rationale: Individuals with alcohol use disorders (AUDs) are at an increased risk of pneumonia and acute respiratory distress syndrome. Data of the lung microbiome in the setting of AUDs are lacking. The objective of this study was to determine the microbial biogeography of the upper and lower respiratory tract in individuals with AUDs compared to non-AUD subjects. Materials and Methods: Gargle, protected bronchial brush, and bronchoalveolar lavage specimens were collected during research bronchoscopies. Bacterial 16S gene sequencing and phylogenetic analysis was performed, and the alterations to the respiratory tract microbiota and changes in microbial biogeography were determined. Results: The microbial structure of the upper and lower respiratory tract was significantly altered in subjects with AUDs compared to controls. Subjects with AUD have greater microbial diversity (p < 0.0001, Effect Size (ES) = 16 ± 1.7 observed taxa) and changes in microbial species relative abundances. Further, microbial communities in the upper and lower respiratory tract displayed greater similarity in subjects with AUDs. Conclusions: Alcohol use is associated with an altered composition of the respiratory tract microbiota. Subjects with AUDs demonstrate convergence of the microbial phylogeny and taxonomic communities between distinct biogeographical sites within the respiratory tract. These results support a mechanistic pathway potentially explaining the increased incidence of pneumonia and lung diseases in patients with AUDs.
Pulmonary fibrosis is characterized by lung fibroblast activation and ECM deposition and has a poor prognosis.Heat shock protein 90 (Hsp90) participates in organ fibrosis and extracellular Hsp90α (eHsp90α) promotes fibroblast activation and migration. This study aimed to investigate whether a selective anti-Hsp90α monoclonal antibody, 1G6-D7, could attenuate lung fibrosis and whether 1G6-D7 presents protective effect by inactivating profibrotic pathway.Our results showed that eHsp90α was increased in mice with BLM-induced pulmonary fibrosis and that 1G6-D7 attenuated inflammation and collagen deposition in the lung. TGF-β1 induced eHsp90α secretion, concomitantly promoting HFL-1 activation and ECM synthesis.1G6-D7-mediated inhibition of eHsp90α significantly blocked these effects,meanwhile inhibiting downstream profibrotic pathways such as ERK, AKT, and P38.Human recombinant (hr)Hsp90α mimicked the effects of TGF-β1, by activating profibrotic pathways and by upregulating LRP-1.Moreover,ERK inhibition effectively blocked the effect of (hr)Hsp90α.In conclusion, 1G6-D7 significantly protects against BLM-induced pulmonary fibrosis by ameliorating fibroblast activation and ECM production,which may be through blocking ERK signaling. Our results suggest a safer molecular therapy, 1G6-D7, in pulmonary fibrosis.
Elevated active plasminogen activator inhibitor 1 (PAI-1) has an adverse effect on the outcomes of intrapleural fibrinolytic therapy (IPFT) in tetracycline-induced pleural injury in rabbits. In order to enhance IPFT with prourokinase (scuPA), two mechanistically distinct approaches to targeting PAI-1 were tested: slowing its reaction with urokinase (uPA) and mAb-mediated PAI-1 inactivation. Removing positively charged residues at the "PAI-1 docking site" (179RHRGGS184->179AAAAAA184) of uPA results in a 60-fold decrease in the rate of inhibition by PAI-1. Mutant prourokinase (0.0625-0.5mg/kg; n=12) showed efficacy comparable to wt-scuPA and did not change IPFT outcomes (p>0.05). Notably, the rate of PAI-1 independent intrapleural inactivation of the mutant uPA was two times higher (p<0.05) than that of the wt-enzyme. Trapping PAI-1 in a "molecular sandwich" type complex with catalytically inactive S195A-tcuPA (0.1 and 0.5mg/kg) did not improve the efficacy of IPFT with scuPA (0.0625-0.5mg/kg; n=11). IPFT failed in the presence of MA-56A7C10 (0.5mg/kg; n=2), which forms a stable intrapleural "molecular sandwich" complex, allowing active PAI-1 to accumulate by blocking its transition to a latent form. In contrast, inactivation of PAI-1 by accelerating the active-to-latent transition mediated by mAb MA-33B8 (0.5mg/kg; n=2) improved the efficacy of IPFT with scuPA (0.25mg/kg). Thus, under conditions of slow (4-8h) fibrinolysis in tetracycline-induced pleural injury in rabbits, only the inactivation of PAI-1, but not a decrease in the rate of its reaction with uPA, enhances IPFT. Therefore, the rate of fibrinolysis, which varies in different pathologic states, could affect the selection of PAI-1 inhibitors to enhance fibrinolytic therapy.
There is no therapeutic intervention proven to prevent the acute respiratory syndrome (ARDS). Novel mechanistic insights into the pathophysiology of ARDS are therefore required. Platelets are implicated in regulating many of the pathogenic processes which occur during ARDS, however the mechanisms remain elusive. The platelet receptor C-type lectin-like 2 (CLEC-2) has been shown to regulate vascular integrity at sites of acute inflammation. Therefore, the purpose of this study was to establish the role of CLEC-2 and its ligand podoplanin in a mouse model of ARDS. Platelet-specific CLEC-2-deficient, as well as alveolar epithelial type I cell (AECI)-specific- or hematopoietic-specific podoplanin deficient mice were established using cre-loxP strategies. Combining these with intratracheal (IT) instillations of lipopolysaccharide (LPS), we demonstrate that arterial oxygen saturation decline in response to IT-LPS in platelet-specific CLEC-2-deficient mice is significantly augmented. An increase in bronchoalveolar lavage (BAL) neutrophils and protein was also observed 48h post IT-LPS, with significant increases in pro-inflammatory chemokines detected in BAL of platelet-specific CLEC-2 deficient animals. Deletion of podoplanin from hematopoietic cells but not AECIs also reduces lung function and increases pro-inflammatory chemokine expression following IT-LPS. Furthermore we demonstrate that following IT-LPS, platelets are present in BAL in aggregates with neutrophils which allows for CLEC-2 interaction with podoplanin expressed on BAL inflammatory alveolar macrophages. Taken together these data suggest that the platelet CLEC-2-podoplanin signaling axis regulates the severity of lung inflammation in mice and is a possible novel target for therapeutic intervention in patients at risk of developing ARDS.
We previously proposed a role for the 2-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy between the 3 TREK isoforms (TREK-1, TREK-2, TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all 3 TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient (triple ko) mice to either room air, 72 hours HO, MV (high and low tidal volume), or a combination of HO+MV, and measured quasi-static lung compliance, BAL protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression, and attempted to prevent HO-induced lung injury by prophylactically administering exogenous surfactant (Curosurf). HO treatment increased lung injury in triple ko but not WT mice, including an elevated LIS, BAL protein concentration, markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO+MV (low TV) further decreased lung compliance in triple ko but not WT mice, and HO+MV (high TV) was lethal for triple ko mice. In triple ko mice, the HO-induced lung injury was associated with decreased surfactant protein A (SPA) and SPC, but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors NF-1, NKX2.1/TTF or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple ko mice.
The early history of cardiac catheterization has many interesting features. First, although it would be natural to assume that the procedure was initiated by cardiologists, two of the three people who shared the Nobel Prize for the discovery were pulmonologists, while the third was a urologist. The primary objective of the pulmonologists, André Cournand and Dickinson Richards, was to obtain mixed venous blood from the right heart so that they could use to use the Fick principle to calculate total pulmonary blood flow. Cournand's initial catheterization studies were prompted by his reading an account by Werner Forssmann who catheterized himself 12 years before. His bold experiment was one of the most bizarre in medical history. In the earliest studies that followed, Cournand and colleagues first passed catheters into the right atrium, then into the right ventricle, and finally the pulmonary artery. At the time, the investigators did not appreciate the significance of the low vascular pressures, nor that what they had done would revolutionize interventional cardiology. Within a year, William Dock predicted that there would be a very low blood flow at the top of the upright lung, and he proposed that this was the cause of the apical localization of pulmonary tuberculosis. The fact that the pulmonary vascular pressures are very low has many implications in lung disease. Cardiac catheterization changed the face of investigative cardiology, and its instigators were awarded the Nobel Prize in 1956.
Levosimendan has a calcium-sensitizing effect in the myocardium and opens adenosine-triphosphate-sensitive potassium channels (KATP) in vascular smooth muscle. Since airway smooth muscle also expresses KATP, we characterized the protective potential of levosimendan against increased airway and respiratory tissue resistances. Animals were administered levosimendan alone (Group L), levosimendan after pretreatment with a KATP channel blocker (glibenclamide, Group LG), glibenclamide only (Group G), or solvent alone (dextrose, Group C). Airway resistance (Raw), tissue damping (G) and elastance were determined by forced oscillations under baseline conditions and following provocation tests with intravenous methacholine (MCh). Cardiac output (CO) was assessed by transpulmonary thermodilution. The same sequence of measurements was then repeated during intravenous infusion of levosimendan in Groups L and LG or glucose in Groups G and C. Sham treatments in Group C and G had no effect on lung responsiveness. However, levosimendan treatment in Group L elevated CO and inhibited the MCh-induced airway responses (Raw changes of 87.8±83[SD]% vs. 24.4±16% at 4μg/kg/min MCh, p<0.001), and in G (35.2±12.7% vs. 25.2±12.9%, p<0.05). The preventive affect of levosimendan against lung constriction vanished in the LG group. Levosimendan exerts a KATP-mediated potential to prevent bronchoconstriction and may prohibit adverse lung peripheral changes both in the small bronchi and the pulmonary parenchyma. The identification of a further pleiotropic property of levosimendan that is related to the pulmonary system is of particular importance for patients with decreased cardiorespiratory reserves, for which simultaneous circulatory support is complemented with prevention of adverse respiratory events.
The acute respiratory distress syndrome (ARDS) is a common and devastating disorder. Alcohol use disorders (AUDs) increase ARDS risk, and worsen outcomes, through mechanisms that may include enhancing pulmonary oxidative stress. Alcohol consumption increases activity of the enzyme xanthine oxidoreductase (XOR) that contributes to production of both reactive oxygen species (ROS) and uric acid, a damage-associated molecular pattern. These by-products have the potential to modulate pro-inflammatory pathways such as those involving cyclooxygenase (COX)-2, and to activate the nucleotide-binding domain, leucine-rich-containing family, pyrin-domain containing-3 (NLRP3) inflammasome. We sought to determine if pulmonary and systemic XOR activity were altered by AUDs. Bronchoscopy with bronchoalveolar lavage (BAL) and blood sampling were performed in otherwise healthy human subjects with AUDs and controls. Uric acid in epithelial lining fluid derived from BAL was substantially higher among individuals with AUDs, and did not normalize after 7 days of abstinence; serum uric acid did not differ across groups. XOR enzyme activity in fresh BAL cells and serum was significantly increased in subjects with AUDs. XOR protein in BAL cells from AUD subjects was increased in parallel with COX-2 expression, and further, mRNA expression of NLRP3 inflammasome components was sustained in LPS-stimulated BAL cells from AUD subjects in conjunction with increased IL-1β. Our data suggest that AUDs augment pulmonary and systemic XOR activity that may contribute to ROS and uric acid generation, promoting inflammation. Further investigations will be necessary to determine if XOR inhibition can mitigate alcohol-associated pulmonary oxidative stress, diminish inflammation, and improve ARDS outcomes.
Mice that globally over-express the transcription factor, Fos-related antigen-2 (Fra-2), develop extensive pulmonary fibrosis and pulmonary vascular remodeling. To determine if these phenotypes are a consequence of ectopic Fra-2 expression in vascular smooth muscle cells and myofibroblasts, we generated mice that over-express Fra-2 specifically in these cells types (α-SMA-rtTA; tetO-Fra-2). Surprisingly, these mice did not develop vascular remodeling or pulmonary fibrosis, but developed a spontaneous emphysema-like phenotype characterized by alveolar enlargement. Secondary septa formation is an important step in the normal development of lung alveoli and α-SMA expressing fibroblasts (myofibroblasts) play a crucial role in this process. The mutant mice showed reduced numbers of secondary septa at P7 and enlarged alveolar size starting at P12, suggesting that the process of secondary septa formation was impaired. Lineage tracing using α-SMA-rtTA mice crossed to a floxed TdTomato reporter revealed that embryonic expression of α-SMA cre marked a population of cells that gave rise to nearly all alveolar myofibroblasts. Comprehensive transcriptome analyses (RNA sequencing) demonstrated that the overwhelming majority of genes whose expression was significantly altered by over-expression of Fra-2 in myofibroblasts encoded secreted proteins, components of the extracellular matrix (ECM) and cell adhesion associated genes, including coordinate upregulation of pairs of integrins and their principal ECM ligands. In addition, primary myofibroblasts isolated from the mutant mice showed reduced migration capacity. These findings suggest that Fra-2 over-expression might impair myofibroblast functions crucial for secondary septation, such as myofibroblast migration across alveoli, by perturbing interactions between integrins and locally produced components of the ECM.
Bronchopulmonary dysplasia (BPD) is characterized by impaired alveolar secondary septation and vascular growth. Exposure to high concentrations of oxygen (hyperoxia) contributes to the development of BPD. Male sex is considered an independent risk factor for the development of BPD. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. We hypothesized that sex-specific modulation of biological processes in the lung under hyperoxic conditions, contributes to sex-based differences. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia (95% FiO2, post-natal day (PND) 1-5: saccular stage of lung development) and euthanized on PND 7 or 21. Pulmonary gene expression was studied using RNA-Seq on the Illumina HiSeq 2500 platform. Analysis of the pulmonary transcriptome revealed differential sex-specific modulation of crucial pathways such as: angiogenesis, response to hypoxia, inflammatory response and p53 pathway. Candidate genes from these pathways were validated at the mRNA level by qPCR. Analysis also revealed sex-specific differences in the modulation of crucial transcription factors. Focusing on the differential modulation of the angiogenesis pathway, we also show sex-specific differential activation of Hif-1α regulated genes using ChIP-qPCR and differences in expression of crucial genes (Vegf, VegfR2, and Phd2) modulating angiogenesis. We demonstrate the translational relevance of our findings by showing that our murine sex-specific differences in gene expression correlate with those from a pre-existing human BPD dataset. In conclusion, we provide novel molecular insights into differential sex-specific modulation of the pulmonary transcriptome in neonatal hyperoxic lung injury and highlight angiogenesis as one of the crucial differentially modulated pathways.
The National Heart Lung and Blood Institute is funding an effort to create a molecular atlas of the developing lung (LungMAP) to serve as a research resource and public education tool. The lung is a complex organ with lengthy development time driven by interactive gene networks and dynamic crosstalk among multiple cell types to control and coordinate lineage specification, cell proliferation, differentiation, migration, morphogenesis and injury repair. A better understanding of the processes that regulate lung development, particularly alveologenesis, will have significant impact on survival rates for premature infants born with incomplete lung development and will facilitate lung injury repair and regeneration in adults. A consortium of four research centers, data coordinating center and human tissue repository will provide high-quality molecular data of developing human and mouse lungs. LungMAP includes mouse and human data for cross-correlation of developmental processes across species. LungMAP is generating foundational data and analysis, creating a web portal for presentation of results and public sharing of datasets, establishing a repository of young human lung tissues obtained through organ donor organizations, and developing a comprehensive lung ontology that incorporates the latest findings of the consortium. The LungMAP website (www.lungmap.net) currently contains over 5000 high resolution lung images, transcriptomic, proteomic, and lipidomic data and provides scientific information to stimulate interest in research careers for young audiences. This paper presents a brief description of research conducted by the consortium, database and portal development and upcoming features that will enhance the LungMAP experience for a community of users.
A mucosal oxidative burst is a hallmark response to pollen exposure that promotes allergic inflammatory responses. Reactive species constituents of oxidative stress signal via the modification of cellular molecules including nucleic acids. One of the most abundant oxidative genomic base damage is 8-oxo-7,8-dihydroguanine (8-oxoG), which is removed from DNA by 8-oxoguanine DNA glycosylase1 (OGG1). OGG1 in complex with 8-oxoG acts as a GDP-GTP exchange factor and induces acute inflammation; however, the mechanism(s) by which OGG1 signaling regulates allergic airway inflammation is not known. Here, we postulate that the OGG1 signaling pathway differentially altered the levels of small regulatory RNAs and increased the expression of T helper 2 (Th2) cytokines in ragweed pollen extract (RWPE)-challenged lungs. To determine this, the lungs of sensitized mice expressing or lacking OGG1 were challenged with RWPE and/or with OGG1's excision product 8-oxoG. The responses in lungs were assessed by next-generation sequencing, as well as various molecular and histological approaches. The results showed that RWPE challenge induced oxidative burst, damage to DNA and activated OGG1 signaling, resulting in the differential expression of 84 microRNAs, which then exacerbated antigen-driven allergic inflammation and histological changes in the lungs. The exogenous administration of the down-regulated let-7b-p3 mimetic or inhibitors of up-regulated miR23a or miR27a decreased eosinophil recruitment, mucus and collagen production via controlling the expression of IL4, IL5, and IL13. Together, these data demonstrate the roles of OGG1 signaling in the regulation of antigen-driven allergic immune responses via differential expressions of microRNAs upstream of Th2 cytokines and eosinophils.
Radiation-induced pulmonary fibrosis (RIPF) is one of the most common side effects of lung cancer radiotherapy. This study was conducted to identify the molecular mechanism responsible for RIPF. We revealed that the transcriptional level of cytochrome P450 2E1 (CYP2E1) was elevated by examining expression profile analysis of RIPF mouse models. We also confirmed that CYP2E1 regulated levels of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) in alveolar epithelial type II (AE2) cells and lung fibroblasts. Inhibition of CYP2E1 via its siRNA or inhibitor significantly attenuated epithelial-to-mesenchymal transition and apoptosis of AE2 cells, as well as myofibroblast formation induced by radiation. Finally, the effects of a CYP2E1 inhibitor on development of RIPF were evaluated by in vivo studies. Taken together, the results of the present study suggest that CYP2E1 is an important mediator of RIPF development that functions by increasing cellular ER stress and ROS levels.
Infants born prematurely often require supplemental oxygen that contributes to aberrant lung development and increased pulmonary morbidity following a respiratory viral infection. We have been using a mouse model to understand how early-life hyperoxia affects the adult lung response to influenza A virus (IAV) infection. Prior studies showed how neonatal hyperoxia (100% oxygen) increased sensitivity of adult mice to infection with influenza A virus (IAV (A/Hong Kong/X31) H3N2) as defined by persistent inflammation, pulmonary fibrosis, and mortality. Since neonatal hyperoxia alters lung structure, we used a novel fluorescently expressing reporter strain of H1N1 IAV (A/Puerto Rico/8/34 mCherry, PR8 mCherry) to evaluate whether it also altered early infection of the respiratory epithelium. Like HKx31, neonatal hyperoxia increased morbidity and mortality of adult mice infected with PR8-mCherry. Whole lung imaging and histology suggested a modest increase in mCherry expression in adult mice exposed to neonatal hyperoxia when compared to room air exposed animals. However, this did not reflect an increase in airway or alveolar epithelial infection when mCherry + cells were identified and quantified by flow cytometry. Instead, a modest increase in the number of CD45+ macrophages expressing mCherry was detected. While neonatal hyperoxia does not alter early epithelial infection to IAV, it may increase the activity of macrophages towards infected cells thereby enhancing early epithelial injury.
Activation of oxytocin receptors has shown benefits in animal models of Obstructive Sleep Apnea (OSA). We tested if nocturnal oxytocin administration could have beneficial effects in OSA patients. 8 patients diagnosed with OSA were administered intranasal oxytocin (40 i.u.). Changes in cardiorespiratory events during sleep, including apnea and hypopnea durations and frequency, risk of event-associated arousals, and heart rate variability were assessed. Oxytocin significantly increased indices of parasympathetic activity, including heart rate variability, total sleep time, and the Post-Polysommogram Sleep Assessment (PPSA) score, an index of self-reported sleep satisfaction. Although the Apnea-Hypopnea Index (AHI) was not significantly changed with oxytocin administration, when apnea and hypopnea events were compared independently, the frequency of hypopneas, but not apneas, were significantly (p<.005) decreased with oxytocin treatment. Both apneas and hypopneas were significantly shortened in duration with oxytocin treatment. Oxytocin treatment significantly decreased the percent of apnea and hypopnea events that were accompanied with an arousal. Oxytocin administration has the potential to restore cardiorespiratory homeostasis and reduce some clinically important (objective and patient-reported) adverse events that occur with OSA. Additional studies are needed to further understand the mechanisms by which oxytocin promotes these changes in cardiorespiratory and autonomic function in OSA patients.
Metabolic reprogramming has been intrinsically linked to macrophage activation. Alveolar macrophages are known to play an important role in the pathogenesis of pulmonary fibrosis. However, systematic characterization of expression profile in these cells is still lacking. Furthermore, main metabolic programs and their regulation of cellular phenotype are completely unknown. In this study, we comprehensively analyzed the expression profile and main metabolic programs in alveolar macrophages from mice with or without experimental pulmonary fibrosis. We found that alveolar macrophages from both bleomycin and active TGF-β1 induced fibrotic mouse lungs demonstrated a primarily pro-fibrotic M2-like profile that was distinct from the well-defined M1 or any of the M2 subtypes. More importantly, we found that fibrotic lung alveolar macrophages assumed augmented glycolysis, which was likely attributed to enhanced expression of multiple key glycolytic mediators. We also found that fatty acid oxidation was upregulated in these cells. However, the pro-fibrotic M2-like profile of fibrotic lung alveolar macrophages was not dependent on fatty acid oxidation and synthesis or lipolysis, but instead on glycolysis, in contrast to the typical IL-4 induced macrophages M(IL-4). Additionally, glutaminolysis, a key metabolic program that has been implicated in numerous pathologies, was not required for the pro-fibrotic M2-like phenotype of these macrophages. In summary, our study identifies a unique expression and metabolic profile in alveolar macrophages from fibrotic lungs and suggests glycolytic inhibition as an effective anti-fibrotic strategy in treating lung fibrosis.
Exposure to hypoxia induces migration and proliferation of pulmonary arterial smooth muscle cells (PASMCs), leading to vascular remodeling and contributing to the development of hypoxic pulmonary hypertension. The mechanisms controlling PASMC growth and motility are incompletely understood, although aquaporin 1 plays an important role. In tumor, kidney and stem cells, AQP1 has been shown to interact with β-catenin, a dual function protein that activates the transcription of crucial target genes (i.e., c-Myc and cyclin D1) related to cell migration and proliferation. Thus, the goal of this study was to examine mechanisms by which AQP1 mediates PASMC migration and proliferation, with a focus on β-catenin. Using primary rat PASMCs from resistance level pulmonary arteries infected with adenoviral constructs containing GFP (control; AdGFP), wild-type AQP1 (AdAQP1) or AQP1 with the C-terminal tail deleted (AdAQP1M), we demonstrated that increasing AQP1 expression using AdAQP1 upregulated β-catenin protein levels and the expression (mRNA and protein) of the known β-catenin targets, c-Myc and cyclin D1. In contrast, infection with AdAQP1M had no effect on any of these variables. Using silencing approaches to reduce β-catenin levels prevented both hypoxia- and AQP1-induced migration and proliferation of PASMCs. Thus, our results indicate that elevated AQP1 levels upregulate β-catenin protein levels, via a mechanism requiring the AQP1 C-terminal tail, enhancing expression of β-catenin targets and promoting PASMC proliferation and migration.
Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/Hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/Hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of crosstalk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/Hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.
Chronic asthma patients experience difficulties even years after the inciting allergen. Although studies in small animal asthma models have enormously advanced progress in uncovering the mechanisms of inception and development of the disease, little is known about the processes involved in the persistence of asthma symptoms in the absence of allergen exposure. Long term asthma mouse models have so far been scarce or not been able to reproduce the findings in patients. Here we used a common ovalbumin induced acute allergic airway inflammation mouse model to study lung function and remodeling after a four months recovery period. We show by x-ray based lung function measurements that the recovered mice continue to show impaired lung function by displaying significant air-trapping compared to controls. High resolution synchrotron phase contrast computed tomography of structural alterations and diaphragm motion analysis suggest that these changes in pulmonary function are due to a pronounced loss in lung elasticity. Histology of lung sections confirmed that this is most likely caused by a decrease in elastic fibers, indicating that remodelling can develop or persist independently of acute inflammation and is closely related to a loss in lung function. Our findings demonstrate that this x-ray based imaging platform has the potential to comprehensively and non-invasively unravel long term effects in preclinical mouse models of AAI and thus benefits our understanding of chronic asthma.
Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a crosslinking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein crosslinking, extracellular matrix collagen accumulation and fibroblast to myofibroblast transformation. We report here that TG2 expression, activity as measured by serotonylated fibronectin and protein crosslinking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein crosslinking activity and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced crosslinking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 siRNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.
IL-4 and IL-13 are major T helper cell (Th) 2 cytokines implicated in the pathogenesis of several lung diseases, including pulmonary fibrosis. In this study, using a novel repetitive intradermal bleomycin model in which mice develop extensive lung fibrosis and a progressive decline in lung function compared to saline-treated control mice, we investigated profibrotic functions of Th2 cytokines. To determine the role of IL-13 signaling in the pathogenesis of bleomycin-induced pulmonary fibrosis, wild-type, IL-13, and IL-4Rα-deficient mice were treated with bleomycin, and lungs were assessed for changes in lung function and pulmonary fibrosis. Histological staining and lung function measurements demonstrated that collagen deposition and lung function decline were attenuated in mice deficient in either IL-13 or IL-4Rα-driven signaling compared to wild-type mice treated with bleomycin. Further, our results demonstrated that IL-13- and IL-4Rα-driven signaling are involved in excessive migration of macrophages and fibroblasts. Notably, our findings demonstrated that IL-13-driven migration involves increased pFAK signaling and F-actin polymerization. Importantly, in vivo findings demonstrated that IL-13 augments MMP2 and MMP9 activity that has also been shown to increase migration and invasiveness of fibroblasts in the lungs during bleomycin-induced pulmonary fibrosis. Together, our findings demonstrate a pathogenic role for Th2-cytokine signaling that includes excessive migration and protease activity involved in severe fibrotic lung disease.
Platelet-derived growth factor (PDGF)-A, which only signals through PDGF-receptor-alpha (PDGFRα) is required for secondary alveolar septal formation. Although PDGFRα distinguishes mesenchymal progenitor cells during the saccular stage, PDGFRα-expressing alveolar cells persist through adulthood. PDGF-A sustains proliferation, limits apoptosis, and maintains alpha-smooth muscle actin (αSMA) containing alveolar cells, which congregate at the alveolar entry ring at postnatal day (P)12. PDGFRα-expressing, αSMA-containing, alveolar cells re-distribute in the elongating septum, suggesting that they migrate to the alveolar entry rings where mechanical tension is higher. We hypothesized that PDGFRα and Ras-related C3 botulinum toxin substrate 1(Rac1) are required for mechanosensitive myofibroblast migration. Spreading of PDGFRα-deficient lung fibroblasts was insensitive to increased rigidity, and their migration was not reduced by Rac1-guanine exchange factor (GEF)-inhibition. PDGFRα-expressing fibroblasts migrated towards stiffer regions within 2-dimensional substrates by increasing migrational persistence (durotaxis). Using a Förster resonance energy transfer (FRET) biosensor for Rac1-GTP, we observed that PDGFRα was required for fibroblast Rac1-responsiveness to stiffness within a 3-dimensional collagen substrate, which by itself increased Rac1-FRET. Rho-GTPase stabilized whereas Rac1-GTPase increased the turnover of focal adhesions. Under conditions which increased Rac1-GTP, PDGFRα signaled through both phosphoinositide-3-kiase (PIK) or Src to engage the Rac1 guanine-exchange factors (GEF)s dedicator of cytokinesis-1 (Dock180) and p21-activated-kinase interacting exchange factor-β (βPIX). In cooperation with collagen fibers, these signaling pathways may guide fibroblasts toward the more rigid alveolar entry ring during secondary septation. Because emphysema and interstitial fibrosis disrupt the parenchymal mechanical continuum, understanding how mechanical factors regulate fibroblast migration could elicit strategies for alveolar repair and regeneration.
The cystic fibrosis transmembrane conductance regulator (CFTR) and the amiloride-sensitive epithelial sodium channels (ENaC) are located in the apical membranes of airway and alveolar epithelial cells. These transporters play an important role in the regulation of lung fluid balance across airway and alveolar epithelia by being the conduits for chloride (Cl-) and bicarbonate (HCO3-) secretion and sodium (Na+) ion absorption, respectively. The functional role of these channels in the respiratory tract is to maintain the optimum volume and ionic composition of the bronchial pericilary fluid (PCL) and alveolar lining fluid (ALF) layers. The PCL is required for proper mucociliary clearance of pathogens and debris and the ALF is necessary for surfactant homeostasis and optimum gas exchange. Dysregulation of ion transport may lead to mucus accumulation, bacterial infections, inflammation, as well as pulmonary edema and compromised respiratory function. Influenza (or flu) in mammals is caused by influenza A and B viruses. Symptoms include dry cough, sore throat and is often followed by secondary bacterial infections, accumulation of fluid in the alveolar spaces and acute lung injury. The underlying mechanisms of flu symptoms are not fully understood. This review summarizes our present knowledge of how influenza virus infections alter airway and alveolar epithelial cell CFTR and ENaC function in vivo and in vitro and the role of these changes in influenza pathogenesis.
A key physiological feature of Acute Respiratory Distress Syndrome (ARDS) is inflammation. Toll-like receptor (TLR) signaling is required to combat the infection that underlies many ARDS cases but also contributes to pathological inflammation. Several TLR signaling pathway genes encoding positive effectors of inflammation also produce alternatively spliced mRNAs encoding negative regulators of inflammation. An imbalance between these isoforms could contribute to pathological inflammation and disease severity. To determine if splicing in TLR pathways is altered in ARDS patients, we monitored alternative splicing of MyD88 and IRAK1, two genes that function in multiple TLR pathways. The MyD88 and IRAK1 genes produce long pro-inflammatory mRNAs (MyD88L and IRAK1) and shorter anti-inflammatory mRNAs (MyD88S and IRAK1c). We quantified mRNA encoding inflammatory cytokines and MyD88 and IRAK1 isoforms in peripheral blood mononuclear cells (PBMCs) from 104 ARDS patients and 30 healthy control subjects. We found that MyD88 pre-mRNA splicing is altered in ARDS patients in a pro-inflammatory direction. We also observed altered MyD88 isoform levels in a second critically ill patient cohort, suggesting that these changes may not be unique to ARDS. Early in ARDS, PBMC IRAK1c levels were associated with patient survival. Despite the similarities in MyD88 and IRAK1 alternative splicing observed in previous in vitro studies, there were differences in how MyD88 and IRAK1 alternative splicing was altered in ARDS patients. We conclude that pre-mRNA splicing of TLR signaling genes is altered in ARDS patients, and further investigation of altered splicing may lead to novel prognostic and therapeutic approaches.
Prostaglandin E2 (PGE2), via cAMP signaling, inhibits a variety of fibroblast functions relevant to fibrogenesis. Among these are their translation of collagen I protein and their differentiation to myofibroblasts. PKA is central to these actions, with cAMP binding to regulatory (R) subunits leading to the release of catalytic subunits. Here we examined the role of specific PKAR subunit isoforms in these inhibitory actions in transforming growth factorβ-1 (TGFβ-1)-stimulated human lung fibroblasts (HLFs). HLFs expressed all four R subunit isoforms. SiRNA-mediated knockdown of subunits PKARIα and PKARIIα had no effect on PGE2 inhibition of either process. However, knockdown of PKARIβ selectively attenuated PGE2 inhibition of collagen I protein expression, whereas knockdown of PKARIIβ selectively attenuated PGE2 inhibition of expression of the myofibroblast differentiation marker, α-smooth muscle actin (α-SMA). cAMP analogs that selectively activate either PKARIβ or PKARIIβ exclusively inhibited collagen I synthesis or differentiation, respectively. In parallel, the PKARIβ agonist (but not a PKARIIβ agonist) reduced phosphorylation of two proteins involved in protein translation, protein kinase B (AKT) and mammalian target of rapamycin (mTOR). By contrast, the PKARIIβ agonist (but not a PKARIβ agonist) reduced levels of the differentiation-associated phosphorylated focal adhesion kinase (p-FAK) and the relative mRNA expression of serum response factor (SRF), a transcription factor necessary for myofibroblast differentiation. Our results demonstrate that cAMP inhibition of collagen I translation and myofibroblast differentiation reflect the actions of distinct PKAR subunits.
Background. Lethal influenza A (H5N1) induces respiratory failure in humans. Although it also causes death at 7 day postinfection (dpi) in mice, the development of the respiratory failure and the viral impact on pre-Botzinger complex (PBC) neurons expressing neurokinin 1 receptor (NK1R), the respiratory rhythm-generator, have not been explored. Methods. Body temperature, weight, ventilation, arterial blood pH and gases were measured at 0, 2, 4, and 6 dpi in control, lethal HK483 and non-lethal HK486 viral infected mice. Immunoreactivities (IR) of PBC NK1R, H5N1 viral nucleoprotein (NP), and active caspase-3 (CASP3, a marker for apoptosis) were detected at 6 dpi. Results. HK483, but not HK486, mice showed following abnormalities: 1) gradual body weight loss and hypothermia; 2) tachypnea at 2-4 dpi and ataxic breathing with long-lasting apneas and hypercapnic hypoxemia at 6 dpi; and 3) viral replication in PBC NK1R neurons with NK1R-IR reduced by 75% and CASP3-IR co-labeled at 6 dpi. Conclusion. Lethal H5N1 viral infection causes tachypnea at the early stage and ataxic breathing and apneas (hypercapnic hypoxemia) leading to death at the late stage. Its replication in the PBC induces apoptosis of local NK1R neurons, contributing to ataxic breathing and respiratory failure.
Fibroblast Growth Factor 9 (FGF9) is necessary for fetal lung development and is expressed by epithelium and mesothelium. We evaluated the role of FGF9 overexpression on adenoviral-induced pleural injury in vivo and determined the biological effects of FGF9 on mesothelial cells in vitro. We assessed the expression of FGF9 and FGF receptors by mesothelial cells in both Human and mouse lungs. Intrapleural injection of an adenovirus expressing human FGF9 (AdFGF9) or a control adenovirus (Adcont) was performed. Mice were sacrificed at day 3, day 5 and day 14. Expression of FGF9 and markers of inflammation and myofibroblastic differentiation was studied by qPCR and immunohistochemistry. In vitro, rat mesothelial cells were stimulated with FGF9 (20ng/ml) and we assessed it effect on proliferation, survival, migration and differentiation. FGF9 was expressed by mesothelial cells in human IPF. FGF receptors, mainly FGFR3, were expressed by mesothelial cells in vivo in human and mice. Adcont instillation induced diffuse pleural thickening appearing at day 5, maximal at day 14. The altered pleura cells strongly expressed alpha-smooth muscle actin and collagen. AdFGF9 injection induced maximal FGF9 expression at day 5 which lasted until day 14. FGF9 overexpression prevented pleural thickening, collagen and fibronectin accumulation, and myofibroblastic differentiation of mesothelial cells. In vitro, FGF9 decreased mesothelial cell migration and inhibited the differentiating effect of TGF-ß1. We conclude that FGF9 has a potential antifibrotic effect on mesothelial cells.
Disruption of the alveolar-capillary barrier is a hallmark of acute respiratory distress syndrome (ARDS) that leads to accumulation protein-rich edema in the alveolar space often resulting in comparable protein concentrations in alveolar edema and plasma and causing deleterious remodeling. Patients who survive ARDS have approximately three-times lower protein concentrations in the alveolar edema than non-survivors, thus, the ability to remove excess protein from the alveolar space may be critical for a positive outcome. We have recently shown that clearance of albumin from the alveolar space is mediated by megalin, a 600 kDa transmembrane endocytic receptor and member of the low-density lipoprotein receptor superfamily. In the currents study, we investigate the molecular mechanisms by which TGF-β, a key molecule of ARDS pathogenesis, drives downregulation of megalin expression and function. TGF-β treatment led to shedding and regulated intramembrane proteolysis of megalin at the cell surface and to a subsequent increase in intracellular megalin c-terminal fragment abundance resulting in transcriptional downregulation of megalin. Activity of classical protein kinase C enzymes and -secretase was required for the TGF-β-induced megalin downregulation. Furthermore, TGF-β-induced shedding of megalin was mediated by matrix metallo-proteases (MMPs)-2, -9 and -14. Silencing of either of these MMPs stabilized megalin at the cell surface after TGF-β treatment and restored normal albumin transport. Moreover, a direct interaction of megalin with MMP-2 and -14 was demonstrated, suggesting that these MMPs may function as novel sheddases of megalin. Further understanding of these mechanisms may lead to novel therapeutic approaches for the treatment of ARDS.
Deficiency of the extracellular matrix (ECM) protein latent transforming growth factor beta (TGFβ) binding protein 4 (LTBP-4) results in lack of intact elastic fibers, which leads to disturbed pulmonary development and lack of normal alveolarization in humans and mice. Formation of alveoli and alveolar septation in pulmonary development requires the concerted interaction of extracellular matrix proteins, growth factors like TGFβ, fibroblasts and myofibroblasts to promote elastogenesis as well as vascular formation in the alveolar septae. To investigate the role of LTBP-4 in this context, lungs of LTBP-4 deficient (Ltbp4-/-) mice were analyzed in close detail. We elucidate the role of LTBP-4 in pulmonary alveolarization and show that three different, interacting mechanisms might contribute to alveolar septation defects in Ltbp4-/- lungs, i) absence of an intact elastic fiber network, ii) reduced angiogenesis and iii) upregulation of TGFβ activity resulting in profibrotic processes in the lung.
Background Macrolides antibiotics have been used effectively in many chronic diseases, especially with Pseudomonas aeruginosa (P. aeruginosa) infection. The mechanisms underlying the therapeutic effects of macrolides in these diseases remain poorly understood. Methods We established a mouse model of chronic lung infection using P. aeruginosa agar-beads, with azithromycin treatment or placebo. Lung injury, bacterial clearance and inflammasome-related proteins were measured. In vitro, the inflammasomes activation induced by flagellin or ATP were assessed in LPS-primed macrophages with or without macrolides treatment. Plasma IL-18 levels were determined from patients who were diagnosed with bronchiectasis isolated with or without P. aeruginosa and treated with azithromycin for three to five days. Results Azithromycin treatment enhanced bacterial clearance and attenuated lung injury in mice chronically infected with P. aeruginosa, which resulted from the inhibition of caspase-1-dependent IL-1β and IL-18 secretion. In vitro, azithromycin and erythromycin inhibited NLRC4 and NLRP3 inflammasomes activation. Plasma IL-18 levels were higher in bronchiectasis patients with P. aeruginosa isolation compared with healthy controls. Azithromycin administration markedly decreased IL-18 secretion in bronchiectasis patients. Conclusions The results of this study reveal that azithromycin and erythromycin exert a novel anti-inflammatory effect by attenuating inflammasomes activation, which suggests potential treatment options for inflammasome-related diseases.
Abstract (242 words) Mitochondrial damage is often overlooked in acute lung injury (ALI), but most of the lung's physiological processes, such as airway tone, muco-ciliary clearance, Va/Q matching, and immune surveillance require aerobic energy provision. Because the cell's processes of mitochondrial quality control (QC) regulate the elimination and replacement of damaged mitochondria to support cell survival, we evaluated mitochondrial biogenesis and mitophagy in the alveolar region of mice in a validated S. aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, extensive oxidative mitochondrial damage was detected along with epithelial cell death. Cell death by TUNEL staining occurred on days 1 and 2 post-inoculation: apoptosis shown by caspase 3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho-MLKL and RIPK1 was present on day 1. Cell death in alveolar type I (AT1) cells assessed by BAL fluid RAGE levels was high post-inoculation, yet in AT2 cells, induction of both mitochondrial biogenesis and mitophagy occurred. These alveolar mitochondrial QC mechanisms were evaluated by locating increases in citrate synthase content, nuclear localization of mitochondrial biogenesis regulators NRF-1 and PGC-1α and increased LC3II/LC3I ratios in AT2 cells. Concomitantly, p62, Pink 1, and Parkin proteins were up-regulated, indicating mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy was often observed within the same cells on the first day. These findings imply that activation of mitochondrial QC programs in damaged AT2 cells promotes cell survival to support return of alveolar function.
Prostaglandins (PG), the products of cyclooxygenase-mediated conversion of arachidonic acid, become upregulated in many situations including allergic response, inflammation, injury, and exhibit variety of biological activities. Previous studies described barrier-enhancing and anti-inflammatory effects of PGE2 and PGI2 on vascular endothelial cells (EC). Yet, the effects of other PG members on EC barrier and inflammatory activation have not been systematically analyzed. This study compared effects of PGE2, PGI2, PGF2α, PGA2, PGJ2 and PGD2 on human pulmonary EC. EC permeability was assessed by measurements of transendothelial electrical resistance and cell monolayer permeability for FITC-labeled tracer. Anti-inflammatory effects of PGs were evaluated by analysis of expression of adhesion molecule ICAM1 and secretion of soluble ICAM1 and cytokines by EC. PGE2, PGI2, and PGA2 exhibited most potent barrier-enhancing effects and most efficient attenuation of thrombin-induced EC permeability and contractile response, while PGI2 effectively suppressed thrombin-induced permeability but was less efficient in attenuation of prolonged EC hyperpermeability caused by interleukin-6 or bacterial wall lipopolysaccharide, LPS. PGD2 showed modest protective effect on EC inflammatory response, while PGF2α and PGJ2 were without effect on agonist-induced EC barrier dysfunction. In vivo, PGE2, PGI2, and PGA2 attenuated LPS-induced lung inflammation, while PGF2α and PGJ2 were without effect. Interestingly, PGD2 exhibited protective effect in the in vivo model of LPS-induced lung injury. This study provides comprehensive analysis of barrier-protective and anti-inflammatory effects of different prostaglandins on lung EC in vitro and in vivo and identifies PGE2, PGI2, and PGA2 as prostaglandins with most potent protective properties.
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality. Cigarette smoke (CS) drives disease development and progression. The epithelial barrier is damaged by CS with increased monolayer permeability. However, the molecular changes that cause this barrier disruption and the interaction between adhesion proteins and the cytoskeleton are not well defined. We hypothesized that CS alters monolayer integrity by increasing cell contractility and decreasing cell adhesion in epithelia. Methods: Normal human airway epithelial cells and primary COPD epithelial cells were exposed to air or CS and measured changes in protein levels. We measured the cortical tension of individual cells and the stiffness of cells in a monolayer. We confirmed that the changes in acute and subacute in vitro smoke exposure reflect protein changes seen in cell monolayers and tissue sections from COPD patients. Results: Epithelial cells exposed to repetitive CS and those derived from COPD patients have increased monolayer permeability. E-cadherin and β-catenin were reduced in smoke exposed cells as well as in lung tissue sections from patients with COPD. Moreover, repetitive CS caused increased tension in individual cells and cells in a monolayer, which corresponded with increased polymerized actin without changes in myosin IIA and IIB total abundance. Conclusion: Repetitive CS exposure impacts the adhesive intercellular junctions and the tension of epithelial cells by increased actin polymer levels, to further destabilize cell adhesion. Similar changes are seen in epithelial cells from COPD patients indicating that these findings likely contribute to COPD pathology.
The intracellular signaling mechanisms through which TGFβ regulates pulmonary development are incompletely understood. Canonical TGFβ signaling involves Smad2/3 phosphorylation, Smad2/3·Smad4 complex formation and nuclear localization, and gene regulation. Here we show that physiologically relevant TGFβ1 levels also stimulate Smad1/5 phosphorylation, typically a mediator of bone morphogenetic protein (BMP) signaling, in mouse pup pulmonary artery smooth muscle cells (mPASMC) and lung fibroblasts and other interstitial lung cell lines. This crosstalk mechanism likely has in vivo relevance because mixed Smad1/5/8·Smad2/3 complexes, which are indicative of TGFβ-stimulated Smad1/5 activation, were detected in the developing mouse lung using a proximity ligation assay. Although mixed Smad complexes have been shown not to transduce nuclear signaling, we determined that TGFβ stimulates nuclear localization of phosphorylated Smad1/5 and induces the expression of prototypical BMP-regulated genes in the mPASMC. Small molecule kinase inhibitor studies suggested that TGFβ-regulated Smad1/5 phosphorylation in these cells is mediated by TGFβ-type I receptors, not BMP-type I receptors, but possibly the accessory ALK1 receptor. Although work by others suggested that ALK1 is expressed exclusively in endothelial cells in the vasculature, we detected ALK1 mRNA and protein expression in mPASMC in vitro and in mouse pup lungs. Moreover, using an anti-murine ALK1 antibody and mPASMC, we determined that ALK1 regulates Smad1/5 phosphorylation by TGFβ. Together, these studies characterize an accessory TGFβ-stimulated BMP R-Smad signaling mechanism in interstitial cells of the developing lung. They also indicate the importance of considering alternate Smad pathways in studies directed at determining how TGFβ regulates newborn lung development.
Pulmonary arterial stiffness is an independent risk factor for mortality in pulmonary hypertension (PH), and plays a critical role in PH pathophysiology. We have recently demonstrated arterial stiffening early in experimental PH, along with evidence for a mechanobiologic feedback loop by which arterial stiffening promotes further cellular remodeling behaviors. Cyclooxygenase-2 (COX-2) and prostaglandin signaling have been implicated in stiffness-mediated regulation, with prostaglandin activity inversely correlated to matrix stiffness and remodeling behaviors in vitro, as well as to disease progression in rodent PH models. The mechanism by which mechanical signaling translates to reduced COX-2 activity in pulmonary vascular cells is unknown. The current work investigated the transcriptional regulators Yes-associated protein (YAP) and WW domain-containing Transcription regulator 1 (WWTR1, aka TAZ), which are known drivers of downstream mechanical signaling, in mediating stiffness-induced changes in COX-2 and prostaglandin activity in pulmonary artery smooth muscle cells (PASMCs). We found that YAP/TAZ activity is increased in PAH PASMCs and experimental PH, and is necessary for the development of stiffness-dependent remodeling phenotypes. Knockdown of YAP and TAZ markedly induces COX-2 expression and downstream prostaglandin production by approximately 3-fold, whereas overexpression of YAP or TAZ reduces COX-2 expression and prostaglandin production to near undetectable levels. Together, our findings demonstrate a stiffness-dependent YAP/TAZ-mediated positive feedback loop that drives remodeling phenotypes in PASMCs via reduced COX-2 and prostaglandin activity. The ability to interrupt this critical mechanobiologic feedback loop and enhance local prostaglandin activity via manipulation of YAP/TAZ signaling presents a highly attractive novel strategy for the treatment of PH.
We explored whether the proteomic analysis of exhaled breath condensate (EBC) may provide biomarkers for non-invasive screening for the early detection of lung cancer (LC). EBC was collected from 192 individuals (49 control (C), 49 risk factor-smoking (S), 46 chronic obstructive pulmonary disease (COPD) and 48 LC (LC)). Using LC-MS/MS, 348 different proteins with a different pattern among the four groups were identified in EBC samples. Significantly more proteins were identified in the EBC from LC compared to other groups (C:12.4±1.3; S:15.3±1; COPD:14±1.6; LC:24.2±3.6; p=0.0001). Furthermore, the average number of proteins identified per sample was significantly higher in LC patients and ROC curve analysis showed an area under the curve of 0.8, indicating diagnostic value. Proteins frequently detected in EBC, such as dermcidin and hornerin, along with others much less frequently detected, such as hemoglobin and histones, were identified. Cytokeratins (KRTs) were the most abundant proteins in EBC samples and levels of KRT6A, KRT6B and KRT6C isoforms were significantly higher in samples from LC patients (p=0.0031, p=0.0011 and p=0.0009, respectively). Moreover, the amount of most KRTs in EBC samples from LC patients showed a significant positive correlation with tumor size. Finally, we used a random forest algorithm to generate a robust model using EBC protein data for the diagnosis of patients with LC where the area under the ROC curve obtained indicated a good classification (82%). Thus, this study demonstrates that the proteomic analysis of EBC samples is an appropriated approach to develop biomarkers for the diagnosis of lung cancer.
CD4+T cell differentiation plays an important role in allergic airway diseases. Tumor necrosis factor receptor 2 (TNFR2) has been shown to regulate CD4+T lymphocyte differentiation, but its role in allergic airway inflammation is not clear. Here we investigated the role of TNFR2 in allergic airway inflammation. Mouse model was generated by immunization with OVA and intranasal administration of TNFR2 antibody. Airway inflammation and CD4+T cell differentiation were measured in vivo and in vitro. Inhibited TNFR2 signaling aggravated airway inflammation and increased the expression of inflammatory cytokines (IL-4, IL-5, IL-17, and TNF-α) in serum and bronchoalveolar lavage fluid (BALF). Impaired TNFR2 signaling promoted Th2 and Th17 polarization but inhibited Th1 and CD4+CD25+ T cells differentiation in vivo. Furthermore, TNFR2 signaling inhibition promoted Th2 and Th17 polarization in vitro, which may through the activation of TNF receptor-associated factor 2 (TRAF2) and nuclear factor (NF)-B signaling. Therefore, our findings indicate that impaired TNF/TNFR2 signaling enhances Th2 and Th17 polarization and aggravates allergic airway inflammation.
Background Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodelling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodelling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Methods Guinea pig precision cut lung slices were pre-incubated with the PDE4-inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 μM) or TGF -β1 (2 ng/mL) for 48 hours. The inhibitory effects on airway smooth muscle remodelling, airway contraction and TGF-β release were investigated. Results Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodelling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodelling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-β1-induced remodelling, but rather inhibited methacholine-induced TGF-β release. Conclusion The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodelling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-β release and bronchoconstriction.
The proper regulation of Zinc (Zn) trafficking proteins and the cellular distribution of Zn is critical for the maintenance of autophagic processes. However, there have been no studies which have examined Zn dyshomeostasis and the disease-related modulation of autophagy observed in the airways afflicted with COPD. We hypothesized that dysregulated autophagy in airway epithelial cells (AEC) is related to Zn dysregulation in cigarette smoke (CS)-induced COPD. We applied a human ex vivo air-liquid interface model, a murine model of smoke-exposure, and human lung tissues, and investigated Zn, ZIP1 and ZIP2 Zn-influx proteins, autophagy (Microtubule-associated 1A/1B-light chain-3 (LC3), Beclin-1), autophagic flux (Sequestosome), apoptosis (Bcl2; X-Linked Inhibitor of Apoptosis (XIAP), Poly (ADP)-ribose Polymerase (PARP)), and inflammation (TSLP, RANTES, and IL-1β). Lung tissues from CS-exposed mice exhibit reduced free-Zn in AEC, with elevated ZIP1 and diminished ZIP2 expression. Interestingly, increased LC3 co-localized with ZIP1, suggesting an autophagic requirement for free-Zn to support its catabolic function. In human AECs, autophagy was initiated, but was unable to efficiently degrade cellular debris, as evidenced by stable Beclin-1 and increased LC3-II, but with a concomitant elevation in Sequestosome. Autophagic dysfunction due to CS-exposure coupled with Zn depletion also induced apoptosis, with the reduction of anti-apoptotic and anti-autophagic proteins Bcl2 and XIAP, and PARP cleavage. This was accompanied by an increase in RANTES, and TSLP, an activator of adaptive immunity. We conclude that the uncoupling of Zn-trafficking and autophagy in AEC may be a fundamental disease-related mechanism for COPD pathogenesis, and provide a new therapeutic target.
Alveolar leukocyte recruitment is a hallmark of acute lung inflammation and involves transmigration of leukocytes through endothelial and epithelial layers. The disintegrin and metalloproteinase (ADAM) 8 is expressed on human isolated leukocytic cells and can be further upregulated on cultured endothelial and epithelial cells by proinflammatory cytokines. By shRNA mediated knockdown we show that leukocytic ADAM8 is required on monocytic THP-1 cells for chemokine-induced chemotaxis as well as transendothelial and transepithelial migration. Furthermore, ADAM8 promotes αL integrin upregulation and THP-1 cell adhesion to endothelial cells. On endothelial cells ADAM8 enhances transendothelial migration and increases cytokine-induced permeability. On epithelial cells the protease facilitates migration in a wound closure assay but does not affect transepithelial leukocyte migration. Blood leukocytes and bone marrow derived macrophages from ADAM8-deficient mice show suppressed chemotactic response. Intranasal application of LPS to mice is accompanied with ADAM8 upregulation in the lung. In this model of acute lung inflammation ADAM8-deficient mice are protected against leukocyte infiltration. Finally, transfer experiments of BMDM in mice indicate that ADAM8 exerts a promigratory function predominantly on leukocytes. Our study provides in vitro and in vivo evidence that ADAM8 on leukocytes holds a proinflammatory function in acute lung inflammation by promoting alveolar leukocyte recruitment.
Chronic obstructive pulmonary disease (COPD) is characterized by unresolved neutrophilic airway inflammation, and is caused by chronic exposure to toxic gases, such as cigarette smoke (CS), in genetically susceptible individuals. Recent data indicate a role for Damage Associated Molecular Patterns (DAMPs) in COPD. Here, we investigated the genetics of CS-induced DAMP release in 28 inbred mouse strains. Subsequently, in lung tissue from a subset of strains the expression of the identified candidate genes was analyzed. We tested whether siRNA-dependent knockdown of candidate genes altered the susceptibility of the human A549 cell line to CS-induced cell death and DAMP release. Furthermore, we tested whether these genes were differentially regulated by CS exposure in bronchial brushings obtained from individuals with a family history indicative of either presence or absence of susceptibility for COPD. We observed that of the 4 DAMPs tested, dsDNA showed the highest correlation with neutrophilic airway inflammation. Genetic analyses identified 11 candidate genes governing either CS-induced or basal dsDNA release in mice. Two candidate genes (Elac2 and Ppt1) showed differential expression in lung tissue upon CS exposure between susceptible and non-susceptible mouse strains. Knockdown of ELAC2 and PPT1 in A549 cells altered susceptibility to CS extract-induced cell death and DAMP release. In bronchial brushings, CS-induced expression of ENOX1 and ARGHGEF11 was significantly different between individuals susceptible or non-susceptible for COPD. Our study shows that genetic variance in a mouse model is associated with CS-induced DAMP release, and that this might contribute to susceptibility for COPD.
Individuals with intrauterine growth restriction (IUGR) are at risk for chronic lung disease. Using a rat model, our previous studies showed that altered lung structure is related to IL-6/STAT3 signaling. As Neuropeptide Y (NPY), a co-neurotransmitter of the sympathetic nerve system, regulates proliferation and immune response, we hypothesized that dysregulated NPY after IUGR is linked to IL-6, impaired myofibroblast function, and alveolar growth. IUGR was induced in rats by isocaloric low-protein diet; lungs were analyzed on embryonic day (E) 21, postnatal day (P) 3, P12 and P23. Finally, primary neonatal myofibroblasts (pnF) and murine embryonic fibroblasts (MEF) were used to assess proliferation, apoptosis, migration, and IL-6 expression. At E21, NPY and IL-6 expression were decreased and AKT/PKC and STAT3/AMPKα signaling reduced, respectively. Early reduction of NPY/IL-6 was associated with increased chord length in lungs after IUGR at P3, indicating reduced alveolar formation. At P23, however, IUGR-rats exhibited a catch-up of body weight and alveolar growth coupled with more proliferating myofibroblasts. These structural findings after IUGR were linked to activated NPY/PKC, IL-6/AMPKα signaling. Complementary, IUGR-pnF showed increased survival, impaired migration and reduced IL-6 compared to control-pnF (Co-pnF). In contrast, NPY induced proliferation, migration and increased IL-6 synthesis in fibroblasts. Additionally, NPY(-/-) mice showed reduced IL-6 signaling and less proliferation of lung fibroblasts. Our study presents a novel role of NPY during alveolarization: NPY regulates (1) IL-6 and lung STAT3/AMPKα signaling, and (2) proliferation and migration of myofibroblasts. These new insights in pulmonary neuro-immune interaction offer potential strategies to enable lung growth.
Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production due to excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex which includes the elastin-binding protein S-gal. In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T helper (Th) and T cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the VGVAPG elastin peptide and 21 days after treatment, local and systemic T lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4+ and CD8+ T cells expressing the cytokines IFN- or IL-17a and the transcription factors Tbet or RORt without effects on IL-4 and Gata3 expression. These effects were maximized when each T cell sub-population was challenged ex vivo with EP and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that during murine emphysema, EP/S-gal interactions contribute to a Th-1 and Th-17 pro-inflammatory T cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response.
Alterations to the pulmonary surfactant system have been observed consistently in ventilation-induced lung injury including composition changes and impairments in the surface tension reducing ability of the isolated extracellular surfactant. However, there is limited information about the effects of VILI on the intracellular form of surfactant, the lamellar body. It is hypothesized that VILI leads to alterations of lamellar bodies numbers and function. To test this hypothesis, rats were randomized to one of three groups, non-ventilated controls, control ventilation and high tidal volume ventilation (VILI). Following physiological assessment to confirm lung injury, isolated lamellar bodies were tested for surfactant function on a constrained sessile drop surfactometer. A separate cohort of animals was used to fix the lungs followed by examination of lamellar body numbers and morphology using transmission electron microscopy. The results showed an impaired ability of reducing surface tension for the lamellar bodies isolated from the VILI group as compared to the two other groups. The morphological assessment revealed that the number, and the relative area covered by, lamellar bodies was significantly decreased in animals with VILI animals as compared to the other groups. It is concluded that VILI causes significant alterations to lamellar bodies. It is speculated that increased secretion causes a depletion of lamellar bodies that cannot be compensated by de novo synthesis of surfactant in these injured lungs.
Histamine is an important mediator of allergic reactions, and mucus hypersecretion is a major allergic symptom. However, the direct effect of histamine on mucus secretion from airway mucosal epithelia has not been clearly demonstrated. TMEM16A is a Ca2+-activated chloride channel, and it is closely related to fluid secretion in airway mucosal epithelia. We investigated whether histamine directly induces fluid secretion from epithelial cells or submucosal glands (SMG) and mechanisms related therewith in allergic airway diseases. In pig airway tissues from nose or trachea, histamine was a potent secretagogue that directly induced strong responses. However, gland secretion from human nasal tissue was not induced by histamine even in allergic rhinitis patients. H1R and H2R were not noted in SMG by in situ hybridization. Cultured primary human nasal epithelial (NHE) cells were used for the measurement of short circuit current changes with the Ussing chamber. Histamine induced slight responses of anion secretions under normal conditions. The response was enhanced by IL-4 stimulation through TMEM16A, which might be related to fluid hypersecretion in allergic rhinitis. Pretreatment with IL-4 augmented the histamine response that was suppressed by a TMEM16A inhibitor. TMEM16A expression was enhanced by 24 h treatment of IL-4 in HNE cells. The expression of TMEM16A was significantly elevated in an allergic rhinitis group, compared to a control group. We elucidated histamine-induced fluid secretions in synergy with IL-4 through TMEM16A in the human airway epithelium. In addition, we observed species differences between pig and human in terms of gland secretion to histamine.
Nicotine is a major component of cigarette smoke. It causes addiction and is used clinically to aid smoke cessation. The aim of the present study is to investigate the effect of nicotine on lipopolysaccharide (LPS)-induced airway hyperreactivity (AHR) and to explore the potential involvement of neuronal mechanisms behind nicotine's effects in murine models in vivo and in vitro. BALB/c mice were exposed to nicotine in vivo via subcutaneous Alzet osmotic minipumps containing nicotine tartate salt solution (24 mg/kg/day) for 28 days. LPS (0.1 mg/ml, 20 µl) were administered intranasally for 3 consecutive days during the end of this period. Lung functions were measured with flexiVent. For the in vitro experiments, mice tracheae were organ-cultured in with either nicotine (10 μM) or vehicle (DMSO, 0.1%) for 4 days. Contractile responses of the tracheal segments were measured in myographs following electric field stimulation (EFS, increasing frequencies 0.2 to 12.8Hz) before and after incubation with 10 µg/ml LPS for 1 hour. Results showed that LPS induced AHR to methacholine in vivo and increased contractile responses to EFS in vitro. Interestingly, long-term nicotine exposure markedly dampened this LPS-induced AHR both in vitro and in vivo. Tetrodotoxin (TTX) inhibited LPS-induced AHR, but did not further inhibit nicotine-suppressed AHR in vivo. In conclusion, long-term nicotine exposure dampened LPS-induced AHR. The effect of nicotine was mimicked by TTX, suggesting the involvement of neuronal mechanisms. This information might be used for evaluating long-term effects of nicotine and further exploring of how tobacco products interact with bacterial airway infections.
Electronic cigarettes (e-cigarettes or e-cigs) are designed to heat and aerosolize mixtures of vegetable glycerin, propylene glycol, nicotine and flavoring additives, thus delivering nicotine by inhalation in the absence of combustion. These devices were originally developed to facilitate smoking cessation and have been available in the United States for over a decade. Since 2010, e-cig use has expanded rapidly, especially among adolescents, despite a paucity of short and long-term safety data. Patterns of use have shifted to include never-smokers and many dual users of e-cigs and combustible tobacco products. Over the last several years, research into the potential toxicities of e-cig aerosols has grown exponentially. In the interim, regulatory policymakers across the world have struggled with how to regulate an increasingly diverse array of suppliers and products, against a backdrop of strong advocacy from users, manufacturers, and tobacco control experts. Herein we provide an updated review of the pulmonary toxicity profile of these devices, summarizing evidence from cell culture, animal models, and human subjects. We highlight the major gaps in our current understanding, emphasize the challenges confronting the scientific and regulatory communities and identify areas that require more research in this important and rapidly evolving field.
We studied acute effects of tumor necrosis factor-alpha (TNFα) on the sensitivity of isolated rat vagal pulmonary sensory neurons. Our results showed: 1) A brief pretreatment with a low dose of TNFα (1.44 nM, 9 min) enhanced the sensitivity of transient receptor potential vanilloid type 1 (TRPV1) receptors in these neurons in two distinct phases: the inward current evoked by capsaicin was amplified (=247%) immediately following the TNFα pretreatment, which gradually declined toward control and then increased again reaching another peak (=384%) after 60-90 min. 2) The immediate phase of this potentiating effect of TNFα was completely abolished by a pretreatment with a selective cyclooxygenase-2 (COX-2) inhibitor, NS-398, whereas the delayed potentiation was only partially attenuated. 3) In sharp contrast, TNFα did not generate any potentiating effect on the responses to non-TRPV1 chemical activators of these neurons. 4) The selectivity of the TNFα action on TRPV1 was further illustrated by the responses to acid (pH 6.0); TNFα did not affect the rapid transient current mediated by acid-sensing ion channels, but significantly augmented the slow sustained current mediated by TRPV1 in the same neurons. 5) In anesthetized rats, a similar pattern of acute sensitizing effects of TNFα on pulmonary C-fiber afferents and the involvement of COX-2 were also clearly shown. In conclusion, a brief pretreatment with TNFα induced both immediate and delayed potentiating effects on the TRPV1 sensitivity in pulmonary sensory neurons, and the production of COX-2 arachidonic acid metabolites plays a major role in the immediate sensitizing effect of TNFα.
Estrogen and secondhand smoke are key risk factors for nonsmoking female lung cancer patients, who frequently have lung adenocarcinoma and show tumor ERα expression. We speculated that estrogen and secondhand smoke might cause harmful effects via ERα signaling. Our results showed that 17β-estradiol (E2), the primary form of endogenous estrogen, exacerbated proliferation, migration, and granzyme B resistance of lung adenocarcinoma cells in an ERα-dependent manner. Cigarette sidestream smoke particulate matter (CSSP), the major component of secondhand smoke, could activate ERα activity dose dependently in human lung adenocarcinoma cells. The estrogenic activity of CSSP was abolished by an ERα-selective antagonist. CSSP regulated the nuclear entry, phosphorylation, and turnover of ERα similarly to E2. Furthermore, CSSP enhanced E2-stimulated ERα activity and Ser118 phosphorylation even when ERα became saturated with E2. Activation of ERα by CSSP required GSK3β activity, but not involving polycyclic aromatic hydrocarbons, reactive oxygen species, calcium, epidermal growth factor receptor, and PI3K/Akt. Although CSSP possessed cytotoxicity, ERα-expressing cells grew and migrated faster than nonexpressing cells upon recovery from CSSP exposure as observed in E2-pretreated cells. Knockdown of ERα by siRNA diminished E2- and CSSP-stimulated cell migration. 21 genes, including SERPINB9, were identified to be upregulated by both E2 and CSSP via ERα. Increased SERPINB9 expression was accompanied with increased resistance to granzyme B-mediated apoptosis. This study demonstrates that estrogen has ERα-dependent tumor-promoting activity. CSSP acts like estrogen and shows a potential to enhance estrogen-induced ERα action.
The phosphatidylinositol 3-kinase (PI3K) pathway is activated in chronic obstructive pulmonary disease (COPD), but the regulatory mechanisms for this pathway are yet to be elucidated. Our aim was to determine the expression and role of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the PI3K pathway, in COPD. PTEN expression and activity were measured in the peripheral lung of COPD patients compared to smoking and non-smoking controls. The direct influence of cigarette smoke extract (CSE) on PTEN expression was assessed using primary lung epithelial cells and a cell line (BEAS-2B) in the presence or absence of L-buthionine-sulfoximine (BSO) to deplete intracellular glutathione. The impact of PTEN knock-down by RNA interference on cytokine production was also examined. In peripheral lung, PTEN protein was significantly decreased in patients with COPD compared to the subjects without COPD (p < 0.001), and positively correlated with the severity of air-flow obstruction (FEV1 % predicted; r = 0.50; p = 0.0012), although no difference was observed in PTEN activity. Conversely, phosphorylated Akt, as a marker of PI3K activation, showed a negative correlation with PTEN protein levels (r = -0.41; p = 0.0042). Both in primary bronchial epithelial cells and BEAS-2B cell line, CSE decreased PTEN protein, which was reversed by N-acetylcysteine treatment. PTEN knock-down potentiated Akt phosphorylation and enhanced production of pro-inflammatory cytokines, such as IL-6, CXCL8, CCL2 and CCL5. In conclusion, oxidative stress reduces PTEN protein levels, which may result in increased PI3K signaling and amplification of inflammation in COPD.
Integrin αvβ5 mediates pulmonary endothelial barrier function and acute lung injury (LI), but its roles in cell apoptosis and autophagy are unclear. Thus, the aims of this study were to investigate the significance of αvβ5 in ischemia/reperfusion (I/R)-induced apoptosis and LI and to explore the relationship between αvβ5 and autophagy. Human pulmonary micro-vascular endothelial cells (HPMVECs) were pretreated with an αvβ5-blocking antibody (ALULA) and challenged with oxygen-glucose deprivation/oxygen-glucose restoration, which mimics I/R; then, cellular autophagy and apoptosis were detected, and cell permeability was assessed. In vivo, mice were pretreated with the autophagy inhibitor chloroquine (CLQ), followed by treatment with ALULA. The mice then underwent operative lung I/R. LI was assessed by performing a pathological examination, calculating the wet/dry lung weight ratio and detecting the bronchial alveolar lavage fluid (BALF) protein concentration. αvβ5 inhibition promoted HPMVEC autophagy under I/R in vitro, alleviated cell permeability, decreased the apoptosis ratio and activated caspase-3 expression. These outcomes were significantly diminished when autophagy was inhibited with a small interfering RNA construct targeting autophagy-related gene 7 (siATG7). Moreover, ALULA pretreatment alleviated I/R-induced LI (I/R-LI), which manifested as a decreased wet/dry lung weight ratio, an altered BALF protein concentration and lung edema. Pre-inhibiting autophagy with CLQ, however, eliminated the protective effects of ALULA on I/R-LI. Therefore, inhibiting αvβ5 effectively ameliorated I/R-induced endothelial cell apoptosis and I/R-LI. This process was dependent on improved autophagy and its inhibitory effects on activated caspase-3.
Lung infections are major causes of acute lung injury (ALI), with limited effective treatment available. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential adaptor regulating Toll-like receptors (TLRs). We recently identified Cullin-5 (Cul-5) as a prominent component in the regulation of TRAF6 polyubiquitination, but its physiological significance in acute lung injury has not been explored. In this study, we investigated the potential role of Cul-5 in regulating ALI using mice receiving intratracheal instillation of LPS. We observed that Cul-5 deficient mice displayed reduced lung injury compared with wild-type mice as evidenced by histological analysis, alveolar neutrophil infiltration and lung liquid accumulation. In addition, inflammatory cytokine expression in bronchoalveolar lavage fluid and lung tissue was also markedly reduced in LPS-treated Cul-5 deficient mice. Interestingly, intratracheal adoptive transfer of Cul-5+/- but not Cul-5+/+ macrophages attenuated neutrophil recruitment, alveolar inflammation, and loss of barrier function in LPS-challenged wild-type mice. Finally, we demonstrated that Cul-5 neddylation following LPS exposure induced Cul-5 and TRAF6 interaction and, thereby, TFAR6 polyubiquitination, leading to NF-B activation and generation of proinflammatory cytokines. Our data show that Nedd8 modification of Cul-5 is required for its interaction with TRAF6 and activation of the TLR4-TRAF6 signaling pathway in LPS-induced acute lung injury, a feature that may be explored for therapeutic intervention.
In bacterial pneumonia, lung damage resulting from epithelial cell injury is a major contributor to the severity of disease, and in some cases, can lead to long-term sequelae, especially in the setting of severe lung injury or ARDS. Leukemia inhibitory factor (LIF), a member of the IL-6 cytokine family, is a critical determinant of lung tissue protection during pneumonia, but the cellular sources of LIF and the signaling pathways leading to its production in the infected lung are not known. Here, we demonstrate that lung epithelium, specifically alveolar type II cells, is the predominant site of LIF transcript induction in pneumonic mouse lungs. Epithelial cell cultures were induced to express LIF by bacteria and by sterile bronchoalveolar lavage fluid from pneumonic mice. Reciprocal bone marrow chimera studies demonstrated that LIF deficiency in the non-hematopoietic compartment, but not LIF deficiency in hematopoietic cells, eliminated LIF induction during pneumonia. Although NF-B RelA (p65) is essential for the expression of many cytokines during pneumonia, its targeted mutation in the lung epithelium was inconsequential for pneumonia-driven LIF induction. However, maximal expression of this epithelial-derived cytokine was dependent upon NF-B RelA in myeloid cells. Overall, our data suggest a signaling axis whereby activation of NF-B RelA in myeloid cells promotes epithelial LIF induction during lung infections, representing a means through which these two cell types collaborate to improve tissue resilience during pneumonia.
Pulmonary hypertension (PH) is a progressive disorder whose cellular pathogenesis involves enhanced smooth muscle cell (SMC) proliferation and resistance to apoptosis signals. Existing evidence demonstrates that the tumor suppressor, programmed cell death 4 (PDCD4) affects patterns of cell growth and repair responses in the systemic vasculature following experimental injury. In the current study, the regulation PDCD4 and its functional effects on growth and apoptosis susceptibility in pulmonary artery smooth muscle cells was explored. We previously demonstrated that pharmacological activation of the nuclear transcription factor, peroxisome proliferator-activated receptor gamma (PPAR), attenuated hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMC) by inhibiting the expression and mitogenic functions of microRNA-21 (miR-21). In the current study, we hypothesize that PPAR stimulates PDCD4 expression and HPASMC apoptosis by inhibiting miR-21. Our findings demonstrate that PDCD4 is reduced in the mouse lung upon exposure to chronic hypoxia (10% O2 x 3-weeks) and in hypoxia-exposed HPASMC (1% O2). HPASMC apoptosis was reduced by hypoxia, by miR-21 overexpression, or by siRNA-mediated PPAR and PDCD4 depletion. Activation of PPAR inhibited miR-21 expression and resultant proliferation, while restoring PDCD4 levels and apoptosis to baseline. Additionally, pharmacological activation of PPAR with rosiglitazone enhanced PDCD4 protein expression and apoptosis in a dose-dependent manner as demonstrated by increased annexin V detection by flow cytometry. Collectively, these findings demonstrate that PPAR confers growth inhibitory signals in hypoxia-exposed HPASMC through suppression of miR-21 and the accompanying de-repression of PDCD4 which augments HPASMC susceptibility to undergo apoptosis.
Airway remodeling in asthma driven by inflammation involves proliferation of epithelial cells and airway smooth muscle (ASM), as well as enhanced extracellular matrix (ECM) generation and deposition, i.e. fibrosis. Accordingly, understanding pro-fibrotic mechanisms is important for developing novel therapeutic strategies in asthma. Recent studies, including our own, have suggested a role for locally-produced growth factors such as brain-derived neurotrophic factor (BDNF) in mediating and modulating inflammation effects. In this study, we explored the pro-fibrotic influence of BDNF in the context of asthma by examining expression, activity and deposition of ECM proteins in primary ASM cells isolated from asthmatic vs. non-asthmatic patients. Basal BDNF expression and secretion, and levels of the high-affinity BDNF receptor TrkB were higher in asthmatic ASM. Exogenous BDNF significantly increased ECM production and deposition, especially of collagen-1 and collagen-3 (less so fibronectin) and the activity of matrix metalloproteinases (MMP-2, MMP-9). Exposure to the pro-inflammatory cytokine TNFα significantly increased BDNF secretion, particularly in asthmatic ASM, whereas no significant changes were observed with IL-13. Chelation of BDNF using TrkB-Fc reversed TNFα-induced increase in ECM deposition. Conditioned media from asthmatic ASM enhanced ECM generation in non-asthmatic ASM which was blunted by BDNF chelation. Inflammation-induced changes in MMP-2, MMP-9, and tissue inhibitor metalloproteinases (TIMP-1, TIMP-2) were reversed in the presence of TrkB-Fc. These novel data suggest ASM as an inflammation-sensitive source of BDNF within human airways, with autocrine effects on fibrosis relevant to asthma.
Exposure to environmental particles during pregnancy increases asthma susceptibility of the offspring. We tested the hypothesis that this transmission continues to F2 and F3 generations and occurs via epigenetic mechanisms. We compared allergic susceptibility of three generations of BALB/c offspring after a single maternal exposure during pregnancy to diesel exhaust particles or concentrated urban air particles. After pregnant dams received intranasal instillations of particle suspensions or control, their F1, F2 and F3 offspring were tested in a low-dose ovalbumin protocol for allergy sensitivity. We found that the elevated asthmatic susceptibility after maternal exposure to particles during pregnancy persists into F2 and, with lesser magnitude, into F3 generations. This was evident from elevated eosinophil counts in bronchoalveolar lavage fluid (BAL), histopathologic changes of allergic airway disease, and increased BAL levels of IL-5 and IL-13. We have previously shown that dendritic cells (DCs) can mediate transmission of risk upon adoptive transfer. We therefore used an enhanced reduced representation bisulfite sequencing protocol to quantify DNA methylation in DCs from each generation. Distinct methylation changes were identified in F1, F2 and F3 DCs. The subset of altered loci shared across the 3 generations were not linked to known allergy genes or pathways, but included a number of genes linked to chromatin modification, suggesting potential interaction with other epigenetic mechanisms (e.g. histone modifications).The data indicate that pregnancy airway exposure to DEP triggers a transgenerationally transmitted asthma susceptibility and suggests a mechanistic role for epigenetic alterations in DCs in this process.
Tuberculosis (TB) is one of the oldest known human diseases and is transmitted by the bacteria, Mycobacterium tuberculosis (Mtb). TB has a rich history with evidence of TB infections dating back to 5,800 B.C. TB is unique in its ability to remain latent in an individual for decades, with the possibility of later reactivation, causing widespread systemic symptoms. Currently, it is estimated that more than one-third of the world's population (about 2 billion people) are infected with Mtb. Prolonged periods of therapy and complexity of treatment regimens, especially in active infection, have led to poor compliance in patients being treated for TB. Therefore, it is vitally important to have a thorough knowledge of the pathophysiology of Mtb to understand the disease progression, as well as to develop novel diagnostic tests and treatments. Alveolar macrophages represent both the primary host cell and the first line of defense against the Mtb infection. Apoptosis and autophagy of macrophages play a vital role in the pathogenesis, and also in the host defense against Mtb. This review will outline the role of these two cellular processes in defense against Mtb with particular emphasis on innate immunity and explore developing therapies aimed at altering host responses to the disease.
Sporadic clinical reports suggested that marijuana smoking induces spontaneous pneumothorax, but no animal models were available to validate these observations and to study the underlying mechanisms. Therefore, we performed a systematic study in CD1 mice as a predictive animal model and assessed the pathophysiological alterations in response to 4-month-long whole body marijuana smoke with integrative methodologies in comparison with tobacco smoke. Bronchial responsiveness was measured with unrestrained whole body plethysmography, cell profile in the bronchoalveolar lavage fluid with flow-cytometry, myeloperoxidase activity with spectrophotometry, inflammatory cytokines with ELISA, and histopathological alterations by light microscopy. Daily marijuana inhalation evoked severe bronchial hyper-reactivity after a week. Characteristic perivascular/peribronchial edema, atelectasis, apical emphysema, neutrophil and macrophage infiltration developed after one month of marijuana smoking, lymphocyte accumulation after 2 months, macrophage-like giant cells, irregular, destroyed bronchial mucosa, goblet cell hyperplasia after 3months, and serious atelectasis, emphysema, obstructed, damaged bronchioles, endothelial proliferation at 4 months. Myeloperoxidase activity, inflammatory cell and cytokine profile correlated with these changes. Airway hyper-responsiveness and inflammation were not altered in mice lacking the CB1 cannabinoid receptor. In comparison, tobacco smoke induced hyper-responsiveness after 2 months, and significantly later developing inflammatory cell infiltration/activation with only mild emphysema. We provide the first systematic and comparative experimental evidence that marijuana causes severe airway hyper-responsiveness, inflammation, tissue destruction and emphysema, which are not mediated by the CB1 receptor.
Innate immune cells of the respiratory tract are the first line of defense against pathogenic and environmental insults. Failure of these cells to perform their immune functions leaves the host susceptible to infection, and may contribute to impaired resolution of inflammation. While combustible tobacco cigarettes have been shown to suppress respiratory immune cell function, the effects of flavored electronic cigarette liquids (e-liquids) and individual flavoring agents on respiratory immune cell responses are unknown. We investigated the effects of seven flavored, nicotine-free e-liquids on primary human alveolar macrophages, neutrophils, and natural killer (NK) cells. Cells were challenged with a range of e-liquid dilutions and assayed for their functional responses to pathogenic stimuli. Endpoints included phagocytic capacity (neutrophils and macrophages), neutrophil extracellular trap formation, proinflammatory cytokine production, and cell mediated cytotoxic response (NK cells). E-liquids were then analyzed via mass spectrometry to identify individual flavoring components. Three cinnamaldehyde-containing e-liquids exhibited dose-dependent, broadly immunosuppressive effects. Quantitative mass spectrometry was used to determine concentrations of cinnamaldehyde in each of the three e-liquids and cells were subsequently challenged with a range of cinnamaldehyde concentrations. Cinnamaldehyde alone recapitulated the impaired function observed with e-liquid exposures and cinnamaldehyde-induced suppression of macrophage phagocytosis was reversed by addition of the small-molecule reducing agent 1,4-dithiothreitol (DTT). We conclude that cinnamaldehyde has the potential to impair respiratory immune cell function, illustrating an immediate need for further toxicological evaluation of chemical flavoring agents to inform regulation governing their use in e-liquid formulations.
Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial pro-fibrotic glycoprotein that is up-regulated in fibrotic lung tissues and that promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 up-regulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional as well as translational levels. Transforming growth factor (TGF)-β1 has long been regarded as a central mediator of tissue fibrosis. Follistatin-like 1 (Fstl1) is a crucial pro-fibrotic glycoprotein that is up-regulated in fibrotic lung tissues and it promotes fibrogenesis via facilitating TGF-β signaling. Here we examined the signaling pathway by which TGF-β1 up-regulates Fstl1 expression in mouse pulmonary fibroblasts. TGF-β1 regulated Fstl1 expression at both the transcriptional and translational levels. Although TGF-β1 rapidly activated the Smad, MAPK, and Akt pathways in lung fibroblasts, only Smad2/3 inhibition eliminated TGF-β1-induced Fstl1 expression. Analysis of the luciferase reporter activity identified a functional c-Jun transcription site in the Fstl1 promoter. Our results suggested a critical role for the Smad3-c-Jun pathway in the regulation of Fstl1 expression by TGF-β1 during fibrogenesis.
During lung-inflation, airspace dimensions are affected non-linearly by both alveolar expansion and recruitment, potentially confounding the identification of emphysematous lung by hyperpolarized helium-3 diffusion magnetic resonance imaging (HP MRI). This study aimed to characterize lung inflation over a broad range of inflation volume and pressure values in two different models of emphysema, as well as in normal lungs. Elastase-treated rats (n=7) and healthy controls (n=7) were imaged with HP MRI. Gradual inflation was achieved by incremental changes to both inflation volume and airway pressure. The apparent diffusion coefficient (ADC) was measured at each level of inflation and fitted to the corresponding airway pressures as the second order response equation, with minimizing residue (2<0.001). A biphasic ADC response was detected, with an initial ADC increase followed by a decrease at airway pressures >18 cmH2O. Discrimination between treated and control rats was optimal when airway pressure was intermediate (between 10-11 cmH2O). Similar findings were confirmed in mice following long-term exposure to cigarette smoke, where optimal discrimination between treated and healthy mice occurred at a similar airway pressure as in the rats. We subsequently explored the evolution of ADC measured at the intermediate inflation level in mice after prolonged smoke exposure, and found a significant increase (P<0.01) in ADC over time. Our results demonstrate that measuring ADC at intermediate inflation enhances the distinction between healthy and diseased lungs, thereby establishing a model that may improve the diagnostic accuracy of future HP gas diffusion studies.
Acute respiratory distress syndrome (ARDS) is a serious, often fatal condition without available pharmacotherapy. While the role of innate cells in ARDS has been studied extensively, emerging evidence suggests that T cells may be involved in disease etiology. Staphylococcus aureus enterotoxins are potent T cell mitogens capable of triggering life-threatening shock. We demonstrate that 2 days after inhalation of S. aureus enterotoxin A, mice developed T cell-mediated increases in vascular permeability, as well as expression of injury markers and caspases in the lung. Pulmonary endothelial cells underwent sequential phenotypic changes marked by rapid activation coinciding with inflammatory events secondary to T cell priming, followed by reductions in endothelial cell number juxtaposing simultaneous T cell expansion and cytotoxic differentiation. Although initial T cell activation influenced the extent of lung injury, CD54 (ICAM-1) blocking antibody administered well beyond enterotoxin exposure substantially attenuated pulmonary barrier damage. Thus, CD54-targeted therapy may be a promising candidate for further exploration into its potential utility in treating ARDS patients.
Emerging evidence indicates that hypnotic anesthetics affect immune function. Many anesthetics potentiate -aminobutyric acid A receptor (GABAAR) activation, and these receptors are expressed on multiple subtypes of immune cells, providing a potential mechanistic link. Like immune cells, airway smooth muscle (ASM) cells also express GABAARs, particularly isoforms containing α4 subunits, and activation of these receptors leads to ASM relaxation. We sought to determine if GABAAR signaling modulates the ASM contractile and inflammatory phenotype of a murine allergic asthma model utilizing GABAAR α4 subunit global knockout (KO; Gabra40/0) mice. Wild type (WT) and Gabra4 KO mice were sensitized with house dust mite (HDM) antigen or exposed to phosphate buffered saline (PBS) intranasally 5 days/week for 3 weeks. Ex vivo tracheal rings from HDM-sensitized WT and Gabra4 KO mice exhibited similar magnitudes of acetylcholine-induced contractile force and isoproterenol-induced relaxation (p=n.s.; n=4). In contrast, in vivo airway resistance (flexiVent) was significantly increased in Gabra4 KO mice (p<0.05, n=8). Moreover, the Gabra4 KO mice demonstrated increased eosinophilic lung infiltration (p<0.05; n=4), and increased markers of lung T cell activation/memory (CD62L low, CD44 high; p<0.01, n=4). In vitro, Gabra4 KO CD4+ cells produced increased cytokines and exhibited increased proliferation after stimulation of the T cell receptor as compared to WT CD4+ cells. These data suggest that the GABAAR α4 subunit plays a role in immune cell function during allergic lung sensitization. Thus, GABAAR α4 subunit-specific agonists have the therapeutic potential to treat asthma via two mechanisms: direct ASM relaxation and inhibition of airway inflammation.
Even with advances in the care of preterm infants, chronic lung disease or bronchopulmonary dysplasia (BPD) continues to be a significant pulmonary complication. Among those diagnosed with BPD, a subset of infants develop severe BPD with disproportionate pulmonary morbidities. In addition to decreased alveolarization, these infants develop obstructive and/or restrictive lung function due to increases in or dysregulation of extracellular matrix proteins. Analyses of plasma obtained from preterm infants during the first week of life indicate that circulating miR-29b is suppressed in infants that subsequently develop BPD, and that decreased circulating miR-29b is inversely correlated with BPD severity. Our mouse model mimics the pathophysiology observed in infants with severe BPD and we have previously reported decreased pulmonary miR-29b expression in this model. The current studies tested the hypothesis that AAV9-mediated restoration of miR-29b in the developing lung will improve lung alveolarization and minimize the deleterious changes in matrix deposition. Pregnant C3H/HeN mice received an intraperitoneal LPS injection on E16 and newborn pups were exposed to 85% oxygen from birth to 14 days of life. On postnatal day 3, AAV9-mir-29b or AAV9-control was administered intranasally. Mouse lung tissues were analyzed for changes in miR-29 expression, alveolarization, and matrix protein levels and localization. Modest improvements in alveolarization were detected in the AAV9-miR29b treated mice at PN28 but treatment completely attenuated defects in matrix protein expression and localization. Our data suggest that miR-29b restoration may be one component of a novel therapeutic strategy to treat or prevent severe BPD in prematurely born infants.
Primary cilia (PC) are solitary cellular organelles that play critical roles in development, homeostasis, and disease pathogenesis by modulating key signaling pathways such as sonic hedgehog and calcium flux. The antenna-like shape of PC enables them also to facilitate sensing of extracellular and mechanical stimuli into the cell, and a critical role for PC has been described for mesenchymal cells such as chondrocytes. However, nothing is known about the role of PC in airway smooth muscle cells (ASMC) in the context of airway remodeling. We hypothesized that PC on ASMCs mediate cell contraction and are thus integral in the remodeling process. We found that PC are expressed on ASMC in asthmatic lungs. Using pharmacological and genetic methods we demonstrated that PC are necessary for ASMC contraction in a collagen gel 3D model both in the absence of external stimulus and in response to the extracellular component hyaluronan. Mechanistically, we demonstrate that the effect of PC on ASMC contraction is to a small extent due to their effect on sonic hedgehog signaling, and to a larger extent due to their effect on calcium influx and membrane depolarization. In conclusion, PC are necessary for the development of airway remodeling by mediating calcium flux and sonic hedgehog signaling.
Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH) but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. While no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV, rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared to healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.
Asthma is a chronic disease related to airway hyper-responsiveness and airway remodeling. Airway remodeling is the important reason of refractory asthma and is associated with differentiation of airway epithelia into myofibroblasts via epithelial-mesenchymal transition (EMT) to increase the process of subepithelial fibrosis. There is growing evidence that autophagy modulates remodeling. However, the underlying molecular mechanisms of these effects are still unclear. In this study, we hypothesized that Follistatin-like 1(FSTL1) promotes EMT and airway remodeling by intensifying autophagy. Using transmission electron microscopy (TEM), double membrane autophagosomes were detected in the airways of patients and mice. More autophagosomes were in asthmatic patients and OVA-challenged mice compared to healthy control. The expression of FSTL1 and Beclin1 were upregulated in the airways of asthmatic patients and OVA-challenged mice, accompany with airway EMT and remodeling. In OVA-challenged Fstl1+/- mice, the degree of airway remodeling and autophagy were decreased compared to control mice. The effects of FSTL1 on autophagy and EMT were also tested in 16HBE cells in vitro. Additionally, inhibition of autophagy by using LY294002 and siRNA-ATG5 reduced the FSTL1-induced EMT in 16HBE cells as measured by E-cadherin, N-cadherin and vimentin expression. In line herewith, administration of LY294002 reduced the expression of autophagy, EMT and airway remodeling markers in FSTL1-challenged WT mice. Taken together, our study suggests that FSTL1 may induce EMT and airway remodeling by activating autophagy. These findings may provide novel avenues for therapeutic research targeting the autophagy and FSTL1 pathway which may be beneficial to patients with refractory asthma.
In airway smooth muscle (ASM) cells, excitation-contraction coupling is accomplished via a cascade of events which connect an elevation of cytosolic Ca2+ concentration ([Ca2+]cyt) with cross-bridge attachment and ATP-consuming mechanical work. Excitation-energy coupling is mediated by linking the elevation in [Ca2+]cyt to an increase in mitochondrial Ca2+ ([Ca2+]mito) which in turn stimulates ATP production. Mitochondria proximity with the sarcoplasmic reticulum (SR) and plasma membrane is thought to be an important mechanism to facilitate mitochondrial Ca2+ uptake. In this regard, mitochondrial movement in ASM may be key in establishing proximity. Mitochondria also move where ATP or Ca2+ buffering is needed. Mitochondrial movement is mediated through interactions with the molecular complex of Miro and Milton, which couples mitochondria to kinesin motors at microtubules. We examined mitochondria movement in human ASM and hypothesized that at basal [Ca2+]cyt levels, mitochondrial movement is necessary to establish proximity of mitochondria with the SR, and that during the transient increase in [Ca2+]cyt induced by agonist stimulation, mitochondrial movement is reduced thereby promoting transient mitochondrial Ca2+ uptake. We further hypothesized that airway inflammation disrupts basal mitochondrial movement via a reduction in Miro and Milton expression, thereby disrupting the ability of mitochondria to establish proximity to the SR and thus reducing transient mitochondrial Ca2+ uptake during agonist activation. The reduced proximity of mitochondria to the SR may affect establishment of transient "hot-spots" of higher [Ca2+]cyt at the sites of SR Ca2+ release that are necessary for mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter.
Pulmonary epithelial cells form the first line of defense of human airways against foreign irritants, and also represent as the primary injury target of these pathogenic assaults. Autophagy is a revolutionary conserved ubiquitous process by which cytoplasmic materials are delivered to lysosomes for degradation when facing environmental and/or developmental changes, and emerging evidence suggests that autophagy plays pivotal but controversial roles in pulmonary epithelial injury. Here we review recent studies focusing on the roles of autophagy in regulating airway epithelial injury induced by various stimuli. Articles eligible for this purpose are divided into two groups according to the eventual roles of autophagy, either protective or deleterious. From the evidences summarized in this review, we draw several conclusions: 1) in all cases when autophagy is decreased from its basal level, autophagy is protective; 2) When autophagy is deleterious, it is generally upregulated by stimulation; 3) a plausible conclusion is that the endosomal/exosomal pathways may be associated with the deleterious function of autophagy in airway epithelial injury, while this needs to be clarified in future investigations.
The alveolar epithelium is composed of type I cells covering most of the gas-blood exchange surface and type II cells secreting surfactant that lowers surface tension of alveoli to prevent alveolar collapse. Here we have identified a subgroup of type II cells expressing higher level of cell surface molecule CD44 (CD44high type II cells) that comprised ~3% of total type II cells in 5-10 week old mice. These cells were preferentially apposed to lung capillaries. They displayed a higher proliferation rate and augmented differentiation capacity into type I cells and the ability to form alveolar organoids compared to CD44low type II cells. More-over, in aged mice of 18-24 months old, the percentage of CD44high type II cells among all type II cells was increased but these cells showed decreased progenitor properties. Thus, CD44high type II cells likely represent a type II sub-population important for constitutive regulating alveolar homeostasis.
Bleomycin-induced lung injury leads to surfactant dysfunction and permanent loss of alveoli due to a remodeling process called collapse induration. Collapse induration also occurs in acute interstitial lung disease and idiopathic pulmonary fibrosis in humans. We hypothesized that surfactant dysfunction aggravates lung injury and early remodeling resulting in collapse induration within 7 days after lung injury. Rats received bleomycin to induce lung injury and either repetitive surfactant replacement therapy (SRT: 100mg Curosurf®/kg BW=surf group)) or saline (0.9% NaCl=saline group). After three (D3) or seven (D7) days, invasive pulmonary function tests were performed to determine tissue elastance (H) and static compliance (Cst). Bronchoalveolar lavage (BAL) was taken for surfactant function, inflammatory markers and protein measurements. Lungs were fixed by vascular perfusion for design-based stereology and electron microscopic analyses. SRT significantly improved minimum surface tension of alveolar surfactant as well as H and Cst at D3 and D7. At D3 decreased inflammatory markers including neutrophilic granulocytes, IL-1β and IL-6 correlated with reduced BAL-protein levels after SRT. Numbers of open alveoli were significantly increased at D3 and D7 in SRT groups while at D7 there was also a significant reduction in septal wall thickness and parenchymal tissue volume. Septal wall thickness and numbers of open alveoli highly correlated with improved lung mechanics after SRT. In conclusion, reduction in surface tension was effective to stabilize alveoli linked with an attenuation of parameters of acute lung injury at D3 and collapse induration at D7. Hence, SRT modifies disease progression to collapse induration.
To test the hypothesis that epoxyeicosatrienoic acids (EETs) facilitate pulmonary responses to hypoxia, male wild type (WT) and soluble epoxide hydrolase knockout (sEH-KO) mice, and WT mice chronically fed a sEH inhibitor (t-TUCB; 1mg/kg/day) were used. Right ventricular systolic pressure (RVSP) was recorded under control and hypoxic conditions. The control RVSP was comparable among all groups. However, hypoxia elicited increases in RVSP in all groups with predominance in sEH-KO and t-TUCB-treated mice. 14,15-EEZE (EET antagonist) attenuated the hypoxia-induced greater elevation of RVSP in sEH-deficient mice, suggesting an EET-mediated increment. Exogenous 5,6-; 8,9- or 14,15-EET (0.05ng/gram body weight) did not change RVSP in any conditions, but 11,12-EET enhanced RVSP under hypoxia. Isometric tension was recorded from pulmonary arteries isolated from WT and sEH-KO mice, vessels that behaved identically in their responsiveness to vasoactive agents and vessel stretch. Hypoxic pulmonary vasoconstriction (HPV, expressed as increases in hypoxic force) was significantly greater in vessels of sEH-KO than WT vessels; the enhanced component was inhibited by EEZE. Treatment of WT vessels with 11,12-EET enhanced HPV to the same level as sEH-KO vessels, confirming EETs as primary players. Inhibition of cyclooxygenases (COXs) significantly enhanced HPV in WT vessels, but attenuated HPV in sEH-KO vessels. Blocking/inhibiting COX-1, prostaglandin H2 (PGH2)/thromboxane A2 (TXA2) receptors and TXA synthase prevented the enhanced HPV in sEH-KO vessels, but had no effects on WT vessels. In conclusion, an EET-dependent alteration in PG-metabolism that favors the action of vasoconstrictor PGH2 and TXA2 potentiates HPV and hypoxia-induced elevation of RVSP in sEH-deficient mice.
Exposure to cadmium (Cd) has been associated with development of chronic obstructive lung disease (COPD). The mechanisms and signaling pathways whereby Cd causes pathological peribronchiolar fibrosis, airway remodeling and subsequent airflow obstruction remain unclear. We aimed to evaluate whether low dose Cd exposure induces vimentin phosphorylation and YAP1 activation leading to peribronchiolar fibrosis and subsequent airway remodeling. Our data demonstrate that Cd induces myofibroblast differentiation and extracellular matrix (ECM) deposition around small (<2 mm in diameter) airways. Upon Cd exposure, α-SMA expression and the production of ECM proteins, including fibronectin and collagen-1, are markedly induced in primary human lung fibroblasts. Cd induces Smad2/3 activation and the translocation of both Smad2/3 and Yes-associated protein 1 (YAP1) into the nucleus. In parallel, Cd induces AKT and cdc2 phosphorylation and downstream vimentin phosphorylation at Ser39 and Ser55, respectively. AKT and cdc2 inhibitors block Cd-induced vimentin fragmentation and secretion in association with inhibition of α-SMA expression, ECM deposition and collagen secretion. Furthermore, vimentin silencing abrogates Cd-induced α-SMA expression and decreases ECM production. Vimentin-deficient mice are protected from Cd-induced peribronchiolar fibrosis and remodeling. These findings identify two specific sites on vimentin that are phosphorylated by Cd and highlight the functional significance of vimentin phosphorylation in YAP1/Smad3 signaling that mediates Cd-induced peribronchiolar fibrosis and airway remodeling.
Airway remodeling including increased airway smooth muscle (ASM) mass is a hallmark feature of asthma and COPD. We previously identified the expression of bitter taste receptors (TAS2Rs) on human ASM cells, and demonstrated that known TAS2R agonists could promote ASM relaxation and bronchodilation, and inhibit mitogen-induced ASM growth. In this study we explored cellular mechanisms mediating the anti-mitogenic effect of TAS2R agonists on human ASM cells. Pre-treatment of ASM cells with TAS2R agonists, chloroquine and quinine, resulted in inhibition of cell survival, which was largely reversed by bafilomycin A1, an autophagy inhibitor. Transmission electron microscope studies demonstrated the presence of double-membrane autophagosomes and deformed mitochondria. In ASM cells, TAS2R agonists decreased mitochondrial membrane potential, increased mitochondrial ROS and mitochondrial fragmentation. Inhibiting dynamin-like protein (DLP1) reversed TAS2R agonist-induced mitochondrial membrane potential change, and attenuated mitochondrial fragmentation and cell death. Furthermore, the expression of mitochondrial protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) and mitochondrial localization of DLP1 were significantly up-regulated by TAS2R agonists. More importantly, inhibiting Bnip3 mitochondrial localization by dominant-negative Bnip3 significantly attenuated cell death induced by TAS2R agonist. Collectively, TAS2R agonists, chloroquine and quinine modulate mitochondrial structure and function resulting in ASM cell death. Furthermore, Bnip3 plays a central role in TAS2R agonist-induced ASM functional changes via a mitochondrial pathway. These findings further establish the cellular mechanisms of anti-mitogenic effects of TAS2R agonists, and identify a novel class of receptors and pathways that can be targeted to mitigate airway remodeling as well as bronchoconstriction in obstructive airway diseases.
Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from 'exaggerated' lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an anti-aging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression whereas Klotho over-expression is protective while Klotho silencing augments AEC mtDNA damage. Compared to wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in WT but not mitochondrial catalase over-expressing (MCAT) mice 3 weeks following exposure to asbestos and that EUK-134, a MnSOD / catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent upon AKT activation arising from upstream fibroblast growth factor receptor 1 (FGFR1) activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.
E-cigarettes are generally thought of as a safer smoking alternative to traditional cigarettes. However, little is known about the effects of e-cigarette liquids (e-liquids) on the lung. Since over 7,000 unique flavors have been identified for purchase in the USA, our goal was to conduct a screen that would test whether different flavored e-liquids exhibited different toxicant profiles. We tested the effects of 13 different flavored e-liquids (with nicotine and propylene glycol/vegetable glycerin (PG/VG) serving as controls) on a lung epithelial cell line (CALU3). Using the MTT assay as an indicator of cell proliferation/viability, we demonstrated a dose-dependent decrease of MTT metabolism by all flavors tested. However, a group of 4 flavors consistently showed significantly greater toxicity compared to the PG/VG control, indicating the potential for some flavors to elicit more harmful effects than others. We also tested the aerosolized 'vapor' from select e-liquids on cells and found similar dose-dependent trends, suggesting that direct e-liquid exposures are a justifiable first-pass screening approach for determining relative e-liquid toxicity. We then identified individual chemical constituents for all 13 flavors using gas chromatography-mass spectrometry. These data revealed that beyond nicotine and PG/VG, the 13 flavored e-liquids have diverse chemical constituents. Since all of the flavors exhibited some degree of toxicity and a diverse array of chemical constituents with little inhalation toxicity available, we conclude that flavored e-liquids should be extensively tested on a case-by-case basis to determine the potential for toxicity in the lung and elsewhere.
Down-regulation of the alveolar macrophage (AM) macrophage receptor with collagenous structure (MARCO) leads to susceptibility to post-influenza bacterial pneumonia, a major cause of morbidity and mortality. We sought to determine whether immunomodulation of MARCO could improve host defense and resistance to secondary bacterial pneumonia. RNAseq analysis identified a striking increase of MARCO expression between days 9 and 11 after influenza infection and indicated important roles for Akt and Nrf2 in MARCO recovery. In vitro, primary human AM-like monocyte-derived macrophages (AM-MDMs) and THP-1 macrophages were treated with IFN to model influenza effects. Activators of Nrf2 (sulforaphane) or Akt (SC79) caused increased MARCO expression and a MARCO-dependent improvement in phagocytosis in IFN-treated cells, and improved survival in mice with post-influenza pneumococcal pneumonia. Transcription factor analysis also indicated a role for transcription factor E-box (TFEB) in MARCO recovery. Overexpression of TFEB in THP-1 cells led to marked increases in MARCO. The ability of Akt activation to increase MARCO expression in IFN-treated AM-MDMs was abrogated in TFEB-knockdown cells, indicating Akt increases MARCO expression through TFEB. Increasing MARCO expression by targeting Nrf2 signaling or Akt-TFEB-MARCO pathway are promising strategies to improve bacterial clearance and survival in post-influenza bacterial pneumonia.
Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Even when patients survive the initial insult, there is significant morbidity and mortality secondary to subsequent pulmonary edema, acute lung injury and nosocomial pneumonia. While the relationship between TBI and secondary pulmonary complications is recognized, little is known about the mechanistic interplay of the two phenomena. Changes in mental status secondary to acute brain injury certainly impair airway- and lung-protective mechanisms. However, clinical and translational evidence suggest that more specific neuronal and cellular mechanisms contribute to impaired systemic and lung immunity that increases the risk of TBI-mediated lung injury and infection. To better understand the cellular mechanisms of that immune impairment, we review here the current clinical data that support TBI-induced impairment of systemic and lung immunity. Furthermore, we also review the animal models that attempt to reproduce human TBI. Additionally, we examine the possible role of damage-associated molecular patterns, the chlolinergic anti-inflammatory pathway and sex dimorphism in post-TBI acute lung injury. In the last part of the review, we discuss current treatments and future pharmacologic therapies, including fever control, tracheostomy and corticosteroids, aimed to prevent and treat pulmonary edema, acute lung injury and nosocomial pneumonia after TBI.
VE-PTP stabilizes endothelial adherens junctions (AJs) through constitutive dephosphorylation of VE-cadherin. Here we investigated the role of STIM1 activation of store-operated Ca2+ entry (SOCE) in regulating adherens junction assembly. We observed that SOCE induced by STIM1 activated Pyk2 in human lung microvascular endothelial cells (ECs) and induced tyrosine phosphorylation of VE-PTP at Y1981. Pyk2-induced tyrosine phosphorylation of VE-PTP promoted Src binding to VE-PTP, Src activation, and subsequent VE-cadherin phosphorylation, and thereby increased the endothelial permeability response. The increase in permeability was secondary to disassembly of AJs. Pyk2-mediated responses were blocked in EC-restricted Stim1 knockout mice indicating the requirement for STIM1 in initiating the signaling cascade. A peptide derived from the Pyk2 phosphorylation site on VE-PTP abolished the STIM1/SOCE-activated the permeability response. Thus, Pyk2 activation secondary to STIM1-induced SOCE causes tyrosine phosphorylation of VE-PTP, and VE-PTP in turn binds to and activates Src, thereby phosphorylating VE-cadherin to increase endothelial permeability through disassembly of AJs. Our results thus identify a novel signaling mechanism by which STIM1-induced Ca2+ signaling activates Pyk2 to inhibit the interaction of VE-PTP and VE-cadherin, and hence increase endothelial permeability. Therefore, targeting the Pyk2 activation pathway may be a potentially important anti-inflammatory strategy.
Supplemental oxygen (O2) increases the risk of lung injury in preterm infants owing to an immature antioxidant system. Our objective was to determine whether impairing antioxidant defense by decreasing glutathione peroxidase 1 (GPx1) gene expression increases the injurious effects of hyperoxia. GPx1+/+ and GPx1-/- C57Bl/6J mice were exposed to 21% O2 (Air) or 40% O2 (Hyperoxia; Hyp) from birth to postnatal day 7 (P7d); they were killed at P7d or maintained in air until adulthood (P56d) to assess short-term and long-term effects, respectively. We assessed lung architecture, three markers of pulmonary oxidative stress (P7d, P56d), macrophages in lung tissue (P7d), immune cells in bronchoalveolar lavage fluid (BALF) (P56d), and GPx1-4 and catalase gene expression in lung tissue (P7d, P56d). At P7d: macrophages were decreased by lack of GPx1 expression, and further decreased by hyperoxia. GPx1 expression was increased in GPx1+/+Hyp mice and decreased in both GPx1-/- groups. At P56d: heme oxygenase-1 was increased by hyperoxia when GPx1 was absent. There were significantly more immune cells from Hyp groups than from the GPx1+/+Air group and greater proportion of lymphocytes in GPx1-/-Hyp. GPx1 expression was significantly decreased in GPx1-/- mice; GPx2-4 and catalase expression was increased in GPx1-/-Hyp mice compared to other groups. Tissue fraction was decreased in GPx1-/-Air mice; bronchiolar smooth muscle was decreased in GPx1-/- mice. GPx1 does not clearly exacerbate hyperoxia-induced increases in oxidative stress or lung injury, but may alter pulmonary immune function. Increased expression of GPx2-4 and catalase in GPx1-/-Hyp mice suggests gene redundancy within the model.
Lung injury can release intracellular actin into the alveolar milieu, and is also associated with increased susceptibility to secondary infections. We investigated the effect of free (extracellular) actin on lung macrophage host defense functions. Western blot analysis demonstrated free actin release into the lung lavage fluids of mouse models of ozone injury, influenza infection and secondary pneumococcal pneumonia, and in samples from patients following burn and inhalation injury. Using levels comparable to those observed in lung injury, we found that free actin markedly inhibited murine lung macrophage binding and uptake in vitro of S. pneumoniae, S. aureus and E. coli e.g., S. pneumoniae, mean % inhibition, actin vs vehicle: 85 ± 0.3 (SD), n = 22, p <.001). Similar effects were observed on the ability of primary human macrophages to bind and ingest fluorescent S. aureus (~75 % inhibition). Plasma gelsolin (pGSN), a protein that functions to bind and cleave actin, restored bacterial binding and uptake by both murine and human macrophages. Scavenger receptor inhibitors reduced binding of fluorescent actin by murine macrophages (fluorescence index (x 10-3) after incubation with vehicle, actin, or actin + polyinosinic acid, respectively: 0.8 ± 0.7, 101.7 ± 50.7, 52.7 ± 16.9, n = 5-6, p < 0.05). In addition, actin binding was reduced in a MARCO / SR-AI/II deficient cell line and by normal AMs obtained from MARCO -/- mice. After release from injured cells during lung injury, free actin likely contributes to impaired host defense by blocking scavenger receptor binding of bacteria. This mechanism for increased risk of secondary infections after lung injury or inflammation may represent another target for therapeutic intervention with pGSN.
Pulmonary surfactant protein-C (SP-C) expression by type II alveolar epithelial cells (AECs) is markedly reduced in diverse types of lung injuries and is often associated with AEC apoptosis. It is unclear whether loss of SP-C contributes to the increased p53 and urokinase-type plasminogen activator (uPA) system cross talk and apoptosis of AECs. We therefore inhibited SP-C expression in human and murine AECs using lentivirus vector expressing shRNA and tested p53 and downstream changes in uPA-fibrinolytic system. Inhibition of SP-C expression in AECs induced p53 and activated caspase-3, indicating AEC apoptosis. We also found that bleomycin or cigarette smoke exposure failed to inhibit SP-C expression or apoptosis in AECs in p53- and plasminogen activator inhibitor-1 (PAI-1)-deficient mice. Depletion of SP-C expression by lentiviral SP-C shRNA in PAI-1-deficient mice failed to induce p53 or apoptosis in AECs, while it increased both AEC p53 and apoptosis in wild type or uPA-deficient mice. SP-C inhibition in AECs also increased in CXCL1 and CXCL2, and their receptor CXCR2 as well as ICAM-1 expression, indicative of a pro-inflammatory response. Overexpression of p53-binding 3'UTR sequences in AECs inhibited PAI-1 induction while maintaining uPA and uPAR protein and mRNA expression. Further, caveolin-1 expression and phosphorylation were increased in AECs indicating an intricate link between caveolin-1 and Src kinase-mediated cell signalling and AEC apoptosis due to loss of SP-C expression through p53 and uPA system-mediated cross-talk. The role of uPA, PAI-1 and p53 in the regulation of AEC apoptosis after injury was also determined in knock out mice.
Although different preclinical models have demonstrated a favorable role for bone marrow derived mesenchymal stem cells (B-MSC) in preventing fibrosis, this protective effect is not observed with late administration of these cells, when fibrotic changes are consolidated. We sought to investigate whether the late administration of B-MSCs overexpressing microRNAs (miRNAs) let-7d (anti-fibrotic) or miR-154 (pro-fibrotic) could alter lung fibrosis in a murine bleomycin model. Using lentiviral vectors we transduced miRNAs (let-7d or miR-154) or a control sequence into human B-MSCs. Overexpression of let-7d or miR-154 was associated with changes in the mesenchymal properties of B-MSCs and in their cytokine expression. Modified B-MSCs were intravenously administered to mice at day 7 after bleomycin instillation and the mice were sacrificed at day 14. Bleomycin-injured animals treated with let-7d cells were found to recover quicker from the initial weight loss compared to the other treatment groups. Interestingly, animals treated with miR-154 cells had the lowest survival rate. Although a slight reduction in collagen mRNA levels was observed in lung tissue from let-7d mice, no significant differences were observed in Ashcroft score and OH-proline. However, the distinctive expression in cytokines and CD45 positive cells in the lung suggest that the differential effects observed in both miRNAs mice groups were related to an effect on the immunomodulation function. Our results establish the use of miRNA-modified mesenchymal stem cells as a potential future research in lung fibrosis.
Pulmonary fibrosis is a severe condition with no cure and limited therapeutic options. Better understanding of its pathophysiology is needed. Recent studies have suggested that pulmonary fibrosis may be driven by accelerated aging-related mechanisms. Sirtuins (SIRTs), particularly SIRT1, -3, and -6, are well-known mediators of aging, however limited data exist on the contribution of sirtuins to lung fibrosis. We assessed the mRNA and protein levels of all seven known sirtuins in primary lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) in comparison with lung fibroblasts from healthy controls. These unbiased tests revealed a tendency for all sirtuins to be expressed at lower levels in fibroblasts from patients compared to controls, but the greatest decrease was observed with SIRT7. Similarly, SIRT7 was decreased in lung tissues of bleomycin-challenged mice. Inhibition of SIRT7 with siRNA in cultured lung fibroblasts resulted in an increase in collagen and alpha-smooth muscle actin (α-SMA). Reciprocally, overexpression of SIRT7 resulted in lower basal and TGF-β-induced levels of COL1A1, COL1A2, COL3A1, and α-SMA mRNAs, as well as collagen and α-SMA proteins. Induced changes in SIRT7 had no effect on endogenous TGF-β mRNA levels or latent TGF-β activation, but overexpression of SIRT7 reduced the levels of Smad3 mRNA and protein. In conclusion, the decline in SIRT7 in lung fibroblasts has a profibrotic effect, which is mediated by changes in Smad3 levels.
Bacterial infection can lead to acidosis of the local microenvironment. While this is believed to exacerbate disease pathogenesis, the mechanisms by which changes in pH alter disease progression are poorly understood. In this report we test the hypothesis that acidosis enhances respiratory epithelial cell death in response to infection with Pseudomonas aeruginosa. Our findings support that acidosis in the context of P. aeruginosa infection results in increased epithelial cell cytotoxicity due to ExoU intoxication. Importantly, enforced maintenance of neutral pH during P. aeruginosa infection demonstrates that cytotoxicity is dependent upon the acidosis. Investigation of the underlying mechanisms revealed that host cell cytotoxicity correlated with increased bacterial survival during an acidic infection which was due to reduced bactericidal activity of host-derived antimicrobial peptides. These findings extend previous reports that the activities of antimicrobial peptides are pH-dependent and provide novel insights into the consequences of acidosis on infection-derived pathology. Therefore, this report provides the first evidence that physiological levels of acidosis increase the susceptibility of epithelial cells to acute Pseudomonas infection and demonstrates the benefit of maintaining pH homeostasis during a bacterial infection.
Airway inflammation is a hallmark of asthma triggering airway smooth muscle (ASM) hyperreactivity and airway remodeling. TNFα increases both agonist-induced cytosolic Ca2+ concentration and force in ASM. The effects of TNFα on ASM force may also be due to an increase in Ca2+ sensitivity, cytoskeletal remodeling and/or changes in contractile protein content. We hypothesized that 24-h exposure TNFα increases ASM force by changing actin and myosin heavy chain (MyHC) content and/or polymerization. Porcine ASM strips were permeabilized with 10% Triton X-100, and force was measured in response to increasing concentrations of Ca2+ (pCa 9.0 to pCa 4.0) in control and TNFα treated groups. Relative phosphorylation of the regulatory myosin light chain, total actin, MLC and myosin heavy chain (MyHC) concentrations were quantified at pCa 9.0, pCa 6.1 and pCa 4.0. Actin polymerization was quantified by the ratio of filamentous to globular actin at pCa 9.0 and pCa 4.0. Total cross-bridge formation was determined by measuring isometric ATP hydrolysis rate at pCa 4.0 using an enzyme-coupled NADH-fluorometric technique. Exposure to TNFα significantly increased force across the range of Ca2+ activation, but did not affect the intrinsic Ca2+ sensitivity of force generation. The TNFα-induced increase in ASM force was associated with an increase in total actin, MLC and MyHC content as well as increased actin polymerization and increase in maximum isometric ATP hydrolysis rate. The results of this study support our hypothesis that TNFα increases force generation in ASM by increasing the number of contractile units (actin-myosin content) contributing to force generation.
Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair responses and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to TGF-β1. Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-β1, and the expression of mesenchymal phenotype and function were analyzed. Pharmacologic protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling were analyzed using siRNA knockdown, inhibitors, and expression of mutant constructs of FAK. Apoptosis was measured using cleaved caspase-3 analysis and TUNEL staining. TGF-β1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of 3D collagen gels. This phenotypic transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD2/3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-β1 induces both EMP and apoptosis in the same cell culture system, but not in the same cell. While blockade of SMAD signaling inhibits EMP, it has a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifts AECs to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGFβ-1 stimulation. TGFβ-1-induced EMP is FAK-dependent; whereas, TGFβ-1-induced apoptosis in AECs is favored when FAK signaling is inhibited.
Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. This association is thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it's unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 co-expressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared to cells co-expressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when co-expressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with co-expression of wt CFTR. However, co-expression with F508del CFTR only increased SLC26A9's interaction with CAL. Mutation of SLC26A9's PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is co-expressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.
Electronic cigarette usage is increasing worldwide, yet there is a paucity of information on the respiratory health effects of electronic cigarette aerosol exposure. This study aimed to assess whether exposure to electronic-cigarette (e-cigarette) aerosol would alter lung function and pulmonary inflammation in mice, and to compare the severity of any alterations with mice exposed to mainstream tobacco smoke. Female BALB/c mice were exposed for 8 weeks to tobacco smoke, medical air (control) or one of 4 different types of e-cigarette aerosol. E-cigarette aerosols varied depending on nicotine content (0 or 12mg/mL) and the main excipient (propylene glycol or glycerin). 24 hours after the final exposure, we measured pulmonary inflammation, lung volume, lung mechanics, and responsiveness to methacholine. Mice exposed to tobacco cigarette smoke had increased pulmonary inflammation and responsiveness to methacholine compared with air controls. Mice exposed to e-cigarette aerosol did not have increased inflammation, but did display decrements in parenchymal lung function at both functional residual capacity and high transrespiratory pressures. Mice exposed to glycerin based e-cigarette aerosols were also hyper-responsive to methacholine regardless of the presence or absence of nicotine. This study shows, for the first time, that exposure to e-cigarette aerosol during adolescence and early adulthood is not harmless to the lungs, and can result in significant impairments in lung function.
To investigate apoptosis as a mechanism of sulfur mustard (SM) inhalation injury in animals, we studied different caspases (caspase-8, -9, -3 and -6) in the lungs from a ventilated rat SM aerosol inhalation model. SM activated all four caspases in cells obtained from bronchoalveolar lavage fluid (BALF) as early as 6 hr after exposure. Caspase-8, which is known to initiate the extrinsic Fas-mediated pathway of apoptosis, was increased 5-fold between 6 to 24 hr, decreasing to the unexposed-control level at 48 hr. The initiator, caspase-9, in the intrinsic mitochondrial pathway of apoptosis as well as the executioner caspases, caspase-3 and -6, all peaked (p<0.01) at 24 hr; caspase-3 and -6 remained elevated, but caspase-9 decreased to unexposed-control level at 48 hr. To study further the Fas pathway, we examined soluble as well as membrane-bound Fas ligand (sFas-L, mFas-L, respectively) and Fas receptor (Fas-R) in both BALF cells and BALF. SFas-L increased significantly at 24 hr after SM exposure in both BALF cells (p<0.01) and BALF (p<0.05). However, mFas-L increased only in BALF cells between 24 to 48 hr (p<0.1, <0.001, respectively). Fas-R increased only in BALF cells by 6 hr (p<0.01) after SM exposure. Apoptosis in SM-inhaled rat lung specimens was also confirmed by both immunohistochemical staining using cleaved caspse-3 and -9 antibodies and TUNEL staining as early as 6 hr in the proximal trachea and bronchi, but not before 48 hr in distal airways. These findings suggest pathogenic mechanisms at the cellular and molecular levels and logical therapeutic target(s) for SM inhalation injury in animals.
Airway smooth muscle (ASM) orientation and morphology determine the ability of the muscle to constrict the airway. In asthma, ASM mass is increased, but it is unknown whether ASM orientation and morphology are altered as well, or whether the remodelling at the source of the mass increase is ongoing. We dissected human airway trees from asthmatic and control lungs. Stained, intact airway sections were imaged in axial projection to show ASM bundle orientation, while cross-sectional histological slides were used to assess ASM area, bundle thickness and ASM bundle to basement membrane distance. We also used these slides to assess cell size, proliferation and apoptosis. We showed that ASM mass increase in cartilaginous airways is primarily the result of an increase of ASM bundle thickness (as measured radially in an airway cross-section) and coincides with an increased distance of the ASM bundles to the airway perimeter. ASM orientation was unchanged in all airways. Apoptosis markers and cell size did not show differences between asthmatics and controls. Our findings show that ASM mass increase likely contributes to the airway constricting capacity of the muscle. Both the increased bundle thickness and increased thickness of the airway wall inwards of the ASM bundles could further enhance this capacity. Turnover of ASM appears to be the same in airways and biopsies, but the lack of correlation between different markers of proliferation casts doubt on the specificity of markers generally used to assess proliferation.
Early-life wheezing associated respiratory tract infection by rhinovirus (RV) is considered a risk factor for asthma development. We have shown that RV infection of 6 day-old BALB/c mice, but not mature mice, induces an asthma-like phenotype which is associated with an increase in the population of type 2 innate lymphoid cells (ILC2s) and dependent on IL-13 and IL-25. We hypothesize that ILC2s are required and sufficient for development of the asthma-like phenotype in immature mice. Mice were infected with RV1B on day 6 of life and treated with vehicle or a chemical inhibitor of retinoic acid receptor-related orphan receptor (ROR)-α, SR3335 (15 mg/kg/day intraperitoneally for 7 days). We also infected Rorasg/sg mice without functional ILC2s. ILC2s were identified as negative for lineage markers and positive for CD25/IL-2Rα and CD127/IL-7Rα. Effects of SR3335 on proliferation and function of cultured ILC2s were determined. Finally, sorted ILC2s were transferred into naïve mice and lungs harvested 14 days later for assessment of gene expression and histology. SR3335 decreased the number of RV-induced lung lineage-negative, CD25+, CD127+ ILC2s in immature mice. SR3335 also attenuated lung mRNA expression of IL-13, Muc5ac and Gob5 as well as mucous metaplasia. We also found reduced expansion of ILC2s in RV-infected Rorasg/sg mice. SR3335 also blocked IL-25 and IL-33-induced ILC2 proliferation and IL-13 production ex vivo. Finally, adoptive transfer of ILC2s led to development of asthma like phenotype in immature and adult mice. RORα-dependent ILC2s are required and sufficient for type 2 cytokine expression and mucous metaplasia in immature mice.-
Asthma is a common disorder characterized, in part, by airway smooth muscle (ASM) hyperresponsiveness. Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel expressed on airway nerve fibers that modulates afferent signals resulting in cough, and potentially bronchoconstriction. In the present study, the TRPV1 transcript was detected by RT-PCR in primary cultured human ASM cells, and the TRPV1 protein was detected in ASM of human trachea by immunohistochemistry. Proximity ligation assays suggest that TRPV1 is expressed in the sarcoplasmic reticulum membrane of human ASM cells in close association with sarco/endoplasmic reticulum Ca2+ ATPase 2. In guinea pig tracheal ring organ bath experiments, the TRPV1 agonist capsaicin led to ASM contraction, but this contraction was significantly attenuated by the sodium-channel inhibitor bupivicaine (N=4, p<0.05) and the NK-2 receptor antagonist GR 159897 (N=4, p<0.05), suggesting that this contraction is neurally-mediated. However, pretreatment of guinea pig and human ASM in organ bath experiments with the TRPV1 antagonist capsazepine inhibited the maintenance phase of an acetylcholine-induced contraction (N=4, p<0.01 for both species). Similarly, capsazepine inhibited methacholine-induced contraction of peripheral airways in mouse precision-cut lung slice (PCLS) experiments (N=4-5, p<0.05). Although capsazepine did not inhibit store-operated calicum entry in mouse ASM cells in PCLS (N=4-7, p=NS), it did inhibit calcium oscillations (N=3, p<0.001). These studies suggest that TRPV1 is expressed on ASM, including the SR, but that ASM TRPV1 activation does not play a significant role in initiation of ASM contraction. However, capsazepine does inhibit maintenance of contraction, likely by inhibiting calcium oscillation.
Our body clock drives rhythms in the expression of genes that have a 24-hour periodicity. BMAL1 is a transcription factor, which is a crucial component in the molecular clock. A number of physiological processes, including immune function are modulated by the circadian clock. Asthma is of particular relevance to the area of circadian control of immunity, since it is a disease with very strong clinical evidence demonstrating regulation by circadian variation. Airway hypersensitivity and asthma attacks are more common at night in humans. The molecular basis for this is unknown and no model of asthma in animals with genetic distortion of the molecular clock exists. In our study we have used mice lacking BMAL1 in myeloid cells (BMAL1-LysM-/-) to determine the role of BMAL1 in allergic asthma. Using the Ovalbumin model of allergic asthma we demonstrated that BMAL1-LysM-/- mice have markedly increased asthma features such as increased lung inflammation demonstrated by drastically higher numbers of eosinophils and increased IL-5 levels in the lung and serum. In vitro studies demonstrated that IL-4 as well as LPS treated macrophages from BMAL1-LysM-/- mice have increased pro-inflammatory chemokine expression and M2 markers compared to wild type controls. This suggests that Bmal1 is a potent negative regulator, in myeloid cells in the context of allergic asthma. Our findings might explain the increase in asthma incidents during the night when BMAL1 expression is low.
Myofibroblasts are important mediators of fibrogenesis, thus blocking fibroblast to myofibroblast differentiation (FMD) may be an effective strategy to treat pulmonary fibrosis (PF). Previously we reported that HDAC4 activity is necessary for TGF-β1-induced human lung FMD. Here we show that TGF-β1 increases NOX4 mRNA and protein expression in normal human lung fibroblasts (NHLFs) and causes nuclear export of HDAC4. Application of the NOX family inhibitor, diphenyleneiodonium chloride (DPI), reduces TGF-β1-induced HDAC4 nuclear export, expression of the myofibroblast marker α-smooth muscle actin (α-SMA), and α-SMA fiber formation. Inhibition of HDAC4 nucleus to cytoplasm translocation using leptomycin B (LMB) had little effect on α-SMA expression, but blocked α-SMA fiber formation. A co-immunoprecipitation assay showed that HDAC4 associates with α-SMA. Moreover LMB abolishes TGF-β1-induced α-SMA fiber formation and cell contraction. Relevant to human pulmonary fibrosis, IPF specimens showed significantly higher NOX4 RNA expression and scant HDAC4 staining within nuclei of fibroblast foci myofibroblasts. Taken together, these results indicate that ROS promote TGF-β1-mediated myofibroblast differentiation and HDAC4 nuclear export. The physical association of HDAC4 with α-SMA suggests that HDAC4 has a role in regulating the α-SMA cytoskeleton arrangement.
In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). Using Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Vegfr2. While best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express Vegfr2 and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. Using an experimental co-culture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of 3-dimensional structures. Both LPS and tyrosine kinase inhibition reduced 3-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.
Abstract: Pompe disease is an autosomal recessive disorder caused by a deficiency of acid alpha-glucosidase (GAA) - an enzyme responsible for hydrolyzing lysosomal glycogen. Deficiency of GAA leads to systemic glycogen accumulation in the lysosomes of skeletal muscle, motor neurons and smooth muscle. Skeletal muscle and motor neuron pathology are known to contribute to respiratory insufficiency in Pompe disease, but the role of airway pathology has not been evaluated. Here we propose that GAA enzyme deficiency disrupts the function of the trachea and bronchi, and this lower airway pathology contributes to respiratory insufficiency in Pompe disease. Using an established mouse model of Pompe disease - the Gaa-/- mouse - we compared histology, pulmonary mechanics, airway smooth muscle function and calcium signaling between Gaa-/- and age matched wild type (WT) mice. Lysosomal glycogen accumulation was observed in the smooth muscle of both the bronchi and the trachea in Gaa-/- but not WT mice. Furthermore, Gaa-/- mice had hyporesponsive airway resistance and bronchial ring contraction to the bronchoconstrictive agents methacholine (Mch) and potassium chloride (KCl), and to a bronchodilator (albuterol). Finally, calcium signaling during bronchiolar smooth muscle contraction was impaired in Gaa-/- mice indicating impaired extracellular calcium influx. We conclude that GAA enzyme deficiency leads to glycogen accumulation in the trachea and bronchi, and impairs the ability of lower airway smooth muscle to regulate calcium and respond appropriately to bronchodilator or constrictors. Accordingly, airway smooth muscle dysfunction may contribute to respiratory impairments in Pompe disease.
A GPCR named FFA4 (also known as GPR120) was found to act as a GPCR for omega-3 polyunsaturated fatty acids. Its expression has been reported in lung epithelial club cells. The authors investigated whether supplementation of the omega-3 fatty acids benefits lung health. Omacor® (7.75 mg kg-1), clinically prescribed preparation of omega-3 fatty acids and FFA4-knockout mice were utilized in a naphthalene-induced mouse model of acute airway injury (one injection of 30 mg kg-1, i.p.). Naphthalene injection induced complete destruction of bronchiolar epithelial cells within a day. Appearance of bronchiolar epithelial cells was observed after 21 days in control mice. It was found, however, that supplementation of omacor accelerated the recovery. The appearance of bronchiolar epithelial cells was observed between 7 and 14 days after naphthalene injury in omacor-treated mice. In isolated club cells, omega-3 fatty acids were found to stimulate cell proliferation and migration but to inhibit cell differentiation. Using pharmacological tools and FFA4-knockout mice, FFA4 was found to be responsible for omega-3 fatty acids-induced proliferation in vitro in club cells. Furthermore, accelerated recovery from naphthalene-induced airway injury in omacor-treated mice was not observed in FFA4-knockout mice in vivo. Present findings indicate that omega-3 fatty acids-induced proliferation of bronchiole epithelial cells through FFA4 is responsible for omacor-induced accelerated recovery from airway injury. Therefore, intermittent administration of omacor needs to be tested for acute airway injury, because omega-3 fatty acids stimulate proliferation but inhibits differentiation of club cells.
Post-natal lung maturation generates a large number of small alveoli, with concomitant thinning of alveolar septal walls, generating a large gas exchange surface area but minimizing the distance traversed by the gases. This demand for a large and thin gas exchange surface area is not met in disorders of lung development, such as bronchopulmonary dysplasia (BPD), histopathologically characterized by fewer, larger alveoli; and thickened alveolar septal walls. Diseases such as BPD are often modeled in the laboratory mouse to better understand disease pathogenesis, or develop new interventional approaches. To date, there are no reports of stereology-based longitudinal studies on post-natal mouse lung development that report dynamic changes in alveoli number or alveolar septal wall thickness during lung maturation. To this end, changes in lung structure were quantified over the first 22 months of post natal life of C57BL/6J mice. Alveolar density peaked at post-natal day (P)39, and remained unchanged at nine months (P274), but was reduced by 22 months (P669). Alveoli continued to be generated, initially at an accelerated rate between P5 and P14, and at a slower rate thereafter. Between P274 and P669, loss of alveoli was noted, without any reduction in lung volume. A progressive thinning of the alveolar septal wall was noted between P5 and P28. Pronounced sex differences were observed in alveoli number in adult, but not juvenile mice, comparing male and female mouse lungs, which were attributed exclusively to the larger volume of male mouse lungs.
The tumor suppressor, WWOX, exhibits regulatory interactions with an array of transcription factors and signaling molecules that are positioned at the well-known crossroads between inflammation and cancer. WWOX is also subject to downregulation by genotoxic environmental exposures, making it of potential interest to the study of lung pathobiology. Knockdown of lung WWOX expression in mice was observed to cause neutrophil influx, and accompanied by a corresponding vascular leak and inflammatory cytokine production. In cultured human alveolar epithelial cells, loss of WWOX expression resulted in increased c-Jun- and IL-8- dependent neutrophil chemotaxis towards cell monolayers. WWOX was observed to directly interact with c-Jun in these cells, and its absence resulted in increased nuclear translocation of c-Jun. Finally, inhibition of c-Jun activating kinase, JNK, abrogated the lung neutrophil influx observed during WWOX knockdown in mice. Altogether, these observations represent a novel mechanism of pulmonary neutrophil influx that is highly relevant to the pathobiology and potential treatment of a number of different lung inflammatory conditions.
Pulmonary complications from stored blood products are the leading cause of mortality related to transfusion. Transfusion related acute lung injury is mediated by antibodies or bioactive mediators, yet, underlying mechanisms are incompletely understood. Sphingolipids such as ceramide regulate lung injury, and their composition changes as a function of time in stored blood. Here, we tested the hypothesis that aged platelets may induce lung injury via a sphingolipid-mediated mechanism. To assess this hypothesis, a two hit mouse model was devised. Recipient mice were treated with 2 mg/kg intraperitoneal lipopolysaccharide (priming) two hours prior to transfusion of 10 mL/kg stored (1-5 days) platelets treated with or without addition of acid sphingomyelinase inhibitor ARC39 or platelets from acid sphingomyelinase deficient mice which both reduce ceramide formation. Transfused mice were examined for signs of pulmonary neutrophil accumulation, endothelial barrier dysfunction, and histological evidence of lung injury. Sphingolipid profiles in stored platelets were analyzed by mass spectrophotometry. Transfusion of aged platelets into primed mice induced characteristic features of lung injury, which increased in severity as a function of storage time. Ceramide accumulated in platelets during storage, but this was attenuated by ARC39 or in acid sphingomyelinase deficient platelets. Compared to wild type platelets, transfusion of ARC39-treated or acid sphingomyelinase deficient aged platelets alleviated lung injury. Aged platelets elicit lung injury in primed recipient mice, which can be alleviated by pharmacological inhibition or genetic deletion of acid sphingomyelinase. Interventions targeting sphingolipid formation represent a promising strategy to increase the safety and longevity of stored blood products.
Exacerbations of chronic obstructive pulmonary disease (COPD) are triggered by viral or bacterial pathogens, with human rhinovirus (HRV) and nontypeable Haemophilus influenzae (NTHI) among the most commonly detected pathogens. Patients who suffer from concomitant viral and bacterial infection have more severe exacerbations. The airway epithelial cell is the initial site of viral and bacterial interactions, and CCL20 is an epithelial chemokine that attracts immature dendritic cells to the airways, and can act as an antimicrobial. As such, it contributes to innate and adaptive immune responses to infection. We used primary cultures of human bronchial epithelial cells and the BEAS-2B cell line to examine the effects of bacterial-viral co-exposure, as well as each stimulus alone, epithelial expression of CXCL8 and, in particular, CCL20. HRV-bacterial co-exposure induced synergistic production of CXCL8 and CCL20 compared to the sum of each stimulus alone. Synergistic induction of CCL20 did not require viral replication and occurred with two different HRV serotypes that use different viral receptors. Synergy was also seen with either NTHI or Pseudomonas aeruginosa. Synergistic induction of CCL20 was transcriptionally regulated. Although NF-B was required for transcription, it did not regulate synergy, but NF-IL-6 did appear to contribute. Among MAP kinase inhibitors studied, neither SB203580 nor PD98059 had any effect on synergy, while U0126 prevented synergistic induction of CCL20 by HRV and bacteria, apparently via "off target" effects. Thus, bacterial-viral co-exposure synergistically increases innate immune responses compared to individual infections. We speculate that this increased inflammatory response leads to worse clinical outcomes.
A thin fluid layer in alveoli is normal and results from a balance of fluid entry and fluid uptake by transepithelial salt and water reabsorption. Conventional wisdom suggests the reabsorption is via epithelial Na+ channels (ENaC), but if all Na+ reabsorption was via ENaC, then amiloride, an ENaC inhibitor, should block AFC. But amiloride blocks only half of AFC. The reason for failure to block is clear from single channel measurements from alveolar epithelial cells: ENaC channels are observed, but another channel is present at the same frequency that is non-selective for Na+ over K+, has a larger conductance, and shorter open and closed times. These two channel types are known as highly-selective channels (HSC) and non-selective cation channels (NSC). HSC channels are made up of three ENaC subunits since knocking down any of the subunits reduces HSC number. NSC channels contain α-ENaC since knocking down α-ENaC reduces the number of NSC (knocking down β- or -ENaC has no effect on NSC, but the molecular composition of NSC channels remains unclear. We show that NSC channels consist of at least one α-ENaC and one or more Acid-Sensing Ion Channel 1 (ASIC1a) proteins. Knocking down either α-ENaC or ASIC1a reduces both NSC and HSC number and no NSC channels are observable in single channel patches on lung slices from ASIC1a knockout mice. AFC is reduced in knockout mice and wet/dry ratio is increased, but the percentage increase in wet/dry ratio is larger than expected based on the reduction in AFC.
Progressive pulmonary fibrosis is a devastating consequence of many acute and chronic insults to the lung. Lung injury leads to alveolar epithelial cell (AEC) death, destruction of the basement membrane and activation of TGFβ. There is subsequent resolution of the injury and a coordinated and concurrent initiation of fibrosis. Both of these processes may involve activation of similar intracellular signaling pathways regulated in part by dynamic changes to the extracellular matrix. Matrix signaling can augment the pro-fibrotic fibroblast response to TGFβ. However, similar matrix/integrin signaling pathways may also be involved in inhibition of ongoing TGFβ-induced AEC apoptosis. Focal adhesion kinase (FAK) is an integrin associated signaling molecule expressed by many cell types. We utilized mice with AEC-specific FAK deletion to isolate the epithelial aspect of integrin signaling in the bleomycin model of lung injury and fibrosis. Mice with AEC-specific deletion of FAK did not exhibit spontaneous lung injury but did have significantly greater TUNEL-positive cells (18.6 vs 7.1) per 200x field, greater BAL protein (3.2 vs 1.8 mg/mL) and significantly greater death (77% vs 19%) after bleomycin injury compared to littermate control mice. Within primary AECs, activated FAK directly associates with caspase8 and inhibit activation of the caspase cascade resulting in less apoptosis in response to TGFβ. Our studies support a model in which dynamic changes to the extracellular matrix after injury promotes fibroblast activation and inhibition of epithelial cell apoptosis in response to TGFβ through FAK activation potentially complicating attempts to non-specifically target this pathway for anti-fibrotic therapy.
In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% v/v) or purified FXa (15-50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270% and 46% respectively (*P<0.05, n=5-8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 μM), or antibodies raised against annexin A2 or PAR-1 (2 μg/mL). FXa-stimulated LFs from IPF patients (n=6) produced twice as much IL-6 as controls (n=10) (*P<0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (*P<0.05, n=4-12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion but lung levels of collagen were not. Our data suggests that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF.
Kv7 potassium channels have recently been found to be expressed and functionally important for relaxation of airway smooth muscle. Previous research suggests that native Kv7 currents are inhibited following treatment of freshly isolated airway smooth muscle cells with bronchoconstrictor agonists, and in intact airways inhibition of Kv7 channels is sufficient to induce bronchiolar constriction. However, the mechanism by which Kv7 currents are inhibited by bronchoconstrictor agonists has yet to be elucidated. In the present study, native Kv7 currents in cultured human trachealis smooth muscle cells (HTSMCs) were observed to be inhibited upon treatment with histamine; inhibition of Kv7 currents was associated with membrane depolarization and an increase in cytosolic Ca2+ ([Ca2+]cyt). The latter response was inhibited by verapamil, a blocker of L-type voltage sensitive Ca2+ channels (VSCCs). Protein kinase C (PKC) has been implicated as a mediator of bronchoconstrictor actions, though the targets of PKC are not clearly established. We found that histamine treatment significantly and dose-dependently suppressed currents through overexpressed wild-type human Kv7.5 (hKv7.5) channels in cultured HTSMCs, and this effect was inhibited by the PKC inhibitor Ro-31-8220 (3 µM). The PKC-dependent suppression of hKv7.5 currents corresponded with a PKC-dependent increase in hKv7.5 channel phosphorylation. Knocking down or inhibiting PKCα, or mutating hKv7.5 serine 441 to alanine, abolished the inhibitory effects of histamine on hKv7.5 currents. These findings provide the first evidence linking PKC activation to suppression of Kv7 currents, membrane depolarization, and Ca2+ influx via L-type VSCCs as a mechanism for histamine-induced bronchoconstriction.
Introduction: Right ventricular dysfunction is associated with numerous smoking-related illnesses including chronic obstructive pulmonary disease (COPD) where it is present even in absence of pulmonary hypertension. It is unknown if exposure to cigarette smoke has direct effects on RV function and cardiac fibroblast proliferation or collagen synthesis. In this study, we evaluated cardiac function and fibrosis in mice exposed to cigarette smoke (CS) and determined mechanisms of smoke-induced changes in cardiac fibroblast signaling and fibrosis. Methods: AKR mice were exposed to cigarette smoke for six weeks followed by echocardiography and evaluation of cardiac hypertrophy, collagen content, and pulmonary muscularization. Proliferation and collagen content were evaluated in primary isolated rat cardiac fibroblasts (CF) exposed to cigarette smoke extract (CSE) or nicotine. Markers of cell proliferation, fibrosis, and proliferative signaling were determined by immunoblot or Sircol collagen assay. Results: Mice exposed to CS had significantly decreased RV function as determined by TAPSE. There were no changes in LV parameters. RV collagen content was significantly elevated but there was no change in RV hypertrophy or pulmonary vascular muscularization. CSE directly increased cardiac fibroblast proliferation and collagen content in CF. Nicotine alone reproduced these effects. CSE and nicotine-induced fibroblast proliferation and collagen content were mediated through α7 nicotinic acetylcholine receptors and were dependent on PKC-α, PKC-, and reduced p38-MAPK phosphorylation. Conclusion: CS and nicotine have direct effects on cardiac fibroblasts to induce proliferation and fibrosis which may negatively affect right heart function.
There is considerable biologic and physiologic heterogeneity among patients who meet standard clinical criteria for acute respiratory distress syndrome (ARDS). In this study, we tested the hypothesis that there exists a sub-group of ARDS patients who exhibit a metabolically distinct profile. We examined undiluted pulmonary edema fluid obtained at the time of endotracheal intubation from 16 clinically phenotyped ARDS patients and 13 control patients with hydrostatic pulmonary edema. Non-targeted metabolic profiling was carried out on the undiluted edema fluid. Univariate and multivariate statistical analyses including principal components analysis (PCA) and partial least squares discriminant analysis (PLSDA) were conducted to find discriminant metabolites. 760 unique metabolites were identified in the pulmonary edema fluid of these 29 patients. We found that a subset of ARDS patients (6/16, 38%) presented a distinct metabolic profile with the overrepresentation of 235 metabolites compared to edema fluid from the other 10 ARDS patients, whose edema fluid metabolic profile was indistinguishable from those of the 13 control patients with hydrostatic edema. This "high metabolite" endotype was characterized by higher concentrations of metabolites belonging to all of the main metabolic classes including lipids, amino acids, and carbohydrates. This distinct group with high metabolite levels in the edema fluid was also associated with a higher mortality rate. Thus, metabolic profiling of the edema fluid of ARDS patients supports the hypothesis that there is considerable biologic heterogeneity among ARDS patients who meet standard clinical and physiologic criteria for ARDS.
We reported defective efferocytosis associated with cigarette smoking and/or airway inflammation in chronic lung diseases including COPD, severe asthma and childhood bronchiectasis. We also showed defects in phagocytosis of non-typeable H. influenzae (NTHi), a common colonizer of the lower airway in these diseases. These defects could be substantially overcome with low-dose Azithromycin; however, chronic usage may induce bacterial resistance. We investigated two novel macrolides, GS-459755 (2'-desoxy-9-(S)-erythromycylamine) and GS-560660 (Azithromycin-based 2'-desoxy molecule) with significantly diminished antibiotic activity against S. aureus, S. pneumonia, M. catarrhalis, and H. influenzae. We tested their effects on efferocytosis, phagocytosis of NTHi, cell viability, receptors involved in recognition of apoptotic cells and/or NTHi (flow cytometry), secreted and cleaved intracellular IL-1β (CBA, immunofluorescence/ confocal microscopy) and NLRP3, using primary alveolar macrophages and THP-1 macrophages ± 10% cigarette smoke extract. Dose response experiments showed optimal pro-phagocytic effects of GS-459755 and GS-560660 at concentrations of 0.5-1µg/mL, comparable to our findings with Azithromycin. Both macrolides significantly improved phagocytosis of apoptotic cells and NTHi (eg, increases in efferocytosis and phagocytosis of NTHi: GS-459755 23% and 22.5% p=0.043; GS-560660 23.5% and 22% p=0.043, respectively). Macrophage viability remained >85% following 24h exposure to either macrolide at concentrations up to 20µg/mL. Secreted and intracellular cleaved IL-1β were decreased with both macrolides with no significant changes in recognition molecules MerTk, SRA1, TLR2/4 or CD36. Particulate cytoplasmic immunofluorescence of NLRP3 inflammasome was also significantly reduced. We conclude that GS-459755 and GS-560660 may be useful for reducing airway inflammation in chronic lung diseases without inducing bacterial resistance.
Ozone causes vagally-mediated airway hyperreactivity and recruits inflammatory cells, including eosinophils, to lungs where they mediate ozone-induced hyperreactivity one day after exposure, but are paradoxically protective three days later. To test the role of newly divided eosinophils in ozone-induced airway hyperreactivity in sensitized and non-sensitized guinea pigs. Non-sensitized and sensitized guinea pigs were treated with 5-bromo-2-deoxyuridine (BrdU) to label newly divided cells and were exposed to air or ozone for 4 hours. One or three days later, vagally-induced bronchoconstriction was measured and inflammatory cells harvested from bone marrow, blood, and bronchoalveolar lavage. Ozone induced eosinophil hematopoiesis. One day post ozone, mature eosinophils dominate the inflammatory response and potentiate vagally-induced bronchoconstriction. However, by three days, newly divided eosinophils have reached the lungs where they inhibit ozone-induced airway hyperreactivity, since depleting them with AbIL-5 or a TNFα antagonist, worsened vagally-induced bronchoconstriction. In sensitized guinea pigs, both ozone-induced eosinophil hematopoiesis and subsequent recruitment of newly divided eosinophils to lungs three days later failed to occur. Thus, mature eosinophils dominated the ozone-induced inflammatory response in sensitized guinea pigs. Depleting these mature eosinophils prevented ozone-induced airway hyperreactivity in sensitized animals. Ozone induces eosinophil hematopoiesis and recruitment to lungs where three days later, newly divided eosinophils attenuate vagally-mediated hyperreactivity. Ozone-induced hematopoiesis of beneficial eosinophils is blocked by a TNFα antagonist, or by prior sensitization. In these animals, mature eosinophils are associated with hyperreactivity. Thus, interventions targeting eosinophils, while beneficial in atopic individuals, may delay resolution of airway hyperreactivity in non-atopic individuals.
In the present study, we investigated the effect of bone morphogenetic protein 4 (BMP4) on PDGF-induced proliferation and collagen synthesis in PASMCs. Normal human PASMCs were incubated with and without PDGF-BB in the absence and presence of BMP4 for 0.5 to 24 h. Then the protein levels of collagen-I, p-Smad2/3, p-Smad1/5, and intracellular active TGFβ1, calpain activity and cell proliferation were measured. The results showed that BMP4 induced an increase in p-Smad1/5 but had no effect on the protein levels of collagen-I, p-Smad2/3, and intracellular active TGFβ1, and calpain activity in PASMCs. Nevertheless, BMP4 attenuated increases in proliferation and protein levels of collagen-I, p-Smad2/3, and intracellular active TGFβ1, and calpain activity in PDGF-BB-treated PASMCs. Moreover, BMP4 increased PKA activity and inhibition of PKA prevented the inhibitory effects of BMP4 on PDGF-BB-induced calpain activation in normal PASMCs. PKA activator forskolin recapitulated the suppressive effect of BMP4 on PDGF-induced calpain activation. Further, BMP4 prevented PDGF-induced decrease in calpain-2 phosphorylation at serine 369 in normal PASMCs. Finally, BMP4 did not attenuate PDGF-induced increases in proliferation, collagen-I protein levels, and calpain activation, and did not induce PKA activation and did not prevent PDGF-induced decrease in calpain-2 phosphorylation at serine 369 in PASMCs from IPAH patients. These data demonstrate that BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in normal PASMCs. The inhibitory effects of BMP4 on PDGF-induced proliferation, collagen synthesis and calpain-2 activation are impaired in PASMCs from PAH patients, which may contribute to pulmonary vascular remodeling in PAH.
Over the past years, a critical role for the immune system and in particular, for mast cells, in the pathogenesis of pulmonary hypertension (PH) has emerged. However, the way in which mast cells promote PH is still poorly understood. Here, we investigated the mechanisms by which mast cells may contribute to PH, specifically focusing on the interaction between the innate and adaptive immune response and the role of B-cells and autoimmunity. Experiments were performed in Sprague Dawley rats and B-cell deficient JH-KO rats in the monocrotaline, sugen-hypoxia and the aortic banding model of PH. Hemodynamics, cell infiltration, IL-6 expression, and vascular remodeling were analyzed. Gene array analyses revealed constituents of immunoglobulins as most prominently regulated mast cell dependent genes in the lung in experimental PH. IL-6 was shown to link mast cells to B-cells, as a) IL-6 was upregulated and colocalized with mast cells and was reduced by mast cell stabilizers, and b) IL-6 or mast cell blockade reduced B-cells in lungs of monocrotaline-treated rats. A functional role for B-cells in PH was demonstrated, in that either blocking B-cells by an anti-CD20 antibody or B-cell deficiency in JH-KO rats attenuated right ventricular systolic pressure and vascular remodeling in experimental PH. We here identify a mast cell - B-cell axis driven by IL-6 as critical immune pathway in the pathophysiology of PH. Our results provide novel insights into the role of the immune system in PH, which may be therapeutically exploited by targeted immunotherapy.
More than 2% of all human genes are coding for a complex system of more than 700 proteases and protease inhibitors. Amongst them, serine proteases play extraordinary diverse functions in different physiological and pathological processes. The Human Airway Trypsin-like protease (HAT), also referred to as TMPRSS11D and serine 11D, belongs to the emerging family of cell surface proteolytic enzymes, the type II transmembrane serine proteases (TTSPs). Through the cleavage of its four major identified substrates, HAT triggers specific responses, notably in epithelial cells, within the pericellular and extracellular environment, including notably inflammatory cytokine production, inflammatory cell recruitment or anticoagulant processes. This review summarizes the potential role of this recently described protease in mediating cell surface proteolytic events, to highlight the structural features, proteolytic activity and regulation, including the expression profile of HAT and discuss its possible roles in respiratory physiology and disease.
Pulmonary artery smooth muscle cell (PASMC) proliferation is one of the hallmark features of hypoxia-induced pulmonary hypertension. With only supportive treatment options available for this life threatening disease, treating and preventing the proliferation of PASMCs is a viable therapeutic option. A key promoter of hypoxia-induced increases in the number of viable human PASMCs is arginase II, with attenuation of viable cell numbers following pharmacologic inhibition or siRNA knockdown of the enzyme. Additionally, increased levels of arginase have been demonstrated in the pulmonary vasculature of patients with pulmonary hypertension. The signaling pathways responsible for the hypoxic induction of arginase II in PASMCs, however, remain unknown. Hypoxia is a recognized activator of adenosine monophosphate-activated protein kinase (AMPK), which is known to be expressed in human PASMCs (hPASMCs). Activation of AMPK by hypoxia has been shown to promote cell survival in PASMCs. In addition, pharmacologic agents targeting AMPK have been shown to attenuate chronic hypoxia-induced pulmonary hypertension in animal models. The present studies tested the hypothesis that hypoxia-induced arginase II expression in hPASMCs is mediated through AMPK signaling. We found that pharmacologic inhibitors of AMPK, as well as siRNA knockdown of AMPKα1, prevented hypoxia-induced arginase II. The hypoxia-induced increase in viable hPASMC numbers was also prevented following both pharmacologic inhibition and siRNA knockdown of AMPK. Furthermore, we demonstrate that overexpression of AMPK induced arginase II protein expression and viable cells numbers in hPASMCs.
Idiopathic pulmonary fibrosis (IPF) is a progressive disease that causes unremitting deposition of extracellular matrix proteins, thus resulting in distortion of the pulmonary architecture and impaired gas exchange. Associated with high morbidity and mortality, IPF is generally refractory to current pharmacological therapies. Lefty A, a potent inhibitor of transforming growth factor (TGF)-β signaling, has been shown to have promising antifibrotic ability in vitro for the treatment of renal fibrosis and other potential organ fibroses. Here, we determined if Lefty A can attenuate bleomycin (BLM)-induced pulmonary fibrosis in vivo based on a novel therapeutic strategy where HEK293 cells are genetically engineered with the Lefty A-associated GFP gene. The engineered HEK293 cells were encapsulated in alginate microcapsules and then subcutaneously implanted in ICR mice that had one week earlier been intratracheally administered BLM to induce pulmonary fibrosis. The severity of fibrosis in lung tissue was assessed using pathological morphology and collagen expression to examine the effect of Lefty A released from the microencapsulated cells. The engineered HEK293 cells with Lefty A significantly reduced the expression of connective tissue growth factor (CTGF) and type I collagen messenger mRNA, lessened the morphological fibrotic effects induced by bleomycin, and increased the expression of matrix metalloproteinase (MMP)-9. This illustrates that engineered HEK293 cells with Lefty A can attenuate pulmonary fibrosis in vivo, thus providing a novel method to treat human pulmonary fibrotic disease and other organ fibroses.
Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF) and correlates with increased rates of lung function decline. Since glucose is a nutrient present in the airways of patients with bacterial airway infections and since insulin controls glucose metabolism, the effect of insulin on CF airway epithelia was investigated to determine the role of insulin receptors and glucose transport in regulating glucose availability in the airway. The response to insulin by human airway epithelial cells was characterized by qPCR, immunoblot, immunofluorescence, and glucose uptake assays. PI3K/Akt signaling and CFTR activity were analyzed by pharmacological and immunoblot assays. We found that normal human primary airway epithelial cells expressed the Glut4 transporter and that application of insulin stimulated cytochalasin B inhibitable glucose uptake consistent with a requirement for glucose transporter translocation. Application of insulin to normal primary human airway epithelial cells promoted airway barrier function as demonstrated by increased transepithelial electrical resistance and decreased paracellular flux of small molecules. This provides the first demonstration that airway cells express insulin-regulated glucose transporters that act in concert with tight junctions to form an airway glucose barrier. However, insulin failed to increase glucose uptake or decrease paracellular flux of small molecules in human airway epithelia expressing F508del-CFTR. Insulin stimulation of Akt1 and Akt2 signaling in CF airway cells was diminished compared to that observed in airway cells expressing wild-type CFTR. These results indicate that the airway glucose barrier is regulated by insulin and is dysfunctional in CF.
The protein concentration of alveolar edema fluid in acute respiratory distress syndrome (ARDS) is dynamic. It reflects alveolar flooding during acute injury as well as fluid and protein clearance over time. We hypothesized that among ARDS patients treated with low tidal volume ventilation, higher concentrations of protein in mini-bronchoalveolar lavage (mBAL) samples would predict slower resolution of lung injury and worse clinical outcomes. Total protein and IgM concentrations in Day 0 mBAL samples from 79 subjects enrolled in the aerosolized albuterol (ALTA) ARDS Network Albuterol trial were measured by colorimetric assay and ELISA respectively. Linear regression models were used to test the association of mBAL proteins with clinical outcomes and measures of length of illness, including ventilator free days (VFDs). Median mBAL total protein concentration was 1740 μg/mL (IQR 890-3170). Each 500 μg/mL increase in Day 0 mBAL total protein was associated with an additional 0.8 VFDs (95%CI 0.05- 1.6, p-value=0.038). Median mBAL IgM concentration was 410 ng/mL (IQR 340-500). Each 50 ng/mL increase in mBAL IgM was associated with an additional 1.1 VFDs (95%CI 0.2- 2.1, p-value=0.022). These associations remained significant and were not attenuated in multivariate models adjusted for age, serum protein concentration, and vasopressor use in the 24 hours prior to enrollment. Thus, higher mBAL total protein and IgM concentrations at Day 0 are associated with more VFDs in patients with ARDS and may identify patients with preserved alveolar epithelial mechanisms for net alveolar fluid clearance.
Rodent pups exposed to hyperoxia develop lung changes similar to bronchopulmonary dysplasia (BPD) in extremely premature infants. Oxidative stress from hyperoxia can injure developing lungs through endoplasmic reticulum (ER) stress. Early caffeine treatment decreases the rate of BPD, but the mechanisms remain unclear. We hypothesized that caffeine attenuates hyperoxia-induced lung injury through its chemical chaperone property. Sprague-Dawley rat pups were raised either in 90% (hyperoxia) or 21% (normoxia) oxygen from postnatal day 1 (P1) to postnatal day 10 (P10) and then recovered in 21% oxygen until P21. Caffeine (20 mg/kg) or normal saline (control) was administered intraperitoneally daily starting from P2. Lungs were inflation fixed for histology or snap frozen for immunoblots. Blood caffeine levels were measured in treated pups at euthanasia and were found to be 18.4±4.9 μg/ml. Hyperoxia impaired alveolar formation, increased ER stress markers and downstream effectors; caffeine treatment attenuated these changes at P10. Caffeine also attenuated the hyperoxia-induced activation of cyclooxygenase-2 and markers of apoptosis. In conclusion, hyperoxia-induced alveolar growth impairment is, in part, mediated by ER stress. Early caffeine treatment protects developing lungs from hyperoxia-induced injury by attenuating ER stress.
β2-microglobulin (β2m), the light chain of major histocompatibility complex class 1 (MHC I), has been identified as a pro-aging factors and involved in the pathogenesis of neurodegenerative disorders by driving cognitive and regenerative impairments. However, little attention has focused on the effect of β2m in development of lung emphysema. Here, we found that concentrations of β2m in plasma were significantly elevated in patients with lung emphysema than those in normal control subjects (1.89 ± 0.12 mg/l vs 1.42 ± 0.06 mg/l, P < 0.01). Moreover, the expression of β2m was significantly higher in lung tissue of emphysema (39.90 ± 1.97% vs 23.94 ± 2.11%, P < 0.01). Immunofluorescence showed that β2m mainly expressed in pro-surfactant protein C positive (pro-SPC+) alveolar epithelial cells and CD14+ macrophages. Exposure of recombinant human β2m and cigarette smoke extract (CSE) in vitro enhanced cellular senescence and inhibited proliferation of A549 cells, which was partially reversed by the presence of anti-β2m antibody. However, anti-β2m antibody didn't attenuate the elevated production of IL-1β, IL-6 and TNF-α in A549 cells exposed to CSE. Immunofluorescence showed that co-localization of β2m and hemochromatosis gene (HFE) protein was observed on A549 cells. These data suggests β2m might participate in development of lung emphysema through inducing lung epithelial cells senescence and proliferation inhibition.
Rationale: Inflammation is a prominent pathologic feature in pulmonary arterial hypertension as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4+ T cells are required for initiating and maintaining inflammation, suggesting these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Objectives: To test the hypothesis that CD4+ T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. Methods: We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 weeks) in wild-type mice and recombination-activating gene 1 knock-out mice (RAG1-/-, lack mature T and B cells). Separate sets of mice were adoptively transferred with CD4+, CD8+, or T helper 17 cells prior to normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. Results: RAG1-/- mice had diminished right ventricular systolic pressure and arterial remodeling compared to wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4+, but not CD8+ T cells, restored the hypertensive phenotype in RAG1-/- mice. Interestingly, RAG1-/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4+ cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. Conclusions: T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension.
Human genome-wide association studies (GWASs) have identified over 50 loci associated with pulmonary function and related phenotypes, yet follow-up studies to determine causal genes or variants are rare. Single nucleotide polymorphisms (SNPs) in serotonin receptor 4 (HTR4) are associated with human pulmonary function in genome-wide association studies and follow-up animal work has demonstrated that Htr4 is causally associated with pulmonary function in mice, although the precise mechanisms were not identified. We sought to elucidate the role of neural innervation and pulmonary architecture in the lung phenotype of Htr4-/- animals. We report here that the Htr4-/- phenotype in mouse is dependent on vagal innervation to the lung. Both, ex vivo tracheal ring reactivity and in vivo flexiVent™ pulmonary functional analyses demonstrate that vagotomy abrogates the Htr4-/- airway hyperresponsiveness phenotype. Hyperpolarized 3He gas magnetic resonance imaging and stereological assessment of wild-type and Htr4-/- mice reveal no observable differences in lung volume, inflation characteristics, or pulmonary-microarchitecture. Finally, control of breathing experiments reveal substantive differences in baseline breathing characteristics between mice with/without functional HTR4 in breathing frequency, relaxation time, flow rate, minute volume, time of inspiration and expiration and breathing pauses. These results suggest that HTR4's role in pulmonary function likely relates to neural innervation and control of breathing.
We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVEC) depends on epidermal growth factor receptor (EGFR) activation. To determine down-stream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVEC would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVEC were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK and arginase II protein levels than did normoxia in hPMVEC. To examine the role of EGFR in these hypoxia-induced changes, hPMVEC were transfected with siRNA against EGFR or a scramble siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVEC were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVEC numbers. hPMVEC were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVEC. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic-induction of arginase II and cellular proliferation.
Pericytes are perivascular PDGFRβ+ stromal cells required for vasculogenesis and maintenance of microvascular homeostasis in many organs. Because of their unique juxtaposition to microvascular endothelium, lung PDGFRβ+ cells are well situated to detect pro-inflammatory molecules released following epithelial injury and promote acute inflammatory responses. Thus, we hypothesized that these cells represent an unrecognized immune surveillance or injury-sentinel interstitial cell. To evaluate this hypothesis, we isolated PDGFRβ+ cells from murine lung and demonstrated that they have characteristics consistent with a pericyte population (referred to as pericyte-like cells for simplicity hereafter). We showed that pericyte-like cells expressed functional Toll-like receptors and upregulated chemokine expression following exposure to bronchoalveolar lavage fluid (BALF) collected from mice with sterile lung injury. Interestingly, BALF from mice without lung injury also induced chemokine expression in pericyte-like cells, suggesting that pericyte-like cells are primed to sense epithelial injury (permeability changes). Following LPS-induced lung inflammation, increased numbers of pericyte-like cells expressed IL-6, CXCL1, CCL2/MCP-1, and ICAM-1 in vivo. Sterile lung injury in pericyte-ablated mice was associated with decreased inflammation compared to normal mice. In summary, we found that pericyte-like cells are immune responsive and express diverse chemokines in response to lung injury in vitro and in vivo. Furthermore, pericyte-like cell ablation attenuated inflammation in sterile lung injury, suggesting that these cells play an important functional role in mediating lung inflammatory responses. We propose a model in which pericyte-like cells function as interstitial immune sentinels, detecting pro-inflammatory molecules released following epithelial barrier damage and participating in recruitment of circulating leukocytes.
Endothelial cell (EC) activation and vascular injury are hallmark features of Acute Lung Injury and Acute Respiratory Distress Syndrome (ALI/ARDS). Caveolin-1 (Cav-1) is highly expressed in pulmonary microvascular ECs and plays a key role in maintaining vascular homeostasis. The aim of this study was to determine whether the lung inflammatory response to Escherichia coli lipopolysaccharide (LPS) promotes priming of ECs via Cav-1 depletion and whether this contributes to the onset of pulmonary vascular remodeling. To test the hypothesis that depletion of Cav-1 primes ECs to respond to profibrotic signals, C57BL6 WT mice (Tie2.Cre-;Cav1lox/lox) were exposed to nebulized LPS (10 mg; 1 hr daily for 4 days) and compared to EC-specific Cav1-/- (Tie2.Cre+;Cav1lox/lox) After 96 hrs of LPS exposure, total lung Cav-1 and BMPRII expression were reduced in WT mice. Moreover, plasma albumin leakage, infiltration of immune cells, and levels of IL-6/IL-6R and TGF-β were elevated in both LPS-treated WT and EC-Cav1-/- mice. Finally, EC-Cav1-/- mice exhibited a modest increase in microvascular thickness basally and moreso upon exposure to LPS (96 hr). EC-Cav1-/- mice and LPS treated WT mice exhibited reduced BMPRII expression and eNOS uncoupling, which along with increased TGF-β promoted TGFβRI-dependent SMAD-2/3 phosphorylation. Finally, human lung sections from patients with ARDS displayed reduced EC Cav-1 expression, elevated TGF-β levels, and severe pulmonary vascular remodeling. Thus, EC Cav-1 depletion, oxidative stress-mediated reduction in BMPRII expression, and enhanced TGFβ-driven SMAD-2/3 signaling promote pulmonary vascular remodeling in inflamed lungs.
It is now clear that in addition to activating several complex kinase pathways (Smad, MAP kinase, PI3 kinase), TGFβ also acts by elevating [Ca2+] within the cytosol of human pulmonary fibroblasts. Ca2+/calmodulin-dependent protein kinase II (CamK II) is also known to regulate gene expression in fibroblasts. In this study, we examined the interactions between calcium signaling, activation of CamK and other kinases, and extracellular matrix (ECM) gene expression. Human pulmonary fibroblasts were cultured and stimulated with artificially-generated Ca2+-pulses in the absence of TGFβ, or with TGFβ (1 nM) or vehicle in the presence of various blockers of Ca2+ signaling. PCR and Western blotting were used to measure gene expression and protein levels, respectively. We found that Ca2+-pulses in the absence of TGFβ increased ECM gene expression in a pulse frequency-dependent manner, and that blocking Ca2+ signaling and the CamK II pathway significantly reduced TGFβ-mediated ECM gene expression, without having any effects on other kinase pathways (Smad, PI3 kinase or MAP kinase). We also found that TGFβ elevated the expression of CamK IIβ and CamK II, while siRNA silencing of those two subtypes significantly reduced TGFβ-mediated expression of collagen A1 and Fibronectin 1. Our data suggest that TGFβ induces the expression of CamK IIβ and CamK II, which in turn are activated by TGFβ-evoked Ca2+ waves in a frequency-dependent manner, leading to increased expression of ECM proteins.
To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1) in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposing to hypoxia, a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE in PASMCs were intensified when TRPC1, STIM1 and Orai1 were upregulated. SOCE in cells transfected with specific small hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1 or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1 and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1 and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.
Pulmonary hypertension (PH) is characterized by increased pulmonary vascular resistance, pulmonary vascular remodeling, and increased pulmonary vascular pressures that often result in right ventricular dysfunction, leading to right heart failure. Evidence suggests that reactive oxygen species (ROS) contribute to PH pathogenesis by altering pulmonary vascular cell proliferation and intracellular signaling pathways. However, the role of mitochondrial antioxidants and oxidant-derived stress signaling in the development of hypoxia-induced PH is largely unknown. Therefore, we examined the role of the major mitochondrial redox regulator, thioredoxin 2 (Trx2). Levels of Trx2 mRNA and protein were examined in human pulmonary arterial endothelial cells (HPAECs) and smooth muscle cells (HPASMCs) exposed to hypoxia, a common stimulus for PH, for 72 hours. Hypoxia decreased Trx2 mRNA and protein levels. In vitro overexpression of Trx2 reduced hypoxia-induced H2O2 production. The effects of increased Trx2 protein level were examined in transgenic mice expressing human Trx2 (TghTrx2) that were exposed to hypoxia (10% O2) for 3 weeks. TghTrx2 mice exposed to hypoxia had exacerbated increases in right ventricular systolic pressures, right ventricular hypertrophy, and increased ROS in the lung tissue. Trx2 overexpression did not attenuate hypoxia-induced increases in Trx2 oxidation or Nox4 expression. Expression of a dominant negative C93S Trx2 mutant that mimics Trx2 oxidation exacerbated hypoxia-induced increases in HPASMC H2O2 levels and cell proliferation. In conclusion, Trx2 overexpression failed to attenuate hypoxia-induced HPASMC proliferation in vitro or hypoxia-induced PH in vivo. These findings indicate that strategies to enhance Trx2 expression are unlikely to exert therapeutic effects in PH pathogenesis.
Background: Prenatal smoke exposure is a risk factor for abnormal lung development and increased sex-dependent susceptibility for asthma and COPD. Birth cohort studies show genome wide DNA methylation changes in children from smoking mothers, but evidence for sex-dependent smoke-induced effects is limited. The insulin-like growth factor (IGF) system plays an important role in lung development. We hypothesized that prenatal exposure to smoke induces lasting changes in promoter methylation patterns of Igf1 and Igf1r, thus influencing transcriptional activity, and contributing to abnormal lung development. Method: We measured and compared mRNA levels along with promoter methylation of Igf1 and Igf1r and their protein concentrations in lung tissue of 30-day-old mice which had been prenatally exposed to cigarette smoke (PSE) or filtered air (control). Body weight at 30 days after birth was measured as global indicator of normal development. Results: Female PSE mice showed lower mRNA levels of Igf1 and its receptor (Igf1: p = 0.05; Igf1r: p = 0.03). Furthermore, CpG site specific methylation changes were detected in Igf1r in a sex-dependent manner and the body weight of female offspring was reduced after prenatal exposure to smoke, while protein concentrations were unaffected. Conclusion: Prenatal exposure to smoke induces a CpG-site specific loss of Igf1r promoter methylation, which can be associated with body weight. These findings highlight the sex-dependent and potentially detrimental effects of in utero smoke exposure on DNA methylation and Igf1 and Igf1r mRNA levels. The observations support a role for Igf1 and Igf1r in abnormal development.
Leonardo da Vinci (1452-1519) enjoys a reputation as one of the most talented people of all time in the history of science and the arts. However little attention has been given to his contributions to physiology. One of his main interests was engineering, and he was fascinated by structural problems, and the flow patterns of liquids. He also produced a large number of ingenious designs for warfare, and a variety of highly original flying machines. But of particular interest to us are his contributions to bioengineering, and how he used his knowledge of basic physical principles to throw light on physiological function. For example he produced new insights into the mechanics of breathing including the action of the ribs and diaphragm. He was the first person to understand the different roles of the internal and external intercostal muscles. He had novel ideas about the airways including the mode of airflow in them. He also worked on the cardiovascular system and had a special interest in the pulmonary circulation. But interestingly he was not able to completely divorce his views from those of Galen, in that although he could not see pores in the interventricular septum of the heart, one of his drawings included them. Leonardo was a talented anatomist who made many striking drawings of the human body. Finally his reputation for many people is based on his paintings including the Mona Lisa that apparently attracts more viewers than any other painting in the world.
The prevailing morbidity and mortality in sepsis are largely due to multiple organ dysfunction (MOD), most commonly lung injury, as well as renal and cardiac dysfunction. Despite recent advances in defining many aspects of the pathogenesis of sepsis-related MOD, including acute respiratory distress syndrome (ARDS), there are currently no effective pharmacological or cell-based treatments for the disease. Human and animal studies have shown that pulmonary thrombosis is common in sepsis-induced ARDS, and pre-clinical studies have shown that anti-coagulation may improve outcome following sepsis challenge. The potential beneficial effect of anti-coagulation on outcome is unconvincing in clinical studies, however, and these discrepancies may arise from the multiple and sometimes opposing actions of thrombosis on the pulmonary endothelium following sepsis. It has been suggested, for example, that mild pulmonary thrombosis prevents escape of bacterial infection into the circulation, while severe thrombosis causes hypoxia and results in pulmonary endothelial damage. Evidence from both human and animal studies has demonstrated the key role of microvascular leakage in determining the outcome of sepsis. In this review, we describe thrombosis-dependent mechanisms that regulate pulmonary endothelial injury and repair following sepsis, including activation of the coagulation cascade by tissue factor, and stimulation of vascular repair by hypoxia-inducible factors. Targeting such mechanisms through anti-coagulant, anti-inflammatory, and reparative methods may represent a novel approach for the treatment of septic patients.
Severe primary graft dysfunction affects 15-20% of lung transplantation recipients and carries a high mortality risk. In addition to known donor, recipient, and perioperative clinical risk factors, numerous biologic factors are thought to contribute to primary graft dysfunction. Our current understanding of the pathogenesis of lung injury and primary graft dysfunction emphasizes multiple pathways leading to lung endothelial and epithelial injury. Biomarkers specific to these pathways can be measured in the plasma, bronchoalveolar lavage fluid, and lung tissue. Clarification of the pathophysiology and timing of primary graft dysfunction could illuminate predictors of dysfunction, allowing for better risk stratification, earlier identification of susceptible recipients, and development of targeted therapies. Here, we review much of what has been learned in biomarker studies about the pathogenesis of primary graft dysfunction, and evaluate the potential prognostic value of biomarkers for primary graft dysfunction at different measurement time-points.
Pulmonary fibrosis contributes to morbidity and mortality in a range of diseases, and there are no approved therapies for reversing its progression. To understand the mechanisms underlying pulmonary fibrosis and assess potential therapies, mouse models are central to basic and translational research. Unfortunately, metrics commonly used to assess murine pulmonary fibrosis require animals to be grouped and sacrificed, increasing experimental difficulty and cost. We examined the ability of three magnetic resonance imaging (MRI)-derived metrics (mean weighted lung signal, percent high signal volume, and signal coefficient of variation) to non-invasively assess lung fibrosis progression and resolution in a doxycycline (Dox) regulatable, transgenic mouse model that overexpresses transforming growth factor alpha (TGF-α) under control of a lung-epithelial-specific promoter. During seven weeks of Dox-treatment, fibrotic lesions were readily observed as high-signal tissue. Mean weighted signal and percent signal volume were found to be the most robust MRI-derived measures of fibrosis, and these metrics correlated significantly with pleural thickness, histology scores, and hydroxyproline content (R=0.75-0.89). When applied longitudinally, percent high signal volume increased by 1.5% week-1 (p<0.001), and mean weighted signal increased at a rate of 0.0065 week-1 (p=0.0062). Following Dox-treatment, lesions partially resolved, with percent high signal volume decreasing by -3.2% week-1 (p=0.0034) and weighted mean signal decreasing at -0.015 week-1 (p=0.0028). Additionally, longitudinal MRI revealed dynamic remodeling in a subset of lesions-a previously unobserved behavior in this model. These results demonstrate MRI can non-invasively assess experimental lung fibrosis progression and resolution and provide unique insights into its pathobiology.
Cigarette smoke usage is prevalent in HIV+ patients and, despite highly active antiretroviral therapy (HAART), these individuals develop an accelerated form of chronic obstructive pulmonary disease (COPD). Studies investigating the mechanisms of COPD development in HIV have been limited by the lack of suitable mouse models. Here we describe a model of HIV induced COPD in wild type mice using EcoHIV, a chimeric HIV capable of establishing chronic infection in immunocompetent mice. A/J mice were infected with EcoHIV and subjected to whole body cigarette smoke exposure. EcoHIV was detected in alveolar macrophages of mice. Compared to uninfected mice, concomitant EcoHIV infection significantly reduced forced expiratory flow (FEF) 50%/forced vital capacity (FVC) and enhanced distal airspace enlargement following cigarette smoke exposure. Lung IL-6, G-CSF, neutrophil elastase, cathepsin G and MMP9 expression was significantly enhanced in smoke-exposed EcoHIV-infected mice. These changes coincided with enhanced IBα, ERK1/2, p-38 and STAT3 phosphorylation and lung cell apoptosis. Thus, the EcoHIV smoke exposure mouse model reproduces several of the pathophysiologic features of HIV-related COPD in humans, indicating that this murine model can be utilized to determine key parameters of HIV-related COPD and to test future therapies for this disorder.
Alveolar fluid clearance mediates perinatal lung transition to air breathing in newborn infants, which is accomplished by epithelial Na+ channels (ENaC) and the Na,K-ATPase. Male sex represents a major risk factor for developing respiratory distress, especially in preterm infants. We previously showed that male sex is associated with reduced epithelial Na+ transport, possibly contributing to the sexual dimorphism in newborn respiratory distress. This study aimed to determine sex-specific effects of sex steroids on epithelial Na+ transport. The effects of testosterone, 5α-dihydrotestosterone (DHT), estradiol and progesterone on Na+ transport and Na+ channel expression were determined in fetal distal lung epithelial (FDLE) cells of male and female rat fetuses by Ussing chamber and mRNA expression analyses. DHT showed a minor effect only in male FDLE cells by decreasing epithelial Na+ transport. However, flutamide, an androgen receptor antagonist, did not abolish the gender imbalance, and testosterone lacked any effect on Na+ transport in male and female FDLE cells. In contrast, estradiol and progesterone increased Na+ transport and Na+ channel expression especially in females and prevented the inhibiting effect of DHT in males. Estrogen receptor inhibition decreased Na+ channel expression and eliminated the sex differences. In conclusion, female sex steroids stimulate Na+ transport especially in females and prevent the inhibitory effect of DHT in males. The ineffectiveness of testosterone suggests that Na+ transport is largely unaffected by androgens. Thus, the higher responsiveness of female cells to female sex steroids explains the higher Na+ transport activity, possibly leading to a functional advantage in females.
Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population(higher incidence and mortality). The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 months) and aged (18 months) mice using a two-hit pre-clinical model. Compared to young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx, levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in beta-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared to control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was post-translationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4and alterations in cellular redox homeostasis. Pharmacologic inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.
Introduction. Although airway mucus dehydration is key to pathophysiology of cystic fibrosis (CF) and other airways diseases, measuring mucus hydration is challenging. We explored a robust method to estimate mucus hydration using sialic acid as a marker for mucin content. Methods. Terminal sialic acid residues from mucins were cleaved by acid hydrolysis from airway samples, and concentrations of sialic acid, urea, and other biomarkers analyzed using mass spectrometry. Results. In mucins purified from human airway epithelial (HAE), sialic acid concentrations after acid hydrolysis correlated with mucin concentrations (r2=0.92). Sialic acid/urea ratios measured from filters applied to the apical surface of cultured HAE correlated to % solids and were elevated in samples from CF HAEs relative to controls (2.2±1.1 vs. 0.93±1.8, p<0.01). Sialic acid/urea ratios were elevated in bronchoalveolar lavage fluid (BALF) from βENaC transgenic mice, known to have reduced mucus hydration, and mice sensitized to house dust mite allergen. In a translational application, elevated sialic acid/urea ratios were measured in BALF from young children with CF who had airway infection relative to those who did not (5.5±3.7 vs.1.9±1.4, p<0.02), and could be assessed simultaneously with established biomarkers of inflammation. Conclusions. The sialic acid/urea ratio performed similarly to % solids, the gold standard measure of mucus hydration. The method proved robust and has potential to serve as flexible techniques to assess mucin hydration, particularly in samples like BALF in which established methods such as % solids cannot be utilized. -
Airway remodeling, a key feature of asthma, alters every layer of the airway wall but most strikingly the airway smooth muscle (ASM) layer. Airway remodeling in asthmatics contributes to fixed airflow obstruction and can amplify airway narrowing caused by ASM activation. Previous modeling studies have shown that the increase in ASM mass has the largest effect on increasing maximal airway narrowing. Simulated heterogeneity in the dimensions and properties of the airway wall can further amplify airway narrowing. Using measurements made on histological sections from donor lungs we show for the first time that there is profound heterogeneity of airway dimensions in both non-asthmatics and asthmatics. Using a mathematical model, we found that this heterogeneity contributes to an increased baseline resistance and elastance in asthmatics as well as a leftward shift in the responsiveness of the airways to a simulated agonist in both non-asthmatics and asthmatics. The ability of heterogeneous wall dimensions to shift the dose response curve is due to an increased susceptibility for the small airways to close. This research confirms that heterogeneity of airway wall dimensions can contribute to exaggerated airway narrowing and provides an actual assessment of the magnitude of these effects.
Bitter taste receptors (T2Rs), a G-protein-coupled receptor family capable of detecting numerous bitter-tasting compounds, have recently been shown to be expressed and play diverse roles in many extraoral tissues. Here we report the functional expression of T2Rs in rat pulmonary sensory neurons. In anesthetized spontaneously breathing rats, intratracheal instillation of T2Rs agonist chloroquine (10 mM, 0.1 ml) significantly augmented chemoreflexes evoked by right-atrial injection of capsaicin, a specific activator for transient receptor potential vanilloid receptor 1 (TRPV1); whereas intravenous infusion of chloroquine failed to significantly affect capsaicin-evoked reflexes. In patch-clamp recordings with isolated rat vagal pulmonary sensory neurons, pretreatment with chloroquine (1-1000 µM, 90 s) concentration-dependently potentiated capsaicin-induced TRPV1-mediated inward currents. Preincubating with diphenitol and denatonium (1 mM, 90 s), two other T2Rs activators, also enhanced capsaicin currents in these neurons but to a lesser extent. The sensitizing effect of chloroquine was effectively prevented by phospholipase C inhibitor U73122 (1 µM), or by protein kinase C inhibitor chelerythrine (10 µM). In summary, our study showed that activation of T2Rs augments capsaicin-evoked TRPV1 responses in rat pulmonary nociceptors, through phospholipase C and protein kinase C signaling pathway.
Various pathophysiological conditions such as surfactant dysfunction, mechanical ventilation, inflammation, pathogen products, environmental exposures and gastric acid aspiration stress lung cells and the compromise of plasma membranes occur as a result. The mechanisms necessary for cells to repair plasma membrane defects have been extensively investigated in the last two decades, and some of these key repair mechanisms are also shown to occur following lung cell injury. As it was theorized that lung wounding and repair are involved in the pathogenesis of acute respiratory distress syndrome (ARDS) and idiopathic pulmonary fibrosis (IPF), in this review, we summarized the experimental evidence of lung cell injury in these two devastating syndromes, discuss relevant genetic, physical and biological injury mechanisms, as well as mechanisms utilized by lung cells for cell survival and membrane repair. Finally, we discuss relevant signalling pathways that may be activated by chronic or repeated lung cell injury as an extension of our cell injury and repair focus in this review. We hope that a holistic view of injurious stimuli relevant for ARDS and IPF could lead to updated experimental models. In addition, parallel discussion of membrane repair mechanisms in lung cells and injury-activated signalling pathways would encourage research to bridge gaps in current knowledge. Indeed, deep understanding of lung cell wounding and repair, and discovery of relevant repair moieties for lung cells should inspire the development of new therapies that are likely preventive and broadly effective for targeting injurious pulmonary diseases.
Glucocorticoids, or corticosteroids, are effective treatments for many chronic inflammatory diseases and, in mild/moderate asthma, long-acting β2-adrenoceptor agonists (LABAs) enhance the efficacy of inhaled corticosteroids (ICSs) more than increasing the ICS dose. In human bronchial epithelial, BEAS-2B, cells, expression of TNFα-induced protein 3 (TNFAIP3), or A20, a dual-ubiquitin ligase that provides feedback inhibition of NF-B, was induced by budesonide, an ICS, formoterol, a LABA, and was further enhanced by budesonide/formoterol combination. The pro-inflammatory cytokine, TNF, induced TNFAIP3 and TNF expression. Whereas subsequent budesonide treatment enhanced TNF-induced TNFAIP3 and reduced TNF expression, formoterol amplified these differential effects. In primary human airway smooth muscle cells, TNFAIP3 expression was induced by TNF. This was largely unaffected by budesonide, but was acutely enhanced by budesonide/formoterol combination. In BEAS-2B cells, TNF recruited RELA, the main NF-B transactivating subunit, to a 3' region of the TNF gene. RELA binding was reduced by budesonide, further reduced by formoterol co-treatment, and was associated with reduced RNA polymerase II recruitment to the TNF gene. This is consistent with reduced TNF expression. TNFAIP3 knockdown enhanced TNF expression in the presence of TNF, TNF plus budesonide and TNF plus budesonide/formoterol combination and confirms feedback inhibition. A luciferase reporter containing the TNF 3' RELA binding region recapitulated TNF inducibility and was inhibited by an IB kinase inhibitor and TNFAIP3 over-expression. Repression of reporter activity by budesonide was increased by formoterol and involved TNFAIP3. Thus LABAs may improve the anti-inflammatory properties of ICSs by augmenting TNFAIP3 expression to negatively regulate NF-B.
Airway mucus hypersecretion contributes to the morbidity and mortality in patients with chronic inflammatory lung diseases. Reducing mucus production is crucial for improving patients' quality of life. The transcription factor SAM-pointed domain-containing Ets-like factor (SPDEF) plays a critical role in the regulation of mucus production, and therefore represents a potential therapeutic target. This study aims to reduce lung epithelial mucus production by targeted silencing SPDEF using the novel strategy epigenetic editing. Zinc fingers and CRISPR/dCas platforms were engineered to target repressors (KRAB, DNA methyltransferases, histone methyltransferases) to the SPDEF promoter. All constructs were able to effectively suppress both SPDEF mRNA and protein expression, which was accompanied by inhibition of downstream mucus-related genes (Anterior gradient 2 (AGR2), Mucin 5AC (MUC5AC)). For the histone methyltransferase G9A, and not its mutant nor other effectors, the obtained silencing was mitotically stable. These results indicate efficient SPDEF silencing and down regulation of mucus related gene expression by epigenetic editing, in human lung epithelial cells. This opens avenues for epigenetic editing as a novel therapeutic strategy to induce long-lasting mucus inhibition.
Emphysema is a typical component of chronic obstructive pulmonary disease (COPD), a progressive and inflammatory airway disease. However, no effective treatment currently exists. Here we show that keratan sulfate (KS), one of the major glycosaminoglycans (GAGs) produced in the small airway, decreased in lungs of cigarette-smoke-exposed mice. In order to confirm the protective effect of KS in the small airway, a disaccharide repeating unit of KS designated L4 ([SO3--6]Galβ1-4[SO3--6]GlcNAc) was administered to two murine models: elastase-induced-emphysema and LPS-induced exacerbation of a cigarette smoke-induced emphysema. Histological and micro-computed tomography (micro-CT) analyses revealed that, in the elastase-induced emphysema model mice, administration of L4 attenuated alveolar destruction. Treatment with L4 significantly reduced neutrophil influx and the levels of inflammatory cytokines, tissue-degrading enzymes (MMPs), and myeloperoxidase (MPO) in bronchoalveolar lavage (BAL) fluid, suggesting that L4 suppressed inflammation in the lung. L4 consistently blocked the chemotactic migration of neutrophils in vitro. Moreover, in the case of the exacerbation model, L4 inhibited inflammatory cell accumulation to the same extent as that of dexamethasone. Taken together, L4 represents one of the potential glycan-based drugs for the treatment of COPD through its inhibitory action against inflammation.
Idiopathic pulmonary fibrosis (IPF) is a well-known age-related disease. However, much less recognized has been the aging associated pathogenesis of this disorder. As we and others previously showed that dysregulation of microRNAs (miRNAs) was an important mechanism involved in pulmonary fibrosis, the role of these molecules in this pathology in the aged population has not been investigated. In this study, by using lung fibrosis model established in old mice, we found that ablation of miR-34a protected aged animals from developing experimental lung fibrosis. miR-34a was upregulated in lung epithelial cells, but not in lung fibroblasts of aged mice, and miR-34a expression was further increased in epithelial cells of the fibrotic lungs of these old animals. We found that miR-34a induced dysfunctions in alveolar epithelial cells (AECs), as evidenced by increased cellular senescence and apoptosis and mitochondrial aberrations. More importantly, these abnormalities were attenuated in AECs of the fibrotic lungs of aged miR-34a-/- mice. We found that miR-34a targeted Sirt1, a master anti-aging regulator, and two key cell cycle modulators E2F3 and cyclin E2 in lung epithelial cells and the repression of these targets was relieved in miR-34a deficient AECs. In summary, our data suggest that elevated AEC miR-34a plays a critical role in the pathogenesis of pulmonary fibrosis in the aged population. Our study also indicates miR-34a be a more precise miRNA target for treating this disease that overwhelmingly affects the people of advanced age.
Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23ºC to 43ºC, or decreasing extracellular pH value from 7.4 to 5.9, enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43ºC)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing extracellular osmotic pressure from 310 to 200 mOsmol/L also increased [Ca2+]cyt and the hypoosmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in normal PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with shRNA) attenuated capsaicin-, acidity- and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt; the capsaicin-induced CREB phosphorylation was significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMCs proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism contributing to the augmented Ca2+ influx and excessive PASMCs proliferation in IPAH patients.
Elevation of hemoglobin concentration, a common adaptive response to high-altitude hypoxia, occurs among Oromo, but is dampened among Amhara highlanders of East Africa. We hypothesized that Amhara highlanders offset their smaller hemoglobin response with a vascular response. We tested this by comparing Amhara and Oromo highlanders at 3700 m and 4000 m to their lowland counterparts at 1200 m and 1700 m. To evaluate vascular responses, we assessed urinary levels of nitrate (NO3–) as a readout of production of the vasodilator nitric oxide and its downstream signal transducer cyclic guanosine monophosphate (cGMP), along with diastolic blood pressure as an indicator of vasomotor tone. To evaluate hematological responses, we measured hemoglobin and percent oxygen saturation of hemoglobin. Amhara highlanders, but not Oromo, had higher NO3– and cGMP compared to their lowland counterparts. NO3– directly correlated with cGMP (Amhara R2=0.25, P<0.0001; Oromo R2=0.30, P<0.0001). Consistent with higher levels of NO3– and cGMP, diastolic blood pressure was lower in Amhara highlanders. Both highland samples had apparent left shift in oxyhemoglobin saturation characteristics, and maintained total oxyhemoglobin content similar to their lowland counterparts. However, deoxyhemoglobin levels were significantly higher, much more so among Oromo than Amhara. In conclusion, the Amhara balance minimally elevated hemoglobin with vasodilatory response to environmental hypoxia, whereas Oromo rely mainly on elevated hemoglobin response. These results point to different combinations of adaptive responses in genetically similar East African highlanders.
Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT but the resultant cell lines did not undergo mucociliary differentiation. We hypothesised that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. CF and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 then their morphology, replication kinetics and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1 transduced basal cells showed normal cell morphology, karyotype and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1 transduced cells were similar to un-transduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.
The signaling crosstalk between the tracheal mesenchyme and epithelium has not been researched extensively leaving a substantial gap of knowledge in the mechanisms dictating embryonic development of the proximal airways by the adjacent mesenchyme. Recently, we have reported that embryos lacking mesenchymal expression of Sox9 did not develop tracheal cartilage rings and showed altered differentiation of the tracheal epithelium. Herein, we propose that tracheal cartilage provides local inductive signals responsible for the proper differentiation, metabolism, and inflammatory status regulation of the tracheal epithelium. The tracheal epithelium of mesenchyme-specific Sox9 mutants showed altered mRNA expression of various epithelial markers such as Pb1fa1, Sftpb, Scgb1a1, and Tff-1. In vitro tracheal epithelial cell cultures confirmed that tracheal chondrocytes secrete factors that inhibit club cell differentiation. Whole gene expression profiling and ingenuity pathway analysis showed that the TNF-a, IFN-g and TGF-b signaling pathways were significantly altered in the Sox9 mutant trachea. Tnf-a and Ifn-g interfered with the differentiation of tracheal epithelial progenitor cells into mature epithelial cell types in vitro. Meanwhile, mesenchymal knockout of Tgf-b1 in vivo resulted in altered differentiation of the tracheal epithelium. Finally, mitochondrial enzymes involved in fat and glycogen metabolism Cox8b and Cox7a1 were strongly up-regulated in the Sox9 mutant trachea, with consequently increased number and size of glycogen storage vacuoles. Our results support an inductive role for tracheal cartilage in the regulation of differentiation and metabolism, and the inflammatory status of the tracheal epithelium by altering the signaling of the TNF-a, IFN-g and TGF-b signaling pathways.
The epithelial sodium channel (ENaC) and the CFTR chloride channel critically regulate airway surface liquid by driving fluid absorption and secretion, respectively. Their functional interplay is complex and incompletely understood. ENaC is a heteromeric channel with three well characterised subunits (α, β, ). In humans, an additional ENaC subunit exists in lung and several other tissues where it may replace the α-subunit to form βENaC. Little is known about the physiological role of βENaC and its possible interaction with CFTR. The aim of the present study was to investigate the effect of human CFTR on human βENaC heterologously expressed in Xenopus laevis oocytes. In oocytes co-expressing βENaC and CFTR the ENaC mediated amiloride-sensitive whole-cell current (Iami) was reduced by ~50% compared to that measured in oocytes expressing βENaC alone. Moreover, basal level of proteolytic ENaC activation was reduced in the presence of CFTR. The inhibitory effect of CFTR on βENaC was due to a combination of decreased average open probability (Po) and reduced channel expression at the cell surface. Interestingly, in oocytes expressing βENaC increasing intracellular [cAMP] by IBMX and forskolin increased Iami by ~50%. This stimulatory effect was not observed for human and rat αβENaC and was independent of CFTR co-expression and co-activation. Experiments with a mutant channel (βS520CENaC) which can be converted to a channel with a Po of nearly one suggested that cAMP activates βENaC by increasing Po. In conclusion our results demonstrate that βENaC is inhibited by CFTR but activated by cAMP.
Force adaptation, a process whereby sustained spasmogenic activation (viz., tone) of airway smooth muscle (ASM) increases its contractile capacity, has been reported in isolated ASM tissues in vitro, as well as in mice in vivo. The objective of the present study was to assess the effect of tone on airway responsiveness in humans. Ten healthy volunteers underwent methacholine challenge on two occasions. One challenge consisted of six serial doses of saline followed by a single high dose of methacholine. The other consisted of six low doses of methacholine five minutes apart followed by a higher dose. The cumulative dose was identical for both challenges. After both methacholine challenges, subjects took a deep inspiration (DI) to total lung capacity as another way to probe ASM mechanics. Responses to methacholine and the DI were measured using multi-frequency forced oscillation technique. Compared to a single high dose, the challenge preceded by tone led to an elevated response measured by respiratory system resistance (Rrs) and reactance (Xrs) at 5 Hz. However, there was no difference in the increase in Rrs at 19 Hz, suggesting a predominant effect on smaller airways. Increased tone also reduced the efficacy of DI, measured by an attenuated maximal dilation during the DI and an increased renarrowing post-DI. We conclude that ASM tone increases small airway responsiveness to inhaled methacholine and reduces the effectiveness of DI in healthy humans. This suggests that force adaptation may contribute to airway hyperesponsiveness and the reduced bronchodilatory effect of DI in asthma.
Small airway fibrosis is a major pathologic feature of chronic obstructive pulmonary disease (COPD) and is refractory to current treatments. Chronic inflammatory cells accumulate around small airways in COPD and are thought to play a major role in small airway fibrosis. Mice deficient in α/β T-cells have recently been shown to be protected from both experimental airway inflammation and fibrosis. In these models, CD4+Th17 cells and secretion of IL-17A are increased. However, a pathogenic role for IL-17 in specifically mediating fibrosis around airways has not been demonstrated. Here a role for IL-17A in airway fibrosis was demonstrated using mice deficient in the IL-17 receptor A (il17ra). Il17ra deficient mice were protected from both airway inflammation and fibrosis in 2 different models of airway fibrosis that employ COPD-relevant stimuli. In these models, CD4+ Th17 are a major source of IL-17A with other expressing cell types including T-cells, type 3 innate lymphoid cells, PMNs, and CD8+ T-cells. Antibody neutralization of IL-17RA or IL-17A confirmed that IL-17A was the relevant pathogenic IL-17 isoform and IL-17RA was the relevant receptor in airway inflammation and fibrosis. These results demonstrate that the IL-17A/IL-17 RA axis is crucial to murine airway fibrosis. These findings suggest that IL-17 might be targeted to prevent the progression of airway fibrosis in COPD.
Asthma is associated with activation of coagulation in the airways. The coagulation system can be initiated via the extrinsic tissue factor dependent pathway or via the intrinsic pathway, in which the central player factor XI (FXI) can be either activated via active factor XII (FXIIa) or via thrombin. We aimed to determine the role of the intrinsic coagulation system, and its possible route of activation, in allergic lung inflammation induced by the clinically relevant human allergen house dust mite (HDM). Wild-type (WT), FXI knockout (KO) and FXII KO mice were subjected to repeated exposure to HDM via the airways, and inflammatory responses were compared. FXI KO mice showed increased influx of eosinophils into lung tissue, accompanied by elevated local levels of the main eosinophil chemoattractant eotaxin. While gross lung pathology and airway mucus production did not differ between groups, FXI KO mice displayed an impaired endothelial/epithelial barrier function, as reflected by elevated levels of total protein and IgM in bronchoalveolar lavage fluid. FXI KO mice had a stronger systemic IgE response with an almost completely absent HDM-specific IgG1 response. The phenotype of FXII KO mice was, except for a higher HDM-specific IgG1 response, similar to that of WT mice. In conclusion, FXI attenuates part of the allergic response to repeated administration of HDM in the airways by a mechanism that is independent of activation via FXII.
Taking a big breath is known to reverse bronchoconstriction induced by bronchochallenge in healthy subjects; this bronchodilatory effect of deep inspiration (DI) is diminished in asthmatics. The mechanism underlying the DI effect is not clear. Observations from experiments using isolated airway smooth muscle (ASM) preparations and airway segments suggest that straining of ASM due to DI could lead to bronchodilation, possibly due to strain-induced reduction in ASM contractility. However, factors external to the lung cannot be excluded as potential causes for the DI effect. Neural reflex initiated by stretch receptors in the lung are known to inhibit the broncho-motor tone and enhance vasodilatation; the former directly reduces airway resistance, and the latter facilitates removal of contractile agonists through the bronchial circulation. If the DI effect is solely mediated by factors extrinsic to the lung, the DI effect would be absent in isolated, non-perfused lungs. Here we examined the DI effect in freshly isolated, non-perfused sheep lungs. We found that imposition of DI on isolated lungs resulted in significant bronchodilation, and this DI effect was present only after the lungs were challenged with a contractile agonist (acetylcholine or histamine), and the effect was independent of the difference in lung volume observed pre- and post-DI. We conclude that a significant portion of the bronchodilatory DI-effect stems from factors internal to the lung related to the activation of ASM.
The alveolar capillary network (ACN) provides an enormously large surface area that is necessary for pulmonary gas exchange. Changes of the ACN during normal or pathological development or in pulmonary diseases are of great functional impact and warrant further analysis. Due to the complexity of the three-dimensional (3D) architecture of the ACN, 2D approaches are limited in providing a comprehensive impression of the characteristics of the normal ACN or the nature of its alterations. Stereological methods offer a quantitative way to assess the ACN in 3D in terms of capillary volume, surface area or number but lack a 3D visualization to interpret the data. Hence, the necessity to visualize the ACN in 3D and to correlate this with data from the same set of data arises. Such an approach requires a large sample volume combined with a high resolution. Here, we present a technically simple and cost efficient approach to create 3D representations of lung tissue ranging from bronchioles over alveolar ducts and alveoli up to the ACN from more than 1mm sample extent to a resolution of less than 1µm. The method is based on automated image acquisition of serially sectioned epoxy resin embedded lung tissue fixed by vascular perfusion and subsequent automated digital reconstruction and analysis of the 3D data. This efficient method may help to better understand mechanisms of vascular development and pathology of the lung.
Elevated levels of reactive oxygen species (ROS) and intracellular Ca2+ play a key role in endothelial barrier dysfunction in acute lung injury (ALI). We previously showed that H2O2-induced increases in intracellular calcium concentrations ([Ca2+]i) in LMVECs involve the membrane Ca2+ channel, transient receptor potential vanilloid-4 (TRPV4), and that inhibiting this channel attenuated H2O2-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H2O2-induced Ca2+ influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacologic inhibition of CD36 on Ca2+ responses to H2O2. H2O2-induced Ca2+ influx was attenuated in LMVEC isolated from mice lacking CD36 (CD36-/-). TRPV4 expression and function was unchanged in LMVEC isolated from WT and CD36-/- mice, as well as mice with deficiency for Fyn (Fyn-/-). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36-/- LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36-/- mice compared to WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate (SSO) did not attenuate H2O2-induced Ca2+ influx. Lastly, we found that CD36-/- mice were protected from ischemia-reperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H2O2-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane in order to facilitate TRPV4 phosphorylation.
Acute lung injury/Acute Respiratory distress syndrome (ALI/ARDS) is an important cause of mortality in critically ill patients. Macrophages play an important role in the pathogenesis of ALI/ARDS. To investigate the role and underlying mechanisms of circulating monocytes and resident alveolar macrophages (AMs) in ALI/ARDS, we depleted circulating monocytes and AMs by clodronate-loaded liposome (CL) in lipopolysaccharide (LPS)-induced ALI/ARDS mouse model. Our results indicated that depletion of circulating monocytes by intravenous (i.v.) injection of CL 2 days prior to intra-tracheal (i.t.) LPS treatment significantly suppressed the acute lung injury in mice with ALI/ARDS, accompanied with significant reduction in neutrophil influx, interleukin-17 (IL-17), monocyte chemoattractant protein 1 (MCP-1), high-mobility group box 1 protein (HMGB1), suppressor of cytokine signaling 3 (SOCS3) and surfactant protein D (SP-D) in the lungs of 2 days LPS-i.t. treated mice. In contrast, depletion of AMs by i.t. delivery of CL enhanced the acute lung injury in association with up-regulation of these mediators. Blocking MCP-1 signaling by intraperitoneal (i.p.) instillation of anti-mouse CCL2 neutralizing antibody significantly reduced acute lung injury and neutrophil influx. In addition, SP-D was up-regulated by mediators released from AMs, because primary murine type II alveolar epithelial cells (AECII) expressed more SP-D after treatment with bronchoalveolar lavage (BAL) from LPS-treated mice or the conditioned media from LPS-treated RAW264.7 cells. The results indicated that circulating monocytes are pro-inflammatory, but AMs have anti-inflammatory functions in the early phase of ALI/ARDS. The study provided a molecular basis for the treatment of ALI/ARDS through modulation of circulating monocytes and AMs.
Respiratory transition at birth involves rapidly clearing fetal lung liquid and preventing efflux back into the lung whilst aeration is established. We have developed a sustained inflation (SIOPT) individualized to volume-response and a dynamic tidal positive end-expiratory pressure (open lung volume, OLV) strategy that both enhance this process. We aimed to compare the effect of each with a group managed with PEEP of 8 cmH2O and no recruitment manoeuvre (No-RM), on gas exchange, lung mechanics, spatiotemporal aeration and lung injury in 127±1d preterm lambs. Forty-eight fetal-instrumented lambs exposed to antenatal steroids were ventilated for 60 min after applying the allocated strategy. Spatiotemporal aeration and lung mechanics were measured with electrical impedance tomography (EIT) and forced-oscillation respectively. At study completion, molecular and histological markers of lung injury were analyzed. Mean (SD) aeration at the end of the SIOPT and OLV groups was 32 (22) and 38 (15) mL/kg, compared to 17 (10) mL/kg (180s) in the No-RM (p=0.024, one-way ANOVA). This translated into better oxygenation at 60 min (p=0.047; two-way ANOVA) resulting from better distal lung tissue aeration in SIOPT and OLV. There was no difference in lung injury. Neither SIOPT nor OLV achieved homogeneous aeration. Histological injury and mRNA biomarker upregulation were more likely in the regions with better initial aeration, suggesting volutrauma. Tidal ventilation or a SI achieve similar aeration if optimized, suggesting that preventing fluid efflux after lung liquid clearance is at least as important as fluid clearance during the initial inflation at birth.
Background and purpose. Sakuranetin is the main isolate flavonoid from Baccharis retusa (Asteraceae) leaves and exhibits anti-inflammatory and anti-oxidative activities. Acute respiratory distress syndrome is an acute failure of the respiratory system for which effective treatment is urgently necessary. This study investigated the preventive and therapeutic effects of sakuranetin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. Experimental approach. Animals were treated with intranasal sakuranetin 30 minutes before or 6 hours after instillation of LPS. Twenty-four hours after ALI was induced, lung function, inflammation, macrophages population markers, collagen fiber deposition, the extent of oxidative stress, and the expression of MMP-9, TIMP-1 and NF-kB were evaluated. Key results. The animals began to show lung alterations six hours after LPS instillation and these changes persisted until 24 hours after LPS administration. Preventive and therapeutic treatment with sakuranetin reduced the neutrophils in the peripheral blood and in the bronchial alveolar lavage. Sakuranetin treatment also reduced macrophages populations, particularly that of M1-like macrophages. In addition, sakurnaetin treatment reduced KC (IL-8 homologue) and NF-B levels, collagen fiber formation, MMM-9 and TIMP-1-positive cells, and oxidative stress in lung tissues compared with LPS animals treated with vehicle. Finally, sakuranetin treatment also reduced total protein, and the levels of TNF-α and IL-1β in the lung. Conclusions and Implications. This study shows that sakuranetin prevented and reduced pulmonary inflammation induced by LPS. Because sakuranetin modulates oxidative stress, the NF-kB pathway and lung function, it may constitute a novel therapeutic candidate to prevent and treat ALI.
Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional PtenSFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and β-catenin expression between cells towards the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in PtenSFTPC-cre mice showed decreased expression of epithelial and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition (EMT) at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5) and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific.
Background: IL-23 has been postulated to be a critical mediator contributing to various inflammatory diseases. Dermatophagoides pteronyssinus (Der p) is one of the most common inhalant allergens. However, the role of IL-23 in Der p-induced mouse asthma model is not well understood, particularly with regard to the development of allergic sensitization in the airways. Objective: To evaluate roles of IL-23 in Der p-sensitization and asthma development. Methods: BALB/c mice were repeatedly administered with Der p intra-nasally to develop Der p-allergic sensitization and asthma. After Der p local administration, changes in IL-23 expression were examined in lung tissues and primary epithelial cells. Anti-IL-23p19 antibody was given during the Der p sensitization period, and its effects were examined. Effects of anti-IL-23p19 antibody at bronchial epithelial levels were also examined in vitro. Results: The expression of IL-23 at bronchial epithelial layers was increased after Der p local administration in mouse. In Der p-induced mouse models, anti-IL-23p19 antibody treatment during allergen sensitization significantly diminished Der p-allergic sensitization and several features of allergic asthma including the production of Th2 cytokines and the population of ILC2 in lungs. The activation of dendritic cells in lung-draining lymph nodes was also reduced by anti-IL-23 treatment. In MLE-12 cells, IL-23 blockade prevented cytokine responses to Der p stimulation, such as IL-1α, GM-CSF, IL-33, and also bone marrow-derived DCs activation. Conclusion: IL-23 is another important bronchial epithelial cell-driven cytokine which may contribute to the development of house dust mite allergic sensitization and asthma.
The efficacy and feasibility of targeting TGF-β in pulmonary fibrosis and lung vascular remodeling in systemic sclerosis (SSc) have not been well elucidated. In this study, we analyzed how blocking TGF-β signaling affects pulmonary abnormalities in fos-related antigen 2 (Fra-2) transgenic mice, a murine model that manifests three important lung pathological features of SSc: fibrosis, inflammation, and vascular remodeling. To interrupt TGF-β signaling in the Fra-2 transgenic mice, we used a pan-TGF-β blocking antibody, 1D11, and transgenic mice in which TGF-β receptor type 2 (Tgfbr2) is deleted from smooth muscle cells and myofibroblasts (α-SMA-CreER; Tgfbr2flox/flox). Global inhibition of TGF-β by 1D11 did not ameliorate lung fibrosis histologically or biochemically, whereas it resulted in a significant increase in the number of immune cells infiltrating in the lungs. In contrast, 1D11 treatment ameliorated the severity of pulmonary vascular remodeling in Fra-2 transgenic mice. Similarly, genetic deletion of Tgfbr2 from smooth muscle cells resulted in improvement in pulmonary vascular remodeling in the Fra-2 transgenic mice as well as a decrease in the number of Ki67-positive vascular smooth muscle cells, suggesting that TGF-β signaling contributes to the development of pulmonary vascular remodeling by promoting the proliferation of vascular smooth muscle cells. Deletion of Tgfbr2 from α-SMA expressing cells had no effect on fibrosis or inflammation in this model. These results suggest that efforts to target TGF-β in SSc will likely require more precision than simply globally inhibiting TGF-β function.
Epidemiologic studies indicate that cigarette smoking (CS) increases the risk and severity of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). The mechanism is not understood at least in part due to lack of animal models that reproduce the key features of CS priming process. In this study, using two strains of mice we characterized a double-hit mouse model of ALI induced by CS priming of injury caused by lipopolysaccharide (LPS). C57BL/6 and AKR mice were pre-exposed to CS briefly (3 hours) or sub-acutely (3 weeks) before intra-tracheal instillation of LPS and ALI was assessed 18 hours after LPS administration by measuring lung static compliance, lung edema, vascular permeability, inflammation and alveolar apoptosis. We found that as little as 3 hours of exposure to CS enhanced LPS-induced ALI in both strains of mice. Similar exacerbating results were observed after 3 weeks of pre-exposure to CS. However, there is a strain difference in susceptibility to CS priming for ALI, with a greater effect in AKR mice. The key features we observed suggest that 3 weeks of CS pre-exposure of AKR mice is a reproducible, clinically relevant animal model that is useful for studying mechanisms and treatment of CS priming for a second hit-induced ALI. Our data also support the concept that increased susceptibility to ALI/ARDS is an important adverse health consequence of CS exposure that needs to be taken into consideration when treating critically ill individuals.
Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a ubiquitous master transcription factor that upregulates antioxidant response elements (AREs)-mediated expression of antioxidant enzyme and cytoprotective proteins. Activation of Nrf2 has been shown to be protective against lung injury. In the lung, diverse stimuli including environmental oxidants, medicinal agents and pathogens can activate Nrf2. Nrf2 translocates to the nucleus and binds to an antioxidant response element (ARE). Through transcriptional induction of ARE-bearing genes encoding antioxidant-detoxifying proteins, Nrf2 induces cellular rescue pathways against oxidative pulmonary injury, abnormal inflammatory and immune responses, and apoptosis. The Nrf2-antioxidant pathway has been shown to be important in the protection against various lung injury including acute lung injury/acute respiratory distress syndrome and bronchopulmonary dysplasia, chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, asthma and allergy, and was widely examined for new therapeutic targets. The current review explores the protective role of Nrf-2 against lung injury and the therapeutic potential in targeting Nrf-2.
Animal dung is a biomass fuel burned by vulnerable populations who cannot afford cleaner sources of energy, such as wood and gas, for cooking and heating their homes. Exposure to biomass smoke is the leading environmental risk for mortality, with over 4 million deaths each year worldwide attributed to indoor air pollution from biomass smoke. Biomass smoke inhalation is epidemiologically associated with pulmonary diseases, including Chronic Obstructive Pulmonary Disease (COPD), lung cancer, and respiratory infections, especially in low and middle-income countries. Yet, few studies have examined the mechanisms of dung biomass smoke-induced inflammatory responses in human lung cells. Here, we tested the hypothesis that dung biomass smoke causes inflammatory responses in human lung cells through signaling pathways involved in acute and chronic lung inflammation. Primary human small airway epithelial cells (SAECs) were exposed to dung smoke at the air-liquid interface using a newly developed, automated and reproducible dung biomass smoke generation system. The examination of inflammatory signaling showed that dung biomass smoke increased the production of several pro-inflammatory cytokines and enzymes in SAECs through activation of the activator protein (AP)-1 and arylhydrocarbon receptor (AhR), but not nuclear factor appa B (NFB) pathways. We propose that the inflammatory responses of lung cells exposed to dung biomass smoke contribute to the development of respiratory diseases.
The aim of this study is to elucidate the role of TRAIL during rhinovirus (RV) infection in vivo. Naïve wild type and TRAIL-deficient (Tnfsf10-/-) BALB/c mice were infected intranasally with RV1B. In separate experiments, Tnfsf10-/- mice were sensitized and challenged via the airway route with house dust mite (HDM) to induce allergic airways disease and then challenged with RVIB or UV-RVIB. Airways hyperreactivity (AHR) was invasively assessed as total airways resistance in response to increasing methacholine challenge and inflammation assessed in bronchoalveolar lavage fluid (BALF) at multiple time points post infection. Chemokines were quantified by ELISA of whole lung lysates and viral load was determined by quantitative RT-PCR and tissue culture infective dose (TCID50). Human airway epithelial cells (BEAS2B) were infected with RV1B and stimulated with recombinant TRAIL or neutralizing anti-TRAIL antibodies and viral titer assessed by TCID50. HDM challenged Tnfsf10-/- mice were protected against RV-induced AHR and had suppressed cellular infiltration in the airways upon RV infection. Chemokine C-X-C-motif ligand 2 (CXCL2) production was suppressed in naïve Tnfsf10-/- mice infected with RV1B, with less RV1B detected 24 hours post-infection. This was associated with reduced apoptotic cell death and a reduction of interferon (IFN)-2/3 but not IFN-α or IFN-β. TRAIL stimulation increased, whereas anti-TRAIL antibodies reduced viral replication in RV1B-infected BEAS2B cells in vitro. In conclusion, TRAIL promotes RV-induced AHR, inflammation and RV1B replication, implicating this molecule and its downstream signaling pathways as a possible target for the amelioration of RV1B-induced allergic and non-allergic lung inflammation and AHR.
Background. Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome (ARDS) in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has never been explored. Methods. C57BL/6 mice were inoculated intranasally with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days post-infection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by UHPLC/MS. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Results. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'diphospho (CDP)-choline and CDP-ethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased sphingomyelin, cholesterol, and protein. Conclusions. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of ARDS in influenza-infected mice.
In many mammals, including humans, removal of one lung (pneumonectomy) results in the compensatory growth of the remaining lung. Compensatory growth involves not only an increase in lung size, but also an increase in the number of alveoli in the peripheral lung; however, the process of compensatory neoalveolarization remains poorly understood. Here, we show that the expression of α-smooth muscle actin (SMA)-a cytoplasmic protein characteristic of myofibroblasts-is induced in the pleura following pneumonectomy. SMA induction appears to be dependent on pleural deformation (stretch) as induction is prevented by plombage or phrenic nerve transection (p<.001). Within 3 days of pneumonectomy, the frequency of SMA+ cells in subpleural alveolar ducts was significantly increased (p<.01). To determine the functional activity of these SMA+ cells, we isolated regenerating alveolar ducts by laser microdissection and analyzed individual cells using microfluidic single-cell quantitative PCR. Single cells expressing the SMA (Acta2) gene demonstrated significantly greater transcriptional activity than endothelial cells or other discrete cell populations in the alveolar duct (p<.05). The transcriptional activity of the Acta2+ cells, including expression of TGF signaling as well as repair-related genes, suggests that these myofibroblast-like cells contribute to compensatory lung growth.
The prevalence of a sedentary (SED) life style combined with calorically rich diets has spurred the rise in childhood obesity which, in turn, translates to adverse health effects in adulthood. Obesity and lack of active (ACT) lifestyle may increase susceptibility to air pollutants. We housed 22 day-old female Long-Evans rats in a cage without (SED) or with a running wheel (ACT). After 10 weeks the rats ran 310 ± 16.3 km (SEM). Responses of SED and ACT rats to whole-body O3 (0, 0.25, 0.5, or 1.0 ppm; 5 hr/day for 2 days) was assessed. Glucose tolerance (GTT) was performed following the first day of O3. ACT rats had less body fat and an improved glucose tolerance (GTT). Ventilatory function (plethysmography) of SED and ACT groups was similarly impaired by O3. Bronchoalveolar lavage fluid (BALF) was collected after the second O3 exposure. SED and ACT rats were hyperglycemic following 1.0 ppm O3. GTT was impaired by O3 in both groups; however, ACT rats exhibited improved recovery to 0.25 and 1.0 ppm O3. BALF cell neutrophils and total cells were similarly increased in ACT and SED groups exposed to 1.0 ppm O3. O3-induced increase in eosinophils was exacerbated in SED rats. Chronic exercise from post-weaning to adulthood improved some of the metabolic and pulmonary responses to O3 (GTT and eosinophils) but several other parameters were unaffected. The reduction in O3-induced rise in BALF eosinophils in ACT rats suggests a possible link between a SED lifestyle and incidence of asthma-related symptoms from O3.
Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor pathway is currently recognized as a counterbalancing mechanism of the renin-angiotensin system in different pathophysiological conditions. We have previously described that treatment with Ang-(1-7) attenuates lung inflammation and remodeling in an experimental model of asthma. In the present study, we investigated whether lack of Mas receptor could alter the inflammatory response in a model of chronic allergic lung inflammation induced by ovalbumin (OVA). MasWT and MasKO mice were subjected to 4 doses of OVA (20µg/mice, i.p.) with 14 days interval. At the 21st day, nebulization with OVA (1%) was started, 3 times/week until the 46th day. Control groups received saline (0.9%, i.p.) and were nebulized with saline (0.9%). MasWT-OVA developed a modest inflammatory response and minor pulmonary remodeling to OVA challenge. Strikingly, MasKO-OVA presented a significant increase in inflammatory cell infiltrate, extracellular matrix deposition, increase in thickening of the alveolar parenchyma, increase in thickening of the smooth muscle layer of the pulmonary arterioles, increase in pro-inflammatory cytokine and chemokines levels in the lungs, characteristic of chronic asthma. Additionally, MasKO-OVA presented an increase in ERK1/2 phosphorylation in comparison to MasWT-OVA. Furthermore, MasKO-OVA showed a worst performance in a test of maximum physical exercise in comparison to MasWT-OVA. Our study shows that effects triggered by Mas receptor are important to attenuate the inflammatory and remodeling processes in a model of allergic lung inflammation in mice. Our data indicate that impairment of Ang-(1-7)/Mas receptor pathway may lead to worsening of the pathophysiological changes of asthma.
Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with a median survival of three years. IPF deteriorates upon viral or bacterial lung infection although pulmonary infection (pneumonia) in healthy lungs rarely induces fibrosis. Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) initiating pro-inflammatory pathways. As TLR4 has already been linked to hepatic fibrosis and scleroderma, we now investigated the role of TLR4 in IPF fibroblasts. Lung tissue sections from IPF patients were analyzed for TLR4 expression. Isolated normal human lung fibroblasts (NL-FB) and IPF fibroblasts (IPF-FB) were exposed to LPS and TGF-β prior to expression analysis of receptors, pro-fibrotic mediators and cytokines. TLR4 is expressed in fibroblast foci of IPF lungs as well as in primary normal human lung fibroblasts (NHLF), NL-FB, and IPF-FB. As a model for a gram-negative pneumonia in the non-fibrotic lung, NL-FB and IPF-FB were co-exposed to LPS and TGF-β. While NL-FB produced significantly less connective tissue growth factor (CTGF) upon co-stimulation compared to TGF-β stimulation alone, IPF-FB showed significantly increased pro-fibrotic markers compared to control fibroblasts after co-stimulation. Although levels of anti-fibrotic prostaglandin E2 (PGE2) were elevated after co-stimulation, they were not responsible for this effect. However, significant downregulation of TGF-β receptor type 1 in control fibroblasts seems to contribute to the reduced pro-fibrotic response in our in vitro model. Normal and IPF fibroblasts thus differ in their pro-fibrotic response upon LPS induced TLR4 stimulation.
Myofibroblast differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-β1 (TGFβ1) is a powerful inducer of myofibroblast differentiation and is implicated in pathogenensis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase, SIRT3 in TGF-β1-induced myofibroblast differentiqtion in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGFβ1 resulted in increased expression of fibrosis markers, smooth muscle alpha-actin (α-SMA), collagen-1 and fibronectin. TGF-β1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized-guanine (8-Oxo-dG), and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-β1 on ROS production and mitochondrial DNA damage, and inhibited TGF-β1-induced myofibroblast differentiation. To determine the anti-fibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared to wild-type controls, SIRT3-KO mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-Oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body SIRT3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of myofibroblast differentiation and lung fibrosis.
Extracorporeal membrane oxygenation (ECMO) is a life-saving treatment for patients with severe, refractory, cardiorespiratory failure. Exposure to the ECMO circuit is thought to trigger/exacerbate inflammation. Determining whether inflammation is due to the patients underlying pathologies or the ECMO circuit is difficult. To discern how different insults contribute to the inflammatory response we developed an ovine model of lung injury and ECMO to investigate the impact of smoke-induced lung injury and ECMO in isolation and cumulatively on pulmonary and circulating inflammatory cells, cytokines and tissue remodeling. Sheep receiving either smoke-induced acute lung injury (S-ALI) or sham injury were placed on veno-venous (VV) ECMO lasting either 2 or 24hrs, with controls receiving conventional ventilation only. Lung tissue, bronchoalveolar fluid and plasma were analyzed by RT-PCR, immunohistochemical staining and zymography to assess inflammatory cells, cytokines and matrix metalloproteinases. Pulmonary compliance decreased in sheep with S-ALI placed on ECMO with increased numbers of infiltrating neutrophils, monocytes and alveolar macrophages compared to controls. Infiltration of neutrophils was also observed with S-ALI alone. RT-PCR studies showed higher expression of MMP2 and MMP9 in S-ALI plus ECMO while IL-6 was elevated at 2hrs. Zymography revealed higher levels of MMP2. Circulating plasma levels of IL-6 were elevated 1-2hrs after commencement of ECMO alone. This data shows that the inflammatory response is enhanced when a host with pre-existing pulmonary injury is placed on ECMO, with increased infiltration of neutrophils and macrophages, the release of inflammatory cytokines and up regulation of matrix metalloproteinases.
Acute lung injury (ALI) is characterized by hypoxemia, enhanced permeability of the air-blood barrier and pulmonary edema. Particularly in the elderly ALI is associated with increased morbidity and mortality. The reasons for this, however, are poorly understood. We hypothesized that age-related changes in pulmonary structure, function and inflammation, lead to a worse prognosis in ALI. ALI was induced in young (2 months) and old (18 months) male C57BL/6 mice by intranasal application of 2.5 mg lipopolysaccharide (LPS)/kg body weight or saline (control mice). After 24 h, lung function was assessed and lungs were either processed for stereological or inflammatory analysis such as bronchoalveolar lavage fluid (BALF) cytometry and qPCR. Both young and old mice developed severe signs of ALI including alveolar and septal edema and enhanced inflammatory BALF cells. However, the pathology of ALI was more pronounced in old compared to young mice with a 6-fold higher BALF protein concentration, twice the number of neutrophils and significantly higher expression of neutrophil chemokine Cxcl1, adhesion molecule Icam-1 and metalloprotease-9, whereas the expression of tight junction protein occludin significantly decreased. The old LPS-mice had thicker alveolar septa due to higher volumes of interstitial cells and extracellular matrix. Tissue resistance and elastance reflected observed changes at ultrastructural level in the lung parenchyma in ALI of young and old mice. In summary, the pathology of ALI with advanced age in mice is characterized by a greater neutrophilic inflammation, leakier air-blood barrier and altered lung function which is in line with findings in elderly patients.
Influenza severity increases with age, with hospitalization and mortality rates during seasonal influenza epidemics being higher in older men than age-matched women. Based on knowledge that with age, circulating testosterone levels decline in males, we hypothesized that reduced testosterone contributes to age-associated increases in influenza severity. A murine model was used to test this hypothesis. As in men, testosterone concentrations were lower in aged (18 months) than young (2 months) male C57BL/6 mice. Following inoculation with influenza A virus (IAV), aged males experienced greater morbidity, clinical disease and pulmonary inflammation than young males, and had lower neutralizing and total anti-influenza IgG antibody responses. Peak titers of virus in the lungs did not differ between aged and young males, but virus clearance was delayed in aged males. In young males, removal of the gonads increased, whereas treatment of gonadectomized males with testosterone reduced, morbidity, clinical illness, and pulmonary pathology, but did not alter viral replication. Treatment of aged males with testosterone improved survival following infection, but did not alter either virus replication or pulmonary pathology. These results indicate that low concentrations of testosterone, whether induced surgically in young males or naturally occurring in aged males, negatively impact the outcome of influenza.
Rationale: Hypoxic pulmonary vasoconstriction (HPV) is the response of the pulmonary vasculature to low levels of alveolar oxygen. HPV improves systemic arterial oxygenation by matching pulmonary perfusion to ventilation during alveolar hypoxia, and is impaired in lung diseases such as the acute respiratory distress syndrome (ARDS) and in experimental models of endotoxemia. Epoxyeicosatrienoic acids (EETs) are pulmonary vasoconstrictors, which are metabolized to less vasoactive dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). We hypothesized that pharmacological inhibition or a congenital deficiency of sEH in mice would reduce the metabolism of EETs and enhance HPV in mice after challenge with lipopolysaccharide (LPS). Methods and Results: HPV was assessed 22h after intravenous injection of LPS by measuring the percentage increase in the pulmonary vascular resistance of the left lung induced by left mainstem bronchial occlusion (LMBO). After LPS-challenge, HPV was impaired in sEH+/+, but not in sEH-/- mice or in sEH+/+ mice treated acutely with a sEH inhibitor. Deficiency or pharmacological inhibition of sEH protected mice from the LPS-induced decrease in systemic arterial oxygen concentration (PaO2) during LMBO. In the lungs of sEH-/- mice, the LPS-induced increase in DHETs and cytokines was attenuated. Conclusions: Deficiency or pharmacologic inhibition of sEH protects mice from LPS-induced impairment of HPV and improves the PaO2 after LMBO. After LPS-challenge, lung EET degradation and cytokine expression were reduced in sEH-/- mice. Inhibition of sEH might prove to be an effective treatment for ventilation-perfusion mismatch in lung diseases such as ARDS.
GRB2-associated-binding protein 1 (Gab1) belongs to Gab adaptor family (Gabs), which integrates multiple signals in response to the epithelial growth factors. Recent genetic studies identified genetic variants of human Gab1 gene as potential risk factors of asthmatic inflammation. However, Gab1 functions in lungs remain largely unknown. Alveolar type-II cells (AT-IIs) are responsible for surfactant homeostasis, and essentially regulate lung inflammation following various injuries. Here, in vitro knockdown of Gab1 was shown to cause a decrease in surfactant proteins (SPs) in AT-IIs. We further examined in vivo Gab1 functions by generating alveolar epithelium-specific Gab1 knockout mice (Gab1/). In vivo Gab1 deficiency leads to a decrease in SP synthesis and the appearance of disorganized lamellar bodies. Histological examination shows no apparent pathological alterations or inflammation in the lung sections obtained from Gab1/, compared with the controls. However, Gab1/ mice demonstrate inflammatory responses during the LPS-induced acute lung injury. Similarly, in mice challenged with bleomycin (BLM), fibrotic lesions were shown to be aggravated in Gab1/. These observations suggest that the abolishment of Gab1 in AT-IIs impairs SP homeostasis, predisposing mice to lung injuries. Additionally, we observed that the production of surfactants in AT-IIs overexpressing Gab1 mutants, in which Shp2 phosphatase and PI3K kinase binding sites have been mutated (Gab1Shp2, Gab1PI3K), has been considerably attenuated. Together, these findings provide the first evidence of the roles of docking protein Gab1 in lungs, adding to our understanding of acute and interstitial lung diseases caused by the disruption of alveolar SP homeostasis.
Chronic epithelial injury triggers a TGFβ-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGFβ induces the mesenchymal cell state, and determined its mechanism. We observe that TGFβ stimulation activates an inflammatory gene program controlled by the NFB/RelA signaling pathway. In the mesenchymal state, NFB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation and activating phosphorylation of the NFB/RelA transcription factor on Ser 276, mediated by a paracrine signal. Phospho-Ser 276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser 2 in its carboxy terminal domain (CTD). RelA-initiated transcriptional elongation is required for expression of the core EMT transcriptional regulators SNAI1, TWIST1 and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGFβ challenge in a mouse model. These data provide a detailed mechanism for how activated NFB and BRD4 control EMT initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validates BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.
Chromatin modifying enzymes mediate DNA methylation and histone modifications upon recruitment to specific target gene loci in response to various stimuli. The key enzymes that regulate chromatin accessibility for maintenance of modifications in DNA and histones, and for modulation of gene expression patterns in response to cigarette smoke (CS), are not known. We hypothesize that CS exposure alters the gene expression patterns of chromatin modifying enzymes, which then affects multiple downstream pathways involved in the response to CS. We have therefore analyzed chromatin modifying enzyme profiles and validated by quantitative real-time PCR (qPCR). We also performed immunoblot analysis of targeted histone marks in C57BL/6J mice exposed to acute and sub-chronic CS, and of lungs from nonsmokers, smokers, and patients with chronic obstructive pulmonary disease (COPD). We found a significant increase in expression of several chromatin modification enzymes, including DNA methyltransferases, histone acetyltransferases, histone methyltransferases and SET domain proteins, histone kinases and ubiquitinases. Our qPCR validation data revealed a significant down-regulation of Dnmt1, Dnmt3a, Dnmt3b, Hdac2, Hdac4, Hat1, Prmt1, and Aurkb. We identified targeted chromatin histone marks (H3K56ac and H4K12ac) which are induced by CS. Thus, CS-induced genotoxic stress differentially affects the expression of epigenetic modulators that regulate transcription of target genes via DNA methylation and site-specific histone modifications. This may have implications in devising epigenetic-based therapies for COPD and lung cancer.
Viral respiratory tract infections are the most common illness in humans. Infection of the respiratory viruses results in accumulation of viral replicative double-stranded RNA (dsRNA), which is one of the important components of infecting viruses for the induction of lung epithelial cell apoptosis and innate immune response, including the production of interferon (IFN). In the present study, we have investigated the regulation of dsRNA-induced airway epithelial cell apoptosis by IFN. We found that transcription factor Runx3 was strongly induced by type-II IFN, slightly by type-III IFN, but essentially not by type-I IFNα in airway epithelial cells. IFN-induced expression of Runx3 was predominantly mediated by JAK-STAT1 pathway and partially by NF-B pathway. Interestingly, Runx3 can be synergistically induced by IFN with a synthetic analog of viral dsRNA polyinosinic-polycytidylic acid (poly(I:C)) or tumor necrosis factor-α (TNFα) through both JAK-STAT1 and NF-B pathways. We further found that dsRNA poly(I:C)-induced apoptosis of airway epithelial cells was mediated by dsRNA receptor Toll-like receptor 3 (TLR3) and was markedly augmented by IFN through the enhanced expression of TLR3 and subsequent activation of both extrinsic and intrinsic apoptosis pathways. Last, we demonstrated that upregulation of Runx3 by IFN promoted TLR3 expression, thus amplifying the dsRNA-induced apoptosis in airway epithelial cells. These novel findings indicate that IFN promotes dsRNA-induced TLR3-dependent apoptosis via upregulation of transcription factor Runx3 in airway epithelial cells. Findings from our study may provide new insights into the regulation of airway epithelial cell apoptosis by IFN during viral respiratory tract infection.
The development of CFTR targeted therapy for cystic fibrosis has generated interest in maximizing membrane residence of mutant forms of CFTR by manipulating interactions with scaffold proteins such as NHERF1. In this study, we explored whether C-terminal sequences in CFTR beyond the PDZ-binding motif influence its interaction with NHERF1. NHERF1 displayed minimal self-association in blot overlays (NHERF1, Kd = 1382 ± 61.1 nM) at concentrations well above physiologic levels, estimated at 240 nM from RNA-Seq and 260 nM by LC-MS/MS in sweat gland, a key site of CFTR function in vivo. However, NHERF1 oligomerized at considerably lower concentrations (10 nM) in the presence of the last 111 amino acids of CFTR (20 nM) in blot overlays and cross-linking assays and in co-immunoprecipitations using differently tagged versions of NHERF1. Deletion and alanine mutagenesis revealed that a six amino acid sequence1417EENKVR1422 and the terminal 1478TRL1480 (PDZ-binding motif) in the C-terminus were essential for the enhanced oligomerization of NHERF1. Full-length CFTR stably expressed in MDCK epithelial cells fostered NHERF1 oligomerization that was substantially reduced (~5 fold) upon alanine substitution of EEN, KVR or EENKVR residues or deletion of the TRL motif. Confocal fluorescent microscopy revealed that the EENKVR and TRL sequences contribute to preferential localization of CFTR to the apical membrane. Together, these results indicate that C-terminal sequences mediate enhanced NHERF1 interaction and facilitate the localization of CFTR; a property that could be manipulated to stabilize mutant forms of CFTR at the apical surface to maximize the effect of CFTR-targeted therapeutics.
Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through rho-kinase (ROCK) activation, ET-1 impairs endothelial cell function, however, whether ET-1-ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intra-peritoneal bleomycin with and without selective ETA (BQ123/BQ610) and ETB (BQ788) receptor blockers, non-selective ET receptor blocker (ETRB) (bosentan) or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT) and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212% and MWT by 140%, and decreased radial alveolar count (RAC) and vessel density by 40% and 44%, respectively (p<0.01 for each comparison) After bleomycin treatment, fasudil and bosentan, partially restored RAC and vessel density and decreased MLI, RV/LV+S and medial wall thickness to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1-ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that non-selective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH.
The pulmonary airways are subdivided into conducting and gas-exchanging airways. The small tree of gas-exchanging airways which is fed by the most distal conducting airway represents an acinus. Very little is known about the development of the number of acini. The goal of this study was to estimate their number throughout rat postnatal development. Right middle rat lung lobes were obtained at postnatal day 4-60, stained with heavy metals, paraffin embedded, and scanned by synchrotron radiation based X-ray tomographic microscopy or imaged using micro computed tomography after critical point drying. The acini were counted by detection of the transitional bronchioles (bronchioalveolar duct junction; BADJ) using morphological criteria (thickness of the walls of airways and appearance of alveoli) during examination of the resulting 3D image stacks. Between postnatal days 4-60, the number of acini per lung remained constant (5840 ± 547 acini), but their volume increased significantly. We conclude that the acini are formed before the end of the saccular stage (before postnatal day 4) and that the developmental increase of the lung volume is achieved by an increase of the acinar volume and not by an increase of their number. Furthermore, our results propose that the bronchioalveolar stem cells, which are residing in the BADJ, are as constant in their location at the BADJ itself.
Bronchopulmonary dysplasia (BPD) is often complicated by pulmonary hypertension (PH). We investigated three biomarkers potentially suitable as screening markers for extremely preterm infants at risk of BPD-associated PH. In this prospective observational cohort study conducted in a tertiary neonatal intensive care unit, 83 preterm infants with BPD born <28 weeks gestation and still inpatients at 36 weeks corrected age received an echocardiogram and blood tests of B-type natriuretic peptide (BNP), troponin I, and YKL-40. Infants were analysed according echocardiographic evidence of tricuspid regurgitation (TR). 30 infants had evidence of TR on echocardiogram at 36 weeks corrected age. Infants with or without TR had similar baseline demographics: mean±SD gestational age 261±12 weeks versus 261±11 weeks, and mean±SD birth weight 830±206g versus 815±187g, respectively. There was no difference in duration of respiratory support. The mean±SD right ventricular systolic pressure of infants with evidence of TR was 40±16 mmHg. BNP was the only biomarker that proved to be significantly higher in infants with evidence of TR: median (IQR) serum level 54.5 (35-105) versus 41.5 (30-59) pg/ml, p=0.043. Subgroup analysis of infants with severe BPD requiring discharge on home oxygen or BPD-related mortality revealed similar results. There was no difference between groups for troponin I and YKL-40. In conclusion, increased serum levels of BNP were associated with evidence of TR at 36 weeks corrected gestational age in extremely preterm infants, suggesting a potential role as a screening biomarker for BPD-associated PH.
Acute respiratory distress syndrome (ARDS) is characterized by inflammatory injury to the alveolar and capillary barriers that results in impaired gas exchange and severe acute respiratory failure. Nuclear orphan receptor Nur77 has emerged as a regulator of gene expression in inflammation and its role in the pathogenesis of ARDS is not clear. The objective of this study is to investigate the potential role of Nur77 and its underlying mechanism in the regulation of endothelin-1 (ET-1) expression in LPS-induced A549 cells and an ARDS rat model. We demonstrate that LPS induced Nur77 expression and nuclear export in A549 cells. Overexpression of Nur77 markedly decreased basal and LPS-induced ET-1 expression in A549 cells, while knockdown of Nur77 increased the ET-1 expression. LPS-induced phosphorylation and nuclear translocation of NF-B and p38 MAPK were blocked by Nur77 overexpression, and was augmented by Nur77 knockdown in A549 cells. In vivo, LPS induced Nur77 expression in lung in ARDS rats. Pharmacological activation of Nur77 by cytosporone B (CsnB) inhibited ET-1 expression in ARDS rats, decreased LPS-induced phosphorylation of NF-B and p38 MAPK and relieved lung, liver and kidney injury. Pharmacological de-activation of Nur77 by 1,1-bis (3'-indolyl)-1-(p-hydroxyphenyl) methane (DIM-C-pPhOH, C-DIM8) had no effect on ET-1 expression and lung injury. These results indicated that Nur77 decreases ET-1 expression by suppressing NF-B and p38 MAPK in LPS-stimulated A549 cells in vitro and in an LPS-induced ARDS rat model, CsnB reduced ET-1 expression and lung injury in ARDS rats.
MicroRNAs play an important role in the development and progression of various diseases, such as idiopathic pulmonary fibrosis (IPF). Although the accumulation of aberrant fibroblasts resistant to apoptosis is a hallmark in IPF lungs, the mechanism regulating apoptosis susceptibility is not fully understood. Here, we investigated the role of miR-29, which is the most downregulated microRNA in IPF lungs and is also known as a regulator of extracellular matrix (ECM), in the mechanism of apoptosis resistance. We found that functional inhibition of miR-29c caused resistance to Fas-mediated apoptosis in lung fibroblasts. Furthermore, experiments using miR-29c inhibitor and miR-29c mimic revealed that miR-29c regulated expression of the death receptor, Fas, and formation of death-inducing signaling complex (DISC) leading to extrinsic apoptosis. The representative profibrotic transforming growth factor (TGF)-β downregulated the expression of miR-29c as well as Fas receptor, and conferred resistance to apoptosis. We also found that introduction of miR-29c mimic abrogated these TGF-β-induced phenotypes of Fas repression and apoptosis resistance. The results presented here suggest that downregulation of miR-29 observed in IPF lungs may be associated with the apoptosis-resistant phenotype of IPF lung fibroblasts via downregulation of Fas receptor. Therefore, restoration of miR-29 expression in IPF lungs could not only inhibit the accumulation of ECM but also normalize the sensitivity to apoptosis in lung fibroblasts, which may be an effective strategy for treatment of IPF.
Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment and polarization. Furthermore, Rac1 modulates both non-canonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.
Lung epithelial cells are suggested to promote pathogen-induced pulmonary inflammation by the release of chemokines, resulting in enhanced recruitment of circulating leukocytes. Recent studies have shown that the interleukin-17C (IL-17C) regulates innate immune functions of epithelial cells in an autocrine manner. The aim of this study was to investigate the contribution of IL-17C to pulmonary inflammation in a mouse model of acute Pseudomonas aeruginosa pneumonia. Infection with P. aeruginosa resulted in an increased expression of IL-17C in lung tissue of wildtype mice. Numbers of neutrophils and the expression of the neutrophil-recruiting chemokines keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) were significantly decreased in lungs of IL-17C-deficient (IL-17C-/-) mice infected with P. aeruginosa at 24 hours. Systemic concentrations of interleukin-6 (IL-6) were significantly decreased in infected IL-17C-/- mice at 24 hours and the survival of IL-17C-/- mice was significantly increased at 48 hours. The expression of IL-17C was reduced in infected mice deficient for interleukin-17A (IL-17A), whereas pulmonary concentrations of IL-17A were not affected by the deficiency for IL-17C. Stimulation of primary alveolar epithelial cells with IL-17A resulted in a significantly increased expression of IL-17C in vitro. Our data suggest that IL-17A-mediated expression of epithelial IL-17C amplifies the release of chemokines by epithelial cells and thereby contributes to the recruitment of neutrophils and systemic inflammation during acute P. aeruginosa pneumonia.
Alveolar epithelial regeneration is essential for resolution of the acute respiratory distress syndrome (ARDS). Although neutrophils have traditionally been considered mediators of epithelial damage, recent studies suggest they promote type II pneumocyte (AT2) proliferation, which is essential for regenerating alveolar epithelium. These studies did not, however, evaluate this relationship in an in vivo model of alveolar epithelial repair following injury. To determine if neutrophils influence alveolar epithelial repair in vivo, we developed a unilateral acid injury model that creates a severe yet survivable injury with features similar to ARDS. Mice that received injections of the neutrophil depleting Ly6G antibody had impaired AT2 proliferation 24 and 72 hours after acid instillation, which was associated with decreased re-epithelialization and increased alveolar protein concentration 72 hours after injury. As neutrophil depletion itself may alter the cytokine response, we questioned the contribution of neutrophils to alveolar epithelial repair in neutropenic granulocyte-colony stimulating factor (G-CSF) -/- mice. We found that the loss of G-CSF recapitulated the neutrophil response of Ly6G treated mice and was associated with defective alveolar epithelial repair, similar to neutrophil-depleted mice, and was reversed by administration of exogenous G-CSF. To approach the mechanisms, we employed an unbiased protein analysis of bronchoalveolar lavage fluid from neutrophil-depleted and neutrophil-replete mice 12 hours after inducing lung injury. Pathway analysis identified significant differences in multiple signaling pathways that may explain the differences in epithelial repair. These data emphasize an important link between the innate immune response and tissue repair in which neutrophils promote alveolar epithelial regeneration.
Chronic neonatal pulmonary hypertension (PHT) frequently results in early death. Systemically administered Rho-kinase (ROCK) inhibitors prevent and reverse chronic PHT in neonatal rats, but at the cost of severe adverse effects, including systemic hypotension and growth restriction. Simvastatin has pleiotropic inhibitory effects on isoprenoid intermediates that may limit activity of RhoA, which signals upstream of ROCK. We therefore hypothesized that statin treatment would safely limit pulmonary vascular RhoA activity and prevent and reverse experimental chronic neonatal PHT via downstream inhibitory effects on pathological ROCK activity. Sprague-Dawley rats in normoxia (room air) or moderate normobaric hypoxia (13% O2) received Simvastatin (2 mg/kg/day i.p.) or vehicle from postnatal days 1-14 (prevention protocol) or from days 14-21 (rescue protocol). Chronic hypoxia increased RhoA and ROCK activity in lung tissue. Simvastatin reduced lung content of the isoprenoid intermediate, farnesyl pyrophosphate, and decreased RhoA/ROCK signaling in the hypoxia-exposed lung. Preventive or rescue treatment of chronic hypoxia-exposed animals with Simvastatin decreased pulmonary vascular resistance, right ventricular hypertrophy, and pulmonary arterial remodeling. Preventive Simvastatin treatment improved weight gain, did not lower systemic blood pressure, and did not cause apparent toxic effects on skeletal muscle, liver or brain. Rescue therapy with Simvastatin improved exercise capacity. We conclude that Simvastatin limits RhoA/ROCK activity in the chronic hypoxia-exposed lung, thus preventing or ameliorating hemodynamic and structural markers of chronic PHT and improving long-term outcome, without causing adverse effects.
Pulmonary infections with non-tuberculous mycobacteria (P-NTM) such as by Mycobacterium avium complex (M. avium) are increasingly found in the elderly, but the underlying mechanisms are unclear. Recent studies suggest that adaptive immunity is necessary but not sufficient for host defense against mycobacteria. Heme oxygenase-1 (HO-1) has been recognized as a critical modulator of granuloma formation and programmed cell death in mycobacterial infections. Old mice (18-21 months) infected with M. avium had attenuated HO-1 response with diffuse inflammation, high burden of mycobacteria, poor granuloma formation, and decreased survival (45%) while young mice (4-6 months) showed tight, well defined granuloma, increased HO-1 expression and increased survival (95%). To further test the role of HO-1 in increased susceptibility to P-NTM infections in the elderly, we used old and young HO-1+/+ and HO-1-/- mice. The transcriptional modulation of the JAK/STAT signaling pathway in HO-1-/- mice due to M. avium infection demonstrated similarities to infected wild type old mice with upregulation of SOCS3 and inhibition of Bcl2. Higher expression of SOCS3 with down regulation of Bcl2, resulted in higher macrophage death via cellular necrosis. Finally, peripheral blood monocytes (PBMCs) from elderly patients with P-NTM also demonstrated attenuated HO-1 responses after M. avium stimulation and increased cell death due to cellular necrosis (9.69% ± 2.02) compared to apoptosis (4.75 % ± 0.98). The augmented risk for P-NTM in the elderly is due in part, to attenuated HO-1 responses, subsequent upregulation of SOCS3 and inhibition of Bcl2, leading to programmed cell death of macrophages, and sustained infection.
The pulmonary endothelium is the target of continuous physiologic and pathologic stimuli that affect its crucial barrier function. The regulation, defense and repair of endothelial barrier function require complex biochemical processes. This review examines the role of endothelial phosphorylating enzymes, kinases, a class with profound, interdigitating influences on endothelial permeability and lung function.
Progressive lung disease with early onset is the main cause of mortality and morbidity in Cystic Fibrosis patients. Here we report a reduction of Sphingosine-1-Phosphate (S1P) in the lung of unchallenged Cftrtm1EUR F508del CFTR mutant mice. This correlates with enhanced infiltration by inducible nitric oxide synthase (iNOS) expressing granulocytes, B- and T-cells. Furthermore, the ratio of macrophage derived dendritic cells (MoDC) to conventional dendritic cells (cDC) is higher in mutant mouse lung, consistent with unprovoked inflammation. Oral application of a S1P-lyase inhibitor (LX2931), increases S1P levels in mutant mouse tissues. This normalizes the lung MoDC/cDC ratio, and reduces B- and T-cell counts. Lung granulocytes are enhanced, but iNOS expression is reduced in this population. Although lung LyC6+ monocytes are enhanced by LX2931, they apparently do not differentiate to MoDC and macrophages. After challenge with bacterial toxins (LPS-fMLP) we observe enhanced levels of pro-inflammatory cytokines TNF-α, KC, IFN and IL-12, and the inducible mucin MUC5AC in mutant mouse lung, evidence of deficient resolution of inflammation. LX2931 does not prevent transient inflammation or goblet cell hyperplasia after challenge, but it reduces MUC5AC and pro-inflammatory cytokine levels towards normal values. We conclude that lung pathology in homozygous mice expressing murine F508del CFTR, which represents the most frequent mutation in CF patients, is characterized by abnormal behavior of infiltrating myeloid cells and delayed resolution of induced inflammation. This phenotype can be partially corrected by a S1P lyase inhibitor, providing a rationale for therapeutic targeting of the S1P signaling pathway in CF patients.
Acute Respiratory Distress Syndrome (ARDS) remains a leading cause of morbidity and mortality in both adult and pediatric intensive care units. A key event in the development of ARDS is neutrophil recruitment into the lungs leading to tissue damage and destruction. Interleukin-8 (IL-8) is the major human chemokine responsible for neutrophil recruitment into the lungs. Protein phosphatase 2A (PP2A) has been shown to be a key regulator of the mitogen-activated protein kinase (MAPK) cascades, which control the production of IL-8. Previously, our lab employed an in vitro model to show that inhibition of PP2A results in an increase in IL-8 production in human alveolar epithelial cells. The objective of this study was to determine whether PP2A regulated this response in vivo by investigating the impact of pharmacologic activation of PP2A on chemokine production, activation of the MAPK cascade and lung injury using endotoxin- and bacterial-challenge models of ARDS in mice. N6-cyclopentyladenosine (N6-CPA) increased PP2A activity and inhibited endotoxin induced cytokine production in a murine alveolar macrophage cell line. N6-CPA pretreatment in mice challenged with intratracheal endotoxin decreased chemokine production, reduced neutrophil infiltration, and attenuated lung injury. Following initiation of lung injury with live Pseudomonas aeruginosa, mice that received N6-CPA four hours following bacterial challenge showed attenuated chemokine production and reduced neutrophil infiltration compared to control mice. Pharmacologic PP2A activation both limited and prevented inflammation and tissue injury in two direct injury models of ARDS. These results suggest modulation of PP2A activity as a therapeutic target in ARDS.
We previously showed that coincident exposure to heat shock (HS; 42°C for 2h) and TNFα synergistically induces apoptosis in mouse lung epithelium. We extended this work by analyzing HS effects on human lung epithelial responses to clinically relevant injury. Cotreatment with TNFα and HS induced little caspase-3 and PARP cleavage in human small airway epithelial cells (SAECs) and A549 and BEAS2B cells. Scratch wound closure rates almost doubled when A549 and BEAS2B cells and air-liquid interface cultures of human bronchial epithelial (NHBE) were heat-shocked immediately post-wounding. Microarray, qRT-PCR, and immunoblotting showed FGF1 to be synergistically induced by HS and wounding. Enhanced FGF1 expression in HS/wounded A549 was blocked by inhibitors of p38 MAPK (SB203580) or heat shock factor (HSF)-1 (KNK-437) and in HSF1-knockout BEAS2B cells. PCR demonstrated FGF1 to be expressed from the two most distal promoters in wounded/heat-shocked cells. Wound closure in heat-shocked A549 cells was reduced by FGF receptor-1/3 inhibition (SU-5402) or FGF1 depletion. Exogenous FGF1 accelerated A549 wound closure in the absence but not presence of HS. In the presence of exogenous FGF1, HS slowed wound closure, suggesting it increases FGF1 expression but impairs FGF1-stimulated wound closure. Frozen sections from normal and IPF lung were analyzed for FGF1 and HSP70 by immunofluorescence confocal microscopy and qRT-PCR. FGF1 and HSP70 mRNA levels were 7.5- and 5.9-fold higher in IPF than normal lung and the proteins co-localized to fibroblastic foci in IPF lung. We conclude that HS signaling may have an important impact on lung injury, healing, and fibrosis.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are diseases with high mortality. Macrophages and neutrophils are responsible for inflammatory responses in ALI and ARDS, which are characterized by excessive production of proinflammatory mediators in bronchoalveolar lavage fluid (BALF) and plasma. Aberrant activation of the JAK/STAT pathway is critical for persistent inflammation in many conditions such as infection and autoimmunity. Given the importance of the STAT3 transcription factor in activating macrophages and neutrophils, and augmenting inflammation, we investigated the therapeutic potential of inhibiting STAT3 activity using the small molecule STAT3 inhibitor, LLL12. Our results demonstrate that LPS induces STAT3 activation in macrophages in vitro and in CD45+CD11b+ cells from BALF in the LPS-induced ALI model in vivo. LLL12 treatment inhibits LPS-induced lung inflammation in the ALI model, which is accompanied by suppression of LPS-induced STAT3 activation, and an inhibition of macrophage and inflammatory cell infiltration in lung and BALF. LLL12 treatment also suppresses expression of proinflammatory genes including IL-1β, IL-6, TNF-α, iNOS, CCL2 and MHC Class II in macrophages and inflammatory cells from BALF and serum as determined by ELISA. Furthermore, hyper-activation of STAT3 in LysMCre-SOCS3fl/fl mice accelerates the severity of inflammation in the ALI model. Both pre and post-LPS administration treatment with LLL12 decreases LPS-induced inflammatory responses in mice with ALI. Importantly, LLL12 treatment attenuates STAT3 phosphorylation in human peripheral blood mononuclear cells induced by plasma from ARDS patients, which suggests the feasibility of targeting the STAT3 pathway therapeutically for ALI and ARDS patients.
Extracellular ATP and other nucleotides are important autocrine/paracrine mediators that regulate diverse processes critical for lung function, including mucocilliary clearance, surfactant secretion and local blood flow. Cellular ATP release is mechanosensitive, however, the impact of physical stimuli on ATP release during breathing has never been tested in intact lungs in real-time and remains elusive. In this pilot study, we investigated inflation-induced ATP release in rat lungs ex-vivo by real-time luciferin-luciferase (LL) bioluminescence imaging coupled with simultaneous infrared tissue imaging to identify ATP-releasing sites. With LL solution introduced into airspaces brief inflation of such edematous lung (1-s, ~20 cmH2O) induced transient (<30s) ATP release in a limited number of air-inflated alveolar sacs during their recruitment/opening. Released ATP reached concentrations of ~10-6 M, relevant for autocrine/paracrine signaling but it remained spatially restricted to single alveolar sacs or their clusters. ATP release was stimulus-dependent, prolonged (100-s) inflation evoked long-lasting ATP release which terminated upon alveoli deflation/de-recruitment while cyclic inflation/suction produced cyclic ATP release. With LL introduced into blood vessels, inflation induced transient ATP release in many small patch-like areas the size of alveolar sacs. Findings suggest that inflation induces ATP release in both alveoli and the surrounding blood capillary network; the functional units of ATP release presumably consist of alveolar sacs or their clusters. Our study demonstrates the feasibility of real-time ATP-release imaging in ex-vivo lungs and provides the first direct evidence of inflation-induced ATP release in lung airspaces and in pulmonary blood capillaries, highlighting the importance of purinergic signaling in lung function.
Recent work from this laboratory showed that endoplasmic reticulum (ER) stress-induced apoptosis of alveolar epithelial cells (AECs) is regulated by the autocrine angiotensin (ANG)II/ANG1-7 system. The proteasome inhibitor MG132 or surfactant protein C (SP-C) BRICHOS domain mutation G100S induced apoptosis in human AECs by activating the pro-apoptotic cathepsin D and reducing anti-apoptotic angiotensin converting enzyme-2 (ACE-2). This study tested the hypothesis that ER stress-induced apoptosis of human AECs might be mediated by influence of the unfolded protein response (UPR) on the autocrine ANGII/ANG1-7 system. A549 cells were challenged with MG132 or SP-C BRICHOS domain mutant G100S to induce ER stress and activation of UPR pathways. The results showed that either MG132 or G100S SP-C mutation activated all 3 canonical pathways of the UPR (IRE1/XBP1, ATF6, and PERK/eIF2α), which led to a significantly increase in cathepsin D or in TACE - an ACE-2 ectodomain shedding enzyme - and eventually caused AEC apoptosis. However, ER stress-induced AEC apoptosis could be prevented by chemical chaperone or by UPR blockers. It is also suggested that ATF6 and IRE1 pathways might play important role in regulation of angiotensin system. These data demonstrate that ER stress induces apoptosis in human AECs through mediation of UPR pathways, which in turn regulate the autocrine ANGII/ANG1-7 system. They also demonstrated that ER stress-induced AEC apoptosis can be blocked by inhibition of UPR signaling pathways.
Cigarette smoke (CS) exposure is a major risk factor for COPD. We investigated whether CS-induced DAMP release or DAMP-mediated inflammation contributes to susceptibility for COPD. Samples, including bronchial brushings were collected from young and old individuals, susceptible and non-susceptible for the development of COPD, before and after smoking, and used for gene profiling and airway epithelial cell (AEC) culture. AECs were exposed to CS extract (CSE) or specific DAMPs. BALB/cByJ and DBA/2J mice were intra-nasally exposed to LL-37 and mitochondrial (mt)DAMPs. Functional gene-set enrichment analysis showed that CS significantly increases the airway epithelial gene-expression of DAMPs and DAMP receptors in COPD patients. In cultured AECs, we observed that CSE induces necrosis and DAMP release, with specifically higher galectin-3 release from COPD-derived compared to control-derived cells. Galectin-3, LL-37 and mtDAMPs increased CXCL8 secretion in AECs. LL-37 and mtDAMPs induced neutrophilic airway inflammation, exclusively in mice susceptible for CS-induced airway inflammation. Collectively, we show that in airway epithelium from COPD patients, the CS-induced expression of DAMPs and DAMP receptors in vivo and the release of galectin-3 in vitro is exaggerated. Further, our studies indicate that a predisposition to release DAMPs and subsequent induction of inflammation may contribute to the development of COPD.
Human lung fibroblasts (HLFs) act as innate immune sentinel cells that amplify the inflammatory response to injurious stimuli. Here, we use targeted lipidomics to explore the hypothesis that HLFs also play an active role in the resolution of inflammation. We detected cyclooxygenase-2 (COX-2)-dependent production of both pro-inflammatory and pro-resolving prostaglandins (PGs) in conditioned culture medium from HLFs treated with a pro-inflammatory stimulus, IL-1β. Among the pro-resolving PGs in the HLF lipidome were several known ligands for peroxisome proliferator-activated receptor gamma (PPAR), a transcription factor whose activation in the lung yields potent anti-inflammatory, anti-fibrotic and pro-resolving effects. We next confirmed the ability of HLF supernatants to activate PPAR using a cell-based luciferase reporter, demonstrating for the first time that HLFs activated with pro-inflammatory IL-1β or cigarette smoke extract (CSE) produce functional PPAR ligands; this phenomenon is temporally-regulated, COX-2- and lipocalin-type PGD synthase-dependent, and enhanced by arachidonic acid supplementation. Finally, we used luciferase reporter assays to show that several of the PGs in the lipidome of activated HLFs independently activate PPAR and/or inhibit NFB. These results indicate that HLFs, as immune sentinels, regulate both pro-inflammatory and pro-resolving responses to injurious stimuli. This novel endogenous resolution pathway represents a new therapeutic target for globally important inflammatory diseases like chronic obstructive pulmonary disease.
Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 hrs) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.
The effect of mechanical forces and focal adhesion kinase (FAK) in regulating the inflammatory responses of airway smooth muscle (ASM) tissues to stimulation with interleukin (IL)-13 were investigated. Canine tracheal tissues were subjected to different mechanical loads in vitro and the effects of mechanical load on eotaxin secretion and inflammatory signaling pathways in response to IL-13 were determined. Eotaxin secretion by tissues in response to IL-13 was significantly inhibited in muscles maintained at a higher (+) load compared to those at a lower (-) load as assessed by ELISA, and Akt activation was also reduced in the higher (+) loaded tissues. Conversely the (+) mechanical load increased activation of the focal adhesion proteins FAK and paxillin in the tissues. The role of FAK in regulating the mechanosensitive responses was assessed by overexpressing FAK-related nonkinase (FRNK) in the tissues, by expressing the FAK kinase-dead mutant FAK Y397F, or by treating tissues with the FAK inhibitor PF-573228. FAK inactivation potentiated Akt activity and increased eotaxin secretion in response to IL-13. FAK-inhibition also suppressed the mechanosensitivity of Akt activation and eotaxin secretion. In addition, FAK inactivation suppressed SmMHC expression induced by the higher (+) mechanical load. The results demonstrate that the imposition of a higher mechanical load on airway smooth muscle stimulates FAK activation, which promotes the expression of the differentiated contractile phenotype and suppresses the synthetic phenotype and the inflammatory responses of the muscle tissue.
Abstract Background/Aims: Epoxyeicosatrienoic acids (EETs) are metabolic products of free arachidonic acid (AA). EETs have anti-inflammatory activities. However, the effect of EETs on cigarette smoke-induced lung inflammation is not clear. Autophagy is believed to be involved in the pathogenesis of COPD. In addition, nuclear erythroid-related factor 2 (Nrf2), is thought to regulate antioxidant defenses in several lung diseases. Besides, interaction between EETs, autophagy and Nrf2 has been reported. The aim of this study was to explore the effect of 14, 15-EET on cigarette smoke condensate (CSC)-induced inflammation in human bronchial epithelial cell line (Beas-2B), and to determine the underlying mechanisms. Methods: Autophagy and its signaling pathway proteins, LC3B, p62, PI3K, AKT, p-AKT, p-mTOR, anti-inflammatory proteins Nrf2, HO-1 were assessed on Western blot analysis. Autophagosomes and autolysosomes were detected by Ad- mRFP-GFP-LC3 transfection. Inflammatory factors (IL-6, IL-8 and MCP-1) were detected by ELISA. Lentiviral vectors carrying p62 shRNA were used to interfere with the expression of p62 to evaluate the effect of p62 on Nrf2 expression. Nrf2 expression was determined on immunocytochemistry. Results: 14, 15-EET significantly reduced the secretion IL-6, IL-8 and MCP-1, and increased Nrf2 and HO-1. In addition, 14, 15-EET inhibited CSC-induced autophagy in Beas-2B cells. The mechanism of anti-inflammatory effect of 14, 15-EET involved inhibition of autophagy and increase in p62 levels, followed by translocation of Nrf2 into the nucleus and upregulated the expression of HO-1. Conclusion: 14, 15-EET protects against CSC-induced lung inflammation by promoting the accumulation of Nrf2 via through inhibition of autophagy.
Patients with congenital diaphragmatic hernia (CDH) suffer from severe pulmonary hypertension due to altered development of the pulmonary vasculature, which is often resistant to vasodilator therapy. Current treatment starts postnatally even though significant differences in the pulmonary vasculature are already present early during pregnancy. We examined the effects of prenatal treatment with the phosphodiesterase-5 inhibitor sildenafil on pulmonary vascular development in experimental CDH starting at a clinical relevant time. The well-established, nitrofen induced CDH rodent model was treated daily with 100 mg/kg sildenafil from day 17.5 until day 20.5 of gestation (E17.5-20.5). Importantly, this timing perfectly corresponds to the developmental stage of the lung at 20 weeks of human gestation, when CDH is detectable by 2D-ultrasonography and/or MRI. At E21.5 pups were delivered by caesarean section and euthanized by lethal injection of pentobarbital. The lungs were isolated and subsequently analyzed using immunostaining, real-time PCR and volume measurements. Prenatal treatment with sildenafil improved lung morphology and attenuated vascular remodeling with reduced muscularization of the smaller vessels. Pulmonary vascular volume was not affected by sildenafil treatment. We show that prenatal treatment with sildenafil within a clinically relevant period improves pulmonary vascular development in an experimental CDH model. This may have important implications for the management of this disease and related pulmonary vascular diseases in human.
Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OxPAPC) attenuates agonist-induced endothelial cell (EC) permeability and increases pulmonary endothelial barrier function via enhancement of both the peripheral actin cytoskeleton and cell junctions mediated by Rac1 and Cdc42 GTPases. This study evaluated the role for the multifunctional Rac1/Cdc42 effector and regulator, IQGAP1, as a molecular transducer of the OxPAPC-mediated EC barrier enhancing signal. IQGAP1 knockdown in endothelial cells by gene-specific siRNA abolished OxPAPC-induced enlargement of VE-cadherin-positive adherens junctions, suppressed peripheral accumulation of actin polymerization regulators, namely cortactin, N-WASP and Arp3, and attenuated remodeling of the peripheral actin cytoskeleton. Inhibition of OxPAPC-induced barrier enhancement by IQGAP1 knockdown was due to suppressed Rac1 and Cdc42 activation. Expression of an IQGAP1 truncated mutant showed that the GTPase regulatory domain (GRD) of IQGAP1 was essential for the OxPAPC-induced membrane localization of cortactin, adherens junction proteins VE-cadherin and p120-catenin as well as for EC permeability response. IQGAP1knockdown attenuated the protective effect of OxPAPC against thrombin-induced cell contraction, cell junction disruption and EC permeability. These results demonstrate for the first time the role of IQGAP1 as a critical transducer of OxPAPC-induced Rac1/Cdc42 signaling to the actin cytoskeleton and adherens junctions which promotes cortical cytoskeletal remodeling and EC barrier protective effects of oxidized phospholipids.
In this study, we investigated the effects of suplatast on acutely dissociated single neurons of sensory and paratracheal ganglia using a patch-clamp technique. Suplatast had little effect on various responses caused by capsaicin, acid, bradykinin, serotonin and adenosine 5'-triphosphate in rat sensory neurons. Suplatast, even at 10-3 M, also did not induce any current at various membrane potentials in rat and guinea pig paratracheal ganglia neurons. Further, acetylcholine- and bradykinin-induced depolarizations were not affected by suplatast. On the other hand, in rat paratracheal ganglia neurons, 10-5 M nicotine-induced current were inhibited by suplatast in a concentration-dependent manner with a 50% inhibitory concentration of 9.86x10-5 M. The effect was noncompetitive and voltage-dependent. Furthermore, the effect was use-independent and not affected by the pretreatment time of suplatast. The results suggested that suplatast may inhibit neurotransmission at the paratracheal ganglia via the inhibition of nicotinic current. Thus, suplatast may attenuate cough production through the improvement of pathological conditions of the lower airway via suppressed acetylcholine release from the postganglionic nerve terminal.
Angiopoietin-like protein 2 (ANGPTL2) is a chronic inflammatory mediator, which when deregulated is associated with various pathologies. However, little is known about its activity in lung. To assess a possible lung function, we generated a rabbit monoclonal antibody that specifically recognizes mouse ANGPTL2 and then evaluated protein expression in mouse lung tissue. We observed abundant ANGPTL2 expression in both alveolar epithelial type I and type II cells and in resident alveolar macrophages under normal conditions. To assess ANGPTL2 function, we compared lung phenotypes in Angptl2 knockout (KO) and wild-type mice but observed no overt changes. We then generated a bleomycin-induced interstitial pneumonia model using wild-type and Angptl2 KO mice. Bleomycin-treated wild-type mice showed specifically upregulated ANGPTL2 expression in areas of severe fibrosing interstitial pneumonia, while Angptl2 KO mice developed more severe lung fibrosis than did comparably treated wild-type mice. Lung fibrosis seen following bone marrow transplant (BMT) was comparable in wild-type or Angptl2 KO mice treated with bleomycin, suggesting that Angptl2 loss in myeloid cells does not underlie fibrotic phenotypes. We conclude that Angptl2 deficiency in lung epithelial cells and resident alveolar macrophages causes severe lung fibrosis seen following bleomycin treatment, suggesting that ANGPTL2 derived from these cell types plays a protective role against fibrosis in lung.
Calcium signaling through store operated channels (SOC) is involved in hypoxic pulmonary hypertension. We determined whether a treatment with 2-aminoethyldiphenylborinate (2-APB), a compound with SOC blocker activity, reduces pulmonary hypertension and vascular remodeling. Twelve newborn lambs exposed to perinatal chronic hypoxia were studied, 6 of them received a 2-APB treatment and the other 6 received vehicle treatment, for 10 days in both cases. Throughout this period, we recorded cardiopulmonary variables and on day 11 we evaluated the response to an acute hypoxic challenge. Additionally, we assessed the vasoconstrictor and vasodilator function in isolated pulmonary arteries as well as their remodeling in lung slices. 2-APB reduced pulmonary arterial pressure at the third and tenth days, cardiac output between the fourth and eighth days, and pulmonary vascular resistance at the tenth day of treatment. The pulmonary vasoconstrictor response to acute hypoxia was reduced by the end of treatment. 2-APB also decreased maximal vasoconstrictor response to the thromboxane mimetic U46619 and endothelin-1 and increased maximal relaxation to 8-Br-cGMP. The maximal relaxation and potency to phosphodiesterase-5 and Rho-kinase inhibition with sildenafil and fasudil respectively, were also increased. Finally, 2-APB reduced the medial and adventitial layers' thickness, the expression of α-actin and the percentage of Ki67+ nuclei of small pulmonary arteries. Taken together, our results indicate that 2-APB reduces pulmonary hypertension, vasoconstrictor responses and pathological remodeling in pulmonary hypertensive lambs. We conclude that SOC targeting may be a useful strategy for the treatment of neonatal pulmonary hypertension, however, further testing of specific blockers is needed.
We previously showed that newborn piglets who develop pulmonary hypertension during exposure to chronic hypoxia have diminished pulmonary vascular NO production and evidence of eNOS uncoupling. Tetrahydrobiopterin (BH4) is a co-factor that promotes eNOS coupling. Current clinical strategies typically invoke initiating treatment after the diagnosis of pulmonary hypertension, rather than prophylactically. The major purpose of this study was to determine whether starting treatment with an oral BH4 compound, Sapropterin Dihydrochloride (Sapropterin), after the onset of pulmonary hypertension would re-couple eNOS in the pulmonary vasculature and ameliorate disease progression in chronically hypoxic piglets. Normoxic (control) and hypoxic piglets were studied. Some hypoxic piglets received oral Sapropterin starting on day 3 of hypoxia and continued throughout an additional 7 days hypoxic exposure. Catheters were placed for hemodynamic measurements and pulmonary arteries were dissected to assess eNOS dimer-to-monomer ratios (a measure of eNOS coupling), NO production, and superoxide (O2•-) generation. Although higher than in normoxic controls, pulmonary vascular resistance was lower in Sapropterin-treated hypoxic piglets than in untreated hypoxic piglets. Consistent with eNOS re-coupling, eNOS dimer-to-monomer ratios and NO production were greater and O2•- generation was less in pulmonary arteries from Sapropterin-treated than untreated hypoxic animals. When started after disease onset, oral Sapropterin treatment inhibits chronic hypoxia-induced pulmonary hypertension at least in part by recoupling eNOS in the pulmonary vasculature of newborn piglets. Rescue treatment with Sapropterin may be an effective strategy to inhibit further development of pulmonary hypertension in newborn infants suffering from chronic cardiopulmonary conditions associated with episodes of prolonged hypoxia.
This study evaluated the pulmonary pathophysiology of the transgenic CFTR "gut-corrected" cystic fibrosis (CF) pigs. Four sows produced 18 piglets of which 11 were stillborn with only 2 animals surviving beyond 2 weeks. Failure to survive beyond the neonatal period by 5 piglets was judged to result from metabolic dysfunction related to genetic manipulation for CFTR gut expression or due to cloning artifact. Plasma analysis showed very low plasma proteins, highly elevated liver enzymes, and severe acidosis. All surviving offspring received furosemide for systemic edema. Physiologic evaluation was performed with lung tissues from the two surviving pigs. Both acetylcholine and forskolin induced mucous liquid secretion that was significantly lower in CF bronchi than non-CF bronchi. The percent non-volatile solids in mucus secreted from CF bronchi was elevated following acetylcholine or forskolin. Mucociliary transport in excised tracheas was reduced in the CF tracheas relative to nonCF tracheas. The diameter of CF tracheas was less than that of non-CF pigs in spite of their greater body weight. Despite exhibiting severe metabolic dysfunction during the neonatal period, this CF animal model appears to express important characteristics of human CF pulmonary disease.
Mechanical ventilation (MV) and oxygen therapy (hyperoxia; HO) comprise the cornerstones of life-saving interventions for patients with Acute Respiratory Distress Syndrome (ARDS). Unfortunately, the side effects of MV and HO include exacerbation of lung injury by barotrauma, volutrauma and propagation of lung inflammation. Despite significant improvements in ventilator technologies and a heightened awareness of oxygen toxicity, besides low tidal volume ventilation few if any medical interventions have improved ARDS outcomes over the past two decades. We are lacking a comprehensive understanding of mechano-transduction processes in the healthy lung and know little about the interactions between simultaneously activated stretch-, HO- and cytokine-induced signaling cascades in ARDS. Nevertheless, as we are unraveling these mechanisms we are gathering increasing evidence for the importance of stretch-activated ion channels (SACs) in the activation of lung resident and inflammatory cells. In addition to the discovery of new SAC families in the lung, e.g. 2-pore domain potassium (K2P) channels, we are increasingly assigning mechano-sensing properties to already known Na+, Ca2+, K+, and Cl- channels. Better insights into the mechano-transduction mechanisms of SACs will improve our understanding of the pathways leading to ventilator-induced lung injury and lead to much needed novel therapeutic approaches against ARDS by specifically targeting SACs. This review (1) summarizes the reasons why the time has come to seriously consider SACs as new therapeutic targets against ARDS, (2) critically analyzes the physiological and experimental factors that currently limit our knowledge about SACs, and (3) outlines the most important questions future research studies need to address.
Chronic obstructive pulmonary disease (COPD) in the U.S. is primarily caused by cigarette smoking. COPD patients are highly susceptible to respiratory infections in part due to alveolar macrophage dysfunction despite a substantial increase in macrophages in the lung. Cadmium (Cd) is a toxic metal that is concentrated within tobacco and accumulates in the lung of smokers. We hypothesized that Cd uptake into macrophages alters immune function thereby impairing the macrophage response to invading pathogens. Our hypothesis was tested by comparing primary human monocytes and macrophages and related cell lines. Strikingly, Cd exposure followed by LPS stimulation resulted in a dose-dependent, significant decrease in nuclear p65 activity in macrophages which was not observed in monocytes. This corresponded with Cd-mediated inhibition of IKKβ in TDMs and an impaired ability to transcribe and release cytokines in response to LPS challenge. These findings provide novel evidence that Cd has the capacity to disrupt macrophage immune function in comparison to monocytes. Importantly, Cd results in immune dysfunction in macrophages through inhibition of the NF-B signaling pathway. Based on these findings, we provide new evidence that Cd contributes to immune dysfunction in the lung of COPD subjects and may increase susceptibility to infection.
Vitamin D deficiency is associated with asthma risk. Vitamin D deficiency may enhance the inflammatory response and we have previously shown that airway remodelling and airway hyperresponsiveness (AHR) is increased in vitamin D-deficient mice. In this study, we hypothesize that vitamin D deficiency would exacerbate house dust mite (HDM)-induced inflammation and alterations in lung structure and function. A BALB/c mouse model of vitamin D deficiency was established by dietary manipulation. Responsiveness to methacholine, airway smooth muscle (ASM) mass, mucus cell metaplasia, lung and airway inflammation and cytokines in bronchoalveolar lavage (BAL) fluid were assessed. Gene expression patterns in mouse lung samples were profiled by RNA-Seq. HDM exposure increased inflammation and inflammatory cytokines in BAL, baseline airway resistance, tissue elastance and ASM mass. Vitamin D deficiency enhanced the HDM-induced influx of lymphocytes into BAL, ameliorated the HDM induced increase in ASM mass and protected against the HDM-induced increase in baseline airway resistance. RNA-Seq identified nine genes that were differentially regulated by vitamin D deficiency in the lungs of HDM-treated mice. Immunohistochemical staining confirmed that protein expression of MID1 and ADM were differentially regulated such that they promoted inflammation, while and HILDPA which is associated with ASM remodelling, was downregulated. Protein expression studies in human bronchial epithelial cells also showed that addition of vitamin D decreased MID1 expression. Differential regulation of these genes by vitamin D deficiency could determine lung inflammation and pathophysiology and suggest that the effect of vitamin D deficiency on HDM-induced allergic airways disease is complex.
Membrane potential (Vm)-, Na+- or Ca2+-sensitive fluorescent dyes were used to analyze changes in Vm or intracellular ion concentrations in airway epithelial cells treated with S. aureus alpha-toxin (Hla), a major virulence factor of pathogenic strains of these bacteria. Gramicidin, a channel forming peptide causing membrane permeability to monovalent cations, a mutated form of Hla, rHla-H35L, which forms oligomers in the plasma membranes of eukaryotic cells but fails to form functional transmembrane pores, or the cyclodextrin-derivative IB201, a blocker of the Hla-pore, were used to investigate the permeability of the pore. Na+- as well as Ca2+-ions were able to pass the Hla-pore and accumulated in the cytosol. The pore-mediated influx of calcium ions was blocked by IB201. Treatment of cells with recombinant Hla resulted in plasma membrane depolarization as well as in increases in the phosphorylation levels of paxillin (signaling pathway mediating disruption of the actin cytoskeleton) and p38 MAP kinase (signaling pathway resulting in defensive actions). p38 MAP kinase phosphorylation, but not paxillin phosphorylation, was elicited by treatment of cells with gramicidin. Although treatment of cells with rHla-H35L resulted in the formation of membrane-associated heptamers, none of these cellular effects were observed in our experiments. This indicates that formation of functional Hla-transmembrane pores is required to induce the cell physiological changes mediated by alpha-toxin. Specifically, the changes in ion equilibria and plasma membrane potential are important activators of p38 MAP kinase, a signal transduction module involved in host cell defense.
Impaired airway homeostasis in COPD could be partly related to Club Cell Secretory Protein (CCSP) deficiency. We hypothesize that CCSP G38A polymorphism is involved and aim to examine the influence of the CCSP G38A polymorphism on CCSP transcription levels and its regulatory mechanisms. CCSP genotype and CCSP levels in serum and sputum were assessed in 66 subjects with stable COPD included in a one-year observational study. 49 of them had an exacerbation. In an in vitro study, the impact on the CCSP promoter of 38G wild type or 38A variant was assessed. BEAS-2B cells were transfected by either the 38G or 38A construct, in the presence/absence of cigarette smoke extract (CSE) or lipopolysaccharides (LPS). Cotransfections with modulating transcription factors, p53 and Nkx2.1, identified by in silico analysis using ConSite and TFSEARCH were conducted. A allele carriers COPD patients had lower serum and sputum CCSP levels, especially among active smokers, and a decreased BODE score. In vitro, baseline CCSP transcription levels were similar between the wild and variant constructs. CSE decreased more profoundly the CCSP transcription level of 38A transfected cells. The opposite effect was observed with p53 cotransfection. LPS stimulation induced CCSP repression in 38A promoter transfected cells. Cotransfection with Nkx2.1 significantly activated the CCSP promoters irrespective of the polymorphism. Circulating CCSP levels are associated with smoking and the CCSP G38A polymorphism. CSE, LPS and the Nkx2.1 and p53 transcription factors modulated the CCSP promoter efficiency. The 38A polymorphism exaggerated the CCSP repression in response to p53 and CSE.
Cystic fibrosis (CF) is an inherited disease associated with chronic, severe lung inflammation, leading to premature death. To develop innovative anti-inflammatory treatments, we need to characterize new cellular and molecular components contributing to the mechanisms of lung inflammation. Here, we focused on the potential role of "transient receptor potential vanilloid-4" (TRPV4), a non-selective, calcium channel. We first showed that 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids, i.e. four natural lipid-based TRPV4 agonists, are present in expectorations of CF patients. We also used both in vitro and in vivo approaches to demonstrate that TRPV4 expressed in airway epithelial cells triggers the secretion of major pro-inflammatory mediators such as chemokines and biologically active lipids, as well as a neutrophil recruitment into lung tissues. We further characterized the contribution of cytosolic phospholipase A2, MAPKs and NF-B in TRPV4-dependent signaling. Finally, we observed an alteration of TRPV4-induced inflammatory response in a CF context, suggesting that TRPV4 is a promising target for the development of new anti-inflammatory treatments for diseases such as CF.
Post-translational modifications add diversity to protein function. Throughout its life cycle, the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) undergoes numerous covalent post-translational modifications (PTMs), including glycosylation, ubiquitination, sumoylation, phosphorylation, and palmitoylation. These modifications regulate key steps during protein biogenesis, such as protein folding, trafficking, stability, function, and association with protein partners, and therefore may serve as targets for therapeutic manipulation. More generally, an improved understanding of molecular mechanisms that underlie CFTR PTMs may suggest novel treatment strategies for CF and perhaps other protein conformational diseases. This review provides a comprehensive summary of co- and post-translational CFTR modifications and their significance with regard to protein biogenesis.
Dendritic cells and CD8+ T cells participate in the pathology of chronic obstructive pulmonary disease, including emphysema, but little is known of the involvement of the CD40/CD40L pathway. We investigated the role of the CD40/CD40L pathway in Tc1 cell differentiation induced by dendritic cells in a mouse model of emphysema, and in vitro. C57BL/6J wildtype and CD40-/- mice were exposed to cigarette smoke (CS) or not (control), for 24 weeks. In vitro experiments involved wildtype and CD40-/- dendritic cells treated with CS extract (CSE) or not. Compared with the control groups, the CS mice (both wildtype and CD40-/-) had a greater percentage of lung dendritic cells and higher levels of major histocompatability complex (MHC) class I molecules and costimulatory molecules CD40 and CD80. Relative to the CS CD40-/- mice, the CS wildtype showed greater signs of lung damage and Tc1 cell differentiation. In vitro, the CSE-treated wildtype cells evidenced more cytokine release (IL-12/p70) and Tc1 cell differentiation than did the CSE-treated CD40-/- cells. Exposure to cigarette smoke increases the percentage of lung dendritic cells and promotes Tc1 cell differentiation via the CD40/CD40L pathway. Blocking the CD40/CD40L pathway may suppress development of emphysema in mice exposed to cigarette smoke.
Epidemiological evidence demonstrates a strong link between postnatal cigarette smoke (CS)-exposure and increased respiratory morbidity in young children. However, how CS induces early onset airways disease in young children and how it interacts with endogenous risk factors remains poorly understood. We, therefore exposed 10 day old neonatal wild-type and βENaC-transgenic mice with cystic fibrosis like lung disease to CS for 4 days. Neonatal wild-type mice exposed to CS demonstrated increased numbers of macrophages and neutrophils in the BALF which was accompanied by increased levels of Mmp12 and Cxcl1. BALF from βENaC-transgenic mice contained greater numbers of macrophages which did not increase following acute CS-exposure, however there was significant increase in airway neutrophilia compared to filtered air transgenic and CS-exposed wild-type controls. Interestingly, wild-type and βENaC-transgenic mice demonstrated epithelial airway and vascular remodeling following CS-exposure. Morphometric analysis of lung sections revealed that CS-exposure caused increased mucus accumulation in the airway lumen of neonatal βENaC-transgenic mice compared to wild-type controls, which was accompanied by an increase in the number of goblet cells and Muc5ac upregulation. We conclude that short-term CS exposure i) induces acute airways disease with airway epithelial and vascular remodeling in neonatal wild-type mice; and ii) exacerbates airway inflammation, mucus hypersecretion and mucus plugging in neonatal βENaC-transgenic mice with chronic lung disease. Our results in neonatal mice suggest that young children may be highly susceptible to develop airways disease in response to tobacco smoke exposure and that adverse effects may be aggravated in children with underlying chronic lung diseases.
CCR2-expressing leukocytes are required for the progression of fibrosis in models of induced lung injury as well as models of bone marrow transplant (BMT)-related idiopathic pneumonia syndrome (IPS). Infection with murid -herpesvirus-68 (HV-68) results in severe pneumonitis and pulmonary fibrosis following syngeneic BMT; however, the roles that various pro-inflammatory leukocyte populations play in this process remain unclear. Deletion of CCR2 in both non-BMT and BMT mice increased early lytic viral replication and resulted in a reduction in the numbers of lung infiltrating GR1+ / F4/80+, and CXCR1+ cells, while maintaining robust neutrophil infiltration. Similarly, in HV-68 infected CCR2-/- BMT mice, recruitment of monocytes and lymphocytes were reduced whereas neutrophil recruitment was increased compared to WT BMT mice. Interestingly, levels of pro-fibrotic IL-17 were increased in infected CCR2 BMT mice in comparison to WT BMT. Further, an increase in lung-associated collagen was detected even though there was an overall decrease in the number of pro-fibrotic CCR2+ fibrocytes detected in the lungs of CCR2-/- BMT mice. These data indicate that, contrary to most models of fibrosis, deletion of CCR2 offers no protection from -herpesvirus-induced pneumonitis and fibrosis, and indeed, CCR2+ cells play a suppressive role during the development of pulmonary fibrosis following -herpesvirus infection post-BMT by limiting IL-17, and collagen production.
Here, we tested the hypothesis that animals with severe pulmonary arterial hypertension (PAH) display increased sensitivity to vascular permeability induced by activation of store operated calcium entry. To test this hypothesis, wild type and TRPC4 knockout Fischer 344 rats were given a single injection of Semaxanib (SU5416; 20 mg/kg) followed by three weeks of exposure to hypoxia (10% oxygen) and a return to normoxia (21% oxygen) for an additional two to three weeks. This Semaxanib/hypoxia/normoxia (i.e. SU5416/hypoxia/normoxia) treatment caused PAH, as evidenced by development of right ventricular hypertrophy, pulmonary artery medial hypertrophy, and occlusive lesions within precapillary arterioles. Pulmonary artery pressure was increased five-fold in Semaxanib/hypoxia/normoxia-treated animals compared to untreated, Semaxanib-treated, and hypoxia treated controls, determined by isolated perfused lung studies. Thapsigargin induced a dose-dependent increase in permeability that was dependent upon TRPC4 in the normotensive perfused lung. This increase in permeability was accentuated in PAH, but not in Semaxanib- or hypoxia-treated, lungs. Fluid accumulated in large perivascular cuffs, and although alveolar fluid accumulation was not seen in histological sections, Evan's blue dye conjugated to albumin was present in bronchoalveolar lavage fluid of hypertensive, but not normotensive, lungs. Thus, PAH is accompanied by a TRPC4-dependent increase in the sensitivity to edemagenic agents that activate store operated calcium entry.
Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is over-expressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated using stably-transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated using Ingenuity Pathway Analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced carcinogenesis.
Airway smooth muscle (ASM) remodeling is a key feature in asthma and includes changes in smooth muscle-specific gene and protein expression. Despite this being a major contributor to asthma pathobiology, our understanding of the mechanisms governing ASM remodeling remains poor. Here, we studied the functional interaction between WNT-11 and TGF-β1 in ASM cells. We demonstrate that WNT-11 is preferentially expressed in contractile myocytes and is strongly upregulated following TGF-β1-induced myocyte maturation. Knock-down of WNT-11 attenuated TGF-β1-induced smooth muscle (sm)-α-actin expression in ASM cells. We demonstrate that TGF-β1-induced sm-α-actin expression is mediated by WNT-11 via RhoA activation and subsequent actin cytoskeletal remodeling, as pharmacological inhibition of either Rho kinase by Y27632 or actin remodeling by latrunculin A attenuated sm-α-actin induction. Moreover, we show that TGF-β1 regulates the nuclear expression of myocardin-related transcription factor-A (MRTF-A) in a Rho kinase-dependent fashion, which in turn, mediates sm-α-actin expression. Finally, we demonstrate that TGF-β1-induced MRTF-A nuclear translocation is dependent on endogenous WNT-11. The present study, thus, demonstrates a WNT-11-dependent Rho kinase-actin-MRTF-A signaling axis that regulates the expression of sm-α-actin in ASM cells.
Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809, and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients.
Cough-related sensory inputs from rapidly-adapting receptors (RARs) and C-fibres are processed by second-order neurons mainly located in the caudal nucleus tractus solitarii (NTS). Both GABAA and glycine receptors have been proven to be involved in the inhibitory control of second-order cells receiving RAR projections. We investigated the role of these receptors within the caudal NTS in the modulation of the cough reflex induced either by mechanical or chemical stimulation of the tracheobronchial tree in pentobarbital sodium-anesthetized, spontaneously breathing rabbits. Bilateral microinjections (30-50 nl) of the receptor antagonists bicuculline and strychnine as well as of the receptor agonists muscimol and glycine were performed. Bicuculline (0.1 mM) and strychnine (1 mM) caused decreases in peak abdominal activity and marked increases in respiratory frequency due to decreases in both inspiratory time (TI) and expiratory time (TE), without concomitant changes in arterial blood pressure. Noticeably, these microinjections induced potentiation of the cough reflex consisting of increases in the cough number associated with decreases either in cough-related TI after bicuculline or in both cough-related TI and TE after strychnine. The effects caused by muscimol (0.1 mM) and glycine (10 mM) were in the opposite direction to those produced by the corresponding antagonists. The results show that both GABAA and glycine receptors within the caudal NTS mediate a potent inhibitory modulation of the pattern of breathing and cough reflex responses. They strongly suggest that disinhibition is one important mechanism underlying cough regulation and possibly provide new hints for novel effective antitussive strategies.
Objective: Micro-RNA (miR)-155 is a novel gene regulator with important roles in inflammation. Herein, our study was aimed at exploring the role of miR-155 in lipopolysaccharide (LPS)-induced acute lung injury(ALI). Measurements and Results: ALI in mice was induced by intratracheally delivered LPS. Loss-of-function experiments performed on miR-155 knockout mice showed that miR-155 gene inactivation protected mice from LPS-induced ALI as manifested preserved lung permeability and reduced lung inflammation compared with wild-type controls. Bone marrow transplantation experiments identified leukocytes, but not lung parenchymal derived miR-155 promoted acute lung inflammation. Real-time PCR analysis showed that the expression of miR-155 in lung tissue was greatly elevated in wild-type mice after LPS stimulation. In situ hybridization showed that miR-155 was mainly expressed in alveolar macrophages. In vitro experiments performed in isolated alveolar macrophages and polarized bone marrow derived macrophages confirmed that miR-155 expression in macrophages was increased in response to LPS stimulation. Conversely, miR-155 gain-of-function in alveolar macrophages remarkably exaggerated LPS-induced acute lung injury. Molecular studies identified the inflammation repressor SOCS-1 as the downstream target of miR-155. By binding to the 3'-UTR of the SOCS-1 mRNA, miR-155 downregulated SOCS-1 expression thus permitting the inflammatory response during lung injury. Finally, we generated a novel miR-155 knockout rat strain and showed that the pro-inflammatory role of miR-155 was conserved in rats. Conclusion Our study identified miR-155 is a pro-inflammatory factor after LPS stimulation, and AM-derived miR-155 has an important role in LPS-induced ALI.
Phospholipase C epsilon (PLC) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. While the role of PLC in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-/- mice to address the role of PLC in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-/- mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNFα, IL-1β, IL-6, MIP2, KC, MCP-1, and GM-CSF), lung PMN infiltration and microvascular leakage and loss of VE-cadherin compared to PLC+/+ mice. These data identify PLC as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC activity in endothelial cells (EC) could contribute in ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC inhibited NF-B activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNFα as well as the non-receptor agonist PMA in EC. Depletion of PLC also inhibited thrombin-induced expression of NF-B target gene, VCAM-1. Importantly, PLC knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC as a novel regulator of EC inflammation and permeability, and show a hitherto unknown role of PLC in the pathogenesis of ALI.
Airway smooth muscle is a major target tissue for glucocorticoid-based asthma therapies, however, molecular mechanisms through which the glucocorticoid receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the NF-kB inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-kB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-kB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-kB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied siRNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-kB contributes to cytokine repression in HASM.
Lung fibrosis negatively impacts on lung function in chronic asthma and is linked to the development of pro-fibrotic macrophage phenotypes. Epidemiological studies have found that lung function benefits from increased consumption of fruit high in polyphenols. We investigated the effect of Boysenberry consumption, in both therapeutic and prophylactic treatment strategies in a mouse model of chronic antigen-induced airways inflammation. Boysenberry consumption reduced collagen deposition and ameliorated tissue remodeling alongside an increase in the presence of CD68+CD206+arginase+ alternatively activated macrophages in the lung tissue. The decrease in tissue remodeling was associated with increased expression of pro-fibrolytic matrix metalloproteinase-9 protein in total lung tissue. We identified alternatively activated macrophages in the mice that consumed Boysenberry as a source of the matrix metalloproteinase-9. Oral Boysenberry treatment may moderate chronic tissue remodeling by supporting the development of pro-fibrolytic alternatively activated macrophages expressing matrix metalloproteinase-9. Regular Boysenberry consumption therefore has the potential to moderate chronic lung remodeling and fibrosis in asthma and other chronic pulmonary diseases.
We have shown that N-methyl-D-aspartate receptors (NMDA-Rs) are receptor-operated calcium entry channels in human airway smooth muscle (HASM) during contraction. Tumor necrosis factor (TNF) augments smooth muscle contractility by influencing pathways that regulate intracellular calcium flux, and can alter NMDA-R expression and activity in cortical neurons and glial cells. We hypothesized that NMDA-R-mediated Ca2+ and contractile responses of ASM can be altered by inflammatory mediators, including TNF. In cultured HASM cells we assessed TNF (10ng/mL, 48h) effect on NMDA-R subunit abundance by quantitative PCR (qPCR), confocal imaging and immunoblotting. We observed dose- and time-dependent changes in NMDA-R composition: increased obligatory NR1 subunit expression, and altered regulatory NR2 and inhibitory NR3 subunits. Measuring intracellular Ca2+ flux in Fura-2 loaded HASM cultures, we observed that TNF exposure enhanced cytosolic Ca2+ mobilization and changed the temporal pattern of Ca2+ flux in individual myocytes induced by NMDA, an NMDA-R selective analog of glutamate. We measured airway responses to NMDA in murine thin cut lung slices (TCLS) from allergen-naïve animals and observed significant airway contraction. However, NMDA acted as a bronchodilator in TCLS from HDM-challenged mice and in allergen-naïve TCLS subjected to TNF exposure. All contractile or bronchodilator responses were blocked by selective NMDA-R antagonist, D-AP5, and bronchodilator responses were prevented by L-NAME (NOS inhibitor) or indomethacin (COX inhibitor). Collectively, we show that TNF augments NMDA-R mediated Ca2+ mobilization in HASM cells,while in multicellular TCLSs, allergic inflammation and TNF exposure leads to NMDA-R mediated bronchodilation. These findings reveal unique contribution of ionotrophic NMDA-R to airway hyperreactivity.
Male sex is considered an independent predictor for the development of bronchopulmonary dysplasia (BPD) after adjusting for other confounders. BPD is characterized by an arrest in lung development with marked impairment of alveolar septation and vascular development. The reasons underlying sexually dimorphic outcomes in premature neonates are not known. In this investigation, we tested the hypothesis that male neonatal mice will be more susceptible to hyperoxic lung injury and will display larger arrest in lung alveolarization. Neonatal male and female mice (C57BL/6) were exposed to hyperoxia (95% FiO2, PND 1-5) and sacrificed on postnatal day (PND) 7 and 21. Extent of alveolarization, pulmonary vascularization, inflammation and modulation of the NF-B pathway were determined and compared to room air controls. Macrophage and neutrophil infiltration was significantly increased in hyperoxia-exposed animals but was increased to a larger extent in males compared to females. Lung morphometry showed a higher mean linear intercept (MLI) and a lower radial alveolar count (RAC) and therefore greater arrest in lung development in male mice. This was accompanied by a significant decrease in the expression of markers of angiogenesis (PECAM1 and VEGFR2) in males after hyperoxia exposure compared to females. Interestingly, female mice showed increased activation of the NF-B pathway in the lungs compared to males. These results support the hypothesis that sex plays a crucial role in hyperoxia-mediated lung injury in this model. Elucidation of the sex-specific molecular mechanisms may aid in the development of novel individualized therapies to prevent/treat BPD.
Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia (CAP). Granzyme A (GzmA) is a serine protease produced by a variety of cell types involved in the immune response. We sought to determine the role of GzmA on the host response during pneumococcal pneumonia. GzmA was measured in bronchoalveolar lavage fluid (BALF) harvested from CAP patients from the infected and contralateral uninfected side, and in lung tissue slides from CAP patients and controls. In CAP patients, GzmA levels were increased in BALF obtained from the infected lung. Human lungs showed constitutive GzmA expression by both parenchymal and non-parenchymal cells. In an experimental setting pneumonia was induced in wild type (WT) and GzmA deficient (GzmA–/–) mice by intranasal inoculation of S. pneumoniae. In separate experiments WT and GzmA–/– mice were treated with natural killer (NK) cell depleting antibodies. Upon infection with S. pneumoniae, GzmA–/– mice showed a better survival and lower bacterial counts in BALF and distant body sites compared to WT mice. Although NK cells showed strong GzmA expression, NK cell depletion did not influence bacterial loads in either WT or GzmA–/– mice. These results implicate that GzmA plays an unfavorable role in host defense during pneumococcal pneumonia by a mechanism that does not depend on NK cells.
The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility (MHC) class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared to standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8+ T cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools such as site-specific inhibitors of the immunoproteasome as well as activity-based probes, however, hold promises as novel therapeutic drugs for respiratory diseases and biomarker profiling, respectively.
The incidence of empyema (EMP) is increasing worldwide, generally occurs with pleural loculation and impaired drainage is often treated with intrapleural fibrinolytic therapy (IPFT) or surgery. A number of IPFT options are used clinically with empiric dosing and variable outcomes in adults. To evaluate the mechanisms governing intrapleural fibrinolysis and disease outcomes, rabbit models of Pasteurella multocida and Streptococcus pneumoniae-induced EMP were generated. The animals were treated with either human tissue (tPA) plasminogen activator or prourokinase (scuPA). Rabbit EMP was characterized by the development of pleural adhesions detectable by chest ultrasonography and fibrinous coating of the pleura. Similar to human EMP, rabbits with EMP accumulated sizable; 20-40 ml fibrinopurulent pleural effusions associated with extensive intrapleural organization, significantly increased pleural thickness, suppression of fibrinolytic and plasminogen activating activities and accumulation of high levels of plasminogen activator inhibitor 1, plasminogen and extracellular DNA. IPFT with tPA (0.145 mg/kg) or scuPA (0.5 mg/kg) was ineffective in rabbit EMP (n=9 and 3 for P. multocida and S. pneumoniae, respectively). 2 mg/kg tPA or scuPA IPFT (n=5) effectively cleared S. pneumoniae-induced EMP collections in 24h with no bleeding observed. While intrapleural fibrinolytic activity for up to 40 min after IPFT was similar for effective and ineffective doses of fibrinolysin, it was lower for tPA compared to scuPA treatments. These results demonstrate the similarities between rabbit and human EMP, the importance of pleural fluid PAI-1 activity and levels of plasminogen in the regulation of intrapleural fibrinolysis and illustrate the dose dependency of IPFT outcomes in EMP.
Background: Mechanical ventilation of preterm lambs causes lung inflammation and injury to the airway epithelium, which is repaired by 15 days after ventilation. In mice, activated basal cells (p63+ KRT14+ KRT8+) initiate injury repair to the trachea, whereas club cells coordinate distal airway repair. In both human and sheep, basal cells line the pseudostratified airways to the distal bronchioles with club cells only present in terminal bronchioles. Hypothesis: Mechanical ventilation causes airway epithelial injury that is repaired through basal cell activation in the fetal lung. Methods: Ewes at 123±1 day GA had the head and chest of the fetus exteriorized and tracheostomy placed. With placental circulation intact, fetal lambs were mechanically ventilated with up to 15 mL/kg for 15 minutes, using 95% N2/5% CO2. Fetal lambs were returned to the uterus for up to 24 h. The trachea, left mainstem bronchi (LMSB), and peripheral lung were evaluated for epithelial injury and cellular response consistent with repair. Results: Peripheral lung tissue had inflammation, pro-inflammatory cytokine production, EGFR ligand up-regulation, increased p63 expression, and proliferation of pro-SPB, TTF-1 positive club cells. In bronchi, KRT14 and KRT8 mRNA increased without increases in Notch pathway mRNA or proliferation. In trachea, mRNA increased for Notch ligands, SPDEF and MUC5B, but not for basal cell markers. Conclusion: A brief period of mechanical ventilation causes differential epithelial activation between trachea, bronchi and peripheral lung. The repair mechanisms identified in adult mice occur at different levels of airway branching in fetal sheep with basal and club cell activation.
Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug or stem cell based therapy is currently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approach the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells, are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of current EC culture approaches.
Lung HO-1 is developmentally regulated with highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared to adults. However, few reports have addressed the functional impact of miRNAs in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) or adult mice (2 months old) were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-UTR of Bach1and regulates its expression and subsequently leads to higher levels of lung HO-1 mRNA compared to adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in the neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its impact on Bach1. This report is the first to show developmental differences in lung miR-196a and its impact on Bach1 and HO-1 expression at baseline and in hyperoxia.
Abstract Hyperoxia-induced lung injury adversely affects ICU patients and neonates on ventilator assisted breathing. The underlying culprit appears to be reactive oxygen species (ROS) -induced lung damage. The major contributor of hyperoxia-induced ROS is activation of the multiprotein enzyme complex NADPH oxidase (Nox). Sphingosine-1-phosphate (S1P) signaling is known to be involved in hyperoxia mediated ROS generation; however, the mechanism(s) of S1P-induced NADPH oxidase activation is unclear. Here, we investigated various steps in the S1P signaling pathway mediating ROS production in response to hyperoxia in lung endothelium. Of the two closely related sphingosine kinases (SphKs)1 and 2, which synthesize S1P from sphingosine, only Sphk1 -/- mice conferred protection against hyperoxia-induced lung injury. S1P is metabolized predominantly by S1P lyase and partial deletion of Sgpl1 (Sgpl1+/-) in mice accentuated lung injury. Hyperoxia stimulated S1P accumulation in human lung microvascular endothelial cells (HLMVECs), and down-regulation of S1P transporter spinster homolog 2 (Spns2) or S1P receptors S1P1 & 2, but not S1P3, using specific siRNA attenuated hyperoxia-induced p47phox translocation to cell periphery and ROS generation in HLMVECs. These results suggest a role for Spns2 and S1P1&2 in hyperoxia mediated ROS generation. In addition, p47phox (phox: phagocyte oxidase) activation and ROS generation was also reduced by PF543, a specific SphK1 inhibitor in HLMVECs. Our data indicate a novel role for Spns2 and S1P1&2 in the activation of p47phox and production of ROS involved in hyperoxia mediated lung injury in neonatal and adult mice.
Systemically-administered bleomycin causes inflammation, arrested lung growth and pulmonary hypertension (PHT) in the neonatal rat, similar to human infants with severe bronchopulmonary dysplasia (BPD). Leukotrienes (LTs) are inflammatory lipid mediators produced by multiple cell types in the lung. The major LTs, LTB4 and cysteinyl LTs, are suggested to contribute to BPD, but their specific roles remain largely unexplored in experimental models. We hypothesized that LTs are increased in bleomycin-induced BPD-like injury and that inhibition of LT production would prevent inflammatory cell influx and thereby ameliorate lung injury. Rat pups were exposed to bleomycin (1 mg/kg/d i.p.) or vehicle (control) from postnatal days 1-14 and were treated with either Zileuton (5-lipoxygenase inhibitor), Montelukast (cysteinyl LT1 receptor antagonist) or SC57461A (LTA4 hydrolase inhibitor) i.p. 10 mg/kg/d. Bleomycin led to increased lung content of LTB4, but not cysteinyl LTs. Bleomycin-induced increases in tissue neutrophils and macrophages and lung contents of LTB4 and tumor necrosis factor-α were all prevented by treatment with Zileuton. Treatment with Zileuton or SC57461A also prevented the hemodynamic and structural markers of chronic PHT, including raised pulmonary vascular resistance, increased Fulton index and arterial wall remodeling. However, neither treatment prevented impaired alveolarization or vascular hypoplasia secondary to bleomycin. Treatment with Montelukast had no effect on macrophage influx, PHT, or on abnormal lung structure. We conclude that LTB4 plays a crucial role in lung inflammation and PHT in experimental BPD. Agents targeting LTB4 or LTB4-mediated signaling may have utility in infants at risk of developing BPD-associated PHT.
High-dose ibuprofen, an effective anti-inflammatory therapy for the treatment of cystic fibrosis (CF), has been shown to preserve lung function in a pediatric population. Despite its efficacy, few patients receive ibuprofen treatment due to potential renal and gastrointestinal toxicity. The mechanism of ibuprofen efficacy is also unclear. We have previously demonstrated that CF microtubules are slower to reform after depolymerization compared to respective wild type controls. Slower microtubule dynamics in CF cells are responsible for impaired intracellular transport and are related to inflammatory signaling. Here, it is identified that high-dose ibuprofen treatment in both CF cell models and primary CF nasal epithelial cells restores microtubule reformation rates to wild-type levels, as well as induce extension of microtubules to the cell periphery. Ibuprofen treatment also restores microtubule dependent intracellular transport monitored by measuring intracellular cholesterol transport. These effects are specific to ibuprofen as other cyclooxygenase inhibitors have no effect on these measures. Effects of ibuprofen are mimicked by stimulation of AMPK and blocked by the AMPK inhibitor compound c. It is concluded that high-dose ibuprofen treatment enhances microtubule formation in CF cells likely through an AMPK-related pathway. These findings define a potential mechanism to explain the efficacy of ibuprofen therapy in CF.
Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3 demonstrating reduced BMPR2 signaling and elevated TGF-β activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared to control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone-marrow derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with IHC showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL-6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF.
The majority of the animal models of Acute Lung Injury (ALI) are focused on the acute phase. This limits the studies of the mechanisms involved in later phases and the effects of long-term treatments. Thus, the goal of this study was to develop an experimental ALI model of aspiration pneumonia, in which diffuse alveolar damage continues for 72 h. Rats were intratracheally instilled with one dose of HCl (0.1 mol/L) followed by another instillation of one dose of LPS (0, 10, 20, 30 or 40 μg/g body weight [b.w.]) two hours later, which models aspiration of gastric contents that progresses to secondary lung injury from bacteria or bacterial products. The rats were sacrificed at 24, 48, and 72 h after the last instillation. The results showed that HCl and LPS at all doses caused activation of inflammatory responses, increased protein permeability and apoptosis in rat lungs at 24 h post-instillation. However, this lung damage was present at 72 h only in rats receiving HCl and LPS at the doses of 30 and 40 μg/g b.w. Mortality (50%) occurred in the first 48 h and only in the rats treated with HCl and LPS at the highest dose (40 μg/g b.w.). In conclusion, intratracheal instillation of HCl followed by LPS at the dose of 30 μg/g b.w. results in severe diffuse alveolar damage that continues at least 72 h. This rat model of aspiration pneumonia-induced ALI will be useful for testing long-term effect of new therapeutic strategies in ALI.
Brain-derived neurotrophic factor (BDNF), a neurotrophin produced by airway smooth muscle (ASM), enhances inflammation effects on airway contractility, supporting the idea that locally produced growth factors influence airway diseases such as asthma. We endeavored to dissect intrinsic mechanisms regulating endogenous as well as inflammation (TNFα)-induced BDNF secretion in ASM of non-asthmatic vs. asthmatic () humans. We focused on specific Ca2+ regulation- and inflammation-related signaling cascades, and quantified BDNF secretion. We find that TNFα enhances BDNF release by ASM cells, via several mechanisms relevant to asthma, including transient receptor potential channels TRPC3 and TRPC6 (but not TRPC1), ERK 1/2, PI3K, PLC and PKC cascades, rho kinase (ROCK), and transcription factors cAMP response element binding protein (CREB) and nuclear factor of activated T cells (NFAT). Basal BDNF expression and secretion are elevated in asthmatic ASM, and increase further with TNFα exposure, involving many of these regulatory mechanisms. We conclude that airway BDNF secretion is regulated at multiple levels, providing a basis for autocrine effects of BDNF under conditions of inflammation and disease, with potential downstream influences on contractility and remodeling.
VX-770 (Ivacaftor) has been approved for clinical usage in CF patients with several CFTR mutations. Yet the binding site(s) on CFTR for this compound and other small molecule potentiators are unknown. We hypothesize that insight into this question could be gained by comparing the effect of potentiators on CFTR channels from different origins, e.g., human, mouse and Xenopus (frog). In the present study, we combined this comparative molecular pharmacology approach with that of computer-aided drug discovery to identify and characterize new potentiators of CFTR, and to explore possible mechanism of action. Our results demonstrate that: (1) VX-770, NPPB, GlyH-101, P1, P2, and P3 all exhibited ortholog-specific behavior in that they potentiated hCFTR, mCFTR, and xCFTR with different efficacies; (2) P1, P2, and P3 potentiated hCFTR in excised macropatches in a manner dependent upon the degree of PKA-mediated stimulation; (3) P1 and P2 did not have additive effects, suggesting that these compounds might share binding sites; and (4) Using a pharmacophore modeling approach, we identified three new potentiators (IOWH-032, OSSK-2, and OSSK-3) that have structures similar to GlyH-101 and that also exhibit ortholog-specific potentation of CFTR. These could potentially serve as lead compounds for development of new drugs for the treatment of cystic fibrosis. The ortholog-specific behavior of these compounds suggest that a comparative pharmacology approach, using cross-ortholog chimeras, may be useful for identification of binding sites on human CFTR.
The expression of toll-like receptor (TLR)-9, a pathogen recognition receptor that recognizes unmethylated CpG sequences in microbial DNA molecules, is linked to the pathogenesis of several lung diseases. TLR9 expression and signaling was investigated in animal and cell models of chronic obstructive pulmonary disease (COPD). Here we observed enhanced TLR9 expression in mouse lungs following exposure to cigarette smoke. Tlr9-/- mice were resistant to cigarette smoke-induced loss of lung function; determined by mean linear intercept, total lung capacity, lung compliance and tissue elastance analysis. Tlr9 expression also regulated smoke-mediated immune cell recruitment to the lung, apoptosis, G-CSF, CXCL5 and MMP-2 expression and protein tyrosine phosphatase 1B (PTP1B) activity in the lung. PTP1B, a phosphatase with anti-inflammatory abilities, was identified to bind to TLR9. In vivo delivery of a TLR9 agonist enhanced TLR9 binding to PTP1B, which inactivated PTP1B. Ptp1b-/- mice had elevated lung concentrations of G-CSF, CXCL5 and MMP-2 and tissue expression of type-1 interferon following TLR9 agonist administration, compared to wild type mice. TLR9 responses were further determined in fully differentiated normal human bronchial epithelial (NHBE) cells isolated from non-smoker, smoker and COPD donors and then cultured at air liquid interface. NHBE cells from smokers and COPD patients expressed more TLR9 and secreted greater levels of G-CSF, IL-6, CXCL5, IL-1β and MMP-2 upon TLR9 ligand stimulation compared to cells from non-smoker donors. Though TLR9 combats infection, our results indicate that TLR9 induction can impact on lung function by inactivating PTP1B and up regulating expression of pro inflammatory cytokines.
Pseudomonas (P.) aeruginosa is a flagellated pathogen frequently causing pneumonia in hospitalized patients and sufferers of chronic lung disease. Here we investigated the role of the common Toll-like receptor (TLR) adaptor myeloid-differentiation factor (MyD)88 in myeloid versus lung epithelial cells in clearance of P. aeruginosa from the airways. Mice deficient for MyD88 in lung epithelial cells (Sftpccre-MyD88-lox mice) or myeloid cells (LysMcre-MyD88-loxmice), and bone marrow chimeric mice deficient for TLR5 (the receptor recognizing Pseudomonas flagellin) in either parenchymal or hematopoietic cells were infected with P. aeruginosa via the airways. Sftpccre-MyD88-lox mice demonstrated a reduced influx of neutrophils into the bronchoalveolar space and an impaired early antibacterial defense after infection with P. aeruginosa, while the response of LysMcre-MyD88-loxmice did not differ from control mice. The immune enhancing role of epithelial MyD88 was dependent on recognition of pathogen-derived flagellin by epithelial TLR5, as demonstrated by an unaltered clearance of mutant P. aeruginosa lacking flagellin from the lungs of Sftpccre-MyD88-lox mice, and an impaired bacterial clearance in bone marrow chimeric mice lacking TLR5 in parenchymal cells. These data indicate that early clearance of P. aeruginosa from the airways is dependent on flagellin-TLR5-MyD88 dependent signaling in respiratory epithelial cells.
17β-estradiol (E2) exerts protective effects on right ventricular (RV) function in pulmonary arterial hypertension (PAH). Since acute exercise-induced increases in afterload may lead to RV dysfunction in PAH, we sought to determine whether E2 allows for superior RV adaptation after an acute exercise challenge. We studied echocardiographic, hemodynamic, structural and biochemical markers of RV function in male and female rats with sugen/hypoxia (SuHx)-induced pulmonary hypertension, as well as in ovariectomized (OVX) SuHx females with or without concomitant E2 repletion (75 μg/kg/d) immediately after 45 min of treadmill running at 75% of individually-determined maximal aerobic capacity (75% VO2 reserve). Compared to males, intact female rats exhibited higher stroke volume and cardiac indices, a strong trend for better RV compliance, and less pronounced increases in indexed total pulmonary resistance. OVX abrogated favorable RV adaptations, whereas E2 repletion after OVX markedly improved RV function. E2's effects on pulmonary vascular remodeling were complex and less robust than its RV effects. Post-exercise hemodynamics in females with endogenous or exogenous E2 were similar to hemodynamics in non-exercised controls, whereas OVX rats exhibited more severely altered post-exercise hemodynamics. E2 mediated inhibitory effects on RV fibrosis and attenuated increases in RV collagen I/III ratio. Pro-apoptotic signaling, eNOS phosphorylation and autophagic flux markers were affected by E2 depletion and/or repletion. Markers of impaired autophagic flux correlated with endpoints of RV structure and function. Endogenous and exogenous E2 exerts protective effects on RV function measured immediately after an acute exercise challenge. Harnessing E2's mechanisms may lead to novel RV-directed therapies.
Exposure to cigarette smoke is known to result in impaired host defense responses and immune suppressive effects. However, the effects of new and emerging tobacco products, such as e-cigarettes, on the immune status of the respiratory epithelium are largely unknown. We conducted a clinical study collecting superficial nasal scrape biopsies, nasal lavage, urine, and serum from non-smokers, cigarette smokers and e-cigarette users and assessed them for changes in immune gene expression profiles. Smoking status was determined based on a smoking history and a 3-4 week smoking diary and confirmed using serum cotinine and urine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels. Total RNA from nasal scrape biopsies were analyzed using the nCounter® Human Immunology v2 Expression panel. Smoking cigarettes or vaping e-cigarettes resulted in decreased expression of immune-related genes. All genes with decreased expression in cigarette smokers (n=53) were also decreased in e-cigarette smokers. Additionally, vaping e-cigarettes was associated with suppression of in a large number of unique genes (n=305). Furthermore, the e-cigarette users showed a greater suppression of genes common with those changed in cigarette smokers. This was particularly apparent for suppressed expression of transcription factors, such as EGR1, which was functionally associated with decreased expression of 5 target genes in cigarette smokers and 18 target genes in e-cigarette users. Taken together, these data indicate that vaping e-cigarettes is associated with decreased expression of a large number of immune-related genes, which are consistent with immune suppression at the level of the nasal mucosa.
Adiponectin, an adipokine, accumulated in lung system via T-cadherin after allergens/ozone challenge. However, the roles of adiponectin on lung pathologies were controversial. Here we reported that adiponectin stimulated expression of inflammatory proteins, cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2), and production of ROS in human alveolar type II A549 cells. AdipoR1/2 involved in adiponectin-activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondria, which further promoted intracellular reactive oxygen species (ROS) accumulation. Protein kinase C (PKC) may involve in adiponectin-activated NADPH oxidase. Similarly, p300 phosphorylation and histone H4 acetylation occurred in adiponectin-challenged A549 cells. Moreover, adiponectin-upregulated cPLA2 and COX-2 expression was significantly abrogated by ROS scavenger (N-acetylcysteine) or the inhibitors of NADPH oxidase (apocynin), mitochondria electron transport chain (rotenone), PKC (Ro31-8220, Gö-6976 and rottlerin) and p300 (garcinol). Briefly, we reported that adiponectin stimulated cPLA2 and COX-2 expression via adipoR1/2-dependent activation of PKC/NADPH oxidase/mitochondria resulting in ROS accumulation, p300 phosphorylation and histone H4 acetylation. These results suggested that adiponectin promoted lung inflammation resulting in exacerbation of pulmonary diseases via upregulating cPLA2 and COX-2 expression together with intracellular ROS production. Understanding the adiponectin signaling pathways on regulating cPLA2 and COX-2 may help develop therapeutic strategies on pulmonary diseases.
Previous work investigating respiratory system mechanics in mice has reported an aging-related increase in compliance and mean linear intercept (Lm). However, these changes were assessed using only a young (2-month-old) and old (20- and 26-month-old) group yet were interpreted to reflect a linear evolution across the lifespan. Therefore, to investigate respiratory system mechanics and lung morphometry across a more complete spectrum of ages, we utilized 2- (100% survival, n=6), 6- (100% survival, n=12), 18- (90% survival, n=12), 24- (75% survival, n=12), and 30- (25% survival, n=12) month-old C57BL/6 mice. We found a non-linear aging-related decrease in respiratory system resistance and increase in dynamic compliance and hysteresis between 2- and 24-month-old mice. However, in 30-month-old mice, respiratory system resistance increased, and dynamic compliance and hysteresis decreased relative to 24-month-old mice. Respiratory system impedance spectra were measured between 1-20.5 Hz at positive end-expiratory pressures (PEEP) of 1, 3, 5, and 7 cmH2O. Respiratory system resistance and reactance at each level of PEEP were increased and decreased, respectively, only in 2-month-old animals. No differences in the respiratory system impedance spectra were observed in 6-, 18-, 24-, and 30-month-old mice. Additionally, lungs were fixed following tracheal instillation of 4% paraformaldehyde at 25 cmH2O and processed for Lm and airway collagen deposition. There was an aging-related increase in Lm consistent with emphysematous-like changes and no evidence of increased airway collagen deposition. Accordingly, we demonstrate non-linear aging-related changes in lung mechanics and morphometry in C57BL/6 mice.
Animal models play a critical role in the study of the acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). One limitation has been the lack of a suitable method for serial assessment of acute lung injury (ALI) in vivo. In this study, we demonstrate the sensitivity of magnetic resonance imaging (MRI) to assess ALI in real-time in rat models of VILI. Sprague-Dawley rats were untreated or treated with intratracheal lipopolysaccharide or PBS. After 48 hours, animals were mechanically ventilated for up to 15 hours to induce VILI. Free induction decay (FID)-projection images were made hourly. Image data were collected continuously for 30 minutes and divided into 13 phases of the ventilatory cycle to make cinematic images. Interleaved measurements of respiratory mechanics were performed using a Flexivent ventilator. The degree of lung infiltration was quantified in serial images throughout the progression or resolution of VILI. MRI detected VILI significantly earlier (3.8 ± 1.6 hours) than detection by altered lung mechanics (9.5 ± 3.9 hours; p=0.0156). Animals with VILI had a significant increase in the Index of Infiltration (p=0.0027), and early regional lung infiltrates detected by MRI correlated with edema and inflammatory lung injury on histopathology. We were also able to visualize and quantify regression of VILI in real-time upon institution of protective mechanical ventilation. MR lung imaging can be utilized to investigate mechanisms underlying the development and propagation of ALI, as well as to test the therapeutic effects of new treatments and ventilator strategies on the resolution of ALI.
Cystic fibrosis (CF) is a life-shortening disease caused by the mutations that generate non-functional CF transmembrane conductance regulator (CFTR) protein. A rare serine-to-tyrosine (S1045Y) CFTR mutation was earlier reported to result in CF-associated fatality. We identified an African American patient with the S1045Y mutation in CFTR, as well as a stop-codon mutation, who has a mild CF phenotype. The underlying mechanism of CF caused by S1045Y-CFTR has not been elucidated. In this study, we determined that S1045Y-CFTR exhibits two-fold attenuated function compared to WT-CFTR. We report that serine-to-tyrosine mutation leads to increased tyrosine phosphorylation of S1045Y-CFTR, followed by recruitment and binding of E3-ubiquitin ligase c-cbl, resulting in enhanced ubiquitination and passage of S1045Y-CFTR into the endosome/lysosome degradative compartment. We demonstrate that inhibition of tyrosine phosphorylation partially rescues S1045Y-CFTR surface expression and function. Based on our findings, it could be suggested that consuming genistein (a tyrosine phosphorylation inhibitor) would likely ameliorate CF symptoms in individuals with S1045Y-CFTR, providing a unique personalized therapy for this rare CF mutation.
Pleural fibrosis is defined as an excessive deposition of extracellular matrix (ECM) components that results in destruction of the normal pleural tissue architecture. It can result from diverse inflammatory conditions, especially tuberculous pleurisy. Pleural mesothelial cells (PMCs) play a pivotal role in pleural fibrosis. Calpain is a family of calcium-dependent endopeptidases, which plays an important role in ECM remodeling. However, the role of calpain in pleural fibrosis remains unknown. In the present study, we found that tuberculous pleural effusion (TPE) induced calpain activation in PMCs and that inhibition of calpain prevented TPE-induced collagen-I synthesis and cell proliferation of PMCs. Moreover, our data revealed that the levels of angiotensin (Ang)-converting enzyme (ACE) were significantly higher in pleural fluid of patients with TPE than those with malignant pleural effusion, and ACE-Ang II in TPE resulted in activation of calpain and subsequent triggering of the PI3K/Akt/NF-B signaling pathway in PMCs. Finally, calpain activation in PMCs and collagen depositions were confirmed in pleural biopsy specimen from patients with tuberculous pleurisy. Together, these studies demonstrated that calpain is activated by renin-angiotensin system in pleural fibrosis, and mediates TPE-induced collagen-I synthesis and proliferation of PMCs via the PI3K/Akt/NF-B signaling pathway. Calpain in PMCs might be a novel target for intervention in tuberculous pleural fibrosis.
We (66) described a non-steroidal anti-inflammatory drug (NSAID) insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis (by calcium independent mitochondrial iPLA2) of oxidized CL (CLox) leading to formation of lyso-CL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAEC) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by Lipoprotein-associated phospholipase A2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species.. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolyso-cardiolipin and dilyso-cardiolipin and oxidized octadecadienoic metabolites including 9- and 13- hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2. Lower doses of non-lethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolyso-cardiolipin or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa infection, 34 +/- 8 molar % (n=6; P<0.02) of circulating CL was oxidized. In addition, molar percent of monolyso-cardiolipin increased by two fold after P. aeruginosa in a subgroup analyzed for these changes. Collectively these studies suggest an important role for: a) oxidation of cardiolipin in pro-inflammatory environments; and b) possible hydrolysis of CLox in extracellular spaces producing lyso-CL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.
Increased pulmonary vascular resistance in Pulmonary Hypertension (PH) is caused by vasoconstriction and obstruction of small pulmonary arteries by proliferating vascular cells. In analogy to cancer, subsets of proliferating cells may be derived from endothelial cells transitioning into a mesenchymal phenotype. To understand phenotypic shifts transpiring within endothelial cells in PH, we injected rats with alkaloid monocrotaline to induce PH and measured lung tissue levels of endothelial-specific protein and critical differentiation marker Vascular Endothelial (VE)-cadherin. VE-cadherin expression by immonoblotting declined significantly twenty-four hours and fifteen days post injection to rebound to baseline at thirty days. There was a concomitant increase in transcriptional repressors Snail and Slug along with a reduction in VE-cadherin mRNA. Mesenchymal markers α-smooth muscle actin and vimentin were upregulated by immunohistochemistry and immunoblotting and α-smooth muscle actin co-localized with endothelial marker Platelet-Endothelial-Cell-Adhesion-Molecule-1 (PECAM-1) by confocal microscopy. Apoptosis was limited in this model, especially in the 24-hour time-point. In addition, monocrotaline resulted in activation of protein kinase B/Akt, endothelial nitric oxide synthase (eNOS), Nuclear Factor (NF)-B and increased lung tissue nitrotyrosine staining. To understand the etiologic relationship between nitrosative stress and VE-cadherin suppression, we incubated cultured rat lung endothelial cells with endothelin-1, a vasoconstrictor and pro-proliferative agent in PAH. This resulted in activation of eNOS, NF-B and Akt, in addition to induction of Snail, downregulation of VE-cadherin and synthesis of vimentin. These effects were blocked by eNOS inhibitor L-NAME. We propose that transcriptional repression of VE-cadherin by nitrosative stress is involved in endothelial- mesenchymal transdifferentiation in experimental PH.
Epigenetic mechanisms, including DNA methylation and histone acetylation, regulate gene expression in idiopathic pulmonary arterial hypertension (IPAH). These mechanisms can modulate expression of extracellular superoxide dismutase (SOD3 or EC-SOD), a key vascular antioxidant enzyme, and loss of vascular SOD3 worsens outcomes in animal models of PAH. We hypothesized that SOD3 gene expression is decreased in patients with IPAH due to aberrant DNA methylation and/or histone deacetylation. We used lung tissue and pulmonary artery smooth muscle cells (PASMC) from subjects with IPAH at transplantation and failed donors (FD). Lung SOD3 mRNA expression and activity was decreased in IPAH vs. FD. In contrast, mitochondrial SOD (Mn-SOD or SOD2) protein expression was unchanged and intracellular SOD activity was unchanged. Using bisulfite sequencing in genomic lung or PASMC DNA, we found the methylation status of the SOD3 promoter was similar between FD and IPAH. Furthermore, treatment with 5-aza-dC did not increase PASMC SOD3 mRNA, suggesting DNA methylation was not responsible for PASMC SOD3 expression. Though total HDAC activity, HAT activity, acetylated histones and acetylated SP1 were similar between IPAH and FD, treatment with two selective class I HDAC inhibitors increased SOD3 only in IPAH PASMC. Class I HDAC3 siRNA also increased SOD3 expression. TSA, a pan-HDAC inhibitor decreased proliferation in IPAH, but not in FD PASMC. These data indicate that histone deacetylation, specifically via class I HDAC3, decreases SOD3 expression in PASMC and HDAC inhibitors may protect IPAH in part by increasing PASMC SOD3 expression.
The development of chronic hypoxia (CH)-induced pulmonary hypertension is associated with increased pulmonary arterial smooth muscle cell (PASMC) Ca2+ influx through acid sensing ion channel 1 (ASIC1) and activation of the Ca2+/calcineurin-dependent transcription factor, nuclear factor of activated T-cells isoform c3 (NFATc3). Whether Ca2+-influx through ASIC1 contributes to NFATc3 activation in the pulmonary vasculature is unknown. Furthermore, both ASIC1 and calcineurin have been shown to interact with the scaffolding protein, protein interacting with C kinase 1 (PICK1). In the present study, we tested the hypothesis that ASIC1 contributes to NFATc3 nuclear translocation in PASMC in a PICK1-dependent manner. Using both ASIC1 knockout (ASIC1-/-) mice and pharmacological inhibition of ASIC1, we demonstrate ASIC1 contributes to CH (1 wk @ 380 mmHg) and endothelin-1 (ET-1; 10-7 M) induced Ca2+ responses and NFATc3 nuclear import in PASMC. The interaction between ASIC1/PICK1/calcineurin was shown using Duolink in situ proximity ligation assay. Inhibition of PICK1, using FSC231, abolished ET-1- and ionomycin-induced NFATc3 nuclear import, but did not alter ET-1-mediated Ca2+ responses suggesting that PICK1 acts downstream of Ca2+ influx. The key findings of the present work are that 1) Ca2+ influx through ASIC1 mediates CH- and ET-1-induced NFATc3 nuclear import and 2) the scaffolding protein, PICK1, is necessary for NFATc3 nuclear import. Together these data provide an essential link between CH-induced ASIC1-mediated Ca2+ influx and activation of the NFATc3 transcription factor. Identification of this ASIC1/PICK1/NFATc3 signaling complex increases our understanding of the mechanisms contributing to the vascular remodeling and increased vascular contractility associated with CH-induced pulmonary hypertension.
Lung endothelial damage contributes to the pathogenesis of acute lung injury. New strategies against lung endothelial barrier dysfunction may provide therapeutic benefits against lung vascular injury. Cell-cell junctions and microtubule cytoskeleton are basic components in maintaining endothelial barrier integrity. HDAC6, a deacetylase primarily localized in the cytoplasm, has been reported to modulate non-nuclear protein function through deacetylation. Both α-tubulin and β-catenin are substrates for HDAC6. Here, we examined the effects of Tubastatin A, a highly selective HDAC6 inhibitor, on TNF-α induced lung endothelial cell barrier disruption and endotoxin-induced pulmonary edema. Selective HDAC6 inhibition by Tubastatin A blocked TNF-α-induced lung endothelial cell hyper-permeability, which was associated with increased α-tubulin acetylation and microtubule stability. Tubastatin A pre-treatment inhibited TNF-α-induced endothelial cell contraction and actin stress fiber formation with reduced myosin light chain phosphorylation. Selective HDAC6 inhibition by Tubastatin A also induced β-catenin acetylation in human lung endothelial cells, which was associated with increased membrane localization of β-catenin and stabilization of adherens junctions. HDAC6 knockdown by siRNA also prevented TNF-α-induced barrier dysfunction and increased α-tubulin and β-catenin acetylation in endothelial cells. Furthermore, in a mouse model of endotoxemia, Tubastatin A was able to prevent endotoxin-induced deacetylation of α-tubulin and β-catenin in lung tissues, which was associated with reduced pulmonary edema. Collectively, our data indicate that selective HDAC6 inhibition by Tubastatin A is a potent approach against lung endothelial barrier dysfunction.
Diesel emissions are the main source of air pollution in urban areas, and diesel exposure is linked with substantial adverse health effects. In vitro diesel exposure models are considered a suitable tool for understanding these effects. Here we aimed to use a controlled in vitro exposure system to whole diesel exhaust to study the effect of whole diesel exhaust concentration and exposure duration on mucociliary differentiated human primary bronchial epithelial cells (PBEC). PBEC cultured at the air-liquid interface (ALI) were exposed for 60 to 375 minutes to three different dilutions of Diesel Exhaust (DE). The DE mixture was generated by an engine at 47 % load, and characterized for particulate matter (PM) size and distribution, chemical and gas composition. Cytotoxicity and epithelial barrier function was assessed, as well as mRNA expression and protein release analysis. DE caused a significant dose-dependent increase in expression of oxidative stress markers (HMOX1, and NQO1; n=4) at 6 h after 150 min exposure. Furthermore, DE significantly increased the expression of the markers of the integrated stress response (ISR) CHOP and GADD34 and of the pro-inflammatory chemokine CXCL8, as well as release of CXCL8 protein. Cytotoxic effects or effects on epithelial barrier function were observed only after prolonged exposures to the highest DE dose. These results demonstrate the suitability of our model, and that exposure dose and duration, and time of analysis post-exposure are main determinants for the effects of diesel exhaust on differentiated primary human airway epithelial cells.
RATIONALE - Patients with pulmonary hypertension (PH) suffer from inspiratory muscle weakness. However, the pathophysiology of inspiratory muscle dysfunction in PH is unknown. We hypothesized that weakness of the diaphragm, the main inspiratory muscle, is an important contributor to inspiratory muscle dysfunction in PH-patients. OBJECTIVES - To combine ex vivo diaphragm muscle fiber contractility measurements with measures of in vivo inspiratory muscle function in chronic thromboembolic pulmonary hypertension (CTEPH) patients. METHODS - To assess diaphragm muscle contractility, function was studied in vivo by maximum inspiratory pressure (MIP), and ex vivo in diaphragm biopsies of the same CTEPH-patients (N=13) obtained during pulmonary endarterectomy. Patients undergoing elective lung surgery served as controls (N=15). Muscle fiber cross sectional area (CSA) was determined in cryosections and contractility in permeabilized muscle fibers. RESULTS - Diaphragm muscle fiber CSA was not significantly different between control and CTEPH-patients in both slow-twitch and fast-twitch fibers. Maximal force generating capacity was significantly lower in slow-twitch muscle fibers of CTEPH-patients, while no difference was observed in fast-twitch muscle fibers. The maximal force of diaphragm muscle fibers correlated significantly with MIP. The calcium sensitivity of force generation was significantly reduced in fast-twitch muscle fibers of CTEPH-patients, resulting in a ~40% reduction of submaximal force generation. The fast skeletal troponin activator CK-2066260 (5µM) restored submaximal force generation to levels exceeding those observed in control subjects. CONCLUSIONS - Diaphragm muscle fiber contractility is hampered in CTEPH-patients and contributes to the reduced function of the inspiratory muscles in CTEPH-patients.
Agricultural dust exposure results in significant lung inflammation, and individuals working in concentrated animal feeding operations (CAFOs) are at risk for chronic airway inflammatory diseases. Exposure of bronchial epithelial cells to aqueous extracts of hog CAFO dusts (HDE) leads to inflammatory cytokine production that is driven by protein kinase C (PKC) activation. cAMP-dependent protein kinase (PKA)-activating agents can inhibit PKC activation in epithelial cells, leading to reduced inflammatory cytokine production following HDE exposure. Beta-2 adrenergic receptor agonists (β2-agonists) activate PKA, and we hypothesized that β2-agonists would beneficially impact HDE-induced adverse airway inflammatory consequences. Bronchial epithelial cells were cultured with short-acting β2-agonist salbutamol or long-acting β2-agonist salmeterol prior to stimulation with HDE. Beta-2-agonist treatment significantly increased PKA activation and significantly decreased HDE-stimulated IL-6 and IL-8 production in a concentration- and time-dependent manner. HDE-induced intracellular adhesion molecule-1 expression and neutrophil adhesion to epithelial cells was significantly reduced with salbutamol treatment. Using an established intranasal inhalation exposure model, salbutamol pretreatment reduced airway neutrophil influx and IL-6, TNF-α, CXCL1, and CXCL2 release in bronchoalveolar lavage fluid following a one-time exposure to HDE. Likewise, when mice were pre-treated daily with salbutamol prior to HDE exposure for 3 weeks, HDE-induced neutrophil influx and inflammatory mediator production was also reduced. The severity of HDE-induced lung pathology in mice repetitively exposed to HDE for 3 weeks was also decreased with daily salbutamol-pre-treatment. Together, these results provide support for future clinical investigations evaluating the utility of β2-agonist therapies in the treatment of airway inflammation associated with CAFO dust exposure.
Deficiency in pulmonary surfactant results in neonatal respiratory distress, and the known genetic mutations in key components of surfactant only account for a small number of cases. Therefore, determining the regulatory mechanisms of surfactant production and secretion, particularly during the transition from prenatal to neonatal stages, is essential for better understanding of the pathogenesis of human neonatal respiratory distress. We have observed significant increase of BMP signaling in neonatal mouse lungs immediately after birth. Using genetically manipulated mice, we then studied the relationship between BMP signaling and surfactant production in neonates. Blockade of endogenous BMP signaling by deleting Bmpr1a (Alk3) or Smad1 from embryonic day 18.5 in perinatal lung epithelial cells resulted in severe neonatal respiratory distress and death, accompanied by atelectasis in histopathology and significant reductions of surfactant protein B and C, as well as Abca3, while their prenatal lung development was not significantly affected. We then identified a new BMP-Smad1 downstream target Nfatc3, which is known as an important transcription activator for surfactant proteins and Abca3. Furthermore, activation of BMP signaling in cultured lung epithelial cells was able to promote endogenous Nfatc3 expression, and also stimulate the activity of a Nfatc3 promoter that contains Smad1 binding site. Therefore, our study suggests that BMP-Alk3-Smad1-Nfatc3 regulatory loop plays an important role in enhancing surfactant production in neonates, possibly helping neonatal respiratory adaptation from prenatal amniotic fluid environment to neonatal air breathing.
Acute lung injury (ALI) and systemic coagulopathy are serious complications of traumatic brain injury (TBI) that frequently leads to poor clinical outcomes. Although the release of Tissue Factor (TF), a potent initiator of the extrinsic pathway of coagulation, from the injured brain is thought to play a key role in coagulopathy after TBI, its function in ALI following TBI remains unclear. In this study, we investigated whether the systemic appearance of TF correlated with ensuing coagulopathy that follows TBI in ALI using an anesthetized rat blunt trauma TBI model. Blood and lung samples were obtained after TBI. Compared to controls, pulmonary edema and increased pulmonary permeability were observed as early as 5 min after TBI without evidence of norepinephrine involvement. Systemic TF increased at 5 min and then diminished 60 min after TBI. Lung injury and alveolar hemorrhaging were also observed as early as 5 min after TBI. A biphasic elevation of TF was observed in the lungs after TBI, and TF positive microparticles (MPs) were detected in the alveolar spaces. Fibrin(ogen) deposition was also observed in the lungs within 60 min after TBI. Additionally, pre-administration of a direct thrombin inhibitor, Refludan, attenuated lung injuries, thus implicating thrombin as a direct participant in ALI after TBI. The results from this study demonstrated that enhanced systemic TF may be an initiator of coagulation activation that contributes to ALI after TBI.
MIF is a pluripotent cytokine associated with several different inflammatory conditions, but its role within lung inflammation and COPD is unclear. This study aimed to examine MIF in both stable COPD and during acute exacerbations (AECOPD). The study included 433 patients with COPD aged 41-76 and 325 controls from the Bergen COPD cohort study. All patients had a FEV1 <80% predicted, FEV1/FVC ratio <0.7 and a smoking history >10 packyears. Serum levels of MIF were compared between the two groups at baseline, and for 149 patients measurements were also done during AECOPD. Linear regression models were fitted with MIF as the outcome variable, adjusted for sex, age, body composition, smoking and Charlson Comorbidity Score (CCS). Median MIF (IQR) in COPD patients was 20.1 (13.5-30.9) compared to 14.9 (11.1-21.6) in controls (p<0.01). MIF was bivariately associated with sex, body composition and CCS (p<0.05 for all). In the regression analysis, MIF was significantly higher in COPD-patients, coefficient 1.32 (p<0.01) and 1.30 (p<0.01) unadjusted and adjusted respectively. In addition, in 146 patients at AECOPD, MIF was significantly elevated, with median 23.2 (14.1-42.3) compared to measures at stable disease 19.3 (12.4-31.3), p<0.01. Serum levels of MIF were significantly higher in COPD patients compared with controls. We also identified an additional increase in MIF-levels at AECOPD.
Patients with obstructive lung diseases commonly undergo bronchodilator reversibility testing during examination of their pulmonary function by spirometry. A positive response is defined by an increase in forced expiratory volume in 1 s (FEV1). FEV1 is a rather non-specific criterion not allowing the regional effects of bronchodilator to be assessed. We employed the imaging technique of electrical impedance tomography (EIT) to visualize the spatial and temporal ventilation distribution in 35 patients with chronic obstructive pulmonary disease at baseline and 5, 10, and 20 min after bronchodilator inhalation. EIT scanning was performed during tidal breathing and forced full expiration maneuver in parallel with spirometry. Ventilation distribution was determined by EIT by calculating the image pixel values of FEV1, forced vital capacity (FVC), tidal volume, peak flow and mean forced expiratory flow between 25% and 75% of FVC. The global inhomogeneity indices of each measure and histograms of pixel FEV1/FVC values were then determined to assess the bronchodilator effect on spatial ventilation distribution. Temporal ventilation distribution was analyzed from pixel values of times needed to exhale 75% and 90% of pixel FVC. Based on spirometric FEV1, significant bronchodilator response was found in 17 patients. These patients exhibited higher post-bronchodilator values of all regional EIT-derived lung function measures in contrast to 'non-responders'. Ventilation distribution was inhomogeneous in both groups. Significant improvements were noted for spatial distribution of pixel FEV1 and tidal volume and temporal distribution in 'responders'. By providing regional data, EIT might increase the diagnostic and prognostic information derived from reversibility testing.
Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) plays a critical role in inflammatory diseases, including allergic airway disease. However, the benefits of inhibiting ER stress in the treatment of allergic airway disease are not well known. Herein, we tested the therapeutic potential of a chemical chaperone, tauroursodeoxycholic acid (TUDCA), in combating allergic asthma, using a mouse model of house dust mite (HDM)-induced allergic airway disease. TUDCA was administered during the HDM-challenge phase (preventive regimen), after the HDM-challenge phase (therapeutic regimen), or therapeutically during a subsequent HDM re-challenge (re-challenge regimen). In the preventive regimen, TUDCA significantly decreased HDM-induced inflammation, markers of ER stress, airway hyperresponsiveness (AHR), and fibrosis. Similarly, in the therapeutic regimen, TUDCA administration efficiently decreased HDM-induced airway inflammation, mucus metaplasia, ER stress markers, and AHR, but not airway remodeling. Interestingly, TUDCA administered therapeutically in the HDM re-challenge regimen markedly attenuated HDM-induced airway inflammation, mucus metaplasia, ER stress markers, methacholine-induced AHR, and airway fibrotic remodeling. These results indicate that the inhibition of ER stress in the lungs through the administration of chemical chaperones could be a valuable strategy in the treatment of allergic airway diseases.
Chronic lung disease of prematurity (CLD) is a frequent sequela of premature birth and oxygen toxicity is a major associated risk factor. Impaired alveolarization, scarring and inflammation are hallmarks of CLD. Mast cell hyperplasia is a feature of CLD but the role of mast cells in its pathogenesis is unknown. We hypothesized that mast cell hyperplasia is a consequence of neonatal hyperoxia and contributes to CLD. Additionally, mast cell products may have diagnostic and prognostic value in preterm infants predisposed to CLD. In order to model CLD neonatal wild type and mast-cell-deficient mice were placed in an O2 chamber delivering hyperoxic gas mixture (FiO2 of 0.8) (HO) for 2 weeks and then returned to room air (RA) for an additional 3 weeks. Aged matched controls were kept in RA (FiO2 of 0.21). Lungs from HO mice had increased numbers of mast cells, alveolar simplification and enlargement and increased lung compliance. Mast-cell-deficiency proved protective by preserving airspace integrity and lung compliance. The mast cell mediators β-hexosaminidase (β-hex), histamine, and elastase increased in the bronchoalveolar lavage fluid of HO wildtype mice. Tracheal aspirate fluids (TAs) from oxygenated and mechanically ventilated preterm infants were analyzed for mast cell products. In TAs from infants with confirmed cases of CLD, β-hex was elevated over time and correlated with FiO2. Mast cell exosomes were also present in the TAs. Collectively, these data show that mast cells play a significant role in hyperoxia-induced lung injury and their products could serve as potential biomarkers in evolving CLD.
Transforming growth factor beta1 (TGF-β1) is involved in regulation of cellular proliferation, differentiation and fibrogenesis, inducing myofibroblast migration and increasing extracellular matrix synthesis. Here, TGF-β1 effects on pulmonary structure and function were analysed. Adenoviral mediated gene-transfer of TGF-β1 in mice lungs was performed and evaluated by design-based stereology, invasive pulmonary function testing and detailed analyses of the surfactant system one and two weeks after gene-transfer. After one week decreased static compliance was linked with a dramatic alveolar derecruitment without edema formation or increase in the volume of septal wall tissue or collagen fibrils. Abnormally high surface tension correlated with down-regulation of surfactant proteins B and C. TTF-1 expression was reduced and using PLA (proximity ligand assay) technology, we found Smad3 and TTF-1 forming complexes in vivo, which are normally translocated into the nucleus of the alveolar epithelial type II cells (AE2C), but in the presence of TGF-β1 remain in the cytoplasm. AE2C show altered morphology, resulting in loss of total apical surface area per lung and polarity. These changes of AE2C were progressive 2 weeks after gene-transfer and correlated with lung compliance. While static lung compliance remained low, the volume of septal wall tissue and collagen fibrils increased two weeks after gene-transfer. In this animal model, the primary effect of TGF-β1 signalling in the lung is downregulation of surfactant proteins, high surface tension, alveolar derecruitment and mechanical stress which precede fibrotic tissue remodelling and progressive loss of AE2C polarity. Initial TTF-1 dysfunction is potentially linked to down-regulation of surfactant proteins.
Ozone is a common, potent oxidant pollutant in industrialized nations. Ozone exposure causes airway hyperreactivity, lung hyperpermeability, inflammation and cell damage in humans and laboratory animals, and exposure to ozone has been associated with exacerbation of asthma, altered lung function, and mortality. The mechanisms of ozone-induced lung injury and differential susceptibility are not fully understood. Ozone-induced lung inflammation is mediated, in part, by the innate immune system. We hypothesized that mannose binding lectin (MBL), an innate immunity serum protein, contributes to the pro-inflammatory events caused by ozone-mediated activation of the innate immune system. Wild-type (Mbl+/+) and MBL deficient (Mbl-/-) mice were exposed to ozone (0.3 ppm) for up to 72 h, and bronchoalveolar lavage fluid (BALF) was examined for inflammatory markers. Compared to Mbl+/+ mice, mean BALF eosinophils, neutrophils and neutrophil attractants CXCL2 (MIP-2) and CXCL5 (LIX) were significantly lower in Mbl-/- mice exposed to ozone. Using genome-wide mRNA microarray analyses, we identified significant differences in expression response profiles and networks at baseline (e.g. NRF2 mediated oxidative stress response) and after exposure (e.g. humoral immune response) between Mbl+/+ and Mbl-/- mice. The microarray data were further analyzed to discover several informative differential response patterns and subsequent gene sets, including antimicrobial response and inflammatory response. These novel findings demonstrate that targeted deletion of Mbl caused differential expression of inflammation-related gene sets basally and after exposure to ozone, and significantly reduced pulmonary inflammation thus indicating an important innate immunomodulatory role of the gene in this model.
Aims: Adaptation of the right ventricle (RV) to increased afterload is crucial for survival in pulmonary hypertension (PH), but it is challenging to assess RV function and identify associated molecular mechanisms. Aim of the current study was to analyze the relationship between invasive and non-invasive parameters of RV morphology and function and associated molecular changes. Methods: The response of mice to normobaric hypoxia was assessed by echocardiography, invasive hemodynamics, histology and molecular analysis. Plasma levels of possibly novel markers of RV remodeling were measured by ELISA in patients with idiopathic pulmonary arterial hypertension (IPAH) and matched healthy controls. Results: Chronic hypoxia-induced PH was accompanied by significantly decreased tricuspid annular plane systolic excursion (TAPSE) and unchanged RV contractility index and tau. RV hypertrophy was present without an increase in fibrosis. There was no change in α- and β-MHC or natriuretic peptides expression. Comparative microarray analysis identified two soluble factors, FGF5 and IL22RA2, as possibly associated with RV remodeling. We observed significantly increased plasma levels of IL22RA2, but not FGF5, in IPAH patients. Conclusions: Hypoxic pulmonary hypertension in a stage of RV remodeling with preserved systolic function is associated with decreased pulmonary vascular compliance, mild diastolic RV dysfunction and significant decrease in TAPSE. Subtle gene expression changes in the RV versus LV upon chronic hypoxia suggest that the majority of changes are due to hypoxia and not due to changes in afterload. Increased IL22RA2 levels might represent a novel RV adaptive mechanism.
Endothelial dysfunction plays a principal role in the pathogenesis of pulmonary arterial hypertension (PAH), which is a fatal disease with limited effective clinical treatments. Mitochondrial dysregulation and oxidative stress are involved in endothelial dysfunction. Peroxisome proliferator-activated receptor- coactivator-1α (PGC-1α) is a key regulator of cellular energy metabolism and a master regulator of mitochondrial biogenesis. However, the roles of PGC-1α in hypoxia-induced endothelial dysfunction are not completely understood. We hypothesized that hypoxia reduces PGC-1α expression and leads to endothelial dysfunction in hypoxia-induced PAH. We confirmed that hypoxia has a negative impact on endothelial PGC-1α in experimental PAH in vitro and in vivo. Hypoxia-induced PGC-1α inhibited the oxidative metabolism and mitochondrial function, whereas sustained PGC-1α decreased reactive oxygen species (ROS) formation, mitochondrial swelling and NF- B activation and increased ATP formation and endothelial nitric oxide synthase (eNOS) phosphorylation. Furthermore, hypoxia-induced changes in the mean pulmonary arterial pressure and right heart hypertrophy were nearly normal after intervention. These results suggest that PGC-1α is associated with endothelial function in hypoxia-induced PAH and that improved endothelial function is associated with improved cellular mitochondrial respiration, reduced inflammation and oxygen stress, and increased PGC-1α expression. Taken together, these findings indicate that PGC-1α may be a new therapeutic target in PAH.
Emphysema is the major component of chronic obstructive pulmonary disease (COPD). During emphysema, elastin breakdown in the lung tissue originates from the release of large amounts of elastase by inflammatory cells. Elevated levels of elastin-derived peptides (EP) reflect massive pulmonary elastin breakdown in COPD patients. Only the EP containing the GXXPG conformational motif with a type VIII β-turn are elastin receptor ligands inducing biological activities. In addition, the C-terminal glycine residue of the GXXPG motif seems a prerequisite to the biological activity. In this study, we endotracheally instilled C57BL/6J mice with GXXPG EP and/or C-terminal glycine deleted-EP whose sequences were designed by molecular dynamics and docking simulations. We investigated their effect on all criteria associated with the progression of murine emphysema. Broncho-alveolar lavages were recovered to analyze cell profiles by flow cytometry and lungs were prepared to allow morphological and histological analysis by immunostaining and confocal microscopy. We observed that exposure of mice to EP elicited hallmark features of emphysema with inflammatory cell accumulation associated with increased matrix metalloproteinases and desmosine expression, and remodeling of parenchymal tissue. We also identified an inactive C-terminal glycine deleted-EP that retains its binding-activity to EBP and that is able to inhibit the in vitro and in vivo activities of emphysema-inducing EP. This study demonstrates that EP are key actors in the development of emphysema, and that they represent pharmacological targets for an alternative treatment of emphysema based upon the identification of EP analogous antagonists by molecular modelling studies.
Impaired vasodilation in persistent pulmonary hypertension of the newborn (PPHN) is characterized by mitochondrial dysfunction. We investigated the hypothesis that decreased nitric oxide (NO) availability leads to impaired mitochondrial biogenesis and function in a lamb model of PPHN induced by prenatal ductus arteriosus constriction. We ventilated PPHN lambs with 100% O2 alone or with inhaled nitric oxide (iNO). We treated pulmonary artery endothelial cells (PAEC) from control and PPHN lambs with detaNONOate, an NO donor. We observed decreased mitochondrial (mt) DNA copy number, electron transport chain (ETC) complex subunit levels and ATP levels in PAEC and lung tissue of PPHN fetal lambs at baseline compared to gestation matched controls. Phosphorylation of AMP-activated kinase (AMPK) and levels of PPAR gamma coactivator 1-alpha (PGC-1α) and sirtuin1, which facilitate mitochondrial biogenesis, were decreased in PPHN. Ventilation with 100% O2 was associated with larger decreases in ETC subunits in the lungs of PPHN lambs compared to unventilated PPHN lambs. INO administration, with weaning of FiO2 partly restored mtDNA copy number, ETC subunit levels and ATP levels. DetaNONoate increased eNOS phosphorylation and its interaction with HSP90, levels of superoxide dismutase 2 (SOD2) mRNA, protein and activity and decreased the mitochondrial superoxide in PPHN-PAEC. Knockdown of eNOS decreased ETC proteins in control PAEC, while detaNONOate increased ETC protein levels and ATP-linked O2 consumption in eNOS knockdown cells and in PPHN-PAEC. We conclude that ventilation with 100% O2 amplifies oxidative stress and mitochondrial dysfunction in PPHN, which are partly improved by supplemental NO and weaning of oxygen.
We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the current study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wildtype mice subjected to single dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wks. Mice deficient in GADD45a (GADD45a -/-) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels and levels of inflammatory cytokine compared to RILI-challenged wildtype animals at 2 and 4 wks. Further, GADD45a-/- mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wks after RILI-challenge relative to wildtype mice while increased RILI susceptibility was observed in both Akt +/- mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a -/- mice. In separate studies, lung fibrotic changes 2 wks after treatment with bleomycin (0.25 U/kg, IT) was significantly increased in GADD45a -/- mice compared to wildtype mice assessed by lung collagen content and histology. These data implicate GADD45a as an important mediator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.
Trans-endothelial hyper-permeability caused by numerous agonists is dependent on Heat shock protein 90 (Hsp90) and leads to endothelial barrier dysfunction (EBD). Inhibition of Hsp90 protects and restores trans-endothelial hyper-permeability. Hyper-acetylation of Hsp90, as by inhibitors of histone deacetylase (HDAC), suppresses its chaperone function and mimics the effects of Hsp90 inhibitors. In this study we assessed the role of HDAC in mediating LPS-induced trans-endothelial hyper-permeability and acute lung injury (ALI). We demonstrate that HDAC inhibition protects against LPS-mediated EBD. Inhibition of multiple HDAC by the general inhibitors panobinostat or trichostatin provided protection against LPS-induced trans-endothelial hyper-permeability, acetylated and suppressed Hsp90 chaperone function and attenuated RhoA activity and signaling crucial to endothelial barrier function. Treatment with the HDAC3-selective inhibitor, RGFP966, or the HDAC6-selective inhibitor tubastatin A, provided partial protection against LPS-mediated trans-endothelial hyper-permeability. Similarly, knock down of HDAC3 and HDAC6 by specific siRNAs provided significant protection against LPS-induced EBD. Furthermore, combined pharmacologic inhibition of both HDAC 3 and 6 attenuated the inflammation, capillary permeability and structural abnormalities associated with LPS-induced ALI in mice. Together these data indicate that HDAC mediate increased trans-endothelial hyper-permeability caused by LPS and that inhibition of HDAC protects against LPS-mediated EBD and ALI by suppressing Hsp90-dependent RhoA activity and signaling.
We tested the hypothesis that suppression of epoxyeicosatrienoic acid (EET) metabolism via genetic knockout of the gene for soluble epoxide hydrolase (sEH-KO), or female-specific downregulation of sEH expression, plays a role in the potentiation of pulmonary hypertension. We used male (M) and female (F) wild type (WT) and sEH-KO mice; the latter have high pulmonary EETs. Right ventricular systolic pressure (RVSP) and mean arterial blood pressure (MABP) in control, and in response to in vivo administration of U46619 (thromboxane analogue), 14,15-EET, and 14,15-EEZE (antagonist of EETs) were recorded. Basal RVSP was comparable among all groups of mice, whereas MABP was significantly lower in F-WT than M-WT mice, and further reduced predominantly in F-KO compared to M-KO mice. U46619 dose-dependently increased RVSP and MABP in all groups of mice. The increase in RVSP was significantly greater and coincided with smaller increases in MABP in M-KO and F-WT mice compared to M-WT mice. In F-KO mice, the elevation of RVSP by U46619 was even higher than M-KO and F-WT mice, associated with the least increase in MABP. 14,15-EEZE prevented the augmentation of U46619-induced elevation of RVSP in sEH-KO mice, whereas, 14,15-EET-induced pulmonary vasoconstriction was comparable in all groups of mice. sEH expression in the lungs was reduced, paralleled with higher levels of EETs in F-WT compared to M-WT mice. In summary, EETs initiate pulmonary vasoconstriction but act as vasodilators systemically. High pulmonary EETs, as a function of downregulation or deletion of sEH, potentiate U46619-induced increases in RVSP in a female-susceptible manner.
The susceptibility to bacterial infections is increased in COPD. This promotes exacerbations. IL-2 triggers CD4+/Th1-cell proliferation, which is important for infection defense. Bacterial endotoxin (LPS) activates MyD88/IRAK and TRIF/IKK/TBK1 pathways via toll-like receptor-4 (TLR4) in Th1-cells. Hypothesis: systemic defects in TLR pathways in CD4+/Th1-cells cause an impairment of IL-2-dependent immune responses to bacterial infections in COPD. Peripheral blood CD4+ T-cells of never-smokers, smokers without COPD and smokers with COPD (each n=10) were ex vivo activated towards Th1 and stimulated with LPS. IL-2, MyD88 and TRIF expression and cell proliferation was analyzed by ELISA, qRT-PCR, BrdU- and trypan blue-staining comparative among the cohorts. IL-2 release from activated T-cells was increased in COPD vs. smokers and never-smokers. LPS reduced IL-2 expression and T-cell proliferation. These effects were increased in COPD vs. never-smokers and inversely correlated with FEV1 [% pred.]. The MyD88/TRIF ratio was decreased in Th1-cells of COPD. The suppression of IL-2 by LPS was abolished by MyD88/IRAK blockade in never-smokers but by TRIF/IKK/TBK1 blockade in COPD. Moxifloxacin restored IL-2 expression and T-cell proliferation in the presence of LPS by blocking p38MAPK. The increased IL-2 release from Th1-cells in COPD might contribute to airway inflammation in disease exacerbations. A switch from MyD88/IRAK to TRIF/IKK/TBK1 signalling amplifies the suppression of IL-2-dependent proliferation of CD4+ T-cells by LPS in COPD. This molecular pathology is of systemic origin, might impair adaptive immune responses and could explain the increased susceptibility to bacterial infections in COPD. Targeting TLR4-downstream signalling, for example, with moxifloxacin, might reduce exacerbation rates.
Bacterial induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome (ARDS). Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized and it depends on which compartment the signal is generated - plasma membrane versus cytosolic - as to whether it is barrier protective or disruptive, respectively. The mammalian soluble adenylyl cyclase (AC) isoform 10 (AC10 or sAC) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. Herein, we tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability is dependent upon extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent upon extracellular bicarbonate: LPS-induced barrier failure and increased permeability is exacerbated in elevated bicarbonate compared to low extracellular bicarbonate. The AC10 inhibitor, KH7, attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase the Kf in the presence of minimal perfusate bicarbonate. When perfusate bicarbonate was increased to the physiological range (24 mM) this revealed the LPS-induced increase in the Kf, which was attenuated by KH7. Further, when PMVECs were treated with LPS for 6-hours there was a dose-dependent increase in AC10 expression. Thus, these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10.
The recruitment and activation of inflammatory cells into the respiratory system is considered a crucial feature in the pathophysiology of chronic obstructive pulmonary disease (COPD). Since dendritic cells (DCs) have a pivotal role in the onset and regulation of immune responses, we investigated the effect of modulating of DC subsets on airway inflammation by acute CS exposure. CS-exposed mice (5 days) were treated with Flt3L (fms-like tyrosine kinase 3 ligand) and 120g8 antibody to increase total DC numbers and deplete plasmacytoid DCs (pDCs), respectively. Flt3L treatment decreased the number of inflammatory cells in the BALF of the smoke-exposed mice and increased these in lung tissue. DC modulation reduced IL-17 and increased IL-10 levels, which may be responsible for the suppression of the BALF cells. Furthermore, depletion of pDCs led to increased infiltration of alveolar macrophages while restricting the presence of CD103+ DCs. This study suggests that DC subsets may differentially and compartment-dependent influence the inflammation induced by CS. pDC may play a role in preventing the pathogenesis of cigarette smoke by inhibiting the alveolar macrophage migration to lung and increasing CD103+ DCs at inflammatory sites to avoid extensive lung tissue damage.
Nitrogen mustard (NM) is a cytotoxic vesicant that causes acute lung injury and fibrosis, accompanied by persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1-7 days later. Splenectomy resulted in an increase in macrophages expressing CCR2, but a decrease in ATR-1α+ cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS+, CD11b+CD43+) macrophages in lungs of NM treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b+CD43- M2 macrophages was observed in SPX rats, with no changes in CD68+, CD163+, CD206+, or YM-1+ M2 macrophages, suggesting distinct origins of M2 subpopulations after NM. Expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2α and 5-HT7 was also reduced in NM-treated SPX rats, when compared to shams, indicating impaired M2 macrophage activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with antiinflammatory activity; moreover, in their absence, proinflammatory/cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis.
High pulmonary vascular resistance (PVR), proximal pulmonary artery (PA) impedance, and right ventricular (RV) afterload due to remodeling contribute to the pathogenesis and severity of pulmonary hypertension (PH). Intra-amniotic exposure to endotoxin (ETX) causes sustained PH and high mortality in rat pups at birth, which is associated with impaired vascular growth and RV hypertrophy in survivors. Treatment of ETX-exposed pups with antenatal vitamin D (vit D) improves survival and lung growth, but the effects of ETX exposure on RV-PA coupling in the neonatal lung is unknown. We hypothesized that intrauterine ETX impairs RV-PA coupling through sustained abnormalities of PA stiffening and RV performance that are attenuated with vit D therapy. Fetal rats were exposed to intra-amniotic injections of ETX, ETX+vit D, or saline at 20 days gestation (term=22days). At postnatal day 14, pups had pressure-volume measurements of the RV and isolated proximal PA, respectively. Lung homogenates were assayed for extracellular matrix (ECM) composition by western blot. We found that ETX lungs contain decreased alpha-elastin, lysyl oxidase, collagen I, and collagen III proteins (P<0.05) compared control and ETX+vit D lungs. ETX exposed animals have increased RV mechanical stroke work (P<0.05 vs. control and ETX+vit D) and elastic potential energy (P<0.05 vs. control and ETX+vit D). Mechanical stiffness and ECM remodeling are increased in the PA (P<0.05 vs. control and ETX+vit D). We conclude that intrauterine exposure of fetal rats to ETX during late gestation causes persistent impairment of RV-PA coupling throughout infancy that can be prevented with early vit D treatment.
Most patients with allergic asthma are sensitized to house dust mite (HDM). The allergenicity of HDM largely depends on disruption of the integrity and pro-inflammatory activation of the airway epithelium. In this study, we hypothesized that Pim1 kinase activity attenuates HDM-induced asthma by preserving airway epithelial integrity. The effects of Pim1 kinase activity on barrier function and release of the pro-inflammatory mediators IL-1α and CCL20 were studied in vitro in 16HBE and primary bronchial epithelial cells (PBECs). Pim1-proficient and deficient mice were exposed to a HDM-driven model of allergic asthma, and airway hyper-responsiveness (AHR) was measured upon metacholine challenge. Airway inflammation and pro-inflammatory mediators in lung tissue and BAL fluid were determined. We observed that inhibition of Pim1 kinase prolongs the HDM-induced loss of barrier function in 16HBE cells and sensitizes PBECs to HDM-induced barrier dysfunction. Additionally, inhibition of Pim1 kinase increased the HDM-induced pro-inflammatory activity of 16HBE cells as measured by IL-1α secretion. In line herewith, HDM exposure induced an enhanced production of the pro-inflammatory chemokines CCL17 and CCL20 in Pim1-deficient mice compared to wild-type controls. While we observed a marked increase in eosinophilic and neutrophilic granulocytes as well as mucus cell metaplasia and AHR to metacholine in mice exposed to HDM, these parameters were independent of Pim1 kinase activity. In contrast, levels of the Th2-cytokines IL-5 and IL-10 were significantly augmented in HDM-treated Pim1-deficient mice. Taken together, our study shows that Pim1 kinase activity maintains airway epithelial integrity and protects against HDM-induced pro-inflammatory activation of the airway epithelium.
Transient receptor potential-3 (TRPC3) channels play a predominant role in forming non-selective cation channels (NSCCs) in airway smooth muscle cells (ASMCs), and are significantly increased in their activity and expression in asthmatic ASMCs. To extend these novel findings, we have explored the regulatory mechanisms that control the activity of TRPC3 channels. Our data for the first time reveal that inositol 1,4,5-trisphosphate (IP3), an important endogenous signaling molecule, can significantly enhance the activity of single NSCCs in ASMCs. The analog of diacylglycerol (another endogenous signaling molecule) 1-oleyl-2-acetyl-sn-glycerol (OAG), 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) and 1-stearoyl-2-linoleoyl-sn-glycerol (SLG) all augments NSCC activity. The effects of IP3 and OAG are fully abolished by lentiviral shRNA-mediated TRPC3 channel knockdown (KD). The stimulatory effect of IP3 is eliminated by heparin, an IP3 receptor (IP3R) antagonist that blocks the IP3-binding site, but not by xestospongin-C, the IP3R antagonist that has no effect on the IP3-binding site. Lentiviral shRNA-mediated KD of IP3R1, IP3R2 or IP3R3 does not alter the excitatory effect of IP3. TRPC3 channel KD greatly inhibits IP3 -induced increase in [Ca2+]i. IP3R1 KD produces a similar inhibitory effect. TRPC3 channel and IP3R1 KD both diminish the muscarinic receptor agonist methacholine-evoked Ca2+ responses. Taken together, we conclude that IP3, the important intracellular second messenger, may activate TRPC3 channels to cause extracellular Ca2+ influx, in addition to opening IP3Rs to induce intracellular Ca2+ release. This novel extracellular Ca2+ entry route may play a significant role in mediating IP3-mediated numerous cellular responses in ASMCs and other cells.
Overwhelming acute inflammation often leads to tissue damage during endotoxemia. In the present study, we investigated the role of Lyn, a member of the Src family tyrosine kinases, in modulating inflammatory responses in a murine model of endotoxemia. We examined lung inflammatory signaling in Lyn knockout (Lyn-/-) mice and wild type littermates (Lyn+/+) during endotoxemia. Our data indicate that Lyn deletion aggravates endotoxin-induced pulmonary inflammation and pro-inflammatory signaling. We found increased activation of pro-inflammatory transcription factor NF-B in the lung tissues of Lyn-/- mice after endotoxin challenge. Furthermore, during endotoxemia, the lung tissues of Lyn-/- mice showed increased inflammasome activation indicated by augmented caspase-1 and IL- 1β cleavage and activation. The aggravated lung inflammatory signaling in Lyn-/- mice was associated with increased production of pro-inflammatory mediators, and elevated matrix metallopeptidase 9 and reduced VE-cadherin levels. Our results suggest that Lyn kinase modulates inhibitory signaling to suppress endotoxin-induced lung inflammation.
In ARDS, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2+]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2+]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on intracellular calcium ([Ca2+]i) in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2+]i through Ca2+ influx and that pharmacologic inhibition of the calcium channel TRPV4 attenuated the H2O2-induced Ca2+ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2+ influx in MLMVECs and HLMVECs with either genetic deletion, silencing or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor, SU-6656, for 24 or 48 h, the H2O2-induced Ca2+ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2+]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.
In hypoxia, mitochondria-generated reactive oxygen species (ROS) not only stimulate accumulation of the transcriptional regulator of hypoxic gene expression, Hif-1, but also cause oxidative base modifications in hypoxic response elements (HREs) of hypoxia-inducible genes. When the hypoxia-induced base modifications are suppressed, Hif-1 fails to associate with the HRE of the VEGF promoter and VEGF mRNA accumulation is blunted. The mechanism linking base modifications to transcription is unknown. Here we determined if recruitment of base excision DNA repair (BER) enzymes in response to hypoxia-induced promoter modifications was required for transcription complex assembly and VEGF mRNA expression. Using ChIP analyses in pulmonary artery endothelial cells, we found that hypoxia-mediated formation of the base oxidation product 8-oxoguanine (8-oxoG) in VEGF HREs was temporally associated with binding of Hif-1α and the BER enzymes Ogg1 and Ref-1/Ape1 and introduction of DNA strand breaks. Hif-1α co-localized with HRE sequences harboring Ref-1/Ape1 but not Ogg1. Inhibition of BER by siRNA-mediated reduction in Ogg1 augmented hypoxia-induced 8-oxoG accumulation and attenuated Hif-1α and Ref-1/Ape1 binding to the VEGF HRE sequences and blunted VEGF mRNA expression. ChIP-seq analysis of 8-oxoG distribution in hypoxic PAECs showed that most of the oxidized base was localized to promoters with virtually no overlap between normoxic and hypoxic data sets. Transcription of genes whose promoters lost 8-oxoG during hypoxia was reduced, while those gaining 8-oxoG was elevated. Collectively, these findings suggest that the BER pathway links hypoxia-induced introduction of oxidative DNA modifications in promoters of hypoxia-inducible genes to transcriptional activation.
Pulmonary Iymphangioleiomyomatosis (LAM), a rare progressive lung disease associated with mutations of the Tuberous Sclerosis Complex (Tsc2) tumor suppressor gene, manifests by neoplastic growth of LAM cells, induction of cystic lung destruction and respiratory failure. LAM severity correlates with up-regulation in serum of the prolymphangiogenic vascular endothelial growth factor D (VEGF-D) that distinguishes LAM from other cystic diseases. The goal of our study was to determine whether Tsc2-deficiency up-regulates VEGF-D and whether axitinib, the FDA-approved small molecule inhibitor of VEGFR signaling, will reduce Tsc2-null lung lesion growth in a mouse model of LAM. Our data demonstrate up-regulation of VEGF-D in the serum and lung lining in mice with Tsc2-null lesions. Progressive growth of Tsc2-null lesions induces recruitment and activation of inflammatory cells and increased nitric oxide production. Recruited cells isolated from the lung lining of mice with Tsc2-null lesions demonstrate up-regulated expression of pro-vasculogenic Vegfa, pro-lymphangiogenic Figf and pro-inflammatory Nos2, Il6, Ccl2 genes. Importantly, axitinib is an effective inhibitor of Tsc2-null lesion growth and inflammatory cell recruitment which correlates with reduced VEGF-D levels in serum and lung lining. Our data demonstrate that pharmacological inhibition of VEGFR signaling with axitinib inhibits Tsc2-null lesion growth, attenuates recruitment and activation of inflammatory cells and reduces VEGF-D levels systemically and in the lung lining. Our study suggests a potential therapeutic benefit of inhibition of VEGFR signaling for treatment of LAM.
The disciplines of physiology and ecology are united by the shared centrality of the concept of homeostasis: the stability of a complex system via internal mechanisms of self-regulation, resilient to external perturbation. In the past decade, these fields of study have been bridged by the discovery of the lung microbiome. The respiratory tract, long considered sterile, is in fact a dynamic ecosystem of microbiota, intimately associated with the host inflammatory response, altered in disease states. If the microbiome is a "newly discovered organ," ecology is the language we use to explain how it establishes, maintains and loses homeostasis. In this essay, we review recent insights into the feedback mechanisms by which the lung microbiome and the host response are regulated in health, and dysregulated in acute and chronic lung disease. We propose three explanatory models supported by recent studies: the adapted island model of lung biogeography, nutritional homeostasis at the host-microbiome interface, and interkingdom signaling and the community stress response.
Despite the greatly expanded knowledge on the regulation of immune response by protein molecules, there is increasing understanding that non-coding RNAs (ncRNAs) are also an integral component of this regulatory network. Abnormal immune response serves a central role in the initiation, progression and exacerbation of inflammatory lung diseases, such as asthma, chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS)/acute lung injury (ALI). Dysregulation of ncRNAs has been linked to various immunopathologies. In this review, we highlighted the role of ncRNAs in the regulation of innate and adaptive immunity, as well as summarized recent findings that ncRNAs participate in the pathogenesis of inflammatory lung diseases via their regulation of pulmonary immunity. We also discussed therapeutic potentials for targeting ncRNAs to treat these lung disorders.
Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI: attenuation of LPS-induced lung injury, but exacerbation of ventilator-induced lung injury (VILI). Due to the critical role of MV in the care of ARDS patients, in the current study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased BAL protein, total cells, neutrophils, and TNFα levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically-relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 hours after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NFB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.
HMGB1 is a damage associated molecular pattern (DAMP) protein that binds toll-like receptors (e.g. TLR4) and the receptor for advanced glycated end products (RAGE). Direct effects of HMGB1 on airway structural cells are not fully known. As epithelial cell responses are fundamental drivers of asthma, including abnormal repair-restitution linked to changes in extracellular matrix (ECM) synthesis, we tested the hypothesis that HMGB1 promotes bronchial epithelial cell wound repair via TLR4 and/or RAGE signalling that regulates ECM (fibronectin and the 2 chain of laminin-5) and integrin protein abundance. To assess impact of HMGB1 we used molecular and pharmacological inhibitors of RAGE or TLR4 signalling in scratch wound, immunofluorescence and immunoblotting assays to assess wound repair, ECM synthesis and phosphorylation of intracellular signalling. HMGB1 increased wound closure and this effect was attenuated by blocking RAGE and TLR4 signalling. HMGB1 induced fibronectin and laminin-5 (2 chain) was diminished by blocking RAGE and/or blunting TLR4 signalling. Similarly, induction of α3 integrin receptor for fibronectin and laminin-5 was also diminished by blocking TLR4 signalling and RAGE. Lastly, rapid and/or sustained phosphorylation of SMAD2, ERK1/2 and JNK signalling modulated HMGB1 induced wound closure. Our findings suggest a role for HMGB1 in human airway epithelial cell repair and restitution via multiple pathways mediated by TLR4 and RAGE that underpin increased ECM synthesis and modulation of cell-matrix adhesion.
SP-D is a pulmonary collectin important in lung immunity. SP-D-deficient mice (Sftpd-/-) are reported to be susceptible to ovalbumin (OVA)- and fungal allergen-induced pulmonary inflammation, while treatment with exogenous SP-D has therapeutic effects in such disease models. β-glucans are a diverse group of polysaccharides previously suggested to serve as fungal ligands for SP-D. We set out to investigate if SP-D could interact with 1,3-β-glucan and attenuate allergic pulmonary inflammation in the presence of 1,3-β-glucan. Allergic airway disease was induced in Sftpd-/- and Sftpd+/+ mice by OVA sensitization and subsequent challenge with OVA, 1,3-β-glucan or OVA/1,3-β-glucan together. Mice in the combined treatment group were further treated with a high dose of recombinant fragment of human SP-D (rfhSP-D). We demonstrated direct interaction between SP-D and 1,3-β-glucan. OVA-induced mucous cell metaplasia was increased in Sftpd-/- mice, supporting previously reported protective effects of endogenous SP-D in allergy. OVA-induced parenchymal CCL11 levels and eosinophilic infiltration in brochoalveolar lavage (BAL) were unaffected by 1,3-β-glucan but were reversed with rfhSP-D treatment. 1,3-β-glucan-treatment did, however, induce pulmonary neutrophilic infiltration and increased TNF-α levels in BAL, independently of OVA-induced allergy. This infiltration was also reversed by treatment with rfhSP-D. 1,3-β-glucan reduced OVA-induced mucous cell metaplasia, Th2 cytokines and IFN- production. rfhSP-D treatment further reduced mucous metaplasia and Th2 cytokine secretion to background levels. In summary, rfhSP-D treatment resulted in attenuation of both allergic inflammation and 1,3-β-glucan mediated neutrophilic inflammation. Our data suggest that treatment with high dose SP-D protects from mold-induced exacerbations of allergic asthma.
Extracellular nucleotides and nucleosides are important signaling molecules in the lung. Nucleotide and nucleoside concentrations in alveolar lining fluid are controlled by a complex network of surface ectonucleotidases. Previously, we demonstrated that influenza A/WSN/33 (H1N1) virus resulted in increased levels of the nucleotide ATP and the nucleoside adenosine in bronchoalveolar lavage fluid (BALF) of wild-type (WT) C57BL/6 mice. Influenza-induced acute lung injury (ALI) was highly attenuated in A1-adenosine receptor-knockout mice. Because AMP hydrolysis by the ecto-5'-nucleotidase (CD73) plays a central role in and is rate-limiting for generation of adenosine in the normal lung, we hypothesized that ALI would be attenuated in C57BL/6-congenic CD73-knockout (CD73-KO) mice. Infection-induced hypoxemia, bradycardia, viral replication, and bronchoconstriction were all moderately increased in CD73-KO mice relative to WT controls. However, post-infection weight loss, pulmonary edema, and parenchymal dysfunction were not altered. Treatment of WT mice with the CD73 inhibitor 5'-(α,β-methylene) diphosphate (APCP) also had no effect on infection-induced pulmonary edema but modestly attenuated hypoxemia. BALF from both CD73-KO mice and APCP-treated WT mice contained more IL-6 and CXCL-10/IP-10, less CXCL-1/KC, and fewer neutrophils than BALF from untreated WT controls. BALF from APCP-treated WT mice also contained less alveolar macrophages and more TGF-β than untreated WT BALF. These results indicate that CD73 is not necessary for development of ALI following influenza A virus infection, and suggest that tissue non-specific alkaline phosphatase may be responsible for increased adenosine generation in the infected lung. However, they do suggest that CD73 has a previously unrecognized immunomodulatory role in influenza.
Background: Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. TGF-β has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-β. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-β induced myofibroblast differentiation. Methods: Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-βand or Gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Results: Gossypol inhibited TGF-β induced expression of the myofibroblast marker α-smooth muscle actin (SMA) in a dose dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3 and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose dependent manner. Furthermore, Gossypol inhibited TGF-βbioactivity in a dose dependent manner. Concurrent treatment with an LDH siRNA increased the ability of Gossypol to inhibit TGF-β induced myofibroblast differentiation. Conclusions: Gossypol inhibits TGF-β induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid and inhibition of TGF-β bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis.
Background: Aspiration is a common cause of lung injury, but it is unclear why some cases are self-limited while others progress to ARDS. Sporadic exposure to more than one insult could account for this variable progression. We investigated whether synergy between airway acid and endotoxin (LPS) amplifies injury severity in mice and whether LPS levels in human patients could corroborate our experimental findings. Methods: C57BL/6 mice aspirated acid (pH 1.3) or normal saline (NS), followed by LPS aerosol or nothing. Lavage fluid (BALF) was obtained 2 to 49 hours later. Mice were injected with FITC-dextran 25 hours after aspiration, connected to a ventilator, and lung elastance (H) measured periodically following deep inflation (DI). Endotracheal and gastric aspirates were also collected from patients in the ICU and assayed for pH and LPS. Results: Lung instability (H following DI) and pressure-volume hysteresis in acid or LPS-exposed mice was greater than in controls, but markedly greater in the combined acid/LPS group. BALF neutrophils, cytokines, protein, and FITC-dextran in the acid/LPS mice were geometrically higher than all other groups. BALF from acid only mice markedly amplified LPS-induced TNF-α production in cultured macrophages. Human subjects had variable airway LPS levels with the highest burden in those at higher risk of aspiration. Conclusions: Acid aspiration amplifies LPS signaling in mice to disrupt barrier function and lung mechanics in synergy. High variation in airway LPS and greater airway LPS burden in patients at higher risk of aspiration could help explain the sporadic progression of aspiration to ARDS.
Chronic tobacco smoking is a major cause of preventable morbidity and mortality world-wide. In the lung, tobacco smoking increases the risk of lung cancer, and also causes chronic obstructive pulmonary disease (COPD), which encompasses both emphysema and chronic bronchitis. E-cigarettes (E-Cigs), or electronic nicotine delivery systems, were developed over a decade ago and are designed to deliver nicotine without combusting tobacco. Whilst tobacco smoking has declined since the 1950s, E-Cig usage has increased, attracting both former tobacco smokers and never smokers. E-Cig liquids (e-liquids) contain nicotine in a glycerol/propylene glycol vehicle with flavorings, which are vaporized and inhaled. To date, neither E-Cig devices, nor e-liquids, are regulated by the Food and Drug Administration (FDA). The FDA has proposed a deeming rule, which aims to initiate legislation to regulate E-Cigs, but the timeline to take effect is uncertain. Proponents of E-Cigs say that they are safe and should not be regulated. Opposition is varied with some opponents proposing that E-Cig usage will introduce a new generation to nicotine addiction, reversing the decline seen with tobacco smoking or that E-Cigs generally may not be safe and will trigger diseases like tobacco. In this review, we shall discuss what is known about the effects of E-Cigs on the mammalian lung and isolated lung cells in vitro. We hope that collating this data will help illustrate gaps in the knowledge of this burgeoning field, directing researchers toward answering whether or not E-Cigs are capable of causing disease.
A sustained first inflation (SI) at birth may aid lung liquid clearance and aeration, but the impact of SI duration relative to the volume-response of the lung is poorly understood. We compared three SI strategies; 1) variable duration defined by attaining volume equilibrium using real-time electrical impedance tomography (EIT; SIplat), 2) 30s beyond equilibrium (SIlong) and 3) short 30s SI (SI30) and 4) positive pressure ventilation without SI (no-SI) on spatio-temporal aeration and ventilation (EIT), gas exchange, lung mechanics and regional early markers of injury in preterm lambs. Fifty-nine fetal-instrumented lambs were ventilated for 60 min after applying the allocated first inflation strategy. At study completion molecular and histological markers of lung injury were analysed. The time to SI volume equilibrium, and resultant volume, were highly variable; mean (SD) 55 (34)s, coefficient of variability 59%. SIplat and SIlong resulted in better lung mechanics, gas exchange and lower ventilator settings than both no-SI and SI30. At 60 min, alveolar-arterial difference in oxygen was a mean (95% CI) 130 (13, 249) higher in SI30 versus SIlong group (2-way ANOVA). These differences were due to better spatio-temporal aeration and tidal ventilation, although all groups showed redistribution of aeration towards the non-dependent lung by 60 min. Histological lung injury scores mirrored spatio-temporal change in aeration and were greatest in SI30 group (p<0.01, Kruskal-Wallis test). An individualized volume-response approach to SI was effective in optimizing aeration, homogeneous tidal ventilation and respiratory outcomes, whilst an inadequate SI duration had no benefit over positive pressure ventilation alone.
Increased serotonin serum levels have been proposed to play a key role in Pulmonary Arterial Hypertension (PAH) by regulating vessel tone and vascular smooth muscle cell proliferation. An intact serotonin system, which critically depends on a normal function of the serotonin transporter (SERT), is required for the development of experimental pulmonary hypertension in rodents exposed to hypoxia or monocrotaline. While these animal models resemble human PAH only with respect to vascular media remodeling, we hypothesized that SERT is likewise required for the presence of lumen-obliterating intima remodeling, a hallmark of human PAH reproduced in the Sugen/hypoxia (SuHx) rat model of severe angioproliferative pulmonary hypertension. Therefore, SERT wild type (WT) and knock-out (KO) rats were exposed to the SuHx-protocol. SERT-KO rats, while completely lacking SERT, were hemodynamically indistinguishable from WT rats. After exposure to SuHx, similar degrees of severe angioproliferative pulmonary hypertension and right ventricular hypertrophy developed in WT and KO rats (right ventricular systolic pressure 60 vs 55 mmHg, intima thickness 38 vs 30%, respectively). In conclusion, despite its implicated importance in PAH, SERT does not play an essential role in the pathogenesis of severe angio-obliterative pulmonary hypertension in rats exposed to SuHx.
Rosiglitazone (RGZ), a Peroxisome Proliferator Activated Receptor (PPAR) ligand, is a novel dilator of small airways in mouse precision cut lung slices (PCLS). In this study, relaxation to RGZ and β-adrenoceptor agonists were compared in trachea from naïve mice and guinea pigs, and trachea and PCLS from a mouse model of chronic allergic airways disease (AAD). Airways were pre-contracted with methacholine before addition of PPAR ligands (RGZ, ciglitazone (CGZ) or 15-deoxy-PGJ2) or β-adrenoceptor agonists (isoprenaline, salbutamol). Effects of T0070907 and GW9662 (PPAR antagonists) or epithelial removal on relaxation were assessed. Changes in force of trachea and lumen area in PCLS were measured using preparations from saline-challenged mice and mice sensitised (days 0, 14) and challenged with ovalbumin (3 times/week, 6 weeks). RGZ and CGZ elicited complete relaxation with greater efficacy than β-adrenoceptor agonists in mouse airways but not guinea pig trachea, while 15-deoxy-PGJ2 did not mediate bronchodilation. Relaxation to RGZ was not prevented by T0070907 or GW9662, or by epithelial removal. RGZ-induced relaxation was preserved in trachea and increased in PCLS after ovalbumin-challenge. Although RGZ was less potent than β-adrenoceptor agonists, its effects were additive with SALB and ISO, and only RGZ maintained potency and full efficacy in maximally contracted airways or after allergen challenge. Acute PPAR-independent, epithelial-independent airway relaxation to RGZ is resistant to functional antagonism and maintained in both trachea and PCLS from a model of chronic AAD. These novel efficacious actions of RGZ support its therapeutic potential in asthma when responsiveness to β-adrenoceptor agonists is limited.
Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 exoUexoT::Tc pUCPexoY; ExoY+) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular endothelial cells (PMVECs) both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP cGMP cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY+ intoxication. In PAECs, intercellular gaps formed within two hours and progressively increased in size up to six hours, when the experiment was terminated. cGMP concentrations increased within one hour post-infection, while cAMP and cUMP concentrations increased within three hours, and cCMP concentrations increased within four hours post-infection. In PMVECs, intercellular gaps did not form until four hours post-infection. Only cGMP and cUMP concentrations increased at three and six hours post-infection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY+ intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.
The induction of allergen-specific T helper 2 (Th2) cells by lung dendritic cells (DCs) is a critical step in allergic asthma development. Airway delivery of purified allergens or microbial products can promote Th2 priming by lung DCs, but how environmentally relevant quantities and combinations of these factors affect lung DC function is unclear. Here, we investigated the ability of house dust extract (HDE), which contains a mixture of environmental adjuvants, to prime Th2 responses against an innocuous inhaled antigen. Inhalational exposure to HDE conditioned lung conventional DCs, but not monocyte-derived DCs, to induce antigen-specific Th2 differentiation. Conditioning of DCs by HDE was independent of Toll-like receptor 4 signaling, indicating that environmental endotoxin is dispensable for programming DCs to induce Th2 responses. DCs directly treated with HDE underwent maturation but were poor stimulators of Th2 differentiation. In contrast, DCs treated with bronchoalveolar lavage fluid (BALF) from HDE-exposed mice induced robust Th2 differentiation. DC conditioning by BALF was independent of the proallergic cytokines IL-25, IL-33 and thymic stromal lymphopoietin (TSLP). BALF treatment of DCs resulted in up-regulation of CD80, but low expression of CD40, CD86 and IL-12p40, which was associated with Th2 induction. These findings support a model whereby environmental adjuvants in house dust indirectly program DCs to prime Th2 responses by triggering the release of endogenous soluble factor(s) by airway cells. Identifying these factors could lead to novel therapeutic targets for allergic asthma.
Acute exposure to ozone (O3), an air pollutant, causes pulmonary inflammation, airway epithelial desquamation, and airway hyperresponsiveness (AHR). Pro-inflammatory cytokines, including interleukin (IL)-6 and ligands of chemokine (C-X-C motif) receptor 2 [keratinocyte chemoattractant (KC) and macrophage inflammatory protein (MIP)-2], tumor necrosis factor (TNF) receptor 1 and 2 (TNF), and type I IL-1 receptor (IL-1α and IL-1β), promote these sequelae. Human resistin, a pleiotropic hormone and cytokine, induces expression of IL-1α, IL-1β, IL-6, IL-8 (the human ortholog of murine KC and MIP-2), and TNF. Functional differences exist between human and murine resistin, yet given the aforementioned observations, we hypothesized that murine resistin promotes O3-induced lung pathology by inducing expression of the same inflammatory cytokines as human resistin. Consequently, we examined indices of O3-induced lung pathology in wild-type and resistin-deficient mice following acute exposure to either filtered room air or O3. In wild-type mice, O3 increased bronchoalveolar lavage fluid (BALF) resistin. Furthermore, O3 increased lung tissue or BALF IL-1α, IL-6, KC, TNF, macrophages, neutrophils, and epithelial cells in wild-type and resistin-deficient mice. With the exception of KC, which was significantly greater in resistin-deficient as compared to wild-type mice, no genotype-related differences in the other indices existed following O3 exposure. O3 caused AHR to acetyl-β-methylcholine chloride (methacholine) in wild-type and resistin-deficient mice. However, genotype-related differences in airway responsiveness to methacholine were non-existent subsequent to O3 exposure. Taken together, these data demonstrate that murine resistin is increased in the lungs of wild-type mice following acute O3 exposure but does not promote O3-induced lung pathology.
Cellular senescence has been associated with the structural and functional decline observed during physiological lung ageing and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the ageing murine lung and following cigarette smoke exposure. We evaluated co-localization of H2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung ageing and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With age, telomere-associated foci increase in small airway epithelial cells of the murine lung, which is accelerated by cigarette smoke exposure. Moreover, telomere-associated foci predict age-dependent emphysema; and late-generation Terc null mice, which harbour dysfunctional telomeres, show early-onset emphysema. We found that cigarette smoke accelerates telomere dysfunction via reactive oxygen species in vitro and may be associated with ATM-dependent secretion of inflammatory cytokines IL-6 and IL-8. We propose that telomeres are highly sensitive to cigarette smoke-induced damage and telomere dysfunction may underlie decline of lung function observed during ageing and in COPD.
Airway diseases and their exacerbations are associated with abnormal circadian rhythms of lung function and inflammatory responses. Circadian rhythms are generated at the cellular level by an autoregulatory feedback loop oscillator of clock gene transcription factors, including the BMAL1:CLOCK activator complex and the repressors PERIOD and CRYPTOCHROME. The key nuclear receptors and transcription factors, REV-ERBα and RORα regulate Bmal1 expression and provide stability to the oscillator. Circadian clock dysfunction is implicated in both immune and inflammatory responses to environmental, inflammatory and infectious agents. Molecular clock function is altered by exposomes, tobacco smoke, lipopolysaccharide, hyperoxia, allergens, bleomycin, and bacterial and viral infections. The deacetylase Sirtuin 1 (SIRT1) regulates the timing of the clock through acetylation of BMAL1 and PER2 and controls the clock-dependent functions which can also be affected by environmental stressors. Environmental agents and redox modulation may alter the levels of REV-ERBα and RORα in lung tissue in association with a heightened DNA damage response, cellular senescence and inflammation. A reciprocal relationship exists between the molecular clock and immune/inflammatory responses in the lungs. Molecular clock function in lung cells may be used as a biomarker of disease severity and exacerbations or assessing the efficacy of chronotherapy for disease management. Here, we provide a comprehensive overview of clock-controlled cellular and molecular functions in the lungs and highlight the repercussions of clock disruption on the pathophysiology of chronic airway diseases and their exacerbations. Further, we highlight the potential for the molecular clock as novel chronopharmacological targets for the management of lung pathophysiology.
Connexin (Cx)43 has been shown to participate in several cardiovascular diseases. Increased vascular permeability is a common and severe complication in sepsis or septic shock. Whether or not Cx43 takes part in the regulation of vascular permeability in severe sepsis is not known and the underlying mechanism has not been described. Using cecal ligation and puncture-induced septic rats and lipopolysaccharide (LPS)-treated vascular endothelial cells (VECs) from pulmonary veins, the role of Cx43 in increased vascular permeability and the relationship to the Rho A/Rock1 pathway were studied. It was shown that vascular permeability in the lungs, kidneys, and mesentery in sepsis rats and LPS-stimulated monolayer pulmonary vein VECs were significantly increased and positively correlated with the increased expression of Cx43 and Rock1 in these organs and cultured pulmonary vein VECs. Connexin inhibitor, carbenoxolone(10mg/kg,iv) and Rock1 inhibitor,Y-27632(2mg/kg,iv) alleviated the vascular leakage of lung, mesentery and kidney in sepsis rats. Over-expressed Cx43 increased the phosphorylation of 20 k Da myosin light chain (MLC20) and the expression of Rock1, and increased the vascular permeability and decreased the transendothelial electrical resistance of pulmonary vein VECs. Cx43 RNA interference decreased the phosphorylation of MLC20 and the expression of Rock1, and decreased LPS-stimulated hyperpermeability of cultured pulmonary vein VECs. The Rock1 inhibitor, Y-27632, alleviated LPS- and over-expressed Cx43-induced hyperpermeability of monolayer pulmonary vein VECs. This report shows that Cx43 participates in the regulation of vascular permeability in sepsis and the he mechanism is related to the Rock1-MLC20 phosphorylation pathway.
Sex differences in the incidence of respiratory diseases have been reported. Women are more susceptible to inflammatory lung disease induced by air pollution, and show worse adverse pulmonary health outcomes than men. However, the mechanisms underlying these differences remain unknown. In the present study, we hypothesized that sex differences in the expression of lung inflammatory mediators affect sex-specific immune responses to environmental toxicants. We focused on the effects of ground-level ozone, a major air pollutant, in the expression and regulation of lung immunity genes. We exposed adult male and female mice to 2 ppm of ozone or filtered air (control) for 3 hours. We compared mRNA levels of 84 inflammatory genes in lungs harvested 4 hours post-exposure using a PCR array. We also evaluated changes in lung histology and bronchoalveolar lavage fluid cell counts and protein content at 24 and 72 hours post exposure. Our results revealed sex differences in lung inflammation triggered by ozone exposure, and in the expression of genes involved in acute phase and inflammatory responses. Major sex differences were found in the expression of neutrophil-attracting chemokines (Ccl20, Cxcl5, and Cxcl2), the pro-inflammatory cytokine interleukin-6, and oxidative stress related enzymes (Ptgs2, Nos2). In addition, the phosphorylation of STAT3, known to mediate IL-6-related immune responses, was significantly higher in ozone-exposed mice. Together, our observations suggest that a differential regulation of the lung immune response could be implicated in the observed increased susceptibility to adverse health effects from ozone observed in women versus men.
Altered extracellular matrix (ECM) protein deposition is a feature in asthmatic airways. Fibronectin (Fn), an ECM protein produced by human bronchial epithelial cells (HBECs), is increased in asthmatic airways. This study investigated the regulation of Fn production in asthmatic or non-asthmatic HBECs, and whether Fn modulated HBEC proliferation and inflammatory mediator secretion. The signaling pathways underlying transforming growth factor (TGF)- β1 regulated Fn production were examined using specific inhibitors for ERK, JNK, p38 MAPK, phosphatidylinositol (PI)3 kinase and activin like kinase (ALK)5. Asthmatic HBECs deposited higher levels of Fn in the ECM than non-asthmatic cells under basal conditions, whilst cells from the two groups had similar levels of Fn mRNA and soluble Fn. TGF-β1 increased mRNA levels, and ECM and soluble forms of Fn but decreased cell proliferation in both cells. The rate of increase in Fn mRNA was higher in non-asthmatic cells. However, the excessive amounts of ECM Fn deposited by asthmatic cells, after TGFβ1 stimulation, persisted compared to non-asthmatic cells. Inhibition of ALK5 completely prevented TGF-β1 induced Fn deposition. Importantly, ECM Fn increased HBECs proliferation and IL-6 release, decreased PGE2 secretion, but had no effect on VEGF release. Soluble Fn had no effect on cell proliferation and inflammatory mediator release. Asthmatic HBECs are intrinsically primed to produce more ECM Fn, which when deposited into the ECM is capable of driving remodeling and inflammation. The increased airway Fn may be one of the key driving factors in the persistence of asthma and represent a novel therapeutic target.
The factors accounting for the pathological maintenance of a high pulmonary vascular (PV) resistance postnatally remain elusive, but neonatal stressors may play a role in this process. Cross fostering in the immediate neonatal period is associated with adult-onset vascular and behavioral changes likely triggered by early-in-life stressors. Hypothesizing that fostering newborn rats induces long lasting PV changes, we evaluated them at 14 days of age during adulthood, and compared the findings with animals raised by their biological mothers. Fostering resulted in reduced maternal-pup contact time, when compared with control newborns. At two weeks of age, fostered rats exhibited reduced pulmonary arterial endothelium-dependent relaxation secondary to downregulation of tissue endothelial nitric oxide synthase (eNOS) expression and tetrahydrobiopterin deficiency-induced uncoupling. These changes were associated with neonatal-onset increased angiotensin II AT1 receptor expression, PV remodeling and right ventricular hypertrophy that persisted into adulthood. The pulmonary arteries of adult fostered rats exhibited a higher contraction dose-response to angiotensin II and thromboxane A2; the latter of which was abrogated by the oxidant scavenger tempol, In conclusion, fostering-induced neonatal stress induces long-standing PV changes modulated via the renin-angiotensin system.
In Chronic Obstructive Pulmonary Disease (COPD), oxidative stress regulates the inflammatory response of bronchial epithelium and monocytes/macrophages through kinase modulation and has been linked to glucocorticoid unresponsiveness. GSK3β inactivation plays a key role in mediating signalling processes upon reactive oxygen species (ROS) exposure. We hypothesized that GSK3β is involved in oxidative stress-induced glucocorticoid insensitivity in COPD. We studied levels of p-GSK3β-ser9, a marker of GSK3β inactivation, in lung sections and cultured monocytes and bronchial epithelial cells of COPD patients, control smokers and non-smokers. We observed increased levels of p-GSK3β-ser9 in monocytes, alveolar macrophages and bronchial epithelial cells from COPD patients and control smokers compared to non-smokers. Pharmacological inactivation of GSK3β did not affect CXCL8 or GM-CSF expression but resulted in glucocorticoid insensitivity in vitro in both inflammatory and structural cells. Further mechanistic studies in monocyte and bronchial epithelial cell lines showed that GSK3β inactivation is a common effector of oxidative stress induced activation of the MEK/ERK-1/2 and PI3K/Akt signalling pathways leading to glucocorticoid unresponsiveness. In primary monocytes, the mechanism involved modulation of histone deacetylase 2 (HDAC2) activity in response to GSK3β inactivation. In conclusion, we demonstrate for the first time that ROS-induced glucocorticoid unresponsiveness in COPD is mediated through GSK3β, acting as a ROS-sensitive hub.
Background: The consequences on lung function and inflammation of alterations in the extracellular matrix affecting the peripheral airway wall in asthma are largely unknown. Hypothesis: Remodeling of collagen and elastic fibers in the peripheral airway wall leads to airway obstruction, and contributes to neutrophilic airway inflammation. Animals: 6 heaves-affected horses and 5 controls. Methods: Large peripheral lung biopsies were obtained from horses with heaves in clinical remission (Baseline) and during disease exacerbation and from age-matched controls. The area of collagen and elastic fiber content in the lamina propria was measured using histological staining techniques and corrected for airway size. Collagen type 1 and type 3 was further assessed from additional horses after postmortem lung samples using immunohistochemistry. The collagen breakdown products PGP and N-α-PGP, were also measured in bronchoalveolar lavage fluids (BALF) using mass spectrometry. Results: In comparison with controls, heaves-affected horses had an increase in collagen (p=0.05) and elastic fiber contents (p=0.04) at baseline. Collagen types 1 and 3 content was also significantly increased in diseased horses (p=0.015), when both collagen types were combined. No further change in collagen content was observed after a 30-day antigenic challenge. Airway collagen at baseline was positively correlated with pulmonary resistance in asthmatic horses (r²=0.78, p=0.03) and elastic fiber content was positively associated with pulmonary elastance in controls (r2=0.95, p= 0.02). No difference between groups was appreciated in PGP and N-α-PGP peptides in BALF. Clinical Importance: Increased airway wall collagen and elastic fiber content may contribute to residual obstruction in the asthmatic airways.
Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension (PAH). MicroRNAs (miRNAs), endogenously expressed small noncoding RNAs, regulate gene expression at the posttranscriptional level. It has previously been shown that miR-17 over-expression in cultured human pulmonary artery smooth muscle cell (hPASMC) resulted in increased viable cell number. Previously, we have found that arginase II promotes hypoxia-induced proliferation in hPASMC. Therefore, we hypothesized that miR-17 would be up-regulated by hypoxia in hPASMC and would result in greater arginase II expression. We found that levels of miR-17-5p and arginase II were significantly greater in cultured hPASMC exposed to 1% O2 for 48 hours than in hPASMC exposed to 21% O2 for 48 hours. Furthermore, inhibiting miR-17-5p expression decreased hypoxia-induced arginase II protein levels in hPASMC. Conversely, overexpressing miR-17-5p resulted in greater arginase II protein levels. Somewhat surprisingly, arginase II inhibition was associated with lower miR-17-5p expression in both normoxic and hypoxic hPASMC, while over-expressing arginase II resulted in greater miR-17-5p expression in hPASMC. These findings suggest that hypoxia-induced arginase II expression is not only regulated by miR-17-5p but also that there is a feedback loop between arginase II and miR-17-5p in hPASMC. We also found that the arginase II-mediated regulation of miR-17-5p was independent of either p53 or c-myc. We also found that L-arginine, the substrate for arginase II and L-ornithine, the amino acid product of arginase II, were not involved in the regulation of miR-17-5p expression.
Patients with idiopathic pulmonary fibrosis (IPF) often do worse following infection, but the cause of the decline is not fully understood. We previously demonstrated that infection with a murine gamma herpes virus (HV-68) could exacerbate established lung fibrosis following administration of fluorescein isothiocyanate. In this study, we anesthetized mice and injected saline or bleomycin intratracheally on day 0. On day 14, mice were anesthetized again and infected with either a Gram negative bacteria (Pseudomonas aeruginosa), or with H1N1 or HV-68 viruses. Measurements were then made on days 15, 21 or 35. We demonstrate that infection with P. aeruginosa does not exacerbate extracellular matrix deposition post-bleomycin. Furthermore, fibrotic mice are effectively able to clear P. aeruginosa infection. In contrast, bleomycin-treated mice develop worse lung fibrosis when infected with HV-68, but not when infected with H1N1. The differential ability of HV-68 to cause increased collagen deposition could not be explained by differences in inflammatory cell recruitment or whole lung chemokine and cytokine responses. Alveolar epithelial cells from HV-68-infected mice displayed increased expression of TGFβ receptor 1, increased SMAD3 phosphorylation and evidence of apoptosis measured by cleaved poly ADP ribose polymerase (PARP). The ability of HV-68 to augment fibrosis required the ability of the virus to reactivate from latency. This property appears unique to HV-68, as the β herpes virus, cytomegalovirus, did not have the same effect.
Cigarette smoking (CS) can impact the immune system and induce pulmonary disorders such as chronic obstructive pulmonary disease (COPD), which is currently the fourth leading cause of chronic morbidity and mortality worldwide. Accordingly, the most significant risk factor associated with COPD is exposure to cigarette smoke. The purpose of the current study is to provide an updated overview of the literature regarding the effect of CS on the immune system and lungs, the mechanism of CS-induced COPD and oxidative stress, as well as the available and potential treatment options for CS-induced COPD. An extensive literature search was conducted on the PubMed/Medline databases to review current COPD treatment research, available in the English language, dating from 1986 to 2014. Studies have investigated the mechanism by which CS elicits detrimental effects on the immune system and pulmonary function through the use of human and animal subjects. A strong relationship among continued tobacco use, oxidative stress, and exacerbation of COPD symptoms are frequently observed in COPD subjects. In addition, therapeutic approaches emphasizing smoking cessation have been developed, incorporating counseling and nicotine replacement therapy. However, the inability to reverse COPD progression establishes the need for improved preventative and therapeutic strategies, such as a combination of intensive smoking cessation treatment and pharmaceutical therapy, focusing on immune homeostasis and redox balance. CS initiates a complex interplay between oxidative stress and the immune response in COPD. Therefore, multiple approaches such as smoking cessation, counseling, and pharmaceutical therapies, targeting inflammation and oxidative stress are recommended for COPD treatment.
Severe bronchospasm refractory to β-agonists continues to cause significant morbidity and mortality in asthmatic patients. We questioned whether chloride channels/transporters are novel targets for the relaxation of airway smooth muscle (ASM). We have screened a library of compounds, derivatives of anthranilic and indanyloxyacetic acid, that were originally developed to antagonize chloride channels in the kidney. We hypothesized that members of this library would be novel calcium activated chloride channel (CaCC) blockers for the airway. The initial screen of this compound library identified 4 of 20 compounds that relaxed a tetraethylammonium chloride (TEA)-induced contraction in guinea pig tracheal rings. The two most effective compounds, compounds 1 and 13, were further studied for their potential to either prevent the initiation of, or relax the maintenance phase of an acetylcholine (Ach)-induced contraction, or potentiate β-agonist mediated relaxation. Both relaxed an established Ach-induced contraction in human and guinea pig ex vivo ASM. In contrast, the prevention of an Ach-induced contraction required the co-pretreatment of the sodium-potassium-chloride cotransporter blocker bumetanide. The combination of compound 13 and bumetanide also potentiated relaxation by the β-agonist isoproterenol in guinea pig tracheal rings. Compounds 1 and 13 hyperpolarized the plasma cell membrane of human ASM cells and blocked spontaneous transient inward currents, a measure of chloride currents in these cells. These functional and electrophysiological data suggest that modulating ASM chloride flux is a novel therapeutic target in asthma and other bronchoconstrictive diseases.
The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging due to the lack of mechanism-based therapeutic approaches. Recent studies have shown that Transient Receptor Potential Vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Post-exposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.
The inflammatory response is a primary mechanism in the pathogenesis of ventilator-induced lung injury. Autophagy is an essential, homeostatic process by which cells break down their own components. We explored the role of autophagy in the mechanisms of mechanical ventilation-induced lung inflammatory injury. Mice were subjected to low (7 ml/kg) or high (28 ml/kg) tidal volume ventilation for 2 h. Bone marrow-derived macrophages transfected with a scrambled or autophagy-related protein 5 small interfering RNA were administered to alveolar macrophage-depleted mice via a jugular venous cannula 30 min before starting ventilation protocol. In some experiments, mice were ventilated in the absence and presence of autophagy inhibitors 3-methyladenine (15 mg/kg, i.p.) or trichostatin A (1 mg/kg, i.p.). Mechanical ventilation with a high tidal volume caused rapid (within minutes) activation of autophagy in the lung. Conventional transmission electron microscopic examination of lung sections showed that mechanical ventilation-induced autophagy activation mainly occurred in lung macrophages. Autophagy activation in the lungs during mechanical ventilation was dramatically attenuated in alveolar macrophage-depleted mice. Selective silencing of autophagy-related protein 5 in lung macrophages abolished mechanical ventilation-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and lung inflammatory injury. Pharmacological inhibition of autophagy also significantly attenuated the inflammatory responses caused by lung hyperinflation. The activation of autophagy in macrophages mediates early lung inflammation during mechanical ventilation via NLRP3 inflammasome signaling. Inhibition of autophagy activation in lung macrophages may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.
Intermittent hypoxia (IH) has been extensively studied during the last decade, primarily as a surrogate model of sleep apnea. However, IH is a much more pervasive phenomenon in human disease, is viewed as a potential therapeutic approach, and has also been used in other disciplines, such as in competitive sports. In this context, adverse outcomes involving cardiovascular, cognitive, metabolic and cancer have emerged in OSA-based studies, while beneficial effects of IH have also been identified. Those a priori contradictory findings may not be as contradictory as initially thought. Indeed, the opposite outcomes triggered by IH can be explained by the specific characteristics of the large diversity of IH patterns applied in each study. The balance between benefits and injury appears to primarily depend on the ability of the organism to respond and activate adaptive mechanisms to IH. In this context, the adaptive or mal-adaptive responses can be generally predicted by the frequency, severity and duration of IH. However, the presence of underlying conditions such as hypertension or obesity, as well as age, sex, or genotypic variance may be important factors tilting the balance between an appropriate homeostatic response and decompensation. Here, the two possible facets of IH as derived from human and experimental animal settings will be reviewed.
We sought to test experimentally whether maternal stress can promote susceptibility to development of asthma-like allergic airways disease in offspring. Normal pregnant mice (day 15) were subjected to a single restraint stress exposure. We subsequently tested their offspring for the development of airway hyperreactivity (AHR) and allergic airway inflammation (AI), after an intentionally suboptimal sensitization protocol. The offspring of stressed mothers showed levels of AI and enhanced airway responses to methacholine comparable to those seen in fully-sensitized and challenged positive control animals; in contrast, minimal effects were seen in control offspring. Restraint stress caused a rapid and large increase in plasma corticosterone levels. Maternal treatment with dexamethasone on day 15 of pregnancy mimicked the stress effect and reproduced the AI and AHR outcomes, while blockade of the stress-induced corticosterone surge with metyrapone pre-treatment of pregnant mice abrogated the effect. We conclude that stress-triggered glucocorticoids during pregnancy can increase susceptibility to allergy in offspring. Since inflammation typically includes a stress hormone response, the results also suggest a common pathway by which various injurious exposures during pregnancy might increase offspring susceptibility to asthma
Apoptosis of alveolar macrophages and their subsequent clearance by neighboring phagocytes are necessary steps in the resolution of acute pulmonary inflammation. We have recently identified that activation of the Fas death receptor on the cell surface of macrophages drives macrophage apoptosis. However, the source of Fas ligand responsible for induction of alveolar macrophage apoptosis is not defined. Given their known role in the resolution of inflammation and ability to induce macrophage apoptosis ex vivo, we hypothesized that T-lymphocytes represented a critical source of Fas ligand. To address this hypothesis, C57BL/6J and lymphocyte deficient (Rag-1 -/-) mice were exposed to intratracheal lipopolysaccharide to induce pulmonary inflammation. Furthermore, utilizing mice expressing non-functional FasL, we adoptively transferred donor lymphocytes into inflamed lymphocyte deficient mice to characterize the effect of lymphocyte derived FasL on alveolar macrophage apoptosis in resolving inflammation. Herein, evidence is presented that lymphocytes expressing Fas ligand enhance alveolar macrophage apoptosis during the resolution of LPS induced inflammation. Moreover, lymphocyte induction of alveolar macrophage apoptosis results in contraction of the alveolar macrophage pool, which occurs in a FasL dependent manner. Specifically, Fas ligand expressing CD8+ T-lymphocytes potently induce alveolar macrophage apoptosis and contraction of the alveolar macrophage pool. Together, these studies identify a novel role for CD8+ T-lymphocytes in the resolution of acute pulmonary inflammation.
Airway epithelial cells are the primary cell type involved in respiratory viral infection. Upon infection, airway epithelium plays a critical role in host defense against viral infection by contributing to innate and adaptive immune responses. Influenza A virus, Rhinovirus, and Respiratory Syncytial virus (RSV) represent a broad range of human viral pathogens that cause viral pneumonia and induce exacerbations of asthma and chronic obstructive pulmonary disease. These respiratory viruses induce airway epithelial production of IL-8, which involves epidermal growth factor receptor (EGFR) activation. EGFR activation involves an integrated signaling pathway that includes NADPH oxidase activation of metalloproteinase, and EGFR pro-ligand release that activates EGFR. Because respiratory viruses have been shown to activate EGFR via this signaling pathway in airway epithelium, we investigated the effect of virus-induced EGFR activation on airway epithelial antiviral responses. CXCL10, a chemokine produced by airway epithelial cells in response to respiratory viral infection, contributes to the recruitment of lymphocytes to target and kill virus-infected cells. While respiratory viruses activate EGFR, the interaction between CXCL10 and EGFR signaling pathways is unclear, and the potential for EGFR signaling to suppress CXCL10 has not been explored. Here, we report that respiratory virus-induced EGFR activation suppresses CXCL10 production. We found that Influenza virus-, Rhinovirus-, and RSV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1-dependent CXCL10 production. In addition, inhibition of EGFR during viral infection augmented IRF1 and CXCL10. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.
Tobacco smoke exposure, the major cause of chronic obstructive pulmonary disease (COPD), instigates a dysfunctional clearance of thick obstructive mucus. However, the mechanism underlying the formation of abnormally viscous mucus remains elusive. We investigated whether nicotine can directly alter the rheological properties of mucin by examining its physicochemical interactions with human airway mucin gels secreted from A549 lung epithelial cells. Swelling kinetics and multiple particle tracking were utilized to assess mucin gel viscosity change when exposed to nicotine. Herein we show that nicotine (≤ 50 nM) significantly hindered post-exocytotic swelling and hydration of released mucins, leading to higher viscosity, possibly by electrostatic and hydrophobic interactions. Moreover, the close association of nicotine and mucins allows airway mucus to function as a reservoir for prolonged nicotine release leading to correlated pathogenic effects. Our results provide a novel explanation for the maltransport of poorly hydrated mucus in smokers. More importantly, this study further indicates that even low concentration nicotine can profoundly increase mucus viscosity and thus highlight the health risks of second-hand smoke exposure.
Exposure to cigarette smoke (CS) is the main risk factor for developing COPD and can induce airway epithelial cell damage, innate immune responses and airway inflammation. We hypothesized that cell survival factors might decrease the sensitivity of airway epithelial cells to CS-induced damage, thereby protecting the airways against inflammation upon CS exposure. Here, we tested whether Pim survival kinases could protect from CS-induced inflammation. We determined expression of Pim kinases in lung tissue, airway inflammation and levels of Keratinocyte-derived Cytokine and several Damage-associated molecular patterns (DAMPs) in bronchoalveolar lavage in mice exposed to CS or air. Human bronchial epithelial BEAS-2B cells were treated with CS extract (CSE) in presence or absence of Pim1 inhibitor, and assessed for loss of mitochondrial membrane potential, induction of cell death and release of HSP70. We observed increased expression of Pim1, but not of Pim2 and Pim3 in lung tissue after exposure to CS. Pim1-deficient mice displayed a strongly enhanced neutrophilic airway inflammation upon CS exposure compared to wild-type controls. Inhibition of Pim1 activity in BEAS-2B cells increased the loss of mitochondrial membrane potential and reduced cell viability upon CSE treatment, while release of HSP70 was enhanced. Interestingly, we observed release of S100A8 but not of dsDNA or HSP70 in Pim1-deficient mice compared to wild-type controls upon CS exposure. In conclusion, we show that expression of Pim1 protects against CS-induced cell death in vitro and neutrophilic airway inflammation in vivo. Our data suggest that the underlying mechanism involves CS-induced release of S100A8 and KC.
The pathogenesis of ventilator-induced lung injury has predominantly been attributed to overdistension or mechanical opening and collapse of alveoli, while mechanical strain on the airways is rarely taken into consideration. Here, we hypothesized that mechanical ventilation may cause significant airway distension which may contribute to the pathologic features of ventilator-induced lung injury. C57BL/6J mice were anesthetized and mechanically ventilated at tidal volumes of 6, 10 or 15 mL/kg body weight. Mice were imaged by flat-panel volume computer tomography, central airways were segmented and rendered in 3D for quantitative assessment of airway distension. Alveolar distension was imaged by intravital microscopy. Functional dead space was analyzed in vivo, and pro-inflammatory cytokine release in isolated, ventilated tracheae. CT scans revealed a reversible, up to 2.5-fold increase in upper airway volume during mechanical ventilation as compared to spontaneous breathing. Airway distension was most pronounced in main bronchi which showed the largest volumes at tidal volumes of 10 mL/kg body weight. Conversely, airway distension in segmental bronchi and functional dead space increased almost linearly, and alveolar distension even disproportionately with higher tidal volumes. In isolated tracheae, mechanical ventilation stimulated the release of the early-response cytokines TNF-α and IL-1β. Mechanical ventilation causes a rapid, pronounced and reversible distension of upper airways in mice that is associated with an increase in functional dead space. Upper airway distension is most pronounced at moderate tidal volumes, while higher tidal volumes redistribute preferentially to the alveolar compartment. Airway distension triggers pro-inflammatory responses, and may thus contribute relevantly to ventilator-induced pathologies.
Cystic Fibrosis Transmembrane conductance Regulator (CFTR) carrying the F508del mutation is retained in endoplasmic reticulum and fails to traffic to the cell surface where it functions as a PKA activated chloride channel. Pharmacological correctors that rescue the trafficking of F508del CFTR may overcome this defect; however the rescued F508del CFTR still displays reduced chloride permeability. Therefore, a combined administration of correctors and potentiators of the gating defect is ideal. We recently found that 4,6,4'-trimethylangelicin (TMA), beside inhibiting the expression of the IL-8 gene in airway cells in which the inflammatory response was challenged with P. aeruginosa, also potentiates the cAMP/PKA-dependent activation of wild type CFTR or F508del CFTR that has been restored to the plasma membrane. Here, we demonstrate that long preincubation with nanomolar concentrations of TMA, is able to effectively rescue both F508del CFTR-dependent chloride secretion and F508del CFTR cell surface expression in both primary or secondary airway cell monolayers homozygous for F508del mutation. The correction effect of TMA seems to be selective for CFTR and persisted for 24 hours after washout. Altogether, the results suggest that TMA, besides its anti-inflammatory and potentiator activities also displays corrector properties.
Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with marked morbidity and mortality. Even though female gender represents one of the most powerful risk factors for PAH, multiple questions about the underlying mechanisms remain, and two "estrogen paradoxes" in PAH exist. First, it is puzzling why estrogens have been found to be protective in various animal models of PAH, whereas PAH registries uniformly demonstrate a female susceptibility to the disease. Second, despite the pronounced tendency for the disease to develop in women, female PAH patients exhibit better survival than men. Recent mechanistic studies in classical as well as in novel animal models of PAH, as well as recent studies in PAH patients have significantly advanced the field. In particular, it is now accepted that estrogen metabolism and receptor signaling, as well as estrogen interactions with key pathways in PAH development, appear to be potent disease modifiers. A better understanding of these interactions may lead to novel PAH therapies. It is the purpose of this review to 1) review sex hormone synthesis, metabolism, and receptor physiology, 2) assess the content in which sex hormones affect PAH pathogenesis, 3) provide a potential explanation for the observed estrogen paradoxes and gender differences in PAH, and 4) identify knowledge gaps and future research opportunities. As the majority of published studies investigated 17beta-estradiol and/or its metabolites, this review will primarily focus on estrogen effects on the pulmonary vasculature and right ventricle. Data for other sex hormones will be discussed very briefly.
In many species submucosal glands are an important source of tracheal mucus, but the extent to which mucociliary clearance (MCC) depends upon gland secretion is unknown. To explore this relationship, we measured basal and agonist-stimulated MCC velocities in ex-vivo tracheas from adult ferrets and compared the velocities with previously measured rates of ferret glandular mucus secretion (Cho, et al. AJP Lung, 2010, 299, L124). Stimulated MCC velocities (mm•min-1, mean ± SE for 10-35 min period post-stimulation) were as follows: 1 μM Carbachol: 19.1±3.3 > 10 μM phenylephrine: 15.3±2.4 10 μM isoproterenol: 15.0±1.9 10 μM forskolin: 14.6±3.1 > 1 μM VIP: 10.2±2.2 >> basal (t15): 1.8 ± 0.3, n = 5-10 for each condition. Synergistic stimulation of MCC was observed between low concentrations of carbachol (100 nM) and isoproterenol (300 nM). Bumetanide inhibited carbachol-stimulated MCC by ~70%, and abolished the increase in MCC stimulated by forskolin + VIP, whereas HCO3--free solutions did not significantly inhibit MCC to either [Ca2+]i or [cAMP]i -elevating agonists. Stimulation and inhibition of MCC and gland secretion differed in several respects: most importantly, elevating [cAMP]i increased MCC much more effectively than expected from its effects on gland secretion; and bumetanide almost completely inhibited [cAMP]i -stimulated MCC while it has a smaller effect on gland secretion. We conclude that changes in glandular fluid secretion are complexly related to MCC and discuss possible reasons for this.
Systemic inflammatory response syndrome (SIRS) is a common clinical condition in ICU patients that can lead to complications including multiple organ dysfunction syndrome (MODS). MODS carries a high mortality rate and it is unclear why some patients resolve SIRS whereas others develop MODS. While oxidant stress has been implicated in the development of MODS, several recent studies have demonstrated a requirement for NADPH oxidase 2 (NOX2)-derived oxidants in limiting inflammation. We recently demonstrated that NOX2 protects against lung injury and mortality in a murine model of SIRS. In the present study, we investigated the role of NOX2-derived oxidants in the progression from SIRS to MODS. Using a murine model of sterile systemic inflammation, we observed significantly greater illness and subacute mortality in gp91phox-/y (NOX2-deficient) mice compared to WT mice. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histology showed multiple organ pathology indicative of MODS in the NOX2-deficient mice and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation.
Postnatal lung development requires coordination of three processes-surface area expansion, microvascular growth, and matrix remodeling. Since normal elastin structure is important for lung morphogenesis, since physiological remodeling of lung elastin has never been defined, and since elastin remodeling is angiogenic, we sought to test the hypothesis that during lung development elastin is remodeled in a defined temporal-spatial pattern, that a novel protease is associated with this remodeling, and that angiogenesis is associated with elastin remodeling. By elastin in situ zymography, lung elastin remodeling increased 24-fold between embryonic day 15.5 (E15.5) and postnatal day 14 (PND14). Remodeling was restricted to major vessels and airways on PND1 with a 7-fold increase in alveolar wall elastin remodeling from PND1 to PND14. By inhibition assays and literature review, we identified chymotrypsin-like elastase 1 (CELA1) as a potential mediator of elastin remodeling. CELA1 mRNA levels increased 12-fold from E15.5 to PND9 and protein levels increased 3.4-fold from E18.5 to PND9. By co-staining experiments, the temporal-spatial pattern of CELA1 expression matched that of elastin remodeling and 58-85% of CELA1 (+) cells were <10 µm from an elastase signal. An association between elastin remodeling and angiogenesis was tested by similar methods. At PND7 and PND14, 60-95% of angiogenin (+) cells were associated with elastin remodeling. Both elastase inhibition and CELA1 silencing impaired angiogenesis in vitro. Our data defines the temporal-spatial pattern of elastin remodeling during lung development, demonstrates an association of this remodeling with CELA1, and supports role for elastin remodeling in regulating angiogenesis.
Interstitial lung disease (ILD) is a well-known adverse effect of mammalian target of rapamycin (mTOR) inhibitors. However, it remains unknown how lung toxicities are induced by mTOR inhibitors. Here, we constructed a mouse model of mTOR inhibitor-induced ILD using temsirolimus and examined the pathogenesis of the disease. Male ICR mice were treated by intraperitoneal injection of different doses of temsirolimus (3 mg/kg/week or 30 mg/kg/week) or vehicle. Temsirolimus treatment increased capillary-alveolar permeability and induced neutrophil infiltration and fibrinous exudate into the alveolar space, indicating alveolar epithelial and/or endothelial injury. It also induced macrophage depletion and accumulation of excessive surfactant phospholipids and cholesterols. Alveolar macrophage depletion is thought to cause surfactant lipid accumulation. To further examine whether temsirolimus has a cytotoxic and/or cytostatic effects on alveolar macrophages and alveolar epithelial cells, we performed in vitro experiments. Temsirolimus inhibited cell proliferation and viability in both alveolar macrophage and alveolar epithelial cells. Temsirolimus treatment caused some signs of pulmonary inflammation, including upregulated expression of several proinflammatory cytokines in both bronchoalveolar lavage cells and lung homogenates, and an increase in lymphocytes in the bronchoalveolar lavage fluid. These findings indicate that temsirolimus has the potential to induce alveolar epithelial injury and to deplete alveolar macrophages followed by surfactant lipid accumulation, resulting in pulmonary inflammation. This is the first study to focus on the pathogenesis of mTOR inhibitor-induced ILD using an animal model.
Pulmonary hypertension (PH) is characterized by elevated pulmonary artery pressure that leads to progressive right heart failure and ultimately death. Injury to endothelium and consequent wound repair cascades have been suggested to trigger pulmonary vascular remodeling, such as that observed during PH. The relationship between injury to endothelium and disease pathogenesis in this disorder remains poorly understood. We and others have shown that in mice, hypoxia-induced mitogenic factor (HIMF, also known as FIZZ1 or RELMα) plays a critical role in the pathogenesis of lung inflammation and the development of PH. In this study, we dissected the mechanism by which HIMF and its human homolog resistin (hRETN) induce pulmonary endothelial cell (EC) apoptosis and subsequent lung inflammation-mediated PH, which exhibits many of the hallmarks of the human disease. Systemic administration of HIMF caused increases in EC apoptosis and interleukin (IL)-4-dependent vascular inflammatory marker expression in mouse lung during the early inflammation phase. In vitro, HIMF, hRETN, and IL-4 activated pulmonary microvascular ECs (PMVECs) by increasing angiopoietin-2 expression and induced PMVEC apoptosis. In addition, the conditioned medium from hRETN-treated ECs had elevated levels of endothelin-1 (ET-1) and caused significant increases in pulmonary vascular smooth muscle cell proliferation. Lastly, HIMF treatment caused development of PH that was characterized by pulmonary vascular remodeling and right heart failure in wild-type mice but not in IL-4 knockout mice. These data suggest that HIMF contributes to activation of vascular inflammation at least in part by inducing EC apoptosis in the lung. These events lead to subsequent PH.
Surfactant protein A (SP-A) plays a vital role in maintaining normal lung function and in host defense. Two genes encode SP-A in humans (SFTPA1, SFTPA2), and several gene variants have been identified for these. We have previously shown that sequence elements of SFTPA1 and SFTPA2 3'UTRs differentially affect translation efficiency in vitro. Polymorphisms at the 3'UTRs of mRNA variants may account for differential binding of miRNAs, a class of small non-coding RNAs that regulate gene expression. In this work, we generated 3'UTR reporter constructs of the SFTPA1 and SFTPA2 variants most frequently found in the population, as well as mutants of a previously described 11-nt indel element (refSNP rs368700152). Reporter constructs were transfected in NCI-H441 cells in the presence or absence of miRNA mimics, and reporter gene expression was analyzed. We found that human miRNA mir-767 negatively affected expression of constructs containing both SFTPA1 and SFTPA2 variants, whereas mir-4507 affected only constructs with 3'UTRs of SFTPA1 variants 6A, 6A3, and 6A4 (not containing the 11-nt element). Three miRNAs (mir-183, mir-449b, and mir-612) inhibited expression of recombinants of SFTPA2 variants, and the SFTPA1 variant 6A2, all containing the 11-nt element. Similar results were obtained for SP-A expression when these miRNAs were transfected in CHO-K1 cells expressing SFTPA1 or SFTPA2 variants, or in NCI-H441 cells (genotype 1A5/1A5-6A4/6A4). Moreover, transfection with a specific antagomir (antagomir-183) reversed the effects of mir-183 on SP-A mRNA levels. Our results indicate that sequence variability at the 3'UTR of SP-A variants differentially affects miRNA regulation of gene expression.
Human airway smooth muscle (HASM) contraction plays a central role in regulating airway resistance in both healthy and asthmatic bronchioles. In vitro studies that investigate the intricate mechanisms that regulate this contractile process are predominantly conducted on tissue culture plastic, a rigid, 2D geometry, unlike the 3D microenvironment smooth muscle cells are exposed to in situ. It is increasingly apparent that cellular characteristics and responses are altered between cells cultured on 2D substrates compared to 3D topographies. Electrospinning is an attractive method to produce 3D topographies for cell culturing as the fibres produced have dimensions within the nanometre range, similar to cells' natural environment. We have developed an electrospun scaffold using the non-degradable, non-toxic, polymer polyethylene terephthalate (PET) composed of uni-axially orientated nanofibres, and have evaluated this topography's effect on HASM cell adhesion, alignment, and morphology. The fibres orientation provided contact guidance enabling the formation of fully aligned sheets of smooth muscle. Moreover, smooth muscle cells cultured on the scaffold present an elongated cell phenotype with altered contractile protein levels and distribution. HASM cells cultured on this scaffold responded to the bronchoconstrictor bradykinin. The platform presented provides a novel in vitro model that promotes airway smooth muscle cell development towards a more in vivo-like phenotype whilst providing topological cues to ensure full cell alignment.
Background: Ozone (O3) is a criteria air pollutant that is associated with numerous adverse health effects, including altered respiratory immune responses. Despite its deleterious health effects, possible epigenetic mechanisms underlying O3-induced health effects remain understudied. MicroRNAs (miRNAs) are epigenetic regulators of genomic response to environmental insults and unstudied in relationship to O3 inhalation exposure. Objectives: To test whether O3 inhalation exposure significantly alters miRNA expression profiles within the human bronchial airways. Methods: Twenty healthy adult human volunteers were exposed to 0.4 ppm O3 for two hours. Induced sputum samples were collected from each subject 48 hours pre-exposure and 6 hours post-exposure for evaluation of miRNA expression and markers of inflammation in the airways. Genome-wide miRNA expression profiles were evaluated using microarray analysis and in silico predicted mRNA targets of the O3-responsive miRNAs were identified and validated against previously measured O3-induced changes in mRNA targets. Biological network analysis was performed on the O3-associated miRNAs and mRNA targets to reveal potential associated response signaling and functional enrichment. Results: Expression analysis of the sputum samples revealed that O3 exposure significantly increased the expression levels of 10 miRNAs, namely miR-132, miR-143, miR-145, miR-199a*, miR-199b-5p, miR-222, miR-223, miR-25, miR-424, and miR-582-5p. The miRNAs and their predicted targets were associated with a diverse range of biological functions and disease signatures, noted among them inflammation and immune-related disease. Conclusions: The present study shows that O3 inhalation exposure disrupts select miRNA expression profiles that are associated with inflammatory and immune response signaling. These findings provide novel insight into epigenetic regulation of responses to O3 exposure.
Oxygen toxicity contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal mice exposed to hyperoxia develop a simplified lung structure that resembles BPD. Sustained activation of the transcription factor NFB and increased expression of protective target genes attenuates hyperoxia-induced mortality in adults. However, the effect of enhancing hyperoxia-induced NFB activity on lung injury and development in neonatal animals is unknown. We performed this study to determine whether sustained NFB activation, mediated through IBβ overexpression, preserves lung development in neonatal animals exposed to hyperoxia. Newborn WT and IBβ overexpressing (AKBI) were exposed to hyperoxia (>95%) or room air from day of life (DOL) 0-14, after which all animals were kept in room air. Survival curves were generated through DOL 14. Lung development was assessed using radial alveolar count (RAC) and mean linear intercept (MLI) at DOL 3 and 28 and pulmonary vessel density at DOL 28. Lung tissue was collected and NFB activity was assessed using Western blot for IB degradation and NFB nuclear translocation. WT mice demonstrated 80% mortality through 14 days of exposure. In contrast, AKBI mice demonstrated 60% survival. Decreased RAC, increased MLI and pulmonary vessel density caused by hyperoxia in WT mice was significantly attenuated in AKBI mice. These findings were associated with early and sustained NFB activation and expression of cytoprotective target genes, including VEGFR2. We conclude that sustained hyperoxia-induced NFB activation improves neonatal survival and preserves lung development. Potentiating early NFB activity after hyperoxic exposure may represent a therapeutic intervention to prevent BPD.
There are 190,600 cases of acute respiratory distress syndrome (ARDS) each year in the United States. Patients with ARDS have alveolar epithelial injury, which may be worsened by high pressure mechanical ventilation. Alveolar type II (ATII) cells are the progenitor cells for the alveolar epithelium and are required to re-establish the alveolar epithelium during the recovery process from ARDS. Lung fibroblasts (FBs) migrate and proliferate early after lung injury and likely are an important source of growth factors for epithelial repair. However, how lung FBs affect epithelial wound healing in the human adult lung has not been investigated in detail. Hepatocyte growth factor (HGF) is known to be released mainly from FBs and to stimulate both migration and proliferation of primary rat ATII cells. HGF is also increased in lung tissue, bronchoalveolar lavage fluid (BALF) and serum in patients with ARDS. Therefore, we hypothesized that HGF secreted by FBs would enhance wound closure in alveolar epithelial cells (AECs). Wound closure was measured using a scratch wound healing assay in primary human AEC monolayers and in a co-culture system with FBs. We found that wound closure was accelerated by FBs mainly through HGF/c-met signaling. HGF also restored impaired wound healing in AECs from the elderly subjects and after exposure to cyclic stretch. We conclude that HGF is the critical factor released from FBs to close wounds in human AEC monolayers and suggest that HGF is a potential strategy for hastening alveolar repair in patients with ARDS.
Epithelial injury is often detected in lung allografts however its relation to rejection pathogenesis is unknown. We hypothesized that sterile epithelial injury can lead to alloimmune activation in the lung. We performed adoptive transfer of mismatched splenocytes into recombinant activating gene 1 (Rag1) deficient mice to induce an alloimmune status, then exposed these mice to naphthalene to induce sterile epithelial injury. We evaluated lungs for presence of alloimmune lung injury, endoplasmic reticulum (ER) stress, and hyaluronan expression, examined the effect of ER stress induction on hyaluronan expression and lymphocyte trapping by bronchial epithelia in vitro, and examined airways from patients with bronchiolitis obliterans syndrome (BOS) and normal controls histologically. We found that Rag1 deficient mice which received mismatched splenocytes and naphthalene injection displayed bronchial epithelial ER stress, peribronchial hyaluronan expression, and lymphocytic bronchitis. Bronchial epithelial ER stress led to the expression of lymphocyte-trapping hyaluronan cables in vitro. Blockade of hyaluronan binding ameliorated naphthalene-induced lymphocytic bronchitis. ER stress was present histologically in over 40% of bronchial epithelia of BOS patients, and associated with subepithelial hyaluronan deposition. We conclude that sterile bronchial epithelial injury in the context of alloimmunity can lead to sustained ER stress, and promote allograft rejection through hyaluronan expression.
Idiopathic pulmonary fibrosis is a progressive and lethal disease, characterized by loss of lung elasticity and alveolar surface area, secondary to alveolar epithelial cell injury, reactive inflammation, proliferation of fibroblasts and deposition of extracellular matrix. The effects of oropharyngeal aspiration of bleomycin in Sprague Dawley rats and C57BL/6 mice, as well as of intra-tracheal administration of ovalbumin to actively sensitized Brown Norway rats on total lung volume as assessed non-invasively by magnetic resonance imaging (MRI) were investigated here. Lung injury and volume were quantified using non-gated or respiratory-gated MRI acquisitions (ultrashort echo time, UTE, or gradient-echo techniques). Lung function of bleomycin-challenged rats was examined additionally using a flexyVent® system. Post-mortem analyses included histology of collagen and hydroxyproline assays. Bleomycin induced an increase of MRI-assessed total lung volume, lung dry and wet weights, hydroxyproline content as well as collagen amount. In bleomycin-treated rats, gated-MRI showed an increased volume of the lung in the inspiratory and expiratory phases of the respiratory cycle, and a temporary decrease of tidal volume. Decreased dynamic lung compliance was found in bleomycin-challenged rats. Bleomycin-induced increase of MRI-detected lung volume was consistent with tissue deposition during fibrotic processes resulting in decreased lung elasticity, while influences by edema or emphysema could be excluded. In ovalbumin-challenged rats, total lung volume quantified by MRI remained unchanged. The somatostatin analogue, SOM230, was shown to have therapeutic effects on established bleomycin-induced fibrosis in rats. This work suggests MRI-detected total lung volume as readout for tissue-deposition in small rodent bleomycin models of pulmonary fibrosis.
Autophagy plays a pivotal role in cellular homeostasis and adaptation to adverse environments, though the regulation of this process remains incompletely understood. We have recently observed that caveolin-1 (Cav-1), a major constituent of lipid rafts on plasma membrane, can regulate autophagy in cigarette smoking-induced injury of lung epithelium, whereas the underlying molecular mechanisms remain incompletely understood. In the present study we found that Cav-1 interacted with and regulated the expression of ATG12-ATG5, an ubiquitin-like conjugation system crucial for autophagosome formation, in lung epithelial Beas-2B cells. Deletion of Cav-1 increased basal and starvation-induced levels of ATG12-ATG5 and autophagy. Biochemical analyses revealed that Cav-1 interacted with ATG5, ATG12, and their active complex ATG12-ATG5. Overexpression of ATG5 or ATG12 increased their interactions with Cav-1, the formation of ATG12-ATG5 conjugate, and the subsequent basal levels of autophagy, but resulted in decreased interactions between Cav-1 and another molecule. Knockdown of ATG12 enhanced the ATG5-Cav-1 interaction. Mutation of the Cav-1 binding motif on ATG12 disrupted their interaction and further augmented autophagy. Cav-1 also regulated the expression of ATG16L, another autophagy protein associating with the ATG12-ATG5 conjugate during autophagosome formation. Altogether these studies clearly demonstrate that Cav-1 competitively interacts with the ATG12-ATG5 system to suppress the formation and function of the latter in lung epithelial cells, thereby providing new insights into the molecular mechanisms by which Cav-1 regulates autophagy, and suggesting the important function of Cav-1 in certain lung diseases via regulation of autophagy homeostasis.
Pulmonary hypertension (PH) is a disease with a poor prognosis characterized by a vascular remodeling process and an increase in pulmonary vascular resistance. While a variety of reports demonstrated that exercise training exerts beneficial effects on exercise performance and quality of life in PH patients it is not known how physical exercise affects vascular remodeling processes occurring in hypoxia-induced PH. Therefore, we investigated the effect of individualized exercise training on the development of hypoxia-induced PH in mice. Training effects were compared to pharmacological treatment with the PDE5 inhibitor Sildenafil or a combination of training plus Sildenafil. Trained mice who received Sildenafil showed a significantly improved walking distance (from 88.9±8.1m to 146.4±13.1m) and maximum oxygen consumption (VO2max from 93.3 ± 2.9% to 105.5 ± 2.2% in combination with Sildenafil, to 102.2 ± 3.0% with Placebo) compared to sedentary controls. Right ventricular systolic pressure, measured by telemetry, was at the level of healthy normoxic animals, whereas right heart hypertrophy did not benefit from training. Most interestingly, the increase in small pulmonary vessel muscularization was prevented by training. Respective counter regulatory processes were detected for the NO-soluble guanylate cyclase-phosphodiesterase system. We conclude that individualized daily exercise can prevent vascular remodeling in hypoxia-induced PH.
Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in 3-dimensional collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 siRNA were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without TGF-ß1 for five days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a non-specific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9 deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9 deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9 deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-ß1 and reduced several TGF-ß1 driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Since TGF-ß1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-ß1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3-dimensional collagen gels mediated by fibroblasts.
We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan rich viral envelope proteins including the hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or, for one viral strain, increased neutralizing activity compared with D325A+R343V. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan site on the head region of the HA and are resistant to inhibition by native SP-D. In contrast, D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains and reduced uptake of Cal09 by epithelial cells. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild type SP-D NCRD did not. All known human pandemic strains have at least one glycan attachment on the HA head and our results indicate that they may be susceptible to inhibition by modified host defense lectins.
Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause Tau phosphorylation and microtubule breakdown. Microtubule breakdown causes inter-endothelial cell gap formation and tissue edema. While ExoY transiently induces inter-endothelial cell gap formation, it remains unclear as to whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for six hours, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY+), one with an inactive ExoY (PA103exoUexoT::Tc pUCPexoYK81M; ExoYK81M), and one that lacks PcrV required for a functional T3SS (PcrV). ExoY+ induced inter-endothelial cell gaps, whereas ExoYK81M and PcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intra-tracheal inoculation of ExoY+ and ExoYK81M caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved one-week following ExoYK81M infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY+ infection. ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury.
Calu-3 is a well differentiated human bronchial cell line with the characteristics of the serous cells of airway submucosal glands. The submucosal glands play a major role in mucociliary clearance because they secrete electrolytes that facilitate airway hydration. Given the significance of both long- and short-term β-adrenergic receptor agonists in the treatment of respiratory diseases, it is important to determine the role of these receptors and their ligands in normal physiological function. The current studies were designed to characterize the effect of epinephrine, the naturally occurring β-adrenergic receptor agonist, on electrolyte transport of the airway serous cells. Interestingly epinephrine stimulated two anion secretory channels, the cystic fibrosis transmembrane conductance regulator and a Ca2+-activated Cl- channel, with the characteristics of TMEM16A, thereby potentially altering mucociliary clearance via multiple channels. Consistent with the dual channel activation, epinephrine treatment resulted in increases in both intracellular cAMP and Ca2+ Furthermore, the current results extend previous reports indicating that the two anion channels are functionally linked.
MARCKS is postulated to regulate the passage of secretory granules through cortical actin in the early phase of exocytosis. There are, however, three proposed mechanisms of action, all of which were derived from studies using synthetic peptides representing either the central phosphorylation site domain, or the upstream, N-terminal domain: it tethers actin to the plasma membrane and/or to secretory granules, and/or it sequesters PIP2. Using MARCKS null mice, we probed for a loss of function secretory phenotype in mast cells harvested from embryonic livers and maturated in vivo (eHMCs). Both WT and MARCKS null eHMCs exhibited full exocytic responses upon FcRI receptor activation with DNP-BSA, whether they were in suspension or adherent. The secretory responses of MARCKS null eHMCs were consistently higher than that of WT cells, but the differences had sporadic statistical significance. The MARCKS null cells exhibited faster secretory kinetics, however, achieving the plateau phase of the response with a t1/2 ~2.5-fold faster. Hence, MARCKS appears to be a non-essential regulatory protein in mast cell exocytosis, but exerts a negative modulation. Surprisingly, the MARCKS N-terminal peptide, MANS, which has been reported to inhibit mucin secretion from airway goblet cells (J Biol Chem 276: 40982-40990, 2001), inhibited hexosaminidase secretion from WT and MARCKS null eHMCs, leading us to re-examine its effects on mucin secretion. Results from studies using peptide inhibitors with human bronchial epithelial cells and with binding assays using purified mucins, suggested that MANS inhibited the mucin binding assay, rather than the secretory response.
Background: Up-regulation of the erythropoietin (EPO) /EPO-receptor (EPOR) system plays protective role against chronic hypoxia-induced pulmonary hypertension (hypoxic PH) through enhancement of endothelial nitric oxide (NO)-mediated signaling. Genistein (Gen), a phytoestrogen, is considered to ameliorate NO-mediated signaling. Objective: We hypothesized Gen attenuates and prevents hypoxic PH. Methods and Results: In vivo, Sprague-Dawley rats raised in a hypobaric chamber were treated with Gen (60mkg·kg-1) for 21 days. Pulmonary hemodynamics and vascular remodeling were ameliorated in Gen-treated hypoxic PH rats. Gen also restored cGMP levels, phosphorylated endothelial NO synthase (eNOS) at Ser1177 and Akt at Ser473 expression in the lungs. Additionally, Gen potentiated plasma EPO concentration and EPOR-positive endothelial cell counts. In experiments with hypoxic PH rats' isolated perfused lungs, Gen caused NO- and phosphatidylinositol 3-kinase (PI3K)/Akt-dependent vasodilation that reversed abnormal vasoconstriction. In vitro, a combination of EPO and Gen increased the phosphorylation of eNOS and the EPOR expression in human umbilical vein endothelial cells under a hypoxic environment. Moreover, Gen potentiated the hypoxic increase in EPO production from human hepatoma cells. Conclusions: We conclude that Gen may be effective for the prevention of hypoxic PH through the improvement of PI3K/Akt-dependent NO-mediated signaling in association with enhancement of the EPO/EPOR system.
Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exerts its beneficial effect, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AEC) to hypoxia (3%O2) plus cytomix, a combination of IL-1β, TNF-α and IFN-. MSC-M was collected from human MSC exposed for 12 hours to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSC in the injured lung. Comparison of paracrine soluble factors in MSC media showed that interleukin-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was 2-fold lower in HCYT-MSC-M compared to MSC-M. In AEC, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS-Isc) and also decreased the number of α-ENaC subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 hours to AEC exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M was unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.
The lungs are among general organs that can undergo irreversible damage from chronic alcohol consumption. Herein, we developed an animal model predisposed for edematous lung injury following chronic ingestion of alcohol in order to better understand the etiology of alcohol related disorders. Using animal modeling, alongside high throughput proteomic and microarray assays, we identified changes in lung protein and transcript in mice and rats, respectively, following chronic alcohol ingestion or a caloric control diet. Liquid chromatography-mass spectrometry (LC-MS/MS) identified several mitochondrial-related proteins in which the expression was up-regulated following long term alcohol ingestion in mice. Consistent with these observations, rat gene chip microarray analysis of alveolar cells obtained from animals maintained on a Lieber-DeCarli liquid alcohol diet confirmed significant changes in mitochondrial-related transcripts in the alcohol lung. Transmission electron microscopy (TEM) revealed significant changes in the mitochondrial architecture in alcohol mice, particularly following lipopolysacharide (LPS) exposure. Chronic alcohol ingestion was also shown to worsen mitochondrial respiration, mitochondrial membrane polarization, and NAD+/NADH ratios in alveolar type 2 cells. In summary, our studies show causal connection between chronic alcohol ingestion and mitochondrial dysfunction; albeit the specific role of each of the mitochondrial-related proteins and transcripts identified in our study requires additional study.
Though many lung diseases are associated with hypoxia, alveolar type II epithelial (ATII) cell impairment, and pulmonary surfactant dysfunction, the effects of oxygen limitation on metabolic pathways necessary to maintain cellular energy in ATII cells have not been studied extensively. This report presents results of targeted assays aimed at identifying specific metabolic processes that contribute to energy homeostasis using primary ATII cells and a model ATII cell line, MLE-15, cultured in normoxic and hypoxic conditions. MLEs cultured in normoxia demonstrated a robust oxygen consumption rate (OCR) coupled to ATP generation and limited extracellular lactate production, indicating reliance on oxidative phosphorylation for ATP production. Pharmacological uncoupling of respiration increased OCR in normoxic cultures to 175% of basal levels, indicating significant spare respiratory capacity. However, when exposed to hypoxia for 20 hours, basal oxygen consumption fell to 60% of normoxic rates and cells maintained only ~50% of normoxic spare respiratory capacity, indicating suppression of mitochondrial function, though intracellular ATP levels remained at near normoxic levels. Moreover, while hypoxic exposure stimulated glycogen synthesis and storage in MLE-15, glycolytic rate (as measured by lactate generation) was not significantly increased in the cells, despite enhanced expression of several enzymes related to glycolysis. These results were largely recapitulated in murine primary ATII, demonstrating MLE-15 suitability for modeling ATII metabolism. The ability of ATII cells to maintain ATP levels in hypoxia without enhancing glycolysis suggests that these cells are exceptionally efficient at conserving ATP in order to maintain bioenergetic homeostasis under O2-limitation.
In vivo imaging is an important tool for pre-clinical studies of lung function and disease. The widespread availability of multimodal animal imaging systems and the rapid rate of diagnostic contrast agent development has empowered researchers to non-invasively study lung function and pulmonary disorders. Investigators can identify, track, and quantify biological processes over time. In this review, we highlight the fundamental principles of bioluminescence, fluorescence, planar X-ray, X-ray computed tomography (CT), magnetic resonance imaging (MRI), and nuclear imaging modalities (such as positron emission tomography and single photon emission computed tomography; PET and SPECT) that have been successfully employed for the study of lung function and pulmonary disorders in a pre-clinical setting. The major principles, benefits, and applications of each imaging modality and technology are reviewed. Limitations and the future prospective of multimodal imaging in pulmonary physiology are also discussed. In vivo imaging bridges molecular biological studies, drug design and discovery, and the imaging field with modern medical practice, and as such, will continue to be a mainstay in biomedical research.
Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically-active NEU3 sialidase.
Oxidative and carbonyl stress is increased in lungs of smokers and patients with chronic obstructive pulmonary disease (COPD), as well as in cigarette smoke (CS)-exposed rodent lungs. We previously showed that sirtuin1 (SIRT1), an anti-aging protein, is reduced in lungs of CS-exposed mice and COPD patients, and that SIRT1 attenuates CS-induced lung inflammation and injury. It is not clear whether SIRT1 protects against CS-induced lung oxidative stress. Therefore, we determined the effect of SIRT1 on lung oxidative stress and antioxidants in response to CS exposure using loss and gain of function approaches, as well as a pharmacological SIRT1 activation by SRT1720. We found that CS exposure increased protein oxidation and lipid peroxidation in lungs of WT mice, which was further augmented in SIRT1 deficient mice. Furthermore, both SIRT1 genetic overexpression and SRT1720 treatment significantly decreased oxidative stress induced by CS exposure. FOXO3 deletion augmented lipid peroxidation products but reduced antioxidants in response to CS exposure, which was not affected by SRT1720. Interestingly, SRT1720 treatment exhibited a similar effect on lipid peroxidation and antioxidants (i.e., MnSOD, HO-1 and NQO-1) in WT and Nrf2 deficient mice in response to CS exposure. This indicates that SIRT1 protects against CS-induced oxidative stress, which is mediated by FOXO3, but is independent of Nrf2. Overall, these findings reveal a novel function of SIRT1, which is to reduce CS-induced oxidative stress, and this may contribute to its protective effects against lung inflammation and subsequent development of COPD.
Chronic allergic asthma leads to airway remodeling and subepithelial fibrosis via mechanisms not fully understood. Airway remodeling is amplified by pro-fibrotic mediators, such as TGF-β1, which plays a cardinal role in various models of fibrosis. We recently have identified a critical role for JNK1 in augmenting the pro-fibrotic effects of TGF-β1, linked to epithelial to mesenchymal transition of airway epithelial cells. To examine the role of JNK1 in HDM-induced airway remodeling, we induced allergic airway inflammation in WT and JNK1-/- mice by administration of HDM extract. WT and JNK1-/- mice were sensitized with intranasal aspirations of HDM extract for 15 days over three weeks. HDM caused similar increases in airway hyperresponsiveness, mucus metaplasia and airway inflammation in WT and JNK1-/- mice. In addition, the pro-fibrotic cytokine TGF-β1 and phosphorylation of Smad3 were equally increased in WT and JNK1-/- mice. In contrast, increases in collagen content in lung tissue induced by HDM were significantly attenuated in JNK1-/- mice compared to WT controls. Furthermore HDM-induced increases of α-SMA protein and mRNA expression as well as the mesenchymal markers HMGA2 and collagen1A1 in WT mice were attenuated in JNK1-/- mice. The let7 family of microRNAs has previously been linked to fibrosis. HDM exposure in WT mice and primary lung epithelial cells resulted in striking decreases in let-7g miRNA that were not observed in mice or primary lung epithelial cells lacking JNK1-/- mice. Overexpression of let-7g in lung epithelial cells reversed the HDM-induced increases in α-SMA. Collectively, these findings demonstrate an important requirement for JNK1 in promoting HDM- induced fibrotic airway remodeling.
The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS), disrupts this balance and enhances mitochondrial dysfunction in the airway. In non-asthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein Drp1 and decreased fusion protein Mfn2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved ROS, activation of ERK, PI3/Akt, PKC and proteasome pathways, as well as transcriptional regulation via factors such as NFB and Nrf2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, while blocking Mfn2 had the opposite, detrimental effect. In ASM from asthmatics, mitochondria exhibited substantial morphological defects at baseline, and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.
Variation in Surfactant Protein D (SP-D) is associated with lung function in tobacco smoke-induced chronic respiratory disease. We hypothesized that the same association exists in the general population and could be used to identify individuals sensitive to smoke-induced lung damage. The association between serum SP-D (sSP-D) and expiratory lung function was assessed in a cross-sectional design in a Danish twin population (N=1,512, 18-72 years old). The adjusted heritability estimates for expiratory lung function, associations between SP-D gene (SFTPD) single-nucleotide polymorphisms or haplotypes, and expiratory lung function were assessed using twin study methodology and mixed-effects models. Significant inverse associations were evident between sSP-D and the forced expiratory volume in 1 second and forced vital capacity in the presence of current tobacco smoking but not in non-smokers. The two SFTPD single-nucleotide polymorphisms, rs1923536 and rs721917, and haplotypes including these single-nucleotide polymorphisms or rs2243539, were inversely associated with expiratory lung function in interaction with smoking. In conclusion, SP-D is phenotypically and genetically associated with lung function measures in interaction with tobacco smoking. The obtained data suggest sSP-D as a candidate biomarker in risk assessments for subclinical tobacco smoke-induced lung damage. The data and derived conclusion warrant confirmation in a longitudinal population following chronic obstructive pulmonary disease initiation and development.
Non-motile primary cilia are recognized as important sensory organelles during development and normal biological functioning. For example, recent work demonstrates that transcriptional regulators of the sonic hedgehog signaling pathway localize to primary cilia and participate in sensing and transducing signals regarding the cellular environment. In contrast, motile cilia are traditionally viewed as mechanical machinery, vital for the movement of solutes and clearance of bacteria and debris, but not participants in cellular sensing and signaling mechanisms. Recently, motile cilia were found to harbor receptors responsible for sensing and responding to environmental stimuli. However, no transcription factors are known to be regulated by cilia localization as a sensing mechanism in vertebrates. Using a mouse model of organic dust-induced airway inflammation, we found that the transcription factor serum response factor (SRF) localizes to motile cilia of airway epithelial cells and alters its localization in response to inflammatory stimuli. Furthermore, inhibition of SRF signaling using the small molecule CCG-1423 reduces organic dust-induced IL-8 release from bronchial epithelial cells, and stimulates cilia beat frequency in ciliated mouse tracheal epithelial cells. Immunohistochemical analyses reveal SRF localizes to the cilia of mouse brain ependymal and ovarian epithelial cells as well. These data reveal a novel mechanism by which a transcription factor localizes to motile cilia and modulates cell activities including cilia motility and inflammation response. These data challenge current dogma regarding motile cilia functioning, and may lead to significant contributions in understanding motile ciliary signaling dynamics, as well as mechanisms involving SRF-mediated responses to inflammation and injury.
The expression of c-kit and its ligand, stem cell factor (SCF), in developing human lung tissue was investigated by immunohistochemistry. Twenty-eight human fetal lungs [range: 13 to 38 gestational weeks, (GW)] and 12 postnatal lungs (range: 1 to 79 years old) were evaluated. We identified c-kit+ cells in the lung mesenchyme as early as 13 GW. These mesenchymal c-kit+ cells in the lung did not express mast cell tryptase or α-smooth muscle actin. However, these cells did express CD34, VEGFR2 and Tie-2, indicating their endothelial lineage. Three-dimensional reconstructions of confocal laser scanning images revealed that c-kit+ cells displayed a closed-end tube formation that did not contain hematopoietic cells. From the pseudoglandular to the canalicular phase, c-kit+ cells appeared to continuously proliferate, to connect with the central pulmonary vessels and finally to develop the lung capillary plexus. The spatial distribution of c-kit and SCF positive cells was also demonstrated, and these cells were shown to be in close association. Our results suggest that c-kit expression the early fetal lungs marks a progenitor population that is restricted to the endothelial lineage. This study also suggests the potential involvement of c-kit signaling in lung vascular development.
Maturation of newly formed vessels is a multi-step phenomenon during which functional endothelial barriers are established. Disruption of vessel integrity is an important feature in many physiological and pathological processes. We previously reported that lymphoblastic leukemia-derived sequence 1 (LYL1) is required for the late stages of post-natal angiogenesis to limit the formation of new blood vessels, notably by regulating the activity of the small GTPase RAP1. In this study, we show that LYL1 is also required during the formation of the mature endothelial barrier in the lungs of adult mice. Specifically LYL1 knockdown in human endothelial cells down-regulated the expression of ARHGAP21 and ARHGAP24, which encode two Rho GTPase-activating proteins, and this was correlated with increased RhoA activity and reorganization of the actin cytoskeleton into stress fibers. Importantly, in lungs of Lyl1-deficient mice, both VE-cadherin and p120-catenin were poorly recruited to endothelial adherens junctions, indicative of defective cell-cell junctions. Consistent with this higher Evans blue dye extravasation, edema and leukocyte infiltration in the lung parenchyma of Lyl1-/- mice than in wild type littermates confirmed that lung vascular permeability is constitutively elevated in Lyl1-/- adult mice. Our data show that LYL1 acts as a stabilizing signal for adherens junction formation by operating upstream of VE-cadherin and of the two GTPases RAP1 and RhoA. As increased vascular permeability is a key feature and a major mechanism of acute respiratory distress syndrome, molecules that regulate LYL1 activity could represent additional tools to modify the endothelial barrier permeability.
Bronchopulmonary dysplasia (BPD), a lung disease of prematurely-born infants, is characterized in part by arrested development of pulmonary alveolae. We hypothesized that heme oxygenase (HO-1) and its byproduct carbon monoxide (CO), which are thought to be cytoprotective against redox stress, mitigate lung injury and alveolar simplification in hyperoxia-exposed neonatal mice, a model of BPD. Three-day old C57BL/6J mice were exposed to air or hyperoxia (FiO2, 75%) in the presence or absence of inhaled CO (250 ppm for 1 h twice daily) for 21 days. Hyperoxic exposure increased mean linear intercept, a measure of alveolar simplification, whereas CO treatment attenuated hypoalveolarization, yielding a normal-appearing lung. Conversely, HO-1 null mice showed exaggerated hyperoxia-induced hypoalveolarization. CO also inhibited hyperoxia-induced pulmonary accumulation of F4/80+, CD11c+ and CD11b+ monocytes and Gr-1+ neutrophils. Further, CO attenuated lung mRNA and protein expression of pro-inflammatory cytokines including the monocyte chemoattractant CCL2 in vivo, and decreased hyperoxia-induced lung type I alveolar epithelial cell CCL2 production in vitro. Hyperoxia-exposed CCL2 null mice, like CO-treated mice, showed attenuated alveolar simplification and lung infiltration of CD11b+ monocytes, consistent with the notion that CO blocks lung epithelial cell cytokine production. We conclude that, in hyperoxia-exposed neonatal mice, inhalation of CO suppresses inflammation and alveolar simplification.
The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring more normal alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs which had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as < 10%/hour), the lungs received either hMSC (5 x106 cells) added to the perfusate or perfusion only as a control. AFC was measured in a different lung lobe at 4 hours. Intravenous hMSC restored AFC in the injured lungs to a normal level. In contrast, perfusion only did not increase AFC. This positive effect on AFC was reduced by intra-bronchial administration of a neutralizing antibody to keratinocyte growth factor (KGF). Thus, intravenous allogeneic hMSCs are effective in restoring the capacity of the alveolar epithelium to remove alveolar fluid at a normal rate, suggesting that this therapy may be effective in enhancing the resolution of pulmonary edema in human lungs deemed clinically unsuitable for transplantation.
Fibrogenesis involves a pathologic accumulation of activated fibroblasts and extensive matrix remodeling. Profibrotic cytokines, such as TGFβ, stimulate fibroblasts to overexpress fibrotic matrix proteins and induce further expression of profibrotic cytokines resulting in progressive fibrosis. Connective tissue growth factor (CTGF) is a profibrotic cytokine that is indicative of fibroblast activation. Epithelial cells are abundant in the normal lung but their contribution to fibrogenesis remains poorly defined. Profibrotic cytokines may activate epithelial cells with protein expression and functions that overlap with the functions of active fibroblasts. We found that alveolar epithelial cells undergoing TGFβ-mediated mesenchymal transition in vitro were also capable of activating lung fibroblasts through production of CTGF. Alveolar epithelial cell expression of CTGF was dramatically reduced by inhibition of Rho signaling. CTGF reporter mice demonstrated increased CTGF promoter activity by lung epithelial cells acutely after bleomycin in vivo. Furthermore, mice with lung epithelial cell-specific deletion of CTGF had an attenuated fibrotic response to bleomycin. These studies provide direct evidence that epithelial cell activation initiates a cycle of fibrogenic effector cell activation during progressive fibrosis. Therapy targeted at epithelial cell production of CTGF offers a novel pathway for abrogating this progressive cycle and limiting tissue fibrosis.
Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations, however their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from PA of hypoxic (VVEC-Hx) compared to control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control, but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.
Acute respiratory distress syndrome (ARDS) is a devastating disease with distinct pathological stages. Fundamental to ARDS is the acute onset of lung inflammation as a part of the body's immune response to a variety of local and systemic stimuli. In patients surviving the inflammatory and subsequent fibroproliferative stages, transition from injury to resolution and recovery is an active process dependent on a series of highly coordinated events regulated by the immune system. Experimental animal models of acute lung injury reproduce key components of the injury and resolution phases of human ARDS, and provide a methodology to explore mechanisms and potential new therapies. Macrophages are essential to innate immunity and host defense, playing a featured role in the lung and alveolar space. Key aspects of their biological response, including differentiation, phenotype, function, and cellular interactions, are determined in large part by the presence, severity, and chronicity of local inflammation. Studies support the importance of macrophages to initiate and maintain the inflammatory response, as well as a determinant of resolution of lung inflammation and repair. We will discuss distinct roles for lung macrophages during early inflammatory and late resolution phases of ARDS using experimental animal models. In addition, each section will highlight human studies that relate to the diverse role of macrophages in initiation and resolution of acute lung injury (ALI) and ARDS.
The epidermal growth factor receptor (EGFR) family of receptors and the transforming growth factor-β (TGF-β) family are active in diverse biological processes and are central mediators in the initiation and maintenance of fibrosis in many diseases. Transforming growth factor-α (TGFα) is a ligand for the EGFR, and doxycycline (Dox)-inducible transgenic mice conditionally expressing TGFα specifically in the lung epithelium develop progressive fibrosis. The αvβ6 integrin is an important in vivo activator of TGF-β activation in the lung. Immunohistochemical analysis of αvβ6 protein expression and bronchoalveolar analysis of TGFβ pathway signaling indicates activation of the αvβ6/TGFβ pathway only at later time points after lung fibrosis was already established in the TGFα model. To determine the contribution of the αvβ6/TGFβ pathway on the progression of established fibrotic disease, TGFα transgenic mice were administered Dox for 4 weeks which leads to extensive fibrosis; these mice were then treated with a function blocking anti-αvβ6 antibody with continued administration of Dox for an additional 4 weeks. Compared to TGFα transgenic mice treated with control antibody, αvβ6 inhibition significantly attenuated pleural thickening and altered the decline in lung mechanics. To test the effects of genetic loss of the β6 integrin, TGFα transgenic mice were mated with β6 null mice and the degree of fibrosis compared in adult mice following 8 weeks of Dox administration. Genetic ablation of the β6 integrin attenuated histologic and physiologic changes in the lungs of TGFα transgenic mice although a significant degree of fibrosis still developed.
Arrested alveolarization is the pathological hallmark of bronchopulmonary dysplasia (BPD), a complication of premature birth. Here, the impact of systemic application of hydrogen sulfide (H2S) on post-natal alveolarization was assessed in a mouse BPD model. Exposure of newborn mice to 85% O2 for 10 days reduced the total lung alveoli number by 56% and increased alveolar septal wall thickness by 29%, as assessed by state-of-the-art stereological analysis. Systemic application of H2S using the slow-release H2S donor GYY4137 for 10 days resulted in pronounced improvement in lung alveolarization in pups breathing 85% O2, compared with vehicle-treated littermates. Although without impact on lung oxidative status, systemic H2S blunted leukocyte infiltration into alveolar airspaces provoked by hyperoxia, and restored normal lung interleukin 10 levels that were otherwise depressed by 85% O2. Treatment of primary mouse alveolar type II (ATII) cells with the rapid-release H2S donor NaHS had no impact on cell viability; however, NaHS promoted ATII cell migration. While exposure of ATII cells to 85% O2 caused dramatic changes in mRNA expression, exposure to either GYY4137 or NaHS had no impact on ATII cell mRNA expression, as assessed by microarray, suggesting that the effects observed were independent of changes in gene expression. The impact of NaHS on ATII cell migration was attenuated by glibenclamide, implicating ion channels; and was accompanied by activation of Akt, hinting at two possible mechanisms of H2S action. These data support further investigation of H2S as a candidate interventional strategy to limit the arrested alveolarization associated with BPD.
Extracellular proteases including matrix metalloproteinases (MMPs) are speculated to play a significant role in chronic lung diseases such as asthma. Although increased protease expression has been correlated with lung pathogenesis, the relationship between localized enzyme activity and disease progression remains poorly understood. We report the application of MMP-2/9 activatable cell-penetrating peptides (ACPPs) and their ratiometric analogs (RACPPs) for in vivo measurement of protease activity and distribution in the lungs of mice that were challenged with the allergen ovalbumin. MMP-2/9 activity was increased > 2-fold in whole, dissected lungs from acutely challenged mice compared to control mice (p = 1.8x10-4). This upregulation of MMP-2/9 activity was localized around inflamed airways with 1.6-fold higher protease-dependent ACPP uptake surrounding diseased airways compared to adjacent, pathologically normal lung parenchyma (p = 0.03). MMP-2/9 activity detected by ACPP cleavage co-localized with gelatinase activity measured with in situ DQ gelatin. For comparison, neutrophil elastase activity and thrombin activity, detected with elastase- and thrombin-cleavable RACPPs, respectively, were not significantly elevated in acutely allergen challenged mouse lungs. The results demonstrate that ACPPs, like the MMP2/9-activated and related ACPPs, allow for real-time detection of protease activity in a murine asthma model, which should improve our understanding of protease activation in asthma disease progression and help elucidate new therapy targets or act as a mechanism for therapeutic drug delivery.
We investigated the role of flow-induced shear stress on the mechanisms regulating surfactant secretion in type II alveolar epithelial cells (ATII) using microfluidic models. Following flow stimulation spanning a range of wall shear stress (WSS) magnitudes, monolayers of ATII (MLE-12 and A549) cells were examined for surfactant secretion by evaluating essential steps of the process including relative changes in the number of fusion events of lamellar bodies (LBs) with the plasma membrane (PM) and intracellular redistribution of LBs. F-actin cytoskeleton and calcium levels were analyzed in A549 cells subjected to WSS spanning 4-20 dyn/cm2. Results reveal an enhancement in LB fusion events with the PM in MLE12 cells upon flow stimulation, while A549 cells exhibit no foreseeable changes in the monitored number of fusion events for WSS levels ranging up to a threshold of ~8 dyn/cm2. Above this threshold, we witness instead a decrease in LB fusion events in A549 cells. Yet, patterns of LB redistribution suggest that WSS can potentially serve as a stimulus for A549 cells to trigger the intracellular transport of LBs towards the cell periphery. This observation is accompanied by a fragmentation of F-actin indicating that disorganization of the F-actin cytoskeleton might act as a limiting factor for LB fusion events. Moreover, we note a rise in cytosolic calcium ([Ca2+]c) levels following stimulation of A549 cells with WSS magnitudes ranging near or above the experimental threshold. Overall, WSS stimulation can influence key components of molecular machinery for regulated surfactant secretion in ATII cells in vitro.
Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/β-catenin-dependent induction of lymphoid enhancer factor (Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred post-transcriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus, we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/β-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing β-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.
The elderly are at much higher risk for developing pneumonia than younger individuals. Pneumonia is a leading cause of death and the third most common reason for hospitalization in the elderly. One reason that the elderly may be more susceptible to pneumonia is a breakdown in the lung's first line of defense, mucociliary clearance. Cilia beat in a coordinated manner to propel out invading microorganisms and particles. Ciliary beat frequency (CBF) is known to slow with aging, however little is known about the mechanism(s). We compared the CBF in BALB/c and C57BL/6 mice aged 2 months, 12 months and 24 months and found that CBF diminishes with age. Both the 12 and 24-month cilia retained their ability to be stimulated by the β2 agonist, procaterol. To help determine the mechanism of ciliary slowing, we measured PKCα and PKC activity. There were no activity differences in PKCα; however, we demonstrate a significantly higher PKC activity in the 12- and 24-month mice than the 2-month mice. The increase in activity is likely due to a nearly 3-fold increase in PKC protein in the lung during aging. To strengthen the connection between PKC activation and ciliary slowing, we treated 2-month tracheas with the PKC agonist, 8-[2-(2-pentylcyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA). We noted a similar decrease in baseline CBF, and the cilia remained sensitive to stimulation with β2 agonists. Conclusions: In this mouse model of aging, we show that the decreases in CBF are related to an increase in PKC activity.
Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) eliminates many epigenetic modifications that characterize differentiated cells. In this study we tested whether functional differences between COPD and non-COPD fibroblasts could be reduced utilizing this approach. Primary fibroblasts from non-COPD and COPD patients were reprogrammed to iPSCs. Reprogrammed iPSCs were positive for oct3/4, nanog and sox2, formed embryoid bodies (EBs) in vitro, and induced teratomas in NOD/SCID mice. Reprogrammed iPSCs were then differentiated into fibroblasts (non-COPD-i and COPD-i, respectively) and were assessed either functionally by chemotaxis and gel contraction or for gene expression by microarrays and compared to their corresponding primary fibroblasts. Primary COPD fibroblasts contracted 3-dimentional (3-D) collagen gels and migrated toward fibronectin less robustly than non-COPD fibroblasts. In contrast, re-differentiated fibroblasts from iPSCs derived from the non-COPD and COPD fibroblasts were similar in response in both functional assays. Microarray analysis identified 1881 genes that were differentially expressed between primary COPD and non-COPD fibroblasts with 605 genes differing by more than 2-fold. After re-differentiation, 112 genes were differentially expressed between COPD-i and non-COPD-i with only three genes by more than 2-fold. Similar findings were observed with miRNA expression: 56 miRNAs were differentially expressed between non-COPD and COPD primary cells; after redifferentiation, only 3 miRNAs were differentially expressed between non-COPD-i and COPD-i fibroblasts. Interestingly, of the 605 genes that were differentially expressed between COPD and non-COPD fibroblasts, 293 genes were changed toward control after re-differentiation. Conclusions: Functional and epigenetic alterations of COPD fibroblasts can be reprogrammed through formation of iPSCs.
Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. As specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Targeted therapies to treat sepsis need to be developed. While an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury and reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1 and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared to non-septic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.
Phagocytosis of the bacterial pathogen Pseudomonas aeruginosa is the primary means by which the host controls bacterially-induced pneumonia during lung infection. Previous studies have identified flagellar swimming motility as a key PAMP recognized by phagocytes in order to initiate engulfment. Correspondingly, loss of flagellar motility is observed during chronic pulmonary infection with P. aeruginosa and this likely reflects a selection for bacteria resistant to phagocytic clearance. However, the mechanism underlying the preferential phagocytic response to motile bacteria is unknown. Here we have identified a cellular signaling pathway in alveolar macrophages and other phagocytes that is specifically activated by flagellar motility. Genetic and biochemical methods were employed to identify that phagocyte PI3K/Akt activation is required for bacterial uptake and, importantly, it is specifically activated in response to P. aeruginosa flagellar motility. Based on these observations, the second important finding that emerged from these studies is that titration of the bacterial flagellar motility results in a proportional activation state of Akt. Therefore, the Akt pathway is responsive to, and corresponds with, the degree of bacterial flagellar motility, is independent of the actin polymerization that facilitates phagocytosis, and determines the phagocytic fate of P. aeruginosa. These findings elucidate the mechanism behind motility-dependent phagocytosis of extracellular bacteria, and support a model whereby phagocytic clearance exerts a selective pressure on P. aeruginosa populations in vivo which contributes to changes in pathogenesis during infections.
Chronic airway diseases are characterized by inflammation and mucus overproduction. The MUC5AC mucin gene is upregulated by the pro-inflammatory cytokine IL1β via activation of CREB in the NCI-H292 cancer cell line and NFB in the HBE1 transformed cell line, with each transcription factor binding to a cognate cis-site in the proximal or distal region, respectively, of the MUC5AC promoter. We utilized primary differentiated human bronchial epithelial (HBE) and A549 lung adenocarcinoma cells to further investigate the contributions of CREB and NFB subunits to the IL1β-induced upregulation of MUC5AC. Data show that ligand binding of IL1β to the IL1β receptor is required to increase MUC5AC mRNA abundance. ChIP analyses show direct binding of CREB to the previously identified CRE site and binding of p65 and p50 subunits to a novel NFB site in a mucin-regulatory domain in the proximal promoter and to a previously identified NFB site in the distal promoter. p50 binds to both NFB sites at 1 h following IL1β exposure, but is replaced at 2 h by p65 in A549 cells and by a p50/p65 heterodimer in HBE cells. Thus, IL1β activates multiple domains in the MUC5AC promoter but exhibits some cell-specific responses, highlighting the complexity of MUC5AC transcriptional regulation. Data show that Dexamethasone, a glucocorticoid that transcriptionally represses MUC5AC gene expression under constitutive conditions, also represses IL1β-mediated upregulation of MUC5AC gene expression. A further understanding of mechanisms mediating MUC5AC regulation should lead to honing of therapeutic approaches for the treatment of mucus overproduction in inflammatory lung diseases.
The phospholipase A2 activity of peroxiredoxin 6 is inhibited by the transition state analogue, 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol (MJ33). This activity is required for the activation of NADPH oxidase, type 2. The present study evaluated the effect of MJ33 on manifestations of acute lung injury. Mice were injected intratracheally (IT) with lipopolysaccharide from E. coli 0111:B4 (LPS, 1 or 5 mg/kg), either concurrently with LPS or 2 h later, and evaluated for lung injury 24 h later. MJ33 inhibited reactive oxygen species (ROS) generation by lungs when measured at 24 h after LPS. LPS at either low or high dose significantly increased lung infiltration with inflammatory cells, secretion of pro-inflammatory cytokines (IL-6, TNF-α) and a chemokine (MIP-2), expression of lung vascular cell adhesion molecule (VCAM), lung permeability (protein in bronchoalveolar lavage fluid, leakage of FITC-dextran, lung wet to dry weight ratio), tissue lipid peroxidation (thiobarbituric acid reactive substances, 8-isoprostanes), tissue protein oxidation (protein carbonyls), and activation of NF-B. MJ33, given either concurrently or 2 h subsequent to LPS, significantly reduced all of these measured parameters. Previous studies of toxicity showed a high margin of safety for MJ33 in the intact mouse. Thus, we have identified MJ33 as a potent, non-toxic, and specific mechanism-based inhibitor of NOX2-mediated ROS generation that protects mice against lung injury associated with inflammation.
We previously observed that transgelin was preferentially expressed in human pulmonary arterial smooth muscle cells (PAMSCs) under hypoxia and that the upregulation of transgelin was independent of hypoxia inducible factor 1α (HIF-1α). Reduced transgelin expression was accompanied by significantly impaired migration ability in vitro. However, the regulation mechanism of transgelin and its function in preventing hypoxic pulmonary hypertension (HPH) was unclear. In the present study, RNA interference with hypoxia inducible factor 2α (HIF-2α) was employed in human PASMCs. Transgelin expression was diminished in HIF-2α-siRNA-treated cells at both the mRNA and protein levels under hypoxia. However, HIF-2α did not transactivate the transgelin promoter directly. TGF-β1 concentration in human PASMCs culture medium was higher under hypoxia, and the accumulated TGF-β1 under hypoxia was regulated by HIF-2α. Furthermore, luciferase and chromatin immunoprecipitation assays indicated that TGF-β1/Smad3 could bind to the transgelin promoter, resulting in increased transgelin expression. In addition to non-intact cellular migration, inhibition of transgelin expression resulted in impaired proliferation in vitro under hypoxia. A lentiviral vector used to inhibit transgelin expression was constructed and intratracheally instilled in rats three weeks prior to hypoxia treatment. Our final results indicated that inhibition of transgelin expression locally could attenuate increased right ventricular systolic pressure and its associated cardiac and pulmonary vessel remodeling under hypoxia. Our findings indicate that HIF-2α upregulates transgelin indirectly and that accumulated TGF-β1 is a mediator in the upregulation of transgelin by HIF-2α under hypoxia. Inhibition of transgelin expression locally could prevent HPH and pulmonary vascular remodeling in vivo.
Pseudomonas aeruginosa is an opportunistic bacterial pathogen responsible for a high incidence of acute and chronic pulmonary infection. These infections are particularly prevalent in patients with chronic obstructive pulmonary disease (COPD) and Cystic Fibrosis (CF): much of the morbidity and pathophysiology associated with these diseases is due to a hyper-susceptibility to bacterial infection. Innate immunity, primarily through inflammatory cytokine production, cellular recruitment, and phagocytic clearance by neutrophils and macrophages, is the key to endogenous control of P. aeruginosa infection. In this review, we highlight recent advances towards understanding the innate immune response to P. aeruginosa, with a focus on the role of phagocytes in control of P. aeruginosa infection. Specifically, we summarize the cellular and molecular mechanisms of phagocytic recognition and uptake of P. aeruginosa, and how current animal models of P. aeruginosa infection reflect clinical observations in the context of phagocytic clearance of the bacteria. Several notable phenotypic changes to the bacteria are consistently observed during chronic pulmonary infections, including changes to mucoidy and flagellar motility, that likely enable or reflect their ability to persist. These traits are likewise examined in the context of how the bacteria avoid phagocytic clearance, inflammation, and sterilizing immunity.
MicroRNAs (microRNAs) are small non-coding RNAs that inhibit protein expression. We have previously shown that the inhibition of the microRNA let-7d in epithelial cells caused changes consistent with Epithelial to Mesenchymal Transition (EMT) both in vitro and in vivo. The aim of this study was to determine whether introduction of let-7d into fibroblasts will alter their mesenchymal properties. Transfection of primary fibroblasts with let-7d caused a decrease in expression of the mesenchymal markers αSMA, N-Cadherin and FSP-1, as well as an increase in the epithelial marker TJP-1. Phenotypic changes were also present, including a delay in wound healing, reduced motility, and proliferation of fibroblasts following transfection. In addition, we examined the effects of transfection on fibroblast responsiveness to TGFβ, an important factor in many fibrotic processes such as lung fibrosis, and found that let-7d transfection significantly attenuated HMGA2 level induction by TGFβ. Our results indicate administration of the epithelial microRNA let-7d can significantly alter the phenotype of primary fibroblasts.
Chronic alcohol abuse increases lung oxidative stress and susceptibility to respiratory infections by impairing alveolar macrophage (AM) function. NADPH oxidases (Nox) are major sources of reactive oxygen species (ROS) in AMs. We hypothesized that treatment with the critical antioxidant glutathione (GSH) attenuates chronic alcohol-induced oxidative stress, by down-regulating Noxes, and restores AM phagocytic function. Bronchoalveolar lavage (BAL) fluid and AMs were isolated from male C57BL/6J mice (8-10 weeks) treated ± ethanol in drinking water (20% w/v, 12 weeks) ± orally gavaged GSH in methylcellulose vehicle (300 mg/kg/day, during week 12). MH-S cells, a mouse AM cell line, were treated ± ethanol (0.08%, 3 d) ± GSH (500 μM, 3 d or last 1 d of ethanol). BAL and AMs were also isolated from ethanol-fed and control mice ± inoculated airway Klebsiella pneumoniae (200 CFUs, 28 hours) ± orally gavaged GSH (300 mg/kg, 24 hours). GSH levels (HPLC), Nox mRNA (qRT-PCR) and protein levels (western blot and immunostaining), oxidative stress (DCFH-DA and Amplex Red), and phagocytosis (S. aureus internalization) were measured. Chronic alcohol decreased GSH levels, increased Nox expression and activity, enhanced oxidative stress, impaired phagocytic function in AMs in vivo and in vitro, and exacerbated K. pneumonia-induced oxidative stress. Although how oral GSH restored GSH pools in ethanol-fed mice is unknown, oral GSH treatments abrogated the detrimental effects of chronic alcohol exposure and improved AM function. These studies provide GSH as a novel therapeutic approach for attenuating alcohol-induced derangements in AM Nox expression, oxidative stress, dysfunction, and risk for pneumonia.
Excessive reactive oxygen/nitrogen species have been associated with the onset, progression and outcome of sepsis, both in pre-clinical and clinical studies. However, the signaling pathways regulating oxidative/nitrative stress in the pathogenesis of sepsis-induced acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are not fully understood. Employing the novel mouse model with genetic deletions of both Cav1 and ADPN (DKO mice), we have demonstrated the critical role of Cav1 and ADPN signaling cross-talk in regulating oxidative/nitrative stress and resulting inflammatory lung injury following LPS challenge. In contrast to the inhibited inflammatory lung injury in Cav1-/- mice, we observed severe lung inflammation and markedly increased lung vascular permeability in DKO mice in response to LPS challenge. Accordingly, the DKO mice exhibited an 80% mortality rate following a sublethal dose of LPS challenge. At basal state, loss of Cav1 and ADPN resulted in a drastic increase of oxidative stress and resultant nitrative stress in DKO lungs. Scavenging of superoxide by pretreating the DKO mice with MnTMPYP (a superoxide dismutase mimetic) restored the inflammatory responses to LPS challenge including reduced lung MPO activity and vascular permeability. Thus, oxidative/nitrative stress collectively modulated by Cav1 and ADPN signalings is a critical determinant of inflammatory lung injury in response to LPS challenge.
KCNQ (Kv7 family) potassium (K+) channels were recently found in airway smooth muscle cells (ASMCs) from rodent and human bronchioles. In the present study we evaluated KCNQ channel expression and their role in constriction/relaxation of rat airways. Real time RT-PCR analysis revealed expression of KCNQ4 > KCNQ5 > KCNQ1 > KCNQ2 > KCNQ3 and patch clamp electrophysiology detected KCNQ currents in rat ASMCs. In precision-cut lung slices (PCLS), the KCNQ channel activator retigabine induced a concentration-dependent relaxation of small bronchioles pre-constricted with methacholine (EC50 = 3.6 ± 0.3 µM). Bronchoconstriction was also attenuated in the presence of two other structurally unrelated KCNQ channel activators: zinc pyrithione (1 μM; 22 ± 7%), and 2,5-dimethylcelecoxib (10 μM; 24 ± 8%). The same three KCNQ channel activators increased KCNQ currents in ASMCs by 2- to 3-fold. The bronchorelaxant effects of retigabine and zinc pyrithione were prevented by inclusion of the KCNQ channel blocker XE991. A long-acting β2-adrenergic receptor agonist, formoterol (10 nM), did not increase KCNQ current amplitude in ASMCs, but formoterol (1-1000 nM) did induce a time- and concentration-dependent relaxation of rat airways, with a notable desensitization during a 30 min treatment or with repetitive treatments. Co-administration of retigabine (10 μM) with formoterol produced a greater peak and sustained reduction of methacholine-induced bronchoconstriction, and reduced the apparent desensitization observed with formoterol alone. Our findings support a role for KCNQ K+ channels in the regulation of airway diameter. Combination of a β2-adrenergic receptor agonist with a KCNQ channel activator may improve bronchodilator therapy.
The pathogenesis of Chronic Obstructive Pulmonary Disease (COPD) remains poorly understood. Cellular senescence and apoptosis contribute to the development of COPD, however, crucial regulators of these underlying mechanisms remain unknown. Macrophage Migration Inhibitory Factor (MIF) is a pleiotropic cytokine that antagonizes both apoptosis and premature senescence and may be important in the pathogenesis of COPD. This study examines the role of MIF in the pathogenesis of COPD. Mice deficient in MIF (Mif -/-), the MIF receptor CD74 (CD74 -/-), and wild type (WT) controls were aged for 6 months. Both Mif -/- and CD74 -/- mice developed spontaneous emphysema by 6 months of age compared to WT mice as measured by lung volume and chord length. This was associated with activation of the senescent pathway markers p53/21 and p16. Following exposure to cigarette smoke, Mif -/- mice were more susceptible to the development of COPD and apoptosis when compared to WT mice. MIF plasma concentrations were measured in a cohort of 224 human participants. Within a subgroup of older current and former smokers (n=72), MIF concentrations were significantly lower in those with COPD [8.8, 95%CI (6.7-11.0)] compared to those who did not exhibit COPD [12.7, 95%CI (10.6-14.8)]. Our results suggest that both MIF and the MIF receptor CD74 are required for maintenance of normal alveolar structure in mice and that decreases in MIF are associated with COPD in human subjects.
Cytosolic phospholipase A2 (cPLA2) plays a pivotal role in mediating agonist-induced arachidonic acid (AA) release for prostaglandin (PG) synthesis during inflammation triggered by tumor necrosis factor-α (TNF-α). However, the mechanisms underlying TNF-α-induced cPLA2 expression in human lung epithelial cells (HPAEpiCs) were not completely understood. Here, we demonstrated that TNF-α induced cPLA2 mRNA and protein expression, promoter activity, and PGE2 secretion in HPAEpiCs. These responses induced by TNF-α were inhibited by pretreatment with the inhibitor of Jak2 (AG490), PDGFR (AG1296), PI3K (LY294002), or MEK1/2 (PD98059) and transfection with siRNA of Jak2, PDGFR, Akt, or p42. We showed that TNF-α markedly stimulated Jak2, PDGFR, Akt, and p42/p44 MAPK phosphorylation, which were attenuated by their respective inhibitors. Moreover, TNF-α stimulated Akt activation via a Jak2/PDGFR pathway in HPAEpiCs. In addition, TNF-α-induced p42/p44 MAPK phosphorylation was reduced by AG1296 or LY294002. On the other hand, TNF-α could induce Akt and p42/p44 MAPK translocation from the cytosol into the nucleus, which was inhibited by AG490, AG1296, or LY294002. Finally, we showed that TNF-α stimulated Elk-1 phosphorylation which was reduced by LY294002 or PD98059. We also observed that TNF-α time-dependently induced p300/Elk-1 and p300/Akt complex formation in HPAEpiCs, which was reduced by AG490, AG1296, or LY294002. The activity of cPLA2 protein up-regulated by TNF-α was reflected on the PGE2 release which was reduced by AG490, AG1296, LY294002, or PD98059. Taken together, these results demonstrated that TNF-α-induced cPLA2 expression and PGE2 release was mediated through a Jak2/PDGFR/PI3K/Akt/p42/p44 MAPK/Elk-1 pathway in HPAEpiCs.
Both phosphodiesterase 5 (PDE5) inhibition and endothelin (ET) receptor blockade have been shown to induce pulmonary vasodilation. However, little is known about the effect of combined blockade of these two vasoconstrictor pathways. Since nitric oxide (NO) exerts its pulmonary vasodilator influence via production of cyclic guanosine monophosphate (cGMP) as well as through inhibition of ET, we hypothesised that interaction between the respective signalling pathways precludes an additive vasodilator effect. We tested this hypothesis in chronically instrumented swine exercising on a treadmill by comparing the vasodilator effect of the PDE5-inhibitor EMD360527, the ETA/ETB-antagonist tezosentan, and combined EMD360527 and tezosentan. In the systemic circulation, vasodilation by tezosentan and EMD360527 was additive, both at rest and during exercise, resulting in ??±?% drop in blood pressure. In the pulmonary circulation, both EMD360527 and tezosentan produced vasodilation. However, tezosentan produced no additional pulmonary vasodilation in the presence of EMD360527, either at rest or during exercise. Moreover, in isolated preconstricted porcine pulmonary small arteries (~300 μm) EMD360527 (1nM-10 μM) induced dose-dependent vasodilation, while tezosentan (1nM-10 μM) failed to elicit vasodilation irrespective of the presence of EMD360527. However, both PDE5-inhibition and 8Br-cGMP, but not cAMP, blunted pulmonary small artery contraction to ET and its precursor Big ET in vitro. In conclusion, in healthy swine, either at rest or during exercise, PDE5-inhibition and the associated increase in cGMP produce pulmonary vasodilation that is mediated in part through inhibition of the ET-pathway, thereby precluding an additive vasodilator effect of ETA/ETB receptor blockade in the presence of PDE5-inhibition.
Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) affect 200,000 people a year in the USA. Pulmonary vascular endothelial (EC) barrier compromise is a hallmark of these diseases. We have recently shown that extracellular adenosine enhances human pulmonary (EC) barrier via activation of adenosine receptors (ARs) in cell cultures. Based on these data, we hypothesized that activation of ARs might exert barrier protective effects in a model of ALI/ARDS in mice. To test this hypothesis we examined the effects of pre- and post-treatment of adenosine and 5'-N-Ethylcarboxamidoadenosine (NECA), a non-selective stable AR agonist, on LPS-induced lung injury. Mice were given vehicle or LPS followed by adenosine, NECA or vehicle instilled via the internal jugular vein (IJV). Post-experiment cell counts, Evans Blue Dye albumin (EBDA) extravasation, levels of proteins and inflammatory cytokines were analyzed. Harvested lungs were used for histology and myeloperoxidase (MPO) studies. Mice challenged with LPS alone demonstrated an inflammatory response typical of ALI. Cell counts, EBDA extravasation as well as levels of proteins and inflammatory cytokines were decreased in adenosine-treated mice. Histology displayed reduced infiltration of neutrophils. NECA had a similar effect on LPS-induced vascular barrier compromise. Importantly, treatment with adenosine or NECA restores lung vascular barrier and inflammation. Furthermore, adenosine significantly attenuated protein degradation of A2A and A3 receptors induced by LPS. Collectively, our results demonstrate that activation of adenosine receptors protects vascular barrier functions and reduced inflammation in LPS-induced ALI.
Effective mucociliary clearance (MCC) depends in part on adequate airway surface liquid (ASL) volume to maintain an appropriate periciliary fluid height that allows normal ciliary activity. Apically expressed large conductance, Ca2+ activated, and voltage-dependent K+ (BK) channels provide an electrochemical gradient for Cl- secretion and thus play an important role for adequate airway hydration. Here we show that interferon- (IFN-) decreases ATP-mediated apical BK activation in normal human airway epithelial cells cultured at the air-liquid interface. IFN- decreased mRNA levels of KCNMA1 but did not affect total protein levels. Since IFN- upregulates DUOX2 and therefore H2O2 production, we hypothesized that BK inactivation could be mediated by BK oxidation. However, DUOX2 knockdown did not affect the IFN- effect on BK activity. IFN- changed mRNA levels of the BK β-modulatory proteins KCNMB2 (increased) and KCNMB4 (decreased) as well as LRRC26 (decreased). Mallotoxin, a BK opener only in the absence of LRRC26, showed that BK channels lost their association with LRRC26 after IFN- treatment. Finally, IFN- caused a decrease in ciliary beating frequency (CBF) that was immediately rescued by apical fluid addition, suggesting that it was due to ASL volume depletion. These data were confirmed with direct ASL measurements using meniscus scanning. Overexpression of KCNMA1, the pore forming subunit of BK, overcame the reduction of ASL volume induced by IFN-. Key experiments were repeated in cystic fibrosis cells and showed the same results. Therefore, IFN- induces mucociliary dysfunction through BK inactivation.
The factors that contribute to pulmonary embolism (PE), a potentially fatal complication of deep vein thrombosis (DVT), remain poorly understood. Whereas fibrin clot structure and functional properties have been implicated in the pathology of venous thromboembolism and the risk for cardiovascular complications, their significance in PE remain incomplete. Therefore we systematically compared and quantified clot formation and lysis time, plasminogen levels, viscoelastic properties, activated factor XIII crosslinking and fibrin clot structure in isolated DVT and PE subjects. Clots made from plasma of PE subjects showed faster clot lysis times with no differences in lag time, rate of clot formation or maximum absorbance of turbidity as compared to DVT. Differences in lysis times were not due to alterations in plasminogen levels. Compared with DVT, clots derived from PE subjects showed accelerated establishment of viscoelastic properties, documented by a decrease in lag time and an increase in the rate of viscoelastic property formation. The rate and extent of fibrin crosslinking by activated factor XIII were similar between clots from DVT and PE subjects. Evaluation by electron microscopy revealed that plasma fibrin clots from PE subjects exhibited lower fiber density compared to those from DVT subjects. These data suggest that clot structure and functional properties differ between DVT and PE subjects and provide insights into mechanisms that may regulate embolization.
Elevated levels of TNF-α have been detected in the airway fluids, which may induce up-regulation of inflammatory proteins. Suppressors of cytokine signaling (SOCS)-3 proteins can be induced by various cytokines and negatively regulated inflammatory responses. Although TNF-α has been shown to induce SOCS-3 expression, the mechanisms underlying TNF-α-induced SOCS-3 expression in human tracheal smooth muscle cells (HTSMCs) remain unclear. Here, we showed that TNF-α induced SOCS-3 expression in HTSMCs and in the airways of mice, which was inhibited by pretreatment with the inhibitor of transcription level (actinomycin D), translation level (cycloheximide), JNK1/2 (SP600125), MEK1/2 (U0126), NADPH oxidase (Nox; apocynin and diphenyleneiodonium chloride), or ROS (N-acetyl-L-cysteine) and transfection with siRNA of JNK1, p47phox, p42, Nox2, or human antigen R (HuR). In addition, TNF-α-stimulated JNK1/2 and p42/p44 MAPK phosphorylation, Nox activation, and ROS generation were inhibited by pretreatment with U0126 or SP600125 and transfection with siRNA of JNK1 or p42. We further showed that TNF-α markedly induced HuR protein expression and translocation from the nucleus to the cytosol, which could stabilize SOCS-3 mRNA. Moreover, TNF-α-enhanced HuR translocation was reduced by transfection with siRNA of p42, JNK1, or p47phox. These results suggested that TNF-α induces SOCS-3 protein expression and mRNA stabilization via a TNFR1/JNK1/2, p42/p44 MAPK/Nox2/ROS-dependent HuR signaling in HTSMCs. Lipopolysaccharide (LPS) has been shown to play a key role in inflammation via induction of adhesion molecules, and then causes airway and lung injury. Moreover, we also demonstrated that overexpression of SOCS-3 protects against LPS-induced adhesion molecules expression and airway inflammation.
Vitamin D (vit D) has anti-inflammatory properties and modulates lung growth, but whether vit D can prevent lung injury after exposure to antenatal inflammation is unknown. We hypothesized that early and sustained vit D treatment could improve survival and preserve lung growth in an experimental model of BPD induced by antenatal exposure to endotoxin (ETX). Fetal rats (E20) were exposed to ETX (10μg), ETX + Vit D (1ng/ml), or saline (control) via intra-amniotic (IA) injections and delivered two days later. Newborn pups exposed to IA ETX received daily intraperitoneal (IP) injections of vit D (1ng/gm) or saline for 14 days. Vit D treatment improved oxygen saturations (78 v. 87%; p <0.001) and postnatal survival (84% vs. 57%; p<0.001) after exposure to IA ETX when compared to IA ETX alone. Postnatal vit D treatment improved alveolar and vascular growth at 14 days by 45% and 25%, respectively (p <0.05). Vit D increased fetal sheep pulmonary artery endothelial cell (PAEC) growth and tube formation by 64% and 44% respectively (p<0.001), and prevented ETX induced reductions of PAEC growth and tube formation. Vit D directly increased fetal alveolar type II cell (ATIIC) growth by 26% (p <0.001) and enhanced ATIIC growth in the presence of ETX induced growth suppression by 73% (p <0.001). We conclude that antenatal vit D therapy improved oxygenation and survival in newborn rat pups and enhanced late lung structure after exposure to IA ETX in vivo, which may partly be due to direct effects on vascular and alveolar growth.
Adiponectin is an adipose-derived hormone with anti-inflammatory activity. Following subacute ozone exposure (0.3 ppm for 24-72 h), neutrophilic inflammation and IL-6 are augmented in adiponectin deficient (Adipo-/-) mice. The IL-17/G-CSF axis is required for this increased neutrophilia. We hypothesized that elevated IL-6 in Adipo-/- mice contributes to their augmented responses to ozone via effects on IL-17A expression. Therefore, we generated mice deficient in both adiponectin and IL-6 (Adipo-/-IL-6-/-) and exposed them to ozone or air. In ozone exposed mice, BAL neutrophils, IL-6, and G-CSF, and pulmonary Il17α mRNA expression were greater in Adipo-/- versus wildtype mice, but reduced in Adipo-/-IL-6-/- versus Adipo-/- mice. IL-17A+ F4/80+ cells and IL-17A+ T-cells were also reduced in Adipo-/-IL-6-/- versus Adipo-/- mice exposed to ozone. Only BAL neutrophils were reduced in IL-6-/- versus WT mice. In wildtype mice, IL-6 was expressed in Gr-1+F4/80-CD11c- cells, whereas in Adipo-/- mice, F4/80+CD11c+ cells also expressed IL-6, suggesting that IL-6 is regulated by adiponectin in these alveolar macrophages. Transcriptomic analysis identified serum amyloid A3 (Saa3), which promotes IL-17A expression, as the gene most differentially augmented by ozone in Adipo-/- versus wildtype mice. After ozone, Saa3 mRNA expression was markedly greater in Adipo-/- versus wildtype mice, but reduced in Adipo-/-/IL-6-/- versus Adipo-/- mice. In conclusion, our data support a pivotal role of IL-6 in the hyperinflammatory condition observed in Adipo-/- mice after ozone exposure, and suggest that this role of IL-6 involves its ability to induce Saa3, IL-17A and G-CSF.
Chronic injury of alveolar lung epithelium leads to epithelial disintegrity in idiopathic pulmonary fibrosis (IPF). We had reported earlier that Grhl2, a transcriptional factor maintains alveolar epithelial cell integrity by directly regulating components of adherens and tight junctions and thus hypothesized an important role of GRHL2 in pathogenesis of IPF. Comparison of GRHL2 distribution at different stages of human lung development showed its abundance in developing lung epithelium and in adult lung epithelium. However, GRHL2 is detected in normal human lung mesenchyme only at early fetal stage (week 9). Similar Mesenchymal re-expression of GRHL2 was also observed in IPF. Immunofluorescence analysis in serial sections from three IPF patients revealed at least two subsets of alveolar epithelial cells (AEC), based on differential GRHL2 expression and the converse fluorescence intensities for epithelial versus mesenchymal markers. Grhl2 was not detected in mesenchyme in IP bleomycin-induced injury as well as in spontaneously occurring fibrosis in HPS1/2 mutant mice, while in contrast in a radiation-induced fibrosis model, with forced Forkhead box M1 (Foxm1) expression, an overlap of Grhl2 with a mesenchymal marker was observed in fibrotic regions. Grhl2's role in alveolar epithelial cell-plasticity was confirmed by altered Grhl2 gene expression analysis in IPF and further by in vitro manipulation of its expression in alveolar epithelial cell lines. Our findings reveal important patho-physiological differences between human IPF and specific mouse models of fibrosis and support a crucial role of GRHL2 in epithelial activation in lung fibrosis and perhaps also in epithelial plasticity.
Creation of bioartificial organs has been enhanced by the development of strategies involving decellularized mammalian lung. Because fibroblasts critically support lung function through a number of mechanisms, study of these cells in the context of the decellularized lung has the potential to improve the structure and function of tissue engineered lungs. We characterized the engraftment and survival of a mouse fibroblast cell line in decellularized rat lung slices and found a time dependent increase in cell numbers assessed by H&E staining, cell proliferation assessed by Ki67 staining and minimal cell death assessed by TUNEL staining. We developed a repopulation index to allow quantification of cell survival that accounts for variation in cell density throughout the seeded scaffold. We then applied this method to the study of mouse lung scaffolds and found that decellularization of presliced mouse lungs produced matrices with preserved alveolar architecture and proteinaceous components including fibronectin, collagens I and IV, laminin, and elastin. Treatment with a β1 integrin neutralizing antibody significantly reduced the repopulation index after twenty-four hours of culture. Treatment with focal adhesion kinase (FAK) inhibitor and extracellular signal-regulated kinase (ERK) inhibitor further reduced initial repopulation scores while treatment with AKT inhibitor increased initial scores. ROCK inhibitor had no discernible effect. These data indicate that initial adhesion and survival of mouse fibroblasts in the decellularized mouse lung occurs in a β1 integrin-dependent, FAK/ERK-dependent manner that is opposed by AKT.
Pigment epithelium-derived factor (PEDF) is a multifunctional protein with important roles in regulation of inflammation and angiogenesis. It is produced by various cell types including endothelial cells (EC). However, the cell autonomous impact of PEDF on EC function needs further investigation. Lung EC prepared from PEDF-deficient (PEDF -/-) mice were more migratory and failed to undergo capillary morphogenesis in Matrigel compared with wild type (PEDF +/+) EC. Although no significant differences were observed in the rates of apoptosis in PEDF -/- EC compared with PEDF +/+ cells under basal or stress conditions, PEDF -/- EC proliferated at a slower rate. PEDF -/- EC also expressed increased levels of proinflammatory markers including vascular endothelial growth factor, inducible nitric oxide synthase, vascular cell adhesion molecule-1, as well as altered cellular junctional organization, and nuclear localization of β-catenin. The PEDF -/- EC were also more adhesive, expressed decreased levels of thrombospondin-2, tenascin-C and osteopontin, and increased fibronectin. Furthermore, we showed lungs from PEDF -/- mice exhibited increased expression of macrophage marker F4/80 along with increased thickness of the vascular walls consistent with a pro-inflammatory phenotype. Together our data suggest that the PEDF expression makes significant contribution to modulation of the inflammatory and angiogenic phenotype of the lung endothelium.
Since avian influenza virus H5N1-induced hypercytokemia plays a key role in acute lung injury, understanding its molecular mechanism is highly desirable for discovering therapeutic targets against H5N1 infection. In the present study, we investigated the role of autophagy in H5N1-induced lung inflammation by using H5N1 hemagglutinin pseudotyped viral particles (H5N1pps). The results showed that H5N1pps significantly induced autophagy both in A549 human lung epithelial cells and in mouse lung tissues, which was primarily due to hemagglutinin (HA) of H5N1 virus. Blocking autophagy with 3-MA (an autophagy inhibitor) or siRNA knockdown of autophagy-related genes (beclin1 and atg5) dramatically attenuated H5N1pp-induced proinflammatory cytokines and chemokines, such as IL-1β, TNF-α, IL-6, CCL2, and CCL5, both in vitro and in vivo. Autophagy-mediated inflammatory responses involved the activation of NF-B and p38 MAPK signaling pathways, which required the presence of clathrin but did not rely on p62 or autophagosome-lysosome fusion. On the other hand, the activation of NF-B also promoted H5N1pp-induced autophagosome formation. This data indicated a positive feedback loop between autophagy and NF-B signaling cascade, which could exacerbate H5N1pp-induced lung inflammation. Our data demonstrated an essential role of autophagy in H5N1pp-triggered inflammatory responses, and targeting the autophagic pathway could be a promising strategy to treat H5N1 virus-caused lung inflammation.
Inhaled nitric oxide (NO) and other cyclic guanosine (cGMP) or adenosine (cAMP) monophosphate-dependent pulmonary vasodilators are often utilized in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome (PPHN). There is in vitro evidence to indicate that NO downregulate the pulmonary vascular response to cGMP-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. Hypothesizing that a synergistic effect is absent, we evaluated newborn and juvenile rat pulmonary arteries to determine the individual and combined vasodilatory effect of cGMP- and cAMP-dependent agonists. In precontracted near-resistance pulmonary arteries, the addition of sildenafil reduced vasorelaxation response to NO donor S-nitroso-N-acetyl penicillamine (SNAP). A similar decrease in SNAP-induced vasodilation was observed in arteries pre-treated with BAY 41-2272 (10-9 M), a soluble guanylate cyclase stimulator, cGMP and its downstream protein kinase activator. cGMP also reduced the vasorelaxant response to the cAMP-dependent forskolin. Inhibition of endogenous vascular NO generation enhanced SNAP-induced relaxation. The present data suggest that the mechanism involved in the cGMP desensitization to other relaxant agonists involves downregulation of the small heat shock protein HSP20 and is evident in rat pulmonary and systemic vascular smooth muscle cells. In newborn rats with chronic hypoxia-induced pulmonary hypertension, the combination of sildenafil and inhaled NO resulted in a lesser reduction in pulmonary vascular resistance, when compared with their individual effect. These data suggest that clinical exposure to one cGMP-dependent pulmonary vasodilator may affect the response to other cGMP- or cAMP-mediated agonists.
Previous reports demonstrated that bleomycin-induced injury of lungs in mice can be improved by the administration of murine multipotent adult stem/progenitor cells (MSCs)from the bone marrow. Recently some of the beneficial effects of MSCs have been explained by the cells being activated by signals from injured tissues to express the inflammation modulating protein TNF-α stimulated gene/protein 6 (TSG-6). In this study,we elected to test the hypothesis that targeting the early phase of bleomycin-induced lung injury with systemic TSG-6 administration may produce therapeutic effects such as preventing the deterioration of lung function and increasing survival by modulation of the inflammatory cascade. Lung injury in C57Bl/6j mice was induced by intratracheal administration of bleomycin. Mice then received intravenous injections of TSG-6 or sham controls. Pulse oximetry was used to monitor changes in lung function. Cell infiltration was evaluated using flow cytometry, cytokine expression was measured using ELISA assays and lungs were assessed for histological attributes. The results demonstrated that intravenous infusion of TSG-6 during the early inflammatory phase decreased cellular infiltration into alveolar spaces. Most importantly, it improved both the subsequent decrease in arterial oxygen saturation levels and the survival of the mice. These findings demonstrated that the beneficial effects of TSG-6 in a model of bleomycin-induced lung injury are largely explained by the protein modulating the early inflammatory phase. Similar phase-directed strategy with TSG-6 or other therapeutic factors that MSCs produce may be useful for other lung diseases and diseases of other organs.
Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and anti-fibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution, and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. Using the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems, and anti-fibrotic activity using the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an anti-fibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids and embryonic lungs at 10 min post-administration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases, or preterm infants with respiratory distress.
The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases, and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88 and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge, and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of Clara cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of Clara cells, since these cells are involved in airway repair. Clara cells may be prevented from differentiating into respiratory epithelial cells due to a lack of IFT88 protein that is necessary to form a single non-motile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.
Background: The lung is an important reservoir of human immunodeficiency virus (HIV). Individuals infected with HIV are more prone to pulmonary infections and chronic lung disorders. We hypothesized that comprehensively profiling the proteomic landscape of bronchoalveolar lavage fluid (BALF) in patients with HIV would provide insights into how this virus alters the lung milieu and contributes to pathogenesis of HIV-related lung diseases. Methods: BALF was obtained from five HIV-negative (HIV-) and six asymptomatic HIV-positive (HIV+) subjects not on anti-retroviral therapy (ART). Each sample underwent shotgun proteomic analysis based on HPLC-tandem mass spectrometry. Differentially expressed proteins between the groups were identified using statistical methods based on spectral counting. Mechanisms of disease were explored using functional annotation to identify overlapping and distinct pathways enriched between the BALF proteome of HIV+ and HIV- subjects. Results: We identified a total of 318 unique proteins in BALF of HIV- and HIV+ subjects. Of these, 87 were differentially up or down-regulated between the two groups. Many of these differentially expressed proteins are known to interact with key HIV proteins. Functional analysis of differentially regulated proteins implicated down-regulation of immune responses in lungs of HIV+ patients. Conclusions: Combining shotgun proteomic analysis with computational methods demonstrated that the BALF proteome is significantly altered during HIV infection. We found that immunity-related pathways are under-represented in HIV+ patients. These findings implicate mechanisms whereby HIV invokes local immunosuppression in the lung and increases the susceptibility of HIV+ patients to develop a wide range of infectious and noninfectious pulmonary diseases.
The hallmark geometric feature of single-walled carbon nanotubes (SWCNT) and carbon nanofibers (CNF) - high length to width ratio - makes them similar to a hazardous agent - asbestos. Very limited data are available concerning long-term effects of pulmonary exposure to SWCNT or CNF. Here we compared inflammatory, fibrogenic and genotoxic effects of CNF, SWCNT or asbestos in mice one year after pharyngeal aspiration. In addition, we compared pulmonary responses to SWCNT by bolus dosing through pharyngeal aspiration and inhalation 5h/day for 4 days, to evaluate the effect of dose rate. The aspiration studies showed that, these particles can be visualized in the lung at one year post-exposure, while some translocate to lymphatics. All these particles induced chronic bronchopneumonia and lymphadenitis, accompanied by pulmonary fibrosis. CNF and asbestos were found to promote the greatest degree of inflammation, followed by SWCNT, while SWCNT were the most fibrogenic of these three particles. Further, SWCNT induced cytogenetic alterations seen as micronuclei formation and nuclear protrusions in vivo. Importantly, inhalation exposure to SWCNT showed significantly greater inflammatory, fibrotic and genotoxic effects than bolus pharyngeal aspiration. Finally, SWCNT and CNF, but not asbestos exposures, increased the incidence of K-ras oncogene mutations in the lung. No increased lung tumor incidence occurred after 1 year post exposure to SWCNT, CNF and asbestos. Overall, our data suggest that long-term pulmonary toxicity of SWCNT, CNF and asbestos - is defined not only by their chemical composition but also by the specific surface area and type of exposure.
Alveolar type 2 (AT2) cells secrete surfactant that forms a protective layer on the lung's alveolar epithelium. Vesicles called, lamellar bodies (LBs) store surfactant. Failure of surfactant secretion, which causes severe lung disease, relates to the manner in which LBs undergo exocytosis during the secretion. However, the dynamics of LBs during the secretion process are not known in intact alveoli. Here, we addressed this question through real-time confocal microscopy of single AT2 cells in live alveoli of mouse lungs. Using a combination of phospholipid and aqueous fluorophores that localize to LBs, we induced surfactant secretion by transiently hyperinflating the lung, and we quantified the secretion in terms of loss of bulk LB fluorescence. In addition, we quantified inter-LB phospholipid flow through determinations of fluorescence recovery after photobleaching. Further, we determined the role of F-actin in surfactant secretion through expression of the fluorescent F-actin probe, Lifeact. Our findings indicate that in AT2 cells in situ, LBs are held in an F-actin scaffold. Although F-actin transiently decreases during surfactant secretion, the LBs remain stationary, forming a chain of vesicles connected by intervesicular channels that convey surfactant to the secretion site on the plasma membrane. This is the first instance of a secretory process in which the secretory vesicles are immobile, but form a conduit for the secretory material.
Acute lung injury is marked by profound influx of activated neutrophils, which have delayed apoptosis, along with fluid accumulation that impairs lung function and causes high mortality. Inflammatory and antimicrobial molecules such as reactive oxygen species from activated neutrophils with prolonged lifespan cause tissue damage and contribute to lung dysfunction. Angiostatin, an endogenous anti-angiogenic molecule, is expressed in the lavage fluid of patients with acute respiratory distress syndrome and modifies neutrophil infiltration in a mouse model of peritonitis. Our aim was to investigate the therapeutic role of angiostatin in acute lung injury. We analyzed bronchoalveolar lavage and lung tissues from C57BL/6 mouse model of E. coli lipopolysaccharide-induced acute lung injury to assess effects of angiostatin treatment. Subcutaneous angiostatin administered at 5 hours post-LPS treatment reduces histological signs of inflammation, protein accumulation, lung Gr1+ neutrophils, myeloperoxidase activity, and expression of phosphorylated p38 MAPK in lung tissues and peripheral blood neutrophils while increasing number of apoptotic cells in the lungs without affecting the levels of MIP-1α, IL-1β, KC and MCP-1 in lavage and lung homogenates at 9 and 24 hours post-LPS treatment. In contrast, angiostatin administered intravenously 5 hours post-LPS treatment did not reduce histological sign of inflammation, BAL cell recruitment and protein concentration at 9 hours of LPS treatment. We conclude that angiostatin administered subcutaneously after LPS challenge inhibits acute lung inflammation up to 24 hours post-LPS treatment.
Perinatal inflammation and the inflammatory cytokine IL-1 can modify lung morphogenesis. To examine the effects of antenatal expression of IL-1β in the distal airway epithelium on fetal lung morphogenesis, we studied lung development and surfactant expression in fetal mice expressing human IL-1β under the control of the surfactant protein (SP)-C promoter. IL-1β-expressing pups suffered respiratory failure and died shortly after birth. IL-1β caused fetal lung inflammation and enhanced the expression of keratinocyte-derived chemokine (KC/CXCL1) and monocyte chemoattractant protein 3 (MCP-3/CCL7), the calgranulins S100A8 and S100A9, the acute-phase protein serum amyloid A3 (SAA3), the chitinase-like proteins Ym1 and Ym2, and pendrin. IL-1β decreased the percentage of the total distal lung area made up of air saccules and the number of air saccules in the lungs of fetal mice. IL-1β inhibited the expression of VEGF-A and of its receptors VEGFR-1 and VEGFR-2. The percentage of the cellular area of the distal lung made up of capillaries was decreased in IL-1β-expressing fetal mice. IL-1β suppressed the production of SP-B and pro-SP-C, and decreased the amount of phosphatidylcholine and the percentage of palmitic acid in the phosphatidylcholine fraction of lung phospholipids, indicating that IL-1β prevented the differentiation of type II epithelial cells. The production of Clara cell secretory protein (CCSP) in the nonciliated bronchiolar (Clara) cells was likewise suppressed by IL-1β. In conclusion, expression of IL-1β in the epithelium of the distal airways disrupted the development of the airspaces and capillaries in the fetal lung and caused fatal respiratory failure at birth.
Ischemia-reperfusion (IR) injury leads to increased mortality and morbidity in lung transplant patients. Lung IR injury involves inflammation contributed by innate immune responses. IL-17 and TNF-α, from iNKT cells and alveolar macrophages, respectively, contribute importantly to lung IR injury. This study tests the hypothesis that IL-17 and TNF-α synergistically mediate CXCL1 (a potent neutrophil chemokine) production by alveolar type II epithelial (ATII) cells via an NADPH oxidase-dependent mechanism during lung IR. Using a hilar clamp model, wild-type and p47phox-/- (NADPH oxidase deficient) mice underwent left lung IR, with or without recombinant IL-17 and/or TNF-α treatment. Wild-type mice undergoing IR treated with combined IL-17 and TNF-α had significantly enhanced lung dysfunction, edema, CXCL1 production and neutrophil infiltration compared to treatment with IL-17 or TNF-α alone. However, p47phox-/- mice had significantly less pulmonary dysfunction, CXCL1 production and lung injury after IR, which was not enhanced by combined IL-17/TNF-α treatment. Moreover, in an acute, in vitro hypoxia-reoxygenation model, murine ATII cells showed a multifold synergistic increase in CXCL1 expression after combined IL-17/TNF-α treatment compared to treatment with either cytokine alone, which was significantly attenuated by an NADPH oxidase inhibitor. Conditioned media transfer from hypoxia-reoxygenation-exposed iNKT cells and macrophages, major sources of IL-17 and TNF-α, respectively, to ATII cells significantly enhanced CXCL1 production, which was blocked by NADPH oxidase inhibitor. These results demonstrate that IL-17 and TNF-α synergistically mediate CXCL1 production by ATII cells after IR, via an NADPH oxidase-dependent mechanism, to induce neutrophil infiltration and lung IR injury.
Progranulin (PGRN) is an anti-inflammatory protein, yet its digestion by neutrophil-derived proteinases generates products that can stimulate epithelial cell lines to secrete the neutrophil chemoattractant IL-8. Since dysregulated neutrophilic inflammation is implicated in the pathophysiology of COPD the possible influence of PGRN and digestion products may be of relevance to understanding and treating inflammation in the disease. PGRN was measured in sputum sol-phase samples from patients with a clinical diagnosis of COPD and chronic sputum production in a clinically stable state, PGRN correlated negatively with bacterial load (CFU/ml) (r=-0.446, p=0.003, n=43) and markers of neutrophilic inflammation including NE (nM) (r=-0.562, p=0.008, n=21) and PR3 (nM) (r=-0.515, p=0.017, n=21). Products of PGRN digestion were detected in sputum sol-phase, and PGRN conversion activity in sputum sol-phase was inhibited with the serine proteinase inhibitor α1-antitrypsin. Digested PGRN at concentrations likely to be present in the airways did not stimulate IL-8 secretion from normal human bronchial epithelial (NHBE) cells. Infection of NHBE cells with live Haemophilus influenzae significantly increased PGRN secretion compared to untreated cells (p=<0.001). The concentration of PGRN relates negatively to the amplified airway inflammation associated with bacterial colonisation in clinically stable COPD. This relationship is driven by the proteolytic action of the neutrophil derived proteinases NE and PR3; the products released by this action are unlikely to stimulate significant IL-8 secretion from epithelial cells in the airways.
Chronic obstructive pulmonary disease (COPD) is characterized by emphysema and chronic bronchitis and is a leading cause of morbidity and mortality worldwide. Tobacco smoke and deficiency in α1-antitrypsin (AAT) are the most prominent environmental and genetic risk factors, respectively. Yet, the pathogenesis of COPD is not completely elucidated. Disease progression appears to include a vicious circle driven by self-perpetuating lung inflammation, endothelial and epithelial cell death and proteolytic degradation of extracellular matrix proteins. Like AAT, serpinB1 is a potent inhibitor of serine proteases including neutrophil elastase and cathepsin G. Because serpinB1 is expressed in myeloid and lung epithelial cells and is protective during lung infections, we investigated the role of serpinB1 in preventing age-related and cigarette smoke-induced emphysema in mice. Fifteen-months-old mice showed increased lung volume and decreased pulmonary function compared to young adult mice (3-months-old), but no differences were observed between serpinB1-deficient (KO) and wild-type (WT) mice. Chronic exposure to secondhand cigarette smoke resulted in structural emphysematous changes compared to respective control mice, but no difference in lung morphometry was observed between genotypes. Of note, the different pattern of stereological changes induced by age and cigarette smoke suggest distinct mechanisms leading to increased airway volume. Finally, expression of intracellular and extracellular protease inhibitors were differently regulated in lungs of WT and KO mice following smoke exposure, however, activity of proteases was not significantly altered. In conclusion, we showed that, while AAT and serpinB1 are similarly potent inhibitors of neutrophil proteases, serpinB1 deficiency is not associated with more severe emphysema.
Factors positively influencing surfactant homeostasis in general and SP-B expression in particular are considered of clinical importance regarding an improvement of lung function in preterm infants. The objective of the study was to identify effects of physiological levels of caffeine on glucocorticoid-mediated SP-B expression in vitro and in vivo. Levels of SP-B and pepsinogen C were quantified by qPCR or immunoblotting in NCI-H441 cells daily exposed to caffeine and/or DEX. In vivo, SP-B expression was analyzed in BAL of preterm sheep exposed to antenatal DEX and/or postnatal caffeine. If DEX and caffeine were continuously present, SP-B mRNA and protein levels were increased for up to 6 days after induction (p<0.05). Additionally, caffeine enhanced SP-B mRNA expression in DEX-pretreated cells (p<0.05). Moreover, caffeine amplified DEX-induced pepsinogen C mRNA expression (p<0.05). After short-term treatment with caffeine in vivo, only slightly higher SP-B levels could be detected in BAL of preterm sheep following antenatal DEX, combined with an increase of arterial oxygen partial pressure (p<0.01). Our data demonstrated that the continuous presence of caffeine in vitro is able to amplify DEX-mediated SP-B expression. In contrast, short-term improvement of lung function in vivo is likely to be independent of altered SP-B transcription and translation. An impact of caffeine on release of surfactant reservoirs from lamellar bodies could however quickly affect SP-B content in BAL, which has to be further investigated. Our findings indicate that caffeine is able to amplify main effects of glucocorticoids which result from changes in surfactant production, maturation, and release.
Our previous studies have shown that the anti-asthma Traditional Chinese Medicine herbal formula ASHMI inhibits acetylcholine-induced contractions of tracheal rings from ovalbumin-sensitized and naïve mice in a β-adrenoceptor independent manner. We sought to determine whether acute in vivo ASHMI administration inhibits airway hyperreactivity (AHR) in a murine model of allergic asthma and acetylcholine induced tracheal ring constriction ex vivo and to elucidate the cellular mechanisms underlying these effects. Ovalbumin-sensitized mice received a single oral ASHMI dose 2 hours prior to intravenous acetylcholine challenge. AHR was determined by invasive airway measurements. Myography was used to determine the effects of ASHMI on acetylcholine-induced constriction of tracheal rings from asthmatic mice with or without epithelial denudation. The effect of cyclooxygenase inhibition and EP2/EP4 receptor blockade on ASHMI attenuation of acetylcholine contractions were evaluated. Tracheal cAMP and PGE2 levels were measured by ELISA. A single acute oral dose of ASHMI dramatically reduced AHR in response to acetylcholine provocation in ovalbumin-sensitized mice (P<0.001). In ex vivo experiments ASHMI significantly and dose-dependently reduced tracheal ring constriction to acetylcholine (P<0.05-0.001) which was epithelium-independent and associated with elevated cAMP levels. This effect was abrogated by cyclooxygenase inhibition or EP2/EP4 receptor blockade. ASHMI also inhibited contraction to high K+ (P<0.001). ASHMI increased tracheal ring PGE2 release in response to acetylcholine or high K+ (P<0.05 for both). ASHMI produced direct and acute inhibition of AHR in vivo, blocked acetylcholine-induced tracheal ring constriction via the EP2/EP4 receptor pathway identifying the mechanism by which ASHMI is an orally active bronchoprotective agent.
Diffuse alveolar hemorrhage (DAH) is characterised by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron and oxidant mediated activation of the transcription factor NF-B. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-B pathway, additionally IL-8 required ERK activation and MCP-1, JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron and redox mediated signaling involving the activation of the NF-B and MAPK pathways. Decompartmentalisation of hemoglobin may be a significant pro-inflammatory stimulus in a variety of lung diseases.
Rationale: The pathogenesis of chronic obstructive pulmonary disease is not fully understood. Objectives: To compare circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease to age, gender and cigarette smoking matched healthy controls. Methods and Measurements: Patients with chronic obstructive pulmonary disease (n=37) and healthy controls (n=19) were matched by age, gender and smoking status. Circulating hematopoietic progenitor cells (CD34+ or CD133+ mononuclear cells) and endothelial progenitor cells (CD34+KDR+ or CD34+CD133+KDR+ mononuclear cells) were quantified by flow cytometry. Endothelial cell-colony forming units from peripheral blood mononuclear cells were quantified in vitro and phenotypic analysis carried out using immunocytochemistry. Main Results: Patients with chronic obstructive pulmonary disease had more circulating mononuclear cells compared to controls (8.4±0.6 versus 5.9±0.4 x109 cells/L; P=0.02). CD34+ hematopoietic progenitor cells were reduced as a proportion of mononuclear cells in patients compared to controls (0.99±0.12 versus 1.9±0.12%; P=0.02), however there were no differences in the absolute number of CD34+, CD34+KDR+ or CD34+CD133+KDR+ cells (P>0.05 for all). Endothelial cell-colony forming units were increased in patients with chronic obstructive pulmonary disease compared to controls (13.7±5.2 versus 2.7±0.9 colonies; P=0.048). Conclusions: In contrast to previous studies the number of circulating progenitor cells were not reduced in patients with chronic obstructive pulmonary disease compared with carefully matched controls. It seems unlikely that circulating endothelial progenitor cells or failure of angiogenesis play a central role in the development of emphysema.
The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the ER folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility and reduced the expression level of wild type ABCA3 in a dose dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that while single motif mutants exhibited a vesicular distribution pattern similar to wild type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability and its disruption results in protein destabilization and proteasomal degradation.
Tumor necrosis factor-α converting enzyme (TACE) is a cell membrane sheddase, expressed in both developmental lung epithelia and mesenchyme. Global abrogation of TACE results in neonatal lethality and multiple organ developmental abnormalities, including dysplastic lung. To further define the roles of TACE in regulating lung development, lung epithelial and/or mesenchymal specific TACE conditional knockout mice were generated. Blockade of TACE function in developing lung epithelial cells caused reduced saccular formation, decreased cell proliferation, as well as reduced mid-distal lung epithelial cell differentiation. In contrast, mesenchymal TACE knockout did not have any phenotypic change in developing lung. Simultaneous abrogation of TACE in both lung epithelial and mesenchymal cells did not result in a more severe lung abnormality. Interestingly, these lung-specific TACE conditional knockout mice were not neonatal lethal, and their lung structures were essentially normal after alveolarization. In addition, TACE conditional knockout in developing cardiomyocytes resulted in non-compaction of ventricular myocardium, as seen in TACE conventional knockout mice. However, these mice were also not neonatal lethal. In conclusion, lung epithelial TACE is essential for promoting fetal lung saccular formation, but not postnatal lung alveolarization in mice. Since the developmental abnormality of either lung or heart induced by TACE deficiency does not directly lead to neonatal lethality, the neonatal death of TACE conventional knockout mice is likely a result of synergistic effects of multiple organ abnormalities.
It is now established that airway smooth muscle (ASM) has roles in determining airway structure and function, well beyond that as the major contractile element. Indeed, changes in ASM function are central to the manifestation of allergic, inflammatory and fibrotic airway diseases in both children and adults, as well as to airway responses to local and environmental exposures. Emerging evidence points to novel signaling mechanisms within ASM cells of different species that serve to control diverse features including 1) [Ca2+]i, contractility and relaxation, 2) cell proliferation and apoptosis, 3) production and modulation of extracellular components, 4) release of pro- vs. anti-inflammatory mediators and factors that regulate immunity as well as the function of other airway cell types such as epithelium, fibroblasts and nerves. These diverse effects of ASM "activity" result in modulation of bronchoconstriction vs. bronchodilation relevant to airway hyperresponsiveness, airway thickening and fibrosis that influences compliance. This perspective highlights recent discoveries that reveal the central role of ASM in this regard, and helps set the stage for future research towards understanding the pathways regulating ASM, and in turn, the influence of ASM on airway structure and function. Such exploration is key to development of novel therapeutic strategies that influence the pathophysiology of diseases such as asthma, COPD and pulmonary fibrosis.
The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney and airway epithelia. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) is a secreted, innate defense protein and an autocrine inhibitor of ENaC that is highly expressed in airway epithelia. While SPLUNC1 has a bactericidal permeability-increasing protein (BPI)-type structure, its N-terminal region lacks structure. Here we found that an eighteen amino acid peptide, S18, which corresponded to residues G22-A39 of SPLUNC1's N-terminus inhibited ENaC activity to a similar degree as full-length SPLUNC1 (~2.5 fold), whilst SPLUNC1 protein lacking this region was without effect. S18 did not inhibit the structurally related acid-sensing ion channels, indicating specificity for ENaC. However, S18 preferentially bound to the β-ENaC subunit in a glycosylation dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Unlike control, CF human bronchial epithelial cultures (HBECs) where airway surface liquid height (ASL) was abnormally low (4.2 ± 0.6 µm), addition of S18 prevented ENaC-led ASL hyperabsorption and maintained CF ASL height at 7.9 ± 0.6 µm, even in the presence of neutrophil elastase, which is comparable to heights seen in normal HBECs. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, suggesting that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the S18 peptide suggests that this peptide may be suitable for treating CF lung disease.
Decreased lung vascular growth and pulmonary hypertension contribute to poor outcomes in congenital diaphragmatic hernia (CDH). Mechanisms that impair angiogenesis in CDH are poorly understood. We hypothesize that decreased vessel growth in CDH is caused by pulmonary artery endothelial cell (PAEC) dysfunction with loss of a highly proliferative population of PAECs (HP-PAEC). PAECs were harvested from near term fetal sheep that underwent surgical disruption of the diaphragm at 60-70 days gestational age. Highly proliferative potential was measured via single cell assay. PAEC function was assessed by assays of growth and tube formation and response to known pro-angiogenic stimuli, vascular endothelial growth factor (VEGF) and nitric oxide (NO). Western blot analysis was used to measure content of angiogenic proteins and superoxide production was assessed. By single cell assay, the proportion of HP-PAEC with growth of >1000 cells was markedly reduced in the CDH PAEC, from 29% (controls) to 1% (CDH) (P < 0.0001). In comparison with controls, CDH PAEC growth and tube formation were decreased by 31% (P = 0.012) and 54% (P < 0.001), respectively. VEGF and NO treatments increased CDH PAEC growth and tube formation. VEGF and VEGF-R2 proteins were increased in CDH PAEC; however, eNOS and extracellular superoxide dismutase proteins were decreased by 29% and 88%, respectively. We conclude that surgically-induced CDH in fetal sheep causes endothelial dysfunction and marked reduction of the HP-PAEC population. We speculate that this CDH PAEC phenotype contributes to impaired vascular growth in CDH.
Female sex predisposes individuals to poorer outcomes during respiratory disorders like cystic fibrosis and influenza-associated pneumonia. A common link between these disorders is dysregulation of alveolar fluid clearance via disruption of epithelial sodium channel (ENaC) activity. Recent evidence suggests that female sex hormones directly regulate expression and activity of alveolar ENaC. In our study, we identified the mechanism by which estradiol (E2) or progesterone (P4) independently regulates alveolar ENaC. Using cell-attached patch clamp, we measured ENaC single channel activity in a rat alveolar cell line (L2) in response to overnight exposure to either E2 or P4. In contrast to P4, E2 increased ENaC channel activity (NPo) through an increase in channel open probability (Po) and an increased number of patches with observable channel activity. Apical plasma membrane abundance of the ENaC α subunit (αENaC) more than doubled in response to E2 as determined by cell surface biotinylation. αENaC membrane abundance was ~3 fold greater in lungs from female rats in proestrus, when serum E2 is greatest, compared to diestrus, when it is lowest. Our results also revealed a significant role for the G-protein coupled estrogen receptor (Gper) to mediate E2's effects on ENaC. Overall, our results demonstrate that E2 signaling through Gper selectively activates alveolar ENaC through an effect on channel gating and channel density, the latter via greater trafficking of channels to the plasma membrane. The results presented herein implicate E2-mediated regulation of alveolar sodium channels in the sex differences observed in the pathogenesis of several pulmonary diseases.
Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by thrombo-fibrotic obstruction of proximal pulmonary arteries. The cellular and molecular mechanisms underlying the pathogenesis remain incompletely understood, although we recently evidenced the potential involvement of the inflammatory marker, C-reactive protein (CRP). We aimed to investigate the intracellular mechanisms induced by CRP in proximal pulmonary arterial endothelial cells (PAEC). PAEC were isolated from vascular material obtained during pulmonary endarterectomy. RNA was extracted from CRP-stimulated PAEC and first stand cDNA was generated. A RT2 profiler PCR Array was used to evaluate the expression of 84 key genes related to NFB-mediated signal transduction. CRP-induced NFB activation was studied. Effects of PDTC, an inhibitor of the NFB pathway, were investigated on CRP-induced adhesion of monocytes to PAEC, adhesion molecule expression, endothelin-1 (ET-1), interleukin-6 (IL-6) and von Willebrand factor (vWF) secretion. Compared to non-stimulated PAEC, serotonin receptor 2B was down-regulated by 25%, inhibitor of NFB kinase subunit epsilon (IKBKE) by 30%, and toll-like receptor 4 and 6 by 18% and 39%, respectively, in CRP-stimulated PAEC. The transcription factor FOS was 3-fold up-regulated. CRP induced RelA/NFBp65 phosphorylation. PDTC dose-dependently inhibited the adhesion of monocytes to CRP-stimulated PAEC. PDTC also inhibited the CRP-induced expression of ICAM-1 at the surface of PAEC. PDTC impaired the secretion of ET-1 by 18% and tended to inhibit the secretion of IL-6 by CRP-stimulated PAEC by 46%. PDTC did not inhibit the CRP-induced secretion of vWF. These results suggest an involvement of the NFB pathway in mediating different effects of CRP on proximal CTEPH-PAEC.
Airway serous secretion is essential for the maintenance of mucociliary transport in airway mucosa, which is responsible for the upregulation of mucosal immunity. Although there are many articles concerning the importance of Toll-like receptors (TLRs) in airway immune systems, the direct relationship between TLRs and airway serous secretion has not been well investigated. Here, we focused on whether TLR5 ligand flagellin, which is one of the components of Pseudomonas aeruginosa, is involved in the upregulation of airway serous secretion. Freshly isolated swine tracheal submucosal gland cells were prepared and the standard patch-clamp technique was applied for measurements of the whole cell ionic responses of these cells. Flagellin showed potentiating effects on these oscillatory currents induced by physiologically relevant low doses of ACh in a dose-dependent manner. These potentiating effects were TLR5-dependent but TLR4-independent. Both nitric oxide (NO) synthase inhibitors and cGMP-dependent protein kinase (cGK) inhibitors abolished these flagellin-induced potentiating effects. Further, TLR5 was abundantly expressed on tracheal submucosal glands. Flagellin/TLR5 signaling further accelerated the intracellular NO synthesis induced by ACh. These findings suggest that TLR5 takes part in the airway mucosal defense systems as a unique endogenous potentiator of airway serous secretions and that NO/cGMP/cGK signaling is involved in this rapid potentiation by TLR5 signaling.
Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder marked by relative resistance to steroids. Inflammation and apoptosis have been suggested to be important mechanisms for COPD. IL-17 superfamily has been associated with chronic inflammation and diminished responses to steroids. It is reasonable to consider that IL-17 may play a role in the pathogenesis of COPD. In this study, we examined IL-17 expression in mice exposed to cigarette smoke (CS), and investigated the contribution of IL-17 to CS-induced inflammation and alveolar cell apoptosis in IL-17-/- mice. After exposing wild-type and IL-17-/- mice to main-stream CS for 4 weeks, IL-17A, but not IL-17F, expression was increased in mice upon CS exposure. Neutrophil infiltration in the lung of IL-17-/- mice was significantly decreased. In IL-17-/- mice, there is reduced expression of IL-6, MIP-2 and MMP12 compared to wild-type mice after CS exposure. The number of apoptotic type II alveolar cells was significantly increased in CS-exposed wild-type mice but not in IL-17-/- mice. The effect of IL-17A on type II alveolar cell apoptosis was confirmed in vitro through either addition of IL-17A or transient knockdown of IL-17A by siRNA transfection in type II alveolar cells. These findings suggest that IL-17A plays an important role in the inflammatory response to CS exposure through increased multiple inflammatory mediators. Moreover, IL-17 may also contribute to type II alveolar cell apoptosis. This study opens a new option in targeting IL-17A to modulate inflammatory response to CS and may be the bases for new therapy for COPD.
Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis related death. Current therapies are not effective in reversing endothelial cell dysfunction; which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide (AICAR) or metformin decreased the LPS-induced increase in permeability, as determined by filtration co-efficient (Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPKα1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPKα1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPKα1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.
Cystic fibrosis-related diabetes (CFRD) is the most common co-morbidity associated with cystic fibrosis (CF), impacting more than half of patients over age 30. CFRD is clinically significant, portending accelerated decline in lung function, more frequent pulmonary exacerbations, and increased mortality. Despite the profound morbidity associated with CFRD, little is known about the underlying CFRD-related pulmonary pathology. Our aim was to develop a murine model of CFRD to explore the hypothesis that elevated glucose in CFRD is associated with reduced lung bacterial clearance. A diabetic phenotype was induced in gut-corrected CFTR knockout mice (CFKO) and their CFTR expressing wild-type littermates (WT) utilizing streptozotocin (STZ). Mice were subsequently challenged with an intratracheal inoculation of P. aeruginosa (PAO1) (75 µL of 1-5x106 cfu/mL) for 18 h. Bronchoalveolar lavage fluid (BALF) was collected for glucose concentration and cell counts. A portion of the lung was homogenized and cultured as a measure of the remaining viable PAO1 inoculum. Diabetic mice had increased airway glucose compared to non-diabetic mice. The ability to clear bacteria from the lung was significantly reduced in diabetic WT mice and control CFKO mice. Critically, bacterial clearance by diabetic CFKO mice was significantly more diminished compared to non-diabetic CFKO mice, despite an even more robust recruitment of neutrophils to the airways. This finding that CFRD mice boast an exaggerated, but less effective, inflammatory cell response to intratracheal PAO1 challenge presents a novel and useful murine model to help identify therapeutic strategies that promote bacterial clearance in CFRD.
We sought to investigate the effects of cockroach allergen (CRA) exposure on the lung macrophage population to determine how different macrophage phenotypes influence exacerbation of disease. CRA exposure caused significantly reduced expression of CD86 on lung macrophages. These effects were not systemic as peritoneal macrophage CD86 expression was not altered. To investigate whether naïve macrophages could reduce asthma-like pulmonary inflammation, autologous peritoneal macrophages were instilled into the airways 24 hours prior to the final CRA challenge. Pulmonary inflammation was assessed by measurement of airway hyper-responsiveness, mucin production, inflammatory cell recruitment, and cytokine production. Cell transfer did not have significant effects in control mice, nor did it affect pulmonary mucin production or airway hyper-responsiveness in control or CRA-exposed mice. However, there was significant reduction in the number of eosinophils recovered in the BAL (5.8x105 vs. 0.88x105), and total cell recruitment to the airways of CRA-exposed mice was markedly reduced (1.1x106 vs. 0.57x106). The reduced eosinophil recruitment was reflected by substantially lower levels of eosinophil peroxidase in the lung, and significantly lower concentrations of eotaxins in BAL (eotaxin 1: 3 pg/ml vs. undetectable; eotaxin 2: 2383 vs. 131 pg/ml) and lung homogenate (eotaxin 1: 1043 vs. 218 pg/ml; eotaxin 2: 10 vs. 1.5 ng/ml). We conclude that CRA decreases lung macrophage CD86 expression. Furthermore, supplementation of the lung cell population with peritoneal macrophages inhibits eosinophil recruitment, achieved through reduction of eotaxin production. These data demonstrate that transfer of naïve macrophages will reduce some aspects of asthma-like pulmonary inflammation in response to CRA.
We showed that stop of flow triggers a mechanosignaling cascade that leads to the generation of reactive oxygen species (ROS); however a mechanosensor coupled to the cytoskeleton that could potentially transduce flow stimulus has not been identified. We showed a role for KATP channel, caveolae (caveolin-1) and NADPH oxidase 2 (NOX2) in ROS production with stop of flow. Based on reports of a mechanosensory complex that includes platelet endothelial cell adhesion molecule (PECAM-1) and initiates signaling with mechanical force, we hypothesized that PECAM-1 could serve as a mechanosensor in sensing disruption of flow. Using lungs in situ, we observed that ROS production with stop of flow was significantly reduced in PECAM-1-/- lungs as compared to lungs from wild type (WT) mice. Lack of PECAM-1 did not affect NOX2 activation machinery or the caveolin-1 expression or caveolae number in the pulmonary endothelium. Stop of flow in vitro triggered an increase in angiogenic potential of WT type pulmonary microvascular endothelial cells (PMVEC) but not of PECAM-1-/- PMVEC. Obstruction of flow in lungs in vivo showed that the neutrophil infiltration as observed in wild type mice was significantly lowered in PECAM-1-/- mice. With stop of flow, WT lungs showed higher expression of the angiogenic marker, vascular endothelial growth factor (VEGF) as compared to untreated (sham) and PECAM-1-/- lungs. Thus PECAM-1 (and caveolae) is part of the mechanosensing machinery that generates superoxide with loss of shear; the resultant ROS potentially drives neutrophil influx and acts as an angiogenic signal.
Chronic hypoxia pulmonary hypertension (CH-PHT) in adulthood is likely to be of fetal origin following intrauterine growth retardation (IUGR). Oxygen (O2)-sensitive voltage-gated potassium channels (Kv channels) in resistance pulmonary artery smooth muscle cells (PASMCs) play an important role in scaling pulmonary artery pressure. Expression and functional changes of Kv channels are determined, in part, by embryonic development. We hypothesized that O2-sensitive Kv channels play an important role in exaggerated CH-PHT following IUGR. We established a rat model of IUGR by restricting maternal food during the entire pregnancy and exposed IUGR rats and their age-matched controls aged12 weeks to hypoxia for 2 weeks. We found that hypoxia exposure significantly induced increased pulmonary artery (PA) pressure and thicker smooth muscle layer in the IUGR group relative to controls. We compared the constriction of the resistance PA to inhibitors of K+ channels, 4-aminopyridine (4-AP), tetraethylammonium (TEA), and barium chloride (BaCl2). Despite the thickness of the smooth muscle layer, the constriction to 4-AP was significantly reduced in the IUGR group exposed to hypoxia. Consistent with these changes in pulmonary vascular reactivity, 2 weeks of hypoxia induced weaker 4-AP-sensitive Kv currents in a single IUGR PASMC. Moreover, after 2 weeks of hypoxia, Kv1.5 expression in resistance PAs decreased significantly in the IUGR group. Overexpression of Kv1.5 in cultured PASMCs could offset hypoxia-induced cell proliferation and hypoxia-inhibited Kv currents in the IUGR group. These results suggest that the inhibited expression of Kv1.5 in PASMCs contribute to the development of exaggerated CH-PHT in IUGR rats during adulthood.
Epidemiologic studies associate environmental cadmium (Cd) exposure with the risk of lung diseases. Although mechanisms are not fully elucidated, several studies demonstrate Cd effects on actin and actin-associated proteins. In a recent study of Cd at concentrations similar to environmental exposures, we found that redox-dependent inflammatory signaling by NF-B was sensitive the actin-disrupting agent, cytochalasin D. The goal of the present study was to use mass spectrometry-based redox proteomics to investigate Cd effects on the actin cytoskeleton proteome and related functional pathways in lung cells at low environmental concentrations. The results showed that Cd under conditions that did not alter total protein thiols or glutathione redox state caused significant oxidation of peptidyl Cys of proteins regulating actin cytoskeleton. Immunofluorescence microscopy of lung fibroblasts and pulmonary artery endothelial cells showed that low dose Cd exposure stimulated filamentous actin formation and nuclear localization of destrin, an actin depolymerizing factor. Taken together, the results show that redox states of peptidyl Cys in proteins associated with actin cytoskeleton pathways are selectively oxidized in lung by Cd at levels thought to occur from environmental exposure.
T cell migration toward sites of antigen exposure is mediated by G protein signaling and is a key function in the development of immune responses. Regulators of G protein signaling (RGS) proteins modulate G protein signaling; however, their role in the regulation of adaptive immune responses has not been thoroughly explored. Herein we demonstrated abundant expression of the Gi/Gq-specific RGS3 in activated T cells, and that diminished RGS3 expression in a T cell thymoma increased cytokine-induced migration. To examine the role of endogenous RGS3 in vivo, mice deficient in the RGS-domain (RGS3RGS ) were generated and tested in an experimental model of asthma. Compared to littermate controls, the inflammation in the RGS3RGS mice was characterized by increased T cell numbers and the striking development of perivascular lymphoid-structures. Surprisingly, while innate inflammatory cells were also increased in the lungs of RGS3RGS mice, eosinophil numbers and Th2 cytokine production was equivalent to control mice. In contrast, T cell numbers in the draining lymph nodes (dLN) were reduced in the RGS3RGS demonstrating a redistribution of T cells from the dLN to the lungs via increased RGS3RGS T cell migration. Together these novel findings show a non-redundant role for endogenous RGS3 in controlling T cell migration in vitro and in an in vivo model of inflammation.
Rationale: Mitochondria are dynamic organelles, which continuously change their shape through fission and fusion. Disruption of mitochondrial dynamics is involved in disease pathology through excessive reactive oxygen species (ROS) production. Accelerated cellular senescence resulting from cigarette smoke (CS) exposure with excessive ROS production has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Hence, we investigated the involvement of mitochondrial dynamics and ROS production in terms of CS extract (CSE)-induced cellular senescence in human bronchial epithelial cells (HBEC). Methods: Mitochondrial morphology was examined by electron microscopy and fluorescence microscopy. Senescence associated beta-galactosidase (SA-β-gal) staining and p21 western blotting of primary HBEC were performed to evaluate cellular senescence. Mitochondrial specific superoxide production was measured by MitoSOX staining. Mitochondrial fragmentation was induced by knockdown of mitochondrial fusion proteins (OPA1 or Mitofusins) by siRNA transfection. N-acetylcysteine (NAC), and Mito-TEMPO were used as antioxidants. Measurements and Main Results: Mitochondria in bronchial epithelial cells were prone to be more fragmented in COPD lung tissues. CSE induced mitochondrial fragmentation and mitochondrial ROS production, which were responsible for acceleration of cellular senescence in HBEC. Mitochondrial fragmentation induced by knockdown of fusion proteins also increased mitochondrial ROS production and percentages of senescent cells. HBEC senescence and mitochondria fragmentation in response to CSE treatment were inhibited in the presence of antioxidants. Conclusions: CSE-induced mitochondrial fragmentation is involved in cellular senescence through the mechanism of mitochondrial ROS production. Hence, disruption of mitochondrial dynamics may be a part of the pathogenic sequence of COPD development
Acrolein, an α, β unsaturated electrophile, is an environmental pollutant released in ambient air from diesel exhausts and cooking oils. This study examines the role of acrolein in altering mitochondrial function and metabolism in lung specific cells. RLE-6TN, H441, and primary alveolar type II (pAT2) cells were exposed to acrolein for 4 h and its effect on mitochondrial oxygen consumption rates was studied by XF Extracellular Flux analysis. Low-dose acrolein exposure decreased mitochondrial respiration in a dose-dependent manner due to alteration in the metabolism of glucose in all the three cell types. Acrolein inhibited glyceraldehyde-3P-dehydrogenase (GAPDH) activity leading to decreased substrate availability for mitochondrial respiration in RLE-6TN, H441, and pAT2 cells; the reduced GAPDH activity was compensated in pAT2 cells by an increase in the activity of glucose-6-phosphate dehydrogen-ase, the regulatory control of the pentose phosphate pathway. The decrease in pyruvate from glucose metabolism resulted in utilization of alternative sources to support mitochondrial en-ergy production: palmitate-BSA complex increased mitochondrial respiration in RLE-6TN and pAT2 cells. The presence of palmitate in alveolar cells for surfactant biosynthesis may prove to be the alternative fuel source for mitochondrial respiration. Accordingly, a decrease in phosphatidylcholine levels and an increase in phospholipase A2 activity were found in the alveolar cells after acrolein exposure. These findings have implications for understanding the decrease in surfactant levels frequently observed in pathophysiological situations with altered lung function following exposure to environmental toxicants.
Bacterial pneumonia is a common and dangerous illness. Mononuclear phagocytes, which comprise monocyte, resident and recruited macrophage and dendritic cell subsets, are critical to anti-microbial defenses, but the dynamics of their recruitment to the lungs in pneumonia is not established. We hypothesized that chemokine-mediated traffic of mononuclear phagocytes is important to defense against bacterial pneumonia. In a mouse model of Klebsiella pneumonia, circulating Ly6Chi, and to a lesser extent, Ly6Clo monocytes expanded in parallel with accumulation of inflammatory macrophages and CD11bhi dendritic cells and plasmacytoid dendritic cells in the lungs, whereas numbers of alveolar macrophages remained constant. CCR2 was expressed by Ly6Chi monocytes, recruited macrophages and airway dendritic cells; CCR6 was prominently expressed by airway dendritic cells; and CX3CR1 was ubiquitously expressed by blood monocytes and lung CD11bhi dendritic cells during infection. CCR2-deficient, but not CCL2-, CX3CR1- or CCR6-deficient animals exhibited worse outcomes of infection. The absence of CCR2 had no detectable effect on neutrophils but resulted in reduction of all subsets of lung mononuclear phagocytes in the lungs, including alveolar macrophages and airway- and plasmacytoid dendritic cells. In addition, absence of CCR2 skewed the phenotype of lung mononuclear phagocytes, abrogating the appearance of M1 macrophages and TNF-producing dendritic cells in the lungs. Taken together, these data define the dynamics of mononuclear phagocytes during pneumonia.
Active sodium transport mediated by epithelial Na+ channel (ENaC) is vital for fetal lung fluid reabsorption at birth and pulmonary edema resolution. Previously, we demonstrated that αENaC expression and activity are downregulated in alveolar epithelial cells by cycloheximide (Chx) and Pseudomonas aeruginosa. The regulatory mechanisms of αENaC mRNA expression by Chx and lipopolysaccharide (LPS) from P. aeruginosa were further studied in the present work. Both agents decreased αENaC mRNA expression to 50% of control values after 4 h. Chx repressed αENaC expression in a dose-dependent manner independently of protein synthesis. Although extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathways were activated by the two treatments, their mechanisms of ENaC mRNA modulation were different. First, activation of the signalling pathways was sustained by Chx but only transiently by LPS. Second, ERK1/2 or p38 MAPK inhibition attenuated the effects of Chx on αENaC mRNA, whereas suppression of both signalling pathways was necessary to alleviate the outcome of LPS on αENaC mRNA. The molecular mechanisms involved in the decrease of αENaC expression were investigated in both conditions. LPS, but not Chx, significantly reduced αENaC promoter activity via the ERK1/2 and p38 MAPK pathways. These results suggest that LPS attenuates αENaC mRNA expression via diminution of transcription, whereas Chx could trigger some post-transcriptional mechanisms. Although LPS and Chx downregulate αENaC mRNA expression similarly and with similar signalling pathways, the mechanisms modulating ENaC expression are different depending on the nature of the cellular stress.
Endothelial cell (EC) inflammation is a central event in the pathogenesis of many pulmonary diseases such as acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS). Alterations in actin cytoskeleton are shown to be crucial for NF-B regulation and EC inflammation. Previously, we have described a role of actin binding protein cofilin, in mediating cytoskeletal alterations essential for NF-B activation and EC inflammation. The present study, describes a dynamic mechanism in which LIM kinase 1 (LIMK1), a cofilin kinase and slingshot-1Long (SSH-1L), a cofilin phosphatase, are engaged by procoagulant and proinflammmatory mediator thrombin to regulate these responses. Our data show that knockdown of LIMK1 destabilizes whereas knockdown of SSH-1L stabilizes the actin filaments through modulation of cofilin phosphorylation; however, in either case thrombin-induced NF-B activity and expression of its target genes (ICAM-1 and VCAM-1) is inhibited. Further mechanistic analyses reveal that knockdown of LIMK1 or SSH-1L each attenuates nuclear translocation and thereby DNA binding of RelA/p65. In addition, LIMK1 or SSH-1L depletion also inhibited RelA/p65 phosphorylation at Ser536, a critical event conferring transcriptional competency to the bound NF-B. However, unlike SSH-1L, LIMK1 knockdown also impairs the release of RelA/p65 from IBα by blocking its phosphorylation/degradation. Interestingly, LIMK1 or SSH-1L depletion failed to inhibit TNFα-induced RelA/p65 nuclear translocation and proinflammatory gene expression. Thus this study provides evidence for a novel role of LIMK1 and SSH-1L in selectively regulating EC inflammation associated with intravascular coagulation.
From the early 17th century the advent of physical and chemical sciences developed two important movements toward the explanation of all vital phenomena: the Iatrochemical and Iatromechanical School. The important research of their representatives as Jan Baptist van Helmont, John Mayow, Robert Boyle, Gian Alfonso Borelli, Richard Lower and Albrecht von Haller, followed by the discovery of the atmospheric gases, provided a fecund soil for the leading work of Lavoisier in respiratory physiology.
Despite the importance of pulmonary veins in normal lung physiology and the pathobiology of pulmonary hypertension with left heart disease (PH-LHD), pulmonary veins remain largely understudied. Difficult to identify histologically, lung venous endothelium or smooth muscle cells display no unique characteristic functional and structural markers that distinguish them from pulmonary arteries. To address these challenges, we undertook a search for unique molecular markers in pulmonary veins. In addition, we addressed the expression pattern of a candidate molecular marker, and analyzed the structural pattern of vascular remodeling of pulmonary veins in a rodent model of PH-LHD and in lung tissue of patients with PH-LHD obtained at time of placement on a left ventricular assist device. We detected urokinase plasminogen activator receptor (uPAR) expression preferentially in normal pulmonary veins of mice, rats, and human lungs. Expression of uPAR remained elevated in pulmonary veins of rats with PH-LHD; however, we also detected induction of uPAR expression in remodeled pulmonary arteries. These findings were validated in lungs of patients with PH-LHD. In selected patients with sequential lung biopsy at the time of removal of the left ventricular assist device, concordant improvement in pulmonary hemodynamics and venous remodeling was observed, indicating potential regression of venous remodeling in response to assist device treatment. Our data indicate that remodeling of pulmonary veins is an integral part of PH-LHD and that pulmonary veins share some key features present in remodeled, yet not normotensive pulmonary arteries.
Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome and acute lung damage. CD162 is an adhesion molecule that has been reported to mediate neutrophil recruitment in acute inflammatory reactions. In this study, the purpose was to investigate the role of CD162 in M1 protein-provoked lung injury. Male C57BL/6 mice were treated with monoclonal antibody directed against CD162 or a control antibody before M1 protein challenge. Edema, neutrophil infiltration, and CXC chemokines were determined in the lung, 4 h after M1 protein administration. Fluorescence intravital microscopy was used to analyze leukocyte-endothelium interactions in the pulmonary microcirculation. Inhibition of CD162 reduced M1 protein-provoked accumulation of neutrophils, edema, and CXC chemokine formation in the lung by more than 54%. Moreover, immunoneutralization of CD162 abolished leukocyte rolling and firm adhesion in pulmonary venules of M1 protein-treated animals. In addition, inhibition of CD162 decreased M1 protein-induced capillary trapping of leukocytes in the lung microvasculature and improved microvascular perfusion in the lungs of M1 protein-treated animals. Our findings suggest that CD162 plays an important role in M1 protein-induced lung damage by regulating leukocyte rolling in pulmonary venules. Consequently, inhibition of CD162 attenuates M1 protein-evoked leukocyte adhesion and extravasation in the lung. Thus, our results suggest that targeting the CD162 might pave the way for novel opportunities to protect against pulmonary damage in streptococcal infections.
Sirtuin1 (SIRT1), a protein/histone deacetylase, protects against the development of pulmonary emphysema. However, the molecular mechanisms underlying this observation remain elusive. The imbalance of tissue inhibitor of matrix metalloproteinases (TIMPs)/matrix metalloproteinases (MMPs) plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. We hypothesized that SIRT1 protects against emphysema by redressing the imbalance between MMPs and TIMPs. To test this hypothesis, SIRT1 deficient and overexpressing/transgenic mice were exposed to cigarette smoke (CS). The protein level and activity of MMP-9 were increased in lungs of SIRT1 deficient mice exposed to CS as compared to WT littermates, which were attenuated by SIRT1 overexpression. SIRT1 deficiency decreased the level of TIMP-1, which was augmented in SIRT1 transgenic mice as compared to WT littermates by CS. However, the level of MMP-2, MMP-12, TIMP-2, TIMP-3, or TIMP-4 was not altered by SIRT1 in response to CS exposure. SIRT1 reduction was associated with imbalance of TIMP-1 and MMP-9 in lungs of smokers and COPD patients. Mass spectrometry and immunoprecipitation analyses revealed that TIMP-1 acetylation on specific lysine residues was increased, whereas its interaction with SIRT1 and MMP-9 was reduced in mouse lungs with emphysema, as well as in lungs of smokers and COPD patients. SIRT1 deficiency increased CS-induced TIMP-1 acetylation, and these effects were attenuated by SIRT1 overexpression. These results suggest that SIRT1 protects against COPD/emphysema via redressing the TIMP-1/MMP-9 imbalance involving TIMP-1 deacetylation. Thus, redressing the TIMP-1/MMP-9 imbalance by pharmacological activation of SIRT1 is an attractive approach in the intervention of COPD.
Rationale: In rodent model systems, the sequential changes in lung morphology resulting from hyperoxic injury are well characterized, and are similar to changes in human acute respiratory distress syndrome (ARDS). In the injured lung, alveolar type two (AT2) epithelial cells play a critical role restoring the normal alveolar structure. Thus characterizing the changes in AT2 cells will provide insights into the mechanisms underpinning the recovery from lung injury. Methods: We apply an unbiased systems level proteomics approach to elucidate molecular mechanisms contributing to lung repair in a rat hyperoxic lung injury model. AT2 cells were isolated from rat lungs at predetermined intervals during hyperoxic injury and recovery. Protein expression profiles were determined by using iTRAQ® with tandem mass spectrometry. Results: Of 959 distinct proteins identified, 183 significantly changed in abundance during the injury-recovery cycle. Gene Ontology enrichment analysis identified cell cycle, cell differentiation, cell metabolism, ion homeostasis, programmed cell death, ubiquitination, and cell migration to be significantly enriched by these proteins. Gene Set Enrichment Analysis of data acquired during lung repair revealed differential expression of gene sets that control multicellular organismal development, systems development, organ development, and chemical homeostasis. More detailed analysis identified activity in two regulatory pathways, JNK and miR 374. A novel Short Time-series Expression Miner (STEM) algorithm identified protein clusters with coherent changes during injury and repair. Conclusion: Coherent changes occur in the AT2 cell proteome in response to hyperoxic stress. These findings offer guidance regarding the specific molecular mechanisms governing repair of the injured lung.
We have employed a simple and robust non-invasive method of continuous in vivo long-term bromodeoxyuridine (BrdU) labelling to analyse lung mesenchymal stromal cell turnover in adult mice in the steady state. Mathematical modelling of BrdU uptake in flow cytometrically sorted CD45negCD31negSca-1pos lung cells following long-term feeding of BrdU to mice in their drinking water reveals that lung mesenchymal stromal cells cycle continuously throughout life. Analysis of BrdU incorporation during long-term feeding, and during chasing (delabeling) following replacement of BrdU-water with normal water shows that the CD45negCD31negSca-1pos lung mesenchymal cell compartment turns over at a rate of approximately 2.26% per day with a time to half-cycled of the compartment of 44 days, with an estimated cell proliferation rate of 0.004/day, and a cell death rate of 0.018/day.
Ion channels perform a variety of cellular functions in lung epithelia. Oxidant- and antioxidant-mediated mechanisms (that is--redox regulation) of ion channels are areas of intense research. Significant progress has been made in our understanding of redox regulation of ion channels since the last Experimental Biology report in 2003. Advancements include: 1) identification of non-phagocytic NADPH oxidases as sources of regulated reactive species (RS) production in epithelia, 2) an understanding that excessive treatment with antioxidants can result in greater oxidative stress, and 3) characterization of novel RS signaling pathways that converge upon ion channel regulation. These advancements, as discussed at the 2013 Experimental Biology Meeting in Boston, MA, impact our understanding of oxidative stress in the lung, and in particular, illustrate that the redox state has profound effects on ion channel and cellular function.
Club cell secretory protein is an indirect phospholipase A2 inhibitor with some immunosuppressive and anti-proliferative properties that is expressed in bronchiolar Club cells. In our murine bone marrow transplant (BMT) model of obliterative bronchiolitis (OB) Club cell secretory protein (CCSP) is diminished; however, its role is unknown. To determine the role of CCSP, B6 wild type (WT) or CCSP deficient (CCSP-/-) mice were lethally conditioned and given allogeneic bone marrow with a sublethal dose of splenic allogeneic T-cells to induce OB. We found that CCSP -/- mice demonstrated a higher mortality following BMT induced OB compared to WT mice. Mice were analyzed 60 days post-BMT for protein expression, pulmonary lung function, and histology. CCSP levels were reduced in WT mice with BMT induced OB and lower levels correlated to decreased lung compliance. CCSP -/- had a higher degree of injury and fibrosis as measured by hydroxyl proline and increased lung resistance. Replacement with recombinant intravenous CCSP partially reversed the weight loss and increased mortality in the CCSP -/- mice. In conclusion, CCSP is decreased in WT mice that develop OB following a BMT. This decrease in CCSP results in lung injury and fibrosis that was reflected in lung mechanics and decreased survival. Replacing CCSP systemically resulted in improved survival. These findings indicate that CCSP has a regulatory role in OB and may have potential as a preventive therapy.
In these studies we determined if progressive pulmonary inflammation associated with aging in surfactant protein-D (Sftpd)-/- mice leads to an exacerbated response to ozone. In Sftpd-/- mice, but not WT mice, increasing age from 8 wk to 80 wk was associated with increasing numbers of enlarged vacuolated macrophages in the lung, along with alveolar wall rupture and increased bronchoalveolar lavage protein and cells, as well as Type II hyperplasia. In Sftpd-/- mice, heme oxygenase+ macrophages also increased with age, together with classically (iNOS+) and alternatively (mannose receptor+, YM-1+, or galectin-3+) activated macrophages. In WT and Sftpd-/- mice, increasing age from 8 wk to 27 wk resulted in reduced lung stiffness, as reflected by decreases in resistance and elastance spectra. This response was blunted in 80 wk old Sftpd-/- mice. Ozone exposure (0.8 ppm, 3 h) caused increases in lung pathology, alveolar epithelial barrier dysfunction, and numbers of iNOS+ macrophages in 8 wk and 27 wk old Sftpd-/-, but not WT mice. Conversely, while increases in alternatively activated macrophages were observed in 8 wk old WT mice following ozone exposure, no changes were observed in Sftpd-/- mice. Ozone also caused alterations in both airway and tissue mechanics in Sftpd-/- mice at 8 wk and 27 wk, but not at 80 wk. These data demonstrate that mild to moderate pulmonary inflammation results in increased sensitivity to ozone; however, in senescent mice, these responses are overwhelmed by the larger effects of age-related increases in baseline inflammation and lung injury.
Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of 8 members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiologic recordings which were blocked using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiologic properties of CaCCs, and they modulate contractile tone in ASM. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.
The receptor for advanced glycation end-products (RAGE) and its soluble forms are predominantly expressed in lung but its physiological importance in this organ is not yet fully understood. As RAGE acts as cell adhesion molecule, we postulated its physiological importance in the respiratory mechanics. Respiratory function using a buffer-perfused isolated lung system and biochemical parameters of the lung were studied in young, adult and old RAGE knock out (RAGE-KO) mice and wild-type (WT) mice. Lungs from RAGE-KO mice showed a significant increase in the dynamic lung compliance and a decrease in the maximal expiratory air flow independent of age-related changes. We also determined lower mRNA and protein levels of elastin in lung tissue of RAGE-KO mice. RAGE deficiency did not influence the collagen protein level, lung capillary permeability and inflammatory parameters (TNF-β, HMGB-1) in lung. Overexpressing RAGE as well as soluble RAGE in lung fibroblasts or co-cultured lung epithelial cells increased the mRNA expression of elastin. Moreover, immunoprecipitation studies indicated a trans interaction of RAGE in lung epithelial cells. Our findings suggest the physiological importance of RAGE and its soluble forms in supporting the respiratory mechanics in which RAGE trans interactions and the influence on elastin expression might play an important role.
Inhalation of particulate matter has presented a challenge to human health for thousands of years. The underlying mechanism for biological effect following particle exposure is incompletely understood. We tested the postulate that particle sequestration of cell and mitochondrial iron is a pivotal event mediating oxidant generation and biological effect. In vitro exposure of human bronchial epithelial cells to silica produced a reduction in intracellular iron which resulted in increases in both the importer divalent metal transporter 1 expression and metal uptake. Diminished mitochondrial 57Fe concentrations following silica exposure confirmed particle sequestration of cell iron. Pre-incubation of cells with excess ferric ammonium citrate increased cell, nuclear, and mitochondrial metal concentrations and prevented significant iron loss from mitochondria following silica exposure. Cell and mitochondrial oxidant generation increased after silica incubation but pre-treatment with iron diminished this generation of reactive oxygen species. Silica exposure activated MAP kinases (ERK and p38) and altered the expression of transcription factors (nF-kappaB and nrf2), pro-inflammatory cytokines (interleukin-8 and -6), and apoptotic proteins. All these changes in indices of biological effect were either diminished or inhibited by cell pre-treatment with iron. Finally, percentage of neutrophils and total protein concentrations in an animal model instilled with silica was decreased by concurrent exposure to iron. We conclude that an initiating event in the response to particulate matter is a sequestration of cell and mitochondrial iron by endocytosed particle. The resultant oxidative stress and biological response after particle exposure are either diminished or inhibited by increasing the cell iron concentration.
Rationale: Intrapleural processing of prourokinase (scuPA) in tetracycline (TCN)-induced pleural injury in rabbits was evaluated to better understand the mechanisms governing successful scuPA-based intrapleural fibrinolytic therapy (IPFT); capable of clearing pleural adhesions in this model. Methods: Pleural fluid (PF) was withdrawn 0-80 min and 24 h after IPFT with scuPA (0-0.5 mg/kg) and activities of free urokinase (uPA), plasminogen activator inhibitor 1 (PAI-1) and uPA complexed with α-macroglobulin (αM) were assessed. Similar analyses were performed using PFs from patients with empyema, parapneumonic, and malignant pleural effusions. Results: The peak of uPA activity (5-40 min) reciprocally correlated with the dose of intrapleural scuPA. Endogenous active PAI-1 (10-20 nM) decreased the rate of intrapleural scuPA activation. The slow step of intrapleural inactivation of free uPA (t1/2β=40±10 min) was dose-independent and 6.7-fold slower than in blood. Up to 260±70 nM of αM/uPA formed in vivo (kass=580±60 M-1s-1). αM/uPA and products of its degradation contributed to durable intrapleural plasminogen activation up to 24 h after IPFT. Active PAI-1, active α2M, and α2M/uPA found in empyema, pneumonia, and malignant PFs demonstrate the capacity to support similar mechanisms in humans. Conclusion: Intrapleural scuPA processing differs from that in the bloodstream and includes (i) dose-dependent control of scuPA activation by endogenous active PAI-1; (ii) two-step inactivation of free uPA with simultaneous formation of αM/uPA; (iii) slow intrapleural degradation of αM/uPA releasing active free uPA. This mechanism offers potential clinically relevant advantages that may enhance the bioavailability of intrapleural scuPA and may mitigate the risk of bleeding complications.
COPD is characterized by abnormal repair in the lung resulting in airway obstruction associated with emphysema and peripheral airway fibrosis. As the presence and degree of airways disease and emphysema varies between COPD patients this may explain the heterogeneity in the response to treatment. It is currently unknown whether and to what extent inhaled steroids can affect the abnormal repair process in the airways and lung parenchyma in COPD. We investigated the effects of fluticasone on TGFβ-and cigarette smoke-induced changes in Smad signaling and extra cellular matrix (ECM) production in airway and parenchymal lung fibroblasts from patients with severe COPD. We showed that TGFβ-induced ECM production by pulmonary fibroblasts, but not activation of the Smad pathway, was sensitive to the effects of fluticasone. Fluticasone induced decorin production by airway fibroblasts, and partly reversed the negative effects of TGFβ treatment. Fluticasone inhibited biglycan production in both airway and parenchymal fibroblasts and procollagen 1 production only in parenchymal fibroblasts, thereby restoring the basal difference in procollagen 1 production between airway and parenchymal fibroblasts. Our findings suggest that the effects of steroids on the airway compartment may be beneficial for patients with severe COPD, i.e. restoration of decorin loss around the airways, while the effects of steroids on the parenchyma may be detrimental as the tissue repair response, i.e. biglycan and procollagen production is inhibited. More research is needed to further disentangle these differential effects of steroid treatment on the different lung compartments and its impact on tissue repair and remodeling in COPD.
Surgical resection of pulmonary tissue exerts a pro-regenerative stretch stimulus in the remaining lung units. Whether this regeneration process reenacts part or whole of lung morphogenesis developmental program remains unclear. To address this question, we analyzed the stretch-induced regenerating lung transcriptome in mice after left pneumonectomy (PNX) in its developmental context. We created a C57BL/6 mice lung regeneration transcriptome time course at 3, 7, 14, 28 and 56 days post-PNX, profiling the cardiac and medial lobes and whole right lung. Prominent expression at days 3 and 7 of genes related to cell proliferation (Ccnb1, Bub1 and Cdk1), extracellular matrices (Col1a1, Eln and Tnc) and proteases (Serpinb2 and Mmp9) indicated regenerative processes that tapered off after 56 days. We projected the post-PNX transcriptomic time course into the transcriptomic principal component space of the C57BL/6 mouse developing lung time series from embryonic day 9.5 to postnatal day 56. All post-PNX samples were localized around late postnatal stage of developing lungs. Shortly after PNX, the temporal trajectory of regenerating lobes and right lung reversed course relative to the developing lungs in a process reminiscent of de-differentiation. This reversal was limited to the later postnatal stage of lung development. The post-PNX temporal trajectory then moves forward in lung development time close to its pre-PNX state after days 28 to 56 in a process resembling re-development. A plausible interpretation is that remaining pulmonary tissue reverts to a more primitive stage of development with higher potential for growth to generate tissue in proportion to the loss.
Rationale: Mechanical ventilation may cause harm by straining lungs at a time they are particularly prone to injury from deforming stress Objective: To define the relative contributions of alveolar overdistension and cyclic recruitment and "collapse" of unstable lung units to membrane wounding of alveolar epithelial cells. Methods: We measured the interactive effects of tidal volume (VT), transpulmonary pressure (PTP) and of airspace liquid on the number of alveolar epithelial cells with plasma membrane wounds in ex vivo mechanically ventilated rat lungs. Plasma membrane integrity was assessed by Propidium Iodide (PI) exclusion in confocal images of subpleural alveoli. Main Results: Cyclic inflations of normal lungs from zero end-expiratory pressure (ZEEP) to 40 cm H2O produced VT's of 56.9±3.1cc/kg and were associated with 0.12±0.12 PI positive cells per alveolus. A preceding tracheal instillation of normal saline (3ml) reduced VT to 49.1±6cc/kg, but was associated with a significantly greater number of wounded alveolar epithelial cells (0.52±0.16 cells per alveolus; p<0.01). Mechanical ventilation of completely saline filled lungs with saline (VT=52cc/kg) to pressures between 10 and 15 cm H2O was associated with the least number of wounded epithelial cells (0.02±0.02 cells per alveolus; p<0.01). In mechanically ventilated, partially saline filled lungs the number of wounded cells increased substantially with VT, but once VT was accounted for, wounding was independent of maximal PTP. Conclusions: Interfacial stress associated with the generation and destruction of liquid bridges in airspaces is the primary biophysical cell injury mechanism in mechanically ventilated lungs.
Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder consisting of chronic bronchitis and/or emphysema. COPD patients suffer from chronic infections, display exaggerated inflammatory responses and a progressive decline in respiratory function. The respiratory symptoms of COPD are similar to those seen in cystic fibrosis (CF), although the molecular basis of the two disorders differs. CF is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride and bicarbonate channel (CFTR), leading to CFTR dysfunction. The majority of COPD cases results from chronic oxidative insults such as cigarette smoke. Interestingly, environmental stresses including cigarette smoke, hypoxia and chronic inflammation have also been implicated in reduced CFTR function, and this suggests a common mechanism that may contribute to both the CF and COPD. Therefore, improving CFTR function may offer an excellent opportunity for the development of a common treatment for CF and COPD. In this article, we review what is known about the CF respiratory phenotype and discuss how diminished CFTR expression-associated ion transport defects may contribute to some of the pathological changes seen in COPD.
Rationale: Mechanical ventilation induces pulmonary apoptosis and inhibits alveolar development in preterm infants, but the molecular basis for the apoptotic injury is unknown. Objective: To determine the signaling mechanism(s) of ventilation(stretch)-induced apoptosis in newborn rat lung. Methods: Seven-day old rats were ventilated with room air for 24 h using moderate tidal volumes (8.5 mL.kg-1). Isolated fetal rat lung epithelial and fibroblast cells were subjected to continuous cyclic stretch (5, 10 or 17% elongation) for up to 12 h. Measurements and Main Results: Prolonged ventilation increased significantly the number of apoptotic alveolar type II cells (i.e. TUNEL-labelling, anti-cleaved caspase-3 immunochemistry) and was associated with increased expression of the apoptotic mediator Fas Ligand (FasL). Fetal lung epithelial cells, but not fibroblasts, subjected to maximal (i.e. 17%, but not lesser elongation) cyclic stretch exhibited increased apoptosis (i.e. nuclear fragmentation; DNA laddering) which appeared to be mediated via the extrinsic pathway (increased expression of FasL and cleaved caspase 3, 7 and 8). The intrinsic pathway appeared not to be involved (minimal mitochondrial membrane depolarization (JC-1 flow analysis) and no activation of caspase-9). Universal caspases inhibition and neutralization of FasL abrogated the stretch-induced apoptosis. Conclusion: Prolonged mechanical ventilation induces apoptosis of alveolar type II cells in newborn rats and the mechanism appears to involve activation of the extrinsic death pathway via the FasL/Fas system.
Nitric oxide (NO) regulates lung development through incompletely understood mechanisms. NO controls pulmonary vascular smooth muscle cell (SMC) differentiation largely through stimulating soluble guanylate cyclase (sGC) to produce cGMP and increase cGMP-mediated signaling. To examine the role of sGC in regulating pulmonary development, we tested whether decreased sGC activity reduces alveolarization in the normal and injured newborn lung. For these studies, mouse pups with gene-targeted sGCα1 subunit truncation were used because we determined that they have decreased pulmonary sGC enzyme activity. sGCα1 knock out (KO) mouse pups were observed to have decreased numbers of small airway structures and lung volume compared with wild type (WT) mice, although lung septation and body weights were not different. However, following mild lung injury caused by breathing 70% O2, the sGCα1 KO mouse pups had pronounced inhibition of alveolarization, as evidenced by an increase in airway mean linear intercept, reduction in terminal airway units, and decrease in lung septation and alveolar openings, as well as reduced somatic growth. Because cGMP regulates SMC phenotype, we also tested whether decreased sGC activity lung reduces myofibroblast differentiation. Cellular markers revealed that vascular SMC differentiation decreased while myofibroblast activation increased in the hyperoxic sCGα1 KO pup lung. These results indicate that lung development, particularly during hyperoxic injury, is impaired in mouse pups with diminished sGC activity. These studies support the investigation of sGC-targeting agents as therapies directed at improving development in the newborn lung exposed to injury.
The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor, S1PR3, are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African and European descent samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Due to limited sample size, conclusive findings could not be drawn in African descent ARDS subjects, however significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two European descent samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter, reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge, and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.
Asthma is a major public health hazard world-wide. Its transgenerational inheritance has been inferred from epidemiologic studies. More recently, using nicotine as a proxy for maternal smoking, we have demonstrated that an asthma-like phenotype can be inherited by rat offspring for up to two generations, i.e., multigenerationally, after the initial intrauterine exposure. We hypothesized that asthma transmission to offspring following perinatal nicotine exposure is not restricted up to F2 generation, but it also extends to subsequent generations. To test this hypothesis, using a well-established rat model of nicotine exposure-induced childhood asthma, we determined if perinatal nicotine exposure of F0 gestating dams would transmit asthma transgenerationally to F3 offspring. We now extend our findings to third generation offspring, including abnormal pulmonary function, particularly as it relates to the occurrence in the upper airway exclusively in males, and to its effects on molecular functional markers (fibronectin and PPAR), previously shown to be consistent with the asthma phenotype, herein expressed in fibroblasts isolated from the lung. These data, for the first time, demonstrate the transgenerational transmission of the asthma phenotype to F3 offspring following perinatal nicotine exposure of F0 dams.
Background and aims: The use of fractional exhaled nitric oxide (FeNO) has been suggested as a quantitative marker for pulmonary arterial hypertension (PAH) in humans. To further characterize FeNO in PAH we investigated this marker in a rodent model. As there is no standardized technique for FeNO measurement in animals, we intended to reduce measuring errors and confounders of an existing published method by mathematical modification and tested its applicability in an NO-regulating therapy concept of PAH. Methods: Thirty-three male Sprague-Dawley rats underwent unilateral pneumonectomy and monocrotaline (P/MCT) injection and were observed for 49 days. A telemetric catheter was introduced into the left pulmonary artery to continuously record mean pulmonary arterial pressure (mPAP) and FeNO was assessed. After 35 days, animals were randomised to receive either oral L-arginine (300mg/kg) in combination with tetrahydrobiopterin (20mg/kg) therapy (n=12) or vehicle (n=11) daily over a period of 14 days. Results: Mean PAP at baseline was 17.19±9.62mmHg, which increased to 53.1±10.63mmHg 28 days after monocrotaline exposure (p<0.001). Using the modified technique there was an inverse correlation between exhaled NO and pulmonary pressures before (r=-0.366, p=0.043) and after MCT (r=-0.363, p=0.038) as well as after therapy administration (r=-0.657, p=0.02). Conclusion: Our modified technique proved robust in a rodent model, since valid and reproducible data were gained and showed an inverse correlation between exhaled NO and mPAP, while the existing method did not.
Proliferation and migration of fibroblasts are vital for fetal lung development. However the regulatory mechanisms are poorly understood. We have previously shown that TROP2 gene expression is closely associated with fetal lung cell proliferation in vivo and that TROP2 knock-down decreases proliferation of fetal lung fibroblasts in culture. We hypothesized that the Trop2 protein also regulates the morphology and motility of fetal lung fibroblasts. Fibroblasts isolated from fetal rat lungs (gestational age E19) adopted a myofibroblast-like morphology in culture. Trop2 protein was localized to lamellipodia. TROP2 siRNA significantly decreased: TROP2 mRNA levels by 77%, the proportion of cells containing Trop2 protein by 70% and cell proliferation by 50%. TROP2 siRNA also decreased the degree of motility as determined by the number of gridlines that cells moved across (2.2 ± 0.2 vs. 3.2 ± 0.2; p < 0.001). TROP2 knock-down altered cell morphology causing a notable absence of lamellipodia and abnormal localisation of components of the cell migration apparatus and it reduced phosphorylated ERK1 and ERK2 levels. In contrast, TROP2 over-expression significantly increased: TROP2 mRNA levels by 40-fold, cell proliferation by 40%, the proportion of cells that were motile by 20% and the number of gridlines that cells moved across (2.1 ± 0.2 vs. 1.6 ± 0.1; p<0.001). Our data suggests that Trop2 regulates cell proliferation and motility and that it does so by regulating the ERK pathway and several critical components of the cell migration apparatus.
Asthma is a disease of the airways with symptoms including exaggerated airway narrowing and airway inflammation. Early asthma therapies utilized methylxanthines to relieve symptoms, in part, by inhibiting cyclic nucleotide phosphodiesterases (PDEs), the enzyme responsible for degrading cAMP. The classification of tissue specific PDE subtypes and the clinical introduction of PDE-selective inhibitors for COPD (i.e roflumilast) have reopened the possibility of using PDE inhibition in the treatment of asthma. Quercetin is a naturally-derived PDE4-selective inhibitor found in fruits, vegetables, and tea. We hypothesized that quercetin relaxes airway smooth muscle via cAMP-mediated pathways and augments β-agonist relaxation. Tracheal rings from male A/J mice were mounted in myographs and contracted with acetylcholine (ACh). Addition of quercetin (100 nM-1 mM) acutely and concentration-dependently relaxed airway rings pre-contracted with ACh. In separate studies, pretreatment with quercetin (100 μM) prevented force generation upon exposure to ACh. In additional studies, quercetin (50 μM) significantly potentiated isoproterenol-induced relaxations. In in vitro assays, quercetin directly attenuated phospholipase C activity, decreased inositol phosphate synthesis, and decreased intracellular calcium responses to Gq-coupled agonists (histamine or bradykinin). Finally, nebulization of quercetin (100 μM) in an in vivo model of airway responsiveness significantly attenuated methacholine-induced increases in airway resistance. These novel data show that the natural PDE4-selective inhibitor, quercetin, may provide therapeutic relief of asthma symptoms and decrease reliance on short-acting β-agonists.
Respiratory muscle-associated stretch has been implicated in normal lung development (fetal breathing movements) and post-pneumonectomy lung growth. To test the hypothesis that mechanical stretch from diaphragmatic contraction contributes to lung growth, we performed left phrenic nerve transections (PNT) in mice with and without ipsilateral pneumonectomy. PNT was demonstrated by asymmetric costal margin excursion and confirmed at autopsy. In mice with two lungs, PNT was associated with a decrease in ipsilateral lung volume (p<.05) and lung weight (p<.05). After pneumonectomy, PNT was not associated with a change in activity level, measureable hypoxemia or altered minute ventilation; however, microCT scanning demonstrated altered displacement and underinflation of the cardiac lobe within the first week after pneumonectomy. Coincident with the altered structural realignment, lung impedance measurements, fitted to the constant phase model, demonstrated elevated airway resistance (p<.05), but normal peripheral tissue resistance (p>.05). Most important, PNT appeared to abrogate compensatory lung growth after pneumonectomy; the weight of the lobes of the right lung were significantly less than pneumonectomy alone (p<.001) and indistinguishable from non-surgical controls (p>.05). We conclude that the cyclic stretch associated with diaphragmatic muscle contraction is a controlling factor in post-pneumonectomy compensatory lung growth.
Sustained lung inflations (SI) at birth may recruit functional residual capacity (FRC). Clinically, SI increase oxygenation and decrease need for intubation in preterm infants. We tested whether a SI to recruit FRC would decrease lung injury from subsequent ventilation of fetal, preterm lambs. The preterm fetus (128±1d gestation) was exteriorized from the uterus, a tracheostomy performed, and fetal lung fluid was removed. While maintaining placental circulation, fetuses were randomized to one of four 15 min interventions: 1) PEEP 8 cmH2O (n=4), 2) 20 sec SI to 50 cmH2O then PEEP 8 cmH2O (n=10), 3) mechanical ventilation at VT 7 mL/kg (n=13) or 4) 20 sec SI then ventilation at VT 7 mL/kg (n=13). Lambs were ventilated with 95%N2/5%CO2 and PEEP 8 cmH2O. Volume recruitment was measured during SI and fetal tissues were collected after an additional 30 min on placental support. SI achieved a mean FRC recruitment of 15 mL/kg (range 8 to 27). 50% of final FRC was achieved by 2 sec, 65% by 5 sec, and 90% by 15 sec, demonstrating prolonged SI times are needed to recruit FRC. SI alone released acute phase proteins into the fetal lung fluid, and increased mRNA expression of pro-inflammatory cytokines and acute phase response genes in the lung. Mechanical ventilation further increased all markers of lung injury. SI prior to ventilation, regardless of the volume of FRC recruited, did not alter the acute phase and pro-inflammatory responses to mechanical ventilation at birth.
Dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) leads to many cellular consequences, including perinuclear accumulation of free cholesterol due to impaired endosomal transport. The hypothesis being tested is that cystic fibrosis related perinuclear cholesterol accumulation due to disrupted endocytic trafficking occurs as a result of reduced microtubule (MT) acetylation. Here, it is identified that acetylated-α-tubulin (Ac-tub) content is reduced by approximately 40% compared to respective wild-type controls in both cultured CF cell models (IB3) and primary Cftr -/- mouse nasal epithelial tissue. Histone deacetylase 6 (HDAC6) has been shown to regulate MT acetylation, which provides reasonable grounds to test its impact on reduced Ac-tub content on CF cellular phenotypes. Inhibition of HDAC6, either through tubastatin treatment or HDAC6 knockdown in CF cells, increases Ac-tub content and results in redistributed free cholesterol and reduced stimulation of NF-B activity. Mechanistically, endoplasmic reticulum (ER) stress, which is widely reported in CF and leads to aggresome formation, is identified as a regulator of MT acetylation. F508del CFTR correction with C18 in primary airway epithelial cells restores MT actyaltion and cholesterol transport. A significant role for phosphotidyl inositol-3 kinase, p110α (PIK3CA) is also identified as a regulator of MT acetylation.
Alveolar epithelial type II (ATII) cells are essential for maintaining normal lung homeostasis because they produce surfactant, express innate immune proteins, and can function as progenitors for alveolar epithelial type I (ATI) cells. Although autocrine production of transforming growth factor (TGF)-β1 has been shown to promote the transdifferentiation of primary rat ATII to ATI cells in vitro, mechanisms controlling this process still remain poorly defined. Here, evidence is provided that Tgf-β1, 2, 3 mRNA and phosphorylated SMAD2 and SMAD3 significantly increase as primary cultures of mouse ATII cells transdifferentiate to ATI cells. Concomitantly, bone morphogenetic protein (Bmp)-2 and 4 mRNA, and phosphorylated SMAD 1/5/8 expression decrease. Exogenously supplied recombinant human TGF-β1 inhibited BMP signaling, and enhanced transdifferentiation by promoting the loss of ATII cell-specific gene expression and weakly stimulating ATI cell-specific gene expression. On the other hand, exogenously supplied recombinant human BMP-4 inhibited TGF-β signaling, and delayed transdifferentiation by inhibiting the gain in ATI cell-specific gene expression and weakly delaying the loss of ATII cell-specific gene expression. In MLE15 cells, siRNA knockdown of TGF-β receptor type-1 (TGFβRI) enhanced basal expression of ATII genes while siRNA knockdown of BMP receptors type-1a and -1b (BMPR1a/b) enhanced basal expression of ATI genes. Together, these results suggest that the rate of ATII cell transdifferentiation is controlled by the opposing actions of BMP and TGF-β signaling that switch during the process of transdifferentiation.
We previously demonstrated that in nasal epithelial cells (NECs) from smokers methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which 5 were differentially methylated in NEC from smokers (CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology-level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased while ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.
Rationale: Asthma is a chronic inflammatory disease of the small airways, with airway hyperresponsiveness (AHR) and inflammation as hallmarks. Recent studies suggest a role for arginase in asthma pathogenesis, possibly because arginine is the substrate for both arginase and NO-synthase (NOS) and because NO modulates bronchial tone and inflammation. Objective: To investigate the importance of increased pulmonary arginase 1 expression on methacholine-induced AHR and lung inflammation in a mouse model of allergic asthma. Methods: Arginase 1 expression in the lung was ablated by crossing Argfl/flwith Tie2Cretg/- mice. Mice were sensitized and then challenged with ovalbumin. Lung function was measured with the Flexivent. Adaptive changes in gene expression, chemokine and cytokine secretion, and lung histology were quantified with qPCR, ELISA, and immunohistochemistry. Results: Arg1 deficiency did not affect the allergic response in lungs and large-airway resistance, but improved peripheral lung function (tissue elastance and resistance), attenuated adaptive increases in mRNA expression of arginine-catabolizing enzymes Arg2 and Nos2, arginine transporters Slc7a1 and Slc7a7, chemokines Ccl2 and Ccl11, cytokines Tnfa and Ifng, mucus-associated epithelial markers Clca3 and Muc5ac, and lung content of IL13 and CCL11. However, expression of Il4, Il5, Il10, and Il13 mRNA, lung content of IL4, IL5, IL10, TNFα, and IFN protein, and lung pathology were not affected. Correlation analysis showed that Arg1 ablation disturbed the coordinated pulmonary response to ovalbumin challenges, suggesting arginine (metabolite) dependence of this response. Conclusion: Arg1 ablation in the lung improved peripheral lung function and affected arginine metabolism, but had little effect on airway inflammation.
Alveolar epithelial damage is a critical event that leads to protein-rich edema in acute lung injury (ALI), but the mechanisms leading to epithelial damage are not completely understood. Cell death by necrosis and apoptosis occurs in alveolar epithelial cells in the lungs of patients with ALI. Fas activation induces apoptosis of alveolar epithelial cells, but its role in the formation of lung edema is unclear. The main goal of this study was to determine whether activation of the Fas/FasL pathway in the lungs could alter the function of the lung epithelium, and the mechanisms involved. The results show that Fas activation alters the alveolar barrier integrity and impairs the ability of the lung alveolar epithelium to reabsorb fluid from the air spaces. This result was dependent on the presence of a normal Fas receptor, and was not affected by inflammation induced by Fas activation. Alteration of the fluid transport properties of the alveolar epithelium was partially restored by β-adrenergic stimulation. Fas activation also caused apoptosis of alveolar endothelial cells, but this effect was less pronounced than the effect on the alveolar epithelium. Thus, activation of the Fas pathway impairs alveolar epithelial function in mouse lungs by mechanisms involving caspase-dependent apoptosis, suggesting that targeting apoptotic pathways could reduce the formation of lung edema in ALI.
Stimulation of MAS oncogene receptor (MAS) or angiotensin (Ang) receptor type 2 (AT2) may be novel therapeutic options for neonatal chronic lung disease (CLD) by counterbalancing the adverse effects of the potent vasoconstrictor angiotensin II, consisting of arterial hypertension (PAH)-induced right ventricular hypertrophy (RVH) and pulmonary inflammation. We determined the cardiopulmonary effects in neonatal rats with CLD of daily treatment during continuous exposure to 100% oxygen for 10 days with specific ligands for MAS (cyclic Ang-(1-7); 10-50 µg/kg/day) and AT2 (dKcAng-(1-7); 5-20 µg/kg/day). Parameters investigated included lung and heart histopathology, fibrin deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in the renin-angiotensin system, inflammation, coagulation and alveolar development. We investigated the role of nitric oxide synthase (NOS) inhibition with N-nitro-L-arginine methyl ester (L-NAME, 25 mg/kg/day) during AT2 agonist treatment. Prophylactic treatment with agonists for MAS or AT2 for 10 days diminished cardiopulmonary injury by reducing alveolar septum thickness and medial wall thickness of small arterioles, and preventing RVH. Both agonists attenuated the pulmonary influx of inflammatory cells, including macrophages (via AT2) and neutrophils (via MAS), but did not reduce alveolar enlargement and vascular alveolar leakage. The AT2 agonist attenuated hyperoxia-induced fibrin deposition. In conclusion, stimulation of MAS or AT2 attenuates cardiopulmonary injury by reducing pulmonary inflammation and preventing PAH-induced RVH, but does not affect alveolar and vascular development in neonatal rats with experimental CLD. The beneficial effects of AT2 activation on experimental CLD were mediated via a NOS-independent mechanism.
Pathologic pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling in pulmonary hypertensive diseases associated with hypoxia. Both the hypoxia-inducible factor (HIF) and PI3K/Akt pathways have been implicated in hypoxia-induced PASMC proliferation. Thioredoxin-1 (Trx1) is a ubiquitously expressed protein that is involved in redox-dependent signaling via hypoxia-inducible factor (HIF) and PI3K-Akt in cancer. The role of Trx1 in PASMC proliferation has not been elucidated. The present studies tested the hypothesis that Trx1 regulates hypoxia-induced PASMC proliferation via HIF and/or PI3K-Akt-dependent mechanisms. Following exposure to chronic hypoxia, our data indicate that Trx1 activity is increased in adult murine lungs. Furthermore, hypoxia-induced increases in cellular proliferation are correlated with increased Trx1 expression, HIF activation, and Akt activation in cultured human PASMC. Both siRNA-mediated knockdown and pharmacologic Trx1 inhibition attenuated hypoxia-induced PASMC proliferation, HIF activation, and Akt activation. While Trx1 knockdown suppressed hypoxia-induced PI3K-Akt activation in PASMC, PI3K-Akt inhibition prevented hypoxia-induced proliferation but had no effect on hypoxia-induced increases in Trx1 or HIF activation. Thus, our findings indicate that Trx1 contributes to hypoxia-induced PASMC proliferation by modulating HIF activation and subsequent PI3K-Akt activation. These novel data suggest that Trx1 might represent a novel therapeutic target to prevent hypoxic PASMC proliferation.
Treatment of acute and chronic pulmonary infections caused by opportunistic pathogen Pseudomonas aeruginosa is limited by the increasing frequency of multidrug bacterial resistance. Here, we describe a novel adjunctive therapy in which administration of a mix of simple sugars - mannose, fucose, and galactose - inhibits bacterial attachment and lung damage, and potentiates standard antibiotic therapy. The sugar mixture inhibits adhesion of non-mucoid and mucoid P. aeruginosa strains to bronchial epithelial cells in vitro. In a murine pneumonia model, treatment with the sugar mixture alone diminishes bacterial dissemination to the subpleural alveoli, lung damage, and neutrophil- and IL-8-driven inflammatory responses. Remarkably, the sugars act synergistically with conventional antibiotics, beta-lactams and quinolones, to further reduce bacterial lung burden and lung damage in murine model of acute pneumonia, and, in an ex vivo infections of live trachea and lung tissues, to further reduce bacterial count. In ex vivo infections and in vitro host-free liquid bacterial culture, the sugars induce rapid but reversible formation of bacterial clusters that exhibit enhanced susceptibility to antibiotics compared to individual bacteria. These suggest a mechanism, at least in part dependent on host cell factor(s), by which the sugars potentiate the efficacy of antibiotics in vivo and ex vivo. Our findings show that sugar inhalation, an inexpensive and safe therapeutic, could be used in combination with conventional antibiotic therapy to more effectively treat P. aeruginosa lung infections.