Plasminogen activation to plasmin protects from lung fibrosis, but the mechanism underlying this antifibrotic effect remains unclear. We found that mice lacking plasminogen activation inhibitor–1 (PAI-1), which are protected from bleomycin-induced pulmonary fibrosis, exhibit lung overproduction of the antifibrotic lipid mediator prostaglandin E2 (PGE2). Plasminogen activation upregulated PGE2 synthesis in alveolar epithelial cells, lung fibroblasts, and lung fibrocytes from saline- and bleomycin-treated mice, as well as in normal fetal and adult primary human lung fibroblasts. This response was exaggerated in cells from Pai1–/– mice. Although enhanced PGE2 formation required the generation of plasmin, it was independent of proteinase-activated receptor 1 (PAR-1) and instead reflected proteolytic activation and release of HGF with subsequent induction of COX-2. That the HGF/COX-2/PGE2 axis mediates in vivo protection from fibrosis in Pai1–/– mice was demonstrated by experiments showing that a selective inhibitor of the HGF receptor c-Met increased lung collagen to WT levels while reducing COX-2 protein and PGE2 levels. Of clinical interest, fibroblasts from patients with idiopathic pulmonary fibrosis were found to be defective in their ability to induce COX-2 and, therefore, unable to upregulate PGE2 synthesis in response to plasmin or HGF. These studies demonstrate crosstalk between plasminogen activation and PGE2 generation in the lung and provide a mechanism for the well-known antifibrotic actions of the fibrinolytic pathway.
Kristy A. Bauman, Scott H. Wettlaufer, Katsuhide Okunishi, Kevin M. Vannella, Joshua S. Stoolman, Steven K. Huang, Anthony J. Courey, Eric S. White, Cory M. Hogaboam, Richard H. Simon, Galen B. Toews, Thomas H. Sisson, Bethany B. Moore, Marc Peters-Golden
Pulmonary fibrosis is a progressive, dysregulated response to injury culminating in compromised lung function due to excess extracellular matrix production. The heparan sulfate proteoglycan syndecan-4 is important in mediating fibroblast-matrix interactions, but its role in pulmonary fibrosis has not been explored. To investigate this issue, we used intratracheal instillation of bleomycin as a model of acute lung injury and fibrosis. We found that bleomycin treatment increased syndecan-4 expression. Moreover, we observed a marked decrease in neutrophil recruitment and an increase in both myofibroblast recruitment and interstitial fibrosis in bleomycin-treated syndecan-4–null (Sdc4–/–) mice. Subsequently, we identified a direct interaction between CXCL10, an antifibrotic chemokine, and syndecan-4 that inhibited primary lung fibroblast migration during fibrosis; mutation of the heparin-binding domain, but not the CXCR3 domain, of CXCL10 diminished this effect. Similarly, migration of fibroblasts from patients with pulmonary fibrosis was inhibited in the presence of CXCL10 protein defective in CXCR3 binding. Furthermore, administration of recombinant CXCL10 protein inhibited fibrosis in WT mice, but not in Sdc4–/– mice. Collectively, these data suggest that the direct interaction of syndecan-4 and CXCL10 in the lung interstitial compartment serves to inhibit fibroblast recruitment and subsequent fibrosis. Thus, administration of CXCL10 protein defective in CXCR3 binding may represent a novel therapy for pulmonary fibrosis.
Dianhua Jiang, Jiurong Liang, Gabriele S. Campanella, Rishu Guo, Shuang Yu, Ting Xie, Ningshan Liu, Yoosun Jung, Robert Homer, Eric B. Meltzer, Yuejuan Li, Andrew M. Tager, Paul F. Goetinck, Andrew D. Luster, Paul W. Noble
Respiratory distress syndrome (RDS), which is the leading cause of death in premature infants, is caused by surfactant deficiency. The most critical and abundant phospholipid in pulmonary surfactant is saturated phosphatidylcholine (SatPC), which is synthesized in alveolar type II cells de novo or by the deacylation-reacylation of existing phosphatidylcholine species. We recently cloned and partially characterized a mouse enzyme with characteristics of a lung lysophosphatidylcholine acyltransferase (LPCAT1) that we predicted would be involved in surfactant synthesis. Here, we describe our studies investigating whether LPCAT1 is required for pulmonary surfactant homeostasis. To address this issue, we generated mice bearing a hypomorphic allele of Lpcat1 (referred to herein as Lpcat1GT/GT mice) using a genetrap strategy. Newborn Lpcat1GT/GT mice showed varying perinatal mortality from respiratory failure, with affected animals demonstrating hallmarks of respiratory distress such as atelectasis and hyaline membranes. Lpcat1 mRNA levels were reduced in newborn Lpcat1GT/GT mice and directly correlated with SatPC content, LPCAT1 activity, and survival. Surfactant isolated from dead Lpcat1GT/GT mice failed to reduce minimum surface tension to wild-type levels. Collectively, these data demonstrate that full LPCAT1 activity is required to achieve the levels of SatPC essential for the transition to air breathing.
James P. Bridges, Machiko Ikegami, Lauren L. Brilli, Xueni Chen, Robert J. Mason, John M. Shannon
Jae Young Choi, Monal Khansaheb, Nam Soo Joo, Mauri E. Krouse, Robert C. Robbins, David Weill, Jeffrey J. Wine
The respiratory tract is continuously exposed to both innocuous airborne antigens and immunostimulatory molecules of microbial origin, such as LPS. At low concentrations, airborne LPS can induce a lung DC–driven Th2 cell response to harmless inhaled antigens, thereby promoting allergic asthma. However, only a small fraction of people exposed to environmental LPS develop allergic asthma. What prevents most people from mounting a lung DC–driven Th2 response upon exposure to LPS is not understood. Here we have shown that lung interstitial macrophages (IMs), a cell population with no previously described in vivo function, prevent induction of a Th2 response in mice challenged with LPS and an experimental harmless airborne antigen. IMs, but not alveolar macrophages, were found to produce high levels of IL-10 and to inhibit LPS-induced maturation and migration of DCs loaded with the experimental harmless airborne antigen in an IL-10–dependent manner. We further demonstrated that specific in vivo elimination of IMs led to overt asthmatic reactions to innocuous airborne antigens inhaled with low doses of LPS. This study has revealed a crucial role for IMs in maintaining immune homeostasis in the respiratory tract and provides an explanation for the paradox that although airborne LPS has the ability to promote the induction of Th2 responses by lung DCs, it does not provoke airway allergy under normal conditions.
Denis Bedoret, Hugues Wallemacq, Thomas Marichal, Christophe Desmet, Florence Quesada Calvo, Emmanuelle Henry, Rodrigue Closset, Benjamin Dewals, Caroline Thielen, Pascal Gustin, Laurence de Leval, Nico Van Rooijen, Alain Le Moine, Alain Vanderplasschen, Didier Cataldo, Pierre-Vincent Drion, Muriel Moser, Pierre Lekeux, Fabrice Bureau
Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8–/– mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8–/– macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.
Kamran Atabai, Sina Jame, Nabil Azhar, Alex Kuo, Michael Lam, William McKleroy, Greg DeHart, Salman Rahman, Dee Dee Xia, Andrew C. Melton, Paul Wolters, Claire L. Emson, Scott M. Turner, Zena Werb, Dean Sheppard
Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the US. Previously, we established an immune-mediated TRALI mouse model, wherein mice with cognate antigen were challenged with MHC class I mAb. In this study, when mice housed in a rodent, specific pathogen–free barrier room were challenged with MHC I mAb, there was significant protection from TRALI compared with nonbarrier mice. Priming mice with LPS restored lung injury with mAb challenge. Using TLR4-deficient bone marrow chimeras, the priming phenotype was restricted to animals with WT hematopoietic cells, and depletion of either neutrophils or platelets was protective. Both neutrophils and platelets were sequestered in the lungs of mice with TRALI, and retention of platelets was neutrophil dependent. Interestingly, treatment with aspirin prevented lung injury and mortality, but blocking the P selectin or CD11b/CD18 pathways did not. These data suggest a 2-step mechanism of TRALI: priming of hematopoietic cells, followed by vascular deposition of activated neutrophils and platelets that then mediate the severe lung injury. Furthermore, our data offer an explanation for the increased incidence of TRALI in patients with immune priming conditions, and we suggest what we believe to be a novel therapeutic approach.
Mark R. Looney, John X. Nguyen, Yongmei Hu, Jessica A. Van Ziffle, Clifford A. Lowell, Michael A. Matthay
Acute lung injury (ALI) is characterized by rapid alveolar injury, inflammation, cytokine induction, and neutrophil accumulation. Although early events in the pathogenesis of ALI have been defined, the mechanisms underlying resolution are unknown. As a model of ALI, we administered intratracheal (i.t.) LPS to mice and observed peak lung injury 4 days after the challenge, with resolution by day 10. Numbers of alveolar lymphocytes increased as injury resolved. To examine the role of lymphocytes in this response, lymphocyte-deficient Rag-1–/– and C57BL/6 WT mice were exposed to i.t. LPS. The extent of injury was similar between the groups of mice through day 4, but recovery was markedly impaired in the Rag-1–/– mice. Adoptive transfer studies revealed that infusion of CD4+CD25+Foxp3+ Tregs as late as 24 hours after i.t. LPS normalized resolution in Rag-1–/– mice. Similarly, Treg depletion in WT mice delayed recovery. Treg transfer into i.t. LPS–exposed Rag-1–/– mice also corrected the elevated levels of alveolar proinflammatory cytokines and increased the diminished levels of alveolar TGF-β and neutrophil apoptosis. Mechanistically, Treg-mediated resolution of lung injury was abrogated by TGF-β inhibition. Moreover, BAL of patients with ALI revealed dynamic changes in CD3+CD4+CD25hiCD127loFoxp3+ cells. These results indicate that Tregs modify innate immune responses during resolution of lung injury and suggest potential targets for treating ALI, for which there are no specific therapies currently available.
Franco R. D’Alessio, Kenji Tsushima, Neil R. Aggarwal, Erin E. West, Matthew H. Willett, Martin F. Britos, Matthew R. Pipeling, Roy G. Brower, Rubin M. Tuder, John F. McDyer, Landon S. King
Inflammation is associated with blood vessel and lymphatic vessel proliferation and remodeling. The microvasculature of the mouse trachea provides an ideal opportunity to study this process, as Mycoplasma pulmonis infection of mouse airways induces widespread and sustained vessel remodeling, including enlargement of capillaries into venules and lymphangiogenesis. Although the mediators responsible for these vascular changes in mice have not been identified, VEGF-A is known not to be involved. Here, we sought to determine whether TNF-α drives the changes in blood vessels and lymphatics in M. pulmonis–infected mice. The endothelial cells, but not pericytes, of blood vessels, but not lymphatics, were immunoreactive for TNF receptor 1 (TNF-R1) and lymphotoxin B receptors. Most TNF-R2 immunoreactivity was on leukocytes. Infection resulted in a large and sustained increase in TNF-α expression, as measured by real-time quantitative RT-PCR, and smaller increases in lymphotoxins and TNF receptors that preceded vessel remodeling. Substantially less vessel remodeling and lymphangiogenesis occurred when TNF-α signaling was inhibited by a blocking antibody or was silenced in Tnfr1–/– mice. When administered after infection was established, the TNF-α–specific antibody slowed but did not reverse blood vessel remodeling and lymphangiogenesis. The action of TNF-α on blood vessels is probably mediated through direct effects on endothelial cells, but its effects on lymphangiogenesis may require inflammatory mediators from recruited leukocytes. We conclude that TNF-α is a strong candidate for a mediator that drives blood vessel remodeling and lymphangiogenesis in inflammation.
Peter Baluk, Li-Chin Yao, Jennifer Feng, Talia Romano, Sonia S. Jung, Jessica L. Schreiter, Li Yan, David J. Shealy, Donald M. McDonald
Uncontrolled activation of the coagulation cascade contributes to the pathophysiology of several conditions, including acute and chronic lung diseases. Coagulation zymogens are considered to be largely derived from the circulation and locally activated in response to tissue injury and microvascular leak. Here we report that expression of coagulation factor X (FX) is locally increased in human and murine fibrotic lung tissue, with marked immunostaining associated with bronchial and alveolar epithelia. FXa was a potent inducer of the myofibroblast differentiation program in cultured primary human adult lung fibroblasts via TGF-β activation that was mediated by proteinase-activated receptor–1 (PAR1) and integrin αvβ5. PAR1, αvβ5, and α-SMA colocalized to fibrotic foci in lung biopsy specimens from individuals with idiopathic pulmonary fibrosis. Moreover, we demonstrated a causal link between FXa and fibrosis development by showing that a direct FXa inhibitor attenuated bleomycin-induced pulmonary fibrosis in mice. These data support what we believe to be a novel pathogenetic mechanism by which FXa, a central proteinase of the coagulation cascade, is locally expressed and drives the fibrotic response to lung injury. These findings herald a shift in our understanding of the origins of excessive procoagulant activity and place PAR1 central to the cross-talk between local procoagulant signaling and tissue remodeling.
Chris J. Scotton, Malvina A. Krupiczojc, Melanie Königshoff, Paul F. Mercer, Y.C. Gary Lee, Naftali Kaminski, John Morser, Joseph M. Post, Toby M. Maher, Andrew G. Nicholson, James D. Moffatt, Geoffrey J. Laurent, Claudia K. Derian, Oliver Eickelberg, Rachel C. Chambers
Pulmonary hypertension (PH) is an unremitting disease defined by a progressive increase in pulmonary vascular resistance leading to right-sided heart failure. Using mice with genetic deletions of caveolin 1 (Cav1) and eNOS (Nos3), we demonstrate here that chronic eNOS activation secondary to loss of caveolin-1 can lead to PH. Consistent with a role for eNOS in the pathogenesis of PH, the pulmonary vascular remodeling and PH phenotype of Cav1–/– mice were absent in Cav1–/–Nos3–/– mice. Further, treatment of Cav1–/– mice with either MnTMPyP (a superoxide scavenger) or l-NAME (a NOS inhibitor) reversed their pulmonary vascular pathology and PH phenotype. Activation of eNOS in Cav1–/– lungs led to the impairment of PKG activity through tyrosine nitration. Moreover, the PH phenotype in Cav1–/– lungs could be rescued by overexpression of PKG-1. The clinical relevance of the data was indicated by the observation that lung tissue from patients with idiopathic pulmonary arterial hypertension demonstrated increased eNOS activation and PKG nitration and reduced caveolin-1 expression. Together, these data show that loss of caveolin-1 leads to hyperactive eNOS and subsequent tyrosine nitration–dependent impairment of PKG activity, which results in PH. Thus, targeting of PKG nitration represents a potential novel therapeutic strategy for the treatment of PH.
You-Yang Zhao, Yidan D. Zhao, Muhammad K. Mirza, Julia H. Huang, Hari-Hara S.K. Potula, Steven M. Vogel, Viktor Brovkovych, Jason X.-J.Yuan, John Wharton, Asrar B. Malik
Idiopathic pulmonary fibrosis (IPF) can lead to the development of secondary pulmonary hypertension (PH) and ultimately death. Despite this known association, the precise mechanism of disease remains unknown. Using a rat model of IPF, we explored the role of the proangiogenic and antiapoptotic growth factor VEGF in the vascular remodeling that underlies PH. In this model, adenoviral delivery of active TGF-β1 induces pulmonary arterial remodeling, loss of the microvasculature in fibrotic areas, and increased pulmonary arterial pressure (PAP). Immunohistochemistry and mRNA analysis revealed decreased levels of VEGF and its receptor, which were inversely correlated with PAP and endothelial cell apoptosis in both the micro- and macrovasculature. Treatment of IPF rats with adenoviral delivery of VEGF resulted in reduced endothelial apoptosis, increased vascularization, and improved PAP due to reduced remodeling but worsened PF. These data show that experimental pulmonary fibrosis (PF) leads to loss of the microvasculature through increased apoptosis and to remodeling of the pulmonary arteries, with both processes resulting in PH. As administration of VEGF ameliorated the PH in this model but concomitantly aggravated the fibrogenic process, VEGF-based therapies should be used with caution.
Laszlo Farkas, Daniela Farkas, Kjetil Ask, Antje Möller, Jack Gauldie, Peter Margetts, Mark Inman, Martin Kolb
Chronic bacterial airway infections are the major cause of mortality in cystic fibrosis (CF). Normal airway defenses include reflex stimulation of submucosal gland mucus secretion by sensory neurons that release substance P (SubP). CFTR is an anion channel involved in fluid secretion and mutated in CF; the role of CFTR in secretions stimulated by SubP is unknown. We used optical methods to measure SubP-mediated secretion from human submucosal glands in lung transplant tissue. Glands from control but not CF subjects responded to mucosal chili oil. Similarly, serosal SubP stimulated secretion in more than 60% of control glands but only 4% of CF glands. Secretion triggered by SubP was synergistic with vasoactive intestinal peptide and/or forskolin but not with carbachol; synergy was absent in CF glands. Pig glands demonstrated a nearly 10-fold greater response to SubP. In 10 of 11 control glands isolated by fine dissection, SubP caused cell volume loss, lumen expansion, and mucus flow, but in 3 of 4 CF glands, it induced lumen narrowing. Thus, in CF, the reduced ability of mucosal irritants to stimulate airway gland secretion via SubP may be another factor that predisposes the airways to infections.
Jae Young Choi, Monal Khansaheb, Nam Soo Joo, Mauri E. Krouse, Robert C. Robbins, David Weill, Jeffrey J. Wine
Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/β-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein–1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.
Melanie Königshoff, Monika Kramer, Nisha Balsara, Jochen Wilhelm, Oana Veronica Amarie, Andreas Jahn, Frank Rose, Ludger Fink, Werner Seeger, Liliana Schaefer, Andreas Günther, Oliver Eickelberg
Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.
Michael T. Borchers, Scott C. Wesselkamper, Victor Curull, Alba Ramirez-Sarmiento, Albert Sánchez-Font, Judith Garcia-Aymerich, Carlos Coronell, Josep Lloreta, Alvar G. Agusti, Joaquim Gea, John A. Howington, Michael F. Reed, Sandra L. Starnes, Nathaniel L. Harris, Mark Vitucci, Bryan L. Eppert, Gregory T. Motz, Kevin Fogel, Dennis W. McGraw, Jay W. Tichelaar, Mauricio Orozco-Levi
Pulmonary hypertension (PH) is a progressive, lethal lung disease characterized by pulmonary artery SMC (PA-SMC) hyperplasia leading to right-sided heart failure. Molecular events originating in pulmonary ECs (P-ECs) may contribute to the PA-SMC hyperplasia in PH. Thus, we exposed cultured human PA-SMC to medium conditioned by P-EC from patients with idiopathic PH (IPH) or controls and found that IPH P-EC–conditioned medium increased PA-SMC proliferation more than control P-EC medium. Levels of FGF2 were increased in the medium of IPH P-ECs over controls, while there was no detectable difference in TGF-β1, PDGF-BB, or EGF levels. No difference in FGF2-induced proliferation or FGF receptor type 1 (FGFR1) mRNA levels was detected between IPH and control PA-SMCs. Knockdown of FGF2 in P-EC using siRNA reduced the PA-SMC growth-stimulating effects of IPH P-EC medium by 60% and control P-EC medium by 10%. In situ hybridization showed FGF2 overproduction predominantly in the remodeled vascular endothelium of lungs from patients with IPH. Repeated intravenous FGF2-siRNA administration abolished lung FGF2 production, both preventing and nearly reversing a rat model of PH. Similarly, pharmacological FGFR1 inhibition with SU5402 reversed established PH in the same model. Thus, endothelial FGF2 is overproduced in IPH and contributes to SMC hyperplasia in IPH, identifying FGF2 as a promising target for new treatments against PH.
Mohamed Izikki, Christophe Guignabert, Elie Fadel, Marc Humbert, Ly Tu, Patricia Zadigue, Philippe Dartevelle, Gerald Simonneau, Serge Adnot, Bernard Maitre, Bernadette Raffestin, Saadia Eddahibi
The bone marrow compartment is enriched in stem and progenitor cells, and an unidentified subpopulation of these cells can contribute to lung epithelial repair. Here we identify this subpopulation and quantitate its relative contribution to injured airway epithelium. A subpopulation of adherent human and murine bone marrow cells that expresses Clara cell secretory protein (CCSP) was identified using flow cytometry. When cultured at the air-liquid interface in ex vivo cultures, Ccsp+ cells expressed type I and type II alveolar markers as well as basal cell markers and active epithelial sodium channels. Ccsp+ cells preferentially homed to naphthalene-damaged airways when delivered transtracheally or intravenously, with the former being more efficient than the latter. Interestingly, naphthalene-induced lung damage transiently increased Ccsp expression in bone marrow and peripheral circulation. Furthermore, lethally irradiated Ccsp-null mice that received tagged wild-type bone marrow contained donor-derived epithelium in both normal and naphthalene-damaged airways. This study therefore identifies what we believe to be a newly discovered cell in the bone marrow that might have airway reconstitution potential in the context of cell-based therapies for lung disease. Additionally, these data could reconcile previous controversies regarding the contribution of bone marrow to lung regeneration.
Amy P. Wong, Armand Keating, Wei-Yang Lu, Pascal Duchesneau, Xinghua Wang, Adrian Sacher, Jim Hu, Thomas K. Waddell
Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-β1. As the ECM critically regulates AEC responses to TGF-β1, we explored the role of the prominent epithelial integrin α3β1 in experimental fibrosis by generating mice with lung epithelial cell–specific loss of α3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through β-catenin has been implicated in EMT; we found that in primary AECs, α3 integrin was required for β-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654–β-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654–β-catenin/pSmad2 complexes and showed accumulation of pY654–β-catenin in myofibroblasts. These findings demonstrate epithelial integrin–dependent profibrotic crosstalk between β-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis.
Kevin K. Kim, Ying Wei, Charles Szekeres, Matthias C. Kugler, Paul J. Wolters, Marla L. Hill, James A. Frank, Alexis N. Brumwell, Sarah E. Wheeler, Jordan A. Kreidberg, Harold A. Chapman
Normal airways homeostatically regulate the volume of airway surface liquid (ASL) through both cAMP- and Ca2+-dependent regulation of ion and water transport. In cystic fibrosis (CF), a genetic defect causes a lack of cAMP-regulated CFTR activity, leading to diminished Cl– and water secretion from airway epithelial cells and subsequent mucus plugging, which serves as the focus for infections. Females with CF exhibit reduced survival compared with males with CF, although the mechanisms underlying this sex-related disadvantage are unknown. Despite the lack of CFTR, CF airways retain a limited capability to regulate ASL volume, as breathing-induced ATP release activates salvage purinergic pathways that raise intracellular Ca2+ concentration to stimulate an alternate pathway to Cl– secretion. We hypothesized that estrogen might affect this pathway by reducing the ability of airway epithelia to respond appropriately to nucleotides. We found that uridine triphosphate–mediated (UTP-mediated) Cl– secretion was reduced during the periovulatory estrogen maxima in both women with CF and normal, healthy women. Estrogen also inhibited Ca2+ signaling and ASL volume homeostasis in non-CF and CF airway epithelia by attenuating Ca2+ influx. This inhibition of Ca2+ signaling was prevented and even potentiated by estrogen antagonists such as tamoxifen, suggesting that antiestrogens may be beneficial in the treatment of CF lung disease because they increase Cl– secretion in the airways.
Ray D. Coakley, Hengrui Sun, Lucy A. Clunes, Julia E. Rasmussen, James R. Stackhouse, Seiko F. Okada, Ingrid Fricks, Steven L. Young, Robert Tarran
Although acute lung injury contributes significantly to critical illness, resolution often occurs spontaneously via activation of incompletely understood pathways. We recently found that mechanical ventilation of mice increases the level of pulmonary adenosine, and that mice deficient for extracellular adenosine generation show increased pulmonary edema and inflammation after ventilator-induced lung injury (VILI). Here, we profiled the response to VILI in mice with genetic deletions of each of the 4 adenosine receptors (ARs) and found that deletion of the A2BAR gene was specifically associated with reduced survival time and increased pulmonary albumin leakage after injury. In WT mice, treatment with an A2BAR-selective antagonist resulted in enhanced pulmonary inflammation, edema, and attenuated gas exchange, while an A2BAR agonist attenuated VILI. In bone marrow–chimeric A2BAR mice, although the pulmonary inflammatory response involved A2BAR signaling from bone marrow–derived cells, A2BARs located on the lung tissue attenuated VILI-induced albumin leakage and pulmonary edema. Furthermore, measurement of alveolar fluid clearance (AFC) demonstrated that A2BAR signaling enhanced amiloride-sensitive fluid transport and elevation of pulmonary cAMP levels following VILI, suggesting that A2BAR agonist treatment protects by drying out the lungs. Similar enhancement of pulmonary cAMP and AFC were also observed after β-adrenergic stimulation, a pathway known to promote AFC. Taken together, these studies reveal a role for A2BAR signaling in attenuating VILI and implicate this receptor as a potential therapeutic target during acute lung injury.
Tobias Eckle, Almut Grenz, Stefanie Laucher, Holger K. Eltzschig