Pleural Mesothelial Cell Differentiation and Invasion in Fibrogenic Lung Injury

Abstract

The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-β1 (TGF-β1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-β1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1–expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression. The origin of the myofibroblast in fibrotic lung disease is uncertain, and no effective medical therapy for fibrosis exists. We have previously demonstrated that transforming growth factor-β1 (TGF-β1) induces pleural mesothelial cell (PMC) transformation into myofibroblasts and haptotactic migration in vitro. Whether PMC differentiation and migration occurs in vivo, and whether this response can be modulated for therapeutic benefit, is unknown. Here, using mice recombinant for green fluorescent protein (GFP) driven by the Wilms tumor-1 (WT-1) promoter, we demonstrate PMC trafficking into the lung and differentiation into myofibroblasts. Carbon monoxide or the induction of heme oxygenase-1 (HO-1) inhibited the expression of myofibroblast markers, contractility, and haptotaxis in PMCs treated with TGF-β1. Intrapleural HO-1 induction inhibited PMC migration after intratracheal fibrogenic injury. PMCs from patients with idiopathic pulmonary fibrosis (IPF) exhibited increased expression of myofibroblast markers and enhanced contractility and haptotaxis, compared with normal PMCs. Carbon monoxide reversed this IPF PMC profibrotic phenotype. WT-1–expressing cells were present within fibrotic regions of the lungs in IPF subjects, supporting a role for PMC differentiation and trafficking as contributors to the myofibroblast population in lung fibrosis. Our findings also support a potential role for pleural-based therapies to modulate pleural mesothelial activation and parenchymal fibrosis progression. Idiopathic pulmonary fibrosis (IPF), the most common idiopathic interstitial pneumonia, is characterized by cellular and structural changes in the parenchyma associated with the proliferation of myofibroblasts and deposition of extracellular matrix components.1Raghu G. Weycker D. Edelsberg J. Bradford W.Z. Oster G. Incidence and prevalence of idiopathic pulmonary fibrosis.Am J Respir Crit Care Med. 2006; 174: 810-816Crossref PubMed Scopus (1050) Google Scholar IPF begins in the subpleural region and extends centrally, resulting in a progressive decline in lung function. The origin of the pathogenic myofibroblast is uncertain. The hallmark lesions of IPF, the fibroblastic foci seen on two-dimensional histopathological slides, were thought of as discrete sites of epithelial injury and repair.2Gross T.J. Hunninghake G.W. Idiopathic pulmonary fibrosis.N Engl J Med. 2001; 345: 517-525Crossref PubMed Scopus (906) Google Scholar Cool et al3Cool C.D. Groshong S.D. Rai P.R. Henson P.M. Stewart J.S. Brown K.K. Fibroblast foci are not discrete sites of lung injury or repair: the fibroblast reticulum.Am J Respir Crit Care Med. 2006; 174: 654-658Crossref PubMed Scopus (168) Google Scholar used three-dimensional reconstruction to demonstrate that these foci are part of a complex, highly interconnected reticulum and suggested the leading edge of the fibroblastic invasion extends from the pleura to the underlying parenchyma like a wave of fibrosis. The pleura is a metabolically active monolayer of mesothelial cells that intimately approximates the lung parenchyma. The close proximity of PMCs to the underlying lung ideally positions them to respond to cytokines, chemokines, and growth factors released during parenchymal stress, injury, infection, or inflammation. The cytokine transforming growth factor-β1 (TGF-β1) is a crucial mediator of epithelial–mesenchymal transition (EMT) and acts as a master switch for induction of fibrosis in many organs, including the lung.4Hills C.E. Squires P.E. TGF-beta1-induced epithelial-to-mesenchymal transition and therapeutic intervention in diabetic nephropathy.Am J Nephrol. 2010; 31: 68-74Crossref PubMed Scopus (170) Google Scholar, 5Meindl-Beinker N.M. Dooley S. Transforming growth factor-beta and hepatocyte transdifferentiation in liver fibrogenesis.J Gastroenterol Hepatol. 2008; 23: S122-S127Crossref PubMed Scopus (102) Google Scholar, 6Yáñez-Mó M. Lara-Pezzi E. Selgas R. Ramírez-Huesca M. Domínguez-Jiménez C. Jiménez-Heffernan J.A. Aguilera A. Sánchez-Tomero J.A. Bajo M.A. Alvarez V. Castro M.A. del Peso G. Cirujeda A. Gamallo C. Sánchez-Madrid F. López-Cabrera M. Peritoneal dialysis and epithelial-to-mesenchymal transition of mesothelial cells [Erratum appeared in N Engl J Med 2005, 353:2827].N Engl J Med. 2003; 348: 403-413Crossref PubMed Scopus (653) Google Scholar PMC transformation into myofibroblasts and haptotactic migration occur in vitro in response to TGF-β1.7Nasreen N. Mohammed K.A. Mubarak K.K. Baz M.A. Akindipe O.A. Fernandez-Bussy S. Antony V.B. Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-beta1 in vitro.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L115-L124Crossref PubMed Scopus (102) Google Scholar The concept of pleural mesothelial–mesenchymal transition (MMT) was investigated in a recent study demonstrating PMC migration into the lung and expression of myofibroblast phenotypic markers. This study demonstrated the presence of PMCs in the lung parenchyma of patients with IPF and a correlation with disease severity and the degree of fibrosis.8Mubarak K.K. Montes-Worboys A. Regev D. Nasreen N. Mohammed K.A. Faruqi I. Hensel E. Baz M.A. Akindipe O.A. Fernandez-Bussy S. Nathan S. Antony V. Parenchymal trafficking of pleural mesothelial cells in idiopathic pulmonary fibrosis.Eur Respir J. 2012; 39: 133-140Crossref PubMed Scopus (65) Google Scholar PMC differentiation into myofibroblasts and subsequent migration may play a crucial role in the development of fibrotic lung disease. Heme oxygenase-1 (HO-1) is the inducible form of the rate-limiting enzyme involved in the degradation of heme with the generation of equimolar quantities of carbon monoxide (CO), iron, and biliverdin.9Tenhunen R. Marver H.S. Schmid R. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc Natl Acad Sci USA. 1968; 61: 748-755Crossref PubMed Scopus (1545) Google Scholar HO-1 induction with subsequent CO production is highly sensitive to numerous stimuli and cellular insults that cause oxidative stress.10Keyse S.M. Tyrrell R.M. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite.Proc Natl Acad Sci USA. 1989; 86: 99-103Crossref PubMed Scopus (1146) Google Scholar Such induction represents a beneficial response to injurious stimuli in diverse diseases, including atherosclerosis, sepsis, and fibrosis. The protective effects of HO-1 and CO are due to anti-inflammatory, antiapoptotic, antioxidant, and antiproliferative properties.11Jarmi T. Agarwal A. Heme oxygenase and renal disease.Curr Hypertens Rep. 2009; 11: 56-62Crossref PubMed Scopus (82) Google Scholar Indeed, HO-1 deficiency has been associated with increased fibrosis, tubular TGF-β1 expression, inflammation, and enhanced EMT in a model of obstructive kidney disease.12Kie J.H. Kapturczak M.H. Traylor A. Agarwal A. Hill-Kapturczak N. Heme oxygenase-1 deficiency promotes epithelial-mesenchymal transition and renal fibrosis.J Am Soc Nephrol. 2008; 19: 1681-1691Crossref PubMed Scopus (84) Google Scholar Up-regulation of HO-1 provides protection against renal injury after unilateral ureteral obstruction and suppression of tubulointerstitial fibrosis via anti-apoptotic pathway modulation.13Kim J.H. Yang J.I. Jung M.H. Hwa J.S. Kang K.R. Park D.J. Roh G.S. Cho G.J. Choi W.S. Chang S.H. Heme oxygenase-1 protects rat kidney from ureteral obstruction via an antiapoptotic pathway.J Am Soc Nephrol. 2006; 17: 1373-1381Crossref PubMed Scopus (72) Google Scholar Additionally, adenoviral transfer of the HO-1 gene, as well as administration of CO and bilirubin, has been associated with suppression of fibrosis in animal models of fibrotic lung disease.14Tsuburai T. Suzuki M. Nagashima Y. Suzuki S. Inoue S. Hasiba T. Ueda A. Ikehara K. Matsuse T. Ishigatsubo Y. Adenovirus-mediated transfer and overexpression of heme oxygenase 1 cDNA in lung prevents bleomycin-induced pulmonary fibrosis via a Fas-Fas ligand-independent pathway.Hum Gene Ther. 2002; 13: 1945-1960Crossref PubMed Scopus (81) Google Scholar, 15Wang H.D. Yamaya M. Okinaga S. Jia Y.X. Kamanaka M. Takahashi H. Guo L.Y. Ohrui T. Sasaki H. Bilirubin ameliorates bleomycin-induced pulmonary fibrosis in rats.Am J Respir Crit Care Med. 2002; 165: 406-411Crossref PubMed Scopus (123) Google Scholar Here, we present definitive evidence for the presence of PMCs in the lung parenchyma of patients with IPF and show that the fibrotic disposition of these cells can be reversed by CO and HO-1 pathway modulation in vitro. Furthermore, using animal models of lung injury, we demonstrate PMC differentiation into myofibroblasts and parenchymal invasion that is inhibited by intrapleural HO-1 induction. Mouse wild-type PMCs of passages between 15 and 35 were used. Cells were grown in F12K medium (catalog no. 11765-054; Life Technologies–Invitrogen, Carlsbad, CA) supplemented with heat-inactivated fetal bovine serum (10%) or in serum-free conditions before (16 hours) and during TGF-β1 treatments. Human PMCs were derived from lung explants from patients diagnosed with IPF undergoing lung transplantation. Cells were cultured in Medium 199 (catalog no. 11150-059; Life Technologies–Invitrogen) supplemented with epidermal growth factor (EGF) and 10% fetal bovine serum or in serum-free conditions without EGF before and during TGF-β1 exposure. Cells were serum-starved for 16 hours and then were treated with 5 ng/mL TGF-β1 (catalog no. 101-B1; R&D Systems, Minneapolis, MN) activated in 4 mmol/L HCl, 0.5% bovine serum albumin. Cells were treated with 5 μmol/L hemin in dimethyl sulfoxide (catalog no. 51280-1G; Sigma-Aldrich, St. Louis, MO) in either serum-free conditions or in 10% fetal bovine serum medium, depending on the experimental design. Cells were treated with 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) at doses of 25 nmol/L or 50 nmol/L, depending on the experiment. The CDDO-Im was provided by Michael Sporn and Karen Liby of Dartmouth Medical School. Tricarbonyldichlororuthenium (II) (CORM-2) was purchased from Sigma-Aldrich (catalog no. 288144-500 MG). Cells were exposed to CORM-2 dissolved in dimethyl sulfoxide (10 μmol/L), which served as a source of CO. Transformed normal pleural mesothelial cells (Met5A) or PMCs from patients with IPF were seeded at a density of 1 × 106 cells/mL. Cells and serum medium were mixed with rat tail collagen reconstituted in 0.1 mol/L acetic acid (catalog no. C7661-50 MG; Sigma-Aldrich) in a 3:1 proportion and seeded in individual wells in a 24-well culture dish. Cells were allowed to acclimatize for 16 hours and then were pretreated with hemin, CDDO-Im, or CORM-2 for 30 minutes, followed by addition of 5 ng/mL TGF-β1; cells were allowed to contract for 48 hours. All protein isolates were prepared with radioimmunoprecipitation assay buffer. Samples were sonicated at 4°C, followed by centrifugation to remove insoluble material, and then were subjected to protein assay using a Micro BCA kit (Pierce; All protein were to followed by with 0.1 mol/L and on 4 to (catalog no. Life Technologies–Invitrogen) using a acid were then to (catalog no. followed by in reconstituted in The were used for no. no. HO-1 no. Life no. no. and (catalog no. All were prepared in reconstituted in and at 4°C, followed by and then with at for hours. The were then in followed by with no. the effects of HO-1 during injury, we used a pleural model in wild-type and mice on a we used a model that in cells were in migration Life as with N. Mohammed K.A. Mubarak K.K. Baz M.A. Akindipe O.A. Fernandez-Bussy S. Antony V.B. Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-beta1 in vitro.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L115-L124Crossref PubMed Scopus (102) Google Scholar the of the were with 1 bovine serum or or were and at in in the presence of The were with and were into The of the was with Medium 199 Technologies–Invitrogen) with fetal bovine PMCs × treated with TGF-β1 with TGF-β1 and either CDDO-Im, or hemin, or with no were seeded into the and for hours at the of medium from the well was The of the were to remove The were and cells were in and with (catalog no. The of cells that was by the of cells on the of the an The are as the haptotactic the of cells the with TGF-β1 acts as an for the PMCs. lungs from 16 patients with IPF, lungs from patients with lungs from patients with fibrosis, and lung from with pulmonary were after lung at the of at with PMCs from lungs were by of the pleura and used in passages to PMCs and Lung were in in and with a before on an was used of The used were as for green fluorescent protein for was used as a was used with and an was used at with and were using a and All human and animal were by the at the of at was from the before in the is a protein that many of the is in including normal pleural is in normal an of PMCs. WT-1–expressing PMCs have been to into the lung parenchyma to form cells of the and cells of the lung during lung J. H. F. D. to and during lung Natl Acad Sci USA. 2008; PubMed Scopus Google Scholar The major role of in differentiation and development has for lung including pulmonary fibrosis. the present using we have demonstrated the presence of cells within the parenchyma of an lung from a with IPF, of the 16 lungs that for cells were from the lungs of patients with interstitial lung fibrosis, and obstructive pulmonary disease not of PMCs with TGF-β1 increased expression of the myofibroblast and has been to myofibroblast activation and fibrogenic to lung injury. generation of hydrogen is for myofibroblast extracellular matrix and R. T. R. S. myofibroblast activation and fibrogenic to lung Med. 2009; PubMed Scopus Google Scholar CDDO-Im, a is a with and that at induces the expression of K. T. N. R. T. Y. G.W. Hill-Kapturczak N. Agarwal A. Sporn The and are of heme oxygenase-1 and PubMed Scopus Google Scholar the effects of HO-1 induction on PMC differentiation and fibrosis, we treated PMCs with TGF-β1 with and without administration of CDDO-Im administration at the of PMC with TGF-β1 an in HO-1 expression and a in and with a HO-1 increased HO-1 and expression of and in PMCs treated with TGF-β1. with a inhibited and expression these demonstrate a role for HO-1 in the of PMC into We to the effects of HO-1 induction and CO on the response to TGF-β1 of PMCs from and patients with PMCs were treated with TGF-β1 or were treated with either hemin, CDDO-Im, or CORM-2 before TGF-β1. normal human was in and in compared with TGF-β1 The PMCs from patients with IPF exhibited contractility, without TGF-β1 with no in for PMC between PMCs and PMCs from patients with with hemin and not with CDDO-Im and a in the addition of CORM-2 to IPF PMCs a in findings to a normal PMC The demonstrate a role for HO-1 induction or CO in the of normal PMCs treated with TGF-β1 and for contractility in PMCs from patients with were in migration cell in response to on a is from which is as cell migration a of represents cell in the extracellular PMCs × were treated with TGF-β1 or with CDDO-Im, or hemin followed by TGF-β1; cells were used as The of cells was and were as the haptotactic with TGF-β1 haptotaxis, which was inhibited by CDDO-Im, and hemin with IPF as demonstrated increased haptotaxis, with increased haptotaxis after with TGF-β1 that was inhibited by hemin or CORM-2 not by CDDO-Im the role of HO-1 in in pleural and parenchymal fibrosis, we intrapleural to and for collagen and demonstrated a pleural fibrotic response in compared with wild-type mice PMC we used mice recombinant for driven by the hours after intratracheal TGF-β1 we cells the lung parenchyma The cells for supporting the concept that PMCs have not into the lung in response to have also differentiation to a myofibroblast the effects of pleural HO-1 induction on parenchymal PMC migration and fibrosis, we treated wild-type mice with either intratracheal or intratracheal with intrapleural mice treated with intratracheal exhibited cells the pleura and in the parenchyma at after treated with intrapleural CDDO-Im at after exhibited the pleura at after Intrapleural HO-1 induction parenchymal PMC migration in response to an fibrotic We expression of and HO-1 in PMCs from patients with IPF, before and after with with or without hemin, or CDDO-Im PMCs from IPF patients exhibited a to and at and also after TGF-β1. with hemin, or CDDO-Im expression of myofibroblast markers the induction of HO-1 in IPF PMCs appeared in in normal human PMCs not The subpleural of fibrosis in IPF is of of interstitial from patients with IPF using three-dimensional reconstruction the fibroblastic foci of interstitial at the leading edge of a reticulum that extends from the pleura to the underlying C.D. Groshong S.D. Rai P.R. Henson P.M. Stewart J.S. Brown K.K. Fibroblast foci are not discrete sites of lung injury or repair: the fibroblast reticulum.Am J Respir Crit Care Med. 2006; 174: 654-658Crossref PubMed Scopus (168) Google Scholar carbon to the of mice with intratracheal in pleural N. G. M. C. induces pleural and subpleural fibrosis in the presence of carbon Respir J. 2010; PubMed Scopus Google Scholar This was associated with progressive subpleural fibrosis, to The that the PMCs myofibroblast and that the matrix within the subpleural The origin of the myofibroblast in IPF is uncertain. pathogenic cells have been to be derived from and epithelial cells that have A. M. Idiopathic pulmonary PubMed Scopus Google Scholar experimental models of pulmonary fibrosis, EMT and to the fibroblast is a to lung H. D. of fibroblasts to bleomycin-induced lung fibrosis.Am J Respir Crit Care Med. 2009; PubMed Scopus Google Scholar The role of EMT and in development and disease is in HO-1 deficiency is associated with increased fibrosis, tubular TGF-β1 expression, inflammation, and enhanced EMT in obstructive kidney disease.12Kie J.H. Kapturczak M.H. Traylor A. Agarwal A. Hill-Kapturczak N. Heme oxygenase-1 deficiency promotes epithelial-mesenchymal transition and renal fibrosis.J Am Soc Nephrol. 2008; 19: 1681-1691Crossref PubMed Scopus (84) Google Scholar PMC transformation into myofibroblasts and haptotactic migration in response to TGF-β1 in N. Mohammed K.A. Mubarak K.K. Baz M.A. Akindipe O.A. Fernandez-Bussy S. Antony V.B. Pleural mesothelial cell transformation into myofibroblasts and haptotactic migration in response to TGF-beta1 in vitro.Am J Physiol Lung Cell Mol Physiol. 2009; 297: L115-L124Crossref PubMed Scopus (102) Google Scholar that PMCs into the lung and myofibroblast phenotypic presence with the severity of fibrosis in K.K. Montes-Worboys A. Regev D. Nasreen N. Mohammed K.A. Faruqi I. Hensel E. Baz M.A. Akindipe O.A. Fernandez-Bussy S. Nathan S. Antony V. Parenchymal trafficking of pleural mesothelial cells in idiopathic pulmonary fibrosis.Eur Respir J. 2012; 39: 133-140Crossref PubMed Scopus (65) Google Scholar play an role in and The mesothelial of the is a major source of cells to the J. origin of and fibroblasts in the PubMed Scopus Google Scholar, J. the to the of the study the of the PubMed Scopus Google Scholar, T. A. Brown of in the using a I. of the PubMed Scopus Google Scholar, T. of the 2002; PubMed Scopus Google Scholar et A. The is a major source of cells of the PubMed Scopus Google Scholar used to demonstrate that mesothelial cells into of major in the and The of PMCs to transformation and migration a crucial role in the recent study of lung development demonstrated that the mesothelial cells the lung to a of cell in the including cells and also myofibroblasts and interstitial J. H. F. D. to and during lung Natl Acad Sci USA. 2008; PubMed Scopus Google Scholar The suggested a common the development of with the of the and that PMCs are and to to cells during injury, or subsequent study provided definitive evidence of the of PMCs by demonstrating differentiation into and cells exposed to an growth the that mesothelial cell differentiation is a potential source of the in and potential in S.M. A. C. H. M. cell differentiation into and Cell Mol Med. PubMed Scopus Google Scholar that WT-1–expressing cells have a to switch between and epithelial H. expression in development and from the gene, Mol PubMed Google Scholar This may that expression is not to the transition between mesothelial and The pleura is a metabolically active monolayer of mesothelial cells that intimately approximates the lung parenchyma. This proximity of PMCs to the underlying lung ideally positions them to respond to released during parenchymal stress, whether or a recent study in demonstrated the of a from the pleura hours after or the with a pleural C. E. S. R. N. with of the pleura as a major of the effects induced by and in the of the J Physiol Lung Cell Mol Physiol. 2006; PubMed Scopus Google Scholar The that occurs between the pleura and parenchyma in the lung may in the and to disease. the present we have demonstrated in vitro PMC differentiation and of a myofibroblast as by expression of markers and including haptotaxis and in response to TGF-β1 Furthermore, induction of HO-1 or with CO PMC differentiation and parenchymal mice also pleural fibrosis in response to intrapleural compared with wild-type cells were present in lung explants from patients with IPF, not in explants from patients with fibrosis or obstructive pulmonary disease not the present study is the to demonstrate phenotypic and in PMCs from patients with IPF, compared with normal PMCs. IPF PMCs a to the as well as a of myofibroblast activation and fibrotic to lung R. T. R. S. myofibroblast activation and fibrogenic to lung Med. 2009; PubMed Scopus Google Scholar IPF PMCs myofibroblast markers, also exhibited increased contractility and for of HO-1 expression in PMCs from patients with IPF was compared with normal an or or in HO-1 expression or in IPF may these patients to fibroblast proliferation and progressive disease. is a potential to HO-1 modulation and CO as therapies for fibrotic lung disease. in have using that HO-1 induction G. T. G. R. S. A. R. S.D. and antiapoptotic effects of heme oxygenase-1 in the 2010; PubMed Scopus Google Scholar and prevents the of liver R. D. J. A. D. J. C. G. G. of heme oxygenase 1 prevents of liver fibrosis in 2012; PubMed Scopus Google Scholar CO also bleomycin-induced lung fibrosis in R. S. S. Choi D. Carbon monoxide bleomycin-induced lung fibrosis.Am J PubMed Scopus Google Scholar by and Choi Heme from to J Respir Cell Mol 2009; PubMed Scopus Google Scholar has the of for HO-1 a study is of Carbon to Idiopathic the present of PMCs with CO in the form of CORM-2 or hemin not or reversed the expression of and also the of IPF PMCs and PMC is to hemin and CORM-2 reversed the increased and haptotaxis seen in CDDO-Im was to the IPF PMCs. This that HO-1 induction may be to against fibrosis in an or HO-1 deficiency also to IPF disease. The of the heme oxygenase may for PMC fibrotic phenotypic The present findings the that intrapleural modulation of HO-1 or of CO to the pleural to in the of IPF by PMC differentiation and parenchymal The Michael Sporn and Karen Liby for the