Harikrishna Tanjore

Activated fibroblasts are key contributors to the fibrotic extracellular matrix accumulation during liver fibrosis. The origin of such fibroblasts is still debated, although several studies point to stellate cells as the principal source. The role of adult hepatocytes as contributors to the accumulation of fibroblasts in the fibrotic liver is yet undetermined. Here, we provide evidence that the pro-fibrotic growth factor, TGF-β1, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of epithelial to mesenchymal transition (EMT). We perform lineage-tracing experiments using AlbCre. R26RstoplacZ double transgenic mice to demonstrate that hepatocytes which undergo EMT contribute substantially to the population of FSP1-positive fibroblasts in CCL4-induced liver fibrosis. Furthermore, we demonstrate that bone morphogenic protein-7 (BMP7), of the which is to of liver in that the of to the of provide evidence the functional of adult hepatocytes in the accumulation of fibroblasts in the fibrotic Furthermore, that EMT is the of liver fibrosis. Activated fibroblasts are key contributors to the fibrotic extracellular matrix accumulation during liver fibrosis. The origin of such fibroblasts is still debated, although several studies point to stellate cells as the principal source. The role of adult hepatocytes as contributors to the accumulation of fibroblasts in the fibrotic liver is yet undetermined. Here, we provide evidence that the pro-fibrotic growth factor, TGF-β1, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of epithelial to mesenchymal transition (EMT). We perform lineage-tracing experiments using AlbCre. R26RstoplacZ double transgenic mice to demonstrate that hepatocytes which undergo EMT contribute substantially to the population of FSP1-positive fibroblasts in CCL4-induced liver fibrosis. Furthermore, we demonstrate that bone morphogenic protein-7 (BMP7), of the which is to of liver in that the of to the of provide evidence the functional of adult hepatocytes in the accumulation of fibroblasts in the fibrotic Furthermore, that EMT is the of liver fibrosis. is still the of to of the liver and functional which is to of such as in the as in such as is as of extracellular matrix extracellular epithelial to mesenchymal growth bone morphogenic extracellular epithelial to mesenchymal growth bone morphogenic which to the of and of Activated fibroblasts are key of fibrosis. the fibrotic such fibroblasts are to and of stellate cells and fibroblasts to of the liver are yet of the in the accumulation of fibroblasts is is that fibroblasts are and studies such as of bone cells and of as contributors to the accumulation of fibroblasts in fibrotic we to the role of adult the epithelial cells of the in the accumulation of fibroblasts in the of hepatocytes to liver is to hepatocytes perform the of liver and of the liver we the that hepatocytes contribute to the accumulation of fibroblasts during liver in to mesenchymal transition is as in which epithelial cells phenotypic and typical of mesenchymal cells such as fibroblasts in cells epithelial and mesenchymal is during as epithelial cells to phenotypic in EMT is to in to which as of which are the of such as in the of fibroblasts is as to the of epithelial and in the fibrotic the adult evidence EMT hepatocytes is studies that EMT is of in the the hepatocytes are in of EMT as cells which mesenchymal and that during hepatocytes contribute to the of cells mesenchymal the we demonstrate that in to population of fibroblasts contribute to the of liver fibrosis. 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RATIONALE: Lung fibroblasts are key mediators of fibrosis resulting in accumulation of excessive interstitial collagen and extracellular matrix, but their origins are not well defined. OBJECTIVES: We aimed to elucidate the contribution of lung epithelium-derived fibroblasts via epithelial-mesenchymal transition (EMT) in the intratracheal bleomycin model. METHODS: Primary type II alveolar epithelial cells were cultured from Immortomice and exposed to transforming growth factor-beta(1) and epidermal growth factor. Cell fate reporter mice that permanently mark cells of lung epithelial lineage with beta-galactosidase were developed to study EMT, and bone marrow chimeras expressing green fluorescent protein under the control of the fibroblast-associated S100A4 promoter were generated to examine bone marrow-derived fibroblasts. Mice were given intratracheal bleomycin (0.08 unit). Immunostaining was performed for S100A4, beta-galactosidase, green fluorescent protein, and alpha-smooth muscle actin. MEASUREMENTS AND MAIN RESULTS: In vitro, primary type II alveolar epithelial cells undergo phenotypic changes of EMT when exposed to transforming growth factor-beta(1) and epidermal growth factor with loss of prosurfactant protein C and E-cadherin and gain of S100A4 and type I procollagen. In vivo, using cell fate reporter mice, approximately one-third of S100A4-positive fibroblasts were derived from lung epithelium 2 weeks after bleomycin administration. From bone marrow chimera studies, one-fifth of S100A4-positive fibroblasts were derived from bone marrow at this same time point. Myofibroblasts rarely derived from EMT or bone marrow progenitors. CONCLUSIONS: Both EMT and bone marrow progenitors contribute to S100A4-positive fibroblasts in bleomycin-induced lung fibrosis. However, neither origin is a principal contributor to lung myofibroblasts.

Evidence of endoplasmic reticulum (ER) stress has been found in lungs of patients with familial and sporadic idiopathic pulmonary fibrosis. We tested whether ER stress causes or exacerbates lung fibrosis by (i) conditional expression of a mutant form of surfactant protein C (L188Q SFTPC) found in familial interstitial pneumonia and (ii) intratracheal treatment with the protein misfolding agent tunicamycin. We developed transgenic mice expressing L188Q SFTPC exclusively in type II alveolar epithelium by using the Tet-On system. Expression of L188Q SFTPC induced ER stress, as determined by increased expression of heavy-chain Ig binding protein (BiP) and splicing of X-box binding protein 1 (XBP1) mRNA, but no lung fibrosis was identified in the absence of a second profibrotic stimulus. After intratracheal bleomycin, L188Q SFTPC-expressing mice developed exaggerated lung fibrosis and reduced static lung compliance compared with controls. Bleomycin-treated L188Q SFTPC mice also demonstrated increased apoptosis of alveolar epithelial cells and greater numbers of fibroblasts in the lungs. With a complementary model, intratracheal tunicamycin treatment failed to induce lung remodeling yet resulted in augmentation of bleomycin-induced fibrosis. These data support the concept that ER stress produces a dysfunctional epithelial cell phenotype that facilitates fibrotic remodeling. ER stress pathways may serve as important therapeutic targets in idiopathic pulmonary fibrosis.

Single-dose intratracheal bleomycin has been instrumental for understanding fibrotic lung remodeling, but fails to recapitulate several features of idiopathic pulmonary fibrosis (IPF). Since IPF is thought to result from recurrent alveolar injury, we aimed to develop a repetitive bleomycin model that results in lung fibrosis with key characteristics of human disease, including alveolar epithelial cell (AEC) hyperplasia. Wild-type and cell fate reporter mice expressing β-galactosidase in cells of lung epithelial lineage were given intratracheal bleomycin after intubation, and lungs were harvested 2 wk after a single or eighth biweekly dose. Lungs were evaluated for fibrosis and collagen content. Bronchoalveolar lavage (BAL) was performed for cell counts. TUNEL staining and immunohistochemistry were performed for pro-surfactant protein C (pro-SP-C), Clara cell 10 (CC-10), β-galactosidase, S100A4, and α-smooth muscle actin. Lungs from repetitive bleomycin mice had marked fibrosis with prominent AEC hyperplasia, similar to usual interstitial pneumonia (UIP). Compared with single dosing, repetitive bleomycin mice had greater fibrosis by scoring, morphometry, and collagen content; increased TUNEL+ AECs; and reduced inflammatory cells in BAL. Sixty-four percent of pro-SP-C+ cells in areas of fibrosis expressed CC-10 in the repetitive model, suggesting expansion of a bronchoalveolar stem cell-like population. In reporter mice, 50% of S100A4+ lung fibroblasts were derived from epithelial mesenchymal transition compared with 33% in the single-dose model. With repetitive bleomycin, fibrotic remodeling persisted 10 wk after the eighth dose. Repetitive intratracheal bleomycin results in marked lung fibrosis with prominent AEC hyperplasia, features reminiscent of UIP.

Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin+ intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ+ cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-β1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis. Intestinal fibrosis is a major complication of Crohn disease (CD), but the precise mechanism by which it occurs is incompletely understood. As a result, specific therapies to halt or even reverse fibrosis have not been explored. Here, we evaluated the contribution of epithelial to mesenchymal transition (EMT) to intestinal fibrosis associated with a mouse model of CD and also human inflammatory bowel disease. Mice administered intrarectal 2,4,6-trinitrobenzene sulfonic acid (TNBS) develop inflammation and fibrosis that resembles CD both histologically and by immunologic profile. We utilized this model to molecularly probe the contribution of EMT to intestinal fibrosis. Additionally, we utilized double-transgenic VillinCre;R26Rosa-lox-STOP-lox-LacZ mice, in which removal of the STOP cassette by Cre recombinase in villin+ intestinal epithelial cells activates permanent LacZ expression, to lineage trace epithelial cells that might undergo EMT upon TNBS administration. TNBS-induced fibrosis is associated with the presence of a significant number of cells that express both epithelial and mesenchymal markers. In the lineage tagged transgenic mice, the appearance of LacZ+ cells that also express the fibroblast marker FSP1 unequivocally demonstrates EMT. Transforming growth factor (TGF)-β1, a known inducer of EMT in epithelial cells, induces EMT in rat intestinal epithelial cells in vitro, and bone morphogenic protein-7, an antagonist of TGF-β1, inhibits EMT and fibrosis both in vitro and in the TNBS-treated mice. Our study demonstrates that EMT contributes to intestinal fibrosis associated with the TNBS-induced model of Crohn colitis and that inhibition of TGF-β1 with recombinant human bone morphogenic protein-7 prevents this process and prevents fibrosis. IntroductionCrohn disease (CD) 5The abbreviations used are: CDCrohn diseaseα-SMAα-smooth muscle actinEMTepithelial to mesenchymal transitionFSP1fibroblast-specific protein 1MTSMasson trichrome stainrhBMP-7recombinant human bone morphogenic protein-7RStopLacZR26-lox-Stop-lox-LacZTGF-βtransforming growth factor-βTNBS2,4,6-trinitrobenzene sulfonic acidTNF-αtumor necrosis factor-αVillin-LacZVillinCre;R26Rosa-lox-Stop-lox-LacZDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumDAPI4′,6-diamidino-2-phenylindoleH&Ehematoxylin and eosinβ-galβ-galactosidase. is a disorder of chronic, transmural inflammation that can affect any part of the gastrointestinal tract from the mouth to the anus (1Podolsky D.K. N. Engl. J. Med. 2002; 347: 417-429Crossref PubMed Scopus (3128) Google Scholar, 2Fiocchi C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar). Chronic inflammation combined with dysregulated wound healing is thought to result in a number of complications including fistula and fibrostenotic stricture formation (3Froehlich F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google Scholar, 4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar). Over one-third of patients with CD undergo surgery to relieve obstruction related to fibrotic strictures, and many will require repeat interventions because of recurrent disease (3Froehlich F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google Scholar).As with fibrosis in other organ systems, intestinal fibrosis is considered to follow a common pathway of fibrogenesis (4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar, 5Pucilowska J.B. Williams K.L. Lund P.K. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G653-G659Crossref PubMed Google Scholar). In what is presumably a protective adaptation, activated fibroblasts are recruited to sites of severe inflammation for the purpose of wound healing. Although fibroblasts are considered a heterogeneous population (6Kalluri R. Zeisberg M. Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3468) Google Scholar, 7Sugimoto H. Mundel T.M. Kieran M.W. Kalluri R. Cancer Biol. Ther. 2006; 5: 1640-1646Crossref PubMed Scopus (517) Google Scholar), those that express fibroblast-specific protein 1 (FSP1) or α-smooth muscle actin (α-SMA) are the main types mediating fibrosis (8Strutz F. Zeisberg M. J. Am. Soc. Nephrol. 2006; 17: 2992-2998Crossref PubMed Scopus (265) Google Scholar, 9Strutz F. Okada H. Lo C.W. Danoff T. Carone R.L. Tomaszewski J.E. Neilson E.G. J. Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (877) Google Scholar, 10Okada H. Danoff T.M. Kalluri R. Neilson E.G. Am. J. Physiol. 1997; 273: F563-F574Crossref PubMed Google Scholar). With persistent inflammation, fibrosis may result from excessive deposition of the extracellular matrix (4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar, 5Pucilowska J.B. Williams K.L. Lund P.K. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G653-G659Crossref PubMed Google Scholar, 11Schuppan D. Ruehl M. Somasundaram R. Hahn E.G. Semin. Liver Dis. 2001; 21: 351-372Crossref PubMed Scopus (428) Google Scholar, 12Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1877) Google Scholar). A better understanding of why and how this happens will likely have a dramatic impact on the treatment of CD and its myriad complications.Epithelial to mesenchymal transition (EMT) is a key contributor to the pool of activated fibroblasts in multiple organ systems including the heart, lung, liver, and kidney (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar, 14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 15Kim K.K. Kugler M.C. Wolters P.J. Robillard L. Galvez M.G. Brumwell A.N. Sheppard D. Chapman H.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13180-13185Crossref PubMed Scopus (1002) Google Scholar, 16Iwano M. Plieth D. Danoff T.M. Xue C. Okada H. Neilson E.G. J. Clin. Invest. 2002; 110: 341-350Crossref PubMed Scopus (1698) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar). EMT is a process in which epithelial cells lose their phenotypic and functional characteristics while acquiring mesenchymal features (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar, 18Zeisberg M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (423) Google Scholar, 19Kalluri R. Weinberg R.A. J. Clin. Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7127) Google Scholar, 20Hay E.D. Zuk A. Am. J. Kidney Dis. 1995; 26: 678-690Abstract Full Text PDF PubMed Scopus (353) Google Scholar). The role of EMT in intestinal fibrosis has yet to be investigated.Although organ fibrosis was once considered irreversible, recent data from animal models of renal, hepatic, and cardiac fibrosis demonstrate the anti-fibrotic effect of recombinant human bone morphogenic protein-7 (rhBMP-7) (14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar, 21Zeisberg M. Nephrol. Dial. Transplant. 2006; 21: 568-573Crossref PubMed Scopus (69) Google Scholar, 22Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1170) Google Scholar). BMP-7 is a member of the transforming growth factor-β (TGF-β) superfamily and is one of 15 proteins with structural and functional homology (21Zeisberg M. Nephrol. Dial. Transplant. 2006; 21: 568-573Crossref PubMed Scopus (69) Google Scholar, 23Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 24Ducy P. Karsenty G. Kidney Int. 2000; 57: 2207-2214Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). BMP-7 was first recognized for its regulatory effect on bone and cartilage formation and is currently employed clinically to accelerate fracture healing (25Cook S.D. Rueger D.C. Clin. Orthop. Relat. Res. 1996; 324: 29-38Crossref PubMed Scopus (151) Google Scholar, 26Friedlaender G.E. Perry C.R. Cole J.D. Cook S.D. Cierny G. Muschler G.F. Zych G.A. Calhoun J.H. LaForte A.J. Yin S. J. Bone Joint. Surg. Am. 2001; 83-A: S151-S158Google Scholar). With respect to organ fibrosis, the anti-fibrotic effect of rhBMP-7 stems from its ability to counteract the pro-fibrotic action of TGF-β1 (17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar, 22Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1170) Google Scholar), which has been implicated in the fibrosis of various organs including the gut (27Fiocchi C. Am. J. Physiol. 1997; 273: G769-G775PubMed Google Scholar). Receptors for BMP-7 are present on colonocytes (28Grijelmo C. Rodrigue C. Svrcek M. Bruyneel E. Hendrix A. de Wever O. Gespach C. Cell Signal. 2007; 19: 1722-1732Crossref PubMed Scopus (70) Google Scholar), and systemically administered rhBMP-7 binds with high affinity to the inflamed colon and prevents inflammation in a rat model of CD induced by rectally administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) (29Maric I. Poljak L. Zoricic S. Bobinac D. Bosukonda D. Sampath K.T. S. J. Physiol. 2003; PubMed Scopus Google Scholar). The of study was to the contribution of EMT to intestinal fibrosis associated with both a mouse model of Crohn colitis and human inflammatory bowel disease to the the anti-fibrotic of rhBMP-7 in the inflamed fibrosis in the of fibrostenotic is a complication of CD and is thought to a result of inflammation and dysregulated wound healing C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar, F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google Scholar). The mechanism by which inflammation to fibrosis is to be but likely EMT to (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar). Although various and have for have impact on fibrosis once it occurs (3Froehlich F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google is a process in which epithelial cells lose their phenotypic and functional characteristics and develop features of the the epithelial and to develop a and R. Weinberg R.A. J. Clin. Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7127) Google Scholar, E.D. 1995; PubMed Scopus Google for EMT epithelial cells in intestinal fibrosis, we transgenic in which intestinal epithelial cells can be by of colitis in the mice, we to demonstrate for the first that EMT contributes to the pool of activated fibroblasts that in a model of Crohn colitis by treatment with fibroblasts are the main of fibrogenesis in the colon in other organs A. S. M. Ther. 2007; PubMed Scopus Google Scholar, J.D. A.B. Am. J. Physiol. PubMed Google Scholar). are a heterogeneous population (6Kalluri R. Zeisberg M. Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3468) Google and the major that are known to fibrosis express FSP1 or (8Strutz F. Zeisberg M. J. Am. Soc. Nephrol. 2006; 17: 2992-2998Crossref PubMed Scopus (265) Google Scholar). fibroblasts have been the fibrogenesis of liver, and (14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 16Iwano M. Plieth D. Danoff T.M. Xue C. Okada H. Neilson E.G. J. Clin. Invest. 2002; 110: 341-350Crossref PubMed Scopus (1698) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google and from EMT M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (423) Google Scholar). In model of the of both and fibroblasts upon disease The effect of treatment with TNBS is dramatic on the population of fibroblasts we used this marker for that one-third of fibroblasts are from intestinal epithelial result is with of EMT in mouse models of M. Kalluri R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and (14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google fibrosis but may be an because the LacZ is in of the Although FSP1 is to epithelial cells that have a fibroblast and of epithelial data the that EMT occurs in the model of TNBS-induced intestinal the anti-fibrotic of rhBMP-7 in this model of a of TNBS with As we and have BMP-7 its anti-fibrotic action in the of disease of the liver, and inhibition of EMT (14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar, 22Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1170) Google Scholar). we that to the pool of fibroblasts in the of intestinal fibrosis and that inhibition of EMT by rhBMP-7 fibrosis. of rhBMP-7 in with TNBS the epithelial and fibrosis in the with have that the TNBS-induced model of Crohn colitis is associated with (29Maric I. Poljak L. Zoricic S. Bobinac D. Bosukonda D. Sampath K.T. S. J. Physiol. 2003; PubMed Scopus Google that of rhBMP-7 its anti-fibrotic action of a pathway in which colonocytes a role (29Maric I. Poljak L. Zoricic S. Bobinac D. Bosukonda D. Sampath K.T. S. J. Physiol. 2003; PubMed Scopus Google Scholar). Our not the that other are to intestinal fibrosis. In this and have been to be in the colon in to BMP-7 L. H. S. D.C. J. Clin. Invest. 2006; PubMed Scopus Google Scholar, C. J. C. X. Proc. Natl. Acad. Sci. U.S.A. 2007; PubMed Scopus Google is associated with a of effect of on TGF-β1 S. C. J. Med. 2007; PubMed Scopus Google Scholar). In this demonstrate that TGF-β1 is a inducer of EMT and that BMP-7 inhibits EMT even while are to of the of and rhBMP-7 in the of inflammation and fibrosis in Although is that treatment with an may stricture formation in patients A. S. D.M. Am. J. 2006; PubMed Scopus Google Scholar, D. G. S. C. Digestion. 2007; PubMed Scopus (19) Google Scholar). In treatment may be and is a of intestinal fibrosis (4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar), and it is the inducer of EMT in various organ systems (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar). we the of TGF-β1 to EMT in rat intestinal epithelial The in in the treatment with TNBS to a inflammatory process that resembles CD both in and S. C. Nat. 2007; PubMed Scopus Google Scholar, M. I. Int. Rev. 2000; 19: PubMed Scopus Google Scholar, F. 2002; Google Scholar), the role of EMT in human inflammatory bowel disease A recent a role for EMT in from a of patients with CD F. C. R. A. Rieder F. Brenmoehl J. S. A. F. Schölmerich J. H. Rogler G. Dis. PubMed Scopus Google Scholar). Our of and in intestinal from patients with inflammatory bowel disease that EMT likely occurs in human intestinal fibrosis The of from patients with CD and colitis the of of human data a role for EMT in human inflammatory bowel disease. the are to CD or a result of inflammation associated with any number of to be of in a and will be to this process it to human intestinal fibrosis in and CD in study demonstrates a role for EMT in the of TNBS-induced intestinal fibrosis. As has been in the kidney and heart, treatment with rhBMP-7 both in and in vitro prevents EMT associated with fibrosis. that rhBMP-7 may be an for intestinal fibrosis in and CD in IntroductionCrohn disease (CD) 5The abbreviations used are: CDCrohn diseaseα-SMAα-smooth muscle actinEMTepithelial to mesenchymal transitionFSP1fibroblast-specific protein 1MTSMasson trichrome stainrhBMP-7recombinant human bone morphogenic protein-7RStopLacZR26-lox-Stop-lox-LacZTGF-βtransforming growth factor-βTNBS2,4,6-trinitrobenzene sulfonic acidTNF-αtumor necrosis factor-αVillin-LacZVillinCre;R26Rosa-lox-Stop-lox-LacZDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumDAPI4′,6-diamidino-2-phenylindoleH&Ehematoxylin and eosinβ-galβ-galactosidase. is a disorder of chronic, transmural inflammation that can affect any part of the gastrointestinal tract from the mouth to the anus (1Podolsky D.K. N. Engl. J. Med. 2002; 347: 417-429Crossref PubMed Scopus (3128) Google Scholar, 2Fiocchi C. Gastroenterology. 1998; 115: 182-205Abstract Full Text Full Text PDF PubMed Scopus (1855) Google Scholar). Chronic inflammation combined with dysregulated wound healing is thought to result in a number of complications including fistula and fibrostenotic stricture formation (3Froehlich F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google Scholar, 4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar). Over one-third of patients with CD undergo surgery to relieve obstruction related to fibrotic strictures, and many will require repeat interventions because of recurrent disease (3Froehlich F. Juillerat P. Mottet C. Pittet V. Felley C. Vader J.P. Gonvers J.J. Michetti P. Digestion. 2007; 76: 113-115Crossref PubMed Scopus (19) Google Scholar).As with fibrosis in other organ systems, intestinal fibrosis is considered to follow a common pathway of fibrogenesis (4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar, 5Pucilowska J.B. Williams K.L. Lund P.K. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G653-G659Crossref PubMed Google Scholar). In what is presumably a protective adaptation, activated fibroblasts are recruited to sites of severe inflammation for the purpose of wound healing. Although fibroblasts are considered a heterogeneous population (6Kalluri R. Zeisberg M. Nat. Rev. Cancer. 2006; 6: 392-401Crossref PubMed Scopus (3468) Google Scholar, 7Sugimoto H. Mundel T.M. Kieran M.W. Kalluri R. Cancer Biol. Ther. 2006; 5: 1640-1646Crossref PubMed Scopus (517) Google Scholar), those that express fibroblast-specific protein 1 (FSP1) or α-smooth muscle actin (α-SMA) are the main types mediating fibrosis (8Strutz F. Zeisberg M. J. Am. Soc. Nephrol. 2006; 17: 2992-2998Crossref PubMed Scopus (265) Google Scholar, 9Strutz F. Okada H. Lo C.W. Danoff T. Carone R.L. Tomaszewski J.E. Neilson E.G. J. Cell Biol. 1995; 130: 393-405Crossref PubMed Scopus (877) Google Scholar, 10Okada H. Danoff T.M. Kalluri R. Neilson E.G. Am. J. Physiol. 1997; 273: F563-F574Crossref PubMed Google Scholar). With persistent inflammation, fibrosis may result from excessive deposition of the extracellular matrix (4Rieder F. Brenmoehl J. Leeb S. Schölmerich J. Rogler G. Gut. 2007; 56: 130-139Crossref PubMed Scopus (230) Google Scholar, 5Pucilowska J.B. Williams K.L. Lund P.K. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G653-G659Crossref PubMed Google Scholar, 11Schuppan D. Ruehl M. Somasundaram R. Hahn E.G. Semin. Liver Dis. 2001; 21: 351-372Crossref PubMed Scopus (428) Google Scholar, 12Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1877) Google Scholar). A better understanding of why and how this happens will likely have a dramatic impact on the treatment of CD and its myriad complications.Epithelial to mesenchymal transition (EMT) is a key contributor to the pool of activated fibroblasts in multiple organ systems including the heart, lung, liver, and kidney (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar, 14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 15Kim K.K. Kugler M.C. Wolters P.J. Robillard L. Galvez M.G. Brumwell A.N. Sheppard D. Chapman H.A. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 13180-13185Crossref PubMed Scopus (1002) Google Scholar, 16Iwano M. Plieth D. Danoff T.M. Xue C. Okada H. Neilson E.G. J. Clin. Invest. 2002; 110: 341-350Crossref PubMed Scopus (1698) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar). EMT is a process in which epithelial cells lose their phenotypic and functional characteristics while acquiring mesenchymal features (13Kalluri R. Neilson E.G. J. Clin. Invest. 2003; 112: 1776-1784Crossref PubMed Scopus (2061) Google Scholar, 18Zeisberg M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (423) Google Scholar, 19Kalluri R. Weinberg R.A. J. Clin. Invest. 2009; 119: 1420-1428Crossref PubMed Scopus (7127) Google Scholar, 20Hay E.D. Zuk A. Am. J. Kidney Dis. 1995; 26: 678-690Abstract Full Text PDF PubMed Scopus (353) Google Scholar). The role of EMT in intestinal fibrosis has yet to be investigated.Although organ fibrosis was once considered irreversible, recent data from animal models of renal, hepatic, and cardiac fibrosis demonstrate the anti-fibrotic effect of recombinant human bone morphogenic protein-7 (rhBMP-7) (14Zeisberg M. Yang C. Martino M. Duncan M.B. Rieder F. Tanjore H. Kalluri R. J. Biol. Chem. 2007; 282: 23337-23347Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar, 21Zeisberg M. Nephrol. Dial. Transplant. 2006; 21: 568-573Crossref PubMed Scopus (69) Google Scholar, 22Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1170) Google Scholar). BMP-7 is a member of the transforming growth factor-β (TGF-β) superfamily and is one of 15 proteins with structural and functional homology (21Zeisberg M. Nephrol. Dial. Transplant. 2006; 21: 568-573Crossref PubMed Scopus (69) Google Scholar, 23Kingsley D.M. Genes Dev. 1994; 8: 133-146Crossref PubMed Scopus (1726) Google Scholar, 24Ducy P. Karsenty G. Kidney Int. 2000; 57: 2207-2214Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). BMP-7 was first recognized for its regulatory effect on bone and cartilage formation and is currently employed clinically to accelerate fracture healing (25Cook S.D. Rueger D.C. Clin. Orthop. Relat. Res. 1996; 324: 29-38Crossref PubMed Scopus (151) Google Scholar, 26Friedlaender G.E. Perry C.R. Cole J.D. Cook S.D. Cierny G. Muschler G.F. Zych G.A. Calhoun J.H. LaForte A.J. Yin S. J. Bone Joint. Surg. Am. 2001; 83-A: S151-S158Google Scholar). With respect to organ fibrosis, the anti-fibrotic effect of rhBMP-7 stems from its ability to counteract the pro-fibrotic action of TGF-β1 (17Zeisberg E.M. Tarnavski O. Zeisberg M. Dorfman A.L. McMullen J.R. Gustafsson E. Chandraker A. Yuan X. Pu W.T. Roberts A.B. Neilson E.G. Sayegh M.H. Izumo S. Kalluri R. Nat. Med. 2007; 13: 952-961Crossref PubMed Scopus (1591) Google Scholar, 22Zeisberg M. Hanai J. Sugimoto H. Mammoto T. Charytan D. Strutz F. Kalluri R. Nat. Med. 2003; 9: 964-968Crossref PubMed Scopus (1170) Google Scholar), which has been implicated in the fibrosis of various organs including the gut (27Fiocchi C. Am. J. Physiol. 1997; 273: G769-G775PubMed Google Scholar). Receptors for BMP-7 are present on colonocytes (28Grijelmo C. Rodrigue C. Svrcek M. Bruyneel E. Hendrix A. de Wever O. Gespach C. Cell Signal. 2007; 19: 1722-1732Crossref PubMed Scopus (70) Google Scholar), and systemically administered rhBMP-7 binds with high affinity to the inflamed colon and prevents inflammation in a rat model of CD induced by rectally administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) (29Maric I. Poljak L. Zoricic S. Bobinac D. Bosukonda D. Sampath K.T. S. J. Physiol. 2003; PubMed Scopus Google Scholar). The of study was to the contribution of EMT to intestinal fibrosis associated with both a mouse model of Crohn colitis and human inflammatory bowel disease to the the anti-fibrotic of rhBMP-7 in the inflamed