Abstract: The crucial role played by the myofibroblast in wound healing and pathological organ remodeling is well established; the general mechanisms of extracellular matrix synthesis and of tension production by this cell have been amply clarified. This review discusses the pattern of myofibroblast accumulation and fibrosis evolution during lung and liver fibrosis as well as during atheromatous plaque formation. Special attention is paid to the specific features characterizing each of these processes, including the spectrum of different myofibroblast precursors and the distinct pathways involved in the formation of differentiated myofibroblasts in each lesion. Thus, whereas in lung fibrosis it seems that most myofibroblasts derive from resident fibroblasts, hepatic stellate cells are the main contributor for liver fibrosis and media smooth muscle cells are the main contributor for the atheromatous plaque. A better knowledge of the molecular mechanisms conducing to the appearance of differentiated myofibroblasts in each pathological situation will be useful for the understanding of fibrosis development in different organs and for the planning of strategies aiming at their prevention and therapy. The crucial role played by the myofibroblast in wound healing and pathological organ remodeling is well established; the general mechanisms of extracellular matrix synthesis and of tension production by this cell have been amply clarified. This review discusses the pattern of myofibroblast accumulation and fibrosis evolution during lung and liver fibrosis as well as during atheromatous plaque formation. Special attention is paid to the specific features characterizing each of these processes, including the spectrum of different myofibroblast precursors and the distinct pathways involved in the formation of differentiated myofibroblasts in each lesion. Thus, whereas in lung fibrosis it seems that most myofibroblasts derive from resident fibroblasts, hepatic stellate cells are the main contributor for liver fibrosis and media smooth muscle cells are the main contributor for the atheromatous plaque. A better knowledge of the molecular mechanisms conducing to the appearance of differentiated myofibroblasts in each pathological situation will be useful for the understanding of fibrosis development in different organs and for the planning of strategies aiming at their prevention and therapy. After tissue injury, fibroblasts differentiate into contractile and secretory myofibroblasts that contribute to tissue repair during wound healing, but that can severely impair organ function when contraction and extracellular matrix (ECM) protein secretion become excessive, such as in hypertrophic scars, scleroderma, and Dupuytren's disease as well as in heart and kidney fibrosis.1Hinz B Formation and function of the myofibroblast during tissue repair.J Invest Dermatol. 2007; 127: 526-537Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar, 2Brown RD Ambler SK Mitchell MD Long CS The cardiac fibroblast: therapeutic target in myocardial remodeling and failure.Annu Rev Pharmacol Toxicol. 2005; 45: 657-687Crossref PubMed Scopus (547) Google Scholar, 3Desmoulière A Darby IA Gabbiani G Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis.Lab Invest. 2003; 83: 1689-1707Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar Moreover, myofibroblasts present in the so-called stroma reaction of epithelial tumors may promote the progression of cancer invasion.4De Wever O Mareel M Role of tissue stroma in cancer cell invasion.J Pathol. 2003; 200: 429-447Crossref PubMed Scopus (855) Google Scholar Here, we discuss the role of myofibroblasts in causing pathological deformation of two vital organs, liver3Desmoulière A Darby IA Gabbiani G Normal and pathologic soft tissue remodeling: role of the myofibroblast, with special emphasis on liver and kidney fibrosis.Lab Invest. 2003; 83: 1689-1707Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar and lung,5Thannickal VJ Toews GB White ES Lynch 3rd, JP Martinez FJ Mechanisms of pulmonary fibrosis.Annu Rev Med. 2004; 55: 395-417Crossref PubMed Scopus (567) Google Scholar and in contributing to the formation of the atheromatous plaque and restenotic lesions.6Zalewski A Shi Y Johnson AG Diverse origin of intimal cells: smooth muscle cells, myofibroblasts, fibroblasts, and beyond?.Circ Res. 2002; 91: 652-655Crossref PubMed Scopus (97) Google Scholar, 7Hao H Gabbiani G Camenzind E Bacchetta M Virmani R Bochaton-Piallat ML Phenotypic modulation of intima and media smooth muscle cells in fatal cases of coronary artery lesion.Arterioscler Thromb Vasc Biol. 2006; 26: 326-332Crossref PubMed Scopus (106) Google Scholar Myofibroblast differentiation is a complex phenomenon that follows distinct patterns in different organs. To counteract therapeutically organ dysfunction caused by myofibroblasts, it is crucial to understand the general molecular pathways regulating their evolution and function to distinguish the mechanisms common to all situations from those specific to a given organ and/or disease. As we shall discuss below, myofibroblasts may have very heterogeneous origins; however, their development follows a well-established sequence of events. In normal conditions, fibroblastic cells exhibit few or no actin-associated cell-cell and cell-matrix contacts and little ECM production.8Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar After tissue injury, they become activated to migrate into the damaged tissue and to synthesize ECM components1Hinz B Formation and function of the myofibroblast during tissue repair.J Invest Dermatol. 2007; 127: 526-537Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar by cytokines locally released from inflammatory and resident cells9Werner S Grose R Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2700) Google Scholar or from malignant epithelial cells.4De Wever O Mareel M Role of tissue stroma in cancer cell invasion.J Pathol. 2003; 200: 429-447Crossref PubMed Scopus (855) Google Scholar Another important stimulus for this phenotypic transition is the change of the mechanical microenvironment; whereas fibroblasts in intact tissue are generally stress-shielded by the crosslinked ECM, this protective structure is lost in the continuously remodeled ECM of injured tissue.8Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar In response to mechanical challenge, fibroblasts acquire contractile stress fibers that are first composed of cytoplasmic actins,8Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar hallmarking the “protomyofibroblast.” Stress fibers are connected to fibrous ECM proteins at sites of integrin-containing cell-matrix junctions10Hinz B Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.Eur J Cell Biol. 2006; 85: 175-181Crossref PubMed Scopus (214) Google Scholar and between cells via de novo established N-cadherin-type adherens junctions.11Hinz B Pittet P Smith-Clerc J Chaponnier C Meister JJ Myofibroblast development is characterized by specific cell-cell adherens junctions.Mol Biol Cell. 2004; 15: 4310-4320Crossref PubMed Scopus (183) Google Scholar These features closely resemble those of cultured fibroblasts that have been mechanically activated by the rigid plastic substrate, whereas stress fibers do not form on very soft culture substrate hydrogels or in compliant collagen gels (Figure 1).10Hinz B Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.Eur J Cell Biol. 2006; 85: 175-181Crossref PubMed Scopus (214) Google Scholar In culture, the protomyofibroblast is a stable phenotype, representing an intermediate step in most in vivo conditions where it proceeds toward the “differentiated myofibroblast” that is characterized by de novo expression of α-smooth muscle actin (α-SMA), its most commonly used molecular marker. Expression of α-SMA in stress fibers confers to the differentiated myofibroblast at least a twofold stronger contractile activity compared with α-SMA-negative fibroblasts in culture.12Hinz B Celetta G Tomasek JJ Gabbiani G Chaponnier C Alpha-smooth muscle actin expression upregulates fibroblast contractile activity.Mol Biol Cell. 2001; 12: 2730-2741Crossref PubMed Scopus (975) Google Scholar It is still unclear how α-SMA generates higher contraction compared with other actin isoforms, but the α-SMA-specific N-terminal amino acid sequence AcEEED plays an important role in this mechanism. Cytoplasmic delivery of this sequence as a peptide selectively removes α-SMA from persisting β-cytoplasmic actin stress fibers and reduces in vivo and in vitro myofibroblast contraction.13Hinz B Gabbiani G Chaponnier C The NH2-terminal peptide of alpha-smooth muscle actin inhibits force generation by the myofibroblast in vitro and in vivo.J Cell Biol. 2002; 157: 657-663Crossref PubMed Scopus (201) Google Scholar At least three local events are needed to generate α-SMA-positive differentiated myofibroblasts: 1) accumulation of biologically active transforming growth factor (TGF) β1, 2) the presence of specialized ECM proteins like the ED-A splice variant of fibronectin, and 3) high extracellular stress, arising from the mechanical properties of the ECM and cell remodeling activity.8Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar Mechanoperception is mediated by specialized cell-matrix junctions, called “fibronexus” in vivo and “supermature focal adhesions” (FAs) in vitro.10Hinz B Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.Eur J Cell Biol. 2006; 85: 175-181Crossref PubMed Scopus (214) Google Scholar Analogously, small N-cadherin-type cell-cell adhesions develop into larger OB-cadherin (cadherin-11)-type junctions during generation of the differentiated myofibroblast in vitro and in vivo.11Hinz B Pittet P Smith-Clerc J Chaponnier C Meister JJ Myofibroblast development is characterized by specific cell-cell adherens junctions.Mol Biol Cell. 2004; 15: 4310-4320Crossref PubMed Scopus (183) Google Scholar It has become increasingly accepted that ECM rigidity determines the size of the cell's anchors, which in turn limits the level of tension generated within stress fibers.10Hinz B Masters and servants of the force: the role of matrix adhesions in myofibroblast force perception and transmission.Eur J Cell Biol. 2006; 85: 175-181Crossref PubMed Scopus (214) Google Scholar Only when substrate stiffness permits formation of supermature FAs (8 to 30 μm long), and thus generation of approximately fourfold greater stress compared with the usual FAs (2 to 6 μm long), does α-SMA become incorporated into pre-existing β-cytoplasmic actin stress fibers; hence, α-SMA can be considered a mechanosensitive protein14Goffin JM Pittet P Csucs G Lussi JW Meister JJ Hinz B Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers.J Cell Biol. 2006; 172: 259-268Crossref PubMed Scopus (559) Google Scholar, 15Wang J Zohar R McCulloch CA Multiple roles of α-smooth muscle actin in mechanotransduction.Exp Cell Res. 2006; 312: 205-214Crossref PubMed Scopus (145) Google Scholar (Figure 1). The myofibroblast cytoskeleton may function as a mechanotransducer translating to biochemical signals involving tyrosine phosphatase and kinase pathways.16Giannone G Sheetz MP Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways.Trends Cell Biol. 2006; 16: 213-223Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar Mechanical force-induced p38 phosphorylation seems to be dependent on an α-SMA stress fiber-dependent pathway that uses a feed-forward amplification loop to synergize force-induced α-SMA expression with p38 activation.15Wang J Zohar R McCulloch CA Multiple roles of α-smooth muscle actin in mechanotransduction.Exp Cell Res. 2006; 312: 205-214Crossref PubMed Scopus (145) Google Scholar Cell adhesion signaling via focal adhesion kinase may represent another central pathway through which biochemical and biophysical ECM signals as well as soluble growth factor signals are integrated.14Goffin JM Pittet P Csucs G Lussi JW Meister JJ Hinz B Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers.J Cell Biol. 2006; 172: 259-268Crossref PubMed Scopus (559) Google Scholar, 17Thannickal VJ Lee DY White ES Cui Z Larios JM Chacon R Horowitz JC Day RM Thomas PE Myofibroblast differentiation by transforming growth factor-β1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase.J Biol Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar The main myofibroblast inducer TGFβ1 up-regulates expression of fibronectin and its integrin receptors in lung fibroblasts; this is closely linked to the activation/phosphorylation of focal adhesion kinase essential for the induction of myofibroblast differentiation.17Thannickal VJ Lee DY White ES Cui Z Larios JM Chacon R Horowitz JC Day RM Thomas PE Myofibroblast differentiation by transforming growth factor-β1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase.J Biol Chem. 2003; 278: 12384-12389Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar At the end of tissue repair, the reconstructed ECM again takes over the mechanical load and myofibroblasts disappear by massive apoptosis8Tomasek JJ Gabbiani G Hinz B Chaponnier C Brown RA Myofibroblasts and mechano-regulation of connective tissue remodeling.Nat Rev Mol Cell Biol. 2002; 3: 349-363Crossref PubMed Scopus (3228) Google Scholar; stress release is a powerful promoter of myofibroblast apoptosis in vivo.18Carlson MA Longaker MT Thompson JS Wound splinting regulates granulation tissue survival.J Surg Res. 2003; 110: 304-309Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar Thus, interrupting the mechanical feedback loop of myofibroblast contraction and gradually increasing ECM tension at the level of stress perception (ie, cell-ECM contacts) is one promising strategy to prevent tissue contracture. An alternative strategy to decrease tissue contracture consists in preventing myofibroblast formation in the first place, requiring knowledge of the myofibroblast origin. Depending on the type of tissue to be remodeled, myofibroblast precursor cells are recruited from different sources; among these, locally residing fibroblasts seem to be the most common.1Hinz B Formation and function of the myofibroblast during tissue repair.J Invest Dermatol. 2007; 127: 526-537Abstract Full Text Full Text PDF PubMed Scopus (1204) Google Scholar Other mesenchymal cells that are discussed to serve as myofibroblast progenitors are pericytes and smooth muscle cells (SMCs) from the vasculature; they seem to play an important role during vessel repair7Hao H Gabbiani G Camenzind E Bacchetta M Virmani R Bochaton-Piallat ML Phenotypic modulation of intima and media smooth muscle cells in fatal cases of coronary artery lesion.Arterioscler Thromb Vasc Biol. 2006; 26: 326-332Crossref PubMed Scopus (106) Google Scholar and have been suggested to contribute to fibrosis in scleroderma.19Rajkumar VS Howell K Csiszar K Denton CP Black CM Abraham DJ Shared expression of phenotypic markers in systemic sclerosis indicates a convergence of pericytes and fibroblasts to a myofibroblast lineage in fibrosis.Arthritis Res Ther. 2005; 7: R1113-R1123Crossref PubMed Google Scholar In addition, bone marrow (BM)-derived circulating cells known as fibrocytes20Abe R Donnelly SC Peng T Bucala R Metz CN Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562Crossref PubMed Scopus (936) Google Scholar have been suggested to represent an alternative source for myofibroblasts during skin wound healing and in liver, lung, and kidney fibrosis, as well as in the stroma reaction to epithelial tumors.21Schmidt M Sun G Stacey MA Mori L Mattoli S Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma.J Immunol. 2003; 171: 380-389Crossref PubMed Scopus (568) Google Scholar, 22Forbes SJ Russo FP Rey V Burra P Rugge M Wright NA Alison MR A significant proportion of myofibroblasts are of bone marrow origin in human liver fibrosis.Gastroenterology. 2004; 126: 955-963Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 23Direkze NC Forbes SJ Brittan M Hunt T Jeffery R Preston SL Poulsom R Hodivala-Dilke K Alison MR Wright NA Multiple organ engraftment by bone-marrow-derived myofibroblasts and fibroblasts in bone-marrow-transplanted mice.Stem Cells. 2003; 21: 514-520Crossref PubMed Scopus (221) Google Scholar, 24Ishii G Sangai T Oda T Aoyagi Y Hasebe T Kanomata N Endoh Y Okumura C Okuhara Y Magae J Emura M Ochiya T Ochiai A Bone-marrow-derived myofibroblasts contribute to the cancer-induced stromal reaction.Biochem Biophys Res Commun. 2003; 309: 232-240Crossref PubMed Scopus (238) Google Scholar Other studies do not support this view as further discussed below.25Hashimoto N Jin H Liu T Chensue SW Phan SH Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (657) Google Scholar, 26Kisseleva T Uchinami H Feirt N Quintana-Bustamante O Segovia JC Schwabe RF Brenner DA Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.J Hepatol. 2006; 45: 429-438Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar Finally, myofibroblasts have been shown to derive from epithelial-mesenchymal transition (EMT).27Zeisberg M Kalluri R The role of epithelial-to-mesenchymal transition in renal fibrosis.J Mol Med. 2004; 82: 175-181Crossref PubMed Scopus (432) Google Scholar, 28Kim KK Kugler MC Wolters PJ Robillard L Galvez MG Brumwell AN Sheppard D Chapman HA Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.Proc Natl Acad Sci USA. 2006; 103: 13180-13185Crossref PubMed Scopus (1040) Google Scholar, 29Xia JL Dai C Michalopoulos GK Liu Y Hepatocyte growth factor attenuates liver fibrosis induced by bile duct ligation.Am J Pathol. 2006; 168: 1500-1512Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar Thus, damaged organs seem to recruit myofibroblast precursors from several sources to satisfy the temporarily high demand of cells with tissue remodeling activity (Figure 2). The presence of stable protomyofibroblasts in normal alveolar septa is well established. The repair process in response to lung injury is characterized by neoformation of differentiated myofibroblasts. In view of its many characteristics that encompass the notable features of fibrosis, such as the elaboration of ECM and expression/activation of TGFβ1,30Zhang K Rekhter MD Gordon D Phan SH Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study.Am J Pathol. 1994; 145: 114-125PubMed Google Scholar, 31Zhang K Gharaee-Kermani M Jones ML Warren JS Phan SH Lung monocyte chemoattractant protein-1 gene expression in bleomycin-induced pulmonary fibrosis.J Immunol. 1994; 153: 4733-4741Crossref PubMed Google Scholar the persistence of the myofibroblast is thought to be of significance in the propagation of fibrosis with evolution to terminal end-stage fibrotic lung disease. Early studies of the origin of the myofibroblast in lung injury and fibrosis suggest several possibilities based on observations of its cytoskeletal phenotype, tissue localization, and in vitro studies. Based on evidence that myofibroblasts arise de novo and on the kinetics of the induction of α-SMA expression, the perivascular and peribronchiolar adventitial fibroblasts are suggested as precursors.30Zhang K Rekhter MD Gordon D Phan SH Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study.Am J Pathol. 1994; 145: 114-125PubMed Google Scholar However, circulating fibrocytes (expressing CD45, CD34, collagen I, and CXCR4) have been reported to migrate to sites of tissue injury and differentiate into myofibroblasts.20Abe R Donnelly SC Peng T Bucala R Metz CN Peripheral blood fibrocytes: differentiation pathway and migration to wound sites.J Immunol. 2001; 166: 7556-7562Crossref PubMed Scopus (936) Google Scholar, 32Phillips RJ Burdick MD Hong K Lutz MA Murray LA Xue YY Belperio JA Keane MP Strieter RM Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (931) Google Scholar Furthermore, other studies using BM chimeric mice, in which the donated BM cells express a marker protein for tracking purposes, as well as human transplant studies, demonstrate that BM-derived progenitors can give rise to lung fibroblasts.25Hashimoto N Jin H Liu T Chensue SW Phan SH Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (657) Google Scholar, 33Bröcker V Langer F Fellous TG Mengel M Brittan M Bredt M Milde S Welte T Eder M Haverich A Alison MR Kreipe H Lehmann U Fibroblasts of recipient origin contribute to bronchiolitis obliterans in human lung transplants.Am J Respir Crit Care Med. 2006; 173: 1276-1282Crossref PubMed Scopus (69) Google Scholar However, the ability of BM-derived fibroblasts to differentiate to myofibroblasts cannot be demonstrated in some studies.25Hashimoto N Jin H Liu T Chensue SW Phan SH Bone marrow-derived progenitor cells in pulmonary fibrosis.J Clin Invest. 2004; 113: 243-252Crossref PubMed Scopus (657) Google Scholar, 26Kisseleva T Uchinami H Feirt N Quintana-Bustamante O Segovia JC Schwabe RF Brenner DA Bone marrow-derived fibrocytes participate in pathogenesis of liver fibrosis.J Hepatol. 2006; 45: 429-438Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar Moreover, another study using α-SMA promoter-driven green fluorescent protein BM chimeric mice indicates that the BM is not a source of progenitor cells for α-SMA-expressing cells.34Yokota T Kawakami Y Nagai Y Ma JX Tsai JY Kincade PW Sato S Bone marrow lacks a transplantable progenitor for smooth muscle type alpha-actin-expressing cells.Stem Cells. 2006; 24: 13-22Crossref PubMed Scopus (57) Google Scholar Thus, the evidence for BM derivation of myofibroblasts in lung fibrosis is controversial, suggesting potentially multiple origins, including intrapulmonary precursors. Additional possibilities for intrapulmonary precursors are suggested by evidence of both endothelial to mesenchymal transition and EMT. Endothelial cells as a source of α-SMA-expressing mesenchymal cells have been shown in the development of the vasculature and when they are stimulated with TGFβ1 in vitro.35Frid MG Kale VA Stenmark KR Mature vascular endothelium can give rise to smooth muscle cells via endothelial-mesenchymal transdifferentiation: in vitro analysis.Circ Res. 2002; 90: 1189-1196Crossref PubMed Scopus (364) Google Scholar Derivation from epithelial cells via EMT has been suggested recently by both in vitro and in vivo studies,28Kim KK Kugler MC Wolters PJ Robillard L Galvez MG Brumwell AN Sheppard D Chapman HA Alveolar epithelial cell mesenchymal transition develops in vivo during pulmonary fibrosis and is regulated by the extracellular matrix.Proc Natl Acad Sci USA. 2006; 103: 13180-13185Crossref PubMed Scopus (1040) Google Scholar, 36Willis BC Liebler JM Luby-Phelps K Nicholson AG Crandall ED du Bois RM Borok Z Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-β1: potential role in idiopathic pulmonary fibrosis.Am J Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar but this could not be demonstrated in another study.37Barth K Reh J Sturrock A Kasper M Epithelial vs myofibroblast differentiation in immortal rat lung cell lines—modulating effects of bleomycin.Histochem Cell Biol. 2005; 124: 453-464Crossref PubMed Scopus (21) Google Scholar The relative contribution by these different potential sources of myofibroblasts requires further study. The mechanism underlying the genesis of the myofibroblast is complex; here, the focus is on downstream effects of myofibroblast-modulating factors on α-SMA transcription, which is particularly well studied in lung fibroblasts. With respect to the Smad signaling pathway, the presence of a Smad3-binding element is essential for myofibroblast differentiation.38Ramirez AM Shen Z Ritzenthaler JD Roman J Myofibroblast transdifferentiation in obliterative bronchiolitis: TGF-β signaling through smad3-dependent and -independent pathways.Am J Transplant. 2006; 6: 2080-2088Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 39Hu B Wu Z Liu T Ullenbruch MR Jin H Phan SH Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (52) Google Scholar However, regulation of the α-SMA gene is more complex and in many respects different from that in SMCs.16Giannone G Sheetz MP Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways.Trends Cell Biol. 2006; 16: 213-223Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 38Ramirez AM Shen Z Ritzenthaler JD Roman J Myofibroblast transdifferentiation in obliterative bronchiolitis: TGF-β signaling through smad3-dependent and -independent pathways.Am J Transplant. 2006; 6: 2080-2088Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 39Hu B Wu Z Liu T Ullenbruch MR Jin H Phan SH Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (52) Google Scholar Additional transcription factors, including C/EBPβ (CCAAT/enhancer-binding protein β), GKLF (gut-enriched Krüppel-like factor), Sp1/Sp3, c-myb, and the downstream effector component of Notch signaling, have been implicated to regulate this gene in a complex and interactive manner.39Hu B Wu Z Liu T Ullenbruch MR Jin H Phan SH Gut-enriched Kruppel-like factor interaction with Smad3 inhibits myofibroblast differentiation.Am J Respir Cell Mol Biol. 2007; 36: 78-84Crossref PubMed Scopus (52) Google Scholar, 40Cogan JG Subramanian SV Polikandriotis JA Kelm Jr, RJ Strauch AR Vascular smooth muscle α-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.J Biol Chem. 2002; 277: 36433-36442Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 41Hu B Wu Z Jin H Hashimoto N Liu T Phan SH CCAAT/enhancer-binding protein β isoforms and the regulation of α-smooth muscle actin gene expression by IL-1β.J Immunol. 2004; 173: 4661-4668Crossref PubMed Scopus (46) Google Scholar, 42Hu B Ullenbruch MR Jin H Gharaee-Kermani M Phan SH An essential role for CCAAT/enhancer binding protein β in bleomycin induced pulmonary fibrosis.J Pathol. 2007; 211: 455-462Crossref PubMed Scopus (31) Google Scholar, 43Noseda M Fu Y Niessen K Wong F Chang L McLean G Karsan A Smooth muscle α-actin is a direct target of Notch/CSL.Circ Res. 2006; 98: 1468-1470Crossref PubMed Scopus (148) Google Scholar In addition to inducers, suppressors such as the liver-enriched inhibitory protein isoform of C/EBPβ may serve to keep the precursor fibroblast in an undifferentiated state under normal homeostasis. Epigenetic regulation is implicated by evidence that inhibitors of DNA methylation or histone deacetylation suppress myofibroblast differentiation in the liver.44Mann J Oakley F Akiboye F Elsharkawy A Thorne AW Mann DA Regulation of myofibroblast transdifferentiation by DNA methylation and MeCP2: implications for wound healing and fibrogenesis.Cell Death Differ. 2007; 14: 275-285Crossref PubMed Scopus (204) Google Scholar Because differentiation is usually accompanied by activation of gene expression, this implies that myofibroblast differentiation is actively suppressed in the quiescent precursor cell by products of certain genes whose expression is suppressed by DNA methylation and deacetylated (or poorly acetylated) histones. Further elucidation is required to understand fully the mechanisms involved in the de novo genesis of the myofibroblast in pulmonary fibrosis. The role of the myofibroblast in pulmonary fibrosis can be extrapolated from its known functional activities in vivo and in vitro. Early observations focus on the expression of α-SMA in prominent stress fibers, suggesting a role in tissue contractility or compliance. The significance of this α-SMA expression, however, seems to extend beyond these mechanical properties, with evidence pointing to important roles in modulating signal transduction and regulation of gene expression, including ECM components.13Hinz B Gabbiani G Chaponnier C The NH2-terminal peptid