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Epithelial–mesenchymal transition in chronic liver disease: Fibrogenesis or escape from death?

  • Massimo Pinzani
    Correspondence
    Tel.: +39 055 4271084; fax: +39 055 417123.
    Affiliations
    Dipartimento di Medicina Interna, Center for Research, High Education and Transfer “DENOThe”, Università degli Studi di Firenze, Viale G.B. Morgagni, 85, 50134 Firenze, Italy
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Open AccessPublished:February 14, 2011DOI:https://doi.org/10.1016/j.jhep.2011.02.001
      The possibility that epithelial–mesenchymal transition (EMT) could contribute to hepatic fibrogenesis in chronic liver diseases as reported in other organs, particularly the kidney, reinforced the concept that activated hepatic stellate cells were not the only key players in the hepatic fibrogenic process and that other cell types, either hepatic (i.e. portal fibroblast) or extrahepatic (bone marrow-derived cells and circulating fibrocytes) could contribute to this process. The possibility of the rapid mobilization of a large amount of fibrogenic cells by EMT after liver tissue injury made this phenomenon a relevant and suitable target for anti-fibrogenic strategies. Following an initial enthusiasm for the discovery of this novel pathway in fibrogenesis and the publication of a several highly quoted papers, more recent research has started to cast serious doubts upon the real relevance of this phenomenon in human fibrogenetic disorders. The debate on the authenticity of EMT or at least on its real contribution to the fibrogenic process has become very animated, sometimes reaching levels of “religious” integralism. The overall result is a general confusion on the meaning and on the definition of several key aspects. The aim of this article is to analyze and discuss the evidence supporting or confuting this possibility in order to reach reasonable and useful conclusions.

      Keywords

      Introduction

      Epithelial–mesenchymal transition or transformation (EMT) is defined as a key developmental program characterized by loss of cell adhesion, repression of E-cadherin expression, and increased cell mobility [
      • Lee J.M.
      • Dedhar S.
      • Kalluri R.
      • Thompson E.W.
      The epithelial–mesenchymal transition: new insights in signaling, development, and disease.
      ]. The term “transition” is currently preferred since it reflects in part the reversibility of the process. Indeed, the phenotypic plasticity afforded by an EMT is characterized by the occurrence of the reverse process, i.e. a mesenchymal–epithelial transition (MET), which involves the conversion of mesenchymal cells to epithelial derivatives. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. Its potential clinical relevance was initially highlighted and characterized in cancer development and metastasis and, more recently, in chronic inflammatory/fibrogenic disorders.

      EMTs: different types for different purposes?

      Upon EMT, epithelial cells assume a more mesenchymal phenotype, characterized by increased cell motility. This possibility does not seem to be restricted to epithelial cells during development or to cancer cells but has been shown to occur in wound healing, tissue regeneration, and organ fibrosis. A classification has been recently proposed to distinguish between these different types of EMT [
      • Kalluri R.
      • Weinberger R.A.
      The basics of epithelial–mesenchymal transition.
      ]: (a) type 1 EMT, associated with implantation, embryo formation, and organ development, is characterized by a program organized to generate diverse cell types that share common mesenchymal phenotypes. In turn, type 1 EMT can generate the primary mesenchyme that has the potential to subsequently undergo a MET to generate secondary epithelia; (b) in type 2 EMT, the program begins as part of a repair-associated event that has been suggested to generate fibroblasts and other related cells in order to reconstruct tissues following trauma and inflammatory injury. Differently from the other types of EMT, type 2 EMT is associated with tissue damage and inflammation and, accordingly, it is abrogated once these causative events cease; (c) type 3 EMT occurs in neoplastic cells that have previously undergone genetic and epigenetic changes, specifically in genes that favor clonal outgrowth and the development of localized tumors. In this context, EMT is elicited by several oncogenic pathways (Src, Ras, integrin, Wnt/beta-catenin and Notch) [
      • Guarino M.
      • Rubino B.
      • Ballabio G.
      The role of epithelial–mesenchymal transition in cancer pathology.
      ]. In particular, Ras-MAPK has been shown to activate two related transcription factors known as Snail and Slug [
      • Zhou B.P.
      • Deng J.
      • Xia W.
      • Xu J.
      • Li Y.M.
      • Gunduz M.
      • et al.
      Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial–mesenchymal transition.
      ]. Both of these proteins are transcriptional repressors of E-cadherin and their expression induces EMT. Activation of the phosphatidylinositol 3’ kinase (PI3K)/AKT axis is also emerging as a central feature of EMT [
      • Larue L.
      • Bellacosa A.
      Epithelial–mesenchymal transition in development and cancer: role of phosphatidylinositol 3′ kinase/AKT pathways.
      ]. Other relevant pathways in EMT observed in the metastatic process include the transcription factor Twist and FOXC2, an important player during embryonic development [
      • Kang Y.
      • Massagué J.
      Epithelial–mesenchymal transitions: twist in development and metastasis.
      ,
      • Hader C.
      • Marlier A.
      • Cantley L.
      Mesenchymal–epithelial transition in epithelial response to injury: the role of Foxc2.
      ], as well as the involvement of microRNAs [
      • Gregory P.A.
      • Bracken C.P.
      • Bert A.G.
      • Goodall G.J.
      MicroRNAs as regulators of epithelial–mesenchymal transition.
      ].
      The possibility that EMT, specifically type 2 EMT, could contribute to hepatic fibrogenesis in chronic liver diseases was suggested by studies that closely followed experimental evidence obtained in other organs and particularly the kidney [
      • Ng Y.Y.
      • Huang T.P.
      • Yang W.C.
      • Chen Z.P.
      • Yang A.H.
      • Mu W.
      • et al.
      Tubular epithelial–myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.
      ,
      • Zeisberg M.
      • Maeshima Y.
      • Mosterman B.
      • Kalluri R.
      Renal fibrosis.
      ,
      • Iwano M.
      • Plieth D.
      • Danoff T.M.
      • Xue C.
      • Okada H.
      • Neilson E.G.
      Evidence that fibroblasts derive from epithelium during tissue fibrosis.
      ]. This biological option strengthened the concept that activated hepatic stellate cells (HSC) were not the only key players in the hepatic fibrogenic process and that other cell types, either hepatic (i.e. portal fibroblast) or extrahepatic (bone marrow-derived cells and circulating fibrocytes) could contribute to this process [
      • Parola M.
      • Pinzani M.
      Hepatic wound repair.
      ,

      Hernandez-Gea V, Friedman SL. Pathogenesis of Liver Fibrosis. Annu Rev Pathol, 2010 [Epub ahead of print] PMID: 21073339.

      ,
      • Pinzani M.
      • Macias-Barragan J.
      Update on the pathophysiology of liver fibrosis.
      ]. The possibility of the rapid mobilization of a large amount of fibrogenic cells by EMT after liver tissue injury made this phenomenon a relevant and suitable target for anti-fibrogenic strategies. Following an initial enthusiasm for the discovery of this novel pathway in fibrogenesis and the publication of a several highly quoted papers, more recent research has started to cast serious doubts about the real relevance of this phenomenon in human fibrogenetic disorders.
      The aim of this article is to analyze and discuss the evidence supporting or confuting this possibility in order to reach reasonable and useful conclusions.

      EMT in fibrogenetic disorders: background issues and “specific” cell markers

      Before addressing specific experimental evidence supporting or not the occurrence of EMT in hepatic fibrogenesis, there are two major pieces of background information that need to be highlighted. The first concerns the almost automatic and uncritical parallels drawn between the morphology and behavior of locomotory or sedentary cells in vitro and in different pathological processes in vivo. In vitro data supporting EMT derive from cells in a highly artificial two-dimensional milieu in which there is no vascular, endocrine, or neurologic contribution. On the other hand, studies performed in three-dimensional matrices also do not recapitulate the real tissue complexity and the results are very much affected by the applied experimental conditions. An additional technical problem concerns the fact that most of the in vitro studies are performed with epithelial cells that in most circumstances belong to transformed cell lines cultured in an abundance of serum factors conditioning the loss of their epithelial features and the progressive acquisition of “mesenchymal” characteristics.
      Within this background, a multitude of studies have shown that epithelial cells, including hepatocytes, when cultured in vitro retain epithelial features including polarity and specific protein expression (i.e. albumin for hepatocytes), but when chronically stimulated with TGF-β1 or serum factors acquire a pattern of gene expression that is somehow typical of myofibroblasts in vivo and in the mesenchyme during development [
      • Fan J.M.
      • Ng Y.Y.
      • Hill P.A.
      • Nikolic-Paterson D.J.
      • Mu W.
      • Atkins R.C.
      • et al.
      Transforming growth factor-beta regulates tubular epithelial–myofibroblast transdifferentiation in vitro.
      ,
      • Gershengorn M.C.
      • Hardikar A.A.
      • Wei C.
      • Geras-Raaka E.
      • Marcus-Samuels B.
      • Raaka B.M.
      Epithelial-to-mesenchymal transition generates proliferative human islet precursor cells.
      ,
      • Kaimori A.
      • Potter J.
      • Kaimori J.Y.
      • Wang C.
      • Mezey E.
      • Koteish A.
      Transforming growth factor-beta1 induces an epithelial-to-mesenchymal transition state in mouse hepatocytes in vitro.
      ,
      • Russ H.A.
      • Ravassard P.
      • Kerr-Conte J.
      • Pattou F.
      • Efrat S.
      Epithelial–mesenchymal transition in cells expanded in vitro from lineage-traced adult human pancreatic beta cells.
      ,
      • Godoy P.
      • Hengstler J.G.
      • Ilkavets I.
      • Meyer C.
      • Bachmann A.
      • Müller A.
      • et al.
      Extracellular matrix modulates sensitivity of hepatocytes to fibroblastoid dedifferentiation and transforming growth factor beta-induced apoptosis.
      ]. These genes are often represented by Slug, Twist, Snail, α-SMA, vimentin, desmin, fibroblast-specific protein 1 (FSP1; also known as S100A4 and MTS-1), and discoidin domain receptor tyrosine kinase 2 (DDR2).
      Some of these markers have been used to identify epithelial cells that are in the midst of undergoing an EMT associated with chronic inflammation. Such cells continue to exhibit epithelial-specific morphology and molecular markers, such as cytokeratin and E-cadherin, but often show the concomitant expression of the fibroblast specific protein-1 (FSP-1) and α-SMA. These aspects have been proposed to represent the intermediate stages of EMT, when epithelial markers continue to be expressed but new mesenchymal markers have already been acquired, and, overall, these observations have led to the notion of the so-called “partial EMT” [
      • Kalluri R.
      • Weinberger R.A.
      The basics of epithelial–mesenchymal transition.
      ].
      A second key issue is the value of different cell markers in defining cell-lineage and, even more relevant, cell function. The most widely used marker identifying myofibroblasts is the cytoskeletal protein α-SMA that is part of the contractile machinery and is involved in cell motility. In adult normal tissue α-SMA expression is mostly restricted to vascular smooth muscle cells, but in most chronic inflammatory and fibrogenic disease states is often found in myofibroblasts of different derivation, and this expression is interpreted as an active involvement of these cells in fibrogenesis (i.e. “activated myofibroblast”). Accordingly, α-SMA cannot be a good lineage marker since its expression is activated by disease states and, in addition, does not denote function since α-SMA expression has little or no role in the synthesis and deposition of fibrillar extracellular matrix (ECM). Regardless of this, there is supportive evidence that epithelial cells express intermediate filaments such as α-SMA and vimentin following tissue injury [
      • Ng Y.Y.
      • Fan J.M.
      • Mu W.
      • Nikolic-Paterson D.J.
      • Yang W.C.
      • Huang T.P.
      • et al.
      Glomerular epithelial–myofibroblast transdifferentiation in the evolution of glomerular crescent formation.
      ,
      • Nitta T.
      • Kim J.S.
      • Mohuczy D.
      • Behrns K.E.
      Murine cirrhosis induces hepatocyte epithelial mesenchymal transition and alterations in survival signaling pathways.
      ]. As it will be further expanded, this is likely due to the engagement of epithelial cells into an adaptive response to injury that includes the activation of several new programs of gene expression leading to the acquisition of a migratory phenotype. An additional popular cell marker is the multifunctional calcium binding protein FSP-1 (member of the S100 family) wrongly defined as “fibroblast-specific”. Indeed, this protein, originally described in malignant epithelial cells as a facilitator of metastatization by regulating the function of contractile proteins [
      • Strutz F.
      • Okada H.
      • Lo C.W.
      • Danoff T.
      • Carone R.L.
      • Tomaszewski J.E.
      • et al.
      Identification and characterization of a fibroblast marker: FSP1.
      ,
      • Schneider M.
      • Hansen J.L.
      • Sheikh S.P.
      S100A4: a common mediator of epithelial–mesenchymal transition, fibrosis and regeneration in diseases?.
      ], is not “fibroblast-specific” and is expressed predominantly by activated macrophages [
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • Duffield J.S.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      ,
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • Hudson T.E.
      • Nowlin B.T.
      • Bonventre J.V.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      ,
      • Le Hir M.
      • Hegyi I.
      • Cueni-Loffing D.
      • Loffing J.
      • Kaissling B.
      Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys.
      ,
      • Inoue T.
      • Plieth D.
      • Venkov C.D.
      • Xu C.
      • Neilson E.G.
      Antibodies against macrophages that overlap in specificity with fibroblasts.
      ]. Therefore, although this protein is expressed in cultured epithelial cells exposed to TGF-β, it cannot be considered as a marker of myofibroblast or epithelial lineage. Finally, the use of other proposed markers such as Snail, Twist, Slug seems to be of limited value since they are readily expressed by epithelial cells, myofibroblasts, and macrophages following tissue injury and, although they may be relevant for cell motility and activation, do not denote cell lineage.
      Overall, in a context of tissue fibrogenesis where type 2 EMT is supposed to occur, the most appropriate marker of myofibroblast function in vivo is the synthesis and deposition of pathological type I collagens [
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • Duffield J.S.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      ,
      • Taura K.
      • Miura K.
      • Iwaisako K.
      • Osterreicher C.H.
      • Kodama Y.
      • Penz-Osterreicher M.
      • et al.
      Hepatocytes do not undergo epithelial–mesenchymal transition in liver fibrosis in mice.
      ]. However, the evaluation of this property is not as easy as it could superficially appear and is mainly hampered by the fact that collagens are predominantly extracellular proteins. Several in vitro studies have shown that different types of epithelial cells, including hepatocytes, activate the Collagen1α1 gene in culture. Similar findings have been obtained in leukocytes and endothelial cells [
      • Pilling D.
      • Fan T.
      • Huang D.
      • Kaul B.
      • Gomer R.H.
      Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts.
      ,
      • Howard B.V.
      • Macarak E.J.
      • Gunson D.
      • Kefalides N.A.
      Characterization of the collagen synthesized by endothelial cells in culture.
      ]. However, all these cell types do not generate Collagen1α1 transcripts in vivo [
      • Lin S.L.
      • Kisseleva T.
      • Brenner D.A.
      • Duffield J.S.
      Pericytes and perivascular fibroblasts are the primary source of collagen-producing cells in obstructive fibrosis of the kidney.
      ,
      • Taura K.
      • Miura K.
      • Iwaisako K.
      • Osterreicher C.H.
      • Kodama Y.
      • Penz-Osterreicher M.
      • et al.
      Hepatocytes do not undergo epithelial–mesenchymal transition in liver fibrosis in mice.
      ]. An additional interpretative problem is related to the fact that some cell types and particularly macrophages are able to internalize ECM with consequent intracellular accumulation of collagen which is not a sign of collagen synthesis. For example, the combination of FSP-1 and intracellular collagen positivity in activated macrophages detected in tissue sections may lead to the wrong identification of epithelial cells undergoing EMT and contributing to fibrogenesis [
      • Pilling D.
      • Fan T.
      • Huang D.
      • Kaul B.
      • Gomer R.H.
      Identification of markers that distinguish monocyte-derived fibrocytes from monocytes, macrophages, and fibroblasts.
      ].
      To summarize these considerations, the expression of several often wrongly defined “myofibroblast-specific” markers is rather irrelevant since the only pathophysiologically relevant feature of fibrogenic cells, irrespective of their origin, is the synthesis and deposition of relevant amounts of fibrillar collagen.

      Developmental rationale for fibrogenic EMT in different tissues: is there a rationale for the liver?

      As already mentioned before, the epic of EMT in the fibrogenic process involving the liver as well as of other organs and tissues was launched by initial evidence obtained in the kidney [
      • Ng Y.Y.
      • Huang T.P.
      • Yang W.C.
      • Chen Z.P.
      • Yang A.H.
      • Mu W.
      • et al.
      Tubular epithelial–myofibroblast transdifferentiation in progressive tubulointerstitial fibrosis in 5/6 nephrectomized rats.
      ,
      • Zeisberg M.
      • Maeshima Y.
      • Mosterman B.
      • Kalluri R.
      Renal fibrosis.
      ,
      • Iwano M.
      • Plieth D.
      • Danoff T.M.
      • Xue C.
      • Okada H.
      • Neilson E.G.
      Evidence that fibroblasts derive from epithelium during tissue fibrosis.
      ]. In this connection, the rationale for type 2 EMT derives from the development paradigm of mesoderm-derived mesenchymal cells developing in epithelial cells according to the process called MET. The entire epithelium of the kidney, including the tubular epithelial cells, is derived from the intermediate mesoderm during the development of the urogenital system via a MET. This represents a peculiar aspect of renal biology: by retaining some imprint of their mesenchymal origins, kidney epithelial cells may be particularly prone to return to this state, via the EMT that occurs in response to inflammatory stress and leads to pathologic fibrosis. While this developmental course is clearly recognized in the kidney, it is not involved in the development of gastrointestinal organs such as the liver and the pancreas, where all the epithelial cells have been shown to derive from the foregut endoderm [
      • Tremblay K.D.
      • Zaret K.S.
      Distinct populations of endoderm cells converge to generate the embryonic liver bud and ventral foregut tissues.
      ,
      • Gu G.
      • Dubauskaite J.
      • Melton D.A.
      Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.
      ] and, in this context, the mesoderm contribution to the development of the liver and pancreas seems to be limited to stromal cells including hepatic and pancreatic stellate cells, respectively [
      • Kolterud A.
      • Wandzioch E.
      • Carlsson L.
      Lhx2 is expressed in the septum transversum mesenchyme that becomes an integral part of the liver and the formation of these cells is independent of functional Lhx2.
      ,
      • Yatoh S.
      • Dodge R.
      • Akashi T.
      • Omer A.
      • Sharma A.
      • Weir G.C.
      • et al.
      Differentiation of affinity-purified human pancreatic duct cells to beta-cells.
      ]. Therefore, a developmental rationale for EMT as a source of myofibroblasts in liver and pancreas is at best very limited.

      The saga of EMT investigation in animal models of liver fibrosis: what have we learned?

      The inadequacy of cell markers for the identification of unequivocal EMT in fibrogenic disorders and the criticisms raised against evidences obtained in vitro prompted major efforts to characterize this potentially relevant phenomenon in animal models of liver fibrosis. The methodological approach used to overcome the limitation of cell surface or cytosolic cell markers is the so-called “lineage tracing” or “fate mapping”, which is based on the concept that once a heritable marker is activated in vivo, it is permanent and not inducible. In other words, the marker will be present in any cell deriving from the labeled cell regardless of differentiation, proliferation, and migration in the context of tissue injury. However, the application of this concept is only achievable with a complex methodology potentially bearing pitfalls and even more complex interpretative issues. For example, the activation of the heritable marker should be a feature only of the cell type to be labeled and should not normally occur in disease. In this context, the most likely candidates to activate a heritable marker are transcription factors that are active developmentally.
      Zeisberg and co-workers were the first to report in vivo evidence for hepatocyte EMT [
      • Zeisberg M.
      • Yang C.
      • Martino M.
      • Duncan M.B.
      • Rieder F.
      • Tanjore H.
      • et al.
      Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition.
      ]. These authors employed a methodology to drive the DNA recombinase Cre under the control of a 2.3 Kb fragment of the albumin promoter/enhancer locus in order to activate the heritable marker LacZ in Rosa26R mice developing CCl4-induced liver fibrosis, so that all hepatocytes and potentially derived cells were irreversibly tagged with β-galactosidase. In this model, recombination was specific for hepatocytes but was, however, incomplete. Regardless of this, the main interpretative issue in this work was related to the sole use of FSP-1 marker to detect scar forming myofibroblastic cells: although it was evident that a detectable percentage of LacZ protein β-gal-positive cells co-expressed FSP-1, there was no conclusive evidence that these cells were effectively myofibroblasts (absence of α-SMA positivity, no evidence of collagen production). This last fundamental issue was subsequently addressed by Taura and co-workers [
      • Taura K.
      • Miura K.
      • Iwaisako K.
      • Osterreicher C.H.
      • Kodama Y.
      • Penz-Osterreicher M.
      • et al.
      Hepatocytes do not undergo epithelial–mesenchymal transition in liver fibrosis in mice.
      ], who employed a similar Alb-Cre transgenic model in the Rosa26R, LacZ expressing reporter mouse with the additional use of the Coll-GFP reporter to simultaneously identify collagen-producing cells and hepatocyte-derived cells in the injured liver. The results of this study suggested that FSP-1 positive cells do not generate collagen in vivo and that collagen producing cells are α-SMA and PDGF-receptor β-positive. These observations, although not related to human chronic liver diseases in terms of both genetic background and injury model strongly argue against the contribution of hepatocyte EMT to liver tissue fibrosis.
      It is a common observation that bile duct basement membranes undergo degradation in fibrogenic liver diseases and that cholangiocytes, the other major hepatic epithelial cell type, assume fibroblast-like, non-cuboidal shapes. Therefore, with the new wave of EMT and liver fibrosis, it became obvious that the next step was to investigate whether or not biliary cells could undergo EMT in chronic liver disease. It is well established that proliferating cholangiocytes within the so-called “ductular reaction” (i.e. “reactive cholangiocytes”), detectable in all types of chronic liver disease, express a variety of pro-fibrogenic growth factors and cytokines (for example PDGF-B chain, TGF-β1, ET 1, MCP-1) and are likely to contribute to fibrosis and inflammation by promoting activation, proliferation, and collagen synthesis in the surrounding pro-fibrogenic cells [
      • Milani S.
      • Herbst H.
      • Schuppan D.
      • Stein H.
      • Surrenti C.
      Transforming growth factors beta 1 and beta 2 are differentially expressed in fibrotic liver disease.
      ,
      • Pinzani M.
      • Milani S.
      • Herbst H.
      • DeFranco R.
      • Grappone C.
      • Gentilini A.
      • et al.
      Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis.
      ,
      • Grappone C.
      • Pinzani M.
      • Parola M.
      • Pellegrini G.
      • Caligiuri A.
      • DeFranco R.
      • et al.
      Expression of platelet-derived growth factor in newly formed cholangiocytes during experimental biliary fibrosis in rats.
      ,
      • Kinnman N.
      • Hultcrantz R.
      • Barbu V.
      • Rey C.
      • Wendum D.
      • Poupon R.
      • et al.
      PDGF-mediated chemoattraction of hepatic stellate cells by bile duct segments in cholestatic liver injury.
      ,
      • Pinzani M.
      • Milani S.
      • De Franco R.
      • Grappone C.
      • Caligiuri A.
      • Gentilini A.
      • et al.
      Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells.
      ,
      • Caligiuri A.
      • Glaser S.
      • Rodgers R.E.
      • Phinizy J.L.
      • Robertson W.
      • Papa E.
      • et al.
      Endothelin-1 inhibits secretin-stimulated ductal secretion by interacting with ETA receptors on large cholangiocytes.
      ,
      • Marra F.
      • DeFranco R.
      • Grappone C.
      • Milani S.
      • Pastacaldi S.
      • Pinzani M.
      • et al.
      Increased expression of monocyte chemotactic protein-1 during active hepatic fibrogenesis: correlation with monocyte infiltration.
      ]. Nevertheless, the possibility of a direct contribution of cholangiocytes to fibrosis via EMT was suggested by the report by Omenetti and co-workers showing in vitro a complete EMT in an immature cholangiocyte cell line treated with activated HSC conditioned medium [
      • Omenetti A.
      • Porrello A.
      • Jung Y.
      • Yang L.
      • Popov Y.
      • Choi S.S.
      • et al.
      Hedgehog signaling regulates epithelial–mesenchymal transition during biliary fibrosis in rodents and humans.
      ]. Concomitantly, other authors reported the co-expression of epithelial and mesenchymal markers in cholangiocytes in human liver obtained from patients with different types of cholestatic disease [
      • Rygiel K.A.
      • Robertson H.
      • Marshall H.L.
      • Pekalski M.
      • Zhao L.
      • Booth T.A.
      • et al.
      Epithelial–mesenchymal transition contributes to portal tract fibrogenesis during human chronic liver disease.
      ,
      • Díaz R.
      • Kim J.W.
      • Hui J.J.
      • Li Z.
      • Swain G.P.
      • Fong K.S.
      • et al.
      Evidence for the epithelial to mesenchymal transition in biliary atresia fibrosis.
      ]. The authenticity of cholangiocyte EMT was recently challenged with the methodology previously used for the investigation of hepatocyte EMT. Along these lines, Scholten and co-workers employed the Cre-Lox technology for lineage tracing and studied several mouse strains expressing Cre under cholangiocyte-, HSC-, or FSP-1-specific promoters in two established models of liver fibrosis, i.e. chronic CCl4 intoxication and common bile duct ligation (BDL) [
      • Scholten D.
      • Osterreicher C.H.
      • Scholten A.
      • Iwaisako K.
      • Gu G.
      • Brenner D.A.
      • et al.
      Genetic labeling does not detect epithelial-to-mesenchymal transition of cholangiocytes in liver fibrosis in mice.
      ]. In this case the fundamental experiment was tracing the fate of cells expressing K19, a bile ductular cell-specific marker, after permanent genetic Cre-mediated labeling of cholangiocytes. The key result of this study was that, although myofibroblast markers were often found in the close proximity of the K19 + progeny of cholangiocytes, the two signals never overlapped in either CCl4 or BDL fibrosis. Based on these and other observations reported in the paper, the authors concluded that cholangiocyte EMT does not occur in their experimental models. Additional data reported in the study by Scholten and co-workers provided parallel evidence that MET, i.e. the transition from myofibroblast to cholangiocyte in the specific experimental context, is also not likely to occur. This latter conclusion is equally of high value since, at some point of this saga, some authors have proposed that, in an ideal perspective of “liver cell panplasticity”, HSC could be transitional cells derived from epithelial cells that have undergone partial EMT [
      • Choi S.S.
      • Diehl A.M.
      Epithelial-to-mesenchymal transitions in the liver.
      ] or even a particular type of oval cell/hepatocyte precursor [
      • Yang L.
      • Jung Y.
      • Omenetti A.
      • Witek R.P.
      • Choi S.
      • Vandongen H.M.
      • et al.
      Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult mouse livers.
      ]. This hypothesis was based, at least in part, on the finding of an adult subpopulation of primary rat HSC expressing the progenitor cell marker CD133 and differentiating into either myofibroblasts or hepatocytes when cultured under different in vitro conditions [
      • Kordes C.
      • Sawitza I.
      • Müller-Marbach A.
      • Ale-Agha N.
      • Keitel V.
      • Klonowski-Stumpe H.
      • et al.
      CD133+ hepatic stellate cells are progenitor cells.
      ]. This latter observation, besides its implications in the issue of MET, raises additional questions on the homogeneity of the HSC population; moreover, the results of the study by Cassiman and co-workers [
      • Cassiman D.
      • Barlow A.
      • Vander Borght S.
      • Libbrecht L.
      • Pachnis V.
      Hepatic stellate cells do not derive from the neural crest.
      ] should also be considered as they clearly showed that HSC do not descend from the neural crest but may derive from multiple sources such as the septum transversum mesenchyme, the endoderm or the mesothelial liver capsule.
      It should be reminded that the methodologies employed in the “lineage tracing” studies bear potential pitfalls. As already highlighted in a recent commentary [
      • Popov Y.
      • Schuppan D.
      Epithelial-to-mesenchymal transition in liver fibrosis: dead or alive?.
      ], the Cre-Lox system is a very elegant tool to demonstrate that lineage conversion does occur but it does not prove conclusively that this does not happen. Going back to the study by Scholten and co-workers [
      • Scholten D.
      • Osterreicher C.H.
      • Scholten A.
      • Iwaisako K.
      • Gu G.
      • Brenner D.A.
      • et al.
      Genetic labeling does not detect epithelial-to-mesenchymal transition of cholangiocytes in liver fibrosis in mice.
      ], since only 40% efficacy of Cre-recombination was achieved, it is possible that only a specific subset of cholangiocytes/ductular cells was traced and, therefore, it is not possible to exclude that EMT may have occurred in the remaining population involved in the ductular reaction characterized by a weak or absent K19 expression and potentially including liver progenitor cells. In order to resolve this important issue, Chu and co-workers undertook lineage tracing studies using Alfp-Cre × Rosa26-yellow fluorescent protein (YFP) mice, a model enabling to track the behavior of virtually all bipotential epithelial progenitors and their progeny in liver injury [

      Chu AS, Diaz R, Hui JJ, Yanger K, Zong Y, Alpini G, et al. Lineage tracing demonstrates no evidence of cholangiocyte epithelial-to-mesenchymal transition in murine models of hepatic fibrosis. Hepatology 2011; Accepted manuscript online: 27 JAN 2011 05:04AM EST|doi:10.1002/hep.24206.

      ]. These authors investigated the co-localization of YFP with the markers such as S100A4, vimentin, α-SMA, or pro-collagen 1α2 in the BDL (2,4, and 8 wks), CCl4 (3 wks), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 2 and 3 wks) models of liver fibrosis. In no case, they found evidence of co-localization although the above “mesenchymal” markers were abundantly present in the peribiliary regions. Therefore, these data add additional evidence against the possible transformation of cholangiocytes and hepatocytes into fibrogenic cells. In any case, it is important to note that the evaluation time-frame in any mouse model is very short when compared to the fibrogenic process occurring in human liver diseases and that mouse models do not display reactive bile ducts with features identical to those present in human disease.
      To conclude this paragraph, it is relevant to delineate the current advancement in injury activated EMT in the kidney, the organ where EMT was first described and where there is indeed the greatest developmental rationale for this process. Using two distinct epithelial cell-specific drivers, two distinct reporters and two different models of renal fibrosis, Humphreys and co-workers found no evidence that epithelial cells become myofibroblasts in vivo [
      • Humphreys B.D.
      • Lin S.L.
      • Kobayashi A.
      • Hudson T.E.
      • Nowlin B.T.
      • Bonventre J.V.
      • et al.
      Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.
      ]. In addition, this study highlighted the possibility that in the kidney all myofibroblasts derive from a discrete population of PDGFRβ+/CD73+ perivascular pericytes that are derived from the metanephric mesenchyme and show similarities with stellate cells of the liver and pancreas [
      • Duffield J.
      Epithelial to mesenchymal transition in injury of solid organs: fact or artifact?.
      ].

      The concept of EMT in organ fibrosis and cancer: need for a more flexible interpretation

      The complexity of EMT and its recognition as a relevant biological phenomenon in organ fibrosis and cancer requires a more flexible and somehow compromising interpretation in order to fit into a logical scheme. Indeed, even in cancer development and metastasis, the occurrence of EMT is currently extensively challenged [
      • Tarin D.
      • Thompson E.W.
      • Newgreen D.F.
      The fallacy of epithelial mesenchymal transition in neoplasia.
      ]. In this context it is argued that disorderly differentiation, loss of cell polarity, loss of lineage specific or tissue specific cytologic features, and acquisition of a migratory/invasive phenotype are typical aspects of carcinoma. In other words, what is called EMT is simply the result of a genetic reprograming of cancer cells aimed at ensuring one of the fundamental aspects of cancer which is cell motility and invasion. The acquisition of cell morphology and the expression of cytoskeletal and surface proteins involved in cell motility typical of mesenchymal cells does not transform a cancer cell into a myofibroblast. Accordingly, EMT in cancer biology is not necessarily seen as a phenomenon where cancer cells differentiate into myofibroblasts to create a tumor-associated fibrotic stroma. This latter is mostly created by the recruitment and activation of local myofibroblasts and circulating fibrocytes in response to soluble factors released by cancer cells and by the disruption of the normal ECM caused by membrane-anchored or secreted metalloproteases in the process of cancer invasion [
      • Räsänen K.
      • Vaheri A.
      Activation of fibroblasts in cancer stroma.
      ,
      • Ota I.
      • Li X.Y.
      • Hu Y.
      • Weiss S.J.
      Induction of a MT1-MMP and MT2-MMP-dependent basement membrane transmigration program in cancer cells by Snail1.
      ].
      In the injury-activated EMT described in chronic fibrogenic disorders, epithelial cells are likely to undergo a similar genetic reprograming which is not caused by genetic mutations typical of cancer but rather due to an adaptive response to injury and to the changes occurring in the milieu of a chronically damaged tissue. As a consequence, epithelial cells acquire features allowing the acquisition of cell motility. When the occurrence of these “transition” features is evaluated with the expression of cell markers classically but unreliably attributed to myofibroblasts, the most likely result is to reach the even more unreliable conclusion that epithelial cells have “cosmetically” transformed into pro-fibrogenic cells. In this framework, the lack of a convincing demonstration of active collagen synthesis remains a critical point supporting the unreliability of this conclusion. Now, a spontaneous question arises: why non-transformed epithelial cells in a context of tissue injury should become motile and “escape” from the original epithelial lining? A possible hypothesis could derive from the so-called “redox-based escape mechanism from death” which has been shown to govern crucial steps of the metastatic process [
      • Pani G.
      • Giannoni E.
      • Galeotti T.
      • Chiarugi P.
      Redox-based escape mechanism from death: the cancer lesson.
      ]. This adaptive response is now recognized to occur also in non-transformed epithelial cells in a context of inflammation, hypoxia, and oxidative stress. Overall, this process can be viewed as an integrated “escape program” from a hostile microenvironment mainly triggered by redox changes but also by the presence of an altered ECM composition and high concentrations of growth factors and cytokines involved in the chronic wound healing reaction. The program is characterized by changes in cell metabolism and cell motility aimed at promoting pro-survival choices and escape from oxidative damage (Fig. 1A). The acquisition of the motile phenotype implies adequate cytoskeletal reorganization with the expression of proteins such as α-SMA and vimentin, the polarization of cytoskeletal structures to the leading edge of movement, integrin clustering and recycling associated with increased affinity for the surrounding ECM components, and the activation of a degradative potential toward these components. This latter feature involves an increased expression and activity of membrane-anchored or secreted metalloproteases, urokinase, heparanase, and matrilysin. In this context, integrin activation through ECM-cell contacts, in addition to being indispensable for cell movement, is also a key factor ensuring resistance to anoikis, a form of programmed cell death which is induced by anchorage-dependent cells detaching from the surrounding ECM. The possible final outcomes of the escape process are basically two (Fig. 1B): (a) the motile epithelial cell reaches a less hostile microenvironment and undergoes a reprograming toward the original lining phenotype (a possibility that, according to the above mentioned “cosmetic” interpretation is viewed as MET), or (b) in the presence of a diffuse and long-lasting hostile milieu, the newly acquired “escaping” features progressively run out and the cell loses the resistance to anoikis with the consequent induction of apoptosis. In both cases there is a clear pathophysiological advantage compared to the fate of non-escaping cells, whose most likely fate is to undergo cell necrosis with further release of reactive oxygen species and other pro-fibrogenic and pro-inflammatory molecules [
      • Friedman S.L.
      Mechanisms of hepatic fibrogenesis.
      ]. Accordingly, what has been defined type 2 EMT could be a process aimed at limiting fibrogenesis rather than being a pro-fibrogenic event.
      Figure thumbnail gr1
      Fig. 1Hypothesis of the “redox-based escape mechanism from death” to explain the acquisition of a migratory phenotype of hepatocytes in a context of tissue injury. (A) Normal polarized lining hepatocytes are exposed to different environmental factors creating a hostile microenvironment. In some hepatocytes, this leads to an epigenetic re-programing leading to polarity disruption, loss of intercellular and ECM adhesions, activation of aerobic glycolysis, and survival pathways. The acquisition of the motile phenotype implies an adequate cytoskeletal reorganization with the expression of proteins such as α-SMA and vimentin, the polarization of cytoskeletal structures to the leading edge of movement and the degradation of basal membrane like-structures through an increased expression and activity of membrane-anchored or secreted metalloproteases, urokinase, heparanase, and matrilysin. Once the cell has “escaped” from the lining, motility is ensured mostly by PI-3K/AKT signaling, integrin clustering, and recycling associated with increased affinity for the surrounding ECM components, as well as the activation of a degradative potential toward these components. In this context, integrin activation through ECM-cell contacts, in addition to being indispensable for cell movement, is also a key factor ensuring resistance to anoikis, a form of programmed cell death which is induced by anchorage-dependent cells detaching from the surrounding ECM. (B) The possible final outcomes of the escape process are basically two: the motile epithelial cell reaches a less hostile microenvironment and undergoes a reprograming toward the original lining phenotype or, in the presence of a diffuse and long-lasting hostile milieu, the newly acquired “escaping” features progressively run out and the cell loses the resistance to anoikis with the consequent induction of apoptosis.
      Figure thumbnail fx1

      Conflict of interest

      The author declared that he does not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

      Acknowledgments

      The author is indebted to Prof. Maurizio Parola, Università di Torino, Turin, Italy and to Dr. Vinicio Carloni, Università di Firenze, Florence, Italy for their expert advice and friendly support in unraveling some the concepts expressed in this article.

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