Reversion of hepatic stellate cell to a quiescent phenotype: From myth to reality?

COMMENTARY ON: Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Kisseleva T, Cong M, Paik Y, Scholten D, Jiang C, Benner C, Iwaisako K, Moore-Morris T, Scott B, Tsukamoto H, Evans SM, Dillmann W, Glass CK, Brenner DA. Proc Natl Acad Sci U S A. 2012 Jun 12;109(24):9448-53. Copyright (2012). Abstract reprinted by permission from the National Academy of Sciences, USA. http://www.ncbi.nlm.nih.gov/pubmed/22566629Abstract: Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl(4)) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl(4)- and alcohol-induced liver fibrosis using Cre-LoxP-based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo. AND Deactivation of hepatic stellate cells during liver fibrosis resolution in mice. Troeger JS, Mederacke I, Gwak GY, Dapito DH, Mu X, Hsu CC, Pradere JP, Friedman RA, Schwabe RF. Gastroenterology. 2012 Oct;143(4):1073-83.e22. Copyright © 2012. Abstract reprinted by permission from the AGA Institute. http://www.ncbi.nlm.nih.gov/pubmed/22750464Abstract: Background & Aims: Activated hepatic stellate cells (HSCs), the main fibrogenic cell type in the liver, undergo apoptosis after cessation of liver injury, which contributes to resolution of fibrosis. In this study, we investigated whether HSC deactivation constitutes an additional mechanism of liver fibrosis resolution. Methods: HSC activation and deactivation were investigated by single-cell PCR and genetic tracking in transgenic mice that expressed a tamoxifen-inducible CreER under control of the endogenous vimentin promoter (Vimentin-CreER). Results: Single-cell quantitative polymerase chain reaction demonstrated activation of almost the entire HSC population in fibrotic livers, and a gradual decrease of HSC activation during fibrosis resolution, indicating deactivation of HSCs. Vimentin-CreER marked activated HSCs, demonstrated by a 6- to 16-fold induction of a membrane-bound green fluorescent protein (mGFP) Cre-reporter after injection of carbon tetrachloride, in liver and isolated HSCs, and a shift in localization of mGFP-marked HSCs from peri-sinusoidal to fibrotic septa. Tracking of mGFP-positive HSCs revealed the persistence of 40%-45% of mGFP expression in livers and isolated HSCs 30-45 days after carbon tetrachloride was no longer administered, despite normalization of fibrogenesis parameters; these findings confirm reversal of HSC activation. After fibrosis resolution, mGFP expression was observed again in desmin-positive peri-sinusoidal HSCs; no mGFP expression was detected in hepatocytes or cholangiocytes, excluding mesenchymal-epithelial transition. Notably, reverted HSCs remained in a primed state, with higher levels of responsiveness to fibrogenic stimuli. Conclusions: In mice, reversal of HSC activation contributes to termination of fibrogenesis during fibrosis resolution, but results in higher responsiveness of reverted HSCs to recurring fibrogenic stimulation. Activation of hepatic stellate cells (HSC) in response to chronic liver injury is a key step in the pathogenesis of liver fibrosis. The activation process is characterized by a phenotypic switch from a quiescent vitamin A-rich to a myofibroblastic fibrogenic phenotype [[1]Lotersztajn S. Julien B. Teixeira-Clerc F. Grenard P. Mallat A. Hepatic fibrosis: molecular mechanisms and drug targets.Ann Rev Pharmacol Toxicol. 2005; 45: 605-628Crossref PubMed Scopus (284) Google Scholar] (Fig. 1). Fibrosis has long been viewed as an irreversible process, but convincing clinical and experimental studies have demonstrated that fibrosis resolution may occur upon eradication of the liver insult [[2]Ellis E.L. Mann D.A. Clinical evidence for the regression of liver fibrosis.J Hepatol. 2012; 56: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar]. Experimental models of fibrosis recovery consistently reported that elimination of activated HSC by apoptosis [[3]Iredale J. Benyon R. Pickering J. McCullen M. Northrop M. Pawley S. et al.Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (940) Google Scholar] or senescence [[4]Krizhanovsky V. Yon M. Dickins R.A. Hearn S. Simon J. Miething C. et al.Senescence of activated stellate cells limits liver fibrosis.Cell. 2008; 134: 657-667Abstract Full Text Full Text PDF PubMed Scopus (1354) Google Scholar] precedes restoration of fibrolytic pathways and regression of fibrosis, suggesting that clearance of activated HSC is a key step in the onset of fibrosis regression. Although culture studies suggested that disappearance of activated HSC may also result from reversal to a quiescent phenotype, whether deactivation may occur in vivo during fibrosis regression remained unsolved. In two recent reports, Kisseleva et al. [[5]Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar] and Troeger et al. [[6]Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar] elegantly address this issue, using a combination of genetic cell fate tracking approach [5Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar, 6Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar], single cell PCR [[6]Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar], and microarray analysis [5Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar, 6Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar]. Both groups used mouse models in which quiescent and activated HSC can be permanently stamped and tracked with a fluorescent label at the onset of fibrosis and during resolution. Kisseleva et al. [[5]Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar] generated a type I collagen promoter-driven Cre-mediated removal of a STOP cassette in a reporter (YFP) mouse strain. Because only activated hepatic myofibroblasts express collagen I, they can be identified by YFP labeling. Liver fibrosis was induced by chronic carbon tetrachloride administration or intragastric alcohol feeding in Cola1(1)Cre-YFP or Cola2(1)Cre-YFP mice, and the fate of YFP-labeled HSC was followed in mice that were allowed to recover for 4–7 weeks. As previously reported, recovery was associated with a marked decrease in the density of α-smooth muscle actin (α-SMA)-positive cells and a parallel normalization of fibrosis parameters. More surprisingly, few apoptotic YFP positive cells were detected (2.6%) whereas hepatic YFP labeling persisted in 50% of cells expressing vitamin A. The low rate of apoptosis compared to previous studies [[3]Iredale J. Benyon R. Pickering J. McCullen M. Northrop M. Pawley S. et al.Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors.J Clin Invest. 1998; 102: 538-549Crossref PubMed Scopus (940) Google Scholar] might be due to the ephemeral nature of apoptosis, but nevertheless, these findings argue for the reversion of a significant number of myofibroblastic HSC to a quiescent state during the recovery phase. Similar findings were obtained by Troeger et al [[6]Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar]. They followed the fate of activated hepatic stellate cells using a bacterial artificial chromososme transgenic mice expressing a tamoxifen-inducible CreER driven by the endogenous promoter for vimentin (an intermediate filament protein highly expressed in myofibroblasts), that were crossed to a fluorescent (mGFP) mouse reporter mice. Activated HSC were tracked in mice exposed to carbon tetrachloride, thioacetamide or bile duct ligation during fibrosis progression and recovery. In addition, FACS sorting and single cell PCR analysis of mGFP labeled cells, isolated at various times after cessation of injury, indicated that the expression of characteristic fibrogenic markers of activated HSC (collagen I, TIMP1, a-SMA, TGF-bR) gradually decreases over time during the recovery period. Intriguingly, comparative gene analysis showed that although reverted HSC reacquire several features of quiescence, they also display specific characteristics with respect to quiescent stellate cells that had never been exposed to a fibrogenic stimulus. Reverted HSC in culture displayed enhanced susceptibility to subsequent activation by TGF-β1 or mitogens [5Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar, 6Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar]. These data indicated that reverted HSC do not fully revert to a quiescent state, but rather retain a preactivated intermediate state (Fig. 1). Interestingly, administration of a second round of carbon tetrachloride after a 6-month recovery period induced more severe fibrosis, compared to mice that received one round of the hepatotoxin, suggesting that reverted HSC may account for more rapid and more severe fibrosis progression upon recurrence of liver injury. Characterization of the mechanisms that maintain HSC in an “intermediate” phenotype is required to fully understand why activated HSC only partially deactivate upon cessation of the insult, but this issue was not addressed in either study. In this respect, whether irreversible changes in the hepatic microenvironment within the injured liver underlie partial phenotype reversion needs to be considered. Indeed, matrix stiffness, cell matrix components such as collagen 1 and integrins, are known to drive and maintain HSC in the activated state [1Lotersztajn S. Julien B. Teixeira-Clerc F. Grenard P. Mallat A. Hepatic fibrosis: molecular mechanisms and drug targets.Ann Rev Pharmacol Toxicol. 2005; 45: 605-628Crossref PubMed Scopus (284) Google Scholar, 7Olsen A.L. Bloomer S.A. Chan E.P. Gaca M.D. Georges P.C. Sackey B. et al.Hepatic stellate cells require a stiff environment for myofibroblastic differentiation.Am J Physiol Gastrointest Liver Physiol. 2011; 301: G110-G118Crossref PubMed Scopus (243) Google Scholar], and it has been reported that irreversible collagen cross-linking persists even after long-term recovery from fibrosis [[8]Popov Y. Sverdlov D.Y. Sharma A.K. Bhaskar K.R. Li S. Freitag T.L. et al.Tissue transglutaminase does not affect fibrotic matrix stability or regression of liver fibrosis in mice.Gastroenterology. 2011; 140: 1642-1652Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar] (Fig. 1). In addition, the role of neighboring cells also warrants further investigation, in particular the liver sinusoidal endothelial cells, which control HSC activation/deactivation [[9]Xie G. Wang X. Wang L. Atkinson R.D. Kanel G.C. Gaarde W.A. et al.Role of differentiation of liver sinusoidal endothelial cells in progression and regression of hepatic fibrosis in rats.Gastroenterology. 2012; 142: e916Abstract Full Text Full Text PDF Scopus (243) Google Scholar]. Altogether, the findings of Kisseleva and Troeger broaden our current view of fibrogenic cell heterogeneity based on their diverse origin, including HSC, portal myofibroblasts, bone marrow-derived fibroblasts and fibrocytes. Indeed, these studies identify hepatic stellate cells as a plastic population that can adopt a wide range of phenotypes, ranging from a fully quiescent to an activated myofibroblastic state, as described for myeloid cells. Nevertheless, a number of issues remain unresolved. In particular, whether the inactivation process is specific to HSC or also affects other liver fibrogenic cells remains to be determined. This is an important point, given the predominant role of specific liver fibrogenic cell populations according to the cause of liver injury, as illustrated by the major contribution of portal myofibroblasts to biliary cirrhosis [[10]Beaussier M. Wendum D. Schiffer E. Dumont S. Rey C. Lienhart A. et al.Prominent contribution of portal mesenchymal cells to liver fibrosis in ischemic and obstructive cholestatic injuries.Lab Invest. 2007; 87: 292-303Crossref PubMed Scopus (129) Google Scholar]. Another major question relates to the characterization of the mechanisms regulating elimination of activated HSC by apoptosis or phenotype reversion, and the functional consequences of the two pathways on fibrosis resolution. In this respect, the study of Kisseleva et al. [[5]Kisseleva T. Cong M. Paik Y. Scholten D. Jiang C. Benner C. et al.Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis.Proc Natl Acad Sci U S A. 2012; 109: 9448-9453Crossref PubMed Scopus (561) Google Scholar] suggests that anti-apoptotic signals such as HSPa1a/b are specifically expressed by HSC undergoing inactivation and participate in the survival of HSCs in culture and in vivo. Although Troger et al. did not confirm these findings [[6]Troeger J.S. Mederacke I. Gwak G.Y. Dapito D.H. Mu X. Hsu C.C. et al.Deactivation of hepatic stellate cells during liver fibrosis resolution in mice.Gastroenterology. 2012; 143: e1022Abstract Full Text Full Text PDF Scopus (360) Google Scholar], these data suggest that the balance between pro and anti-apoptotic proteins may represent one of the key mechanisms that govern the fate of activated HSC. Finally, determining whether the reverted phenotype represents an ultimate state or if inactivated cells can further evolve towards a quiescent phenotype will also be a critical issue to be addressed. These two convergent studies provide compelling experimental evidence supporting in vivo HSC reversion during fibrosis recovery (Fig. 1). The question as to the causal relationship between HSC reversion and fibrosis resolution still remains open as well as to the respective roles of apoptotic vs. reverted HSCs in the recovery process. Determination of the clinical relevance of the current findings is another major issue that might prove challenging, given the current lack of assays available for HSC fate tracking in humans. This work was supported by the INSERM,the Université Paris-Est, and the Agence Nationale de la Recherche. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.