Transcriptional Profiling Reveals Novel Markers of Liver Fibrogenesis

Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression, representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. Serial analysis of gene expression was performed on RNA isolated from quiescent, activated, and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor-binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice, whereas it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs it could be a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver. Activated hepatic stellate cells (HSC) that transdifferentiate to myofibroblasts in the injured liver are responsible for scar formation that leads to fibrosis and eventually cirrhosis. To investigate the gene expression profile during different stages of this process, we performed serial analysis of gene expression, representing a quantitative and qualitative description of all expressed genes. Stellate cells were isolated from human livers and cultured. Serial analysis of gene expression was performed on RNA isolated from quiescent, activated, and transdifferentiated HSC. Comparison of the three resulting transcriptomes showed that less than 5% of all genes changed significantly in expression. Established markers of liver fibrosis showed enhanced expression in accordance with the transdifferentiation process. In addition, induction was seen for several genes not yet recognized to be involved in liver fibrosis, such as insulin-like growth factor-binding proteins (IGFBP) and antagonists of bone morphogenic proteins: follistatin and gremlin. The induction of these genes was validated in vivo in mice developing liver fibrosis. The expression of IGFBPs and gremlin was measurable in the livers of these mice, whereas it was low or undetectable in control mice without liver fibrosis. Since gremlin modulates the activity of bone morphogenic growth factors, it may represent a novel pathway and a target for therapeutic intervention and together with IGFBPs it could be a specific marker of liver fibrosis. In conclusion, the comparison of the three transcriptomes of (activated) stellate cells reveals novel genes involved in fibrogenesis and provides an appreciation of the sequence and timing of the fibrotic process in liver. Hepatic fibrosis is the common response to most chronic liver injuries like viral hepatitis, parasitic infection, metabolic or autoimmune diseases, congenital abnormalities, and drugs or alcohol abuse (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1902) Google Scholar, 2Friedman S.L. J. Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar). It is characterized by increased production of components of the extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; HSC, hepatic stellate cell(s); SAGE, serial analysis of gene expression; IGFBP, insulin-like growth factor-binding protein; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor β; Mdr2(-/-), disrupted Mdr2 P-glycoprotein gene; RT-qPCR, reverse transcription quantitative PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; IGF, insulin-like growth factor; IGFBP-rP, insulin-like growth factor binding protein related protein; BIGH3, transforming growth factor β-induced 68 kDa. like fibril-forming collagens I and III, proteoglycans, fibronectins, and hyaluronic acid (3Gressner A.M. Eur. J. Clin. Chem. Clin. Biochem. 1991; 29: 293-311PubMed Google Scholar). The hepatic stellate cells (HSC) are the primary source of excess ECM accumulation in liver fibrosis. HSC in normal human liver comprise roughly one-third of the nonparenchymal cell population or about 5–8% of total liver cells (4Hautekeete M.L. Geerts A. Virchows. Archiv. 1997; 430: 195-207Crossref PubMed Scopus (269) Google Scholar). HSC are the main storage site for retinoids (5Blomhoff R. Wake K. FASEB J. 1991; 5: 271-277Crossref PubMed Scopus (352) Google Scholar) and are situated in the sinusoid within the subendothelial space of Disse in close contact with the hepatocytes. During the development of liver fibrosis, HSC undergo a process of activation, resulting in a reduced retinoid storage capacity and transdifferentiation to a myofibroblast-like phenotype that is characterized by expression of α-smooth muscle actin (α-SMA). Actually, this myofibroblast-like phenotype is not limited to the liver but also is a prominent feature of fibrosis in other tissues, including pancreas, kidney, lung, and skin (6Serrini G. Gabbiani O. Exp. Cell Res. 1999; 250: 273-283Crossref PubMed Scopus (506) Google Scholar). The HSC activation process is the result of a complex interplay between the different hepatic cell types through cytokines, growth factors, and oxidative stress signals (7Gressner A.M. J. Hepatol. 1995; 22: 28-36Crossref PubMed Google Scholar). The cellular transformation that develops gradually in vivo can be mimicked in vitro by short term culture of HSC on plastic, providing a model to study the intra- and extracellular determinants that regulate the transformation/activation process. The hallmarks of activation include excessive cellular proliferation and an abundant ECM protein production that is not counteracted by increased ECM degradation (8Arthur M.J. Digestion. 1998; 59: 376-380Crossref PubMed Scopus (66) Google Scholar). Another major feature of the activation process is the responsiveness of stellate cells to cytokines, resulting in expression of the platelet-derived growth factor receptor (9Wong L. Yamasaki G. Johnson R.J. Friedman S.L. J. Clin. Invest. 1994; 94: 1563-1569Crossref PubMed Scopus (266) Google Scholar, 10Pinzani M. Gentilini A. Caligiuri A. De Franco R. Pellegrini G. Milani S. Marra F. Gentilini P. Hepatology. 1995; 21: 232-239PubMed Google Scholar) and members of TGF-β receptor family. Platelet-derived growth factor is a major mitogen for stellate cells (11Marra F. Choudhury G.G. Pinzani M. Abboud H.E. Gastroenterology. 1994; 107: 1110-1117Abstract Full Text PDF PubMed Scopus (52) Google Scholar), and TGF-β appears to be the primary fibrogenic cytokine (12Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar, 13Tiggelman A.M. Boers W. Linthorst C. Sala M. Chamuleau R.A. J. Hepatol. 1995; 23: 307-317Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Although several genes play a role in the activation of HSC, it is yet not clear which specific genes are responsible for the initiation and perpetuation of the fibrotic response. To identify genes involved in the transformation of HSC into myofibroblasts, we performed transcriptional profiling during different stages of activation. For this purpose, we have chosen to use the technique of serial analysis of gene expression (SAGE) (14Velculescu V.E. Zhang L. Vogelstein B. Kinzler K.W. Science. 1995; 270: 484-487Crossref PubMed Scopus (3626) Google Scholar), which allows the effective monitoring of tens of thousands of genes without prior knowledge of the genes and their expression. This technology provides both a quantitative and qualitative measurement of gene transcription by any cell type or tissue. Under the auspices of the Cancer Genome Anatomy Project, an analysis of differential gene expression by SAGE in normal and tumor tissues and normal and malignant cell types has been applied (available on the World Wide Web at www.ncbi.nlm.nih.gov/SAGE/and cgap.nci.nih.gov/SAGE/). This project represents over 1.2 × 106 unique SAGE tags from more than 300 libraries of human origin and has been designed to assist in unraveling the molecular basis of cancer. A similar analysis of quiescent and activated HSC might provide into the development of hepatic fibrosis and could be used as a for fibrotic in other tissues (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1902) Google Scholar, 2Friedman S.L. J. Hepatol. 2003; 38: S38-S53Abstract Full Text Full Text PDF PubMed Google Scholar, G. Gabbiani O. Exp. Cell Res. 1999; 250: 273-283Crossref PubMed Scopus (506) Google Scholar). we a gene expression in human hepatic stellate cells in three the quiescent activated and the transdifferentiated the hepatic we have validated the induction of several genes by and in we the induction of these genes in vivo in an model of liver fibrosis. mice C.M. R. Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar), and control mice were with a control The with and were the mice were was and livers were for were performed of the on and were isolated from of normal human liver for or from human liver as (12Casini A. Pinzani M. Milani S. Grappone C. Galli G. Jezequel A.M. Schuppan D. Rotella C.M. Surrenti C. Gastroenterology. 1993; 105: 245-253Abstract Full Text PDF PubMed Google Scholar, A.M. Boers W. Linthorst C. Sala M. Chamuleau R.A. J. Hepatol. 1995; 23: Full Text PDF PubMed Scopus Google Scholar). a with HSC were from other liver nonparenchymal cells by over of The of HSC in these was as by of and of A.M. Boers W. Linthorst C. Sala M. Chamuleau R.A. J. Hepatol. 1995; 23: Full Text PDF PubMed Scopus Google Scholar). RNA was for the of the SAGE of quiescent HSC. To activated stellate these cells were for on culture in with and To transdifferentiated hepatic stellate cells or myofibroblasts, the cells were phenotype was by of and α-smooth muscle actin with from and was a and at the RNA was SAGE SAGE libraries were the SAGE (14Velculescu V.E. Zhang L. Vogelstein B. Kinzler K.W. Science. 1995; 270: 484-487Crossref PubMed Scopus (3626) Google Scholar). including of the SAGE technique can be on the World Wide Web at In of was isolated from of total RNA from human hepatic stellate cells or transdifferentiated human hepatic stellate and were performed The was with for at and into both were to different both the both were with for at of the both were and The resulting was with of and were with and on a to the The were to between and were isolated from an into the site of and into cells between and were with and with for the of and was on an a and SAGE analysis was performed a World Wide in the of R. F. M. 2000; PubMed Scopus Google Scholar), for of tags from sequence and on the human gene The three SAGE libraries were F. PubMed Scopus Google Scholar), and were as with less than To study and expression, the was used on the World Wide Web at or The normal liver was from these Web at analysis on stages of stellate cell activation was performed on for the SAGE analysis with The used are in To comparison with the SAGE expression were to the of of used for quantitative of from human stellate cells and in a to in the RNA was isolated from livers and to reverse the of several fibrotic markers was with the used are in To for in reverse transcription all were to the of of used for quantitative of from protein in a SAGE in Hepatic Stellate for the three stages of transdifferentiation is in tags represent the of tags for The unique tags represent the of the and tags in the total In unique identify a to and more tags can identify the unique tags were less than that the expression of the genes recognized by these tags is a of the tags was more than in quiescent, in activated, and in the The of the tags was of the of the in the three stages of transdifferentiation of hepatic stellate cells to myofibroblasts as by serial analysis of gene to unique to unique to unique to unique in a of SAGE of is the of the isolated HSC. of the SAGE markers for or The of tags for at this any of this on the be analysis of the three SAGE libraries that was seen for more than of the unique tags This that during activation of human stellate cells and transformation into less than 5% of all expressed genes a in expression. In the of the quiescent human stellate genes were with a significantly expression with both other representing different genes were in isolated stellate cells in both stages that the expression of these genes to be during stellate cell activation. genes are unique for quiescent stellate these tags were not in a SAGE of a normal liver. For the expression to more during stellate cell activation. The of tags of these genes was reduced in activated cells with that in quiescent whereas tags were in the transdifferentiated cells genes of which the expression is during activation to be as markers of quiescent stellate of may play a role in the in stellate In the activated stellate genes were significantly expressed with the quiescent genes were in the quiescent that their expression is to stellate cell activation genes were significantly more expressed in the activated HSC than in the were in the myofibroblasts In the activated genes were which were both in and in transdifferentiated stellate cells increased expression during the that could play a role in the transdifferentiation of stellate tags in the activated stellate cells were not in a SAGE of a normal that are specific for this cell in the HSC, expression of genes was significantly different from that in both other tags were in the the have a tags in these cells were not in a SAGE of a normal liver. representing different genes were not expressed in quiescent stellate cells but a low expression in the activated stellate cells as in The of these genes that may have a role in the activation of these of these tags were not in normal the of hepatic genes expressed in the myofibroblasts could play a role in these transdifferentiated stellate of of SAGE we the expression of markers of activated stellate including involved in matrix like and inhibitor inhibitor type and their and genes for proteins like actin and and α-smooth muscle in the expression of most of the markers of fibrosis activation the matrix and and their and whereas not a in expression. The in the myofibroblasts of an involved in the of is in with the increased matrix production in fibrotic liver. In a SAGE of a normal human liver of total of which are the expression of all these genes was into that a of from total liver be from stellate this of tags that the expression of these genes is low in stellate cells in normal liver. The expression of GAPDH, which as a gene is used as a is expression of and markers for transdifferentiation of hepatic stellate cells to to of tags I I to of tags in a of HSC of which the expression was different and which could be as markers of HSC transformation are in is the in expression of several insulin-like growth factor-binding proteins (IGFBP) and proteins and were all during transformation of HSC, and the expression of in the hepatic myofibroblasts is as showed the expression in the of the transdifferentiation process. IGFBPs a low expression that not significantly between the three stages or were the and expression of novel markers for transdifferentiation of hepatic stellate cells to to of tags 68 to of tags in a genes that are and to be of are members of the TGF-β gremlin and is not in normal stellate whereas in myofibroblasts, tags were This induction a role of this of the in stellate cell activation. genes are during the transdifferentiation 68 and The is not in the SAGE libraries of quiescent and activated HSC. To identify markers for liver fibrosis, we SAGE for these novel genes with a SAGE of a normal human liver. In this tags were for several markers and including and that these proteins are specific for (activated) stellate cells and could be markers for liver fibrosis. of SAGE by genes three genes that are markers of HSC activation, and genes that could be as for by analysis the The of the SAGE are in the expression as in the three SAGE libraries from quiescent HSC, activated HSC, and transdifferentiated HSC, To be to SAGE with the both are to the expression of SAGE that the expression is during stellate cell activation, this in a of the The expression of and was in the hepatic The by and SAGE were For all markers and the expression by was with that by expression profile of and novel markers for transdifferentiation of hepatic stellate cells to myofibroblasts as by quantitative the stages of was isolated from the to and by quantitative were in with the The expression of the different genes is to the expression of the gene The genes were as markers of α-SMA, and novel markers of the genes were follistatin and in of of novel markers of liver fibrosis by SAGE were in isolated from human stellate cells and activated in To investigate the of these novel markers in we to study their induction in an model for fibrosis. the model that develops a fibrotic liver a C.M. R. Gastroenterology. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis that of all mice fibrosis. In the liver of type mice the was To the development of fibrosis in these we and as markers of liver fibrosis. In addition, we the induction of three novel and The expression of all genes was by in total liver The expression of was in the livers of the mice The expression of was not in normal but in the fibrotic a but clear was of showed that in the fibrotic liver this gene was expressed at about of S. The three novel markers were in the fibrotic For novel and we were to the of induction in fibrotic which were and and gremlin expression was in all but of the normal whereas a clear was of from the fibrotic livers to the low or expression in normal liver not of the of induction of gremlin. we that in the fibrotic about of gremlin were of S. The of these three genes in vivo that as novel markers for hepatic The term is used to the and of expressed by a population of specific cells V.E. Zhang L. W. Vogelstein J. P. Vogelstein B. Kinzler K.W. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The of this study was to such a profile for hepatic stellate cells during and in vitro transformation from quiescent to activated transdifferentiated liver For this purpose, we use of RNA from human HSC in and The resulting expression more than genes and a and of the genes involved in the activation process. the to genes of which the expression It that this genes that play an role in stellate cell activation. of the of the activated HSC a cell that is involved in of components of the extracellular matrix as in for transformation from quiescent HSC to activated myofibroblasts are α-smooth muscle actin and as by G. S. 59: Scopus Google Scholar, Gabbiani G. Friedman S.L. J. Google Scholar). This is in accordance with the expression of these genes in the of activated human HSC. the expression of genes involved in I III, in the of activated stellate cells is as During fibrosis, the and of these fibril-forming collagens is increased with that of Schuppan D. G. Gastroenterology. Full Text PDF PubMed Google Scholar). The expression of an involved in is also in accordance with an enhanced formation of the the expression of of and that matrix degradation be The enhanced expression of result in the of to a role in the degradation of collagens and the induction of inhibitor type during transdifferentiation that matrix degradation be inhibitor type the activation of and as such the of into The induction of these three result in a of the fibril-forming collagens and I M.J. R. G. Hepatology. PubMed Google Scholar) and to an increased of these matrix components of the expression of is low in the liver of the of of S. This low expression to with the seen in the of HSC for This that of the stellate cells in the fibrotic liver have not yet transdifferentiated into In this it is that SAGE that HSC for in an activated these cells for a enhanced the expression of the genes. A novel in SAGE is the expression of insulin-like growth factor-binding proteins stellate cell activation, in the IGFBPs and In the proliferation of HSC is enhanced by S. K. A.M. 1999; PubMed Scopus Google Scholar, G. F. A. A. L. G. P. G. L. A. F. Hepatology. 1999; 29: PubMed Scopus Google Scholar). In production of by HSC has to liver fibrosis M. R.J. M. J. B. J. A. C. B. J. J. Hepatol. Full Text Full Text PDF PubMed Scopus Google Scholar). to with liver S. S. C.M. A. J. PubMed Scopus Google Scholar). Since IGFBPs and the induction of these binding proteins may fibrosis. IGFBPs have been which in molecular and binding for and 1999; PubMed Scopus Google Scholar). 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The of and in the SAGE that both are markers for stellate cell activation. both specific for stellate markers for liver fibrosis. in vivo in the mice the of both the development of liver fibrosis, their as a novel in vivo Another is that genes for transforming growth factor gremlin and are in transdifferentiated are not in the SAGE of a normal human liver and may be novel and specific markers for fibrosis both are may be as markers of is a acid of the binding to bone morphogenic gremlin their activity and as such a role in growth and during development D. 2003; PubMed Scopus Google Scholar). increased expression of this factor was also in several of M. R. Exp. PubMed Scopus Google Scholar). showed induction of gremlin in activated human stellate cells and in the fibrotic livers of bone morphogenic the development of fibrosis and may fibrosis in the M. C. F. R. J. 2003; PubMed Scopus Google Scholar). This that induction of gremlin may have a that of gremlin expression could be for from liver or fibrosis. but can also several bone morphogenic with a It has been that follistatin is both in with and in with S. J. PubMed Scopus Google Scholar). A was to hepatic stellate cells the expression of and W. PubMed Scopus Google Scholar). In that follistatin not the of A but also the of TGF-β on the expression of type I follistatin of from HSC. In that the increased expression of follistatin could have an on stellate cell activation. A showed that follistatin reduced fibrosis in with not a in HSC proliferation but also reduced of with S. J. W. J. PubMed Scopus Google Scholar). increased expression of genes for proteins and TGF-β was in transdifferentiated In normal expression of these genes is not or at a low has been of an cell with TGF-β and has been with the role of and protein is in the gene are with and in mice, expression of is in the of tissues all of the development stages M. S. S. D. K. C. M. Biochem. Res. 2000; PubMed Scopus (52) Google Scholar). also as was isolated as a gene in cells and to the family. at and that role as an in cellular cells of like and M. K. Cell PubMed Scopus Google Scholar). In this the expression of in the transdifferentiated HSC is also is an of the TGF-β pathway involved in of TGF-β from the TGF-β to the of transcriptional A. J. PubMed Scopus Google Scholar). The induction of these may also have a role in the transdifferentiation of stellate In conclusion, we SAGE libraries of human hepatic stellate cells in three of activation and the gene expression In to proteins to be in hepatic fibrosis, a of were that were not yet with HSC we showed that the induction of both and novel also in The of these novel may not provide into the development of liver fibrosis but may also markers for liver the comparison of the transcriptomes of quiescent, activated HSC and transdifferentiated myofibroblasts provides an appreciation of the sequence and timing of the fibrotic process in D. Geerts and P. for with