Gene Expression Profiles During Hepatic Stellate Cell Activation in Culture and In Vivo

Abstract

Background & Aims: Following hepatic injury, hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts and to promote hepatic fibrogenesis. In this study, we determine gene expression changes in 3 different models of HSC activation and investigate whether HSC culture activation reproduces gene expression changes of HSC in vivo activation. Methods: HSCs were isolated by density centrifugation and magnetic antibody cell sorting from normal mice, CCl4-treated mice, and mice that underwent bile duct ligation (BDL). Gene expression was analyzed by microarray and confirmed by polymerase chain reaction and Western blot analysis. Results: Two thousand seventy-three probe sets were differentially expressed in at least 1 of 3 models of HSC activation, including novel genes that encode proinflammatory and antiapoptotic mediators; transcription factors; cell surface receptors; and cytoskeleton components such as CXCL14, survivin, septin 4, osteopontin, PRX1, LMCD1, GPR91, leiomodin, and anillin. BDL- and CCl4-activated HSCs showed highly correlated gene expression patterns, whereas culture activation only partially reproduced the gene expression changes observed during BDL- and CCl4-induced activation. Coculture with Kupffer cells or lipopolysaccharide treatment during culture activation shifted the expression of most examined genes toward the pattern observed during in vivo activation, suggesting a role for these factors in the microenvironment that drives HSC activation. Conclusions: The almost identical HSC gene expression patterns after BDL or CCl4 treatment indicate that HSCs exert similar functions in different types of liver injury. Because culture activation does not properly regulate gene expression in HSCs, in vivo activation should be considered the gold standard for the study of HSC biology. Background & Aims: Following hepatic injury, hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts and to promote hepatic fibrogenesis. In this study, we determine gene expression changes in 3 different models of HSC activation and investigate whether HSC culture activation reproduces gene expression changes of HSC in vivo activation. Methods: HSCs were isolated by density centrifugation and magnetic antibody cell sorting from normal mice, CCl4-treated mice, and mice that underwent bile duct ligation (BDL). Gene expression was analyzed by microarray and confirmed by polymerase chain reaction and Western blot analysis. Results: Two thousand seventy-three probe sets were differentially expressed in at least 1 of 3 models of HSC activation, including novel genes that encode proinflammatory and antiapoptotic mediators; transcription factors; cell surface receptors; and cytoskeleton components such as CXCL14, survivin, septin 4, osteopontin, PRX1, LMCD1, GPR91, leiomodin, and anillin. BDL- and CCl4-activated HSCs showed highly correlated gene expression patterns, whereas culture activation only partially reproduced the gene expression changes observed during BDL- and CCl4-induced activation. Coculture with Kupffer cells or lipopolysaccharide treatment during culture activation shifted the expression of most examined genes toward the pattern observed during in vivo activation, suggesting a role for these factors in the microenvironment that drives HSC activation. Conclusions: The almost identical HSC gene expression patterns after BDL or CCl4 treatment indicate that HSCs exert similar functions in different types of liver injury. Because culture activation does not properly regulate gene expression in HSCs, in vivo activation should be considered the gold standard for the study of HSC biology. See editorial on page 2059.Hepatic stellate cells (HSCs) constitute approximately 8%–14% of cells in the normal liver and are the primary site for retinoid storage in the body.1Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1877) Google Scholar, 2Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4020) Google Scholar Following liver injury, HSCs transdifferentiate from lipocyte-like cells into contractile and highly proliferative myofibroblast-like cells.1Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1877) Google Scholar, 2Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4020) Google Scholar Activation of HSCs is considered a crucial event that promotes increased extracellular matrix (ECM) production and hepatic fibrosis.1Friedman S.L. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury.J Biol Chem. 2000; 275: 2247-2250Crossref PubMed Scopus (1877) Google Scholar, 2Bataller R. Brenner D.A. Liver fibrosis.J Clin Invest. 2005; 115: 209-218Crossref PubMed Scopus (4020) Google Scholar Currently, the molecular signals that are activated during HSC activation are only incompletely understood. Transforming growth factor β (TGF-β) and platelet-derived growth factor (PDGF) are key cytokines that drive HSC activation and proliferation following hepatocellular injury, but other mediators including angiotensin II, leptin, endothelin, insulin, insulin-like growth factor, and lipopolysaccharide (LPS) as well as factors released by Kupffer cells additionally contribute to HSC activation.3Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (490) Google Scholar, 4Saxena N.K. Ikeda K. Rockey D.C. Friedman S.L. Anania F.A. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice.Hepatology. 2002; 35: 762-771Crossref PubMed Scopus (354) Google Scholar, 5Rockey D.C. Chung J.J. Endothelin antagonism in experimental hepatic fibrosis Implications for endothelin in the pathogenesis of wound healing.J Clin Invest. 1996; 98: 1381-1388Crossref PubMed Scopus (183) Google Scholar, 6Svegliati-Baroni G. Ridolfi F. Di Sario A. Casini A. Marucci L. Gaggiotti G. Orlandoni P. Macarri G. Perego L. Benedetti A. Folli F. Insulin and insulin-like growth factor-1 stimulate proliferation and type I collagen accumulation by human hepatic stellate cells: differential effects on signal transduction pathways.Hepatology. 1999; 29: 1743-1751Crossref PubMed Scopus (272) Google Scholar, 7Paik Y.H. Schwabe R.F. Bataller R. Russo M.P. Jobin C. Brenner D.A. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells.Hepatology. 2003; 37: 1043-1055Crossref PubMed Scopus (548) Google Scholar, 8Isayama F. Hines I.N. Kremer M. Milton R.J. Byrd C.L. Perry A.W. McKim S.E. Parsons C. Rippe R.A. Wheeler M.D. LPS signaling enhances hepatic fibrogenesis caused by experimental cholestasis in mice.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1318-G1328Crossref PubMed Scopus (99) Google Scholar, 9Seki E. De Minicis S. Osawa Y. Österreicher C.H. Brenner D.A. Schwabe R.F. TLR4 mediates hepatic fibrosis by down-regulating TGF-β pseudoreceptor BAMBI and enhancing TGF-β signaling.Hepatology. 2006; 44: 225AGoogle Scholar, 10Friedman S.L. Arthur M.J. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors.J Clin Invest. 1989; 84: 1780-1785Crossref PubMed Scopus (349) Google Scholar, 11Rivera C.A. Bradford B.U. Hunt K.J. Adachi Y. Schrum L.W. Koop D.R. Burchardt E.R. Rippe R.A. Thurman R.G. Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G200-G207PubMed Google Scholar, 12Duffield J.S. Forbes S.J. Constandinou C.M. Clay S. Partolina M. Vuthoori S. Wu S. Lang R. Iredale J.P. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair.J Clin Invest. 2005; 115: 56-65Crossref PubMed Scopus (1258) Google Scholar, 13Nieto N. Oxidative-stress and IL-6 mediate the fibrogenic effects of rodent Kupffer cells on stellate cells.Hepatology. 2006; 44: 1487-1501Crossref PubMed Scopus (179) Google Scholar The majority of studies investigate the HSC activation process by culturing purified HSCs on plastic surfaces of cell culture dishes as a surrogate for the molecular events that occur during HSC activation in the injured liver. Culture activation of HSC up-regulates activation markers such as α-smooth muscle actin (α-SMA) and collagen α1(I) and is accompanied by a loss of retinoids. However, it is not known whether culture activation of HSCs can reproduce the underlying changes in gene expression of in vivo HSC activation and whether it represents a suitable model to study HSC activation. Moreover, it remains elusive whether all types of liver injury lead to the activation of a conserved and largely identical genetic program in HSCs or whether HSC activation results in a stimulus-specific activation of HSCs with certain gene expression patterns during one type of hepatic injury and different gene expression patterns in other types of hepatic injury. To answer these questions, we performed microarray studies and compared gene expression profiles between 2 different models of HSC in vivo activation and HSC culture activation.Materials and MethodsCell Isolation, Purification, and CultureMouse HSCs were isolated from normal and fibrotic liver by collagenase-pronase perfusion and subsequent density centrifugation on Nycodenz gradients as described previously.3Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (490) Google Scholar, 14Siegmund S.V. Seki E. Osawa Y. Uchinami H. Cravatt B.F. Schwabe R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver.J Biol Chem. 2006; 281: 10431-10438Crossref PubMed Scopus (88) Google Scholar For each isolation, HSCs from 3 male Balb/c mice were loaded onto 1 Nycodenz gradient.3Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (490) Google Scholar, 14Siegmund S.V. Seki E. Osawa Y. Uchinami H. Cravatt B.F. Schwabe R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver.J Biol Chem. 2006; 281: 10431-10438Crossref PubMed Scopus (88) Google Scholar Kupffer cells were depleted by magnetic antibody sorting (MACS; Miltenyi Biotech, Auburn, CA) using F4/80 (eBioscience, San Diego, CA) and CD11b (Pharmingen, San Diego, CA) antibodies. Purity was tested by retinoid autofluorescence and exceeded 95% in all isolations. For in vivo HSC activation, mice underwent BDL for 2 weeks or 4 intraperitoneal injections of CCl4 (0.5 μL dissolved 1:3 in olive oil, every 3 days). HSCs were cultured for 20 hours (“quiescent HSCs” or “in vivo-activated HSCs”) or for 5 days (“culture-activated HSCs”) in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), glutamine, HEPES buffer, and antibiotics. For some experiments, HSCs were isolated from transgenic mice that express green fluorescent protein (GFP) under the collagen α1(I) promoter.15Magness S.T. Bataller R. Yang L. Brenner D.A. A dual reporter gene transgenic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations.Hepatology. 2004; 40: 1151-1159Crossref PubMed Scopus (204) Google Scholar To judge contamination of HSC isolations with Kupffer cells, endothelial cells, and hepatocytes, primary Kupffer cells, endothelial cells, and hepatocytes were isolated from mouse liver and served as reference value (100%). Kupffer cells were isolated as described above. Hepatocytes and endothelial cells were isolated as described previously.14Siegmund S.V. Seki E. Osawa Y. Uchinami H. Cravatt B.F. Schwabe R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver.J Biol Chem. 2006; 281: 10431-10438Crossref PubMed Scopus (88) Google Scholar, 16Liu S. Premont R.T. Kontos C.D. Zhu S. Rockey D.C. A crucial role for GRK2 in regulation of endothelial cell nitric oxide synthase function in portal hypertension.Nat Med. 2005; 11: 952-958Crossref PubMed Scopus (198) Google Scholar All animal procedures were approved by the Columbia University Institutional Animal Care and Use Committee and meet guidelines of the National Institutes of Health.HSC-Kupffer Cell Coculture and LPS TreatmentKupffer cells were isolated by collagenase-pronase perfusion as described above followed by a 15% Nycodenz gradient and MACS-based positive selection using F4/80- and CD11b-specific antibodies. HSCs and Kupffer cells were cocultured in a noncontact-dependent manner by adding 2.5 × 106 Kupffer cells to a 0.4 μmol/L pore cell culture insert to 1 × 106 HSCs plated in a 6-well plate and exchanging one third of the media daily until RNA extraction at day 5. To investigate the effect of LPS on gene expression, HSCs were activated for 3 days treated with Escherichia coli LPS (100 ng/mL; Sigma-Aldrich Co., St. Louis, MO) for 6 hours, followed by RNA extraction. To investigate the effects of LPS on protein expression, HSCs were isolated, and LPS (100 ng/mL) was added daily to the media during the 5-day culture activation, followed by protein extraction. Each experiment was based on 3 independent HSC and Kupffer cell isolations.Flow Cytometric AnalysisOne day after isolation, HSCs were scraped into phosphate-buffered saline, followed by Fc-receptor blockade, incubation with allophycocyanin-conjugated anti-F4/80, flurorescein isothiocyanate (FITC)-conjugated anti-CD11b, and 2 washes. After gating viable cells, expression of F4/80 and CD11b was analyzed on channels FL1 and FL4 using FACS Calibur (Becton Dickinson, Franklin Lakes, NJ).RNA Isolation, Labeling, Microarray Data Analysis, and Quantitative Polymerase Chain ReactionRNA was extracted by a combination of Trizol and RNeasy columns (Qiagen, Valencia, CA). Microarray analysis was performed using the 1-cycle complementary DNA (cDNA) synthesis kit, 1-cycle in vitro labeling kit, and mouse genome 430 2.0 array gene chips (all Affymetrix, Santa Clara, CA) according to the manufacturer’s instructions. Chips were scanned with a Genechip Scanner 3000 7G (Affymetrix). Data normalization was done by the GCRMA algorithm. Data analysis was performed using Genespring GX 7.3 (Agilent, Palo Alto, CA). All groups (culture-activated HSCs, n = 3; BDL-activated HSCs, n = 3; CCl4-activated HSCs, n = 3) were compared with quiescent HSCs (n = 5). Significant changes in gene expression were identified by unpaired t test and subsequent Benjamini–Hochberg correction using a P value of .05 as criterion of statistical significance. Correlation was investigated by Spearman rank correlation using SPSS 13.0 software for Windows (SPSS Inc., Chicago, IL). Differential gene expression was confirmed by quantitative real-time polymerase chain reaction (qPCR) using commercially available primer-probe sets (Applied Biosystems, Foster City, CA) as described previously.17Schwabe R.F. Sakurai H. IKKβ phosphorylates p65 at S468 in transactivation domain 2.FASEB J. 2005; 19: 1758-1760Crossref PubMed Scopus (65) Google Scholar All samples were DNAse treated before reverse transcription and quantified by comparing threshold cycle values to a serial-dilution standard curve.Western Blot AnalysisWestern blots were performed using 10% to 15% SDS polyacrylamide gels as previously described.14Siegmund S.V. Seki E. Osawa Y. Uchinami H. Cravatt B.F. Schwabe R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver.J Biol Chem. 2006; 281: 10431-10438Crossref PubMed Scopus (88) Google Scholar, 17Schwabe R.F. Sakurai H. IKKβ phosphorylates p65 at S468 in transactivation domain 2.FASEB J. 2005; 19: 1758-1760Crossref PubMed Scopus (65) Google Scholar After blocking, membranes were incubated with antibodies to survivin (Abcam, Cambridge, MA) and to osteopontin and TIMP-1 (both R&D Systems, Minneapolis, MN), followed by incubation with horseradish peroxidase-conjugated secondary antibodies and enhanced chemoluminescence detection.ResultsCombination of Density Centrifugation and MACS Achieves High-Purity HSCsIsolation of HSCs from normal mouse liver usually results in at least 95% purity.3Bataller R. Schwabe R.F. Choi Y.H. Yang L. Paik Y.H. Lindquist J. Qian T. Schoonhoven R. Hagedorn C.H. Lemasters J.J. Brenner D.A. NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis.J Clin Invest. 2003; 112: 1383-1394Crossref PubMed Scopus (490) Google Scholar, 14Siegmund S.V. Seki E. Osawa Y. Uchinami H. Cravatt B.F. Schwabe R.F. Fatty acid amide hydrolase determines anandamide-induced cell death in the liver.J Biol Chem. 2006; 281: 10431-10438Crossref PubMed Scopus (88) Google Scholar In HSCs isolated from bile duct-ligated mice, we detected a high degree of Kupffer cell contamination similar to the levels previously reported for HSC isolation from CCl4-treated rats.18Kristensen D.B. Kawada N. Imamura K. Miyamoto Y. Tateno C. Seki S. Kuroki T. Yoshizato K. Proteome analysis of rat hepatic stellate cells.Hepatology. 2000; 32: 268-277Crossref PubMed Scopus (204) Google Scholar To achieve the high purity required for microarray analysis, we employed MACS-based Kupffer cell depletion after Nycodenz density centrifugation. F4/80- and CD11b-based Kupffer cell depletion strongly decreased the expression levels of macrophage markers F4/80, CD11b, and CD68 (Figure 1A and B) and was thus employed for all HSC isolations used in this study. To exclude contamination with other cell types in HSCs isolated by this method, we additionally performed real-time PCR for the hepatocyte marker albumin and the endothelial cell marker CD31. We found less than 0.05% hepatocyte contamination and less than 0.2% endothelial cell contamination in HSCs isolated from normal or fibrotic livers (Figure 1C and D). Moreover, virtually all cells isolated by this method displayed fluorescent retinoid droplets (Figure 1E). All 3 methods of HSC activation resulted in a phenotype of activated HSCs and up-regulation of collagen in virtually all HSCs as visualized by GFP expression in HSCs isolated from mice that express GFP under the collagen α1(I) promoter (Figure 1E).Figure 1Combination of collagenase-pronase based density centrifugation and MACS achieves high purity of quiescent and activated hepatic stellate cells. (A and B) Hepatic stellate cells were isolated from normal mice and mice that underwent BDL for 2 weeks and were either depleted of CD11b- and F4/80-expressing cells or left untreated. Expression of CD11b and F4/80 was analyzed by flow cytometric analysis (A). Expression of CD68 in quiescent HSCs (“Q”), culture-activated HSCs (“A”), BDL-activated HSCs (“B”), and CCl4-activated HSCs (“C”) was measured by quantitative real-time PCR (B). Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison to Kupffer cells (“KC” = 100%). (C and D) Contamination of Kupffer cell-depleted HSCs with hepatocytes (C) and endothelial cells (D) was measured by quantitative real-time PCR for albumin and CD31, respectively. Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison with primary hepatocytes (“Hep” = 100%) and primary hepatic endothelial cells (“EC” = 100%). (E) To demonstrate purity and activation status, HSCs were isolated from transgenic mice expressing GFP under the collagen α1(I) promoter. GFP and retinoid autofluorescence were checked 1 day after isolation in quiescent HSC and HSC isolated from bile duct-ligated (“BDL”) and CCl4-treated mice and after 5 days in culture-activated HSCs.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Gene Expression Pattern of Culture-Activated HSCs Is Different From That of BDL- and CCl4-Activated HSCsTwo thousand seventy-three probe sets were more than 2-fold up- or down-regulated in at least 1 of the 3 groups of activated HSCs when compared with quiescent HSCs (Supplementary Table 1; see Supplemental Table 1 online at www.gastrojournal.org). Gene expression patterns in all groups showed high similarity between different isolations confirming consistent purity of isolations and microarray quality (Figure 2A). Although BDL- and CCl4-activated HSCs displayed an almost identical pattern of up- and down-regulated genes, gene expression in culture-activated HSCs overlapped only partially with BDL- and CCl4-activated HSCs (Figure 2A). To investigate further the extent to which gene expression differed between culture-activated and in vivo-activated HSCs, we correlated the levels of down- and up-regulation of all 45,000 probe sets between the different models of HSC activation. Although correlation between culture-activated HSC and either BDL- or CCl4-activated HSCs was significant, the level of correlation was relatively weak at r = 0.22 and r = 0.17, respectively (Figure 2B). In contrast, correlation between BDL- and CCl4-activated HSCs was much higher at r = 0.76. Next, we compared up- and down-regulated genes among all groups of activated HSCs. Accordingly, the Venn diagram of all 2073 differentially regulated genes showed that only 56% of up-regulated and 63% of down-regulated genes from culture-activated HSCs were concordantly up- and down-regulated in BDL- and/or CCl4-activated HSCs (Figure 2C). Accordingly, many genes were up- or down-regulated in in vivo-activated HSCs but not in culture-activated HSCs (Supplementary Table 2; see Supplemental Table 2 online at www.gastrojournal.org) and vice versa (Supplementary Table 3; see Supplemental Table 3 online at www.gastrojournal.org). In contrast, BDL- and CCl4-activated HSCs showed a strong overlap, and more than 95% and 99% of probe sets of BDL- and CCl4-activated HSCs, respectively, were concordantly regulated in at least 1 of the 2 other groups of activated HSCs (Figure 2C). Even though some genes were significantly up-regulated in BDL-activated but not in CCl4-activated HSCs and vice versa, only very few genes showed a strong up- or down-regulation in one model of in vivo activation but not the other (Supplementary Tables 4 and 5; see Supplemental Tables 4 and 5 online at www.gastrojournal.org). Pathway analysis revealed that differentially expressed genes were involved in the regulation of proliferation, transcription, and signal transduction, and a smaller percentage in inflammation, cell death, adhesion, cytoskeletal organization, development, and metabolism, but did not show differences between culture-activated and in vivo-activated HSCs using these categories (Supplementary Figure 1; see Supplemental Figure 1 online at www.gastrojournal.org).Figure 2The gene expression pattern of culture-activated HSCs is different from that of BDL- and CCl4-activated HSCs. Hepatic stellate cells were isolated from normal mice and mice that underwent BDL for 2 weeks or 4 CCl4 injections. HSCs were considered culture activated after 5 days of culture in 10% fetal bovine serum on uncoated cell culture dishes. After isolation of mRNA and generation of cRNA, microarray was performed. (A) Shown is a representative graph of 3 separate HSC isolations per group (“1,” “2,” and “3”) containing all genes that were more than 2-fold up-regulated (shown in red) or down-regulated (shown in blue) in at least 1 of the 3 groups of activated HSCs in comparison with quiescent HSCs (t test, P < .05 followed by Benjamini–Hochberg correction). (B) The relative expression of all 45,000 probe sets of the array was correlated among culture-activated HSCs, BDL-activated HSCs, and CCl4-activated HSCs using Spearman rank correlation. (C) The Venn diagram shows overlapping patterns of probe sets that were significantly (P < .05) and at least 2-fold up-regulated and down-regulated in 1 of the 3 groups of activated HSCs. Probe sets that were 2-fold up-regulated or down-regulated in one group were considered down-regulated or up-regulated in the other groups if they showed at least 1.5-fold up-regulation or down-regulation.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of Novel Genes Associated With HSC ActivationTo assess the validity of the obtained data, we analyzed whether typical HSC activation markers were up-regulated in the microarray and additionally examined their expression by quantitative real-time PCR. Using this method, we confirmed the strong up-regulation of α-SMA and collagen α1(I) messenger RNA (mRNA) levels (Figure 3A and Supplementary Table 1; see Supplemental Table 1 online at www.gastrojournal.org). In addition, we discovered differential expression of a large number of genes whose expression has not been associated with HSC activation and confirmed their expression by real-time PCR (Figure 3A). We identified genes involved in proliferation (Ki 67), cell survival (survivin, septin 4), cytoskeletal organization and contraction (plexin C1, anillin, leiomodin), inflammation (CXCL14, osteopontin), ECM organization and degradation (Col8a1, MMP10), and lipid metabolism (cholesterol 25-hydroxylase) as well as transcription factors (PRX1, LMCD1), cell surface receptors (adenosine receptor 2b, GPR91), bone morphogenetic protein BMP5, and neural markers (synaptotagmin, neurotrimin) (Figure 3A and Supplementary Table 1; see Supplemental Table 1 online at www.gastrojournal.org). Although the inhibitor of apoptosis protein (IAP) family member surviving was up-regulated during HSC activation, other IAP members such as c-IAP1, c-IAP2, and XIAP were not up-regulated (Figure 3A). Expression patterns of several of these genes were confirmed by Western blot analysis. The expression pattern of all investigated proteins was similar to our PCR and microarray analysis data, with survivin being up-regulated in both culture- and in vivo-activated HSCs, TIMP-1 being up-regulated in in vivo- but not in culture-activated HSCs, and osteopontin being down-regulated in culture-activated HSCs but not in in vivo-activated HSCs (Figure 3B).Figure 3Identification of previously known and novel differentially regulated genes during HSC activation in vivo and in culture. (A) Expression of differentially regulated genes was confirmed in quiescent HSCs (“Q”), culture-activated HSCs (“A”), BDL-activated HSCs (“B”), and CCl4-activated HSCs (“C”) by qPCR. Results were normalized to 18S and are expressed as fold induction ± standard error of the mean in comparison with quiescent HSCs. Each group of HSC samples consisted of at least 3 different isolations. (B) Expression of selected genes was confirmed by Western blot analysis. Each Western blot is based on HSCs isolated from 3 normal, BDL-, or CCl4-treated mice and is representative of at least 2 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Coculture With Kupffer Cells or Exposure to LPS Corrects the Gene Expression Pattern of Culture-Activated HSCsCulture activation of HSCs lacks the microenvironment that is typical of HSC activation in vivo. The presence of Kupffer cells and exposure to LPS have been demonstrated to be critical for HSC activation and fibrogenesis in vivo.7Paik Y.H. Schwabe R.F. Bataller R. Russo M.P. Jobin C. Brenner D.A. Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells.Hepatology. 2003; 37: 1043-1055Crossref PubMed Scopus (548) Google Scholar, 8Isayama F. Hines I.N. Kremer M. Milton R.J. Byrd C.L. Perry A.W. McKim S.E. Parsons C. Rippe R.A. Wheeler M.D. LPS signaling enhances hepatic fibrogenesis caused by experimental cholestasis in mice.Am J Physiol Gastrointest Liver Physiol. 2006; 290: G1318-G1328Crossref PubMed Scopus (99) Google Scholar, 9Seki E. De Minicis S. Osawa Y. Österreicher C.H. Brenner D.A. Schwabe R.F. TLR4 mediates hepatic fibrosis by down-regulating TGF-β pseudoreceptor BAMBI and enhancing TGF-β signaling.Hepatology. 2006; 44: 225AGoogle Scholar, 10Friedman S.L. Arthur M.J. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors.J Clin Invest. 1989; 84: 1780-1785Crossref PubMed Scopus (349) Google Scholar, 11Rivera C.A. Bradford B.U. Hunt K.J. Adachi Y. Schrum L.W. Koop D.R. Burchardt E.R. Rippe R.A. Thurman R.G. Attenuation of CCl(4)-induced hepatic fibrosis by GdCl(3) treatment or dietary glycine.Am J Physiol Gastrointest Liver P