- Email: [email protected]
325 Endotoxin-induced hsc activation involves pdgfr and egfr recruitment
04C. Molecular and cellular biology these liposomes, both M6P and scavenger receptors are involved. Targeting anti-fibrotic drugs to the cell types, which have regulatory functions in the fibrotic process, may offer a powerful strategy for therapeutic intervention.
['3"-~ "I'VVO TYPES OF HISTOLOGICALLY N O R M A L LIVER CAN HAVE MARKED DIFFERENCE IN GENE EXPRESSION PROFILES: IMPORTANCE OF AN ADEQUATE NORMAL TISSUE CONTROL IN GENE EXPRESSION STUDIES T. Asselahl'2, I. Bieche 2, I. Laurendeau 2, V. Paradis 3, D. Saadoun 1, D. Valla 1, M. Vidaud 2'4, R Marcellin 1. 1Service d'H@atologie &
INSERM CRB3, University of Paris VIL AP-HP H@ital Beaujon, Clichy, France," 2Laboratoire de Gdndtique Moldculaire, EA 361& Facultd de Pharmacie, Paris' K France," 3Service d 'Anatomie Pathologique, H@ital Beaujon, 4Service de Bioehimie, H@ital Beaujon, France Background and Aims: Gene expression profiling technologies allow the
analysis of gene networks whose expression is associated with specific pathological conditions in comparison with normal tissue. We made the hypothesis that histological normal tissue obtained in 2 different conditions (i.e; percutaneous or surgical liver biopsies), usually used as normal controls in gene expression studies, could have different liver gene expression profiles. Patients and Methods: Group A: Surgical liver biopsies, with general anesthesia, of non-tumoral liver (14 patients): during surgery for liver metastasis of colorectal cancer or for liver benign tumors. Group B: Percutaneous liver biopsies with local anesthesia (14 patients) obtained from patients with mild elevation of serum alanine aminotransferase (ALT) in whom all causes of liver disease were ruled out. All these 28 (group A&B) liver tissue specimens were histologically normal (i.e. absence of inflammation, fibrosis and pathological pattern). We used real-time quantitative RT-PCR assays to compare the mRNA expression of 240 selected genes in these 2 groups. Results: The expression of 29 (12%) of the 240 genes was significantly different between surgical (A) and percutaneous (B) pools specimens; 26 genes were up-regulated and 3 were down-regulated (pool A versus pool B). The most notable changes in gene expression mainly occurred in the inflammatory response genes family. The 7 most discriminatory up-regulated genes in individual samples in A in comparison with B were: PTGS2, THBS1, PAIl, CXCR4, JUN, FOS, ILS. The gene most discriminatory down-regulated was IHH. For example, we observed a higher or lower expression of a specific gene (i.e ILS) in HCV infection, if comparison was done with histologically normal controls obtained percutaneously or surgically, respectively. Conclusion: Our study has demonstrated that histologically normal liver tissue obtained in 2 different conditions (i.e; percutaneous or surgical liver biopsies) have marked difference in gene expression profile although all specimens were histologically normal. Therefore, this study outlines the importance of an adequate selection of histologically normal controls to prevent aberrant results.
[•]
REGULATION OF S U P P R E S S O R OF CYTOKINE SIGNALING 3 m R N A STABILITY BY TNF-~ INVOLVES ACTIVATION OF THE M K K 6 / P 3 8 M A P K CASCADE
C. Ehlting 1, F. Schapel2, E.D. BrenndSrfer 1, R.J. Matthes 1, P.C. Heinrich 2, D. Hfiussinger1, J.G. Bode 1. 1Department of Gastroenterology,
Hepatology and Infectiology, Heinrieh-Heine University, Diisseldorf Germany," :Institute of Biochemistry, University Hospital of the RWTH Aachen, Aaehen, Germany Background and Aims: The potential of some pro-inflammatory mediators to inhibit gpl30 dependent signaling by enhancing SOCS3 expression in macrophages, such as liver macrophages, represents an important molecular switch to abrogate signals with anti-inflammatory properties
(e) HSCs and fibrosis
$125
in order to perpetuate the inflammatory processes. Dis-balanced pro- and anti-inflammatory activities are causative for many inflammatory diseases. Hence, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by pro-inflammatory mediators. In this study we investigate SOCS3 expression initiated by the pro-inflammatory cytokine TNF-0~. Methods: The following methods were used: cell culture, reporter-gene assay, transfection, northern and western blot analysis. Results: In contrast to IL-6, TNF-c~ increases SOCS3-expression by stabilizing SOCS3 mRNA. Activation of the MKK6/p38MAPK-cascade is required for TNF-c~-mediated stabilization of SOCS3 mRNA and enhanced SOCS3 protein expression. Moreover, destabilization of SOCS3 mRNA by the zink-finger protein tristetraprolin and therefore inhibition of SOCS3protein expression is antagonized by activation of the MKK6/p38MAPK pathway. Tristetraprolin acts through an AU rich element containing region within the 31 untranslated region of the SOCS3 mRNA. Conclusions: In contrast to IL-6, TNF-~ regulates SOCS3 expression in macrophages on the level of mRNA stability employing activation of the MKK6/p38MAPK cascade thereby inhibiting the mRNA destabilizing activity of TTE
~ ' - ~ ENDOTOXIN-INDUCED HSC ACTIVATION INVOLVES PDGFR A N D EGFR RECRUITMENT R Brun 1'2, D. Martines 2, M. Scarpa 1'2, G. Pah~1, I. Castagliuolo 1.
1Department of Histology, Microbiology and Medical Bioteehnologies, 2Department of Gastroenterological Sciences, University of Padua, Padua, Italy Background and aims: Recent evidence has suggested that bacterial cell wall products can induce a pro-inflammatory and pro-fibrogenic phenotype in hepatic stellate cells (HSCs), a key cell population implicated in the pathogen:sis and progression of liver diseases. Endotoxins directly bind to HSCs through specific membrane receptors, namely CD14, TLR4 and TLR2, activating complex signal cascade pathways, involving ERK1, and leading to pro-inflammatory genes transcription. Since sequential phosphorylation of Raf-I/MEK/ERK is also elicited by activation of PDGFR-[J and EGFR, protein kinase receptors involved in cell proliferation and fibrosis, in this study we investigated whether PDGFR-[J and EGFR are involved in HSCs response to endotoxins. Methods: HSCs were isolated from Balb/c mice and used at passage two. Serum starved cells were exposed to lipopolysaccharide (LPS) or lipoteichoic acid (LTA) 10 gg/ml for 0.5 24 hrs to assess pro-inflammatory cytokine release (MCP1 and IL-6) by ELISA and ERK1 phosphorylation by immunoprecipitation and Western Blot. Results: Following LPS and LTA exposure, HSCs secreted significant amounts of IL-6 (10.9• and 2.9• respectively) and MCP-1 (9.7• and 5.6• ng/ml, respectively). However, pretreatment of HSCs with AG1295, a specific PDGFR-[J inhibitor or AG835, an EGFR inhibitor, caused a significant decrease of LPS- and LTA-induced IL6 and MCP1 release (P <0.05). In addition, simultaneous PDGFR-[J and EGFR inhibition with AG1478 (200 gM), further decreased IL-6 and MCP1 release following LPS and LTA stimulation (P <0.02). To asses whether receptor clustering was involved in EGFR and PDGFR-[J activation following HSCs exposure to endotoxins, we pre-treated HSCs with nystatin (60 gg/ml), a lipid raft disrupting agent. Indeed, nystatin treatment significantly decreased IL-6 and MCP1 secretion following LPS and LTA stimulation (P < 0.02). Furthermore, by immunoprecipitation and Western blot analysis we showed that ERK1 phosphorylation in HSCs exposed to LPS or LTA was significantly reduced by AG1478 or nystatin pre-treatment. Conclusions: We suggest that endotoxin induced HSCs pro-inflammatory phenotype involves EGFR and PDGFR-[J signalling and organization in supramolecular structures (signalling platforms). We speculate that signalling platforms involving growth factor receptors may represent an
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attractive target for therapy in liver diseases characterized by excessive fibrotic tissue deposition.
F•
PPAR-y L I G A N D PIOGLITAZONE D O E S NOT P R E V E N T HEPATIC FIBROSIS IN MICE
A. Da Silva Morais, J. Abarca-Quinones, P. Stfirkel, Y. Horsmans, I.A. Leclercq. Laboratoire de Gastro-Ent~rologie, Universit~ Catholique
de Louvain (UCL), Brussels', Belgium Background and Aims: Hepatic fibrosis results from activation of quiescent HSC into extracellular matrix producing, proliferative and contractile myofibroblast-like cells. Peroxisome proliferator-activated receptor gamma (PPAR-y) has been suggested to play a key role in the control of HSC activation. Indeed, in rats, PPAR-y expression is depleted in activated HSC and PPAR-y ligands inhibit the activation of HSC as well as prevent hepatic fibrosis in vivo. Also, forced expression of PPAR-y into activated HSC restores the quiescent phenotype. Here we evaluate the impact of PPAR-y agonist Pioglitazone (PGZ) on hepatic fibrogenesis in mice both in vitro and in vivo. Methods: Primary HSCs from Balb/C mice were cultured, with or without PGZ, on plastic dishes. Collagen Ial mRNA, R-smooth-muscle actin (aSMA) and desmin protein expression were measured to assess activation. PPAR-y mRNA and protein expression as well as the expression of the PPAR-y regulated genes CD36 and aP2 were evaluated to determine the biological effect of PGZ treatment. In in vivo experiments, mice were divided in 3 groups treated for 4 weeks with vehicle, CC14 (IR 400 L 3• or CC14+PGZ as a 0.01% PGZ-supplemented diet (wt/wt). Results: In vitro, mRNA expression of PPAR-~ was stable in HSC cultured up to day 9. However, PPAR-? protein was only found in quiescent and early activated HSC (up to 5 days) there after, protein expression decreased dramatically. Exposure of HSC to PGZ from day 3 (at a time when PPAR-y is expressed) to day 7 maintained the expression of PPAR-y ?and stimulated the expression of PPAR-y-regulated gene CD36, but did not alter induction of collagen Ial mRNA or aSMA. In vivo, PGZ treatment induced adiponectin, CD36 and aP2 mRNA in adipose tissue and increased serum adiponectin concentrations, but PGZ treatment did not decrease the severity of hepatic fibrosis induced by CC14. Conclusion: Our data demonstrate that, although having anti-fibrotic properties in rats, PPAR-? stimulation by PGZ does not prevent activation of HSC in vitro, nor hepatic fibrogenesis in vivo in mice. Direct antifibrotic effect of such substances remains to be demonstrated in humans.
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O P I O I D D E P E N D E N T M O D U L A T I O N OF LIVER FIBROGENESIS: A ROLE F O R HEPATIC R E D O X STATE
M.R. Ebrahimkhanil., S. Kiani 2, T. Kendall 3, S.M. Tavangar4, A. Shariftabrizi 2, E Oakley 3, A.R. Mani 1, A.R. Dehpom2, D.A. Mann 3, K.R Moore 1. 1The UCL Institute of Hepatology, Department of Medicine,
Royal Free and University College Medical School, University College London, London, UK," 2Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran," SLiver Group, Southampton General Hospital, University of Southampton, Southampton, UK,"4Department of Pathology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Background: Opioid peptides are known to decrease hepatic GSH synthesis, and induce liver injury. Increased endogenous opioids are well recognised in patients and animals with liver disease. We have recently shown that the opioid antagonist naltrexone decreases plasma transaminases in animals with biliary obstruction. The aim of this study was to investigate the role of opioids in liver fibrogenesis and its underlying mechanisms. Methods: Liver fibrosis was assessed using sirius red staining and hepatic hydroxyproline level in bile duct ligated (BDL) or sham operated rats given either naltrexone or saline for 28 days. Liver matrix metalloproteinase-2
(MMP-2) activity, a smooth muscle actin (a-SMA) and CD45 (leukocyte common antigen) immunostaining were performed in liver sections. The redox state of the liver was assessed by measurement of hepatic GSH/GSSG and S-nitrosothiols by reductive chemiluminescense-based assay and esterified F2-isoprostane concentration by gas chromatography mass spectrophotometry. To assess the effects of opioids on hepatic stellate cells (HSC), different subtypes of opioid receptors were characterized in cultured HSC by reverse-transcription polymerase chain reaction (RT-PCR), and the effects of selective opioid agonists or antagonists on the cellular proliferation of HSC in culture were investigated using [3H] thymidine incorporation assay. Results: Bile duct ligation led to extensive hepatic fibrosis and opioid receptor blockade significantly attenuated hepatic collagen accumulation and MMP-2 activity in BDL rats (P<0.01). a-SMA staining of liver sections demonstrated reduction in the number of activated HSC after naltrexone treatment in animals with cirrhosis (P <0.05). Activated rat HSC showed a weak expression of (31-opioid receptor mRNA. However, none of the selective opioid-receptor agonists or antagonists had any effect on HSC proliferation in vitro. The development of biliary cirrhosis was associated with an altered redox state in the liver with decreased hepatic GSH/GSSG, and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalized by treatment with naltrexone respectively. Leukocyte infiltration assessed by CD45 immunostaining and liver F2-isoprostane concentration were unaffected by chronic opioid receptor blockade in BDL rats. Conclusions: These data suggest that endogenous opioids contribute to liver fibrogenesis by altering the hepatic redox state and the regulation of redox sensitive genes.
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R E D U C E D CHOLESTATIC LIVER I N J U R Y IN MICE DELETED F O R INSULIN-LIKE G R O W T H FACTOR 1 R E C E P T O R IN HEPATOCYTES
A. Cadoret 1, C. Rey 1, D. Wendum2, M. Holzenberger 3, C. Housset 1.
11NSERM U680, Facultd de Mddecine St-Antoine, Paris, France," :Service d 'Anatomopathologie, Hdpital St-Antoine, Paris, France," 3INSERM U515, Hdpital St-Antoine, Paris', France Background and Aim: In all types of liver injury, there is a complex balance between hepatocyte cell death, regenerative and fibrogenic responses. Activation of the IGF-1R signaling pathway contributes to the proliferation of hepatocytes during post-hepatectomy liver regeneration. The aim of this study was to examine the role of IGF signaling on other aspects of liver tissue repair, i.e. hepatocyte cell death and fibrogenesis, using a conditional knockout mouse in which IGF-1R is deleted in hepatocytes. Methods: To specifically invalidate the IGF-1R in hepatocytes, mice homozygously floxed on IGF-1R alleles were bred with mice that expressed the Cre recombinase under the control of albumin promoter and a-fetoprotein enhancer (LIGFREKO mice). LIGFREKO mice and control littermates underwent bile duct ligation (BDL) and were investigated 3 days later. Serum concentrations of transaminases (ALAT/ASAT) and of bile acids were measured. Histological analysis was performed on paraffine embedded, hematoxykin-eosin-stained liver sections and hepatocyte necrotic areas was measured by morphometric analysis. Hepatocyte proliferation was assessed by Ki67 immunolabeling, mRNA expression of R-smooth muscle actin (a-SMA), collagen I and TGF-~I was quantified by real-time RT-PCR using Light Cycler technique. Results: No difference in ALAT/ASAT nor in bile acid concentrations was found in comparing LIGFREKO mice with controls. Moreover, hepatocytes proliferation was similarly induced in both 3 days-BDL LIGFREKO and control mice. However, the LIGFREKO mice exhibited significantly less extensive parenchymal bile infarct and hepatocyte necrosis as compared to control mice. The expression of a-SMA, collagen I and TGF-[Jl was also reduced by 40 50% in 3 days-BDL LIGFREKO compared with control mice.
['3"-~ "I'VVO TYPES OF HISTOLOGICALLY N O R M A L LIVER CAN HAVE MARKED DIFFERENCE IN GENE EXPRESSION PROFILES: IMPORTANCE OF AN ADEQUATE NORMAL TISSUE CONTROL IN GENE EXPRESSION STUDIES T. Asselahl'2, I. Bieche 2, I. Laurendeau 2, V. Paradis 3, D. Saadoun 1, D. Valla 1, M. Vidaud 2'4, R Marcellin 1. 1Service d'H@atologie &
INSERM CRB3, University of Paris VIL AP-HP H@ital Beaujon, Clichy, France," 2Laboratoire de Gdndtique Moldculaire, EA 361& Facultd de Pharmacie, Paris' K France," 3Service d 'Anatomie Pathologique, H@ital Beaujon, 4Service de Bioehimie, H@ital Beaujon, France Background and Aims: Gene expression profiling technologies allow the
analysis of gene networks whose expression is associated with specific pathological conditions in comparison with normal tissue. We made the hypothesis that histological normal tissue obtained in 2 different conditions (i.e; percutaneous or surgical liver biopsies), usually used as normal controls in gene expression studies, could have different liver gene expression profiles. Patients and Methods: Group A: Surgical liver biopsies, with general anesthesia, of non-tumoral liver (14 patients): during surgery for liver metastasis of colorectal cancer or for liver benign tumors. Group B: Percutaneous liver biopsies with local anesthesia (14 patients) obtained from patients with mild elevation of serum alanine aminotransferase (ALT) in whom all causes of liver disease were ruled out. All these 28 (group A&B) liver tissue specimens were histologically normal (i.e. absence of inflammation, fibrosis and pathological pattern). We used real-time quantitative RT-PCR assays to compare the mRNA expression of 240 selected genes in these 2 groups. Results: The expression of 29 (12%) of the 240 genes was significantly different between surgical (A) and percutaneous (B) pools specimens; 26 genes were up-regulated and 3 were down-regulated (pool A versus pool B). The most notable changes in gene expression mainly occurred in the inflammatory response genes family. The 7 most discriminatory up-regulated genes in individual samples in A in comparison with B were: PTGS2, THBS1, PAIl, CXCR4, JUN, FOS, ILS. The gene most discriminatory down-regulated was IHH. For example, we observed a higher or lower expression of a specific gene (i.e ILS) in HCV infection, if comparison was done with histologically normal controls obtained percutaneously or surgically, respectively. Conclusion: Our study has demonstrated that histologically normal liver tissue obtained in 2 different conditions (i.e; percutaneous or surgical liver biopsies) have marked difference in gene expression profile although all specimens were histologically normal. Therefore, this study outlines the importance of an adequate selection of histologically normal controls to prevent aberrant results.
[•]
REGULATION OF S U P P R E S S O R OF CYTOKINE SIGNALING 3 m R N A STABILITY BY TNF-~ INVOLVES ACTIVATION OF THE M K K 6 / P 3 8 M A P K CASCADE
C. Ehlting 1, F. Schapel2, E.D. BrenndSrfer 1, R.J. Matthes 1, P.C. Heinrich 2, D. Hfiussinger1, J.G. Bode 1. 1Department of Gastroenterology,
Hepatology and Infectiology, Heinrieh-Heine University, Diisseldorf Germany," :Institute of Biochemistry, University Hospital of the RWTH Aachen, Aaehen, Germany Background and Aims: The potential of some pro-inflammatory mediators to inhibit gpl30 dependent signaling by enhancing SOCS3 expression in macrophages, such as liver macrophages, represents an important molecular switch to abrogate signals with anti-inflammatory properties
(e) HSCs and fibrosis
$125
in order to perpetuate the inflammatory processes. Dis-balanced pro- and anti-inflammatory activities are causative for many inflammatory diseases. Hence, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by pro-inflammatory mediators. In this study we investigate SOCS3 expression initiated by the pro-inflammatory cytokine TNF-0~. Methods: The following methods were used: cell culture, reporter-gene assay, transfection, northern and western blot analysis. Results: In contrast to IL-6, TNF-c~ increases SOCS3-expression by stabilizing SOCS3 mRNA. Activation of the MKK6/p38MAPK-cascade is required for TNF-c~-mediated stabilization of SOCS3 mRNA and enhanced SOCS3 protein expression. Moreover, destabilization of SOCS3 mRNA by the zink-finger protein tristetraprolin and therefore inhibition of SOCS3protein expression is antagonized by activation of the MKK6/p38MAPK pathway. Tristetraprolin acts through an AU rich element containing region within the 31 untranslated region of the SOCS3 mRNA. Conclusions: In contrast to IL-6, TNF-~ regulates SOCS3 expression in macrophages on the level of mRNA stability employing activation of the MKK6/p38MAPK cascade thereby inhibiting the mRNA destabilizing activity of TTE
~ ' - ~ ENDOTOXIN-INDUCED HSC ACTIVATION INVOLVES PDGFR A N D EGFR RECRUITMENT R Brun 1'2, D. Martines 2, M. Scarpa 1'2, G. Pah~1, I. Castagliuolo 1.
1Department of Histology, Microbiology and Medical Bioteehnologies, 2Department of Gastroenterological Sciences, University of Padua, Padua, Italy Background and aims: Recent evidence has suggested that bacterial cell wall products can induce a pro-inflammatory and pro-fibrogenic phenotype in hepatic stellate cells (HSCs), a key cell population implicated in the pathogen:sis and progression of liver diseases. Endotoxins directly bind to HSCs through specific membrane receptors, namely CD14, TLR4 and TLR2, activating complex signal cascade pathways, involving ERK1, and leading to pro-inflammatory genes transcription. Since sequential phosphorylation of Raf-I/MEK/ERK is also elicited by activation of PDGFR-[J and EGFR, protein kinase receptors involved in cell proliferation and fibrosis, in this study we investigated whether PDGFR-[J and EGFR are involved in HSCs response to endotoxins. Methods: HSCs were isolated from Balb/c mice and used at passage two. Serum starved cells were exposed to lipopolysaccharide (LPS) or lipoteichoic acid (LTA) 10 gg/ml for 0.5 24 hrs to assess pro-inflammatory cytokine release (MCP1 and IL-6) by ELISA and ERK1 phosphorylation by immunoprecipitation and Western Blot. Results: Following LPS and LTA exposure, HSCs secreted significant amounts of IL-6 (10.9• and 2.9• respectively) and MCP-1 (9.7• and 5.6• ng/ml, respectively). However, pretreatment of HSCs with AG1295, a specific PDGFR-[J inhibitor or AG835, an EGFR inhibitor, caused a significant decrease of LPS- and LTA-induced IL6 and MCP1 release (P <0.05). In addition, simultaneous PDGFR-[J and EGFR inhibition with AG1478 (200 gM), further decreased IL-6 and MCP1 release following LPS and LTA stimulation (P <0.02). To asses whether receptor clustering was involved in EGFR and PDGFR-[J activation following HSCs exposure to endotoxins, we pre-treated HSCs with nystatin (60 gg/ml), a lipid raft disrupting agent. Indeed, nystatin treatment significantly decreased IL-6 and MCP1 secretion following LPS and LTA stimulation (P < 0.02). Furthermore, by immunoprecipitation and Western blot analysis we showed that ERK1 phosphorylation in HSCs exposed to LPS or LTA was significantly reduced by AG1478 or nystatin pre-treatment. Conclusions: We suggest that endotoxin induced HSCs pro-inflammatory phenotype involves EGFR and PDGFR-[J signalling and organization in supramolecular structures (signalling platforms). We speculate that signalling platforms involving growth factor receptors may represent an
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attractive target for therapy in liver diseases characterized by excessive fibrotic tissue deposition.
F•
PPAR-y L I G A N D PIOGLITAZONE D O E S NOT P R E V E N T HEPATIC FIBROSIS IN MICE
A. Da Silva Morais, J. Abarca-Quinones, P. Stfirkel, Y. Horsmans, I.A. Leclercq. Laboratoire de Gastro-Ent~rologie, Universit~ Catholique
de Louvain (UCL), Brussels', Belgium Background and Aims: Hepatic fibrosis results from activation of quiescent HSC into extracellular matrix producing, proliferative and contractile myofibroblast-like cells. Peroxisome proliferator-activated receptor gamma (PPAR-y) has been suggested to play a key role in the control of HSC activation. Indeed, in rats, PPAR-y expression is depleted in activated HSC and PPAR-y ligands inhibit the activation of HSC as well as prevent hepatic fibrosis in vivo. Also, forced expression of PPAR-y into activated HSC restores the quiescent phenotype. Here we evaluate the impact of PPAR-y agonist Pioglitazone (PGZ) on hepatic fibrogenesis in mice both in vitro and in vivo. Methods: Primary HSCs from Balb/C mice were cultured, with or without PGZ, on plastic dishes. Collagen Ial mRNA, R-smooth-muscle actin (aSMA) and desmin protein expression were measured to assess activation. PPAR-y mRNA and protein expression as well as the expression of the PPAR-y regulated genes CD36 and aP2 were evaluated to determine the biological effect of PGZ treatment. In in vivo experiments, mice were divided in 3 groups treated for 4 weeks with vehicle, CC14 (IR 400 L 3• or CC14+PGZ as a 0.01% PGZ-supplemented diet (wt/wt). Results: In vitro, mRNA expression of PPAR-~ was stable in HSC cultured up to day 9. However, PPAR-? protein was only found in quiescent and early activated HSC (up to 5 days) there after, protein expression decreased dramatically. Exposure of HSC to PGZ from day 3 (at a time when PPAR-y is expressed) to day 7 maintained the expression of PPAR-y ?and stimulated the expression of PPAR-y-regulated gene CD36, but did not alter induction of collagen Ial mRNA or aSMA. In vivo, PGZ treatment induced adiponectin, CD36 and aP2 mRNA in adipose tissue and increased serum adiponectin concentrations, but PGZ treatment did not decrease the severity of hepatic fibrosis induced by CC14. Conclusion: Our data demonstrate that, although having anti-fibrotic properties in rats, PPAR-? stimulation by PGZ does not prevent activation of HSC in vitro, nor hepatic fibrogenesis in vivo in mice. Direct antifibrotic effect of such substances remains to be demonstrated in humans.
[•-]
O P I O I D D E P E N D E N T M O D U L A T I O N OF LIVER FIBROGENESIS: A ROLE F O R HEPATIC R E D O X STATE
M.R. Ebrahimkhanil., S. Kiani 2, T. Kendall 3, S.M. Tavangar4, A. Shariftabrizi 2, E Oakley 3, A.R. Mani 1, A.R. Dehpom2, D.A. Mann 3, K.R Moore 1. 1The UCL Institute of Hepatology, Department of Medicine,
Royal Free and University College Medical School, University College London, London, UK," 2Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran," SLiver Group, Southampton General Hospital, University of Southampton, Southampton, UK,"4Department of Pathology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Background: Opioid peptides are known to decrease hepatic GSH synthesis, and induce liver injury. Increased endogenous opioids are well recognised in patients and animals with liver disease. We have recently shown that the opioid antagonist naltrexone decreases plasma transaminases in animals with biliary obstruction. The aim of this study was to investigate the role of opioids in liver fibrogenesis and its underlying mechanisms. Methods: Liver fibrosis was assessed using sirius red staining and hepatic hydroxyproline level in bile duct ligated (BDL) or sham operated rats given either naltrexone or saline for 28 days. Liver matrix metalloproteinase-2
(MMP-2) activity, a smooth muscle actin (a-SMA) and CD45 (leukocyte common antigen) immunostaining were performed in liver sections. The redox state of the liver was assessed by measurement of hepatic GSH/GSSG and S-nitrosothiols by reductive chemiluminescense-based assay and esterified F2-isoprostane concentration by gas chromatography mass spectrophotometry. To assess the effects of opioids on hepatic stellate cells (HSC), different subtypes of opioid receptors were characterized in cultured HSC by reverse-transcription polymerase chain reaction (RT-PCR), and the effects of selective opioid agonists or antagonists on the cellular proliferation of HSC in culture were investigated using [3H] thymidine incorporation assay. Results: Bile duct ligation led to extensive hepatic fibrosis and opioid receptor blockade significantly attenuated hepatic collagen accumulation and MMP-2 activity in BDL rats (P<0.01). a-SMA staining of liver sections demonstrated reduction in the number of activated HSC after naltrexone treatment in animals with cirrhosis (P <0.05). Activated rat HSC showed a weak expression of (31-opioid receptor mRNA. However, none of the selective opioid-receptor agonists or antagonists had any effect on HSC proliferation in vitro. The development of biliary cirrhosis was associated with an altered redox state in the liver with decreased hepatic GSH/GSSG, and increased concentrations of hepatic S-nitrosothiols, which were partially or completely normalized by treatment with naltrexone respectively. Leukocyte infiltration assessed by CD45 immunostaining and liver F2-isoprostane concentration were unaffected by chronic opioid receptor blockade in BDL rats. Conclusions: These data suggest that endogenous opioids contribute to liver fibrogenesis by altering the hepatic redox state and the regulation of redox sensitive genes.
•
R E D U C E D CHOLESTATIC LIVER I N J U R Y IN MICE DELETED F O R INSULIN-LIKE G R O W T H FACTOR 1 R E C E P T O R IN HEPATOCYTES
A. Cadoret 1, C. Rey 1, D. Wendum2, M. Holzenberger 3, C. Housset 1.
11NSERM U680, Facultd de Mddecine St-Antoine, Paris, France," :Service d 'Anatomopathologie, Hdpital St-Antoine, Paris, France," 3INSERM U515, Hdpital St-Antoine, Paris', France Background and Aim: In all types of liver injury, there is a complex balance between hepatocyte cell death, regenerative and fibrogenic responses. Activation of the IGF-1R signaling pathway contributes to the proliferation of hepatocytes during post-hepatectomy liver regeneration. The aim of this study was to examine the role of IGF signaling on other aspects of liver tissue repair, i.e. hepatocyte cell death and fibrogenesis, using a conditional knockout mouse in which IGF-1R is deleted in hepatocytes. Methods: To specifically invalidate the IGF-1R in hepatocytes, mice homozygously floxed on IGF-1R alleles were bred with mice that expressed the Cre recombinase under the control of albumin promoter and a-fetoprotein enhancer (LIGFREKO mice). LIGFREKO mice and control littermates underwent bile duct ligation (BDL) and were investigated 3 days later. Serum concentrations of transaminases (ALAT/ASAT) and of bile acids were measured. Histological analysis was performed on paraffine embedded, hematoxykin-eosin-stained liver sections and hepatocyte necrotic areas was measured by morphometric analysis. Hepatocyte proliferation was assessed by Ki67 immunolabeling, mRNA expression of R-smooth muscle actin (a-SMA), collagen I and TGF-~I was quantified by real-time RT-PCR using Light Cycler technique. Results: No difference in ALAT/ASAT nor in bile acid concentrations was found in comparing LIGFREKO mice with controls. Moreover, hepatocytes proliferation was similarly induced in both 3 days-BDL LIGFREKO and control mice. However, the LIGFREKO mice exhibited significantly less extensive parenchymal bile infarct and hepatocyte necrosis as compared to control mice. The expression of a-SMA, collagen I and TGF-[Jl was also reduced by 40 50% in 3 days-BDL LIGFREKO compared with control mice.