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303 THE 70 KDA PEROXISOMAL MEMBRANE PROTEIN IS INVOLVED IN THE ACTIVATION OF HEPATIC STELLATE CELLS
S118
Poster Session − Thursday, April 23
302 TGF-BETA-DEPENDENT PROLIFERATION ARREST OF HEPTOCYTES AND DUCTULAR REACTION CONTRIBUTE TO HBV-ASSOCIATED LIVER FIBROGENESIS VIA NOTCH SIGNALING H.-L. Weng1 , J. Li1 , L. Ciuclan1 , C. Meyer1 , T. Huang2 , P. Mertens3 , M. Singer1 , S. Dooley1 . 1 Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany; 2 Department of Internal Medicine, Zhenhai Lianhua Hospital, Ningbo, China; 3 Department of Nephrology and Clinical Immunology, RWTH-University Hospital, Aachen, Germany E-mail: [email protected] Background and Aims: Hepatic progenitor cells (HPC) instead of normal hepatocytes become the main population of regenerative hepatocytes, promoting a periportal ductular reaction (DR), which may contribute to periportal fibrogenesis in chronic liver disease in association with HCV and NASH. We hypothesize that a TGF-beta/Notch signalling-dependent alteration of the replicative pathway in hepatocytes and subsequent DR play critical roles in HBV-associated fibrogenesis and cirrhosis. Methods: 105 biopsied liver tissues with chronic HBV infection were stained for cytokeratin-7 (CK7) to quantify DR and the number of HPCs, and for p21 to assess hepatocyte replicative arrest. Results: CK7 positivity is significantly correlated with inflammatory grade (r = 0.642, P < 0.00001), fibrotic stage (r = 0.688, P < 0.00001) and Smad2phosphorylation, a marker for activated TGF-beta signaling (r = 0.453, P < 0.01). p21 positive hepatocytes were at large found in advanced fibrotic and cirrhotic liver tissues, indicating that hepatocyte proliferative arrest is not a premise of HPC activation and DR in HBV-associated liver damage. In CCl4 -damaged Smad7 Exon-1 knock out mice with enhanced TGF-beta signaling, CK7 positive liver cells and p21 staining in hepatocytes was significantly increased when compared to control animals. In cultured hepatocytes, TGF-beta treatment significantly increased Notch-1 and Jagged-1 protein expression. In addition, TGF-beta-induced p21 promoter activation and p21 protein expression were remarkably blunted by gamma-secretase inhibitor GSI (a broad-range inhibitor for Notch receptor activation), indicating that Notch signaling is critical in TGF-beta-induced hepatocyte replicative arrest. Finally, we observed that treatment with IFN-gamma, a TGF-beta signaling antagonist, in 18 patients with chronic HBV infection remarkably reduced CK7 positive cell number, further supporting involvement of TGF-beta in generation of HPCs and DR in chronic HBV infection. Conclusions: TGF-beta plays a critical role in the altered replicative pathway of hepatocytes via a Notch-dependent pathway in HBV-associated liver damage. TGF-beta induces HPC activation and DR, which contributes to HBV-related liver fibrogenesis. 303 THE 70 KDA PEROXISOMAL MEMBRANE PROTEIN IS INVOLVED IN THE ACTIVATION OF HEPATIC STELLATE CELLS J. Woudenberg, F.A.J. van den Heuvel, K.P. Rembacz, S. Dunning, T.E. Woudenberg-Vrenken, M. Buist-Homan, H. Moshage, K.N. Faber. Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, The Netherlands E-mail: [email protected] Background and Aims: Peroxisomes are organelles that are particularly enriched in the liver. They are involved in a large variety of cellular functions, including fatty acid b-oxidation and bile acid biosynthesis. Peroxisomes are abundant in hepatocytes. Their putative function in other liver cell-types has not been analyzed yet. Typical peroxisomal proteins in hepatocytes are the antioxidant enzyme catalase, the ATP-bindingcassette-transporters Adrenoleukodystrophy protein (ALDP) and 70 kDa peroxisomal membrane protein (PMP70) and peroxins that are required for peroxisome biogenesis. Here, we studied the expression and subcellular location of peroxisomal proteins in transdifferentiating hepatic stellate cells (HSCs), with special focus on PMP70.
Methods: Primary rat HSCs were isolated and analyzed after a 4 hattachment period (quiescent-qHSCs) and after 1, 3, 7 and 14 days in culture (activated-aHSCs). Messenger RNA and protein expression were measured by quantitative PCR and western blotting, respectively. RNA interference was used to reduce PMP70 expression in aHSCs. The subcellular location of a-smooth muscle actin (aSMA), calnexin (marker for endoplasmic reticulum-ER), Pex14p, catalase, ALDP and PMP70 were analyzed by immunofluorescence microscopy and in transiently transfected aHSCs containing DsRed-labeled peroxisomes. Results: All peroxisomal marker proteins are expressed in HSCs. Pex14p, PMP70 and ALDP are transiently induced during HSC activation. Pex14p and catalase are predominantly peroxisomal in aHSCs. In contrast, ALDP is predominantly present in the ER. Remarkably, most PMP70 resides in cellular fibers, with minor amounts in peroxisomes. Silencing of PMP70 expression leads to strongly reduced levels of the activation markers aSMA and desmin, without affecting collagen 1a1 expression. Conclusions: PMP70 is required for activation of HSCs in which it resides at an abnormal subcellular location. Our observations suggest a specialized function for PMP70 in the fibrotic liver. 304 LENTIVIRAL-MEDIATED ACE2 RNAI AGGRAVATES LIVER INJURY AND LIVER FIBROGENESIS Q. Huang, Q. Xie, C.-C. Shi, L.-Y. Lin, H. Wang, G.-D. Zhao, N.-N. Jia, X.-G. Xiang, B.-D. Gong, H. Yu. Department of Infectious Disease, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China E-mail: [email protected] Background and Aims: The rennin angiotensin system (RAS) plays an important role in hepatic fibrogenesis. A new homologue of Angitensin converting enzyme -ACE2 has been identified to be negative regulator of the RAS by degrading ang II to ang1−7.The expression of ACE2 is increased in BDL rat and patient with chronic HCV infection. But the role of ACE2 remains elusive in liver fibrosis. We constructed lentivectors expressing ACE2 specific short hairpin RNAs (shRNAs) to investigate the role of ACE2 in experimental hepatic fibrosis in vivo. Methods: We selected siRNA sequences of ACE2 and design of shRNAs, then cloning and validation of shRNAs, and generate lentivectors expressing ACE2specificshRNAs(LVsh-ACE2). The lentiviral vector carried GFP as a marker. Male SD rats received carbon tetrachloride (CCl4) by subcutaneous injections every three days for six consecutive weeks, and meantime they were also injected LVsh-ACE2 to rats’ liver by intra-portal vein. A negative control shRNA (mock group) or cell medium (positive group) were used as controls. All rats were sacrificed and analyzed at various time points. Liver pathology was analyzed by HE and Sirius staining. Real-time PCR was performed to determine gene expression of ACE2, a-SMA, TIMP-1, Collagena1. Serum samples were used to test ALT and AST. Results: After lentivirus administration GFP expression was detectable at the two week and lasted for 9 week by Confocal fluorescence microscope. GFP expression was nearly deteced in the liver. Real-time PCR analysis revealed that ACE2 mRNA of LVsh-ACE2 group were reduced 90.1%, 73.2%, 74.8% compared with controls at the two-, four-, and six-week time points. a-SMA protein and mRNA level increased 3.11, 2.74 and 2.88 fold in LVsh-ACE2 group compared with controls. TIMP-1, Collagena1 mRNA level were significantly higher in LVsh-ACE2 group compared with controls. ALT and AST level significantly upregulated after lentivirus administration. Conclusions: Lentivector-based short hairpin RNAs can efficient and stable knock down of ACE2. ACE2 possesses tissue-protective role and improve liver fibrogenesis. Acknowledgements: This work was supported by a grant (07jc14044) from the Shanghai Science and Technology Association Development Foundation and by a grant (20070032) from Liver Fibrosis Foundation of Wang Bao En.
Poster Session − Thursday, April 23
302 TGF-BETA-DEPENDENT PROLIFERATION ARREST OF HEPTOCYTES AND DUCTULAR REACTION CONTRIBUTE TO HBV-ASSOCIATED LIVER FIBROGENESIS VIA NOTCH SIGNALING H.-L. Weng1 , J. Li1 , L. Ciuclan1 , C. Meyer1 , T. Huang2 , P. Mertens3 , M. Singer1 , S. Dooley1 . 1 Molecular Alcohol Research in Gastroenterology, Department of Medicine II, Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany; 2 Department of Internal Medicine, Zhenhai Lianhua Hospital, Ningbo, China; 3 Department of Nephrology and Clinical Immunology, RWTH-University Hospital, Aachen, Germany E-mail: [email protected] Background and Aims: Hepatic progenitor cells (HPC) instead of normal hepatocytes become the main population of regenerative hepatocytes, promoting a periportal ductular reaction (DR), which may contribute to periportal fibrogenesis in chronic liver disease in association with HCV and NASH. We hypothesize that a TGF-beta/Notch signalling-dependent alteration of the replicative pathway in hepatocytes and subsequent DR play critical roles in HBV-associated fibrogenesis and cirrhosis. Methods: 105 biopsied liver tissues with chronic HBV infection were stained for cytokeratin-7 (CK7) to quantify DR and the number of HPCs, and for p21 to assess hepatocyte replicative arrest. Results: CK7 positivity is significantly correlated with inflammatory grade (r = 0.642, P < 0.00001), fibrotic stage (r = 0.688, P < 0.00001) and Smad2phosphorylation, a marker for activated TGF-beta signaling (r = 0.453, P < 0.01). p21 positive hepatocytes were at large found in advanced fibrotic and cirrhotic liver tissues, indicating that hepatocyte proliferative arrest is not a premise of HPC activation and DR in HBV-associated liver damage. In CCl4 -damaged Smad7 Exon-1 knock out mice with enhanced TGF-beta signaling, CK7 positive liver cells and p21 staining in hepatocytes was significantly increased when compared to control animals. In cultured hepatocytes, TGF-beta treatment significantly increased Notch-1 and Jagged-1 protein expression. In addition, TGF-beta-induced p21 promoter activation and p21 protein expression were remarkably blunted by gamma-secretase inhibitor GSI (a broad-range inhibitor for Notch receptor activation), indicating that Notch signaling is critical in TGF-beta-induced hepatocyte replicative arrest. Finally, we observed that treatment with IFN-gamma, a TGF-beta signaling antagonist, in 18 patients with chronic HBV infection remarkably reduced CK7 positive cell number, further supporting involvement of TGF-beta in generation of HPCs and DR in chronic HBV infection. Conclusions: TGF-beta plays a critical role in the altered replicative pathway of hepatocytes via a Notch-dependent pathway in HBV-associated liver damage. TGF-beta induces HPC activation and DR, which contributes to HBV-related liver fibrogenesis. 303 THE 70 KDA PEROXISOMAL MEMBRANE PROTEIN IS INVOLVED IN THE ACTIVATION OF HEPATIC STELLATE CELLS J. Woudenberg, F.A.J. van den Heuvel, K.P. Rembacz, S. Dunning, T.E. Woudenberg-Vrenken, M. Buist-Homan, H. Moshage, K.N. Faber. Department of Gastroenterology and Hepatology, University Medical Center Groningen, Groningen, The Netherlands E-mail: [email protected] Background and Aims: Peroxisomes are organelles that are particularly enriched in the liver. They are involved in a large variety of cellular functions, including fatty acid b-oxidation and bile acid biosynthesis. Peroxisomes are abundant in hepatocytes. Their putative function in other liver cell-types has not been analyzed yet. Typical peroxisomal proteins in hepatocytes are the antioxidant enzyme catalase, the ATP-bindingcassette-transporters Adrenoleukodystrophy protein (ALDP) and 70 kDa peroxisomal membrane protein (PMP70) and peroxins that are required for peroxisome biogenesis. Here, we studied the expression and subcellular location of peroxisomal proteins in transdifferentiating hepatic stellate cells (HSCs), with special focus on PMP70.
Methods: Primary rat HSCs were isolated and analyzed after a 4 hattachment period (quiescent-qHSCs) and after 1, 3, 7 and 14 days in culture (activated-aHSCs). Messenger RNA and protein expression were measured by quantitative PCR and western blotting, respectively. RNA interference was used to reduce PMP70 expression in aHSCs. The subcellular location of a-smooth muscle actin (aSMA), calnexin (marker for endoplasmic reticulum-ER), Pex14p, catalase, ALDP and PMP70 were analyzed by immunofluorescence microscopy and in transiently transfected aHSCs containing DsRed-labeled peroxisomes. Results: All peroxisomal marker proteins are expressed in HSCs. Pex14p, PMP70 and ALDP are transiently induced during HSC activation. Pex14p and catalase are predominantly peroxisomal in aHSCs. In contrast, ALDP is predominantly present in the ER. Remarkably, most PMP70 resides in cellular fibers, with minor amounts in peroxisomes. Silencing of PMP70 expression leads to strongly reduced levels of the activation markers aSMA and desmin, without affecting collagen 1a1 expression. Conclusions: PMP70 is required for activation of HSCs in which it resides at an abnormal subcellular location. Our observations suggest a specialized function for PMP70 in the fibrotic liver. 304 LENTIVIRAL-MEDIATED ACE2 RNAI AGGRAVATES LIVER INJURY AND LIVER FIBROGENESIS Q. Huang, Q. Xie, C.-C. Shi, L.-Y. Lin, H. Wang, G.-D. Zhao, N.-N. Jia, X.-G. Xiang, B.-D. Gong, H. Yu. Department of Infectious Disease, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China E-mail: [email protected] Background and Aims: The rennin angiotensin system (RAS) plays an important role in hepatic fibrogenesis. A new homologue of Angitensin converting enzyme -ACE2 has been identified to be negative regulator of the RAS by degrading ang II to ang1−7.The expression of ACE2 is increased in BDL rat and patient with chronic HCV infection. But the role of ACE2 remains elusive in liver fibrosis. We constructed lentivectors expressing ACE2 specific short hairpin RNAs (shRNAs) to investigate the role of ACE2 in experimental hepatic fibrosis in vivo. Methods: We selected siRNA sequences of ACE2 and design of shRNAs, then cloning and validation of shRNAs, and generate lentivectors expressing ACE2specificshRNAs(LVsh-ACE2). The lentiviral vector carried GFP as a marker. Male SD rats received carbon tetrachloride (CCl4) by subcutaneous injections every three days for six consecutive weeks, and meantime they were also injected LVsh-ACE2 to rats’ liver by intra-portal vein. A negative control shRNA (mock group) or cell medium (positive group) were used as controls. All rats were sacrificed and analyzed at various time points. Liver pathology was analyzed by HE and Sirius staining. Real-time PCR was performed to determine gene expression of ACE2, a-SMA, TIMP-1, Collagena1. Serum samples were used to test ALT and AST. Results: After lentivirus administration GFP expression was detectable at the two week and lasted for 9 week by Confocal fluorescence microscope. GFP expression was nearly deteced in the liver. Real-time PCR analysis revealed that ACE2 mRNA of LVsh-ACE2 group were reduced 90.1%, 73.2%, 74.8% compared with controls at the two-, four-, and six-week time points. a-SMA protein and mRNA level increased 3.11, 2.74 and 2.88 fold in LVsh-ACE2 group compared with controls. TIMP-1, Collagena1 mRNA level were significantly higher in LVsh-ACE2 group compared with controls. ALT and AST level significantly upregulated after lentivirus administration. Conclusions: Lentivector-based short hairpin RNAs can efficient and stable knock down of ACE2. ACE2 possesses tissue-protective role and improve liver fibrogenesis. Acknowledgements: This work was supported by a grant (07jc14044) from the Shanghai Science and Technology Association Development Foundation and by a grant (20070032) from Liver Fibrosis Foundation of Wang Bao En.