- Email: [email protected]
114 AUTOPHAGY: A NOVEL REGULATOR OF HEPATIC STELLATE CELL ACTIVATION AND FIBROGENESIS
ORAL PRESENTATIONS Results: Most frequent etiology was non-alcoholic (63%). MELD score was 16 (6–31) and Child–Pugh 9±2. Fourteen (17.5%) patients developed HRS, all had LVDD grade 1 or 2. In the univariate analysis, 12 variables had predictive value for the development of HRS: portal pressure gradient, mean arterial pressure (MAP), cardiac output, ejection fraction, auricular and left ventricle diameter, LVDD parameters [early diastolic velocity at mitral annulus (Ea) and early diastolic velocity transmitral ratio (E/Ea)] and endogenous vasoactive system activity. Multivariate analysis showed that only MAP and the presence of LVDD expressed by Ea, were related to the appearance of HRS. Episodes of HRS-type1 developed in 9 (64%) patients with LVDD-2, and 5 (36%) with LVDD-1 (p < 0.001). Renal function normalization (complete response) happened in 2 (40%) with LVDD-1, and in 1 (11%) with LVDD-2. All 11 patients, whose renal function deteriorated, died. Multivariate analysis showed that the presence of LVDD-2 was associated independently with survival. Conclusion: The presence of LVDD in advanced cirrhosis is a risk factor for development HRS. Their severity is associated with poor prognosis in treatment response of HRS type1 and lower survival.
Parallel Session: FIBROGENESIS EXPERIMENTAL
114 AUTOPHAGY: A NOVEL REGULATOR OF HEPATIC STELLATE CELL ACTIVATION AND FIBROGENESIS V. Hernandez-Gea1 , Z. Ghiassi-Nejad1 , R. Gordon1 , I. Fiel1 , Z. Yue1 , M.J. Czaja2 , S. Friedman1 . 1 Mount Sinai School of Medicine, 2 Albert Einstein College of Medicine, New York, NY, USA E-mail: [email protected] Introduction: Features of hepatic stellate cell (HSC) activation during liver fibrosis are well characterized and tightly coordinated. Remarkably, some of these features are also hallmarks of autophagy, a highly regulated cellular response evolved to maintain energy homeostasis during cellular stress or enhanced metabolic demand. Hypothesis: Autophagy regulates HSC activation. Aim: To analyze the role of autophagy in HSC activation in hepatic fibrosis. Methods: Autophagy was blocked in vitro in both immortalized and primary murine and human HSC by 3-methyladenine (3MA, 10 mM), chloroquine or lentiviral silencing of the autophagy genes Atg7 and Atg 5. Effects on autophagy induction were assessed following nutrient deprivation and TGFb incubation. To establish the contribution of autophagy to HSC in vivo autophagic activity was assessed by LC3B-I conjugation to LC3B-II and by ultrastructural evidence of autophagic vacuoles in C5BI6 mice after liver injury due to CCl4 or bile duct ligation (BDL). We also generated a transgenic mouse with HSC-specific deletion of Atg7 and analyzed their response to liver injury as assessed by histology, serum liver enzymes and fibrogenic mRNA expression. Results: In vitro blockade of autophagy significantly inhibited HSC activation based on both reduced expression of mRNAs for Cola1 (I), Cola2 (I), b-PDGFR, MMP2 by qRT-PCR, and reduced fibrogenic protein expression by Western blot. Autophagy inhibition also markedly increased intracellular lipid content detected by Oil red-O, suggesting reversion to a more quiescent phenotype. Conversely, nutrient deprivation and TGFb administration stimulated HSC activation and fibrogenic gene expression. Autophagy levels were increased during HSC activation in vivo following CCl4 and BDLinduced liver injury. Heterozygous mice lacking one allele of Atg7 in HSCs had attenuated hepatic fibrosis after CCl4, despite slightly increased hepatocyte injury, reinforcing the role of autophagy by
HSCs during fibrogenesis in vivo and the potential role of HSCs in modifying hepatocellular damage. Conclusion: Our data establish a contribution of autophagy to the regulation of HSC activation, providing a novel framework for understanding hepatic fibrosis that could translate into new targets for therapy. 115 CXCL9 ATTENUATES EXPERIMENTALLY INDUCED ANGIOGENESIS AND LIVER FIBROSIS IN MICE H. Sahin1 , M.M. Zaldivar1 , P. Schmitz1 , F. Kiessling2 , C. Trautwein1 , H.E. Wasmuth1 . 1 Medizinische Klinik III, 2 Department of Experimental Molecular Imaging, Universit¨ atsklinikum Aachen, Aachen, Germany E-mail: [email protected] Background: Previous in vivo and in vitro findings suggest that the chemokine receptor CXCR3 and its ligand CXCL9 are a chemokinemediated pathway which mediates anti-fibrogenic effects within the liver (Wasmuth et al. Gastroenterology 2009). Neoangiogenesis critically contributes to liver fibrosis but the specific role of antiangiogenic chemokines in liver scarring is still unclear. Notably, CXCL9 has been shown to inhibit angiogenesis. We here investigate the role of neoangiogenesis in wild-type and CXCR3−/− mice after CCl4 treatment and the feasibility of attenuation of experimental liver fibrosis and angiogenesis by systemic administration of CXCL9. Methods: Liver fibrosis was induced by intraperitoneal injections of CCl4 in CXCR3−/− and wild-type mice for 6 weeks. In a separate experiment, CXCL9 (1 mg/day) or vehicle administered daily concomitantly with CCl4 to wild-type mice. Angiogenesis was assessed by immunofluorescence staining of CD31 and by in vivo molecular imaging of VEGF receptor-2 (VEGFR-2) using fluorescence tomography. Fibrosis was assessed by morphological analysis of Sirius red staining and by liver hydroxyproline concentration. Recruitment of interferon-g-positive-T-cells to the liver was quantified by FACS analysis. In vitro, migration (Boyden chamber) as well as proliferation (BrdU assay) of endothelial cells was analyzed after stimulation with VEGF in the presence or absence of CXCL9. Results: Compared to wild-type mice, CXCR3 deficient mice displayed a significantly increased fibrosis and angiogenesis within the liver (p < 0.01) as determined by the number of CD31 positive vessels after chronic injury. This increased angiogenesis in CXCR3−/− mice was also reflected by increased VEGFR2 levels in vivo. Notably, the systemic treatment of CXCL9 led to a significant attenuation of angiogenesis as well as of liver fibrosis (both p < 0.05). FACS analysis revealed no alteration in recruitment of interferon-g-positive-Tcells into the liver. Functionally, CXCL9 inhibited the VEGF induced migration and proliferation of endothelial cells (both p < 0.05) in vitro. Discussion: Fibrosis progression in mice is strongly associated with increased neoaniogenesis. This can be attenuated by systemic administration of CXCL9, suggesting that the systemic administration of this chemokine might be a novel therapeutic approach in liver diseases. 116 STIMULATION OF LIVER REGENERATION AND AMELIORATION OF FIBROSIS BY ANTAGONISM OF A 5-HT2B /ERK/JUND/TGFb1 SIGNALING PATHWAY IN HEPATIC STELLATE CELL F. Oakley, L. Murphy, M. Wright, D. Mann. Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, UK E-mail: fi[email protected] Background: The molecular processes that stimulate regeneration of lost and damaged functional tissue mass in the injured liver are complex and involve the interplay of factors from parenchymal and non-parenchymal cells. Here we investigated the contribution
Journal of Hepatology 2011 vol. 54 | S45–S60
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Parallel Session: FIBROGENESIS EXPERIMENTAL
114 AUTOPHAGY: A NOVEL REGULATOR OF HEPATIC STELLATE CELL ACTIVATION AND FIBROGENESIS V. Hernandez-Gea1 , Z. Ghiassi-Nejad1 , R. Gordon1 , I. Fiel1 , Z. Yue1 , M.J. Czaja2 , S. Friedman1 . 1 Mount Sinai School of Medicine, 2 Albert Einstein College of Medicine, New York, NY, USA E-mail: [email protected] Introduction: Features of hepatic stellate cell (HSC) activation during liver fibrosis are well characterized and tightly coordinated. Remarkably, some of these features are also hallmarks of autophagy, a highly regulated cellular response evolved to maintain energy homeostasis during cellular stress or enhanced metabolic demand. Hypothesis: Autophagy regulates HSC activation. Aim: To analyze the role of autophagy in HSC activation in hepatic fibrosis. Methods: Autophagy was blocked in vitro in both immortalized and primary murine and human HSC by 3-methyladenine (3MA, 10 mM), chloroquine or lentiviral silencing of the autophagy genes Atg7 and Atg 5. Effects on autophagy induction were assessed following nutrient deprivation and TGFb incubation. To establish the contribution of autophagy to HSC in vivo autophagic activity was assessed by LC3B-I conjugation to LC3B-II and by ultrastructural evidence of autophagic vacuoles in C5BI6 mice after liver injury due to CCl4 or bile duct ligation (BDL). We also generated a transgenic mouse with HSC-specific deletion of Atg7 and analyzed their response to liver injury as assessed by histology, serum liver enzymes and fibrogenic mRNA expression. Results: In vitro blockade of autophagy significantly inhibited HSC activation based on both reduced expression of mRNAs for Cola1 (I), Cola2 (I), b-PDGFR, MMP2 by qRT-PCR, and reduced fibrogenic protein expression by Western blot. Autophagy inhibition also markedly increased intracellular lipid content detected by Oil red-O, suggesting reversion to a more quiescent phenotype. Conversely, nutrient deprivation and TGFb administration stimulated HSC activation and fibrogenic gene expression. Autophagy levels were increased during HSC activation in vivo following CCl4 and BDLinduced liver injury. Heterozygous mice lacking one allele of Atg7 in HSCs had attenuated hepatic fibrosis after CCl4, despite slightly increased hepatocyte injury, reinforcing the role of autophagy by
HSCs during fibrogenesis in vivo and the potential role of HSCs in modifying hepatocellular damage. Conclusion: Our data establish a contribution of autophagy to the regulation of HSC activation, providing a novel framework for understanding hepatic fibrosis that could translate into new targets for therapy. 115 CXCL9 ATTENUATES EXPERIMENTALLY INDUCED ANGIOGENESIS AND LIVER FIBROSIS IN MICE H. Sahin1 , M.M. Zaldivar1 , P. Schmitz1 , F. Kiessling2 , C. Trautwein1 , H.E. Wasmuth1 . 1 Medizinische Klinik III, 2 Department of Experimental Molecular Imaging, Universit¨ atsklinikum Aachen, Aachen, Germany E-mail: [email protected] Background: Previous in vivo and in vitro findings suggest that the chemokine receptor CXCR3 and its ligand CXCL9 are a chemokinemediated pathway which mediates anti-fibrogenic effects within the liver (Wasmuth et al. Gastroenterology 2009). Neoangiogenesis critically contributes to liver fibrosis but the specific role of antiangiogenic chemokines in liver scarring is still unclear. Notably, CXCL9 has been shown to inhibit angiogenesis. We here investigate the role of neoangiogenesis in wild-type and CXCR3−/− mice after CCl4 treatment and the feasibility of attenuation of experimental liver fibrosis and angiogenesis by systemic administration of CXCL9. Methods: Liver fibrosis was induced by intraperitoneal injections of CCl4 in CXCR3−/− and wild-type mice for 6 weeks. In a separate experiment, CXCL9 (1 mg/day) or vehicle administered daily concomitantly with CCl4 to wild-type mice. Angiogenesis was assessed by immunofluorescence staining of CD31 and by in vivo molecular imaging of VEGF receptor-2 (VEGFR-2) using fluorescence tomography. Fibrosis was assessed by morphological analysis of Sirius red staining and by liver hydroxyproline concentration. Recruitment of interferon-g-positive-T-cells to the liver was quantified by FACS analysis. In vitro, migration (Boyden chamber) as well as proliferation (BrdU assay) of endothelial cells was analyzed after stimulation with VEGF in the presence or absence of CXCL9. Results: Compared to wild-type mice, CXCR3 deficient mice displayed a significantly increased fibrosis and angiogenesis within the liver (p < 0.01) as determined by the number of CD31 positive vessels after chronic injury. This increased angiogenesis in CXCR3−/− mice was also reflected by increased VEGFR2 levels in vivo. Notably, the systemic treatment of CXCL9 led to a significant attenuation of angiogenesis as well as of liver fibrosis (both p < 0.05). FACS analysis revealed no alteration in recruitment of interferon-g-positive-Tcells into the liver. Functionally, CXCL9 inhibited the VEGF induced migration and proliferation of endothelial cells (both p < 0.05) in vitro. Discussion: Fibrosis progression in mice is strongly associated with increased neoaniogenesis. This can be attenuated by systemic administration of CXCL9, suggesting that the systemic administration of this chemokine might be a novel therapeutic approach in liver diseases. 116 STIMULATION OF LIVER REGENERATION AND AMELIORATION OF FIBROSIS BY ANTAGONISM OF A 5-HT2B /ERK/JUND/TGFb1 SIGNALING PATHWAY IN HEPATIC STELLATE CELL F. Oakley, L. Murphy, M. Wright, D. Mann. Liver Group, Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, UK E-mail: fi[email protected] Background: The molecular processes that stimulate regeneration of lost and damaged functional tissue mass in the injured liver are complex and involve the interplay of factors from parenchymal and non-parenchymal cells. Here we investigated the contribution
Journal of Hepatology 2011 vol. 54 | S45–S60
S51