- Email: [email protected]
1044 DLL4 IS A CRITICAL MEDIATOR IN HBV-ASSOCIATED LIVER FIBROGENESIS
POSTERS the progression of liver fibrosis. Further experiments will address the effects of mitochondrial uncoupling on the inhibition of in vivo models of liver fibrosis. 1044 DLL4 IS A CRITICAL MEDIATOR IN HBV-ASSOCIATED LIVER FIBROGENESIS Y. Liu, C. Meyer, I. Ilkavets, Q. Li, H. Shen, A. Muller, ¨ S. Dooley, H.-L. Weng. Molecular Hepatology – Alcohol Dependent Diseases, II. Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Germany E-mail: [email protected] Background and Aims: Mutation of Jagged-1 gene, a Notch ligand, leads to Alagille syndrome. However, patients with Alagille syndrome usually do not progress into serious liver fibrosis. In the present study, we investigated the role of Notch ligands in liver fibrogenesis. Methods: The expression of Notch ligands, including Jagged1, Jagged2, Delta-like ligand (Dll)1, Dll3 and Dll4, was measured in 130 patientes with chronic HBV infection by immunohistochemistry. The effect of Notch ligands was as well examined in TGF-b treated hepatic stellate cells (HSCs). Results: Immunohistochemistry analysis revealed positive expression of Jagged1, Dll3 and Dll4 in fibrotic liver tissues from chronic HBV infected patients. Distinct from Jagged1 and Dll3, which majorly located in cholangiocates and hepatocytes, positive Dll4 demonstrated in sinusoidal cells and portal tracts. Confocol microscopy analysis confirmed these Dll4-positive cells were a-smooth muscle actin (a-SMA) positive, indicating that Dll4 was upregulated in activated HSCs or myofibroblasts in chronic HBV infected patients. Dll4 positive liver cells remarkably correlated with inflammatory grade (r = 0.6, P < 0.001) and fibrotic stage (r = 0.68, P < 0.001) in HBV infected patients. In vitro, knock-down of Dll4 by using siRNA significantly increased TGF-b induced protein expression of connective growth factor, but decreased protein level of a-SMA in CFSC cells, a cell line from CCl4 -induced cirrhotic rat HSCs. Conclusions: Dll4 plays a crucial role in liver fibrogenesis maybe via influencing TGF-b dependent activation of HSCs and extracelluar matrix production. 1045 PIRFENIDONE MAINTAINS NUCLEAR NRF2 ACTIVATION AND REDUCES PROINFLAMMATORY AND FIBROGENIC EFFECTS INDUCED BY OXIDATIVE STRESS IN HUMAN HEPATIC STELLATE CELLS (HSC) J. Macias1 , A. Caligiuri1 , E. Novo2 , M. Parola2 , J. Armendariz´ Borunda3 , M. Pinzani1 . 1 Dipartimento di Medicina Interna, Universit` a degli Studi di Firenze, Florence, 2 Dipartimento di Medicina e Oncologia Sperimentale, Universit` a degli Studi di Torino, Turin, Italy; 3 Instituto de Biolog´ıa Molecular y Gen´ omica, Universidad de Guadalajara, Guadalajara, Mexico E-mail: [email protected] Background: In chronic liver diseases, the interaction between reactive oxygen species (ROS) and hepatic stellate cells (HSC) results in potent pro-fibrogenic and pro-inflammatory effects. Studies in animal models and in patients with chronic HCV hepatitis have demonstrated that Pirfenidone (PFD) exerts antifibrotic effects. Although PFD may acts through anti-inflammatory and proantioxidant actions, the key cellular and molecular mechanisms are still incompletely understood. Aim: To explore the mechanisms of PFD action in activated human HSC subjected to oxidative stress and their relationship with inflammatory and fibrogenic pathways, in particular, the effects of PFD on the compartmentalisation of the transcriptional factor NRF2, a master regulator of antioxidant responses.
Methods and Results: Activated human HSC were incubated with two intracellular ROS-inducer agents: 2-methyl-1,4naphthoquinone (menadione or MEN) and 2,3-dimethoxy-1,4naphthoquinone (DMNQ). PFD was able to inhibit dose-dependently (10–1000 mM) MEN- and DMNQ- or PDGF-induced pro-fibrogenic actions, including cell proliferation, cell motility as well as de novo synthesis of COL1A1, TGF-b and TIMP-1 fibrogenic genes and IL-6 and TNF inflammatory genes. These effects were associated with NRF2 nuclear translocation, which was assessed by western blotting and confocal microscopy, thus reducing pro-fibrogenic effects triggered by ROS. In addition, incubation of human HSC with PFD, stimulated a dose- and time-dependent activation of several antioxidant genes, (glutamyl cysteine synthetase catalytic and regulatory subunits, glutamyl and heme oxygenase 1) whose activity may contribute to the down-regulation of ROS-induced pro-fibrogenic and pro-inflammatory effects. Also by DCFH-DA conversion, PFD showed a reduction of MEN- or DMNQ- ROS generation, indicating that PFD counteracts oxidative-stress and cell damage. Conclusions: PFD exerts its beneficial effects both directly, by counteracting ROS-induced pro-fibrogenic signalling, and indirectly, by regulating the biosynthesis of antioxidant proteins through its ability to increase expression of antioxidant genes. Thus, PFD anti-oxidant action supports its anti-fibrogenic and antiinflammatory effects, more studies should be addressed to improve our understanding of the underlying mechanisms and as an approach for its use in more therapeutic trials. 1046 ROLE OF ANGIOPOIETIN RECEPTOR TIE-2 IN THE INVASION OF HUMAN HEPATIC STELLATE CELLS INDUCED BY HEPATITIS C VIRUS S. Mart´ın-V´ılchez1 , Y. Rodr´ıguez-Munoz ˜ 2 , R. Lopez-Rodr´ ´ ıguez2 , 3 ´ Hernandez-Bartolom A. ´ e´ 2 , F. Molina-Jimenez ´ , M. TraperoMarugan ´ 2 , R. Moreno-Otero1 , P. Sanz-Cameno1 . 1 Liver Unit, Hospital Universitario de la Princesa, CIBERehd Instituto de Salud Carlos III, 2 Liver Unit, 3 Molecular Biology Unit, Hospital Universitario de la Princesa, Madrid, Spain E-mail: [email protected] Introduction: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Tie-2, a tyrosine kinase receptor essential for vascular development and maintenance, interacts with a family of ligands known as angiopoietins. Actually, these molecules have been implicated in proinflammatory cells recruitment to the surrounding tumour area, favouring tumour growth. Activated hepatic stellate cells (HSCs) are found around hepatocellular carcinoma, thus HSCs expression of Tie-2 may play an important role in HSCs migration and invasion to the damaged area that, together with their capacity to remodelate extracellular matrix, could favour pathological angiogenesis associated with chronic hepatitis C progression. Aim: To explore Tie-2 receptor ability to modulate HSCs activation and invasion mediated by HCV. Methods: HSCs line LX-2, was incubated for with conditioned media from selectable genomic HCV RNAs cells (replicons) derived from the human hepatoma cell line Huh-7. HSCs activation markers, alfa-smooth muscle actin (a-SMA) and collagen-I (COL-I), expression were studied by Western Blot. HSCs metalloprotease-2 (MMP-2) activity and expression was tested by zimography. HSCs invasion capacity was assessed, co-culturing LX2 cells with different HCV replicons or the parental Huh-7 cells, using transwell inserts collagen-coated. Tie-2 expression in HSCs was investigated in kinetic activation assays and in HSCs exposed to conditioned media. Tie-2 implications in HSCs activation and invasion capacity were analyzed using a Tie-2 neutralizing antibody, both in the conditioned media and in the transwell assays.
Journal of Hepatology 2011 vol. 54 | S363–S534
S415
Methods and Results: Activated human HSC were incubated with two intracellular ROS-inducer agents: 2-methyl-1,4naphthoquinone (menadione or MEN) and 2,3-dimethoxy-1,4naphthoquinone (DMNQ). PFD was able to inhibit dose-dependently (10–1000 mM) MEN- and DMNQ- or PDGF-induced pro-fibrogenic actions, including cell proliferation, cell motility as well as de novo synthesis of COL1A1, TGF-b and TIMP-1 fibrogenic genes and IL-6 and TNF inflammatory genes. These effects were associated with NRF2 nuclear translocation, which was assessed by western blotting and confocal microscopy, thus reducing pro-fibrogenic effects triggered by ROS. In addition, incubation of human HSC with PFD, stimulated a dose- and time-dependent activation of several antioxidant genes, (glutamyl cysteine synthetase catalytic and regulatory subunits, glutamyl and heme oxygenase 1) whose activity may contribute to the down-regulation of ROS-induced pro-fibrogenic and pro-inflammatory effects. Also by DCFH-DA conversion, PFD showed a reduction of MEN- or DMNQ- ROS generation, indicating that PFD counteracts oxidative-stress and cell damage. Conclusions: PFD exerts its beneficial effects both directly, by counteracting ROS-induced pro-fibrogenic signalling, and indirectly, by regulating the biosynthesis of antioxidant proteins through its ability to increase expression of antioxidant genes. Thus, PFD anti-oxidant action supports its anti-fibrogenic and antiinflammatory effects, more studies should be addressed to improve our understanding of the underlying mechanisms and as an approach for its use in more therapeutic trials. 1046 ROLE OF ANGIOPOIETIN RECEPTOR TIE-2 IN THE INVASION OF HUMAN HEPATIC STELLATE CELLS INDUCED BY HEPATITIS C VIRUS S. Mart´ın-V´ılchez1 , Y. Rodr´ıguez-Munoz ˜ 2 , R. Lopez-Rodr´ ´ ıguez2 , 3 ´ Hernandez-Bartolom A. ´ e´ 2 , F. Molina-Jimenez ´ , M. TraperoMarugan ´ 2 , R. Moreno-Otero1 , P. Sanz-Cameno1 . 1 Liver Unit, Hospital Universitario de la Princesa, CIBERehd Instituto de Salud Carlos III, 2 Liver Unit, 3 Molecular Biology Unit, Hospital Universitario de la Princesa, Madrid, Spain E-mail: [email protected] Introduction: Hepatitis C virus (HCV) infection is a major cause of chronic liver disease. Tie-2, a tyrosine kinase receptor essential for vascular development and maintenance, interacts with a family of ligands known as angiopoietins. Actually, these molecules have been implicated in proinflammatory cells recruitment to the surrounding tumour area, favouring tumour growth. Activated hepatic stellate cells (HSCs) are found around hepatocellular carcinoma, thus HSCs expression of Tie-2 may play an important role in HSCs migration and invasion to the damaged area that, together with their capacity to remodelate extracellular matrix, could favour pathological angiogenesis associated with chronic hepatitis C progression. Aim: To explore Tie-2 receptor ability to modulate HSCs activation and invasion mediated by HCV. Methods: HSCs line LX-2, was incubated for with conditioned media from selectable genomic HCV RNAs cells (replicons) derived from the human hepatoma cell line Huh-7. HSCs activation markers, alfa-smooth muscle actin (a-SMA) and collagen-I (COL-I), expression were studied by Western Blot. HSCs metalloprotease-2 (MMP-2) activity and expression was tested by zimography. HSCs invasion capacity was assessed, co-culturing LX2 cells with different HCV replicons or the parental Huh-7 cells, using transwell inserts collagen-coated. Tie-2 expression in HSCs was investigated in kinetic activation assays and in HSCs exposed to conditioned media. Tie-2 implications in HSCs activation and invasion capacity were analyzed using a Tie-2 neutralizing antibody, both in the conditioned media and in the transwell assays.
Journal of Hepatology 2011 vol. 54 | S363–S534
S415