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383 BREVISCAPINE ATTENUATES THE CONTRACTILE FORCE OF RAT HEPATIC STELLATE CELLS INDUCED BY ENDOTHELIN-1 AND DECREASES PORTAL VEIN PRESSURE IN CIRRHOTIC RATS
POSTERS in 113 HBV infected liver tissues. Primary rat hepatic stellate cells (HSC) and the CFSC cell line were used to examine the effects of Notch ligands in liver fibrogenesis. Results: Microarray analyses revealed upregulated Notch-related genes, e.g. Jagged-1 and Jagged-2, in HBV associated fibrotic liver tissues. IHC demonstrated remarkable positive staining for three Notch ligands, Jagged1, Dll3 and Dll4, in cirrhotic liver tissues compared to controls. In addition, Jagged-1 and Dll4 were confirmed to localise in activated HSC, the main collagen producing liver cell type, by confocal microscopy. Dll4 and Jagged-1 staining displayed an opposing trend in HBV infected patients. Dll4 IHC score correlated with inflammatory grades (r = 0.60, P < 0.0001) and fibrotic stages (r = 0.68, P < 0.0001) in 113 HBV infected patients, whereas 69% of specimens from cirrhotic patients were Dll4 immunostaining negative. In contrast, sinusoidal cell positive Jagged-1 staining was mainly observed in cirrhotic patients (54%), but only 20% non-cirrhotic patients showed Jagged-1 positive staining in sinusoidal cells. In vitro, blocking Notch signalling by g-secretase inhibitor significantly reduced expression of collagen I in HSCs. Treatment of HSCs and CFSCs with recombinant Dll4 protein decreased TGF-b1-dependent collagen I and connective tissue growth factor (CTGF) expression. On the other hand, recombinant Jagged-1 protein incubation ameliorated the effects of Dll4 on collagen I and CTGF expression. Conclusions: Notch ligands contribute to liver fibrogenesis. Dll4 and Jagged-1 play opposing effects on the regulation of fibrosisassociated genes. 382 OSTEOPONTIN INDUCES DUCTULAR REACTION AND CONTRIBUTES TO LIVER FIBROSIS A. Lopategi1 , X. Wang1 , Y. Lu1 , N. Kitamura1 , X. Ge1 , R. Urtasun1 , T.-M. Leun1 , I. Fiel2 , N. Nieto1 . 1 Division of Liver Diseases, Department of Medicine, 2 Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA E-mail: [email protected] Background and Aim: A key feature in the pathogenesis of liver fibrosis is ductular reaction (DR). Thus far, the mechanism for the onset of DR and its ability to drive scarring still remain unknown. Previous work from our laboratory identified osteopontin (OPN) as a matricellular protein highly induced during druginduced liver injury in hepatocytes, oval cells (OC), biliary epithelial cells (BEC) and hepatic stellate cells (HSC) and demonstrated that OPN up-regulates collagen-I expression in HSC via integrin av b3 engagement and activation of the PI3K-pAkt-NFúB signaling pathway (Hepatology, PMID:21953216); thus, we hypothesized that OPN could drive fibrogenesis by promoting DR, which could signal to HSC and enhance scarring. Results: OPN was sensitive to reactive oxygen species (ROS) in BEC and OC. Opn−/− mice were protected from thioacetamide (TAA) and bile duct ligation (BDL)-induced liver injury as shown by H&E staining, the pathology scores and ALT activity. Recombinant OPN (rOPN) decreased hepatocyte proliferation rates in vitro and the hepatocyte Ki67 index was greater in TAA-treated or BDL Opn−/− than in WT mice. In contrast, rOPN increased BEC proliferation, invasion and migration in vitro. Moreover, OC expansion, the DR score and cytokeratin-19 (CK19) staining were lower in TAA-treated and BDL Opn−/− than in WT mice, suggesting that OPN activates the OC compartment. Overall, the TAA-treated and BDL Opn−/− developed less fibrosis than WT mice. Lastly, co-culture of HSC with BEC showed increased collagen-I and OPN protein expression compared to HSC in mono-culture. Conclusion: OPN emerges as a key matricellular cytokine driving DR and contributing to scarring and liver fibrosis.
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383 BREVISCAPINE ATTENUATES THE CONTRACTILE FORCE OF RAT HEPATIC STELLATE CELLS INDUCED BY ENDOTHELIN-1 AND DECREASES PORTAL VEIN PRESSURE IN CIRRHOTIC RATS C. Lu1 , H. Xu1 , J. Ping1 , L. Xu1,2 . 1 Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 2 Institute of Liver Diseases, University of Traditional Chinese Medicine, Shanghai, China E-mail: [email protected] Background and Aims: The contraction of hepatic stellate cells (HSCs) is one of important mechanism of portal hypertension in liver cirrhosis. Breviscapine is an ingredient of Erigeron breviscapus (Vant.) Hand-Mazz. In this article, we investigated the effect of Breviscapine on inhibiting contraction of rat HSCs induced by Endothelin-1 (ET-1) and the effect on decreasing portal vein pressure in cirrhotic rats. Methods: HSCs were isolated from Sprague-Dawley rats. Contractility of HSCs and myosin light chain (MLC) phosphorylation were evaluated using collagen gel contraction assay and western blot analysis respectively. Fluo-3/AM and FITC-labeled palloidin were utilized to detect intracellular concentration of calcium and F-actin organization. Cirrhosis model were established by administrated intraperitoneally with 0.5% of dimethylnitrosamine (DMN) in rats for 4 weeks and then subcutaneous injected with Breviscapine for 3 weeks. The alteration of liver pathology was observed, concentration of hepatic hydroxyproline and the portal vein pressure was measured. Results: ET-1(10 nM) induced a significant contraction of HSCs In vitro. The lattice area of collagen gel on which HSCs planted was 49.96±4.46% compared to 80.58±3.26% in controls. 10 mM and 100 mM Breviscapine attenuated the contractile, showing an area of 74.33±5.5% and 67.39±3.47% (vs ET-1 group, P < 0.01). Breviscapine also ablated F-actin polymerization, decreased phosphorylation of MLC and reduced the intracellular concentration of calcium in HSCs induced by ET-1. Such results were also obtained with applying specific MLCK inhibitor ML-7, ROCK inhibitor Y-27632 or L-type calcium channel blocker Nifedipine. Liver injury, inflammation and collagen fiber deposition in the liver tissue of DMN model rats were relieved by Breviscapine In vivo. Portal vein pressure was lower in high-dose (20 mg/kg) Breviscapine group (13.40±1.23 mmHg) and in middle-dose (10 mg/kg) Breviscapine group (13.70±3.08 mmHg) than that of controls (17.60±2.66 mmHg, P < 0.05). The hepatic hydroxyproline level was also reduced in high-dose Breviscapine group (610.08±119.85 mg/g liver) compared to controls (737.82±154.88 mg/g liver, P < 0.05). Conclusions: Breviscapine could anti-fibrosis and decrease portal vein pressure in cirrhotic rats. The effect mechanisms of Breviscapine were related with inhibiting ET-1-stimulated contraction of HSCs. This inhibitory effect on contractility of HSCs is attributed to the rearrangement of stress fibers, and reduction of MLC phosphorylation and intracellular concentration of calcium. 384 HEPCIDIN KNOCKOUT MICE AS A MODEL OF IRON-OVERLOAD ASSOCIATED LIVER FIBROSIS DEVELOPMENT M. Lunova1 , S. Vaulont2 , C. Lackner3 , P. Strnad1 . 1 Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany; 2 2Institut Cochin, INSERM U1016, Universit´e Paris Descartes, Paris, France; 3 Institute of Pathology, Medical University of Graz, Graz, Austria E-mail: [email protected] Background and Aims: Hepcidin is the central regulator of iron homeostasis. Disrupted hepcidin signalling results in hereditary hemochromatosis and iron overload in chronic liver disorders. While the association between iron overload and development of end-stage liver disease is well established, the understanding of the
Journal of Hepatology 2012 vol. 56 | S71–S224
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383 BREVISCAPINE ATTENUATES THE CONTRACTILE FORCE OF RAT HEPATIC STELLATE CELLS INDUCED BY ENDOTHELIN-1 AND DECREASES PORTAL VEIN PRESSURE IN CIRRHOTIC RATS C. Lu1 , H. Xu1 , J. Ping1 , L. Xu1,2 . 1 Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, 2 Institute of Liver Diseases, University of Traditional Chinese Medicine, Shanghai, China E-mail: [email protected] Background and Aims: The contraction of hepatic stellate cells (HSCs) is one of important mechanism of portal hypertension in liver cirrhosis. Breviscapine is an ingredient of Erigeron breviscapus (Vant.) Hand-Mazz. In this article, we investigated the effect of Breviscapine on inhibiting contraction of rat HSCs induced by Endothelin-1 (ET-1) and the effect on decreasing portal vein pressure in cirrhotic rats. Methods: HSCs were isolated from Sprague-Dawley rats. Contractility of HSCs and myosin light chain (MLC) phosphorylation were evaluated using collagen gel contraction assay and western blot analysis respectively. Fluo-3/AM and FITC-labeled palloidin were utilized to detect intracellular concentration of calcium and F-actin organization. Cirrhosis model were established by administrated intraperitoneally with 0.5% of dimethylnitrosamine (DMN) in rats for 4 weeks and then subcutaneous injected with Breviscapine for 3 weeks. The alteration of liver pathology was observed, concentration of hepatic hydroxyproline and the portal vein pressure was measured. Results: ET-1(10 nM) induced a significant contraction of HSCs In vitro. The lattice area of collagen gel on which HSCs planted was 49.96±4.46% compared to 80.58±3.26% in controls. 10 mM and 100 mM Breviscapine attenuated the contractile, showing an area of 74.33±5.5% and 67.39±3.47% (vs ET-1 group, P < 0.01). Breviscapine also ablated F-actin polymerization, decreased phosphorylation of MLC and reduced the intracellular concentration of calcium in HSCs induced by ET-1. Such results were also obtained with applying specific MLCK inhibitor ML-7, ROCK inhibitor Y-27632 or L-type calcium channel blocker Nifedipine. Liver injury, inflammation and collagen fiber deposition in the liver tissue of DMN model rats were relieved by Breviscapine In vivo. Portal vein pressure was lower in high-dose (20 mg/kg) Breviscapine group (13.40±1.23 mmHg) and in middle-dose (10 mg/kg) Breviscapine group (13.70±3.08 mmHg) than that of controls (17.60±2.66 mmHg, P < 0.05). The hepatic hydroxyproline level was also reduced in high-dose Breviscapine group (610.08±119.85 mg/g liver) compared to controls (737.82±154.88 mg/g liver, P < 0.05). Conclusions: Breviscapine could anti-fibrosis and decrease portal vein pressure in cirrhotic rats. The effect mechanisms of Breviscapine were related with inhibiting ET-1-stimulated contraction of HSCs. This inhibitory effect on contractility of HSCs is attributed to the rearrangement of stress fibers, and reduction of MLC phosphorylation and intracellular concentration of calcium. 384 HEPCIDIN KNOCKOUT MICE AS A MODEL OF IRON-OVERLOAD ASSOCIATED LIVER FIBROSIS DEVELOPMENT M. Lunova1 , S. Vaulont2 , C. Lackner3 , P. Strnad1 . 1 Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany; 2 2Institut Cochin, INSERM U1016, Universit´e Paris Descartes, Paris, France; 3 Institute of Pathology, Medical University of Graz, Graz, Austria E-mail: [email protected] Background and Aims: Hepcidin is the central regulator of iron homeostasis. Disrupted hepcidin signalling results in hereditary hemochromatosis and iron overload in chronic liver disorders. While the association between iron overload and development of end-stage liver disease is well established, the understanding of the
Journal of Hepatology 2012 vol. 56 | S71–S224