- Email: [email protected]
96 SEMAPHORIN7A, A NEW PLAYER IN HEPATIC FIBROSIS: AN IN VITRO AND IN VIVO STUDY
ORAL PRESENTATIONS glucose supplementation, implying inefficient mitochondrialderived energy production. Caveolins can act independently of caveolae and are found in non-caveolar regions of caveolaecontaining cells including ER, Golgi and possibly mitochondria. Mitochondria can be closely apposed to plasma membrane caveolae and caveolin-1 has been detected in liver mitochondria. Since mitochondrial cholesterol modulates hepatocellular susceptibility to TNF/Fas, our aim was to examine the role of caveolin-1 in the regulation of mitochondrial cholesterol and fate of hepatocytes following TNF/Fas challenge. Methods: Primary hepatocytes and isolated mitochondria were prepared from wild-type and caveolin-1 knockout mice (KO). Oxygen consumption, GSH, free cholesterol levels, mitochondrial membrane permeabilization and subsequent release of cytochrome c and Smac/Diablo were analyzed in isolated mitochondria, while susceptibility to TNF/Fas-mediated apoptosis was examined in primary hepatocytes. Results: Impaired state 3 respiration and decreased acceptor control ratio were observed in mitochondria from caveolin-1 KO mice compared to wild-type mice. tBid+Bax-induced release of cytochrome c and Smac/Diablo was greater in isolated mitochondria from caveolin-1 KO mice than from wild-type mice. Moreover, these findings were accompanied by higher mitochondrial free cholesterol levels (30–50%) and GSH depletion (40–60%). Consistent with the latter results, hepatoyctes from caveolin-1 KO mice were more susceptible to Fas-mediated cytochrome c release and apoptosis with respect to wild-type mice, an outcome that was prevented upon GSH ethyl ester pretreatment. Finally, mitochondrial cholesterol extraction by methylcyclodextrin improved mitochondrial respiration and prevented tBid+Bax-mediated cytochrome c release. Conclusions: These findings indicate a novel role of caveolin-1 in the hepatocellular susceptibility to TNF/Fas-mediated apoptosis by regulating mitochondrial cholesterol trafficking and GSH homeostasis, implying that caveolin-1 could play a role in liver diseases, such as alcoholic and non-alcoholic steatohepatitis. 96 SEMAPHORIN7A, A NEW PLAYER IN HEPATIC FIBROSIS: AN IN VITRO AND IN VIVO STUDY S. De Minicis1 , C. Rychlicki1 , L. Agostinelli1 , L. Trozzi1 , C. Candelaresi1 , S. Saccomanno1 , R. Bataller2 , E. Juez2 , M. Marzioni1 , A. Benedetti1 , G. Svegliati-Baroni1 . 1 Gastroenterology, University of Ancona, Ancona, Italy; 2 Liver Unit, Hospital Clinic of Barcelona, Barcelona, Spain E-mail: [email protected] Introduction: Semaphorin7a (Sema7a) is a membrane-anchored protein that regulates axonal growth, immune and inflammatory response. Recent evidences have confirmed an effect of Sema7a on the regulation of bleomycin-induced pulmonary fibrosis. However, no information is available on Sema7a expression in the liver and its potential role in the regulation of hepatic fibrosis. Aims: To evaluate Sema7a expression in the liver and its role in liver fibrosis. Methods: Liver injury was induced in vivo by CCl4 i.p.injection in wild type and Sema7a KO-mice. Kupffer cells, hepatic stellate cells (HSCs), hepatocytes and endothelial cells were isolated by a nycodenz-based gradient method from mice livers. Sema7a over-expression-vector in HSCs was performed by AdEasy system. Sema7a expression was evaluated by westernblot, immunofluorescence staining and quantitative real-time PCR (qRT-PCR) methods. Inflammation and fibrogenic markers were evaluated by qRT-PCR in vitro and in vivo. Human liver samples of normal and cirrhotic liver (virus, alcohol, NASH) were evaluated for Sema7a expression by qRT-PCR. S44
Results: Sema7a expression was increased (9–10 folds) in human fibrotic-liver samples versus normal-liver. Among the different cell types in the liver, Sema7a is mainly expressed in HSCs. Doublelabel confocal immunofluorescence shows Sema7a and aSMA colocalization in fibrotic liver. Sema7a expression was significantly increased (3–4 folds) during liver injury and (3 folds) in activated HSCs (5-days culture) versus quiescent HSCs (overnight culture), as demonstrated by qRT-PCR and immunofluorescence staining. In addition, TGFb-stimulated HSCs show increased expression of Sema7a. Moreover, over-expression of Sema7a-vector in HSCs shows a significant increase of the fibrogenic marker collagen a1(I) (1.8 folds) versus HSCs treated with target-vector. Sema7a KO-mice developed a significantly reduced collagen a1(I) gene expression, liver fibrosis and HSC activation after CCl4 treatment versus wild type-mice, as demonstrated by qRT-PCR, Sirius-Red staining and western-blot for aSMA. Conclusions: This study represents the first demonstration of Sema7a expression in the liver and of its increase in the course of liver fibrosis, both in mouse and in humans. Sema7a is mainly expressed in HSCs versus other cell types in the liver. The reduced fibrosis and HSC activation in Sema7a KO-mice indicate that it plays a critical role in regulating the fibrogenetic process. 97 THE BENEFICIAL EFFECT OF ANGIOTENSIN-BLOCKING AGENT ON LIVER FIBROSIS IN PATIENTS WITH ALCOHOLIC LIVER DISEASE: A RANDOMIZED CONTROLLED TRIAL M.Y. Kim1 , M.Y. Cho2 , S.K. Baik1 , C.S. Won1 , J.W. Byun1 , H.J. Park1 , H.J. Choi1 , H.K. Chun1 , S.Y. Park1 , Y.H. Kwon1 , G.J. Cheon3 , Y.D. Kim3 . 1 Department of Internal Medicine and Institute of Basic Medical Science, 2 Department of Pathology, Yonsei University Wonju College of Medicine, Wonju, 3 Department of Internal Medicine, Kangneung Asan Hospital, Ulsan University College of Medicine, Kangneung, Republic of Korea E-mail: [email protected] Background and Aims: Recent studies have shown that reninangiotensin system is implicated in hepatic fibrogenesis in vitro and in-vivo, however no study examine liver fibrosis via histology in human with alcoholic liver disease. We prospectively studied the antifibrotic effect of angiotensin II type 1 receptor blocking agent (ARB) in patients with alcoholic liver disease. Methods: Patients with alcoholic liver fibrosis (≥F2) were randomized to receive either ARB, candesartan 8 mg with ursodeoxycholic acid (UDCA, 600 mg/day) (n = 37), or UDCA alone (n = 36) as control for 6 months. All enrolled patients underwent liver biopsies twice at baseline and 6 months later for measurement of fibrosis score, area of fibrosis and a-smooth muscle actin (SMA) positive by immunohistochemical staining, hydroxyproline as quantitative measurement of fibrosis. Transforming growth factor b (TGFb), collagen, angiotensin type I receptor (AT1-R), tissue inhibitor of metalloproteinase-1 (TIMP-1), Rac1, p22phox which represent oxidant stress, in liver parenchyma by real time RT PCR were also measured before and after 6 months therapy. Fibrosis was evaluated by one liver pathologist who was blinded to clinical data of patients, using the Laennec fibrosis scoring system which divides fibrosis stage 4 as subclass 4A, 4B and 4C according to thickness of fibrotic septa. In addition, all patients were closely monitored for alcohol intake and any patient who intake alcohol was dropped out. Results: Candesartan reduced the fibrosis score according to the Laennec system from 3.5±1.7 to 3.1±1.1 compared to controls (P < 0.05). Candesartan also reduced the fibrosis area from 11.2±6.0 to 8.2±5.1 and hydroxyproline levels (mg/g liver tissue) from 7.8±2.6 to 5.9±1.9 significantly compared to controls (P < 0.05). In addition, the relative expression of TGF-b1, collagen-1, AT1-R, TIMP-1, Rac1 and p22phox by real-time RT-PCR were decreased in
Journal of Hepatology 2010 vol. 52 | S43–S54
Results: Sema7a expression was increased (9–10 folds) in human fibrotic-liver samples versus normal-liver. Among the different cell types in the liver, Sema7a is mainly expressed in HSCs. Doublelabel confocal immunofluorescence shows Sema7a and aSMA colocalization in fibrotic liver. Sema7a expression was significantly increased (3–4 folds) during liver injury and (3 folds) in activated HSCs (5-days culture) versus quiescent HSCs (overnight culture), as demonstrated by qRT-PCR and immunofluorescence staining. In addition, TGFb-stimulated HSCs show increased expression of Sema7a. Moreover, over-expression of Sema7a-vector in HSCs shows a significant increase of the fibrogenic marker collagen a1(I) (1.8 folds) versus HSCs treated with target-vector. Sema7a KO-mice developed a significantly reduced collagen a1(I) gene expression, liver fibrosis and HSC activation after CCl4 treatment versus wild type-mice, as demonstrated by qRT-PCR, Sirius-Red staining and western-blot for aSMA. Conclusions: This study represents the first demonstration of Sema7a expression in the liver and of its increase in the course of liver fibrosis, both in mouse and in humans. Sema7a is mainly expressed in HSCs versus other cell types in the liver. The reduced fibrosis and HSC activation in Sema7a KO-mice indicate that it plays a critical role in regulating the fibrogenetic process. 97 THE BENEFICIAL EFFECT OF ANGIOTENSIN-BLOCKING AGENT ON LIVER FIBROSIS IN PATIENTS WITH ALCOHOLIC LIVER DISEASE: A RANDOMIZED CONTROLLED TRIAL M.Y. Kim1 , M.Y. Cho2 , S.K. Baik1 , C.S. Won1 , J.W. Byun1 , H.J. Park1 , H.J. Choi1 , H.K. Chun1 , S.Y. Park1 , Y.H. Kwon1 , G.J. Cheon3 , Y.D. Kim3 . 1 Department of Internal Medicine and Institute of Basic Medical Science, 2 Department of Pathology, Yonsei University Wonju College of Medicine, Wonju, 3 Department of Internal Medicine, Kangneung Asan Hospital, Ulsan University College of Medicine, Kangneung, Republic of Korea E-mail: [email protected] Background and Aims: Recent studies have shown that reninangiotensin system is implicated in hepatic fibrogenesis in vitro and in-vivo, however no study examine liver fibrosis via histology in human with alcoholic liver disease. We prospectively studied the antifibrotic effect of angiotensin II type 1 receptor blocking agent (ARB) in patients with alcoholic liver disease. Methods: Patients with alcoholic liver fibrosis (≥F2) were randomized to receive either ARB, candesartan 8 mg with ursodeoxycholic acid (UDCA, 600 mg/day) (n = 37), or UDCA alone (n = 36) as control for 6 months. All enrolled patients underwent liver biopsies twice at baseline and 6 months later for measurement of fibrosis score, area of fibrosis and a-smooth muscle actin (SMA) positive by immunohistochemical staining, hydroxyproline as quantitative measurement of fibrosis. Transforming growth factor b (TGFb), collagen, angiotensin type I receptor (AT1-R), tissue inhibitor of metalloproteinase-1 (TIMP-1), Rac1, p22phox which represent oxidant stress, in liver parenchyma by real time RT PCR were also measured before and after 6 months therapy. Fibrosis was evaluated by one liver pathologist who was blinded to clinical data of patients, using the Laennec fibrosis scoring system which divides fibrosis stage 4 as subclass 4A, 4B and 4C according to thickness of fibrotic septa. In addition, all patients were closely monitored for alcohol intake and any patient who intake alcohol was dropped out. Results: Candesartan reduced the fibrosis score according to the Laennec system from 3.5±1.7 to 3.1±1.1 compared to controls (P < 0.05). Candesartan also reduced the fibrosis area from 11.2±6.0 to 8.2±5.1 and hydroxyproline levels (mg/g liver tissue) from 7.8±2.6 to 5.9±1.9 significantly compared to controls (P < 0.05). In addition, the relative expression of TGF-b1, collagen-1, AT1-R, TIMP-1, Rac1 and p22phox by real-time RT-PCR were decreased in
Journal of Hepatology 2010 vol. 52 | S43–S54