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Leptin as a profibrogenic factor in the liver
AI014 AASLD ABSTRACTS
3593 LEPTIN AS A PROFIBROGENIC FACTOR IN THE LIVER. Kenichi Ikejima, Hajime Honda, Miyoko Hirose, Toru Fukuda, Tsuneo Kitamura, Yoshiyuki Takei, Nobuhiro Sato, Dept of Gastroenterology, luntendo Univ, Tokyo, lapan. Leptin is an obese gene product which is mainly produced by adipose tissue and regulates food intake. Recently it was reported that hepatic stellate cells produce leptin during transactivation in vitro. Further, we have demonstrated that leptin is also produced by activated stellate cells in vivo in the fibrotic liver induced by thioacetamide in the rat (Ikejima et aI., Hepatology, 30: 492A, 1999). However. the role of leptin in liver fibrogenesis has not been elucidated. The purpose of this study, therefore, was to clarify whether leptin and its signaling pathways are involved in fibrogenesis in the liver. Methods: Male Zucker rats, in which functionalleptin receptors are deficient, and their lean littermates were used in this study. Both Zucker rats and lean controls 5 weeks after birth were separated into two groups randomly, and thioacetamide (TAA, 200 mglkg BW) was injected intraperiotneally 3 times per week for 8 weeks. Liver histology was examined by Azan staining, and al(l) prpcollagen mRNA in the liver was measured using an RNase protection assay. Results: As expected, the control liver after 8-week TAA treatment showed remarkable fibrosis with regenerating nodule formation. In contrast, in Zucker rats, which lack leptin receptors, fibrosis in the liver was very weak and normal liver architecture was well preserved. The steady state mRNA levels of al(l) procollagen in the liver were increased largely in the lean control, however, this increase was prevented dramatically to the levels about 1/5 of lean controls. Conclusions: TAA-induced liver fibrosis was prevented dramatically in Zucker rats, which lack leptin receptors, indicating that the signaling through leptin receptors most likely plays a pivotal role in the development of fibrosis in the liver. It is postulated that leptin produced by transactivated hepatic stellate cells works as a local cytokine that augments extracellular matrix synthesis in the liver. The regulation of leptin production from transactivated hepatic stellate cells and leptin receptor-mediated signaling might provide an important therapeutic strategy for the prevention and/or regression of liver fibrosis. 3594 GENE THERAPY FOR RAT LIVER CIRRHOSIS BY NAKED HEPATOCYTE GROWTH FACTOR GENE TRANSFFER INTO LIVER. Masaharu Takeuchi, Hiranari Kanemura, Takahiro Ueki, Kiyoshi Horiguchi, Yasukane Asano, liro Fujimoto, Hyogo Coli of Medicine, Nishinomiya, lapan. Background/Aim: Hepatocyte growth factor(HGF) is recently shown to have anti-fibrogenic effect and therapeutic effect for liver cirrhosis in rat. We developed a simple and highly effective gene transfection approach to liver using naked plasmid DNA of HGF to treat rat liver cirrhosis. MethodslResults: Liver cirrhosis is induced by dimethylnitrosamine(DMN) is characterized by hepatocyte death and hyper-accumulation of fibrous component that mimics human cirrhosis. 200g of naked plasmid DNA of human-HGF gene or LacZ gene was injected into liver via hepatic artery with 25% mannitol after liver cirrhosis has been established. In LacZ transfected rats, hepatocyte as well as nonparenchymal liver cells were stained with X-gal staining. By enzyme immunoassay, h-HGF protein was detected 10 fold higher in HGF gene transfected rat liver than those of LacZ transfected control rat. Azan-Mallory/hematoxillin staining revealed that fibrosis in both periportal and centrilobular liver diminished and deformation of the liver acinus decreased two weeks after the HGF gene transfection. HGF inhibited hepatocyte apoptosis and potently stimulated hepatocyte mitosis. Moreover, HGF improved portal hypertension in cirrhosis rat (18 6cmH 2 0 in LacZ transfected rats. vs. 9 4cmHzO in HGF transfected rats.). Conclusion: Our present results indicate that HGF gene therapy using naked plasmid DNA may be translate into a useful clinical regimen for the treatment of patients with liver cirrhosis. 3643 A NOVEL RAT MODEL OF CHRONIC FIBROSING CHOLANGITIS INDUCED BY LOCAL ADMINISTRATION OF A HAPTEN REAGENT INTO THE DILATED BILE DUCT IS ASSOCIATED WITH INCREASED TNF-A PRODUCTION AND ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES (ANCA). Thomas Orth, Markus F. Neurath, Peter Schirmacher, Peter R. Galle, Werner 1. Mayet, I Medicine Clin, Mainz, Germany; I Medicine Klink, Mainz, Germany; Inst fuer Pathology, Koeln, Germany. The cholangiopathies represent a group of hepatobiliary diseases in which bile duct epithelial cells are targets for a variety of destructive processes, including immune mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by local administration of the hapten reagent 2,4,6 trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. The common bile duct was dilated due to a mild stenosis in 8 weeks old female Lewis rats. Low doses of TNBS (50 mg 1 kg) 1ethanol 1 NaCI were injected during a second laparotomy. TNBS - treatment reproducibly resulted in chronic fibrosing cholangitis. The cholangitis started directly after administration of TNBS and was observed over the entire study period of 12 weeks. The bile ducts of animals with chronic fibrosing cholangitis showed intra- and extrahepatic irregularities, beading and strictures in retrograde cholangiography. Immunohistochemical staining in
GASTROENTEROLOGY Vol. 118, No.4
TNBS - treated animals showed an increased number of macrophages in the first two weeks and an increased number of CD3+ T lymphocytes between two and four weeks. Moreover, there was an increased expression of MHC class II antigens especially in the portal tracts after TNBS treatment. There was increase in the spontaneous IFN--y, TNF-a and IL-l 0 production of liver derived mononuclear cells. AP levels remained higher in the TNBS - treated animals versus the ethanol 1 NaCl injected animals throughout the study period. In TNBS - treated rats antineutrophil cytoplasmic antibodies (ANCA) were found between one and twelve weeks after TNBS injection. Autoantibody specificities were of IgG type and directed against myeloperoxidase, catalase and actin as detected by ELISA and Western-Blot. In conclusion. we established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiografic, serologic and immunologic similarities to human primary sclerosing cholangitis. This animal model may be used to investigate pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease. 3644 INCREASED LIVER DAMAGE AFTER BILE DUCT LIGATION IN INTERFERON-GAMMA RECEPTOR DEFICIENT MICE: INTERFERON-GAMMA AS A PROTECTIVE FACTOR. Tom van der Poll, Fiebo I. ten Kate, Sander 1. van Deventer, Miguel E. Sewnath, Huug Obertop, Dirk 1. Gouma, Acad Med Ctr Amsterdam, Amsterdam, Netherlands. Septic complications after gastrointestinal procedures occur more frequently in patients with obstructive jaundice (OJ) than in non-jaundiced patients. This may be related to an increased susceptibility to endotoxin (LPS) and an increased inflammatory response to stress factors such as surgery. Interferon-v (IFN·oy) is a proinflammatory cytokine, secreted by activated T-Iymphocytes and natural killer cells, that plays an important role in infection and inflammation. IFN-oy exerts it biological effects by an interaction with the IFN-oy receptor (IFN-oyR). To determine the role of IFN-oy in the inflammatory cascade during OJ, clinical and histological effects of bile duct ligation (bdl) or sham in IFN-oyR deficient (IFN-oyR-I-) and wildtype (wt) 129SV mice were investigated. Mice were sacrificed on day 0, 1, 2, 3, 4, 5, 6, or 7 after bdl/sham (n=8/group), or underwent a survival study (n=12/group). Liver was harvested and collected in 0.4% formaldehyde for histology with routine HE staining, and heparin plasma was sampled for liver chemistry using a Hitachi auto-analyzer. All values are mean (± SEM). Statistics by Wilcoxon and One Way ANOVA. During the 7 days after bdl, IFN-oyR-I- and wt mice showed similar weight loss (5.6 ± 0.6 g and 7.0 ± 1.2g resp, NS), while none of the mice died. Sham operated mice did not show weight loss. Seven days after bdl, IFN-oyR-Imice had more severe jaundice and liver injury than wt mice, as reflected by higher bilirubin (421 ± 48 JLM vs. 271 ± 60 JLM, P<0.05), alkaline phosphatase (1088 ± 63 UIL vs. 782 ± 63 UIL' P<0.05), ALAT (1165 ± 202 UIL vs. 622 ± 113 UIL, P<0.05 and ASAT levels (2273 ± 465 UIL vs. 697 ± 98 UIL, P<0.05). In addition, the liver/body weight ratio was higher in IFN-oyR-I- than in wt mice at this time point (60 ± 7 JLg/mg vs. 43 ± 6 JLg/mg, P<0.05). Upon histological examination, livers of IFN-yR-I- mice showed more severe inflammation and necrosis than wt mice (P<0.05), although the extent of fibrosis and ductular proliferation was similar. Moreover, after bdl significant mortality occurred in IFN-oyR-I(5/12) compared with wt mice (0/12) (P 0.05). In the second data-set, 5A carriage (90.4% v 72.7%, P = 0.012, OR 3.5) and 5A allelic frequency (65.4% v 48.5%, P = 0.005, OR = 2.0) were
3593 LEPTIN AS A PROFIBROGENIC FACTOR IN THE LIVER. Kenichi Ikejima, Hajime Honda, Miyoko Hirose, Toru Fukuda, Tsuneo Kitamura, Yoshiyuki Takei, Nobuhiro Sato, Dept of Gastroenterology, luntendo Univ, Tokyo, lapan. Leptin is an obese gene product which is mainly produced by adipose tissue and regulates food intake. Recently it was reported that hepatic stellate cells produce leptin during transactivation in vitro. Further, we have demonstrated that leptin is also produced by activated stellate cells in vivo in the fibrotic liver induced by thioacetamide in the rat (Ikejima et aI., Hepatology, 30: 492A, 1999). However. the role of leptin in liver fibrogenesis has not been elucidated. The purpose of this study, therefore, was to clarify whether leptin and its signaling pathways are involved in fibrogenesis in the liver. Methods: Male Zucker rats, in which functionalleptin receptors are deficient, and their lean littermates were used in this study. Both Zucker rats and lean controls 5 weeks after birth were separated into two groups randomly, and thioacetamide (TAA, 200 mglkg BW) was injected intraperiotneally 3 times per week for 8 weeks. Liver histology was examined by Azan staining, and al(l) prpcollagen mRNA in the liver was measured using an RNase protection assay. Results: As expected, the control liver after 8-week TAA treatment showed remarkable fibrosis with regenerating nodule formation. In contrast, in Zucker rats, which lack leptin receptors, fibrosis in the liver was very weak and normal liver architecture was well preserved. The steady state mRNA levels of al(l) procollagen in the liver were increased largely in the lean control, however, this increase was prevented dramatically to the levels about 1/5 of lean controls. Conclusions: TAA-induced liver fibrosis was prevented dramatically in Zucker rats, which lack leptin receptors, indicating that the signaling through leptin receptors most likely plays a pivotal role in the development of fibrosis in the liver. It is postulated that leptin produced by transactivated hepatic stellate cells works as a local cytokine that augments extracellular matrix synthesis in the liver. The regulation of leptin production from transactivated hepatic stellate cells and leptin receptor-mediated signaling might provide an important therapeutic strategy for the prevention and/or regression of liver fibrosis. 3594 GENE THERAPY FOR RAT LIVER CIRRHOSIS BY NAKED HEPATOCYTE GROWTH FACTOR GENE TRANSFFER INTO LIVER. Masaharu Takeuchi, Hiranari Kanemura, Takahiro Ueki, Kiyoshi Horiguchi, Yasukane Asano, liro Fujimoto, Hyogo Coli of Medicine, Nishinomiya, lapan. Background/Aim: Hepatocyte growth factor(HGF) is recently shown to have anti-fibrogenic effect and therapeutic effect for liver cirrhosis in rat. We developed a simple and highly effective gene transfection approach to liver using naked plasmid DNA of HGF to treat rat liver cirrhosis. MethodslResults: Liver cirrhosis is induced by dimethylnitrosamine(DMN) is characterized by hepatocyte death and hyper-accumulation of fibrous component that mimics human cirrhosis. 200g of naked plasmid DNA of human-HGF gene or LacZ gene was injected into liver via hepatic artery with 25% mannitol after liver cirrhosis has been established. In LacZ transfected rats, hepatocyte as well as nonparenchymal liver cells were stained with X-gal staining. By enzyme immunoassay, h-HGF protein was detected 10 fold higher in HGF gene transfected rat liver than those of LacZ transfected control rat. Azan-Mallory/hematoxillin staining revealed that fibrosis in both periportal and centrilobular liver diminished and deformation of the liver acinus decreased two weeks after the HGF gene transfection. HGF inhibited hepatocyte apoptosis and potently stimulated hepatocyte mitosis. Moreover, HGF improved portal hypertension in cirrhosis rat (18 6cmH 2 0 in LacZ transfected rats. vs. 9 4cmHzO in HGF transfected rats.). Conclusion: Our present results indicate that HGF gene therapy using naked plasmid DNA may be translate into a useful clinical regimen for the treatment of patients with liver cirrhosis. 3643 A NOVEL RAT MODEL OF CHRONIC FIBROSING CHOLANGITIS INDUCED BY LOCAL ADMINISTRATION OF A HAPTEN REAGENT INTO THE DILATED BILE DUCT IS ASSOCIATED WITH INCREASED TNF-A PRODUCTION AND ANTINEUTROPHIL CYTOPLASMIC AUTOANTIBODIES (ANCA). Thomas Orth, Markus F. Neurath, Peter Schirmacher, Peter R. Galle, Werner 1. Mayet, I Medicine Clin, Mainz, Germany; I Medicine Klink, Mainz, Germany; Inst fuer Pathology, Koeln, Germany. The cholangiopathies represent a group of hepatobiliary diseases in which bile duct epithelial cells are targets for a variety of destructive processes, including immune mediated damage. We describe a novel rat model of chronic fibrosing cholangitis induced by local administration of the hapten reagent 2,4,6 trinitrobenzenesulfonic acid (TNBS) into the dilated bile duct. The common bile duct was dilated due to a mild stenosis in 8 weeks old female Lewis rats. Low doses of TNBS (50 mg 1 kg) 1ethanol 1 NaCI were injected during a second laparotomy. TNBS - treatment reproducibly resulted in chronic fibrosing cholangitis. The cholangitis started directly after administration of TNBS and was observed over the entire study period of 12 weeks. The bile ducts of animals with chronic fibrosing cholangitis showed intra- and extrahepatic irregularities, beading and strictures in retrograde cholangiography. Immunohistochemical staining in
GASTROENTEROLOGY Vol. 118, No.4
TNBS - treated animals showed an increased number of macrophages in the first two weeks and an increased number of CD3+ T lymphocytes between two and four weeks. Moreover, there was an increased expression of MHC class II antigens especially in the portal tracts after TNBS treatment. There was increase in the spontaneous IFN--y, TNF-a and IL-l 0 production of liver derived mononuclear cells. AP levels remained higher in the TNBS - treated animals versus the ethanol 1 NaCl injected animals throughout the study period. In TNBS - treated rats antineutrophil cytoplasmic antibodies (ANCA) were found between one and twelve weeks after TNBS injection. Autoantibody specificities were of IgG type and directed against myeloperoxidase, catalase and actin as detected by ELISA and Western-Blot. In conclusion. we established a novel rat model of chronic fibrosing cholangitis with histologic, cholangiografic, serologic and immunologic similarities to human primary sclerosing cholangitis. This animal model may be used to investigate pathomechanisms of chronic cholangitis without concomitant inflammatory bowel disease. 3644 INCREASED LIVER DAMAGE AFTER BILE DUCT LIGATION IN INTERFERON-GAMMA RECEPTOR DEFICIENT MICE: INTERFERON-GAMMA AS A PROTECTIVE FACTOR. Tom van der Poll, Fiebo I. ten Kate, Sander 1. van Deventer, Miguel E. Sewnath, Huug Obertop, Dirk 1. Gouma, Acad Med Ctr Amsterdam, Amsterdam, Netherlands. Septic complications after gastrointestinal procedures occur more frequently in patients with obstructive jaundice (OJ) than in non-jaundiced patients. This may be related to an increased susceptibility to endotoxin (LPS) and an increased inflammatory response to stress factors such as surgery. Interferon-v (IFN·oy) is a proinflammatory cytokine, secreted by activated T-Iymphocytes and natural killer cells, that plays an important role in infection and inflammation. IFN-oy exerts it biological effects by an interaction with the IFN-oy receptor (IFN-oyR). To determine the role of IFN-oy in the inflammatory cascade during OJ, clinical and histological effects of bile duct ligation (bdl) or sham in IFN-oyR deficient (IFN-oyR-I-) and wildtype (wt) 129SV mice were investigated. Mice were sacrificed on day 0, 1, 2, 3, 4, 5, 6, or 7 after bdl/sham (n=8/group), or underwent a survival study (n=12/group). Liver was harvested and collected in 0.4% formaldehyde for histology with routine HE staining, and heparin plasma was sampled for liver chemistry using a Hitachi auto-analyzer. All values are mean (± SEM). Statistics by Wilcoxon and One Way ANOVA. During the 7 days after bdl, IFN-oyR-I- and wt mice showed similar weight loss (5.6 ± 0.6 g and 7.0 ± 1.2g resp, NS), while none of the mice died. Sham operated mice did not show weight loss. Seven days after bdl, IFN-oyR-Imice had more severe jaundice and liver injury than wt mice, as reflected by higher bilirubin (421 ± 48 JLM vs. 271 ± 60 JLM, P<0.05), alkaline phosphatase (1088 ± 63 UIL vs. 782 ± 63 UIL' P<0.05), ALAT (1165 ± 202 UIL vs. 622 ± 113 UIL, P<0.05 and ASAT levels (2273 ± 465 UIL vs. 697 ± 98 UIL, P<0.05). In addition, the liver/body weight ratio was higher in IFN-oyR-I- than in wt mice at this time point (60 ± 7 JLg/mg vs. 43 ± 6 JLg/mg, P<0.05). Upon histological examination, livers of IFN-yR-I- mice showed more severe inflammation and necrosis than wt mice (P<0.05), although the extent of fibrosis and ductular proliferation was similar. Moreover, after bdl significant mortality occurred in IFN-oyR-I(5/12) compared with wt mice (0/12) (P