Hepatocyte growth factor promotes compensatory hypertrophy and makes major hepatectomy possible in liver cirrhosis

Parallel Session 4: Molecular Hepatology, Regeneration and Cancer the presence of HGF and EGF. Levels of Jaggedl increased during cell culture as detected by Western blot analyses. Following TGF-alpha treatment we detected an early increase and a later decrease in the Jaggedl levels. Minimal Notchl-protein was detected in fresh hepatocytes followed by a transient increase and a sustained decrease. This increase was suppressed by TGF-alpha. Our results also show that Jaggedl is well expressed in rat liver and down-regulated 48 h after partial hepatectomy. This coincides with the peak of biliary proliferation and TGF-alpha levels after partial hepatectomy. Only minor changes were seen in Notchl levels after partial hepatectomy. The above results indicate that cell-cell interaction via Notch/Jagged signaling pathway varies as a result of cell proliferation or liver regeneration. We hypothesize that this pathway may negatively regulate cell proliferation during liver regeneration especially of the biliary component.

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HEPATOCYTE GROWTH FACTOR PROMOTES COMPENSATORY HYPERTROPHY AND MAKES MAJOR HEPATECTOMY POSSIBLE IN LIVER CIRRHOSIS

Toshimi Kaido 1, Hideaki Oe 2, Masayuki Imamura 2. 1Department of

Surgery, Otsu Municipal Hospital, Otsu; 2Department of Surgery and Surgical Basic Science, Kyoto University School of Medicine, Kyoto, Japan In this study, we examined whether HGF is useful in accelerating compensatory hypertrophy caused by portal branch ligation in normal, jaundiced, and cirrhotic rats and also investigated the possibility of major hepatectomy in liver cirrhosis. Normal, obstructive jaundiced, and dimethylnitrosamineinduced cirrhotic rats underwent portal ligation of the left lateral and median branches, supplying approximately 70% of the total volume of the liver. Simultaneously, the animals were continuously treated with either recombinant human HGF (rhHGF) or vehicle alone via an intraperitoneally implanted osmotic pump. Two and 4 days after portal ligation, the degree of compensatory hypertrophy in unoccluded lobes was examined by measuring the wet weight ratios of the unoccluded lobes to the whole liver and the 5-bromo-2'-deoxyuridine labeling index of hepatocytes in each group. To examine the possibility of major hepatectomy in cirrhotic liver, the occluded lobes were resected 4 days after portal ligation. The HGF treatment significantly increased the wet weight ratios and the DNA synthesis in nonoccluded lobes 2 and 4 days after portal ligation in each liver. The HGF treatment markedly suppressed the posthepatectomy liver dysfunction and improved the survival rate of the cirrhotic rats at 48 h after the major hepatectomy. Continuous rhHGF administration accelerated compensatory hypertrophy in various types of liver and made extensive hepatectomy possible in liver cirrhosis

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HUMAN UPA GENE THERAPY AS A CO-ADYUVANT ELEMENT OF BILIO-DIGESTIVE ANASTOMOSIS IN THE TREATMENT OF SECONDARY BILIARY CIRRHOSIS

Juan Armendariz-Borunda I , Alejandra Miranda 1, Silvia Salgado l, Miriam Bueno I , Jose Vera I , Estuardo Aguilar-Cordova 2. llnstituto de

Biologia Molecular y Terapia Genica, CUCS, Universidad de Guadalajara, Guadalajara, Mexico; 2Harvard Gene Therapy Initiative, Boston MA, USA We have previously shown (Salgado et, al., MOLECULAR THERAPY 2 (6): 545-551, 2000), that one single injection of an adenoviral vector bearing a modified cDNA coding for a non-secreted form of human urokinasePlasminogen Activator (Ad-huPA) efficiently reverts CCIA-induced liver cirrhosis which resembles human alcoholic cirrhosis. Here, we established an experimental model of secondary biliary cirrhosis by ligating and sectioning the common bile-duct in Wistar rats for 4 weeks. Then, we performed a bilio-digestive anastomosis to drain the clogged bile, though an important degree of fibrosis was still noticeable. After this surgical procedure, an injection of 6 x 1011 viral particles/kg of Ad-huPA was adminis-

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tered via iliac vein to cirrhotic animals. After ten days, a significant 40% decrease in fibrosis was noted as compared with rats treated with a control Ad-GFP adenovector (p < 0.001). Also, a decrease in total and direct bilirubins was dramatic, though ALT and AST did not diminished at the same extent. Hepatic cells proliferation was measured with an anti-PCNA antibody and Ad-huPA treated animals had a greater labeling index as well as abundant mitotic figures as compared with Ad-GFP-animal, indicating hepatic regeneration. Finally, all these cellular events were reflected in decreased ascitis and gastric varices in Ad-huPA improved animals. Hence, we believe that urokinase-Plasminogen Activator gene therapy might be useful as a co-adjuvant therapy to bilio-digestive anastomosis in the treatment of human secondary biliary cirrhosis.

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PRENEOPLASTIC MARKERS OF HEPATOCARCINOGENESIS IN HEPATITIS B VIRUS CARRIERS

Y.R. Jin 1.2, H.W.L. Hann 2, Marcy Clayton ~, Anne Bussard 4, Catherine Liu 4, Mark Feitelson 1,3. 1Department of Pathology, Anatomy

and Cell Biology; 2Department of Medicine; 3Department of Microbiology and Immunology; 4jefferson Medical, College, Thomas Jefferson University, Philadelphia PA, USA Hepatitis B virus (HBV) carriers are at high risk for the development of hepatocellular carcinoma (HCC), but there are no convenient markers of early detection. Since HBV encoded X antigen (HBxAg) likely contributes to HCC by up- or down-regulation of host gene expression, X positive and negative cells were made and subjected to differential display. When specific ELISAs were constructed measuring differentially expressed antigen and corresponding antibody (Ab) in serum samples from chronically infected patients, HBxAg triggers the development of Abs to several differentially regulated genes. In serial serum samples collected from carriers, 8 out of 11 cases tested (73%) developed Abs to one or more of these over-expressed proteins up to a year prior to the clinical diagnosis of HCC. Cross-sectional studies with other patients showed <20% of serum sampies from HBV carriers with or without chronic liver disease had Abs to at least two markers, while 70% of carriers with HCC were Ab positive. The results suggest that these Abs may detect early HCC among HBV carriers.

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BIOLOGICAL FEATURES OF HEPATOCELLULAR CARCINOMA EXPRESSING VARIANT ESTROGEN RECEPTORS TRANSCRIPTS

Alessandra Colantoni 1,2 Nicola De Maria 1, Mauro Manno I , Simona Lambertini 1, Ilva Ferretti l, Alberto Bagni 3, Federico Manentil, Erica Villa I . 1Division of Gastroenterology - University of Modena &

Reggio Emilia; SDepartment of Pathology- University of Modena & Reggio Emilia, Italy; 2Department of Medicine - Loyola University of Chicago - Maywood, IL, USA Hepatocellular carcinoma (HCC) expresses predominantly either the wild type form of estrogen receptor (ER) alpha (wtER) or an exon 5 deleted ER variant form (vER) that keeps the transcriptional activity of the receptor. HCC vER+ is extremely aggressive and the median survival of patients with HCC vER+ is significantly lower than that of those with wtER+ tumors. Aim of the study was to determine whether HCCs vER+ differ from wtER+ in terms of biological features. Cell proliferation, cyclin D1 expression, TGF betal production and metalloproteinase (MMP) 2 activity and MMP9 levels (index of local invasiveness) were measured in 30 HCC wtER+ and 30 vER+. A portion of HCCs was used for immunohistochemistry (Ki67 and PCNA, indexes of proliferation, cyclin D1 and MMP-9) and frozen samples were used for ER determination by RT-PCR and ELISA assays (TGF and MMP-2). Proliferation levels and the expression of cyclin D1 were greater in vER + than wtER+ HCCs (p = 0.001). The production of TGF betal differed between the two groups of HCC and MMPs levels and activity were lower in HCC wtER+ than HCC vER+. These data indicate that the presence of vER identifies HCC with a more pronounced disruption