232A AASLD ABSTRACTS HEPATOLOGY October 1995

50 1 EXPRESSION OF HEPARIN-BINDING EGF-LIKE GROWTH FACTOR IN THE INJURED RAT LIVERS TREATED WITH CARBON TETRACHLORIDE AND D-GALACTOSAMINE S Kiso I. S Kawata 1. S Tamura t. N Ito 1. H Tsushima l. A Yamada 1. O Oshikawa l. S Tamai I. S Hieashivama 2, N Tani~uchi 2. Y Matsuzawa t tSecond Dept. of Int. Med., 2Dept. of Biochem., Osaka University Medical School, Osaka 565, Japan

We have recently reported that heparin-binding EGF-like growth factor (HB-EGF) is a new hepatotrophic factor in regenerating rat liver after partial hepatectomy. To clarify the participation of HB-EGF in liver regeneration after hepatic injury, we examined the expression of HB-EGF in the liver after hepatotoxin-induced liver injury. MATERIALS AND METHODS (1) Male Sprague-Dawley rats weighing about 200gm were used. Experimental hepatitis was induced by intraperitoneal injection Of CC14 (0.5ml/250g b.w.) or D- galactosamine(250mg/kg b.w.). (2) Their liver were sequentially removed (0,6,12,18,24,36,48, and 72 hours). Poly(A) + RNAs were extracted from the livers of normal, and hepatotoxins-treated rats. (3) Northern blot analysis was performed with these 5 pg of poly(A) + RNAs using [32p] dCTP-labeled rat HB-EGF eDNA. (4) Membrane fraction obtained from the liver was chromatographed on the heparin columns. The concentrated samples were electrophoresed on 15% SDS-polyacryamide gel. Proteins were transferred to a nitrocellulose membrane and detected with antibody to HB-EGF. RESULTS (1)The level of HB-EGF mRNA was very low in normal rat liver,.but increased markedly in the liver of rats treated with CC14 and reached a maximum at 6 h. (2) HB-EGF mRNA level increased markedly in the liver of rats treated with D-galactosamine and reached to the maximum level at 18 h. (3) By using Western blot analysis, HB-EGF protein in the liver of CC14-treated rats (at 6 h) increased about 3.4- fold over normal. CONCLUSION The level of HB-EGF gene markedly increased in the injured rat liver. These results indicated that HB-EGF may play a role in liver regeneration after injury by hepatotoxins.

502 RECOVERY OF RATS WITH D-GALACTOSI~41NE-INDUCED ACUTE LIVER FAILURE AFTER INJECTION OF THE LIVER GROWTH FACTOR, LGF. JJ Dfaz-GiL, C Riga i C Hachfn. RN Cereceda, HC guijarro, R Navided, R garcfa-Ca~ero~ M de Foro~da i JP~rez de Die~o, C Tr| t la, P Escart~n. C[inica Puerta de Hierro and Universidad Comptutense, Nadrid, Spain.

Hale Wistar rats (160 g bw) were injected with 2. 6 g O-galactosamine (D*gat)/kg I:~ in a Single injection. Under these Conditions, ice detected over 50% ntortatity within 72 hr. Tv, entwfour hours after D-GaL administratiofl~ we injected LDf, 8 #g/rat in a single ~njection, into one group of animals. Rats were sacrificed 48 hr after D-Rat injection (nx:caent of maximum hepatic necrosis), and the findings in those treated wi~h D-Gal only, group A, were compared with those correspand~ng to group B, injected uith D-Gat ÷ LGF.

Taking as 10~g the vatues of parameters of healthy rats, the levels for animals in group A were AST = 1756%, ALl = 1831%, aLcatine phosphatase = 233%, totat serum proteins = 72%, atl~min = S4X, total bi l i rubin = 3.21 mR/dr. The corresponding values for rats of group B were: AST = 90%, ALT = 135%, alkaline phosphatase ~94%, total proteins = 95%, albumin = 99'~, total b i l i rubin = 0.15 mg/dl. Levels of total Ca in Liver tissue were: A = 277%, and B = 112%. Values for malondiaLdehyde (MDA), end product of l ipid peroxidati~, were: A = 189%, and 8 = 97% (n = 8, p < 0.001). The GSH/G$SG ratio in l iver tissue, an index for prevention of oxidative stress, was 67% in group A and 95% in group B. Liver ONA synthesis, measured by fl- thyrMdine incorporation into DNA, was 939% in rats of group A, and rose to 2065~ in rats of group B after LGF injection.

Light microscopy of Liver biopsies of group A rats showed generalized acide- phit ie hepatoceltutar necrosis, more intense in central areas. Inflammatory in- f i l t ra t ion around portal areas was also observed, as welt as abundant Kupffer celts with cellular debris in digestion. In rats of group B, hepatic architecture was signif icantly restored, following a centro:portal gradient, with a remarkable re- version of the effects produced by D-Gal.

ALl these data demonstrate that LgF is capable of correcting the deleterious effects caused i n i t i a l l y by D-Gal in the l iver (maxinvams of Ca and HDA are detect- able in the f i r s t 12 hr), d~Je to i ts intense act iv i ty as both a free radical scav- enger and a Growth Factor. Furthermore, these data suggest that LgF could be a useful tool in situations of acute hepatic fai lure.

503 CELLULAR LOCALIZATION OF HEPATOCYTE GROWTH FACTOR IN RAT AND HUMAN LIVER TISSUES. H Herbst, T Cramer , M Bauer. D Schunnan = Instltut of Pathology and *Department of Gastroenterology, Klinikum Benjamin Franklin. Free University, Berlin, Germany.

Hepatocyte regeneration following parenchymal injury is stimulated by a variety of growth factors, most prominently epidermal growth factor (EGF) and hepatocyte growth factor/scatter factor (HGF). The HGF-receptur is identical to the c-met proto-oneogene product expressed on hepatocytes. The cellular sources of HGF in the liver are ill-def'med. Studies on cell lines and primary cell cultures have implicated lipocytes, endothelial cells, macrophages, lymphocytes and other cell types in the generation of HGF in the liver. We have tested the cellular origin of HGF in rat and human liver by isotopic ilt sire hybridization combined with immunohistology for: ceU-type specific markers in normal and fibrotic rat liver (CCt 4- intoxicationl and human fibrotic/cirrhotic livers. In rats with acute CC14-injury, HGF transcripts became detectable 6 hours after intoxication in sinusoidal cells with prominent expression adjacent: to tbe area of parenchymal damage. Transcript levels remained constant until 12 hours and decreased subsequently. In rats receiving repeated CCl4-injections , highest transcript levels were found at 12 hours after each last injection. HGF-specific signals were predominantly found in desmin-positive cells, i.e. lipocytes. Human livers with moderate to severe fibrosis and cirrhosis displayed variable transcript levels in sinusoidal mesenchymal cells, all of which were labeled by antibodies against vimentin. Antibodies against (z-smooth muscle actin stained variable proportions of these cells, whereas no co- localization was noted for HGF and antigens CD31 (endothelial cells), CD68 (macrophages), and CD45 (lymphocytes). In conclusion, (activated) lipocytes are the principal sources of HGF in the liver and express this factor in a distinct temporal and spatial pattern.

504 EXPRESSION OF C-MET mRNA AND TGF-J31 mRNA CORRELATES WITH DUCTULAR PROLIFERATION AND FIBROSIS IN GRAFTED RAT LIVERS

M Zhao, D Zhao, AM Wheatley, RR Fdis, JA Laissue, A Zimmermann. Depts. of Pathology, Visceral and Transplantation Surgery, and Institute of Clinical and Experimental Research, University of Berne, Switzerland.

Ductular proliferation (DP) accompanied by septal fibrosis (SF) occurs in many l iver diseases. The pathogenesis of both, DP and SF, is not well known, but growth factors may play a significant role. Recently we showed that DP and SF occurred in syngeneic rat l iver transplantation without graft artedalization(NOLT) but not in rearterialized grafts (AOL'F) ( Hepatology 1995; 21:1353-1360). Here we used these models each in a non-rajection (NR) and rejection(AR) configuration to establish if (i) DP is associated with an upregulation of c-met m R N A and (ii) SF with TGF-J]I mRNA expression. Methods: NPJNOLT and NR/AOLT transplants were performed using a male Lewis rat combination while a DA to Lewis combination was used for AR, resulting in 4 groups, NR-NOLT (N=5), NR-AOLT (N=4), AR-NOL T (N=6) , AR-AOLT (N=10). No immunosupprassion was used. Animals were sacrif iced st day 10 post- OLT. Morphometry of the livers was performed. RNA was extracted and mRNA's quantified by Northern blotting using c-met and TGF-J]I probes. Localization of c-met mRNA was assessed by in situ hybridization (ISH). Results: c-met mRNA was not detected in the NR-AOLT group, but was moderately expressed in the NR-NOLT group showing no l iver cell damage but DP and SF. c-met mRNA was strongly elevated in both the AR-AOLT and AR-NOLT groups where hepatocyte damage, infiltration, DP and SF were in evidence, c-met mRNA levels correlated with the DP volume density (p<0.01) in NR-NOLT, AR-AOLT and AR-NOLT. By use of !SH, c-met mRNA was found to be localized in hepatocytes and DP cells. Levels of TGF-B1 mRNA correlated with the degree of infiltration, venous endothelitis, DP and S F i n the AR groups, and with DP and SF in the NR-NOLT group (p<0.05). Conclusion: The findings indicate that c-met-and TGF-131 play a significant role in the pathogenesis of DP and SF, respectively, in rat l iver grafts.

Supported in part by SNF Nr.3200-042555.