2 7 8 A A A S L D A B S T R A C T S HEPATOLOGY O c t o b e r 1995

6 8 5 IN'l'l~,JgUllN I ~ l g , / ~ n KxTKACBIJ, ULAR MATRIX DEGRADATION AND PLAMilINOGI~ ACTIVATOR ACTIVITY [ ] ~ FROM CIRRHOTIC RATS. L Rodriguez-Fragoso a, MP Gonz~ilez ~, and P rdurlel *. "Departamento de Gastroenterologia, instituto Nacional de la NutrJcf6n Salvador ZubirAn, M6xJco, D.F. and ~epartamento de Fatmacologia y Toxicologia, Centre de lnvestiguci6n y de Estudios Avanzados del LP.N., M6xlco, D.F., M6xico.

It has been demonstrated that lnterferons prevent liver collagen deposition by an antiflbrogcnie mechanism acting on mRNA procollagcn regulation. The aim of the present work was to study if interferon was also able to decrease collagen by increasing its degradation. Fibrosis was induced in male Wistar rats by double ligation and section of the common bile duct. Interferon-a (100 000 IU/rat, S.C.) was administered tc t

bile duct llguted rats dally after surgery for four weeks. Interferon Increased the

SHAM s ~.sq t222~N,M*,FN capacity of the liver

t~-~eoL to degrade type I i i , 0 ~ ~ . . . . . and Ill collagen and

matrigel; in addition, * .~ o.5] [..~ ~ p l a s m l n o g e n

activator activity o was also increased.

Since plasminogens

* | . . key participants in ~ 2 the balance of ~ protcolytic activities

M -~ ~ that regulate c]F~-] ~ extraeellular matrix

degradation, its increase may be another antiflbrotic (proteolytic) mechanism of interferon.

686 HEPATIC EXPRESSION OF u-SMOOTH MUSCLE ACTIN IN CHRONIC HEPATITIS C: CORRELATION WITH DISEASE ACTIVITY AND CItA_NGES wITH THERAPY. JE Poulos( E M BrtmF. CG Jannev 2. BR Bacon p. andAMDiBiscee l i e j. ]Division of Gastroenterology and Hepatology, 2Dept. of Pathology, St. Louis University School of Medicine, St. Louis, Me.

Activation of hepatic lipocytes results in increased synthesis of matrix proteins with subsequent fibrogenesis, and is associated with the expression of a-smooth muscle actin (SMA). Aims: To assess SMA expression by immunohistoehemistry in liver biopsies from patients with chronic hepatitis C, and to evaluate its relationship to disease severity and response to interferon therapy. Methods: Twelve patients (9 male), mean age 44 yrs, with elevated serum aminotransferases. HCV RNA and anti-HCV positivity for > 6 mos. were studied. The degree of liver injury and fibrosis was scored using the system described by Ludwig. SMA was detected by immunoperoxidase staining of formalin-fixed liver sections using a specific monoclonal antibody. Immunoreactivity was scored semi-quantitatively in acinar zones and regenerative nodules as follows: (< 1%, 0; 1-10%, I; 10-30%, 2; > 30%, 3). Results: SMA was detected in hepatic parenchyma in 8 of 12 patients (67%). There was a distinct gradient in staining: zone 3 greater than zone I (mean score: zone 3 = 2.1, zone 1 = 1.3). There was no correlation with disease activity or severity (grade, stage, or serum ALT). All 12 patients were treated with alpha interferon (3 mu TIW for 6 mos); 6 had a biochemical response with ALT decreasing to normal. Repeat liver biopsy at the end of 6 months of therapy indicated that a biochemical response was associated with significant improvement in the grade of inflammatory activity (mean grade: pre-IFN = 2.0 to post-IFN = 0.91, p = 0.008). Furthermore, acinar SMA staining decreased in 5 of the 6 responders (mean: 1.9 to 1.2, p < 0.05): while non-responders had no overall change in either necroinflammatory activity or in SMA staining. Conclusions: SMA is expressed in the livers of patients with chronic hepatitis C, suggesting that lipocyte activation occurs during hepatic inflammation and fibrosis associated with chronic viral hepatitis. A response to therapy was associated with a decrease in SMA, suggesting that a decrease in HCV replication may reduce lipocyte activation and the risk of hepatic fibrogenests.

687 EXPRESSION AND POTENTIAL ROLE OF ENDOTltELIN (ET) IN CHOLESTATIC LIVER INJURY. C Housset. A Laudat, ~ and ~ . INSERM U402, CHU Saint-Antoine, Paris and the Liver Center Laboratory, University of Californi a , San Francisco.

Circulating endothelin (ET) levels have been reported to be elevated in patients with cirrhosis. Previous work demonstrated abundant ET receptors on hepatic lipocytes and potent functional effects of the ETs on lipocytes (PNAS 90:9266,1993), suggesting that lipoc2/tes may he a major target of ET in the liver. Because ETs act as a paracrme factors, we hypothesized that increased ET production in injured liver may have effects on lipocytes. Further, since lipocytes play a key role in hepatic fibrogenesis, ET-I may be important in this process. The aim of this study was to investigate the expression of ET-1 in liver injury, and to elucidate effects of ET-1 on hepatic lipocytes. Methods: Liver injury was induced in male Sprague- Dawley rats by ligation of the common bi le duct. Hepatic lipocytes were isolated by denslt':/ centrifugation and sinusoidal endothelial cells by centrifugal elutriatlon. PreproET-1 mRNA was quanfitated by RNase protection assay. ET-I (peptide) was measured by radioimmunoassay. Lipocyte proliferation was assessed by vitiated thymidine incorporation and smooth muscle a actin (a marker Of lipocyte activation) was detected by immunoblot. Resul ts : PreproET-1 mRNA increased from undetectable levels in normal whole liver extracts to markedly elevated levels after bile duct ligation. ET-1 (peptide) increased from undetectable levels in normal liver extracts to 0.76, 3.25 and 7.85 pg/mg tissue 3, 10 and 21 days after bile duct ligation, respectively. PreproET-1 mRNA increased by 5-fold and 2-fold, 3 days after bile duct ligation in lipocytes and sinusoidal endothelial cells, respectively. In cultured lioocytes under serum starved conditions, ET-1 (2 x 10gM) resulted in a 1.5"-fold increase in thymidine incorporation, consistent with enhanced cellular proliferation. ET- /a lso induced cellular spreading and resulted in over a 7-fold increase in smooth muscle a actin, both features consistent with stimulation of their activation. In contrast, in myofibroblast-like lipocytes, ET-I resulted in a 5-fold reduction in serum-induced thymidine incorporation. Conclusion: Endothelin-1 (preproET-I mRNA and peptide) increases dramatically after experimental liver injury and appears to be derived from sinusoidal endothelial cells and lipocytes. This cytokine has l~elatively potent stimulating effects on the initiating events in ]ipocyte activation, but an inhibitory effect on proliferation of myofibroblast-like cells. These data suggest that endothehn-1 may play a pivotal role, possibly via autocrine or paracrine effects, in the pathogenesis and regulation of hepatic fibrosis.

688 GROWTH INHIBITION OF MYOFIBROBLASTLIKE ITO CELLS BY ENDOTHELIN-1 : A cAMP-MEDIATED EFFECT INVOLVING INHIBITION OF JUN KINASE AND MAP KINASE PATHWAYS. A Mallat 1, A - M Pr~auxL. L Fouassierl. P Mavier 1. D Dhumeauxl,,._C.C Bradham2, D Brenner 2, D Rau(aste 3. C Serradeil-Le Gala. S Lotersztain 1. IINSERM U99, H6pital Henri Mender 94010 Cr~teil, France. 2Sanofi Recherche, 31036 Toulouse, France, 3University of North Carolina, Chapel Hill, NC, USA.

Activated Ito cells (myofibroblastlike Ito cells, MFBIC) have been shown to play a preponderant role in the development of liver fibrosis. We have previously shown that binding of endothelin-1 (ET-1) to ETB receptors inhibits proliferation of human MFBIC (A. Manat et al., J Clin Invest, 1995, in press). The aim of this s tudy was to investigate the transduction pathway involved in the growth inhibitory effect of ET-1.

Human MFBIC were obtained by outgrowth from explants of non tumoral liver taken during hepatectomy and characterized by positive staining for desmin and smooth muscle cc-actin.

ET-1 caused a selective reduction of c-jun mRNA induction by serum wi th no modification of c-fos and krox 24 mRNA induction. In addition, ET-1 decreased activation of both MAP kinase (MAPK) and Jun kinase (JNK) by serum. Cyclic AMP acted as the second messenger since : (a) ET-1 caused a 13-fold elevation of intracellular cyclic AMP ; (b) an adenylyl cyclase inhibitor, SQ22536, b lunted the growth inhibitory effect of ET-1 ; (e) like ET-1, the adenylyl cyclase activator forskolin (FK) inhibited serum-stimulated cell proliferation by 60 %; (d) FK also inhibited serum-stimulated MAPK and JNK activities and (e) caused a selective decrease of c-jun mRNA induction. Finally, there was a 5-fold up-regulation of ET receptors in the presence of FK, suggesting the existence of a positive feedback regulation by cAMP.

In conclusion, these results indicate that cAMP is the intraceltular messenger responsible for growth inhibitory effects of ET,-1 towards MFBIC, and that the transduction mechanism involves inhibition of both the MAPK and the JNK pathways. In addition, our findings suggest that the use of pharmacological agents that inhibit cyclic AMP production may prove of benefit in limiting the extension of the fibrotic process during chronic liver diseases.