280A AASLD ABSTRACTS HEPATOLOGY October 1995

693 MAPKINASE AND LIPID-DERIVED SIGNAL TRANSDUCTION CASCADES IN HEPATIC STELLATE CELL PROLIFERATION. HL Reeves, AD Burt, CP Day. Dept of Medicine University ofNewcastie U.K.

Hepatic stellate cells (HSCs) are the principal effectors of hepatic fibrogenesis. In response to injury or prolonged culture on plastic they proliferate and transform from quiescent cells into myofibroblast-like cells. Studies in fibroblast cell lines have indicated important roles for both the MAPkinase cascade and the lipid-derived second messenger phosphatidic acid (PA) in the mitogeuic response to peptide growth factors. We have investigated the relative importance of these two signal transduction pathways in the proliferation of qu!escent and transformed HSCs in response to the established mitogens PDGF and TGFa, HSCs were isolated from male Wistar rats and plated in 20% FCS. At 36h (quiescent) or %10 days (transformed) cells were placed into 0% FCS for 24hrs prior to incubation with growth factors. Proliferation was assessed by [3H]thymidine incorporation and MAPkirmse activity by phosphorylation of myelin basic protein. For estimation of PA, cells were labelled with [3H]myriatate tbr 3h prior to incubation with growth factors. Radiolabelled lipids were extracted and separated by TLC. PA was identified by ' 12 staining and quantified by liquid scintillation counting. In transformed HSCs stimulation of proliferation was greater with PDGF (30,~ml) than with 10riM TGFa (PDGF 632-%40% of control, TGFct 1402-6%). MAPkinase activity, expressed as a % increase of conirol, was stimulated by both PDGF ( 91+1l at 2rain, 379-+3 at 5rain, 252-+20 at 15rain, 162-+30 at 30rain ) and TGFct ( 8(~-13 at 2rain, 273+_5i at 5 rain, 143+2 at 15 rain, 88-+11 at 30 rain). PA, expressed as % increase of control, was elevated in response to PDGF (28~-7 at 2rain, 8ffi-_10 at 15rain), hut not TGFct (6-+6 at 2rain, 4-+4 at 15 rain). In quiescent HSCs there was no significant proliferation in response to either PDGF or TGE~ and no increase in either MAPkinsse activity or PA. Conclusions (i)These results suggest either that PA-induced signal cascades are more important than MAPkinsse cascades in HSC proliferation or that stimulation of both pathways is required for optimal mitogenesis. (ii)Tbe absence of a proliferative response to growth factors in quiescent HSCs seems likely to be due in part to the absence of stimulation of either signal cascade in these cells.

694 NEW PROLYL 4-HYDROXYLASE INHIBITOR INHIBITS PIG SERUI4-1NDUCED LIVER FIBROSIS BY PREVENTING IT0 CELL ACTIVATION. Y Matsumura, I 8aknida, S Wasaki, A Naontomi, T Kimura K Tani~wa, K Tamura, M Yas~, K 0kita First Department of Intsrrml bledioine, Yamaguchi University, Ube, Yamaguohi 755 Japan

The aim of this study was Te investigate the effect and mechanism of fibrosuppression by a newly synthesized prolyl 4 - hydroxylase inhibitor {HOE 077, I, 4-pyridine dicarboxylio acid bis [ ( 2 -met/qoxyethyl amide)~ } on pig serum-induced liver cirrhosis in the rat. Methods Male Wistar rate received O.5ml of pig serum twnoe a week for 8 weeks with 0. 50, or 100 ppm of HOE 077. At the end of the experiment, hydroxyproline content of the liver. ALT and AST were measured. Histological stalns used were HE, azan, and a staLn for ~ smooth muscle ao~in (~SMA, a marker of activated Ito cells). Electron microscopy was also performed. Messenger RNA expresslon of type | pro- collagen was ex~tmined by Northern blot analysis. Morphometrio 8iQalysie of (~Sl~-posltlVe cells and fibers with azan staining was assessed as percent area of the tissue speolmen using an image analysis system Bern*Its Bats that received plg serum for 8 weeks showed an increased liver hydroxyproline content of 518_+59Rg/g(n=50). 100ppm of HOE 077 reduced this increase to 155_+2S~g/g(n=50, P<0.01 ) in parallel with improved histological findings on azan stainLng (4.42"+2.85 vs. 2.94 ±2.52%, P< 0.05). I 00 ppm of HOE 077 reduced mBNA expresslon of ~I(|) prooollagen and percent area of ~SMA positive cells from 5.00--+2.1995 to 2.05_+ 1.92~. Electron micr0soopy revealed that 1 00 ppm of HOE 077 prevented the loss of fat droplets and reduction of rough el~doplasmio retieulum. Co~Iusion A prolyl 4-hydroxylase inhibitor (HOE 077) prevented pig serum-induced liver fibrosis by inhibiting Ito cell activation.

695 PENTOXIFYLLINE INHIBITS THE PROLIFERATION AND THE F I B R O G E N I C PROPERTIES OF H U M A N HE P AT IC MYOFIBROBLASTIC CELLS. A.-M Prdaux. A. Mallat. D. Dhumeaux. P. Mavier. INSERM U99, HSpital Henri Mondor, 94010 Cr6teil, France.

Hepatic myofibroblast-like cells, mostly derived from Ito cells, play a major role in the development of liver fibrosis. Pentoxifylline (PTX) exhibits growth inhibitory properties and decreases collagen synthesis in human dermal fibroblasts. The aim of the present study was to test the effects of PTX on : 1) the proliferation of human hepatic myofibroblastic cells (MFC), and 2) the synthesis of collagen and matrix metalloproteinase-2 (MMP-2) by these cells. MFC were obtained by outgrowth froin explants obtained from non tumoral liver taken during hepatectomy and characterized by specific c~toskeletal markers. Cell proliferation was quantified by measuring [ H]thymidine incorporation into DNAI Collagen and MMP-2 secretion were assessed by t31[ H]hydroxyproline incorporation and gelatin zymography, respectively. The mRNAs for type I and type III procollagens and for MMP-2 were analyzed by Northern blotting. PTX markedly decreased DNA synthesis. The inhibition was 54% at a concentration of 50 ~tg/ml and reached 97% at 500 ~tg/ml. Independently of this antiproliferative effect, PTX affected collagen secretion that was reduced by 24% and 67% at concentrations of 100 and 500 p.g/ml, respectively. This antifibrogenic effect occurred at a pretranslational level, as demonstrated by a decrease in type I and III procollagen mRNA levels. In contrast, PTX had no effect on the expression of MMP-2. In conclusion, PTX exhibits potent antiproliferative and antifibrogenic effects toward MFC. These results suggest that pentoxifylline might have therapeutic implications in chronic liver disease.

696 EFFECT OF PDGF, PENTOXIFYLLINE AND METABOLITE-1 ON COLLAGEN PRODUCTION IN PORCINE HEPATIC STELLATE CELLS. R.A. Isbmcker and T.C. Peterson. Dalhousie University, Departments of Phaimacology and Medicine, Halifax, Nova Scotia, Canada

Proliferation of fibroblasts and accumulation of extracellular matrix are two major events occurring in fibrotic liver disease. Ito cells, also known as hepatic stellate cells (HSC), are the major collagen producing cells of the liver and become proliferative following activation by eytokincs. Whether proliferation and extracellular matrix productio n are regulated by the same, or different, cytokines is not known. PDGF was previously shown to cause proliferation of non-confluent fibroblasts in vitro.

3 Incorporation of H-proline into collagenase digestion-susceptible protein was used to assay the effect of PDGF B/B on collagen production by confluent porcine hepatic steUate cells. PDGF was shown to induce collagen production by HSCs 2.3 fold above basal levels with maximum effect occurring by 20 ng/ml. Cell counts revealed no significant difference in HSC numbers between those exposed to 32 ng/ml PDGF B/B, or those grown in basal medium (p<0.05) indicating that increased collagen production can occur in the absence of proliferation. Pentoxifylline (FrX) was previously shown to inhibit PDGF-stimulated proliferation of fibroblasts. F r x and its metabolite (M1; 3,7-dimethyl- 1-(5-hydroxyhexyl)xanthine) inhibited collagen production in PDGF B/B-stimulated porcine HSCs. ICso for PTX and M1 was 100 ug/ml and 15 ug/ml, respectively. Both of the ICso concentrations inhibited collagen production levels below those of unstimulated HSCs. These results demonstrate that PDGF stimulates increased collagen production by HSCs in the absence of an effect on proliferation and that PTX is able to block the stimulatory effects of PDGF. (Supported by the Medical Research Council of Canada, RAI holds a graduate studentship from the Canadian Liver Foundation)