$22 Clinical and Research Forum/International Hepatology Communications 3 Suppl. (199S) $5-$36

F8-1 TRAGETED INHIBITION OF TYPE I PROCOLLAGEN SYNTHESIS BY ANTISENSE DNA OLIGONUCLEOTIDES. C.H.Wu, C.M.Wahon and G.Y.Wu. Dept. of Med. Univ. of Connecticut School of Medicine, Farmington, CT, USA

"lhe overproduction of collagen and its deposition into the extracellular matrix is characteristics of hepatic fibrosis. OBJEC17VE: To use targeted antisense oligonucleotide (ASO) DNA directed to specific Type l procollagen mRNA sequences to inhibit cellular procollagen production. METHODS: Six phos-phorotiolate ASOs directed towards the rat a 1(I) and a 2(I) mRNAs were chosen based on the prediaed secondary structure using the FoldRNA program. ASOs were bound to an asialoorosomucoid-polylysine (AsORPL) carrier. We showed previously that DNA bound to AsORPL can be targeted specifically to cells expressing the asialoglycoprotein receptor (AsGR). 3T3-AsGR cells were treated with ASO-AsORPL complexes. Collagenase digestion and Nonhero blot analysis were performed. RESULTS: We found that two of the ASOs (oligos C and E) complexes were most effective at inhibiting collagen synthesis. Newly synthesized collagen was decreased by 66% and 49%, respectively, in cells treated with ASO C or ASO E complex. Noncollagen protein synthesis was not affected by any of the AS(3 complexes tested. Northern blots showed that type I procollagen mRNA level decreased by 67% (ASO C complex) and 73% (ASO E complex). Type I procollagen mRNA level was not effected by treatment with a random ASO complex. In addition, treatment with AsORPL alone or ASO alone did not affect type 1 procollagen mRNA level, fl-actin mRNA remained unchanged in cells treated with either ASO C or ASO E complexes. CONCLUSION: Cell specific type procollagen production can be inhibited by targeting. ASOs directed against specific sequence of the mRNA. (Supported hi pan by grants from Targetech Inc. and Alcohol Bev. Med. Res. Foundation).

F8-2 TRANSCRIPTIONAL ACTIVATION OF ct2(l) COLLAGEN PROMOTER FOLLOWING ACUTE LIVER INJURY -ANALYSIS USING TRANSGENIC MOUSE-

Y. Inagaki, T. Nemoto, H, Kawai, M. Unoera, K. Kobayashi I st Dept. of Internal Medicine, School of Medicine, Kanazawa University Kanazawa, Japan

We have previously identified the promoter region of ct2(1) collagen gene (COL1A2) that is essential for both the basal transcription and TGF-I~-elicited stimulation of the gene in skin fibroblasts and fat-storing cells. In order to clarity if the same promoter region is activated following acute liver injury in vivo, several transgenic mouse lines have been

generated in which the -313 to +54 COLIA2 promoter sequence linked to the bacterial 13-galactosidase gene (LacZ) has been transmitted. Integration of the transgene was analyzed by Southern blot hybridization using tail DNA. Acute liver injury was introduced by injecting 0.1ml/kg BW of CCI 4 intraperitoneally. Liver tissues were excised 48 hr and 7 days after CCI4 injection, and the continuous sections were subjected to both the regular HE stain and the X-Gal stain detecting LacZ expression. Liver tissues 48 hr after CC] 4 injection showed centrilobular zonal necrosis, and LacZ expression was detected only in the centrilobular necrotic areas but not in the hepatocytes around the portal tracts. In parallel with the recovery from acute liver injury, LacZ expression was decreased 7 days after injection. It is therefore suggested that transient activation of the COL1A2 promoter containing the TGF-~-responsive element is a critical event to produce collagen following acute liver injury.

T H E R O L E O F ITO CELLS IN HEPATIC F8-3 F I B R O S I S A N D P O R T A L H Y P E R T E N S I O N

Mass imo Pinzani. Ist i tuto di Medicina Interna, Centro Interuniversi tar io di Fisiopatologia Epatica, Universita ' di Firenze, 1-50134, Florence, Italy

Ito cells, peris inusoidal mesenchymal elements, have recently been shown to play multiple physiological and pathophysiological roles. In particular, several in vivo and in vitro studies have clearly indicated that Ito cells play a relevant role in the progress ion of liver fibrogenesis. More recently, the at tent ion has been focussed on the mechanisms leading to Ito cell activation, prol iferat ion and synthesis of extracellular matrix components . A m o n g other soluble factors potentially involved in these processes, t ransforming ~rowth factor-il l (TGF-fl) and platelet-derived growth tactor ( P D G F ) have been shown to act in a paracrine, and possibly autocrine, fashion on Ito cells, thus perpe tua t ing their activated state. The potential relevance of these in vitro findings is suppor ted by the observat ion of a marked overexpression of bo th TGF-fl and P D G F in animal models of liver f ibrogenesis and in chronic liver diseases in humans . In addition, Ito cells have been shown to play an active role by p romot ing chemotaxis and long- term survival of infiltrating inf lammatory ceils. In addit ion to their key role in liver fibrogenesis, I to cells, on the basis of similarities with pericytes regulating b lood flow in o ther organs, have b e e n p roposed to funct ion as liver-specific pericytes. Indeed, current studies indicate that I to cells isolated f rom rat or h u m a n liver and main ta ined in culture contract in response to several vasoconstrict ing stimuli, and may therefore play a role in the regulat ion of sinusoidal blood flow in normal liver and in condit ions of increased resistance to portal flow.

F8-4 TGF-~ AND HEPATIC FIBROSIS M.A. Zorn, R.

Santos, S. Degli Esposti. Department of Medicine, Jefferson Medical College, Philadelphia, PA, USA

To our Ic~towledge, we were the first to describe the role of TGF-~I in liver disease [Hepatology 7:1028, 1987]. Since then, we have elucidated the importance of this cytokine in in vitro studies, animal models, and in man. For example, when three animal models of hepatic fibrosis were examined, they all exhibited increased levels of TGF-[B I gene expression at times somewhat preceding the increase in collagen synthesis. Treatment of cultured Ito cells with TGF-[~I resulted in a greater than 3-fold increase m total collagen content, and in type I procollagen mRNA levels. Moreover, TGFq31 treatment induced an increase in TGFq31 mRNA content in cultured cells, suggesting an autocrine stimulus of collagen synthesis. Confirming that these findings in model systems have relevance and validity in man is crucial. Our studies, as well as the work of other investigators, have clearly demonstrated increased TGF-131 expression in the livers of patients with active fibrosis. Recently, two other members of the TGF-~ family, TGF-[$2 and 133, have

been shown to have somewhat different physiologic roles and regulatory

~ athways than does I~l. Therefore, we have also assessed the role of TGF- isoforms in hepatic fibrogenesis. Northern blot hybridization analysis of

liver tissue from rats treated with CC14 showed large increases in the expression of all three isoforms during the period of maximum fibrogenesis. Freshly isolated nonparenchymal liver cells showed TGF-131 mRNA transcripts, but no ~2 or ~3 transcripts. However, when lto cells or a combination of Kupffer and endothelial ceils were cultured for one week, they expressed all three TGF-~ isoforms. When cocultures of nonparenchymal hepatic ceils were treated with either TGF-132 or TGF-133, they had increased expression of several matrix proteins. Thirty patients with alcoholic liver disease had pereataneous liver biopsies performed for diagnostic purposes. An unused portion of each biopsy was employed to quantitate the gene expression of TGF-~I, TGF-[~2, and TGF-~3, by RT- PCR. The gene expression for all three isoforms was significantly increased in these patients when compared to a normal control group.