HEPATOLOGY Vol. 22, No. 4, Pt . 2, 1995 A A S L D A B S T R A C T S 221A

457 ALTERED PULMONARY EXPRESSION OF VASOACTIVE MEDIATORS IN A RAT MODEL OF HEPATOPULMONARY SYNDROME. MB Fallon. GA Abrams. YF Cben. S OnariL. G Garcia-Cardena*. WC Sessa*. JW McGrath. Dept. of Internal Medicine, University of Alabama Birmingham, Birmingham, AL and *Dept. of Pharmacology, Yale University, New Haven, CT.

Hepatopulmonary syndrome (HPS) is characterized by abnormal arterial oxygenation due to intrapulmonary vasodilatation in patients with liver disease and/or portal hypertension. Chronic common bile duct ligation (CBDL) in the rat results in many features similar to human HPS including abnormal gas exchange and a loss of hypoxia-induced pulmonary vasoconstriction and serves as an animal model (Chang and Ohara, Am Rev Resp Dis, 1992, JCI, 1994). The AIM of this study was to assess the pulmonary expression of various vasoactive mediators, which might alter intrapulmonary vascular tone in the CBDL rat model of HPS. METHODS: 6 Sham and 6 CBDL rats were studied 4-5 weeks after surgery. Atrial naturetic peptide (ANP), endothelin-1 (ET-I), endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNnS) were analyzed. Steady state mRNA levels were measured by Northern analysis and tissue protein levels by Western analysis (eNOS, iNnS) or RIA (ANP, ET-1). Plasma ANP and ET-1 levels were measured by RIA and nitric oxide (NO) by chemiluminescence. RESULTS: Pulmonary ANP mRNA levels increased 5 fold (p<0.05)and protein levels increased 3 fold (8.6 vs 2:7 ng/gm, p<0.0001) in CBDL rats compared to sham rats. Plasma ANP levels were also elevated (88 vs. 46 pg/mL p< 0.007). Pulmonary ET-1 mRNA levels increased 7 fold (p<0.001) and plasma ET-1 levels 9 fold (55 vs. 5.9 pg/ml, p<0.05) but pulmonary protein levels were unchanged. Pulmonary eNOS mRNA levels were not altered after CBDL, though pulmonary protein levels rose 2.2 fold (p<0.02). No changes in pulmonary iNnS mRNA and protein or plasma NO levels were observed after chronic CBDL. CONCLUSIONS: These findings demonstrate that 1) the levels of both vasodilatory (ANP, eNOS) and vasoconstrictive (El'-1 ) mediators are increased in the CBDL model of HPS and 2) an increase in vasodilatory mediators predominates in the lung. We propose that an imbalance of vasoactive substances in the lung in chronic liver disease may be important in the pathogenesis of lIPS.

4 5 8 IS OXYGEN TENSION A MODULATOR OF HEPATIC GENE EXPRESSION IN VIVO? S W¢l?~ter, J June. AJ Thornton and JJ G~mucio. Division of Gastroenterology, Department of Medicine, VA Medical Center and The University of Michigan, Ann Arbor, MI.

Evidence that oxygen gradients develop in hepatic sinusoids has been provided by Wansgenic experiments performed with the human erythropoeitin gene. Under conditions of hypoxia, in vivo, the transgene is induced in hepatocytes located in the distal two thirds of the cell plate (Blood 1991;77:2497). To assess whether an endogenous liver gene, the CYP2B1 gene, expressed in the distal two thirds of the cell plate, was modulated by oxygen tension, rats were subjected to various level s of bypoxia in vivo. Blood pH, gases and pressure were continuously monitored. After exposure to 4% oxygen for 30 min., there was a 2-3 fold induction of the CYP2B 1 mRNA, as established by semiquantitative analysis of Northern blots. Subsequently, we tested the hypothesis that hypoxia, similar to phenobarbital-induction, increases the level of an "inducer" poly(A)+RNA-protein which acts on the CYP2B 1 gene. Poly(A)+RNA was isolated from rats subjected to 4% hypoxia and from controls. The effects of poly(A)+RNA on the rate of CYP2B I gene transcription was tested in the coupled transcription-translation Xenopus oocyte system. 30 Xenopus laevis oocytes were injected (intranoclear injection) with one of six DNA constructs comprising nested deletions of the initial -430bp of the CYP2B1 gene 5' flanking region linked to the chlorarnpbenicol acetyl transferase (CAT) gene. The CAT activity of constructs containing at least -290bp of CYP2B 1 was increased 2-3 fold by the injection (into the vegetal pole of the oocyte) of poly(A)+RNA extracted from livers of rats exposed to 4% oxygen for 30 min, compared to control poly(A)+RNA. Constructs comprising -270bp or smaller did not respond to the hypoxia-poly(A)+RNA injection. The hypoxia responsive region contains a palindrome, between -274 and -263bp, encompassing a putative hypoxia responsive element found in other oxygen-regulated genes, We propose that hypoxia stimulates the production of a liver poly(A)+RNA-protein capable of modifying the transcriptional activity of the CYP2B 1 gene, possibly by interaction with specific hypoxia-responsive sequences. This observation suggests that oxygen sensing mechanisms exist in bepatocytes and that oxygen tension in the sinusoid may play a major role in the modulation of hepatic gene expression in vivo.

459 CELL-SPECIFIC TRANSCRIPTION OF THE COLLAGEN al(I)GENE IN HEPATIC STELLATE CELLS. K Houglam. P Bornstein. and M Chojkier. Dept. of Medicine, VAMC, and Center for Molecular Genetics University of California, San Diego, CA 92161, and Depts. of Biochemistry and Medicine, University of Washington, Seattle, WA 98195.

In order to identify the critical c/s-elements of the collagen ,~I(D gene in stellate cells, we used animals bearing collagen cx(I) 5' regulatory regions- human growth hormone (hGH) transgenes. We found that in the liver, the steady state P0ol ofhGH reporter mRNA was consistently stimulated by CCI,, in parallel to the endogenous collagen od(I) gene, whether the transgene contained the 0.44 or 2.3 kb 5' collagen cq(I) flanking sequence, and in the presence or absence of the first intron (bases +292 to +1607). Because stellate ceils are activated, and proliferate following CC14-induced hepatic injury, we also established that expression of the collagen transgene was increased per cell, eliminating the confounding variable of stellate cell proliferation. Fnthermore, the expression of collagen ,1(I) in activated cultured stellate cells (day 10) was about forty-fold higher than in quiescent stellate cells (day 1). The endogenous collagen gene expression was paralleled by the transcriptional activation of the -440COL-hGH transgene. Thus, the transactivation of collagen ~1(I) gene in cultured, activated stellate cells requires only 0.44kb of the 5' regulatory region, and resembles the regulation of these cells in the CCl4-treated animal. In contrast, in skin and tendon the - 2.3 kb region is necessary to direct a high level of transcription of the hGH reporter gene. Nuclear extracts from activated, but not from quiescent, stellate cells form a single high affinity complex with the cis-element -82 to -ll0bp (FP1) and with a NF1 consensus oligonucleotide. The FP1 complex was competed by a NF1 cis-element, but neither by a C/EBP or a SP1 cognate domain. Mutation of this site markedly decreased the transcription from a collagen cq(I) promoter-CAT reporter gene in primary activated stellate ceils. These data indicate that the 0.44 kb 5' region of the collagen ,1(I) gene is sufficient for maximal transactivation of this gene in stellate cells, and that the FP1 sequence (presumably by its interaction with NF1) is critical for transcriptional activation.

4 6 0 ENDOTHELIN (ET) ANTAGONISM IN EXPERIMENTAL HEPATIC FIBROSIS: IMPLICATIONS FOR ENDOTHELIN IN THE PATHOGENESIS OF WOUND HEALING. DC Rockev and JJ Chung. Liver Center Laboratory, University of C a l i f o ~ n c i s c o .

fuThe ETs are a family of potent vasoconstrictors with diverse biologic nctions. Three isotypes exist, ET-1, ET-2 and ET-3 which bind with

different arhnity to me two major enuotheim receptors, ET A and ET B. We have previously_ demonstrated abundant ET receptors on h~patic lipocy_tes (PNA'S 90:9256,1993) anq increaseu preproET-1 mRN-A expression during liver iniury. Hepatic lipocytes undergo activation with liver iniury, !eading to ,enhanced extrace-lh]ar m a r x proctuction. We testeff tile nypomesis mat increasea t~x prouuction uuring tiver injury may contribute to iaeoatic fibrogenesis via stimulation of lioocyte acttvation. Methods: Hepatic lipocytes were isolated as describ.ed. Liver fibrosis was induced in ma].e Sprague-Dawley rats by bile duct ligation (8 days) and gavage with carbon tetrachloride (CC14, 4doses @ 0.5 ml/kg over 20 days-). Bosentan (Roche, Switzerland), a mixed ET,/ET B receptor antagonist, was administered orally (100 mg/kg/day) 'during liver injury. For animals receiving CC14 fibrosis was evaquated using a modified Knodell scoring index. Type I c611agen mRNA was cluantitated by RNase protection assay. Smooth.musc!e a actin (a marker o'f lipocyte activation), was detected 5y immunoiaistocgemistry and immunoblot. Results: In Cultured lipocytes, ET-1 or sararotoxin S6C markedly induced smooth muscle a actin expression and bosentan inhibited this response. In both fibrosis models, bosentan reduced extracellUlar matrix deposition as assessed by reticulin stain. For the CC14 model, fibrosis scores were 2.2 + 0.41 vs. 1.2 + 0.23 for control and bosentan treated animals res .p.ectively (p <0.05, n=3). Collagen I mRNA expression in whole liver ani:t lipocytes is shown below. mRNA Source Bile Duct Ligation Carbon Tetrachloride ]

[ Control Bosentan Control Bosentan I * + * + Whole liver ~ ~ ~

Lipocyte 100~_9 "19.5+5 100-!-_22 "23.1+9 Type I collagen mRNA levels were normalized to an internal standard mRNA (S-14)

land the value for control was arbitrarily set to 100. *p < 0.05 for control compared to I Ibosentan, n = 3 for each condition. | By immunoblot, smooth muscle a actin was reduced in lipocytes to 56% and 39% or control in the bile duct ligation and CC14 models, resp.ectively (p <0.05, n=3). Immunocytochemicaq results were similar. C o n c l u s i o n : In culture endothelin sttmulated lipocyte activation was inhibited by bosentan. ]~n two different models of'liver inju.ry and fibrosis, antazonism of endothelin inhibited lipocyte activation and ameliorated the fiSrosing response to injury. Because the liver's response to injury constitutes a paradigm for wound healing in other organs, the data imply that endothelin may contribute to the fibrogenic response in diverse forms of tissue injury.