Human hepatic progenitor cells and reactive bile ductules express the muscarinic acetylcholine receptor type 3

Category 3: Molecular and cell biology (gene expression, signalling, fibrosis) Results: Staging improved in 58% SR, 12.5% RR and 11.5% NR. Any SR worsen fibrosis, against 8.3% RR and 7.7% NR. There was a correlation between PIIIP and HAI before (n = 71 rs = 0.41 p < 0.0004) and after IFN (n = 62 rs = 0.58 p < 0.01). Before IFN, MDA was elevated (11.67 SD 4.05 nmol/ml, p < O.Ol), correlating with staging (rs = 0.51 p < 0.03) and PIIIP (rs = 0.53 p < 0.03) and t-SH decreased (6.72SD1.28 micromol/g. protein p < 0.001). After IFN, MDA decreased (9.28 SD 2.16 p < 0.01) and t-SH increased (7.72 SD 1.4 p < 0.01). Conclusions: Results confirmed the anti-fibrotic properties of interferon and the presence of oxidative stress during HCV infection. Anti-oxidants could be useful, associated to interferon.

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223

EXPRESSION OF HEPATOCELLULAR TRANSPORTERS LONG-TERM CULTURED SMALL HEPATOCYTES

IN

PI. Meier, M. Sidler Pfindler, B. Stieger. Department ofMedicine, University Hospital, Zurich, Switzerland Background/Aims: Primary cultured hepatocytes rapidly dedifferentiate with increasing culture time. Small hepatocytes (SH), a subpopulation of hepatocytes, retain a proliferative potential in culture. Therefore, the expression of hepatocellular transport systems for uptake and secretion of cholephilic compounds was investigated to determine the differentiation status of long-term cultured SH. Methods: SH were isolated and cultured (Hepatology 29; 111, 1999). Basolateral uptake systems (Na+/taurocholate cotransporting-polypeptide, Ntcp; Organic-anion-transporting-polypeptides, Oatpl, 2, 4) and canalicular export pumps (bile salt export pump, Bsep; multidrugresistance-associated-protein 2, Mrp2) were assayed in SH cultures at different time points with real time PCR (Taqman system) for mRNA levels and with immunofluorescence (IF) for protein levels. Results: In cultures of SH, the appearance of small colonies of hepatocyte-like cells was observed starting about 1 week after initiation of culture. After a lag-time of 3 to 4 weeks, mRNAs for Ntcp, 0atp2, 0atp4, Bsep and Mrp2 increased 3- to 4-fold relative to 18s RNA at 6 to 7 weeks. mRNA for Oatpl was barely detectable throughout the experimental period. mRNA encoding albumin increased in the same time 8- to lo-fold. Concomitant with this increase, transporters could be detected at the protein level. Importantly, IF of Bsep and Mrp2 showed a time dependent polar expression pattern in hepatocyte colonies. At stages with well-differentiated expression of Mrp2, fluorescein secretion into a tubular network was observed. Conclusions: SH proliferate in culture to form hepatccyte colonies expressing hepatocellular transport systems in a polarized manner. Hence, proliferating SH develop into differentiated hepatocytes and are a useful model to study hepatocyte differentiation.

I 352

CARIPORIDE, A SELECTIVE NA+/H+ EXCHANGE INHIBITOR, DECREASES LIVER FIBROSIS IN THE RAT

A. Di Sario, E. Bendia, G. Svegliati Baroni, M. Marzioni, F. Ridolfi, U. Schindler, L. Trozzi, A. Benedetti. Department of Gastroenterology, University of Ancona, Italy The Na+/l-I’ exchanger plays an important role in the pathophysiology of liver fibrosis, mainly mediating the proliferative effect of PDGF. Cariporide is a novel and very selective Na+/H+ exchange inhibitor. We therefore evaluated the potential antifibrotic effect of this drug in isolated rat hepatic stellate cells (HSC) and in the dimethylmtrosamine (DMN) model of liver fibrosis. PDGF-induced HSC proliferation (27.6 f 3 vs 8.6 f 1.5% BrdU positive cells in controls; P < 0.001) was significantly inhibited by cariporide starting at a concentration of 100 nM (18 f 2.7%; P < O.Ol), with a maximal effect at 10 PM (8.9 f 1.6%; P < 0.001). Incubation with cariporide did not inhibit PDGF-induced ERKl and ERK2 activation, as well as p70S6k (a downstream component of the PI-3 kinase pathway) phosphorylation, but reduced PDGF-induced

81

activation of the Na+/H’ exchanger, with a maximal effect at 10 PM (JH+ = 9.0 f 1.6 vs 16.7 f 2.8 mmol/min in PDGF-stimulated HSC; P < 0.01). Rats treated with DMN (10 mg/kg) for 5 weeks received a diet containing or not 6 parts per million of cariporide (Aventis Pharma, Frankfurt, Germany). No toxic effects of cariporide were observed in control rats. Cariporide treatment reduced the degree of liver injury, as determined by ALT values (76 f 15 vs 128 f 18 U/L; P i 0.02). This was associated with both reduced HSC proliferation (8.7 f 2.0 vs 26.4 f 3.4 ce11s/mm*; P < 0.001) and collagen deposition (4.2 f 1.1 vs 20.7 f 3.4%; P < O.OOl), as determined by morphometrical evaluation of a-SMA/PCNA positive cells and of % of Sirius-Red positive parenchyma, respectively. These results confirm that reduction of liver fibrosis can be obtained through a selective inibition of the Na+/H+ exchanger.

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377

ESTABLISHMENT OF IMMORTALIZED HEPATOCYTE-LIKE CELL LINES FROM PS&DEFICIENT MICE

T. Pollicino, EM. Spagnoli, M. Weiss, G. Raimondo, M. Buendia. Dipartimento Medicina Interna, Universita di Messina; Dipartimento Biotecnologia Cellulare ed Ematologia, Universita La Sapienza, Roma, Italy; Unite’de Genetique de la Differentiation, Znstitut Pasteur Paris; Unite’de Recombination et Expression Genetique, lnstitut Pasteur Paris, France Long-term cultures of primary mammalian hepatocytes usually survive for some weeks, but do not proliferate and lose their differentiated phenotypes. Established cell lines able to express liver-specific functions were derived from hepatocellular carcinomas and hepatoblastomas, and cannot be used to investigate mechanisms of tumorigenesis. We succeeded in establishing immortalized mouse hepatocyte cell lines retaining most features of mature, differentiated liver cells. These cells were derived from adult p53-deficient mice and maintained for more than two years. All cell lines displayed the polyclonal shape typical of epithelial cells, and expressed cytokeratins 8, 18 and 19, E-cadherin, l - and l -catenins, and the tight junction ZO-1 protein. Ultrastructural examination revealed desmosomes and cytoplasmic organelles including mitochondria, smooth and rough endoplasmic reticulum and Golgi, that are characteristic of hepatocytes. Furthermore, bile canalicular-like structures were sealed by tight junctions between adjacent cells. A faint expression of various liver-enriched transcription factors (HNFs, CYEBPs)could be detected in all cell lines that, however, did not express other liver marker proteins such as albumin and o-fibrinogen. All cells exhibited both anchorage-dependent growth and contact inhibition, and they failed to grow in soft agar even after long-term culture showing that they were not tumorigenic. Interestingly, the p53-deficient hepatocyte cell lines were highly resistant to various apoptotic stimuli, including serum and growth factor deprivation and doxorubicin treatment. These data indicate that loss of ~53 function in hepatocytes can stimulate continuous cell growth, but not malignant transformation. These cells may represent a useful tool to explore tissue-specific gene expression and protein function as well as hepatocarcinogenesis process.

I 392

HUMAN HEPATIC PROGENITOR CELLS AND REACTIVE BILE DUCTULES EXPRESS THE MUSCARINIC ACETYLCHOLINE RECEPTOR TYPE 3

L. Libbrecht, D. Cassiman, V. Desmet, C. Denef, T. Roskams. University of Leuven, Belgium Neural and neuroendocrine factors such as chromogranin A and neural cell adhesion molecule are expressed by human hepatic ‘progenitor cells’ and reactive bile ductules. These factors probably play a role in the proliferation of hepatic ‘progenitor cells’. Recent results show that the cholinergic system stimulates the growth of proliferating cholangiocytes in rat liver via binding to the muscarinic acetylcholine receptor type 3

Poster Sessions

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(AchM3R). Therefore, we studied the expression of AchM3R in human liver diseases characterized by activation of hepatic ‘progenitor cells’ by immunohistochemistry (IH) and double-staining for AchM3R and cytokeratin (CK) 7 and by RT-PCR for AchM3R. We used frozen tissue of near-normal donor liver (n = 5), focal nodular hyperplasia (n = 2), chronic liver diseases in fibrotic or cirrhotic stage (HCV: n = 10, alcohol: n = 5, PBC: n = 8, PSC: n = 2) and liver with submassive necrosis (n = 3). In near-normal liver, only bile ducts were immunoreactive for AchM3R. IH and double-staining in liver diseases revealed that hepatic ‘progenitor cells’, reactive bile ductules and intermediate hepatocyte-like cells which were immunoreactive for CK7 were also immunoreactive for AchM3R. RT-PCR confirmed the presence of AchM3R mRNA transcripts in liver homogenates made from frozen tissue. Our results show that human hepatic ‘progenitor cells’ and reactive bile ductules express AchM3R. Since the cholinergic system influences proliferation in rat liver, these findings may have clinical consequences for liver transplant patients and may lead to a better understanding of the pathobiology of hepatic progenitor cells.

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LOW PROLIFERATIVE RESPONSE OF HUMAN HEPATOCYTES TO GROWTH FACTORS DUE TO LACK OF CYCLIN D

D.M. Runge, D. Jgger, G.W. Schute, S.C. Strom, G.K. Michalopoulos, D. Runge. Ingenium Pharmaceuticals AG., Innere Medizin, Universitiit Halle, Germany; Dept. of Pathology, University of Pittsburgh, USA

Hepatocyte Growth Factor (HGF) and Epidermal Growth Factor (EGF) induce proliferation and de-differentiation in cultured rat hepatocytes. Human hepatocyte cultured under similar conditions show a low proliferative response but stay differentiated for at least 6 weeks. They express EGF-receptor, c-MET, urokinase and its receptor (uPAR). EGF and HGF can be co-precipitated with their respective receptor and activation of HGF is likely to occur since HGF co-precipitates with the uPA/uPAR complex. To evaluate the differences between rat and human hepatocytes in culture we analyzed downstream players in the growth factor mediated signaling cascade. Jun-N-terminal kinases (JNKs) as well as extracellular regulated kinases ERK-1 and ERK-2 were expressed in hepatocyte cultures of both species at constant levels for more than 10 days. HGF and EGF administration to gowth factor depleted cultures led to time dependent phosphorylation of members of both kinase families with maximal activation at 5 min. In addition, APl-DNA-binding activity was prsent in both species indicating that growth factor mediated signal transduction via activation of jun/fos was maintained. Analysis of cellc ycle regulators revealed differences between rat and human hepatocyte cultures: While in rat hepatocytes Cyclin D was expressed at constant levels, no Cyclin D was detectable in human hepatocyte cultures. This strongly suggests that lack of this important cell cycle regulator is a major reason for the low proliferative response of human hepatocytes in culture

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CORRELATION OF HEPATIC MATRIX GENE EXPRESSION IN DIAGNOSTIC BIOPSIES WlTH SURROGATE SERUM MARKERS OF FIBROGENESIS AND FIBROLYSIS

D. Axelos, M. Voelker, H. Schoepper, M. Plommer, E.G. Hahn, D. Schuppan. Friedrich-Alexander-Universitaet Erlangen-Nuernberg; Medizinische Klinik I, Germany

In hepatic fibrogenesis an increased synthesis and deposition of matrix proteins is accompanied by a reduced expression and activity of fibrolytic proteases. In addition, inhibitors of matrix metalloproteinases (TIMP- 1, TIMP-2) are upregulated. Data that clearly show how far surrogate serum markers can reflect hepatic fibrogenensis or fibrolysis are missing. Background:

Methods: RNA was isolated from liver-biopsies of 48 patients and reverse-transcribed. Procollagen I, TIMP-1 and MMP-2 mRNA were quantitated in relation to GAPDH mRNA by real time (TaqMan)-PCR. Results were correlated with the corresponding paired serum-levels of TIMP-I, MMP-2, MMP-9, tenascin, hyaluronan, collagen IV and collagen VI, as determined by automated ELISAs. Serum marker TIMP-1 MMP-2 MMPTenascin Hyaluronan Collagen IV Collagen VI

Procollagen I mRNA

TIMP-1 IllRNA

MMP-2 mRNA

0.36 0.37 -0.10 0.67 0.28 0.35 0.31

0.61 0.30 0.10 0.53 0.54 0.5 0.3

0.23 0.25 0.00 0.30 0.27 0.26 0.14

Conclusion: 1) Hepatic procollagen I mRNA expression correlates best with serum-levels of tenascin; 2) Serum-levels of TIMP-1, hyaluronan and tenascin correlated well with hepatic expression of TIMP-1, a key promoter of fibrogenesis; 3) Whereas tenascin and TIMP-1 appear to qualify as serum markers of hepatic fibrogenesis, markers of fibrolysis remain to be defined.

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TRANSCRIPTION OF LEPTIN GENE IN SINUSOIDAL ENDOTHELIAL RAT LIVER CELLS

H. Rodriguez-Hemandez ‘, M.R. Reyes ‘, B. Escalante ‘, F. Posadas’.

1Food & Nutrition Research Centel; Faculty of Medicine U.J.E.D., Durango; 2 CINVESTAX I2 N., Mexico Leptin is a cytokine involved in food intake; recently its proangiogenic effect has been disclosed. In liver leptin production by stellate cells and leptin receptors in sinusoidal endothelial cells have been shown; leptin’s role in this site remains speculative. Endothelial cells are key components of capillarization and angiogenesis in liver fibrosis. Aim: To study leptin gene expression by sinusoidal endothelial liver cells. Methods: Sinusoidal endothelial rat liver cells were cultured in Dulbecco’s MEM with 10% FBS or adult BS. Leptin and leptin-receptor gene transcription was studied by RT-PCR from total RNA with tbe Superscript RT-PCR kit. Primers were designed from mRNA coding sequences in GenBank. RT-PCR products were analyzed in agarose gels. Results: Sinusoidal endothelial cells transcribed leptin gene; the intensity of the signal was dependent on the cultureconditions; with FBS, a clear leptin amplicon was observed after 35 PCR cycles; when adult BS was employed, a bare signal was observed after 40 PCR cycles. Leptin-receptor amplicon was consistently observed independent of FBS. Conclusions: Sinusoidal endothelial rat liver cells can achieve in vitro leptin gene transcription. In vivo leptin of endothelial origin in the liver could be linked to proangiogenic events. Leptin represents an intervention point for fibrotic liver disease and studies on this issue are warranted. (Grant 1216P from CONACYT-Mexico. MRR is recipient of the fellowship “Don Alfonso Espeleta Torrijos”).

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LAP CONTROLS ITS OWN TRANSCRIPTION ELEMENT IN ITS PROMOTER REGION

BY A NEW

M. Niehof, L. Zender, S. Kubicka, M.P. Manns, C. Trautwein. Dep. of Gastroenterology & Hepatology, MH-Hannoveq Germany

LAP (liver activating protein) contributes to hepatocyte-specific gene expression. The activity of LAP can be regulated at the transcriptional and posttranslational level or by protein-protein interaction. Here we show that LAP controls its own transcription. Transfection experiments