- Email: [email protected]
[311] URSODEOXYCHOLIC ACID, BUT NOT NORURSODEOXYCHOLIC ACID REVERSES TAUROLITHOCHOLIC ACID-INDUCED CHOLESTASIS IN RAT LIVER
POSTERS
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Results: Balu-I inhibited [3H]-TCA uptake by human OATPIBI (-95%), OATPIB3 (-15%) and rat Oatplal (-50%), Oatpla4 (-35%) and Oatplb2 (-40%) transporters, but had no effect on [3H]-TCA uptake by NTCP. In rat receiving Balu-I, a marked increase in bile flow (+70%, p 0.05), higher than that induced by TCA (+20%, p i 0.05), was found. This enhanced choleretic ability was probably accounted for by the fact that critical micellar concentration of Balu-1 was 5-fold lower than that of TCA. Phalloidin administration decreased basal (-60%, p i 0.05) and TCA-stimulated bile flow (-55%, p < 0.05), without affecting total bile acid output. Cholestasis was accompanied by signs of liver necrosis, nephrotoxicity and haematuria. In contrast, in Balu-l -treated animals, phalloidin-induced reduction in bile flow was only of 15% and haematuria or signs of hepatic or kidney injury were not observed. Conclusion. Balu-l is able to protect against phalloidin hepatotoxicity, probably due to delayed hepatic uptake and enhanced biliary secretion of the toxin.
13091 IMPACT OF COLITIS ON BILE SECRETION AND HEPATOBILIARY TRANSPORTER EXPRESSION IN MICE: POSSIBLE IMPLICATIONS FOR PATHOGENESIS OF SCLEROSING CHOLANGITIS J. Jahnel’, M. Wagner’, C. Langne?, C. Hoegenauer’, P. Fickert’, D. Silbert’ , J. Gumhold’, A. Fuchsbichleg, H. Denk2, M. Trauner’ . I Laboratory of Experimental and Molecular Hepatology, Dioision of‘ Gastroenterology and Heputology, Department of‘ Internal Medicine, Medical linioersiiy Graz, Graz; ’Institut of‘ Pathology, Medical University Gruz, Graz, Auxtria E-mail: [email protected] Background and Aims: The pathogenetic link between ulcerative colitis and primary sclerosing cholangitis (SC) remains poorly understood. Translocation of lipopolysaccharide (LPS) or other bacterial products could contribute to cholestasis via inflammation-induced changes in hepatobiliary transporter gene expression. We therefore examined the effects of acute colitis on bile secretion and expression of proinflammatory mediators, hepatobiliary transporters and their regulatory nuclear receptors in mice. Furthermore we tested the potential role of chronic colitis as “second hit” in heterozygous multidrug resistance gene 2 knockout mice (Mdr2+’-) which contrary to homozygotes (Mdr2-’-) do not spontaneously develop SC. Methods: Acute colitis was induced in C57/BL6 mice with 3% dextran sulfate sodium (DSS) for 7 days. Chronic colitis in Mdr2”- and wildtype Mdr2+’+ was induced by 5 cycles of DSS. In acute colitis, bile flow/composition and hepatic mRNA expression (RT-PCR) was compared with LPS treated animals (15mgikg BW i.p.). The impact of chronic colitis on bile formation and liver histology was compared between Mdr2+’+ and animals. Results: Acute DSS colitis increased biliary output of bile acids (BA; 210%, p i 0 . 0 5 ) and phospholipids (PL; 210%, p i 0 . 0 5 ) but not of cholesterol (1 13%), glutathione (95%) and bile flow (1 14%, n x ) . Mdrla (1.7-fold, p < 0.05) and Ostbeta (1 .%fold, p < 0.05) mRNA was induced, while other transporters (Ntcp, Bsep, Mrp2- 4, Ostbeta, AbcgS, Abcg8, Oatp I - 4, Mdrl b, Mdr2), nuclear receptors (RXRa, FXR, CAR, PXR) or proinflammatory mediators (TNFa, iNOS) remained unchanged. In comparison, LPS reduced Ntcp (29%), Bsep (22‘%), Mrp2 (52%) and induced Mdrlb (1.7-fold), TNFa (6.8-fold) and iNOS (12-fold; all p < 0.05). BA/PL ratios did not change in acute (7.84 vs. 8.68 in controls) and chronic DSS colitis ( 1 0.27 vs. 7.73) but decreased after LPS (2.19). Chronic colitis induced mild portal inflammation in Mdr2+’- but not Mdr2+’+. However, the t i l l histological picture of SC was not observed. Conclusions: LPS but not acute DSS-induced colitis reduced hepatobiliary gene expression and bile formation. Although chronic colitis in Mdr2+’mice led to mild portal inflammation, SC was not induced. This suggests that genetic Mdr2 defects may not represent a major pathogenetic factor for development of SC in colitis.
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13101 CELL CYCLE QUIESCENCE IS ASSOCIATED WITH THE HEPATOBILIARY GENE EXPRESSION IN HUMAN INTRAHEPATIC BlLlARY EPITHELIAL CELL LINES IN VITRO A. Komori’,2, S. Fujiwara’,2, Y. Aiba’,2, M. Nalcamura’,2, S . Shimoda3, H. Fujiolca’,2, K. Migita’,2, M. Tto’,2, K. Yano’,2, H. Yatsuhashi’,2, H. lshibashi’ >2. ‘Clinical Research Center: NHO National Nagasaki Medical Center: Omnra, Nagasaki; ’Departnzent of‘ Hepatology, Nagasaki University Graduate School of Biomedical Sciences, Omnra, Nagasaki; ”Department of Medicine and Biosystenzic Science, Kynshu linioersity Graduate School of Medical Sciencex, Fuhioku, Japan E-mail: [email protected] Background and Aims: Molecular signature of the regeneration of intrahepatic biliary tree after their wound remains elusive; transcriptomal and phenotypic changes from the terminally differentiated to the dedifferentiated state, as well as from the proliferating to the re-quiescent state, of intrahepatic biliary epithelial cells have not been extensively defined. The aim of the study was to compare the gene expression pattern of human intrahepatic biliary epithelial cell lines (HIBEC) between proliferating and quiescent state in vitro, to elucidate their plasticity in gene expression. Methods: Human epithelial antigen positive (HEA) HIBEC were purified from resected normal adult human livers and propagated in the medium containing 10% FBS and growth factors (EGF and HGF). The forced quiescent condition of HTBEC culture was either monolayer in serum/growth factors-free medium (SFM) on laminin, or floating spheroid culture in ultra low attachment vessels. The mRNA expression of biliary and of hepatocyte markers were assessed by semi-quantitative RT-PCR and Taqman PCR. Human Oligo Microarray Kit (Agilent, CA) was used to characterize the transcriptomal changes in HIBEC. Results: Microarray analysis defined the growth factors/serum deprivationassociated quiescent transcriptome in HIBECs: Down-regulation of a set of cell-cycle associated genes (e.g. cyclin A2 as well as BI/B2) and up-regulation of biliary and epithelial genes (e.g. Cytokeratin (CK) 19, aquaporin I , E-cadherin) were some of the facets. Increase in CK19 and E-cadherin mRNAs were also observed i n the anchorage-independent floating spheroid, and the restoration of these mRNAs in HTBEC in SFM was reversed by the addition of EGF andor serum, suggesting that epithelial-to mesenchymal transition (EMT) occurred in their propagation in vitro. Surprisingly, the forced quiescence were accompanied by the induction of albumin and hepatocyte-specific genes (i.e. G6PC, TO) as well, recapitulating transcriptomally transitional state of HIBEC in vitro. Conculsions: HTBEC gain the hepatobiliary, more-epithelial gene expression in the course of quiescence in vitro. This pattern might be reminiscent of the hepatobiliary phenotype of cells of the finer branches of the biliary tree in inflamed liver, i.e. ductular reaction. Additional environmental cue should be required for the proper re-differentiation of HTBEC in vivo.
13111 URSODEOXYCHOLIC ACID, BUT NOT NORURSODEOXYCHOLIC ACID REVERSES TAUROLITHOCHOLIC ACID-INDUCED CHOLESTASIS IN RAT LIVER S . Maitz’, G.U. Denk’, R. Wimmer’, P. Tnvernizzi2, A.F. Hofmann3, U. Beuers’ . ’Department of Medicine 2 Grosshadern, linioersity of’ Mnnich, Munich, Germany; 2Dioision o f Internal Medicine, Sun Paolo of School of Medicine, Uniuer~xit?,of Milan, Milan, Italy; 3De~~artment Medicine, University of Califtwnia, Sun Diego, USA E-mail: [email protected] -
Background and Aims: Norursodeoxycholic acid (norUDCA) exerts therapeutic effects superior to ursodeoxycholic acid (UDCA) in Mdr2 (Abcb4) knockout mice (Mdr2-’-) that develop chronic progressive sclerosing cholangitis (Gastroenterology 2006; 130:465). Taurolithocholic acid
04B. MOLECULAR AND CELLULAR BIOLOGY (TLCA)-induced cholestasis represents a well-established posttranscriptional model of hepatocellular cholestasis (JBC 2003;278: I78 10). It was the aim of this study to further unravel norUDCA's potentially beneficial effects in cholestasis by comparing its effects to those of UDCA in the model of TLCA-induced cholestasis in isolated perfused rat livers. Methods: The effect of norUDCA (25 pmol/l) on bile flow and biliary secretion of the Mrp2 (Abcc2) substrate, dinitrophenyl-glutathione (GSDNP), was studied in comparison to equimolar UDCA in presence or absence of TLCA (1 0 ymol/l) in isolated rat livers perfused for 115 min with Krebs-Henseleit-bicarbonte buffer (JBC 2003;278:17810). Bile acid administration was started after 45 min, and chlorodinitrobenzene (CDNB, 30 ymolil), the precursor of GS-DNP, was administered from min 65 to 75. Bile secretion was determined gravimetrically and GS-DNP secretion fluorometrically. Results: Administration of UDCA or norUDCA led to a comparable increase of bile secretion in isolated perfused rat livers in comparison to DMSO controls (DMSO, 65.2+2.0 yl/50min/g liver; 0.01 vs. DMSO; UDCA, norUDCA, 136.2&22.6 y1/50min/g liver, p i 0.01 vs. DMSO; mean+SD, n = 4 , each, 165.7f40.9 yli50minlg liver, p i ANOVA) whereas GS-DNP secretion remained unchanged (DMSO, 1037+79 nmoli50minig liver; norUDCA, 876+333 nmol/50minig liver; UDCA, 838+258 nmoli50minlg liver). TLCA (10 ymolil) markedly reduced bile flow (5.5f2.1 yl/50min/g liver) and GS-DNP secretion (52&14 nmol/50min/g liver). Interestingly, UDCA, but not norUDCA reversed TLCA-induced impairment of bile flow (TLCA+UDCA, 47.7+ 15.0 yli50minlg liver, p i 0.0 I vs. TLCA; TLCA+norUDCA, 8.9f3.0 yli50minig liver, n.s. vs. TLCA) and GS-DNP secretion (TLCA+UDCA, 174&72 nmol/50 min/g liver, p iO.0 1 vs TLCA; TLCA+norUDCA, 39&19 nmol/50min/g liver, n x ) . Conclusion: UDCA, but not norUDCA exerts anticholestatic effects at the level of the hepatocyte in the experimental model of TLCA-induced cholestasis in rats. It is intriguing to speculate that combined therapy with UDCA and norUDCA may be superior to monotherapy in biliary disorders in which cholangiocyte as well as hepatocyte dysfunction contribute to disease progression and liver failure.
13121 VEGF PLAYS A MAJOR ROLE IN MEDIATING ESTROGENS PROLIFERATIVE EFFECTS ON HUMAN INTRAHEPATIC CHOLANGIOCARCINOMA M.G. Mancino', A. Mancino', A. Torrice', M.C. Bragazzi', 1. Stammegna', G. Alpini3, P. Onori4, A. Franchitto', E. Gaudio', A.F. Attili' , D. Alvaro' ,'. 'Department of Clinical Medicine, Diuision of Gustroenterology, linioersiiy "La Sapienzu ", Rome; Departnzent of Clinical Medicine, Division of Ga,str~)enter~)l~)~y, Uniuer~xihi ' L a Sapienzu" Polo Pontino, Rome, Italy; 'Scott & White and The Texas A & M Unioersity System Health Science Center College of Medicine, Temple, TX, 1JSA; 4Department of'Experimentu1 Medicine, Section of' Human & Clinical Anatomy, State linioersity of L'Aquib; 'Departnwnt of' Anatomy, linioersity of' Rome, Italy E-mail: [email protected]
Background: Estrogens and VEGF induce the proliferation of neoplastic cells expressing their receptors (ER and VEGFR) through the synergism with growth factors and the induction of neo-angiogenesis. We previously demonstrated that human intrahepatic cholangiocarcinoma expresses ER and that estrogens induce the proliferation of human cholangiocarcinoma cell lines. Aims: The aims of our study were to evaluate in human cholangiocarcinoma: (i) the expression of VEGF and VEGFR; (ii) estrogens effects on VEGFR expression and (iii) the role played by VEGF in mediating the proliferative effects of estrogens. Methods: The study was performed in biopsies of human intrahepatic cholangiocarcinoma (n=5) and in HUH-28 cell line derived from human intrahepatic cholangiocarcinoma. The expression of VEGF-A and
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VEGF-C and VEGFR were evaluated by immunohistochemistry (IHC) or western blot. Cell proliferation was measured by both PCNA westem-blot and MTS proliferation assay. Results: Biopsies of human intrahepatic cholangiocarcinoma were strongly and uniformly positive for VEGF-A and -C at the IHC examination. HUH-28 cells were positive for VEGF-A, VEGF-C, VEGFR-1-2-3 and 17P-estradiol markedly enhanced (VEGF-A 3 4 f l '% ; VEGF-C 57f4'X; VEGFR-I 70f5%; VEGFR2 517&12?4; VEGFR3 102fl0?4) their expression in association with the induction of cell proliferation. VEGF administration stimulated HUH-28 cells proliferation at a similar extent than serum redmission but into a lower extent than I7(i-estradiol. 17(i-estradiol induced the secretion of VEGF (from 0 to 180 pgiml, ELISA) in the supernatant of HUH-28 cells. The stimulatory effect of 17(i-estradiol on the protein expression of VEGF and VEGFR was blocked by ER (Icil 82,780) or IGFl-R (ctlR3) antagonists, indicating an effect mediated by a coupling of ER and IGFl -R. Finally VEGF-Trap, a receptor-based VEGF inhibitor, decreased by 70% 17[3-estradiol proliferative effect on HUH-28 cells indicating that most of the proliferative effect of estrogens on human cholangiocarcinoma is mediated by VEGF induction. Conclusions: Estrogens stimulate the growth of human intrahepatic cholangiocarcinoma by inducing the expression and secretion of VEGF that plays a major role in mediating estrogens proliferative effects. Thus strategies based on the antagonism of ER and/or VEGF could help in delaying the progression of cholangiocarcinoma.
13131 MECHANISMS OF HUMAN HEPATOBILIARY TRANSPORTER GENE REGULATION IN THE HEPATOCYTE-LIKE CELL LINE HepaRG 1.V Martin, S. Voigt, S. Strauch, C. Trautwein, A. Geier. Department of Internal Medicine Ill, Uniuersity Hospital Auchen, RWTH, Aachm, Germany E-mail: [email protected]
Background and Aim: Functional studies on human hepatic gene regulation are limited due to the lack of suitable models. Currently used human hepatoma cell lines such as HepG2 or Huh7 are deficient in several transporters such as NTCP or BSEP whereas primary human hepatocytes and freshly prepared liver slices are limited in availability. The highly differentiated hepatic cell line HepaRG, which exhibits a hepatocyte-like equipment with various CYP P450s, nuclear receptors and a broad range of hepatobiliary transporters, represents an ideal model system to study human transporter gene regulation (Levee et al. 2006, Aninat et al. 2006). The aim of the present study was to functionally characterize inflammatory and bile salt-induced mechanisms of human transporter gene regulation in the unique HepaRG cell system (kindly provided by Biopredic). Methods: Highly differentiated HepaRG cells maintained in DMSO containing medium were treated with either I 00ngiml LPS or 1 Ongiml IL- I(i or I OyM taurocholate (exemplary for inflammatory conditions or bile salt overload) for 16 hours. RNA, total and nuclear protein were prepared and analyzed by real-time RT-PCR and Western blotting. Results: Upon LPS-treatment NTCP mRNA expression decreased to 16% whereas BSEP expression only showed a moderate decline compared to untreated controls. Effects of IL-lfi treatment were even more pronounced (NTCP 3% and BSEP 10%). NTCP mRNA was paralled by HNF4 mRNA levels of 17%. MRP2 is down-regulated by inflammatory signals to 46% at the protein level whereas mRNA remained unchanged. MRP3 mRNA is increased I .5-fold while MRP4 expression remained stable under these conditions. Taurocholate treatment moderately suppressed NTCP expression while BSEP, MRP3 and MRP2 were unaffected. In contrast, MRP4 mRNA expression was upregulated 9-fold and paralleled by 7-fold increased PXR levels while other nuclear receptors such as FXR, CAR and RXR are decreased. Conclusions: Human bile salt transporters NTCP and BSEP are suppressed by similar inflammatory mechanisms as their rat counterparts.
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Results: Balu-I inhibited [3H]-TCA uptake by human OATPIBI (-95%), OATPIB3 (-15%) and rat Oatplal (-50%), Oatpla4 (-35%) and Oatplb2 (-40%) transporters, but had no effect on [3H]-TCA uptake by NTCP. In rat receiving Balu-I, a marked increase in bile flow (+70%, p 0.05), higher than that induced by TCA (+20%, p i 0.05), was found. This enhanced choleretic ability was probably accounted for by the fact that critical micellar concentration of Balu-1 was 5-fold lower than that of TCA. Phalloidin administration decreased basal (-60%, p i 0.05) and TCA-stimulated bile flow (-55%, p < 0.05), without affecting total bile acid output. Cholestasis was accompanied by signs of liver necrosis, nephrotoxicity and haematuria. In contrast, in Balu-l -treated animals, phalloidin-induced reduction in bile flow was only of 15% and haematuria or signs of hepatic or kidney injury were not observed. Conclusion. Balu-l is able to protect against phalloidin hepatotoxicity, probably due to delayed hepatic uptake and enhanced biliary secretion of the toxin.
13091 IMPACT OF COLITIS ON BILE SECRETION AND HEPATOBILIARY TRANSPORTER EXPRESSION IN MICE: POSSIBLE IMPLICATIONS FOR PATHOGENESIS OF SCLEROSING CHOLANGITIS J. Jahnel’, M. Wagner’, C. Langne?, C. Hoegenauer’, P. Fickert’, D. Silbert’ , J. Gumhold’, A. Fuchsbichleg, H. Denk2, M. Trauner’ . I Laboratory of Experimental and Molecular Hepatology, Dioision of‘ Gastroenterology and Heputology, Department of‘ Internal Medicine, Medical linioersiiy Graz, Graz; ’Institut of‘ Pathology, Medical University Gruz, Graz, Auxtria E-mail: [email protected] Background and Aims: The pathogenetic link between ulcerative colitis and primary sclerosing cholangitis (SC) remains poorly understood. Translocation of lipopolysaccharide (LPS) or other bacterial products could contribute to cholestasis via inflammation-induced changes in hepatobiliary transporter gene expression. We therefore examined the effects of acute colitis on bile secretion and expression of proinflammatory mediators, hepatobiliary transporters and their regulatory nuclear receptors in mice. Furthermore we tested the potential role of chronic colitis as “second hit” in heterozygous multidrug resistance gene 2 knockout mice (Mdr2+’-) which contrary to homozygotes (Mdr2-’-) do not spontaneously develop SC. Methods: Acute colitis was induced in C57/BL6 mice with 3% dextran sulfate sodium (DSS) for 7 days. Chronic colitis in Mdr2”- and wildtype Mdr2+’+ was induced by 5 cycles of DSS. In acute colitis, bile flow/composition and hepatic mRNA expression (RT-PCR) was compared with LPS treated animals (15mgikg BW i.p.). The impact of chronic colitis on bile formation and liver histology was compared between Mdr2+’+ and animals. Results: Acute DSS colitis increased biliary output of bile acids (BA; 210%, p i 0 . 0 5 ) and phospholipids (PL; 210%, p i 0 . 0 5 ) but not of cholesterol (1 13%), glutathione (95%) and bile flow (1 14%, n x ) . Mdrla (1.7-fold, p < 0.05) and Ostbeta (1 .%fold, p < 0.05) mRNA was induced, while other transporters (Ntcp, Bsep, Mrp2- 4, Ostbeta, AbcgS, Abcg8, Oatp I - 4, Mdrl b, Mdr2), nuclear receptors (RXRa, FXR, CAR, PXR) or proinflammatory mediators (TNFa, iNOS) remained unchanged. In comparison, LPS reduced Ntcp (29%), Bsep (22‘%), Mrp2 (52%) and induced Mdrlb (1.7-fold), TNFa (6.8-fold) and iNOS (12-fold; all p < 0.05). BA/PL ratios did not change in acute (7.84 vs. 8.68 in controls) and chronic DSS colitis ( 1 0.27 vs. 7.73) but decreased after LPS (2.19). Chronic colitis induced mild portal inflammation in Mdr2+’- but not Mdr2+’+. However, the t i l l histological picture of SC was not observed. Conclusions: LPS but not acute DSS-induced colitis reduced hepatobiliary gene expression and bile formation. Although chronic colitis in Mdr2+’mice led to mild portal inflammation, SC was not induced. This suggests that genetic Mdr2 defects may not represent a major pathogenetic factor for development of SC in colitis.
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13101 CELL CYCLE QUIESCENCE IS ASSOCIATED WITH THE HEPATOBILIARY GENE EXPRESSION IN HUMAN INTRAHEPATIC BlLlARY EPITHELIAL CELL LINES IN VITRO A. Komori’,2, S. Fujiwara’,2, Y. Aiba’,2, M. Nalcamura’,2, S . Shimoda3, H. Fujiolca’,2, K. Migita’,2, M. Tto’,2, K. Yano’,2, H. Yatsuhashi’,2, H. lshibashi’ >2. ‘Clinical Research Center: NHO National Nagasaki Medical Center: Omnra, Nagasaki; ’Departnzent of‘ Hepatology, Nagasaki University Graduate School of Biomedical Sciences, Omnra, Nagasaki; ”Department of Medicine and Biosystenzic Science, Kynshu linioersity Graduate School of Medical Sciencex, Fuhioku, Japan E-mail: [email protected] Background and Aims: Molecular signature of the regeneration of intrahepatic biliary tree after their wound remains elusive; transcriptomal and phenotypic changes from the terminally differentiated to the dedifferentiated state, as well as from the proliferating to the re-quiescent state, of intrahepatic biliary epithelial cells have not been extensively defined. The aim of the study was to compare the gene expression pattern of human intrahepatic biliary epithelial cell lines (HIBEC) between proliferating and quiescent state in vitro, to elucidate their plasticity in gene expression. Methods: Human epithelial antigen positive (HEA) HIBEC were purified from resected normal adult human livers and propagated in the medium containing 10% FBS and growth factors (EGF and HGF). The forced quiescent condition of HTBEC culture was either monolayer in serum/growth factors-free medium (SFM) on laminin, or floating spheroid culture in ultra low attachment vessels. The mRNA expression of biliary and of hepatocyte markers were assessed by semi-quantitative RT-PCR and Taqman PCR. Human Oligo Microarray Kit (Agilent, CA) was used to characterize the transcriptomal changes in HIBEC. Results: Microarray analysis defined the growth factors/serum deprivationassociated quiescent transcriptome in HIBECs: Down-regulation of a set of cell-cycle associated genes (e.g. cyclin A2 as well as BI/B2) and up-regulation of biliary and epithelial genes (e.g. Cytokeratin (CK) 19, aquaporin I , E-cadherin) were some of the facets. Increase in CK19 and E-cadherin mRNAs were also observed i n the anchorage-independent floating spheroid, and the restoration of these mRNAs in HTBEC in SFM was reversed by the addition of EGF andor serum, suggesting that epithelial-to mesenchymal transition (EMT) occurred in their propagation in vitro. Surprisingly, the forced quiescence were accompanied by the induction of albumin and hepatocyte-specific genes (i.e. G6PC, TO) as well, recapitulating transcriptomally transitional state of HIBEC in vitro. Conculsions: HTBEC gain the hepatobiliary, more-epithelial gene expression in the course of quiescence in vitro. This pattern might be reminiscent of the hepatobiliary phenotype of cells of the finer branches of the biliary tree in inflamed liver, i.e. ductular reaction. Additional environmental cue should be required for the proper re-differentiation of HTBEC in vivo.
13111 URSODEOXYCHOLIC ACID, BUT NOT NORURSODEOXYCHOLIC ACID REVERSES TAUROLITHOCHOLIC ACID-INDUCED CHOLESTASIS IN RAT LIVER S . Maitz’, G.U. Denk’, R. Wimmer’, P. Tnvernizzi2, A.F. Hofmann3, U. Beuers’ . ’Department of Medicine 2 Grosshadern, linioersity of’ Mnnich, Munich, Germany; 2Dioision o f Internal Medicine, Sun Paolo of School of Medicine, Uniuer~xit?,of Milan, Milan, Italy; 3De~~artment Medicine, University of Califtwnia, Sun Diego, USA E-mail: [email protected] -
Background and Aims: Norursodeoxycholic acid (norUDCA) exerts therapeutic effects superior to ursodeoxycholic acid (UDCA) in Mdr2 (Abcb4) knockout mice (Mdr2-’-) that develop chronic progressive sclerosing cholangitis (Gastroenterology 2006; 130:465). Taurolithocholic acid
04B. MOLECULAR AND CELLULAR BIOLOGY (TLCA)-induced cholestasis represents a well-established posttranscriptional model of hepatocellular cholestasis (JBC 2003;278: I78 10). It was the aim of this study to further unravel norUDCA's potentially beneficial effects in cholestasis by comparing its effects to those of UDCA in the model of TLCA-induced cholestasis in isolated perfused rat livers. Methods: The effect of norUDCA (25 pmol/l) on bile flow and biliary secretion of the Mrp2 (Abcc2) substrate, dinitrophenyl-glutathione (GSDNP), was studied in comparison to equimolar UDCA in presence or absence of TLCA (1 0 ymol/l) in isolated rat livers perfused for 115 min with Krebs-Henseleit-bicarbonte buffer (JBC 2003;278:17810). Bile acid administration was started after 45 min, and chlorodinitrobenzene (CDNB, 30 ymolil), the precursor of GS-DNP, was administered from min 65 to 75. Bile secretion was determined gravimetrically and GS-DNP secretion fluorometrically. Results: Administration of UDCA or norUDCA led to a comparable increase of bile secretion in isolated perfused rat livers in comparison to DMSO controls (DMSO, 65.2+2.0 yl/50min/g liver; 0.01 vs. DMSO; UDCA, norUDCA, 136.2&22.6 y1/50min/g liver, p i 0.01 vs. DMSO; mean+SD, n = 4 , each, 165.7f40.9 yli50minlg liver, p i ANOVA) whereas GS-DNP secretion remained unchanged (DMSO, 1037+79 nmoli50minig liver; norUDCA, 876+333 nmol/50minig liver; UDCA, 838+258 nmoli50minlg liver). TLCA (10 ymolil) markedly reduced bile flow (5.5f2.1 yl/50min/g liver) and GS-DNP secretion (52&14 nmol/50min/g liver). Interestingly, UDCA, but not norUDCA reversed TLCA-induced impairment of bile flow (TLCA+UDCA, 47.7+ 15.0 yli50minlg liver, p i 0.0 I vs. TLCA; TLCA+norUDCA, 8.9f3.0 yli50minig liver, n.s. vs. TLCA) and GS-DNP secretion (TLCA+UDCA, 174&72 nmol/50 min/g liver, p iO.0 1 vs TLCA; TLCA+norUDCA, 39&19 nmol/50min/g liver, n x ) . Conclusion: UDCA, but not norUDCA exerts anticholestatic effects at the level of the hepatocyte in the experimental model of TLCA-induced cholestasis in rats. It is intriguing to speculate that combined therapy with UDCA and norUDCA may be superior to monotherapy in biliary disorders in which cholangiocyte as well as hepatocyte dysfunction contribute to disease progression and liver failure.
13121 VEGF PLAYS A MAJOR ROLE IN MEDIATING ESTROGENS PROLIFERATIVE EFFECTS ON HUMAN INTRAHEPATIC CHOLANGIOCARCINOMA M.G. Mancino', A. Mancino', A. Torrice', M.C. Bragazzi', 1. Stammegna', G. Alpini3, P. Onori4, A. Franchitto', E. Gaudio', A.F. Attili' , D. Alvaro' ,'. 'Department of Clinical Medicine, Diuision of Gustroenterology, linioersiiy "La Sapienzu ", Rome; Departnzent of Clinical Medicine, Division of Ga,str~)enter~)l~)~y, Uniuer~xihi ' L a Sapienzu" Polo Pontino, Rome, Italy; 'Scott & White and The Texas A & M Unioersity System Health Science Center College of Medicine, Temple, TX, 1JSA; 4Department of'Experimentu1 Medicine, Section of' Human & Clinical Anatomy, State linioersity of L'Aquib; 'Departnwnt of' Anatomy, linioersity of' Rome, Italy E-mail: [email protected]
Background: Estrogens and VEGF induce the proliferation of neoplastic cells expressing their receptors (ER and VEGFR) through the synergism with growth factors and the induction of neo-angiogenesis. We previously demonstrated that human intrahepatic cholangiocarcinoma expresses ER and that estrogens induce the proliferation of human cholangiocarcinoma cell lines. Aims: The aims of our study were to evaluate in human cholangiocarcinoma: (i) the expression of VEGF and VEGFR; (ii) estrogens effects on VEGFR expression and (iii) the role played by VEGF in mediating the proliferative effects of estrogens. Methods: The study was performed in biopsies of human intrahepatic cholangiocarcinoma (n=5) and in HUH-28 cell line derived from human intrahepatic cholangiocarcinoma. The expression of VEGF-A and
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B ) BlLlARY TRACT PATHOPHYSIOLOGY
S123
VEGF-C and VEGFR were evaluated by immunohistochemistry (IHC) or western blot. Cell proliferation was measured by both PCNA westem-blot and MTS proliferation assay. Results: Biopsies of human intrahepatic cholangiocarcinoma were strongly and uniformly positive for VEGF-A and -C at the IHC examination. HUH-28 cells were positive for VEGF-A, VEGF-C, VEGFR-1-2-3 and 17P-estradiol markedly enhanced (VEGF-A 3 4 f l '% ; VEGF-C 57f4'X; VEGFR-I 70f5%; VEGFR2 517&12?4; VEGFR3 102fl0?4) their expression in association with the induction of cell proliferation. VEGF administration stimulated HUH-28 cells proliferation at a similar extent than serum redmission but into a lower extent than I7(i-estradiol. 17(i-estradiol induced the secretion of VEGF (from 0 to 180 pgiml, ELISA) in the supernatant of HUH-28 cells. The stimulatory effect of 17(i-estradiol on the protein expression of VEGF and VEGFR was blocked by ER (Icil 82,780) or IGFl-R (ctlR3) antagonists, indicating an effect mediated by a coupling of ER and IGFl -R. Finally VEGF-Trap, a receptor-based VEGF inhibitor, decreased by 70% 17[3-estradiol proliferative effect on HUH-28 cells indicating that most of the proliferative effect of estrogens on human cholangiocarcinoma is mediated by VEGF induction. Conclusions: Estrogens stimulate the growth of human intrahepatic cholangiocarcinoma by inducing the expression and secretion of VEGF that plays a major role in mediating estrogens proliferative effects. Thus strategies based on the antagonism of ER and/or VEGF could help in delaying the progression of cholangiocarcinoma.
13131 MECHANISMS OF HUMAN HEPATOBILIARY TRANSPORTER GENE REGULATION IN THE HEPATOCYTE-LIKE CELL LINE HepaRG 1.V Martin, S. Voigt, S. Strauch, C. Trautwein, A. Geier. Department of Internal Medicine Ill, Uniuersity Hospital Auchen, RWTH, Aachm, Germany E-mail: [email protected]
Background and Aim: Functional studies on human hepatic gene regulation are limited due to the lack of suitable models. Currently used human hepatoma cell lines such as HepG2 or Huh7 are deficient in several transporters such as NTCP or BSEP whereas primary human hepatocytes and freshly prepared liver slices are limited in availability. The highly differentiated hepatic cell line HepaRG, which exhibits a hepatocyte-like equipment with various CYP P450s, nuclear receptors and a broad range of hepatobiliary transporters, represents an ideal model system to study human transporter gene regulation (Levee et al. 2006, Aninat et al. 2006). The aim of the present study was to functionally characterize inflammatory and bile salt-induced mechanisms of human transporter gene regulation in the unique HepaRG cell system (kindly provided by Biopredic). Methods: Highly differentiated HepaRG cells maintained in DMSO containing medium were treated with either I 00ngiml LPS or 1 Ongiml IL- I(i or I OyM taurocholate (exemplary for inflammatory conditions or bile salt overload) for 16 hours. RNA, total and nuclear protein were prepared and analyzed by real-time RT-PCR and Western blotting. Results: Upon LPS-treatment NTCP mRNA expression decreased to 16% whereas BSEP expression only showed a moderate decline compared to untreated controls. Effects of IL-lfi treatment were even more pronounced (NTCP 3% and BSEP 10%). NTCP mRNA was paralled by HNF4 mRNA levels of 17%. MRP2 is down-regulated by inflammatory signals to 46% at the protein level whereas mRNA remained unchanged. MRP3 mRNA is increased I .5-fold while MRP4 expression remained stable under these conditions. Taurocholate treatment moderately suppressed NTCP expression while BSEP, MRP3 and MRP2 were unaffected. In contrast, MRP4 mRNA expression was upregulated 9-fold and paralleled by 7-fold increased PXR levels while other nuclear receptors such as FXR, CAR and RXR are decreased. Conclusions: Human bile salt transporters NTCP and BSEP are suppressed by similar inflammatory mechanisms as their rat counterparts.