100 NOTCH1 SIGNALING IN LIVER SINUSOIDAL ENDOTHELIAL CELLS IS ESSENTIAL FOR HOMEOSTASIS OF THE HEPATIC MICROCIRCULATION

ORAL PRESENTATIONS 100 NOTCH1 SIGNALING IN LIVER SINUSOIDAL ENDOTHELIAL CELLS IS ESSENTIAL FOR HOMEOSTASIS OF THE HEPATIC MICROCIRCULATION S. Rothweiler1 , M.T. Dill1 , V. Djonov2 , R. Hlushchuk2 , L. Tornillo3 , L. Terracciano3 , M. Heim1,4 , D. Semela1,4 . 1 Department of Biomedicine, University Basel, Basel, 2 Institute of Anatomy, University Fribourg, Fribourg, 3 Institute of Pathology, University Hospital Basel, 4 Division of Gastroenterology and Hepatology, University Hospital Basel, Basel, Switzerland E-mail: [email protected] Background: Notch signaling plays a pivotal role in embryonic vascular development as well as in normal vascular remodeling. We have reported that Notch1 KO in mice leads to nodular regenerative hyperplasia (NRH). The pathogenesis of NRH is still unclear. The aim of our study is to elucidate the role of Notch1 signaling in NRH focusing on liver sinusoidal endothelial cells (LSECs). Methods: We used MxCre Notch1 lox/lox mice as a tissueunspecific conditional KO mouse model. A hepatocyte-specific KO was created by crossing Notch1 lox/lox with AlbCre+/− mice. Morphological assessment of the liver was performed (proliferation by BrdU IHC, hepatic stellate cell (HSC) activation by alpha-SMA IHC). Morphology of hepatic vasculature was assessed by scanning electron microscopy (SEM) of vascular casts. Portal vein pressure was measured in anesthetized KO mice via a pressure transducer. In vitro angiogenesis was studied using murine, immortalized LSECs on matrigel with primary hepatocyte conditioned medium (CM) from either Notch1 KO mice or control mice or with transformed hepatocyte CM. Results: MxCre induced KO mice developed NRH within 14 days after deletion of Notch1 in the absence of fibrosis or HSC activation. BrdU staining showed a persistently increased proliferation rate of LSECs in MxCre Notch1 KO mice (p = 0.0058). SEM of vascular casts showed that loss of Notch1 leads to dedifferentiation and dramatic vascular remodeling of the hepatic sinusoidal microvasculature with increased branching and diameter (p < 0.001) as well as active intussusceptive angiogenesis. MxCre Notch1 KO mice developed portal hypertension (p = 0.0041) and upregulated endothelial CD34 as marker of capillarisation. In contrast, hepatocyte-specific Notch1 KO mice were phenotypically normal. Accordingly, no difference was found in LSEC proliferation and tube formation with Notch1 CM compared to other CM. Conclusions: Notch1 signaling is required for vascular homeostasis of hepatic sinusoids by inducing quiescence and differentiation of LSEC in adult mice. Disruption of Notch1 pathway leads to LSEC proliferation, vascular remodeling and portal hypertension without fibrosis or HSC activation. The lack of phenotypic changes in hepatocyte-specific Notch1 KO mice suggests that the development of NRH in our model is secondary to vascular remodeling induced by loss of Notch1 signaling in LSEC. 101 HEPATOCYTE-MEDIATED CELL KILLING IS FACILITATED BY THE ASIALOGLYCOPROTEIN RECEPTOR C.S. Guy, S.L. Rankin, T.I. Michalak. Molecular Virology and Hepatology Research Group, Faculty of Medicine, Memorial University, St. John’s, NL, Canada E-mail: [email protected] Background and Aims: It has been shown that both primary and cultured hepatocytes can act as cytotoxic effectors and kill contacted cells via both CD95 ligand (CD95L)-CD95- and perforin-dependent pathways (Hepatology 2006; 43:1231 and 2008; 47:1691). It was also demonstrated that progressing chronic as well as resolved experimental hepadnaviral hepatitis are associated with augmented hepatocyte-mediated cell killing (in press). However, it remained undetermined whether hepatocyteS46

mediated cell elimination is indiscriminant or specifically directed towards cells targeted for killing. It was also unknown whether hepatocytes could directly kill autologous lymphocytes. Materials and Methods: Normal mouse primary hepatocytes, normal woodchuck WCM-260 hepatocyte line and human HepG2 cells served as cytotoxic effectors in JAM cytotoxic DNA fragmentation assay (Hepatology 2006; 43:1231). Murine splenocytes, peripheral blood mononuclear cells (PBMC) and affinity-purified CD4+CD25− T cells activated or not with phytohemaglutinin (PHA), as well as CD95-bearing P815 cells and CD95-deficient K562 cells were used as targets. In some experiments, cell targets were pretreated with neuraminidase (0.02–0.5 U/ml) to remove sialic acid residues from the cell surface glycoproteins or asilofetuin (ASF) was included to block activity of the hepatocyte asialoglycoprotein receptor (ASGPR). Silencing of ASGPR gene encoding the major receptor subunit was facilitated with the gene-specific siRNA. Scrambled siRNA was used as a control. Results: Desialylation of surface glycoproteins rendered cell targets more susceptible to hepatocyte-mediated killing. Usage of a competitive ligand ASF and siRNA-mediated knockdown of the hepatocyte ASGPR, confirmed that this receptor is involved in the recognition of multiple target cell types, including activated lymphocytes. Conclusions: These data demonstrate that hepatocytes are capable of elimination of autologous activated immune cells, such as CD4+ T cells. They suggest that hepatocyte-mediated cell killing is reliant upon the recognition of terminally desialylated glycoproteins on the surface of the cell targets. This study adds a further dimension to the role of the hepatic ASGPR and provides additional evidence that hepatocytes could be active contributors to intrahepatic immune regulation and they may moderate the local inflammatory response. 102 PIVOTAL EFFECTS OF TOLL-LIKE RECEPTOR-9 ACTIVATION IN HEPATIC FIBROSIS J. Amer1 , J.H. Axelrod2 , E. Khvalevsky2 , S. Doron1 , L. Abu Tair1 , Y. Nechemia2 , E. Galun2 , R. Safadi1 . 1 Liver Unit, Gastroenterology Institute, Division of Medicine, 2 Goldyne Savad Institute of Gene Therapy, Hadassah University Medical Center, Jerusalem, Israel E-mail: [email protected] Background: CD8-subsets mediate hepatic-fibrosis & NK-cells have an anti-fibrotic effect. Oligodeoxynucleotides (ODNs) with immunomodulatory-motifs acting through toll-like-receptor-9 (TLR9) to stimulate NK-cells. TLR9−/− mice decreased hepaticfibrosis. Aims: To evaluate TLR9-effects in lymphocyte-interactions of hepatic-fibrosis model. Methods: Carbon-tetrachloride (CCl4 ) hepatic-fibrosis-model was induced in C57Bl/6 wild-type (WT) mice (groups A-D). During the last 2 weeks, mice were weekly treated by hydrodynamicbased tail-vein transfection with eithersaline (group-A), 20 mg-CpG ODN (group-B), 20 mg-pGEM7 (group-C) and were compared to CCl4 (group-D) & naïve (Group-E) controls. Hepatic-fibrosis was evaluated in wild-type (WT) and TLR9−/− . Isolated-splenocytes from WT and TLR9−/− naïve-donors were separately transferred either to irradiated WT or TLR9−/− recipients. Recipients underwent/didn’t fibrosis-induction. Results: Hepatic-fibrosis significantly increased in all fibroticgroups compared to naïves in all experiments. WT-fibroticgroups (A&D) displayed equivalent serum-ALT levels and liveralpha-Smooth-Muscle expressions. Compared to group-D, fibrosissevirity & serum-ALT-levels significantly decreased in ODN-treated WT-groups (B&C). CD8-subset significantly increased in fibrotic groups (A&D), thereafter decreased in treated groups (B&C). CD4-

Journal of Hepatology 2010 vol. 52 | S43–S54