AASLD Abstracts CpG islands in a region around the translation start site of the gene were examined by BGS. We found that 4 out of 9 low SULF1 expressing HCC cell lines had CpG methylation rates between 18-67%; while 5 similarly low SULF1 expressing primary HCC tumors showed methylation between 30-91%. In addition, in two HCC cell lines with methylation rates of 18% and 67% respectively, treatment with 5-aza-2′ deoxycytidine resulted in restoration of sulfatase activity and increased sensitivity to cisplatin-induced apoptosis. Conclusions: Gene methylation was implicated in down-regulation of SULF1 in 4 HCC cell lines and 5 primary HCC tumors. Restoration of sulfatase activity by treatment with demethylation agent resulted in increased sensitivity to cisplatin-induced apoptosis. Therefore, in a subset of HCC, demethylating agents can enhance the effect of chemotherapy by targeting the SULF1 gene. (Supported by NIH Grants CA100882 and CA100882-03S1). 577 HCV-Induced Oxidative Stress Suppresses Basal and Il6-Stimulated Hepcidin Expression in Human Hepatoma Cell Lines Kouichi Miura, Kojiro Taura, Yuzo Kodama, David A. Brenner Background: Chronic hepatitis C infection is characterized by iron accumulation in the liver, which is proposed as a trigger that promotes fibrosis and subsequent occurrence of liver cancer. However, the mechanism by which HCV regulates iron metabolism is poorly under- stood. Hepcidin, an acute phase response protein produced by hepatocytes, inhibits iron absorption from the duodenum and maintains iron storage in macrophages. The present study aimed to elucidate the mechanism by which HCV regulates hepcidin expression. Materials and Methods: HCV replicon cells, HuH7 (parental cells) and HuH7.5, a cell line in which HCV was eradicated by interferon treatment, were cultured. In addition, HuH7 cells were infected with adenoviruses expressing HCV core or NS3-5 protein. We examined the relationship between hepcidin mRNA expression and HCV-induced oxidative stress by measuring DCF, an H2O2-senstive probe. We examined the expression and DNA binding activity of transcriptional factors involved in hepcidin gene regulation, including C/EBPα, hypoxia inducible factors (HIFs) and STAT3 by real-time PCR, Western blot and chromatin immunoprecipitation assay. Results: Hepcidin mRNA expression was significantly lower in HCV replicon cells than in HuH7 and HuH7.5 cells. HCV core protein but not NS3-5 protein decreased hepcidin mRNA expression. Hepcidin mRNA expression was restored by anti-oxidants in HCV replicon cells and in HCV core infected HuH7, which produced much more reactive oxygen species. C/EBPα, a positive hepcidin regulator, were equivalent in protein expression in all cell lines. However, C/EBPα binding to the hepcidin promoter was weaker in HCV replicon cells, which was recovered by anti-oxidants. In contrast, HCV replicon cells strongly expressed HIF-1α and HIF-2α, negative regulators of hepcidin, which were decreased by anti-oxidants. siRNA targeted for HIF mRNAs restored hepcidin expression. These results indicate that oxidative stress reduces basal hepcidin expression in HCV replicon cells. Then we tested IL6, a potent hepcidin inducer. Hepcidin expression increased 35-fold in HuH7 and 17-fold in HuH7.5 but only 4-fold in HCV replicon cells. Although STAT3 phosphorylation in response to IL6 were equivalent in all cell line, STAT3 binding to the hepcidin promoter was weak in HCV replicon cells, which was recovered by anti-oxidants. IL6 combined with an anti-oxidant induced 11-fold hepcidin expression in HCV replicon cells. Conclusion: HCV-induced oxidative stress suppresses basal and IL6- stimulated hepcidin expression. Then low hepcidin expression may induce iron accumulation in the liver of patients with chronic hepatitis C. 578 Human Serum Amyloid P (Hsap) Inhibits Bile Duct Ligation Induced Liver Fibrosis in Mice Tatiana Kisseleva, George Notas, Kojiro Taura, Yuzo Kodama, Samuele De Minicis, Mike Kramer, David P. Hesson, Tim Pelura, David A. Brenner BACKGROUND: Liver fibrosis develops in response to chronic liver injury and is character- ized by extensive deposition of extracellular matrix proteins, mostly collagen type I. Activation of quiescent hepatic stellate cells (HSCs) into collagen producing myofibroblasts and recruit- ment of inflammatory cells into the injured liver are critical steps in development of hepatic fibrosis. Human Serum Amyloid P (hSAP), a member of the pentraxin family that includes C-reactive protein (CRP), has been previously shown to attenuate differentiation and prolifera- tion of fibrocytes, CD45+ collagen type I+ bone marrow-derived cells, and inhibit cardiac fibrosis and bleomycin-induced lung fibrosis in mice. AIM: The aim of this study is to investigate anti-fibrogenic and hepatoprotective properties of hSAP in liver fibrosis. METHODS: Liver injury was induced in wild type and Collagen-GFP reporter mice, expressing GFP under control of the collagen-α1(I) promoter/enhancer, by bile duct ligation (BDL). Mice were divided into two groups and injected every other day after BDL either with hSAP (250 μg/mouse, 7 injections) or PBS . 14 days after BDL liver tissue were analyzed for the expression of fibrogenic markers by fluorescent microscopy and RT-PCR. The effect of hSAP on collagen expressing cells was also tested In Vitro by culturing quiescent HSCs in the presence or absence of hSAP. RESULTS: Fluorescent microscopy revealed that hSAP inhibited the expression of collagen-α1(I) (45-50%) in liver tissues of Collagen-GFP+ BDL- mice. Hepatic fibrosis was reduced 50% by hSAP administration, as detected by Masson TriChrome staining. Moreover, administration of hSAP significantly inhibited BDL-induced tissue damage (hepatocyte necrosis, proliferation of the bile ducts), indicating that hSAP exhibits not only anti-fibrogenic but also hepatoprotective properties. These findings were confirmed by RT-PCR analysis. Collagen-α1(I) mRNA, α-SMA and TIMP-1 mRNA expression were reduced in hSAP treated mice. Moreover, expression of IL-6, an acute phase response cytokine, was also downregulated by hSAP. However, the levels of TNF-α, IL-1β and TGF- β1, the major pro-fibrogenic cytokine mostly produced by macrophages, were not affected by hSAP administration. These findings suggest that hSAP potentiates its anti-fibrogenic effect through inhibition of collagen producing cells rather than inflammatory cells. In Vitro, 30 μg/ml, was sufficient to inhibit HSCs activation. CONCLUSIONS: hSAP has an anti- fibrotic and hepatoprotective effect in a BDL model of liver fibrosis in mice. Anti-fibrotic effects of hSAP result in part to its direct effects on HSCs and fibrocytes. A-768 AASLD Abstracts 683 Hepatocyte Priming and Proliferation in Response to Partial Hepatectomy Is Impaired in the eNOS Knockout Mice Yu Mei, Hongdan Sun, Bryan Tackett, Sundararajah Thevananther Background: Partial hepatectomy (PH) induces a well-orchestrated cascade of signaling events necessary for the initial priming phase, effective cell cycle progression, and proliferation of hepatocytes which culminates in liver regeneration. However, the identity of critical factor(s) responsible for the initiation of hepatocyte priming and proliferation in response to PH remains unknown. Nitric oxide (NO) is a well-known pleiotropic agent influencing multiple aspects of hepatic physiology and pathophysiology and generated via the activation of inducible and endothelial nitric oxide synthase isoforms (iNOS and eNOS), each with distinct modes of activation and co-factor requirements in the liver. The specific role of eNOS- mediated signaling in liver regeneration however, is currently unknown. Therefore, the purpose of this study was to test the hypothesis that eNOS plays a critical role in the induction of hepatocyte priming and proliferation in response to partial hepatectomy. Methods: PH (70%) was performed on 12 week old adult male wild-type (WT) and eNOS knockout mice (KO), both raised on C57BL6/J background. Remnant livers were harvested at 30 min for the analysis of priming events, and at 24, 45, and 72h for the analysis of cell-cycle progression. In order to characterize the early priming events, nuclear proteins were analyzed for the phosphorylation and activation of immediate early gene c-Jun and induction of activator protein 1 (AP-1) DNA-binding activity. Cell cycle progression and proliferation were evalu- ated by analyzing BrdU incorporation and induction of cyclins and PCNA in the remnant livers. Results: PH induced robust activation of c-Jun phosphorylation and AP-1 activity in the remnant livers of WT. The induction of c-Jun phosphorylation (0.6 fold, P<0.01) and AP-1 activity (0.4 fold, p<0.05) was impaired in the KO as compared to the WT (1.0). Correspondingly, hepatocyte proliferative response to PH was impaired in the remnant livers of KO mice, as evidenced by impaired BrdU incorporation (0.3 fold at 45 hrs; p< 0.05), expression of cyclin A (0.3 fold at 45h; P<0.01) and PCNA (0.8 and 0.4 folds at 45h and 72 h respectively; p<0.05) as compared to WT (1.0). Conclusions: Our findings suggest that eNOS activation plays a critical role in the initiation of hepatocyte priming and elicitation of a robust hepatocyte proliferative response to PH. The results highlight a hitherto unidenti- fied role for NO and eNOS-specific signaling as key mediators of liver regeneration, independ- ent of the known iNOS-mediated pathways, with implications for our understanding of multifactorial mediators of liver growth and repair after injury. 684 C133+ Liver Cancer Stem Cells from Methionine Adenosyl Transferase 1A Deficient Mice Demonstrate Resistance to TGF-β Induced Apoptosis Carl B. Rountree, Wei Ding, Shelly C. Lu Background: Methionine adenosyltransferase (MAT) is an essential enzyme that catalyzes S- adenosylmethionine biosynthesis. Hepatic MAT activity falls during chronic liver injury, and mice lacking MAT1A develop hepatocellular carcinoma (HCC) spontaneously by 18 months. We have previously demonstrated that CD133+ CD45- oval cells from old MAT1A KO mice represent a liver cancer stem cell population. The transforming growth factor-β (TGF-β) pathway constitutes a central signaling network in cell differentiation, proliferation, motility, apoptosis, and tumorgenesis. However, the role of TGF-β in oval cell to cancer stem cell progression is unknown. Our current work tests the response of these tumorgenic liver stem cells to TGF-β. Methods: Single CD133+ CD45- oval cells were isolated by flow cytometry and expanded as five separate clones from pre-malignant 18 month old MAT1A KO mice. Gene (qPCR) and protein (Immuno-Blot) expression analysis was conducted on all clones. Cells were plated in soft agar to demonstrate anchorage independent growth. TGF-β was administered at 20 ng/ml in-vitro. Results: All five clones demonstrated expression of hepato- cyte (AFP) and cholangiocyte (cholangiocyte cytokeratin) markers by immunohistochemistry with co-localization in 33%+4 of cells, indicating a robust oval cell population. Rapid anchorage independent growth was demonstrated in all clones. Gene expression analysis confirmed the bi-lineage potential of 100% of these clones, which demonstrated hepatocyte markers: Albumin, αFP, A1AT, HNF4a, and cholangiocyte markers: CK-19 and Biliary glycoprotein, as well as oval cell genes ABCG2, HNF3b, HNF1, and Hepatocyte Growth Factor receptor (HGFr) c-MET. HGF stimulation resulted in ERK phosphorylation, indicating functional HGF/c-MET signal pathway. BRDU pulse demonstrated that 50.8%+5% of cell entering S phase of cell cycle after 1 hour, a high level of proliferation. TGF-β stimulation resulted in 90% apoptosis after 12 hours, as demonstrated by DNA ladder. Of the remaining viable cells, the number entering the cell cycle was reduced by 85%. When C133+ cancer stem cells were compared to CD133- cells from the same population, the CD133+ cells demonstrated resistance to TGF-β induced apoptosis, with a 25% reduction in the number of aopototic cells compared to CD133- cells. Furthermore, C133- cell demonstrated a substantial decrease in phospho-ERK when compared to C133+ cells after TGF-β stimulation. Conclusion: CD133+ liver cancer stem cells from MAT1A KO mice demonstrate resistance to TGF-β induced apoptosis compared to CD133- cells. This resistance indicates a mechanism for liver cancer stem cell survival and proliferation. 685 Glial Fibrillary Acidic Protein(+) Progenitors Regenerate Adult Livers Liu Yang, Youngmi Jung, Jiawen Huang, Alessia Omenetti, Rafal P. Witek, Steve S. Choi, Gianfranco Alpini, Anna Mae Diehl Background and Aims: When hepatocyte proliferation is inhibited, progenitors regenerate adult livers. The mechanisms involved remain obscure. We investigated whether myofibrobl- astic hepatic stellate cells (MF-HSC) and ductular cells that accumulate in damaged liver share a common lineage and are precursors of hepatocytes. Because MF-HSC derive from GFAP-expressing cells, we hypothesized that GFAP(+) cells are common progenitors of all three cell types. Methods: We generated GFAP-Cre/reporter mice to evaluate GFAP, Cre,