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766

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The extracellular proteinase inhibitor gene Expi is a candidate gene for hepatic fibrosis susceptibility

Roman Liebe, Rabea A. Hall, Steven Dooley, Frank Lammert;
Department of Medicine II, Saarland University, Homburg, Germany

Aim: To identify new contributors towards increased susceptility for hepatic fibrosis progression, we mapped quantitative trait loci (QTLs) that determine hepatocellular susceptibility to profibrogenic transforming growth factor (TGF)-β signalling in cultured primary hepatocytes. We availed of cells from the murine reference population BXD, recombinant inbred offspring of the mouse strains C57BL/6J and DBA/2J, which differ in fibrosis susceptibility (Andreuxet al. Cell 2012). A common 6 Mb locus on chromosome 1 1 modulated TGF-β-induced total cell death in vitro and histological fibrosis stage after CCl4 challenge in vivo. Methods: The effects of genetic loci within a 6 Mb phenotypic QTL on hepatic gene expression following short-term (24 hrs) liver damage by ethanol or chronic CCl4 challenge (6 wks) were assessed using expression QTL (eQTL) analysis. A mouse with targeted Expi knockout was provided by the EUCOMM repository. Homozygous disruption of the Expi gene by insertion of a neo cassette was verified by genomic PCR and resulted in no detectable phenotype in untreated mice. Results: eQTL mapping of correlations between SNPs within the QTL and transcript abundance in liver identified a variant at 83.5 Mb that co-segregated significantly with expression variation. Analysis of amino acid exchanges near the major expression-associated SNP indicated candidacy of the extracellular proteinase inhibitor Expi for both traits. Cellular damage assessment of knockout hepatocytes following 48 hrs treatment with TGF-β suggested higher susceptibility in Expi knockout cells. Quantification of hepatic collagen contents following 6 weeks of CCl4 challenge was indicative of higher fibrosis rates in wild-type mice as compared to knockout animals. Conclusions: The combination of QTL mapping in vitro and in vivo with eQTL analysis identifies novel susceptibility genes for fibrogenesis. Knockout of the candidate gene Expi results in differential susceptibility to TGF-β-induced cell death and hepatic collagen contents after CCl4 challenge, consistent with a potential novel role of this extracellular proteinase inhibitor in acute and chronic liver injury.

Disclosures:

The following people have nothing to disclose: Roman Liebe, Rabea A. Hall, Steven Dooley, Frank Lammert

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Loss of Gab1 scaffolding adaptor protein in the hepato- cytes exacerbates cholestasis-induced liver fibrosis in mice

Takashi Kizu, Yuichi Yoshida, Kunimaro Furuta, Satoshi Ogura, Mayumi Egawa, Norihiro Chatani, Mina Hamano, Hisao Ezaki, Yoshihiro Kamada, Shinichi Kiso, Tetsuo Takehara;
Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka, Japan

Background and Aims: Chronic cholestatsis induces liver injury and eventually leads to liver fibrosis. Various receptor tyrosine kinases (RTKs)-mediated signaling, such as HGF/c-Met, has been shown to be involved in this process. Grb2-associated binder 1 (Gab1) is a scaffolding adaptor protein that acts downstream of RTKs and has been shown to be required for hepatocyte proliferation during liver regeneration. However, the role of Gab1 in liver fibrosis progression during chronic cholestatsis has remained unclear. The aim of this study was to elucidate this issue using cholestasis-induced mouse liver fibrosis model. Methods: Hepatocyte-specific Gab1 knockout (KO) mice were generated using Cre-loxP system. KO and wild type (WT) mice were subjected to bile duct ligation (BDL) to induce cholestasis-induced liver fibrosis. Results KO mice had an increased number of apoptotic hepatocytes (p<0.05) and a decreased number of proliferating hepatocytes (p<0.05) compared with WT mice at 5 days after BDL. KO mice also showed an increase in the number of infiltrating neutrophils and macrophages. These data indicates that hepatic loss of Gab1 enhanced liver injury and inflammation. We next examined liver fibrosis of these mice at 10 days after BDL. KO mice developed more severe liver fibrosis with a 2-fold increase in the fibrosis area assessed by picrosirius red staining (p<0.05) and a 1.5-fold increase in hepatic hydroxyproline content (p<0.05). αSMA staining also showed enhanced activation of hepatic stallate cells in KO mouse liver. Consistent with this, KO mice demonstrated an increased expression of fibrosis related genes, such as Col 1α, ACTA2 orTGFβ1 (p<0.05). This abnormal liver fibrosis in KO mice was associated with increased tyrosine phosphorylation of c-Met, which might be a result of negative feedback due to hepatic loss of Gab1, a key signal transducer of HGF/c-Met signaling. Finally, cDNA microarray analysis identified chemokine CCL5, which has been shown to have a fibrosis-promoting activity in recent reports, as an up-regulated gene in the liver of KO mice at 1 0 days after BDL. Further validation by qRT-PCR demonstrated that KO mouse livers displayed a 5-fold increase in gene expression of CCL5 (p<0.05). Moreover, administration of CCL5 antagonist significantly improved liver fibrosis in KO mice, indicating that the induction of CCL5 in KO mice was functional. Conclusion: Loss of Gab1 in the hepatocytes exacerbates liver fibrosis after BDL in mice. Gab1 might protect from liver fibrosis via suppression of CCL5 in the liver.

Disclosures:

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.

The following people have nothing to disclose: Takashi Kizu, Yuichi Yoshida, Kunimaro Furuta, Satoshi Ogura, Mayumi Egawa, Norihiro Chatani, Mina Hamano, Hisao Ezaki, Yoshihiro Kamada, Shinichi Kiso

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Hedgehog-Induced Serine Biogenesis is Required for Activation of Hepatic Stellate Cells

Yuping Chen, Gregory A. Michelotti, Guanhua Xie, Cynthia A. Moylan, Anna Mae Diehl;
Division of Gastroenterology, Duke University Medical Center, Durham, NC

Background: Hepatic stellate cells (HSCs) transit from a non-pro-liferative quiescent (Q) to a proliferative myofibroblastic (MF) phenotype during liver fibrosis. Hedgehog (Hh) signaling regulates HSC reprogramming by shifting energy utilization towards aerobic glycolysis, correlating with lipid depletion and lactate accumulation. Recent studies revealed that serine biogenesis also contributes importantly to energy homeostasis by regulating levels of α-ketoglutarate, a key intermediate of the tricar-boxylic acid cycle. Since key intermediates are common to both glycolytic and amino acid metabolic pathways, we tested the hypothesis that serine biogenesis regulates HSC reprogramming in a Hh-dependent manner. Methods: In vitro: Differentially expressed genes for serine biogenesis were identified by microarray analysis of mouse Q-HSCs and MF-HSCs and validated by qRT-PCR and western blot (WB). HSCs were cultured in media supplemented with either glucose or glutamine and cellular proliferation, migration, and invasion were quantified. Hh signaling was perturbed by overexpression of GLI1 or GLI2 or by Cre-mediated deletion of Smoothened (Smo) in HSCs isolated from Smo-LoxP mice. In vivo: Serine biogenesis was examined by qRT-PCR, WB, and immunohistochemistry in three liver fibrosis models (methionine choline deficient (MCD) diet, carbon tetrachloride (CCl4), and bile duct ligation (BDL)). Hh signaling was perturbed in αSMA-Cre/ERT2-Smo conditional knockout mice subjected to BDL-injury. Results: In vitro: Genes encoding enzymes for serine biogenesis and glutaminolysis were highly induced in MF-HSC versus Q-HSC, including several rate limiting enzymes (Phgdh 20-fold;Psat1 3.1-fold;Gls1 44-fold) and c-Myc (7.5-fold) (p<0.001). GLI overexpression increased expression of these key genes (all p<0.001), while genetic ablation of Smo expression in primary MF-HSC decreased expression. Perturbation of serine metabolism by depleting either glucose or glutamine resulted in dramatic reduction of cell proliferation, migration and invasion in cultured HSCs relative to HSCs grown under standard conditions (p<0.05). In vivo: Consistent with in vitro results, all markers of serine biogenesis were elevated in mice exposed to both acute (CCl4 and BDL) and chronic (MCD diet) fibrogenic injuries versus control mice. Conditional inhibition of Hh signaling in αSMA(+) cells repressed whole liver Phgdh expression, correlating with reduced collagen expression and liver fibrosis. Conclusion: HSC reprogramming requires a Hh-regulated metabolic transition that leads to preferential enhancement of serine biogenesis, likely impacting levels of key metabolic intermediates.

Disclosures:

Anna Mae Diehl - Consulting: Bristol Myers Squibb, Synergy, GlaxoSmithKline, Norgine; Grant/Research Support: GlaxoSmithKline

The following people have nothing to disclose: Yuping Chen, Gregory A. Michelotti, Guanhua Xie, Cynthia A. Moylan

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Lack of secreted frizzled-related protein 5 (Sfrp5), an adipocytokine, enhanced carbon-tetrachloride-induced liver fibrosis in mice

Norihiro Chatani1, Yoshihiro Kamada1, Takashi Kizu1, Satoshi Ogura1, Kunimaro Furuta1, Mayumi Egawa1, Mina Hamano1, Hisao Ezaki1, Shinichi Kiso1, Akihiko Shimono2, Noriyuki Ouchi3, Yuichi Yoshida1, Tetsuo Takehara1;
1Gastroenterology and Hepa-tology, Osaka University Graduate School of Medicine, Suita, Japan; 2Reseach and development Dept., TransGenic Inc., Kobe, Japan; 3Molecular Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan

Background and Aims: It is well known that obesity enhances liver fibrosis progression through adipocytokine dysregulation. Recently, secreted frizzled-related protein 5 (Sfrp5), a non-canonical Wnt signal (Wnt5a) inhibitor, was identified as a novel adipocytokine. Sfrp5 expression in adipose tissue was reduced in obese (Science 2010). It has been reported that Wnt5a activates c-Jun N-terminal kinase (JNK), and JNK activation augments liver fibrosis progression. Moreover, the hepatic gene expression of Wnt5a was reported to be upregulated in fibrosis model mice. Sfrp5 would play important roles in liver fibrosis prevention. To elucidate this issue, we investigated the roles of Sfrp5 in liver fibrosis using Sfrp5 knockout (KO) mice. Methods: (1) Acute liver injury model; To investigate the degree of carbon-tetrachloride (CCl4) induced acute liver injury, male KO and C57BL6J (WT) mice (8 weeks old) were used. Mice were each injected with a dose of CCl4 (0.5 ml/kg/bw) intraperitoneally. At the time of 6, 12, 24, 48, and 72 hours after CCl4 injection, mice were sacrificed. (2) Liver fibrosis model; To compare the degree of CCl4 induced liver fibrosis, male KO and WT mice (8∼12 weeks old) were injected with a dose of CCl4 (0.5 ml/kg/bw) intraperitoneally twice a week for 6 weeks. At the end of each experiment, mice were sacrificed. (3) In vitro study; Hepatic stellate cells (HSCs) were isolated from WT mice liver by collagenase perfusion method. We investigated the effects of recombinant Wnt5a on the HSC proliferation, migration, fibrotic gene expression, and nuclear translocation of Smad3. We simultaneously investigated the inhibitory effects of Sfrp5 on these Wnt5a functions. Results: (1) Acute liver injury induced no significant differences in liver histology and serum ALT levels between WT and KO mice. However, KO mice showed more augmented hepatic JNK phosphorylation than WT mice at the time of 6 and 12 hours after CCl4 injection. (2) Hepatic fibrosis area evaluated by Sirius-red staining was significantly increased in KO mice compared with in WT mice (p<0.005). In addition, the number of α-smooth muscle actin positive cells significantly increased in KO mice liver. (3) Wnt5a induced JNK phosphorylation and Smad3 nuclear translocation in HSCs. Fibrosis related gene expression (transforming growth factor-β, collagen Iα1) was upregulated by Wnt5a. Moreover, both proliferation and migration were also enhanced by Wnt5a. Co-administration of Sfrp5 with Wnt5a cancelled these effects of Wnt5a on HSCs. Conclusion: The findings indicate that the lack of Sfrp5 enhanced carbon-tetrachloride-induced liver fibrosis in mice through Wnt5a signal inhibition.

Disclosures:

Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD K.K.

The following people have nothing to disclose: Norihiro Chatani, Yoshihiro Kamada, Takashi Kizu, Satoshi Ogura, Kunimaro Furuta, Mayumi Egawa, Mina Hamano, Hisao Ezaki, Shinichi Kiso, Akihiko Shimono, Noriyuki Ouchi, Yuichi Yoshida

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β-catenin-mediated CXCL1 and CXCL10 secretion by fibrocystin-defective cholangiocytes regulates peribiliary recruitment of fibrocytes in congenital hepatic fibrosis

Luca Fabris1,3, Luigi Locatelli2, Davide Viganò2, Maria De Mat-teis1, Romina Fiorotto3, Roberto Scirpo2, Stuart D. Morton1, Massi-miliano Cadamuro1, Carlo Spirli3, Mario Strazzabosco2,3;
1Dep. of Surgery, Oncology and Gastroenterology, University of Padova, Padova, Italy; 2Department of Surgery and Interdisciplinary Medicine, University of Milan-Bicocca, Milan, Italy; 3Section of Digestive Diseases, Yale University, New Haven, CT

BACKGROUND AND AIM Congenital Hepatic Fibrosis (CHF) is a genetic liver disease caused by mutations in PKHD1, a gene encoding for Fibrocystin, a ciliary protein expressed by cholangiocytes. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis and portal hypertension. In CHF mechanisms of portal fibrosis are unknown. We have recently reported that Pkhd1-/- mice present: 1)activation of b-catenin signaling in cystic cholangiocytes; 2)increased per-icystic infiltrate of CD45+/Collagen type1 (Col1)+ cells, reminiscent of fibrocytes. Fibrocytes are monocyte-derived cells, involved in several fibrosing conditions, but their role in liver scarring is controversial. b-catenin is emerging as a regulator of inflammation, therefore we hypothesized that a b-catenin-dependent chemokine production by cholangiocytes drives recruitment of fibrocytes in Pkhd1-/- mice. METHODS In Pkhd1 -/- mice we investigated: a)a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); b)the effects of two different b-catenin inhibitors (ICG-001, 25uM, or quercetin, 50uM) on the expression of CXCL1 and CXCL10 (RT-PCR); c)the immunohistochemi-cal expression of CD45/Col1 (fibrocyte markers) and aSMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with portal fibrosis (Sirius-red) in liver samples at 1-12 months; d)the effects of cholangiocyte conditioned media (CM) and CXCL1+CXCL10 on WEHI265.1-monocyte chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1 (A1)). WT mice served as controls. RESULTS Pkhd1-/- cholangiocytes secreted significantly higher basolateral levels of CXCL1 and CXCL1 0 as compared to WT. Expression of CXCL1 and CXCL10 was significantly inhibited in cells treated with ICG-001 and quercetin. Pkhd1-/- mice showed progressive fibrosis, but portal accumulation of aSMA+ cells was evident only after 9 months. In contrast, we observed an early peribiliary recruitment of CD45+/Col1+ cells, whose number strongly correlated with the Sirius-red area (r=0.89,p<0.01). CM from Pkhd1-/-cholangiocytes, as well as CXCL1+CXCL10 stimulated both migration and expression of COL1 (A1) mRNA in WEHI265.1 cells, consistent with transdifferentiation of fibrocytes. CONCLUSIONS In Pkhd1-/- mice progressive portal accumulation of CD45+/COL1 + cells (fibrocytes) correlates with portal fibrosis and cholangiocyte secretion of increased levels of CXCL1 and CXCL10 in a b-catenin-dependent way. This novel mechanism may underlay the recruitment of monocytes and their transdifferentiation into fibrocytes and consequently promote fibrosis deposition.

Disclosures:

The following people have nothing to disclose: Luca Fabris, Luigi Locatelli, Davide Viganò, Maria De Matteis, Romina Fiorotto, Roberto Scirpo, Stuart D. Morton, Massimiliano Cadamuro, Carlo Spirli, Mario Strazzabosco

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The pluripotency involved factors, Lin28, KLF4, and Notch 1 are targets of neuronal miRNAs in hepatic stellate cells

Jia Huang1, Andrea Noetel1, Moritz Perrech2, Ingo Strack1, Jürgen Hescheler2, Reinhard Buettner1, Tomo Saric2, Margarete Odenthal1;
1Institute for Pathology, University Hospital Cologne, Cologne, Germany;2Department of Neurophysiology, University of Cologne, Cologne, Germany

Background and Aim: Myofibroblastic hepatic stellate cells (HSC) are the central cell types of liver fibrosis due to their excessive matrix production. By deep sequencing of the Ago2 interacting transcriptome we have demonstrated that neuronal miR-9, miR-125b, miR -128 are involved in regulation of chemokine signaling during myofibroblastic HSC activation (Noetel et al., 201 3). In the present study, we aimed to analyse further putative targets of the Ago2 neuronal miRNA complex, namely Notch 1, KLF4, and Lin28. Methods: Notch 1, KLF4, and Lin28 gene expression was quantified in primary rat HSC cultures by real time PC R. 3∽UTR sites of Notch 1, KLF4, and Lin28 transcripts, putatively targeted by neuronal miR-9, miR-125b, miR-128, were cloned downstream to a luciferase reporter gene and used for reporter assays in myofibroblastic HSC treated with miRNA mimics or with scrambled miRNA. Notch 1 and Lin28 were overexpressed by lentiviral vector transduction and the influence on HSC was determined by expression profiling using PCR arrays. Results: Since the pluripotency involved factors, Notch 1, KLF4, and Lin28 were suggested to be targets of posttranscriptional neuronal miRNA repression, we investigated the expression of these mediators during myofibroblastic HSC transition. Whereas the neuronal miRNAs miR-9, miR-125b, and miR-128 were highly increased, the putative targets Notch 1 and Lin28, but not KLF4 were markedly decreased during myofibroblastic differentiation of HSC. Next, we analysed the 3∽UTR of Notch1, KLF4, and Lin28 transcripts for putative binding sites of the three neuronal miRNAs. Reporter assays of the luciferase constructs, harboring the putative 3∽UTR sites of Notch1, KLF4, and Lin28 mRNA in comparison to the corresponding mutants definitively demonstrate the inhibitory interaction of all neuronal miRNAs to Lin28 and Notch1, and of miR-128 to KLF4 mRNA. Furthermore, neuronal miRNA over-expression in HSC resulted in a prominent decrease of Notch1 and Lin28. In order to proof the impact of the neuronal miRNA Notch1 axis, lentiviral overexpression was performed. Expression profiling of Notch1 overexpressing cells revealed that members of the Notch signaling pathway were highly upregu-lated whereas some fibrogenic mediators are downregulated. Conclusion: Upregulation of neuronal miR-9, miR-125b, and miR-128 during myofibroblastic transition and the identified interaction with factors involved in pluripotency and cell differentiation suggests a prominent role of these miRNAs in HSC differentiation and activation during fibrosis.

Disclosures:

The following people have nothing to disclose: Jia Huang, Andrea Noetel, Moritz Perrech, Ingo Strack, Jürgen Hescheler, Reinhard Buettner, Tomo Saric, Margarete Odenthal

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Anaphylatoxin C5a modulates Hepatic Stellate Cell migration

Dola Das1, Jazmine Danner1, Mark A. Barnes2, Laura E. Nagy1,2;
1 Pathobiology, Lerner Research Institute, CCF, Cleveland, OH; 2Molecular Medicine, Case Western Reserve University, Cleveland, OH

C5a and its cognate receptor C5aR are critical modulators of both liver immunity and liver fibrosis. However, the molecular mechanism for the cross talk between the complement system and liver fibrosis is not well understood. C5a is a potent chemokine regulating the migration of cells in the innate immune system. Since the migration of hepatic stellate cells (HSC) is a hallmark of liver fibrosis, we hypothesized that C5a contributes to fibrosis by regulating HSC migration. Primary cultures of mouse HSC were activated in response to culture on plastic, marked by increased expression of alpha smooth muscle actin (αSMA) and collagen 1A1 (Col1A1) mRNA. Expression of mRNA and protein for the C5aR also increased during HSC activation in culture. To study if C5aR expression also increased during in vivo activation of HSC, hepatic fibrosis was induced in mice expressing GFP under the control of the collagen promoter by exposure to carbon tetrachloride. FACS analysis of GFP expressing HSC revealed an increased expression of C5aR, similar to that observed in HSC activated in culture. To understand the functional significance of C5aR expression in activated HSC activation, we next investigated whether C5a influenced HSC activation or migration. Challenge of HSCs with C5a during culture had no effect on expression of αSMA and Col1A1, suggesting that C5a did not influence HSC activation. Another important characteristic of HSC is their migratory capacity, primarily mediated by the chemokine MCP-1 and platelet derived growth factor (PDGF). Since C5a is a potent chemokine, we hypothesized that C5a would stimulate HSC migration. To test this hypothesis, wound healing cell migration assay was carried out. C5a enhanced HSC migration almost as efficiently as PDGF. C5a also stimulated the expression of MCP-1. C5a-induced cell migration was slower, but not completely inhibited, in presence of 227016, an MCP-1 receptor antagonist, suggesting C5a-induced migration occurs via MCP-1 dependent and independent mechanisms. Furthermore, C5a did not increase Ki67 nuclear staining, indicating wound healing was independent of cell proliferation. Taken together, these data reveal a novel mechanism for the interaction between complement and hepatic fibrosis, and suggest that C5a and its receptors are possible therapeutic targets in the treatment of liver fibrosis.

Disclosures:

The following people have nothing to disclose: Dola Das, Jazmine Danner, Mark A. Barnes, Laura E. Nagy

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Hepatocytes-derived microparticles released during lipotoxicity induce hepatic stellate cells activation and migration

Davide Povero1, Akiko Eguchi1, Chiara Busletta2, Erica Novo2, Maurizio Parola2, Ariel E. Feldstein1;
1Department of Pediatrics, University of California San Diego, La Jolla, CA; 2Department of Clinical and Biological Sciences, University of Torino, Torino, Italy

Hepatic fibrosis represents the most worrisome histopathologic feature in non-alcoholic steatohepatitis (NASH) and it suggests a more severe and progressive liver damage. Understanding the mechanisms linking NASH to fibrogenesis is essential for defining potential novel therapeutic strategies. We have recently demonstrated (EASL 2013) that hepatocyte-derived microparticles (MPs) are released in the bloodstream during experimental NASH and their levels strongly correlate with severity of liver fibrosis. Here we tested the hypothesis that MPs released by hepatocytes during lipotoxicity alters hepatic stellate cells (HSC) biology resulting in its activation. Methods. For induction of lipotoxicity, the human hepatoma cells (HepG2) were treated with a saturated free fatty acids (FFA) including palmitic acid, or stearic acid, for up to 24hrs with various concentrations (0.25 to 0.50 mM). MPs and MP-free supernatant were isolated from cell-free supernatants by ultracentrifugation and quantitated by FACS analysis. Hepatocytes-derived MPs, MP-free supernatants, MP+Vanin-1 neutralizing antibody (VNN1 nAb) and controls were used to treat HSC (LX2 and primary human HSC) for 6 and 24hrs. Migration was assessed by Boyden's chamber and wound healing response while HSC activation was determined by quantitation of the expression of pro-fibrogenic markers. Internalization of MPs into the HSCs was studied by immunofluorescence. Results. Exposure of HSC with hepatocytes-derived MPs resulted in significant increase expression of key pro-fibrogenic genes, including α-SMA, TIMP1 and Collagen-I (p<0.01) and proliferation (MPs vs. MP-free supernatant, p<0.04). Exposure of primary HSC and LX2 cells to MPs released by hepatocyte during lipotoxicity resulted in a significant phenotypic change characteristic of their activation, with increased migration (MPs vs. MP-free supernatant, p<0.001) and wound healing response (MPs vs. MP-free supernatant, p<0.002) mainly after 24hrs of incubation. MPs internalization by HSC was crucial for the MP effects and was mediated at least in part through a Vanin-1 -dependent mechanism. Indeed activation and migration of HSC were significantly abrogated by neutralizing Vanin-1 on the MPs by a specific neutralizing antibody (MP vs. MP+VNN1 nAb, p<0.04). Conclusion. Our study demonstrates that MPs released from dying hepatocytes during lipotoxicity are critical signals that contribute to HSC activation in a process dependent on Vanin-1 expression. These results provide a mechanistic link between MPs and liver fibrosis and has important implications for development of novel diagnostic and therapeutic strategies for patients with this condition.

Disclosures:

Maurizio Parola - Independent Contractor: Shire Pharmaceutical Ltd, Basingstoke, UK

The following people have nothing to disclose: Davide Povero, Akiko Eguchi, Chiara Busletta, Erica Novo, Ariel E. Feldstein

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PDGF receptor alpha is required for SMAD dependent TGF-beta signaling in hepatic stellate cells

Chunsheng Liu, Vijay Shah, Ningling Kang; Gastroenterology Research Unit, Mayo Clinic, Rochester, MN

Introduction: PDGF and TGF-β contribute to hepatic stellate cell (HSC) activation process under pathological conditions such as metastatic tumor growth in the liver. PDGFs regulate the proliferation and migration of HSCs and TGF-β induces transdiffer-entiation of quiescent HSCs into myofibroblasts. It is believed that different intracellular adaptor proteins downstream of PDGF and TGF-β receptors making these two signaling pathways functionally divergent. For example, AKT, MAPK and PLCγ are major signal intermediates of PDGF receptor tyrosine kinases and SMADs serve this role for TGF-β receptor serine/threonine kinases. Although TGF-β can also activate AKT and MAPK to some extent, evidence to support a convergence of these two independent signaling pathways for HSC activation remains scarce. Hypothesis: We tested a hypothesis that PDGF receptors may participate in the TGF-β signaling by binding to TGF-β receptors in HSCs. Methods: Lentiviral vectors encoding PDGF receptor alpha (PDGFRα) or beta (PDGFRβ) were used to knockdown PDGFRα or PDGFRβ in HSCs. HSCs were treated with TGF-β and phosphorylation of SMAD2, ERK and AKT was determined by Western blot analysis. Receptor interactions were determined by immunoprecipitation (IP) and plasma membrane TGF-β receptor II (TβRII) was quantitated and biotinylation of cell surface proteins. Results: Knockdown of PDGFRα but not PDGFRβ drastically reduced TGF-β induced phosphorylation of SMAD2 in HSCs. This was specific for SMAD dependent TGF-β signaling since knockdown of PDGFRα did not reduce TGF-β phosphorylation of ERK or AKT, a readout for SMAD independent TGF-β signaling. Knockdown of PDGFRα did not change the total SMAD2 protein levels but increased TβRII protein levels. Biotinylation study revealed that knockdown of PDGFRα induced accumulation of TβRII on the plasma membrane of HSCs. Additionally, we found that that PDGFRα formed a protein complex with TGF-β receptors upon TGF-β stimulation and that PDGFRα knockdown inhibited TGF-|3 induced TβRI/TβRII interactions as determined by IP. These data suggest that PDGFRα knockdown may inhibit TGF-β signaling by blocking the interaction and trafficking of TGF-β receptors into the early endosomes, where SMADs were phos-phorylated by TGF-β receptor kinases. Conclusion: PDGFRα is required for TGF-β induced TβRI/TβRII interactions and subsequent SMAD dependent intracellular signaling events. Our identification of PDGFRα in TGF-β receptor complexes highlights a convergence of PDGF and TGF-β receptor mediated signaling pathways and PDGFRα as a therapeutic target for liver metastasis and other settings of HSC activation.

Disclosures:

The following people have nothing to disclose: Chunsheng Liu, Vijay Shah, Ningling Kang

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Cell Type-Dependent Role of Class III Alcohol Dehydro- genase during Hepatic Fibrogenesis in Mice

Hyon-Seung Yi1, Young-Sun Lee1, Wonhyo Seo1, So Yeon Kim1, Jong-Min Jeong1, Won-IL Jeong1,2;
1 Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea; 2KAIST Institute for the Biocentury, Daejeon, Republic of Korea

Background and Aims: Recently, the important roles of retinols and their metabolites have been emphasized in immune responses and metabolic disorders. However, exact roles of retinols stored in HSCs have not been cleared yet, especially in HSCs and hepatic immune cells such as NK cells during hepatic fibrogenesis. Moreover, the critical enzyme responsible for retinol metabolism in HSCs and NK cells has not been elucidated. Thus, we identified a specific retinol metabolizing enzyme, alcohol dehydrogenase 3 (ADH3) and also investigated the roles of ADH3 in HSCs and NK cells respectively in liver fibrosis. Methods: Liver fibrosis was induced by bile duct ligation (BDL) or carbon tetrachloride (CCl4) treatment for 2 weeks in mice. To inhibit retinol metabolism, 4-methylpyrazole (4-MP), a broad ADH inhibitor, was administered to mice. In vitro, HSCs and NK cells were isolated or co-cultured. 4-MP treatment and siRNA targeting ADH3 gene were used for assessing the roles of ADH3 in HSCs and NK cells. Moreover, using ADH3-chimeric mice, we demonstrated the reciprocal functions of ADH3 on HSCs and NK cells in liver fibrosis. Results: In vitro, only ADH3 expression was identified in HSCs and NK cells although hepatocytes expressed several different types of retinol metabolizing enzymes. Inhibition of ADH3 by 4-MP and siRNA reduced expression of fibrotic mediators such as collagen and TGF-beta in HSCs. However, α-SMA expression was not changed. In NK cells, direct treatment of retinols suppressed interferon-γ (IFN-γ) production and cytotoxicity of NK cells against activated HSCs. In contrast, ADH3 inhibition in NK cells increased their cytotoxicity against activated HSCs via enhanced production of IFN-γ during co-culturing. In vivo experiments, inhibition of retinol metabolism by 4-MP or whole ADH3 gene depletion ameliorated liver fibrosis in both BDL and CCl4-treated mice. Freshly isolated HSCs and NK cells from 4-MP-treated and ADH3-depleted mice showed less expression of fibrotic mediators and enhanced expression of IFN-γ respectively than those of control or wild type mice. Using ADH3-chimeric mice, we also confirmed that ADH3-depleted NK cells attenuated CCl4-induced liver fibrosis via enhanced production of IFN-γ. Conclusions: Based on our findings, we could speculate that ADH3 is a critical enzyme for retinol metabolism in both HSCs and NK cells. However, it plays as a positive regulator for the activation of HSCs but a negative one for NK cells. Therefore, cell-type specific role of ADH3 may give rise to a new potential therapeutic target in liver fibrosis.

Disclosures:

The following people have nothing to disclose: Hyon-Seung Yi, Young-Sun Lee, Wonhyo Seo, So Yeon Kim, Jong-Min Jeong, Won-IL Jeong

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Novel mouse model of PSC-like liver disease with rapid progression of severe fibrosis, early onset portal hypertension and accelerated tumorigenesis

Naoki Ikenaga1, Susan B. Liu1, Deanna Sverdlov1, Qingen Ke2, Peter M. Kang2, Yury Popov1;
1Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Boston, MA; 2Division of Cardiology, Beth Israel Deaconess Medical Center, Boston, MA

BACKGROUND/AIMS: Robust mouse models of human disease are essential for therapeutic target discovery and preclinical drug testing. We previously characterized the Mdr2 (Abcb4)-/- mouse on the FVB genetic background (Mdr2-/-.FVB) as a reproducible genetic model of spontaneous chronic biliary liver disease closely resembling human PSC. However, liver disease in this model is relatively mild and fibrosis progression is slow compared to the human disease. We aimed to improve this model by moving the knockout into a fibrosis-sus-ceptible genetic background. METHODS: We generated novel congenic Mdr2 (Abcb4)-/- mice on a fibrosis-susceptible genetic background (BALB/c) by conventional backcross/inter-cross for 1 2 generations. Liver fibrosis in Mdr2-/-.BALB/c mice was directly compared to the parental strain (Mdr2-/-.FVB) in histology, biochemical determination of collagen, and fibrosis-related mRNA levels from 4 weeks to up to 1 year of age. Direct measurement of portal pressure was performed by inserting a micro-tip pressure monitor into the portal vein of anesthetized mouse. Liver tumors were evaluated macroscopically and microscopically. RESULTS: Mdr2-/-.BALB/c mice spontaneously developed periductular onion-skin type fibrotic lesions and pronounced ductular reaction starting from 4 weeks of age. When compared to the parental strain, Mdr2-/-.BALB/c mice demonstrated a dramatically accelerated liver fibrosis with about a 3 times faster collagen deposition (1793±78 vs. 687±55 ug hydroxyproline/liver in parental strain Mdr2-/-.FVB) and showed signs of bridging fibrosis (early signs of cirrhosis) histologically by the age of 12 weeks. This was accompanied by an early onset of severe portal hypertension in Mdr2-/- BALB/c (p < 0.001; 11.1 ±0.2 mmHg at age of 8 weeks vs. 7.3 ± 0.1 mmHg in Mdr2-/- FVB). Aggressive fibrosis in Mdr2-/-.BALB/c mice was associated with a dramatic increase in several pro-fibrogenic transcripts such as procolla-gen α1(I), TGFβ2 and TIMP-1 (2-4 fold above parental strain levels). While the development of liver tumors in Mdr2-/-.FVB starts from 10 months, Mdr2-/- BALB/c developed liver tumors as early as 7 months of age, with a greater tumor burden compared to Mdr2-/-.FVB at age 12 months (p < 0.05). CONCLUSIONS: Mdr2-/-.BALB/c mice demonstrate unprecedented degree and rapidity of hepatic fibrosis progression among any reported mouse models. Disease progression in Mdr2-/-.BALB/c is associated with early onset portal hypertension and accelerated primary liver cancer, which is a clinically relevant complication of cirrhosis. This new model will facilitate development ofantifibrotic drugs and mechanisms of study in biliary fibrosis progression.

Disclosures:

Peter M. Kang - Grant/Research Support: Abbott Labs

Yury Popov - Consulting: Gilead Sciences, Inc, Ymir Genomics; Grant/Research Support: Gilead Sciences, Inc

The following people have nothing to disclose: Naoki Ikenaga, Susan B. Liu, Deanna Sverdlov, Qingen Ke

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LXRs Balance Retinoid Storage in Stellate Cells Through Rab18, a Novel all-trans Retinoic Acid (ATRA) Responsive Lipid Droplet Associated Protein

Fiona O'Mahony1, Kevin W. Wroblewski1, Jihane Benhammou1, Sheila M. O'Byrne2, Hongfeng Jiang2, William S. Blaner2, Simon W. Beaven1;
1Division of Digestive Diseases, UCLA, Los Angeles, CA; 2Medicine, Columbia University, New York, NY

Quiescent hepatic stellate cells (HSCs) store retinoid in lipid droplets, but lose these droplets as they “activate” in response to liver injury. Whether this is required for full acquisition of the fibrotic program is unclear. We previously showed that liver X receptors (LXRs) are an important determinant for stellate cell activation. We found that Lxrαβ-/- HSCs are intrinsically pro-inflammatory and have a “super-sized” lipid droplet. The aim of this study was to determine whether LXRs link cholesterol to retinoid storage or metabolism. We hypothesized that Lxrαβ-/-HSCs are primed for activation because of increased retinyl ester storage and derivative retinoic acid receptor (RAR) signaling. Methods: Stellate cells were purified from wild-type (WT) and Lxrαβ-/- mice (10 per genotype) by sequential in situ perfusion of Pronase/collagenase, then density gradient ultra-centrifugation. Cells were collected during culture activation (ex vivo, 1, 2, 3, and 5 days) and analyzed by HPLC to quantify retinoid. Transcriptional profiling for each day was separately performed using Affymetrix gene arrays. Gene expression was determined by qPCR. Results: HPLC analysis showed Lxrαβ-/-HSCs store twice as much retinyl ester as WT at baseline. Both genotypes lose their retinoid over 5-7 days in culture, but the kinetics of lipid droplet loss are accelerated in Lxrαβ-/- HSCs, correlating with an earlier induction of fibrotic genes (Col1a1, Acta2). Increased RAR target gene expression in Lxrαβ-/- cells (Rarb, Crabp1) shows that a functional RAR ligand is present. ATRA potentiates Acta2 gene expression in both primary and immortalized stellate cells (LX-2). Microarray analysis identified an 8-fold increase in Rab1 8 (a lipid droplet associated protein) in Lxrαβ-/- HSCs during early activation. We show that Rab1 8 expression is inversely regulated by RAR (up) and LXR (down) ligands. Knockdown of Rab1 8 by siRNA in primary stellate cells decreases Acta2 gene expression and retards the loss of the large, auto-fluorescent lipid droplets, even several days into culture activation. Conclusion: Lxrαβ-/- stellate cells are overloaded with retinyl esters, have increased RAR signaling (even during cellular quiescence), and are ‘frame shifted'towards earlier activation. This work directly ties the kinetics of lipid droplet loss with fibrotic activation. We demonstrate for the first time that cholesterol and retinoid metabolism are intimately linked in stellate cells. The fulcrum of this link appears to be the novel ATRA-responsive gene, Rab1 8. We anticipate that Rab1 8 interference may have significant therapeutic benefit in ameliorating liver fibrosis.

Disclosures:

The following people have nothing to disclose: Fiona O'Mahony, Kevin W. Wrob-lewski, Jihane Benhammou, Sheila M. O'Byrne, Hongfeng Jiang, William S. Blaner, Simon W. Beaven

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Genetic Ablation of M1 Muscarinic Receptors (M1R) Attenuates Azoxymethane (AOM)-Induced Murine Liver Injury by TRAIL-R2-Mediated Hepatic Stellate Cell (HSC) Apoptosis

Vikrant Rachakonda1, Nathalie H. Urrunaga1, Ravirajsinh Jadeja1, Daniel Ahmad1, Leon McLean1, William S. Twaddell2, Kunrong Cheng1, Neeraj K. Saxena1, Jean-Pierre Raufman1, Sandeep Khu-rana1;
1 Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD; 2Department of Pathology, University of Maryland School of Medicine, Baltimore, MD

We previously demonstrated that M1 R regulates AOM-induced chronic liver injury in mice. AOM-treated M1 R-deficient (Chrm1-/-) mice had decreased gross liver nodularity, fibrosis and ductular hyperplasia compared to AOM-treated wild type (WT) mice (Gastroenterol 142: S-973). Chrm1 ablation reduced HSC activation and proliferation via down-regulation of receptors for transforming growth factor-β and platelet derived growth factor (Hepatology 564, 766A). Previous investigations have implicated TRAIL-R2-mediated HSC apoptosis in fibrosis resolution (Gut 2001; 48: 548, Hepatology 2003; 37: 87). Aim: To elucidate the role of M1 R on HSC apoptosis as a hepatoprotective mechanism in AOM-induced chronic murine liver injury. Methods: Chrm1-/- (N=29) and WT (N=25) male 129SvEvxCFl mice were treated with AOM (10 mg/kg/wk ip X 6 wks) or PBS. Livers were harvested 14 weeks after the last injection, and mRNA was extracted to measure expression of apoptotic factors and their receptors (TNFα, TNFα-R1, TRAIL, TRAIL-R2/DR5, Fas and FasL). Dual staining for alpha-smooth muscle actin (αSMA) and TUNEL was performed on liver sections to quantify activated HSC apoptosis. Results: TNFα expression was similar in PBS-treated Chrm1-/- and WT mice. TNFα-R1 was modestly reduced in PBS-treated Chrm1-/- mice (p<0.001). M1 R deficiency attenuated AOM-induced up-regu-lation of TNFα (2.41 ±0.12 vs. 5.10±0.64 [expressed as fold-PBS-treated-WT-mice], p<0.01). In PBS-treated mice, there was no baseline difference in Fas and FasL expression and after AOM treatment Fas and FasL expression increased modestly only in WT mice. PBS-treated Chrm 1-/- compared to WT mice had reduced TRAIL expression (0.20±0.02 vs. 1.00±0.15, p<0.001). After AOM exposure TRAIL expression decreased only in WT mice (1.00±0.22 vs.0.54±0.17, p<0.05). In PBS-treated mice, M1 R deficiency did not alter TRAIL-R2 expression. However, in Chrm1-/- mice AOM treatment stimulated a large increase in TRAIL-R2 expression compared to WT mice (32.24±7.12 vs. 1.82±0.37, p<0.001). Consistent with this observation, there was a significant increase in the proportion of TUNEL-positive HSC in AOM-treated Chrm1-/- compared to WT mice (48.4% ± 5.3% vs. 22.46% ± 3.10%, p<0.001). Conclusion: In AOM-treated mice, M1 R deficiency is associated with up-regulated TRAIL-R2 expression and enhanced HSC apoptosis. These findings provide mechanistic insight into the effect of M1 R ablation on hepatic fibrosis resolution.

Disclosures:

The following people have nothing to disclose: Vikrant Rachakonda, Nathalie H. Urrunaga, Ravirajsinh Jadeja, Daniel Ahmad, Leon McLean, William S. Twaddell, Kunrong Cheng, Neeraj K. Saxena, Jean-Pierre Raufman, Sandeep Khurana

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Differential regulation of liver fibrosis progression and regression by pharmacological inhibition of M2 macrophage polarization through IL-4Ralpha1

Shih-Yen Weng1, Kornelius Padberg1, Yong Ook Kim1, Xiao-Yu Wang1, Michael L. McCaleb2, Jeff R. Crosby2, DetlefSchuppan1,3;
1 Institute of Molecular and Translational Medicine, University Medicine, Johannes Gutenberg University, Mainz, Germany, Mainz, Germany; 2Isis Pharmaceuticals, Carlsbad, CA; 3Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Progression and Regression of liver fibrosis have been linked with the innate immune system, especially macrophages. Depending on stimulation by different cytokines or LPS, macrophages can differentiate into M1 (classically activated) and a spectrum of M2 (alternatively activated) macrophages. The roles of M1 and M2 in liver fibrosis progression or regression are largely unexplored. We used the models of liver fibrosis progression (6 weeks of CCl4 by oral gavage) and spontaneous regression after withdrawal of the toxin for 4 weeks in C57BL6 mice, and the model of Mdr2KO mice (spontaneous biliary fibrosis progression) to assess the role of M2 and their manipulation in liver fibrosis progression and reversal. In mice with CCL4-induced liver fibrosis, expression of the M2-inducing, IL-4/IL-13 responsive IL-4Ralpha1 was increased during progression, but strongly decreased after 2 weeks of spontaneous fibrosis regression. In Mdr2 KO mice, expression of the IL-4Ralpha1 gradually increased until age 6-wk, and decreased thereafter. For functional characterization of M2 macrophages, neutralizing IL-4Ralpha antisense oligonu-cleotide (ASO) was tested in vitro (murine RAW macrophages) and in vivo via the intraperitoneal route. The specific ASO but not an irrelevant control ASO suppressed IL-4Ralpha expression in RAW macrophages by 90% at 2μg/ml. When given to CCL4-treated mice twice weekly at 40 mg/kg i.p. from wk-2 to w-4 during the regression phase, the ASO suppressed hepatic expression of IL-4Ralpha1 by 47%, and collagen deposition as determined by Sirius Red staining by 30%. Concomitantly, ASO treatment decreased the M2 markers Arg1 and Mrc1 and increased the M1 markers CCL3, MMP-8 and MMP-9, and profibrogenic procollagen alpha1 (I) RNA. Serum ALT was increased 4-fold in ASO-treated vs untreated mice. Mdr2 KO mice that received the ASO from week 6 to week 10 also showed a similar shift from the M2 to the M1 phenotype, a similar regulation of the above genes and a significant increase in ALT. We showed that, somewhat unexpectedly, suppression of M2 macrophage differentiation ameliorates fibrosis during fibrosis progression and regression, addressing the IL-4Ralpha1 with specific inhibitors may be a novel M2 macrophage targeted approach to inhibit liver fibrosis.

Disclosures:

Michael L. McCaleb - Employment: Isis Pharmaceuticals; Stock Shareholder: Isis Pharmaceuticals

Jeff R. Crosby- Employment: ISIS Pharmaceuticals

Detlef Schuppan - Advisory Committees or Review Panels: Aegerion, Eli Lilly, Gilead; Consulting: Boehringer-Ingelheim, Isis, Takeda; Grant/Research Support: Boehringer-Ingelheim

The following people have nothing to disclose: Shih-Yen Weng, Kornelius Padberg, Yong Ook Kim, Xiao-Yu Wang

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Branched-chain amino acids improve liver fibrosis and reduce tumorigenesis in mice fed an atherogenic high-fat diet by suppressing PDGFR-β/ERK signaling

Kai Takegoshi1, Masao Honda1, Hikari Okada1, Hajime Sunagozaka1, Naoto Matsuzawa3, Toshinari Takamura2, Takuji Tanaka4, Shuichi Kaneko1;
1Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 2Disease Control and Homeostasis, Kanazawa University Graduate School of Medicine, Kanazawa, Japan; 3Nutrition, UC Davis, Davis, CA; 4Tohkai Cytopathology Institute: Cancer Research and Prevention, Gifu, Japan

Background and Aim: Branched-chain amino acids (BCAAs) reportedly improve event-free survival in patients with liver cirrhosis and inhibit liver carcinogenesis in heavier patients with liver cirrhosis. However, the mechanisms underlying these effects remain unclear. The aim of this study was to determine the effect of BCAAs on liver steatosis, fibrosis, and tumorigenesis in mice fed an atherogenic high-fat (Ath HF) diet. Methods: Male mice (8 weeks old) were divided into 3 groups, and each group was fed one of the following diets for 12, 30, or 60 weeks: basal diet, Ath HF diet, and Ath HF diet containing 3% BCAAs. The degree of hepatic steatosis and fibrosis and tumor number were calculated. The expression of fibrosis-related genes was evaluated by immunohistochemical staining, realtime polymerase chain reaction, and Western blotting. Lx2 cells activated by recombinant TGF-β were treated with BCAAs or transfected with Raptor siRNA to examine the mechanisms underlying the effects of BCAAs. Results: Mice fed the Ath HF diet for 12 or 30 weeks developed liver steatosis and fibrosis, whereas mice fed the Ath HF diet containing BCAAs showed markedly reduced steatosis and fibrosis. The expression of collagen I/IV, αSMA, TGF-β, PDGFR-β, and pERK was suppressed in mice fed the Ath HF diet containing BCAAs compared with mice fed only the Ath HF diet. After 60 weeks, 71 % of mice fed the Ath HF diet developed liver tumors. BCAAs reduced tumor incidence to 25%. In Lx-2 cells, recombinant TGF-β induced the expression of collagen I, PDGFR-β, and pERK, whereas BCAAs suppressed the expression of these genes in a dose-dependent manner. In Lx-2 cells transfected with Raptor siRNA, the expression of collagen I and PDGFR-β was increased. Conclusions: BCAAs improved liver fibrosis and reduced tumorigenesis in mice fed the Ath HF diet, possibly by suppressing PDGFR-β/ERK signaling. We believe these findings may have a significant therapeutic impact on nonalcoholic steatohepatitis.

Disclosures:

Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Aji-nomoto Co., Inc, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Kai Takegoshi, Masao Honda, Hikari Okada, Hajime Sunagozaka, Naoto Matsuzawa, Toshinari Takamura, Takuji Tanaka

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Identification of CD248 as a potential therapeutic target in liver fibrosis

Victoria Aldridge1, Annika Wilhelm1, Abhilok Garg1, Amy J. Nay-lor2, Janine Fear1, Neil C. Henderson3, Christopher D. Buckley2, Philip N. Newsome1;
1Centre for Liver Research, University of Birmingham, Birmingham, United Kingdom; 2Centre for Translational Inflammation Research, University of Birmingham, Birmingham, United Kingdom; 3University of Edinburgh, Edinburgh, United Kingdom

CD248 is a stromal cell marker expressed on fibroblasts and pericytes. We hypothesised that CD248 expression may be upregulated in liver fibrosis and thatCD248 knockout (ko) mice will develop less fibrosis than equivalent wild-type (WT) mice. Methods: CD248 expression was studied by immunostaining and quantitative PCR in both normal and diseased human/murine liver tissue and isolated hepatic stellate cells (HSC). Chronic liver injury was induced in CD248 ko and wild-type (WT) controls by bi-weekly injections of carbon tetrachlo-ride (CCl4) for 8 or 12 weeks. Liver fibrosis was quantified by picrosirius red staining (PSR) and qPCR for Collagen I and a-smooth muscle actin (α-SMA). Expression of platelet derived growth factor receptor (PDGFR) on hepatic stellate cells and their response to PDGF stimulation was studied by CyQUANT proliferation assay and c-fos analysis. Results: CD248 expression was seen in normal human and mouse liver but was significantly increased in liver injury on both immunostaining and gene expression. CD248 was found to be co-expressed with a range of fibroblast/HSC markers including desmin, vimentin, αSMA and GFAP in murine and human liver sections. CD248 expression was restricted to isolated murine and human HSC (co-stained with GFAP, desmin and αSMA) and was not seen on isolated hepatocytes, biliary endothelial cells or sinusoidal endothelial cells. PSR staining of liver tissue after chronic CCl4 injury revealed less fibrosis in CD248ko mice compared to wt mice as assessed by digital morphometric analysis of PSR stained sections (56.4 & 51.1% less; p<0.001 and p<0.05 at 8 & 12 weeks respectively) and qPCR for Collagen I (65.3 & 64.0% less; p<0.05 at 8 & 12 weeks respectively) & αSMA (62.5 & 86.1% less; p=0.01 and p<0.05 at 8 & 12 weeks respectively). Isolated HSC from WT and CD248ko mice expressed PDGFR α and β at similar levels. As expected PDGF-BB induced proliferation of WT HSC (80.5% proliferation), whereas CD248ko HSC did not demonstrate a proliferative response to PDGF-BB (10.3%). Abrogated PDGF signalling in CD248ko HSC was confirmed by significantly reduced c-fos expression compared to WT HSC at gene level (0.03+/-0.01 vs 0.22+/-0.06, p<0.05). Conclusion Our data indicate that CD248 is upregulated in liver fibrosis and its expression is restricted to stellate cells and myofibroblasts in both murine and human settings. We demonstrate that genetic knockout of CD248 reduces susceptibility to liver fibrosis by reducing PDGF-BB mediated proliferation of HSC. These data highlight CD248 as a potential novel therapeutic target in fibrotic liver disease.

Disclosures:

Philip N. Newsome - Grant/Research Support: Novo Nordisk

The following people have nothing to disclose: Victoria Aldridge, Annika Wilhelm, Abhilok Garg, Amy J. Naylor, Janine Fear, Neil C. Henderson, Christopher D. Buckley

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Disruption of retinol metabolism in hepatic stellate cells ameliorates Concanavalin A-mediated hepatitis of mice via regulating intrahepatic Tregs

Young-Sun Lee1, Hyon-Seung Yi1, Wonhyo Seo1, So Yeon Kim1, Jong-Min Jeong1, Won-IL Jeong1,2;
1Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea; 2KAIST Institute for the Biocentury, Daejeon, Republic of Korea

Study's purpose: Hepatic stellate cells (HSCs) play an important role not only in liver fibrosis but also in inflammation by regulating hepatic immune cells. Although HSCs store most of body retinols and their metabolites (retinoic acids) are critical in immune responses, there are few reports about the role of hepatic retinols in inflammatory disease. Therefore, we investigated the effects of HSC's retinols on Concanavalin A (Con A)-induced hepatitis of mice. Methods: To induce acute hepatitis in mice, Con A (12 μg/g) was injected to mice via tail vein with or without the pretreatment of 4-methylpyrazole (4-MP) (10 μg/g) 3 hours before Con A injection to block retinol metabolism. Mice were sacrificed at 0, 3, 12 and 24 hours after Con A treatment. Hepatocytes, HSCs, liver mononuclear cells and Tregs were isolated for ex vivo and in vitro experiments. HSCs and Tregs were treated with interferon-γ (IFN-γ) under the presence of 4-MP or not. Migration assay of Tregs was also performed during co-culturing. Results: After Con A treatment, liver injuries increased and peaked at 24 hour. However, 4-MP treatment significantly reduced liver injuries by decreasing IFN-γ production. In FACS analyses, the population of Tregs in 4-MP-treated livers significantly increased, whereas IL-17 producing cells inversely decreased at 12 and 24 hours compared with those of vehicle-treated livers of mice. Freshly isolated HSCs and liver mononuclear cells in vehicle-treated mice showed increased gene expression of retinol metabolic enzymes and IFN-γ respectively, which was significantly reduced in 4-MP-treated mice. Freshly isolated hepatocytes showed less expression of IFN-γ receptors in 4-MP treated mice. In vitro co-culturing Tregs with HSCs, 4-MP treatment to HSCs enhanced migration and function of Tregs by up-regulated expression of CCL2, IL-1 0 and IL-6. In addition, the migration of Tregs to HSCs was decreased as CCR2 and CCL2 were depleted in Tregs and HSCs respectively. Furthermore, 4-MP treatment increased survival rate of mice (50%) compared with that of vehicle-treated group (33%) in Con A-induced fulminant hepatitis. Conclusion: In Con A-mediated hepatitis, disruption of retinol metabolism in HSCs might protect liver injuries via Treg-mediated decreased effects of IFN-γ. Therefore, the regulation of retinol metabolism in HSCs could be a new therapeutic target for immune-mediated hepatitis.

Disclosures:

The following people have nothing to disclose: Young-Sun Lee, Hyon-Seung Yi, Wonhyo Seo, So Yeon Kim, Jong-Min Jeong, Won-IL Jeong

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In tra hepatic alkaline phosphatase attenuates LPS-medi- ated effects during liver fibrosis

Marlies Schippers1, Eduard Post1, Aysegul Cetintas1, Catharina Reker-Smit1, Madhu S. Malo2, Richard A. Hodin2, Jose L. Millan3, Leonie Beljaars1, Klaas Poelstra1;
1Dept. of Pharmacokinetics, Toxicology & Targeting, University of Groningen, Groningen, Netherlands; 2Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA; 3Sanford-Burnham Medical Research Institute, Sanford Children's Health Research Center, La Jolla, CA

Background and Aim: Alkaline phosphatase (AP) activity is increased during fibrogenesis and is used as a marker for many liver diseases including liver fibrosis. We found that this enzyme is able to dephosphorylate LPS. Phosphate groups in the Lipid A moiety of LPS greatly determine the biological effects of this molecule, so we hypothesized that alkaline phosphatase represents an LPS-detoxifying enzyme within the liver. Methods: First we examined the effects of di-phosphoryl lipid A (representing the native form in wild type LPS) and mono-phosphoryl lipid A on macrophages and fibroblasts (RAW264.7, 3T3 and LX2 cells) in vitro. We then examined alkaline phosphatase and LPS-dephosphorylating activity in normal and fibrotic livers of CCL4- treated mice and human patients. We also studied the effect of a 2-week administration of Calf Intestinal AP (500 mU/mouse/injection) on macrophages and fibroblasts in mice with CCl4-induced established fibrosis (8 weeks model). Finally, intestinal AP knock-out C57BL/6 mice (iAP KO) were examined at 6 weeks of age and compared to age-matched wild-type. Results: Whereas diphosphoryl Lipid A strongly activated RAW cells, 3T3 and LX2 cells as reflected by enhanced expression of pro-inflammatory cytokines, adhesion molecules and MHC Class II, monophosphoryl lipid A induced no such activities in either cell type. Significant dephosphorylating activity of diphosphoryl lipid A and wild type LPS was found in fibrotic livers of both mice and man. This activity co-localized with intra-hepatic AP activity. CD68-positive macrophages and α-SMA-positive cells were found to express AP activity. Administration of AP to CCL4-exposed fibrotic mice significantly attenuated staining for desmin which was associated with a reduction in the expression of the M2 macrophage marker YM-1. In contrast, iAP KO mice displayed higher intrahepatic expression levels of fibrogenic markers (PAR-1, Collagen I) paralleled by an increase in YM-1 staining relative to WT. So, lack of intestinal AP stimulates fibrogenesis within the liver. Conclusions: Based on our in vitro studies it can be concluded that removal of just one phosphate from the Lipid A moiety of LPS abrogates many biological effects of LPS. The detection of LPS-dephosphorylating activity in fibrotic livers of mice and man may therefore represent the upregulation of a protective enzyme. The in vivo effects of exogenous AP on fibroblasts and macrophages in fibrotic mice and our observations in i-AP KO mice further support the hypothesis that alkaline phosphatase activity attenuates LPS-mediated effects within the fibrotic liver.

Disclosures:

Klaas Poelstra - Consulting: BiOrion Technologies BV; Grant/Research Support: BiOrion Technologies BV; Stock Shareholder: BiOrion Technologies BV

The following people have nothing to disclose: Marlies Schippers, Eduard Post, Aysegul Cetintas, Catharina Reker-Smit, Madhu S. Malo, Richard A. Hodin, Jose L. Millan, Leonie Beljaars

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Interaction between the HIV-envelope protein gp120 and the chemokine receptor CCR5 mediates activation of the NALP3-inflammasome complex in human hepatic stellate cells

Andrea Cappon1, Raffaele Bruno2, Sandra Gessani3, Andrea Masotti4, Fabio Marra1;
1University of Florence, Florence, Italy; 2University of Pavia, Pavia, Italy; 3Istituto Superiore di Sanita, Rome, Italy; 4Ospedale Pediatrico Bambino Gesu, Rome, Italy

Patients with HCV/HIV co-infection show a faster progression of hepatic fibrosis and more severe inflammation. The HIV envelope protein gp120 has been previously shown to modulate different aspects of hepatic stellate cell (HSC) biology, including directional migration and expression of profibrogenic cytokines. These effects were mediated, at least in part, via activation of the chemokine receptor CCR5. Recent work has identified the NALP3 inflammasome as a critical pathway in the generation of proinflammatory signals during liver injury. Moreover, IL-1 generated through this pathway exerts profibrogenic activities through the modulation of TIMP-1 and MMP9. Aim of this study was to evaluate the role of CCR5 in mediating inflammasome activation in response to gp120, in cells involved in liver tissue repair, including HSC. Myofibroblastic human HSCs were isolated from normal human liver tissue and cultured on plastic until fully activated. PBMC were separated from human whole blood. Gene expression was measured by qPCR. Protein IL-1 β protein levels were assayed by ELISA. HSCs or PBMC were exposed to 500 ng/ml recombinant M-tropic gp120 (CN54) for 2, 8 and 24 hours showed a time-dependent, significant up-regulation of pycard and NALP3, critical proteins for the assembly of NALP3-dependent inflammasome, and of cas-pase-1 and IL-1 β. gp120 efficiently induced secretion of mature IL-1β, as shown by ELISA tests performed on the supernatants of both cell types. Notably, pre-incubation of HSCs with TAK779, a CCR5 receptor antagonist or with neutralizing α-CCR5 antibody reverted gp120-mediated IL-1 β production, indicating a primary role for this receptor in the activation of inflammasome complex. Interestingly, when HSC were stimulated with CCL5 (RANTES), a natural agonist of CCR5, we also found a significant up-regulation of IL-1 b, confirming that CCR5 may mediate activation of this inflammatory complex in HSC. In conclusion, HIV-gp1 20 significantly increases the expression of components of the NALP3 inflammasome pathway in human HSC and PBMC, through activation of CCR5. These data identify a novel mechanism by which HIV-gp1 20 may directly influence hepatic necroinflammation and fibrosis during HCV/HIV coinfection, i.e. through increased production of IL-1 β. Moreover, these data establish for the first time a direct link between the inflammasome complex, HIV proteins and CCR5. We thank aid-sreagent.org for the kind gift of gp120.

Disclosures:

Fabio Marra - Advisory Committees or Review Panels: Abbott; Consulting: Bayer Healthcare, Gilead; Grant/Research Support: ViiV

The following people have nothing to disclose: Andrea Cappon, Raffaele Bruno, Sandra Gessani, Andrea Masotti

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Lack of Matrix Metalloproteinase-8 Ameliorates Liver Fibrosis Progression and Promotes Fibrosis Regression

Yong Ook Kim1, Matthias Stoll1, Shih-Yen Weng1, Kyoung-Sook Park1, Benhard Hebich1, Rosario Heck1, Yury Popov2, Swaantje Hamdi1, Detlef Schuppan1,2;
1Institute of Molecular and Transla-tional Medicine, University Medicine, Johannes-Gutenberg-University, Mainz, Germany; 2Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Matrix metalloproteinases (MMPs) are involved in various processes such as modulation of inflammation, tissue remodeling and collagen turnover. MMP-8 plays a yet ill-defined role in liver fibrogenesis and fibrolysis. Thus, we investigated the role of MMP-8 in toxin-induced liver fibrosis and spontaneous fibrosis regression using MMP-8 knock-out mice. Six week old female MMP-8 KO mice (n=10/group,C57/BL6 background) were treated according to an optimized CCl4 and TAA fibrosis-induction protocol for 4 and 8 weeks. For fibrosis regression, mice were harvested 5 days, 2 weeks, and 4 weeks after discontinuation of toxin treatment. Parameters of liver function, fibrosis (hydroxyproline, Sirius red morphometry) and transcript levels related to fibrogenesis, fibrolysis and inflammation were determined at sacrifice. After CCl4 and TAA treatment for 4 weeks livers of MMP-8 KO mice did not show significant difference in liver morphology or Sirius red staining. However, after 8 weeks collagen accumulation in TAA-treated MMP-8 KO mice was significantly decreased compared to their wild type controls. AST, ALT and ALP, but also IL-10 and IL-13 production were significantly lower in CCl4 treated MMP-8 KO mice. Both CCl4 and TAA treated MMP-8 KO mice demonstrated an up-regulation of MMP-9 and IL-1 0 mRNA and a significant down-regulation of profibrogenic TGFβ1, COL α1(I), and MMP-2 mRNA compared to WT controls. Both at 4 and 8 weeks, significant upregulation was observed for the chemokines CCL3 (>1.2 fold) and CCL5 (2-4 fold). TAA treated mice experienced a mild spontaneous fibrosis regression compared to non-regressing CCl4 treated mice after 4 weeks. Notably, MMP-8 KO mice showed a more pronounced fibrosis regression than their WT controls. Accordingly, profibrogenic gene expression (COLα1(I), α-SMA, and MMP-2) was clearly downregulated only in the WT mice during fibrosis regression. During regression MMP-8 KO mice showed a higher activation of chemokines and chemokine receptors that induce e.g. macrophage recruitment such as CCL3, CCR7, and CXCR3. There was no difference in the transcript level of IL-4α1 receptor, the major receptor for alternative macrophage polarization, in all treatment groups of WT and MMP-8 KO mice. We show that MMP-8 adversely modulates liver fibrosis progression and regression in two models. MMP-8 promotes fibrosis progression by decreasing MMP-9 and IL-10 production. During fibrosis regression, MMP-8 appears to mitigate favourable tissue remodeling by decreased recruitment and activation of fibrolytic immunocytes. This may be related to MMP-8 functioning as direct or indirect inactivator of cetain chemokines and chemokine receptors.

Disclosures:

Yury Popov - Consulting: Gilead Sciences, Inc, Ymir Genomics; Grant/Research Support: Gilead Sciences, Inc

Detlef Schuppan - Consulting: Boehringer Ingelheim, Aegerion, Gilead, Gen-zyme, GSK, Pfizer, Takeda, Sanofi Aventis, Silence

The following people have nothing to disclose: Yong Ook Kim, Matthias Stoll, Shih-Yen Weng, Kyoung-Sook Park, Benhard Hebich, Rosario Heck, Swaantje Hamdi

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IL-17A Enhances Liver Fibrosis through Upregulation of the TGF-β Receptor on Hepatic Stellate Cells

Thomas Fabre1,3, Hassen Kared1, Scott L. Friedman2, Naglaa H. Shoukry1,4;
1Centre de Recherche du CHUM, Hôpital St Luc, Montreal, QC, Canada; 2Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY; 3Département de micro-biologie et immunologie, Université de Montréal, Montreal, QC, Canada; 4Département de médecine, Faculté de médecine, Université de Montréal, Montreal, QC, Canada

Background: Activation of hepatic stellate cells (HSCs) is a key event in the initiation of hepatic fibrosis, characterized by enhanced extracellular matrix (ECM) production and altered degradation. Activation of HSCs can be modulated by cytokines produced by immune cells. Recent reports implicate the pro-inflammatory cytokine IL-17A, in liver fibrosis progression during hepatitis B virus infection and alcoholic hepatitis. IL-17A can enhance fibrosis indirectly by increasing the recruitment of pro-inflammatory macrophages and monocytes to the liver IL-17A is produced mainly by Th17 cells, NK and NKT cells, which are enriched in the liver. We hypothesized that IL-1 7A may also play a direct role by enhancing activation of HSC. Aim: The aim of this study was to characterise the effect of IL-1 7A exposure on activation of HSC and induction of fibrogenic signaling in these cells Methods: The human HSC line LX2 and primary human HSCs were stimulated with increasing doses of IL-17A and compared to TGF-β and PBS-treated cells as positive and negative controls, respectively. Activation of HSCs was evaluated by qRT-PCR for alpha-smooth muscle actin (α-SMA), collagen type I (COL1A1) and tissue inhibitor of matrix metalloproteinase I (TIMP-I). Results were correlated with protein expression by western blots and picro-Sir-ius red staining for collagen deposition. Cell surface expression of the cytokine receptors TGF-β-RII and IL-17RA was evaluated by flow cytometry. Signaling through the TGF-β receptor was evaluated by examining phosphorylation of SMAD2/3 by Western following cytokine stimulation. Results: IL-1 7A alone did not induce activation of HSC. However, IL-1 7A sensitized HSCs to the action of suboptimal doses of TGF-β as confirmed by strong induction of α-SMA, collagen type I and TIMP-I gene expression and protein production. IL-1 7A specifically up-regulated and stabilized the cell surface expression of TGF-β-RII following stimulation. Pretreatment of HSCs with IL-1 7A enhanced signaling through the TGF-β-RII as observed by increased phosphorylation of SMAD2/3 in response to stimulation with sub-optimal doses of TGF-β. Conclusion: Our results suggest a novel pro-fibrotic function for IL-1 7A through sensitization of HSCs to the action of TGF-β. IL-1 7A acts through up-regulation and stabilization of the TGF-β-RII leading to increased SMAD2/3 signaling. These findings represent a novel example of cooperative signaling between an immune cytokine and a fibrogenic receptor.

Disclosures:

Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical, Sanofi-Aventis; Consulting: Abbott Laboratories, Conatus Pharm, Exalenz, Genenetch, Glaxo Smith Kline, Hoffman-La Roche, Intercept Pharma, Isis Pharmaceuticals, Melior Discovery, Nitto Denko Corp., Debio Pharm, Synageva, Gilead Pharm., Ironwood Pharma, Alnylam Pharm, Tokai Pharmaceuticals, Bristol Myers Squibb, Takeda Pharmaceuticals, Nimbus Discovery, Isis Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira Pharm, Vaccinex Therapeutics; Stock Shareholder: Angion Biomedica

The following people have nothing to disclose: Thomas Fabre, Hassen Kared, Naglaa H. Shoukry

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Neo-epitopes of extracellular matrix remodelling in serum reflect antifibrotic therapy

Robert Schierwagen1, Diana J. Leeming2, Sabine Klein1, Mette J. Nielsen2, Tilman Sauerbruch1, Aleksander Krag3, Morten A. Kars-dal2, Jonel Trebicka1;
1Internal Medicine I, University of Bonn, Bonn, Germany; 2Fibrosis Biology and Biomarkers, Nordic Bio-science, Herlev, Denmark; 3Department of Gastroenterology, University Hospital Odense, Odense, Denmark

Background Progression of liver fibrosis is characterized by synthesis and degradation of extracellular matrix (ECM). Matrix-metalloproteinases (MMP) cleave collagen fibres at a specific site generating soluble fragments of ECM (neo-epitopes). The levels of these neo-epitopes may reflect the stage of liver fibrosis and could allow the monitoring of anti-fibrotic therapies. Here we analyzed these neo-epitopes as read-out for a liver directed therapy with statins. Methods Bile duct ligation (BDL) was performed on wildtype rats, which received atorvas-tatin (15mg/kg*d) for one week starting at one, two, three, four and five weeks after BDL (T1-T5), while controls remained untreated. Hepatic fibrosis was analyzed by immunohisto-chemistry and hepatic hydroxyproline content. TGFβ levels were measured by RT-PCR. Proteolytic activity of MMP-2 was examined by zymography. Levels of type I, III, IV and VI collagen degradation by MMP activity (C1M, C3M, C4M and C6M) and formation of type III and IV collagen (PRO-C3 and P4NP7S) markers were assessed by specific ELISAs in serum probes. Results Serum markers of ECM neo-epitopes reflected significantly the remodelling of the ECM in the liver and were able to distinguish between early (T1-T3) and severe fibrosis (T4-T5). Statin treatment was associated with significantly lower levels of neo-epitopes, especially when therapy was initiated in the stage of severe fibrosis (T4-T5). Furthermore, the neo-epi-tope markers were correlated to hepatic expression of profi-brotic cytokines TGFb1 and TGFb2. The formation markers PRO-C3 and P4NP7S as well as degradation markers C4M and C6M correlated significantly with MMP-2 activity in rats with severe fibrosis. Discussion Determination of ECM neo-epitopes in serum allowed us to distinguish between mild and severe fibrosis and to assess ECM remodeling. With respect to the results during statin therapy, neo-epitopes might serve as read-out for efficacy of anti-fibrotic treatment.

Disclosures:

Diana J. Leeming - Employment: Nordic Bioscience

Mette J. Nielsen -Grant/Research Support: Nordic Bioscience A/S

Morten A. Karsdal - Stock Shareholder: Nordic Bioscience

The following people have nothing to disclose: Robert Schierwagen, Sabine Klein, Tilman Sauerbruch, Aleksander Krag, Jonel Trebicka

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Selective inhibition of lysyl oxidase like 2 (LOXL2) using a therapeutic monoclonal antibody suppresses the progression of biliary fibrosis in novel PSC-like mouse model

Naoki Ikenaga1, Shuhei Yoshida1, Susan B. Liu1, Jeanhee Chung1, Deanna Sverdlov1, Derek Marshall2, Vivian Barry2, Victoria Smith2, Maria Kovalenko2, Satyajit Karnik2, Nezam H. Afdhal1, Yury Popov1;
1Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Boston, MA; 2Gilead Sciences, Foster City, CA

BACKGROUND/AIMS: LOXL2 is a key enzyme that promote-scross-linking of collagen type I and is expected to be a novel therapeutic target for liver fibrosis. The efficacy of LOXL2 inhibitor on panlobular fibrosis has been previously demonstrated in hepatotoxin-induced models, however the efficacy in biliary-type fibrosis is not known. We studied the therapeutic efficacy of a novel anti-LOXL2 monoclonal antibody in two mouse models of primary sclerosis cholangitis (PSC)-like biliary fibrosis. METHODS: We developed an improved mouse model resembling human PSC with rapidly progressive fibrosis and early-onset portal hypertension by backcrossing the Mdr2 mutation on a fibrosis susceptible background (BALB/c). Anti-LOXL2 therapeutic antibody (AB0023mAB, 30mg/kg) or control antibody (M64, 30mg/kg) were administered i.p. twice a week in Mdr2-/-.BALB/c mice (n = 10 per group) from age 4 weeks to 8 weeks, and in C57BL/6 mice fed 3,5- diethoxycarbonyl- 1,4-dihydrocollidine (DDC)- diet for 4 weeks (n=9-11 per group). Non-selective lysyl oxidase inhibitor beta-animopropionitrile (BAPN, 100mg/kg/day, i.p.) was administered for comparison. Liver fibrosis was evaluated by histology, biochemical determination of collagen, and analysis of profibrogenic gene expression by qRT-PCR. RESULTS: Immunohistochemical analysis revealed that LOXL2 was virtually absent from healthy liver, but was strongly induced in periductular fibrotic areas in mice with biliary fibrosis. Anti-LOXL2 treatment significantly reduced hepatic collagen deposition by 28% in Mdr2-/- BALB/c mice compared to control antibody (p = 0.0021) and BAPN treatment (p = 0.0012). Results were validated in a second, mechanistically different model of DDC-induced biliary fibrosis, where anti-LOXL2 treatment reduced hepatic collagen content by 23% (p = 0.0151). BAPN treatment showed similar efficacy to anti-LOXL2 mAB in the DDC model, but was ineffective in Mdr2-/-.BALBc model. CONCLUSIONS: A therapeutic anti-LOXL2 antibody significantly inhibited the progression of liver fibrosis in two mouse models of biliary fibrosis, outperforming non-selective LOX inhibition. Feasibility of antibody targeting of LOXL2 to prevent the ongoing biliary liver fibrosis, such as PSC, should be evaluated in clinical trials.

Disclosures:

Derek Marshall - Employment: Gilead Sciences

Vivian Barry- Employment: Gilead Sciences, Inc.; Stock Shareholder: Gilead Sciences, Inc.

Victoria Smith - Employment: Gilead Sciences Inc Satyajit Karnik - Employment: Gilead Sciences

Nezam H. Afdhal - Consulting: Merck, Vertex, Idenix, GlaxoSmithKline, Spring-bank, Gilead, Pharmasett, Abbott; Grant/Research Support: Merck, Vertex, Idenix, GlaxoSmithKline, Springbank, Gilead, Pharmasett, Abbott

Yury Popov - Consulting: Gilead Sciences, Inc, Ymir Genomics; Grant/Research Support: Gilead Sciences, Inc

The following people have nothing to disclose: Naoki Ikenaga, Shuhei Yoshida, Susan B. Liu, Jeanhee Chung, Deanna Sverdlov, Maria Kovalenko

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Expression and function of methylthioadenosine phos- phorylase in chronic liver disease

Barbara Czech1, Katja Dettmer2, Daniela Valletta1, Wolfgang E. Thasler3, Martina Müller1, Peter J. Oefner2, Anja Bosserhoff2, Claus Hellerbrand1;
1University Hospital Regensburg, Regensburg, Germany; 2University Regensburg, Regensburg, Germany; 3Ludwig-Maximilians-University, Munich, Germany

Methylthioadenosine phosphorylase (MTAP) the rate-limiting enzyme in the methionine and adenine salvage pathway catalyzes the phosphorylation of 5-deoxy-5-(methylthio)denosine (MTA) which is a by-product of polyamine synthesis. The aim of this study was to assess MTAP expression and function during the progression of chronic liver disease. Methods: MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry. Results: MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs. Conclusion: MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis.

Disclosures:

Martina Müller - Grant/Research Support: Novartis

The following people have nothing to disclose: Barbara Czech, Katja Dettmer, Daniela Valletta, Wolfgang E. Thasler, Peter J. Oefner, Anja Bosserhoff, Claus Hellerbrand

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Inhibition of the progression of biliary and panlobular fibrosis in vivo by C12-200-siProcollagen α1 (I) RNA in lipid-like carriers

Carolina Jimenez Calvente1, Alfica Sehgal2, Yong Ook Kim1, DetlefSchuppan1,3;
1Alnylam Pharmaceuticals, Boston, MA;2Institute of Molecular and Translational Medicine, Mainz, Germany; 3Beth Israel Deaconess Medical Center, Boston, MA

Introduction: Hepatic fibrosis, a wound-healing response to chronic liver injury, is characterized by excess production and deposition of extracellular matrix (ECM). There are currently no approved antifibrotic therapies for liver fibrosis. The demonstration that hepatic fibrosis in animals and humans can regress when collagen synthesis is inhibited suggests that fibrosis may be reversible once hepatic procollagen-α1 (I) mRNA is suppressed. Methods: Mice deficient in the phospholipid flippase (Mdr2) show progressive biliary fibrosis and mice treated with increasing doses of CCL4 longterm develop panlobular cirrhosis. Optimized siRNAs directed against the transcripts of the major scar tissue protein, procollagen α1(I) (siCol1 A1), were encapsulated in C12-200 lipid particles (LPs) (Love KT et al PNAS 2009). siGFP (green fluorescent protein)-LPs served as negative control. Groups of 5 mice were treated with increasing doses of CCL4 by oral gavage for 8 weeks three times weekly. One day after the last CCL4 treatment, mice received four injections of PBS, siCol1 A1-LPs or control siGFP-LPs at 0.1 and 0.2 mg per kg BW for 4 weeks. 8 week old Mdr2KO mice (n=7-8) and their nonfibrotic wild-type controls received 4 injections of PBS, siCol1 A1-LPs or control siGFP-LPs for 2 weeks (doses of 0.4 and 0.8 mg siRNA per kg BW). 24 h after the last injection of siRNA-LPs, liver collagen content was quantified via hydroxyproline (Hyp) and Sirius red morphometry. Fibrosis related transcript levels were determined by quantitative RT-PCR. α-SMA, CD68 and TLR4 were identified by IHC. Results: Compared to the GFP control, siCol1 A1-LPs significantly suppressed the expression of procollagen α1 (I) by 80% and 60% in Mdr2ko and CCL4 mice, respectively. Total collagen deposition was significantly reduced by 25% in both models after treatment with siCol1 A1-LPs. In mice with CCl4-induced fibrosis the α-SMA positive area was reduced by 57 %. Parameters of hepatic inflammation (ALT and AST) and kidney function (crea-tinine) remained unchanged in both models compared to siGFP-LPs. Populations of CD68 and TLR4 expressing cells were not significantly changed, compared to the groups treated with siGFP -LPs or PBS, indicating the absence of innate immune stimulation by the siRNA and/or the LPs. Conclusion: C12-200 LPs loaded with siRNA to procollagen I and likely other fibro-genic transcripts are a promising approach to inhibit liver fibrosis progression and promote its regression in vivo.

Disclosures:

Alfica Sehgal - Employment: Alnylam Pharmaceuticals

Detlef Schuppan - Consulting: Boehringer Ingelheim, Aegerion, Gilead, Gen-zyme, GSK, Pfizer, Takeda, Sanofi Aventis, Silence

The following people have nothing to disclose: Carolina Jimenez Calvente, Yong Ook Kim

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Effective delivery of C12-200 lipid-like carriers to the liver and cells implicated in fibrogenesis in mice with biliary and panlobular liver fibrosis

Carolina Jimenez Calvente1, Alfica Sehgal2, Mustafa Diken3, Detlef Schuppan1,4;
1Institute of Molecular and Translational Medicine, Mainz, Germany; 2Alnylam Pharmaceuticals, Boston, MA; 3TRON (Translational Oncology), Mainz, Germany; 4Beth Israel Deaconess Medical Center, Boston, MA

Introduction: Liver fibrosis results from the excessive accumulation of extracellular matrix including collagens. The development of effective antifibrotic agents is in hampered by a lack of highly specific and liver targeted drugs. Small interfering RNA (siRNA) is a powerful tool for post-transcriptional gene silencing. The systemic delivery of naked siRNAs is fraught with obstacles, such as degradation by serum and tissue nucleases, inefficient endocytosis by tissue cells and rapid excretion via the kidneys. Lipid-like particles (LPs) could permit efficient delivery of siRNA, minimizing interference with blood cells and plasma, thus having low or no side effects. We could show that C12-200 LPs when loaded with siRNA to procollagen α1 (I) induce a 50-80% target knockdown in liver fibrosis models in vivo. However, distribution and cell-specific uptake of these LPs remained unkown. Methods: 3 mg of C12-200 LPs were linked to 50 μg of DiR near infrared (NIR) or DiI lipid dyes. One hour later, the unbound dye molecules were removed by centrifuga-tion. Lipid dye binding and the final composition of the liposo-mal dispersion was confirmed by light scattering flow cytometry and fluorescent microscopy. Immediately after labeling, Mdr2 deficient and CCL4-fibrotic mice, and their wildtype or untreated littermates, were subjected to a single i.v. injection of DiR- or DiI-labeled C12-200 LPs. Their in vivo distribution was determined by NIR-in vivo imaging and their co-localization with liver cell subsets was determined by fluorescent IHC at 30 min, 4, 24, and 48 h post-injection. Results: 30 min after injection, 90% of administered DiR-labeled C1 2-200 LPs were found in the liver of Mdr2KO and CCL4-treated mice and their nonfi-brotic controls. Maximum hepatic accumulation was found at 24 h. DiI-labeled C12-200 LPs localized differently in the two fibrosis models. At 24 h in Mdr2KO mice, 11%, 35%, 15%, 65% and 5% of hepatocytes (Albumin+), mesenchymal cells (vimentin+), activated HSCs (α-SMA+), Kupffer (CD68+) and endothelial cells (CD31+), respectively, took up DiI-labeled C12-200 LPs, whereas in their wildtype controls or CCL4-untreated littermates these percentages were much lower (5%, 1.5%, 1.5%, and 2%, respectively). In CCl4-treated mice, 8%, 7%, 5%, 23% and 5% of hepatocytes, mesenchymal cells, activated HSCs, Kupffer and endothelial cells, respectively, co-localized with DiI-labeled C12-200 LPs. Conclusion: C12-200 LPs are specifically retained in the liver and distribute to/fuse mainly with activated HSC and Kupffer cells. This explains their remarkable antifibrotic effects when loaded with siRNA directed to profibrotic genes in vivo.

Disclosures:

Alfica Sehgal - Employment: Alnylam Pharmaceuticals

Detlef Schuppan - Consulting: Boehringer Ingelheim, Aegerion, Gilead, Gen-zyme, GSK, Pfizer, Takeda, Sanofi Aventis, Silence

The following people have nothing to disclose: Carolina Jimenez Calvente, Mustafa Diken

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Previous episodes of liver injury, but not aging, enhance pro-fibrotic response in mice

Charlotte Selvais, Valérie Lebrun, Yves J. Horsmans, Isabelle A. Leclercq;
laboratory of hepato-gastroenterology, Institut de Recherche Expérimentale et Clinique, Université catholique de Lou-vain, Brussels, Belgium

Background and Aim: In humans with chronic viral hepatitis C, fibrosis develops faster and is more severe in patients who are infected at old age compared to those infected at a younger age. Our aim was to determine whether old age influence fibrosis development in a mouse model. Methods: Young (7 weeks old) and old (15 months old) BALB/c mice were injected CCl4 3 times a week for 4 weeks and fibrosis compared. In a second experiment, response to a given fibrogenic stimulus (CCl4 3 times a week for 2 weeks) was compared between naïve mice and mice that had previously experienced and recovered from CCl4-induced fibrosis (3 rounds of 2 weeks CCl4, 3 times a weeks, separated by 4 weeks recovery, called naïve/test CCl4 and Fib/CCl4, respectively). Histological and molecular markers of fibrosis, matrix remodeling and inflammation were analyzed. Results: Young and old mice developed similar significant fibrosis with similar collagen deposition and Collagen type I and αSMA mRNA expression in the 2 age groups. However, macrophage infiltration and expression of profibro-genic TGFβ1 were higher in old than young mice. Thus, the magnitude of liver scarring in response to a given insult is similar irrespective of the age, but at the expense of increased inflammatory and pro-fibrogenic responses in older ones. We then compared the response to a test CCl4 regimen in naïve mice (naïve/test CCl4) and in mice previously exposed to CCl4 (3 rounds of 2 weeks CCl4 separated by 4 weeks recovery, called Fib/ test CCl4). We sacrificed a group of mice at the end of this induction/recovery period (Fib/-) and confirmed ad inte-gro recovery of liver architecture. After test CCl4 stimulus, mice previously exposed to CCl4 (Fib/test CCl4) exhibited enhanced bridging fibrosis, confirmed by Sirius red staining and mor-phometry compared to naïve/test CCl4 mice. Up-regulation of pro-fibrogenic genes COLL-I, αSMA and TGFβ1, macrophage infiltration (F4/80 and CD68 IHC and PCR) and M1/M2 polarization (PCR) were not significantly different between these 2 groups. However, by analyzing the gene expression and activity of remodeling factors, the MMP/TIMP relative balance was tilted towards matrix degradation in naïve/test CCl4 but not in Fib/test CCl4 livers consistent with increased accumulation of scarring tissue as a result of reduced matrix degradation rather than increased deposition in mice with previous experience of fibrosis. Conclusion: Age per se does not potentiate liver fibrosis development. By contrast, previous exposure to a pro-fibrotic stimulus enhances the fibrotic response to a given stimuli later in life.

Disclosures:

Yves J. Horsmans - Consulting: helssinn, johnson&johnson, roche, merck, ipsen, helssinn, johnson&johnson, roche, merck, ipsen, helssinn, johnson&johnson, roche, merck, ipsen, helssinn, johnson&johnson, roche, merck, ipsen; Grant/Research Support: gsk, astra-zeneca, gsk, astra-zeneca, gsk, astra-zeneca, gsk, astra-zeneca; Speaking and Teaching: bms, bms, bms, bms

The following people have nothing to disclose: Charlotte Selvais, Valérie Lebrun, Isabelle A. Leclercq

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Direct Contribution of Mitochondorial Oxidative Stress to Hepatic Fibrogenesis

Tadashi Moro1,3, Sachie Nakao1, Hideaki Sumiyoshi1, Takamasa Ishii2, Masaki Miyazawa2, Naoaki Ishii2, Yutaka Inagaki1;
Department of Regenerative Medicine, Tokai University School of Medicine, Isehara, Japan; 2Department of Molecular Life Science, Tokai University School of Medicine, Isehara, Japan; 3Research Laboratory, Minophagen Pharmaceutical Co. Ltd., Zama, Japan

Background & Aims: Oxidative stress is considered to play a key role in the progression of chronic liver diseases such as hepatitis C and non-alcoholic steatohepatitis. However, since both the production of oxidative stress and liver fibrosis may merely represent the consequences of cell damage caused by the primary disease, it is virtually unknown whether oxidative stress is linked directly to fibrogenesis. Here we examined the direct effects of oxidative stress on hepatic fibrogenesis by using “Tet-mev-1 mouse”, in which mitochondrial reactive oxygen species can be induced by doxycycline (Dox)-regulable expression of mutant succinate dehydrogenase. Methods: Hepatocytes obtained from wild type or Tet-mev-1 mice were treated with different Dox concentrations. Production of reactive oxygen species and decreased membrane potential of mitochondria were examined by using dichlorofluoresceine diacetate (DCFDA) and tetramethyl rhodamine methyl ester (TMRM), respectively. Both wild type and Tet-mev-1 mice were administered 0.4 mg/mL of Dox for 1 year, and subsequently fed either normal diet chow or high fat/high sucrose (HFHS) diet. Liver tissues obtained from these mice were subjected to histopatho-logical examination and microarray gene expression analyses. Hepatocytes isolated from the same mice were co-cultured with hepatic stellate cells (HSC) prepared from transgenic col1a2 luciferase reporter mice (COL/LUC). Results: DCFDA fluorescent intensities were significantly increased after 1 and 1 0 μg/mL of Dox treatment of hepatocytes from Tet-mev-1 mice, but not control animals. Mitochondrial membrane potential was decreased in Dox-treated Tet-mev-1 hepatocytes as compared with untreated cells. Histopathological examination revealed considerable infiltration of inflammatory cells around the portal tracts, but no apparent cell damage, in Dox-treated Tet-mev-1 mouse liver. Microarray analyses indicated that c-JUN/c-fos expression was markedly elevated in liver tissues from Dox-treated Tet-mev-1 mice as compared with control animals. In addition, feeding of Tet-mev-1 mice with HFHS diet synergisti-cally enhanced a number of chemokine and growth factor signals such as CCR2/CCR5, FGF, TGF-beta and IL-1. Moreover, luciferase assays of COL/LUC HSC lysates indicated that col1a2 promoter activity was increased 3.5-fold after co-culture with hepatocytes from Dox-treated Tet-mev-1 mice as compared with hepatocytes from Dox-treated wild type animals. Conclusion: Tet-mev-1 mice provide good in vivo and in vitro models to evaluate direct contribution of oxidative stress to hepatic fibrogenesis without any secondary effects of cell damage caused by the primary disease.

Disclosures:

The following people have nothing to disclose: Tadashi Moro, Sachie Nakao, Hideaki Sumiyoshi, Takamasa Ishii, Masaki Miyazawa, Naoaki Ishii, Yutaka Inagaki

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Osteopontin induces Ductular Reaction Contributing to Liver Fibrosis

Xiaodong Wang1, Aritz Lopategi1, Yongke Lu1, Naoto Kitamura1, Xiaodong Ge1, Raquel Urtasun1, Tung Ming Leung1, M. Isabel Fiel2, Natalia Nieto1;
1Division of Liver Diseases, liver, medicine, New York, NY; 2Pathology, Mount Sinai medical school, New York, NY

Background: A key feature in the pathogenesis of liver fibrosis is ductular reaction, mainly characterized by proliferation of biliary epithelial cells. The mechanism for the onset of ductular reaction and its possible role in scar formation remains unknown. Osteopontin (OPN) is a soluble cytokine and a matrix-associated protein constitutively expressed in cells within the periportal region highly induced in liver injury. OPN could enable cells to sense molecular patterns associated with liver injury and trigger signals that are required for oval cell expansion, ductular reaction and fibrogenesis to occur. Since we previously showed OPN is highly induced in biliary epithelial cells during drug-induced liver injury and OPN up-regulates colla-gen-I expression in hepatic stellate cells, we hypothesized that OPN could drive the fibrogenic response by promoting ductular reaction and as a result signal to hepatic stellate cells to enhance scarring. Methods: Liver fibrosis was induced in WT and Opn-/- mice by administration of thioacetamide in the drinking water for 4 months or by bile duct ligation for 8 days. In vitro studies were performed with HSC, hepatocytes or biliary epithelial cells. Results: OPN was sensitive to reactive oxygen species in biliary epithelial cells and in oval cells. Opn-/-mice were protected from thioacetamide and bile duct ligation induced liver injury as shown by H&E staining, pathology scores and serum ALT activity. Recombinant OPN (rOPN) decreased the hepatocyte proliferation rate in vitro and the hepatocyte Ki67 index was greater in thioacetamide-treated or in bile duct ligated Opn-/- mice than in WT mice. In contrast, rOPN increased biliary epithelial cell proliferation and motility in vitro. Oval cell expansion, the ductular reaction score and cytokeratin-1 9 and transforming growth factor-β immunostain-ing were lower in thioacetamide-treated and bile duct ligated Opn-/- mice than in WT mice, suggesting that OPN activates the oval cell compartment and induces ductular reaction. Overall, thioacetamide-treated and bile duct ligated Opn-/- mice developed less liver fibrosis and injury than WT mice. Primary hepatic stellate cells co-cultured with biliary epithelial cells showed increased collagen-I and OPN protein expression compared to hepatic stellate cells in mono-culture. There was up-reg-ulation of transforming growth factor-β in biliary epithelial cells and blocking OPN, transforming growth factor-β or both reduced collagen-I expression in hepatic stellate cells. Conclusion: OPN emerges as a key matricellular cytokine driving ductular reaction and contributing to scarring and liver fibrosis via transforming growth factor-β.

Disclosures:

The following people have nothing to disclose: Xiaodong Wang, Aritz Lopategi, Yongke Lu, Naoto Kitamura, Xiaodong Ge, Raquel Urtasun, Tung Ming Leung, M. Isabel Fiel, Natalia Nieto

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Mechanisms of hepatic fibrogenesis in autosomal recessive polycystic kidney disease/congenital hepatic fibro-sis: role of CTGF

James Weemhoff1, Genea Edwards1, Prachi C. Borude1, Darren P. Wallace2, Udayan Apte1, Michele T. Pritchard1;
1Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 2Internal Medicine-Nephrology, University of Kansas Medical Center, Kansas City, KS

Congenital hepatic fibrosis (CHF), the most common extra-hepatic manifestation of autosomal recessive polycystic kidney disease (ARPKD), is associated with excessive extracellular matrix (ECM) deposition which encapsulates ductal plate cell-derived cysts. The precise mechanisms of hepatic cystogenesis and associated CHF are not known. Therapeutic options for ARPKD/CHF are extremely limited. Here, making use of the polycystic kidney (PCK) rat which harbors the same mutation found in ARPKD patients, we characterized the development of hepatic fibrosis from post natal day (PND) 0 to 3 months after birth. Sprague-Dawley (SD) rats were used as controls. Liver to body weight ratios were greater in PCK rats compared to controls, consistent with the development and growth of intrahep-atic cysts. At three months, PCK rats had increased hepatic mRNA accumulation of αSMA (myofibroblast marker), type I collagen, elastin (portal fibroblast marker), desmin (hepatic stellate cell marker) and connective tissue growth factor (CTGF) compared to SD rats. Consistent with those findings, 3-month old PCK rats exhibited increased type 1 collagen, Sirius red staining and CTGF protein relative to SD rats. Time course analysis revealed that the peak hepatic mRNA accumulation of αSMA, Col 1a 1, CTGF and elastin was at PND 10-20. Hepatic αSMA protein also peaked at PND 10. Hepatic CTGF mRNA and protein was induced in PCK rats at PND5, peaked at PND10 and remained increased throughout the time course. While cysts were observable PND 0, diffuse ECM deposition around hepatic cysts revealed by Sirius red staining began PND 5 in PCK rats; diffuse Sirius red staining increased until PND 20. In PND 30 and 3-month old PCK rats, Sirius red staining became intense and compressed around proliferating cysts. The increased hepatic fibrosis observed in PCK rat livers was corroborated by observations made in human PKD liver samples. Collectively, these data suggest that initiation of fibrogenesis in PCK rats occurs during early postnatal period and involves both portal fibroblast- and hepatic stellate cell-derived myofibroblasts. Furthermore, these data suggest that CTGF may be a driving force behind CHF in the PKC rat and reveal CTGF as a potential therapeutic target. These studies were supported by grants to U.A. and D.P.W. (P50 DK057301-11) and M.T.P (P20 GM103549 and R00 AA017918).

Disclosures:

The following people have nothing to disclose: James Weemhoff, Genea Edwards, Prachi C. Borude, Darren P. Wallace, Udayan Apte, Michele T. Pritchard

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15 -Deoxy-Δ12,14-prostaglandin J2 Inhibits Recruitment of Bone Marrow-Derived Mesenchymal Stem Cells in Liver Fibrosis Induced by Carbon Tetrachloride in Mice

Xin Liu, Shuangshuang Jia, Ping Mai, Le Yang, Lin Yang, Lin Wang, Liying Li;
Department of Cell Biology, Capital Medical University, Beijing, China

Background: Bone marrow-derived mesenchymal stem cells (BMSCs) can migrate to damaged liver and differentiate to myofibroblasts during liver fibrosis. Blocking BMSCs recruitment may be an effective strategy to stop or even reverse liver fibrosis. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a natural peroxisome proliferator-activated receptor gamma (PPAR-γ) ligand, has been implicated as a new antifibrotic compound with possible clinical applications. This study aimed to evaluate the effects of 15d-PGJ2 on BMSCs migration in CCl4-induced liver fibrosis in mice, and investigated the mechanism underlying this process. Methods: Mice were leathally irradiated and received bone marrow transplants from enhanced green fluorescent protein transgenic mice. CCl4 was used to induce liver fibrosis with/without 15d-PGJ2 administration. CD166+ and CD44+ cells in liver tissue were analyzed by flow cytometric (FACS) and immunofluorescent staining to identify BMSCs. The mRNA expressions of fibrotic markers, including procollagen α1 (I), procollagen α1 (III), and α-smooth muscle actin in liver tissue were examined by real-time RT-PCR. Moreover, hepatic collagen deposition was evaluated by morphometric analysis of Sirius red staining. The production of reactive oxygen species (ROS) was examined by probe DCFH-DA. PPAR-γ distribution was analyzed by immunofluorescence and high content screening. Results: FACS and immunofluorescent staining analysis for CD166+ and CD44+ showed that the proportion of BMSCs in CCl4-induced fibrotic mouse liver decreased markedly after 1 5d-PGJ2 administration. In vitro, 15d-PGJ2 (1-5 μM) caused a dose-dependent decrease in the migration of primary BMSCs. Neither PPAR-γ agonists nor antagonist had an effect on BMSCs migration. However, 15d-PGJ2 promoted intracellular ROS production, and its inhibitory effect on BMSCs migration was abrogated by general ROS inhibitor, NAC, indicating 15d-PGJ2 reduced BMSCs migration through ROS formation, not PPAR-γ. Furthermore, we measured the mean of PPAR-γ fluorescence intensity in the nucleoplasmic and cytoplasmic area and analyzed the ratio of nucleus/cytoplasm by high content screening analysis, showing that 15d-PGJ2 did not alter PPAR-γ distribution. In vivo, 15d-PGJ2 administration ameliorated CCl4-induced hepatic fibrosis, as demonstrated by the reduced mRNA expression of fibrotic markers and decreased liver collagen deposition. Conclusion: 15d-PGJ2 significantly decreases recruitment of BMSCs toward the damaged liver, which involves ROS generation and independently of PPAR-γ. Therefore, 1 5d-PGJ2 may represent an effective antifibrotic target through inhibiting the homing of BMSCs.

Disclosures:

The following people have nothing to disclose: Xin Liu, Shuangshuang Jia, Ping Mai, Le Yang, Lin Yang, Lin Wang, Liying Li

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A long-acting GLP-1 agonist exenatide suppresses inflammation and fibrosis in models of NASH and biliary fibrosis

Xiao-Yu Wang1, Shih-yen Weng1, Thomas Klein2, Yong Ook Kim1, Detlef Schuppan1,3;
1Institute of Molecular and Translational Medicine, Mainz, Germany; 2Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany; 3Division of Gastroen-terology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Background and aims: Non-alcoholic steatohepatitis (NASH) is now regarded as the most common cause of abnormal liver function tests in most countries. Anti-inflammatory and especially antifibrotic therapies for NASH are urgently needed.Glucagon-like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion, delays gastric emptying and exhibits other antihyperglycemic actions following its release into the circulation from the gut. We examined the effect of a long-acting GLP-1 agonist exenatide (BYDUREON, BY) on inflammation and fibrosis in models of fibrotic NASH and biliary fibrosis. Methods: BY was administered twice weekly by subcutaneous injection at 0.4or 2 mg per kg BW to Mdr2KO mice from week 7-1 1 of age, and to 8 week old C57BL/6 mice fed a methionine and choline deficient diet (MCD) for 4 weeks. Hepatic fibrosis was assessed by morphometric analysis of sirius redstained collagen and measurement of hydrox-yproline content. Serum biochemistries were determined by an autoanalyzer, and hepatic inflammation was assessed by semi-quantitative immunohistochemistry. Fibrosis and inflammation related transcript levels were quantified by quantitative realtime polymerase chain reaction (qPCR). Results: In mice on the MCD diet, 0.4 more than 2.0 mg/kg BY causeda significant reduction of hepatic steatosis, inflammation and a 30%reduc-tion in total collagen content compared to untreated controls.BYsignificantly decreased fibrosis related transcripts such as αSMA, procollagenα1 (I),TGFβ1, TIMP-1, but also of putatively fibrolyticMMP-8,MMP-9 and -13. BY also suppressed inflammation related transcripts such as CD68, CCL3, and TNFα, and increased (anti-inflammatory) Arg1 transcripts. In Mdr2 -/- mice, 0.4 mg/kg BYsignificantly lowered liver collagen content, decreased MMP-13 but increased Arg1 transcripts. Conclusions: A long-acting GLP-1 agonist which is already in clinical use for treatment of type 2 diabetesreduced parameters of hepatic steatosis, inflammation and fibrosis, without negative effects on weight gain, supporting its usefulness to treat human NASH and liver fibrosis.

Disclosures:

Detlef Schuppan - Advisory Committees or Review Panels: Aegerion, Eli Lilly, Gilead; Consulting: Boehringer-Ingelheim, Isis, Takeda; Grant/Research Support: Boehringer-Ingelheim

The following people have nothing to disclose: Xiao-Yu Wang, Shih-yen Weng, Thomas Klein, Yong Ook Kim

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Hedgehog signaling regulates the regression of fibrosis by placenta-derived stem cells in CCl4-induced fibrosis models of rat

Jeongeun Hyun1, Gi Jin Kim2, Youngmi Jung1;
1Biological Science, Pusan National university, Pusan, Republic of Korea; 2Biomedical Science, Cha University, seoul, Republic of Korea

Placenta-derived stem cells (PDSCs) have been focused as a cell source for liver regeneration. Emerging evidence provides the anti-fibrotic effect of PDSCs on liver fibrosis. However, underlying mechanisms on the effect of PDSCs on liver fibrogenesis remain unclear. The hedgehog (Hh) signaling pathway orchestrates tissue reconstruction in the damaged liver. Recently, micro (mi) RNA-125b is reported to regulate smoothened (smo), Hh signaling activator. Hence, we hypothesized that miRNA-125b mediated Hh signaling pathway might regulate liver regeneration by PDSCs. Male rats injected with carbon tetrachloride 4 for 9 weeks were transplanted with human PDSCs (Tx) or saline (Non-Tx), and sacrificed at 2 and 3 weeks after transplantation. Biochemical analysis was performed for accessing the expression of miRNAs, Hh signaling molecules and fibrotic markers. Sirius red staining was used for evaluating fibrosis. The expression of miRNA-125b was a significantly higher in PDSCs than normal liver tissue. In Tx group, as miRNA-125b was up-regulated, its target, smo, and Hh-target gene, Gli2, were down-regulated compared to Non-Tx group. miRNA-125b was harbored by PDSCs. Production of Sonic Hh (Shh, one of Hh ligands) by hepatocytes was rarely detected in the PDSCs-transplanted livers, whereas shh-expressing hepatocytes were significantly accumulated in the liver from Non-Tx group. Pancytokeratin and SOX9 (hepatic progenitor cell markers)-expressing cells also showed the greater reduction in Tx group, compared to Non-Tx group. The expression of TGF-β, fibrosis-stimulating factor, and fibrotic markers, such as α-sma, collagen type 1α1 (col 1 α1), s100a4 and vimentin was lower in Tx group than Non-Tx group. Histopathological analysis revealed the greater reduction of collagen deposition in Tx rats. In addition, indian Hh (Ihh), another Hh ligand, showed the significant increase in Tx group, implying that distinct role of Ihh from Shh on liver regeneration. Those results demonstrated that the up-regulation of miRNA-125b was associated with the decreased activation of Hh signaling pathway, which contributed to the regression of fibrosis, suggesting that Hh signaling might be related with liver regeneration by PDSCs.

Disclosures:

The following people have nothing to disclose: Jeongeun Hyun, Gi Jin Kim, Youngmi Jung

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The role of the calcium-activated potassium channel KCa3.1 in a murine model of hepatic fibrosis

Linda S. Møller1,2, Matteo Biagini3, Annette D. Fialla1, Ove B. Schaffalitzky de Muckadell1, Jonel Trebicka4, Ralf Köhler2,5;
1Department of medical gastroenterology, Odense University Hospital, Odense, Denmark; 2Institute of Molecular Medicin, University of Southern Denmark, Odense, Denmark; 3Department of Pathology, Odense University Hospital, Odense, Denmark; 4Department of Internal Medicin, University of Bonn, Bonn, Germany; 5Aragon Institute of Health Sciences, Zaragoza, Spain

BACKGROUND: Activation of hepatic stellate cells (HSC) is a hallmark in liver fibrogenesis. The activated HSC show increased proliferation, contraction and collagen production. The Ca2+-activated K+-channel KCa3.1 (IK) plays a role in cell proliferation by enhancing intracellular Ca2+ and affecting cell cycle progression, as well as regulation of cell volume, and it modulates T lymphocyte activation, and thereby inflammation. Interestingly, IK exerts anti-fibrotic properties in murine renal fibrosis. In this study we evaluated the role of the channel in experimental liver fibrosis. MATERIAL AND METHODS: Male 3 months old IK-knockout mice (IK-KO) and corresponding wild type littermates (IK-WT), received CCl4 1 ml/kg diluted in peanut oil ip. twice weekly. Animals were sacrificed after 4, 8, 12 and 16 weeks of CCl4 intoxication (n= 6-10 per group). Liver fibrosis and inflammation were analysed by histology (Masson-trichrome, METAVIR-score), qRT-PCR (collagen 1, alpha-SMA, TGFβ1, MMP-2, TIMP-2, FSP-1, CD8) and hepatic hydroxyproline content. RESULTS: Histologic blinded evaluation of fibrosis and inflammation showed the expected progression of fibrosis over the study period. Interestingly, there was a trend towards more fibrosis in the IK-KO in early fibrosis stages and reaching significance after 12 and 16 weeks. The hydroxyproline levels confirmed the histological finding, but only statistically significant at 12 weeks. Inflammation score was increased after 12 and 16 weeks (statistically significant only after 12 weeks). qRT-PCR analysis revealed higher mRNA levels in IK-KO of collagen 1, TGFβ1, MMP-2, TIMP-2, FSP-1. CD8 and alfa-SMA increased, but there were no difference between WT and IK-KO. The table below marks groups and genes with statistically significant differences (upwards arrow means higher levels in knockouts). CONCLUSION: This study delivers the first evidence that deficiency of the KCa3.1 channel results in more fibrosis and inflammation in the CCl4 induced murine fibrosis model. The exact role of the KCa3.1 channel in hepatic fibrogenesis remains to be established. But the presence of this channel might be beneficial in severe hepatic fibrosis and might offer a new target for anti-fibrotic therapies. Further studies are ongoing to elucidate the mechanism underlying these processes.

qPCR differences

Table 1. 
 4 weeks8 weeks12 weeks16 weeks
collagen 1[UPWARDS ARROW]   
TGFβl [UPWARDS ARROW]  
MMP-2[UPWARDS ARROW][UPWARDS ARROW] [UPWARDS ARROW]
TIMP-2[UPWARDS ARROW]  [UPWARDS ARROW]
FSP-l[UPWARDS ARROW][UPWARDS ARROW] [UPWARDS ARROW]

Disclosures:

The following people have nothing to disclose: Linda S. Møller, Matteo Biagini, Annette D. Fialla, Ove B. Schaffalitzky de Muckadell, Jonel Trebicka, Ralf Köhler

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5 -methyl-1-phenyl-2-(1H)-pyridone modulates proin- flammatory and fibrogenic phenotype of human hepatic stellate cells through activation of antioxidant system pathways

Jose Macias-Barragan1,2, Alessandra Caligiuri2, Jose Vera-Cruz1, Jesus Garcia-Banuelos1, Silvia Lucano-Landeros1, Adriana M. Salazar Montes1, Margarita Montoya-Buelna2, Belinda C. Gomez-Meda1, Massimo Pinzani3, Juan Armendáriz-Borunda1;
1Instituto de Biologia Molecular y Terapia Genica, University of Guadalajara, Guadalajara, Mexico; 2Dipartimento di Medicina Interna. Denothe, Università di Firenze, Florence, Italy; 3Institute of Liver and Digestive Health, University Collage London, London, United Kingdom

Background. Hepatic stellate cells (HSC) are perisinusoidal cells of the liver, located in the space of Disse between hepatocytes and sinusoidal endothelial cells. In normal liver are described as being in a quiescent state containing lipid droplets storing vitamin A. When liver is damaged they change into an activated state that is characterized by proliferation, contractility and chemotaxis. HSC profibrogenic cytokines are key targets of anti-fibrotic therapies. 5-methyl-1-phenyl-2-(1 H)-pyridone or pirfenidone (PFD) is a small molecule indicated for treatment of chronic inflammation and fibrogenesis. Oxidative stress is directly involved in the onset of hepatic fibrosis by HSC activation. Aim. In order to identify whether anti-inflammatory and anti-fibrogenic effects of PFD are related to activation of the endogenous antioxidant system, HSC were incubated with PDGF or 2-methyl-1 ,4-naphthoquinone (MEN) a ROS-inducer. Methods and Results. PFD was able to inhibit PDGF or MEN-induced profibrogenic actions, including cell proliferation, cell motility and de novo synthesis of Collagen type I, TGFβ, TIMP-1, IL-1 and TNFα. These effects were associated with an increase of nuclear Nrf2 assessed by western blotting and con-focal microscopy. Because PFD activates JNK, which stimulates Nrf2 transcriptional factor, through siRNA-mediated silencing we examined downstream antioxidant targets as antioxidant enzymes. JNK blockade by siRNA and SP600125 down-regulates Nrf2 activation. Also PFD induced a dose- and time-dependent activation of several antioxidant genes, (Glutamyl cysteine synthetase catalytic subunit, Glutamyl cysteine syn-thetase regulatory subunit and Heme oxygenase 1) and increase glutathione content, whose activity may contribute to the down-regulation of ROS-induced profibrogenic and pro-inflammatory effects. Conclusion. These results provide molecular insights in anti-fibrogenic immunmodulatory action of PFD by counteracting ROS-induced pro-fibrogenic signalling, and by regulation of the biosynthesis of antioxidant proteins. This study indicates that activation of the antioxidant system plays

an essential role in the modulation of inflammatory and fibrogenic cytokines in HSC.

Disclosures:

The following people have nothing to disclose: Jose Macias-Barragan, Alessandra Caligiuri, Jose Vera-Cruz, Jesus Garcia-Banuelos, Silvia Lucano-Landeros, Adriana M. Salazar Montes, Margarita Montoya-Buelna, Belinda C. Gomez-Meda, Massimo Pinzani, Juan Armendáriz-Borunda

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The antifibrinolytic drug tranexamic acid reduces liver injury and fibrosis in a mouse model of chronic bile duct injury

NikitaJoshi1,2, Bryan Copple2, Kurt Williams1, James P. Luyendyk1;
1 Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, MI; 2Pharmacology and Toxicology, Michigan State University, East Lansing, MI

We have shown previously that robust hepatic fibrin(ogen) deposition accompanies the development of chronic alpha-naph-thylisothiocyanate (ANIT)-induced cholestatic liver injury in mice. Similarly, hepatic fibrin deposition is evident in livers of patients with cholestatic liver disease. Despite being a conspicuous feature of chronic liver disease, the role of fibrin(ogen) deposition in liver injury and fibrosis is not completely understood. Of interest, we found that mice lacking circulating fib-rinogen (and hepatic fibrin deposits) developed increased liver injury when fed a diet containing 0.025% ANIT (ANIT diet), suggesting a hepatoprotective effect of fibrin in cholestatic liver injury. We tested the hypothesis that administration of the antifibrinolytic drug tranexamic acid would reduce ANIT diet-induced liver injury and fibrosis in mice. Compared to wild type C57Bl/6J mice fed control diet (AIN-93M), wild type mice fed ANIT diet developed liver injury characterized by multifocal hepatocellular necrosis, inflammation, biliary hyperplasia and peribiliary fibrosis. Liver inflammation and fibrosis were more severe in mice fed the ANIT diet for 4 weeks compared to 2 weeks. Prophylactic administration of tranexamic acid (1200 mg/kg, ip, bid) significantly reduced hepatocellular necrosis in mice fed ANIT diet for two weeks. Despite reducing necrosis, tranexamic acid did not impact hepatic expression of profibrogenic genes or peribiliary collagen deposition at this time. In mice fed the ANIT diet for 4 weeks, treatment with tranexamic acid for the last 14 days of the ANIT exposure increased hepatic fibrin deposition and suppressed profibrogenic gene induction in liver. Moreover, tranexamic acid treatment significantly reduced type 1 collagen deposition in mice fed ANIT diet for 4 weeks. This reduction in fibrosis occurred in the absence of an effect on integrin β6 mRNA expression, the latter a key component of the αVβ6 integrin, which activates latent TGF-β in this model. Taken together, the results suggest that inhibition of fibrinolysis and stabilization of hepatic fibrin reduces liver injury and fibrosis in mice fed ANIT diet. These studies highlight a potential hepatoprotective function of fibrin(ogen) in chronic cholestatic liver injury.

Disclosures:

The following people have nothing to disclose: Nikita Joshi, Bryan Copple, Kurt Williams, James P. Luyendyk

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The impact of Toll like receptor 4 and Its Ligands on the Gene Expression Network of Hepatic Stellate Cells

Yangyang Ouyang1, Chengzhao Lin2,Jie Lin3, Yirong Cao1, Yuan-qing Zhang1, Shiyao Chen1, Jiyao Wang1, Luonan Chen3, Scott L. Friedman4, Jinsheng Guo1;
1Digestive Diseases, Zhong Shan Hospital, Shanghai, China; 2Institute of Biomedical Sciences, Fu Dan University, SHanghai, China; 3Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences,, Chinese Academy of Sciences, SHanghai, China; 4Division of Liver Diseases, Mount Sinai School of Medicine, New York, NY

Background & Aims: Toll-like receptor 4 (TLR4) signaling in response to lipopolysacchride (LPS), or high mobility group box 1 (HMGB1), a damage-associated endogenous ligand contributes to the activation of hepatic stellate cells (HSC). We investigated the impact of TLR4 signaling on the gene expression network of HSC to identify key regulatory molecules. Methods: Wild type (JS1) and TLR4 knockout (JS2) HSCs were stimulated with saline vehicle (control), 100ng/ml LPS, or 100ng/ml HMGB1 for 24 hours. mRNAs were hybridized on 4644K Agilent whole mouse genome oligo microarray chips for gene expression analysis. Gene interaction and co-expression networks were built on the base of ontology and pathway analysis by KEGG. Topology analysis was used to obtain the main functional modules of TLR4-dependent common differential expression genes. The differential Gene expression was verified by RT-PCR, ELISA and/or Western Blot. Results: Gene expression profiles are markedly different between JS1 and JS2 cells under basal or stimulated condition with TLR4 ligands. Differentially expressed genes that were verified included those linked to fibrogenesis (Col I, Col III, FN1), matrix remodeling (MMP2), growth factors (VEGFD, FGF7, IGF, FIGF), chemokines (CXCL12, CXCR7), inflammation and immunity (IL6), and transcription (Jun B, SP1, Stat3). Signaling pathways up-regulated in JS1 cells compared to JS2 include focal adhesion, p53, NOD-like receptor, mTOR, chemokine, and Jak-STAT. Whereas multiple MHC molecules, MAPK kinases, Prkca, Pik3r3, and Ikbkb were the key regulatory factors in LPS responsiveness in JS1, molecules involved in HMGB1 responsiveness include Prkca, Pik3r3, Herc1, JAK1, ODC1, Traf6, and MAPK kinases. The gene interaction and co-expression networks in TLR4 null cells post LPS or HMGB1 stimulation were significantly simpler and lacked core regulatory factors. Among the 452 common differentially expressed genes in JS1 versus JS2 in response to LPS or HMGB1, there were 29 functional modules identified by topology analysis, which were linked to signaling transduction, extracellular matrix remodeling, growth factors and receptors, chemokine ligands and receptors, stress response, cell growth and apoptosis, and lipid metabolism. Conclusion: There are complex gene expression alterations when TLR4 is absent from HSC. The signaling event via TLR4 regulates a wide spectrum of HSC functions, including inflammatory, fibrogenic, chemotactic properties, as well as the cell growth and metabolism. These finding emphasizes the complex cascades downstream of TLR4 in HSC with significant consequence on the cell biology and function.

Disclosures:

Scott L. Friedman - Advisory Committees or Review Panels: Pfizer Pharmaceutical, Sanofi-Aventis; Consulting: Abbott Laboratories, Conatus Pharm, Exalenz, Genenetch, Glaxo Smith Kline, Hoffman-La Roche, Intercept Pharma, Isis Pharmaceuticals, Melior Discovery, Nitto Denko Corp., Debio Pharm, Synageva, Gilead Pharm., Ironwood Pharma, Alnylam Pharm, Tokai Pharmaceuticals, Bristol Myers Squibb, Takeda Pharmaceuticals, Nimbus Discovery, Isis Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira Pharm, Vaccinex Therapeutics; Stock Shareholder: Angion Biomedica

The following people have nothing to disclose: Yangyang Ouyang, Chengzhao Lin, Jie Lin, Yirong Cao, Yuanqing Zhang, Shiyao Chen, Jiyao Wang, Luonan Chen, Jinsheng Guo

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Nuclear expression of AQP2 in hepatic stellate cells - a novel localization for a water transport protein

Ori Rackovsky1, Tingfang Lee1, Youngmin A. Lee1, Charles E. Rogler2, Leslie E. Rogler2, Yedidya Saiman1, Feng Hong1, Scott L. Friedman1;
1Medicine, Icahn School of Medicine at Mount Sinai, New York, NY; 2Medicine, Albert Einstein College of Medicine, Bronx, NY

A single nucleotide polymorphism (SNP) in the 3' untranslated region (UTR) of the aquaporin 2 (AQP2) gene (c.3002G>C [rs2878771]) has been linked to delayed fibrosis progression in chronic HCV (Huang et al, Hepatology, 2007). Our aim was to explore mechanisms underlying this SNP's association with fibrosis. Methods: PCR and Western blotting were used to confirm AQP2 expression in HSCs, immunohistochemistry to evaluate AQP2 expression in normal and fibrotic human liver, and immunocytochemistry to evaluate AQP2 expression in primary human and LX2 HSCs. Luciferase reporter assays were used to evaluate mRNA binding in close proximity to the WT and SNP variants. RNA secondary structure were predicted for WT and SNP AQP2 mRNAs through the rnafold web server. Results: AQP2 was expressed in primary human and LX2 HSCs by PCR, Western blotting and immunofluorescence. By immunohistochemistry there was a significant increase in AQP2 expression in non-parenchymal cells of fibrotic liver compared to normal liver. Surprisingly, AQP2 was present in the nucleus of HSCs by immunofluorescence, an intracellular location not previously reported in any cell type. This finding was further confirmed by nuclear/ cytoplasmic fractionation and Western blotting. There was no difference in miRNA binding to WT or SNP variants. RNA secondary centroid structure prediction of WT compared to SNP variant AQP2 mRNA showed divergent predicted structures. Discussion: The expression of AQP2 in HSCs, as well as increased expression of AQP2 in non-parenchymal cells of fibrotic liver, suggest a role for AQP2 in HSC activation, and therefore abrogation of AQP2 function in the development of fibrosis might have a protective effect. The unexpected finding of nuclear localization of AQP2 is unique and further studies are currently underway to determine whether this results in abrogation of normal AQP2 function in HSCs.

Disclosures:

Scott L. Friedman -Advisory Committees or Review Panels: Pfizer Pharmaceutical, Sanofi-Aventis; Consulting: Abbott Laboratories, Conatus Pharm, Exalenz, Genenetch, Glaxo Smith Kline, Hoffman-La Roche, Intercept Pharma, Isis Pharmaceuticals, Melior Discovery, Nitto Denko Corp., Debio Pharm, Synageva, Gilead Pharm., Ironwood Pharma, Alnylam Pharm, Tokai Pharmaceuticals, Bristol Myers Squibb, Takeda Pharmaceuticals, Nimbus Discovery, Isis Pharmaceuticals; Grant/Research Support: Galectin Therapeutics, Tobira Pharm, Vaccinex Therapeutics; Stock Shareholder: Angion Biomedica

The following people have nothing to disclose: Ori Rackovsky, Tingfang Lee, Youngmin A. Lee, Charles E. Rogler, Leslie E. Rogler, Yedidya Saiman, Feng Hong

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Alleviation of liver damage and hepatic fibrosis by oral administration of Imm 124E colostrums in Carbon tetra-chloride (CCl4) model is mediated by decrease of intra-hepatic F4/80 macrophages activation

Maias Abd Alrahem1, Lida Zolotarov1, Yehudit Shabat1, Areej A. Khatib2, Meir Mizrahi1;
1Gastroenterology and liver disease institute, Hadassah medical center, Jerusalem, Israel; 2Department of Pathology and Laboratory Medicine, Augusta Victoria Hospital, East Jerusalem, Israel

Hepatic fibrosis development requires the coordinated actions of several cell type including Kupffer cells. Imm 124E colostrum exerts an immunomodulatory effect and alleviates target organ damage in different animal models. Aim: To determine the efficacy of oral administration of Imm 124E colostrum to mice undergoing treatment with CCl4 to prevent hepatic damage and fibrosis by modulating hepatic F4/80 macrophages . Methods: Liver injury was induced by intraperitonealy (IP) administration of CCl4 (0.5 mL/kg). Control mice in group A were treated only with IP CCl4 treatment, Mice in groups B were treated only with oral Imm 124E colostrum (IgG-enhanced fraction of Enterotoxigenic E.coli colostrum Immuron, Australia) and group C were orally treated with Imm 124E colostrums and IP CCl4, al groups were treated for 30 days. Mice were followed for liver injury by ALT and AST, Bilirubin serum levels, body, liver and spleen weight, liver pathology, western blot for alpha SMA, FACS for F4/80 levels and immunehistochimestry for F4/80. Results: Oral administration of Imm 124E was effective in. alleviation of liver injury as was determined by the following measures: A decrease in liver enzymes was noted between the different study groups at day 30 with ALT levels 4376, 28, 52, u/L, ; AST levels 1409, 57, 95 u/L,; Bilirubin levels 2.42 1.28 and 1.55, for groups A, B, and C respectively (p<0.0001). Body weight was different between the groups: 27.1 8, 31.26 and 29.8 grams for groups A, B, and C respectively (p<0.001), spleen and liver weight were also different between the study groups 0.17. 0.08. 0.1 grams for spleen and 1.33, 1.71 and 1.51 grams for liver weight for groups A, B, and C respectively (p<0.001). Liver pathology staining with trichrom blue and Masson red showed differences in: Peripor-tal Necro-inflammatory Changes 2.6, 0, 1.6, Bridging and Confluent Necrosis: 1, 0.16, And 0.8. Focal (Spotty) Lobular Necrosis and hepatocellular apoptosis 1.6,, 0.66, 1. Portal Inflammation 2.4, 0.66, 1.4 and Fibrosis score - Metavir 3.4, 0, 1.8 for groups A, B, and C respectively (p<0.001). These effects were associated with decrease number of F4/80 in the liver 17.98 vs. 13.24 for group A and C respectively (p<0.05) measured by FACS and immunehistochimestry. Alpha SMA levels were also decreased in group C compared with group A (p<0.05) Conclusion: Oral administration of Imm 124E exerts an immunomodulatory effect in mice treated with CCl4. The regulatory effect and suppression of F4/80 macrophages was associated with alleviation liver damage and fibrosis in these treated mice.

Disclosures:

The following people have nothing to disclose: Maias Abd Alrahem, Lida Zolotarov, Yehudit Shabat, Areej A. Khatib, Meir Mizrahi

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Exploring the role of WNT-5A in liver fibrogenesis

Leonie Beljaars1, Sara Daliri1, Christa Dijkhuizen1,2, Klaas Poel-stra1, Reinoud Gosens2;
1Pharmacokinetics, Toxicology and Targeting, University of Groningen, Groningen, Netherlands; 2Molecular Pharmacology, University of Groningen, Groningen, Netherlands

WNT-5A is a secreted growth factor that belongs to the non-canonical members of the Wingless-related MMTV-integration family. Although WNT proteins are predominantly associated with embryogenesis, they are also important in disease progression. Previous studies described regulation of WNT-5A by the fibrogenic growth factor TGFβ. This interaction warrants further studies into the role of WNT-5A in liver fibrosis. Although gene-array studies identified increased WNT5 in fibrotic livers, the functional role of WNT-5A in this liver disease is not described yet. Therefore, our aim was to elucidate its role in liver fibrosis. We studied the expression of WNT-5A in mouse and human livers in more detail and examined the relation between WNT-5A and various fibrosis-associated growth factors, cytokines and extracellular matrix proteins. WNT-5A and collagen I expression in normal and fibrotic mouse and human livers were analyzed with RT-PCR, Western blot, and immuno-histochemistry. The effects of cytokines/growth factors on WNT-5A and collagen I expression in LX2 cells were analyzed with RT-PCR and Western blot. The effects of WNT-5A on the expression of matrix proteins were determined after incubation of LX2 cells with WNT-5A siRNA in the absence and presence of TGFβ. WNT-5A gene and protein expression was significantly higher in fibrotic mouse and human livers compared to normal. Immunohistochemical analysis showed that WNT-5A expression was found in fibrotic collagen-rich areas of mouse and human livers. WNT-5A staining co-localized with desmin staining in these areas. In vitro studies with myofibroblasts showed that WNT-5A expression was significantly increased after incubation with TGF-β while PDGF-BB and pro-inflammatory cytokines (IL1β and TNFα) did not change WNT-5A expression. After silencing of WNT-5A in myofibroblasts, using WNT-5A siRNA, reduced levels of collagen I, vimentin, and fibronectin in TGF-β-stimulated LX2 cells were found as compared to transfection controls. Interestingly, the antifibrotic cytokine IFNγ suppressed WNT-5A and collagen type I expressions in vitro. In addition, hepatic WNT-5A and collagen Ievels were significantly reduced in CCL4 exposed (8 weeks) mice treated with (targeted) IFNγ as compared to untreated fibrotic mice. In conclusion, WNT-5A is significantly upregulated in fibrotic livers in particular in myofibroblasts in fibrotic bands. WNT-5A expression in myofibroblasts is induced by TGFβ and is involved in the regulation of various fibrotic matrix proteins. Targeted IFNγ therapy reduces hepatic WNT-5A expression and ameliorates liver fibrosis. These results identify WNT-5A as a potential new antifibrotic drug target.

Disclosures:

Klaas Poelstra - Consulting: BiOrion Technologies BV; Grant/Research Support: BiOrion Technologies BV; Stock Shareholder: BiOrion Technologies BV

The following people have nothing to disclose: Leonie Beljaars, Sara Daliri, Christa Dijkhuizen, Reinoud Gosens

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Circulating endothelial progenitor cells enhance the proliferation of hepatic stellate cells (HSCs) and increase liver fibrosis in bile duct ligated rats

Skand Gupt1, Mohsin Hasan2, Neha Sharma2, Vaibhav Dixit1, Deepti Vyas1, Dinesh M. Tripathi2, Savneet Kaur1, Nirupma Tre-hanpati2, Shiv K. Sarin2;
1Gautam Buddha University, Greater Noida, India; 2Institute of liver and hepatobiliary Sciences, New Delhi, India

Background and Aim: Recent in vitro studies from our lab have reported that bone-marrow derived circulating endothelial progenitor cells (EPCs) interact with liver sinusoidal endothelial cells and enhance angiogenesis via paracrine mediators in cir-rhotic patients. However whether these cells also interact with hepatic stellate cells (HSCs) and contribute to liver fibrosis remains unknown. In the current study, we evaluated the effect of EPC-secreted angiogenic factors on the functions of HSCs under in vitro conditions and also the effect of EPC transplantation on liver fibrosis in bile duct ligated (BDL) cirrhotic rat models in vivo. Methodology: For the in vitro studies, HSCs were isolated from normal rats after liver perfusion, cultured and characterized by alpha-SMA staining. Circulating human EPCs were isolated from blood and characterized by CD34 and vegfr2 staining. HSCs were then co-cultured with conditioned media (CM) obtained from EPCs. The proliferation of HSCs was analyzed by MTT assay and the level of an important angiogenic factor secreted by HSCs, vascular endothelial growth factor (VEGF) was evaluated by ELISA in presence of EPC-CM. For in vivo studies, rat models (n= 1 0) were prepared by identifying and ligating the bile duct. EPCs isolated from human blood, were cultured ex vivo and transplanted in treated group of BDL rats (n=5) via the tail vein, three weeks after bile duct ligation. The untreated group of rats (n= 5) received only saline. Rats were sacrificed one week after the transplantation of cells. Fibrosis was evaluated by histopathology of the liver tissues from untreated and EPC-treated rats. The expression of an important fibrogenic marker, alpha-SMA was analyzed in EPC-treated and untreated animals by western blotting. Results: HSCs co-cultured with EPC-CM showed significantly increased proliferation in comparison to that observed in HSCs alone (P< 0.05). However, VEGF levels in HSCs didn't show a significant change in presence of EPC-CM. The in vivo histopathology studies revealed an increase of fibrosis from stage 2 to stage 3 (bridging fibrosis) in EPC-treated rats as compared to the untreated rats. Also, the expression of the fibrosis marker, alpha-SMA present on activated HSCs was about 1.2 fold higher in EPC-treated rats as compared to the untreated rats (p<0.05). Conclusion: The study suggests that EPCs may contribute to liver fibrosis by enhancing the proliferation of HSCs. Further studies are underway to evaluate the association of EPC-mediated angiogenesis with liver fibrosis.

Disclosures:

The following people have nothing to disclose: Skand Gupt, Mohsin Hasan, Neha Sharma, Vaibhav Dixit, Deepti Vyas, Dinesh M. Tripathi, Savneet Kaur, Nirupma Trehanpati, Shiv K. Sarin

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Vitamin D inhibits development of liver fibrosis in animal model but cannot ameliorate established cirrhosis

Shirley Abramovitch1, Efrat Sharvit2, Yosef Weisman3, Eli Bra-zowski4, Shimon Reif1,5;
1Hadassah medical center, Jerusalem, Israel; 2Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel; 3Sourasky Medical Center, Tel-Aviv, Israel; 4Pathology, Sourasky Medical Center, Tel-Aviv, Israel; 5Faculty of Medicine, Hebrew University, Jerusalem, Israel

Background and aims: 1,25(OH)2D3, the active form of vitamin D has anti-proliferative and anti-fibrotic effect on hepatic stellate cells. Our aim was to investigate the potential of 1,25(OH)2D3 to inhibit the development of liver fibrosis and to ameliorate established fibrosis in vivo. Methods: The anti-fibrotic effect of 1,25(OH)2D3 was investigated in thioac-etamide (TAA) model (as a preventive treatment and as a remedial treatment) and in a bile duct ligation model. In the preventive model, rats received simultaneously intra-peritoneum injection of TAA and/or 1,25(OH)2D3, for 10 weeks. In the remedial model, rats were treated with TAA for 1 0 weeks and then received 1,25(OH)2D3 or saline for eight weeks. Fibrotic score was determined by Masson staining. Collagen I, α-smooth muscle actin (αSMA), tissue inhibitor of metallopro-teinase (TIMP1), platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) expression were measured by western blot analysis and real-time PCR. Hypercalemia was detected by chemistry measurements. Results: Preventive treatment of 1,25(OH)2D3 significantly suppressed liver fibrosis both macroscopically and microscopically and significantly lowered the fibrotic score of TAA+1,25(OH)2D3 group compared to the TAA group. 1,25(OH)2D3 significantly inhibited expression of PDGF and TGF-β by ∼50% and suppressed the expression of collagen Iα1, TIMP1 and αSMA by ∼3, 2, 3 fold, respectively. In contrast, 1,25(OH)2D3 was inefficient to ameliorate established liver fibrosis. Furthermore, administration of 1,25(OH)2D3 to BDL rats, led to high mortality rate probably caused by hypercalcemia. Conclusion: 1,25(OH)2D3 may be considered as a potential preventive treatment in an in-vivo model but failed to ameliorate established cirrhosis

Disclosures:

The following people have nothing to disclose: Shirley Abramovitch, Efrat Sharvit, Yosef Weisman, Eli Brazowski, Shimon Reif

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In vivo migration of BM-derived circulating angiogenic cells (CACs) to liver in chronic liver disease

Arpita Banik1,4, Savneet Kaur3, Nirupma Trehanpati1, Ashok Mukhopadhyay4, Shiv K. Sarin2;
1Research, Institute of Liver and Biliary Sciences, New Delhi, India; 2Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi, India; 3School of Biotechnology, Gautam Buddha University, Noida, India; 4Stem cell Biology Lab, National Institute of Immunology, New Delhi, India

Background: Bone marrow (BM) -derived stem cells contributes to liver fibrosis in conditions of chronic liver injury. Formation of new blood vessels during hepatic chronic wound healing may lead to progression of fibrosis to cirrhosis. Therefore, it is important to investigate the BM- derived circulating angiogenic cells (CACs) in the progression of fibrosis to cirrhosis in chronic liver diseases (CLDs). Aim: To investigate to the migration of CACs in fibrotic or cirrhotic liver during chronic liver disease. Materials and Methods: Bone marrow chimera of C57BL/6J mouse was established by intra bone marrow transplantation of GFP+ BM cells from enhanced green fluorescent protein (eGFP) -expressing [C57BL/6-Tg(UBC-GFP)30Scha/J] in lethally irradiated donor mice. Chimerism was confirmed by flow cytometry after 21 days of transplantation. Chronic liver disease model was generated by injecting carbontetrachloride (CCl4) 0.8 ml/kg intra peritoneally twice a week for 30 days while the age-matched chimeric animals received PBS (controls). . After a month, CACs mobilisation were detected by CD-34+ and FLK-1 + cells (CACs markers) in peripheral blood. Co-expression of GFP+ cells with CD-31+ was analysed in liver tissue by immunofluorescence to determine the contribution of BM-derived endothelial progenitors in vasculogenesis. Tissues were also stained with hematoxylin/eosin and Sirius red to analyse morphological changes and collagen deposition in liver. Results: After 21 days post-surgery of BM-GFP cells, the percentage of GFP+ cells (chimerism) was 69±2.3 %. Further, on CCl4 injury, liver tissue showed significant fibrosis with increased hepatic inflammation, necrosis and collagen deposition with bridge formation. Ishak scoring of 1-2 was observed on day 14 and 3-4 was observed on day 25. After one and two weeks of CCl4 injury, percentage of GFP+ cells increased from 69±2.3 % to 82±1.9 % and 94.35±3.1 % in the blood respectively. Flk-1 +/CD34+ cells in blood were also increased from 0.02±0.01 % to 0.2±0.04 % and 0.24±0.01% after 1 and 2 weeks of injury. Immunofluroscence of the liver sections showed co-localization of CD-31+/GFP+ cells indicating the mobilization of CACs from BM to the liver. Conclusion: Our result shows the migration of CD-31+/GFP+ CACs from bone marrow to liver during fibrosis. The CACs may contribute to vascular repair and are capable of accelerating the recovery of liver injury. Further studies are needed to define the CACs role in arresting or reverencing the fibrosis

Disclosures:

The following people have nothing to disclose: Arpita Banik, Savneet Kaur, Nirupma Trehanpati, Ashok Mukhopadhyay, Shiv K. Sarin

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DSS-induced Chronic Colitis Aggravates Inflammation and Fibrogenesis in Mice with CCl4-induced hepatic Fibrosis

Xiaolan Zhang, Yufeng Liu, Libo Zheng, Guochao Niu, Hong Zhang, Jinbo Guo, Guozun Zhang, Huicong Sun;
Dept of Gas-troentology, The Second Hospital of Hebei Medical University, Shi-jiazhuang City, China

Background: Inflammatory bowel disease (IBD) is found to be associated with several kinds of liver disease. The purpose of this study is to investigate the role of combination with dextran sodium sulfate (DSS) in hepatitis and fibrosis in mice treated by with CCl4. Methods: Male C57BL/6 mice were grouped as follows: Control group (n=1 0), DSS group (n=1 0), Olive oil group (n=10), CCl4 group (n=10) and CCl4+DSS group (n=10). Severity of colitis was evaluated by disease activity index (DAI), colon length, colon pathology score, myeloperoxidase (MPO) and histopathology. Haematoxylin and eosin (H&E) staining, Sirius red staining and Masson's trichrome (MT) staining were used to detect liver histopathological changes. Pro-inflammatory cytokines in both colon and liver tissues including TNF-α, IFN-γ and IL-17A were detected by immunohistochemical staining, western blot and real-time Q-PCR, respectively. The protein and mRNA expressions of TGF-β1, α-SMA, collagen I, collagen III, MMP-2 and TIMP-2 in liver tissues were observed by immunohistochemical staining, western blot and real-time Q-PCR, respectively. Results: DSS treatment led to increased BW loss, higher DAI score, shortened colon length, elevated MPO activity, and worsened histologic inflammation in colon. Moreover, TNF-α, IFN-γ and IL— 1 7A expressions in both colon and liver tissues were all enhanced in DSS group. Hepatitis was also found in DSS group as well as CCl4 group and CCl4+DSS group by histological analysis. However, comparing with CCl4 group, hepatitis in CCl4+DSS were more severe, reflected by histology and pro-inflammation cytokines expressions. Increased hepatic fibrosis was observed by Sirius red staining and MT staining, as well as higher levels TGF-β1, α-SMA, collagen I, collagen type III, TIMP-2 and lower level of MMP-2 in liver tissue. Conclusions: The DSS-induced mouse colitis may promote hepatic inflammation and fibrosis in mice treated by CCl4.

Disclosures:

The following people have nothing to disclose: Xiaolan Zhang, Yufeng Liu, Libo Zheng, Guochao Niu, Hong Zhang, Jinbo Guo, Guozun Zhang, Huicong Sun