POSTERS study demonstrates a role for mechano-sensitive signalling by the transcriptional regulators Yes associated protein (YAP) and WW domain-containing transcription regulator protein 1 (TAZ) during HSC activation. Methods: Expression was assayed by qPCR, immunoblot and immunocytochemistry of primary HSCs. Acrylamide gels were used to model altered matrix stiffness. Chromatin immunoprecipitation (ChIP) identified YAP binding sites. Results: During activation of HSCs, expression of Yap, Taz and their DNA binding partner Tead was increased. Yap was localised to the nucleus of HSCs positive for profibrotic markers alpha-smooth muscle actin, collagen 1 and Sox9. In contrast, HSCs cultured on soft substrates appeared quiescent-like (characterised by autofluorescence) with inactive Yap sequestered in the cytoplasm. Abrogation of Yap and Taz using siRNA reduced fibrotic gene expression. Hippo signalling (an alternative mechanism regulating Yap/Taz activation) via Lats1 and Lats2 has no effect on fibrotic gene expression in HSCs, confirming a role for mechano-transduction via Yap/Taz in HSC activation in vitro. Moreover, chemical inhibition of the Tead-Yap interaction caused reduced expression of Yap targets (Gli2, Ctgf ) and fibrotic markers (collagen, Sox9). ChIP identified Sox9 as a novel downstream Yap target. Conclusions: These data indicate a functional role for mechano- transduction by Yap/Taz during HSC activation. Capitalizing on this finding, particularly the ability to pharmacologically disrupt Yap target gene interaction may provide insight into novel therapeutic strategies. P618 INFLAMMATORY RESPONSE OF PRIMARY MOUSE HEPATIC STELLATE CELLS TO LIPOPOLYSACCHARIDE IN VITRO R. Liebe1, K. Breitkopf-Heinlein1, U. Albrecht2, M. Thomas3, V. Costina1, P. Findeisen1, J. Bode2, S. Dooley1. 1Heidelberg University, Medical Faculty Mannheim, Mannheim, 2Uniklinikum Du¨sseldorf, Du¨sseldorf, 3IKP Stuttgart, Stuttgart, Germany E-mail:
[email protected] Background and Aims: Bacterial lipopolysaccharide mediates the acute inflammatory response upon infection. In liver, Kupffer cells (KC) and hepatic stellate cells (HSC) express the (LPS) receptor TLR4 and are the central providers of the “cytokine storm” for the hepatocyte acute phase response (APR). Our aim was to delineate in greater detail the crosstalk of HSC with hepatocytes in this setting. Methods: We treated cultured HSC and mice with LPS and quantified gene expression of each 45 inflammatory and fibrogenic candidate genes by qPCR. Cytokine release of LPS treated HSC into the culture supernatant was assessed by ELISA. Total proteome changes in LPS treated HSC were characterised by MALDI-TOF. Results: HSC showed a substantial increase in the expression of inflammatory markers upon LPS-stimulation, with 15 of 45 inflammatory markers upregulated, particularly Cxcl2, Ccl2, IL10 and IL-1beta. The fibrogenic response was comparatively weak with only 7/45 genes significantly upregulated. Analysis of secreted inflammatory mediators by ELISA showed a fulminant increase by LPS, particularly for Ccl2, TNF-alpha and IL-1beta. Proteome analysis in LPS-treated HSC revealed pronounced metabolic alterations (e.g. Fructose-bisphosphate aldolase A, Hexokinase-2), substantial increases in excreted proteins (CTGF, PAI1, Thromobospondin) and Integrins, and fulminant downregulation of various Keratin and Actin subspecies. Conclusions: Isolated HSC (without contact to KC) show a strong and rapid pro-inflammatory rather than pro-fibrogenic response following LPS challenge that is accompanied (and possibly enabled) by substantial changes in metabolism and cellular scaffold and adhesion molecules. We hypothesize the response of HSC to LPS is critical for KC activation and hepatocyte APR. P619 INHIBITION OF FIBRONECTIN DEPOSITION IMPROVES EXPERIMENTAL LIVER FIBROSIS E. Altrock1,2, C. Sens1,2, N. Kawelke1,2, S. Dooley3, J. Sottile4, I.A. Nakchbandi1,2. 1Max-Planck Institute for Biochemistry, Martinsried, 2Institute for Immunology, University of Heidelberg, Heidelberg, 3Department of Medicine I, University of Heidelberg at Mannheim, Mannheim, Germany; 4Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States E-mail:
[email protected] Background and Aims: Common pathogenic steps in liver fibrosis are inflammation and accumulation of extracellular matrix proteins including collagen, which lead to disruption of tissue microarchitecture and liver dysfunction. Adequate fibronectin fibril formation is required for collagen matrix deposition in several cell types in vitro. We therefore hypothesized that preventing fibronectin fibril assembly will result in decreased collagen matrix accumulation, and hence diminish liver injury associated with fibrosis. Methods: In vitro studies on hepatic stellate cells confirmed that the fibronectin assembly inhibitor, pUR4 suppresses accumulation of both fibronectin and collagen in the extracellular matrix, but does not affect hepatic stellate cell activation, collagen mRNA expression, and availability or activity of TGF-b. Induction of fibrosis over 4 weeks using DMN was therefore combined with treatment with pUR4. Results: pUR4 normalized the amount of fibrotic tissue that accumulated with injury, and improved liver function. Testing pUR4 in an alternative fibrosis model by administering carbon tetrachloride (CCl4) confirmed the effect. Specifically, pUR4 treatment decreased collagen and fibronectin matrix accumulation. Most interestingly, we did not detect any changes in Kupffer (F4/80+) or a-smooth muscle actin expressing hepatic stellate cell numbers. Further, there was no impact on mRNA expression of TGF-b or TNF-a. Conclusions: In summary, a peptide that blocks fibronectin deposition results in decreased collagen accumulation and improved liver function during liver fibrogenesis. Thus, fibronectin matrix modulation offers a therapeutic benefit in preclinical models of liver fibrosis. P620 THE AMPK-RELATED KINASE, Nuak2 IS MODULATED BY THE ACTIVATION PROCESS AND REGULATES MOTILITY OF HEPATIC STELLATE CELLS (HSC) C. Tosti-Guerra1, A. Caligiuri1, A. Provenzano1, K. Rombouts2, C. Bertolani1, M. Pinzani2, F. Marra1. 1University of Florence Dipartimento di Medicina Interna, Florence, Italy; 2Institute of Digestive and Liver Health, UCL, London, United Kingdom E-mail: fabio.marra@unifi.it Background and Aims: AMPK related kinases (ARKs) are energy sensors and controllers of cellular structure. Nuak2 is a novel member of this family, and has different effects on cell motility and cytoskeletal organization depending on the cell types. AMPK is expressed in HSC, and negatively modulates their profibrogenic features. However no information is available on the involvement of Nuak2 during HSC activation or in the biology of these cells. Aim of this study was to investigate whether Nuak2 is expressed in HSC and its possible involvement in HSC activation and functions. Methods: HSC were isolated from normal rat liver and activated by culture on plastic in the presence or absence of AICAR, an AMPK activator. Knockdown of Nuak2 was achieved by siRNA. Cell migration was evaluated in modified Boyden Chambers. Results: Incubation with AICAR, an activator of ARKs, blocked the activation process of primary HSC and caused disruption of the Journal of Hepatology 2014 vol. 60 | S215–S359 S275 POSTERS actin cytoskeleton, resulting in altered cell adhesion and spreading, but without effects on viability. HSC were found to express the alpha1 and the alpha2 subunits of AMPK, but also the Nuak2, a recently characterized ARK. Expression of AMPK subunits and of Nuak2 was high in freshly isolated HSC and decreased during trans- activation to a myofibroblastic phenotype. In fully activated HSC, down-regulation of Nuak2 was associated with a marked increase in cell migration, together with changes in the expression of several molecules implicated in cytoskeletal organization. Conclusions: ARKs, including Nuak2, are modulated during the activation process of HSC and regulate cytoskeletal organization and cell motility. P621 INTEGRIN b1 REGULATES HEPATIC STELLATE CELL ACTIVATION VIA THE ACTIN MYOSIN CYTOSKELETON K. Martin, J. Pritchett, E. Harvey, V. Athwal, J. Llewellyn, C. Streuli, L. Zeef, N. Hanley, K. Piper Hanley. University of Manchester, Manchester, United Kingdom E-mail:
[email protected] Background and Aims: Liver fibrosis is characterised by excessive extracellular matrix deposition from hepatic stellate cells (HSCs) as they become activated in response to injury. The integrin receptor system is key to ECM mediated signalling. This study identified integrin b1 (Itgb1) playing a central role in HSC activation through regulating contractile actin cytoskeletal proteins relevant to mechanosensitive signalling via yes associated protein 1 (Yap1). Methods: HSCs were culture activated on plastic. Expression was assayed by qPCR, immunoblotting and immunocytochemistry. Cell migration, contraction and proliferation were assayed. Expression microarray was analysed with limitations of p < 0.05 and fold change >1.5. CreER-loxP and siRNA were used for gene knockout/down. Gene regulation was analysed by chromatin immunoprecipitation and luciferase assay. Results: In activated HSCs, Itgb1 was upregulated similar to profibrotic markers Sox9, type I collagen (ColI) and a-smooth muscle actin (a-Sma). Genetic deletion of Itgb1 resulted in a more quiescent phenotype; with reduced migration, contraction, proliferation and expression of profibrotic factors. Expression microarray highlighted myosin regulatory light chain 9 (Myl9) and Yap1, as downstream effectors of Itgb1 signalling. Inactive Yap1 was sequestered in the cytoplasm in Itgb1-null HSCs, in contrast to active nuclear localisation in control HSCs. Moreover, Yap1 was capable of binding to, and regulating, Myl9 in activated HSCs. Conclusions: These data suggest Itgb1 is an important regulator of HSC activation at the heart of intrinsic cytoskeleton signalling with implications for increased migration and contractility in liver fibrosis. Understanding Itgb1 signalling may reveal new therapeutic targets for the treatment of liver fibrosis. P622 MODELLING HCV REPLICATION IN AN INFLAMED LIVER ENVIRONMENT: A NEW ROLE FOR STROMAL EXPRESSED VAP-1 TO REGULATE VIRAL REPLICATION S.K. Galsinh1, C. Weston2, A. Barnes1, I.A. Rowe1,2, G. Muirhead2, D. Smith3, D. Adams2, C. Buckley4, B. Peter1, J.A. McKeating1,2. 1Centre for Human Virology, School of Immunity and Infection, 2NIHR Centre for Liver Disease, Birmingham University, Birmingham, United Kingdom; 3Biotie Therapies Corp., Turku, Finland; 4Centre for Translational Inflammation Research, School of Immunity and Infection, Birmingham University, Birmingham, United Kingdom E-mail:
[email protected] Background and Aims: Hepatitis C virus (HCV) is a major cause of global morbidity and mortality. An estimated 170 million individuals are infected worldwide and a significant proportion will develop cirrhosis and hepatocellular carcinoma. While hepatocytes are the major site of viral replication the role of non-parenchymal cells in the viral lifecycle is limited. Methods: Stroma or myofibroblasts define the microenvironment of the liver and given their close proximity to hepatocytes in vivo, we established co-culture systems to study their role in the HCV lifecycle. Results: Liver myofibroblasts inhibit HCV replication in adjacent hepatocytes in a cell-contact dependent manner by limiting genome replication and particle entry. Importantly, this observation is not restricted to HCV and lentiviral pseudotypes expressing murine leukemia virus glycoproteins show limited infection of fibroblast-hepatocyte co-cultures, suggesting a general perturbation of membrane protein dynamics. Vascular adhesion protein-1 (VAP-1), a primary amine oxidase prevalent in the diseased liver, has been implicated in the establishment and persistence of hepatitis. Activated liver myofibroblasts express VAP-1 and we therefore evaluated the effect of recombinant VAP1 (rVAP-1) on HCV replication. We demonstrate that rVAP-1 induces a dose- dependent inhibition of HCV entry and replication, identifying a new paracrine pathway for myofibroblasts to regulate HCV replication. Data will be presented addressing the mechanism of action of VAP-1 in the HCV lifecycle. Conclusions: These observations uncover a new role for VAP-1 to regulate HCV replication and provide new therapeutic avenues for treating both the underlying inflammatory response and viral replication in chronic hepatitis. P623 NCU-G1GT /GT MICE: A LONG-LIVED MODEL FOR LIVER FIBROSIS X.Y. Kong1, C.K. Nesset1, C. Schjalm1, E.M. Løberg2, A.C. Rustan3, G.H. Thoresen3, E.T. Kase3, W. Eskild1. 1Department of Biosciences, University of Oslo, 2Department of Neuroradiology, Oslo University Hospital Ullevaal, 3Department of Pharmaceutical Biosciences, University of Oslo, Oslo, Norway E-mail:
[email protected] Background and Aims: Liver fibrosis is a result of sustained injury to the liver. Although etiologies vary considerably, the progression and manifestations of this condition are very similar [1]. NCU- G1, an uncharacterized protein, was initially described as a nuclear protein [2], but later identified as a lysosomal protein [3]. Dysfunction of lysosomal proteins may lead to a number of genetic diseases (4). The phenotypic consequences vary, but may include chronic liver damage [4]. Recently, we created and characterized a viable mouse model without any detectable expression of NCU-G1 [5]. The predominant phenotype is spontaneous liver injury, which progresses into fibrosis within 1 month of age. The condition progresses slowly, and these animals have been kept till the age of 18 months. Most established fibrosis models require external provocations, and the number of transgenic models is limited. Here we present a novel transgenic mouse model, which spontaneously develops liver fibrosis. Methods: Liver sections were analyzed microscopically after H/E-staining, AFOG-staining and a-SMA-labelling. Excess collagen depositions were confirmed by Sirius-red quantification. Gene expression of fibrosis related genes was assessed by qPCR and gelatine-zymography. Results: Analyses of fibrotic livers show inflammation, activated HSCs and increased depositions of fibrous collagen. Elevated expression of the fibrosis inducer TGF-b and several other fibrogenic genes indicates an ongoing fibrogenesis. Conclusions: Our novel mouse model displays all hallmarks of fibrosis, and could become a potential new model for studying this disease. Reference(s) [1] Friedman (2008) [2] Steffensen et al. (2007) S276 Journal of Hepatology 2014 vol. 60 | S215–S359