- Email: [email protected]
403 EXPRESSION AND FUNCTION OF THE ATYPICAL CADHERIN FAT1 IN HEPATIC FIBROSIS
POSTERS 401 CATHEPSIN B OVEREXPRESSION DUE TO ACID SPHINGOMYELINASE ABLATION PROMOTES LIVER FIBROSIS IN NIEMANN PICK DISEASE N. Tarrats1 , A. Moles1 , J.C. Fernandez-Checa1,2 , M. Mari1 . 1 Instituto Investigaciones Biomedicas Barcelona, CSIC, Liver Unit, Hospital Clinic, IDIBAPS, Ciberehd, Barcelona, Spain; 2 Southern California Research Center for ALPD, Keck School of Medicine, Los Angeles, CA, USA E-mail: [email protected] Niemann Pick disease (NPD) is a lysosomal storage disease caused by the loss of acid sphingomyelinase (ASMase) that features neurodegeneration and liver disease. Since ASMase knockout mice models NPD and our previous findings revealed that ASMase activates cathepsins B/D (CtsB/D), our aim was to investigate the expression and processing of CtsB/D in hepatic stellate cells (HSC) from ASMase null mice and their role in liver fibrosis. Surprisingly, HSC from ASMase knockout mice exhibit increased basal level and activity of CtsB as well as its in vitro processing in culture, paralleling the enhanced expression of fibrogenic markers a-SMA (alpha-smooth muscle actin), TGF-b and Col1A1 (pro-collagena1(I)). Moreover, pharmacological inhibition of CtsB blunted the expression of a-SMA, Col1A1 and proliferation of HSC cells from ASMase knockout mice. Consistent with the enhanced activation of CtsB in HSC from ASMase null mice, the in vivo liver fibrosis induced by chronic treatment with CCl4 increased in ASMase null compared to wild-type mice, an effect that was reduced upon CtsB inhibition. In addition to liver, the enhanced proteolytic processing of CtsB was also observed in brain and lung of ASMase knockout mice, suggesting that the overexpression of CtsB may underlie the phenotype of NPD. In conclusion, these findings reveal a functional relationship between ASMase and CtsB and that the ablation of ASMase leads to the enhanced processing and activation of CtsB. Therefore, targeting CtsB may be of relevance in the treatment of liver fibrosis in patients with NPD. 402 A ROLE FOR AUTOPHAGY DURING HEPATIC STELLATE CELL ACTIVATION L.F.R. Thoen1 , E.L.M. Guimar˜aes1 , L. Dolle´ 1 , I. Mannaerts1 , M. Najimi2 , E. Sokal2 , L.A. van Grunsven1 . 1 Vrije Universiteit Brussel, 2 Universit´e Catholique de Louvain, Brussel, Belgium E-mail: [email protected] Background and Aims: Autophagy is a metabolic process by which cells degrade and metabolize own constituents, e.g. intracellular organelles and proteins, with many connections to human disease and physiology. We studied the role of autophagy in hepatic stellate cell (HSC) activation, a key event in liver fibrogenesis. Methods: Analysis of the autophagic flux during in vitro activation of primary mouse HSCs was performed using a DsRed-GFP-LC3B encoding plasmid. The effect of autophagy inhibition by bafilomycin A1 on the in vitro activation process of human and mouse HSCs was examined by measuring proliferation, presence of activation markers by RT-qPCR, immunofluorescence and western blotting. Analysis of lipid droplet and microtubule-associated protein light chain 3 beta (LC3B) colocalization in the presence of PDGF-BB was investigated by immunocytochemistry. Results: A significant increased autophagic flux was observed during culture-induced mouse HSC activation. Treatment with autophagy inhibitor bafilomycin A1 results in a significant decreased proliferation and expression of activation markers in mouse and human HSCs. In addition, a significant increased amount of large lipid droplets was observed in bafilomycin A1-treated mouse HSCs. Besides this, lipid droplet and LC3B colocalization was increased after PDGF-BB treatment in quiescent HSCs. Conclusions: During HSC activation, autophagic flux is increased. The demonstration of partly inhibition of in vitro HSC activation
after treatment with an autophagy inhibitor unveils a potential new therapeutic strategy for liver fibrosis. The observation of LC3B and lipid droplet colocalization in quiescent HSCs after PDGFBB treatment and the increased amount of large lipid droplets after seven days of bafilomycin A1 treatment suggests a role for autophagy in lipid droplet metabolism. Additional experiments that address this role for autophagy in HSCs will be presented. 403 EXPRESSION AND FUNCTION OF THE ATYPICAL CADHERIN FAT1 IN HEPATIC FIBROSIS D. Valletta1 , B. Czech1 , M. Saugspier1 , C. Dorn1 , A. Bosserhoff2 , C. Hellerbrand1 . 1 University Hospital Regensburg, 2 University Regensburg, Regensburg, Germany E-mail: [email protected] The activation of hepatic stellate cells (HSCs) is a key event of hepatic fibrosis, and previous studies found that cadherin expression is altered in activated HSCs. Fat1 is an atypical cadherin of more than 500kDa. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development The aim of this study: Was to assess the expression and function of Fat1 in liver fibrosis. Methods and Results: Quantitative PCR, Western Blotting and immunohistochemical analysis revealed that hepatic Fat1 expression was increased in different murine models of chronic liver injury (bile duct ligation, chronic CCl4 treatment and two dietary models of non-alcoholic fatty liver disease (NAFLD)), as well as in patients with liver cirrhosis of different origin and patients with NAFLD of different degree. The increased FAT1 expression paralleled fibrosis progression, and quantitative PCR and Western blotting revealed increased Fat1 expression in HSCs during in vitro activation. Hypoxia and formation of free oxygen radicals (ROS) further increased FAT1 expression, while ROS-scavengers inhibited both basal as well as hypoxia induced FAT1 expression in activated HSCs. The half life of Fat1 protein in activated HSCs was determined to be more than 7 days but transfection with FAT1 siRNA led to an almost complete suppression of Fat1 in activated HSC after 14 days. Activated HSCs with with Fat1 suppression revealed significantly lower activity of the transcription factor NFkappaB, and FAT1 downregulation in activated HSCs significantly reduced resistance towards apoptosis as assessed by FACS and analysis of caspase-3 activity. Conclusion: These findings indicate that increased FAT1 expression in activated HSCs functionally affects fibrosis. Interfering with (the mechanisms leading to) FAT1 induction in HSCs appears as novel therapeutic strategy to prevent the progression of chronic liver disease. 404 CULTURING ALTERNATIVELY ACTIVATED MACROPHAGES ONTO TYPE III COLLAGEN CAUSES THE DEVELOPMENT OF AN INTERMEDIATE CLASSIC/ALTERNATIVE PHENOTYPE M. van Deemter, P. Ramachandran, A. Pellicoro, T.T. Gordon-Walker, J. Schrader, J.P. Iredale. MRC/Centre for Inflammation Research, Queen’s Medical Research Institute, Edinburgh, UK E-mail: [email protected] Background and Aims: In general, alternatively activated (AA) macrophages are assumed to be profibrotic and classically activated (CA) macrophages proinflammatory. However, a range of intermediate forms exists. Through extracellular matrix (ECM) binding receptors such as integrins, cells can distinguish between different ECM proteins and adapt their phenotype accordingly. Our aim was to determine changes in the phenotype of AA macrophages after plating them onto different ECM proteins, and the pathways involved therein. Methods: CA or AA murine macrophages were plated into culture plates coated with type I collagen or type III collagen, and harvested
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after treatment with an autophagy inhibitor unveils a potential new therapeutic strategy for liver fibrosis. The observation of LC3B and lipid droplet colocalization in quiescent HSCs after PDGFBB treatment and the increased amount of large lipid droplets after seven days of bafilomycin A1 treatment suggests a role for autophagy in lipid droplet metabolism. Additional experiments that address this role for autophagy in HSCs will be presented. 403 EXPRESSION AND FUNCTION OF THE ATYPICAL CADHERIN FAT1 IN HEPATIC FIBROSIS D. Valletta1 , B. Czech1 , M. Saugspier1 , C. Dorn1 , A. Bosserhoff2 , C. Hellerbrand1 . 1 University Hospital Regensburg, 2 University Regensburg, Regensburg, Germany E-mail: [email protected] The activation of hepatic stellate cells (HSCs) is a key event of hepatic fibrosis, and previous studies found that cadherin expression is altered in activated HSCs. Fat1 is an atypical cadherin of more than 500kDa. It engages in several functions, including cell polarity and migration, and in Hippo signaling during development The aim of this study: Was to assess the expression and function of Fat1 in liver fibrosis. Methods and Results: Quantitative PCR, Western Blotting and immunohistochemical analysis revealed that hepatic Fat1 expression was increased in different murine models of chronic liver injury (bile duct ligation, chronic CCl4 treatment and two dietary models of non-alcoholic fatty liver disease (NAFLD)), as well as in patients with liver cirrhosis of different origin and patients with NAFLD of different degree. The increased FAT1 expression paralleled fibrosis progression, and quantitative PCR and Western blotting revealed increased Fat1 expression in HSCs during in vitro activation. Hypoxia and formation of free oxygen radicals (ROS) further increased FAT1 expression, while ROS-scavengers inhibited both basal as well as hypoxia induced FAT1 expression in activated HSCs. The half life of Fat1 protein in activated HSCs was determined to be more than 7 days but transfection with FAT1 siRNA led to an almost complete suppression of Fat1 in activated HSC after 14 days. Activated HSCs with with Fat1 suppression revealed significantly lower activity of the transcription factor NFkappaB, and FAT1 downregulation in activated HSCs significantly reduced resistance towards apoptosis as assessed by FACS and analysis of caspase-3 activity. Conclusion: These findings indicate that increased FAT1 expression in activated HSCs functionally affects fibrosis. Interfering with (the mechanisms leading to) FAT1 induction in HSCs appears as novel therapeutic strategy to prevent the progression of chronic liver disease. 404 CULTURING ALTERNATIVELY ACTIVATED MACROPHAGES ONTO TYPE III COLLAGEN CAUSES THE DEVELOPMENT OF AN INTERMEDIATE CLASSIC/ALTERNATIVE PHENOTYPE M. van Deemter, P. Ramachandran, A. Pellicoro, T.T. Gordon-Walker, J. Schrader, J.P. Iredale. MRC/Centre for Inflammation Research, Queen’s Medical Research Institute, Edinburgh, UK E-mail: [email protected] Background and Aims: In general, alternatively activated (AA) macrophages are assumed to be profibrotic and classically activated (CA) macrophages proinflammatory. However, a range of intermediate forms exists. Through extracellular matrix (ECM) binding receptors such as integrins, cells can distinguish between different ECM proteins and adapt their phenotype accordingly. Our aim was to determine changes in the phenotype of AA macrophages after plating them onto different ECM proteins, and the pathways involved therein. Methods: CA or AA murine macrophages were plated into culture plates coated with type I collagen or type III collagen, and harvested
Journal of Hepatology 2012 vol. 56 | S71–S224
S161