Delta-Like Homolog 1 (DLK1), Profibrogenic Protein Overexpressed in CCL4 Rat Liver Fibrosis

POSTER PRESENTATIONS including EPCR, glycosaminoglycans and TFPI. The aim of this study was to revisit the coagulation function of patients with cirrhosis using an assay taking into account endothelial cells. Methods: We included 64 patients with cirrhosis (Child-Pugh A, n = 22; B, n = 19; C, n = 23) and 30 healthy controls. The cause of cirrhosis was excessive alcohol consumption (n = 26), hepatitis C (n = 20), NASH (n = 8), hepatitis B (n = 4), and other (n = 6). We performed thrombin generation assay using 1 pM of tissue factor, 4 μM of phospholipids, with or without thrombomodulin (2 nM). We also assessed thrombin generation on cultured endothelial cells (HUVECs) (5 pM of tissue factor; 4 of μM phospholipids). We measured thrombomodulin and EPCR expression by HUVECs incubated with the plasma of patients or controls using flow cytometry. Results: In the absence of endothelial cells, endogenous thrombin potential (ETP) was similar in patients and in controls (1062 vs. 1081 nM.min, respectively; p = 0.96). Velocity was higher in patients than in controls (38 vs. 21 nM/min; p < 0.001). In the presence of thrombomodulin, patients had higher ETP (768 vs. 295 nM/min; p < 0.001) and velocity (36 vs. 14 nM/min; p < 0.001) than controls. The ratio of ETP without and with thrombomodulin was higher in patients than in controls (0.7 vs. 0.3; p < 0.001). However, in the presence of HUVECs, ETP was lower in patients than in controls (1182 vs. 1513 nM.min; p < 0.001), but velocity was higher (95 vs. 62 nM/ min; p = 0.001). Thrombomodulin and EPCR expression by HUVECs was not affected by the incubation with the plasma of patients as compared to controls. Conclusions: When endothelial cells are present, the plasma of patients with cirrhosis induces less thrombin generation than the plasma of controls. Yet, thrombin is quickly generated. These results show that the coagulation changes associated with cirrhosis are complex and are not recapitulated by the resistance to thrombomodulin. FRI-022 KINETICS OF PULMONARY ANGIOGENESIS IN MOUSE COMMON BILE DUCT LIGATION-INDUCED LIVER FIBROSIS S. Raevens1, L. Devisscher1, A. Paridaens1, E. Bogaerts1, S. Lefere1, Y.-P. Vandewynckel1, C. Casteleyn2, K. Bracke3, T. Maes3, X. Verhelst1, H. Van Vlierberghe1, C. Van Steenkiste1, A. Geerts1, I. Colle1. 1 Hepatology and Gastroenterology, Ghent University, Ghent; 2Laboratory of Applied Veterinary Morphology, Department of Veterinary Sciences, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Wilrijk; 3Laboratory for Translational Research in Obstructive Pulmonary Diseases, Department of Respiratory Medicine, Ghent University Hospital, Ghent, Belgium E-mail: [email protected] Background and Aims: Hepatopulmonary syndrome (HPS) is a severe pulmonary complication of liver disease for which no medical treatment is available. In rats, common bile duct ligation (CBDL) has been documented as a model for human HPS, which is characterized by pathological pulmonary angiogenesis. Studies in genetically modified mice could offer opportunities for further research, however, in this species the development of pulmonary angiogenesis in biliary cirrhosis has not been outlined yet. We aimed to elucidate the temporal changes in proangiogenic signature of hepatic and pulmonary vasculature after CBDL in mice and in addition identify potential proangiogenic factors contributing to the pathogenesis of HPS. Methods: Male Swiss mice underwent CBDL or sham surgery and were sacrificed at a weekly basis for 6 consecutive weeks. Pulmonary inflammation was studied by cytology on broncho-alveolar lavage fluid, myeloperoxidase assay and luminex bead based assay. Liver and lungs were collected for protein analysis and histology to assess liver fibrosis and hepatic and pulmonary angiogenesis. Scanning electron microscopy was performed on vascular corrosion casts to visualize pulmonary vasculature during cirrhosis ex vivo.

Results: CBDL progressively induced liver fibrosis from week 1 (F0-1) to 6 (F4). This was accompanied by a gradual increase in hepatic immunopositivity for Endoglin and von Willebrand Factor, two markers of endothelial cell activation ( p < 0.0001). Hepatic levels of vascular endothelial growth factor (VEGF), VEGF receptor 1 and 2 were significantly increased at week 6, whereas placental growth factor (PlGF), which is exclusively involved in pathological angiogenesis, was already upregulated at week 2 ( p < 0.0001). In the pulmonary compartment, CBDL resulted in neutrophil infiltration and increased pro-inflammatory mediators from week 2 to 6 (all p < 0.001). Pulmonary immunoreactivity for Endoglin and von Willebrand Factor progressively increased from week 4 to 6, while PlGF was already increased from week 2 onwards (all p < 0.0001). Scanning electron microscopy revealed regions of abnormal vascular architecture, mainly located at the pleural side, decreased intercapillary distance ( p < 0.001) and increased capillary density ( p < 0.05) in lungs of cirrhotic mice. Conclusions: CBDL in mice is associated with pathological pulmonary angiogenesis and may represent a model for human HPS. In addition, we point to PlGF as an early indicator of pathological hepatic and pulmonary angiogenesis. FRI-023 DELTA-LIKE HOMOLOG 1 (DLK1), PROFIBROGENIC PROTEIN OVEREXPRESSED IN CCL4 RAT LIVER FIBROSIS S. Carvajal1, V. Reichenbach1, D. Oró1, S. Marfà1, G. Fernández-Varo1,2, S. Lamas3, W. Jiménez1,2. 1Service of Biochemistery and Molecular Genetics, Hospital Clinic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Institut ̀ iques August Pi i Sunyer (IDIBAPS); 2Department d’Investigacions Biomed of Physiological Sciences I, University of Barcelona, Barcelona; 3 Laboratorio Mixto Consejo Superior de Investigaciones Científicas (CSIC)-Fundación Renal Iñigo Alvarez de Toledo, Centro de Biología Molecular Severo Ochoa, Madrid, Spain E-mail: [email protected] Background and Aims: Delta-like homolog 1 (DLK1) is a transmembrane protein that can be cleaved by ADAM-17 and released as a soluble protein. Recent investigations provide evidences that the soluble fragment of DLK1 protein acts as a contributor to liver fibrosis through promoting activation of hepatic stellate cells. However, little is known about the relationship between DLK1 expression and fibrosis progression in liver disease. Methods: 50 Wistar rats were included in the study. 35 rats were induced to fibrosis by CCl4 inhalation for 16–30 weeks to obtain animals with different levels of liver fibrosis, and 15 healthy rats were used as control. Serum samples were obtained and tissue samples were also collected from liver, heart, brain, kidney, lung, spleen and aorta. Standard renal and liver function test and systemic hemodynamics were assessed in all animals. Liver fibrosis was determined by Sirius Red staining. Gene and protein expression of DLK1 and other profibrogenic factors were analyzed by RT-PCR and Western Blot, respectively. Additionally, soluble DLK1 serum levels were measured by ELISA. Results: Rats were classified in 4 groups according to liver fibrosis content as: control rats, mild fibrotic rats, severe fibrotic rats and cirrhotic rats. Hemodynamic and biochemical parameters confirmed an increased loss of hepatic function as liver fibrosis evolves. A progressive increment of DLK1 gene expression was observed in liver, raising 90 fold in cirrhotic livers compared to controls. This was an organ specific phenomenon, since no changes were observed in DLK1 expression in the other organs analyzed. It was also observed an increase of DLK1 protein in liver correlated with the fibrosis stage, showing also a positive correlation with protein amount of profibrogenic factors (AII and TGF-β). Furthermore, serum DLK1 levels were found increased in fibrotic and cirrhotic rats compared to control animals, demonstrating a relationship between liver fibrosis intensity and serum DLK1 levels.

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POSTER PRESENTATIONS Conclusions: These results show that hepatic DLK1 expression is closely related with fibrosis severity. Its overexpression give raise to an increase of DLK1 soluble form that is known to act as a profibrogenic factor during the liver fibrosis process. FRI-024 SPLENECTOMY AMPLIFY THE THERAPEUTIC POTENTIAL OF ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELLS VIA STROMAL CELL-DERIVED FACTOR-1 AND HEPATOCYTE GROWTH FACTOR T. Wei-Ping1, A. Tomohiko1, H. Makoto1. 1Department of Disaster and Emergency Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan E-mail: [email protected] Background and Aims: Clinical studies suggest that splenectomy improves liver function in liver cirrhotic patients, but the influence of splenectomy on stem cell infusion is poorly understood. The present work investigated the influence of splenectomy on cell infusion and elucidated the possible mechanism. Methods: Syngeneic Adipose tissue-Derived mesenchymal Stem Cells (ADSCs) were infused into carbon tetrachloride-induced liver cirrhosis rats received with or without splenectomy, followed by assessment of the in vivo distribution of ADSCs and pathological changes 5 days later. Stromal cell-Derived Factor-1 (SDF-1) and Hepatocyte Growth Factor (HGF) expression were investigated in splenectomized liver cirrhotic patients and rats. A direct co-culture model was employed to clarify the relationship among ADSCs, hepatocytes, and Hepatic Stellate Cells (HSCs).

Results: Splenectomy prior to ADSCs infusion improved liver function and suppressed fibrosis progression more efficiently than ADSCs infusion alone in experimental cirrhosis model (Serum albumin: 4.68 ± 0.3 vs. 3.6 ± 0.4 g/dL. Alanine aminotransferase: 98.6 ± 24.6 vs. 208 ± 54.4 U/L; liver fibrosis area: 4.4 ± 0.3 vs. 7.8 ± 0.7% as compared with ADSCs infusion alone group, p < 0.05). Moreover, bio-imaging analysis revealed that more ADSCs recruit into liver in splenectomy group. Double Immunofluorescence staining confirmed that ADSCs secrete HGF efficiency in liver, the infused ADSCs integrated into fibrosis septa and participated in regeneration more efficiently in splenectomized rats. More importantly, serum SDF-1 and HGF levels after splenectomy were increased in liver cirrhotic patients (SDF-1 concentration: 2244.9 ± 235.1 pre-splenectomy vs. 2877.9 ± 371.9 pg/mL post-splenectomy; HGF concentration: 2296.1 ± 378.9 pre-splenectomy vs. 3242.9 ± 342.3 pg/mL post-splenectomy, p < 0.05) and rats. The upregulated

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SDF-1 and HGF significantly facilitated ADSCs motility, migration and proliferation in vitro. And C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration. Direct coculture with ADSCs led to promotion of hepatocytes proliferation and HSCs apoptosis. In addition, the activation of c-jun N-terminal kinase-p53 signaling pathway mediate HGF-induced HSCs apoptosis. Conclusions: Splenectomy prior to cell infusion enhanced the therapeutic potential of ADSCs infusion on liver cirrhosis rats, which involved the upregulation of SDF-1 and HGF after splenectomy. FRI-359 A DIET COMBINING HIGH FAT AND GLUCOSE-FRUCTOSE SYRUP INDUCES OBESITY, NON-ALCOHOLIC STEATOHEPATITIS AND PORTAL HYPERTENSION IN A NEW ANIMAL MODEL T. García-Lezana1,2, M. Martell1,2, N. Ezkurdia1, M. Torres-Arauz1, M. T. Salcedo3, J. Genescà1,2, S. Augustin1,2. 1Liver Unit, Hospital Universitario Vall d’Hebron – Institut de Recerca Vall d’Hebron (VHIR); 2 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD); 3Servicio de Anatomía Patológica, Hospital Vall d’Hebron, Barcelona, Spain E-mail: [email protected] Background and Aims: The prevalence of non-alcoholic steatohepatitis (NASH) is rapidly increasing in the context of the current obesity epidemics. Recent data in patients associate obesity with an accelerated progression of portal hypertension. However, the physiopathological link between both conditions remains unclear. The aims of our study are the development and characterization of an animal model of portal hypertension in diet-induced obesity and NASH. Methods: Sprague-Dawley rats were fed with high fat diet (30% fat, 1% Cholesterol) and high glucose-fructose syrup (42 g/L) (HFGFS) or control diet + tap water (CD). At week 8, both groups underwent a minimal portal vein ligation (PVL, 16G) or sham surgery, generating 4 groups (CD-sham, CD-PVL, HFGFS-Sham, HFGFS-PVL). After 10 days, still on diet, hemodynamic parameters were measured in vivo and serum and liver samples collected for biochemical and histological analysis.

Results: HFGFS induced a significant gain in weight (248.31 ± 44.92 vs CD: 220.41 ± 35.76; p = 0.047) and insulin resistance (HOMA-IR in HFGFS-sham 0.29 ± 0.15 vs 0.94 ± 0.27 in controls). HFGFS also induced steatosis, ballooning and NASH (defined as NAFLD activity score >3) in a higher proportion of individuals when compared to controls (0% NASH in CD-Sham vs. 100% HFGFS-Sham and 90% HFGFS-PVL, p = 0.002 (HFD-sham vs CD-sham) and p = 0.009 (HFDPVL vs CD-sham, respectively), However, there were no significant differences in fibrosis between groups. Finally, HFGFS was associated

Journal of Hepatology 2016 vol. 64 | S425–S630