534 BACTERIAL LIPOPOLYSACCHARIDE (LPS) INHIBITS CB2 RECEPTOR EXPRESSION IN HUMAN MONOCYTIC CELLS

POSTERS biliary cirrhosis disease (Clin. Sci. 2007; 112(3): 167–74). In these experiments we have found abnormal intracellular Ca2+ homeostasis and alterations in the metabolism of homocysteine (Hcy) that may contribute to those platelet alterations (J. Hepatology 2009, S280). The role of ROS in this platelet hyperactivity is not known. Objective: Investigate agonist-induced production of ROS in platelets of bile duct ligated rats and the effects of chronic folic acid treatment (an essential cofactor in the metabolism of homocysteine (Hcy)). Methods: Cirrhosis was induced in rats by bile duct ligation (BDL) and sham-operated rats were used as control. The experiments were performed after 21 days (without ascitis) and 28 days (BDL group with ascitis) after surgery. Platelet rich plasma aggregation in response to ADP (5mM) was analyzed using a lumiaggregometer. To analyze ROS, by fluorescence spectroscopy, platelet suspensions were washed and incubated with CM-H2 DCFDA acetyl ester and stimulated with thrombin (0.3U/mL), ADP (5mM) and homocysteine (Hcy, 50–200mM). In some experiments, sham and BDL rats were treated with folic acid (8 mg/kg/day in drinking water). Results: Platelet stimulation with thrombin and Hcy induced an increase in ROS production that was of greater magnitude in platelet from BDL rats than in control. Ascitis reduced aggregation response and increased ROS production. Chronic treatment with folic acid decreased more platelet aggregation and ROS production in BDL than in control group. Conclusion: Folic acid show antiaggregant actions in cirrhotic rats due in part to attenuation of a high ROS production and might be used for prevention of venous thromboembolism in liver cirrhosis. This study has been supported by grant from Ministerio de Educacion ´ y Ciencia (SAF2006–9127). 534 BACTERIAL LIPOPOLYSACCHARIDE (LPS) INHIBITS CB2 RECEPTOR EXPRESSION IN HUMAN MONOCYTIC CELLS 1 J. Ros1 , V. Reichenbach1 , J. Munoz-Luque ˜ , M. Morales1 , M. Navasa2 , 1 1 W. Jimenez ´ . Biochemistry and Molecular Genetics Service, 2 Liver Unit-Institut de Malaties Digestives, Hospital Clinic i Provincial de Barcelona, IDIBAPS, CIBERehd, University of Barcelona, Barcelona, Spain E-mail: [email protected] Background and Aims: Endocannabinoids are a family of substances with a wide array of physiological effects related to fibrogenesis, vasodilation and inflammation. Two different types of receptors CB1 and CB2 mediate these effects. The latter is located in the cells of the immune system and is responsible for the antiinflammatory effects of endocannabinoids. Cirrhotic patients have increased susceptibility to bacterial infection, which is associated with altered host-defense response mechanisms. In the present study we considered the possibility that impaired expression of CB2 receptor in monocytic cells of cirrhotic patients could be involved in the pathogenesis of this phenomenon. Methods: To ascertain whether bacterial wall derived products modulate CB2 expression in human monocytic cells, we measured the changes induced by LPS (10 ng/mL and 1ug/mL) on mRNA and protein abundance of this receptor in a differentiated human monocytic cell line (U937 cells) by real-time PCR and Western blot, respectively. To assess the functional significance of the changes induced by LPS on CB2 expression, a migration assay towards anandamide (AEA, 1 uM) or 2-arachidonoylglycerol (2AG, 1 uM) was then performed on U937 cells exposed to LPS or vehicle. Finally, to determine the clinical relevance of our findings, mRNA of CB2 receptor was quantified in peripheral monocytes and peritoneal macrophages of cirrhotic patients with or without spontaneous bacterial peritonitis (SBP). S214

Results: Exposure to LPS resulted in a dose dependent decrease in both, mRNA and protein CB2 receptor abundance in U937 cells. The endogenous CB2 ligands, AEA and 2-AG, produced a significant increase in the migratory activity of U937 cells that reverted when the experiments were performed in the presence of LPS. Circulating monocytes of cirrhotic patients showed a significant diminished mRNA expression of CB2 compared to control subjects. Moreover, very low levels of this transcript were found in peritoneal macrophages of cirrhotic patients with ascites, being fully suppressed when analyzed in patients with SBP. Conclusion: These results demonstrate that LPS inhibits CB2 receptor expression in human monocytic cells and suggest that the endocannabinoid system could be involved in the pathogenesis of the altered host-defense response mechanisms in human cirrhosis. 535 HIGH BILIRUBIN REDUCES CELL SURVIVAL AND DIFFERENTIATION OF PRIMARY HUMAN OSTEOBLASTS. IS THIS EFFECT APPLICABLE TO THE SERUM OF CHRONIC CHOLESTATIC PATIENTS? S. Ruiz-Gaspa` 1 , A. Martinez-Ferrer1 , A. Enjuanes1 , P. Peris1 , M.J. Mertinez de Osaba1 , L. Alvarez1 , A. Monegal1 , A. Combalia1 , 1 N. Guanabens ˜ , A. Pares ´ 2 . 1 Metabolic Bone Diseases Unit and Liver Unit, CIBERehd. Hospital Clinic. IDIBAPS. Iniversiti of Barcelona, 2 Liver Unit, Hospital Cl´ınic, CIBERhed, IDIBAPS, University of Barcelona, Barcelona, Spain E-mail: [email protected] Background and Aims: It has been suggested that hyperbilirubinemia impairs osteoblast proliferation, assessed by a bioassay that measures plasma mitogenic activity. Although low bone formation is considered to be the main feature in osteoporosis associated with cholestatic diseases, the effects of retained substances in chronic cholestasis on bone cells have been scarcely studied. Therefore, we have performed studies on human osteoblasts aimed at analyzing the cell viability and differentiation in two experimental conditions: 1. bilirubin at different concentrations and times; 2. serum samples from cholestatic patients with high and normal values of bilirubin and healthy controls, at different concentrations and times. Methods: Assays were performed on human osteoblastic cells cultured from trabecular bone pieces obtained from patients undergoing hip replacement for osteoarthritis. Cell viability was measured using a colorimetric assay (WST-1) and cell differentiation was assessed by measuring the alkaline phosphatase (ALP) activity. Results: The addition of bilirubin decreased cell viability by 36 and 56% at concentrations of 50 and 100 mM, respectively (p ≤ 0.002). These effects were observed in cultures up to 48 h. and were partially prevented by fetal bovine serum. Moreover, bilirubin (1000 mM) had a toxic effect, since cell survival was <20% at all times and media conditions (<0.001). Bilirubin (50 mM) decreased ALP activity by 13.5% at 72 h (p = 0.04), and by 25%, 53% and 56% at 24, 48 and 72 h, respectively when the concentration of bilirubin was 100 mM (p < 0.001). Serum from jaundiced patients with total bilirubin concentrations of 6.6, 33 and 66 mM decreased osteoblast viability at 48 h by 27%, 26% and 34%, respectively (p < 0.03). No effects on cell viability were observed with serum of cholestatic patients with normal bilirubin levels and with serum from healthy controls. Serum from jaundiced patients significantly decreased ALP activity from 72 hours at all bilirubin concentrations (6.6, 33 and 66 mM). Conclusion: High bilirubin causes harmful effects on human osteoblasts, decreasing cell survival and differentiation. The inhibitory effects of serum from cholestatic patients with hiperbilirubinemia on the viability and differentiation of osteoblasts

Journal of Hepatology 2010 vol. 52 | S183–S317