Abstract 5224: CB1 Receptor Inhibition Leads to Decreased Vascular AT1 Receptor Expression, Inhibition of Oxidative Stress and Improved Endothelial Function

Abstract

Inhibition of the cannabinoid receptor CB1 (CB1-R) exerts numerous effects such as modulation of blood pressure, insulin sensitivity and lipid concentrations. However, direct vascular effects of CB1-R inhibition remain unclear. Methods and Results: CB1-R expression was validated in vascular smooth muscle cells (VSMC) and aortic tissue of mice (real-time RT-PCR). Treatment of cultured VSMC with the selective CB1-R antagonist rimonabant resulted in reduced angiotensin II (ang II)-mediated reactive oxygen species (ROS) production (DCF fluorescence) and NADPH oxidase activity (lucigenin-enhanced chemiluminescence). Because rimonabant inhibited ang II-induced but not basal ROS production, and ang II activates NADPH oxidase through ang II type-1 receptor (AT1-R) stimulation, we analyzed AT1-R expression in VSMC. CB1-R inhibition with rimonabant or AM251 led to down-regulation of AT1-R protein expression (Western blot), stimulation with the CB1-R agonist CP55,940 resulted in AT1-R up-regulation, and co-incubation with CP55,940 abolished the effect of rimonabant, indicating that AT1-R expression is directly regulated by the CB1-R. The expression of rac1 and MnSOD was not affected. VSMC showed no significant expression of the CB2-R under basal conditions (real-time PCR, Western blot), and CB2-R inhibition had no impact on AT1-R expression in VSMC. To investigate the relevance of CB1-R inhibition in vivo, apolipoprotein E-deficient (ApoE−/−) mice were treated with cholesterol-rich diet and rimonabant or vehicle for 7 weeks. Consistent with the in vitro findings, CB1-R inhibition decreased AT1-R expression (real-time RT-PCR, Western blot), NADPH oxidase activity, and ROS production (L-012 chemiluminescence) in the aortic wall. Importantly, CB1-R inhibition improved endothelium-dependent vasodilation of isolated aortic segments (organ chamber experiments), indicating a functional relevance in vivo. Conclusions: CB1-R inhibition leads to decreased vascular AT1-R expression, NADPH oxidase activity and ROS production in vitro and in vivo. This antioxidative effect is associated with improved endothelial function in ApoE−/− mice, indicating beneficial direct vascular effects of CB1-R inhibition.