Endothelial dysfunction is a key event in atherosclerosis, leading to reduced endothelial nitric oxide release and thus impaired vasodilation. Endocannabinoids exhibit a variety of cardiovascular effects, including vasodilation in isolated blood vessels and hypotension in anesthetized rodents. The underlying mechanisms are less clear and may involve endothelial-dependent or -independent mechanisms. In the present study, we took advantage of genetically modified mice with enhanced endocannabinoid anandamide (AEA) levels due to deficiency of its major degrading enzyme fatty acid amide hydrolase (FAAH). Our aim was to study the effect of elevated AEA levels on endothelial function in the apolipoprotein E knockout (ApoE-/-) mouse model of atherosclerosis.Methods and Results
ApoE-/- and ApoE-/- FAAH-/- mice were maintained during 10 weeks with high cholesterol diet. Aortic rings of 2-3 mm length were isolated, placed in a myograph and stretched to a passive tension of 5mN. Endothelium-dependent and —independent contractions were measured using phenylephrine (PE) dose-response (from 10e-10 to 10e-6 M). ApoE-/- FAAH-/- mice displayed reduced endothelium-dependent contractile response compared to ApoE-/- mice (Emax 4.4 vs 7.4 mN, p < 0.01). Then, endothelium-dependent and -independent relaxations were assessed by concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, of matched PE-induced precontraction. ApoE-/- FAAH-/- mice showed a significant decreased of Ach EC50 (400 vs 250 nM, p < 0.01) suggesting their greater sensitivity to the endothelium-dependent relaxation. Moreover, western blot analysis demonstrated increased aortic endothelial nitric oxide synthase (eNOS) expression in ApoE-/- FAAH-/- mice. Further in vitro studies with human umbilical vein endothelial cells (HUVEC) revealed enhanced phosphorylation of eNOS and Akt, a kinase directly phosphorylating eNOS, as well as an increased nitric oxide (NO) release in response to stimulation with the stable AEA analogue methanandamide.Conclusion
Our data showed for the first time in a mouse model of atherosclerosis, that the endocannabinoid AEA improves vasodilation through an endothelium-dependent mechanism which seemed to be mediated by the activation of the eNOS pathway and the release of NO.