Mechanisms of endothelial cell activation by endocannabinoid 2-arachidonoylglycerol

Abstract

Abstract Background Endothelial dysfunction promotes atherogenesis, vascular inflammation, and thrombus formation. Reendothelialization after angioplasty is required in order to restore vascular function and to prevent stent thrombosis. The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation. Earlier studies have demonstrated the relevance of this endocannabinoid in human pathophysiology during coronary artery disease and in murine experimental atherogenesis. However, evidence on the impact of 2-AG on endothelial cell function remains scarce. Methods Endothelial repair was studied in two treatment groups of wildtype mice following electrical denudation of the common carotid artery. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184, which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received DMSO. The residual endothelial gap at five days was visualized by Evan's blue staining in either group. In vitro, the effect of 2-AG on human coronary artery endothelial cell (HCAEC) viability was assessed by an XTT-based assay. Endothelial activation was studied by an adhesion assay of THP-1 monocytes to 2-AG-preconditioned HCAEC. Activation of HCAEC adhesion molecules was characterized by flow cytometry. Results Elevated 2-AG levels significantly impaired reendothelialization in wildtype mice following electrical injury of the common carotid artery, resulting in a residual denudation at 5 days of 2291±286 μm vs. 1505±223 μm (n=18–19; p<0.05). In vitro, 2-AG significantly reduced viability of HCAEC at 24 hours (0.31±0.10 vs. 1.00±0.08; n=3; p<0.01). Finally, 2-AG promoted HCAEC activation resulting in a significant increase in THP-1 monocyte adhesion to HCAEC following pre-treatment of HCAEC with 2-AG (0.17±0.03 THP-1 cells per HCAEC vs. 0.07±0.01 THP-1 cells per HCAEC; n=3; p<0.05). Adhesion molecules E-selectin, ICAM-1 and VCAM-1, that are known to be regulated by 2-AG in the venous endothelium, remained unchanged in arterial endothelial cells. Besides, HCAEC migration, ROS-production, expression of NADPH oxidases and secretion of inflammatory cytokines were unaffected by 2-AG. Conclusion Elevated 2-AG levels hamper endothelial repair and impair HCAEC proliferation while facilitating adhesion of monocytes. Intriguingly, the underlying mechanisms in the arterial vascular bed appear distinct from venous endothelium. Given that 2-AG is elevated during coronary artery disease in humans, 2-AG might impair reendothelialization after angioplasty and thus impact on clinical outcomes. Funding Acknowledgement Type of funding source: None