Aims: The endocannabinoid system has emerged as an important lipid signaling system in various pathophysiological conditions, including metabolic disorders and atherosclerosis. Since endothelial dysfunction plays a critical role in the early stages of atherosclerosis, we here aim to study the effect of endothelial cell-specific CB1 deficiency on atherosclerotic plaque development and composition.

Methods: We generated endothelial cell-specific CB1 knockout mice by breeding BmxCreERT2 with CB1-flox mice. The transgenic mice were under ApoE-/- background and received 4 or 16 weeks Western Diet (WD; 0.15% cholesterol). Immune status and atherosclerotic plaque progression were assessed by flow cytometry, qPCR, Western Blot, and histology.

Results: Endothelial CB1 deficiency (EC-CB1-KO) in female mice attenuated plaque development in the aortic roots (EC-CB1-WT 752478 ± 27719, n=9; EC-CB1-KO 631143 ± 29851, n=11; P<0.01) and abdominal aortas, accompanied by more stable plaque phenotype with increased collagen at advanced stage (16 weeks WD). The effect of endothelial CB1 deficiency on lesion development was not observed in male mice, suggesting that endothelial CB1 might play a sex-specific role in atherosclerosis. Female EC-CB1-KO mice exhibited a significant decrease in aortic adhesion molecule ICAM-1 and VCAM-1 expression. Moreover, ex vivo imaging of carotid arteries via two-photon microscopy revealed less DIL-oxLDL uptake by CB1-deficient endothelium (WT 5.75 x10^-3± 4.98x10^-4, n=5; KO 3.345 x 10^-3 ± 7.69 x 10^-4, n=6; P<0.05; particles per cell length). Immunofluorescence staining revealed a significant reduction of aortic endothelial caveolin-1 (CAV-1) expression in female EC-CB1-KO mice, which might provide a mechanistic explanation for reduced endothelial lipid uptake and consequently increased plasma cholesterol levels observed in EC-CB1-KO mice. Treatment of atherosclerotic mice with the peripheral CB1 antagonist JD-5037 reduced plaque progression, endothelial adhesion molecule and CAV-1 expression again only in females, but not males, suggesting that peripheral CB1 antagonism might represent a novel sex-specific therapeutic target.

Conclusion: Our results indicate that endothelial CB1 contributes to atherosclerosis by modulating endothelial oxLDL uptake and vascular inflammation, possibly involving CAV-1 dependent transcytosis. The underlying mechanisms and sex-specific effects of CB1 signaling deserve further investigation.