Abstract Body (Do not enter title and authors here):
Background: Beyond their established roles in hemostasis and thrombosis, platelets are increasingly recognized for their contribution to chronic thrombo-inflammatory conditions within the vascular system, including atherosclerosis. Platelets actively participate in systemic responses by modulating their microenvironment through the exocytosis of cytokines and signaling molecules. Overexpression of VAMP8, the key v- SNARE protein involved in platelet exocytosis, has been linked to platelet hyperactivity and an elevated risk of coronary events in humans. This study investigates the role of VAMP8 in the progression of atherosclerotic lesions.
Methods and Results: To confirm platelet involvement in atherosclerosis, washed platelets from healthy WT mice were treated with 0.05 U/mL thrombin, and the platelet releasate was analyzed using highly-multiplexed CodePlex chip secretome proteomics. Our findings confirmed that platelets secrete several cytokines and chemokines associated with atherosclerosis, notably IL-1β. To further investigate the impact of impaired platelet exocytosis on atherosclerosis, we crossed VAMP8-deficient (VAMP8^-/-) mice with ApoE-deficient (ApoE^-/-) mice to create a hypercholesterolemic double- knockout (ApoE^-/-/VAMP8^-/-) model. These mice were fed a Western diet for 12 weeks to induce atherosclerosis. Atherosclerotic lesions were manually traced on the intimal surface from the ascending aorta to the descending thoracic aorta, 1 mm distal to the left subclavian artery, using Nikon NIS-Elements software (en face method). Our data indicated that VAMP8 deficiency significantly suppressed atherosclerosis development in male ApoE^-/-/VAMP8^-/- mice. We further validated these results by inducing hypercholesterolemia in mice aged 8-12 weeks via an adeno-associated viral (AAV) vector expressing a gain-of-function mutation (D377Y) in mouse PCSK9, followed by a Western diet. Atherosclerotic lesion development was also markedly reduced in VAMP8^-/- mice in this model. Plasma total cholesterol levels did not differ between WT and VAMP8^-/- groups in either model. Additionally, bulk RNA-seq analysis of washed WT versus VAMP8^-/- platelets revealed significant downregulation of atherosclerosis- associated and inflammatory pathways, suggesting a potential impact on cargo release and its role in atherosclerosis development.
Conclusion: VAMP8 deletion significantly reduces atherosclerosis and affects the release of inflammatory cargo from platelets.