Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis

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Abstract
BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage–specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence ( P P FGFR2 , PPP2CA , and ADAM17 in human carotid artery smooth muscle cells and WNT5A , APP , and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.
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