Conserved and Divergent Modulation of Calcification in Atherosclerosis and Aortic Valve Disease by Tissue Extracellular Vesicles

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Abstract
Background: Fewer than 50% of patients develop calcification of both atherosclerotic plaques and aortic valves, implying differential pathogenesis. While circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs associate with early mineralization, but their contents, function, and contributions to disease remain unknown. Results: Global proteomics of human carotid artery endarterectomies and calcified aortic valves from a total of 27 donors/patients revealed significant over-representation of proteins with vesicle-associated pathways/ontologies common to both diseases. We exploited enzymatic digestion, serial (ultra)centrifugation and OptiPrep density-gradient separation to isolate EV populations from diseased arteries and valves. Mass spectrometry found 22 EV marker proteins to be highly enriched in the four least-dense OptiPrep fractions while extracellular matrix proteins predominated in denser fractions, as confirmed by CD63 immunogold electron microscopy and nanoparticle tracking analysis. Proteomics and miRNA-sequencing of OptiPrep-enriched tissue EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target genes. Pathway networks of proteins and miR targets common to artery and valve tissue EVs revealed a shared regulation of Rho GTPase and MAPK intracellular signaling cascades. 179 proteins and 5 miRs were significantly altered between artery and valve EVs; multi-omics integration determined that EVs differentially modulated cellular contraction and p53-mediated transcriptional regulation in diseased vascular vs. valvular tissue. Conclusions: Our findings delineate a strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Multi-omics and network approaches implicated tissue-resident EVs in human cardiovascular disease.
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