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

Abstract

53 54 Background: Fewer than 50% of patients develop calcification of both atherosclerotic plaques 55 and aortic valves, implying differential pathogenesis. While circulating extracellular vesicles 56 (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs associate with early 57 mineralization, but their contents, function, and contributions to disease remain unknown. 58 59 Results: Global proteomics of human carotid artery endarterectomies and calcified aortic valves 60 from a total of 27 donors/patients revealed significant over-representation of proteins with 61 vesicle-associated pathways/ontologies common to both diseases. We exploited enzymatic 62 digestion, serial (ultra)centrifugation and OptiPrep density-gradient separation to isolate EV 63 populations from diseased arteries and valves. Mass spectrometry found 22 EV marker proteins 64 to be highly enriched in the four least-dense OptiPrep fractions while extracellular matrix 65 proteins predominated in denser fractions, as confirmed by CD63 immunogold electron 66 microscopy and nanoparticle tracking analysis. Proteomics and miRNA-sequencing of OptiPrep-67 enriched tissue EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target 68 genes. Pathway networks of proteins and miR targets common to artery and valve tissue EVs 69 revealed a shared regulation of Rho GTPase and MAPK intracellular signaling cascades. 179 70 proteins and 5 miRs were significantly altered between artery and valve EVs; multi-omics 71 integration determined that EVs differentially modulated cellular contraction and p53-mediated 72 transcriptional regulation in diseased vascular vs. valvular tissue. 73 74 Conclusions: Our findings delineate a strategy to isolate, purify, and study protein and RNA 75 cargoes from EVs entrapped in fibrocalcific tissues. Multi-omics and network approaches 76 implicated tissue-resident EVs in human cardiovascular disease. 77 78 4 Keywords 79 80 Extracellular vesicles, exosomes, atherosclerosis, vascular calcification, calcific aortic valve 81 disease, proteomics, transcriptomics, miRNA, multi-omics integration, network analysis 82 83 84 Background 85 86Cardiovascular calcification directly correlates with incidence of cardiovascular events such as 87 myocardial infarction, stroke, and heart failure, and strongly predicts morbidity and mortality.(1) 88While aberrant mineralization of arteries and heart valves share numerous risk factors, only 25-89 50% of patients develop both vascular and valvular calcification, implying that they involve 90 differential drivers.(2) Histopathological studies described these two conditions as being grossly 91 comparable (3) and there has been little subsequent study of this apparent epidemiological 92 paradox. Despite the histological similarities, pharmacological therapies such as statins have 93 been successful in mitigating inflammation and lowering cholesterol in patients with 94 atherosclerosis but they failed to improve outcomes for aortic valve stenosisleaving patients 95 without effec...

Proinflammatory Matrix Metalloproteinase-1 Associates With Mitral Valve Leaflet Disruption Following Percutaneous Mitral Valvuloplasty

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Network-Guided Multiomic Mapping of Aortic Valve Calcification

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Small particles with large impact: Insights into the unresolved roles of innate immunity in extracellular vesicle‐mediated cardiovascular calcification

“…We have recently taken a similar approach to assess the vesiculome of diseased carotid and valvular tissue. 158 We established a method to isolate tissue resident EVs from human surgically resected fibrocalcific cardiovascular tissues. In calcified aortic valves and carotid endarterectomy specimens, tissue entrapped EVs could be isolated using a combination of approaches to overcome the challenges presented by the complex and fibrous nature of the tissue.…”

“…We have recently taken a similar approach to assess the vesiculome of diseased carotid and valvular tissue 158 . We established a method to isolate tissue resident EVs from human surgically resected fibrocalcific cardiovascular tissues.…”