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Updated: Jul 24, 2025
Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models
Published on: January 27, 2023
Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve
Mark C Blaser1, Fabrizio Buffolo1, Arda Halu1,2
1Center for Interdisciplinary Cardiovascular Sciences, Cardiovascular Division, Department of Medicine (M.C.B., F.B., A.H., M.E.T., F.S., H.H., C.L.C., L.A.S., S.K.A., M.A.R., T.P., S.A.S., M.A., E.A.).
Insights
Tissue-entrapped extracellular vesicles (EVs) play a role in cardiovascular calcification. This study reveals tissue-specific EV cargoes linked to calcific signaling pathways in arteries and valves, offering new insights into disease mechanisms.
Area of Science:
- Cardiovascular Biology
- Extracellular Vesicle Research
- Proteomics and Transcriptomics
Background:
- Aortic valve calcification and atherosclerosis often have different causes.
- Circulating extracellular vesicles (EVs) are cardiovascular disease biomarkers, but tissue-EV roles in mineralization are unknown.
Purpose of the Study:
- To investigate the cargoes, functions, and disease contributions of tissue-entrapped EVs in cardiovascular calcification.
- To compare proteomic and transcriptomic profiles of EVs from diseased carotid arteries and aortic valves.
Main Methods:
- Disease-stage proteomics on human carotid plaques and aortic valves.
- Isolation and vesiculomics (proteomics and small RNA-sequencing) of tissue-EVs.
- Network analyses and gene validation in primary cells.
Main Results:
- Proteomes of carotid plaques and aortic valves showed significant convergence but retained unique proteins.
- Disease progression altered EV protein and microRNA content, implicating shared pathways.
- Tissue-specific EV cargoes were linked to Notch and Wnt signaling, driving calcification.
Conclusions:
- This study is the first to compare proteomics of human carotid plaques and aortic valves, identifying distinct disease drivers.
- A novel vesiculomics strategy was developed to analyze EVs within fibrocalcific tissues.
- Tissue EVs play a significant role in modulating cardiovascular disease progression.
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<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules 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.
