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. Author manuscript; available in PMC: 2024 Aug 22.
Published in final edited form as: Circulation. 2023 Jul 10;148(8):661–678. doi: 10.1161/CIRCULATIONAHA.122.063402

Figure 3: Tissue EVs are Highly Enriched in the Least-Dense Fractions from Density Gradient Separation.

Figure 3:

A and C, From 15-fraction survey experiments, raw abundance by mass spectrometry of selected proteins from intact human carotid artery plaques and calcified aortic valves demonstrated that extracellular vesicle marker proteins (Annexin A2, CD63, CD81, MFGE8 [lactadherin], HSP70–2) were highly enriched in the four least-dense fractions of both tissue types; n=3 carotid artery plaque and 4 calcified aortic valve donors. Enrichment of globular collagens (Collagen VIA1, VIA2, VIA3), fibrillar collagens (Collagen IA1, IA2, IIIA1), and other components of the extracellular matrix (ECM; FBN1, VCAN) was identified in denser fractions. B and D, Nanoparticle tracking analysis confirmed the characteristic presence of particles ~150–200 nm in diameter in fractions 1–4 of both intact carotid artery plaques and calcified aortic valves (n=3 per tissue type), while mean particle size remained consistent between fractions (mean±SEM). E, Representative CD63-labelled immunogold transmission electron microscopy (TEM) identified CD63+ membrane-bound EVs in fractions 1–4 (arrows) from intact carotid artery plaques (top) and calcified aortic valves (bottom); bar=100 nm. Consistent with mass spectrometry-derived protein abundance in A/C, TEM showed abundant globular collagens in fractions 5–10 (arrowheads) and fibrillar collagens in the most-dense fractions (open arrows).