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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 7: Integration of EV Multi-Omics Identifies Modulators of Calcification.

Figure 7:

A and B, Overlap of KEGG, Reactome, and BioCarta pathways enriched amongst tissue EV proteins and unique gene targets of tissue EV miRs that were differentially-enriched between intact normal and diseased carotid arteries (A) and aortic valves (B). N=6 normal carotid arteries, n=4 diseased carotid artery atherosclerotic plaques, n=6 normal aortic valves, n=4 diseased calcified aortic valves. C and D,Protein-protein interaction networks further prioritized candidate EV-derived calcification modulators: constituents of selected overlapping carotid artery (C, blue nodes; FGFR2, PPP2CA, ADAM17) and aortic valve pathways (D, green nodes; WNT5A, APP, APC) that were significantly altered by disease progression in EV multi-omics had high betweenness-centrality scores in their respective networks. Node diameter corresponds to node degree. E and G, Relative mRNA expression levels of FGFR2, PPP2CA, ADAM17, WNT5A, APP, and APC vs. GAPDH in primary human carotid artery smooth muscle cells (hCtASMCs, E) and human aortic valvular interstitial cells (hVICs, G) after 6 days in normal medium (NM) incubated with scrambled siRNA (siSCR) or siRNA targeting FGFR2, PPP2CA, ADAM17, WNT5A, APP, and APC (siFGFR2, siPPP2CA, siADAM17, siWNT5A, siAPP, siAPC) demonstrated robust knockdown of target gene expression; n=1 donor per cell type, triplicate wells per donor, duplicate qPCR reactions averaged per well; mean±SD; *p<0.05, ***p<0.001, ****p<0.0001. F and H, Representative Alizarin red staining of target knockdown in hCtASMCs (F) and hVICs (H) at days 14–21 in NM or pro-calcifying medium (PM) culture. I and J, Quantification of solubilized Alizarin red stain in hCtASMCs (I) and hVICs (J) treated as in F and H for 14 and 21 days confirmed that inhibition of molecules identified by integration of EV multi-omics in A-D significantly modulated calcification of human vascular and valvular cells; n=3 hCtASMC donors and 3 hVIC donors, duplicate wells averaged per timepoint per donor; *p<0.05, **p<0.01. Grey bars indicate the minimum to maximum intensity range of the NM siSCR condition.