HIV Promotes Atherosclerosis via Circulating Extracellular Vesicle MicroRNAs

Figures

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  Figure 1. PLHIV have increased atherosclerosis and decreased ECFCs levels. (A). cIMT in PLHIV is higher than that in HIVneg subjects (* p < 0.05). (B). Circulating ECFC levels as determined by CFUs in PLHIV are lower than in HIVneg individuals (*** p < 0.001). PLHIV (N = 74) and HIVneg subjects (N = 23). Statistical test used: student’s t-test.

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  Figure 2. HIVposEVs increase atherosclerosis burden in apoE−/− mice. (A). Representative images of H&E and Oil Red O staining of aortic sections from mice treated with HIVposEVs, HIV PLdepEVs, HIVnegEVs or PBS. Mice treated with HIVposEVs have the most severe atherosclerotic lesions. (B). The ratio of plaque area/total lumen area in the four treatment groups (* p < 0.05). N = 5 animals per group. Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Scale bars: 10×, 200 µm; 40×, 50 µm.

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  Figure 3. HIVposEVs treatment results in impaired vascular repair/rejuvenation in vivo. (A,B). β-gal staining shows a greater number of senescent cells (black arrows show examples of positive cells, stained in blue) in the aortas of apoE−/− mice treated with HIVposEVs. (C,D). The TUNEL assay shows more apoptotic cells (examples of positive cells are pointed by white arrows. Positive cells are red) in the vascular wall of aorta from apoE−/− mice treated with HIVposEVs than mice treated with HIV PLdepEVs, HIVnegEVs and PBS. Each group had five animals (* p < 0.05). Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Enlargements of the representative images are available in Supplementary Materials. Scale bars: 200 µm.

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  Figure 4. HIVposEVs promote lin− BMC senescence and impair lin− BMC functions in vivo and in vitro. (A,B). Lin− BMCs isolated from HIVposEVs-treated apoE−/− mice show increased β-gal staining, as compared with lin− BMCs from mice treated with HIV PLdepEVs, HIVnegEVs or PBS. (C,D). Lin− BMCs from HIVposEVs-treated apoE−/− mice show slower migration than cells from mice treated with HIV PLdepEVs, HIVnegEVs or PBS. Dashed lines in panel C mark the edges of the scrach in the cell monolayer. (E). Lin− BMCs from HIVposEVs-treated apoE−/− mice display reduced proliferation in comparison with control treatments. (F,G). Lin− BMCs isolated from 3-week-old untreated apoE−/− mice were treated in vitro with HIVposEVs for 96 h, which resulted in increased percentage of senescent cells (β-gal staining) than lin− BMCs treated with HIV PLdepEVs, HIVnegEVs or PBS. For the in vivo experiment, each group had five animals. For the in vitro experiment, all experiments were performed in triplicate and repeated thrice (* p < 0.05, ** p < 0.001). Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Scale bars: (A,F): 200 µm; (C): 400 µm.

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  Figure 5. HIVposEVs treatment results in impaired migration and diminish proliferation in ECs in vitro. Mouse aortic ECs isolated from 3-week-old apoE−/− mice exposed to HIVposEVs show slower migration (A,B) and lower cell proliferation after 96 h of exposure (C), compared with HIV PLdepEVs, HIVnegEVs and PBS. Dashed lines in panel C mark the edges of the scrach in the cell monolayer. The experiments were performed in triplicate and repeated thrice (* p < 0.05). Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Scale bars: 400 µm.

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  Figure 6. Tailored EVs containing antagomirs for let-7b-5p and miR-103a-3p abrogate the effects of HIVposEVs in lin− BMC senescence in vitro. (A). Representative images of lin− BMCs incubated with HIVposEVs or HIVposEVs plus miRZip-Ctr show increased percentages of β-gal positivity. By contrast, lin− BMCs exposed to miRZip-103a-3pTEVs and miRZip-let-7b-5p TEVs combined with HIVposEVs (1:1 ratio) for 96 h show similar percentages of β-gal positivity relative to cells exposed to PBS. (B). Quantification analysis shows the statistically significant changes among these treatment groups (* p < 0.05 compared to PBS treatment). The experiments were performed in triplicate and repeated thrice. Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Scale bars: 200 µm.

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  Figure 7. Hmga2 with 3′UTR deletion attenuates HIVposEVs effect in lin− BMC senescence. (A,B). Expression and quantification of Hmga2 in lin− BMCs transduced with different expression vectors. (C). Cellular senescence is measured by β-gal staining. Let-7b EVs partially recapitulate the effects of HIVposEVs on lin− BMCs. In Hmga2-3′UTRdel-overexpressing lin− BMCs, the effects of HIVposEVs on β-gal expression were partially blocked, similar to co-incubation with miRZip-let-7b-5p TEVs, whereas Hmga2-3′UTRdel overexpression almost completely blocked the effects of let-7b EVs. Furthermore, wt Hmga2 overexpression modestly attenuated the effects of HIVposEVs and let-7b EVs. The experiments were performed in triplicate and repeated thrice. Statistical tests used: Kruskal–Wallis and Dunn’s Multiple Comparison Test as a post test. Representative images for the β-gal experiment are available in the Supplementary Materials (* p < 0.05).