Plasma CD16⁺Extracellular Vesicles Associate with Carotid Artery Intima-Media Thickness in HIV⁺Adults on Combination Antiretroviral Therapy

Figures

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  FIG 1. Characterizing EVs in biological samples for their relative size, absolute count, and cell of origin. (A) Schematic of the method for isolation and analysis of EVs. (B) SSC height (SSC-H) dot plot showing sensitivity to detect a mix of polystyrene beads 100 to 1,000 nm in diameter, a size range used to set EV gates on the LSRII flow cytometer. (C) Representative plots showing EVs expressing surface markers from their cell of origin.

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  FIG 2. Circulating EVs expressing monocyte markers are related to carotid artery intima-media thickness in HIV-infected individuals. (A) Scatterplots demonstrating the association between the right common carotid (cIMTCCA) and the right carotid bifurcation (cIMTBIF) intima-media thickness. (B and C) Correlation of EVs expressing lymphocyte- (B) and monocyte-associated markers (C) with cIMT, evaluated using Spearman’s rank correlation coefficient test. After correcting for multiple testing with an FDR of <0.1, CD16+ EVs remained associated with cIMT. Dots represent individual participants.

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  FIG 3. EVs expressing monocyte-associated markers are correlated with plasma soluble inflammatory markers. Scatterplots demonstrating the relationship between levels of EVs expressing monocyte-associated markers and levels of soluble C-reactive protein (A), serum amyloid A (B), and myeloperoxidase (C). Spearman correlation coefficient (R) and significance (P) are indicated in the corresponding boxes. These results remained significant after correcting for multiple testing with an FDR of <0.1. Dots represent individual patients.

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  FIG 4. Spearman rank correlations between all EV subtypes. (A) Spearman correlation matrix showing the relationship between all EV subtypes based on their cellular origin. Positive correlations are displayed in red. (B) Scatterplots demonstrating the strong correlations of surface markers with each other on EVs. These results remained significant after correcting for multiple testing with an FDR of <0.1. Dots represent individual patients.

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  FIG 5. Apoptosis and necrosis rates of HUVECs after cell exposure to EVs. (A and B) Schematic of the EV isolation method (A) and apoptosis assay (B). (C) Representative graph of nanoparticle tracking analysis showing the EV particle-size distribution generated by ultracentrifugation of plasma. EV preparations from three uninfected participants were measured, each with similar size distribution. (D) Levels of the non-EV-associated protein albumin in the original plasma and EV samples were determined by ELISA (n = 5 HIV-uninfected donors and 5 HIV-infected participants). (E) Representative flow cytometry plots for cells without treatment and treated with EVs or 10% ethanol (EtOH). (F) P values were determined by using one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison post hoc test. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.