Plasma extracellular vesicle modulate immune cell transcriptional responses following acute myocardial infarction

Figures

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  Figure 1. Characterization of plasma EV in MI patients EVs were isolated from the plasma of MI patients at hospital presentation and at 3-month follow-up. (A) EV-Array analysis of canonical EV-associated markers shows significant enrichment at MI presentation (n = 17 patients paired at both timepoints). (B) EV-cell-associated markers were also enriched at presentation with MI. White in the heatmaps denotes the baseline (normalized to 1), corresponding to the 3-month follow-up value. (C) Total EV concentrations measured by NTA of SEC and ultracentrifugation-purified plasma EVs. (D and E) Spaghetti plots showing EV size and particle concentration profiles at the individual patient level across the two timepoints (n = 19 patients paired at both timepoints). (E) Western blotting confirmed the presence of EV markers (CD9 and Syntenin-1), absence of cellular contaminants (GM130, ATP5A, and Histone H3), and depletion of the plasma Apo B. (F) TEM images confirmed typical cup-shaped EV morphology. Scale bars, 500 nm or 100 nm. (G and H) Single-particle interferometric reflectance imaging (NanoView) confirmed tetraspanin-positive EVs (CD9, CD63, and CD81), (H) with associated size and concentration…

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  Figure 2. Small RNA analysis of plasma EV in MI patients (A) Volcano plot illustrating differentially expressed small RNAs (miRNAs, snoRNAs, etc.) in plasma EVs from MI patients at presentation with MI compared to 3 months later (n = 10 patients paired at both timepoints). Non-significant (NS) RNAs are shown in gray, with log2 fold change (FC) in green, p value < 0.05 in blue, and adjusted p value < 0.05 with log2 FC in red. (B) TissueAtlas expression analysis for miRNA-101-5p. (C) TissueAtlas expression analysis for miRNA-320b, suggesting potential cardiovascular endothelial cell origin. (D) RT-qPCR analysis of miRNA-126-3p, miRNA-320b, and miRNA-101-5p in HUVECs cell pellets normalized to miR-103a-3p, confirming endothelial association. Data are group means ± SD. N = 8 biological replicates per group.

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  Figure 3. Enrichment of miRNA-320b in endothelial cell-derived EV following inflammatory stimulation HUVECs were cultured under control (black) or TNF-α-stimulated (red) conditions to model endothelial inflammation. (A) Total and viable cell counts at study endpoint confirm similar cell numbers across conditions. (B) Soluble VCAM-1 levels in supernatants were significantly increased following TNF-α stimulation, confirming proinflammatory activation. (C and D) Single-particle interferometric reflectance imaging (NanoView) detected CD9, CD63, and CD81-positive EVs in both conditions. Scale bars, 10 μm. (D) Total captured EV concentrations were significantly higher in TNF-α-stimulated cultures. (E) Average particle diameters of captured EVs did not differ significantly. (F) NTA showed elevated particle concentrations in EV preparations from TNF-α-treated HUVECs. (G) Size distribution profiles of EVs from control and TNF-α conditions. (H) EV counts were normalized to total and live cell numbers, confirming increased release under inflammatory stimulation. (I) Western blot analysis of HUVEC-derived EVs confirmed enrichment of canonical EV markers (TSG101, Syntenin-1, and CD9), absence of…

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  Figure 4. Identification of miRNA-320b mRNA targets and pathway analysis (A) Venn diagram showing the overlap of predicted miRNA-320b targets identified using miRWalk, TargetScan, and miRDB databases. (B) GO biological process analysis of overlapping miRNA-320b targets revealed significant enrichment of pathways related to homophilic cell adhesion via plasma membrane adhesion molecules and cell-cell adhesion. (C) Functional validation of miRNA-320b EV activity in a monocyte adhesion assay. HUVEC-derived EVs (from TNF-α-stimulated cells) enriched in miRNA-320b significantly enhanced THP-1 monocyte adhesion to TNF-α-activated endothelial cell monolayers compared with control EVs. Representative fluorescence microscopy images are shown. Scale bars, 20 μm. Data are presented as group means ± SD. N = 4–5 biological replicates per group. Statistical analysis: (C) one-way ANOVA with Tukey’s post hoc test. ∗∗∗∗p < 0.0001.

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  Figure 5. Enrichment of miRNA-320b-mRNA targets in immune cell transcriptomes following MI (A and B) GO biological pathway analysis showing the overlap between miRNA-320b-mRNA targets and DEG in: (A) peripheral blood monocytes following MI, highlighting pathways such as positive regulation of cytokine production and myeloid leukocyte activation; and (B) in peripheral blood neutrophils, showing overlap in biological pathways with miRNA-320b-mRNA targets. (C) Comparison of enriched GO biological pathway terms in monocyte and neutrophil transcriptomes with miRNA-320b-mRNA targets, identifying common pathways such as cytokine production and cell adhesion.

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  Figure 6. Impact of inflammatory endothelial cell-derived extracellular vesicles (EC-EVs) on monocyte-derived macrophage cytokine production Human CD14+ monocytes were differentiated into macrophages, pretreated with EC-EVs from control or TNF-α-stimulated HUVECs, and subsequently activated under M1 polarizing conditions (LPS and IFN-γ). (A and B) RT-qPCR analysis showed significantly increased IL6 and TNF mRNA expression in macrophages treated with inflammatory EC-EVs (red) compared to untreated controls (black) (N = 4 biological donors per group). (C and D) In contrast, ELISA of cell culture supernatants revealed reduced secretion of IL-6 and TNF-α protein in TNF-α-EC-EV-treated macrophages under the same M1 conditions (N = 6 biological donors per group). This discrepancy suggests post-transcriptional modulation of cytokine production by inflammatory EC-EVs. Data are presented as group means ± SD. Statistical analysis: two-way ANOVA with Tukey’s post hoc test. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 7. Modulation of monocyte response to EC-EVs (A) MA plot showing DEG in monocytes from MI patients compared to controls, highlighting significantly altered genes in red. (B) Venn diagram showing the intersection of miRNA-320b-mRNA targets with DEG in MI monocytes. (C and D) Comparison of DEG profiles in M1-stimulated monocytes and (D) GO biological pathway from MI and control patients. N = 3 patients per group/condition.

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  Figure 8. Transcriptomic activation of PBMCs by plasma EVs from MI patients (A and B) MA plot showing DEG in PBMCs treated with plasma EVs from (A) control (NSTEMI) patients and (B) MI patients, red dots are significantly altered genes. (C and E) Venn diagram showing the overlap of DEG induced by control and MI plasma EVs in PBMCs. MA plot showing DEG in PBMCs treated with plasma EVs from (D) control (NSTEMI) plasma EV and (E) MI patient plasma EV 3 months post-MI. (F–I) GO biological pathway analysis comparing pathways enriched by MI plasma EVs at presentation, 3 months post-MI, and shared pathways. MA plot showing DEG in PBMCs treated with (G) control plasma EVs and (H) MI patient plasma EV at presentation and (I) control (NSTEMI) plasma EV and (J) MI patient plasma EV 3 months post-MI followed by M1-stimulation. N = 5 PBMC donors per group.