Therapeutic effects of platelet-derived extracellular vesicles on viral myocarditis correlate with biomolecular content

Figures

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  Figure 1. Characterization revealed platelet-derived EVs (PEV) and premenopausal (pm)PEV display typical extracellular vesicle (EV) profiles. PEV and pmPEV were reconstituted at 11.2 mg/mL or 8.0 mg/mL, respectively, as per manufacturer instructions. For characterization, size-exclusion chromatography (SEC) was performed on samples to provide a smaller noise to particle ratio. Fractions 7-11 from SEC were analyzed for size distribution using (A) nanoparticle tracking analysis, which was also measured by tunable resistive pulse sensing (TRPS) in Supplementary Figures 1C–E . TRPS was also used to measure (B) zeta potential, shown as mean ± SD. (C) Western blot analysis was performed to test for positive and negative EV markers, full blots found in Supplementary Figures 1A, B . (D) ELISAs and (E) EV multiplex assays were performed on samples in duplicate after reconstitution and without SEC to appropriately estimate relative abundance of positive and negative markers of EVs. (F, G) PEV and pmPEV samples were visualized with transmission electron microscopy for morphology (magnification 150,000X, Scale bars: 100 nm); widefield images found in Supplementary Figures 1F–I . Visualization was also…

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  Figure 2. Treatment with PEV or pmPEV decreased myocardial inflammation and fibrosis. EVs from healthy men and women (PEV), or from healthy premenopausal women (pmPEV) (0.25 mL of 1010 EVs/mL), or control (0.25 mL of 1X PBS) were given ip to male BALB/c 8-week-old mice on days -1, 0, 1 pi with 103 PFU of CVB3 given ip on day 0. Representative photos of (A) pericarditis, (C) myocarditis, (E) fibrosis at day 10 pi; Scale bars: 200 µm. Quantification of inflammation (B, D) or fibrosis (E) was performed on individual hearts. Data show mean ± SEM for 10 mice/group, one-way ANOVA with Dunnett’s multiple comparison, *, p < 0.05; **, p < 0.01; ***, p < 0.001 (see Supplementary Table 3 ).

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  Figure 3. PEV and pmPEV globally downregulate immune and fibrosis markers, but more changes occur with PEV. EVs from healthy men and women (PEV), or from healthy premenopausal women (pmPEV) (0.25 mL of 1010 NPs/mL) or control (0.25 mL of 1X PBS) were given to male BALB/c 8-week-old mice ip on days -1, 0, 1 pi with 103 PFU of CVB3 given ip on day 0. Relative gene expression (RGE) from whole hearts by qRT-PCR at day 10 pi was used to assess (A, B) acute myocarditis immune markers and (C, D) fibrosis markers indicated on the left axis of each matrix in individual experiments compared to the housekeeping gene Hprt (see Supplementary Figure 3 for individual data). Data show mean RGE for 10 mice/group, one-way ANOVA with Dunnett’s multiple comparison were conducted for each marker but are depicted separately as PEV or pmPEV vs. control; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (see Supplementary Figure 3 , Supplementary Table 5 ).

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  Figure 4. Initial assessment of PEV versus pmPEV proteome. (A) Principal component analysis. (B) Hierarchal clustering. (C) Volcano plot of significantly different proteins.

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  Figure 5. Proteomic analysis of PEV versus pmPEV reveal immune-specific enrichment in PEV and pmPEV. (A) Cytoscape pathway shows super-clusters of proteins in PEV (green) versus pmPEV (purple). Differentially enriched proteins between (B) PEV and (C) pmPEV with adjusted p value < 0.01.

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  Figure 6. PEV and pmPEV share a proteome with other platelet/plasma EV products. (A) 4-way Venn diagram shows shared proteome of PEV and pmPEV compared to 3 other platelet EV products reported in the literature. (B) Cytoscape pathway visualization depicts super-clusters of pathways from the shared proteome of all five of these platelet/plasma EV products. The 12 superclusters visualized include: cdc42 signal transduction, immune process response, cell junction disassembly, factor insulin growth, signalling receptor binding, regulation transport positive, plasma lipoprotein particle, binding lipid complex, transporter activity transfer, platelet intracellular vesicle, skin extracellular matrix, and encapsulating structure collagen.

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  Figure 7. PEV and pmPEV enriched for anti-inflammatory miRs, but pmPEV had a more proinflammatory signature. (A) Principal component analysis. (B) Hierarchical clustering of miRs. (C) Hierarchal clustering of differentially expressed miRs.

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  Figure 8. Comparison of miR targets between PEV and pmPEV. Mechanistic effects of miRs reported in the literature identified in PEV (blue) versus pmPEV (purple) that influence key pathways in the pathogenesis of viral myocarditis. References and descriptions of miRs found in Tables 3 , 4 . This figure was created using BioRender.com.