IL-10 partly mediates the ability of MSC-derived extracellular vesicles to attenuate myocardial damage in experimental metabolic renovascular hypertension

Figures

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  Figure 1. Characterization and tracking of swine MSC-derived EVs. (A, B). NTA showed the size distribution and quantity of EVs from 15×106 MSCs and IL-10-KD MSCs. (C, D). qPCR detected the IL-10 mRNA expression in MSCs and their daughter EVs. n = 6 (E, F). EVs tracking in the heart sections and comparable numbers of PKH-26-labeled EVs between RVH+EVs and RVH+IL-10-KD EVs. Blue is DAPI-stained cell nuclei. Scale bar = 20µm. Data are expressed as mean ± SD. **p < 0.01, ***p < 0.001 between groups (Student’s t-test following normality and log-normality tests). MSCs, mesenchymal stem cells; EVs, extracellular vesicles; NTA, Nanoparticle Tracking Analysis; RVH, renovascular hypertension; KD, knockdown. ns, no significance.

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  Figure 2. Systemic characteristics, cardiac and renal function in the study groups. Systemic characteristics, including the (A) degree of stenosis, (B) bodyweight, and (C) PRA at week 16. (D–H) Cardiac function and SCr at week 16. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, double-headed arrow with ‘ns’ indicate no statistical significance among the included groups (One-way ANOVA followed by either the Bonferroni posttest or Two-stage step-up method of Benjamini, Krieger and Yekutieli posttest as appropriate). (I–K) Single-kidney GFR, kidney volume and normalized GFR in all groups. ‘ns’ no significance, *p < 0.05, **p < 0.01: compared two kidneys in particular; ‘NS’ no significance, a p < 0.05, aaa p < 0.001, b p < 0.05, bb p < 0.01 compared with single-kidney in the Lean group; c p < 0.05 compared to stenotic kidneys in RVH group (Student’s t-test when normally distributed, otherwise a nonparametric test; One-way ANOVA followed by an appropriate posttest test). (L) MAP was elevated at 12 and 16 weeks in all RVH groups. aa p < 0.01 compared with Lean group at week 12, bb p < 0.01, bbb p < 0.001 compared with Lean group at week 16 (One-way ANOVA followed by posttest). PRA,…

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  Figure 3. EVs modulated inflammation in the heart tissue. (A) Representative images (40×) of immunofluorescence staining showed that EVs induced macrophage polarization towards M2 phenotype and increased Tregs while IL-10-KD EVs had opposite effect. M1 [CD68+(green)/iNOS+(red)], M2 [CD68+(green)/Arg-1+(red)] and Treg [CD4+(green)/CD25+(red)/Foxp3+ (far red)]. Scale bar=50µm. (B–E) Quantitative analysis of M1, M2, M1/M2 ratio and Treg. (F–I) qPCR showed mRNA level of inflammatory cytokines in the heart tissue. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 between groups (One-way ANOVA followed by either the Bonferroni posttest or Two-stage step-up method of Benjamini, Krieger and Yekutieli posttest). iNOS, inducible nitric oxide synthase; Arg-1, Arginase-1. ns, no significance.

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  Figure 4. IL-10-KD EVs reversed the effects of WT-EVs on alleviating myocardial fibrosis and increasing capillary density. (A, C) Representative Masson’s trichrome staining (40×) and quantification of myocardial fibrosis; (B, D) Representative heart sections stained with H&E (40×), showing capillaries (yellow arrows) and quantification of capillary density (numbers/field). (E, F) Myocardial protein expression of VEGF and its quantification. Scale bar = 50µm. Data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 between groups (One-way ANOVA followed by either the Bonferroni posttest or Two-stage step-up method of Benjamini, Krieger and Yekutieli posttest). H&E, Hematoxylin-eosin; VEGF, vascular endothelial growth factor. ns, no significance.

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  Figure 5. Proliferation ability of naïve CD3+ T-cells cocultured with EVs or IL-10-KD-EVs was assayed using the CTFR method by Flow cytometry (n = 6 biological replicates). (A) naïve T-cells without ConA stimulation; (B) ConA stimulated the proliferation of T-cells that were then cocultured with (C) NC-EVs, (D) Lipo-EVs, (E) WT-EVs, or (F) IL-10-KD-EVs. (G) Comparison of proliferation of ConA-stimulated T-cells. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 between groups, double-headed arrow with ‘ns’, no statistical significance among the indicated groups (One-way ANOVA followed by either the Bonferroni posttest or Benjamini, Krieger and Yekutieli posttest). CTFR, CellTrace Far Red; ConA, concanavalin-A; NC, negative control with empty vector; Lipo, lipofectamine, P1, T-cell passage 1, the original T-cell; P2, T-cell passage 2, the proliferated T-cell.

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  Figure 6. IL-10-KD-EVs failed to stimulate migration or attenuate inflammatory cytokine expression in CAECs. (A) Representative immunofluorescence images (40×) for surface markers of CAEC. (B) Cell migration ability examined using the wound healing assay under microscopy (10×). (C, D) Wound width and confluency quantified at 0h and 36h. (E–G) qPCR showed inflammatory cytokines in CAECs cocultured with vehicle, NC-EVs, Lipo-EVs, WT-EVs or IL-10-KD-EVs. Scale Bar = 50µm. *p < 0.05, **p < 0.01, ***p < 0.001 between groups, double-headed arrow with ‘ns’ meant no statistical significance among the included groups (One-way ANOVA followed by either the Bonferroni posttest or Benjamini, Krieger and Yekutieli posttest). CAECs, coronary artery endothelial cells.