Extracellular Vesicles Derived From Regeneration Associated Cells Preserve Heart Function After Ischemia-Induced Injury

Figures

• 

  Figure 1. Characterization of extracellular vesicles. (A–C) Nano tracking particle analysis of EV characteristics such as size and quantity. (D) Representative transmission-electron microscopy figures showing the lipid bilayer structure of EVs. Distinct EV biomarkers expression profiles such as CD9, CD63, and Alix of (E,F) RACev and (G) MSCev. Statistical significance was determined using Mann-Whitney U test. The results are presented as mean ± SEM (n = 4–7 per group).

• 

  Figure 2. RACev enhanced endothelial cell proliferation in a dose-dependent manner. (A,B) 5 *105 RACs-derived EVs (500K) increased the cell numbers and (B,C) growth area in culture dishes. (D) The miRs responsible for proliferation and anti-apoptosis were markedly upregulated in RACev vs. MSCev. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 for RACev-500K vs. other groups. Statistical significance was determined using one way ANOVA followed by Tukey's multiple comparison test. The results are presented as mean ± SEM (n = 7–10 per group).

• 

  Figure 3. RACev transplantation improved cardiac function. (A) Schematic of in vivo experiment design. (B–D) Representative echocardiographic images of the Control, MSCev, and RACev groups. (E,F) The RACev transplanted group showed preserved ejection fraction and fractional shortening at 4 weeks. *P < 0.05; ***P < 0.001; ****P < 0.0001 vs. the Control group; ##P < 0.01; ###P < 0.001 vs. the MSCev group. Scale bar = 100 μm Statistical significance was determined using 2-way ANOVA followed by Tukey's multiple comparison post-hoc test. The results are presented as mean ± SEM. (n = 8–10 per group). EchoCG, echocardiography; EV, extracellular vesicles; M-IRI, myocardial ischemic reperfusion injury; Tx, transplantation; 500K is 5 x 105 either RACs or MSCs-derived EVs.

• 

  Figure 4. RACev transplantation preserved mitral regurgitation (MR) and interstitial fibrosis. (A) Representative mitral regurgitation echocardiographic 2D image. (B) At 4 weeks, mitral regurgitation incidences were higher in the Control and MSCev-treated groups than the RACev group. (C) Mitral regurgitation caused end-systolic volume increase in the control and MSCev-treated groups but not in the RACev group. (D, E) Interstitial fibrosis accumulation was greater in the control group than in the MSCev and RACev groups. (F) Anti-fibrosis miRs were significantly upregulated in RACev vs. MSCev. Furthermore, miR-133a-3p and miR-133b are expressed in RACev but not MSCev (G) Previously defined anti-inflammation related miRs such as miR10a-3p and miR-24-2-5p were expressed only in RACev group (RACev vs. MSCev). (H) Cardiomyogenesis-related miR significantly upregulated in RACev vs. MSCev, and miR-208a-3p and miR499a-5p detected in RACev group but not in MSCev. Scale bar = 100μm *P < 0.05; **P < 0.01; ***P < 0.001 vs. the MSCev group. Statistical significance was determined using 2-way ANOVA (for MR and ESV) or one-way ANNOVA (for interstitial fibrosis) followed by Dunn's or Tukey‘s multiple…

• 

  Figure 5. RACev derived angiomiRs enhanced angiogenesis in infarcted tissues. (A,B) Microvascular density was enhanced in myocardial infarcted tissue in RACev transplanted group. Cardiac capillaries were stained with FITC conjugated Isolectin B4 (green) (C) Mechanistically, enhanced capillary formation in injured myocardium may couple with well-known angiomiRs (miR-126-3p and 5p, miR-195-5p, etc.) delivery by RACev. Scale bar = 50μm. *P < 0.05; **P < 0.01; ***P < 0.001 vs. MSCev group; Statistical significance was determined using Kruskal-Wallis followed by Dunn's multiple comparisons post-hoc test. Results are presented as the mean ± SEM (n = 8–10 per group).

• 

  Figure 6. RACev preferentially accumulates to the ischemic myocardium. Representative histological findings of infarcted tissue (A) in control groups; (B) MSCev showed less infiltration in the ischemic myocardium, whereas (C) large amount of RACev accumulated into the infarcted tissue, and the labeled some RACev (red color) were internalized into the cardiomyocyte nuclei (yellow arrows; nuclei stained with Hoechst). (D,E) in vivo imaging system (IVIS) imaging showed large accumulation of labeled RACev into the infarcted left anterior ventricle. All experiments were repeated twice with the same results. Statistical significance was determined using the Mann-Whitney U test. The results are presented as mean ± SEM (n = 6 per group).

• 

  Figure 7. Immunomodulatory effect of RACev. (A) The body weights of all three groups were similar across the study period. (B) Spleen weight mass at days 4 and 28 days after myocardial ischemia reperfusion injury was not different among the three groups. (C–F) Peripheral blood-derived CD4, CD8, Treg, and CD11b/c cells were not elevated, indicating immune tolerance effect of RACev. (G–K) Spleen-derived immune reaction or response biomarkers such as CD4, TNK, iNK, and antigen-presenting cells were evaluated and did not change among the three groups. Statistical significance was determined using the One-way ANNOVA followed by Dunn's multiple comparisons post-hoc test. The results are presented as mean ± SEM (n = 8–10 per group).