Adipose-Derived Mesenchymal Stem Cells-Derived Exosomes Carry MicroRNA-671 to Alleviate Myocardial Infarction Through Inactivating the TGFBR2/Smad2 Axis

Fig. 1. Identification of the adMSCs and the Exo. a Expression of the MSC-surface positive marker proteins CD29, CD44, and CD99 and the negative marker protein CD45 in the acquired adMSCs examined by flow cytometry; b Osteogenic differentiation and adipogenic differentiation potentials of the adMSCs examined by alizarin red staining and Oil red O staining, respectively; c Morphology of the extracted particles observed under the TEM; d Particle size distribution examined by a NTA; e Expression of the exosome surface marker proteins determined by western blot analysis; f Uptake of exosomes by cardiomyocytes observed by the PHK26 labeling. Three independent experiments were performed. Representative images are presented.

Fig. 2. Exo treatment protects cardiomyocytes against OGD-induced damage. a Viability of cardiomyocytes at different time points after OGD treatment determined by the MTT assay; b Apoptosis of cardiomyocytes at different time points after OGD treatment examined by flow cytometry; c Viability of cardiomyocytes after Exo treatment examined by the MTT assay; d Apoptosis of cardiomyocytes after Exo treatment detected by flow cytometry; e Protein levels of apoptosis-related factors (Bax and Bcl-2) and the fibrosis-related factor α-SMA in OGD- and Exo-treated cardiomyocytes quantified by western blot analysis; f Production of pro-inflammatory cytokines (IL-6 and TNF-α) in OGD- and Exo-treated cardiomyocytes determined using ELISA kits. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by one-way ANOVA (a–d) or two-way ANOVA (e, f); *p < 0.05, **p < 0.01 vs. 0 h; #p < 0.05 vs. control group; &p < 0.05 vs. PBS group.

Fig. 3. Exo treatment enhances miR-671 expression in cardiomyocytes. a Top 10 differentially expressed miRNAs in cardiomyocytes before and after Exo treatment using a miRNA microarray analysis; b Expression of miR-671 in cardiomyocytes after OGD and Exo treatment determined by RT-qPCR. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by one-way ANOVA (b); *p < 0.05 vs. control group; #p < 0.05 vs. PBS group.

Fig. 4. Downregulation of miR-671 blocks the protective functions of Exo on cardiomyocytes. a miR-671 expression in the exosome precipitates and the supernatant after centrifugation using RT-qPCR; b Transfection efficiency of miR-671 inhibitor in adMSCs examined by RT-qPCR; c miR-671 expression in the exosomes determined by RT-qPCR; d Viability of OGD-cardiomyocytes after Exo-NC or Exo-inhibitor treatment determined by the MTT assay; e Apoptosis of OGD-cardiomyocytes after Exo-NC or Exo-inhibitor treatment examined by flow cytometry; f Protein levels of apoptosis-related factors (Bax and Bcl-2) and the fibrosis-related factor α-SMA in OGD-cardiomyocytes after Exo-NC or Exo-inhibitor treatment determined by western blot analysis; g Production of pro-inflammatory cytokines (IL-6 and TNF-α) in OGD-cardiomyocytes after Exo-NC or Exo-inhibitor treatment measured using ELISA kits. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by unpaired t test (a–e) or two-way ANOVA (f, g); ***p < 0.001 vs. supernatant; #p < 0.05 vs. NC inhibitor group; &p < 0.05 vs. Exo-NC group.

Fig. 5. miR-671 directly targets TGFBR2. a Putative target transcripts of miR-671 predicted using five bioinformatic systems; b A KEGG pathway enrichment analysis based on the above predicted mRNAs; c Expression of miR-671 and the mRNA expression of NF2, FBXW11, DLG, TGFBR2, DVL3, and YWHAZ in mouse cardiomyocytes after miR-671 mimic transfection examined by RT-qPCR; d Putative binding sequence between TGFBR2 and miR-671 and the mutant binding sequence for the construction of luciferase vectors; e Binding relationship between TGFBR2 and miR-671 validated through a dual luciferase assay; f Enrichment of miR-671 and TGFBR2 fragments in the compounds pulled down by anti-Ago2 examined by the RIP assay. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by two-way ANOVA (c, e, and f); *p < 0.05 vs. NC mimic group; ##p < 0.01 vs. anti-IgG.

Fig. 6. Exosomal miR-671 targets TGFBR2 and reduces Smad2 phosphorylation. a Protein levels of TGFBR2, Smad2 and the phosphorylation of Smad2 in each group of cardiomyocytes examined by western blot analysis; b Viability of cardiomyocytes after si-TGFBR2 transfection measured by the MTT assay; c Apoptosis of cardiomyocytes after si-TGFBR2 transfection examined by flow cytometry; d Protein levels of apoptosis-related factors (Bax and Bcl-2) and the fibrosis-related factor α-SMA in cardiomyocytes after si-TGFBR2 transfection determined by western blot analysis; e Production of pro-inflammatory cytokines (IL-6 and TNF-α) in cardiomyocytes after si-TGFBR2 transfection determined using ELISA kits. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by unpaired t test (b, c) or two-way ANOVA (a, d, and e); *p < 0.05 vs. Control group; #p < 0.05 vs. OGD group; &p < 0.05 vs. Exo-NC group; @p <0.05 vs. Exo-inhibitor + si-NC group.

Fig. 7. Exosomal miR-671 alleviates fibrosis and cell apoptosis in myocardial tissues in mice with MI. a miR-671 expression in murine myocardial tissues nearby the infarcted area detected using RT-qPCR; b Protein levels of TGFBR2 and p-Smad2 in murine myocardial tissues nearby the infarcted area examined by IHC staining; c Concentrations of IL-6 and TNF-α in the homogenate of murine myocardial tissues examined by ELISA kits; d Myocardial fibrosis in mouse examined by masson's trichrome staining; e Cell apoptosis in murine myocardial tissues examined by TUNEL assay. N = 8 in each group; representative images are provided. Data were collected from three experiments and exhibited as mean ± SEM. Differences were analyzed by one-way ANOVA (a, d, and e) or two-way ANOVA (b, c); *p < 0.05 vs. sham group; #p < 0.05 vs. Exo-NC group.

Fig. 8. A diagram for the molecular mechanism. AdMSC-derived exosomal miR-671 directly targets the TGFBR2/Smad2 axis, therefore alleviating OGD-induced damage on mouse cardiomyocytes in vitro and myocardial injury in model mice with MI.