Exosomes derived from mir-214-3p overexpressing mesenchymal stem cells promote myocardial repair

Fig. 1. Characterization of huMSCs and exosomes which are derived from huMSCs. (A-B) Morphology and multiple differentiation potential of huMSCs. Scale bar = 100 μm. (C) Fluorescence microscopy image of lentivirus transfection in Ctrl-MSC and miR-214OE-MSCs groups. Scale bar = 20 μm. (D) The expression of miR-214-3p in cells and exosomes was verified by real-time PCR. (n = 3 biological replicates for each group). (E) Cup-shaped morphology of purified Ctrl-Exo and miR-214OE- Exo assessed by TEM. (F) NTA representative screen shot video; the bright white point represents a particle movement. (G) The particle size distribution and particle concentration were analyzed by nanoparticle tracking analysis. (H) The exosomal protein markers of TSG101, CD63, and CD81 in Ctrl-Exo and miR-214OE-Exo groups. (I) Confocal images of Dil labeled exosomes (red fluorescence) were endocytosed by HUVECs and NRCMs 6 and 24 h after incubation. Scale bar = 20 μm

Fig. 2. Effects of miR-214OE-Exo on HUVECs and NRCMs. (A, D) Migration of HUVECs cultured with PBS, Ctrl-Exo and miR-214OE-Exo was showed in Figure A & D. Scale bar = 100 μm. (n = 3 biological replicates for each group). (B, E) Tube formation of HUVECs incubated with PBS, Ctrl-Exo and miR-214OE-Exo and quantification analysis. Scale bar = 100 μm. (n = 3 biological replicates for each group). (C, F) Representative images of the apoptosis determined by TUNEL staining (red) in NRCMs among different groups and Quantitative analysis of the percentage of the apoptotic NRCMs among the different groups. Scale bar = 50 μm. Continuous and categorical variables were described by means ± SEM and percentages, respectively. n = 3 for each group. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001

Fig. 3. Distribution of miR-214-loaded exosomes in infarcted hearts. (A) Flow chart of in vivo experiment design. (B-C) The expression level of CD31 (B) and α-actin (C) protein was detected by immunofluorescence 6 h after intramyocardial injection of Dil labeled exosomes. The white arrow indicate the injection site. The yellow arrow indicates that exosomes were absorbed by endothelial cells and cardiomyocytes. Yellow squares indicate the region near exothelium and yellow boundaries show the region near myocardium. Scale bar = 50 μm. (D) FISH analysis in infarcted LV myocardium of SD Rats. Cy3-miR-214 probe (red) was used to detect miR-214. Concentrated miR-214 was indicated by yellow arrowheads. Scale bar = 20 μm

Fig. 4. miR-214OE-Exo effectively preserved cardiac function in rats with MI in vivo. (A) Representative images of M-mode echocardiography captured 28 days after MI between different groups. (B) The representative Masson’s trichrome-stained myocardial cross-Sect. 4 weeks after MI with the injection of PBS, Ctrl-Exo, and miR-214OE-Exo. Red frame indicates the infarct zone and yellow frame indicates the border zone. Scale bar = 20 μm. (C) HE staining at the border zone 28 days post MI. (D-E) Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) measured by echocardiography 28 days after MI. (n = 5 animals for each group). (F) Quantitative analysis of heart fibrosis between different groups. (n = 5 animals for each group)

Fig. 5. miR-214OE-Exo promoted angiogenesis and cardiomyocyte survival in infarcted hearts. (A) Neovascularization at the border zone 28 days after MI was evaluated by staining with CD31 (red) and nuclei (blue). Scale bar = 50 μm. (B) α-SMA positively stained arterioles at the border zone 4 weeks after MI. Scale bar = 50 μm. (C) Representative images showing the terminal deoxynucleotidyl transferase-mediated dUTP nick end‐labeling (TUNEL) positive cells in the heart tissue among the different groups. Scale bar = 50 μm. (D) Cleaved Caspase-3 staining at the border zone 28 days post MI. Scale bar = 50 μm. (E and F) Quantitative analysis of the CD31(E) and α-SMA(F) density in the heart tissue of rats from the different groups. (n = 5 animals for each group). (G) Quantitative analysis of the apoptotic rates among the different groups. (n = 5 animals for each group). (H) Quantitative analysis of cleaved-caspase-3+ cells at the border zone among the different groups. (n = 5 animals for each group)

Fig. 6. Exosomal miR-214-3p attenuated cardiomyocyte injury via regulation of p-AKT by targeting PTEN. (A) Western blot analysis showing the level of p-AKT、AKT、Bcl2 and Bax among the different groups. (B) Neonatal rat cardiomyocytes (NRCMs) were treated with miR-214OE-Exo or miR-214OE-Exo + MK2206, then exposed to H/SD. Western blot analysis showing the level of p-AKT、AKT、Bcl2 and Bax among the different groups. (C) Quantitative analysis of the level of p-AKT、AKT、Bcl2 and Bax among the different groups. (n = 3 biological replicates for each group). (D) Quantitative analysis of the level of p-AKT、AKT、Bcl2 and Bax in Fig. 6B among the different groups. (n = 3 biological replicates for each group). (E) The predicted miR-214 targeting sequence in the 3’-UTR of PTEN. (F) Western blot analysis showing the PTEN levels after administration of Ctrl-Exo or miR-214OE-Exo in HUVECs. (n = 3 biological replicates for each group). (G) Western blot analysis of PTEN after administration of Ctrl-Exo or miR-214OE-Exo in NRCMs. (n = 3 biological replicates for each group). (H) Luciferase reporter assay was performed to confirm the target gene of miR-214. (n = 3 biological replicates for each group)

Fig. 7. miR-214OE-Exo promote migration, tube formation and apoptosis inhibition by targeting PTEN. (A) Expression level of PTEN in HUVECs after transfection with plasmids containing Vector or PTEN. (n = 3 biological replicates for each group). (B) Expression level of PTEN in HUVECs after transfection with Vector or PTEN followed by administration miR-214OE-Exo. (n = 3 biological replicates for each group). (C-D) Tube formation assays were utilized to verify the functional role of PTEN on angiogenesis in HUVECs. Scale bar = 100 μm. (n = 3 biological replicates for each group). (E-F) Scratch assays were used to validate the functional effect of PTEN on migration of HUVECs. Scale bar = 100 μm. (n = 3 biological replicates for each group). (G-H) The apoptosis rate of HUVECs transfected with plasmids containing Vector or PTEN followed by administration miR-214OE-Exo was detected by Annexin V/FITC/PI double-staining flow cytometry. (n = 3 biological replicates for each group). (I-J) The apoptosis rate of NRCMs transfected with plasmids containing Vector or PTEN followed by administration miR-214OE-Exo was detected by Annexin V/FITC/PI double-staining flow cytometry. (n = 3 biological…