IFN-γ enhances the efficacy of mesenchymal stromal cell-derived exosomes via miR-21 in myocardial infarction rats

Fig. 1. Characterization of Exos derived from MSCs. A Cup-shaped morphology of purified Ctrl-Exo and IFN-γ-Exo assessed by TEM. B Representative images of western blot showing the exosomal protein markers in Ctrl-Exo and IFN-γ-Exo groups. C The particle size distribution and particle concentration were analyzed by nanoparticle tracking analysis (n = 3). Confocal images showed that red fluorescence of dye DiI labeled Exos were endocytosed by HUVECs D and H9c2 E 6 and 24 h after incubation. Scale bar = 20 μm

Fig. 2. IFN-γ-Exo exhibited better protective effects on HUVECs and H9c2 cardiomyocytes than Ctrl-Exo in vitro. A TUNEL analysis for H9c2 cells (n = 3). Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei. Scale bars = 50 μm. B Flow cytometric analysis (n = 3). C Western blot analyzed Bax, Bcl2, and cleaved-caspase-3 protein levels in hypoxic and ischemic H9c2 cells incubated with PBS, Ctrl-Exo and IFN-γ-Exo. Relative protein levels are presented as the average expression normalized to β-Tubulin (n = 3). D Migration was monitored for 6 and 12 h after scratching in HUVECs cultured with PBS, Ctrl-Exo and IFN-γ-Exo (n = 3). E Tube formation of HUVECs incubated with PBS, Ctrl-Exo, and IFN-γ-Exo (n = 3). F TUNEL analysis for HUVECs (n = 3). Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei. Scale bars = 50 μm. Data are presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s correction. *P < 0.05; **P < 0.01; ***P < 0.001, NS not significant

Fig. 3. IFN-γ-Exo effectively preserved cardiac function in rats with MI in vivo. A The flowchart of experimental design in vivo. B DiI-labeled Exos were injected into the infarcted heart of rats for 6 h (50 μg Exos per rat). Representative images of post-MI heart sections stained with DiI-labeled Exos (red), cTNT(green), and DAPI (blue), Scale bar = 20 μm. C Representative echocardiographic images showed heart function among the different groups on the 14th and 28th day following MI and quantitative analysis of left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) among the different groups (n = 6 for the sham group, n = 5 for MI+PBS group, n = 6 for MI+Ctrl-Exo group, n = 6 for MI+IFN-γ-Exo group). D Masson’s trichrome stained myocardial sections on the 28th day following MI in rats treated with PBS, Ctrl-Exo, and IFN-γ-Exo. Scar tissue and viable myocardium were identified in blue and red, respectively (n = 4). Data were presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 4. IFN-γ-Exos were better at inhibiting apoptosis or promoting angiogenesis than IFN-γ-Exo in vivo. (A) Representative fluorescence images of macrophages in the border zone of ischemic hearts stained with CD68 (red) and α-actinin (green) (3 random fields per anima). (B) Representative photographs showing the TUNEL-positive cells (green) in the heart tissue (red) among the different groups. Quantitative analysis of the apoptotic rate at the border zone in CMs and non-CMs among the different groups (3 random fields per animal). (C) Representative fluorescence images of blood vessels in the border zone of ischemic hearts stained with CD31 (red) and α-actinin (green) (3 random fields per animal). Data are presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 5. miR-21 was a key component in IFN-γ-Exo-induced cardioprotection. Quantitative real-time PCR (qRT-PCR) analysis of miR-21 level in control and IFN-γ-primed MSCs (A) and Exos (B) (n = 3). C Quantitative real-time PCR (qRT-PCR) analysis of miR-21 level among groups at 3 days post-MI. (n = 6) (D) Heat map based on mRNAs sequence values (red represents high expression and the green represents low expression) between control and IFN-γ-primed MSCs. E Bioinformatic analysis network of the transcription factors interacting with the promoter of miR‐21. The yellow circle represents hsa‐miR‐21, and the blue and red circles represent transcription factors. F STAT1, STAT2, and FOXC1 were present in both up-regulated transcription factors and bioinformatics prediction analysis results. G qRT-PCR analysis of STAT1 level in MSCs treated with control and IFN-γ (n = 3). H qRT-PCR analysis of miR-21-5p level in MSCs treated with control, IFN-γ, IFN-γ + si-STAT1 (n = 3). I Luciferase reporter assay was used to detect the miR-21 promoter-reporter activity in 293T cells transfected with vector or STAT1 (n = 3). Data were presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA…

Fig. 6. MiR-21 directly targeted BTG2 in H9c2 cells. A Predicted miR-21 target sequence in BTG2-3′ UTRs. Target sequences of BTG2-3′ UTRs were mutated. B Luciferase assay of 293T cells transfected with BTG2-3′ UTR-WT or BTG2-3′ UTR-Mut reporter together with mimics NC or miR-21-5p mimics (n = 3). C qRT-PCR analysis of BTG2 level in H9C2 cells treated with mimics NC and miR-21-5p mimics (n = 3). D Western blot analyzed BTG2 protein levels in H9c2 cells treated with control, mimics NC and miR-21-5p mimics. Relative protein levels were presented as the average expression normalized to β-Tubulin (n = 3). E qRT-PCR analysis of BTG2 level in H9c2 cells treated with IFN-γ-Exo and si-IFN-γ-Exo (n = 3). Data were presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 7. Overexpression of BTG2 aggravated OGD-induced injury damage and reversed the protective effect of miR-21. A Western blot analyzed Bax, Bcl2, and cleaved-caspase-3 protein levels in hypoxic and ischemic H9c2 cells treated with PBS, vector and BTG2. Relative protein levels were presented as the average expression normalized to GAPDH (n = 3) and TUNEL analysis (n = 3) B Green, TUNEL-positive nuclei; blue, DAPI-stained nuclei. Scale bars = 50 μm. And flow cytometric analysis (n = 3) (C). D H9c2 cells treated with control, OGD, OGD + miR-21 mimics and OGD + miR-21 mimics + BTG2. Relative Bax, Bcl2, and cleaved-caspase-3 protein levels among different groups were presented as the average expression normalized to β-Tubulin (n = 3). Data were presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Bonferroni’s correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

Fig. 8. Exosomal miR-21 derived from IFN-γ-primed MSCs improved cardiac function after infarction by promoting angiogenesis and inhibiting apoptosis