Nicorandil-Pretreated Mesenchymal Stem Cell-Derived Exosomes Facilitate Cardiac Repair After Myocardial Infarction via Promoting Macrophage M2 Polarization by Targeting miR-125a-5p/TRAF6/IRF5 Signaling Pathway

Figures

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  Figure 1. Characterization of MSC-exo. (A) surface markers of bone marrow MSC detected by flow cytometry were shown as CD45−CD11−CD90+CD29+. (B) Representative Western blot images of exosomal protein markers (Alix and Tsg101) and MSC markers (CD73). (C) MSC-exo and MSCNIC-exo manifested cup-shaped morphology under TEM. Scale bar = 200 μm. (D) The particle size and concentration were analyzed by nanoparticle tracking analysis. No significant difference was found between MSC-exo and MSCNIC-exo. (E) Representative confocal images suggested that exosomes were endocytosed by BMDM. Scale bar = 10 μm.

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  Figure 2. MSCNIC-exo strengthened cardiac repair in AMI rats. (A) Representative M-mode echocardiogram in different groups 4 weeks post-AMI. (B) Cardiac function including LVEF, LVFS, LVEDV, and LVESV were assessed 4 weeks post-AMI (n=9–10). (C and D) Representative images of heart cross-sections stained with Masson’s trichrome staining (C) and Sirius Red staining (D) 28 days after MI. Scale bar = 2000 μm. (E and F) Quantification of infarct size in C (E) and collagen area in D (F) (n=8–10). (G) Representative HE staining images of the border zone of the infarcted heart. Scale bar= 100 μm. (H) IL-6 expression levels in the infarct border zone tissue of rat hearts using ELISA (n=5). (I and J) Representative images of CD31 positively stained capillaries (I) and α-SMA positively stained arterioles (J) at the border zone. Scale bar= 50 μm. (K and L) Quantification of capillaries density in I (K) and arteriole density in J (L) (5 random fields per animal; n=4 for each group). All data are presented as mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey’s test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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  Figure 3. MSCNIC-exo polarized macrophage toward M2 phenotype both in vivo and in vitro. (A) M2 macrophage at the border zone on day 3 post-MI was identified by labeling with CD206 (green) and CD68 (red) and nuclei (blue). (B) Quantification of the CD206+CD68+/CD68+ ratio of each group in A (5 random fields per animal; n=5 for each group). (C and D) mRNA expression levels of M1 macrophage markers IL-1β, IL-6, and TNF-α in infarcted myocardium at day 3 after AMI (n=5). (E) mRNA expression levels of M2 macrophage markers Mrc1, Tgfb1, IL-4R, and Arg1 in infarcted hearts at day 3 after AMI (n=5). (F and G) Representative western blot image (F) and quantification of protein expressions (G) of Arg1 in infarcted hearts at day 3 after AMI (n=5). (H) IL-1β and TNF-α levels in the supernatants of different group using ELISA (n=3) (I) IL-4, Vegfa and IL-10 levels in the supernatants of different group using ELISA (n=3). (J) mRNA levels of M2 macrophage markers Mrc1, Tgfb1, IL-4R, IL-10, Vegfa, and Arg1 in BMDM of each group (n=6). (K) mRNA expression levels of M1 macrophage markers IL-1β, and TNF-α in BMDM (n=6). (L) Representative western blot image of Arg1 expression in BMDM. (M) Quantification of…

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  Figure 4. Differential expressed exosomal miRNA expression and miR-125a-5p exerted as a main effector for cardiac repair capacity of MSCNIC-exo. (A) Heatmap of differentially expressed miRNAs between MSC-exo and MSCNIC-exo (n=3). (B) Volcano plot showing log2 (Fold change) on the x-axis and -log10 (P value) on the y-axis (n=3). (C) miRNAs upregulated in MSCNIC-exo which engaged in inflammation modulation or cardiac repair. (D and E) miR-125a-5p expression levels in MSC (D) (n =5) and exosome (E) (n =6). (F) miR-125a-5p expression levels in MSC or MSCNIC transfected with miR-125a-5p mimics, inhibitors, or its negative control (NC) (n = 4). (G) Representative images of echocardiograms at 28 days post-AMI. (H) Analysis of LVEF, LVFS, LVEDV, and LVESV 4 weeks post-AMI (n = 9–10). (I and J) Representative images of Masson’s trichrome staining (I) and Sirius red staining (J) 4 weeks post-AMI. Scale bar = 2000 μm. (K and L) Analysis of Masson’s trichrome staining (K) and Sirius red staining (L) at 4 weeks post-AMI (n=7). (M) Representative HE staining images of the border zone of the infarcted heart. Scale bar= 100 μm. (N and O) Representative images of CD31 positively stained capillaries (N) and…

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  Figure 5. miR-125a-5p played a crucial role in MSCNIC-exo mediated M2 macrophage polarization both in vivo and in vitro. (A) M2 macrophage at the border zone 3 days post-AMI was identified by staining with CD206 (green) and CD68 (red) and nuclei (blue). (B) Quantification of the CD206+CD68+/CD68+ ratio of each group in (E)(5 random fields per animal; n=5 for each group). (C) mRNA expression of M2 macrophage markers Mrc1, Tgfb1, IL-4R, and Arg1 in infarcted hearts 3 days post-AMI (n=5). (D) mRNA expression of M1 macrophage markers IL-1β, IL-6, and TNF-α in infarcted hearts 3 days post-injury (n=5). (E) Representative western blot image of Arg1 expression in infarcted hearts. (F) Quantification of protein expressions of Arg1 in infarcted hearts 3 days post-AMI (n=5). (G) mRNA expression levels of M2 macrophage markers Mrc1, Tgfb1, IL-4R, and Arg1 in BMDM (n=5). (H) mRNA expression levels of M1 macrophage markers IL-1β and TNF-α in BMDM (n=5). (I) Representative western blot image of Arg1 and Mrc1 expression in BMDM. (J and K) Quantification of protein expression levels of Mrc1 (J) and Arg1 (K) in BMDM (n=5). All data are presented as mean ± SEM. Statistical analysis was performed with…

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  Figure 6. miR-125a-5p participated in the polarization of M2 macrophage by regulating TRAF6. (A) miR-125a-5p expression levels in M1 and M2 macrophage (n=6). (B) miR-125a-5p expression levels in BMDM transfected with miR-125a-5p mimics, inhibitors, or its negative control (NC) (n = 3). (C and D) mRNA expression of M1 macrophage markers (IL-1β and TNF-α) (C) and M2 markers (Mrc1, Tgfb1, IL-4R, and Arg1) (D) in BMDM after transfected with miR-125a-5p mimics, inhibitors, or corresponding NC (n=5). (E–G) Representative western blot image (E) and quantification of protein expression (F and G) of M2 markers (Mrc1 and Arg1) in BMDM of each group (n=5). (H) Relative luciferase activity of miR-125a-5p mimics-treated HEK293T cells which transfected with TRAF6-wild-type or TRAF6-mutant plasmids was determined. (I) TRAF6 mRNA expression level in BMDM (n=4). (J and K) Representative western blot image (J) and quantification of the protein expression level (K) of TRAF6 in BMDM (n=5). (L) TRAF6 mRNA expression level in BMDM after transfected with miR-125a-5p mimics, inhibitors or corresponding NC (n=5). (M–O) Representative western blot image (M) and quantification of protein expression of TRAF6 and IRF5…

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  Figure 7. IRF5 may be a down-stream effector of TRAF6 in M2 macrophage polarization. (A) TRAF6 mRNA expression level in BMDMs after si-TRAF6 or corresponding NC transfection (n=4). (B–D) Representative western blot image (B) and quantification of the protein expression level of TRAF6 (C) (n=4) and IRF5 (D) (n=6) in BMDM after transfected with si-TRAF6 or corresponding NC. (E and F) mRNA expression of M2 markers (Mrc1 and Arg1) in BMDM (n=6). (G and H) mRNA expression of M1 markers (IL-1β and TNF-α) in BMDM (n=5–6). (I and J) Representative western blot image (I) and quantification of protein expression levels of Arg1 (J) in BMDM (n=4). All data are presented as mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey’s test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.