Exosomal miR‐218‐5p/miR‐363‐3p from Endothelial Progenitor Cells Ameliorate Myocardial Infarction by Targeting the p53/JMY Signaling Pathway

Figure 1. Differential miRNA expression profiles between EPCs and EPC-Exos. (a) Hierarchical clustering of differentially expressed miRNAs between EPCs and EPC-Exos, which are displayed on a scale from green (low) to red (high). (b) Hierarchical clustering heat map of the clearly distinct differentially expressed miRNAs in EPCs and EPC-Exos, ranging from red (relatively high miRNA expression) to blue (relatively low miRNA expression). The miRNAs in the heat map are clustered based on the relative expression patterns. (c) The relative expression levels of sixteen differentially expressed miRNAs (eight upregulated and eight downregulated) in EPC-Exos compared with EPCs were measured with qRT-PCR. The data are presented as the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, EPC-Exos versus EPCs.

Figure 2. miR-218-5p and miR-363-3p promoted angiogenesis and proliferation of CFs. (a) The relative expression levels of miR-6087, miR-218-5p, and miR-363-3p mimics and inhibitors were measured with qRT-PCR. The data are presented as the mean ± SD (n = 3). ∗∗P < 0.01 and ∗∗∗P < 0.001, mimic-Exo or inhibitor-Exo versus NC-Exo. (b) Tube formation capability and relative tube length were increased in the miR-218-5p and miR-363-3p mimic-Exo groups and decreased in inhibitor-Exo groups. The data are presented as the mean ± SD (n = 3). ∗P < 0.05 and ∗∗∗P < 0.001, mimic-Exo or inhibitor-Exo versus NC-Exo. (c) CCK8 assay to detect the effect of exosomal miR-218-5p and miR-363-3p on cell proliferation. The data are presented as the mean ± SD (n = 3). ∗#P < 0.05 and ∗∗P < 0.01, mimic-Exo or inhibitor-Exo versus NC-Exo. (d) BrdU labelling and flow cytometry assay to detect the effect of exosomal miR-218-5p and miR-363-3p on cell proliferation. The data are presented as the mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, mimic-Exo or inhibitor-Exo versus NC-Exo.

Figure 3. miR-218-5p and miR-363-3p promoted mesenchymal-endothelial transition in CFs. (a) A phagocytosis assay was performed to detect the engulfment of Dil-Ac-LDL and binding to endothelial-specific lectin FITC-UEA-1 by CFs treated with mimic-218-Exo or inhibitor-218-Exo. Magnification: 120x. (b) A phagocytosis assay was performed to detect the engulfment of Dil-Ac-LDL and binding to endothelial-specific lectin FITC-UEA-1 by CFs treated with mimic-363-Exo or inhibitor-363-Exo. Magnification: 120x. (c) qRT-PCR detection of the relative expression levels of CD31, VEGFR-2, α-SMA, and vimentin in CFs treated with mimic-218-Exo or inhibitor-218-Exo. The data are presented as the mean ± SD (n = 3). ∗∗P < 0.01 and ∗∗∗P < 0.001, mimic-Exo or inhibitor-Exo versus NC-Exo. (d) qRT-PCR detection of the relative expression levels of CD31, VEGFR-2, α-SMA, and vimentin in CFs treated with mimic-363-Exo or inhibitor-363-Exo. The data are presented as the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, mimic-Exo or inhibitor-Exo versus NC-Exo.

Figure 4. Immunofluorescence staining to detect the relative expression levels of α-SMA (a), vimentin (b), CD31 (c), and VEGFR-2 (d) in CFs treated with miR-218-5p and miR-363-3p mimics and inhibitors. The photographs were taken with a laser confocal microscope (Zeiss, LSM700), magnification: 120x.

Figure 5. Western blot analysis to detect the relative expression levels of CD31, VEGFR-2, α-SMA, and vimentin in CFs treated with miR-218-5p (a) and miR-363-3p (b) mimic-Exo and inhibitor-Exo. The data are presented as the mean ± SD (n = 3). ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001, mimic-Exo or inhibitor-Exo versus NC-Exo.

Figure 6. p53/JMY as a target of miR-218-5p and miR-363-3p. (a) The predicted binding sites for miR-218-5p in the p53 promoter. (b) Schematic diagram of pGL3-Basic vector construction harboring p53 promoter binding sites. (c) Relative luciferase activity (F/R) assay for miR-218-5p binding to the p53 promoter. cel-miR-39 mimic served as the negative control. The data are presented as the mean ± SD (n = 3), ∗∗P < 0.01, miR-218-5p mimic versus NC or cel-miR-39 mimic. (d) The predicted binding sites for miR-363-3p in the 3′UTR of JMY. (e) Schematic diagram of psi-Check2 vector construct harboring the 3′UTR of JMY binding sites. (f) Relative luciferase activity (R/F) assay for miR-363-3p in the 3′UTR of JMY. The data are presented as the mean ± SD (n = 3), ∗∗P < 0.01, miR-363-3p mimic versus NC or cel-miR-39 mimic. cel-miR-39 mimic served as the negative control. (g) qRT-PCR to detect the relative expression levels of p53 and JMY in CFs in the mimic-Exo and inhibitor-Exo groups. The data are presented as the mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, mimic-Exo or inhibitor-Exo versus NC-Exo.

Figure 7. Effect of miR-218-5p and miR-363-3p on p53/JMY. (a, c) Immunofluorescence staining for the relative expression levels of p53 and JMY in CFs, in the miR-218-5p/miR-363-3p mimic-Exo and inhibitor-Exo groups. (b, d) Western blot analysis to detect the relative expression levels of p53 and JMY in miR-218-5p/miR-363-3p mimic-Exo and inhibitor-Exo groups in CFs, respectively. The photographs were taken with a laser confocal microscope (Zeiss, LSM700), magnification: 120x. The data are presented as the mean ± SD (n = 3). ∗P < 0.05 and ∗∗P < 0.01, mimic-Exo or inhibitor-Exo versus NC-Exo. (e) Western blot analysis to detect the relative expression levels of p53 and JMY in si-p53 and OE-JMY CF groups, respectively. The data are presented as the mean ± SD (n = 3). ∗∗P < 0.01, si-p53 or OE-JMY group versus mimic-218-Exo or mimic-363-Exo group. (f) The tube formation capability and relative tube length were decreased in the si-p53 and OE-JMY groups. The data are presented as the mean ± SD (n = 3). ∗∗P < 0.01, si-p53 or OE-JMY group versus mimic-218-Exo or mimic-363-Exo group. (g) BrdU labelling and flow cytometry assay to detect the effect of si-p53 and OE-JMY on cell proliferation. The data…

Figure 8. Effect of exosomal miR-218-5p and miR-363-3p on cardiac features and function in MI rats. (a) A representative ECG of LCA-induced MI rats in the different groups (sham, model, miR-218-5p, and miR-363-3p mimic-Exo). (b) TTC staining of heart tissues from the sham, model, and mimic-Exo groups. The data are presented as the mean ± SD (n = 10). ∗∗P < 0.01 and ∗∗∗P < 0.001, mimic-Exo versus model. (c, d) qRT-PCR to detect the relative expression levels of miR-218-5p, miR-363-3p, p53, and JMY in the mimic-Exo groups. (e) The protein expression of p53, JMY, collagen-1/3, and Timp-1-4 in the different groups. The data are presented as the mean ± SD (n = 10). ∗P < 0.05 and ∗∗P < 0.01, mimic-Exo versus model.

Figure 9. Exosomal miR-218-5p and miR-363-3p alleviate LCA-induced myocardial infarction. (a) H&E, Masson, and Van Gieson staining of heart tissues treated with EPC-Exos containing miR-218-5p mimic or miR-363-3p mimic. Magnification: 40x. (b) Immunofluorescence staining for the relative expression levels of α-SMA in heart tissues treated with EPC-Exos containing miR-218-5p mimic or miR-363-3p mimic. Magnification: 40x. (c) Immunohistochemistry for vWF in heart tissues treated with EPC-Exos containing miR-218-5p mimic or miR-363-3p mimic. (d) The number of vessels/field area in the remodeled left ventricle. The data are presented as the mean ± SD (n = 3). ∗∗P < 0.01, mimic versus model.