Extracorporeal Cardiac Shock Wave-Induced Exosome Derived From Endothelial Colony-Forming Cells Carrying miR-140-3p Alleviate Cardiomyocyte Hypoxia/Reoxygenation Injury via the PTEN/PI3K/AKT Pathway

Figures

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  FIGURE 1. Characterization of rat bone-derived endothelial colony-forming cells (ECFCs). (A) On day 3, day 7, day14, and day 21 of cellular morphology under an inverted microscope, scale bar = 100 μm. (B) Double staining of ECFCs positive for Dil-AcLDL and FITC-UEA-1 under fluorescent microscope, scale bar = 20 μm. (C) Cell surface markers (CD31, CD34, CD133, VEGFR-2, CD14, and CD45) of ECFCs were identified by flow cytometry. (D) Tube formation assay of ECFCs was evaluated under an inverted microscope, left: scale bar = 100 μm; right: scale bar = 50 μm.

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  FIGURE 2. Characterization of exosomes derived from ECFCs. (A-B). exosome morphology was evaluated via transmission electron microscopy, scale bar = 100–200 nm. (C-D) The size distribution of exosomes was analyzed by nanoparticle tracking analysis. The diameter of exosomes ranges mainly from 30 to 150 nm. (E) Western blot analysis of protein levels of CD9, CD63, CD81, and calnexin in cell lysis and ECFCs-exo. (F) H9c2 cells were incubated with PKH26 (positive control), PKH26-labeled exosomes, and unlabeled exosomes, nuclei were counterstained with DAPI, and the red fluorescence of PKH-26 in cells was traced by fluorescence microscope, scale bar = 20 μm.

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  FIGURE 3. Extracorporeal cardiac shock wave (ECSW)-induced ECFCs-exo protected cardiomyocytes against hypoxia/reoxygenation injury. (A) The cell viability of H9c2 cells after hypoxia/reoxygenation (H/R) injury treated with CON-exo and SW-exo at different concentrations from 0 to 40 µg/ml. *p < 0.05, **p < 0.01, within group; ##p < 0.01, between groups. (B–E) Quantitative analysis of the cell viability (B) by cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) activity (C) by reagent kit, cell apoptosis rate (D) by flow cytometry, and the production of reactive oxygen species (ROS) (E) by dichlorodihydrofluorescein diacetate (DCFH-DA) staining were shown for each group, including NC, H/R, and H9c2 cells after H/R injury treated with PBS, CON-exo, and SW-exo. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, E: scale bar = 50 μm.

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  FIGURE 4. ECSW-induced ECFCs-exo promoted the expression of Bcl-2 and inhibited the expressions of Bax, cleaved caspase-3, and NF-κB p65 in H9c2 cells. (A) Western blotting was used to detect the expression of Bcl-2, Bax, cleaved caspase-3, and NF-κB p65 in cardiomyocytes, normalized to the GAPDH protein. (B–E) Protein bands were analyzed using ImageJ software. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01.

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  FIGURE 5. Characterization of miRNA sequences in SW-exo and CON-exo, and functional enrichment analysis. (A) Relative amounts of expression for 18 miRNAs enriched in SW-exo and CON-exo are displayed in a heat-map (p < 0.05, n = 4). (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis of differential target genes was displayed in a scatter plot. (C) Venn diagrams of detected miRNAs (SW-exo vs. CON-exo) shows a large overlap between the two groups. (D) Mapping of miR-140-3p and target gene network in the PI3K/AKT signaling pathway was drawn by Cytoscape software. (E) The relative expression of miR-140-3p in ECFCs and ECFCs-exo were detected by qRT-PCR normalized to U6 (n = 3). *p < 0.05, **p < 0.01.

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  FIGURE 6. SW-exo mainly delivered pivotal miR-140-3p to protect cardiomyocytes against hypoxia/reoxygenation injury. (A–D) Quantitative analysis of the cell viability (A) by CCK-8 assay, LDH activity (B) by reagent kit, cell apoptosis rate (C) by flow cytometry, and the production of ROS (D) by DCFH-DA staining are shown for each group, including H9c2 cells after H/R injury treated with miR-140-3p mimic, mimic NC, miR-140-3p inhibitor, inhibitor NC, and SW-exo. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01, E: scale bar = 50 μm.

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  FIGURE 7. SW-exo delivered pivotal miR-140-3p to H9c2 cells for promoting the expression of Bcl-2 and inhibiting the expressions of Bax, cleaved caspase-3, and NF-κB p65. (A) Western blotting was used to detect the expression of Bcl-2, Bax, cleaved caspase-3, and NF-κB p65 in cardiomyocytes, normalized to the GAPDH protein. (B–E) Protein bands were analyzed using ImageJ software. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01.

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  FIGURE 8. SW-exo containing miR-140-3p inhibited the expression of PTEN. (A) Profile of reporter vectors expressing luciferase fused with the 3′UTR of the PTEN wild type (WT) or mutant (MUT). (B) Potential binding sites (red) for miR-140-3p on PTEN 3′UTR are predicted, and mutated sequences are presented. (C) The relative luciferase activity was measured by dual-luciferase reporter gene assay (n = 3). (D) The relative expression of miR-140-3p and PTEN mRNA in H9c2 cells after transfected with miR-140-3p mimic or mimic NC, miR-140-3p inhibitor, or inhibitor NC were detected by qRT-PCR normalized to U6 and GAPDH (n = 3). *p < 0.05, **p < 0.01.

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  FIGURE 9. SW-exo exerted regulatory effect on the PTEN/PI3K/AKT signaling pathway via miR-140-3p in H9c2 cells. (A–D) Western blot was used to detect the expression of PTEN, p-PI3K, PI3K, p-AKT, and AKT, normalized to the GAPDH protein. (E, F) The relative expression of miR-140-3p and PTEN mRNA in H9c2 cells were detected by qRT-PCR, normalized to U6 and GAPDH. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01.

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  FIGURE 10. miR-140-3p inhibitor reversed the effect of SW-exo by blunting the PTEN/PI3K/AKT signaling pathway in H9c2 cells. (A–D) Western blot was used to detect the expression of PTEN, p-PI3K, PI3K, p-AKT, and AKT, normalized to the GAPDH protein. (E, F) The relative expression of miR-140-3p and PTEN mRNA in cardiomyocytes were detected by qRT-PCR, normalized to U6 and GAPDH. Data are presented as mean ± SD, n = 3. *p < 0.05, **p < 0.01.