miR-182/183-Rasa1 axis induced macrophage polarization and redox regulation promotes repair after ischemic cardiac injury

Figures

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  Fig. 1. Functional assessment after CBSCs and CBSC-EV transplantation post-MI. A. Schema for experimental design; Mice received Saline, CBSCs or CBSC-EV treatment right after myocardial infarction surgery. Serial echocardiography measurements were performed at baseline, 1 week, 2 weeks, 4 weeks and 6 weeks post-MI. B. Kaplan-Meier curves of survival rate post-MI. C. Reduction in infarct size in CBSCs and CBSC-EV treated animals determined by Masson's Trichrome staining in heart tissues, **p < 0.01, ***p < 0.001. D. Ejection Fraction and Fractional Shortening was improved in mice treated with CBSCs and CBSC-EV compared to saline 2–6 weeks post-MI. (n = 6 animals per group), *p < 0.05, **p < 0.01, CBSCs versus Saline, †p < 0.01 CBSCs-EV versus Saline. E. Representative images of vector diagrams by speckle tracking analysis were shown. F. Heart weight to body weight ratio at terminal. With cardiomyocyte cross-sectional area (CSA) determined with wheat germ agglutinin (WGA) staining in left ventricle boarder zone (LV-BDZ), left ventricle remote zone (LV-RMTZ) and right ventricle (RV) respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  Fig. 2. CBSCs and CBSC-EV modulated cytokine profile and restricted cell deaths acutely post-MI. A. Plasma analysis of cytokine protein levels 24 h after MI; 1) CXCL10, 2) CXCL1, 3) M-CSF, 4) CCL2, 5) CCL12, 6) CXCL9, 7) SDF-1, ****p < 0.0001. B. Gene expression levels of pro-inflammatory cytokines such as IL-18, IL-6, and anti-inflammatory cytokines such as IL-10 in infarct border zone area 24 h after MI (n = 5 animals in Saline, CBSCs, CBSC-EV treated groups), ****p < 0.0001. C. Myocardium apoptotic cells 2 days after MI measured by TUNEL staining (TUNEL: green; DAPI: blue; α-sarcomeric actin: red). **P < 0.01 versus saline. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 3. CBSCs and CBSC-EV promote pro-reparative immune responses in the myocardium post-MI. A. Percentage of CD86 (M1 macrophage marker) positive cells and CD206 (M2 macrophage marker) positive cells among total DAPI determined by immune fluorescent staining of left ventricular heart tissues 5-day post-MI, *p < 0.05，**p < 0.01, ***p < 0.001. B. Schema of cardiac immune cell isolation and Flow cytometry quantification performed 5, 7 or 14-day post-MI. C. Percentage of CD45, CD206 and CD86 positive cells respectively determined by Flow cytometry, ***p < 0.001 (n = 3 animals per group). D. CBSCs and CBSC-EV treatment increased CD3 positive cells infiltration after MI measured by immunofluorescence analysis. E. Percentage of CD8, CD4 in total CD45 positive cell population and FoxP3 positive cells in CD4 positive cell population 14 days post-MI determined by Flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus saline.

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  Fig. 4. CBSCs derived extracellular vesicles (CBSC-EV) regulates bone marrow derived macrophages (BMDMs) polarization and phagocytosis in vitro. A. CBSC-EV treated bone marrow derived macrophages (BMDMФs) showed increased CD206+ population compared to control RPMI media without LPS stimulation determined by Flow cytometry, *p < 0.05. B. Under LPS stimulation, CBSC-EV treated BMDMs showed decreased CD86+ macrophage population, increased CD163/CD206 double positive macrophages and enhanced phagocytosis capacity determined by Flow cytometry, *p < 0.05, ****p < 0.0001. C. Expression levels of pro- and anti-inflammatory genes determined by qRT-PCR with or without LPS stimulation in BMDMs, ***p < 0.001, ****p < 0.0001.

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  Fig. 5. CBSCs derived extracellular vesicles (CBSC-EV) possess significantly different transcriptome profile compared with extracellular vesicles isolated from endothelial progenitor cells (EPCs) and cardiosphere-derived cells (CDCs). A. Principal component analysis (PCA) of the transcriptome profiles of CBSC-EV, CDC-EV and EPC-EV. B. Number of differentially expressed genes (DEGs) when comparing CBSC-EV versus EPC-EV and CDC-EV respectively. Venn diagram depicted the number of overlapped DEGs between comparisons. C. Top GO derived from gene ontology enrichment analysis of molecular function comparing CBSC-EV versus EPC-EV and CDC-EV respectively. D. Pie-charts depicted the compositions of DEGs, miRNA counts 9.9% and 9.7% of DEGs respectively. E. Volcano plots representing relative gene expression levels with fold change (x-axis) and p value (y-axis) comparing CBSC-EV versus EPC-EV or CDC-EV. MiR-182 and miR183 rank top of the most significant upregulated genes.

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  Fig. 6. CBSCs derived extracellular vesicles (CBSC-EV) modulates macrophage polarization through miR-182/183. A. Gene expression levels of IL6, CD86, IL10, IL18 and Spp1 from bone marrow derived macrophages (BMDMs) in culture media (NT), or after treated with LPS with miRNA negative control or miR-182/183 mimics. B. Percentage of F4/80 positive cells, CD86 and CD80 positive cells in F4/80+ populations were determined with Flow cytometry analysis. C. Percentage of phagocytotic beads positive cells and CD206 positive cells in F4/80+ populations determined with Flow cytometry analysis. D. Gene expression levels of IL6, CD86, IL10, IL18 and Spp1 BMDMs in culture media (NT), or after treated with LPS and CBSC-EV with or without miR-182/183 inhibitors. E. Percentage of CD86, CD206 and phagocytosis beads (FITC) positive cells were determined with Flow cytometry analysis. F. Seahorse assay determined the OXPHOS rate in BMDMs treated without LPS or with 24 h LPS treatment, that have either miRNA negative control or miR-182/183 mimics treatment. G. Mitochondrial ROS and cellular ROS level from miRNA negative control and miR-182/183 mimics pre-treated BMDMs after 3 h LPS treatment. *p < 0.05, **p <…

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  Fig. 7. miR-182/183 inhibits Rasa1 and activates downstream pathways. A. Ras p21 activator protein 1 (Rasa1) is the predicted target gene of miR-182-5p. Inhibition of Rasa1 by miR-182/183 was confirmed by luciferase assay in HEK293 cells. B. Protein level of Rasa1, Akt and phosphorylated-Akt (p-Akt) in BMDMs after treated with LPS and miRNA negative control of miR-182/183 mimics were quantified. BMDMs without LPS treatment served as non-treatment (NT) control. C. Protein level of pERK, c-Myc, catalase and SOD2 in BMDMs after treated with LPS and miRNA negative control of miR-182/183 mimics were quantified. BMDMs without LPS treatment served as non-treatment (NT) control. D. Illustration of proposed signaling pathways of miR-182/183. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 8. Illustration of the reparative mechanisms of CBSC-EV.