Atorvastatin-pretreated mesenchymal stem cell-derived extracellular vesicles promote cardiac repair after myocardial infarction via shifting macrophage polarization by targeting microRNA-139-3p/Stat1 pathway

Figures

• 

  Fig. 1. The characterization of atorvastatin-pretreated bone marrow MSC-derived EVs (MSCATV-EV) in rats. A The schematic protocol of EVs isolated from rat bone marrow MSCs with or without ATV pretreatment. B Flow cytometry analysis of surface markers on rat bone marrow MSCs. C Morphology of MSCs treated with DMSO or ATV under the microscope. D Transmission electron microscopy scanning for the morphology of EVs. E Western blot analysis for the protein markers of EVs. F Western blot analysis for the protein expressions of Arg1 (a marker of M2 macrophages) and iNOS (a marker of M1 macrophages) in rat BMDMs treated with different concentrations (2 μg/mL, 20 μg/mL, 40 μg/mL) of MSC-EV or MSCATV-EV for 24 h. G Western blot analysis for the protein expressions of Arg1 and iNOS in rat BMDMs stimulated with LPS for 6 h and then incubated with 40 μg/mL MSC-EV or MSCATV-EV for another 48 h. Arg1, arginase 1; ATV, atorvastatin; BMDMs, bone marrow-derived macrophages; DMSO, dimethyl sulfoxide; EV, extracellular vesicle; IMDM, Iscove’s Modified Dulbecco’s Medium; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells

• 

  Fig. 2. The superior effects of MSCATV-EV on ventricular function, MI size, macrophage infiltration and M2 polarization in AMI rats. A Representative M-mode echocardiogram of each group on day 28 after AMI. B The dynamic changes in LVEF, LVFS, LVESV, and LVEDV of each group on days 0, 3, 7, 28 after AMI, n = 6–7 per group. C Representatives of heart cross sections with Masson trichrome staining on day 28. D Quantification of infarct size of each group in C, n = 6–7 per group. E Representative confocal images of CD68+ or CD206+ macrophages within the infarct border zone on day 3 post-AMI. F Quantification of CD68+ or CD206+ macrophages in E, n = 6-7 per group. G Western blot analysis of iNOS and Arg1 expressions in infarcted myocardium of each group. H Quantification of iNOS and Arg1 expressions in G, n = 3 per group. I The mRNA expressions of M1 (iNOS, IL-12, TNF-α) and M2 (Arg1, IL-10, CD206) macrophage markers in infarcted zone measured by qPCR, n = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Arg1, arginase 1; AMI, acute myocardial infarction; ATV, atorvastatin; EV, extracellular vesicle; IL, interleukin; iNOS, inducible nitric oxide synthase; LVEDV, left…

• 

  Fig. 3. The effects of MSCATV-EV and MSC-EV on macrophage polarization shifts in vitro. A PKH-67 labeled MSCATV-EV or MSC-EV (green) intake by rat BMDMs (red). B Western blot analysis for protein expressions of iNOS and Arg1 in BMDMs. C Quantification of iNOS and Arg1 expressions in B, n = 3 per group. D qPCR analysis for mRNA expressions of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in BMDMs described in B, n = 4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Arg1, arginase 1; AMI, acute myocardial infarction; ATV, atorvastatin; BMDMs, bone marrow-derived macrophages; EV, extracellular vesicle; IL, interleukin; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MSCs, mesenchymal stem cells; qPCR, quantitative polymerase chain reaction; TNF-α, tumor necrosis factor alpha

• 

  Fig. 4. Small RNA sequencing analysis identifies miR-139-3p as an extracellular vesicle miRNA candidate. A, B Hierarchical clustering heat map (A) and volcano plot (B) showing 9 differentially expressed miRNAs between MSC-EV and MSCATV–EV. C Verification for the mRNA expressions in MSC-EV and MSCATV–EV by qPCR analysis, n = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001. D, E GO (D) and KEGG (E) enrichment analysis of target genes of three upregulated miRNAs (miR-139-3p, miR-320-3p, miR-501-3p) between MSC-EV and MSCATV–EV. F Interaction network between target genes of three upregulated miRNAs (miR-139-3p, miR-320-3p, miR-501-3p) and upregulated genes in patients suffered from first acute myocardial infarction (GEO: GSE24591). G Interaction network between target genes of three upregulated miRNAs (miR-139-3p, miR-320-3p, miR-501-3p) and downregulated genes in human monocyte-derived M2 macrophages (GEO: GSE32164). H The intersection between the three upregulated miRNAs of miR-139-3p, miR-320-3p, miR-501-3p, and 25 downregulated miRNAs in patients suffered from first acute myocardial infarction (GEO: GSE24591) is miR-139-3p. ATV, atorvastatin; EV, extracellular vesicle; GEO, gene…

• 

  Fig. 5. miR-139-3p is an effector of MSCATV–EV-mediated macrophage polarization by targeting Stat1 in vitro. A Schematic protocol of isolating MSCATV-EV-miR inhibitor and MSC-EV-miR mimic and incubating LPS-pretreated rat BMDMs. B The protein expressions of macrophage M1 marker iNOS and M2 marker Arg1 in the BMDMs as described in A. C Quantification of iNOS and Arg1 expressions in B, n = 3 per group. D qPCR analysis for the mRNA expressions of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in the BMDMs described in A, n = 3–4 per group. E Schematic protocol of miR-139-3p mimics being directly transfected into LPS-pretreated BMDMs. F The protein expressions of macrophage M1 marker iNOS and M2 marker Arg1 in the BMDMs described in E. G Quantification of iNOS and Arg1 expressions in F, n = 3 per group. H qPCR analysis for the mRNA expressions of macrophage markers (M1: iNOS, IL-12, TNF-α; M2: Arg1, IL-10, CD206) in the BMDMs described in E, n = 3–4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. I, J The protein expression of Stat1 in rat BMDMs transfected with miR-139-3p mimics or inhibitors, n = 3 per group. K, L The protein expressions of iNOS, Arg1,…

• 

  Fig. 6. MSCATV-EV transfer of miR-139-3p enhances M2 macrophage polarization in infarcted region of AMI rats. A Representative confocal images of CD68+ (M1) and CD206+ (M2) macrophages within the infarct zone on day 3 after AMI. B Quantification of CD68+ or CD206+ macrophages in A, n = 6–7 per group. C Representative M-mode echocardiogram of each group on day 28 after AMI. D Comparisons of LVEF and LVFS in each group based on the echocardiographic images, n = 6–7 per group. *P < 0.05, **P < 0.01, ***P < 0.001. E Representatives cross sections of AMI heart with Masson trichrome staining on day 28. F Quantification of infarct size of each group in E, n = 6–7 per group. G Western blot analysis of iNOS, Arg1, and p-Stat1 expressions in infarct myocardium of each group. H Quantification of iNOS, Arg1, and p-Stat1 expressions in G, n = 3 per group. I The mRNA expressions of macrophage M1 (iNOS, IL-12, TNF-α) and M2 (Arg1, IL-10, CD206) markers in infarcted region measured by qPCR, n = 3-4 per group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. MSCATV-EV-miR inhibitor, MSCATV-EV with miR-139-3p inhibitor; MSC-EV-miR mimic, MSC-EV with miR-139-3p mimic. Arg1, arginase 1; AMI, acute…

• 

  Fig. 7. Schematic summary of our main findings. Intramyocardial injection of rat bone marrow MSCATV-EV in border zone delivers the upregulated miR-139-3p into infiltrated macrophages and promotes M2 macrophage polarization by suppressing Stat1 expression and activation, facilitating cardiac repair after infarction. ATV, atorvastatin; EVs, extracellular vesicles; MSCs, mesenchymal stem cells; Stat1, signal transducer and activator of transcription 1