Exosomes from SIRT1-Overexpressing ADSCs Restore Cardiac Function by Improving Angiogenic Function of EPCs

Abstract

Acute myocardial infarction (AMI) is one of the leading causes of mortality in cardiovascular diseases. The aim of this study was to investigate whether exosomes from Sirtuin 1 (SIRT1)-overexpressing adipose-derived stem cells (ADSCs) had a protective effect on AMI. The expression of C-X-C chemokine receptor type 7 (CXCR7) was significantly downregulated in peripheral blood endothelial progenitor cells (EPCs) from AMI patients (AMI-EPCs) compared with that in healthy donors, which coincided with impaired tube formation. The exosomes from SIRT1 overexpression in ADSCs (ADSCs-SIRT1-Exos) increased the expression of C-X-C motif chemokine 12 (CXCL12) and nuclear factor E2 related factor 2 (Nrf2) in AMI-EPCs, which promoted migration and tube formation of AMI-EPCs, and overexpression of CXCR7 helped AMI-EPCs to restore the function of cell migration and tube formation. Moreover, CXCR7 was downregulated in the myocardium of AMI mice, and knockout of CXCR7 exacerbated AMI-induced impairment of cardiovascular function. Injection of ADSCs-SIRT1-Exos increased the survival and promoted the recovery of myocardial function with reduced infarct size and post-AMI left ventricular remodeling, induced vasculogenesis, and decreased AMI-induced myocardial inflammation. These findings showed that ADSCs-SIRT1-Exos may recruit EPCs to the repair area and that this recruitment may be mediated by Nrf2/CXCL12/CXCR7 signaling.

Keywords: CXCL12; Nrf2; Sirtuin 1; acute myocardial infarction; adipose-tissue-derived stem cells; chemokine receptor CXCR7; exosomes.

Graphical abstract

Figure 1

AMI Reduced the Expression of CXCR7 in EPCs and Decreased the Angiogenesis Function of EPCs (A) Flow cytometry analysis of the expression of CD34/CD133/CD45 (green lines) in the peripheral blood EPCs of healthy subjects (control) and AMI patients, which were compared with isotype controls (red lines). (B) Expression of CXCR7 of EPCs was detected by western blots. (C) The western blot results were normalized to β-actin. ∗p < 0.05, compared to the control group. (D) The angiogenic function of EPCs was evaluated by tube formation assays. Scale bars, 100 μm. (E) The tube lengths were measured, and the control-EPCs were normalized to 1. ∗∗p < 0.01, compared to the control group.

Figure 2

Exosomes from ADSCs Promoted EPC Migration and Tube Formation, and Upregulation of CXCR7 Helped AMI-EPCs Restore Cell Migration and Tube Formation (A and B) EPCs from healthy controls (control-EPCs) were transfected with interfering plasmid or lentiviral interfering CXCR7 plasmid, while EPCs from acute myocardial infarction (AMI) patients (AMI-EPCs) were transfected with lentiviral plasmid or lentiviral overexpression of the CXCR7 plasmid, and cultured for 48 h. (A) Expression of CXCR7 was detected by western blotting. (B) The western blot results were normalized to β-actin. ∗∗p < 0.01, compared to the non-transfected cells of control- or AMI-EPCs. (C) Cell migration was measured using Transwell assays. The upper chamber contained EPCs; the lower chamber contained DMEM containing 10% FBS or ADSCs with or without pretreatment with 2.5 μM GW4869 for 8 h. Scale bars, 100 μm. (D) Migrated cells were calculated. ∗p < 0.05, compared to the DMEM control group; ^#p < 0.05, compared to the ADSC-treated group. (E and F) The indicated EPCs treated with supernatants of ADSCs with or without pretreatment with GW4869. (E) The tube formation assay. Scale bars, 100 μm. (F) The tube lengths were measured. The control-EPCs transfected with siRNA were normalized to 1. ∗p < 0.05, compared to the untreated group; ^#p < 0.05, compared to the ADSCs-supernatant-treated group.

Figure 3

Direct Transfer of SIRT1 from ADSCs to EPCs Using Exosome Delivery (A) Transmission electron microscopy of exosomes from ADSCs (ADSCs-Exos group) or ADSCs overexpressing SIRT1 (ADSCs-SIRT1-Exos group). Scale bars, 100 nm. (B) Nanoparticle tracking analysis of the exosome diameters (in nanometers). (C) Expression of SIRT1 and specific exosomal surface markers (CD63 and TSG101) were detected by western blotting of ADSCs and exosomes. (D) Localization of PKH26-labeled ADSCs-Exos (red) in AMI-EPCs visualized by confocal microscopy at 0 and 24 h post-ADSCs-Exos incubation. Scale bars, 10 μm. (E) Fluorescence microscopy analysis was performed to assay the green fluorescent signals in AMI-EPCs co-cultured with ADSCs-Exos with or without GFP-labeled SIRT1 treatment. Scale bars, 10 μm.

Figure 4

Exosomes from ADSCs Overexpressing SIRT1 Restored AMI-EPC Migration and Tube Formation (A) Cell migration was measured using Transwell assays. Upper chamber: AMI patient endothelial progenitor cells (EPCs) with or without pretreatment with 200 μg/mL ADSCs-Exos or ADSCs-SIRT1-Exos for 24 h; lower chamber: DMEM containing 10% FBS. Scale bars, 100 μm. (B) The number of migrated cells was calculated. ∗p < 0.05; ∗∗p < 0.01, compared with the untreated group; ^#p < 0.05, compared with the ADSCs-Exo-treated group. (C–H) EPCs from AMI patients were pretreated with ADSCs-Exos or ADSCs-SIRT1-Exos for 24 h. (C) Tube formation assay. Scale bars, 100 μm. (D) The tube lengths were measured. The AMI-EPCs without any treatment were normalized to 1. ∗p < 0.05; ∗∗p < 0.01, compared with the untreated group; ^#p < 0.05, compared with the ADSCs-Exos-treated group. (E) Expressions of Nrf2 and CXCL12 in AMI-EPCs were detected by western blotting. (F and G) The western blot results were normalized to β-actin; (F) Nrf2 and (G) CXCL12. ∗∗p < 0.01, compared with the untreated group. (H) Acetylated Nrf2 levels were measured by immunoprecipitation.

Figure 5

Injection of Exosomes from ADSCs Overexpressing SIRT1 Helped Restore Cardiac Function in AMI of WT mice but Not in CXCR7^−/− Mice AMI was produced by surgical ligation of the left anterior descending (LAD) coronary artery. LAD artery ligation or sham surgery was performed in mice; injection of phosphate-buffered saline (PBS; vehicle), ADSCs-Exos, or ADSCs-SIRT1-Exos was performed after AMI surgery, and myocardial tissue samples were collected at 28 days after surgery. (A and B) Western blot analyses of the expression levels of CXCR7. The CXCR7 levels were normalized to β-actin. ∗p < 0.05, compared with the sham group. (C) Survival analysis of mice treated as indicated each day after surgery. (D–I) Echocardiographic and hemodynamic measurements of the left ventricular enddiastolic dimension (LVEDD) (D); left ventricular endsystolic dimension (LVESD) (E); fractional shortening (FS) (F); ejection fraction (EF) (G); rates of maximal rise in left ventricular pressure (+dP/dt) (H); and the rate of maximal fall in left ventricular pressure (−dP/dt) (I). ∗p < 0.05; ∗∗p < 0.01, compared with the sham group; ^#p < 0.05, compared with the AMI + vehicle group of WT mice; ^&p < 0.05, compared with the AMI + vehicle group of WT mice; n = 6 per group.

Figure 6

Injection of Exosomes from ADSCs Overexpressing SIRT1 Reduced the Infarct Size and Post-AMI Left Ventricular Remodeling (A) Cardiac structures in the groups as revealed by Masson’s trichrome straining. Scale bars, 100 μm. (B and C) Quantitative analysis of the fibrosis area (B) and infarct size (C). *p < 0.05; **p < 0.01, compared with the AMI + vehicle group; ^#p < 0.05, compared with the AMI + ADSCs-Exo group; n = 6 per group. n.d., not determined.

Figure 7

Injection of Exosomes from ADSCs Overexpressing SIRT1 Decreased AMI-Induced Myocardial Inflammation (A) Macrophage density was assessed using Mac3 staining. Scale bars, 100 μm. (B) Quantification of infiltrated macrophages per area in frozen sections of infarcted hearts at days 3 and 7 after AMI surgery. ^#p < 0.05; ^##p < 0.01, compared with the AMI + vehicle group; ^&p < 0.05, compared with the AMI + ADSCs-Exos group; n = 6 per group. (C–E) The levels of TNF-α (C), IL-1β (D), and IL-10 (E) measured in heart homogenates after AMI or sham surgery for 24 h. ∗p < 0.05; ∗∗p < 0.01, versus the sham group; ^#p < 0.05, compared with the AMI + vehicle group; ^&p < 0.05, compared with the AMI + ADSCs-Exos group; n = 6 per group.

Figure 8

Injection of Exosomes from ADSCs Overexpressing SIRT1-Induced Vasculogenesis (A and B) Arteriole density from α-SMA staining. Representative images (A, Scale bars, 100 μm.) and quantification (B) of arteriole density in the peri-infarcted myocardium at 28 days after AMI, analyzed by α-SMA staining. ^#p < 0.05; ^##p < 0.01, compared with the AMI + vehicle group; ^&p < 0.05, compared with the AMI + ADSCs-Exos group; n = 6 per group. (C and D) Capillary density from vWF-positive vessels; representative images (C, Scale bars, 100 μm.) and quantification (D). The blood vessel density is indicated as the vessel number per square millimeter. ^#p < 0.05; ^##p < 0.01, compared with the AMI + vehicle group; ^&p < 0.05, compared with the AMI + ADSCs-Exos group; n = 6 per group.