Suppression of oxidative stress in endothelial progenitor cells promotes angiogenesis and improves cardiac function following myocardial infarction in diabetic mice

Abstract

Myocardial infarction is a major contributor to morbidity and mortality in diabetes, which is characterized by inadequate angiogenesis and consequent poor blood reperfusion in the diabetic ischemic heart. The aim of the present study was to investigate the effect that oxidative stress in endothelial progenitor cells (EPCs) has on cardiac angiogenesis in diabetic mice. EPCs derived from diabetic mice revealed reductions in superoxide dismutase (SOD) expression levels and activity compared with those from normal mice. An endothelial tube formation assay showed that angiogenesis was markedly delayed for diabetic EPCs, compared with normal controls. EPCs subjected to various pretreatments were tested as a cell therapy in a diabetic mouse model of myocardial infarction. Induction of oxidative stress in normal EPCs by H₂O₂ or small interfering RNA-mediated knockdown of SOD reduced their angiogenic activity in the ischemic myocardium of the diabetic mice. Conversely, cell therapy using EPCs from diabetic mice following SOD gene overexpression or treatment with the antioxidant Tempol normalized their ability to promote angiogenesis. These results indicate that decreased expression levels of SOD in EPCs contribute to impaired angiogenesis. In addition, normalization of diabetic EPCs by ex vivo SOD gene therapy accelerates the ability of the EPCs to promote angiogenesis and improve cardiac function when used as a cell therapy following myocardial infarction in diabetic mice.

Keywords: angiogenesis; diabetes; endothelial progenitor cells; myocardial infarction; superoxide dismutase.

Figure 1.

Hyperglycemia increased ROS levels and reduced the expression of antioxidant enzymes in EPCs isolated from the peripheral blood of control or diabetic mice. Cells were tested for (A) ROS production by the determination of DHE intensity, (B) Mn-SOD mRNA levels by RT-qPCR, (C) Mn-SOD protein levels by western blotting, and (D) Mn-SOD activity using a kit. Data are expressed as mean ± standard error of the mean (n=5 per group). *P<0.05 vs. control. ROS, reactive oxygen species; EPC, endothelial progenitor cell; DHE, dihydroethidium; Mn-SOD, manganese superoxide dismutase; RT-qPCR, reverse transcription-polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2.

EPC function was damaged in diabetic mice. EPCs were isolated from the peripheral blood of control or diabetic mice. Cultured EPCs were measured for (A) production of VEGF and bFGF in medium by enzyme-linked immunosorbent assay, (B) tube formation using Matrigel and (C) migratory response by expanding EPCs toward stromal cell-derived factor-1 gradients and measured by modified Transwell chamber migration assay. Data are expressed as mean ± standard error of the mean (n=5 per group). Magnification, ×400. *P<0.05, vs. the control. EPC, endothelial progenitor cell; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor.

Figure 3.

Hyperglycemia reduced tube formation of EPCs via oxidative stress. Cultured EPCs from diabetic mice were (A) treated with 100 µM Tempol for 24 h or (B) infected with Ad-Mn-SOD for 48 h. Tube formation was determined using Matrigel. Data are expressed as the mean ± standard error of the mean (n=5 per group). Magnification, ×400. *P<0.05, vs. the control. EPC, endothelial progenitor cell; GFP, green fluorescent protein; Ad, adenovirus; Mn-SOD, manganese superoxide dismutase.

Figure 4.

Induction of oxidative stress in EPCs attenuated angiogenesis in the ischemic myocardium of diabetic mice. Cultured EPCs from control mice (A) pre-treated with 1 µM H₂O₂ for 24 h or (B) transfected with Mn-SOD siRNA for 48 h were transplanted into diabetic mice after myocardial infarction. Angiogenesis was assayed by immunohistochemical staining of CD31 in the myocardium. Expression levels of Mn-SOD were assayed by western blotting. Data are expressed as means ± standard error of the mean (n=5 per group). Magnification, ×400. *P<0.05, vs. control. EPC, endothelial progenitor cell; siRNA, small interfering RNA; Mn-SOD, manganese superoxide dismutase; CD, cluster of differentiation.

Figure 5.

Induction of oxidative stress in EPCs attenuated recovery of heart function in diabetic mice. Cultured EPCs from control mice (A) pre-treated with 1 µM H₂O₂ for 24 h or (B) transfected with Mn-SOD siRNA for 48 h were transplanted into diabetic mice following myocardial infarction. Heart function was determined by B ultrasound (upper) and sLVID and dLVID values (lower) were determined. Data are expressed as means ± standard error of the mean (n=5 per group). *P<0.05, vs. the control. EPC, endothelial progenitor cells; siRNA, small interfering RNA; sLVID, left ventricular end-systolic diameter; dLVID, left ventricular end-diastolic diameter; Mn-SOD, manganese superoxide dismutase.

Figure 6.

Suppression of oxidative stress in EPCs improved heart function and promoted angiogenesis in the diabetic hearts of mice following myocardial infarction. Cultured EPCs from diabetic mice (A) pre-treated with 100 µM Tempol for 24 h or (B) infected with Ad-Mn-SOD for 48 h were transplanted into diabetic mice following myocardial infarction. Heart function was determined by B ultrasound. Angiogenesis was assayed by immunohistochemical staining of CD31 in the myocardium. Data are expressed as mean ± standard error of the mean (n=5 per group). Magnification, ×400. *P<0.05, vs. the control. EPC, endothelial progenitor cell; sLVID, left ventricular end-systolic diameter; dLVID, left ventricular end-diastolic diameter; Ad, adenovirus; GFP, green fluorescent protein; Mn-SOD, manganese superoxide dismutase.