Application of peripheral-blood-derived endothelial progenitor cell for treating ischemia-reperfusion injury and infarction: a preclinical study in rat models

Abstract

Background: Our aim was to explore the therapeutic effects of peripheral blood-derived endothelial progenitor cells (PB-EPC) in cardiac ischemia-reperfusion infarction models in rats and in in vitro culture systems.

Methods: Rat models of ischemia reperfusion and myocardial infarction were developed using male, Sprague-Dawley rats. Cardiomyocyte and endothelial cell cultures were also established. Therapeutic effects of PB-EPCs were examined in vivo and in vitro in both models. Rats underwent either cardiac ischemia-reperfusion (n = 40) or infarction (n = 56) surgeries and were transplanted with genetically modified EPCs. Treatment efficacy in the ischemia-reperfusion group was measured by infarct size, myocardial contraction velocity, and myeloperoxidase activity after transplantation. Cardiomyocyte survival and endothelial cell apoptosis were investigated in vitro. Vascular growth-associated protein expression and cardiac function were evaluated in the myocardial infarction group by western blot and echocardiography, respectively.

Results: Infarct size and myeloperoxidase activity were significantly decreased in the ischemia-reperfusion group, whereas myocardial contractility was significantly increased in the EPC and Tβ4 groups compared with that in the control group. In contrast, no differences were found between EPC + shRNA Tβ4 and control groups. Rates of cardiomyocyte survival and endothelial cell apoptosis were significantly higher and lower, respectively, in the EPC and Tβ4 groups than in the control group, whereas no differences were found between the EPC + shRNA Tβ4 and control group. Four weeks after myocardial infarction, cardiac function was significantly better in the EPC group than in the control group. Expressions of PDGF, VEGF, and Flk-1 were significantly higher in EPC group than in control group.

Conclusions: Study findings suggest that PB-EPCs are able to protect cardiomyocytes from ischemia-reperfusion or infarction-induced damage via a Tβ4-mediated mechanism. EPCs may also provide protection through increased expression of proteins involved in mediating vascular growth. Autologous peripheral-blood-derived EPCs are readily available for efficient therapeutic use without the concerns of graft rejection.

Figure 1

The effect of EPC and Tβ4 in a myocardiac infarction model. Infarct size (A) and area at risk (AAR: B) in rats 72 hours after ligating the LAD coronary artery. Rats were treated with: endothelial progenitor cells (EPCs) (150 μL, 5 × 10⁶ cells per rat); EPCs transfected with Tβ4 short hairpin RNA (shRNA) (150 μL, 5 × 10⁶ cells per rat); EPCs transfected with scrambled (SC) shRNA (150 μL, 5 × 10⁶ cells per rat); or 6 mg Tβ4. Control rats were untreated. *P < 0.05 compared with the control group; †P < 0.05 compared with the Tβ4 group; ‡P < 0.05 compared with the EPC group; § P < 0.05 compared with the EPC + Tβ4 shRNA group. Data are presented as mean ± standard deviation (n = 8 per group).

Figure 2

Measurement of cardiac functions and myeloperoxidase activities. Increase in the peak rate of intraventricular pressure increase (+dP/dtmax: A), decrease in the peak rate of intraventricular pressure (−dP/dtmax: B) in rats 72 hours, and cardiac myeloperoxidase activity (MPO: C) in rats 24 hours after ischemia-reperfusion injury. Rats were treated with: endothelial progenitor cells (EPCs) (150 μL, 5 × 10⁶ cells per rat); EPCs transfected with Tβ4 short hairpin RNA (shRNA) (150 μL, 5 × 10⁶ cells per rat); EPCs transfected with scrambled (SC) shRNA (150 μL, 5 × 10⁶ cells per rat); or 6 mg Tβ4. Control rats were untreated. *P < 0.05 compared with the control group; †P < 0.05 compared with the Tβ4 group; ‡P < 0.05 compared with the EPC group; §P < 0.05 compared with the EPCs + SC shRNA group. Data are presented as mean ± standard deviation (n = 8 per group).

Figure 3

Measuring EPC function in an in vitro assay. Cardiomyocyte survival (A) and endothelial cell apoptosis (B) after exposure to hypoxia-reoxygenation (18 h-4 h) ischemia-reperfusion. Cells were incubated with: endothelial progenitor cells (EPCs); EPCs transfected with Tβ4 short hairpin RNA (shRNA); EPCs transfected with scrambled (SC) shRNA in transwell plate; Tβ4 were added directly into culturing well. Control cells were not treated beside hypoxia-reoxygenation *P < 0.05 compared with the control group; †P < 0.05 compared with the Tβ4 group; ‡P < 0.05 compared with the EPC group; §P < 0.05 compared with the EPCs + SC shRNA group. Data are presented as mean ± standard deviation (n = 6 per group).

Figure 4

EPC treatment induced cardioprotection related proteins expression. Protein expression of platelet-derived growth factor (PDGF: A), vascular endothelial growth factor (VEGF: B), fetal liver kinase-1 (Flk-1: C), fibroblast growth factor-17 (FGF-17: D), fibroblast growth factor receptor-2 (FGFR-2: E), Tbx-18 (F), and β-Catenin (G) in heart tissue from rats 24 h after myocardial infarction. Rats were treated with endothelial progenitor cells (EPCs) or were untreated (controls). Protein expression levels were determined by Western blot using heart tissue harvested from non-infarct areas. Density units were normalized to the GAPDH loading control. *P < 0.05 compared with the EPC group. Data are presented as mean ± standard deviation (n = 6 per group).