Cardiac shockwave therapy improves myocardial function in patients with refractory coronary artery disease by promoting VEGF and IL-8 secretion to mediate the proliferation of endothelial progenitor cells

Abstract

Cardiac shockwave therapy (CSWT) is a potential and effective remedy to promote revascularization in the ischemic myocardium of patients with refractory coronary heart disease (CHD). The technique is both safe and non-invasive; however, the underlying molecular mechanism remains unclear. The aim of this study was to evaluate the efficacy of CSWT in treating CHD patients and investigate a potential mechanism. A total of 26 patients with CHD were enrolled in the study, and CSWT was performed over a 3-month period. The efficacy of CSWT was assessed using several clinical parameters. Peripheral blood (PB) was collected prior to and following treatment. The number of circulating endothelial progenitor cells (EPCs) in the PB was counted using a flow cytometer, and the levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), stromal cell-derived factor 1 and matrix metalloproteinase 9 in the PB were analyzed. Mononuclear cells were isolated from the PB and cultured in vitro. The EPCs and EPC-colony forming units (EPC-CFUs) in the PB mononuclear cell culture were counted using an inverted phase contrast microscope. Following CSWT, the tested clinical parameters were significantly improved. The levels of circulating EPCs, VEGF and IL-8 in the PB were significantly increased, as were the EPCs and EPC-CFUs from the PB mononuclear cell culture. We suggest that EPC proliferation, mediated by VEGF and IL-8 secretion, may be among the potential mechanisms associated with CSWT.

Keywords: cardiac shockwave therapy; coronary artery disease; endothelial progenitor cells; interleukin-8; vascular endothelial growth factor.

Figures

Figure 1.

Identification of EPCs. (A) Red indicates cells positively marked with Dil-acLDL (magnification, ×200); (B) green indicates cells positively marked with FITCUEA-1 (magnification, ×200); (C) overlap of (A) and (B), with yellow indicating the differentiating EPCs (magnification, ×200). After 7 days' culure the adherent cells were incubated with Dil-acLDL (2.4 mg/l) for 1 h at 37°C, and the Dil-acLDL uptake was then detected. If uptake was visible, the cells were fixed using 2% paraformaldehyde and rinsed with phosphate-buffered saline, prior to the addition of FITCUEA-1 (10 mg/l) and incubation for another 1 h at 37°C. Images were captured using a confocal laser scanning microscope. EPC, endothelial progenitor cell; Dil-acLDL, Dil-labeled acetylated low-density lipoprotein; FITCUEA-1, fluorescein isothiocyanate-Ulex europaeus agglutinin-1.

Figure 2.

Effect of CSWT on the numbers of EPCs from PB mononuclear cell culture in vitro. (A) Pre-CSWT (magnification, ×100); (B) post-CSWT (magnification, ×100). Blood was drawn from the upper-arm veins of the patients with coronary heart disease 1 day before CSWT and 30 days after CSWT. PB mononuclear cells were obtained by density gradient centrifugation. After 7 days' culture in vitro, EPCs were counted using an inverted phase contrast microscope. CSWT, cardiac shockwave therapy; EPC, endothelial progenitor cell; PB, peripheral blood.

Figure 3.

Effect of CSWT on the numbers of EPC-CFUs from PB mononuclear cell culture in vitro. (A) Pre-CSWT (magnification, ×100); (B) post-CSWT (magnification, ×100). Blood was drawn from the upper-arm veins of the patients with coronary heart disease 1 day before CSWT and 30 days after CSWT. PB mononuclear cells were obtained by density gradient centrifugation. After 7 days' culture in vitro, EPC-CFUs were counted using an inverted phase contrast microscope. CSWT, cardiac shockwave therapy; EPC-CFU, endothelial progenitor cell-colony forming unit; PB, peripheral blood.

Figure 4.

Effect of CSWT on the numbers of EPCs and EPC-CFUs from PB mononuclear cell culture in vitro. Blood was drawn from the upper-arm veins of the patients with coronary heart disease 1 day before CSWT and 30 days after CSWT. PB mononuclear cells were obtained by density gradient centrifugation and cultured for 7 days in vitro. EPC-CFUs and EPCs were counted in 3 randomly selected visual fields using an inverted phase contrast microscope. Identification of EPCs was performed using a confocal laser scanning microscope. Data are presented as the mean ± standard deviation (n=26). ##P<0.001, compared with pre-treatment. CSWT, cardiac shockwave therapy; EPC-CFU, endothelial progenitor cell-colony forming unit; PB, peripheral blood.

Figure 5.

Effect of CSWT on EPCs in the PB. Blood was drawn from the upper-arm veins of the patients with coronary heart disease 1 day before CSWT and 30 days after CSWT. EPCs in the PB were identified using a flow cytometer. The following formula was used: EPCs (%) = (experimental tube EPCs - control tube EPCs)/mononuclear cells × 100. Data are presented as the mean ± standard deviation (n=26). ***P