Implantation of autologous murine endothelial progenitor cells promotes neovascularization in ischemic myocardium

Quansheng XING; Silin PAN; Long SUN

ABSTRACT

BACKGROUND:

Under certain condition, circulating endothelial progenitor cells (EPCs) can differentiate into endothelial cells, and further participate in angiogenesis.

OBJECTIVE:

The goal of this study was to investigate the feasibility and efficacy of peripheral blood-derived EPCs in promoting angiogenesis in the ischemic myocardium, in order to provide a new cell implanting method for the treatment of coronary heart disease.

DESIGN:

A randomized controlled experiment.MATERIALS Sixty male Sprague-Dawley (SD) rats, of clean grade, weighing (340±20) g, were provided by Qingdao Laboratory Animal Center. These animals were randomly divided into 2 groups with 30 rats in each experimental group and control group. In each group, ten rats were separately observed 2, 4 and 8 weeks after EPCs being injected. The protocol was conducted in accordance with animal ethics guidelines for the use and care of animals.

METHODS:

This study was carried out in the Qingdao Key Laboratory of Medical Biological Technology between May 2003 and September 2004. After SD rats in the experimental group were anesthetized, peripheral blood was taken.Mononuclear cells were harvested by density gradient centrifugation. CD31, CD34, Flk-1 and von Willebrand disease factor immunofluorescence staining positive EPCs were harvested by adding the defined media of vascular endothelial growth factors and basic fibroblast growth factor. Myocardial ischemia was induced by ligation of murine left anterior descending coronary artery. Autologous EPCs isolated from the peripheral blood of each animal were infused to ischemic myocardium. In the control groups,cell culture media were infused, and the other procedures were the same as those in the experimental group. Two, four and eight weeks after ligation, all animals were sacrificed by overdose anesthesia, and heart tissue sections were made.MAIN OUTCOME

MEASURES:

①After haematoxylin-eosin staining, myocardial structure changes were observed under an optical microscope. ②Cells positive for factor Ⅷ were numbered and then to calculate the total number of cells in each visual field for evaluating microvessel density with a German ZEISS Axiotron image analyzer.

RESULTS:

All the 60 SD rats were involved in the final analysis, without deletion. ①Myocardial structure was relatively disarrayed in the control groups, with collagens and fibroblasts substituted for cardiomyocytes. In the marginal infarct areas, cardiomyocytes were irregular and partial cells were significantly hypertrophied. As compared with control group, myocardial collagen fibers fused less, and tissue structure got more disarranged in the experimental group. Microvessel density in the implanted area was obviously increased. ②In the 2nd, 4th and 8th weeks after EPC infusion, microvessel density at the ischemic myocardium was significantly higher than that at the corresponding time point in the control group (P ＜ 0.01). In the EPCs groups, microvessel density tended to statistically increase with time going (P ＜ 0.05). No prominent difference was observed in the three control groups (P＞ 0.05).

CONCLUSION:

Relatively purified EPCs can be obtained by certain procedure of isolation and culture from rat peripheral blood. Intramyocardial implantation of autologous EPCs promotes microangiogenesis and has a protective effect on ischemic myocardial tissue.