Circulating Endothelial Progenitor Cell Transplantation Improves Neovascularization In Rabbits With Myocardial Infarction

Part IObjective To provide methods and cell resource for therapeutic neovascularization of myocardial infarction.Methods Mononuclear cells from human peripheral blood were isolated by a density-gradient centrifugation method and incubated in M199 medium containing 20% fetal calf serum and 4 μg/ml bovine pituitary extract. Markers for endothelial cells were identified by flow cytometry system. MTT assay was used to study the activity of proliferation.The morphological characteristics of attaching cells were also studied.Results After 7 days of culture,the cells showed the appearance of cell clusters,spindle-shaped and attaching cells,and cord-like structures. 54.2% ±8.6% of total adherent cells were CD31/VWF duoble positive by flow cytometry analysis. There were significant differences in the proliferation activity day 5 and day 7 compared with that of day 3 in culture by MTT assay (P<0.001) .Conclusion Endothelial progenitor cells can be isolated from peripheral blood and expanded in vitro.More cells will be needed for clinical application.Part IIObjective To investigate the effect on cell therapy with autologous circulating EPC for improving neovascularization in the infarcted myocardium.Mathods After the left anterior desecending artery was ligated,rabbits were administrated simvastatin(lmg. kg"1. d"1) for 23 days.Mononuclear cells were isolated from 20 ml of blood 48h after operation,and incubated in endothelial medium.On the ninth day of model reproducing,the rabbits were randomized to receive a directly intravascular injection of either Ml99 medium (simvastatin group,n=7) or cultured adherent cells (EPC group, 1 X 106/kg body weight,n=8) labeled with 5-bromo-2-deoxyuridine (BrdU).2 weeks later.histologic analysis was performed to study neovascularization induced by cell therapy. The mobilization effect of simvastatin on EPC was also studied by flow cytometry analysis. In addition, EPC proliferation,migration and adhesive capacity were studied by MTT assay,modified Boyden chamber assay and adhesion assay to test the influence of simvastatin on cultured adherent cells.Results Adherent cells at day 7 of culture showed the characterization of EPC;simvastatin promoted EPC proliferation,migration and adhesive capacity in culture (P<0.001,0.05,0.01, respectively); simvastatin increased the number of circulating mononuclear cells that express endothelial cell lineage markers (simvastatin group vs control group, /)<0.01); BrdU immunohistologic analysis showed that transplanted cells can express in ischemic myocardium;There was a significant increase in the density of capillaries in EPC group(P<0.05).Conclusion Intravascular injection of cultured EPC can augment the density of...