Endothelial nitric oxide synthase as a marker for human endothelial progenitor cells

Endothelial progenitor cells (EPCs) have been proposed as a promising tool for therapeutic neovascularization, vascular repair, tumor pathology and tissue engineering, though their identification is still a subject of much discussion. EPCs consist of two different subpopulations, termed endothelial cell (EC)-like cells and endothelial outgrowth cells (EOCs). Both types of EPCs are derived from mononuclear cells, but they have different characteristics. Our aim was to characterize and compare the two types of EPCs to find reliable biological features of EPCs that can be used for identification of EPCs. In this study, human peripheral blood mononuclear cells were isolated by density gradient centrifugation and cultured on fibronectin-coated culture plates. While adherent cells were maintained, EC-like cells appeared within 4-7 days of culture, and EOCs developed after 2-3 weeks of culture. EOCs, which were characterized by high proliferation potential, were able to form capillary tubes on Matrigel, but not EC-like cells, despite the higher concentrations of three angiogenic cytokines, vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8, in the conditioned medium of EC-like cells. In contrast, endothelial nitric oxide synthase (eNOS) was expressed in both types of EPCs, and both cell types could produce nitric oxide (NO), as judged by measuring the total amounts of nitrites and nitrates in culture media. In conclusion, the expression of eNOS and the production of NO could be used as common biological features to identify EPCs. These findings provide new insights into the identification of EPCs.