Effects of Ox-LDL on number and activity of circulating endothelial progenitor cells

Backgrounds: Endothelial dysfunction is thought to play a crucial role in the pathogenesis of atherosclerosis induced by ox-LDL. Recently, a variety of evidence suggested that endothelial progenitor cells (EPCs) participated in neovascularization and reendothelialization. However, effects of ox-LDL on EPCs number and activity are ill understood. Methods: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with ox-LDL (to make a series of final concentrations: 25 mug/mL, 50 mug/mL, 100 mug/mL, 200 mug/mL), native LDL (100 mug/mL) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Results: Incubation of isolated human EPCs with ox-LDL decreased the number of EPCs in concentration-dependent manner, maximum at 200 mug/mL (approximately 70% reduction, P < 0.001). In time-course experiments performed with an ox-LDL concentration of 100 mug/mL, decrease of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (approximately 50% reduction, P < 0.01). In addition, ox-LDL dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. Conclusion: The results of the present study defined a novel mechanism of action of ox-LDL: the reduction of EPCs with decreased functional activity.