Effects of oxidative low-density lipoprotein on number and function of endothelial progenitor cells from peripheral blood

Objective To observe the effects of oxidative low density lipoprotein (ox LDL) on number and function of endothelial progenitor cells (EPCs) from peripheral blood. Methods Total mononuclear cells (MNC) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin coated culture dishes. After incubation for 7 days, attached cells were collected and stimulated with different cencentrations of ox LDL (25, 50, 100 and 200 μg/ml), native LDL (100 μg/ml) or vehicle control for different time (6, 12, 24 and 48 h). EPC were characterized and adherent cells which were double positive for DiLDL uptake and lectin binding by direct fluorescent staining were observed under a laser scanning confocal microscope. EPC were further identified by demonstrating the expression of CD34, VEGFR 2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPC were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPC adhesion assay was performed by replating those on fibronectin coated dishes, then adherent cells were counted. Results Incubation of isolated human MNC with ox LDL decreased the number of EPC in a concentration dependent manner, being maximum at 200 μg/ml (approximately 70% reduction, P0.01). In time course experiments performed with a concentration of 100 μg/ml ox LDL, the decrease of EPC number became apparent at 12 h and the maximal decrease was at 24 h (approximately 50% reduction, P0.01). In addition, ox LDL impaired proliferative, migratory, adhesive and in vitro vasculogenesis capacities of EPC in dose and time dependent manners. Conclusion Incubation of EPC with ox LDL reduces the number of EPC and impaires their function.