Effect of nicotine on number and activities of human endothelial progenitor cells

AIM To investigate whether nicotine has influences on human endothelial progenitor cells (EPC) number and activities, which results in postnatal neovascularization. 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 7 d cultured, attached cells were stimulated with nicotine (final concentrations: 10 -12 , 10 -10 , 10 -8 , 10 -6 and 10 -4 mol·L -1 ) or vehicle control for the respective time points(12, 18, 24, 32 and 48 h). EPC were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPC were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry . EPC proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPC adhesion assay was performed by replating those on fibronectin-coated dishes, and then adherent cells were counted. RESULTS Incubation of isolated human MNC with nicotine concentration-dependently increased EPC number, proliferative, migratory , adhesive activity and in vitro vasculogenesis at nicotine concentrations of 10 -12 -10 -8 mol·L -1 . The peak effects on EPC were observed at concentrations of nicotine 10 -8 mol·L -1 . In addition, nicotine (10 -8 mol·L -1 ) time-dependently increased EPC number and activity. However, cytotoxicity was seen at high nicotine concentrations (10 -6 mol·L -1 ). CONCLUSION Nicotine has complex effects on EPC, including proliferation and activities at nicotine concentrations of 10 -12 -10 -8 mol·L -1 . However, higher nicotine concentrations induces cytotoxicity.