Effect of batroxobin on the number and the function of in vitro cultured endothelial progenitor cells

Objective To investigate whether batroxobin(DF-521)augments the number of endothelial progenitor cells(EPCs)and promotes EPCs proliferation,migration and adhesion.Methods Mononuclear cells(MNCs)were isolated from peripheral blood by Ficoll density gradient centrifugation.The cells were then plated on fibronectin-coated culture dishes.After being cultured for 7 d,the attached cells were stimulated with DF-521(final concentration:0.05,0.1,0.2 BU/ml)for 6,12,24 and 48 h.EPCs were characterized as adherent cells double positive for DiI-LDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope.EPCs were further documented by demonstrating the expression of CD34,VEGFR-2 and CD133 with flow cytometry.Proliferation and migration of EPCs were assayed by MTT assay and modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating it on fibronectin-coated dishes and the adherent cells were then counted.Results Compared with the controls,the number of EPCs increased significantly with increasing the concentration of DF-521 used for stimulation.The increase reached the maximum 24 h after administration of 0.1 BU/ml DF-521(2-fold increase,P0.05).In addition,DF-521 also promoted proliferation,migration and adhesion of EPCs.Conclusion The results of the present study define a novel mechanism of the action of DF-521:augmentation of number of EPCs with enhanced functional activity in vitro.