Effects Of Salvianolic Acid On Proliferation,Adhesion And NO Secretion Activity Of Human Peripheral Endothelial Progenitor Cells Cultured In Vitro

Object:To investigate the isolation, culture, differentiation and identification of endothelial progenitor cells (EPCs) from human peripheral blood and observe the effects on proliferation,adhesion and nitric oxide(NO) secretion activity of EPCs cultured in different concentrations of salvianolic acid in vitro.Method:The mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers by Ficoll density gradient centrifugation, and then the cells were planted on the human fibronectin (FN) coated culture dishes. The cells were suspended in endothelial basal medium (EBM-2) supplemented with EGM-2-MV-SingleQuots. We observed morphological changes with inverted phase contrast microscope. After 7 days of culture,the adherent cells were subjected to immunofluorescence and immunocytochemistry to analyze the expression of DiL-acLDL,FITC-UEA-1 and KDR. EPCs were characterized as adherent cells double positive for DiL-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. After cultered for 7 days ,EPCs were randomized into 6 groups: control group,simvastatin group and different concentrations of salvianolic acid groups(0.5,2.5,5,10mg/L). After different periods of culturing,the ability of cell proliferation was assayed with MTT assay, counting adherent cells assayed the adhesion activity of EPCs, the NO content was measured in the cell culture medium by nitrate reductase method to find the effect on cell secretion activity.Result:1-2×106 MNCs were isolated in one ml human peripheral blood,the cells were small and shaped round, suspended in the endothelial basal medium. Cluster attaching cells appeared in the second day. We could see the cell colony, formed by the spindle-like adherent cells radiating towards periphery in the fifth day of culture. After 7 days culture,there were more and more spindle-like attaching cells appeared, and formed cord-like or duct-like structure.Fluorescence microscopy revealed absorption DiL-acLDL and FITC-UEA-1, DiL-acLDL uptake in the cells were red,cells lectin binding were green, double positive cells appearing yellow. EPCs were characterized as adherent cells double positive for DiL-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were furter documented by demonstrating the expression of KDR. We and other investigators have previously demonstrated that endothelial progenitor cells isolated in this fashion also exhibitmany other endothelial characteristics, including expression of CD31, CD34 ,Von Willebrand factor (vWF), VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR-2), and so on.Incubation of EPCs with Salvianolic acid increased the number of EPCs, with a maximum at 5mg/L after 24 hours (P<0.01).In addition, Salvianolic acid promotes EPCs proliferative, adhesive and NO secreting capacity.Conclusion:EPCs can be isolated and cultured from mononuclear cells from human peripheral blood through the method of Ficoll gradient centrifugation.Salvianolic acid can promote EPCs augmentation and enhance its proliferation, adhension and NO secreting function.It's likely to be a new mechanism of EPCs for therapy ischemic disease.