Effect Of Simvastatin On The Functions Of Human Umbilical Cord Blood-derived Late Endothelial Progenitor Cells

Objective:1. Endothelial progenitor cells (EPCs) paticipate in neoangiogenesis in the physiologicaland pathological conditions of the body. Now, it is still difficult to define an EPC acccurately,EPCs cultured in one investigator are not the same as that cultured in other study group. They atleast contain early EPCs and late EPCs. As late EPCs have stronger ability of proliferation andangiogenesis, they are more promising in the cell transplantation therapy. In the first part of thisstudy, we plan to culture late EPCs from the human umbilical cord blood and compare differentseparation methods and culture conditions during the culture process in order to get more EPCsfor clinic applications.2. Statins not only have lipid-lowering effect on cardiovascular disease, but also havepleiotropic effects independent of its lipid-lowering effect. While it is well known that statinscan improve neoangiogenesis of early EPCs, their effect on late EPCs are not known. In thesecond part of this study, we will culture human umbilical cord blood-derived late EPCs withdifferent concentrations of simvastatin to investigate the effect of simvastatin on the functions oflate EPCs.Methods:1. Optimize the culture conditions of late EPCs in the following methods:①The number of survival mononuclear cells (MNCs) were compared by differentisolation methods (density gradient centrifugation and hydroxyethyl starch sedimentation);②The morphologies, the surface markers and the functions of two types of EPCs obtainedby EGM-2MV medium and complete medium M199were compared, which also could identifyEPCs;③The appearance time and number of late EPCs clones were evaluated by differentconcentration of fetal bovine serum (5%,10%and15%) and initial medium renewal time(24h,48h,72h and96h)..2. The good growth state of late EPCs of second generation were treated with simvastatin ina series of concentrations (10-8Mol/L、10-7Mol/L、10-6Mol/L、10-5Mol/L) for24h,48h,72h andthe effect of simvastatin on functions of late EPCs were analyzed by the proliferation,adhesion and tubule-formation assay.Results:1.The outcome of the optimization for late EPCs①Compared with hydroxyethyl starch sedimentation, more survival MNCs were obtainedunder the density gradient centrifugation (P<0.05).②MNCs cultured in EGM-2MV could be differentiated into late EPCs, while in M199medium could be differentiated into early EPCs.Comparison of the two EPCs were as follows:Cell morphologies: late EPCs developed an endothelial shape and appeared clonescomposed of well-circumscribed monolayers of cobblestone-appearing cells; early EPCsshowed spindle-like and formed clusters comprised of a central cord of round cells surroundedby spindle-shaped cells.Surface markers: late EPCs highly expressed endothelial cell-surface antigens CD31, CD34,eNOS, KDR and VE-Cadherin, lowly expressed CD133identified on endothelial andhematopoietic progenitor cells and not expressed the hematopoietic cell-specific surface antigensCD45and CD14; early EPCs highly expressed the hematopoietic cell-specific surface antigensCD45and CD14, could express KDR and nearly not express CD133and CD31.Cell functions: late EPCs showed active proliferative capacity and formed capillary-likestructures in Matrigel; early EPCs showed low proliferative capacity and did not formcapillary-like structures in Matrigel. Both of them could incorporate DiI-acetylated low densitylipoprotein (DiI-Ac-LDL) and bind FITC-ulex-lectin (FITC-UEA-I).③Earlier initial late EPCs clones emergent and more clones were obtained under thefollowing conditions:10%FBS and48-72h as initial medium renewal time.2. Compared with control, lower concentrations of simvastatin (10-8Mol/L、10-7Mol/L、10-6Mol/L) significantly promoted cell proliferation, adhesion and in vitro vascularization and theconcentration of10-7Mol/L was the most prominent, while higher concentration (10-5Mol/L)inhibited the functions above (P<0.05). simvastatin increased the proliferative capacity of EPCsin a time dependent manner, but adhesion and tubule-formation activity was the most prominentat48h (P<0.05).Conclusion:1. By comparison, we got optimal culture method of late EPCs, which laid a foundation for follow-up study and provide reference in the applications of late EPCs for ischemic diseases,cell transplantation treatment and vascular tissue engineering．2. Simvastatin has promoting effect on late EPCs which provides a new theoretical basis forits clinical benefit in the treatment of cardiovascular disease.