Effects Of Simvastatin On Number And Activity Of Endothdlal Progenitor Cells From Peripheral Blood

Objective:We investigated the effects of simvastatin on endothelial progenitor cell(EPC)number,proliferation,migration, adhesion,senescence and apoptosis.Methods:Total mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation,and then the cells were plated on fibronectincoated culture dishes.After 7 days cultured,attached cells were stimulated with simvastatin of different concentrations(0.0001,0.001,0.01,0.1,1.0μmol/L)..EPCs were characterized as adherent cells positive for both DiI-acLDL and UEA-I by direct fluorescent staining.EPC number was counted under an inverted microscope.EPC proliferation was analysed by MTT assay.EPC migration was detected by a modified Boyden chamber assay.EPC adhesion assay was performed by counting adherent cells on fibronectin-coated culture dishes.EPC senescence was assessed by both senescence-associated-β-galactosidase staining and DAPI staining.EPC apoptosis was evaluated by flow cytometric analysis. Results:Simvastatin not only increased EPC number,but also promote proliferative capacity,migratory capacity and adhesive capacity of EPCs significantly(P＜0.01 or P＜0.05)with a maximal effect at 0.01 mol/L. However,the enhancement of simvastatin on EPC capacity was reversed by the PI3K inhibitor Ly294002.Furthermore,the inhibition of EPC senescence occurred at 0.01μmol/L of simvastatin.Simvastatin exhibited a trend toward reducing apoptosis of EPCs under ischema-induced condition in the present study.Conclusion:Our findings indicate that simvastatin increase EPC number,improve EPC proliferation,promote EPC migration,enhances EPC adhesion and delays the onset of senescence of EPCs.The proliferative,promigratory,proadhesive effects of simvastatin on EPC are demonstrating the involvement of the PI3K-Akt pathway. Objective:The aim of this present study is to investigate the impacts of combinatorial simvastatin administration and endothelial progenitor cell(EPC)transplantation on therapeutic angiogenesis in an athymic nude mouse model of hind limb ischemia.Methods:Athymic nude mice were divided into 4 groups(n=10/group):vehicle administration plus PBS injection(control),simvastatin administration plus PBS injection(simvastatin),vehicle administration plus EPC transplantation(EPC),and Simvastatin administration plus EPC transplantation(combination).Results:The laser Doppler perfusion index was significantly higher in the simvastatin group,the EPC group and the combination group than in the control group(0.59±0.06,0.68±0.07 and 0.81±0.07 versus 0.46±0.05,respectively,P＜0.05 or P＜0.01) on day 10 after treatment and showed further improvement afterwards (0.74±0.07,0.80±0.07 and 0.91±0.06 versus 0.57±0.06,respectively, P＜0.05 or P＜0.01)on day 21.The LDPI index was the highest in the combination group among the 4 groups.The capillary/muscle fiber ratio was the highest in the combination group,followed by the EPC group,the simvastatin group and the control group(0.69±0.07,0.49±0.05 and 0.45±0.04 versus 0.26±0.03,respectively,P＜0.01).Importantly,the percentage of TUNEL-positive cells markedly decreased in the simvastatin group(25.62±2.62%),the EPC group(23.43±2.55%)and the combination group(14.43±1.42%)compared with the control group (37.24±3.83%,P＜0.01).The percentage of apoptotic cells was the lowest in the combination group among the 4 group.The percentage of Bax positive cells was significantly reduced in the simvastatin group,the EPC group and the combination group compared with the control group (39.03±4.27%,35.51±3.60%and 20.84±2.05%versus 65.14±6.47%, respectively,P＜0.01).The percentage of Bcl-2 positive cells was higher in the simvastatin group,the EPC group and the combination group than in the control group(34.70±3.52%,37.24±3.74%and 68.16±6.25% versus 17.29±1.81%,respectively,P＜0.01).The percentage of Bax-positive cells was the lowest(Figure 6 B)and the percentage of Bcl-2-positive cells was the highest(Figure 6 C)in the combination group among the 4 groups.Moreover,the combination therapy exhibited the highest efficacy of increasing the ratio of phospho-Akt to Akt among the 4 groups.Conclusions:The simvastatin and EPC combination therapy promotes powerful angiogenesis in hindlimb ischemia.The combination therapy not only inhibites apoptosis of ischemic skeletal muscle cells partially via downregulation of Bax and upregulation of Bcl-2,but also activates Akt phosphorylation significantly.These efficacies may be mediated by the angiogenic potency of simvastatin, EPCs,and by the beneficial effects of simvastatin on transplanted EPCs as well.