Platelet-derived Microparticles Affects Vascular Endothelial Growth Factor Expression And The Mechanism In Endothelial Cells

Objective:Detect vascular endothelial growth factor induce by platelet-derived microparticles (PMPs) in human umbilical vein endothelial cells(HUVECs) and its downstream transduction pathway. Study on the difference of vascular endothelial growth factor(VEGF) expression in HUVECs induced by different concentration of PMPs and different intervention time. The aim of our study was to discuss the possible mechanism of PMPs effecting on VEGF releasing and its clinical significance in endothelial cells.Methods:Applying the method of flow cytometry (FCM) to isolate platelet-derived microparticles (PMPs) from platelet poor plasma(PPP), and staine with fluorescin isothiocyanate monoclonal antibody against CD61, applying flow cytometry (FCM) method to detect the quantification of PMPs, and the tolal protein concentration of PMPs was determined using Semi-automatic Biochemical Analyzer. Human umbilical vein endothelial cells were maintained at 37℃,5%CO2 humidified atmosphere, in DMEM low glucose, supplemented with 10% heat inactivated fetal calf serum and 1% penicillin/streptomycin, for at least 3-4 passage of HUVECs, cells were separated and seeded into six pore plate (pore size 9.6 cm2, 1×105/ml cells). PMPs intervene HUVECs until an 100% cells confluent monolayer was formed,the HUVECs attachment number is about 2.5×106 each pore. Different concentrations of PMPs intervene HUVECs and incubate for different time.There are four cell pores each group, collecting the cells after different intervention time(2,4,24hours) of PMPs. We used semi-quantitative reverse transcription polymerase chain reaction techniques to measure VEGF,phosphatidylinositol-3 kinase(PI3K),extracellular signal-regulated kinase(ERK)and VEGF typeⅡreceptor (KDR) mRNA levels.Results:High density of PMPs can be obtained from PPP after activation of adenosine diphosphate (ADP) and detected by FCM.VEGF expression in HUVECs induced by different concentration of PMPs and different intervention time, semi-quantitative reverse transcription polymerase chain reaction techniques measures VEGF,PI3K,ERK and KDR mRNA levels. VEGF mRNA leverl in control group(non intervention group) was significantly higher than the four different concentration of PMPs groups(P<0.05).In contrast,KDR mRNA leverl in control group(non intervention group) was significantly lower than the four different concentration of PMPs groups(P<0.05).ERK mRNA leverl in 50μg/mLPMPs group was significantly higher than the other four groups(P<0.05), PI3K mRNA leverl in 100μg/mLPMPs group was significantly higher than the other groups(P=0.016), we can detect that KDR,ERK,PI3K mRNA leverls turn out ascend trend along with the concentration of PMPs.Just as the effect of of different concentration PMPs on mRNA expression, VEGF mRNA leverl in control group(non intervention group) was significantly higher than the 24 hours intervention time group(P<0.05).KDR mRNA leverl in the 4hour and 24hour groups were significantly higher than control group (P<0.05). PI3K mRNA leverl in 24hour group was significantly higher than the control group(P<0.05).Conclusions:Abundant PMPs can be obtained from PPP after activation of adenosine diphosphate (ADP). PMPs interfere on the HUVECs, PMPs may release generous VEGF,which result in decreased expression of VEGF in HUVECs by negative feedback and increased expression of VEGF typeⅡreceptor(KDR)and the two keyenzymes in KDR Downstream Signaling Transduction Pathways,that is to say, PMPs may induce the biological processes of blood vessel and angiogenesis via PI3K/Akt and MAPK/ERK signal transduction pathways, and play an important role in proliferation of HUVECs and Vascular Smooth Miscle Cell, PMPs have important significance on the process of neovascularization in ischemia myocardium and the formation of atherosclerotic plaque.