In Vitro Study Of The Impacts And Mechanism Of Platelets-Derived Microparticles On HUVECs

BackgroundMicroparticle is a carrier of intercellular signal transduction,which included proteins,mRNAs,miRNAs and other bioactive substances.It plays an important role in the pathophysiological process such as cell proliferation,apoptosis,migration and so on.Platelets are the major source of circulating microparticles.Clinical studies have shown that in patients with coronary heart disease,atrial fibrillation and other cardiovascular diseases,there is a significant increase of microparticles in the circulation.And microparticles are closely related to the process of cardiovascular diseases.Our previous studies have shown that the expression profiling of miRNAs in microparticles derived from activated platelets stimulated by thrombin changed significantly.PMP can be absorbed by endothelial cells and may regulate phenotype of cells through post-transcriptional pathway.MicroRNA is a length of about 21-23 nucleotides of single-stranded non-coding RNA molecules,which regulates gene expression by specifically binding to the m RNA sequence,inducing transforming or degrading mRNA.With the progress of the study,the role of microRNAs in regulation of the pathophysiological process has be taken more and more seriously.It has the potential to be diagnostic biomarkers and targets of biological treatments in the future.ObjectiveThe aim of our study is to establish and improve the separation,extraction system and identification methods of platelet-derived microparticles,and compare the changes of membrane protein of microparticles harvested from platelets stimulated by vortex and thrombin.Then we try to explore the effects of platelet-derived microparticles stimulated by thrombin on the apoptosis,migration,ROS,secretion of NO and inflammatory factors of HUVECs.Then we try to carry out initial exploration in the effects of PMP on the miRNAs expression profile,the impacts and mechanisms of miRNA on the above-mentioned phenotype of HUVECs.MethodsThe platelet-derived microparticles stimulated by vortex and thrombin were obtai ned by gradient centrifugation.The counts,membrane proteins,size and inner struct ure of microparticles were determined by flow cytometry and transmission electron microscope.HUVECs apoptosis were induced by H2O2.The effects of microparticles on HUVECs apoptosis were tested by flow cytometry with Annexin V-FITC/PI stai ning.The effects of microparticles on the migration of HUVECs were detected by cell wound healing and transwell assay.The effects of microparticles on the NO an d inflammatory factors secretion of HUVECs were evaluated by Griess and Elisa as say respectively.The impacts of microparticles on ROS production of HUVECs were determined by ROS kit and Image J software.The changes of expression of miR NAs in HUVECs were detected by real-time PCR assay.Then we chose the micro RNA which have the most change to transfect into HUVECs,and detected the effe cts on the above phenotype of HUVECs.The potential targeted genes of miRNAs were predicted by targetscan and literature review,then tested by dual-luciferase rep orter.Results(1)Flow cytometry showed that platelets could be activated and generate a large amount of microparticles either stimulated by thrombin or vortex,and aspirin could effectively inhibit the generation of platelets-derived microparticles.High purity plateletsderived microparticles could be obtained by gradient centrifugation.The surface membrane proteins of microparticles were complicated and the propotion of CD63 was significantly different on the surface of microparticles stimulated by vortex or thrombin(55.38±5.27% vs 43.50±3.86%,p<0.05).Transmission electron microscopy revealed that microparticles have diversiform structures and electron density,and complex components such as ? granules,glycogen granules,mitochondria and so on.(2)The platelets-derived microparticles stimulated by thrombin could promote cell apoptosis induced by oxidative stress(25.59±9.50% vs 12.42±1.23 %,p<0.05),ROS production(0.140±0.010/pixel vs 0.109±0.010/pixel,p<0.05)and secretion of IL-1?(2.67±0.87 pg/ml vs 1.25±0.41 pg/ml,p<0.05)of HUVECs.On the contrary,they could inhibit the migration(wound scratch assay:47.99±4.65% vs 61.68±2.80%,p<0.05;Transwell assay: 123±15 vs 206±14,p<0.01)and NO secretion(27.68±1.70?mol/L vs 34.13±1.47?mol/L,p<0.01)of HUVECs.(3)The miR-223(150.60±29.63 vs 1.00±0.75,p<0.001)and miR-320(20.84±18.58 vs 1.07±0.98,p<0.05)in HUVECs were significantly increased after co-incubation with platelets-derived microparticles.The miR-223 could promote cell apoptosis induced by oxidative stress(22.45±2.34% vs 12.95±0.36%,p<0.001),and inhibit the migration of HUVECs(wound scratch assay:66.66±4.21% vs 71.59±4.02%,p<0.05;Transwell assay:151±17 vs 215±29,p<0.05),but had no apparently effects on ROS production,secretion of inflammatory factors and NO of HUVECs.We predict NRF1,ATP7 A and ZEB1 three target genes of mi R-223 based on targetscan software and literature review.MiR-223 has no significant impact on NRF1,ATP7 A and ZEB1 at the mRNA level.Dual luciferase reporter gene excluded NRF1 as a target gene of miR-223.Conclusion(1)Platelets are easy to be activated and could derive a large amount of microparticles which have complex compositions and structures and different sizes after being activated.High purity platelets-derived microparticles could be obtained by gradient centrifugation.Microparticles derived from platelets stimulated by thrombin or vortex have a significant difference on CD63.(2)Platelets-derived microparticles could promote apoptosis,ROS production,and secretion of inflammatory factors of HUVECs,but have an opposite influence on migration and NO secretion of HUVECs.(3)A large amount of miR-223 could be transferred into HUVECs through platelets-derived microparticles.Overexpression of miR-223 could promote apoptosis and inhibit migration of HUVECs.NRF1 is not a target gene of miR-223.