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Endothelial microparticles (EMPs) are capable of transferring active molecules, such as microRNA, to neighboring cells and regulate the biological functions of these recipient cells, which are considered as the new messengers in cell-cell communication. Our previous study found that microRNA-19b in plasma microparticles of patients with unstable angina was obviously up-regulated, which partially results from the increase of circulating EMPs. Nevertheless, the role of EMPs-mediated transfer of microRNA-19b in atherosclerosis has not yet been elucidated. The aim of the study was to investigate the Endothelial microparticles released from Human umbilical vein endothelial cells(HUVECs) which contains different levels of microRNA-19b and its effects on HUVECs migration and angiogenesis, so that molecular mechanism of microRNA-19b in endothelial microparticles can be further illustrate in atherosclerosis.
(1) Stimulated HUVECs with different concentration and different time of hypoxia. Collect microparticles to detect content using flow cytometry after gradient centrifugation of cell culture. The level of microRNA-19b in endothelial microparticles was detected by quantitative PCR to determine the optimal concentration and time which continuous stimulating to release microparticles;(2) The endothelial cells were labeled with Calcein-M and the dynamic release of microparticles was observed by confocal microscopy. The microparticles was acquired from cell culture by gradient centrifugation after FAM-microRNA-19b was transfected into endothelial cells, than co cultured with endothelial cells to observe green fluorescent microRNA-19b by confocal microscopy;(3) Incubated with different levels of microRNA-19b (normal, increased, decreased) in microparticles, endothelial cells performed different cell functions, including A: cell migration: Scratch test and Transwell test; B: angiogenesis: tube formation assay;(4) The potential target genes of microRNA-19b were predicted using several online programs with databases, including TargetScan, miRanda and PicTar. In order to identify the target genes regulate cellular biological effects, the expression of mRNA and protein levels in cells was detected by quantitative PCR and Western blots assay.
(1) Microparticles released from HUVECs contains microRNA-19b, the optimal concentration for endothelial cells to release microRNA-19b is 37°C,3％O2,5％CO2, 12 hours is the optimum stimulus time; (2)Confocal microscopy was used to dynamically monitor the release of endothelial microparticles and their fluorescent labeled microRNA-19b into endothelial cells; (3)Endothelial microparticles with high level of microRNA-19b from HUVECs could suppress migration of endothelial cells; (4) Endothelial microparticles with high level of microRNA-19b from HUVECs could impairs HUVEC tube formation; (5) ARHGAP5 and TGFβ2 are target of microRNA-19b,which modulate cell migration and angiogenesis of HUVECs.
(1) Microparticles released from HUVECs contain microRNA-19b, which act as intercellular communication messenger into endothelial cells; (2) HUVECs continued to release microparticles of the highest level under the stimulus condition as37°C,3％O2,5％CO2 in 12h; (3) Endothelial microparticles with high microRNA-19b from HUVECs could inhibit endothelial cell migration and angiogenesis; (4) RHGAP5 and TGFβ2 are target of microRNA-19b,which modulate the function of HUVECs.