Effect of Endothelial Microparticles Induced by Hypoxia on Migration and Angiogenesis of Human Umbilical Vein Endothelial Cells by Delivering MicroRNA-19b

摘要

Background:Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells,which are involved in the development of atherosclerosis.Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients.Hypoxia is involved in atherosclerosis as a critical pathological stimulus.However,it still remains unclear whether the increase ofmiR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b-wrapped within EMPs-stimulates hypoxia on vascular endothelial cells.This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells.Methods:Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and arranged to harvest EMPs in two parts:the first part consisted of EMPcontrol and EMPhypoxia and the second part included EMPvehicle,EMPNc mimic,and EMPmiR-19b mimic.Cell migration was detected by scratch migration and transwell chamber migration.Angiogenesis was assessed by tube formation assays.Furthermore,we predicted the target gene of miR-19b by bioinformatics analysis,and luciferase assay was used to verify the targeted gene of miR-19b.Data were analyzed by one-way analysis of variance.Student's t-test was used when two groups were compared.Results:Compared with EMP l-and EMphypoxia-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs.28.37 ± 1.40,P ＜ 0.001) and transwell chamber migration assay (83.00 ± 3.46 vs.235.00 ± 16.52,P ＜ 0.01),the number of tube formations was markedly reduced by 70％ in the EMPhypoxia group (P ＜ 0.001) in vitro analysis of HUVECs.Meanwhile,a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMPmiR-19b mimic was observed.This effect might be due to the delivery of miR-19b in EMPs.Transforming growth factor-β2 (TGFβ2) was predicted to be one of the target genes of miR-19b,and we further confirmed that TGFβ2 was a direct target gene of miR-19b using the luciferase assay.The expression of TGFβ2 in HUVECs was inhibited by treatment with EMPhypoxia and EMPmiR-19b mimic.Conclusions:MiR-19b in EMPs induced by hypoxia could reduce endothelial cell migration and angiogenesis by downregulating TGFβ2 expression,which may have inhibited the progression of atherosclerosis.