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 microRN...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 of miR-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 culturedin 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’st-test was used when two groups were compared.Results: Compared with EMPcontrol- 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 thatTGFβ2 was a direct target gene of miR-19b using the luciferase assay. The expression ofTGFβ2 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.展开更多
基金grants from the National Natural Science Foundation of China (Nos.81770356, 81470473,and 81600340)and the Capital Health Research and Development of Special (No.2016-2-4083).
文摘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 of miR-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 culturedin 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’st-test was used when two groups were compared.Results: Compared with EMPcontrol- 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 thatTGFβ2 was a direct target gene of miR-19b using the luciferase assay. The expression ofTGFβ2 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.
