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Objective To investigate the effects of microvesicles(MVs) derived from hypoxia/reoxygenation(H/R)-treated human umbilical vein endothelial cells(HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings.Methods H/R injury model was established to induce HUVECs to release H/R-EMVs.H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium.H/R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry.Thoracic aortic rings of rats were incubated with 2.5,5,10,20 μg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours,and their endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.The nitric oxide(NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent.The expression of endothelial NO synthase(eNOS) and phosphorylated eNOS(p-eNOS,Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting.Furthermore,the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit.Results H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation.The membrane vesicles(< 1 urn) induced by H/R were CD144 positive.ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner(P<0.05,P<0.01).The expression of total eNOS(t-eNOS)was not affected by H/R-EMVs.However,the expression of p-eNOS decreased after treated with H/R-EMVs.The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings(P<0.01).Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner.The mechanisms included a decrease in NO production,p-eNOS expression and an increase in oxidative stress.
Objective To investigate the effects of microvesicles (MVs) derived from hypoxia / reoxygenation (H / R) -treated human umbilical vein endothelial cells (HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings. Methods H / R injury model was established to induce HUVECs to release H / R-EMVs.H / R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H / R-EMVs were characterized using 1 urn latex beads and anti-PE-CD144 by flow cytometry. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 μg / ml H / R-EMVs derived from H / R-treated HUVECs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) or endothelium- independent relaxation in response to sodium nitroprusside (SNP) was recorded in vitro. The nitric oxide (NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent. The expression of endothelial NO synthase (eNOS) and phosphorylated eNOS ( p-eNOS, Ser-1177) in the thoracic aorti c rings of rats were detected by Western blotting. Frthermore, the levels of SOD and MDA in H / R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit. Results H / R-EMVs were induced by H / R-treated HUVECs and isolated by ultracentrifugation. The membrane vesicles (<1 urn) induced by H / R were CD144 positive. ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H / R-EMVs treatment in a concentration the expression of total eNOS was not affected by H / R-EMVs.However, the expression of p-eNOS decreased after treated with H / R-EMVs (P <0.05, P <0.01) .The activity of SOD decreased and the level of MDA increased in H / R-EMVs treated rat thoracic aortic rings (P <0.01) .Conclusion ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H / R-EMVs in a concentration-dependent manner. the mechanisms included a decrease in NO production, p-eNOS expression and an increase in oxidative stress.