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 ring...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.展开更多
Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and exp...Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10^(-7)-10^(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P<0.05, P<0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P<0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.展开更多
Amperometric studies have indicated that substance P as well as NMDA stimulates release of NO in rat aortic rings. These data have been confirmed by functional observations of vaso-relaxant action of NMDA within norad...Amperometric studies have indicated that substance P as well as NMDA stimulates release of NO in rat aortic rings. These data have been confirmed by functional observations of vaso-relaxant action of NMDA within noradrenaline pre-contracted aortic rings, supporting the presence of NMDA receptor in rat aortic rings. It is known that the enzyme endothelial NO synthase (eNOS) mediates vasodilatation not only in rats, but also in C57BL6 mice aortic ring, indicating that in this blood vessel NO is the endogenous endothelium-derived vasodilator. In this work, amperometry together with specifically nitrites insensitive micro-biosensors have been applied to examine the effect of NMDA and substance P upon NO release in rat and in two strains of mice aortic rings. The electrochemical data monitored demonstrate that NMDA mediates vascular relaxation via NO in rats but not in mice. These results are supported by functional data, therefore they suggest that NMDA receptors are “not responding” within these experimental conditions in mice aortic rings.展开更多
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China(20101202110005)the Natural Science Foundation of Tianjin(11JCZDJC18300)+1 种基金the Research Foundation of Tianjin Municipal Education Commission(20110106)the National Key Basic Research Program of China(973 Program, 2011CB933100)
文摘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.
基金supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (20101202110005)the Natural Science Foundation of Tianjin (11JCZDJC18300)the Research Foundation of Tianjin Municipal Education Commission (20110106)
文摘Objective: To investigate the effect of Iptakalim(Ipt) preventing injury of endothelial microvesicles(EMVs) derived from hypoxia/reoxygenation(H/R)-treated HUVECs on the relaxation of rat thoracic aortic rings and explore the underlying mechanism. Methods: H/R injury model was established to release H/R-EMVs from HUVECs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized by using Transmission Electron Microscope(TEM). Thoracic aortic rings of rats were incubated with 10^(-7)-10^(-3 )mol/L Ipt and co-cultured with 10 μg/ml H/R-EMVs for 4 hours, and their endothelium- dependent relaxation in response to acetylcholine(ACh) was recorded in vitro. The nitric oxide(NO) production of ACh-treated rat thoracic aortic rings was measured by using Griess reagent. The expression of endothelial NO synthase(e NOS), phosphorylated e NOS(p-e NOS, Ser-1177), serine/threonine kinas(Akt) and phosphorylated Akt(p-Akt, Ser-473) in the thoracic aortic rings of rats was detected by Western blotting. Results: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The isolated H/R-EMVs subjected to TEM revealed small, rounded vesicles(100–1 000 nm) surrounded by a membrane. H/R-EMVs impaired relaxation induced by ACh of rat thoracic aortic rings significantly. Compared with H/R-EMVs treatment individually, relaxation and NO production of rat thoracic aortic rings were increased by Ipt treatment in a concentration-dependent manner(P<0.05, P<0.01). The expression of total e NOS(t-e NOS) and total Akt(t-Akt) was not affected by Ipt or H/R-EMVs. However, the expression of p-e NOS and p-Akt increased after treated with Ipt(P<0.01). Conclusion: Based on H/R-EMVs treatment, ACh induced endothelium-dependent relaxation of rat thoracic aortic rings was ameliorated by Ipt in a concentration-dependent manner. The mechanisms involved the increase in NO production, p-e NOS and p-Akt expression.
文摘Amperometric studies have indicated that substance P as well as NMDA stimulates release of NO in rat aortic rings. These data have been confirmed by functional observations of vaso-relaxant action of NMDA within noradrenaline pre-contracted aortic rings, supporting the presence of NMDA receptor in rat aortic rings. It is known that the enzyme endothelial NO synthase (eNOS) mediates vasodilatation not only in rats, but also in C57BL6 mice aortic ring, indicating that in this blood vessel NO is the endogenous endothelium-derived vasodilator. In this work, amperometry together with specifically nitrites insensitive micro-biosensors have been applied to examine the effect of NMDA and substance P upon NO release in rat and in two strains of mice aortic rings. The electrochemical data monitored demonstrate that NMDA mediates vascular relaxation via NO in rats but not in mice. These results are supported by functional data, therefore they suggest that NMDA receptors are “not responding” within these experimental conditions in mice aortic rings.
