| Vascular endothelial dysfunction is in the initial and core process of the pathogenesis of cardiovascular diseases. It is a major abnormality in many diseases such as acute myocardial ischemia, diabetes, and atherosclerosis. Recent studies indicated that patients with diabetes, atherosclerosis and ischemic heart disease and many other cardiovascular diseases have increased circulating levels of microvesicles(MVs). These circulating MVs contain MVs released from activated or apoptotic endothelial cells. MVs released from endothelial cells(EMVs) not only a biomarker but also play a critical role in the pathogenesis of vascular endothelial dysfunction. Myocardial ischemia/reperfusion(I/R), as the pathophysiologic basis of coronary artery diseases, induced severe injury, apoptosis and necrosis on myocytes. However, it is not clear whether hypoxia/reoxygenation induced EMVs(H/R-EMVs) play an important role on endothelial dysfunction. Whether H/R-EMVs can induce impairment of endothelium relaxation and the underlying mechanisms have not been elucidated. In the current research, the hypoxia/reoxygenation(H/R) injury model of HUVECs was established in vitro to mimic I/R injury in vivo. The H/R-EMVs derived from this model was used to induce effects on the endothelium relaxation of thoracic aortic rings of rats. The underlying mechanisms were studied.Methods:1. Preparation of hypoxia/reoxygenation injury model in HUVECsHUVECs were cultured with DMEM containing 10% FBS for 24 hours. Then supernatants of subconfluent HUVECs were replaced with a mimic hypoxic buffer. HUVECs were put into hypoxic chamber. The gas mixture containing 95% N2 and 5% CO2 were filled into chamber at a rate of 20 L/min. HUVECs were exposed to the anoxic condition in the hypoxic chamber for 12 h and then to normoxia for 4 h at 37oC, then cell viability was tested by MTT assay. H/R injury model was built.2. Preparation of H/R-EMVs, flow cytometric analysis and protein quantification of H/R-EMVsH/R-EMVs were collected from Hypoxic buffer through ultracentrifugation and identified by flow cytometry. H/R-EMVs were characterized using 1 μm calibrationbeads and anti-PE-CD144 antibody by flow cytometry. Absolute number of H/R-EMVs was calculated with 2 μm standard beads, and protein contents of H/R-EMVs were quantified by BCA assay.3. Preparation of thoracic aortic rings of ratsMale Wistar rats(body wt 250±10 g) were chosen. The thoracic cavity was opened. Rat thoracic aorta obtained from rats were carefully removed and placed in Krebs solutions. Aorta were cleared from periadventitial tissue and cut into 3-4 mm rings.4. Preparation of thoracic aortic rings of rats treated with H/R-EMVs Thoracic aortic rings of rats were cultured with DMEM containing 10%Thoracic aortic rings of rats were cultured with DMEM containing 10% FBS and were divided into control group, H/R-EMVs 2.5, 5 10, 20 μg/m L groups randomly. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 μg/m L H/R-EMVs derived from H/R-treated HUVECs for 4 hours. The Control group incubated with D-Hank’s solution.5. Relaxations of thoracic aortic rings of rats induced by ACh or SNPThoracic aortic rings of rats were placed in 10 m L tissue chambers filled with Krebs solution and adjust basal resting tension 2.0 g. Then aortic rings were treated with phenylephrine(PE, 10-6 mol/L) to make the aortic rings contract. When aortic rings arrived at the stable maximal contractile response after 15 min the endothelium-dependent relaxation in response to acetylcholine(ACh) or endothelium-independent relaxation in response to sodium nitroprusside(SNP) was recorded in vitro.6. Measurement of NO productionThe nitric oxide(NO) production of thoracic aortic rings of rats was measured using Griess reagent.7. Western blot for detecting the expression of t-eNOS and p-eNOSThe expression of total endothelial NO synthase(t-e NOS) and phosphorylated e NOS(p-e NOS, Ser-1177) of thoracic aortic rings of rats was detected by Western blot.8. Measurement of SOD, MDA productionThe activity of SOD and level of MDA in H/R-EMVs-treated thoracic aorticrings of rats were measured using SOD and MDA kit.Results:1. Hypoxia/reoxygenation was used to establish the injury model of HUVECsHUVECs showed shrinkage and distortion after 12 hours of hypoxia and 4 hours of reoxygenation(H12/R4). Compared with control, viability of HUVECs was decreased significantly(73.73%±2.61% vs 100%±0%, P<0.05). With moderate decrease of cell viability, experimental results stable, H12/R4 was appropriate for induction of H/R model on HUVECs.2. Isolation and characterization of H/R-EMVsAfter ultracentrifuge, there is small white sedimentation in the bottom of the ultracentrifuge tubes which refers to H/R-EMVs. Microvesicles induced by H/R were identified by Flow Cytometry were CD144 positive with a size of <1 μm. The concentration of H/R-EMVs was 23017±322/μL, and the pelleted H/R-EMVs protein was quantified at 0.35±0.1 μg/μL.3. Effects of H/R-EMVs on ACh or SNP-induced relaxationCompared with control(93.59%±7.53%), the relaxation of aortic rings induced by a cumulative increments of ACh was impaired by H/R-EMVs at the concentrations of 5, 10, 20 μg/m L(Emax: 81.11%±6.12%, 67.71%±5.96%, 43.50%±9.35% for 5, 10, 20 μg/m L, respectively, P<0.05, P<0.01) significantly. The relaxation of aortic rings induced by ACh was impaired by H/R-EMVs in a dose-dependent manner at the concentration range of 5-20 μg/m L. There was no significant different of the aortic rings relaxation between H/R-EMVs 2.5 μg/m L group(Emax: 99.53%±2.47%) and control. Endothelium-independent relaxation induced by SNP was similar between control and H/ R-EMVs treated groups4. Effects of H/R-EMVs on NO productionWestern blot showed that compared with control(84.64±4.37 μmol), NO production of rat thoracic aortic rings were decreased by H/R-EMVs at the concentrations of 5, 10, 20 μg/m L(72.61±0.81, 34.58±1.97, 21.45±1.16 μmol, P<0.01) significantly. The NO production decreased by H/R-EMVs in a dose-dependent manner at the concentration range of 5-20 μg/m L. There was no significant different of the NO production of aortic rings between H/R-EMVs 2.5μg/m L group(88.26±4.68 μmol) and control.5. Effects of H/R-EMVs on t-e NOS and p-e NOS expressionCompared with control, the expression of p-e NOS of rat thoracic aortic rings decreased by H/R-EMVs at the concentrations of 5, 10, 20 μg/m L significantly. The expression of p-e NOS was decreased by H/R-EMVs in a dose-dependent manner at the concentration range of 5-20 μg/m L. The expression of t-e NOS was not affected by H/R-EMVs. The ratio of p-e NOS/t-e NOS was decreased. There was no significant different of the p-e NOS of rat thoracic aortic rings between H/R-EMVs 2.5 μg/m L group and control.6. Effects of H/R-EMVs on SOD, MDA productionCompared with control(SOD: 52.22±1.25 U/mg prot; MDA: 1.91±0.11 μmol/mg prot), the activity of SOD decreased and the level of MDA increased of rat thoracic aortic rings by H/R-EMVs at the concentrations of 5, 10, 20 μg/m L:(Activity of SOD: 23.44±0.99, 17.28±0.63, 10.88±0.54 U/mg prot; Level of MDA: 3.27±0.19, 8.08±0.19, 10.22±0.17 μmol/mg prot, P<0.01) significantly. The activity of SOD was increased and the level of MDA decreased by H/R-EMVs in a dose-dependent manner at the concentration range of 5-20 μg/m L. There was no significant different of activity of SOD and level of MDA of rat thoracic aortic rings between H/R-EMVs 2.5 μg/m L(SOD: 51.5±2.12 U/mg prot; MDA: 1.97±0.13 μmol/mg prot, P<0.01)group and control.Conclusion:1. Stable H12/R4 injury model of HUVECs was established.2. H/R-EMVs were generated from HUVECs under the condition of H12/R4, and characterized as CD144+ vesicles with diameter <1 μm. And the pelleted H/R-EMVs protein was quantified at 0.35±0.1 μg/μL.3. ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner at the concentration range of 5-20 μg/m L.4. The NO production of thoracic aortic rings of rats was decreased by H/R-EMVs in a concentration-dependent manner at the concentration range of 5-20 μg/m L. The mechanisms of the impairment of ACh induced relaxation of thoracicaortic rings of rats included a decrease of NO production.5. The expression of p-e NOS thoracic aortic rings of rats was decreased by H/R-EMVs in a concentration-dependent manner at the concentration range of 5-20 μg/m L. The expression of t-e NOS was not affect by H/R-EMVs. The decrease of NO production may due to the decrease expression of p-e NOS.6. The activity of SOD decreased and level of MDA increased of thoracic aortic rings of rats by H/R-EMVs in a concentration-dependent manner at the concentration range of 5-20 μg/m L. The mechanism of the impairment of ACh induced relaxation of thoracic aortic rings of rats included an increase in oxidative stress. |
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