title: Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings.
abstract: 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 μm 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 μm) 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).
  
  
  CONCLUSIONS
  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.
authors.forename: Shao-Xun
authors.initials: SX
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhang
authors.forename: Man
authors.initials: M
authors.lastname: Shang
authors.forename: Su
authors.initials: S
authors.lastname: Wei
authors.forename: Miao
authors.initials: M
authors.lastname: Liu
authors.forename: Yi-Lu
authors.initials: YL
authors.lastname: Wang
authors.forename: Meng-Xiao
authors.initials: MX
authors.lastname: Zhang
authors.forename: Yan-Na
authors.initials: YN
authors.lastname: Wu
authors.forename: Ming-Lin
authors.initials: ML
authors.lastname: Liu
authors.forename: Jun-Qiu
authors.initials: JQ
authors.lastname: Song
authors.forename: Yan-Xia
authors.initials: YX
authors.lastname: Liu
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000502:physiology*; D015687:Cell Hypoxia; D004730:Endothelium, Vascular / Q000502:physiology*; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology*; D006801:Humans; D066298:In Vitro Techniques; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D018384:Oxidative Stress; D051381:Rats
chemical_list: D009599:Nitroprusside; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D000109:Acetylcholine