title: Pyridoxine inhibits endothelial NOS uncoupling induced by oxidized low-density lipoprotein via the PKCα signalling pathway in human umbilical vein endothelial cells.
abstract: OBJECTIVE
  One key mechanism for endothelial dysfunction is endothelial NOS (eNOS) uncoupling, whereby eNOS generates superoxide (O(2) (•-) ) rather than NO. We explored the effect of pyridoxine on eNOS uncoupling induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs) and the potential molecular mechanism.
  
  
  METHODS
  HUVECs were incubated with ox-LDL with/without pyridoxine, N(G) -nitro-L-arginine methylester (L-NAME), chelerythrine chloride (CHCI) or apocynin. Endothelial O(2) (•-) was measured using lucigenin chemiluminescence, and O(2) (•-) -sensitive fluorescent dye dihydroethidium (DHE). NO levels were measured by chemiluminescence, PepTag Assay for non-radioactive detection of PKC activity, depletion of PKCα and p47phox by siRNA silencing and the states of phospho-eNOS Thr495, total-eNOS, phospho-PKCα/βII, total PKC, phospho-PKCα, total PKCα and p47phox were measured by Western blot.
  
  
  RESULTS
  Ox-LDL significantly increased O(2) (•-) production and reduced NO levels released from HUVECs; an effect reversed by eNOS inhibitor, L-NAME. Pyridoxine pretreatment significantly inhibited ox-LDL-induced O(2) (•-) generation and preserved NO levels. Pyridoxine also prevented the ox-LDL-induced reduction in phospho-eNOS Thr495 and PKC activity. These protective effects of pyridoxine were abolished by the PKC inhibitor, CHCI, or siRNA silencing of PKCα. However, depletion of p47phox or treatment with the NADPH oxidase inhibitor, apocynin, had no influence on these effects. Also, cytosol p47phox expression was unchanged by the different treatments.
  
  
  CONCLUSIONS
  Pyridoxine mitigated eNOS uncoupling induced by ox-LDL. This protectant effect was related to phosphorylation of eNOS Thr495 stimulated by PKCα, not via NADPH oxidase. These results provide support for the use of pyridoxine in ox-LDL-related vascular endothelial dysfunction.
authors.affiliation: Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics, Atherosclerosis Research Centre, Nanjing Medical University, Nanjing, China.
authors.forename: Liping
authors.initials: L
authors.lastname: Xie
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Wen
authors.initials: W
authors.lastname: Zhang
authors.forename: Qiongyu
authors.initials: Q
authors.lastname: Mi
authors.forename: Xiaozhen
authors.initials: X
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Tang
authors.forename: Qi
authors.initials: Q
authors.lastname: Chen
authors.forename: Albert
authors.initials: A
authors.lastname: Ferro
authors.forename: Yong
authors.initials: Y
authors.lastname: Ji
mesh_terms: D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D051571:Protein Kinase C-alpha / Q000378:metabolism*; D011736:Pyridoxine / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D014803:Vitamin B Complex / Q000494:pharmacology*
chemical_list: D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D013481:Superoxides; D014803:Vitamin B Complex; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051571:Protein Kinase C-alpha; D011736:Pyridoxine