title: Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers.
abstract: BACKGROUND
  Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs).
  
  
  RESULTS
  52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers' serum but not to non-smokers' serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamate-cysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers' serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs.
  
  
  CONCLUSIONS
  In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion.
authors.affiliation: Department of Medicine, Section of Internal Medicine D, University of Verona, Verona, Italy. anna.frattapasini@univr.it
authors.forename: Anna
authors.initials: A
authors.lastname: Fratta Pasini
authors.forename: Anna
authors.initials: A
authors.lastname: Albiero
authors.forename: Chiara
authors.initials: C
authors.lastname: Stranieri
authors.forename: Mattia
authors.initials: M
authors.lastname: Cominacini
authors.forename: Andrea
authors.initials: A
authors.lastname: Pasini
authors.forename: Chiara
authors.initials: C
authors.lastname: Mozzini
authors.forename: Paola
authors.initials: P
authors.lastname: Vallerio
authors.forename: Luciano
authors.initials: L
authors.lastname: Cominacini
authors.forename: Ulisse
authors.initials: U
authors.lastname: Garbin
mesh_terms: D000328:Adult; D015153:Blotting, Western; D020134:Catalytic Domain; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism / Q000503:physiopathology*; D005260:Female; D005721:Glutamate-Cysteine Ligase / Q000235:genetics / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000378:metabolism; D008074:Lipoproteins / Q000097:blood; D008297:Male; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010713:Phosphatidylcholines / Q000097:blood / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D044967:Serum; D012907:Smoking / Q000097:blood / Q000503:physiopathology*; D055815:Young Adult
chemical_list: C468114:1-palmitoyl-2-arachidonyl-3-phosphorylcholine; D003470:Culture Media; D008074:Lipoproteins; D051267:NF-E2-Related Factor 2; D010713:Phosphatidylcholines; D017382:Reactive Oxygen Species; D051547:Heme Oxygenase-1; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione