title: Dual role of endothelial nitric oxide synthase in oxidized LDL-induced, p66Shc-mediated oxidative stress in cultured human endothelial cells.
abstract: BACKGROUND
  The aging gene p66Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66Shc activation.
  
  
  RESULTS
  Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66Shc at Ser-36, resulting in an enhanced production of superoxide anion (O2-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66Shc phosphorylation, suggesting that basal NO production inhibits O2- production. oxLDL-induced, p66Shc-mediated O2- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66Shc activation and reduced O2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66Shc activation, and reduced O2- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.
  
  
  CONCLUSIONS
  The present study reports that in primary cultured HAEC treated with oxLDL, p66Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.
authors.affiliation: Cardiology, University Heart Center, University Hospital Zürich and Center for Molecular Cardiology, Campus Schlieren, University of Zurich, Zurich, Switzerland; Center for Integrative Human Physiology (ZHIP), University of Zurich, Zurich, Switzerland; Biomedical Research Center, Zhongshan Hospital, Fudan University, Shanghai, China.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Cardiology, University Heart Center, University Hospital Zürich and Center for Molecular Cardiology, Campus Schlieren, University of Zurich, Zurich, Switzerland; Center for Integrative Human Physiology (ZHIP), University of Zurich, Zurich, Switzerland.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Lüscher
authors.affiliation: Cardiology, University Heart Center, University Hospital Zürich and Center for Molecular Cardiology, Campus Schlieren, University of Zurich, Zurich, Switzerland; Center for Integrative Human Physiology (ZHIP), University of Zurich, Zurich, Switzerland.
authors.forename: Giovanni G
authors.initials: GG
authors.lastname: Camici
mesh_terms: D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects*; D006801:Humans; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects*; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013481:Superoxides / Q000378:metabolism
chemical_list: D008077:Lipoproteins, LDL; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; C099252:oxidized low density lipoprotein; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; C003402:sapropterin