title: The p47phox- and NADPH oxidase organiser 1 (NOXO1)-dependent activation of NADPH oxidase 1 (NOX1) mediates endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction in a streptozotocin-induced murine model of diabetes.
abstract: OBJECTIVE
  We have previously shown that NADPH oxidase (NOX) lies upstream of uncoupled endothelial nitric oxide synthase (eNOS), which is known to occur in diabetic endothelium. However, it remains unclear which specific NOX isoform(s) is responsible for eNOS uncoupling and endothelial dysfunction in diabetic mouse models. The aim of the present study was to test the hypothesis that one or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS, which has been shown to occur in patients with diabetes to contribute to endothelial dysfunction.
  
  
  METHODS
  Diabetes was induced by streptozotocin administration. The N (ω)-nitro-L-arginine methyl ester (L-NAME)-sensitive superoxide production of aortic segments, reflective of eNOS uncoupling activity, was determined by electron spin resonance.
  
  
  RESULTS
  The L-NAME-sensitive superoxide production was more than doubled in wild-type diabetic mice, implicating uncoupling of eNOS. This was abolished in diabetic p47 ( phox-/-) (also known as Ncf1 (-/-)) mice, but preserved in Nox2 (-/y) (also known as Cybb (-/-)) mice made diabetic. The eNOS uncoupling activity was markedly attenuated in diabetic mice transfected with Nox1 or Nox1 organiser 1 (Noxo1) short interfering RNA (siRNA), and abolished in Nox1 (-/y) diabetic mice. Diabetes-induced impairment in endothelium-dependent vasorelaxation was also significantly attenuated in the Nox1 (-/y) mice made diabetic. By contrast, Nox4 siRNA, or inhibition of mitochondrial complex I or III with rotenone or siRNA, respectively, had no effect on diabetic uncoupling of eNOS. Overexpression of Dhfr, or oral administration of folic acid to improve dihydrofolate reductase (DHFR) function, recoupled eNOS in diabetes to improve endothelial function.
  
  
  CONCLUSIONS
  Our data demonstrate for the first time that the p47(phox) and NOXO1-dependent activation of NOX1, but not that of NOX2, NOX4 or mitochondrion, mediates diabetic uncoupling of eNOS. NOX1-null mice are protected from diabetic endothelial dysfunction. Novel approaches to inhibit NOX1 and/or improve DHFR function, may prove to have therapeutic potential for diabetic endothelial dysfunction.
authors.affiliation: Division of Molecular Medicine, Cardiovascular Research Laboratories, Departments of Anesthesiology and Medicine, David Geffen School of Medicine, University of California Los Angeles, 650 Charles E Young Drive, Los Angeles, CA 90095, USA.
authors.forename: J Y
authors.initials: JY
authors.lastname: Youn
authors.forename: L
authors.initials: L
authors.lastname: Gao
authors.forename: H
authors.initials: H
authors.lastname: Cai
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D014664:Vasodilation
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C472287:Noxo1 protein, mouse; D011506:Proteins; D052250:Nitric Oxide Synthase Type III; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D019255:NADPH Oxidases; C000614711:NOX1 protein, mouse; C060505:neutrophil cytosolic factor 1