Conclusions/interpretation
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.
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/interpretation: 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.