Abstract 20061: NADPH Oxidase 1, not 2 or 4, Mediates Diabetic Uncoupling of eNOS
Introduction: We have previously shown that NAD(P)H oxidase (NOX) lies upstream of uncoupled eNOS in angiotensin II (Ang II) treated endothelial cells, and that Ang II induces eNOS uncoupling in diabetic mice. We have also recently demonstrated that folic acid potently recouples eNOS in Ang II-induced hypertension, via upregulation of tetrahydrobiopterin salvage enzyme dihydrofolate reductase (DHFR).
Hypothesis: One or more NOX isoform(s) mediate(s) diabetic uncoupling of eNOS. Mitochondrion however does not contribute to uncoupling of eNOS in diabetes.
Methods and Results: Diabetes was induced by streptozotocin administration. Superoxide levels were determined by electron spin resonance. Transfection of mice with siRNAs was achieved by repetitive tail vein injection of siRNAs-Invivofectaimine mixture. The L-NAME sensitive superoxide production, reflective of eNOS uncoupling activity, was provoked in the wild type diabetic aortas, but not in the p47phox deficient diabetic aortas, implicating an intermediate role of p47phox-dependent NOX in diabetic uncoupling of eNOS. The eNOS uncoupling activity was preserved in the NOX2 null diabetic mice, but diminished by NOX1 or NOXO1 (NADPH oxidase organizer 1) siRNA transfection of diabetic mice. In contrast, NOX4 siRNA was ineffective in attenuating diabetic uncoupling of eNOS. Inhibition of mitochondrial complex I or III with Rotenone or siRNA targeting complex III Rieske Fe-S center, had no effect in preventing diabetic uncoupling of eNOS. Oral administration of folic acid abolished eNOS uncoupling in diabetic mice, presumably via restoration of DHFR, which is deficient in diabetic mice to mediate eNOS uncoupling.
Conclusions: Our present data for the first time demonstrate that NOX1, but not NOX2, NOX4 or mitochondrion, mediates diabetic uncoupling of eNOS. Folic acid may prove to be vascular protective in diabetes by effectively recoupling eNOS.
- © 2010 by American Heart Association, Inc.