3′,4′-Dihydroxyflavonol Reduces Superoxide and Improves Nitric Oxide Function in Diabetic Rat Mesenteric Arteries

Background: 3',4'-Dihydroxyflavonol (DiOHF) is an effective antioxidant that acutely preserves nitric oxide (NO) activity in the presence of elevated reactive oxygen species (ROS). We hypothesized that DiOHF treatment (7 days, 1 mg/kg per day s.c.) would improve relaxation in mesenteric arteries from diabetic rats where endothelial dysfunction is associated with elevated oxidant stress. Methodology/Principal Findings: In mesenteric arteries from diabetic rats there was an increase in ROS, measured by L-012 and 2',7'-dichlorodihydrofluorescein diacetate fluorescence. NADPH oxidase-derived superoxide levels, assayed by lucigenin chemiluminescence, were also significantly increased in diabetic mesenteric arteries (diabetes, 4892 +/- 946 counts/mg versus normal 2486 +/- 344 counts/mg, n = 7-10, p<0.01) associated with an increase in Nox2 expression but DiOHF (2094 +/- 300 counts/mg, n = 10, p<0.001) reversed that effect. Acetylcholine (ACh)-induced relaxation of mesenteric arteries was assessed using wire myography (pEC(50) = 7.94 +/- 0.13 n = 12). Diabetes significantly reduced the sensitivity to ACh and treatment with DiOHF prevented endothelial dysfunction (pEC(50), diabetic 6.86 +/- 0.12 versus diabetic+DiOHF, 7.49 +/- 0.13, n = 11, p<0.01). The contribution of NO versus endothelium-derived hyperpolarizing factor (EDHF) to ACh-induced relaxation was assessed by evaluating responses in the presence of TRAM-34+apamin+iberiotoxin or N-nitro-L-arginine+ODQ respectively. Diabetes impaired the contribution of both NO (maximum relaxation, R-max diabetic 24 +/- 7 versus normal, 68 +/- 10, n = 9-10, p<0.01) and EDHF (pEC(50), diabetic 6.63 +/- 0.15 versus normal, 7.14 +/- 0.12, n = 10-11, p<0.01) to endothelium-dependent relaxation. DiOHF treatment did not significantly affect the EDHF contribution but enhanced NO-mediated relaxation (R-max 69 +/- 66, n = 11, p<0.01). Western blotting demonstrated that diabetes also decreased expression and increased uncoupling of endothelial NO synthase (eNOS). Treatment of the diabetic rats with DiOHF significantly reduced vascular ROS and restored NO-mediated endothelium-dependent relaxation. Treatment of the diabetic rats with DiOHF also increased eNOS expression, both in total and as a dimer. Conclusions/Significance: DiOHF improves NO activity in diabetes by reducing Nox2-dependent superoxide production and preventing eNOS uncoupling to improve endothelial function.