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In postmenopausal women, the risk of diabetic cardiovascular disease drastically increases compared with that of men or premenopausal women. However, the mechanism of this phenomenon has not yet been clarified. We hypothesized that the beneficial effects of estrogen on endothelial function may be relevant to protection against hyperglycemia-induced vascular derangement. Bovine aortic endothelial cells were incubated for 72 h in the presence and absence of the physiological concentration of 17␤-estradiol (17␤-E2) under normal and highglucose conditions. The presence of 17␤-E2 significantly counteracted the reduction in basal nitric oxide production under high-glucose conditions. This finding was associated with the recovery of endothelial nitric-oxide synthase (eNOS) protein expression, tetrahydrobiopterin (BH4) levels, and the activity and gene expression of GTP cyclohydrolase I (GTPCH-I), a rate-limiting enzyme for BH4 synthesis. Both the gene transfer of estrogen receptor ␣ using adenovirus and treatment with the protein kinase C inhibitor bisindolylmaleimide I significantly enhanced the effects of 17␤-E2 treatment under high-glucose conditions, whereas these effects were abolished by the estrogen receptor antagonist ICI 182,780 (faslodex). Transfection of small-interfering RNA targeting eNOS resulted in a marked reduction in GTPCH-I mRNA under both normal and highglucose conditions, but this reduction was strongly reversed by 17␤-E2. These results suggest that the activation of ER␣ with 17␤-E2 can counteract high-glucose-induced down-regulation of eNOS and GTPCH-I in endothelial cells. Therefore, estrogen deficiency may result in an exaggeration of hyperglycemiainduced endothelial dysfunction, leading to the development of cardiovascular disease in postmenopausal diabetic women.

The long-term benefits of nitroglycerin therapy are limited by tolerance development. Understanding the precise nature of mechanisms underlying nitroglycerin-induced endothelial cell dysfunction may provide new strategies to prevent tolerance development. In this line, we tested interventions to prevent endothelial dysfunction in the setting of nitrate tolerance. When bovine aortic endothelial cells (BAECs) were continuously treated with nitric oxide (NO) donors, including nitroglycerin, over 2-3 days, basal production of nitrite and nitrate (NO(x)) was diminished. The diminished basal NO(x) levels were mitigated by intermittent treatment allowing an 8-h daily nitrate-free interval during the 2- to 3-day treatment period. Addition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin restored the basal levels of NO(x) that were decreased by continuous nitroglycerin treatment of BAECs. Apocynin caused significant improvement of increased mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in BAECs given nitroglycerin continuously over the treatment period. Apocynin also reduced endothelial production of reactive oxygen species (ROS) after continuous nitroglycerin treatment. These results showed an essential similarity to the effects of a nitrate-free interval. Application of the NOS inhibitor N(omega)-nitro- l-arginine methyl ester caused a recovery effect on basal NO(x) and eNOS expression but was without effect on ROS levels in continuously NO donor-treated BAECs. In conclusion, the present study characterized abnormal features and functions of endothelial cells following continuous NO donor application. We suggest that inhibition of NADPH oxidase, by preventing NO donor-induced endothelial dysfunction, may represent a potential therapeutic strategy that confers protection from nitrate tolerance development.

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