Constitutive nitric oxide synthase expression in retinal vascular endothelial cells is suppressed by high glucose and advanced glycation end products.

Abstract

It has been suggested that increased production of nitric oxide (NO), a potent endothelium-derived vasodilator, may be responsible for increased blood flow in the retinal and renal vascular beds in early diabetes. However, NO-mediated vasodilation has been reported as impaired in diabetes, and there is evidence that the synthesis and release of NO by the vascular endothelium may be flawed in this condition. We examined the effect of high ambient glucose and exposure to exogenous glycated proteins on NO synthesis in cultured retinal microvascular endothelial cells (RMECs), using a polarographic sensor to measure released NO gas. Nitrite (the stable end product of the reaction between NO and molecular oxygen) was measured in tissue culture supernatants. The expression of vascular endothelial constitutive nitric oxide synthase (eNOS), which is responsible for NO synthesis in endothelial cells, was studied by Western blot analysis and Northern hybridization experiments. A dose-dependent reduction of NO synthesis by RMECs occurred 5 days after exposure to 15 and 25 mmol/l glucose, and concomitantly we found that accumulation of nitrite in culture supernatants of high-glucose exposed cells was also reduced. Coincubation of endothelial cells with inhibitors of protein kinase C (PKC) increased the accumulation of nitrite but did not restore it to the levels obtained when cells were cultured in 5 mmol/l glucose. The expression of eNOS by RMECs was markedly reduced by 5 days of exposure to 25 mmol/l glucose and glycated albumin. This study implicates the PKC pathway, which is known to be upregulated on exposure to high ambient glucose concentrations, as a possible factor in the inhibition of eNOS expression in RMECs. This study also suggests that glycated proteins may be involved in the pathogenesis of vascular endothelial dysfunction by modulating the nitric oxide synthase (NOS)/NO pathway in retinal vascular endothelial cells.

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets

“…It has been hypothesized recently that iNOS expression in macrophages and other tissues after experimental animals have been treated with endotoxin might be responsible for the inhibition of insulin action in that condition and that it might contribute to the development of TNF-a-induced skeletal-muscle insulin resistance in obesitylinked diabetes [22]. Moreover, both high glucose and advanced glycation end-products have been shown to modify the expression of cNOS in retinal vascular endothelial cells [23].…”

Early impairment of large artery structure and function in Type I diabetes mellitus

“…Finally it could be due to the intravascular pressure load because mechanical stretching of a vessel wall also stimulates its tissue components to undergo cell replication and growth [44]. Indeed, all these factors are likely to be involved because markers of altered glucose metabolism (which negatively modulates nitric oxide secretion) [45], daily insulin doses and blood pressure all showed some correlation with arterial distensibility and wall thickness. Although carotid artery and aortic distensibility and wall thickness correlated with blood pressure but not with glycated haemoglobin, this was, however, reversed for radial artery distensibility.…”

A new look at the heart in diabetes mellitus: from ailing to failing

“…As suggested more recently by observations in cultured retinal microvascular cells, hyperglycaemiainduced PKC up-regulation impairs NO-mediated vasodilatation in diabetes by inhibiting constitutive NO synthase (cNOS) expression [132]. Protein kinase C has been suggested to regulate renal haemodynamics by increasing or decreasing NO production dependent on cell type, tissue location and duration of diabetes [126,133,134].…”