Various neurohumoral mediators and mechanical forces acting on the innermost layer of blood vessels, the endothelium, are involved in the regulation of the vascular tone. The main pathway of vasoregulation involves the activation of the constitutive, endothelial isoform of NO synthase (eNOS) resulting in NO production (53). Endothelium-depen-dent vasodilatation is frequently used as a reproducible and accessible parameter to probe endothelial function in various pathophysiological conditions. It is well established that endothelial dysfunction, in many diseases, precedes, predicts, and predisposes for the subsequent, more severe vascular alterations. Endothelial dysfunction has been documented in various forms of diabetes, and even in prediabetic individuals (52,54-58). The pathogenesis of this endothelial dysfunction involves many components including increased polyol pathway flux, altered cellular redox state, increased formation of diacylglycerol, and the subsequent activation of specific PKC isoforms, and accelerated nonenzymatic formation of advanced glycation end-products (AGE) (59-61). Many of these pathways, in concert, trigger the production of oxygen- and nitrogen-derived oxidants and free radicals, such as superoxide anion and peroxynitrite, which play a significant role in the pathogenesis of the diabetes-associated endothelial dysfunction (59,60,62).
The cellular sources of reactive oxygen species (ROS) such as superoxide anion are multiple and include AGEs, nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidases, the mitochondrial respiratory chain, xanthine oxidase, the arachidonic acid cascade (lipoxygenase and cycloxygenase), and microsomal enzymes (60,63).
Superoxide anion may quench NO, thereby reducing the efficacy of a potent endothe-lium-derived vasodilator system that participates in the homeostatic regulation of the vasculature, and evidence suggests that during hyperglycemia, reduced NO availability exists (64). Hyperglycemia-induced superoxide generation contributes to the increased expression of NAD(P)H oxidase, which in turn generate more superoxide anion. Hyperglycemia also favors, through the activation of NF-kB an increased expression of iNOS, which may increase the generation of NO (65,66).
Superoxide anion interacts with NO, forming the strong oxidant peroxynitrite (ONOO-), which attacks various biomolecules, leading to—among other processes—the production of a modified amino acid, nitrotyrosine (67). Although nitrotyrosine was initially considered a specific marker of peroxynitrite generation, other pathways can also induce tyrosine nitration. Thus, nitrotyrosine is now generally considered a collective index of reactive nitrogen species, rather than a specific indicator of peroxynitrite formation (68,69). The possibility that diabetes is associated with increased nitrosative stress is supported by the recent detection of increased nitrotyrosine plasma levels in type 2 diabetic patients (70) and iNOS-dependent peroxynitrite production in diabetic platelets (71). Nitrotyrosine formation is detected in the artery wall of monkeys during hyperglycemia (72) and in diabetic patients during an increase of postprandial hyperglycemia
(73). In a recent study we have demonstrated increased nitrotyrosine immunoreactivity in microvasculature of type 2 diabetic patients (52). In the same study significant correlations were observed between nitrotyrosine immunostaining intensity and fasting blood glucose, HbA1c, ICAM, and vascular cellular adhesion molecule (VCAM).
The toxic action of nitrotyrosine is supported by the evidence that increased apoptosis of endothelial cells, myocytes and fibroblasts in heart biopsies from diabetic patients
(74), in hearts from streptozotocin (STZ)-induced diabetic rats (75), and in working hearts from rats during hyperglycemia (76), and the degree of cell death and/or dysfunction show a correlation with levels of nitrotyrosine found in those cells. There is also evidence that nitrotyrosine can also be directly harmful to endothelial cells (77). Additionally, high glucose-induced oxidative and nitrosative stress alters prostanoid profile in human endothelial cells (78,79). Recent studies have suggested that increased oxidative and nitrosative stress is involved in the pathogenesis of diabetic microvascular injury in retinopathy, nephropathy, and neuropathy (80-85).
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