Alteration of ETs has been demonstrated in both type I and type II diabetes. Although a number of studies can be cited that provide contradictory reports of plasma ET levels in diabetic patients, it should be noted that these peptides act in both an autocrine and paracrine fashion. Therefore, plasma levels may not provide an adequate assessment of their biological activity . In both animal and human diabetes, use of ET antagonists may be more revealing in terms of the consequences of ET alteration. We and others have demonstrated that in endothelial cells and in several target organs of diabetic complications, ETs are upregulated and mediate structural and functional alterations .
The mechanisms by which sustained hyperglycemia leads to upregulation of ETs include activation of PKC, augmented polyol pathway and pseudohypoxia, oxidative stress, elaboration of growth factors, and alteration of vasoactive factors such as NO. Mechanisms and consequences of ET alteration in diabetes are diagrammed in Figure 1. We will briefly describe the possible mechanism by which these biochemical anomalies may lead to alteration of the ET system in diabetes.
PKC activation has been demonstrated in both diabetic micro- and macrovasculopathy . High glucose levels can
Oxidative Stress n
induce de novo synthesis of DAG and activation of PKC. PKC has been implicated in mediating several important vascular functions such as regulation of blood flow, vascular permeability, expansion of extracellular matrix, and in the elaboration of various growth factors and cytokines. Studies have demonstrated an interactive relationship between PKC and ETs. We have previously demonstrated inhibition of high glucose-induced ET upregulation by both general (chelerythrine) PKC inhibitor and specific (LY379196) PKCß inhibitor . PKC activation may also regulate several other growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor, epidermal growth factor, insulin-like growth factor, and fibroblast growth factor. Elaboration of these growth factors may also mediate PKC-induced ET alteration in diabetes.
Polyol pathway has been implicated in several chronic diabetic complications . High intracellular glucose levels overwhelm the glycolytic pathway and lead to enzymatic conversion of glucose to sorbitol via aldose reductase (AR). Sorbitol is subsequently metabolized to fructose by sorbitol dehydrogenase. AR activity requires oxidation of NADPH whereas sorbitol dehydrogenase requires reduction of NAD+. Therefore, increased flux through the polyol pathway leads to alteration of NADH:NAD+ and NADPH: NADP+. Such imbalance in redox state may cause endothelial dysfunction secondary to hyperglycemia . Interestingly, the augmented polyol pathway may reduce NO synthesis, which also requires NADPH. Impairment of the NO system may lead to ET alteration, as NO has been shown to regulate ETs.
Glucose and other reducing sugars such as glucose 6-phosphate, trioses, and fructose may react nonenzymatically with amino groups of proteins. Advanced glycation end products (AGEs) may also be produced from glycating dicarbonyl compounds such as 3-dioxyglucosone, methyl-glyoxal, and glyoxals. AGEs were first thought to mark senescent proteins for degradation. However, in recent years, numerous AGE receptors (RAGEs) have been identified. Binding of AGEs to AGE receptors may mediate intra-cellular signaling and cause upregulation of growth factors such as ET-1 and VEGF . In addition, AGE formation has been shown to reduce NO, which would further lead to ET alteration.
Oxidative Stress and NO
Increased oxidative stress due to glucose autoxidation, AGE/RAGE interaction, and NO generation has been implicated in the pathogenesis of diabetic complications. In cultured endothelial cells as well as several target organs of diabetic complications, NO synthase mRNA has been shown to be upregulated. However, diabetic patients exhibit impaired endothelium-dependent relaxation. Several theories have been proposed to reconcile these contradictory results. It is interesting to note that concurrent with increased NO synthase expression is increased production of free radicals. Activation of various lipoxygenase enzymes, secondary to hyperglycemia, may promote scavenging of NO by superoxide anions. This interaction yields highly reactive peroxynitrite and hydroxyl radicals. Sequestration of NO by superoxide anions could also contribute to reduced NO bioactivity and availability leading to upregulation of ETs. Increased oxidative stress has also been demonstrated to mediate PKC activation, AGE formation, augmented polyol pathway and sorbitol accumulation, and NF-kB activation in endothelial cells. Such anomalies may further alter the ET system in diabetes. Recently, poly (ADP-ribose) polymerase (PARP), an enzyme well known for polymerizing ADP-ribose in DNA backbone synthesis, has been implicated in ET upregulation. Use of various PARP inhibitors was shown to prevent diabetes-induced alteration of ETs in kidney tissues . Whether diabetes-induced PARP activation leads to ET upregulation in other target tissues, such as the retina, remains to be determined.
Insulin represents another factor leading to ET alteration which could be of significance to microvascular complications in type II diabetes. Insulin has been shown to up-regulate ET peptide and receptor expression in vascular endothelial and smooth muscle cells . Administration of insulin also increases plasma ET levels in both humans and animals. In addition, hyperinsulinemia has been linked to accelerated macroangiopathy in diabetic patients. The role of insulin, however, in ET alteration and microangiopathy still remains to be determined.
Several other factors may be of importance in augmented ET-1 expression in diabetes. A large number of studies indicate the role of angiotensin II and transforming growth factor-ß (TGF-ß) in the development of diabetic micro- and macroangiopathy. Angiotensin II is mitogenic for smooth muscle cells and can lead to increased extracellular matrix (ECM) protein synthesis. Recent reports indicate that angiotensin II possibly mediates mitogenic and fibrogenic effects via ET system. Angiotensin II has also been shown to increase synthesis and secretion of ET-1 from vascular endothelial cells. In addition, an interactive relationship between TGF-ß and ET has been established. These findings suggest multiple signaling pathways leading to alteration of ET-1 in chronic diabetes.
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