Renu A. Kowluru

Oxidative stress and diabetic retinopathy.

Oxygen metabolism is essential for sustaining aerobic life, and normal cellular homeostasis works on a fine balance between the formation and elimination of reactive oxygen species (ROS). Oxidative stress, a cytopathic consequence of excessive production of ROS and the suppression of ROS removal by antioxidant defense system, is implicated in the development of many diseases, including Alzheimers disease, and diabetes and its complications. Retinopathy, a debilitating microvascular complication of diabetes, is the leading cause of acquired blindness in developed countries. Many diabetes-induced metabolic abnormalities are implicated in its development, and appear to be influenced by elevated oxidative stress; however the exact mechanism of its development remains elusive. Increased superoxide concentration is considered as a causal link between elevated glucose and the other metabolic abnormalities important in the pathogenesis of diabetic complications. Animal studies have shown that antioxidants have beneficial effects on the development of retinopathy, but the results from very limited clinical trials are somewhat ambiguous. Although antioxidants are being used for other chronic diseases, controlled clinical trials are warranted to investigate potential beneficial effects of antioxidants in the development of retinopathy in diabetic patients.

Abnormalities of Retinal Metabolism in Diabetes or Experimental Galactosemia. IV. Antioxidant Defense System

Activities of enzymes that protect the retina from reactive oxygen species were investigated in experimentally diabetic rats and experimentally galactosemic rats, two animal models known to develop vascular lesions consistent with diabetic retinopathy. Diabetes or experimental galactosemia of 2 months duration significantly decreased the activities of glutathione reductase and glutathione peroxidase in the retina while having no effect on the glutathione synthesizing enzymes glutathione synthetase and gamma-glutamyl cysteine synthetase. Activities of two other important antioxidant defense enzymes-superoxide dismutase (SOD) and catalase-also were decreased (by more than 25%) in retinas of diabetic rats and galactosemic rats. Administration of supplemental antioxidants, vitamins C and E, for the 2 months prevented the diabetes-induced impairment of antioxidant defense system in the retina. In experimentally galactosemic rats, the supplemental antioxidants were not as effective: SOD activity was normalized, but the enzymes of the glutathione redox cycle were only partly restored, and the subnormal catalase activity was unaffected. Diabetes or experimental galactosemia results in significant impairment of the antioxidant defense system in the retina, and exogenous antioxidant supplementation can help alleviate the subnormal activities of antioxidant defense enzymes.

Abnormalities of Retinal Metabolism in Diabetes or Experimental Galactosemia. III. Effects of Antioxidants

Effects of antioxidants on hyperglycemia-induced alterations of retinal metabolism were evaluated in rats diabetic or experimentally galactosemic for 2 months. Oxidative stress was estimated by measuring lipid peroxides (measured as thiobarbituric acid reactive substances [TEARS]) in retina and plasma. Erythrocyte osmotic fragility, another measure of oxidative stress, also was determined in the same groups of rats. In diabetic rats, TBARS were elevated by 74% in retina and 87% in plasma. In galactose-fed rats, TBARS were significantly elevated in retina (P < 0.05), but were normal in plasma. The administration of supplemental dietary ascorbic acid and α-tocopherol acetate for 2 months prevented the elevation of retinal TBARS and the decrease of Na+-K+- ATPase and calcium ATPase activities in retinas of diabetic animals without having any beneficial effect on plasma TBARS. In galactosemic rats, these antioxidants had a partial beneficial effect on the activity of retinal Na+-K+-ATPase, but failed to have any effect on calcium ATPase. The beneficial effects of antioxidants in diabetes and experimental galactosemia were not caused by the amelioration of hyperglycemia or retinal polyol accumulation. Erythrocyte osmotic fragility was increased by more than twofold in diabetes, but was normal in experimental galactosemia, and antioxidants prevented diabetes- induced increases in erythrocyte osmotic fragility. Diabetes-induced increased oxidative stress and subnormal ATPase activities in the retina can be inhibited by dietary supplementation with antioxidants.

Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase

Abstract Retinal mitochondria become dysfunctional in diabetes and the production of superoxide radicals is increased; over-expression of MnSOD abrogates mitochondrial dysfunction and prevents the development of diabetic retinopathy. The mitochondrial DNA (mtDNA) is particularly prone to oxidative damage. The aim of this study is to examine the role of MnSOD in the maintenance of mtDNA. The effect of MnSOD mimic, MnTBAP or over-expression of MnSOD on glucose-induced alterations in mtDNA homeostasis and its functional consequence was determined in retinal endothelial cells. Exposure of retinal endothelial cells to high glucose increased mtDNA damage and compromised the DNA repair machinery. The gene expressions of mitochondrial-encoded proteins of the electron transport chain complexes were decreased. Inhibition of superoxide radicals by either MnTBAP or by over-expression of MnSOD prevented mtDNA damage and protected mitochondrial-encoded genes. Thus, the protection of mtDNA from glucose-induced oxidative damage is one of the plausible mechanisms by which MnSOD ameliorates the development of diabetic retinopathy.

BENEFICIAL EFFECT OF ZEAXANTHIN ON RETINAL METABOLIC ABNORMALITIES IN DIABETIC RATS

PURPOSE Oxidative damage and growth factors are implicated in the pathogenesis of retinopathy in diabetes. Recent studies have shown that two dietary carotenoids, lutein and zeaxanthin (Zx), that are specifically concentrated within ocular tissues, may play important roles in maintaining their integrity. This study is to evaluate the potential protective effects of Zx against retinal oxidative damage and growth factors in diabetes. METHODS A group of rats received normal powdered diet or powdered diet supplemented with 0.02% or 0.1% Zx soon after induction of diabetes. Age-matched normal rats served as control subjects. At 2 months of diabetes, oxidative stress, vascular endothelial cell growth factor (VEGF), and intercellular adhesion molecule (ICAM)-1 were quantified in the retina. RESULTS Zx supplementation prevented diabetes-induced increase in retinal damage, and increases in VEGF and ICAM-1. The levels of lipid peroxide, oxidatively modified DNA, electron transport complex III, nitrotyrosine, and mitochondrial superoxide dismutase were similar in the retinas of Zx-treated diabetic rats and normal control rats, and these values were significantly different from those obtained from diabetic rats without any supplementation. In the same rats, Zx also prevented diabetes-induced increases in retinal VEGF and ICAM-1. Both 0.02% and 0.1% Zx had similar effects on diabetes-induced retinal abnormalities, and these effects were achieved without ameliorating the severity of hyperglycemia. However, Zx administration failed to prevent a diabetes-induced decrease in retinal GSH levels. CONCLUSIONS Zx significantly inhibits diabetes-induced retinal oxidative damage and elevation in VEGF and adhesion molecule, all abnormalities that are associated with the pathogenesis of diabetic retinopathy. The results suggest that Zx supplementation has the potential to inhibit the development of retinopathy in diabetic patients.

Diabetes-induced metabolic abnormalities in myocardium: effect of antioxidant therapy.

Effects of hyperglycemia (both diabetes and experimental galactosemia) on cardiac metabolism have been determined. In addition, the effect of supplemental antioxidants on these hyperglycemia-induced abnormalities of cardiac metabolism has been investigated. Diabetes or experimental galactosemia of 2 months duration in rats significantly increased oxidative stress in myocardium, as demonstrated by elevation of thiobarbituric acid reactive substances (TBARS) and lipid fluorescent products in left ventricle. Activity of protein kinase C (PKC) was elevated in the myocardium, and the activities of (Na,K)-ATPase and calcium ATPases were subnormal. Administration of supplemental antioxidants containing a mixture of ascorbic acid, Trolox; α-tocopherol acetate, N-acetyl cysteine, β-carotene, and selenium prevented both the diabetes-induced and galactosemia-induced elevation of oxidative stress and PKC activity, and inhibited the decreases of myocardial (Na,K)-ATPase and calcium ATPases. The results show that these metabolic abnormalities are not unique to diabetes per se, but are secondary to elevated blood hexose levels, and supplemental antioxidants inhibit these metabolic abnormalities. Our findings suggest that antioxidants inhibit abnormal metabolic processes that may contribute to the development of cardiac disease in diabetes, and offer a potential clinical means to inhibit cardiac abnormalities in diabetes.

Role of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy and Its Regulation by H-Ras

PURPOSE Diabetes activates a small molecular weight G-protein, H-Ras, in the retina and its capillary cells, and H-Ras activation is implicated in the apoptosis of retinal capillary cells. Matrix metalloproteinase (MMP)-9 is regulated by H-Ras, and in diabetes its activation is associated with increased vascular permeability. The goal of this study was to investigate the role of sustained activation of MMP-9 in the pathogenesis of diabetic retinopathy and to illustrate the mechanism through which it is upregulated in diabetes. METHODS Retinal MMP-9 activation and its tissue inhibitor, TIMP-1, were quantified in streptozotocin-induced diabetic rats. Inhibition of H-Ras by simvastatin on diabetes-induced activation of H-Ras was evaluated. The mechanism by which diabetes regulates retinal MMP-9 was confirmed by determining the effect of genetic or pharmacologic regulation of H-Ras on its activation in retinal endothelial cells. RESULTS In rats, MMP-9 was activated and expression of TIMP-1 was decreased in the retina and its microvasculature at both 2 months and 12 months of diabetes. In retinal endothelial cells, high glucose activated MMP-9, and inhibition of its activation (by pharmacologic inhibitor or siRNA) ameliorated accelerated apoptosis. Inhibition of H-Ras, both in diabetic rats (simvastatin) and in isolated endothelial cells (H-Ras siRNA), abrogated the activation of MMP-9 and prevented the reduction of TIMP-1. CONCLUSIONS Hyperglycemia-induced activation of MMP-9 accelerates apoptosis of retinal capillary cells, a phenomenon that predicts the development of diabetic retinopathy, and the activation of MMP-9 is downstream of H-Ras. Characterizing the role of MMP-9 in the development of diabetic retinopathy will help explore novel molecular targets for future pharmacological interventions.

Abnormalities of retinal metabolism in diabetes or experimental galactosemia. VI. Comparison of retinal and cerebral cortex metabolism, and effects of antioxidant therapy

Metabolic abnormalities observed in retina and in cerebral cortex were compared in diabetic rats and experimentally galactosemic rats. Diabetes or experimental galactosemia of 2 months duration significantly increased oxidative stress in retina, as shown by elevation of retinal thiobarbituric acid reactive substances (TBARS) and subnormal activities of antioxidant defense enzymes, but had no such effect in the cerebral cortex. Activities of sodium potassium adenosine triphosphatase [(Na,K)-ATPase] and calcium ATPase became subnormal in retina as well as in cerebral cortex. In contrast, protein kinase C (PKC) activity was elevated in retina but not in cerebral cortex in the same hyperglycemic rats. Dietary supplementation with an antioxidant mixture (containing ascorbic acid, Trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, and selenium) prevented the diabetes-induced and galactosemia-induced elevation of retinal oxidative stress, the elevation of retinal PKC activity and the decrease of retinal ATPases. In cerebral cortex, administration of the antioxidant diet also prevented the diabetes-induced decreases in (Na,K)-ATPase and calcium ATPases, but had no effect on TBARS and activities of PKC and antioxidant-defense enzymes. The results indicate that retina and cerebral cortex differ distinctly in their response to elevation of tissue hexose, and that cerebral cortex is more resistant than retina to diabetes-induced oxidative stress. The greater resistance to oxidative stress in cerebral cortex, as compared to retina, is consistent with the resistance of cerebral cortex to microvascular disease in diabetes, and with a hypothesis that oxidative stress contributes to microvascular disease in diabetes. Dietary supplementation with these antioxidants offers a means to inhibit multiple hyperglycemia-induced retinal metabolic abnormalities.

Glyceraldehyde-3-phosphate dehydrogenase in retinal microvasculature: implications for the development and progression of diabetic retinopathy.

PURPOSE Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been hypothesized as a mediator in the activation of multiple pathways implicated in the pathogenesis of diabetic retinopathy. The objective of this study was to understand the mechanism by which high glucose inactivates GAPDH in retinal microvascular cells. METHODS Bovine retinal endothelial cells (BRECs), transfected with GAPDH, were incubated in 20 mM glucose. The effect of the overexpression of GAPDH on its activity, apoptosis, and upstream signaling pathways, protein kinase C, and hexosamine pathways was determined. The effect of the inhibitors of nitration and ribosylation on GAPDH activity, its nuclear translocation and reversal of glucose insult was also evaluated. RESULTS High glucose decreased GAPDH activity, expression, and nuclear translocation. Overexpression of GAPDH prevented glucose-induced inhibition of its activity, nuclear translocation, apoptosis, and activation of protein kinase C and hexosamine pathways. Inhibitors of nitration and ribosylation ameliorated glucose-induced inhibition of GAPDH, and their addition during the normal glucose exposure that followed high glucose levels had a beneficial effect on GAPDH activity and the degree of nitration and ribosylation. CONCLUSIONS In hyperglycemia, GAPDH in retinal microvascular cells is inhibited by its covalent modifications, and this activates multiple pathways implicated in the pathogenesis of diabetic retinopathy. The agents that can directly target modification of GAPDH have potential in inhibiting the development and in arresting the progression of diabetic retinopathy.

Erythrocyte sodium-potassium ATPase activity and thiol metabolism in genetically hyperglycemic mice

Erythrocyte sodium pump activity, osmotic fragility, and thiol status were measured in genetically hyperglycemic (db/db) mice and compared with their nondiabetic littermates (db/m). The data showed no major differences in these parameters. However, erythrocytes from streptozotocin (Stz)-induced diabetic rats had significantly lower activity of sodium pump and thiols with an almost fourfold increase in osmotic fragility as compared with erythrocytes from nondiabetic rats. Sorbinil (an aldose reductase inhibitor) treatment of Stz-diabetic rats normalized all these lesions, suggesting a key role for polyol pathway. However, sorbitol levels in erythrocytes from db/db and db/m mice were undetectable. The data suggest that in db/db mice, the relative lack of polyol pathway, a potential consumer of NADPH, may provide erythrocytes with optimal NADPH for glutathione reductase system, thus maintaining normal GSH levels even at the height of hyperglycemia. Thus, the genetically hyperglycemic mice may serve as a useful model to study diabetes related complications without involving polyol pathway.