INTRODUCTION

As has been explained in earlier chapters, development of diabetic retinopathy follows a pattern similar to that seen with ischemic retinopathy, beginning with a period of vascular dysfunction and breakdown of the blood-retinal barrier, which may be followed by active proliferation of new vessels in the retina and vitreous (for review, see (1, 2) ). Large-scale clinical trials have demonstrated that hyperglycemia is the primary pathogenic factor in the development of diabetic complications, including diabetic retinopathy (3, 4). While the specific mechanisms by which elevated blood glucose causes tissue injury in the diabetic retina are not fully understood, studies in animal models as well as in clinical specimens have shown that diabetic retinopathy is associated with increases in inflammatory mediators, including VEGF, TNFα, IL-6, IL-1β, and MCP-1 (5–10). This results in increased endothelial cell expression of adhesion molecules, such as ICAM-1 and PECAM, and with leukocyte accumulation and attachment to the retinal blood vessels (leukostasis) (11). Leukostasis is thought to contribute to increases in vascular permeability and subsequent neovascularization. During retinopathy, leukocytes become activated (12) and are thought to influence retinal edema, ischemia, and angiogenesis (13). Macrophages are also important participants in the inflammatory process (14) and have been implicated in retinal neovascularization in models of ischemic retinopathy (15, 16). Microglial cells (resident macrophages) are also activated during diabetes (7, 17). Activated monocytes, neutrophils, and microglial cells are all important sources of oxidative stress.

The association between oxidative stress and the progression of diabetes and its complications has been recognized for over 20 years (18). Increased production of reactive oxygen species (ROS) has been strongly implicated in the pathogenesis of diabetic retinopathy (for review, see (2, 19) ). However, in spite of overwhelming evidence supporting the damaging consequences of oxidative stress and its established role in experimental models of diabetes, the results of large-scale clinical trials with classic antioxidants have failed to show any benefit for diabetic patients (for review, see (20) ). The disappointing results of antioxidant trials in patients underline the importance of identifying the specific sites and sources of oxidative stress in the tissues of diabetic patients. In this chapter we will summarize the current perspective on how diabetes induces oxidative stress in the retina, how diabetes-induced oxidative stress may lead to the development of diabetic retinopathy and consider therapeutic approaches for preventing the onset and progression of the retinal complications of diabetes.

SOURCES OF OXIDATIVE STRESS IN THE DIABETIC RETINA

Overview

The term “oxidative stress” refers to the condition in which there is a serious imbalance between the production of oxidants (including both reactive oxygen species (ROS) and reactive nitrogen species (RNS)), and antioxidant defense, leading to potential tissue damage (for review, see (21)). Prominent members of the ROS group are superoxide, hydroxyl radical, and peroxy radical. Key members of the RNS group are nitric oxide, peroxynitrite, and their derivatives. Oxidative reactions are essential for mechanisms of host defense mediated by neutrophils, macrophages, and other cells of the immune system. However,

when oxidants are overproduced they cause tissue injury and cell death. In normal conditions, antioxidants are present in tissues to neutralize free radicals and prevent excessive oxidative stress (22, 23). Antioxidants have been variably defined as substances that, when present at low concentrations compared to those of an oxidizable substrate, significantly delay or inhibit oxidation of the substrate (24) or as a metabolic intermediate, i.e., a substrate that protects biological tissues from free-radical damage and is able to be recycled or regenerated by biological reductants (25). A variety of compounds (flavonoids, uric acid, bilirubin, albumin, vitamin E, vitamin C, α-lipoic acid, and glutathione) and various enzymes (catalase, superoxide dismutase, glutathione peroxidase) have been described as antioxidants.

Formation of ROS is increased in diabetes/hyperglycemia, and is directly related to the vascular dysfunction and complications of diabetes. Several initiating events and sources of ROS in diabetes have been described and include disruption of the mitochondrial electron transport chain, formation of advanced glycation end (AGE) products, auto-oxidation of glucose, flux through the aldose reductase/polyol pathway, uncoupling of NOS, and activation of PKC and NAD(P)H oxidase (26, 27). Overproduction of superoxide can lead to scavenging of NO, reducing its bioavailability and to production of peroxynitrite and other reactive species, with subsequent vascular dysfunction and pathology (28). The primary source of ROS is considered to be overproduction of superoxide anion by the mitochondrial electron transport chain which then initiates superoxide production by other sources (27). However, it is quite likely that several sources of superoxide production are activated by hyperglycemia and that the activities of these sources are connected and increased through a series of positive feedback relationships (Fig. 1). The contributing events and ROS sources will be discussed, some only briefly as they are the subjects of other chapters in this book.

Mitochondrial Electron Transport Chain (ETC)

Hyperglycemia can disrupt normal mitochondrial function and substantially increase superoxide production. This action appears to occur through the increased hyperglyc- emia-derived electron donors mostly from the citric acid cycle – NADH and FADH2 – which increase electron flow through the ETC complexes as well as the efflux of protons from the mitochondrial matrix across the inner mitochondrial membrane by complexes I, III, and IV. This leads to a substantial increase in mitochondrial membrane potential and the preferential inhibition of electron flow through complex III. This inhibition disrupts normal ETC electron flow and promotes the leak of electrons leading to formation of superoxide (29). The increase in superoxide formation leads to oxidative damage of mitochondrial and cellular lipids, proteins, and nucleic acids which contribute heavily to the pathology of hyperglycemic/diabetic state. These damaging events are amplified by the fact that free-radical defense mechanisms such as superoxide dismutase, catalase, glutathione peroxidase, and levels of the intracellular antioxidant GSH are also substantially compromised during diabetes (for review, see (19)). The key role of mitochondrial ROS formation in diabetes-induced oxidative damage in the retina has been demonstrated by recent studies using transgenic mice that overexpress mitochondrial SOD. These experiments showed that overexpressing mitochondrial SOD protects the retina from diabetes-induced oxidative stress (30). Moreover, studies using the same

Hyperglycemia

VEGF Inflammation

Diabetic Retinopathy

Fig. 1. Sources of ROS and RNS in the diabetic retina.

mice confirmed that retinal mitochondria undergo oxidative damage in diabetes and that complex III is a source of increased superoxide formation. Most importantly, the retinas of these mice were protected from diabetes-induced capillary degeneration, indicating the key role of mitochondrial ROS formation in this pathological process (31).

Advanced Glycation End (AGE) Product Formation

Hyperglycemia is the initiating event in formation of AGEs as gluco-carbonyl adducts with amino acids (such as lysine) and can involve auto-oxidation of glucose to glyoxal. Besides being able to crosslink a number of proteins and alter their physical properties, AGEs interact with a specific receptor, RAGE to activate PKC-δ and subsequently NADPH oxidase (32). AGEs appear to activate NADPH oxidase in neural cells through activation of PKC- δ. (33). Studies in patients, animals, and tissue culture models have clearly demonstrated the role of AGE formation in the complications of diabetes (34,35).

Cyclo-oxygenase (COX)

Expression of the inducible form of COX, COX-2 rises markedly with diabetes, high glucose, and oxidant stress (36, 37). This greater COX-2 expression causes an increased rate of conversion of prostaglandin G2 to prostaglandin H2, which increases ROS formation through the functionally linked peroxidase activity of COX-2. This results in exacerbation

of oxidative stress (38). COX-2 inhibition has been noted to protect against diabetic neuropathy in STZ-diabetic mice (39). Treatment with aspirin or other nonsteroidal anti-inflammatory inhibitors of COX-1, COX-2, and prostaglandin synthesis has been shown to prevent signs of vascular lesions in the retinas of diabetic animals (40, 41), suggesting that this pathway could be important in the development of diabetic retinopathy.

Flux Through Aldose Reductase (AR) Pathway

AR is the first step in the polyol pathway. In hyperglycemia conditions, AR reduces glucose to sorbitol and consumes NADPH. Since regeneration of the antioxidantreduced glutathione requires NADPH, the active AR pathway can increase levels of ROS. Conversion of sorbitol to fructose requires reduction of NAD+ to NADH, leading to higher levels of oxidized triose phosphates, precursors of AGEs and of diacylglycerol (DAG) through α-glycerol-3-phosphate. Thus, activities through the AGE and PKC pathways are enhanced. The potential role of the AR pathway in the development of diabetic retinopathy is supported by studies in experimental animal models and endothelial cells treated with high glucose (42, 43).

Activation of Protein Kinase C (PKC)

PKC has many isoforms which are activated by diacylglycerol (DAG). De novo synthesis of DAG comes largely from glycolytic intermediates and stepwise acylation of glycerol-3-phosphate. All the classic and novel PKC isoforms are activated by DAG, but primarily the ß and α isoforms appear to be involved in diabetes (44). Studies have shown increased levels of DAG in vascular tissue of diabetic subjects (45) as well as elevated DAG levels in cultured vascular cells exposed to high glucose. Hyperglycemia can also indirectly activate PKC through activation of AGE receptors and polyol pathway products. DAG can also be synthesized through the phospholipase pathways activated by growth factors, cytokines, and hormones, such as angiotensin II, which are elevated in diabetes (46–48). PKC can also be activated by peroxynitrite, superoxide, and high amounts of NO (49).

Endothelial NO Synthase (eNOS)

Uncoupled NOS can be a source of superoxide production. When the cellular supply of its substrate, L-arginine or the required co-factor tetrahydrobiopterin is limited, NOS becomes uncoupled and utilizes molecular oxygen as a principal substrate, producing superoxide instead of NO (50–52). Superoxide can combine rapidly with NO to form peroxynitrite or other oxidants (53). L-Arginine can be limited by several means. A state of imbalance between L-arg availability and NOS activity can occur when cellular transport of L-arginine is inhibited, as with oxidative stress associated with cardiovascular disease. Conditions of prolonged and elevated NOS activity (54, 55), reduced recycling of L-citrulline back to L-arginine (56), and/or elevated catabolism of L-arginine by arginase (57–59) can also reduce L-arginine availability to NOS. Diabetes has been shown to reduce cellular transport of L-arginine and enhance vascular and hepatic arginase activity (60).