Superoxide Dismutase (SOD)

The SODs are ubiquitous components of cellular antioxidant systems and effectively protect retinal tissue against free-radical oxidation of membrane phospholipids. Different isoforms of SOD are located at different sites within the cell. CuZn-SOD is located in both the cytoplasm and the nucleus. In contrast MnSOD is found only in the mitochondria, but can be released into the extracellular space (for review, see (99)). The SODs act as a major defense system against the cytotoxic effects of superoxide radicals by catalyzing the conversion of superoxide anion to oxygen and hydrogen peroxide. The activity and the expression of SOD are downregulated in the retinas of diabetic or galactosemic rats (79, 112). It has also been shown that diabetes causes decreases in CuZnSOD mRNA in human pericytes but that levels of MnSOD mRNA were not affected (110). Studies in diabetic rats have shown that therapies that inhibit the development of retinopathy, including aminoguanidine and antioxidants, also prevent diabetes-induced decreases in retinal SOD levels and normalize SOD activity (for review, see (19)). Furthermore, treatment with SOD mimetics and overexpression of MnSOD protect the retina from diabetes-induced oxidative stress and prevent glucose-induced mitochondrial dysfunction and apoptosis of retinal capillary cells (30, 31, 113).

Catalase

Catalase catalyzes the conversion of hydrogen peroxide to water and oxygen and thus protects against hydrogen peroxide-mediated oxidative damage. The enzyme also has peroxidase activity and reacts with organic peroxides and hydrogen donors to form water and organic alcohols. It is located mainly in cellular peroxisomes and to some extent in the cytosol. The enzyme is especially important in conditions where content of GSH is limited or when activity of GPx in diminished. The effects of diabetes on catalase activity in the retina are somewhat contradictory. Studies have shown modest increases in catalase activity in the diabetic rat retina (112), whereas activity in the diabetic mouse retina is apparently decreased (114).

EFFECTS OF OXIDATIVE STRESS IN THE DIABETIC RETINA

Overview

Recent studies have identified ROS as key second messengers in multiple signaling pathways that initiate diverse biological responses (for review, see (115, 116)). First, ROS can modify the activity of redox-sensitive protein kinases (such as members of the MAPK family, Akt, PKC, PKD, and JAK (Janus kinase)) either indirectly via inactivation of tyrosine phosphatases or in some cases by direct activation. Second, ROS can alter the activity of redox-sensitive transcription factors such as AP-1 (activator protein 1), NF-kB (nuclear factor kB), HIF-1 (hypoxia-inducible factor 1), and STAT (signal transducer and activator of transcription). This latter effect can occur directly or secondary to altered activity of upstream kinases. Third, ROS can modulate the activity of redoxsensitive molecules such as thioredoxin. Fourth, ROS can directly affect the function of enzymes, receptors, or ion channels. Finally, ROS-mediated production of inflammatory cytokines such as TNF-α may in turn increase NADPH oxidase activity and expression, thereby completing the vicious circle of inflammation (117).

Growth Factors and Cytokines

Increases in oxidative stress have been linked to increased production of VEGF upon high glucose treatment in vitro and in the diabetic retina (84, 118–121). The mechanisms by which oxidative stress contributes to VEGF overexpression are not fully understood. However, inhibiting NOS or scavenging peroxynitrite has been shown to prevent signs of diabetic retinopathy in rats (73, 105), suggesting that formation of reactive nitrogen species plays a role in the pathology. Studies using tissue culture models suggest that high glucose-induced peroxynitrite formation increases VEGF expression by a mechanism involving the activation of STAT3 (122, 123). Studies have shown that constitutive activation of STAT3 is correlated with increased rates of VEGF expression and angiogenesis (124–128). Because VEGF stimulation of retinal microvascular endothelial cells can induce its own expression via the activation of STAT3 (123, 129), it appears likely that the effects of diabetes in causing pathological overgrowth of the retinal microvasculature are due in part to VEGF’s actions in triggering its autocrine expression. VEGF autocrine production in the microvascular endothelium has been described in hypoxia, brain tumors, when the cell-to-cell junctions are disrupted or during in vitro angiogenesis induced by AGE products (127, 130).

PEDF is a noninhibitory member of the serpin superfamily. It was first discovered as a neurotrophic factor, but it is now known to function as an endogenous inhibitor of angiogenesis and a blocker of VEGF-induced permeability (131–134). VEGF and PEDF appear to have a reciprocal relationship in the eyes of patients with proliferative diabetic retinopathy in that levels of VEGF are increased whereas levels of PEDF are decreased (135). The protective role of PEDF in preventing retinopathy has been supported by studies showing that intravitreal injection of PEDF significantly reduces vascular hyperper- meabilityinmodelsofdiabetesandoxygen-inducedretinopathy.Thepermeability-blocking effect was correlated with decreased levels of retinal inflammatory factors, including VEGF, VEGF receptor-2, MCP-1, TNF-α, and ICAM-1 (136). In cultured retinal capillary endothelial cells, PEDF significantly decreased TNF-α and ICAM-1 expression induced by hypoxia. These protective actions of PEDF may involve an antioxidant function in that PEDF has been shown to protect cultured retinal pericytes from AGE-induced injury through its antioxidative properties (137). It has also been shown to block angiotensin II signaling and to inhibit TNF-alpha-induced IL-6 expression in endothelial cells by suppressing NADPH oxidase-mediated ROS generation (138, 139). PEDF was also found to inhibit AGE-induced retinal vascular hyperpermeability by blocking ROS-mediated expression of VEGF (140) and to block ROS-induced apoptosis and dysfunction of cultured retinal pericytes (141). Further evidence supporting an antioxidant action of PEDF comes from studies of ocular fluids from patients with proliferative diabetic retinopathy which showed that levels of PEDF are positively correlated with total antioxidant capacity (142, 143).

Diabetic retinopathy exhibits signs of chronic inflammatory disease (144). As has been explained in the section on sources of ROS, iNOS expression is increased in retinas of diabetic patients and experimental animal models, and inhibiting iNOS or knocking out the iNOS gene protects against diabetic retinopathy (75, 81). Production of large amounts of NO and ROS can trigger a variety of inflammatory reactions. Extracellular release of superoxide, produced in leukocytes as a respiratory burst, is an important mechanism of pathogen killing and also leads to endothelial damage resulting in

increased vascular permeability as well as cell death. Intracellular production of NO and ROS also can promote the release of other mediators of inflammation. ROS can increase chemokine and cytokine expression, which can increase adhesion molecule expression on both the endothelium and the inflammatory cells, thus affecting inflammatory cell recruitment to the sites of vascular damage. Recent studies in animal and tissue culture models indicate that diabetesor high glucose-induced increases in expression of VEGF and ICAM-1 as well as retinal leukostasis and breakdown of the blood-retinal barrier depend critically on the activity of NADPH oxidase in triggering the activation of STAT3 (89, 122). The role of NADPH oxidase in diabetes-induced retinal vascular inflammation has been confirmed by studies showing that diabetes or high glucose increase NADPH oxidase expression in the vascular wall and that inhibition of NADPH oxidase or deletion of its catalytic subunit NOX2 reduces signs of vascular inflammation in the diabetic retina (88).

Cytoxicity

Studies in clinical specimens and animal models have shown that retinal capillary cells undergo accelerated apoptosis prior to the appearance of clinical signs of diabetic retinopathy (79, 145). Experimental diabetes or treatment of endothelial cells or pericytes with high glucose has been shown to result in increased levels of oxidative stress and activation of caspase 3 and NF-κB, suggesting a causal relationship between oxidative stress and vascular injury (for review, see (19)). Studies showing that overexpression of mitochondrial SOD reduces oxidative stress, protects the retina from diabetes-induced abnormalities in the mitochondria, and prevents vascular pathology strongly support the role of mitochondrial-derived ROS in diabetic vascular injury (31).

Nearly 50 years ago, Bloodworth proposed that diabetic retinopathy is not just a disease of the vasculature but a multifactorial disease involving the retinal neurons and glia (146). Early histopathologic studies noted the loss of neurons in patients with diabetic retinopathy. Since then, studies using electroretinography, dark adaptation, contrast sensitivity, and color vision tests have conclusively demonstrated that neuroretinal function is compromised before the onset of vascular lesions in humans (for review, see (147, 148)). While extensive research effort has been focused on defining the vascular pathology in the diabetic retina, neurodegenerative changes also occur. These include increased apoptosis of ganglion cells; glial cell reactivity, microglial activation, and altered glutamate metabolism. The metabolic factors that lead to this neuronal cell death have been suggested to include loss of insulin-mediated trophic support (149–151) and/ or injury due to accumulation of excess hexosamines (152), tumor necrosis factor-alpha (7, 153), or glutamate (for review, see (147)). Studies showing that treatments that target formation of ROS exert neuroprotective effects suggest that diabetes-induced oxidative stress also has a key role in the pathogenesis of the neuronal degeneration (154–155).

Müller cells undergo reactive gliosis following acute retinal injury or chronic neuronal stress (156). Gliosis is characterized by glial cell proliferation, changes in cell shape due to alterations in intermediate filament production (GFAP), and secretion of NO and VEGF (for review, see (157)). The progression of gliosis in diabetic retina has been correlated with increases in ROS/RNS formation (158–161) as well as with increased levels of inflammatory mediators (162, 163).