Oxidative Stress

the intracellular pool of reduced glutathione (GSH) (Fig. 3). Nuclear magnetic resonance studies of rat lens exposed in vitro to both high glucose levels and oxidants indicate competition between the polyol pathway and the glutathione reductase pathway for NADPH (102). However, in some tissues, rapid regeneration of NADPH is possible through the action of the pentose phosphate pathway (PPP) (103), as well as by cytoplasmic malic enzyme and NADP+-dependent isocitrate dehydrogenase, e.g., (104). The latter two enzyme activities are reported to be “high” in rat retinal tissue, with NADP+- dependent isocitrate dehydrogenase activity (Fig. 3) 8-fold higher per retina than malic enzyme (105). Depletion of NADPH or GSH has not been observed in the retina of rats with a short (6 weeks) duration of diabetes (101). It must be noted, however, that after such short diabetes duration there is also no evidence of diabetes-induced toxicity for relevant retinal cell types. Apoptosis of retinal capillary cells is not yet detectable (106), and Müller glial cells do not show reactive characteristics (C. Gerhardinger, unpublished observations).

In the second step of the pathway, persistent utilization of NAD+ by SDH can lead to an increased ratio of NADH/NAD+, a condition that has been termed “pseudohypoxia” (107). Numerous investigators have observed increased free cytoplasmic NADH/NAD+ (calculated from retinal lactate/pyruvate) in retinas from diabetic rats and retinas exposed to high glucose in vitro when compared to normal retinas (108–112). Discordant results (113, 114) seem likely to be due to methodological differences. Elevated free cytoplasmic NADH/NAD+ has been linked to a multitude of metabolic and signaling changes that contribute to oxidative stress and changes in gene expression (115, 116). For example, excess NADH can provide substrate for NAD(P)H oxidase, which, in the presence of oxygen, generates superoxide and related intracellular oxidant species (117) (Fig. 3). Superoxide reacts nonenzymatically with nitric oxide to produce the powerful oxidant, peroxynitrite (118) (for simplicity not shown in Fig. 3); thus, nitric oxide levels also play a key role in retinopathy (119). Elevated cytoplasmic unbound NADH as well as cytoplasmic pyruvate can also transmit reducing equivalents into the mitochondrion via mitochondrial transporters and shuttles and accelerate electron transport within the mitochondrial membrane, a process also linked to superoxide production (120) (Fig. 3). In addition increased oxidative stress can reversibly convert xanthine dehydrogenase to superoxide-generating xanthine oxidase (Fig. 3) (121), an enzyme found in human and bovine inner retinal capillaries and in human cones (122).

Excess polyol pathway activity can contribute to oxidative stress also by interfering with upregulation of antioxidant defenses. Peripheral white blood cells or fibroblasts from diabetic patients with retinopathy and nephropathy, but not from uncomplicated diabetic patients or healthy individuals, when exposed to high glucose in vitro failed to induce mRNAs for antioxidant defense enzymes including catalase, glutathione peroxidase, and cytoplasmic superoxide dismutase (123, 124). In white blood cells, the defect correlated inversely with the AR genotype, i.e., the high AR expression genotype manifested low antioxidant mRNA induction. Moreover, an essentially normal response of antioxidant mRNAs was restored by treatment in vitro with ARI zopolrestat (124). Thus, it appears that increased flux through the polyol pathway can interfere with the induction or upregulation of antioxidant defense enzymes (Fig. 3), especially in cells from individuals prone to the complications of diabetes. Consistent with these findings in human cells, treatment with fidarestat, an ARI structurally distinct from zopolrestat,

prevented oxidative stress and allowed upregulation of antioxidant defense enzymes in the retina of diabetic rats (101). Importantly, fidarestat did not prevent oxidative stress caused in cultured retinal endothelial cells by three different pro-oxidants under normal glucose conditions, demonstrating that the ARI did not have direct antioxidant activity. Rather, the antioxidant effect of the ARI in the diabetic rat retina results in all likelihood from inhibiting elevated metabolic flux through retinal aldose reductase (see also the sections “AR Knockout Mice” and “Effects of ARIs in Experimental Diabetic Retinopathy”).

Activation of Protein Kinase C

Sustained elevation of NADH/NAD+ under hyperglysolic conditions coupled with oxidative and poly(ADP-ribose) polymerase-mediated inhibition of glyceraldehyde-3- phosphate dehydrogenase (125), favors production of diacylglycerol which activates protein kinase C (PKC). Accordingly, AR inhibition prevents high glucose-induced diacylglycerol production and PKC activation in vascular smooth muscle cells (126) and rat glomeruli (127), and glucose-induced PKC activation in human mesangial cells (128). PKC can further activate the superoxide-producing NAD(P)H oxidase complex (129) (Fig. 3).

Generation of AGE Precursors

The fructose produced by the polyol pathway, which can directly fructosylate proteins and induce cross-linking more rapidly than glucose (130), can enter in the formation of fructose-3-phosphate and 3-deoxyglucosone. These are powerful glycating agents and AGE precursors (131), and their formation is prevented by ARI treatment in both the erythrocytes of diabetic patients (132, 133) and the lens of diabetic rats (134). Moreover, 3-deoxyglucosone has been shown to inactivate intracellular enzymes important in the detoxification of oxidant species (133). The retina of experimentally diabetic rats shows accumulation of AGEs colocalized with AGE receptors (135), and interaction of AGEs with their receptor generates oxidative stress (136, 137). Excess polyol pathway activity may thus contribute to oxidative stress also through the generation of AGE precursors.

Proinflammatory Events and Apoptosis

Reactive oxygen species, functioning both as signaling and damaging molecules, are known to trigger proinflammatory responses (138) as well as apoptosis (139) (Fig. 3). It may thus be expected that chronically enhanced polyol pathway activity in diabetes will contribute to both these types of events. Of the proinflammatory events described in diabetic retinopathy, a few have been examined in relation to polyol pathway activity. AR inhibitors have shown thus far to prevent in experimentally diabetic rats increased expression of leukocyte adhesion molecules (140) and complement activation (16). Apoptosis is a prominent phenomenon in the diabetic retina (141,142), and AR inhibitors have successfully prevented in diabetic rats apoptosis of both retinal neurons and vascular cells (16, 19, 140).