Formation of peroxynitrite and superoxide can also cause NOS uncoupling due to oxidation of tetrahydrobiopterin, leading to further formation of superoxide. It has also been reported that hydrogen peroxide, a byproduct of superoxide, can itself stimulate eNOS activity (61). This may result in a positive feedback loop, stimulating further increases in superoxide production. Further evidence of ROS involvement in eNOS uncoupling and vascular dysfunction has been provided by experiments showing that native LDL enhances superoxide formation in blood vessels and that this effect can be reversed by acute treatment with supplemental L-arginine (62). Additionally, blood vessels dysfunctional because of oxidized LDL exposure regain their ability to vasodilate in response to serotonin or acetylcholine upon treatment with L-arginine (63, 64). Thus, supplemental L-arginine treatment may ameliorate vascular injury by limiting superoxide formation. However, a number of studies in animals and humans have found no benefit or worsening of adverse outcomes when administration of supplemental L-arginine is prolonged (65, 66). These negative outcomes may be related to the action of L-arginine in increasing arginase activity (67). A study with rabbits showed that chronic administration of L-arginine for 3 days caused decreased NO production in response to acetylcholine which was associated with increased arginase activity in both liver and aorta. In contrast, chronic treatment with L-citrulline for the same period was beneficial in supporting NO production (68). L-Citrulline, a byproduct in the formation of NO, is recycled back to L-arginine, contributing to sustained L-arginine supply for NOS activity (69). L-Citrulline is also an allosteric inhibitor of arginase (70). Therefore, L-citrulline may suppress arginase activity.

Certain eNOS polymorphisms in humans have been associated with severe diabetic retinopathy (71). The eNOS4b/b polymorphism, which increases NOS expression and activity, is associated with severe diabetic retinopathy. In contrast, persons with the eNOS4a/a homozygous deletion in which NOS expression and activity are reduced have absent or background diabetic retinopathy (72). It is possible that those with a high eNOS expression phenotype are more prone to eNOS uncoupling when L-arginine availability and BH4 levels are reduced. An uncoupled highly expressed and activated enzyme would produce higher levels of peroxynitrite through a combination of superoxide and NO, leading to increased VEGF levels (73), a major feature of diabetic retinopathy.

Inducible NOS (iNOS)

Excessive NOS activity and peroxynitrite formation may also be involved in vascular and cellular injury associated with diabetic retinopathy. Peroxynitrite can modify protein and lipid structure via multiple mechanisms (74). These include nitration of tyrosine residues or thiol oxidation, which can alter cell signaling events, and DNA strand breakage, which leads to activation of the nuclear enzyme poly- ADP-ribose polymerase (PARP). Studies showing that diabetes-induced activation of PARP, leukostasis, and formation of acellular capillaries in the retina are all accompanied by increases in the nitration of tyrosine residues of retinal proteins imply a role for peroxynitrite in diabetic retinopathy (75). These effects are blocked in mice deficient in inducible NOS, suggesting that the activity of this enzyme has a critical role in the development of diabetic retinopathy. The potential role of peroxynitrite and protein

tyrosine nitration in retinopathy is further supported by studies showing that knocking out eNOS prevents vaso-obliteration in the mouse model of ischemic retinopathy (76) and by data showing that treatment with aminoguanidine, which inhibits inducible NOS (iNOS) and reduces AGE, also inhibits the development of microvascular lesions of diabetic retinopathy in animals (77–79).

In contrast with the constitutive isoforms of NOS (eNOS and nNOS), the activity of iNOS is regulated primarily at the transcriptional level. NO formation by iNOS is independent of agonist stimulation and does not require a rise in intracellular calcium. iNOS is expressed in macrophages, microglia, glial cells, neurons, and vascular cells of the retina and can play multiple roles in inflammatory response. During inflammation, iNOS is upregulated by multiple stimuli, for example cytokines such as IL-1β activate NFκB, which upregulates the transcription of iNOS. In macrophages, monocytes, and other cells the induction of iNOS and the presence of L-arginine are sufficient to initiate the generation of NO. Kinetics of NO production by iNOS differs greatly from the production by eNOS or nNOS in that iNOS produces very large, toxic amounts of NO in a sustained manner, whereas the constitutive NOS isoforms produce NO within seconds and its activities are direct and short acting.

There are multiple intracellular mechanisms through which NO may act as a proinflammatory mediator (for review, see (80) ). When it is produced by activated macrophages, NO kills microorganisms and nitrosylates macromolecules. Large amounts of “inflammatory NO” from myeloid cells are usually generated side by side with large amounts of superoxide anion. As explained these two can form peroxynitrite which mediates cytotoxic effects of NO, such as DNA damage, LDL oxidation, isoprostane formation, tyrosine nitration, and modification of enzyme activity.

The role of iNOS in diabetic retinopathy has been supported by numerous experiments showing beneficial effects of iNOS inhibitors in blocking signs of diabetic retinopathy (for review, see (81) ). Moreover, recent studies with mice have shown that iNOS deletion prevents the development of vascular lesions and blocks the loss of neuronal cells in the diabetic retina (75). While the results of these experiments are promising, it is important to remember that NO made by iNOS is of benefit to host defense reactions by contributing to microbial killing. The exact role of iNOS-derived NO in diabetic retinopathy awaits further elucidation and evaluation

NADPH Oxidase

NADPH oxidase is considered to be a major source of ROS in diabetes. Recently, the NOX family of NADPH oxidases has emerged as a major source of ROS induction (82). Studies have shown that both the mitochondria and the NADPH oxidase are involved in the sustained accumulation of ROS in a serum-withdrawal model of cell death (83). Importantly, it was found that the mitochondria and the NADPH oxidase do not act independently but rather function in a cooperative manner to extend the production of ROS. Data showing increased activity of NADPH oxidase in diabetic patients and animals and in high glucose-treated endothelial cells (84–87) suggest that NADPH oxidase is an important source of hyperglycemia-induced ROS formation. Recent studies indicate that superoxide production by NADPH oxidase has a primary role in VEGF expression and vitreoretinal neovascularization in a mouse model for ischemic retinopathy