VEGF’s function as an endothelial cell survival factor is well established (164). However, VEGF also appears to have a role in promoting neuronal cell survival. Genetic studies in mice with a deletion of the hypoxia response element in the VEGF gene promoter have shown that mice with reduced VEGF levels develop adult-onset motor neuron degeneration similar to that seen in patients with amyotrophic lateral sclerosis (165). VEGF has also been found to reduce neuronal injury in stroke (165). The mechanism(s) of these effects are not yet clear, but may involve both a direct action on neural cells that express VEGFR1 and 2 as well as an indirect action in promoting angiogenesis and reducing tissue ischemia. In the developing retina VEGFR-1 and -2 are expressed specifically in Muller glial cells. Studies in the developing retina showed that inhibiting the activity of VEGFR1 and 2 in the avascular regions of the developing neural retina results in a loss of cells in the inner retinal layers, suggesting that retinal neurons and/or glial cells may be VEGF dependent (166).

Paradoxically, even though levels of VEGF and VEGFR2 are increased in the diabetic retina, VEGF’s prosurvival function is compromised in that endothelial cells, neurons, and glial cells undergo apoptosis (145,147,164,167). These observations suggest that VEGF prosurvival signaling is altered by the diabetic state. VEGF activation of VEGFR2 transduces prosurvival signals via the PI3-kinase/Akt signaling pathway (168). However, VEGF also activates p38 MAP kinase, which is a known modulator of proapoptotic signals in endothelial cells (169). Blockade of VEGF-mediated activation of PI3 kinase or Akt signaling can lead to increases in apoptosis by enhancing the activation of p38 MAP kinase (170). Studies in retinal endothelial cells have shown a similar phenomenon of accelerated apoptosis even in the presence of exogenous VEGF when cells are exposed to high glucose or oxidative stress (171,172). This proapoptotic effect is associated with activation of p38 MAP kinase, inhibition of Akt-kinase, and tyrosine nitration of the regulatory subunit of PI3 kinase p85 (171). Given that p85 is a known target for peroxynitriteinduced nitration on tyrosine which blocks its interaction with the PI3 kinase catalytic subunit p110 (173), these data suggest that peroxynitrite can alter cell survival responses mediated by PI3-kinase. More work is needed to determine whether this mechanism also plays a role in ROS-mediated impairment of neuronal and glial cell survival function.

THERAPEUTIC STRATEGIES FOR REDUCING OXIDATIVE STRESS

Overview

As has been explained in the section “Antioxidants in Diabetic Retinopathy,” formation of ROS is increased in diabetes and is directly related to the complications of diabetes. A number of treatments that reduce levels of oxidative stress have also shown promise in reducing signs of diabetic retinopathy in experimental models (Fig. 2). Therapies with potential actions in reducing ROS will be discussed in this section, some only briefly as they are the subjects of other chapters in this book.

Antioxidants

The causal role of oxidants in diabetic retinopathy is well established, and antioxidant therapy has shown great promise when tested in tissue culture and experimental animal models. However, antioxidant agents that scavenge formed oxidants have not

Diabetic Retinopathy

Fig. 2. Therapeutic strategies for reducing ROS and RNS in the diabetic retina.

proven to be very effective clinically. Treatment with vitamin E (α-tocopherol) for 4 months raised retinal blood flow in patients with type I diabetes and mild or no retinopathy (174), but other large studies failed to show a beneficial effect. Chronic treatment with vitamin E also failed to decrease cardiovascular events in a large study with a high percentage of diabetic patients (20). A major limitation of treatment with vitamin E or other antioxidants is that these therapies scavenge already-formed oxidants, but do not prevent their formation.

PKC Inhibitors

As has been noted, PKC has been clearly established as both a source and a target of reactive oxygen species in diabetic retinopathy. Clinical trials testing the efficacy of the PKC-beta inhibitor ruboxistaurin have supported the hypothesis that PKC activation, especially the β isoform, plays an important role in the development of diabetic macular edema (for review, see (45)). However, while ruboxistaurin treatment was found to improve visual acuity in patients with diabetic macular edema, clinical trials showed that it did not reduce or reverse the progression of diabetic macular edema or prevent the development of proliferative diabetic retinopathy (175).

Inhibitors of the Renin-Angiotensin System

Studies in animal and tissue culture models have implicated the renin-angiotensin system (RAS) in the development and progression of retinal vascular diseases, including

diabetic retinopathy. A phase I trial investigating the effect of a single intraocular injection of an adenoviral vector-expressing human PEDF in patients with advanced choroidal neovascularization due to age-related macular degeneration has been completed with promising results (193). Further study is needed to determine the efficacy of PEDF in preventing diabetic retinopathy.

Cannabinoids

Recent studies indicate that cannabinoids may also be useful in reducing oxidative stress. Cannabinoids have a variety of potentially beneficial properties including antiinflammatory (194) and antioxidant actions (195). Several synthetic, nonpsychoactive cannabinoids have shown promising results in the reducing oxidative stress, suppressing inflammation, and inhibiting neurotoxicity in conditions of central nervous system injury. Phase 3 clinical trials have demonstrated the efficacy and safety of dexanabinol in the treatment of traumatic brain injury due to its abilities to antagonize N-methyl- D-aspartate receptors, scavenge reactive oxygen species, and suppress inflammation (196). Another synthetic, nonpsychoactive compound, cannabidiol (CBD), has been found to block neuronal damage resulting from cerebral ischemia (197). CBD has recently been approved for the treatment of inflammation, pain, and spasticity in patients with multiple sclerosis. A recent study demonstrated potent antioxidant and antiinflammatory effects of CBD where it blocked the effects of high glucose in increasing mitochondrial superoxide generation, NF-kappa B activation, nitrotyrosine formation, upregulation of iNOS and adhesion molecules ICAM-1 and VCAM-1, transendothelial migration of monocytes, and monocyte-endothelial adhesion in human coronary endothelial cells (198). CBD has also been shown to have potent neuroprotective actions in the retina (199). Studies showing that CBD also prevents diabetes-induced neurotoxicity and preserves blood-retinal barrier function in experimental diabetes suggest that it could also be useful in the treatment of diabetic retinopathy (155).

Cyclo-oxygenase-2 (COX-2) Inhibitors

As has been outlined in the section “Antioxidants in Diabetic Retinopathy,” COX-2 expression is increased during diabetes (37) and elevated COX-2 increases ROS formation (38). COX-2 inhibition has been noted to protect against diabetic neuropathy in animals (39). High doses of aspirin, a nonselective inhibitor of both COX-1 and-2, have been reported to prevent some signs of diabetic retinopathy in diabetic patients and experimental animals (78, 200, 201). However, other clinical trials showed that treatment with high-dose aspirin did not prevent the development of high-risk proliferative retinopathy and did not reduce the risk of visual loss, nor did it increase the risk of vitreous hemorrhage (202). Clinical trials are in progress to evaluate the effectiveness of celecoxib on proliferative diabetic retinopathy (179). Unfortunately, chronic use of selective COX-2 inhibitors has been associated with increased risks of adverse cardiovascular events (203).

Peroxisome Proliferator-Activated Receptor g Ligands

Thiazolidinediones and glitazones are insulin-sensitizer agents that bind to and activate the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). Although these