risk of neovascularization in branch retinal vein occlusion (16). In 1948, Michaelson proposed that a diffusible angiogenic “factor X” released from areas of hypoxic retina, is responsible for neovascularization in diabetic retinopathy, as well as in other ischemic retinopathies (2). In the 1990s, VEGF emerged as a strong candidate for “factor X,” and subsequent research has strongly established VEGF as a major stimulator of retinal neovascularization in the ischemic retinopathies, including proliferative diabetic retinopathy.

Interest in VEGF’s role in eye disease arose from earlier studies of VEGF’s systemic role, including its contribution to tumor angiogenesis. VEGF is a homodimeric glycoprotein that is both a vasopermeability (17) and an angiogenesis factor (18,19). It was initially denoted as vasopermeability factor (VPF) based on its ability to increase microvascular permeability (17). Indeed, in one assay of dermal microvascular permeability, VEGF was found to be 50,000 times as potent, on a molar basis, as histamine (20). VEGF is mitogenic primarily for vascular endothelial cells (19). The expression of VEGF has been found to be greatly increased in rapidly growing, highly vascularized tumors (21), and inhibition of VEGF with a monoclonal antibody inhibited tumor growth in vivo (22). Hypoxia induces VEGF expression in tumors and glial myogenic tumor cell lines (23). VEGF binds two high affinity cell-surface tyrosine kinase receptors, in particular fms-like tyrosine kinase (Flt) and fetal live kinase 1 (Flk-1), deneted VEGFR-1 (Flt-1) and VEGFR-2 (KDR/ FIk-7) both of which are expressed on vascular endothelial cells. VEGF and its receptors are critical for normal embryological development, and even heterozygous knockout of the VEGF gene results in embryonic lethality due to impairment of developmental angiogenesis (24,25). These characteristics of VEGF suggested that it might play a major role in mediating the microvascular complications observed in diabetic retinopathy, since they are also characterized by tissue ischemia, angiogenesis, and vascular permeability. Subsequent extensive studies in ocular cells, animal models, and patients have confirmed this, and they are detailed in the following paragraphs.

REGULATION OF VEGF EXPRESSION IN THE RETINA

Regulation of VEGF in Proliferative Diabetic Retinopathy

VEGF is produced by many cell types within the eye, including retinal pigment epithelial cells, pericytes, endothelial cells, glial cells, Muller cells, and ganglion cells (26, 27). In the context of diabetic retinopathy, VEGF upregulation was first appreciated in the proliferative stage. In the mid-1990s, clinical studies demonstrated significantly increased intraocular concentrations of VEGF in specimens from patients with proliferative retinopathies, including diabetic retinopathy. In one investigation, 210 specimens of ocular fluid (vitreous and/or aqueous) were collected from 164 patients undergoing intraocular surgery, including 143 samples from patients with diabetes (28). The patients with diabetes had different stages of retinopathy, including no retinopathy, nonproliferative retinopathy, quiescent proliferative retinopathy, and active proliferative retinopathy. VEGF concentrations were significantly elevated in both the vitreous and aqueous of patients with active proliferative diabetic retinopathy. In contrast, VEGF concentrations were low in a control group of patients with no neovascular disorder, and in diabetic patients with no retinopathy, nonproliferative retinopathy, or quiescent proliferative retinopathy. Six patients successfully underwent laser photocoagulation treatment for

active proliferative retinopathy. In these six patients, intraocular VEGF concentrations were decreased by an average of 75% after treatment, as compared to before treatment (28). Another study demonstrated similar findings measuring VEGF concentrations in vitreous specimens from 8 patients with PDR as compared to 12 control patients with no neovascular disorder (29). These results have subsequently been corroborated by numerous studies.

Upregulation of VEGF levels has also been observed in animal models of retinal neovascularization. In the mouse model of oxygen-induced retinopathy, retinal VEGF RNA expression was increased by threefold within 12 h after commencement of relative retinal hypoxia, and the increase in VEGF expression persisted during the development of retinal neovascularization (30). VEGF RNA was markedly increased in the inner nuclear layer, and confocal immunohistochemistry studies demonstrated that the VEGF-producing cells had a Muller-like morphology. Similar upregulation of VEGF was found in rat (31) and cat models of retinal neovascularization (32, 33), as well as a primate model of iris neovascularization induced by laser occlusion of branch retinal veins (34). These models all exhibit a temporal relationship between VEGF expression and ocular angiogenesis, with increased VEGF expression after the onset of retinal hypoxia, but before the development of neovascularization.

The role of hypoxia as an important stimulus for VEGF upregulation is supported by cell-culture studies. Hypoxia induces VEGF RNA expression in various ocular cell types (27). Furthermore, VEGF induction is reversible upon return of the cells to normoxia. An important mediator of hypoxia-induced upregulation of VEGF expression is HIF-1, a hypoxia-induced transcription factor that is known to stimulate the transcription of multiple genes upregulated by hypoxia (35). HIF-1 has been demonstrated to play an important role in the activation of VEGF transcription in cultured cells subjected to hypoxia (36). Strong support for the involvement of HIF-1 in VEGF upregulation was provided by a study of mice in which the VEGF gene was replaced by a mutant VEGF gene containing a deletion of the HIF-1 binding site (hypoxia-response element) from the promoter region. Both wild-type and mutant mice were subjected to oxygen-induced retinopathy. In contrast to the wild-type mice, retinal VEGF RNA levels were not increased in the mutant mice. In addition, the mutant mice had significantly less retinal neovascularization (37).

As noted earlier, laser photocoagulation has been demonstrated to reduce intraocular VEGF levels in patients with active proliferative retinopathy (28). This has been corroborated in a study of aqueous specimens in patients with PDR undergoing panretinal photocoagulation (38). In addition, immunohistochemical studies of postmortem eyes from individuals at different stages of diabetic retinopathy demonstrated that the intensity of VEGF immunostaining in diabetic retinas that had undergone laser photocoagulation (and exhibited no preretinal neovascularization) was similar to that in diabetic retinas without overt retinopathy (39). A reduction in pro-angiogenic factors had long been suspected to underlie the therapeutic benefits of laser photocoagulation for PDR. The basis for this might be that the laser destroys cells responsible for production of pro-angiogenic factors including VEGF. However, a more attractive hypothesis is that laser-induced destruction of photoreceptor cells results in reduced oxygen consumption of the outer retina, allowing delivery of oxygen from the choriocapillaris to the inner layers of the retina. This idea is supported by studies of a mouse model of genetically induced photoreceptor

degeneration. In this study, the mice did not develop retinal neovascularization when subjected to the experimental protocol of oxygen-induced retinopathy (40). In addition, these mice did not exhibit the expected upregulation of retinal VEGF expression. A final idea concerning the basis for the therapeutic benefit of laser photocoagulation is the possible increase in levels of anti-angiogenic factors.

Regulation of VEGF in Nonproliferative Diabetic Retinopathy

Although upregulation of VEGF in diabetic retinopathy was first reported in the proliferative phase, it has become increasingly appreciated that VEGF levels are also elevated in nonproliferative diabetic retinopathy. Increased VEGF levels have been described in several studies of postmortem eyes of patients with NPDR compared with nondiabetic controls (39–42). Furthermore, immunopositivity of VEGF was demonstrated in eyes with no anatomic evidence of retinal nonperfusion (42). Increased VEGF levels have also been demonstrated in the vitreous of patients with nonproliferative diabetic retinopathy, particularly in the setting of macular edema (43). Similarly, several investigators have demonstrated an increase in retinal VEGF levels in animal models of diabetic retinopathy (44–46).

Several studies have provided a biochemical basis for VEGF upregulation in NPDR, linking this phenomenon to oxidative stress, and an increase in advanced glycation endproducts, two well-known consequences of diabetes in the retina. VEGF is upregulated by reactive oxygen intermediates in vitro (47), and increased levels of reactive oxygen intermediates have been correlated with increased VEGF in diabetic rodents (48). Furthermore, diabetes-induced increases in retinal VEGF protein are significantly reduced by antioxidant treatment (49), strongly supporting the upregulation of VEGF by oxidative stress. Advanced glycation endproducts have also been demonstrated to upregulate VEGF expression in vitro. Interestingly, this AGE-induced upregulation was inhibited by antioxidant treatment (50), further supporting the role of oxidative stress.

FUNCTIONAL ROLE OF VEGF IN RETINAL NV AND PROLIFERATIVE

DIABETIC RETINOPATHY

In light of its important role in tumor angiogenesis, the evidence for dramatic upregulation of intraocular VEGF levels in PDR led to experiments to confirm its functional importance in retinal neovascularization. VEGF inhibition studies in animal models have established a causal relationship for VEGF in ocular neovascular processes. Several VEGF inhibitory molecules have been evaluated, including VEGF receptor chimeric proteins, neutralizing antibodies, and antisense phosphorothioate oligodeoxynucleotides.

VEGF receptor chimeric proteins were constructed containing the entire extracellular domain of VEGFR1 (the Flt receptor) or VEGFR2 (the Flk-1 receptor) joined with the heavy chain of IgG (51). These chimeric proteins bind VEGF with the same affinity as the native receptors, and can therefore act as competitors for VEGF binding. Injection of these chimeric proteins into the mouse model of oxygen-induced retinopathy (discussed earlier) was performed just when the retinas became hypoxic. Retinal neovascularization was significantly reduced by either single or dual injections of either chimeric protein, with a mean suppression of approximately 50% (52) (Fig. 3).

Fig. 3. Soluble VEGF receptor-IgG chimeric proteins reduce histologically evident ischemia-induced retinal neovascularization. Retinal ischemia was induced in C57BL/6J mice. The right eye of each mouse was injected with 250 ng of human CD4-IgG control chimeric protein on P12 and P14 (left). The left eye received intravitreal injections of 250 ng of human Flt-IgG chimera at the same times (right). Paraffin-embedded, periodic acid/Schiff reagent, and hematoxylin-stained 6 m serial sections were obtained. Typical findings from corresponding retinal locations from both eyes of the same mouse are shown and are representative of all animals studied. Vascular cell nuclei internal to the inner limiting membrane represent areas of retinal neovascularization and are indicated with arrows. No vascular cell nuclei anterior to the internal limiting membrane are observed in normal, unmanipulated animals (from (52) with permission). × 50.

Antisense phosphorothioate oligodeoxynucleotides were also studied in the same model of oxygen-induced retinopathy. Two different VEGF antisense molecules were found to reduce retinal levels of VEGF protein by 40–66%, while decreasing new blood vessel growth by 25 and 31%, respectively, as compared to sense or noncomplementary mRNA controls (53). Further investigation of VEGF inhibition was performed in a primate model of iris neovascularization, using VEGF neutralizing antibodies. In this study, intravitreal injections of VEGF neutralizing antibody administered every other day for 2 weeks resulted in inhibition of iris neovascularization as assessed by fluorescein iris angiograms (54).

These initial studies strongly suggested that VEGF has a significant role in mediating retinal neovascularization in general and PDR in particular. Notably, the studies did not achieve complete inhibition of retinal neovascularization, suggesting either insufficient delivery of VEGF inhibitor or the role of other growth factors. In addition, it remains a significant question whether VEGF by itself is sufficient to stimulate retinal neovascularization. Intravitreal sustained release of VEGF-induced transient retinal NV in rabbits, but not primates (55). Intraocular injections of VEGF in monkeys resulted in multiple vascular changes, including capillary nonperfusion, vessel dilation, and tortuosity, and disruption of the blood–retinal barrier. Preretinal neovascularization was observed in the peripheral retina that originated from superficial veins and venules, but neovascularization was not observed in the posterior pole (56). Transgenic mice in which VEGF is produced by the photoreceptors exhibited very significant neovascularization that grew from the deep capillary bed of the retina and extended into the subretinal space, but not preretinal neovascularization (perhaps not surprising, since the VEGF was produced by the photoreceptors in this model) (57). Conceivably, the ability of VEGF to stimulate preretinal neovascularization may depend on its levels and sustained presence. On the other hand, the presence of other pro-angiogenic factors or a reduction in anti-angiogenic molecules may be required. Nevertheless, the initial animal studies