of tortuous, beaded vessels as well as microaneurysms, endothelial cell hyperplasia, and neovascular glaucoma, demonstrating an important role for VEGF in ocular neovascular disease. The remainder of this section concentrates on more recent experiments that have examined the inflammatory nature of VEGF in promoting both the BRB breakdown and the ischemia-mediated neovascularization that are characteristic of DR.

VEGF as a Mediator of Diabetes-associated Retinal Leukostasis

and BRB Breakdown

As discussed earlier, DR is associated with a chronic elevation of retinal leukostasis, which has in turn been identified as a major contributor to endothelial cell injury and BRB breakdown. Like the studies examining the roles of ICAM-1 and its cognate integrins in these processes, studies of VEGF have employed the STZ-induced diabetic rat, and they have been further supplemented by experiments examining the effect of intravitreous VEGF in nondiabetic animals.

Within a week of STZ induction, retinal expression of VEGF mRNA was elevated by 3.2-fold with respect to controls (Fig. 10A) (79), together with the concomitant increases in vascular permeability, BRB breakdown (79), and elevated expression of ICAM-1 (80) that have been described earlier. These diabetes-induced increases were significantly reduced by the systemic administration of a VEGF receptor fusion protein that blocks the bioactivity of VEGF (79, 80); in fact, STZ-mediated upregulation of ICAM-1 expression was essentially eliminated (Fig. 10B), suggesting that in this model VEGF is responsible for much of the subsequent ICAM-1-mediated leukostasis. In a parallel experiment, intravitreous injection of VEGF in nondiabetic rats also resulted in increased retinal leukocyte adhesion (Fig. 11), together with retinal ICAM-1 upregulation and increased vascular permeability (81). Both retinal vascular permeability (Fig. 12A) and leukocyte accumulation (Fig. 12B) could be inhibited by a systemically administered anti-ICAM-1 antibody (81). Thus, VEGF elevations, whether caused by diabetes or intravitreous injection, led to the development of similar retinal pathologic consequences. Taken together, these data support a model in which diabetes-induced elevations of retinal VEGF lead to upregulation of retinal expression of ICAM-1 and increased leukostasis. As described earlier, the final outcome of these events is leukocyte-mediated vascular damage.

VEGF164/165 as a Proinflammatory Cytokine

Data from experiments with rodent models have suggested that only one VEGF isoform VEGF165 acts as an especially pathogenic proinflammatory cytokine. These experiments have focused on VEGF164 and VEGF120 (corresponding to human VEGF165 and VEGF121, respectively). Experiments comparing intravitreous injection of VEGF164 and VEGF120 in nondiabetic rats demonstrated that VEGF164 was approximately twice as effective in promoting increased retinal ICAM-1 expression, leukocyte adhesion, and BRB breakdown (82). Moreover, in diabetic animals, selective inhibition of VEGF164, through intravitreous injection of pegaptanib, an RNA aptamer that binds VEGF164 while sparing VEGF120, significantly inhibited retinal leukostasis and BRB breakdown. The inhibition of BRB breakdown by pegaptanib was particularly marked in early diabetes (2 weeks

Fig. 11. Vascular endothelial growth factor (VEGF) induces retinal leukostasis. Appearance of a normal rat retina before (A) and 48h after (B) intravitreous injection of 50 ng VEGF; retinal leukostasis was assessed by acridine orange leukocyte fluorography. Numerous static leukocytes are visible (white dots) as well as vessel dilation and tortuosity. Scale bar: 100 m. (Reprinted from Miyamoto et al. 2000 (81) with permission from the American Society for Investigative Pathology.)

after induction; Fig. 13A), but was still evident in established diabetes at 3 months (Fig. 13B) (82). The suppression effected by intravitreous pegaptanib in these experiments was comparable to that described earlier in the studies with the VEGF receptor

fusion protein that binds to all VEGF isoforms (79, 80), suggesting that VEGF164/165 is responsible for much of the retinal vasculopathy that results from the diabetes-induced

elevation of VEGF.

Additional support for the inflammatory nature of VEGF164/165 has come from the retinopathy of prematurity model of ischemic neovascularization in which the expres-

sion of VEGF164 was markedly enhanced compared to VEGF120 (55). Intravitreous injection of pegaptanib or the VEGF receptor fusion protein inhibited leukocyte adhesion by approximately 50%; both also dramatically inhibited pathologic vascularization (Fig. 14A) (55). Unlike the fusion protein, which also inhibited physiologic retinal revascularization, intravitreous pegaptanib spared this process (Fig. 14B) (55). In part, the enhanced pathogenicity of VEGF164/165 in the ischemic ocular neovascularization model

may reflect the greater potency of VEGF164/165 compared to VEGF120/121 in acting as a chemoattractant for infiltrating macrophages (58), which are known to amplify pathologic

neovascularization, as discussed earlier.

TUMOR NECROSIS FACTOR-α

TNF-α is a key proinflammatory cytokine that has been implicated in several immunological disorders (83). The main cellular source of TNF-α is macrophages (84), although other immune cells such as T lymphocytes (85), neutrophils (86), and a variety of other cell types, including endothelial cells, can synthesize TNF-α as well (84). Stimulation of its cognate receptors by TNF-α can lead to a multitude of cellular responses, including the recruitment of leukocytes and monocytes, induction of apoptosis, stimulation of adhesion molecule expression, and stimulation of synthesis and release of a variety of other cytokines and inflammatory mediators (84). In addition to its actions in mediating specific pathologic features of diabetes, including nephropathy

Fig. 12. Effect of anti-intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody on permeability and leukostasis after intravitreous vascular endothelial growth factor (VEGF) injection. Rats receiving intravitreous VEGF had a 3.2-fold increase in vascular permeability (A), as measured by assessing radioactive albumin permeation into retinal tissue; systemic administration of an anti–ICAM-1 antibody significantly reduced vascular leakage when compared to a control antibody (P < 0.0001). Similar increases in VEGF-mediated leukostasis (B) also were blocked with the anti–ICAM-1 antibody. NS = not significant. (Reprinted from Miyamoto et al. 2000 (81) with permission from the American Society for Investigative Pathology).

Fig. 13. Pegaptanib, an antivascular endothelial growth factor (VEGF)165 aptamer, reduces diabetic blood–retinal barrier (BRB) breakdown. In 2-week diabetic rats (A), intravitreous treatment with pegaptanib resulted in an 82.6% reduction of BRB breakdown when compared to polyethylene glycol (PEG) alone (P < 0.01). In established diabetes (B), there was 55% inhibition of BRB breakdown with pegaptanib (P < 0.01). BRB breakdown was assessed by a fluorescein-conjugated dextran method; data represent mean ± standard deviation. (Reproduced from Ishida et al. 2003 (82) with permission from Investigative Ophthalmology & Visual Science. Copyright 2003 by Investigative Ophthalmology & Visual Science.)

(84) and retinopathy, TNF-α has also been found to contribute to the induction of pancreatic β-cell apoptosis in mice (87) and to insulin resistance in adipose tissue (88).

Evidence supporting a role for TNF-α in DR comes from studies demonstrating elevations of TNF-α in ocular fibrovascular membranes (15), platelets (89), and plasma