RNA was still observed outside blood vessels. Isolated Muller cells were found to express both VEGFR1 and R2 (92). Pharmacologic inhibition of VEGFR1 and R2 from P0 to P9 with a small molecule antagonist, SU5416, resulted in cell loss in the inner nuclear layer and ganglion cell layer, as compared with control mice (92). This suggests that VEGF receptor activity may be important for normal retinal neural development.

VEGF and Neuroprotection

Research over the past few years indicates that VEGF has an additional role in neuroprotection (93). VEGF stimulated axonal outgrowth and increased survival of both neurons and satellite cells in experiments with cultured superior cervical ganglia and dorsal root ganglia (94). In explants of the ventral mesencephalon, VEGF treatment promoted the growth and survival of dopaminergic neurons and astrocytes (95). Intriguing in vivo studies have implicated VEGF as a possible cause of motor neuron degeneration. A study of knock-in mice was performed, in which the VEGF gene promoter contained a deletion of the HIF-1 binding site (hypoxia-response element). These mice exhibited adult-onset motor neuron degeneration that was reminiscent of ALS. This neurodegeneration appeared to result from reduced neural vascular perfusion (96). In addition, VEGF treatment of cultured motor neuron cells significantly reduced apoptosis induced by hypoxia, oxidative stress, and serum deprivation, indicating direct neuroprotective effects of VEGF. This survival effect was blocked by antibodies to neuropilin-1 and VEGFR2 (96). Notably, variations in the VEGF gene that lower systemic VEGF expression increase the risk of ALS in humans (97).

MODULATION OF VEGF ACTION BY OTHER GROWTH FACTORS

Although VEGF clearly plays a major role in stimulating retinal neovascularization, it is likely that other growth factors also play important roles, either independently or in concert with VEGF. For instance, a critical effect in VEGF modulation has been demonstrated for growth hormone (GH) and insulin-like growth factor-1 (IGF-1). IGF-1, which is known to mediate the growth promoting aspects of GH, has been demonstrated to modulate VEGF induction of endothelial cell signaling. In retinal endothelial cells, IGF-1 controlled the maximal VEGF stimulation of Akt (78). In addition, antagonism of the IGF-1 receptor significantly inhibited VEGF stimulation of ERK1/2 in retinal endothelial cells (98). These experiments suggest that IGF-1 plays a permissive role in VEGF action on angiogenesis. The importance of the GH/ IGF-1 axis in retinal neovascularization is highlighted by studies using the oxygeninduced retinopathy model. In this model, retinal neovascularization was significantly decreased both in transgenic mice expressing a growth hormone antagonist gene and in normal mice treated with an inhibitor of growth hormone secretion (99). Of note, GH inhibition did not reduce retinal expression of VEGF or VEGFR2. This inhibition of retinal neovascularization was reversed by systemic administration of IGF-1 (99). Further experiments demonstrated that an IGF-1 receptor antagonist also suppressed retinal neovascularization in this model (98).

A second growth factor that appears to modulate VEGF action on endothelial cells is placental growth factor, or PlGF. Placental growth factor is a member of the VEGF

family of structurally related growth factors (100). In contrast to VEGF-A (which binds both VEGFR-1 and VEGFR-2), PlGF specifically binds VEGFR-1. PlGF has been reported to enhance, or “amplify,” VEGF-driven angiogenesis (101), increasing VEGF induction of endothelial cell survival, migration, and proliferation. A molecular basis for this synergism was reported, in which PlGF activation of VEGFR1 results in intermolecular transphosphorylation of VEGFR2, thus amplifying VEGF-driven angiogenesis via VEGFR2 (102). The potential importance of PlGF in PDR has been highlighted by experiments using mice deficient in PlGF, which have demonstrated that PlGF is required for pathologic retinal neovascularization in the oxygen-induced retinopathy model (101). Furthermore, PlGF levels are significantly increased in the vitreous in PDR as compared to nonneovascular disease (103, 104), and retinal PlGF protein is increased in the oxygen-induced retinopathy model (105).

The ability of other growth factors to modulate VEGF action suggests that these growth factors might themselves be therapeutic targets for proliferative diabetic retinopathy. Indeed, somatostatin analogs that inhibit the GH/IGF-1 axis have been under investigation for the treatment of diabetic retinopathy.

CONCLUSION

The major role that VEGF plays in retinal neovascularization is now beyond question, in light of the preponderance of clinical, preclinical, and basic research studies. For this reason, the development and use of therapeutics targeting VEGF and its downstream actions is a promising approach for current and future treatment of proliferative diabetic retinopathy. At the same time, the multiple actions of VEGF warrant an awareness of the potential side effects of therapies targeting this molecule. In addition, it is highly likely that additional growth factors play important roles in retinal neovascularization in DR. Such growth factors might potentially have synergistic effects with VEGF or act at different steps in the neovascular process. Therefore, it will be important to continue investigations into the role of other growth factors, which themselves may serve as additional targets for therapy.

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