Pathological angiogenesis, or the production of new capillary vessels from preexisting vasculature, within the eye is a serious event that often leads to blindness. Upregulation of vascular endothelial growth factor (VEGF) has been linked to neovascularization in the eye, suggesting that it could be a suitable target to inhibit angiogenic changes. This work investigated whether the presence of a proven antiangiogenic factor, the soluble variant of the VEGF receptor, sFlt-1, in the anterior chamber is sufficient to inhibit new vessel formation in the cornea in an animal model of corneal neovascularization. A recombinant adenovirus vector that can mediate efficient in vivo gene transfer and expression in ocular cells was selected as a delivery agent. We have shown that after the injection of Ad.betagal into the anterior chamber of normal and cauterized rat eyes, corneal endothelial cells and cells of the trabecular meshwork were efficiently transduced and that beta-galactosidase (beta-Gal) expression was maintained up to 10 days postinjection. Cauterization significantly increased the amount of immunoreactive VEGF in vehicle- or Ad.null-injected animals (t test, p < 0.001 and p < 0.001, respectively). However, when cauterization was combined with Ad.sflt injection there was no statistically significant increase in the amount of immunoreactive VEGF (p = 0.12). The injection of Ad.sflt into the anterior chamber slowed or inhibited VEGF-induced angiogenic changes. After cauterization, 100% of uninjected and vehicle-injected and 82% of Ad.null-injected animals developed moderate to severe corneal angiogenesis in contrast to 18% of Ad.sflt-injected animals. These in vivo results suggest that the transient presence of antiangiogenic agents in the anterior chamber can be successfully used to inhibit the development of corneal angiogenesis.

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Diabetic retinopathy, one of the most serious complications of long-term diabetes, could clinically be divided into two stages: 1) background retinopathy that does not cause visual impairment and 2) proliferative retinopathy, which is a potentially blinding condition. This study aims to investigate the correlation between enhancement of vascular endothelial growth factor (VEGF) expression and neovascular changes. A binary recombinant adeno-associated virus construct producing green fluorescent protein (GFP) and VEGF under the control of the human cytomegalovirus promoter, recombinant adeno-associated virus (rAAV).VEGF.GFP, was produced and injected into the subretinal space of C57BL mice. GFP expression was tracked by fluorescence fundus photography, and VEGF expression was confirmed by immunohistochemistry and enzyme-linked immunoassay. Neovascular changes were monitored by fluorescein angiography and histology and by quantifying the number of inner retinal vessels. GFP expression was found in 100% of injected eyes, and vascular changes were detected in 9 of 10 rAAV.VEGF.GFPinjected eyes. Of these, four demonstrated microaneurysms and five showed moderate to severe leakage. There was a statistically significant increase in blood vessel number in the inner nuclear layer (P < 0.03) and dilatation of retinal veins (P < 0.05). This work has demonstrated that the development of different stages of diabetic retinopathy is closely correlated with an increased VEGF level in the retina. Diabetes 52: 857-863, 2003

Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed.