Adenovirus-Mediated Overexpression of VEGF in the Retinal Pigment Epithelium Increases the Size of Laser-Induced Choroidal Neovascularization in Mice

Abstract

Purpose: : Subretinal injection of adenovirus expressing VEGF has been reported to cause choroidal neovascularization (CNV) in mice and rats. However, injection of empty adenovirus also causes CNV. In both cases, CNV occurs at or near the injection site, suggesting that focal disruption of Bruch's membrane during the procedure is a contributing factor. Quantifying the area of CNV, and thereby determining the effect of overexpressed VEGF, has been challenging, mainly because of the variability in the extent of tissue damage at the injection site. We generated a mouse model which combines the adenoviral system for delivering VEGF with the precision of laser photocoagulation to quantify the effect of VEGF overexpression on CNV.

Methods: : One microliter of 2 x10⁵ pfu of adenovirus expressing mouse VEGF₁₆₄ (Ad-mVEGF₁₆₄-ZsG) or 2 x 10⁵ pfu of empty adenovirus (Ad-ZsG) were injected into the subretinal space of 7-week-old C57Bl/6 mice via a trans-scleral approach. Three days after injection, laser shots were fired within the adenoviral-infected area (which fluoresced green under the slit lamp due to the expression of a ZsGreen1 reporter) and outside of the infected area. Seven days after laser, rhodamine-concanavalin A was injected IV to label the vasculature, and mice were euthanized. RPE-choroidal-scleral flatmounts were prepared, fluorescent images of each CNV lesion were captured, and the CNV area was determined with Axiovision software.

Results: : Overexpression of VEGF resulted in a 75% increase in the size of laser-induced CNV lesions outside of the adenoviral-infected area (mean Ad-ZsG CNV area = 10,977 µm²; mean Ad-mVEGF₁₆₄-ZsG CNV area = 19,250 µm²; p < 0.001). Laser shots fired into the adenoviral-infected area failed to induce CNV, likely because of the residual subretinal fluid interfering with photocoagulation.

Conclusions: : Overexpression of VEGF does promote CNV, and the magnitude of the effect can be quantified using a combined adenoviral/laser-induced CNV mouse model. This combined model can now be used to assess the effects of other secreted factors on CNV.

Keywords: age-related macular degeneration • adenovirus • vascular endothelial growth factor