Vascular endothelial growth factor expression in choroidal neovascularization in rats.

Abstract

The pathogenesis of choroidal neovascularization is largely unknown. We investigated vascular endothelial growth factor (VEGF) expression in laser-induced choroidal neovascularization (CNV) in rats. Intense krypton laser photocoagulation was applied to the posterior poles of the eyes of pigmented rats to induce CNV, which was confirmed by fluorescein angiography and histopathology. The eyeballs were enucleated 1, 3, 7, 14 and 28 days after laser photocoagulation. Cryostat sections were prepared for immunofluorescence staining using anti-VEGF and macrophage marker (ED1) antibodies. The posterior segments of eyeballs pooled from photocoagulated and control rats were submitted for immunoprecipitation and immunoblotting by the anti-VEGF antibody, and reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of VEGF mRNA. Very weak immunoreactivity for anti-VEGF antibody was found in the ganglion cell layer, inner nuclear layer, and retinal pigment epithelium (RPE) in the normal retina. In the development of CNV, strong positive staining for anti-VEGF antibody was found in photocoagulated areas in the subretinal space and choroid. Double immunofluorescence staining showed that many cells in lasered lesions were positive both for anti-VEGF and macrophage marker ED1 antibody staining in the early stage of this model. Immunoblots showed a positive band for the VEGF molecule in treated but not control animals. RT-PCR results demonstrated upregulation of VEGF transcripts in the CNV model compared with normal animals. Our findings showed the upregulation of VEGF expression in experimentally induced CNV, where it may be involved in promoting choroidal angiogenesis. Macrophages may be one of the main sources of VEGF in the early stage of the disease.