Evidence for Ciliary Epithelium as a Source of Vascular Endothelial Growth Factor

Abstract

Abstract: : Purpose: To investigate the ciliary body as a potential source for the production of vascular endothelial growth factor (VEGF) in eyes with neovascular glaucoma. Methods: We processed 8 human globes enucleated for uncontrolled neovascular glaucoma and, as control, 8 age–matched human normal eyes for tissue sectioning. We used a rabbit anti–VEGF (AB2) antibody and avidin–biotin peroxidase for immunodetection of VEGF protein, and hybridization in situ to detect its mRNA. Briefly, VEGF (human VEGF121) cDNA was cut with EcoR1 and transcribed in vitro with T7 polymerase or cut with BamIII and transcribed with T3 polymerase for generation of antisense and sense (control) probes, respectively. After prehybridization, the tissue sections were hybridized overnight with the prepared probes. The tissue sections were incubated with alkaline phosphate–conjugated digoxigenin antibody and in substrate solution for probe detection. Two independent observers masked to the identity of the tissue source examined the sections by light microscopy. Results: VEGF immunostaining was minimal in the ciliary tissues of control eyes. In eyes with neovascular glaucoma, a strongly positive immunoreaction was observed in the nonpigmented epithelial cells of the ciliary processes. Negative control sections, in which nonimmune serum was substituted for the anti–VEGF antibody, showed no immunostaining. Minimal or no staining for VEGF mRNA was detected in ocular tissues of control eyes. In contrast, eyes with neovascular glaucoma displayed strong staining for VEGF mRNA in the nonpigmented ciliary epithelium. VEGF mRNA expression was also observed in the ganglion cell, inner nuclear and outer nuclear layers of eyes with rubeosis iridis. Adjacent sections hybridized with VEGF sense probe revealed minimal background staining. Conclusion: Our findings identify an extraretinal source for the synthesis of VEGF in eyes with uncontrolled neovascular glaucoma and could account for increased levels of VEGF protein in the aqueous humor that we reported previously (Ophthalmology 105:232–7, 1998). It may also provide an explanation why pan–retinal photocoagulation applied to the ischemic retina does not induce regression of iris neovascularization in all patients with venous thrombosis and/or diabetic retinopathy.

Keywords: growth factors/growth factor receptors • ciliary body • in situ hybridization