Expression of vascular endothelial growth factor in rabbit ocular surface epithelia

Purpose. We have previously demonstrated that the status of corneal vascularization during corneal wound healing can be modulated by the epithelial phenotypes, i.e., corneal epithelium is angiostatic whereas conjunctival epithelium is angiogenic. In this study, we further investigated whether Vascular Endothelial Growth Factor (VEGF) is present in the ocular surface epithelia to modulate the process of corneal vascularization. Methods. For in vitro study, conditioned media from explant cultures of normal and vascular rabbit ocular surface epithelia were collected. ELISA (R & D system) and Western blots with a monoclonal anti-human VEGF antibody (R & D system) were used to assay VEGF. For in vivo study, normal and vascular ocular surface epithelia were collected by surgical debridement. Expression of mRNA of VEGF was studied by RT-PCR and Southern blots. The primer pairs and oligoprobes were designed from previously published highly conserved gene sequences. Results. VEGF was found to be present in all supernatants from normal and vascular corneas, limbus and conjunctiva. VEGF concentrations (ng/ml) in normal cornea, limbus, conjunctiva and vascular cornea were 7.8±0.6, 20.6±0.9, 21.7±1.6 and 18.4±0.4, respectively. Western blots revealed a 42 KD band, consistent with reported molecular weight of VEGF. By RT-PCR, mRNA of VEGF was noted to be preferentially expressed by the limbus and conjunctival derivatives such as vascular corneas. The specificity of PCR products of VEGF was confirmed by Southern blots. Conclusions. The results indicate that VEGF is expressed preferentially in the conjunctival derivatives of ocular surface epithelia and to a much less extent in the normal cornea. Such a finding further implicates that VEGF may serve as one of the angiogenic cytokines responsible for conjunctivalization-induced corneal vascularization.