In situ Ablation of Pigment Epithelium Derived Factor (PEDF) Does Not Breach Corneal Avascularity

Abstract

Abstract: : Purpose:To study whether Pigment Epithelium Derived Factor (PEDF) is essential for corneal avascularization. Methods: An siRNA expression cassette against mouse PEDF was developed and cloned into the pSEC Neo vector. A scrambled sequence served as negative control. The insert had its own H1 promoter expressing siRNA hairpin. Positive controls to induce corneal neovascularization included VEGF165 cloned into pBLAST and siRNA against soluble FLT in pSEC Neo. The plasmids were injected into the mouse corneas and the corneas were harvested at the 2, 5, 8, and 14 day intervals. RNA was isolated from the corneas and semi–quantitative RT PCR were done using primers designed against PEDF and 18S Ribosomal RNA, to assess the downregulation of PEDF expression. Corneas were also mounted on glass slides and immunohistochemical staining was done using CD31 FITC labeled monoclonal antibodies to assess angiogenesis. Results:PEDF was suppressed by RNA interference upto the level of 75%, 80%, 73% and 75 % on 2^nd , 5^th, 8^th and 14^th day respectively after pSEC.siRNA.PEDF injection as indicated by comparison of PEDF and 18 S Ribosomal RNA gene expression in the semi quantitative RT PCR results. pBLAST VEGF 165 induced blood vessel formation spatially correlated with the injection track. Mice injected with pSEC.siRNA.PEDF or pSEC.negative control siRNA corneas did not exhibit angiogenesis. Conclusions: PEDF is not essential for the maintenance of the avascularity of the cornea, as its knockdown by RNA interference does not induce corneal angiogenesis.

Keywords: cornea: basic science • neovascularization • immunohistochemistry