Effect of APNpI on VEGF expression. (A) RT-PCR products for VEGF in RPE-choroid-sclera of naïve, APNpI and PBS treated rats (n=6 rats in each group) at 10 post laser injury. APNpI decreased VEGF mRNA (327 bp) expression. A strong band for β-actin (335 bp) indicates equal amounts of RNA in each lane. (C) Densitometric analysis of PCR products. Western blot analysis of RPE-choroid tissue shows that laser treatment increases expression of VEGFA (23 kDa) in RPE-choroid compared to naïve control (B, D). Real-time PCR analysis of VEGFA mRNA expression in choroidal endothelial cells showed significant down regulation of VEGFA after APNpI (80 µg/mL) treatment (E). (F–H) Representative images of IHC staining for VEGF (green color). (I–K) Merged DIC (black and white), VEGF (green) and Isolectin IB4 (red) images helps recognize RPE cells (pigmented cells in upper part of each image) and vessels (non pigmented oval shaped structures, positively marked with Isolectin IB4 in red color) and laser injured area. Quantification of mean intensity of fluorescence in RPE (L), choroidal vessels (M) and the rest of the choroid or subretinal tissue (N) was done. VEGF expression significantly increased in all investigated structures at day 10 post laser injury (L, M, N). APNpI administration decreased expression of VEGF in RPE cells, vessels and laser injured area compared to PBS treated group. Bar for all images is 25 µm.