Localized AAV-PEDF gene transfer reduces corneal neovascularization significantly in rabbits in vivo via selective apoptosis

Abstract

Purpose: Corneal neovascularization (CNV) is a major cause of global blindness. We hypothesized that localized AAV-Pigment Epithelium Derived Factor (PEDF) gene therapy would eliminate CNV in rabbits in vivo by restoring critical balance between pro-and anti-angiogenic factors and causing apoptosis in neovessels via Fas-Fas ligand signaling.

Methods: New Zealand White rabbits were used. Topical alkali (1N NaOH) application for 1min on the central cornea produced CNV. PEDF gene therapy was administered into rabbit stroma in vivo via topical AAV5 titer (100μl; 5x10¹²vg/ml) using defined technique: (a) 30min after alkali-burn (b) 3day after alkali-burn or (c) 1day prior to alkali-burn. Slitlamp biomicroscopy, H&E staining, stereomicroscope and immunofluorescence determined differential changes in CNV, vessels number, length and area, keratocyte density, apoptosis, and overall ocular health. Immunoblotting and qPCR quantified PEDF and Fas ligand expressions. Imaging data was analyzed with NIH ImageJ and Adobe Photoshop.

Results: Localized AAV-PEDF gene transfer into keratocytes significantly increased PEDF levels in rabbit cornea in vivo (>4 fold; p<0.01). AAV-PEDF therapy given eyes exhibited dramatically reduced vasculature, vessel density, vessel-size (length and thickness) in rabbit cornea. AAV-PEDF therapy given 30min after injury showed 86-89% (p<0.001), 3days after injury exhibited 63-69% (p<0.001), and 1day prior to injury 95% (p<0.001) in morphometric analysis. A remarkably less and small-diameter blood vessel in PEDF-delivered corneas than the no-PEDF controls were detected by H&E and immunofluorescence. Detection of 2.5 fold increased Fas-ligand (p<0.05) in PEDF-delivered rabbit corneas suggested that PEDF over-expression increases Fas-ligand significantly. Detection of double-labeled lectin+ and TUNEL+ cells in vessels suggested CNV resolution via apoptosis. Quantification of pro-and anti-angiogenic factors is underway.

Conclusions: Localized tissue-targeted AAV-PEDF gene therapy has translational potential to cure corneal neovascularization. Selective apoptosis in neovessels via Fas-Fas ligand pathway is the likely mechanism. More studies are warranted.