Cleavage of Fas Ligand Regulates Vessel Leakage in Laser Induced CNV

Abstract

Purpose: : In age-related macular degeneration (AMD) choroidal neovascularization (CNV) is the main cause of severe visual loss, in part due to vessel leakage that causes retinal edema. Fas ligand (FasL) has been shown to be an important inhibitor of neovascularization by inducing apoptosis in Fas+ endothelial cells. However, FasL is a membrane-bound protein (mFasL) that can be cleaved by metalloproteases to release a soluble form (sFasL). Membrane FasL is pro-apoptotic and pro-inflammatory, while sFasL is anti-apoptotic and anti-inflammatory. FasL is constitutively expressed in immune privileged sites and within the eye is found in the retina and RPE cells. Preventing FasL cleavage by treating with doxycyline to inhibit MMPs has been shown to reduce CNV. We extended these studies by examining the effect of FasL cleavage in vessel leakage using a membrane-only FasL mouse (ΔCS.1) in which the FasL metalloproteinase cleavage sites in exon 2 was mutated. ΔCS.1 mice only express mFasL and no sFasL.

Methods: : A mouse model of laser-induced CNV was used in which four laser spots were placed in each quadrant using a red krypton laser. Groups of WT B6 and ΔCS.1 mice received laser treatment and at days 5 and 14 post-treatment high-resolution SD-OCT and fluorescein angiography (FA) were performed. Choroidal flat-mounts and retinal sections were analyzed histologically.

Results: : Laser-induced damage led to significantly increased vessel leakage in WT, but not in mFasL- only mice, as observed by FA 5 and 14 days post treatment. Reduced vessel leakage was not due to a smaller retinal lesion in mFasL- only mice, since SD-OCT analysis revealed equal size lesions in WT and mFasL- only mice.

Conclusions: : Laser treatment of ΔCS.1 mice that express only mFasL resulted in significantly less vessel leakage. These data indicate that cleavage of FasL and production of sFasL is pathogenic and contributes to vessel leakage and retinal edema.

Keywords: age-related macular degeneration • choroid: neovascularization • inflammation