Vascular normalization: A novel method for inhibiting vascular leakage in age-related macular degeneration

Abstract

Purpose: Fas ligand (FasL) has been shown to inhibit neovascularization by inducing apoptosis in Fas+ endothelial cells. However, using a knock-in mouse in which the FasL cleavage sites were mutated to prevent cleavage of mFasL (ΔCS mice) we previously demonstrated that mFasL alone neither inhibits CNV lesions, nor prevents vascular leakage in a mouse model of laser-induced CNV. Unexpectedly, we observed that sFasL is also required for inhibition of vascular leakage.The current studies elucidate the mechanism by which the two forms of FasL work together to inhibit vascular leakage in a mouse model of laser-induced CNV.

Methods: Laser CNV was induced in WT mice (B6.FasL^+/+) that express low mFasL and low sFasL, and B6.FasL^ΔCS/+ mice which are heterozygous for the knock-in mutation and thus express more mFasL and less sFasL as compared to WT mice. Vascular leakage was assessed by fluorescein angiography and CNV lesion size by optical coherence tomography. Vessel maturation was assessed in choroidal whole mounts perfused with FITC dextran and stained for collagen IV, CD31 (endothelial cells), and NG2 (pericytes). Quantitative PCR was performed for pro-and antiangiogenic factors. Choroidal explant matrigel cultures were used to examine sprouting and maturation of choroidal vessels.

Results: Reduced vascular leakage was observed at all time points in B6.FasL^ΔCS/+ mice as compared to WT mice. Confocal analysis revealed well perfused and highly organized vessels in CNV lesions of B6.FasL^ΔCS/+ mice and staining with CD31 and NG2 revealed increased co-localization of endothelial cells and pericytes. The mature vascular phenotype in B6.FasL^ΔCS/+ mice correlated with increased mRNA expression of PDGFβ, PDGFrβ, and TGFβ1, factors that promote vessel stabilization via recruitment and differentiation of pericytes. In addition, a reduced VEGF/PEDF ratio was observed in in B6.FasL^ΔCS/+ mice as compared to WT mice. Choroidal explants from B6.FasL^ΔCS/+ mice also showed accelerated outgrowth of vessels with a highly reticular phenotype and increased co-localization of CD31 with NG2.

Conclusions: The optimal ratio of mFasL and sFasL (high mFasL/low sFasL) promotes vessel maturation and prevents vascular leakage via accelerated recruitment and differentiation of pericytes and reduced VEGF/PEDF ratio. These data support vascular normalization as a novel approach to preventing vascular leakage in AMD.