Frizzled-4 Is Required For Retinal Angiogenesis And Maintenance Of The Blood-Retina Barrier

Abstract

Purpose: : Mice deficient in the secreted protein NORRIN or its receptor FRIZZLED-4 (FZD4) exhibit incomplete vascularization of the neural retina. However, due to early retinal vascular defects in the knockout models it has not been possible to study FZD4 contribution in ocular neovascular disease. To further understand the role of this signaling pathway in physiological and pathological angiogenesis, we generated a monoclonal antibody that neutralizes FZD4 function in vivo.

Methods: : Antibodies were generated by immunizing FZD4 knockout mice with the FZD4 ectodomain. A monoclonal antibody (1.99.25) was discovered that antagonizes NORRIN-induced β-catenin accumulation in vitro. 1.99.25 and an isotype matched negative control antibody were evaluated in models of developmental retinal angiogenesis (DRA), oxygen-induced retinopathy (OIR), retinal angiomatous proliferation (RAP) and laser-induced choroidal neovascularization (CNV). We also investigated the role of FZD4 in maintaining the blood-retina barrier in normal adult mice.

Results: : Administration of 1.99.25 to wild type newborn pups recapitulated the FZD4 knockout phenotype. In the in vivo models tested, 1.99.25 inhibited both physiological and pathological angiogenesis within the retina but did not affect pathological angiogenesis in the choroid. Inhibition of FZD4 in developing vascular networks resulted in up-regulation of PLVAP, a marker of fenestrated vascular endothelium that is normally lost upon assumption of tight barrier function. In the adult neural retina, treatment with 1.99.25 reinstated PLVAP expression in the deep capillary bed and enabled passage of small and large molecules through the blood-retina barrier.

Conclusions: : These results demonstrate that FZD4 is required for physiological and pathological angiogenesis in the retina and regulation of retinal endothelial cell differentiation. Furthermore, we show that FZD4 is critical in maintaining the integrity of the mature blood-retina barrier.

Keywords: retinal neovascularization • development • proliferative vitreoretinopathy