Phosphatidylserine (PS) is exposed in choroidal neovascular endothelium, and PS-targeting antibodies inhibit choroidal angiogenesis in vivo and ex vivo

Abstract

Purpose: Despite improved clinical outcomes in wet AMD brought about by anti-VEGF agents, results are still suboptimal. Additional drugs directed against different targets in the neovascular process may allow for combination therapies that improve efficacy and reduce treatment burden. The aminophospholipid phosphatidylserine (PS) may be such a target: it is almost exclusively localized to the inner leaflet of the cell membrane’s lipid bilayer in normal cells, but becomes exposed on the outer membrane in tumor neovasculature. Our goals were to: 1) determine if there is exposure of PS in choroidal neovascularization (CNV), and 2) evaluate if PS-targeting antibodies can treat CNV.

Methods: CNV was induced in B6J mice using a laser model. First, to determine if PS was exposed in the vascular endothelium of CNV lesions, we stained for PS and for ICAM-2. Confocal fluorescence microscopy was used to evaluate stained CNV lesions in RPE-choroid-scleral flat mounts, and fluorescence microscopy was used to evaluate stained CNV cross-sections. We then tested whether PS-targeting drugs could inhibit CNV growth. After induction of CNV, mice were treated either with intravitreal or intraperitoneal injections of PS-targeting antibodies vs control antibodies. CNV lesion size was then determined by ICAM-2 staining. Finally, a choroidal sprouting assay was used to determine the effect of PS targeting antibodies in a non-laser model of choroidal angiogenesis.

Results: We were able to demonstrate exposure of PS in CNV lesions, and its absence in normal choroidal and retinal vessels. PS-staining had a vascular pattern, and co-localized with ICAM-2 staining, demonstrating that PS was exposed on the choroidal neovascular endothelium. Two different PS-targeting antibodies showed a strong and reproducible inhibition of CNV when administered either intravitreally or i.p (p<0.01 vs. control). This anti-neovascular effect was also corroborated by a reduction (P<0.05) in choroidal sprouting in a non-laser ex vivo model. We also report that CNV lesions treated with PS-targeting antibodies accumulate increased density of microglia (P<0.05).

Conclusions: PS is exposed in choroidal neovasculature in both laser-induced CNV and ex vivo choroidal sprouting models. PS-targeting antibodies may be of therapeutic value in CNV.