Activated Protein C Induces Stabilization of the Outer Retinal Barrier and Inhibits Laser Induced Choroidal Neovascularization in a Murine Model
Authors: Livnat Tami , Nisgav Yael , Budnik Ivan , Weinberger Yehonatan , Dardik Rima , Kenet Gili , Weinberger Dov 
Published: October 2019

Abstract
Introduction: Age-related macular degeneration (AMD) is considered one of the main causes of severe vision loss in older adults. Wet AMD is characterized by choroidal neovascular membrane developed under or into the sensory retina, breaking the outer blood-retina barrier (oBRB) and causing leakage and bleeding. Protein C (PC) is a plasma serine protease zymogen whose active form, activated protein C (APC), exerts diverse homeostatic cell signaling pathways apart from its anticoagulant activity. APC activities are mediated mainly through endothelial PC receptor (EPCR) and protease activated receptor (PAR)-1. APC was recently shown to act as a novel ligand for Tie2, a tyrosine kinase receptor playing an important role in angiogenesis. Thus, APC may modulate pathological angiogenesis in relevant ocular conditions, such as choroidal neovascularization (CNV), via its interaction with Tie2. To the best of our knowledge, there are no studies addressing the potential use of APC as oBRB stabilizer and CNV inhibitor.
Aim: To assess the protective effect of APC as a retinal barrier stabilizer and its ability to reduce CNV growth, leakage and penetration from the choroid into the sensory retina. Moreover, to study the role of Tie2 receptor upon APC mode of action in the retina.
Methods: CNV development was induced in C57BL/6J mice by laser applications. Immediately following CNV induction APC (0.1-10 µg/µl) was injected intravitrealy. Seven to 14 days later, the retina was separated from the underlying tissues and the 2 segments were flattened: sclera-choroid-retinal pigment epithelium (RPE) segment and the photoreceptors-sensory retina segment. Blood vessels were stained using CD31 (an endothelial marker) or FITC-dextran and analyzed by using a confocal microscopy. Fluorescein angiography (FA) was performed to detect leakage from CNV. The ability of APC to stabilize the oBRB and prevent leakage was studied using ARPE-19 tissue culture (simulating the oBRB). FITC-Dextran leakage through the cultured cell monolayers was measured and the tight junction protein, Zonula occludens-1 (ZO1) cellular distribution was followed using immunostaining. Tie2 Kinase inhibitor and anti-Tie2 antibodies were used for Tie2 inhibition in the murine and cell culture models, respectively.
Results: Quantitative analysis of the CNV area and volume revealed that 7-14 days after laser applications, APC treatment resulted in a statistically significant reduction in CNV growth, area and volume as compared to saline. Moreover, APC inhibited the penetration of CNV from the choroid throughout the RPE into the sensory retina in a dose-dependent manner. Figure 1 summarize the quantitative assessment of APC suppression of blood vessel penetration from the choroid (Fig.1A) to the sensory retina segment (Fig.1B). FA analysis showed that APC significantly suppressed the leakage of fluorescein from the laser lesion and preserved the retinal architecture. Incubation of RPE cells with APC resulted in dose dependent translocation of ZO1 to the cell membrane region, accompanied by reduction in permeability throughout the RPE monolayer. ZO1 translocation formed a typical ZO1 honeycomb pattern that is characteristic for the stabilization of tight junctions (Fig.1C). Inhibition of Tie2, in the laser induced CNV mice model and in RPE cell culture, significantly decreased APC protective activities in both models.
Conclusions: Several Chorio-retinal pathologies are accompanied by damage to the oBRB and pathological growth of CNV. Our present data provide evidence that APC can protect the choroid and the retina from CNV growth, can suppress leakage from the newly formed choroidal blood vessels and may act as oBRB stabilizer. The protective function of APC in the eye provides direction for new strategies to treat chorio-retinal diseases affected by abnormal oBRB function and CNV growth. Our preliminary results warrant further investigation to extend the data regarding the potential use of APC as a novel anti-CNV treatment and oBRB protector.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.