The protective effect of activated protein C (APC) on cell permeability and laser-induced CNV progression

Abstract

Purpose : Choroidal neovascularization (CNV) is the leading cause of severe vision loss in various ocular diseases such as age-related macular degeneration (AMD), angioid streaks and high myopia. A rupture of Bruch’s membrane which follows by a damage to the blood retinal barrier (BRB) induces CNV formation and progression. Activated protein C (APC) is a plasma serine protease with systemic anticoagulant, anti-inflammatory and antiapoptotic activity. Our aim is to explore the barrier protective effect of APC treatment in both an in vitro and a laser-induced CNV animal models.

Methods : In vitro model: human retinal pigment epithelial cells (ARPE-19) were cultured for a month to achieve definite polarity properties. APC-induced cell permeability was evaluated based on spectrophotometric monitoring of the transport across the cell layer of labeled dextran. Cellular localization of the tight junction protein Zonula Occludens 1 (ZO-1) was studied using immunofluorescence staining with anti-ZO1 antibody. In vivo model: CNV was induced by indirect diode laser photocoagulation on male C57BL/6J mice. Immediately following injury mice were injected intravitreally with 1µl APC at 1µg/animal or 1µl bevacizumab at 25 µg/animal. CNV area of flat choroid was determined by using anti CD31 antibody immunofluorescence staining on days 5 and 14 post laser injury. CNV area was evaluated by image J.

Results : In vitro: APC induced translocation of ZO-1 protein to the ARPE-19 cell membrane and reduced RPE permeability as compared to untreated cells. In vivo: APC treatment dramatically reduced CNV area. The CNV area on days 5 and 14 post laser application, as indicated by CD31 staining was 34% and 50%, respectively as compared to untreated control eyes. APC effect was to a similar extant as compared to bevacizumab outcome.

Conclusions : APC was found to reduce cell permeability in RPE cells, and change the re-organization of ZO1 into a membranal position. This is the first study to show that intravitreal injection of APC lead to a significant reduction in CNV area compared to the treatment of choice, bevacizumab. This study offers an innovative approach that may lead to the development of novel therapeutic strategies targeting CNV. APC should be considered as a good candidate for such strategies though future studies are warrant to fully explore its beneficial potential.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.