Reengineering Human Bruch's Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium.

Abstract

We have shown previously that Bruch's membrane (BM) aging decreases retinal pigment epithelium (RPE) phagocytosis. Herein, we determine the effects of BM reengineering on RPE phagocytosis. BM explants were dissected from young and old donor eyes. Some old BM explants were reengineered by cleaning with Triton X-100 and/or coating with extracellular matrix (ECM) ligands. ARPE-19 cell-derived ECM (ARPE-ECM) modified ("aged") by sodium nitrite was subjected to similar treatments. ARPE-19 cells were then cultured to confluence onto the different surfaces. Fluorescently-labeled bovine rod outer segments (ROS) were fed to cells with or without αVβ5 integrin antibody. Image acquisition and phagocytosis quantification was performed by fluorescence microscopy and ImageJ analysis. Cleaning old donor-derived BM with detergent does not increase the uptake of ROS, but a combination of cleaning and coating with ECM ligands significantly increases RPE phagocytosis (54.9 ± 6.2 vs. 83.5 ± 6.5 arbitrary units; P < 0.05) to levels closer to young donor BM (123.6 ± 9.9 arbitrary units). Similar effects were observed on nitrite-modified ARPE-ECM subjected to the same treatments. Incubation of αVβ5 blocking antibody with ROS significantly decreased RPE phagocytosis. The detrimental effects of aging BM on RPE phagocytosis can be reversed by reengineering the BM surface with detergent cleaning and recoating with ECM ligands. These results demonstrate that the therapeutic success of transplanted RPE cells may require, at least in part, reengineering of diseased BM to make it a more suitable environment for attachment, survival and proper functioning of the RPE.