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

Abstract

Purpose: 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.Methods: 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 aVb5 integrin antibody. Image acquisition and phagocytosis quantification was performed by fluorescence microscopy and ImageJ analysis.Results: 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 6.2 vs. 83.5 6 6.5 arbitrary units; P , 0.05) to levels closer to young donor BM (123.6 6 9.9 arbitrary units). Similar effects were observed on nitrite-modified ARPE-ECM subjected to the same treatments. Incubation of aVb5 blocking antibody with ROS significantly decreased RPE phagocytosis. Conclusions: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.Translation Relevance: 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.

Stem cell-derived retinal pigment epithelium from patients with age-related macular degeneration exhibit reduced metabolism and matrix interactions

“…We devised a model for age‐related changes to Bruch's membrane that is based on nonenzymatic nitration of extracellular matrix (ECM) that allows one to study RPE dysfunction in a compromised subretinal environment. The tissue culture method was validated ex vivo using aged or diseased Bruch's membrane …”

“…The tissue culture method was validated ex vivo using aged or diseased Bruch's membrane. 19,[32][33][34][35] A new generation of culture models uses human-induced pluripotent stem cells (iPSCs) that are generated from adult somatic cells harvested from patients with AMD and other macular dystrophies. 29,36,37 These studies have shown that complement and inflammatory markers are upregulated in iPSC-derived RPE cells from patients with macular degenerations.…”

“…Preparation of RPE cell-derived ECM and nitrite-modified ECM RPE cell-derived ECM plates were prepared from ARPE-19 cells (used within five passages of obtaining them from the American Type Culture Collection (ATCC; Manassas, Virginia, www.atcc.org), as described previously 26,32,35. ECM on 96-well plates were used to create two experimental plating surfaces (nontreated ECM and nitritemodified ECM).…”

Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD)

“…For example, aging of human BM, or damage induced by surgical manipulation of choroidal neovascularization, can reduce the ability of transplanted RPE cells to attach to human BM, survive after transplantation, and proliferate to repopulate this structure [42,45,48,49]. Aging of human BM also reduces the ability of human RPE cells seeded on this surface to ingest rod outer segments [50]. Thus, it is clear that in the advanced stages of AMD, namely GA, age-related changes to BM can adversely affect the successful transplantation of cells.…”

“…Thus, it is clear that in the advanced stages of AMD, namely GA, age-related changes to BM can adversely affect the successful transplantation of cells. Interestingly, we have previously shown that non-enzymatic nitration of the basement membrane is a relevant model system to study BM pathology and can affect RPE dysfunction, such as altered VEGF secretion, phagocytic ability, and expression of complement regulatory proteins in a manner that mimics the effects of BM aging in AMD [50,51,52,53]. We have further shown that cleaning and coating the surface of BM or nitrite-modified extracellular matrix (ECM) with such proteins as laminin, fibronectin, and vitronectin can reverse the effects of damage associated with an aged and/or diseased BM [52,54].…”