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Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model

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Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model Subretinal pigment epithelial deposition of drusen components including hydroxyapatite in a primary cell culture model Pilgrim, M. G., Lengyel, I., Lanzirotti, A., Newville, M., Fearn, S., Emri, E., Knowles, J. C., Messinger, J. D., Read, R. W., Guidry, C., & Curcio, C. A. (2017). Subretinal pigment epithelial deposition of drusen components including hydroxyapatite in a primary cell culture model. Investigative Ophthalmology and Visual Science, 58(2), 708-719. https://doi.org/10.1167/iovs.16-21060 Published in: Investigative Ophthalmology and Visual Science Document Version: Publisher's PDF, also known as Version of record Queen's University Belfast - Research Portal: Link to publication record in Queen's University Belfast Research Portal Publisher rights Copyright 2017 the authors. This is an open access article published under a Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits distribution and reproduction for non-commercial purposes, provided the author and source are cited. General rights Copyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights. Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact [email protected]. Download date:30. Sep. 2021 Retinal Cell Biology Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model Matthew G. Pilgrim,1,2 Imre Lengyel,1,3 Antonio Lanzirotti,4 Matt Newville,4 Sarah Fearn,5 Eszter Emri,1,3 Jonathan C. Knowles,2 Jeffrey D. Messinger,6 Russell W. Read,6 Clyde Guidry,6 and Christine A. Curcio6 1UCL Institute of Ophthalmology, University College London, London, United Kingdom 2Division of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, University College London, London, United Kingdom 3Centre for Experimental Medicine, School of Medicine, Dentistry and Biomedical Science, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom 4Center for Advanced Radiation Sources, The University of Chicago, Chicago, Illinois, United States 5Department of Materials, Imperial College London, London, United Kingdom 6Department of Ophthalmology, University of Alabama School of Medicine, Birmingham, Alabama, United States Correspondence: Christine A. Cur- PURPOSE. Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals cio, Department of Ophthalmology, that form between the basal lamina of the RPE and the inner collagenous layer of Bruch’s EyeSight Foundation of Alabama Vi- membrane are hallmarks of early AMD. We examined whether cultured RPE cells could sion Research Laboratories, 1670 produce extracellular deposits containing all of these molecular components. University Boulevard, Room 360, University of Alabama School of METHODS. Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were Medicine, Birmingham, AL 35294- cultured on Transwell membranes for up to 6 months. Deposit composition and structure 0019, USA; were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray [email protected]. diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohisto- chemistry. CG and CAC are joint senior authors. RESULTS. Apparently functional primary RPE cells, when cultured on 10-lm-thick inserts with MCP and IL are joint first authors. 0.4-lm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, Submitted: November 4, 2016 proteins, and trace elements, without outer segment supplementation, by 12 weeks. Accepted: December 22, 2016 CONCLUSIONS. The data suggest that sub-RPE deposit formation is initiated, and probably Citation: Pilgrim MG, Lengyel I, Lan- regulated, by the RPE, as well as the loss of permeability of the Bruch’s membrane and zirotti A, et al. Subretinal pigment choriocapillaris complex associated with age and early AMD. This cell culture model of early epithelial deposition of drusen com- AMD lesions provides a novel system for testing new therapeutic interventions against sub- ponents including hydroxyapatite in a RPE deposit formation, an event occurring well in advance of the onset of vision loss. primary cell culture model. Invest Keywords: hydroxyapatite, lipids, x-ray fluorescence, secondary ion mass spectrometry, x-ray Ophthalmol Vis Sci. 2017;58:708– 719. DOI:10.1167/iovs.16-21060 diffraction ge-related macular degeneration (AMD), the third largest and BLinD confer risk for progression to neovascularization and A cause of vision loss globally,1 is expected to affect 288 RPE cell death.6 Therefore, a strategy to prevent progression to million individuals worldwide by 2040.2 The end stages of AMD late AMD is to modulate deposit formation and clearance. A are geographic atrophy and neovascular AMD. At present, there is better understanding of the molecular events leading to the no treatment for geographic atrophy.3 End-stage neovascular retention of drusen components is essential for the success of AMD can be treated with regular injections of VEGF inhibitors,4 a this strategy. A model system that recapitulates as many aspects treatment that is costly to health care systems, burdensome for of drusen and BLinD formation as possible would accelerate patients,5 and possibly contributory to further atrophy.4 There- progress toward these goals. fore, novel therapeutic intervention strategies should target early Sources of the molecular components of drusen and BLinD stages of AMD to prevent progression to advanced stages. are not yet established, although candidates exist. An early Early AMD is characterized by the presence of hallmark hypothesis suggests that these lesions are formed from lesions that form between the basal lamina of the RPE and the byproducts of photoreceptor outer segment phagocytosis by inner collagenous layer of Bruch’s membrane (BrM). These sub- the RPE.7 However, it was later postulated that a lipid-recycling RPE deposits can be focal (drusen) or diffuse (basal linear pathway, driven by diet, of RPE-secreted lipoproteins is deposit [BLinD]) and are distinct from a diffuse thickening of responsible.8 Johnson et al.9 showed that highly differentiated the RPE basal lamina called basal laminar deposits (BLamD). and well-polarized primary human foetal RPE cells (hfRPE) Although drusen and BLinD are minimally present in normal secreted several deposit-associated proteins without exposure aged human eyes, they are abundant in eyes with AMD. Drusen to outer segments or choroidal blood flow. This supports the Copyright 2017 The Authors iovs.arvojournals.org j ISSN: 1552-5783 708 This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/936033/ on 02/01/2017 Drusen in a Dish IOVS j February 2017 j Vol. 58 j No. 2 j 709 idea that some components of deposits originate from the RPE Scientific, Waltham, MA, USA) with Epithelial Cell Medium and/or components derived from the culture medium. (EpiCM) (ScienCell), completed with 2% fetal bovine serum Proteomic analysis of isolated human drusen also implicated (ScienCell), 1:100 mL/mL epithelial cell growth supplement circulating plasma as a protein source.10 (ScienCell), and 107 U/L penicillin/10 g/L streptomycin Regardless of source(s), it is important to understand what (ScienCell). For our experiments, cells at passage 2 were triggers the retention of lipids and proteins in the sub-RPE cultured either on 10-lm-thick Transwell inserts with a pore space. One potential mechanism is binding of RPE-produced diameter of 0.4 lm (Corning; Thermo Fisher Scientific) coated lipids and lipoproteins to extracellular matrix elements with Geltrex extracellular matrix (Thermo Fisher Scientific) or including proteoglycans in BrM, a mechanism similar to plasma on 100-lm-thick Transwell inserts with a pore diameter of 0.45 lipoproteins binding to arterial intima in atherosclerosis.11 A lm (Millipore; Thermo Fisher Scientific) coated with laminin second mechanism is retention of proteins via formation of (Sigma-Aldrich Corp., St. Louis, MO, USA), in MEM-a modifica- large oligomers,12 a process that is accelerated in the presence tions as previously described.22 of trace metals like zinc.13,14 A third mechanism is binding of proteins to the hydroxyapatite (HAP) spherules recently Immunohistochemistry of Porcine Cells identified in the sub-RPE space and in drusen of humans.15 Any of these or a combination may be operative. Immunofluorescence was performed on flat mount and cross Published cell culture models for sub-RPE deposit forma- sections of porcine RPE cells using methods for cells on 21 tion9,16,17 each identified some, but not all, of the major coverslips. Cross sections shown in Figure 1 were 10 lm molecular constituents of human sub-RPE deposits, lipids,18,19 thick and were generated using a cryostat (Leica CM 3050 S; hydroxyapatite,15 trace elements,13,14 and proteins,10,20
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