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K. Sun, H. Cai, T. H. Tezel, H. J. Kaplan, L. V. Del Priore; Reengineering Human Bruch’s Membrane Increases Rod Outer Segment Phagocytosis by Human Retinal Pigment Epithelium (RPE). Invest. Ophthalmol. Vis. Sci. 2007;48(13):2181.
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We have shown previously that aging of human Bruch’s membrane decreases RPE phagocytosis of rod outer segments; in separate studies, we demonstrated that reengineering human Bruch’s membrane with detergent cleaning and subsequent coating with extracellar matrix (ECM) ligands increases the ability of cultured RPE to attach to and repopulate the inner aspects of human Bruch’s. Herein we determine the effect of cleaning with or without subsequent addition of extracellular matrix (ECM) ligands to Bruch’s membrane on the phagocytic function of RPE.
Explants of aged Bruch's membrane (donor > 55 years) were cleaned with Triton X-100; some explants were then coated with a mixture of ECM ligands [laminin (330 microg/mL), fibronectin (250 microg/mL), and vitronectin (33 microg/mL)]. ARPE-19 cells were plated onto Bruch’s membrane and cultured to confluence for up to 14 days. Adult bovine rod outer segments (ROS) purified by sucrose gradient centrifugation were labeled with 10 microg /ml fluorescein isothiocyanate and fed to the RPE. RPE were examined by fluorescence microscopy and flow cytometry to quantify ROS phagocytosis.
The uptake rates of ROS between untreated and Triton-X treated explants are similar (54.9 + 6.18 vs. 57.2 + 8.48 arbitrary units, respectively; p> 0.05). The highest phagocytosis rate is observed after combined cleaning and ECM protein coating (83.5 + 6.45 arbitrary units; p < 0.05 compared to untreated or Triton-X cleaned explants).
Detergent cleaning alone of older Bruch’s membrane does not increase the ability of RPE to phagocytose rod outer segments, but ECM protein coating preceded by cleaning increases phagocytosis significantly. Our results suggest aging of human Bruch’s membrane decreases RPE phagocytosis of ROS, indicating that critical RPE functions are modulated by age-related alterations of the Bruch’s membrane. Herein, we demonstrated that rejuvenation of the aged Bruch’s membrane can restore RPE phagocytic function.
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