title: Bruch's membrane aging decreases phagocytosis of outer segments by retinal pigment epithelium.
abstract: OBJECTIVE
  We have shown previously that aging of human Bruch's membrane affects the attachment, survival and gene expression profile of the overlying retinal pigment epithelium (RPE). Herein we determine the effects of Bruch's membrane aging on RPE phagocytosis of rod outer segments.
  
  
  METHODS
  Explants of human Bruch's membrane were prepared from cadaver donor eyes (aged 9-81years) within 48 h of death, and 6 mm punches were embedded with the basal lamina in a 96-well plate. Approximately 50,000 ARPE-19 cells per well were seeded onto the explant surface and cultured for two weeks until they reached confluence. In addition, ARPE-19 were also seeded onto RPE-derived extracellular matrix (RPE-ECM) that was unmodified or modified by nonenzymatic nitration. Bovine rod outer segments were purified by sucrose gradient centrifugation, labeled with 10 ug/ml fluorescein isothiocyanate, and added to ARPE-19 cultured on Bruch's membrane or RPE-ECM for 24 h. Phagocytic activity was quantified by flow cytometry of harvested cells.
  
  
  RESULTS
  The ability of RPE to phagocytose rod outer segments decreased as a function of aging of Bruch's membrane; mean phagocytotic activity of ARPE-19 on younger Bruch's membrane was significantly higher than on older Bruch's membrane (129.7 +/- 34.8 versus 67.4 +/- 4.2 arbitrary units, respectively; p<0.01). Nitrite treatment of RPE-ECM decreased rod outer segment phagocytosis compared to untreated RPE-ECM and mimicked the effects of aging of human Bruch's membrane.
  
  
  CONCLUSIONS
  Aging of human Bruch's membrane decreases rod outer segment phagocytosis by ARPE-19. This effect can be mimicked by nonenzymatic nitration of extracellular matrix in vitro. Our observations may have implications for understanding the role of aging changes within Bruch's membrane on pathogenesis of age-related macular degeneration and other disorders.
authors.affiliation: Department of Ophthalmology, Harkness Eye Institute, Columbia University, New York, NY 10032, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Sun
authors.forename: Hui
authors.initials: H
authors.lastname: Cai
authors.forename: Tongalp H
authors.initials: TH
authors.lastname: Tezel
authors.forename: David
authors.initials: D
authors.lastname: Paik
authors.forename: Elizabeth R
authors.initials: ER
authors.lastname: Gaillard
authors.forename: Lucian V
authors.initials: LV
authors.lastname: Del Priore
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000375:Aging / Q000502:physiology; D000818:Animals; D016570:Bruch Membrane / Q000166:cytology / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects / Q000502:physiology; D002648:Child; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D009566:Nitrates / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*
chemical_list: D009566:Nitrates