June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Hydroquinone Sensitizes Human RPE Cells to Basal Complement Attack by the Alternative Pathway
Author Affiliations & Notes
  • Ping Yang
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Peter Baciu
    Allergan Inc, Irvine, CA
  • Glenn Jaffe
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Footnotes
    Commercial Relationships Ping Yang, None; Peter Baciu, Allergan (F); Glenn Jaffe, Heidelberg Engineering (C), Regeneron Pharmaceuticals (F), Neurotech USA (C), Abbott (C), Psivida (F), Pfizer (C), Bayer (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1185. doi:
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      Ping Yang, Peter Baciu, Glenn Jaffe; Hydroquinone Sensitizes Human RPE Cells to Basal Complement Attack by the Alternative Pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1185.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Complement activation and oxidative stress have been increasingly implicated in the pathogenesis of AMD. Complement activation products have been detected in Bruch’s membrane and drusen from patients, indicating that in AMD, complement attack likely occurs primarily on the RPE basal surface. Herein, we describe a model used to investigate the effect of oxidative stress on complement-mediated RPE cell injury, whereby complement attack was initiated with an RPE-specific priming antibody on the basal surface of differentiated human RPE cells.

Methods: ARPE-19 cells and RPE cells from 2 donors were cultured for one month on collagen-coated porous supports in DMEM-F12 containing 1% FBS. Transepithelial resistance (TER) was recorded, apical microvilli and tight junctions were identified by transmission electron microscope, and ZO-1 was examined by immuno-fluorescent staining. Cytokeratin-18 staining was used to confirm the epithelial nature of RPE cells. The cells were basally exposed for 2 hours to media with or without hydroquinone (HQ), then basally primed with a sheep anti-ARPE-19 antibody (as a complement-fixing antibody) for 1 hour followed by a 5-hour basal incubation with C1q-depleted human serum (initiating complement attack through alternative pathway). Anti-ARPE-19 antibody binding efficiency was evaluated by Western blot. Tetrazolium salt WST-1 and lactate dehydrogenase release were used to assess cell viability and cell permeability, respectively.

Results: Cytokeratin-18 and ZO-1 were detected in ARPE-19 cells and RPE cells from 2 donors. After 3 weeks, the TER in RPE cells from the 51 year-old donor was similar to that observed in ARPE-19 cells. RPE cells from the 61 year-old donor produced a lower resistance when compared to ARPE-19 cells. Apical microvilli and tight junctions were identified after 4 weeks. Basal-delivered priming antibody bound to RPE cells. Basal complement attack slightly decreased cell viability and HQ significantly enhanced complement-mediated cell injury by increasing cell permeability and decreasing cell viability as compared to cells treated with HQ and complement alone (P≤0.05).

Conclusions: Oxidative stress enhances basal surface complement-induced RPE cell death. This approach, which more closely simulates in vivo complement attack on the RPE basal surface is useful to study the functional effects of complement and complement/oxidant interactions.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 634 oxidation/oxidative or free radical damage  
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