May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Complement Regulatory Protein CD46/MCP Inhibits Complement Activation and VEGF Release From Human Retinal Pigmental Epithelial Cells (RPE)
Author Affiliations & Notes
  • H. Cai
    Ophthalmology, Columbia University Medical Center, New York, New York
  • H. J. Kaplan
    Ophthalmology, University of Louisville, Louisville, Kentucky
  • T. H. Tezel
    Ophthalmology, University of Louisville, Louisville, Kentucky
  • L. V. Del Priore
    Ophthalmology, Columbia University Medical Center, New York, New York
  • Footnotes
    Commercial Relationships  H. Cai, None; H.J. Kaplan, None; T.H. Tezel, None; L.V. Del Priore, None.
  • Footnotes
    Support  Research to Prevent Blindness, Robert L. Burch III Fund, Retina Society, Hickey’s Family Foundation and the Foundation Fighting Blindness.
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 5160. doi:https://doi.org/
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    • Get Citation

      H. Cai, H. J. Kaplan, T. H. Tezel, L. V. Del Priore; Complement Regulatory Protein CD46/MCP Inhibits Complement Activation and VEGF Release From Human Retinal Pigmental Epithelial Cells (RPE). Invest. Ophthalmol. Vis. Sci. 2008;49(13):5160. doi: https://doi.org/.

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

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Abstract

Purpose: : Recent attention has been focused on the role of the complement system in the pathogenesis of age-related macular degeneration (AMD). CD46 is a complement regulatory protein located on the basal surface of the retinal pigment epithelium; the gene for CD46 is located at the same sub-band on the chromosome (1q32) as complement factor H (CFH) within a complex of immunoregulatory genes. The purpose of this study is to determine the effects of CD46 on complement activation and its implications on angiogenesis.

Methods: : Human ARPE19 cells were cultured to confluence in a cell culture insert. CD46 and CFH siRNAs were designed and synthesized from commercial sources and used to knock down CD46 or CFH protein expression in ARPE19. Amine was used to transfect the siRNA oligos into cultured ARPE19. After 48 hours of CD46 siRNA transfection, we collected fluid from the apical and basal surface of the ARPE19 monolayer and fixed the monolayer itself for immune cytochemistry. VEGF levels were determined by ELISA; fixed samples were immune stained for CD46 and VEGF. Complement activation assay were used to monitor the effect of knockdown of CD46 protein expression. Various signal transduction pathway related antibodies and DNA microarray of CD46-and CFH- knockdown-RPE samples were used to elucidate the possible molecular mechanism.

Results: : After 48 hours of siRNA transfection, CD46 protein expression in ARPE19 was decreased to 60% of the control level. CD46 knockdown ARPE19 increased complement activation by two-fold. CD46 knockdown ARPE19 also increased the protein expression and secretion of VEGF on the basal side of ARPE19 culture. The analyses of gene expression profiles between CD46- and CHF-knockdown ARPE19 point to alterations in various cellular pathways for regulating cell proliferation, immune responses and angiogenesis.

Conclusions: : The results suggest that CD46 on RPE cells may play an important role in regulating the activation of complement and the homeostasis of angiogenesis to maintain the normal function of the retina. Alterations in CD46 may lead to secondary changes in the levels of VEGF on the basal side of the RPE, and may thus contribute to the pathogenesis of choroidal neovascularization.

Keywords: retinal degenerations: cell biology • immunomodulation/immunoregulation • retinal pigment epithelium 
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