April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
Complement Attack by the Alternative Pathway Increases Gene Expression and Protein Expression of ER Stress Markers in Human RPE Cells
Author Affiliations & Notes
  • Angel Long
    Duke University School of Medicine, Durham, NC
  • Michelle Bao
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Ping Yang
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Glenn J Jaffe
    Ophthalmology, Duke University Eye Center, Durham, NC
  • Footnotes
    Commercial Relationships Angel Long, None; Michelle Bao, None; Ping Yang, None; Glenn Jaffe, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5994. doi:https://doi.org/
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      Angel Long, Michelle Bao, Ping Yang, Glenn J Jaffe; Complement Attack by the Alternative Pathway Increases Gene Expression and Protein Expression of ER Stress Markers in Human RPE Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5994. doi: https://doi.org/.

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

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Abstract

Purpose: We have previously shown that oxidative stress and alternative pathway (AP) complement attack, both of which have been implicated in age-related macular degeneration (AMD), synergistically damage RPE cells. While oxidative stress has been shown to induce ER stress in RPE cells, the role of complement activation in ER stress has not yet been explored. This study was undertaken to clarify the role of AP complement activation on ER stress in RPE cells.

Methods: Cultured RPE cells were incubated with serum-free MEM or 125 uM hydroquinone (HQ) for 1.5 hours, primed with a sheep anti-ARPE-19 antibody (Allergan, Inc.) for 30 minutes, and then incubated in either 6% C1q-depleted human serum or 6% heat inactivated normal human serum for 2, 3, 4.5, and 6 hours. Quantitative RT-PCR was performed to test the ER stress markers CHOP, PERK, and GRP78 gene expression levels. Western blot was performed to compare the protein expression of CHOP in cells primed with the anti-ARPE-19 antibody to that of untreated age-matched cells at 2, 3 and 6 hours.

Results: Both CHOP and PERK gene expression at 2 and 3 hours was up-regulated over 2-fold and over 4-fold after treatment with AP complement attack alone and in combination with HQ 125 uM, respectively. By 4.5 hours, the up-regulation of these genes had decreased. GRP78 gene expression increased at 3 hours, and its up-regulation persisted at 4.5 hours. CHOP protein levels were increased over untreated controls by AP complement attack alone and in combination with HQ 125 uM. These increased protein levels were diminished by 6 hours.

Conclusions: AP complement attack on human RPE cells, either alone or in combination with hydroquinone, increases ER stress marker gene expression and protein levels. The effect of AP complement activation and HQ on ER stress markers was additive. These results suggest that oxidant-enhanced AP complement-mediated ER stress marker activation may induce RPE cell death and contribute to AMD.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium • 533 gene/expression  
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