June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Cytoplasmic Hybrids of ARPE-19 Cells and Mitochondria from Patients with Age-related Macular Degeneration Accurately Model Reactive Oxygen Species Hallmarks Found In Vivo
Author Affiliations & Notes
  • Jacob Dohl
    Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, United States
  • Kevin Schneider
    Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, United States
  • Shari Atilano
    Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, United States
  • Alan Bao
    Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, United States
  • M.Cristina Kenney
    Pathology and Laboratory Medicine, University of California Irvine, Irvine, California, United States
  • Footnotes
    Commercial Relationships   Jacob Dohl None; Kevin Schneider None; Shari Atilano None; Alan Bao None; M.Cristina Kenney None
  • Footnotes
    Support  National Eye Institute R01 EY027363
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 479 – A0016. doi:
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      Jacob Dohl, Kevin Schneider, Shari Atilano, Alan Bao, M.Cristina Kenney; Cytoplasmic Hybrids of ARPE-19 Cells and Mitochondria from Patients with Age-related Macular Degeneration Accurately Model Reactive Oxygen Species Hallmarks Found In Vivo. Invest. Ophthalmol. Vis. Sci. 2022;63(7):479 – A0016.

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

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Abstract

Purpose : Age-related Macular Degeneration (AMD) is the leading cause of vision impairment among the elderly in the United States, necessitating novel therapies to prevent vision loss; however, AMD research lacks accessible in vivo and in vitro models. Cytoplasmic hybrids (cybrids), generated through fusing mitochondria-rich platelets from AMD patients with mitochondrially-depleted retinal pigment epithelial cells (ARPE-19), provide a novel and accessible in vitro model for AMD research. A major hallmark of AMD is an increase in reactive oxygen species (ROS). We hypothesized the AMD cybrids would show elevated ROS levels as well as downstream effects of ROS, including mitochondrial DNA (mtDNA) damage, and elevated anti-inflammatory protein expression levels.

Methods : Cybrids from age-, sex-, and mtDNA haplogroup-matched patients with dry AMD (AMD, n=6) and without (Ctrl, n=6) were grown under standard culture conditions. ROS levels were determined using DCFDA fluorescence as well as CellROX Deep Red Reagent intensity (ThermoFisher). mtDNA copy number was determined through qPCR using multiplexed Taqman 18S and ND2assays (ThermoFisher), while fragmentation was qualitatively assessed through long extension PCR (LX-PCR). Western blotting was performed using HSP70 and Actin antibodies (Cell Signaling Technology) at a concentration of 1:1000 and 1:5000, respectively. A two tailed Student’s T test was used for statistical analysis between groups.

Results : AMD cybrids showed a 140% increase in ROS levels (p<0.05) and a 3-fold increase in mtDNA copy number compared to controls (p<0.05). In addition, LX-PCR demonstrated increased levels of fragmentation across the entire mitochondrial genome. Western blotting showed AMD cybrids had a 1.7-fold increase in HSP70, a key chaperonin which is upregulated during cell stress conditions (p<0.01).

Conclusions : Taken together, our findings suggest that AMD cybrids exhibit elevated ROS levels, similar to observations found in vivo, which may contribute to increased antioxidant response and damage to the mitochondrial genome. Further investigations are underway to determine if the cybrid model reflects other in vivo hallmarks of AMD.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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