June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Differential Susceptibility of Primary Human Adult vs. Fetal RPE Cultures to AMD-Relevant Insults
Author Affiliations & Notes
  • Qitao Zhang
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • David Reed
    The Ohio State University Wexner Medical Center, Columbus, Ohio, United States
  • Feriel K Presswalla
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Carol Charniga
    Neural Stem Cell Institute, Rensselaer, New York, United States
  • Melissa Calton
    Department of Genetics, Stanford University, Stanford, California, United States
  • Jeffrey Stern
    Neural Stem Cell Institute, Rensselaer, New York, United States
  • Sally Temple
    Neural Stem Cell Institute, Rensselaer, New York, United States
  • Douglas Vollrath
    Wu Tsai Neurosciences Institute, Stanford University, Stanford, California, United States
  • David N Zacks
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Debra A Thompson
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Jason Miller
    Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan, United States
  • Footnotes
    Commercial Relationships   Qitao Zhang, None; David Reed, None; Feriel Presswalla, None; Carol Charniga, None; Melissa Calton, None; Jeffrey Stern, None; Sally Temple, None; Douglas Vollrath, None; David Zacks, None; Debra Thompson, None; Jason Miller, None
  • Footnotes
    Support  Vision Research Core Grant Funded by NEI; Departmental Grant from Research to Prevent Blindness; Kellogg Eye Center Pre-Residency Fellowship; M2014137 (BrightFocus); R01EY025790; P30EY026877; T32EY20485.
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 2708. doi:
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    • Get Citation

      Qitao Zhang, David Reed, Feriel K Presswalla, Carol Charniga, Melissa Calton, Jeffrey Stern, Sally Temple, Douglas Vollrath, David N Zacks, Debra A Thompson, Jason Miller; Differential Susceptibility of Primary Human Adult vs. Fetal RPE Cultures to AMD-Relevant Insults. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2708.

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

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Abstract

Purpose : Age-related macular degeneration (AMD) is characterized by progressive retinal pigment epithelial (RPE) dysfunction and eventual atrophy. Numerous pathogenic mechanisms have been proposed, including accumulation of lipofuscin, shifts in RPE metabolism, and alterations in the RPE’s underlying extracellular matrix. Our goal is to identify pathways in RPE that contribute to resiliency and can be pharmacologically manipulated to protect RPE in AMD.

Methods : Primary human adult RPE (ahRPE) and fetal RPE (hfRPE) were cultured based on established protocols. Lipofuscin-like granules accumulate through repeated feedings of photo-oxidized outer segments to cultures. Glycolysis and mitochondrial function were assayed by a Seahorse Analyzer. Calcium deposition in the extracellular matrix was determined using a variety of standard stains. Gene expression differences between ahRPE and hfRPE were assessed by RNASeq using DESEQ2 for differential expression and iDEP90 with both GSEA and PGSEA for pathway analysis.

Results : Lipofuscin-like granule accumulation was significantly higher in ahRPE compared to hfRPE, despite more ingestion of OS in the hfRPE group. Further, lipofuscin-like granules are associated with a reduction in oxidation consumption rate, a marker of mitochondrial capacity, only in ahRPE. RNASeq comparing ahRPE with hfRPE demonstrates generally similar expression of RPE-specific genes, but differences in fatty acid degradation, ketogenesis, and lysosomal function that may account for hfRPE’s resistance to lipofuscin accumulation and toxicity. RNASeq also revealed an ectopic extracellular calcification program robustly expressed in ahRPE only. Ongoing studies are determining whether the basolateral extracellular matrix is more calcified in ahRPE compared to hfRPE cultures.

Conclusions : Compared to hfRPE, ahRPE demonstrates increased susceptibility to several AMD-relevant stresses. Initial RNASeq analysis also suggests ahRPE may uniquely express a basolateral extracellular membrane calcification program that could account for calcified drusen, a significant risk factor for AMD progression. Ongoing studies are cataloging other AMD-relevant insults that preferentially affect ahRPE over hfRPE. Comparative gene expression and secreted metabolomic analysis are being used to identify pathways contributing to hfRPE’s resiliency.

This is a 2021 ARVO Annual Meeting abstract.

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