Chronic oxidative stress upregulates Drusen-related protein expression in adult human RPE stem cell-derived RPE cells: A novel culture model for dry AMD

David M Rabin,Richard L Rabin,Sally Temple,Timothy A Blenkinsop,Jeffrey H Stern

doi:10.18632/aging.100516

David M Rabin, Richard L Rabin + Show 3 more

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https://doi.org/10.18632/aging.100516

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Abstract

The goal of this study was to examine changes in the expression of transcripts and proteins associated with drusen in Age-related Macular Degeneration (AMD) after exposing human retinal pigment epithelium (hRPE) cells to chronic oxidative stress. Primary adult human RPE cells were isolated from cadaveric donor eyes. The subpopulation of RPE stem cells (RPESCs) was activated, expanded, and then differentiated into RPE progeny. Confluent cultures of RPESC-derived hRPE and ARPE-19 cells were exposed to a regimen of tert-butylhydroperoxide (TBHP) for 1-5 days. After treatment, gene expression was measured by quantitative PCR (qPCR), protein expression was assessed by immunocytochemistry and transepithelial resistance and cell toxicity were measured. hRPE cells exposed to a regimen of TBHP for 5 days upregulate expression of several molecules identified in drusen, including molecular chaperones and pro-angiogenic factors. 5-day TBHP treatment was significantly more effective than 1-day treatment at eliciting these effects. The extent of hRPE response to 5-day treatment varied significantly between individual donors, nevertheless, 6 transcripts were reliably significantly upregulated. ARPE-19 cells treated with the same 5-day stress regime did not show the same pattern of response and did not upregulate this group of transcripts. RPESC-derived hRPE cells change significantly when exposed to repeated oxidative stress conditions, upregulating expression of several drusen-related proteins and transcripts. This is consistent with the hypothesis that hRPE cells are competent to be a source of proteins found in drusen deposits. Our results suggest that donor-specific genetic and environmental factors influence the RPE stress response. ARPE-19 cells appear to be less representative of AMD-like changes than RPESC-derived hRPE. This adult stem cell-based system using chronic TBHP treatment of hRPE represents a novel in vitro model useful for the study of drusen formation and dry AMD pathophysiology.

Highlights

Age‐related Macular Degeneration (AMD) is a highly prevalent disease that is the leading cause of vision loss for patients over 65 years of age in first world countries [1,2,3,4,5,6]
We found that αB-crystallin was highly expressed in RPE cells immediately adjacent to drusen, further demonstrating that in AMD, the RPE cells change to express proteins associated with the disease [17]
ARPE-19 cells were cultured in the same conditions as donor-derived hRPESCs and produced monolayers that closely resembled the cobblestone morphology of human retinal pigment epithelium (hRPE) cells (Fig. 1E, F)

Summary

Introduction

AMD is a highly prevalent disease that is the leading cause of vision loss for patients over 65 years of age in first world countries [1,2,3,4,5,6]. An early sign of AMD is the formation of sub-RPE deposits termed drusen, which can accumulate over time leading to RPE dysfunction, cell death and loss of central vision. The majority of AMD patients, approximately 85%, have the 'dry’ form of the disease, which is associated with drusen and defined by the absence of neovascular ingrowth. Proteomic analysis of human drusen has identified a number of proteins that differ in expression in patients with and without an AMD diagnosis [11,12,13]. Drusen from donors without a diagnosis of AMD had lower to undetectable levels of crystallins and proteins related to amyloid-β (Aβ) production [11,12,13,14]

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