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

Abstract

PurposeThe 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.MethodsPrimary 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.ResultshRPE 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.ConclusionsRPESC-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.

TAK1 Inhibition Accelerates Cellular Senescence of Retinal Pigment Epithelial Cells

“…Moreover, it was recently shown that senescence is associated with drusen formation. 27 Our present data demonstrate that TAK1 is a key player in the regulation of senescence in RPE cells. Inhibition of its kinase activity results in abundant presence of senescent RPE cells, demonstrating increased SA-b-gal and enhanced cellcycle arrest at G0/G1.…”

Biphasic regulation of lysosomal exocytosis by oxidative stress

“…Fig 2B shows that rotenone suppressed levels of lysosomal exocytosis as well (72% of control levels, n=9, p<0.05). These tBHP and rotenone concentrations are routinely used to study the effects of oxidative stress in neuronal cultures in vivo and in vitro [19, 49, 50, 55]. …”

Antiapoptotic Properties of α-Crystallin–Derived Peptide Chaperones and Characterization of Their Uptake Transporters in Human RPE Cells

“…23 As well, ARPE-19 and adult RPE cultures may have differential responses to oxidant challenge. 24 In addition, use of hfRPE cells may have an advantage over adult human RPE cells in that, adult human RPE cells show an age-dependent decline in mitochondrial function and antioxidant potency. 25 In the present study, we found that mini-chaperone peptides derived from aA and aB-crystallins exhibited antiapoptotic properties.…”