April 2014
Volume 55, Issue 13
ARVO Annual Meeting Abstract  |   April 2014
Induction of Necrotic Cell death by Oxidative Stress in Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • Jakub Hanus
    Cell and Molecular Biology, Tulane University, New Orleans, LA
  • Hongmei Zhang
    Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX
  • Zhigao Wang
    Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX
  • Qinghua Liu
    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX
  • Shusheng Wang
    Cell and Molecular Biology, Tulane University, New Orleans, LA
    Department of Ophthalmology, Tulane University, New Orleans, LA
  • Footnotes
    Commercial Relationships Jakub Hanus, None; Hongmei Zhang, None; Zhigao Wang, None; Qinghua Liu, None; Shusheng Wang, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 633. doi:
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      Jakub Hanus, Hongmei Zhang, Zhigao Wang, Qinghua Liu, Shusheng Wang; Induction of Necrotic Cell death by Oxidative Stress in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):633.

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

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Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, mostly pointing to a dominant role of apoptosis. However, apoptosis normally doesn’t induce immune response, which is contradictory to the strong immune and inflammatory response in dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress using ARPE19 cells and mouse primary RPE cells.


ARPE-19 cell line or primary mouse RPE cells were treated with different concentration of H2O2 or tBHP and analyzed in different time points for cell death hallmarks: membrane permeability, nucleus condensation and chromatin fragmentation, TUNEL assay, caspase 3 and PARP cleavage, ATP levels, release of HMGB1, activation of RIP3 kinase.


Characteristic features of apoptosis, including DNA fragmentation, Caspase 3 activation, were not observed during RPE cell death induced by either H2O2 or tBHP. Cell death was prevented by RIPK1 inhibitor necrostatin but not Caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, RIPK3 aggregation, release of HMGB1 from nucleus, plasma membrane breakdown indicated by PI staining, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Interestingly RPE cells subjected to oxidative stress were TUNEL positive. The necrotic nature of RPE death is consistent with the release of nuclear protein HMGB1 into the cytoplasm and cell medium, which induces inflammatory gene expression in neighboring healthy RPE cells. Similar features: lack of DNA fragmentation, RIPK3 aggregation and HMGB1 release from nucleus were observed in mouse RPE cells ex vivo. Interestingly, pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells.


In contrast to the current dogma that apoptosis is a major mechanism of oxidative stress-induced RPE death, our results unequivocally show that, necrosis is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing necrotic RPE death may be a viable approach for late stage dry AMD, especially geographic atrophy.

Keywords: 412 age-related macular degeneration • 701 retinal pigment epithelium  

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