Abstract
Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. 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, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.
Highlights
retinal pigment epithelium (RPE) of the retina is essential for retinal homeostasis by transporting nutrients and waste for the retinal photoreceptor cells
In an effort to clarify the mechanism of oxidative stressinduced RPE cell death, we found that molecular features of apoptosis, autophagy or pyroptosis were not observed in RPE cells in response to H2O2 or tert-butyl hydroperoxide (tBHP) treatment
Sub-confluent ARPE-19 cells were treated with H2O2 or tBHP, and cell viability was measured by MTT assay 24 h later
Summary
RPE of the retina is essential for retinal homeostasis by transporting nutrients and waste for the retinal photoreceptor cells. Two major types of cell death, apoptosis and necrosis, have been delineated in response to oxidative stress.[19] Apoptosis is characterized by cytoplasm shrinkage, chromatin condensation and fragmentation, apoptotic body formation, caspase activation and maintenance of the plasma membrane. Necrotic cells can release multiple pro-inflammatory factors, including high mobility group protein B1 (HMGB1) proteins, to activate inflammatory response.[21] Besides apoptosis and necrosis, autophagy and pyroptosis are linked to cell death. In an effort to clarify the mechanism of oxidative stressinduced RPE cell death, we found that molecular features of apoptosis, autophagy or pyroptosis were not observed in RPE cells in response to H2O2 or tBHP treatment. Our data provide compelling evidence that necrosis is a major type of cell death in response to oxidative stress, highlighting the potential of therapeutic targeting RPE cell necrosis in GA
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