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S. Jiang, M. Orr, S.E. Moriarty, P. Sternberg, Jr, D.P. Jones; Oxidant–induced apoptosis in human retinal pigment epithelial cells: Dependence upon extracellular redox state . Invest. Ophthalmol. Vis. Sci. 2004;45(13):766.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The redox states of thiol/disulfide pools (glutathione/glutathione disulfide and cysteine/cystine) in human plasma become oxidized with age. Because of the central role of thiols in protein function, cell signaling and defense against oxidative stress, this oxidation could contribute to age–related disease development. The current study was to test whether variation in extracellular redox potential (Eh) over the physiologic range occurring in human plasma affects oxidant–induced apoptosis in normal human retinal pigment epithelial (hRPE) cells, the cells initially lost in age–related macular degeneration (AMD). Methods: hRPE cells were incubated in culture medium with controlled variations of thiol–disulfide established by adding different concentrations of cysteine (Cys) and cystine (CySS) into CySS–free medium. Oxidant tert–butylhydroperoxide (tBH) was then added to induce apoptosis in hRPE cells. Apoptosis was assessed by measuring DNA cleavage with TUNEL and phosphatidylserine exposure with Annexin–V staining. Cytotoxicity was determined by the cleavage of a tertrazolium salt WST–1. Mitochondrial potential was measured by flow cytometry using rhodamine 123. Release of cytochrome c from mitochondria, activation of caspase 3, and expression of total Fas and FasL were determined by Western blot using specific antibodies. Cell surface Fas and FasL were analyzed by flow cytometry. Results: hRPE cells were incubated in culture medium with controlled variation of Eh established over the range of –16 mV (most oxidized) to –158 mV (most reduced). Apoptosis induced by tBH was significantly less at reduced values (Eh < –89 mV) compared to oxidized values (Eh > –55 mV). The loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 3 following tBH were all increased under more oxidized conditions. However, extracellular redox state did not affect expression of Fas or FasL on hRPE cells. Conclusions: These results show that oxidation of plasma thiol–disulfide redox state, as occurs in aging humans, increases hRPE cell susceptibility to oxidant–induced apoptosis by a mechanism involving the intrinsic mitochondrial pathway. The results suggest that oxidation of plasma thiol–disulfide redox state could contribute to development of AMD by altering the sensitivity of hRPE cells to oxidant–induced apoptosis.
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