March 2005
Volume 46, Issue 3
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Retinal Cell Biology  |   March 2005
Oxidant-Induced Apoptosis in Human Retinal Pigment Epithelial Cells: Dependence on Extracellular Redox State
Author Affiliations
  • Shunai Jiang
    From the Departments of Ophthalmology and
  • Siobhan E. Moriarty-Craige
    From the Departments of Ophthalmology and
    Medicine, Emory University School of Medicine, Atlanta, Georgia; and the
  • Michael Orr
    Medicine, Emory University School of Medicine, Atlanta, Georgia; and the
  • Jiyang Cai
    Medicine, Emory University School of Medicine, Atlanta, Georgia; and the
    Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee.
  • Paul Sternberg, Jr
    From the Departments of Ophthalmology and
    Vanderbilt Eye Institute, Vanderbilt University, Nashville, Tennessee.
  • Dean P. Jones
    From the Departments of Ophthalmology and
    Medicine, Emory University School of Medicine, Atlanta, Georgia; and the
Investigative Ophthalmology & Visual Science March 2005, Vol.46, 1054-1061. doi:10.1167/iovs.04-0949
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      Shunai Jiang, Siobhan E. Moriarty-Craige, Michael Orr, Jiyang Cai, Paul Sternberg, Jr, Dean P. Jones; Oxidant-Induced Apoptosis in Human Retinal Pigment Epithelial Cells: Dependence on Extracellular Redox State. Invest. Ophthalmol. Vis. Sci. 2005;46(3):1054-1061. doi: 10.1167/iovs.04-0949.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. To test whether variation in extracellular cysteine (Cys) redox potential (Eh) over the physiologic range occurring in human plasma affects oxidant-induced apoptosis in cultured human retinal pigment epithelial (hRPE) cells.

methods. The hRPE cells were incubated in culture medium with Eh established over the range of −16 mV (most oxidized) to −158 mV (most reduced) by adding different concentrations of Cys and cystine (CySS) with constant total Cys equivalents. Apoptosis was induced with tert-butylhydroperoxide (tBH).

results. The hRPE cells were sensitized to tBH-induced apoptosis in the more oxidized extracellular conditions (Eh > −55 mV) compared with the reduced conditions (Eh < −89 mV). Loss of mitochondrial membrane potential (Δψm), release of cytochrome c, and activation of caspase 3 after tBH treatments all increased under the more oxidized conditions. However, the extracellular redox state did not affect expression of Fas or FasL in hRPE cells.

conclusions. The hRPE cells that are exposed to a more oxidized extracellular redox environment have increased susceptibility to oxidant-induced apoptosis through the intrinsic mitochondrial pathway, which could contribute to an age-related decline in cell populations in the retina and thereby provide a potential mechanism for the degenerative changes that are associated with age-related macular degeneration (ARMD).

The redox regulatory mechanisms are distinct among various cellular and extracellular compartments. Each compartment contains different pools of thiol/disulfide couples, such as glutathione (GSH)/glutathione disulfide (GSSG), cysteine (Cys)/cystine (CySS), and dihydrolipoic acid/lipoic acid. 1 The functions of these redox couples are dependent on both the ratio and total concentrations of the reduced and oxidized forms, the inherent electron donating/accepting characters that are defined by the relative standard potential (E0) values of the respective couples, and the kinetics of interactions of the components. GSH is the predominant cellular thiol and is maintained at millimolar concentrations with redox potentials ranging from −260 to −200 mV. 2 In human plasma, the GSH concentration is <4 μM, and the GSH/GSSG redox state is much more oxidized than the intracellular redox state—for example, about −140 mV. The plasma concentrations of CySS and Cys are 40 to 50 μM and 8 to 10 μM, respectively. 3 The redox potential of the plasma Cys/CySS pool is −80 ± 9 mV and is more oxidized than GSH/GSSG. 3 Decreased concentrations of plasma Cys and GSH have been implicated in a number of human diseases such as Alzheimer’s and Parkinson’s, diabetes, cystic fibrosis, and HIV infection. 4 5 6 7  
Considerable evidence is available to show that thiols protect against oxidant-induced apoptosis, and this has often been linked to maintenance of cellular GSH pools. 8 9 GSH functions in elimination of peroxides and lipid peroxidation as well as direct reaction with free radicals. 10 11 12 Although protection against oxidative stress clearly can occur by detoxification of reactive oxygen intermediates, accumulating evidence indicates that low-molecular-weight thiols also have a fundamental role through effects on protein thiol/disulfide redox state. 11 13 Changes in the thiol/disulfide redox state alter signal transduction, DNA and RNA synthesis, protein synthesis, enzyme activation, and cell cycle regulation. 11 13 14  
One common aging disease, age-related macular degeneration (ARMD), is the leading cause of blindness in elderly Americans. 15 One theory is that oxidative damage contributes to disease onset and progression. 16 This theory is supported by epidemiologic studies that have demonstrated that high serum carotenoid levels are associated with a decreased risk of neovascular ARMD 17 and that a combination of vitamin C, vitamin E, β-carotene, and zinc significantly reduced the risk of advanced ARMD and associated vision loss. 18 Specifically, vision loss in ARMD occurs through photoreceptor damage in the macula, with abnormalities in the retinal pigment epithelium (RPE) and Bruch’s membrane being the hallmark of the disease. 19 Studies have demonstrated that apoptotic cells are present in the RPE of eyes with ARMD, indicating that apoptosis in RPE cells could contribute to ARMD. 20 21 Because of its anatomic location, the RPE is primed for oxidant and free radical production and has an extraordinary need for antioxidant protection. 16 22 23 24 The GSH antioxidant system may play a large role in RPE protection, but the RPE has limited capacity to take up exogenous GSH. 25 Therefore, the maintenance of cellular GSH levels in RPE is largely dependent on GSH synthesis. The age-related decline of plasma Cys is associated with an oxidation of both the Cys/CySS and GSH/GSSG redox states in human plasma. 3 6 26 This decline may compromise the antioxidant function of RPE by limiting the GSH synthesis and render RPE cells susceptible to oxidative stress, which may serve as one mechanism of RPE injury in ARMD. Accordingly, there is a general consensus that oxidative damage to RPE contributes to the initiation and progression of ARMD. 22 23 24  
Our previous in vitro studies have demonstrated that pro-oxidants can cause apoptosis through both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways in cultured human RPE (hRPE) cells. 27 28 29 In the extrinsic pathway, FasL binds its receptor, Fas, which belongs to the tumor necrosis factor receptor superfamily. Binding to Fas triggers a signal transduction pathway, ultimately ending in apoptosis in cells that express Fas. 30 In addition, the mitochondria play a central role in regulation of the apoptotic pathway. An early event in this pathway is the mitochondrial permeability transition (MPT), which includes loss of mitochondrial membrane potential (Δψm). MPT activation results in release of cytochrome c, which triggers caspase activation and apoptosis. 31 32 33 Both mechanisms work to ensure that apoptosis occurs in times of stress, and both may be important in degenerative changes associated with aging and age-related disease. 
We recently found that the redox states of human plasma GSH and Cys pools become oxidized in association with increased age. 26 If such oxidation were to alter the sensitivity of cells to apoptosis, the thiol/disulfide redox state of plasma may be a central determinant of age-related degenerative processes. The present study tested the hypothesis that controlled variation in extracellular redox potential (Eh) over the physiological range would affect the sensitivity of hRPE cells to oxidant-induced apoptosis. The results show that hRPE cells were more susceptible to apoptosis under more oxidized redox potentials, which we established by varying the Cys and CySS concentrations. The intrinsic pathway for apoptosis, measured by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase 3, varied as a function of extracellular redox state; whereas no redox-dependent effects were observed for the death receptor Fas/FasL system. Thus, oxidant-induced apoptosis in hRPE cells signaled by the mitochondrial pathway is sensitive to the extracellular Cys/CySS redox state. 
Materials and Methods
Cell Culture
hRPE cell cultures were established from donor eyes as previously described. 25 Because cell lines from different individuals differ in characteristics, each experiment was performed on cell lines from at least three individuals. For the study, eyes from four donors were used. The ages of the donors were 74, 70, 39 and 41 years. The study protocol conformed to the guidelines in the Declaration of Helsinki for research involving human tissue. No history of ARMD was documented for any of the donors. After the hRPE culture was established, there was no correlation between the ages of the donor and the cellular redox status. More than 90% of the cells were initially pigmented and showed typical epithelial cell morphology under the light microscope, although with time they gradually lost their pigmentation. Cells were routinely grown in complete Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin in a 37°C humidified incubator in an atmosphere of 5% CO2. They were passaged every 7 days. RPE cells between passages 6 and 10 were used throughout the study. 
Preparation of Media with Different Thiol-Disulfide Redox Status
The Cys-free DMEM used for these studies did not contain CySS but was otherwise identical with regular DMEM, which contains 200 μM CySS. Addition of 10% FBS to the Cys-free DMEM provided 0.3 μM Cys and 3.2 μM CySS, as measured by HPLC. To generate the desired range of extracellular redox states, we added various concentrations of Cys and CySS to cyst(e)ine-free DMEM with 10% FBS, as shown in Table 1 . Medium pH was adjusted to 7.4 after the addition of Cys and CySS. The concentrations of Cys and CySS were chosen to approximate the human plasma thiol/disulfide pool but still allow for cellular utilization. The ratios between Cys and CySS were varied to achieve predicted Eh from 0 to −190 mV, with the total Cys/CySS pool size (the total cysteine moieties (Cys) + (2 × CySS), equal to 200 μM. In all experiments, cells were seeded in complete DMEM on plates or dishes and cultured for 2 days. Cells were then washed once with phosphate-buffer saline (PBS) and incubated in fresh DMEM media containing different concentrations of Cys and CySS. 
Apoptosis Assay
Apoptosis was induced by adding 300 μM tBH for the indicated time in DMEM medium with different ratios of Cys and CySS. DNA cleavage, which commonly occurs in apoptosis, was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) with a kit (In Situ Cell Death Detection Kit with Fluorescein; Roche Molecular Biochemicals, Mannheim, Germany) followed by flow cytometry. Phosphatidylserine exposure, another apoptotic feature, was determined with an annexin V-FITC kit (TACS; Trevigen, Gaithersburg, MD). Briefly, cells were resuspended in 100 μL of binding buffer containing 10 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 1 mg/mL annexin-V-FITC, and propidium iodide. After 15 minutes of incubation at room temperature in the dark, 400 μL of binding buffer was added, and cells were then analyzed by flow cytometry. 
Cytotoxicity
A colorimetric assay based on the cleavage of the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2,5-tetrazolio]-1,3-benzene disulfonate) to formazan by mitochondrial dehydrogenases of viable cells was performed (Roche Molecular Biochemicals). Data are shown as the percentage of the untreated control. 
Measurement of Δψm
To measure Δψm, cells were incubated with 1 μM rhodamine 123 for 15 minutes in culture medium at 37°C. The cells were washed once with PBS and immediately analyzed by flow cytometry. 
Cytosolic Extracts for Western Blot Analysis of Cytochrome c and Caspase 3 Activities
A digitonin-permeabilization technique was used to release cytosol from cells. 34 35 Briefly, cells were washed once with PBS and then resuspended in a solution containing 70 mM Tris and 250 mM sucrose (pH 7.0). Digitonin was added to provide a minimum concentration resulting in 95% cell staining with 0.2% trypan blue (∼0.05 μg/mL final concentration). Cells were immediately centrifuged at 3000 rpm for 10 minutes at 4°C, and the supernatant was collected as cytosol. The amount of protein in the cytosol was measured by the Lowry method, and 20 μg of protein was loaded onto each lane of a 15% SDS-polyacrylamide gel. The separated proteins were blotted to a 0.45 μM polyvinylidene difluoride (PVDF) membrane (Hybond; Amersham Pharmacia Biotech, Piscataway, NJ). Nonspecific binding was blocked by incubation in 5% nonfat milk with 0.1% Tween 20 in PBS overnight at 4°C. The membrane was then stained with antibodies against cytochrome c (BD Biosciences-PharMingen, San Diego, CA) or caspase 3 (Oncogene Research Products, San Diego, CA) diluted 1:1000 in PBS containing 5% nonfat milk and 0.1% Tween 20 for 1 hour at room temperature with gentle agitation. After being washed three times with PBS containing 0.1% Tween 20, the membrane was incubated with 1:3000 diluted horseradish peroxidase–coupled anti-mouse IgG for 1 hour at room temperature. The specific protein was then detected by chemiluminescence (Renaissance Western blot Chemiluminescence Reagent; NEN Life Science Products, Boston, MA). 
FasL and Fas Expression
Expression of FasL and Fas on RPE cells was detected as described previously. 22 For flow cytometric detection of cell surface FasL, cells were sequentially stained with 2.5 μg/mL rabbit anti-FasL antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and 2.5 μg/mL FITC-conjugated anti-rabbit IgG (Pierce, Rockford, IL). For detection of cell surface Fas, cells were incubated with phycoerythrin (PE)-conjugated anti-Fas antibody (BD Biosciences-PharMingen). Cells stained with isotypically matched control IgG and FITC-conjugated secondary antibodies and cells stained with PE-conjugated isotypically matched control Ig were run in parallel as negative controls for FasL and Fas staining, respectively. The cells were then analyzed by flow cytometer. 
Expression of total FasL and total Fas was determined by Western blot analysis. Cells were lysed in a boiling solution containing 10% glycerol, 250 mM Tris (pH 6.8), 4% SDS, and 2% β-mercaptoethanol. The cell lysates were boiled for 5 minutes and centrifuged at 10,000g for 5 minutes at 4°C. The supernatants (100 μg) were subjected to 12% SDS-polyacrylamide gel electrophoresis followed by transfer to PVDF membranes, and immunoblotted with antibodies against FasL and Fas (1:1000 dilution; Transduction Laboratories, Lexington, KY). FasL and Fas proteins were detected using horseradish peroxidase–labeled secondary antibody and enhanced chemiluminescence (NEN Life Science Products). 
Measurement of Acid Soluble Thiols
The hRPE cells were treated as indicated and then extracted with 5% perchloric acid/0.2 M boric acid. For medium samples, an equal volume of 10% perchloric acid/0.2 M boric acid was added to precipitate the protein. The acid soluble Cys, CySS, GSH, and GSSG were derivatized with iodoacetic acid and dansyl chloride and measured on an HPLC system (2695 Alliance; Waters, Milford, MA), as described. 28 35 The solvent used for the mobile phase was 80% methanol. Samples were loaded onto a propylamine column (YMC Pack NH2; Waters) and was eluted with a gradient of sodium acetate. 
Statistical Analysis
Each experiment was performed at least three times with three different RPE cell lines. One-way ANOVA was used to determine whether the means of different groups were significantly different (P < 0.05). Results are expressed as the mean ± SEM. 
Results
Stabilization of Extracellular Cys/CySS Redox Status by hRPE Cells
The media with different Eh of Cys/CySS were prepared by adding various concentrations of Cys and CySS as shown in Table 1 . The hRPE cells were incubated in these media and, at different time points, the media were collected and measured for concentrations of Cys and CySS by HPLC. As shown in Figure 1 , in more reduced media (−158, −199, and −89 mV), Cys levels were rapidly decreased and stabilized to 10 μM by 5 hours. There was little Cys in the more oxidized medium (−55, −30, and −16 mV) at the beginning of incubation. However, the concentrations of Cys slowly rose during the culture and by 24 hours, they reached 10 μM. CySS, the oxidized form of Cys, gradually declined in all media except in the most reduced medium (−158 mV). In the latter case, CySS was increased first and reached a peak at 5 hours, then slowly decreased during further incubation. When the extracellular Eh for Cys/CySS was calculated, the oxidized media became more reduced while reduced media became more oxidized as the incubations progressed. By 24 hours, an Eh (Cys/CySS) of −70 mV was achieved in all media, regardless of the starting redox status. The −70 mV value is the same as measured Eh of Cys/CySS in normal human plasma. 3 This shows that hRPE cells have the capability to control the extracellular redox environment and can adjust the oxidized and reduced media into the normal range of redox state. 
We then examined whether variation in extracellular redox affects intracellular redox states. The hRPE cells were incubated in the culture media with different concentrations of Cys/CySS. At different time points, cells were harvested and intracellular GSH/GSSG and Cys/CySS contents were measured by HPLC. As shown in Figure 2 , during the 24-hour incubation, despite differences in extracellular Cys/CySS, the intracellular GSH remained the same except under the extreme redox conditions. At 5 and 10 hours, no significant difference in intracellular Eh (GSH/GSSG) was seen in hRPE cells incubated in media with different extracellular Eh (Cys/CySS). The values of intracellular Eh approximated −220 mV, a normal value observed in cultured proliferating hRPE cells. 28 At 1 hour, hRPE cells cultured in the most reduced condition (−158 mV) tended to have more reduced intracellular Eh for GSH/GSSG. At 24 hour, hRPE cells in the most oxidized condition (−16 mV) tended to be more oxidized. However, the differences did not reach statistical significance. Thus, variations in the extracellular Cys/CySS redox had no significant effect on the intracellular GSH/GSSG redox state. 
Stimulation of tBH-Induced Apoptosis by Oxidized Redox State of Cys/CySS
We have shown that the oxidant tBH causes hRPE cell death mediated by apoptosis, with activation by both the intrinsic mitochondrial pathway 29 and the extrinsic Fas/FasL pathway. 27 To determine whether culture in media with different extracellular Cys/CySS resulted in altered sensitivities to tBH-induced injury of hRPE cells, the cells were incubated in media with different Eh for 24 hours and then treated with tBH. As shown in Figure 3A , tBH exhibited less cytotoxicity as detected in hRPE cells using the TUNEL assay in cells incubated in the most reduced conditions (−119 and −158 mV) compared with the oxidized conditions. Quantification of the differences in sensitivity showed that under conditions in which tBH induced minimal apoptosis at −119 and −158 mV, the same treatment induced apoptosis in 40% of the cells at −30 to −16 mV (Fig. 3B) . Similar results were obtained when hRPE cells were incubated in media with different Eh only for 1 hour, treated with tBH, and assayed by annexin V staining (Fig. 3C) . Thus, the results show that the sensitivity of hRPE cells to tBH-induced apoptosis is dependent on the extracellular Cys/CySS redox state. 
No Changes in Fas and FasL Expression in Cells Incubated in Different Extracellular Eh
As increased expression and activation of the Fas pathway is involved in tBH-induced apoptosis in hRPE cells, 27 we examined whether the change in susceptibility to apoptosis due to extracellular redox resulted from changes in the expression of Fas and FasL. The hRPE cells were cultured in different extracellular Cys/CySS redox states for various time periods. Cell surface Fas and FasL expression was determined by flow cytometry, and total (cell surface and intracellular) Fas and FasL were determined by Western blot. As shown in Figure 4 , cell surface and total Fas were not changed in response to alterations in extracellular Eh. Similarly, FasL expression was not affected by changing extracellular Cys/CySS redox. Consequently, the increased sensitivity to oxidant-induced apoptosis was not due to increased expression of the Fas/FasL system. 
Effects of Extracellular Eh in tBH-Induced Mitochondrial Membrane Potential (Δψm) Loss, Cytochrome c Release, and Caspase 3 Activation
We then studied the intrinsic pathway of apoptosis to determine whether changes in mitochondrial signaling could account for the increased susceptibility to tBH-induced apoptosis in hRPE cells cultured under the more oxidized conditions. Our previous study showed that in tBH-induced apoptosis, a decrease of Δψm was observed as early as 2 hours, followed by release of cytochrome c from mitochondria and activation of caspase 3 by 4 hours. 29 To determine whether there was a difference in loss of Δψm, cells were incubated in culture media with controlled variation in Eh for 1 hour and then treated with tBH. The Δψm loss was considerably greater under the most oxidized conditions (Fig. 5) . Loss of the Δψm is associated with cytochrome c release in the intrinsic pathway for activation of apoptosis. Measurement of cytochrome c loss after tBH treatment showed that release was stimulated under more oxidized conditions (Fig. 6) . To determine whether this increased release of cytochrome c was associated with caspase activation, Western blot analyses of cleavage of Poly(ADP-ribose) polymerase (PARP) was performed. Increased cleavage of the PARP was detected in hRPE cells cultured in more oxidized media (Fig. 6) . Thus, the data show that cells exposed to more oxidized extracellular Cys/CySS redox state are more susceptible to oxidant-induced apoptosis, and this increased susceptibility involves the intrinsic mitochondrial pathway for apoptosis. 
Discussion
Our present study of hRPE function in various extracellular redox conditions has shown that hRPE cells have the ability to modulate the extracellular redox pool. They were able to change the reduced and oxidized media to the redox state of −70 mV (Fig. 1) , a value detected in normal human plasma. For those experiments, we used 80% to 90% confluent hRPE cells that were seeded onto 60-mm dishes with 4 mL of medium. The estimated cell volume was <5% of the medium volume. Although rapid oxidation of Cys to CySS may contribute to the change of extracellular redox to some extent, it could not explain the adjustment of extracellular redox to −70 mV in all media regardless of their initial redox status. When the media were placed alone (without hRPE cells) in the incubator for 24 hours, the Eh of the most oxidized medium (−16 mV) was −3 mV, whereas the Eh of the most reduced medium (−158 mV) became −24 mV. The media incubated alone had Cys concentrations around 1 μM, whereas the media cultured with hRPE cells had 5 to 10 times higher concentrations of Cys. Moreover, compared with the media incubated alone, the media cultured with hRPE cells had relatively lower CySS concentrations. Thus, the observed redox changes (Fig. 1)were not solely caused by air oxidation of Cys in the media. The results indicate that an important characteristic of hRPE cells is that they regulate the thiol/disulfide redox state in their extracellular environment. 
In spite of this ultimate stabilization of extracellular redox, the data show that a more oxidized extracellular Cys/CySS sensitized hRPE to oxidant-induced apoptosis. We previously found that both GSH and Cys protect hRPE cells from tBH-induced cell death but the mechanisms involved are different. 25 Although Cys can protect cells from oxidative injury by elevating intracellular GSH, GSH is not transported into the cells and may act on the cell membrane of hRPE. The present research and previous studies have shown that variation in extracellular Cys/CySS results in little change in cellular GSH and GSSG concentrations. 36 The measured GSSG levels had nearly 20% variations between experimental replicates, due to limitations of the analytical procedures. However, the results (Fig. 2and Ref. 36 ) consistently supported the interpretation that extracellular Cys/CySS redox was not linked to intracellular GSH/GSSG redox. Therefore, this suggests a regulatory mechanism specifically controlled by extracellular Cys/CySS redox and also implies that the extracellular redox sensitivity involves other signal transduction systems in addition to the expected thiol-dependent antioxidant effects. 
Exogenously added GSH or N-acetyl cysteine (NAC) can effectively downregulate cell surface Fas and FasL expression in hRPE cells and therefore inhibit Fas-mediated apoptosis. 27 However, the present studies show that change in extracellular Cys/CySS redox does not affect Fas or FasL expression. Instead, the data show that the altered sensitivity to apoptosis is mediated by the mitochondrial pathway, as evidenced by mitochondrial membrane potential loss, cytochrome c release, and caspase 3 activation. Because this occurs without detectable change in cellular GSH/GSSG redox, the data indicate that the response may be mediated by cell signaling components in the plasma membrane. Such an interpretation is consistent with recent models of cell survival pathways, such as those involving insulin-like growth factor receptor (IGFR), AKT, and forkhead transcription factors, which serve to orchestrate cell survival and apoptosis signaling. 37 38  
Accumulating evidence indicates that oxidation of the extracellular thiol/disulfide redox state not only provides a biomarker of oxidative stress, 8 9 11 12 but also contributes to disease development by inhibiting cell proliferation 8 and decreasing resistance to infection. 4 5 39 Cys concentration in plasma declines with age and, along with an increase of CySS, results in an oxidation of the redox value for Cys/CySS. 3 6 34 When controlled for age, conditions of oxidative stress, such as cigarette smoking 40 and high-dose chemotherapy 41 result in oxidation of plasma Cys/CySS and GSH/GSSG redox states. Oxidation of the plasma thiol/disulfide redox state has also been associated with type 2 diabetes 6 and cardiovascular disease. 42 Results from the present study suggest that a common consequence of the plasma redox changes is to sensitize affected cells to mitochondria-mediated apoptosis. 
In addition to clinical studies, in vitro oxidation of extracellular redox state occurs in association with growth arrest, cellular differentiation, and apoptosis. 36 28 43 44 The present study shows that oxidized thiol/disulfide redox state enhances the sensitivity of RPE cells to oxidant-induced apoptosis. In our experiments, we have used a standard model of oxidative stress to postmortem cultured hRPE cells that has been replicated by other laboratories. We recognize that these cells are capable of active division and that this may affect their susceptibility to injury. Data from other laboratories indicate that differentiation status of the cultured cells may influence the cellular response to oxidative stress. 45 In fact, we have developed an alternative model in which we age the hRPE cells in vitro and are able to render them postmitotic. 28 In this setting, which is likely to mimic the in vivo scenario more closely, the hRPE cells demonstrate even greater sensitivity to oxidative injury than in the model chosen for this project. Those cells also showed more oxidized intracellular GSH redox. Thus, both intracellular and extracellular redox status contribute to the sensitivity of RPE to oxidative stress. Although we used only the chemical oxidant tBH for most of the studies, a similar redox regulatory mechanisms may also be applied to other acute toxicities, such as light-induced damage to the retina. 
In conclusion, this study shows that hRPE cells can regulate their extracellular redox state, but that oxidation of the extracellular thiol/disulfide redox state increases hRPE cell susceptibility to oxidative injury. This indicates that a shift in the extracellular redox state in vivo could play an important role in RPE cell function and contribute to apoptosis of RPE cells undergoing oxidative stress. Because of this, the extracellular thiol/disulfide redox state may represent a physiologic environment in which other oxidative challenges, such as infection, activation of the immune system, or other stress conditions, result in enhanced RPE cell loss and development of ARMD. Consequently, manipulation of the plasma thiol/disulfide redox state to a more reduced level may decrease the sensitivity of the retina and RPE to apoptosis from oxidative stress. 
 
Table 1.
 
Preparation of DMEM with Different CyS/CySS Redox Potential
Table 1.
 
Preparation of DMEM with Different CyS/CySS Redox Potential
CyS (μM) CySS (μM) Eh (mV)
200 0 −158
80 60 −119
40 80 −89
14 93 −55
4 98 −30
0 100 −16
Figure 1.
 
Control of extracellular Cys/CySS redox status by cultured hRPE cells. Cells were incubated in media with Eh levels of Cys/CySS ranging from −16 to −158 mV. The time-dependent changes in concentrations of Cys and CySS were measured, and Eh levels were calculated based on the Nernst Equation. Data points show the mean ± SEM of results in four experiments.
Figure 1.
 
Control of extracellular Cys/CySS redox status by cultured hRPE cells. Cells were incubated in media with Eh levels of Cys/CySS ranging from −16 to −158 mV. The time-dependent changes in concentrations of Cys and CySS were measured, and Eh levels were calculated based on the Nernst Equation. Data points show the mean ± SEM of results in four experiments.
Figure 2.
 
Intracellular GSH/GSSG redox status in hRPE cells cultured in media with different Eh. The cellular GSH and GSSG were measured at the indicated time points. Data points show the mean ± SEM of four separate experiments, using different hRPE cell lines.
Figure 2.
 
Intracellular GSH/GSSG redox status in hRPE cells cultured in media with different Eh. The cellular GSH and GSSG were measured at the indicated time points. Data points show the mean ± SEM of four separate experiments, using different hRPE cell lines.
Figure 3.
 
Increased susceptibility to apoptosis in cells incubated in the oxidized conditions. The hRPE cells were incubated in the culture media with various Eh for either 24 hours (A, B) or 1 hour (C). Cells were untreated or treated with 300 μM tBH for 4 hour. (A) Apoptosis was assessed by examining DNA cleavage with TUNEL. Representative data of three separate experiments are shown. (B) Cytotoxicity was measured through the cleavage of WST-1. Data points show the mean ± SEM of results in six experiments. *P < 0.05 vs. the control medium with Eh of −16 mV. (C) Cells were stained with annexin-V for phosphatidylserine exposure. Data from one representative of three separate experiments are shown.
Figure 3.
 
Increased susceptibility to apoptosis in cells incubated in the oxidized conditions. The hRPE cells were incubated in the culture media with various Eh for either 24 hours (A, B) or 1 hour (C). Cells were untreated or treated with 300 μM tBH for 4 hour. (A) Apoptosis was assessed by examining DNA cleavage with TUNEL. Representative data of three separate experiments are shown. (B) Cytotoxicity was measured through the cleavage of WST-1. Data points show the mean ± SEM of results in six experiments. *P < 0.05 vs. the control medium with Eh of −16 mV. (C) Cells were stained with annexin-V for phosphatidylserine exposure. Data from one representative of three separate experiments are shown.
Figure 4.
 
Expression of FasL and Fas in hRPE cells incubated in different redox status. (A) Flow cytometry measurement of cell surface FasL and Fas expression. SMFI: specific mean fluorescence intensity = mean fluorescence intensity of cells stained with antibody − mean fluorescence intensity of cells stained with isotype matched Ig. Data points show the mean ± SEM from 18 (FasL) and 17 (Fas) separate experiments. No significant changes were detected. (B) Western blot analysis of total FasL and Fas expression. Data from one representative of four separate experiments are shown.
Figure 4.
 
Expression of FasL and Fas in hRPE cells incubated in different redox status. (A) Flow cytometry measurement of cell surface FasL and Fas expression. SMFI: specific mean fluorescence intensity = mean fluorescence intensity of cells stained with antibody − mean fluorescence intensity of cells stained with isotype matched Ig. Data points show the mean ± SEM from 18 (FasL) and 17 (Fas) separate experiments. No significant changes were detected. (B) Western blot analysis of total FasL and Fas expression. Data from one representative of four separate experiments are shown.
Figure 5.
 
Effects of Eh in tBH-induced Δψm loss. hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were untreated or treated with 300 μM tBH for 2 or 4 hours, stained with rhodamine123, and analyzed by flow cytometry. Data from one representative of five separate experiments are shown.
Figure 5.
 
Effects of Eh in tBH-induced Δψm loss. hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were untreated or treated with 300 μM tBH for 2 or 4 hours, stained with rhodamine123, and analyzed by flow cytometry. Data from one representative of five separate experiments are shown.
Figure 6.
 
Effects of Eh in tBH-induced cytochrome c release and PARP cleavage. The hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were subsequently treated with 300 μM tBH for 2, 4, and 6 hours. Release of cytochrome c and PARP cleavage were detected by Western blot analyses. Data from one representative of three separate experiments are shown.
Figure 6.
 
Effects of Eh in tBH-induced cytochrome c release and PARP cleavage. The hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were subsequently treated with 300 μM tBH for 2, 4, and 6 hours. Release of cytochrome c and PARP cleavage were detected by Western blot analyses. Data from one representative of three separate experiments are shown.
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Figure 1.
 
Control of extracellular Cys/CySS redox status by cultured hRPE cells. Cells were incubated in media with Eh levels of Cys/CySS ranging from −16 to −158 mV. The time-dependent changes in concentrations of Cys and CySS were measured, and Eh levels were calculated based on the Nernst Equation. Data points show the mean ± SEM of results in four experiments.
Figure 1.
 
Control of extracellular Cys/CySS redox status by cultured hRPE cells. Cells were incubated in media with Eh levels of Cys/CySS ranging from −16 to −158 mV. The time-dependent changes in concentrations of Cys and CySS were measured, and Eh levels were calculated based on the Nernst Equation. Data points show the mean ± SEM of results in four experiments.
Figure 2.
 
Intracellular GSH/GSSG redox status in hRPE cells cultured in media with different Eh. The cellular GSH and GSSG were measured at the indicated time points. Data points show the mean ± SEM of four separate experiments, using different hRPE cell lines.
Figure 2.
 
Intracellular GSH/GSSG redox status in hRPE cells cultured in media with different Eh. The cellular GSH and GSSG were measured at the indicated time points. Data points show the mean ± SEM of four separate experiments, using different hRPE cell lines.
Figure 3.
 
Increased susceptibility to apoptosis in cells incubated in the oxidized conditions. The hRPE cells were incubated in the culture media with various Eh for either 24 hours (A, B) or 1 hour (C). Cells were untreated or treated with 300 μM tBH for 4 hour. (A) Apoptosis was assessed by examining DNA cleavage with TUNEL. Representative data of three separate experiments are shown. (B) Cytotoxicity was measured through the cleavage of WST-1. Data points show the mean ± SEM of results in six experiments. *P < 0.05 vs. the control medium with Eh of −16 mV. (C) Cells were stained with annexin-V for phosphatidylserine exposure. Data from one representative of three separate experiments are shown.
Figure 3.
 
Increased susceptibility to apoptosis in cells incubated in the oxidized conditions. The hRPE cells were incubated in the culture media with various Eh for either 24 hours (A, B) or 1 hour (C). Cells were untreated or treated with 300 μM tBH for 4 hour. (A) Apoptosis was assessed by examining DNA cleavage with TUNEL. Representative data of three separate experiments are shown. (B) Cytotoxicity was measured through the cleavage of WST-1. Data points show the mean ± SEM of results in six experiments. *P < 0.05 vs. the control medium with Eh of −16 mV. (C) Cells were stained with annexin-V for phosphatidylserine exposure. Data from one representative of three separate experiments are shown.
Figure 4.
 
Expression of FasL and Fas in hRPE cells incubated in different redox status. (A) Flow cytometry measurement of cell surface FasL and Fas expression. SMFI: specific mean fluorescence intensity = mean fluorescence intensity of cells stained with antibody − mean fluorescence intensity of cells stained with isotype matched Ig. Data points show the mean ± SEM from 18 (FasL) and 17 (Fas) separate experiments. No significant changes were detected. (B) Western blot analysis of total FasL and Fas expression. Data from one representative of four separate experiments are shown.
Figure 4.
 
Expression of FasL and Fas in hRPE cells incubated in different redox status. (A) Flow cytometry measurement of cell surface FasL and Fas expression. SMFI: specific mean fluorescence intensity = mean fluorescence intensity of cells stained with antibody − mean fluorescence intensity of cells stained with isotype matched Ig. Data points show the mean ± SEM from 18 (FasL) and 17 (Fas) separate experiments. No significant changes were detected. (B) Western blot analysis of total FasL and Fas expression. Data from one representative of four separate experiments are shown.
Figure 5.
 
Effects of Eh in tBH-induced Δψm loss. hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were untreated or treated with 300 μM tBH for 2 or 4 hours, stained with rhodamine123, and analyzed by flow cytometry. Data from one representative of five separate experiments are shown.
Figure 5.
 
Effects of Eh in tBH-induced Δψm loss. hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were untreated or treated with 300 μM tBH for 2 or 4 hours, stained with rhodamine123, and analyzed by flow cytometry. Data from one representative of five separate experiments are shown.
Figure 6.
 
Effects of Eh in tBH-induced cytochrome c release and PARP cleavage. The hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were subsequently treated with 300 μM tBH for 2, 4, and 6 hours. Release of cytochrome c and PARP cleavage were detected by Western blot analyses. Data from one representative of three separate experiments are shown.
Figure 6.
 
Effects of Eh in tBH-induced cytochrome c release and PARP cleavage. The hRPE cells were incubated in the culture media with various Eh for 1 hour. Cells were subsequently treated with 300 μM tBH for 2, 4, and 6 hours. Release of cytochrome c and PARP cleavage were detected by Western blot analyses. Data from one representative of three separate experiments are shown.
Table 1.
 
Preparation of DMEM with Different CyS/CySS Redox Potential
Table 1.
 
Preparation of DMEM with Different CyS/CySS Redox Potential
CyS (μM) CySS (μM) Eh (mV)
200 0 −158
80 60 −119
40 80 −89
14 93 −55
4 98 −30
0 100 −16
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