Abstract
purpose. To determine whether keratoconus (KC) corneal fibroblast cultures have increased reactive oxygen species (ROS) production and are more susceptible to stress-related challenges.
methods. Normal (n = 9) and KC (n = 10) stromal fibroblast cultures were incubated in either neutral- or low-pH conditions, with or without hydrogen peroxide. Catalase activities were measured with a fluorescent substrate assay. Superoxide and ROS/reactive nitrogen species (RNS) productions were determined with an amine-reactive green-dye assay and 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay, respectively. Cell viability was analyzed by a dye-exclusion assay. Caspase 3 activity was measured by a fluorochrome inhibitor of caspase (FLICA) assay. A cationic (green) dye was used to measure the mitochondrial membrane potential (ΔΨm).
results. KC fibroblasts had increased superoxide and ROS/RNS production (6.2-fold, P < 0.001 and 1.8-fold, P < 0.001, respectively) and catalase activity (P < 0.01) with higher concentrations of H2O2 compared with normal cultures (P = 0.16). After a low-pH stress challenge, KC fibroblasts maintained higher ROS/RNS levels (3.3-fold, P < 0.02), showed higher caspase-3 activity (7.5-fold, P < 0.02) and decreased ΔΨm (2.6-fold, P < 0.04), and had decreased cell viability (37%, P < 0.005 vs. 20%, P < 0.27) compared with normal fibroblasts.
conclusions. Under identical conditions, KC fibroblasts had increased basal generation of ROS/RNS and were more susceptible to stressful challenges (low-pH and/or H2O2 conditions) than were normal fibroblasts. In addition, the stressed KC fibroblasts possessed characteristics similar to those found in the intact KC corneas (increased catalase activity, ROS production, and apoptosis). These properties may play a role in the pathogenesis of KC.
Keratoconus (KC) is a leading indication for corneal transplantation, with a reported incidence of 1 in 2000 individuals.
1 2 3 4 Clinically, it is characterized as a noninflammatory thinning disorder of the cornea that leads to irregular corneal astigmatism and decreased visual acuity.
4 5 6 The KC corneal stroma may become less than one quarter its normal thickness, which leads to extensive distortion. Mechanisms of this extreme thinning appear to be related to increased proteinase activity,
7 8 9 10 11 12 13 along with decreased proteinase inhibitors.
14 15 At this time, the mechanisms for increased proteinase activity, including matrix metalloproteinases (MMPs), are not understood, but may be related to oxidative stress within the KC corneas.
16 17 18 It has been shown that oxidative stress elements, in particular nitric oxide and peroxynitrites, can degrade tissue inhibitors of matrix metalloproteinases (TIMPs) and activate MMP-2.
16 As such, the oxidative stress involvement does not contradict the important role that MMPs or other degradative enzymes have in this disorder, but in fact they may be interrelated.
In normal corneas the reactive oxygen species/reactive nitrogen species (ROS/RNS) are eliminated by various antioxidant enzymes, including catalase, which converts H
2O
2 to water and oxygen.
19 20 21 Our previous study found a twofold increase in levels of catalase RNA and activity in KC corneas compared with age-matched normal corneas.
22 Because catalase is the major antioxidant enzyme functioning to eliminate H
2O
2,
19 20 21 these findings imply elevated levels of H
2O
2 within KC corneas. The consequences of increased H
2O
2 levels within the cornea or corneal cell cultures are not known. In other cell systems, H
2O
2 can have significant effects on cellular properties, including mitochondrial (mt)DNA damage, decreased intracellular adenosine triphosphate (ATP), apoptosis, and cell death.
23 24 25 26 Cells that are conditioned to be resistant to H
2O
2 exposure show significantly elevated catalase levels.
27 28 29 30 31 We wanted to determine the effect(s) of a H
2O
2 challenge on normal and KC fibroblasts in vitro, including the effects on catalase levels, which are increased in intact KC corneas.
22
There are significant relationships among oxidative stress, ROS/RNS elements, and mitochondria. The accumulation of ROS/RNS can cause a vicious cycle of damage to the mtDNA, leading to mitochondrial dysfunction, decreased oxidative phosphorylation (OXPHOS) and increased ROS/RNS production.
32 33 Normal tissues can have 4% to 5% of the consumed mitochondrial oxygen converted to superoxides and H
2O
2. These byproducts are usually eliminated by antioxidant enzymes, many of which are abnormal in KC corneas.
22 34 35 36 37 38 39 Therefore, it is evident that mitochondria are both a major source and a target for ROS/RNS. Recent studies have shown that KC corneas have increased levels of acquired mitochondria DNA (mtDNA) deletions and mutations compared to age-matched normal corneas.
40 The triad of mitochondrial dysfunction, ROS production, and oxidative stress may play a role in the pathogenesis of KC.
22 40 In the present investigation, we examined whether one element of this triad, that is ROS production, is abnormal in KC fibroblast cultures.
Our working hypothesis was that KC fibroblasts have an elevated ROS/RNS burden and, as a result, are more susceptible to stress-related challenges. Using six different assays, we demonstrate that in response to stress-related challenges, the KC corneal fibroblast cultures have increased ROS/RNS production, catalase activity, and caspase-3 activity along with loss of ΔΨm and cell viability compared to normal fibroblasts. Any of the KC fibroblasts basal properties were similar to the normal fibroblasts treated with high concentrations of H2O2. Moreover, these KC fibroblasts possessed oxidative stress–related properties similar to those in the intact KC corneas. These characteristics imply that the KC cells have inherent defects that are retained during cell isolation and culture. The data support our overall hypothesis that oxidative stress, including mitochondrial damage and ROS production, are factors that may contribute to KC pathogenesis.
ROS/RNS production was measured with the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; Invitrogen), which detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions. Normal and KC stromal fibroblasts were plated in 24-well plates. After 24 hours, cells were washed with Dulbecco’s phosphate-buffered saline (D-PBS) and incubated for 1 hour at 37°C in D-PBS at either pH 7.0 (normal, n = 5 and KC, n = 3) or pH 5.0 (normal, n = 6 and KC, n = 6). Another set of cultures were placed in D-PBS (pH 5.0) with or without H2O2 (normal, n = 6 and KC, n = 5). The cells were washed with D-PBS and incubated with 10 μM H2DCFDA in D-PBS for 30 minutes at 37°C. ROS/RNS production was measured with the scanning unit (excitation λ = 488 nm, emission λ = 520 nm, FMBIO III; Hitachi). The summarized data were from triplicate cultures of normal and KC fibroblasts. Data were analyzed using an unpaired Student’s t-test.
Superoxide anion production was measured with an amine-reactive green-dye assay (Oxyburst Green reagent; Invitrogen). Fibroblasts were plated in either 24-well plates (normal, n = 3 and KC, n = 3) or two-well chamber slides (normal, n = 2 and KC, n = 2). After 24 hours, media were removed, the cells washed with D-PBS (Invitrogen), incubated for 1 hour with D-PBS (pH 5.0), washed again with D-PBS, and incubated for 30 minutes with 10 μM of the dye. The fluorescence intensity was measured with the scanning unit (excitation λ = 488 nm, emission λ = 520 nm; FMBIO III; Hitachi).
Images of superoxide production by cultured fibroblasts were captured with an Inverted Fluorescence Microscope (Leica, Deerfield, IL) with a digital camera (Optronics MicroFire; McBain Instruments, Chatsworth, CA). The entire experiment was repeated twice. For the purpose of this article, ROS/RNS refers to the elements measured by the H2DCFDA method and superoxide refers to the elements measured by the green-dye assay (Oxyburst; Invitrogen).
Normal (n = 2) and KC (n = 2) fibroblasts were plated on 24-well plates for 24 hours. Cultures were treated for 1 hour, with or without H2O2, as in the assay for ROS/RNS production. The cultures were rinsed with PBS-EDTA and then incubated at 37°C for 1 and 3 hours in MEM with 10% FBS. A FLICA solution (Immunochemistry Technologies, LLC, Bloomington, MN) was added to the cultures and incubated an additional hour. The caspase-3 activity was measured as fluorescence intensity (excitation λ = 490 nm, emission λ = 520 nm; FMBIO III scanner; Hitachi). The experiment was performed in triplicate and repeated twice. Statistics were analyzed by two-tailed Student’s t-test.
Loss of the membrane potential (ΔΨm) is a hallmark for cellular apoptosis and was measured with a mitochondrial membrane potential detection kit (JC-1; Cell Technology, Minneapolis, MN) which contains a cationic dye (5,5′6,6′-tetrachloro-1 to 1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide) that fluoresces red in the mitochondria of healthy cells. When the mitochondrial membrane potential collapses, the cationic dye remains in the cytoplasm as green fluorescence. The ratio of red fluorescence to green fluorescence is higher in healthy cells and decreases in apoptotic cells.
Normal (n = 3) and KC (n = 3) corneal fibroblasts were plated on 24-well plates for 24 hours and incubated for 1 hour at 37°C in low-pH stress conditions. After the cells were rinsed with PBS, the media (MEM with 10% FBS) were added and incubated for another hour at 37°C. The JC-1 reagent was added and incubated for 15 minutes. Cells were washed in PBS, red fluorescence was measured at 532-nm excitation and 580-nm emission, and green fluorescence at 488-nm excitation and 520-nm emission on the fluorescence scanner. The ratios of red to green fluorescence were calculated and the data analyzed by unpaired Student’s t-test.
At unstressed neutral pH conditions and stressed (low-pH and/or H2O2) conditions, the cultured KC corneal fibroblasts had significantly increased ROS/RNS production and higher catalase activity than did the normal fibroblasts. Moreover, in low-pH stress conditions, the KC fibroblasts showed increased caspase-3 activity and decreased ΔΨm and cell viability, findings consistent with ongoing apoptosis via the mitochondrial pathway. In addition, the cultured KC fibroblasts exhibited properties similar to the intact KC corneas (increased catalase activities, ROS production and apoptosis). Furthermore, we speculate that the KC fibroblasts have inherent, possibly mitochondrial and/or genetic defects retained during tissue culture.
In this study, the KC fibroblast cultures had higher catalase activity after H
2O
2 treatment than did normal cells. This is consistent with increased catalase activity in the intact KC corneas compared with the normal corneas.
22 In addition, other ocular cell types respond similarly to H
2O
2 treatment. In human lens epithelial cells, catalase was induced by as little as 50 μM H
2O
2.
45 When lenses are treated with H
2O
2, epithelial cell death and cataract formation can occur.
24 25 In vitro, after H
2O
2 exposure the human lens epithelial cells and RPE cells show mtDNA damage, decreased mitochondrial membrane potential and redox function, and loss of cell viability.
23 46 Although corneal fibroblasts are reportedly more resistant to H
2O
2 treatment than are human RPE cells,
23 we suspect that similar mechanisms of cell damage may occur in the KC cell cultures.
Abnormalities of antioxidant enzymes other than catalase are known to occur in KC corneas. For example, increased levels of cathepsins B, G, and V/L2 along with other lysosomal enzymes are found in KC corneas.
11 47 48 49 Cathepsins can elevate hydrogen peroxide levels, thus increasing oxidative stress and apoptosis.
50 51 52 53 54 KC corneas also have decreased levels of extracellular superoxide dismutase (EC-SOD, SOD3) activity
39 that may lead to higher levels of superoxides. In addition, the KC corneas have increased glutathione
S-transferase activity and decreased aldehyde dehydrogenase class 3 (ALDH3A1) activity compared with normal corneas.
38 Therefore, there are multiple potential sources of ROS/RNS elements that could contribute directly to the overall oxidative damage of KC corneas. In addition, accumulation of ROS/RNS can impact cells by increasing degradative enzyme activities and decreasing collagen synthesis,
16 17 18 which may be important for a thinning disorder such as KC.
At the neutral- and low-pH conditions, the KC fibroblast cultures had significantly higher levels of ROS/RNS production when compared with normal cultures. These increased levels were consistently found in the experiments conducted at pH 7, pH 5
(Fig. 2) , and pH 6 (data not shown). In normal fibroblast cultures, the change from neutral-pH to low-pH conditions resulted in a 4.4-fold decline in ROS/RNS production, whereas the KC cultures showed only a 2.3-fold decrease. At the neutral-pH conditions, the cells are likely more metabolically active, thereby generating higher overall ROS/RNS levels through the mitochondrial electron transport system.
55 Although low doses of ROS are beneficial in cellular processes, higher ROS levels are associated with aging and diseases.
56 57 58 59 60 61 62 63 64 65 66 67 In general, ROS production may be either from excessive electrons released during dysfunctional OXPHOS, forming higher levels of superoxides
32 68 or decreased elimination of ROS elements related to abnormal antioxidant enzyme activities.
22 34 35 36 37 38 39 KC corneas reportedly have damaged mtDNA
40 that can lead to decreased OXPHOS and increased ROS/RNS formation. Also altered antioxidant enzyme activities are common in KC corneas.
22 34 35 36 37 38 39 Further studies are needed to determine the mechanisms which lead to this ROS/RNS accumulation.
There is controversy as to whether the H
2DCFDA assay can be used to measure superoxide or H
2O
2 formation conclusively in cells undergoing oxidative stress.
69 However, there are fundamental differences between the present investigation and that of Rota et al.
69 as they studied basic chemical reactions in the absence of cells or any biological system. In contrast, we investigated the responses of primary corneal fibroblasts in vitro. We feel confident in our results using two different assays (H
2DCFDA and Oxyburst; Invitrogen) for measurements of two different groups of ROS/RNS (H
2O
2/peroxyl radicals/peroxynitrite anions and superoxide anions), which consistently showed elevated production in KC cultures.
Another potential problem with the DCFH-DA method was a recent study that questioned the site of interaction of DCFH with ROS/RNS components.
70 It concluded that because the H
2O
2 and superoxides penetrated or crossed the lipid bilayer to react with lipophilic substrates, the interactions between DCFH and H
2O
2/superoxides might be at the lipid bilayer rather than the aqueous intracellular phase.
70 Our study was not designed to address the site of interactions but rather to compare the relative ROS/RNS production between normal and KC fibroblast cultures. Moreover, the H
2DCFDA method is used commonly in research of oxidative stress and free radical biology including in the eye field.
71 72 73 We believe that although the H
2DCFDA assay may not be the perfect measurement for ROS formation, it is accepted in the literature and can provide a relative measurement between cells or tissues. In addition, even if the ROS/superoxide data were omitted, our study still demonstrates that KC fibroblast cultures have increased caspase-3 activity, loss of ΔΨm, and decreased cell viability, which are consistent with higher oxidative stress levels.
Significantly increased caspase-3 activity and ΔΨm loss, consistent with apoptosis, were found in KC corneal fibroblasts after low-pH stress challenges compared with normal cultures. Our results are consistent with reports that the intact KC corneas have high levels of apoptosis in epithelium > endothelium > stromal cells,
74 75 along with a statistically significant decline in anterior and posterior keratocyte densities, as measured by in vivo confocal microscopy.
76 Our findings also agree with reports that stress-induced apoptosis is mediated through the mitochondrial pathway.
77 78 79 We suspect that lowering the extracellular pH temporarily causes passive H
+ influx and decreases the intracellular pH. The H
+ is subsequently removed by the increased activity of the Na
+/H
+ exchange. The cell must expend energy (ATP) to eliminate the Na
+ and readjust the intracellular pH to normal, processes that place high metabolic demands on mitochondrial function. We speculate that the KC mitochondrial function is already compromised and that this additional demand cannot be met, thereby initiating apoptotic events. However, the specific factors initiating the higher levels of caspase-3 and ΔΨm loss in KC fibroblasts are not yet clear.
Clinically, we do not think that the successful use of contact lenses (which may cause hypoxia, acidosis and/or cellular stress) for KC treatment is inconsistent or untenable with our findings that KC fibroblasts are more susceptible to oxidative stress and acidification than are normal fibroblasts. The events that we describe are occurring at a molecular level in stressed tissue culture conditions. When stromal keratocytes are in the in vivo corneal milieu, they may have a blunted or modified response to acidification or hypoxia, or the conditions may not be as severe as that found in our tissue culture system. We believe that additional studies must be completed to further test our oxidative stress hypothesis.
In summary, in the present study, KC fibroblasts treated under conditions identical to normal fibroblasts had significantly increased ROS production, caspase-3 activity, and ΔΨm loss and decreased cell viability. Our findings suggest that KC cells have a higher baseline ROS burden that makes the cells more susceptible to additional oxidative stress than are normal fibroblasts. We hypothesize that these properties play a role in the pathogenesis of KC.
Supported by National Eye Institute Grant EY06807, the Schoellerman Charitable Foundation, the Discovery Eye Foundation, Research to Prevent Blindness Foundation, the Guenther Foundation, the Iris and B. Gerald Cantor Foundation, and the Skirball Molecular Ophthalmology Program.
Submitted for publication June 28, 2005; revised November 8 and December 7, 2005; accepted March 3, 2006.
Disclosure:
M. Chwa, None;
S.R. Atilano, None;
V. Reddy, None;
N. Jordan, None;
D.W. Kim, None;
M.C. Kenney, None
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: M. Cristina Kenney, Department of Ophthalmology, University of California Irvine, Medical Center, 101 The City Drive, Orange CA 92868;
[email protected].
Table 1. Characteristics of Normal and Keratoconus Corneas
Table 1. Characteristics of Normal and Keratoconus Corneas
| Normal | Age | Gender | Cause of Death | Keratoconus | Age | Gender |
| 1 | 33 | M | Gunshot wound | 1 | 27 | F |
| 2 | 25 | F | Respiratory arrest | 2 | 28 | M |
| 3 | 48 | M | Spinal fracture | 3 | 32 | M |
| 4 | 34 | M | Acute MI | 4 | 43 | M |
| 5 | 33 | M | Gunshot wound | 5 | 30 | M |
| 6 | 24 | M | N/A | 6 | 25 | M |
| 7 | 46 | M | N/A | 7 | 31 | F |
| 8 | 44 | M | MVA | 8 | 43 | M |
| 9 | 44 | F | N/A | 9 | 43 | F |
| | | | 10 | 37 | F |
| Mean age | 36.8 | | | Mean age | 33.9 | |
LoisN, KowalVO, CohenEJ, et al. Indications for penetrating keratoplasty and associated procedures, 1989–1995. Cornea. 1997;16:623–629.
[PubMed]CursiefenC, KuchleM, NaumannGO. Changing indications for penetrating keratoplasty: histopathology of 1,250 corneal buttons. Cornea. 1998;17:468–470.
[CrossRef] [PubMed]LiuE, SlomovicAR. Indications for penetrating keratoplasty in Canada, 1986–1995. Cornea. 1997;16:414–419.
[PubMed]BronAJ. Keratoconus. Cornea. 1988;7:163–169.
[PubMed]KrachmerJH, FederRS, BelinMW. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol. 1984;28:293–322.
[CrossRef] [PubMed]BrownD, ChwaMM, OpbroekA, KenneyMC. Keratoconus corneas: increased gelatinolytic activity appears after modification of inhibitors. Curr Eye Res. 1993;12:571–581.
[CrossRef] [PubMed]SmithVA, EastyDL. Matrix metalloproteinase 2: involvement in keratoconus. Eur J Ophthalmol. 2000;10:215–226.
[PubMed]SmithVA, HohHB, LittletonM, EastyDL. Over-expression of a gelatinase A activity in keratoconus. Eye. 1995;9:429–433.
[CrossRef] [PubMed]SawaguchiS, YueBY, SugarJ, GilboyJE. Lysosomal enzyme abnormalities in keratoconus. Arch Ophthalmol. 1989;107:1507–1510.
[CrossRef] [PubMed]ZhouL, SawaguchiS, TwiningSS, et al. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci. 1998;39:1117–1124.
[PubMed]KenneyMC, BrownDJ, RajeevB. Everett Kinsey lecture. The elusive causes of keratoconus: a working hypothesis. CLAO J. 2000;26:10–13.
[PubMed]CollierSA. Is the corneal degradation in keratoconus caused by matrix- metalloproteinases?. Clin Exp Ophthalmol. 2001;29:340–344.
[CrossRef] OpbroekA, KenneyMC, BrownD. Characterization of a human corneal metalloproteinase inhibitor (TIMP-1). Curr Eye Res. 1993;12:877–883.
[CrossRef] [PubMed]SawaguchiS, TwiningSS, YueBY, et al. Alpha-1 proteinase inhibitor levels in keratoconus. Exp Eye Res. 1990;50:549–554.
[CrossRef] [PubMed]BrownDJ, LinB, ChwaM, et al. Elements of the nitric oxide pathway can degrade TIMP-1 and increase gelatinase activity. Mol Vis. 2004;10:281–288.
[PubMed]SiwikDA, PaganoPJ, ColucciWS. Oxidative stress regulates collagen synthesis and matrix metalloproteinase activity in cardiac fibroblasts. Am J Physiol Cell Physiol. 2001;280:C53–C60.
[PubMed]BrenneisenP, BrivibaK, WlaschekM, et al. Hydrogen peroxide (H2O2) increases the steady-state mRNA levels of collagenase/MMP-1 in human dermal fibroblasts. Free Radic Biol Med. 1997;22:515–524.
[CrossRef] [PubMed]SpectorA, LiD, MaW, et al. Differential amplification of gene expression in lens cell lines conditioned to survive peroxide stress. Invest Ophthalmol Vis Sci. 2002;43:3251–3264.
[PubMed]YamadaM, HashinakaK, InazawaJ, AbeT. Expression of catalase and myeloperoxidase genes in hydrogen peroxide-resistant HL-60 cells. DNA Cell Biol. 1991;10:735–742.
[CrossRef] [PubMed]LinF, JacksonVE, GirottiAW. Amplification and hyperexpression of the catalase gene in selenoperoxidase-deficient leukemia cells. Arch Biochem Biophys. 1995;317:7–18.
[CrossRef] [PubMed]KenneyMC, ChwaM, AtilanoSR, et al. Increased levels of catalase and cathepsin V/L2 but decreased TIMP-1 in keratoconus corneas: evidence that oxidative stress plays a role in this disorder. Invest Ophthalmol Vis Sci. 2005;46:823–832.
[CrossRef] [PubMed]BallingerSW, Van HoutenB, JinGF, et al. Hydrogen peroxide causes significant mitochondrial DNA damage in human RPE cells. Exp Eye Res. 1999;68:765–772.
[CrossRef] [PubMed]SpectorA, WangGM, WangRR, et al. A brief photochemically induced oxidative insult causes irreversible lens damage and cataract. I. Transparency and epithelial cell layer. Exp Eye Res. 1995;60:471–481.
[CrossRef] [PubMed]SpectorA. Oxidative stress-induced cataract: mechanism of action. FASEB J. 1995;9:1173–1182.
[PubMed]WangX, SimpkinsJW, DykensJA, CammarataPR. Oxidative damage to human lens epithelial cells in culture: estrogen protection of mitochondrial potential, ATP, and cell viability. Invest Ophthalmol Vis Sci. 2003;44:2067–2075.
[CrossRef] [PubMed]SpectorA, MaW, SunF, et al. The effect of H2O2 and tertiary butyl hydroperoxide upon a murine immortal lens epithelial cell line, alphaTN4–1. Exp Eye Res. 2002;75:573–582.
[CrossRef] [PubMed]CarperD, JohnM, ChenZ, et al. Gene expression analysis of an HO-resistant lens epithelial cell line. Free Radic Biol Med. 2001;31:90–97.
[CrossRef] [PubMed]SpitzDR, ElwellJH, SunY, et al. Oxygen toxicity in control and H2O2-resistant Chinese hamster fibroblast cell lines. Arch Biochem Biophys. 1990;279:249–260.
[CrossRef] [PubMed]BojesHK, SureshPK, MillsEM, et al. Bcl-2 and Bcl-xL in peroxide-resistant A549 and U87MG cells. Toxicol Sci. 1998;42:109–116.
[PubMed]BhuyanKC, BhuyanDK. Superoxide dismutase of the eye: relative functions of superoxide dismutase and catalase in protecting the ocular lens from oxidative damage. Biochim Biophys Acta. 1978;542:28–38.
[CrossRef] [PubMed]YakesFM, Van HoutenB. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc Natl Acad Sci USA. 1997;94:514–519.
[CrossRef] [PubMed]WallaceDC. Mitochondrial defects in neurodegenerative disease. Ment Retard Dev Disabil Res Rev. 2001;7:158–166.
[CrossRef] [PubMed]RichterC. Oxidative damage to mitochondrial DNA and its relationship to ageing. Int J Biochem Cell Biol. 1995;27:647–653.
[CrossRef] [PubMed]ChanceB, SiesH, BoverisA. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979;59:527–605.
[PubMed]TurrensJF, BoverisA. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980;191:421–427.
[PubMed]GondhowiardjoTD, van HaeringenNJ. Corneal aldehyde dehydrogenase, glutathione reductase, and glutathione S-transferase in pathologic corneas. Cornea. 1993;12:310–314.
[CrossRef] [PubMed]GondhowiardjoTD, van HaeringenNJ, Volker-DiebenHJ, et al. Analysis of corneal aldehyde dehydrogenase patterns in pathologic corneas. Cornea. 1993;12:146–154.
[CrossRef] [PubMed]BehndigA, KarlssonK, JohanssonBO, et al. Superoxide dismutase isoenzymes in the normal and diseased human cornea. Invest Ophthalmol Vis Sci. 2001;42:2293–2296.
[PubMed]AtilanoSR, CoskunP, ChwaM, et al. Accumulation of mitochondrial DNA damage in keratoconus corneas. Invest Ophthalmol Vis Sci. 2005;46:1256–1263.
[CrossRef] [PubMed]KenneyMC, ChwaM, OpbroekAJ, BrownDJ. Increased gelatinolytic activity in keratoconus keratocyte cultures: a correlation to an altered matrix metalloproteinase-2/tissue inhibitor of metalloproteinase ratio. Cornea. 1994;13:114–124.
[CrossRef] [PubMed]BealesMP, FunderburghJL, JesterJV, HassellJR. Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: maintenance of the keratocyte phenotype in culture. Invest Ophthalmol Vis Sci. 1999;40:1658–1663.
[PubMed]JesterJV, BarryPA, LindGJ, et al. Corneal keratocytes: in situ and in vitro organization of cytoskeletal contractile proteins. Invest Ophthalmol Vis Sci. 1994;35:730–743.
[PubMed]JarrettSG, BoultonME. Antioxidant up-regulation and increased nuclear DNA protection play key roles in adaptation to oxidative stress in epithelial cells. Free Radic Biol Med. 2005;38:1382–1391.
[CrossRef] [PubMed]GoswamiS, SheetsNL, ZavadilJ, et al. Spectrum and range of oxidative stress responses of human lens epithelial cells to H
2O
2 insult. Invest Ophthalmol Vis Sci. 2003;44:2084–2093.
[CrossRef] [PubMed]LiangFQ, GodleyBF. Oxidative stress-induced mitochondrial DNA damage in human retinal pigment epithelial cells: a possible mechanism for RPE aging and age-related macular degeneration. Exp Eye Res. 2003;76:397–403.
[CrossRef] [PubMed]WhitelockRB, FukuchiT, ZhouL, et al. Cathepsin G, acid phosphatase, and alpha 1-proteinase inhibitor messenger RNA levels in keratoconus corneas. Invest Ophthalmol Vis Sci. 1997;38:529–534.
[PubMed]KenneyMC, ChwaM, LinB, et al. Identification of cell types in human diseased corneas. Cornea. 2001;20:309–316.
[CrossRef] [PubMed]BrookesNH, LohIP, CloverGM, et al. Involvement of corneal nerves in the progression of keratoconus. Exp Eye Res. 2003;77:515–524.
[CrossRef] [PubMed]ZhaoM, AntunesF, EatonJW, BrunkUT. Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis. Eur J Biochem. 2003;270:3778–3786.
[CrossRef] [PubMed]CirmanT, OresicK, MazovecGD, et al. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem. 2004;279:3578–3587.
[PubMed]IshisakaR, UtsumiT, KannoT, et al. Participation of a cathepsin L-type protease in the activation of caspase-3. Cell Struct Funct. 1999;24:465–470.
[CrossRef] [PubMed]IshisakaR, UtsumiK, UtsumiT. Involvement of lysosomal cysteine proteases in hydrogen peroxide-induced apoptosis in HL-60 cells. Biosci Biotechnol Biochem. 2002;66:1865–1872.
[CrossRef] [PubMed]GuicciardiME, DeussingJ, MiyoshiH, et al. Cathepsin B contributes to TNF-alpha-mediated hepatocyte apoptosis by promoting mitochondrial release of cytochrome c. J Clin Invest. 2000;106:1127–1137.
[CrossRef] [PubMed]JezekP, HlavataL. Mitochondria in homeostasis of reactive oxygen species in cell, tissues, and organism. Int J Biochem Cell Biol. 2005;37:2478–2503.
[CrossRef] [PubMed]MatesJM, Perez-GomezC, Nunez de CastroI. Antioxidant enzymes and human diseases. Clin Biochem. 1999;32:595–603.
[CrossRef] [PubMed]WallaceDC. Mitochondrial diseases in man and mouse. Science. 1999;283:1482–1488.
[CrossRef] [PubMed]BusciglioJ, YanknerBA. Apoptosis and increased generation of reactive oxygen species in Down’s syndrome neurons in vitro. Nature. 1995;378:776–779.
[CrossRef] [PubMed]BusciglioJ, PelsmanA, WongC, et al. Altered metabolism of the amyloid beta precursor protein is associated with mitochondrial dysfunction in Down’s syndrome. Neuron. 2002;33:677–688.
[CrossRef] [PubMed]DruzhynaN, NairRG, LeDouxSP, WilsonGL. Defective repair of oxidative damage in mitochondrial DNA in Down’s syndrome. Mutat Res. 1998;409:81–89.
[CrossRef] [PubMed]ArbuzovaS, HutchinT, CuckleH. Mitochondrial dysfunction and Down’s syndrome. Bioessays. 2002;24:681–684.
[CrossRef] [PubMed]OgawaO, PerryG, SmithMA. The “Down’s” side of mitochondria. Dev Cell. 2002;2:255–256.
[CrossRef] [PubMed]McCordJM. The evolution of free radicals and oxidative stress. Am J Med. 2000;108:652–659.
[CrossRef] [PubMed]DattaK, SinhaS, ChattopadhyayP. Reactive oxygen species in health and disease. Natl Med J India. 2000;13:304–310.
[PubMed]GoodPF, WernerP, HsuA, et al. Evidence of neuronal oxidative damage in Alzheimer’s disease. Am J Pathol. 1996;149:21–28.
[PubMed]PetitPX, SusinSA, ZamzamiN, et al. Mitochondria and programmed cell death: back to the future. FEBS Lett. 1996;396:7–13.
[CrossRef] [PubMed]RichterC. Oxidative stress, mitochondria, and apoptosis. Restor Neurol Neurosci. 1998;12:59–62.
[PubMed]WallaceDC. A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found Symp. 2001;235:247–263.discussion 63–66
[PubMed]RotaC, ChignellCF, MasonRP. Evidence for free radical formation during the oxidation of 2′-7′-dichlorofluorescin to the fluorescent dye 2′-7′-dichlorofluorescein by horseradish peroxidase: possible implications for oxidative stress measurements. Free Radic Biol Med. 1999;27:873–881.
[CrossRef] [PubMed]AfriM, FrimerAA, CohenY. Active oxygen chemistry within the liposomal bilayer. Part IV: Locating 2′,7′-dichlorofluorescein (DCF), 2′,7′-dichlorodihydrofluorescein (DCFH) and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in the lipid bilayer. Chem Phys Lipids. 2004;131:123–133.
[CrossRef] [PubMed]MaherP, HannekenA. The molecular basis of oxidative stress-induced cell death in an immortalized retinal ganglion cell line. Invest Ophthalmol Vis Sci. 2005;46:749–757.
[CrossRef] [PubMed]SreekumarPG, KannanR, YaungJ, et al. Protection from oxidative stress by methionine sulfoxide reductases in RPE cells. Biochem Biophys Res Commun. 2005;334:245–253.
[CrossRef] [PubMed]TchahH, KimMJ, KimTI, et al. Regulation of 1-cys peroxiredoxin expression in the process of stromal wound healing after photorefractive keratectomy. Invest Ophthalmol Vis Sci. 2005;46:2396–2403.
[CrossRef] [PubMed]KaldawyRM, WagnerJ, ChingS, SeigelGM. Evidence of apoptotic cell death in keratoconus. Cornea. 2002;21:206–209.
[CrossRef] [PubMed]KimWJ, RabinowitzYS, MeislerDM, WilsonSE. Keratocyte apoptosis associated with keratoconus. Exp Eye Res. 1999;69:475–481.
[CrossRef] [PubMed]HollingsworthJG, EfronN, TulloAB. In vivo corneal confocal microscopy in keratoconus. Ophthalmic Physiol Opt. 2005;25:254–260.
[CrossRef] [PubMed]JiangS, Moriarty-CraigeSE, OrrM, et al. Oxidant-induced apoptosis in human retinal pigment epithelial cells: dependence on extracellular redox state. Invest Ophthalmol Vis Sci. 2005;46:1054–1061.
[CrossRef] [PubMed]YangJH, LeWD, BasingerSF, et al. Mechanisms of apoptosis in human retinal pigment epithelium induced by TNF-alpha in conditions of heavy metal ion deficiency. Invest Ophthalmol Vis Sci. 2005;46:1039–1046.
[CrossRef] [PubMed]CharlesI, KhalyfaA, KumarDM, et al. Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. Invest Ophthalmol Vis Sci. 2005;46:1330–1338.
[CrossRef] [PubMed]