May 2006
Volume 47, Issue 5
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Cornea  |   May 2006
Increased Stress-Induced Generation of Reactive Oxygen Species and Apoptosis in Human Keratoconus Fibroblasts
Author Affiliations
  • Marilyn Chwa
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
  • Shari R. Atilano
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
  • Vinitha Reddy
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
  • Nicole Jordan
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
  • Dae W. Kim
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
  • M. Cristina Kenney
    From the Department of Ophthalmology, University of California Irvine Medical Center, Irvine, California.
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1902-1910. doi:10.1167/iovs.05-0828
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      Marilyn Chwa, Shari R. Atilano, Vinitha Reddy, Nicole Jordan, Dae W. Kim, M. Cristina Kenney; Increased Stress-Induced Generation of Reactive Oxygen Species and Apoptosis in Human Keratoconus Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2006;47(5):1902-1910. doi: 10.1167/iovs.05-0828.

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

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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 H2O2 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 H2O2, 19 20 21 these findings imply elevated levels of H2O2 within KC corneas. The consequences of increased H2O2 levels within the cornea or corneal cell cultures are not known. In other cell systems, H2O2 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 H2O2 exposure show significantly elevated catalase levels. 27 28 29 30 31 We wanted to determine the effect(s) of a H2O2 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 H2O2. 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. 
Materials and Methods
Corneal Fibroblast Cultures
Age-matched normal corneas (n = 9, Table 1 ) were received within 24 hours after death from the National Disease Research Interchange (Philadelphia, PA). Keratoconus corneas (n = 10, Table 1 ) were received within 24 hours after penetrating keratoplasty. Informed consent was obtained from participants, and the study was performed according to the tenets of the Declaration of Helsinki for research involving human subjects. Normal and KC stromal cells were isolated and cultured as described previously. 41 Cells were cultured in minimal essential medium (MEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Omega Scientific Inc., Tarzana, CA). For individual experiments, third-passage fibroblasts 42 43 were used. Cells were plated overnight in 24-well plates (1 × 105), 6-well plates (4.3 × 105), 2-well chamber slides (1 × 105), or 60-mm dishes (5 × 105). 
In this study, the fibroblast cultures were stressed by using two different mechanisms. The first stress challenge was exposure to low pH conditions for 1 hour, to increase the metabolic demand for the cells to maintain their intracellular pH. The second stress method was exposure to H2O2, a major ROS element that induces oxidative stress response of the cell. Levels of 50 to 100 μM H2O2 have been reported to be physiologic, 26 whereas 10 μM H2O2 for 24 hours is sublethal. 44 In the literature, the most frequently used H2O2 concentrations are 50 to 200 μM for 1- to 24-hour incubations, and these were the ranges used in our studies. 
Catalase Activity Assay
KC (n = 3) and normal (n = 3) fibroblast cultures were plated into six-well plates overnight, rinsed with serum-free MEM and then incubated at 37°C for 1 hour in serum-free MEM with H2O2 (50, 100, 200, or 400 μM). In general, the rate of H2O2 disappearance is very rapid, with more than one-half of the H2O2 being eliminated by 20 minutes and the remainder by 1 hour. 23 Cultures were rinsed, protein was isolated in phosphate buffer (pH 7.2), and protein concentrations were measured by a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Inc., Rockford, IL). Catalase activities were measured with a fluorescent substrate assay (Amplex Red Catalase kit; Invitrogen, Carlsbad, CA) using a fluorescence image scanner (excitation λ = 535 nm, emission λ = 590 nm; FMBIO III; Hitachi, Yokohama, Japan). Each concentration was assayed in triplicate. A linear regression statistical analysis was performed to test whether the slope was significantly different for normal and KC cultures (GraphPad Software, Inc., San Diego, CA). P <0.05 was considered significant. 
Detection of ROS/RNS Production
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 Detection
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). 
Cell Viability Assay
The cell viabilities of the normal and KC fibroblast cultures were measured by a dye-exclusion assay. Duplicate normal (n = 3) and KC (n = 3) fibroblasts were plated onto a six-well plate and treated without or with H2O2 for 1 hour. Viable cells were measured by trypan blue dye-exclusion assay by cell counter (Beckman-Coulter, Inc., Fullerton, CA). Statistics were analyzed with the two-tailed Student’s t-test. 
Caspase-3 Assay
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. 
Mitochondrial Membrane Potential Measurements
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. 
Results
Figure 1shows increased catalase activity by KC fibroblast cultures after H2O2 treatment. In the KC fibroblast cultures, the slope for the catalase activity (as measured by linear regression plotting) was steeper after H2O2 treatment (P < 0.01) compared with the normal cultures (P = 0.16). The relative activities for catalase activities in the untreated, 200 μM H2O2-treated, and 400 μM H2O2-treated cultures were 100%, 120%, and 142%, for the normal fibroblasts, respectively, and 100%, 256%, and 294%, in the KC cultures, respectively. 
At both neutral-pH and low-pH stress conditions, the KC fibroblasts cultures had significantly higher levels of ROS/RNS formation than did normal cultures. In neutral-pH conditions (Fig. 2A) , the KC fibroblast cultures (n = 3) had a 1.8-fold increased production of ROS/RNS compared with normal (n = 5) fibroblast cultures (34,940 ± 484 vs. 18,070 ± 2,087; P < 0.001). Figure 2Bshows that after exposure to low-pH conditions, the KC fibroblast cultures (n = 7) have a 3.3-fold increase of ROS/RNS production compared with the normal (n = 6) cultures (17,200 ± 2,913 vs. 4,103 ± 1,161; P < 0.02 cells). These are all downstream elements in the oxidative stress pathways. 
We also wanted to determine whether superoxide anions, which are upstream and can be generated from oxidative phosphorylation within the mitochondria, were also increased in the KC cultures. The kit (Oxyburst Green; Invitrogen), which detects only superoxide anions, was used to quantify and visualize the superoxide production in normal and KC fibroblasts in low-pH conditions (Figs. 2C 2D) . The KC fibroblast cultures (n = 3) showed a 6.2-fold increase in superoxide production compared with the normal (n = 3) cultures (654 ± 290 vs. 4079 ± 143, P < 0.0012). By cytochemistry, normal fibroblasts showed minimal staining indicative of the presence of superoxides, whereas the KC fibroblasts were positive throughout the cytoplasm (Fig. 2D)
With the KC fibroblasts producing higher levels of superoxides and ROS/RNS, we speculated that the increased burden would lead to loss of cell viability. The cultures were exposed to low-pH conditions with or without 200 or 400 μM H2O2 and both ROS/RNS production and cell viability were measured (Fig. 3) . The normal fibroblast cultures (n = 6) showed a statistically significant increase in ROS production that corresponded to increasing the H2O2 concentrations (*P < 0.05; Fig. 3A ). The ROS/RNS levels of KC fibroblast cultures (n = 5) after low-pH conditions were already significantly higher than the low-pH–stressed normal cultures (**P < 0.02) and the additional exogenous H2O2 did not further increase ROS/RNS formation (Fig. 3A) . Note that the low-pH–stressed KC fibroblasts had levels of ROS/RNS formation that were similar to that of normal fibroblasts treated with low-pH and 200 μM H2O2
The KC fibroblast cultures (n = 3) had a greater loss of cell viability than did normal cultures (n = 3) when exposed to low-pH stress conditions. Figure 3Bshows the significant loss of cell viability with increased concentrations of H2O2. An equal number of cells (i.e., total cells in Fig. 3B ) was plated in normal (n = 3) and KC fibroblast cultures (n = 3). After exposure to low-pH conditions, the KC fibroblast cultures had a 37% significant decrease in cell viability compared with the total cells plated (2.7 × 105 ± 0.27 × 105 vs. 4.29 × 105 ± 0.003 × 105; *P < 0.005). The low-pH–treated normal cultures showed a 20% decline compared with the total number of cells originally plated (3.46 × 105 ± 0.65 × 105 vs. 4.29 × 105 ± 0.003 × 105; P < 0.27). After treatment with 200 or 400 μM H2O2, the cell viability decreased in both the normal cultures (20% and 12% viability; **P < 0.03, respectively) and KC cultures (13% and 6% viability; ***P < 0.003, respectively) compared with the low-pH–treated cultures. 
The KC fibroblast cultures had increased levels of caspase-3 activity compared with the normal cultures (Fig. 4) . Because the KC fibroblasts had higher ROS levels and a greater loss of cell viability, we hypothesized that these cells are undergoing an apoptosis-like process. Therefore, we measured the caspase-3 levels after a low-pH challenge, with and without exposure to 200 or 400 μM H2O2. Figure 4Ashows that the low-pH–treated KC fibroblast cultures had significantly higher caspase-3 activity compared with normal cultures (5590 ± 1276 vs. 740 ± 478, P < 0.02). The normal fibroblast cultures showed an incremental increase in the caspase-3 levels that correlated with the higher H2O2 levels (Fig. 4A) . The KC cultures consistently showed higher levels of caspase-3 activity than did the normal cultures. It is interesting to note that the untreated KC fibroblast cultures had levels of caspase-3 similar to those in the 400-μM H2O2-treated normal cultures. Figure 4Bshows cytochemistry images of the low-pH 200-μM and 400-μM H2O2–treated cultures. The KC cultures had an increased number of caspase-3-positive cells compared with the normal cultures in all categories examined. 
The low-pH–stressed KC fibroblasts showed loss of ΔΨm compared with normal cultures by cationic dye staining (JC-1 assay; Cell Technology; Fig. 5 ). The red fluorescence represents live cells and the green fluorescence shows cells that are undergoing apoptosis. Quantification of the red–green fluorescence ratio shows normal fibroblasts with a mean fluorescence ratio of 3.433 ± 0.7055, whereas the KC cultures were 2.6-fold lower (1.333 ± 0.333, P < 0.04). 
Discussion
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 H2O2 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 H2O2 treatment. In human lens epithelial cells, catalase was induced by as little as 50 μM H2O2. 45 When lenses are treated with H2O2, epithelial cell death and cataract formation can occur. 24 25 In vitro, after H2O2 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 H2O2 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 H2DCFDA assay can be used to measure superoxide or H2O2 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 (H2DCFDA and Oxyburst; Invitrogen) for measurements of two different groups of ROS/RNS (H2O2/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 H2O2 and superoxides penetrated or crossed the lipid bilayer to react with lipophilic substrates, the interactions between DCFH and H2O2/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 H2DCFDA 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 H2DCFDA 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. 
 
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
Figure 1.
 
The KC fibroblast cultures had increased catalase activity in response to H2O2 treatment compared with normal cultures. The linear regression plot of the slope for catalase activity in KC fibroblast cultures increased significantly (P < 0.01) with higher concentrations of H2O2, whereas the normal cultures did not (P = 0.16). For the normal fibroblast cultures, the relative activities for catalase in the untreated and 200 and 400 μM H2O2 were 100%, 120%, and 142%, respectively. The KC cultures were 100%, 256%, and 294%, respectively. H2O2, hydrogen peroxide; NL, normal; KC, keratoconus.
Figure 1.
 
The KC fibroblast cultures had increased catalase activity in response to H2O2 treatment compared with normal cultures. The linear regression plot of the slope for catalase activity in KC fibroblast cultures increased significantly (P < 0.01) with higher concentrations of H2O2, whereas the normal cultures did not (P = 0.16). For the normal fibroblast cultures, the relative activities for catalase in the untreated and 200 and 400 μM H2O2 were 100%, 120%, and 142%, respectively. The KC cultures were 100%, 256%, and 294%, respectively. H2O2, hydrogen peroxide; NL, normal; KC, keratoconus.
Figure 2.
 
KC fibroblast cultures had increased levels of ROS and superoxides compared to normal cultures. (A) In neutral pH conditions, the KC fibroblast cultures had a 1.8-fold increased production of ROS compared with age-matched normal fibroblast cultures (34,940 ± 484 vs. 18,070 ± 2,087; **P < 0.001). (B) In low pH conditions, the KC fibroblast cultures showed a 3.3-fold higher production of ROS than did the normal cultures (17,200 ± 2,913 vs. 4,103 ± 1,161; *P < 0.02). (C) In low-pH conditions, the KC fibroblast cultures had a 6.2-fold higher superoxide production than did the normal fibroblasts (**P < 0.001). (D) KC fibroblasts were positive for superoxides as identified with the green-dye assay (Oxyburst; Invitrogen). NL cultures had minimal superoxide staining. NL, normal; KC, keratoconus; *,**Statistically significant compared with NL.
Figure 2.
 
KC fibroblast cultures had increased levels of ROS and superoxides compared to normal cultures. (A) In neutral pH conditions, the KC fibroblast cultures had a 1.8-fold increased production of ROS compared with age-matched normal fibroblast cultures (34,940 ± 484 vs. 18,070 ± 2,087; **P < 0.001). (B) In low pH conditions, the KC fibroblast cultures showed a 3.3-fold higher production of ROS than did the normal cultures (17,200 ± 2,913 vs. 4,103 ± 1,161; *P < 0.02). (C) In low-pH conditions, the KC fibroblast cultures had a 6.2-fold higher superoxide production than did the normal fibroblasts (**P < 0.001). (D) KC fibroblasts were positive for superoxides as identified with the green-dye assay (Oxyburst; Invitrogen). NL cultures had minimal superoxide staining. NL, normal; KC, keratoconus; *,**Statistically significant compared with NL.
Figure 3.
 
KC fibroblast cultures exposed to low-pH stress had higher levels of ROS formation and decreased cell viability compared to normal cultures. (A) Low-pH–stressed normal fibroblasts showed increased ROS production that corresponded to increasing H2O2 concentrations (*P < 0.05). In KC fibroblast cultures, the baseline ROS activity was significantly greater than in the normal cells (**P < 0.02). With the addition of H2O2, ROS formation did not increase further. (B) Compared with the total cells originally plated, the low-pH–stressed KC fibroblast cultures had a 37% decrease in cell viability (P < 0.005). The low-pH–stressed normal cultures showed a 20% decline. After treatment with 200 or 400 μM H2O2, cell viabilities decreased in both the normal cultures (20% and 12% viability; **P < 0.03, respectively) and KC cultures (13% and 6% viability; ***P < 0.003, respectively) compared to the low-pH–treated cultures. H2O2 = hydrogen peroxide. NL, normal; KC. keratoconus.
Figure 3.
 
KC fibroblast cultures exposed to low-pH stress had higher levels of ROS formation and decreased cell viability compared to normal cultures. (A) Low-pH–stressed normal fibroblasts showed increased ROS production that corresponded to increasing H2O2 concentrations (*P < 0.05). In KC fibroblast cultures, the baseline ROS activity was significantly greater than in the normal cells (**P < 0.02). With the addition of H2O2, ROS formation did not increase further. (B) Compared with the total cells originally plated, the low-pH–stressed KC fibroblast cultures had a 37% decrease in cell viability (P < 0.005). The low-pH–stressed normal cultures showed a 20% decline. After treatment with 200 or 400 μM H2O2, cell viabilities decreased in both the normal cultures (20% and 12% viability; **P < 0.03, respectively) and KC cultures (13% and 6% viability; ***P < 0.003, respectively) compared to the low-pH–treated cultures. H2O2 = hydrogen peroxide. NL, normal; KC. keratoconus.
Figure 4.
 
KC fibroblast cultures had increased caspase-3 activity compared with normal cultures. (A) Low-pH–treated KC fibroblast cultures had significantly higher caspase-3 activity than did normal cultures (5590 ± 1276 vs. 740 ± 478; *P < 0.02). KC cultures consistently showed higher levels of caspase-3 activity than did normal cultures. (B) Cytochemistry images of the H2O2-treated cells showed that an increased number of KC fibroblasts were FLICA positive, representing increased caspase-3 activity compared with normal cells. H2O2, hydrogen peroxide. NL, normal; KC, keratoconus; *Statistically significant difference between designated groups.
Figure 4.
 
KC fibroblast cultures had increased caspase-3 activity compared with normal cultures. (A) Low-pH–treated KC fibroblast cultures had significantly higher caspase-3 activity than did normal cultures (5590 ± 1276 vs. 740 ± 478; *P < 0.02). KC cultures consistently showed higher levels of caspase-3 activity than did normal cultures. (B) Cytochemistry images of the H2O2-treated cells showed that an increased number of KC fibroblasts were FLICA positive, representing increased caspase-3 activity compared with normal cells. H2O2, hydrogen peroxide. NL, normal; KC, keratoconus; *Statistically significant difference between designated groups.
Figure 5.
 
KC fibroblasts showed loss of ΔΨm compared with normal cultures. After a low-pH stress challenge, KC fibroblasts had a 2.6-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The normal fibroblast red-to-green ratio was 3.433 ± 0.705 compared with the KC fibroblast ratio with 1.333 ± 0.0333, P < 0.04.
Figure 5.
 
KC fibroblasts showed loss of ΔΨm compared with normal cultures. After a low-pH stress challenge, KC fibroblasts had a 2.6-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The normal fibroblast red-to-green ratio was 3.433 ± 0.705 compared with the KC fibroblast ratio with 1.333 ± 0.0333, P < 0.04.
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Figure 1.
 
The KC fibroblast cultures had increased catalase activity in response to H2O2 treatment compared with normal cultures. The linear regression plot of the slope for catalase activity in KC fibroblast cultures increased significantly (P < 0.01) with higher concentrations of H2O2, whereas the normal cultures did not (P = 0.16). For the normal fibroblast cultures, the relative activities for catalase in the untreated and 200 and 400 μM H2O2 were 100%, 120%, and 142%, respectively. The KC cultures were 100%, 256%, and 294%, respectively. H2O2, hydrogen peroxide; NL, normal; KC, keratoconus.
Figure 1.
 
The KC fibroblast cultures had increased catalase activity in response to H2O2 treatment compared with normal cultures. The linear regression plot of the slope for catalase activity in KC fibroblast cultures increased significantly (P < 0.01) with higher concentrations of H2O2, whereas the normal cultures did not (P = 0.16). For the normal fibroblast cultures, the relative activities for catalase in the untreated and 200 and 400 μM H2O2 were 100%, 120%, and 142%, respectively. The KC cultures were 100%, 256%, and 294%, respectively. H2O2, hydrogen peroxide; NL, normal; KC, keratoconus.
Figure 2.
 
KC fibroblast cultures had increased levels of ROS and superoxides compared to normal cultures. (A) In neutral pH conditions, the KC fibroblast cultures had a 1.8-fold increased production of ROS compared with age-matched normal fibroblast cultures (34,940 ± 484 vs. 18,070 ± 2,087; **P < 0.001). (B) In low pH conditions, the KC fibroblast cultures showed a 3.3-fold higher production of ROS than did the normal cultures (17,200 ± 2,913 vs. 4,103 ± 1,161; *P < 0.02). (C) In low-pH conditions, the KC fibroblast cultures had a 6.2-fold higher superoxide production than did the normal fibroblasts (**P < 0.001). (D) KC fibroblasts were positive for superoxides as identified with the green-dye assay (Oxyburst; Invitrogen). NL cultures had minimal superoxide staining. NL, normal; KC, keratoconus; *,**Statistically significant compared with NL.
Figure 2.
 
KC fibroblast cultures had increased levels of ROS and superoxides compared to normal cultures. (A) In neutral pH conditions, the KC fibroblast cultures had a 1.8-fold increased production of ROS compared with age-matched normal fibroblast cultures (34,940 ± 484 vs. 18,070 ± 2,087; **P < 0.001). (B) In low pH conditions, the KC fibroblast cultures showed a 3.3-fold higher production of ROS than did the normal cultures (17,200 ± 2,913 vs. 4,103 ± 1,161; *P < 0.02). (C) In low-pH conditions, the KC fibroblast cultures had a 6.2-fold higher superoxide production than did the normal fibroblasts (**P < 0.001). (D) KC fibroblasts were positive for superoxides as identified with the green-dye assay (Oxyburst; Invitrogen). NL cultures had minimal superoxide staining. NL, normal; KC, keratoconus; *,**Statistically significant compared with NL.
Figure 3.
 
KC fibroblast cultures exposed to low-pH stress had higher levels of ROS formation and decreased cell viability compared to normal cultures. (A) Low-pH–stressed normal fibroblasts showed increased ROS production that corresponded to increasing H2O2 concentrations (*P < 0.05). In KC fibroblast cultures, the baseline ROS activity was significantly greater than in the normal cells (**P < 0.02). With the addition of H2O2, ROS formation did not increase further. (B) Compared with the total cells originally plated, the low-pH–stressed KC fibroblast cultures had a 37% decrease in cell viability (P < 0.005). The low-pH–stressed normal cultures showed a 20% decline. After treatment with 200 or 400 μM H2O2, cell viabilities decreased in both the normal cultures (20% and 12% viability; **P < 0.03, respectively) and KC cultures (13% and 6% viability; ***P < 0.003, respectively) compared to the low-pH–treated cultures. H2O2 = hydrogen peroxide. NL, normal; KC. keratoconus.
Figure 3.
 
KC fibroblast cultures exposed to low-pH stress had higher levels of ROS formation and decreased cell viability compared to normal cultures. (A) Low-pH–stressed normal fibroblasts showed increased ROS production that corresponded to increasing H2O2 concentrations (*P < 0.05). In KC fibroblast cultures, the baseline ROS activity was significantly greater than in the normal cells (**P < 0.02). With the addition of H2O2, ROS formation did not increase further. (B) Compared with the total cells originally plated, the low-pH–stressed KC fibroblast cultures had a 37% decrease in cell viability (P < 0.005). The low-pH–stressed normal cultures showed a 20% decline. After treatment with 200 or 400 μM H2O2, cell viabilities decreased in both the normal cultures (20% and 12% viability; **P < 0.03, respectively) and KC cultures (13% and 6% viability; ***P < 0.003, respectively) compared to the low-pH–treated cultures. H2O2 = hydrogen peroxide. NL, normal; KC. keratoconus.
Figure 4.
 
KC fibroblast cultures had increased caspase-3 activity compared with normal cultures. (A) Low-pH–treated KC fibroblast cultures had significantly higher caspase-3 activity than did normal cultures (5590 ± 1276 vs. 740 ± 478; *P < 0.02). KC cultures consistently showed higher levels of caspase-3 activity than did normal cultures. (B) Cytochemistry images of the H2O2-treated cells showed that an increased number of KC fibroblasts were FLICA positive, representing increased caspase-3 activity compared with normal cells. H2O2, hydrogen peroxide. NL, normal; KC, keratoconus; *Statistically significant difference between designated groups.
Figure 4.
 
KC fibroblast cultures had increased caspase-3 activity compared with normal cultures. (A) Low-pH–treated KC fibroblast cultures had significantly higher caspase-3 activity than did normal cultures (5590 ± 1276 vs. 740 ± 478; *P < 0.02). KC cultures consistently showed higher levels of caspase-3 activity than did normal cultures. (B) Cytochemistry images of the H2O2-treated cells showed that an increased number of KC fibroblasts were FLICA positive, representing increased caspase-3 activity compared with normal cells. H2O2, hydrogen peroxide. NL, normal; KC, keratoconus; *Statistically significant difference between designated groups.
Figure 5.
 
KC fibroblasts showed loss of ΔΨm compared with normal cultures. After a low-pH stress challenge, KC fibroblasts had a 2.6-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The normal fibroblast red-to-green ratio was 3.433 ± 0.705 compared with the KC fibroblast ratio with 1.333 ± 0.0333, P < 0.04.
Figure 5.
 
KC fibroblasts showed loss of ΔΨm compared with normal cultures. After a low-pH stress challenge, KC fibroblasts had a 2.6-fold loss of ΔΨm, as measured by the ratio of red fluorescence (live cells) to green fluorescence (apoptotic cells). The normal fibroblast red-to-green ratio was 3.433 ± 0.705 compared with the KC fibroblast ratio with 1.333 ± 0.0333, P < 0.04.
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
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