|Molecular Vision 2007;
Received 15 September 2007 | Accepted 10 December 2007 | Published 17 December 2007
Sustained stress response after oxidative stress in trabecular meshwork cells
Paloma B. Liton,
David L. Epstein,
Department of Ophthalmology, Duke University, Durham, NC
Correspondence to: Dr. Pedro Gonzalez, Department of Ophthalmology, Duke University Medical Center, Erwin Rd., Box 3802, Durham, NC, 27710; Phone: (919) 681 5995; FAX: (919) 684 8983; email: firstname.lastname@example.org
Purpose: To investigate the mechanisms by which chronic oxidative stress may lead to a sustained stress response similar to that previously observed in the trabecular meshwork (TM) of glaucoma donors.
Methods: Porcine TM cells were treated with 200 μM H2O2 twice a day for four days and were allowed to recover for three additional days. After the treatment, TM cells were analyzed for generation of intracellular reactive oxygen species (iROS), mitochondrial potential, activation of NF-κB, and the expression of inflammatory markers IL-1α, IL-6, IL-8, and ELAM-1. Potential sources of iROS were evaluated using inhibitors for nitric oxide, nitric oxide synthetase, cyclooxygenase, xanthine oxidase, NADPH oxidase, mitochondrial ROS, and PKC. The role of NF-κB activation in the induction of inflammatory markers was evaluated using the inhibitors Lactacystin and BAY11-7082.
Results: Chronic oxidative stress simulated by H2O2 exposure of porcine TM cells resulted in the sustained production of iROS by the mitochondria. Inhibition of mitochondrial iROS had a significant inhibitory effect on the activation of NF-κB and the induction of IL-1α, IL-6, IL-8, and ELAM-1 triggered by chronic oxidative stress. Inhibition of NF-κB partially prevented the induction of IL-1α, IL-8, and ELAM-1, but not IL-6.
Conclusions: Chronic oxidative stress in TM cells induced iROS production in mitochondria. This increase in iROS may contribute to the pathogenesis of the TM in glaucoma by inducing the expression of inflammatory mediators previously observed in glaucoma donors as well as the levels of oxidative damage in the tissue.
Glaucoma is a major cause of irreversible blindness, affecting more than 70 million individuals worldwide . Elevated intraocular pressure (IOP) is a major risk factor in the development of glaucoma  and in the progression of glaucomatous damage . High IOP usually occurs as a result of an increase in aqueous humor outflow resistance in TM. The specific mechanisms leading to the failure of the TM to maintain normal levels of aqueous humor outflow resistance are not yet understood.
It has been reported that glaucoma is characterized by the sustained activation of a tissue-specific stress response in the cells of the TM. Such a stress response includes the sustained activation of NF-κB and the expression of inflammatory markers such as interleukin (IL)-1α and vascular endothelial leukocyte-adhesion molecule (ELAM)-1 .
It has been recently reported that treatment of porcine TM cells with an acute treatment with H2O2 (1 mM concentration) induces the expression of ELAM-1 , suggesting that oxidative stress could contribute to the expression of this protein in POAG. A contributing role for oxidative stress in the morphologic and physiologic alterations in the aqueous outflow pathway in aging and glaucoma has been hypothesized for a long time and is supported by some experimental evidence [6-16].
Sublethal oxidative damage has been shown to result in the induction of inflammatory markers in several cell types [17-19]. Sublethal oxidative damage has also been shown to lead to a prolonged increase in the endogenous generation of iROS in several cell types [20-23]. An increase in iROS generation has the potential to result in sustained activation of NF-κB, which is likely to induce the expression of proinflammatory markers. Therefore, we investigated whether chronic oxidative stress in TM cells can lead to increased production of iROS and whether, in turn, this would result in sustained activation of a stress response involving sustained activation of NF-κB and the expression of inflammatory markers similar to that observed in POAG. We also analyzed the potential sources of iROS generation induced by chronic oxidative stress in porcine TM cells.
Porcine trabecular meshwork cell culture
TM tissue from fresh porcine eyes was digested in 10 mg collagenase/20 mg bovine serum albumin (BSA)/5 ml phosphate buffer saline (PBS) solution. The cells were plated on gelatin coated 10 cm Petri dishes and maintained at 37 °C in a humidified atmosphere of 5% CO2 in TM culture medium. The TM culture medium was low glucose Dulbecco's Modified Eagle Medium (DMEM) with L-glutamine and 110 mg/l sodium pyruvate, supplemented with 10% fetal bovine serum (FBS), 100 μM nonessential amino acids, 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate. All reagents were obtained from Invitrogen Corporation (Carlsbad, CA).
Lactacystin (Lact, L6785), BAY11-7082 (BAY, B5556), Dibenziodolium chloride (DPI, D2926), Oxypurinol (Oxy, O4502), Indomethacin (Indo, I7378), Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, N5751), Apocynin (Apo, A10809), Aminoguanidine bicarbonate salt (AMG, A7259), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (Fccp, C2920), Chelerythrine Chloride (Chele, C2932), and 30% Hydrogen peroxide solution (H2O2, 31642) were commercially obtained from Sigma-Aldrich (St. Louis, MO). 5,5',6,6'-tetrachloro-1, 1',3,3'-tetracthylbenzimidazolylcarbocyanine iodide (JC-1, M34152) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, D-399) were purchased from Molecular Probes (Carlsbad, CA)
Porcine TM cells (passage 4-5) were treated with H2O2 200 \mu M in DMEM containing 10% FBS, twice a day, for four days. To differentiate from acute stress responses to oxidative challenge, TM cells were allowed a recovery time of three days after the H2O2 treatment. The medium was changed with fresh DMEM containing 10% FBS on the first recovery day. For iROS assay, inhibitors were pretreated 1 h in a serum free condition followed by H2DCFDA incubation. For realtime Q-PCR and NF-κB activity assay, inhibitors were used 24 h before RNA and protein extractions. For IL-6 and IL-8 ELISA assay, supernatant was collected at the end of the recovery day.
Preparation of cytosolic and nuclear extracts
Cells were trypsinized and washed twice with cold PBS and lysed with 200 μl cytosolic lysis buffer (10 mM Tris-HCl [pH7.4], 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM dTT, and freshly added proteinase inhibitor cocktail), incubated on ice for 15 min and vortexed 10 s. Cytosolic proteins were collected after centrifugation at 1,500 xg for 5 min at 4 °C. The pellet was washed once with wash buffer (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, and 0.2 mM EDTA), resuspended with nuclear lysis buffer (20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA with freshly added protease inhibitor cocktail), and shaken in an orbital shaker for 30 min at 4 °C. Nuclear proteins were collected after centrifugation at 10,000 xg for 10 min at 4 °C. Protein concentration was measured using the Micro BCA Protein assay kit (23235; Pierce, Rockford, IL) following the instruction.
NoShift II NF-κB assay
Nuclear NF-κB activities were determined with the NoShift II Transcription Factor (71680-3; Novagen, Inc., Madison, WI) following the manufacturer's instructions. Briefly, 3 μg of nuclear protein was mixed with Binding solution and incubated for 20 min at RT. Following additional incubation with Binding Reagent for 20 min at RT, digestion buffer or Digestion Reagent was added to each reaction. The reaction mixtures were incubated at 37 °C for 20 min and then transferred to NoShift II Capture Plate for a 45 min hybridization period at RT with vigorous shaking. After washing four times with Wash Buffer, Detection Reagent was added to each well and the plate was incubated 30 min at RT with shaking. Following a second set of four washes, Chemiluminescent Substrate was added to each well and incubated 30 min at 37 °C. Light intensity was then measured with a microplate chemiluminometer, and the results were adjusted for negative control reading and recorded as relative light units (RLUs).
RNA extraction and real-time quantitative polymerase chain reaction
Total RNA from porcine TM cells was isolated using the RNeasy kit (Qiagen Inc., Valencia, CA) following the manufacturer's protocol. First strand cDNA was synthesized using oligodT primer and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Real-time Q-PCR was performed using iQ SYBR Green Supermix (BioRad, Hercules, CA) in the BioRad iCycler iQ system (BioRad, Hercules, CA). Fold expression changes were determined using the iCycle iQ system software. β-Actin served as an internal standard of mRNA expression. The sequences of the primers used for the amplifications are indicated in Table 1.
Assay of iROS
The production of iROS was measured by DCFH oxidation as described previously, with minor modifications . Briefly, 10 mM H2DCFDA was dissolved in methanol and was diluted 500 fold in HBSS to give a 20 μM concentration of H2DCFDA. H2O2 treated and control cells in 96-well plates were treated with inhibitors and their specific vehicles for 1 h, and then exposed to H2DCFDA for an additional 1 h. After incubation, the fluorescence was read at 485 nm excitation and 530 nm emission on a fluorescence plate reader.
Measurement of mitochondrial membrane potential (Δψm)
Mitochondrial membrane potential was monitored in cells loaded with 2 μM of JC-1 dye according to the manufacturer instructions. JC-1 is a positively charged fluorescent compound, which is taken up by mitochondria proportionally to the inner mitochondrial membrane potential. When a critical concentration is reached, JC-1 monomers (fluorescent green) form aggregates (fluorescent red). Briefly, cells in six-well plates treated with or without H2O2 were trypsinized and pellet by centrifuge. The cells were then washed with PBS once, loaded with JC-1 in 1 ml of PBS to final concentration of 2 μM, and incubated at 37 °C, 5% CO2 for 15 min. The cells were pelleted and washed again with PBS, then resuspended in 200 μl of PBS. The cells were analyzed on a flow cytometer with 488 nm excitation using emission filters appropriate for Alexa Fluor 488 dye and R-phycoerythrin.
Quantification of cytokines by enzyme-linked immunosorbent assay (ELISA)
IL-6 and IL-8 protein levels in cell culture supernatant were determined using the Quantikine porcine ELISA kits (P6000 and P8000; R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The optical density was read using a microplate reader setting to 450 nm with 540 nm correction.
The data were presented as the mean ±SD. For multiple comparisons of groups, ANOVA was used. Statistical significance between groups was assessed by paired or unpaired Student's t test, with Bonferroni's correction. A value of p<0.05 was considered statistically significant
Chronic oxidative stress induced the activation of NF-κB and upregulation of inflammatory markers IL-6, ELAM-1, IL-1α, and IL-8
As indicated in Methods, TM cells were treated with H2O2 (200 μM) twice a day for four days and then they were allowed three days of recovery. After this treatment, TM cells showed a fourfold increase (p<0.01, compared to control) in the levels of activation of NF-κB analyzed by NoShift II NF-κB Assay (Figure 1A). The activation of NF-κB was accompanied by a significant upregulation of inflammatory mediators including IL-6, ELAM-1, IL-1α, and IL-8 (Figure 1B,C).
Oxidative stress results in increased production of iROS and loss of Δψm
As shown in Figure 1D, cells subjected to oxidative stress showed a significant (p<0.01) increase of 25% in iROS production measured with H2DCFDA. This increase in iROS generation was accompanied by a significant (p<0.01) decrease of 30% in mitochondrial membrane potential measured by JC-1 (Figure 1D).
Sources of iROS production after oxidative stress
Inhibition of nitric oxide with L-NAME, nitric oxide synthetase with AMG, cyclooxygenase (COX) with Indo, and xanthine oxidase with Oxy, did not have any significant effect on the induction of iROS (Figure 2A). The NADPH oxidase inhibitor DPI and the PKC inhibitor Chele exerted some inhibitory effect on the total iROS in a dose dependent manner (14%, 25%, and 34% for Chele, and 9%, 13%, and 28% for DPI; p<0.05 and 0.01; Figure 2B,C). The highest effect on iROS production was obtained by inhibiting mitochondrial ROS generation with Fccp (45%, 49%, and 51% reduction; p<0.001; Figure 2D).
Effect of PKC inhibition on the induction of iROS
The PKC inhibitor, Chele showed some effect on the production of iROS (Figure 2B). However, Chele did not have any significant effect on the induction of IL-6, IL-8, IL-1α, and ELAM-1 (data not shown).
Role of iROS in induction of NF-κB, IL-6, ELAM-1, IL-1α, and IL-8
Inhibition of iROS production by Fccp had a significant effect on the reduction of NF-κB activities (43.3%, p<0.01), as well as on the decrease of IL-6 (49.3%, p<0.01), ELAM-1 (84.2%, p<0.001), IL-1α (67.3%, p<0.01) and IL-8 (98.1%, p<0.01) after oxidative stress (Figure 3).
NF-κB activation is involved in the induction of ELAM-1, IL-1α, and IL-8
The two NF-κB inhibitors tested (Lact 10 μM and BAY 5 μM) significantly decreased the induction of ELAM-1 (Lact: 36.2%, p<0.05; BAY: 44.4%, p<0.01) and IL-1α (Lact: 55.9%, p<0.01; BAY: 40%, p<0.05). BAY significantly reduced IL-8 expression, but Lact did not (Lact: 27.6%, p=0.07; BAY: 88.8%, p<0.01). However, none of the NF-κB inhibitors had any significant effect on the induction of IL-6 after oxidative stress (Figure 4).
In the present study, we found that chronic H2O2 treatment resulted in sustained activation of NF-κB and the upregulation of proinflammatory markers, including ELAM-1, IL-1α, IL-6, and IL-8. Our data shows that chronic H2O2 treatment resulted in increased generation of iROS, a key mediator of the observed activation of NF-κB and the induction of inflammatory mediators. A similar increase in iROS production after exogenous H2O2 treatment has been observed in several cell types [20-22,25] and appears to be a feature of sublethal damaged and senescent cells . Different mechanisms have been implicated in the endogenous generation of ROS after an oxidative stress challenge. These mechanisms include the activation of NADPH oxidase (NOX)  to produce anion superoxide radical (O2-) [21,25], intracellular mitochondrial H2O2 generation , xanthine oxidase , uncoupled endothelial nitrite oxide synthase (eNOS) , and COX .
The observed lack of effect of the inhibitors for xanthine oxidase with Oxy, eNOS with L-NAME, AMG, and COX with Indo, suggests that none of these enzymes may contribute significantly to the increase in iROS production after oxidative challenge in TM cells.
The induction of iROS was partially inhibited by the NOX inhibitor DPI. DPI is a large spectrum inhibitor of electron transporters including not only various NOX enzymes, but also mitochondrial oxidase and xanthine oxidase . Therefore, the effects of DPI could potentially be attributed to inhibitory effects on mitochondrial and xanthine oxidase ROS production. However, since the inhibitor of xanthine oxidase, Oxy, did not have any effect on iROS generation, it is unlikely that the effect observed with DPI would result from xanthine oxidase inhibition. Similarly, the inhibitory effects of DPI on iROS generation can not be attributed to mitochondrial inhibition since inhibition of ROS generation occurred in the submicromolar range, while DPI inhibition of mitochondrial oxidase occurs with an IC50 of approximately 10 μM DPI [30,31]. In addition, while inhibition of iROS generation by the mitochondrial inhibitor Fccp also blocked the activation of NF-κB and upregulation of proinflammatory markers, treatment with DPI had no significant effect in any of these variables. In comparison with DPI, the NOX inhibitor Apo did not have any effect on iROS induction. This result is not necessarily surprising since Apo is known to require myeloperoxidase-dependent metabolism for full activity and therefore does not effectively inhibit NOX activity in all cell types . Therefore, our data suggests that H2O2 treatment may lead to some activation of NOX that should contribute to the total increase in ROS generation, but is not responsible for the sustained activation of NF-κB and the expression of inflammatory markers. In contrast, the mitochondrial inhibitor Fccp, which is a protonophore (H+ ionophore) and uncoupler of oxidative phosphorylation in mitochondria, was capable of reducing iROS production in a dose-dependent manner. Treatment with Fccp inhibited the induction of NF-κB, IL-1α, IL-6, IL-8, and ELAM-1, suggesting that the production of iROS by the mitochondria plays a key role in the activation of NF-κB and proinflamatory markers that result from chronic oxidative stress in TM cells.
The activation of NF-κB appears to mediate the induction of IL-1α, IL-8, and ELAM-1, but not that of IL-6. Our results showed that neither of the two NF-κB inhibitors tested had any effect on IL-6 induction after oxidative stress. Therefore, endogenous ROS may activate other pathways different from NF-κB that also contribute to the induction of inflammatory mediators after oxidative stress including the induction of IL-6.
PKC has been implicated in the activation of iROS production by NOX and mitochondria in several cell types [33-35]. Treatment of TM cells with Chele, a PKC inhibitor, resulted in partial inhibition of iROS generation with no effect observed on the induction of inflammatory markers. Therefore, although activation of PKC might contribute to the induction of iROS production, it does not appear to be the exclusive mechanism leading to iROS generation and is not necessary to activate the induction of inflammatory mediators. Therefore, alternative mechanisms should be involved in the activation of iROS generation.
It has been proposed that while short-term activation of inflammatory mediators such as IL-1α and IL-6 may help to decrease IOP [36,37], their chronic activation may exert pathologic affects on the TM and contribute to the progression of glaucoma . Therefore, the observed induction of iROS production resulting form oxidative stress may constitute mechanisms by which oxidative stress could contribute to the pathogenesis of glaucoma through the sustained activation of inflammatory mediators.
In addition to the upregulation of proinflammatory cytokines, the observed induction of iROS after chronic oxidative stress also has the potential to contribute by itself to pathophysiological changes of the outflow pathway by increasing the levels of oxidative damage in the cells. Consistent with this concept, our data showed a decrease in mitochondria potential following this chronic H2O2 treatment (Figure 2). Furthermore, the mitochondria damage and generation of the secondary ROS was persistent since loss of mitochondria potential is irreversible by Fccp (data not shown).
In summary, our results show that chronic oxidative stress leads to the endogenous production of ROS by the mitochondria in TM cells, which in turn induces a sustained stress response characterized by activation of NF-κB and expression of inflammatory markers. The endogenous production of ROS by TM cells resulting from oxidative stress might thus contribute to the pathogenesis of the TM in glaucoma by inducing the expression of inflammatory mediators previously observed in the TM of glaucoma donors and by increasing the levels of oxidative damage in the tissue.
This work was supported by NEI EY01894, NEI EY016228, NEI EY05722, and Research to Prevent Blindness. We also gratefully thank Dr. Ping Yang's technical support for this project.
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