Sustained stress response after oxidative stress in trabecular meshwork cells

Abstract

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 microM 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-kappaB, and the expression of inflammatory markers IL-1alpha, 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-kappaB 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-kappaB and the induction of IL-1alpha, IL-6, IL-8, and ELAM-1 triggered by chronic oxidative stress. Inhibition of NF-kappaB partially prevented the induction of IL-1alpha, 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.

Figure 1

Induction of proinflammatory factors by chronic H₂O₂ treatment. TM cells were treated with H₂O₂ 200 μM twice a day for four days and then they were allowed three days to recover. A: Activation of NF-κB measured by NoShift II NF-κB assay. Data represents the mean values of NF-κB activation in relative light units (rlu) ±SD of n=3. B: Realtime Q-PCR analysis of the induction of IL-1α, IL-6, IL-8, and ELAM-1. For each individual sample, the expression level of each gene was first normalized with that of β-actin and then the relative differences between groups were expressed as mean fold changes compared with the controls ±SD of n=3. C: Protein levels of IL-6 and IL-8 in cell culture media assessed by ELISA. Data represent means of protein concentration ±SD of n=6. D: Induction of iROS and decrease of Δψ_m detected by H₂DCFDA and JC-1. Data showed a percentage of control ±SD (n=5) in iROS treated with H₂O₂. This increase in iROS was associated with a reduction of percentage ±SD in Δψ_m (n=3). An asterisk means that p<0.05; a double asterisk means that p<0.01; and three asterisks mean that p<0.001 (compared to nontreated control).

Figure 2

Sources of iROS in pTM cells induced by H₂O₂ treatment. Production of iROS after H₂O₂ treatment was determined by H₂DCFDA. A:The following inhibitors were added to the cultures 1 h before H₂DCFDA incubation: Chele; 5 μM, DPI; 10 μM, Apo; 10 μM, Fccp; 10 μM, L-NAME; 1 mM, AMG; 10 μM, Oxyp; 20 μM, and Indo; 200 μM. The data represent the mean ±SD (n=3) of iROS percentage change treated with inhibitors compared to controls treated with the same vehicles in which the inhibitors were prepared. Additional experiments were conducted to evaluate the dose response to Chele (B), DPI (C), and Fccp (D). An asterisk means p<0.05; a double asterisk means p<0.01; and three asterisks mean p<0.001 (n=3).

Figure 3

Role of iROS generation in the induction of NF-κB and proinflammatory mediators. The activation of NF-κB measured by the NoShift II NF-κB assay and the induction of IL-6, IL-8, IL-1α, and ELAM-1 measured by real-time Q-PCR, were evaluated in pTM cells treated with H₂O₂ in the presence or absence of 5 μM Fccp. The data show percentage changes in cultures treated with Fccp and H₂O₂ compared to those treated with vehicle (ethanol) and H₂O₂. A double asterisk means p<0.01; three asterisks mean p<0.001 (n=3).

Figure 4

Role of NF-κB activation in the induction of proinflammatory mediators. The levels of induction of IL-6, IL-8, IL-1α, and ELAM-1 three days after H₂O₂ treatment were evaluated by realtime Q-PCR in the presence or absence of the NF-κB inhibitors Lact 10 μM and BAY 5 μM. The data show percentage changes in cultures treated with NF-κB inhibitors (Lact and BAY) and H₂O₂ compared to those treated with vehicle (DMSO) and H₂O₂. An asterisk means p<0.05; a double asterisk means p<0.01 (n=3–4).