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Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors.

McElnea EM, Quill B, Docherty NG, Irnaten M, Siah WF, Clark AF, O'Brien CJ, Wallace DM - Mol. Vis. (2011)

Bottom Line: Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control.MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC.Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

View Article: PubMed Central - PubMed

Affiliation: 1Institute of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.

ABSTRACT

Purpose: Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH.

Methods: Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca(2+)](i)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively.

Results: Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR.

Conclusions: Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

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Hydrogen peroxide treatment of NLC cells causes a down-regulation in expression of Ca2+ extrusion system (NCX-1, PMCA-1, and PMCA-4) expression. NLC cells obtained from 3 donors (N1, N2, and N3) were treated with 200 µM H202 for 1 h, RNA isolated and gene expression of NCX-1, PMCA-1, and PMCA-4 was analyzed by quantitative PCR. Control (C) NLC cells were included for expressional comparison and assigned the arbitrary value of 1. Introduction of H2O2 significantly reduced expression of Ca2+ extrusions systems (NCX-1, PMCA-1, and PMCA-4) in some cell lines. Although, not all of the reductions are statistically significant; most approach significance and exhibit a downward trend. Results are representative of 3 individual experiments and significant results are denoted by * (p<0.05).
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f5: Hydrogen peroxide treatment of NLC cells causes a down-regulation in expression of Ca2+ extrusion system (NCX-1, PMCA-1, and PMCA-4) expression. NLC cells obtained from 3 donors (N1, N2, and N3) were treated with 200 µM H202 for 1 h, RNA isolated and gene expression of NCX-1, PMCA-1, and PMCA-4 was analyzed by quantitative PCR. Control (C) NLC cells were included for expressional comparison and assigned the arbitrary value of 1. Introduction of H2O2 significantly reduced expression of Ca2+ extrusions systems (NCX-1, PMCA-1, and PMCA-4) in some cell lines. Although, not all of the reductions are statistically significant; most approach significance and exhibit a downward trend. Results are representative of 3 individual experiments and significant results are denoted by * (p<0.05).

Mentions: Previous studies have shown that oxidative stress (hydrogen peroxide) can alter the expression of Ca2+ channels [34,37,39] thus, we investigated the redox modulation of Ca2+ transport proteins in NLC cells following exposure to H202 (Figure 5). Cells were treated with 200 µM H202 for 1 h before RNA isolation and assessment of Ca2+ extrusion transporter expression by RT–PCR. Results demonstrate that NLC cells subjected to H202-induced oxidative stress results in a down-regulation of NCX-1, PMCA-1, and PMCA-4. While some donors show a non-statistical decrease, all approached significance.


Oxidative stress, mitochondrial dysfunction and calcium overload in human lamina cribrosa cells from glaucoma donors.

McElnea EM, Quill B, Docherty NG, Irnaten M, Siah WF, Clark AF, O'Brien CJ, Wallace DM - Mol. Vis. (2011)

Hydrogen peroxide treatment of NLC cells causes a down-regulation in expression of Ca2+ extrusion system (NCX-1, PMCA-1, and PMCA-4) expression. NLC cells obtained from 3 donors (N1, N2, and N3) were treated with 200 µM H202 for 1 h, RNA isolated and gene expression of NCX-1, PMCA-1, and PMCA-4 was analyzed by quantitative PCR. Control (C) NLC cells were included for expressional comparison and assigned the arbitrary value of 1. Introduction of H2O2 significantly reduced expression of Ca2+ extrusions systems (NCX-1, PMCA-1, and PMCA-4) in some cell lines. Although, not all of the reductions are statistically significant; most approach significance and exhibit a downward trend. Results are representative of 3 individual experiments and significant results are denoted by * (p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102025&req=5

f5: Hydrogen peroxide treatment of NLC cells causes a down-regulation in expression of Ca2+ extrusion system (NCX-1, PMCA-1, and PMCA-4) expression. NLC cells obtained from 3 donors (N1, N2, and N3) were treated with 200 µM H202 for 1 h, RNA isolated and gene expression of NCX-1, PMCA-1, and PMCA-4 was analyzed by quantitative PCR. Control (C) NLC cells were included for expressional comparison and assigned the arbitrary value of 1. Introduction of H2O2 significantly reduced expression of Ca2+ extrusions systems (NCX-1, PMCA-1, and PMCA-4) in some cell lines. Although, not all of the reductions are statistically significant; most approach significance and exhibit a downward trend. Results are representative of 3 individual experiments and significant results are denoted by * (p<0.05).
Mentions: Previous studies have shown that oxidative stress (hydrogen peroxide) can alter the expression of Ca2+ channels [34,37,39] thus, we investigated the redox modulation of Ca2+ transport proteins in NLC cells following exposure to H202 (Figure 5). Cells were treated with 200 µM H202 for 1 h before RNA isolation and assessment of Ca2+ extrusion transporter expression by RT–PCR. Results demonstrate that NLC cells subjected to H202-induced oxidative stress results in a down-regulation of NCX-1, PMCA-1, and PMCA-4. While some donors show a non-statistical decrease, all approached significance.

Bottom Line: Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control.MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC.Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

View Article: PubMed Central - PubMed

Affiliation: 1Institute of Ophthalmology, Mater Misericordiae University Hospital, Dublin, Ireland.

ABSTRACT

Purpose: Oxidative stress is implicit in the pathological changes associated with glaucoma. The purpose of this study was to compare levels of oxidative stress in glial fibrillary acid-negative protein (GFAP) lamina cribrosa (LC) cells obtained from the optic nerve head (ONH) region of 5 normal (NLC) and 4 glaucomatous (GLC) human donor eyes and to also examine mitochondrial function and calcium homeostasis in this region of the ONH.

Methods: Intracellular reactive oxygen species (ROS) production was examined by a thiobarbituric acid reactive substances (TBARS) assay which measures malondialdehyde (MDA), a naturally occurring product of lipid peroxidation and is used as an indicator of oxidative stress. Mitochondrial membrane potential (MMP) and intracellular calcium ([Ca(2+)](i)) levels were evaluated by flow cytometry using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetrabenzimidazolecarbocyanine iodide) and fluo-4/AM probes respectively. Anti-oxidant and Ca(2+) transport system gene and protein expression were determined by real time polymerase chain reaction (RT-PCR) using gene-specific primer/probe sets and western immunoblotting, respectively.

Results: Intracellular ROS production was increased in GLC compared to NLC (27.19 ± 7.05 µM MDA versus 14.59 ± 0.82 µM MDA, p < 0.05). Expression of the anti-oxidants Aldo-keto reductase family 1 member C1 (AKR1C1) and Glutamate cysteine ligase catalytic subunit (GCLC) were significantly lower in GLC (p = 0.02) compared to NLC control. MMP was lower in GLC (57.5 ± 6.8%) compared to NLC (41.8 ± 5.3%). [Ca(2+)](i) levels were found to be higher (p < 0.001) in GLC cells compared to NLC. Expression of the plasma membrane Ca(2+)/ATPase (PMCA) and the sodium-calcium (NCX) exchangers were lower, while intracellular sarco-endoplasmic reticulum Ca(2+)/ATPase 3 (SERCA) expression was significantly higher in GLC compared to NLC. Subjection of NLC cells to oxidative stress (200 µM H(2)0(2)) reduced expression of Na(+)/Ca2(+) exchanger 1 (NCX 1), plasma membrane Ca2+ ATPase 1 (PMCA 1), and PMCA 4 as determined by RT-PCR.

Conclusions: Our data finds evidence of oxidative stress, mitochondrial dysfunction and impaired calcium extrusion in GLC cells compared to NLC cells and suggests their importance in the pathological changes occurring at the ONH in glaucoma. Future therapies may target reducing oxidative stress and / or [Ca(2+)](i).

Show MeSH
Related in: MedlinePlus