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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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Related in: MedlinePlus

Expression of Ca2+ extrusion and storage systems are altered in GLC cells. Western blot analyses of Ca2+ extrusion and storage systems in NLC cells (n=3 cell lines) and GLC cells (n=3 cell lines). Protein lysates were examined for differential expression of the calcium extrusion components, PMCA, NCX and the SERCA storage pump. Overall, PMCA-1 and PMCA-4 expression is down regulated in GLC cells when compared to NLC cells, a similar pattern is also observed for NCX-1 and NCX-3. Expression of SERCA2 and SERCA3 is significantly upregulated in GLC cells compared to NLC cells. n=3 for NLC and GLC cells. Actin is used as a loading control for this experiment.
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f4: Expression of Ca2+ extrusion and storage systems are altered in GLC cells. Western blot analyses of Ca2+ extrusion and storage systems in NLC cells (n=3 cell lines) and GLC cells (n=3 cell lines). Protein lysates were examined for differential expression of the calcium extrusion components, PMCA, NCX and the SERCA storage pump. Overall, PMCA-1 and PMCA-4 expression is down regulated in GLC cells when compared to NLC cells, a similar pattern is also observed for NCX-1 and NCX-3. Expression of SERCA2 and SERCA3 is significantly upregulated in GLC cells compared to NLC cells. n=3 for NLC and GLC cells. Actin is used as a loading control for this experiment.

Mentions: Based on our observation that intracellular Ca2+ levels are elevated in GLC cells (Figure 3) we determined whether there was differential gene expression of Ca2+ exchangers/pumps involved in the maintenance of Ca2+ homeostasis in GLC compared with NLC cells (Table 1) and confirmed the results at the protein level (Figure 4).


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)

Expression of Ca2+ extrusion and storage systems are altered in GLC cells. Western blot analyses of Ca2+ extrusion and storage systems in NLC cells (n=3 cell lines) and GLC cells (n=3 cell lines). Protein lysates were examined for differential expression of the calcium extrusion components, PMCA, NCX and the SERCA storage pump. Overall, PMCA-1 and PMCA-4 expression is down regulated in GLC cells when compared to NLC cells, a similar pattern is also observed for NCX-1 and NCX-3. Expression of SERCA2 and SERCA3 is significantly upregulated in GLC cells compared to NLC cells. n=3 for NLC and GLC cells. Actin is used as a loading control for this experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Expression of Ca2+ extrusion and storage systems are altered in GLC cells. Western blot analyses of Ca2+ extrusion and storage systems in NLC cells (n=3 cell lines) and GLC cells (n=3 cell lines). Protein lysates were examined for differential expression of the calcium extrusion components, PMCA, NCX and the SERCA storage pump. Overall, PMCA-1 and PMCA-4 expression is down regulated in GLC cells when compared to NLC cells, a similar pattern is also observed for NCX-1 and NCX-3. Expression of SERCA2 and SERCA3 is significantly upregulated in GLC cells compared to NLC cells. n=3 for NLC and GLC cells. Actin is used as a loading control for this experiment.
Mentions: Based on our observation that intracellular Ca2+ levels are elevated in GLC cells (Figure 3) we determined whether there was differential gene expression of Ca2+ exchangers/pumps involved in the maintenance of Ca2+ homeostasis in GLC compared with NLC cells (Table 1) and confirmed the results at the protein level (Figure 4).

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