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W. F. Siah, D. M. Wallace, A. F. Clark, J. G. Flanagan, C. J. O'Brien; The Relationship Between Oxidative Stress, Calcium Homeostasis and Extracellular Matrix Remodeling in Human Lamina Cribrosa Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2705.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular matrix (ECM) remodeling at the optic nerve head (ONH) may be responsible for retinal ganglion cell axon loss in glaucoma. Our group has shown that lamina cribrosa (LC) cells which form the ONH, when subjected to mechanical strain are pro-fibrotic. LC cells from glaucoma patients (GLC) have a raised basal and stretch-induced cytosolic free Ca2+ levels and Ca2+-dependent maxi-K+ channel activity when compared to normal LC (NLC) cells. The main purpose of this study is to understand the link between oxidative stress and Ca2+ homeostasis in ECM remodeling in glaucoma. We aim to study the expression of Ca2+ channels such as plasma membrane Ca2+ ATPase (PMCA), sodium-calcium exchanger (NCX) and Na+/K+-ATPase (NKA) in NLC using an oxidative stress model.
Primary NLC and GLC were grown to confluency and treated with a range of hydrogen peroxide (H2O2) concentrations (200 µM-600 µM) for 60 minutes. The effect of calcium ionophore (A23187) treatment on NLC cells was also studied. Quantitative real-time PCR (RT-PCR) using gene-specific primers for PMCA, NCX, NKA and ECM, was performed in the treated and non-treated NLC cells. This was compared with GLC cells. Brain cDNA was used as a positive control for gene expression. Results were confirmed at the protein level by Western blot.
We found downregulation of PMCA in NLC cells after treatment with H2O2. NCX isoform 1 and NKA were not expressed in LC cells but present in brain cDNA. PMCA and NCX-1 levels were present in both NLC and GLC on Western blotting. ECM gene expressions were altered in NLC cells following treatment with H2O2 and A23187, and confirmed by Western blot.
This study has found that Ca2+ dysregulation and abnormal ECM remodelling at the ONH may be explained by oxidative stress. This provides potential therapy to regulate ECM remodelling at the ONH by means of targeting oxidative stress or regulating Ca2+ homeostasis.
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