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A. L. Gramajo, A. Neekhra, S. Luthra, M. Chwa, S. R. Atilano, D. J. Brown, B. D. Kuppermann, M. C. Kenney; Mitochondrial Oxidative Stress Induced by 7-Ketocholesterol in Retinal Pigment Epithelial (ARPE-19) and Microvascular Endothelial Cells (HMVEC) Cultures. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5072.
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The relationship between cholesterol and retinal diseases is well known, but the pathway used by oxysterols to damage retinal structures remains to be elucidated. This study was designed to assess oxysterol-induced mitochondrial DNA (mtDNA) damage and mitochondrial dysfunction in human retinal pigment epithelial (ARPE- 19) and microvascular endothelial (HMVEC) cell lines in culture.
ARPE-19 and HMVEC cultures were grown in DMEM-F12 and M131 supplemented with Microvascular Growth Supplement, respectively. Cultures were treated for 6 and 24 hrs with 20 to 40µg/ml of the oxysterol 7-ketocholesterol (7kCh) and total DNA was extracted. Long-extension-polymerase chain reaction (LX-PCR) was performed to amplify the mtDNA genome and the product separated by electrophoresis on a 0.8% agarose gel stained with ethidium bromide. The mtDNA to nuclear DNA (nDNA) ratio was assessed by PCR to amplify the mtDNA gene ND2 and the genomic 18S product. The JC-1 assay was used to measure mitochondrial membrane potential (ΔΨm). The loss of the ΔΨm is a hallmark for cellular apoptosis.
The intact mtDNA following LX-PCR appeared as a single ~16.2 kb band. In ARPE-19 and HMVEC cultures at 24 hrs, LX-PCR showed a significant decrease in the ~16.2 kb mtDNA band after treatment with 7kCh. In addition, the ΔΨm in the 7kCh-treated ARPE-19 cultures showed a 2.2-fold decrease compared to control cultures (p< 0.001). The HMVEC cultures also showed a decrease in the ND2/18S ratio at 6 and 24hrs (p< 0.001).
Mitochondrial DNA damage and mitochondrial dysfunction are observed in both the retinal pigment epithelial ARPE-19 and microvascular HMVEC cell lines, when treated with 7kCh. These observations suggest that the mitochondrial pathways can be implicated in the pathogenesis of retinal diseases caused by oxysterol-induced oxidative stress.
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