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Manish Mishra, Renu A. Kowluru; Epigenetic Modification of Mitochondrial DNA in the Development of Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2015;56(9):5133-5142. doi: https://doi.org/10.1167/iovs.15-16937.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal mitochondria are dysfunctional in diabetes, and mitochondrial DNA (mtDNA) is damaged and its transcription is compromised. Our aim was to investigate the role of mtDNA methylation in the development of diabetic retinopathy.
Effect of high glucose (20 mM) on mtDNA methylation was analyzed in retinal endothelial cells by methylation-specific PCR and by quantifying 5-methylcytosine (5mC). Dnmt1 binding at the D-loop and Cytb regions of mtDNA was analyzed by chromatin immunoprecipitation. The role of mtDNA methylation in transcription and cell death was confirmed by quantifying transcripts of mtDNA-encoded genes (Cytb, ND6, and CoxII) and apoptosis, using cells transfected with Dnmt1-small interfering RNA (siRNA), or incubated with a Dnmt inhibitor. The key parameters were validated in the retinal microvasculature from human donors with diabetic retinopathy.
High glucose increased mtDNA methylation, and methylation was significantly higher at the D-loop than at the Cytb and CoxII regions. Mitochondrial accumulation of Dnmt1 and its binding at the D-loop were also significantly increased. Inhibition of Dnmt by its siRNA or pharmacologic inhibitor ameliorated glucose-induced increase in 5mC levels and cell apoptosis. Retinal microvasculature from human donors with diabetic retinopathy presented similar increase in D-loop methylation and decrease in mtDNA transcription.
Hypermethylation of mtDNA in diabetes impairs its transcription, resulting in dysfunctional mitochondria and accelerated capillary cell apoptosis. Regulation of mtDNA methylation has potential to restore mitochondrial homeostasis and inhibit/retard the development of diabetic retinopathy.
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