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D. P. Dash, R. Pringle, A. W. Stitt; The Glyoxalase System Modulates Advanced Glycation Mediated Pathology in Retinal Müller Glia. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1368. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
During diabetes retinal Müller glia show increased glycolytic metabolism which may produce high intracellular levels of methylglyoxal (MGO). MGO is an important precursor for rapid formation of advanced glycation endproducts (AGEs) although many cells possess the glyoxalase enzyme system that can detoxify this AGE-forming agent. Recent evidence suggests that Müller glia exposed to high glucose show MGO-derived adduct formation on endogenous proteins that regulate angiopoietin-2 transcription. We have investigated if modulation of GLO-1 in Müller glia in vitro can influence AGE accumulation and alter VEGF mRNA expression - two linked pathogenic responses that are important in diabetic retinopthy initiation and progression.
We used the human Müller cell line MIO-M1. To over express GLO-I in these cells, a construct was transfected by electroporation. To knockdown the expression of GLO I in Müller glia, pre-designed siRNAs were co-transfected with the GLO-I construct. Post transfection analysis was carried out at mRNA (qRT-PCR) and protein (western blot) level. AGEs were analysed using an MGO-derived AGE-specific monoclonal antibody using immunocytochemistry. Muller glia are a significant source of retinal VEGF so mRNA levels of this growth factor was quantified in the various cells following exposure to high glucose (HG)(25mM) for 10 days or exogenous MGO 175uM for 10 days.
MIO-M1 Müller glia constitutively expressed GLO-I at the mRNA and protein level. GLO-I could be over-expressed in transfected cells (>2 fold) and significantly knocked down using siRNA (~95%). Müller glia grown in HG showed markedly elevated lactate levels suggesting a metabolic switch towards glycolysis. HG or treated with MGO also showed significant cytoplasmic accumulation of AGEs when compared to appropriate controls. AGE levels were supra-elevated when GLO-I activity was knocked down. VEGF expression was also significantly increased in high glucose or MGO-exposed cells when compared to controls. However, when GLO-I was knocked-down VEGF increased by a further x30 and x60 fold respectively.
This study has shown that enhanced glycolytic activity of retinal Muller glia in the context of diabetes can lead to significant AGE accumulation. The protective role of GLO-1 has been shown in these cells, because reduced enzymatic activity in these cells leads to supra-high MGO-derived AGEs and concomitant increases in VEGF.
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