Protection Against Methylglyoxal-derived Ages By Regulation Of Glyoxalse-1 Expression Prevents Retinal Neuroglial And Vasodegenerative Pathology

Abstract

Purpose: : The reactive dicarbonyl methylglyoxal (MG) forms as a metabolite of glycolysis and is an important precursor for rapid formation of advanced glycation endproducts (AGEs). Normally, MG is detoxified by the glyoxalase (GLO) enzyme system and this limits MG-derived AGE accumulation. Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using diabetes-induction in a transgenic rat model that over-expresses GLO-1 we have investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy.

Methods: : Transgenic animals were developed by over-expression of full length GLO-1 cDNA. Diabetes was induced using STZ in wild-type (WT) and GLO-1 rats and the animals sacrificed after 12 or 24 weeks of hyperglycaemia respectively. AGE-formation in retina was determined by immunofluorescence. Müller glia dysfunction was determined using by evaluating GFAP immunoreactivity and spatial localisation of the potassium channel Kir4.1. The formation of acellular capillaries was investigated by the lack of co-localisation of lectin and collagen IV immunoreactivity in retinal flat mounts. Müller cells were treated with siRNA against GLO-1 and formation of the MG-derived AGE adducts carboxyethyl-lysine (CEL) and MG-hydroimidazalone-1 (MGH1) were quantified by mass spectrometry.

Results: : GLO-1 over-expression prevented CEL and MGH1 accumulation in the diabetic retina when compared to WT diabetic counterparts (p<0.01). Diabetes-related increases in Müller glial GFAP expression and loss of Kir4.1 at the vascular endfeet were significantly prevented by GLO-1 over-expression (p<0.05) at both 12 and 24 weeks time points. GLO-1 diabetic animals showed significantly fewer acellular capillaries than WT diabetics (P<0.001) at 24 weeks diabetes. GLO-1 knock-down effectively reduced accumulation of MGO derived AGEs (CEL and MGH1), especially in high glucose conditions.

Conclusions: : This study has shown that detoxification of MG reduces AGE adduct accumulation which, in turn, can prevent formation of key retinal neuroglial and vascular lesions as diabetes progresses. MG-derived AGEs appear to play an important role in diabetic retinopathy.

Keywords: diabetic retinopathy • Muller cells • retina