Protection against methylglyoxal-derived AGEs by regulation of glyoxalase 1 prevents retinal neuroglial and vasodegenerative pathology
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- Berner, A.K., Brouwers, O., Pringle, R. et al. Diabetologia (2012) 55: 845. doi:10.1007/s00125-011-2393-0
Methylglyoxal (MG) is an important precursor for AGEs. Normally, MG is detoxified by the glyoxalase (GLO) enzyme system (including component enzymes GLO1 and GLO2). Enhanced glycolytic metabolism in many cells during diabetes may overpower detoxification capacity and lead to AGE-related pathology. Using a transgenic rat model that overexpresses GLO1, we investigated if this enzyme can inhibit retinal AGE formation and prevent key lesions of diabetic retinopathy.
Transgenic rats were developed by overexpression of full length GLO1. Diabetes was induced in wild-type (WT) and GLO1 rats and the animals were killed after 12 or 24 weeks of hyperglycaemia. Nε-(Carboxyethyl)lysine (CEL), Nε-(carboxymethyl)lysine (CML) and MG-derived-hydroimidazalone-1 (MG-H1) were determined by immunohistochemistry and by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS). Müller glia dysfunction was determined by glial fibrillary acidic protein (GFAP) immunoreactivity and by spatial localisation of the potassium channel Kir4.1. Acellular capillaries were quantified in retinal flat mounts.
GLO1 overexpression prevented CEL and MG-H1 accumulation in the diabetic retina when compared with WT diabetic counterparts (p < 0.01). Diabetes-related increases in Müller glial GFAP levels and loss of Kir4.1 at the vascular end-feet were significantly prevented by GLO1 overexpression (p < 0.05) at both 12- and 24-week time points. GLO1 diabetic animals showed fewer acellular capillaries than WT diabetic animals (p < 0.001) at 24 weeks’ diabetes.
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 play an important role in diabetic retinopathy.
KeywordsAGEs Diabetic complications Diabetic retinopathy Glyoxalase 1 Methylglyoxal Pathogenesis Retina
Glial fibrillary acidic protein
Receptor for AGEs
Ultra-performance liquid chromatography tandem mass spectrometry
Diabetic retinopathy is a complex condition with a multifactorial pathogenesis. Among several hyperglycaemia-mediated pathogenic mechanisms that may contribute to this diabetic complication is the formation and accumulation of AGEs . Clinical studies have demonstrated that serum levels of Nε-(carboxy-methyl)lysine (CML), pentosidine and methylglyoxal (MG)-derived hydroimidazolone-1 (MG-H1) are associated with disease progression [2, 3, 4]. However, serum AGEs can be variable and evidence suggests that intracellular and matrix-immobilised AGEs are more robust biomarkers . For example, Genuth et al. have demonstrated that crosslinking AGEs on long-lived skin proteins are significantly associated with progression of diabetic retinopathy . Beyond clinical correlates, AGEs and related advanced lipoxidation end-products (ALEs) accumulate in the retinal vasculature and Müller glia during diabetes [6, 7, 8], and AGE inhibitors show efficacy in reducing retinal lesions in diabetic animal models [8, 9, 10, 11].
AGEs can form directly from the reaction of glucose with amino groups or from the reaction with α-oxoaldehydes such as glyoxal (GO), MG and 3-deoxyglucosone (3-DG). These highly reactive intermediates arise from both chemical and metabolic pathways and can lead to rapid adduct formation on lysine and arginine residues . α-Oxoaldehydes occur at elevated levels in cells exposed to high glucose and also in diabetic serum, and are probably the most important source of intra- and extracellular AGEs [13, 14]. For example, MG can give rise to Nε-(carboxyethyl)lysine (CEL), argpyrimidine and MG-H1, which have been identified at elevated levels in diabetic tissues [14, 15].
α-Oxoaldehydes are a normal product of metabolism, and cells possess a range of endogenous enzyme systems that ‘detoxify’ these AGE precursors. For example, a glutathione-dependent glyoxalase complex (formed from glyoxalase I [GLO1] and glyoxalase II [GLO2] components) protects cells by converting GO and MG to d-lactate . The capacity of this enzyme system to limit AGEs is demonstrated in endothelial cells which were transfected to overexpress GLO-1 and subsequently showed less accumulation of MG-derived adducts . This was accompanied by protection against high glucose-mediated dysfunction [17, 18]. Overexpression of GLO1 in diabetic rats has been shown to be protective against AGE formation and oxidative stress in muscle , and also prevent abnormalities in endothelium-dependent vasorelaxation . Likewise, regulation of GLO1 activity in cells can prevent diabetes-related cell dysfunction [21, 22].
MG-derived AGEs are important in diabetic retinopathy. It has been demonstrated that retinal levels of MG-H1 increase by 279% following 24 weeks of diabetes , and that MG-H1 correlates with diabetic retinopathy . In retinal cells, MG induces retinal pericyte dysfunction  and GLO1 has been shown to protect against premature death of pericytes and endothelial cells following high glucose exposure [22, 25].
Given that MG-derived adducts are important clinically and that prevention of this pathway has therapeutic potential, we hypothesise that MG-derived AGEs play a key role in neuroglial and microvascular degenerative pathology during the early and intermediate stages of diabetic retinopathy. If this is the case, modulation of the glyoxalase detoxification system could be protective and prevent formation of key retinal lesions during diabetes.
Diabetic rat model
Rats transgenic for human GLO1 were developed by overexpression of the full-length GLO1 cDNA under control of the cytomegalovirus enhancer/chicken β-actin promoter system and transgene expression in various tissues was confirmed by PCR. GLO1-overexpressing rats were crossed with wild-type (WT) Wistar rats to obtain enough GLO1 progeny for the experiment. The rats were obtained from the Nippon Seibutsu Zairyo Center (Saitama, Japan) and studies using these animals have been recently published [19, 20]. All animal studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments involving rats were reviewed and approved by the Ethics Committee for Animal Care and Use of Maastricht University, the Netherlands.
A total of 55 animals were included in the study (31 WT and 24 GLO1 rats). At the age of 10 weeks, both rat groups were rendered diabetic by a tail-vein injection of streptozotocin (STZ) (65 mg/kg in citrate buffer for 3 months’ duration of diabetes, and 45 mg/kg for 6 months’ duration) to prevent severe illness. At 1 and 12 weeks post diabetes induction, blood glucose was measured and only rats with a blood glucose >15 mmol/l at both time points were included in the study. Diabetes was maintained for 3 and 6 months prior to death.
Rat retinal immunohistology
Enucleated eyes were fixed in 4% paraformaldehyde (PFA) for 30 min, stored in PBS containing 0.1% sodium azide and shipped from the University of Maastricht, the Netherlands to Belfast for investigation. The posterior eye-cup was separated from the globes and the retinas then dissected into two. The half retinas from each sample population were immediately mounted in cryo-embedding compound (BDH, Poole, UK) in a bath of iso-pentane surrounded with dry ice. Samples were stored at −20°C until used. Prior to sectioning, samples were allowed to equilibrate for approximately 20 min in a cryostat. Retinas were vertically sectioned at 12 or 20 μm and then collected on Superfrost/Plus microscope slides (Menzel-Glaser, Braunschweig, Germany) and stored at −20°C until use. The other half retinas were used as flat mount preparations (see Staining for acellular capillaries).
Frozen retinal sections were thawed at room temperature and then circled with a DAKO pen (DAKO, Glostrup, Denmark) to provide a barrier for the solutions applied to the sections. They were rehydrated by the application of 200 μl of PBS (3 × 10 min). Samples were blocked (PBS, pH 7.4, 10% goat serum, 0.1% Tritron X-100) for 1 h in a humidity chamber, followed by incubation in a range of primary antibodies including: MG-H1, GLO1 (BioMac, Leipzig, Germany), glial fibrillary acidic protein (GFAP) (Dako), CEL and CML (Cosmobio, Carlsbad, CA, USA), and Kir4.1 (Alomone, Jerusalem, Israel). All primary antibodies were diluted in blocking solution overnight at 4°C. A series of 4 × 10 min washes in PBS was followed by a 1-h incubation at room temperature in an appropriate alexa fluor secondary antibody in the dark (Molecular Probes, Paisley, UK). Sections were washed again 4 × 10 min in PBS and later incubated with DAPI nuclear stain for 5 min, followed by a series of 3 × 5 min washes. Sections were coverslipped with vectashield (Vector Laboratories, Peterborough, UK). Control experiments were performed by addition of secondary antibody to observe the extent of non-specific antibody binding and secondary antibody autofluorescence.
Fluorescence was either visualised using a Nikon TE-2000 inverted microscope fitted with Nikon C1 confocal system (Nikon, Surrey, UK) or an epi-fluorescence microscope (Nikon Eclipse E400; Nikon). Images were taken from central regions of the retina. For each primary antibody used in the study, the sample with brightest fluorescence was measured first, and confocal settings were held constant when recording images of all subsequent samples. Images were processed and analysed using either ImageJ software (NIH, Bethesda, MD, USA), NIS Elements software (Nikon) or Velocity software (Mountain View, CA, USA). Immunofluorescence was measured by the intensity of the pixels above a threshold, which was defined as 4 SDs above the mean background fluorescence intensity measured from regions devoid of retinal tissue. A user-defined frame was drawn around each layer of the retina and the average fluorescence intensity above the threshold calculated. Either the left or the right eye was analysed for each rat. Six tissue sections were examined per retina.
Ultra-performance liquid chromatography tandem mass spectrometry analysis
The AGE adducts CML, CEL and MG-H1 were measured in eyes and retina with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MSMS) as described in detail previously . Liquid chromatography was performed at 30°C using an Acquity UPLC BEH C18, 1.7 μm, 2.1 × 100 mm column (Waters, Milford, MA, USA), and the Micromass Quattro Premier XE Tandem Mass Spectrometer (Waters) was used in electrospray-positive multiple reaction monitoring mode.
RNA isolation, reverse transcription and real-time quantitative PCR was performed on retinas isolated from the 3-month experimental groups. The approach was as described previously . Transcripts investigated included Vegf, Tgfβ-1 and -2 (also known as Tgfb1 and -2), Ctgf, Icam1, Gal3 and Timp1.
Acellular capillary quantification
Retina flat mounts were prepared for immunofluorescence evaluation as previously described . The retinal flat mounts were stained with biotinylated isolectin GS-IB4 overnight (Sigma, Dorset, UK) or for collagen IV immunoreactivity (Acris Antibodies, Germany). Appropriate ligand (streptavidin Alexa Fluor 488) and secondary antibody were used (Alexa Fluor 568 goat anti-rabbit IgG) (both from Molecular Probes). DAPI (Sigma) was also added to visualise the nuclear layers of the retina. Stained retinas were imaged using confocal microscopy (Eclipse TE2000-U; Nikon). Five regions were taken at ×20 in the central and peripheral retina for collagen IV and lectin.
Data were expressed as the mean values ± SEM. Statistical differences in the mean were assessed using the unpaired Student’s t test or ANOVA. All the statistical analyses were performed using Graph pad InStat 3.0 (GraphPad Software, San Diego, CA, USA).
GLO1 expression in rat retina
GLO1 overexpression reduces retinal AGEs during diabetes
GLO1 overexpression prevents Müller glia dysfunction during diabetes
GLO1 overexpression in diabetic animals alters retinal expression of key transcripts
GLO1 protects against capillary closure
The relationship between raised intracellular MG, rapid AGE formation and the detoxifying potential of the glyoxalase system has been the focus of intensive study in the area of diabetic complications and ageing [17, 18, 32]. In the context of diabetic retinopathy, the current study has demonstrated that GLO1 overexpression in diabetic rats prevents hyperglycaemia-induced formation of MG-derived AGEs in the neural retina and prevents Müller glia dysfunction and protects against capillary degenerative pathology. This follows on from previous reports in diabetic rats in which GLO1 overexpression protected against elevated tissue and systemic circulating levels of AGEs and oxidative stress markers , and also prevented hyperglycaemia-mediated impairment of arterial vasorelaxation .
Accumulation of AGEs in the diabetic retina is an established phenomenon and their inhibition can effectively prevent key aspects of diabetic retinopathy [8, 9] . Although AGEs form in the retina as diabetes progresses, their precise derivation and the relative importance of various adducts remains unclear. The efficacy of benfotiamine in preventing lesions of diabetic retinopathy by addressing hyperglycaemia-mediated oxidative stress and associated elevations of intracellular MG suggests that this α-oxoaldehyde is an important source of AGEs . To further dissect the importance of MG-derived AGEs in diabetic retinas, the current study has combined immunohistochemistry and UPLC-MSMS to show that CEL, CML and MG-H1 are elevated when compared with non-diabetic controls, and that these adducts appear to accumulate in the retina, particularly in Müller glia. Furthermore, the evidence that GLO1 overexpression can prevent retinal cell dysfunction and death indicates that MG and GO are not only major precursors for AGEs in the diabetic retina but also that they impact on disease progression.
MG-derived adduct formation also occurs alongside the ‘classic’ Maillard chemistry and other pathways such as myeloperoxidase-mediated protein modification via glycolaldehyde , which also contribute to the ‘AGE-load’ during diabetes. However, MG-H1 derived from MG, GO and 3-DG appear to be the major AGEs quantitatively measured in whole retina . It has also been shown that MG-derived adducts accumulate around the retinal blood vessels of diabetic mice . The current study indicates that Müller glia may be an important retinal cell-type for AGE formation in the diabetic retina. In the current study, galectin 3, a protein that binds AGEs and has been shown to be associated with blood retinal barrier dysfunction in diabetes , is upregulated in WT diabetic rats. Interestingly, GLO1 overexpression attenuates this diabetes-induced response. Such data indicate that further studies on the role of GLO1 in retinal vasopermeability during diabetes are warranted.
A range of AGE adducts may also bind to the receptor for AGEs (RAGE). RAGE is known to be highly expressed by Müller glia in the diabetic retina  and, although ligands such as S100B are important in this context, it is possible that receptor activation by MG-derived AGEs could be playing an important role in diabetic retinopathy. Ongoing research is seeking to examine the pathophysiological implications of RAGE and MG-derived AGEs in Müller glia and their contribution to retinal lesions during diabetes.
GLO1 is part of an enzyme complex that requires reduced glutathione (GSH) as a co-factor and it is possible that following genetic overexpression its detoxification ability would be limited by available GSH. Previous studies have suggested that GSH and oxidised GSH do not change in diabetic retina, although enzymatic activity of superoxide dismutase, glutathione peroxidase, glutathione reductase and glutathione transferase are all reduced in diabetic rats compared with controls . We have previously published that GLO1 activity remains high in many tissues of these transgenic rats (including the eye) , and the observation that MG-related retinal AGEs are concomitantly reduced suggests that GSH is not limiting in the retina of these transgenic rats.
In addition to the glyoxalase-mediated detoxification, the aldose reductase (AR) pathway also plays a role in regulating intracellular levels of MG . Indeed, MG acts as an aldehyde substrate of AR, which also requires GSH for enzymatic activity , although both enzyme systems appear to react differently to the diabetic milieu. For example in diabetic lens, GLO1 activity goes down, whereas AR is enhanced, and the AGE inhibitor pyridoxamine corrects both these responses . In many tissues where GSH is high and AR activity low, the glyoxalase system may be the main detoxifier of MG . Müller glia contain appreciable levels of intracellular GSH where it has antioxidant activity and is also linked to glutamate transporter activity . Therefore, it is possible that AR and glyoxalase enzymes could both contribute to MG detoxification in the retina. How AR and glyoxalase enzymatic interactions contribute overall to retinal AGEs during diabetes requires further study.
Dysfunction of retinal Müller glia during diabetes has been described previously [31, 40] and manifests as upregulation of GFAP, cytokine expression and impaired protection against retinal excitotoxicity [41, 42, 43]. Winkler et al. have demonstrated that Müller glia exposed to high glucose conditions in vitro produce excess lactate, indicative of increased glycolytic flux , which would suggest enhanced susceptibility to MG and GO toxicity. In the current study, GLO1 overexpression protected against changes in GFAP production as a robust stress response by Müller glia associated with AGEs and associated lipid modifications . In addition, GLO1 overexpression also prevented the diabetes-linked mis-localisation of Kir4.1, which is a phenomenon also impacting on aquaporin 4 channel function in Müller glia [31, 45] and has already been linked to AGE accumulation . Such upset of retinal K+ clearance and hydration balance could promote oedema and neuronal hyper-excitation as diabetes progresses . Kir4.1 has numerous arginine residues, although it remains unknown if this channel could be directly modified by MG with functional consequences for channel function or its normal association with α-syntrophin .
One of the most significant findings of the current study is that GLO1 overexpression protects against retinal capillary degeneration over 6 months of diabetes duration. We cannot be definitive if this phenomenon links completely to the observed Müller glia dysfunction established after 3 months of diabetes and ongoing after 6 months. However, such a link would seem likely as these glia play a major role in vascular integrity in the retina and disruption of the normal vascular-glial interactions, for example in ion exchange at the Müller glia end-feet , would impact on capillary degeneration. In addition, the importance of MG in endothelial cell damage during diabetes is established  and it has been previously demonstrated that GLO1 can regulate dysfunction of these cells during high glucose exposure in vitro [17, 18]. In the current in vivo study, it is likely that GLO1 upregulation maintains the retinal capillary network by a combination of reducing MG-derived AGEs in Müller glia and endothelial cells.
In conclusion, our study demonstrates that elevating GLO1 offers protection against diabetic retinopathy and the data are consistent with a strong link between elevated MG, AGE formation and cell damage. Recently, it has also been demonstrated that the angiotensin type II receptor inhibitor candesartin can increase GLO1 activity, prevent formation of MG-derived AGEs and protect against key retinopathic lesions in vivo . Therefore, there is considerable hope that enzymatic detoxification of AGE precursors could be a useful therapeutic target to prevent diabetic complications such as retinopathy.
This work was funded by the Juvenile Diabetes Research Foundation (JDRF), Fight for Sight UK, a Dutch Diabetes Foundation Grant 2005.11.013 and Grant 2005.00.042 from the Diabetes Fonds Nederland. The authors would like to acknowledge the expert technical support of I. Vogels.
All authors contributed to analysis and interpretation of data, were involved in drafting the article and approved the final submission. In addition, AWS and CS contributed to the conception and design of the research and directed the overall study.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.