Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Homodimerization Is Essential for the Receptor for Advanced Glycation End Products (RAGE)-mediated Signal Transduction

The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that binds to diverse ligands and initiates a downstream proinflammatory signaling cascade. RAGE activation has been linked to diabetic complications, Alzheimer disease, infections, and cancers. RAGE is known to mediate cell signaling and downstream proinflammatory gene transcription activation, although the precise mechanism surrounding receptor-ligand interactions is still being elucidated. Recent fluorescence resonance energy transfer evidence indicates that RAGE may form oligomers on the cell surface and that this could be related to signal transduction. To investigate whether RAGE forms oligomers, protein-protein interaction assays were carried out. Here, we demonstrate the interaction between RAGE molecules via their N-terminal V domain, which is an important region involved in ligand recognition. By protein cross-linking using water-soluble and membrane-impermeable cross-linker bis(sulfosuccinimidyl) suberate and nondenaturing gels, we show that RAGE forms homodimers at the plasma membrane, a process potentiated by S100B and advanced glycation end products. Soluble RAGE, the RAGE inhibitor, is also capable of binding to RAGE, similar to V peptide, as shown by surface plasmon resonance. Incubation of cells with soluble RAGE or RAGE V domain peptide inhibits RAGE dimerization, subsequent phosphorylation of intracellular MAPK proteins, and activation of NF-kappa B pathways. Thus, the data indicate that dimerization of RAGE represents an important component of RAGE-mediated cell signaling.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)

Aims/hypothesis: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia.

Methods: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting.

Results: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia.

Conclusions/interpretation: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.

AGEs, RAGE, and Diabetic Retinopathy

Diabetic retinopathy is a major diabetic complication with a highly complex etiology. Although there are many pathways involved, it has become established that chronic exposure of the retina to hyperglycemia gives rise to accumulation of advanced glycation end products (AGEs) that play an important role in retinopathy. In addition, the receptor for AGEs (RAGE) is ubiquitously expressed in various retinal cells and is upregulated in the retinas of diabetic patients, resulting in activation of pro-oxidant and proinflammatory signaling pathways. This AGE-RAGE axis appears to play a central role in the sustained inflammation, neurodegeneration, and retinal microvascular dysfunction occurring during diabetic retinopathy. The nature of AGE formation and RAGE signaling bring forward possibilities for therapeutic intervention. The multiple components of the AGE-RAGE axis, including signal transduction, formation of ligands, and the end-point effectors, may be promising targets for strategies to treat diabetic retinopathy.

RAGE Regulates Immune Cell Infiltration and Angiogenesis in Choroidal Neovascularization

Purpose: RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD).

Methods: RAGE null (RAGE−/−) mice and age-matched wild type (WT) control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV) lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs) towards S100B was investigated.

Results: RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001). RAGE−/− mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05). S100B mRNA was upregulated in the lasered WT retina but not RAGE−/− retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE−/− mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE−/− mice when compared to WT counterparts (p<0.001). A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05–0.01) but this was not apparent in cells isolated from RAGE−/− mice.

Conclusions: RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.

Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice

Aims/hypothesis
The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy.
Methods
Diabetes was induced in wild-type (WT) and Rage −/− mice (also known as Ager −/− mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. 

Results
Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage −/− mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage −/− mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05–0.001). Rage −/− mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. 

Conclusions/interpretation Rage −/− in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.

Impaired retinal angiogenesis in diabetes: role of advanced glycation end products and galectin-3

Suppression of angiogenesis during diabetes is a recognized phenomenon but is less appreciated within the context of diabetic retinopathy. The current study has investigated regulation of retinal angiogenesis by diabetic serum and determined if advanced glycation end products (AGEs) could modulate this response, possibly via AGE-receptor interactions. A novel in vitro model of retinal angiogenesis was developed and the ability of diabetic sera to regulate this process was quantified. AGE-modified serum albumin was prepared according to a range of protocols, and these were also analyzed along with neutralization of the AGE receptors galectin-3 and RAGE. Retinal ischemia and neovascularization were also studied in a murine model of oxygen-induced proliferative retinopathy (OIR) in wild-type and galectin-3 knockout mice (gal3(-/-)) after perfusion of preformed AGEs. Serum from nondiabetic patients showed significantly more angiogenic potential than diabetic serum (P <0.0001) and within the diabetic group, poor glycemic control resulted in more AGEs but less angiogenic potential than tight control (P <0.01). AGE-modified albumin caused a dose-dependent inhibition of angiogenesis (P <0.001), and AGE receptor neutralization significantly reversed the AGE-mediated suppression of angiogenesis (P <0.01). AGE-treated wild-type mice showed a significant increase in inner retinal ischemia and a reduction in neovascularization compared with non-AGE controls (P <0.001). However, ablation of galectin-3 abolished the AGE-mediated increase in retinal ischemia and restored the neovascular response to that seen in controls. The data suggest a significant suppression of angiogenesis by the retinal microvasculature during diabetes and implicate AGEs and AGE-receptor interactions in its causation.

Upregulation of Oxidative Stress Markers in Human Microvascular Endothelial Cells by Complexes of Serum Albumin and Digestion Products of Glycated Casein

The extent of absorption of dietary advanced glycation end products (AGEs) is not fully known. The possible physiological impact of these absorbed components on inflammatory processes has been studied little and was the aim of this investigation. Aqueous solutions of bovine casein and glucose were heated at 95 degrees C for 5 h to give AGE-casein (AGE-Cas). Simulated stomach and small intestine digestion of AGE-Cas and dialysis (molecular mass cutoff of membrane = 1 kDa) resulted in a low molecular mass (LMM) fraction of digestion products, which was used to prepare bovine serum albumin (BSA)-LMM-AGE-Cas complexes. Stimulation of human microvascular endothelial cells with BSA-LMM-AGE-Cas complexes significantly increased mRNA expression of the receptor of AGE (RAGE), galectin-3 (AGE-113), tumor necrosis factor alpha, and a marker of the mitogen-activated protein kinase pathway (MAPK-1), as well as p65NF-kappa B activation. Cells treated with LMM digestion products of AGE-Cas significantly increased AGE-R3 mRNA expression. Intracellular reactive oxygen species production increased significantly in cells challenged with BSA-LMM-AGE-Cas and LMM-AGE-Cas. In conclusion, in an in vitro cell system, digested dietary AGEs complexed with serum albumin play a role in the regulation of RAGE and down-stream inflammatory pathways. AGE-R3 may protect against these effects.

The role of advanced glycation end products in retinal ageing and disease

The retina is exposed to a lifetime of potentially damaging environmental and physiological factors that make the component cells exquisitely sensitive to age-related processes. Retinal ageing is complex and a raft of abnormalities can accumulate in all layers of the retina. Some of this pathology serves as a sinister preamble to serious conditions such as age-related macular degeneration (AMD) which remains the leading cause of irreversible blindness in the Western world.

The formation of advanced glycation end products (AGEs) is a natural function of ageing but accumulation of these adducts also represents a key pathophysiological event in a range of important human diseases. AGEs act as mediators of neurodegeneration, induce irreversible changes in the extracellular matrix, vascular dysfunction and pro-inflammatory signalling. Since many cells and tissues of the eye are profoundly influenced by such processes, it is fitting that advanced glycation is now receiving considerable attention as a possible pathogenic factor in visual disorders.

This review presents the current evidence for a pathogenic role for AGEs and activation of the receptor for AGEs (RAGE) in initiation and progression of retinal disease. It draws upon the clinical and experimental literature and highlights the opportunities for further research that would definitively establish these adducts as important instigators of retinal disease. The therapeutic potential for novel agents that can ameliorate AGE formation of attenuate RAGE signalling in the retina is also discussed.