The biology of the receptor for advanced glycation end products and its ligands

Receptor for advanced glycation end products (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands includes advanced glycation end products (AGEs), amyloid fibrils, amphoterins and S100/calgranulins. The overlapping distribution of these ligands and cells overexpressing RAGE results in sustained receptor expression which is magnified via the apparent capacity of ligands to upregulate the receptor. We hypothesize that RAGE-ligand interaction is a propagation factor in a range of chronic disorders, based on the enhanced accumulation of the ligands in diseased tissues. For example, increased levels of AGEs in diabetes and renal insufficiency, amyloid fibrils in Alzheimer's disease brain, amphoterin in tumors and S100/calgranulins at sites of inflammation have been identified. The engagement of RAGE by its ligands can be considered the 'first hit' in a two-stage model, in which the second phase of cellular perturbation is mediated by superimposed accumulation of modified lipoproteins (in atherosclerosis), invading bacterial pathogens, ischemic stress and other factors. Taken together, these 'two hits' eventuate in a cellular response with a propensity towards tissue destruction rather than resolution of the offending pathogenic stimulus. Experimental data are cited regarding this hypothesis, though further studies will be required, especially with selective low molecular weight inhibitors of RAGE and RAGE knockout mice, to obtain additional proof in support of our concept.     

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.     

Advanced glycation endproducts alter functions and promote apoptosis in endothelial progenitor cells through receptor for advanced glycation endproducts mediate overpression of cell oxidant stress

Endothelial progenitor cells (EPCs) play an important role in preventing atherosclerosis. The factors that regulate the function of EPCs are not completely clear. Increased formation of advanced glycation endproducts (AGEs) is generally regarded as one of the main mechanisms responsible for vascular damage in patients with diabetes and atherosclerosis. AGEs lead to the generation of reactive oxygen species (ROS) and part of the regenerative capacity of EPCs seems to be due to their low baseline ROS levels and reduced sensitivity to ROS-induced cell apoptosis. Therefore, we tested the hypothesis that AGEs can alter functions and promote apoptosis in EPCs through overpress cell oxidant stress. EPCs, isolated from bone marrow, were cultured in the absence or presence of AGEs (50, 100, and 200 μg/ml). A modified Boyden’s chamber was used to assess the migration of EPCs and the number of recultured EPCs was counted to measure the adhesiveness function. MTT assay was used to determine the proliferation function. ROS were analyzed using the ROS assay kit. A spectrophotometer was used to assess superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activity, and PCR was used to test mRNA expression of SOD and GSH-PX. SiRNA was used to block receptor for advanced glycation endproducts (RAGEs) expression. Apoptosis was evaluated by Annexin V immunostaining and TUNEL staining. Co-culturing with AGEs increases ROS production, decreases anti-oxidant defenses, overpresses oxidant stress, inhibits the proliferation, migration, and adhesion of EPCs, and induces EPCs apoptosis. In addition, these effects were attenuated during block RAGE protein expression by siRNA. AGEs may serve to impair EPCs functions through RAGE-mediate oxidant stress, and promote EPCs sensitivity toward oxidative-stress-mediated apoptosis, which indicates a new pathophysiological mechanism of disturbed vascular adaptation in atherosclerosis and suggests that lower levels of AGEs might improve the success of progenitor cell therapy.     

The shedding-derived soluble receptor for advanced glycation endproducts sustains inflammation during acute Pseudomonas aeruginosa lung infection

BackgroundThe membrane-bound isoform of the receptor for advanced glycation end products (FL-RAGE) is primarily expressed by alveolar epithelial cells and undergoes shedding by the protease ADAM10, giving rise to soluble cleaved RAGE (cRAGE). RAGE has been associated with the pathogenesis of several acute and chronic lung disorders. Whether the proteolysis of FL-RAGE is altered by a given inflammatory stimulus is unknown.Pseudomonas aeruginosa causes nosocomial infections in hospitalized patients and is the major pathogen associated with chronic lung diseases.MethodsP. aeruginosa was injected in Rage −/− and wild-type mice and the impact on RAGE expression and shedding, levels of inflammation and bacterial growth was determined.ResultsAcute P. aeruginosa lung infection in mice induces a reduction of the active form of ADAM10, which determines an increase of FL-RAGE expression on alveolar cells and a concomitant decrease of pulmonary cRAGE levels. This was associated with massive recruitment of leukocytes and release of pro-inflammatory factors, tissue damage and relocation of cRAGE in the alveolar and bronchial cavities. The administration of sRAGE worsened bacterial burden and neutrophils infiltration. RAGE genetic deficiency reduced the susceptibility to P. aeruginosa infection, mitigating leukocyte recruitment, inflammatory molecules production, and bacterial growth.ConclusionsThese data are the first to suggest that inhibition of FL-RAGE shedding, by affecting the FL-RAGE/cRAGE levels, is a novel mechanism for controlling inflammation to acute P. aeruginosa pneumonia. sRAGE in the alveolar space sustains inflammation in this setting.General significanceRAGE shedding may determine the progression of inflammatory lung diseases.     

An update on advanced glycation endproducts and atherosclerosis

Advanced glycation endproducts (AGEs) are a group of modified molecular species formed by nonenzymatic reactions between the aldehydic group of reducing sugars with proteins, lipids, or nucleic acids. Formation and accumulation of AGEs are related to the aging process and are accelerated in diabetes. AGEs are generated in hyperglycemia, but their production also occurs in settings characterized by oxidative stress and inflammation. These species promote vascular damage and acceleration of atherosclerotic plaque progression mainly through two mechanisms: directly, altering the functional properties of vessel wall extracellular matrix molecules, or indirectly, through activation of cell receptor-dependent signaling. Interaction between AGEs and the key receptor for AGEs (RAGE), a transmembrane signaling receptor which is present in all cells relevant to atherosclerosis, alters cellular function, promotes gene expression, and enhances the release of proinflammatory molecules. The importance of the AGE-RAGE interaction and downstream pathways, leading to vessel wall injury and plaque development, has been amply established in animal studies. Moreover, the deleterious link of AGEs with diabetic vascular complications has been suggested in many human studies. Blocking the vicious cycle of AGE-RAGE axis signaling may be essential in controlling and preventing cardiovascular complications. In this article, we review the pathogenetic role of AGEs in the development, progression and instability of atherosclerosis, and the potential targets of this biological system for the prevention and treatment of cardiovascular disease.     

Localization of the ezrin binding epitope for advanced glycation endproducts

Glycated proteins/advanced glycation endproducts contribute to the development of diabetic complications but the precise pathway from glycated proteins to complications is still being delineated. The ezrin, radixin and moesin protein family is a new class of advanced glycation endproduct-binding protein and we hypothesize that advanced glycation endproducts mediate some of their detrimental effects leading to diabetic complications by inhibiting ezrin's actions. Our previous study revealed that glycated proteins bind to the N-terminal domain of ezrin (aa 1–324) and this study further defines the ezrin binding epitope. Binding of glycated albumin to recombinant N-ezrin deletion constructs (aa 1–280, 1–170 and 1–144) and glutathione-S-transferase-N-ezrin fusion proteins, (aa 200–324 and 270–324) was analysed using ligand and far Western blotting, and surface plasmon resonance. Glycated albumin binding was markedly reduced on removal of amino acids 280–324, while binding was preserved in the fusion proteins. A series of peptides based on residues 280–324 was synthesized and those containing residues 277–299 of ezrin bound maximally. Peptide binding to glycated albumin was glycation-specific. An ezrin peptide (aa 277–299) dose-dependently reversed the inhibitory effect of glycated albumin on ezrin (1–324) phosphorylation in vitro, suggesting that binding of advanced glycation endproducts to ezrin changes the conformation of the latter sufficiently to alter binding interactions distant from the advanced glycation endproduct-binding site. This may have consequences for subcellular ezrin localization and signalling pathways. Altogether, these studies provide important structural knowledge for developing peptide antagonists that may be therapeutically useful in preventing advanced glycation endproduct:ezrin interactions in diabetes.     

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014).In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.     

Receptor for advanced glycation endproducts mediates neutrophil migration across intestinal epithelium

Receptor for advanced glycation endproducts (RAGE) is an Ig superfamily cell surface receptor that interacts with a diverse array of ligands associated with inflammatory responses. In this study, we provide evidence demonstrating that RAGE is involved in inflammatory responses in the intestines. We showed that RAGE is expressed in intestinal epithelial cells, primarily concentrated at the lateral membranes close to the apical cell junction complexes. Although RAGE expression was low in epithelium under normal conditions, this protein was up-regulated after treatment with the inflammatory cytokines IFN-gamma and/or TNF-alpha. RAGE expression was also elevated in colon tissue samples from patients with inflammatory bowel diseases. Using in vitro transmigration assays, we found that RAGE mediates neutrophil (polymorphonuclear leukocytes (PMN)) adhesion to, and subsequent migration across, intestinal epithelial monolayers. This activity appears to be mediated by the binding of RAGE to the PMN-specific beta(2) integrin CD11b/CD18. Thus, these results provide a novel mechanism for the regulation of PMN transepithelial migration and may suggest a new therapeutic target for intestinal inflammation.     

De-N-glycosylation or G82S mutation of RAGE sensitizes its interaction with advanced glycation endproducts

Interactions between advanced glycation endproducts (AGE) and the receptor for AGE (RAGE) have been implicated in the development of diabetic vascular complications. RAGE has two N-glycosylation sites in and near the AGE-binding domain, and G82S mutation in the second N-glycosylation motif was recently reported in human. In this study, we examined whether de-N-glycosylation or G82S of RAGE affect its ability to bind AGE and cellular response to AGE. Recombinant wild-type, de-N-glycosylation and G82S RAGE proteins were produced in COS-7 cells, purified and assayed for ligand-binding abilities. De-N-glycosylation at N81 and G82S mutation decreased Kd for glycolaldehyde-derived AGE to three orders of magnitude lower levels compared with wild-type. AGE-induced upregulation of VEGF mRNA was significantly augmented in endothelial cell-derived ECV304 cells expressing de-N-glycosylated and G82S RAGE when compared with wild-type expressor. Exposure to low glucose resulted in the appearance of RAGE proteins of deglycosylated size in wild-type RAGE-expressing cells and significantly enhanced glycolaldehyde-derived AGE-induced VEGF mRNA expression. De-N-glycosylation or G82S mutation of RAGE increases affinity for AGE ligands, and may sensitize cells or conditions with it to AGE.     

Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1^?/?), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGE_RQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGE_RQ/AA and RAGE-Diaph1^?/? cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.     

Blockade of receptor for advanced glycation endproducts: a new target for therapeutic intervention in diabetic complications and inflammatory disorders

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

Respiratory insufficiency in patients with myasthenia gravis

We evaluated 20 patients who required prolonged mechanical ventilation for respiratory failure associated with myasthenia gravis. All 20 patients survived and were weaned from the ventilator after 3 to 14 days (mean 6.5 days) of respiratory support. Progressing bulbar symptoms and respiratory infection were the most frequent causes of the myasthenic crisis. During a period of assisted mechanical ventilation, anticholinesterase medication was interrupted and the patients were treated with steroids and antibiotics. Plasmapheresis may be considered in the management of myasthenic crisis.     

Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies

Myasthenia gravis (MG) is an antibody-mediated autoimmune disease of the neuromuscular junction. In approximately 80% of patients, auto-antibodies to the muscle nicotinic acetylcholine receptor (AChR) are present. These antibodies cause loss of AChR numbers and function, and lead to failure of neuromuscular transmission with muscle weakness. The pathogenic mechanisms acting in the 20% of patients with generalized MG who are seronegative for AChR-antibodies (AChR-Ab) have not been elucidated, but there is evidence that they also have an antibody-mediated disorder, with the antibodies directed towards another, previously unidentified muscle-surface-membrane target. Here we show that 70% of AChR-Ab-seronegative MG patients, but not AChR-Ab-seropositive MG patients, have serum auto-antibodies against the muscle-specific receptor tyrosine kinase, MuSK. MuSK mediates the agrin-induced clustering of AChRs during synapse formation, and is also expressed at the mature neuromuscular junction. The MuSK antibodies were specific for the extracellular domains of MuSK expressed in transfected COS7 cells and strongly inhibited MuSK function in cultured myotubes. Our results indicate the involvement of MuSK antibodies in the pathogenesis of AChR-Ab-seronegative MG, thus defining two immunologically distinct forms of the disease. Measurement of MuSK antibodies will substantially aid diagnosis and clinical management.     

The role of acetylcholine receptor antibodies in myasthenia gravis.

Myasthenia gravis is an autoimmune disease of man characterized by remitting and relapsing muscle fatigability. Although the etiology and pathogenesis are incompletely understood, the presence of circulating antibodies directed against the nicotinic acetylcholine (ACh) receptor in 80--90% of patients with myasthenia gravis and the identification of immune complexes at their neuromuscular junction have helped explain the altered neuromuscular transmission. The ACh receptor antibodies do not block access of ACh to the receptor, but do decrease the number of receptors by accelerating their degradation both in rat myotube cultures and in vivo models. In vitro these antibodies play a major role in myasthenia gravis. However, correlations of antibody titers with the clinical state following thymectomy or in neonatal myasthenia suggest that host factors may be equally important in determining whether the ACh receptor antibodies will result in clinical myasthenia.     

Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.     

Advanced glycation endproducts are increased in rheumatoid arthritis patients with controlled disease

Introduction  

Advanced glycation end products (AGEs) are produced and can accumulate during chronic inflammation, as might be present in patients with rheumatoid arthritis (RA). AGEs are involved in the development of cardiovascular disease. The aim of this study is to evaluate whether AGEs are increased in patients with long-standing RA and whether AGE accumulation is related to disease activity, disease severity and measures of (premature) atherosclerosis, such as endothelial activation, endothelial dysfunction and intima media thickness (IMT).