A comprehensive review on the advanced glycation end products detection: From conventional to advanced approaches.

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

Advanced glycation endproducts in food and their effects on health

Modulation of the biophysical and biochemical properties of collagen by glycation for tissue engineering applications.

Dietary glycotoxins exacerbate progression of experimental fatty liver disease

This is the first part of a bipartite review that summarizes the rising knowledge on the molecular mechanisms underlying the action of advanced glycation endproducts (AGEs) and their contribution to diabetic complications and vascular disease. While the first part presented here focusses on AGE formation, the second part will describe the AGE-protein/receptor interactions and their role in mediating AGE-dependent intracellular signalling. Nonenzymatic glycation, in which reducing sugars are covalently attached to free amino groups and ultimately form AGEs, has been found to occur during normal aging and at accelerated rate in diabetes mellitus. Oxidation, accompanying glycation in vivo, further supports chemical modifications. AGE formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. AGE modifications do not only change the physicochemical properties of the afflicted molecules, but also induce cellular signalling, activation of transcription factors and subsequent gene expression in vitro and in vivo. AGEs elicit a wide range of cell-mediated responses that might contribute to the pathogenesis of diabetic complications, vascular and renal disease and Alzheimer's disease. Substances that inhibit AGE formation, reduce oxidative stress or destroy already formed crosslinks may limit the progression of disease and may offer new tools for therapeutic interventions in the therapy of AGEs mediated disease.

Advanced glycation endproducts (AGEs) represent one of the many types of chemical modifications that occur with age in long-lived proteins. AGEs also accumulate in pathologies such as diabetes, cardiovascular diseases, neurodegeneration and cancer. Mast cells are major effectors of acute inflammatory responses that also contribute to the progression of chronic diseases. Here we investigated interactions between AGEs and mast cells. Histamine secretion from AGEs-stimulated mast cells was measured. Involvement of a receptor for AGEs, RAGE, was assessed by PCR, immunostaining and use of inhibitors of RAGE. Production of reactive oxygen species (ROS) and cytokines was measured. Advanced glycation endproducts dose-dependently induced mast cell exocytosis with maximal effects being obtained within 20 s. RAGE mRNA was detected and intact cells were immunostained by a specific anti-RAGE monoclonal antibody. AGEs-induced exocytosis was inhibited by an anti-RAGE antibody and by low molecular weight heparin, a known RAGE antagonist. RAGE expression levels were unaltered after 3 h treatment with AGEs. AGE-RAGE signalling in mast cells involves Pertussis toxin-sensitive G(i)-proteins and intracellular Ca(2+) increases as pretreatment with Pertussis toxin, caffeine, 2-APB and BAPTA-AM inhibited AGE-induced exocytosis. AGEs also rapidly stimulated ROS production. After 6 h treatment with AGEs, the pattern of cytokine secretion was unaltered compared with controls. Advanced glycation endproducts activated mast cells and may contribute to a vicious cycle involving generation of ROS, increased formation of AGEs, activation of RAGE and to the increased low-grade inflammation typical of chronic diseases.

High resolution mass spectrometric strategies in drug discovery for the investigation of covalent and non covalent interactions Dott. Danilo De Maddis PhD tutor: Prof. Giancarlo Aldini The research work here described was focused on the set-up and application of analytical methods for studying compounds effective as inhibitors of the advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs) as well as as antagonists of the receptors of AGEs (RAGE). AGEs and ALEs represent a quite complex and heterogeneous class of compounds that are formed by different mechanisms, by heterogeneous precursors and can be formed either exogenously or endogenously. AGEs represent a class of covalently modified proteins generated by oxidative and non-oxidative pathways, involving sugars or their degradation products. The term ALEs includes a variety of covalent adducts which are generated by the non-enzymatic reaction of reactive carbonyl species (RCS), produced by lipid peroxidation and lipid metabolism, with the nucleophilic residues of macromolecules, especially proteins. AGEs and ALEs share some common properties, for example, both consist of non-enzymatic, covalently modified proteins and oxidative stress is often (although not always) involved in the mechanism of their formation. Moreover some AGEs and ALEs have the same structure, since they arise from common precursors, as in the case of carboxymethyllysine (CML) which is generated by glyoxal, which in turn is formed by both lipid and sugar oxidative degradation pathways [1]. Besides being considered as reliable biomarkers of oxidative damage, as well as predictors and prognostic factors, more recently, AGEs and ALEs have also been recognized as important pathogenetic factors of some oxidative based diseases, as supported by the following facts: 1) a strict correlation between the amount of AGEs/ALEs in tissues and fluids and disease states has been found, in both animal and human subjects; 2) a substantial amount of literature is now available reporting the molecular and cellular pathogenic mechanisms for the AGEs/ALEs involvement in the onset and progression of different oxidative-based diseases including diabetes [2], chronic renal failure [3], cardiovascular diseases [4] and neurological disorders [5]. The AGEs/ALEs damaging effect is mediated by different mechanisms, including the dysfunction of the proteins undergoing the oxidative modification, protein polymerization, signal transduction, immunoresponse and RAGE activation. Some of the biological effects are due to the loss of function of the target proteins undergoing the covalent modification, such as in the case of extracellular matrix proteins that lose their elastic and mechanical functions when modified as AGEs/ALEs and in particular, when cross-links are involved [6]. Other examples of a direct damaging effect of AGEs/ALEs can be ascribed to the covalent modification of enzymes and receptors that lose their activity due to the covalent modification involving the catalytic…

Advanced glycation end products (AGE) such as N-ε-carboxy-ethyl-lysine (CEL), N-ε-carboxy-methyl-lysine (CML), imidazolone, methyl-glyoxal-lysine dimer (MOLD), glyoxal-lysine dimer (GOLD), pyrraline and pentosidine have been imparted in the development and worsening of complications of diabetes. They are also involved in atherosclerosis, normal aging process, arthritis, cancer and progression of age-related neurodegenerative diseases like Alzheimer`s disease. Endogenously, they are formed by nonenzymatic glycation by aldoses/ketoses to form intermediate precursor that were slowly converted into AGE. A positive correlation was observed with the level of AGEs formation and progression of the diseases. Exogenously, they formed in foods when they were cooked at very high temperature. AGEs can interact with the cell surface receptors of AGE (RAGE) to release cytokines, free radicals as well as directly modify the extracellular matrix and action of hormones. Hence, the mechanism of AGE association with pathogenesis of diseases can be ascribed mainly to the generated cytokines and free radicals. Second type of receptors such as AGE receptor-1, 2 and 3 were more specific and involved in their detoxification and clearance. Therapeutic agents were used to inhibit AGEs formation, traps the reactive carbonyl intermediate precursors, interfering with Amadori`s products, cross-link breaker and low molecular weight inhibitors of RAGE had been described as well. Despite the several therapeutic agents described so far, none of them have proven to be recommended for clinical use. Furthermore, no methods or standard units were accepted universally to measure AGEs are existing. This review discusses AGEs formation, association with diseases and therapeutic agents to alleviate them.

The high incidence of vascular complications in patients with diabetes mellitus remains incompletely understood. Several metabolic or endocrine abnormalities have been postulated as possible triggers for micro and macroangiopathies. This review article focuses on the consequences of hyperglycemia, leading to the formation of advanced glycation endproducts (AGE), on vascular function. Advanced glycation endproducts are the product of the binding of aldoses onto free amino groups of proteins or lipoproteins, which, after molecular rearrangement, result in a class of molecules of a brown color and specific fluorescence. Different cell membrane proteins have been shown to bind AGE and the best characterized receptor for AGE has been named RAGE. The AGE receptor is present on different cell types including endothelial cells, smooth muscle cells, lymphocytes and monocytes. Experimental studies have revealed that the binding of AGE to RAGE produces an activation of monocytes and endothelial cells. Activated endothelial cells produce interleukin and express vascular cell adhesion molecule and tissue factor. Advanced glycation endproducts, when infused into animals, induce an increase in vascular permeability. The blockade of RAGE by specific antibodies corrects the hypermeability observed in diabetic animals. The prevention of AGE formation by aminoguanidine treatment improves the microvascular lesions found in diabetic animals either in the retina or the glomerus. The infusion of recombinant RAGE in diabetic animals corrects hyperpermeability. The colocalization of RAGE and AGE at the microvascular site of the injury suggests that their interaction may play a significant role in the pathogenesis of diabetic vascular lesions.

Advanced glycation end-products (AGE) can promote chronic kidney disease (CKD) progression and CKD-related morbidities. The soluble receptor for AGE (sRAGE) is a potential biomarker of inflammation and oxidative stress. Here, we explored the role of AGE, glycated albumin, sRAGE and its different forms, cRAGE and esRAGE, as prognostic factors for mortality in 111 advanced CKD patients. The median follow-up time was 39 months. AGE were quantified by fluorescence, sRAGE and its forms by ELISA. Malnutrition was screened by the Malnutrition Inflammation Score (MIS). The Cox proportional hazards regression model was used to assess the association of variables with all-cause mortality. Mean levels of sRAGE, esRAGE and cRAGE were 2318 ± 1224, 649 ± 454 and 1669 ± 901 pg/mL. The mean value of cRAGE/esRAGE was 2.82 ± 0.96. AGE were 3026 ± 766 AU and MIS 6.0 ± 4.7. eGFR correlated negatively with AGE, sRAGE, esRAGE and cRAGE, but not with cRAGE/esRAGE. Twenty-eight patients died. No difference was observed between diabetic and non-diabetic patients. Starting dialysis was not associated with enhanced risk of death. AGE, esRAGE and cRAGE/esRAGE were independently associated with all-cause mortality. AGE, esRAGE and cRAGE/esRAGE may help to stratify overall mortality risk. Implementing the clinical evaluation of CKD patients by quantifying these biomarkers can help to improve patient outcomes.

A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis

Advanced glycation end-products (AGEs) are formed from the so-called Amadori products by rearrangement followed by other reactions giving rise to compounds bound irreversibly. The structure of some of them is shown and the mechanism of formation is described. Several AGE binding molecules (Receptors for AGE, RAGE) are known and it is thought that many of the effects caused by AGEs are mediated by RAGE. Some of these were shown to be toxic, and called TAGE. The mechanism of detoxification of glyoxal and methylglyoxal by the glyoxalase system is described and also the possibility to eliminate glycated proteins by deglycation enzymes. Compounds able to inhibit AGEs formation are also taken into consideration.

The advanced glycation end products (AGEs) are organic molecules formed in any living organisms with a great variety of structural and functional properties. They are considered organic markers of the glycation process. Due to their great heterogeneity, there is no specific test for their operational measurement. In this review, we have updated the most common chromatographic, colorimetric, spectroscopic, mass spectrometric, and serological methods, typically used for the determination of AGEs in biological samples. We have described their signaling and signal transduction mechanisms and cell epigenetic effects. Although mass spectrometric analysis is not widespread in the detection of AGEs at the clinical level, this technique is highly promising for the early diagnosis and therapeutics of diseases caused by AGEs. Protocols are available for high-resolution mass spectrometry of glycated proteins although they are characterized by complex machine management. Simpler procedures are available although much less precise than mass spectrometry. Among them, immunochemical tests are very common since they are able to detect AGEs in a simple and immediate way. In these years, new methodologies have been developed using an in vivo novel and noninvasive spectroscopic methods. These methods are based on the measurement of autofluorescence of AGEs. Another method consists of detecting AGEs in the human skin to detect chronic exposure, without the inconvenience of invasive methods. The aim of this review is to compare the different approaches of measuring AGEs at a clinical perspective due to their strict association with oxidative stress and inflammation.

BackgroundWe recently reported strong associations between eight skin collagen AGEs and two solubility markers from skin biopsies obtained at DCCT study closeout and the long-term progression of microvascular disease in EDIC, despite adjustment for mean glycemia. Herein we investigated the hypothesis that some of these AGEs (fluorescence to be reported elsewhere) correlate with long-term subclinical cardiovascular disease (CVD) measurements, i.e. coronary artery calcium score (CAC) at EDIC year 7–9 (n = 187), change of carotid intima-media thickness (IMT) from EDIC year 1 to year 6 and 12 (n = 127), and cardiac MRI outcomes at EDIC year 15–16 (n = 142).MethodsSkin collagen AGE measurements obtained from stored specimens were related to clinical data from the DCCT/EDIC using Spearman correlations and multivariable logistic regression analyses.ResultsSpearman correlations showed furosine (early glycation) was associated with future mean CAC (p < 0.05) and CAC >0 (p = 0.39), but not with CAC score <100 vs. >100. Glucosepane and pentosidine crosslinks, methylglyoxal hydroimidazolones (MG-H1) and pepsin solubility (inversely) correlated with IMT change from year 1 to 6(all P < 0.05). Left ventricular (LV) mass (cMRI) correlated with MG-H1, and inversely with pepsin solubility (both p < 0.05), while the ratio LV mass/end diastolic volume correlated with furosine and MG-H1 (both p < 0.05), and highly with CML (p < 0.01). In multivariate analysis only furosine (p = 0.01) was associated with CAC. In contrast IMT was inversely associated with lower collagen pepsin solubility and positively with glucosepane,ConclusionsIn type 1 diabetes, multiple AGEs are associated with IMT progression in spite of adjustment for A1c implying a likely participatory role of glycation and AGE mediated crosslinking on matrix accumulation in coronary arteries. This may also apply to functional cardiac MRI outcomes, especially left ventricular mass. In contrast, early glycation measured by furosine, but not AGEs, was associated with CAC score, implying hyperglycemia as a risk factor in calcium deposition perhaps via processes independent of glycation.Trial registration: Registered at Clinical trial reg. nos. NCT00360815 and NCT00360893, http://www.clinicaltrials.gov

We recently reported strong associations between eight skin collagen AGEs and two solubility markers from skin biopsies obtained at DCCT study closeout and the long-term progression of microvascular disease in EDIC, despite adjustment for mean glycemia. Herein we investigated the hypothesis that some of these AGEs (fluorescence to be reported elsewhere) correlate with long-term subclinical cardiovascular disease (CVD) measurements, i.e. coronary artery calcium score (CAC) at EDIC year 7–9 (n = 187), change of carotid intima-media thickness (IMT) from EDIC year 1 to year 6 and 12 (n = 127), and cardiac MRI outcomes at EDIC year 15–16 (n = 142). Skin collagen AGE measurements obtained from stored specimens were related to clinical data from the DCCT/EDIC using Spearman correlations and multivariable logistic regression analyses. Spearman correlations showed furosine (early glycation) was associated with future mean CAC (p 0 (p = 0.039), but not with CAC score 100. Glucosepane and pentosidine crosslinks, methylglyoxal hydroimidazolones (MG-H1) and pepsin solubility (inversely) correlated with IMT change from year 1 to 6(all P < 0.05). Left ventricular (LV) mass (cMRI) correlated with MG-H1, and inversely with pepsin solubility (both p < 0.05), while the ratio LV mass/end diastolic volume correlated with furosine and MG-H1 (both p < 0.05), and highly with CML (p < 0.01). In multivariate analysis only furosine (p = 0.01) was associated with CAC. In contrast IMT was inversely associated with lower collagen pepsin solubility and positively with glucosepane, In type 1 diabetes, multiple AGEs are associated with IMT progression in spite of adjustment for A1c implying a likely participatory role of glycation and AGE mediated crosslinking on matrix accumulation in coronary arteries. This may also apply to functional cardiac MRI outcomes, especially left ventricular mass. In contrast, early glycation measured by furosine, but not AGEs, was associated with CAC score, implying hyperglycemia as a risk factor in calcium deposition perhaps via processes independent of glycation. Trial registration: Registered at Clinical trial reg. nos. NCT00360815 and NCT00360893, http://www.clinicaltrials.gov