Advanced glycation end products (AGEs) and their receptors (RAGEs) in diabetic vascular diseaseby P. Marchetti, Italy

Piero MARCHETTI, MD, PhDDepartment of Endocrinologyand MetabolismUniversity of PisaPisa, ITALY

Increasing evidence demonstrates that advanced glycation end products (AGEs) play a pivotal role in the development and progression of diabetic vascular damage. AGEs are generated as a result of chronic hyperglycemia. Then, following the interaction with receptors for advanced glycation end products (RAGEs), a series of events leading to vessel damage are elicited and perpetuated, which include oxidative stress, increased inflammation, and enhanced extracellular matrix accumulation. Whereas targeting glycemic control and treating additional risk factors, such as obesity, dyslipidemia, and hypertension, are mandatory to reduce chronic complications and prolong life expectancy in diabetic patients, drug therapy tailored to reducing the deleterious effects of the AGE-RAGE interaction is being actively investigated and showing signs of promise.Medicographia. 2009;31:257-265 (see French abstract on page 265)

Accelerated atherosclerosis is the leading cause of morbidity and mortality in patients with diabetes.1 Several mechanisms, including endothelial cell damage, platelet activation and aggregation, hypercoagulability, and impaired fibrinolysis, are involved in the pathogenesis of a thrombogenic diathesis in diabetes.1 Among various biochemical pathways implicated in diabetic vascular complications, the process of formation and accumulation of advanced glycation end products (AGEs) and their mode of action play a major role.2-4 AGEs are generated in the diabetic milieu as a result of chronic hyperglycemia and enhanced oxidative stress. Then, via pathways also involving receptor-dependent signals, they promote the development and progression of cardiovascular disease. These compounds interact with receptors, such as RAGEs (receptors for advanced glycation end products), to induce oxidative stress, increase inflammation by promoting nuclear factor-B (NFB) activation, and enhance extracellular matrix accumulation.5-7 These biological effects translate into accelerated plaque formation and increased cardiac fibrosis, with consequent effects on cardiac function. In this article, we will deal with the biology of AGEs and RAGEs, with particular emphasis on their role in diabetes. Strategies to reduce the deleterious effects of the AGE-RAGE interaction will also be discussed.Advanced glycation end products (AGEs)Advanced glycation end products (AGEs) are modifications of proteins or lipids that become nonenzymatically glycated and oxidized after contact with aldose sugars. In other words, they are the result of a chain of chemical reactions which follow an initial glycation reaction. The intermediate products are known as Schiff base, Amadori, and Maillard products, after the researchers who first described them. Initially, glycation involves covalent reactions between free amino groups of amino acids, such as lysine, arginine, or protein terminal amino acids and sugars (glucose, fructose, ribose, etc), to create, first, the Schiff base and then Amadori products, of which the best known are HbA1c (Figure 1) and fructosamine (fructoselysine). Additional reactions occur successively.

Figure 1. Formation of glycated hemoglobin A1c (HbA1c). HbA1c is an Amadori product and is formed through the intermediate Schiff base step.AGE formation from fructoselysine involves the nonoxidative dissociation of fructoselysine to form new reactive intermediates that again modify proteins to form AGEs of various different chemical structures (Figure 2). Alternatively, fructoselysine decays and releases its carbohydrate moiety either as glucose or as the more reactive hexoses, such as 3-deoxyglucosone, which themselves may modify proteins. In addition, it has recently been found that glucose can auto-oxidize to form reactive carbonyl compounds (glyoxal and methylglyoxal) which can react with proteins to form glycoxidation products (Figure 2). In addition to this, products of oxidative stress, such as peroxynitrite, can also induce the formation of carboxymethyl lysine by oxidative cleavage of Amadori products and/or the generation of reactive dicarbonyl compounds from glucose (Figure 2). Thus, AGEs can arise from glucose and lipids through several, partially intermingling reactions. Once formed, they may damage cellular structures via a number of mechanisms, including the formation of cross-links between key molecules in the basement membrane of the extracellular matrix (ECM) and the interaction of AGEs with RAGEs on cell surfaces, thus altering cellular functions.2-7

Accumulation of AGEs in the ECM occurs on proteins with a slow turnover rate, with the formation of cross-links that can trap other local macromolecules. In this way, AGEs alter the properties of the large matrix proteins collagen, vitronectin, and laminin. AGE cross-linking on type I collagen and elastin causes an increase in the area of ECM, resulting in increased stiffness of the vasculature. Glycation results in increased synthesis of type III collagen, type V collagen, type VI collagen, laminin, and fibronectin in the ECM, most likely via upregulation of transforming growth factor- pathways. Formation of AGEs on laminin results in reduced binding to type IV collagen, reduced polymer elongation, and lower binding of heparan sulfate proteoglycan. Glycation of laminin and type I and type IV collagens, key molecules in the basement membrane, causes inhibited adhesion to endothelial cells for both matrix glycoproteins. In addition, it has been suggested that AGE formation leads to a reduction in the binding of collagen and heparan to the adhesive matrix molecule vitronectin. AGE-induced alterations of vitronectin and laminin may explain the reduction in binding of heparan sulfate proteoglycan, a stimulant of other matrix molecules in the vessel wall, to the diabetic basement membrane. As for the role of lipids, glycated low-density lipoprotein (LDL) reduces nitric oxide (NO) production and suppresses uptake and clearance of LDL through its receptor on endothelial cells.

Schematic representation of the formation of some common advanced glycation end products (AGEs).It must also be kept in mind that AGEs can be absorbed through diet.8 In this regard, foods high in protein and fat, such as meat, cheese, and egg yolk, are rich in AGEs, whereas those high in carbohydrates have the lowest amount of AGEs. In addition, increased cooking temperatures, through broiling and frying, and increased cooking times lead to an increased amount of AGEs. A diet heavy in AGEs results in proportional elevations in serum AGE levels and increased cross-linking in patients with diabetes, whereas, conversely, dietary AGE restriction causes a marked reduction in serum AGEs in healthy subjects.9-11Receptor for AGEs (RAGE)RAGE is a member of the immunoglobulin superfamily of receptors. The human RAGE gene is on chromosome 6 in the major histocompatibility complex between genes for class II and class III. It is composed of 11 exons and a 3_UTR region, and common variants have been described.12 For example, the Gly82Ser polymorphism in exon 3 is located in the ligand- binding V-domain of RAGE (see below), and has been studied to assess its role in subjects with vascular disease. It was found that cells bearing the Ser82 isoform displayed higher ligand affinity resulting in increased activation of the proinflammatory proteins TNF-, IL-6, and MMP-9.13 In contrast, the 374T/A polymorphism in the promoter region of the RAGE gene has been shown to exert protective effects. In diabetic patients with the mutation, there was a lower incidence of coronary heart disease, acute myocardial infarction, and peripheral vascular disease, and, in nondiabetic individuals, the presence of the polymorphism was associated with a reduced risk of coronary artery disease.6,14At the protein level, RAGE is an approximately 45-kDa protein. It has an extracellular component, consisting of two Ctype (constant) domains preceded by one V-type (variable) immunoglobulin-like domain (Figure 3). RAGE has a single transmembrane domain followed by a cytosolic tail. The V domain in the N-terminus is important in ligand binding, and the cytosolic tail is critical for RAGE-induced intracellular signaling. In addition to full-length RAGE, truncated forms have also been described (due to mRNA splice variants). In particular, one variant protein (N-truncated type) lacks the V-type immunoglobulin domain, but it is otherwise identical to full-length RAGE and is retained in the plasma membrane.

Figure 3. products (RAGE). Adapted from reference 6: Basta G. Atherosclerosis. 2008;196:9-21. Copyright 2008, Elsevier, Ltd.However, since the V-type immunoglobulin domain is deleted, this RAGE form shows impaired ability to bind ligands. In addition, forms of RAGE lacking both the cytosolic and the transmembrane domains have been described. These forms of RAGE are, therefore, secreted extracellularly, can be detected in circulating blood, and are called soluble receptors for advanced glycation end products (sRAGEs).5-7 This is of importance since sRAGEs can bind their ligands in the circulation, thus preventing the adverse intracellular events of the AGE-RAGE axis (see below).It has to be kept in mind, however, that RAGEs also bind ligands other than AGEs.5-7 Shortly after its discovery, structural analysis of the ligand- RAGE interaction revealed that the receptor recognized three-dimensional structures, such as sheets and fibrils, rather than specific amino acid sequences (ie, primary structures). As a matter of fact,RAGEs bind amyloid- peptide (which accumulates in Alzheimers disease) and amyloid A (which accumulates in systemic amyloidosis). Further ligands of RAGE are S100/calgranulins, a family of closely related calcium-binding polypeptides that accumulate extracellularly at sites of chronic inflammation. An additional proinflammatory ligand of RAGE is the DNA-binding protein HMGB1 (amphoterin), which is released by cells undergoing necrosis. Finally, RAGEs also interact with surface molecules on bacteria and leukocytes. Thus, RAGEs have a large repertoire of ligands, making this receptor crucial at the crossroad between diabetes, inflammation, and vascular disease.Cellular effects of the AGE-RAGE interactionRAGE is expressed in many tissues and is most abundant in the heart, lung, skeletal muscle, and vessel wall. In addition, it is present in monocytes/macrophages and lymphocytes. In vessels, it is located in the endothelium and in smooth muscle cells. Physiologically, the receptor might play a role in developmental processes, at least as shown in a few experimental models. For example, RAGE activation contributes to axonal sprouting that accompanies neuronal development, while reduction of functional regeneration of the sciatic nerve occurs after blockade of RAGE.15,16 However, RAGE-/- mice demonstrate neither obvious neuronal deficits nor overt behavior abnormalities, indicating that RAGE may contribute to neuronal development, but that there are redundant systems that substitute for this receptor in its absence.16Intriguingly, it has been demonstrated that activation of RAGE can promote cell survival through increased expression of the antiapoptotic protein Bcl-2.15 However, whereas nanomolar concentrations of ligand induced trophic effects in RAGE-expressing cells, micromolar concentrations caused apoptosis in a manner that appeared to depend on oxidative stress.15 For both of these outcomes, the cytoplasmic domain of RAGE was required, as cells lacking the cytosolic tail were unresponsive. After being highly expressed during embryonic development, RAGE is downregulated in most organs during normal life. With aging, RAGE expression increases again, possibly due to the accumulation of RAGE ligands, which upregulate receptor expression. In the cases of diabetes, inflammation, and atherosclerosis, there is marked induction of RAGE due to the action of its ligands and to several mediators from activated inflammatory cells.5-7,16,17 In turn, the binding of ligands to RAGE induces further upregulation of the receptor (positive feedback), leading to a vicious circle. Unsurprisingly, one of the locations where RAGE expression is enhanced is in the diabetic atherosclerotic plaque (particularly at the vulnerable regions of the plaque and in macrophages), where it colocalizes with cyclooxygenase 2, microsomal prostaglandin E2, and metalloproteases.The most important pathological consequence of RAGE interaction with its ligands is the activation of several intracellular pathways, leading to the induction of oxidative stress and a broad spectrum of signaling mechanisms, schematically represented in Figure 4. The interactions lead to prolonged inflammation, mainly as a result of the RAGE-dependent expression of proinflammatory cytokines and chemokines. In the vasculature, the first pathological consequence of RAGE interaction with its ligands is the induction of increased intracellular reactive oxygen species (ROS), the generation of which is linked, at least in part, to the activation of the NAD(P)H-oxidase system. In addition, in endothelial cells, mitochondrial sources of ROS are also involved, following the AGE-RAGE interaction. Experimental evidence demonstrates that RAGE dependent modulation of gene expression and cellular properties depends upon signal transduction. Based on the intensity and duration of stimulation, diverse signaling pathwaysmay be triggered (Figure 4), including p21ras, erk1/2, mitogen-activated protein kinases (MAPKs), p38 and SAPK/JNK MAPKs, PI3K, and the JAK/STAT pathway. The downstream consequence of these changes is the activation of key transcription factors (nuclear factor-B [NFB], in particular), which in turn cause induction of molecules with damaging actions on the cells (Figure 4). In human endothelial cells, RAGE activation enhances the expression of adhesion molecules, including VCAM-1, ICAM-1, and E-selectin. AGE bound to RAGE on the endothelium also determines alterations to the surface antithrombotic properties of flowing blood, as shown by a reduction in thrombomodulin expression and the concomitant induction of tissue factor expression that confers procoagulant properties. The interaction of AGEs with RAGEs in monocytes induces their activation to macrophages, which manifests with the induction of platelet-derived growth factor, insulin-like growth factor 1, and proinflammatory cytokines, such as IL-1 and TNF-. In addition to all this, AGE-RAGE interaction promotes monocyte chemotaxis and, at the level of smooth muscle cells, is associated with increased cellular proliferation. Viewed together, these findings indicate that the AGE-RAGE interaction elicits and potentiates inflammatory responses through the enhanced generation of reactive oxygen species, proinflammatory adhesion molecules, and cytokines, causing continued amplification of inflammatory events.

Figure 5. RAGE (receptor for advanced glycation end products)expression is higher in plaques from type 2 diabetic patients. Adapted from reference 27: Cipollone F, Iezzi A, Fazia M, et al. Circulation. 2003; 108:1070-1077. Copyright 2003, American Heart Association, Inc.AGE, RAGE, and diabetesIt has long been recognized that increased HbA1c (a precursor of AGEs) levels are associated with a higher incidence of vascular complications and reduced life expectancy in diabetic patients. In addition, intervention studies to reduce HbA1c lead to lower micro- and macrovascular lesions and a reduced death rate over several years.18,19Serum levels of AGEs in patients with type 2 diabetes and coronary heart disease are higher than those in patients without heart disease and correlate with the severity of the coronary syndrome.3,4,20 Furthermore, AGE levels are higher in type 2 diabetic patients with peripheral artery occlusive disease compared with those without it. Serum levels of AGE in type 1 diabetic patients are associated with decreased isovolumetric relaxation time of the left ventricle, a marker of left ventricular diastolic dysfunction.21 AGEs are also related to other other features of cardiovascular disease, such as carotid stenosis, peripheral artery occlusive disease, increased pulse pressure, and a low ankle-brachial index.3,4,22 Unsurprisingly, other studies have demonstrated that serum AGE level is a predictor for heart failure and new cardiac events.3,4 In addition, work has shown that high AGE levels correlate with poor outcomes, as demonstrated by adverse cardiac events in patients after cardiac surgery, prolonged ventilation times, and longer stays in intensive care units.3,4 Finally, in diabetic patients receiving cardiac stents, an elevated level of serum AGEs appears to be an independent risk factor for the development of angiographic restenosis.23In terms of relationship with life expectancy, it has been reported that increased serum levels of AGE predicted increased total cardiovascular and coronary mortality in women with type 2 diabetes during a follow-up period of 18 years.24 AGE level remained a strong predictor of survival even after adjustment for confounding factors, including C-reactive protein.At a pathological level,25,26 when atherosclerotic plaques retrieved from human subjects were studied, it was found that, compared to nondiabetics, plaques from diabetic subjects had increased RAGE expression, especially in smooth muscle cells and in macrophages within the lesion (Figure 5).27 In a prospective study, type 2 diabetic patients were randomized to treatment with diet alone or with diet plus the addition of a statin for four months before carotid endarterectomy.28 The expression of AGEs and RAGEs as well as myeloperoxidase, NFB, cyclooxygenase 2, and metalloproteinases 2 and 9 was significantly lower in the plaques of statin-treated patients. Fewer macrophages, T cells, and HLA-DRexpressing cells were noted in the lesions of these subjects. Notably, the ex- pression of RAGE in statin-treated, plaque-derived macrophages can be restored by in vitro incubation with AGEs. Additional findings from plaques retrieved from type 2 diabetic patients include larger necrotic cores and a correlation between RAGE expression on macrophages and apoptotic smooth muscle cells.25-29 Altogether, the findings indicate that the AGE-RAGE axis may compromise cell survival and, thereby, promote mechanisms linked to plaque destabilization.

Figure 6. NF B activation by AGEs is reduced by the presenceof gliclazide. *P