Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.

Advanced glycation end products (AGEs) form by the interaction of aldoses with proteins and the subsequent molecular rearrangements of the covalently linked sugars, eventuating in a diverse group of fluorescent compounds of yellow-brown color. This heterogeneous class of nonenzymatically gh/cated proteins or lipids is found in the plasma and accumulates in the vessel wall and tissues even in normal aging. As a consequence of hypergrycemia, AGE formation and deposition are much enhanced in diabetes, in which their presence has been linked to secondary complications, especially microvascular disease. This review summarizes the cellular interactions of AGEs and describes the central role of a novel receptor for AGE (RAGE). RAGE, an immunoglobulin superfamily member, mediates the binding of AGEs to endothelial cells and mononuclear phagocytes, interacts with a lactoferrin-like polypeptide that also binds AGEs, and appears to activate intracellular signal transduction mechanisms con-W hen proteins or lipids are exposed to aldose sugars, they undergo nonenzymatic glycation and oxidation. 17 Initially, this leads to the formation of early glycation products, Schiff bases, and Amadori products, the best known of which is hemoglobin A lc for its use in monitoring glucose control over days or weeks in patients with diabetes. 8 The early glycation products are still in equilibrium with plasma glucose, and when glucose levels fall, the early glycation products can dissociate to the native proteins.'* Alternatively, if glycation continues, further largely undefined molecular rearrangements occur, resulting in formation of the irreversible advanced glycation end products (AGEs). The latter constitute a heterogeneous class of structures that are yellowbrown pigments, fluoresce, tend to form cross-links, generate reactive oxygen intermediates, and recognize a class of receptors on cellular surfaces. 1 -7 - 913 Certain AGEs have been characterized in detail, including pentosidine and W-(carboxymethyl)lysine. 1417 Received April 27, 1994; revision accepted June 24, 1994.

Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverses, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE-and dia- IntroductionExposure of proteins or lipids to reducing sugars results in nonenzymatic glycation and oxidation. Initially, reversible early glycation adducts, Schiff bases and Amadori products, form on free amino groups (1). Further complex molecular rearrangements produce irreversible advanced glycation end products (AGEs):1 heterogeneous structures of yellow-brown color, characteristic fluorescence, and a propensity to form cross-links, which generate reactive oxygen intermediates and interact with specific cellular receptors (1-4). The presence of AGEs in tissue has been linked to development of vasculopathy, especially in the setting of diabetes (1, 3). AGE-modified adducts on long-lived proteins in extracellular matrix alter basement membrane structure by trapping plasma macromolecules and by increasing vessel wall rigidity through formation of cross-links (3). The principal means through which AGEs influence cellular properties is by binding to specific receptors (4-6), the best characterized of which is the receptor for AGEs (RAGE), a member of the immunoglobulin superfamily expressed by endothelial cells (ECs), smooth muscle cells, and mononuclear phagocytes (7), cells central to both vascular homeostasis and the pathogenesis of vascular lesions. A potential role for RAGE in vascular dysfunction is suggested by two lines of evidence: ( a ) engagement of AGEs by cellular RAGE affects critical properties of these cells in a manner contributory to vascular dysfunction (4); and ( b ) there is enhanced expression of RAGE in diabetic vasculopathy and in arteriosclerotic and other vascular lesions, such as inflammatory vasculitides (8).Increased vascular permeability is characteristic of diabetic vasculopathy (9), even at the earliest stages in which microalbuminuria may be the only harbinger of vascular complications yet to come (10). As ECs are the critical guardians of vascular barrier function, we postulated that AGEs in plasma or the subendothelium would promote vascular hyperpermeability by interacting with RAGE. We demonstrate that when diabetic rat red cells bearing AGEs are infused into normal animals, increased vascular permeability results, an effect which is prevented by blockade of RAGE. Hyperperme...

Vascular complications are an important cause of morbidity and mortality in patients with diabetes. The extent of vascular complications has been linked statistically to enhanced adherence ofdiabetic erythrocytes to endothelial cells (ECs) and to the accumulation of a class of glycated proteins termed advanced glycation end products (AGEs). We Nonenzymatic glycation ofproteins, such as hemoglobin, has been shown to provide a useful index for management of patients with diabetes (1). The ultimate result of the nonenzymatic glycation and oxidation of proteins is formation of advanced glycation end products (AGEs), whose presence in plasma and tissues has been linked to the development of complications in diabetics (2-5). The cellular interactions of AGEs are mediated by receptors/cell surface binding proteins identified on endothelial cells (ECs) and mononuclear phagocytes (MPs), engagement of which leads to perturbation of cellular functions (3,(6)(7)(8). Our studies have characterized an integral membrane protein, receptor for AGE (RAGE), a newly discovered member of the immunoglobulin superfamily, which has a central role in mediating the interactions of AGEs with cellular surfaces (7-9).We previously showed that erythrocytes from diabetic patients exhibited enhanced binding to cultured endothelium (10). We hypothesized that, dependent on the duration of exposure of erythrocytes to plasma hyperglycemia, AGE modification oferythrocyte surface membrane proteins could occur, allowing them to bind and thereby to modulate properties of RAGE-expressing vessel wall cells. Our studies demonstrate that the molecular basis of the increased adherence of diabetic erythrocytes results largely from AGEs on the erythrocyte surface interacting with EC RAGE. This results in the induction of oxidant stress in the endothelium, potentially modulating expression of a spectrum ofgenes that could contribute to the pathogenesis of vascular complications.MATERIALS AND METHODS Subjects. The group of patients (n = 18 each for the normal and diabetic subjects) was comparable in age, duration of diabetes, fasting blood glucose, and hemoglobin Alc levels.Erythrocytes from two patients homozygous for sickle cell disease were also studied.Erythrocyte Adhesion Assay. Cultured human umbilical vein ECs were prepared and assayed as described (10-12).The specific activity of 51Cr-labeled erythrocytes for normal and diabetic erythrocytes was 3750 ± 260 and 3820 ± 253 cpm per mg of hemoglobin, respectively. The adhesion ratio (AR) was calculated as follows: AR = (cpm of diabetic erythrocytes)/(cpm of normal erythrocytes). An AR value of 1 represents the adhesion observed with normal erythrocytes. Where indicated, either erythrocytes or ECs were preincubated with soluble RAGE (sRAGE) or antibodies and EC nuclear extracts were prepared (13).Preparation of AGE-Modified Proteins, AGE Binding Proteins, and Antisera. AGE albumin was prepared and characterized as described (3,7,8). AGE binding proteins were purified from bovine lung (7) and cons...

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.

Receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface molecules and engages diverse ligands relevant to distinct pathological processes. One class of RAGE ligands includes glycoxidation products, termed advanced glycation end products, which occur in diabetes, at sites of oxidant stress in tissues, and in renal failure and amyloidoses. RAGE also functions as a signal transduction receptor for amyloid beta peptide, known to accumulate in Alzheimer disease in both affected brain parenchyma and cerebral vasculature. Interaction of RAGE with these ligands enhances receptor expression and initiates a positive feedback loop whereby receptor occupancy triggers increased RAGE expression, thereby perpetuating another wave of cellular activation. Sustained expression of RAGE by critical target cells, including endothelium, smooth muscle cells, mononuclear phagocytes, and neurons, in proximity to these ligands, sets the stage for chronic cellular activation and tissue damage. In a model of accelerated atherosclerosis associated with diabetes in genetically manipulated mice, blockade of cell surface RAGE by infusion of a soluble, truncated form of the receptor completely suppressed enhanced formation of vascular lesions. Amelioration of atherosclerosis in these diabetic/atherosclerotic animals by soluble RAGE occurred in the absence of changes in plasma lipids or glycemia, emphasizing the contribution of a lipid- and glycemia-independent mechanism(s) to atherogenesis, which we postulate to be interaction of RAGE with its ligands. Future studies using mice in which RAGE expression has been genetically manipulated and with selective low molecular weight RAGE inhibitors will be required to definitively assign a critical role for RAGE activation in diabetic vasculopathy. However, sustained receptor expression in a microenvironment with a plethora of ligand makes possible prolonged receptor stimulation, suggesting that interaction of cellular RAGE with its ligands could be a factor contributing to a range of important chronic disorders.

The role of chronic hyperglycemia in the development of diabetic microvascular complications and in neuropathy has been clearly established by intervention studies. However, the biochemical or cellular links between elevated blood glucose levels, and the vascular lesions remain incompletely understood. This review focuses on the consequences of hyperglycemia on the formation of advanced glycation end‐products (AGEs), and on the role of AGEs and of their specific receptors (RAGE) in the functional and anatomical alterations of the vascular wall. AGEs are formed during the Mailiard reaction by the binding of aldoses on free NH2 groups of proteins, which, after a cascade of molecular rearrangements, result in molecules of brown color and specific fluorescence. Experimental studies have indicated that the binding of AGEs to RAGE activates cells, particularly monocytes and endothelial calls. Activated endothelial cells produce cytokines, and express adhesion molecules and tissue factor. The role of AGEs in increased oxidative stress, and in the functional alterations in vascular tone control observed in diabetes, in part related to a reduction in nitric oxide, is also discussed. The microvascular retinal, glomerular and nerve lesions induced by experimental diabetes in animals are prevented by an inhibitor of AGEs formation, aminoguanidine. The administration in diabetic animals of recombinant RAGE, which hinders AGEs‐RAGE interaction, prevents hyperpermeability and vascular lesions. These data suggest a central role of AGEs and RAGE in the development of chronic complications of diabetes.

AGE binding to RAGE stimulated mesothelial cell activity, and resulted in the overexpression of VCAM-1, a structure for leukocyte adhesion. The AGE-RAGE interaction resulted in HPMC activation, which may promote local inflammation, and thus is implicated in the peritoneal injury found in long-term PD patients.