The receptor for advanced glycation end products (RAGE), a newly-identified member of the immunoglobulin superfamily, mediates interactions of advanced glycation end product (AGE)-modified proteins with endothelium and other cell types. Survey of normal tissues demonstrated RAGE expression in situations in which accumulation of AGEs would be unexpected, leading to the hypothesis that under physiologic circumstances, RAGE might mediate interaction with ligands distinct from AGEs. Sequential chromatography of bovine lung extract identified polypeptides with M r values of Ϸ12,000 (p12) and Ϸ23,000 (p23) which bound RAGE. NH 2 -terminal and internal protein sequence data for p23 matched that reported previously for amphoterin. Amphoterin purified from rat brain or recombinant rat amphoterin bound to purified sRAGE in a saturable and dose-dependent manner, blocked by anti-RAGE IgG or a soluble form of RAGE (sRAGE). Cultured embryonic rat neurons, which express RAGE, displayed dose-dependent binding of 125 I-amphoterin which was prevented by blockade of RAGE using antibody to the receptor or excess soluble receptor (sRAGE). A functional correlate of RAGE-amphoterin interaction was inhibition by anti-RAGE F(ab) 2 and sRAGE of neurite formation by cortical neurons specifically on amphoterin-coated substrates. Consistent with a potential role for RAGEamphoterin interaction in development, amphoterin and RAGE mRNA/antigen were co-localized in developing rat brain. These data indicate that RAGE has physiologically relevant ligands distinct from AGEs which are likely, via their interaction with the receptor, to participate in physiologic processes outside of the context of diabetes and accumulation of AGEs.Incubation of proteins or lipids with aldose sugars results in nonenzymatic glycation and oxidation (1-7). Following formation of the reversible early glycation products, Schiff bases and Amadori products, further complex molecular rearrangements result in irreversible advanced glycation end products (AGEs). 1Factors favoring nonenzymatic glycation include delayed protein turnover, as in amyloidoses, accumulation of macromolecules with high lysine content, and situations with elevated glucose levels, as in diabetes. AGE formation occurs during normal aging, and at an accelerated rate in diabetes, in which their accumulation in the plasma and vessel wall has been speculated to underlie the pathogenesis of vasculopathy (1, 2, 4).One of the principal means through which AGEs impact on cellular elements is through interaction with cellular binding proteins. Although there are several possible cell-associated polypeptides with which AGEs might interact (8, 9), our work has focussed on the receptor for AGEs (RAGE), as its expression in endothelium, vascular smooth muscle, mononuclear phagocytes, and the central nervous system suggests strategic loci for interaction with the glycated ligands (10, 11). The potential pathophysiologic relevance of AGE-RAGE interaction was emphasized by studies demonstrating that blockade of RAGE pr...

Vascular cell adhesion molecule-1 (VCAM-1), an inducible cell-cell recognition protein on the endothelial cell surface (EC), has been associated with early stages of atherosclerosis. In view of the accelerated vascular disease observed in patients with diabetes, and the enhanced expression of VCAM-1 in diabetic rabbits, we examined whether irreversible advanced glycation endproducts (AGEs), could mediate VCAM-1 expression by interacting with their endothelial cell receptor (receptor for AGE, RAGE). Exposure of cultured human ECs to AGEs induced expression of VCAM-1, increased adhesivity of the monolayer for Molt-4 cells, and was associated with increased levels of VCAM-1 transcripts. The inhibitory effect of anti-RAGE IgG, a truncated form of the receptor (soluble RAGE) or N-acetylcysteine on VCAM-1 expression indicated that AGE-RAGE-induced oxidant stress was central to VCAM-1 induction. Electrophoretic mobility shift assays on nuclear extracts from AGE-treated ECs showed induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter, which was blocked by anti-RAGE IgG or N-acetylcysteine. Soluble VCAM-1 antigen was elevated in human diabetic plasma. These data are consistent with the hypothesis that AGE-RAGE interaction induces expression of VCAM-1 which can prime diabetic vasculature for enhanced interaction with circulating monocytes. (J. Clin. Invest. 1995.96:1395-1403

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...

We hypothesized that one mechanism underlying advanced periodontal disease in diabetes may involve oxidant stress in the gingiva, induced by the effects of Advanced Glycation Endproducts (AGEs), the irreversible products of non-enzymatic glycation and oxidation of proteins and lipids which accumulate in diabetic plasma and tissue. Infusion of AGE albumin, a prototypic ligand, into mice resulted in increased generation of thiobarbituric acid reactive substances (TBARS) compared with infusion of non-glycated albumin in the gingiva, as well as in the lung, kidney and brain. Pretreatment of the animals with the antioxidants probucol or N-acetylcysteine (NAC) prevented the generation of TBARS in the gingiva. Affinity-purified antibody to AGEs demonstrated increased immunoreactivity for AGEs in the vasculature and connective tissues of the gingiva in streptozotocin-induced diabetic mice compared to non-diabetic controls. Increased immunoreactivity for AGEs was also demonstrated in the gingiva of diabetic humans compared with non-diabetic individuals via immunohistochemistry and ELISA. Consistent with these data, immunohistochemistry for heme oxygenase-1, a marker of enhanced oxidant stress, was increased in the gingival vasculature of diabetic mice and humans compared with non-diabetic controls. These data suggest that AGEs present in diabetic gingiva may be associated with a state of enhanced oxidant stress, a potential mechanism for accelerated tissue injury.

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.