AGEs Tumor biology Amphoterin albumin prepared in vitro (7). Its extracellular domain consists of three immunoglobulin-like regions, one “V”-type followed by two “C”-type (8). RAGE contains a single transmembrane-spanning domain and a 43–amino acid cytosolic tail. Structure-function studies have shown that the V-domain is critical for ligand binding and that the cytosolic tail is essential for RAGE-mediated intracellular signalling (Figure 1). A truncated form of RAGE, which lacks the cytosolic tail, remains firmly embedded in the membrane. Although this form of the receptor is competent to bind the usual complement of RAGE ligands, it acts as a dominant negative (termed “DN-RAGE”) receptor, and its expression strikingly suppresses RAGE-mediated signaling, even in cells bearing the full-length form (9, 10). RAGE is expressed at low levels in normal tissues and vasculature. However, the receptor becomes upregulated wherever its ligands accumulate (11–13). In diabetic vessels, for example, RAGE ligands include AGEs of at least two types,(carboxymethyl) lysine adducts and hydroimidazolones (14), and S100/calgranulins as well. RAGE expression is increased in endothelium, smooth muscle cells, and infiltrating mononuclear phagocytes in diabetic vasculature. The overlapping expression of RAGE and its ligands led us to consider the possibility that RAGE mediates cellular activation in diabetic vessels. Indeed, studies in cell culture show that AGE-RAGE interaction alters cellular properties important in vascular homeostasis (15–17). For example, following engagement of RAGE by AGEs, endothelia increase their expression of VCAM-1, tissue factor, and IL-6, and their permeability to macromolecules (18–20). AGE-RAGE–mediated activation of the transcription factor NF-κB is likely to explain these observations, at least in part (20, 21). In mononuclear phagocytes, RAGE activates expression of cytokines and growth factors and induces cell migration in response to soluble AGEs, whereas haptotaxis occurs with immobilized ligands (22). These considerations led us to propose the following two-hit model for RAGE-mediated perturbation of cellular properties in diabetic vasculature: the presence of ligands for RAGE changes properties of the vasculature, resulting in a basal state of activation/dysfunction (first hit; Figure 2); with a superimposed stimulus, such as deposition of oxidized lipoproteins, infection, or ischemia, cellular responses favor derangement of vascular function and tissue damage, rather than restitution of homeostasis (second hit; Figure 2). Diabetic macrovascular disease provides an especially important situation to test this model, as epidemiologic studies have suggested that factors other than glycemia and those traditionally associated with atherosclerosis contribute to pathogenesis. For these studies, our initial experimental system employed atherosclerosis-prone mice (apoE-null animals in the C57BL/6 background) treated with streptozotocin (23), a β-cell toxin that causes insulin deficiency. In contrast to euglycemic apoE-null mice, diabetic animals display advanced atherosclerosis already evident by 14 weeks of age. Diabetic vasculature in the apoE-null animals shows an increased number and area of atherosclerotic lesions, as well as enhanced complexity, with fibrous caps, cholesterol clefts, and necrosis. Levels of AGEs and RAGE are increased in diabetic vasculature, as is expression of tissue factor, VCAM-1, and matrix metalloproteinases (MMPs) 2 and 9. To deter-