Ann Marie Schmidt

N ε-(Carboxymethyl)Lysine Adducts of Proteins Are Ligands for Receptor for Advanced Glycation End Products That Activate Cell Signaling Pathways and Modulate Gene Expression

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure,N ε-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-κB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.

Receptor for Advanced Glycation End Products Mediates Inflammation and Enhanced Expression of Tissue Factor in Vasculature of Diabetic Apolipoprotein E–Null Mice

Abstract—Advanced glycation end products (AGEs) and their cell surface receptor, RAGE, have been implicated in the pathogenesis of diabetic complications. Here, we studied the role of RAGE and expression of its proinflammatory ligands, EN-RAGEs (S100/calgranulins), in inflammatory events mediating cellular activation in diabetic tissue. Apolipoprotein E– null mice were rendered diabetic with streptozotocin at 6 weeks of age. Compared with nondiabetic aortas and kidneys, diabetic aortas and kidneys displayed increased expression of RAGE, EN-RAGEs, and 2 key markers of vascular inflammation, vascular cell adhesion molecule (VCAM)-1 and tissue factor. Administration of soluble RAGE, the extracellular domain of the receptor, or vehicle to diabetic mice for 6 weeks suppressed levels of VCAM-1 and tissue factor in the aorta, in parallel with decreased expression of RAGE and EN-RAGEs. Diabetic kidney demonstrated increased numbers of EN-RAGE–expressing inflammatory cells infiltrating the glomerulus and enhanced mRNA for transforming growth factor-&bgr;, fibronectin, and &agr; 1 (IV) collagen. In mice treated with soluble RAGE, the numbers of infiltrating inflammatory cells and mRNA levels for these glomerular cytokines and components of extracellular matrix were decreased. These data suggest that activation of RAGE primes cells targeted for perturbation in diabetic tissues by the induction of proinflammatory mediators.

Endothelial Cell Injury

In conventional cardiac surgery, use of cardiopulmonary bypass (CPB) initiates a series of events best-characterized as a “whole body inflammatory response,” with activation of coagulation, fibrinolysis, and inflammatory cascades (1–3). Perturbation and activation of the endothelium are central in this response (4–6). However, with the emergence of minimally invasive techniques that allow coronary artery bypass grafts to be performed on a beating heart without CPB, it is likely that diminished acute systemic endothelial activation will result.

Calreticulin and Thrombosis

This chapter concerns the interaction of calreticulin with coagulation factors and vascular endothelium, as well as the anticoagulant actions of exogenously administered calreticulin. These observations1 were quite unexpected, in that the properties of calreticulin elucidated in previous studies have pertained to its intracellular/extracellular actions unrelated to thrombosis. Calreticulin has been characterized as a ~55-kDa multifunctional Ca2+ binding protein which possesses three distinct structural domains; the N-domain spans the first 200 residues near the amino terminus; the P-domain (enriched in proline residues) comprises the next 131 residues; and the C-domain, containing many acidic residues, spans the carboxyl-terminal quarter of the protein (Chapter 2).2 Although calreticulin appears to function largely as a high-capacity Ca2+ binding protein, there is abundant experimental data to suggest that it has other functions as well. It has recently been suggested that its localization to the endoplasmic reticulum (Chapter 3) (particularly rough endoplasmic reticulum3) may be the result of its putative function as a molecular chaperone, either aiding in folding of nascent polypeptides, or aiding in the retention of protein subunits prior to assembly (Chapter 4).4 In fact, calreticulin possesses a carboxyl-terminal KDEL sequence, which acts as a retention signal for proteins destined to remain in the lumen of the endoplasmic reticulum. Although the rough endoplasmic reticulum is the preponderant location for calreticulin, it appears in some cells to also localize to the nuclear envelope,5 and may play an active role in gene transcription (Chapter 6).6