Faculty Bibliography

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

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 glycated proteins or lipids is found in the plasma and accumulates in the vessel wall and tissues even in normal aging. As a consequence of hyperglycemia, 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 consequent to its interaction with the glycated ligand. RAGE is expressed by ECs, mononuclear phagocytes, smooth muscle cells, mesangial cells, and neurons, indicating a potential role in the regulation of their properties in homeostasis and/or their dysfunction in the development of diabetic complications. Since AGEs have been shown to generate reactive oxygen intermediates, tethering of AGEs to the cell surface by their receptors focuses oxidant stress on cellular targets, resulting in changes in gene expression and the cellular phenotype. The discovery of RAGE and development of reagents to block its interaction with AGEs should provide insights into the role of this ligand-receptor interaction in the pathogenesis of diabetic complications and, potentially, atherosclerosis

Advanced glycation end products (AGEs), formed as the result of the extended interaction of proteins with ketoses, modulate central properties of endothelial cells and mononuclear phagocytes by interacting with a cell surface binding site comprised of a novel integral membrane protein (receptor for AGE = RAGE) and a lactoferrin-like polypeptide (LF-L), the latter having sequence identity to lactoferrin (LF). To further understand this cellular binding site, the interaction of RAGE with LF-L and LF was characterized. By ligand blotting and a solid state competitive binding assay, 125I-LF-L and 125I-LF bound to RAGE immobilized on nitrocellulose membranes or polypropylene tubes in a time-dependent and reversible manner, demonstrating a high affinity component with Kd approximately 100 pM. The interaction of 125I-LF-L and 125I-LF with RAGE was independent of iron in LF and was competed by addition of an excess of unlabeled carboxyl-terminal portion of LF. Cross-linking studies with purified 125I-LF-L and RAGE, in the presence of disuccinimidyl suberate, showed a new, slowly migrating band, corresponding to a complex of RAGE and LF-L, and cross-linking on mouse aortic endothelial cells showed two new slowly migrating bands on immunoblotting visualized with both anti-RAGE IgG and anti-LF-L IgG. These data lead us to propose that the endothelial cell surface binding site for AGEs consists of LF-L bound noncovalently to RAGE anchored in the cell membrane

Attack by reactive oxygen intermediates, common to many kinds of cell/tissue injury, has been implicated in the development of diabetic and other vascular diseases. Such oxygen-free radicals can be generated by advanced glycation end products (AGEs), which are nonenzymatically glycated and oxidized proteins. Since cellular interactions of AGEs are mediated by specific cellular binding proteins, receptor for AGE (RAGE) and the lactoferrin-like polypeptide (LF-L), we tested the hypothesis that AGE ligands tethered to the complex of RAGE and LF-L could induce oxidant stress. AGE albumin or AGEs immunoisolated from diabetic plasma resulted in induction of endothelial cell (EC) oxidant stress, including the generation of thiobarbituric acid reactive substances (TBARS) and resulted in the activation of NF-kappa B, each of which was blocked by antibodies to AGE receptor polypeptides and by antioxidants. Infusion of AGE albumin into normal animals led to the appearance of malondialdehyde determinants in the vessel wall and increased TBARS in the tissues, activation of NF-kappa B, and induction of heme oxygenase mRNA. AGE-induced oxidant stress was inhibited by pretreatment of animals with either antibodies to the AGE receptor/binding proteins or antioxidants. These data indicate that interaction of AGEs with cellular targets, such as ECs, leads to oxidant stress resulting in changes in gene expression and other cellular properties, potentially contributing to the development of vascular lesions. Further studies will be required to dissect whether oxidant stress occurs on the cell surface or at an intracellular locus

Advanced glycation end products (AGEs), the final products of nonenzymatic glycation and oxidation of proteins, are found in the plasma and accumulate in the tissues during aging and at an accelerated rate in diabetes. A novel integral membrane protein, termed receptor for AGE (RAGE), forms a central part of the cell surface binding site for AGEs. Using monospecific, polyclonal antibody raised to human recombinant and bovine RAGE, immunostaining of bovine tissues showed RAGE in the vasculature, endothelium, and smooth muscle cells and in mononuclear cells in the tissues. Consistent with these data, RAGE antigen and mRNA were identified in cultured bovine endothelium, vascular smooth muscle, and monocyte-derived macrophages. RAGE antigen was also visualized in bovine cardiac myocytes as well as in cultures of neonatal rat cardiac myocytes and in neural tissue where motor neurons, peripheral nerves, and a population of cortical neurons were positive. In situ hybridization confirmed the presence of RAGE mRNA in the tissues, and studies with rat PC12 pheochromocytes indicated that they provide a neuronal-related cell culture model for examining RAGE expression. Pathological studies of human atherosclerotic plaques showed infiltration of RAGE-expressing cells in the expanded intima. These results indicate that RAGE is present in multiple tissues and suggest the potential relevance of AGE-RAGE interactions for modulating properties of the vasculature as well as neural and cardiac function, prominent areas of involvement in diabetes and in the normal aging process

Nonenzymatic glycation of proteins occurs at an accelerated rate in diabetes and can lead to the formation of advanced glycation end products of proteins (AGEs), which bind to mononuclear phagocytes (MPs) and induce chemotaxis. We have isolated two cell surface-associated binding proteins that mediate the interaction of AGEs with bovine endothelial cells. One of these proteins is a new member of the immunoglobulin superfamily of receptors (termed receptor for AGEs or RAGE); and the second is a lactoferrin-like polypeptide (LF-L). Using monospecific antibodies to these two AGE-binding proteins, we detected immunoreactive material on Western blots of detergent extracts from human MPs. Radioligand-binding studies demonstrated that antibody to the binding proteins blocked 125I-AGE-albumin binding and endocytosis by MPs. Chemotaxis of human MPs induced by soluble AGE-albumin was prevented in a dose-dependent manner by intact antibodies raised to the AGE-binding proteins, F(ab')2 fragments of these antibodies and by soluble RAGE. When MP migration in response to N-formyl-Met-Leu-Phe was studied in a chemotaxis chamber with AGE-albumin adsorbed to the upper surface of the chamber membrane, movement of MPs to the lower compartment was decreased because of interaction of the glycated proteins with RAGE and LF-L on the cell surface. The capacity of AGEs to attract and retain MPs was shown by implanting polytetrafluoroethylene (PTFE) mesh impregnated with AGE-albumin into rats: within 4 d a florid mononuclear cell infiltrate was evident in contrast to the lack of a significant cellular response to PTFE with adsorbed native albumin. These data indicate that RAGE and LF-L have a central role in the interaction of AGEs with human mononuclear cells and that AGEs can serve as a nidus to attract MPs in vivo

Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins

A linear vasopressin antagonist, Phaa-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti-vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (Kd = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (Ki = 2.1 nM), and affinities for the rat kidney- and adenohypophysis-vasopressin receptors were much lower (Ki = 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V1a receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.

In response to epidermal growth factor (EGF) stimulation, the intrinsic protein tyrosine kinase of EGF receptor is activated, leading to tyrosine phosphorylation of several cellular substrate proteins, including the EGF receptor molecule itself. To test the mechanism of EGF receptor autophosphorylation in living cells, we established transfected cell lines coexpressing a kinase-negative point mutant of EGF receptor (K721A) with an active EGF receptor mutant lacking 63 amino acids from its carboxy terminus. The addition of EGF to these cells caused tyrosine phosphorylation of the kinase-negative mutant by the active receptor molecule, demonstrating EGF receptor cross-phosphorylation in living cells. After internalization the kinase-negative mutant and CD63 have separate trafficking pathways. This limits their association and the extent of cross-phosphorylation of K721A by CD63. The coexpression of the kinase-negative mutant together with active EGF receptors in the same cells suppressed the mitogenic response toward EGF as compared with that in cells that express active receptors alone. The presence of the kinase-negative mutant functions as a negative dominant mutation suppressing the response of active EGF receptors, probably by interfering with EGF-induced signal transduction. It appears, therefore, that crucial events of signal transduction occur before K721A and active EGF receptors are separated by their different endocytic itineraries.

Ligand binding to the membrane receptor for EGF induces its clustering and internalization. Both receptor and ligand are then degraded by lysosomal enzymes. A kinase defective point mutant (K721A) of EGF receptor undergoes internalization similarly to the wild-type receptor. However, while internalized EGF molecules bound to either the wild-type or mutant receptors are degraded, the K721A mutant receptor molecules recycle to the cell surface for reutilization. To investigate the mechanism of receptor trafficking, we have established transfected NIH-3T3 cells coexpressing the kinase-negative mutant (K721A) together with a mutant EGF receptor (CD63) with active kinase. CD63 was chosen because it behaves like wild-type EGF receptor with respect to biological responsiveness and cellular routing but afforded immunological distinction between kinase active and inactive mutants. Although expressed in the same cells, the two receptor mutants followed their separate endocytic itineraries. Like wild-type receptor, the CD63 mutant was downregulated and degraded in response to EFG while the kinase-negative mutant K721A returned to the cell surface for reutilization. Intracellular trafficking of EGF receptor must be determined by a sorting mechanism that specifically recognizes EGF receptor molecules according to their intrinsic kinase activity.