Characterization of the advanced glycation end-product receptor complex inhuman vascular endothelial cells

Advanced glycation end products (AGEs) have been implicated as causal facto rs in the vascular complications of diabetes and it is known that these pro ducts interact with cells through specific receptors. The AGE-receptor comp lex, originally described as p60 and p90, has been characterised in hemopoi etic cells and the component proteins identified and designated AGE-R1, -R2 and -R3. In the current study we have characterised this receptor in human umbilical vein endothelial cells (HUVECs) and elucidated several important biological properties which may impact on AGE mediated vascular disease. I -125-AGE-BSA binding to HUVEC monolayers was determined with and without va rious cold competitors. The synthetic AGE, 2-(2-furoyl)-4(5)-furanyl-1H-imi dazole (FFI)-BSA, failed to compete with AGE-BSA binding unlike observation s already reported in hemopoietic cells, The ability of I-AGE-BSA to bind t o separated HUVEC plasma membrane (PM) proteins was also examined and the b inding at specific bands inhibited by antibodies to each component of the A GE-receptor complex. Western blotting of whole cell and PM fractions, befor e and after exposure to AGE-BSA, revealed that AGE-RI, -R2 and -R3 are subj ect to upregulation upon exposure to their ligand, a phenomenon which was a lso demonstrated by immunofluorescence of non-permeabilised cells. mRNA exp ression of each AGE-receptor component was apparent in HUVECs, with the AGE -RE and -R3 gene expression being upregulated upon exposure to AGEs in a ti me-dependent manner. A phosporylation assay in combination with AGE-RE immu noprecipitation demonstrated that this component of the receptor complex is phosphorylated by acute exposure to AGE-BSA. These results indicate the pr esence of a conserved AGE-receptor complex in vascular endothelium which de monstrates subtle differences to other cell-types. In response to AGE-modif ied molecules, this complex is subject to upregulation, while the AGE-R, co mponent also displays increased phosphorylation possibly leading to enhance d signal transduction. (C) 1999 Academic Press.