Differential effect of plasma or erythrocyte AGE-ligands of RAGE on expression of transcripts for receptor isoforms

Binding of advanced glycation end-products (AGEs) to the receptor for AGEs (RAGE) contributes to diabetic vascular complications. RAGE transcript splicing generates membrane-bound proteins [full-length (FL) and N-truncated (Nt)] and a soluble protein [endogenous secretory (esRAGE)] that may act as a decoy. We tested the effect of AGE-ligands on the regulation of RAGE isoforms and the consequences on red blood cell (RBC) adhesion.

RAGE isoforms were measured by real-time RT-PCR assay, using a LightCycler System, in human umbilical vein endothelial cells (HUVECs), incubated with either characterized AGEs [Nvarepsilon-(carboxymethyl)lysine human serum albumin (CML-HSA) and methylglyoxal-modified HSA (MG-HSA)] or with RBCs from diabetic patients (DRBCs). Inhibition of RAGE access was achieved by using blocking either anti-RAGE antibodies or recombinant RAGE. Adhesion of DRBCs to endothelium was measured under flow conditions using HUVECs stimulated with MG-HSA or CML-HSA. Antibodies directed to RBC membrane proteins were tested for blocking DRBC adhesion in static conditions.

MG-HSA stimulated the expression of membrane-bound RAGE (FL+Nt) and esRAGE transcripts to similar extents, while CML-HSA and DRBC more selectively induced mRNA for FL and Nt-RAGE. Anti-RAGE antibody inhibited the effect of glycated proteins. Stimulation of HUVECs with CML-HSA enhanced DRBC adhesion, while MG-HSA had no effect. CD233 (band 3) was glycated in DRBC membrane, and anti-CD233 antibodies inhibited the adhesion of DRBCs, as did the anti-RAGE and anti-AGE antibodies.

Receptor engagement by distinct AGEs differentially enhances expression of RAGE isoforms and DRBC adhesion. The CML-adduct, by facilitating adhesion, has more deleterious effects than MG-derived AGEs.