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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Grossin
N
N
Vascular and Cellular Biology Laboratory, University of Paris-7 Denis-Diderot, France.
Wautier
M-P S
MP
Picot
J
J
Stern
D M
DM
Wautier
J-L T
JL
D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D002448:Cell Adhesion; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000503:physiopathology; D003925:Diabetic Angiopathies / Q000517:prevention & control; D004730:Endothelium, Vascular / Q000378:metabolism / Q000503:physiopathology; D004912:Erythrocytes / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation; D006442:Glycated Hemoglobin / Q000032:analysis; D017127:Glycation End Products, Advanced / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D006442:Glycated Hemoglobin; D017127:Glycation End Products, Advanced; D008024:Ligands; D020033:Protein Isoforms; D012333:RNA, Messenger; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D012709:Serum Albumin; C454201:SLC4A1 protein, human