Binding of receptor for advanced glycation end products (RAGE) ligands is not sufficient to induce inflammatory signals: lack of activity of endotoxin-free albumin-derived advanced glycation end products

Abstract

Aims/hypothesis. Activation of the receptor for advanced glycation end products (RAGE) reportedly triggers cellular responses implicated in the pathogenesis of diabetes, such as increasing vascular cell adhesion molecule-1 (VCAM-1) expression on vascular endothelial cells and inducing TNF-α secretion by mononuclear cells. The objective of this study was to evaluate whether RAGE binding affinity of AGEBSAs and cellular activation correlate. Methods. To produce AGEs with varying glycation, bovine albumin AGEs were prepared with 500 mmol/l of glucose, fructose or ribose at times of incubation from 1 to 12 weeks. In addition, AGE-BSA was generated using either glyoxylic acid or glycolaldehyde. Cellular binding of the AGE-BSAs and the effect on endothelial cell VCAM-1 expression were studied in RAGE-expressing human microvascular endothelial cell line-4 cells. Induction of TNF-α secretion was assessed using RAGE-expressing human peripheral blood mononuclear cells (PBMCs).Results. Cellular binding of the different AGE preparations correlated well with RAGE affinity. Interestingly, we found that the AGE preparations, which were essentially endotoxin free (≤0.2 ng/mg protein), were incapable of inducing VCAM-1 or TNF-α secretion regardless of RAGE binding affinity, AGE concentration or incubation time. In contrast, the reported RAGE ligand S100b was confirmed to induce VCAM-1 expression on endothelial cells and TNF-α secretion by PBMCs after 24 h of treatment. Conclusions/interpretation. The results of this study suggest that AGE modification and high RAGE binding affinity are not sufficient to generate pro-inflammatory signalling molecules. Thus, RAGE binding affinity of AGE-BSAs does not seem to correlate with cellular activation. Our findings using AGEs with strong RAGE-binding properties indicate that AGEs may not uniformly play a role in cellular activation.

Advanced Glycation in macrophages induces intracellular accumulation of 7-ketocholesterol and total sterols by decreasing the expression of ABCA-1 and ABCG-1

“…These events were also related to the elevated generation of reactive oxygen species in glycolaldehyde-modified albumin-treated macrophages, since treatment with aminoguanidine that reduced ROS generation was able to restore ABCA-1 protein content [21]. AGE-albumin also primers macrophages to inflammation which is related to the diminished cholesterol exportation to apolipoprotein A-I [22]. Although we were not able to detect by immunoblot CML derivatives in ABCA-1 and ABCG-1, at this point we cannot completely exclude that part of the changes observed in the content and activity of ABCA-1 and ABCG-1 relates to GAD modification of both transporters.…”

Insulin Resistance, Hyperglycemia, and Atherosclerosis

“…These findings are most likely explained by the ability of RAGE to bind several different ligands, such as S100/calgranulins and high-mobility group box 1, which are elevated in a large number of inflammatory diseases, including diabetes (Yan et al, 2010; Soro-Paavonen et al, 2008). The relative importance of AGEs as RAGE activators in diabetes is therefore still unknown, in part due to the difficulty of specifically blocking AGE-RAGE interactions without blocking RAGE binding to its other ligands in vivo and to issues related to preparing pure and physiologically relevant AGE preparations (Valencia et al, 2004). Thus, while endothelial or possibly macrophage RAGE activation may conspire with diabetes to promote early/mid stage atherosclerosis, it is as yet uncertain whether RAGE activation is an important mediator of hyperglycemia-induced atherosclerosis per se .…”

Role of Glycated Proteins in the Diagnosis and Management of Diabetes: Research Gaps and Future Directions

“…RAGE is expressed on the surface of several cells, including endothelial and renal cells, raising intriguing hypotheses about the role of RAGE in the pathophysiology of specific diabetes complications. Nevertheless, some studies cast doubt on whether AGE-modified proteins activate RAGE (87). Proteolysis of RAGE leads to a truncated soluble form of RAGE (sRAGE) (66), which is found in serum and can be measured by a commercially available ELISA.…”