S100A9-RAGE Axis Accelerates Formation of Macrophage-Mediated Extracellular Vesicle Microcalcification in Diabetes Mellitus

Abstract

Objective: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P <0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe −/− S100a9 −/− mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. Conclusions: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.

Compositional change of gut microbiome and osteocalcin expressing endothelial progenitor cells in patients with coronary artery disease

“…It could be postulated that increased levels of OCN-expressing cells accompanied by dysbiosis-induced inflammation might mediate this bone-vascular axis, requiring future investigation. A recent study also suggested that hyperglycemic condition might promote secretion of proinflammatory and osteogenic factors from macrophages, which might be associated with increased levels of OCN-expressing EPCs in diabetic patients [ 45 ].…”

S100A8/A9 in Myocardial Infarction: A Promising Biomarker and Therapeutic Target

“…A genome-wide comparative bioinformatics analysis of gene expression in monocytes stimulated with S100A8 revealed overexpression of specific functional clusters related to the inflammatory response, leukocyte activation, cell migration, and the NF-κB signal transduction pathway ( Fassl et al, 2015 ). Recombinant S100A9 induces the transcription of proinflammatory mediators in macrophages, and the response can be blocked by a RAGE antagonist or S100A9 silencing ( Kawakami et al, 2020 ).…”

“…Moreover, a significant upregulation of RAGE results in sustained NF-κB activation ( Andrassy et al, 2006 ), triggering a continuous inflammatory response and generating key proinflammatory mediators. S100A8/A9 might stimulate this positive feedback loop by engaging with RAGE ( Kawakami et al, 2020 ), driving adverse cardiac remodeling post I/R. Upon ligation with its various receptors, S100A8/A9 can regulate CM death and cytokine release from innate immune cells in response to I/R injury ( Figure 3 ).…”

Inflammation Meets Metabolism Roles: for the Receptor for Advanced Glycation End Products Axis in Cardiovascular Disease

“…RAGE ligand S100A9 was linked to calcification mechanisms through high glucose treatment of macrophages. When human primary macrophages were cultured in the presence of high glucose conditions, the secretion of RAGE ligand S100A9 and expression of RAGE in those cells was noted to increase [ 87 ]. Recombinant S100A9 was employed to directly test its effects on macrophages; S100A9 induced expression of osteogenic factors and the production of extracellular vesicles that contained high calcific potential (on account of high alkaline phosphatase activity); this was prevented by blockade of RAGE or silencing RNAs targeting S100a9 .…”