The RANKL/RANK/OPG Signaling Pathway Mediates Medial Arterial Calcification in Diabetic Charcot Neuroarthropathy

摘要

OBJECTIVE-The receptor activator of nuclear factor-KB (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG) signaling pathway (RANKL/RANK/OPG signaling) is implicated in the osteolysis associated with diabetic Charcot neuroarthropathy (CN); however, the links with medial arterial calcification (MAC) seen in people with CN are unclear. This study aimed to investigate the role of RANKL/OPG in MAC in patients with CN. RESEARCH DESIGN AND METHODS-Enzyme-linked im-munosorbent assay and Bio-plex multiarray technology were used to quantify a range of cytokines, including RANKL and OPG in sera from 10 patients with diabetes, 12 patients with CN, and 5 healthy volunteers. Human tibial artery segments were immuno-histochemically stained with Alizarin red and human RANKL antibody. Human vascular smooth muscle cells (VSMCs) were also explanted from arterial segments for in vitro studies. RESULTS-We demonstrate colocalization and upregulation of RANKL expression in areas displaying MAC. Systemic levels of RANKL, OPG, and inflammatory cytokines (interleukin-8, gran-ulocyte colony-stimulating factor) were elevated in those with CN compared with diabetic patients and healthy control subjects. Human VSMCs cultured in CN serum showed accelerated osteo-blastic differentiation (alkaline phosphatase activity) and mineralization (alizarin red staining) compared with cells treated with diabetic or control serum (P < 0.05). Coincubation with OPG, the decoy receptor for RANKL, attenuated osteogenic differentiation of VSMCs and was independent of a high calcium-phosphate milieu. The accelerated mineralization induced by RANKL and CN serum correlated with nuclear translocation of nuclear factor-KB, a process abrogated by OPG. CONCLUSIONS-Our data provide direct evidence that RANKL/ RANK/OPG signaling is modulated in patients with CN and plays a role in vascular calcification. This study highlights this pathway as a potential target for intervention.