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

• Published in: Diabetes (New York, N.Y.) Vol. 60; no. 8; pp. 2187 - 2196
• Main Authors: NDIP, Agbor, WILLIAMS, Alfred, JUDE, Edward B, SERRACINO-INGLOTT, Ferdinand, RICHARDSON, Steve, SMYTH, J. V, BOULTON, Andrew J. M, ALEXANDER, M. Yvonne
• Format: Journal Article
• Language: English
• Published: Alexandria, VA American Diabetes Association 01.08.2011
• Subjects: Aged; Amputation; Angiogenesis; Biological and medical sciences; Calcification; Calcification (Physiology); Calcinosis - etiology; Cell Differentiation; Cells, Cultured; Cellular signal transduction; Charcot-Marie-Tooth Disease - physiopathology; Complications; Cytokine receptors; Cytokines; Diabetes; Diabetes. Impaired glucose tolerance; Diabetic Nephropathies - physiopathology; Endocrine pancreas. Apud cells (diseases); Endocrinopathies; Etiopathogenesis. Screening. Investigations. Target tissue resistance; Female; Genetic aspects; Granulocyte Colony-Stimulating Factor - blood; Granulocytes; Growth factors; Humans; Indexing in process; Interleukin-8 - blood; Joint diseases; Male; Medical sciences; Middle Aged; Mineralization; Mortality; Muscle, Smooth, Vascular - cytology; Muscle, Smooth, Vascular - metabolism; Osteoprotegerin - physiology; Patients; Physiological aspects; RANK Ligand - physiology; Receptor Activator of Nuclear Factor-kappa B - physiology; Research design; Signal Transduction - physiology; Smooth muscle; Veins & arteries; United Kingdom; Endocrinopathy; Calcification; Signal transduction; Diabetes mellitus
• ISSN: 0012-1797; 1939-327X
• DOI: 10.2337/db10-1220

The receptor activator of nuclear factor-κB (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. Enzyme-linked immunosorbent 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 immunohistochemically stained with Alizarin red and human RANKL antibody. Human vascular smooth muscle cells (VSMCs) were also explanted from arterial segments for in vitro studies. We demonstrate colocalization and upregulation of RANKL expression in areas displaying MAC. Systemic levels of RANKL, OPG, and inflammatory cytokines (interleukin-8, granulocyte 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 osteoblastic 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-κB, a process abrogated by OPG. 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.