title: The RANKL/RANK/OPG signaling pathway mediates medial arterial calcification in diabetic Charcot neuroarthropathy.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  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.
authors.affiliation: Department of Medicine and Diabetes, Manchester Royal Infirmary, Manchester, UK. agbor.ako@manchester.ac.uk
authors.forename: Agbor
authors.initials: A
authors.lastname: Ndip
authors.forename: Alfred
authors.initials: A
authors.lastname: Williams
authors.forename: Edward B
authors.initials: EB
authors.lastname: Jude
authors.forename: Ferdinand
authors.initials: F
authors.lastname: Serracino-Inglott
authors.forename: Steve
authors.initials: S
authors.lastname: Richardson
authors.forename: J V
authors.initials: JV
authors.lastname: Smyth
authors.forename: Andrew J M
authors.initials: AJ
authors.lastname: Boulton
authors.forename: M Yvonne
authors.initials: MY
authors.lastname: Alexander
mesh_terms: D000368:Aged; D002114:Calcinosis / Q000209:etiology; D002454:Cell Differentiation; D002478:Cells, Cultured; D002607:Charcot-Marie-Tooth Disease / Q000503:physiopathology*; D003928:Diabetic Nephropathies / Q000503:physiopathology*; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000097:blood; D006801:Humans; D016209:Interleukin-8 / Q000097:blood; D008297:Male; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D053244:Osteoprotegerin / Q000502:physiology*; D053245:RANK Ligand / Q000502:physiology*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D016209:Interleukin-8; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D016179:Granulocyte Colony-Stimulating Factor