title: RANKL enhances macrophage paracrine pro-calcific activity in high phosphate-treated smooth muscle cells: dependence on IL-6 and TNF-α.
abstract: BACKGROUND
  Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines.
  
  
  METHODS
  We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification.
  
  
  RESULTS
  Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification.
  
  
  CONCLUSIONS
  RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.
authors.affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA.
authors.forename: Kate Ann
authors.initials: KA
authors.lastname: Deuell
authors.forename: Andrea
authors.initials: A
authors.lastname: Callegari
authors.forename: Cecilia M
authors.initials: CM
authors.lastname: Giachelli
authors.forename: Michael E
authors.initials: ME
authors.lastname: Rosenfeld
authors.forename: Marta
authors.initials: M
authors.lastname: Scatena
mesh_terms: D000818:Animals; D002114:Calcinosis / Q000150:complications / Q000209:etiology*; D018920:Coculture Techniques; D016207:Cytokines; D015850:Interleukin-6 / Q000378:metabolism* / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology; D053244:Osteoprotegerin / Q000096:biosynthesis; D010710:Phosphates / Q000494:pharmacology*; D053245:RANK Ligand / Q000096:biosynthesis / Q000494:pharmacology*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000096:biosynthesis; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D016207:Cytokines; D015850:Interleukin-6; D053244:Osteoprotegerin; D010710:Phosphates; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D014409:Tumor Necrosis Factor-alpha