title: RANKL promotes osteoblastic activity in vascular smooth muscle cells by upregulating endothelial BMP-2 release.
abstract: Receptor activator of nuclear factor kappa beta-ligand (RANKL) is thought to promote vascular calcification (VC) by inducing osteoblastic behaviour in vascular smooth muscle cells (VSMC) in an ill-defined process. The present study assessed whether RANKL affects pro-osteoblastic paracrine signalling between human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) using both conditioned media transfer and cell co-culture experimental approaches.
  
  
  
  For initial experiments (6-well format), HAEC-conditioned media was harvested following 72h exposure to RANKL, and transferred to reporter HASMCs with/without noggin, an inhibitor of pro-osteoblastic bone morphogenetic protein (BMP) paracrine signalling. In further experiments, HAECs and HASMCs were co-cultured within the CellMax(®) Duo, a perfusing bioreactor unit that mimics the flow-mediated co-interaction of these cells within the arterial wall, and RANKL was added to the perfusing media for 72h. At the conclusion of each experiment markers of osteoblastic activity were measured in HASMCs, including alkaline phosphatase (ALP) activity, mRNA levels of ALP and Runx2, as well as BMP-2 and BMP-4 concentrations. RANKL increased BMP-2 release from HAECs, while exposure of HASMCs to RANKL-treated HAEC-conditioned media induced osteoblastic behaviour in HASMCs, an effect prevented by noggin. Within the CellMax(®) Duo bioreactor, the addition of RANKL to the intraluminal HAECs also produced an increase in BMP-2 and increased osteoblastic behaviour within the co-cultured HASMC population.
  
  
  
  RANKL promotes VC by inducing BMP-2 release from HAECs, which in turn appears to act in a paracrine fashion on the adjacent HASMC population to increase osteoblastic activity.
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland. Electronic address: drcdavenport@gmail.com.
authors.forename: Colin
authors.initials: C
authors.lastname: Davenport
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Emma
authors.initials: E
authors.lastname: Harper
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland.
authors.forename: Hannah
authors.initials: H
authors.lastname: Forde
authors.affiliation: Conway Institute, University College Dublin, Dublin, Ireland.
authors.forename: Keith D
authors.initials: KD
authors.lastname: Rochfort
authors.affiliation: School of Health and Human Performance, Dublin City University, Dublin, Ireland; Centre for Preventive Medicine, Dublin City University, Dublin, Ireland.
authors.forename: Ronan P
authors.initials: RP
authors.lastname: Murphy
authors.affiliation: Department of Academic Endocrinology, Beaumont Hospital, Dublin, Ireland.
authors.forename: Diarmuid
authors.initials: D
authors.lastname: Smith
authors.affiliation: School of Biotechnology, Dublin City University, Dublin, Ireland; Centre for Preventive Medicine, Dublin City University, Dublin, Ireland.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Cummins
mesh_terms: D000328:Adult; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism*; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000000981:diagnostic imaging*; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects*; D053245:RANK Ligand / Q000494:pharmacology*; D055815:Young Adult
chemical_list: C525718:BMP2 protein, human; D055396:Bone Morphogenetic Protein 2; D053245:RANK Ligand
keywords: BMP-2; Calcification; Endothelial; Osteoprotegerin; RANKL