title: Calcifying nanoparticles promote mineralization in vascular smooth muscle cells: implications for atherosclerosis.
abstract: BACKGROUND
  Nano-sized complexes of calcium phosphate mineral and proteins (calcifying nanoparticles [CNPs]) serve as mineral chaperones. Thus, CNPs may be both a result and cause of soft tissue calcification processes. This study determined if CNPs could augment calcification of arterial vascular smooth muscle cells in vitro.
  
  
  METHODS
  CNPs 210 nm in diameter were propagated in vitro from human serum. Porcine aortic smooth muscle cells were cultured for up to 28 days in medium in the absence (control) or presence of 2 mM phosphate ([P] positive calcification control) or after a single 3-day exposure to CNPs. Transmission electron-microscopy was used to characterize CNPs and to examine their cellular uptake. Calcium deposits were visualized by light microscopy and von Kossa staining and were quantified by colorimetry. Cell viability was quantified by confocal microscopy of live-/dead-stained cells and apoptosis was examined concurrently by fluorescent labeling of exposed phosphatidylserine.
  
  
  RESULTS
  CNPs, as well as smaller calcium crystals, were observed by transmission electron-microscopy on day 3 in CNP-treated but not P-treated cells. By day 28, calcium deposits were visible in similar amounts within multicellular nodules of both CNP- and P-treated cells. Apoptosis increased with cell density under all treatments. CNP treatment augmented the density of apoptotic bodies and cellular debris in association with mineralized multicellular nodules.
  
  
  CONCLUSIONS
  Exogenous CNPs are taken up by aortic smooth muscle cells in vitro and potentiate accumulation of smooth-muscle-derived apoptotic bodies at sites of mineralization. Thus, CNPs may accelerate vascular calcification.
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, MN, USA.
authors.forename: Larry W
authors.initials: LW
authors.lastname: Hunter
authors.affiliation: Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA.
authors.forename: Jon E
authors.initials: JE
authors.lastname: Charlesworth
authors.affiliation: Lincoln University, Christchurch, New Zealand ; Izon Science Ltd., Christchurch, New Zealand.
authors.forename: Sam
authors.initials: S
authors.lastname: Yu
authors.affiliation: Department of Internal Medicine, Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN, USA ; Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Lieske
authors.affiliation: Department of Surgery, Mayo Clinic, Rochester, MN, USA ; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.
authors.forename: Virginia M
authors.initials: VM
authors.lastname: Miller
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism; D059587:Calcifying Nanoparticles / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism*; D013552:Swine
chemical_list: D059587:Calcifying Nanoparticles; D002118:Calcium
keywords: biomineralization; hydroxyapatite; protein-mineral complex; vascular calcification