title: Effect of particle size on hydroxyapatite crystal-induced tumor necrosis factor alpha secretion by macrophages.
abstract: Macrophages may promote a vicious cycle of inflammation and calcification in the vessel wall by ingesting neointimal calcific deposits (predominantly hydroxyapatite) and secreting tumor necrosis factor (TNF)alpha, itself a vascular calcifying agent. Here we have investigated whether particle size affects the proinflammatory potential of hydroxyapatite crystals in vitro and whether the nuclear factor (NF)-kappaB pathway plays a role in the macrophage TNFalpha response. The particle size and nano-topography of nine different crystal preparations was analyzed by X-ray diffraction, Raman spectroscopy, scanning electron microscopy and gas sorbtion analysis. Macrophage TNFalpha secretion was inversely related to hydroxyapatite particle size (P=0.011, Spearman rank correlation test) and surface pore size (P=0.014). A necessary role for the NF-kappaB pathway was demonstrated by time-dependent I kappaB alpha degradation and sensitivity to inhibitors of I kappaB alpha degradation. To test whether smaller particles were intrinsically more bioactive, their mitogenic activity on fibroblast proliferation was examined. This showed close correlation between TNFalpha secretion and crystal-induced fibroblast proliferation (P=0.007). In conclusion, the ability of hydroxyapatite crystals to stimulate macrophage TNFalpha secretion depends on NF-kappaB activation and is inversely related to particle and pore size, with crystals of 1-2 microm diameter and pore size of 10-50 A the most bioactive. Microscopic calcific deposits in early stages of atherosclerosis may therefore pose a greater inflammatory risk to the plaque than macroscopically or radiologically visible deposits in more advanced lesions.
authors.affiliation: British Heart Foundation Cardiovascular Medicine Unit, Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
authors.forename: Imad
authors.initials: I
authors.lastname: Nadra
authors.affiliation: Department of Materials, Imperial College London, Prince Consort Road, London SW7 2BP, UK.
authors.forename: Aldo R
authors.initials: AR
authors.lastname: Boccaccini
authors.affiliation: British Heart Foundation Cardiovascular Medicine Unit, Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
authors.forename: Pandelis
authors.initials: P
authors.lastname: Philippidis
authors.affiliation: Department of Clinical Pharmacology, The Royal College of Surgeons and Mater Misericordiae University Hospital, Dublin, Ireland.
authors.forename: Linda C
authors.initials: LC
authors.lastname: Whelan
authors.affiliation: Department of Clinical Pharmacology, The Royal College of Surgeons and Mater Misericordiae University Hospital, Dublin, Ireland.
authors.forename: Geraldine M
authors.initials: GM
authors.lastname: McCarthy
authors.affiliation: British Heart Foundation Cardiovascular Medicine Unit, Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK.
authors.forename: Dorian O
authors.initials: DO
authors.lastname: Haskard
authors.affiliation: British Heart Foundation Cardiovascular Medicine Unit, Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, Du Cane Road, London W12 ONN, UK. Electronic address: r.landis@imperial.ac.uk.
authors.forename: R Clive
authors.initials: RC
authors.lastname: Landis
mesh_terms: D050197:Atherosclerosis / Q000503:physiopathology; D001672:Biocompatible Materials / Q000737:chemistry / Q000494:pharmacology*; D002114:Calcinosis / Q000503:physiopathology; D002478:Cells, Cultured; D017886:Durapatite / Q000737:chemistry / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D052816:Foreskin / Q000166:cytology; D006801:Humans; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D016328:NF-kappa B / Q000378:metabolism; D010316:Particle Size; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism*
chemical_list: D001672:Biocompatible Materials; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D017886:Durapatite