title: Circulating myeloid calcifying cells have antiangiogenic activity via thrombospondin-1 overexpression.
abstract: Myeloid calcifying cells (MCCs) represent a subpopulation of human monocytes with procalcific potential and are characterized by coexpression of osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, an in-depth proteomic investigation of MCCs based on fluorescence-activated cell sorting, protein extraction and digestion, isobaric tag for relative and absolute quantitation labeling, fractionation, and analysis on matrix-assisted laser desorption/ionization-time of flight/time of flight and LTQ Orbitrap mass spectrometers identified and quantified more than 700 proteins and revealed pathways activated in OC(+)BAP(+) MCCs compared with those in OC(-)BAP(-) cells. Among proteins referable to angiogenesis, the thrombospondin-1 pathway was markedly up-regulated in MCCs vs. control cells. Up-regulation of the thrombospondin-1 pathway was confirmed by a genome-wide transcriptional analysis. Using in vitro and in vivo angiogenesis assays, we found that freshly isolated MCCs and cultured MCCs display an antiangiogenic function by means of both paracrine activity (conditioned medium) and altered spatial localization in cocultures with endothelial cells. Thrombospondin-1 inhibition by antibody-mediated neutralization or gene knockdown restored the angiogenic activity of OC(+)BAP(+) MCCs toward normal values and abolished the antiangiogenic effects of MCC conditioned medium. These data indicate that circulating MCCs exert antiangiogenic activity by virtue of their overexpression of thrombospondin-1. The study highlights the successful identification and validation of a pathogenic pathway by a gold standard proteomic/transcriptomic analysis of blood cells.
authors.affiliation: 2Department of Medicine, Division of Metabolic Diseases, University Hospital of Padova, Via Giustiniani 2, 35100 Padova, Italy. gianpaolo.fadini@unipd.it.
authors.forename: Lisa
authors.initials: L
authors.lastname: Menegazzo
authors.forename: Mattia
authors.initials: M
authors.lastname: Albiero
authors.forename: Renato
authors.initials: R
authors.lastname: Millioni
authors.forename: Serena
authors.initials: S
authors.lastname: Tolin
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.forename: Nicol
authors.initials: N
authors.lastname: Poncina
authors.forename: Paolo
authors.initials: P
authors.lastname: Tessari
authors.forename: Angelo
authors.initials: A
authors.lastname: Avogaro
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Fadini
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic; D002196:Capillaries / Q000378:metabolism / Q000502:physiology; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D051379:Mice; D022423:Myeloid Cells / Q000378:metabolism* / Q000502:physiology; D018919:Neovascularization, Physiologic; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D019899:Paracrine Communication; D020543:Proteome / Q000378:metabolism; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D020543:Proteome; D019700:Thrombospondin 1; D015675:Osteocalcin; D000469:Alkaline Phosphatase
keywords: atherosclerosis; endothelial cells; inflammation; stem cells