2Department of Medicine, Division of Metabolic Diseases, University Hospital of Padova, Via Giustiniani 2, 35100 Padova, Italy.
FASEB J. 2013 Nov;27(11):4355-65. doi: 10.1096/fj.12-223719. Epub 2013 Jul 30.
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.
骨髓钙化成骨细胞 (MCCs) 代表了具有成骨潜能的人单核细胞亚群，其特征是骨钙素 (OC) 和骨碱性磷酸酶 (BAP) 的共表达。在此，通过荧光激活细胞分选、蛋白质提取和消化、相对和绝对定量标记的同量异位标记、基于基质辅助激光解吸/电离-飞行时间/飞行时间和 LTQ Orbitrap 质谱仪的分馏和分析，对 MCCs 进行了深入的蛋白质组学研究，鉴定和定量了超过 700 种蛋白质，并揭示了 OC(+)BAP(+)MCCs 与 OC(-)BAP(-)细胞相比激活的途径。在可归属于血管生成的蛋白质中，血小板反应蛋白-1 途径在 MCCs 中明显上调。通过全基因组转录分析证实了血小板反应蛋白-1 途径的上调。通过体外和体内血管生成测定，我们发现新鲜分离的 MCCs 和培养的 MCCs 通过旁分泌活性（条件培养基）和与内皮细胞共培养时空间定位的改变来发挥抗血管生成功能。通过抗体介导的中和或基因敲低抑制血小板反应蛋白-1 恢复了 OC(+)BAP(+)MCCs 的血管生成活性至正常水平，并消除了 MCC 条件培养基的抗血管生成作用。这些数据表明，循环 MCCs 通过过度表达血小板反应蛋白-1发挥抗血管生成活性。该研究通过血细胞的金标准蛋白质组学/转录组学分析成功鉴定和验证了一条致病途径。
