Kristensen Lars P, Larsen Martin R, Mickley Hans, Saaby Lotte, Diederichsen Axel C P, Lambrechtsen Jess, Rasmussen Lars M, Overgaard Martin
Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
J Proteomics. 2014 Apr 14;101:141-53. doi: 10.1016/j.jprot.2013.12.011. Epub 2013 Dec 22.
Atherosclerosis is a chronic disease of the arterial wall that is recognized as the leading cause of mortality and morbidity worldwide. There is an eminent need for better biomarkers that can aid in patient care before the onset of the first cardiovascular event. We used quantitative proteomics to identify proteins with altered concentrations in plasma samples from four groups: 1) Individuals without cardiovascular symptoms and without the presence of coronary calcium, 2) individuals without cardiovascular symptoms, but with high amounts of coronary calcium, 3) individuals operated because of atherosclerotic diseases, and 4) individuals with an acute coronary syndrome. Immunoassays and SRM-MS were used for single patient verification of candidate proteins. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed an increased expression profile from groups 1 to 4. The top-most elevated protein, vinculin (Vcl) displayed a similar profile. Immunoassays confirmed the expression profile of apo(a) and CRP. A 5-plex SRM-MS assay for Vcl, SAA, CRP, apo(a) and thrombospondin-4 (TSP-4) was developed for multiplex verification in all 120 individual samples. The 5-plex SRM assay confirmed a statistically significant up-regulation of Vcl in the acute coronary syndrome group.
The aim of this study was to identify new candidate plasma markers of atherosclerosis manifestations, which may develop into screening-, diagnostic- or monitoring biomarkers for risk stratification of cardiovascular disease (CVD). At present no studies have elucidated the proteomic changes that occur along with several stages and manifestations of atherosclerotic disease. By using 4-plex iTRAQ, we identified and quantified proteins with altered concentrations in pooled plasma samples from 120 individuals from four middle-aged groups. Proteins involved in cardiovascular diseases i.e. serum amyloid protein A (SAA), C-reactive protein (CRP), and apolipoprotein(a) [apo(a)] displayed an increased expression profile along with increased manifestations of CVD. A novel candidate marker was identified as vinculin (Vcl), a multi-protein linker that connects cell-matrix adhesions and cell-cell adhesions to the actin-based cytoskeleton. Immuno- and SRM-assays were used for single patient validation of candidate proteins. While further studies needs to address the role of Vcl in the development of atherosclerosis, the combined data provided in this report offers a catalog of the proteomic changes that occurs in plasma over several stages and manifestations of atherosclerotic disease.
动脉粥样硬化是一种动脉壁的慢性疾病，被认为是全球范围内死亡率和发病率的主要原因。迫切需要更好的生物标志物，以在首次心血管事件发生前辅助患者护理。我们使用定量蛋白质组学来鉴定四组血浆样本中浓度发生变化的蛋白质：1）无心血管症状且无冠状动脉钙化的个体；2）无心血管症状但冠状动脉钙化程度高的个体；3）因动脉粥样硬化疾病接受手术的个体；4）患有急性冠状动脉综合征的个体。免疫测定和SRM-MS用于对候选蛋白质进行单患者验证。参与心血管疾病的蛋白质，即血清淀粉样蛋白A（SAA）、C反应蛋白（CRP）和载脂蛋白（a）[apo（a）]，从第1组到第4组呈现出表达增加的趋势。升高最明显的蛋白质，纽蛋白（Vcl）呈现出类似的趋势。免疫测定证实了apo（a）和CRP的表达趋势。开发了一种用于Vcl、SAA、CRP、apo（a）和血小板反应蛋白-4（TSP-4）的5重SRM-MS测定法，用于对所有120个个体样本进行多重验证。5重SRM测定法证实急性冠状动脉综合征组中Vcl有统计学意义的上调。
本研究的目的是鉴定动脉粥样硬化表现的新候选血浆标志物，这些标志物可能发展成为用于心血管疾病（CVD）风险分层的筛查、诊断或监测生物标志物。目前尚无研究阐明随着动脉粥样硬化疾病的几个阶段和表现而发生的蛋白质组学变化。通过使用4重iTRAQ，我们鉴定并定量了来自四个中年组的120名个体的混合血浆样本中浓度发生变化的蛋白质。参与心血管疾病的蛋白质，即血清淀粉样蛋白A（SAA）、C反应蛋白（CRP）和载脂蛋白（a）[apo（a）]，随着CVD表现的增加而呈现出表达增加的趋势。一种新的候选标志物被鉴定为纽蛋白（Vcl），它是一种多蛋白连接物，将细胞-基质粘附和细胞-细胞粘附连接到基于肌动蛋白的细胞骨架上。免疫测定和SRM测定用于对候选蛋白质进行单患者验证。虽然需要进一步研究来探讨Vcl在动脉粥样硬化发展中的作用，但本报告提供的综合数据提供了一份在动脉粥样硬化疾病的几个阶段和表现中血浆中发生的蛋白质组学变化的目录。
