title: Plasma proteome profiling of atherosclerotic disease manifestations reveals elevated levels of the cytoskeletal protein vinculin.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Lars P
authors.initials: LP
authors.lastname: Kristensen
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Larsen
authors.affiliation: Department of Cardiology, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Hans
authors.initials: H
authors.lastname: Mickley
authors.affiliation: Department of Cardiology, Odense University Hospital, Odense, Denmark.
authors.forename: Lotte
authors.initials: L
authors.lastname: Saaby
authors.affiliation: Department of Cardiology, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Axel C P
authors.initials: AC
authors.lastname: Diederichsen
authors.affiliation: Department of Cardiology, Odense University Hospital, Svendborg, Denmark.
authors.forename: Jess
authors.initials: J
authors.lastname: Lambrechtsen
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark; Centre for Individualized Medicine in Arterial Diseases, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark. Electronic address: Lars.Melholt.Rasmussen@ouh.regionsyddanmark.dk.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Rasmussen
authors.affiliation: Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark; Institute of Clinical Research, University of Southern Denmark, Odense, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: Overgaard
mesh_terms: D000368:Aged; D050197:Atherosclerosis / Q000378:metabolism*; D015415:Biomarkers / Q000032:analysis / Q000097:blood; D001798:Blood Proteins / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D055442:Metabolome; D008875:Middle Aged; D020543:Proteome / Q000032:analysis / Q000378:metabolism*; D015854:Up-Regulation; D016596:Vinculin / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D003598:Cytoskeletal Proteins; D020543:Proteome; D016596:Vinculin
keywords: Atherosclerosis; Immunoassay; Plasma biomarker; Proteomics; Selected-reaction-monitoring; Vinculin