Article pubs.acs.org/jpr
Deep Proteome Profiling of Circulating Granulocytes Reveals Bactericidal/Permeability-Increasing Protein as a Biomarker for Severe Atherosclerotic Coronary Stenosis Onno B. Bleijerveld,†,‡ Patrick Wijten,†,# Salvatore Cappadona,†,# Elizabeth A. McClellan,§,# Ayse N. Polat,† Reinout Raijmakers,† Jan-Willem Sels,∥ Loes Colle,‡ Simona Grasso,† Henk W. van den Toorn,† Bas van Breukelen,† Andrew Stubbs,§ Gerard Pasterkamp,‡ Albert J.R. Heck,† Imo E. Hoefer,*,‡ and Arjen Scholten*,† †
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute of Pharmaceutical Sciences, Utrecht University and Netherlands Proteomics Centre, Utrecht, The Netherlands ‡ Experimental Cardiology Laboratory, University Medical Center Utrecht, Utrecht, The Netherlands § Department of Bioinformatics, Erasmus Medical Center, Rotterdam, The Netherlands ∥ Department of Cardiology, Catharina Hospital Eindhoven, Eindhoven, The Netherlands S Supporting Information *
ABSTRACT: Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system. We therefore employed proteomics to search for biomarkers associated with severe atherosclerotic coronary lumen stenosis in circulating leukocytes. In a twophase approach, we first performed in-depth quantitative profiling of the granulocyte proteome on a small pooled cohort of patients suffering from chronic (sub)total coronary occlusion and matched control patients using stable isotope peptide labeling, two-dimensional LC−MS/MS and data-dependent decision tree fragmentation. Over 3000 proteins were quantified, among which 57 candidate biomarker proteins remained after stringent filtering. The most promising biomarker candidates were subsequently verified in the individual samples of the discovery cohort using label-free, single-run LC−MS/MS analysis, as well as in an independent verification cohort of 25 patients with total coronary occlusion (CTO) and 19 matched controls. Our data reveal bactericidal/permeability-increasing protein (BPI) as a promising biomarker for severe atherosclerotic coronary stenosis, being down-regulated in circulating granulocytes of CTO patients. KEYWORDS: ETD, HCD, CID, data-dependent decision tree, biomarkers, atherosclerosis, granulocytes, bactericidal/permeability-increasing protein
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INTRODUCTION Coronary artery disease (CAD) and stroke are the two leading causes of mortality worldwide, accounting for over 20% of all deaths, with the number of cardiovascular events strongly increasing in both Western society and developing countries.1 The underlying pathological process of this growing burden of cardiovascular disease is atherosclerosis, a chronic inflammatory disease of the arterial wall, which is initially characterized by endothelial dysfunction. In response to turbulent flow, dyslipidemia, hypertension or proinflammatory mediators, activated endothelium provokes early plaque formation by adhesion of platelets and leukocytes2 and subsequent transmigration of leukocytes to subendothelial regions.3 During atheroprogression, development of more advanced plaques is © XXXX American Chemical Society
driven by a continuous influx of leukocytes into the atherosclerotic lesion. Mature plaques are characterized by a lipid-rich “necrotic core” composed of cell debris and a fibrous cap composed of smooth muscle cells and matrix proteins. An increase in matrix-degrading factors over time can cause thinning of the fibrous cap, eventually leading to plaque rupture, subsequent thrombus formation and arterial occlusion, resulting in acute coronary syndrome (unstable angina or myocardial infarction) or stroke. When coronary atherosclerosis progresses over time without giving rise to acute coronary syndrome (ACS), patients Received: May 11, 2012
A
dx.doi.org/10.1021/pr3004375 | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
enrolled in the Circulating Cells study (see the Acknowledgment) were included in the discovery cohort of this study. Five patients with 1-vessel coronary artery disease and (sub)total stenosis (≥90%) of 1 coronary segment were selected on the basis of the angiographic description. Five age- and sex-matched patients without significant coronary atherosclerosis served as control. Table 1 summarizes the baseline characteristics of the
eventually may develop total coronary occlusions, defined as 99−100% lumen diameter stenosis with absent antegrade flow (TIMI 0/1).4 Patients with CTO (i.e., when a total luminal occlusion is present for at least 3 months) typically have collateralization of the distal vessel on coronary angiography, but these collaterals may not provide sufficient blood flow to the myocardial bed, resulting in ischemia and stable anginal symptoms.5 Among patients who have a clinical indication for coronary angiography, the incidence of CTO has been reported to be as high as 15−30%.4,5 In many cases, however, CTOs can remain clinically silent and hence unnoticed. On the background of the recent studies on the favorable outcome after revascularization therapy of CTOs, markers for the detection of (sub)total coronary occlusions are warranted. When it comes to risk prediction for coronary heart disease, the only “biomarkers” currently used by both general practitioners and clinicians are LDL and HDL cholesterol and triglycerides.6 However, plasma cholesterol concentrations are poor risk predictors, as more than half of all vascular events occur in individuals with below-average total cholesterol concentrations.7 Several candidate serum biomarkers reflecting inflammation, oxidative stress and thrombosis have been evaluated as potential clinical tools for improving risk prediction.8,9 However, the additive value of plasma biomarkers such as C-reactive protein and B-type natriuretic peptide, along with existing risk scores (e.g., Framingham Risk Score, FRS), so far remains minimal.10 Over the past decade, considerable efforts have been made to search for biomarkers employing proteomics. As an alternative to the notoriously difficult plasma analyses, investigation of the diseased vasculature has also been proposed, including coronary artery segments, plaque specimens and the intimal layer of atherosclerotic coronaries.11−13 However, given the inflammatory nature of atherosclerosis and the poor accessibility of plaque tissue in patients, circulating cells (leukocytes and platelets in particular) are logical and interesting alternative sources to investigate.14 Besides the well-studied role of granulocytes in acute inflammatory processes, evidence revealing a previously underappreciated role in atherosclerosis is accumulating:15,16 systemic inflammation involving granulocyte activation is demonstrated to be associated with unstable conditions of CAD.17 An increased number of circulating neutrophils is a well-known risk factor for future cardiovascular outcomes,18,19 and infiltrated granulocytes are detectable in culprit lesions of CAD patients.20 This indicates the potential of circulating granulocytes as sources of biomarkers for atherosclerosis. Here we present a platform for the discovery and verification of novel biomarkers for atherosclerosis in circulating granulocytes from patients with CTO. Using quantitative proteomics to screen for biomarkers in circulating granulocytes from patients with CTO and matching controls, we discovered bactericidal/permeability-increasing protein (BPI) as a promising biomarker associated with severe atherosclerotic coronary stenosis.
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Table 1. Characteristics of CTO Patients (n = 5) and Controls (n = 5) Included in the Discovery Phasea
age BMI diabetes active smoking statin use platelet inhibitor use
(sub)total occlusion
nonsignificant atherosclerosis
p-value
58 ± 13.5 27.85 ± 2.47 0/5 1/5 5/5 5/5
60 ± 12.1 27.44 ± 5.05 1/5 1/5 4/5 4/5
0.834 0.465 1.000 1.000 1.000 1.000
a
Age and BMI are indicated with the mean and standard deviation; a Mann−Whitney U test was used for the p-values for numerical variables, and a Fisher’s exact test was used for the p-values for categorical variables.
study subjects. In the verification cohort of this study, 25 CTO (TIMI flow 0 or 1) patients and 19 matched control patients (including both males and females) enrolled in the Circulating Cells study were selected on the basis of the same inclusion criteria as described for the discovery cohort (see Supporting Information Table S7). All patients enrolled in the clinical study presented with symptoms of stable angina or unstable angina. All samples were collected directly after insertion of the arterial sheath used for the coronary catheterization procedure to exclude any potential effect of the intervention on granulocyte protein expression. Patients with (sub)total occlusions in both the discovery and the validation cohort all suffered from stable angina. Thus, none of the samples were collected after an acute myocardial ischemic period. Isolation and Storage of Granulocytes
Granulocytes were isolated essentially as described by Boyum et al.,21 with slight modifications. Briefly, EDTA blood samples (blood samples collected in EDTA-coated tubes) were centrifuged at 160g for 15 min without brake. The plasma fraction was removed, and the blood cell pellet was diluted with PBS before loading on top of 15 mL of Ficoll-paque Plus density gradient medium in 50 mL Leucosep tubes. Blood cells were centrifuged in a swinging bucket rotor at 1000g for 15 min at room temperature. After supernatant aspiration and removal of the lymphocyte/PBMC-rich interphase, the erythrocyte/ granulocyte fraction was collected, transferred to a new tube and incubated in sterile red blood cell (RBC) lysis buffer (NH4Cl (8.4 g/L), KHCO3 (1 g/L) in ddH2O, pH 7.4) for 10 min on ice. Cells were spun down at 330g for 5 min at 4 °C and resuspended in RBC lysis buffer once more, followed by another centrifuge step. The resulting cell pellet was washed with a large volume of cold PBS and centrifuged at 330g for 5 min at 4 °C. Finally, the granulocyte cell pellet was resuspended in PBS and diluted with cold serum-free cell freezing medium with 8.7% DMSO (Sigma Aldrich) and stored in liquid nitrogen until analysis.
EXPERIMENTAL SECTION
Subjects
All samples used in this study were obtained with approval of the Internal Review Boards of the respective institutions and after receiving signed, informed consent from patients. Ten male patients with clinical symptoms of coronary artery disease B
dx.doi.org/10.1021/pr3004375 | J. Proteome Res. XXXX, XXX, XXX−XXX
Journal of Proteome Research
Article
Sample Preparation
Data Analysis: Identification and Quantitation
The stored granulocyte samples were thawed and centrifuged at 14000g for 10 min at 4 °C. The pellet was reconstituted in a lysis buffer containing 100 mM Tris, 10 mM DTT, 2% SDS at pH 8.0 with Complete Mini protease inhibitor (Roche Diagnostics, Mannheim, Germany) and PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics) and heated at 95 °C for 5 min. Lysates were subjected to ultrasonication using a Labsonic M Ultrasonic Homogenizer (Sartorius Stedim Biotech, Nieuwegein, The Netherlands): 3 times 30 s of pulses (100% amplitude, 80% interval) were performed with 30 s intervals, followed by 60 s of continuous pulsing (100% amplitude, 100% interval). The remaining cell debris was removed by centrifugation at 14000g for 10 min at room temperature. For the discovery phase, two sample pools (chronic (sub)total coronary occlusion (≥90%), n = 5; and controls, n = 5) were generated, consisting of 25 μg of protein per patient adding up to 125 μg of protein per pool. Proteins were then reduced, alkylated and digested using the FASP approach in which the lysis buffer is exchanged to 8 M urea pH 8.0 as described previously in order to remove the SDS present in the sample.22 Digestion was performed for 4 h with Lys-C (Wako, Richmond, VA, USA), after which the mixture was diluted 4-fold to 2 M urea and digested with trypsin (Promega, Madison, WI, USA) at 37 °C overnight. Finally, the sample was acidified with formic acid to a final concentration of 5%. Tryptic peptides were desalted using Sep-Pak C18 cartridges (Waters Corporation, Milford, MA). The peptides were subsequently labeled on-column with stable isotope dimethyl labeling as described previously,23 the CTO patient pool was labeled “intermediate”, whereas the control pool received the “light” label. Labeling efficiency was checked by LC−MS/MS before mixing the pools in a 1:1 ratio.
Peak lists were generated from the raw data files using the Proteome Discoverer software package version 1.3.339 (Thermo Scientific, Bremen, Germany). Peptide identification was performed by searching the peak list against a concatenated target-decoy database containing the human sequences in the Uniprot database (release 2010_12, 41032 sequences) supplemented with a common contaminants database using the Mascot search engine version 2.3 (Matrix Science, London, U. K.) via the Proteome Discoverer interface. The search parameters included the use of trypsin as proteolytic enzyme allowing up to a maximum of 2 missed cleavages. Carbomidomethylation of cysteines was set as a fixed modification, whereas oxidation of methionines, the dimethyl “light” and “intermediate” labels on N-termini and lysine residues were set as variable modifications. Precursor mass tolerance was initially set at 50 ppm, while fragment mass tolerance was set at 0.6 Da for CID fragmentation (in the ion trap), and at 0.05 Da for HCD and ETD fragmentation (in the Orbitrap). Subsequently, the peptide identifications were filtered for true mass accuracy 20 until an FDR
