CLINICAL RESEARCH: CORONARY ARTERY DISEASE
Increased Levels of Neutrophil-Activating Peptide-2 in Acute Coronary Syndromes
Possible Role of Platelet-Mediated Vascular Inflammation
Camilla Smith, MD*,
Jan K. Damås, MD, PhD*,
Kari Otterdal, MSc*,
Erik Øie, MD, PhD , ,
Wiggo J. Sandberg, MSc*,
Arne Yndestad, PhD*,
Torgun Wæhre, MD, PhD*,
Hanne Scholz, PhD*,
Knut Endresen, MD, PhD,
Peder S. Olofsson, MD||,
Bente Halvorsen, PhD*,
Lars Gullestad, MD, PhD,
Stig S. Frøland, MD, PhD*, ,
Gøran K. Hansson, MD, PhD|| and
Pål Aukrust, MD, PhD*,,*
* Research Institute for Internal Medicine, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway
Institute for Surgical Research, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway
Department of Cardiology, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway
Section of Clinical Immunology and Infectious Diseases, Rikshospitalet University Hospital, University of Oslo, Oslo, Norway
|| Department of Medicine and Centre for Molecular Medicine, Karolinska University Hospital, Stockholm, Sweden
Manuscript received April 21, 2006;
revised manuscript received June 6, 2006,
accepted June 19, 2006.
* Reprint requests and correspondence: Dr. Pål Aukrust, Section of Clinical Immunology and Infectious Disease, Rikshospitalet University Hospital, N-0027 Oslo, Norway (Email: pal.aukrust{at}rikshospitalet.no).
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Abstract
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OBJECTIVES: We sought to investigate the role of the CXC chemokine neutrophil-activating peptide-2 (NAP-2) in atherogenesis and plaque destabilization.
BACKGROUND: Chemokines are involved in atherogenesis, but the role of NAP-2 in atherosclerotic disorders is unclear. Based on its potential pro-atherogenic properties, we hypothesized a pathogenic role for NAP-2 in coronary artery disease.
METHODS: We tested this hypothesis by differential experimental approaches including studies in patients with stable (n = 40) and unstable angina (n = 40) and healthy control subjects (n = 20).
RESULTS: The following results were discovered: 1) patients with stable, and particularly those with unstable, angina had markedly raised plasma levels of NAP-2 compared with control subjects, accompanied by increased expression of CXC receptor 2 in monocytes; 2) platelets, but also peripheral blood mononuclear cells (PBMCs), released large amounts of NAP-2 upon stimulation, with a particularly prominent PBMC response in unstable angina; 3) NAP-2 protein was detected in macrophages and smooth muscle cells of atherosclerotic plaques and in monocytes and platelets of coronary thrombi; 4) in vitro, recombinant and platelet-derived NAP-2 increased the expression of adhesion molecules and chemokines in endothelial cells; and 5) whereas aspirin reduced plasma levels of NAP-2, statin therapy increased NAP-2 with stimulating effects both on platelets and leukocytes.
CONCLUSIONS: Our findings suggest that NAP-2 has the potential to induce inflammatory responses within the atherosclerotic plaque. By its ability to promote leukocyte and endothelial cell activation, such a NAP-2-driven inflammation could promote plaque rupture and acute coronary syndromes.
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Abbreviations and Acronyms
| | CAD = coronary artery disease | | ELISA = enzyme-linked immunosorbent assay | | HUVEC = human umbilical vein endothelial cell | | IL = interleukin | | LPS = lipopolysaccharide | | MCP = monocyte chemoattractant protein | | MI = myocardial infarction | | NAP = neutrophil-activating peptide | | oxLDL = oxidized low-density lipoprotein | | PBMC = peripheral blood mononuclear cell | | PCI = percutaneous coronary intervention | | PHA = phytohemagglutinin | | PRP = platelet-rich plasma | | rh = recombinant human | | SMC = smooth muscle cell | | STEMI = ST-segment elevation myocardial infarction | | VCAM = vascular cellular adhesion molecule |
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Chemokines are involved in atherogenesis and plaque destabilization by activating and directing leukocytes into atherosclerotic lesion (1). Enhanced expression of monocyte chemoattractant protein (MCP)-1 is found within atherosclerotic plaques in humans, and targeted disruption of the genes for MCP-1 and its receptor significantly decrease atherosclerotic lesion formation in mice prone to develop atherosclerosis (1). Although much focus has been directed against the role of CXC chemokines in inflammatory lung disease, recent studies in gene-modified mice suggest that these chemokines (e.g., interleukin [IL]-8/CXC receptor 2 [CXCR2]) may also be involved in atherogenesis (2). Although the role of chemokines in atherosclerosis has been extensively studied in various animal models, their involvement in human coronary artery disease (CAD) has not been fully clarified.
Chemokines can be produced by a variety of cells including leukocytes, endothelial cells, and platelets. Upon activation, platelets release significant amounts of various chemokines such as regulated on activation normally T-cell expressed and secreted (RANTES), promoting inflammation in adjacent leukocytes and endothelial cells (3). Activated platelets also release large quantities of neutrophil-activating peptide-2 (NAP-2) through proteolytic conversion from its precursors beta-thromboglobulin and connective tissue-activating protein III (4). Through interaction with its receptors CXCR1 and CXCR2, NAP-2 induces chemotaxis of neutrophils and monocytes and neutrophil degranulation (4). Although several studies support a pathogenic role for its receptors in atherogenesis (1), few studies have examined the role of NAP-2 in human CAD.
Based on its potential pro-atherogenic properties, we hypothesized a pathogenic role for NAP-2 in atherogenesis and plaque destabilization. Herein we investigated this hypothesis by differential experimental approaches.
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Methods
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Patients and control subjects.
Patients with angina pectoris were consecutively recruited into the study (Table 1). All patients with unstable angina (n = 40) had experienced ischemic chest pain at rest within the preceding 48 h (i.e., Braunwald’s class IIIB), but with no evidence of myocardial necrosis by enzymatic criteria. Transient ST-T segment depression and/or T-wave inversion was present in all cases. All patients with stable angina (n = 40) had stable effort angina of >6 months duration and a positive exercise test. The diagnosis of CAD was confirmed in all patients by coronary angiography showing at least 1-vessel disease (>50% narrowing of luminal diameter). Exclusion criteria were myocardial infarction (MI) or thrombolytic therapy in the previous month, concomitant inflammatory diseases such as infections and autoimmune disorders, and liver or kidney disease. In patients undergoing percutaneous coronary intervention (PCI), all blood samples were taken before this procedure. Blood samples were taken >12 h after the last dosage of low molecular weight heparin. Control subjects in the study were 20 healthy blood donors (Table 1). Informed consent for participation in the study was obtained from all individuals. All parts of the study were approved by the local ethical committee. Blood samples (platelet-poor plasma) were collected as previously described (5).
Isolation of cells.
Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized blood by Isopaque-Ficoll (Lymphoprep; Nycomed, Oslo, Norway) gradient centrifugation. Further separation of CD14+ monocytes and CD3+ T-cells was performed as described elsewhere (6).
Cell culture experiments.
Peripheral blood mononuclear cells were incubated in 96-well trays (2 x 106/ml; Costar, Cambridge, Massachusetts), in medium alone (RPMI 1640 with 2 mmol/l L-glutamine and 25 mmol/l HEPES buffer [Gibco, Paisley, United Kingdom]) supplemented with 10% fetal calf serum (Sigma, St. Louis, Missouri) or stimulated with phytohemagglutinin (PHA) (Murex, Dratford, United Kingdom; final dilution 1:100) or lipopolysaccharide (LPS) from Escherichia coli O26:B6 (Sigma; 10 ng/ml). In some experiments, cells were incubated with the atorvastatin metabolite ortho-hydroxy atorvastatin (gift from Pfizer, New York, New York). The human monocytic cell line THP-1 (American Type Culture Collection, Rockville, Maryland) was differentiated into macrophages and cultured as described (7), with or without oxidized low-density lipoprotein (oxLDL, 20 µg/ml). Human aortic smooth muscle cells (SMCs) were obtained from PromoCell (Heidelberg, Germany) and grown in SMC Growth Medium 2 with complete supplement mix (PromoCell). At experimental start, the cells were cultured with and without oxLDL (20 µg/ml) in 24-well plates (1.5 x 105 cells/ml; Costar). Primary human umbilical vein endothelial cells (HUVECs) were obtained from umbilical cord veins and cultured as described (5). Human umbilical vein endothelial cells were stimulated with oxLDL (20 µg/ml) or different concentrations of recombinant human NAP-2 (rhNAP-2; R&D Systems, Minneapolis, Minnesota) and rhIL-8 (R&D Systems) with and without anti-human CXCR1 and anti-human CXCR2 antibodies or isotype-matched control mouse IgG2A (50 µg/ml for all antibodies; R&D Systems). At different time points, cell-free supernatants and cell pellets from PBMCs, THP-1 cells, vascular SMCs, and HUVECs were harvested and stored at –80°C. In the HUVEC cultures, the confluent cell layer was prepared for cellular enzyme-linked immunosorbent assay (ELISA). Low-density lipoprotein was isolated from human endotoxin-free heparin plasma and oxidatively modified by Cu2+ ions (7). The endotoxin levels of all stimulants and culture media were <10 pg/ml (Limulus Amebocyte Assay, Nelson Laboratories, Salt Lake City, Utah).
Chemotaxis assay.
Chemotactic activities were estimated in 24-well Transwell plates (Costar) using polycarbonate membranes with 8 µm pore size (Falcon; Becton Dickinson Labware, Bedford, Massachusetts). The lower chamber contained 800 µl RPMI 1640 with 0.5% bovine serum albumin, 20 mmol/l N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), pH 7.4 (buffer A), and rhNAP-2 (R&D Systems). To estimate random migration, the chemokine was omitted in negative control experiments. Freshly isolated monocytes (2.5 x 105/200 µl buffer A) were loaded into the upper chamber, incubated at 37°C for 90 min, and the cells attached to the side of the polycarbonate membrane in contact with the cell suspension were removed. After fixation with 1% glutaraldehyde, the migrated cells adhering to the membranes were stained with crystal violet and counted under an inverted microscope.
Stimulation of platelet-rich plasma (PRP).
Citrated PRP was incubated at room temperature after addition of 100 µl/l of the thrombin receptor agonist peptide SFLLRN (The Biotechnology Centre of Oslo, Oslo, Norway) or Tris-buffered saline only (8). At baseline and after 30 min, PRP was centrifuged at 10,000 g for 10 min, and platelet-free plasma and platelet pellets (with Tris-buffered saline) were stored separately at –80°C. In some experiments, PRP was pre-incubated with ortho-hydroxy atorvastatin for 30 min before addition of SFLLRN. Platelet pellets were lysed (5), and the NAP-2 levels were analyzed in the lysates. The increase in chemokine levels (ng/108 platelets) was expressed as the concentration in platelet-free plasma at the end of the experiments minus the concentration at baseline.
Incubation of HUVECs with platelet extracts.
Preparation of extract from resting platelets was performed as previously described (5). The platelet extract, prepared from a solution of 109 platelets/ml, was added to confluent HUVECs in 96-well trays for stimulation for 5 h. To block for platelet-derived NAP-2-mediated effects, a NAP-2 neutralizing and an irrelevant isotype-matched antibody (20 µg/ml; R&D Systems) were added to the platelet extract before exposing it to HUVECs. The anti–NAP-2 antibody dose-dependently inhibits rhNAP-2 bioactivity with no cross-reactivity with other platelet-derived cytokines, but we cannot totally exclude that this antibody could interfere with the NAP-2 precursors.
Real-time quantitative reverse transcription-polymerase chain reaction.
Total RNA was extracted from PBMCs, monocytes, and T-cells using RNeasy columns (Qiagen, Hilden, Germany), subjected to DNase I treatment (RQI DNase; Promega, Madison, Wisconsin), and stored in RNA storage solution (Ambion, Austin, Texas) at –80°C. Primers were designed using the Primer Express software, version 2.0 (Applied Biosystems, Foster City, California). Primer sequences could be provided by request. Quantification of mRNA was performed using the ABI Prism 7000 (Applied Biosystems) (6). Gene expression of the housekeeping gene beta-actin (Applied Biosystems) was used for normalization.
Tissue sampling of thrombus materials.
In 8 patients with acute ST-segment elevation myocardial infarction (STEMI) undergoing PCI, thrombi at the site of the occlusion were aspirated immediately after crossing the lesion with the guidewire (5). The aspirated solid material was fixed in 4% paraformaldehyde and embedded in paraffin.
Tissue sampling from carotid plaque.
Atherosclerotic plaques representing type VI lesions were obtained from patients undergoing carotid endarterectomy due to transient ischemic attacks (9). As controls, non-atherosclerotic human renal artery samples were taken from patients undergoing nephrectomy due to non-metastatic renal carcinoma.
Immunohistochemistry.
Paraformaldehyde-fixed sections of thrombus material and acetone-fixed sections of carotid plaques and renal arteries were stained using monoclonal mouse anti-human NAP-2, mouse anti-human CXCR1, mouse anti-human CXCR2 (all from R&D Systems), mouse anti-human CD41 (Immunotech, Marseille, France) antibodies, and affinity purified polyclonal mouse anti-human monocyte/macrophage (calprotectin) IgG (MCA874G, Serotec, Oxford, United Kingdom). The primary antibodies were followed by biotinylated anti-mouse IgG (Vector Laboratories, Burlingame, California). The immunoreactivities were further amplified using avidin-biotin-peroxidase complexes (Vector Laboratories). Diaminobenzidine was used as the chromogen in a metal-enhanced system (Pierce Chemical, Rockford, Illinois). The sections were counterstained with hematoxylin. Omission of the primary antibody served as negative control.
ELISAs.
Total cellular expression of E-selectin and vascular cellular adhesion molecule (VCAM)-1 was measured by ELISA on fixed adherent HUVECs (5). Levels of NAP-2, MCP-1, IL-8, and RANTES were measured by ELISAs (R&D Systems).
Statistical analysis.
For comparison of 2 groups, the Mann-Whitney U test (2-tailed) was used. When more than 2 groups were compared, 1-way analysis of variance followed by Scheffe’s post hoc test for statistical significance was used. For comparisons within the same individuals, the Wilcoxon signed rank test was used. Probability values (2-sided) were considered significant at value of <0.05.
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Results
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NAP-2 levels in platelet-poor plasma.
Both patients with stable (n = 40) and particularly those with unstable (n = 40) angina had significantly increased plasma levels of NAP-2 comparing healthy control subjects (n = 20) (Fig. 1A). Although the CAD patients used medications with potential platelet inhibitory effects (e.g., aspirin and warfarin), they still had raised NAP-2 levels. Despite the use of more aggressive platelet inhibition (i.e., clopidogrel and glycoprotein IIb/IIIa antagonists) in the unstable angina patients, they still had markedly raised NAP-2 levels. Although heparin may enhance platelet aggregation induced by some platelet agonists (e.g., epinephrine), it impairs platelet activation in response to thrombin and collagen (10), and it is very unlikely that the difference in NAP-2 levels between stable and unstable angina do not merely reflect differences in the use of heparin (Table 1). Some of the patients had additional risk factors that could interfere with platelet activation (e.g., diabetes and hypertension), but the same pattern of NAP-2 levels was observed even if these patients were excluded from the study. There was a higher proportion of women in the unstable compared with the stable angina group (Table 1), but we found the same pattern of NAP-2 levels in both genders.
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Figure 1 (A) Shows plasma levels of neutrophil-activating peptide (NAP)-2 in 40 patients with unstable angina pectoris (AP), 40 patients with stable AP, and 20 healthy control subjects. (B and C) show mRNA levels for the NAP-2 receptors CXCR2 (B) and CXCR1 (C) relative to beta-actin mRNA in monocytes from 12 patients with unstable AP, 12 patients with stable AP, and 10 healthy control subjects. Data are mean values ± SEM. *p < 0.05 and ***p < 0.001 versus healthy control subjects; #p < 0.05 versus stable AP. One-way analysis of variance followed by Scheffe’s post hoc test for statistical significance was used for statistical comparisons.
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The expression of NAP-2 receptors in monocytes and T-cells.
When examining monocytes from 12 patients with unstable angina, 12 patients with stable angina, and 10 healthy control subjects, we found that the increase in NAP-2 in unstable angina was accompanied by enhanced gene expression of its corresponding receptors CXCR1 and CXCR2 in monocytes from these patients (Figs. 1B and 1C). In contrast, these receptors were down-regulated in T-cells from unstable angina patients, but not in T-cells from stable angina patients, comparing healthy control subjects (data not shown) possibly reflecting down-regulation of the receptors due to persistent exposure to their ligands or redistribution of CXCR1/2-positive T-cells to the plaque in unstable angina.
The cellular source of NAP-2 in unstable angina.
The release of NAP-2 from platelets
As expected, platelets released large amounts of NAP-2 into the supernatants upon SFLLRN stimulation in both stable angina patients (n = 12), unstable angina patients (n = 12), and healthy control subjects (n = 10) (Fig. 2A). Although there were no differences in the SFLLRN-stimulated release of NAP-2 between patients and control subjects, platelet pellets from unstable angina patients, but not from those with stable angina, contained significantly higher levels of NAP-2 than platelet pellets from healthy control subjects (2.2 ± 0.4 vs. 1.9 ± 0.1 µg/108 platelets, p < 0.05).
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Figure 2 (A) Shows neutrophil-activating peptide (NAP)-2 levels in platelet-free plasma of unstimulated (Unstim) and SFLLRN-stimulated (100 µM) platelets (incubated for 30 min) in 12 patients with unstable angina pectoris (AP) (black bars), 12 patients with stable AP (striped bars), and 10 healthy control subjects (white bars). (B) Shows the NAP-2 levels in Unstim and in lipopolysaccharide (LPS) (10 ng/ml) and phytohemagglutinin (PHA) (final dilution 1:100) stimulated peripheral blood mononuclear cells supernatants after culturing for 20 h in 12 patients with unstable AP (black bars), 12 patients with stable AP (striped bars), and 10 healthy control subjects (white bars). Data are mean ± SEM. *p < 0.05 and **p < 0.01 versus unstimulated cells; p < 0.05 versus healthy control subjects. One-way analysis of variance followed by Scheffe’s post hoc test for statistical significance was used for statistical comparisons.
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The release of NAP-2 from PBMCs
Neutrophil-activating peptide-2 is considered to be a predominantly platelet-derived chemokine, but also PBMCs released increased amounts of this chemokine upon PHA and LPS stimulation (Fig. 2B). Although the increase in PBMCs from healthy control subjects (n = 10) and stable angina patients (n = 12) was rather modest and non-significant, respectively, cells from those with unstable angina (n = 12) released large amount of NAP-2, showing a significant higher LPS- and PHA-mediated NAP-2 response than in healthy control subjects (Fig. 2B). An increase in NAP-2 was also seen at the mRNA level, suggesting that PBMCs have the ability to produce NAP-2 (data not shown).
The release of NAP-2 from vascular SMCs and THP-1 macrophages
We next examined the ability of oxLDL to induce NAP-2 release in other cell types known to be involved in atherogenesis, and oxLDL significantly induced NAP-2 release comparing unstimulated cells in both THP-1 macrophages (0.094 ± 0.028 vs. 0.169 ± 0.035 ng/ml, p < 0.05) and vascular SMCs (0.028 ± 0.010 vs. 0.106 ± 0.026 ng/ml, p < 0.05), but not in HUVECs. However, the amount of NAP-2 was rather modest compared with the release in platelets and PBMCs (Fig. 2).
Expression of NAP-2 and its receptors in human atherosclerotic lesions.
When examining the expression of NAP-2 and its receptors in atherosclerotic plaques from 4 patients undergoing carotid endarterectomy surgery because of symptomatic carotid stenosis, positive platelet staining was observed, mainly near the luminal surface of the plaque, accompanied by NAP-2 immunoreactivity. However, strong NAP-2 immunostaining was also seen in other parts of the plaque, in areas with predominantly calprotectin-positive macrophages, and in vascular SMCs (data not shown), without accompanying platelet staining (Fig. 3). Neutrophil-activating peptide-2 immunoreactivity was accompanied by immunostaining of its corresponding receptors in the same regions. Immunostaining of NAP-2 and its receptors was not seen in non-atherosclerotic renal arteries (n = 4) (Fig. 3).
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Figure 3 Photomicrographs showing staining and localization of neutrophil-activating peptide-2, CXCR1, and CXCR2 in plaque sections of left carotid artery. Plaque sections stained with omission of the primary antibody as control demonstrated no immunostaining of any of the cellular elements (A). Fairly strong immunostaining of neutrophil-activating peptide-2 (B), CXCR1 (C), and CXCR2 (D) was co-localized to areas with calprotecin-positive macrophages (E). No immunostaining was seen in sections from non-atherosclerotic renal arteries when staining for neutrophil-activating peptide-2, CXCR1, or CXCR2 (F). Original magnification x 400.
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Expression of NAP-2 in arterial thrombosis.
We next examined the expression of NAP-2 in thrombus material obtained from 8 patients with STEMI undergoing primary PCI. Immunostaining of the material removed from the site of plaque rupture showed large amounts of NAP-2 in platelets and calprotectin-positive monocytes (Fig. 4). Also, CXCR1 and CXCR2 showed strong immunostaining in these samples, not only in calprotectin-positive monocytes, but also in areas with aggregated platelets (Fig. 4).
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Figure 4 Photomicraphs showing staining and localization of neutrophil-activating peptide-2, CXCR1, and CXCR2 in thrombus material removed from the site of plaque rupture in ST-segment elevation myocardial infarction patients undergoing percutaneous coronary intervention. Thrombus material stained with omission of the primary antibody as control demonstrated no immunostaining of any of the cellular elements (A). Fairly strong immunostaining of neutrophil-activating peptide-2 (B), CXCR1 (C), and CXCR2 (D) was seen in monocytes (arrows) and in areas with platelets (*). (E) Demonstrates thrombus material with CD41-positive platelets, and (F) demonstrates calprotecin-positive monocytes. Original magnification x 400.
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Effects of rhNAP-2 on monocyte chemotaxis.
To map any pathogenic consequences of the enhanced NAP-2 levels in unstable angina, we examined the ability of NAP-2 to promote chemotaxis of monocytes from unstable angina patients (n = 5) and healthy control subjects (n = 5). Recombinant NAP-2 significantly enhanced monocyte chemotaxis in a dose-dependent manner (1 to 100 ng/ml) in both unstable angina patients and control subjects with a more marked response in unstable angina (40 ± 6 vs. 65 ± 8, migrated cells/field at 100 ng/ml NAP-2; p < 0.05).
Effect of rhNAP-2 on adhesion molecules and chemokines in endothelial cells.
As shown in Figures 5A and 5B, rhNAP-2 significantly increased the protein levels of E-selectin and VCAM-1 in HUVECs. Concomitantly, NAP-2 strongly induced the release of MCP-1 and IL-8 in these cells (Figs. 5C and 5D). Neutralizing antibodies against CXCR1 and CXCR2 attenuated the NAP-2-induced increase in IL-8 levels by 41% and 43%, respectively, indicating that this NAP-2-mediated effect involves ligation of both receptors (Fig. 5E). A similar pattern was seen for MCP-1 (data not shown). Recombinant NAP-2-activated HUVECs showed a similar pattern of MCP-1 release as was seen after rhIL-8 stimulation (data not shown), suggesting that the inflammatory potential of NAP-2 may be comparable to that of IL-8.
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Figure 5 The effects of different concentrations of recombinant human neutrophil-activating peptide (rhNAP)-2 on E-selectin (A), vascular cellular adhesion molecule (VCAM)-1 (B), monocyte chemoattractant protein (MCP)-1 (C), and interleukin (IL)-8 (D) in human umbilical vein endothelial cells after culturing for 5 (A and B) and 24 h (C and D). (E) Shows the effect of neutralizing antibodies against CXCR1 and CXCR2 (p = 0.06) receptors on the rhNAP-2-induced (250 ng/ml) release of IL-8 in human umbilical vein endothelial cells supernatants after culturing for 24 h. No down-regulatory effect was seen when a control antibody of the same isotype and concentration was added (data not shown). (F) Shows the ability of a neutralizing antibody against NAP-2 and a control antibody of the same isotype and concentration (IgG1), to attenuate the effect of the platelet extract-mediated increase of IL-8 release in human umbilical vein endothelial cell supernatants after culturing for 5 h. Data are mean values ± SEM of 6 separate experiments. *p < 0.05 and **p < 0.001 versus unstimulated (Unstim) cells; #p < 0.05 versus cells stimulated with rhNAP-2 (E) or platelet extract (PE) (F). The Wilcoxon signed rank test (simple comparisonwise p values) was used for statistical comparisons. OD = optical density.
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Platelet-derived NAP-2 promotes inflammation in endothelial cells.
We next examined if platelet-derived NAP-2 could mimic some of the inflammatory responses induced by rhNAP-2. When HUVECs were incubated with platelet extracts, we found a marked up-regulation of IL-8 (Fig. 5F). Whereas an unspecific antibody had only minor effect on IL-8 expression, neutralizing antibody against NAP-2 significantly attenuated the effect of platelet extract on IL-8 level, suggesting that it involves platelet-derived NAP-2 (Fig. 5F). Similar pattern was seen for the platelet extract-mediated effect on MCP-1 (data not shown).
Effects of aspirin and statins on NAP-2 levels.
Finally, we examined the effects of aspirin and statins on NAP-2. Aspirin (160 mg every day) significantly down-regulated plasma levels of NAP-2 when given for 7 days in 11 healthy control subjects (Fig. 6A). In contrast, statins significantly increased plasma levels of NAP-2 after 6 months of therapy in CAD patients receiving simvastatin (20 mg every day, n = 21) or atorvastatin (80 mg every day, n = 14) (11), with similar effect in both treatment groups (Fig. 6B). The enhancing effect of statins on NAP-2 levels was seen also in vitro with increasing effects of ortho-hydroxy atorvastatin on gene expression of NAP-2 in PBMCs (Fig. 6C) and on the release of NAP-2, but not the release of RANTES, in SFLLRN-stimulated (100 µM) PRP from 5 healthy control subjects (Fig. 6D).
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Figure 6 (A) Shows the effect of aspirin (160 mg every day for 7 days) on plasma levels of neutrophil-activating peptide (NAP)-2 in 12 healthy control subjects. Baseline level is set to 100% for each individual. (B) Shows plasma levels of NAP-2 in coronary artery disease patients before and after 6 months of therapy with simvastatin (20 mg every day, n = 21) or atorvastatin (80 mg every day, n = 14), with similar effect in both treatment groups. (B) Shows pooled data from both treatment groups (means ± SEM). (C) Shows the effect of ortho-hydroxy atorvastatin (AT; 10 µmol/l) on the gene expression of NAP-2 in peripheral blood mononuclear cells from 5 healthy control subjects after culturing for 20 h (mean ± SEM). (D) Shows the effect of AT (10 µmol/l) on SFLLRN-stimulated (100 µmol/l) release of NAP-2 in platelet-rich plasma (incubated for 90 min) from 6 healthy control subjects. *p < 0.05 and **p < 0.01 versus unstimulated; #p < 0.05 versus SFLLRN-stimulated platelets. The Wilcoxon signed rank test (simple comparisonwise p values) was used for statistical comparisons.
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Discussion
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We report increased NAP-2 levels in angina patients with particularly high levels in those with unstable disease, possibly reflecting enhanced release not only from platelets, but also from other cells involved in atherogenesis such as monocytes/macrophages and vascular SMCs. The relationship between raised NAP-2 levels and unstable disease was further supported by our immunohistochemical analyses showing increased NAP-2 expression on platelets and monocytes within thrombus material obtained at the site of plaque rupture in patients with STEMI undergoing PCI. CXCR1 and CXCR2 have previously been shown to play a pathogenic role in atherosclerotic disorders, mainly thought to reflect their interaction with IL-8 (3,4). Our findings suggest that also another ligand for these receptors (i.e., NAP-2) could be involved in atherogenesis and plaque destabilization. It may be argued that the high NAP-2 level is a consequence rather than a cause of unstable angina. However, recent studies describe acute coronary syndromes as a continuous process involving multiple ruptured atherosclerotic plaques, not only in the culprit lesion (12). Hence, although not the initial event, NAP-2-driven inflammation may contribute to the progression of patient instability representing an amplification factor in this process.
Platelets have been regarded as the only cellular source of NAP-2, but also monocytes/macrophages have recently been found to express NAP-2 (13). Herein, we extend these findings by showing that PBMCs, THP-1 macrophages, and vascular SMCs release NAP-2 upon various stimuli such as oxLDL, and the release of NAP-2 from PBMCs was particularly enhanced in unstable angina patients. Although we cannot at present determine whether the elevated NAP-2 release reflects increased synthesis, enhanced generation of NAP-2 from its precursors, or both (14), our data clearly show the ability of other cells than platelets to generate NAP-2. Our findings of NAP-2 protein in macrophages and SMCs within atherosclerotic carotid plaques and in monocytes of thrombus material obtained from the site of plaque rupture during MI further support that these cells have the capacity to express NAP-2 and demonstrate that they do so in atherothrombosis.
Up-regulation of adhesion molecules and chemokines are important steps in the recruitment and activation of leukocytes into the atherosclerotic lesions (1). Herein, we show that NAP-2 is a potent inducer of E-selectin and VCAM-1 as well as the chemokines MCP-1 and IL-8 in endothelial cells. This effect was induced not only by rhNAP-2, but also by platelet-derived NAP-2. We found that monocytes from unstable angina patients showed enhanced chemotactic responses to NAP-2, suggesting that the increased CXCR2 expression in monocytes from these patients affects their functional responses. Our findings imply that the raised NAP-2 levels in unstable angina could contribute to enhanced vascular inflammation within the atherosclerotic and unstable lesion.
Experimental as well as some clinical data indicate that statins may confer cardiovascular benefits beyond their lipid-lowering activity. These pleiotropic effects of statins function, in part, by attenuating inflammatory responses (11). However, our findings suggest that these medications also may exert inflammatory properties as also has been reported by others (15). Thus, both aggressive and conventional statin treatment significantly enhanced plasma levels of NAP-2 in CAD patients after 6 months of therapy. Such a NAP-2-enhancing potential was also supported by our in vitro experiments showing that ortho-hydroxy atorvastatin induced NAP-2 gene expression in PBMCs and NAP-2 release in SFLLRN-activated platelets. The lack of effect on the release of RANTES, another chemokine that is released from alpha-granules, suggests that statins may increase NAP-2 release from platelets by enhancing the conversion from its precursors or by attenuating NAP-2 degradation. Although we clearly do not argue against a beneficial role for statins in CAD, these results suggest that other medications with another anti-inflammatory profile should be investigated in these patients. Forthcoming studies should also examine if this NAP-2-enhancing effect is restricted to lipophilic statins.
The present study has some limitations. Relatively few patients were examined, and the patients groups were to a certain extent heterogeneous, for example with regard to the use of platelet inhibitors. However, despite the use of more aggressive platelet inhibition in the unstable angina patients, they still had markedly raised NAP-2 levels compared with both healthy control subjects and stable angina patients suggesting that our data represent an underestimation rather than an overestimation of the "real" NAP-2 levels in these patients.
Our findings suggest that NAP-2 has the potential to induce an inflammatory response within the atherosclerotic plaque. By its ability to promote leukocyte and endothelial cell activation within the vessel wall, such a NAP-2-driven inflammation could ultimately lead to plaque rupture and acute coronary syndromes. Such a pathogenic NAP-2 loop, involving interactions between platelets, leukocytes, and endothelial cells, could represent a potential target for therapy in CAD.
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Footnotes
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This work was supported by grants from the Norwegian Council of Cardiovascular Research, Swedish Heart-Lung Foundation, Research Council of Norway, the University of Oslo, and Medinnova Foundation. Drs. Dam s and Otterdal contributed equally to this work.
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References
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