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
Institute for Surgical Research
Department of Cardiology
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
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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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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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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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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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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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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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