Increased Levels of Leukocyte-Derived MMP-9 in Patients with Stable Angina Pectoris

Objective: There is a growing interest for matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in plasma as novel biomarkers in coronary artery disease (CAD). We aimed to identify the sources of MMP-8, MMP-9, TIMP-1 and TIMP-2 among peripheral blood cells and further explore whether gene expression or protein release was altered in patients with stable angina pectoris (SA). Methods: In total, plasma MMP-9 was measured in 44 SA patients and 47 healthy controls. From 10 patients and 10 controls, peripheral blood mononuclear cells (PBMC) and neutrophils were isolated and stimulated ex vivo. MMPs, TIMPs and myeloperoxidase were measured in plasma and supernatants by ELISA. The corresponding gene expression was measured by real-time PCR. Results: Neutrophils were the dominant source of MMP-8 and MMP-9. Upon moderate stimulation with IL-8, the neutrophil release of MMP-9 was higher in the SA patients compared with controls (p,0.05). In PBMC, the TIMP-1 and MMP-9 mRNA expression was higher in SA patients compared with controls, p,0.01 and 0.05, respectively. There were no differences in plasma levels between patients and controls except for TIMP-2, which was lower in patients, p,0.01. Conclusion: Measurements of MMPs and TIMPs in plasma may be of limited use. Despite similar plasma levels in SA patients and controls, the leukocyte-derived MMP-9 and TIMP-1 are significantly altered in patients. The findings indicate that th

Detection of apoptosis in patients with coronary artery disease : Assessment of temporal patterns and potential sources

The atherosclerotic process and its consequences are considered driven by an imbalance between pro- and ant-inflammatory actions. One contributing factor in this scenario is an altered regulation of apoptosis, which affects both immune, vascular and myocardial cells. The general aim of this thesis was to measure soluble markers of apoptosis in peripheral venous blood, in various clinical stages of coronary artery disease (CAD) and to further identify possible sources with specific focus on natural killer (NK) cell apoptosis and myocardial ischemia-reperfusion (IR)-injury. There was evidence of an increased apoptosis of NK cells, but not T cells, in the circulation of CAD patients. Spontaneous NK cell apoptosis and the cells´ sensitivity to oxidative stress in the form of oxidized lipids ex vivo, were increased. Findings were thus suggestive of an enhanced apoptosis contributing to the reduced NK cell activity seen in CAD. However, we could not verify that oxidative stress in the circulation was a driving force behind this loss. Soluble forms of the cell surface bound receptors of apoptosis include soluble (s) Fas and sFas ligand (L). They are detected in plasma and used as surrogate markers of apoptosis. Here we investigated the relationship between these markers and NK cell apoptosis and NK cell levels, in a 12 month longitudinal study on CAD patients. Plasma levels of sFasL correlated with increased susceptibility to NK cell apoptosis ex vivo but also with the levels of NK cells in the circulation after a coronary event. NK cells undergoing apoptosis ex vivo were also found to be a major source of sFasL themselves, indicating potential usefulness of sFasL in monitoring changes in NK cell levels. Apoptosis is suggested to be a key event in IR-injury, resulting in increased infarct size, left ventricular (LV) dysfunction, remodeling and heart failure. We investigated soluble markers of apoptosis in relation to these parameters in a ST-elevation myocardial infarction (STEMI) population. In addition to sFas and sFasL, we also measured tumor necrosis factor (TNF) receptor (R) I and II in this study. Acute phase levels of sTNFRI and sTNFRII, but not sFas or sFasL, correlated to cardiac MR (CMR) measures of infarct size and LV-dysfunction at 4 months after the ischemic event. Also, the soluble markers of apoptosis were correlated with matrix metalloproteinase (MMP)-2, a mechanistic trigger for cardiomyocyte apoptosis, further strengthening the role of apoptosis in IR-injury. Finally we explored the temporal patterns of soluble markers of apoptosis after an MI and, furthermore, investigated possible differences between patients presenting with a non(N)-STEMI versus STEMI. The sTNFRI/II and the sFas/sFasL pathways of apoptosis showed different temporal changes indicating diverse roles of these two systems. NSTEMI and STEMI patients however, shared these temporal patterns pointing to apoptosis as equally involved in either infarct type. Furthermore sTNFRs, but not sFas/sFasL correlated to levels of cytokine interleukin (IL)-6 illustrating the overlapping role TNF signaling in inflammation and apoptosis, while again suggesting differences between the TNF and the Fas/FasL systems during myocardial IR--‐injury

Soluble TNF Receptors Are Associated with Infarct Size and Ventricular Dysfunction in ST-Elevation Myocardial Infarction

Objectives The aim of the study was to investigate circulating markers of apoptosis in relation to infarct size, left ventricular dysfunction and remodeling in an ST-elevation myocardial infarction (STEMI) population undergoing primary percutaneous coronary intervention (PCI). Background Immediate re-opening of the acutely occluded infarct-related artery via primary PCI is the treatment of choice in STEMI to limit ischemia injury. However, the sudden re-initiation of blood flow can lead to a local acute inflammatory response with further endothelial and myocardial damage, so-called reperfusion injury. Apoptosis is suggested to be a key event in ischemia-reperfusion injury, resulting in LV-dysfunction, remodeling and heart failure. Methods The present study is a prespecified substudy of the F.I.R.E. trial. We included 48 patients with STEMI undergoing primary PCI. Blood samples were collected prior to PCI and after 24 hours. Plasma was separated for later analysis of soluble tumor necrosis factor receptor (sTNFR) 1, sTNFR2, sFas and sFas ligand (sFasL) by ELISA. Infarct size, left ventricular (LV) dysfunction and remodeling were assessed by cardiac magnetic resonance imaging at five days and four months after STEMI. Results The levels of sTNFR1 at 24 h as well as the relative increases in sTNFR1 and sTNFR2 over 24 h showed consistent and significant correlations with infarct size and LV-dysfunction at four months. Moreover, both sTNFRs correlated strongly with Troponin I and matrix metalloproteinase (MMP)-2 measurements. Soluble Fas and sFasL did not overall correlate with measures of infarct size or LV-dysfunction. None of the apoptosis markers correlated significantly with measures of remodeling. Conclusions In STEMI patients, circulating levels of sTNFR1 and sTNFR2 are associated with infarct size and LV dysfunction. This provides further evidence for the role of apoptosis in ischemia-reperfusion injury.Funding Agencies|Swedish Heart-Lung Foundation||</p

Soluble TNF Receptors Are Associated with Infarct Size and Ventricular Dysfunction in ST-Elevation Myocardial Infarction

Objectives The aim of the study was to investigate circulating markers of apoptosis in relation to infarct size, left ventricular dysfunction and remodeling in an ST-elevation myocardial infarction (STEMI) population undergoing primary percutaneous coronary intervention (PCI). Background Immediate re-opening of the acutely occluded infarct-related artery via primary PCI is the treatment of choice in STEMI to limit ischemia injury. However, the sudden re-initiation of blood flow can lead to a local acute inflammatory response with further endothelial and myocardial damage, so-called reperfusion injury. Apoptosis is suggested to be a key event in ischemia-reperfusion injury, resulting in LV-dysfunction, remodeling and heart failure. Methods The present study is a prespecified substudy of the F.I.R.E. trial. We included 48 patients with STEMI undergoing primary PCI. Blood samples were collected prior to PCI and after 24 hours. Plasma was separated for later analysis of soluble tumor necrosis factor receptor (sTNFR) 1, sTNFR2, sFas and sFas ligand (sFasL) by ELISA. Infarct size, left ventricular (LV) dysfunction and remodeling were assessed by cardiac magnetic resonance imaging at five days and four months after STEMI. Results The levels of sTNFR1 at 24 h as well as the relative increases in sTNFR1 and sTNFR2 over 24 h showed consistent and significant correlations with infarct size and LV-dysfunction at four months. Moreover, both sTNFRs correlated strongly with Troponin I and matrix metalloproteinase (MMP)-2 measurements. Soluble Fas and sFasL did not overall correlate with measures of infarct size or LV-dysfunction. None of the apoptosis markers correlated significantly with measures of remodeling. Conclusions In STEMI patients, circulating levels of sTNFR1 and sTNFR2 are associated with infarct size and LV dysfunction. This provides further evidence for the role of apoptosis in ischemia-reperfusion injury.Funding Agencies|Swedish Heart-Lung Foundation||</p

Baseline, procedural and outcome measures of the study population (n = 46).

<p>SD, standard deviation; PCI, percutaneous coronary intervention; TIMI, Thrombolysis In Myocardial Infarction; LVEF, left-ventricular ejection fraction; EDVI, end-diastolic volume index; ESVI, end-systolic volume index.</p

Plasma levels of sTNFR1, sTNFR2, sFAS, sFASL, MMPs, TIMPs and MPO during the first 24 hours after STEMI and reperfusion treatment (n = 46).

<p>Values are given as median (25^th, 75^th percentile). P-values are shown for Wilcoxon signed ranks test. sTNFR1, soluble tumour necrosis factor receptor 1; sTNFR2, soluble tumour necrosis factor receptor 2; sFAS, soluble FAS; sFASL, soluble FAS ligand; MMP-2, matrix metalloproteinase-2; TIMP, tissue inhibitor of metalloproteinase; MPO, myeloperoxidase; STEMI, ST-elevation myocardial infarction.</p

Correlations between sTNFR1, sTNFR2, sFAS and sFASL and measures of infarct size.

<p>Values are given as rho-values from Spearman test.</p>*<p>p<0.05.</p><p>sTNFR1, soluble tumour necrosis factor receptor 1; sTNFR2, soluble tumour necrosis factor receptor 2; sFAS, soluble FAS; sFASL, soluble FAS ligand; LGE, late gadolinium enhancement zone.</p

Correlations between sTNFR1, sTNFR2, sFAS and sFASL and measures of left ventricular dysfunction and remodeling.

<p>Values are given as rho-values from Spearman test.</p>*<p>p<0.05.</p><p>sTNFR1, soluble tumour necrosis factor receptor 1; sTNFR2, soluble tumour necrosis factor receptor 2; sFAS, soluble FAS; sFASL, soluble FAS ligand; LVEF, left ventricular ejection fraction; dEDVI, change in end-diastolic volume index from 5 days to 4 months; dESVI, change in end-systolic volume index from 5 days to 4 months.</p

Soluble Fas ligand is associated with natural killer cell dynamics in coronary artery disease

Objective: Apoptosis of natural killer (NK) cells is increased in patients with coronary artery disease (CAD) and may explain why NK cell levels are altered in these patients. Soluble forms of Fas and Fas ligand (L) are considered as markers of apoptosis. Here, we investigated whether plasma levels of Fas and FasL were associated with NK cell apoptosis and NK cell levels in CAD patients. Methods: Fas and FasL in plasma were determined by ELISA in 2 cohorts of CAD patients; one longitudinal study measuring circulating NK cells and apoptotic NK cells by flow cytometry 1 day, 3 months and 12 months after a coronary event and one cross-sectional study measuring NK cell apoptosis ex vivo. Both studies included matched healthy controls. Fas and FasL were also determined in supernatants from NK cells undergoing cytokine-induced apoptosis in cell culture. Results: In the 12-month longitudinal study, plasma FasL increased by 15% (p less than 0.001) and NK cell levels by 31% (p less than 0.05) while plasma Fas did not change. Plasma FasL and NK cell levels were significantly related at 3 months and 12 months, r = 0.40, p less than 0.01. Furthermore, plasma FasL, but not plasma Fas, correlated with NK cell apoptosis ex vivo in CAD patients, r = 0.54, p less than 0.05. In vitro, cytokine-induced apoptosis of NK cells resulted in abundant release of FasL. Conclusion: In CAD patients, FasL in plasma is associated with both apoptotic susceptibility of NK cells and dynamic changes in circulating NK cells. NK cells are also themselves a potential source of soluble FasL. Our findings link NK cell status to a soluble marker with possible atheroprotective effects thereby supporting a beneficial role of NK cells in CAD

Characteristics of the subpopulations of patients with stable angina pectoris (SA) and controls selected for the ex vivo assays.

<p>Data are given as median (inter-quartile range).</p