a Department of Cardiology, University Hospital Antwerp (UIA), Edegem, Belgium
b Department of Immunology, University Hospital Antwerp (UIA), Edegem, Belgium
c Department of Medical Statistics, University Hospital Antwerp (UIA), Edegem, Belgium
* Correspondence to: Viviane Conraads, MD, PhD, Department of Cardiology, University Hospital Antwerp, Wilrijkstraat 10, 2650 Edegem, Belgium. Tel: +32 3 821 46 72; fax: +32 3 825 08 48
E-mail address: Viviane.Conraads{at}uza.uia.ac.be
Received 20 November 2002; revised 14 September 2003; accepted 19 September 2003
Abstract
Aims The observation that low total cholesterol predicted poor survival in patients with chronic heart failure, has questioned the beneficial effect of lipid-lowering in this population. This study investigated the relation between lipoprotein concentrations and the levels of interleukin (IL)-6, tumour necrosis factor (TNF)- and the soluble TNF-
receptors in patients with chronic heart failure due to coronary artery disease (CAD) or idiopathic dilated cardiomyopathy (IDCM).
Methods and results Seventy-one patients with chronic heart failure due to CAD (n=36) or IDCM (n=35) were enrolled. Plasma concentrations of lipoproteins, IL-6, TNF-, soluble TNF-
receptor 1 (sTNFR1) and 2 (sTNFR2) were measured. Total cholesterol/HDL-cholesterol (CHOL/HDL) correlated with levels of TNF-
(r=0.24, P=0.035), sTNFR1 (r=0.32, P=0.008) and sTNFR2 (r=0.37, P=0.002). In the CAD group, CHOL/HDL and triglycerides (TG) correlated with sTNFR2 (r=0.48, P=0.005 for CHOL/HDL, r=0.40, P=0.015 for TG). No relation was found between lipoproteins and cytokines or sTNF-
receptors for IDCM patients (P>0.1).
Conclusion An atherogenic lipid profile favoured the inflammatory process in patients with heart failure due to CAD. No relation between lipoproteins and cytokines was detected in case of IDCM.
Key Words: Heart failure Lipoproteins Cytokines Inflammation
1. Introduction
The benefit of hypolipidaemic drugs in coronary heart disease has become evident from large clinical trials. By contrast, the effect of lipid lowering therapy in patients with heart failure has not been specifically investigated. Interestingly, the recent observation that low total cholesterol predicted poor clinical outcome in patients with heart failure has questioned the concept that lipid lowering would be beneficial in this population.1On the contrary, the inverse relationship of cholesterol with tumour necrosis factor (TNF)- in serum in that study, suggests that modification of a presumed atherogenic lipid profile could be even harmful in patients with established heart failure.
There has been growing evidence that a disturbed proinflammatory cytokine network may contribute to the pathogenesis and progression of heart failure.26Although causality has not been explicitly confirmed, it has been shown that this chronic inflammatory state has prognostic significance.4,5,7Elevated circulating levels of soluble CD14 (sCD14), interleukin-6 (IL-6), TNF- and in particular the soluble TNF-
receptors appear to be stronger predictors of mortality than more traditional prognostic factors, such as NYHA class, left ventricular ejection fraction, VE/VCO2slope, maximal oxygen consumption and wasting.5
The purpose of the present study was, therefore, to explore the relationship between lipoprotein concentrations and the levels of circulating IL-6, TNF- and the soluble TNF-
receptors (soluble TNF-
receptor 1 [sTNFR1] and soluble TNF-
receptor [sTNFR2]) in patients with heart failure of mixed aetiology.
2. Methods
2.1. Subjects
Seventy-one consecutive patients with chronic heart failure due to coronary artery disease (CAD, n=36) or idiopathic dilated cardiomyopathy (IDCM, n=35) participated in this cross-sectional study. All patients were stable with regard to symptoms and therapy for at least 1 month. Demographic characteristics of the patients are shown in Table 1. Patients were on standard medical treatment consisting of ACE-inhibitors (90%), diuretics (86%), spironolactone (69%), digoxin (44%), beta-blockers (55%), low dose aspirin (35%) and amiodarone (20%). Exclusion criteria were active infection, allergy, rheumatoid disease, cancer, treatment with anti-inflammatory drugs, severe renal failure (creatinine >2mg/dl). Treatment with fibrates or statins at baseline was not allowed. Fifteen age-matched subjects served as a healthy control group (no chronic underlying disease, no cardiac history, no medication, no allergic condition).
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2.2. Study design
Fasting blood samples were collected between 8 and 9a.m. into ethylenediaminetetraacetic acid (EDTA) tubes (Vacutainer®, Becton and Dickinson, Meylan, France). Plasma was separated by centrifugation and aliquots were stored at 20°C.
2.3. Laboratory measurements
Concentrations of IL-6, TNF-, sTNFR1 and sTNFR2 were measured using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's specifications (Quantikine, R&D Systems, sensitivity: 0.7pg/ml for IL-6, 1.5pg/ml for sTNFR1, 1pg/ml for sTNFR2). A high sensitivity kit (Quantikine HS, R&D Systems, sensitivity 0.18pg/ml) was used to measure TNF-
. All samples were run-in duplicate. Cytokine levels were determined in the control population.
Total cholesterol (CHOL) and triglycerides (TG) were quantified by reflometry with Vitros 750 assays, using dry slide technology (Ortho Clinical Diagnostics, Beerse, Belgium). HDL- and LDL-cholesterol were measured on Hitachi 912 with direct homogenous enzymatic assays, using HDL-C plus and LDL-plus reagents (Roche Diagnostics GmbH, Mannheim, Germany). High sensitive C-reactive protein (CRP) was analysed bynephelometry.
2.4. Statistical analyses
Data are given as median+range. Because cytokine data were not normally distributed, non-parametric tests were chosen for statistical analyses. Comparisons of numerical data between groups of patients were carried out with the non-parametric MannWhitney U test. Pair wise comparisons were carried out using the Wilcoxon matched pair signed rank test. Categorical/binary data were analysed using a Chi-square test. Correlations were determined using Spearman's rank correlation test. P-values <0.05 were considered statistically significant. However, due to multiple comparisons, particular attention should be directed towards lower p values (i.e., P<0.01).
3. Results
Plasma concentrations of cytokines, soluble TNF- receptors and CRP were significantly elevated in the total population versus controls (P>0.5). At baseline, CRP, TNF-
and sTNFR2 levels were higher for the CAD patients (Table 1). Although CHOL levels were comparable, LDL and CHOL/HDL were higher, whereas HDL was significantly lower in the CAD group. Comparison for the total population according to functional class showed significantly elevated CRP (P=0.002), sedimentation rate (P=0.0007), IL-6 (P=0.004), TNF-
(P=0.02), sTNFR1 (P<0.0001) and sTNFR2 (P<0.0001) levels in patients with NYHA class >III versus NYHA <III. No differences were found for lipoproteins. For the CAD group, differences according to NYHA class persisted for CRP (P=0.007), sedimentation rate (P=0.02), IL-6 (P=0.04), TNF-
(P<0.0001), sTNFR1 (P=0.0003) and sTNFR2 (P<0.0001), whereas for the IDCM patients, the sedimentation rate (P=0.02), sTNFR1 (P=0.002) and sTNFR2 (P=0.002) were significantly different.
TNF- correlated with both soluble TNF-
receptors (r=0.51, P<0.0001 for sTNFR1, r=0.55, P<0.0001 for sTNFR2).
For the total population, there was an inverse relation between HDL and the two soluble TNF- receptors (HDL versus sTNFR1: r=0.30, P=0.009, HDL versus sTNFR2: r=0.26, P=0.03). CHOL/HDL correlated with TNF-
(r=0.24, P=0.035) and with both receptors (CHOL/HDL versus sTNFR1: r=0.32, P=0.008, CHOL/HDL versus sTNFR2: r=0.37, P=0.002). There was no relation between IL-6 and lipoprotein concentrations. For the CAD group, the relations between sTNFR2, CHOL/HDL (Fig. 1) and TG (Fig. 2) were significant (CHOL/HDL versus sTNFR2: r=0.48, P=0.005, TG versus sTNFR2: r=0.40, P=0.015). A trend for a positive correlation was also seen between TG, CHOL/HDL and TNF-
(TG versus TNF-
: r=0.3, P=0.073, CHOL/HDL versus TNF-
: r=0.32, P=0.060), between CHOL/HDL and sTNFR1 (r=0.32, P=0.06) and between CHOL and sTNFR2 (r=0.31, P=0.062) (Table 2). No significant relations between lipoproteins, cytokines and TNF-
receptors were found in the IDCM group (P>0.1).
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In this cross-sectional study, an atherogenic lipid profile favoured the inflammatory process in patients with chronic heart failure due to coronary artery disease (CAD), whereas no relation between lipoproteins and proinflammatory cytokines was observed in case ofidiopathic dilated cardiomyopathy (IDCM).
4.1. Inflammation and heart failure: do lipoproteins matter?
The presence of low-grade chronic inflammation in patients with chronic heart failure has been widely recognized.26Rauchhaus et al. underscored the prognostic importance of IL-6, TNF-, sCD14, sTNFR1 and sTNFR2 in patients with heart failure.5The authors identified sTNFR1 as the strongest prognostic factor, independent of other established markers of heart failure severity. In a smaller study from Ferrari et al.3and in the analysis of the cytokine database from the VEST trial,6sTNFR2 was the most powerful independent indicator of short-term mortality.
In the recently published endotoxin-lipoproteinhypothesis,7the possible beneficial effects of hypolipaemic drugs in patients with heart failure have been questioned. The rationale for the latter conjecture was derived from both in-vitro and in-vivo experiments712in which lipoproteins were shown to bind and neutraliseendotoxin (lipopolysaccharide [LPS]), a well-knownstrong stimulator for the release of inflammatory cytokines by monocytes and macrophages. Pajkrt et al.13demonstrated an anti-inflammatory effect (reduction of TNF, IL-6 and IL-8) of reconstituted HDL during human endotoxaemia. A downregulation of CD14, the main LPS receptor on monocytes, was also shown. No comparable investigations have been undertaken in chronic heart failure models. However, Rauchhaus and colleagues reported a negative correlation between CHOL, LDL and TG on the one hand and TNF-, sTNFR1 and sTNFR2 on the other hand.1The strongest relations were found between sCD14/CHOL, TNF-
and the two soluble TNF-receptors. Comparing TNF-
and TNF-
receptor concentrations to a ratio in which the numerator and the nominator are interdependent, however, is questionable. Moreover, substituting LPS by sCD14 ignores the relevance of other endotoxin receptors such as the ß2-integrins. Vredevroe et al.14showed that lower levels of CHOL, LDL and TG were predictors of mortality only in patients with idiopathic dilated cardiomyopathy; no such relation was found in patients with ischemic heart disease.
The results in the present study clearly deviate. Although HDL correlated inversely with sTNFR1 and sTNFR2, higher CHOL/HDL was found in patients with elevated TNF-receptor concentrations. At first glance, this result seems to reinforce the idea of a protective effect of HDL through LPS neutralization. However, the demonstration of a stronger relationship between the CHOL/HDL ratio and both sTNFR1 and sTNFR2 receptors allows still an alternative explanation.
When the total population was divided according to aetiology, no such relation was found between lipoprotein levels and cytokines or receptors for the IDCM group, whereas the relation between sTNFR2 on the one hand, and CHOL/HDL and TG were significant. Importantly, these findings are in line with the analysis of the cytokine database from the Vesnarinone Trial (VEST) which showed higher IL-6, TNF- and TNF-
receptor levels in chronic heart failure patients with ischemic cardiomyopathy.6The fact that baseline TNF-
and sTNFR2 receptors levels were higher for the CAD group underscores this finding. Although a causal relation between the lipid profile and inflammation intensity remains to be demonstrated, the fact that TNF-
receptor levels are proportionately elevated as a function of the atherogenicity of the lipid profile is, at least, suggestive. However, other mechanistic explanations warrant discussion. Myocardial ischaemia is a known trigger for myocardial TNF and IL-6 biosynthesis. It is therefore plausible that episodes of ischemia contribute to tissue genesis of these cytokines with peripheral spillover leading to higher circulating concentrations. Alternatively, the higher levels of cytokines may reflect a more general inflammatory process that is characteristic of atherosclerotic heart disease.15
In the present study, patients with CAD were characterized by a more unfavourable lipid profile, when compared to the IDCM group. As was suggested in the endotoxin-lipoprotein hypothesis, the complex balance between the endotoxin-neutralising effect of lipoproteins on the one hand, and their proinflammatory effect in the setting of atherosclerotic disease on the other hand, could be relevant. Disturbance of this balance in favour of inflammation in patients with CAD could possibly explain the association between lipoproteins and cytokines in ischaemic patients, while in the IDCM group such an association could not be found in the present study.
The finding that low CHOL predicted mortality in patients with chronic heart failure seemingly contradicts the results of the present observation.1,14However, discrepancies might have resulted from differences in patient demographics between the studies. When going into detail in the paper by Rauchhaus et al.,1it is important to stress the fact that ischaemic patients had lipoprotein concentrations similar to the non-ischaemic group. In the population we studied, HDL, LDL and CHOL/HDL significantly differed between the two groups. LDL levels in the CAD patients enrolled in our study, were higher than those included in the study by Rauchhaus et al. (mean LDL 153mg/dl versus 135mg/dl, P=0.02). Similar to our findings, Vredevroe et al.14showed higher CHOL and LDL levels in the CAD patients. Contradictory to Rauchhaus and co-workers, these authors demonstrated that in the IDCM group only, lower lipid levels predicted mortality.
Concerning the relation between lipid profiles and cytokine activation, it is not surprising that very ill, malnourished patients have low lipoprotein levels. Therefore, the coincidence of low lipid profiles with high cytokine levels does not necessarily prove an endotoxin-lipoprotein mediated relationship. Alternatively, both findings could be mere markers of severe disease. Although prognostic data from our cohort of patients are not available at the present time, the data provided by our cross-sectional study, suggest that in patients with chronic heart failure due to atherosclerosis, lower lipid levels might be beneficial, whereas in the non-ischaemic patients, cytokine activation is independent of lipoprotein concentrations.
4.2. Limitations
As could be expected, patients with IDCM in this study presented with significantly lower LDL, higher HDL levels and a lower CHOL/HDL ratio. Therefore, from the presently studied cohort, comparable relations between cytokines and lipoproteins in non-ischemic chronic heart failure patients with a more pronounced pro-atherogenic lipid profile cannot be ruled out. Although not statistically significant, we cannot ignore the fact that patients with CAD were older then patients included in the IDCM group. This might have influenced cytokine levels and their different relationship with lipoproteins.
On the basis of the large numbers of multiple comparisons, it cannot be excluded that some of the correlations are significant by chance.
5. Conclusion
The endotoxin-lipoprotein hypothesis cautions the use of lipid-lowering drugs in patients with chronic heart failure. However, the positive correlation between a pro-atherogenic lipid profile and proinflammatory cytokines in the present study suggests possible benefit in patients with heart failure due to CAD. In addition, anti-inflammatory effects of statins and in particular, the inhibition of monocyte produced proinflammatory cytokines, have been documented in several in vitro and in vivo experiments.1618Several studies have recently shown significant benefit of treatment with statins early in the course of an acute coronary syndrome.1921Proposed mechanisms are reduced inflammation, plaque stabilisation, inhibition of thrombosis and restoration of endothelial function. Many of these effects are produced independent of lipid-lowering. Recent work has unravelled lipid-unrelated pathways by which statins interfere with the function and activation of monocytes and macrophages.22Through inhibition of the mevalonate pathway, statins reduce levels of isoprenoid intermediates such as farnesyl pyrophosphate and geranylgeranylpyrophosphate. Isoprenoids post-translationally prenylate several G proteins, such as Ras and Rho, enabling signal transduction from these membrane-associated proteins. Interference with the mevalonate trail has been held responsible for the statin-related control of iNOS, eNOS and cytokine expression,22,23the pro-apoptotic effect on smooth muscle cells,24the inhibition of MCP-1 production in mononuclear cells,25the reduction in Ox-LDL induced macrophage growth26and alterations in monocyte chemotaxis and neutrophil-endothelial interactions.27
At present, there is indeed a clear need for a prospective placebo-controlled trial of statin therapy in patients with chronic heart failure.28
References