Arne Yndestad

Systemic inflammation in heart failure--the whys and wherefores.

Patients with chronic heart failure (HF) are characterized by systemic inflammation, as evident by raised circulating levels of several inflammatory cytokines with increasing levels according to the degree of disease severity. In addition to the myocardium itself, several tissues and cells can contribute to this inflammation, including leukocytes, platelets, tissue macrophages and endothelial cells. Although the mechanisms for the systemic inflammation is unknown, both infectious (e.g., endotoxins) and non-infectious (e.g., oxidative stress and hemodynamic overload) events could be operating, also including activation of Toll-like receptors as well as interaction with the neurohormone system. A growing body of evidence suggests that this systemic inflammation in chronic HF may play a role in the development and progression of this disorder, not only by promoting myocardial dysfunction, but also by inducing pathogenic consequences in other organs and tissues, thereby contributing to additional aspects of the HF syndrome such as cachexia, endothelial dysfunction and anemia. Although this inappropriate immune activation and inflammation could represent a new target for therapy in patients with chronic HF, the anti-tumor necrosis factor trials have been disappointing, and future research in this area will have to more precisely identify the most important mechanisms and actors in the immunopathogenesis of chronic HF in order to develop better immunomodulating agents for this disorder.

Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure.

AIMS Neutrophil gelatinase-associated lipocalin (NGAL or lipocalin-2) is a glycoprotein with bacteriostatic properties. Growing evidence suggests that NGAL may also be involved in cell survival, inflammation, and matrix degradation. We therefore aimed to investigate the role of NGAL in heart failure (HF). METHODS AND RESULTS Our main findings were (i) patients with acute post-myocardial infarction (MI) HF (n = 236) and chronic HF (n = 150) had elevated serum levels of NGAL (determined by enzyme immunoassay), significantly correlated with clinical and neurohormonal deterioration, (ii) in patients with HF following acute MI, elevated NGAL levels of at baseline were associated with adverse outcomes (median of 27 months follow-up), (iii) in a rat model of post-MI HF, NGAL/lipocalin-2 gene expression was increased in the non-ischaemic part of the left ventricle primarily located to cardiomyocytes, (iv) strong NGAL immunostaining was found in cardiomyocytes within the failing myocardium both in experimental and clinical HF, (v) interleukin-1beta and agonists for toll-like receptors 2 and 4, representing components of the innate immune system, were potent inducers of NGAL/lipocalin-2 in isolated neonatal cardiomyocytes. CONCLUSION Our demonstration of enhanced systemic and myocardial NGAL expression in clinical and experimental HF further support a role for innate immune responses in the pathogenesis of HF.

Dysregulated Osteoprotegerin/RANK Ligand/RANK Axis in Clinical and Experimental Heart Failure

Background—Persistent inflammation appears to play a role in the development of heart failure (HF). Osteoprotegerin (OPG), the receptor activator of nuclear factor-&kgr;B (RANK), and RANK ligand (RANKL) are newly discovered members of the tumor necrosis factor superfamily that are critical regulators in bone metabolism but appear also to be involved in immune responses. We hypothesized that the OPG/RANK/RANKL axis could be involved in the pathogenesis of heart failure (HF), and this hypothesis was investigated in both experimental and clinical studies. Methods and Results—Our main and novel findings were as follows: (1) In a rat model of postinfarction HF, we found persistently increased gene expression of OPG, RANK, and RANKL in the ischemic part of the left ventricle (LV) and, for OPG, in the nonischemic part that involved both noncardiomyocyte and in particular cardiomyocyte tissue. (2) Enhanced myocardial protein levels of OPG, RANK, and RANKL, in particular, were also seen in human HF, and using immunohistochemistry, we localized these mediators to cardiomyocytes within the LV in both experimental and clinical HF. (3) In human HF, we also found increased systemic expression of RANKL (T cells and serum) and OPG (serum), with increasing levels according to functional, hemodynamic, and neurohormonal disease severity. (4) RANKL increased total matrix metalloproteinase activity in human fibroblasts, which indicates a matrix-degrading net effect and suggests a potential mechanism by which enhanced RANKL expression in HF may contribute to LV dysfunction. Conclusions—These findings suggest a potential role for known mediators of bone homeostasis in the pathogenesis of HF and possibly represents new targets for therapeutic intervention in this disorder.

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure

OBJECTIVES Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.

Inflammatory Cytokines in Heart Failure: Mediators and Markers

Evidence from both experimental and clinical trials indicates that inflammatory mediators are of importance in the pathogenesis of chronic heart failure (HF) contributing to cardiac remodeling and peripheral vascular disturbances. Several studies have shown raised levels of inflammatory cytokines such as tumor necrosis factor (TNF)α, interleukin (IL)-1β and IL-6 in HF patients in plasma and circulating leukocytes, as well as in the failing myocardium itself. There is strong evidence that these mediators are involved in processes leading to cardiac remodeling such as hypertrophy, fibrosis and apoptosis. Some of these cytokines can also give useful prognostic information as reliable biomarkers in this disorder. In general, immunomodulating treatments have, with a few exceptions, been neutral or even harmful. However, the negative results of anti-TNF studies, for instance, do not necessarily argue against the ‘cytokine hypothesis’. These studies just underscore the challenges in developing treatment modalities that can modulate the cytokine network in HF patients and result in beneficial net effects. Future studies should identify the crucial actors and their mechanisms of action in the immunopathogenesis of chronic HF and, in particular, clarify the balance between adaptive and maladaptive effects of these molecules. Such studies are a prerequisite for the development of new treatment strategies that target inflammatory and immunopathogenic mechanisms in HF. In this review article, these issues are thoroughly discussed, and we also argue for the possibility of future therapeutic targets such as mediators in innate immunity, chemokines and mediators in matrix remodeling.

The NLRP3 inflammasome is up-regulated in cardiac fibroblasts and mediates myocardial ischaemia–reperfusion injury

AIMS Nucleotide-binding oligomerization domain-Like Receptor with a Pyrin domain 3 (NLRP3) is considered necessary for initiating a profound sterile inflammatory response. NLRP3 forms multi-protein complexes with Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) and Caspase-1, which activate pro-interleukin-1β (IL-1β) and pro-IL-18. The role of NLRP3 in cardiac cells is not known. Thus, we investigated the expression and function of NLRP3 during myocardial ischaemia. METHODS AND RESULTS Myocardial infarction (MI) was induced in adult C57BL/6 mice and Wistar rats by ligation of the coronary artery. A marked increase in NLRP3, IL-1β, and IL-18 mRNA expression was found in the left ventricle after MI, primarily located to myocardial fibroblasts. In vitro studies in cells from adult mice showed that myocardial fibroblasts released IL-1β and IL-18 when primed with lipopolysaccharide and subsequently exposed to the danger signal adenosine triphosphate, a molecule released after tissue damage during MI. When hearts were isolated from NLRP3-deficient mice, perfused and subjected to global ischaemia and reperfusion, a marked improvement of cardiac function and reduction of hypoxic damage was found compared with wild-type hearts. This was not observed in ASC-deficient hearts, potentially reflecting a protective role of other ASC-dependent inflammasomes or inflammasome-independent effects of NLRP3. CONCLUSION This study shows that the NLRP3 inflammasome is up-regulated in myocardial fibroblasts post-MI, and may be a significant contributor to infarct size development during ischaemia-reperfusion.

Inflammatory and anti‐inflammatory cytokines in chronic heart failure: Potential therapeutic implications

Persistent inflammation, involving increased levels of inflammatory cytokines, seems to play a pathogenic role in chronic heart failure (HF) by influencing heart contractility, inducing hypertrophy and promoting apoptosis, contributing to myocardial remodeling. While several stimuli may be operating such as heat shock proteins, microbial antigens and oxidative stress, it seems that the inflammatory response to these stimuli may represent a common final pathogenic pathway in HF regardless of the initial event. Traditional cardiovascular drugs appear to have little influence on the cytokine network, and immunomodulatory therapy has emerged as a possible new treatment modality in HF. Several animal studies and also some clinical studies have suggested that downregulation of inflammation may improve cardiac performance. However, the results from the placebo‐controlled anti‐tumor necrosis factor studies suggest no improvement or even adverse effects of such therapy. Although somewhat disappointing, these negative results do not necessarily argue against the ‘cytokine hypothesis’. These studies just underscore the challenges in developing treatment modalities that can modulate the cytokine network in HF patients resulting in beneficial net effects. Further research will have to identify more precisely the most important actors in the immunopathogenesis of chronic HF in order to develop more specific immunomodulating agents for this disorder.

Enhanced T-cell expression of RANK ligand in acute coronary syndrome: possible role in plaque destabilization.

Objective—Based on its role in inflammation and matrix degradation, we hypothesized a role for osteoprotegerin (OPG), RANK, and RANK ligand (RANKL) in coronary artery disease. Methods and Results—We examined the expression of various members of the OPG/RANKL/RANK axis in patients with stable and unstable angina and in the atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice. Our findings were: (1) Serum levels of OPG were raised in patients with unstable angina (n=40), but not in those with stable angina (n=40), comparing controls (n=20); (2) mRNA levels of RANKL were increased in T-cells in unstable angina patients accompanied by increased expression of RANK in monocytes; (3) strong immunostaining of OPG/RANKL/RANK was seen within thrombus material obtained at the site of plaque rupture during acute myocardial infarction; (4) OPG/RANKL/RANK was expressed in the atherosclerotic plaques of apoE−/− mice, with RANKL located specifically to the plaques; and (5) RANKL enhanced the release of monocyte chemoattractant peptide-1 in mononuclear cells from unstable angina patients, and promoted matrix metalloproteinase (MMP) activity in vascular smooth muscle cells. Conclusions—We show enhanced expression of the OPG/RANKL/RANK system both in clinical and experimental atherosclerosis, with enhanced T-cell expression of RANKL as an important feature of unstable disease.

Increased Expression of Interleukin-1 in Coronary Artery Disease With Downregulatory Effects of HMG-CoA Reductase Inhibitors

Background—Inflammation is important in atherogenesis. Interleukin (IL)-1 is the prototypic inflammatory cytokine. We hypothesized a dysbalance between inflammatory and anti-inflammatory mediators in the IL-1 family in coronary artery disease (CAD) and a possible modulation of these mediators by HMG-CoA inhibitors (statins). Methods and Results—In a microarray screening experiment examining peripheral blood mononuclear cells (PBMCs) from 6 CAD patients and 4 healthy control subjects, IL-1β was identified as 1 of 25 genes whose expression were upregulated in CAD and downregulated by statins. In the following, we studied the role of IL-1β and related mediators in CAD. Our major findings were as follows. (1) Although mRNA levels of IL-1&agr; and IL-1β were markedly reduced in PBMCs from CAD patients after 6 months of simvastatin (20 mg/d, n=15) and atorvastatin (80 mg/d, n=15) therapy, the reduction in IL-1 receptor antagonist (IL-1Ra) was more modest. Statins also reduced the spontaneous release of IL-1β and IL-1Ra from PBMCs in CAD patients. (2) mRNA levels of IL-1&agr;, IL-1β, and IL-1Ra were increased in PBMCs from patients with stable (n=20) and unstable (n=20) angina compared with healthy control subjects (n=15). Although the unstable patients had particularly high levels of IL-1β and IL-1&agr;, IL-1Ra was not correspondingly increased. (3) IL-1β induced release of proatherogenic cytokines from PBMCs, whereas atorvastatin partly abolished this effect. Conclusions—Our findings suggest that cytokines in the IL-1 family may represent therapeutic targets in CAD. The ability of statins to modulate these cytokines in an anti-inflammatory direction underscores their immunomodulatory potential.

Chemokines and Cardiovascular Risk

Based on the importance of inflammation in atherogenesis, recent work has focused on whether plasma markers of inflammation can noninvasively diagnose and prognosticate atherosclerotic disorders. Although several studies support an important pathogenic role of chemokines in atherosclerosis, potentially representing attractive therapeutic targets in atherosclerotic disorders, this does not necessarily mean that chemokines are suitable parameters for risk prediction. In fact, the ability to reflect upstream inflammatory activity, stable levels in individuals, and high stability of the actual protein (eg, long half-life and negligible circadian variation) are additional important criteria for an ideal biomarker in cardiovascular disease. Although plasma/serum levels of certain chemokines (eg, interleukin- 8/CXCL8 and monocyte chemoattractant protein-1/CCL2) have shown to be predictive for future cardiac events in some studies, their role as clinical biomarkers is unclear, and their ability to predict subclinical atherosclerosis has been disappointing. Further prospective studies, including a larger number of patients, are needed to make any firm conclusion. Based on the participation of several chemokines in atherogenesis, it is possible that in the future, combined measurements of multiple chemokines could reveal as a “signature of disease” that can serve as a highly accurate method to assess for the presence of atherosclerotic disease.