Erik Øie

Increased Expression of Visfatin in Macrophages of Human Unstable Carotid and Coronary Atherosclerosis Possible Role in Inflammation and Plaque Destabilization

Background— Although the participation of inflammation in atherogenesis is widely recognized, the identification of the different components has not been clarified. In particular, the role of inflammation in plaque destabilization is not fully understood. Methods and Results— Our main findings were as follows: (1) In a microarray experiment, we identified visfatin, one of the most recently identified adipokines, as a gene that was markedly enhanced in carotid plaques from symptomatic compared with plaques from asymptomatic individuals. This finding was confirmed when carotid plaques from 7 patients with asymptomatic and 14 patients with symptomatic lesions were examined with real-time reverse transcription polymerase chain reaction. (2) Immunohistochemistry showed that visfatin was localized in areas that were rich in lipid-loaded macrophages. (3) The relationship between visfatin and unstable lesions was also found in patients with coronary artery disease, demonstrating a strong visfatin immunostaining in lipid-rich regions within the material obtained at the site of plaque rupture in patients with acute myocardial infarction. (4) Both oxidized low-density lipoprotein and tumor necrosis factor-&agr; increased visfatin expression in THP-1 monocytes, with a particularly enhancing effect when these stimuli were combined. (5) Visfatin increased matrix metalloproteinase-9 activity in THP-1 monocytes and tumor necrosis factor-&agr; and interleukin-8 levels in peripheral blood mononuclear cells. Both of these effects were abolished when insulin receptor signaling was blocked. Conclusions— Our findings suggest that visfatin should be regarded as an inflammatory mediator, localized to foam cell macrophages within unstable atherosclerotic lesions, that potentially plays a role in plaque destabilization.

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.

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.

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.

Role of inflammation in the progression of heart failure.

Chronic heart failure (HF) is a disorder characterized in part by immune activation and inflammation. Thus, patients with HF have elevated levels of a number of inflammatory cytokines, both in the circulation and in the failing heart itself. Several mechanisms for this immune activation, which are not mutually exclusive, have been suggested, including neurohormonal activation, hemodynamic overload, and activation of the innate immune system secondary to cardiac stress. Importantly, experimental studies have shown that inflammatory cytokines such as tumor necrosis factor-α, interleukin-1β, and monocyte chemoattractant peptide-1 may contribute to the development and progression of HF by promoting myocardial hypertrophy, activating matrix metalloproteinases, provoking contractile dysfunction, and inducing apoptosis. However, inflammatory cytokines may also have adaptive and cardioprotective effects. This important aspect of cytokine biology must be kept in mind when designing new immunomodulatory treatment modalities in HF.

Elevated Levels of Activin A in Heart Failure Potential Role in Myocardial Remodeling

Background—Although modulation of inflammatory processes has been suggested as a new treatment modality in heart failure (HF), our knowledge about abnormalities in the cytokine network during HF is still limited. On the basis of a previous cDNA array study examining peripheral blood mononuclear cells from HF patients, we hypothesized a role for activin A, a member of the transforming growth factor (TGF)-&bgr; superfamily, in the pathogenesis of HF. Methods and Results—This study had 4 main and novel findings. First, serum levels of activin A were significantly elevated in patients with HF (n=86) compared with healthy control subjects (n=20), with increasing levels according to disease severity as assessed by clinical, hemodynamic, and neurohormonal parameters. Second, compared with control subjects, HF patients, as determined by real-time quantitative reverse transcriptase polymer chain reaction, also had markedly increased gene expression of the activin A subunit activin &bgr;A in T cells but not in monocytes. Third, in a rat model of HF, we demonstrated a concerted induction of the gene expression of activin &bgr;A and activin receptors IA, IB, IIA, and IIB after myocardial infarction. Immunohistochemical analysis localized activin A solely to cardiomyocytes. Finally, activin A markedly increased gene expression of mediators involved in infarction healing and myocardial remodeling (ie, atrial natriuretic peptide, brain natriuretic peptide, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, transforming growth factor-&bgr;1, and monocyte chemoattractant protein-1) in neonatal rat cardiomyocytes. Conclusions—Together with our demonstration of activin A–induced gene expression in neonatal cardiomyocytes of mediators related to myocardial remodeling, the expression pattern of activin A during clinical and experimental HF suggests an involvement of this cytokine in the pathogenesis of HF.

Dickkopf-1 Enhances Inflammatory Interaction Between Platelets and Endothelial Cells and Shows Increased Expression in Atherosclerosis

Objective—Based on the emerging importance of the wingless (Wnt) pathways in inflammation and vascular biology, we hypothesized a role for Dickkopf-1 (DKK-1), a major modulator of Wnt signaling, in atherogenesis and plaque destabilization. Methods and Results—We report increased levels of DKK-1 in experimental (ApoE−/− mice) and clinical (patients with coronary artery disease [n=80] and patients with carotid plaque [n=47]) atherosclerosis, both systemically (serum) and within the lesion, with particularly high levels in advanced and unstable disease. We identified platelets as an important cellular source of DKK-1 as shown by in vitro experiments and by immunostaining of thrombus material obtained at the site of plaque rupture in patients with acute ST-elevation myocardial infarction, with strong immunoreactivity in platelet aggregates. Our in vitro experiments identified a role for platelet- and endothelial-derived DKK-1 in platelet-dependent endothelial activation, promoting enhanced release of inflammatory cytokines. These inflammatory effects of DKK-1 involved inhibition of the Wnt/&bgr;-catenin pathway and activation of nuclear factor &kgr;B. Conclusion—Our findings identify DKK-1 as a novel mediator in platelet-mediated endothelial cell activation. The demonstration of enhanced DKK-1 expression within advanced carotid plaques may suggest that this DKK-1-driven inflammatory loop could be operating within the atherosclerotic lesion.