Hans Geir Eiken

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

OBJECTIVESnChemokines 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.nnnMETHODSnWe 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.nnnRESULTSnOur 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.nnnCONCLUSIONnThe 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.

Stromal Cell–Derived Factor-1α in Unstable Angina Potential Antiinflammatory and Matrix-Stabilizing Effects

Background—Chemokines play a pathogenic role in atherogenesis and plaque destabilization by activating and directing leukocytes into the atherosclerotic plaque. However, stromal cell–derived factor (SDF)-1 was recently found to have antiinflammatory effects, and we hypothesized that this chemokine could play a beneficial role in coronary artery disease. Methods and Results—Plasma levels of SDF-1&agr; were significantly decreased in patients with stable (n=30) and unstable angina (n=30) compared with healthy control subjects (n=20), particularly in those with unstable disease. By flow cytometry and RNase protection assay, we found decreased surface expression but increased gene expression of the SDF-1&agr; receptor CXCR-4 in peripheral blood mononuclear cells (PBMC) from patients with stable angina and patients with unstable angina. In vitro, SDF-1&agr; (500 ng/mL) reduced both unstimulated and endotoxin/mitogen-stimulated mRNA and protein levels of monocyte chemoattractant protein-1, interleukin-8, matrix metalloproteinase-9, and tissue factor while increasing tissue inhibitor of metalloproteinases-1 in PBMC from patients with unstable angina. The SDF-1&agr;–mediated suppression of monocyte chemoattractant protein-1 and interleukin-8 appears to involve cAMP/protein kinase A type I–dependent pathways. Finally, although SDF-1&agr; suppressed the spontaneous release of these inflammatory mediators in unstable angina, enhancing effects were seen in unstimulated PBMC from healthy control subjects, possibly reflecting that PBMC in unstable angina are preactivated in vivo. Conclusions—In contrast to several other chemokines, our findings suggest that SDF-1&agr;, at least in high concentrations, may mediate antiinflammatory and matrix-stabilizing effects in unstable angina. These effects may promote plaque stabilization, and therapeutic intervention that enhances SDF-1&agr; activity could potentially be beneficial in acute coronary syndromes.

Enhanced gene expression of chemokines and their corresponding receptors in mononuclear blood cells in chronic heart failure—modulatory effect of intravenous immunoglobulin ☆

OBJECTIVESnWe sought to study the gene expression of chemokines and their corresponding receptors in mononuclear blood cells (MNCs) from patients with chronic heart failure (CHF), both of which were cross-sectional and longitudinal studies during therapy with intravenous immunoglobulin (IVIg).nnnBACKGROUNDnWe have recently demonstrated that IVIg improves left ventricular ejection fraction (LVEF) in patients with CHF. Based on the potential pathogenic role of chemokines in CHF, we hypothesized that the beneficial effect of IVIg may be related to a modulatory, effect on the expression of chemokines and their receptors in MNCs.nnnMETHODSnWe examined: 1) the gene expression of C, CC and CXC chemokines and their receptors in MNCs from 20 patients with CHF and 10 healthy blood donors; and 2) the expression of these genes in MNCs from 20 patients with CHF randomized in a double-blind fashion to therapy with IVIg or placebo for 26 weeks.nnnRESULTSnOur main findings in CHF were: 1) markedly raised gene expression of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and interleukin (IL)-8; 2) enhanced gene expression of their corresponding receptors; 3) modulation in a normal direction of this abnormal chemokine and chemokine receptor gene expression during IVIg, but not during placebo therapy; 4) down-regulation of MIP-1alpha, MIP-1beta and IL-8 during IVIg at the protein level in plasma; and 5) a correlation between down-regulation of MIP-1alpha gene expression and improved LVEF during IVIg therapy.nnnCONCLUSIONSnOur results further support a pathogenic role for chemokines in CHF and suggest that IVIg may represent a novel therapeutic approach, with the potential to improve LVEF in patients with CHF, possibly by modulatory effects on the chemokine network.

Increased gene expression of tumor necrosis factor superfamily ligands in peripheral blood mononuclear cells during chronic heart failure.

OBJECTIVEnInflammation may play a pathogenic role in chronic heart failure (CHF). The objective of the study was to characterise the imbalance in the cytokine network in CHF.nnnMETHODSncDNA expression arrays were used to analyse the gene expression of cytokines and related mediators in peripheral blood mononuclear cells (PBMC) from CHF patients (n=8) and healthy controls (n=8). Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the gene expression of individual genes in additional 12 patients and eight controls.nnnRESULTSnFrom 375 genes, 34 were upregulated and two downregulated in CHF patients in the cDNA expression array experiments. Regulated genes included chemokines/-receptors, members of the transforming growth factor beta superfamily, orphan receptors and in particular several members of the tumor necrosis factor (TNF) superfamily. Thus, 4-1BB ligand (L), APRIL, CD27L, CD40L, FasL, LIGHT, TRAIL-receptor 4 were upregulated, while TRAIL-receptor 3 was downregulated. Real-time quantitative RT-PCR confirmed significantly upregulated gene expression of APRIL, LIGHT, FasL and CD27L in CHF patients and showed in addition significantly enhanced gene expression of TNFalpha and TRAIL.nnnCONCLUSIONnThe present study demonstrates differential gene expression in PBMC of several members of the cytokine network in CHF. In particular, the enhanced expression of several ligands in the TNF superfamily may reflect a potential pathogenic role of these cytokines in CHF.

Myocardial gene expression of leukaemia inhibitory factor, interleukin-6 and glycoprotein 130 in end-stage human heart failure.

Background Studies in different animal models and plasma analyses in humans suggest that members of the interleukin‐6 (IL‐6) cytokine family may be involved in the pathogenesis of congestive heart failure (CHF). Accordingly, we have examined IL‐6‐related cytokines in chronic CHF in humans by analysing gene and protein expression in myocardium derived from patients with end‐stage heart failure and donor hearts.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells

Summary.u2002 CXC‐chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC‐chemokines in the inflammatory interactions between oxidized (ox‐) low‐density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without ‘traditional’ risk factors and 15 carefully matched controls. Our main findings were: (a) ox‐LDL stimulated the release of the CXC‐chemokines interleukin (IL)‐8, ENA‐78 and GRO‐α from PBMC, particularly in CAD. (b) In platelets, ox‐LDL induced release of ENA‐78 and, when combined with SFLLRN, also of GRO‐α, with significantly higher response in CAD. (c) Platelet‐rich plasma, especially when costimulated with ox‐LDL, enhanced the release of IL‐8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL‐8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox‐LDL, platelets and PBMC in CAD patients involving CXC‐chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Monocyte chemoattractant protein-1 enhances and interleukin-10 suppresses the production of inflammatory cytokines in adult rat cardiomyocytes.

Objective Chemokines control the migration of leukocytes to inflamed tissue, and in particular monocyte chemoattractant protein (MCP)-1 has been implicated in the pathogenesis of several cardiovascular disorders such as chronic heart failure (CHF) and myocarditis. We hypothesised that MCP-1 may directly contribute to an inflammatory response in the cardiomyocytes, and in the present study we examined in adult rat cardiomyocytes: (i) the effect of tumour necrosis factor (TNF)a on MCP-1 production, (ii) the effect of MCP-1 on production of other inflammatory cytokines, and (iii) if the anti-inflammatory cytokine interleukin (IL)-10 could suppress any TNFα-induced MCP-1 production. Methods We used enzyme immunoassays, RNase protection assays and slot blot analysis to measure protein and mRNA levels of various cytokines in adult rat cardiomyocyte cultures. Results (i) We found a ∼6.4-fold increase of the MCP-1 level accompanied by an increase in MCP-1 mRNA accumulation in cardiomyocyte cultures after TNFα stimulation. (ii) In contrast, TNFα had no effect on IL-10 and only a modest effect on IL-1β and IL-6 levels in these cells. (iii) Importantly, MCP-1 stimulated inflammatory response in cardiomyocytes by enhancing IL-1β and IL-6 levels in these cells as found at both the protein and mRNA level. (iv) Co-stimulation with IL-10 resulted in a ∼55 % reduction in TNFα-stimulated MCP-1 levels in cardiomyocyte culture supernatants. Conclusion The present study demonstrates for the first time that MCP-1 can directly affect cardiomyocytes, and we introduce MCP-1 as a potential enhancer and IL-10 as a potential suppresser of inflammatory responses within the myocardium.

Cytokine networks are pre-activated in T cells from HIV-infected patients on HAART and are under the control of cAMP.

Objective: Cytokines seem to play a critical role in HIV infection. The cAMP/protein kinase A (PKA) type I pathway is shown to be hyper-activated and contributes to T-cell immune dysfunction in HIV infection. Here, we analysed firstly the levels of cytokine gene expression in unstimulated CD3+T cells from HIV-infected patients on HAART, and secondly the regulation of cytokine and cytokine-related genes by cAMP agonist and antagonist in anti-CD3 activated T cells in order to understand their effects on cytokine networks. Methods: Cytokine Macro Array and real-time RT–PCR techniques were used to study cytokine gene expression in T cells of HIV-positive patients. Results: Of the cytokine-related genes analysed 45% were expressed at twofold or higher levels in unstimulated T cells from HIV-infected patients as compared with healthy controls, and one-third of these genes were hypo-responsive upon activation as compared with controls. Furthermore, cAMP modulated levels of expression of a number of cytokine-related genes differently in patient and control T cells. CXCR4, CCR5 and amphiregulin were up-regulated by cAMP agonist, whereas other cytokine-related genes including macrophage inflammatory protein 1β, tumour necrosis factor-α and lymphotoxin-β were markedly down-regulated by cAMP agonist in T cells from both HIV-infected patients and controls. Moreover, members of the chemokine/chemokine receptor family were over-represented among genes regulated by cAMP agonist/antagonist in patient T cells. Conclusions: Our data indicate that T cells from HIV-infected patients are in a pre-activated state and that a set of cytokine genes is hypo-responsive to activation and under tonic regulation by cAMP in these T cells.

Myocardial gene expression of inflammatory cytokines after heart transplantation in relation to the development of transplant coronary artery disease

Long-term success of cardiac transplantation is mainly limited by the development of transplant coronary artery disease (CAD); it is generally accepted that it is immune mediated, involving cytokines and growth factors. We show that development of transplant CAD is associated with a particular cytokine profile in myocardial biopsies characterized by a late (i.e., 1 year) increase in tumor necrosis factor-alpha and interferon-gamma gene expression, which precede and potentially contribute to the development of allograft vasculopathy, further supporting a role for inflammation and the pathogenesis of transplant CAD in humans.

Enhanced detection of mutations in BRCA1 exon 11 using restriction endonuclease fingerprinting-single-strand conformation polymorphism

A novel approach to mutation screening in the large exon 11 (comprising 3427xa0bp) of the human BRCA1 gene is presented. Restriction endonuclease fingerprinting single-strand conformation polymorphism (REF-SSCP) is based on repeated detection of DNA sequence variants in different restriction endonuclease fragments, and we evaluated the method using blood samples from 25 Norwegian patients with hereditary breast/ovarian cancer. We compared REF-SSCP to constant denaturant gel electrophoresis (CDGE) and to the protein truncation test (PTT). REF-SSCP detected 12 different DNA variants. Four of these were not detected by CDGE, and only one variant detected by CDGE was missed by REF-SSCP. PTT detected 4 of these 13 variants. REF-SSCP was subsequently applied to a second patient series (Swedish, n=20). A total of 14 different DNA variants were detected by REF-SSCP, 6 of which were truncating mutations (PTT detected only 4). Nonsense and frameshift mutations that are putative breast/ovarian cancer mutations, were detected in 7 of the 25 Norwegian and 9 of the 20 Swedish patients.