Wiggo J. Sandberg

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.

Expression of Fractalkine (CX3CL1) and its Receptor, CX3CR1, Is Elevated in Coronary Artery Disease and Is Reduced During Statin Therapy

Objective—Recent data derived primarily from studies in animal models suggest that fractalkine (CX3CL1) and its cognate receptor, CX3CR1, play a role in atherogenesis. We, therefore, hypothesized that enhanced CX3CL1/CX3CR1 expression may promote atherogenesis in patients with coronary artery disease (CAD). Methods and Results—We examined the plasma levels of CX3CL1 and CX3CR1 expression in peripheral blood mononuclear cells (PBMC) in various CAD populations (30 patients with previous myocardial infarction, 40 patients with stable angina, 40 patients with unstable angina, and a total of 35 controls) and used various experimental approaches to characterize CX3CL1-mediated leukocyte responses. We found that the plasma levels of CX3CL1 are greatly increased in CAD, particularly in unstable disease. The parallel increase of CX3CR1 expression in PBMC was predominantly attributable to an expansion of the CX3CR1+CD3+CD8+ T cell subset and was associated with enhanced chemotactic, adhesive, and inflammatory responses to CX3CL1. Statin therapy for 6 months reduced the expression of CX3CL1 and CX3CR1, reaching statistical significance for both parameters only during aggressive (atorvastatin, 80 mg qd) but not conventional (simvastatin, 20 mg qd) therapy. Consequently, the functional responses of the PBMC to CX3CL1 including migration, adhesion, and secretion of interleukin-8 were attenuated by the treatments. Conclusion—Our results suggest that the CX3CL1/CX3CR1 dyad may contribute to atherogenesis and plaque destabilization in human CAD.

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.

Enhanced Expression of the Homeostatic Chemokines CCL19 and CCL21 in Clinical and Experimental Atherosclerosis. Possible Pathogenic Role in Plaque Destabilization

Objective—Based on their role in T-cell homing into nonlymphoid tissue, we examined the role of the homeostatic chemokines CCL19 and CCL21 and their common receptor CCR7 in coronary artery disease (CAD). Methods and Results—We performed studies in patients with stable (n=40) and unstable (n=40) angina and healthy controls (n=20), in vitro studies in T-cells and macrophages, and studies in apolipoprotein-E–deficient (ApoE−/−) mice and human atherosclerotic carotid plaques. We found increased levels of CCL19 and CCL21 within the atherosclerotic lesions of the ApoE−/− mice, in human atherosclerotic carotid plaques, and in plasma of CAD patients. Whereas strong CCR7 expression was seen in T-cells from murine and human atherosclerotic plaques, circulating T-cells from angina patients showed decreased CCR7 expression. CCL19 and CCL21 promoted an inflammatory phenotype in T-cells and macrophages and increased matrix metalloproteinase (MMP) and tissue factor levels in the latter cell type. Although aggressive statin therapy increased CCR7 and decreased CCL19/CCL21 levels in peripheral blood from CAD patients, conventional therapy did not. Conclusions—The abnormal regulation of CCL19 and CCL21 and their common receptor in atherosclerosis could contribute to disease progression by recruiting T-cells and macrophages to the atherosclerotic lesions and by promoting inflammatory responses in these cells.

Low numbers of regulatory T cells in common variable immunodeficiency: association with chronic inflammation in vivo

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. Abnormalities of CD4+CD25highforkhead box P3 (FoxP3)+ regulatory T cells (Treg) have been associated with autoimmune and inflammatory disorders, and we hypothesized that CVID might be characterized by Treg abnormalities. CD3+ cells from patients and controls were analysed for the expression of FoxP3 mRNA by real time reverse transcription–polymerase chain reaction (RT–PCR). Peripheral blood mononuclear cells from CVID patients and controls were stained for Treg markers, analysed by flow cytometry and compared to clinical characteristics. The main findings were: (i) CVID patients had significantly decreased expression of FoxP3 mRNA and decreased proportions of CD4+CD25highFoxP3+ cells compared to controls; (ii) CVID patients with splenomegaly had even lower proportions of Treg compared to other patients and controls; (iii) serum levels of the inflammatory marker neopterin were correlated negatively with the proportions of Treg within the CVID population, while there was no significant association with bronchiectasis. We have demonstrated decreased proportions of Treg in CVID patients, particularly in those with signs of chronic inflammation. Decreased proportions of TReg are suggested to be pathogenetically important in autoimmunity, and our results suggest that TReg may have a similar role in CVID.

A Potential Role of the CXC Chemokine GROα in Atherosclerosis and Plaque Destabilization

Objective—We examined the role of the CXCR2 ligand growth-related oncogene (GRO) α in human atherosclerosis. Methods and Results—GROα levels were examined by enzyme immunoassay, real-time quantitative RT-PCR, and cDNA microarrays. The in vitro effect of statins on GROα was examined in endothelial cells and THP-1 macrophages. Our main findings were: (1) GROα was among the 10 most differentially expressed transcripts comparing peripheral blood mononuclear cells (PBMCs) from patients with coronary artery disease (CAD) and healthy controls. (2) Both patients with stable (n=41) and particularly those with unstable (n=47) angina had increased plasma levels of GROα comparing controls (n=20). (3) We found increased expression of GROα within symptomatic carotid plaques, located to macrophages and endothelial cells. (4) GROα enhanced the release of matrix metalloproteinases in vascular smooth muscle cells, and increased the binding of acetylated LDL in macrophages. (5) Atorvastatin downregulated GROα levels as shown both in vitro in endothelial cells and macrophages and in vivo in PBMCs from CAD patients. (6) The effect on GROα in endothelial cells involved increased storage and reduced secretion of GROα. Conclusions—GROα could be involved in atherogenesis and plaque destabilization, potentially contributing to inflammation, matrix degradation, and lipid accumulation within the atherosclerotic lesion.

Enhanced Plasma Levels of LIGHT in Unstable Angina Possible Pathogenic Role in Foam Cell Formation and Thrombosis

Background— Numerous studies have demonstrated the ability of oxidized LDL [(ox)LDL] to promote an inflammatory response in macrophages. Several inflammatory mediators have been reported to increase after oxLDL stimulation of such cells, but their relative importance is still unknown. In the present study, we used microarrays to identify genes in THP-1 macrophages that were upregulated by oxLDL. Methods and Results— Our main findings were as follows. In a microarray screening experiment, we identified LIGHT, a ligand in the tumor necrosis factor superfamily, as one of the genes that were markedly upregulated in oxLDL-stimulated THP-1 macrophages. We showed significantly raised plasma levels of LIGHT in patients with stable angina (n=40) and particularly in those with unstable angina (n=40) compared with healthy controls (n=20), which underscores the clinical relevance of the in vitro finding. We also showed that LIGHT enhanced lipid accumulation in oxLDL-stimulated THP-1 macrophages, possibly through upregulation of class A scavenger receptor (SR-A). This increased lipid accumulation was accompanied by enhanced expression of tissue factor and plasminogen activator inhibitor-1, as well as enhanced thrombin formation, transforming macrophages into a prothrombotic phenotype. The LIGHT-mediated increase in SR-A, tissue factor, and plasminogen activator inhibitor-1 was also seen in human monocyte-derived macrophages. Finally, the LIGHT-mediated enhancement of SR-A and TF expression appears to involve nuclear factor-&kgr;B activation. Conclusions— These findings suggest that LIGHT could serve as a molecular link between lipid metabolism, inflammation, and thrombus formation, which are all features of atherosclerotic plaques.

High levels and inflammatory effects of soluble CXC ligand 16 (CXCL16) in coronary artery disease: down-regulatory effects of statins

AIMS CXC ligand 16 (CXCL16) may be involved in inflammation and lipid metabolism, and we hypothesized a role for this chemokine in coronary artery disease (CAD). METHODS AND RESULTS We performed clinical studies in CAD patients as well as experimental studies in cells with relevance to atherogenesis [i.e. endothelial cells, vascular smooth muscle cells (SMC), and peripheral blood mononuclear cells (PBMC)]. We also examined the ability of HMG-CoA reductase inhibitors (statins) to modulate CXCL16 levels both in vivo and in vitro. Our main findings were: (i) patients with stable (n = 40) and unstable (n = 40) angina had elevated plasma levels of CXCL16 compared with controls (n = 20); (ii) low-dose simvastatin (20 mg qd, n = 15) and high-dose atorvastatin (80 mg qd, n = 9) down-regulated plasma levels of CXCL16 during 6 months of therapy; (iii) in vitro, atorvastatin significantly decreased the interleukin (IL)-1beta-mediated release of CXCL16 from PBMC and endothelial cells; (iv) attenuating effect of atorvastatin on the IL-1beta-mediated release of CXCL16 in PBMC seems to involve post-transcriptional modulation as well as down-regulation of CXCL16 release through inhibition of the protease a disintegrin and metalloproteinase 10 (ADAM10); (v) soluble CXCL16 increased the release of IL-8, monocyte chemoattractant peptide 1, and matrix metalloproteinases in vascular SMC and increased the release of IL-8 and monocyte chemoattractant peptide 1 in PBMC, with particularly enhancing effects in cells from CAD patients. CONCLUSION Our findings suggest that soluble CXCL16 could be linked to atherogenesis not only as a marker of inflammation, but also as a potential inflammatory mediator.

Tumor necrosis factor superfamily molecules in acute coronary syndromes

Abstract Accumulating evidence suggests that inflammatory pathways play an essential role in all stages of atherogenesis. Inflammatory processes are not only involved in plaque progression, but seem also to play a critical role in plaque rupture. Members of the tumor necrosis factor (TNF) superfamiliy are potent regulators of inflammation and cell survival and consist of 20 ligands that signal through 29 different receptors. Several lines of evidence suggest that TNF-related molecules are involved in the development of acute coronary syndromes (ACS). Most, convincing evidence exists for CD40 ligand-CD40 interaction, but several other members of the TNF superfamily seem also to be involved in this immune-mediated promotion of plaque instability, including LIGHT, receptor activator of nuclear factor κB ligand, and TNF-α. These plaque destabilization pathways involve the bidirectional interaction between platelets and endothelial cells/monocytes, activation of vascular smooth muscle cells, and co-stimulatory effects on T cells, promoting inflammation, thrombus formation, matrix degradation, and apoptosis. TNF-related pathways could contribute to the non-resolving inflammation that characterizes atherosclerosis, representing pathogenic loops that are operating during plaque rupture and the development of ACS. These TNF-related molecules could also represent attractive new targets for therapy in this disorder.

Activation of Notch signaling in cardiomyocytes during post-infarction remodeling.

Abstract Objective. Notch signaling is crucial for cell-to-cell interaction during cardiovascular development and may influence differentiation, proliferation, and apoptotic events. We investigated whether Notch signaling is activated during myocardial remodeling in heart failure (HF). Design. Myocardial gene expression and localization of Notch receptors (Notch1-4) and ligands (Jagged1-2, and Delta-like (Dll)-1 and 4) were investigated in rats with HF after induction of myocardial infarction and in humans with HF. Results. All Notch receptors and ligands investigated and Notch intracellular domain (NICD) were present in rat and human myocardial tissue and in cardiomyocytes with differences in their relative expression levels and altered expression levels in failing vs. non-failing myocardium. In isolated rat cardiomyocytes, Notch3 and Notch4 appeared to be the major Notch receptors, and Notch3 and Notch4 mRNA levels and NICD-3 and -4 in cardiomyocytes were upregulated in chronic HF (p < 0.05), indicating increased Notch3 and Notch4 signaling. Conclusion. The Notch signaling system is present in the cardiomyocytes and activated in HF, indicating a role of Notch signaling during myocardial remodeling in HF.