Interleukin 27 is increased in carotid atherosclerosis and promotes NLRP3 inflammasome activation.

AIM: Interleukin-27 (IL-27) is involved in different inflammatory diseases; however, its role in atherosclerosis is unclear. In this study we investigated the expression of IL-27 and its receptor in patients with carotid atherosclerosis and if IL-27 could modulate the inflammatory effects of the NLRP3 inflammasome in vitro. METHODS: Plasma IL-27 was measured by enzyme immunoassay in patients with carotid stenosis (n = 140) and in healthy controls (n = 19). Expression of IL-27 and IL-27R was analyzed by quantitative PCR and immunohistochemistry in plaques from patients and in non-atherosclerotic vessels. THP-1 monocytes, primary monocytes and peripheral blood mononuclear cells (PBMCs) were used to study effects of IL-27 in vitro. RESULTS: Our main findings were: (i) Plasma levels of IL-27 were significantly elevated in patients with carotid atherosclerotic disease compared to healthy controls. (ii) Gene expression of IL-27 and IL-27R was significantly elevated in plaques compared to control vessels, and co-localized to macrophages. (iii) In vitro, IL-27 increased NLRP3 inflammasome activation in monocytes with enhanced release of IL-1 ß. CONCLUSIONS: We demonstrate increased levels of IL-27 and IL-27R in patients with carotid atherosclerosis. Our in vitro findings suggest an inflammatory role for IL-27, which can possibly be linked to atherosclerotic disease development.

Statins affect the presentation of endothelial chemokines by targeting to multivesicular bodies.

BACKGROUND: In addition to lowering cholesterol, statins are thought to beneficially modulate inflammation. Several chemokines including CXCL1/growth-related oncogene (GRO)-α, CXCL8/interleukin (IL)-8 and CCL2/monocyte chemoattractant protein (MCP)-1 are important in the pathogenesis of atherosclerosis and can be influenced by statin-treatment. Recently, we observed that atorvastatin-treatment alters the intracellular content and subcellular distribution of GRO-α in cultured human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the mechanisms involved in this phenomenon. METHODOLOGY/ PRINCIPAL FINDINGS: The effect of atorvastatin on secretion levels and subcellular distribution of GRO-α, IL-8 and MCP-1 in HUVECs activated by interleukin (IL)-1ß were evaluated by ELISA, confocal microscopy and immunoelectron microscopy. Atorvastatin increased the intracellular contents of GRO-α, IL-8, and MCP-1 and induced colocalization with E-selectin in multivesicular bodies. This effect was prevented by adding the isoprenylation substrate GGPP, but not the cholesterol precursor squalene, indicating that atorvastatin exerts these effects by inhibiting isoprenylation rather than depleting the cells of cholesterol. CONCLUSIONS/ SIGNIFICANCE: Atorvastatin targets inflammatory chemokines to the endocytic pathway and multivesicular bodies and may contribute to explain the anti-inflammatory effect of statins at the level of endothelial cell function.

Thrombin generation and platelet activation related to subintimal percutaneous transluminal angioplasty.

OBJECTIVE: The purpose of this study was to measure the in vivo platelet activation and thrombin generation in arterial blood after passing a subintimal conduit. METHODS: Subintimal percutaneous transluminal angioplasty (SPTA) is a technique where a subintimal channel is created, allowing recanalization of long peripheral arterial occlusion. From 10 patients with intermittent claudication, undergoing successful SPTA for femoropopliteal occlusive disease, we collected antecubital venous blood samples immediately before treatment, preprocedural arterial blood samples taken at the entry level proximal to the vessel occlusion, and subsequently at the reentry level after successful recanalization. Venous follow-up blood samples were taken after 24 hours. Plasma concentrations of ß-thromboglobulin (ß-TG), RANTES, and Prothrombin fragment (F1 + 2), were determined by immunoassay. Fibrinogen binding to platelets, leukocyte-platelet adhesion, and P-selectin were determined by flow cytometry. RESULTS: We found a statistically significant transluminal increase in the plasma concentrations of RANTES, ß-TG and F1 + 2 (p = 0.002, 0.001 and 0.001 respectively), which all normalized within 24 hours. Platelet-leukocyte aggregates significantly decreased after 24 hours compared with preprocedural and preentry levels (3.26% versus 5.26 %, p = 0.017). P-selectin expression on circulating platelets was statistically significantly increased in the blood sample taken at the re-entry level compared with the pre-procedural and pre-entry level (p = 0.007). After 24 hours there was no statistically significant difference to pre-procedural levels. There was no significant change in platelet fibrinogen binding at any levels. CONCLUSION: When passing a subintimal conduit, in vivo sampled blood demonstrated an extremely rapid and substantial uniform platelet activation and thrombin generation.

Activation of platelets by Aspergillus fumigatus and potential role of platelets in the immunopathogenesis of Aspergillosis.

Aspergillus fumigatus is the most frequent cause of invasive mold infections worldwide. Platelets contribute to inflammation and promote thrombosis, characteristically seen in aspergillosis, and might be involved both in antifungal defense and in the histopathological process. In the experiments reported here, in vitro activation of platelets by conidia, swollen conidia, and hyphae from A. fumigatus was assessed by flow cytometry and enzyme immunoassays. THP-1 monocytes and human monocytes with and without platelets were cultured with hyphae from A. fumigatus, and the release of interleukin-8 (IL-8) was measured by enzyme immunoassays. A. fumigatus potently induced the expression of CD62-p and CD63 and the release of CD40 ligand, RANTES, and Dickkopf homolog 1 in platelets, with particularly enhancing effects of hyphae compared with conidia. The hypha-mediated activation of platelets further enhanced the release of IL-8 both in THP-1 monocytes and in human adherent monocytes. In conclusion, we have found that A. fumigatus is a potent inducer of platelet-mediated inflammation, potentially promoting protective as well as harmful responses during aspergillosis.

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/beta-catenin pathway and activation of nuclear factor kappaB. CONCLUSIONS: 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.

Variability in aggregometry response before and after initiation of clopidogrel therapy.

BACKGROUND: Evaluation of clopidogrel therapy by in vitro methods has limitations which may be of clinical importance. We wanted to explore the variability in aggregometry response in aspirin sensitive patients before and after initiation of clopidogrel therapy. METHODS: ADP 9.37 microM, AA 1.2mM and TRAP 25 mM stimulated light transmissions aggregometry (LTA) were performed twice before (Exams 1 and 2; 3 weeks apart)-and within one year after-initiation of clopidogrel therapy (Exam 3) in 79 patients treated with PCI. Repeated ADP aggregometry was also performed in 16 healthy volunteers in order to estimate LTA measurement error. RESULT: Inter-individual differences in ADP aggregation e.g. at Exam 1 were substantial (range 17-77%, SD 15.8%). Intra-individual changes between Exams 1 and 2 were significant (-27 to +36%, SD 14.6%, p<0.05). Inter-individual differences at Exam 3 (on clopidogrel treatment) were larger than expected from Exams 1 and 2 (p<0.01). AA aggregation was the same before and during clopidogrel treatment. In controls, inter-individual differences were smaller at ADP 10 than at ADP 5 microM. CONCLUSIONS: Inter-individual differences in ADP aggregation were significant both before and during clopidogrel therapy, and there were significant intra-individual variations over time. Therefore, prediction of aggregometry response before or during clopidogrel therapy based on single tests may be unreliable. Inter-individual differences in healthy controls are smaller at high concentrations of ADP, and comparisons of aggregometry response should be performed with caution unless ADP concentrations are standardized.

Elevated levels of activin A in clinical and experimental pulmonary hypertension.

Activin A, a member of the transforming growth factor (TGF)-beta superfamily, is involved in regulation of tissue remodeling and inflammation. Herein, we wanted to explore a role for activin A in pulmonary hypertension (PH). Circulating levels of activin A and its binding protein follistatin were measured in patients with PH (n = 47) and control subjects (n = 14). To investigate synthesis and localization of pulmonary activin A, we utilized an experimental model of hypoxia-induced PH. In mouse lungs, we also explored signaling pathways that can be activated by activin A, such as phosphorylation of Smads, which are mediators of TGF-beta signaling. Possible pathophysiological mechanisms initiated by activin A were explored by exposing pulmonary arterial smooth muscle cells in culture to this cytokine. Elevated levels of activin A and follistatin were found in patients with PH, and activin A levels were significantly related to mortality. Immunohistochemistry of lung autopsies from PH patients and lungs with experimental PH localized activin A primarily to alveolar macrophages and bronchial epithelial cells. Mice with PH exhibited increased pulmonary levels of mRNA for activin A and follistatin in the lungs, and also elevated pulmonary levels of phosphorylated Smad2. Finally, we found that activin A increased proliferation and induced gene expression of endothelin-1 and plasminogen activator inhibitor-1 in pulmonary artery smooth muscle cells, mediators that could contribute to vascular remodeling. Our findings in both clinical and experimental studies suggest a role for activin A in the development of various types of PH.

Raised LIGHT levels in pulmonary arterial hypertension: potential role in thrombus formation.

RATIONALE: Thrombus formation and inflammation are involved in the pathogenesis of pulmonary arterial hypertension (PAH), and LIGHT (Lymphotoxin-like Inducible protein that competes with Glycoprotein D for Herpesvirus entry mediator on T lymphocytes) has been shown to promote vascular inflammation. OBJECTIVES: We sought to investigate the role of the tumor necrosis factor superfamily ligand LIGHT in the pathogenesis of PAH. METHODS: We studied 73 patients with severe PAH and 10 control subjects. LIGHT and pro- and antithrombotic markers were assessed by enzyme immunoassays. MEASUREMENTS AND MAIN RESULTS: (1) Patients with idiopathic PAH (n = 21), patients with PAH related to risk factors or associated conditions (n = 31), and those with chronic thromboembolic PAH (n = 21) all had raised serum levels of LIGHT compared with control subjects (n = 10). (2) LIGHT levels in femoral artery were significantly related to mortality in the patients with PAH. (3) Immunostaining of LIGHT and its receptors was seen in alveolar macrophages, vascular smooth muscle cells, and endothelial cells in lungs from patients with PAH. (4) Thirteen patients received prostacyclin infusion (3 mo), and all showed hemodynamic improvement, accompanied by decreased LIGHT levels. (5) Prostacyclin abolished the release of LIGHT from activated platelets in vitro, suggesting that the decrease in LIGHT during prostacyclin therapy could involve direct effects on platelets. (6) LIGHT increased tissue factor and plasminogen activator inhibitor type 1 and decreased thrombomodulin levels in endothelial cells, inducing a prothrombotic state in these cells. CONCLUSIONS: Our findings suggest prothrombotic effects of LIGHT in PAH involving endothelium-related mechanisms, potentially contributing to the progression of this disorder.

Platelet-derived LIGHT induces inflammatory responses in endothelial cells and monocytes.

Traditionally, platelets are known to play an important role in hemostasis, thrombosis, and wound healing, but increasing evidence suggests that activated platelets also may promote inflammation. Platelet-induced modulation of inflammation seems to involve platelet expression of ligands in the tumor necrosis factor (TNF) superfamily such as CD40 ligand and Fas ligand. The present study demonstrates that LIGHT, another member of the TNF superfamily, is associated with platelets and is released as a soluble ligand on platelet activation. The release of LIGHT involves GP IIb/IIIa-dependent mechanisms and action of metal-dependent proteases as well as intracellular processes such as actin polymerization. We also report that platelet-derived LIGHT is biologically active and can induce an inflammatory response in monocytes and particularly within endothelial cells measured as up-regulation of adhesion molecules and release of chemokines. Moreover, we demonstrate that thrombus material, obtained at the site of plaque rupture in patients with acute myocardial infarction, contains platelet-associated LIGHT, suggesting that LIGHT-mediated inflammation also is operating in vivo within an inflamed and thrombotic vessel wall. The data may suggest a pathogenic role for platelet-derived LIGHT in atherogenesis and plaque destabilization as well as in other inflammatory disorders involving leukocyte infiltration into the vessel wall.

Effect of activated platelets on expression of cytokines in peripheral blood mononuclear cells - potential role of prostaglandin E2.

Platelets may act as inflammatory cells. To study the effects of soluble and cell-bound platelet factors on the expression of several cytokines and related mediators in leukocytes, peripheral blood mononuclear cells (PBMC) were incubated with platelet-free supernatants from SFLLRN-activated platelet-rich plasma (PRP) or SFLLRN-activated PRP in itself. Our main findings were: (i) the gene expression of several chemokines and some cytokines were markedly increased by both activated PRP and supernatants, as also confirmed at the protein level for IL-6, IL-8 and MIP-1alpha; (ii) the selective protein kinase A type I (PKAI) antagonist Rp-8-Br-cAMP reduced this platelet-induced expression of IL-6, IL-8 and MIP-1alpha in PBMC, suggesting a role of cAMP/PKAI mediated mechanisms in this interaction; (iii) PGE(2) dose-dependently increased the release of IL-6, IL-8 and MIP-1alpha from PBMC mimicking the effect of activated platelets. Furthermore, activated platelets released comparable amounts of PGE(2), suggesting that platelet-derived PGE2 could interact with PBMC in co-cultures; (iv) IL-10 inhibited the platelet-inducing effect on IL-6, IL-8 and MIP-1alpha in PBMC, and notably, the addition PGE2 totally abolished this IL-10 effect suggesting that the suppressive effect of IL-10 on the plateletinduced activation of PBMC might at least partly involve PGE(2) related mechanisms. The present study supports a view of platelets as inflammatory cells, and suggests a potential role of platelet-derived PGE(2) in platelet-induced inflammatory responses.

Release of soluble CD40 ligand after platelet activation: studies on the solubilization phase.

sCD40L is released from platelets as a soluble, proteolyzed form of CD40 ligand (CD40L; CD154) which is exposed on the surface after platelet activation. Ethylenediaminetetraacetate (EDTA), the CD40-blocking antibody G28-5, and GPIIb-IIIa antagonists are known to inhibit the solubilization when added prior to activation. It is assumed that the surface expression of CD40L is a result of a separate fast process and that the solubilization is secondary to this. The release of sCD40L in this solubilization phase has been studied; that is, inhibitory substances were added to platelet-rich plasma (PRP) 10 min after addition of the activation agonist (100 microM SFLLRN), at which time the secretion phase was over as tested with beta-thromboglobulin (beta-TG). G28-5 (10 microg/ml) and EDTA (5 mM) inhibited the solubilization phase which did not require the presence of an activation agonist. Prostaglandin E1 (PGE1; 20 microM) and cytochalasin D (C8273; 60 and 100 microM), which exert their effects intracellularly, inhibited the solubilization even in the presence of abciximab (ReoPro; 40 microg/ml). The intracellular effect was not related to CD40L-containing microparticles as demonstrated by ultracentrifugation. Intracellular alkalinization by preincubation of PRP with 20 mM NH4Cl for 60 min resulted in a small but reproducible reduction in the amount of extracellular sCD40L. SFLLRN induced solubilization of CD40L also from the platelets of a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa, albeit at a lower rate than from normal platelets, and fibrinogen enhanced the solubilization from washed normal platelets. The data show that the solubilization of CD40L not only depends on reactions on the platelet surface but also that intracellular structures are engaged even during the solubilization phase.