Summary. Background: Platelets release the immune-modulating lipid sphingosine-1-phosphate (S1P). However, the mechanisms of platelet S1P secretion are not fully understood.Objectives: The present study investigates the function of thromboxane (TX) for platelet S1P secretion during platelet activation and the consequences for monocyte chemotaxis. Methods: S1P was detected using thin-layer chromatography in [ 3 H]sphingosine-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays. Results: Release of S1P from platelets was stimulated with protease-activated receptor-1-activating peptide (PAR-1-AP, 100 lM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S1P release. Oral ASA (500-mg single dose or 100 mg over 3 days) attenuated S1P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S1P release was increased by the TX receptor (TP) agonist U-46619, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S1P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-1-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S1P-dependent manner via S1P receptors-1 and -3. These effects were inhibited by ASA-pretreatment of platelets. Conclusions: TX synthesis and TP activation mediate S1P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

Objectives This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)–induced myocarditis. Background An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ). Methods To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 105 plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry. Results Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction. Conclusions PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus.

The emission of composite-particles is studied in the reaction p+Au at Ep=2.5 GeV, in addition to neutrons and protons. Most particle energy spectra feature an evaporation spectrum superimposed on an exponential high-energy, non-statistical component. Comparisons are first made with the predictions by a two-stage hybrid reaction model, where an intra-nuclear cascade (INC) simulation is followed by a statistical evaporation process.The high-energy proton component is identified as product of the fast pre-equilibrium INC, since it is rather well reproduced by the INCL2.0 intra-nuclear cascade calculations simulating the first reaction stage. The low-energy spectral components are well understood in terms of sequential particle evaporation from the hot nuclear target remnants of the fast INC. Evaporation is modeled using the statistical code GEMINI. Implementation of a simple coalescence model in the INC code can provide a reasonable description of the multiplicities of high-energy composite particles such as 2–3H and 3He. However, this is done at the expense of 1H which then fails to reproduce the experimental energy spectra

The data suggest a molecular circuit involving the MALAT1-mascRNA system, interactions between MALAT1 and NEAT1, and key immune effector molecules, cumulatively impacting upon the development of atherosclerosis. It appears reasonable to look for therapeutic targets in this circuit and to screen for anomalies in the NEAT1-MALAT1 region in humans, too, as possible novel disease risk factors.

A signaling molecule which is involved in proliferation and migration of malignant cells is the lipid mediator sphingosine-1-phosphate (S1P). There are hints for a potential role of S1P signaling in malignant brain tumors such as glioblastoma multiforme (GBM) which is characterized by a poor prognosis. Therefore, a comprehensive expression analysis of S1P receptors (S1P1-S1P5) and S1P metabolizing enzymes in human GBM (n = 117) compared to healthy brain (n = 10) was performed to evaluate their role for patient's survival. Furthermore, influence of S1P receptor inhibition on proliferation and migration were studied in LN18 GBM cells. Compared to control brain, mRNA levels of S1P1, S1P2, S1P3 and S1P generating sphingosine kinase-1 were elevated in GBM. Kaplan-Meier analyses demonstrated an association between S1P1 and S1P2 with patient's survival times. In vitro, an inhibitory effect of the SphK inhibitor SKI-II on viability of LN18 cells was shown. S1P itself had no effect on viability but stimulated LN18 migration which was blocked by inhibition of S1P1 and S1P2. The participation of S1P1 and S1P2 in LN18 migration was further supported by siRNA-mediated silencing of these receptors. Immunoblots and inhibition experiments suggest an involvement of the PI3-kinase/AKT1 pathway in the chemotactic effect of S1P in LN18 cells.In summary, our data argue for a role of S1P signaling in proliferation and migration of GBM cells. Individual components of the S1P pathway represent prognostic factors for patients with GBM. Perspectively, a selective modulation of S1P receptor subtypes could represent a therapeutic approach for GBM patients and requires further evaluation.

Sphingosine-1-phosphate (S1P) is a versatile lipid signaling molecule and key regulator in vascular inflammation. S1P is secreted by platelets, monocytes, and vascular endothelial and smooth muscle cells. It binds specifically to a family of G-protein-coupled receptors, S1P receptors 1 to 5, resulting in downstream signaling and numerous cellular effects. S1P modulates cell proliferation and migration, and mediates proinflammatory responses and apoptosis. In the vascular barrier, S1P regulates permeability and endothelial reactions and recruitment of monocytes and may modulate atherosclerosis. Only recently has S1P emerged as a critical mediator which directly links the coagulation factor system to vascular inflammation. The multifunctional proteases thrombin and FXa regulate local S1P availability and interact with S1P signaling at multiple levels in various vascular cell types. Differential expression patterns and intracellular signaling pathways of each receptor enable S1P to exert its widespread functions. Although a vast amount of information is available about the functions of S1P and its receptors in the regulation of physiological and pathophysiological conditions, S1P-mediated mechanisms in the vasculature remain to be elucidated. This review summarizes recent findings regarding the role of S1P and its receptors in vascular wall and blood cells, which link the coagulation system to inflammatory responses in the vasculature.

Altogether, Pim1 could be a novel therapeutic target, which should be further analyzed to improve the outcome of patients with aggressive GBM.

Objective-The platelet P2Y12 ADP receptor is a well-known target of thienopyridine-type antiplatelet drugs. This study is the first to describe increased transcriptional expression of a functionally active P2Y12 in response to thrombin in human vascular smooth muscle cells (SMC). Methods and Results-On exposure to thrombin, P2Y12 mRNA was transiently increased, whereas total protein and cell surface expression of P2Y12 were markedly increased within 6 hours and remained elevated over 24 hours. This effect was mediated by activation of nuclear factor B. Preincubation with thrombin significantly enhanced the efficacy of the P2Y receptor agonist 2-methylthio-ADP to induce interleukin 6 expression and SMC mitogenesis. Effects induced by 2-methylthio-ADP were prevented by RNA interference-mediated knockdown of P2Y12 and a selective P2Y12-antagonist R-138727, the active metabolite of prasugrel. In addition, positive P2Y12 immunostaining was shown in SMC of human carotid artery plaques and was found to colocalize with tissue factor, the rate-limiting factor of thrombin formation in vivo. Conclusion-These data suggest that the P2Y12 receptor not only is central to ADP-induced platelet activation but also may mediate platelet-independent responses, specifically under conditions of enhanced thrombin formation, such as local vessel injury and atherosclerotic plaque rupture. Key Words: atherosclerosis Ⅲ thrombin Ⅲ P2Y12 receptor Ⅲ inflammation Ⅲ vascular smooth muscle P 2Y12 ADP receptor antagonists, such as the thienopyridines, are widely used as antiplatelet drugs. 1,2 Besides their role as inhibitors of platelet aggregation, there is increasing evidence that these compounds also exert antiinflammatory actions. Reduced plasma levels of inflammatory markers, such as CD40 ligand, C-reactive protein, P-selectin, and platelet-leukocyte aggregates, have been shown with P2Y12 antagonists in atherothrombotic patients, 3 and platelet P2Y12 has also been shown recently to influence vessel wall responses to injury and thrombosis. 4 Thus far, these effects have been attributed solely to the antiplatelet action and have led to the concept that antiplatelet agents exert their antiinflammatory actions via platelet-related mechanisms. 5 The ADP receptor P2Y12 was originally identified in platelets and in the brain. 6 It is a member of the P2 receptor family, which consists of the ion-channel P2X and the G-protein-coupled P2Y receptors. 7 P2 receptors are activated by the adenine nucleotides ATP and ADP, which can be released from platelets, endothelial cells, sympathetic nerve terminals, and immune cells. 8 P2 receptors have been shown to be involved in a variety of inflammatory processes, such as allergen-driven lung inflammation. 9 In addition, the P2Y12 receptor has been associated with enhanced cell growth in certain brain tumors. 10 Recently, P2Y12 was found to be expressed in vascular smooth muscle cells (SMC), 11 and a role in vessel contraction was suggested. 12 However, no functional changes were seen after thienopyridine tr...