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Prenylcysteine Oxidase 1 (PCYOX1) is an enzyme involved in the degradation of prenylated proteins. It is expressed in different tissues including vascular and blood cells. We recently showed that the secretome from Pcyox1 -silenced cells reduced platelet adhesion both to fibrinogen and endothelial cells, suggesting a potential contribution of PCYOX1 into thrombus formation. Here, we show that in vivo thrombus formation after FeCl 3 injury of the carotid artery was delayed in Pcyox1 -/- mice, which were also protected from collagen/epinephrine induced thromboembolism. The Pcyox1 -/- mice displayed normal blood cells count, vascular procoagulant activity and plasma fibrinogen levels. Deletion of Pcyox1 reduced the platelet/leukocyte aggregates in whole blood, as well as the platelet aggregation, the alpha granules release, and the α IIb β 3 integrin activation in platelet-rich plasma, in response to adenosine diphosphate (ADP) or thrombin receptor agonist peptide (TRAP). Washed platelets from the Pcyox1 -/- and WT animals showed similar phosphorylation pathway activation, adhesion ability and aggregation. The presence of Pcyox1 -/- plasma impaired agonist-induced WT platelet aggregation. Our findings show that the absence of PCYOX1 results in platelet hypo-reactivity and impaired arterial thrombosis, and indicates that PCYOX1 could be a novel target for antithrombotic drugs.

Cigarette smoke (CS) increases the incidence of atherothrombosis, the release of prostaglandin (PG) E2, and the amount of tissue factor (TF). The link between PGE2 and TF, and the impact of this interaction on CS-induced thrombosis, is unknown. Plasma from active smokers showed higher concentration of PGE2, TF total antigen, and microparticle-associated TF (MP-TF) activity compared with never smokers. Similar results were obtained in mice and in mouse cardiac endothelial cells (MCECs) after treatment with aqueous CS extracts (CSEs) plus IL-1β [CSE (6.4 puffs/L)/IL-1β (2 μg/L)]. A significant correlation between PGE2 and TF total antigen or MP-TF activity were observed in both human and mouse plasma or tissue. Inhibition of PGE synthase reduced TF in vivo and in vitro and prevented the arterial thrombosis induced by CSE/IL-1β. Only PG E receptor 1 (EP1) receptor antagonists (SC51089:IC50 ∼ 1 μM, AH6809:IC50 ∼ 7.5 μM) restored the normal TF and sirtuin 1 (SIRT1) levels in MCECs before PGE2 (EC50 ∼ 2.5 mM) or CSE/IL-1β exposure. Similarly, SIRT1 activators (CAY10591: IC50 ∼ 10 μM, resveratrol: IC50 ∼ 5 μM) or prostacyclin analogs (IC50 ∼ 5 μM) prevented SIRT1 inhibition and reduced TF induced by CSE/IL-1β or by PGE2. In conclusion, PGE2 increases both TF expression and activity through the regulation of the EP1/SIRT1 pathway. These findings suggest that EP1 may represent a possible target to prevent prothrombotic states., (© FASEB.)

Brain-derived neurothrophic factor (BDNF) is a neurotrophin expressed in different tissues and cells, including neurons, endothelial cells, leukocytes, megakaryocytes and platelets. Modifications of BDNF in plasma and/or in serum are associated with neurodegenerative and psychiatric disorders, cardiovascular diseases, metabolic syndrome and with mortality risk. Indeed, changes in blood levels of BDNF may reflect those of its tissue of origin and/or promote pathological dysfunctions. The measurement of BDNF amount in plasma or in serum has been characterized with particular attention in the impact of different anti-coagulants, clotting duration, temperature (≲21 °C) and delay in blood sample centrifugation as well as in stability of storage. However, the influences of normothermic conditions (37 °C) and of clotting duration on BDNF levels in human serum have not been investigated yet. Here, we showed that time and temperature during serum preparation could be taken into consideration to assess the association and/or impact of BDNF levels in the occurrence of pathological conditions. [ABSTRACT FROM AUTHOR]

Cigarette smoke (CS) increases the incidence of atherothrombosis, the release of prostaglandin (PG) E2, and the amount of tissue factor (TF). The link between PGE2and TF, and the impact of this interaction on CS-induced thrombosis, is unknown. Plasma from active smokers showed higher concentration of PGE2, TF total antigen, and microparticle-associated TF (MP-TF) activity compared with never smokers. Similar results were obtained in mice and in mouse cardiac endothelial cells (MCECs) after treatment with aqueous CS extracts (CSEs) plus IL-1β [CSE (6.4 puffs/L)/IL-1β (2 µg/L)]. A significant correlation between PGE2 and TF total antigen or MP-TF activity were observed in both human and mouse plasma or tissue. Inhibition of PGE synthase reduced TF in vivo and in vitro and prevented the arterial thrombosis induced by CSE/IL-1β. Only PG E receptor 1 (EP1) receptor antagonists (SC51089:IC50 ~ 1 µM, AH6809:IC50 ~ 7.5 µM) restored the normal TF and sirtuin 1 (SIRT1) levels in MCECs before PGE2 (EC50 ~ 2.5 mM) or CSE/IL-1β exposure. Similarly, SIRT1 activators (CAY10591: IC50 ~ 10 µM, resveratrol: IC50 ~ 5 µM) or prostacyclin analogs (IC50 ~ 5 µM) prevented SIRT1 inhibition and reduced TF induced by CSE/IL-1β or by PGE2. In conclusion, PGE2 increases both TF expression and activity through the regulation of the EP1/SIRT1 pathway. These findings suggest that EP1 may represent a possible target to prevent prothrombotic states. [ABSTRACT FROM AUTHOR]

Tobacco smoke (TS) interacts with interleukin-1β (IL-1β) to modulate generation of reactive oxygen species (ROS) and expression of cyclooxygenase-2. We explored molecular mechanisms by which TS/IL-1β alters expression and activity of microsomal-prostaglandin E synthase-1 (mPGES-1) and of prostacyclin synthase (PGIS) in mouse cardiac endothelial cells. TS (EC50 ~5 puffs/L) interacting with IL-1β (2µg/L) upregulates PGE2 production and mPGES-1 expression, reaching a plateau at 4-6 h, but down-regulates pro- stacyclin (PGI2) release by increasing IL-1β-mediated PGIS tyrosine nitration. Inhibition of NADPH-oxidase, achieved pharmacologically and/or by silencing its catalytic subunit p47phox, or exogenous PGI2 (carbaprostacyclin; IC50 ~5 µM) prevents production of both ROS and PGE2, and negativelymodulatesmPGES-1 expression induced by TS/IL-1β. Moreover, inhibiting PGI2, either using PGIS siRNA and/or CAY10441 (EC50 ~20 nM), a PGI2 receptor antagonist, increases NADPH-oxidase activation, mPGES-1 synthesis, and PGE2 production. Finally, lower PGI2 levels associated with higher PGIS tyrosine nitration, p47phox translocation to the membrane (an index of activation of NADPH-oxidase), and mPGES-1 expression and activity were detected in cardiovascular tissues of ApoE-/- mice exposed to cigarette smoke compared to control mice. In conclusion, cigarette smoke in association with cytokines alters the balance between PGI2/PGE2, reducing PGI2 production and increasing synthesis and activity of mPGES-1 via NADPH-oxidase activation, predisposing to development of pathological conditions. [ABSTRACT FROM AUTHOR]

Aims Cigarette smoking engenders inflammation and endothelial dysfunction, processes implicated in atherothrombotic disease. We hypothesized that an interaction between inflammatory cytokines in smokers' blood and circulating components of cigarette smoke is necessary to induce reactive oxygen species (ROS) and cyclooxygenase-2 (COX-2) in endothelium. We then explored the molecular mechanisms involved in these effects. Methods and results Serum from nine healthy active smokers (AS) compared with serum from nine non-smokers (NS) showed higher levels of interleukin-1beta (IL-1β) and tumour necrosis factor-alpha (TNF-α) and a greater ability to induce ROS production, p47phox translocation to the plasma membrane, and COX-2 mRNA and protein expression in endothelial cells (ECs). Similar results were obtained in vivo and in vitro after treatment with aqueous extracts of cigarette smoke plus IL-1β and TNF-α(TS/IL-1β/TNF-α). In ECs increased ROS production and COX-2 mRNA induced by serum from AS correlated positively with their serum levels of IL-1β and TNF-α. Moreover, a positive correlation was observed between ROS generation and COX-2 mRNA. Simultaneous immuno-neutralization of IL-1β and TNF-α prevented endothelial dysfunction induced by serum from AS. Inhibitors of NADPH oxidase and/or p47phox siRNA diminished ROS production and COX-2 expression as well as phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and Akt mediated either by AS serum or by TS/IL-1β/TNF-α. Finally, direct inhibition of p38MAPK and Akt activity also abolished COX-2 expression mediated by both types of stimuli. Our results suggest a crucial role played by interactions between inflammatory cytokines and tobacco smoke in the induction of endothelial dysfunction. [ABSTRACT FROM AUTHOR]