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.

Background-Selective inhibitors of cyclooxygenase (COX)-2 increase the risk of myocardial infarction and thrombotic events, but the responsible mechanisms are not fully understood. Methods and Results-We found that ferric chloride-induced arterial thrombus formation was significantly greater in COX-2 knockout compared with wild-type mice. Cross-transfusion experiments excluded the likelihood that COX-2 knockout platelets, despite enhanced aggregation responses to collagen and thrombin, are responsible for increased arterial thrombus formation in COX-2 knockout mice. Importantly, we observed that COX-2 deletion decreased prostacyclin synthase and production and peroxisome proliferator-activated receptor-and sirtuin-1 (SIRT1) expression, with consequent increased upregulation of tissue factor (TF), the primary initiator of blood coagulation. Treatment of wild-type mice with a prostacyclin receptor antagonist or a peroxisome proliferator-activated receptor-␦ antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-␦ agonist completely reversed the thrombotic phenotype in COX-2 knockout mice, restoring normal SIRT1 levels and reducing TF activity. Furthermore, inhibition of SIRT1 increased TF expression and activity and promoted generation of occlusive thrombi in wild-type mice, whereas SIRT1 activation was sufficient to decrease abnormal TF activity and prothrombotic status in COX-2 knockout mice. Conclusions-Modulation of SIRT1 and hence TF by prostacyclin/peroxisome proliferator-activated receptor-␦ pathways not only represents a new mechanism in controlling arterial thrombus formation but also might be a useful target for therapeutic intervention in the atherothrombotic complications associated with COX-2 inhibitors. (Circulation. 2012; 126:1373-1384.)Key Words: blood coagulation Ⅲ carotid arteries Ⅲ prostacyclin Ⅲ signal transduction Ⅲ thrombosis P rostanoids are a family of bioactive lipid mediators formed from arachidonic acid by cyclooxygenases (COXs; COX-1 and COX-2). 1 Prostanoids are involved in a variety of physiological activities, including platelet aggregation, vasorelaxation and vasoconstriction, local inflammatory response, and leukocyte-endothelial cell adhesion. 2,3 In this context, prostanoids modulate the pathogenesis of vascular diseases such as arterial thrombosis and atherosclerosis. 4 The role of COX-2 in atherothrombosis is complex. This enzyme generates not only proinflammatory, but also anti-inflammatory prostanoids, such as prostacyclin (PGI 2 ), which, under certain conditions, may counteract platelet-derived thromboxane (TXA 2 ). 5 PGI 2 is a potent vasodilator and an inhibitor of platelet aggregation, leukocyte adhesion, and vascular smooth muscle cell proliferation. 6 These actions of PGI 2 are mediated through specific cell surface receptors known as PGI 2 receptors (IPs). Each IP is a 7-membrane-spanning G-protein-coupled receptor that elicits cAMP p...

Activation of platelets, alteration in coagulation pathways, and changes in vessel wall protein expression in BDNFMet/Met mice recapitulate well the features occurring in the anxiety/depression condition. Furthermore, our data suggest that the BDNFVal66Met polymorphism contribute to the individual propensity for arterial thrombosis related to AMI.

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 (EC(50) ∼5 puffs/L) interacting with IL-1β (2 μg/L) up-regulates PGE(2) production and mPGES-1 expression, reaching a plateau at 4-6 h, but down-regulates prostacyclin (PGI(2)) 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 PGI(2) (carbaprostacyclin; IC(50) ∼5 μM) prevents production of both ROS and PGE(2), and negatively modulates mPGES-1 expression induced by TS/IL-1β. Moreover, inhibiting PGI(2), either using PGIS siRNA and/or CAY10441 (EC(50) ∼20 nM), a PGI(2) receptor antagonist, increases NADPH-oxidase activation, mPGES-1 synthesis, and PGE(2) production. Finally, lower PGI(2) 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 PGI(2)/PGE(2), reducing PGI(2) production and increasing synthesis and activity of mPGES-1 via NADPH-oxidase activation, predisposing to development of pathological conditions.