Patrizia Amadio

Cyclooxygenase-2–Derived Prostacyclin Regulates Arterial Thrombus Formation by Suppressing Tissue Factor in a Sirtuin-1–Dependent-Manner

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-&dgr; antagonist, which predisposes to arterial thrombosis, decreased SIRT1 expression and increased TF activity. Conversely, exogenous prostacyclin or peroxisome proliferator-activated receptor-&dgr; 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-&dgr; 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.

Vascular pentraxin 3 controls arterial thrombosis by targeting collagen and fibrinogen induced platelets aggregation

Aim The long pentraxin PTX3 plays a non-redundant role during acute myocardial infarction, atherosclerosis and in the orchestration of tissue repair and remodeling during vascular injury, clotting and fibrin deposition. The aim of this work is to investigate the molecular mechanisms underlying the protective role of PTX3 during arterial thrombosis. Methods and results PTX3 KO mice transplanted with bone marrow from WT or PTX3 KO mice presented a significant reduction in carotid artery blood flow following FeCl3 induced arterial thrombosis (− 80.36 ± 11.5% and − 95.53 ± 4.46%), while in WT mice transplanted with bone marrow from either WT or PTX3 KO mice, the reduction was less dramatic (− 45.55 ± 1.37% and − 53.39 ± 9.8%), thus pointing to a protective effect independent of a hematopoietic cells derived PTX3. By using P-selectin/PTX3 double KO mice, we further excluded a role for P-selectin, a target of PTX3 released by neutrophils, in vascular protection played by PTX3. In agreement with a minor role for hematopoietic cell-derived PTX3, platelet activation (assessed by flow cytometric expression of markers of platelet activation) was similar in PTX3 KO and WT mice as were haemostatic properties. Histological analysis indicated that PTX3 localizes within the thrombus and the vessel wall, and specific experiments with the N-terminal and the C-terminal PTX3 domain showed the ability of PTX3 to selectively dampen either fibrinogen or collagen induced platelet adhesion and aggregation. Conclusion PTX3 interacts with fibrinogen and collagen and, by dampening their pro-thrombotic effects, plays a protective role during arterial thrombosis.

BDNFVal66met polymorphism: a potential bridge between depression and thrombosis.

Aims Epidemiological studies strongly suggest a link between stress, depression, and cardiovascular diseases (CVDs); the mechanistic correlation, however, is poorly understood. A single-nucleotide polymorphism in the BDNF gene (BDNFVal66Met), associated with depression and anxiety, has been proposed as a genetic risk factor for CVD. Using a knock-in mouse carrying the BDNFVal66Met human polymorphism, which phenocopies psychiatric-related symptoms found in humans, we investigated the impact of this SNP on thrombosis. Methods and results BDNFMet/Met mice displayed a depressive-like phenotype concomitantly with hypercoagulable state and platelet hyperreactivity. Proteomic analysis of aorta secretome from BDNFMet/Met and wild-type (WT) mice showed differential expression of proteins involved in the coagulation and inflammatory cascades. The BDNF Met allele predisposed to carotid artery thrombosis FeCl3-induced and to death after collagen/epinephrine injection. Interestingly, transfection with BDNFMet construct induced a prothrombotic/proinflammatory phenotype in WT cells. SIRT1 activation, using resveratrol and/or CAY10591, prevented thrombus formation and restored the physiological levels of coagulation and of platelet markers in BDNFMet/Met mice and/or cells transfected with the Met allele. Conversely, inhibition of SIRT1 by sirtinol and/or by specific siRNA induced the prothrombotic/proinflammatory phenotype in WT mice and cells. Finally, we found that BDNF Met homozygosity is associated with increased risk of acute myocardial infarction (AMI) in humans. Conclusion 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 regulates the expression and activity of microsomal prostaglandin E synthase-1: role of prostacyclin and NADPH-oxidase

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 (2 μg/L) up‐regulates PGE2 production and mPGES‐1 expression, reaching a plateau at 4–6 h, but down‐regulates prostacyclin (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 negatively modulates mPGES‐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.—Barbieri, S. S., Amadio, P., Gianellini, S., Zacchi, E., Weksler, B. B., Tremoli, E. Tobacco smoke regulates the expression and activity of microsomal prostaglandin E synthase‐1: role of prostacyclin and NADPH‐oxidase. FASEB J. 25, 3731–3740 (2011). www.fasebj.org

Role of thromboxane-dependent platelet activation in venous thrombosis: Aspirin effects in mouse model.

Recent trials suggest that Aspirin (ASA) reduces the incidence of venous thromboembolism in human. However, the molecular mechanisms underlying this effect are still unclear. In this study we assessed the effects of ASA in venous thrombosis mouse model induced by inferior vena cava (IVC) ligation and we investigated the mechanisms responsible for this effect. ASA (3mg/kg daily for 2 days) treatment decreased the thrombus size, the amounts of tissue factor activity in plasma microvesicles (TF-MP) and the levels of 2,3-dinor Thromboxane B2 (TXB-M) in urine compared to control mice. Interestingly, the thrombus size positively correlated with both TF-MP activity and TXB-M. In addition, positive correlation was observed between TF-MP activity and TXB-M. A reduced number of neutrophils and monocytes, and of TF-positive cells accompanied to a lower amount of fibrin and neutrophil extracellular traps (NETs) were also found in thrombi of ASA-treated mice. Similar results were obtained when mice were treated 24h before IVC ligation with SQ29548 (1mg/kg), a selective thromboxane receptor antagonist. In addition, transfusion of platelets in SQ29548 treated-mice excluded the likelihood of a redundant role of platelet-TP receptor in this context. Finally, incubation of macrophages and neutrophils with SQ29548 prevented TF activity and/or NETs formation induced by supernatant of activated platelets or by IBOP, a selective thromboxane analogue. In conclusion, ASA, suppressing TXA2, prevents macrophages and neutrophils activation and markedly reduces thrombus size with a mechanism most likely dependent of the inhibition of TF activity and NETs formation. These results provide a new link between platelet-produced thromboxane and the occurrence of venous thrombosis.

Production of prostaglandin E2 induced by cigarette smoke modulates tissue factor expression and activity in endothelial cells

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:IC5o ~ 1 μM, AH6809:IC5o ~ 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.—Amadio, P., Baldassarre, D., Tarantino, E., Zacchi, E., Gianellini, S., Squellerio, L., Amato, M., Weksler, B. B., Tremoli, E., Barbieri, S. S. Production of prostaglandin E2 induced by cigarette smoke modulates tissue factor expression and activity in endothelial cells. FASEB J. 29, 4001‐4010 (2015). www.fasebj.org

Effect of cigarette smoke on monocyte procoagulant activity: Focus on platelet-derived brain-derived neurotrophic factor (BDNF)

Abstract Cigarette smoke (CS) activates platelets, promotes vascular dysfunction, and enhances Tissue Factor (TF) expression in blood monocytes favoring pro-thrombotic states. Brain-derived neurotrophic factor (BDNF), a member of the family of neurotrophins involved in survival, growth, and maturation of neurons, is released by activated platelets (APLTs) and plays a role in the cardiovascular system. The effect of CS on circulating levels of BDNF is controversial and the function of circulating BDNF in atherothrombosis is not fully understood. Here, we have shown that human platelets, treated with an aqueous extract of CS (CSE), released BDNF in a dose-dependent manner. In addition, incubation of human monocytes with BDNF or with the supernatant of platelets activated with CSE increased TF activity by a Tropomyosin receptor kinase B (TrkB)-dependent mechanism. Finally, comparing serum and plasma samples of 12 male never smokers (NS) and 29 male active smokers (AS) we observed a significant increase in microparticle-associated TF activity (MP-TF) as well as BDNF in AS, while in serum, BDNF behaved oppositely. Taken together these findings suggest that platelet-derived BDNF is involved in the regulation of TF activity and that CS plays a role in this pathway by favoring a pro-atherothrombotic state.

Abnormal megakaryopoiesis and platelet function in cyclooxygenase-2-deficient mice

Previous studies suggest that cyclooxygenase-2 (COX-2) might influence megakaryocyte (MK) maturation and platelet production in vitro. Using a gene deletion model, we analysed the effect of COX-2 deficiency on megakaryopoiesis and platelet function. COX-2-/- mice (10-12 weeks old) have hyper-responsive platelets as suggested by their enhanced aggregation, TXA2 biosynthesis, CD62P and CD41/CD61 expression, platelet-fibrinogen binding, and increased thromboembolic death after collagen/epinephrine injection compared to wild-type (WT). Moreover, increased platelet COX-1 expression and reticulated platelet fraction were observed in COX-2-/- mice while platelet count was similar to WT. MKs were significantly reduced in COX-2-/- bone marrows (BMs), with high nuclear/cytoplasmic ratios, low ploidy and poor expression of lineage markers of maturation (CD42d, CD49b). However, MKs were significantly increased in COX-2-/- spleens, with features of MK maturation markers which were not observed in MKs of WT spleens. Interestingly, the expression of COX-1, prostacyclin and PGE2 synthases and prostanoid pattern were modified in BMs and spleens of COX-2-/- mice. Moreover, COX-2 ablation reduced the percentage of CD49b+ cells, the platelet formation and the haematopoietic stem cells in bone marrow and increased their accumulation in the spleen. Splenectomy decreased peripheral platelet number, reverted their hyper-responsive phenotype and protected COX-2-/- mice from thromboembolism. Interestingly, fibrosis was observed in spleens of old COX-2-/- mice (28 weeks old). In conclusion, COX-2 deletion delays BM megakaryopoiesis promoting a compensatory splenic MK hyperplasia, with a release of hyper-responsive platelets and increased thrombogenicity in vivo. COX-2 seems to contribute to physiological MK maturation and pro-platelet formation.

Prostaglandin-endoperoxide synthase-2 deletion affects the natural trafficking of Annexin A2 in monocytes and favours venous thrombosis in mice

Deep-vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism (VTE) and death. The role of prostaglandin-endoperoxide synthase (PTGS)2 in arterial thrombosis has been well established, whereas its impact in venous thrombosis remains unclear. Here, we showed that PTGS2 deletion predisposes to venous thrombosis as suggested by greater clot firmness and clot elasticity, by higher plasma levels of functional fibrinogen, factor VIII and PAI-1 activity, and proved by bigger thrombi detected after inferior vena cava ligation (IVCL) compared to WT mice. PTGS2-/- thrombi have greater fibrin content, higher number of F4/80+, TF+ and ANXA2+ cells, and lower S100A10+ cells. Remarkably, monocyte depletion reduced thrombus size in mutant mice, suggesting an important role of PTGS2-/- monocytes in this experimental setting. Interestingly, PTGS2 deletion reduced membrane ANXA2, and total S100A10, promoted assembly of ANXA2/p50NF-kB complex and its nuclear accumulation, and induced TF in peritoneal macrophages, whereas ANXA2 silencing decreased dramatically TF. Finally, Carbaprostacyclin treatment prevented venous thrombus formation induced by IVCL in mutant mice, reduced the ANXA2 binding to p50NF-kB subunit and its nuclear trafficking, and decreased TF in PTGS2-/- macrophages. PTGS2 deletion, changing the natural distribution of ANXA2 in monocytes/macrophages, increases TF expression and activity predisposing to venous thrombosis. Interestingly, Carbaprostacyclin treatment, inhibiting nuclear ANXA2 trafficking, controls monocyte TF activity and prevents DVT occurrence. Our data are of help in elucidating the mechanisms by which PTGS2 inhibition increases DVT risk, and suggest a new role for ANXA2 in venous thrombosis.

Apocynin Prevents Abnormal Megakaryopoiesis and Platelet Activation Induced by Chronic Stress

Environmental chronic stress (ECS) has been identified as a trigger of acute coronary syndromes (ACS). Changes in redox balance, enhanced reactive oxygen species (ROS) production, and platelet hyperreactivity were detected in both ECS and ACS. However, the mechanisms by which ECS predisposes to thrombosis are not fully understood. Here, we investigated the impact of ECS on platelet activation and megakaryopoiesis in mice and the effect of Apocynin in this experimental setting. ECS induced by 4 days of forced swimming stress (FSS) treatment predisposed to arterial thrombosis and increased oxidative stress (e.g., plasma malondialdehyde levels). Interestingly, Apocynin treatment prevented these alterations. In addition, FSS induced abnormal megakaryopoiesis increasing the number and the maturation state of bone marrow megakaryocytes (MKs) and affecting circulating platelets. In particular, a higher number of large and reticulated platelets with marked functional activation were detected after FSS. Apocynin decreased the total MK number and prevented their ability to generate ROS without affecting the percentage of CD42d+ cells, and it reduced the platelet hyperactivation in stressed mice. In conclusion, Apocynin restores the physiological megakaryopoiesis and platelet behavior, preventing the detrimental effect of chronic stress on thrombosis, suggesting its potential use in the occurrence of thrombosis associated with ECS.