Repurposing of a Thromboxane Receptor Inhibitor Based on a Novel Role in Metastasis Identified by Phenome-Wide Association Study.

Although new drug discoveries are revolutionizing cancer treatments, repurposing existing drugs would accelerate the timeline and lower the cost for bringing treatments to cancer patients. Our goal was to repurpose CPI211, a potent and selective antagonist of the thromboxane A2-prostanoid receptor (TPr), a G-protein-coupled receptor that regulates coagulation, blood pressure, and cardiovascular homeostasis. To identify potential new clinical indications for CPI211, we performed a phenome-wide association study (PheWAS) of the gene encoding TPr, TBXA2R, using robust deidentified health records and matched genomic data from more than 29,000 patients. Specifically, PheWAS was used to identify clinical manifestations correlating with a TBXA2R single-nucleotide polymorphism (rs200445019), which generates a T399A substitution within TPr that enhances TPr signaling. Previous studies have correlated 200445019 with chronic venous hypertension, which was recapitulated by this PheWAS analysis. Unexpectedly, PheWAS uncovered an rs200445019 correlation with cancer metastasis across several cancer types. When tested in several mouse models of metastasis, TPr inhibition using CPI211 potently blocked spontaneous metastasis from primary tumors, without affecting tumor cell proliferation, motility, or tumor growth. Further, metastasis following intravenous tumor cell delivery was blocked in mice treated with CPI211. Interestingly, TPr signaling in vascular endothelial cells induced VE-cadherin internalization, diminished endothelial barrier function, and enhanced transendothelial migration by tumor cells, phenotypes that were decreased by CPI211. These studies provide evidence that TPr signaling promotes cancer metastasis, supporting the study of TPr inhibitors as antimetastatic agents and highlighting the use of PheWAS as an approach to accelerate drug repurposing.

Neuroprotective effects of thromboxane receptor antagonist SQ 29,548 after pilocarpine-induced status epilepticus in mice.

Thromboxane A2 (TXA2) is an important eicosanoid in the cardiovascular system, and increasing evidence suggests that TXA2 receptors (TPs) and their ligands may constitute valuable tools for the development of neuroprotective drugs. However, the role of TPs on seizure-induced damage has not been investigated. Therefore, we evaluated the effects of SQ 29,548, a potent and selective TP antagonist-on neuromotor performance, neurodegeneration, reactive astrocytosis, and c-Fos protein immunoreactivity after pilocarpine-induced status epilepticus (SE) in mice. Adult C57BL/6 mice received intracerebroventricular SQ 29,548 injections 90 min and 24 h after pilocarpine-induced SE. We found that SQ 29,548 prevented the impairment of neuromotor performance (Neuroscore test) 48 h after pilocarpine-induced SE. Data analysis suggested the existence of two subgroups of SQ 29,548-treated post-SE animals. Eight out of 12 SQ 29,548-treated animals displayed Neuroscore values identical to those of vehicle-treated controls, and were considered SQ 29,548 responders. However, 4 out of 12 SQ 29,548-treated animals did not show any improvement in Neuroscore values, and were considered SQ 29,548 non-responders. Treatment with SQ 29,548 attenuated SE-induced increase in the number of FJC- or GFAP-positive cells in the hippocampus of SQ 29,548 responders. In addition, SQ 29,548 prevented the SE-elicited increase of c-Fos immunoreactivity in the hippocampus. In summary, our results suggest that the TP antagonist (SQ 29,548) improves neurological outcome after pilocarpine-induced SE in mice. The existence of SQ 29,548 responders and non-responders was suggested by results from the Neuroscore test. Additional studies are needed to understand the mechanisms underlying these findings, as well as the potential uses of TP antagonists in the treatment of seizure-induced damage.

Sildenafil (Viagra®) Prevents Cox-1/ TXA2 Pathway-Mediated Vascular Hypercontractility in ApoE-/- Mice.

BACKGROUND/AIMS: The atherosclerotic apolipoprotein E-deficient (apoE-/-) mouse exhibits impaired vasodilation and enhanced vasoconstriction responsiveness. The objectives of this study were: a) to determine the relative contribution of cyclooxygenases (Cox-1 and Cox-2), thromboxane A2 (TXA2) and endothelin-1 (ET-1) to enhancing vascular hyperresponsiveness in this model of atherosclerosis and b) to investigate the beneficial effects of the phosphodiesterase 5 inhibitor sildenafil on this endothelial dysfunction. METHODS: Adult male apoE-/- mice were treated with sildenafil (40 mg/kg/day, for 3 weeks) and compared with non-treated ApoE-/- and wild-type mice. The beneficial effects of sildenafil on vascular contractile response to phenylephrine (PE) in aortic rings were evaluated before and after incubation with Cox-1 (SC-560) or Cox-2 (NS-398) inhibitors or the TP antagonist SQ-29548, and on contractile responsiveness to ET-1. RESULTS: ApoE-/- mice exhibited enhanced vasoconstriction to PE (Rmax ∼35%, p<0.01), which was prevented by treatment with sildenafil. The enhanced PE-induced contractions were abolished by both Cox-1 inhibition and TP antagonist, but were not modified by Cox-2 inhibition. Aortic rings from ApoE-/- mice also exhibited enhanced contractions to ET-1 (Rmax ∼30%, p<0.01), which were attenuated in sildenafil-treated ApoE-/- mice. In addition, we observed augmented levels of vascular proinflammatory cytokines in ApoE-/- mice, which were partially corrected by treatment with sildenafil (IL-6, IL-10/IL-6 ratio and MCP-1). CONCLUSION: The present data show that the Cox-1/TXA2 pathway prevails over the Cox-2 isoform in the mediation of vascular hypercontractility observed in apoE-/-mice. The results also show a beneficial effect of sildenafil on this endothelial dysfunction and on the proinflammatory cytokines in atherosclerotic animals, opening new perspectives for the treatment of other endothelium-related cardiovascular abnormalities.

Effects of BM-573 on Endothelial Dependent Relaxation and Increased Blood Pressure at Early Stages of Atherosclerosis.

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3 µM, 1 hour) and chronic (10 mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.

Multitargeting of selected prostanoid receptors provides agents with enhanced anti-inflammatory activity in macrophages.

A polypharmacologic approach to prostanoid based anti-inflammatory therapeutics was undertaken in order to exploit both the anti- and proinflammatory properties attributed to the various prostanoid receptors. Multitargeting of selected prostanoid receptors yielded a prototype compound, compound 1 (AGN 211377), that antagonizes prostaglandin D2 receptors (DPs) DP1 (49) and DP2 (558), prostaglandin E2 receptors (EPs) EP1 (266) and EP4 (117), prostaglandin F2α receptor (FP) (61), and thromboxane A2 receptor (TP) (11) while sparing EP2, EP3, and prostaglandin I2 receptors (IPs); Kb values (in nanomoles) are given in parentheses. Compound 1 evoked a pronounced inhibition of cytokine/chemokine secretion from lipopolysaccharide or TNF-α stimulated primary human macrophages. These cytokine/chemokines included cluster of designation 40 receptor (CD40), epithelial-derived neutrophil-activating protein 78 (ENA-78), granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), IL-8, IL-18, monocyte chemotactic protein-1 (CCL2) (MCP-1), tissue plasminogen activator inhibitor (PAI-1), and regulated on activation, normal T cell expressed and secreted (RANTES). In contrast, the inhibitory effects of most antagonists selective for a single receptor were modest or absent, and selective EP2 receptor blockade increased cytokine release in some instances. Compound 1 also showed clear superiority to the cyclooxygenase inhibitors diclofenac and rofecoxib. These findings reveal that blockade of multiple prostanoid receptors, with absent antagonism of EP2 and IP, may provide more effective anti-inflammatory activity than global suppression of prostanoid synthesis or highly selective prostanoid receptor blockade. These investigations demonstrate the first working example of prostanoid receptor polypharmacology for potentially safer and more effective anti-inflammatory therapeutics by blocking multiple proinflammatory receptors while sparing those with anti-inflammatory activity.

Regulation of cAMP Intracellular Levels in Human Platelets Stimulated by 2-Arachidonoylglycerol.

We demonstrated that in human platelets the endocannabinoid 2-arachidonoylglycerol (2-AG) decreased dose- and time-dependently cAMP intracellular levels. No effect on cAMP decrease induced by 2-AG was observed in the presence of the adenylate cyclase inhibitor SQ22536 as well in platelets pretreated with the thromboxane A2 receptor antagonist, SQ29548 or with aspirin, inhibitor of arachidonic acid metabolism through the cyclooxygenase pathway. An almost complete recovering of cAMP level was measured in platelets pretreated with the specific inhibitor of phosphodiesterase (PDE) 3A, milrinone. In platelets pretreated with LY294002 or MK2206, inhibitors of PI3K/AKT pathway, and with U73122, inhibitor of phospholipase C pathway, only a partial prevention was shown. cAMP intracellular level depends on synthesis by adenylate cyclase and hydrolysis by PDEs. In 2-AG-stimulated platelets adenylate cyclase activity seems to be unchanged. In contrast PDEs appear to be involved. In particular PDE3A was specifically activated, as milrinone reversed cAMP reduction by 2-AG. 2-AG enhanced PDE3A activity through its phosphorylation. The PI3K/AKT pathway and PKC participate to this PDE3A phosphorylation/activation mechanism as it was greatly inhibited by platelet pretreatment with LY294002, MK2206, U73122, or the PKC specific inhibitor GF109203X. Taken together these data suggest that 2-AG potentiates its power of platelet agonist reducing cAMP intracellular level.

Prostanoid-mediated contractions of the carotid artery become Nox2-independent with aging.

Aging is a major risk factor for carotid artery disease that may lead to stroke and dementia. Vascular effects associated with aging include increased vasomotor tone, as well as enhanced contractility to endothelial vasoconstrictor prostanoids and reduced nitric oxide (NO) bioactivity partly due to increased oxidative stress. We hypothesized that vascular NADPH oxidase (Nox)-derived superoxide may be involved in prostanoid- and NO-related functional aging. NO-mediated relaxations and prostanoid-mediated contractions to acetylcholine as well as phenylephrine-dependent contractions were investigated in the carotid artery from young (4 months) and aged mice (24 months). Gene expression of Nox subunits and endothelial NO synthase (eNOS) was determined in the carotid artery and aorta. In young mice, the thromboxane-prostanoid receptor antagonist SQ 29,548 fully blocked acetylcholine-induced contractions while reducing responses to phenylephrine by 75 %. The Nox2-targeted inhibitor Nox2ds-tat and the superoxide scavenger tempol reduced acetylcholine-stimulated, prostanoid-mediated contractions by 85 and 75 %, respectively, and phenylephrine-dependent contractions by 45 %. Unexpectedly, in aged mice, the substantial Nox2-dependent component of acetylcholine- and phenylephrine-induced, prostanoid-mediated contractions was abolished. In addition, endothelium-dependent, NO-mediated relaxations were impaired with aging. The expression of Nox subunits was greater in the aorta compared with the carotid artery, in which Nox1 was undetectable. eNOS gene expression was reduced in the aorta of aged compared to young mice. In conclusion, aging decreases prostanoid-mediated contractility in the carotid artery involving a loss of Nox2 activity and is associated with impaired endothelium-dependent, NO-mediated relaxation. These findings may contribute to a better understanding of the pathophysiology of carotid artery disease and the aging process.

Prostaglandin E2 inhibits mast cell-dependent bronchoconstriction in human small airways through the E prostanoid subtype 2 receptor.

BACKGROUND: Inhaled prostaglandin (PG) E2 might inhibit asthmatic responses, but the mechanisms involved remain undefined. OBJECTIVE: We sought to characterize the direct and indirect effects of PGE2 on human small airways with particular reference to the receptors mediating the responses. METHODS: Contraction and relaxation were studied in isolated human bronchi with an inner diameter of 1 mm or less. RESULTS: Low concentrations of PGE2 (0.01-1 µmol/L) relaxed the bronchi precontracted by histamine. The bronchodilator response was inhibited by the E prostanoid (EP) subtype 4 receptor antagonist ONO-AE3-208 but unaffected by the EP2 receptor antagonist PF-04418948. Higher concentrations of PGE2 (10-100 µmol/L) contracted the small airways. However, the TP receptor agonists U-46,619, PGF2α, and PGD2 were more potent than PGE2. Moreover, the bronchoconstrictor responses to PGE2 and all other tested prostanoids, including the EP1/EP3 receptor agonist 17-phenyl trinor PGE2 and the partial FP receptor agonist AL-8810, were uniformly abolished by the TP receptor antagonist SQ-29,548. In the presence of TP and EP4 antagonists, PGE2 inhibited the mast cell-mediated bronchoconstriction resulting from anti-IgE challenge. Measurement of the release of histamine and cysteinyl leukotrienes documented that this bronchoprotective action of PGE2 was mediated by the EP2 receptor, unrelated to bronchodilation, and increased with time of exposure. CONCLUSION: The pharmacology of PGE2 in isolated human small airways was different from its profile in animal models. This first demonstration of powerful EP2 receptor-mediated inhibition of IgE-dependent contractions in human airways introduces a new selective target for the treatment of asthma. This EP2 control of mast cell-mediated bronchoconstriction is presumably exaggerated in patients with aspirin-exacerbated respiratory disease.

Novel antiplatelet agents in acute coronary syndrome.

For more than 10 years, dual antiplatelet therapy with aspirin and clopidogrel has remained the cornerstone of treatment for patients with acute coronary syndrome (ACS). The novel oral P2Y purinoceptor 12 (P2Y12)-receptor inhibitors prasugrel and ticagrelor were approved by the FDA for clinical use in 2009 and 2011, respectively. These agents have a faster-acting, more-potent, and more-predictable antiplatelet effect than clopidogrel, which translates into improved clinical outcomes in patients with ACS, albeit at the expense of an increased risk of bleeding. However, some patients continue to experience adverse ischaemic events despite treatment with aspirin and a P2Y12-receptor antagonist, because platelets can remain activated via pathways not inhibited by these agents, such as the protease-activated receptor (PAR)-1 platelet activation pathway stimulated by thrombin. Emerging antiplatelet therapies that might address these limitations include intravenous P2Y12 antagonists, oral PAR-1 antagonists, and thromboxane-receptor inhibitors. In this Review, we provide an overview of these novel antiplatelet drugs, including newly approved agents and emerging compounds currently under clinical development, and also discuss evolving concepts and unmet needs related to antiplatelet therapy for the treatment of ACS.

Involvement of the TP receptor in TNF-α-induced endothelial tissue factor expression.

BACKGROUND: Thromboxane (TX) A2, prostaglandin endoperoxides and F2-isoprostanes exert their effects through a TX-prostanoid (TP) receptor, also expressed in endothelial cells. We investigated a role of the TP receptor in the endothelial expression of tissue factor (TF), a key trigger to thrombosis. METHODS AND RESULTS: Human umbilical vein endothelial cells (HUVEC) exposed to the TP receptor agonist U46619 featured a concentration-dependent increase in TF surface exposure and procoagulant activity. HUVEC pre-incubation with the TP receptor antagonist S18886, followed by stimulation with either U46619 or tumor necrosis factor-α (TNF-α), attenuated TF surface exposure and activity compared with stimulated control. Aspirin or indomethacin, while inhibiting cyclooxygenase (COX)-1 and -2 activities, did not mimic this effect. Probing of underlying mechanisms by selective pharmacological and gene silencing experiments showed that S18886 reduced U46619- or TNF-α-induced TF expression inhibiting ROS production, NAD(P)H oxidase and PKC activation. In addition, S18886 also inhibited ERK activation in the presence of both U46619 and TNF-α alone, while inhibition of JNK activation only occurred in the presence of U46619. CONCLUSION: The endothelial TP receptor contributes to TF surface exposure and activity induced not only by known TP receptor agonists, but also by TNF-α. Such findings expand the therapeutic potential of TP receptor inhibition.

Thromboxane A2 exerts promoting effects on cell proliferation through mediating cyclooxygenase-2 signal in lung adenocarcinoma cells.

BACKGROUND: Lung cancer concerns a worldwide health problem and the efficacy of available treatments is unsatisfactory. Recently, thromboxane A2 (TXA2) synthase (TXAS) and receptor (TXA2R) have been documented to play a role in lung cancer development. Therefore, dual TXA2R modulator (i.e., the dual blocker of TXAS and TXA2R) may be more efficacious to kill lung tumor cells than single TXAS inhibitor or TXA2R antagonism. The close relationship between cyclooxygenase (COX)-2 and TXAS also raises whether or how TXA2 contributes to the oncogenic activity of COX-2. This study is therefore conducted to answer these questions. METHODS: Various inhibitors and siRNA were used to evaluate the roles of TXA2 and COX-2 in the proliferation and apoptosis of lung adenocarcinoma cells. Cell proliferation was detected using both MTS ELISA and BrdU labeling ELISA. Cell cycle distribution and apoptosis were examined by flow cytometric analysis. TXB2 level, reflecting the biosynthesis of TXA2, was detected by peroxidase-labeled TXB2 conjugates using an enzyme immunoassay kit. Western blotting was performed to evaluate many biomarkers for cell cycles, apoptosis and proliferation. The levels of COXs were screened by reverse transcriptase and real-time quantitative PCR. RESULTS: We found either single TXAS inhibitor/TXA2R antagonist or the dual TXA2 modulators offered a similar inhibition on cell proliferation. Moreover, inhibition of TXA2 arrested cells at the G2/M phase and induced apoptosis. It is further demonstrated that TXA2 was able to function as a critical mediator for tumor-promoting effects of COX-2 in lung adenocarcinoma cells. CONCLUSION: The present study has for the first shown that dual TXA2 modulators and the single blocker of TXAS or TXA2R offer a similar inhibitory role in lung adenocarcinoma cell proliferation and that the tumor-promoting effects of COX-2 can largely be relayed by TXA2. Thus, TXA2 should be regarded as a critical molecule in COX-2-mediated tumor growth and a valuable target against lung cancer.

Pharmacodynamic effects of EV-077 in patients with diabetes mellitus and coronary artery disease on aspirin or clopidogrel monotherapy: results of an in vitro pilot investigation.

Patients with diabetes mellitus (DM) have increased propensity to generate thromboxane A2 (TXA2) and other eicosanoids which can contribute to their heightened platelet reactivity. EV-077 is a potent thromboxane receptor antagonist and thromboxane synthase inhibitor and thus represents an attractive therapy in patients with DM. However, the effects of EV-077 on pharmacodynamic (PD) profiles in patients with DM and coronary artery disease (CAD) while on antiplatelet therapy is poorly explored and represented the aim of this in vitro pilot investigation. Patients with DM and stable CAD (n = 10) on low-dose aspirin (81 mg/day) were enrolled and then switched to clopidogrel (75 mg/day) monotherapy for 7-10 days. PD assessments were conducted while on aspirin and on clopidogrel using light transmittance aggregometry following stimuli with U-46619 [TXA2 stable analogue (7 µM)], arachidonic acid [AA (1 mM)], collagen (3 µg/mL) and adenosine diphosphate [ADP (5 µM and 20 µM)] with and without in vitro EV-077. EV-077 completely inhibited U-46619-stimulated platelet aggregation (p = 0.005 for both aspirin and clopidogrel) and also showed a significant reduction of collagen-induced aggregation (aspirin p = 0.008; clopidogrel p = 0.005). EV-077 significantly reduced AA-induced platelet aggregation in clopidogrel (p = 0.009), but not aspirin (p = 0.667) treated patients. Ultimately, EV-077 significantly reduced ADP-mediated platelet aggregation in both aspirin (ADP 5 µM p = 0.012; ADP 20 µM p = 0.032) and clopidogrel (ADP 5 µM p = 0.007; ADP 20 µM p = 0.008) treated patients. In conclusion, in DM patients with CAD on aspirin or clopidogrel monotherapy, in vitro EV-077 exerts potent platelet inhibitory effects on multiple platelet signaling pathways. These data support that EV-077 has only additive platelet inhibiting effects on top of standard antiplatelet therapies. These findings warrant further investigation in ex vivo settings.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells.

The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.

Thromboxane inhibitors attenuate inflammatory and fibrotic changes in rat liver despite continued ethanol administrations.

BACKGROUND: Thromboxane levels are increased in rats fed ethanol (EtOH), whereas thromboxane inhibitors reduce alcoholic liver injury. The aim of this study is to determine whether thromboxane inhibitors could attenuate the already established alcoholic liver injury. METHODS: Rats were fed EtOH and liquid diet for 6 weeks by intragastric infusion to induce liver injury after which EtOH was continued for 2 more weeks, and the rats were treated with either a thromboxane synthase inhibitor (TXSI) or a thromboxane receptor antagonist (TXRA). Liver pathology, lipid peroxidation, nuclear factor-kappa-B (NF-κB) activity, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and transforming growth factor-beta1 (TGF-ß(1) ) were evaluated. RESULTS: Administration of fish oil and EtOH caused fatty liver, necrosis, inflammation and fibrosis accompanied by increased in lipid peroxidation, NF-κB activity, and expression of TNF-α, COX-2, and TGF-ß(1) . Treatment with the thromboxane inhibitors ameliorated a certain level of the pathological and biochemical abnormalities. In particular, TXSI in addition to reducing necrosis, inflammation and fibrosis also decrease the severity of fatty liver. CONCLUSIONS: Thromboxane inhibitors attenuated the alcoholic liver injury, inflammation and fibrotic changes despite continued EtOH administration. Inhibition of the production of thromboxane by thromboxane inhibitor and receptor antagonists may be a useful treatment strategy in clinical alcoholic liver disease.

Acute effects of prostaglandin E1 and E2 on vascular reactivity and blood flow in situ in the chick chorioallantoic membrane.

The chick chorioallantoic membrane (CAM) subserves gas exchange in the developing embryo and shell-less culture affords a unique opportunity for direct observations over time of individual blood vessels to pharmacologic interventions. We tested a number of lipids including prostaglandins PGE(1&2) for vascular effects and signaling in the CAM. Application of PGE(1&2) induced a decrease in the diameter of large blood vessels and a concentration-dependent, localized, reversible loss of blood flow through small vessels. The loss of flow was also mimicked by misoprostol, an agonist for 3 of 4 known PGE receptors, EP(2-4), and by U46619, a thromboxane mimetic. Selective receptor antagonists for EP(3) and thromboxane each partially blocked the response. This is a first report of the effects of prostaglandins on vasoreactivity in the CAM. Our model allows the unique ability to examine simultaneous responses of large and small vessels in real time and in vivo.

New perspectives in antiplatelet therapy.

Platelet activation is a complex mechanism of response to vascular injury and atherothrombotic disease, leading to thrombus formation. A wide variety of surface receptors -integrins, leucine-rich family receptors, G protein coupled receptors, tyrosine kinase receptors- and intraplatelet molecules support and regulate platelet activation. They are potential targets of antiplatelet therapy for the prevention and treatment of arterial thrombosis. Despite the overall clinical benefit of established antiplatelet drugs targeting cyclooxigenase-1 (COX-1), glycoprotein integrin αIIbß3, and the purinergic P2Y(12) receptor of adenosine diphosphate, a significant proportion of treated patients continue to experience recurrent ischaemic events. This may be in partly attributed to insufficient inhibition of platelet activation. In addition, it should not be underestimated that these drugs are not immune from bleeding complications. The substantial progress in understating the regulation of platelet activation has played a key role in the development of novel antiplatelet agents. Current examples of drug under development and evaluation include: novel P2Y(12) receptor inhibitors (prasugrel, ticagrelor, cangrelor, and elinogrel), thrombin receptor PAR-1 antagonists (vorapaxar, atopaxar), new integrin glycoprotein IIb/IIIa inhibitors, and inhibitors targeting the thromboxane receptor (TP), phosphodiesterases, the collagen receptor glycoprotein VI, and intraplatelet signalling molecules. This review summarizes the mechanisms of action and current clinical evaluation of these novel antiplatelet agents.

Antiplatelet therapy in patients with diabetes mellitus.

Platelets are key players in arterial thrombosis, and oral antiplatelet therapy is a cornerstone in the treatment and prevention of cardiovascular events. However, although currently approved antiplatelet drugs have proved successful in reducing cardiovascular events, platelet-dependent thrombosis remains an important cause of morbidity and mortality in patients with coronary artery disease. It is well-known that patients with diabetes mellitus (DM) have an increased risk of cardiovascular events and, therefore, understanding the mechanism of action and safety profile of antiplatelet drugs in this high-risk population is of particular interest. There is considerable inter-individual variation in the efficacy of established antiplatelet drugs, and high on-treatment platelet reactivity is associated with an increased risk of cardiovascular events, thus prompting the search for novel drugs against platelet-dependent thrombosis. New antiplatelet treatment strategies include drugs with more efficient and reversible platelet inhibition. This review discusses selective inhibitors of the platelet cyclooxygenase enzyme, thienopyridine and non-thienopyridine inhibitors of the platelet adenosine diphosphate receptor, phosphodiesterase inhibitors, and protease-activated receptor antagonists. An overview of currently available antiplatelet drugs is provided, focusing on benefits and limitations in patients with DM. Furthermore, the rationale for new oral antiplatelet drugs under development is discussed with particular focus on the potential role of these drugs to improve cardiovascular outcomes in patients with DM.

Thromboxane prostanoid receptor activation amplifies airway stretch-activated contractions assessed in perfused intact bovine bronchial segments.

A deep inspiration (DI) produces bronchodilation in healthy individuals. Conversely, in asthmatics, DIs are less effective in producing bronchodilation and can cause more rapid airway renarrowing and even bronchoconstriction in moderate to severe asthmatics. It is noteworthy that the manner by which a DI is able to cause bronchoconstriction via a stretch-activated contraction (R(stretch)) is thought to correlate positively with airway inflammation. Asthmatic airway inflammation is associated with increased production of thromboxane A(2) (TxA(2)) and subsequent thromboxane prostanoid (TP) receptor activation, causing the heightened contractility of airway smooth muscle. In this study, we sought to investigate the effect of TxA(2) on airway R(stretch) by using bovine bronchial segments. In brief, these intact bronchial segments (2 mm in diameter) were dissected, side branches were ligated, and the tissues were mounted horizontally in an organ bath. R(stretch) was elicited by varying the transmural pressure under isovolumic conditions. Using a pharmacological approach, we showed a reduced R(stretch) response in tissues pretreated with indomethacin, a cyclooxygenase inhibitor, a result mimicked by pretreatment with the TP-selective receptor antagonist 4-(Z)-6-(2-o-chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl)hexenoic acid (ICI 192605) and the selective p42/p44 mitogen-activated protein kinase inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD 95089) and by airway epithelial denudation. 9,11-Dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619), a TP receptor agonist, elicited enhanced R(stretch) responses in a dose-dependent manner. Pretreatment with 6-isopropoxy-9-oxoxanthene-2-carboxylic acid (AH 6809), a prostaglandin E (EP) receptor 1/prostaglandin D2 (DP)-selective receptor antagonist, and 9α,15R-dihydroxy-11.ß-fluoro-15-(2,3-dihydro-1H-inden-2-yl)-16,17,18,19,20-pentanor-prosta-5Z,13E-dien-1-oic acid (AL 8810), a prostaglandin F (FP)-selective receptor antagonist, had no effect, suggesting EP, DP, and FP receptor activation is not involved in amplifying airway smooth muscle R(stretch). These data suggest a role for TP receptor activation and epithelial release of TxA(2) in amplifying airway R(stretch), thus providing novel insights into mechanisms regulating the DI-induced bronchoconstriction seen in asthmatics.

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

BACKGROUND AND PURPOSE: Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH: We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS: Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS: TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone.

Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation.

Docosahexaenoic acid (DHA), an important component of marine lipids, exhibits anti-inflammatory activity related to some of its oxygenated metabolites, such as neuroprotectin/protectin D1 [NPD1/PD1; 10(R),17(S)-dihydroxy-docosa-4Z,7Z, 11E,13E,15Z,19Z-hexaenoic acid] produced through the 15-lipoxygenase pathway. However, other metabolites from DHA can be produced through this pathway, and other polyunsaturated fatty acids (PUFAs) of nutritional value may be oxygenated as well. Their biological activities remain unknown. Isomers of protectin D1 were synthesized using soybean lipoxygenase and tested for their ability to inhibit human blood platelet aggregation. A geometric isomer called PDX, previously described with the 11E,13Z,15E geometry, instead of 11E,13E,15Z in PD1, inhibited platelet aggregation at submicromolar concentrations when induced by either collagen, arachidonic acid, or thromboxane. The inhibition occurred at the level of both the cyclooxygenase activity and thromboxane receptor site. Interestingly, all the metabolites tested exhibiting the E,Z,E-conjugated triene were active, whereas E,E,Z trienes (as in PD1) or all-trans (E,E,E) trienes were inactive. We conclude that PDX and other oxygenated products from PUFAs of nutritional interest, having the E,Z,E-conjugated triene motif and collectively named poxytrins (PUFA oxygenated trienes), might have antithrombotic potential.