Comparative study on the effect of aspirin, TP receptor antagonist and TxA2 synthase inhibitor on the vascular tone of human saphenous vein and internal mammary artery.

AIMS: Thromboxane (TxA2) is synthesized from arachidonic acid (AA) via thromboxane synthase (TxS) enzyme and induces vasoconstriction via TP receptor. Our aim is to compare the effects of aspirin, TxS inhibitor and TP receptor antagonist on vascular reactivity of bypass grafts (saphenous vein and internal mammary artery). MAIN METHODS: Using isolated organ bath, saphenous vein and internal mammary artery preparations were incubated with TP receptor antagonist, TxS inhibitor, aspirin, IP or EP4 receptor antagonist. Then prostaglandin (PG)E2, PGF2α, phenylephrine and AA were administered in concentration-dependent manner. The expression of prostanoid receptor and PGI2 synthase (PGIS) enzyme was determined by Western Blot. KEY FINDINGS: TP receptor antagonist inhibited the contraction induced by PGE2, PGF2α, and AA but not that induced by phenylephrine in both types of vessels. Aspirin increased phenylephrine-induced contraction only in internal mammary artery and decreased AA-induced contraction in saphenous vein. TxS inhibitor decreased both PGE2 and AA-induced contraction in both types of vessels. This decrease was reversed by co-incubation of TxS inhibitor and IP/EP4 receptor antagonists. The expressions of EP3 receptor and PGIS enzyme were greater in internal mammary artery compared to saphenous vein while IP and TP receptors expressed at similar levels. SIGNIFICANCE: TP receptor antagonist and TxS inhibitor are more effective to reduce contraction induced by different spasmogens in comparison to aspirin. Our results suggest that TP receptor antagonist and TxS inhibitor might have an advantage over aspirin due to their preventive effect on increased vascular reactivity observed in post-operative period of coronary artery bypass grafting.

Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo.

Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic vein blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.

Effect of pharmaceutical interventions targeting thromboxane receptors and thromboxane synthase in cardiovascular and renal diseases.

The present review focuses on the roles of thromboxane A2 (TxA2) in arterial thrombosis, atherogenesis, vascular stent-related ischemic events and renal proteinuria. Particular emphasis is laid on therapeutic interventions targeting the TxA2 (TP) receptors and TxA2 synthase (TS), including dual TP-receptor antagonists and TS inhibitors. Their significant inhibitory efficacies on arterial thrombogenesis, atherogenesis, restenosis after stent placement, vasoconstriction and proteinuria indicate novel and improved treatments for cardiovascular and selected renal diseases. New therapeutic interventions of the TxA2 pathway may also be beneficial for patients with poor biological antiplatelet drug response, for example, to aspirin and/or clopidogrel. These new TP/TS agents offer novel improved treatments to efficiently and simultaneously interfere with thrombogenesis and atherogenesis, and to enlarge the existing panel of platelet inhibitors for efficient prophylaxis and treatment of arterial thrombosis and renal proteinuria.

Antiplatelet effect of NQ12: a possible mechanism through the arachidonic acid cascade.

NQ12, an antithrombotic agent, has been reported to display a potent antiplatelet activity. This study was undertaken to reveal the effect of NQ12 on rabbit platelet aggregation and signal transduction involved in the arachidonic acid (AA) cascade. NQ12 concentration-dependently suppressed collagen-, AA-, and U46619-induced rabbit platelet aggregation, with IC(50) values of 0.71 +/- 0.2, 0.82 +/- 0.3, and 0.45 +/- 0.1 microM, respectively. In addition, the concentration-response curve of U46619 was shifted to the right after NQ12 treatment, indicating an antagonism on thromboxane (TX) A(2) receptors. The collagen-stimulated AA liberation was inhibited by NQ12 in the same pattern as its inhibition of platelet aggregation. Further study revealed that NQ12 potently suppressed AA-mediated TXA(2) formation, but had no effect on the PGD(2) production, indicating an inhibitory effect on TXA(2) synthase, which was supported by a TXA(2) synthase activity assay indicating that NQ12 concentration-dependently inhibited TXA(2) formation converted from PGH(2). On the other hand, the AA-stimulated 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formation was also suppressed by NQ12. Taken together, these results suggest that NQ12 has a potential to inhibit TXA(2) synthase activity and TXA(2) receptors, and it can modulate AA liberation as well as 12-HETE formation in platelets. This may be a convincing mechanism for the antithrombotic action of NQ12.

Radical scavenging glycoprotein inhibiting cyclooxygenase-2 and thromboxane A2 synthase from aloe vera gel.

An active glycoprotein fraction containing 58 % protein was isolated from Aloe vera gel by precipitation with 55 % ammonium sulfate followed by gel permeation using DEAE-Sephacel A-25, Sepharose 6B and Sephadex G-50 columns in a yield of 3 x 10 -3 %. The glycoprotein fraction showed a single band corresponding to a subunit of verectin at the same position when stained with both Coomassie brilliant blue and periodic acid-Schiff reagents on 18 % SDS-PAGE. The molecular weight (14 kDa) was confirmed by Sephadex G-50 column chromatography. The glycoprotein fraction showed a radical scavenging activity against superoxide anion generated by the xanthine-xanthine oxidase system as well as inhibition of cyclooxygenase-2 and reduction of thromboxane A 2 synthase level in vitro.

Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility.

Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

Effects of camonagrel, a selective inhibitor of platelet thromboxane synthase, on the platelet-subendothelium interaction.

The aim of this study was to compare the effects of a new thromboxane synthase inhibitor, camonagrel, on platelet aggregation and platelet-subendothelium interaction under flow conditions, in comparison with a standard thromboxane synthase inhibitor (dazoxiben) and a cyclooxygenase inhibitor (acetylsalicylic acid). With respect to platelet aggregation in whole blood, the 50% inhibitory concentrations (IC(50)) of camonagrel were between 318 and 797 micromol/l after induction with collagen and adenosine 5'-diphosphate, respectively. For inhibition of thromboxane B(2) synthesis, the IC(50) values were 868 +/- 68 micromol/l; prostaglandin E(2) was inhibited only by acetylsalicylic acid (IC(50) for camonagrel >2,000 micromol/l), and the leukocyte 6-keto-PGF(1alpha) level was increased by camonagrel. The greatest reduction in percentage subendothelial surface occupied by platelets (mainly in the thrombi) after blood perfusion was seen after incubation with camonagrel in the range of concentrations that inhibited collagen-induced platelet aggregation. In conclusion, camonagrel reduced platelet-subendothelium interaction under flow conditions, showing this effect in a range of concentrations lower than in inhibition of platelet aggregation.

KW-4679, an antiallergic drug, inhibits the production of inflammatory lipids in human polymorphonuclear leukocytes and guinea pig eosinophils.

The effects of (Z)-11-[(3-dimethylamino)propylidene]-6,11-dihydrodibenz [b.e.]oxepin-2-acetic acid monohydrochloride (KW-4679), an orally active antiallergic drug, on the production of platelet-activating factor (PAF), leukotriene (LT) and thromboxane (TX) induced by Ca2+ ionophore A23187 were examined. KW-4679 at 10 microM reduced the amount of cell-associated PAF by 52.8% in human polymorphonuclear leukocytes (PMNs). KW-4679 (1-100 microM) also inhibited the release of both LTB4 and TXB2, a stable metabolite of TXA2, by human PMNs in a concentration-dependent manner, but did not influence the release of beta-glucuronidase. The 50% inhibitory concentration (IC50) values for LTB4 and TXB2 release were 5.9 and 6.0 microM, respectively. In guinea pig eosinophils, KW-4679 inhibited the release of peptide LTs at a concentration higher than 10 microM (IC50 = 66.9 microM). KW-4679 failed to inhibit PAF acetyltransferase, 5-lipoxygenase and TX synthase, but inhibited the arachidonic acid release by human PMNs in a concentration-dependent manner in a similar concentration as that inhibiting production or release of lipid mediators (IC50 = 19.5 microM). These results indicate that KW-4679 suppresses LTs and TX release and PAF formation by reducing arachidonic acid release from phospholipids, probably through interference with phospholipase A2. The inhibitory action of KW-4679 on PAF, LT and TX production is a beneficial effect of an antiallergic drug.