Thromboxane Receptor Antagonist Research Articles

Platelet aggregation response to cyclooxygenase inhibition and thromboxane receptor antagonism using impedance aggregometry: A pilot study.

Impedance aggregometry is an alternative to light transmission aggregometry that allows analysis of platelet function in whole blood samples. We hypothesized (1) impedance aggregometry would produce repeatable results, (2) inhibition of cyclooxygenase with aspirin would attenuate aggregation responses to collagen and abolish the aggregation response to arachidonic acid (AA), and (3) thromboxane receptor antagonism (terutroban) would attenuate the aggregation response to AA. Venous blood was obtained from 11 participants three times separated by at least 2 weeks. One sample followed 7-day-aspirin intervention (81 mg once daily; ASA), the others no intervention (control). Aggregation was induced using 1 μg/mL collagen ([col 1]), 5 μg/mL collagen ([col 5]), and 50 mM AA via impedance aggregometry to determine total aggregation (AUC) analyzed for intra-test repeatability, inter-test repeatability, intervention (ASA or control), and incubation (saline or terutroban). [col 1] showed high intra-test (p ≤ 0.03 visit 1 and 2) and inter-test repeatability (p < 0.01). [col 5] and AA showed intra- ([col 5] p < 0.01 visit 1 and 2; AA p < 0.001 visit 1 and 2) but not inter-test repeatability ([col 5] p = 0.48; AA p = 0.06). ASA attenuated AUC responses to [col 1] (p < 0.01), [col 5] (p = 0.03), and AA (p < 0.01). Terutroban attenuated AUC in response to AA (p < 0.01). [col 1] shows sufficient repeatability for longitudinal investigations of platelet function. [col 5] and AA may be used to investigate mechanisms of platelet function and metabolism at a single time point.

Abstract 2097: Jak2 V617f Clonal Hematopoiesis Promotes Arterial Thrombosis Via Platelet Activation And Cross-talk

JAK2 V617F ( JAK2 VF ) clonal hematopoiesis (CH) is associated with a marked increase in atherothrombotic cardiovascular disease (CVD). The role of JAK2 VF CH in arterial thrombosis has not been assessed. Using mice simulating JAK2 VF CH, we showed accelerated carotid artery thrombosis, with increased platelet counts and activation and increased leukocytes and red blood cells. There was increased platelet activation in both Jak2 VF and wild type (WT) platelets in Jak2 VF CH suggesting cross-talk between mutant and WT platelets. To assess platelet-specific effects, we used Gp1ba-Cre (VFGp1ba) to selectively express Jak2 VF in platelets. VFGp1ba increased platelet counts to a similar level as in 20% Jak2 VF CH mice but had no effect on leukocyte counts. Like Jak2 VF CH, VFGp1ba increased platelet activation and accelerated arterial thrombosis. Mechanistically, Jak2 VF platelets showed 2.8-fold up-regulation of COX-1, increased activation of cytosolic phospholipase A2, an enzyme important for arachidonic acid generation, and increased thromboxane A2 production. Conditioned media from activated Jak2 VF platelets induced WT platelet activation that was reversed by a thromboxane receptor antagonist. Low dose aspirin ameliorated carotid thrombosis in VFGp1ba and Jak2 VF CH but not in WT control mice. This study shows increased arterial thrombosis in Jak2 VF CH related to Jak2 VF platelet activation and cross-talk with WT platelets via thromboxane, suggesting a potential beneficial role of aspirin treatment in JAK2 VF CH.

Small Molecules for the Treatment of Long-COVID-Related Vascular Damage and Abnormal Blood Clotting: A Patent-Based Appraisal.

People affected by COVID-19 are exposed to, among others, abnormal clotting and endothelial dysfunction, which may result in deep vein thrombosis, cerebrovascular disorders, and ischemic and non-ischemic heart diseases, to mention a few. Treatments for COVID-19 include antiplatelet (e.g., aspirin, clopidogrel) and anticoagulant agents, but their impact on morbidity and mortality has not been proven. In addition, due to viremia-associated interconnected prothrombotic and proinflammatory events, anti-inflammatory drugs have also been investigated for their ability to mitigate against immune dysregulation due to the cytokine storm. By retrieving patent literature published in the last two years, small molecules patented for long-COVID-related blood clotting and hematological complications are herein examined, along with supporting evidence from preclinical and clinical studies. An overview of the main features and therapeutic potentials of small molecules is provided for the thromboxane receptor antagonist ramatroban, the pan-caspase inhibitor emricasan, and the sodium-hydrogen antiporter 1 (NHE-1) inhibitor rimeporide, as well as natural polyphenolic compounds.

Jak2V617F clonal hematopoiesis promotes arterial thrombosis via platelet activation and cross talk

AbstractJAK2V617F (JAK2VF) clonal hematopoiesis (CH) has been associated with atherothrombotic cardiovascular disease (CVD). We assessed the impact of Jak2VF CH on arterial thrombosis and explored the underlying mechanisms. A meta-analysis of 3 large cohort studies confirmed the association of JAK2VF with CVD and with platelet counts and adjusted mean platelet volume (MPV). In mice, 20% or 1.5% Jak2VF CH accelerated arterial thrombosis and increased platelet activation. Megakaryocytes in Jak2VF CH showed elevated proplatelet formation and release, increasing prothrombogenic reticulated platelet counts. Gp1ba-Cre–mediated expression of Jak2VF in platelets (VFGp1ba) increased platelet counts to a similar level as in 20% Jak2VF CH mice while having no effect on leukocyte counts. Like Jak2VF CH mice, VFGp1ba mice showed enhanced platelet activation and accelerated arterial thrombosis. In Jak2VF CH, both Jak2VF and wild-type (WT) platelets showed increased activation, suggesting cross talk between mutant and WT platelets. Jak2VF platelets showed twofold to threefold upregulation of COX-1 and COX-2, particularly in young platelets, with elevated cPLA2 activation and thromboxane A2 production. Compared with controls, conditioned media from activated Jak2VF platelets induced greater activation of WT platelets that was reversed by a thromboxane receptor antagonist. Low-dose aspirin ameliorated carotid artery thrombosis in VFGp1ba and Jak2VF CH mice but not in WT control mice. This study shows accelerated arterial thrombosis and platelet activation in Jak2VF CH with a major role of increased reticulated Jak2VF platelets, which mediate thromboxane cross talk with WT platelets and suggests a potential beneficial effect of aspirin in JAK2VF CH.

Effects of the thromboxane receptor antagonist S18886 in the porcine coronary circulation

Thromboxane A2 (TxA 2 ) is a potent coronary vasoconstrictor that has been implicated in promoting decreases in myocardial perfusion in a variety of (patho)-physiologic conditions. S18886 is a promising orally-active TxA 2 receptor antagonist currently approved for investigational clinical use. However, the coronary vascular effects of S18886 are unknown and its specificity and affinity for the thromboxane receptor in the coronary circulation remain unclear. We tested the hypothesis that administration of S18886 dose-dependently attenuates coronary vasoconstriction to the TxA 2 mimetic U46619 without influencing coronary responses to prostaglandin F 2α , acetylcholine, or smooth muscle depolarization (K + ). Experiments to test this hypothesis were performed in male (n = 5) and female (n = 6) domestic swine. Hearts were excised and the left circumflex coronary artery isolated, cleaned of periadventitial fat, and cut into 3 mm rings. Isometric tension of coronary artery rings was measured in response to log order increments of U46619 (1 nM to 1 μM) with and without S18886 (0.1-100 nM). Similar isometric studies were conducted with prostaglandin F 2α (10 nM-10 μM), acetylcholine (0.1-10 μM), and KCl (5-90 mM). U46619 induced concentration dependent increases in tension development of isolated coronary artery rings (average EC50 of 42 ± 19 nM). Incubation of coronary arteries with S18886 (1 nM) significantly attenuated coronary vasoconstriction to U46619 resulting in a rightward shift of the EC50 to 187 ± 38 nM (P &lt; 0.02). Vehicle had no effect on U46619-induced contractions. Higher concentrations of S18886 (10nM and 100nM) dose-dependently reduced U46619-induced contractions by 77% ± 3 and 93% ± 6 respectively. S18886 (1 nM) antagonized coronary vasoconstriction of prostaglandin F2α (10 μM) by 68% ± 5 (P &lt; 0.0001) but had no effect on either acetylcholine or KCl-induced contraction. Data from this investigation indicate that S18886 is an effective antagonist of U46619-induced vasoconstriction in the porcine coronary circulation. While S18886 does not influence coronary smooth muscle response to either acetylcholine or activation of L-type Ca 2+ channels, attenuation of prostaglandin F 2α suggests the antagonists specificity may extend beyond TxA 2 receptor signaling. NIH R01HL158723 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TXA2 mediates LPA1-stimulated uterine contraction in late pregnant mouse

Lysophosphatidic acid (LPA) is known to increase uterine contraction in the estrus cycle and early pregnancy, however, the effect of LPA in late pregnant uterus and its mechanisms are not clear. In the present study, we show the LPA receptor subtypes expressed and the mechanism of LPA-induced contractions in late pregnant mouse uterus. We determined the relative mRNA expression of LPA receptor genes by quantitative PCR and elicited log concentration-response curves to oleoyl-L-α-LPA by performing tension experiments in the presence and absence of nonselective and selective receptor antagonists and inhibitors of the TXA2 pathway. LPA1 was the most highly expressed receptor subtype in the late pregnant mouse uterus and LPA1/2/3 agonist (Oleoyl-L-α LPA) elicited increased contractions in this tissue that had lesser efficacy compared to oxytocin. LPA1/3 antagonist, Ki-16425, and a potent LPA1 antagonist (AM-095) significantly inhibited the LPA-induced contractions. Further, the nonselective COX inhibitor, indomethacin, and potent thromboxane A2 synthase inhibitor, furegrelate significantly impaired LPA-induced contractions. Moreover, selective thromboxane receptor (TP) antagonist, SQ-29548, and Rho kinase inhibitor, Y-27632 almost eliminated LPA-induced uterine contractions. LPA1 stimulation elicits contractions in the late pregnant mouse uterus using the contractile prostanoid, TXA2 and may be targeted to induce labor in uterine dysfunctions/ dystocia.

The thromboxane receptor antagonist NTP42 promotes beneficial adaptation and preserves cardiac function in experimental models of right heart overload.

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary artery pressure leading to right ventricular (RV) failure. While current PAH therapies improve patient outlook, they show limited benefit in attenuating RV dysfunction. Recent investigations demonstrated that the thromboxane (TX) A2 receptor (TP) antagonist NTP42 attenuates experimental PAH across key hemodynamic parameters in the lungs and heart. This study aimed to validate the efficacy of NTP42:KVA4, a novel oral formulation of NTP42 in clinical development, in preclinical models of PAH while also, critically, investigating its direct effects on RV dysfunction. The effects of NTP42:KVA4 were evaluated in the monocrotaline (MCT) and pulmonary artery banding (PAB) models of PAH and RV dysfunction, respectively, and when compared with leading standard-of-care (SOC) PAH drugs. In addition, the expression of the TP, the target for NTP42, was investigated in cardiac tissue from several other related disease models, and from subjects with PAH and dilated cardiomyopathy (DCM). In the MCT-PAH model, NTP42:KVA4 alleviated disease-induced changes in cardiopulmonary hemodynamics, pulmonary vascular remodeling, inflammation, and fibrosis, to a similar or greater extent than the PAH SOCs tested. In the PAB model, NTP42:KVA4 improved RV geometries and contractility, normalized RV stiffness, and significantly increased RV ejection fraction. In both models, NTP42:KVA4 promoted beneficial RV adaptation, decreasing cellular hypertrophy, and increasing vascularization. Notably, elevated expression of the TP target was observed both in RV tissue from these and related disease models, and in clinical RV specimens of PAH and DCM. This study shows that, through antagonism of TP signaling, NTP42:KVA4 attenuates experimental PAH pathophysiology, not only alleviating pulmonary pathologies but also reducing RV remodeling, promoting beneficial hypertrophy, and improving cardiac function. The findings suggest a direct cardioprotective effect for NTP42:KVA4, and its potential to be a disease-modifying therapy in PAH and other cardiac conditions.

Macrophage 12(S)-HETE Enhances Angiotensin II-Induced Contraction by a BLT2 (Leukotriene B4 Type-2 Receptor) and TP (Thromboxane Receptor)-Mediated Mechanism in Murine Arteries.

12/15-LO (12/15-lipoxygenase), encoded by Alox15 gene, metabolizes arachidonic acid to 12(S)-HETE (12-hydroxyeicosatetraenoic acid). Macrophages are the major source of 12/15-LO among immune cells, and 12/15-LO plays a crucial role in development of hypertension. Global Alox15- or macrophage-deficient mice are resistant to Ang II (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to Ang II-mediated arterial constriction and thus to development of Ang II-induced hypertension. Ang II constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nmol/L) alone was without effect; however, it significantly enhanced Ang II-induced constriction. The presence of wild-type macrophages also enhanced the Ang II-induced constriction, while Alox15-/- macrophages did not. Using this model, pretreatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-mediated, Ang II receptor-2-mediated and superoxide-mediated enhancing effect of 12(S)-HETE on Ang II constrictions. The role of superoxide was confirmed using aortas from p47phox-/- mice where 12(S)-HETE failed to enhance constriction to Ang II. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE or Ang II increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of Ang II constriction. Both Ang II-induced hypertension and the enhancing effect of 12(S)-HETE on Ang II contractions were eliminated by a BLT2 (leukotriene B4 receptor-2) antagonist. These results outline a mechanism where the macrophage 12/15-LO pathway enhances the action of Ang II. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of Ang II in part by promoting endothelial synthesis of a superoxide-derived TP agonist.

The Role of 20-HETE, COX, Thromboxane Receptors, and Blood Plasma Antioxidant Status in Vascular Relaxation of Copper-Nanoparticle-Fed WKY Rats.

Recently, the addition of copper nanoparticles (NPs) in a daily diet (6.5 mg/kg) was studied in different animal models as a possible alternative to ionic forms. Male Wistar–Kyoto rats (24-week-old, n = 11) were fed with copper, either in the form of carbonate salt (Cu6.5) or metal-based copper NPs (NP6.5), for 8 weeks. The third group was fed with a half dose of each (NP3.25 + Cu3.25). The thoracic aorta and blood plasma was studied. Supplementation with NP6.5 decreased the Cu (×0.7), Cu/Zn-ratio (×0.6) and catalase (CAT, ×0.7), and increased Zn (×1.2) and superoxide dismutase (SOD, ×1.4). Meanwhile, NP3.25 + Cu3.25 decreased the Cu/Zn-ratio (×0.7), and CAT (×0.7), and increased the daily feed intake (×1.06). Preincubation with either the selective cyclooxygenase (COX)-2 inhibitor, or the non-selective COX-1/2 inhibitor attenuated vasodilation of rat thoracic aorta in the NP6.5 group exclusively. However, an increased vasodilator response was observed in the NP6.5 and NP3.25 + Cu3.25 group of rats after preincubation with an inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) formation, and the thromboxane receptor (TP) antagonist. Significant differences were observed between the NP6.5 and NP3.25 + Cu3.25 groups of rats in: dietary intake, acetylcholine-induced vasodilation, and response to COX-inhibitors. Copper NPs in a standard daily dose had more significant effects on the mechanism(s) responsible for the utilization of reactive oxygen species in the blood plasma with the participation of prostanoids derived from COX-2 in the vascular relaxation. Dietary copper NPs in both doses modified vasodilation through the vasoconstrictor 20-HETE and the TP receptors.

The thromboxane receptor antagonist NTP42 improves haemodynamics, inhibits remodelling, and restores function within the cardiopulmonary system in preclinical pulmonary arterial hypertension models

Abstract Background Pulmonary arterial hypertension (PAH) is a rare and fatal disease characterised by a progressive increase of pulmonary artery pressure leading to right heart failure. While current PAH therapies provide symptomatic relief and some improvement in quality of life, they show limited benefit in preventing disease progression and often have severe side effects. Recent studies by us reported that a novel thromboxane A2 receptor (TP) antagonist NTP42 attenuated multiple disease hallmarks of PAH in two preclinical models of the disease. Purpose The aim of this study was to assess the efficacy of a novel oral formulation of NTP42 developed for pharmaceutical use on monocrotaline (MCT)-induced PAH in rats across a range of cardiopulmonary parameters, and when compared with standard of care (SOC) PAH therapies. It also aimed to evaluate the expression of the TP, the target receptor for NTP42, in tissues from the MCT-induced PAH model and related disease models. Methods PAH was induced by subcutaneous injection of 60 mg/kg MCT. Rats were randomly assigned to 7 groups: (1) No MCT; (2) MCT Only; (3) MCT+NTP42 Formulation (1 mg/kg); (4) MCT+Sildenafil (50 mg/kg); (5) MCT+Macitentan (30 mg/kg), (6) MCT+Selexipag (1 mg/kg), and (7) MCT+Riociguat (5 mg/kg), where in all cases twice-daily oral dosing was initiated 7 days post-MCT and continued for 22 days. Results The results show that the formulated NTP42 reduced the severity of MCT-induced PAH as determined from haemodynamic measurements of mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), Fulton's Index of cardiac hypertrophy and histological assessments of pulmonary vascular remodelling, pulmonary oedema, inflammation and fibrosis, and right ventricular fibrosis. In addition, these assessments showed that NTP42 inhibited these effects at least to a similar extent relative to the SOC drugs Sildenafil, Macitentan, Selexipag and Riociguat. Notably, unlike most of the PAH SOCs, NTP42 had no adverse effects on liver weight and or on hepatic histopathology. Importantly, NTP42 had no effects on either the systemic mean arterial pressure (mAP) or heart rate (HR). Furthermore, while abundant expression of the target TP was noted in numerous cell and tissue types of both cardiac and pulmonary tissue, there were highly significant upregulations of TP expression observed following PAH-induction (RV, &amp;gt;2-fold, p=0.0002). Conclusions Taken together, these data demonstrate that NTP42 significantly attenuates MCT-induced PAH and to a similar or greater extent relative to market leader PAH SOC drugs. Notably, we reveal that expression of the TP, the target receptor for NTP42, was shown to be highly significantly upregulated throughout the cardiopulmonary system, validating the potential importance of this largely ignored PAH target ripe for pharmaceutical intervention. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): EU Horizon 2020 SME Instrument

Phytochemical Analysis of Phenolics, Sterols, and Terpenes in Colored Wheat Grains by Liquid Chromatography with Tandem Mass Spectrometry.

The colored grain of wheat (Triticum aestivum L.) contains a large number of polyphenolic compounds that are biologically active ingredients. The purpose of this work was a comparative metabolomic study of extracts from anthocyaninless (control), blue, and deep purple (referred to here as black) grains of seven genetically related wheat lines developed for the grain anthocyanin pigmentation trait. To identify target analytes in ethanol extracts, high-performance liquid chromatography was used in combination with Bruker Daltonics ion trap mass spectrometry. The results showed the presence of 125 biologically active compounds of a phenolic (85) and nonphenolic (40) nature in the grains of T. aestivum (seven lines). Among them, a number of phenolic compounds affiliated with anthocyanins, coumarins, dihydrochalcones, flavan-3-ols, flavanone, flavones, flavonols, hydroxybenzoic acids, hydroxycinnamic acids, isoflavone, lignans, other phenolic acids, stilbenes, and nonphenolic compounds affiliated with alkaloids, carboxylic acids, carotenoids, diterpenoids, essential amino acids, triterpenoids, sterols, nonessential amino acids, phytohormones, purines, and thromboxane receptor antagonists were found in T. aestivum grains for the first time. A comparative analysis of the diversity of the compounds revealed that the lines do not differ from each other in the proportion of phenolic (53.3% to 70.3% of the total number of identified compounds) and nonphenolic compounds (46.7% to 29.7%), but diversity of the compounds was significantly lower in grains of the control line. Even though the lines are genetically closely related and possess similar chemical profiles, some line-specific individual compounds were identified that constitute unique chemical fingerprints and allow to distinguish each line from the six others. Finally, the influence of the genotype on the chemical profiles of the wheat grains is discussed.

Efficacy of the thromboxane receptor antagonist NTP42 alone, or in combination with sildenafil, in the sugen/hypoxia-induced model of pulmonary arterial hypertension

NTP42 is a novel antagonist of the thromboxane A2 receptor (TP) in development for the treatment of pulmonary arterial hypertension (PAH). Recent studies demonstrated that NTP42 and TP antagonism have a role in alleviating PAH pathophysiology. However, the efficacy of NTP42 when used in combination with existing PAH therapies has not yet been investigated. Herein, the Sugen 5416/hypoxia (SuHx)-induced PAH model was employed to evaluate the efficacy of NTP42 when used alone or in dual-therapy with Sildenafil, a PAH standard-of-care. PAH was induced in rats by injection of Sugen 5416 and exposure to hypoxia for 21 days. Thereafter, animals were treated orally twice-daily for 28 days with either vehicle, NTP42 (0.05 mg/kg), Sildenafil (50 mg/kg), or NTP42+Sildenafil (0.05 mg/kg + 50 mg/kg, respectively). While Sildenafil or NTP42 mono-therapy led to non-significant reductions in the SuHx-induced rises in mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RSVP), combined use of NTP42+Sildenafil significantly reduced these increases in mPAP and RVSP. Detailed histologic analyses of pulmonary vessel remodelling, right ventricular hypertrophy and fibrosis demonstrated that while NTP42 and Sildenafil in mono-therapy resulted in significant benefits, NTP42+Sildenafil in dual-therapy showed an even greater benefit over either drug used alone. In summary, combined use of NTP42+Sildenafil in dual-therapy confers an even greater benefit in treating or offsetting key aetiologies underlying PAH. These findings corroborate earlier preclinical findings suggesting that, through antagonism of TP signalling, NTP42 attenuates PAH pathophysiology, positioning it as a novel therapeutic for use alone or in combination therapy regimens.

The Characterization of Ground Raspberry Seeds and the Physiological Response to Supplementation in Hypertensive and Normotensive Rats.

This study aimed to evaluate the protective role of ground raspberry seeds (RBS) as a source of polyphenols and essential fatty acids on blood plasma enzymatic antioxidant status, lipid profile, and endothelium-intact vasodilation during physiological and pathological conditions. Young normotensive Wistar–Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) at ten weeks of age were fed with either a control diet or were supplemented with added 7% RBS for six weeks (n = 6). The main component of RBS was dietary fiber (64%) and the main polyphenols were ellagitannins (1.2%) and flavan-3-ols (0.45%). Irrespective of the rat model, ground RBS decreased liver enzyme aspartate aminotransferase (0.9-fold) and hydrogen peroxide scavenging capacity (Catalase, 0.9-fold). In supplemented SHRs, preincubation with inducible nitric oxide synthase (iNOS) inhibitor 1400W, nonselective cyclooxygenase (COX) inhibitor indomethacin, selective COX-2 inhibitor NS-398, prostacyclin (PGI2) synthesis inhibitor tranylcypromine (TCP), thromboxane receptor (TP) antagonist SQ-29548, thromboxane synthesis inhibitor furegrelate, and 20-HETE synthesis inhibitor HET0016 induced the same relaxant response to acetylcholine as in the nonsupplemented control group. In supplemented WKYs, atherogenic index was decreased (0.8-fold), while iNOS and COX-2-derived PGI2 increased acetylcholine-induced vasodilation. These effects of ground RBS may constitute a potential mechanism for preventing cardiovascular diseases.

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.

P3670Efficacy of the novel thromboxane receptor antagonist NTP42 alone, or in combination with Sildenafil, in the sugen/hypoxia-induced model of pulmonary arterial hypertension

Abstract Pulmonary arterial hypertension (PAH) is characterized by an elevated pulmonary vascular resistance resulting from excessive pulmonary arterial vasoconstriction and vascular remodelling, ultimately leading to right ventricular hypertrophy and right heart failure. NTP42 is a novel antagonist of the thromboxane (TX)A2 receptor (TP), currently in development for the treatment of PAH. Signalling through the TP, TXA2 is a potent vasoconstrictor, a driver of platelet aggregation and a pro-mitogenic and pro-inflammatory mediator. Moreover, the TP also mediates the actions of the isoprostane 8-iso-prostaglandin F2 alpha, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat many of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, fibrosis and inflammation. The aim of the study was to evaluate the effects of NTP42 when used alone, or as a dual-therapy in combination with a PAH standard-of-care (Sildenafil). Alongside control animals maintained in normoxia, PAH was induced in rats by injection of Sugen5416 (20 mg/kg) and exposure to hypoxia (10% O2) for 21 days. Thereafter, animals were returned to normoxia and treated for 28 days with either vehicle, NTP42 (0.05 mg/kg BID), Sildenafil (50 mg/kg BID), or NTP42+Sildenafil (0.05 mg/kg + 50 mg/kg BID, respectively). While the standard-of-care PAH drug Sildenafil or NTP42 when used in mono-therapy led to non-significant reductions in the SuHx-induced rises in mean pulmonary arterial pressure (mPAP) or right ventricular systolic pressure (RSVP), combined use of NTP42+Sildenafil in dual-therapy significantly reduced these increases in mPAP and RVSP. Moreover, NTP42+Sildenafil also significantly reduced the SuHx-induced increase in cardiac hypertrophy. Detailed morphometric analysis of vessel remodelling confirmed that while both NTP42 and Sildenafil mono-therapy resulted in significant benefits, combined use of NTP42+Sildenafil showed an even greater benefit over either drug used in mono-therapy. Moreover, a multiparameter score of key PAH cardiac and pulmonary disease indices show that NTP42+Sildenafil when used in combination results in a highly significant treatment benefit, indicating a potential synergistic effect. Assessment of key hemodynamic, cardiac hypertrophy and pulmonary vascular remodelling parameters shows equivalent or greater efficacy of the TP antagonist NTP42 compared with the standard-of-care Sildenafil when used as monotherapies in the SuHx-induced preclinical model of PAH. Combined use of both drugs in dual therapy form confirms an even greater benefit in treating or offsetting the key aetiologies underlying PAH. These findings suggest that NTP42 and antagonism of TP signalling may alleviate PAH pathophysiology, representing a novel therapeutic for use in mono- dual- or triple-therapy regimens. Acknowledgement/Funding H2020 SME-Instrument Grant 822258

Evidence for the induction of Th2 inflammation by group 2 innate lymphoid cells in response to prostaglandin D2 and cysteinyl leukotrienes in allergic rhinitis.

Group 2 innate lymphoid cells (ILC2s) play important roles in allergic inflammation. However, their roles in the pathophysiology of allergic rhinitis (AR) are poorly understood. Prevalence of ILC2s in the inferior nasal turbinate (INT) tissues and the activating mechanisms of ILC2s were examined in patients with house dust mite (HDM)-induced AR. Eighteen patients with HDM-induced AR and 13 control subjects were recruited. Fresh INT tissues and peripheral blood mononuclear cells (PBMCs) were analysed using flow cytometry. Nasal lavage fluids (NLF) were collected at 10minutes after the nasal provocation test (NPT) with HDM disc, and released mediators were measured by ELISA. Sorted ILC2s were cultured and stimulated with mediators associated with AR. The prevalence of ILC2s was significantly increased in nasal mucosa of patients with HDM-induced AR, and it was positively correlated with the number of infiltrating eosinophils. ILC2s in the INT tissues expressed a prostaglandin D2 (PGD2 ) receptor, chemoattractant receptor-homologous molecule-expressed TH2 cells (CRTH2) and a cysteinyl leukotriene (cysLTs) receptor, CysLT1. After NPT, the number of eosinophils and concentrations of PGD2 and cysLTs were significantly increased in the NLF from AR patients. PGD2 and cysLTs significantly induced IL-5 production from cultured PBMC-derived ILC2s dose-dependently. PGD2 -induced and cysLTs-induced productions of IL-5 and IL-13 from ILC2s were completely inhibited by ramatroban, a dual CRTH2 and thromboxane receptor antagonist, and montelukast, a CysLT1 antagonist, respectively. PGD2 -CRTH2 and cysLTs-CysLT1 axes may activate tissue-resident ILC2s to produce Th2 cytokines, IL-5 and IL-13, leading to the development of allergic inflammation in AR.

Detrimental effects of 2-arachidonoylglycerol on whole blood platelet aggregation and on cerebral blood flow after a focal ischemic insult in rats.

2-Arachidonoylglycerol (2-AG) is a major modulator of blood flow and platelet aggregation and a potential neuroprotectant. The present study investigated, for the first time, the effects of 2-AG on cerebral blood flow (CBF) in the first critical hours during middle cerebral artery occlusion (MCAO) and on platelet aggregation in rats. Adult male Sprague-Dawley rats ( n = 30) underwent permanent MCAO under isoflurane anesthesia and were randomly assigned to receive either 2-AG (6 mg/kg iv), monoacylglycerol lipase inhibitor JZL-184 (10 mg/kg iv), or vehicle ( n = 6 rats/group) treatment. CBF and cardiovascular responses were measured, by a blinded investigator, for up to 4 h. In separate experiments, platelet aggregation by 2-AG (19-300 µM) was assessed by whole blood aggregometry ( n = 40). 2-AG and JZL-184 significantly increased the severity of the CBF deficit versus vehicle (20.2 ± 8.8% and 22.7 ± 6.4% vs. 56.4 ± 12.1% of pre-MCAO baseline, respectively, P < 0.05) but had no effect on blood pressure or heart rate. While JZL-184 significantly increased the number of thrombi after MCAO, this did not reach significance by 2-AG. 2-AG induced platelet aggregation in rat whole blood in a similar manner to arachidonic acid and was significantly reduced by the cyclooxygenase inhibitors indomethacin and flurbiprofen and the thromboxane receptor antagonist ICI 192,605 ( P < 0.05). This is the first study showing that 2-AG increases the severity of the CBF deficit during MCAO, and further interrogation confirmed 2-AG-induced platelet aggregation in rats. These findings are important because 2-AG had previously been shown to exert neuroprotective actions and therefore force us to reevaluate the circumstances under which 2-AG is beneficial. NEW & NOTEWORTHY 2-Arachidonoylglycerol (2-AG) has neuroprotective properties; however, the present study revealed that 2-AG increases the severity of the cerebral blood flow deficit during middle cerebral artery occlusion in rats. Further interrogation showed that 2-AG induces platelet aggregation in rats. These findings force us to reevaluate the circumstances under which 2-AG is beneficial.

Nonsteroidal Anti-Inflammatory Drugs: Exploiting Bivalent COXIB/ TP Antagonists for the Control of Cardiovascular Risk.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are some of the most widely prescribed or dispensed over the counter analgesics and antipyretics that act by inhibiting prostaglandins and thromboxane synthesis. After the identification of a second isoform of COX, the pharmaceutical research focused on developing COX-2- selective drugs (COXIBs) considered as second generation NSAIDs that would retain the anti-inflammatory and analgesic activities of traditional NSAID without blunting the gastrointestinal cytoprotection sustained by COX1-derived products such as PGE2. However, while several clinical trials confirmed a gastrointestinal safer profile of COXIBs vs unselective COX inhibitors, increasing evidence for potential cardiovascular risk associated with COXIBs rapidly emerged. Today, there are no really safe NSAIDs to be used in chronic pain and anti-inflammatory treatments, as an adequate therapy associated with a minimal gastrointestinal damage and cardiovascular toxicity is yet to be developed. Here, we present evidences that combining the anti-aggregating and antiatherotrombotic activities of a thromboxane receptor antagonist with the antiinflammatory activity of a COXIB we could obtain a new multitarget drug providing protection against the harmful activities mediated by the COXIB component, yet exploiting its recognized therapeutic advantages as a gastrointestinal-safer anti-inflammatory drug. We also summarize recent progress achieved in this field of research and possible new strategies to obtain a new bivalent compound. This possible third-generation NSAID with a safer pharmacological profile, will have all the pharmacological characteristics for the long-term therapy of chronic disorders such as inflammatory diseases or selected forms of cancer.

Antagonist of thromboxane A2 receptor by SQ29548 lowers DOCA-induced hypertension in diabetic rats

Diabetes is one of high risk factors for cardiovascular diseases, including atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to hypertension in diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were monitored by western blot. Endothelial function was determined by organ bath. High glucose (HG) or thromboxane A2 mimetic U46619 significantly reduced the levels of p-eNOS and p-Akt in cultured HUVECs, which were reversed by inhibition of TPr. HG/U46619-induced reductions of p-eNOS and p-Akt were accompanied with increases of total and phosphorylated tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). PTEN siRNA restored Akt-eNOS signaling in cells treated with HG. In rats, streptozotocin-induced hyperglycemia was associated with aortic PTEN upregulation and reductions of p-Akt and p-eNOS. TPr antagonist SQ29548 ablated these alterations and reduced blood pressure in rats with DOCA-induced hypertensive. In conclusion, hyperglycemia activates thromboxane A2 receptor to augment DOCA-induced high blood pressure in rats via the PTEN-Akt-eNOS signaling.

High oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production in the rat mesenteric artery.

Whether high oxygen is harmful to the vascular function is unclear. The present study examined if high oxygen modifies vasodilator effect of cysteine via enhanced oxidative stress and thromboxane production. Rat mesenteric arteries with endothelium at 95 or 50% oxygen were subjected to isometric force recordings, measurement of thromboxane B2 levels, determination of superoxide and peroxynitrite levels and evaluation of NADPH oxidase subunit protein expression, respectively. L-cysteine (0.01-3mM) constricted or dilated arteries at 95 and 50% oxygen, respectively. Thromboxane receptor antagonist SQ-29,548 (1μM) abolished the constriction at 95% oxygen. L-cysteine (3mM) increased levels of thromboxane B2 in arteries upon 95% oxygen application. L-cysteine relaxed arteries treated with superoxide inhibitor tiron (2mM) or NADPH oxidase inhibitor gp91ds-tat (1μM) irrespective of the oxygen concentration while ATP-sensitive K(+) channel inhibitor glibenclamide (1μM) and cystathionine-γ-lyase (CSE) inhibitor DL-propargylglycine (10mM) similarly abolished the relaxation. L-cysteine (3mM) with 95% oxygen augmented levels of superoxide as well as nitrotyrosine within the artery, concomitantly with enhanced membrane protein expression of NADPH oxidase subunit p47phox. The higher concentration of oxygen attenuates L-cysteine-induced vasodilation via superoxide production mediated by NADPH oxidase along with thromboxane A2 production, resulting in vasoconstriction. The increased levels of superoxide, as well as peroxynitrite, coexist with the impaired vasodilation related to ATP-sensitive K(+) channels and CSE. Higher oxygen with plasma cysteine may cause oxidative stress and vasoconstrictor prostanoid production in blood vessels.