Thromboxane A2 Modulates de novo Synthesis of Adrenal Corticosterone in Mice via p38/14-3-3γ/StAR Signaling.

Prostanoids are endogenous lipid bioactive mediators that play essential roles in physiological processes such as glucocorticoid secretion. Here, it is found that the thromboxane (Tx)A2 receptor (TP) is highly expressed in the adrenal cortex of mice. Both global and adrenocortical-specific deletion of the TP receptor lead to increased adiposity in mice by elevating corticosterone synthesis. Mechanistically, the TP receptor deletion increases the phosphorylation of steroidogenic acute regulatory protein (StAR) and corticosterone synthesis in adrenal cortical cells by suppressing p-p38-mediated phosphorylation of 14-3-3γ adapter protein at S71. The activation of the p38 in the adrenal cortical cells by forced expression of the MKK6EE gene attenuates hypercortisolism in TP-deficient mice. These observations suggest that the TxA2/TP signaling regulates adrenal corticosterone homeostasis independent of the hypothalamic-pituitary-adrenal axis and the TP receptor may serve as a promising therapeutic target for hypercortisolism.

Doxorubicin alters G-protein coupled receptor-mediated vasocontraction in rat coronary arteries.

Doxorubicin (Doxo)-associated cardio-and vasotoxicity has been recognised as a serious complication of cancer chemotherapy. The purpose of this novel paper was to determine the effect of Doxo on G-protein coupled receptor (GPCR)-mediated vasocontraction located on vascular smooth muscle cells. Rat left anterior descending artery segments were incubated for 24 h with 0.5 µM Doxo. The vasocontractile responses by activation of endothelin receptor type A (ETA) and type B (ETB), serotonin receptor 1B (5-HT1B) and thromboxane A2 prostanoid receptor (TP) were investigated by a sensitive myography using specific agonists, while the specificity of the GPCR agonists was verified by applying selective antagonists (i.e. ETA and ETB agonist = 10- 14-10- 7.5 M endothelin-1 (ET-1); ETA antagonist = 10 µM BQ123; ETB agonists = 10- 14-10- 7.5 M sarafotoxin 6c (S6c) and ET-1; ETB antagonist = 0.1 µM BQ788; 5-HT1B agonist = 10- 12-10- 5.5 M 5-carboxamidotryptamine (5-CT); 5-HT1B antagonist = 1 µM GR55562; TP agonist = 10- 12-10- 6.5 M U46619; TP antagonist = 1 µM Seratrodast). Our results show that 0.5 µM Doxo incubation of LAD segments leads to an increased VSMC vasocontraction through the ETB, 5-HT1B and TP GPCRs, with a 2.2-fold increase in ETB-mediated vasocontraction at 10- 10.5 M S6c, a 2.0-fold increase in 5-HT1B-mediated vasocontraction at 10- 5.5 M 5-CT, and a 1.3-fold increase in TP-mediated vasocontraction at 10- 6.5 M U46619. Further studies unravelling the involvement of intracellular GPCR signalling pathways will broaden our understanding of the Doxo-induced vasotoxicity, and thus pave the way to mitigate the adverse effects by potential implementation of adjunct therapy options.

Thromboxane A2/thromboxane A2 receptor axis facilitates hepatic insulin resistance and steatosis through endoplasmic reticulum stress in non-alcoholic fatty liver disease.

BACKGROUND AND PURPOSE: Defective insulin signalling and dysfunction of the endoplasmic reticulum (ER), driven by excessive lipid accumulation in the liver, is a characteristic feature in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Thromboxane A2 (TXA2 ), an arachidonic acid metabolite, is significantly elevated in obesity and plays a crucial role in hepatic gluconeogenesis and adipose tissue macrophage polarization. However, the role of liver TXA2 /TP receptors in insulin resistance and lipid metabolism is largely unknown. EXPERIMENTAL APPROACH: TP receptor knockout (TP-/- ) mice were generated and fed a high-fat diet for 16 weeks. Insulin sensitivity, ER stress responses and hepatic lipid accumulation were assessed. Furthermore, we used primary hepatocytes to dissect the mechanisms by which the TXA2 /TP receptor axis regulates insulin signalling and hepatocyte lipogenesis. KEY RESULTS: TXA2 was increased in diet-induced obese mice, and depletion of TP receptors in adult mice improved systemic insulin resistance and hepatic steatosis. Mechanistically, we found that the TXA2 /TP receptor axis disrupts insulin signalling by activating the Ca2+ /calcium calmodulin-dependent kinase II γ (CaMKIIγ)-protein kinase RNA-like endoplasmic reticulum kinase (PERK)-C/EBP homologous protein (Chop)-tribbles-like protein 3 (TRB3) axis in hepatocytes. In addition, our results revealed that the TXA2 /TP receptor axis directly promoted lipogenesis in primary hepatocytes and contributed to Kupffer cell inflammation. CONCLUSIONS AND IMPLICATIONS: The TXA2 /TP receptor axis facilitates insulin resistance through Ca2+ /CaMKIIγ to activate PERK-Chop-TRB3 signalling. Inhibition of hepatocyte TP receptors improved hepatic steatosis and inflammation. The TP receptor is a new therapeutic target for NAFLD and metabolic syndrome.

Investigating the Mechanisms Underlying U46619-Induced Contraction on Porcine Lower Esophageal Sphincter.

Gastroesophageal reflux disease (GERD) is associated with an incompetent lower esophageal sphincter (LES), resulting in the reflux of gastric contents into the esophagus. U46619, a thromboxane A2 (TXA2) receptor agonist, induces contractions in various smooth muscles. Therefore, this study aimed to investigate the effects and mechanisms of action of U46619 on the porcine LES. To achieve this, contractions of the clasp and sling strips of the porcine LES, induced by U46619, were measured using isometric transducers. Furthermore, the contractile mechanism of U46619 in the porcine LES was investigated by pretreating the strips with atropine (a muscarinic receptor antagonist), tetrodotoxin (a neuronal sodium channel blocker), nifedipine (a calcium channel blocker), and Ca2+-free Krebs-Henseleit solution. Additionally, reverse transcription polymerase chain reaction (PCR) and immunohistochemistry (IHC) were performed to determine the presence of the TXA2 receptor in porcine LES. The results of this study demonstrated that U46619 caused marked concentration-dependent contractions in both porcine sling and clasp strips. The mechanism of U46619-induced contraction of the porcine LES was found to be related to calcium channels. Furthermore, the reverse transcription PCR analysis and IHC revealed that the TXA2 receptor was expressed in the clasp and sling fibers of porcine LES. Consequently, this study suggests that U46619 mediates the contraction of porcine LES through calcium channels and has potential as a therapeutic approach for treating GERD. SIGNIFICANCE STATEMENT: This study establishes that U46619 induces concentration-dependent contractions in porcine LES, primarily mediated by calcium channels. The presence of the TXA2 receptor in LES clasp and sling fibers is confirmed. These findings highlight U46619's potential as a GERD therapeutic by targeting calcium channels for LES contraction modulation.

Inhibition of TP signaling promotes endometriosis growth and neovascularization.

Endometriosis is highly dependent on angiogenesis and lymphangiogenesis. Prostaglandin E2, an arachidonic acid metabolite, has been shown to promote the formation of new blood and lymphatic vessels. However, the role of another arachidonic acid metabolite, thromboxane A2 (TXA2) in angiogenesis and lymphangiogenesis during endometriosis remains largely unexplored. Using a murine model of ectopic endometrial transplantation, fragments from the endometrium of WT donor mice were transplanted into the peritoneal walls of recipient WT mice (WT→WT), resulting in an increase in both the area and density of blood and lymphatic vessels. Upon transplantation of endometrial tissue from thromboxane prostanoid (TP) receptor (TXA2 receptor)­deficient (TP­/­) mice into TP­/­ mice (TP­/­â†’TP­/­), an increase in implant growth, angiogenesis, and lymphangiogenesis were observed along with upregulation of pro­angiogenic and lymphangiogenic factors, including vascular endothelial growth factors (VEGFs). Similar results were obtained using a thromboxane synthase (TXS) inhibitor in WT→WT mice. Furthermore, TP­/­â†’TP­/­ mice had a higher number of F4/80+ cells than that of WT→WT mice, with increased expression of genes related to the anti­inflammatory macrophage phenotype in endometrial lesions. In cultured bone marrow (BM)­derived macrophages, the levels of VEGF­A, VEGF­C, and VEGF­D decreased in a TP­dependent manner. Furthermore, TP signaling affected the polarization of cultured BM­derived macrophages to the anti­inflammatory phenotype. These findings imply that inhibition of TP signaling promotes endometrial implant growth and neovascularization.

Preparing to strike: Acute events in signaling by the serpentine receptor for thromboxane A2.

Over the last two decades, awareness of the (patho)physiological roles of thromboxane A2 signaling has been greatly extended. From humble beginnings as a short-lived stimulus that activates platelets and causes vasoconstriction to a dichotomous receptor system involving multiple endogenous ligands capable of modifying tissue homeostasis and disease generation in almost every tissue of the body. Thromboxane A2 receptor (TP) signal transduction is associated with the pathogenesis of cancer, atherosclerosis, heart disease, asthma, and host response to parasitic infection amongst others. The two receptors mediating these cellular responses (TPα and TPß) are derived from a single gene (TBXA2R) through alternative splicing. Recently, knowledge about the mechanism(s) of signal propagation by the two receptors has undergone a revolution in understanding. Not only have the structural relationships associated with G-protein coupling been established but the modulation of that signaling by post-translational modification to the receptor has come sharply into focus. Moreover, the signaling of the receptor unrelated to G-protein coupling has become a burgeoning field of endeavor with over 70 interacting proteins currently identified. These data are reshaping the concept of TP signaling from a mere guanine nucleotide exchange factors for Gα activation to a nexus for the convergence of diverse and poorly characterized signaling pathways. This review summarizes the advances in understanding in TP signaling, and the potential for new growth in a field that after almost 50 years is finally coming of age.

In silico analysis of single nucleotide polymorphism (rs34377097) of TBXA2R gene and pollen induced bronchial asthma susceptibility in West Bengal population, India.

Introduction: Prevalence of asthma is increasing steadily among general population in developing countries over past two decades. One of the causative agents of broncho-constriction in asthma is thromboxane A2 receptor (TBXA2R). However few studies of TBXA2R polymorphism were performed so far. The present study aimed to assess potential association of TBXA2R rs34377097 polymorphism causing missense substitution of Arginine to Leucine (R60L) among 482 patients diagnosed with pollen-induced asthma and 122 control participants from West Bengal, India. Also we performed in-silico analysis of mutated TBXA2R protein (R60L) using homology modeling. Methods: Clinical parameters like Forced expiratory volume in 1 second (FEV1), FEV1/Forced vital capacity (FVC) and Peak expiratory flow rate (PEFR) were assessed using spirometry. Patients' sensitivity was measured by skin prick test (SPT) against 16 pollen allergens. Polymerase chain reaction-based Restriction fragment length polymorphism was done for genotyping. Structural model of wild type and homology model of polymorphic TBXA2R was generated using AlphaFold2 and MODELLER respectively. Electrostatic surface potential was calculated using APBS plugin in PyMol. Results: Genotype frequencies differed significantly between the study groups (P=0.03). There was no significant deviation from Hardy-Weinberg equilibrium in control population (χ2=1.56). Asthmatic patients have significantly higher frequency of rs34377097TT genotype than control subjects (P=0.03). SPT of patients showed maximum sensitivity in A. indica (87.68%) followed by C. nusifera (83.29%) and C. pulcherima (74.94%). Significant difference existed for pollen sensitivity in adolescent and young adult (P=0.01) and between young and old adult (P=0.0003). Significant negative correlation was found between FEV1/FVC ratio and intensity of SPT reactions (P<0.0001). Significant association of FEV1, FEV1/FVC and PEFR was observed with pollen-induced asthma. Furthermore, risk allele T was found to be clinically correlated with lower FEV1/FVC ratio (P=0.015) in patients. Our data showed R60L polymorphism, which was conserved across mammals, significantly reduced positive electrostatic charge of polymorphic protein in cytoplasmic domain thus altered downstream pathway and induced asthma response. Discussion: The present in-silico study is the first one to report association of TBXA2R rs34377097 polymorphism in an Indian population. It may be used as prognostic marker of clinical response to asthma in West Bengal and possible target of therapeutics in future.

Electroacupuncture Treats Myocardial Infarction by Influencing the Regulation of Substance P in the Neurovascular to Modulate PGI2/TXA2 Metabolic Homeostasis via PI3K/AKT Pathway: A Bioinformatics-Based Multiomics and Experimental Study.

Acute myocardial infarction (AMI) is the most severe form of coronary heart disease caused by ischemia and hypoxia. The study is aimed at investigating the role of neuropeptides and the mechanism of electroacupuncture (EA) in acute myocardial infarction (AMI) treatment. Compared with the normal population, a significant increase in substance P (SP) was observed in the serum of patients with AMI. PGI2 expression was increased in the SP-treated AMI mouse model, and TXA2 expression was decreased. And PI3K pathway-related genes, including Pik3ca, Akt, and Mtor, were upregulated in myocardial tissue of SP-treated AMI patients. Human cardiomyocyte cell lines (HCM) treated with SP increased mRNA and protein expression of PI3K pathway-related genes (Pik3ca, Pik3cb, Akt, and Mtor). Compared to MI control and EA-treated MI rat models, Myd88, MTOR, Akt1, Sp, and Irak1 were differentially expressed, consistent with in vivo and in vitro studies. EA treatment significantly enriched PI3K/AKT signaling pathway genes within MI-associated differentially expressed genes (DEGs) according to Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, it was confirmed by molecular docking analysis that PIK3CA, AKT1, and mTOR form stable dockings with neuropeptide SP. PI3K/AKT pathway activity may be affected directly or indirectly by EA via SP, which corrects the PGI2/TXA2 metabolic imbalance in AMI. MI treatment is now better understood as a result of this finding.

Seratrodast, a thromboxane A2 receptor antagonist, inhibits neuronal ferroptosis by promoting GPX4 expression and suppressing JNK phosphorylation.

More than 30 % of individuals with epilepsy are refractory to currently available drugs, highlighting the urgent need to develop novel candidate drugs. Accumulating evidence implicates the key role of ferroptosis in the pathophysiology of epileptic seizuresand its potential as a new drug target. Drug repurposing is a promising strategy for the rapid generation of new candidate drugs from the market drugs with new therapeutic indications, such as the best-selling drug thalidomide. Herein, we reported the discovery of Seratrodast, a market drug of thromboxane A2 receptor antagonist as a new ferroptosis inhibitor (IC50: 4.5 µmol·L-1). Seratrodast could reduce lipid ROS production, regulate the system xc-/glutathione (GSH)/glutathione peroxidase 4 (GPX4) axis, and inhibit JNK phosphorylation and p53 expression. In addition, Seratrodast elevated GPX4 expression and decreased JNK phosphorylation in pentylenetetrazole-induced seizures in mice. Seratrodast increased the latency of seizures and reduced seizure duration in pentylenetetrazole-induced seizures. Our results suggest Seratrodast might be either a ferroptosis inhibitor or a novel lead compound for further optimization of novel drug discovery.

Cyclosporin A up-regulated thromboxane A2 receptor through activation of MAPK and NF-κB pathways in rat mesenteric artery.

Cyclosporin A (CsA) is an immunosuppressant used in transplantation patients and inflammatory diseases. CsA-induced local vasoconstriction can lead to serious side effects including nephrotoxicity and hypertension. However, the underlying mechanisms are not fully understood. Mesenteric artery rings of rats were cultured with CsA and specific inhibitors for mitogen-activating protein kinases (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. A sensitive myograph recorded thromboxane (TP) receptor-mediated vasoconstriction. Protein levels of key signaling molecules were assessed by Western blotting. The results show that CsA up-regulated the TP receptor expression with the enhanced vasoconstriction in a dose- and time-dependent manner. Furthermore, the blockage of MAPKs or NF-κB activation markedly attenuated CsA-enhanced vasoconstriction and the TP receptor protein expression. Rats subcutaneously injected with CsA for three weeks showed increased blood pressure in vivo and increased contractile responses to a TP agonist ex vivo. CsA also enhanced TP receptor, as well as p-ERK1/2, p-p38, p- IκBα, p-NF-κB P65 protein levels and decreased IκBα protein expression, demonstrating that CsA induced TP receptor enhanced-vasoconstriction via activation of MAPK and NF-κB pathways. In conclusion, CsA up-regulated the expression of TP receptors via activation of MAPK and NF-κB pathways. The results may provide novel options for prevention of CsA-associated hypertension.

Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells.

We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.

Sensory neuron inositol 1,4,5-trisphosphate receptors contribute to chronic mechanoreflex sensitization in rats with simulated peripheral artery disease.

The mechanoreflex is exaggerated in patients with peripheral artery disease (PAD) and in a rat model of simulated PAD in which a femoral artery is chronically (∼72 h) ligated. We found recently that, in rats with a ligated femoral artery, blockade of thromboxane A2 (TxA2) receptors on the sensory endings of thin fiber muscle afferents reduced the pressor response to 1 Hz repetitive/dynamic hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). Conversely, we found no effect of TxA2 receptor blockade in rats with freely perfused femoral arteries. Here, we extended the isolated mechanoreflex findings in "ligated" rats to experiments evoking dynamic hindlimb skeletal muscle contractions. We also investigated the role played by inositol 1,4,5-trisphosphate (IP3) receptors, receptors associated with intracellular signaling linked to TxA2 receptors, in the exaggerated response to dynamic mechanoreflex and exercise pressor reflex activation in ligated rats. Injection of the TxA2 receptor antagonist daltroban into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic contraction in ligated but not "freely perfused" rats. Moreover, injection of the IP3 receptor antagonist xestospongin C into the arterial supply of the hindlimb reduced the pressor response to 1 Hz dynamic stretch and contraction in ligated but not freely perfused rats. These findings demonstrate that, in rats with a ligated femoral artery, sensory neuron TxA2 receptor and IP3 receptor-mediated signaling contributes to a chronic sensitization of the mechanically activated channels associated with the mechanoreflex and the exercise pressor reflex.

Thromboxane A2 receptors contribute to the exaggerated exercise pressor reflex in male rats with heart failure.

Mechanical and metabolic signals associated with skeletal muscle contraction stimulate the sensory endings of thin fiber muscle afferents and produce reflex increases in sympathetic nerve activity and blood pressure during exercise (i.e., the exercise pressor reflex; EPR). The EPR is exaggerated in patients and animals with heart failure with reduced ejection fraction (HF-rEF) and its activation contributes to reduced exercise capacity within this patient population. Accumulating evidence suggests that the exaggerated EPR in HF-rEF is partially attributable to a sensitization of mechanically activated channels produced by thromboxane A2 receptors (TxA2 -Rs) on those sensory endings; however, this has not been investigated. Accordingly, the purpose of this investigation was to determine the role played by TxA2 -Rs on the sensory endings of thin fiber muscle afferents in the exaggerated EPR in rats with HF-rEF induced by coronary artery ligation. In decerebrate, unanesthetized rats, we found that injection of the TxA2 -R antagonist daltroban (80 µg) into the arterial supply of the hindlimb reduced the pressor response to 30 s of electrically induced 1 Hz dynamic hindlimb muscle contraction in HF-rEF (n = 8, peak ∆MAP pre: 22 ± 3; post: 14 ± 2 mmHg; p = 0.01) but not sham (n = 10, peak ∆MAP pre: 13 ± 3; post: 11 ± 2 mmHg; p = 0.68) rats. In a separate group of HF-rEF rats (n = 4), we found that the systemic (intravenous) injection of daltroban had no effect on the EPR (peak ΔMAP pre: 26 ± 7; post: 25 ± 7 mmHg; p = 0.50). Our data suggest that TxA2 -Rs on thin fiber muscle afferents contribute to the exaggerated EPR evoked in response to dynamic muscle contraction in HF-rEF.

Molecular Pathomechanisms of Impaired Flow-Induced Constriction of Cerebral Arteries Following Traumatic Brain Injury: A Potential Impact on Cerebral Autoregulation.

(1) Background: Traumatic brain injury (TBI) frequently occurs worldwide, resulting in high morbidity and mortality. Here, we hypothesized that TBI impairs an autoregulatory mechanism, namely the flow-induced constriction of isolated rat middle cerebral arteries (MCAs). (2) Methods: TBI was induced in anaesthetized rats by weight drop model, and then MCAs were isolated and transferred into a pressure-flow chamber. The internal diameter was measured by a video-microscopy. (3) Results: In MCAs from intact rats, increases in flow and pressure + flow elicited constrictions (-26 ± 1.9 µm and -52 ± 2.8 µm, p < 0.05), which were significantly reduced after TBI or in the presence of thromboxane-prostanoid (TP receptor) antagonist SQ 29,548. Flow-induced constrictions were significantly reduced by HET0016, inhibitor of cytochrome P450 4A (CYP450 4A). Arachidonic acid, (AA, 10-7 M), and CYP-450 4A metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) elicited constrictions of intact MCA (-26 ± 2.3% and -31 ± 3.6%), which were significantly reduced after TBI (to 11 ± 1.3% and -16 ±2.5%). The TP receptor agonist U46619 (10-7 M) elicited substantial constrictions of MCA from intact rats (-21 ± 3.3%), which were also significantly reduced, after TBI (to -16 ± 2.4%). (4) Conclusions: Flow-induced constrictor response of MCA is impaired by traumatic brain injury, likely due to the reduced ability of cytochrome P450 4A to convert arachidonic acid to constrictor prostaglandins and the mitigated sensitivity of thromboxane-prostanoid receptors.

Roles of Thromboxane Receptor Signaling in Enhancement of Lipopolysaccharide-Induced Lymphangiogenesis and Lymphatic Drainage Function in Diaphragm.

[Figure: see text].

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.

Mechanism of thromboxane receptor-induced vasoconstriction in human saphenous vein.

Saphenous vein (SV) is one of the most widely used graft material in patients undergoing coronary artery bypass graft surgery (CABG). Thromboxane A2 (TXA2) is implicated in graft failure by inducing vasoconstriction and platelet aggregation. The aim of this study is to investigate the mechanism involved in TXA2-induced vasoconstriction in human SV. The role of different inhibitors and blockers on U46619 (TXA2-mimetic)-induced vasoconstriction is investigated by using an isolated organ bath system. Relaxation responses to several mediators are evaluated in SV pre-contracted with U46619 and compared with those pre-contracted with phenylephrine. Our results demonstrate that U46619-induced contraction is completely blocked by myosin light chain kinase inhibitor ML-9 or TP receptor antagonist BAY u3405. Furthermore, U46619-induced contraction is partially inhibited by phospholipase C inhibitor U73122, protein kinase C inhibitor calphostin C, Rho-kinase inhibitor Y-27632, L-type calcium channel blocker nifedipine, store-operated channel inhibitor SKF96365 or removal of extracellular calcium. Relaxation responses to NO donor (sodium nitroprusside), guanylate cyclase (GC) stimulator (riociguat), phosphodiesterase (PDE) inhibitors (sildenafil, IBMX), adenylate cyclase (AC) activator (forskolin) and acetylcholine (ACh) are markedly reduced when U46619 is used as a pre-contraction agent. Our results demonstrate that influx of extracellular Ca2+ (through L-type calcium channels and store-operated calcium channels) and intracellular Ca2+ release together with Ca2+ sensitization (through Rho-kinase activation) are necessary components for TXA2-induced vasoconstriction in SV. Moreover, more pronounced decrease in vasorelaxation induced by several mediators (SNP, riociguat, sildenafil, IBMX, forskolin, and ACh) in the presence of U46619 when compared with phenylephrine suggests that there is a crosstalk between the TP receptor signaling pathway and PDE, AC, GC enzymes. We believe that the investigation of mechanism of the TXA2-induced vasoconstriction in SV will provide additional information for the prevention of SV graft failure.

Thromboxane A2 receptors mediate chronic mechanoreflex sensitization in a rat model of simulated peripheral artery disease.

The exercise pressor reflex is a feedback autonomic and cardiovascular control mechanism evoked by mechanical and metabolic signals within contracting skeletal muscles. The mechanically sensitive component of the reflex (the mechanoreflex) is exaggerated in patients with peripheral artery disease (PAD) and in a rat model of simulated PAD in which a femoral artery is chronically ligated. Products of cyclooxygenase enzyme activity have been shown to chronically sensitize the mechanoreflex in PAD, but the identity of the muscle afferent receptors that mediate the sensitization is unclear. We hypothesized that injection of the endoperoxide 4 receptor (EP4-R) antagonist L161982 or the thromboxane A2 receptor (TxA2-R) antagonist daltroban into the arterial supply of the hindlimb would reduce the pressor response to repetitive, dynamic hindlimb skeletal muscle stretch (a model of isolated mechanoreflex activation) in rats with a femoral artery that was ligated ~72 h before the experiment but not in rats with freely perfused femoral arteries. We found that EP4-R blockade had no effect on the pressor response (peak Δmean arterial pressure) to stretch in freely perfused (n = 6, pre: 14 ± 2, post: 15 ± 2 mmHg, P = 0.97) or ligated (n = 8, pre: 29 ± 4, post: 29 ± 6 mmHg, P = 0.98) rats. In contrast, TxA2-R blockade had no effect on the pressor response to stretch in freely perfused rats (n = 6, pre: 16 ± 3, post: 17 ± 4 mmHg, P = 0.99) but significantly reduced the response in ligated rats (n = 11, pre: 29 ± 4, post: 17 ± 5 mmHg, P < 0.01). We conclude that TxA2-Rs contribute to chronic mechanoreflex sensitization in the chronic femoral artery-ligated rat model of simulated PAD.NEW & NOTEWORTHY We demonstrate that thromboxane A2 receptors, but not endoperoxide 4 receptors, on the sensory endings of thin fiber muscle afferents contribute to the chronic sensitization of the muscle mechanoreflex in rats with a ligated femoral artery (a model of simulated peripheral artery disease). The data may have important implications for our understanding of blood pressure control during exercise in patients with peripheral artery disease.

Regulation of liver regeneration by prostaglandin E2 and thromboxane A2 following partial hepatectomy in rats.

The implication of prostaglandin E2 (PGE2) and thromboxane A2 (TXA2) in the striking process of liver regeneration has been previously reported. However, their exact roles and downstream signals have not been utterly revealed. Therefore, the present study was conducted to explore whether inhibition of cyclooxygenase-2 (COX-2)-derived PGE2 by celecoxib and blocking of TXA2 action by seratrodast could alter the progression of liver regeneration after 70% partial hepatectomy (PHx) in rats. Celecoxib (20 mg/kg/day) and seratrodast (2 mg/kg/day) were given orally 1 h before PHx and then daily till the end of experiment (1, 3, or 7 days after the operation). Interestingly, celecoxib-treated rats showed a further increase in interleukin-6, p65 nuclear factor κB, and phosphorylated signal transducer and activator of transcription 3 as compared with PHx control rats. Furthermore, the liver contents of growth factors as well as ß-catenin and cyclin D1protein expressions were also enhanced by celecoxib. Accordingly, celecoxib significantly improved hepatic proliferation as indicated by the increase in Ki67 expression and liver index. Contrariwise, seratrodast hindered the normal regeneration process and completely abolished the proliferative effect of celecoxib. In conclusion, TXA2 has a major role in liver regeneration that could greatly mediate the triggering effect of celecoxib on hepatocytes proliferation following PHx.