Thromboxane Synthetase Research Articles

Exploring the mechanism of the antithrombotic effects of Pueraria lobata and Pueraria lobata var. thomsonii based on network pharmacology

Ethnopharmacological relevancePueraria lobata (Willd.) Ohwi and Pueraria lobata var. thomsonii (Benth.) Maesen are nutritious medicine food homology plants that are widely used in the food and health products industry and are excellent natural materials for the development of new health foods, with great potential for domestic and foreign markets. Clinically, P. lobata and P. thomsonii are used to treat coronary heart disease, atherosclerosis, cerebral infarction and other cardiovascular diseases, and antithrombotic actions may be their core effect in the treatment of thrombotic diseases. However, the underlying mechanisms of the antithrombotic properties of P. lobata and P. thomsonii have not been clarified. MethodsFirst, P. lobata and P. thomsonii were identified by high-performance liquid chromatography (HPLC). An arteriovenous bypass thrombosis rat model was established. Thrombus dry‒wet weight, platelet accumulation rate and the four coagulation indices, including activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT) and fibrinogen (FIB), were detected in plasma to manifest the P. lobata and P. thomsonii antithrombotic function. Network pharmacology and molecular docking methods were used to obtain key targets and verify reliability. David 6.8 was used for GO and KEGG analyses to explore pathways and potential targets for P. lobata and P. thomsonii antithrombotic functions. Prostaglandin I2 (PGI2), thromboxane A2 (TXA2), cyclooxygenase 2 (COX-2), myeloperoxidase (MPO) and endothelial nitric oxide synthase (eNOS) were tested by enzyme-linked immunosorbent assay (ELISA). ResultsThe results indicated that P. lobata and P. thomsonii can reduce thrombus dry‒wet weight and platelet accumulation in rats and inhibit TT, APTT, FIB, and PT. A comprehensive network pharmacology approach successfully identified 9 active ingredients in P. lobata and P. thomsonii. The main active ingredients include polyphenols, amino acids and flavonoids. A total of 15 antithrombotic function targets were obtained, including 3 key targets (PTGS2, NOS3, MPO). Pathway analysis showed 10 significant related pathways and 29 biological processes. P. lobata and P. thomsonii inhibited platelet aggregation by upregulating PGI2 and downregulating TXA2, inhibited PTGS2 to reduce inflammation, and increased the level of eNOS to promote vasodilation. In addition, P. lobata and P. thomsonii alleviated oxidative stress by increasing SOD levels and significantly decreasing MDA contents. ConclusionThe results of the study further clarify the antithrombotic mechanism of action of P. lobata and P. thomsonii, which provides a scientific basis for the development of new drugs for thrombogenic diseases and lays the foundation for the development of P. lobata and P. thomsonii herbal resources and P. lobata and P. thomsonii health products.

Abstract P1004: Macrophage Apoptotic Cell Receptor Mertk Triggers The Synthesis Of Arachidonic Acid Derivatives To Promote Cardiac Regeneration

Introduction: Macrophages (MΦs) are indispensable for regeneration of the mammalian neonatal heart. However, the underlying mechanisms by which neonatal MΦs sense the injured cardiac milieu and promote regeneration remain obscure. Utilizing single-cell transcriptomics, we previously observed a regenerative neonatal cardiac MΦ population that distinctly upregulates MerTK, an apoptotic cell receptor, compared to adult cardiac MΦs. Objective: We hypothesized that MerTK is required to program neonatal cardiac MΦs to secrete specialized pro-“regenerative” lipids to induce cardiomyocyte proliferation leading to heart regeneration. Methods: Postnatal day 1 and adult mice were subjected to cardiac injury via ligation of the left anterior descending artery. Single-cell transcriptomics, lipid-omics, and molecular approaches were utilized to determine the mechanisms by which neonatal MΦs regulate cardiac regeneration. Results: To determine the necessity of MΦ MerTK for cardiac regeneration, we employed a myeloid-specific knockout of MerTK ( mMertk -/- ) which failed to regain homeostatic cardiac function. We next performed single-cell transcriptomics on Mertk +/+ and Mertk -/- neonates to determine how MerTK activation reprograms neonatal MΦs for regeneration. Mertk +/+ MΦs significantly upregulated genes associated with arachidonic acid metabolism. Hypothesizing that arachidonic acid derivatives may be key signaling molecules for regeneration, we performed a targeted lipid-omics screen which revealed an increase in Thromboxane B 2 (TXB 2 ) in regenerating hearts but not within non-regenerative Mertk -/- hearts. Furthermore, addition of a TXA 2 agonist to neonatal cardiomyocytes in vitro significantly increases their proliferation by regulating the YAP/TAZ signaling. Alternatively, inhibition of thromboxane synthase in neonatal MΦs or thromboxane receptor on cardiomyocytes by small molecule inhibitors significantly abrogated the increase in cardiomyocyte proliferation by efferocytic Mertk +/+ neonatal MΦs. Conclusions: Altogether, our data suggest that MerTK programs neonatal cardiac MΦs to secrete TXA 2 to induce cardiomyocyte proliferation via the HIPPO-YAP/TAZ pathway prompting neonatal cardiac regeneration.

TBXAS1 Gene Polymorphism Is Associated with the Risk of Ischemic Stroke of Metabolic Syndrome in a Chinese Han Population.

Objective To investigate the association between thromboxane A synthase 1 (TBXAS1) gene polymorphism and metabolic syndrome (MS) and explore whether gene polymorphism could act as biomarkers in MS and its components or whether it could play a role in MS-related damage. Methods A total of 3072 eligible subjects were obtained, of which 1079 cases were controls and 1993 cases were MS patients. Subjects were followed up for 5 years, and the endpoint were recorded. The gene polymorphism of TBXAS1 was detected by using the Sequenom MassArray method. Results Significant differences were observed in ischemic stroke and NC_000007.14: g.139985896C>T (P < 0.05). The incidence of ischemic stroke was significantly higher in T allele carriers than in C (P < 0.05). C allele was the protective factor of the onset of ischemic stroke. There were negative interactions between C allele and waist circumference (WC), systolic blood pressure (SBP), diastolic blood pressure (DBP), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and fasting plasma glucose (FPG). Conclusion These findings suggest that NC_000007.14: g.139985896C>T was related to the incidence of ischemic stroke in the whole and MS population, and individuals who carry the C allele have a reduced risk of ischemic stroke, which may be used as a promising biomarker of disease risk in patients with MS.

P809: MODULATION OF ARACHIDONIC ACID PATHWAY BY IBUPROFEN RESULTS IN COMPLETE HEMATOLOGICAL RESPONSE IN GHOSAL HEMATODIAPHYSEAL DYSPLASIA WITHOUT NEED FOR CORTICOSTEROID

Background: Ghosal hematodiaphyseal dysplasia (GHDD) is a rare autosomal recessive disorder characterized by pancytopenia and increased bone density of the long bones. First described in families of Indian and Middle Eastern descent, subsequent studies identified the genetic lesion in this disease as due to loss of function mutations in the TBXAS1 gene encoding thromboxane synthase, a key component of the arachidonic acid pathway responsible for the formation of thromboxane A2 from prostaglandin H2. As well as pancytopenia, laboratory features include platelet aggregation defects and increase in osteoblastic activity, the latter being the basis of increased bone density in patients with Ghosal syndrome. Simultaneously, loss of thromboxane synthase activity may be associated with shunting of prostaglandin precursors away from production of thromboxane into production of pro-inflammatory prostaglandins. This may be the mechanism responsible for the observed marrow suppression seen in these patients. We describe a 3 year old female of non-consanguineous parents of Filipino origin who presented with marked pancytopenia. Next Generation Sequencing bone marrow failure gene panel revealed a novel homozygous mutation in TBXAS1. Subsequent DXA scanning revealed increased bone mineral density consistent with the diagnosis (Z -score +2.4 for lumbar spine (L1-4)) although clinical sequelae of bony disease were not apparent. Aims: While multiple case reports document that these patients have hematological response to long term corticosteroid therapy, we hypothesized that inhibition of cyclooxygenase (COX) upstream of thromboxane synthase in the arachidonic acid pathway may be effective in this condition. Methods: Patient was treated with ibuprofen at regular therapeutic doses (approximately 30 mg/kg/24 hours daily initially) and serial full blood counts were monitored. Results: The patient exhibited a rapid and complete response to ibuprofen with normalization of full blood counts. The dose of ibuprofen was reduced to 10 mg/kg/day, and hematologic response is maintained on low dose after one year.The treatment is well tolerated with no side effects. Image:Summary/Conclusion: Treatment of GHDD with ibuprofen is a novel approach that offers clear potential benefits in comparison to the known adverse effects profile of long term corticosteroids. Bone density scan and markers are in process to ascertain the impact of this treatment on bone density, although this may take longer to show beneficial effects. The treatment was easily tolerated and well managed by the patient and family at home with minimum disruption to her quality of life.

Thromboxane-Prostanoid Receptor Signaling Drives Persistent Fibroblast Activation in Pulmonary Fibrosis.

Rationale: Although persistent fibroblast activation is a hallmark of idiopathic pulmonary fibrosis (IPF), mechanisms regulating persistent fibroblast activation in the lungs have not been fully elucidated. Objectives: On the basis of our observation that lung fibroblasts express TBXA2R (thromboxane-prostanoid receptor) during fibrosis, we investigated the role of TBXA2R signaling in fibrotic remodeling. Methods: We identified TBXA2R expression in lungs of patients with IPF and mice and studied primary mouse and human lung fibroblasts to determine the impact of TBXA2R signaling on fibroblast activation. We used TBXA2R-deficient mice and small-molecule inhibitors to investigate TBXA2R signaling in preclinical lung fibrosis models. Measurements and Main Results: TBXA2R expression was upregulated in fibroblasts in the lungs of patients with IPF and in mouse lungs during experimental lung fibrosis. Genetic deletion of TBXA2R, but not inhibition of thromboxane synthase, protected mice from bleomycin-induced lung fibrosis, thereby suggesting that an alternative ligand activates profibrotic TBXA2R signaling. In contrast to thromboxane, F2-isoprostanes, which are nonenzymatic products of arachidonic acid induced by reactive oxygen species, were persistently elevated during fibrosis. F2-isoprostanes induced TBXA2R signaling in fibroblasts and mediated a myofibroblast activation profile due, at least in part, to potentiation of TGF-β (transforming growth factor-β) signaling. In vivo treatment with the TBXA2R antagonist ifetroban reduced profibrotic signaling in the lungs, protected mice from lung fibrosis in three preclinical models (bleomycin, Hermansky-Pudlak mice, and radiation-induced fibrosis), and markedly enhanced fibrotic resolution after bleomycin treatment. Conclusions: TBXA2R links oxidative stress to fibroblast activation during lung fibrosis. TBXA2R antagonists could have utility in treating pulmonary fibrosis.

Classification models and SAR analysis on thromboxane A2 synthase inhibitors by machine learning methods

ABSTRACT Thromboxane A2 synthase (TXS) is a promising drug target for cardiovascular diseases and cancer. In this work, we conducted a structure-activity relationship (SAR) study on 526 TXS inhibitors for bioactivity prediction. Three types of descriptors (MACCS fingerprints, ECFP4 fingerprints, and MOE descriptors) were utilized to characterize inhibitors, 24 classification models were developed by support vector machine (SVM), random forest (RF), extreme gradient boosting (XGBoost), and deep neural networks (DNN). Then we reduced the number of fingerprints according to the contribution of descriptors to the models, and constructed 16 extra models on simplified fingerprints. In general, Model_4D built by DNN algorithm and 67 bits MACCS fingerprints performs best. The prediction accuracy of the model on the test set is 0.969, and Matthews correlation coefficient (MCC) is 0.936. The distance between compound and model (dSTD-PRO) was used to characterize the application domain of the model. In the test set of Model_4D, dSTD-PRO of 91.5% compounds is lower than the corresponding training set threshold (threshold0.90 = 0.1055), and the accuracy of these compounds is 0.983. In addition, the important descriptors were summarized and further analyzed. It showed that aromatic nitrogenous heterocyclic groups were beneficial to improve the bioactivity of TXS inhibitors.

THROMBOXANE-DEPENDENT CORONARY VASOCONSTRICTION IN OBESE MICE: ROLE OF PEROXYNITRITE

Objective: Obesity leads to chronic oxidative stress promoting the development of cardiovascular diseases including endothelial dysfunction, which might occur through the cyclooxygenase (COX) pathway. This study aimed to investigate the role of peroxynitrite in the generation of COX-derived products and its role in coronary vascular responses. Design and method: We evaluated arachidonic acid (AA)-dependent coronary vascular responses in isolated hearts from control and obese C57BL/6 mice and explored COX metabolism using the COX-2 inhibitor indomethacin and 1-benzylimidazole, inhibitor of thromboxane synthase (TXS). COX, TXS, and TXA2 receptor (TP) protein expression was assessed in heart homogenates. The release of TXA2 in the coronary circulation was also evaluated. In addition, the possible involvement of peroxynitrite on increased coronary response was explored by detecting peroxynitrite by coumarin boronic acid (CBA) and 3-nitrotyrosine protein expression. Alterations in TXS, and TP receptor protein expression were also evaluated in heart homogenates from obese mice treated with the peroxynitrite scavenger FeTMPyP. Results: Arachidonic acid (AA) dependent coronary vasoconstriction in isolated hearts, increased from 31.06 ± 2.67 mmHg in control mice to 88.99 ± 6.09 mmHg in obese mice. Both, indomethacin and 1-benzylimidazole reduced the vasoconstriction response in control and obese mice. TXA2 released during AA-induced vasoconstriction phase was increased in heart perfusates from 76.1 ± 18.42 pg/mL in control mice to 203.6 ± 29.8 pg/mL in obese mice. COX- 2, TXS and TP receptor protein expression was increased in hearts from obese mice (1.96 ± 0.25, 0.70 ± 0.07, 1.22 ± 0.19 arbitrary units, respectively) compared with control mice (0.17 ± 0.02; 0.06 ± 0.02; and 0.29 ± 0.06 arbitrary units, respectively). Moreover, nitrotyrosine and peroxynitrite levels were higher in obese mice, compared with control mice. Increase in coronary vasoconstriction, TXA2 release as well as protein expression was prevented with FeTMPyP treatment in obese mice. Conclusions: Peroxynitrite-dependent induction of COX-2 and TXS protein expression in heart tissue from obese mice may lead to increased TXA2 coronary circulation, thereby increasing coronary vasoconstriction.

Imbalanced prostanoid release mediates cigarette smoke-induced human pulmonary artery cell proliferation

BackgroundPulmonary hypertension is a common and serious complication of chronic obstructive pulmonary disease (COPD). Studies suggest that cigarette smoke can initiate pulmonary vascular remodelling by stimulating cell proliferation; however, the underlying cause, particularly the role of vasoactive prostanoids, is unclear. We hypothesize that cigarette smoke extract (CSE) can induce imbalanced vasoactive prostanoid release by differentially modulating the expression of respective synthase genes in human pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs), thereby contributing to cell proliferation.MethodsAqueous CSE was prepared from 3R4F research-grade cigarettes. Human PASMCs and PAECs were treated with or without CSE. Quantitative real-time RT-PCR and Western blotting were used to analyse the mRNA and protein expression of vasoactive prostanoid syhthases. Prostanoid concentration in the medium was measured using ELISA kits. Cell proliferation was assessed using the cell proliferation reagent WST-1.ResultsWe demonstrated that CSE induced the expression of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostanoid synthesis, in both cell types. In PASMCs, CSE reduced the downstream prostaglandin (PG) I synthase (PGIS) mRNA and protein expression and PGI2 production, whereas in PAECs, CSE downregulated PGIS mRNA expression, but PGIS protein was undetectable and CSE had no effect on PGI2 production. CSE increased thromboxane (TX) A synthase (TXAS) mRNA expression and TXA2 production, despite undetectable TXAS protein in both cell types. CSE also reduced microsomal PGE synthase-1 (mPGES-1) protein expression and PGE2 production in PASMCs, but increased PGE2 production despite unchanged mPGES-1 protein expression in PAECs. Furthermore, CSE stimulated proliferation of both cell types, which was significantly inhibited by the selective COX-2 inhibitor celecoxib, the PGI2 analogue beraprost and the TXA2 receptor antagonist daltroban.ConclusionsThese findings provide the first evidence that cigarette smoke can induce imbalanced prostanoid mediator release characterized by the reduced PGI2/TXA2 ratio and contribute to pulmonary vascular remodelling and suggest that TXA2 may represent a novel therapeutic target for pulmonary hypertension in COPD.

Increased COX-2 after ureter obstruction attenuates fibrosis and is associated with EP2 receptor upregulation in mouse and human kidney.

AimCyclooxygenase‐2 (COX‐2) activity protects against oxidative stress and apoptosis early in experimental kidney injury. The present study was designed to test the hypothesis that COX‐2 activity attenuates fibrosis and preserves microvasculature in injured kidney. The murine unilateral ureteral‐obstruction (UUO) model of kidney fibrosis was employed and compared with human nephrectomy tissue with and without chronic hydronephrosis.MethodsFibrosis and angiogenic markers were quantified in kidney tissue from wild‐type and COX‐2−/− mice subjected to UUO for 7 days and in human kidney tissue. COX‐enzymes, prostaglandin (PG) synthases, PG receptors, PGE2, and thromboxane were determined in human tissue.ResultsCOX‐2 immunosignal was observed in interstitial fibroblasts at baseline and after UUO. Fibronectin, collagen I, III, alpha‐smooth muscle actin, and fibroblast specific protein‐1 mRNAs increased significantly more after UUO in COX‐2−/− vs wild‐type mice. In vitro, fibroblasts from COX‐2−/− kidneys showed higher matrix synthesis. Compared to control, human hydronephrotic kidneys showed (i) fibrosis, (ii) no significant changes in COX‐2, COX‐1, PGE2‐, and prostacyclin synthases, and prostacyclin and thromboxane receptor mRNAs, (iii) increased mRNA and protein of PGE2‐EP2 receptor level but unchanged PGE2 tissue concentration, and (iv) two‐ to threefold increased thromboxane synthase mRNA and protein levels, and increased thromboxane B2 tissue concentration in cortex and outer medulla.ConclusionCOX‐2 protects in the early phase against obstruction‐induced fibrosis and maintains angiogenic factors. Increased PGE2‐EP2 receptor in obstructed human and murine kidneys could contribute to protection.

Activation of Nrf2 in Mice Causes Early Microvascular Cyclooxygenase-Dependent Oxidative Stress and Enhanced Contractility.

Nuclear factor erythroid factor E2-related factor 2 (Nrf2) transcribes antioxidant genes that reduce the blood pressure (BP), yet its activation with tert-butylhydroquinone (tBHQ) in mice infused with angiotensin II (Ang II) increased mean arterial pressure (MAP) over the first 4 days of the infusion. Since tBHQ enhanced cyclooxygenase (COX) 2 expression in vascular smooth muscle cells (VSMCs), we tested the hypothesis that tBHQ administration during an ongoing Ang II infusion causes an early increase in microvascular COX-dependent reactive oxygen species (ROS) and contractility. Mesenteric microarteriolar contractility was assessed on a myograph, and ROS by RatioMaster™. Three days of oral tBHQ administration during the infusion of Ang II increased the mesenteric microarteriolar mRNA for p47phox, the endothelin type A receptor and thromboxane A2 synthase, and increased the excretion of 8-isoprostane F2α and the microarteriolar ROS and contractions to a thromboxane A2 (TxA2) agonist (U-46,619) and endothelin 1 (ET1). These were all prevented in Nrf2 knockout mice. Moreover, the increases in ROS and contractility were prevented in COX1 knockout mice with blockade of COX2 and by blockade of thromboxane prostanoid receptors (TPRs). In conclusion, the activation of Nrf2 over 3 days of Ang II infusion enhances microarteriolar ROS and contractility, which are dependent on COX1, COX2 and TPRs. Therefore, the blockade of these pathways may diminish the early adverse cardiovascular disease events that have been recorded during the initiation of Nrf2 therapy.

Effects of human TFPI and CD47 expression and selectin and integrin inhibition during GalTKO.hCD46 pig lung perfusion with human blood.

Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n=7) were compared to GalTKO.hCD46 lungs (reference, n=5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti-histamine, and anti-GPIb integrin-blocking Fab, and were pre-treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL-1 and GMI-1271 (P- and E-selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n=6 hTFPI.hCD47, n=3 reference). All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype. Expression of hTFPI.hCD47 on porcine lung may be useful as part of an integrated strategy to prevent neutrophil adhesion and platelet activation that are associated with xenograft injury. Additionally, targeting canonical selectin and integrin adhesion pathways reduced PVR elevation associated with hTFPI.hCD47 expression, but did not significantly attenuate neutrophil or platelet sequestration. We conclude that other adhesive mechanisms mediate the residual sequestration of human formed blood elements to pig endothelium that occurs even in the context of the multiple genetic modifications and drug treatments tested here.

Investigation of the characteristic binding mode of prostaglandin endoperoxide H2 formed by cyclooxygenase with a group of terminal prostanoid synthases

Free arachidonic acid mobilized from membrane glycerophospholipids by the cognate phospholipases is transformed into prostaglandin endoperoxide H2 (PGH2) by cyclooxygenase (COX), a common substrate for a group of terminal prostanoid synthases. COXs are thought to exhibit distinct functional coupling with these enzymes, which include prostaglandin E2, prostaglandin D2, prostaglandin F2α, prostacyclin, and thromboxane synthases. Three distinctive PGE2 synthases catalyzing nonoxidative rearrangement of PGH2 to PGE2 are identified, which are cytosolic PGES, two membrane-bound PGESs.

A novel prostaglandin I2 agonist, ONO-1301, attenuates liver inflammation and suppresses fibrosis in non-alcoholic steatohepatitis model mice

BackgroundONO-1301 is a novel long-lasting prostaglandin (PG) I2 mimetic with inhibitory activity on thromboxane (TX) A2 synthase. This drug can also induce endogenous prostaglandin (PG)I2 and PGE2 levels. Furthermore, ONO-1301 acts as a cytokine inducer and can initiate tissue repair in a variety of diseases, such as pulmonary hypertension, pulmonary fibrosis, cardiac infarction, and obstructive nephropathy. In this study, our aim was to evaluate the effect of ONO-1301 on liver inflammation and fibrosis in a mouse model of non-alcoholic steatohepatitis (NASH).MethodsThe therapeutic effects of ONO-1301 against liver damage, fibrosis, and occurrence of liver tumors were evaluated using melanocortin 4 receptor-deficient (Mc4r-KO) NASH model mice. The effects of ONO-1301 against macrophages, hepatic stellate cells, and endothelial cells were also evaluated in vitro.ResultsONO-1301 ameliorated liver damage and fibrosis progression, was effective regardless of NASH status, and suppressed the occurrence of liver tumors in Mc4r-KO NASH model mice. In the in vitro study, ONO-1301 suppressed LPS-induced inflammatory responses in cultured macrophages, suppressed hepatic stellate cell (HSC) activation, upregulated vascular endothelial growth factor (VEGF) expression in HSCs, and upregulated hepatocyte growth factor (HGF) and VEGF expression in endothelial cells.ConclusionsThe results of our study highlight the potential of ONO-1301 to reverse the progression and prevent the occurrence of liver tumors in NASH using in vivo and in vitro models. ONO-1301 is a multidirectional drug that can play a key role in various pathways and can be further analyzed for use as a new drug candidate against NASH.

Aspirin suppresses breast cancer metastasis to lung by targeting anoikis resistance.

Clinical evidence recently suggests that the regular use of aspirin is associated with a lower risk of breast cancer metastasis, but mechanisms remain unclear. Resistance to anoikis has been implicated in malignant transformation and metastasis. Here, we investigated whether aspirin might prevent breast cancer metastasis to lung by targeting anoikis resistance. Aspirin sensitized breast cancer cells to anoikis in vitro and lowered the circulating tumor cells as well as distant metastasis in vivo. Mechanistically, thromboxane A2 (TXA2) pathway was identified as the relevant molecular target for aspirin in anoikis sensitization. Upon detachment, both thromboxane A2 receptor (TP) and thromboxane A2 synthase 1 (TBXAS1) were up-regulated in metastatic breast cancer cells, conferred anoikis resistance through persistent activation of Akt, thereby facilitated breast cancer metastasis to lung. Consistently, either knockdown of TP in cancer cells or genetic deletion of TP in mice protected against lung metastasis in vivo. Collectively, TXA2 pathway plays a critical role in anoikis resistance and might serve as potential target for chemoprevention of breast cancer metastasis.

Thromboxane Mobilizes Insect Blood Cells to Infection Foci

Innate immune responses are effective for insect survival to defend against entomopathogens including a fungal pathogen, Metarhizium rileyi, that infects a lepidopteran Spodoptera exigua. In particular, the fungal virulence was attenuated by cellular immune responses, in which the conidia were phagocytosed by hemocytes (insect blood cells) and hyphal growth was inhibited by hemocyte encapsulation. However, the chemokine signal to drive hemocytes to the infection foci was little understood. The hemocyte behaviors appeared to be guided by a Ca2+ signal stimulating cell aggregation to the infection foci. The induction of the Ca2+ signal was significantly inhibited by the cyclooxygenase (COX) inhibitor. Under the inhibitory condition, the addition of thromboxane A2 or B2 (TXA2 or TXB2) among COX products was the most effective to recover the Ca2+ signal and hemocyte aggregation. TXB2 alone induced a microaggregation behavior of hemocytes under in vitro conditions. Indeed, TXB2 titer was significantly increased in the plasma of the infected larvae. The elevated TXB2 level was further supported by the induction of phospholipase A2 (PLA2) activity in the hemocytes and subsequent up-regulation of COX-like peroxinectins (SePOX-F and SePOX-H) in response to the fungal infection. Finally, the expression of a thromboxane synthase (Se-TXAS) gene was highly expressed in the hemocytes. RNA interference (RNAi) of Se-TXAS expression inhibited the Ca2+ signal and hemocyte aggregation around fungal hyphae, which were rescued by the addition of TXB2. Without any ortholog to mammalian thromboxane receptors, a prostaglandin receptor was essential to mediate TXB2 signal to elevate the Ca2+ signal and mediate hemocyte aggregation behavior. Specific inhibitor assays suggest that the downstream signal after binding TXB2 to the receptor follows the Ca2+-induced Ca2+ release pathway from the endoplasmic reticulum of the hemocytes. These results suggest that hemocyte aggregation induced by the fungal infection is triggered by TXB2 via a Ca2+ signal through a PG receptor.

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

AimsThromboxane (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 methodsUsing 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 findingsTP 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. SignificanceTP 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.

The role of thromboxane prostanoid receptor signaling in gastric ulcer healing.

The process of gastric ulcer healing includes cell migration, proliferation, angiogenesis and re‐epithelialization. Platelets contain angiogenesis stimulating factors that induce angiogenesis. Thromboxane A2 (TXA2) not only induces platelet activity but also angiogenesis. This study investigated the role of TXA2 in gastric ulcer healing using TXA2 receptor knockout (TPKO) mice. Gastric ulcer healing was suppressed by treatment with the TXA2 synthase inhibitor OKY‐046 and the TXA2 receptor antagonist S‐1452 compared with vehicle‐treated mice. TPKO showed delayed gastric ulcer healing compared with wild‐type mice (WT). The number of microvessels and CD31 expression were lower in TPKO than in WT mice, and TPKO suppressed the expression of transforming growth factor beta (TGF‐β) and vascular endothelial growth factor A (VEGF‐A) in areas around gastric ulcers. Immunofluorescence assays showed that TGF‐β and VEGF‐A co‐localized with platelets. Gastric ulcer healing was significantly reduced in WT mice transplanted with TPKO compared with WT bone marrow. These results suggested that TP signalling on platelets facilitates gastric ulcer healing through TGF‐β and VEGF‐A.

8-iso-prostaglandin E2 induces nasal obstruction via thromboxane receptor in murine model of allergic rhinitis.

Thromboxane receptor (TP) mediates nasal obstruction, a typical symptom of allergic rhinitis. Since it has been reported that several types of eicosanoids, such as non-enzymatic oxidation product of arachidonic acid isoprostane, act as a TP ligand, there is a possibility that some other eicosanoids contribute to the TP-mediated nasal obstruction. The aim of this study is to investigate the mechanisms of TP-mediated nasal obstruction. Intranasal challenges of ovalbumin (OVA) induced nasal obstruction in mice. Pharmacological blockade of TP receptor but not thromboxane A2 synthase inhibited OVA-induced nasal obstruction. Simultaneous analysis of eicosanoids in nasal lavage fluid and the responses in trans-endothelial resistance suggested that 8-iso-prostaglandin E2 (PGE2 ) can be a candidate for TP ligand. Intranasal challenge of 8-iso-PGE2 induced vascular hyperpermeability and nasal obstruction in TP receptor-dependent manner. Wholemount immunostaining of nasal septum mucosa revealed that 8-iso-PGE2 increased plasma leakage accompanied by distention of venous sinusoids. This study shows that 8-iso-PGE2 is a contributor in TP-mediated nasal obstruction in mice.

Thromboxane A2 synthase inhibition ameliorates endothelial dysfunction, memory deficits, oxidative stress and neuroinflammation in rat model of streptozotocin diabetes induced dementia

RationaleVascular dementia (VaD) is the second leading cause of dementia worldwide. It is very important to find the possible pharmacological agents which may be useful in management and therapy of VaD. ObjectivesThe present study investigates the effect of ozagrel, a selective thromboxane A2 (TXA2) synthase inhibitor, in a rat model of VaD. MethodsSingle intraperitoneal injection of streptozotocin [STZ, (50 mg/kg)] was administered to Wistar rats to induced diabetes-associated vascular endothelial dysfunction and memory impairment. Morris water maze (MWM) test was employed to assess learning and memory. Endothelial dysfunction was assessed in the isolated aorta by observing endothelial-dependent vasorelaxation and levels of serum nitrite. Various biochemical and histopathological estimations were also performed. ResultsSTZ treatment produced endothelial dysfunction, impairment of learning and memory, reduction in body weight and serum nitrite/nitrate, and increase in serum glucose, brain oxidative stress (increased brain thiobarbituric acid reactive species and decreased reduced glutathione levels), brain acetylcholinesterase activity and brain myeloperoxidase activity. Further a significant rise in brain tumor necrosis factor-α & interleukin-6 levels and brain neutrophil infiltration were also observed. Treatment of ozagrel (10 & 20 mg/kg, p. o.)/donepezil (0. 5 mg/kg, i.p., serving as standard) ameliorated STZ induced endothelial dysfunction; memory deficits; biochemical and histopathological changes. ConclusionsIt may be concluded that ozagrel markedly improved endothelial dysfunction; learning and memory; biochemical and histopathological alteration associated with STZ induced dementia and that TXA2 can be considered as an important therapeutic target for the management of VaD.

1213-P: A Novel Relationship between Thromboxane A2 Receptor and Obesity-Related Inflammation

Animal models of obesity have shown that the expression of Thromboxane A2 Receptor (tbxa2r) and thromboxane synthase (Tbxas1) are increased in adipose tissue (AT) and are associated with insulin resistance and inflammation. Objective: To compare Tbxa2r gene expression in the AT of human subjects with different degrees of obesity and to determine the association between AT Tbxa2r with BMI and markers of AT inflammation. Methods: Subcutaneous AT samples were collected from lean (BMI 40) subjects. Gene expression of Tbxa2r was analyzed in AT and compared among different groups. Correlation analysis to determine the association between AT Tbxa2r and obesity and obesity-related inflammation. Results: The mRNA levels of Tbxa2r were significantly higher in obese compared to normal subjects (P Conclusion: Signaling of TxA2 is higher in AT in obesity and is associated with BMI as well as inflammatory genes. Tbxa2r may be a potential therapeutic target for obesity-linked diseases including type 2 DM and CV disease. Disclosure D. Alcaraz alvarez: None. S. Viswanathan: None. T. Gopal: None. N. Kumar: None. K. Kelley: None. C. Desouza: Advisory Panel; Self; Bayer AG, Novo Nordisk, Consultant; Self; AstraZeneca.