Thromboxane A2 Receptor Research Articles

PM2.5 causes vascular hyperreactivity through the upregulation of the thromboxane A2 receptor and activation of MAPK pathways.

Airborne fine particulate matter (PM2.5) is a major cardiovascular disease environmental risk factor. However, the underlying mechanism of action is not fully understood. Thromboxane is widely known as an important vasoconstrictor substance that binds to G-protein-coupled receptors (GPCR) in arteries and is involved in various cardiovascular diseases. This study examined the effect of PM2.5 on thromboxane A2 receptor (TP) in the mesenteric arteries and the underlying intracellular signal mechanisms (by focusing on the mitogen-activated protein kinase (MAPK) pathway). Rat mesenteric artery segments were exposed to PM2.5 in the presence of MAPK pathway inhibitors. The contractile reactivity of mesenteric arteries was analyzed using wire myography. The mRNA and protein expression of TP receptor and MAPK pathway molecules were detected by real-time PCR and Western blot. Mesenteric artery receptor localization was assessed by immunohistochemistry. The results showed that TP receptor-mediated maximum contraction response was achieved after exposing arteries to 1.0μg/mL PM2.5 for 16h (Emax: 228 ± 16% of K+). Moreover, inhibitor U0126 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), and SP600125 (JNK inhibitor) depressed the increased TP receptor-mediated contractile responses (reduced rage were 17.9 ~ 59.6%). These inhibitors also decreased the increased mRNA expression and protein of the TP receptor induced by PM2.5 (reduced by more than 50% and 46%, respectively). The immunoreactivity of increased TP receptor expression was primarily localized in the cytoplasm. In addition, phosphorylation quantitative analysis showed that in the presence of MAPK inhibitors, the PM2.5-induced phosphorylation of ERK1/2, p38, and JNK protein increased by more than 30.0 ~ 130.3%. These results suggest that PM2.5 upregulates the TP receptor of rat mesenteric arteries through activation of the ERK1/2, p38, and JNK MAPK pathways.

Thromboxane A2 G Protein-Coupled Receptor Production and Crystallization for Structure Studies.

G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands.

ATP-sensitive K<sup>+</sup> channel activation in the brain is involved in elevation of plasma levels of adrenaline

Recently, we reported that restraint stress-induced elevation of plasma levels of adrenaline, but not of noradrenaline, was suppressed by intravenous and intracerebroventricular (i.c.v.) administration of glucose via brain thromboxane A2-dependent mechanisms. (Yamaguchi et al., 2022). Since glucose can be converted to ATP in the brain cells, in the present study, we investigate whether ATP-sensitive K+ channel (KATP channel) activity in the brain is involved in changes of plasma levels of catecholamines in urethane anesthetized rats. I.c.v. administered diazoxide (20 µg/rat), a KATP channel opener, and A-769662 (25 µg/rat), an AMPK activator, elevated plasma levels of adrenaline, but not noradrenaline. In contrast, i.c.v administered glibenclamide (25 µg/rat), a KATP channel antagonist, did not alter plasma levels of adrenaline. I.c.v. pretreatment with SQ29548 (10 µg/rat), a thromboxane A2 receptor antagonist, attenuated diazoxide- and A-7969662-induced elevation of plasma levels of adrenaline. These results suggest that KATP channel activation in the brain might trigger the activation of thromboxane A2 receptor, resulting in elevation of plasma levels of adrenaline.

Arterial thromboxane A2-induced transient contraction after IL-1β exposure

The involvement of thromboxane A2 (TXA2) in systemic inflammation and infection is well recognized. However, there are few reports on the involvement of prostanoids in warm shock (the initial pathology of sepsis). Previous studies showed that interleukin (IL)-1β causes a rapid inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mediated relaxation in peripheral blood vessels during warm shock. Furthermore, a transient contraction was seen before this relaxation occurred. The present study aimed to elucidate the mechanism of this transient contraction. We measured isometric tension changes in the superior mesenteric arteries from normal male Wistar rats by adding IL-1β at the point of maximum contraction by phenylephrine (Ph). The same study was performed for each vessel pretreated with various inhibitors, including SQ29548, a TXA2 receptor antagonist, 30 min before Ph contraction. In addition, the concentration of thromboxane B2 (TXB2) in SMA was measured by probe electrospray ionization. Treatment of endothelial vessels with cyclooxygenase 1 (COX1)/2 inhibitors SC560/NS398 and TXA2 receptor antagonist SQ29548 suppressed IL-1β–induced transient contractions. This transient contraction reaction was derived from TXA2. Additionally, gene expression of COX2/TXA2 synthetase and the concentration of TXB2 were significantly increased in IL-1β-exposed vessels. It was demonstrated for the first time in inflamed blood vessels that endothelial cell-derived COX2/TXA2 is induced before iNOS and causes transient contractions. TXA2 may be considered an early sign of warm shock or as a biological defense mechanism in the early stages of septic shock.

Mice Lacking PECAM-1 and Ceacam1 Have Enhanced Platelet Secretion and Thrombus Growth: Novel Link with PAR4.

The Ig-ITIM bearing receptors, PECAM-1 and CEACAM1, have been shown net negative regulators of platelet-collagen interactions and hemiITAM signaling pathways. In this study, a double knockout (DKO) mouse was developed with deleted PECAM-1 and CEACAM1 to study their combined contribution in platelet activation by glycoprotein VI, C-type lectin-like receptor 2, protease activated receptor (PAR4), ADP purinergic receptors, and thromboxane receptor (TP) A2 pathways. In addition, their collective contribution was examined in thrombus formation under high shear and microvascular thrombosis using in vivo models. DKO platelets responded normally to ADP purinergic receptors and the TP A2 pathway. However, DKO platelets released significantly higher amounts of P-selectin compared with hyper-responsive Pecam-1-/- or Ceacam1-/- versus wild-type (WT) upon stimulation with collagen-related peptide or rhodocytin. In contrast, DKO platelets showed increased amounts of P-selectin exposure upon stimulation with PAR4 agonist peptide or thrombin but not Pecam-1-/- , Ceacam1-/- , or WT platelets. Blockade of phospholipase C (PLC) or Rho A kinase revealed that DKO platelets enhanced α-granule release via PAR4/Gαq/PLC signaling without crosstalk with Src/Syk or G12/13 signaling pathways. Severely delayed clot retraction in vitro was observed in DKO phenotype. The DKO model revealed a significant increase in thrombus formation compared with the hyper-responsive Ceacam1-/- or Pecam-1-/- versus WT phenotype. DKO platelets have similar glycoprotein surface expression compared with Pecam-1-/- , Ceacam1-/- , and WT platelets. This study demonstrates that PECAM-1 and CEACAM1 work in concert to negatively regulate hemiITAM signaling, platelet-collagen interactions, and PAR4 Gαq protein- coupled signaling pathways. Both PECAM-1 and CEACAM1 are required for negative regulation of platelet activation and microvascular thrombosis in vivo.

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.

15-hydroxy eicosadienoic acid is an exacerbating factor for nasal congestion in mice.

Allergic rhinitis (AR) is one of the most common allergic inflammatory diseases worldwide. In AR, increased blood flow and vascular permeability in nasal mucosa cause rhinorrhea and nasal congestion. We investigated the role of an 11Z,14Z-eicosadienoic acid-derived metabolite, 15-hydroxy-11Z,13Z-eicosadienoic acid (15-HEDE), in functional changes in vasculature and nasal congestion in AR. Repeated intranasal administration of Ovalbumin (OVA) caused AR symptoms, such as sneezing and nasal congestion, in mice. OVA administration increased the level of 15-HEDE in nasal lavage fluid, which reached approximately 0.6ng/ml after ten OVA treatments. Upon measuring vascular contraction, treatment with 0.1-3μM 15-HEDE did not cause contraction in mouse aortae, while it dilated aortae that were pre-contracted by thromboxane receptor stimulation. Pretreatment with the voltage-gated K+ (KV ) channel inhibitor 4-aminopyridine significantly inhibited the 15-HEDE-induced vascular relaxation. Intravital imaging showed that administration of 1μg 15-HEDE dilated blood vessels, and Mile's assay demonstrated that this administration also caused dye leakage, indicating vascular hyperpermeability in mouse ears. Computed tomography scanning and morphological study revealed that administration of 3μg 15-HEDE narrowed nasal passages and thickened nasal mucosa in mice. Finally, we confirmed that treating mice with 3μg 15-HEDE caused rhinitis symptoms, such as abdominal breathing, and reduced respiratory frequency, suggesting nasal congestion. 15-HEDE caused vasodilation by activating KV channels and increased vascular permeability, which may lead to nasal congestion. Furthermore, 15-HEDE might be a new lipid mediator that exacerbates nasal congestion in AR.

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.

Abstract 10294: Endothelial Pannexin1-TRPV4 Signaling Regulates Pulmonary Arterial Pressure

The pulmonary endothelial cells (EC) dilate small pulmonary arteries (PAs) and maintain low pulmonary arterial pressure (PAP). Importantly, the loss of EC function contributes to elevated PAP in pulmonary hypertension (PH). Recent studies show that Pannexin 1 (Panx1), an ATP efflux pathway at the cell membrane, is a critical regulator of endothelial function. However, the impact of endothelial Panx1 (Panx1 EC ) on PA contractility and PAP is unknown. We observed that the extracellular ATP (eATP) levels are reduced in PAs from inducible, EC-specific Panx1 -/- (Panx1 EC -/- ) mice, identifying Panx1 EC as an important ATP release pathway in PAs. Our recently published findings demonstrated that eATP dilates PAs via activation of Ca 2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4 EC ) ion channels. Therefore, we hypothesized that endothelial Panx1—eATP—TRPV4 channel signaling dilates PAs and lowers resting PAP. Apyrase (10 U/mL), an ATP hydrolyzing enzyme, reduced the activity of TRPV4 EC sparklets (individual Ca 2+ influx signals through TRPV4 EC channels) in PAs, implying a constitutive regulation of TRPV4 EC channels by ATP. The activity of TRPV4 EC sparklets was lower in PAs from Panx1 EC -/- mice than controls, identifying Panx1 EC as a novel promoter of TRPV4 EC channel activity. Pressure myography studies in PAs (~ 50 μm) from Panx1 EC -/- and TRPV4 EC -/- mice showed higher constriction to thromboxane A2 receptor agonist U46619 (1-300 nM) compared to PAs from controls. Finally, right ventricular systolic pressure was higher in Panx1 EC -/- and TRPV4 EC -/- mice compared to the respective controls. These data indicated that Panx1 EC —eATP—TRPV4 EC channel signaling lowers PA contractility and PAP. ATP is an endogenous activator of purinergic receptor signaling. Inhibition of P2Y1 and P2X receptors did not alter ATP activation of TRPV4 EC sparklets in PAs. However, P2Y2 receptor inhibition and EC-specific P2Y2 receptor deletion abolished ATP activation of TRPV4 EC sparklets. Together, these data identify a novel Panx1 EC —eATP—P2Y2R EC signaling pathway that enhances TRPV4 EC channel activity and lowers PAP. Individual elements of this pathway may provide novel therapeutic targets for treating pulmonary vascular disorders.

Structure-Activity Relationship Analysis of Analogs of Rhosin, a RhoA Inhibitor, Reveals a New Generation of Improved Antiplatelet Agents

Platelet activation and aggregation play a key role in mediating hemostasis and thrombosis. The antiplatelet therapies currently available in the market are associated with a high risk of hemorrhage and are mostly irreversible in suppressing platelet activity; hence, there is a need to develop better therapeutic agents. Previous genetic and pharmacological studies have implicated the small GTPase RhoA in multiple platelet signaling pathways. We devised a lead RhoA activity-specific inhibitor, Rhosin/G04, based on the structure-function relationship of RhoA interaction with its activator, guanine nucleotide exchange factor (GEF) (Figure 1A). Rhosin/G04 binds to RhoA directly with micromolar affinity at a surface groove that is essential for GEF recognition and blocks GEF-mediated GTP loading to RhoA. Rhosin/G04 inhibits platelet spreading on fibrinogen and thrombin-induced platelet aggregation, mimicking effects of RhoA gene targeting. In the current work, we have utilized the inhibitory activity of G04 for platelet activation and its biochemical activity to define its structure-activity relationship (SAR) and to understand its mechanism of action in an effort to improve efficacy and druggability.The structure of G04 in a groove of RhoA interaction was hypothesized based on the docking studies using Molsoft ICM-Pro. Cincinnati Children's Hospital Medical Center's compound library of over 360,000 chemicals was scanned for G04 analogs by similarity and substructure searches. In the initial screen, a human platelet aggregation assay was performed at both a low concentration (1 µg/ml) and a high concentration (5 µg/ml) of collagen. The first round similarity search resulted in a set of 7 compounds (Set-1), from which, compound 177629 showed significantly enhanced potency relative to G04 (Figure 1B). The second round of similarity searches for compounds more closely related to 177629 (Set-2) identified 14 compounds. The third-round search for other related compounds (Set-3) led to 9 additional compounds that add to the understanding of the SAR. The compounds that showed enhanced antiplatelet activity were examined for their potency and selectivity in in vitro biochemical binding assays and in suppressing RhoA-GTP formation and downstream phosphorylation of myosin light chain (p-MLC) signaling in platelets. The active compounds were further examined for their anti-platelet activities under diverse stimuli including thrombin, ADP, U46619 (a stable thromboxane receptor agonist), and arachidonic acid.The most active compounds from Set-1, Set-2, and Set-3 inhibited platelet aggregation by at least 70% and showed IC 50 values below 6 µM. Of these compounds, 12 showed significantly greater potency than the initial compound, G04. The most active compounds were 177618, 177619, 177628, 177629, 177633, and 177634. These compounds specifically inhibited RhoA activity and blocked p-MLC. SAR analyses led us to believe that the quinoline is optimally attached to the hydrazine at the 4-position. The halogen (choloro- or trifluoromethyl-) substitution at the 7- or 8- position improved activity, and the 7- position may be slightly favored. The aryl group is considerably variable with similar potency between the indole, methylphenyl, and dichlorophenyl- groups.Rhosin/G04 is the R enantiomer (i.e. Rhosin is R-G04), so its S enantiomer, S-G04 was also evaluated (Figure 1C). S-G04 is significantly more potent than R-G04 in inhibiting collagen-stimulated RhoA-GTP formation and aggregation of platelets, and its effect is completely reversible by washing the platelets. Finally, R-G04 and S-G04 showed differential inhibition of arachidonic acid and U46619 stimulated primary and secondary aggregation, highlighting the potential utilities of the inhibitors in dissecting different platelet activation mechanisms. S-G04 is active in inhibiting thrombin, ADP, U46619, and arachidonic acid-mediated platelet activation at submicromolar concentration, suggesting a broad role of RhoA signaling in integrating platelet signal cross talk.In summary, evaluation of Rhosin/R-G04 analogs in a platelet activity screen identified a new generation of improved small-molecule RhoA inhibitors, including an enantiomer with significantly improved efficacy. These analog studies of novel anti-platelet agents provide a new approach to effectively and reversibly manipulate platelet activities. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs.

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

The Role of Thromboxane in the Course and Treatment of Ischemic Stroke: Review.

Cardiovascular diseases are currently among the leading causes of morbidity and mortality in many developed countries. They are distinguished by chronic and latent development, a course with stages of worsening of symptoms and a period of improvement, and a constant potential threat to life. One of the most important disorders in cardiovascular disease is ischemic stroke. The causes of ischemic stroke can be divided into non-modifiable and modifiable causes. One treatment modality from a neurological point of view is acetylsalicylic acid (ASA), which blocks cyclooxygenase and, thus, thromboxane synthesis. The legitimacy of its administration does not raise any doubts in the case of the acute phase of stroke in patients in whom thrombolytic treatment cannot be initiated. The measurement of thromboxane B2 (TxB2) in serum (a stable metabolic product of TxA2) is the only test that measures the effect of aspirin on the activity of COX-1 in platelets. Measurement of thromboxane B2 may be a potential biomarker of vascular disease risk in patients treated with aspirin. The aim of this study is to present the role of thromboxane B2 in ischemic stroke and to present effective therapies for the treatment of ischemic stroke. Scientific articles from the PubMed database were used for the work, which were selected on the basis of a search for “thromboxane and stroke”. Subsequently, a restriction was introduced for works older than 10 years, those concerning animals, and those without full text access. Ultimately, 58 articles were selected. It was shown that a high concentration of TXB2 may be a risk factor for ischemic stroke or ischemic heart disease. However, there is insufficient evidence to suggest that thromboxane could be used in clinical practice as a marker of ischemic stroke. The inclusion of ASA in the prevention of stroke has a beneficial effect that is associated with the effect on thromboxane. However, its insufficient power in 25% or even 50% of the population should be taken into account. An alternative and/or additional therapy could be a selective antagonist of the thromboxane receptor. Thromboxane A2 production is inhibited by estrogen; therefore, the risk of CVD after the menopause and among men is higher. More research is needed in this area.

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 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.

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, >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

2-Methoxyestradiol Ameliorates Angiotensin II-Induced Hypertension by Inhibiting Cytosolic Phospholipase A2α Activity in Female Mice.

[Figure: see text].

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.

Endothelial mineralocorticoid receptor ablation confers protection towards endothelial dysfunction in experimental diabetes in mice.

With diabetes comes a significant risk of macrovascular and microvascular complications. Circulating aldosterone levels increase in patients with diabetes. Aldosterone can directly affect vascular function via activation of the mineralocorticoid receptor (MR). We hypothesized that aldosterone via endothelial MR impairs endothelial function in a murine model of experimental diabetes. Endothelial cell-specific mineralocorticoid receptor knockout MRflox/flox ; Tie2-Cre mice (ECMR-KO) and wild-type FVB littermates were subjected to an experimental type-1 diabetic model by low dose streptozotocin injections (55mg/kg/day) for five consecutive days. After 10weeks of diabetes, second-order mesenteric resistance arteries were perfused ex vivo to evaluate vessel contractility and endothelial function. The effect of ex vivo incubation with aldosterone with and without the antagonist, spironolactone was determined. Diabetic ECMR-KO and wild-type mice had similar, elevated, plasma aldosterone concentration while only diabetic wild-type mice displayed elevated urine albumin excretion and cardiac and kidney hypertrophy at 10weeks. There were no differences in contraction (Emax and EC50 ) to thromboxane receptor agonist (U46619) and elevated K+ between groups. Wild-type diabetic mice showed impaired acetylcholine (ACh)-dependent relaxation, while diabetic ECMR-KO mice had intact ACh-mediated relaxation. Aldosterone incubation ex vivo impaired ACh mediated relaxation and rendered responses similar to diabetic WT arteries. Direct, ex vivo aldosterone effects were absent in ECMR-KO animals. Ex vivo inhibitory effects of aldosterone on endothelial relaxation in arteries from WT were abolished by spironolactone. These findings show that endothelial cell mineralocorticoid receptor activation accounts for diabetes-induced systemic endothelial dysfunction in experimental diabetes and may explain the cardiovascular protection by MR antagonists in diabetes.

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.