Glucagon-like Peptide-1 Receptor Pathway Attenuates Platelet Activation in Aspirin-Exacerbated Respiratory Disease.

Platelets are key contributors to allergic asthma and aspirin-exacerbated respiratory disease (AERD), an asthma phenotype involving platelet activation and IL-33-dependent mast cell activation. Human platelets express the glucagon-like peptide-1 receptor (GLP-1R). GLP-1R agonists decrease lung IL-33 release and airway hyperresponsiveness in mouse asthma models. We hypothesized that GLP-1R agonists reduce platelet activation and downstream platelet-mediated airway inflammation in AERD. GLP-1R expression on murine platelets was assessed using flow cytometry. We tested the effect of the GLP-1R agonist liraglutide on lysine-aspirin (Lys-ASA)-induced changes in airway resistance, and platelet-derived mediator release in a murine AERD model. We conducted a prospective cohort study comparing the effect of pretreatment with liraglutide or vehicle on thromboxane receptor agonist-induced in vitro activation of platelets from patients with AERD and nonasthmatic controls. GLP-1R expression was higher on murine platelets than on leukocytes. A single dose of liraglutide inhibited Lys-ASA-induced increases in airway resistance and decreased markers of platelet activation and recruitment to the lung in AERD-like mice. Liraglutide attenuated thromboxane receptor agonist-induced activation as measured by CXCL7 release in plasma from patients with AERD and CD62P expression in platelets from both patients with AERD (n = 31) and nonasthmatic, healthy controls (n = 11). Liraglutide, a Food and Drug Administration-approved GLP-1R agonist for treatment of type 2 diabetes and obesity, attenuates in vivo platelet activation in an AERD murine model and in vitro activation in human platelets in patients with and without AERD. These data advance the GLP-1R axis as a new target for platelet-mediated inflammation warranting further study in asthma.

Alpha-linolenic acid selectively inhibits the contraction of pig coronary arteries mediated through prostanoid TP receptors.

We examined the inhibitory effects of α-linolenic acid (ALA) on the contractions of pig coronary arteries. ALA concentration-dependently inhibited the contractions elicited by U46619 and prostaglandin F2α without affecting those elicited by 80 mM KCl, histamine, acetylcholine, and serotonin. ALA rightward shifted the concentration-response curve of U46619, and Schild plot analysis revealed that ALA competitively antagonized U46619. Furthermore, ALA inhibited the increase in intracellular Ca2+ concentration caused by TP receptor stimulation but not that caused by FP receptor stimulation. These results suggest that ALA behaves as a selective antagonist of TP receptors in coronary arteries.

Trial of thromboxane receptor inhibition with ifetroban: TP receptors regulate eicosanoid homeostasis in aspirin-exacerbated respiratory disease.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is the triad of asthma, nasal polyposis, and respiratory reactions to COX-1 inhibitors. Overproduction of cysteinyl leukotrienes and underproduction of prostaglandin E2 (PGE2) are hallmarks of AERD. A mouse model predicted a key role for the thromboxane-prostanoid (TP) receptor in AERD. OBJECTIVE: Our aim was to determine whether ifetroban, a TP receptor antagonist, attenuates aspirin-induced respiratory symptoms in patients with AERD. METHODS: A total of 35 patients with AERD completed a 4-week double-blinded, placebo-controlled trial of ifetroban and underwent an oral aspirin challenge. The primary outcome was change in the provocative dose of aspirin that caused a 2-point increase in Total Nasal Symptom Score. Changes in lung function, eicosanoid levels, and platelet and mast cell activation were assessed. Cultured human nasal fibroblasts were stimulated with or without the TP agonist U46619 and assayed for prostanoid production. RESULTS: Ifetroban was well tolerated in AERD and did not change the mean 2-point increase in Total Nasal Symptom Score (P = .763). Participants taking ifetroban had greater aspirin-induced nasal symptoms and a greater decline in FEV1 value than did participants receiving placebo (-18.8% ± 3.6% with ifetroban vs -8.4% ± 2.1% with placebo [P = .017]). Four weeks of ifetroban significantly increased urinary leukotriene E4 levels and decreased nasal PGE2 levels compared with placebo. Peak aspirin-induced urinary thromboxane levels correlated with peak urinary leukotriene E4 and prostaglandin D2 metabolite levels in participants taking ifetroban. U46119 significantly potentiated the production of PGE2 by cultured nasal fibroblasts from subjects with AERD but not by cultured nasal fibroblasts from controls without polypoid sinusitis. CONCLUSION: Contrary to our hypothesis, TP receptor blockade worsened aspirin-induced reactions in AERD, possibly by exacerbating dysregulation of the eicosanoid system. TP signaling on stromal cells may be critical to maintaining PGE2 production when COX-2 function is low.

Prostaglandin F2α evokes vasoconstrictor and vasodepressor activities that are both independent of the F prostanoid receptor.

The F prostanoid receptor (FP), which accounts for the therapeutic effect of PGF2α in uterine atony that leads to postpartum hemorrhage and maternal morbidity, could possibly mediate vasoconstrictor effect in small or resistance arteries to elevate blood pressure that limits the clinical use of the agent in patients with cardiovascular disorders. This study aimed to test the above hypothesis with genetically altered mice. Ex vivo and in vivo experiments were performed on control wild-type (WT) mice and mice with deficiencies in FP (FP-/- ) or thromboxane (Tx)-prostanoid receptor (the original receptor of TxA2 ; TP-/- ), and/or those with an additional deficiency in E prostanoid receptor-3 (one of the vasoconstrictor receptors of PGE2 ; EP3-/- ). Here, we show that PGF2α indeed evoked vasoconstrictor responses in the above-mentioned tissues of WT mice, which were however unaltered by FP-/- . Interestingly, such contractile responses were reversed into dilations by TP-/- /EP3-/- . A similar pattern of results was observed with the pressor effect of PGF2α under in vivo conditions. However, TP-/- alone (which could largely remove the contractile responses) did not result in relaxation to PGF2α . Also, either the ex vivo vasodilator effect or the in vivo depressor response of PGF2α obtained after the removal of TP and EP3-mediated actions was unaltered by FP-/- . Therefore, both the ex vivo vasoconstrictor action in small or resistance arteries and the systemic pressor effect of PGF2α can reflect vasoconstrictor activities derived from the non-FP receptors TP and EP3 outweighing a concurrently activated dilator effect, which is again independent of FP.

A Thromboxane A2 Receptor-Driven COX-2-Dependent Feedback Loop That Affects Endothelial Homeostasis and Angiogenesis.

BACKGROUND: TP (thromboxane A2 receptor) plays an eminent role in the pathophysiology of endothelial dysfunction and cardiovascular disease. Moreover, its expression is reported to increase in the intimal layer of blood vessels of cardiovascular high-risk individuals. Yet it is unknown, whether TP upregulation per se has the potential to affect the homeostasis of the vascular endothelium. METHODS: We combined global transcriptome analysis, lipid mediator profiling, functional cell analyses, and in vivo angiogenesis assays to study the effects of endothelial TP overexpression or knockdown/knockout on the angiogenic capacity of endothelial cells in vitro and in vivo. RESULTS: Here we report that endothelial TP expression induces COX-2 (cyclooxygenase-2) in a Gi/o- and Gq/11-dependent manner, thereby promoting its own activation via the auto/paracrine release of TP agonists, such as PGH2 (prostaglandin H2) or prostaglandin F2 but not TxA2 (thromboxane A2). TP overexpression induces endothelial cell tension and aberrant cell morphology, affects focal adhesion dynamics, and inhibits the angiogenic capacity of human endothelial cells in vitro and in vivo, whereas TP knockdown or endothelial-specific TP knockout exerts opposing effects. Consequently, this TP-dependent feedback loop is disrupted by pharmacological TP or COX-2 inhibition and by genetic reconstitution of PGH2-metabolizing prostacyclin synthase even in the absence of functional prostacyclin receptor expression. CONCLUSIONS: Our work uncovers a TP-driven COX-2-dependent feedback loop and important effector mechanisms that directly link TP upregulation to angiostatic TP signaling in endothelial cells. By these previously unrecognized mechanisms, pathological endothelial upregulation of the TP could directly foster endothelial dysfunction, microvascular rarefaction, and systemic hypertension even in the absence of exogenous sources of TP agonists.

Pharmacological study on the enhancing effects of U46619 on guinea pig urinary bladder smooth muscle contraction induced by acetylcholine and α,ß-methylene ATP and the possible involvement of protein kinase C.

We examined whether U46619 (a prostanoid TP receptor agonist) could enhance the contractions of guinea pig urinary bladder smooth muscle (UBSM) in response to acetylcholine (ACh) and an ATP analog (α,ß-methylene ATP (αß-MeATP)) through stimulation of the UBSM TP receptor and whether protein kinase C (PKC) is involved. U46619 (10-7 M) markedly enhanced UBSM contractions induced by electrical field stimulation and ACh/αß-MeATP (3 × 10-6 M each), the potentiation of which was completely suppressed by SQ 29,548 (a TP receptor antagonist, 6 × 10-7 M). PKC inhibitors did not attenuate the ACh-induced contractions enhanced by U46619 although they partly suppressed the U46619-enhanced, αß-MeATP-induced contractions. While phorbol 12-myristate 13-acetate (PMA, a PKC activator, 10-6 M) did not enhance ACh-induced contractions, it enhanced αß-MeATP-induced contractions, an effect that was completely suppressed by PKC inhibitors. αß-MeATP-induced contractions, both with and without U46619 enhancement, were strongly inhibited by diltiazem. U46619/PMA enhanced 50 mM KCl-induced contractions, the potentiation of which was partly/completely attenuated by PKC inhibitors. These findings suggest that U46619 potentiates parasympathetic nerve-associated UBSM contractions by stimulating UBSM TP receptors. PKC-increased Ca2+ influx through voltage-dependent Ca2+ channels may partially play a role in purinergic receptor-mediated UBSM contractions enhanced by TP receptor stimulation.

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-ß (transforming growth factor-ß) 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.

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-ß) and vascular endothelial growth factor A (VEGF-A) in areas around gastric ulcers. Immunofluorescence assays showed that TGF-ß 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-ß 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.

Endothelium-dependent contraction: The non-classical action of endothelial prostacyclin, its underlying mechanisms, and implications.

Although commonly thought to produce prostacyclin (prostaglandin I2 ; PGI2 ) that evokes vasodilatation and protects vessels from the development of diseases, the endothelial cyclooxygenase (COX)-mediated metabolism has also been found to release substance(s) called endothelium-derived contracting factor(s) (EDCF) that causes endothelium-dependent contraction and implicates in endothelial dysfunction of disease conditions. Various mechanisms have been proposed for the process; however, the major endothelial COX metabolite PGI2 , which has been classically considered to activate the I prostanoid receptor (IP) that mediates vasodilatation and opposes the effects of thromboxane (Tx) A2 produced by COX in platelets, emerges as a major EDCF in health and disease conditions. Our recent studies from genetically altered mice further suggest that vasomotor reactions to PGI2 are collectively modulated by IP, the vasoconstrictor Tx-prostanoid receptor (TP; the prototype receptor of TxA2 ) and E prostanoid receptor-3 (EP3; a vasoconstrictor receptor of PGE2 ) although with differences in potency and efficacy; a contraction to PGI2 reflects activities of TP and/or EP3 outweighing that of the concurrently activated IP. Here, we discuss the history of endothelium-dependent contraction, evidences that support the above hypothesis, proposed mechanisms for the varied reactions to endothelial PGI2 synthesis as well as the relation of its dilator activity to the effect of another NO-independent vasodilator mechanism, the endothelium-derived hyperpolarizing factor. Also, we address the possible pathological and therapeutic implications as well as questions remaining to be resolved or limitations of our above findings obtained from genetically altered mouse models.

Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles.

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10-5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10-6 M) and ONO-8130 (a prostanoid EP1 receptor antagonist, 10-7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10-7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10-5 M) or L-798,106 (a prostanoid EP3 receptor antagonist, 10-8 M). NP-1815-PX (10-5-10-4 M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F2α (PGF2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10-5-10-4 M) strongly suppressed the U46619-induced increase in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10-4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

The Role and Regulation of Thromboxane A2 Signaling in Cancer-Trojan Horses and Misdirection.

Over the last two decades, there has been an increasing awareness of the role of eicosanoids in the development and progression of several types of cancer, including breast, prostate, lung, and colorectal cancers. Several processes involved in cancer development, such as cell growth, migration, and angiogenesis, are regulated by the arachidonic acid derivative thromboxane A2 (TXA2). Higher levels of circulating TXA2 are observed in patients with multiple cancers, and this is accompanied by overexpression of TXA2 synthase (TBXAS1, TXA2S) and/or TXA2 receptors (TBXA2R, TP). Overexpression of TXA2S or TP in tumor cells is generally associated with poor prognosis, reduced survival, and metastatic disease. However, the role of TXA2 signaling in the stroma during oncogenesis has been underappreciated. TXA2 signaling regulates the tumor microenvironment by modulating angiogenic potential, tumor ECM stiffness, and host immune response. Moreover, the by-products of TXA2S are highly mutagenic and oncogenic, adding to the overall phenotype where TXA2 synthesis promotes tumor formation at various levels. The stability of synthetic enzymes and receptors in this pathway in most cancers (with few mutations reported) suggests that TXA2 signaling is a viable target for adjunct therapy in various tumors to reduce immune evasion, primary tumor growth, and metastasis.

Isoprostanes evoke contraction of the murine and human detrusor muscle via activation of the thromboxane prostanoid TP receptor and Rho kinase.

Local or systemic inflammation can severely impair urinary bladder functions and contribute to the development of voiding disorders in millions of people worldwide. Isoprostanes are inflammatory lipid mediators that are upregulated in the blood and urine by oxidative stress and may potentially induce detrusor overactivity. The aim of the present study was to investigate the effects and signal transduction of isoprostanes in human and murine urinary bladders in order to provide potential pharmacological targets in detrusor overactivity. Contraction force was measured with a myograph in murine and human urinary bladder smooth muscle (UBSM) ex vivo. Isoprostane 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the murine UBSM, which was abolished in mice deficient in the thromboxane prostanoid (TP) receptor. The responses remained unaltered after removal of the mucosa or incubation with tetrodotoxin. Smooth muscle-specific deletion of Gα12/13 protein or inhibition of Rho kinase by Y-27632 decreased the contractions. In Gαq/11-knockout mice, responses were reduced and in the presence of Y-27632 abolished completely. In human UBSM, the TP agonist U-46619 evoked dose-dependent contractions. Neither atropine nor the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid decreased the effect, indicating that TP receptors directly mediate detrusor muscle contraction. 8-iso-PGE2 and 8-iso-PGF2α evoked dose-dependent contraction in the human UBSM, and these responses were abolished by the TP antagonist SQ-29548 and were decreased by Y-27632. Our results indicate that isoprostanes evoke contraction in murine and human urinary bladders, an effect mediated by the TP receptor. The G12/13-Rho-Rho kinase pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target in detrusor overactivity.NEW & NOTEWORTHY Voiding disorders affect millions of people worldwide. Inflammation can impair urinary bladder functions and contribute to the development of detrusor overactivity. The effects and signal transduction of inflammatory lipid mediator isoprostanes were studied in human and murine urinary bladders ex vivo. We found that isoprostanes evoke contraction, an effect mediated by thromboxane prostanoid receptors. The G12/13-Rho-Rho kinase signaling pathway plays a significant role in mediating the contraction and therefore may be a potential therapeutic target.

Vitamin D Deficiency Reduces Vascular Reactivity of Coronary Arterioles in Male Rats.

BACKGROUND: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. METHODS: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. RESULTS: Thromboxane A2 (TXA2)-agonist induced reduced vasoconstriction, testosterone (T) and 17-ß-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. CONCLUSIONS: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA2 and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.

Differential properties of E prostanoid receptor-3 and thromboxane prostanoid receptor in activation by prostacyclin to evoke vasoconstrictor response in the mouse renal vasculature.

Vasomotor reactions of prostacyclin (prostaglandin I2 ; PGI2 ) can be collectively modulated by thromboxane prostanoid receptor (TP), E-prostanoid receptor-3 (EP3), and the vasodilator I prostanoid receptor (IP). This study aimed to determine the direct effect of PGI2 on renal arteries and/or the whole renal vasculature and how each of these receptors is involved. Experiments were performed on vessels or perfused kidneys of wild-type mice and/or mice with deficiency in TP (TP-/- ) and/or EP3. Here we show that PGI2 did not evoke relaxation, but instead resulted in contraction of main renal arteries (from ~0.001-0.01 µM) or reduction of flow in perfused kidneys (from ~1 µM); either of them was reversed into a dilator response in TP-/- /EP3-/- counterparts. Also, we found that in renal arteries although it has a lesser effect than TP-/- on the maximal contraction to PGI2 (10 µM), EP3-/- but not TP-/- resulted in relaxation to the prostanoid at 0.01-1 µM. Meanwhile, TP-/- only significantly reduced the contractile activity evoked by PGI2 at ≥0.1 µM. These results demonstrate that PGI2 may evoke an overall vasoconstrictor response in the mouse renal vasculature, reflecting activities of TP and EP3 outweighing that of the vasodilator IP. Also, our results suggest that EP3, on which PGI2 can have a potency similar to that on IP, plays a major role in the vasoconstrictor effect of the prostanoid of low concentrations (≤1 µM), while TP, on which PGI2 has a lower potency but higher efficacy, accounts for a larger part of its maximal contractile activity.

Prostaglandin E2 sequentially activates E-prostanoid receptor-3 and thromboxane prostanoid receptor to evoke contraction and increase in resistance of the mouse renal vasculature.

Although recognized to have an in vivo vasodepressor effect blunted by the vasoconstrictor effect of E-prostanoid receptor-3 (EP3), prostaglandin E2 (PGE2 ) evokes contractions of many vascular beds that are sensitive to antagonizing the thromboxane prostanoid receptor (TP). This study aimed to determine the direct effect of PGE2 on renal arteries and/or the whole renal vasculature and how each of these two receptors is involved in the responses. Experiments were performed on isolated vessels and perfused kidneys of wild-type mice and/or mice with deficiency in TP (TP-/- ), EP3 (EP3-/- ), or both TP and EP3 (TP-/- /EP3-/- ). Here we show that PGE2 (0.001-30 µM) evoked not only contraction of main renal arteries, but also a decrease of flow in perfused kidneys. EP3-/- diminished the response to 0.001-0.3 µM PGE2 , while TP-/- reduced that to the prostanoid of higher concentrations. In TP-/- /EP3-/- vessels and perfused kidneys, PGE2 did not evoke contraction but instead resulted in vasodilator responses. These results demonstrate that PGE2 functions as an overall direct vasoconstrictor of the mouse renal vasculature with an effect reflecting the vasoconstrictor activities outweighing that of dilation. Also, our results suggest that EP3 dominates the vasoconstrictor effect of PGE2 of low concentrations (≤0.001-0.3 µM), but its effect is further added by that of TP, which has a higher efficacy, although activated by higher concentrations (from 0.01 µM) of the same prostanoid PGE2 .

Low-dose Aspirin prevents hypertension and cardiac fibrosis when thromboxane A2 is unrestrained.

Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A2 in their pathophysiology remains unclear. We investigated the systemic TXA2 biosynthesis in vivo and gene expression of its receptor TP in 22 essential hypertension patients and a mouse model of salt-sensitive hypertension. The contribution of platelet TXA2 biosynthesis on enhanced blood pressure (BP) and overload-induced cardiac fibrosis was explored in mice by treating with low-dose Aspirin, resulting in selective inhibition of platelet cyclooxygenase (COX)-1-dependent TXA2 generation. In essential hypertensive patients, systemic biosynthesis of TXA2 [assessed by measuring its urinary metabolites (TXM) reflecting predominant platelet source] was enhanced together with higher gene expression of circulating leukocyte TP and TGF-ß, vs. normotensive controls. Similarly, in hypertensive mice with prostacyclin (PGI2) receptor (IP) deletion (IPKO) fed with a high-salt diet, enhanced urinary TXM, and left ventricular TP overexpression were detected vs. normotensive wildtype (WT) mice. Increased cardiac collagen deposition and profibrotic gene expression (including TGF-ß) was found. Low-dose Aspirin administration caused a selective inhibition of platelet TXA2 biosynthesis and mitigated enhanced blood pressure, cardiac fibrosis, and left ventricular profibrotic gene expression in IPKO but not WT mice. Moreover, the number of myofibroblasts and extravasated platelets in the heart was reduced. In cocultures of human platelets and myofibroblasts, platelet TXA2 induced profibrotic gene expression, including TGF-ß1. In conclusion, our results support tailoring low-dose Aspirin treatment in hypertensive patients with unconstrained TXA2/TP pathway to reduce blood pressure and prevent early cardiac fibrosis.

Expression of ADP receptor P2Y12, thromboxane A2 receptor and C-type lectin-like receptor 2 in cord blood-derived megakaryopoiesis.

The ADP receptor P2Y12, the thromboxane A2 receptor (TXA2R) and the C-type lectin-like receptor 2 (CLEC-2) mediate platelet activation by different mechanisms. Only little is known about the expression of the receptors in human megakaryopoiesis. Our study aimed to establish a flow cytometry (FC) method for the measurement of P2Y12, TXA2R, and CLEC-2 on platelets of healthy donors and to monitor receptor expression in ex vivo megakaryopoiesis. We determined mean fluorescence intensity (MFI) values of FITC, PE, or APC labeled antibodies binding to the receptors on platelets of 90 healthy donors. For cord blood-derived megakaryopoiesis (CBMK) differentiation of CD34+ cells was induced by IL-3, SCF, and TPO. At 6 time points between day 0 and day 21 of cell culture the MFI values for CD34, CD41, CD61, P2Y12, TXA2R, and CLEC-2 were measured. Quantitative PCR was used for relative quantification of the corresponding mRNA. Transcription factor (TF) binding sites were predicted by in silico analysis of the genes. Platelets showed expectable high MFI values for the platelet marker CD41 (13,716 median MFI). Lower MFI was found for P2Y12 (2,847 median MFI) and CLEC-2 (1,211 median MFI), whereas, binding of the TXA2R antibody revealed even higher values (21,458 median MFI) than CD41. In CBMK the CD34+ cells were negative for P2Y12, TXA2R, and CLEC-2 at day 0. A maximum of 21-fold and 6-fold increase of P2Y12 and TXA2R MFI values, respectively, was found on day 14 to 17. MFI for CLEC-2 increased by 58-fold within the first week and reached a maximum of 1,572-fold increase within the first two weeks of CBMK. Very similar results were obtained on the RNA level. The differential regulation of receptor expression in CBMK was further supported by significant differences in the numbers and types of TF binding sites. P2Y12 and TXA2R, both upregulated only to a low extent in CBMK, probably, are dispensable for megakaryopoiesis. Furthermore, we speculate that CLEC-2 strongly upregulated in early CMBK is important for megakaryopoiesis.

TP and/or EP3 receptors mediate the vasoconstrictor and pressor responses of prostaglandin F2α in mice and/or humans.

The vasoconstrictor and/or pressor effects of prostaglandin (PG)F2α participate in the development of vascular pathologies and limit the clinical use of the agent. This study aimed to determine the receptor types responsible for the vasoconstrictor activity of PGF2α and whether they mediate the pressor response evoked by the prostanoid under in vivo conditions. Experiments were performed on genetically altered mice and/or on vessels from these mice or humans. Here we show that deletion of the thromboxane-prostanoid receptor (TP-/-) abolished or drastically diminished the contraction to PGF2α in isolated mouse vessels (some of which were resistance arteries) and reduced the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In accordance, TP antagonism abolished the contraction in small arteries of human omentum. Further deletion of E prostanoid receptor type 3 (EP3-/-) removed the PGF2α-evoked contraction that remained in some TP-/- arteries and added to the effect of TP-/- on the elevation in blood pressure evoked by the prostanoid under in vivo conditions. In contrast, the uterine contraction to PGF2α mediated via the F prostanoid receptor (FP) was unaltered in TP-/-/EP3-/- mice. These results demonstrate that the non-FP receptors TP and/or EP3 mediate the vasoconstrictor and pressor effects of PGF2α, which are still of concern under clinical conditions.-Liu, B., Li, J., Yan, H., Tian, D., Li, H., Zhang, Y., Guo, T., Wu, X., Luo, W., Zhou, Y. TP and/or EP3 receptors mediate the vasoconstrictor and pressor responses of prostaglandin F2α in mice and/or humans.

No effect of endoperoxide 4 or thromboxane A2 receptor blockade on static mechanoreflex activation in rats with heart failure.

NEW FINDINGS: What is the central question of this study? Do endoperoxide 4 and thromboxane A2 receptors, which are receptors for cyclooxygenase products of arachidonic metabolism, on thin fibre muscle afferents play a role in the chronic mechanoreflex sensitization present in rats with heart failure with reduced ejection fraction (HF-rEF)? What is the main finding and its importance? The data do not support a role for endoperoxide 4 receptors or thromboxane A2 receptors in the chronic mechanoreflex sensitization in HF-rEF rats. ABSTRACT: We investigated the role of cyclooxygenase metabolite-associated endoperoxide 4 receptors (EP4-R) and thromboxane A2 receptors (TxA2 -R) on thin fibre muscle afferents in the chronic mechanoreflex sensitization in rats with myocardial infarction-induced heart failure with reduced ejection fraction (HF-rEF). We hypothesized that injection of either the EP4-R antagonist L-161,982 (1 µg) or the TxA2 -R antagonist daltroban (80 µg) into the arterial supply of the hindlimb would reduce the increase in blood pressure and renal sympathetic nerve activity (RSNA) evoked in response to 30 s of static hindlimb skeletal muscle stretch (a model of isolated mechanoreflex activation) in decerebrate, unanaesthetized HF-rEF rats but not sham-operated control rats (SHAM). Ejection fraction was significantly reduced in HF-rEF (45 ± 11%) compared to SHAM (83 ± 6%; P < 0.01) rats. In SHAM and HF-rEF rats, we found that the EP4-R antagonist had no effect on the peak increase in mean arterial pressure (peak ΔMAP SHAM n = 6, pre: 15 ± 7, post: 15 ± 9, P = 0.99; HF-rEF n = 9, pre: 30 ± 11, post: 32 ± 15 mmHg, P = 0.84) or peak increase in RSNA (peak ΔRSNA SHAM pre: 33 ± 14, post: 47 ± 31%, P = 0.94; HF-rEF, pre: 109 ± 47, post: 139 ± 150%, P = 0.76) response to stretch. Similarly, in SHAM and HF-rEF rats, we found that the TxA2 -R antagonist had no effect on the peak ΔMAP (SHAM n = 7, pre: 13 ± 7, post: 19 ± 14, P = 0.15; HF-rEF n = 14, pre: 24 ± 13, post: 21 ± 13 mmHg, P = 0.47) or peak ΔRSNA (SHAM pre: 52 ± 43, post: 57 ± 67%, P = 0.94; HF-rEF, pre: 108 ± 93, post: 88 ± 72%, P = 0.30) response to stretch. The data do not support a role for EP4-Rs or TxA2 -Rs in the chronic mechanoreflex sensitization in HF-rEF.