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Background: Abnormal accumulation of senescent cells in the vessel wall leads to a compromised vascular function contributing to vascular aging. Soluble DPP4 (dipeptidyl peptidase 4; sDPP4) secretion from visceral adipose tissue is enhanced in obesity, now considered a progeric condition. sDPP4 triggers vascular deleterious effects, albeit its contribution to vascular aging is unknown. We aimed to explore sDPP4 involvement in vascular aging, unraveling the molecular pathway by which sDPP4 acts on the endothelium. Methods: Human endothelial cell senescence was assessed by senescence-associated β-galactosidase assay, visualization of DNA damage, and expression of prosenescent markers, whereas vascular function was evaluated by myography over human dissected microvessels. In visceral adipose tissue biopsies from a cohort of obese patients, we explored several age-related parameters in vitro and ex vivo. Results: By a common mechanism, sDPP4 triggers endothelial cell senescence and endothelial dysfunction in isolated human resistance arteries. sDPP4 activates the metabotropic receptor PAR2 (protease-activated receptor 2), COX-2 (cyclooxygenase 2) activity, and the production of TXA 2 (thromboxane A 2 ) acting over TP (thromboxane receptor) receptors (PAR2–COX-2–TP axis), leading to NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3) inflammasome activation. Obese patients exhibited impaired microarterial functionality in comparison to control nonobese counterparts. Importantly, endothelial dysfunction in obese patients positively correlated with greater expression of DPP4, prosenescent, and proinflammatory markers in visceral adipose tissue nearby the resistance arteries. Moreover, when DPP4 activity or sDPP4-induced prosenescent mechanism was blocked, endothelial dysfunction was restored back to levels of healthy subjects. Conclusions: These results reveal sDPP4 as a relevant mediator in early vascular aging and highlight its capacity activating main proinflammatory mediators in the endothelium that might be pharmacologically tackled.

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.

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 A

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, &lt, 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-β-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.

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT-qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D

The F2-isoprostane 8-iso-PGF

The F prostanoid receptor (FP), which accounts for the therapeutic effect of PGF

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.

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

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 AngII (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to AngII-mediated arterial constriction and thus to development of AngII-induced hypertension. AngII constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nM) alone was without effect; however, it significantly enhanced AngII-induced constriction. The presence of wild type macrophages also enhanced the AngII-induced constriction, while Alox15(−/−) macrophages did not. Using this model, pre-treatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-, AngII receptor-2- and superoxide-mediated enhancing effect of 12(S)-HETE on AngII constrictions. The role of superoxide was confirmed using aortas from p47(phox−/−) mice where 12(S)-HETE failed to enhance constriction to AngII. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE and/or AngII increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of AngII constriction. Both AngII-induced hypertension and the enhancing effect of 12(S)-HETE on AngII contractions were eliminated by a BLT2 (leukotriene B(4) receptor-2) antagonist. These results outline a mechanism where the macrophage-12/15-LO pathway enhances the action of AngII. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of AngII in part by promoting endothelial synthesis of a superoxide-derived TP agonist.

Thromboxane (TxAUsing 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)ETP receptor antagonist inhibited the contraction induced by PGETP 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 risk of thrombotic events dramatically increases with age and may be accelerated in women by the cessation of endogenous estrogen production at menopause. Patients at risk of thrombosis are prescribed dual anti-platelet therapy (DAPT; aspirin and a P2Y12 antagonist) and are encouraged to participate in regular physical activity, as these modalities improve nitric oxideand prostacyclin-mediated inhibition of platelet aggregation. Methods: We assessed prostacyclin sensitivity as well as basal platelet reactivity with and without in vitro DAPT before and after an 8-week high-intensity exercise training program in 13 healthy, sedentary postmenopausal women.The training intervention consisted of three 1 h sessions per week. Isolated platelets were analyzed for thromboxane A2 receptor, thromboxane A2 synthase, cyclooxygenase-1, and prostacyclin receptor protein expression. Additionally, plasma 6-keto prostaglandin F1α and thromboxane B2 levels weredetermined. Results: Exercise training made platelets more sensitive to the inhibitory effects of prostacyclin on thromboxane-, collagen-, and adenosine 5′-diphosphate (ADP)-induced aggregation, as well as thrombin-receptor activator peptide 6- and ADP-induced aggregation with DAPT. However, there was no change in protein expression from isolated platelets or plasma thromboxane B2 andprostacyclin levels following training. Conclusion: Together, these findings emphasize the importance of promoting physical activity as a tool for reducing thrombotic risk in postmenopausal women and suggest that training status should be considered when prescribing DAPT in this cohort.

Excessive mitochondrial fission in podocytes serves as a central hub for the pathogenesis of diabetic nephropathy (DN), and the thromboxane/prostaglandin receptor (TP receptor) plays a potential role in DN. However, regulation of the TP receptor during mitochondrial dynamics disorder in podocytes remains unknown. Here, we firstly reported novel mechanistic details of TP receptor effects on mitochondrial dynamics in podocytes under diabetic conditions. Expression of the TP receptor was significantly upregulated in podocytes under diabetic conditions both in vivo and in vitro. S18886 attenuated podocyte mitochondrial fission, glomerular injury and renal dysfunction in diabetic mice. Furthermore, inhibition of the TP receptor by both genetic and pharmacological methods dramatically reduced mitochondrial fission and attenuated podocyte injury induced by high glucose through regulating dynamin-related protein 1 (Drp1) phosphorylation and its subsequent translocation to mitochondria. In contrast, TP receptor overexpression and application of TP receptor agonist U46619 in these podocytes showed the opposite effect on mitochondrial fission and podocyte injury. Furthermore, treatment with Y27632, an inhibitor of Rho-associated kinase1 (ROCK1), significantly blunted more fragmented mitochondria and reduced podocyte injuries in podocytes with TP receptor overexpression or after U46619 treatment. Finally, pharmacological inhibition of Drp1 alleviated excessive mitochondrial fragmentation and podocyte damage in TP receptor overexpressing podocytes. Our data suggests that increased expression of the TP receptor can occur in a human cultured podocyte cell line and in podocytes derived from streptozotocin (STZ)-induced diabetic mice, which contributes to mitochondrial excessive fission and podocyte injury via ROCK1-Drp1 signaling.

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 A

Airborne fine particulate matter (PM

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.

We examined the potential stimulatory effects of U46619 (a prostanoid TP receptor agonist) and five prostanoids on the contractile activities of urinary bladder smooth muscle (UBSM), focusing on the role of the TP receptor and its associated CaChanges in the basal tone and spontaneous contractile activity (amplitude and frequency) of isolated guinea pig UBSM were measured isotonically. The presence of TP receptors in UBSM was examined by RT-qPCR and immunofluorescence.U46619, prostaglandin (PG) EProstanoids can enhance UBSM contractile activities and thus may be endogenous candidates for induction of detrusor overactivity. The TP receptor and TP-receptor-activated VDCCs/SOCCs are key molecules responsible for these effects.

Metabolic syndrome (MetS) is associated with alterations in coronary vascular smooth muscle and endothelial function. The current study examined the contractile response of the isolated coronary arterioles to serotonin in pigs with and without MetS and investigated the signaling pathways responsible for serotonin-induced vasomotor tone. The MetS pigs (8-weeks old) were fed with a hyper-caloric, fat/cholesterol diet and the control animals (lean) were fed with a regular diet for 12 weeks (n = 6/group). The coronary arterioles (90–180 μm in diameter) were dissected from the harvested pig myocardial tissues and the in vitro coronary arteriolar response to serotonin was measured in the presence of pharmacological inhibitors. The protein expressions of phospholipase A2 (PLA2), TXA2 synthase, and the thromboxane-prostanoid (TP) receptor in the pigs’ left ventricular tissue samples were measured using Western blotting. Serotonin (10−9–10−5 M) induced dose-dependent contractions of coronary-resistant arterioles in both non-MetS control (lean) and MetS pigs. This effect was more pronounced in the MetS vessels compared with those of non-MetS controls (lean, P

α-CGRP is synthesized by sensory nerves in the dermis and its release can cause vasodilation and local inflammation. Its vasorelaxant effects are based on the direct activation of smooth muscle and endothelial cells, as well as the activation of mast cells causing the release of vasoactive and proinflammatory mediators. Here, we show that in the capsaicin model for neurogenic inflammation, capsaicin-induced edema formation is mediated by α-CGRP and mast cells, but is absent in thromboxane receptor-deficient mice. Capsaicin treatment of mice induced a thromboxane synthesis, which was mediated by α-CGRP and mast cells. Fittingly, α-CGRP induced thromboxane synthesis in mast cells and the thromboxane receptor agonist I-BOP caused edema formation independently of mast cells, suggesting that mast cells are the source of thromboxane. Most importantly, I-BOP-induced edema formation was mediated by α-CGRP and I-BOP was able to stimulate through calcineurin the α-CGRP release from peripheral neurons. Likewise, the signaling pathway, including α-CGRP, thromboxane receptor, and mast cells, also mediated capsaicin-induced mechanical hypersensitivity, a common symptom of capsaicin treatment. Taken together, the thromboxane-induced α-CGRP release from neurons forms a positive feedback loop causing prolonged α-CGRP release and edema formation during capsaicin-induced neurogenic inflammation.

Background Several lipid metabolites in cerebrospinal fluid are correlated with poor outcomes in aneurysmal subarachnoid haemorrhage. Most of these metabolites bind to ubiquitous thromboxane–prostaglandin (TP) receptors, causing vasoconstriction and inflammation. Here, we evaluated terutroban (TBN), a specific TP receptor antagonist, for the prevention of post-haemorrhage blood-brain barrier disruption, neuronal apoptosis and delayed cerebral hypoperfusion. Methods The rat double subarachnoid haemorrhage model was produced by twice injecting (days 1 and 2) autologous blood into the cisterna magna. Seventy-eight male Sprague-Dawley rats were assigned to experimental groups. Rats exposed to subarachnoid haemorrhage were allocated to no treatment (SAH group) or TBN treatment by gastric gavage during the first 5 days after haemorrhage (SAH+TBN group). Control rats received artificial cerebrospinal fluid injections (CSF group). Sham-operated rats with or without TBN administration were also studied. Body weight and Garcia neurological scores were assessed on day 2 and day 5. We used nanoscale single-photon emission computed tomography (nanoSPECT) to measure brain uptake of three radiolabelled agents: 99mTechnetium-diethylenetriaminepentacetate (99mTc-DTPA), which indicated blood-brain barrier permeability on day 3, 99mTechnetium-annexin V-128 (99mTc-Anx-V128), which indicated apoptosis on day 4, and 99mTechnetium-hexamethylpropyleneamineoxime (99mTc-HMPAO), which indicated cerebral perfusion on day 5. Basilar artery narrowing was verified histologically, and cerebral TP receptor agonists were quantified. Results 99mTc-DTPA uptake unveiled blood-brain barrier disruption in the SAH group. TBN mitigated this disruption in the brainstem area. 99mTc-Anx-V128 uptake was increased in the SAH group and TBN diminished this effect in the cerebellum. 99mTc-HMPAO uptake revealed a global decreased perfusion on day 5 in the SAH group that was significantly counteracted by TBN. TBN also mitigated basilar artery vasoconstriction, neurological deficits (on day 2), body weight loss (on day 5) and cerebral production of vasoconstrictors such as Thromboxane B2 and Prostaglandin F2α. Conclusions Based on in vivo nanoscale imaging, we demonstrated that TBN protected against blood-brain barrier disruption, exerted an anti-apoptotic effect and improved cerebral perfusion. Thus, TP receptor antagonists showed promising results in treating post-haemorrhage neurovascular events.

Enhanced platelet activation has been reported in patients with essential hypertension and heart failure. The possible contribution of platelet-derived thromboxane (TX)A

Hypertension is one of the most common complications of chronic kidney disease (CKD). Some research has indicated that changes in large artery function especially caused by thromboxane A2 (TXA2) may be a novel factor acting to induce hypertension in CKD. We studied the 5/6 nephrectomy rat model and measured serum levels of creatinine (Cr), calcium (Ca), phosphorus (P), TXA2-stable metabolites (thromboxane B2, TXB2), and caudal artery pressure after nephrectomy. The tension variations in thoracic aortas were measured after stimulating by vasoconstrictor/vasodilator using the cumulative concentration administration method and then tested the expression of TXA2 receptors in the thoracic aortas through western blots. The CKD rats developed uremia, electrolyte imbalances，and hypertension. They also exhibited a significant increase in TXB2 concentration. The aortic rings of CKD rats showed an increased contraction response to U46619 (a TXA2 analogue) and the expression of TXA2 receptors also enhanced. In the meanwhile, the diastolic function decreased in the CKD group. Our results demonstrate that the impairment of artery contractile function caused by the increase of TXA2 receptors on the wall of aortic rings may be involved in hypertension in CKD rats.

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.

The primary purpose of this investigation was to determine the role played by endoperoxide 4 receptors (EP4-R) and thromboxane A(2) receptors (TxA(2)-R) during isolated dynamic muscle mechanoreflex activation in rats with heart failure with reduced ejection fraction (HF-rEF) and sham-operated healthy controls. We found that injection of the EP4-R antagonist L-161,982 (1μg) into the arterial supply of the hindlimb had no effect on the peak pressor response to dynamic hindlimb muscle stretch in HF-rEF (n=6, peak ΔMAP pre: 27±7; post: 27±4 mmHg; P=0.99) or sham (n=6, peak ΔMAP pre: 15±3; post: 13±3 mmHg; P=0.67) rats. In contrast, injection of the TxA(2)-R antagonist daltroban (80μg) into the arterial supply of the hindlimb reduced the pressor response to dynamic hindlimb muscle stretch in HF-rEF (n=11, peak ΔMAP pre: 28±4; post: 16±2 mmHg; P=0.02) but not sham (n=8, peak ΔMAP pre: 17±3; post: 16±3; P=0.84) rats. Our data suggest that TxA(2)-Rs on thin fibre muscle afferents contribute to the exaggerated mechanoreflex in HF-rEF.

This study was to determine how endothelium-dependent contractions (EDCs) change in iliac arteries of Wistar-Kyoto (WKYs) and spontaneously hypertensive rats (SHRs) during the transition from adolescence to adulthood and the underlying mechanism(s). We also aimed to elucidate effects of L-798106, an EP3 receptor antagonist, on EDCs and the blood pressure increase in adolescent SHRs. Blood vessels were isolated for functional and biochemical analyses. EDCs were comparable in adolescent iliac arteries of both strains, and contractions to ACh, prostacyclin (PGI2), the EP3 receptor agonist sulprostone and the TP receptor agonist U46619 in adult vessels were less prominent compared with those in the adolescents, while the attenuation of vasoconstrictions to ACh, PGI2 or U46619 with age was to a lesser extent in SHRs. PGI2 production was decreased to a similar level in adult arteries. TP and EP3 expressions were downregulated in adult vessels, whereas the extent of TP downregulation was less in SHRs. L-798106 partially suppressed the vasoconstrictions to U46619 and attenuated EDCs to a greater extent than SQ29548, and administration of L-798106 blunted the blood pressure increase with age in prehypertensive SHRs. These results demonstrate the comparable EDCs in iliac arteries of the adolescents are decreased in the adults, but relatively larger EDCs in adult SHRs can be a reflection of differential downregulation of TP and EP3 receptors during the transition from adolescence to adulthood. Also, our data suggest that blockade of both TP and EP3 receptors starting from the prehypertensive stage suppresses EDCs and the development of hypertension in SHRs.

We investigated whether sex differences exist in cyclooxygenase-dependent effects on membrane potential and relaxation in mice mesenteric resistance arteries. Mesenteric small arteries of 9- to 12-week-old, male and female, wild-type mice, db/+ mice and diabetic db/db mice were mounted in myographs for measurements of isobaric diameter and smooth muscle cell membrane potential. Acetylcholine caused smaller dilation of arteries from male db/+ mice compared to arteries from female db/+ mice. In the presence of the NO synthase inhibitor N ω-nitro-L-arginine methyl ester (L-NAME), acetylcholine-induced dilation of arteries from males increased in the presence of indomethacin and the COX-1-specific inhibitor FR122047. The presence of indomethacin was also associated with a more negative membrane potential in arteries from males. In arteries from db/db mice, no sex differences were seen. In arteries from male but not female wild-type mice, the thromboxane receptor blocker SQ29,548 increased relaxation to acetylcholine. In contrast to arteries from female mice, COX (most likely COX-1)-derived prostanoids and activation of thromboxane receptors counteract acetylcholine vasodilatation probably through increased smooth muscle depolarization in arteries from male mice. In mice with diabetes and pronounced endothelial dysfunction, inhibition of COX did not enhance acetylcholine vasodilatation.

We investigated the role of cyclooxygenase metabolite-associated endoperoxide 4 receptors (EP4-R) and thromboxane A(2) receptors (TxA(2)-R) on thin fiber 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 TxA(2)-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, unanesthetized 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

The ADP receptor P2Y

Aim By activating prostacyclin receptors (IP receptors), prostacyclin (PGI2 ) exerts cardiovascular protective effects such as vasodilation and inhibition of vascular smooth muscle cell (VSMC) proliferation. However, IP receptors are dysfunctional under pathological conditions, and PGI2 produces detrimental effects that are opposite to its physiological protective effects via thromboxane-prostanoid (TP) receptors. This attempted to investigate whether or not IP receptor dysfunction facilitates the shift of PGI2 action. Methods The effects of PGI2 and its stable analog iloprost on VSMC phenotypic transformation and proliferation were examined in A10 cells silencing IP receptors, in human aortic VSMCs (HAVSMCs) knocked down IP receptor by CRISPR-Cas9, or in HAVSMCs transfected with a dysfunctional mutation of IP receptor IPR212C . Results PGI2 /iloprost treatment stimulated cell proliferation, upregulated synthetic proteins and downregulated contractile proteins, suggesting that PGI2 /iloprost promotes VSMC phenotypic transformation in IP-deficient cells. The effect of PGI2 /iloprost was prevented by TP antagonist S18886 or TP knockdown, indicating that the VSMC detrimental effect of PGI2 is dependent on TP receptor. RNA sequencing and Western blotting results showed that RhoA/ROCKs, MEK1/2 and JNK signalling cascades were involved. Moreover, IP deficiency increased the distribution of TP receptors at the cell membrane. Conclusion PGI2 induces VSMC phenotypic transformation when IP receptors are impaired. This is attributed to the activation of TP receptor and its downstream signaling cascades, and to the increased membrane distribution of TP receptors. The VSMC detrimental effect of PGI2 medicated by IP dysfunction and TP activation might probably exacerbate vascular remodelling, accelerating cardiovascular diseases.

BackgroundNTP42is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy ofNTP42in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs.MethodsPAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar–Kyoto rats. Animals were assigned into groups: 1. ‘No MCT’; 2. ‘MCT Only’; 3. MCT + NTP42(0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT.ResultsFrom haemodynamic assessments, NTP42reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover,NTP42was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals.ConclusionsThese findings suggest thatNTP42and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

Objective Activation of thromboxane A2 synthase (TXAS)/thromboxane A2 (TXA2)/thromboxane prostanoid (TP) receptor leads to arterial constriction, platelet aggregation and vascular injury. We attempted to characterize the microvascular dysfunction in ischaemia/reperfusion injury using genetically modified TXAS−/−, TP−/− and TXAS−/−TP−/− mice. Approach and Results The cardiac micro-circulation and electrocardiograms were evaluated from B6, TXAS−/−, TP−/− and TXAS−/−TP−/− mice in response to intravenous saline, endothelin-1, U46619 (a TXA2 agonist) and myocardial ischaemia/reperfusion injury. Cardiac function was investigated with myocardial permeability, the troponin I concentration and the infarct size. Myocardial TXAS, TP, endothelial nitric oxide (NO) synthase (eNOS), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOx4), 4-hydroxynonenal, interleukin (IL)-1β, cell apoptosis, coronary effluent thromboxane B2 (TXB2) and superoxide anions (O2 −) and NO concentrations were measured. Mice mesenteric reactivity in response to various drugs was assessed by wire myography. In vivo fluorescent platelet adhesiveness to the mesenteric arterial endothelium after FeCl3 stimulation was examined. In B6 mice, ischaemia/reperfusion significantly increased levels of ST-segment elevation, myocardial TXAS, TP, NOx4, IL-1β, apoptosis, coronary endothelin-1, TXB2, O2 − release and the infarct size, with concomitant decreases in eNOS, NO concentrations and cardiac micro-circulation. These effects were remarkably depressed in TXAS−/−, TP−/− and TXAS−/−TP−/− mice. Aspirin treatment or depletion of the TXAS, TP or TXAS/TP gene significantly attenuated the exaggerated vascular reactivity by vasoconstrictors and vasodilators and efficiently reduced platelet adhesion to the mesenteric endothelium under FeCl3 stimulation. Conclusion Inhibiting TXAS/TXA2/TP signalling confers microvascular protection against oxidative injury in both cardiac and mesenteric arteries.

Increasing evidence suggests that prostanoid receptors and their ligands may constitute valuable tools for development of new antiepileptic drugs. Thromboxane A2 (TXA2) is a major eicosanoid in cardiovascular homeostasis. TXA2 exerts its action through the specific G protein-coupled TXA2 receptor (TP). In addition to its crucial role in the cardiovascular system, TXA2 and TPs play a role in the brain. Nevertheless, previously identified roles have been limited to cell protection of neurotoxicity, and the role of TPs on seizure activity was not investigated. Here we evaluated the effect of potent and selective TP agonist U-46619 on seizures induced by pentylenetetrazol (PTZ). Adult C57BL/6 mice received increasing doses of U-46619 (0, 30, 100 or 300 μg/kg). After 30 min we measured the latencies to myoclonic and generalized seizures induced by PTZ (60 mg/kg). We found that U-46619 increased the latency to PTZ-induced myoclonic jerks and tonic-clonic seizures. Moreover, U-46619 increased the immunocontent of phosphorylated Ser657 at protein kinase C (PKC) alpha subunit, indicating PKC activation in the hippocampus and cerebral cortex. Levels of TPs were not altered by the agonist. Administration of a TP antagonist, SQ 29,548, did not alter seizures and did not blunt the anticonvulsant-like effect of the agonist. In summary, we showed that a potent and selective TP agonist, U-46619, increased seizure latency in mice. Activation of PKC signaling pathways may underlie the anticonvulsant-like effect. Further investigation is needed to understand the potential of TPs in seizure treatment.

Aim Enhancement of thromboxane A2 (TXA2) synthase (TXAS) activity, TXA2 release, and thromboxane prostanoid (TP) receptor activation leads to vasoconstriction and oxidative injury. We explored whether genetic deletion of TXAS/TXA2/TP signalling may reduce renal ischaemia/reperfusion (I/R) injury in mice. Materials and Methods Renal haemodynamics and function were evaluated in TXAS+/+TP+/+ (wild-type, WT), TXAS−/− (TXS−/−), TP−/− and TXAS−/−TP−/− (double knockout, dKO) mice in response to intravenous TXA2 mimetic-U46619 and 45-minute renal ischaemia and 4-hour reperfusion injury. We examined renal TXAS and TP expression, blood urea nitrogen (BUN) and creatinine, reactive oxygen species (ROS) amount, pro-inflammatory cytokines and pathophysiologic mechanisms, including apoptosis, autophagy and pyroptosis under I/R injury. Results Renal I/R enhanced the levels of TXAS, TP, nuclear factor-κB, nicotinamide adenine dinucleotide phosphate oxidase gp91, Bax/Bcl-2/caspase-3/apoptosis, Beclin-1/LC3-II/autophagy, caspase-1/gasdermin D/interleukin-1β/pyroptosis, renal thromboxane B2 (TXB2) concentration, ROS amount, plasma BUN, creatinine and TXB2 and decreased renal endothelial nitric oxide synthase expression in WT mice. All these enhanced parameters were significantly decreased in three KO mice. Intravenous U46619 significantly decreased renal microcirculation and enhanced gp91 and Bax/Bcl-2 in WT and TXS−/− but not TP−/− in dKO mice. I/R significantly decreased renal microcirculation in all mice; however, the time for recovery to baseline renal blood flow level was significantly shortened in TXS−/−, TP−/−and dKO mice versus WT mice. Blockade of TXAS/TP signalling attenuated I/R-enhanced pro-inflammatory cytokine profile. Conclusion Blockade of TXAS/TXA2/TP signalling confers renal protection against I/R injury through the actions of anti-oxidation, anti-inflammation, anti-apoptosis, anti-autophagy and anti-pyroptosis.

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.

Thromboxane A2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations.

Background Muscular dystrophy ( MD ) causes a progressive cardiomyopathy characterized by diffuse fibrosis, arrhythmia, heart failure, and early death. Activation of the thromboxane‐prostanoid receptor ( TP r) increases calcium transients in cardiomyocytes and is proarrhythmic and profibrotic. We hypothesized that TP r activation contributes to the cardiac phenotype of MD , and that TP r antagonism would improve cardiac fibrosis and function in preclinical models of MD . Methods and Results Three different mouse models of MD (mdx/utrn double knockout, second generation mdx/ mTR double knockout, and delta‐sarcoglycan knockout) were given normal drinking water or water containing 25 mg/kg per day of the TP r antagonist ifetroban, beginning at weaning. After 6 months (10 weeks for mdx/utrn double knockout), mice were evaluated for cardiac and skeletal muscle function before euthanization. There was a 100% survival rate of ifetroban‐treated mice to the predetermined end point, compared with 60%, 43%, and 90% for mdx/utrn double knockout, mdx/mTR double knockout, and delta‐sarcoglycan knockout mice, respectively. TP r antagonism improved cardiac output in mdx/utrn double knockout and mdx/ mTR mice, and normalized fractional shortening, ejection fraction, and other parameters in delta‐sarcoglycan knockout mice. Cardiac fibrosis in delta‐sarcoglycan knockout was reduced with TP r antagonism, which also normalized cardiac expression of claudin‐5 and neuronal nitric oxide synthase proteins and multiple signature genes of Duchenne MD. Conclusions TP r antagonism reduced cardiomyopathy and spontaneous death in mouse models of Duchenne and limb‐girdle MD . Based on these studies, ifetroban and other TP r antagonists could be novel therapeutics for treatment of the cardiac phenotype in patients with MD .

Sphingosine kinases (SphK) catalyse the formation of sphingosine-1-phosphate (S1P) and play important roles in the cardiovascular, nervous and immune systems. We have shown before that G

Thromboxane A2 (TXA2) is an important eicosanoid in the cardiovascular system, and increasing evidence suggests that TXA2 receptors (TPs) and their ligands may constitute valuable tools for the development of neuroprotective drugs. However, the role of TPs on seizure-induced damage has not been investigated. Therefore, we evaluated the effects of SQ 29,548, a potent and selective TP antagonist-on neuromotor performance, neurodegeneration, reactive astrocytosis, and c-Fos protein immunoreactivity after pilocarpine-induced status epilepticus (SE) in mice. Adult C57BL/6 mice received intracerebroventricular SQ 29,548 injections 90 min and 24 h after pilocarpine-induced SE. We found that SQ 29,548 prevented the impairment of neuromotor performance (Neuroscore test) 48 h after pilocarpine-induced SE. Data analysis suggested the existence of two subgroups of SQ 29,548-treated post-SE animals. Eight out of 12 SQ 29,548-treated animals displayed Neuroscore values identical to those of vehicle-treated controls, and were considered SQ 29,548 responders. However, 4 out of 12 SQ 29,548-treated animals did not show any improvement in Neuroscore values, and were considered SQ 29,548 non-responders. Treatment with SQ 29,548 attenuated SE-induced increase in the number of FJC- or GFAP-positive cells in the hippocampus of SQ 29,548 responders. In addition, SQ 29,548 prevented the SE-elicited increase of c-Fos immunoreactivity in the hippocampus. In summary, our results suggest that the TP antagonist (SQ 29,548) improves neurological outcome after pilocarpine-induced SE in mice. The existence of SQ 29,548 responders and non-responders was suggested by results from the Neuroscore test. Additional studies are needed to understand the mechanisms underlying these findings, as well as the potential uses of TP antagonists in the treatment of seizure-induced damage.

Advanced glycation end products (AGEs) play a pivotal role in vascular functions under various pathophysiological conditions. Although uridine diphosphate (UDP) is an important extracellular nucleotide, the relationship between AGEs and UDP regarding their effect on vascular functions remains unclear. Therefore, we investigated the effects of AGE-bovine serum albumin (AGE-BSA) on UDP-mediated responses in rat thoracic aorta and carotid arteries. In rat thoracic aorta, UDP-induced relaxation was observed and this relaxation was similar between control (1.0 v/v% PBS) and AGE-BSA-treated (0.1 mg/mL for 60 min) groups. In contrast, contraction but not relaxation was obtained following UDP application to carotid arteries with and without endothelia; contraction was greater in the AGE-BSA-treated group than in the control group. The difference in UDP-induced contraction between the two groups was not abolished by the use of a nitric oxide synthase (NOS) inhibitor, whereas it was abolished by the use of cyclooxygenase (COX), thromboxane synthase (TXS), and thromboxane-prostanoid (TP) receptor antagonist. Further, the difference in UDP-induced contraction was not abolished by the use of a cPLA2 inhibitor, whereas it was abolished by the use of an iPLA2 inhibitor. UDP increased TXA2 release in both groups, and its level was similar in both groups. Moreover, the release of PGE2, PGF2α, and PGI2 was similar among the groups. Under NOS inhibition, TP receptor agonist–induced contraction increased in the AGE-BSA-treated group (vs. control group). In conclusion, the increase in UDP-induced carotid arterial contraction by AGE-BSA can be attributed to an increase in the COX/TXS/TP receptor pathway, particularly, TP receptor signaling.

Sinusoidal obstruction syndrome (SOS) is a major complication of chemotherapy and hematopoietic stem cell transplantation. The early stage of SOS is characterized by liver sinusoidal endothelial cell (LSEC) injury accompanied by platelet aggregation. Thromboxane A

Although recognized to have an in vivo vasodepressor effect blunted by the vasoconstrictor effect of E-prostanoid receptor-3 (EP3), prostaglandin E