Thromboxane-prostanoid Receptor Antagonist Research Articles

Effects of antagonism of thromboxane-prostanoid receptor on alcohol-associated liver disease

Backgrounds: Alcohol-associated liver disease (ALD) is one of the leading causes of liver diseases. Thromboxane-prostanoid receptor (TP-R) is widely expressed in the liver and is activated by thromboxane A2 (TXA2). A previous study reported an increase in TP-R mRNA level in liver after ethanol feeding. Preclinical studies have documented that TP-R antagonists exert a protective effect against cardiovascular and liver diseases. In particular, TP-R antagonist is reported to attenuate ALD in rodents. However, the mechanism(s) by which antagonizing TP-R attenuates ALD remains unclear. Methods: Herein, we used the chronic plus binge ethanol feeding model. Briefly, male, C57BL/6 wild-type mice (8-week) were fed a Lieber-Decarli ethanol (5% v/v) diet (ET, n = 10) or isocaloric control diet (CON, n = 6) for ten days followed by a single binge administration of ethanol or maltose-dextrin via oral gavage. A cohort of ethanol-fed mice received SQ 29,548, a TP-R antagonist (ET+SQ, n = 8) by oral administration. Results: Western blot analysis showed that the protein level of thromboxane A2 synthase 1 ( P<0.05), responsible for TXA2 production, was upregulated in the mouse liver upon ethanol exposure. Of note, ethanol feeding significantly increased plasma aspartate transferase (AST) level ( P<0.05), which was abolished by SQ administration ( P<0.05). Moreover, compared with CON mice, ET mice showed an increase in liver protein levels of pro-inflammatory markers, including TNFa ( P<0.05), cluster of differentiation 68 (CD68, P<0.001), and vascular cell adhesion protein 1 ( P<0.05). In addition, the pro-fibrogenic markers including collagen type 1 alpha 1 chain (COL1A1, P<0.05) and matrix metalloproteinase 9 (MMP9, P<0.01) were also increased in ET mice. However, these alterations were not found in ET+SQ mice compared to their controls. In particular, ET+SQ mice exhibited a marked reduction in hepatic protein levels of CD68 ( P<0.001), COL1A1 ( P<0.001), and MMP9 ( P<0.01) versus ET mice. Interestingly, we noted that ethanol consumption decreases the protein level of NADH:ubiquinone oxidoreductase subunit B8 (NDUFB8), a component of mitochondrial complex I, in mouse liver ( P<0.05), which was reversed by SQ administration( P<0.05). Conclusion: These findings suggest that blockade of TP-R activity attenuates ethanol-induced liver inflammation and fibrosis possibly through improving the function of mitochondrial complex I. This study was supported by the NIH-NIAAA P50 award-Alcohol Center of Research-Nebraska (P50AA030407-5130). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes.

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

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

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. Amouse model predicted a key role for the thromboxane-prostanoid (TP) receptor in AERD. Our aim was to determine whether ifetroban, a TP receptor antagonist, attenuates aspirin-induced respiratory symptoms in patients with AERD. 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. 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. 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.

Abstract P2021: Blockade Or Deletion Of The Thromboxane/Prostanoid Receptor Reduces Right Ventricular Stiffness To Maintain Function In Right Ventricular Pressure Overload

The increased afterload of pulmonary arterial hypertension (PAH) impairs right ventricular function, as the RV struggles to adapt to increased pressure with remodeling and fibrosis. RV failure is the primary cause of death in PAH, and a frequent cause of death in pulmonary hypertension secondary to pulmonary embolism, emphysema, pulmonary fibrosis, and left-heart failure. There is currently no RV-specific therapeutic to maintain function and prolong patient life. During PAH, cardiomyocytes upregulate cell-surface expression of the G αq /IP 3 -coupled thromboxane/prostanoid receptor (TPr), which is activated by multiple endogenous ligands increased in PAH, and leads to calcium influx. TPr activation is also pro-fibrotic in multiple cell types and models. Previous studies have shown reduced fibrosis following TPr antagonism in mouse models of short-term pulmonary hypertension; however this effect has been variable in long-term studies. To more precisely determine the functional effects of TPr activation in RV pressure overload, we used pulmonary arterial banding (PAB) for a direct, sustained increase in pressure, antagonism or deletion of the TPr, and murine cardiac MRI for simultaneous evaluation of the RV and LV. TPr antagonism in a long-term PAB model improved RV ejection fraction and restored LV volume and output by preventing septal bulging and LV eccentricity. This occurred with even short-term antagonism, and was associated with decreased fibrosis in the septum and RV insertion points. Similar results were seen following PAB of universal or cardiomyocyte-specific TPr knockout mice; in all cases, decreasing TPr activation during RV pressure overload reduced RV stiffness even when RV fibrosis remained unchanged. In long-term pressure overload, the improved compliance with antagonism normalized expression of Yap/Taz-associated genes. These results suggest that TPr activation contributes to cardiomyocyte mechanotransduction and deleterious remodeling in response to the RV pressure overload of pulmonary hypertension, and that antagonism of the TPr may preserve RV function to prolong life in PAH patients.

Impaired UTP-induced relaxation in the carotid arteries of spontaneously hypertensive rats.

Uridine 5'-triphosphate (UTP) has an important role as an extracellular signaling molecule that regulates inflammation, angiogenesis, and vascular tone. While chronic hypertension has been shown to promote alterations in arterial vascular tone regulation, carotid artery responses to UTP under hypertensive conditions have remained unclear. The present study investigated carotid artery responses to UTP in spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY). Accordingly, our results found that although UTP promotes concentration-dependent relaxation in isolated carotid artery segments from both SHR and WKY after pretreatment with phenylephrine, SHR exhibited significantly lower arterial relaxation responses compared with WKY. Moreover, UTP-induced relaxation was substantially reduced by endothelial denudation and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine in both SHR and WKY. The difference in UTP-induced relaxation between both groups was abolished by the selective P2Y2 receptor antagonist AR-C118925XX and the cyclooxygenase (COX) inhibitor indomethacin but not by the thromboxane-prostanoid receptor antagonist SQ29548. Furthermore, we detected the release of PGE2, PGF2α, and PGI2 in the carotid arteries of SHR and WKY, both at baseline and in response to UTP. UTP administration also increased TXA2 levels in WKY but not SHR. Overall, our results suggest that UTP-induced relaxation in carotid arteries is impaired in SHR perhaps due to impaired P2Y2 receptor signaling, reductions in endothelial NO, and increases in the levels of COX-derived vasoconstrictor prostanoids.

Abstract 443: Blockade of the Thromboxane/prostanoid Receptor Prevents ECG Abnormalities in RV Pressure Overload

The increased afterload of pulmonary arterial hypertension (PAH) impairs right ventricular function and ultimately leads to failure, as the RV struggles to adapt to increased pressure with remodeling and fibrosis. During PAH, cardiomyocytes upregulate cell-surface expression of the G protein-coupled thromboxane/prostanoid receptor (TPr). Increased myofibroblast and immune cell populations may also contribute to the enhanced TPr expression seen in the PAH RV. Activation of the cardiomyocyte TPr increases intracellular calcium via G αq /IP 3 ; activation of the receptor in other cells leads to fibrosis and vasoconstriction. Preventing signaling through the TPr prevents RV fibrosis in murine models of PAH without affecting arterial pressure. Because infusion of TPr agonist can cause arrhythmia in anesthetized rabbits, and we have previously found that RV pressure overload causes sustained increases in end-diastolic calcium in RV cardiomyocytes that is blocked with TPr antagonist, we hypothesized that endogenous TPr activation can lead to conduction abnormalities in RV pressure overload. Here, we used pulmonary arterial banding (PAB) of female mice to induce fixed pressure overload of the RV. Sham-operated or PAB mice were treated with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban and were evaluated at 4 weeks past PAB. RV ejection fraction was similarly depressed in vehicle- and antagonist-treated mice, although spontaneous running, RV fibrosis, and RV relaxation time were improved in PAB mice given ifetroban. ECG abnormalities in PAB mice confirmed a prolonged relaxation and suggested delays in repolarization. These were abolished with TPr antagonism. PAB altered RV expression and localization of connexin-43 (Cx43) in vehicle-treated, but not ifetroban-treated mice. Cx43 derangement is associated with impaired cell-to-cell electrical conduction and impulse propagation. Compiled, our findings suggest that endogenous TPr activation produces alterations in RV calcium handling, signaling, and cell-cell junctions that contribute to early failure in pressure overload. Therapeutic TPr antagonism may prevent this deleterious remodeling and prolong survival in patients with PAH.

NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension

BackgroundNTP42 is 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) A2 is 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 of NTP42 in 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, NTP42 reduced 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, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals.ConclusionsThese findings suggest that NTP42 and 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.

Abstract 824: Thromboxane/Prostanoid Receptor Activation Increases Calpain-Mediated Proteolysis and Alters Calcium Handling and Fibrosis Following Right Ventricular Pressure Overload

In pulmonary arterial hypertension (PAH), the right ventricle undergoes remodeling and fibrosis as it struggles to adapt to the increased pressure overload. RV dysfunction and failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid receptor (TPr) is expressed in vascular smooth muscle, myofibroblasts, and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiomyocyte TPr increases intracellular calcium via G αq /IP 3 ; activation of the receptor in other cells leads to fibrosis and vasoconstriction. The TPr is activated by isoprostane as well as thromboxane, which suggests that the receptor could contribute to deleterious remodeling during cardiac stress. Our previous studies demonstrate that TPr antagonism prevents RV fibrosis and dilatation in murine models of PAH, without affecting pressures. Because the TPr can signal through Gq, we hypothesized that its activation in PAH causes changes in cardiomyocyte calcium-handling proteins which contribute to remodeling and failure. In this study, we used pulmonary arterial banding (PAB) to induce fixed pressure overload of the RV. Mice were treated for 4 weeks past PAB with normal drinking water or water containing 25 mg/kg/day of the TPr antagonist ifetroban, and either underwent pressure-volume catheterization and whole RV evaluation, or cardiomyocytes were isolated for calcium handling and protein. PAB caused an increase in cardiomyocyte resting (end-diastolic) intracellular calcium, which was ameliorated in mice given TPr antagonist. The increased intracellular calcium following PAB was associated with increased activity of the calcium-activated protease calpain, also blocked with TPr antagonism. There was no decrease in caffeine-mediated release of calcium from the sarcoplasmic reticulum (SR) at 4 weeks past PAB, and phosphorylation of phospholamban was increased, suggesting compensation to drive calcium into the SR. Our findings suggest that TPr activation produces alterations in RV calcium handling, signaling, and calpain activity that contribute to deleterious remodeling and early failure in pressure overload. Therapeutic TPr antagonism may help preserve RV function in patients with PAH.

Increased coronary arteriolar contraction to serotonin in juvenile pigs with metabolic syndrome

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 < 0.05]. Serotonin-induced contraction of the MetS vessels was significantly inhibited in the presence of the selective PLA2 inhibitor quinacrine (10−6 M), the COX inhibitor indomethacin (10−5 M), and the TP receptor antagonist SQ29548 (10−6 M), respectively (P < 0.05). MetS exhibited significant increases in tissue levels of TXA2 synthase and TP receptors (P < 0.05 vs. lean), respectively. MetS is associated with increased contractile response of porcine coronary arterioles to serotonin, which is in part via upregulation/activation of PLA2, COX, and subsequent TXA2, suggesting that alteration of vasomotor function may occur at an early stage of MetS and juvenile obesity.

Endothelium-dependent and endothelium-independent effects of 1-nitro-2-propylbenzene on rat aorta.

A group of nitro compounds contains a benzene ring in a short aliphatic chain with the NO2 group, property that supposedly favors its vasodilator profile. In this study, we evaluated in isolated rat aorta the effects of 1-nitro-2-propylbenzene (NPB), a nitro compound containing the NO2 in the aromatic ring. In aorta precontracted with KCl, NPB (1-3000μm) induced full endothelium-independent relaxation. In endothelium-intact preparations, phenylephrine-induced contractions were fully relaxed by NPB, effect unaltered by N(ω)-nitro-L-arginine methyl ester (L-NAME) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In the concentration range of 30-300μm, NPB slightly but significantly potentiated the phenylephrine-induced contraction. Such potentiation was unaltered by the thromboxane-prostanoid receptor antagonist seratrodast, but was abolished by endothelium removal or by preincubation of endothelium-intact preparations with L-NAME, ODQ or by ruthenium red and HC-030031, blockers of subtype 1 of ankyrin transient receptor potential (TRPA1 ) channels. Verapamil exacerbated the potentiating effect of NPB. The potentiating effect was undetectable in preparations precontracted by 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U-46619). Relaxation was reduced by ruthenium red while it was enhanced by HC-030031. In conclusion, NPB has vasodilator properties but with a mechanism of action distinct from its analogues. Contrary to other nitro compounds, its relaxing effects did not involve recruitment of the guanylyl cyclase pathway. NPB has also endothelium-dependent potentiating properties on phenylephrine-induced contractions, a phenomenon that putatively required a role of endothelial TRPA1 channels. The present findings reinforce the notion that the functional group NO2 in the aliphatic chain of these nitro compounds determines favorably their vasodilator properties.

Abstract 397: Preventing Cardiomyopathy of Muscular Dystrophy Through Antagonism of the Thromboxane/Prostanoid Receptor

Muscular dystrophy causes mechanical damage and increased membrane permeability of cardiomyocytes, leading to progressive cardiomyopathy and diffuse myocardial fibrosis that typically begins in the free wall of the LV. Cardiac dysfunction is a primary cause of death in Duchenne (DMD) and other muscular dystrophy (MD) patients. Activation of the thromboxane/prostanoid (TP) receptor increases calcium transients in cardiomyocytes, causes arrhythmia, and is pro-fibrotic. We thus hypothesized that TP receptor activation contributes to the cardiac phenotype of MD, and that blockade of the TP receptor would improve cardiac fibrosis and function in mouse MD models. We gave 3 different mouse models of MD either normal drinking water or water containing 25 mg/kg/day of the TP receptor antagonist ifetroban, from weaning to the predetermined endpoint. TP receptor antagonism improved 10-week survival from 60% to 100% in utrophin/dystrophin double knockout mice, a model of severe DMD, and increased cardiac output compared with surviving vehicle-treated mice. In the mdx/mTR mouse model of DMD, treatment with ifetroban likewise improved 6-month survival from 43% to 100% and increased cardiac output. Finally, we examined delta-sarcoglycan knockout (dSG KO) male mice, a model of limb-girdle muscular dystrophy (LGMD) that replicates the DMD cardiac phenotype with improved survival. TP receptor antagonism normalized fractional shortening, ejection fraction, and LVSP in dSG KO mice, and decreased plasma ANP. However, it had no effect on the contraction deficits of isolated cardiomyocytes other than to normalize the slowed relaxation of dSG KO. Ifetroban-treated mice had improved myocardial, but not skeletal muscle fibrosis. This was most noticeable in the LV free wall and occurred in conjunction with decreased TGF-beta activity and normalized plasma WBC. Typical of DMD cardiomyopathy, dSG KO hearts had reduced expression of neuronal nitric oxide synthase (nNOS) and claudin-5, which was improved with TPr antagonism. The results of our studies indicate that TP receptor activation may contribute to MD cardiomyopathy, and oral antagonism of the TP receptor may be a novel therapeutic for the cardiac phenotype in DMD and LGMD patients.

Abstract 261: Activation of the Thromboxane/Prostanoid Receptor Contributes to Elevated End-Diastolic Calcium in Cardiomyocytes and Cardiac Fibrosis Following Right Ventricular Pressure Overload

Like its systemic counterpart, pulmonary arterial hypertension (PAH) results in remodeling and fibrosis of the right ventricle as it attempts to adapt to the increased pressure overload. This eventually leads to contractile dysfunction, and RV failure is the primary cause of death in PAH patients. The G protein-coupled thromboxane/prostanoid (TP) receptor is expressed in vascular smooth muscle and immune cells, and is upregulated in cardiomyocytes following PAH. Activation of the cardiac TP receptor increases cardiomyocyte intracellular calcium and can lead to arrhythmias. We previously reported that oral treatment with the TP receptor antagonist ifetroban prevents RV fibrosis in a mouse pressure overload model of PAH. Here, we investigate the effects of TP receptor activation on calcium handling in RV cardiomyocytes and explore treatment of established RV remodeling with ifetroban, compared with prevention. Fixed pressure overload of the RV via pulmonary arterial banding (PAB) caused an increase in contractility and resting (end-diastolic) intracellular calcium in individual cardiomyocytes after 3 weeks; this occurred in conjunction with RV dilation, fibrosis, and stiffness. Surprisingly, total calcium content of the sarcoplasmic reticulum was increased following PAB. Antagonism with ifetroban decreased formation of fibrosis in a time-dependent manner. However, treatment with antagonist following establishment of RV fibrosis still prevented the cardiomyocyte increase in end-diastolic calcium. This suggests a multi-factorial contribution of the TP receptor in the RV response to PAH. Further studies continue to analyze changes in calcium-dependent signaling, as well as the contribution of the cardiomyocyte TP receptor to both fibrosis and calcium handling.

Molecular docking, molecular modeling, and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists.

Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes. Graphical abstract ᅟ.

Targeting the PGD2/CRTH2/DP1 Signaling Pathway in Asthma and Allergic Disease: Current Status and Future Perspectives

Prostaglandin D2 (PGD2) released by degranulating mast cells is believed to play a key role in orchestrating mechanisms of inflammation in allergies and asthma. The biological effects of PGD2 are mediated by D-prostanoid (DP1), CRTH2 (DP2), and thromboxane prostanoid (TP) receptors. The CRTH2 receptor is involved in induction of migration and activation of T helper type 2 (Th2) lymphocytes, eosinophils, and basophils; up-regulation of adhesion molecules; and promotion of pro-inflammatory Th2-type cytokines (interleukin [IL]-4, 5, 13), whereas the DP receptor is associated with relaxation of smooth muscles, vasodilation, inhibition of cell migration, and apoptosis of eosinophils. A number of CRTH2/PGD2 receptor antagonists have been investigated in asthma and allergic diseases. The CRTH2 antagonist (OC000459) or dual CRTH2 and TP receptor antagonist (ramatroban) were effective in reducing eosinophilia, nasal mucosal swelling, and clinical symptoms of allergic rhinitis, with the latter drug registered for clinical use in this indication. OC000459 and setipiprant reduced the late but not early phase of response in an allergen challenge in atopic asthmatics. In persistent asthma, some molecules induced limited improvement in lung function, quality of life, and asthma symptoms (OC000459, BI671800), but in other trials with AMG 853 and AZ1981 these findings were not confirmed. The clear discrepancy between animal studies and clinical efficacy of CRTH2 antagonism in allergic rhinitis, and lack of efficacy in a general cohort of asthmatics, highlight the issue of patient phenotyping. There is no doubt that the PGD2/CATH2/DP1 pathway plays a key role in allergic inflammation and further studies with selective or combined antagonisms in well defined cohorts of patients are needed.

Antagonism of the thromboxane-prostanoid receptor is cardioprotective against right ventricular pressure overload.

Right ventricular (RV) failure is the primary cause of death in pulmonary arterial hypertension (PAH) and is a significant cause of morbidity and mortality in other forms of pulmonary hypertension. There are no approved therapies directed at preserving RV function. F-series and E-series isoprostanes are increased in heart failure and PAH, correlate to the severity of disease, and can signal through the thromboxane-prostanoid (TP) receptor, with effects from vasoconstriction to fibrosis. The goal of these studies was to determine whether blockade of the TP receptor with the antagonist CPI211 was beneficial therapeutically in PAH-induced RV dysfunction. Mice with RV dysfunction due to pressure overload by pulmonary artery banding (PAB) were given vehicle or CPI211. Two weeks after PAB, CPI211-treated mice were protected from fibrosis with pressure overload. Gene expression arrays and immunoblotting, quantitative histology and morphometry, and flow cytometric analysis were used to determine the mechanism of CPI211 protection. TP receptor inhibition caused a near normalization of fibrotic area, prevented cellular hypertrophy while allowing increased RV mass, increased expression of antifibrotic thrombospondin-4, and blocked induction of the profibrotic transforming growth factor β (TGF-β) pathway. A thromboxane synthase inhibitor or low-dose aspirin failed to replicate these results, which suggests that a ligand other than thromboxane mediates fibrosis through the TP receptor after pressure overload. This study suggests that TP receptor antagonism may improve RV adaptation in situations of pressure overload by decreasing fibrosis and TGF-β signaling.

Prostanoid-mediated contractions of the carotid artery become Nox2-independent with aging.

Aging is a major risk factor for carotid artery disease that may lead to stroke and dementia. Vascular effects associated with aging include increased vasomotor tone, as well as enhanced contractility to endothelial vasoconstrictor prostanoids and reduced nitric oxide (NO) bioactivity partly due to increased oxidative stress. We hypothesized that vascular NADPH oxidase (Nox)-derived superoxide may be involved in prostanoid- and NO-related functional aging. NO-mediated relaxations and prostanoid-mediated contractions to acetylcholine as well as phenylephrine-dependent contractions were investigated in the carotid artery from young (4 months) and aged mice (24 months). Gene expression of Nox subunits and endothelial NO synthase (eNOS) was determined in the carotid artery and aorta. In young mice, the thromboxane-prostanoid receptor antagonist SQ 29,548 fully blocked acetylcholine-induced contractions while reducing responses to phenylephrine by 75 %. The Nox2-targeted inhibitor Nox2ds-tat and the superoxide scavenger tempol reduced acetylcholine-stimulated, prostanoid-mediated contractions by 85 and 75 %, respectively, and phenylephrine-dependent contractions by 45 %. Unexpectedly, in aged mice, the substantial Nox2-dependent component of acetylcholine- and phenylephrine-induced, prostanoid-mediated contractions was abolished. In addition, endothelium-dependent, NO-mediated relaxations were impaired with aging. The expression of Nox subunits was greater in the aorta compared with the carotid artery, in which Nox1 was undetectable. eNOS gene expression was reduced in the aorta of aged compared to young mice. In conclusion, aging decreases prostanoid-mediated contractility in the carotid artery involving a loss of Nox2 activity and is associated with impaired endothelium-dependent, NO-mediated relaxation. These findings may contribute to a better understanding of the pathophysiology of carotid artery disease and the aging process.

A vasoconstrictor response to COX-1-mediated prostacyclin synthesis in young rat renal arteries that increases in prehypertensive conditions.

This study aimed to determine whether prostacyclin (PGI2) functions as an endothelium-derived contracting factor (EDCF) in young rat renal arteries, and, if so, we wanted to examine the underlying mechanism(s) and how it changes in prehypertensive conditions. Vessels from Wistar-Kyoto (WKY) and prehypertensive spontaneously hypertensive rats (SHRs) of 25-28 days of age were isolated for functional and biochemical analyses. Result showed that following NO synthase (NOS) inhibition PGI2 and the thromboxane-prostanoid (TP) receptor agonist U-46619 evoked contractions in young WKY renal arteries that were similar to those in prehypertensive SHRs. Meanwhile, the endothelial muscarinic receptor agonist ACh evoked an endothelium-dependent contraction under NOS-inhibited conditions and a production of the PGI2 metabolite 6-keto-PGF1α; both were sensitive to cyclooxygenase (COX) and/or COX-1 inhibition but higher in prehypertensive SHRs than in young WKYs. Interestingly, in WKY renal arteries PGI2 did not evoke relaxation even after TP receptor antagonism that diminished the contraction evoked by the agonist. Indeed, PGI2 (IP) receptors were not detected in the vessel with Western blot. Moreover, we noted that treatment with the nonselective COX inhibitor indomethacin, which was started at the prehypertensive stage, blunted the elevation of systolic blood pressure and reduced the heart-to-body ratio in SHR within 2 mo of treatment. These results demonstrate that due to scarcity of IP receptors, PGI2, which is derived mainly from COX-1-mediated metabolism, acts as an EDCF in young WKY renal arteries, and it increases in prehypertensive conditions. Also, our data revealed that COX inhibition starting from the prehypertensive stage has an antihypertensive effect in young SHRs.

Abstract 335: Prostaglandin I 2 Receptor Blockade Contracts Rat Renal Arteries and Facilitates Endothelium-Dependent Contractions.

Endothelium-dependent contractions are augmented in human hypertension and in isolated preparations, including renal arteries, from hypertensive animals. Renal vasomotor tone is essential in the regulation of arterial blood pressure and renal arteries are highly sensitive to vasoconstrictor prostanoids including prostacyclin, identified as endothelium-derived contracting factor (EDCF) activating thromboxane prostanoid (TP) receptors. However, the mechanisms underlying increased EDCF responses in hypertension are not completely resolved. Isometric tension was measured, in wire myographs, in renal arterial rings from spontaneously hypertensive (SHR) and age (38-52 weeks)-matched Wistar Kyoto (WKY) rats. Responses were compared under control conditions and after incubation with indomethacin (10 -5 mol/L, cyclooxygenase inhibitor), ketanserin (10 -6 mol/L, 5-HT 2 receptor antagonist), CAY10441 (10 -6 mol/L, prostaglandin I 2 [IP] receptor antagonist), and S18886 (10 -7 mol/L, TP receptor antagonist). All experiments were performed in the presence of L-NAME (3 x 10 -4 mol/L) to exclude effects of endothelium-derived NO. Surprisingly, CAY10441 (10 -10 to 10 -6 mol/L) contracted renal arterial rings from both strains; the contraction at the highest concentration was significantly larger in SHR (15±0.7 mN) than in WKY (10±1.5 mN) preparations ( n =10, P &lt;0.05). This phenomenon was blunted by indomethacin, ketanserin, but not S18886. After washout and re-incubation, EDCF responses to acetylcholine (10 -8 to 10 -4 mol/L) were similar between SHR and WKY preparations in the presence of CAY10441 ( E max 72.6±5.3% and 67.4±3.8% KCl, n =8), but significantly larger than under control conditions ( n =4-8, P &lt;0.05). S18886 prevented EDCF responses in the absence but not in the presence of CAY10441. Contractions to prostacyclin [in the presence of indomethacin] were facilitated by CAY10441 in rings from both strains and no longer prevented by S18886, although the TP receptor antagonist abolished contractions to U46619 (10 -11 to 3 x 10 -6 mol/L). These data suggest that: a) inhibitory IP receptors reduce renal vasomotor tone; b) IP and TP receptors form heterodimers; and c) endogenous serotonin contributes to the action of prostacyclin in rat renal arteries.

Endothelin-1 but not angiotensin II contributes to functional aging in murine carotid arteries

AimsAging is a major risk factor for carotid artery disease and stroke. Endothelin-1 (ET-1) and angiotensin II (Ang II) are important modifiers of vascular disease, partly through increased activity of NADPH oxidase and vasoconstrictor prostanoids. Since the renin–angiotensin and endothelin systems become activated with age, we hypothesized that aging affects NADPH oxidase- and prostanoid-dependent contractions to ET-1 and Ang II. Main methodsCarotid artery rings of young (4month-old) and old (24month-old) C57BL6 mice were pretreated with the NO synthase inhibitor L-NAME to exclude differential effects of NO. Contractions to ET-1 and Ang II were determined in the presence and absence of the NADPH oxidase-selective inhibitor gp91ds-tat or the thromboxane-prostanoid receptor antagonist SQ 29,548. Gene expression of endothelin and angiotensin receptors was measured by qPCR. Key findingsAging reduced ET-1-induced contractions and diminished ETA but increased ETB receptor gene expression levels. Gp91ds-tat inhibited contractions to ET-1 in young and to a greater extent in old animals, whereas SQ 29,548 had no effect. Ang II-induced contractions were weak compared to ET-1 and unaffected by aging, gp91ds-tat, and SQ 29,548. Aging had also no effect on AT1A and AT1B receptor gene expression levels. SignificanceAging in carotid arteries decreases ETA receptor gene expression and responsiveness to ET-1, which nevertheless becomes increasingly dependent upon NAPDH oxidase activity with age; responses to Ang II and gene expression of its receptors are however unaffected. These findings suggest that physiological aging differentially regulates functional responses to G protein-coupled receptor agonists and the signaling pathways associated with their activation.

Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.