Thromboxane Prostanoid Receptor Research Articles

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.

Endothelin-1-Induced Microvascular ROS and Contractility in Angiotensin-II-Infused Mice Depend on COX and TP Receptors.

(1) Background: Angiotensin II (Ang II) and endothelin 1 (ET-1) generate reactive oxygen species (ROS) that can activate cyclooxygenase (COX). However, thromboxane prostanoid receptors (TPRs) are required to increase systemic markers of ROS during Ang II infusion in mice. We hypothesized that COX and TPRs are upstream requirements for the generation of vascular ROS by ET-1. (2) Methods: ET-1-induced vascular contractions and ROS were assessed in mesenteric arterioles from wild type (+/+) and knockout (−/−) of COX1 or TPR mice infused with Ang II (400 ng/kg/min × 14 days) or a vehicle. (3) Results: Ang II infusion appeared to increase microvascular protein expression of endothelin type A receptors (ETARs), TPRs, and COX1 and 2 in COX1 and TPR +/+ mice but not in −/− mice. Ang II infusion increased ET-1-induced vascular contractions and ROS, which were prevented by a blockade of COX1 and 2 in TPR −/− mice. ET-1 increased the activity of aortic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and decreased superoxide dismutase (SOD) 1, 2, and 3 in Ang-II-infused mice, which were prevented by a blockade of TPRs. (4) Conclusion: Activation of vascular TPRs by COX products are required for ET-1 to increase vascular contractions and ROS generation from NADPH oxidase and reduce ROS metabolism by SOD. These effects require an increase in these systems by prior infusion of Ang II.

2090-P: Role of Thromboxane-Prostanoid Receptor in Obesity Related Insulin Resistance

Background: Thromboxane-Prostanoid Receptor (TP-R) is implicated in several chronic inflammatory disorders. Our preclinical animal studies showed that TP-R plays a critical role in obesity linked Insulin Resistance (IR). Aim: To determine the link between TP-R and obesity and/or obesity-associated inflammation/IR in human subjects. Methods: We collected PBMCs from lean normal and obese IR subjects and analyzed the expression of various inflammatory genes including TP-R, Thromboxane A2 Synthase (TXA2S), TNFα, and MCP-1. We obtained subcutaneous adipose tissue biopsies from obese subjects and analyzed the TP-R mRNA expression and its correlation with inflammatory cytokines mediating IR. Results: TP-R mRNA expression is significantly increased in PBMCs of obese IR subjects compared to lean normal subjects (P&amp;lt;0.04 by Wilcoxon rank-sum test). In addition, PBMC TP-R expression is positively correlated with the expression of TXA2S, a gene involved in thromboxane A2 synthesis (n=29, Spearman’s r=0.36, P=0.05). Moreover, TP-R expression is positively correlated with body weight, BMI and fat mass (Figure). In AT, TP-R expression is positively correlated with IL-8 expression (n=31, r=0.49, P&amp;lt;0.005). Conclusion: Our data demonstrate that TP-R expression is positively correlated with markers of obesity and/or IR and support our hypothesis that TP-R can be used as a biomarker or a therapeutic target for obesity related IR. Disclosure T. Thangavelu: None. S. Viswanathan: None. C. Desouza: Consultant; Self; Novo Nordisk A/S, Sanofi US. Funding National Institute of General Medical Sciences; Great Plains Institutional Developmental Award for Clinical and Translational Research

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.

116 - Thromboxane prostanoid receptor signaling in the urinary bladder smooth muscle

116 - Thromboxane prostanoid receptor signaling in the urinary bladder smooth muscle

Mechanisms of U46619-induced contraction in mouse intrarenal artery.

Thromboxane A2 (TXA2 ) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca2+ ]i ) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo-4/AM dye and a confocal laser scanning microscopy. The results show that the U46619-induced vasoconstriction of renal artery was completely blocked by a TXA2 receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI-PLC) inhibitor U73122 at 10μmol/L and partially inhibited by a Phosphatidylcholine - specific phospholipase C (PC-PLC) inhibitor D609 at 50μmol/L. Moreover, the U46619-induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10μmol/L, and a selective PKCδ inhibitor rottlerin at 10μmol/L. In addition, the PKC-induced vasoconstriction was partially inhibited by a Rho-kinase inhibitor Y-27632 at 10μmol/L and was further completely inhibited together with a putative IP3 receptor antagonist and store-operated Ca2+ (SOC) entry inhibitor 2-APB at 100μmol/L. On the other hand, U46619-induced vasoconstriction was partially inhibited by L-type calcium channel (Cav1.2) inhibitor nifedipine at 1μmol/L and 2-APB at 50 and 100μmol/L. Last, U46619-induced vasoconstriction was partially inhibited by a cell membrane Ca2+ activated C1- channel blocker 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) at 50 and 100μmol/L. Our results suggest that the U46619-induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane-prostanoid receptors and its downstream signaling pathway.

Anaphylatoxin C3a induces vasoconstriction and hypertension mediated by thromboxane A2 in mice

IntroductionSeveral recent reports indicate a marked crosstalk between innate immunity and vasoregulation. Anaphylatoxins C3a and C5a have been reported to induce vasoactive effects, but the mechanism of their actions is obscure. Earlier studies reported increased plasma levels of C3 in cardiovascular diseases, especially pulmonary hypertension. Therefore, complement activation may contribute to changes of the vascular tone and reactivity under pathophysiological conditions. In our present study we investigated the effects of C3a, the activate cleavage product of the most abundant complement protein of the human circulation, on the arterial tone and blood pressure as well as the signaling pathways involved.Materials and methodsThoracic aortic segments were isolated from adult male C57Bl/6 wild type (WT) and knockout (KO) mice deficient in either thromboxane prostanoid receptor (TP KO) or cyclooxygenase 1 (COX1 KO). Isometric tension changes of vascular segments were measured via myography. We studied the effects of C3a by using C3a (63–77) peptide fragment, a specific C3a receptor agonist. Vascular responses were quantitated as percent of reference contraction induced by 124 mM K+. In addition, we performed experiments monitoring blood pressure (BP) changes in vivo. In these experiments zymosan (30 mg/kg, i.v.), a direct complement activator was administered intravenously, after cannulation of carotid artery and jugular vein in anesthetized (pentobarbital, 90 mg/kg i.p.) mice. Blood samples were taken from other groups of WT mice and plasma TXB2 levels were measured 5 minutes after treatment with saline or zymosan.ResultsC3a (63–77) evoked a pronounced vasoconstriction in WT vessels (33±5%), which effect remained unaltered in the absence of endothelium (37±7%). The vessels of COX1 KO mice showed a decreased response (10±2%), and the C3a fragment caused a similarly reduced constriction in TP KO vessels (4±1%). In our in vivo experiments the administration of zymosan led to an increase of BP in WT mice (with an average of 14±5 mmHg), whereas causing a drop of 22±3 mmHg in TP KO mice's BP. In accordance, zymosan caused a two‐fold increase in the plasma level of thromboxane B2 in WT animals.ConclusionsOur experiments showed, that C3a (63–77) causes vasoconstriction in a non‐endothelium‐dependent manner, and this effect is mediated by COX1 and TP. These results propose that C3a‐mediated vasoconstriction is mediated by smooth muscle derived thromboxane A2. In addition, C3a (63–77) induce hypertension, which is also mediated by thromboxane A2. These results altogether indicate that C3a is involved in the regulation of vascular tone and/or reactivity via stimulating thromboxane A2 release.Support or Funding InformationGrant support: Semmelweis Research and Innovation Fund (STIA‐M‐17); Ministry of Human Capacities of Hungary (ÚNKP‐18‐2‐I‐SE‐12, FIKP‐2018 and EFOP‐3.6.3‐VEKOP‐16‐2017‐00009) and the Hungarian NRDIO (OTKA K‐115623, K‐125174 and NVKP_16‐1‐2016‐0042)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prostaglandin D2 receptor antagonists in allergic disorders: safety, efficacy, and future perspectives

ABSTRACTIntroduction: Prostaglandin D2 (PGD2) is a major cyclooxygenase mediator that is synthesized by activated human mast cells and other immune cells. The biological effects of PGD2 are mediated by D-prostanoid (DP1), DP2 (CRTH2) and thromboxane prostanoid (TP) receptors that are expressed on several immune and non-immune cells involved in allergic inflammation. PGD2 exerts various proinflammatory effects relevant to the pathophysiology of allergic disorders. Several selective, orally active, DP2 receptor antagonists and a small number of DP1 receptor antagonists are being developed for the treatment of allergic disorders.Areas covered: The role of DP2 and DP1 receptor antagonists in the treatment of asthma and allergic rhinitis.Expert opinion: Head-to-head studies that compare DP1 antagonists with the standard treatment for allergic rhinitis are necessary to verify the role of these novel drugs as mono- or combination therapies. Further clinical trials are necessary to verify whether DP2 antagonists as monotherapies or, more likely, as add-on therapies, will be effective for the treatment of different phenotypes of adult and childhood asthma. Long-term studies are necessary to evaluate the safety of targeted anti-PGD2 treatments.

Genetic Depletion of Thromboxane A2/Thromboxane-Prostanoid Receptor Signalling Prevents Microvascular Dysfunction in Ischaemia/Reperfusion Injury.

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. 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. Inhibiting TXAS/TXA2/TP signalling confers microvascular protection against oxidative injury in both cardiac and mesenteric arteries.

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

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

Impaired vasocontractile responses to adenosine in chorionic vessels of human term placenta from pregnant women with pre-existing and gestational diabetes

There is clinical and experimental evidence for altered adenosine signalling in the fetoplacental circulation in pregnancies complicated by diabetes, leading to adenosine accumulation in the placenta. However, the consequence for fetoplacental vasocontractility is unclear. This study examined contractility to adenosine of chorionic vessels from type 1 diabetes mellitus, gestational diabetes mellitus and normal pregnancies. Chorionic arteries and veins were isolated from human placenta from normal, gestational diabetes mellitus and type 1 diabetes mellitus pregnancies. Isometric tension recording measured responses to adenosine and the thromboxane A2 analogue U46619 (thromboxane A2 mediates fetoplacental vasoconstriction to adenosine). Adenosine and thromboxane prostanoid receptor protein expression was determined by immunoblotting. Adenosine elicited contractions in chorionic arteries and veins which were impaired in both gestational diabetes mellitus and type 1 diabetes mellitus. Contractions to potassium chloride were unchanged. Adenosine A2A and A2B receptor protein levels were not different in gestational diabetes mellitus and normal pregnancies. Contractions to U46619 were unaltered in gestational diabetes mellitus arteries and increased in type 1 diabetes mellitus arteries. Overnight storage of vessels restored contractility to adenosine in gestational diabetes mellitus arteries and normalized contraction to U46619 in type 1 diabetes mellitus arteries. These data are consistent with the concept of aberrant adenosine signalling in diabetes; they show for the first time that this involves impaired adenosine contractility of the fetoplacental vasculature.

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.

P2622Ageing-related endothelial dysfunction in the femoral vein is mediated by cyclooxygenases: Role of thromboxane prostanoid receptors

P2622Ageing-related endothelial dysfunction in the femoral vein is mediated by cyclooxygenases: Role of thromboxane prostanoid receptors

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.

Role of thromboxane prostanoid (TP) receptor in the facilitation of angiogenesis and the healing of gastric ulcers

Role of thromboxane prostanoid (TP) receptor in the facilitation of angiogenesis and the healing of gastric ulcers

PPAR-α agonists acutely inhibit Ca2+-independent PLA2 to reduce H2O2-induced contractions in aortae of spontaneously hypertensive rats.

Hypertension is associated with endothelial dysfunction, which favors the release of endothelium-derived contracting factors, including vasoconstrictor prostanoids and reactive oxygen species. Peroxisome proliferator-activated receptor-α (PPAR-α) agonists, clinically used as lipid-lowering drugs, possess antioxidant properties and exert beneficial effects in the vascular system. The present study aimed to identify the mechanism(s) underlying the acute effects of the PPAR-α agonists Wy14643 and fenofibate on endothelium-dependent contractions, in particular those related to oxidative stress, in the aorta of the spontaneously hypertensive rat (SHR). Aortic rings with and without endothelium of male SHRs and normotensive Wistar-Kyoto rats were suspended in organ chambers for isometric tension measurements and homogenized for enzyme activity assays. Contractions to acetylcholine in quiescent SHR aortae with endothelium were reduced by tiron (superoxide anion scavenger), diethyldithiocarbamic acid (superoxide dismutase inhibitor), and acute treatment with either Wy14643 or fenofibrate. Similarly to contractions evoked by acetylcholine, H2O2-induced increases in tension in SHR aortae involved, in succession, phospholipase A2 (PLA2), cyclooxygenase, and thromboxane-prostanoid receptors. Wy14643 or fenofibrate, by decreasing the activity of endothelial Ca2+-independent PLA2, attenuated the contractions to H2O2. In conclusion, the increased oxidative stress in the SHR aorta (mainly increased production of H2O2 and its partially reduced product, hydroxyl radical) contributed to acetylcholine-induced, endothelium-dependent contractions; PPAR-α agonists likely inhibit the H2O2-mediated contractions by inhibiting endothelial Ca2+-independent PLA2. The present study highlights the prospective therapeutic effects of PPAR-α agonists in improving endothelial function in hypertension and other vascular implications due to oxidative stress. NEW & NOTEWORTHY Peroxisome proliferator-activated receptor-α agonists, which are used clinically as lipid-lowering drugs, acutely reduce H2O2-induced contractions in aortae of hypertensive rats by inhibiting the activity of endothelial Ca2+-independent phospholipase A2. These vascular effects of peroxisome proliferator-activated receptor-α agonists suggest that they may help to prevent vascular complications under pathological conditions associated with oxidative stress.

Endothelium-derived contraction in a model of rheumatoid arthritis is mediated via angiotensin II type 1 receptors

A role for endothelium-derived constricting factors (EDCF), and the angiotensin II type 1 receptor (AT1R) pathway, in the vascular impairment found in the rat Freund's complete adjuvant (FCA)-model of rheumatoid arthritis (RA) was examined. FCA arthritis was induced in rats±losartan. Vehicle-treated rats served as controls. Knee-joint swelling and red blood cell (RBC) aggregation were measured as indicators of inflammation and endothelium reactivity assessed by response to acetylcholine (ACh) on aortic rings. Results show that knee-joint swelling and RBC aggregation were elevated in the FCA+vehicle group and restored to control levels in the FCA+losartan-treated animals. ACh-induced relaxation of aortic rings taken from FCA+vehicle animals was significantly impaired compared to vehicle-controls and this vasoreactivity was restored to control levels in the FCA+losartan-treated group. Further examination of aorta from the FCA+vehicle animals revealed an EDCF that was reliant on cyclooxygenase-2 (but not cyclooxygenase-1), generation of superoxide anion generation (but not hydrogen peroxide) and activation of thromboxane-prostanoid receptor. Losartan administration in vivo or ex vivo (to aortic rings) prevented the generation of the EDCF. In summary, this is the first evidence of an EDCF in a model of RA and identifies this mechanism as potentially significant in the cardiovascular disorder associated with the disease.

1, 6-di-O-caffeoyl-β-D-glucopyranoside, a natural compound from Callicarpa nudiflora Hook impairs P2Y12 and thromboxane A2 receptor-mediated amplification of platelet activation and aggregation

BackgroundPlatelet activation and subsequent accumulation at sites of vascular injury perform a central role in thrombus formation, which is believed to be the trigger of several cardiovascular diseases, such as atherosclerosis, myocardial infarction and strokes. In this sense, the search for agents that are capable of blocking platelets aggregation has important implications for these diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicine herb for eliminating stasis to subdue swelling and hemostasis. Our previous study found several compounds extracted from this herb, including 1, 6-di-O-caffeoyl-β-D-glucopyranoside (CGP), showed inhibitory effects on adenosine diphosphate (ADP) induced platelet aggregation. PurposeThe aim of current study is confirmation of the anti-platelet effects and elucidation of the probable mechanisms. MethodsThe experiments were performed on platelet rich plasma freshly isolated from SD rat. ADP, U46619 or arachidonic acid (AA) induced platelet aggregation assay were performed to evaluate the anti-platelet properties of CGP. Activated αIIbβ3 integrin abundance, serotonin (5-HT) secretion, thromboxane A2 (TXA2) synthesis was determined to assess the effects of CGP on platelet activation. Furthermore, RhoA and PI3K/Akt/GSK3β signal transduction were analyzed by Western Blotting assay. In addition, radiolabelled ligand binding assay was involved to evaluate the ability of CGP binding to thromboxane prostanoid (TP) and P2Y12 receptors. ResultsCGP inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid (AA), significantly. Furthermore, it is also found that LGP exhibited obvious inhibitory effects on αIIbβ3 integrin activation, serotonin (5-HT) secretion from granule and thromboxane A2 (TXA2) synthesis. Next, we found that CGP suppressed RhoA and PI3K/Akt/GSK3β signal transduction. Data from radiolabelled ligand binding assay showed that CGP displayed apparent competing effects on TP and P2Y12 receptors. ConclusionCollectively, the data presented here demonstrated that CGP, a natural compound from Callicarpa nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual-receptor inhibition on P2Y12 and TP receptors.

Abstract P344: Nuclear Factor E2-related Factor 2 (nrf2) Causes Early Microvascular Endothelial Dysfunction and Increased Adma by Activation of the Cox2/tp Receptor Pathway in Mice Infused With Angiotensin II for Three Days

Background: Activation of Nrf2 by tert-buthylhydroquinone (tBHQ) prevents hypertension, oxidative stress, endothelial dysfunction and ADMA in mice infused with angiotensin II (Ang II) for 12 days. However, Nrf2 activation with bardoxolone methyl increased cardiovascular events in diabetic patients within one week. Since we found that tBHQ activated cyclooxygenase (COX)-2 within 3 days, we tested the hypothesis that short-term tBHQ administration to Ang II infused mice increases microvascular dysfunction via COX dependent of PGs and TxA 2 that activate thromboxane-prostanoid receptors (TP-Rs). Methods: Mesenteric resistance arterioles(MRAs)were isolated from mice infused for 3 days with ANG II (400 ng/kg/min) or vehicle and given oral tBHQ (0.1% of water) or vehicle (n=6 mice/group). Endothelial derived relaxation factor (EDRF) was assessed by a myograph and NO and ROS by RatioMaster TM . Results: Compared to vehicle, Ang II infused mice given tBHQ had increased (P&lt;0.05) conscious mean arterial pressure (135±6 vs 115±7 mmHg), urinary 8-Iso prostane (1.5±0.4 vs 1.1±0.2ng/mg creatinine), and decreased EDRF (17± 3 vs 23± 2%) and NO (0.23 ± 0.02 vs 0.35 ± 0.02 Δunits), and enhanced ( P&lt;0.05, ) cellular ROS (0.27 ± 0.02 vs 0.12 ± 0.03 Δunits), mitochondria ROS (0.24 ± 0.02 vs 0.1 ± 0.02 Δunit) and microvascular asymmetric dimethylarginine (ADMA, 77 ± 6 vs 55 ± 7 nmol/mg protein). All these effects of tBHQ were prevented in COX-1 knockout mice drinking parecoxib (COX-2 inhibitor) and in TPR and Nrf2 knockout mice. Conclusions: Activation of Nrf2 increases short term Ang II-induced increases in BP, oxidative stress, ADMA and endothelial dysfunction. These depend on signaling via COX 1 + 2 and TP receptors. Our studies reveal novel dual effects of Nrf2 in Ang II infused mice: increased BP, ROS, ADMA and endothelial dysfunction within 3 days secondary to activation of COX/TPR signaling follow by the opposite effects of Nrf2 on BP, ADMA and vascular function within 12 days. Therefore, blockade of COX-2/TPRs pathway during initiation of Nrf2 therapy may prevent the devastating early adverse effects that have prevented its use in hypertension or diabetes.