Expression Of Thromboxane-prostanoid Receptors Research Articles

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.

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<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<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

Thromboxane Prostanoid Receptors Enhance Contractions, Endothelin-1, and Oxidative Stress in Microvessels From Mice With Chronic Kidney Disease

Cardiovascular disease is frequent in chronic kidney disease and has been related to angiotensin II, endothelin-1 (ET-1), thromboxane A2, and reactive oxygen species (ROS). Because activation of thromboxane prostanoid receptors (TP-Rs) can generate ROS, which can generate ET-1, we tested the hypothesis that chronic kidney disease induces cyclooxygenase-2 whose products activate TP-Rs to enhance ET-1 and ROS generation and contractions. Mesenteric resistance arterioles were isolated from C57/BL6 or TP-R+/+ and TP-R-/- mice 3 months after SHAM-operation (SHAM) or surgical reduced renal mass (RRM, n=6/group). Microvascular contractions were studied on a wire myograph. Cellular (ethidium: dihydroethidium) and mitochondrial (mitoSOX) ROS were measured by fluorescence microscopy. Mice with RRM had increased excretion of markers of oxidative stress, thromboxane, and microalbumin; increased plasma ET-1; and increased microvascular expression of p22(phox), cyclooxygenase-2, TP-Rs, preproendothelin and endothelin-A receptors, and increased arteriolar remodeling. They had increased contractions to U-46,619 (118 ± 3 versus 87 ± 6, P<0.05) and ET-1 (108 ± 5 versus 89 ± 4, P<0.05), which were dependent on cellular and mitochondrial ROS, cyclooxygenase-2, and TP-Rs. RRM doubled the ET-1-induced cellular and mitochondrial ROS generation (P<0.05). TP-R-/- mice with RRM lacked these abnormal structural and functional microvascular responses and lacked the increased systemic and the increased microvascular oxidative stress and circulating ET-1. In conclusion, RRM leads to microvascular remodeling and enhanced ET-1-induced cellular and mitochondrial ROS and contractions that are mediated by cyclooxygenase-2 products activating TP-Rs. Thus, TP-Rs can be upstream from enhanced ROS, ET-1, microvascular remodeling, and contractility and may thereby coordinate vascular dysfunction in chronic kidney disease.

Maternal Obesity Impairs Specific Regulatory Pathways in Human Myometrial Arteries1

Obese women (body mass index ≥30 kg/m(2)) are at greater risk than normal weight women of pregnancy complications associated with maternal and infant morbidity, particularly the development of cardiovascular disease and metabolic disorders in later life; why this occurs is unknown. Nonpregnant, obese individuals exhibit systemic vascular endothelial dysfunction. We tested the hypothesis that obese pregnant women have altered myometrial arterial function compared to pregnant women of normal (18-24 kg/m(2)) and overweight (25-29 kg/m(2)) body mass index. Responses to vasoconstrictors, U46619 (thromboxane mimetic) and arginine vasopressin, and vasodilators, bradykinin and the nitric oxide donor sodium nitroprusside, were assessed by wire myography in myometrial arteries from normal weight (n = 18), overweight (n = 18), and obese (n = 20) women with uncomplicated pregnancies. Thromboxane-prostanoid receptor expression was assessed using immunostaining in myometrial arteries of normal weight and obese women. Vasoconstriction and vasodilatation were impaired in myometrial arteries from obese women with otherwise uncomplicated pregnancies. Disparate agonist responses suggest that vascular function in obese women is not globally dysregulated but may be specific to thromboxane and nitric oxide pathways. Because obesity rates are escalating, it is important to identify the mechanisms underlying impaired vascular function and establish why some obese women compensate for vascular dysfunction and some do not. Future studies are needed to determine whether central adiposity results in an altered endocrine milieu that may promote vascular dysfunction by altering the function of perivascular adipose tissue.

A vasoconstrictor role for cyclooxygenase-1-mediated prostacyclin synthesis in mouse renal arteries

This study was to determine whether prostacyclin [prostaglandin I2 (PGI2)] evokes mouse renal vasoconstriction and, if so, the underlying mechanism(s) and how its synthesis via cyclooxygenase-1 (COX-1) influences local vasomotor reaction. Experiments were performed on vessels from C57BL/6 mice and/or those with COX-1 deficiency (COX-1(-/-)). Results showed that in renal arteries PGI2 evoked contraction more potently than in carotid arteries, where COX-1 is suggested to mediate prominent endothelium-dependent contraction. A similar result was observed with the thromboxane-prostanoid (TP) receptor agonist U46619. However, in renal arteries TP receptor antagonism, which inhibited the contraction, did not result in any relaxation in response to PGI2. Moreover, we noted that the endothelial muscarinic receptor agonist ACh evoked an increase in the production of the PGI2 metabolite 6-keto-PGF1α, which was prevented by endothelial denudation or COX-1(-/-). Interestingly, COX-1(-/-) was further found to abolish a force development that was sensitive to TP receptor antagonism and result in enhanced relaxation evoked by ACh following NO synthase inhibition. Also, in renal arteries the COX substrate arachidonic acid evoked a vasoconstrictor response, which was again abolished by COX-1(-/-). Meanwhile, nonselective COX inhibition did not show any effect in vessels from COX-1(-/-) mice. Thus, in mouse renal arteries, high expression of TP receptors together with little functional involvement from the vasodilator PGI2 receptors results in a potent vasoconstrictor effect evoked by PGI2. Also, our data imply that endogenous COX-1-mediated PGI2 synthesis leads to vasoconstrictor activity and this could be an integral part of endothelium-derived mechanisms in regulating local renal vascular function.

Thromboxane-prostanoid receptor expression and antagonism in dextran-sodium sulfate-induced colitis

In the current study of murine colitis, the potential roles of thromboxane and the thromboxane-prostanoid (TP) receptor were investigated, in as much as thromboxane signaling has been implicated in human inflammatory bowel disease. Colitis was induced in C57BL/6 mice via ingestion of dextran sodium sulfate (DSS), with or without co-administration of the thromboxane synthase inhibitor ozagrel (25 mg/kg/day) or the TP receptor antagonist vapiprost (2.5 mg/kg/day). Immunohistochemistry of colonic tissue demonstrated a DSS-induced increase in TP receptor expression, but not of thromboxane synthase. Moreover, tissue levels of the metabolite thromboxane B(2) were unchanged by DSS. Vapiprost, but not ozagrel, partially attenuated histologic signs of inflammation induced by DSS, with vapiprost allowing a smaller increase in colon weight per unit length than ozagrel. Vapiprost also tended to attenuate DSS-induced alterations in intestinal transit. In summary, TP receptor antagonism was more effective than thromboxane synthase inhibition in alleviating DSS-induced colitis in mice.

Chronic Inhibition of Nitric-Oxide Synthase Potentiates Endothelium-Dependent Contractions in the Rat Aorta by Augmenting the Expression of Cyclooxygenase-2

Acute inhibition of nitric-oxide synthase (NOS) unmasks the release of endothelium-derived contracting factors (EDCFs). The present study investigated whether chronic inhibition of NOS modulates endothelium-dependent contractions. Eighteen-week-old male Sprague-Dawley rats were treated by daily gavage for 6 weeks with the NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) (60 mg/kg) or vehicle (distilled water; 1 ml/kg). Chronic treatment with L-NAME increased arterial blood pressure. Isometric tension was measured in aortic rings with or without endothelium. Endothelium-dependent relaxations to acetylcholine and the calcium ionophore 5-(methylamino)-2-[(2R,3R,6S,8S,9R,11R)-3,9,11-trimethyl-8-[(1S)-1-methyl-2-oxo-2-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazolecarboxylic acid (A23187) were reduced in preparations from L-NAME-treated rats. The reduction in relaxation to A23187 was partially reversed by L-arginine (1 mM). In quiescent aortic rings, A23187 caused contractions in the presence of L-NAME and intact endothelium. The A23187-induced contractions were greater in rings from the L-NAME-treated rats than in those from the control group. These contractions were abolished by the cyclooxygenase (COX)-2 inhibitor N-[2-cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS-398) and the thromboxane-prostanoid (TP) receptor antagonist 3-((6R)-6-{[(4-chlorophenyl)sulfonyl]amido}-2-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)propanoate (S18886), but not by the COX-1 inhibitor 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560). Chronic L-NAME treatment reduced the level of nitric oxide in the plasma but increased COX activity in the aortic rings. Western blotting and immunohistochemical staining showed that endothelial NOS expression was reduced in the aortae of the chronic L-NAME-treated group. COX-1 expression was augmented slightly, whereas COX-2 expression was up-regulated markedly. The TP receptor expression was comparable with control. These experiments demonstrate that chronic NOS inhibition increases endothelium-dependent contractions of the rat aorta by inducing COX-2 expression and augmenting the production of EDCF.

Involvement of COX-1 in A3 adenosine receptor-mediated contraction through endothelium in mice aorta

We investigated whether A(3) adenosine receptor (A(3)AR) is involved in endothelium-mediated contraction through cyclooxygenases (COXs) with the use of wild-type (WT) and A(3) knockout (A(3)KO) mice aorta. A(3)AR-selective agonist, Cl-IBMECA, produced a concentration-dependent contraction (EC(50): 2.9 +/- 0.2 x 10(-9) M) in WT mouse aorta with intact endothelium (+E) and negligible effects in A(3)KO +E aorta. At 10(-7) M, contractions produced by Cl-IBMECA were 29% in WT +E, while being insignificant in A(3)KO +E aorta. Cl-IBMECA-induced responses were abolished in endothelium-denuded tissues (-E), in both WT and A(3)KO aorta. A(3)AR gene and protein expression were reduced by 74 and 72% (P < 0.05), respectively, in WT -E compared with WT +E aorta, while being undetected in A(3)KO +E/-E aorta. Indomethacin (nonspecific COXs blocker, 10(-5) M), SC-560 (specific COX-1 blocker, 10(-8) M), SQ 29549 (thromboxane prostanoid receptor antagonist, 10(-6) M), and furegrelate (thromboxane synthase inhibitor, 10(-5) M) inhibited Cl-IBMECA-induced contraction significantly. Cl-IBMECA-induced thromboxane B(2) production was also attenuated significantly by indomethacin, SC-560, and furegrelate in WT +E aorta, while having negligible effects in A(3)KO +E aorta. NS-398 (specific COX-2 blocker) produced negligible inhibition of Cl-IBMECA-induced contraction in both WT +E and A(3)KO +E aorta. Cl-IBMECA-induced increase in COX-1 and thromboxane prostanoid receptor expression were significantly inhibited by MRS1523, a specific A(3)AR antagonist in WT +E aorta. Expression of both A(3)AR and COX-1 was located mostly on endothelium of WT and A(3)KO +E aorta. These results demonstrate for the first time the involvement of COX-1 pathway in A(3)AR-mediated contraction via endothelium.

Metabolite ligands of estrogen receptor-β reduce primate coronary hyperreactivity

Previous reports showed that 17beta-estradiol implants attenuate in vivo coronary hyperreactivity (CH), characterized by long-duration vasoconstrictions (in coronary angiographic experiments), in menopausal rhesus monkeys. Prolonged Ca2+ contraction signals that correspond with CH in coronary vascular muscle cells (VMC) to the same dual-constrictor stimulus, serotonin + the thromboxane analog U-46619, in estrogen-deprived VMC were suppressed by >72 h in 17beta-estradiol. The purpose of this study was to test whether an endogenous estrogen metabolite with estrogen receptor-beta (ER-beta) binding activity, estriol (E3), suppresses in vivo and in vitro CH. E3 treatment in vivo for 4 wk significantly attenuated the angiographically evaluated vasoconstrictor response to intracoronary serotonin + U-46619 challenge. In vitro treatment of rhesus coronary VMC for >72 h with nanomolar E3 attenuated late Ca2+ signals. This reduction of late Ca2+ signals also appeared after >72 h of treatment with subnanomolar 5alpha-androstane-3beta,17beta-diol (3beta-Adiol), an endogenous dihydrotestosterone metabolite with ER-beta binding activity. R,R-tetrahydrochrysene, a selective ER-beta antagonist, significantly blocked the E3- and 3beta-Adiol-mediated attenuation of late Ca2+ signal increases. ER-beta and thromboxane-prostanoid receptor (TPR) were coexpressed in coronary arteries and aorta. In vivo E3 treatment attenuated aortic TPR expression. Furthermore, in vitro treatment with E3 or 3beta-Adiol downregulated TPR expression in VMC, which was blocked for both agonists by pretreatment with R,R-tetrahydrochrysene. E3- and 3beta-Adiol-mediated reduction in persistent Ca2+ signals is associated with ER-beta-mediated attenuation of TPR expression and may partly explain estrogen benefits in coronary vascular muscle.

Medroxyprogesterone Acetate and Dihydrotestosterone Induce Coronary Hyperreactivity in Intact Male Rhesus Monkeys

Coronary hyperreactivity (CH), characterized by persistent severe vasoconstrictions in response to vasoconstrictor challenge, is oppositely influenced by progesterone (P) and medroxyprogesterone acetate (MPA) treatment in surgically menopausal primates. In this study we tested whether multiweek MPA or dihydrotestosterone (DHT) exposure induced CH in intact male rhesus monkeys. Coronary angiographic experiments with intracoronary serotonin and the thromboxane A(2) analog U46619 stimulated brief vasoconstriction (for 1-3 min) in large epicardial coronaries in untreated male monkeys. In contrast, MPA- and DHT-treated monkeys displayed long-duration constrictions (>5 min), with significantly greater reductions in the minimal diameters of epicardial coronaries. Immunocytochemistry demonstrated androgen receptors (AR) and P receptors in aorta and coronary arteries, and immunocytochemistry and Western blotting showed AR and P receptors in rhesus coronary vascular muscle cells. In vivo, MPA or DHT increased thromboxane prostanoid (TP) receptor expression in the aorta. In vitro, MPA or DHT increased, whereas P did not change, TP receptor expression in primary coronary vascular muscle cell. This MPA- or DHT-mediated increase in TP receptor expression was attenuated by the AR antagonist flutamide. MPA or DHT induction of CH in intact adult male primates, hypothesized to occur via androgenic up-regulation of vascular muscle TP receptor expression, could predispose to CH-mediated myocardial ischemia.