Thromboxane A2 Mimetic U46619 Research Articles

Human Disabled-2 regulates thromboxane A2 signaling for efficient hemostasis in thrombocytopenia.

Understanding platelet protein functions facilitates better assessment of platelet disorders. Megakaryocyte lineage-restricted human Disabled-2 knock-in (hDAB2-KI) mice are generated to delineate the functions of hDab2, a regulator of platelet function, in the control of bleeding associated with thrombocytopenia. Here we show that hDab2-KI mice with thrombocytopenia display decreased bleeding time when compared to the control mice. hDab2 augments thromboxane A2 (TxA2) mimetic U46619- but not other agonists-stimulated granule secretion, integrin activation, and aggregation at a lower platelet concentration in vitro. Binding of hDab2 to phosphatidic acid (PA) facilitates formation of the PA-hDab2-AKT complex leading to an increase in U46619-stimulated AKT-Ser473 phosphorylation and the first wave of ADP/ATP release. Consistent with these findings, hDab2 expression in platelets from patients with immune thrombocytopenic purpura is positively correlated with U46619-stimulated ATP release, which in turn inversely correlated with their bleeding tendency. hDab2 appears crucial in regulating bleeding severity associated with thrombocytopenia by a functional interplay with ADP/ATP release underlying TxA2 signaling.

6-Nitrodopamine is an endogenous selective dopamine receptor antagonist in Chelonoidis carbonaria aorta

Chelonoidis carbonaria aortic rings present endothelium-derived release of dopamine, noradrenaline, adrenaline and 6-nitrodopamine (6-ND). Here it was investigated whether 6-ND release is coupled to nitric oxide (NO) synthesis and its action on the vascular smooth muscle reactivity.Basal release of 6-ND from aortic rings in the absence and presence of the NO synthesis inhibitor L-NAME was quantified by LC-MS-MS. Aortic rings were suspended vertically between two metal hooks in 10-mL organ baths containing Krebs-Henseleit's solution and attached to isometric transducers. The tissues were allowed to equilibrate for 1 h before starting the experiments.The release of 6-ND was significantly reduced by previous incubation with L-NAME. 6-ND (up to 300 μM) had no contractile activity in the aortic rings. 6-ND (1, 3 and 10 μM) produced significant rightward shifts of the concentration-response curves to dopamine in endothelium-intact (pA2 6.09) and L-NAME pre-treated endothelium-intact (pA2 7.06) aortic rings. Contractions induced by noradrenaline and adrenaline were not affected by pre-incubation with 6-ND. The EFS (16 Hz)-induced aortic contractions were significantly inhibited by incubation with 6-ND (10 μM).In the thromboxane A2 mimetic U-46619 (30 nM) pre-contracted endothelium intact aortic rings, 6-ND (1 nM–1 μM) and the dopamine D2-receptor antagonist haloperidol (1 nM-1 μM) induced concentration-dependent relaxations. The relaxations were not present in endothelium-removed aortic rings but they were not affected by incubation with L-NAME in endothelium-intact aortic rings.The results indicate that the synthesis of this novel catecholamine in Chelonoidis carbonaria aortic rings is coupled to NO release and that 6-ND acts as a highly selective dopamine D2-like receptor antagonist.

Does high on-treatment platelet aggregability reflect poor individual response to clopidogrel?

IntroductionOn-treatment platelet aggregability represents the major form of functional assessment for patients treated with P2Y12 receptor antagonists, with “high” on-treatment platelet aggregability (HTPA) predicting thrombotic risk. However HTPA reflects a variable combination of pre-treatment hyperaggregability and poor response to P2Y12 antagonists. We have previously shown that integrity of platelet adenylate cyclase/cAMP signaling, assessed with PGE1, is a strong predictor of individual responses to clopidogrel. We therefore sought to determine the extent to which HTPA reflects impaired platelet responsiveness to clopidogrel. MethodsUsing data from our previous investigations of acute and sub-acute effects of clopidogrel, we analyzed the relationship between on-treatment aggregability and acute/steady state responsiveness to clopidogrel, utilizing ADP, the thromboxane A2 mimetic U46619, and thrombin receptor-activating peptide (TRAP) as pro-aggregants. The relationship between anti-aggregatory response to PGE1 and both on-treatment and pre-treatment aggregability was also examined. Results and conclusionsWith all 3 pro-aggregants, (1) response to clopidogrel after 4 h, as measured by ΔADP response, exhibits a strong inverse relationship with on-treatment aggregation, with a similarly inverse relationship between pre-treatment PGE1 response and on-treatment aggregability; (2) there is a weaker inverse relationship between clopidogrel response and pre-treatment platelet aggregability, and a significant inverse relationship between pre-treatment PGE1 response and pre-treatment platelet aggregability. Furthermore, pre-treatment PGE1 response also predicts on-treatment platelet aggregability in response to ADP at steady state. Thus, HTPA largely represents clopidogrel resistance.

Mechanism underlying the vasodilation induced by diosmetin in porcine coronary artery

Diosmetin is a flavonoid present naturally in citrus fruit. Plants containing diosmetin have been reported to have anti-hypertensive and vasorelaxant effects. Therefore, experiments were carried out to study the effects of diosmetin in segments of the porcine coronary artery (PCA).PCA rings were mounted for isometric tension recording in isolated tissue baths and pre-contracted with the thromboxane A2 mimetic U46619 or KCl. Cumulative concentration response curves to diosmetin were then carried out in the presence or absence of inhibitors or activators of different signaling pathways. The effect on calcium channels was determined by investigating the effect of a single concentration of diosmetin (30 μM) on calcium-induced contractions or contractions to BAY K8644.Diosmetin caused a concentration-dependent relaxation after pre-contraction with U46619 or KCl, which was unaffected by removal of the endothelium. Tetraethylammonium (TEA), and 4-aminopyridine (4-AP), but not barium chloride, caused significant inhibition of the diosmetin-mediated vasorelaxation, indicating a role for potassium channels. Diosmetin inhibited calcium-induced contractions and contractions to the L-type calcium channel opener BAY K8644. Furthermore, diosmetin inhibited the contractions in response to caffeine, cyclopiazonic acid and ionomycin, indicating a general effect on calcium-induced contractions. Contractions in response to the protein kinase C (PKC) activator Phorbol 12-myristate 13-acetate (PMA) were also inhibited by diosmetin, suggesting that it may inhibit a calcium-activated PKC isoform.In summary, diosmetin produced significant vasodilatory effects. The data indicate a role for potassium channels as well as an effect on calcium-induced contractile pathways, possible through inhibition of PKC.

Aldosterone impairs coronary adenosine-mediated vasodilation via reduced functional expression of Ca2+-activated K+ channels

Elevated plasma aldosterone (Aldo) levels are associated with greater risk of cardiac ischemic events and cardiovascular mortality. Adenosine-mediated coronary vasodilation is a critical cardioprotective mechanism during ischemia; however, whether this response is impaired by increased Aldo is unclear. We hypothesized that chronic Aldo impairs coronary adenosine-mediated vasodilation via downregulation of vascular K+ channels. Male C57BL/6J mice were treated with vehicle (Con) or subpressor Aldo for 4 wk. Coronary artery function, assessed by wire myography, revealed Aldo-induced reductions in vasodilation to adenosine and the endothelium-dependent vasodilator acetylcholine but not to the nitric oxide donor sodium nitroprusside. Coronary vasoconstriction to endothelin-1 and the thromboxane A2 mimetic U-46619 was unchanged by Aldo. Additional mechanistic studies revealed impaired adenosine A2A, not A2B, receptor-dependent vasodilation by Aldo with a tendency for Aldo-induced reduction of coronary A2A gene expression. Adenylate cyclase inhibition attenuated coronary adenosine dilation but did not eliminate group differences, and adenosine-stimulated vascular cAMP production was similar between Con and Aldo mice. Similarly, blockade of inward rectifier K+ channels reduced but did not eliminate group differences in adenosine dilation whereas group differences were eliminated by blockade of Ca2+-activated K+ (KCa) channels that blunted and abrogated adenosine and A2A-dependent dilation, respectively. Gene expression of several coronary KCa channels was reduced by Aldo. Together, these data demonstrate Aldo-induced impairment of adenosine-mediated coronary vasodilation involving blunted A2A-KCa-dependent vasodilation, independent of blood pressure, providing important insights into the link between plasma Aldo and cardiac mortality and rationale for aldosterone antagonist use to preserve coronary microvascular function.NEW & NOTEWORTHY Increased plasma aldosterone levels are associated with worsened cardiac outcomes in diverse patient groups by unclear mechanisms. We identified that, in male mice, elevated aldosterone impairs coronary adenosine-mediated vasodilation, an important cardioprotective mechanism. This aldosterone-induced impairment involves reduced adenosine A2A, not A2B, receptor-dependent vasodilation associated with downregulation of coronary KCa channels and does not involve altered adenylate cyclase/cAMP signaling. Importantly, this effect of aldosterone occurred independent of changes in coronary vasoconstrictor responsiveness and blood pressure.

Paying the Toll for Inflammation

Paying the Toll for Inflammation

MAPKs-mediated modulation of the myocyte voltage-gated K+ channels is involved in ethanol-induced rat coronary arterial contraction

Acute coronary arterial spasm is contributory to ethanol-induced heart ischemic events. The present experiments were designed to study contractive effect of ethanol in isolated rat coronary arteries (RCAs) stimulated mildly with vasoconstrictors and involvement of voltage-gated potassium (KV) channels and mitogen-activated protein kinases (MAPKs) of rat coronary arterial smooth muscle cell (RCASMC) in the spasm. The vascular tension was recorded with a wire myograph. KV currents of single freshly isolated RCASMC were assessed with patch clamp. Phosphorylation of RCASMC MAPKs (p38 MAPK and p44/42 MAPK) was assayed by Western blots. Mild stimulation, which was defined as 5–20% of 60 mM KCl-induced contraction, with thromboxane A2 mimetic U46619, endothelin-1 or KCl tremendously increased contractive response of RCAs to ethanol (0.8–8.0 mg/ml). Ethanol (8 and 25 mg/ml) reduced the maximal KV currents by 53.6% and 56.6% respectively without concentration-dependence. Both ethanol (8.0 mg/ml) and U46619 (0.3 μM) enhanced phosphorylation of p38 MAPK and p44/42 MAPK and there was a pronounced synergism between them. MAPK pathway inhibition with p38 MAPK inhibitor SB239063 and p44/42 MAPK inhibitor PD98059 depressed ethanol-induced contraction by 22–55% in the stimulated RCAs and almost abrogated ethanol-induced reduction in KV currents. The present results for the first time demonstrated that ethanol induces potent vasoconstriction in stimulated RCAs and depresses KV currents in RCASMCs. It is suggested that MAPKs pathways play an important role in the etiology of ethanol-induced coronary artery diseases.

Vasoconstrictor stimulus determines the functional contribution of myoendothelial feedback to mesenteric arterial tone.

In isolated resistance arteries, endothelial modulation of vasoconstrictor responses to α1 -adrenoceptor agonists occurs via a process termed myoendothelial feedback: localized inositol trisphosphate (InsP3 )-dependent Ca2+ transients activate intermediate conductance Ca2+ -activated K+ (IKCa ) channels, hyperpolarizing the endothelial membrane potential to limit further reductions in vessel diameter. We demonstrate that IKCa channel-mediated myoendothelial feedback limits responses of isolated mesenteric arteries to noradrenaline and nerve stimulation, but not to the thromboxane A2 mimetic U46619 or to increases in intravascular pressure. In contrast, in the intact mesenteric bed, although responses to exogenous noradrenaline were limited by IKCa channel-mediated myoendothelial feedback, release of NO and activation of endothelial small conductance Ca2+ -activated K+ (SKCa ) channels in response to increases in shear stress appeared to be the primary mediators of endothelial modulation of vasoconstriction. We propose that (1) the functional contribution of myoendothelial feedback to arterial tone is determined by the nature of the vasoconstrictor stimulus, and (2) although IKCa channel-mediated myoendothelial feedback may contribute to local control of arterial diameter, in the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction. Constriction of isolated resistance arteries in response to α1 -adrenoceptor agonists is limited by reciprocal engagement of inhibitory endothelial mechanisms via myoendothelial feedback. In the current model of feedback, agonist stimulation of smooth muscle cells results in localized InsP3 -dependent Ca2+ transients that activate endothelial IKCa channels. The subsequent hyperpolarization of the endothelial membrane potential then feeds back to the smooth muscle to limit further reductions in vessel diameter. We hypothesized that the functional contribution of InsP3 -IKCa channel-mediated myoendothelial feedback to limiting arterial diameter may be influenced by the nature of the vasoconstrictor stimulus. To test this hypothesis, we investigated the functional role of myoendothelial feedback in modulating responses of rat mesenteric resistance arteries to the adrenoceptor agonist noradrenaline, the thromboxane A2 mimetic U46619, increases in intravascular pressure and stimulation of perivascular sympathetic nerves. In isolated arteries, responses to noradrenaline and stimulation of sympathetic nerves, but not to U46619 and increases in intravascular pressure, were modulated by IKCa channel-dependent myoendothelial feedback. In the intact mesenteric bed perfused under conditions of constant flow, responses to exogenous noradrenaline were modulated by myoendothelial feedback, but shear stress-induced release of NO and activation of endothelial SKCa channels appeared to be the primary mediators of endothelial modulation of vasoconstriction to agonists and nerve stimulation. Thus, we propose that myoendothelial feedback may contribute to local control of diameter within arterial segments, but at the level of the intact vascular bed, increases in shear stress may be the major stimulus for engagement of the endothelium during vasoconstriction.

Resolvin E1, resolvin D1 and resolvin D2 inhibit constriction of rat thoracic aorta and human pulmonary artery induced by the thromboxane mimetic U46619

The ω-6 fatty acid-derived lipid mediators such as prostanoids, thromboxane and leukotrienes have well-established roles in regulating both inflammation and smooth muscle contractility. Resolvins are derived from ω-3 fatty acids and have important roles in promoting the resolution of inflammation, but their activity on smooth muscle contractility is unknown. We investigated whether resolvin E1 (RvE1), resolvin D1 (RvD1) and resolvin D2 (RvD2) can modulate contractions of isolated segments of rat thoracic aorta (RTA) or human pulmonary artery (HPA) induced by the α1 -adrenoceptor agonist phenylephrine or the stable thromboxane A2 mimetic U46619. Contractile responses in RTA and HPA were measured using wire myography. Receptor expression was investigated by immunohistochemistry. Constriction of RTA segments by U46619, but not by phenylephrine, was significantly inhibited by pretreatment for 1 or 24h with 10-100nM RvE1, RvD1 or RvD2. The inhibitory effect of RvE1 was partially blocked by a chemerin receptor antagonist (CCX832). RvE1 at only 1-10nM also significantly inhibited U46619-induced constriction of HPA segments, and the chemerin receptor, GPR32 and FPR2/ALX were identified in HPA smooth muscle. These data suggest that resolvins or their mimetics may prove useful novel therapeutics in diseases such as pulmonary arterial hypertension, which are characterized by increased thromboxane contractile activity.

Antagonist of thromboxane A2 receptor by SQ29548 lowers DOCA-induced hypertension in diabetic rats

Diabetes is one of high risk factors for cardiovascular diseases, including atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to hypertension in diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured. The phosphorylated levels of endothelial nitric oxide synthase (eNOS) and Akt were monitored by western blot. Endothelial function was determined by organ bath. High glucose (HG) or thromboxane A2 mimetic U46619 significantly reduced the levels of p-eNOS and p-Akt in cultured HUVECs, which were reversed by inhibition of TPr. HG/U46619-induced reductions of p-eNOS and p-Akt were accompanied with increases of total and phosphorylated tumor suppressor phosphatase and tensin homolog on chromosome 10 (PTEN). PTEN siRNA restored Akt-eNOS signaling in cells treated with HG. In rats, streptozotocin-induced hyperglycemia was associated with aortic PTEN upregulation and reductions of p-Akt and p-eNOS. TPr antagonist SQ29548 ablated these alterations and reduced blood pressure in rats with DOCA-induced hypertensive. In conclusion, hyperglycemia activates thromboxane A2 receptor to augment DOCA-induced high blood pressure in rats via the PTEN-Akt-eNOS signaling.

Hyperglycemia via activation of thromboxane A2 receptor impairs the integrity and function of blood-brain barrier in microvascular endothelial cells.

Diabetes is one of high risk factors for cardio- and cerebra-vascular diseases, including stroke, atherosclerosis and hypertension. This study was conducted to elucidate whether and how thromboxane receptor (TPr) activation contributes to blood-brain barrier (BBB) dysfunction in diabetes. Human brain microvascular endothelial cells (HBMECs) were cultured. The levels of phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 (p-eNOS) and Akt at Ser473 (p-Akt) were assayed by western blot. Exposure of HBMECs to either high glucose (HG) or thromboxane A2 (TxA2) mimetic U46619, significantly reduced p-eNOS and p-Akt. These effects were abolished by pharmacological or genetic inhibitors of TPr. HG/U46619-induced suppressions of eNOS and Akt phosphorylation were accompanied by upregulation of PTEN and Ser380/Thr382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt-eNOS signaling in the face of TPr activation or HG. The small GTPase, Rho, was also activated by HG stimulation, and pretreatment of HBMECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued HG-impaired Akt-eNOS signaling. In STZ-injected rats, we found that hyperglycemia dramatically increased the levels of PTEN and PTEN-Ser380/Thr382/383 phosphorylation, reduced both levels of p-eNOS and p-Akt, and disrupted BBB function assayed by Evans blue staining, which were abolished by SQ29548 treatment. We conclude that hyperglycemia activates thromboxane A2 receptor to impair the integrity and function of blood-brain barrier via the ROCK-PTEN-Akt-eNOS pathway.

Sensitivity to the thromboxane A2 analog U46619 varies with inner diameter in human stem villous arteries

IntroductionThe vascular resistance of stem villous arteries is determined by the balance between different contractile and relaxant agents and in the utero-placental circulation. Thromboxane A2 (TxA2), prostaglandin F2α (PGF2α) and endothelin-1 (ET-1) are considered to be among the most important contractile factors. However, it is not known if their contractile effects are consistent along the villous tree. We hypothesized that the sensitivity to different agonists could be influenced by artery diameter and thus that their contribution to placental vascular resistance may differ. MethodsUsing an isometric wire myograph, the contractility and sensitivity (pD2) to the thromboxane A2 mimetic U46619, PGF2α and ET-1 were investigated in isolated human stem villous arteries and human uterine fundus and isthmus arteries obtained from healthy, pregnant women who had experienced uncomplicated pregnancy. ResultsIn fetal arteries, the pD2 values for U46619 correlated positively with arterial diameter with no such dependence observed for ET-1 and PGF2α. In maternal arteries, pD2 remained constant for all the agonists tested despite highly variable vessel diameter. DiscussionA selective decrease in sensitivity to TxA2 receptor stimulation was observed with decreasing vascular diameter in human stem villous arteries. The contractile factors PGF2α and ET-1 show no such relationship.

Systemic vasoconstriction modulates the responses of pulmonary vasculature and airway to vasoconstrictors in anesthetized rats

ABSTRACTPurpose: The physiological responses of the pulmonary vasculature and airway to various vasoconstrictors were studied using isolated perfused lungs and pulmonary arteries, but these responses were not systematically studied in in vivo rats. We determined these responses and modulating effects of systemic circulation in anesthetized rats. Methods: We measured directly pulmonary arterial pressure (PAP), left atrial pressure (LAP), aortic blood flow, and airway pressure (AWP) to determine pulmonary vascular resistance (PVR), following injections of angiotensin II (ANG II), endothelin-1 (ET-1), vasopressin, phenylephrine and thromboxane A2 mimetic U46619 in anesthetized SD rats. Results: ANG II, phenylephrine and vasopressin at high doses caused strong systemic vasoconstriction and left heart overload, resulting in a transient increase in LAP and pulmonary congestion, which consequently decreased PVR. Nonetheless, prior to LAP elevation, PVR was slightly but significantly increased by ANG II and phenylephrine. In contrast, ET-1 and U46619 substantially increased PVR in the absence of LAP elevation, while vasopressin did not increase PVR. In separate experiments, PAP and AWP increased when LAP was forcedly elevated. AWP was increased by U46619 through bronchoconstriction and by the other agents through increased LAP-induced pulmonary congestion. Conclusion: Airway constriction is induced by U46619, and pulmonary vasoconstriction is induced strongly by U46619 and ET-1, and weakly by ANG II and phenylephrine, but not by vasopressin in anesthetized rats. ANG II, vasopressin and phenylephrine exert indirectly a transient pulmonary vasodilatory action due to pulmonary congestion evoked by strong systemic vasoconstriction, which may account for weak pulmonary pressor responses to these agents.

Abstract 624: Resolvin E1 Inhibits Thromboxane-Induced Contraction of Rat Aorta and Human Pulmonary Artery

Thromboxane is a vasoactive omega-6-derived lipid implicated in hypertension. Omega-3 fatty acid derivatives including resolvin (Rv) E1 have important roles in the resolution of inflammation, but their ability to mediate vasomotor activity is unknown. We assessed whether RvE1 modulates contraction of rat thoracic aorta and human pulmonary artery (HPA) in vitro. Rats were culled by CO2 inhalation and cervical dislocation, and the thoracic aorta was removed. HPA were obtained with informed consent from lung tissue of surgical patients at Southampton General Hospital. Segments of aorta or HPA were incubated in DMEM-F12 with or without RvE1 (10 nM, 100 nM or 300 nM) for 1 or 24 hours, and then mounted on a wire myograph. Mounted segments were bathed at 37[[Unable to Display Character: ]]C in Kreb’s buffer, gassed (95%/5% O2/CO2) and set to 1.5 g of baseline tension. Functional integrity was confirmed by a contractile response to 125 mM KPSS. After washing to restore baseline tension, a cumulative concentration-response curve was defined to the stable thromboxane A2 mimetic U46619 (1 nM - 1 μM). Contractions are expressed as percent of KPSS response and differences determined by two-way ANOVA and by pairwise comparisons. In rat aorta, treatment with RvE1 (10 nM) significantly shifted the U46619 curve rightwards, increasing the EC50 value 13-fold from 0.13 μM to 1.75 μM after 1 hour (n=5), and 4-fold from 0.093 μM to 0.41 μM after 24 hours (n=5). In contrast, 300 nM RvE1 caused no significant change in EC50 (1 hour 0.11 μM; 24 hours 0.097 μM, n=5). In HPA segments, RvE1 (10 nM, 1 hour) increased the U46619 EC50 value 7-fold from 0.0079 μM to 0.054 μM (n=3), with a preliminary experiment suggesting a similar outcome after 24 hours. Higher concentrations of RvE1 had no significant effect, suggesting the relaxant action of RVE1 has a bell-shaped response curve. In preliminary experiments, RvE1 inhibited U-46619 contractions of bronchiolar airways from the same human donors. Resolvin E1 is known for its immunomodulatory actions on leukocytes and also inhibits U46619-induced aggregation of platelets, but our results are the first evidence that this pro-resolution mediator can also inhibit contractions of intact vasculature from rat and human lung induced by a thromboxane mimetic.

Synthetic analogues of flavonoids with improved activity against platelet activation and aggregation as novel prototypes of food supplements

We investigated the ability of quercetin and apigenin to modulate platelet activation and aggregation, and compared the observed efficacy with that displayed by their synthetic analogues 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 1–4, and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones, 5–7. Platelet aggregation was explored through a spectrophotometric assay on platelet-rich plasma (PRP) treated with the thromboxane A2 mimetic U46619, collagen and thrombin in presence/absence of various bioisosteres of flavonoids (12.5–25–50–100μM). The platelet density, (mean platelet component, MPC), was measured by the Advia 120 Hematology System as a marker surrogate of platelet activation. The induced P-selectin expression, which reflects platelet degranulation/activation, was quantified by flow cytometry on PRP.Our synthetic compounds modulated significantly both platelet activation and aggregation, thus turning out to be more effective than the analogues quercetin and apigenin when tested at a concentration fully consistent with their use in vivo. Accordingly, they might be used as food supplements to increase the efficacy of natural flavonoids.

Cardiopulmonary Bypass Is Associated With Altered Vascular Reactivity of Isolated Pulmonary Artery in a Porcine Model: Therapeutic Potential of Inhaled Tezosentan

Whereas it is established that endothelin-1 elicits sustained deleterious effects on the cardiovascular system during cardiopulmonary bypass (CPB), presently it remains unknown whether the inhaled administration of the dual ETA and ETB antagonist tezosentan prevents the development of pulmonary endothelial dysfunction. A prospective, randomized laboratory investigation. University research laboratory. Landrace swine. Three groups of animals underwent a 90-minute period of full bypass followed by a 60-minute period of reperfusion. Among treated groups, one received tezosentan through inhalation prior to CPB, whereas the other one received it intravenously at weaning from CPB; the third group remained untreated. Pulmonary vascular reactivity studies, realized on a total of 285 rings, were performed in all groups, including 1 sham. The contractility of pulmonary arteries to prostaglandin F2α and to the thromboxane A2 mimetic U46619 was preserved in animals submitted to CPB. By contrast, there were significant increases both in the maximal contraction to endothelin-1 and in the plasma levels of the peptide 60minutes after reperfusion. Tezosentan administered by inhalation or intravenously did not prevent the development of pulmonary CPB-associated endothelial dysfunction. However, while hemodynamic disturbances were improved with both routes, the inhaled administration had a beneficial effect on oxygen parameters over intravenous administration. Despite the blockade of the endothelin-1 pathway with tezosentan, the development of the pulmonary endothelial dysfunction associated with CPB still occurred. However, only the inhalation route had a significant impact on gas exchange during CPB.

Enhanced contractile responses to Ang II and Thromboxane by placental soluble fms-like tyrosine kinase (sFlt-1) in human omental arteries

Enhanced contractile responses to Ang II and Thromboxane by placental soluble fms-like tyrosine kinase (sFlt-1) in human omental arteries

Thromboxane A2 mediates apoptosis in cardiomyocytes via IP3

We have shown that the inflammatory mediator thromboxane A2 (TxA2) can induce ventricular arrhythmias that were associated with increased intracellular calcium in cardiomyocytes. We were able to prevent both the arrhythmias and the changes in intracellular calcium by inhibiting inositol trisphosphate (IP3) signaling. In this investigation, we sought to determine if TxA2 receptor (TxA2R) activation would trigger apoptosis in isolated adult mouse cardiomyocytes. Our hypothesis is that TxA2R‐stimulation induces IP3 formation and subsequent IP3 receptor activation triggers apoptosis. We found that TxA2Rs were expressed in the ventricles and that there was a concentration dependent increase in cell death (Trypan blue and MTT assays) following treatment with the TxA2 mimetic U46619 (0.1–10 μM). We have confirmed that TxA2‐induced death was apoptotic via cleaved caspase‐3 and TUNEL staining. Treatment of cells with the TxA2R antagonist SQ29548 and the IP3 inhibitors Gentamycin and 2‐APB eliminated the increase in apoptosis by U46619. In conclusion, our data suggests that: 1) TxA2‐induced cell death is mediated in part by apoptosis and 2) the IP3 pathway is a novel mediator of apoptosis in cardiomyocytes. These findings may provide important therapeutic targets for inflammatory‐induced cardiac apoptosis that can lead to heart failure. (AHA 11SDG5330016 to MJW)

Nitroxyl (HNO) suppresses vascular Nox2 oxidase activity

Nox2 oxidase activity underlies the oxidative stress and vascular dysfunction associated with several vascular-related diseases. We have reported that nitric oxide (NO) decreases reactive oxygen species production by endothelial Nox2. This study tested the hypothesis that nitroxyl (HNO), the redox sibling of NO, also suppresses vascular Nox2 oxidase activity. Specifically, we examined the influence of two well-characterized HNO donors, Angeli’s salt and isopropylamine NONOate (IPA/NO), on Nox2-dependent responses to angiotensin II (reactive oxygen species production and vasoconstriction) in mouse cerebral arteries. Angiotensin II (0.1μmol/L)-stimulated superoxide (measured by lucigenin-enhanced chemiluminescence) and hydrogen peroxide (Amplex red fluorescence) levels in cerebral arteries (pooled basilar and middle cerebral (MCA)) from wild-type (WT) mice were ~60% lower (P<0.05) in the presence of either Angeli’s salt (1μmol/L) or IPA/NO (1μmol/L). Similarly, phorbyl 12,13-dibutyrate (10μmol/L; Nox2 activator)-stimulated hydrogen peroxide levels were ~40% lower in the presence of IPA/NO (1μmol/L; P<0.05). The ability of IPA/NO to decrease superoxide levels was reversible and abolished by the HNO scavenger l-cysteine (3mmol/L; P<0.05), but was unaffected by hydroxocobalamin (100μmol/L; NO scavenger), ODQ (10μmol/L; soluble guanylyl cyclase (sGC) inhibitor), or Rp-8-pCPT-cGMPS (10μmol/L; cyclic guanosine monophosphate (cGMP)-dependent protein kinase inhibitor). Angiotensin II-stimulated superoxide was substantially less in arteries from Nox2-deficient (Nox2−/y) versus WT mice (P<0.05). In contrast to WT, IPA/NO (1μmol/L) had no effect on superoxide levels in arteries from Nox2−/y mice. Finally, angiotensin II (1–1000μmol/L)-induced constriction of WT MCA was virtually abolished by IPA/NO (1μmol/L), whereas constrictor responses to either the thromboxane A2 mimetic U46619 (1–100 nmol/L) or high potassium (122.7mmol/L) were unaffected. In conclusion, HNO suppresses vascular Nox2 oxidase activity via a sGC–cGMP-independent pathway. Thus, HNO donors might be useful therapeutic agents to limit and/or prevent Nox2-dependent vascular dysfunction.

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E2 (PGE2) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3 µM) used alone or in combination with the P2Y12 antagonist cangrelor (1 µM), both without and with aspirin (100 µM). Platelet aggregation and P-selectin expression were measured in whole blood (n = 10) following stimulation with the thromboxane A2 (TXA2) mimetic U46619 (0.3 or 1 µM) in combination with either the EP3 agonist sulprostone (0.1 µM), or PGE2 (1 µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n = 42) were randomly assigned to receive no background treatment, clopidogrel (300 mg loading dose plus 75 mg daily) or clopidogrel and aspirin (75 mg daily) for 10 days alongside DG-041 (200 mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y12 antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y12 blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.