Thromboxane A2 Receptor Agonist U46619 Research Articles

Deletion of vascular thromboxane A2 receptors and its impact on angiotensin II-induced hypertension and atherosclerotic lesion formation in the aorta of Ldlr-deficient mice

The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.

Sex differences in middle cerebral artery reactivity and hemodynamics in 6-month-old Sprague-Dawley rats independent from systemic arterial stiffness or blood pressure

Cerebrovascular disease (CVD) is a major risk factor for developing cognitive impairment. Young women are protected against CVD compared with older women or men, however the underlying mechanisms are not well understood. Greater peripheral arterial stiffness has been linked to increased cerebral flow pulsatility and cognitive impairment. In this study we determined whether sex differences in middle cerebral artery (MCA) reactivity are associated with changes in cerebral [MCA, intracranial internal carotid artery (ICA)] or systemic [common carotid artery (CCA), thoracic aorta (TAo)] arterial resistance and stiffness in six month-old Sprague-Dawley (SD) rats. No differences in systolic or diastolic blood pressures measured by tail-cuff method were observed between sexes. Heart rate was higher in the female versus male SD (438±9 vs. 343±10 bpm, p<0.05, n=8-12). The pulsatility (PI) and resistance indexes (RI) of left MCA and left intracranial ICA were calculated as follows: PI=(Peak systolic velocity (Vs)-End-diastolic velocity (Vd))/Vmean, or RI=(Vs-Vd)/Vs (Vevo LAZR, FUJIFILM VisualSonics). Left MCA PI and RI indexes were lower in the female versus male SD (PI: 0.75±0.09 vs. 1.17±0.11; RI: 0.49±0.04 vs. 0.63±0.03, all p<0.05; n=7-8), while no differences in left ICA blood flow (n=4-9) or resistance (n=7-10) were observed between sexes. There were no differences in arterial stiffness of TAo or left CCA evaluated by pulse wave velocity (PWV) measurements. To investigate vascular reactivity, left MCA segments were isolated and mounted on a wire Multi Myograph (DMT-USA). Data were acquired and analyzed with Powerlab 8 and LabChart v8 (ADInstruments). Female left MCA had lower contraction to potassium (K+: 0.6±0.1 vs. 1±0.2 mN/mm, p<0.05), but similar maximal contraction (109±10 vs. 116±6 %KMAX, p>0.05) and sensitivity (7.3±0.2 vs. 7.2±0.2, p>0.05) to thromboxane A2 receptor agonist U46619. Pre-incubation with indomethacin lowered maximal response (97±5 vs. 116±6 %KMAX, p<0.05) and sensitivity (6.8±0.1 vs. 7.2±0.2, p<0.05) to U46619 in male MCA, with no effect in female MCA (Male: 109±9 vs. 113±7 %KMAX; Female: 7.3±0.2 vs. 7.3±0.2). MCA collagen deposition (by trichrome staining) was similar between sexes, however eNOS immunoreactivity tended to increase in the female versus male MCA (7.3±2 vs. 1.2±0.6; n=3-4, p=0.07). No sex differences in MCA COX-2 expression were found. In summary, lower PI and RI of MCA in 6-month-old females suggests decreased cerebrovascular resistance compared with age-matched male SD. Increased eNOS expression may contribute to the lower contraction observed in female MCA, whereas vasoconstrictor prostaglandins appear to have a greater role in male versus female MCA. Our results demonstrate sex differences in MCA vascular reactivity associated with a protective functional profile of MCA in 6-month-old female versus male SD rats independent from changes in systemic arterial stiffness or blood pressure. Studies were supported by the WFUSM CVSC Pilot Award and NIH 5R01HL155420. This is the full abstract presented at the American Physiology Summit 2023 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.

Dolutegravir potentiates platelet activation by a calcium-dependent, ionophore-like mechanism

Dolutegravir is a highly potent HIV integrase strand transfer inhibitor that is recommended for first-line anti-retroviral treatment in all major treatment guidelines. A recent study has shown that people taking this class of anti-retroviral treatment have a substantially higher risk of early-onset cardiovascular disease, a condition shown previously to be associated with increased platelet reactivity. To date, few studies have explored the effects of dolutegravir on platelet activation. Accordingly, the current study was undertaken with the primary objective of investigating the effects of dolutegravir on the reactivity of human platelets in vitro. Platelet-rich plasma, isolated platelets, or buffy coat cell suspensions prepared from the blood of healthy adults were treated with dolutegravir (2.5–10 µg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin, or a thromboxane A2 receptor agonist U46619. Expression of platelet CD62P (P-selectin), formation of heterotypic neutrophil:platelet aggregates, and calcium (Ca2+) fluxes were measured using flow cytometry and fluorescence spectrometry, respectively. Dolutegravir caused dose-related potentiation of ADP-, thrombin- and U46619-activated expression of CD62P by platelets, as well as a significant increases in formation of neutrophil:platelet aggregates. These effects were paralleled by a spontaneous, receptor-independent elevation in cytosolic Ca2+ that appears to underpin the mechanism by which the antiretroviral agent augments the responsiveness of these cells to ADP, thrombin and U46619. The most likely mechanism of dolutegravir-mediated increases in platelet cytosolic Ca2+ relates to a combination of lipophilicity and divalent/trivalent metal-binding and/or chelating properties of the anti-retroviral agent. These properties are likely to confer ionophore-type activities on dolutegravir that would promote movement of Ca2+ across the plasma membrane, delivering the cation to the cytosol where it would augment Ca2+-dependent intracellular signaling mechanisms. These effects of dolutegravir may lead to hyper-activation of platelets which, if operative in vivo, may contribute to an increased risk for cardiometabolic co-morbidities.

Effects of fasudil on blood–brain barrier integrity

BackgroundCerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen–glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model.MethodsBBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6 h OGD/24 h reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells.ResultsWe found that treatment with 0.3–30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes.ConclusionsOur findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

Thromboxane A2 receptor activation via Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction inhibits angiogenic sprouting of human endothelial cells

We could previously show that thromboxane A2 receptor (TP) activation inhibits the angiogenic capacity of human endothelial cells, but the underlying mechanisms remained unclear. Therefore, the aim of this study was to elucidate TP signal transduction pathways relevant to angiogenic sprouting of human endothelial cells. To clarify this matter, we used RNAi-mediated gene silencing as well as pharmacological inhibition of potential TP downstream targets in human umbilical vein endothelial cells (HUVEC) and VEGF-induced angiogenic sprouting of HUVEC spheroids in vitro as a functional read-out. In this experimental set-up, the TP agonist U-46619 completely blocked VEGF-induced angiogenic sprouting of HUVEC spheroids. Moreover, in live-cell analyses TP activation induced endothelial cell contraction, sprout retraction as well as endothelial cell tension and focal adhesion dysregulation of HUVEC. These effects were reversed by pharmacological TP inhibition or TP knockdown. Moreover, we identified a TP-Gα13-RhoA/C-ROCK-LIMK2-dependent signal transduction pathway to be relevant for U-46619-induced inhibition of VEGF-mediated HUVEC sprouting. In line with these results, U-46619-mediated TP activation potently induced RhoA and RhoC activity in live HUVEC as measured by FRET biosensors. Interestingly, pharmacological inhibition of ROCK and LIMK2 also normalized U-46619-induced endothelial cell tension and focal adhesion dysregulation of HUVEC. In summary, our work reveals mechanisms by which the TP may disturb angiogenic endothelial function in disease states associated with sustained endothelial TP activation.

Abstract 10294: Endothelial Pannexin1-TRPV4 Signaling Regulates Pulmonary Arterial Pressure

The pulmonary endothelial cells (EC) dilate small pulmonary arteries (PAs) and maintain low pulmonary arterial pressure (PAP). Importantly, the loss of EC function contributes to elevated PAP in pulmonary hypertension (PH). Recent studies show that Pannexin 1 (Panx1), an ATP efflux pathway at the cell membrane, is a critical regulator of endothelial function. However, the impact of endothelial Panx1 (Panx1 EC ) on PA contractility and PAP is unknown. We observed that the extracellular ATP (eATP) levels are reduced in PAs from inducible, EC-specific Panx1 -/- (Panx1 EC -/- ) mice, identifying Panx1 EC as an important ATP release pathway in PAs. Our recently published findings demonstrated that eATP dilates PAs via activation of Ca 2+ influx through endothelial transient receptor potential vanilloid 4 (TRPV4 EC ) ion channels. Therefore, we hypothesized that endothelial Panx1—eATP—TRPV4 channel signaling dilates PAs and lowers resting PAP. Apyrase (10 U/mL), an ATP hydrolyzing enzyme, reduced the activity of TRPV4 EC sparklets (individual Ca 2+ influx signals through TRPV4 EC channels) in PAs, implying a constitutive regulation of TRPV4 EC channels by ATP. The activity of TRPV4 EC sparklets was lower in PAs from Panx1 EC -/- mice than controls, identifying Panx1 EC as a novel promoter of TRPV4 EC channel activity. Pressure myography studies in PAs (~ 50 μm) from Panx1 EC -/- and TRPV4 EC -/- mice showed higher constriction to thromboxane A2 receptor agonist U46619 (1-300 nM) compared to PAs from controls. Finally, right ventricular systolic pressure was higher in Panx1 EC -/- and TRPV4 EC -/- mice compared to the respective controls. These data indicated that Panx1 EC —eATP—TRPV4 EC channel signaling lowers PA contractility and PAP. ATP is an endogenous activator of purinergic receptor signaling. Inhibition of P2Y1 and P2X receptors did not alter ATP activation of TRPV4 EC sparklets in PAs. However, P2Y2 receptor inhibition and EC-specific P2Y2 receptor deletion abolished ATP activation of TRPV4 EC sparklets. Together, these data identify a novel Panx1 EC —eATP—P2Y2R EC signaling pathway that enhances TRPV4 EC channel activity and lowers PAP. Individual elements of this pathway may provide novel therapeutic targets for treating pulmonary vascular disorders.

Intrauterine\ufeff growth restriction weakens anticontractile influence of NO in coronary arteries of adult rats

Intrauterine growth restriction (IUGR) is one of the most common pathologies of pregnancy. The cardiovascular consequences of IUGR do not disappear in adulthood and can manifest themselves in pathological alterations of vasomotor control. The hypothesis was tested that IUGR weakens anticontractile influence of NO and augments procontractile influence of Rho-kinase in arteries of adult offspring. To model IUGR in the rat, dams were 50% food restricted starting from the gestational day 11 till delivery. Mesenteric and coronary arteries of male offspring were studied at the age of 3 months using wire myography, qPCR, and Western blotting. Contractile responses of mesenteric arteries to α1-adrenoceptor agonist methoxamine as well as influences of NO and Rho-kinase did not differ between control and IUGR rats. However, coronary arteries of IUGR rats demonstrated elevated contraction to thromboxane A2 receptor agonist U46619 due to weakened anticontractile influence of NO and enhanced role of Rho-kinase in the endothelium. This was accompanied by reduced abundance of SODI protein and elevated content of RhoA protein in coronary arteries of IUGR rats. IUGR considerably changes the regulation of coronary vascular tone in adulthood and, therefore, can serve as a risk factor for the development of cardiac disorders.

Regulation of Pulmonary Arterial Pressure by Endothelial Pannexin1–TRPV4 Channel Signaling

Endothelial cells (EC) dilate small pulmonary arteries (PAs) and lower the resting pulmonary arterial pressure (PAP). Moreover, the loss of endothelial function is a key contributor to elevated PAP in pulmonary hypertension. Therefore, an in-depth understanding of endothelial mechanisms controlling PAP is necessary for designing novel therapeutic strategies against pulmonary hypertension. Pannexin 1 (Panx1), an ATP efflux pathway on the EC membranes, has emerged as a crucial controller of endothelial function. However, the impact of endothelial Panx1 (Panx1EC) on PA contractility and PAP is not known. Recent studies support the idea that endothelial transient receptor potential vanilloid 4 (TRPV4EC) channels dilate small PAs, and are activated by extracellular ATP (eATP). Since Panx1EC is an ATP efflux pathway, we hypothesized that Panx1EC-TRPV4EC channel signaling dilates PAs and lowers resting PAP. Right ventricular systolic pressure (RVSP), an indirect indicator of PAP, was higher in inducible, EC-specific Panx1 (Panx1EC-/-) and TRPV4 channel knockout (TRPV4EC-/-) mice compared to the respective control mice. The activity of TRPV4EC channels was recorded as individual Ca2+ influx signals (TRPV4EC sparklets) in fluo 4-loaded en face PAs using spinning disk confocal imaging. The TRPV4EC sparklet activity was lower in PAs from Panx1EC-/- mice than the control mice, identifying Panx1EC as a novel promoter of TRPV4EC channel activity. Apyrase (10 U/mL), an ATP hydrolyzing enzyme, also reduced the activity of TRPV4EC sparklets in PAs from control mice but not Panx1EC-/- mice. Pressurized PAs (15 mm Hg) from Panx1EC-/- and TRPV4EC-/- mice showed higher constriction to thromboxane A2 receptor agonist U46619 (1-300 nM) compared to PAs from the respective control mice. These data indicated that Panx1EC-eATP-TRPV4EC signaling lowers PA contractility and PAP. Consistent with previous findings that eATP dilates PAs via purinergic P2Y2 receptor (P2Y2R) signaling, ATP activation of TRPV4EC channels was lost in PAs from inducible P2Y2EC-/- mice. Together, these data identify a novel Panx1EC-eATP-P2Y2REC signaling pathway that promotes TRPV4EC channel activity in PAs. Panx1, P2Y2R, and TRPV4 channels have been shown to localize with the scaffolding protein caveolin 1 (Cav-1), raising the possibility that Cav-1EC may provide a signaling scaffold for Panx1EC-P2Y2REC-TRPV4EC signaling in PAs. Accordingly, P2Y2REC-activation of TRPV4EC channels was lost in PAs from inducible Cav-1EC-/- mice, and these mice showed higher resting RVSPs compared to the control mice. Additionally, in situ proximity ligation assay confirmed the nanometer proximity of Cav-1EC with Panx1EC, P2Y2REC, and TRPV4EC channel in PAs. Overall, our findings identify Cav-1EC-dependent Panx1EC-P2Y2REC-TRPV4EC channel pathway that dilates PAs and lowers PAP. Targeting individual elements of this pathway may provide novel therapeutic options in pulmonary vascular disorders.

Simvastatin causes pulmonary artery relaxation by blocking smooth muscle ROCK and calcium channels: Evidence for an endothelium-independent mechanism.

Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction. In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the α1–adrenergic agonist phenylephrine (5 μM) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 μM) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 μM), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.

Increase in the constrictor effects of Rho-kinase in skeletal muscle and coronary arteries of rats with chronic hypothyroidism

Aim. The deficit of thyroid function is known to be accompanied by an increase in the overall peripheral vascular resistance. This work tested the hypothesis that long-term hypothyroidism leads to an increase in the vasoconstrictor effect of Rho-kinase in skeletal muscle and heart resistance arteries of adult rats.Materials and methods.Male Wistar rats consumed the antithyroid drug propylthiouracil (PTU) in drinking water (0.025%), starting at 10 weeks of age. The rats of the control group received PTU-free water. After 14 weeks, the contractile responses of the gastrocnemius muscle arteries (to the α1-adrenoceptor agonist methoxamine) and the septal coronary artery (to the thromboxane A2 receptor agonist U46619) were isometrically recorded. The contribution of the Rho-kinase to the arterial contractile responses was assessed using inhibitor Y27632 (3 μM).Results.The consumption of propylthiouracil was accompanied by a marked decrease of thyroid hormone concentrations and an increase in total cholesterol serum level as well as a decrease in body weight. Maximal contractile responses of studied arteries were also reduced in hypothyroid rats. However, basal tone and reactivity to the moderate concentrations of agonists in arteries of hypothyroid rats were increased compared to control animals. Y27632 significantly weakened the contractile responses of the arteries and negated the differences between the two groups of rats.Conclusion.Chronic hypothyroidism leads to an increase in the activity of the Rho-kinase signaling pathway in the arteries of the gastrocnemius muscle and heart, which results in the increase of the spontaneous tone of the arteries and their reactivity to agonists.

Anticonvulsant-like effect of thromboxane receptor agonist U-46619 against pentylenetetrazol-induced seizures

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

Defined Structure-Activity Relationships of Boswellic Acids Determine Modulation of Ca2+ Mobilization and Aggregation of Human Platelets by Boswellia serrata Extracts.

Boswellic acids constitute a group of unique pentacyclic triterpene acids from Boswellia serrata with multiple pharmacological activities that confer them anti-inflammatory and anti-tumoral properties. A subgroup of boswellic acids, characterized by an 11-keto group, elevates intracellular Ca2+ concentrations [Ca2+]i and causes moderate aggregation of human platelets. How different BAs and their mixtures in pharmacological preparations affect these parameters in activated platelets has not been addressed, so far. Here, we show that boswellic acids either antagonize or induce Ca2+ mobilization and platelet aggregation depending on defined structural determinants with inductive effects predominating for a B. serrata gum resin extract. 3-O-Acetyl-11-keto-β-boswellic acid potently suppressed Ca2+ mobilization (IC50 = 6 µM) and aggregation (IC50 = 1 µM) when platelets were activated by collagen or the thromboxane A2 receptor agonist U-46619, but not upon thrombin. In contrast, β-boswellic acid and 3-O-acetyl-β-boswellic acid, which lack the 11-keto moiety, were weak inhibitors of agonist-induced platelet responses, but instead they elicited elevation of [Ca2+]i and aggregation of platelets (≥ 3 µM). 11-Keto-β-boswellic acid, the structural intermediate between 3-O-acetyl-11-keto-β-boswellic acid and β-boswellic acid, was essentially inactive independent of the experimental conditions. Together, our study unravels the complex agonizing and antagonizing properties of boswellic acids on human platelets in pharmacologically relevant preparations of B. serrata gum extracts and prompts for careful evaluation of the safety of such extracts as herbal medicine in cardiovascular risk patients.

NO-mediated anticontractile effect of the endothelium is abolished in coronary arteries of adult rats with antenatal/early postnatal hypothyroidism

IntroductionThyroid hormones are essential for proper development of many systems and organs, including circulatory system. Thyroid deficiency during pregnancy may affect the cardiovascular function in children early on and later in adulthood. However, long-term effects of early thyroid deficiency are poorly understood. We hypothesized that antenatal/early postnatal hypothyroidism will influence anticontractile effect of NO in coronary arteries of adult rats. Design and methodsTo model antenatal/early postnatal hypothyroidism dams were treated with 6-propyl-2-thiouracil (PTU) in drinking water (0.0007%, w/v) from the first day of pregnancy till 2 weeks after delivery. Control females were supplied with pure water. Their male offspring was grown up till the age of 10–12 weeks. Systolic blood pressure was measured using tail cuff method. Septal coronary arteries were isolated and studied in wire myograph. Blood serum thyroid hormones concentrations (ELISA) and NO metabolites level (Griess method) were evaluated. ResultsAt the age of 10–12 weeks thyroid hormones, TSH concentrations, NO metabolites and systolic blood pressure level didn't differ between groups. Arterial responses to acetylcholine and exogenous NO-donor DEA/NO were similar in control and PTU groups. Along with that, in control rats endothelium denudation strongly potentiated basal tone of arteries and their contractile responses to thromboxane A2 receptor agonist U46619. The effects of endothelium denudation were absent in PTU rats indicating that anticontractile effect of endothelium is abolished in their arteries. Further, NO-synthase inhibitor L-NNA (100 μM) caused significant elevation of basal tone and increased U46619-induced contraction of endothelium-intact arteries only in control rats, while had no effect in PTU group. ConclusionsOur data demonstrate that NO-mediated anticontractile effect of endothelium is eliminated in coronary arteries of adult rats, which suffered from antenatal/early postnatal hypothyroidism. Therefore, maternal thyroid hormones deficiency may have detrimental consequences in adult offspring including coronary circulation pathologies, despite normal blood levels of thyroid hormones.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets.

BackgroundVasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.MethodsWashed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.ResultsSurprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.ConclusionsWe identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Smooth muscle filamin A is a major determinant of conduit artery structure and function at the adult stage.

Human mutations in the X-linked FLNA gene are associated with a remarkably diverse phenotype, including severe arterial morphological anomalies. However, the role for filamin A (FlnA) in vascular cells remains partially understood. We used a smooth muscle (sm)-specific conditional mouse model to delete FlnA at the adult stage, thus avoiding the developmental effects of the knock-out. Inactivation of smFlnA in adult mice significantly lowered blood pressure, together with a decrease in pulse pressure. However, both the aorta and carotid arteries showed a major outward hypertrophic remodeling, resistant to losartan, and normally occurring in hypertensive conditions. Notably, arterial compliance was significantly enhanced in the absence of smFlnA. Moreover, reactivity of thoracic aorta rings to a variety of vasoconstrictors was elevated, while basal contractility in response to KCl depolarization was reduced. Enhanced reactivity to the thromboxane A2 receptor agonist U46619 was fully reversed by the ROCK inhibitor Y27632. We discuss the possibility that a reduction in arterial stiffness upon smFlnA inactivation might cause a compensatory increase in conduit artery diameter for normalization of parietal tension, independently of the ROCK pathway. In conclusion, deletion of smFlnA in adult mice recapitulates the vascular phenotype of human bilateral periventricular nodular heterotopia, culminating in aortic dilatation.

Abstract P130: The Influence of Maternal Obesity on Perivascular Adipose Tissue Function in Offspring

Objective: Maternal obesity pre-programs offspring to develop obesity and associated cardiovascular disease although the underlying mechanism is currently unknown. This study investigated the effect of maternal obesity on perivascular adipose tissue (PVAT) regulation of resistance artery tone. Design and method: 8 week old female SD rats were fed a 10% fat diet (controls) or 45% fat obesogenic diet (HFD) for 12 weeks before mating, then continued on their respective diets during pregnancy and lactation. Male offspring were provided with 10% fat diet until sacrifice at 12 weeks old. PVAT-intact or -denuded mesenteric arteries (250-300μm internal diameter) from offspring were mounted on a wire myograph. Cumulative concentration-response curves were constructed to the thromboxane A2 receptor agonist U46619 (10nM-3μM) ± 10μM A769662, an activator of AMP-activated kinase (AMPK) or 100μM L-NMMA, a nitric oxide synthase (NOS) inhibitor. Results: Body weight, systolic and diastolic blood pressure were significantly increased in HFD dams compared to age-matched controls (391±11 vs 348±12 g; 154±1 vs 145±1 mmHg; 116±1 vs 103±103 mmHg respectively) but no differences were observed between offspring. However, fat pads and insulin levels were increased in both HFD dams versus controls (13.4±1.6 vs 6.5±0.8 g; 1.18±0.09 vs 0.76±0.14 nmol/L respectively) and their offspring (7.2±0.3 vs 6.0±0.2 g; 0.56±0.15 vs 0.17±0.05 nmol/L respectively) versus controls. PVAT exerted an anti-contractile effect in artery segments from offspring of control dams (p<0.001), an effect which was lost in offspring of HFD dams. AMPK activation decreased contractility of both PVAT-denuded and -intact arteries from control offspring (p<0.01; p<0.0001 respectively, n=8); this effect was abolished in PVAT-intact vessels of offspring of HFD dams. Inhibition of NOS increased contractility of both PVAT-denuded and -intact arteries from control offspring (p<0.0001 and p<0.0001 respectively, n=8), revealing a contractile effect of PVAT in control offspring (p<0.01, n=8) but not in arteries from offspring of HFD dams. Conclusions: In summary, the diminished anti-contractile effects of PVAT in 12 week old offspring of HFD dams may be due to reduced AMPK and nitric oxide activity.

Enhanced parenchymal arteriole tone and astrocyte signaling protect neurovascular coupling mediated parenchymal arteriole vasodilation in the spontaneously hypertensive rat.

Functional hyperemia is the regional increase in cerebral blood flow upon increases in neuronal activity which ensures that the metabolic demands of the neurons are met. Hypertension is known to impair the hyperemic response; however, the neurovascular coupling mechanisms by which this cerebrovascular dysfunction occurs have yet to be fully elucidated. To determine whether altered cortical parenchymal arteriole function or astrocyte signaling contribute to blunted neurovascular coupling in hypertension, we measured parenchymal arteriole reactivity and vascular smooth muscle cell Ca(2+) dynamics in cortical brain slices from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats. We found that vasoconstriction in response to the thromboxane A2 receptor agonist U46619 and basal vascular smooth muscle cell Ca(2+) oscillation frequency were significantly increased in parenchymal arterioles from SHR. In perfused and pressurized parenchymal arterioles, myogenic tone was significantly increased in SHR. Although K(+)-induced parenchymal arteriole dilations were similar in WKY and SHR, metabotropic glutamate receptor activation-induced parenchymal arteriole dilations were enhanced in SHR. Further, neuronal stimulation-evoked parenchymal arteriole dilations were similar in SHR and WKY. Our data indicate that neurovascular coupling is not impaired in SHR, at least at the level of the parenchymal arterioles.

Lemon grass (Cymbopogon citratus (D.C) Stapf) polyphenols protect human umbilical vein endothelial cell (HUVECs) from oxidative damage induced by high glucose, hydrogen peroxide and oxidised low-density lipoprotein

The aromatic herb Cymbopogon citratus Stapf is widely used in tropical and subtropical countries in cooking, as a herbal tea, and in traditional medicine for hypertension and diabetes. Some of its properties have been associated with the in vitro antioxidant effect of polyphenols isolated from their aerial parts. However, little is known about C. citratus effects on endothelial cells oxidative injury. Using chromatographic procedures, a polyphenol-rich fraction was obtained from C. citratus (CCF) and their antioxidant properties were assessed by cooper-induced LDL oxidation assay. The main constituents of the active CCF, identified by high-performance liquid chromatography with diode-array detection and mass spectrometry (HPLC-DAD–MS), were chlorogenic acid, isoorientin and swertiajaponin. CCF 10 and 100μg/ml diminishes reactive oxidative species (ROS) production in human umbilical vein endothelial cell (HUVECs), challenged with high d-glucose (60% inhibition), hydrogen peroxide (80% inhibition) or oxidised low-density lipoprotein (55% inhibition). CCF 10 or 100μg/ml did not change nitric oxide (NO) production. However, CCF was able to inhibit vasoconstriction induced by the thromboxane A2 receptor agonist U46619, which suggest a NO-independent vasodilatador effect on blood vessels. Our results suggest that lemon grass antioxidant properties might prevent endothelial dysfunction associated to an oxidative imbalance promoted by different oxidative stimuli.

194 ANTI-CONTRACTILE VASCULAR RESPONSES TO 5’ AMP-ACTIVATED PROTEIN KINASE ACTIVATION; THE ROLE OF PERIVASCULAR ADIPOSE TISSUE AND THE INFLUENCE OF AGING

Introduction 5’ AMP-activated protein kinase (AMPK) is a serine/threonine kinase with a plethora of vital physiological roles, including regulation of energy metabolism. AMPK is present in the three major components of the vasculature: smooth muscle (VSM), the endothelium and perivascular adipose tissue (PVAT) and is known to augment bioavailability of the endogenous vasodilator nitric oxide (NO) via activation of endothelial nitric-oxide synthase (eNOS). Although it is known that AMPK can modulate VSM and endothelial function, it is unknown whether AMPK can modulate the anti-contractile effects of PVAT, despite the expression of both AMPK and eNOS by perivascular adipocytes. Aging is an independent risk factor for cardiovascular disease and is associated with altered arterial contractility. It is known that AMPK activity in skeletal muscle is reduced with advanced aging, however it is unknown whether the effects of AMPK on arterial contractility are modulated by age. Therefore we investigated the effects of activation of AMPK on arterial contractility in the presence and absence of PVAT and whether these effects are modulated by aging. Methods and Results Third order mesenteric endothelium-intact arteries were harvested from male Wistar rats aged 3 months old (m.o.) and 24 m.o. and contractility to increasing concentrations of the thromboxane A2 receptor agonist U46619 (10 nM-3 µM) was assessed via wire-myography in the presence and absence of PVAT and the AMPK activator A769662 (10 µM). Results (expressed as mean±SE) were analysed using two-way ANOVA (Bonferroni post-hoc). In 3 m.o. rat arteries, A769662 caused a reduction in subsequent contractions to increasing concentrations of U44619, in both the presence of PVAT (max increase in tension (1–3 µM U46619, mN/mm) : control 0.55±0.07 (n=3); +A769662 0.37±0.04 (n=4); p=0.0018) and absence of PVAT (control 1.20±0.52 (n=6); +A769662 0.60±0.22 (n=3)). However in 24 m.o. rat arteries, A769662 only caused a reduction in the presence of PVAT (control 0.88±0.15 (n=8); +A769662 0.55±0.17 (n=8); p=0.0054), and had no effect in PVAT-denuded vessels (control 0.85±0.11 (n=8); +A769662 0.83±0.11 (n=8)). Summary young animals. In older animals, however, AMPK activation can only elicit this effect in the presence of PVAT. These findings are consistent with differential changes in the expression and/or signalling of AMPK in the VSM and endothelium compared to PVAT with age. Further studies are currently being undertaken to investigate the mechanisms behind these differences.

Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis

Background Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined. Methods We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist U46619. Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured. Results No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P<0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P<0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P<0.05). Conclusions The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.