Downregulation of thromboxane A2 and angiotensin II type 1 receptors associated with aging-related decrease in internal anal sphincter tone.

Aging-associated decrease in internal anal sphincter (IAS) tone (AADI) is a major contributor in the rectoanal incontinence (RI). To determine the pathogenesis of AADI, we investigated the effect of aging on GPCR activation and related downstream signaling. We particularly investigated two GPCRs that characterize IAS smooth muscle cells (SMCs): thromboxane A2 and angiotensin II type 1. Two groups of Fischer 344 rats (6-month-old [young group] and 26-month-old [old group]) were employed to determine the GPCR function by isometric contraction, the expressions of GPCRs, and their downstream regulatory signaling proteins (regulator of G-protein signaling 2, RGS2; GPCR Kinase 5, GRK5; and ß-arrestin, Arrb2) using RT-PCR, qPCR, and western blot analyses. We used reversible biotinylation to monitor the GPCR trafficking using SMCs. Aging selectively attenuated thromboxane A2 and Ang II-induced IAS contraction. RT-PCR, qPCR, and WB data revealed a significant decrease in the expressions of the GPCRs and increase in the expression of RGS2, GRK5, and Arrb2. The increased GPCR internalization and decreased recycling under aging were validated by reversible biotinylation. We conclude that downregulation of GPCR, accompanied by upregulation of regulatory proteins, plays an important role in receptor desensitization and may be important underlying mechanisms of RI in certain aging patients.

ß-Arrestin-2-ERK1/2 cPLA2α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain.

LPS has been shown to elicit neuroinflammation associated with the up-regulation of the eicosanoid pathway in animal models; however, the regulatory mechanisms of TLR4 in brain neuroinflammatory conditions remain elusive. ß-Arrestins are key regulators of the GPCR signaling pathway and are involved in the leukotriene B4-induced leukocyte migration to initiate inflammatory response. However, the roles of ß-arrestins in eicosanoid regulation and related diseases are not clear. To address this issue, we conducted a study to investigate the effect of TLR4 on the eicosanoid pathway in ischemic stroke brain and to explore the underlying molecular regulation mechanism. Cerebral ischemia was produced by occlusion of the middle cerebral artery, followed by reperfusion for 24 h. We demonstrated that knockout of TLR4 improves ischemic stroke brain associated with eicosanoid down-regulation. Interestingly, genetic disruption of ß-arrestin-2 failed to decrease neuroinflammation in the damaged brain of TLR4-/- mice, which indicates the requirement of ß-arrestin-2 for TLR4 knockdown protection. Further study showed that the negative regulation of phosphorylated (phospho-)ERK1/2 and phospho-cytosolic phospholipase A2 α (cPLA2α) by TLR4 deficiency was eliminated by genetic disruption of ß-arrestin-2. In addition, ß-arrestin-2 deficiency reversed the reduction of colocalization of phospho-ERK1/2 with phospho-cPLA2α in TLR4-/- mice following ischemic stroke. Mechanistic studies indicated that ß-arrestin-2 specifically colocalized and associated with ERK1/2 to prevent ERK1/2-dependent cPLA2α activation following ischemic injury, and ß-arrestin-2 deficiency blocked the negative regulation of phospho-ERK1/2, revived the association of phospho-ERK1/2 with phospho-cPLA2α, and subsequently increased the prostaglandin E2 and thromboxane A2 production remarkably. Our findings may provide novel insights that ß-arrestin-2 is responsible for ischemic brain improvement in TLR4-/- mice via negative regulation of eicosanoid production.-Xiang, Y., Wei, X., Du, P., Zhao, H., Liu, A., Chen, Y. ß-Arrestin-2-ERK1/2 cPLA2α axis mediates TLR4 signaling to influence eicosanoid induction in ischemic brain.

Increased Levels of Txa2 Induced by Dengue Virus Infection in IgM Positive Individuals Is Related to the Mild Symptoms of Dengue.

The inflammatory process plays a major role in the prognosis of dengue. In this context, the eicosanoids may have considerable influence on the regulation of the Dengue virus-induced inflammatory process. To quantify the molecules involved in the cyclooxygenase and lipoxygenase pathways during Dengue virus infection, plasma levels of thromboxane A2, prostaglandin E2 and leukotriene B4; mRNA levels of thromboxane A2 synthase, prostaglandin E2 synthase, leukotriene A4 hydrolase, cyclooxygenase-2 and 5-lipoxygenase; and the levels of lipid bodies in peripheral blood leukocytes collected from IgM-positive and IgM-negative volunteers with mild dengue, and non-infected volunteers, were evaluated. Dengue virus infection increases the levels of thromboxane A2 in IgM-positive individuals as well as the amount of lipid bodies in monocytes in IgM-negative individuals. We suggest that increased levels of thromboxane A2 in IgM-positive individuals plays a protective role against the development of severe symptoms of dengue, such as vascular leakage.

Citalopram inhibits platelet function independently of SERT-mediated 5-HT transport.

Citalopram prevents serotonin (5-HT) uptake into platelets by blocking the serotonin reuptake transporter (SERT). Although some clinical data suggest that selective serotonin reuptake inhibitors (SSRIs) may affect haemostasis and thrombosis, these poorly-characterised effects are not well understood mechanistically and useful in vitro data is limited. We sought to determine whether the inhibitory effects of citalopram on platelets are mediated via its pharmacological inhibition of 5-HT transport. We quantified the inhibitory potency of (RS)-, (R)- and (S)-citalopram on platelet function. If SERT blockade is the primary mechanism for citalopram-mediated platelet inhibition, these potencies should show quantitative congruence with inhibition of 5-HT uptake. Our data show that citalopram inhibits platelet aggregation, adhesion and thromboxane production with no difference in potency between (R)- and (S)-isomers. By contrast, citalopram had a eudysmic ratio of approximately 17 (S > R) for SERT blockade. Furthermore, nanomolar concentrations of citalopram inhibited 5-HT uptake into platelets but had no effect on other platelet functions, which were inhibited by micromolar concentrations. Our data indicate that citalopram-induced inhibition of platelets in vitro is not mediated by blockade of 5-HT transport. This raises a new question for future investigation: by what mechanism(s) does citalopram inhibit platelets?

Thromboxane A2 Activates YAP/TAZ Protein to Induce Vascular Smooth Muscle Cell Proliferation and Migration.

The thromboxane A2 receptor (TP) has been implicated in restenosis after vascular injury, which induces vascular smooth muscle cell (VSMC) migration and proliferation. However, the mechanism for this process is largely unknown. In this study, we report that TP signaling induces VSMC migration and proliferation through activating YAP/TAZ, two major downstream effectors of the Hippo signaling pathway. The TP-specific agonists [1S-[1α,2α(Z),3ß(1E,3S*),4 α]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) and 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619) induce YAP/TAZ activation in multiple cell lines, including VSMCs. YAP/TAZ activation induced by I-BOP is blocked by knockout of the receptor TP or knockdown of the downstream G proteins Gα12/13 Moreover, Rho inhibition or actin cytoskeleton disruption prevents I-BOP-induced YAP/TAZ activation. Importantly, TP activation promotes DNA synthesis and cell migration in VSMCs in a manner dependent on YAP/TAZ. Taken together, thromboxane A2 signaling activates YAP/TAZ to promote VSMC migration and proliferation, indicating YAP/TAZ as potential therapeutic targets for cardiovascular diseases.

Apelin: an antithrombotic factor that inhibits platelet function.

Apelin peptide and its receptor APJ are directly implicated in various physiological processes ranging from cardiovascular homeostasis to immune signaling. Here, we show that apelin is a key player in hemostasis with an ability to inhibit thrombin- and collagen-mediated platelet activation. Mice lacking apelin displayed a shorter bleeding time and a prothrombotic profile. Their platelets exhibited increased adhesion and a reduced occlusion time in venules, and displayed a higher aggregation rate after their activation by thrombin compared with wild-type platelets. Consequently, human and mouse platelets express apelin and its receptor APJ. Apelin directly interferes with thrombin-mediated signaling pathways and platelet activation, secretion, and aggregation, but not with ADP and thromboxane A2-mediated pathways. IV apelin administration induced excessive bleeding and prevented thrombosis in mice. Taken together, these findings suggest that apelin and/or APJ agonists could potentially be useful adducts in antiplatelet therapies and may provide a promising perspective for patients who continue to display adverse thrombotic events with current antiplatelet therapies.

Regulation of tissue factor gene expression in monocytes and endothelial cells: Thromboxane A2 as a new player.

Tissue factor (TF) is the primary activator of the coagulation cascade. Under normal conditions, endothelial cells (ECs) and blood cells, such as monocytes, do not express TF. However, bacterial lipopolysaccharide (LPS) induces TF expression in monocytes and this leads to disseminated intravascular coagulation during endotoxemia and sepsis. A variety of stimuli induce TF expression in ECs in vitro, although it is unclear how much TF is expressed by the endothelium in vivo. LPS induction of TF gene expression in monocytic cells and ECs is mediated by various intracellular signaling pathways and the transcription factors NF-ĸB, AP-1 and Egr-1. In contrast, vascular endothelial cell growth factor (VEGF) induces TF gene expression in ECs via the transcription factors NFAT and Egr-1. Similarly, oxidized phospholipids (such as 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine) induce TF expression in ECs and possibly monocytes via NFAT and Egr-1. Thromboxane A2 (TXA2) can now be added to the list of stimuli that induce TF gene expression in both monocytes and ECs. Interestingly, inhibition of the TX-prostanoid (TP) receptor also reduces TF expression in with tumor necrosis factor (TNF)-α stimulated ECs and LPS stimulated monocytes, which suggests that TP receptor antagonist may be useful in reducing pathologic TF expression in the vasculature and blood.

n-Butanol soluble fraction of the water extract of Chinese toon fruit ameliorated focal brain ischemic insult in rats via inhibition of oxidative stress and inflammation.

AIM OF THE STUDY: Toona sinensis Roem. (Meliaceae; Toona sinensis; Chinese toon) is a type of arbor that is widely distributed in Asia. The fruits of Toona sinensis Roem has been traditionally recognized for treatment of cerebrovascular diseases. To evaluate the potential clinical use of the fruits of Toona sinensis Roem, we determined the dose dependence of the neuroprotective efficacy in a focal cerebral ischemic reperfusion model of rats and explored the underlying mechanisms. MATERIALS AND METHODS: Rats were subjected to occlusion of the middle cerebral artery (MCAO) by a nylon filament and treated with different doses (20mg/kg and 30 mg/kg) of n-butanol soluble fraction of the water extract of Chinese toon fruit or the vehicle for 1 week before induction of ischemia, s.i.d.. RESULTS: n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced in a dose-dependent manner the ischemia-induced cerebral infarct and edema volume and attenuated neurological deficits observed at 6h point after ischemia. n-Butanol soluble fraction of the water extract of Chinese toon fruit reduced the levels of nitrate, nitrite, lipid peroxidation, cyclooxygenase-1, thromboxane in post-ischemic brain. n-Butanol soluble fraction of the water extract of Chinese toon fruit adjusted the elevation of the activity of glutathione peroxidase and superoxide dismutase in ischemic brain. CONCLUSIONS: The present study was the first evidence of effectiveness of n-butanol soluble fraction of the water extract of Chinese toon fruit in the rat stroke models, as it reduced infarct volume, inhibited the oxidative stress and inflammation.

Thromboxane A2 mediates iron-overload cardiomyopathy in mice through calcineurin-nuclear factor of activated T cells signaling pathway.

BACKGROUND: Recent studies demonstrated that iron overload could enhance the production of arachidonic acid and prostanoid, suggesting a causal connection between these signals and iron-overload cardiomyopathy. However, information regarding the downstream signaling is limited. Because thromboxane A2 (TXA2) and prostacyclin are the 2 major prostanoids in the cardiovascular system, and TXA2 plays a major role in vascular atherosclerosis and has pro-inflammatory characteristics, we intended to elucidate the role of TXA2 in iron-overload cardiomyopathy. METHODS AND RESULTS: A 4-week iron loading protocol was instituted for both TXAS gene-deleted (TXAS(-/-)) mice and wild-type (WT) mice, with less severe cardiac fibrosis and preserved normal left ventricular contraction in the TXAS(-/-) mice. Inflammatory profiles, including MCP-1, TNF-α, IL-6, ICAM-1, and myeloperoxidase activity were also lower in the TXAS(-/-) as compared with WT littermates. TXAS supplement to the iron-injured TXAS(-/-) mice re-aggravated cardiac inflammation. Using a TXA2 analog, U46619, for NFAT reporter luciferase activity on cardiomyoctes, and intraperitonal injection of U46619 into nuclear factor of activated T cells (NFAT)-luciferase transgenic mice demonstrated that U46619 increase NFAT expression, and this expression, as well as TNF-α expression, can be blocked by TXA2 receptor antagonist (SQ29548), NFAT-SiRNA, calcineurin inhibitor, or calcium chelator. Finally, intraperitoneal injection of the TNF-α antibody, infliximab, into iron-injured mice decreased TXAS expression and attenuated cardiac fibrosis. CONCLUSIONS: TXA2 mediates iron-overload cardiomyopathy through the TNF-α-associated calcineurin-NFAT signaling pathway.

Histamine, carbachol, and serotonin induce hyperresponsiveness to ATP in guinea pig tracheas: involvement of COX-2 pathway.

Extracellular ATP promotes an indirect contraction of airway smooth muscle via the secondary release of thromboxane A2 (TXA2) from airway epithelium. Our aim was to evaluate if common contractile agonists modify this response to ATP. Tracheas from sensitized guinea pigs were used to evaluate ATP-induced contractions before and after a transient contraction produced by histamine, carbachol, or serotonin. Epithelial mRNA for COX-1 and COX-2 was measured by RT-PCR and their expression assessed by immunohistochemistry. Compared with the initial response, ATP-induced contraction was potentiated by pretreatment with histamine, carbachol, or serotonin. Either suramin (antagonist of P2X and P2Y receptors) plus RB2 (antagonist of P2Y receptors) or indomethacin (inhibitor of COX-1 and COX-2) annulled the ATP-induced contraction, suggesting that it was mediated by P2Y receptor stimulation and TXA2 production. When COX-2 was inhibited by SC-58125 or thromboxane receptors were antagonized by SQ-29548, just the potentiation was abolished, leaving the basal response intact. Airway epithelial cells showed increased COX-2 mRNA after stimulation with histamine or carbachol, but not serotonin, while COX-1 mRNA was unaffected. Immunochemistry corroborated this upregulation of COX-2. In conclusion, we showed for the first time that histamine and carbachol cause hyperresponsiveness to ATP by upregulating COX-2 in airway epithelium, which likely increases TXA2 production. Serotonin-mediated hyperresponsiveness seems to be independent of COX-2 upregulation, but nonetheless is TXA2 dependent. Because acetylcholine, histamine, and serotonin can be present during asthmatic exacerbations, their potential interactions with ATP might be relevant in its pathophysiology.

Low-dose aspirin delays an inflammatory tumor progression in vivo in a transgenic mouse model of neuroblastoma.

Tumor-associated inflammation is a driving force in several adult cancers and intake of low-dose aspirin has proven to reduce cancer incidence. Little is known about tumor-associated inflammation in pediatric neoplasms and no in vivo data exists on the effectiveness of low-dose aspirin on established tumors. The present study employs the transgenic TH-MYCN mouse model for neuroblastoma (NB) to evaluate inflammatory patterns paralleling tumor growth in vivo and low-dose aspirin as a therapeutic option for high-risk NB. Spontaneously arising abdominal tumors were monitored for tumor-associated inflammation ex vivo at various stages of disease and homozygous mice received daily low-dose aspirin (10mg/kg) using oral gavage or no treatment, from 4.5 to 6 weeks of age. Using flow cytometry, a transition from an adaptive immune response predominated by CD8(+) T cell in early neoplastic lesions, towards enrichment in immature cells of the innate immune system, including myeloid-derived suppressor cells, dendritic cells and tumor-associated macrophages, was detected during tumor progression. An M1 to M2 transition of tumor-associated macrophages was demonstrated, paralleled by a deterioration of dendritic cell status. Treatment with low-dose aspirin to mice homozygous for the TH-MYCN transgene significantly reduced the tumor burden (P < 0.01), the presence of tumor-associated cells of the innate immune system (P < 0.01), as well as the intratumoral expression of transforming growth factor-ß, thromboxane A2 (P < 0.05) and prostaglandin D2 (P < 0.01). In conclusion, tumor-associated inflammation appears as a potential therapeutic target in NB and low-dose aspirin reduces tumor burden in the TH-MYCN transgenic mouse model of NB, hence warranting further studies on aspirin in high-risk NB.

Prostaglandin E2 deficiency uncovers a dominant role for thromboxane A2 in house dust mite-induced allergic pulmonary inflammation.

Prostaglandin E(2) (PGE(2)) is an abundant lipid inflammatory mediator with potent but incompletely understood anti-inflammatory actions in the lung. Deficient PGE(2) generation in the lung predisposes to airway hyperresponsiveness and aspirin intolerance in asthmatic individuals. PGE(2)-deficient ptges(-/-) mice develop exaggerated pulmonary eosinophilia and pulmonary arteriolar smooth-muscle hyperplasia compared with PGE(2)-sufficient controls when challenged intranasally with a house dust mite extract. We now demonstrate that both pulmonary eosinophilia and vascular remodeling in the setting of PGE(2) deficiency depend on thromboxane A(2) and signaling through the T prostanoid (TP) receptor. Deletion of TP receptors from ptges(-/-) mice reduces inflammation, vascular remodeling, cytokine generation, and airway reactivity to wild-type levels, with contributions from TP receptors localized to both hematopoietic cells and tissue. TP receptor signaling ex vivo is controlled heterologously by E prostanoid (EP)(1) and EP(2) receptor-dependent signaling pathways coupling to protein kinases C and A, respectively. TP-dependent up-regulation of intracellular adhesion molecule-1 expression is essential for the effects of PGE(2) deficiency. Thus, PGE(2) controls the strength of TP receptor signaling as a major bronchoprotective mechanism, carrying implications for the pathobiology and therapy of asthma.

Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism.

Thromboxane A(2) (TXA(2)) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA(2) receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro- and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA(2) synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro- and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues.

An important role for Akt3 in platelet activation and thrombosis.

The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3(-/-) mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A2 (TXA2), but not collagen or VWF. In contrast, platelets from Akt1(-/-) or Akt2(-/-) mice are defective in platelet activation induced by thrombin, TXA2, and VWF, but only Akt1(-/-) platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3(-/-) platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3ß (GSK-3ß) at Ser9, which is known to inhibit GSK-3ß function. Thus, Akt3 is important in inhibiting GSK-3ß. Accordingly, treatment of Akt3(-/-) platelets with a GSK-3ß inhibitor rescued the defect of Akt3(-/-) platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3ß may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3(-/-) mice showed retardation in FeCl3-induced carotid artery thrombosis in vivo. Thus, Akt3 plays an important and distinct role in platelet activation and in thrombosis.

Up-regulation of thromboxane A2 impairs cerebrovascular eNOS function in aging atherosclerotic mice.

We previously reported that in healthy mouse cerebral arteries, endothelial nitric oxide synthase (eNOS) produces H2O2, leading to endothelium-dependent dilation. In contrast, thromboxane A2 (TXA2), a potent pro-oxidant and pro-inflammatory endogenous vasoconstrictor, is associated with eNOS dysfunction. Our objectives were to elucidate whether (1) the cerebrovascular eNOS-H2O2 pathway was sensitive to oxidative stress associated with aging and dyslipidemia and (2) TXA2 contributed to cerebral eNOS dysfunction. Atherosclerotic (ATX = LDLR(-/-); hApoB(+/+)) and wild-type (WT) control mice were used at 3 and 12 months old (m/o). Three-m/o ATX mice were treated with the cardio-protective polyphenol catechin for 9 months. Dilations to ACh and the simultaneous eNOS-derived H2O2 production were recorded in isolated pressurized cerebral arteries. The age-associated decrease in cerebral eNOS-H2O2 pathway observed in WT was premature in ATX mice, decreasing at 3 m/o and abolished at 12 m/o. Thromboxane synthase inhibition by furegrelate increased dilations at 12 months in WT and at 3 and 12 months in ATX mice, suggesting an anti-dilatory role of TXA2 with age hastened by dyslipidemia. In addition, the non-selective NADP(H) oxidase inhibitor apocynin improved the eNOS-H2O2 pathway only in 12-m/o ATX mice. Catechin normalized the function of this pathway, which became sensitive to L-NNA and insensitive to furegrelate or apocynin; catechin also prevented the rise in TXA2 synthase expression. In conclusion, the age-dependent cerebral endothelial dysfunction is precocious in dyslipidemia and involves TXA2 production that limits eNOS activity. Preventive catechin treatment reduced the impact of endogenous TXA2 on the control of cerebral tone and maintained eNOS function.

Pericytes in capillaries are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain.

Modern functional imaging techniques of the brain measure local hemodynamic responses evoked by neuronal activity. Capillary pericytes recently were suggested to mediate neurovascular coupling in brain slices, but their role in vivo remains unexplored. We used two-photon microscopy to study in real time pericytes and the dynamic changes of capillary diameter and blood flow in the cortex of anesthetized mice, as well as in brain slices. The thromboxane A(2) analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α (U46619), induced constrictions in the vicinity of pericytes in a fraction of capillaries, whereas others dilated. The changes in vessel diameter resulted in changes in capillary red blood cell (RBC) flow. In contrast, during brief epochs of seizure activity elicited by local administration of the GABA(A) receptor antagonist, bicuculline, capillary RBC flow increased without pericyte-induced capillary diameter changes. Precapillary arterioles were the smallest vessels to dilate, together with penetrating and pial arterioles. Our results provide in vivo evidence that pericytes can modulate capillary blood flow in the brain, which may be important under pathological conditions. However, our data suggest that precapillary and penetrating arterioles, rather than pericytes in capillaries, are responsible for the blood flow increase induced by neural activity.

Maternal levels of prostacyclin, thromboxane, ICAM, and VCAM in normal and preeclamptic pregnancies.

PROBLEM: To evaluate whether impaired endothelial function and endothelial inflammatory response occur in parallel in the women with preeclampsia. METHOD OF STUDY: Venous blood was drawn from normal (n=40) and severe preeclamptic (sPE) (n=40) pregnant women when they were admitted to the L&D Unit and 24 hrs after delivery. Plasma and serum samples were extracted and measured for 6-keto PGF1α and TXB(2) (stable metabolites of PGI2 and TXA2), and intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) by ELISA. Data are analyzed by Mann-Whitney test and paired t-test. The statistical significance is set as P<0.05. Results Plasma 6-keto PGF1α levels were significantly reduced at admission and 24hr after delivery in sPE compared to normal pregnant controls, P<0.01. The ratio of 6-keto PGF1α and TXB(2) was significant less in sPE than that in normal pregnant controls before delivery. There was no significant difference for ICAM and VCAM levels between normal and patients with sPE before and after delivery. CONCLUSION: Maternal 6-keto PGF1α levels and the ratio of 6-keto PGF1α and TXB(2) were decreased in patients with sPE compared to normal pregnant controls. In contrast, maternal ICAM and VCAM levels were not different between the two groups. These data suggest that serum ICAM and VCAM levels may not be sensitive inflammatory biomarkers for preeclampsia.

Role of glycoprotein Ibalpha mobility in platelet function.

Incubation at 0 degrees C is known to expose b- N -acetyl-D-glucosamine residues on glycoprotein (GP) Ibalpha inducing receptor clustering and alpha(M)beta(2)-mediated platelet destruction by macrophages. Here we show that incubation at 0/37 degrees C (4 hours at 0 degrees C, followed by 1 hour at 37 degrees C to mimic cold-storage and post-transfusion conditions) triggers a conformational change in the N -terminal flank (NTF, amino acids, aa 1-35) but not in aa 36-282 of GPIbalpha as detected by antibody binding. Addition of the sugar N -acetyl-D-glucosamine (GN) inhibits responses induced by 0/37 degrees C. Incubation at 0 degrees C shifts GPIbalpha from the membrane skeleton to the cytoskeleton. Different GPIbalpha conformations have little effect on VWF/ristocetin-induced aggregation, but arrest of NTF change by GN interferes with agglutination and spreading on a VWF-coated surface under flow. Strikingly, incubation at 0/37 degrees C initiates thromboxane A(2) formation through a von Willebrand factor (VWF)-independent and GPIbalpha-dependent mechanism, as confirmed in VWF- and GPIbalpha-deficient platelets. We conclude that the NTF change induced by 0/37 degrees C incubation reflects clustering of GPIbalpha supports VWF/ristocetin-induced agglutination and spreading and is sufficient to initiate platelet activation in the absence of VWF.

Involvement of the thromboxane A2 receptor in the regulation of steroidogenic acute regulatory gene expression in murine Leydig cells.

Recent studies suggested an involvement of thromboxane A2 in cyclooxygenase-2-dependent inhibition of steroidogenic acute regulatory (StAR) gene expression. The present study further investigated the role of thromboxane A2 receptor in StAR gene expression and steroidogenesis in testicular Leydig cells. The thromboxane A2 receptor was detected in several Leydig cell lines. Blocking thromboxane A2 binding to the receptor using specific antagonist SQ29548 or BM567 resulted in dose-dependent increases in StAR protein and steroid production in MA-10 mouse Leydig cells. The results were confirmed with Leydig cells isolated from rats. StAR promoter activity and StAR mRNA level in the cells were also increased after the treatments, suggesting an involvement of the thromboxane A2 receptor in StAR gene transcription. Furthermore study indicated that blocking the thromboxane A2 receptor reduced dosage sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 protein, a transcriptional repressor of StAR gene expression. Specific binding of the antagonists to the receptors on cellular membrane was demonstrated by binding assays using (3)H-SQ29548 and binding competition between (3)H-SQ29548 and BM567. Whereas SQ29548 enhanced cAMP-induced StAR gene expression, in the absence of cAMP, it was unable to increase StAR protein and steroidogenesis. However, when the receptor was blocked by the antagonist, subthreshold levels of cAMP were able to induce maximal levels of StAR protein expression, suggesting that blocking the thromboxane A2 receptor increase sensitivity of MA-10 cells to cAMP stimulation. Taken together, the results from the present and previous studies suggest an autocrine loop, involving cyclooxygenase-2, thromboxane A synthase, and thromboxane A2 and its receptor, in cyclooxygenase-2-dependent inhibition of StAR gene expression.

Group IVA cytosolic phospholipase A2 (cPLA2alpha) and integrin alphaIIbbeta3 reinforce each other's functions during alphaIIbbeta3 signaling in platelets.

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) catalyzes release of arachidonic acid from glycerophospholipids, leading to thromboxane A(2) (TxA(2)) production. Some platelet agonists stimulate cPLA(2)alpha, but others require fibrinogen binding to alphaIIbbeta3 to elicit TxA(2). Therefore, relationships between cPLA(2)alpha and alphaIIbbeta3 were examined. cPLA(2)alpha and a cPLA(2)alpha binding partner, vimentin, coimmunoprecipitated with alphaIIbbeta3 from platelets, independent of fibrinogen binding. Studies with purified proteins and with recombinant proteins expressed in CHO cells determined that the interaction between cPLA(2)alpha and alphaIIbbeta3 was indirect and was dependent on the alphaIIb and beta3 cytoplasmic tails. Fibrinogen binding to alphaIIbbeta3 caused an increase in integrin-associated cPLA(2)alpha activity in normal platelets, but not in cPLA(2)alpha-deficient mouse platelets or in human platelets treated with pyrrophenone, a cPLA(2)alpha inhibitor. cPLA(2)alpha activation downstream of alphaIIbbeta3 had functional consequences for platelets in that it was required for fibrinogen-dependent recruitment of activated protein kinase Cbeta to the alphaIIbbeta3 complex and for platelet spreading. Thus, cPLA(2)alpha and alphaIIbbeta3 interact to reinforce each other's functions during alphaIIbbeta3 signaling. This provides a plausible explanation for the role of alphaIIbbeta3 in TxA(2) formation and in the defective hemostatic function of mouse or human platelets deficient in cPLA(2)alpha.