Anji white tea relaxes precontracted arteries, represses voltage-gated Ca2+ channels and voltage-gated K+ channels in the arterial smooth muscle cells: Comparison with green tea main component (-)-epigallocatechin gallate.

ETHNOPHARMACOLOGICAL RELEVANCE: Tea (Camellia sinensis) is a favorite drink worldwide. Tea extracts and green tea main component (-)-epigallocatechin gallate (EGCG) are recommended for various vascular diseases. Anji white tea is a very popular green tea. Its vascular effect profile, the mechanisms, and the contribution of EGCG to its integrated effect need elucidation. AIM: To characterize the vasomotion effects of Anji white tea and EGCG, and to explore possible involvement of voltage-gated Ca2+ channels (VGCCs) and voltage-gated K+ (Kv) channels in their vasomotion effects. MATERIALS AND METHODS: Anji white tea water soaking solution (AJWT) was prepared as daily tea-making process and concentrated to a concentration amounting to 200 mg/ml of dry tea leaves. The tension of rat arteries including aorta, coronary artery (RCA), cerebral basilar artery (CBA), intrarenal artery (IRA), intrapulmonary artery (IPA) and mesenteric artery (MA) was recorded with myographs. In arterial smooth muscle cells (ASMCs) freshly isolated from RCA, the levels of intracellular Ca2+ were measured with Ca2+-sensitive fluorescent probe fluo 4-AM, and Kv currents were recorded with patch clamp. The expressions of VGCCs and Kv channels were assayed with RT-qPCR and immunofluorescence staining. RESULTS: At 0.4-12.8 mg/ml of dry tea leaves, AJWT profoundly relaxed all tested arteries precontracted with various vasoconstrictors about half with a small transient potentiation on the precontractions before the relaxation. KCl-induced precontraction was less sensitive than precontractions induced by phenylephrine (PE), U46619 and serotonin (5-HT). IPA was less sensitive to the relaxation compared with other arteries. AJWT pretreatment for 1 h, 24 h and 72 h time-dependently inhibited the contractile responses of RCAs. In sharp contrast, at equivalent concentrations according to its content in AJWT, EGCG intensified the precontractions in most small arteries, except that it induced relaxation in PE-precontracted aorta and MA, U46619-precontracted aorta and CBA. EGCG pretreatment for 1 h and 24 h did not significantly affect RCA contractile responses. In RCA ASMCs, AJWT reduced, while EGCG enhanced, intracellular Ca2+ elevation induced by depolarization which activates VGCCs. Patch clamp study showed that both AJWT and EGCG reduced Kv currents. RT-qPCR and immunofluorescence staining demonstrated that both AJWT and EGCG reduced the expressions of VGCCs and Kv channels. CONCLUSION: AJWT, but not EGCG, consistently induces vasorelaxation. The vasomotion effects of either AJWT or EGCG vary with arterial beds and vasoconstrictors. Modulation of VGCCs, but not Kv channels, contributes to AJWT-induced vasorelaxation. It is suggested that Anji white tea water extract instead of EGCG may be a promising food supplement for vasospastic diseases.

Effect of Cichoric Acid on Colorectal Cancer: Impact on Migration, Epithelial-Mesenchymal Transition, and Proliferation via RhoA/ROCK Signaling Pathway Modulation.

BACKGROUND: Colorectal cancer (CRC) is a common malignancy with high morbidity and mortality. To improve CMC prognosis, research must identify safe and effective natural drugs that improve the proliferation, migration, and epithelial mesenchymal transition (EMT) processes of CRC. The purpose of this paper is to understand how cichoric acid (CA) impacts CRC proliferation, metastasis, and EMT of CRC by adjusting the Ras homolog family member A (RhoA)/RHO-associated coiled coil protein kinase (ROCK) pathway. METHODS: Human Colon Cancer Cells (HCT116) cells were randomly divided into Control (blank medium treatment), low concentration CA (CA-L), medium concentration CA (CA-M), high concentration CA (CA-H), and high-concentration CA+RhoA activator U46619 (CA-H+U46619) groups. Cell proliferation, migration and invasion, and apoptosis were evaluated with cell counting kit-8 (CCK-8) assay, transwell assay, and flow cytometry, respectively. The expression of RhoA, ROCK, and EMT-associated proteins were detected by Western Blot. The CRC transplanted tumor model of nude mice was constructed, and the mice were grouped into low-dose CA (CA-Low, 15 mg/kg CA), high-dose CA (CA-High, 30 mg/kg CA), high-dose CA+RhoA activator U46619 (CA-High+U46619, 30 mg/kg CA+10 mM U46619), and Model groups at random, with 12 mice in each group. Tumor volume, mass, and inhibition rate were measured and calculated, and the pathological changes of tumor in nude mice were detected by hematoxylin-eosin (HE) staining. RESULTS: Compared with Control, the optical density of cells at 450 nm (OD450) value (48 h, 72 h), cell migration number, cell invasion number, RhoA, ROCK1, N-cadherin, vimentin protein expression levels of HCT116 cells were reduced in CA-M and CA-H groups; however, E-cadherin level and apoptosis rate were increased (p < 0.05). In the CA-High group, we observed a significant decrease (p < 0.05) in both tumor volume and mass in nude mice. Additionally, the tumor tissue cells exhibited better organization, reduced size, reduced tumor and vascular tissue hyperplasia, and decreased infiltration of inflammatory cells. U46619 decreased the retardation of CA on the proliferation, EMT, and migration of CRC tumor cells as well as the growth of transplanted CRC tumors in nude mice. CONCLUSIONS: CA may reduce CRC migration, proliferation, and EMT by inhibiting the activation of the RhoA/ROCK signaling pathway.

Increased Coronary Contraction to Thromboxane A2 in Cardiac Surgery Patients With Poorly Controlled Hypertension.

INTRODUCTION: Cardioplegia and cardiopulmonary bypass (CP/CPB) alters coronary arteriolar response to thromboxane A2 (TXA2) in patients undergoing cardiac surgery. Comorbidities, including hypertension (HTN), can further alter coronary vasomotor tone. This study investigates the effects of HTN on coronary arteriolar response to TXA2 pre and post-CP/CPB and cardiac surgery. MATERIALS AND METHODS: Coronary arterioles pre and post-CP/CPB were dissected from atrial tissue samples in patients with no HTN (NH, n = 9), well-controlled HTN (WC, n = 12), or uncontrolled HTN (UC, n = 12). In-vitro coronary microvascular reactivity was examined in the presence of TXA2 analog U46619 (10-9-10-4M). Protein expression of TXA2 receptor in the harvested right atrial tissue samples were measured by immunoblotting. RESULTS: TXA2 analog U46619 induced dose-dependent contractile responses of coronary arterioles in all groups. Pre-CPB contractile responses to U46619 were significantly increased in microvessels in the UC group compared to the NH group (P < 0.05). The pre-CP/CPB contractile responses of coronary arterioles were significantly diminished post-CP/CPB among the three groups (P < 0.05), but there remained an increased contractile response in the microvessels of the UC group compared to the WC and NH groups (P < 0.05). There were no significant differences in U46619-induced vasomotor tone between patients in the NH and WC groups (P > 0.05). There were no differences in expression of TXA2R among groups. CONCLUSIONS: Poorly controlled HTN is associated with increased contractile response of coronary arterioles to TXA2. This alteration may contribute to worsened recovery of coronary microvascular function in patients with poorly controlled HTN after CP/CPB and cardiac surgery.

Ontogenesis of functional platelet subpopulations from preterm and term neonates to adulthood: The PLINIUS study.

Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, which is mainly reflected by the stage-specific expression of hemoglobin chains. Perinatal alterations in thrombopoiesis are poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adulthood. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks), 10 term neonates, 8 infants (<2 years), 5 children (2-13 years), and 9 adults (>13 years). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that the expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced but correlated with decreased platelet size, indicating a normal surface density. Although CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP, or U46619 was unaltered or only slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted but showed a continuous increase until adulthood, correlating with the expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis using automated clustering revealed that neonates presented with a CD63+/PAC-1- pattern, followed by a continuous increase in CD63+/PAC-1+ platelets until adulthood. Our findings revealed that the number of platelet-monocyte and platelet-neutrophil aggregates, but not platelet-lymphocyte aggregates, is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that the platelet phenotype and function evolve continuously from neonates to adulthood.

Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1.

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

Increased thromboxane/prostaglandin receptors contribute to high glucose-induced podocyte injury and mitochondrial fission through ROCK1-Drp1 signaling.

Excessive mitochondrial fission in podocytes serves as a central hub for the pathogenesis of diabetic nephropathy (DN), and the thromboxane/prostaglandin receptor (TP receptor) plays a potential role in DN. However, regulation of the TP receptor during mitochondrial dynamics disorder in podocytes remains unknown. Here, we firstly reported novel mechanistic details of TP receptor effects on mitochondrial dynamics in podocytes under diabetic conditions. Expression of the TP receptor was significantly upregulated in podocytes under diabetic conditions both in vivo and in vitro. S18886 attenuated podocyte mitochondrial fission, glomerular injury and renal dysfunction in diabetic mice. Furthermore, inhibition of the TP receptor by both genetic and pharmacological methods dramatically reduced mitochondrial fission and attenuated podocyte injury induced by high glucose through regulating dynamin-related protein 1 (Drp1) phosphorylation and its subsequent translocation to mitochondria. In contrast, TP receptor overexpression and application of TP receptor agonist U46619 in these podocytes showed the opposite effect on mitochondrial fission and podocyte injury. Furthermore, treatment with Y27632, an inhibitor of Rho-associated kinase1 (ROCK1), significantly blunted more fragmented mitochondria and reduced podocyte injuries in podocytes with TP receptor overexpression or after U46619 treatment. Finally, pharmacological inhibition of Drp1 alleviated excessive mitochondrial fragmentation and podocyte damage in TP receptor overexpressing podocytes. Our data suggests that increased expression of the TP receptor can occur in a human cultured podocyte cell line and in podocytes derived from streptozotocin (STZ)-induced diabetic mice, which contributes to mitochondrial excessive fission and podocyte injury via ROCK1-Drp1 signaling.

Effects of fasudil on blood-brain barrier integrity.

BACKGROUND: Cerebral 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. METHODS: BBB 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. RESULTS: We 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. CONCLUSIONS: Our findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

Dexmedetomidine reduces dextran sulfate sodium (DSS)-induced NCM460 cell inflammation and barrier damage by inhibiting RhoA/ROCK signaling pathway.

OBJECTIVE: This study investigated the role of dexmedetomidine (DEX) in dextran sulfate sodium (DSS)-induced NCM460 cells. MATERIAL AND METHODS: The viability and apoptosis of NCM460 cells treated with DEX with or without DSS were detected by CCK-8 and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay. The level of inflammatory factors and expression of inflammation-related proteins, tight junction proteins and Ras homolog gene family, member A/Rho-associated coiled-coil containing protein kinase (RhoA/ROCK) signaling-related proteins in NCM460 cells treated with DEX and/or U46619 (RhoA/ROCK agonist) and/or DSS were detected by the respective enzyme-linked immunosorbent assay (ELISA) kits and Western blot analysis. The permeability of NCM460 monolayers was examined with transepithelial electrical resistance (TEER) assay. RESULTS: DEX had no effect on NCM460 cell viability. However, DEX improved the viability and barrier damage and suppressed the apoptosis and inflammation of DSS-induced NCM460 cells. Correspondingly, the expression of inflammation-related proteins was reduced and the expression of tight junction proteins was increased in DSS-induced NCM460 cells after treatment with DEX. In addition, RhoA/ROCK signaling was activated in NCM460 cells induced by DSS, which was suppressed by DEX. The protective effects of DEX on DSS-indued NCM460 cells were reversed by U46619. CONCLUSION: DEX improved viability and barrier damage while suppressed apoptosis and inflammation in DSS-indued NCM460 cells by inhibiting RhoA/ROCK signaling pathway.

Docosahexaenoic acid and eicosapentaenoic acid strongly inhibit prostanoid TP receptor-dependent contractions of guinea pig gastric fundus smooth muscle.

The inhibitory effects of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and linoleic acid (LA) on the contractions induced by five prostanoids and U46619 (a TP receptor agonist) were examined in guinea pig gastric fundus smooth muscle (GFSM). Tension changes were isometrically measured, and the mRNA expression of prostanoid receptors was measured by RT-qPCR. DHA and EPA significantly inhibited contractions induced by the prostanoids and U46619, whereas LA inhibited those induced by prostaglandin D2 and U46619. The mRNA expression levels of the prostanoid receptors were TP ≈ EP3  >> FP > EP1 . The inhibition by DHA, EPA, and LA was positively correlated with that by SQ 29,548 (a TP receptor antagonist) but not with that by L-798,106 (an EP3 receptor antagonist). DHA and EPA suppressed high KCl-induced contractions by 35% and 25%, respectively, and the contractions induced by the prostanoids and U46619 were suppressed by verapamil, a voltage-dependent Ca2+ channel (VDCC) inhibitor, by 40%-85%. Although LA did not suppress high KCl-induced contractions, it suppressed U46619-induced contractions in the presence of verapamil. However, LA did not show significant inhibitory effects on U46619-induced Ca2+ increases in TP receptor-expressing cells. In contrast, LA inhibited U46619-induced contractions in the presence of verapamil, which was also suppressed by SKF-96365 (a store-operated Ca2+ channel [SOCC] inhibitor). These findings suggest that the TP receptor and VDCC are targets of DHA and EPA to inhibit prostanoid-induced contractions of guinea pig GFSM, and SOCCs play a significant role in LA-induced inhibition of U46619-induced contractions.

Inhibition of Full Smooth Muscle Contraction in Isolated Human Detrusor Tissues by Mirabegron Is Limited to Off-Target Inhibition of Neurogenic Contractions.

Mirabegron is used for treatment of storage symptoms in overactive bladder (OAB) caused by spontaneous bladder smooth muscle contractions. However, owing to limitations in available studies using human tissues, central questions are still unresolved, including mechanisms underlying improvements by mirabegron and its anticontractile effects in the detrusor. Here, we assessed concentration-dependent mirabegron effects on contractions of human detrusor tissues in frequency-response curves and concentration-response curves for different cholinergic and noncholinergic agonists. Detrusor tissues were sampled from patients undergoing radical cystectomy. Contractions were induced by electric field stimulation (EFS) and by cumulative concentrations of cholinergic agonists, endothelin-1, and the thromboxane A2 analog U46619. EFS-induced contractions were inhibited using 10 µM mirabegron, but not using 1 µM. Inhibition by 10 µM mirabegron was resistant to the ß 3-adrenergic antagonist L-748,337. Concentration-dependent contractions by carbachol were not inhibited by 1 µM or 10 µM mirabegron. Concentration-response curves for methacholine were slightly right-shifted by 10 µM, but not 1 µM mirabegron. Concentration-dependent contractions by endothelin-1 or U46619 were not changed by mirabegron. In contrast, the muscarinic antagonist tolterodine right-shifted concentration-response curves for carbachol and methacholine and inhibited EFS-induced contractions. In conclusion, inhibition of neurogenic contractions in isolated detrusor tissues by mirabegron requires concentrations highly exceeding known plasma levels during standard dosing and the known binding constant (Ki values) for ß 3-adrenoceptors. Full contractions by cholinergic agonists, endothelin-1, and U46619 are not affected by therapeutic concentrations of mirabegron. Improvements of storage symptoms are most likely not imparted by inhibition of ß 3-adrenoceptors in the bladder wall itself. SIGNIFICANCE STATEMENT: Mirabegron is used for overactive bladder (OAB) treatment, but the underlying mechanisms are unclear, and preclinical and clinical findings are controversial due to limitations in available studies. Our findings suggest that inhibition of detrusor contractions by mirabegron is limited to neurogenic contractions, which requires unphysiologic concentrations and does not involve ß 3-adrenoceptors. Mechanisms accounting for improvements of OAB by mirabegron are located outside the urinary bladder.

ALOX12 mutation in a family with dominantly inherited bleeding diathesis.

The arachidonic acid (AA) cascade plays a significant role in platelet aggregation. AA released from membrane phospholipids is metabolized by cyclooxygenase (COX) pathway to thromboxane A2 (TXA2) or by 12S-lipoxygenase (ALOX12) to 12-hydroperoxyeicosatetraenoic acid (12-HPETE). In contrast to a well-known role of the COX pathway in platelet aggregation, the role of ALOX12 is not well understood. Platelets of ALOX12-deficient mice exhibit increased sensitivity for ADP-induced aggregation. However, recent evidence strongly suggests a significant role of ALOX12 in platelet aggregation and calcium signaling. 12-HPETE potentiates thrombin- and thromboxane-induced platelet aggregation, and calcium signaling. Inhibition experiments of ALOX12 demonstrated decreased platelet aggregation and calcium signaling in stimulated platelets. We studied a family with a dominantly inherited bleeding diathesis using next-generation sequencing analysis. Platelet aggregation studies revealed that the proband's platelets had defective aggregation responses to ADP, TXA2 mimetic U46619, collagen, and AA, normal affinity of TXA2 receptor for U46619, and normal induction of GTPase activity upon stimulation with U46619. However, the production of inositol 1,4,5-triphosphate (IP3) was only increased up to 30% of the control upon U46619 stimulation, suggesting a defect in phospholipase C-ß2 (PLCB2) activation downstream from TXA2 receptors. Affected family members had no mutation of PLCB2, but had a heterozygous c.1946A > G (p.Tyr649Cys) mutation of ALOX12. ALOX12 activity in platelets from the affected members was decreased to 25-35% of the control. Our data strongly suggested that a heterozygous c.1946A > G ALOX12 mutation was a disease-causing mutation; however, further experiments are required to confirm the pathogenesis of ALOX12 mutation in platelet aggregation.

Biochemical characterization of AeD7L2 and its physiological relevance in blood feeding in the dengue mosquito vector, Aedes aegypti.

Aedes aegypti saliva facilitates blood meal acquisition through pharmacologically active compounds that prevent host hemostasis. Among these salivary proteins are the D7s, which are highly abundant and have been shown to act as scavengers of biogenic amines and eicosanoids. In this work, we performed comparative structural modeling, characterized the binding capabilities, and assessed the physiological functions of the Ae. aegypti salivary protein AeD7L2 compared to the well-characterized AeD7L1. AeD7L1 and AeD7L2 show different binding affinities to several biogenic amines and biolipids involved in host hemostasis. Interestingly, AeD7L2 tightly binds U-46619, the stable analog of thromboxane A2 (KD  = 69.4 nm), which is an important platelet aggregation mediator, while AeD7L1 shows no binding. We tested the ability of these proteins to interfere with the three branches of hemostasis: vasoconstriction, platelet aggregation, and blood coagulation. Pressure myography experiments showed these two proteins reversed isolated resistance artery vasoconstriction induced by either norepinephrine or U-46619. These proteins also inhibited platelet aggregation induced by low doses of collagen or U-46619. However, D7 long proteins did not affect blood coagulation. The different ligand specificity and affinities of AeD7L1 and AeD7L2 matched our experimental observations from studying their effects on vasoconstriction and platelet aggregation, which confirm their role in preventing host hemostasis. This work highlights the complex yet highly specific biological activities of mosquito salivary proteins and serves as another example of the sophisticated biology underlying arthropod blood feeding.

Functional investigations of thromboxane synthase (CYP5A1) in lipid bilayers of nanodiscs.

CYP5A1 is a membrane-associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH2 ) into thromboxane A2 (TXA2 ), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER-bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug-metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small-molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.

Liver kinase B1 is required for thromboxane receptor-dependent nuclear factor-κB activation and inflammatory responses.

OBJECTIVE: Thromboxane A2 receptor (TPr) has been reported to trigger vascular inflammation. Nuclear factor κ B (NF-κB) is a known transcription factor. The aims of the present study were to determine the contributions of NF-κB activation to TPr-triggered vascular inflammation and elucidate the mechanism(s) underlying TPr activation of NF-κB. APPROACH AND RESULTS: The effects of TPr activators, [1S-[1 alpha,2 alpha(Z),3beta(1E,3S*), 4 alpha]]-7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP) and U46619, on NF-κB activation, phosphorylation of rhoA/rho-associated kinases and liver kinase B1, cell adhesion and migration, proliferation, and endothelium-dependent vasorelaxation were assayed in cultured human umbilical vein endothelial cells, human monocytes, or isolated mouse aortas. Exposure of human umbilical vein endothelial cells to TPr agonists I-BOP and U46619 induced dose-dependent and time-dependent phosphorylation of inhibitor of κB α in parallel with aberrant expression of inflammatory markers cyclooxygenase-2, inducible nitric oxide synthase, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Inhibition of NF-κB by pharmacological or genetic means abolished TPr-triggered expression of inflammatory markers. Consistently, exposure of human umbilical vein endothelial cells to either I-BOP or U46619 significantly increased phosphorylation of inhibitor of κB α, I kappaB kinase, rhoA, rho-associated kinases, and liver kinase B1. Pretreatment of human umbilical vein endothelial cells with the TPr antagonist SQ29548 or rho-associated kinases inhibitor Y27632 or silencing of the LKB1 blocked TPr-enhanced phosphorylation of inhibitor of κB α and its upstream kinase, I kappaB kinase. Finally, exposure of isolated mouse aortas to either U46619 or I-BOP enhanced NF-κB activation and vascular inflammation in parallel with reduced endothelium-dependent relaxation in intact vessels. CONCLUSIONS: TPr stimulation instigates aberrant inflammation and endothelial dysfunction via rho-associated kinases/liver kinase B1/I kappaB kinase-dependent NF-κB activation in vascular endothelial cells.

Site-directed mutations and the polymorphic variant Ala160Thr in the human thromboxane receptor uncover a structural role for transmembrane helix 4.

The human thromboxane A2 receptor (TP), belongs to the prostanoid subfamily of Class A GPCRs and mediates vasoconstriction and promotes thrombosis on binding to thromboxane (TXA2). In Class A GPCRs, transmembrane (TM) helix 4 appears to be a hot spot for non-synonymous single nucleotide polymorphic (nsSNP) variants. Interestingly, A160T is a novel nsSNP variant with unknown structure and function. Additionally, within this helix in TP, Ala160(4.53) is highly conserved as is Gly164(4.57). Here we target Ala160(4.53) and Gly164(4.57) in the TP for detailed structure-function analysis. Amino acid replacements with smaller residues, A160S and G164A mutants, were tolerated, while bulkier beta-branched replacements, A160T and A160V showed a significant decrease in receptor expression (Bmax). The nsSNP variant A160T displayed significant agonist-independent activity (constitutive activity). Guided by molecular modeling, a series of compensatory mutations were made on TM3, in order to accommodate the bulkier replacements on TM4. The A160V/F115A double mutant showed a moderate increase in expression level compared to either A160V or F115A single mutants. Thermal activity assays showed decrease in receptor stability in the order, wild type>A160S>A160V>A160T>G164A, with G164A being the least stable. Our study reveals that Ala160(4.53) and Gly164(4.57) in the TP play critical structural roles in packing of TM3 and TM4 helices. Naturally occurring mutations in conjunction with site-directed replacements can serve as powerful tools in assessing the importance of regional helix-helix interactions.

BM-573 inhibits the development of early atherosclerotic lesions in Apo E deficient mice by blocking TP receptors and thromboxane synthase.

Atherosclerosis is the principal cause of mortality in industrialized countries. Its development is influenced by several mediators of which thromboxane A(2) (TXA(2)) and 8-iso-PGF(2α) have recently received a lot of attention. This study aimed to investigate the effect of a dual thromboxane synthase inhibitor and thromboxane receptor antagonist (BM-573) and ASA on lesion formation in apolipoprotein E-deficient mice. The combination of ASA and BM-573 was also studied. Plasma measurements demonstrated that the treatments did not affect body weight or plasma cholesterol levels. BM-573, but not ASA, significantly decreased atherogenic lesions as demonstrated by macroscopic analysis. Both treatments alone inhibited TXB(2) synthesis but only BM-573 and the combination therapy were able to decrease firstly, plasma levels of soluble intracellular adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) and secondly, the expression of these proteins in the aortic root of Apo E. These results were confirmed in endothelial cell cultures derived from human saphenous vein endothelial cells (HSVECs). In these cells, BM-573 also prevented the increased mRNA expression of ICAM-1 and VCAM-1 induced by U-46619 and 8-iso-PGF(2α). Our results show that a molecule combining receptor antagonism and thromboxane synthase inhibition is more efficient in delaying atherosclerosis in Apo E(-/-) mice than sole inhibition of TXA(2) formation.

The function and three-dimensional structure of a thromboxane A2/cysteinyl leukotriene-binding protein from the saliva of a mosquito vector of the malaria parasite.

The highly expressed D7 protein family of mosquito saliva has previously been shown to act as an anti-inflammatory mediator by binding host biogenic amines and cysteinyl leukotrienes (CysLTs). In this study we demonstrate that AnSt-D7L1, a two-domain member of this group from Anopheles stephensi, retains the CysLT binding function seen in the homolog AeD7 from Aedes aegypti but has lost the ability to bind biogenic amines. Unlike any previously characterized members of the D7 family, AnSt-D7L1 has acquired the important function of binding thromboxane A(2) (TXA(2)) and its analogs with high affinity. When administered to tissue preparations, AnSt-D7L1 abrogated Leukotriene C(4) (LTC(4))-induced contraction of guinea pig ileum and contraction of rat aorta by the TXA(2) analog U46619. The protein also inhibited platelet aggregation induced by both collagen and U46619 when administered to stirred platelets. The crystal structure of AnSt-D7L1 contains two OBP-like domains and has a structure similar to AeD7. In AnSt-D7L1, the binding pocket of the C-terminal domain has been rearranged relative to AeD7, making the protein unable to bind biogenic amines. Structures of the ligand complexes show that CysLTs and TXA(2) analogs both bind in the same hydrophobic pocket of the N-terminal domain. The TXA(2) analog U46619 is stabilized by hydrogen bonding interactions of the ω-5 hydroxyl group with the phenolic hydroxyl group of Tyr 52. LTC(4) and occupies a very similar position to LTE(4) in the previously determined structure of its complex with AeD7. As yet, it is not known what, if any, new function has been acquired by the rearranged C-terminal domain. This article presents, to our knowledge, the first structural characterization of a protein from mosquito saliva that inhibits collagen mediated platelet activation.

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery.

BACKGROUND AND PURPOSE: Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery. EXPERIMENTAL APPROACH: Segments of the porcine coronary artery were prepared for either isometric tension recording or determination of cyclic GMP content. The effect of quercetin and metabolites on submaximal responses to U46619 was examined in the presence and absence of substance P, bradykinin, forskolin, sodium nitroprusside (SNP) and glyceryl trinitrate (GTN). KEY RESULTS: Quercetin and quercetin 3'-sulphate inhibited endothelin and U46619-induced contractions with greater potency (three- to fivefold) against the former, while quercetin 3-glucoronide was inactive. Quercetin enhanced both the cyclic GMP content of the artery (threefold) and cyclic GMP-dependent relaxations to GTN and SNP (two to threefold), but forskolin-induced relaxations were unaffected. Although the effect of quercetin was qualitatively similar to that noted for UK-114,542, a selective inhibitor of phosphodiesterase 5, it was still evident against SNP-induced relaxations in the presence of 10 nM UK-114,542. Quercetin and quercetin 3'-sulphate significantly reduced the development of GTN-associated 'tolerance'. CONCLUSIONS AND IMPLICATIONS: Quercetin and quercetin 3'-sulphate inhibited receptor-mediated contractions of the porcine isolated coronary artery by an endothelium-independent action. Quercetin selectively enhanced cyclic-GMP-dependent relaxations by a mechanism not involving phosphodiesterase 5 inhibition. In addition, quercetin and quercetin 3'-sulphate opposed GTN-induced tolerance in vitro, which may be beneficial for patients treated for angina pectoris.

An increased regional blood flow precedes mesenteric inflammation in rats treated by a phosphodiesterase 4 inhibitor.

The study was undertaken to assess the hemodynamic effects induced by a single dose of the phosphodiesterase 4 (PDE4) inhibitor, CI-1044, which is known to cause mesenteric vascular alterations in rats. In the present study, an administration of 160 mg/kg of CI-1044 caused perivascular and interstitial inflammation, with infiltrates of admixed neutrophils and macrophages but without evidence of vascular necrosis (ileum, 15/20 rats; duodenum + jejunum, 7/20 rats). Four hours after administration, blood pressure was decreased (- 13%). A fluorescent microsphere technique demonstrated that, in these conditions, cardiac output was doubled (+ 100%) and total peripheral resistance was decreased (- 54%). The largest increases in blood flow were measured in the duodenum (+ 101%), in the jejunum (+ 110%), and in the ileum (+ 192%). Therefore, the mesentery was the most sensitive organ affected by the drug and, within this area, parts with the highest incidence of vascular alteration were those which had shown the highest increase in flow. In addition, isolated precontracted mesenteric resistance arteries dissected from untreated animals were fully relaxed when incubated with increasing concentrations of CI-1044 up to 2.5 x 10(-5)M. At this latter concentration, contractile abilities and sensitivities to the physiological agonist noradrenaline (NA) and to the thromboxane analogue U46619 were significantly attenuated (- 28 and - 27%, respectively). This effect could lead to a decreased response to NA and possibly to other agonists in vivo consistent with the vasodilation observed with the microsphere technique. These data provide evidence that the PDE4 inhibitor CI-1044 induces changes of vascular tone that could lead to histological alterations in the mesenteric area.

Effects of platelet-activating factor and thromboxane A2 on isolated perfused guinea pig liver.

Lipid mediators, thromboxane A2 (TxA2) and platelet-activating factor (PAF), are potent vasoconstrictors, and have been implicated as mediators of liver diseases, such as ischemic-reperfusion injury. We determined the effects of a TxA2 analogue (U-46619) and PAF on the vascular resistance distribution and liver weight (wt) in isolated guinea pig livers perfused with blood via the portal vein. The sinusoidal pressure was measured by the double occlusion pressure (P(do)), and was used to determine the pre- (R(pre)) and post-sinusoidal (R(post)) resistances. U-46619 and PAF concentration-dependently increased the hepatic total vascular resistance (R(t)). The minimum concentration at which significant vasoconstriction occurs was 0.001 microM for PAF and 0.1 microM for U-46619. Moreover, the concentration of U-46619 required to increase R(t) to the same magnitude is 100 times higher than PAF. Thus, the responsiveness to PAF was greater than that to U-46619. Both agents increased predominantly R(pre) over R(post). U-46619 caused a sustained liver weight loss. In contrast, PAF also caused liver weight loss at lower concentrations, but it produced liver weight gain at higher concentrations (2.5 +/- 0.3 per 10g liver weight at 1 microM PAF), which was caused by substantial post-sinusoidal constriction and increased P(do). In conclusion, both TxA2 and PAF contract predominantly the pre-sinusoidal veins. TxA2 causes liver weight loss, while PAF at high concentrations increases liver weight due to substantial post-sinusoidal constriction in isolated guinea pig livers.