Thromboxane A2 Mimetic U46619 Research Articles

Abstract 4013: The positive role of TPγ in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells

Abstract Many studies have shown that 4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK), the most potent carcinogen present in tobacco smoke, can induce lung tumor growth. We previously demonstrated that the activation of extracellular regulated protein kinases (ERK) and cAMP response element-binding protein (CREB) is responsible for the pro-survival and proliferative effects of thromboxane A2 (TxA2) and its receptor (TP) in the human lung cancer cells stimulated with NNK (reported in oncogene journal). In current study, we further determined the role of cyclooxygenase(COX)-2/TxA2/TP pathway in lung cancer cell growth mediated by NNK. TP exists as two isoforms, ≤ and β, in human beings. Western blotting showed that TPα, but not TPα, was widely expressed in a series of human lung cells. NCI-H23 and CRL-2066, which express both TPγ and TPα, were selected as models in this study. COX-2 inhibitor NS398 could significantly reduce NNK-stimulated TxA2 synthesis. Moreover, NS398 and BM567, an agent combining TxA2 synthase inhibition and TP antagonism, had the similar effects to reverse NNK-induced activation of ERK and CREB and the increase in cell growth. Furthermore, the administration of TxA2 mimetic U46619 could almost reconstituted NNK-induced ERK and CREB activation in the presence of COX-2-siRNA. Collectively, these findings suggest that in lung cancer cells stimulated with NNK, TxA2 is mainly derived from COX-2 and acts as a key mediator for tumor-promoting effects of COX-2. Interestingly, TP antagonist SQ29548 could inhibit NNK-induced COX-2 protein expression and TxA2 production, indicating that TP is able to modulate NNK-induced COX-2 expression and activity, thereby constituting an auto-amplification mechanism of TxA2 synthesis in lung cancer cells stimulated with NNK. We further examined which TP isoform contributes to the NNK effects. We observed that the protein expression of TPα, but not TPα, was time-dependently increased by NNK. Moreover, the activation of ERK and CREB and the production of COX-2 and TxA2 by NNK were potentiated in cells transfected with pcDNA3-TPγ as compared with cells transfected with control vector or pcDNA3-TPα, and such effects could be reversed by SQ29548. However, in cells transfected with pcDNA3-TPα, the COX-2, TxA2, phospho-ERK and phospho-CREB levels were similar to that seen in the cells transfected with control vector, and neither NNK nor SQ29548 had any additional effects when compared to the control cells. Taken together, these data strongly suggest that TPγ rather than TPγ is, at least in part, responsible for the induction of COX-2, TxA2 and cell growth by NNK in lung cancer cells. Our study implies that targeting TxA2 and its receptor TPγ may represent a promising strategy for prevention of smoking-associated lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4013. doi:1538-7445.AM2012-4013

Angiotensin II receptor antagonist reduces subsequent uterine arterial dysfunction in pregnant offspring of protein-restricted rat dams

A low-protein diet (LPD) during pregnancy induces vascular dysfunction and hypertension in the offspring, prevented by administration of an angiotensin II type 1 (AT(1)) receptor antagonist in early life to the offspring. Whether such protection extends to subsequent pregnancy is unknown; we therefore hypothesized that administration of a specific AT(1) receptor antagonist (losartan) in early life to offspring of LPD dams would improve vascular dysfunction in their uterine arteries when they, in turn, were pregnant. Pregnant rats were randomly divided into two dietary groups fed a control (C) or protein-restricted (R) diet throughout pregnancy. Between two and 10 weeks postnatally, female offspring (F(1)) were randomly assigned to drink either pure tap water (CO, RO) or water with losartan (CL, RL). Offspring were mated and killed on gestational day 19 or 20 in order to investigate uterine artery function. In pregnant offspring, vasoconstriction of the uterine arteries to phenylephrine (PE) and the thromboxane A2 mimetic U46619 was greater in RO than CO (F(1)). Responses to both antagonists were suppressed in RL (F(1)). Relaxation to sodium nitroprusside was increased in RO versus CO and suppressed in RL versus RO (F(1)). Administration of an AT(1) receptor antagonist to offspring during the suckling and juvenile period improves the uterine vascular dysfunction in pregnancy induced by prior maternal LPD during their development. Such treatment may contribute to decreasing the transmitted risks of maternal malnutrition from offspring to the subsequent generation.

Determination of efficacy of linotroban by inducing a reduction of renal inulin/para-aminohippuric acid clearances with the thromboxane A2 mimetic U-46619 in the conscious female rat.

Linotroban (CAS 141443-73-4), a potent and selective thromboxane (TXA2) receptor antagonist, is known as a novel antithrombotic agent. It is suggested that pharmacological inhibition of TXA2 synthesis or action merits continued evaluation in the treatment of a variety of renal diseases. The aim of this study was the determination of efficacy of this new TXA2 receptor antagonist by assessing its effect on the reduction in inulin and para-aminohippuric acid (PAH) clearances induced by the TX/prostaglandin endoperoxide mimetic U-46619 in the conscious female rats. The following doses (3, 10, 30 mg/kg/24 h) of linotroban mixed with 720 micrograms TX-mimetic U-46619/kg/24 h were administered via osmotic pumps at a delivery rate of 10 microliters/h, implanted s.c. during 72 h. Rats of the U-46619 group were administered 720 micrograms U-46619/kg/24 h alone as described above, controls received 3.5% NaHCO3, respectively. Inulin/PAH clearances were determined at the end of the 4-h clearance period (68 h-72 h). Summarizing the data, glomerular filtration rate (GFR) and PAH clearances were reduced significantly by U-46619. When linotroban (3, 10 or 30 mg/kg/24 h) was added to U-46619 the GFR and PAH clearance were reversed to values that showed no significant differences to the controls.

Thromboxane and isoprostane share the same prostanoid receptors to increase human placental tone

As the placenta is devoid of autonomic innervation, umbilical-placental vascular tone should be under the control of tissue and humoral factors. Among the numerous stimuli capable of challenging the placental circulation, we propose that prostanoids could be responsible for the regulation of placental vascular tone. Consequently, we measured vasomotor responses to the thromboxane A2 (TXA2) mimetic U-46619 and the isoprostane 8-iso-prostaglandin E2 (8-isoPGE2) in the human placental vasculature. Placental tissues were collected from normotensive women after elective caesarean delivery. Cotyledons were set up in a perfusion system, whereas chorionic arteries were prepared as rings and installed in glass-jacketed tissue baths. The effects of U-46619 and 8-isoPGE2 were measured in the absence and presence of blockers of TXA2 receptors (TP), SQ29,548 and ICI192,605, and of PGE2 receptors (EP), AH6809. The influence of nitric oxide (NO) was assessed with NG-nitro-L-arginine methyl ester (L-NAME). U-46619 and 8-isoPGE2 markedly increased perfusion pressure in cotyledons and tension in chorionic arteries. Dose-response curves to both prostanoids were competitively shifted to the right by all antagonists, but to different extents. L-NAME had no significant impact on the dose-response curves to U-46619. The effects of U-46619 and 8-isoPGE2 were found to be mediated by both TP and EP. The presence of these receptors and the actions exerted by their agonists support our postulate that prostanoids play an important regulatory role in placental vascular tone and resistance. NO, however, does not seem to be involved.

Expansion of the Human Adipose-Derived Stromal Vascular Cell Fraction Yields a Population of Smooth Muscle-Like Cells with Markedly Distinct Phenotypic and Functional Properties Relative to Mesenchymal Stem Cells

Adipose tissue contains a heterogeneous cell population composed of endothelial cells, adipocytes, smooth muscle cells (SMC), and mesenchymal progenitors and stromal cells that meet the criteria put forth by the International Society for Cellular Therapy as defining mesenchymal stem cells (MSC). In this study, we expanded the stromal vascular fraction (SVF) of human adipose tissue and characterized the resulting adherent primary cell cultures by quantitative reverse transcription-polymerase chain reaction, antigen expression, protein fingerprinting, growth kinetics, in vitro tri-lineage differentiation bioactivity, and functional responses to small molecules modulating SMC-related developmental pathways and compared the results to those obtained with functionally validated MSC cultures. SVF-derived initial cultures (P0) were expanded in a defined medium that was not optimized for MSC growth conditions, neither were recombinant cytokines or growth factors added to the media to direct differentiation. The adherent cell cultures derived from SVF expansion under these conditions had markedly distinct phenotypic and biological properties relative to functionally validated MSC cultures. SVF-derived adherent cell cultures retained characteristics consistent with the SMC subpopulation within adipose tissue--phenotype, gene, and protein expression--that were independent of passage number and source of SVF (n=4 independent donors). SVF-derived cells presented significantly less robust in vitro tri-lineage differentiation bioactivity relative to validated MSC. Expanded SVF cells and MSC had opposite responses to the thromboxane A2 mimetic U46619, demonstrating an unambiguous functional distinction between the two cell types. Taken together, these data support the conclusions that SVF cells expanded under the conditions described in these studies are accurately described as adipose-derived SMC and represent a cellular subpopulation of adipose SVF that is separate and distinct from other classes of adipose-derived cells.

Mode of action of P2Y12 antagonists as inhibitors of platelet function

P2Y(12) receptor antagonists are antithrombotic agents that inhibit platelet function by blocking the effects of adenosine diphosphate (ADP) at P2Y (12)receptors. However, some P2Y(12) receptor antagonists may affect platelet function through additional mechanisms. It was the objective of this study to investigate the possibility that P2Y(12) antagonists inhibit platelet function through interaction with G-protein-coupled receptors other than P2Y(12) receptors. We compared the effects of cangrelor, ticagrelor and the prasugrel active metabolite on platelet aggregation and on phosphorylation of vasodilator-stimulated phosphoprotein (VASP). We compared their effects with those of selective IP, EP4 and A2A agonists, which act at Gs-coupled receptors. All three P2Y(12) antagonists were strong inhibitors of ADP-induced platelet aggregation but only partial inhibitors of aggregation induced by thrombin receptor activating peptide (TRAP) or the thromboxane A2 mimetic U46619. Further, after removing ADP and its metabolites using apyrase and adenosine deaminase, the P2Y(12) antagonists produced only minor additional inhibition of TRAP or U46619-induced aggregation. Conversely, the Gs-coupled receptor agonists always produced strong inhibition of aggregation irrespective of whether ADP was removed. Other experiments using selective receptor agonists and antagonists provided no evidence of any of the P2Y(12) antagonists acting through PAR1, TP, IP, EP4, A2A or EP3 receptors. All three P2Y (12)antagonists enhanced VASP-phosphorylation to a small and equal extent but the effects were much smaller than those of the IP, EP4 and A2A agonists. The effects of cangrelor, ticagrelor and prasugrel on platelet function are mediated mainly through P2Y(12)receptors and not through another G-protein-coupled receptor.

Previous gestational diabetes impairs long-term endothelial function in a mouse model of complicated pregnancy

Women who develop gestational diabetes mellitis (GDM) display endothelial dysfunction up to 1 yr after pregnancy, despite a return to normoglycemia. It is unknown whether this dysfunction was preexisting or whether GDM pregnancy leads to long-term endothelial dysfunction. A mouse model that spontaneously develops GDM (Lepr(db/+)) was used to determine whether the endothelial dysfunction that develops during GDM is evident in later life. Heterozygous and wild-type (WT) controls were allowed to litter once, then age to 9-10 mo, and were compared with virgin controls. Vascular function of small mesenteric arteries was assessed using wire myography. Concentration response curves to the thromboxane A(2)mimetic U46619 and the endothelium-dependent vasodilator methacholine were constructed. Superoxide production and peroxynitrite formation was also measured. Mice with previous GDM displayed blood glucose concentrations similar to previously pregnant WT mice (8.0 +/- 0.1 vs. 7.1 +/- 0.3 mmol/l, P > 0.05). Arteries from mice with previous GDM displayed increased sensitivity to U46619 (EC(50) 5.2 +/- 0.7 vs. 45.2 +/- 1.0 nmol/l, P < 0.01) and impaired endothelium-dependent relaxation compared with WT controls (29 +/- 8 vs. 58 +/- 16 percent relaxation, P < 0.05). This was associated with increased superoxide production (93.3 +/- 2.3 vs. 64.6 +/- 1.6 mean fluorescence intensity, P < 0.001) and increased peroxynitrite formation (173.5 +/- 11.0 vs. 57.4 +/- 16.2 mean fluorescence intensity, P < 0.01) compared with virgin controls. In summary, endothelial dysfunction was evident in mice with previous GDM compared with previously healthy pregnant mice or virgin controls. These data suggest that GDM affects endothelial function and may contribute to an increased risk of cardiovascular disease.

The role of prostanoid receptors in mediating the effects of PGE2 on human platelet function

The effects of prostaglandin E2 (PGE2) on platelet function are believed to be the result of opposing mechanisms that lead to both enhancement and inhibition of platelet function. Enhancement of platelet function is known to be via EP3 receptors linked to Gi and inhibition of adenylyl cyclase. However, the receptors involved in inhibition of platelet function have not been fully defined. Here we have used measurements of platelet aggregation, calcium signaling and P-selectin expression to assess platelet function induced by platelet activating factor (PAF), thrombin receptor activating peptide (TRAP-6) and the thromboxane A2 mimetic U46619 respectively, to determine the effects of PGE2 and of selective prostanoid receptor agonists on platelet function. Their effects on vasodilator-stimulated phosphoprotein (VASP) phosphorylation were also determined. We also assessed the ability of selective prostanoid receptor antagonists to modify the effects of PGE2. The agonists and antagonists used were iloprost (IP agonist), ONO-DI-004 (EP1 agonist), ONO-AE1-259 (EP2 agonist), sulprostone (EP3 agonist), ONO-AE1-329 (EP4 agonist), CAY10441 (IP antagonist), ONO-8713 (EP1 antagonist), DG-041 (EP3 antagonist) and ONO-AE3-208 (EP4 antagonist). Using the agonists available to us we demonstrated that EP3, EP4 and IP receptors elicit functional responses in platelets. The EP3 receptor agonist promoted platelet aggregation, calcium signaling and P-selectin expression and this was associated with a reduction in VASP phosphorylation. Conversely agonists acting at IP and EP4 receptors inhibited platelet function and this was associated with an increase in VASP phosphorylation. The effects on platelet function and VASP phosphorylation of the selective prostanoid receptor antagonists used in conjunction with PGE2 were consistent with PGE2 interacting with EP3 receptors to enhance platelet function and with EP4 receptors (but not IP receptors) to inhibit platelet function. This is the first demonstration of the involvement of EP4 receptors in platelet responses to PGE2.

Role of Rho-kinase in mediating contraction of chicken embryo femoral arteries

Rho-kinase-dependent Ca2+ sensitization is an essential process for contraction of mammalian vascular smooth muscle but the information about its effects in non-mammalian vessels is scarce. We aimed to investigate, using the Rho-kinase inhibitor hydroxyfasudil, the potential role of the Rho-kinase pathway of Ca2+ sensitization in depolarization- and agonist-mediated contraction of chicken embryo (at day 19 of the 21 days of incubation) femoral arteries. Contraction elicited by KCl (125 mM) comprised two phases (phasic and tonic contraction), both of which were abolished in the absence of extracellular Ca2+. Hydroxyfasudil (10 μM) left the initial phasic component nearly intact but abolished the tonic component. Hydroxyfasudil also induced a marked impairment of the contractions elicited by phenylephrine (PE), the thromboxane A2 mimetic U46619, and endothelin-1. In contrast, inhibition of protein kinase C (PKC) by chelerythrine did not affect KCl- or PE-induced contractions, indicating lack of participation of PKC-mediated Ca2+ sensitization. Incubation under chronic hypoxia (15% O2 from day 0) impaired embryonic growth but did not significantly affect hydroxyfasudil-mediated relaxation. In summary, our findings are indicative of a role for Rho-kinase activity in depolarization- and agonist-induced force generation in chicken embryo femoral arteries.

REGIONAL DIFFERENCES IN THE MODULATORY ROLE OF THE EPITHELIUM IN SHEEP AIRWAY

1. The airway epithelium may modulate smooth muscle responsiveness via the release of biologically active substances, such as nitric oxide (NO) and prostaglandins. Based on regional differences in structure and function described for the airway epithelium, we performed a comparative study on the responsiveness of sheep isolated, epithelium-intact or -denuded, first- to fourth-order bronchi to acetylcholine (ACh). 2. We performed contractility studies using KCl or cholinergic stimuli in the presence or absence of NO or prostaglandin-related drugs in epithelium-intact and epithelium-denuded bronchial strips obtained from all four airway regions. We also studied the expression of NO synthase (NOS), using the NADPH-diaphorase staining technique, and the effect of airway epithelium removal on the synthesis of NO metabolites in the different bronchi orders. 3. There was no difference in the response of first- to fourth-order epithelium-intact bronchi to ACh (1 nmol/L-100 mmol/L) or KCl (5-100 mmol/L). Removal of the epithelium had no effect on ACh-induced contractions of first- and second-order bronchi, but increased responses of third- and fourth-order bronchi to ACh. The NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) increased ACh-induced contractions of fourth-order epithelium-intact bronchi only. The NO donor sodium nitroprusside (1 nmol/L-1 mmol/L) equally relaxed 1 micromol/L carbachol-precontracted epithelium-denuded first- and fourth-order bronchi. 4. Although NAPDH-diaphorase staining demonstrated no regional differences in NOS expression, basal levels of NO metabolites were 4.5-fold greater in fourth- compared with second-order epithelium-intact bronchi. 5. The cyclo-oxygenase inhibitor indomethacin (10 micromol/L) had no effect on ACh-induced contractions of first- to fourth-order epithelium-intact bronchi, but decreased responses of fourth-order epithelium-denuded bronchi to ACh. The contractile effect of the thromboxane A(2) mimetic U-46619 (1 nmol/L-10 micromol/L) was greater in fourth- compared with first-order epithelium-denuded bronchi. 6. In conclusion, the sheep airway epithelium exhibits regional differences in its modulatory role and this is particularly apparent in small bronchi.

Endotoxin Induced Chorioamnionitis Prevents Intestinal Development during Gestation in Fetal Sheep

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis.Chorioamnionitis was induced at different gestational ages (GA). Animals were sacrificed at low GA after 2d or 14d exposure to chorioamnionitis. Long term effects of 30d exposure to chorioamnionitis were studied in near term animals after induction of chorioamnionitis. The cellular distribution of tight junction protein ZO-1 was shown to be underdeveloped at low GA whereas endotoxin induced chorioamnionitis prevented the maturation of tight junctions during later gestation. Endotoxin induced chorioamnionitis did not induce an early (2d) inflammatory response in the gut in preterm animals. However, 14d after endotoxin administration preterm animals had increased numbers of T-lymphocytes, myeloperoxidase-positive cells and gammadelta T-cells which lasted till 30d after induction of chorioamnionitis in then near term animals. At early GA, low intestinal TLR-4 and MD-2 mRNA levels were detected which were further down regulated during endotoxin-induced chorioamnionitis. Predisposition to organ injury by ischemia was assessed by the vascular function of third-generation mesenteric arteries. Endotoxin-exposed animals of low GA had increased contractile response to the thromboxane A2 mimetic U46619 and reduced endothelium-dependent relaxation in responses to acetylcholine. The administration of a nitric oxide (NO) donor completely restored endothelial dysfunction suggesting reduced NO bioavailability which was not due to low expression of endothelial nitric oxide synthase.Our results indicate that the distribution of the tight junctional protein ZO-1, the immune defence and vascular function are immature at low GA and are further compromised by endotoxin-induced chorioamnionitis. This study suggests that both prematurity and inflammation in utero disturb fetal gut development, potentially predisposing to postnatal intestinal pathology.

Thromboxane A2-induced Bi-directional Regulation of Cerebral Arterial Tone

Myosin light chain phosphatase plays a critical role in modulating smooth muscle contraction in response to a variety of physiologic stimuli. A downstream target of the RhoA/Rho-kinase and nitric oxide (NO)/cGMP/cyclic GMP-dependent kinase (cGKI) pathways, myosin light chain phosphatase activity reflects the sum of both calcium sensitization and desensitization pathways through phosphorylation and dephosphorylation of the myosin phosphatase targeting subunit (MYPT1). As cerebral blood flow is highly spatio-temporally modulated under normal physiologic conditions, severe perturbations in normal cerebral blood flow, such as in cerebral vasospasm, can induce neurological deficits. In nonpermeabilized cerebral vessels stimulated with U-46619, a stable mimetic of endogenous thromboxane A2 implicated in the etiology of cerebral vasospasm, we observed significant increases in contractile force, RhoA activation, regulatory light chain phosphorylation, as well as phosphorylation of MYPT1 at Thr-696, Thr-853, and surprisingly Ser-695. Inhibition of nitric oxide signaling completely abrogated basal MYPT1 Ser-695 phosphorylation and significantly increased and potentiated U-46619-induced MYPT1 Thr-853 phosphorylation and contractile force, indicating that NO/cGMP/cGKI signaling maintains basal vascular tone through active inhibition of calcium sensitization. Surprisingly, a fall in Ser-695 phosphorylation did not result in an increase in phosphorylation of the Thr-696 site. Although activation of cGKI with exogenous cyclic nucleotides inhibited thromboxane A2-induced MYPT1 membrane association, RhoA activation, contractile force, and regulatory light chain phosphorylation, the anticipated decreases in MYPT1 phosphorylation at Thr-696/Thr-853 were not observed, indicating that the vasorelaxant effects of cGKI are not through dephosphorylation of MYPT1. Thus, thromboxane A2 signaling within the intact cerebral vasculature induces "buffered" vasoconstrictions, in which both the RhoA/Rho-kinase calcium-sensitizing and the NO/cGMP/cGKI calcium-desensitizing pathways are activated.

Thromboxane A2modulates migration, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A(2) (Tx(2)) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of alpha-smooth muscle actin (alpha-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and alpha-SMA expression. These results suggest that TxA2 plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.

N-Methyl-D-Aspartate (NMDA) Antagonists—S(+)-ketamine, Dextrorphan, and Dextromethorphan—Act as Calcium Antagonists on Bovine Cerebral Arteries

Ketamine, an intravenous anesthetic and a major drug of abuse, is a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist. Ketamine's enantiomer, S(+)-ketamine, acts stereoselectively on neuronal NMDA receptors. The purpose of this in vitro study was to compare the direct effects of S(+)-ketamine, 2 other noncompetitive NMDA receptor antagonists (dextrorphan and dextromethorphan), and the calcium entry blocker nimodipine on the cerebral vasculature, using bovine middle cerebral arteries as an experimental model. Arterial rings were mounted in isolated tissue chambers equipped with isometric tension transducers to obtain pharmacologic dose-response curves. In the absence of exogenous vasoconstrictors, the NMDA antagonists or nimodipine had negligible effects on cerebral arterial tone. When rings were preconstricted with either potassium or the stable thromboxane A2 mimetic U46619, the NMDA antagonists and nimodipine each produced dose-dependent relaxation. Prior endothelial stripping had no effect on subsequent drug-induced relaxation of K+-constricted rings. In Ca2+-deficient media containing either potassium or U46619, the NMDA antagonists and nimodipine each produced competitive inhibition of subsequent Cainduced constriction. In additional experiments, arterial strips were mounted in isolated tissue chambers to directly measure calcium uptake, using 45calcium (45Ca) as a radioactive tracer. The NMDA antagonists and nimodipine each blocked potassium-stimulated or U46619-stimulated Ca2+ uptake into arterial strips. These results indicate that S(+)-ketamine, dextrorphan, and dextromethorphan, like nimodipine, directly dilate cerebral arteries by acting as calcium antagonists; they all inhibit 45Ca uptake through both potential-operated (potassium) and receptor-operated (U46619) channels in cerebrovascular smooth muscle.

Isoprostanes Inhibit Vascular Endothelial Growth Factor–Induced Endothelial Cell Migration, Tube Formation, and Cardiac Vessel Sprouting In Vitro, As Well As Angiogenesis In Vivo via Activation of the Thromboxane A 2 Receptor

Isoprostanes are endogenously formed end products of lipid peroxidation. Furthermore, they are markers of oxidative stress and independent risk markers of coronary heart disease. In patients experiencing coronary heart disease, impaired angiogenesis may exacerbate insufficient blood supply of ischemic myocardium. We therefore hypothesized that isoprostanes may exert detrimental cardiovascular effects by inhibiting angiogenesis. We studied the effect of isoprostanes on vascular endothelial growth factor (VEGF)-induced migration and tube formation of human endothelial cells (ECs), and cardiac angiogenesis in vitro as well as on VEGF-induced angiogenesis in the chorioallantoic membrane assay in vivo. The isoprostanes 8-iso-PGF(2alpha), 8-iso-PGE(2), and 8-iso-PGA(2) inhibited VEGF-induced migration, tube formation of ECs, and cardiac angiogenesis in vitro, as well as VEGF-induced angiogenesis in vivo via activation of the thromboxane A(2) receptor (TBXA2R): the specific TBXA2R antagonists SQ-29548, BM 567, and ICI 192,605 but not the thromboxane A(2) synthase inhibitor ozagrel blocked the effect of isoprostanes. The isoprostane 8-iso-PGA(2) degraded into 2 biologically active derivatives in vitro, which also inhibited EC tube formation via the TBXA2R. Moreover, short hairpin RNA-mediated knockdown of the TBXA2R antagonized isoprostane-induced effects. In addition, Rho kinase inhibitor Y-27632 reversed the inhibitory effect of isoprostanes and the thromboxane A(2) mimetic U-46619 on EC migration and tube formation. Finally, the various isoprostanes exerted a synergistic inhibitory effect on EC tube formation. We demonstrate for the first time that isoprostanes inhibit angiogenesis via activation of the TBXA2R. By this mechanism, isoprostanes may contribute directly to exacerbation of coronary heart disease and to capillary rarefaction in disease states of increased oxidative stress.

Contractile smooth muscle cells derived from hair-follicle stem cells

We hypothesized that hair-follicle stem cells can differentiate toward smooth contractile muscle cells, providing an autologous cell source for cardiovascular tissue regeneration. Smooth muscle progenitor cells (SMPCs) were obtained from ovine hair follicles using a tissue-specific promoter and fluorescence-activated cell sorting. Hair-follicle smooth muscle progenitor cells (HF-SMPCs) expressed several markers of vascular smooth muscle including alpha-actin, calponin, myosin heavy chain (MHC), caldesmon, smoothelin, and SM22. HF-SMPCs were highly proliferative and showed high clonogenic potential without any signs of chromosomal abnormalities as evidenced by karyotype analysis. HF-SMPCs compacted fibrin hydrogels to a similar extent as vascular smooth muscle cells from ovine umbilical veins (V-SMCs), indicating the development of the force-generating machinery. In addition, cylindrical tissue equivalents prepared with HF-SMPCs displayed significant contractility in response to vasoactive agonists including KCl and the thromboxane A2 mimetic U46619, suggesting that these cells had developed receptor and non-receptor-mediated pathways of contractility. Finally, transforming growth factor-beta1 promoted differentiation of HF-SMPCs toward a mature SMC phenotype as suggested by increased expression of MHC and enhanced matrix compaction. Our results suggest that hair follicles may be an easily accessible, autologous, and rich source of functional SMPC for cardiovascular tissue engineering and regenerative medicine.

The Effect of a Mast Cell Degranulating Agent on Vascular Resistance in the Human Placental Vascular Bed and on the Tone of Isolated Placental Vessels

The objective of this study is to investigate the effect of a mast cell degranulating agent, compound 48/80, on vascular resistance in the perfused human placenta and on the tone of isolated human chorionic vessels. Human placenta was obtained from term nonlaboring women undergoing cesarean delivery. Placental vascular bed perfusion pressure and isometric tension for segments of chorionic plate artery and vein were obtained in response to potassium chloride, compound 48/80, a mast cell stabilizer (cromolyn), and thromboxane A2 mimetic (U46619). Compound 48/80 significantly increased perfusion pressure in isolated human placental cotyledons. This effect was significantly potentiated further after induction of active vascular tone by thromboxane A2 mimetic U46619. Cromolyn significantly attenuated responses to compound 48/80 in these preparations. Compound 48/80 also significantly increased tone in isolated human chorionic artery and vein rings, and responses were abolished by cromolyn. In conclusion, degranulation of placental and intravascular mast cells by compound 48/80 leads to the release of vasoconstrictive substances. This could impair placental blood flow and result in growth restriction in fetuses of women with type l hypersensitivity reactions.

Orchidectomy increases the formation of prostanoids and modulates their role in the acetylcholine-induced relaxation in the rat aorta

This study examines the effect of endogenous male sex hormones on thromboxane A2 (TXA2), prostaglandin (PG) I2, PGF(2 alpha), and PGE I2 release, as well as their role in acetylcholine (ACh)-mediated relaxation in the aorta. Aortic segments from orchidectomized and control male Sprague-Dawley rats were used to measure COX-2 protein expression. ACh-induced relaxation of these segments was also determined in the absence and presence of the COX-2 inhibitor NS-398, the TXA2 synthesis inhibitor furegrelate, the PGI2 synthesis inhibitor tranylcypromine (TCP), or the thromboxane-prostanoid (TP) receptor antagonist SQ-29 548. Furthermore, TXA2, PGI2, PGF(2 alpha), and PGE2 release as well as the vasomotor effect of exogenous TXA2, PGI2, PGF(2 alpha), and PGE2 were measured. COX-2 expression was increased in aortas from orchidectomized rats. NS-398 did not modify the ACh-induced relaxation in arteries from both control or orchidectomized rats. Furegrelate did not modify the ACh-induced relaxation in aortas from control animals but, in aortas from orchidectomized rats, it increased that response. TCP decreased the ACh-induced relaxation in both groups. The TP receptor antagonist, SQ29 548 failed to modify ACh-induced relaxation in aortas from either rat group. Pre-incubating arteries from orchidectomized rats with TCP plus furegrelate did not modify the decrease in the ACh response induced by TCP alone, but this response was restored by co-incubation of TCP plus SQ29 548. ACh-induced TXA2, PGI2, PGF(2 alpha), and PGE2 release were increased by orchidectomy. The presence of furegrelate plus TCP increased the ACh-induced PGE2 release more in arteries from orchidectomized than in those from control rats. The contractile responses induced by the TXA2 mimetic U-46619 or by exogenous PGF(2 alpha) were similar in arteries from control and orchidectomized rats, while those induced by exogenous PGE2 were increased in arteries from orchidectomized rats; the vasodilator response induced by exogenous PGI2 was decreased in arteries from orchidectomized rats. These data show that endogenous male sex hormone deprivation increases COX-2 expression, the release of TXA2, PGI2, PGF(2 alpha), and PGE2 and the contractile response induced by exogenous PGE2 and TXA2, while it decreases the relaxation induced by exogenous PGI2. Despite the predominance of vasoconstrictor prostanoids derived from COX-2 in aortas from orchidectomized rats, the ACh-induced relaxation remains increased.

Abstract 1287: TRPV4-Deficient Mice Exhibit Impaired Endothelium-Dependent Relaxation Induced by Acetylcholine in Vitro and in Vivo

Agonist-induced Ca 2+ entry in endothelial cells is important for the synthesis and release of vasoactive factors, although mechanisms of Ca 2+ entry remain largely unknown. Emerging evidence suggests that the transient receptor potential vanilloid 4 (TRPV4) channel, a Ca 2+ -permeant TRP channel, is expressed in endothelial cells and may be involved in the regulation of vascular tone. Here we investigated the potential role of TRPV4 channels in acetylcholine-induced vasodilation in vitro and in vivo using the TRPV4 knockout (TRPV4 −/− ) mice model. Carotid arteries were isolated and preconstricted with the thromboxane A2 mimetic U46619. Concentration-dependent relaxations to acetylcholine (10 −9 –10 −5 M) were markedly reduced in carotids of TRPV4 −/− vs. wild-type (WT) mice (maximal relaxations of 31±12% vs 53±4%, respectively; n=4 mice). There was no significant change in the ED50 for Ach. In both WT and TRPV4 −/− , acetylcholine-induced relaxations were blocked and converted to constrictions by the NO synthase inhibitor L-NAME (maximal relaxations of −25±6% and −24±7%, respectively). There was no difference in papaverine-induced relaxations between WT and TRPV4 −/− mice (maximal relaxations of 93±3% vs. 90±3%, respectively). U46619 caused similar contractions in carotid arteries from those mice. We also compared in vivo vasodilator effects of acetylcholine by measuring changes in blood pressure in those animals. Intravenous administration of acetylcholine (15 ng/gm bolus) decreased blood pressure by 32±6 mmHg in WT mice (from 90±15 to 57±10 mmHg; n=6), whereas blood pressure was reduced by only 10 mmHg in TRPV4 −/− mice (from 67±6 to 56±4 mmHg; n=12). Acetylcholine caused similar reductions in heart rate in WT and TRPV4 −/− mice, with mean changes of 365±57 and 292±40 beats/min, respectively. We conclude that the endothelium-dependent vasodilator response to acetylcholine is reduced both in vitro and in vivo in TRPV4 −/− mice, and these findings may provide novel insight into the mechanisms of Ca 2+ entry evoked by chemical agonists in endothelial cells. The paradoxically lower baseline blood pressure in TRPV4 −/− mice requires further investigation.

Properties of the Human Umbilical Vein as a Living Scaffold for a Tissue-Engineered Vessel Graft

Umbilical cords are usually discarded after delivery, even though they contain a set of functional vessels. We investigated whether the human umbilical vein (HUV) is suitable as a storable scaffold for the tissue engineering of small-caliber vessel grafts. Isolated HUVs were cryopreserved by freezing or vitrification. The reaction of the vessels to vasoactive compounds and the mechanical properties were determined in an organ bath. Mitochondrial metabolism, release of antithrombotic compounds, and platelet adhesion were measured on the luminal vessel surface. Seeding with endothelial cells was tested on denuded HUVs. The vessels showed a weak response to norepinephrine but were readily contracted by serotonin and by the thromboxane A2 mimetic U46619. Endothelium-dependent vasorelaxation was weak, reaching significance only for histamine. However, the vessels relaxed to sodium nitroprusside, and to acetylcholine if sandwiched with human saphenous vein. Cryopreservation did not change the mechanical properties in the relevant tension range. Vasoconstriction to potassium chloride and serotonin were reduced after freezing (22.9 ± 7.6%, 27.7 ± 10.2%) and after vitrification (2.6 ± 5.8%, 4.3 ± 7.1%). The mitochondrial metabolism was also attenuated after freezing (57.9 ± 25.9%) and after vitrification (21.7 ± 6.7%). Prostacyclin release was elevated after both cryopreservation procedures (4.0-fold, 3.9-fold), whereas there was no significant change in the adhesion of platelets. Denuded HUVs could readily be seeded with isolated endothelial cells before and after freezing. We conclude that HUV is suitable as a storable living scaffold with antithrombogenic properties.