Thromboxane A2 Receptor Activation Research Articles

Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model.

Extracellular protein disulfide isomerases (PDIs), including PDI, endoplasmic reticulum protein 57 (ERp57), ERp72, ERp46, and ERp5, are required for invivo thrombus formation in mice. Platelets secrete PDIs upon activation, which regulate platelet aggregation. However, platelets secrete only ∼10% of their PDI content extracellularly. The intracellular role of PDIs in platelet function is unknown. Here, we aim to characterize the role of ERp5 (gene Pdia6) using platelet conditional knockout mice, platelet factor 4 (Pf4) Cre+/ERp5floxed (fl)/fl. Pf4Cre+/ERp5fl/fl mice developed mild macrothrombocytopenia. Platelets deficient in ERp5 showed marked dysregulation of their ER, indicated by a twofold upregulation of ER proteins, including PDI, ERp57, ERp72, ERp46, 78 kilodalton glucose-regulated protein (GRP78), and calreticulin. ERp5-deficient platelets showed an enhanced ER stress response to exvivo and invivo ER stress inducers, with enhanced phosphorylation of eukaryotic translation initiation factor 2A and inositol-requiring enzyme 1 (IRE1). ERp5 deficiency was associated with increased secretion of PDIs, an enhanced response to thromboxane A2 receptor activation, and increased thrombus formation invivo. Our results support that ERp5 acts as a negative regulator of ER stress responses in platelets and highlight the importance of a disulfide isomerase in platelet ER homeostasis. The results also indicate a previously unanticipated role of platelet ER stress in platelet secretion and thrombosis. This may have important implications for the therapeutic applications of ER stress inhibitors in thrombosis.

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

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

ATP-sensitive K<sup>+</sup> channel activation in the brain is involved in elevation of plasma levels of adrenaline

Recently, we reported that restraint stress-induced elevation of plasma levels of adrenaline, but not of noradrenaline, was suppressed by intravenous and intracerebroventricular (i.c.v.) administration of glucose via brain thromboxane A2-dependent mechanisms. (Yamaguchi et al., 2022). Since glucose can be converted to ATP in the brain cells, in the present study, we investigate whether ATP-sensitive K+ channel (KATP channel) activity in the brain is involved in changes of plasma levels of catecholamines in urethane anesthetized rats. I.c.v. administered diazoxide (20 µg/rat), a KATP channel opener, and A-769662 (25 µg/rat), an AMPK activator, elevated plasma levels of adrenaline, but not noradrenaline. In contrast, i.c.v administered glibenclamide (25 µg/rat), a KATP channel antagonist, did not alter plasma levels of adrenaline. I.c.v. pretreatment with SQ29548 (10 µg/rat), a thromboxane A2 receptor antagonist, attenuated diazoxide- and A-7969662-induced elevation of plasma levels of adrenaline. These results suggest that KATP channel activation in the brain might trigger the activation of thromboxane A2 receptor, resulting in elevation of plasma levels of adrenaline.

Impairment of amyloid precursor protein alpha-processing in cerebral microvessels of type 1 diabetic mice

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A2 (TXA2). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

Activation of thromboxane A2 receptors mediates endothelial dysfunction in diabetic mice

ABSTRACTBackground: Diabetes is one of high-risk factors for cardiovascular disease. Improvement of endothelial dysfunction in diabetes reduces vascular complications. However, the underlying mechanism needs to be uncovered. This study was conducted to elucidate whether and how thromboxane A2 receptor (TPr) activation contributes to endothelial dysfunction in diabetes. Methods and Results: Exposure of human umbilical vein endothelial cells (HUVECs) to either TPr agonists, two structurally related thromboxane A2 (TxA2) mimetics, significantly reduced phosphorylations of endothelial nitric oxide synthase (eNOS) at Ser1177 and Akt at Ser473. These effects were abolished by pharmacological or genetic inhibitors of TPr. TPr-induced suppression of eNOS and Akt phosphorylation was accompanied by upregulation of PTEN (phosphatase and tension homolog deleted on chromosome 10) and Ser380/Thr382/383 PTEN phosphorylation. PTEN-specific siRNA restored Akt–eNOS signaling in the face of TPr activation. The small GTPase, Rho, was also activated by TPr stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase (ROCK) inhibitor, rescued TPr-impaired Akt–eNOS signaling. In mice, streptozotocin-induced diabetes was associated with aortic PTEN upregulation, PTEN-Ser380/Thr382/383 phosphorylation, and dephosphorylation of Akt (at Ser473) and eNOS (at Ser1177). Importantly, administration of TPr antagonist blocked these changes. Conclusion: We conclude that TPr activation impairs endothelial function by selectively inactivating the ROCK–PTEN–Akt–eNOS pathway in diabetic mice.

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

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

GPVI and Thromboxane Receptor on Platelets Promote Proinflammatory Macrophage Phenotypes during Cutaneous Inflammation

Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.

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),3β(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.

Abstract 1618: Thromboxane A2 receptor controls migration and metastasis of triple-negative breast cancer cells

Abstract Background: Triple negative breast cancers (TNBCs) have poor prognosis and there are no effective targeted therapies available. Thromboxane A2 receptor (TBXA2R) is a G protein coupled receptor whose ligand is a prostanoid produced by cyclooxygenase and thromboxane A2 synthase. There are two isoforms of TBXA2R as results of alternative splicing. TBXA2Rα and β differ in the cytoplasmic tail distal at amino acid residue 328. High expression of TBXA2R has been linked with poor progression free survival of breast cancer cells, but it is unknown how TBXA2R contributes to the progression of breast cancers and which isoform(s) is involved. Methods: TBXA2R expression in breast cancer was determined by RT-PCR, Western blot and immunohistochemistry using isoform specific primer sets or antibodies. Small hairpin RNAs and CRISPR-CAS9 were used to knock down or knock out TBXA2R expression. Tumor cell migration was evaluated by wound healing and Boyden chamber assays. Cell proliferation and survival was evaluated by MTS and trypan blue based cell counting. Xenograft models were used to assess breast tumor growth, progression, and metastasis. Results: While TBXA2Rα is ubiquitously expressed in breast cancer cells, TBXA2Rβ is selectively expressed in triple negative breast cancer. Activation of TBXA2R had no discernible effects on proliferation or survival of MDA-MB-231 cells. However, TBXA2R activation led to rapid activation of RhoA and cytoskeletal reorganizations of TNBC cells. TBXA2R activation also led to rapid activation of protein kinase N1 (PKN1), a kinase frequently amplified in TNBCs. Inhibition or depletion of TBXA2R led to reduced tumor cell migration in vitro and prevented breast cancer metastasis in vivo. Further analyses found that inhibition or depletion of TBXA2R compromised the ability of TNBC cells to extravasate in an animal model. Conclusions: Our data suggest that TBXA2R can contribute to TNBC progression and metastasis by controlling tumor cell cytoskeleton reorganization and motility. Citation Format: Yuanqin Zhang, Xuejing Zhang, Daotai Nie. Thromboxane A2 receptor controls migration and metastasis of triple-negative breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1618.

Calcium desensitisation in late polymicrobial sepsis is associated with loss of vasopressor sensitivity in a murine model.

BackgroundSepsis is characterised by diminished vasopressor responsiveness. Vasoconstriction depends upon a balance: Ca2+-dependent myosin light-chain kinase promotes and Ca2+-independent myosin light-chain phosphatase (MLCP) opposes vascular smooth muscle contraction. The enzyme Rho kinase (ROK) inhibits MLCP, favouring vasoconstriction. We tested the hypothesis that ROK-dependent MLCP inhibition was attenuated in late sepsis and associated with reduced contractile responses to certain vasopressor agents.MethodsThis is a prospective, controlled animal study. Sixteen-week-old C57/BL6 mice received laparotomy or laparotomy with caecal ligation and puncture (CLP). Antibiotics, fluids and analgesia were provided before sacrifice on day 5. Vasoconstriction of the femoral arteries to a range of stimuli was assessed using myography: (i) depolarisation with 87 mM K+ assessed voltage-gated Ca2+ channels (L-type, Cav1.2 Ca2+ channels (LTCC)), (ii) thromboxane A2 receptor activation assessed the activation state of the LTCC and ROK/MLCP axis, (iii) direct PKC activation (phorbol-dibutyrate (PDBu), 5 μM) assessed the PKC/CPI-17 axis independent of Ca2+ entry and (iv) α1-adrenoceptor stimulation with phenylephrine (10−8 to 10−4 M) and noradrenaline (10−8 to 10−4 M) assessed the sum of these pathways plus the role of the sarcoplasmic reticulum (SR). ROK-dependent MLCP activity was indexed by Western blot analysis of P[Thr855]MYPT. Parametric and non-parametric data were analysed using unpaired Student's t-tests and Mann-Whitney tests, respectively.ResultsROK-dependent inhibition of MLCP activity was attenuated in both unstimulated (n = 6 to 7) and stimulated (n = 8 to 12) vessels from mice that had undergone CLP (p < 0.05). Vessels from CLP mice demonstrated reduced vasoconstriction to K+, thromboxane A2 receptor activation and PKC activation (n = 8 to 13; p < 0.05). α1-adrenergic responses were unchanged (n = 7 to 12).ConclusionsIn a murine model of sepsis, ROK-dependent inhibition of MLCP activity in vessels from septic mice was reduced. Responses to K+ depolarisation, thromboxane A2 receptor activation and PKC activation were diminished in vitro whilst α1-adrenergic responses remained intact. Inhibiting MLCP may present a novel therapeutic target to manage sepsis-induced vascular dysfunction.

Cardiac thromboxane A2 receptor activation does not directly induce cardiomyocyte hypertrophy but does cause cell death that is prevented with gentamicin and 2-APB.

BackgroundWe have previously shown that the thromboxane (TXA2) receptor agonist, U46619, can directly induce ventricular arrhythmias that were associated with increases in intracellular calcium in cardiomyocytes. Since TXA2 is an inflammatory mediator and induces direct calcium changes in cardiomyocytes, we hypothesized that TXA2 released during ischemia or inflammation could also cause cardiac remodeling.MethodsU46619 (0.1-10 μM) was applied to isolated adult mouse ventricular primary cardiomyocytes, mouse ventricular cardiac muscle strips, and cultured HL-1 cardiomyocytes and markers of hypertrophy and cell death were measured.ResultsWe found that TXA2 receptors were expressed in ventricular cardiomyocytes and were functional via calcium imaging. U46619 treatment for 24 h did not increase expression of pathological hypertrophy genes (atrial natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin) and it did not increase protein synthesis. There was also no increase in cardiomyocyte size after 48 h treatment with U46619 as measured by flow cytometry. However, U46619 (0.1-10 μM) caused a concentration-dependent increase in cardiomyocyte death (trypan blue, MTT assays, visual cell counts and TUNEL stain) after 24 h. Treatment of cells with the TXA2 receptor antagonist SQ29548 and inhibitors of the IP3 pathway, gentamicin and 2-APB, eliminated the increase in cell death induced by U46619.ConclusionsOur data suggests that TXA2 does not induce cardiac hypertrophy, but does induce cell death that is mediated in part by IP3 signaling pathways. These findings may provide important therapeutic targets for inflammatory-induced cardiac apoptosis that can lead to heart failure.

Thromboxane A2 receptor-mediated release of matrix metalloproteinase-1 (MMP-1) induces expression of monocyte chemoattractant protein-1 (MCP-1) by activation of protease-activated receptor 2 (PAR2) in A549 human lung adenocarcinoma cells

Matrix metalloproteinases (MMPs) and monocyte chemoattractant protein-1 (MCP-1, CCL2) are known to be upregulated in many tumors. Their roles in tumor invasion and metastasis are being uncovered. How they are related to each other and involved in tumor progression remains to be determined. Earlier it was reported that I-BOP-initiated activation of thromboxane A2 receptor (TP) induced the release of MMP-1, MMP-3, and MMP-9 from lung cancer A549 cells overexpressing TPα (A549-TPα). Herein it was found that MMP-1, but not MMP-3 or MMP-9, induced the expression of MCP-1 in A549 cells. Conditioned medium (CM) from I-BOP activated, MMP-1 siRNA pretreated A549-TPα cells induced greatly attenuated expression of MCP-1 in A549 cells indicating that MMP-1 in the CM contributed significantly to the expression of MCP-1. MMP-1 was shown to activate protease-activated receptor 2 (PAR2) instead of commonly assumed PAR1 to increase the expression of MCP-1 in A549 cells. This conclusion was reached from the following findings: (1) expression of MCP-1 induced by trypsin, a PAR2 agonist, and also PAR2 agonist peptide, was inhibited by a PAR2 antagonist; (2) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was blocked by a PAR2 antagonist but not by other PAR antagonists; (3) expression of MCP-1 induced by MMP-1 and by CM from I-BOP activated A549-TPα cells was attenuated significantly by pretreatment of cells with PAR2-siRNA. These results suggest that PAR2 is a novel MMP-1 target mediating MMP-1-induced signals in A549 lung cancer cells.

Activation of Thromboxane A2 Receptor (TP) Increases the Expression of Monocyte Chemoattractant Protein -1 (MCP-1)/Chemokine (C-C motif) Ligand 2 (CCL2) and Recruits Macrophages to Promote Invasion of Lung Cancer Cells

Thromboxane synthase (TXAS) and thromboxane A2 receptor (TP), two critical components for thromboxane A2 (TXA2) signaling, have been suggested to be involved in cancer invasion and metastasis. However, the mechanisms by which TXA2 promotes these processes are still unclear. Here we show that TXA2 mimetic, I-BOP, induced monocyte chemoattractant protein -1(MCP-1)/chemokine (C-C motif) ligand 2 (CCL2) expression at both mRNA and protein levels in human lung adenocarcinoma A549 cells stably over-expressing TP receptor α isoform (A549-TPα). The induction of MCP-1 was also found in other lung cancer cells H157 and H460 that express relatively high levels of endogenous TP. Using specific inhibitors of several signaling molecules and promoter/luciferase assay, we identified that transcription factor SP1 mediates I-BOP-induced MCP-1 expression. Furthermore, supernatants from I-BOP-treated A549-TPα cells enhanced MCP-1-dependent migration of RAW 264.7 macrophages. Moreover, co-culture of A549 cells with RAW 264.7 macrophages induced expression of MMPs, VEGF and MCP-1 genes, and increased the invasive potential in A549 cells. These findings suggest that TXA2 may stimulate invasion of cancer cells through MCP-1-mediated macrophage recruitment.

Interleukin 10 knockout frail mice develop cardiac and vascular dysfunction with increased age

Cardiovascular dysfunction is a primary independent predictor of age-related morbidity and mortality. Frailty is associated with activation of inflammatory pathways and fatigue that commonly presents and progresses with age. Interleukin 10 (IL-10), the cytokine synthesis inhibitory factor, is an anti-inflammatory cytokine produced by immune and non-immune cells. Homozygous deletion of IL-10 in mice yields a phenotype that is consistent with human frailty, including age-related increases in serum inflammatory mediators, muscular weakness, higher levels of IGF-1 at midlife, and early mortality. While emerging evidence suggests a role for IL-10 in vascular protection, a clear mechanism has not yet been elucidated. MethodsIn order to evaluate the role of IL-10 in maintenance of vascular function, force tension myography was utilized to access ex-vivo endothelium dependent vasorelaxation in vessels isolated from IL-10 knockout IL-10(tm/tm) and control mice. Pulse wave velocity ((PWV), index of stiffness) of vasculature was measured using ultrasound and blood pressure was measured using the tail cuff method. Echocardiography was used to elucidated structure and functional changes in the heart. ResultsMean arterial pressures were significantly higher in IL-10(tm/tm) mice as compared to C57BL6/wild type (WT) controls. PWV was increased in IL-10(tm/tm) indicating stiffer vasculature. Endothelial intact aortic rings isolated from IL-10(tm/tm) mice demonstrated impaired vasodilation at low acetylcholine doses and vasoconstriction at higher doses whereas vasorelaxation responses were preserved in rings from WT mice. Cyclo-oxygenase (COX-2)/thromboxane A2 inhibitors improved endothelial dependent vasorelaxation and reversed vasoconstriction. Left ventricular end systolic diameter, left ventricular mass, isovolumic relaxation time, fractional shortening and ejection fraction were all significantly different in the aged IL-10(tm/tm) mice compared to WT mice. ConclusionAged IL-10(tm/tm) mice have stiffer vessels and decreased vascular relaxation due to an increase in eicosanoids, specifically COX-2 activity and resultant thromboxane A2 receptor activation. Our results also suggest that aging IL-10(tm/tm) mice have an increased heart size and impaired cardiac function compared to age-matched WT mice. While further studies will be necessary to determine if this age-related phenotype develops as a result of inflammatory pathway activation or lack of IL-10, it is essential for maintaining the vascular compliance and endothelial function during the aging process. Given that a similar cardiovascular phenotype is present in frail, older adults, these findings further support the utility of the IL-10(tm/tm) mouse as a model of frailty.

Isoprostanes as physiological mediators of transition to newborn life: novel mechanisms regulating patency of the term and preterm ductus arteriosus

Increased oxygen tension at birth regulates physiologic events that are essential to postnatal survival, but the accompanying oxidative stress may also generate isoprostanes. We hypothesized that isoprostanes regulate ductus arteriosus (DA) function during postnatal vascular transition. Isoprostanes were measured by gas chromatography-mass spectrometry. DA tone was assessed by pressure myography. Gene expression was measured by quantitative PCR. Oxygen exposure was associated with increased 8-iso-prostaglandin (PG)F2α in newborn mouse lungs. Both 8-iso-PGE2 and 8-iso-PGF2α induced concentration-dependent constriction of the isolated term DA, which was reversed by the thromboxane A2 (TxA2) receptor antagonist SQ29548. SQ29548 pretreatment unmasked an isoprostane-induced DA dilation mediated by the EP4 PG receptor. Exposure of the preterm DA to 8-iso-PGE2 caused unexpected DA relaxation that was reversed by EP4 antagonism. In contrast, exposure to 8-iso-PGF2α caused preterm DA constriction via TxA2 receptor activation. Further investigation revealed the predominance of the TxA2 receptor at term, whereas the EP4 receptor was expressed and functionally active from mid-gestation onward. This study identifies a novel physiological role for isoprostanes during postnatal vascular transition and provide evidence that oxidative stress may act on membrane lipids to produce vasoactive mediators that stimulate physiological DA closure at birth or induce pathological patency of the preterm DA.

Abstract C45: Validation of thromboxane A2 receptor (TP) as a target to block breast cancer metastasis.

Abstract The receptor of thromboxane A2 (TXA2), TP, is a seven transmembrane, G-protein-coupled receptor. Increased expression of TP at RNA level was found to correlate with a poor prognosis in breast cancer patients but it is unknown how TP expression and activities are involved in breast cancer progression. Here we report that TP is expressed in metastatic breast cancer cells at both RNA and protein levels. Treatment of breast cancer MDA-MB-231 cells by U46619, a TP agonist, induced cell body contraction. The U46619-induced contraction was blocked by SQ29548, a high-affinity TP antagonist. Further studies found that small GTPase RhoA was activated by TP activation and that pretreatment of MDA-MB-231 cells with Y27632, a Rho kinase (ROCK) inhibitor, blocked U46619-induced cell contraction. The requirement of RhoA activation in U46619 induced cell contraction was further confirmed by the ability of dominant negative RhoA to block cell contraction induced by U46619. MDA-MB-231 cells with TP knockdown displayed a reduced percentage of contraction under the treatment of U46619 and TXA2 agonist I-BOP as well as attenuated invasion ability. When injected into mice, MDA-MB-231 cells with TP depleted did not present significant changes in primary tumor formation or growth. However, TP depletion reduced the ability of MDA-MB-231 cells to metastasize to the lung and the liver in an experimental metastasis model. We further demonstrated that TP depletion reduced the lung colonization ability of MDA-MB-231 cells by blocking active extravasation. Taken together, TP is expressed in breast cancer and that activation of TP has profound effects on tumor cell cytoskeleton and contraction through activation of RhoA. Inhibition or depletion of TP reduces the metastatic dissemination of breast cancer cells in vivo by blocking the extravasation of tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C45.

Abstract 3483: Activation of thromboxane A2 receptor-alpha promotes tumor growth and angiogenesis through expression of VEGF in prostate cancer

Abstract The primary objective of this study is to investigate the role of TXA2R signaling in prostate cancer progression and metastasis. Thromboxane A2 (TXA2) is a major arachidonic acid metabolite that signals through TXA2 receptors to induce platelet aggregation and smooth muscle contraction. TXA2 receptors (TP) are expressed as two different isoforms in humans- TP-alpha (TPα) and TP-beta (TPβ), which share both common and distinct signaling pathways. Previous studies have established the important role played by TXA2R isoforms in several cancers such as bladder, lung and breast cancer. But the role played by TXA2R signaling in prostate cancer (PCa) progression is not well understood. Our data indicates that human prostate cancer cell lines such as PC3, DU145, PC3M and LNCaP only express the TPα isoform but not the TPβ isoform. Also expression of TPα was higher in PCa cells compared to normal prostate epithelial cells. Results from the angiogenesis array indicated that activation of TXA2 receptors using TXA2 mimetic-IBOP resulted in an increase in angiogenesis related proteins such as VEGF, MMP9 and uPA in DU145 cells. To further confirm the role of TPα activation on VEGF, which is one of the most potent and predominant angiogenic factor, Real-time PCR and ELISA for human VEGF was performed that showed an increased expression of VEGF in response to IBOP in DU145 cells. To further understand the mechanisms behind TPα mediated VEGF expression, immunoblotting was performed on DU145 cells lysates treated with IBOP in order to analyze the phosphorylation status of downstream targets with phospho-specific antibodies. Activation of TXA2 signaling by IBOP did result in phosphorylation of downstream targets such as Src kinase, EGFR kinase and ERK. To verify whether EGFR kinase is involved in TPα induced VEGF expression, we pre-treated these cells with an EGFR kinase inhibitor AG1478 before treatment with IBOP. Results from Real-time PCR and ELISA suggest that inhibition of EGFR kinase did result in reduced VEGF mRNA and protein expression that confirmed its role in TPα mediated angiogenesis. Over expression of TPα in another PCa cell line, PC3 (PC3-TPα) that has lower endogenous expression of TPα also resulted in an increase in VEGF expression. Increased angiogenesis was further verified by a preliminary in vivo study that showed mice injected with PC3-TPα cells exhibited greater tumor growth and increased neovascularization compared to mice with PC3-Neo cells. Taken together, these data suggests that, activation of TPα receptor in PCa cells induced the expression of pro angiogenic factors such as VEGF and hence might play an important role in prostate cancer progression and metastasis. Acknowledgements: This research was supported by NIH Grant 1R01 CA114051-01A1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3483. doi:10.1158/1538-7445.AM2011-3483

Abstract LB-364: G(alpha)12 is required for thromboxane A2 to regulate tumor cell motility

Abstract Introduction: Thromboxane (TX) A2 is a prostaglandin produced by metabolism of arachidonic acid through cyclooxygenase and thromboxane synthase. TXA2 is biologically active, mainly through activating its cognate, seven transmembrane, G protein coupled receptor. We previously reported that thromboxane A2 receptor (TP) was expressed in prostate cancer, and further activation of this receptor elicited cell contraction and modulated tumor cell motility through regulating small GTPase RhoA (Nie et al., Cancer Res 68: 115–121, 2008). This study aims to identify G alpha protein(s) involved in thromboxane A2 signaling in tumor cell motility. Methods: The expression of G alpha proteins in the PC3MM cells was studied by real-time PCR. Cell contraction assay was performed by staining cells with TRITC phalliodin and evaluated under an epifluorescence microscope. Tumor cell motility and invasion were evaluated using wound healing assay and transwell Boyden Chamber assay. To determine the roles of G alpha protein in thromboxane A2 signaling, we expressed different alleles of G alpha proteins (wild type, constitutive active) in PC3MM cells and then the subsequent effects on cell contractions was determined. We also depleted G alpha protein expression by short hairpin RNA and examined subsequent changes in cell contraction and migration after activation of TP. Results: PC3 MM cell line had the endogenous level of all the G alpha protein (G alpha 12, G alpha i1, G alpha 11, G alpha 13), but not G alpha q. Overexpressionof G alpha 12/13 increased cell contraction after activation of thromboxane A2 receptor with U46619. Expression of constitutively active G alpha 12 by itself was sufficient to cause cell contraction, regardless whether TP is activated or not. Depletion of G alpha 12 via shRNAs reduced cell contraction after TP activation. Tumor cells with G alpha 12 depleted had tumor cell motility and invasiveness. Conclusion: The findings indicate that G alpha 12 is required for TP to induce cell contraction and to regulate tumor cell motility. The studies suggest G alpha 12 may be a promising target of intervention to reduce tumor cell motility and metastasis in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-364. doi:10.1158/1538-7445.AM2011-LB-364

Potassium Channel Regulation of Thromboxane A2 Receptor Function

G-protein coupled receptors (GPCRs) and the large-conductance, voltage- and Ca2+ -activated K+ channel (MaxiK), are commonly expressed at the plasma membrane of cells. GPCR activation results in activation or inhibition of MaxiK channel activity depending on the type of GPCR. For example, activation of Thromboxane A2 receptor leads to MaxiK channel inhibition. We now investigate whether MaxiK channel alpha subunit (Slo1) can talk back and regulate Thromboxane A2 receptor (TP) function. To this end, we analyzed two downstream processes of activated TP: activation of the monomeric G-protein Rho and stimulation of intracellular calcium release.

Impaired Contractile Response Of Human Peripheral Arterioles To TXA-2 After Cardiopulmonary Bypass

Introduction: Cardiopulmonary bypass (CPB) is often associated with peripheral vasomotor dysfunction. This is thought to lead to decreased vascular tone, hypotension, and an increased need for vasopressor support. We investigated the contractile response to the vasoconstrictor thromboxane A2 (TXA-2), the blockade of TXA-2 receptors and the inhibition of phospholipase-C (PLC) and phospholipase-A2 (PLA-2) in the human peripheral microvasculature pre- and post- CPB. We also examined the effect of CPB on the gene expression of skeletal muscle TXA-2 and TXA-2 synthase. Methods: Chest wall skeletal muscle tissue was harvested pre- and post-CPB from patients undergoing coronary artery bypass grafting. Skeletal muscle arterioles (90-180 micrometer in diameter) were dissected from the harvested tissue and placed in an organ bath containing Krebs solution. Constractile response of the peripheral arterioles to the TXA-2 analog U46619, with and without the highly selective TXA-2 receptor antagonist, SQ 29548, the specific PLC inhibitor U73122 or the selective PLA-2 inhibitor, quinacrine was assessed by videomicroscopy. TXA-2 receptor and TXA-2 synthase gene expression were examined using microarray analysis. Results: The post-CPB contractile response of peripheral arterioles to TXA-2 analog U46619 (10−6M) was significantly impaired compared with pre-CPB (28.5±2% vs. 42.7±3.6 %, P=0.002). The presence of TXA-2 antagonist SQ 29548 (10−6 M) prevented the contractile response to U46619. Pretreatment with the PLC inhibitor U73122 (10−5M) significantly inhibited the U46619-induced contractile response as well. Addition of the PLA-2 inhibitor,quinacrine (10−6M) failed to affect U46619-induced contraction. Microarray analysis showed no significant changes in the gene expression of TXA-2 receptor and TXA-2 synthase. Conclusions: CPB decreases the contractile response of human peripheral arterioles to TXA-2 analog U46619. The contractile response to the TXA-2 analog U46619 is via activation of TXA-2 receptors and PLC, but not of PLA-2. These results provide novel mechanisms for TXA-2-induced contraction and signaling in peripheral vasomotor dysfunction after CPB and cardiac surgery.