Altered expression of eNOS, prostacyclin synthase, prostaglandin G/H synthase, and thromboxane synthase in porcine aortic endothelial cells after exposure to human serum-relevance to xenotransplantation.

Under normal conditions, the activity of platelets is stringently and precisely balanced between activation and quiescent state. This guarantees rapid hemostasis and avoids uncontrolled thrombosis. However, excessive platelet activation and resulting thrombotic microangiopathy are frequently observed in pig-to-primate xenotransplantation models. Endothelium-derived inhibitory mechanisms play an important role in regulation of platelet activation. These mainly include nitric oxide (NO), prostacyclin PGI2 , and adenosine, which are synthesized by endothelial NO synthases (eNOS), prostacyclin synthase, and CD39/CD73, respectively. We investigated whether endothelium-derived regulatory mechanisms are affected in porcine aortic endothelial cells (PAECs) after exposure to human serum. In the present study, exposure of PAECs or porcine iliac arteries to human serum suppressed gene expression of eNOS and prostacyclin synthase, while induced gene expression of prostaglandin G/H synthase and thromboxane synthase. Simultaneously, exposure to human serum reduced NO and PGI2 production in PAEC culture supernatants. Thus, human serum altered the balance of endothelium-derived inhibitory mechanisms in PAECs, which may indicate a regulatory mechanism of excessive platelet activation in pig-to-primate xenotransplantation.

Metabolomic analysis revealed the role of DNA methylation in the balance of arachidonic acid metabolism and endothelial activation.

Arachidonic acid (AA) metabolism plays an important role in vascular homeostasis. We reported that DNA hypomethylation of EPHX2 induced a pro-inflammatory response in vascular endothelial cells (ECs). However, the change in the whole AA metabolism by DNA methylation is still unknown. Using a metabolomic approach, we investigated the effect of DNA methylation on the balance of AA metabolism and the underlying mechanism. ECs were treated with a DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-AZA), and AA metabolic profiles were analyzed. Levels of prostaglandin D2 (PGD2) and thromboxane B2 (TXB2), metabolites in the cyclooxygenase (COX) pathway, were significantly increased by 5-AZA treatment in ECs resulting from the induction of PGD2 synthase (PTGDS) and thromboxane A synthase 1 (TBXAS1) expression by DNA hypomethylation. This phenomenon was also observed in liver and kidney cell lines, indicating a universal mechanism. Pathophysiologically, homocysteine, known to cause DNA demethylation, induced a similar pattern of the change of AA metabolism. Furthermore, 5-AZA activated ECs, as evidenced by the upregulation of adhesion molecules. Indomethacin, a COX inhibitor, reversed the effects of 5-AZA on the levels of PGD2 and TXB2, EC activation and monocyte adhesion. In vivo, the plasma levels of PGD2 and TXB2 and the expression of In vivo PTGDS and TBXAS1 as well as adhesion molecules were increased in the aorta of the mice injected with 5-AZA. In conclusion, using a metabolomic approach, our study uncovered that DNA demethylation increased AA metabolites PGD2 and TXB2 by upregulating the expression of the corresponding enzymes, which might contribute to the DNA hypomethylation-induced endothelial activation.

Trypanosoma cruzi infection and endothelin-1 cooperatively activate pathogenic inflammatory pathways in cardiomyocytes.

Trypanosoma cruzi, the causative agent of Chagas' disease, induces multiple responses in the heart, a critical organ of infection and pathology in the host. Among diverse factors, eicosanoids and the vasoactive peptide endothelin-1 (ET-1) have been implicated in the pathogenesis of chronic chagasic cardiomyopathy. In the present study, we found that T. cruzi infection in mice induces myocardial gene expression of cyclooxygenase-2 (Cox2) and thromboxane synthase (Tbxas1) as well as endothelin-1 (Edn1) and atrial natriuretic peptide (Nppa). T. cruzi infection and ET-1 cooperatively activated the Ca(2+)/calcineurin (Cn)/nuclear factor of activated T cells (NFAT) signaling pathway in atrial myocytes, leading to COX-2 protein expression and increased eicosanoid (prostaglandins E(2) and F(2α), thromboxane A(2)) release. Moreover, T. cruzi infection of ET-1-stimulated cardiomyocytes resulted in significantly enhanced production of atrial natriuretic peptide (ANP), a prognostic marker for impairment in cardiac function of chagasic patients. Our findings support an important role for the Ca(2+)/Cn/NFAT cascade in T. cruzi-mediated myocardial production of inflammatory mediators and may help define novel therapeutic targets.

Thromboxane-induced contractile response of human coronary arterioles is diminished after cardioplegic arrest.

BACKGROUND: We investigated the contractile response of human coronary microvasculature to thromboxane A-2 (TXA-2), with and without the blockade of TXA-2 receptors or the inhibition of phospholipase-C (PLC) or of protein kinase C-α (PKC-α) in the human coronary microvasculature before and after cardioplegia, followed by reperfusion (CP/Rep). Protein/gene expression and localization of TXA-2 receptors, TXA-2 synthase, PLC, and other TXA-2-related proteins was also examined. METHODS: Right atrial tissue was harvested before and after cold blood cardioplegia, followed by about 10 minutes of reperfusion, from 28 patients undergoing cardiac operations. Coronary arterioles (90 to 170 µm in diameter) were dissected from the harvested tissue. RESULTS: The post-CP/Rep contractile response of coronary arterioles to TXA-2 analog U-46619 was significantly impaired vs pre-CP/Rep (p<0.05). The TXA-2 receptor antagonist SQ-29548 (10(-6) M) prevented the contractile response to U-46619 (p<0.05). Pretreatment with the PLC inhibitor U73122 (10(-6) M) significantly inhibited the U-46619-induced contractile response (p<0.05). Administration of the PKC-α inhibitor safingol failed to affect U-46619-induced contraction. Total protein levels and gene expression of TXA-2 receptors, TXA-2 synthase, PLC-ß3, phospho-PLC-ß3, PLC-γ1, and phospho-PLC-γ1 were not altered after CP/Rep. Confocal microscopy showed no significant differences in the expression of TXA-2 receptors or PLC-ß3 in the microcirculation. TXA-2 receptors and PLC-ß3 were both present in smooth muscle and endothelium. CONCLUSIONS: Cardioplegia/Rep decreases the contractile response of human coronary arterioles to TXA-2 soon after cardiac operations. The contractile response to the TXA-2 analog U-46619 is through activation of TXA-2 receptors and PLC.

4-Methylnitrosamino-1-3-pyridyl-1-butanone (NNK) promotes lung cancer cell survival by stimulating thromboxane A2 and its receptor.

The role of thromboxane A(2) (TxA(2)) in smoking-associated lung cancer is poorly understood. This study was conducted to study the role of TxA(2) in smoking carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-promoted cell survival and growth in human lung cancer cells. We found that NNK increased TxA(2) synthase (TxAS) expression and thromboxane B(2) (TxB(2)) generation in cultured lung cancer cells, the result of which was supported by the increased level of TxAS in lung cancer tissues of smokers. Both TxAS-specific inhibitor furegrelate and TxA(2) receptor antagonist SQ29548 completely blocked NNK-mediated cell survival and growth via inducting apoptosis. TxA(2) receptor agonist U46619 reconstituted a near-full survival and growth response to NNK when TxAS was inhibited, affirming the role of TxA(2) receptor in NNK-mediated cell survival and growth. Suppression of cyclic adenosine monophosphate response element binding protein (CREB) activity by its small interference RNA blocked the effect of NNK. Phosphatidylinositol 3-kinase (PI3K)/Akt and extracellular signal-regulated kinase (ERK) also had a positive role. Altogether, our results have revealed that NNK stimulates TxA(2) synthesis and activates its receptor in lung cancer cells. The increased TxA(2) may then activate CREB through PI3K/Akt and extracellular ERK pathways, thereby contributing to the NNK-promoted survival and growth of lung cancer cells.

Expression of thromboxane synthase, prostacyclin synthase and thromboxane receptor in atherosclerotic lesions: correlation with plaque composition.

OBJECTIVES: Prostaglandins, such as thromboxane A(2) (TxA(2)) and prostacyclin (PGI(2)), are bioactive lipid mediators that are implicated in the pathogenesis of atherosclerosis. In the current study, we tested the hypothesis that thromboxane synthase (TXAS), prostacyclin synthase (PGIS) and thromboxane receptor (TP) are expressed within the atherosclerotic lesion. METHODS: Atherosclerotic aorta segments were obtained from low-density lipoprotein receptor deficient (LDL r-KO) mice on a high fat diet. Expression levels of TXAS, PGIS and TP were evaluated by real-time quantitative reverse transcription PCR, and immunohistochemistry; TxA(2) and PGI(2) biosynthesis was also assayed. RESULTS: After 8 weeks on the fat diet, aortic arches from LDL r-KO mice showed a significant increase in PGIS, TXAS, TP mRNA, TxA(2) and PGI(2) levels, when compared with controls. By contrast, after 16 weeks on the high fat diet PGIS and PGI(2) were significantly reduced, whereas TXAS and TP message and protein and TxA(2) levels were further and significantly increased in the atherosclerotic tissues when compared with the 8-week group. These changes correlated with the cellular composition of the atherosclerotic lesions. CONCLUSIONS: TXAS, PGIS and TP are all present within the atherosclerotic lesion areas, their levels change during progression of atherogenesis and contribute to TxA(2) and PGI(2) formation.

Altered coronary microvascular serotonin receptor expression after coronary artery bypass grafting with cardiopulmonary bypass.

OBJECTIVE: We evaluated roles of serotonin 1B and 2A receptors, thromboxane synthase and receptor, and phospholipases A(2) and C in response to cardiopulmonary bypass. METHODS: Patients' atrial tissues were harvested before and after cardiopulmonary bypass with cardioplegia (n = 13). Coronary microvessels were assessed for vasoactive response to serotonin with and without inhibitors of serotonin 1B and 2A receptors and phospholipases A(2) and C. Expressions of serotonin receptor messenger RNA were determined with reverse transcriptase polymerase chain reaction. Expressions of serotonin receptors and thromboxane A(2) receptor and synthase proteins were determined with immunoblotting and immunohistochemistry. RESULTS: Microvessel exposure to serotonin elicited 7.3% +/- 2% relaxation before bypass, changing to contraction of -19.2% +/- 2% after bypass (P <.001). Additions of specific serotonin 1B receptor antagonist and inhibitor of phospholipase A(2) resulted in significantly decreased contraction, -8.6% +/- 1% (P < .001) and 2.8% +/- 3% (P = .001), respectively. Serotonin 1B receptor messenger RNA expression increased 1.82 +/- 0.34-fold after bypass (p = .044); serotonin 2A receptor messenger RNA expression did not change. Serotonin 1B but not 2A receptor protein expression increased after bypass by 1.35 +/- 0.7-fold (P = .0413). Thromboxane synthase and receptor expressions were unchanged after bypass. Serotonin 1B receptor increased mainly in arterial smooth muscle. There were no appreciable differences in arterial expressions of thromboxane synthase or receptor. CONCLUSIONS: Serotonin-induced vascular dysfunction after cardiopulmonary bypass with cardioplegic arrest may be mediated by increased expression of serotonin 1B receptor and subsequent phospholipase A(2) activation in myocardial coronary smooth muscle.

Prostanoid production in Saccharomyces cerevisiae provides a novel assay for nonsteroidal anti-inflammatory drugs.

Prostanoids are a large family of lipid mediators originating from prostaglandin H synthase (PGHS) activity on the 20-carbon polyunsaturated fatty acids dihomo-gamma-linolenic acid (DGLA), arachidonic acid (AA) and eicosapentaenoic acid. The two mouse PGHS isoforms, PGHS-1 and PGHS-2, were expressed in Saccharomyces cerevisiae (yeast), as was a signal-peptide-deleted version of PGHS-1 (PGHS-1MA). PGHS-1 showed high activity with both AA and DGLA as substrate, whereas PGHS-2 activity was high with DGLA but low with AA. Signal peptide removal reduced the activity of PGHS-1MA by >50% relative to PGHS-1, but the residual activity indicated that correct targeting to the lumen of the endoplasmic reticulum may not be necessary for enzyme function. Coexpression of PGHS-1 with cDNAs encoding mouse prostaglandin I synthase and thromboxane A synthase, and with Trypanosoma brucei genomic DNA encoding prostaglandin F synthase in AA-supplemented yeast cultures resulted in production of the corresponding prostanoids, prostaglandin I(2), thromboxane A(2) and prostaglandin F(2alpha). The inhibitory effects of nonsteroidal anti-inflammatory drugs (NSAIDs) on prostanoid production were tested on yeast cells expressing PGHS-1 in AA-supplemented culture. Dose-dependent inhibition of prostaglandin H(2) production by aspirin, ibuprofen and indomethacin demonstrated the potential utility of this simple expression system in screening for novel NSAIDs.

Cyclooxygenase and prostaglandin synthases in atherosclerosis: recent insights and future perspectives.

Cyclooxygenase (COX) is the key enzyme in the conversion of arachidonic acid to prostanoids, lipid mediators involved in several physiological and pathological processes. Two COX isoenzymes have been characterized, COX-1 and COX-2, that differ in terms of regulatory mechanisms of expression, tissue distribution, substrate specificity, and preferential coupling to upstream and downstream enzymes. Both isoforms play fundamental roles in atherothrombosis; however, whereas the function of COX-1 in this setting is well established, the role of COX-2 remains unclear. Indeed, the intracellular pathways regulating COX-2 induction appear numerous and complicated, varying between cell types and cellular stimulus. In recent years a long series of studies has been performed with the aim of clarifying the role of COX-2 in atherothrombosis, with the major finding that the COX-2 expression pattern in arterial vessels may be associated with either protective or plaque-destabilizing phenotypes according to the downstream synthase that couples with COX-2. In this review we summarize the role of COX-2 as well as the different downstream synthases in atherosclerosis and atherothrombosis. Finally, we briefly review the controversial vascular effects on prostanoid inhibition by COX-2 inhibitors.

Up-regulated thromboxane production in the rat liver with biliary obstruction does not contribute to promote hepatic injury.

This study sought to determine whether in vivo inhibition of thromboxane A2 (TXA2) action contribute to attenuate hepatic damage after bile duct ligation (BDL). Male Wistar rats were assigned to sham operation or BDL. At the time of operation, infusion pump with saline, ozagrel natrium (TXA2 synthase inhibitor), or SQ29548 (TXA2 receptor antagonists) was implanted in the abdominal cavity. Plasma alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, and total bilirubin levels were measured at 4 days after the operation. The levels of plasma TXB2, a stable metabolite of TXA2, were significantly increased after BDL. Gene expression of TXA2 synthase was also significantly upregulated in the liver. Nonetheless, either an inhibition of TXA2 synthesis by ozagrel natrium or a blockade of TXA2 receptor by SQ29548 has no effect in every measured parameter related to hepatic function. These results indicated that despite a highly increased production in the liver, TXA2 is not directly related to the hepatic injury in BDL rats.

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium.

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.

Role of COX-2, thromboxane A2 synthase, and prostaglandin I2 synthase in papillary thyroid carcinoma growth.

The development of papillary thyroid carcinoma is influenced by many factors including genetic alterations, growth factors, and physical agents such as radiation. Arachidonic acid and its derivatives including prostaglandins (PG) and thromboxane along with the enzymes involved in their synthesis have been shown to influence the growth of various tumors. We analyzed the immunoreactivity for cyclooxygenase-2 (COX-2) and mRNA expression levels of the enzymes COX-2, thromboxane A(2) (TXA(2)) synthase, and PGI(2) synthase by RT-PCR in papillary carcinomas and matching normal tissues to determine the role of these enzymes in the development of papillary thyroid carcinomas. A papillary thyroid carcinoma cell line TPC-1 was also studied in vitro to determine the role of the specific COX-2 inhibitor NS-398 on COX-2 and vascular endothelial growth factor-A, since COX-2 also has a role in regulating tumor angiogenesis. RT-PCR analysis showed significant increases in TXA(2) synthase mRNA levels in papillary thyroid carcinomas compared to normal thyroid tissues. Although COX-2 mRNA levels were generally increased in papillary carcinomas, the differences were not statistically significant. There were no significant differences in PGI(2) synthase mRNA levels. COX-2 protein expression was greater in papillary carcinoma compared to normal thyroid tissues; however, the levels were quite variable. In vitro studies with a COX-2 inhibitor, NS-398, showed inhibition of tumor growth along with increased levels of COX-2 and vascular endothelial growth factor-A mRNA expression. These results indicate that specific enzyme levels in the PG synthesis pathway such as TXA(2) synthase are increased in papillary thyroid carcinomas. COX-2 also has a role in papillary thyroid growth, since a specific inhibitor of COX-2 regulates papillary thyroid carcinoma cell proliferation. These results implicate several enzymes in the synthesis of prostanoids as regulators of thyroid papillary carcinoma proliferation and suggest that increased levels of expression of these enzymes may play a role in the pathogenesis of these tumors.

Expression of cyclooxygenase-2 and thromboxane synthase in non-neoplastic and neoplastic thyroid lesions.

Cyclooxygenase-2 (COX-2) and thromboxane synthase (TBXAS) are important enzymes involved in the arachidonic acid pathway and synthesis of prostaglandins. We examined COX-2 and TBXAS immunoreactivity in 150 surgically resected thyroid specimens using immunohistochemistry to determine expression in benign and malignant thyroid lesions and to examine their roles in thyroid tumor progression. Papillary thyroid carcinomas and follicular carcinomas expressed higher levels of COX-2 compared to follicular adenomas and adenomatous nodules. We showed for the first time that TBXAS was expressed in thyroid tissues, with higher levels in papillary carcinomas compared to non-neoplastic and benign thyroid tissues. Western blot was performed on seven thyroid samples. These results indicate that both COX-2 and TBXAS are expressed in benign and malignant thyroid tissues. Although some malignant thyroid tumors showed higher levels of COX-2 expression, COX-2 and TBXAS are probably not useful in the immunohistochemical diagnosis of thyroid malignancies. However, the expression of both COX-2 and TBXAS by thyroid tissue may provide insight into the role of these enzymes in progression from benign to malignant thyroid tumors.

Analysis of Cox-2 and thromboxane synthase expression in pituitary adenomas and carcinomas.

Recent studies have examined the role of cyclooxygenase-2 (Cox-2) expression in normal pituitaries and pituitary adenomas and have suggested a role for Cox-2 in the regulation of angiogenesis in the pituitary. Thromboxane synthase (TBXAS), which catalyzes the synthesis of thromboxane A2, is one of the downstream enzymes in Cox metabolism and appears to play a role in the regulation of invasiveness and angiogenesis in some neoplasms. To analyze the role of Cox-2 and TBXAS in pituitary tumor progression, we examined normal pituitaries (n = 8), pituitary adenomas (n = 174), and pituitary carcinomas (n = 7) for expression of Cox-2 and TBXAS by immunohistochemistry. Weak Cox-2 and moderate TBXAS expression was present in normal pituitary cells. Most pituitary adenomas showed increased expression of both Cox-2 and TBXAS. Pituitary tumors as a whole, but particularly carcinomas, showed greater Cox-2 expression than did normal pituitaries. Pituitary adenomas and carcinomas also showed greater staining for TBXAS when compared to normal pituitary. Nonfunctional adenomas had significantly higher levels of TBXAS expression compared to functional adenomas (p = 0.017). Adenomas and carcinomas showed similar degrees of staining for TBXAS. In summary, TBXAS appears to be one of the up-regulated downstream enzymes of Cox metabolism in pituitary tumors. Both Cox-2 and TBXAS may play an important role in pituitary tumor development and progression.

Differential expression of thromboxane synthase in prostate carcinoma: role in tumor cell motility.

Arachidonic acid metabolism through cyclooxygenase, lipoxygenase, or P-450 epoxygenase pathways can generate a variety of eicosanoids. Thromboxane synthase (TxS) metabolizes the cyclooxygenase product, prostanglandin H(2), into thromboxane A(2) (TXA(2)), which can cause vessel constriction, platelet activation, and aggregation. Here we demonstrate that human prostate cancer (PCa) cells express enzymatically active TxS and that this enzyme is involved in cell motility. In human PCa cell lines, PC-3, PC-3M, and ML-2 cells expressed higher levels of TxS than normal prostate epithelial cells or other established PCa cell lines such as DU145, LNCaP, or PPC-1. We cloned and sequenced the full-length TxS cDNA from PC-3 cells and found two changes in the amino acid residues. Immunohistochemical analysis of tumor specimens revealed that expression of TxS is weak or absent in normal differentiated luminal, or secretory cells, significantly elevated in less differentiated or advanced prostate tumors, and markedly increased in tumors with perineural invasion. TxS expressed in PC-3 cells was enzymatically active and susceptible to carboxyheptal imidazole, an inhibitor of TxS. The biosynthesis of TXA(2) in PC-3 cells was dependent on COX-2, and to a lesser extent, COX-1. Treatment of PC-3 cells with a COX-1 selective inhibitor, piroxicam, reduced TXA(2) synthesis by approximately 40%, while the COX-2 specific inhibitor NS398 reduced TXA(2) production by approximately 80%. Inhibition of TxS activity or blockade of TXA(2) function reduced PC-3 cell migration on fibronectin, while having minimal effects on cell cycle progression or survival. Finally, increased expression of TxS in DU145 cells increased cell motility. Our data suggest that human PCa cells express TxS and that this enzyme may contribute to PCa progression through modulating cell motility.

Functional and biochemical evaluation of platelet aspirin resistance after coronary artery bypass surgery.

BACKGROUND: Aspirin inhibits platelet activation and reduces atherothrombotic complications in patients at risk of myocardial infarction and stroke. However, a sufficient inhibition of platelet function by aspirin is not always achieved. The causes of this aspirin resistance are unknown. METHODS AND RESULTS: Patients undergoing coronary artery bypass grafting (CABG) have a high incidence of aspirin resistance. To evaluate functional and biochemical responses to aspirin, platelet-rich plasma was obtained before and at days 1, 5, and 10 after CABG. Thromboxane formation, aggregation, and alpha-granule secretion were effectively inhibited by 30 or 100 micromol/L aspirin in vitro before CABG, but this inhibition was prevented or attenuated after CABG. Whereas the inhibition of thromboxane formation and aggregation by aspirin in vitro partly recovered at day 10 after CABG, oral aspirin (100 mg/d) remained ineffective. The inducible isoform of cyclooxygenase in platelets, COX-2, has been suggested to confer aspirin resistance. In fact, immunoreactive COX-2 was increased 16-fold in platelets at day 5 after CABG, but the COX-2 selective inhibitor celecoxib did not alter aspirin-resistant thromboxane formation. By contrast, the combined inhibitor of thromboxane synthase and thromboxane receptor antagonist terbogrel equally prevented thromboxane formation of platelets obtained before (control) and after CABG. CONCLUSIONS: Platelet aspirin resistance involves an impairment of both in vivo and in vitro inhibition of platelet functions and is probably due to a disturbed inhibition of platelet COX-1 by aspirin.

Cellular localization of thromboxane synthase in ovine spinal cord and hindbrain.

We and others have demonstrated that endogenously-produced prostanoids modify the function of the hypothalamus-pituitary-adrenal (HPA) axis. We have demonstrated that exogenously-administered thromboxane mimetic stimulates ACTH secretion in fetal sheep, and that the endogenous production of thromboxane modifies the HPA response to cardiovascular stress. The purpose of this study was to identify the structures within the fetal and adult ovine medulla and hindbrain which express immunoreactive thromboxane synthase. Using immunohistochemical techniques, we demonstrated thromboxane synthase immunostaining in regions important for cardiovascular afferent signaling (nucleus tractus solitarius, ventrolateral medulla) in both cell bodies and axons. Thromboxane synthase was also apparent in neuroanatomical locations which are consistent with afferent and efferent projections from the cerebellum. We observed staining in the superior cerebellar peduncle in the rostal pons, in the corticopontocerebellar fibers, and in Purkinje cells. The enzyme was found in motor regions, including the dorsal motor nucleus of the vagus nerve, and in the motor neurons of the dorsal column of the spinal cord. In addition to the apparent neuronal staining, there was positive staining in the ventricular ependymal cells. We conclude that, consistent with physiological evidence, thromboxane synthase is present in brain regions which are important for afferent and efferent cardiovascular signaling.

COX-2-dependent cardiac failure in Gh/tTG transgenic mice.

Gh is a GTP binding protein that couples to the thromboxane receptor (TP), but also functions as tissue transglutaminase II (tTG). A transgenic mouse model was generated in which Gh was overexpressed (GhOE) in ventricular myocytes under the control of the alpha-myosin heavy chain promoter. Heart rate was elevated and both blood pressure and left ventricular ejection fraction were depressed in GhOEs. Left ventricular mass was increased, consistent with genetic and ultrastructural evidence of hypertrophy. Fibrosis and apoptosis were also augmented. Survival declined disproportionately in older GhOEs. Cardiomyocyte expression of COX-2, thromboxane synthase (TxS), and the receptors for TxA2 (the TP), PGF2alpha (the FP), and PGI2 (the IP) were upregulated and urinary 8,12-iso-iPF2alpha-VI,2,3-dinor-6-keto-PGF1alpha and 2,3-dinor-thromboxane B2 were increased in GhOEs, reflecting increased lipid peroxidation and cyclooxygenase (COX) activation. Selective COX-2 inhibition, TP antagonism, and suppression of lipid peroxidation each rescued the cardiac phenotype. Infusion of an FP agonist exacerbated the phenotype, whereas administration of an IP agonist improved cardiac function. Directed cardiac overexpression of Gh/tTG causes both TG activation and increased TP/Gh-dependent signaling. The COX-2-dependent increase in TxA2 generation augments cardiac hypertrophy, whereas formation of PGI2 by the same isozyme ameliorates the phenotype. Oxidant stress may contribute, via regulation of COX-2 expression and/or ligation of the TP and the FP by isoprostanes. Gh/tTG activation regulates expression of COX-2 and its products may differentially modulate cardiomyocyte commitment to cell death or survival.

Intraluminal pressure modulates eicosanoid enzyme expression in vascular endothelium of intact human conduit vessels at physiological levels of shear stress.

OBJECTIVE: Biosynthesis of eicosanoid metabolites in blood vessels regulates vascular tone and platelet function. We investigated whether intraluminal pressure modulates gene and protein expression of key eicosanoid enzymes in intact human conduit vessels and/or release of their vasoactive metabolites. METHODS: Paired segments of human umbilical veins were perfused under laminar flow for 1.5, 3 and 6 h at high versus low intraluminal pressure (40/20 mmHg) with identical shear stress (10 dyn/cm(2)). Endothelial cell mRNAs encoding cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostaglandin synthase (PGS), and thromboxane synthase (TXS) were measured by quantitative real-time RT-PCR. Secretion of PGI2 and TXA2 to the perfusion medium was measured by enzyme immunoassay of their metabolites 6-keto-prostaglandin F(1alpha) and TXB2. RESULTS: Intraluminal pressures were 39.9 +/- 0.02 and 20.0 +/- 0.03 mmHg (P < 0.0001) in high and low pressure circuits, and shear stress levels were 10.6 +/- 0.60 and 9.7 +/- 0.36 dyn/cm(2) (NS, not significant). COX-1 mRNA was significantly up-regulated after 1.5 h of high pressure stimulation and continued up to 3 h, but fell thereafter significantly below baseline after 6 h. COX-2 mRNA was initially significantly down-regulated, followed by a significant up-regulation after 6 h. Gene expressions of PGS and TXS were significantly induced after 6 h of high pressure perfusion. High pressure depressed the production of PGI(2) (P < 0.05) but did not alter TXA(2) formation. CONCLUSIONS: Intraluminal pressure has differential effects on gene and protein expression of key eicosanoid enzymes and biosynthesis of prostanoid metabolites in intact human conduit vessels. The new, computerized biomechanical perfusion system may be a useful tool to elucidate specific effects of various biomechanical forces on intact mammalian conduit vessels.

CD44-mediated cyclooxygenase-2 expression and thromboxane A2 production in RAW 264.7 macrophages.

OBJECTIVE AND DESIGN: CD44 is the major cell surface receptor for hyaluronan (HA) on macrophages. Stimulation of macrophages via the HA-CD44 pathway leads to the enhanced expression of inflammatory gene products, including cytokines, chemokines, and adhesion molecules. We have examined whether activation of CD44 by crosslinking is capable of activating the cyclooxygenase (COX) and prostaglandin (PG)/thromboxane (TX) pathway in cultured macrophages. MATERIALS AND METHODS: CD44 was crosslinked on RAW 264.7 mouse macrophages using specific rat anti-mouse CD44 monoclonal antibodies and anti-rat IgG. Total RNA was extracted and subjected to RT-PCR analysis for genes of the PG/TX synthetic pathway. Supernatants were analyzed for PGE2 and TXB2 using specific ELISAs. RESULTS: Transcripts for COX-1, COX-2, TX synthase (TXS), and PGE2 synthase (PGES) were all constitutively expressed in the mouse macrophage cell line RAW 264.7. Crosslinking of CD44 markedly enhanced COX-2 and weakly increased TXS mRNA, whereas COX-1 and PGES mRNA did not change significantly in these cells. Crosslinking of CD44 selectively increased the production of TXB2 but not PGE2. CONCLUSIONS: These findings suggest that the activation of the CD44 pathway plays a unique role in PG synthesis. Activation of this pathway results in enhanced TXA2 but not PGE2 production. This leads to an imbalance of the TXA2/PGE2 profile which favors a proinflammatory and vasoconstrictory response.