Cardiac glycosides regulate endothelial tissue factor expression in culture.

BACKGROUND: Tissue factor (TF) plays an important role in acute coronary syndromes and stent thrombosis. This study investigates whether Na(+)/K(+)-ATPase regulates TF expression in human endothelial cells. METHODS AND RESULTS: Ouabain inhibited tumor necrosis factor (TNF)-alpha-induced endothelial TF protein expression; maximal inhibition occurred at 10(-5) mol/L, reached more than 70%, and was observed throughout the 5 hours stimulation period. The decrease in protein expression was paralleled by a reduced TF surface activity. Similarly, lowering of extracellular potassium concentration inhibited TNF-alpha-induced TF protein expression. In contrast, ouabain did not affect TNF-alpha-induced expression of full-length TF mRNA for up to 5 hours of stimulation; instead, expression of alternatively-spliced TF mRNA was upregulated after 3 and 5 hours of stimulation. Ouabain did not affect TNF-alpha-induced activation of the MAP kinases p38, extracellular signal-regulated kinase (ERK), and c-Jun terminal NH(2) kinase; activation of Akt and p70S6 kinase remained unaltered as well. Similar to the MAP kinases, ouabain did not affect TNF-alpha-induced degradation of IkappaB-alpha. Ouabain had no effect on TF protein degradation. CONCLUSIONS: Na(+)/K(+)-ATPase is required for protein translation of endothelial TF in culture. This observation provides novel insights into posttranscriptional regulation of TF expression.

Dimethyl sulfoxide inhibits tissue factor expression, thrombus formation, and vascular smooth muscle cell activation: a potential treatment strategy for drug-eluting stents.

BACKGROUND: Subacute stent thrombosis is a major clinical concern, and the search for new molecules to cover stents remains important. Dimethyl sulfoxide (DMSO) is used for preservation of hematopoietic progenitor cells and is infused into patients undergoing bone marrow transplantation. Despite its intravenous application, the impact of DMSO on vascular cells has not been assessed. METHODS AND RESULTS: In human endothelial cells, monocytes, and vascular smooth muscle cells (VSMC), DMSO inhibited tissue factor (TF) expression and activity in response to tumor necrosis factor-alpha or thrombin in a concentration-dependent manner. DMSO did not exert any toxic effects as assessed by phase-contrast microscopy, trypan blue exclusion, and lactate dehydrogenase release. Real-time polymerase chain reaction revealed that inhibition of TF expression occurred at the mRNA level. This effect was mediated by reduced activation of the mitogen-activated protein kinases c-Jun terminal NH2 kinase (51+/-6%; P=0.0005) and p38 (50+/-3%; P<0.0001) but not p44/42 (P=NS). In contrast to TF, DMSO did not affect expression of TF pathway inhibitor or plasminogen activator inhibitor-1. In vivo, DMSO treatment suppressed TF activity (41%; P<0.002) and prevented thrombotic occlusion in a mouse carotid artery photochemical injury model. DMSO also inhibited VSMC proliferation (70%; P=0.005) and migration (77%; P=0.0001) in a concentration-dependent manner; moreover, it prevented rapamycin and paclitaxel-induced upregulation of TF expression. CONCLUSIONS: DMSO suppresses TF expression and activity, as well as thrombus formation; in addition, it inhibits VSMC proliferation and migration. Given its routine use in modern clinical practice, we propose DMSO as a novel strategy for coating drug-eluting stents and treating acute coronary syndromes.

Paclitaxel enhances thrombin-induced endothelial tissue factor expression via c-Jun terminal NH2 kinase activation.

Paclitaxel is used on drug-eluting stents because it inhibits proliferation of vascular cells. Stent thrombosis remains a concern with this compound, particularly with higher dosages. This study investigates the effect of paclitaxel on tissue factor (TF) expression in human endothelial cells. Paclitaxel enhanced thrombin-induced endothelial TF protein expression in a concentration- and time-dependent manner. A concentration of 10(-5) mol/L elicited a 2.1-fold increase in TF protein and a 1.6-fold increase in TF surface activity. The effect was similar after a 1 hour as compared with a 25-hour pretreatment period. Real-time polymerase chain reaction revealed that paclitaxel increased thrombin-induced TF mRNA expression. Paclitaxel potently activated c-Jun terminal NH2 kinase (JNK) as compared with thrombin alone, whereas the thrombin-mediated phosphorylation of p38 and extracellular signal-regulated kinase remained unaffected. Similar to paclitaxel, docetaxel enhanced both TF expression and JNK activation as compared with thrombin alone. The JNK inhibitor SP600125 reduced thrombin-induced TF expression by 35%. Moreover, SP600125 blunted the effect of paclitaxel and docetaxel on thrombin-induced TF expression. Paclitaxel increases endothelial TF expression via its stabilizing effect on microtubules and selective activation of JNK. This observation provides novel insights into the pathogenesis of thrombus formation after paclitaxel-eluting stent deployment and may have an impact on drug-eluting stent design.

Extracellular ATP determines 11beta-hydroxysteroid dehydrogenase type 2 activity via purinergic receptors.

Hypertension and sodium retention are features of a diminished 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2). The activity of this enzyme is reduced in various disease states with abnormal renal sodium retention and hypertension, including preeclampsia. ATP release to the extracellular compartment is observed with shear stress, inflammation, and placental ischemia. It was hypothesized that ATP downregulates 11beta-HSD2 activity. For that purpose, cell lines from different tissues that previously were used to study the regulation of 11beta-HSD2 were investigated: JEG-3, a vascular trophoblastic; LLCPK1, a renal tubular; and SW620, a colonic epithelial cell line. The 11beta-HSD2 activity, assessed by the conversion of 3H-cortisol to cortisone, was reversibly reduced during incubation with ATP or its stable analogue ATPgammaS in intact JEG-3 and LLCPK1, but not in SW620 cells. In JEG-3 cells, the purinergic antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid but not suramin reversed the inhibition. Incubation with UTP and ADP and their degradation products including adenosine and alpha,beta-methylene-ATP did not inhibit 11beta-HSD2 activity. In contrast, 11beta-HSD2 activity increased almost 2.5-fold after incubation with 2'-methylthio-ATP. This indicates a bidirectional regulation by nucleotides via purinergic receptors. In JEG-3 cells, ATP/ATPgammaS did not alter 11beta-HSD2 promoter activity but reduced 11beta-HSD2 protein and mRNA concentration and half-life, suggesting a posttranscriptional regulation. In conclusion, ATP inhibits cell type specifically via purinergic receptors the expression and activity of the 11beta-HSD2 by a posttranscriptional mechanism.

Evidence for compromised aldosterone synthase enzyme activity in preeclampsia.

BACKGROUND: In normal pregnancy, an increased aldosterone (Aldo) concentration coincides with volume expansion. In preeclampsia, Aldo levels are low despite intravascular volume depletion. The present investigation aimed to characterize the compromised Aldo synthesis in preeclampsia, and to identify the molecular basis hereof. METHODS: We recruited 66 pregnant women (24 uneventful, 42 preeclamptic). Genomic DNA was isolated from peripheral blood leukocytes. Urine samples were obtained for gas chromatography-mass spectroscopic measurements of steroid hormones reflecting apparent Aldo synthase (CYP11B2) and 11-hydroxylase (CYP11B1) activities. Polymerase chain reaction (PCR)-based screening for CYP11B2 mutations was performed by SSCP, restriction analysis, and sequencing. RESULTS: CYP11B1 activity was unaltered, but reduction of mean tetrahydro (TH)-Aldo excretion by a factor of 3.9 indicated a diminished CYP11B2 activity in preeclampsia. Accordingly, the ratios of (TH-11-dehydrocorticosterone [A]+TH-corticosterone [B]+5alpha-THB) to (TH-cortisone +TH-cortisol [F]+5alpha-THF) and of 18-OH-THA to THAldo were increased in preeclampsia 2.6- and 15.2-fold, respectively, indicating reduced Aldo synthesis due to diminished methyl oxidase (MO) activity. A lower percentage of women with normal pregnancies had CYP11B2 mutations when compared to preeclamptic women (P < 0.05). Eight polymorphisms were detected, two of which were non-amino acid conserving. Of those, the mutation V386A, earlier found to jeopardize MO activity, was exclusively observed in preeclampsia (0% vs. 17%; P < 0.05). CONCLUSION: Aldo deficiency due to a compromised MO step of Aldo synthesis favors extracellular volume depletion, and may account for an increased risk of placental hypoperfusion and consecutive development of preeclampsia. The sole presence of mutation V386A in preeclamptic mothers may identify a subgroup with an increased risk to develop preeclampsia during pregnancy.

Angiotensin II regulates 11beta-hydroxysteroid dehydrogenase type 2 via AT2 receptors.

BACKGROUND: In preeclampsia, cortisol degradation by the enzyme 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) is compromised, which enhances intracellular cortisol availability. This leads to vasoconstriction and renal sodium retention with volume expansion, thus increasing blood pressure. An augmented availability of angiotensin II (Ang II) predisposes to preeclampsia. Some effects of Ang II are mediated by the mitogen-activated protein kinase (MAPK) cascade, which also regulates 11beta-HSD2 activity. Therefore, we hypothesized that Ang II regulates 11beta-HSD2. METHODS: The human choriocarcinoma cell line JEG-3, which expresses the 11beta-HSD2 isoenzyme, was used. 3H-cortisol/cortisone conversion assays and mRNA analyses by reverse transcription-polymerase chain reaction (RT-PCR) were performed. Cells were stimulated with Ang II and the effect was modulated by Ang II type 1 (AT1) and AT2 receptor blockers DUP753 or L-158809 and PD-123319, respectively. In order to elucidate the signaling cascade, the MAPK kinase inhibitors PD-098059 and U-0126 were probed. The impact of a modulated 11beta-HSD2 activity was assessed by determining the effect of cortisol on AT1 receptor mRNA. RESULTS: Ang II reduced mRNA and activity of 11beta-HSD2 mainly by a post-transcriptional mechanism. This Ang II effect was abrogated by AT2, but not by AT1 receptor blockade. Mitogen-activated protein (MAP) kinase kinase (MAPKK) inhibitors reversed the Ang II effect. Dexamethasone augmented the mRNA expression of AT1 receptors. Cortisol enhanced AT1 receptor mRNA expression when the 11beta-HSD2 activity was reduced either by Ang II or by glycyrrhetinic acid, an 11beta-HSD2 inhibitor. CONCLUSION: Ang II decreases the activity of 11beta-HSD2 by an AT2 receptor- and MAPK-dependent mechanism. The decreased activity of 11beta-HSD2 increases the intracellular availability of cortisol, which might be relevant for the pathogenesis of hypertension and preeclampsia.