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1.
Cardiovasc Res ; 73(4): 806-12, 2007 Mar 01.
Article in English | MEDLINE | ID: mdl-17266944

ABSTRACT

OBJECTIVES: The aim of this study was to investigate the effect of the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) on the ionizing radiation (IR)- and tumor necrosis factor-alpha (TNF-alpha) induced tissue factor (TF) expression and its release from human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were irradiated with a single dose of either 5 Gy or 10 Gy and stimulated with TNF-alpha (10 ng/mL) in the presence or absence of PDTC and NAC, respectively. Quantitative real-time PCR, ELISA, and TF activity measurements were performed, including TF activity in the supernatant. Apoptosis was detected by flow cytometric active caspase-3 measurement and formation of reactive oxygen species (ROS) by chemiluminescence. RESULTS: We demonstrated a thus far uninvestigated persistent induction of TF expression in HUVECs after treatment with IR and TNF-alpha. Combined stimulation with IR and TNF-alpha led to an immense shedding of microparticle-associated TF which was positively correlated with apoptosis and ROS formation. Antioxidative pre-treatment reduced not only apoptosis and ROS formation, but also the release of thrombogenic microparticles. CONCLUSIONS: Antioxidative treatment inhibited apoptosis and shedding of microparticles, thereby reducing thrombogenicity. Thus, antioxidants may help to prevent late thrombosis after antiproliferative treatment when used in combination with anticoagulants.


Subject(s)
Acetylcysteine/pharmacology , Antioxidants/pharmacology , Endothelial Cells/metabolism , Pyrrolidines/pharmacology , Thiocarbamates/pharmacology , Thromboplastin/metabolism , Analysis of Variance , Apoptosis , Caspase 3/analysis , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/radiation effects , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Luminescent Measurements , Radiation, Ionizing , Reactive Oxygen Species/analysis , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thromboplastin/analysis , Tumor Necrosis Factor-alpha/pharmacology , Umbilical Veins
2.
Trends Cardiovasc Med ; 16(5): 177-82, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16781952

ABSTRACT

Alternatively spliced tissue factor (asTF) has recently been discovered as a soluble form of tissue factor (TF), which circulates in blood and exhibits procoagulant activity. This soluble TF variant expanded the concept of circulating TF by a further element. Up to 30% of the TF antigen found in circulating blood was proposed to be derived from alternative splicing. We showed that cytokines induced the expression of asTF and the release from endothelial cells. The use of plasma asTF as a clinical marker for an inflammation-associated dysregulated hemostasis may therefore be a novel approach in predicting the patients' prognosis. This review covers the latest findings in the field of soluble TF focusing on asTF and its potential role besides the one in coagulation.


Subject(s)
Alternative Splicing , Blood Coagulation , Hemostasis/physiology , Thromboplastin/metabolism , Animals , Humans , Protein Isoforms/blood , Protein Isoforms/chemistry , Thromboplastin/chemistry
4.
Circ Res ; 96(12): 1233-9, 2005 Jun 24.
Article in English | MEDLINE | ID: mdl-15920023

ABSTRACT

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-alpha and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-alpha (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-alpha and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223+/-0.0069 versus 0.0012+/-0.0006 for control, P<0.001 and 0.0022+/-0.0004 versus 0.0012+/-0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-alpha stimulation, supporting factor Xa generation after relipidation (6.55+/-1.13 U versus 2.99+/-0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.


Subject(s)
Blood Coagulation , Endothelial Cells/metabolism , Inflammation/blood , Interleukin-6/pharmacology , Thromboplastin/biosynthesis , Tumor Necrosis Factor-alpha/pharmacology , Cells, Cultured , Humans , Lipoproteins/analysis , Lipoproteins/physiology , Phospholipids/physiology , RNA, Messenger/analysis , Thromboplastin/genetics , p38 Mitogen-Activated Protein Kinases/physiology
5.
J Am Coll Cardiol ; 45(7): 1081-9, 2005 Apr 05.
Article in English | MEDLINE | ID: mdl-15808768

ABSTRACT

OBJECTIVES: We investigated the myocardial localization and expression of tissue factor (TF) and alternatively spliced human tissue factor (asHTF) in patients with dilated cardiomyopathy (DCM). BACKGROUND: Tissue factor is expressed in cardiac muscle and may play a role in maintaining myocardial structure. METHODS: Myocardial biopsies were obtained from patients with a normal or mildly impaired ejection fraction (EF) (> or =50%) and moderate to severely reduced EF (<50%). Explanted DCM hearts were also examined. Myocardial TF expression level was assessed by real-time polymerase chain reaction, TF protein by enzyme-linked immunosorbent assay, and localization by immunohistochemistry. RESULTS: We report the identification of asHTF in the human myocardium: it was located in cardiomyocytes and endothelial cells. Quantification of myocardial TF messenger ribonucleic acid in DCM revealed a decrease in the TF/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio (1.76 x 10(-1) +/- 6.08 x 10(-2) for EF > or =50% [n = 19] vs. 1.06 x 10(-1) +/- 5.26 x 10(-2) for EF <50% [n = 27]; p < 0.001) and asHTF/GAPDH ratio (13.91 x 10(-5) +/- 11.20 x 10(-5) for EF > or =50% vs. 7.17 x 10(-5) +/- 3.82 x 10(-5) for EF <50%; p = 0.014). Tissue factor isoform expression level was also decreased in explanted DCM hearts (p < 0.01; n = 12). Total TF protein was reduced by 26% in DCM (p < 0.05). The TF/GAPDH ratio correlated positively with the EF (r = 0.504, p < 0.0001). Immunohistochemistry showed TF localized to the sarcolemma and Z-bands of the cardiomyocytes in patients with normal EF, whereas TF was found in the cardiomyocytic cytosol around the nucleus in DCM. CONCLUSIONS: Tissue factor was down-regulated in the myocardium of DCM patients. The reduction in TF expression and change in localization may influence cell-to-cell contact stability and contractility, thereby contributing to cardiac dysfunction in DCM.


Subject(s)
Cardiomyopathy, Dilated/metabolism , Thromboplastin/metabolism , Blotting, Western , Cardiomyopathy, Dilated/pathology , Case-Control Studies , DNA Primers , Female , Gene Expression , Humans , Immunohistochemistry , Male , Middle Aged , Myocardium/metabolism , Myocardium/pathology , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Severity of Illness Index , Thromboplastin/genetics
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