Induction of Endothelial Tissue Factor by Endotoxin and Its Precursors

1993 ◽  
Vol 70 (04) ◽  
pp. 702-706 ◽  
Author(s):  
Charles F Moldow ◽  
Ronald R Bach ◽  
Katherine Staskus ◽  
Paul D Rick
Keyword(s):  
Tissue Factor ◽  
Lipid A ◽  
Umbilical Vein ◽  
Structural Determinants ◽  
Monophosphoryl Lipid A ◽  
Maximal Induction ◽  
Umbilical Vein Endothelial Cells ◽  
Acyloxyacyl Hydrolase ◽  
Endothelial Tissue ◽  
Specific Mrna

SummaryThe structural determinants of lipopolysaccharide required for the induction of tissue factor in human umbilical vein endothelial cells were studied. Intact lipid A was essential for the induction of tissue factor whereas the incomplete lipid A precursors lipid IVA and lipid X, as well as monophosphoryl lipid A and acyloxyacyl hydrolase-treated lipopolysaccharide, were unable to induce tissue factor and tissue factor specific mRNA. However, the lipid A precursor, lipid IVA, was able to inhibit LPS-mediated induction of tissue factor; structural determinants distal to lipid A were found to be required for maximal induction of tissue factor activity and tissue factor mRNA. The presence of serum in the assay was found to amplify but was not obligate for tissue factor induction by LPS.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

Lysophosphatidylcholine Decreases the Synthesis of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

1998 ◽  
Vol 79 (01) ◽  
pp. 217-221 ◽  
Author(s):  
Koichi Kokame ◽  
Toshiyuki Miyata ◽  
Naoaki Sato ◽  
Hisao Kato
Keyword(s):  
Endothelial Cells ◽  
Mrna Expression ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Mrna Levels ◽  
Down Regulation ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

SummaryThrombotic complications are frequently associated with atherosclerosis. Lysophosphatidylcholine (LPC), a component accumulated in oxidatively modified LDL (ox-LDL), is known to play a crucial role in the initiation and progression of atherosclerotic vascular lesions. Since a vascular anticoagulant, tissue factor pathway inhibitor (TFPI), has the function of regulating the initial reaction of tissue factor (TF)-induced coagulation, we investigated the effect of LPC on TFPI synthesis in cultured human umbilical vein endothelial cells (HUVEC). The treatment of HUVEC with LPC for 24 h decreased TFPI antigen levels in both the culture medium and the cell lysate in a dose-dependent manner. Northern blot analysis revealed that LPC caused a time-dependent decrease in the TFPI mRNA levels. The levels of TFPI antigen and mRNA were decreased to 72% and 38%, respectively, by the incubation with 50 μM LPC for 24 h. The down-regulation by LPC of TFPI mRNA expression was not observed in the presence of cycloheximide, suggesting that protein synthesis was involved in the suppression of TFPI mRNA expression. The TFPI mRNA levels in actinomycin D-treated cells were relatively stable, indicating that the down-regulation of TFPI mRNA by LPC would be partly explained by the enhanced mRNA destabilization. In contrast to the significant down-regulatory effects of LPC on TFPI expression, LPC did not induce TF mRNA expression in HUVEC. These results indicate that LPC accumulated in the atherosclerotic vascular wall would suppress endothelial TFPI synthesis, reducing the antithrombotic property of endothelial cells.

scholarly journals Association of Tissue Factor Pathway Inhibitor With Human Umbilical Vein Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3568-3578 ◽  
Author(s):  
John-Bjarne Hansen ◽  
Randi Olsen ◽  
Paul Webster
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Coagulation Factors ◽  
Coagulation System ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

AbstractTissue factor pathway inhibitor (TFPI) is a serine protease inhibitor of the extrinsic coagulation system, synthesized in endothelial cells, which has recently been shown to play an important role in the regulation of activated coagulation factors at the endothelial cell surface. In the present study we investigated the subcellular localization and metabolism of TFPI in human umbilical vein endothelial cells (HUVEC). Immunocytochemical labeling of HUVEC with anti-TFPI showed specific labeling associated with the cell surface and with many intracellular organelles including the Golgi complex. Further characterization of these organelles was performed by colocalizing the anti-TFPI with 3-(2,4-dinitroanilino)′-amino-N-methyldipropylamine (DAMP; to demonstrate low pH), mannose phosphate receptor (endosomes), and LAMP 1 (late endocytic compartments). TFPI also colocalized with antibodies to the human transferrin receptor, a marker for early endocytic, recycling compartment. Endogenous TFPI colocalized with biotin in intracellular vesicles during endocytosis after biotinylation of the cell surface, which indicated that TFPI was being co-internalized with the surface biotin. The binding of exogenously added 125I-TFPI increased linearly to HUVEC over the concentration range of 0 to 32 nmol/L without saturation, the binding was not affected by up to a thousand-fold molar excess of unlabeled TFPI, and heparin inhibited the binding dose dependently. An intact C-terminal domain was important for the interaction between TFPI and the cell surface of HUVEC, because less than 10% of a C-terminal truncated form of TFPI (TFPI1-161 ) was bound after addition of equimolar concentrations of full-length TFPI. Exogenously added 125I-TFPI was not degraded in HUVEC during 4 hours at 37°C. The presence of TFPI in endocytic and recycling compartments support the hypothesis that endogenous, membrane-anchored TFPI could be internalized for subsequent recycling back to the cell surface.

scholarly journals The principal eosinophil peroxidase product, HOSCN, is a uniquely potent phagocyte oxidant inducer of endothelial cell tissue factor activity: a potential mechanism for thrombosis in eosinophilic inflammatory states

Blood ◽  
2006 ◽  
Vol 107 (2) ◽  
pp. 558-565 ◽  
Author(s):  
Jian-Guo Wang ◽  
Shawn A. Mahmud ◽  
Jacob A. Thompson ◽  
Jian-Guo Geng ◽  
Nigel S. Key ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Umbilical Vein ◽  
Hypereosinophilic Syndrome ◽  
Eosinophil Peroxidase ◽  
Cell Tissue ◽  
Reactive Oxidant ◽  
Tf Gene ◽  
Umbilical Vein Endothelial Cells ◽  
Kinase Pathway

AbstractIn vivo, bromide (Br–), nitrite (NO2–), and thiocyanate (SCN–) compete for oxidation by eosinophil peroxidase (EPO) and H2O2, yielding, respectively, HOBr, NO2·, and HOSCN. We have recently shown that SCN– is the strongly preferred substrate for EPO in vivo and that HOSCN, in contrast with other EPO-generated oxidants and HOCl, is a relatively weak, cell-permeant, sulfhydryl (SH)–reactive oxidant. We here show that HOSCN is a uniquely potent (up to 100-fold) phagocyte oxidant inducer of tissue factor (TF) activity in human umbilical vein endothelial cells (HUVECs). This induction is attributable to transcriptional up-regulation of TF gene expression dependent upon both activation of the p65/c-Rel TF-κB transcription factor and activity of the ERK1/2 kinase pathway upstream of Egr-1 and was markedly further enhanced in the presence of wortmannin, an inhibitor of the PI3 kinase/Akt pathway. HOSCN also markedly activates the proinflammatory p65/p50 NF-κB pathway. Based on these findings we hypothesize that HOSCN generated by adherent and infiltrating eosinophils may provoke the development of a prothrombotic and proinflammatory endothelial/endocardial phenotype that promotes the pronounced thrombotic diathesis characteristic of the hypereosinophilic syndrome.

Effects of Fluvastatin on the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2253-2253
Author(s):  
Keiko Maruyama ◽  
Eriko Morishita ◽  
Hiroki Torishima ◽  
Akiko Sekiya ◽  
Hidesaku Asakura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Protein Expression ◽  
Western Blot ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
The Other ◽  
Mrna And Protein Expression ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

Abstract Abstract 2253 OBJECTIVE: 3-Hydroxyl-3-methyl coenzyme A reductase inhibitors (statins) inhibit the production of mevalonate and other isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP). Statins can protect the vasculature from inflammation and atherosclerosis caused by cholesterol-dependent and cholesterol-independent mechanisms. The latest investigations show that statins modulate the expression of genes related to inflammation, blood coagulation and fibrinolysis in cultured endothelial cells. Tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. The aim of this study was to elucidate the effects of fluvastatin, lipophilic statin, on expressions of TFPI in human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were incubated for 24 h in culture medium including fluvastatin (0.1, 1.0, 10.0 μM). The expression of TFPI mRNA and protein was evaluated by western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. To identify which product of statin reaction is necessary for the effect of fluvastatin, HUVECs were incubated for 24h with fluvastatin with mavalonate, FPP, or GGPP. On the other hand, it is known that fluvastatin increase nitric oxide (NO) bioavailability. To determine whether fluvastatin induced NO affects TFPI mRNA and protein expression, HUVECs were incubated for 24h with fluvastatin with NG-Nitro-L-arginine methyl ester, hydrochloride (L-NAME: specific inhibitor of NO synthase). Additionally, to determine whether fluvastatin affects p38MAPK, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), and protein kinase C (PKC) pathways, HUVECs were incubated for 24h with fluvastatin with the inhibitors of p38MAPK (SB203580), JNK (SP600125), MEK (U0126), PI3K (LY294002), and PKC (GF109203). The expression of TFPI mRNA and protein was evaluated by western blot. RESULTS: Fluvastatin increased TFPI mRNA and protein expression (1μM: p<0.01, 10μM: p<0.05; Figure 1). This fluvastatin-dependent up-regulation of TFPI was prevented by mevalonate and geranylgeranylphosphate (GG-PP). In contrast, the addition of L-NAME did not alter induction of TFPI expression by fluvastatin. Similarly, Y-27632 (Rho kinase inhibitor) and NSC23766 (Rac1 inhibitor) were ineffective. Additionally, the inhibitors of p38MAPK, PI3K, and PKC prevented fluvastatin-dependent up-regulation. On the other hand, the inhibitors of JNK and MEK were ineffective. CONCLUSIONS: This study suggests that fluvastatin significantly increases TFPI mRNA and protein expression, and this effect of fluvastatin is accompanied by the activation of p38 MAPK, PI3K, and PKC pathways. Therefore, this effect may play an important role in preventing cardiovascular events. Disclosures: No relevant conflicts of interest to declare.

Recombinant factor VIIa enhances deposition of platelets with congenital or acquired αIIbβ3 deficiency to endothelial cell matrix and collagen under conditions of flow via tissue factor–independent thrombin generation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1864-1870 ◽  
Author(s):  
Ton Lisman ◽  
Sultana Moschatsis ◽  
Jelle Adelmeijer ◽  
H. Karel Nieuwenhuis ◽  
Philip G. De Groot
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Recombinant Factor Viia ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Novel Approach ◽  
Platelet Deposition ◽  
Platelet Aggregate ◽  
Umbilical Vein Endothelial Cells ◽  
Thrombin Formation

A novel approach to treat bleeding episodes in patients with Glanzmann thrombasthenia (GT) and perhaps also in patients receiving αIIbβ3 inhibitors is the administration of recombinant factor VIIa (rFVIIa). The mechanism of action of rFVIIa in these patients is, however, still unclear. We studied the effect of rFVIIa-mediated thrombin formation on adhesion of αIIbβ3-deficient platelets under flow conditions. Adhesion of αIIbβ3-deficient platelets to the extracellular matrix (ECM) of stimulated human umbilical vein endothelial cells or to collagen type III was studied using a model system with washed platelets and red cells. When αIIbβ3-deficient platelets were perfused over the surface at arterial shear rate for 5 minutes, a low surface coverage was observed (GT platelets, mean ± SEM, 37.5% ± 5.0%; normal platelets preincubated with an RGD-containing peptide, 7.4% ± 2.1%). When rFVIIa, together with factors X and II, was added to the perfusate, platelet deposition significantly increased (GT platelets, mean ± SEM, 67.0% ± 4.3%; normal platelets preincubated with an RGD-containing peptide, 48.2% ± 2.9%). The same effect was observed when normal platelets were pretreated with the commercially available anti-αIIbβ3 drugs abciximab, eptifibatide, or tirofiban. It was shown that tissue factor–independent thrombin generation (presumably induced by binding of rFVIIa to adhered platelets) was responsible for the increase in platelet deposition. In conclusion, defective adhesion of αIIbβ3-deficient platelets to ECM can be restored by tissue factor–independent rFVIIa-mediated thrombin formation. The enhanced generation of platelet procoagulant surface facilitates fibrin formation, so that lack of platelet aggregate formation might be compensated for.

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