Localization of Tissue Factor Pathway Inhibitor In Endothelial Cells: Pitfalls and Challenges

1999 ◽  
Vol 5 (S2) ◽  
pp. 1124-1125
Author(s):  
R. Olsen ◽  
J.-B. Hansen ◽  
P. Webster
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Light And Electron Microscopy ◽  
Coagulation Factors ◽  
Endocytic Pathway ◽  
Coagulation System ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

Tissue Factor Pathway Inhibitor (TFPI) is a potent inhibitor of the extrinsic coagulation system, synthesized in endothelial cells. Membrane bound TFPI has recently been shown to play an important role in the degradation of activated coagulation factors in the endothelium. Comparison of subcellular TFPI localization patterns and TFPI production between Human Umbilical Vein Endothelial Cells (HUVEC) and an immortalized cell line (ECV 304) are presented here, as are the essential protocol modifications that we used to obtain labeling results by both light- and electron microscopy. We show that ECV 304 cells are similar to HUVEC in that TFPI is present in the Golgi complex (fig. 1) as well as in the endocytic pathway of ECV cells. Additionally, they secrete higher levels of TFPI into the culture medium than HUVEC. A colocalization of anti-TFPI with endocytosed BSA-gold, as well as with accumulated DAMP, demonstrated the presence of TFPI in the endocytic pathway.

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.

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.

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.

Crucial roles of GATA-2 and SP1 in adrenomedullin-affected expression of tissue factor pathway inhibitor in human umbilical vein endothelial cells exposed to lipopolysaccharide

2007 ◽  
Vol 97 (05) ◽  
pp. 839-846 ◽  
Author(s):  
Wen Wang ◽  
Shu-Yu Zu ◽  
Wei Liu ◽  
Zi-Qiang Zhu ◽  
Guang-Jin Zhu
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Mutational Analysis ◽  
Umbilical Vein ◽  
Regulatory Sequences ◽  
Mobility Shift ◽  
Human Umbilical Vein ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

SummaryAdrenomedullin (ADM) is a vasodilator peptide that has a variety of effects, including antithrombotic activities and resistant roles to lipopolysaccharide (LPS)-induced septic shock. During sepsis,LPS triggers the development of disseminated intravascular coagulation (DIC) via the tissue factor-dependent pathway of coagulation.It is unknown whether the antithrombotic activities of ADM contribute to its resistance to sepsis. In the present study, we investigated the effects of ADM on tissue factor pathway inhibitor (TFPI) (primary anticoagulant factor) expression in human umbilical vein endothelial cells (HUVECs) exposed to LPS,and the possible underlying mechanism for these effects.Exposure of HUVECs to LPS for 12 hours caused significant decrease of TFPI protein activities and mRNA expression.These effects were abolished by treatment withADM (10–10 to 10–6 M), cAMP analogue and calcium antagonist. Accordingly, cAMP antagonist inhibited the counteraction effect of ADM on LPS in TFPI expression. Electrophoresis mobility shift assay (EMSA) and Western blot analysis showed that the protein level of GATA-2 and SP1 transcriptional factors and their binding to the corresponding regulatory sequences decreased by LPS treatment. And these effects of LPS were antagonized by ADM. Promoter- reporter assays and mutational analysis also confirmed the roles of GATA-2 and SP1 motifs from –1247 to –381bp promoter sequence in TFPI inducible expression. Taken together, these results indicate that ADM antagonizes the effect of LPS on TFPI expression, which is mediated by affecting transcriptional factor GATA-2 and SP1 through cAMP and calcium signaling pathway.

Tissue factor and tissue factor pathway inhibitor levels in trophoblast cells: implications for placental hemostasis

2004 ◽  
Vol 92 (10) ◽  
pp. 776-786 ◽  
Author(s):  
Benjamin Brenner ◽  
Tamar Katz ◽  
Yohei Miyagi ◽  
Naomi Lanir ◽  
Anat Aharon
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Maternal Blood ◽  
Placental Function ◽  
Protein Levels ◽  
Tissue Factor Pathway ◽  
Low Levels ◽  
Umbilical Vein Endothelial Cells

SummaryThe placenta is a highly vascularized organ with fetal and maternal blood supply. Syncytiotrophoblasts (STB), which line the placenta villous are possibly involved in local hemostatic mechanisms.The aim of this study was to evaluate the levels of tissue factor (TF) and its inhibitors, tissue factor pathway inhibitor (TFPI,TFPI-2), in STB model within hemostatic and inflammatory environments. Human primary STB cell cultures were characterized by vascular and hormonal markers. TF and TFPI mRNA expression, protein levels and activity were determined and compared to human umbilical vein endothelial cells (HUVEC). High levels of TF were demonstrated in STB cells compared to low levels in HUVEC. In contrast, STB expressed lower TFPI levels than HUVEC. LPS and TNFα increased the high constitutive TF in STB, whereas LPS and IL-1α further reduced TFPI levels. The procoagulant tendency of STB identified by us may reflect the physiological need for immediate inhibition of hemorrhage in the placental inter-villous spaces in basal and inflammatory conditions.This hemostatic balance may be critical for normal placental function and pregnancy outcome.

scholarly journals Further Insight Into the Heparin-Releasable and Glycosylphosphatidylinositol-Lipid— Anchored Forms of Tissue Factor Pathway Inhibitor

2008 ◽  
Vol 14 (3) ◽  
pp. 267-278 ◽  
Author(s):  
Paul E.R. Ellery ◽  
Kathy Hardy ◽  
Robert Oostryck ◽  
Murray J. Adams
Keyword(s):  
Phospholipase C ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Fold Increase ◽  
Activity Levels ◽  
Dependent Manner ◽  
Tissue Factor Pathway ◽  
Umbilical Vein Endothelial Cells

The release of tissue factor pathway inhibitor (TFPI) from human umbilical vein endothelial cells (HUVECs) was investigated using heparin and phospholipase C. The experiment included incubating HUVECs with 0, 1, or 10 U/mL heparin diluted in Dulbecco Modified Eagle's Medium plus 5% fetal calf serum for 1 or 24 hours. A statistically significant increase in TFPI activity levels was seen at 1 hour, but not at 24 hours. A 20-fold increase in the release of TFPI after phospholipase C treatment of HUVECs was demonstrated, confirming that it is glycosylphosphatidylinositol-lipid (GPI) anchored. Sequential treatment of HUVECs with phospholipase C and heparin was performed, and a trend was observed where GPI-anchored TFPI levels were increased after 1 hour of pretreatment with heparin but were decreased after 24 hours. Serum is a requirement for the heparin-dependent release of TFPI from HUVECs. Heparin pretreatment of HUVECs may affect levels of GPI anchored TFPI in a time and dose-dependent manner.

scholarly journals Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix

2021 ◽  
Author(s):  
Julie A. Peterson ◽  
Susan A. Maroney ◽  
Nicholas D. Martinez ◽  
Alan E. Mast
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Surface ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Umbilical Vein ◽  
Human Kidney ◽  
Laminin 5 ◽  
C Terminus ◽  
Tissue Factor Pathway

Objective: Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα. Approach and Results: ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures. Conclusions: ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.

Subcellular Localozation of TFPI in Human Umbilical Vein Endothelial Cells (HUVEC)

1996 ◽  
Vol 54 ◽  
pp. 44-45
Author(s):  
Randi Olsen ◽  
Paul Webster ◽  
John Bjarne Hansen
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Factor Viia ◽  
Umbilical Vein ◽  
Factor Vii ◽  
Coagulation System ◽  
Regulatory Function ◽  
Human Umbilical Vein ◽  
Main Site ◽  
Umbilical Vein Endothelial Cells

Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor which regulates tissue factor (TF) induced blood coagulation by inhibiting the action of factor VIIa-TF complexes and factor Xa. In plasma, more than 80 % of the TFPI is bound to lipoproteins, 10 -20 % is carrier free, whereas platelets contains a small amount of TFPI. A major pool is probably assosiated with the vascular endothelium (1). This pool may have a crucial regulatory function because the endothelial surface serves as a binding site for factor VII/VIIa, mediating the activation of the extrinsic coagulation system.The normal distribution of TFPI within human umbilical vein endothelial cells (HUVEC) was examined on ultrathin cryosections labeled with specific antibodies and colloidal gold probes (2,3). Label was seen associated with the cell surface and with many intracellular structures. The surface label was patchy, the signal intensity varied between cells and between different parts of the same cell. Intracellular TFPI was found in many compartments including the Golgi complex (fig.l) a finding that was expected as these cells are the main site of TFPI synthesis, and endocytic organelles. Anti TFPI-antibodies were seen over organelles that also contained 3-(2,4-Dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), a compound that accumulates in organelles with low lumenal pH. DAMP was visualized using anti-DNP antibodies (fig.2). Structures that contained exclusively TFPI or DAMP were also seen, suggesting that TFPI did not enter all endocytic organelles and was not exclusively localized to low pH compartments. The TFPI-antibodies also colocalized with specific markers of endosomes (MPR) (fig.3) and lysosomes (LAMP-1) (fig.4)(4). TFPI colocalized with both, demonstrating the appearence of TFPI also in these endocytic compartments.

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