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.

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.

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.

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.

Novel Protein Encoded by C6orf105 Regulates Tissue Factor Pathway Inhibitor Expression and Function In Human Endothelial Cells In Normal Conditions and During Androgen Stimulation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 348-348
Author(s):  
Cristina Lupu ◽  
Hua Zhu ◽  
Jonathan Wren ◽  
Florea Lupu
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Western Blot ◽  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Anticoagulant Activity ◽  
Uncharacterized Protein ◽  
Tissue Factor Pathway ◽  
Anticoagulant Function ◽  
And Function

Abstract Abstract 348 Cardiovascular disease (CVD) and thrombotic complications (deep vein thrombosis/venous thromboembolism, DVT/VTE) represent major health problems, with men having higher rates of clinical events than women. Tissue Factor Pathway Inhibitor (TFPI) is the key natural inhibitor of coagulation: it neutralizes factor Xa (FXa) and inhibits tissue factor-factor VIIa (TF-FVIIa) in the presence of FXa. In vivo most of TFPI is in endothelial cells (EC), reversibly bound to yet unidentified receptors, and glycosyl phosphatidylinositol-floated in caveolae and/or lipid raft microdomains. Intravascular thrombosis occurs frequently in older people, especially associated with cancer, diabetes, or CVD. TF is directly involved in tumor hypercoagulability, angiogenesis and metastasis. Cell-associated TFPI is the most physiologically significant inhibitor of the TF-FVIIa- triggered coagulation pathway; nevertheless, very few mechanisms/factors that could regulate the natural expression of TFPI have been identified so far. Here we describe androgen treatment of EC as a novel way to preserve and/or enhance a healthy vascular function, particularly related to the regulation of TFPI-dependent anticoagulant function of the endothelium. Our hypothesis is that a yet uncharacterized protein encoded by C6orf105 is a novel regulator of TFPI expression and function in EC, both in native conditions and during androgen stimulation. “In silico” data mining using global meta-analysis of publicly available NCBI's Gene Expression Omnibus 2-channel human microarray datasets identified C6orf105 as highly co-expressed with TFPI and following a parallel co-regulation. The uncharacterized protein has 230-aa, Mr ∼27 kDa, 5–6 predicted transmembrane domains and has sequence similarities with members of the androgen-inducible genes family. We tentatively named it TFPI-Regulating Factor (TFPI-RF). Real-time qPCR and western blot confirmed robust expression of TFPI-RF in EC in culture (HUVEC and EA.hy926 hybrid cell line). By immunofluorescence (IMF) TFPI-RF appears both on the cell surface and intracellularly co-localizing with TFPI and caveolin-1 (cav-1). Post-transcriptional (siRNA) down-regulation of TFPI-RF decreased TFPI, both as protein (∼2-times) and as anticoagulant activity (∼3-fold), apparently by reducing the co-localization of the TF-FVIIa-FXa-TFPI complex with cav-1. Over-expression of TFPI-RF in HUVEC and EA.hy926 led to enhanced co-localization of TFPI-RF with TFPI, and increased TFPI mRNA and anticoagulant activity (∼2-times). Western blot of cellular fractions after extraction with Triton X-114 and temperature-induced phase separation revealed the presence of TFPI and TFPI-RF in detergent-insoluble fractions, which suggests predominant lipid raft association. IMF illustrates TFPI-RF co-clustering with TFPI and cav-1 or GM1 (raft marker) in live EC incubated with anti-TFPI antibody or Cholera Toxin-B, respectively. The effect of androgens was studied by incubating EC with 30 nM dehydrotestosterone (DHT) or equivalent testosterone-BSA (cell-impermeable). 1-h incubation led to 2-times enhanced TFPI activity, increased co-localization of the quaternary complex with cav-1 and TFPI-RF, and enhanced exposure of TFPI and TFPI-RF on the cell surface. 24-h treatment with DHT up-regulates the expression of both TFPI (2-fold) and TFPI-RF (3-fold), as well as the TFPI inhibitory activity against FXa. DHT failed to enhance TFPI activity in TFPI-RF siRNA EC. Our results reveal a novel mechanism of up-regulation of the anticoagulant activity of endogenous TFPI in response to physiological levels of androgen. While the precise role of androgens in the ageing process is unclear, it is believed that androgen replacement could have beneficial influence on the declining functions in the elderly. Our data could expand on the effects of androgens on the haemostatic function of the endothelium and discover new roles for novel proteins like C6orf105/TFPI-RF in enhancing the endothelial anticoagulant function. These may open possibilities to manipulate the cellular endogenous TFPI and/or other intrinsic factors to counteract pro-thrombotic states associated with CVD, DVT/VTE, sepsis and cancer. Disclosures: No relevant conflicts of interest to declare.

Curcumin Prevents Cytokine-Mediated Tissue Factor Pathway Inhibitor Down-Regulation in Human Endothelial Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1202-1202
Author(s):  
Keiko Maruyama ◽  
Eriko Morishita ◽  
Yukie Goto ◽  
Akiko Sekiya ◽  
Hidesaku Asakura ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Western Blotting ◽  
Tissue Factor Pathway Inhibitor ◽  
The Other ◽  
Tnf Alpha ◽  
Down Regulation ◽  
Protein Levels ◽  
Other Hand ◽  
Tissue Factor Pathway

Abstract Abstract 1202 Objective: Curcumin (diferuloyl methane), an active component of the spice turmeric, has been shown to exhibit anti-inflammatory and antioxidant activities in addition to an anticartinogenic activity in vitro and in vivo. Furthermore, we reported that curcumin inhibited the induction of tissue factor (TF) expression in human umbilical vein endothelial cells (HUVECs) at 52nd ASH 2010. Therefore, curcumin may ameliorate hyper-coagulable state associated with inflammation or oxidative stress. On the other hand, tissue factor pathway inhibitor (TFPI) which is expressed by endothelial cells plays a crucial role in hemostasis by regulating TF-induced initiation of coagulation. This study examined whether curcumin modulates the expression of TFPI in HUVECs. Methods: HUVECs were pretreated with curcumin at the concentration of 20 μM for 3h, washed and stimulated with tumor necrosis factor-alpha (TNF-alpha, 10 ng/ml) for additional 12 or 24h. The mRNA and protein levels of TFPI in the cultured HUVECs were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting, respectively. To determine whether curcumin affects the MAPK signaling pathways, the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) in the HUVECs were analyzed with western blotting. Additionally, to determine whether curcumin affects nuclear factor-kappa B (NF-kB) pathway, nuclear and cytoplasmic fractions were extracted and protein levels were determined by western blotting for NF-kB (p65), p-IkB and IkB. Results: After TNF-alpha stimulation, TFPI mRNA levels were approximately decreased by 40% compared to the control (p<0.05; Figure 1B). Similarly to the mRNA expression, TFPI protein levels were decreased (Figure 1A). On the other hand, pretreatment of HUVECs with curcumin significantly suppresses TNF-alpha-induced TFPI mRNA and protein down-regulation (p<0.05; Figure 1A, B). Curcumin inhibited TNF-alpha-induced activation of p38MAPK, ERK1/2, and JNK. Moreover, curcumin inhibits TNF-alpha-induced IkB activation in HUVECs. And, translocation of NF-kB from the cytosol into the nucleus by TNF-alpha was inhibited by curcumin. Conclusions: These results indicate that curcumin may suppress the TNF-alpha-induced TFPI down-regulation via NF-kB pathways. Thus, curcumin may offer a novel antithrombotic option for treatment of the hypercoagulable state associated with inflammation. Disclosures: No relevant conflicts of interest to declare.

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.

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