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

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.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614242 ◽

1998 ◽

Vol 79 (01) ◽

pp. 217-221 ◽

Cited By ~ 13

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.

Download Full-text

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

Blood ◽

10.1182/blood.v118.21.2253.2253 ◽

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.

Download Full-text

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

Thrombosis and Haemostasis ◽

10.1160/th04-01-0033 ◽

2004 ◽

Vol 92 (10) ◽

pp. 776-786 ◽

Cited By ~ 52

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.

Download Full-text

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

Blood ◽

10.1182/blood.v90.9.3568 ◽

1997 ◽

Vol 90 (9) ◽

pp. 3568-3578 ◽

Cited By ~ 20

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.

Download Full-text

Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to inflammatory mediators

Blood ◽

10.1182/blood.v79.12.3219.3219 ◽

1992 ◽

Vol 79 (12) ◽

pp. 3219-3226 ◽

Cited By ~ 68

Author(s):

A Ameri ◽

MN Kuppuswamy ◽

S Basu ◽

SP Bajaj

Keyword(s):

Endothelial Cells ◽

Blood Serum ◽

Tissue Factor ◽

Inflammatory Mediators ◽

Whole Blood ◽

Tissue Factor Pathway Inhibitor ◽

Binding Motif ◽

Cultured Endothelial Cells ◽

The Media ◽

Tissue Factor Pathway

Abstract We recently proposed that endothelium may represent the primary physiologic site of synthesis of the tissue factor pathway inhibitor (TFPI). In support of this conclusion, we have now found that the poly(A)+ RNAs obtained from rabbit and bovine lung tissues contain abundant amounts of TFPI messenger RNAs (mRNAs), whereas the poly(A)+ RNAs obtained from the liver of these animals contain less than 5% of that found in the lung tissues. Because inflammatory mediators are known to upregulate tissue factor (TF) expression by the endothelium, we have examined the effect of these agents on the TFPI expression by the cultured endothelial cells. When cultured human umbilical vein endothelial cells were stimulated (in 10% fetal bovine serum) with phorbol myristate acetate (PMA), endotoxin, interleukin-1, or tumor necrosis factor-alpha, the TF mRNA increased approximately 7- to 10- fold within 2 to 4 hours. Unstimulated cells constitutively expressed TFPI mRNA and its levels either did not change or increased slightly (up to 1.5-fold) upon stimulation with these inflammatory agents. TF mRNA abruptly declined to a negligible level and the TFPI mRNA returned essentially to the basal level at approximately 24 hours. The membrane- bound TF clotting activity of induced cells peaked between 4 and 8 hours, and finally declined. The cumulative TFPI activity secreted into the media was either unchanged or slightly higher in the induced cell cultures as compared with that present in the noninduced cultures. Endothelial cells were also cultured in 10% heat-inactivated human serum derived from plasma or whole blood. TFPI secreted into the media containing whole blood serum was consistently higher (approximately 1.5- fold at 8 hours) than that secreted into the media supplemented with serum obtained from plasma lacking the formed elements; these cells also expressed similarly increased levels of TFPI mRNA. Moreover, PMA- stimulated cells cultured in whole blood serum expressed modestly increased levels of TFPI mRNA (approximately 1.5-fold); supernatants from these cells also contained similarly increased TFPI activity. Cumulatively, our data indicate that, unlike thrombomodulin and fibrinolytic enzymes synthesized by the endothelial cells, TFPI synthesis is not downregulated and may be slightly upregulated during an inflammatory response. Inspection of the 5′ flanking region of the TFPI gene showed a conserved GATA-binding motif located approximately 400 bp upstream of the proposed transcription initiation site(s). This motif by binding to the GATA-2 transcriptional factor may keep the endothelium in an ‘on’ state for constitutive expression of TFPI.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins

Thrombosis and Haemostasis ◽

10.1160/th07-10-0623 ◽

2008 ◽

Vol 99 (03) ◽

pp. 576-585 ◽

Cited By ~ 19

Author(s):

Mathieu Provençal ◽

Marisol Michaud ◽

Édith Beaulieu ◽

David Ratel ◽

Georges-Étienne Rivard ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Tissue Factor ◽

Focal Adhesion ◽

Tissue Factor Pathway Inhibitor ◽

Endothelial Cell Migration ◽

Cell Functions ◽

Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range,TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glio- blastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion,suggesting an alteration of focal adhesion complex integrity. Accordingly,we observed thatTFPI inhibited the phosphorylation of focal adhesion kinase and paxillin,two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.

Download Full-text

Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

Blood ◽

10.1182/blood-2014-10-604587 ◽

2015 ◽

Vol 125 (9) ◽

pp. 1488-1496 ◽

Cited By ~ 34

Author(s):

Cristina Puy ◽

Erik I. Tucker ◽

Anton Matafonov ◽

Qiufang Cheng ◽

Keith D. Zientek ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Factor X ◽

Factor Xi ◽

Extrinsic Pathway ◽

Fibrin Formation ◽

Key Points ◽

Tissue Factor Pathway ◽

Factor X Activation

Key Points Activated factor XI binds and proteolyzes tissue factor pathway inhibitor. Activated factor XI promotes factor X activation generation and fibrin formation through the inactivation of tissue factor pathway inhibitor from platelets and on endothelial cells.

Download Full-text