Tissue Factor Pathway Inhibitor mRNA Expression Research Articles

Effects of andrographolide on the expression of procoagulant and fibrinolytic inhibition factors in rat type II alveolar epithelial cells stimulated by lipopolysaccharide

To determine the effect of andrographolide (AD) on the expression of procoagulant and fibrinolytic inhibitory factors in rat type II alveolar epithelial cells (AEC II) stimulated by lipopolysaccharide (LPS). The AEC II cells RLE-6TN in the logarithmic growth phase were divided into 5 groups: the normal control (NC) group, the LPS group, and the 6.25, 12.5, and 25 mg/L AD groups (AD 6.25 group, AD 12.5 group, AD 25 group). The NC group was cultured with RPMI 1640 conventional medium. In the LPS group, 5 mg/L LPS was added to the RPMI 1640 conventional medium for stimulation. Cells in the AD groups were treated with 6.25, 12.5, and 25 mg/L AD in advance for 1 hour and then given LPS to stimulate the culture. The cells and cell culture supernatant were collected 24 hours after LPS stimulation. The protein and mRNA expressions of tissue factor (TF), tissue factor pathway inhibitor (TFPI), and plasminogen activator inhibition-1 (PAI-1) in cells were detected by Western blotting and real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The levels of procollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT), antithrombin III (AT-III) and activated protein C (APC) in the cell supernatant were detected by enzyme linked immunosorbent assay (ELISA). Compared with the NC group, the protein and mRNA expressions of TF and PAI-1 in the LPS group were significantly increased, and the protein and mRNA expressions of TFPI were significantly reduced. At the same time, the levels of PIIIP and TAT in the cell supernatant were significantly increased, the levels of AT-III, APC were significantly reduced. Compared with the LPS group, the protein and mRNA expressions of TF and PAI-1 in AD 6.25 group, AD 12.5 group, AD 25 group were significantly reduced [TF/GAPDH: 0.86±0.08, 0.45±0.04, 0.44±0.04 vs. 1.32±0.10, TF mRNA (2-ΔΔCt): 2.59±0.25, 2.27±0.05, 1.95±0.04 vs. 4.60±0.26, PAI-1/GAPDH: 2.11±0.07, 1.45±0.04, 0.86±0.09 vs. 2.56±0.09, PAI-1 mRNA (2-ΔΔCt): 3.50±0.22, 2.23±0.29, 1.84±0.09 vs. 6.60±0.27, all P < 0.05], while the protein and mRNA expressions of TFPI were significantly increased [TFPI/GAPDH: 0.78±0.05, 0.81±0.03, 0.84±0.07 vs. 0.36±0.02, TFPI mRNA (2-ΔΔCt): 0.46±0.09, 0.69±0.07, 0.91±0.08 vs. 0.44±0.06, all P < 0.05]. Also the levels of PIIIP and TAT in the cell supernatant were significantly reduced, and the levels of AT-III and APC were significantly increased [PIIIP (μg/L): 13.59±0.23, 12.66±0.23, 10.59±0.30 vs. 15.82±0.29, TAT (ng/L): 211.57±6.41, 205.69±4.04, 200.56±9.85 vs. 288.67±9.84, AT-III (μg/L): 102.95±3.86, 123.92±2.63, 128.67±1.67 vs. 92.93±3.36, APC (μg/L): 1 188.95±14.99, 1 366.12±39.93, 1 451.15±29.69 vs. 1 145.55±21.07, all P < 0.05]. With the increase of the dose of AD, the above-mentioned promotion and inhibition effects became more obvious. In the AD 25 group, TF, PAI-1 protein and mRNA expressions decreased, TFPI mRNA expression increased, PIIIP level in the supernatant decreased and AT-III, APC levels increased compared with AD 6.25 group, the difference was statistically significant, and the decrease of PAI-1 protein expression and PIIIP level in the supernatant were also statistically significant compared with AD 12.5 group. Andrographolide in the dose range of 6.25-25 mg/L can dose-dependently inhibit the expression and secretion of procoagulant and fibrinolytic inhibitor-related factors in AEC II cells RLE-6TN stimulated by LPS, and promote the secretion of anticoagulant factors. 25 mg/L has the most obvious effect.

Transcription factor FOXP3: A repressor of the TFPI gene?

Single nucleotide polymorphisms (SNPs) may play an important role in the risk of certain diseases. We have previously shown that the -287T/C SNP of the tissue factor pathway inhibitor (TFPI) gene promoter region exerts differential impact on TFPI mRNA expression; the C allele being associated with higher TFPI expression, which in turn is associated with reduced risk of thrombosis. In the present study, we aimed to reveal the underlying molecular mechanisms using human embryonic kidney 293 (HEK293) and Michigan Cancer Foundation-7 (MCF7) cells that both express TFPI. Transfecting the cells with luciferase reporter gene constructs containing the TFPI promoter with either the T or the C allele of -287T/C resulted in increased luciferase activity with the C allele relative to the T allele. Three potential candidate transcription factors for binding to the two -287 alleles were predicted using the ALGGEN PROMO software, and results from electrophoretic mobility shift assays indicated that forkhead box protein 3 (FOXP3), initially identified as a functional marker of T regulator cells, bound more specifically to the T allele compared with the C allele. By chromatin immunoprecipitation assays analysis it was confirmed that FOXP3 was able to bind to the DNA region that contains the SNP. Knockdown or overexpression of FOXP3 resulted in increased or decreased TFPI levels, respectively, in both cell types. In conclusion, this study indicates that FOXP3 most likely is involved in the increased levels of TFPI observed with the -287C allele and also that FOXP3 might be a repressor for TFPI expression.

A novel hypoxia response element regulates oxygen-related repression of tissue factor pathway inhibitor in the breast cancer cell line MCF-7

BackgroundHypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. AimTo explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. Methods and resultsQuantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at −170 to +21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5′-AAACAGGA-3′ for HIF-2α/EPAS1 within the TFPI promoter located at −45 to −38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. ConclusionsThis study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors.

Abstract 13974: Loss of Endothelial Primary Cilia Promotes Thrombus Formation in Mice

Introduction: Recent evidence suggest that endothelial cilia may suppress anti-inflammatory signaling and protect against atherosclerosis. Given that atherosclerosis predisposes to thrombosis, we hypothesized that endothelial cilia may also play a fundamental role in thrombogenesis. Hypothesis: We hypothesize that endothelial cilia is important for protection against thrombosis, and that loss of endothelial cilia promotes thrombus formation in mice. Methods: We focused on the intraflagellar transport 88 (IFT88) gene since endothelial deletion of IFT88 has been associated with decreased atherosclerotic lesions in a murine model of atherosclerosis. Key thrombosis-associated molecules were evaluated in IFT88-silenced human umbilical vein endothelial cells (HUVECs). Thrombi formation were monitored in the laser-injured cremasteric arteries of 8 week old male endothelial cell (EC) specific IFT88 knockout (EC-IFT88 -/- ) mice. Results: Compared to scrambled siRNA-treated HUVECs, those exposed to siIFT88 exhibited lower mRNA and protein levels of tissue factor pathway inhibitor (TFPI) and thrombomodulin, two key regulators of blood coagulation (-27% and -20%, respectively; N=3; p &lt; 0.05). We next examined the effect of IFT88 loss on thrombosis in mice. Upon laser-induced injury, we determined, via mean fluorescence intensity, that although the thrombi formed in the cremasteric arteries of EC-IFT88 -/- mice were similar in size to those produced in the wild type (WT) mice (2.2 ± 0.6 and 2.0 ± 0.3 AU, respectively; x10 6 ; N=32 thrombi; p=0.8), the average rate at which thrombi developed was significantly faster in the EC-IFT88 -/- mice relative to that in WT mice (97 ± 11 and 57 ± 3 AU/s, respectively; N=32 thrombi; p&lt;0.001). Conclusions: Loss of endothelial IFT88 results in decreased TFPI mRNA and protein expression in HUVECs. EC-IFT88 -/- mice exhibited earlier thrombi formation in our laser injury model of thrombosis. These results collectively suggest that endothelial cilia intricately regulates thrombus formation in a TFPI-dependent manner and represents a previously unrecognized target to modulate thrombosis susceptibility.

Fluvastatin Upregulates the Expression of Tissue Factor Pathway Inhibitor in Human Umbilical Vein Endothelial Cells.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are cholesterol-lowering drugs with a variety of pleiotropic effects including antithrombotic properties. Tissue factor pathway inhibitor (TFPI), which is produced predominantly in endothelial cells and platelets, inhibits the initiating phase of clot formation. We investigated the effect of fluvastatin on TFPI expression in cultured endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with fluvastatin (0-10μM). The expression of TFPI mRNA and antigen were detected by RT-PCR and western blotting, respectively. The effects of mevalonate intermediates, small GTP-binding inhibitors, and signal transduction inhibitors were also evaluated to identify which pathway was involved. A luciferase reporter assay was performed to evaluate the effect of fluvastatin on TFPI transcription. The stability of TFPI mRNA was estimated by quantitating its levels after actinomycin D treatment. Fluvastatin increased TFPI mRNA expression and antigen in HUVECs. Fluvastatin-induced TFPI expression was reversed by co-treatment with mevalonate or geranylgeranylpyrophosphate (GGPP). NSC23766 and Y-27632 had no effect on TFPI expression. SB203580, GF109203, and LY294002 reduced fluvastatin-induced TFPI upregulation. Moreover, fluvastatin did not significantly affect TFPI promoter activity. TFPI mRNA degradation in the presence of actinomycin D was delayed by fluvastatin treatment. Fluvastatin increases endothelial TFPI expression through inhibition of mevalonate-, GGPP-, and Cdc42-dependent signaling pathways, and activation of the p38 MAPK, PI3K, and PKC pathways. This study revealed unknown mechanisms of the anticoagulant effect of statins and gave a new insight to its therapeutic potential for the prevention of thrombotic diseases.

Effect of α-melanocyte stimulating hormone and its novel analogue on the production of tissue factor pathway inhibitor in mice with endotoxemia

To evaluate the effect of α-melanocyte stimulating hormone (α-MSH) and its novel analogue STY39 on the production of tissue factor pathway inhibitor (TFPI) in mice with endotoxemia. Female BALB/c mice were randomly divided into eight groups with 9 mice in each group. Endotoxemia was reproduced by intraperitoneal injection of lipopolysaccharide (LPS, 25 μg/kg) and D-galactosamine (D-Gal, 100 mg/kg). The animals of the control group were given phosphate buffered solution (PBS) instead. In the experimental groups, the mice were injected intraperitoneally with 2.5 mg/kg α-MSH or STY39 at 1, 2 or 3 hours following LPS injection. The orbital blood was collected at different time points, and tissues of lung, liver, and kidney were collected 8 hours after the administration of LPS. The plasma TFPI levels were determined by enzyme linked immunosorbent assay (ELISA), and the expression of TFPI mRNA in different tissues was determined with reverse transcription-polymerase chain reaction (RT-PCR). The plasma TFPI levels began to increase (11.84±1.55 μg/L) in the endotoxemia mice 4 hours after LPS challenge and reached the peak (23.49 ± 1.12 μg/L) at 8 hours. α-MSH or STY39 treatment at 1, 2 or 3 hours after LPS challenge could significantly increase the TFPI content, with the best drug effect at 1 hour after LPS challenge (the blood was collected 8 hours after LPS challenge, α-MSH group: 58.79±2.67 μg/L vs. 28.49±1.69 μg/L, STY39 group: 71.08±2.13 μg/L vs. 28.49±1.69 μg/L, both P<0.01), and the effect of STY39 was better than that of α-MSH (P<0.01). A small amount of TFPI mRNA expression was observed in each tissue of the healthy mice. After LPS challenge, TFPI mRNA expression was increased in all the tissues, especially in the lung, liver and kidney. α-MSH or STY39 treatment at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the lung and liver (A value, α-MSH in lung: 51.10±2.89 vs. 32.43±2.51, STY39 in lung: 72.11±3.48 vs. 32.43±2.51; α-MSH in liver: 43.21±2.12 vs. 29.29±2.06, STY39 in liver: 66.82±1.76 vs. 29.29±2.06, both P<0.01). The treatment with STY39 at 1 hour after LPS challenge could significantly up-regulate the expression of TFPI mRNA in the kidney (A value: 45.21±1.80 vs. 30.44±2.23, P<0.01), but the treatment with α-MSH had no obvious effect (A value: 24.61±1.98 vs. 30.44±2.23, P>0.05). The enhancing effect of early administration of STY39 on TFPI mRNA expression in the lung, liver and kidney tissues of endotoxemia mice was more powerful than that of α-MSH (all P<0.01). The early administration of α-MSH or STY39 may up-regulate TFPI production in the mice with endotoxemia, and the effect of STY39 is superior to α-MSH.

Tumour expresion of tissue factor and tissue factor pathway inhibitor in ovarian cancer- relationship with venous thrombosis risk

Ovarian cancer is known to display a particular association with venous thromboembolism (VTE) with reports up to 42% of patients developing thromboembolic complications. Tissue Factor (TF) and its inhibitor Tissue Factor Pathway Inhibitor (TFPI) have been implicated in VTE risk in cancer. The aim of this study was to measure tumour derived TF and TFPI and to investigate their potential role in VTE in ovarian cancer patients. TF and TFPI mRNA expression was measured using TaqMan real time PCR in 99 ovarian tumour samples. Nineteen cases complicated by VTE were matched to 19 cases without VTE. TF and TFPI protein levels were measured using ELISA and immunohistochemistry was used to localize TF expression. The role of TF expression on overall survival was also determined. TF mRNA and protein expression was increased in tumours from patients with clear cell carcinoma (p<0.001). TF protein expression was also increased in endometroid carcinoma (P<0.01) compared with benign tumours. TFPI mRNA expression was increased in clear cell carcinoma (P<0.01). TF mRNA and antigen level was increased in malignant tumours of patients who developed VTE compared with matched malignant õtumours of patients who remained thrombosis free (P<0.01). There was no difference in TFPI expression between the two groups. TF expression in ovarian cancer is significantly higher in patients who develop VTE. TF expression was increased in clear cell ovarian cancer and endometroid cancer and this may explain the higher risk of VTE in these subgroups. TF derived from these tumours may be the trigger for VTE in ovarian cancer.

PM.06 Low Molecular Heparin Within the Uteroplacental Unit

BackgroundPerturbation of the uteroplacental haemostasis has been implicated in placenta mediated pregnancy complications in thrombophilic women. LMWH may be effective in altering local thrombin production in the uteroplacental compartment.AimWe determined...

Circulating monocytes mirror the imbalance in TF and TFPI expression in carotid atherosclerotic plaques with lipid-rich and calcified morphology

BackgroundThrombogenicity of atherosclerotic plaque largely depends on plaque morphology and their content of tissue factor (TF) and tissue factor pathway inhibitor (TFPI). The relationship between morphological composition of plaque (lipid-rich or calcified) and expression of TF and TFPI in circulating blood monocytes and within the plaques is not characterized. ObjectiveTo investigate whether lipid-rich (echolucent) or calcified (echogenic) morphology of carotid atherosclerotic plaques is associated with differences in TF and TFPI expression in circulating blood monocytes and within carotid atherosclerotic plaques. MethodsWe studied levels of monocyte TF and TFPI mRNA and protein expression and association with traditional risk factors for atherosclerosis in asymptomatic subjects with echolucent (n=20) or echogenic (n=20) carotid plaques, or controls without carotid atherosclerosis (n=20) determined by ultrasonography. Sections of calcified or lipid-rich carotid plaques obtained from symptomatic patients were assessed for TF and TFPI antigen expression. ResultsTF and TFPI surface presentation, surface TF/TFPI ratio, and TF activity were higher in monocytes obtained from subjects with echolucent than with echogenic plaques or controls without carotid atherosclerosis. Multiple regression analyses revealed inverse association between serum apoA1 and monocyte surface TF antigen expression (p=0.007), and positive association between serum apoB and monocyte surface TFPI expression (p=0.028). Sections from lipid-rich carotid plaques contained 2.5-fold more TF and 1.5-fold more TFPI antigens relative to calcified lesions, also yielding a higher TF/TFPI ratio. ConclusionsOur findings indicate that circulating monocytes of asymptomatic individuals with echolucent lipid-rich carotid atherosclerosis express an imbalance between TF and TFPI expression cohering with changes found within advanced carotid atherosclerotic plaques obtained from symptomatic patients.

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

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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Characterization of the Procoagulant Potential of Endothelial Colonies Forming Cells and Comparison with Two Cell Therapy Products: Relevance for Vascular Cell Therapy

Abstract 5317 Background:Endothelial colonies forming cells (ECFC) are good candidates for cell-based therapy in cardiovascular diseases. Concerns have been raised about the potential risks of ECFC-based cell therapy, in terms of thrombogenicity, particularly in inflammatory conditions. We published that cord blood ECFC (cb-ECFC) express Tissue Factor (TF) in response to TNFα. TF/FVIIa interaction did not modify in vitro the non-procoagulant properties of TNFα stimulated cb-ECFC [Cuccuini et al, JTH 2010]. We also reported, in a monocyte model, a co-expression of TF and metalloproteinase MMP-9, with possible consequences in terms of haemostasis and angiogenesis [Poitevin et al, JTH 2008].Aim of the study: The objective of this study was: 1- to further evaluate the procoagulant potential of ECFC and the capacity to exhibit MMP activity; 2- to evaluate whether TF and MMP could be detected in two different cell therapy products (CTP). Methods:ECFC were obtained in culture after isolation of CD34+ cells from cord blood (as previously described). CTP were obtained from an ongoing clinical trial evaluating cell therapy to improve vascularization in chronic critical limb ischemia (NIH NCT00533104). Two different CTP were analyzed: 1- Peripheral blood mononuclear cells (PB-MNC) obtained by cytapheresis without any mobilization (n= 11); 2- Bone marrow mononuclear cells (BM-MNC) (n= 11). CTP are characterized in flow cytometry, in terms of cell composition, including CD34+ cells; CD34+/CD133+/VEGF-R2+ cells were considered as endothelial progenitor cells. Results:cb-ECFC can express procoagulant TF in response to TNFa (10 ng/mL). The expression of TF was compared with purified monocytes, CD34+ cells, mature endothelial cells (HUVEC), PB-MNC and BM-MNC. TF mRNA were detected in CD34+ cells. As microparticles (MPs) are strongly procoagulant, we evidenced that cb-ECFC produced CD146+/Annexin V+/TF+ MPs in response to TNFa. These MPs can trigger thrombin generation. The analysis of conditioned supernatants of cb-ECFC in response to TNFa indicated that the majority of TF is borne by MP, but a small amount of soluble TF is present, in the absence of MP. This soluble TF does not induce thrombin generation. Using Q-PCR, we showed that cb-ECFC expressed Tissue Factor Pathway Inhibitor (TFPI). This expression of TFPI mRNA was not increased by TNFa. The analysis of TNFa-conditioned cb-ECFC supernatants showed that the amount of TFPI protein is a 20 fold lower than plasmatic levels of free-TFPI.We next examined the presence of TF mRNA in both CTP. TF mRNA were estimated to a 2 fold higher in BM-MNC than in cb-ECFC (baseline). On the contrary, TF mRNA were a 5 fold lower in PB-MNC than in cb-ECFC. In both CTP, the alternatively spliced form (as-TF) represented a very minor part of TF. The percentage of CD14+ cells was respectively 17 % and 26 % in BM-MNC and in PB-MNC. The amount of TF mRNA in PB-MNC was not statistically different from unstimulated purified monocytes. On the contrary, TF mRNA were a 7 fold higher in BM-MNC in comparison with PB-MNC or monocytes. The percentage of CD34+ cells was low: 2.6 % in BM-MNC and 0.1 % in PB-MNC. TF mRNA were higher in CD34+ cells than in BM-MNC (3 fold) and in PB-MNC (27 fold).As we previously reported a co-regulation of TF with MMP, we first analyzed the expression of MMP-2 and MMP-9 in cb-ECFC. Using Q-PCR, we found that cb-ECFC strongly expressed MMP-2 whereas the expression of MMP-9 was very weak. This profile was comparable in cb-ECFC and HUVEC. Using zymography, we confirmed the expression of MMP-2 but not MMP-9, in response to TNFa. We next analyzed CTP. In BM-MNC, MMP-2 and MMP-9 were expressed at a very high level. In PB-MNC, MMP-2 and MMP-9 could be detected but at a very low level. Conclusion:Taken together, our results show that ECFC, BM-MNC and PB-MNC can express TF. The procoagulant activity of ECFC is also the consequence of MP formation in inflammatory conditions. The profile of MMP in CTP remains to be clarified in terms of cell origin and possible influence on thrombotic and angiogenic properties. In our hands, the inhibition of TF can limit procoagulant activity with no effect on proangiogenic properties in vitro. In terms of clinical relevance, one single patient treated with PB-MNC, developed a deep vein thrombosis at the site of injection. It is therefore mandatory to evaluate the procoagulant potential of CTP in the context of vascular repair. Disclosures:No relevant conflicts of interest to declare.

Combined effects of irbesartan and carvedilol on expression of tissue factor and tissue factor pathway inhibitor in rats after myocardial infarction

The objective of this study was to investigate the effects of irbesartan, carvedilol, and irbesartan plus carvedilol on the expression of tissue factor (TF) and tissue factor pathway inhibitor (TFPI) mRNA and protein in rat myocardium after myocardial infarction (MI). MI was induced in male Wistar rats by ligation of the anterior descending branch of the left coronary artery. Irbesartan at 50mg/kg/day, carvedilol at 1mg/kg/day, irbesartan plus carvedilol, or placebo was administered intragastrically; expression of TF and TFPI mRNA and protein was determined by RT-PCR and Western blot analysis. The relative left ventricle weights were lower in all three treatment groups than in the placebo group, with the lowest relative weight in the irbesartan plus carvedilol group (P<0.001). The size of the infarcted area was lower in the carvedilol and the combined groups than in the placebo group (P<0.001). The levels of expression of TF and TFPI mRNA and protein were lower in the combined group than in the placebo group or the carvedilol group (P<0.001). Treatment with irbesartan plus carvedilol reduced the expression of TF and TFPI mRNA and protein after MI in rats, and combined treatment with both agents had greater effects than the single agents alone. These findings suggest that the beneficial effects of these drugs may include anticoagulation and that combined therapy with both agents is an option that should be evaluated further.

The role of TFPI in regulation of TF-induced thrombogenicity on the surface of human monocytes

Introduction Although the procoagulant reactivity of monocytes largely depends on expression and cell surface presentation of tissue factor (TF), little is known about the impact of tissue factor pathway inhibitor (TFPI) on regulation of TF function on the monocyte surface. Materials and methods Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy subjects and cryopreserved. We investigated TF and TFPI mRNA expression by reverse transcription-quantitative real-time PCR (RT-qPCR), surface presentation by flow cytometry and confocal microscopy, and TFPI-mediated regulation of TF functional activity on the surface of resting and LPS-stimulated PBMCs by TF activity assay and Calibrated Automated Thrombogram (CAT) assay. Results Unstimulated PBMCs contained nearly no TF, but detectable TFPI protein levels. TFPI mRNA levels were 2-fold higher than TF, and the TFPIα mRNA isoform expression was higher than TFPIβ. LPS stimulation caused a parallel and sustained upregulation of both TFPI isoforms, concomitant with increased surface presentation of TFPI antigen. Stronger, but transient upregulation of TF mRNA and surface antigen was observed at 6 hrs of LPS stimulation. After LPS stimulation TF and TFPI were co-localized in the same areas of the monocyte membrane. Pre-incubation of PBMCs with anti-TFPI IgG significantly enhanced TF activity, shortened Lag-time, and increased thrombin generation. TFPI-dependent inhibition of TF was more prominent in resting than in LPS-stimulated cells. Conclusions Our results support the concept that surface TFPI is an important regulator of procoagulant reactivity of human monocytes.

Expression of tissue factor pathway inhibitor (TFPI) in human breast and colon cancer tissue

Activation of blood coagulation, a phenomenon frequently observed in breast and colon cancer patients, contributes to tumour progression. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF), while tissue factor pathway inhibitor (TFPI) is the main inhibitor of the TF-dependent pathway of blood coagulation. Previous immunohistochemical studies revealed no expression of TFPI in human cancer cells. The aim of the study was to evaluate the expression of TFPI protein and mRNA in breast and colon cancer tissues. A total of 108 cancer tissues (from primary tumours and metastatic lymph nodes) were obtained from 87 patients during surgical treatment. Immunohistochemical studies using a polyclonal anti-TFPI antibody were performed including a semiquantitative analysis. The in situ hybridisation method employed single-stranded DNA oligonucleotide (probe sequence: 5'Biotin-CCACCATACTTGAAACGTTCACACT-Biotin3') directed against TFPI mRNA. Strong or medium expression of TFPI protein was observed in cancer cell bodies in all breast cancers and in most (39/66 cases) colon cancers examined. Weaker expression of TFPI was detected in cancer cells localised in lymph node metastatic foci of breast cancer. Endothelial cells were also TFPI-positive. TFPI mRNA was demonstrated in all cases of breast and in approximately 80% cases of colon cancer cells. TFPI mRNA and protein are present in association with colon and breast cancer cells, suggesting that the protein may play a role in cancer biology. The presence of TFPI in association with breast cancer cells localised in regional lymph nodes may indicate its role in lymphatic spread.

Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

Tissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-alpha (TNF-alpha)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HUVECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-alpha-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-alpha, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp on TNF-alpha-induced TF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IkappaB-alpha was decreased by Adp, but phosphorylation of p44/42 MAPK, SAPK/JNK, and p38 MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor-kappaB (NF-kappaB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-alpha-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY294002, suggesting that Akt activation might inhibit TF expression induced by TNF-alpha. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-kappaB through stabilization of IkappaB-alpha and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

Characterization of TFPI and TFPI-beta in Mouse Tissues.

Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor. The first two Kunitz domains efficiently inhibit factor VIIa and factor Xa and thereby abrogate tissue factor initiated blood coagulation. Two isoforms of TFPI have been identified. Full-length TFPI is thought to indirectly associate with endothelium via binding to a GPI-anchored co-receptor. TFPI-beta is produced by alternative splicing of full-length TFPI. It consists of the first two Kunitz domains followed by a unique C-terminal region with a direct GPI-anchor attachment site. The physiological significance of TFPI-beta is not known. We performed 3′RACE of mouse placental cDNA to determine if additional isoforms of TFPI exist. These studies identified only TFPI and TFPI-beta. Of importance, a full-length form of TFPI containing a direct GPI-anchor attachment site was not identified. This finding supports the hypothesis that full-length TFPI binds to endothelium indirectly through association with a GPI-anchored co-receptor. In order to identify potential physiological roles for TFPI and TFPI-beta, we analyzed mRNA expression and protein production in mouse tissues. Message for both TFPI and TFPI-beta was present in all tissues examined. Real time PCR showed TFPI message 2.5- to 65-fold higher than TFPI-beta message in the different tissues (heart (2.5)>lung (5.1)>testicle (5.8)>spleen (9.3)>thymus (10)>uterus (11)>kidney (26)> liver (35)> placenta (37)>brain (65)). The high relative amount of TFPI-beta in heart, lung and testicle suggest that it may have an important physiological role in these tissues. In situ hybridization studies did not identify vascular bed specific expression of TFPI or TFPI-beta. These studies demonstrated that TFPI and TFPI-beta localize predominantly within endothelial cells with identical staining patterns in all tissues studied. Western blot analysis for TFPI and TFPI-beta in detergent lysates of the different tissues demonstrated the presence of full-length TFPI in all tissues in amounts proportional to the vascularization of the tissue (placenta>>heart>lung>spleen>kidney>liver>>>brain).TFPI-beta was not observed by western blot analysis in any of the tissues indicating that TFPI-beta message may be transcriptionally repressed. Brain contains much less TFPI than other tissues, yet has high amounts of tissue factor. The low TFPI in the brain could be due to either low numbers of endothelial cells within the brain or to low expression of TFPI by brain endothelial cells. We used real time PCR to compare the level of TFPI and TFPI-beta mRNA expression to that of the endothelial specific proteins VE-cadherin and PECAM. When compared to these markers, the relative amount of TFPI mRNA brain is similar to that present in the heart and lung, indicating that TFPI likely has an important role in prevention of thrombosis within the cerebral vasculature.

TFPI (Tissue Factor Pathway Inhibitor) Is Present in Breast Cancer Tumor Cells.

Abnormalities in hemostatic system are common in breast cancer patients. The principal initiator of blood coagulation activation in cancer patients is tissue factor (TF). Tissue factor plays an important role in the processes of angiogenesis, invasion and metastatic dissemination. Tissue factor pathway inhibitor (TFPI) is the main inhibitor of TF-dependent pathway of blood coagulation. The catalytic activity of the TF/VIIa complex triggers activation of factor X, consequently thrombin generation and finally fibrin formation. Thrombin and fibrin facilitate cancer progression. Previous immunohistochemical studies revealed no expression of TFPI in cancer cells of different malignant tumor types (Werling et al, Thromb Haemost 1993; 69: 366–369) The aim of the study was to evaluate the expression of TFPI protein and mRNA in tumor cells of breast cancer.Breast cancer tissues were obtained during surgical treatment of 35 women. The patients undergoing surgery had not received any previous anticancer therapy. Tumor fragments were preserved in 4% paraformaldehyde/phosphate buffered saline and than embedded in paraffin. Immunohistochemical studies (avidin-biotin complex-ABC - technique) were performed using polyclonal antibodies against TFPI. In situ hybridization method employed single-stranded DNA oligonucleotide (probe sequence: 5′Biotin-CATCGCCCAGTGCAGCCTCCGTCAG-Biotin3′) directed against locus NM 006287 of TFPI mRNA.Strong expression of TFPI protein was observed in cancer cell bodies in examined specimens. Weak expression of the above protein was observed in tumor infiltrating macrophages. Normal cells revealed much weaker staining for TFPI than cancer cells. Similarly, mRNA for TFPI was observed in cancer cells of all breast cancer tissues.The above data indicate that TFPI is present in neoplastic cells of breast cancer and may play a role in neutralizing TF procoagulant and signaling functions.

Relative quantification of glycosaminoglycan-induced upregulation of TFPI-mRNA expression in vitro

Objective Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type serine proteinase inhibitor that plays a central role in the extrinsic pathway of blood coagulation and is mainly expressed by endothelial cells. In this study we examined the in vitro effects of heparin and other glycosaminoglycans on TFPI mRNA-expression in cultivated human endothelial ( Ea.hy 926) and in chondrosarcoma ( SW 1353) cells. Methods We used a LightCycler-based method for relative quantification of the TFPI-mRNA expression before and after stimulation. The cells were stimulated with different concentrations of heparin (with and without addition of protamin), heparan sulfate (HS) and chondroitin-6-sulfate (CS). Cells were harvested after incubation times of 4, 8 and 24 h, total RNA was isolated, and cDNA was synthesized and quantified relatively to a constantly expressed housekeeping gene. Results Stimulation of Ea.hy 926 cells with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) caused a time- and dose-dependent upregulation of TFPI-mRNA expression with LMWH showing the stronger effect. In contrast to this, HS led to a strongly and CS to a slightly decreased TFPI-mRNA expression. SW 1353 cells which were stimulated with LMWH/UFH and HS/CS did not show a significant up- or downregulative effect. Conclusion Our results show that we have developed a versatile method for the relative quantification of TFPI-mRNA expression. As a conclusion, the determined heparin-induced upregulation of TFPI-mRNA expression can be considered a major component of the modulation of the anticoagulant properties of the endothelium.

Alterations in the expression of tissue factor and tissue factor pathway inhibitor induced by the onset of cardiac hypertrophy

Tissue factor (TF), a transmembrane glycoprotein and its regulator, tissue factor pathway inhibitor (TFPI), are the major regulatory of the extrinsic pathway of blood coagulation. TF is a multifunctional protein that can also induce changes in the patterns of gene expression. TF expression is thought to be up-regulated during the onset of cardiac hypertrophy. We examined the expression of TF and TFPI at the onset of cardiac hypertrophy. Hypertrophy was induced surgically via abdominal aortic constriction with a 0.5 mm (OD) needle. TF and TFPI expression was examined at 1, 3, 7 and 14 days post-surgery, in isolated cardiomyocytes and left ventricular tissue using semi-quantitative RT-PCR and western blot analysis. Progressive cardiac hypertrophy was confirmed by the expression of atrial natriuretic factor (ANF). TFPI mRNA expression in isolated cardiomyocytes peaked on day 7 in the constricted animals, but normalised by day 14. TF expression in the isolated cardiomyocytes was undetectable throughout the 14 days. In contrast, in left ventricular tissue TFPI expression transiently increased within 24 h with a second, ore persistent wave at day 7, while TF expression remained constant throughout. TFPI protein expression in the isolated cardiomyocytes decreased over the period of investigation, whereas in left ventricular tissue, expression peaked at day 7 returning to the original level by day 14. TF protein expression remained at baseline level for the first 7 days in the experimental group and then doubled by the 14th day. Thus, the altered expressions of TF and TFPI in the hypertrophied heart reflect an imbalance in haemostatic and tissue homeostatic mechanisms, arising during the onset of cardiac hypertrophy.

Impact of native VLDL on tissue factor pathway inhibitor in endothelial cells and interactions between TFPI and lipoprotein lipase

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of tissue factor (TF)-induced blood coagulation. A positive association between very low density lipoproteins (VLDLs) and TFPI has been reported in vivo. In contrast, one in vitro study indicates that TFPI may enhance lipoprotein lipase (LPL) activity, thereby increasing triglyceride hydrolysis. The current study was conducted to investigate how native VLDL influenced the synthesis and release of TFPI in endothelial cells, and how TFPI affected the LPL-induced hydrolysis of VLDL in vitro and at the endothelial surface. A spontaneously transformed immortal endothelial cell line (ECV304) and primary coronary artery cells (CoEc) were used, and VLDL was isolated from healthy volunteers by density gradient ultracentrifugation. Sequential free fatty acid (FFA) measurements were used to evaluate the kinetics of the LPL-induced hydrolysis. The levels of TFPI mRNAs in the stimulated cells were determined by quantitative real-time reverse transcription-ploymerase chain reaction (qPCR) using the ABI PRISM 7700 Sequence Detection System. Stimulation of ECV304 cells for 24 hours with native VLDL (0-100 microg/mL) caused a dose-dependent increase of TFPI in the medium (6.7-23.8 ng/10(6) cells, P < 0.001), without affecting the cellular content of TFPI. The expression of TFPI mRNA was significantly upregulated after 10 minutes of stimulation with n-VLDL. Both recombinant TFPI (r-TFPI) and LPL showed a dose-dependent binding to ECV 304 cells without saturation, and no competitive binding interactions between LPL and TFPI were observed at the endothelial surface. The addition of increasing concentrations of r-TFPI to ECV 304 cells, preincubated with LPL, did not affect the hydrolysis of VLDL triglycerides. The maximal reaction velocity (V(max)) of LPL-induced hydrolysis of n-VLDL was not affected by the addition of increasing concentrations of r-TFPI to the reaction mixture in vitro. The current experimental study indicates an upregulation of TFPI synthesis and release by VLDL. LPL-induced hydrolysis of VLDL in vitro was not influenced by TFPI neither in suspension nor at the endothelial surface.