组织因子表达下调影响阿霉素诱导人胶质母细胞瘤细胞凋亡的研究

目的研究组织因子（TF）对阿霉素诱导人胶质母细胞瘤细胞凋亡的影响。方法以免疫印迹法检测TF表达。分子生物学方法构建TFsiRNA-pSUPER表达载体,以脂质体介导进行基因转染。以水溶性四唑盐法检测阿霉素的细胞毒性。以印迹法检测阿霉素诱导的Caspase-3、PARP的裂解。以Hochest33342染色荧光显微镜检测凋亡细胞。结果人胶质母细胞瘤细胞系U373MG高表达TF。转染TFsiRNA-pSUPER表达载体的U373MG细胞及对照细胞分别以阿霉素处理,可见转染细胞较对照细胞Caspase-3、PARP的裂解增强。以不同浓度阿霉素处理48h后,可见转染后的U373MG细胞的存活数较对照细胞明显减少（P〈0.05）。以Hochest33342染色检测到转染后的U373MG细胞经阿霉素处理后的凋亡细胞数显著增多（P〈0.05）。结论人胶质母细胞瘤U373MG中TF异常高表达的下调,可增强阿霉素诱导的细胞凋亡。肿瘤细胞中TF的异常高表达可能影响肿瘤细胞对化疗药物的耐受性。     

The intersubject variability of tissue factor mRNA production in human monocytes--relation with the toll-like receptor 4

OBJECTIVE: Tissue factor (TF) is known as the primary initiator of blood coagulation. Previous studies have suggested a considerable variation in the monocytic lipopolysaccharide (LPS) stimulated TF antigen levels and procoagulant activities between different individuals. Our aim with the present study was to investigate the replicability of LPS induced TF mRNA production in a series of standardised experiments with the purpose to identify putative factors influencing the TF high and low response. RESULTS: A constant and reproducible production of LPS induced TF mRNA was identified in five high responders and three low responders (out of 42 individuals) and followed-up in three subsequent experiments performed over 2 years. The LPS induced TF mRNA production correlated with the corresponding expressions of interleukin-8, tumor necrosis factor-alfa and interleukin-1 beta, indicating a common pathway with the TF high and low response. A strong and significant correlation between the LPS induced TF and toll-like receptor 4 mRNA expressions was subsequently identified and replicated. CONCLUSIONS: We demonstrated a high and low responder phenomenon of LPS induced TF mRNA in human monocytes. The production of toll-like receptor 4 mRNA was significantly enhanced in TF high responders.     

Validation of a novel tissue factor assay in experimental human endotoxemia

BACKGROUND: Nuclear factor kappa B (NF-kappa B) activation and tissue factor (TF) expression may contribute to lethality in sepsis. Inappropriate in vivo expression of TF is likely responsible for fibrin deposition in sepsis-associated disseminated intravascular coagulation (DIC). Clinical assessment of TF expression has remained a major challenge. No point-of-care assays are currently available to measure the level of TF activity in whole blood. The current study examined the suitability of the TiFaCT assay as a point-of-care assay to detect TF expression. METHODS: 30 healthy male volunteers received 2 ng/kg of LPS. Tissue factor-dependent coagulation was quantified with a novel assay called tissue factor clotting time (TiFaCT), and by measurement of activation markers of downstream coagulation. RESULTS: Ex vivo addition of anti-TF antibodies to blood slightly increased clotting times at 0-24 h (p<0.01) indicating that some tissue factor activity was present in whole blood at any time. LPS bolus infusion decreased TiFaCT clotting time by -23% compared to baseline (p<0.01), when in vivo clotting increased, as demonstrated by a 10-fold increase in prothrombin fragment levels (F1+2). Ex vivo incubation with LPS considerably shortened TiFaCT (from 1000s to 400s as compared to control incubation; p<0.01). This effect was blunted at 2-4 h after LPS infusion (i.e. the time of monocytopenia), but twofold enhanced 24 h after LPS challenge (p<0.01). CONCLUSIONS: In summary, the TiFaCT assay was validated in our in vivo model of LPS-induced coagulation. It detected minute quantities of circulating TF even at baseline. TiFaCT is shortened at times of in vivo thrombin generation.     

Suppression of HUVEC tissue factor synthesis by antisense oligodeoxynucleotide

Tissue factor (TF) is an important regulator and effector molecule of coagulation. It is primary known as a cofactor for factor VIIa-mediated triggering of blood coagulation, which proceeds in a cascade of extracellular reactions, ultimately resulting in thrombin formation. In sepsis, expression of TF by activated monocytes, macrophages and endothelial cells may lead to disseminated intravascular coagulation. Further studies have suggested that TF also plays non-haemostatic roles in blood vessel development, tumor angiogenesis, metastasis and inflammation. In the present study we examined the feasibility of inhibiting lipopolysaccharide (LPS)-induced TF expression in cultured human umbilical vein endothelial cells (HUVECs) using a modified phosphorothioate antisense oligodeoxynucleotide targeted to the TF mRNA. CD31 receptor-mediated endocytosis was used as a means of delivering TF antisense oligomer to HUVECs. This DNA carrier system consists of anti-CD31 antibody conjugated to the antisense. Co-exposure of HUVECs with TF antisense and LPS resulted in 54.6 ± 3.2% suppression of TF activity when compared with control LPS stimulated cells. The antisense also reduced the LPS-induced TF mRNA level. Control experiments with TF sense and mismatched antisense oligomers were performed to exclude non-specific inhibitory effects. The cytotoxicity of the antisense oligomer conjugate was also evaluated. Results demonstrate that this TF antisense oligomer specifically suppressed the synthesis of biologically active endothelial TF and that antisense oligomers might represent a useful tool in the investigation of endothelial TF function/biology.     

Regulation by DDAH/ADMA pathway of lipopolysaccharide-induced tissue factor expression in endothelial cells

Previous studies have shown the regulatory effect of nitric oxide (NO) on endotoxin-induced tissue factor (TF) in endothelial cells. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production in vivo and in vitro. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endogenous NO production. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the effect of lipopolysaccharide (LPS) on TF expression in endothelial cells. Human umbilical vein endothelial cells (HUVECs) were treated with LPS (1 microg/ml) to induce TF expression. Exogenous ADMA significantly enhanced the increase in both TF mRNA level and activity induced by LPS, whereas L-arginine, the NOS substrate, markedly attenuated the LPS-induced TF increment. LPS markedly increased the level of ADMA in cultured medium and decreased DDAH activity in endothelial cells, and over-expression of DDAH2 could significantly suppress LPS-induced TF increment in endothelial cells. LPS could increase intracellular reactive oxygen species (ROS) production and activate nuclear factor-kappaB, which were enhanced by exogenous ADMA and attenuated by either L-arginine or overexpression of DDAH2. Therefore, our present results for the first time suggest that the DDAH/ADMA pathway can regulate LPS-induced TF expression via ROS-nuclear factor-kappaB-dependent pathway in endothelial cells.     

Human platelets do not express tissue factor

Background

The controversy about the expression of tissue factor (TF) in platelet after de novo synthesis prevail despite many groups recognize that platelet isolation, assays and reagents, particularly non-specific antibodies, may account for the diversity. In this study the potential of TF expression was evaluated using immune-purified human platelets and employing a very sensitive and highly specific TF activity assay.

Methods

Isolated platelets in plasma anti-coagulated with Fragmin were subjected to stimulation by LPS plus PMA, IgG antibody or TRAP and tested for TF activity.

Results

Platelets stimulated with LPS plus PMA for 4 hours expressed trace amounts of TF like activity (PCA), not inhibited by anti-TF antibody (0.2 ± 0.1 mU/ml blood). Platelets, not immune-adsorbed to remove monocytes, showed significant TF activity (2.0 ± 0.9 mU/ml blood) that was nearly abolished by anti-TF antibody. IgG antibody from patient with lupus anticoagulant failed to enhance the trace amount of PCA as compared to the control in contrast to high TF activity induced in monocytes (0.4 ± 0.1 mU/ml blood versus 27.5 ± 10.5 mU/10⁶ cells) showing that activation of complement is not mediating TF expression. Platelet subjected to TRAP activation for 10 min possessed only trace amounts of PCA that was not inhibited by anti-TF antibody and slightly enhanced by anti-TFPI antibody.

Conclusions

It is concluded that platelets free of monocytes do not express TF activity when stimulated by LPS or activated complement factors, implying no role for Toll like receptor (TLR4) as suggested recently. There is no evidence of TF activity associated with platelets as a result of rapid and dynamic process.     

Inhibition of lipopolysaccharide-induced tissue factor expression in monocytes by urinary trypsin inhibitor in vitro and in vivo

Tissue factor (TF) plays a critical role in the pathogenesis of disseminated intravascular coagulation (DIC) observed in patients with septic shock. Urinary trypsin inhibitor (UTI), a multivalent protease inhibitor, is currently used for treatment of patients with septic shock. This study was undertaken to determine whether UTI reduces LPS-induced coagulation abnormalities by inhibiting lipopolysaccharide (LPS)-induced expression of TF by monocytes. UTI inhibited LPS-induced increases in both TF activities and TF mRNA expression in monocytes without affecting the viability. Although activation of nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1) and extracellular signal-regulated kinase (ERK)1/2 were shown to be critically involved in LPS-induced increases in TF activities in isolated monocytes, UTI inhibited phosphorylation of ERK1/2 and decreased expression of early growth response factor-1 (Egr-1) induced by LPS without affecting the activation of NF-kappaB and AP-1. UTI inhibited both the expression of TF mRNA in whole blood, increases in TF activities in mononuclear cells, and increases in serum levels of fibrin and fibrinogen degradation products (E) in rats given LPS without affecting the number of monocytes in the peripheral blood. Taken together these results strongly suggested that UTI might reduce LPS-induced coagulation abnormalities in rats by inhibiting TF expression in monocytes through inhibition of Egr-1 expression.     

Monocyte IL-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles

Lipopolysaccharide (LPS)-stimulated monocytes are known to have a procoagulant effect. This property is currently explained by the fact that monocytes, in response to LPS, can express tissue factor (TF) and undergo a process of membrane microvesiculation. Interleukin-10 (IL-10) has been shown to downregulate TF expression and inhibit procoagulant activity (PCA). In order to further characterize the inhibitory effect of IL-10 on LPS-induced PCA, we used the integrated system of analysis of kinetics of thrombin generation in normal plasma (thrombinography). For this, we developed an original method of elutriation allowing to obtain a highly purified monocyte preparation, under endotoxin-free conditions. Thrombin generation was measured using a highly sensitive and specific fluorogenic method which we adapted to inhibit the contact factor pathway. Results show that recombinant human IL-10 decreased the kinetics of thrombin generation in a dose-dependent manner. Furthermore, the inhibition of endogenous IL-10 released by monocytes in response to LPS is associated with an increase in the kinetics of thrombin generation. We demonstrated that this effect was a consequence of the up-regulation of TF expression and TF-bound microparticle release. In conclusion, we report that IL-10 can regulate thrombin generation in conditions close to physiology as allowed by thrombinography, and that endogenous IL-10 regulates TF expression and release of active TF-bound microparticles by a negative feed back loop through IL-10 receptor alpha.     

Discrepancy between tissue factor activity and tissue factor expression in endotoxin-induced monocytes is associated with apoptosis and necrosis

Tissue factor (TF), the main initiator of blood coagulation, contributes to the manifestation of disseminated intravascular coagulation following septic shock in meningococcal infection. Since a direct relationship between disease severity and lipopolysaccharide (LPS) concentration in the circulation has been shown, we hypothesized that the procoagulant and cytotoxic effects of endotoxin also in vitro were related to its concentration. In vitro studies, however, have frequently used much higher LPS concentrations than those observed in clinical samples. Using elutriation-purified human monocytes, we observed that LPS up to 1000 ng/ml exerted a concentration-dependent increase in TF activity (tenase activity, fibrin formation in plasma). Although there was a dose-dependent increase in TF activity, there was not a concomitant increase in TF expression at LPS concentrations above 1 ng/ml (flow cytometry, Western blotting, TF mRNA). Flow cytometry revealed that this discrepancy between TF activity and TF expression at endotoxin concentrations above 1 ng/ml, coincided with an LPS dose-dependent increase in cell surface phosphatidylserine (PS), considered to promote coagulation. The increased PS expression was associated with an increased number of 7-AAD-positive cells indicating cell death. We conclude that enhancement of monocyte procoagulant activity in vitro by high concentrations of LPS may result from increased PS exposure due to apoptosis and necrosis. Therefore, the LPS concentrations used to examine monocyte procoagulant activity in vitro, should be carefully chosen.     

Differential expression of tissue factor mRNA and protein expression in murine sepsis. The role of the granulocyte revisited

Tissue factor (TF) is a transmembrane protein, which is essential for initiation of the coagulation cascade. TF has been reported to play an important role in the progression of endotoxin (lipopolysaccharide, LPS)-mediated endotoxemia, being induced in numerous tissues, such as kidney, spleen and lung. We developed and validated a rabbit anti-murine TF peptide antiserum to localize TF protein in a murine sepsis model. TF protein distribution was compared to localization of TF mRNA and fibrin deposits, the ultimate resultant of procoagulant TF activity. Evident LPS mediated TF mRNA induction was observed in the tubular area at the cortico-medullar junction in the kidney, and TF activity was increased after 6 hours of endotoxemia. In the spleen, however, TF mRNA was induced in the interfollicular region upon LPS injection, corresponding to increased TF protein in the same area. The clusters of TF-protein positive cells in the spleen are predominantly granulocytes, but no TF mRNA expression was observed within these cells. Based on these observations and the presence of TF-protein positive granulocytes after splenectomy, we hypothesize that granulocytes take-up TF for transport to other locations in order to initiate fibrin formation or to induce pro-inflammatory gene expression upon interaction with factor VIIa.     

A simplified and low-cost one-stage chromogenic assay for tissue factor dependent procoagulant activity of endothelial cells

We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS), were incubated with 50 μl of diluted normal human plasma (NHP) and 50 μl of Factor Xa-specific chromogenic substrate (CBS 31–39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31–39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10⁴ EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31–39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.     

Role of protease-activated receptor 1 in tumor metastasis promoted by tissue factor

Tissue factor (TF) is a transmembrane glycoprotein that complexes with factor VIIa to initiate blood coagulation. We previously reported that expression of high levels of TF in a human melanoma cell line promotes metastasis. Both the cytoplasmic domain of TF and its extracellular domain complexed with factor VIIa are required for the metastatic effect. To further explore the mechanism of TF-mediated metastasis, we investigated the possibility that a protease-activated receptor (PAR) might play a role. For this purpose, we first determined the expression levels of the known PARs (PAR1-4) in a human melanoma cell line, SIT1, that has low endogenous levels of TF and low metastatic potential. We found negligible levels of all of the known PARs and transfection of this cell line with human TF cDNA did not alter expression of the known PARs. To study the possible role of PAR1 in TF-mediated metastasis, we prepared a panel of transfected cell lines with varying levels of TF and PAR1. Our studies show that TF promotes metastasis by a pathway that does not involve high expression of known PARs by tumor cells. In addition, while overexpression of PAR1 is insufficient to induce metastasis in cells with low TF expression, it enhances the metastatic potential of cells with high TF expression, indicating a possible synergy between TF and PAR1 in promoting metastasis.     

Measurement of tissue factor activity in whole blood

High circulating levels of the procoagulant molecule tissue factor (TF) are associated with thrombosis in a variety of diseases including unstable angina, cancer, and sepsis. Currently, there are no clinical assays to measure the level of TF activity in whole blood. We present an assay called Tissue Factor Clotting Time ("TiFaCT") that detects fibrin formation in human blood. The mean baseline clotting time in a healthy population was 472 +/- 94 s (mean +/- SD, n = 150). Bacterial lipopolysaccharide (LPS or endotoxin) shortened the clotting time in a time-dependent manner. Inhibitory anti-TF antibodies prolonged the clotting time of LPS-stimulated blood, indicating that the shortened clotting time was due to induction of TF expression. Patients with unstable angina had shortened mean baseline clotting time (284 +/- 86, n = 13) compared with healthy volunteers (474 +/- 98, n = 30), suggesting that these patients had elevated levels of circulating TF. The TiFaCT assay should prove clinically useful in quantifying the levels of circulating TF in patients at risk of thrombosis.     

Detection of endogenous tissue factor levels in plasma using the calibrated automated thrombogram assay

Background

The calibrated automated thrombogram (CAT) assay measures thrombin generation in plasma.

Objective

Use the CAT assay to detect endogenous tissue factor (TF) in recalcified platelet-rich plasma (PRP) and platelet-free plasma (PFP).

Methods

Blood from healthy volunteers was collected into citrate and incubated at 37 °C with or without lipopolysaccharide (LPS) for 5 hours. PRP and PFP were prepared and clotting was initiated by recalcification. Thrombin generation was measured using the CAT assay.

Results

The lag time (LT) was significantly shortened in PRP prepared from LPS-treated blood compared with untreated blood (10 ± 3 min versus 20 ± 6 min), and this change was reversed by the addition of inactivated human factor VIIa. LPS stimulation did not change the peak thrombin. Similar results were observed in PFP (21 ± 4 min versus 35 ± 5 min). LPS stimulation also significantly reduced the LT of PRP and PFP derived from blood containing citrate and a factor XIIa inhibitor. Finally, a low concentration of exogenous TF shortened the LT of PFP prepared from unstimulated, citrated blood without affecting the peak thrombin.

Conclusion

Changes in LT in the CAT assay can be used to monitor levels of endogenous TF in citrated plasma.     

Monocyte tissue factor response is decreased in patients with hyperlipidemia

Monocytes are potent regulators of blood coagulation through the expression of tissue factor (TF) on stimulation and of tissue factor pathway inhibitor (TFPI), a selective inhibitor of TF pathway. As hyperlipidemia can modify some monocyte functions, we compared the TF and TFPI expression by circulating monocytes and the plasma TFPI levels between 65 healthy normolipemic controls and 38 nontreated hyperlipemic patients. TF and TFPI relationships with plasma lipoproteins are also examined. TF and TFPI expression were evaluated in peripheral mononuclear cells after isolation from blood by density gradient centrifugation and after short culture with or without lipopolysaccharide (LPS). TF and TFPI activity and antigen were measured in mononuclear cell lysates using amidolytic assay and enzyme-linked immunosorbent assay, respectively. TFPI activity and antigen were measured in plasma using the same methods. Plasma factor VII (FVII) activity and antigen were also determined. LPS-stimulated monocyte TF activity and antigen were lower in hyperlipidemic patients than in controls (0.0001相似文献   

Increased expression of TF in BMP-7-treated human mononuclear cells depends on activation of select MAPK signaling pathways

Bone morphogenetic protein (BMP)-7 regulates atherosclerotic plaque calcification, and it contributes to increased thrombogenicity of lipid-rich lesions by enhancement of TF expression in monocytes/macrophages by unknown mechanism. Since Erk1/2, JNK and p38 mitogen activated protein kinases (MAPKs) regulate TF expression, we studied involvement of MAPK pathways in BMP-7-induced activation of TF in human mononuclear cells (MNCs). Whole blood from healthy volunteers was treated with BMP-7, MNCs were isolated, and TF expression was assessed by western blot (WB) and In-Cell Western assay. Phosphorylation and nuclear translocation of Smad1/5/8 in response to BMP-7 stimulation of MNCs was evaluated by WB and confocal microscopy. Activation of MAPKs was judged by measuring the levels of phoshorylated Erk1/2, JNK, and p38 in the lysates of MNCs. The impact of Erk1/2 and p38 activation was studied by use of PD98059 and SB203580 inhibitors, respectively. Stimulation of whole blood with BMP-7 increased the levels of TF in the lysates of MNCs by 7-fold as compared to 12-fold after LPS stimulation. It was followed by elevation in TF fuctional activity. It was accompanied by elevated levels of phosphorylated Smad 1/5/8 and nuclear translocation of Smad1/5/8 proteins. Treatment of whole blood with BMP-7 led to a phosphorylation of Erk1/2, JNK and p38 MAPKs. BMP-7-induced TF expression was partially inhibited by Erk1/2 inhibitor, whereas TF expression was completely abolished by p38 inhibitor. BMP-7-dependent activation of TF in human MNCs by BMP-7 is accompanied by activation of canonic Smad1/5/8 signaling pathway and depends on activation of Erk1/2 and p38.     

Fibrin down-regulates LPS- and PMA-induced tissue factor expression by blood mononuclear cells

Several studies indicate that fibrin may play a functional role in inflammation by modulating a variety of cellular functions. We investigated the effect of fibrin on tissue factor (TF) production by blood mononuclear cells (MNC). Citrated human blood was recalcified and incubated at 37 degrees C for 1-4 h. The resulting clot was lysed by the addition of tissue plasminogen activator (t-PA) and MNC were isolated by density gradient centrifugation. A control blood sample was processed in the same way but omitting calcium addition and clot formation. Clot- and blood-derived MNC did not express detectable TF activity and antigen whatever the incubation time. Clot-derived MNC, however, generated on average 5 fold less TF (activity and antigen) than control cells, when stimulated with lipopolysaccharide (LPS, I microg/ml) for 3 h at 37 degrees C. A reduced TF response of clot-derived cells was also observed at mRNA level as indicated by RT-PCR and in situ hybridization. The effect was dependent on the incubation time within the clot, could not be reversed by enhancing LPS concentration or by adding serum, and was maintained if LPS was replaced by the tumor promoter PMA. A reduced TF response was also found when washed MNC were incorporated for 1 h at 37 degrees C within purified fibrin but not when the cells were incubated with fibrinogen, thrombin or fibrin split products alone. indicating that contact with fibrin was responsible for the inhibition of TF production. Fibrin-induced down-regulation of TF response to LPS and PMA by MNC may represent a negative feed-back aimed at limiting excessive blood clotting activation in immunoinflammatory diseases.