TF mRNA Research Articles

Human megakaryocytes confer tissue factor to a subset of shed platelets to stimulate thrombin generation.

Tissue factor (TF), the main activator of the blood coagulation cascade, has been shown to be expressed by platelets. Despite the evidence that both megakaryocytes and platelets express TF mRNA, and that platelets can make de novo protein synthesis, the main mechanism thought to be responsible for the presence of TF within platelets is through the uptake of TF positive microparticles. In this study we assessed 1) whether human megakaryocytes synthesise TF and transfer it to platelets and 2) the contribution of platelet-TF to the platelet hemostatic capacity. In order to avoid the cross-talk with circulating microparticles, we took advantage from an in vitro cultured megakaryoblastic cell line (Meg-01) able to differentiate into megakaryocytes releasing platelet-like particles. We show that functionally active TF is expressed in human megakaryoblasts, increased in megakaryocytes, and is transferred to a subset of shed platelets where it contributes to clot formation. These data were all confirmed in human CD34pos-derived megakaryocytes and in their released platelets. The effect of TF silencing in Meg-megakaryoblasts resulted in a significant reduction of TF expression in these cells and also in Meg-megakaryocytes and Meg-platelets. Moreover, the contribution of platelet-TF to the platelet hemostatic capacity was highlighted by the significant delay in the kinetic of thrombin formation observed in platelets released by TF-silenced megakaryocytes. These findings provide evidences that TF is an endogenously synthesised protein that characterises megakaryocyte maturation and that it is transferred to a subset of newly-released platelets where it is functionally active and able to trigger thrombin generation.

The Megalobrama amblycephala transferrin and transferrin receptor genes: Molecular cloning, characterization and expression during early development and after Aeromonas hydrophila infection

Both transferrin (TF) and transferrin receptor (TFR) play vital roles in iron homeostasis, which is essential for cellular growth and survival. Besides, TF and TFR are also reported to be involved in immune response against bacterial infection. In the present study, we cloned and characterized the Tf and TfR genes in Megalobrama amblycephala. The M. amblycephala Tf gene contained 17 exons and 16 introns, encoding 651 amino acids, while the M. amblycephala TfR gene contained 18 exons and 17 introns, encoding 768 amino acids. In healthy fish, Tf mRNA was most abundant in the liver, and TfR was highly expressed in the blood and brain. During early development, the expression of Tf increased from 12 hpf (hour post fertilization) to 26 hpf, followed by a diminution at 32 hpf, then increased significantly to the peak level at 2 dph (day post hatching). The expression pattern of TfR was similar to that of Tf, fluctuating from 0 hpf to 32 hpf and dramatically increasing to the peak at 2 dph. Additionally, both Tf and TfR genes responded to Aeromonas hydrophila infection, by increasing their expression in the liver, spleen and kidney at both mRNA and protein levels, indicating that they were involved in M. amblycephala immune response. Immunohistochemical analysis and Prussian blue staining verified the internalization of TF-receptor system with bound-iron in the liver of M. amblycephala.

Influence of FcγRIIA H131R Polymorphism on the Risk of Thrombosis in HIT and Variable Effect of Normal IgG on Platelet Activation

Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of IgG antibodies that activate platelets via FcγRIIA receptors. HIT is associated with thrombotic complications occurring in about 50% of affected patients.Moreover, the response of platelets to anti-PF4/H IgG varies from one subject to another. This variability, partly explained by gene polymorphisms affecting FcγRIIA signaling (Rollin et al, Blood 2012), might also depend on variations in the binding of HIT antibodies to FcγRIIA receptors.Objective. The influence of FcγRIIA H131R polymorphism on the risk of HIT and thrombosis was investigated in parallel with the effect of normal IgG on the platelet response to HIT antibodies.Patients and Methods. A cohort of 89 patients with definite HIT (PF4-specific ELISA and serotonin release assay both positive) and two control groups with (Abpos ; n=160) or without (Abneg ; n=179) significant levels of PF4-specific antibodies after cardiac surgery were studied. Platelet aggregation tests (PAT) were performed in 59 healthy donors using HIT plasma samples or 5B9, a humanized chimeric monoclonal antibody specific to PF4/H complexes that we have recently developed which fully mimics the effects of human HIT antibodies. The influence of normal IgG and IgG subclasses on platelet aggregation was also evaluated according to the FcγRIIA H131R polymorphism. Finally, tissue factor mRNA levels and plasma phospholipid-dependent procoagulant activity were measured after addition of HIT antibodies and heparin in the whole blood collected from healthy donors with different FCGR2A genotypes.Results. Genotypes and allele frequencies were similar in the HIT patients and both Abneg and Abpos controls. However, interestingly, homozygous FcγRIIA 131RR subjects were more frequent among the HIT patients who had developed thrombotic complications compared to those without thrombosis (34.5% vs. 8.5%, respectively, p=0.008). The FcγRIIA 131RR genotype therefore appeared to increase the risk of thrombosis in HIT significantly (OR = 5.9; 95% CI 1.7-20). This association was not found in two other cohorts of patients who had developed ischemic stroke or venous thromboembolism in a medical context other than HIT, thus indicating that the impact of FcγRIIA H131R polymorphism on the risk of thrombosis was specific to HIT. PAT performed with platelet-rich plasma (PRP) showed that the lag time measured in response to 5B9 with heparin (5B9/H) was significantly longer with FcγRIIA 131HH platelets compared to RR platelets (p=0.03). Importantly, this variable platelet response to 5B9/H related to the FcγRIIA polymorphism was no longer detectable when PAT were performed with washed platelets, when the IgG fraction had been removed from the plasma, and when collagen was used as inducer. On the other hand, the addition of human polyvalent IgG to IgG-depleted PRP inhibited the aggregation induced by 5B9/H, but lower concentrations of IgG were required to achieve this effect in FcγRIIA 131HH subjects compared to RR donors. Importantly, this variable effect of normal IgG on platelet aggregation induced by HIT antibodies was shown to depend mainly on the IgG2 subclass, which binds to the H131 isoform more effectively, whereas IgG1 exerted a similar inhibitory effect in RR and HH homozygous donors. Finally, a stronger increase in TF mRNA levels was also evidenced in homozygous FcγRIIARR donors compared to homozygous FcγRIIAHH donors after incubation of 5B9 and heparin in whole blood. Moreover, this effect was associated with a greater relative shortening of plasma clotting time indicating that the release of procoagulant phospholipid microparticles was more pronounced in RR subjects in response to HIT antibodies (p=0.04).Conclusion. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to an increased cell activation induced by antibodies to PF4/H with a lower inhibitory effect of endogenous IgG, especially of the IgG2subclass. DisclosuresNo relevant conflicts of interest to declare.

PARP-14 combines with tristetraprolin in the selective posttranscriptional control of macrophage tissue factor expression

AbstractTissue factor (TF) (CD142) is a 47 kDa transmembrane cell surface glycoprotein that triggers the extrinsic coagulation cascade and links thrombosis with inflammation. Although macrophage TF expression is known to be regulated at the RNA level, very little is known about the mechanisms involved. Poly(adenosine 5′-diphosphate [ADP]-ribose)-polymerase (PARP)-14 belongs to a family of intracellular proteins that generate ADP-ribose posttranslational adducts. Functional screening of PARP-14–deficient macrophages mice revealed that PARP-14 deficiency leads to increased TF expression and functional activity in macrophages after challenge with bacterial lipopolysaccharide. This was related to an increase in TF messenger RNA (mRNA) stability. Ribonucleoprotein complex immunoprecipitation and biotinylated RNA pull-down assays demonstrated that PARP-14 forms a complex with the mRNA-destabilizing protein tristetraprolin (TTP) and a conserved adenylate-uridylate-rich element in the TF mRNA 3′ untranslated region. TF mRNA regulation by PARP-14 was selective, as tumor necrosis factor (TNF)α mRNA, which is also regulated by TTP, was not altered in PARP-14 deficient macrophages. Consistent with the in vitro data, TF expression and TF activity, but not TNFα expression, were increased in Parp14−/− mice in vivo. Our study provides a novel mechanism for the posttranscriptional regulation of TF expression, indicating that this is selectively regulated by PARP-14.

Potential detrimental effect of soy isoflavones on testis sertoli cells

To determine the effect of soy isoflavones on cell proliferation and the transcription levels of follicle-stimulating hormone receptor (FSHR), inhibin α (INHα), INHβB, androgen binding protein (ABP), transferrin (Tf) and vimentin in testis sertoli cells in SD rats. Sertoli cells were cultured in vitro, exposed to daidzein at 0.03, 0.3, 3, and 30 μmol/L and genistein at 0.05, 0.5, 5 and 50 μmol/L, respectively. MTT was used to detect the proliferation of sertoli cells. Real-time PCR was used to detect the relative mRNA expressions of FSHR, INHα, INHβB, ABP, Tf and vimentin. Compared with control groups, cell proliferation and the relative mRNA expression levels of INHβB and ABP in the treated cells showed no significant alternation. The INHα mRNA expression levels were increased in 0.3 and 3 μmol/L Dai and 0.05 μmol/L Gen, while the mRNA expression levels of FSHR were downregulated in 30 μmol/L Dai and Gen at all concentrations. Tf mRNA expression levels were downregulated in 30 μmol/L Dai and 5 μmol/L and 50 μmol/L Gen, and the mRNA expression levels of vimentin were downregulated in 3 and 30 μmol/L Dai and 50 μmol/L Gen. Soy Isoflavones may have potential detrimental effect on the male reproductive system, as they may impact the function of sertoli cells by downregulating the transcription levels of some important proteins.

Air pollution upregulates endothelial cell procoagulant activity via ultrafine particle-induced oxidant signaling and tissue factor expression.

Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure.

Regulatory effect of iron regulatory protein-2 on iron metabolism in lung cancer.

Iron metabolism plays an important role in the pathogenesis of lung cancer. This study aimed to investigate the effect of gene silencing of iron regulatory protein-2 (IRP2) on mRNA and protein expression of transferrin (Tf), transferrin receptor (TfR), and ferritin (Fn) in A549 lung cancer cells. A549 cells were cultured and divided into a liposome control group, a liposome + oligonucleotide (SCODN) control group, and a Lipofectamine + antisense oligonucleotide (ASODN) group. RT-PCR and Western blotting were used to detect mRNA and protein expression of Tf, TfR, and Fn. We found no significant change in Tf mRNA expression among the 3 groups (P = 0.078). TfR and Fn mRNA expressions in the ASODN group notably decreased compared to the liposome and SCODN groups (P < 0.01). IRP2 and TfR protein expressions in the ASODN group were significantly lower than in the liposome or SCODN groups (P < 0.05), whereas no significant change in Tf protein expression was observed between the 3 groups (P = 0.088). Fn protein expression in the ASODN group was significantly higher than in the liposome or SCODN group (P < 0.05). IRP2 can regulate the expression of TfR and Fn by changing its own protein expression and thereby regulate iron metabolism.

Increased Inflammatory Markers and Their Correlations To Clinical Complications In Hemoglobin SC Disease

Hemoglobin SC (HbSC) disease is the second most prevalent hemoglobinopathy after sickle cell anemia (SCA – homozygous HbSS). Despite its high prevalence, most of the knowledge about the pathophysiology of HbSC is inferred from studies focused primarily on SCA. In general, HbSC is considered a milder form of SCA. Chronic inflammatory activity is clearly seen in SCA, but not much is known about the inflammation present in HbSC. In the present study we aimed to evaluate inflammatory markers in HbSC patients and their associations with clinical and laboratory characteristics of the disease. This was a cross-sectional study performed on a cohort of 56 HbSC patients (mean age of 41 years), 39 SCA patients (mean age of 34 years), and 24 healthy age-matched controls. All of the patients were in steady state, with no history of painful crisis, hospitalization or transfusions during the preceding 3 months. None of the patients were in use of hydroxyurea. We evaluated the inflammatory markers, tumor necrosis factor-alpha (TNF-α) and interleukin 8 (IL8). Levels of inflammatory markers were correlated with hemolysis markers, blood counts, coagulation markers (tissue factor expression [TF], thrombin-antithrombin complex [TAT], d-dimer [DD]) and endothelial activation marker (soluble thrombomodulin [sTM]). TNF-α, IL8, TAT, DD and sTM were all measured by ELISA. Leukocyte TF mRNA expression was analyzed by real time quantitative PCR. Statistical analyses were performed by Mann-Whitney's U test and Spearman's rank correlation test. HbSC patients presented higher TNF-α levels than those of controls (2.72 vs. 0 pg/mL; P <0.0001), which were similar to the levels seen in SCA patients (2.72 vs. 2.74 pg/mL; P = 1.0). IL8 levels were similar between HbSC patients and controls (2.54 vs. 2.29 pg/mL; P = 0.16) and also similar between HbSC and SCA patients (2.54 vs. 2.82 pg/mL; P = 0.45). In the analyses of the HbSC cohort, IL8 levels presented significant positive correlations with: leukocyte (r=0.4; P=0.02), monocyte (r=0.5; P=0.001), and platelet counts (r=0.6; P=0.0002); and hemolysis markers: indirect bilirubin (r=0.4; P=0.04) and lactate dehydrogenase levels (r=0.4, P=0.04). TNF-α levels presented no significant correlations with laboratory markers. We also evaluated associations between IL8 and TNF-α levels and clinical complications; stroke, pulmonary arterial hypertension, acute thoracic syndrome, retinopathy, osteonecrosis, leg ulcers and autosplenectomy. HbSC patients with osteonecrosis had significantly higher IL8 levels (3.8 vs. 2.4; P=0.01) when compared with patients without this complication. IL8 levels were also higher in patients with autosplenectomy (3.8 vs. 2.0; P=0.0003). Our results indicate that HbSC disease patients present elevation of inflammation markers, similar to alterations seen in SCA. In our cohort, patients with higher peripheral blood counts and a more intense hemolytic activity, such as patients with autoesplenectomy, have higher levels of inflammatory markers. Our data suggest that inflammation may be a factor contributing to the pathophysiology of a very prevalent chronic complication of HbSC disease, osteonecrosis. Studies focusing on the pathophysiology of HbSC disease are lacking, and in view of the high prevalence of this disease we believe it should be the focus of future studies. Disclosures:No relevant conflicts of interest to declare.

Heme Potently Stimulates Tissue Factor Expression By Peripheral Blood Monocytes: A Novel Mechanism For Thrombosis In Intravascular Hemolytic Diseases

Intravascular hemolytic diseases such as sickle cell anemia as well as diseases such as sepsis/DIC in which intravascular hemolysis occurs are frequently complicated by micro- and macrovascular thrombosis but mechanisms underlying this association are unclear. Plasma heme levels in sickle cell anemia are typically 5-30 µM. LPS is a prototypic and potent agonist for peripheral blood monocyte tissue factor expression and by this mechanism contributes to thrombosis in sepsis. Because heme, like LPS, signals by binding to and activating TLR4, we hypothesized that heme would also stimulate tissue factor expression in monocytes and so promote thrombosis in sickle cell anemia. We isolated human peripheral blood mononuclear cells (PBMC) and monocytes, incubated them 4 hours in the presence or absence of 10 µM hemin or 10 ng/ml LPS, made whole cell lysates by freeze-thawing and sonication and measured tissue factor activity using a one-stage clotting assay employing recalcified citrated human plasma. The assay was standardized with recombinant human tissue factor and the tissue factor dependence of the assay was confirmed using the neutralizing anti-human tissue factor antibody ATF. Hemin stimulated PBMC TF activity ca. 40-fold (range 6- to 296-fold) from 53 to 1885 pg/ml/million cells, an extent comparable to that of LPS (1939 pg/ml/million cells). In isolated monocytes heme increased TF activity ca. 70-fold (range 25-115-fold) to 9800 pg/ml/million cells, again comparable to LPS (8500 pg/ml/million cells.) Heme stimulation of monocyte TF expression did not reflect endotoxin contamination of our hemin preparation because it was 1) reproduced with a preparation of hemin made for infusion into patients with intermittent porphyria that had no detectable endotoxin; 2) unaffected by addition of 1 µg polymyxin B, which abrogated LPS stimulation; 3) blocked by 15 µM hemopexin, a high affinity heme-binding protein. qRT-PCR of TF expression in monocytes shows a 140-350-fold increase in TF mRNA levels over baseline between 2 and 4 hours after exposure to heme. Examining pathways for heme signaling, we demonstrate that heme stimulation of TF expression in monocytes is impaired > 90% by inhibitors of TLR4 (TAK242), NADPH oxidase (diphenylene iodonium), PKC (5 µM calphostin) and ERK ½ (10 µM U-0126), impaired 75% by the p50 NF-kB inhibitor 10 µg/ml andrographolide and unaffected by 100 nM wortmannin, an AKT/PI3K pathway inhibitor. We conclude that heme, at concentrations found in intravascular hemolytic diseases such as sickle cell anemia, is a highly potent stimulant of blood monocyte TF transcriptional expression dependent upon signaling through TLR4, PKC, NADPH oxidase, ERK ½ and NF-kB. By this direct mechanism heme may promote thrombosis and contribute to the pathogenesis of sickle cell anemia and other diseases characterized by intravascular hemolysis, including sepsis/DIC. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells

IntroductionTissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells.MethodshESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis.ResultsWe first found that TF was expressed only in trophoblasts and granulocyte–monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway.ConclusionsThe miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.

The role of fibrin matrices and tissue factor in early-term trophoblast proliferation and spreading

Fibrin deposition in placenta is a common phenomenon which can be triggered by villous injury and coagulation activation. Fibrin abnormalities (hypo/dysfibrinogenemia) and factor XIII deficiency are associated with infertility and pregnancy loss. While trophoblasts are known to grow on fibrin matrices, the role of this protein in trophoblast repair processes remains unclear. We hypothesize that fibrin may have an essential role in trophoblast remodeling. Morphology and spreading of primary early-term human trophoblasts and villi explants were investigated on various fibrin components. Cross-linking of matrices was evaluated by D-dimer assay. TF procoaguant activity, protein and mRNA levels in cells and villi were determined by chromogenic assay, ELISA, immunohistochemistry and reverse-transcription PCR (RT-PCR). Fibrin but not fibrinogen, thrombin or fibronectin caused increased trophoblast proliferation and spreading. Trophoblasts cultured on factor XIII (FXIII) depleted fibrin caused their increased proliferation and spreading, associated with cross-linking. FXIII addition further increased this effect, while cell culturing on active FXIII without fibrin retained cellular proliferation. Decreased TF activity, antigen and RNA expression were demonstrated in fibrin-cultured trophoblasts and villi explants, compared to matrigel explants. Results obtained demonstrate distinct mechanisms underlying fibrin cross-linking, which can affect trophoblast proliferation. The excess of fibrin deposits may be limited by the decrease in TF levels, thus enabling adequate placental perfusion. These findings demonstrate fibrin importance for placental repair and may partly explain poor pregnancy outcome associated with certain fibrinogen/fibrin abnormalities and FXIII deficiency.

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...

Hypoxia dysregulates fibrinolytic pathways in cultured human mesothelial cells (HMCs): Implications for intraabdominal adhesion formation

Abdominal surgery induces mesothelial injury and the extravasation of a fibrinous exudate into the peritoneum. The ensuing deposition of a fibrin matrix serves as a precursor to adhesions. Our aim was to determine the mechanism(s) by which injury‐induced hypoxia and/or inflammation contribute to the formation of the fibrin matrix, the precursor of adhesions. Cultured HMCs were treated with either an inflammatory cytokine cocktail of TGFβ+IL‐1β+IFN‐γ (C), the hypoxia mimetic dimethyloxaloglycine (D) or both, and mRNA levels for vascular endothelial growth factor (VEGF; extravasation), protein c receptor (PrCR; inhibits Protein C activation), thrombomodulin (TM; inhibits thrombin), and tissue factor (TF; activates thrombin) were measured at 1, 3, 6, 12 &amp; 18 hrs via RT‐PCR. VEGF mRNA was increased by both D (peak: 6hr, 8±2.3 fold vs. control), and C (peak 12 hr: 7±0.1 fold, both p&lt;.01) at all times, and the combination was additive. Hypoxia reduced PrCR mRNA levels to a min at 18 hr (0.5±0.06 fold, p&lt;.05) and elevated TF mRNA levels to a max at 18 hr (3.5±0.9 fold; p&lt;.05). TM mRNA levels were decreased by D and D+C to a min at 18 hr (0.25±0.06 fold, p&lt;.05). These data show that hypoxia impairs fibrinolytic pathways in HMCs and may promote adhesion formation. Funded by Cooper‐Tyler Endowment, Department of Surgery, Boston University Medical Center.

TFPIα and TFPIβ are expressed at the surface of breast cancer cells and inhibit TF-FVIIa activity

BackgroundTissue factor (TF) pathway inhibitor-1 (TFPI) is expressed in several malignant tissues- and cell lines and we recently reported that it possesses anti-tumor effects in breast cancer cells, indicating a biological role of TFPI in cancer. The two main splice variants of TFPI; TFPIα and TFPIβ, are both able to inhibit TF-factor VIIa (FVIIa) activity in normal cells, but only TFPIα circulates in plasma. The functional importance of TFPIβ is therefore largely unknown, especially in cancer cells. We aimed to characterize the expression and function of TFPIα, TFPIβ, and TF in a panel of tumor derived breast cancer cell lines in comparison to normal endothelial cells.MethodsTFPIα, TFPIβ, and TF mRNA and protein measurements were conducted using qRT-PCR and ELISA, respectively. Cell-associated TFPI was detected after phosphatidylinositol-phospholipase C (PI-PLC) and heparin treatment by flow cytometry, immunofluorescence, and Western blotting. The potential anticoagulant activity of cell surface TFPI was determined in a factor Xa activity assay.ResultsThe expression of both isoforms of TFPI varied considerably among the breast cancer cell lines tested, from no expression in Sum149 cells to levels above or in the same range as normal endothelial cells in Sum102 and MDA-MB-231 cells. PI-PLC treatment released both TFPIα and TFPIβ from the breast cancer cell membrane and increased TF activity on the cell surface, showing TF-FVIIa inhibitory activity of the glycosylphosphatidylinositol- (GPI-) anchored TFPI. Heparin treatment released TFPIα without decreasing the cell surface levels, thus indicating the presence of intracellular storage pools of TFPIα in the breast cancer cells.ConclusionGPI-attached TFPI located at the surface of breast cancer cells inhibited TF activity and could possibly reduce TF signaling and breast cancer cell growth locally, indicating a therapeutic potential of the TFPIβ isoform.

Evaluation of the hypolipidemic activity of 6,7-dimethoxycoumarin on placental tissue factor mRNA expression in experimental anti-phospholipid syndrome

Background:Anti-phospholipid syndrome is a thrombogenic and systemic autoimmune disorder that influences fetal life throughout gestation period. Over expression of tissue factor on the surface of monocyte(s) is reported to be a major causative agent in inducing anti-phospholipid antibody-mediated placental thrombosis and fetal loss in pregnant women. The over expression of tissue factor is proposed to be due to high levels of blood cholesterol and oxidized lipoproteins.Objective:In this study, we report the lipid-lowering property and anti-tissue factor activity of one of the naturally occurring coumarin derivates 6,7-dimethoxycoumarin, found aplenty in Chinese medicinal plant Artemisia scoparia, and its effect on tissue factor mRNA expression in experimental anti-phospholipid syndrome.Materials and Methods:Adult female mice were immunized with cardiolipin and beta-2-glycoprotein-1 to induce experimental anti-phospholipid syndrome. Female mice with high titer of aCL were allowed to mate with male, and the female mice were treated with 6,7-dimethoxycoumarin on a daily dose of 5 mg/kg body weight from day 3 to day 15 of gestation. On day 18 of pregnancy, all the animals were dissected to measure biochemical parameters in blood, and TF mRNA expression levels were measured in placenta.Results:Treatment with 6,7-dimethoxycoumarin significantly reduced the levels of cholesterol and plasma lipids by its potent hypolipidemic property, which eventually reduced the over-expression tissue factor at mRNA levels in placenta. We believe that further studies in animal model would reveal the potential therapeutic properties of 6,7-dimethoxycoumarin against anti-phospholipid syndrome.Conclusion:The 6,7-dimethoxycoumarin is capable to reduce the expression of TF in placenta at the mRNA level and thrombus generation indirectly by its potent anti-TF and anti-oxidant activities.

Hypoxia/reoxygenation stress increases markers of vaso-occlusive crisis in sickle SAD mice

In sickle cell disease, the factors involved in vasoocclusive crisis (VOC) include the sickling of red blood cells (RBC), abnormal blood rheology, inflammation, vascular adhesion, oxidative stress, coagulation, and vascular tone modulation. The aim of this study was to further characterize the molecular response of some factors involved in VOC by inducing a hypoxia/reoxygenation stress in sickle SAD mice. Results show that a hypoxia/reoxygenation stress in SAD mice can induce: (i) a decrease in reticulocytes count, and mean corpuscular volume along with an increase in lactate dehydrogenase (p = 0.07) and sickled cell proportion; (ii) a significant increase in lung VCAM-1, ICAM-1, IL-1β, ET-1, eNOS, and TF mRNA associated with an increase in VCAM-1 expression on lung endothelium; (iii) a rise in cardiac oxidative stress with increased lipid oxidation and decreased anti-oxidant enzyme activities, and (iv) an increase in plasma TNF-α and IL-6 and a decrease in plasma ET-1. In SAD mice, hypoxia/reoxygenation stress induces hemolysis that, together with oxidative stress, inflammation, vascular adhesion, and coagulation, may induce vascular occlusion and consequently RBC sickling. The present results give the kinetics of VOC molecular markers in SAD mice which may aid in testing the efficiency of new therapeutic processes against VOC.

The role of tissue factor and protease‐activated receptor 2 in endometriosis

Little is known about the roles of TF and PAR-2 in endometriosis. This article investigated the expression of TF and PAR-2 in ectopic and eutopic endometrium with endometriosis and their relationship with the menstrual cycle. Ectopic and eutopic endometrium tissues from 42 women with ovarian endometrioma and endometrium tissues from 20 women without endometriosis were obtained. All the samples were assessed for TF and PAR-2 protein location using immunohistochemistry and for relative TF and PAR-2 mRNA expression using real-time florescent quantitative polymerase chain reaction (FQ-PCR). Total TF and PAR-2 expression were significantly higher in ectopic and eutopic endometrium of women with endometriosis when compared with controls. Moreover, TF expression in ectopic and eutopic endometrium and PAR-2 expression in ectopic endometrium were significantly increased through the whole menstrual cycle. However, in eutopic endometrium with endometriosis, PAR-2 expression only in secretory phase was higher than its cycle-matched normal controls. There is no such difference in the proliferative phase. The abnormal co-upregulated expression of TF and PAR-2 in eutopic and ectopic endometrium may affect the development and growth of endometriotic lesions and highlighted the pathologic role of TF and PAR-2 in eutopic endometrium in endometriosis.

Opposite regulation by PI3K/Akt and MAPK/ERK pathways of tissue factor expression, cell-associated procoagulant activity and invasiveness in MDA-MB-231 cells

BackgroundTissue factor (TF), an initiator of blood coagulation, participates in cancer progression and metastasis. We recently found that inhibition of MAPK/ERK upregulated both full length TF (flTF) and soluble isoform TF (asTF) gene expression and cell-associated TF activity in breast cancer MDA-MB-231 cells. We explored the possible mechanisms, especially the possible interaction with EGFR and PI3K/Akt pathways.MethodsA plasmid containing TF promoter −2174 ~ +128 plus luciferase reporter gene was introduced into MDA-MB-231 cells to evaluate TF promoter activity. In order to study the interaction of these pathways, ERK inhibitor (PD98059), PI3K inhibitors (LY294002, wortmannin), Akt inhibitor (A6730), and EGFR inhibitor (erlotinib) as well as the corresponding siRNAs were used to treat MDA-MB-231 cells, and ovarian cancer OVCAR-3 and SKOV-3 cells. Quantitative PCR and western blot were used to determine TF expression. One stage clotting assays were used to measure pro-coagulation activity of the MDA-MB-231 cells.ResultsWe show that PI3K inhibitors LY294002, wortmannin and A6730 significantly inhibited TF promoter activity, and reduced TF mRNA and protein levels due to the inhibition of Akt phosphorylation. In contrast, ERK inhibitor PD98059 and ERK siRNA enhanced TF promoter activity by 2.5 fold and induced an increase in TF mRNA and protein levels in a dose dependent manner in these cells. The PI3K/Akt pathway was shown to be involved in PD98059-induced TF expression because the induction was inhibited by PI3K/Akt inhibitors. Most interestingly, the EGFR inhibitor erlotinib and EGFR siRNA also significantly suppressed PD98059- or ERK siRNA-induced TF promoter activity and TF protein expression. Similar results were found with ovarian cancer cells SKOV-3 and OVCAR-3. Furthermore, in MDA-MB-231, mRNA levels of asTF were regulated in a similar way to that of TF in response to the cell treatment.ConclusionsThis study showed a regulatory mechanism in which MAPK/ERK signals inhibit EGFR/PI3K/Akt-mediated TF expression in breast cancer MDA-MB-231 cells. The same regulation was observed in ovarian cancer OVCAR-3 and SKOV-3 cells. Interestingly, we observed that both flTF and asTF could be regulated in a parallel manner in MDA-MB-231. As the PI3K/Akt pathway and EGFR regulate TF expression in cancer cells, targeting these signaling components is expected to potentially inhibit TF expression-associated tumor progression.

Effect and mechanism of rosuvastatin on the expression of tissue factor in cultured human monocytemacrophages cell induced by oxidized low density lipoprotein

Objective To investigate the effects and mechanism of rosuvastatin on the expression of tissue factor in cultured human monocyte-macrophages cells which was induced by oxidized low density lipoprotein(ox-LDL).Methods The human monocyte-macrophages cells were divided into four groups:control group,ox-LDL group,Poly-inosine monophosphate (Poly Ⅰ) group,rosuvastatin group.The expression of LOX-1mRNA and TF mRNA was assayed by RT-PCR.The ELISA was performed to determine the protein concentration of TF.Results Compared with control group,the expression of LOX-1 mRNA and TF mRNA was increased in the ox-LDL group[ (3.25156±0.15772) vs (1±0) ; (2.522451±0.138967) vs (1±0) ],and it was in a concentration-dependent manner (P＜0.01).Compared with the expression of LOX-1 mRNA in the Poly-inosine monophosphate group and rosuvastatin group,TF mRNA were decreased in the ox-LDL group[ (2.95139±0.157253) vs(3.25156±0.15772) ; (2.877343±0.156558) vs(3.25156±0.15772) ; (1.811956±0.169699) vs (2.522451±0.138967) ; (1.687701±0.174647) vs (2.522451±0.138967)],and it was in a concentration-dependent manner(P＜0.05).Compared with control group,the expression of TF in the ox-LDL group was increased [(207.7233±1.154701) vs (184.8467±0.871799) ],and it was in a concentration-dependent manner (P＜0.01).Compared with the Poly-inosine monophosphate group and rosuvastatin group [(197.8733±1.505003) vs (207.7233±1.154701) ;(202.9567±2.722744)vs(207.7233±1.154701) ],the expression of TF in the ox-LDL group were decreased,and it was in a concentration-dependent manner (P＜0.05).Conclusions LOX-1 may be responsible for the expression of TF in human monocyte-macrophages cells induced by ox-LDL.Rosuvastatin is able to down-regulate the expression of LOX-1mRNA in human monocyte-macrophages cells through oxLDL,and TF mRNA and TF expression can be reduced. Key words: Fluorobenzenes/PD; Pyrimidines/PD; Sulfonamides/PD; Mononuclear phagocyte system/ME/DE; Thromboplastin/ME

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.