Tissue Factor Expression Levels Research Articles

The effect of GnRH-a on the angiogenesis of endometriosis.

Neoangiogenesis is necessary for adhesion and invasiveness of endometriotic lesions in women affected by endometriosis. Vascular endothelial growth factor (VEGF) is one of the main components of angiogenesis and is part of the major pathway tissue factor (TF)-protease activated receptor-2 (PAR-2)-VEGF that leads to neoangiogenesis. Specificity protein 1 (SP1) is a transcriptional factor that has recently been studied for its crucial role in angiogenesis via a specific pathway. We hypothesize that by blocking angiogenetic pathways we can suppress endometriotic lesions. Gonadotrophin-releasing hormone-agonists (GnRH-a) are routinely used, especially preoperatively, in endometriosis. It would be of great interest to clarify which angiogenetic pathways are affected and, thereby, pave the way for further research into antiangiogenetic effects on endometriosis. We used quantitative real-time polymerase chain reaction (qRT-PCR) to study mRNA expression levels of TF, PAR-2, VEGF, and SP1 in endometriotic tissues of women who underwent surgery for endometriosis and received GnRH-a (leuprolide acetate) preoperatively. VEGF, TF, and PAR-2 expression is significantly lower in patients who received treatment (p < 0,001) compared to those who did not, whereas SP1 expression is not altered (p = 0.779). GnRH-a administration does affect some pathways of angiogenesis in endometriotic lesions, but not all of them. Therefore, supplementary treatments that affect the SP1 pathway of angiogenesis should be developed to enhance the antiangiogenetic effect of GnRH-a in patients with endometriosis. Clinicaltrial.gov ID: NCT06106932.

Time Course of Priming Effect of TF Inducers on Synergistic TF Expression and Intra-Cellular Gap Formation of Human Vascular Endothelial Cells via the Extrinsic Coagulation Cascade.

Chronic spontaneous urticaria (CSU) is characterized by daily recurring wheal and flare with itch for more than 6 weeks. The extrinsic coagulation system has been shown to be activated in correlation with CSU severity. We have reported that tissue factor (TF), a trigger of the extrinsic coagulation cascade, is synergistically expressed on vascular endothelial cells by simultaneous stimulation with TF inducers (TFI), followed by activation of the extrinsic coagulation cascade and hyper permeability in vitro. However, vascular endothelial cells are not likely to be simultaneously stimulated by multiple TFIs under physiological conditions. Therefore, in order to know whether sequential, rather than simultaneous, stimuli with interval may induce synergistic activation of TF, we investigated the time course of the priming effects of each TFI for synergistic TF expression in vascular endothelial cells (HUVECs). We stimulated HUVECs with a TFI (first stimulation) and then stimulated cells with another TFI at indicated time points (second stimulation) and detected TF expression and activity. The TF expression induced by simultaneous stimulation diminished in a few hours. However, both synergistic enhancement of TF expression and activation level of the coagulation cascade were detected even when the second stimulation was added 18 or 22 h after the first stimulation. Thus, the priming effect of TFI for synergistic TF expression may persist for a half day or longer.

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement

T cell engaging bispecific antibody (TCB) is an effective immunotherapy for cancer treatment. Through co-targeting CD3 and tumor-associated antigen (TAA), TCB can redirect CD3+ T cells to eliminate tumor cells regardless of the specificity of T cell receptor. Tissue factor (TF) is a TAA that involved in tumor progression. Here, we designed and characterized a novel TCB targeting TF (TF-TCB) for the treatment of TF-positive tumors. In vitro, robust T cell activation, tumor cell lysis and T cell proliferation were induced by TF-TCB. The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB, and was related to TF expression level of tumor cells. In vivo, in both tumor cell/human peripheral blood mononuclear cells (PBMC) co-grafting model and established tumor models with poor T cell infiltration, tumor growth was strongly inhibited by TF-TCB. T cell infiltration into tumors was induced during the treatment. Furthermore, efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors. For the first time, our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.

Evaluation of tissue factor expression on monocytes in the patients with sepsis

Sepsis is nearly always associated with some type of haemostatic disorder. The factors that play main causal role in pathogenesis of these processes are pro-inflammatory cytokines, vascular endothelium, platelets, leukocytes, and tissue factor (TF) expressed on these cells, which is always in an active state. Given a potential relationship between the blood clotting and pathophysiology of sepsis, TF may be considered a biomarker for early diagnosis, risk stratification, and prognosis of disease outcome in sepsis. Objective – to study quantitative content (CD14+CD142+) and the levels of TF expression on monocytes in the patients with sepsis, to analyze the dependence of these parameters on the severity of multiple organ dysfunction according to the SOFA scale, and disease outcomes.67 patients with sepsis were examined. The severity of multiple organ dysfunction/failure was assessed by means of the SOFA score (Sepsis-related Organ Failure Assessments, Sequential Organ Failure Assessment). All the patients were divided in 2 groups based on the severity of their condition and extent of organ failure. Group 1 (n = 30) included the patients diagnosed with sepsis and severe organ dysfunction of 6 points on the SOFA scale; Group 2 (n = 37) consisted of the patients with sepsis and organ dysfunction of 6 points according to the SOFA scores. Blood sampling from patients was made within initial 48 hours after admission and diagnosis. Quantitative content (CD14+CD142+) and the level of expression of tissue factor on monocytes were investigated by flow cytometry. We have found that the content of (CD14+CD142+) cells was significantly higher in patients with sepsis than in healthy individuals (6.03±1.05% vs 0.24±0.02%, p = 0.001), being higher in more severe organ dysfunction (SOFA) vs less severe cases (SOFA) (6.50±0.98% versus 4.42±0.36%, p = 0.05). High level of TF expression on monocytes showed a direct correlation (r 0.71; p = 0.05) with severity of organ dysfunction (SOFA), and it was associated (p = 0.004) with lethal outcome of the disorder. These results suggest that expression of tissue factor on monocytes can serve as a biomarker reflecting the degree of systemic inflammation in sepsis, thus being a criterion for predicting clinical severity and outcome of the disease in patients with sepsis.

Thrombin and factor Xa promote premature senescence and remodeling in areas at risk of the left appendage and in atrial endothelial cells: potential modulation by SGLT1 and/or SGLT2 inhibition

Abstract Introduction Atrial fibrillation (AF), the most common cardiac arrhythmia, promotes prothrombotic responses particularly in the distal part of the left atrial appendage (LAA) characterized by reduced flow and low shear stress. AF occurrence and duration are closely associated with endothelial dysfunction and left atrium remodeling. The contribution of coagulation factors thrombin and factor Xa (FXa) to LA remodeling remains poorly studied. This study investigated whether thrombin and FXa promote endothelial dysfunction and profibrotic and prothrombotic responses in the distal and proximal parts of the LAA. Since SGLT2 inhibitors have shown pronounced cardiac protection, the contribution of SGLT2 was examined. Methods LAAs were harvested from porcine hearts. Proximal (low stasis, high shear) and distal (high stasis, low shear) parts were cut and incubated with a coagulation factor (thrombin or FXa). Left atrial endothelial cells (AECs) were collected enzymatically, cultured and used at passage 1. The level of oxidative stress was assessed using the redox-sensitive probe dihydroethidium (DHE), fibrosis by Sirius red histological staining, senescence-associated β-galactosidase (SA-β-gal) activity using X-gal staining in tissues and the fluorogenic substrate C12FDG and flow cytometry in cells. The expression of target proteins was detected by immunofluorescence staining and Western blot analysis. Results The distal part of LAA displayed higher levels of oxidative stress in the endothelium and the atrial wall than the proximal part. SA-β-gal activity, ICAM-1, VCAM-1, and MMP-9 staining was more pronounced in the endothelium of the distal part compared to the proximal part whereas eNOS staining was decreased. The distal LAA wall showed higher levels of fibrosis. Western blot analysis showed higher expression levels of tissue factor (TF), VCAM-1, MCP-1, MMP-9, TGF-β and SGLT-2 in the distal part compared to the proximal one. Both thrombin and FXa increased these signals to a greater extent in the distal part of the LAA whereas the eNOS expression level was decreased. Exposure of AECs to either thrombin or FXa induced a pro-oxidant response and increased the expression level of ICAM-1, TGF-β, MMP-2, MMP-9, SGLT1 and SGLT-2. Thrombin promoted SA-β-gal activity in AECs. The selective SGLT-2 inhibitor empagliflozin and the dual SGLT1/2 inhibitor sotagliflozin prevented the pro-oxidant response and SA-β-gal activity in response to thrombin. Conclusion Thus, the distal part of the LAA is prematurely affected by endothelial dysfunction and senescence associated with a pro-oxidant, pro-remodeling and pro-inflammatory response that is further increased by thrombin and FXa. Hence, the coagulation cascade appears to prematurely promote atrial endothelium and wall dysfunction predominantly in areas at risk that may pave the way to thrombus formation. The findings also suggest a potential protective effect of SGLT1 and/or SGLT2 inhibition on atrial dysfunction. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): Unrestricted research grant from Boehringer Ingelheim Pharma GmbH &amp; Co. KG, Biberach, Germany

Tissue factor promotes airway pathological features through epithelial-mesenchymal transition of bronchial epithelial cells in mice with house dust mite-induced asthma

It has recently been shown that expression levels of tissue factor (TF) are high in the serum and peripheral blood mononuclear cells of patients with asthma. However, whether TF impacts airway inflammation and remodelling in asthma remains unknown. The aim of this study was to investigate the effect of TF in asthma airway inflammation and remodelling using a house dust mite (HDM)-induced chronic asthma model and human bronchial epithelial (16HBE) cells. A chronic asthma model was constructed in BALB/c mice by the intranasal instillation of HDM. Mice were treated with short hairpin TF (shTF), and airway inflammation and remodelling features of asthma and epithelial-mesenchymal transition (EMT) were assessed. 16HBE cells were induced by transforming growth factor-β1 (TGF-β1) and HDM in the presence or absence of shTF; then, EMT markers and invasion and migration ability were determined. TF expression increased in the lung tissue and 16HBE cells when exposed to HDM. TF downregulation in the lung significantly reduced airway hyperresponsiveness, eosinophil inflammation, the EMT process, and levels of interleukin (IL)-4, IL-6, IL-13, and TGF-β1 in bronchoalveolar lavage fluid of asthmatic mice. Moreover, TF downregulation inhibited migration and incursion and decreased the expression levels of fibronectin 1 and TGF-β1, but increased the expression of E-cadherin in HDM- and TGF-β1-stimulated 16HBE cells. These results demonstrated that TF promoted airway pathological features by enhancing the EMT of bronchial epithelial cells both in vitro and in mice with house dust mite-induced asthma.

Expression of tissue factor and tissue factor pathway inhibitors during ovulation in rats: a relevance to the ovarian hyperstimulation syndrome

BackgroundBlood coagulation has been associated with ovulation and female infertility. In this study, the expression of the tissue factor system was examined during ovulation in immature rats; the correlation between tissue factor and ovarian hyperstimulation syndrome (OHSS) was evaluated both in rats and human follicular fluids.MethodsOvaries were obtained at various times after human chorionic gonadotropin (hCG) injection to investigate the expression of tissue factor system. Expression levels of ovarian tissue factor, tissue factor pathway inhibitor (Tfpi)-1 and Tfpi-2 genes and proteins were determined by real-time quantitative polymerase chain reaction (qPCR), and Western blot and immunofluorescence analyses, respectively. Expression levels of tissue factor system were also investigated in ovaries of OHSS-induced rats and in follicular fluid of infertile women.ResultsThe expression of tissue factor in the preovulatory follicles was stimulated by hCG, reaching a maximum at 6 h. Tissue factor was expressed in the oocytes and the preovulatory follicles. Tfpi-2 mRNA levels were mainly increased by hCG in the granulosa cells whereas the mRNA levels of Tfpi-1 were decreased by hCG. Human CG-stimulated tissue factor expression was inhibited by the progesterone receptor antagonist. The increase in Tfpi-2 expression by hCG was decreased by the proliferator-activated receptor γ (PPARγ) antagonist. Decreased expression of the tissue factor was detected in OHSS-induced rats. Interestingly, the tissue factor concentrations in the follicular fluids of women undergoing in vitro fertilization were correlated with pregnancy but not with OHSS.ConclusionsCollectively, the results indicate that tissue factor and Tfpi-2 expression is stimulated during the ovulatory process in rats; moreover, a correlation exists between the levels of tissue factor and OHSS in rats but not in humans.

Type 1 interferon aggravates lipopolysaccharide-induced sepsis through upregulating Caspase-11 and Gasdermin D.

This study aimed to investigate the mechanism of type I interferon (IFN) in aggravating sepsis in bacterial infection, focusing on the roles of Caspase-11 (Casp11) and Gasdermin D (Gsdmd) in this process. Type I interferons, including IFNα and IFNβ, were used to treat peritoneal macrophage harvested from wild-type or IFNα/βR1 knockout (KO) mice, of which the levels of Casp11 and Gsdmd were monitored using real-time polymerase chain reaction (RT-PCR) and Western blot, the exposure to phosphatidylserine was monitored by flow cytometry, and tissue factor (TF) activation was assessed by RT-PCR and TF chromogenic assay. Endotoxemia in wild-type mice led to upregulation of Casp11 and Gsdmd in myeloid cells, which in contrast was attenuated in IFNα/βR1 KO mice. IFNα or IFNβ treatment led to dose-dependent upregulation of Casp11 and Gsdmd in peritoneal macrophages harvested from wild-type mice, but induced negligible changes in IFNα/βR1 KO mice. Type I IFN promoted phosphatidylserine exposure in peritoneal macrophage from wild-type mice but not IFNα/βR1 KO mice. Type I IFN induced insignificant changes of TF expression levels in both wild-type mice and IFNα/βR1 KO mice, but the TF activity was markedly increased in wild-type mice after type I IFN treatment. Our data suggested that the upregulation of Casp11 and Gsdmd in myeloid cells and macrophages induced by endotoxemia was reliant on the expression of IFNα/βR1. IFNα or IFNβ treatment efficiently upregulated Casp11 and Gsdmd, phosphatidylserine exposure, and TF activity of macrophages. Therefore, type I IFN could aggravate sepsis through upregulating Casp11 and Gsdmd.

Berberine dose-dependently inhibits the expression of procoagulant and fibrinolytic inhibitory factors in lipopolysaccharide-induced rat type II alveolar epithelial cells

To observe the effects of berberine on procoagulant and fibrinolytic inhibitory factors produced by rat type II alveolar epithelial cell (AEC II) induced by lipopolysaccharide (LPS). AEC II cells (RLE-6TN cells) were cultured in vitro, and the cells in logarithmic growth phase were collected. The cytotoxicity text of berberine was detected by cell counting kit-8 (CCK-8) to determine the drug concentration range according to inhibition concentration of half cells (IC50). The RLE-6TN cells were divided into five groups, the cells in blank control group were cultured in DMEM; the cells in LPS group were stimulated with 5 mg/L LPS; and the cells in berberine pretreatment groups were pretreated with 20, 50 and 80 μmol/L berberine for 1 hour, and then were co-cultured with 5 mg/L LPS. The cells were collected after LPS induced for 24 hours. The protein and mRNA expression levels of tissue factor (TF), tissue factor pathway inhibitor (TFPI) and plasminogen activator inhibitor-1 (PAI-1) in the cells were detected by Western blotting and real-time fluorescence quantification reverse transcription-polymerase chain reaction (RT-qPCR). The levels of activated protein C (APC), precollagen III peptide (PIIIP), thrombin-antithrombin complex (TAT) and antithrombin III (AT III) in the cell supernatant were measured by enzyme linked immunosorbent assay (ELISA). According to the inhibition rate curve, the IC50 of berberine on RLE-6TN cells was 81.16 μmol/L. Therefore, 20, 50 and 80 μmol/L were selected as the intervention concentration of berberine. Compared with the blank control group, the expression and secretion of procoagulant and fibrinolytic inhibitory factors were abnormal in RLE-6TN cells after LPS induced for 24 hours. The protein and mRNA expression levels of TF and PAI-1 in the LPS group were significantly increased, but the protein and mRNA expression levels of TFPI were significantly decreased. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly decreased, while the levels of PIIIP and TAT were significantly increased. After pretreatment with berberine, the abnormal expression and secretion of procoagulant and fibrinolytic inhibitory factors induced by LPS were corrected in a dose-dependent manner, especially in 80 μmol/L. Compared with the LPS group, the protein and mRNA expression levels of TF and PAI-1 in the berberine 80 μmol/L group were significantly decreased [TF protein (TF/GAPDH): 0.45±0.02 vs. 0.55±0.03, TF mRNA (2-ΔΔCt): 0.39±0.08 vs. 1.48±0.11, PAI-1 protein (PAI-1/GAPDH): 0.37±0.02 vs. 0.64±0.04, PAI-1 mRNA (2-ΔΔCt): 1.14±0.29 vs. 4.18±0.44, all P < 0.01] and those of TFPI were significantly increased [TFPI protein (TFPI/GAPDH): 0.53±0.02 vs. 0.45±0.02, TFPI mRNA (2-ΔΔCt): 0.94±0.08 vs. 0.40±0.05, both P < 0.01]. Meanwhile, the levels of APC and AT III in the cell supernatant were significantly increased [APC (μg/L): 1 358.5±26.0 vs. 994.2±23.1, AT III (μg/L): 118.0±7.4 vs. 84.4±2.7, both P < 0.01], while those of PIIIP and TAT were significantly decreased [PIIIP (μg/L): 11.2±0.4 vs. 18.6±0.9, TAT (ng/L): 222.1±2.8 vs. 287.6±7.0, both P < 0.01]. Berberine could inhibit the LPS-induced expressions of procoagulant and fibrinolytic inhibitory factors in rat AEC II cells and promote the expressions of anticoagulant factors in a dose-dependent manner. Berberine may be a new therapeutic target for alveolar hypercoagulability and fibrinolysis inhibition in acute respiratory distress syndrome (ARDS).

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

Down-regulation of tissue factor inhibits invasion and metastasis of non-small cell lung cancer.

Objective: Tissue factor (TF) is clinically identified as a marker for the detection of various types of cancer as well as the prediction of prognosis for cancer patients. This present study aims to explore the possibility and feasibility to use plasma TF as a biomarker for the prediction of prognosis of patients with non-small cell lung cancer (NSCLC).Methods: A total of 100 patients with NSCLC at stage I to IV was included in the study, in whom the expression of plasma TF was detected. The Cox proportional-hazards regression model was then used to analyze the collected information, attempting to identify how patients' overall survival (OS) was associated with the expression of plasma TF. To verify the function of TF in invasion and metastasis, the expression of plasma TF was downregulated by SiRNA both in vivo and in vitro.Results: The expression of plasma TF in NSCLC patients was related to the diagnosis age of the patient. It was noted that patients with high TF expression levels tended to have worse OS performance, which implied that TF could be used as a marker for patients with stage I-IV NSCLC (HR = 2.030, 95% CI = 1.21-3.398, P = 0.007). TF down-regulation inhibited the growth of tumor in vitro as well as the metastasis and invasion of NSCLC cells in vivo.Conclusion: Both in vivo and in vitro, the invasion and migration of NSCLC cells are suppressed by TF knockdown. TF has the potential to become an effective biomarker for the prediction of prognosis of patients with stage I-IV NSCLC.

Anti‑tissue factor antibody‑mediated immuno‑SPECT imaging of tissue factor expression in mouse models of pancreatic cancer

Tissue factor (TF) has emerged as a critical factor in oncogenic events, leading to the development of TF‑targeted diagnostic and therapeutic approaches. A non‑invasive imaging method to evaluate target molecule expression with high sensitivity and high quantitative ability is imperative for selecting the appropriate patients for TF‑targeted therapy. To elucidate the potential of 111In‑labeled anti‑TF antibody 1849 (111In‑1849) as an immuno‑single photon emission computed tomography (SPECT) probe targeting TF, we evaluated TF‑dependent in vitro binding as well as in vivo biodistribution and tumor accumulation of 111In‑1849 in pancreatic cancer cells/models with varying TF expression levels. TF expression levels in five human pancreatic cancer cell lines, BxPC‑3, BxPC‑3‑TF‑knockout (BxPC‑3‑TFKO), Capan‑1, PSN‑1 and SUIT‑2, were examined by immunofluorescence. Binding of 111In‑1849 to each cell line was assessed. Biodistribution and imaging studies were also conducted in tumor‑bearing mice. Furthermore, the relationship of TF expression with cell binding and tumor uptake was analyzed. In the immunofluorescence studies, BxPC‑3 exhibited the highest TF expression, followed by Capan‑1, PSN‑1, SUIT‑2 and BxPC‑3‑TFKO. Cell binding assays revealed that BxPC‑3 cells had the highest 111In‑1849 binding, followed by PSN‑1, Capan‑1 and SUIT‑2; no binding was detected in BxPC‑3‑TFKO cells. The BxPC‑3 xenograft was clearly visualized on 111In‑1849 SPECT/CT, and the highest uptake was detected on day 4. The biodistribution of 111In‑1849 on day 4 revealed that tumor uptake ranged from 8.68 to 50.58% of the injected dose per gram of tissue; BxPC‑3 had the highest uptake and SUIT‑2 had the lowest. TF expression was significantly associated with cell binding (R2=0.79, P<0.05) and tumor uptake (R2=0.92, P<0.01). The association of 111In‑1849 uptake with TF expression suggests the potential application of non‑invasive imaging with radiolabelled 1849 for selecting the appropriate patients who would likely respond to TF‑targeted therapies in clinical practice.

Clinical Significance of TF and VEGF Expressions on Peripheral CD14 Positive Monocytes in Patients with Diffuse Large B Cell Lymphoma

To investigate the clinical significance of tissue factor (TF) and vascular endothelial growth factor (VEGF) expression on peripheral blood CD14 positive monocytes in patients with diffuse large B cell lymphoma (DLBCL). The expressions of TF and VEGF on peripheral CD14+ monocytes in 41 patients with DLBCL (DLBCL group) before chemotherapy and after 4 chemotherapeutic courses, and in 20 healthy subjects (control group) were detected by flow cytometry respectively, meanwhile, the relationship of the expression of TF and VEGF with international prognostic indexes (IPI) and short-term effects were analysed. The expression levels of TF and VEGF on peripheral CD14+ monocytes in DLBCL group were significantly higher than those in control group (P<0.01), and a positive correlation was found between the two groups (r=0.755, P<0.01). The expression of TF and VEGF on CD14+ monocytes in patients with prognostic risk factors significantly increased as compared with those in patients without prognostic risk factors (P<0.05), but there were no significant differences of TF and VEGF expressions on CD14+ monocytes in DLBCL group with different sex, age, subtypes (P＞0.05). As compared with patients without prognostic risk factors, the expression levels of TF and VEGF on CD14+ monocytes of patients with prognostic risk factors significantly increased (P<0.05). The expression of TF and VEGF on CD14+ monocytes in DLBCL group showed an increasing tendency along with the increase of IPI index (P<0.01). The expression levels of TF and VEGF on CD14+ monocytes in remission group before chemotherapy were lower than those in non-remission group (P<0.01); after chemotherapy, the expression levels of TF and VEGF on CD14+ monocytes in remission group were lower than those before chemotherapy (P<0.05), while the TF and VEGF expression levels in non-remission group were no singnificauly different from TF and VEGF levels before chemtherapy (P>0.05), the survival of patients in group with low expression of TF and VEGF was superior to that in group with high expression of TF and VEGF (P<0.05). The paripheral blood CD14+ monocytes in DLBCL patients highly express the TF and VEGF, which relate with IPI, therapeutic efficacy and survival, thus the TF and VEGF expression levels are of reference significance for evaluating the therapeutic efficacy and prognosis of patients.

The expression changes and correlation analysis of high mobility group box-1 and tissue factor in the serum of rats with sepsis.

To investigate the expression changes of high mobility group box-1 (HMGB-1) and tissue factor (TF) and their correlation in the serum of sepsis rat models. 30 rats were divided into the sham-operated group, 15 rats were in the control group. The cecal ligation and puncture method was used to make the animal model with abdominal infection induced by sepsis. There were 15 rats in the sepsis group among which they were divided into 3 subgroups at different time points after modeling (after 6 hours, 12 hours, 24 hours). Cardiac function indicators of the rats in each subgroup were monitored, including heart rate (HR), left ventricular end-diastolic pressure (LVEDP) and left ventricular developed pressure (LVDP), and enzyme-linked immunosorbent assay (ELISA) was used to test the changes of the expression levels of HMGB-1 and TF in the serum of the rats after 6 hours, 12 hours, 24 hours. Pearson correlation analysis was used to analyze the correlation between HMGB-1 and TF. HR and LVEDP of the rats in the sepsis group were significantly higher than those of the rats in the control group. The differences were statistically significant (p<0.050). LVDP of the rats in the sepsis group was markedly lower than that of the rats in the control group. The differences were statistically significant (p<0.050). The expressions of HMGB-1 and TF of the rats in the subgroups of the sepsis group were higher than those of the rats in the control group after 6 hours, 12 hours, 24 hours; the expression levels of HMGB-1 and TF of the rats with sepsis increased with time. The differences were statistically significant (p<0.050). When the expressions of HMGB-1 and TF of the rats in the sepsis group were compared with each other within the group the differences were significantly different (p<0.050). The expressions of HMGB-1 and TF in the subgroups at the 24th hour were significantly higher than those at the 6th hour. The differences were statistically significant (p<0.050). The differences of the expression of TF of the rats in the control group were not statistically significant (p>0.050). There was a significant positive correlation between HMGB-1 and TF of the rats in the sepsis group (r=0.772, p=0.002). The expression levels of HMGB-1 and TF of the rats with sepsis gradually increased with time, and the level of HMGB-1 was positively correlated with the level of TF.

Protease-activated receptor 2 exacerbates cisplatin-induced nephrotoxicity.

Acute kidney injury (AKI) is associated with hypercoagulability. Tissue factor/factor VIIa complex and factor Xa in the coagulation cascade activate protease-activated receptor 2 (PAR2). Previously, we have shown that PAR2-mediated inflammation aggravates kidney injury in models of diabetic kidney disease and adenine-induced renal fibrosis. However, the role of PAR2 in AKI remains unclear. To clarify the role of PAR2, we administered cisplatin, one of the most common causal factors of AKI, to wild-type and PAR2-deficient mice. The expression levels of tissue factor and PAR2 were significantly increased in the kidneys of mice that were administered cisplatin. A lack of PAR2 corrected the levels of plasma blood urea nitrogen and creatinine as well as ameliorated the acute tubular injury score in the kidney. A lack of PAR2 corrected the infiltration of neutrophils and the gene expression levels of proinflammatory cytokines/chemokines in these mouse kidneys. Similarly, apoptotic markers, such as cleaved caspase-3-positive area and Bax/Bcl2 ratio, were attenuated via PAR2 deletion. Thus, elevated PAR2 exacerbates cisplatin nephrotoxicity, and targeting PAR2 is a novel therapeutic option that aids in the treatment of patients with cisplatin-induced AKI.

Positive crosstalk between EGFR and the TF-PAR2 pathway mediates resistance to cisplatin and poor survival in cervical cancer.

Cisplatin-based chemoradiation is the standard treatment for cervical cancer, but chemosensitizing strategies are needed to improve patient survival. EGFR (Epidermal Growth Factor Receptor) is an oncogene overexpressed in cervical cancer that is involved in chemoresistance. Recent studies showed that EGFR upregulates multiple elements of the coagulation cascade, including tissue factor (TF) and the protease-activated receptors (PAR) 1 and 2. Moreover, many G protein-coupled receptors, including PARs, have been implicated in EGFR transactivation. However, the role of coagulation proteins in the progression of cervical cancer has been poorly investigated. Herein we employed cervical cancer cell lines and The Cancer Genome Atlas (TCGA) database to evaluate the role of EGFR, TF and PAR2 in chemoresistance. The SLIGKL-NH2 peptide (PAR2-AP) and coagulation factor VIIa (FVIIa) were used as PAR2 agonists, while cetuximab was used to inhibit EGFR. The more aggressive cell line CASKI showed higher expression levels of EGFR, TF and PAR2 than that of C33A. PAR2 transactivated EGFR, which further upregulated cyclooxygenase-2 (COX2) expression. PAR2-AP decreased cisplatin-induced apoptosis through an EGFR- and COX2-dependent mechanism. Furthermore, treatment of CASKI cells with EGF upregulated TF expression, while treatment with cetuximab decreased the TF protein levels. The RNA-seq data from 309 TCGA samples showed a strong positive correlation between EGFR and TF expression (P = 0.0003). In addition, the increased expression of EGFR, PAR2 or COX2 in cervical cancer patients was significantly correlated with poor overall survival. Taken together, our results suggest that EGFR and COX2 are effectors of the TF/FVIIa/PAR2 signaling pathway, promoting chemoresistance.

Tissue Factor Enhances the Alveolar Epithelial Barrier Integrity during Acute Lung Injury

IntroductionWe have previously reported that lung epithelial tissue factor (TF) knockout (TFΔEpi) mice have increased alveolar capillary barrier permeability. Ultrastructurally, these mouse lungs have profound morphological defects in epithelial attachment to the basement membrane. Adhesion/migration are regulated in part by integrins which bind the extracellular matrix proteins to modulate cytoskeletal dynamics. In cancer cells, TF binds β1 integrin and increases cell migration, a finding that suggests a role for non‐coagulative mechanisms of TF in cell adhesion and migration. We hypothesize that TF‐β1 integrin binding promotes alveolar epithelial barrier integrity through cell adhesion and migration during acute lung injury (ALI).MethodTF overexpressing (TF+Epi) and knockout (TFΔEpi) mice, along with TF knockout A549 cells (A549ΔTF) were used to test whether lung epithelial TF is critical for the integrity of the alveolar epithelial barrier. TF+Epi mice were treated with intratracheal PBS or LPS and lung permeability was measured by bronchoalveolar lavage (BAL) fluid protein level. Alveolar type II epithelial cells (ATII) were isolated from TFΔEpi and TF+Epi mice to measure β1 integrin protein levels. A549ΔTF cells were treated with LPS (100ug/mL) or PBS for 24 hours before western blot of β1 integrin expression. Migration was assessed in wild‐type and A549ΔTF cells using Electric Cell‐substrate Impedance Sensing (ECIS) on cell monolayers. Adhesion was assayed by plating wild‐type and A549ΔTF cells on matrigel and quantifying the absorbance of crystal violet stained adherent cells.ResultsTF+Epi mice had decreased lung permeability (BAL protein 303 μg/mL +/− 62 versus 416 +/− 137 in control, p=0.025) following LPS challenge. A549ΔTF cells had significantly decreased cell adhesion (Figure) (absorbance 0.308 +/− 0.119 versus 0.603 +/− 0.246 in wild type, *p&lt;0.001) along with decreased migration (p=0.017) compared to control A549 cells with wild type TF expression levels. A549ΔTF cells had decreased β1 integrin protein levels (relative expression 0.017 +/− 0.01 versus 0.61 +/− 0.02 control, p=0.036) following LPS challenge. ATII cells isolated from TFΔEpi mice also have decreased β1 integrin protein levels.ConclusionsTF contributes to alveolar barrier integrity in part through a mechanism dependent on β1 integrin. These data provide support that lung epithelial TF may play a non‐coagulative role in protection during ALI. This novel role for TF in maintaining alveolar epithelial barrier integrity offers a potential therapeutic target during ALI.Support or Funding InformationVanderbilt University Initiative for Maximizing Student Diversity funding through the NIH, Nashville Veterans Affairs Medical Center, Funding: HL090785, HL103836This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.

AimsSirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI).Methods and resultsUsing a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3−/− mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3−/− compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3−/− mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3−/− bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3−/− mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3−/− neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01).Conclusions Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

ImmunoPET imaging of tissue factor expression in pancreatic cancer with 89Zr-Df-ALT-836

Overexpression of tissue factor (TF) has been associated with increased tumor growth, tumor angiogenesis, and metastatic potential in many malignancies, including pancreatic cancer. Additionally, high TF expression was shown to strongly correlate with poor prognoses and decreased survival in pancreatic cancer patients. Herein, we exploited the potential targeting of TF for positron emission tomography (PET) imaging of pancreatic cancer. The TF-targeted tracer was developed through radiolabeling of the anti-human TF monoclonal antibody (ALT-836) with 89Zr. The tracer was characterized by fluorescence microscopy and flow cytometry assays in BXPC-3 and PANC-1 cells, two pancreatic cancer cell lines with high and low TF expression levels, respectively. Non-invasive PET scans were acquired in tumor-bearing mice injected with 89Zr-Df-ALT-836. Additionally, ex vivo biodistribution, blocking, and histological studies were performed to establish the affinity and specificity of 89Zr-Df-ALT-836 for TF in vivo. 89Zr-labeling of Df-ALT-836 was achieved in high yield and good specific activity. Flow cytometry and microscopy studies revealed no detectable difference in TF-binding affinity between ALT-836 and Df-ALT-836 in vitro. Longitudinal PET scans unveiled a lasting and prominent 89Zr-Df-ALT-836 uptake in BXPC-3 tumors (peak at 31.5±6.0%ID/g at 48h post-injection; n=3), which was significantly abrogated (2.3±0.5%ID/g at 48h post-injection; n=3) when mice were pre-injected with a blocking dose (50mg/kg) of unlabeled ALT-836. Ex vivo biodistribution data confirmed the accuracy of the PET results, and histological analysis correlated high tumor uptake with in situ TF expression. Taken together, these results attest to the excellent affinity and TF-specificity of 89Zr-Df-ALT-836. With elevated, persistent, and specific accumulation in TF-positive BXPC-3 tumors, PET imaging using 89Zr-Df-ALT-836 promises to open new avenues for improving future diagnosis, stratification, and treatment response assessment in pancreatic cancer patients.

Effect of Agkistrodon Acutus Venom PCA on HUVEC Activity

To investigate the effects of Agkistrodon acutus venom protein C activator(PCA) on ultrastructure of human umbilical vein endothelial cells(HUVEC), and the levels of tissue factor(TF), vascular von Willebrand factor (vWF) and endothelin-1 secreted by HUVEC and to clarify the anti-thrombotic mechanism of PCA. The experiments were divided into control group(DMEM), LPS group (LPS 0.1 µg/ml), PCA group(PCA 1 µg/ml) and PCA+LPS group (1 µg/ml PCA+ 0.1 µg/ml LPS). The morphology of endoplasmic reticulum, mitochondria and the number of autophagosome in HUVEC were observed by transmission electron microscopy. The TF, vWF and ET-1 were measured in the medium of each group by ELISA; RT-PCR was used to detect mRNA expression level of vWF and ET-1 in cells; and the protein expression level of TF in cells was detected by Western blot. Compared with the control group, the ultrastructural changes of HUVEC in the LPS group included the cell membrane getting rough, swelling of mitochondria and endoplasmic reticulum, and autophagosome increase, however, the ultrastructure differences between PCA and control group were not significant. Compared with the ultrastructure of HUVECs in LPS group, the swelling of mitochondria and endoplasmic reticulum disappeared in the LPS+PCA group, and the number of autophagosome decreased obviously. Compared with the control group, the content of ET-1, vWF and TF in cell culture supernatant, and the protein expression level of vWF, ET-1 gene and TF protein were significantly increased in LPS group (P<0.05); the expression levels of the 3 factors in the cell culture supernatant and cells in PCA group were not significantly different from the control group (P>0.05). The expression levels of TF, vWF and ET-1 in LPS group were significantly lower than those in LPS+PCA group (P<0.05). PCA(1 µg/ml) can reduce the ultrastructural changes of HUVEC induced by LPS, and inhibit the increase of TF, vWF and ET-1 secretion from HUVEC induced by LPS.