High Tissue Factor Expression Research Articles

Tissue Factor Maintains Lung Epithelial Barrier Integrity in Acute Lung Injury Through Cell Adhesion

Background. Acute Respiratory Distress Syndrome (ARDS) is a common cause of acute respiratory failure. While loss of lung epithelial barrier integrity is a pathologic mechanism of ARDS, little is known about the factors that regulate epithelial barrier integrity in the lung. Our previously published mouse studies showed that deletion of alveolar type II epithelial cell Tissue Factor (TF) caused impaired lung barrier integrity in models of Acute Lung Injury (ALI). TF is an integral membrane protein that both initiates the extrinsic coagulation cascade and serves several non-coagulant functions. Notably, TF promotes cell adhesion through interactions with cell surface integrin heterodimers, comprised of individual α and β integrin subunits. As such, we hypothesize that epithelial TF is necessary for maintaining lung barrier integrity and that its overexpression is protective in ALI through increased cell adhesion. Methods. To determine whether supraphysiologic overexpression of TF in the lung epithelium can enhance barrier integrity we created a novel transgenic mouse in which TF was inducibly overexpressed in the lung epithelium (TFEpi+) using a CMV-TetO promoter and crossed with SPC-rtTA59 mice. High alveolar epithelial TF expression compared to wild-type littermates (WT) was confirmed through immunohistochemistry and western blot analysis after one week of doxycycline in drinking water. To induce ALI, mice were intranasally infected with 2000 colony forming units of Klebsiella pneumoniae (KP) or PBS control. At 24-hours post infection, mice were euthanized, lung tissue was collected, and a bronchoalveolar lavage (BAL) was performed. BAL protein, clot time, and leukocyte influx were measured. Lung wet-to-dry weight ratios and bacterial burden were measured. To better understand TF regulation of β1 integrin function, TF-knockout A549 cells were created using CRISPR and cultured on specific β1 integrin ligands (Collagen I, IV, Laminin-511, and Fibronectin). On-cell western blots and crystal violet were used to assess cell surface β1 integrin expression and cell adhesion. Results. TFEpi+ mice showed higher lung TF protein expression (median 38031 [IQR 25033, 57948] vs 1801 [IQR 804.3, 2083] expression normalized to total protein; p=0.0016) compared to WT. TFEpi+ mice infected with KP showed lower BAL protein (mean 248.51 [SD 98.03] vs 366.3 [SD 159.8] μg/ml; p=0.0035) and lung wet-to-dry weight ratios (mean 4.27 [SD 0.37] vs 5.29 [SD 0.75]; p=0.0152) compared to WT. Bacterial burden and BAL inflammatory cell counts were higher, while BAL clot time was lower in infected WT mice compared to control. However, these outcomes did not differ between infected TFEpi+ and WT mice. Loss of TF in alveolar epithelial cells reduced cell surface β1 integrin expression and cell adhesion to Laminin-511. Conclusion: Alveolar epithelial TF overexpression is protective for maintaining lung barrier integrity independent of coagulation and inflammation. Further, cell adhesion studies suggest that this is potentially mediated through increased a6β1 epithelial cell adhesion. AHA Postdoctoral Fellowship, National Institute of Health Grants (HL150783, I01BX002288). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

EML4-ALK fusion protein in Lung cancer cells enhances venous thrombogenicity through the pERK1/2-AP-1-tissue factor axis

BackgroundAccumulating evidence links the echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangement to venous thromboembolism (VTE) in non-small cell lung cancer (NSCLC) patients. However, the corresponding mechanisms remain unclear.MethodHigh-throughput sequencing analysis of H3122 human ALK-positive NSCLC cells treated with ALK inhibitor/ dimethyl sulfoxide (DMSO) was performed to identify coagulation-associated differential genes between EML4-ALK fusion protein inhibited cells and control cells. Sequentially, we confirmed its expression in NSCLC patients’ tissues and in the plasma of a subcutaneous xenograft mouse model. An inferior vena cava (IVC) ligation model was used to assess clot formation potential. Additionally, pathways involved in tissue factor (TF) regulation were explored in ALK-positive cell lines H3122 and H2228. Statistical significance was determined by Student t-test and one-way ANOVA using SPSS.ResultsSequencing analysis identified a significant downregulation of TF after inhibiting EML4-ALK fusion protein activity in H3122 cells. In clinical NSCLC cases, TF expression was increased especially in ALK-positive NSCLC tissues. Meanwhile, H3122 and H2228 with high TF expression exhibited shorter plasma clotting time and higher TF activity versus ALK-negative H1299 and A549 in cell culture supernatant. Mice bearing H2228 tumor showed a higher concentration of tumor-derived TF and TF activity in plasma and the highest adjusted IVC clot weights. Limiting EML4-ALK protein phosphorylation downregulated extracellular regulated protein kinases 1/2 (ERK1/2)-activating the protein-1(AP-1) signaling pathway and thus attenuated TF expression.ConclusionEML4-ALK fusion protein may enhance venous thrombogenicity by regulating coagulation factor TF expression. There was potential involvement of the pERK1/2-AP-1 pathway in this process.

Therapeutic Strategies Focused on Cancer-Associated Hypercoagulation for Ovarian Clear Cell Carcinoma.

Simple SummaryOvarian clear cell carcinoma (OCCC) has a poor prognosis in advanced cases and displays resistance to standard treatments for epithelial ovarian cancer. OCCC shows a specific clinical characteristic of a high incidence of cancer-associated thromboembolism. In this review, we focused on the association between cancer-related hypercoagulation and molecular biology in OCCC. Moreover, we summarized potential drugs targeting hypercoagulation to contribute to novel therapies for OCCC.Ovarian clear cell carcinoma (OCCC) is associated with chemotherapy resistance and poor prognosis, especially in advanced cases. Although comprehensive genomic analyses have clarified the significance of genomic alterations such as ARID1A and PIK3CA mutations in OCCC, therapeutic strategies based on genomic alterations have not been confirmed. On the other hand, OCCC is clinically characterized by a high incidence of thromboembolism. Moreover, OCCC specifically shows high expression of tissue factor and interleukin-6, which play a critical role in cancer-associated hypercoagulation and may be induced by OCCC-specific genetic alterations or the endometriosis-related tumor microenvironment. In this review, we focused on the association between cancer-associated hypercoagulation and molecular biology in OCCC. Moreover, we reviewed the effectiveness of candidate drugs targeting hypercoagulation, such as tissue factor- or interleukin-6-targeting drugs, anti-inflammatory drugs, anti-hypoxia signaling drugs, anticoagulants, and combined immunotherapy with these drugs for OCCC. This review is expected to contribute to novel basic research and clinical trials for the prevention, early detection, and treatment of OCCC focused on hypercoagulation.

Tissue Factor Expression in Colorectal Adenocarcinoma: Association with Angiogenesis and Clinical and Pathological Aspects

Abstract Introduction Tissue factor (TF) expression has been described in various neoplasms and was correlated with angiogenesis and metastases. Objectives To describe TF expression in colorectal cancers, correlating it with microvessel density and clinical and pathological variables. Methods Immunohistochemistry was used to determine TF expression and microvessel density. The Student t-test was used to compare high and low TF expression with microvessel density and with age. The chi-squared test was used for other comparisons, and Kaplan-Meier curves were used for survival analyses. Results Forty-three patients were operated with curative intent. Their mean age was 58.1 ± 12.6 years old, and 62.8% were male. The rectum was the most common location (60,4%), and most tumors reached the serosa and peri-intestinal fat (72.1%). Lymph nodes were positive in 46.5%, and 72.1% of the tumors were moderately differentiated adenocarcinomas. Death occurred in 27.6 ± 12.8 months in 51.1% of the patients who had recurrence. Tissue factor expression was intense in 88.4%. There was a positive correlation between TF expression and microvessel density (p = 0.02), and between TF and older age (p < 0.01). There was no correlation between TF expression and other variables (gender, histological type, penetration into the intestinal wall, and lymphatic and systemic metastases). Tissue factor expression did not correlate with survival. Conclusion Tissue factor expression correlated with increased microvessel density and older age. Further studies are necessary to ascertain the clinical relevance of TF in colorectal cancer.

Expression and role of interleukin-1β and associated biomarkers in deep vein thrombosis.

Lower extremity deep vein thrombosis (DVT) is a common peripheral vascular disease, in which inflammation plays an important role. The aim of the present study was to investigate the expression and role of inflammatory factors in DVT. A rat model of venous thrombosis of the lower extremities was established through venous ligation surgery. The rats were examined at 2, 8, 24, 48 and 72 h after the induction of inferior venous stenosis and compared with control and sham surgery groups. The serum levels of interleukin-1β (IL-1β), tissue factor (TF) and xanthine oxidase (XOD) were measured using ELISAs. The morphology of the DVT tissue was observed by hematoxylin and eosin staining. Circulating endothelial cells (CECs) in peripheral blood were counted by flow cytometry. Reverse transcription-quantitative PCR and western blotting were used to detect mRNA and protein expression, respectively. The serum levels of IL-1β, TF and XOD exhibited no significant differences between the control and sham surgery groups. However, those in the rat model of DVT presented an upward trend from 2 to 24 h and peaked at 24 h, with a significant difference from the respective levels in the control and sham surgery groups. The histopathological analysis revealed the presence of red and mixed thrombi in the rats 2-48 h following the induction of inferior venous stenosis group with inflammatory cell infiltration in the vascular wall. Thrombus formation was evident after 72 h. While significant difference was observed in the number of CECs in the peripheral blood between the control and sham surgery groups, the number of peripheral blood CECs in the rats with inferior venous stenosis group increased from 8 to 72 h, with significant differences among these groups. The mRNA levels of IL-1β, TF, XOD and NF-κB in the tissues peaked at 24 h, with significant differences compared with those in the control and sham surgery groups. In addition, the protein expression level of NF-κB increased from 2 to 72 h. In conclusion, these results suggest that the high expression of IL-1β, TF, XOD and NF-κB may promote thrombus formation.

Extracellular Vesicle-Associated Tissue Factor Activity in Prostate Cancer Patients with Disseminated Intravascular Coagulation.

Simple SummaryDisseminated intravascular coagulation (DIC) may occur in patients with advanced prostate cancer. In the present study, we detected elevated extracellular vesicle (EV)-associated tissue factor (TF) activity in the plasma of prostate cancer patients with DIC compared with matched prostate cancer patients without DIC and healthy individuals. TF-exposing EVs from DIC patients were highly coagulant in a clotting assay. In in vitro co-culture experiments, EV-TF activity was increased by interactions between a TF-exposing prostate cancer cell line (DU145), peripheral blood mononuclear cells (PBMCs), and platelets. Data from this study contribute to the understanding of the pathogenesis of prostate cancer-related DIC.Patients with advanced prostate cancer may develop fulminant disseminated intravascular coagulation (DIC). Circulating extracellular vesicles (EVs)-exposing tissue factor (TF), the initiator of the coagulation cascade, may play an important role. We included 7 prostate cancer patients with DIC, 10 age- and stage-matched cancer controls without DIC, and 10 age-matched healthy male individuals. EV-TF activity was highly elevated in prostate cancer patients with DIC (11.40 pg/mL; range: 4.34–27.06) compared with prostate cancer patients without DIC (0.09 pg/mL; range: 0.00–0.30, p = 0.001) and healthy controls (0.18 pg/mL; range: 0.09–0.54; p = 0.001). Only EVs from patients with DIC reduced fibrin clot formation time of pooled plasma in a TF-dependent manner. Next, we performed in vitro co-culture experiments including EVs derived from a prostate cancer cell line with high (DU145) and low (LNCaP) TF expression, peripheral blood mononuclear cells (PBMCs), and platelets. Co-incubation of DU145 EVs with PBMCs and platelets significantly increased EV-TF activity in conditioned medium and induced TF activity on monocytes. No such effects were seen in co-culture experiments with LNCaP EVs. In conclusion, the findings indicate that elevated EV-TF activity plays a role in the development of prostate-cancer-related DIC and may result from interactions between tumor-derived EVs, monocytes, and platelets.

Tissue factor expression and tumor-infiltrating T lymphocytes in ovarian carcinomas and their association with venous thromboembolism.

Ovarian cancer is a known risk factor of venous thromboembolism (VTE). Thrombogenic factor expression and lymphocytic infiltrate have been reported in endometriosis and ovarian cancers. We reviewed 30 cases of ovarian carcinomas (high grade serous carcinoma, 10; endometrioid carcinoma, 10; clear cell carcinoma (CCC), 10) and 16 endometriotic lesions. We immunohistochemically investigated the expressions of tissue factor (TF), podoplanin, P-selectin, and number of CD4 and CD8 positive lymphocytes in cancer tissue and endometriotic lesions, along with their relationship with VTE. The expression of TF was higher in CCC. The TF expression and the number of CD8 positive cells were higher in cancer tissues with VTE than in those without VTE. The podoplanin or P-selectin expression did not differ among histological types or between cases with and without VTE. Our results demonstrated a high TF expression and intraepithelial CD8 cells in CCC, which were associated with VTE. The results suggest that infiltrating lymphocytes may affect TF expression that, in turn, influences VTE.

Higher Tissue Factor (TF) Expression in the Lungs of COVID-19 Pneumonia Patients Than Patients with Acute Respiratory Distress Syndrome: Association with Thrombi Formation

A substantial proportion of patients with severe COVID-19 pneumonia develop thrombosis (both venous and arterial) via an undefined mechanism. Systemic elevation of high levels of D-dimer, a marker of coagulation activation and thrombolysis, is evident in almost all advanced COVID-19 patients and is associated with disease severity and mortality. However, the extrinsic factors that initiate blood coagulation in COVID-19 is not clear. Because tissue factor (TF) is the prime initiator of the extrinsic coagulation cascade, and because it is expressed and exposed under inflammatory conditions leading to vascular damage, we tested the hypothesis that higher TF expression is responsible for thrombi formation in the lungs of patients with severe COVID-19.To this end, we examined autopsy lung tissues from five COVID-19 pneumonia patients with acute respiratory syndrome (ARDS) who required ICU hospitalization, with 4 of the 5 patients requiring mechanical ventilation before they died, and five controls with acute ARDS caused by bacterial pneumonia, one with a prior influenza infection, and all on mechanical ventilation. Histological findings revealed all the COVID-19 lungs had characteristics of ARDS, including DAD with hyaline deposition and inflammatory cell invasion. Immunofluorescence staining showed TF expression throughout all lung tissues and in many blood vessels that were filled with thrombi with either fibrin, activated platelets, or both. TF expression was significantly higher in COVID-19 than control ARDS lung tissues (1.2 ± 0.3% in COVID-19 vs. 0.52 ± 0.2% in ARDS controls (p=0.004). Fibrin-enriched thrombi areas were higher in COVID-19 cases than in controls (1.6 ± 0.36% vs. 0.94 ± 0.4%; p=0.008) and correlated with TF expression (R2=0.4, p=0.02). Platelet factor 4 (PF4)-enriched thrombi areas were also higher in COVID-19 lung, but this trend was not statistically significant (0.94 ± 0.4% in COVID-19 and 0.54 ± 0.3% in controls; p=0.09), although it did, however, correlate with TF expression (R2=0.4, p=0.02). Many thrombi were in close proximity to TF-expressing areas in both COVID-19 and ARDS pneumonia controls.Dual RNA in situ hybridization with SARS-CoV-2 and TF fluorescence probes showed variable viral and TF mRNA expression. Increased TF mRNA expression was seen in COVID-19 vs. control lung (0.77 ± 0.4 % vs. 0.31 ± 0.15 %, p=0.05). TF mRNA expression correlated with viral mRNA in COVID-19 patients (R2=0.78, p=0.01). High-resolution images identified both sporadic and clustered SARS-CoV-2, and some areas co-localized with TF mRNA expression.We conclude that higher TF expression might be responsible for fibrin formation and platelet activation in the lungs of both COVID-19 and ARDS controls. Our observation of higher TF expression in COVID-19 patients was documented by two very sensitive methods, RNA in situ hybridization and immunostaining with very specific antibodies against TF and mRNA probes. Its correlation with SARS-CoV-2 mRNA suggests that SARS-CoV-2 infection induces both de novo gene transcription and protein synthesis in the lungs of COVID-19 patients. Thus, TF-initiated extrinsic coagulation might be responsible for the critical thrombi formation observed in many COVID-19 cases, rendering TF a potential therapeutic target.DisclosuresLaurence:Alexion Pharmaceuticals: Honoraria, Research Funding.

Prognostic significance of tissue factor in patients with pancreatic cancer: a systematic review protocol.

IntroductionPancreatic cancer is a highly aggressive digestive system tumour with poor prognosis. Venous thromboembolism (VTE) is a well-known complication of pancreatic cancer, and tissue factor (TF) contributes to the generation of a hypercoagulable state and thrombotic disease in pancreatic cancer. Several studies showed that an elevated TF level was related to the development of VTE and influenced the survival of patients with pancreatic cancer. Thus, we wish to conduct a systematic review of literature to clarify the prognostic significance of TF in pancreatic cancer.Methods and analysisStudies comparing the circulating microparticle-associated TF (MP TF) level between patients who had pancreatic cancer with and without VTE will be included to evaluate the roles of TF in VTE development. Studies comparing the survival data between patients with high TF expression and low TF expression will also be included to explore the association of TF expression with patient survival. The outcomes are plasma MP TF level and survival endpoints (overall and progression-free survival), respectively. Primary studies of any type published in English will be included. Two reviewers will search Medline, EMBASE and Cochrane databases from inception to June 2020, retrieve relevant studies, and independently select the literatures and extract data from the included studies. The quality of each included study will be assessed by the Newcastle-Ottawa Scale score. The HR and 95% CI of each study will be pooled for survival outcome, and the standardised mean difference (SMD) with 95% CIs will be used for continuous outcomes. If meta-analysis is inappropriate, the result will only be reported qualitatively. Subgroup and sensitivity analyses will be considered to identify sources of heterogeneity. The Grades of Recommendation, Assessment, Development and Evaluation method will be applied to assess the level of evidence of this systematic review.Ethics and disseminationThere are no concerning ethical issues. The results will be published.PROSPERO registration numberCRD42019133665.

Cell therapies can bring insult to injury

Some cell therapies increase the risk of thromboembolism in trauma patients due to high tissue factor expression.

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.

ET-05 PRECLINICAL STUDY OF AN ANTI-HUMAN TISSUE FACTOR ANTIBODY-DRUG CONJUGATE IN A MALIGNANT GLIOMA XENOGRAFT MODEL

Whereas macromolecules such as antibody hardly extravasate from normal blood vessels compared with low molecular agents, macromolecules leak from tumor vasculature because of the enhanced permeability. As a result, macromolecules selectively accumulate at tumor sites. We apply this phenomenon, known as the enhanced permeability and retention effect (EPR effect), to drug delivery for cancer therapy. Drug delivery system (DDS) based on the EPR effect is called the passive targeting. On the other hand, DDS based on antigen-antibody or ligand-receptor interaction is the active targeting. Antibody-drug conjugate (ADC), antibody conjugated with antitumor agents, retains both of the passive and active targeting functions. Tissue factor (TF), an initiator in the extrinsic pathway of blood coagulation, is overexpressed in various cancers including malignant glioma. To target the molecule in tumor sites, we have produced several anti-TF monoclonal antibodies. Previously, we evaluated tumor accumulation of an indium-111-labeled anti-TF antibody in an orthotopic glioma xenograft model with high expression of TF by single photon emission computed tomography/computed tomography (SPECT/CT). The imaging study showed that anti-TF antibody significantly accumulated in the tumor compared with control antibody (P < 0.01). The finding suggests that blood-brain barrier in brain tumors is broken and antibodies accumulate in tumors by utilizing both of the passive and active targeting. In this study, to prepare anti-TF ADC, we conjugated monomethyl auristatin E (MMAE), a microtubule inhibitor, to humanized anti-TF antibody. The anti-TF ADC recognized TF expressed in glioma cells and showed potent cytocidal activity against human glioma cell lines depending on TF expression. In addition, we evaluated in vivo antitumor effect of the ADC in a mouse model subcutaneously inoculated with TF-overexpressing glioma cells. Anti-TF ADC showed a significant higher antitumor effect compared with control ADC (P = 0.015).

P38/TF/HIF-α Signaling Pathway Participates in the Progression of CIPN in Mice

Chemotherapy induced peripheral neuropathy (CIPN) is a serious adverse effect of chemotherapeutics with limited pathogenetic mechanism been known. Whether microcirculatory disturbance is involved in CIPN has not been reported. Considering that tissue factor (TF) is an endogenous coagulation factor, we hypothesize CIPN may be induced by the high expression of TF in macrophages and sciatic nerve, which induces the molecular signal related to ischemia and hypoxia. Oxaliplatin (L-OHP) was used to establish CIPN model. Von Frey Hairs was used to measure nociception. The murine macrophage cell line Raw 264.7 was used for cell experiments. Gelatin zymography and western blotting were used to measure the activity or expression of protein. TF expression and MMP-9/2 activity in sciatic nerve and blood are significantly increased by L-OHP. L-OHP increased the release of HSP70 from macrophage and enhanced the expression of p-p38 and HIF-1α in vivo and in vitro. Hirudin significantly suppressed the overexpression of p38, HIF-1α and activation of MMP-9/2 induced by L-OHP and attenuated CIPN in mice. This study suggests that a novel HSP70-TLR-4-p38-TF-HIF-1a axis may play a pivotal role in the pathological process of CIPN. It is also shown that the use of anticoagulant Hirudin can inhibit the above mechanisms and improve CIPN.

15-epi-lipoxin A4 inhibits TNF-α-induced tissue factor expression via the PI3K/AKT/ NF-κB axis in human umbilical vein endothelial cells

Inflammation and coagulation are two important processes implicated in venous thromboembolism (VTE). 15-epi-lipoxin A4 (15-epi-LXA4) is the epimer of LXA4, a small lipid molecule, is known to play a key role in the resolution of inflammation. This study aimed to demonstrate whether 15-epi-LXA4 could suppress the inflammatory factor tumor necrosis factor-alpha (TNF-α)-induced upregulation of tissue factor (TF), an important regulator of the blood coagulation cascade, and evaluated the possible underlying mechanisms. We found that 15-epi-LXA4 not only reduced the up-regulation of mRNA and antigens, but also lowered the activity of TF (elevated by TNF-α) in primary culture of human umbilical vein endothelial cells (pc-HUVECs). In addition, 15-epi-LXA4 suppressed the activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway, induced by TNF-α, in pc-HUVECs. 15-epi-LXA4 also inhibited the binding of NF-κB on the TF promoter, which is otherwise enhanced by TNF-α. The role of 15-epi-LXA4 in regulating TNF-α-induced effects was enhanced by the PI3K inhibitor and prevented by the PI3K activator. In conclusion, 15-epi-LXA4 lowered the TNF-α-induced high TF expression and activity by suppressing PI3K/AKT signaling activation, thereby reducing the binding capacity of NF-κB on the TF promoter in pc-HUVECs.

A unique protein kinase C‐dependent pathway for tissue factor downregulation in pericytes

Essentials Many mediators increase tissue factor (TF) expression in a wide variety of cell types. The only known example of TF downregulation is by pericytes during wound healing angiogenesis. Downregulation of TF mRNA and protein in cultured pericytes is Protein Kinase C (PKC) dependent. Pericyte TF regulation is unique, since PKC mediates increased TF in all other cell types tested. SUMMARY: Background Embryonic and tumor-associated angiogenesis are linked to elevated expression of the procoagulant transmembrane receptor tissue factor (TF). In contrast, we have reported that high baseline TF expression by perivascular cells (pericytes) is dramatically reduced during angiogenesis at sites of wound healing. This is the only setting in which active TF downregulation has been reported, thus revealing a novel mechanism of TF regulation. Objectives To define the mechanisms underlying the unique pattern of TF expression in pericytes. Methods TF expression in primary cultures of human pericytes is not altered by angiogenic cytokines or growth factors, but is actively downregulated by phorbol 12-myristate 13-acetate (PMA). We characterized TF transcription, protein stability and trafficking in response to PMA. Results Exposure to PMA reduced TF mRNA synthesis and shortened the half-life of TF protein from 11h to 4.5h. Addition of PMA rapidly triggered endocytosis of cell surface TF, followed by degradation in lysosomes. Cell surface TF coagulant activity was maintained until internal stores were depleted. Reduction of TF transcription, TF endocytosis and enhanced degradation of TF protein were all blocked by broad-spectrum inhibitors of protein kinaseC (PKC). This was a surprising finding, because PKC activation increases TF expression in other cell types that have been tested. Conclusions The unique PKC-dependent pathway of TF downregulation in pericytes suggests that TF downregulation may play a functional role in angiogenesis. Distinct pathways regulating pathological and physiological TF expression could be utilized to modulate TF expression for therapeutic purposes.

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.

A study on the relationship between tissue factor expression and liver damage in diabetic patients with hepatitis C virus-related cirrhosis

Objective The aim of this study was to identify a possible role for tissue factor (TF) in the pathogenesis of hepatic damage in patients with hepatitis C virus (HCV)-related cirrhosis with type 2 diabetes mellitus (T2DM). Background HCV infection and T2DM are both prevalent diseases worldwide. The exact mechanism by which type 2 diabetes worsens liver function is needed to be clarified. TF is a key link that connects hemostatic, immune, and inflammatory processes. Materials and methods The study included 80 participants divided into three groups: 30 patients with HCV-related cirrhosis and T2DM, 30 cirrhotic patients, and 20 healthy controls. Flow cytometry analysis was used for measuring TF (CD142) and the costimulatory molecule (CD86) on lipopolysaccharide-activated monocytes (CD14 positive cells) in peripheral blood in different studied groups. Results Our results have shown higher TF and CD86 expression in cirrhotic patients with T2DM compared with those with cirrhosis and healthy controls (34.06 ± 5.99, 24.65 ± 6.76 and 11.90 ± 3.04 for TF and 72.09 ± 12.57, 49.38 ± 15.07 and 9.35 ± 3.07 for CD86, respectively; P Conclusion On the basis of our data, we suggest a possible role for TF in the pathogenesis of liver damage through exaggerating the inflammatory process. This can, at least partially, explain the worsened liver function in HCV-related cirrhosis in patients with T2DM.

Nitric Oxide Mediates Downregulation of Tissue Factor Expression in Primary Human Pericytes through a p38 MAPK Signaling Pathway

Tissue factor (TF) is a high-affinity receptor for FVII/FVIIa that serves as a key initiator of hemostasis and is thought to also play a functional role in angiogenesis. Elevated TF expression has been linked to upregulated angiogenesis in malignant tumors, while reducing TF expression in experimental tumor models results in decreased angiogenesis. Although these data suggest that high TF expression is critical for angiogenesis, we have reported that TF expression declines significantly in pericytes that surround angiogenic vessels at sites of wound healing. This is the only known example of active TF downregulation, suggesting that pericytes regulate their expression of TF by a unique mechanism. Additionally, TF expression increases in response to many mediators, yet none that decrease TF expression have been described. The goal of this study was to characterize TF downregulation in pericytes and identify mediators of this process.We have previously shown that TF expression in primary cultures of human pericytes is not affected by treatment with various growth factors or inflammatory stimuli, but decreases significantly in response to phorbol 12-myristate 13-acetate (PMA). PMA triggers degradation of TF protein and inhibition of TF mRNA synthesis, both in a Protein Kinase C (PKC)- dependent manner. To identify other signaling molecules in this pathway we used chemical inhibitors to block the activity of signaling molecules downstream of PKC before adding PMA to pericyte cultures. Inhibition of NF-kB, ERK1/2, AKT, JNK, and p38 MAPK did not block degradation of TF protein. However, pericytes that received a p38 inhibitor (SB202190) alone demonstrated significant reduction of TF mRNA. Treatment with SB202190 followed by PMA produced an additive effect on TF mRNA reduction. Western blotting showed that prolonged PMA treatment (>4 hours) produced a sustained decrease in p38 phosphorylation. These data suggest that PMA inhibits p38 activity, and that p38 confers stability to TF mRNA.We have previously found that basic Fibroblast Growth Factor (bFGF) triggers downregulation of pericyte TF a co-culture system with human microvascular endothelial cells. However, transferring bFGF-conditioned endothelial cell media to pericytes cultured alone failed to reproduce TF loss. bFGF has been shown to stimulate nitric oxide (NO) production, and both bFGF and NO have been linked to angiogenesis. This led us to consider NO as a potential labile mediator of TF downregulation. ±6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Pericytes treated with DETA NO demonstrated sustained p38 phosphorylation for up to 8 hours. Taken together, these data suggest that DETA NO downregulates TF protein but maintains basal levels of TF mRNA, potentially in a p38-dependent manner.Based on these data, we hypothesize that the p38 signaling axis is a key component of a unique pathway of TF regulation in pericytes, and that endothelial nitric oxide contributes to downregulation of pericyte TFin vivo at sites of physiologic angiogenesis. DisclosuresHoffman:Novo Nordisk A/S: Consultancy, Honoraria, Research Funding.

Expression of tissue factor in human cervical carcinoma tissue.

The present study aimed to investigate tissue factor (TF) expression in cervical cancer and explore its association with disease progression. A total of 258 cervical cancer tissues and their adjacent normal tissues were collected between September 2014 and September 2016. TF expression was detected in the tissue samples by immunohistochemistry and western blot analysis. Associations between the expression of TF and clinical stage, differentiation status and metastasis of cancer cells were examined. The mean immunohistochemistry score of TF expression in cervical cancer tissues was 2.86±1.76, which was significantly increased compared with the adjacent normal tissues (0.28±0.45). The expression of TF was also significantly associated with the clinical stage, lymph node metastasis and distant metastasis of cancer cells. Immunohistochemistry staining and western blot analysis demonstrated that TF expression in cervical cancer tissues significantly increased as the clinical stage increased. TF expression in tumor tissues from patients with lymph node metastasis was significantly increased compared with samples from patients without lymph node metastasis. TF expression was also significantly increased in patients with distant metastasis compared with those without. In conclusion, TF is highly expressed in cervical cancer tissues and high expression of TF may enhance the invasion and metastasis of cervical cancer cells.

RNA Interference Mediated Silencing of Tissue Factor in Human Umbilical Vein Endothelial Cells

Objective: To construct to silence tissue factor (TF) expression in Human Um-bilical Vein Endothelial Cells (HUVECs) for providing evidence of the gene therapy and prevention of coagulation dysfunction in placental abruption (PA) neonates. Methods: Cultured HUVECs in vitro were divided into control group and PA group, each including non-prevention, scramble siRNA and TF-siRNA subgroups. pENTRTM/U6-shRNA/TF vector expressing TF were constructed and transfected into HUVECs. The mRNA expression of TF was tested with RT-PCR, and TF protein expression was detected with immunofluorescence staining. Results: Monolayer HUVECs with short-rod and short spindle shaped were adherent to the bottom, forming paving stone arrangement. The TF mRNA expression was significantly different between control group and PA group (P < 0.01) and among different subgroups (P < 0.01). In non-prevention subgroups, significant difference was observed in TF mRNA expression between control group and PA group (P < 0.05). Significant difference in TF mRNA expression was found in false-prevention subgroups (P < 0.05). The TF mRNA expression was markedly different among different subgroups in control (P < 0.01), and the similar result among different PA subgroups (P < 0.01). In both control and PA groups, the TF mRNA expression was the lowest after TF silencing. Immunofluorescence staining showed high TF expression in HUVECs in false-prevention subgroups. In scramble siRNA group, the TF protein expression reduced as compared to non-prevention group and reduced dramatically after TF silencing as compared to control. Conclusion: pENTRTM/U6-TF-shRNA is able to significantly inhibit the TF mRNA and protein expression in HUVECs from healthy neonates and PA neonates.