Downregulation Of Tissue Factor Research Articles

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.

PARP1 deficiency protects against hyperglycemia-induced neointimal hyperplasia by upregulating TFPI2 activity in diabetic mice

Diabetes mellitus (DM) promotes neointimal hyperplasia, characterized by dysregulated proliferation and accumulation of vascular smooth muscle cells (VSMCs), leading to occlusive disorders, such as atherosclerosis and stenosis. Poly (ADP-ribose) polymerase 1 (PARP1), reported as a crucial mediator in tumor proliferation and transformation, has a pivotal role in DM. Nonetheless, the function and potential mechanism of PARP1 in diabetic neointimal hyperplasia remain unclear. In this study, we constructed PARP1 conventional knockout (PARP1−/−) mice, and ligation of the left common carotid artery was performed to induce neointimal hyperplasia in Type I diabetes mellitus (T1DM) mouse models. PARP1 expression in the aorta arteries of T1DM mice increased significantly and genetic deletion of PARP1 showed an inhibitory effect on the neointimal hyperplasia. Furthermore, our results revealed that PARP1 enhanced diabetic neointimal hyperplasia via downregulating tissue factor pathway inhibitor (TFPI2), a suppressor of vascular smooth muscle cell proliferation and migration, in which PARP1 acts as a negative transcription factor augmenting TFPI2 promoter DNA methylation. In conclusion, these results suggested that PARP1 accelerates the process of hyperglycemia-induced neointimal hyperplasia via promoting VSMCs proliferation and migration in a TFPI2 dependent manner.

MiR-19a-3p downregulates tissue factor and functions as a potential therapeutic target for sepsis-induced disseminated intravascular coagulation

Sepsis-induced disseminated intravascular coagulation (DIC) is a common life-threatening terminal-stage disease with high mortality. This study aimed to identify effective miRNAs as therapeutic targets for DIC. Bioinformatics and luciferase reporter gene analyses were performed to predict miR-19a-3p and validate that it targets tissue factor (TF). Quantitative real-time PCR was used to detect the expression of miR-19a-3p and TF, and TF procoagulant activity was determined using the chromogenic substrate method. Western blotting was used to detect the protein levels of TF, AKT serine/threonine kinase (AKT), extracellular regulated protein kinases (ERK), nuclear factor kappa B (NF-κB) P65, NFKB inhibitor alpha (IκB-a) and their phosphorylated counterparts in cell experiments. Furthermore, a rat model was established to explore the potential of miR-19a-3p in DIC treatment. As a result, a human clinical study revealed that miR-19a-3p was downregulated and that TF was upregulated in neonates with sepsis-induced DIC compared with those in the control group. The luciferase reporter assay showed that TF was a direct target of miR-19a-3p. Cell experiments verified that the mRNA and protein levels of TF, and the p-AKT/AKT, p-Erk/Erk, p-P65/P65, p-IκB-a/IκB-a ratios, and TF procoagulant activity were significantly decreased in lipopolysaccharide (LPS) -induced human peripheral blood mononuclear cells (PBMCs) and human umbilical vein endothelial cells (HUVECs) inhibited by overexpression of miR-19a-3p, and that miR-19a-3p regulating TF was dependent on the NF-kB and AKT pathways. In vivo, miR-19a-3p injection into DIC rats suppressed the mRNA expression of TF; more importantly, significant improvements in coagulation function indicators and in histopathologies of lung and kidney were observed. In conclusion, miR-19a-3p may suppress DIC by targeting TF and might be a potential therapeutic target in treating sepsis-induced DIC.

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.

Ref-1 protects against FeCl3-induced thrombosis and tissue factor expression via the GSK3β-NF-κB pathway.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

Deletion of Glutathione S-Transferase Omega 1 Activates Type I Interferon Genes and Downregulates Tissue Factor.

GST omega 1 (GSTO1) is an atypical GST isoform that is overexpressed in several cancers and has been implicated in drug resistance. Currently, no small-molecule drug targeting GSTO1 is under clinical development. Here we have validated GSTO1 as an impactful target in oncology. Transcriptional profiling coupled with proteomics uncovered novel pharmacodynamic markers and cellular pathways regulated by GSTO1. CRISPR/Cas9 GSTO1 knockout (KO) cell lines failed to form tumors or displayed growth delay in vivo; they also formed smaller 3D spheroids in vitro. Multiomics analysis in GSTO1 KO cells found a strong positive correlation with cell adhesion molecules and IFN response pathways and a strong negative correlation with Myc transcriptional signature. In addition, several clinically used drugs showed significant synthetic lethality with loss or inhibition of GSTO1. Transcription and protein expression of tissue factor (gene name, F3) were downregulated in response to GSTO1 KO. F3 is associated with poor patient survival and promotion of tumor progression in multiple cancers and is a known risk factor for metastasis. Transcription of F3 was regulated by IL1β, whose secretion decreased upon inhibition of GSTO1, suggesting that IL1β links GSTO1 expression and F3 transcription. In summary, our results implicate GSTO1 as a potential therapeutic target in cancer and offer new mechanistic insights into its significant role in cancer progression. SIGNIFICANCE: These findings validate GSTO1 as a therapeutic target in cancer and implicate GSTO1 in the modulation of tumor growth, immune responses, and expression of F3.

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.

Huisheng Oral Solution exerts anti-tumor effects by downregulating tissue factor and inhibiting the expression of metastasis-related factors, CD44, MMP2, and VEGF

Background: This study aimed to investigate the anti-tumor effects of Huisheng Oral Solution (HSOS) on the promotion of blood circulation to dispel blood stasis, the inhibition of metastasis and inflammation, the pathogenesis of tumor progression, and to provide guidelines for using HSOS in clinical settings. Methods: Eight-month-old Lewis lung carcinoma (LLC)-bearing C57BL/6 mice were orally administered HSOS (0.25 mL/d) for 21 d starting on day 2 of model generation. One hour after the final administration, their eyeballs were dissected out to collect serum to determine tissue factor (TF) and interleukin-6 (IL-6) levels via ELISA. Blood was collected intracardially with an anticoagulant to determine fibrinogen levels, using an automated blood coagulation analyzer. The mice were euthanized via cervical dislocation and their thymi, spleens, and tumors were extracted for weight measurement and organ index calculation. CD44 and MMP2 expression and VEGF protein and mRNA expression in tumor tissues were detected using immunohistochemical (IHC) and RT-qPCR assays, respectively. Lastly, the effect of HSOS on the migration ability of A549 lung carcinoma cells was investigated using in vitro scratch assay. Results: HSOS significantly downregulated TF and Fib in tumor-bearing mice. HSOS inhibited the overexpression of CD44, MMP2, and VEGF, and the migration ability of tumor cells. Moreover, the pro- inflammatory cytokine (IL-6) was significantly downregulated, but the thymic and splenic indices increased. Conclusions: HSOS might exert anti-tumor effects by improving hypercoagulability in tumor-bearing mice, inhibiting tumor cell metastasis, alleviating inflammatory responses, and enhancing immune function.

Huisheng Oral Solution exerts anti-tumor effects by downregulating tissue factor and inhibiting the expression of metastasis-related factors, CD44, MMP2, and VEGF.

BackgroundThis study aimed to investigate the anti-tumor effects of Huisheng Oral Solution (HSOS) on the promotion of blood circulation to dispel blood stasis, the inhibition of metastasis and inflammation, the pathogenesis of tumor progression, and to provide guidelines for using HSOS in clinical settings.MethodsEight-month-old Lewis lung carcinoma (LLC)-bearing C57BL/6 mice were orally administered HSOS (0.25 mL/d) for 21 d starting on day 2 of model generation. One hour after the final administration, their eyeballs were dissected out to collect serum to determine tissue factor (TF) and interleukin-6 (IL-6) levels via ELISA. Blood was collected intracardially with an anticoagulant to determine fibrinogen levels, using an automated blood coagulation analyzer. The mice were euthanized via cervical dislocation and their thymi, spleens, and tumors were extracted for weight measurement and organ index calculation. CD44 and MMP2 expression and VEGF protein and mRNA expression in tumor tissues were detected using immunohistochemical (IHC) and RT-qPCR assays, respectively. Lastly, the effect of HSOS on the migration ability of A549 lung carcinoma cells was investigated using in vitro scratch assay.ResultsHSOS significantly downregulated TF and Fib in tumor-bearing mice. HSOS inhibited the overexpression of CD44, MMP2, and VEGF, and the migration ability of tumor cells. Moreover, the pro-inflammatory cytokine (IL-6) was significantly downregulated, but the thymic and splenic indices increased.ConclusionsHSOS might exert anti-tumor effects by improving hypercoagulability in tumor-bearing mice, inhibiting tumor cell metastasis, alleviating inflammatory responses, and enhancing immune function.

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.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium and the modulatory mechanism of D39

Non‐muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor‐α (TNF‐α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose‐dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF‐κB p65 nuclear translocation and I B degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3 and suppressed NF‐κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF‐α‐stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model. Moreover, D39 inhibited tissue factor expression and procoagulant activities in HUVECs and decreased thrombus weight in inferior venacava‐ligated mice dose‐dependently. Serial affinity chromatography and molecular docking analysis suggested that D39 bound to NMMHC IIA. In HEK293T cells, D39 inhibited tissue factor expression evoked by NMMHC IIA overexpression. This effect was blocked by NMMHC IIA knockdown in HUVECs. D39 inhibited dissociation of NMMHC IIA from TNFR2, which subsequently modulated the Akt/GSK3β–NF‐κB signalling pathways. Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β‐NF‐κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders. In addition, we identified a new natural product that targeted NMMHC IIA, as a potential treatment for thrombotic disorders and other vasculopathies.Support or Funding InformationThe present research was supported by the National Natural Science Foundation of China (Grant No. 81274131 and 81601034), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Abstract 288: Nitric Oxide Mediates Active Downregulation of Tissue Factor Expression in Human Pericytes

Tissue Factor (TF) is a transmembrane protein that not only expresses pro-coagulant enzymatic activity, but also mediates signal transduction by several second messenger pathways. It plays an obvious role in hemostasis and thrombosis, as well as modulating angiogenesis, inflammation, immune responses and progression of malignancy and metastasis. TF is normally expressed by a limited subset of cell types. It can also be induced on other cells by a plethora of mediators, mostly linked to inflammation and immune responses. By contrast, TF has only been shown to be actively downregulated in a single circumstance: we have reported that constitutive expression of TF by pericytes around dermal vessels disappears during angiogenesis associated with wound healing. This suggests that downregulation of pericyte TF may play a functional role in the process of angiogenesis. We previously reported that TF protein in cultured human pericytes is significantly reduced 8 hours after treatment with phorbol 12-myristate 13-acetate (PMA) (94.71% reduction + 2.18%, p&lt;0.001). This loss of TF protein is facilitated by reduction of TF mRNA. Downregulation of TF protein and mRNA depends on Protein Kinase C. The goal of the current study was to identify a physiologic mediator of pericyte TF downregulation. Treatment of human pericytes with growth factors and cytokines involved in wound healing did not change TF expression. Co-culture of human pericytes with human microvascular endothelial cells in transwells in the presence of basic fibroblast growth factor (bFGF) resulted in downregulation of pericyte TF. However, conditioned media from endothelial cells grown in the presence of bFGF did not trigger a decrease of TF in pericytes. bFGF has been shown to stimulate NO production, and both bFGF and NO have been linked to angiogenesis. This led us to consider nitric oxide (NO) as a potential labile mediator of TF downregulation. TF protein was reduced 8 hours after treatment with DETA/NO (44% reduction + 6.1%, p&lt;0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Our working hypothesis is that TF downregulation in pericytes is partially mediated by endothelial cell-derived NO, but is also mediated by additional PKC-dependent pathways.

Abstract 642: Specific Protein Kinase C Isoforms Are Critical Mediators of Physiologic Downregulation of Perivascular Tissue Factor

Tissue factor (TF) is a procoagulant and transmembrane receptor for FVII(a) that is upregulated in pathological conditions. We recently demonstrated that perivascular TF is downregulated around angiogenic vessels near a cutaneous wound. The goal of this study was to identify mechanisms that mediate TF loss. Primary cultures of human pericytes express high levels of TF. TF expression was lost during culture with phorbol-12 myristate 13-acetate (PMA) for 8 hours, and was maintained for 24 hours. This model recapitulates the pattern of downregulation observed in vivo . Using qRT-PCR we assessed changes in TF gene expression in response to PMA. TF mRNA decreased 4- and 6-fold at 8 and 12 hours after treatment (p&lt;0.01), and remained 2-fold lower in treated cells after 24 hours (p&lt;0.05). Inhibiting de novo transcription with actinomycin D showed that degradation of TF mRNA was similar in PMA- and vehicle-treated groups (p=ns). Thus, downregulation of TF mRNA occurs primarily through inhibition of its synthesis. We next identified a physiologic mediator of TF downregulation using specific inhibitors against PMA-responsive signaling proteins. Two different inhibitors against Protein Kinase C (PKC) were used: Go6983, which inhibits isoforms α, β, δ, ε, μ, and ζ, and GFX, which inhibits α, β, ε, and γ. Both inhibitors significantly attenuated PMA-mediated transcriptional downregulation (p&lt;0.001). Based on overlap of inhibited isoforms, this suggests a minimal role for ζ, μ, δ, and γ, while one or more of the α, β, and ε isoforms appear to be critical mediators of TF mRNA synthesis inhibition. Since the timing of TF protein loss is not fully explained by transcriptional inhibition, we investigated the role of protein degradation in TF loss. When protein synthesis was inhibited by cyclohexamide, addition of PMA shortened the half-life of pericyte TF from 11 hours to 5 hours (p&lt;0.001). This indicates that increased protein degradation contributes to PMA-induced loss of TF expression. Both Go6983 and GFX significantly attenuated TF protein degradation also. Taken together, our data show that TF downregulation is mediated by transcriptional inhibition and protein degradation, and PKC α, β, and ε have emerged as potential mediators of both mechanisms.

Abstract 377: Phorbol 12-myristate 13-acetate Downregulates Tissue Factor Gene Expression in Human Pericytes

Tissue factor (TF) is a transmembrane receptor for factor VII/VIIa that plays a central role in hemostasis and angiogenesis. While TF is generally upregulated in pathologic conditions, we have reported that TF is downregulated in pericytes around vessels near a healing cutaneous wound. As the first demonstration of physiologic downregulation of TF, this finding suggested a potential means of modulating TF expression for therapeutic purposes. The goal of this study was to contribute to implementation of future therapies by elucidating mechanisms that regulate loss pericyte TF. To identify mediators of TF downregulation we utilized primary cultures of human placental pericytes to screen growth factors involved in wound healing including TGFβ, bFGF, VEGF, PDGF and ANG2. None significantly reduced pericyte TF expression as assessed by western blotting. We next tested agents that directly activate signaling pathways. Phorbol 12-myristate 13-acetate (PMA) triggered ~60% reduction in TF protein 4 hours after treatment. Complete loss of TF occurred by 8 hours (p&lt;0.001) and remained until the experiment was terminated 24 hours after PMA addition. These results suggested that TF loss is mediated, at least in part, by downregulation of TF gene expression. We utilized qRT-PCR to determine the effects of PMA on synthesis of TF transcripts 4, 8, 12, and 24 hours after treatment. TF mRNA levels were unchanged 4 hours after treatment. However, TF transcripts decreased 4 and 6 fold 8 hours and 12 hours after PMA, respectively (p&lt;0.01). 24 hours after treatment the amount of TF mRNA rebounded, yet remained 2 fold lower than the control (p&lt;0.05). To determine if the decrease in TF transcripts is caused by mRNA destabilization, pericytes were treated with actinomycin D prior to PMA. Degradation of TF transcripts in PMA-treated cells was similar to that of DMSO-treated control cells at all time points (p=ns), indicating that PMA-mediated downregulation of TF expression occurs primarily through inhibition of mRNA synthesis rather than through destabilization of existing transcripts. In conclusion, our results demonstrate a role for transcriptional downregulation in PMA-mediated loss of pericyte TF protein, findings that pave the way for future in vivo studies of wound healing.

Oestrogens Downregulate Tissue Factor Pathway Inhibitor through Oestrogen Response Elements in the 5'-Flanking Region.

Oestrogens influence the pathology and development of hormone-sensitive breast cancers. Tissue factor pathway inhibitor (TFPI) has been shown to be associated with breast cancer pathogenesis. Recently, we found TFPI mRNA levels to be significantly reduced by oestrogens in a breast cancer cell line (MCF7), a process mediated through the oestrogen receptor alpha (ERα). The aim of the present study was to investigate the mechanism(s) by which oestrogens may regulate TFPI at the transcriptional level. The TFPI 5’-flanking region contains three oestrogen response element (ERE) half-sites at positions -845, -769 and -50. Constructs containing the wild type or mutated ERE half-sites of the TFPI 5’-flanking region were generated in a luciferase reporter gene vector and transiently co-transfected with an ERα expression vector into HEK293 cells and subsequently treated with oestrogens. We found that luciferase activity was significantly downregulated after oestrogen stimulation in cells transfected with the wild type construct, an effect that was abolished by mutating either ERE half-sites. Electrophoretic mobility shift assay suggested direct and specific interaction of ERα with the ERE half-sites in the TFPI 5’-flanking region. Chromatin immunoprecipitation showed that ERα was recruited to the region -899 to -578 of the TFPI 5’-flanking region in vivo, where the ERE half-sites -845 and -769 are located. Our results indicate that ERα can interact with all three ERE half-sites in the TFPI 5’-flanking region and thus participate in the repression of oestrogen mediated TFPI transcription in breast cancer cells.

Downregulation of Tissue Factor in Pericytes By a Protein Kinase C-Dependent Mechanism

Tissue factor (TF) is a transmembrane protein in the type II cytokine receptor family that serves as a cofactor for factor VIIa activity, and is essential for normal initiation of hemostasis. Its expression is generally upregulated in the settings of inflammation, immune activation, tissue injury and malignancy. TF has biological functions in addition to its role hemostasis. In malignancy, high expression of TF by tumor cells promotes local angiogenesis. By contrast, we have reported that expression of perivascular TF is downregulated within 24 hours after wounding, and remains low around angiogenic vessels during physiologic wound healing. The mechanism(s) of physiologic downregulation of TF, and its role in physiologic angiogenesis have not been previously characterized. Therefore, the objective of this study was to identify mechanisms contributing to TF downregulation in primary human pericytes. We determined that loss of TF in human pericytes can be recapitulated in vitro by treating pericyte cultures with phorbol 12-myristate 13-acetate (PMA). The time course and degree of TF downregulation over 24 hours, as shown in the figure, is similar to that observed within the first 24 hours in vivo. Since PMA is a potent activator of Protein Kinase C (PKC), we determined the role of PKC in PMA-mediated TF loss by pretreating pericytes with the broad spectrum PKC inhibitor, Go 6983, prior to stimulation with PMA. PKC inhibition eliminated PMA-induced TF loss, supporting the conclusion that the PMA effect occurs through a PKC-dependent mechanism. PKCa has been shown to induce phosphorylation of the TF cytoplasmic tail, which results in budding of TF-rich microvesicles from the plasma membrane. PMA also triggers activation of the cell-surface protease ADAM17. Based on domain homology with other targets, ADAM17 could potentially cleave and release the extracellular domain of TF from the cell surface. To determine if TF loss occurs through its release from the cell by either mechanism we utilized biotin labeling of surface TF, streptavidin pull down, and western blot analysis to assess trafficking of TF into the culture media. Our results show that surface TF significantly decreases within 2 hours of PMA treatment. However, we failed to detect biotinylated TF in the culture media. Isolation of microvesicles from the media followed by western blotting for TF showed that pericytes do release TF-associated microvesicles at a basal rate, but this rate is not affected by PMA. Taken together, these data show that PMA induces downregulation of TF in primary human pericytes through a PKC-dependent mechanism, and that this mechanism does not stimulate release of TF from the cell in either microvesicles or by proteolytic cleavage of the TF extracellular domain. Our working hypothesis is that PMA triggers internalization of surface TF via activation of protein kinase C, leading to its subsequent degradation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Nasal priming with immunobiotic Lactobacillus rhamnosus modulates inflammation-coagulation interactions and reduces influenza virus-associated pulmonary damage.

To evaluate the effect of the nasal administration of live and heat-killed Lactobacillus rhamnosus CRL1505 (Lr1505) on immune-coagulative response during influenza virus (IFV) infection to improve survival and reduce lung injury. Six-week-old BALB/c mice were treated with live or heat-killed Lr1505 by the nasal route during two consecutive days. Treated and untreated control mice were then nasally challenged with IFV. Both viable and non-viable Lr1505 protected infected mice by reducing pulmonary injury and lung viral loads trough several mechanisms: (a) Inflammatory cytokines were efficiently regulated allowing higher clearance of virus and reduction of inflammatory lung tissue damage, associated to higher levels of the regulatory cytokine IL-10. (b) The antiviral immune response was enhanced with improved levels of type I interferons, CD4(+)IFN-γ(+) lymphocytes, and lung CD11c(+)CD11b(low)CD103(+) and CD11c(+)CD11b(high)CD103(-) dendritic cells. (c) The procoagulant state was reversed mainly by down-regulating tissue factor expression and restoring thrombomodulin levels in lung. The capacity of Lr1505 to improve the outcome of IFV infection would be related to its ability to beneficially modulate lung TLR3-triggered immune response. Our work is the first to demonstrate the ability of an immunobiotic strain to beneficially modulate inflammation-coagulation interactions during IFV infection. Interestingly, non-viable L. rhamnosus CRL1505 was as effective as the viable strain to beneficially modulate respiratory antiviral immune response.

SiRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation.

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA, but not IIB or IIC, inhibited TF expression, activated Akt/GSK3β and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model.Our findings provide solid evidence that inhibition of NMMHC II, most likely NMMHC IIA, impedes TF expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.