Tissue Factor Gene Expression Research Articles

Hydroxytyrosol Alleviates Methotrexate-Induced Pulmonary Fibrosis in Rats: Involvement of TGF-β1, Tissue Factor, and VEGF.

Methotrexate (MTX) is an indispensable drug used for the treatment of many autoimmune and cancerous diseases. However, its clinical use is associated with serious side effects, such as lung fibrosis. The main objective of this study is to test the hypothesis that hydroxytyrosol (HT) can mitigate MTX-induced lung fibrosis in rats while synergizing MTX anticancer effects. Pulmonary fibrosis was induced in the rats using MTX (14 mg/kg/week, per os (p.o.)). The rats were treated with or without HT (10, 20, and 40 mg/kg/d p.o.) or dexamethasone (DEX; 0.5 mg/kg/d, intraperitoneally (i.p.)) for two weeks concomitantly with MTX. Transforming growth factor beta 1 (TGF-β1), interleukin-4 (IL-4), thromboxane A2 (TXA2), vascular endothelial growth factor (VEGF), 8-hydroxy-2-deoxy-guanosine (8-OHdG), tissue factor (TF) and fibrin were assessed using enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and RT-PCR. Pulmonary fibrosis was manifested by an excessive extracellular matrix (ECM) deposition and a marked increase in TGF-β1 and IL-4 in lung tissues. Furthermore, cotreatment with HT or dexamethasone (DEX) significantly attenuated MTX-induced ECM deposition, TGF-β1, and IL-4 expression. Similarly, HT or DEX notably reduced hydroxyproline contents, TXA2, fibrin, and TF expression in lung tissues. Moreover, using HT or DEX downregulated the gene expression of TF. A significant decrease in lung contents of VEGF, IL-8, and 8-OHdG was also observed in HT + MTX- or DEX + MTX -treated animals in a dose-dependent manner. Collectively, the results of our study suggest that HT might represent a potential protective agent against MTX-induced pulmonary fibrosis.

TF/PAR2 Signaling Axis Supports the Protumor Effect of Neutrophil Extracellular Traps (NETs) on Human Breast Cancer Cells.

Neutrophil extracellular traps (NETs) have been implicated in several hallmarks of cancer. Among the protumor effects, NETs promote epithelial-mesenchymal transition (EMT) in different cancer models. EMT has been linked to an enhanced expression of the clotting-initiating protein, tissue factor (TF), thus favoring the metastatic potential. TF may also exert protumor effects by facilitating the activation of protease-activated receptor 2 (PAR2). Herein, we evaluated whether NETs could induce TF expression in breast cancer cells and further promote procoagulant and intracellular signaling effects via the TF/PAR2 axis. T-47D and MCF7 cell lines were treated with isolated NETs, and samples were obtained for real-time PCR, flow cytometry, Western blotting, and plasma coagulation assays. In silico analyses were performed employing RNA-seq data from breast cancer patients deposited in The Cancer Genome Atlas (TCGA) database. A positive correlation was observed between neutrophil/NETs gene signatures and TF gene expression. Neutrophils/NETs gene signatures and PAR2 gene expression also showed a significant positive correlation in the bioinformatics model. In vitro analysis showed that treatment with NETs upregulated TF gene and protein expression in breast cancer cell lines. The inhibition of ERK/JNK reduced the TF gene expression induced by NETs. Remarkably, the pharmacological or genetic inhibition of the TF/PAR2 signaling axis attenuated the NETs-induced expression of several protumor genes. Also, treatment of NETs with a neutrophil elastase inhibitor reduced the expression of metastasis-related genes. Our results suggest that the TF/PAR2 signaling axis contributes to the pro-cancer effects of NETs in human breast cancer cells.

Isoquercetin for thromboinflammation in sickle cell disease: a randomized double-blind placebo-controlled trial

AbstractData from a small trial in patients with cancer suggest that isoquercetin (IQ) treatment lowered thrombosis biomarkers and prevented clinical thrombosis, but, to our knowledge, no studies of IQ have been conducted to target thromboinflammation in adults with sickle cell disease (SCD). We conducted a randomized, double-blind, placebo-controlled trial in adults with steady-state SCD (hemoglobin SS [HbSS], HbSβ0thal, HbSβ+thal, or HbSC). The primary outcome was the change in plasma soluble P-selectin (sP-selectin) after treatment compared with baseline, analyzed in the intention-to-treat population. Between November 2019 and July 2022, 46 patients (aged 40 ± 11 years, 56% female, 75% under hydroxyurea treatment) were randomized to receive IQ (n = 23) or placebo (n = 23). IQ was well tolerated and all the adverse events (AEs; n = 21) or serious AEs (n = 14) recorded were not attributable to the study drug. The mean posttreatment change for sP-selectin showed no significant difference between the treatment groups (IQ, 0.10 ± 6.53 vs placebo, 0.74 ± 4.54; P = .64). In patients treated with IQ, whole-blood coagulation (P = .03) and collagen-induced platelet aggregation (P = .03) were significantly reduced from the baseline. Inducible mononuclear cell tissue factor gene expression and plasma protein disulfide isomerase reductase activity were also significantly inhibited (P = .003 and P = .02, respectively). Short-term fixed-dose IQ in patients with SCD was safe with no off-target bleeding and was associated with changes from the baseline in the appropriate direction for several biomarkers of thromboinflammation. The trial was registered at www.clinicaltrials.gov as #NCT04514510.

Temozolomide and Lomustine Induce Tissue Factor Expression and Procoagulant Activity in Glioblastoma Cells In Vitro

Glioblastoma (GBM) patients have one of the highest risks of venous thromboembolism (VTE), which is even further increased upon treatment with chemotherapy. Tissue factor (TF) is the initiator of the extrinsic coagulation pathway and expressed by GBM cells. In this study, we aimed to examine the effect of routinely used chemotherapeutic agents Temozolomide (TMZ) and Lomustine (LOM) on TF procoagulant activity and expression in GBM cells in vitro. Three human GBM cell lines (U-251, U-87, U-118) were exposed to 100 µM TMZ or 30 µM LOM for 72 h. TF procoagulant activity was assessed via an FXa generation assay and TF gene and protein expression through qPCR and Western blotting. The externalization of phosphatidylserine (PS) was studied using Annexin V flow cytometry. Treatment with TMZ and LOM resulted in increased procoagulant activity in all cell lines. Furthermore, both agents induced procoagulant activity in the supernatant and tumor-cell-secreted extracellular vesicles. In line, TF gene and protein expression were increased upon TMZ and LOM treatment. Additionally, PS externalization and induction of inflammatory-associated genes were observed. Overall, the chemotherapeutic modalities TMZ and LOM induced procoagulant activity and increased TF gene and protein expression in all GBM cell lines tested, which may contribute to the increased VTE risk observed in GBM patients undergoing chemotherapy.

Acute vaping in a golden Syrian hamster causes inflammatory response transcriptomic changes.

E-cigarette vaping is a major aspect of nicotine consumption, especially for children and young adults. Although it is branded as a safer alternative to cigarette smoking, murine and rat models of subacute and chronic e-cigarette vaping exposure have shown many proinflammatory changes in the respiratory tract. An acute vaping exposure paradigm has not been demonstrated in the golden Syrian hamster, and the hamster is a readily available small animal model that has the unique benefit of becoming infected with and transmitting respiratory viruses, including SARS-CoV-2, without genetic alteration of the animal or virus. Using a 2-day, whole body vaping exposure protocol in male golden Syrian hamsters, we evaluated serum cotinine, bronchoalveolar lavage cells, lung, and nasal histopathology, and gene expression in the nasopharynx and lung through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Depending on the presence of nonnormality or outliers, statistical analysis was performed by ANOVA or Kruskal-Wallis tests. For tests that were statistically significant (P < 0.05), post hoc Tukey-Kramer and Dunn's tests, respectively, were performed to make pairwise comparisons between groups. In nasal tissue, RT-qPCR analysis revealed nicotine-dependent increases in gene expression associated with type 1 inflammation (CCL-5 and CXCL-10), fibrosis [transforming growth factor-β (TGF-β)], nicotine-independent increase oxidative stress response (SOD-2), and a nicotine-independent decrease in vasculogenesis/angiogenesis (VEGF-A). In the lung, nicotine-dependent increases in the expression of genes involved in the renin-angiotensin pathway [angiotensin-converting enzyme (ACE), ACE2], coagulation (tissue factor, Serpine-1), extracellular matrix remodeling (MMP-2, MMP-9), type 1 inflammation (IL-1β, TNF-α, and CXCL-10), fibrosis (TGF-β and Serpine-1), oxidative stress response (SOD-2), neutrophil extracellular traps release (ELANE), and vasculogenesis and angiogenesis (VEGF-A) were identified. To our knowledge, this is the first demonstration that the Syrian hamster is a viable model of e-cigarette vaping. In addition, this is the first report that e-cigarette vaping with nicotine can increase tissue factor gene expression in the lung. Our results show that even an acute exposure to e-cigarette vaping causes significant upregulation of mRNAs in the respiratory tract from pathways involving the renin-angiotensin system, coagulation, extracellular matrix remodeling, type 1 inflammation, fibrosis, oxidative stress response, neutrophil extracellular trap release (NETosis), vasculogenesis, and angiogenesis.

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Abstract Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P &amp;lt; 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P &amp;lt; 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P &amp;lt; 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P &amp;lt; 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P &amp;lt; 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P &amp;lt; 0.01; CLI-095: −100 ± 6,6 %, n = 6, P &amp;lt; 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P &amp;lt; 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

Binding of gamma-glutamyl transferase to TLR-4 allows the activation of tissue factor expression in human monocytes

Abstract Background Gamma-glutamyl transferase (GGT) plays a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of Tissue Factor (TF) gene expression, the initiator of the clotting cascade. GGT may modulate TF expression, an assumption verified by previous studies carried out in human Peripheral Blood Mononuclear Cells (PBMCs). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic activity since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. Purpose To assess whether GGT-induced TF stimulation is a consequence of binding to Toll-Like Receptor (TLR)-4 and activation of NF-κB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from a leukemia patient, were incubated with hrGGT (0.5 ng/μl for PBMCs and 1ng/μl for THP-1). LPS-Rs (0.5 ng/μl for PBMCs and 1 ng/μl for THP-1), CLI-095 (3x10–6 M) and BAY-11-7082 (10–5 M) were used to block TLR-4 receptors, TLR4 signaling and NF-κB respectively. TF pro-coagulant activity (PCA) was assessed using of StartMax coagulometer and results were expressed in pg/ml after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110±70 to 510±43, n=7, p&amp;lt;0.01) and THP-1 cells (PCA from 170±64 to 460±80, n=15, p&amp;lt;0.001).In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72±17% n=4, p&amp;lt;0.01) a TLR-4 antagonist, CLI-095 (PCA:-74±34%, n=7, p&amp;lt;0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71±32%, n=7, p&amp;lt;0.001), a NF-κB inhibitor. Similar results were obtained in THP-1 cells [LPS-Rs: −76±15%, n=6, p&amp;lt;0.01; CLI-095: −100±6,6%, n=6, p&amp;lt;0.01; BAY-11-7082: −100±2,1%, n=6, p&amp;lt;0.01]. hrGGT activates NF-κB in hTLR4-positive HEK cell lines while doesn't induces effect in TLR4-negative HEK cells. Conclusions Besides confirming the cytokine-Like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells, these data identify for the first time the possible role of TLR-4 as the receptor of GGT and NfkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque. Funding Acknowledgement Type of funding sources: None.

JCAD enhances arterial thrombosis by regulating endothelial plasminogen activator inhibitor-1 and tissue factor expression

Abstract Introduction Arterial thrombosis underlies most acute CV events. Variants of the Junctional cadherin 5 associated (JCAD) locus were consistently shown to associate with increased risk of acute coronary syndrome. Being a component of cell junctions, JCAD protein is highly expressed in endothelial cells and was shown to promote atherosclerosis by acting on the Hippo pathway through LATS2 kinase. Purpose This project investigated the effect of JCAD in arterial thrombosis by using an established in vivo mouse model of carotid injury. The translational value of animal findings was assessed in primary human aortic endothelial cells (HAECs) as well as in CV patients. Methods JCAD knock-out (Jcad−/−) mice were exposed to photochemically-induced carotid artery endothelial injury to trigger thrombosis. Primary HAECs treated with JCAD small-interfering RNA (si-JCAD), LATS2-silencing RNA (si-LATS2) or control siRNA (si-SCR) were employed for in vitro assays. Plasma JCAD was measured in patients with chronic coronary syndrome (CCS) or ST-elevation myocardial infarction (STEMI). Results Compared to wild-type, Jcad−/− mice displayed reduced thrombus formation as underlined by delayed time to occlusion following endothelial-specific carotid damage. Suggesting a blunted activation of the extrinsic coagulation cascade, Jcad−/− animals showed reduced tissue factor (TF) protein expression and activity in carotid artery lysates (Fig. 1). Increased thrombus embolization episodes and D-dimer further suggested an increased activation of the fibrinolytic system in Jcad−/− mice. Indeed, Jcad−/− mice displayed reduced vascular expression of the fibrinolysis inhibitor plasminogen activator inhibitor (PAI)-1. In contrast, platelets aggregation in response to collagen and thrombin was similar in Jcad−/− and Jcad+/+ mice (Fig. 1). In line with the in vivo data, JCAD-silencing of HAECs inhibited TF and PAI-1 gene and protein expression. In accordance with previous literature, JCAD-silenced HAECs displayed increased levels of LATS2 Kinase, which blunts the Hippo pathway by increasing YAP phosphorylation. Yet, double JCAD and LATS2 silencing did not retrieve the phenotype of control HAECs. Of interest, si-JCAD HAECs showed increased levels of Akt phosphorylation, known to downregulate procoagulant expression and to directly phosphorylate YAP. Treatment with the Akt inhibitor Wortmannin prevented the effect of JCAD silencing on TF and PAI-1 indicating a causative role for this pathway (Fig. 2). Recapitulating in vitro findings, p-Akt and p-YAP levels were higher in arterial tissue of Jcad−/− animals as compared to WT (Fig. 1). Patients with STEMI showed significantly higher plasma levels of JCAD as compared to CCS (Fig. 2). Conclusions JCAD promotes arterial thrombosis by selectively modulating coagulation and fibrinolysis, but not platelet aggregation through endothelial TF and PAI-1. Our findings support the importance of JCAD as a novel therapeutic target for CV prevention. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation

Defining the Balance between the Expression of Procoagulant, Anticoagulant and Angiogenic Molecules in Myeloma Plasma Cells

Introduction: Multiple myeloma is a clonal B-cell malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow and other tissues. This disease significantly increases the risk of venous thromboembolic disorders. The interplay between Myeloma Plasma Cells (MPCs) with coagulation and angiogenesis is currently under investigation. It has been shown that MPCs promotes angiogenesis through VEGF expression. Whether MPCs express procoagulant and/or anticoagulant proteins remains to be shown.Aim: To determine the expression of tissue factor (TF), tissue factor pathway inhibitor 1 (TFPI 1), tissue factor pathway inhibitor 2 (TFPI 2), vascular endothelial growth factor (VEGF-A) and thrombomodulin (TM) by MPCs.Methods: Human MPCs (RPMI 8226) and human umbilical vein endothelial cells (HUVECs) that were used as control were cultured according to standard procedures. Total RNA was extracted and reversely transcribed as previously described. Briefly, 1 μg cDNA was PCR amplified using the Go TAq® qPCRMaster mix (Promega) and the Mx3000P qPCR system according to the manufacturer's protocol. Quantitative RT-PCR was used to measure the relative mRNA levels using TaqMan assays for VEGF-A, TFPI 1, TFPI 2, TF or TM (Sigma Aldrich). Ct values were normalized against the endogenous control B-Actine (from Sigma Aldrich).Relative mRNA expression was calculated using the comparative threshold method (1/PUISSANCE (2 (Ct (gene)-Ct (endogenous control))).Results: MPCs exhibited 4-fold higher TF expression compared to HUVECs but significantly lower expression of TFPI1 and TFPI2. For both MPCs and HUVECs, the expression of TFPI 1 was more pronounced compared to the expression of TF gene. Interestingly, MPCs showed lower expression of TFPI 2 compared to TF. Therefore, the TF/TFPI 1 ratio was higher in MPCs, reflecting a potentially increased TF activity compared to HUVECs. MPCs and HUVECs expressed comparable levels of TM. Finally, VEGF expression was significantly higher in the MPCs as compared to HUVECs. Results are summarized in Table 1.Conclusions. Plasmocytes from human multiple myeloma cells are characterized by a marked proangiogenic potential driven by high gene expression of VEGF which is accompanied by increased expression of TF. However, TF expression seems to be at least partially compensated by increased expression of TFPIs leading to the assumption that the TF-dependent procoagulant properties of plasmocytes are restricted by a balanced increase in TFPI. These data indicate that TF expression by myeloma cells may be associated with cell proliferation rather than a procoagulant [Display omitted] DisclosuresGarderet:Amgen: Honoraria; Takeda: Honoraria.

Antisense Tissue Factor Oligodeoxynucleotides Protected Diethyl Nitrosamine/Carbon Tetrachloride-Induced Liver Fibrosis Through Toll Like Receptor4-Tissue Factor-Protease Activated Receptor1 Pathway.

Tissue factor (TF) is a blood coagulation factor that has several roles in many non-coagulant pathways involved in different pathological conditions such as angiogenesis, inflammation and fibrogenesis. Coagulation and inflammation are crosslinked with liver fibrosis where protease-activated receptor1 (PAR1) and toll-like receptor4 (TLR4) play a key role. Antisense oligodeoxynucleotides are strong modulators of gene expression. In the present study, antisense TF oligodeoxynucleotides (TFAS) was evaluated in treating liver fibrosis via suppression of TF gene expression. Liver fibrosis was induced in rats by a single administration of N-diethyl nitrosamine (DEN, 200 mg/kg; i. p.) followed by carbon tetrachloride (CCl4, 3 ml/kg; s. c.) once weekly for 6 weeks. Following fibrosis induction, liver TF expression was significantly upregulated along with liver enzymes activities and liver histopathological deterioration. Alpha smooth muscle actin (α-SMA) and transforming growth factor-1beta (TGF-1β) expression, tumor necrosis factor-alpha (TNF-α) and hydroxyproline content and collagen deposition were significantly elevated in the liver. Blocking of TF expression by TFAS injection (2.8 mg/kg; s. c.) once weekly for 6 weeks significantly restored liver enzymes activities and improved histopathological features along with decreasing the elevated α-SMA, TGF-1β, TNF-α, hydroxyproline and collagen. Moreover, TFAS decreased the expression of both PAR1 and TLR4 that were induced by liver fibrosis. In conclusion, we reported that blockage of TF expression by TFAS improved inflammatory and fibrotic changes associated with CCl4+DEN intoxication. In addition, we explored the potential crosslink between the TF, PAR1 and TLR4 in liver fibrogenesis. These findings offer a platform on which recovery from liver fibrosis could be mediated through targeting TF expression.

The receptor for advanced glycation end products is a sensor for cell-free heme.

Heme's interaction with Toll-like receptor 4 (TLR4) does not fully explain the proinflammatory properties of this hemoglobin-derived molecule during intravascular hemolysis. The receptor for advanced glycation end products (RAGE) shares many features with TLR4 such as common ligands and proinflammatory, prothrombotic, and pro-oxidative signaling pathways, prompting us to study its involvement as a heme sensor. Stable RAGE-heme complexes with micromolar affinity were detected as heme-mediated RAGE oligomerization. The heme-binding site was located in the V domain of RAGE. This interaction was Fe3+ -dependent and competitive with carboxymethyllysine, another RAGE ligand. We confirmed a strong basal gene expression of RAGE in mouse lungs. After intraperitoneal heme injection, pulmonary TNF-α, IL1β, and tissue factor gene expression levels increased in WT mice but were significantly lower in their RAGE-/- littermates. This may be related to the lower activation of ERK1/2 and Akt observed in the lungs of heme-treated, RAGE-/- mice. Overall, heme binds to RAGE with micromolar affinity and could promote proinflammatory and prothrombotic signaling invivo, suggesting that this interaction could be implicated in heme-overload conditions.

LIM-only protein FHL2 attenuates vascular tissue factor activity, inhibits thrombus formation in mice and FHL2 genetic variation associates with human venous thrombosis.

Bleeding disorders and thrombotic complications are major causes of morbidity and mortality with many cases being unexplained. Thrombus formation involves aberrant expression and activation of tissue factor (TF) in vascular endothelial and smooth muscle cells. Here, we sought to identify factors that modulate TF gene expression and activity in these vascular cells. The LIM-only protein FHL2 is a scaffolding protein that modulates signal transduction pathways with crucial functions in endothelial and smooth muscle cells. However, the role of FHL2 in TF regulation and thrombosis remains unexplored. Using a murine model of venous thrombosis in mesenteric vessels, we demonstrated that FHL2 deficiency results in exacerbated thrombus formation. Gain- and loss-of-function experiments revealed that FHL2 represses TF expression in endothelial and smooth muscle cells through inhibition of the transcription factors nuclear factor κB and activating protein-1. Furthermore, we observed that FHL2 interacts with the cytoplasmic tail of TF. In line with our in vivo observations, FHL2 decreases TF activity in endothelial and smooth muscle cells whereas FHL2 knockdown or deficiency results in enhanced TF activity. Finally, the FHL2 single nucleotide polymorphism rs4851770 was associated with the risk of venous thrombosis in a large population of venous thrombosis cases and control subjects from 12 studies (INVENT consortium). Altogether, our results highlight functional involvement of FHL2 in TF-mediated coagulation and identify FHL2 as a novel gene associated with venous thrombosis in humans.

Oxidized low-density lipoproteins induce tissue factor expression in T-lymphocytes via activation of lectin-like oxidized low-density lipoprotein receptor-1.

T-lymphocytes plays an important role in the pathophysiology of acute coronary syndromes. T-cell activation in vitro by pro-inflammatory cytokines may lead to functional tissue factor (TF) expression, indicating a possible contribution of immunity to thrombosis. Oxidized low-density lipoproteins (oxLDLs) are found abundantly in atherosclerotic plaques. We aimed at evaluating the effects of oxLDLs on TF expression in T cells and the role of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1). CD3+ cells were isolated from healthy volunteers. Gene, protein, and surface expression of TF, as well as of LOX-1, were assessed at different time-points after oxLDL stimulation. To determine whether oxLDL-induced TF was LOX-1 dependent, T cells were pre-incubated with an LOX-1 inhibiting peptide (L-RBP) or with an anti-LOX-1 blocking antibody. To exclude that TF expression was mediated by reactive oxygen species (ROS) generation, oxLDL-stimulated T cells were pre-incubated with superoxide dismutase + catalase or with 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol), an intracellular free radical scavenger. Finally, to determine if the observed findings in vitro may have a biological relevance, the presence of CD3+/TF+/LOX-1+ cells was evaluated by immunofluorescence in human carotid atherosclerotic lesions. oxLDLs induced functionally active TF expression in T cells in a dose- and time-dependent manner, independently on ROS generation. No effect was observed in native LDL-treated T cells. LOX-1 expression was also induced by oxLDLs in a time- and dose-dependent manner. Pre-incubation with L-RBP or anti-LOX-1 antibody almost completely inhibited oxLDL-mediated TF expression. Interestingly, human carotid plaques showed significant infiltration of CD3+ cells (mainly CD8+ cells), some of which were positive for both TF and LOX-1. oxLDLs induce functional TF expression in T-lymphocytes in vitro via interaction of oxLDLs with LOX-1. Human carotid atherosclerotic plaques contain CD3+/CD8+cells that express both TF and LOX-1, indicating that also in patients these mechanisms may play an important role.

Anticoagulant effect of wogonin against tissue factor expression

Tissue factor (TF) is the primary cause of atherothrombosis, the rupture of atherosclerotic plaques with subsequent thrombosis, leading to acute cardiovascular events, such as myocardial infarction and stroke. Wogonin (Wog) is an active component of Scutellaria baicalensis, used for inflammatory diseases, atherosclerosis, and hyperlipidemia. The anticoagulant effect of Wog on TF expression remains unexplored. In this study, we have investigated the effects of Wog on TF gene expression and its underlying molecular mechanism in human vascular endothelial cells (ECs). We found that Wog dose-dependently inhibited PMA-enhanced TF mRNA, protein, and activity in ECs. This inhibition was attributed to its decreasing nuclear accumulations of transcription factors, phospho-c-Jun and early growth response-1(Egr-1), not nuclear factor-κB (NF-κB), through blocking extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. Reduction by Wog of Egr-1 nuclear level and Egr-1/DNA binding activity was associated with its inhibition of Egr-1 de novo synthesis. Wog as well as inhibitors to ERK and JNK suppressed TF promoter activity and protein expression in reporter gene and Western blot analyses. Furthermore, it also exhibited anticoagulant function by inhibiting TF expression and activity in tumor necrosis factor-alpha (TNF-α)- and lipopolysaccharide (LPS)-treated ECs and THP-1 cells. These results suggest that Wog inhibits ERK/Egr-1- and JNK/AP-1-mediated transactivation of TF promoter activity, leading to downregulation of TF expression and activity induced by inflammatory mediators. Wog targeting pathological TF expression without affecting its basal level may be a safer templet in the development of anticoagulant agent for cardiovascular thrombotic diseases related to atherothrombosis.

Evaluation of Tissue Factor -603 A/G Promoter Gene Polymorphism in Thrombotic Processes in Patients with Antiphospholipid Syndrome

<strong>Objective</strong>: Major clinical manifestations of Antiphospholipid syndrome (APS) are trombosis and thrombosis related complications. Tissue factor (TF) serves primary initiatory of the in vivo coagulation. TF gene expression may be induced in endothelial cells to initiate thrombus formation. In this study, we investigated the frequency of TF 603A/G promoter gene polymorphism in patients with antiphospholipid syndrome (APS) and its effects on TF expression and thrombotic events. <br> <strong>Materials and Methods</strong>: This study included 64 patients with APS (APS patient with thrombosis arterial and venous (n=38), antiphospholipid antibody-positive patients without thrombosis (n=26) and 44 healthy controls (HCs). Plasma TF levels and antiphospholipid antibodies were measured by ELISA method. Lupus anticoagulant was measured by activated partial thromboplastin time and kaolin clotting time. TF-603 A/G polymorphism was investigated by PCR method. <br> <strong>Results</strong>: Our results have showed that patients with APS, TF levels were found in patients significantly higher than the healthy control group (p: 0.006, p&lt;0.001, respectively). Compared between the two patient groups, TF level was significantly higher in the patient with thrombosis group compared to the patient without thrombosis group (p: 0.027). <br> <strong>Conclusion</strong>: In this study, we suggest that thrombotic complications observed in patients with APS are associated with increased TF expression.&nbsp;

Tissue Factor and Cancer: Regulation, Tumor Growth, and Metastasis.

There is a strong relationship between tissue factor (TF) and cancer. Many cancer cells express high levels of both full-length TF and alternatively spliced (as) TF. TF expression in cancer is associated with poor prognosis. In this review, the authors summarize the regulation of TF expression in cancer cells and the roles of TF and asTF in tumor growth and metastasis. A variety of different signaling pathways, transcription factors and micro ribonucleic acids regulate TF gene expression in cancer cells. The TF/factor VIIa complex enhances tumor growth by activating protease-activated receptor 2 signaling and by increasing the expression of angiogenic factors, such as vascular endothelial growth factor. AsTF increases tumor growth by enhancing integrin β1 signaling. TF and asTF also contribute to metastasis via multiple thrombin-dependent and independent mechanisms that include protecting tumor cells from natural killer cells. Finally, a novel anticancer therapy is using tumor TF as a target to deliver cytotoxic drugs to the tumor. TF may be useful in diagnosis, prognosis, and treatment of cancer.

NFκB Inhibition Mitigates Serum Amyloid A-Induced Pro-Atherogenic Responses in Endothelial Cells and Leukocyte Adhesion and Adverse Changes to Endothelium Function in Isolated Aorta.

The acute phase protein serum amyloid A (SAA) is associated with endothelial dysfunction and early-stage atherogenesis. Stimulation of vascular cells with SAA increases gene expression of pro-inflammation cytokines and tissue factor (TF). Activation of the transcription factor, nuclear factor kappa-B (NFκB), may be central to SAA-mediated endothelial cell inflammation, dysfunction and pro-thrombotic responses, while targeting NFκB with a pharmacologic inhibitor, BAY11-7082, may mitigate SAA activity. Human carotid artery endothelial cells (HCtAEC) were pre-incubated (1.5 h) with 10 μM BAY11-7082 or vehicle (control) followed by SAA (10 μg/mL; 4.5 h). Under these conditions gene expression for TF and Tumor Necrosis Factor (TNF) increased in SAA-treated HCtAEC and pre-treatment with BAY11-7082 significantly (TNF) and marginally (TF) reduced mRNA expression. Intracellular TNF and interleukin 6 (IL-6) protein also increased in HCtAEC supplemented with SAA and this expression was inhibited by BAY11-7082. Supplemented BAY11-7082 also significantly decreased SAA-mediated leukocyte adhesion to apolipoprotein E-deficient mouse aorta in ex vivo vascular flow studies. In vascular function studies, isolated aortic rings pre-treated with BAY11-7082 prior to incubation with SAA showed improved endothelium-dependent vasorelaxation and increased vascular cyclic guanosine monophosphate (cGMP) content. Together these data suggest that inhibition of NFκB activation may protect endothelial function by inhibiting the pro-inflammatory and pro-thrombotic activities of SAA.

Cleavage of Anti-PF4/H IgG Antibodies By Ides, and Potential Benefit in the Treatment of Heparin-Induced Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a severe drug-adverse event due to platelet-activating antibodies (Abs) directed against platelet factor 4 (PF4)/heparin (H) complexes. In most patients, HIT Abs are IgG that directly activate platelets and monocytes in the presence of heparin via FcγRIIA receptors. The interaction between the Fc fragment of anti-PF4/H IgG and FcγRIIA is thus a key step for cellular activation in HIT. Several bacterial proteases such as IdeS (IgG-degrading enzyme of Streptococcus pyogenes) are cleaving IgG in the lower hinge region of heavy chain leading to the formation of single cleaved IgG (scIgG) and then of Fab'2. Importantly, cleavage of IgG by IdeS can abolish their ability to bind FcγR and suppress the cellular effects resulting from this interaction.The aim of this study was therefore to evaluate whether anti-PF4/H IgG cleavage by IdeS could inhibit cell activation induced by HIT antibodies, and their pathogenicity. To achieve this objective, we studied the effects of IdeS on platelet responses to 5B9, a monoclonal chimeric anti-PF4/H IgG1 recently developed in our laboratory, and which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017).IdeS was demonstrated to quickly (6 minutes) cleave purified 5B9 IgG, leading to the formation of sc5B9, without any reduction in its binding ability to PF4/H complex. However, flow cytometry experiments showed that heparin-dependent binding of sc5B9 to platelets and FcγRIIA was dramatically reduced compared to those of uncleaved 5B9. In addition, functional assays (serotonin release assays and platelet aggregation tests) also confirmed that sc5B9 was unable to induce platelet activation and aggregation in the presence of heparin. Incubation of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood containing 5B9 IgG or HIT plasma samples also lead in every sample tested to the cleavage of anti-PF4/H Abs, which fully abolished their capacity to induce heparin-dependent platelet aggregation, as demonstrated by impedance aggregometry (Multiplate analyzer). As expected, no effect of IdeS was observed on platelet aggregation induced by collagen (1 µg/mL), or ADP (10 µM). Moreover, tissue factor (TF) gene expression induced in monocytes by 5B9 in the presence of heparin was also completely abolished after addition of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood, whereas no inhibitory effect of this protease on TF expression induced by LPS was evidenced. We also showed that platelet aggregation and fibrin formation induced by 5B9 with heparin was completely inhibited after IdeS treatment when whole blood was perfused in vWF-coated microfluidic channels with shear rates similar to those of venous flow (500s-1). Finally, IdeS was also showed to prevent efficiently thrombocytopenia and hypercoagulability (with no increase in thrombin/anti-thrombin plasma levels) induced by 5B9 in transgenic mice expressing human PF4 and FcγRIIA receptors, when previously treated by this protease (0.5 µg/g) before IV injection of heparin.In conclusion, the cleavage of anti-PF4/H IgG by IdeS prevents heparin-dependent cellular activation induced by HIT antibodies, thereby reducing their pathogenicity. Therefore, injection of IdeS could be considered as a potential treatment in patients with severe HIT, particularly in those who necessitate emergent cardiac surgery with cardiopulmonary bypass and thus anticoagulation with unfractionated heparin, which remains the safest and easiest anticoagulant to be used in this specific surgical procedure. DisclosuresNo relevant conflicts of interest to declare.

P1845The pro-inflammatory signaling lipid sphingosine-1-phosphate regulates gene and protein expression of both tissue factor and plasminogen activator inhibitor-1 in differentiated fat cells

P1845The pro-inflammatory signaling lipid sphingosine-1-phosphate regulates gene and protein expression of both tissue factor and plasminogen activator inhibitor-1 in differentiated fat cells

Structural and cellular mechanisms of peptidyl-prolyl isomerase Pin1-mediated enhancement of Tissue Factor gene expression, protein half-life, and pro-coagulant activity.

Tissue Factor is a cell-surface glycoprotein expressed in various cells of the vasculature and is the principal regulator of the blood coagulation cascade and hemostasis. Notably, aberrant expression of Tissue Factor is associated with cardiovascular pathologies such as atherosclerosis and thrombosis. Here, we sought to identify factors that regulate Tissue Factor gene expression and activity. Tissue Factor gene expression is regulated by various transcription factors, including activating protein-1 and nuclear factor-κ B. The peptidyl-prolyl isomerase Pin1 is known to modulate the activity of these two transcription factors, and we now show that Pin1 augments Tissue Factor gene expression in both vascular smooth muscle cells and activated endothelial cells via activating protein-1 and nuclear factor-κ B signaling. Furthermore, the cytoplasmic domain of Tissue Factor contains a well-conserved phospho-Ser258-Pro259 amino-acid motif recognized by Pin1. Using co-immunoprecipitation and solution nuclear magnetic resonance spectroscopy, we show that the WW-domain of Pin1 directly binds the cytoplasmic domain of Tissue Factor. This interaction occurs via the phospho-Ser258-Pro259 sequence in the Tissue Factor cytoplasmic domain and results in increased protein half-life and pro-coagulant activity. Taken together, our results establish Pin1 as an upstream regulator of Tissue Factor-mediated coagulation, thereby opening up new avenues for research into the use of specific Pin1 inhibitors for the treatment of diseases characterized by pathological coagulation, such as thrombosis and atherosclerosis.