Tissue Factor mRNA Expression Research Articles

Sars-Cov-2 Spike Protein Triggers Inflammasome-Mediated Tissue Factor Activity

Introduction: An exaggerated inflammatory response both locally and systemically mediated by innate immune signaling is a near universal feature of severe COVID-19 illness and has been associated with multi-organ damage and thrombosis. As part of an antiviral response, activation and formation of the intracellular NLRP3 inflammasome leads to expression of proinflammatory IL-1 family cytokines. In macrophages, the NLRP3 inflammasome is linked to the release of tissue factor (TF) bearing microvesicles. Multiple studies provide evidence of NLRP3 inflammasome activation in COVID-19 including increased IL-1β in monocytes, sera, and bronchoalveolar lavage fluid from COVID-19 patients. Patient monocytes bear features of inflammasome activation, which correlate with disease severity (Potere et al. Cardiovascular Research 2023). Given the central role of thromboinflammation in COVID-19, we explored the ability of various coronavirus spike proteins to increase TF activity in a monocytic cell line. Mechanistically, we sought to explore the role of the NLRP3 inflammasome in SARS-CoV-2 spike protein triggered tissue factor generation. Methods: The SARS-CoV-2 α variant S1 subunit (S1α) and Omicron variant BA.1.1.529 S1 (S1O) recombinant proteins were obtained from Raybiotech and OC43, a non-COVID respiratory human coronavirus, recombinant spike protein (OC43 S) from Sino Biological. THP-1 cells at a concentration of 2x10 6/mL were incubated with spike proteins at 37°C in RPMI, supplemented with 10% fetal bovine serum. A specific NLRP3 inflammasome inhibitor (MCC950) was incubated with select samples for 1 hour following addition of the spike protein. The cells were harvested, subjected to RNA extraction and cDNA synthesis by reverse transcription. TF, NLRP3, caspase (CASP)-1, and IL-1β relative mRNA expression was quantified using RT-PCR and normalized to GADPH expression. For quantifying microparticle TF activity, cells were incubated with the spike proteins for 6 hours then microparticles were isolated from the supernatant after centrifugation at 20,000 g for 30 minutes at 4°C, washed and TF activity was measured using a two-stage chromogenic assay kit (Abcam). A monoclonal anti-TF antibody (Santa Cruz) was used to confirm specificity for TF generation. Experiments were performed in triplicate. Data is presented as mean ± standard deviation. The student t test was used to assess the difference between unpaired groups with a value of p <.05 considered statistically significant. Results: Our previous data showed that S1α, but not S1O or OC43 S, leads to increased TF mRNA expression in THP-1 cells in a dose dependent manner. Here we show TF microparticle activity was increased with S1α (20 µg/mL) as compared to incubation of THP-1 cells with S1O or OC43 spike proteins (20 µg/mL), which were comparable to the activity of cells alone ( Figure 1). After treatment with S1α (20 µg/mL) for 1 hour, we found increased expression of TF (10-fold), NLRP3 (2-fold), CASP-1 (3-fold), and IL-1β (3.5-fold). Treatment with S1O and OC43 S did not lead to a significant change in relative mRNA expression of TF, NLRP3, CASP-1, and IL-1β. Following incubation with the S1α spike protein for 1 hour, cells were re-plated in fresh media containing 10 µM MCC950. Treatment with MCC950 significantly reduced expression of TF, NLRP3, CASP-1, and IL-1β ( Figure 2). Conclusion: We confirmed that TF expression induced by the S1α spike protein leads to an increase in TF activity, as assessed by microparticle generation. The NLRP3 inflammasome is a mediator of coagulopathy induced by the spike protein, and TF, CASP-1, and IL-1β expression are abrogated after specific NLRP3 inflammasome inhibition. TF activity and inflammation induced by the coronavirus spike proteins in vitro correlates with the respective clinical phenotype and disease severity of the variants. This suggests that the effects of SARS-CoV-2 on TF and inflammasome activation derive in part from the spike protein, and these assays may be useful in predicting the effects of spike protein variants.

PB0713 Increased Tissue Factor mRNA Expression is Associated with High Plasma Levels of Activated Factor VII-Antithrombin Complex and Predicts Mortality in Primary Liver Cancer

PB0713 Increased Tissue Factor mRNA Expression is Associated with High Plasma Levels of Activated Factor VII-Antithrombin Complex and Predicts Mortality in Primary Liver Cancer

Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability.

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes. Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production. CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway. The findings of the present study have further clarified the procoagulant properties of CNT.

Administration of spermidine attenuates concanavalin A-induced liver injury.

A previous study revealed that treatment with the anticoagulant heparin attenuated concanavalin A (ConA)-induced liver injury. The administration of spermidine (SPD) increased urokinase-type plasminogen activator (uPA) levels in the serum. uPA is clinically used for the treatment of some thrombotic diseases such as cerebral infarction. Therefore, SPD may attenuate ConA-induced liver injury that is exacerbated by blood coagulation. The present study investigated the effect of SPD on liver injury in mice with autoimmune hepatopathy induced by ConA. A model of liver injury was created by intravenous injection of ConA into mice. SPD was administered in free drinking water and was biochemically and pathologically examined over time. The administration of SPD to ConA-treated mice significantly reduced liver injury. However, SPD treatment upregulated the mRNA expression of TNF-α and IFN-ϒ in the livers of ConA-treated mice. In contrast, the mRNA expression of tissue factor in the livers of SPD-treated mice was decreased after ConA injection. The frequency of lymphocytes and lymphocyte activation were not affected by SPD administration in ConA-treated mice. SPD treatment increased uPA levels in the serum and decreased the level of D-dimer in ConA-treated mice. Moreover, SPD decreased fibrin in the livers of ConA-treated mice. These results indicated that SPD treatment increased anticoagulant ability by increasing of uPA and attenuated ConA-induced liver injury.

Deletion of Smad7 Ameliorates Intestinal Inflammation and Contributes to Fibrosis.

Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-β compared with non-IBD controls. SMAD7 negatively regulates TGF-β signaling. An earlier study aiming to target Smad7 showed a lack of clinical benefit. It remains unknown whether inhibition of SMAD7 is beneficial in specific settings of IBD. We evaluated the effect of Smad7 deficiency on inflammation, fibrogenesis, and wound healing. For the initiation of fibrosis in Smad7-/- (Smad7Δex-I) CD-1 mice, the dextran sodium sulfate-induced chronic colitis model and the heterotopic transplantation model of fibrosis were used. Wound closure of fibroblasts from Smad7-/- mice was determined using culture inserts and electric cell-substrate impedance sensing in vitro. In dextran sodium sulfate-induced chronic colitis, Smad7 deficiency was associated with ameliorated inflammation, as evidenced by decreased clinical score, histological score, and myeloperoxidase activity. Absence of SMAD7 decreased T-cell accumulation in colonic tissue and tumor necrosis factor (TNF) mRNA expression levels. Smad7-/- mice showed a significant increase in hydroxyproline and collagen content, as well as ColIVa1 mRNA expression. Wild type mice transplanted with terminal ileum from Smad7-/- mice in the heterotopic animal model for intestinal fibrosis showed a significant increase in collagen content and protein expression of α-smooth muscle actin. Smad7 deficiency is associated with a decrease in intestinal inflammation and an increase in fibrosis. Targeting SMAD7 constitutes a potential new treatment option for IBD; progression of disease-associated fibrosis should be considered.

Evaluation of the mechanisms of heme-induced tissue factor activation: Contribution of innate immune pathways.

Hemolytic diseases such as Sickle Cell Disease (SCD) are characterized by a natural propensity for both arterial and venous thrombosis. The ability of heme to induce tissue factor (TF) activation has been shown both in animal models of SCD, and in human endothelial cells and monocytes. Moreover, it was recently demonstrated that heme can induce coagulation activation in the whole blood of healthy volunteers in a TF-dependent fashion. Herein, we aim to further explore the cellular mechanisms by which heme induces TF-coagulation activation, using human mononuclear cells, which have been shown to be relevant to in vivo hemostasis. TF mRNA expression was evaluated by qPCR and TF procoagulant activity was evaluated using a 2-stage assay based on the generation of activated factor X (FXa). Heme was capable of inducing both TF expression and activation in a TLR4-dependent pathway. This activity was further amplified after TNF-α-priming. Our results provide additional details on the mechanisms by which heme is involved in the pathogenesis of hypercoagulability in hemolytic diseases.

The Plasma Level of IL-1β As a Biomarker of Angiopathy in Systemic Chronic Active EBV Infection

Background and aim: Systemic chronic active Epstein-Barr virus infection (sCAEBV) is an intractable rare disease revealing persistent systemic inflammation with clonal proliferation of EBV-infected T- or NK-cells. 25% of sCAEBV patients accompany angiopathy such as aneurysm and vasculitis. (Yonese et al. Blood advances. 2020). Because angiopathy degrades patients' quality of life and is one of the main causes of death in sCAEBV, it is crucial to clarify the mechanisms of angiopathy development in sCAEBV. Interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) are reported to be involved in angiopathy onset. We investigated if IL-1β and TNFα play roles to induce vascular endothelial cell damage in sCAEBV with angiopathy.Methods: sCAEBV patients were diagnosed when meeting all of the following four conditions: 1) inflammation persisting for more than 3 months, 2) increasing EBV-DNA in peripheral blood (PB) or in diseased tissue, 3) EBV infected T- or NK-cells, 4) not applying to other known diseases. We measured the plasma concentration of cytokines by high sensitivity cytokine beads assay (MILLIPLEX ®MAP Kit). We isolated EBV-infected cells and monocytes by magnetic beads (MACS ® Cell Separation). Ea.hy926, a human umbilical vein endothelial cell line, was used as a vascular endothelial cell model.Results: Samples from 17 sCAEBV patients (infected cell types: CD4 in 7; CD8 in 1; and CD56 in 9) and 8 healthy donors were examined. We detected elevated levels of IL-1β in 4 out of 17 sCAEBV patient's plasma. Interestingly, among the 4, 3 had clinically associated angiopathy: 1 aneurysm, 1 vasculitis, and 1 intracranial vascular lesions with multiple cerebral infarctions. The EBV-infected cells of these 3 patients were CD4-positive cells. The TNFα concentration of patients' plasma was higher than that of healthy donors, but there was no correlation with angiopathy. The mRNA of IL-1β in the EBV-infected cells of patients with high plasma IL-1β was not enhanced compared to that of patients with undetectable plasma IL-1β. In one patient with high plasma IL-1β, the level of IL-1β mRNA of the monocytes was 17.2 times higher than the level of the same patient's EBV-infected cells. IL-1β inhibited the proliferation of Ea.hy926 cells. IL-1β upregulated the mRNA expression of Tissue factor (TF) as well as Plasminogen activator inhibitor-1 (PAI-1) and suppressed the mRNA of Thrombomodulin (TM) in Ea.hy926 cells.Discussion: IL-1β originally exists in immunocompetent cells such as CD4-positive cells in the form of precursor protein (pro-IL-1β). In the occurrence of inflammation, pro-IL-1β is cleaved by caspase-1 activated by the inflammasome, and the production and the secretion of IL-1β is induced without mRNA elevation. The infected cells of sCAEBV patients with high level plasma IL-1β and vascular lesion were all CD4-positive. Consequently, we suspect that EBV-infected, CD4-positive cells are responsible for the production of IL-1β. Likewise, we detected high expression of IL-1β in monocytes derived from a patient with high plasma IL-1β. Based on these facts, we set two hypotheses on the production of IL-1β in sCAEBV. One is the production in EBV-infected cells by the cleavage of pro-IL-1β. The other is the production through transcription and translation in monocytes. IL-1β suppressed the proliferation of Ea.hy926 cells. In the same cells, IL-1β also induced TF and PAI-1 expression and suppressed TM expression. These results suggest that IL-1β may induce vascular damage and blood coagulation to cause angiopathy.Conclusion: In sCAEBV, IL-1β may be a biomarker of angiopathy. IL-1β may be a therapeutic target to treat sCAEBV accompanying vascular lesions. DisclosuresArai: Abbvie GK: Honoraria; BMS: Honoraria; Chugai Pharmaceutical Co Ltd: Honoraria, Research Funding; Eisai Co Ltd: Research Funding; Abbott Japan LLC: Honoraria; Kyowa Kirin Co ltd: Honoraria, Research Funding; Ono Pharmaceutical Co ltd: Honoraria, Research Funding; Nippon Shinyaku Co Ltd: Honoraria, Research Funding; Otsuka Pharmaceutical Co ltd: Research Funding; Novartis Pharma KK: Honoraria; Takeda Pharmaceuticals Co Ltd: Honoraria, Research Funding; Shionogi & Co ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Sanofi KK: Honoraria; Pfizer Japan Inc: Honoraria; Astellas Pharma Inc: Honoraria.

Impact of LMWH and Specific Factor Xa Inhibitors, Apixaban and Fondaparinux, on Cancer Cell Biology and Procoagulant Properties of Cancer Microenvironment

Background Cancer patients with venous thromboembolism (VTE) or at risk of VTE are treated with antithrombotic agents. Cancer cells express procoagulant properties and induce hypercoagulability in the microenvironment, that could impact the efficiency of the antithrombotic agents.Aims In the present study, we investigated the interaction between antithrombotic agents with pancreatic cancer cells, as well as with their microenvironment. The impact of apixaban, fondaparinux, enoxaparin and tinzaparin on the procoagulant properties of pancreatic cancer cells BXPC3 was examinated. Reciprocally, we also investigated the impact of BXPC3 on the potency of these antithrombotic agents.Methods BXPC3 cells (400 cells/μl) were exposed for 48 hours to apixaban (2 µg/ml), fondaparinux (2 µg/ml), enoxaparin, tinzaparin (2 anti-Xa IU/ml) or NaCL (control). Then, conditioned media (CM) and BXPC3 cells were harvested, separated and put in contact with normal platelet-poor plasma (PPP). Subsequently, thrombin generation (TG) was assessed using Thrombogram-Thrombinoscope® assay (Diagnostica Stago). Cells' viability was also assessed with the MTT assay. Gene expression for Tissue Factor (TF), Vascular Endothelial Growth Factor (VEGF), Thrombospondin 1 (THSB1) was assessed with RT-qPCR at the cells exposed or not to the antithrombotic agents. Expression of TF protein and activity of cancer cells was assessed using ELISA method. Residual anti-Xa activity in CM was measured using specific amidolytic assays for each antithrombotic agent.Results Apixaban, fondaparinux, enoxaparin, and tinzaparin significantly reduced cell viability by 25%, 12%, 14%, and 11% respectively. In the control experiment non treated BXPC3 cells enhanced TG. Pre-treatment of BXPC3 with the antithrombotic agents did not significantly modify their capacity to trigger and enhance TG. Among the studied agents only apixaban resulted in significant decrease of TF mRNA expression. However, protein expression of TF was not significantly modified by any of the antithrombotic agents. VEGF's mRNA expression was significantly decreased by fondaparinux and enoxaparin. THBS1's mRNA expression was significantly increased by apixaban. The concentrations of the anti-Xa activity of fondaparinux, enoxaparin and tinzaparin in the CM obtained at 48h after exposure of cells were reduced by 27%, 48% and 26% respectively as compared to those initially added in the culture medium. In contrast, the concentration of apixaban in the CM did not significantly change. The CM obtained by cells exposed to apixaban, fondaparinux, enoxaparin and tinzaparin inhibited TG by 70%, 30%, 40% and 90% respectively.Conclusion. Antithrombotic agents reduced the viability of BXPC3 cells. Among the studied agents, apixaban had the most pronounced effect on cells' viability. The antithrombotic agents had a potential downregulating effect on the proangiogenetic properties of BXPC3 via the decrease of VEGF gene expression (fondaparinux and enoxaparin) and enhancement of THBS1 gene expression (apixaban). Nevertheless, preincubation of BXPC3 with the antithrombotic agents did not alter the expression of TF protein and their effect on thrombin generation. Moreover, BXPCE exerted a “degradation” effect on LMWH and fondaparinux. Apixaban appeared to escape from this effect of the cancer cells. A significant inhibitory effect on thrombin generation was exerted by the residual concentrations of the antithrombotic agents in the microenvironment of cancer cells. The ensemble of these data highlight for the first time that the presence of antithrombotic agents in cancer cell microenvironment alters the biology of cancer cells and offer a constant antithrombotic effect in the microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Substrate stiffness modulates endothelial cell function via the YAP-Dll4-Notch1 pathway

Endothelial cells adapt their functions as a consequence of sensing extracellular substrate stiffness; these alterations allow them to maintain their vascular structure and function. Substrate stiffness-mediated yes-associated protein 1 (YAP) activation plays an important role in mechano-transduction and pro-angiogenic phenotype of endothelial cells, and Delta-like ligand 4 (Dll4)-Notch1 signaling is closely related to angiogenesis; however, the impact of substrate stiffness-mediated interrelation of these pathways on endothelial cell functions remains elusive. We confirmed that endothelial cells on softer substrates not only elongate cellular aspects but also attenuate YAP activation compared to cells on stiffer substrates. Endothelial cells on softer substrates also upregulate the vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 mRNA expression that is enhanced by VEGF stimulation. We determined that endothelial cells on softer substrates increased Dll4 expression, but not Notch1 expression, via YAP signaling. Moreover, endothelial cells on soft substrates induced not only VEGFRs upregulation but also suppression of pro-inflammatory interleukin-6 and plasminogen activator inhibitor-1 mRNA expression and the facilitation of anti-coagulant thrombomodulin and pro-coagulant tissue factor mRNA expression. Our results suggest that endothelial cells activate the YAP-Dll4-Notch signaling pathway in response to substrate stiffness and dictate cellular function.

Convallatoxin, the primary cardiac glycoside in lily of the valley (Convallaria majalis), induces tissue factor expression in endothelial cells.

BackgroundConvallotoxin (CNT), present in lily of the valley (Convallaria majalis), is a toxin that causes food poisoning among humans and companion animals. Although various symptoms of CNT poisoning have been well described, hypercoagulability owing to CNT is only empirically known among some veterinarians, and the underlying mechanism remains to be elucidated. CNT exerts cytotoxic effects on endothelial cells.ObjectivesThis study aimed to determine whether CNT induces the expression of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade, in endothelial cells and leads to a hypercoagulable state.MethodsHuman umbilical vein endothelial cells (HUVECs) were used for in vitro experiments. HUVECs were treated with or without CNT (50 and 100 nM) for 4 h. Phosphate‐buffered saline was used as a control. Cell viability was determined using the WST‐8 assay. Quantitative real‐time polymerase chain reaction was performed to determine TF mRNA expression. TF protein expression was observed using a laser scanning confocal microscope.ResultsThe viability of HUVECs significantly reduced after CNT treatment compared with that of non‐treated cells (p < 0.05). Moreover, a significant increase in TF mRNA and protein expression was observed after 4 h of CNT treatment. CNT elicited these effects in a dose‐dependent manner.ConclusionsTF expression induced by CNT in endothelial cells can contribute to the development of a hypercoagulable state. The present study partially revealed the mechanisms underlying the CNT‐induced hypercoagulable state. The findings can contribute to the development of a novel therapy for lily of the valley poisoning.

Vascular endothelial tissue factor contributes to trimethylamine N-oxide-enhanced arterial thrombosis.

Gut microbiota and their generated metabolites impact the host vascular phenotype. The metaorganismal metabolite trimethylamine N-oxide (TMAO) is both associated with adverse clinical thromboembolic events, and enhances platelet responsiveness in subjects. The impact of TMAO on vascular Tissue Factor (TF) in vivo is unknown. Here, we explore whether TMAO-enhanced thrombosis potential extends beyond TMAO effects on platelets, and is linked to TF. We also further explore the links between gut microbiota and vascular endothelial TF expression in vivo. In initial exploratory clinical studies, we observed that among sequential stable subjects (n = 2989) on anti-platelet therapy undergoing elective diagnostic cardiovascular evaluation at a single-site referral centre, TMAO levels were associated with an increased incident (3 years) risk for major adverse cardiovascular events (MACE) (myocardial infarction, stroke, or death) [4th quartile (Q4) vs. Q1 adjusted hazard ratio (HR) 95% confidence interval (95% CI), 1.73 (1.25-2.38)]. Similar results were observed within subjects on aspirin mono-therapy during follow-up [adjusted HR (95% CI) 1.75 (1.25-2.44), n = 2793]. Leveraging access to a second higher risk cohort with previously reported TMAO data and monitoring of anti-platelet medication use, we also observed a strong association between TMAO and incident (1 year) MACE risk in the multi-site Swiss Acute Coronary Syndromes Cohort, focusing on the subset (n = 1469) on chronic dual anti-platelet therapy during follow-up [adjusted HR (95% CI) 1.70 (1.08-2.69)]. These collective clinical data suggest that the thrombosis-associated effects of TMAO may be mediated by cells/factors that are not inhibited by anti-platelet therapy. To test this, we first observed in human microvascular endothelial cells that TMAO dose-dependently induced expression of TF and vascular cell adhesion molecule (VCAM)1. In mouse studies, we observed that TMAO-enhanced aortic TF and VCAM1 mRNA and protein expression, which upon immunolocalization studies, was shown to co-localize with vascular endothelial cells. Finally, in arterial injury mouse models, TMAO-dependent enhancement of in vivo TF expression and thrombogenicity were abrogated by either a TF-inhibitory antibody or a mechanism-based microbial choline TMA-lyase inhibitor (fluoromethylcholine). Endothelial TF contributes to TMAO-related arterial thrombosis potential, and can be specifically blocked by targeted non-lethal inhibition of gut microbial choline TMA-lyase.

Rivaroxaban protects from the oxysterol-induced damage and inflammatory activation of the vascular endothelium

ABSTRACT Background Rivaroxaban is one of the direct factor Xa inhibitors. Its function in the inactivated coagulation cascade is unclear. The aim of the study was to assess the effect of rivaroxaban on the endothelial integrity and inflammatory properties of endothelial cells stimulated by 25-hydroxycholesterol (25-OHC). Methods HUVECs were stimulated with 25-OHC, rivaroxaban and 25-OHC+ rivaroxaban. HUVEC integrity and permeability were measured using the xCELLigence system and paracellular flux assay. The mRNA expression of tissue factor, ICAM-1, VEGF, IL-33, MCP-1, TNF-α was analyzed in the real-time PCR. Apoptosis and viability were measured by flow cytometry. The VEGF protein concentration was assessed by ELISA. The confocal microscope was used to evaluate the expression of VE-cadherin in endothelial cells. Results 25-OHC decreased endothelial cell integrity and increased the mRNA expression of IL-33, tissue factor, ICAM-1, MCP-1, VEGF, TNF-α as compared to unstimulated controls. Following the stimulation with rivaroxaban, HUVEC restored integrity disrupted by 25-OHC (p < .01). In HUVECs pre-stimulated with oxysterol, rivaroxaban decreased mRNA expression of IL-33, TNF-α, chemokines MCP-1, ICAM-1, VEGF and tissue factor (p < .01). Rivaroxaban 100 mg/ml+25-OHC increased the VE-cadherin expression in endothelium as compared to 25-OHC (p < .05). Conclusion Our finding suggests that rivaroxaban may restore the endothelial barrier and inhibit the inflammatory activation caused by oxysterol in vitro.

P067 The effect of a probiotic mix on mucosal healing and fibrotic responses in healthy colonic subepithelial myofibroblasts

Abstract Background Various probiotic strains play a positive role in Inflammatory Bowel Disease and might support mucosal healing. Colonic Subepithelial Myofibroblasts (SEMFs) have a pivotal role in mucosal healing and fibrosis, by migrating to the trauma region and secreting collagen. Our study aimed to investigate whether different strains of probiotics can affect the migration capacity and the fibrotic phenotype of SEMFs isolated from healthy individuals. Methods The probiotic mix of Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium lactis and Saccharomyces boulardii was reconstituted in SEMFs culture medium, they were identified by Gram staining and their viability was assessed by Trypan Blue staining. Primary SEMFs were isolated from colonic biopsies from healthy individuals, and stimulated with 102, 104 or 108 cfu/ml of the mix for 6h or 48h; in 6h total RNA was collected and mRNA expression of collagen type I, III, fibronectin, tissue factor (TF) and α-SMA was measured by quantitative PCR, and in 48h their effect on migration rate through the Wound Healing Assay and collagen secretion via Sircol was assessed. Results Trypan Blue confirmed that the majority of the probiotic mix was alive, and all three bacterial strains and the yeast were stained Gram positive. Stimulation of SEMFs with the probiotic mix resulted in different effects depending on its concentration; 102 cfu/ml downregulated protein collagen secretion (3.4μg/ml, IQR: 2.7–5.0, p&amp;lt;0.01 vs control 6.3μg/ml, IQR: 5.2–6.7) and α-SMA mRNA production (0.7-fold, IQR: 0.7–0.8, p&amp;lt;0.01), 104 cfu/ml upregulated the Fibronectin mRNA expression (1.6-fold, IQR: 1.2–1.8, p&amp;lt;0.0001) and 108 cfu/ml downregulated collagen type I (0.2-fold, IQR: 0.1–0.8, p&amp;lt;0.01) and III mRNAs (0.3-fold, IQR: 0.1–0.8, p&amp;lt;0.01). In addition, all three concentrations resulted in the overall upregulation of TF mRNA expression. Regarding the effect of the probiotic mix on SEMFs migration capacity, 104 cfu/ml of the probiotic mix resulted in increased migration rate by 133.8% (IQR: 102.8–150.2, p&amp;lt;0.01) after 24h, while the use of 102 cfu/ml and 108 cfu/ml decreased it by 58.2% (IQR: 34.8–79.7, p&amp;lt;0.01) and by 46.8% (IQR: 34.3–72.1, p&amp;lt;0.01) respectively. Conclusion This study provides evidence that the probiotic strains L. plantarum, L. acidophillus, B. lactis and S. boulardii remain viable in SEMFs’ culture medium and have the ability to interact with and alter the behavior of SEMFs. Moreover, the effect of the probiotic mix on SEMFs fibrotic phenotype is dependent of the concentration of the probiotics used. Further preclinical research is needed in order to identify the effect of each strain, as well as the suitable dose and combinations in various pathological conditions.

Synthesis and analysis of small molecules to restrain the function of tissue factor within tumour cells.

Introduction: The restriction of prolyl-protein cis/trans isomerase 1 (Pin1) activity has been shown to prevent the release of tissue factor (TF) leading to the accumulation of the latter protein within the cell. This study tested the ability of novel small molecules to inhibit Pin1, suppress TF activity and release, and induce cellular apoptosis. Methods: Four compounds were designed and synthesised based on modification of 5-(p-methoxyphenyl)-2-methylfuran-3-carbonyl amide and the outcome on MDA-MB-231 and primary cells examined. These compounds contained 3-(2-naphthyl)-D-alanine (4a), D-tryptophan (4b), D-phenylalanine (4c), and D-tyrosine (4d) at the amino-termini. Results: Treatment of cells with compound 4b and 4d reduced the cell-surface TF activity after 60 min on MDA-MB-231 cells. Incubation with compound 4d also reduced TF antigen on the cell surface and its incorporation into microvesicles, while compounds 4a and 4b significantly increased TF release. None of the four compounds significantly altered the total amount of TF antigen or TF mRNA expression. Compound 4b and 4d also suppressed the binding of Pin1 to TF-cytoplasmic domain peptide. However, compound 4d reduced while compound 4b increased the Pin1 isomerase activity. Finally, treatment with compound 4b and 4d reduced the cell numbers, increased nuclear localisation of p53, Bax protein and bax mRNA expression and induced cellular apoptosis in MDA-MB-231 but not primary endothelial cells. Conclusions: In conclusion, we have identified small molecules to regulate the function of TF within cells. Two of these compounds may prove to be beneficial in moderating TF function specifically and restrain TF-mediated tumour growth without detrimental outcomes on normal vascular cells.

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.

Histamine- or vascular endothelial growth factor-induced tissue factor expression and gap formation between vascular endothelial cells are synergistically enhanced by lipopolysaccharide, tumor necrosis factor-α, interleukin (IL)-33 or IL-1β.

The pathogenesis of chronic spontaneous urticaria (CSU), also called chronic idiopathic urticaria, has been considered to be associated with the activation of the extrinsic blood coagulation cascade. However, the trigger for the extrinsic coagulation cascade in patients with CSU remains unclear. We previously reported that histamine and lipopolysaccharide (LPS) synergistically induced the expression of tissue factor (TF), a trigger for the extrinsic coagulation cascade, in human umbilical vein endothelial cells (HUVEC). Because the elevation of tumor necrosis factor (TNF)-α, interleukin (IL)-33, IL-1β and vascular endothelial growth factor (VEGF) in serum has also been observed in patients with CSU, we examined the effects of LPS, TNF-α, IL-33, IL-1β, VEGF and histamine on TF expression in HUVEC by reverse transcription polymerase chain reaction and flow cytometry, as well as examining the activity that triggers the extrinsic coagulation cascade and induces intercellular gap formation of HUVEC in the presence of plasma by Actochrome® TF activity assay and impedance sensor, respectively. The expression of TF mRNA and surface protein of TF on HUVEC in response to histamine or VEGF were synergistically enhanced by the treatment with LPS, TNF-α, IL-33 or IL-1β. Moreover, the activation of the extrinsic coagulation pathway and intercellular gap formation of HUVEC in response to histamine or VEGF were also synergistically increased in the presence of TNF-α and LPS. Thus, TF expression on vascular endothelial cells was strongly enhanced by co-stimulation with CSU-related molecules in blood. Blocking a common pathway of LPS, TNF-α, IL-33 and IL-1β, and/or that of VEGF and histamine may be an effective therapeutic measure for patients with severe and refractory CSU.

Expression of tissue factor mRNA in thrombosis associated with antiphospholipid syndrome.

Tissue factor (TF) is a procoagulant protein associated with increased risk of thrombotic events in antiphospholipid syndrome (t-APS). The mechanisms by which TF levels are increased in APS have not yet been established. The aim of this study was to investigate whether TF mRNA expression is associated with TF levels and thrombosis in APS. We compared levels of circulating TF and high sensitivity C-reactive protein (hs-CRP) between t-APS and controls (individuals without thrombosis). The association between TF mRNA expression, quantified by real time quantitative polymerase chain reaction, and t-APS was accessed using regression analysis. We included 41 controls and 42t-APS patients, mean age was 41years old (SD 14) in both groups. Hs-CRP and TF levels were higher in t-APS patients (mean hs-CRP levels 0.81mg/dL [SD 1.88] and median TF levels 249.0pg/mL [IQR 138.77-447.61]) as compared to controls (mean hs-CRP levels 0.24mg/dL [SD 0.26] and median TF levels 113.0pg/mL [IQR 81.17-161.53]; P = 0.02 and P < 0.0001, respectively). There was no correlation between TF mRNA expression and TF levels in t-APS (r -0.209, P = 0.19). TF mRNA expression was not associated with t-APS (adjusted OR 1.16; 95%CI 0.72-1.87). Despite circulating TF levels being higher in patients with t-APS than in controls, TF mRNA expression was similar between groups. The results demonstrate that TF mRNA expression is not associated with levels of circulating TF and hypercoagulability in t-APS.

Aryl Hydrocarbon Receptor Activation and Tissue Factor Induction by Fluid Shear Stress and Indoxyl Sulfate in Endothelial Cells.

Endogenous agonists of the transcription factor aryl hydrocarbon receptor (AHR) such as the indolic uremic toxin, indoxyl sulfate (IS), accumulate in patients with chronic kidney disease. AHR activation by indolic toxins has prothrombotic effects on the endothelium, especially via tissue factor (TF) induction. In contrast, physiological AHR activation by laminar shear stress (SS) is atheroprotective. We studied the activation of AHR and the regulation of TF by IS in cultured human umbilical vein endothelial cells subjected to laminar fluid SS (5 dynes/cm2). SS and IS markedly increased the expression of AHR target genes PTGS2 (encoding for COX2), AHRR, CYP1A1, and CYP1B1, as well as F3 (encoding for TF), in an AHR-dependent way. IS amplified SS-induced TF mRNA and protein expression and upregulation of AHR target genes. Interestingly, tyrosine kinase inhibition by genistein decreased SS- but not IS-induced TF expression. Finally, the increase in TF expression induced by laminar SS was not associated with increased TF activity. In contrast, IS increased TF activity, even under antithrombotic SS conditions. In conclusion, IS and SS induce AHR activation and AHR-dependent TF upregulation by different mechanisms. Impairment of the antithrombotic properties of shear stressed endothelium by toxic AHR agonists could favor cardiovascular diseases in CKD.

Anti-\u03b22-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies exert similar pro-thrombotic effects in peripheral blood monocytes and endothelial cells

PurposeThe introduction of the anti-phosphatidylserine/prothrombin (aPS/PT) antibodies among the routinely investigated anti-phospholipid (aPL) antibodies led to an improvement in anti-phospholipid syndrome (APS) laboratory diagnostic performance; however, their pathogenic mechanism is still substantially undefined. To support clinical data and future inclusion as possible new criteria antibodies, we designed a head-to-head study to directly compare the procoagulant effects sustained in vitro by aPS/PT to those sustained by anti-β2-glycoprotein I (aβ2GpI) domain 1-specific antibodies.MethodsBlood donors-derived monocytes and endothelial cells (HUVEC) were stimulated with lipopolysaccharides (LPS) alone or in combination with the IgG fractions isolated from the serum of six APS patients, positive only for aβ2GpI or for aPS/PT antibodies. As control, cells were incubated with LPS plus the IgG isolated from blood donors. Tissue factor (TF) mRNA expression was measured after four hours incubation by real-time PCR. Nitric oxide (NO) levels were measured in cells supernatant after 16 h incubation by colorimetric assay.ResultsaPS/PT and aβ2GpI IgG antibodies fractions showed comparable ability to enhance LPS-induced TF mRNA expression, either in monocytes and in HUVEC. Compared to LPS alone, we found that NO levels are strongly overproduced in HUVEC treated with LPS plus aβ2GpI and aPS/PT IgG fractions.ConclusionsOur data support the significant and independent role of aPS/PT in the pathogenesis of the thrombotic events in APS patients, possibly adding new light to the therapeutic management of cases characterized by the sole presence of aPS/PT IgG antibodies.

The impact of glucocorticoid on coagulation of smoke inhalation induced lung injury

Objective To explore the impact of glucocorticoid on coagulation through administrating on rats with smoke inhalation. Methods Totally 150 male S-D rats were randomly (random number) divided into 5 groups: control group (ambient air inhalation), smoke group (smoke inhalation for 30 min), smoke+high dosage methyl prednisolone group(MP 40 mg/kg, intraperitoneal injection, s+HMP group), smoke+medium dosage MP (4 mg/kg) group (s+MMP group), smoke+low dosage MP (0.4 mg/kg) group (s+LMP group) (all n=30). Survival rates were calculated 24 h after smoke inhalation. Lung tissues were collected for histopathology and wet to dry (W/D) ratio. Arterial blood was collected for blood gas test. Coagulation factors in lung and plasma were tested. Results Survival rates of three MP groups were markedly improved compared with the smoke group (all P<0.05), and was significantly higher in the medium dosage group(85.17%) than those in the low and high dosage groups (65.73% and 60.07%, all P<0.05). The W/D ratio and blood gas test were markedly improved in the high and medium groups (all P<0.05). Tissue factor (TF) and thrombin-antithrombin complex (TAT-c) in bronchoalveolar lavage fluid (BALF) increased dramatically after SI (P<0.01, P=0.005) with a remarkable drop of factor Ⅱ (FⅡ) (P=0.007), all of which were attenuated by MP with dosage dependence. The mRNA expression of TF increased dramatically after SI and recovered significantly with MP administration, while the expression of thrombomodulin (TM) recovered in the opposite direction with MP, all of which were in a dosage dependent manner. TF, fibrinogen (FIB), TAT-c increased significantly in plasma after smoke inhalation (P<0.01, P=0.027, P=0.005). FⅧ% increased with MP administration and TF was raised by high dosage MP compared with the smoke group. FIB and TAT-c were decreased in all MP groups, which were significant higher in the high and middle dosage groups. The change of TM and endothelial cell protein C receptor (EPCR) in circulation were similar with FIB or TAT-c with or without MP. Protein C (PC%) and antithrombin (ATⅢ%) dropped dramatically after SI, high and middle dosages of MP could restore the activity significantly, while low dosage would restore ATⅢ% but not PC%. Conclusions Glucocorticoid can significantly improve local and systemical coagulation disorder caused by smoke inhalation, and high- and medium-dosage hormones are effective. The regulation of hormones on the coagulation system is an important mechanism in the treatment of smoke inhalation induced lung injury. Key words: Smoke inhalation; Acute lung injury; Coagulation; Glucocorticoid; Thrombomodulin