Overexpression Of Tissue Factor Research Articles

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

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

An MRI Radiomics Approach to Predict the Hypercoagulable Status of Gliomas.

Venous thromboembolic events are frequent complications of Glioblastoma Multiforme (GBM) and low-grade gliomas (LGGs). The overexpression of tissue factor (TF) plays an essential role in the local hypercoagulable phenotype that underlies these complications. Our aim was to build an MRI radiomics model for the non-invasive exploration of the hypercoagulable status of LGG/GBM. Radiogenomics data from The Cancer Genome Atlas (TCGA) and REMBRANDT (Repository for molecular BRAin Neoplasia DaTa) cohorts were used. A logistic regression model (Radscore) was built in order to identify the top 20% TF-expressing tumors, considered to be at high thromboembolic risk. The most contributive MRI radiomics features from LGG/GBM linked to high TF were identified in TCGA using Least Absolute Shrinkage and Selection Operator (LASSO) regression. A logistic regression model was built, whose performance was analyzed with ROC in the TCGA/training and REMBRANDT/validation cohorts: AUC = 0.87 [CI95: 0.81-0.94, p < 0.0001] and AUC = 0.78 [CI95: 0.56-1.00, p = 0.02], respectively. In agreement with the key role of the coagulation cascade in gliomas, LGG patients with a high Radscore had lower overall and disease-free survival. The Radscore was linked to the presence of specific genomic alterations, the composition of the tumor coagulome and the tumor immune infiltrate. Our findings suggest that a non-invasive assessment of the hypercoagulable status of LGG/GBM is possible with MRI radiomics.

Tissue factor overexpression promotes resistance to KRAS-G12C inhibition in non-small cell lung cancer

The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.

From Infection to Death: An Overview of the Pathogenesis of Visceral Leishmaniasis.

Kala-azar, also known as visceral leishmaniasis (VL), is a disease caused by Leishmania infantum and L. donovani. Patients experience symptoms such as fever, weight loss, paleness, and enlarged liver and spleen. The disease also affects immunosuppressed individuals and has an overall mortality rate of up to 10%. This overview explores the literature on the pathogenesis of preclinical and clinical stages, including studies in vitro and in animal models, as well as complications and death. Asymptomatic infection can result in long-lasting immunity. VL develops in a minority of infected individuals when parasites overcome host defenses and multiply in tissues such as the spleen, liver, and bone marrow. Hepatosplenomegaly occurs due to hyperplasia, resulting from parasite proliferation. A systemic inflammation mediated by cytokines develops, triggering acute phase reactants from the liver. These cytokines can reach the brain, causing fever, cachexia and vomiting. Similar to sepsis, disseminated intravascular coagulation (DIC) occurs due to tissue factor overexpression. Anemia, hypergammaglobulinemia, and edema result from the acute phase response. A regulatory response and lymphocyte depletion increase the risk of bacterial superinfections, which, combined with DIC, are thought to cause death. Our understanding of VL's pathogenesis is limited, and further research is needed to elucidate the preclinical events and clinical manifestations in humans.

Tissue factor overexpression in triple-negative breast cancer promotes immune evasion by impeding T-cell infiltration and effector function

Triple-negative breast cancer (TNBC) remains a most deadly human malignancy with limited response to chemotherapy, targeted therapy and immunotherapy. Tumor immunoenvironment plays an increasingly important role in therapy outcome. Tissue factor (TF) is the target of the FDA-approved ADC Tivdak. HuSC1-39 is the parent antibody of MRG004A, a clinical stage TF-ADC (NCT04843709). Here, we employed HuSC1-39 (termed “anti-TF”) to investigate the role of TF in regulating immune-tolerance in TNBC. We found that patients with aberrant TF expression had a poor prognosis and low immune effector cell infiltration, characterizing as “cold tumor”. In the 4T1 TNBC syngeneic mouse model, knockout of tumor cell TF inhibited tumor growth and increased tumor infiltration of effector T cell, which was not dependent on the clotting inhibition. In an immune-reconstituted M-NSG mouse model of TNBC, anti-TF inhibited tumor growth, which was further enhanced by a dual-targeting anti-TF&TGFβR fusion protein. There were diminished P-AKT and P-ERK signaling and profound tumor cell death in treated tumors. Transcriptome analyses and immunohistochemistry revealed a dramatically improved tumor immunoenvironment including the increase of effector T cells, decrease of Treg cells and the transformation of tumor into “hot tumor”. Moreover, employing qPCR analysis and T cell culture, we further demonstrated that TF expression in tumor cells is sufficient to block the synthesis and secretion of T cell-recruiting chemokine CXCL9/10/11. Treatment of TF-high TNBC cells with anti-TF or TF-knockout all stimulated CXCL9/10/11 production, promoted T cell migration and effector function. Thus, we have identified a new mechanism of TF in TNBC tumor progression and therapy resistance.

Inflammasome activation mediated by oxidised low-density lipoprotein in patients with sleep apnoea and early subclinical atherosclerosis.

Atherosclerosis is a common comorbidity of obstructive sleep apnoea (OSA) patients, caused by the interaction of dyslipidaemia and systemic inflammation. The OSA pro-inflammatory response is mediated by NLRP3 inflammasome activation, which requires a priming signal mediated by intermittent hypoxia (IH) and an activation signal provided by soluble stimulus present in plasma. Our objectives were to study oxidised low-density lipoprotein (oxLDL) expression in OSA patients with or without early subclinical atherosclerosis (eSA) as well as its contribution to NLRP3 activation and tissue factor (TF) release. We analysed oxLDL, key components of the NLRP3 inflammasome cascade and TF in plasma and monocytes from OSA patients and non-apnoeic subjects, with or without eSA as determined by increased carotid intima-media thickness without the appearance of atherosclerotic plaques. The oxLDL contribution to NLRP3 inflammasome activation was assessed using in vitro models. High levels of oxLDL were identified in plasma from OSA patients, particularly in those with eSA, as well as an overexpression of NLRP3 cascade components and TF. Furthermore, in vitro models showed that both oxLDL and plasma from OSA patients with eSA act synergistically with IH as a priming and activation signal of NLRP3 that enhances the inflammatory response, pyroptosis and TF release. OSA patients with eSA exhibit NLRP3 activation by IH and the presence of oxLDL capable of releasing TF, constituting a pathway for the interaction between dyslipidaemia and systemic inflammation in the development of atherosclerotic lesions.

HMG-CoA Reductase Inhibitors and the Modification of Risk for Disseminated Intravascular Coagulation in Patients with Sepsis

Background: Disseminated intravascular coagulation (DIC) is a highly morbid syndrome characterized by uncontrolled activation of coagulation leading to the consumption of clotting factors and platelets and increasing risk for life-threatening hemorrhage. Sepsis is one of the underlying disorders that can provoke DIC. The mechanism of DIC is thought to involve tissue factor overexpression, defective anticoagulation pathways, endothelial and monocytic cytokines, oxidation, and calpains. HMG-CoA reductase inhibitors (statins) have demonstrated anti-inflammatory and mild anti-coagulant properties effecting the pathways proposed to mediate DIC. Objectives: The aim of this study is to evaluate whether septic patients who take statin medications are at reduced risk for the development of DIC. Methods: In a retrospective cohort study, 120,397 critically ill patients with sepsis with and without shock were identified within a three year period from 2019 to 2021. Patients were stratified by whether they were medicated with low, medium, high intensity statins or none. Odds ratios for the development of DIC were calculated. Results: Overall, 2795 patients would develop DIC. The odds ratio for the development of DIC were .700 (95% CI .617-.794), .615 (95% CI .529-.714), and .901 (95% CI .635-1.277) when comparing high, moderate, and low intensity statin therapy to no statin therapy. Conclusion: The use of moderate and high intensity statins correlated with a significantly reduced association between critically ill septic patients and the development of DIC. More research is needed to clarify the potential for this class of medication to be protective against DIC. This research was supported (in whole or in part) by HCA Healthcare and/or an HCA Healthcare affiliated entity. The views expressed in this publication represent those of the author(s) and do not necessarily represent the official views of HCA Healthcare or any of its affiliated entities.

Ebola virus infection induces a delayed type I IFN response in bystander cells and the shutdown of key liver genes in human iPSC-derived hepatocytes.

SummaryLiver damage and an exacerbated inflammatory response are hallmarks of Ebola virus (EBOV) infection. Little is known about the intrinsic response to infection in human hepatocytes and their contribution to inflammation. Here, we present an induced pluripotent stem cell (iPSC)-derived hepatocyte-like cell (HLC) platform to define the hepato-intrinsic response to EBOV infection. We used this platform to show robust EBOV infection, with characteristic ultrastructural changes and evidence for viral replication. Transcriptomics analysis revealed a delayed response with minimal early transcriptomic changes, followed by a general downregulation of hepatic function and upregulation of interferon signaling, providing a potential mechanism by which hepatocytes participate in disease severity and liver damage. Using RNA-fluorescence in situ hybridization (FISH), we showed that IFNB1 and CXCL10 were mainly expressed in non-infected bystander cells. We did not observe an inflammatory signature during infection. In conclusion, iPSC-HLCs are an immune competent platform to study responses to EBOV infection.

Tanshinone IIA Inhibits Tissue Factor Expression Induced by Thrombin in Human Umbilical Vein Endothelial Cells via PAR-1 and p38 MAPK Signaling Pathway

Background: The potential signaling pathway of TSA suppressing TF expression induced by thrombin was unknown. Thus, the transcription of TF in HUVECs and the expressions of DCF, phospho-p38 MAPK, NADPH oxidase 4, PAR-1, and NF-κB were detected in our study. Methods: HUVECs were randomly divided into control group, thrombin-treated group (with 5 U/mL of thrombin), and 4 TSA-treated groups (with 5 U/mL of thrombin plus TSA with 4 different concentrations of 1 μg/mL, 10 μg/mL, 100 μg/mL, and 1 mg/mL, respectively). Results: After incubation with thrombin for 6 h at 37°C, the results showed increased TF mRNA, TF procoagulant activity, and antigen of TF in HUVECs of thrombin-treated group (p < 0.01); however, they were restored by TSA in a dose-dependent manner (p < 0.01). In addition, reactive oxygen species (ROS), phospho-p38 MAPK, NADPH oxidase 4, NF-κB, and PAR-1 expressed more intensively, and phosphorylated Akt decreased obviously in HUVECs after thrombin stimulation (p < 0.01); however, they were reversed to different extents by TSA in a dose-dependent manner (p < 0.01). Conclusions: Study suggests that TSA inhibits TF expression induced by thrombin in cultured HUVECs, and the potential signaling pathway of which is TSA interrupts the activation of PAR-1 and NADPH oxidase as well as derivative ROS generation, thereafter suppresses the activation of NF-κB, the upstream signal molecule of TF, via hampering phosphorylation of p38 MAPK and dephosphorylation of Akt, and finally inhibits thrombin-induced TF overexpression.

Arterial and venous involvement in Behçet's syndrome: a narrative review.

Behçet syndrome (BS) is a unique type of vasculitis that affects veins and arteries of all sizes, leading to recurrent vascular events, mostly venous thrombosis. The prevalence of venous thromboembolism in BS patients ranges between 15 and 40%. Thrombosis is usually an early manifestation leading to diagnosis of BS in up to 40% of patients. BS is per se a model of inflammation-induced thrombosis. The primary autoimmune response activates lymphocytes that in turn produce a cytokine cascade that activates neutrophils, which modify the secondary structure of fibrinogen making it less susceptible to plasmin-induced lysis. This leads to endothelial dysfunction, platelet activation and overexpression of tissue factor leading to inflammatory thrombi, usually attached to the wall. The pathogenesis of thrombosis is especially relevant to direct the specific treatment, that is based on immunosuppression rather than anticoagulation. Superficial vein thrombosis (SVT) and deep vein thrombosis (DVT) are the most common form of thrombosis in BS, but thrombosis in atypical sites (cava vein, suprahepatic veins, intracardiac thrombus) and arterial involvement can also occur. We assessed the latest update of the European League Against Rheumatism recommendations for the management of BS. Vascular Behçet treatment is usually based of immunosuppressants, and the role of anticoagulation remains controversial. The use of interventional and surgical procedures should be carefully evaluated, due to the risk of triggering a vascular pathergy phenomenon.

Modulation of Cellular NAD+ Attenuates Cancer-Associated Hypercoagulability and Thrombosis via the Inhibition of Tissue Factor and Formation of Neutrophil Extracellular Traps.

Cancer-associated thrombosis is the second-leading cause of mortality in patients with cancer and presents a poor prognosis, with a lack of effective treatment strategies. NAD(P)H quinone oxidoreductase 1 (NQO1) increases the cellular nicotinamide adenine dinucleotide (NAD+) levels by accelerating the oxidation of NADH to NAD+, thus playing important roles in cellular homeostasis, energy metabolism, and inflammatory responses. Using a murine orthotopic 4T1 breast cancer model, in which multiple thrombi are generated in the lungs at the late stage of cancer development, we investigated the effects of regulating the cellular NAD+ levels on cancer-associated thrombosis. In this study, we show that dunnione (a strong substrate of NQO1) attenuates the prothrombotic state and lung thrombosis in tumor-bearing mice by inhibiting the expression of tissue factor and formation of neutrophil extracellular traps (NETs). Dunnione increases the cellular NAD+ levels in lung tissues of tumor-bearing mice to restore the declining sirtuin 1 (SIRT1) activity, thus deacetylating nuclear factor-kappa B (NF-κB) and preventing the overexpression of tissue factor in bronchial epithelial and vascular endothelial cells. In addition, we demonstrated that dunnione abolishes the ability of neutrophils to generate NETs by suppressing histone acetylation and NADPH oxidase (NOX) activity. Overall, our results reveal that the regulation of cellular NAD+ levels by pharmacological agents may inhibit pulmonary embolism in tumor-bearing mice, which may potentially be used as a viable therapeutic approach for the treatment of cancer-associated thrombosis.

Bile acid-induced tissue factor activity in hepatocytes correlates with activation of farnesoid X receptor

Bile acids (BA) have been found to promote coagulation by increasing tissue factor (TF) activity. The contribution of elevated BA levels and cholestasis to TF decryption within the liver parenchyma and the role of farnesoid X receptor (FXR) in this process remain unclear. We investigated the effects of BA on TF activity and thrombin generation in hepatocytes and correlated these effects with activation of FXR-dependent signaling and apoptosis. HepG2 cells and primary hepatocytes were incubated with chenodeoxycholic acid (CDCA), glycochenodeoxycholic acid (GCDCA), ursodeoxycholic acid (UCDA), or the synthetic FXR agonist GW4064 for 24 h. MTT tests demonstrated cell viability throughout experiments. TF activity was tested via factor Xa generation and thrombin generation was measured by calibrated automated thrombography. Increased TF activity alongside enhanced thrombin generation was observed with CDCA and GW4064 but not with GCDCA and UDCA. TF activity was substantially reduced when FXR activation was blocked with the antagonist DY 268. Quantitative polymerase chain reaction revealed upregulation of FXR target genes only by CDCA and GW4064. Western blot analysis and fluorescence microscopy showed no TF overexpression arguing for TF decryption. Caspase 3 activity measurements and flow cytometric analysis of Annexin V binding showed no signs of apoptosis. Long-term exposure of hepatocytes to nontoxic BA may cause intracellular FXR overstimulation, triggering TF decryption irrespective of the amphiphilic properties of BA. The effect of BA on TF activation correlates with the molecule's ability to enter the cells and activate FXR. TF decryption occurs independently of apoptotic mechanisms.The potential of various bile acids to induce procoagulant tissue factor (TF) activity in viable HepG2 cells and primary human hepatocytes was investigated. Increased TF activity correlated with the molecules' ability to enter the cells and activate the farnesoid X receptor (FXR) suggesting a crucial role of FXR in bile acid-mediated TF activity within the liver parenchyma.

Bleeding and Thrombosis: Insights into Pathophysiology of Bothrops Venom-Related Hemostasis Disorders.

Toxins from Bothrops venoms targeting hemostasis are responsible for a broad range of clinical and biological syndromes including local and systemic bleeding, incoagulability, thrombotic microangiopathy and macrothrombosis. Beyond hemostais disorders, toxins are also involved in the pathogenesis of edema and in most complications such as hypovolemia, cardiovascular collapse, acute kidney injury, myonecrosis, compartmental syndrome and superinfection. These toxins can be classified as enzymatic proteins (snake venom metalloproteinases, snake venom serine proteases, phospholipases A2 and L-amino acid oxidases) and non-enzymatic proteins (desintegrins and C-type lectin proteins). Bleeding is due to a multifocal toxicity targeting vessels, platelets and coagulation factors. Vessel damage due to the degradation of basement membrane and the subsequent disruption of endothelial cell integrity under hydrostatic pressure and tangential shear stress is primarily responsible for bleeding. Hemorrhage is promoted by thrombocytopenia, platelet hypoaggregation, consumption coagulopathy and fibrin(ogen)olysis. Onset of thrombotic microangiopathy is probably due to the switch of endothelium to a prothrombotic phenotype with overexpression of tissue factor and other pro-aggregating biomarkers in association with activation of platelets and coagulation. Thrombosis involving large-caliber vessels in B. lanceolatus envenomation remains a unique entity, which exact pathophysiology remains poorly understood.

Effect of heparanase inhibitor on tissue factor overexpression in platelets and endothelial cells induced by anti‐β2‐GPI antibodies

BackgroundAnti‐phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity associated with the presence of “anti‐phospholipid antibodies.” Thrombosis may be the result of a hypercoagulable state related to activation of endothelial cells and platelets by anti‐β2‐glycoprotein I (β2‐GPI) antibodies. Anti‐β2‐GPI antibodies induce a proinflammatory and procoagulant phenotype in these cells that, after activation, express tissue factor (TF), the major initiator of the clotting cascade, playing a role in thrombotic manifestations. Moreover, TF expression may also be induced by heparanase, an endo‐β‐D‐glucuronidase, that generates heparan sulfate fragments, regulating inflammatory responses. ObjectivesIn this study we analyzed, in human platelets and endothelial cells, the effect of a new symmetrical 2‐aminophenyl‐benzazolyl‐5‐acetate derivative (RDS3337), able to inhibit heparanase activity, on signal transduction pathways leading to TF expression triggered by anti‐β2‐GPI. MethodsPlatelets and endothelial cells were incubated with affinity purified anti‐β2‐GPI after pretreatment with RDS3337. Cell lysates were analyzed for phospho‐interleukin‐1 receptor‐associated kinase 1 (IRAK1), phospho‐p65 nuclear factor kappa B (NF‐κB) and TF by western blot. In addition, platelet activation and secretion by ATP release dosage were evaluated. ResultsIRAK phosphorylation and consequent NF‐κB activation, as well as TF expression triggered by anti‐β2‐GPI treatment were significantly prevented by previous pretreatment with RDS3337. In the same vein, pretreatment with RDS3337 prevented platelet aggregation and ATP release triggered by anti‐β2‐GPI antibodies. ConclusionThese findings support the view of heparanase involvement in a prothrombotic state related to APS syndrome, suggesting a novel target to regulate overexpression of procoagulant protein(s).

Loss of RAR-α and RXR-α and enhanced caspase-3-dependent apoptosis in N-acetyl-p-aminophenol-induced liver injury in mice is tissue factor dependent.

Tissue factor (TF) activates the coagulation system and has an important role in the pathogenesis of various diseases. Our previous study stated that retinoid receptors (RAR-α and RXR-α) are released as a lipid droplet in monocrotaline/lipopolysaccharide-induced idiosyncratic liver toxicity in mice. Herein, the interdependence between the release of retinoid receptors RAR-α and RXR-α and TF in N-acetyl-p-aminophenol (APAP)-induced mice liver toxicity, is investigated. Serum alanine transaminase (ALT) level, platelet and white blood cells (WBCs) counts, protein expression of fibrin, TF, cyclin D1 and cleaved caspase-3 in liver tissues are analyzed. In addition, histopathological evaluation and survival study are also performed. The results indicate that using of TF-antisense (TF-AS) deoxyoligonucleotide (ODN) injection (6 mg/kg), to block TF protein synthesis, significantly restores the elevated level of ALT and WBCs and corrects thrombocytopenia in mice injected with APAP. TF-AS prevents the peri-central overexpression of liver TF, fibrin, cyclin D1 and cleaved caspase-3. The release of RXR-α and RAR-α droplets, in APAP treated sections, is inhibited upon treatment with TF-AS. In conclusion, the above findings designate that the released RXR-α and RAR-α in APAP liver toxicity is TF dependent. Additionally, the enhancement of cyclin D1 to caspase-3-dependent apoptosis can be prevented by blocking of TF protein synthesis.

Role of tissue-factor bearing extracellular vesicles released from ovarian cancer cells in platelet aggregation in vitro and venous thrombosis in mice

ObjectiveVenous thromboembolism (VTE) contributes to morbidity and mortality in women with ovarian cancer. Underlying mechanisms of venous thrombosis in ovarian cancer are not well-understood. The aim of this study was to identify the potential role of tissue factor (TF)-bearing extracellular vesicles (EVs) originated from cancer cells in venous thrombosis in ovarian cancer models. MethodsWe examined the expression of TF on EVs generated by ovarian cancer cells and the effect of TF-positive EVs on platelet aggregation. Furthermore, we performed TF-knockdown or induced TF-overexpression in ovarian cancer cell lines and examined the effects of EVs obtained from these cells on platelet aggregation. We examined the effect of TF-bearing EVs originated from ovarian cancer cells on venous thrombosis in a mouse model of inferior vena cava (IVC) stenosis. ResultsTF was expressed in several ovarian cancer cell lines. EVs derived from ovarian cancer cell lines expressing TF promoted platelet aggregation. TF-knockdown in TF-high CAOV3 and OVCAR8 ovarian cancer cells delayed platelet aggregation induced by their EVs in vitro. Conversely, TF overexpression in TF-low A2780 and HeyA8 cells shortened platelet aggregation time induced by their EVs. EVs from TF-overexpressing A2780 ​cells enhanced thrombosis formation in the IVC stenosis model and resulted in a larger clot burden as compared to EVs from A2780 control cells. ConclusionsTF expression in ovarian cancer cell-derived EVs promoted platelet aggregation and thrombosis in preclinical models. These findings may have implications for reducing VTE rates in women with ovarian cancer.

Role of Tissue Factor in the Pathogenesis of COVID-19 and the Possible Ways to Inhibit It.

COVID-19 (Coronavirus Disease 2019) is a highly contagious infection and associated with high mortality rates, primarily in elderly; patients with heart failure; high blood pressure; diabetes mellitus; and those who are smokers. These conditions are associated to increase in the level of the pulmonary epithelium expression of angiotensin-converting enzyme 2 (ACE-2), which is a recognized receptor of the S protein of the causative agent SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). Severe cases are manifested by parenchymal lung involvement with a significant inflammatory response and the development of microvascular thrombosis. Several factors have been involved in developing this prothrombotic state, including the inflammatory reaction itself with the participation of proinflammatory cytokines, endothelial dysfunction/endotheliitis, the presence of antiphospholipid antibodies, and possibly the tissue factor (TF) overexpression. ARS-Cov-19 ACE-2 down-regulation has been associated with an increase in angiotensin 2 (AT2). The action of proinflammatory cytokines, the increase in AT2 and the presence of antiphospholipid antibodies are known factors for TF activation and overexpression. It is very likely that the overexpression of TF in COVID-19 may be related to the pathogenesis of the disease, hence the importance of knowing the aspects related to this protein and the therapeutic strategies that can be derived. Different therapeutic strategies are being built to curb the expression of TF as a therapeutic target for various prothrombotic events; therefore, analyzing this treatment strategy for COVID-19-associated coagulopathy is rational. Medications such as celecoxib, cyclosporine or colchicine can impact on COVID-19, in addition to its anti-inflammatory effect, through inhibition of TF.

The crucial roles of coagulation factors in inducing brown adipose tissue dysfunction and systemic metabolic disorder in obesity

Abstract The prevalence of obesity is increasing worldwide. Obese individuals are predisposed to cardio-metabolic disorders. Brown adipose tissue (BAT) is an active metabolic organ abundant with mitochondria, and studies suggest a potential role of BAT in the maintenance of metabolic health in rodents and humans. Metabolic stress causes BAT dysfunction, but the underlying mechanisms are largely unknown. Coagulation factor Xa (FXa) is critically involved in a coagulation cascade, and it is also known to mediate biological effects by the activation of protease-activated receptor (PAR)-signaling. Accumulating evidence shows that PAR1 contributes to tissue remodeling in cardiovascular system. Analyzing deposited microarray data, we found transcripts for coagulation factors including factor VII (F7), factor X (F10), and PAR1 receptor were increased in BAT from obese mice. Here we show a previously unknown role of FXa-PAR signaling in promoting BAT dysfunction and systemic metabolic disorder in a murine dietary obese model. Imposing a high fat diet (HFD) on C57BL/6NCr mice led to a marked increase in tissue factor (TF), coagulation factor VII and FXa in BAT. TF-FVIIa (activated form of FVII)-FXa complex is known to activate PAR1, and we found a significant increase in PAR1 expression in BAT upon metabolic stress. Administration of a FXa inhibitor ameliorated BAT whitening, improved thermogenic response and systemic glucose intolerance upon dietary obesity. Fxa inhibition reduced reactive oxygen species (ROS) level in BAT. In contrast, administration of warfarin did not show any phenotype in BAT. BAT specific TF and PAR1 over-expression model showed significant whitening of this tissue, which was associated with systemic glucose intolerance. We generated BAT specific PAR1 KO mice. BAT-PAR1 KO mice exhibited re-browning of BAT along with reduced ROS level in this tissue. In BAT-PAR1 KO mice, glucose intolerance and thermogenic response under a metabolically stressed condition were ameliorated. In differentiated brown adipocytes, FXa markedly increased mitochondrial ROS and reduced mitochondrial membrane potential. Inhibition of PAR1 ameliorated FXa-induced mitochondrial ROS production and reduction in membrane potential. We also found that plasma FXa level did not increase in obese mice as well as in obese individuals. These results suggest the previously unknown role of coagulation systems in promoting BAT dysfunction, leading to systemic metabolic disorders. Maintenance of BAT homeostasis through the suppression of FXa-PAR1 signaling would become a new therapeutic target for obesity and diabetes. Funding Acknowledgement Type of funding source: None

NRF-1 and HIF-1α contribute to modulation of human VDAC1 gene promoter during starvation and hypoxia in HeLa cells

VDAC (Voltage Dependent Anion Channel) is a family of pore forming protein located in the outer mitochondrial membrane. Its channel property ensures metabolites exchange between mitochondria and the rest of the cell resulting in metabolism and bioenergetics regulation, and in cell death and life switch. VDAC1 is the best characterized and most abundant isoform, and is involved in many pathologies, as cancer or neurodegenerative diseases. However, little information is available about its gene expression regulation in normal and/or pathological conditions. In this work, we explored VDAC1 gene expression regulation in normal conditions and in the contest of some metabolic and energetic mitochondrial dysfunction and cell stress as example. The core of the putative promoter region was characterized in terms of transcription factors responsive elements both by bioinformatic studies and promoter activity experiments. In particular, we found an abundant presence of NRF-1 sites, together with other transcription factors binding sites involved in cell growth, proliferation, development, and we studied their prevalence in gene activity. Furthermore, upon depletion of nutrients or controlled hypoxia, as detected in various pathologies, we found that VDAC1 transcripts levels were significantly increased in a time related manner. VDAC1 promoter activity was also validated by gene reporter assays. According to PCR real-time experiments, it was confirmed that VDAC1 promoter activity is further stimulated when cells are exposed to stress. A bioinformatic survey suggested HIF-1α, besides NRF-1, as a most active TFBS. Their validation was obtained by TFBS mutagenesis and TF overexpression experiments. In conclusion, we experimentally demonstrated the involvement of both NRF-1 and HIF-1α in the regulation of VDAC1 promoter activation at basal level and in some peculiar cell stress conditions.

Potential role for tissue factor in the pathogenesis of hypercoagulability associated with in COVID-19.

In December 2019, a new and highly contagious infectious disease emerged in Wuhan, China. The etiologic agent was identified as a novel coronavirus, now known as Severe Acute Syndrome Coronavirus-2 (SARS-CoV-2). Recent research has revealed that virus entry takes place upon the union of the virus S surface protein with the type I transmembrane metallo-carboxypeptidase, angiotensin converting enzyme 2 (ACE-2) identified on epithelial cells of the host respiratory tract. Virus triggers the synthesis and release of pro-inflammatory cytokines, including IL-6 and TNF-α and also promotes downregulation of ACE-2, which promotes a concomitant increase in levels of angiotensin II (AT-II). Both TNF-α and AT-II have been implicated in promoting overexpression of tissue factor (TF) in platelets and macrophages. Additionally, the generation of antiphospholipid antibodies associated with COVID-19 may also promote an increase in TF. TF may be a critical mediator associated with the development of thrombotic phenomena in COVID-19, and should be a target for future study.