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

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

Download Full-text

Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Blood ◽

10.1182/blood-2012-06-437772 ◽

2012 ◽

Vol 120 (10) ◽

pp. 2133-2143 ◽

Cited By ~ 143

Author(s):

Roxane Darbousset ◽

Grace M. Thomas ◽

Soraya Mezouar ◽

Corinne Frère ◽

Rénaté Bonier ◽

...

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Blood Coagulation ◽

Thrombus Formation ◽

Coagulation Cascade ◽

Factor Xii ◽

Long Time ◽

Platelet Accumulation

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

Download Full-text

Fluid Shear Stress Attenuates Tumor Necrosis Factor-α–Induced Tissue Factor Expression in Cultured Human Endothelial Cells

Blood ◽

10.1182/blood.v91.11.4164 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4164-4172 ◽

Cited By ~ 26

Author(s):

Yutaka Matsumoto ◽

Yohko Kawai ◽

Kiyoaki Watanabe ◽

Kazuo Sakai ◽

Mitsuru Murata ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Tumor Necrosis Factor ◽

Tissue Factor ◽

Shear Force ◽

Tumor Necrosis ◽

Shear Stresses ◽

Dependent Manner ◽

Tnf Α ◽

Necrosis Factor

Abstract Hemodynamic forces modulate various endothelial cell functions under gene regulation. Previously, we have shown that fibrinolytic activity of endothelial cells is enhanced by the synergistic effects of shear stress and cytokines. In this study, we investigated the effect of shear stress on tumor necrosis factor (TNF)-α–induced tissue factor (TF) expression in cultured human umbilical vein endothelial cells (HUVECs), using a modified cone-plate viscometer. Shear stresses at physiological levels reduced TNF-α (100 U/mL)–induced TF expression at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner, whereas shear stress itself did not induce TF expression in HUVECs. TF expressed on the cell surfaces measured by flow cytometry using an anti-TF monoclonal antibody (HTF-K180) was also decreased to one third by shear force applied at 18 dynes/cm2 for 15 hours before and 6 hours after TNF-α stimulation. Furthermore, functional activity of TF, as assessed by the activation of factor X in the presence of FVIIa and Ca2+, was also decreased by shear application. However, the stability of TF mRNA was not decreased in the presence of shear stress. These results suggest that shear force acts as an important regulator of TF expression in endothelium at the transcriptional level.

Download Full-text

Fluid Shear Stress Attenuates Tumor Necrosis Factor-α–Induced Tissue Factor Expression in Cultured Human Endothelial Cells

Blood ◽

10.1182/blood.v91.11.4164.411k29_4164_4172 ◽

1998 ◽

Vol 91 (11) ◽

pp. 4164-4172

Author(s):

Yutaka Matsumoto ◽

Yohko Kawai ◽

Kiyoaki Watanabe ◽

Kazuo Sakai ◽

Mitsuru Murata ◽

...

Keyword(s):

Endothelial Cells ◽

Shear Stress ◽

Tumor Necrosis Factor ◽

Tissue Factor ◽

Shear Force ◽

Tumor Necrosis ◽

Shear Stresses ◽

Dependent Manner ◽

Tnf Α ◽

Necrosis Factor

Hemodynamic forces modulate various endothelial cell functions under gene regulation. Previously, we have shown that fibrinolytic activity of endothelial cells is enhanced by the synergistic effects of shear stress and cytokines. In this study, we investigated the effect of shear stress on tumor necrosis factor (TNF)-α–induced tissue factor (TF) expression in cultured human umbilical vein endothelial cells (HUVECs), using a modified cone-plate viscometer. Shear stresses at physiological levels reduced TNF-α (100 U/mL)–induced TF expression at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner, whereas shear stress itself did not induce TF expression in HUVECs. TF expressed on the cell surfaces measured by flow cytometry using an anti-TF monoclonal antibody (HTF-K180) was also decreased to one third by shear force applied at 18 dynes/cm2 for 15 hours before and 6 hours after TNF-α stimulation. Furthermore, functional activity of TF, as assessed by the activation of factor X in the presence of FVIIa and Ca2+, was also decreased by shear application. However, the stability of TF mRNA was not decreased in the presence of shear stress. These results suggest that shear force acts as an important regulator of TF expression in endothelium at the transcriptional level.

Download Full-text

Plasmin-induced expression of cytokines and tissue factor in human monocytes involves AP-1 and IKKβ-mediated NF-κB activation

Blood ◽

10.1182/blood.v97.12.3941 ◽

2001 ◽

Vol 97 (12) ◽

pp. 3941-3950 ◽

Cited By ~ 102

Author(s):

Tatiana Syrovets ◽

Marina Jendrach ◽

Angela Rohwedder ◽

Almut Schüle ◽

Thomas Simmet

Keyword(s):

Tissue Factor ◽

Nuclear Translocation ◽

Lipid Mediator ◽

Mrna Levels ◽

Dependent Manner ◽

Human Monocytes ◽

Mobility Shift ◽

Concentration Dependent Manner ◽

Iκb Kinase ◽

Tnf Α

It was previously shown that plasmin activates human peripheral monocytes in terms of lipid mediator release and chemotactic migration. Here it is demonstrated that plasmin induces proinflammatory cytokine release and tissue factor (TF) expression by monocytes. Plasmin 0.043 to 1.43 CTA U/mL, but not active site-blocked plasmin, triggered concentration-dependent expression of mRNA for interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), and TF with maximum responses after 4 hours. Plasmin-mediated mRNA expression was inhibited in a concentration-dependent manner by the lysine analoguetrans-4-(aminomethyl)cyclohexane-1-carboxylic acid (t-AMCA). Increases in mRNA levels were followed by concentration- and time-dependent release of IL-1α, IL-1β and TNF-α and by TF expression on monocyte surfaces. Neither cytokines nor TF could be detected when monocytes were preincubated with actinomycin D or cycloheximide. Electrophoretic mobility shift assays indicated plasmin-induced activation of NF-κB; DNA-binding complexes were composed of p50, p65, and c-Rel, as shown by supershift experiments. Nuclear translocation of NF-κB/Rel proteins coincided with IκBα degradation. At variance with endotoxic lipopolysaccharide, plasmin elicited the rapid degradation of another cytoplasmic NF-κB inhibitor, p105. Proteolysis of NF-κB inhibitors was apparently due to transient activation of IκB kinase (IKK) β that reached maximum activity at 1 hour after plasmin stimulation. In addition, AP-1 binding was increased in plasmin-treated monocytes, with most complexes composed of JunD, c-Fos, and FosB. These findings further substantiate the role of plasmin as a proinflammatory activator of human monocytes and reveal an important new link between the plasminogen-plasmin system and inflammation.

Download Full-text

The Modulation of Nrf-2/HO-1 Signaling Axis by Carthamus tinctorius L. Alleviates Vascular Inflammation in Human Umbilical Vein Endothelial Cells

Plants ◽

10.3390/plants10122795 ◽

2021 ◽

Vol 10 (12) ◽

pp. 2795

Author(s):

Yun Jung Lee ◽

Yong Pyo Lee ◽

Chang Seob Seo ◽

Eun Sik Choi ◽

Byung Hyuk Han ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Adhesion Molecule ◽

Nuclear Translocation ◽

Vascular Inflammation ◽

Vascular Complications ◽

Carthamus Tinctorius ◽

Dependent Manner ◽

Tnf Α ◽

Signaling Axis

Carthamus tinctorius L., known as safflower, has been used in traditional treatment for cardiovascular, cerebrovascular, and diabetic vascular complications. We proposed to investigate how the ethanol extract of Carthamus tinctorius L. (ECT) can be used ethnopharmacologically and alleviate vascular inflammatory processes under cytokine stimulation in human vascular endothelial cells. Using the optimized HPLC method, six markers were simultaneously analyzed for quality control of ECT. Pretreatment with ECT (10–100 μg/mL) significantly reduced the increase of leukocyte adhesion to HUVEC by TNF-α in a dose-dependent manner. Cell adhesion molecules (CAMs) such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial cell selectin (E-selectin) are decreased by ECT. In addition, ECT significantly suppressed TNF-α-induced oxidative stress referring to reactive oxygen species (ROS) production. p65 NF-κB nuclear translocation and its activation were inhibited by ECT. Furthermore, pretreatment of ECT increased the HO-1 expression, and nuclear translocation of Nrf-2. These data suggest the potential role of ECT as a beneficial therapeutic herb in vascular inflammation via ROS/NF-kB pathway and the regulation of Nrf-2/HO-1 signaling axis is involved in its vascular protection. Thus, further study will be needed to clarify which compound is dominant for protection of vascular diseases.

Download Full-text

KLF11 Protects against Venous Thrombosis via Suppressing Tissue Factor Expression

Thrombosis and Haemostasis ◽

10.1055/s-0041-1735191 ◽

2021 ◽

Author(s):

Wenying Liang ◽

Haocheng Lu ◽

Jinjian Sun ◽

Guizhen Zhao ◽

Huilun Wang ◽

...

Keyword(s):

Venous Thrombosis ◽

Tissue Factor ◽

Gene Transcription ◽

Loss Of Function ◽

Expression Change ◽

Causative Gene ◽

Tnf Α ◽

Gene Assay ◽

Tf Gene

AbstractKrüppel-like factors (KLFs) play essential roles in multiple biological functions, including maintaining vascular homeostasis. KLF11, a causative gene for maturity-onset diabetes of the young type 7, inhibits endothelial activation and protects against stroke. However, the role of KLF11 in venous thrombosis remains to be explored. Utilizing stasis-induced murine deep vein thrombosis (DVT) model and cultured endothelial cells (ECs), we identified an increase of KLF11 expression under prothrombotic conditions both in vivo and in vitro. The expression change of thrombosis-related genes was determined by utilizing gain- and loss-of-function approaches to alter KLF11 expression in ECs. Among these genes, KLF11 significantly downregulated tumor necrosis factor-α (TNF-α)-induced tissue factor (TF) gene transcription. Using reporter gene assay, chromatin immunoprecipitation assay, and co-immunoprecipitation, we revealed that KLF11 could reduce TNF-α-induced binding of early growth response 1 (EGR1) to TF gene promoter in ECs. In addition, we demonstrated that conventional Klf11 knockout mice were more susceptible to developing stasis-induced DVT. These results suggest that under prothrombotic conditions, KLF11 downregulates TF gene transcription via inhibition of EGR1 in ECs. In conclusion, KLF11 protects against venous thrombosis, constituting a potential molecular target for treating thrombosis.

Download Full-text

PERTURBATION OF ENDOTHELIAL CELLS INDUCES THROMBIN FORMATION IN FLOWING BLOOD RESULTING IN PLATELET AGGREGATION ON THEIR MATRIX

10.1055/s-0038-1644733 ◽

1987 ◽

Author(s):

Jaap Jan Zwaginga ◽

Martin J W IJsseldijk ◽

Philip G de Groot ◽

Jan J Sixma

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Thrombus Formation ◽

Strong Increase ◽

Prolonged Incubation ◽

Perfusion Studies ◽

The Matrix ◽

Flowing Blood ◽

Thrombin Formation

Studies which try to measure the contribution of the vessel wall to thrombin formation in flowing blood and the consequences of the formed thrombin for thrombus formation can not be performed with native blood because the perfusion system itself causes thrombin formation. Therefore, we have collected blood into^a solution of low molecular weight heparin (20 U/ml, Fragmin , Kabi Vitrum) and used this blood for perfusion studies over endothelial cells (EC) and their extracellular matrix (ECM) in a flat perfusiqn chamber (J.Lab.Clin.Med. 1983, 102, 522-532).Cells (2 x 105 ) and matrices (3.2 cm2) wetje perfused with 12 ml blood for 5 min at a shear rate of 1300 s . No fibrinopeptide A (FPA) generation in the perfusate was seen after perfusion over EC, ECM or empty coverslips. Treatment of EC for 4 hours with 4-phorbol 12-myristate 13-acetate (PMA 20 µg/ml) caused a strong increase in FPA generation (50 ng/ml) which was decreased again after a 16 hour incubation (25 ng/ml). ECM of EC's treated for 4 h with PMA showed a comparable FPA generation as their EC's (40 ng/ml), however, the FPA generation increased with prolonged incubation with PMA. FPA generation was completely blocked by hirudin and with a specific antibody against FVIIa (Dr. Mertens, CLB). By incubation of the matrix with an antitissue factor serum (Dr. Bertina, AZL), FPA generation could be blocked. The effect of the locally formed thrombin on platelet adhesion to ECM of 16 h PMA treated EC was evaluated and showed that 47.1 ± 9.3 (SD)% of the surface was covered with platelets of which 22.0 ± 9.3% consisted of small (< 5 µm) and 3.1 ± 3.4% of larger (5-10 µm) aggregates. EM studies showed degranulation of platelets and fibrin formation around the aggregates. ECM of untreated EC showed a similar coverage 41.5 ± 5.5% but only 6.7% small aggregates. Anti FVIIa and anti tissue factor decreased aggregate height and number to the values found with untreated ECM; adhesion remained unchanged. These data indicate that PMA perturbation of EC induces a tissue factor dependent thrombin formation which is associated with the induction of local thrombus formation. This system may serve as an in vitro model for thrombosis.

Download Full-text

Novel interaction of antioxidant-1 with TRAF4: role in inflammatory responses in endothelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00264.2019 ◽

2019 ◽

Vol 317 (6) ◽

pp. C1161-C1171

Author(s):

Archita Das ◽

Varadarajan Sudhahar ◽

Masuko Ushio-Fukai ◽

Tohru Fukai

Keyword(s):

Endothelial Cells ◽

High Fat Diet ◽

Nuclear Translocation ◽

Inflammatory Responses ◽

Inflammatory Diseases ◽

Dependent Manner ◽

Oxygen Species ◽

High Fat ◽

Inflammatory Gene Expression ◽

Tnf Α

NADPH oxidase (NOX)-derived reactive oxygen species (ROS) and copper (Cu), an essential micronutrient, have been implicated in vascular inflammatory diseases. We reported that in proinflammatory cytokine TNF-α-stimulated endothelial cells (ECs), cytosolic Cu chaperone antioxidant-1 (Atox1) functions as a Cu-dependent transcription factor for the NOX organizer p47phox, thereby increasing ROS-dependent inflammatory gene expression. However, the role and mechanism of Atox1 nuclear translocation in inflamed ECs remain unclear. Using enface staining and nuclear fractionation, here we show that Atox1 was localized in the nucleus in inflamed aortas from ApoE−/− mice with angiotensin II infusion on a high-fat diet, while it was found in cytosol in those from control mice. In cultured human ECs, TNF-α stimulation promoted Atox1 nuclear translocation within 15 min, which was associated with Atox1 binding to TNF-α receptor-associated factor 4 (TRAF4) in a Cu-dependent manner. TRAF4 depletion by siRNA significantly inhibited Atox1 nuclear translocation, p47phox expression, and ROS production as well as its downstream VCAM1/ICAM1 expression and monocyte adhesion to inflamed ECs, which were rescued by overexpression of nuclear targeted Atox1. Furthermore, Atox1 colocalized with TRAF4 at the nucleus in TNF-α-stimulated inflamed ECs and vessels. In summary, Cu-dependent Atox1 binding to TRAF4 plays an important role in Atox1 nuclear translocation and ROS-dependent inflammatory responses in TNF-α-stimulated ECs. Thus the Atox1-TRAF4 axis is a novel therapeutic target for vascular inflammatory disease such as atherosclerosis.

Download Full-text

Tissue Factor Expression in Bovine Endothelial Cells Induced by Pasteurella haemolytica Lipopolysaccharide and Interleukin-1

Veterinary Pathology ◽

10.1177/030098589403100107 ◽

1994 ◽

Vol 31 (1) ◽

pp. 55-60 ◽

Cited By ~ 14

Author(s):

M. A. Breider ◽

Z. Yang

Keyword(s):

Endothelial Cells ◽

Tissue Factor ◽

Interleukin 1 ◽

Cellular Tissue ◽

Pasteurella Haemolytica ◽

Dependent Manner ◽

Tissue Factor Expression ◽

Vascular Thrombosis ◽

Factor Expression

Pasteurella haemolytica in cattle produces fibrinohemorrhagic pleuropneumonia characterized by extensive pulmonary microvascular thrombosis and parenchymal necrosis. The purpose of this in vitro study was to determine if P. haemolytica lipopolysaccharide (LPS) promotes vascular thrombosis by inducing a procoagulant state in vascular endothelial cells. After treatment of confluent monolayers of bovine pulmonary artery endothelial cells with various concentrations of either P. haemolytica LPS or Escherichia coli LPS, the procoagulant activity of the endothelial cells was determined using a chromogenic assay dependent on cellular tissue factor expression. The LPS treatment induced significant increases in cellular tissue factor expression in a LPS concentration- and time-dependent manner. Highest levels of tissue factor were present at 22 hours after treatment, although high LPS concentrations induced moderate tissue factor levels at 5 hours after treatment. Interleukin-1 also induced tissue factor expression in endothelial cells and enhanced the LPS-induced effects. This interleukin-1 effect could be diminished by concurrent use of an interleukin-1 receptor antagonist. These results demonstrate that LPS and cytokine promotion of a procoagulant state in endothelial cells occurs in vitro. Similar mechanisms may play a role in P. haemolytica-mediated pulmonary vascular thrombosis.

Download Full-text

Adiponectin inhibits tissue factor expression and enhances tissue factor pathway inhibitor expression in human endothelial cells

Thrombosis and Haemostasis ◽

10.1160/th08-02-0124 ◽

2008 ◽

Vol 100 (08) ◽

pp. 291-300 ◽

Cited By ~ 20

Author(s):

Yi-Jian Chen ◽

Li-Qun Zhang ◽

Guang-Ping Wang ◽

Hui Zeng ◽

Ben Lü ◽

...

Keyword(s):

Endothelial Cells ◽

Protein Expression ◽

Mrna Expression ◽

Tissue Factor ◽

Tissue Factor Pathway Inhibitor ◽

Inhibitory Effect ◽

Dependent Manner ◽

Tnf Α ◽

Tissue Factor Pathway ◽

Expression And Activity

SummaryTissue factor (TF) plays a pivotal role in thrombus formation and atherogenesis in acute coronary syndrome. Tissue factor pathway inhibitor (TFPI) is a specific physiological inhibitor of TF/ FVIIa complex that regulates TF-induced coagulation. Adiponectin (Adp) is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Adp inhibits inflammatory cytokine and adhesion molecules expression, and it can prevent endothelial dysfunction. In this study, we investigated the effects of Adp on tumor necrosis factor-α (TNF-α)-induced expression of TF and TFPI in human umbilical vein endothelial cells (HU-VECs), and the signaling transduction pathways involved. It was found that Adp significantly inhibited both TF protein expression and activity in TNF-α-stimulated HUVECs. In the meanwhile, it increased TFPI protein expression and activity for about two folds. Adp also inhibited TF mRNA expression induced by TNF-α, but had no effect on TFPI mRNA expression. The inhibitory effect of Adp onTNF-α-inducedTF expression was prevented by pretreatment with Rp-cAMPs, a PKA inhibitor. Adp increased intracellular cAMP content and PKA activity levels in a dose-dependent manner. Phosphorylation of IκB-α was decreased by Adp, but phosphorylation of p44/42MAPK, SAPK/ JNK, and p38MAPK were not affected. These results suggested that Adp inhibits TF expression through inhibition of a PKA dependent nuclear factor- κB (NF-κB) signaling pathway. It was also found that adiponectin promoted Akt and AMP-activated protein kinase phosphorylation. The inhibitory effect of Adp on TNF-α-induced TF synthesis was abrogated in part by pretreatment with the PI3kinase inhibitor LY 294002, suggesting that Akt activation might inhibit TF expression induced by TNF-α. The inhibitory effect of Adp is almost completely abrogated by inhibition of both the cAMP/PKA pathway and PI3K/Akt pathway. In conclusion, our data indicated that inhibition of NF-κB through stabilization of IκB-α and activation of Akt phosphorylation may mediate the inhibitory effect of Adp on TF expression; but the enhancement effect of Adp on the TFPI production might occur via translational rather than transcriptional regulation.

Download Full-text