Endothelial Tissue Factor Research Articles

Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting.

Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.

PCSK9 aggravated carotid artery stenosis in ApoE-/- mice by promoting the expression of tissue factors in endothelial cells via the TLR4/NF-κB pathway

Atherosclerosis, a chronic inflammatory disease, is the most relevant cause of carotid artery stenosis. Vascular endothelial cells (ECs) play a significant role in the development of atherosclerosis. In this chronic inflammatory environment, we aimed to investigate whether PCSK9 could mitigate atherosclerosis progression by reducing tissue factor expression in ECs via in vivo and in vitro assays. In vivo, we investigated the effect of PCSK9 inhibition on preventing atherosclerotic lesion formation in ApoE-/- mice fed a western diet. The results showed that inhibiting PCSK9 could significantly downregulate the protein expression of tissue factor (TF) in ECs to reduce the area of atherosclerotic plaques. In vitro, we incubated human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS). We found that LPS-induced TF elevation was suppressed by a PCSK9 inhibitor at both the mRNA and protein levels and that the TLR4/NF-κB pathway was also suppressed by a PCSK9 inhibitor. With respect to plasma samples from patients with carotid artery stenosis, we also demonstrated that the expression of TF was positively correlated with that of PCSK9. Thus, in addition to regulating lipid metabolism, the regulation of endothelial cell TF expression through the TLR4/NF-κB pathway may be a potential mechanism of PCSK9 in promoting atherosclerotic carotid stenosis.

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

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

JAK-STAT inhibition reduces endothelial prothrombotic activation and leukocyte–endothelial proadhesive interactions

BackgroundVascular activation is characterized by increased proinflammatory, pro thrombotic, and proadhesive signaling. Several chronic and acute conditions, including Bcr-abl-negative myeloproliferative neoplasms (MPNs), graft-vs-host disease, and COVID-19 have been noted to have increased activation of the janus kinase (JAK)-signal transducer and downstream activator of transcription (STAT) pathways. Two notable inhibitors of the JAK-STAT pathway are ruxolitinib (JAK1/2 inhibitor) and fedratinib (JAK2 inhibitor), which are currently used to treat MPN patients. However, in some conditions, it has been noted that JAK inhibitors can increase the risk of thromboembolic complications. ObjectivesWe sought to define the anti-inflammatory and antithrombotic effects of JAK-STAT inhibitors in vascular endothelial cells. MethodsWe assessed endothelial activation in the presence or absence of ruxolitinib or fedratinib by using immunoblots, immunofluorescence, qRT-PCR, and function coagulation assays. Finally, we used endothelialized microfluidics perfused with blood from normal and JAK2V617F+ individuals to evaluate whether ruxolitinib and fedratinib changed cell adhesion. ResultsWe found that both ruxolitinib and fedratinib reduced endothelial cell phospho-STAT1 and STAT3 signaling and attenuated nuclear phospho-NK-κB and phospho-c-Jun localization. JAK-STAT inhibition also limited secretion of proadhesive and procoagulant P-selectin and von Willebrand factor and proinflammatory IL-6. Likewise, we found that JAK-STAT inhibition reduced endothelial tissue factor and urokinase plasminogen activator expression and activity. ConclusionsBy using endothelialized microfluidics perfused with whole blood samples, we demonstrated that endothelial treatment with JAK-STAT inhibitors prevented rolling of both healthy control and JAK2V617F MPN leukocytes. Together, these findings demonstrate that JAK-STAT inhibitors reduce the upregulation of critical prothrombotic pathways and prevent increased leukocyte–endothelial adhesion.

Vascular endothelial growth factor, tissue factor, coagulation and fibrinolysis markers in slow-flow vascular malformations: a prospective study of treatment with sirolimus.

Slow-flow vascular malformations (VMs), especially those with venous components, can be complicated by localized intravascular coagulopathy (LIC), responsible for pain and impaired quality of life. Several studies have shown the effectiveness of mTOR inhibitors (especially sirolimus) on slow-flow VMs but its effect on coagulation has been poorly studied, especially in children. Our study shows that venous and combined VMs are associated with coagulation abnormalities and provides novel evidence that sirolimus improves coagulopathy in venous malformations. However we did not clearly evidence predictive biomarkers of response to sirolimus but this is the first study attempting to highlight predictive markers of response to sirolimus.

Von Willebrand Factor and ADAMTS-13 Are Associated with the Severity of COVID-19 Disease.

Coagulopathy in COVID-19 patients is presumably based on systemic hypercoagulation with the inflammatory response. As a result of endothelial dysfunction, tissue factor and von Willebrand factor (vWF) are released into the blood stream, which leads to prothrombinase activation. The vWF/ADAMTS-13 ratio can be used for monitoring the severity of the disease. This observational prospective study included 141 patients with COVID-19. In patients with mild COVID-19 (group 1), the assessment was performed on the 3rd–7th day of illness (point 1) and 14–28 days after recovery (point 2). In patients with moderate (groups 2) and severe (group 3) COVID-19, the assessment was performed during hospitalization (point 1) and after 14 days (point 2). The vWF:RCo/ADAMTS-13:activity (point 1), vWF/ADAMTS-13 (point 2) and vWF:RCo/ADAMTS-13:activity (point 2) ratios were significantly higher in patients with moderate and severe COVID-19. Moreover, in these patients, both ratios increased after recovery (point 2), which is a negative prognostic factor of thrombotic complications. Thus, COVID-19 is characterized by a decrease in the concentration and activity of ADAMTS-13 metalloproteinase, especially in patients with the severe form of COVID-19. A decrease in ADAMTS-13 activity results in an increase in vWF concentration and activity so the ratio of vWF to ADAMTS-13 changes significantly.

Fluorometholone inhibits high glucose-induced cellular senescence in human retinal endothelial cells.

Diabetic retinopathy (DR) is a common diabetic complication that severely impacts the life quality of diabetic patients. Recently, cellular senescence in human retinal endothelial cells (HRECs) induced by high glucose has been linked to the pathogenesis of DR. Fluorometholone (FML) is a glucocorticoid drug applied in the treatment of inflammatory and allergic disorders of the eye. The objective of the present study is to investigate the protective function of FML on high glucose-induced cellular senescence in HRECs. The in vitro injury model was established by stimulating HRECs with 30mm glucose. After evaluating the cytotoxicity of FML in HRECs, 0.05% and 0.1% FML were used as the optimal concentration in the entire experiment. It was found that the excessive released inflammatory factors including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8) in HRECs induced by high glucose were significantly suppressed by FML, accompanied by the inhibitory effects on the expression levels of vascular endothelial growth factor (VEGF) and tissue factor (TF). Declined telomerase activity and enhanced senescence-associated β-galactosidase (SA-β-gal) activity were found in high glucose-challenged HRECs, which were dramatically alleviated by FML, accompanied by the inactivation of the p53/p21 and retinoblastoma (Rb) signaling. Interestingly, FML ameliorated high glucose-induced dephosphorylation of Akt. Lastly, the protective effects of FML against high glucose-induced cellular senescence in HRECs were abolished by the co-treatment of the PI3K/Akt signaling inhibitor LY294002, suggesting the involvement of this pathway. Taken together, these data revealed that FML-inhibited high glucose-induced cellular senescence mediated by Akt in HERCs, suggesting a novel molecular mechanism of FML.

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

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

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.

A thrombin-PAR1/2 feedback loop amplifies thromboinflammatory endothelial responses to the viral RNA analogue poly(I:C)

AbstractActivation of blood coagulation and endothelial inflammation are hallmarks of respiratory infections with RNA viruses that contribute significantly to the morbidity and mortality of patients with severe disease. We investigated how signaling by coagulation proteases affects the quality and extent of the response to the TLR3-ligand poly(I:C) in human endothelial cells. Genome-wide RNA profiling documented additive and synergistic effects of thrombin and poly(I:C) on the expression level of many genes. The most significantly active genes exhibiting synergistic induction by costimulation with thrombin and poly(I:C) included the key mediators of 2 critical biological mechanisms known to promote endothelial thromboinflammatory functions: the initiation of blood coagulation by tissue factor and the control of leukocyte trafficking by the endothelial-leukocyte adhesion receptors E-selectin (gene symbol, SELE) and VCAM1, and the cytokines and chemokines CXCL8, IL-6, CXCL2, and CCL20. Mechanistic studies have indicated that synergistic costimulation with thrombin and poly(I:C) requires proteolytic activation of protease-activated receptor 1 (PAR1) by thrombin and transactivation of PAR2 by the PAR1-tethered ligand. Accordingly, a small-molecule PAR2 inhibitor suppressed poly(I:C)/thrombin–induced leukocyte-endothelial adhesion, cytokine production, and endothelial tissue factor expression. In summary, this study describes a positive feedback mechanism by which thrombin sustains and amplifies the prothrombotic and proinflammatory function of endothelial cells exposed to the viral RNA analogue, poly(I:C) via activation of PAR1/2.

Expression of Concern: Molecular mechanism whereby paraoxonase-2 regulates coagulation activation through endothelial tissue factor in rat haemorrhagic shock model.

Expression of Concern: Molecular mechanism whereby paraoxonase-2 regulates coagulation activation through endothelial tissue factor in rat haemorrhagic shock model.

The protective effects of naproxen against interleukin-1β (IL-1β)- induced damage in human umbilical vein endothelial cells (HUVECs)

ABSTRACT Non-steroidal anti-inflammatory drugs (NSAIDs) are one of the most widely used medications in the world. Naproxen is an NSAID with relatively low selectivity for cyclooxygenase-2 (COX-2), thereby having decreased risk for cardiovascular (CV) events. However, it is unclear whether naproxen might provide protection against atherosclerosis, an underlying cause of numerous cardiovascular diseases (CVDs). In the present study, we exposed human umbilical vein endothelial cells to interleukin-1β (IL-1β), a key cytokine involved in atherogenesis, with or without naproxen. Our findings indicate that naproxen could protect against IL-1β-induced damage by improving cell viability and preventing cell death. Additionally, naproxen suppressed the expression of the cytokines IL-6, IL-12, and tumor necrosis factor-α (TNF-α), and downregulated the expression of vascular endothelial growth factor (VEGF) and tissue factor (TF) induced by IL-1β. Importantly, naproxen also inhibited the attachment of monocytes to endothelial cells, which was achieved through Krüppel-like factor 6 (KLF6)-mediated reduced expression of intracellular adhesion molecule-1 (ICAM-1) and E-selectin. These findings suggest that naproxen may aid in the prevention of atherosclerosis by exerting cardioprotective effects beyond low COX-2-selectivity.

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.

The P2Y2 Nucleotide Receptor Mediates Tissue Factor Expression in Human Monocytes

Recently we reported that in human coronary artery endothelial cells, activation of the P2Y2 receptor induces up‐regulation of tissue factor (TF), a key initiator of the coagulation cascade. However, it has been shown by others that monocyte TF is more important than endothelial TF in terms of provoking pro‐thrombotic state. Thus, we aimed to study whether human monocytes express P2Y2 receptor and its role in control of TF expression. RT‐PCR and receptor activity assays revealed that among the eight P2Y nucleotide receptors, the P2Y2 subtype was selectively and functionally expressed in human monocytic THP‐1 cells. Consistent with this, stimulation of the THP‐1 cells by ATP or UTP at micromolar concentration dramatically increased TF protein expression measured by Western blotting, which was abolished by AR‐C118925XX, a selective P2Y2 receptor antagonist, suggesting a key role for P2Y2 receptor. In addition, UTP or ATP treatment induced a rapid accumulation of TF mRNA, which was maximal at 2h and preceded with an increased TF pre‐mRNA, indicating enhanced TF gene transcription. However, we also observed a significant increase in TF mRNA stability after UTP and ATP treatment. Furthermore, activation of the monocyte P2Y2 receptor by a low dose of UTP or ATP (1μM) significantly activated the MAPK pathways including ERK1/2, JNK, and p38, along with their downstream transcription factors including c‐Jun, c‐Fos and ATF‐2, whereas blocking the ERK1/2, JNK or p38 pathways respectively, all significantly suppressed P2Y2 receptor‐mediated TF expression. These findings demonstrate for the first time that the P2Y2 receptor mediates TF expression in human monocytes through new mechanisms involving ERK1/2, JNK and p38, and that both transcriptional and posttranscriptional mechanisms contribute to a pro‐thrombotic status in monocytes through the P2Y2‐TF axis, highlighting monocyte P2Y2 receptor may be a new drug target for the prevention and/or treatment of relevant thrombotic diseases.Support or Funding InformationSupported by NIH funding 1R01HL125279‐01A1 (JS)

Molecular mechanism whereby paraoxonase-2 regulates coagulation activation through endothelial tissue factor in rat haemorrhagic shock model.

We investigated the molecular mechanism of paraoxonase-2 (PON-2) in regulating blood coagulation activation in rats with haemorrhagic shock through endothelial tissue factor (TF). Thirty adult Sprague Dawley rats were randomly divided into three groups: healthy control group (group A), the haemorrhagic shock PON-2 treatment group (group B), and the haemorrhagic shock group (group C). After the model was established, blood was withdrawn from the inferior vena cava of all rats. The difference in plasma thrombomodulin (TM) levels of the three groups was determined by Western blotting. The expression of transcription factors Egr-1 and Sp1 was detected by Western blotting assays. reverse transcription-polymerase chain Reaction (RT-PCR) was used to determine the mRNA expression of t-PA, PAI-1, TM, and PON-2 in the serum of three groups of rats. Endothelial TF was measured by enzyme linked immunosorbent assay (ELISA), and coagulation assay was used to detect the activity of coagulation factor VIII. Histopathological examination of the arteries of the rats was performed. The molecular mechanism of PON-2 in regulating blood coagulation activation in haemorrhagic shock model rats by endothelial tissue factor was analysed. The expression of thrombin was determined by electrophoresis. Compared with the healthy control group, the expression of TM in groups B and C decreased, both 188.64 ± 12.47 and 137.48 ± 9.72, respectively, with a significant difference. The mRNA expression of TM and PON was determined by RT-PCR. The mRNA expression of TM and PON in group B was 0.97 ± 0.07 and 1.14 ± 0.09, compared with the control group, and the mRNA expression of TM and PON in group C was 0.86 ± 0.38 and 1.12 ± 0.41, both of which increased, and there were significant differences. By measuring the expression of endothelial TF, the expression of TF in groups B and C was elevated to 12.69 ± 1.07 and 11.59 ± 0.87, with significant differences. The enzyme activities of PON-2 in groups B and C, which were 110.34 ± 14.37 and 52.37 ± 8.06, respectively, were increased compared with the healthy control group and there were significant differences. PON-2 regulates the activation of coagulation in rats with haemorrhagic shock by regulating the expression of endothelial tissue-related genes such as plasma TM and endothelial TF under hypoxic and ischaemic conditions.

Protease Activity in Vascular Disease.

Protease Activity in Vascular Disease.

Neutrophil Extracellular Traps Induce Endothelial Cell Activation and Tissue Factor Production Through Interleukin-1α and Cathepsin G

Objective- Coronary artery thrombosis can occur in the absence of plaque rupture because of superficial erosion. Erosion-prone atheromata associate with more neutrophil extracellular traps (NETs) than lesions with stable or rupture-prone characteristics. The effects of NETs on endothelial cell (EC) inflammatory and thrombogenic properties remain unknown. We hypothesized that NETs alter EC functions related to erosion-associated thrombosis. Approach and Results- Exposure of human ECs to NETs increased VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA and protein expression in a time- and concentration-dependent manner. THP-1 monocytoid cells and primary human monocytes bound more avidly to NET-treated human umbilical vein ECs than to unstimulated cells under flow. Treatment of human ECs with NETs augmented the expression of TF (tissue factor) mRNA, increased EC TF activity, and hastened clotting of recalcified plasma. Anti-TF-neutralizing antibody blocked NET-induced acceleration of clotting by ECs. NETs alone did not exhibit TF activity or acceleration of clotting in cell-free assays. Pretreatment of NETs with anti-interleukin (IL)-1α-neutralizing antibody or IL-1Ra (IL-1 receptor antagonist)-but not with anti-IL-1β-neutralizing antibody or control IgG-blocked NET-induced VCAM-1, ICAM-1, and TF expression. Inhibition of cathepsin G, a serine protease abundant in NETs, also limited the effect of NETs on EC activation. Cathepsin G potentiated the effect of IL-1α on ECs by cleaving the pro-IL-1α precursor and releasing the more potent mature IL-1α form. Conclusions- NETs promote EC activation and increased thrombogenicity through concerted action of IL-1α and cathepsin G. Thus, NETs may amplify and propagate EC dysfunction related to thrombosis because of superficial erosion.

Paraoxonase-2 regulates coagulation activation through endothelial tissue factor

AbstractOxidative stress and inflammation of the vessel wall contribute to prothrombotic states. The antioxidative protein paraoxonase-2 (PON2) shows reduced expression in human atherosclerotic plaques and endothelial cells in particular. Supporting a direct role for PON2 in cardiovascular diseases, Pon2 deficiency in mice promotes atherogenesis through incompletely understood mechanisms. Here, we show that deregulated redox regulation in Pon2 deficiency causes vascular inflammation and abnormalities in blood coagulation. In unchallenged Pon2−/− mice, we find increased oxidative stress and endothelial dysfunction. Bone marrow transplantation experiments and studies with endothelial cells provide evidence that increased inflammation, indicated by circulating interleukin-6 levels, originates from Pon2 deficiency in the vasculature. Isolated endothelial cells from Pon2−/− mice display increased tissue factor (TF) activity in vitro. Coagulation times were shortened and platelet procoagulant activity increased in Pon2−/− mice relative to wild-type controls. Coagulation abnormalities of Pon2−/− mice were normalized by anti-TF treatment, demonstrating directly that TF increases coagulation. PON2 reexpression in endothelial cells by conditional reversal of the knockout Pon2 cassette, restoration in the vessel wall using bone marrow chimeras, or treatment with the antioxidant N-acetylcysteine normalized the procoagulant state. These experiments delineate a PON2 redox-dependent mechanism that regulates endothelial cell TF activity and prevents systemic coagulation activation and inflammation.

Tumor necrosis factor-α levels and non-surgical bleeding in continuous-flow left ventricular assist devices

Non-surgical bleeding (NSB) due to angiodysplasia is common in left ventricular assist device (LVAD) patients. Thrombin-induced angiopoietin-2 (Ang-2) expression in LVAD patients leads to altered angiogenesis and is associated with lower angiopoietin-1 (Ang-1) and increased NSB. However, the mechanism for decreased Ang-1, made by pericytes, is unknown and the origin of thrombin in LVAD patients is unclear. We hypothesized that high tumor necrosis factor-α (TNF-α) levels in LVAD patients induce pericyte apoptosis, tissue factor (TF) expression and vascular instability. We incubated cultured pericytes with serum from patients with heart failure (HF), LVAD or orthotopic heart transplantation (OHT), with or without TNF-α blockade. We performed several measurements: Ang-1 expression was assessed by reverse transcript-polymerase chain reaction (RT-PCR) and pericyte death fluorescently; TF expression was assessed by RT-PCR in cultured endothelial cells incubated with patient plasma with or without TNF-α blockade; and TF expression was assessed in endothelial biopsy samples from these patients by immunofluorescence. We incubated cultured endothelial cells on Matrigel with patient serum with or without TNF-α blockade and determined tube formation by microscopy. Serum from LVAD patients had higher levels of TNF-α, suppressed Ang-1 expression in pericytes, and induced pericyte death, and there was accelerated endothelial tube formation compared with serum from patients without LVADs. TF was higher in both plasma and endothelial cells from LVAD patients, and plasma from LVAD patients induced more endothelial TF expression. All of these effects were reversed or reduced with TNF-α blockade. High levels of TNF-α were associated with increased risk of NSB. Elevated TNF-α in LVAD patients is a central regulator of altered angiogenesis, pericyte apoptosis and expression of TF and Ang-1.

Evening primrose oil or forskolin ameliorates celecoxib-enhanced upregulation of tissue factor expression in mice subjected to lipopolysaccharide-induced endotoxemia.

Celecoxib, a selective cyclooxygenase-2 inhibitor, produces thrombotic events in patients predisposed to cardiovascular risk factors. One theory reported an increase in endothelial expression of tissue factor (TF) as a predisposing factor. This work explored the effect of evening primrose oil (EPO), a source of prostaglandin E1, and forskolin (a cyclic adenosine monophosphate stimulator) against the prothrombotic effect of celecoxib in mice. Lipopolysaccharide mouse model of endotoxemia was used to induce an upregulation of TF activity. Male mice received celecoxib (25mg/kg), celecoxib plus EPO, or celecoxib plus forskolin for 4weeks and then subjected to a prothrombotic challenge in the form of an intraperitoneal injection of lipopolysaccharide. Results showed an increase in plasma TF activity, endothelial TF expression, and thrombin-antithrombin (TAT) but lower antithrombin III (ATIII) level in mice that received celecoxib in comparison to those that received the vehicle. Adding EPO or forskolin to celecoxib regimen significantly decreased the prothrombotic effect of celecoxib. A positive correlation (r=0.8501) was found between TF activity and TAT. Co-administration of EPO or forskolin decreased the activity of TF and mitigated the prothrombotic effect of celecoxib. Therefore, these combinations may have the utility to abrogate the prothrombotic adverse effect of celecoxib in clinical setting.