Thrombomodulin Expression In Endothelial Cells Research Articles

Epigenetic repression of THBD transcription by BRG1 contributes to deep vein thrombosis

BackgroundDeep vein thrombosis (DVT) with its major complication, pulmonary embolism, is a global health problem. Endothelial dysfunction is involved in the pathogenesis of DVT. We have previously demonstrated that endothelial specific deletion of Brahma-related gene 1 (BRG1) ameliorates atherosclerosis and aneurysm in animal models. Whether endothelial BRG1 contributes to DVT development remains undetermined. MethodsDVT was induced in mice by ligation of inferior vena cava. Deletion of BRG1 in endothelial cells was achieved by crossing the Cdh5-ERT-Cre mice with the Brg1loxp/loxp mice. ResultsHere we report that compared to the wild type mice, BRG1 conditional knockout (CKO) mice displayed substantially decreased DVT susceptibility characterized by decreased weight and size of thrombus and reduced immune infiltration. In endothelial cells, thrombomodulin (THBD) expression was significantly decreased by TNF-α stimulation, while BRG1 knockdown or inhibition recovered THBD expression. Further analysis revealed that BRG1 deficiency decreased the CpG methylation levels of the THBD promoter induced by TNF-α. Mechanistically, BRG1 directly upregulated DNMT1 expression after TNF-α treatment in endothelial cells. More importantly, administration of a small-molecule BRG1 inhibitor PFI-3 displayed potent preventive and therapeutic potentials in the DVT model. ConclusionsOur findings implicate BRG1 as an important regulator of DVT pathogenesis likely through epigenetic regulation of THBD expression in endothelial cells and provide translational proof-of-concept for targeting BRG1 in DVT intervention.

Endothelial thrombomodulin downregulation caused by hypoxia contributes to severe infiltration and coagulopathy in COVID-19 patient lungs.

SummaryBackgroundThromboembolism is a life-threatening manifestation of coronavirus disease 2019 (COVID-19). We investigated a dysfunctional phenotype of vascular endothelial cells in the lungs during COVID-19.MethodsWe obtained the lung specimens from the patients who died of COVID-19. The phenotype of endothelial cells and immune cells was examined by flow cytometry and immunohistochemistry (IHC) analysis. We tested the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the endothelium using IHC and electron microscopy.FindingsThe autopsy lungs of COVID-19 patients exhibited severe coagulation abnormalities, immune cell infiltration, and platelet activation. Pulmonary endothelial cells of COVID-19 patients showed increased expression of procoagulant von Willebrand factor (VWF) and decreased expression of anticoagulants thrombomodulin and endothelial protein C receptor (EPCR). In the autopsy lungs of COVID-19 patients, the number of macrophages, monocytes, and T cells was increased, showing an activated phenotype. Despite increased immune cells, adhesion molecules such as ICAM-1, VCAM-1, E-selectin, and P-selectin were downregulated in pulmonary endothelial cells of COVID-19 patients. Notably, decreased thrombomodulin expression in endothelial cells was associated with increased immune cell infiltration in the COVID-19 patient lungs. There were no SARS-CoV-2 particles detected in the lung endothelium of COVID-19 patients despite their dysfunctional phenotype. Meanwhile, the autopsy lungs of COVID-19 patients showed SARS-CoV-2 virions in damaged alveolar epithelium and evidence of hypoxic injury.InterpretationPulmonary endothelial cells become dysfunctional during COVID-19, showing a loss of thrombomodulin expression related to severe thrombosis and infiltration, and endothelial cell dysfunction might be caused by a pathologic condition in COVID-19 patient lungs rather than a direct infection with SARS-CoV-2.FundingThis work was supported by the Johns Hopkins University, the American Heart Association, and the National Institutes of Health.

VEGF-Induced Endothelial Podosomes via ROCK2-Dependent Thrombomodulin Expression Initiate Sprouting Angiogenesis.

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Antithrombotic Effects of Nur77 and Nor1 Are Mediated Through Upregulating Thrombomodulin Expression in Endothelial Cells.

Thrombomodulin is highly expressed on the lumenal surface of vascular endothelial cells (ECs) and possesses potent anticoagulant, antifibrinolytic, and anti-inflammatory activities in the vessel wall. However, the regulation of thrombomodulin expression in ECs remains largely unknown. In this study, we characterized nuclear receptor 4A family as a novel regulator of thrombomodulin expression in vascular ECs. We demonstrated that both nuclear receptors 4A, Nur77 and Nor1, robustly increase thrombomodulin mRNA and protein levels in human vascular ECs and in mouse liver tissues after adenovirus-mediated transduction of Nur77 and Nor1 cDNAs. Moreover, Nur77 deficiency and knockdown of Nur77 and Nor1 expression markedly attenuated the basal and vascular endothelial growth factor165-stimulated thrombomodulin expression. Mechanistically, we found that Nur77 and Nor1 increase thrombomodulin expression by acting through 2 different mechanisms. We showed that Nur77 barely affects thrombomodulin promoter activity, but significantly increases thrombomodulin mRNA stability, whereas Nor1 enhances thrombomodulin expression mainly through induction of Kruppel-like factors 2 and 4 in vascular ECs. Furthermore, we demonstrated that both Nur77 and Nor1 significantly increase protein C activity and inhibit tumor necrosis factor α-induced prothrombotic effects in human ECs. Deficiency of Nur77 increases susceptibility to arterial thrombosis, whereas enhanced expression of Nur77 and Nor1 protects mice from arterial thrombus formation. Our results identified nuclear receptors 4A as novel regulators of thrombomodulin expression and function in vascular ECs and provided a proof-of-concept demonstration that targeted increasing expression of Nur77 and Nor1 in the vascular endothelium might represent a novel therapeutic approach for the treatment of thrombotic disorders.

Effect of simvastatin on Kruppel-like factor2, endothelial nitric oxide synthase and thrombomodulin expression in endothelial cells under shear stress

Aims Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) and fluid wall shear stress have been reported to modulate the expression of genes related to inflammation, blood coagulation, thrombosis, and vascular constriction in cultured endothelial cells. In this study, we investigated the combined effect of laminar shear stress (LSS) and statins on endothelial cell gene expression. Main methods Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) mRNA and protein expression were evaluated in human abdominal aortic endothelial cells (HAAEC) treated with simvastatin (0.1, 1 or 10 µM) at various levels of LSS (0, 1.25, 12.5 or 25 dynes/cm 2). Key findings As expected, simvastatin and LSS separately enhanced KLF2, eNOS, and TM mRNA expressions. The combination of simvastatin and LSS resulted in significantly higher mRNA levels of all three genes compared to cells treated with LSS only. The highest KLF2, eNOS, and TM mRNA levels were detected at 10 µM simvastatin and 25 dynes/cm 2. Under these conditions, eNOS and TM protein levels were also elevated. Combining LSS and simvastatin produced an overall additive increase in KLF2, eNOS, and TM mRNA. Treatment of the endothelial cells with 10 µM simvastatin and 200 µM mevalonate completely eliminated the effect of simvastatin. Significance Our results suggest an additive increase in KLF2, eNOS, and TM expressions when simvastatin and LSS are combined. These results may help to explain the proposed non-lipid lowering benefits of statins observed in the clinic.

Thrombin Downregulates Thrombomodulin Expression and Activity in Primary Human Endothelial Cells

Thrombomodulin (TM) is a cell surface anticoagulant glycoprotein that plays a key role in the protein C pathway. TM expression in endothelial cells may be modulated by a variety of extracellular signals. Most notably, TM has been shown to be downregulated by inflammatory mediators, such as tumor necrosis factor-alpha and lipopolysaccharide. The objective of this study was to determine the effect of thrombin on TM expression and activity. Thrombin resulted in reduced TM in primary cultures of human endothelial cells by approximately 40% at the level of mRNA, protein, and activity. These effects were blocked by the thrombin inhibitor hirudin. These results suggest that activation of the coagulation cascade may result in a positive-feedback loop consisting of thrombin-mediated repression of TM-dependent protein C activation.

Thrombomodulin expression in endothelial cells after contact with bone cement

The expression of thrombomodulin after contact with CMW ® 1 bone cement extracts was studied in human umbilical vein endothelial cells. Cement extracts after 1 h and 7-day curing induced no significant variations in thrombomodulin antigen levels and in mRNA expression. Significant increase of thrombomodulin was observed when endothelial cells were treated with all-trans retinoic acid (ATRA). ATRA induced the increase of thrombomodulin also in cells incubated with cement extracts. These results suggest that CMW ® 1 bone cement does not impair the expression of thrombomodulin in endothelial cells.

Regulation of Thrombomodulin Expression by All-trans Retinoic Acid and Tumor Necrosis Factor-α: Differential Responses in Keratinocytes and Endothelial Cells

Thrombomodulin is a cell-surface anticoagulant glycoprotein expressed by vascular endothelial cells and epidermal keratinocytes. Thrombomodulin expression in endothelial cells is regulated by retinoic acid and tumor necrosis factor-alpha (TNF), agents that also modulate epidermal differentiation. We examined thrombomodulin function and regulation of thrombomodulin expression by all-trans retinoic acid (ATRA) and TNF in human keratinocytes and endothelial cells. Untreated keratinocytes and endothelial cells expressed thrombomodulin of comparable activity and apparent thrombin affinity. Incubation of keratinocytes with 10 mumol/L ATRA for 24 hours increased thrombomodulin activity 5.4 +/- 0.9-fold (mean +/- SE), with equivalent increases observed in thrombomodulin protein (5.5 +/- 2.1-fold) and mRNA (4.2 +/- 1.2-fold). Incubation of keratinocytes with 1.0 nmol/L TNF markedly increased expression of keratinocyte transglutaminase, but had no effect on thrombomodulin activity, protein, or mRNA. In endothelial cells, ATRA produced a small increase in thrombomodulin activity (1.9 +/- 0.1-fold), and incubation with TNF for 24 hours decreased thrombomodulin activity 83% +/- 7%. The activity profile of keratinocyte thrombomodulin exhibited a distinct maximum near 1.0 mmol/L Ca2+. These results demonstrate that keratinocyte thrombomodulin is regulated by retinoids and Ca2+, but not by TNF, and that regulation of thrombomodulin expression differs in keratinocytes and endothelial cells.

Blood coagulation equilibrium in rat liver microcirculation as evaluated by endothelial cell thrombomodulin and macrophage tissue factor

The regulatory mechanisms of microcirculation might differ in the liver from other organs, because macrophages are resident in the hepatic sinusoids and sinusoidal endothelial cells are unique in shape and function. Thrombomodulin expression in endothelial cells and tissue factor activity in isolated macrophages were studied in the liver and lung of rats. In normal rats, the thrombomodulin expression was minimal in hepatic sinusoids, but prominent in pulmonary capillaries, while the tissue factor activity in the presence of endotoxin was higher in pulmonary macrophages than in Kupffer cells, although the levels in the absence of endotoxin were comparable in both cells. The tissue factor activity in hepatic macrophages was increased after priming of the cells with Corynebacterium parvum or after induction of liver necrosis or cirrhosis with carbon tetrachloride. In the necrotic or cirrhotic liver, increased thrombomodulin expression was seen along capillaries extending in necrotic areas and regenerating nodules, but this increase was minimal in the Corynebacterium parvum-treated rat liver. Blood coagulation equilibrium in microcirculation regulated by endothelial cells and macrophages may differ between the liver and lung. Such equilibrium in the liver may vary depending on pathological status.

Possible cause of primary graft non-function after orthotopic liver transplantation: a hypothesis with rat models.

The cause of primary graft non-function after orthotopic liver transplantation (OLTX) is uncertain, but its clinical features are similar to fulminant hepatic failure with coagulopathy. In preserved livers, endothelial cell damage in the hepatic sinusoids is characteristic, indicating that fibrin deposition in the hepatic sinusoids, which can cause massive hepatic necrosis, might occur in the liver after OLTX. When rat liver was preserved in University of Wisconsin solution at 1 degree C, detachment of endothelial linings into sinusoidal lumens developed with fat-storing cell damage after 18 h. In this liver, hepatic macrophages were activated after reperfusion. Tissue factor activity in hepatic macrophages isolated from livers after OLTX was significantly increased compared to the control liver and this increase was enhanced by addition of endotoxin. In the preserved and transplanted livers, thrombomodulin expression in endothelial cells disappeared and fibrin deposition was seen in the hepatic sinusoids. Intravenous infusion therapy with antithrombin III attenuated liver injury 24 h after OLTX following preservation for 18 h. These results suggest that intravascular coagulation in the hepatic sinusoids associated with liver injury occurs in the liver after OLTX following cold preservation. This coagulopathy may be caused by sinusoidal endothelial cell damage due to regulatory imbalance in coagulation as a result of increased tissue factor activity in hepatic macrophages and decreased thrombomodulin activity in sinusoidal endothelial cells. Fat-storing cell damage may also contribute to the endothelial cell damage. A hypothesis regarding the cause of primary graft non-function after OLTX is proposed.

Fluid Shear Stress Increases the Expression of Thrombomodulin by Cultured Human Endothelial Cells

Endothelial cells (ECs) cultured from human umbilical vein were exposed to medium flow in a flow-loading chamber, and changes in thrombomodulin (TM) expression were examined by flow cytometry and enzyme linked immunosorbent assay with monoclonal antibody. The expression of TM antigen was increased time- and shear stress-dependently by flow, and when exposed to a shear stress of 15 dynes/cm 2 for 24 hr, it increased to approximately 200% of the stationary control level. Reverse transcriptase-polymerase chain reaction showed that TM mRNA levels in ECs also increased in response to flow. TM mRNA began to increase one hour after the application of shear stress of 15 dynes/cm 2 and reached a maximum (approximately 330% of stationary control) after eight hours. These results, demonstrating an up-regulating effect of flow on TM expression in ECs, suggest that shear stress may be an important modulator of intravascular blood coagulation.

Hepatic sinusoids may differ from pulmonary capillaries in coagulability in rats: Evaluation by tissue factor activity in macrophages and thrombomodulin expression in endothelial cells

Hepatic sinusoids may differ from pulmonary capillaries in coagulability in rats: Evaluation by tissue factor activity in macrophages and thrombomodulin expression in endothelial cells

Effect of protein kinase inhibitors on tissue factor and thrombomodulin expression in endothelial cells and monocytes: [formula omitted], M.CI Laplace, A. Lalé, F. Dol, A. Dumas and J.M. Herbert, Sanofi Recherche, Toulouse France

Effect of protein kinase inhibitors on tissue factor and thrombomodulin expression in endothelial cells and monocytes: [formula omitted], M.CI Laplace, A. Lalé, F. Dol, A. Dumas and J.M. Herbert, Sanofi Recherche, Toulouse France

Expression of Thrombomodulin by Smooth Muscle Cells in Culture: Different Effects of Tumor Necrosis Factor and Cyclic Adenosine Monophosphate on Thrombomodulin Expression by Endothelial Cells and Smooth Muscle

Thrombomodulin (TM), a critical component of the protein C anticoagulant pathway, has previously been localized to endothelial cells (EC), but not smooth muscle cells (SMC) of the blood vessel wall. We demonstrate that cultured rat, bovine, as well as human SMC, but not blood vessel wall smooth muscle tissue, possess significant functional levels of TM and TM mRNA. Cyclic adenosine monophosphate stimulates TM expression in cultured SMC, but not EC, while tumor necrosis factor suppresses TM expression in EC but not cultured SMC. We postulate that following acute or chronic EC injury, luminal SMC can express TM, and are therefore able to protect the damaged blood vessel from thrombosis.

Expression of thrombomodulin by smooth muscle cells in culture: different effects of tumor necrosis factor and cyclic adenosine monophosphate on thrombomodulin expression by endothelial cells and smooth muscle cells in culture

Thrombomodulin (TM), a critical component of the protein C anticoagulant pathway, has previously been localized to endothelial cells (EC), but not smooth muscle cells (SMC) of the blood vessel wall. We demonstrate that cultured rat, bovine, as well as human SMC, but not blood vessel wall smooth muscle tissue, possess significant functional levels of TM and TM mRNA. Cyclic adenosine monophosphate stimulates TM expression in cultured SMC, but not EC, while tumor necrosis factor suppresses TM expression in EC but not cultured SMC. We postulate that following acute or chronic EC injury, luminal SMC can express TM, and are therefore able to protect the damaged blood vessel from thrombosis.