Thrombomodulin In Endothelial Cells Research Articles

Reduced Production of JAK2V617F-positive Microparticles at Mild Hypothermia.

The Philadelphia chromosome-negative (Ph-) myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise from abnormal growth of blood cells in the bone marrow. Patients with MPNs are at increased risk for life-threatening thromboembolic complications. The detection of JAK2V617F in endothelial cells (ECs) brought a new perspective to the research of thromboembolic events. However, the mechanisms by which the mutation contributes to risk have yet to be entirely understood. Consequently, the objective of this study was to investigate how JAK2V617F impacts endothelial cells by considering thermoregulation. We applied our previously created model for EC that was genetically modified with JAK2 wild type (WT)-GFP and JAK2V617F-GFP lentiviruses; the cells were cultured for 48 h at 37°C for normothermia and 32°C for mild hypothermia. We examined the effect of thermoregulation on infection efficiency and the expression of cell surface markers, including endothelial protein C receptor (EPCR), thrombomodulin (TM), and tissue factor (TF), which are related to the coagulation pathways. Furthermore, the microparticle production from the genetically modified EC (EMPs) was analyzed. We found suppression of the expression of coagulation factors, including EPCR, TM, and TF in JAK2V617F positive ECs under mild hypothermia. JAK2V617F-positive ECs showed slightly higher EMP production under mild hypothermia. Although the molecular mechanisms of the thermal effects on the tumor microenvironment with JAK2V617F and its effect on EMP production and coagulation are not known yet, the therapy-oriented effect of thermoregulation might be considered in future studies.

Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19. Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU. Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry. Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction. Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

The mechanism by which enoxaparin sodium–high-viscosity bone cement reduces thrombosis by regulating CD40 expression in endothelial cells

ObjectivePMMA bone cement leads to the development of local thrombi. Our study found that ES-PMMA bone cement, a novel material, can reduce local thrombosis. We used a simple and reproducible animal model to confirm the reduction in local thrombosis and preliminarily explored the associated molecular mechanism.MethodsNew Zealand rabbits, which were used to model thrombosis using extracorporeal carotid artery shunts, were divided into the following three groups, with 10 rabbits in each group: the sham group, PMMA group and ES-PMMA group. Four hours after modelling, experimental samples were collected, and the degree of thrombosis was compared between the groups. The expression of thrombomodulin in endothelial cells was quantified in vascular tissues samples.ResultsThrombosis was observed in the PMMA group and ES-PMMA group but not in the sham group. The thrombosis weight was 0.00732 ± 0.00089 g/cm in the PMMA group and 0.00554 ± 0.00077 g/cm in the ES-PMMA group (P < 0.001). Quantitative real-time polymerase chain reaction (RT–qPCR) and Western blotting revealed that the expression of CD40, which can regulate thrombosis in vascular endothelial cells, was significantly lower in the ES-PMMA group than in the PMMA group.ConclusionCompared with PMMA bone cement, ES-PMMA bone cement can reduce local thrombosis by decreasing the expression of the thrombus-associated regulatory protein CD40 in vascular endothelial cells.

Thrombomodulin is essential for maintaining quiescence in vascular endothelial cells

Thrombomodulin (TM) is a thrombin receptor on endothelial cells that is involved in promoting activation of the anticoagulant protein C pathway during blood coagulation. TM also exerts protective anti-inflammatory properties through a poorly understood mechanism. In this study, we investigated the importance of TM signaling to cellular functions by deleting it from endothelial cells by CRISPR-Cas9 technology and analyzed the resultant phenotype of TM-deficient (TM-/- ) cells. Deficiency of TM in endothelial cells resulted in increased basal permeability and hyperpermeability when stimulated by thrombin and TNF-α. The loss of the basal barrier permeability function was accompanied by increased tyrosine phosphorylation of VE-cadherin and reduced polymerization of F-actin filaments at cellular junctions. A significant increase in basal NF-κB signaling and expression of inflammatory cell adhesion molecules was observed in TM-/- cells that resulted in enhanced adhesion of leukocytes to TM-/- cells in flow chamber experiments. There was also a marked increase in expression, storage, and release of the von Willebrand factor (VWF) and decreased storage and release of angiopoietin-2 in TM-/- cells. In a flow chamber assay, isolated platelets adhered to TM-/- cells, forming characteristic VWF-platelet strings. Increased VWF levels and inflammatory foci were also observed in the lungs of tamoxifen-treated ERcre-TMf/f mice. Reexpression of the TM construct in TM-/- cells, but not treatment with soluble TM, normalized the cellular phenotype. Based on these results, we postulate cell-bound TM endows a quiescent cellular phenotype by tightly regulating expression of procoagulant, proinflammatory, and angiogenic molecules in vascular endothelial cells.

Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia

Thrombomodulin (THBD) is a transmembrane protein that regulates endothelial function by 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C activation, and 3) down-regulating complement activation by inactivating C3a and C5a (PMID 29866818). Reduced THBD function has been implicated in several vasculopathies (PMID 29866818). Oxidative damage increases cleaved non-functional THBD in circulation and increased circulating non-functional THBD predicts the severity of organ damage in thrombotic microangiopathies (PMID 11190905). Vascular endothelial dysfunction is a hallmark feature of sickle cell disease (SCD) that leads to acute and chronic organ damage and may, in part, be mediated by hemolysis (PMID 28248201). Hemolysis leads to release of cell-free hemoglobin which may lead to vasculopathy through consumption of NO, activation of TLR4 pathways, and direct oxidative damage. We investigated whether decreased THBD activity may be implicated in the pathophysiology of SCD vasculopathy in endothelial cells, transgenic sickle mice, and in a prospective cohort of patients with sickle cell anemia. In vitro: We exposed endothelial cells (EA.hy926, ATTC® CRL-2922TM; Manassas, VA) to incremental doses of cell-free hemoglobin. With higher doses of cell-free hemoglobin we observed reduced surface endothelial cell THBD activity at 6 hours of incubation, assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin (Figure 1A). In conjunction with the reduced endothelial THBD activity, there were increased concentrations of cleaved THBD in the supernatant by ELISA (R&amp;D Systems, Minneapolis, MN) (Figure 1B). Transgenic sickle mice: At 6 months of age, transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine) had higher plasma concentrations of cleaved non-functional THBD versus hemoglobin AA mice (Figure 2A). Furthermore, staining of the glomerular microvasculature demonstrated decreased endothelial-bound THBD (Figure 2B). Patients with sickle cell anemia: We evaluated whether plasma concentrations of cleaved non-functional THBD are predictive of acute multiorgan failure syndrome (PMID 8109600) in a cohort of 103 SCD patients recruited into a longitudinal kidney cohort study. Clinical and laboratory variables and plasma samples were obtained at the time of enrolment and the patients were monitored prospectively for acute multiorgan failure syndrome. Hemoglobinuria was defined by urine dipstick positive for blood with &amp;lt; 2 red blood cells/high power field. The median age of this cohort was 35 years (interquartile range, 28 - 44 years), 46% were female, 87% had hemoglobin SS genotype, and 47% were on hydroxyurea at the time of enrolment. In cross-sectional analysis, plasma THBD concentrations were greater in patients with hemoglobinuria, a marker of intravascular hemolysis-derived cell-free hemoglobin in circulation exceeding scavenging capability and filtering through the glomerulus, versus without hemoglobinuria (6.1 ± 0.6 µg/mL vs. 3.6 ± 0.4 µg/mL, P = 0.004) (Figure 3A). With a median follow up of 5.5 years (interquartile range, 1.4 - 5.9 years), 18 (17%) SCD patients had a multiorgan failure event. SCD patients with a multiorgan failure event were older (43 vs. 34 years, P = 0.01) but without significant differences in sex, SCD genotype, or hydroxyurea therapy. After adjusting for age, baseline THBD concentrations predicted a greater risk for multi-organ failure syndrome (log-transformed OR 4.0, 95% CI: 1.2 - 13.3; P = 0.01) (Figure 3B). In conclusion, circulating non-functional THBD, a protein that normally functions to reduce vasculopathy when bound to the endothelium, is increased after endothelial cell exposure to incremental doses of hemoglobin in vitro, in hemoglobin SS vs. AA mice, and in SCD patients with versus without hemoglobinuria. Furthermore, circulating THBD is a biomarker that predicted the risk for multi-organ failure syndrome on longitudinal follow up in SCD patients. Future studies investigating the role of THBD in SCD vasculopathy may help improve our understanding for the catastrophic multiorgan failure syndrome and therapies to augment endothelial cell THBD function may guide future intervention practices. Figure 1 Disclosures Gordeuk: Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding. Saraf:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

Antiretroviral Therapy Does Not Correct the Increased Thrombin Generation and Platelet Hyperactivity Associated with HIV

Background: Human Immunodeficiency Virus (HIV) infection is associated with an increased risk of thrombosis, and treatment with antiretroviral therapy (ART) does not decrease this risk. While the mechanism leading to thrombosis is unclear, HIV infection is associated with decreased plasma anticoagulant proteins, most commonly protein S (PS), expression of tissue factor (TF) on monocytes, platelet hyperactivity, and thrombocytopenia. Despite the prevalence of PS deficiency, its pathologic consequences are unclear because PS concentration does not correlate with thrombin generation in the standard calibrated automated thrombography (CAT) assay. As increased plasma thrombin generation is a strong predictor of mortality in these patients, we sought to determine which of the described procoagulant effects of HIV is associated with thrombin generation in this population. Method: Blood was collected from 27 consenting HIV+ patients (16 naive samples collected from patients on first diagnosis and 11 samples from patients on ART) and 10 healthy controls. Results: 63% (17/27) of HIV+ plasma samples were deficient in PS, compared to the control samples, as determined by a total PS ELISA (p=0.034). PS functions as a cofactor for the anticoagulant enzyme activated protein C (APC), whose activation requires endothelial cell thrombomodulin (TM). Thus, to correlate plasma PS with thrombin generation, we established a modified CAT assay that is sensitive to PS concentration, by supplementing plasma samples with 20nM TM. Plasma PS did not correlate with thrombin generation in the absence of TM, consistent with previous reports. In the presence of TM, there was a negative correlation, with decreased PS being associated with increased thrombin peak height (p=0.004) and endogenous thrombin potential (p=0.003). Since 2.5% of total circulating PS is stored in platelets and released upon platelet stimulation, we quantified platelet PS by immunoblotting, and found no correlation with plasma PS concentration, consistent with the physiologically distinct origins of these two pools. HIV is reported to promote TF expression in monocytes, and monocytes can release TF-bearing microvesicles into plasma, so we also measured circulating TF activity, but found no difference between the patients and controls. As anticipated, TF correlated with a shorter lag time for thrombin generation, but only when no exogenous TF was added (p=0.0097). Treatment with ART resulted in undetectable viral load and also decreased plasma markers of inflammation, including TNF-α, IL-6, and IL-1β, to the levels observed in controls. However, there was no effect of ART on PS concentration (p=0.521), and platelet-leukocyte aggregates (PLA) remained elevated in patients on ART (19.66%) compared to controls (4.62%, p=0.0001). Platelet hyperactivity, as measured by PLA, was independent of platelet count, and did not correlate with platelet or plasma PS concentration. Interestingly, the use of ART had no impact on plasma PS or TF, but did correlate with greater thrombin generation in assays performed in the absence of TM, and thus the absence of PS activity (10% higher total thrombin, p=0.039), suggesting a PS-independent procoagulant effect of ART. Conclusion: PS deficiency in HIV patients correlates with higher thrombin generation and likely contributes to the thrombotic risk associated with this condition, while microvesicle TF, which does not differ from that seen in healthy controls, is unlikely to be a contributing factor. ART treatment decreases viral load and inflammation, but does not decrease platelet hyperactivity or restore plasma PS concentration and is not associated with any decrease in thrombin generation. Rather, one or more of the ART drugs appear to be further increasing thrombin generation through a PS-independent mechanism. In summary, it appears that multiple, independent factors contribute to the thrombotic risk associated with HIV, including platelet hyperactivity, plasma PS, and the ART treatment itself. Disclosures No relevant conflicts of interest to declare.

Cell-surface HSP70 associates with thrombomodulin in endothelial cells.

Heat shock protein-70 (HSP70) is crucial for proteostasis and displays cell-protective effects. Meanwhile, enhanced levels of cell-surface (cs) and secreted HSP70 paradoxically associate with pathologic cardiovascular conditions. However, mechanisms regulating csHSP70 pool are unknown. We hypothesized that total and csHSP70 expressions are modulated by hemodynamic forces, major contributors to endothelial pathophysiology. We also investigated whether thrombomodulin, a crucial thromboresistance cell-surface protein, is a csHSP70 target. We used proteomic/western analysis, confocal microscopy, and cs-biotinylation to analyze the pattern and specific characteristics of intracellular and csHSP70. HSP70 interaction with thrombomodulin was investigated by confocal colocalization, en face immunofluorescence, proximity assay, and immunoprecipitation. Thrombomodulin activity was assessed by measured protein C activation two-step assay. Our results show that csHSP70 pool in endothelial cells (EC) exhibits a peculiar cluster-like pattern and undergoes enhanced expression by physiological arterial-level laminar shear stress. Conversely, total and csHSP70 expressions were diminished under low shear stress, a known proatherogenic hemodynamic pattern. Furthermore, total HSP70 levels were decreased in aortic arch (associated with proatherogenic turbulent flow) compared with thoracic aorta (associated with atheroprotective laminar flow). Importantly, csHSP70 co-localized with thrombomodulin in cultured EC and aorta endothelium; proximity ligation assays and immunoprecipitation confirmed their physical interaction in EC. Remarkably, immunoneutralization of csHSP70 enhanced thrombomodulin activity in EC and aorta ex vivo. Overall, proatherogenic hemodynamic forces promote reduced total HSP70 expression, which might implicate in disturbed proteostasis; meanwhile, the associated decrease in cs-HSP70 pool associates with thromboresistance signaling. Cell-surface HSP70 (csHSP70) expression regulation and csHSP70 targets in vascular cells are unknown. We showed that HSP70 levels are shear stress-modulated and decreased under proatherogenic conditions. Remarkably, csHSP70 binds thrombomodulin and inhibits its activity in endothelial cells. This mechanism can potentially explain some deleterious effects previously associated with high extracellular HSP70 levels, as csHSP70 potentially could restrict thromboresistance and support thrombosis/inflammation in stress situations.

Decreased Expression of Thrombomodulin in Endothelial Cells by Fibroblast Growth Factor-23/α-Klotho.

Chronic kidney disease (CKD) has been known to be a state of excessive fibroblast growth factor-23 (FGF23) and α-Klotho deficiency. Patients undergoing hemodialysis have an increased mortality risk associated with cardiovascular disease and endothelial dysfunction. The mechanism responsible for the relationship of FGF23 to endothelial damage in these patients has been unclear. On the other hands, increasing evidences have demonstrated that thrombomodulin (TM) plays an important role in the endothelial barrier. Here, we report the suppression of membrane TM, in a dose-dependent manner, in human umbilical vein endothelial cells after FGF23 and FGF23/α-Klotho stimulation. In addition, the levels of soluble TM, which reflect endothelial cell injury, were much higher in cell supernatants after FGF23 and FGF23/α-Klotho stimulation than in the control supernatant. This study indicates a possible mechanism by which excessive levels of FGF23 are involved in endothelial TM disruption, which has been implicated as a potential cardiovascular risk factor in patients with CKD, especially in HD patients.

P1.04-023 Thrombomodulin Inhibits the Growth and Angiogenesis of Human Lung Cancer via Blocking VEGFR2-Mediated JAK/STAT3 Signaling Pathway

Angiogenesis has been an attractive target for drug therapy because of its key role in the growth and metastatic spread of malignant tumor. Thrombomodulin has been shown to possess anti-inflammatory and vascular endothelial protection activities. However, its roles in tumor angiogenesis are unknown. The aim of this study was to investigate the roles of thrombomodulin in tumor angiogenesis and its anticancer activities. ex vivo aortic ring angiogenesis sprouting assay was used to detect neo-vascularization. Western blotting was performed to examine STAT3 signaling cascade. Thrombomodulin significantly inhibited human umbilical vascular endothelial cell (HUVEC) proliferation, migration and tube formation in vitro and blocked vascular endothelial growth factor (VEGF)-triggered neo-vascularization. VEGF receptor (VEGFR) 2 mediated-Janus Kinase 2/STAT3 signaling pathway was significantly inhibited by thrombomodulin in endothelial cells. In addition, the constitutively activated STAT3 protein, and the expression of STAT3-dependent target genes, including cyclin D1, c-Myc, Bcl-xL, and VEGF were also down-regulated in response for thrombomodulin in human lung cancer cells. Consistent with the above findings, thrombomodulin inhibited tumor cell cycle progression and induced cell apoptosis in vitro. Therefore, our provided the first evidence that thrombomodulin inhibited tumor angiogenesis and growth via inhibiting VEGFR2-mediated JAK/STAT3 signaling pathway with the potential of a drug candidate for cancer therapy.

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions.

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2ng/ml and as early as 8hrs after exposure. TM activates protein C by altering thrombin’s substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells’ ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone’s effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Local extension of HMGB1 in atherosclerotic lesions of human main cerebral and carotid arteries.

High mobility group box 1 protein (HMGB1) is a non-histone chromosomal protein which is highly conserved, ubiquitous, and widely distributed. HMGB1 has multiple functions in the nucleus, including the maintenance of nucleosome structure, the regulation of gene transcription, and involvement in DNA recombination. HMBG1 is currently recognized to have a wide range of potential functions and pathological relevance. HMGB1 is released into the extracellular space from necrotic cells and from activated macrophages. HMGB1 binds to the receptor for advanced glycation end products, resulting in the induction of inflammatory cytokines, and to endothelial cell thrombomodulin. HMGB1 neutralization may also reduce the development of atherosclerosis and ameliorate brain infarction. We investigated the immunolocalization of HMGB1 in atherosclerotic lesions of human cerebral and carotid arteries using a specific antibody, and confirmed the detailed expression and cell type localization using double immunofluorolabeling. In the main cerebral arteries, this anti-HMGB1 antibody intensely immunolabeled both normal morphological vascular smooth muscle cells (VSMCs) within the tunica media and infiltrating VSMCs within the intima of thickened fibrous cap plaques. Endothelial cells were also positive for HMGB1. In carotid plaques, HMGB1-like immunoreactivity (IR) was intense in macrophages, although this IR decreased with increasing cell size. Medium-sized foam cells (50-150 μm) were the most intensely stained. This IR was also observed in the nuclei of foam cells and VSMCs. These findings may provide a basis for understanding the association of HMGB1 with atherosclerotic lesions of the cerebral and carotid arteries, and for constructing strategies to counteract atherosclerosis with anti-HMGB1 antibody.

Histone and DNA Methylation–Mediated Epigenetic Downregulation of Endothelial Kruppel-Like Factor 2 by Low-Density Lipoprotein Cholesterol

Low-density lipoprotein (LDL) cholesterol induces endothelial dysfunction and is a major modifiable risk factor for coronary heart disease. Endothelial Kruppel-like Factor 2 (KLF2) is a transcription factor that is vital to endothelium-dependent vascular homeostasis. The purpose of this study is to determine whether and how LDL affects endothelial KLF2 expression. LDL downregulates KLF2 expression and promoter activity in endothelial cells. LDL-induced decrease in KLF2 parallels changes in endothelial KLF2 target genes thrombomodulin, endothelial NO synthase, and plasminogen activator inhibitor-1. Pharmacological inhibition of DNA methyltransferases or knockdown of DNA methyltransferase 1 prevents downregulation of endothelial KLF2 by LDL. LDL induces endothelial DNA methyltransferase 1 expression and DNA methyltransferase activity. LDL stimulates binding of the DNA methyl-CpG-binding protein-2 and histone methyltransferase enhancer of zeste homolog 2, whereas decreases binding of the KLF2 transcriptional activator myocyte enhancing factor-2, to the KLF2 promoter in endothelial cells. Knockdown of myocyte enhancing factor-2, or mutation of the myocyte enhancing factor-2 site in the KLF2 promoter, abrogates LDL-induced downregulation of endothelial KLF2 and thrombomodulin, and KLF2 promoter activity. Similarly, knockdown of enhancer of zeste homolog 2 negates LDL-induced downregulation of KLF2 and thrombomodulin in endothelial cells. Finally, overexpression of KLF2 rescues LDL-induced clotting of platelet-rich plasma on endothelial cells. LDL represses endothelial KLF2 expression via DNA and histone methylation. Downregulation of KLF2 by LDL leads to a dysfunctional, hypercoagulable endothelium.

Production of cloned pigs expressing human thrombomodulin in endothelial cells

For long-term xenograft survival, coagulation control is one of the remaining critical issues. Our attention has been directed toward human thrombomodulin (hTM), because it is expected to exhibit the following beneficial effects on coagulation control and cytoprotection: (i) to solve the problem of molecular incompatibility in protein C activation; (ii) to exert a role as a physiological regulator, only when thrombin is formed; (iii) to suppress direct prothrombinase activity; and (iv) to have anti-inflammatory properties. hTM gene was transfected into pig (Landrace/Yorkshire) fibroblasts using pCAGGS expression vector and pPGK-puro vector. After puromycin selection, only fibroblasts expressing a high level of hTM were collected by cell sorting and then applied to nuclear transfer. Following electroactivation and subsequent culture, a total of 1547 cleaved embryos were transferred to seven surrogate mother pigs. Two healthy cloned piglets expressing hTM were born, successfully grew to maturity and produced normal progeny. Immunohistochemical staining of organs from F1 generation pigs demonstrated hTM expression in endothelial cells as well as parenchymal cells. High expression was observed particularly in endothelial cells of kidney and liver. Aortic endothelial cells from cloned pigs were found to express hTM levels similar to human umbilical vein endothelial cells (HUVEC) and to make it possible to convert protein C into activated protein C. The blockade of human endothelial cell protein C receptor (hEPCR) significantly reduced APC production in HUVEC, but not in hTM-PAEC. Although no bleeding tendency was observed in hTM-cloned pigs, activated partial thromboplastin time (APTT) was slightly prolonged and soluble hTM was detected in pig plasma. hTM was expressed in platelets and mononuclear cells, but not in RBC. Cloned pigs expressing hTM in endothelial cells at a comparable level to HUVEC were produced. As complete suppression of antigen-antibody reaction in the graft is essential for accurate assessment of transgene related to coagulation control, production of genetically engineered pigs expressing hTM and complement regulatory protein based on galactosyltransferase knockout is desired.

The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen

AbstractLewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM—TM domain 1 (rTMD1)—in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.

The effect of simvastatin on the survival of ischaemic skin flap: An experimental study in rats

Thrombomodulin (TM) is down-regulated from the vascular endothelial surfaces and corresponds to disturbed dermal blood flow and microthrombus formation in the ischaemic skin flap. We examined the therapeutic potential of simvastatin which up-regulates endothelial cell TM expression and activity in the dorsal ischaemic skin flap model. The study was carried out on 30 rats, divided into three groups. Group 1 was treated with simvastatin at a dose of 5mgkg(-1) day(-1) by intraperitoneal injection. Group 2 was treated with 1mgkg(-1) day(-1) with a phosphate-buffered saline for 7 days. Group 3 was the control group. Tissue blood flow, vascularisation and the survival rate of the skin flaps from each group were compared. The mean surviving area of group 1 was higher than groups 2 and 3 (p<0.05). The blood flow change rate did not decrease in the treatment group in contrast to the control groups at 3cm and 5cm (p<0.05). Microangiography demonstrated decreased flap vascularity in groups 2 and 3. There was no evidence of necrosis or positive peroxidase staining for TM in group 1 at 3cm and 5cm., although it was negative in groups 2 and 3. In this study, it was demonstrated that simvastatin prevented shedding of endothelial TM and contributed to flap survival.

Effect of C5a on expression of thrombomodulin in endothelial cells in vitro

To investigate the effect of C5a on the expression of thrombomodulin (TM) in human umbilical vein endothelial cells (HUVECs). HUVECs cultured in vitro were stimulated with C5a for 8, 12, 16, 20 hours in a concentration of 200 microg/L, and also with different concentrations of 100, 200, 300 microg/L for 12 hours respectively. The levels of both mRNA and protein expression of TM was detected by real-time polymerase chain reaction (PCR) and Western blotting respectively, and the dose and time dependent effects of C5a on the expression of TM were evaluated. C5a down-regulated the TM expression at both protein and mRNA level. The down-regulation was time-dependent [(93.11+/- 1.57) x10(-2) , (71.05+/-3.39)x10(-2), (65.48+/-4.28)x10(-2), (62.69+/-4.03)x10(-2)at protein level and (301.71+/-80.40)x10(-6), (38.29+/-20.24)x10(-6), (8.82+/-2.66)x10(-6), (7.05+/-0.80)x10(-6) at mRNA level, all P<0.05] and dose-dependent [(113.25+/-3.97)x10(-2), (80.18+/-2.56)x10(-2), (73.22+/- 4.36)x10(-2) at protein level and (401.77+/-20.46)x10(-6), (31.12+/-3.51)x10(-6), (18.19+/-1.46)x10(-6) at mRNA level , all P<0.05]. When concentration of C5a at 300 microg/L was used to stimulate HUVECs for longer than 12 hours, the lowering of TM at protein level was slowed down obviously. And concentration of C5a at 200 microg/L was used to stimulate HUVECs for 12 hours, the lowering of TM at mRNA level was slowed down obviously. C5a can depress the gene expression of TM, and then affect the protein's translation. By this means, C5a can lead to hypercoagulability and inflammatory injuries in sepsis.

Modulation of endothelial cell thrombomodulin by PPAR ligands — Variation according to environment

Introduction Thrombomodulin (TM) is an important anti-coagulant protein that is down-regulated on endothelial cells overlying atherosclerotic plaques. We investigated the effects of the peroxisome proliferator-activated receptor (PPAR) ligands, fenofibrate and rosiglitazone, on the expression of TM ex vivo by advanced carotid atheromas, and in vitro by endothelial cells. Methods Adjacent carotid atheroma biopsies were incubated in vehicle control or PPAR ligand in explant culture for 4 days. Human aortic endothelial cells were incubated with PPAR ligands in vitro. TM expression was measured by Western blotting and flow cytometry. TM activity was assessed by generation of activated protein C. Results The PPAR-α activator, fenofibrate, up-regulated total TM expression within carotid explants by 1.7-fold ( P < 0.001) with no effect on activity. Rosiglitazone treatment had no effect on protein levels but reduced activity by 73% of the control ( P < 0.05). We noted disparate effects of PPAR ligands in atheroma samples from different patients and postulated that the response of endothelial cells to medication was influenced by the atheromatous environment. Incubation of human aortic endothelial cells with fenofibrate alone led to a dose-dependent increase in TM expression ( P < 0.05), however, in the presence of oxidized LDL a dose-dependent reduction in TM expression was induced by fenofibrate ( P < 0.05). Conclusions The ability of fenofibrate to increase endothelial cell and carotid atheroma TM protein expression suggests a potential therapeutic role for this medication. The response to PPAR ligands likely varies depending on the exact constituents of individual atherosclerotic plaques, such as the relative amount of oxidized LDL.

Thrombin's faces revealed: cellular effects include induction of neoangiogenesis

Thrombin's faces revealed: cellular effects include induction of neoangiogenesis

Statins increase thrombomodulin expression and function in human endothelial cells by a nitric oxide-dependent mechanism and counteract tumor necrosis factor alpha-induced thrombomodulin downregulation.

Expression of functionally active thrombomodulin (TM) on the luminal surface of endothelial cells is critical for vascular thromboresistance. TM maintains thrombohemorrhagic homeostasis by forming a complex with thrombin, which subsequently loses its procoagulant properties and instead activates protein C. Acquired deficiency of endothelial TM is of particular pathophysiological significance in sepsis and related disorders. We show here that two different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), atorvastatin and simvastatin, strongly increase the expression and functional activity of TM in human umbilical vein endothelial cells, human coronary artery endothelial cells, and EA.hy926 endothelial cells. The increase in endothelial TM conferred by statin was prevented by the addition of mevalonic acid, geranylgeranyl-pyrophosphate, and nitric oxide scavenger, and was mimicked by the addition of a specific inhibitor of geranylgeranyl transferase, as well as by nitric oxide donors. Moreover, statin counteracted tumor necrosis factor alpha-induced downregulation of endothelial cell TM. The increase in endothelial cell TM activity in response to statin constitutes a novel pleiotropic (non-lipid-related) effect of these commonly used compounds, and may be of clinical significance in disorders where deficient endothelial TM and protein C activation play a pathophysiological role.

Association between decreased pulmonary endothelial cell thrombomodulin and local fibrin deposition in pneumonia.

Thrombomodulin (TM) plays an important role in anticoagulation by forming a complex with thrombin, which subsequently activates protein C. TM is inactivated and downregulated by inflammatory cell mediators. This study examined whether bronchopneumonia is associated with changes in TM immunoreactivity, and whether a decrease in TM is accompanied by evidence of hypercoagulability, i.e. local deposition of fibrin. Double antibody staining for TM and fibrin was performed on lung tissue sections from patients who had died of pneumonia and from patients who had died rapidly, secondary to trauma. Inflammatory changes were assessed histologically and immunohistochemically using antibodies against interleukin-1alpha, tumor necrosis factor-alpha, and myeloperoxidase. Areas with bronchopneumonia exhibited markedly decreased endothelial TM staining of alveolar walls and small vessels. These changes were associated with prominent fibrin immunoreactivity. Some areas exhibited mild to moderate inflammation with little fibrin deposition and variable amounts of TM in adjacent vessels. This study is the first to relate changes of TM immunoreactivity levels to fibrin deposition in a human disease process. These data may have implications for pulmonary pathophysiology in patients with bronchopneumonia.