Increased Thrombomodulin Expression Research Articles

S1P receptor-1 signaling reduces arterial thrombosis by upregulation of endothelial thrombomodulin expression

Abstract Background Sphingosine-1-phosphate (S1P) is a bioactive lipid known for its cardioprotective effects. The extent to which S1P also has effects on arterial thrombosis has not yet been fully elucidated. Another important factor in arterial thrombosis is thrombomodulin (TM), an important endothelial antithrombotic factor. To what extent S1P, TM, and, in addition, platelet adhesion as an initial part influences arterial thrombosis is unclear and the aim of this work. Methods The influence of S1P on human umbilical cord venous endothelial cells (HUVECs) was investigated in cell culture. To investigate effects of S1P and TM on platelet adhesion, flow chamber experiments were performed. In addition, murine arterial thrombus formation was analyzed by intravital microscopy. Results S1P increased TM expression on HUVECs. This effect was abolished by S1P receptor1 (S1PR1) inhibition with W146 (S1P: 1.081 ± 0.091 vs. S1P + W146: 0.951 ± 0.073, normalized to control, p<0.0001). Sphingosine kinase-2 deficient mice (SphK1-/-) lack the kinase primarily producing sphingosine. Accordingly, these animals have less S1P. Here, TM expression was significantly lower in the aorta compared to wild-type mice (WT: 207.2 ± 45.8 pg/g aorta vs. SphK1-/- 124.5 ± 40.2 pg/g aorta, p<0.0001). S1P treatment decreased platelet adhesion from whole blood to endothelial cells in the flow chamber (Con: 100.00 ± 28.09% vs. S1P: 80.91 ± 22.85%, p<0.0001). W146 reversed this effect by S1PR1 inhibition (S1P: 80.91 ± 22.85%, vs. S1P + W146: 118.60 ± 33.65%, p=0.0067). Platelet adhesion to endothelial cells was assessed in the presence of a TM-neutralizing antibody. The TM antibody abolished the anti-adhesive effects of S1P (S1P: 54.79 ± 29.59% vs. S1P+AB: 105.54 ± 35.43%, p=0.0174). TM inhibited intravital murine arterial thrombus formation (time to first occlusion: con: 9.38 ± 1.02 min vs. TM: 60 ± 0 min, p=0.0286). Furthermore, the duration of the first occlusion was significantly shorter and the embolic rate as a marker for thrombus stability was higher. Conclusion We demonstrated that S1P inhibits arterial thrombosis via regulation of endothelial TM. In particular, platelet adhesion to endothelial cells is reduced. This effect is mediated via S1PR1. The extent to which S1PR1 represents a potential antithrombotic target must be clarified in future studies.

Chondrocyte Thrombomodulin Protects against Osteoarthritis.

Osteoarthritis (OA) is a prevalent form of arthritis that affects over 32.5 million adults worldwide, causing significant cartilage damage and disability. Unfortunately, there are currently no effective treatments for OA, highlighting the need for novel therapeutic approaches. Thrombomodulin (TM), a glycoprotein expressed by chondrocytes and other cell types, has an unknown role in OA. Here, we investigated the function of TM in chondrocytes and OA using various methods, including recombinant TM (rTM), transgenic mice lacking the TM lectin-like domain (TMLeD/LeD), and a microRNA (miRNA) antagomir that increased TM expression. Results showed that chondrocyte-expressed TM and soluble TM [sTM, like recombinant TM domain 1 to 3 (rTMD123)] enhanced cell growth and migration, blocked interleukin-1β (IL-1β)-mediated signaling and protected against knee function and bone integrity loss in an anterior cruciate ligament transection (ACLT)-induced mouse model of OA. Conversely, TMLeD/LeD mice exhibited accelerated knee function loss, while treatment with rTMD123 protected against cartilage loss even one-week post-surgery. The administration of an miRNA antagomir (miR-up-TM) also increased TM expression and protected against cartilage damage in the OA model. These findings suggested that chondrocyte TM plays a crucial role in counteracting OA, and miR-up-TM may represent a promising therapeutic approach to protect against cartilage-related disorders.

Minimal Change Disease Is Associated With Endothelial Glycocalyx Degradation and Endothelial Activation

IntroductionMinimal change disease (MCD) is considered a podocyte disorder triggered by unknown circulating factors. Here, we hypothesized that the endothelial cell (EC) is also involved in MCD.MethodsWe studied 45 children with idiopathic nephrotic syndrome (44 had steroid sensitive nephrotic syndrome [SSNS], and 12 had biopsy-proven MCD), 21 adults with MCD, and 38 healthy controls (30 children, 8 adults). In circulation, we measured products of endothelial glycocalyx (EG) degradation (syndecan-1, heparan sulfate [HS] fragments), HS proteoglycan cleaving enzymes (matrix metalloprotease-2 [MMP-2], heparanase activity), and markers of endothelial activation (von Willebrand factor [vWF], thrombomodulin) by enzyme-linked immunosorbent assay (ELISA) and mass spectrometry. In human kidney tissue, we assessed glomerular EC (GEnC) activation by immunofluorescence of caveolin-1 (n = 11 MCD, n = 5 controls). In vitro, we cultured immortalized human GEnC with sera from control subjects and patients with MCD/SSNS sera in relapse (n = 5 per group) and performed Western blotting of thrombomodulin of cell lysates as surrogate marker of endothelial activation.ResultsIn circulation, median concentrations of all endothelial markers were higher in patients with active disease compared with controls and remained high in some patients during remission. In the MCD glomerulus, caveolin-1 expression was higher, in an endothelial-specific pattern, compared with controls. In cultured human GEnC, sera from children with MCD/SSNS in relapse increased thrombomodulin expression compared with control sera.ConclusionOur data show that alterations involving the systemic and glomerular endothelium are nearly universal in patients with MCD and SSNS, and that GEnC can be directly activated by circulating factors present in the MCD/SSNS sera during relapse.

Cerebral cavernous malformations form an anticoagulant vascular domain in humans and mice

AbstractCerebral cavernous malformations (CCMs) are common brain vascular dysplasias that are prone to acute and chronic hemorrhage with significant clinical sequelae. The pathogenesis of recurrent bleeding in CCM is incompletely understood. Here, we show that central nervous system hemorrhage in CCMs is associated with locally elevated expression of the anticoagulant endothelial receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR). TM levels are increased in human CCM lesions, as well as in the plasma of patients with CCMs. In mice, endothelial-specific genetic inactivation of Krit1 (Krit1ECKO) or Pdcd10 (Pdcd10ECKO), which cause CCM formation, results in increased levels of vascular TM and EPCR, as well as in enhanced generation of activated protein C (APC) on endothelial cells. Increased TM expression is due to upregulation of transcription factors KLF2 and KLF4 consequent to the loss of KRIT1 or PDCD10. Increased TM expression contributes to CCM hemorrhage, because genetic inactivation of 1 or 2 copies of the Thbd gene decreases brain hemorrhage in Pdcd10ECKO mice. Moreover, administration of blocking antibodies against TM and EPCR significantly reduced CCM hemorrhage in Pdcd10ECKO mice. Thus, a local increase in the endothelial cofactors that generate anticoagulant APC can contribute to bleeding in CCMs, and plasma soluble TM may represent a biomarker for hemorrhagic risk in CCMs.

IKKβ regulates endothelial thrombomodulin in a Klf2‐dependent manner

Endothelial thrombomodulin (TM) is critically involved in anticoagulation, anti-inflammation, cytoprotection and normal fetal development. Tumor necrosis factor alpha (TNFα) suppresses TM expression. TNFα has been shown to down-regulate TM partly via activation of nuclear factor kappa B (NF-κB). However, because the TM promoter lacks an NF-κB binding site, the direct involvement of NF-κB has been controversial. We investigated the role of the upstream regulatory serine kinase, inhibitory kappa-B kinase-β (IKKβ), in TM expression and function with or without TNFα treatment. Inhibition of IKKβ was achieved by specific chemical inhibitors, siRNA or shRNA. TM expression was assessed by qRT-PCR, Western blot, flow cytometry, luciferase reporter assay and chromatin immune-precipitation (ChIP) assay. TM function was estimated by generation of activated protein C (APC). NF-κB activation was determined by immunocytochemistry. IKKβ inhibition increased TM expression and function, and attenuated TNFα-mediated TM down-regulation. In contrast, inhibition of downstream canonical NF-κB protein family members p50 and p65 (RelA) failed to up-regulate TM expression and did not affect IKKβ inhibition-mediated TM over-expression. However, knockdown of cRel and RelB, family members of the canonical and non-canonical NF-κB pathway, respectively, resulted in TM over-expression. IKKβ inhibition caused over-expression, increased promoter activity and enhanced binding of Krüppel-like factor 2 (Klf2) to the TM promoter, which positively regulates TM expression. Finally, knockdown of Klf2 completely attenuated IKKβ inhibition-mediated TM up-regulation. We conclude that IKKβ regulates TM in a Klf2-dependent manner.

Thrombomodulin expression regulates tumorigenesis in bladder cancer.

BackgroundThe identification of potential tumor markers will help improve therapeutic planning and patient management. Thrombomodulin (TM) is a sensitive urothelial marker. TM was reported to be one of the endogenous anti-metastatic factors and has diagnostic and prognostic values for the progression of carcinoma. In the present study, we examine the role of TM in bladder cancer.MethodsWe studied the role of TM in tumor behavior and related signaling pathways in vitro using the human bladder cancer cell lines HT1376, HT1197, J82 and T24, and in vivo using animal models. We also selected clinical specimens from 100 patients with bladder cancer for immunohistochemical staining to evaluate the predictive capacity of TM in tumor invasiveness.ResultsThe data revealed that positive immunoreactivity for TM was inversely correlated with clinical stage and DNA methyltransferase 1 immunoreactivity. Decreased TM expression could predict the aggressive tumor growth and advanced clinical stage in bladder cancer. When TM was inhibited, tumor growth rate and invasion ability were augmented in vitro and in vivo. The underlying changes included increased cell proliferation, enhanced epithelial-mesenchymal transition (EMT) and angiogenesis. Moreover, inhibition of NF-κB activation significantly increased TM expression and attenuated tumor aggressiveness in bladder cancer.ConclusionsTM plays an important role in bladder cancer tumor aggressiveness in vitro and in vivo and is a clinically significant predictor that may represent a suitable therapeutic target for bladder cancer.

Thrombomodulin, a novel molecule regulating inorganic phosphate-induced vascular smooth muscle cell calcification

Hyperphosphatemia has emerged as a cardiovascular risk factor that stimulates calcification in vessels. We explored molecules that were induced by inorganic phosphate (Pi) at an early stage in vascular smooth muscle cells (VSMC). In the present study, we examined the role of thrombomodulin (TM) in Pi-induced VSMC calcification based on the results of DNA microarray analysis. Both mRNA and protein expression of TM were markedly augmented in Pi-induced calcification. Conversely, knockdown of TM by siRNA significantly inhibited calcification, in addition to Pi-induced apoptosis which plays critical roles in VSMC calcification. We further found that TM suppressed both of mRNA and protein expression of growth arrest-specific gene 6 (Gas6), a key molecule regulating apoptosis. Recombinant extracellular epidermal growth factor (EGF)-repeat domain of TM exaggerated calcification and this effect was abrogated by a neutralizing antibody for EGF receptor, suggesting that the cleaved and secreted form of TM may activate EGF receptor. We also found that downregulation of Gas6 by TM/EGF receptor axis was mediated by ERK in VSMC calcification. In the aorta of adenine-fed rat, a typical medial calcification model with hyperphosphatemia, we found that TM expression was increased. Furthermore, in human calcified aorta, increased TM expression was also observed. These results indicate that TM is a novel molecule that promotes apoptosis and vascular calcification by regulation of Gas6, presumably via EGF receptor/ERK axis.

Thrombomodulin and the brain

In this issue of Neurology ®, Giwa et al.1 report robust expression of thrombomodulin (TM) in small vessel disease, i.e., in small brain arteries or arterioles that exhibit the typical hyalinized thickening characteristic of chronic hypertensive changes. Using immunohistochemistry of postmortem brain tissue, the authors demonstrate extensive presence of TM in these small arterial vessels, with substantial association between extent of extent of vessel sclerosis and TM expression. Not only was small vessel TM expression significantly less in age-matched controls, but younger subjects with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy also exhibited increased TM expression in small caliber arterial vessels. Enhanced expression of TM thus appears to be characteristic of cerebral small vessel disease, and the authors suggest that the TM in this setting may be both compensatory and protective. It would be an understatement to say that TM is not commonly discussed within neurologic circles. Within the field of vascular biology, however, TM remains an intense focus of attention, with a recent review article even referring to this molecule as the “protectorate God of the vasculature.”2 TM is an integral membrane protein routinely found in vascular endothelium of …

Ets-1 mediates platelet-derived growth factor-BB-induced thrombomodulin expression in human vascular smooth muscle cells

Thrombomodulin (TM), a potent anticoagulant, is not detected in quiescent vascular smooth muscle cells (VSMCs). In diseased vessels, VSMC expresses TM, but the mechanisms are unclear. This study examined molecular mechanisms for TM expression in VSMCs. Platelet-derived growth factor-BB (PDGF-BB) induced TM expression in cultured human aortic VSMCs. PDGF-induced TM is functional in activating protein C. TM induction was eliminated by inhibitors of Src kinase, phosphatidylinositol 3-kinase (PI3-kinase), and mammalian target of rapamycin (mTOR) and by expressing dominant-negative Akt while expressing active Akt-stimulated TM expression. PDGF-BB activated the TM promoter, and the deletion of a sequence segment -394/-255 drastically reduced TM promoter activity. Transcription factor E26 transformation-specific sequence-1 (Ets-1) was upregulated by PDGF-BB in a PI3-kinase- and mTOR-dependent manner. RNA interference of Ets-1 inhibited PDGF induction of TM, and overexpressing Ets-1 increased TM expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay detected increased Ets-1 binding to the TM promoter after PDGF treatment. Following carotid artery ligation of C57/BL6 mice, PDGF-BB and TM were co-expressed in the media and neointima. In VSMCs, PDGF-BB stimulates TM expression that is mainly mediated by Ets-1 via the Src kinase/PI3-kinase/Akt/mTOR signalling pathway. Furthermore, PDGF-BB may regulate TM expression in VSMCs during vascular remodelling.

Impact of Short-Term Administration of High-Density Lipoproteins and Atorvastatin on Atherosclerosis in Rabbits

This study investigates effects of short-term administration of high-density lipoproteins (HDL) and a statin on atherosclerosis in cholesterol-fed rabbits. Effects of HDL apolipoprotein and phospholipid composition have also been investigated. Aortic atherosclerosis was established over 17 weeks in 46 rabbits by balloon denudation and cholesterol feeding. During the past 5 days of the cholesterol-feeding period, animals received: (1) no treatment; (2) oral atorvastatin 5 mg/kg on each of the 5 days; or (3) infusions of HDL (8 mg/kg apolipoprotein A-I) on days 1 and 3 of the treatment phase. After euthanization, lesion size and composition were assessed by histological and immunohistochemical analysis. HDL (but not atorvastatin) reduced lesion size by 36% (P<0.05). The ratio of smooth muscle cells to macrophages in the lesions increased 2.6-fold in animals infused with HDL (P<0.05) and 4-fold in those receiving atorvastatin (P<0.01). HDL and atorvastatin reduced matrix metalloproteinase (MMP)-9 expression by 42% (P<0.05) and 45% (P<0.03), respectively. HDL increased thrombomodulin expression 2-fold (P<0.03). The beneficial effects on lesion area and plaque cellular composition were influenced by HDL phospholipid and apolipoprotein composition. Infusing small amounts of HDL rapidly reduces lesion size and is comparable to atorvastatin in promoting a stable plaque phenotype.

Statins and Blood Coagulation

The 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. There is growing evidence that treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation. Accordingly, statin use has been associated with impairment of several coagulant reactions catalyzed by this enzyme. Moreover, evidence indicates that statins, via increased thrombomodulin expression on endothelial cells, may enhance the activity of the protein C anticoagulant pathway. Most of the antithrombotic effects of statins are attributed to the inhibition of isoprenylation of signaling proteins. These novel properties of statins, suggesting that these drugs might act as mild anticoagulants, may explain, at least in part, the therapeutic benefits observed in a wide spectrum of patients with varying cholesterol levels, including subjects with acute coronary events. The HMG-CoA reductase inhibitors (statins) have been shown to exhibit several vascular protective effects, including antithrombotic properties, that are not related to changes in lipid profile. Treatment with statins can lead to a significant downregulation of the blood coagulation cascade, most probably as a result of decreased tissue factor expression, which leads to reduced thrombin generation.

New nonlipid effects of statins and their clinical relevance in cardiovascular disease.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, statins, have been demonstrated to reduce cardiovascular morbidity and mortality in patients with a wide range of cholesterol levels. Numerous cholesterol-independent effects of statins that may limit atherosclerosis are probably related to inhibition of the geranylgeranylation of GTP-binding intracellular signaling proteins and involve: improved vasoreactivity, mostly through increased NO bioavailability; decreased expression of proinflammatory cytokines (interleukin-6, interleukin-1 beta, tumor necrosis factor alpha), C-reactive protein, chemokines, matrix metalloproteinases, and tissue factor with the subsequent inhibition of thrombin generation; reduced platelet activity; increased thrombomodulin expression; enhanced fibrinolysis, regulation of angiogenesis and immunomodulation. However, the clinical relevance of multiple protective effects induced by statins has not been clarified yet.

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.

Influence of pentoxifylline on membrane thrombomodulin levels in endothelial cells submitted to hypoxic conditions.

Thrombomodulin (TM) expression at the surface of endothelial cells is upregulated by cAMP analogues, whereas hypoxic conditions induce a decrease in this expression. Pentoxifylline (PTX) enhances the intracellular levels of cAMP in human umbilical vein endothelial cells (HUVECs). We therefore investigated the influence of this drug on TM expression in cells submitted to hypoxia. TM expression was quantified at the surface of HUVECs in the presence or absence of PTX during 18 h of incubation. Membrane TM was immunolabeled with a fluorescent monoclonal antibody and the expression was quantified by flow cytometric analysis. Our preliminary data revealed that PTX at 0.01 microM significantly increased TM expression levels compared with cells in hypoxia without the drug. Therefore, at this low dose PTX appears able to counterbalance the procoagulant effect of hypoxia.