Endothelial Thrombomodulin Research Articles

Macro- and microscale variables regulate stent haemodynamics, fibrin deposition and thrombomodulin expression

Drug eluting stents are associated with late stent thrombosis (LST), delayed healing and prolonged exposure of stent struts to blood flow. Using macroscale disturbed and undisturbed fluid flow waveforms, we numerically and experimentally determined the effects of microscale model strut geometries upon the generation of prothrombotic conditions that are mediated by flow perturbations. Rectangular cross-sectional stent strut geometries of varying heights and corresponding streamlined versions were studied in the presence of disturbed and undisturbed bulk fluid flow. Numerical simulations and particle flow visualization experiments demonstrated that the interaction of bulk fluid flow and stent struts regulated the generation, size and dynamics of the peristrut flow recirculation zones. In the absence of endothelial cells, deposition of thrombin-generated fibrin occurred primarily in the recirculation zones. When endothelium was present, peristrut expression of anticoagulant thrombomodulin (TM) was dependent on strut height and geometry. Thinner and streamlined strut geometries reduced peristrut flow recirculation zones decreasing prothrombotic fibrin deposition and increasing endothelial anticoagulant TM expression. The studies define physical and functional consequences of macro- and microscale variables that relate to thrombogenicity associated with the most current stent designs, and particularly to LST.

Ulcerative Colitis and Limb Loss: A Case Report

Introduction: Protein C is a precursor of a vitamin K-dependent serine protease, which is activated by thrombin in the presence of endothelial cofactor, thrombomodulin and endothelial cell protein C receptor. Case Presentation: A 33-year old woman with acute exacerbation of ulcerative colitis was found to have protein C deficiency (serum protein C activity estimated as 35% of the normal activity). Protein C deficiency was diagnosed after the onset of several arterial thromboemboli complications (brachiocephalic and popliteal artery thrombosis) during her hospital admission for exacerbation of ulcerative colitis (UC). Therefore, upper limb below elbow and lower limb below knee amputation were performed for her. Conclusions: After total proctocolectomy, serum protein C activity remained as 35% of the normal activity, so the patient used warfarin as lifelong anticoagulant. Investigation of other family members revealed normal thrombophilia screening tests.

Anticoagulant effects of statins and their clinical implications

There is evidence indicating that statins (3-hydroxy-methylglutaryl coenzyme A reductase inhibitors) may produce several cholesterol-independent antithrombotic effects. In this review, we provide an update on the current understanding of the interactions between statins and blood coagulation and their potential relevance to the prevention of venous thromboembolism (VTE). Anticoagulant properties of statins reported in experimental and clinical studies involve decreased tissue factor expression resulting in reduced thrombin generation and attenuation of pro-coagulant reactions catalysed by thrombin, such as fibrinogen cleavage, factor V and factor XIII activation, as well as enhanced endothelial thrombomodulin expression, resulting in increased protein C activation and factor Va inactivation. Observational studies and one randomized trial have shown reduced VTE risk in subjects receiving statins, although their findings still generate much controversy and suggest that the most potent statin rosuvastatin exerts the largest effect.

Regulation Of Hematopoietic Stem Cell Trafficking By The Coagulation Pathway

Hematopoeitic stem and progenitor cells (HSPC) dynamically switch between a quiescent, non-motile mode in the bone marrow (BM), to an active state, in which they proliferate, differentiate and egress to the circulation. Injection of the coagulation protease thrombin induced rapid HSPC mobilization to the blood via activation of its major receptor, protease activated receptor 1 (PAR1) on BM hematopoietic and stromal cells. We hypothesized that coagulation factors control stem cells fate in the BM. We examined if thrombin is generated in the murine BM and found by immunohistochemistry prothrombin associated with bone lining osteoblasts in the endosteum region. These cells also highly express osteopontin which induces stem cell quiescence and retention. Cleavage of osteopontin by thrombin or by osteoclast derived cathepsin K induces stem cell mobilization. In addition, a unique structure of multinucleated CD45+ cell clusters in the trabecular-rich area of the murine femoral metaphysis express the cell surface receptor Tissue Factor (TF), a potent initiator of the coagulation cascade leading to thrombin generation. These clusters were found adjacent to multinucleated TRAP (tatrate resistant acid phosphate) positive active osteoclasts. In vitro, we found that immature osteoclasts expressed TF in cell fusion areas, suggesting that osteoclast maturation also activates the coagulation thrombin/PAR1 axis, thus mediating HSPC recruitment to the circulation. Supporting this notion, bleeding which prompts a hemostatic response and thrombin production, is a strong inducer of osteoclasts activation and HSPC mobilization. In addition, injection of bacterial lipopolysaccharides (LPS) is known to activate osteoclasts and induce HSPC mobilization (Kollet et al Nat Med 06). We found that LPS injection upregulated TF expression by CD45+ myeloid cells in the murine BM. LPS treatment provoked massive HSPC mobilization, which was attenuated by PAR1 inhibition. To further address the role of thrombin in stem cell maintenance, we targeted prothrombin in vivo by applying Antisense Oligonucleotides (ASO) knockdown technology, previously shown to induce a dose- and time-dependent up to 90% reduction of prothrombin mRNA levels in the murine liver (Monia et al Blood 2010). Prothrombin depletion altered the BM niche microenvironment by expanding the mesenchymal stem and progenitor (MSPC) population and the long-term repopulating CD34-/ROSlow/LSK HSPC population in the BM. In untreated mice, TF was also expressed by a small MSPC population, suggesting that the bone stomal compartment may also contribute to the regulation of HSPC mobilization upon demand. To further asses the role of thrombin generation in HSPC development, we examined the involvement of the endothelial cell receptor Thrombomodulin (TM) that is pivotal for the anticoagulant pathway which mediates activation of protein C. TM protein is expressed by BM small blood vessels resembling sinusoids and by neighboring MSPC. By immunohistochemistry, we also detected activated protein C on the same blood vessels. A mouse model with a mutation in the TM gene (TMPro/Pro) is characterized by reduced capacity for activated protein C generation which in turn increases thrombin levels in these mice. We found increased circulating hematopoietic stem cells in TMPro/Pro mice, suggesting that chronically increased basal levels of thrombin generation can promote HSC egress. Conversely, short term (5 day) intermittent treatment of mice with low dose thrombin that mainly causes activated protein C formation in vivo, display higher levels of CD34-/ROSlow/LSK and EPCR+LSK stem cells in the BM, indicating additional roles for the anticoagulant pathway in BM stem cell pool maintenance. In summary, our results provide evidence that the activator of the coagulation cascade, TF, and coagulation factors Thrombin and activated protein C are present in the BM and regulate and integrate functions of hematopoietic stem and progenitor cells and BM stromal progenitor cells. Disclosures: Crosby: Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other. Monia:Isis pharmaceuticals: the ASO for prothrombin was obtained from Isis pharmaceuticals Other.

A Comparison Of Hemostatic and Inflammatory Markers In Overt and Non-Overt DIC

▪Disseminated intravascular coagulation (DIC) is a complex pathophysiologic syndrome with marked alterations in the hemostatic system, which may lead to microvascular thrombosis and multi-organ failure. The International Society of Thrombosis and Hemostasis (ISTH) classifies DIC into two distinct categories; overt and non-overt DIC (NO). Overt DIC is characterized by a decompensated hemostatic system whereas non-overt is characterized as a stressed, compensated system. DIC is a frequently observed in sepsis patients. An association between the hemostatic aberrations and inflammation has been previously reported. Therefore we studied the level of hemostatic activation and inflammation in patients with sepsis and suspected DIC enrolled in the ART-123, Phase 2b study. The level of inflammatory mediators such as anaphylatoxin C5a (C5a), procalcitonin (PCT), interleukin 6 (IL-6), interleukin 10 (IL-10), myeloperoxidase (MPO), and circulating levels of hemostatic markers including protein C inhibitor (PCI), plasminogen activator inhibitor 1 (PAI-1), and protein C (Pr C) were evaluated in 617 subjects enrolled in a randomized, double-blind, placebo-controlled, Phase-2b study evaluating the safety and efficacy of recombinant thrombomodulin (ART-123) in subjects with sepsis and suspected DIC. When the subjects with overt DIC and with NO DIC were compared, the PrC level was lower and PCT, PAI-1, IL-6 and IL-10 levels were higher, in the overt DIC subjects (Table). The upregulation of the inflammatory cytokines observed in this study may contribute to the decreased levels of Pr C. Inflammatory cytokines may dysregulate the endothelial thrombomodulin resulting impaired hemostatic regulation. Pr C levels are also low due to consumption and impaired synthesis, especially in the overt DIC patients. These results suggest that profiling these mediators may be useful in the diagnostic and prognostic management of sepsis and differentiation between overt and non-overt DIC. Disclosures:Hoppensteadt:Asahi Kasei Pharma: Research Funding. Tsuruta:Asahi Kasei Pharma: Employment. Hirman:Asahi Kasei Pharma: Consultancy. Kaul:Asahi Kasei Pharma: Employment. Osawa:Asahi Kasei Pharma: Employment. Fareed:Asahi Kasei Pharma: Research Funding.BiomarkerGroupNMeanSEMMedianP ValuePr C (%)Normal4094.52.491.80.0043Overt9824.71.828.30.0043NO15746.52.637.00.0043PCTNormal240.260.020.270.0001(ng/ml)Overt9836.87.011.220.0001NO51918.031.45.330.0001PAI-1Normal3120.62.118.7<0.0001(ng/ml)Overt98232.925.0137.5<0.0001NO519147.18.676.0<0.0001Il-6Normal306.80.536.10.0003(pg/ml)Overt981017.6121.7374.40.0003NO519576.739.6174.70.0003IL-10Normal3210.42.43.50.0049(pg/ml)Overt98187.339.146.00.0049NO518108.114.831.00.0049

Single Nucleotide Polymorphisms Within The Thrombomodulin Gene (THBD) Predict Risk Of Non-Relapse Mortality In Patients With Graft-Versus-Host Disease

Purpose Acute graft-versus-host disease (GVHD) is a major complication after allogeneic stem cell transplantation (allo-SCT). With the current treatment approaches focussing on escalating immunosuppression, mortality rates of therapy-refractory disease courses are approx. 80-90%, underlining the medical need for novel therapeutic concepts. The pathophysiology of refractory GVHD is incompletely understood, however, recent evidence points towards a crucial role for endothelial damage. We have previously shown that steroid-refractory GVHD associates with increasing serum thrombomodulin (TM) levels 1, 2 and loss of endothelial TM expression 3, a constellation that indicates endothelial stress. However prior to allo-SCT, baseline TM levels did not predict steroid-refractory GVHD or survival. We hypothesized that endothelial vulnerability caused by single nucleotide polymorphisms within the TM gene (THBD) is a risk factor for refractory GVHD predicting outcome after allo-SCT. Patients and Methods Seven SNPs within the THBD gene were studied (rs1962.TC, rs1042579.TC, rs1042580.AG, rs3176123.TG, rs3176124GA, rs3176126.GA and rs3176134.CT) in a training cohort of 465 allografted patients. The relevant genotypes were then re-assessed in an independent validation cohort (n=386). Results Allele frequencies of seven THBD SNPs allowed meaningful statistical correlation with outcome after allo-SCT. An increased risk of non-relapse mortality (NRM) was associated with three SNPs: rs1962.CC, rs1042579.TT (455V) and rs1042580.GG. When patients were divided into risk cohorts (one vs. no high-risk SNP), a strong correlation with NRM was observed in both cohorts (training cohort: p=0.002, HR 2.32 CI 1.36-3.95; validation cohort: p=0.007, HR 2.13, CI 1.23-3.70) (Figure 1). NRM was predicted by THBD SNPs in particular for patients who later develop GVHD (validation cohort: p=0.00056, HR 3.03 CI 5.68-1.61, training cohort: p=0.012, HR 2.38 CI 1.21-4.69) but not in patients without GVHD. In contrast, THBD SNPs did not predict incidence of acute GVHD. Multivariate analyses adjusting for clinical variables confirmed the independent effect of THBD SNPs on NRM in both cohorts. Conclusions Our results demonstrate that THBD-SNPs predict mortality of GVHD patients without influencing GVHD incidence. This supports our hypothesis that endothelial vulnerability contributes to therapy refractory courses of the disease. Rather than focussing on the escalation of combinations of (endothelial cell toxic) immunosuppressive drugs, therapeutic approaches aiming at endothelial protection should be evaluated. 1. Dietrich S, Falk CS, Benner A, et al. Endothelial vulnerability and endothelial damage are associated with risk of graft-versus-host disease and response to steroid treatment. Biol Blood Marrow Transplant 2013; 19(1): 22-7. 2. Luft T, Dietrich S, Falk C, et al. Steroid-refractory GVHD: T-cell attack within a vulnerable endothelial system. Blood 2011; 118(6): 1685-92. 3. Andrulis M, Dietrich S, Longerich T, et al. Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease. Haematologica 2012; 97(11): 1674-7. Disclosures: Dreger: Riemser Pharma : Consultancy, Honoraria, Research Funding.

Tissue Factor Pathway Inhibitor Modulates Fibrin Deposition In The Brain In a Manner That Is Independent Of Thrombomodulin and Activated Protein C

BackgroundFibrin deposition in cerebral vessels contributes to the pathogenesis of stroke and vascular dementia. Under quiescent conditions fibrin deposition is prevented or limited by natural anticoagulants of the endothelium. Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two major endothelial anticoagulants and act on different portions of the clotting cascade. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and FXa. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. Both proteins are reported to have low expression in the brain and their function in prevention of cerebral vascular fibrin deposition is unclear. This is emphasized by findings from murine models of decreased TFPI and TM (TMpro/pro) activity. TFPI null embryos have fibrin deposition in the brain and liver, while TMpro/pro mice have fibrin deposition in the lungs, heart, spleen and liver, but the brain is protected from fibrin deposition even following lethal LPS doses. Interestingly, TFPI heterozygosity combined with TMpro/pro results in cerebral vascular fibrin. Here, we report a detailed analysis of TFPI expression in the brain vasculature, the consequences of vascular TFPI deficiency in adult mice, and the capacity of APC to compensate for TFPI deficiency. Methods and ResultsReal time PCR studies demonstrated that TFPI expression in total brain is lower than in other tissues in comparison to the RPL-19 housekeeping gene. However, when the endothelial-specific genes, VE-cadherin or CD31 were used as housekeeping genes, brain TFPI expression was higher than that of lung endothelium (TFPIa: ∼9 fold higher; TFPIb: ∼5-fold higher). In situ hybridization studies revealed that TFPI and TM are expressed on endothelium throughout the brain parenchyma. In addition, TFPI was expressed within the granule layer of the cerebellum. TFPI null embryos succumb to embryonic lethality with necrotic lesions and fibrin deposition in the brain. To determine if increased TM-APC anticoagulant activity would rescue TFPI null embryos, a transgene over expressing human activated protein C (hHPC) was bred into heterozygous TFPI mice, which were then bred to determine if TFPI null mice expressing hHPC survive embryogenesis. However, the hHPC transgene did not rescue TFPI null embryos. Furthermore, TFPI null embryos had similar brain lesions and brain fibrin deposition regardless of the presence of the hHPC transgene. A hematopoietic cell transplant model system was used to examine the consequences of TFPI deficiency in the adult mouse brain. Mice transplanted with hematopoietic cells lacking TFPI had cerebral vascular fibrin deposition that was not observed in mice transplanted with hematopoietic cells producing normal amounts of TFPI. Interestingly, the fibrin was often found at sites of endothelial injury as demonstrated by co-localization with anti-C1qA, which binds to phosphatidylserine on damaged endothelium. ConclusionsTFPI prevents necrotic brain lesions and cerebral fibrin deposition in developing mouse embryos and over expression of hHPC is not sufficient to compensate for the absence of TFPI. Adult mice lacking TFPI in hematopoietic cells have increased amounts of fibrin deposited at sites of cerebral vascular injury despite having normal endothelial TM. These data indicate that the distinct function of TFPI in the cerebral vasculature is not compensated by the anticoagulant activity of TM in regulating cerebral vascular thrombosis. Disclosures:Mast:Novo Nordisk: Honoraria, Research Funding.

Rapamycin modulates anti- and pro-thrombotic gene expressions via Kruppel-like factor 2: in vitro studies

Purpose: Stent thrombosis (ST) is one of severe complications after sirolimus-eluting stent (SES) implantation. However, the underlying transcriptional mechanisms of this adverse effect have less been investigated. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with rapamycin and gene expression profiling including Krupple-like factor 2 (KLF2), tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), endothelial nitric oxide synthase (eNOS), thrombomodulin (TM), and mammalian target of rapamycin (mTOR) were determined. Cells infected with adenovirus-KLF2 (Ad-KLF2) were stimulated by rapamycin and the expressions of the preceding genes were also examined. At same time, the effects of cell lysates on blood clotting time were evaluated in vitro. Results: Rapamycin enhanced TF, PAI-1 but inhibited KLF2, eNOS and TM expressions in a dose-dependent manner, which were antagonized by FK-506, a competitor of rapamycin. Overexpression of KLF2 strongly reversed rapamycin-induced effects on preceding gene expressions. Meantime, KLF2 overexpression increased the time of blood clotting in vitro. Conclusion: Rapamycin resulted in an imbalance of anti- and pro- thrombotic gene expression profiling, which was reversed by overexpression of KLF2, suggesting that that the KLF2 pathway might be involved in ST after SES implantation.

Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects

Thrombomodulin (TM) is a 557-amino acid protein with a broad cell and tissue distribution consistent with its wide-ranging physiological roles. When expressed on the lumenal surface of vascular endothelial cells in both large vessels and capillaries, its primary function is to mediate endothelial thromboresistance. The complete integral membrane-bound protein form displays five distinct functional domains, although shorter soluble (functional) variants comprising the extracellular domains have also been reported in fluids such as serum and urine. TM-mediated binding of thrombin is known to enhance the specificity of the latter serine protease toward both protein C and thrombin activatable fibrinolysis inhibitor (TAFI), increasing their proteolytic activation rate by almost three orders of magnitude with concomitant anticoagulant, antifibrinolytic, and anti-inflammatory benefits to the vascular wall. Recent years have seen an abundance of research into the cellular mechanisms governing endothelial TM production, processing, and regulation (including flow-mediated mechanoregulation)—from transcriptional and posttranscriptional (miRNA) regulation of TM gene expression, to posttranslational processing and release of the expressed protein—facilitating greater exploitation of its therapeutic potential. The goal of the present paper is to comprehensively review the endothelial/TM system from these regulatory perspectives and draw some fresh conclusions. This paper will conclude with a timely examination of the current status of TM's growing therapeutic appeal, from novel strategies to improve the clinical efficacy of recombinant TM analogs for resolution of vascular disorders such as disseminated intravascular coagulation (DIC), to an examination of the complex pleiotropic relationship between statin treatment and TM expression.

Reciprocal antagonism between inflammation and the protein C system

Protein C is a vitamin K-dependent serine protease secreted by the hepatocytes as an inactive zymogen and activated by thrombin bound to endothelial thrombomodulin. An endothelial protein C receptor (EPCR) is involved in both activation and enhancement of protein C activity, resulting in proteolytic degradation of clotting factors Va and VIIIa, thereby providing an efficient anticoagulant mechanism. Evidence was also provided that proinflammatory cytokines would impair the endothelia-mediated activation and activity of the Protein C system by inducing an internalization and proteolytic degradation of thrombomodulin and by shedding EPCR from the surface of endothelial cells membrane. Clinical and experimental studies also emphasized that an inflammatory acute phase reaction is accompanied by a commuted hepatic protein synthesis leading to an increase of plasma fibrinogen, factor VIII:C and of α1 protease inhibitor, while the plasma level of protein C zymogen decrease. On the other hand infusions of activated protein C were reported to protect from a toxico-septic shock by exerting not only anticoagulant but also anti-inflammatory effects.

EFFECT OF CIGARETTE SMOKE EXTRACT ON THE EXPRESSION OF THROMBOMODULIN IN HUMAN ENDOTHELIAL CELLS

ObjectivesCigarette smoking cause triggering of coronary thrombosis. However, the mechanism by which smoking cause arterial thrombosis remains unknown. Thrombomodulin (TM) is constitutively expressed on the endothelial cell surface. TM has...

Free fatty acids inhibit TM–EPCR expression through JNK pathway: an implication for the development of the prothrombotic state in metabolic syndrome

Metabolic syndrome is associated with significant hypercoagulable prothrombotic tendency; however, the mechanism for the prothrombotic state is not completely understood. We hypothesize that higher circulating plasma free fatty acids (FFAs) in metabolic syndrome inhibit the endothelial thrombomodulin (TM)-endothelial protein C receptor (EPCR) pathway, thereby promoting thrombus formation. Human umbilical vein endothelial cells were cultured in media supplemented with various doses of palmitic acid (PA), in the presence or absence of JNK inhibitor, and the expression of TM and EPCR was measured by western blot. The thrombotic state of high fat fed C57BL/6J mice was examined by tail bleeding time and deep venous thrombosis (DVT) model. As a result, PA inhibited the expression of TM and EPCR in endothelial cells, and this effect was blunted by inhibiting JNK signaling. High fat diet fed mice had higher level of circulating FFAs and exhibited prothrombotic state, evidenced by increased tail bleeding time and enlarged thrombotic size in DVT model, compared to the control diet fed mice. Hence, FFAs inhibit TM-EPCR-Protein C system in endothelial cells through activating JNK signaling, which may be a mechanism for the prothrombotic state in metabolic syndrome.

Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.

Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis

AbstractThrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

Abstract 358: Cyclic Strain-Induced Release of Thrombomodulin from Human Aortic Endothelial Cells

Introduction: Loss of thrombomodulin (TM), a cofactor in the protein C anti-coagulant pathway, through a combination of reduced TM expression and elevated TM release, is a key feature of endothelial dysfunction leading to vascular diseases. Reduction in endothelial TM levels for example is viewed as a major cause of cyclic strain-induced early vein graft failure. In this respect, our understanding of the mechanistic relationship between vessel stretch and TM expression/release in endothelial cells is very limited, with a particular shortage of in vitro models. In this study, we investigated how cyclic circumferential strain negatively regulates thrombomodulin levels in human aortic endothelial cells (HAECs) in vitro. Methods: A Flexercell® Tension-Plus™ FX-4000T™ system (Flexcell International Corp., Hillsborough, NC) was routinely employed to subject HAECs to equibiaxial cyclic strain over a range of doses (0, 2.5 and 7.5% stretch; 60 cycles/min; cardiac waveform) and times (0-48 hours). TM expression was analyzed via Western blotting and qRT-PCR, whilst TM release was monitored by ELISA. Results: (i) Cyclic strain of HAECs led to significant force- and time-dependent reductions in TM expression (mRNA and protein) in conjunction with elevated TM release into media; (ii) Inclusion of either GM6001 (25 μM) or apocynin (10 mM) into strain experiments to block MMP-2/9 and NADPH oxidase activities, respectively, had no effect on strain-induced TM release; (iii) Control studies demonstrated that treatment of HAECs with neither TNF-α (0, 10, 100 ng/ml) nor oxLDL (10, 50, 145.7 μg/ml) increased TM release from HAECs - however, oxLDL treatment was found to dose-dependently potentiate cyclic-strain-induced TM release. Conclusions: Cyclic strain negatively regulates TM levels in endothelial cells in vitro. Moreover, release of TM from HAECs does not appear to involve the strain-dependent induction of either MMP-2/9 proteolytic activity or NADPH oxidase-dependent superoxide production. Finally, the cyclic strain-induced release of TM appears to be potentiated by inflammatory atherogenic conditions such as high oxLDL levels.

Neuropathologic evidence of endothelial changes in cerebral small vessel disease

Cerebral small vessel disease (SVD) is common in aged brains and causes lacunar stroke, diffuse white matter lesions (leukoaraiosis), and vascular cognitive impairment. The pathogenesis is unknown. Endothelial dysfunction is a possible causal factor, and circulating markers of endothelial activation (intercellular adhesion molecule-1, thrombomodulin) and inflammation (interleukin [IL]-6) are elevated in patients with SVD. In this case-control study, we tested whether brain endothelial ICAM1, thrombomodulin, and IL-6 are altered in SVD. We examined small penetrating cerebral arteries of pathologically diagnosed SVD cases, aged controls without SVD, young control cases with no brain pathology, and cases with early-onset hereditary SVD (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy [CADASIL]). All tissues had minimal cerebral amyloid angiopathy or other Alzheimer pathology. Thrombomodulin immunoreactivity was present in all aged SVD, aged control, and CADASIL cases, primarily in small artery endothelium. Thrombomodulin was augmented in aged SVD cases compared with aged controls (p = 0.012) and in vessels with higher sclerotic index (an indicator of SVD severity; p < 0.01). Thrombomodulin was sparse/absent in young controls. Endothelial ICAM1 and IL-6 were rarely seen, and were not related to SVD. Our data suggest that cerebral endothelial activation in deep penetrating arteries is not associated with SVD. Endothelial thrombomodulin increased with SVD severity, and CADASIL data suggest that this may be a cerebral response to SVD. Elevated thrombomodulin may be a protective agent in SVD. Our data confirm endothelial involvement in SVD.

Inhibition of transforming growth factor-β restores endothelial thromboresistance in vein grafts

Thrombosis is a major cause of the early failure of vein grafts (VGs) implanted during peripheral and coronary arterial bypass surgeries. Endothelial expression of thrombomodulin (TM), a key constituent of the protein C anticoagulant pathway, is markedly suppressed in VGs after implantation and contributes to local thrombus formation. While stretch-induced paracrine release of transforming growth factor-β (TGF-β) is known to negatively regulate TM expression in heart tissue, its role in regulating TM expression in VGs remains unknown. Changes in relative mRNA expression of major TGF-β isoforms were measured by quantitative polymerase chain reaction (qPCR) in cultured human saphenous vein smooth muscle cells (HSVSMCs) subjected to cyclic stretch. To determine the effects of paracrine release of TGF-β on endothelial TM mRNA expression, human saphenous vein endothelial cells (HSVECs) were co-cultured with stretched HSVSMCs in the presence of 1D11, a pan-neutralizing TGF-β antibody, or 13C4, an isotype-control antibody. Groups of rabbits were then administered 1D11 or 13C4 and underwent interpositional grafting of jugular vein segments into the carotid circulation. The effect of TGF-β inhibition on TM gene expression was measured by qPCR; protein C activating capacity and local thrombus formation were measured by in situ chromogenic substrate assays; and VG remodeling was assessed by digital morphometry. Cyclic stretch induced TGF-β(1) expression in HSVSMCs by 1.9 ± 0.2-fold (P < .001) without significant change in the expressions of TGF-β(2) and TGF-β(3). Paracrine release of TGF-β(1) by stretched HSVSMCs inhibited TM expression in stationary HSVECs placed in co-culture by 57 ± 12% (P = .03), an effect that was abolished in the presence of 1D11. Similarly, TGF-β(1) was the predominant isoform induced in rabbit VGs 7 days after implantation (3.5 ± 0.4-fold induction; P < .001). TGF-β(1) protein expression localized predominantly to the developing neointima and coincided with marked suppression of endothelial TM expression (16% ± 2% of vein controls; P < .03), a reduction in situ activated protein C (APC)-generating capacity (53% ± 9% of vein controls; P = .001) and increased local thrombus formation (3.7 ± 0.8-fold increase over vein controls; P < .01). External stenting of VGs to limit vessel distension significantly reduced TGF-β(1) induction and TM downregulation. Systemic administration of 1D11 also effectively prevented TM downregulation, preserved APC-generating capacity, and reduced local thrombus in rabbit VGs without observable effect on neointima formation and other morphometric parameters 6 weeks after implantation. TM downregulation in VGs is mediated by paracrine release of TGF-β(1) caused by pressure-induced vessel stretch. Systemic administration of an anti-TGF-β antibody effectively prevented TM downregulation and preserved local thromboresistance without negative effect on VG remodeling.

Effects of sirolimus upon endothelial thrombotic function of human umbilical vein endothelial cells

To explore the effects of sirolimus upon endothelial thrombotic function of human umbilical vein endothelial cells (HUVEC). Sirolimus was added into the in vitro cultured HUVEC at the concentrations of 0 (control), 0.1, 1 and 10 ng/ml. At 2, 4, 8, 12 and 24 h post-incubation, the cells were harvested for determination of tissue factor (TF), plasminogen activator inhibitor (PAI-1), endothelial nitric oxide synthase (eNOS) and thrombomodulin (TM) expression by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The clotting functions of HUVEC were assayed by an automated coagulation analyzer. Sirolimus induced the expressions of TF and PAI-1 and inhibited the expressions of eNOS and TM in a concentration-dependent manner. The maximal change of mRNA expression was observed at 8 h and remained up to at least 24 h. And the most marked change of protein expression (65% reduction in eNOS expression, 52% reduction in TM, 1.7-fold increase in PAI-1 and 2.8-fold increase in TF) was at 12 h. The clotting time in sirolimus group (89 s ± 9 s) was significantly shorter than that in control group (152 s ± 17 s, P = 0.005). Sirolimus induces endothelial antithrombotic dysfunction and shortens the clotting time through an elevated expression of prothrombotic genes TF and PAI-1 and a lowered expression of antithrombotic genes eNOS and TM. It may be one of mechanisms of thrombosis after the implantation of sirolimus-eluting stents.

Sources of Thrombomodulin in Pre-Eclampsia: Renal Dysfunction or Endothelial Damage?

A plethora of evidence exists to show that endothelial perturbations underlie many of the clinical features of pre-eclampsia (P-EC), and consequently the markers signifying endothelial disturbance exhibit a rise in plasma. Among others, plasma thrombomodulin (TM) level rises significantly. TM is a transmembrane glycoprotein expressed abundantly on the endothelium of the microvasculature. It neutralizes the thrombotic potential of thrombin, mediating this anticoagulant effect through activation of protein C. Endothelial injury results in a localized loss of TM with a focal impairment of protein C activation and resultant thrombotic tendency. Mainly expressed on the endothelial cells, a small amount of TM is found in plasma with levels rising in certain pathological conditions. Although elevation in levels of TM can be due to endothelial TM proteolysis secondary to endothelial insult, ineffective clearance may account for this in renal and hepatic dysfunction. In P-EC not only is there ongoing endothelial injury, but renal function is also affected. To establish the cause of elevated TM level in P-EC, three groups of pregnant women were investigated. It appears that the elevation in plasma TM is not related to renal or hepatic dysfunction in P-EC. It is more likely that plasma TM is derived from placental or vascular endothelial cells subsequent to activation or damage, confirming the hypothesis that damage to vascular endothelial cells is the primary underlying cause of P-EC.

The Lectin-Like Domain of Thrombomodulin Protects Against Diabetic Nephropathy by Inhibiting Complement Activation

AbstractAbstract 654 Introduction:Thrombomodulin (TM) is type 1 transmembrane glycoprotein predominately expressed on vascular endothelial cells. TM consists of distinct structural domains, which enable interaction with multiple ligands and thus modulation of coagulation, fibrinolysis, complement activation, inflammation, cell proliferation, and apoptosis. We have previously shown that TM protects against diabetic nephropathy (dNP) through activation of protein C (PC), which inhibits glomerular apoptosis. Recent studies showed that TM mediates cytoprotective effects independent of PC activation through it's lectin-like domain (LLD). The LLD directly interferes with complement activation and protect against arthritis. dNP is associated with an infiltration of inflammatory cells, enhanced cytokine production, and complement activation raising the question whether endothelial TM may protect against DN through a dual mechanism depending on (a) PC activation and (b) complement inhibition through the LLD. Methods:Experimental dNP was evaluated in mice with targeted deletion of the LLD of TM (TMLed/Led mice). Persistent hyperglycemia was induced using streptozotocin in TMLed/Led mice and in control wild type mice (wt). Subgroups of mice were treated with low molecular heparin (LMWH) to inhibit complement activation and coagulation activation, fondaparinux to inhibit coagulation activation, or treated with minocycline to inhibit glomerular apoptosis. After 28 weeks albuminuria was determined and mice were sacrificed to isolated tissues and blood samples for analyses. Results:Albuminuria, histological indices of diabetic nephropathy and glomerular C3 deposition were increased in diabetic wt mice and further increased in diabetic TMLed/Led mice. Only treatment with LMWH, but not with fondaparinux corrected the aggravated DN in TMLed/Led mice, despite equal anticoagulation in both groups. Glomerular complement depositions (immunohistochemistry) were significantly reduced following in vivo LMWH treatment, but no in fondaparinux treated mice. In vitro, endothelial cells expressing a TM mutant lacking the LLD had diminished capacity to bind and inactivate C3b and were thus sensitive to complement mediated cell lyses. Of note, apoptosis (TUNEL) and expression levels of apoptosis regulators (p53, Bax/Blc-2 ratio, Western) did not differ between untreated or LMWH/fondaparinux treated diabetic TMLed/Led mice, indicating that the protective effect of the LLD is independent of apoptosis inhibition. Consistently, inhibition of apoptosis with minocycline failed to normalize albuminuria or prevent histological indices of dNP in diabetic TMLed/Led mice. Likewise, we did not observe difference in HMGB-1 levels within the kidney or in plasma sample, and inhibition of HMGB-1 using sodium Tanshinon II asilate failed to reduce dNP in diabetic TMLed/Led mice. Discussion and Conclusion:We identify a novel pathway through which endothelial TM protects against dNP. TM protects against dNP by inhibiting complement activation through its LLD. This function is independent of the recently identified activated PC dependent inhibition of glomerular apoptosis. Thus, endothelial TM prevents dNP through two independent pathways. These results provide further experimental support for a causative role of endothelial dysfunction for DN. Disclosures:No relevant conflicts of interest to declare.