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Brill A.,Immune Disease Institute | Brill A.,Harvard University | Yesilaltay A.,Massachusetts Institute of Technology | De Meyer S.F.,Immune Disease Institute | And 9 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2012

Objective-Deep vein thrombosis (DVT) and pulmonary embolism are frequent causes of morbidity and mortality. The goal of our study was to determine whether plasma high-density lipoprotein (HDL), which inversely correlates with the risk of cardiovascular events, affects DVT. Methods and Results-Using a murine DVT model of inferior vena cava stenosis, we demonstrated that deficiency of the HDL receptor, scavenger receptor class B type I (SR-BI), promotes venous thrombosis. As SR-BI mice have increased plasma cholesterol levels and abnormal HDL particles, we tested SR-BI mice with an SR-BI liver transgene that normalizes both parameters. These mice also exhibited increased susceptibility to DVT, indicating a protective role of extrahepatic SR-BI. Mice lacking the major HDL apolipoprotein apoA-I or endothelial nitric oxide synthase (eNOS) (a downstream target of endothelial SR-BI signaling) also had a prothrombotic phenotype. Intravenous infusion of human apoA-I, an HDL component and SR-BI ligend, prevented DVT in wild-type but not SR-BI or eNOS mice, suggesting that its effect is mediated by SR-BI and eNOS. Intravenous apoA-I infusion abolished histamine-induced platelet-endothelial interactions, which are important for DVT initiation. Conclusion-An apoA-I (HDL)-SR-BI-eNOS axis is highly protective in DVT and may provide new targets for prophylaxis and treatment of venous thrombosis. © 2012 American Heart Association, Inc.


Vets S.,Catholic University of Leuven | Kimpel J.,Georg Speyer Haus | Kimpel J.,Innsbruck Medical University | Volk A.,Georg Speyer Haus | And 9 more authors.
Molecular Therapy | Year: 2012

Lens epithelium-derived growth factor (LEDGF/p75) is an essential cofactor of HIV integration. Both stable overexpression of the C-terminal part of LEDGF/p75 (LEDGF325-530) containing the integrase (IN)-binding domain (IBD) and stable knockdown (KD) of LEDGF/p75 are known to inhibit HIV infection in laboratory cell lines. Here, primary human CD4 + T-cells were transduced with lentiviral vectors encoding LEDGF 32525-530, the interaction-deficient mutant LEDGF325-530 D366N, or a hairpin depleting LEDGF/p75 and challenged with HIV. Maximal protection of primary T-cells from HIV infection was obtained after LEDGF 325-530 overexpression reducing HIV replication 40-fold without evidence of cellular toxicity. This strategy was subsequently evaluated in the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model. Threefold reduction in mean plasma viral load was obtained in mice engrafted with CD4 + T-cells expressing LEDGF325-530 in comparison with engraftment with LEDGF325-530 D366N cells. Four weeks after transplantation with LEDGF325-530 D366N cells, 70% of the CD4 + cells were lost due to ongoing HIV replication. However, in mice transplanted with LEDGF325-530 cells only a 20% decrease in CD4 + cells was measured. Liver and spleen sections of LEDGF325-530 mice contained less HIV than LEDGF 325-530 D366N mice as measured by p24 antigen detection. LEDGF 325-530 overexpression potently inhibits HIV replication in vivo and protects against HIV mediated cell killing of primary CD4 + T-cells. © The American Society of Gene & Cell Therapy.


De Meyer S.F.,Immune Disease Institute | De Meyer S.F.,Childrens Hospital Boston | De Meyer S.F.,Harvard University | De Meyer S.F.,Laboratory for Thrombosis Research | And 5 more authors.
Stroke | Year: 2012

Thrombus formation is of paramount importance in the pathophysiology of acute ischemic stroke. Current antithrombotics used to treat or prevent cerebral ischemia are only moderately effective or bear an increased risk of severe bleeding. von Willebrand factor (VWF) has long been known to be a key player in thrombus formation at sites of vascular damage. While the association between VWF and coronary heart disease has been well studied, knowledge about the role of VWF in stroke is much more limited. However, in recent years, an increasing amount of clinical and preclinical evidence has revealed the critical involvement of VWF in stroke development. This review summarizes the latest insights into the pathophysiologic role of VWF-related processes in ischemic brain injury under experimental conditions and in humans. Potential clinical merits of novel inhibitors of VWF-mediated platelet adhesion and activation as powerful and safe tools to combat thromboembolic disorders including ischemic stroke are discussed. Preclinical and clinical evidence illustrates an important role of VWF in ischemic stroke, suggesting that VWF could become a promising target in stroke therapy. © 2011 American Heart Association, Inc.


Barsam S.J.,New York Medical College | Barsam S.J.,University College London | Psaila B.,New York Medical College | Psaila B.,Imperial College London | And 9 more authors.
Blood | Year: 2011

This study investigated the immature platelet fraction (IPF) in assessing treatment effects in immune thrombocytopenia (ITP). IPF was measured on the Sysmex XE2100 autoanalyzer. The mean absolute-IPF (A-IPF) was lower for ITP patients than for healthy controls (3.2 vs 7.8 × 109/L, P < .01), whereas IPF percentage was greater (29.2% vs 3.2%, P < .01). All 5 patients with a platelet response to Eltrombopag, a thrombopoietic agent, but none responding to an anti-FcγRIII antibody, had corresponding A-IPF responses. Seven of 7 patients responding to RhoD immuneglobulin (anti-D) and 6 of 8 responding to intravenous immunoglobulin (IVIG) did not have corresponding increases in A-IPF, but 2 with IVIG and 1 with IVIG anti-D did. This supports inhibition of platelet destruction as the primary mechanism of intravenous anti-D and IVIG, although IVIG may also enhance thrombopoiesis. Plasma glycocalicin, released during platelet destruction, normalized as glycocalicin index, was higher in ITP patients than controls (31.36 vs 1.75, P = .001). There was an inverse correlation between glycocalicin index and A-IPF in ITP patients (r2 = -0.578, P = .015), demonstrating the relationship between platelet production and destruction. Nonresponders to thrombopoietic agents had increased megakaryocytes but not increased A-IPF, suggesting that antibodies blocked platelet release. In conclusion, A-IPF measures real-time thrombopoiesis, providing insight into mechanisms of treatment effect. © 2011 by The American Society of Hematology.


Verschuere T.,Catholic University of Leuven | Toelen J.,Catholic University of Leuven | Maes W.,Laboratory for Thrombosis Research | Maes W.,Catholic University of Leuven | And 11 more authors.
International Journal of Cancer | Year: 2014

Galectin-1 is a glycan-binding protein, which is involved in the aggressiveness of glioblastoma (GBM) in part by stimulating angiogenesis. In different cancer models, galectin-1 has also been demonstrated to play a pivotal role in tumor-mediated immune evasion especially by modulating cells of the adaptive immune system. It is yet unknown whether the absence or presence of galectin-1 within the glioma microenvironment also causes qualitative or quantitative differences in innate and/or adaptive antitumor immune responses. All experiments were performed in the orthotopic GL261 mouse high-grade glioma model. Stable galectin-1 knockdown was achieved via transduction of parental GL261 tumor cells with a lentiviral vector encoding a galectin-1-targeting miRNA. We demonstrated that the absence of tumor-derived but not of host-derived galectin-1 significantly prolonged the survival of glioma-bearing mice as such and in combination with dendritic cell (DC)-based immunotherapy. Both flow cytometric and pathological analysis revealed that the silencing of glioma-derived galectin-1 significantly decreased the amount of brain-infiltrating macrophages and myeloid-derived suppressor cells (MDSC) in tumor-bearing mice. Additionally, we revealed a pro-angiogenic role for galectin-1 within the glioma microenvironment. The data provided in this study reveal a pivotal role for glioma-derived galectin-1 in the regulation of myeloid cell accumulation within the glioma microenvironment, the most abundant immune cell population in high-grade gliomas. Furthermore, the prolonged survival observed in untreated and DC-vaccinated glioma-bearing mice upon the silencing of tumor-derived galectin-1 strongly suggest that the in vivo targeting of tumor-derived galectin-1 might offer a promising and realistic adjuvant treatment modality in patients diagnosed with GBM. What's new? Galectin-1 is a glycan-binding protein that plays a major role in the aggressiveness of glioblastomas (GBMs), via a number of different mechanisms. In different types of tumors, galectin-1 has been demonstrated to contribute to tumor-mediated immune evasion. Whether glioma-derived galectin-1 also contributes to glioma-mediated immune evasion is unknown, however. In this study, the authors found that glioma-derived galectin-1 plays an important role in the regulation of myeloid cell accumulation within the tumor microenvironment. They also found that silencing of galectin-1 could improve glioma-bearing mice survival. Intratumoral silencing of galectin-1 may therefore offer a promising adjuvant treatment modality for patients with high-grade glioma. © 2013 UICC.


Soenen S.J.,Laboratory of BioNanoColloids | De Meyer S.F.,Laboratory for Thrombosis Research | Dresselaers T.,Biomedical NMR Unit MoSAIC | Velde G.V.,Biomedical NMR Unit MoSAIC | And 5 more authors.
Biomaterials | Year: 2011

The use of contrast material to stimulate magnetic resonance imaging (MRI) of migrating cells has become an important area of research. In the present study, cationic magnetoliposomes (MLs) were used to magnetically label human blood outgrowth endothelial cells (BOECs) and follow their homing by magnetic resonance imaging (MRI). The biodistribution and functional integration capacity of BOECs, which have shown extensive promise as gene delivery vehicles, have thus far only rarely been investigated. MLs were avidly internalized by BOECs giving clear MRI contrast in phantom studies and the magnetic labeling did not affect cell proliferation, viability, morphology or homeostasis and elicited only minor reactive oxygen species levels. Intravenous injection of labeled BOECs was compared with injection of free MLs and unlabeled BOECs, resulting in homing of BOECs toward the liver and spleen, which was confirmed by histology. The MLs used offer great potential for cellular tracking studies by MRI when low levels of widely distributed cells are present. In particular, the use of these MLs will allow to evaluate the efficacy of new methods to enhance BOEC homing and integration to optimize their use as efficient vehicles for gene therapy. © 2011 Elsevier Ltd.


Maes W.,Laboratory for Thrombosis Research | Verschuere T.,Catholic University of Leuven | Van Hoylandt A.,Catholic University of Leuven | Boon L.,Bioceros | Van Gool S.,Catholic University of Leuven
Clinical and Developmental Immunology | Year: 2013

The recruitment and activation of regulatory T cells (Tregs) in the micro-environment of malignant brain tumors has detrimental effects on antitumoral immune responses. Hence, local elimination of Tregs within the tumor micro-environment represents a highly valuable tool from both a fundamental and clinical perspective. In the syngeneic experimental GL261 murine glioma model, Tregs were prophylactically eliminated through treatment with PC61, an anti-CD25 mAb. This resulted in specific elimination of CD4+CD25hiFoxp3+ Treg within brain-infiltrating lymphocytes and complete protection against subsequent orthotopic GL261 tumor challenge. Interestingly, PC61-treated mice also showed a pronounced infiltration of CD11b+ myeloid cells in the brain. Phenotypically, these cells could not be considered as Gr-1+ myeloid-derived suppressor cells (MDSC) but were identified as F4/80+ macrophages and granulocytes. © 2013 Wim Maes et al.


De Maeyer B.,Laboratory for Thrombosis Research | De Meyer S.F.,Laboratory for Thrombosis Research | Feys H.B.,Laboratory for Thrombosis Research | Pareyn I.,Laboratory for Thrombosis Research | And 3 more authors.
Journal of Thrombosis and Haemostasis | Year: 2010

Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentied. Methods: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13-/- mouse. Results: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT andM) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo. In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains inuence proteolysis of platelet-decorated VWF strings in vivo. © 2010 International Society on Thrombosis and Haemostasis.


De Meyer S.F.,Harvard University | De Meyer S.F.,Childrens Hospital Boston | De Meyer S.F.,Laboratory for Thrombosis Research | Suidan G.L.,Harvard University | And 5 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2012

Objective-Recently, a growing number of studies have revealed a prothrombotic and cytotoxic role for extracellular chromatin. Cerebral ischemia/reperfusion injury is characterized by a significant amount of cell death and neutrophil activation, both of which may result in the release of chromatin. The goal of this study was to assess the effect of extracellular chromatin in ischemic stroke using a mouse model of transient middle cerebral artery occlusion. Methods and Results-Similar to reports in stroke patients, we observed increased levels of circulating nucleosomes and DNA after ischemic stroke in mice. In addition, we observed that general hypoxia also augmented extracellular chromatin. We hypothesized that targeting extracellular chromatin components would be protective in ischemic stroke. Indeed, treatment with recombinant human DNase 1 significantly improved stroke outcome. Neutralization of histones using an antihistone antibody was also protective as evidenced by smaller infarct volumes, whereas increasing levels of extracellular histones via histone infusion exacerbated stroke outcome by increasing infarct size and worsening functional outcome. Conclusion-Our results indicate that extracellular chromatin is generated and is detrimental during cerebral ischemia/reperfusion in mice. Targeting DNA and histones may be a new therapeutic strategy to limit injury resulting from ischemic stroke. © 2012 American Heart Association, Inc.


Brill A.,Childrens Hospital Boston | Brill A.,Harvard University | Fuchs T.A.,Childrens Hospital Boston | Fuchs T.A.,Harvard University | And 12 more authors.
Journal of Thrombosis and Haemostasis | Year: 2012

Summary. Background:Upon activation, neutrophils can release nuclear material known as neutrophil extracellular traps (NETs), which were initially described as a part of antimicrobial defense. Extracellular chromatin was recently reported to be prothrombotic in vitro and to accumulate in plasma and thrombi of baboons with experimental deep vein thrombosis (DVT). Objective:To explore the source and role of extracellular chromatin in DVT. Methods:We used an established murine model of DVT induced by flow restriction (stenosis) in the inferior vena cava (IVC). Results:We demonstrate that the levels of extracellular DNA increase in plasma after 6h IVC stenosis, compared with sham-operated mice. Immunohistochemical staining revealed the presence of Gr-1-positive neutrophils in both red (RBC-rich) and white (platelet-rich) parts of thrombi. Citrullinated histone H3 (CitH3), an element of NETs' structure, was present only in the red part of thrombi and was frequently associated with the Gr-1 antigen. Immunofluorescent staining of thrombi showed proximity of extracellular CitH3 and von Willebrand factor (VWF), a platelet adhesion molecule crucial for thrombus development in this model. Infusion of Deoxyribonuclease 1 (DNase 1) protected mice from DVT after 6h and also 48h IVC stenosis. Infusion of an unfractionated mixture of calf thymus histones increased plasma VWF and promoted DVT early after stenosis application. Conclusions:Extracellular chromatin, likely originating from neutrophils, is a structural part of a venous thrombus and both the DNA scaffold and histones appear to contribute to the pathogenesis of DVT in mice. NETs may provide new targets for DVT drug development. © 2011 International Society on Thrombosis and Haemostasis.

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