B Robaye

Université Libre de Bruxelles, Bruxelles, Brussels Capital Region, Belgium

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Publications (38)147.03 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND/OBJECTIVES: We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor, and P2Y12 receptor deficiency in vivo. METHODS: DVT was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor, or vehicle and in P2Y12 -/- mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. RESULTS: Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin-, and ticagrelor-pretreated wild-type and P2Y12 -/- mice developed only small thrombi. After injection of the contrast agent, growing thrombi delineated clearly as negative contrast in CEUS. Thrombus size in CEUS after 25 minutes was significantly smaller in enoxaparin- (0.3±0.1mm(2) ) and ticagrelor-treated (0.5±0.1mm(2) ) wild-type and in P2Y12 -/- mice (0.4±0.1mm(2) ) as compared to vehicle-treated wild-type mice (2.0±0.3mm(2) ) in the maximal sagittal plane (p < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. CONCLUSIONS: CEUS allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y12 receptor in early DVT formation. © 2013 International Society on Thrombosis and Haemostasis.
    Journal of Thrombosis and Haemostasis 03/2013; · 6.08 Impact Factor
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    ABSTRACT: The clearance of tissue debris by microglia is a crucial component of maintaining brain homeostasis. Microglia continuously survey the brain parenchyma and utilize extracellular nucleotides to trigger the initiation of their dynamic responses. Extracellular uridine diphosphate (UDP), which leaks or is released from damaged neurons, has been reported to stimulate the phagocytotic activity of microglia through P2Y(6) receptor activation. However, the intracellular mechanisms underlying microglial P2Y(6) receptor signals have not been identified. In this study, we demonstrated that UDP stimulation induced immediate and long-lasting dynamic movements in the cell membrane. After 60 min of UDP stimulation, there was an upregulation in the number of large vacuoles formed in the cell that incorporate extracellular fluorescent-labeled dextran, which indicates microglial macropinocytosis. In addition, UDP-induced vacuole formation and continuous membrane motility were suppressed by the protein kinase D (PKD) inhibitors, Gö6976 and CID755673, unlike Gö6983, which is far less sensitive to PKD. The inhibition of PKD also reduced UDP-induced incorporation of fluorescent-labeled dextran and soluble β-amyloid and phagocytosis of microspheres. UDP induced rapid phosphorylation and membrane translocation of PKD, which was abrogated by the inhibition of protein kinase C (PKC) with Gö6983. However, Gö6983 failed to suppress UDP-induced incorporation of microspheres. Finally, we found that inhibition of PKD by CID755673 significantly suppressed UDP-induced engulfment of IgG-opsonized microspheres. These data suggest that a PKC-independent function of PKD regulates UDP-induced membrane movement and contributes to the increased uptake of extracellular fluid and microspheres in microglia.
    Glia 04/2012; 60(7):1094-105. · 5.07 Impact Factor
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    ABSTRACT: Extracellular nucleotides have widespread effects and various cell responses. Whereas the effect of a purine nucleotide (ATP) and a pyrimidine nucleotide (UTP) on myocardial infarction has been examined, the role of different purine and pyrimidine nucleotides and nucleosides in cardioprotection against hypoxic stress has not been reported. To investigate the role of purine and pyrimidine nucleotides and nucleosides in protective effects in cardiomyocytes subjected to hypoxia. Rat cultured cardiomyocytes were treated with various extracellular nucleotides and nucleosides, before or during hypoxic stress. The results revealed that GTP or CTP exhibit cardioprotective ability, as revealed by lactate dehydrogenase (LDH) release, by propidium iodide (PI) staining, by cell morphology, and by preserved mitochondrial activity. Pretreatment with various P2 antagonists (suramin, RB-2, or PPADS) did not abolish the cardioprotective effect of the nucleotides. Moreover, P2Y₂ -/- , P2Y₄ -/-, and P2Y₂ -/-/P2Y₄ -/- receptor knockouts mouse cardiomyocytes were significantly protected against hypoxic stress when treated with UTP. These results indicate that the protective effect is not mediated via those receptors. We found that a wide variety of triphosphate and diphosphate nucleotides (TTP, ITP, deoxyGTP, and GDP), provided significant cardioprotective effect. GMP, guanosine, and ribose phosphate provided no cardioprotective effect. Moreover, we observed that tri/di-phosphate alone assures cardioprotection. Treatment with extracellular nucleotides, or with tri/di-phosphate, administered under normoxic conditions or during hypoxic conditions, led to a decrease in reactive oxygen species production. Extracellular tri/di-phosphates are apparently the molecule responsible for cardioprotection against hypoxic damage, probably by preventing free radicals formation.
    Biochemical pharmacology 03/2011; 81(10):1219-27. · 4.25 Impact Factor
  • Bone 01/2011; 48. · 3.82 Impact Factor
  • Hypertension. 12/2010; 56(6):1169.
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    ABSTRACT: Extracellular ATP contributes to the pathogenesis of asthma via signalling at purinergic receptors. However, the precise purinergic receptors subtypes mediating the pro-asthmatic effects of ATP have not been identified, yet. In vivo studies were performed using the OVA-alum model. Functional expression of the P2Y(2) purinergic receptor subtype on human monocyte-derived dendritic cells and eosinophils was investigated using real-time PCR, migration assays, and production of reactive oxygen species. Compared to wild-type animals P2Y(2) -/- mice showed reduced allergic airway inflammation which can be explained by defective migration of blood myeloid DCs towards ATP in vitro and in vivo, whereas the influence of ATP on maturation and cytokine production was not changed. Additionally, ATP failed to induce migration of bone marrow-derived eosinophils from P2Y(2) R-deficient animals. The relevance of our findings for humans was confirmed in functional studies with human monocyte-derived DCs and eosinophils. Interestingly, stimulation of human DCs derived from allergic individuals with house dust mite allergen induced functional up-regulation of the P2Y(2) R subtype. Furthermore, eosinophils isolated from asthmatic individuals expressed higher levels of P2Y(2) R compared to healthy controls. This was of functional relevance as these eosinophils were more sensitive to ATP-induced migration and production of reactive oxygen metabolites. In summary, P2Y(2) R appears to be involved in asthmatic airway inflammation by mediating ATP-triggered migration of mDCs and eosinophils, as well as reactive oxygen species production. Together our data suggest that targeting P2Y(2) R might be a therapeutic option for the treatment of asthma.
    Allergy 09/2010; 65(12):1545-53. · 5.88 Impact Factor
  • Bone 01/2010; 47. · 3.82 Impact Factor
  • Purinergic Signalling 01/2010; 6(1):144-144. · 2.64 Impact Factor
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    ABSTRACT: Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice. Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2. NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.
    Cardiovascular research 08/2009; 85(1):204-13. · 5.80 Impact Factor
  • Atherosclerosis Supplements - ATHEROSCLER SUPPL. 01/2009; 10(2).
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    ABSTRACT: Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na(+) channel (ENaC)-mediated Na(+) absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y(2) and a luminal P2Y(4) receptor, stimulate K(+) secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na(+) absorption in distal colonic mucosa of mice treated on a low Na(+) diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V (te)): -13.7 +/- 1.9 mV (lumen negative), transepithelial resistance (R (te)): 24.1 +/- 1.8 Omega cm(2), equivalent short circuit current (I (sc)): -563.9 +/- 63.8 microA/cm(2) (n = 21). Amiloride completely inhibited I (sc) to -0.5 +/- 8.5 microA/cm(2). Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I (sc) by 160.7 +/- 29.7 microA/cm(2) (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC(50) values of 10 microM and 3 microM respectively. In P2Y(2) knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na(+) absorption was absent. In contrast, in P2Y(4) KO mice the inhibitory effect of luminal UTP on Na(+) absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na(+) diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y(2) and P2Y(4) receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y(2) receptor mediates inhibition of electrogenic Na(+) absorption.
    Pflügers Archiv - European Journal of Physiology 10/2007; 454(6):977-87. · 4.87 Impact Factor
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    ABSTRACT: Based on pharmacological criteria, we previously suggested that in the mouse aorta endothelium-dependent relaxation by nucleotides is mediated by P2Y1 (ADP), P2Y2 (ATP) and P2Y6 (UDP) receptors. For UTP it was unclear whether P2Y2, P2Y6 or yet another subtype was involved. Therefore, in view of the lack of selective purinergic agonists and antagonists we used P2Y2-deficient mice to clarify the action of UTP. Thoracic aorta segments (width 2 mm) of P2Y2-deficient and wild type (WT) mice were mounted in organ baths to measure isometric force development and intracellular calcium signalling. Relaxations evoked by ADP, UDP and acetylcholine were identical in knockout and WT mice indicating that the receptors for these agonists function normally. P2Y2-deficient mice showed impaired ATP- and ATPS-evoked relaxation, suggesting that in WT mice ATP and ATPS activate predominantly the P2Y2-subtype. The ATP/ATPS-evoked relaxation and calcium signals in the knockout mice were partially rescued by P2Y1, since they were sensitive to 2′-deoxy-N6-methyladenosine3′,5′-bisphosphate (MRS2179), a P2Y1 selective antagonist. In contrast to ATP, the UTP-evoked relaxation was not different among knockout and WT mice. Moreover, the action of UTP was not sensitive to MRS2179. Therefore, the action of UTP is probably mediated mainly by a P2Y6 (like) receptor subtype. In conclusion, we demonstrated that ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2. But this P2Y2 receptor has apparently no major role in UTP-evoked relaxation. The vasodilator effect of UTP is probably mediated mainly by a P2Y6 (like) receptor.
    Fundamental and Clinical Pharmacology 01/2006; 20(3):332-332. · 1.99 Impact Factor
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    ABSTRACT: Extracellular nucleotides are important regulators of epithelial ion transport, frequently exerting their action from the luminal side. Luminal P2Y receptors have previously been identified in rat distal colonic mucosa. Their activation by UTP and ATP stimulates K+ secretion. The aim of this study was to clarify which of the P2Y receptor subtypes are responsible for the stimulated K+ secretion. To this end P2Y2 and P2Y4 knock-out mice were used to measure distal colonic ion transport in an Ussing chamber. In mouse (NMRI) distal colonic mucosa, luminal UTP and ATP with similar potency induced a rapid and transient increase of the transepithelial voltage (V(te)) (UTP: from -0.81 +/- 0.23 to 3.11 +/- 0.61 mV, n = 24), an increase of equivalent short circuit current (I(sc)) by 166.9 +/- 22.8 microA cm(-2) and a decrease of transepithelial resistance (R(te)) from 29.4 +/- 2.4 to 23.5 +/- 2.0 Omega cm2. This effect was completely inhibited by luminal Ba2+ (5 mm, n = 5) and iberiotoxin (240 nm, n = 6), indicating UTP/ATP-stimulated K+ secretion. RT-PCR analysis of isolated colonic crypts revealed P2Y2, P2Y4 and P2Y6 specific transcripts. The luminal UTP-stimulated K+ secretion was still present in P2Y2 receptor knock-out mice, but significantly reduced (DeltaV(te): 0.83 +/- 0.26 mV) compared to wild-type littermates (DeltaV(te): 2.08 +/- 0.52 mV, n = 9). In P2Y4 receptor knock-out mice the UTP-induced K+ secretion was similarly reduced. Luminal UTP-stimulated K+ secretion was completely absent in P2Y2/P2Y4 double receptor KO mice. Basolateral UTP showed no effect. In summary, these results indicate that both the P2Y2 and P2Y4 receptors are present in the luminal membrane of mouse distal colonic mucosa, and stimulation of these receptors leads to K+ secretion.
    The Journal of Physiology 05/2005; 564(Pt 1):269-79. · 4.38 Impact Factor
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    ABSTRACT: Apoptosis induction and micronuclei formation were compared following cytotoxic treatments in two rat glioma differing in p53 integrity. In vitro, micronuclei emergence but not apoptosis was linked to the p53 mutated status. In vivo, micronuclei assays were more sensitive to evaluate DNA damage induced by chemotherapy in a p53-mutated solid tumour.
    British Journal of Cancer 09/2003; 89(4):727-9. · 5.08 Impact Factor
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    ABSTRACT: Through the cAMP signaling pathway, TSH stimulates thyroid follicular cell proliferation, differentiation, and function. Although the autocrine production of IGF-I in the thyroid gland suggests an important physiological function for this factor in these processes, the exact role of the IGF-I/IGF-I receptor system in vivo remains unclear. Although the mitogenic action of TSH requires the presence of IGF-I or insulin in primary culture of dog and human thyroid cells, IGF-I has an effect equal to and independent of the effect of TSH on cell proliferation in rat thyroid cell lines and may even be the main growth regulator in this case. To investigate the in vivo function of the IGF-I/IGF-I receptor system, transgenic mice overexpressing human IGF-I, IGF-I receptor, or both in the thyroid were generated. Adult transgenic mice did not present external signs of thyroid dysfunction, but mice overexpressing both transgenes had significantly increased gland weight and follicular lumen area. A decreased TSH level together with a slightly increased serum T(4) concentration and increased thyroidal iodine uptake were also observed, suggesting that IGF-I and IGF-I receptor stimulate thyroid function to some extent in vivo.
    Endocrinology 01/2002; 142(12):5131-9. · 4.72 Impact Factor
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    ABSTRACT: Cytotoxic lymphocytes may induce apoptosis in their target cells by the FasL (Fas ligand) pathway or the perforin/granzyme B pathway. It has been shown that Fas-expressing colon carcinoma (CC) cells are resistant to FasL-mediated apoptosis. The aims of this study were to determine whether CC cells are also resistant to perforin/granzyme B and whether the FasL resistance lies upstream of caspase-3 activation. The resistance of the Fas-expressing rat CC531s cells to the FasL pathway was confirmed by treating them with recombinant human soluble FasL, using rat hepatocytes as a positive control. The intracellular delivery of granzyme B by sublytic concentrations of perforin, on the other hand, resulted in many features of apoptosis (chromatin condensation, nucleus fragmentation, loss of microvilli and internucleosomal DNA fragmentation) within 3 h. Since both the FasL and perforin/granzyme B pathways converge at caspase-3, we measured caspase-3 activity to learn whether the FasL resistance was due to failure to activate this crucial executioner. Caspase-3 activation occurred in CC531s cells after perforin/granzyme B treatment, but not after the addition of recombinant FasL. Furthermore, we showed that caspase-3 activity is involved in the execution of perforin/granzyme-B-induced apoptosis in CC531 s cells, since the cell-permeable caspase-3 inhibitor Z-DEVD-FMK abrogated DNA fragmentation. Together, these results suggest that CC cells are sensitive to perforin/granzyme-B-induced apoptosis by activating caspase-3 and FasL resistance lies upstream of this executioner caspase.
    Cancer Immunology and Immunotherapy 07/2001; 50(4):212-7. · 3.64 Impact Factor
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    ABSTRACT: Recently, it has been shown that ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells (DC); the effect of ATP was reproduced by hydrolysis-resistant derivatives of ATP and was blocked by suramin, suggesting the involvement of a P2 receptor, but the particular subtype involved was not identified. In this report we confirm that ATP and various derivatives synergize with TNF-alpha and LPS to induce the maturation of human monocyte-derived DC, as revealed by up-regulation of the CD83 marker and the secretion of IL-12. The rank order of potency of various analogs (AR-C67085 > adenosine 5'-O-(3-thiotriphosphate) = 2'- and 3'-O-(4-benzoyl-benzoyl) ATP > ATP > 2-methylthio-ATP) was close to that of the recombinant human P2Y11 receptor. Furthermore, these compounds activated cAMP production in DC, in a xanthine-insensitive way, consistent with the involvement of the P2Y11 receptor, which among P2Y subtypes has the unique feature of being dually coupled to phospholipase C and adenylyl cyclase activation. The involvement of the P2Y11/cAMP/protein kinase A signaling pathway in the nucleotide-induced maturation of DC is supported by the inhibitory effect of H89, a protein kinase A inhibitor. Taken together, our results demonstrate that ATP activates DC through stimulation of the P2Y11 receptor and subsequent increase in intracellular cAMP.
    The Journal of Immunology 06/2001; 166(12):7172-7. · 5.52 Impact Factor
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    ABSTRACT: In order to understand the role of actin microfilaments in the apoptotic process, we followed their evolution during tumor necrosis factor-alpha (TNF)-induced apoptosis in bovine aortic endothelial (BAE) cells. Using Western blotting analysis and immunofluorescence microscopy, we observed that the actin microfilaments network was disrupted in apoptotic cells. Depolymerization of F-actin was concomitant with internucleosomal DNA degradation and with the morphological changes associated with apoptotic cell death. However, using the actin microfilament disrupting agent, cytochalasin, we present evidence that the formation of blebs leading to apoptotic cell fragmentation requires neopolymerization of actin. Indeed, in the presence of cyochalasin, induction of apoptosis (internucleosomal DNA degradation) in BAE cells by TNF and cycloheximide was not associated with these classical morphological markers of apoptosis. Moreover, when added to BAE cells showing incipient apoptotic fragmentation, cytochalasin E reversed this process. We also observed an accumulation of actin at the basis of the apoptotic bodies in formation in these cells. Together, these results suggest that the actin network of flattened cells is disrupted concomitantly to the morphological modifications associated to the apoptotic cell death, and that the cytochalasin-sensitive reorganisation of actin is required to the formation of apoptotic blebs.
    Journal of Cellular Physiology 09/2000; 184(2):239-45. · 4.22 Impact Factor
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    ABSTRACT: Nucleotides are ubiquitous intercellular messengers whose actions are mediated by specific receptors. Since the first clonings in 1993, it is known that nucleotide receptors belong to two families: the ionotropic P2X receptors and the metabotropic P2Y receptors. Five human P2Y receptor subtypes have been cloned so far and a sixth one must still be isolated. In this review we will show that they differ by their preference for adenine versus uracil nucleotides and triphospho versus diphospho nucleotides, as well as by their transduction mechanisms and cell expression.
    Cellular Signalling 07/2000; 12(6):351-60. · 4.30 Impact Factor
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    ABSTRACT: The cytoskeleton undergoes dramatic changes during apoptosis and many cytoskeletal proteins are known to be degraded during this process. The number of proteases found to be involved in apoptosis is growing but the role of the proteolysis they cause remains poorly understood. This report describes for the first time that myosin heavy chain is cleaved in aortic endothelial cell apoptosis induced either by tumour necrosis factor-alpha or okadaic acid. The cleavage was specific since a well-defined major 97 kDa fragment of myosin heavy chain was produced. The intermediate filament component vimentin was also cleaved into well-defined fragments (31, 28 and 23 kDa). Kinetic studies showed that proteolysis occurred concomitantly with the morphological changes associated with apoptosis, i.e. cellular condensation and fragmentation in apoptotic bodies. These data suggest that the degradation of myosin and vimentin could be involved in the execution of the morphological alterations observed during apoptotic cell death.
    Cell Proliferation 05/2000; 33(2):101-14. · 2.27 Impact Factor

Publication Stats

956 Citations
147.03 Total Impact Points

Institutions

  • 1989–2013
    • Université Libre de Bruxelles
      • • Erasme Hospital
      • • Department of Interdisciplinary Research
      • • Institute of Interdisciplinary Research in human and molecular Biology (IRIBHM)
      • • Institute of Molecular Biology and Medicine (IBMM)
      • • Laboratory of Oncology and Experimental Surgery
      Bruxelles, Brussels Capital Region, Belgium
  • 1990–2006
    • Free University of Brussels
      • Institute of Interdisciplinary Research (IRIBHM)
      Bruxelles, Brussels Capital Region, Belgium
  • 1995
    • Unité Inserm U1077
      Caen, Lower Normandy, France