Nous avons identifié l’expression du récepteur des angiopoïétines, le récepteur Tie2, à la surface des neutrophiles humains. De plus, nous avons démontré qu’Ang1 et Ang2 induisent des activités pro-inflammatoires sur les neutrophiles, comme l’adhésion aux cellules endothéliales (CEs) et la synthèse du facteur d’activation plaquettaire (PAF). Puisque le PAF augmente la viabilité des neutrophiles et que les angiopoïétines modulent la survie des CEs, nous avons voulu évaluer l’effet des angiopoïétines sur la survie des neutrophiles. Des neutrophiles humains ont été isolés à partir du sang de donneurs sains en accord avec le comité d’éthique de l’Institut de cardiologie de Montréal. La viabilité des neutrophiles a été mesurée par cytométrie en flux à l’aide de marqueurs d’apoptose et de nécrose. Un traitement avec des témoins positifs, soit l’interleukine 8 (IL-8; 25 nM) ou le PAF (100 nM), a augmenté la survie basale des neutrophiles de 34 et 27%, respectivement. De plus, un traitement avec Ang1 (1 pM – 10 nM) a augmenté la survie des neutrophiles jusqu’à 35%, alors qu’Ang2 n’a eu aucun effet. La combinaison de l’IL-8 ou du PAF avec Ang1 (10 nM) a eu un effet additif sur la viabilité des neutrophiles et a augmenté la survie de 56 et 60%, respectivement. Un prétraitement avec des anticorps bloquants contre l’IL-8 a permis d’inhiber l’activité anti-apoptotique de l’IL-8 et d’Ang1 de 92 et 80%, respectivement. Ainsi, notre étude est la première à démontrer la capacité d’Ang1 à prolonger la viabilité des neutrophiles, qui est principalement causée par la relâche d’IL-8. / We reported the expression of angiopoietin receptor Tie2 on the surface of human neutrophils. In addition, we reported that Ang1 and Ang2 are both capable to promote pro-inflammatory activities in neutrophils, namely their adhesion onto endothelial cells (ECs) and platelet-activating factor (PAF) synthesis. PAF is known to promote pro-survival activity on neutrophils and since both angiopoietins can modulate ECs viability, we addressed whether Ang1 and/or Ang2 could modulate neutrophil viability. Human neutrophils were isolated from blood of healthy volunteers in accordance with the guidelines of the Montreal Heart Institute’s ethical committee. Neutrophil viability was assessed by flow cytometry using apoptotic and necrotic markers. Treatment with anti-apoptotic mediators such as interleukin 8 (IL-8; 25 nM) and PAF (100 nM) increased neutrophil basal viability by 34 and 27%, respectively. In addition, treatment with Ang1 (1 pM – 10 nM) increased neutrophil viability by up to 35%, while Ang2 had no effect. Combination of IL-8 or PAF with Ang1 (10 nM) provided an additive effect on neutrophil viability and further increased viability by 56 and 60%, respectively. Pretreatment of the neutrophils with blocking anti-IL-8 antibodies inhibited the anti-apoptotic effect of IL-8 and Ang1 by 92 and 80%, respectively. In summary, our data are the first one to report Ang1 pro-survival activity on neutrophils, which is mainly driven through IL-8 release.

L'épilepsie du lobe temporal est fréquente et souvent pharmacorésistante. L'épileptogenèse est imputée à la mort neuronale, l'inflammation ou au déséquilibre de la neurotransmission. Récemment, la perméabilité vasculaire a été reconnue comme une cause de crises d'épilepsie. Dans un modèle d'épilepsie chronique, nous avons montré une angiogenèse associant vascularisation, surexpression de VEGF, perte de protéines des jonctions serrées et perméabilité de la BHE. L'observation des immunoglobulines G (IgGs) comme marqueurs de perméabilité vasculaire nous a permis de découvrir que les IgGs s'accumulent dans les neurones. Nous avons alors étudié le rôle de ces protéines dans l'épileptogenèse. Ensuite, afin de corréler la perméabilité de la BHE à l'épileptogenèse, nous avons étudié le kindling, un modèle dans lequel les crises sont induites mais pas spontanées. Nous n'avons observé aucun remaniement vasculaire, si ce n'est une dérégulation transitoire de deux protéines de jonctions serrées. La comparaison de ces deux modèles confirme la contribution de la dérégulation de la BHE dans la genèse des crises et la désigne comme une nouvelle cible thérapeutique. / Temporal lobe epilepsy is the most frequent form of pharmacoresistant epilepsies. Epileptogenesis is commonly imputed to neuronal loss, inflammation and an imbalance in neurotransmission. Now, vascular permeability was shown to participate in epileptic seizures generation. In a model of chronic epilepsy, we showed a neo-vascularisation associated with VEGF over expression, loss of tight junction proteins and BBB permeability. The use of immunoglobulins G (IgGs) as markers of permeability vascular allowed us to discover that the IgGs accumulates in neurones. We then studied the role of these proteins in epileptogenesis. Then, to correlate BBB permeability to epileptogenesis, we studied the kindling, a model in which seizures are induced but never spontaneous. We observed no vascular remodeling, except for a transient deregulation of tight junctions proteins. The comparison of these models confirms the contribution of BBB deregulation and points it as new therapeutic target.

No description available.

Abstract Venous malformations (VMs) are localized defects in vascular morphogenesis which can seriously impede or even threaten the patient’s life. VMs are characterized by enlarged, torturous vein-like channels lined by unevenly distributed smooth muscle cells. A large number of mutations in the endothelial TIE2 receptor tyrosine kinase have been found from more than half of the lesions screened, thus providing a common genetic cause. TIE2 has a crucial role in vascular development, remodeling and quiescence. However, the molecular and cellular abnormalities caused by TIE2-mutations in endothelial cells and how they relate to VM formation have been unknown. The aim of this study was to examine how VM-specific mutations affect the molecular characteristics of TIE2-receptor downstream signaling and cellular functions. Because no effective treatment has been available for VMs, a better understanding of the molecular basis of their pathology should enable the development of more potent and non-invasive treatments as well as provide a better understanding of vascular morphogenesis in general. The results demonstrate that the TIE2-VM forms have both common and specific effects on TIE2 and the endothelial cells (ECs) expressing them. Mutation-induced TIE2 autoactivation leading to loss of normal EC monolayer organization due to extracellular matrix (ECM) fibronectin deficiency was found to be a common change. This was shown to occur through chronic activation of the mitogen-activated protein kinase (MAPK) pathway, which also caused activation of the proteolytic plasminogen system. Also, most mutations altered TIE2 trafficking and angiopoietin ligand regulated TIE2 functions, albeit through different mechanisms. Using RNA-screening we showed that the most common sporadic TIE2-VM mutation dysregulates genes affecting vascular development, cell migration and ECM remodeling. PDGFB, a major attractant of vascular mural cells, was found to be strongly attenuated due to chronic activation of Akt, which also increases EC survival, by the TIE2 mutant receptors. To conclude, the results in this thesis reveal genetic, molecular and cellular alterations which may potentiate VM formation. This data provides new information on the pathological mechanisms behind abnormal vascular morphogenesis and should assist the development of new molecular treatment strategies for VM patients. / Tiivistelmä Laskimoepämuodostumat ovat paikallisia verisuoniston kehityksen häiriöitä. Riippuen niiden koosta ja anatomisesta sijainnista ne voivat aiheuttaa merkittävää haittaa. Epämuodostumat koostuvat laajentuneista, laskimonkaltaisista verisuonista, joissa sileiden lihassolujen kerros on puutteellisesti järjestäytynyt. Yli puolessa tutkituista laskimoepämuodostumista havaitaan mutaatioita verisuonten sisäpinnan endoteelisoluissa ilmenevässä TIE2 reseptorityrosiinikinaasissa, joka säätelee verisuonten kehitystä, muokkausta ja fysiologista toimintaa. TIE2-mutaatioiden aiheuttamia molekyyli- ja solutason muutoksia tai niiden yhteyttä epämuodostumien syntyyn ei ole aikaisemmin tunnettu. Tämän tutkimuksen tarkoituksena oli selvittää, miten laskimoepämuodostumista löydetyt mutaatiot vaikuttavat TIE2-reseptorin toimintaan molekyyli- ja solutasolla sekä TIE2-reseptorista alkavaan solunsisäiseen viestintään. Koska pysyvää hoitomuotoa laskimoepämuodostumille ei tunneta, voisi tieto niiden taustalla olevista patologisista mekanismeista edesauttaa parempien, ei-kajoavien hoitomuotojen kehittämisessä ja antaa myös yleisesti uutta tietoa verisuoniston kehityksestä. Väitöskirjan tulokset osoittavat, että mutaatiot vaikuttavat TIE2-reseptoriin ja sitä ilmentäviin endoteelisoluihin mutaatioille yhteisillä sekä mutaatiokohtaisilla tavoilla. Mutaatioille tyypillinen TIE2-reseptorin ligandista riippumaton aktivaatio aiheutti aktivaation nousun myös TIE2:sta alavirtaan olevissa viestinvälittäjissä. Tämä puolestaan johti fibronektiini-proteiinin häviämiseen soluväliaineesta, sileitä lihassoluja säätelevän PDGFB-kasvutekijän ilmenemisen laskuun ja solujen ohjelmoidun solukuoleman vähenemiseen. Useimmat tutkitut mutaatiot muuttivat myös TIE2-reseptorin sijaintia soluissa häiriten TIE2:n angiopoietiini-ligandien säätelemiä toimintoja usean eri mekanismin kautta. Transkriptomin laajuiset RNA-tutkimukset osoittivat monien verisuonten kehitykseen, solujen liikkumiseen ja soluväliaineen muokkaukseen liittyvien geenien ilmentymisen muuttuneen. Lopputuloksena tutkimus paljasti geeni-, molekyyli-, ja solutason muutoksia, jotka saattavat vaikuttaa laskimoepämuodostumien syntyyn. Tulokset antavat lisätietoa sairautta aiheuttavista mekanismeista verisuoniston kehityksen häiriöiden taustalla ja ovat hyödyksi kehitettäessä uusia lääkkeitä laskimoepämuodostumien molekulaarisia hoitoja varten.

Les neutrophiles peuvent synthétiser et relâcher l’angiopoïétine 1 (Ang1), un facteur de croissance cytosolique impliqué dans l’angiogenèse, capable d’induire plusieurs activités pro-inflammatoires chez les neutrophiles. Ces derniers induisent la synthèse et la relâche de neutrophil extracellular traps (NETs), des filaments d’ADN nucléaires décondensés transportant des protéines telles que l’élastase, la myélopéroxydase (MPO), la protéinase (PR3) et la calprotectine (S100A8/S100A9) qui contribuent à la réponse immunitaire innée afin de combattre les pathogènes (i.e. bactéries). Les NETs ont des effets pro-inflammatoires, pro-thrombotiques, jouant un rôle dans la dysfonction endothéliale, et ont récemment été retrouvés dans l’insuffisance cardiaque (IC) et le diabète de type 2 (T2DM). Le but de cette étude est de déterminer la synthèse et la relâche d’Ang1 chez des patients T2DM et insuffisants cardiaques avec fraction d’éjection préservée (HFpEF) (stables ou en décompensation aigue (ADHFpEF)) avec ou sans T2DM en comparant à des volontaires sains (VS) comme groupe témoin. Nos résultats démontrent qu’en absence de traitement (PBS) et avec un traitement avec LPS, les niveaux de NETs augmentent chez les patients ADHFpEF + T2DM comparé aux VS. Nous observons également que le LPS, le PMA ou A23187 augmentent la synthèse de l’Ang1 (de 150 à 250%) chez des VS et cet effet est amplifié chez les patients T2DM et IC. L’Ang1 est relâchée à 100% par les neutrophiles de tous les groupes à l’étude et ne se lient pas sur les NETs, comparé à la calprotectine. Notre étude suggère que les patients en insuffisance cardiaque aigue (ADHFpEF + T2DM) synthétisent et relâchent plus de NETs et que l’exocytose de l’Ang1 est indépendante de la synthèse et la relâche de NETs. / Neutrophils can induce the synthesis and release of angiopoietin 1 (Ang1), a cytosolic growth factor involved in angiogenesis capable of inducing several neutrophil-driven pro-inflammatory activities. Neutrophils also synthesize and release neutrophil extracellular traps (NETs) composed of decondensed nuclear DNA filaments carrying proteins such as neutrophil elastase (NE), myeloperoxidase (MPO), proteinase 3 (PR3) and calprotectin (S100A8/S100A9) which contribute to the innate immune response to combat pathogens (e.g., bacteria). NETs are also implicated in various pathological conditions through pro-inflammatory, pro-thrombotic properties, leading to endothelial dysfunction, and have recently been found in heart failure (HF) and type 2 diabetes (T2DM). The purpose of this study is to determine the synthesis and release of Ang1 in patients with T2DM and HF with preserved ejection fraction (HFpEF) (stable or acute decompensated (ADHFpEF)) with or without T2DM, compared to healthy volunteers (HV) as control group. Our results show that in absence of agonist (PBS) and after LPS treatment, NETs levels are increased in ADHFpEF + T2DM patients compared to HV. We also observed that LPS, PMA or A23187 treatments increase the synthesis of Ang1 (from 150 to 250%) in HV and this effect is amplified in T2DM and HF patients. Ang1 is completely released (100%) by neutrophils isolated from all cohorts and does not bind to NETs compared to calprotectin. Our study suggests that severely ill patients with decompensated heart failure (ADHFpEF + T2DM) synthesize and release more NETs and that Ang1 exocytosis is independent from NETs synthesis.

No description available.

Diversas doenças estão associadas com a formação de novos vasos a parti vasos pré-existentes, ou angiogênese. Dentre elas está a aterosclerose (Griffioen & Molema, 2000). Pesquisas recentes demonstram que a hipercolesterolemia, que têm um papel importante na fisiologia da aterosclerose, também pode prejudicar a ação de fatores angiogênicos (Jang et.al., 2000). A hipercolesterolemia que é decorrente de aumento de LDL no plasma ocasiona um aumento no tempo de permanência desta partícula na circulação (Yasunobu, 2001). Contudo, a LDL pode sofrer modificação na circulação, dando origem a uma subfração mais eletronegativa da LDL, a LDL (-). A LDL (-) pode prejudicar cada etapa da angiogênese, desregulando a função endotelial (Tai et. al., 2006). Em nosso estudo, vimos que apesar da LDL (-) ter estimulado a miga celular, esta partícula inibiu a formação de túbulos in vitro. A LDL (-) não foi capa afetar a angiogênese in vivo. / A large number of diseases is associated with formation of new blood vessels out of pre-existing capillaries, or angiogenesis. These diseases include the atherosclerosis (Griffioen & Molema, 2000). Resents researches demonstrate that the hypercholesterolemia, that have a important role in the physiology of the atherosclerosis, can impaired the angiogenesis (Jang et. al., 2000) . The hypercholesterolemia that is decurrente of high levels of LDL in the plasma causes an increase in the time of permanence this particle in the circulation (Yasunobu, 2001). However, the LDL can to suffer modification in the circulation, giving rise to a subfration more eletronegative from LDL, the LDL (-). The LDL (-) could impair each one of the steps of the angiogenesis, thereby dysregulating endothelial function (Tai et. al., 2006). In our study, see that despite the LDL (-) have stimulated the cell migration, this particle inhibited the Tube formation in vitro. The LDL (-) didn\'t affect the angiogenesis in vivo.

Diversas doenças estão associadas com a formação de novos vasos a parti vasos pré-existentes, ou angiogênese. Dentre elas está a aterosclerose (Griffioen & Molema, 2000). Pesquisas recentes demonstram que a hipercolesterolemia, que têm um papel importante na fisiologia da aterosclerose, também pode prejudicar a ação de fatores angiogênicos (Jang et.al., 2000). A hipercolesterolemia que é decorrente de aumento de LDL no plasma ocasiona um aumento no tempo de permanência desta partícula na circulação (Yasunobu, 2001). Contudo, a LDL pode sofrer modificação na circulação, dando origem a uma subfração mais eletronegativa da LDL, a LDL (-). A LDL (-) pode prejudicar cada etapa da angiogênese, desregulando a função endotelial (Tai et. al., 2006). Em nosso estudo, vimos que apesar da LDL (-) ter estimulado a miga celular, esta partícula inibiu a formação de túbulos in vitro. A LDL (-) não foi capa afetar a angiogênese in vivo. / A large number of diseases is associated with formation of new blood vessels out of pre-existing capillaries, or angiogenesis. These diseases include the atherosclerosis (Griffioen & Molema, 2000). Resents researches demonstrate that the hypercholesterolemia, that have a important role in the physiology of the atherosclerosis, can impaired the angiogenesis (Jang et. al., 2000) . The hypercholesterolemia that is decurrente of high levels of LDL in the plasma causes an increase in the time of permanence this particle in the circulation (Yasunobu, 2001). However, the LDL can to suffer modification in the circulation, giving rise to a subfration more eletronegative from LDL, the LDL (-). The LDL (-) could impair each one of the steps of the angiogenesis, thereby dysregulating endothelial function (Tai et. al., 2006). In our study, see that despite the LDL (-) have stimulated the cell migration, this particle inhibited the Tube formation in vitro. The LDL (-) didn\'t affect the angiogenesis in vivo.