Sickle cell anemia is a hematological disorder that is caused by a single point mutation in the beta-globin chain of hemoglobin. It results in several complications related to the small and large vessels in patients with the disease. Large vessel complications include cerebral infarcts, which are observed in children under ten years old. The mechanism behind this complication is not completely understood. It is the goal of this project to begin to understand the role sickle erythrocytes may play in causing endothelial dysfunction as a precursor to sickle related complications. The hypothesis of this work is that exposure of large vessel endothelium to sickle erythrocytes causes an increase in endothelial permeability through loosening of adherens junctions. In the first goal of this work, bovine aortic endothelial cells (BAECs) are grown on coverslips and exposed to sickle erythrocytes for 5 minutes and either immediately fixed or incubated in 30 minutes and then fixed. Immunofluorescent studies labeling VE cadherin show changes in VE cadherin dynamics, suggesting sickle erythrocytes may be involved in this observation. Next, BAECs were grown on transwell inserts and exposed to sickle erythrocytes for 5 minutes. The erythrocytes are washed off and the BAEC are incubated with 10,000 MW dextran conjugated to lucifer yellow or FITC-BSA or to determine BAEC permeability. When dextran is used as the test molecule, endothelial permeability did not show a significant change from baseline. However, when BSA is used as the test molecule, increases in endothelial permeability are observed. Explanations into the differences between the transport mechanisms of the two molecules are discussed. These experiments show changes in VE cadherin localization due to sickle erythrocyte exposure. This may cause increases in endothelial permeability and an experimental model and preliminary studies are performed. This study provides potential mechanisms to explain the changes in VE cadherin localization and provide suggestions for further studies to test the effect of sickle erythrocytes on endothelial permeability. This work provides a strong foundation for continuing studies on the effects of sickle erythrocytes on endothelial dysfunction within the confines of sickle related complications.

Vascular endothelial cells (VECs) play an essential role in regulating the passage of macromolecules and cells between the blood stream and underlying tissues. The second messenger 3’, 5’ cyclic adenosine monophosphate (cAMP) regulates numerous events in VECs, including permeability. Since human VECs express several distinct cAMP-hydrolyzing phosphodiesterases (PDEs), and these are the only enzymes that catalyze the inactivation of cAMP, we investigated if selective pharmacological inhibition of PDEs could impact VEC permeability. Interestingly, we found that PDE4 inhibitors decreased human aortic VEC (HAEC) permeability and PDE4 and PDE3 inhibitors decreased human microvascular VEC (HMVEC) permeability. Consistent with a role for both protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in regulating VEC permeability, selective activators of these enzymes significantly decreased permeability. Since neither PDE4 nor PDE3 inhibitors significantly increased cAMP in these cells, our data are consistent with the idea that PDE inhibition causes small localized increases in “pools” of cAMP that regulate permeability. In order to test if PDE4 enzymes could act locally on pools of cAMP that regulated permeability, we selectively isolated the adherens junctional protein VE-cadherin from confluent monolayers of HAECs or HMVECs, and immunoblotted these isolates for cAMPeffectors and PDEs. Briefly, we found that each PKA-II, EPAC1, and a PDE4D variant, but not PDE3 enzymes, each could be isolated in VE-cadherin-based complexes from these cells. These novel findings identify PKA-II, EPAC1, and PDE4D as members of VE-cadherin-based signaling complexes in human VECs and are consistent with the idea that localized cAMP-signaling regulates permeability in these cells. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2007-09-14 15:52:20.216

Adherens junctions are cadherin-dependent structures that mediate intercellular signaling and structural integrity of the endothelial barrier. Formins are a highly conserved family of cytoskeletal remodeling proteins whose activity has been implicated in regulating adherens junction formation in other cell-types. Therefore, we tested the hypothesis that formin activity is essential for adherens junction assembly in endothelial cells. A small-molecule formin inhibitor (smiFH2) was used to determine the effect of formin inhibition on junction formation using an in vitro vascular permeability assay. We determined that smiFH2 treatment caused a dose-dependent inhibition of junction formation. We used siRNAs to knockdown expression of the seven formins shown to be expressed in TIME cells and determined that individual knockdown of FHOD1, FHOD3 and Dia1 significantly increased the permeability of the endothelial monolayer. Interestingly, FMNL2 knockdown actually potentiated barrier function. Knockdown of the remaining formins had little or no effect on junction formation. Knockdown of FHOD3 had the greatest inhibitory effect on junction assembly; VE-cadherin protein levels were decreased in FHOD3-depleted cells. The FHOD3 knockdown cells were also elongated in comparison to controls and formed thin linear adherens junctions and few focal adherens junctions. In contrast, the morphology of FMNL2-depleted cells did not appear obviously different from controls. In conclusion, our results suggest that multiple formins play diverse roles in adherens junction formation and maintenance in endothelial cells.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24529号 / 医博第4971号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙橋 良輔, 教授 浅野 雅秀, 教授 辻川 明孝 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

La croissance tumorale et le processus de métastatisation dépendent de la néoformation de vaisseaux sanguins ou néoangiogenèse. Parmi les molécules intervenant dans ce processus, l'adrenomédul1ine (AM) est un peptide, dont l'expression est corrélée à l'agressivité de certaines tumeurs, et qui représente un maillon «clé» dans les interactions entre les cellules tumorales et les cellules du microenvironnement. Les résultats spectaculaires qu'offre le traitement des xénogreffes de cellules issues de glioblastomes (GBM) humains par les anticorps dirigés contre l'AM ou son récepteur sont très encourageants, puisque la tumeur traitée régresse en quelques semaines, la vascularisation tumorale s'en trouve touchée de manière spécifique. C'est dans ce contexte, que nous avons choisi de poursuivre notre travail sur les mécanismes d'action de l'AM dans la néoangiogenèse. Grâce à des études in vitro et in vivo, nous avons pu montrer que l'AM est impliquée dans plusieurs étapes de la néoangiogenèse tumorale : migration des cellules endothéliales, stabilisation des contacts endothéliaux et endothélio-péricytaires, recrutement des cellules mésenchymateuses. Nos résultats démontrent que nous sommes en présence d'une molécule d'AM qui agit sur diverses cibles moléculaires et cellulaires, régulant la stabilité du complexe d”adhésion intercellulaire VE-cadhérine/-caténine, nécessaire à la protection des interactions homotypiques et hétérotypiques de l°endothélium nouvellement formé. Ainsi, l'étude des mécanismes d'action de l'AM réalisée pennettra d'établir ue stratégie thérapeutique autour de l'AM. / Tumoral growth and process of metastatization depend on the formation of new blood vessels or angiogenesis. Among the molecules implicated in this process, adrenomedullin (AM) is a peptide, which expression is correlated with the aggressiveness of tumors, and which represents a "key" link in the interactions between tumoral cells and the microenvironment cells. The spectacular results offered by the treatment of human glioblastoma (GBM) xenograft by antibodies directed against the AM or its receptor are very encouraging, as the treated tumor declines in some weeks, and the tumoral vascularization is also touched in a specific way. In this context, we chose to pursue our work on the mechanisms of action of AM in angiogenesis. In vitro and in vivo studies showed that AM is involved in several stages of tumoral angiogenesis : migration of endothelial cells, stabilization of endothelial contacts, stabilization of the pericyte coverage, recruitment of multipotent cells. Our results demonstrate that we are in presence of a molecule of AM which acts on diverse molecular and cellular targets, regulating the stability of the VE-cadherin/β-catenin complex, required for the protection of the homotypics and heterotypics interactions of the newly formed endothelium. The study of the mechanisms of action of AM realized will allow us to establish a therapeutic strategy around AM.

L'endothelium vasculaire constitue la principale barrière entre le sang et les tissus régulant le passage de macromolécules et de cellules circulantes. Longtemps considéré comme une monocouche passive, l'endothélium joue d'importants rôles dans la régulation de la pression sanguine, de l'hémostase, des réponses immunitaires et inflammatoires. L'adhérence cellule/cellule est initiée dans l'endothélium vasculaire par des interactions homophiliques entre molécules de VE-cadhérine (= jonctions adhérentes). La dynamique de la jonction et du cytosquelette est importante pour le remodelage des jonctions intercellulaires qui a lieu au cours l'angiogenèse, de la vasculogenèse et lors de la réparation de l'endothélium. C'est pourquoi la détermination des mécanismes moléculaires sous-jacents est indispensable à la comprehension de phénomènes physiopathologiques (angiogenèse et progression tumorales, inflammation...). D'après la littérature, la protéine EPLIN intervient dans la formation du complexe E-cadhérine/alpha-caténine/EPLIN et stabilise l'actine corticale. Actuellement décrite comme spécifique des modèles épithéliaux, EPLIN peut-elle intervenir dans la liaison du complexe à base de VE-cadhérine au cytosquelette d'actine? De plus, il paraît essentiel de comprendre le rôle de cette protéine dans les cellules car son expression est fortement diminuée dans la plupart des cancers alors qu'à l'inverse sa surexpression bloque la prolifération cellulaire. / The endothelium forms the main barrier regulating the passage of macromolecules and circulating cells between the blood and tissue. Historically viewed as a passive vascular lining, vascular endothelium plays important roles in the regulation of vascular pressure, hemostasis, immune and inflammatory responses. In vascular endothelium, cell/cell adhesion is mediated by homophilic interactions of VE-cadherin molecules (= adherens junctions). Cytoskeleton and junction dynamics are important for intercellular junctions remodelling that occurs during angiogenesis, vasculogenesis and endothelium repair. So, determining the underlying molecular mechanisms is essential for the comprehension of pathologic phenomena such as angiogenesis, tumor progression or inflammation. We learn from the literature that EPLIN is involved in E-cadherin/α-catenin/EPLIN complex formation and cortical actin stabilization. Usually described as a protein specific of epithelial models, we wondered if EPLIN is able to link VE-cadherin complex to actin cytoskeleton. Furthermore, it seems essential to understand its cellular role since it is downregulated in many cancers while in contrast its overexpression blocks cell proliferation.

L’angiogenèse, indispensable à la mise en place d’un réseau vasculaire fonctionnel, est au cœur des stratégies thérapeutiques des pathologies ischémiques. L’hypoxie, caractérisant ces tissus ischémiques, est un stimulus majeur de l’angiogenèse, en induisant l’expression de facteurs de croissance tels que le VEGF et de protéines de la matrice extracellulaire endothéliale. Nous avons identifié la protéine ANGPTL4, comme une cible majeure de l’hypoxie et ayant des effets opposés au VEGF sur la perméabilité vasculaire. Le but de cette thèse a consisté en l’analyse du rôle d’ANGPTL4 sur la formation de capillaire et l’organisation des jonctions adhérentes dans un contexte dépendant du VEGF. J’ai démontré que le VEGF stimule la formation d’un dense réseau capillaire 3D alors qu’ANGPTL4 induit la formation de capillaires étroits et peu ramifiés. ANGPTL4 réduit la taille du réseau de capillaire induit par le VEGF en limitant le nombre de bourgeons, de branchements et la largeur des capillaires. ANGPTL4 renforce l’intégrité des capillaires formés en présence de VEGF en préservant des jonctions adhérentes stables. J’ai démontré qu’ANGPTL4 limite les processus de migration 3D et de prolifération induits par le VEGF. L’analyse de la voie de signalisation VEGF/ANGPTL4 a montré une potentialisation par ANGPTL4 de la phosphorylation Y1175 du VEGFR2, impliqué dans l’internalisation de VEGFR2. En conclusion, ce modèle révèle un effet d’ANGPTL4 dépendant du contexte 3D, qui stimule les processus d’angiogenèse en absence de VEGF et qui contrecarre la morphogenèse induite par le VEGF en renforçant l’intégrité des jonctions adhérentes et en régulant la signalisation en aval du VEGFR2. / Angiogenesis, by promoting new functional capillaries, is a main target of therapeutic strategies of ischemic pathologies. Ischemic tissues are characterized by hypoxic environment, which stimulates angiogenesis by inducing expression and secretion of growth factors such as VEGF and by remodeling endothelial extracellular matrix. Our team identified ANGPTL4 as a hypoxia-induced target and characterized its counteracting effect on VEGF-induced vascular permeability. This PhD study therefore aimed to decipher the role of ANGPTL4 on angiogenesis, capillary architecture and adherens junction (VE-cadherin) organization in a VEGF-dependent context. I demonstrated that VEGF induced formation of branched capillaries forming a dense 3D network while ANGPTL4 enhanced the formation of unbranched and tight capillaries. Remarkably, ANGPTL4 reduces VEGF-induced angiogenesis, by limiting branching and widening of the capillaries. Furthermore, ANGPTL4 regulates the local VE-cadherin patterning during the sprouting process by maintaining lateral linear structures and limiting the VEGF-induced formations involved in the migratory capacities. I demonstrated that ANGPTL4 limited VEGF-induced 3D endothelial cell migration and proliferation. Analysis of VEGF/ANGPTL4 signaling pathway pointed out that ANGPTL4 enhanced phosphorylation of Y1175 VEGFR2, known to enhance internalization of VEGFR2. In conclusion, this study modeled the 3D context-dependent effect of ANGPTL4 that stimulates angiogenesis in absence of VEGF whereas it counteracts VEGF-induced endothelial morphogenesis by regulating VEGFR2 trafficking and strengthening adherens junctions.

La fonction de barrière de l'endothélium vasculaire est affectée par des modifications de la cadhérine des cellules endothéliales (VE-cadhérine) telles que la phosphorylation sur tyrosine dans son domaine cytoplasmique et le clivage de son domaine extracellulaire (sVE). Ce travail s'est articulé en deux parties : 1- Etude de ces modifications dans le contexte de la polyarthrite rhumatoïde (PR), et les mécanismes sous-tendus. Ce travail a permis de montrer que la VE-cadhérine est une cible du TNFα, une cytokine essentielle dans la PR, qui induit de la libération de sVE de façon dépendante de l'activité kinase de Src, suggérant l'implication d'un mécanisme de phosphorylation sur tyrosine dans ce processus. De plus, la VE-cadhérine est aussi la cible des protéases de la matrice extracellulaire telles que MMP-2. L'application de ces données fondamentales à la clinique a permis de montrer que sVE était retrouvée dans le sang de patients PR (n=63) et que son taux était corrélé à l'activité de la maladie. Ainsi, le dosage de sVE est d'intérêt dans la prise en charge des patients. 2-Etude de la phosphorylation de la VE-cadhérine dans un contexte d'angiogenèse hormono-régulée au cours du cycle ovarien chez la souris. Les résultats ont montré que le site Y685 de la VE-cadhérine est phosphorylé dans l'ovaire et l'utérus de souris de façon régulée pendant le cycle, ce qui permet de proposer ce modèle physiologique pour étudier la phosphorylation de la VE-cadhérine in vivo. L'analyse de souris knock-in Y685F de la VE-cadhérine (VE-Y685F) a montré que l'absence du site confère une perméabilité accrue dans l'ovaire et l'utérus mais aussi dans les petits capillaires de la peau. De plus, dans deux modèles d'induction d'arthrite, les souris VE-Y685F ont présenté un taux de sVE plus élevé que les contrôles. Au total, sVE et la phosphorylation de la VE-cadhérine ont un vaste champ d'application dans le traitement de la PR ainsi que pour le développement de nouvelles thérapies pouvant s'étendre à d'autres pathologies vasculaires. / The vascular endothelium barrier function is influenced by vascular endothelial cadherin (VE-cadherin) modifications such as its cytoplasmic tail tyrosine phosphorylation and its ecto-domain cleavage (sVE). The work reported herein was divided into two parts: 1- Study of VE-cadherin modifications and the mechanisms underlying these ones in the rheumatoid arthritis context. This work demonstrated that VE-cadherin is a target of TNFα, a highly important cytokine in rheumatoid arthritis (RA) pathogenesis. We found TNFα to induce sVE shedding in a Src kinase dependent manner, suggesting the involvement of phosphorylation mechanism in this process. In addition, this VE-cadherin cleavage requires matrix metalloproteinase activities such as that of MMP-2. Applying these fundamental data to clinical study showed that sVE was detected in sera of 63 RA patients and its titer was correlated with the disease activity. This finding suggests an obvious interest for assaying sVE in RA therapies. 2- Study of VE-cadherin tyrosine phosphorylation in a context of hormones-regulated angiogenesis during mouse estrous cycle. The results showed VE-cadherin phosphorylation at Y685 that was regulated along mouse estrous cycle allowing to proposing mouse estrous cycle as a physiological model for studying VE-cadherin phosphorylation in vivo. The analysis of VE-cadherin Y685F knock-in mice (VE-Y685F) showed that these mice exhibit a higher vascular permeability in uterus and ovaries and the skin small capillaries compared to wild-type animals. Moreover, VE-Y685F mice presented higher levels of soluble VE-cadherin compared to wild-type counterparts in two induced arthritis model. Altogether, sVE and VE-cadherin phosphorylation present an array of interests for RA therapies as well as developing new therapeutic tools in RA and other vascular diseases.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.

A lagarta da espécie Lonomia obliqua é medicamente importante, cujo veneno, presente nas espículas, causa uma síndrome hemorrágica caracterizada por equimoses, alterações da coagulação, dentre outros sintomas. Isto sugere a presença de peptídeos bioativos com potencial farmacêutico, devido à capacidade de modular o comportamento das células endoteliais. O objetivo deste estudo é analisar os potenciais efeitos do veneno de Lonomia obliqua na angiogênese. Uma linhagem celular endotelial (HUVEC) foi exposta a diferentes concentrações do extrato de espículas da Lonomia obliqua (Lonomia obliqua Bristle extract - LOBE) 5 μg/mL, 10 μg/mL, 20 μg/mL e 50 μg/mL. Empregando citometria de fluxo, observou-se que nenhuma das doses afetou o ciclo celular, viabilidade ou apoptose das células endoteliais após 24h de exposição. Os esferóides das células HUVEC foram plaqueados numa matriz 3D de colágeno e observou-se que LOBE (10 μg/mL, 20 μg/mL e 50 μg/mL) induz um aumento na migração celular, consistente com o processo de angiogênese. A análise da dinâmica da VE-caderina indica que a exposição imediata a LOBE (10 μg/mL) induz um desprendimento da junção célula-célula, o que corrobora com a hemorragia observada nas vítimas de envenenamento. Através de espectrometria de massa, observou-se que LOBE possui vários potenciais peptídeos bioativos. Grupos destes peptídeos foram isolados por fracionamento com metanol a partir do veneno bruto. Os peptídeos presentes, em cada uma das 10 frações, foram caracterizados por espectrometria de massa e foram analisados os efeitos de cada fração sobre a angiogênese. Os resultados sugerem que alguns dos efeitos do envenenamento por Lonomia obliqua são devidos à presença de peptídeos bioativos que modulam o comportamento das células endoteliais. / The caterpillar of the species Lonomia obliqua is medically important, whose venom present in the bristles leads to an hemorrhagic syndrome characterized by ecchymosis, coagulation disorders and others symptoms. This suggests the presence of bioactive peptides with pharmaceutical potencial due to the ability to modulate the behavior of endothelial cells. The aim of this study is to analyze the potential effects of Lonomia obliqua venom on angiogenesis. An endothelial cell line (HUVEC) was exposed to different concentrations (5 μg/mL, 10 μg/mL, 20 μg/mL and 50 μg/mL) of Lonomia obliqua bristle extract (LOBE). Using flow cytometry, it was observed that none of the doses affected endothelial cell cycle, cell viability or apoptosis after 24h of exposition. Spheroids of HUVEC cells were plated in a 3D-collagen matrix and it was observed that LOBE (10 μg/mL, 20 μg/mL and 50 μg/mL) induced an increase on cell migration consistent with the angiogenesis process. Analysis of VE-cadherin dynamics indicates that the immediate exposition to LOBE (10 μg/mL) induced a loosening of cell-cell junction, which corroborates with the hemorrhage observed in the victims. By mass spectroscopy, it was observed that LOBE possesses several potentially bioactive peptides. Groups of these peptides were isolated by a methanol-based fractioning of the crude venom. The peptides present in each of the 10 fractions were characterized by mass spectroscopy and it was analyzed the effects of each fraction on angiogenesis. The results suggest that some of the effects of Lonomia obliqua envenomation are due to the presence of bioactive peptides that modulate the behavior of endothelial cells.