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L’obstruction des vaisseaux est responsable d’ischémie tissulaire dans différents territoires périphériques, cardiaques et cérébraux. Un des mécanismes d’adaptation du tissu à l’ischémie est la formation de néo-vaisseaux. Plusieurs données récentes mettent en évidence un rôle de la voie de signalisation Wnt/Frizzled (Fzd) dans le développement vasculaire. Le travail de cette thèse s’est focalisé sur l’étude du récepteur Frizzled7 (Fzd7) et de son rôle dans la formation des vaisseaux. Le modèle des corps embryoïdes, un modèle de différenciation des cellules souches embryonnaires vers le phénotype endothélial, nous a permis de démontrer que Fzd7 était exprimé au cours des différentes étapes de différenciation endothéliale. Des études sur des cellules endothéliales matures nous ont permis de montrer que Fzd7 régulait différentes propriétés des cellules endothéliales dont la migration et la formation de tubes endothéliaux, mais pas la prolifération. De plus, Fzd7 participe à la stabilité des jonctions cellulaires en interagissant avec la VE-cadhérine. Concernant les mécanismes moléculaires mis en jeu par Fzd7, nous avons pu montrer que celui-ci était capable d’activer la voie Wnt/PCP via la phosphorylation de la protéine JNK. Enfin, une étude in vivo dans le modèle du poisson Zèbre, nous a permis de mettre en évidence un rôle de Fzd7 dans la formation des vaisseaux intersomitiques. La deuxième partie de ce travail concerne le rôle de la voie Wnt/Fzd dans les propriétés angiogèniques des cellules souches mésenchymateuses (CSM). L’objectif de cette étude était de définir comment les CSM participaient à la formation des vaisseaux et si le système Wnt/Frizzled était nécessaire. Nous avons pu montrer que sFRP1, un modulateur de la voie Wnt, améliore la fonction cellulaire des CSM et contribue à la maturation des néo-vaisseaux. De plus, nous avons pu montrer que les CSM implantées dans un modèle d’ischémie du membre inférieur amélioraient la réponse angiogénique lorsque celles-ci étaient préconditionnées en hypoxie. Nous avons mis en évidence le rôle de Wnt4 dans ce processus. / The obstruction of the vessels is responsible for ischemia in various outlying areas, heart and brain. One of the mechanisms of tissue adaptation to ischemia is the formation of neo-vessels. Several recent data show a role of Wnt/Frizzled (Fzd) pathway in vascular development.The work of this thesis focused on the study of receptor Frizzled7 (Fzd7) and its role in vessel formation. Using the model of embryoid bodies, a model of embryonic stem cell differentiation toward the endothelial lineage, we demonstrated that Fzd7 was expressed during different stages of endothelial cell differentiation. Studies on mature endothelial cells have shown that Fzd7 regulates endothelial cells properties including migration and endothelial tube formation but not proliferation. In addition, Fzd7 participates in the stability of cell junctions by interacting with VE-cadherin. Regarding the molecular mechanisms involved in Fzd7 signalling, we could show that this receptor was capable of activating the Wnt/PCP pathway via phosphorylation of JNK protein. Finally, an in vivo study in zebrafish model, allowed us to highlight a role of Fzd7 in intersomitic vessel formation.The second part of this work concerns the role of the Wnt/Fzd pathway in the angiogenic properties of mesenchymal stem cells (MSC). The objective of this study was to determine how CSM participated in vessel formation and if the Wnt/Frizzled pathway was necessary. We show that sFRP1, a modulator of the Wnt pathway, improves cellular function of MSCs and contributes to the maturation of neo-vessels. In addition, we have shown that MSCs implanted in a model of lower limb ischemia improved the angiogenic response when they were preconditioned by hypoxia. We have highlighted the role of Wnt4 in this process.

Le carcinome hépatocellulaire (CHC) est un cancer de très mauvais pronostic, disposant de peu d’options thérapeutiques. Il est urgent de développer de nouvelles stratégies thérapeutiques. Notre équipe a montré antérieurement que l’expression du récepteur Frizzled-7 (FZD7) dans le CHC jouait un rôle dans le contrôle du phénotype cancéreux. Le but de ma thèse était de développer une stratégie pour inhiber le signal FZD7 et d’étudier l’effet anti-tumoral potentiel d’une telle inhibition. A l’aide d’une approche peptidique inhibitrice de la liaison du récepteur avec son adaptateur cytoplasmique (Dishevelled), j’ai pu démontrer que l’inhibition du signal FZD7 exerce un effet anti-tumoral sur des lignées cellulaires de CHC, mais également in vivo, dans un modèle murin transgénique de CHC. J’ai élucidé une partie des mécanismes de cet effet anti-tumoral en étudiant l’impact du peptide sur les voies qui sont potentiellement activées par FZD7 dans des lignées cellulaires de CHC. Cette approche peptidique m’a permis de valider l’inhibition de FZD7 comme cible thérapeutique pour le CHC. Parallèlement, nous avons développé une collaboration avec l’entreprise IMAXIO qui a mis au point un test double-hybride permettant de cribler des molécules inhibitrices d’une interaction. Nous avons identifié 8 molécules chimiques qui inhibent l’interaction FZD7/Dishevelled. La poursuite de ce projet va permettre d’identifier, parmi ces 8 molécules, celles qui ont un potentiel anti-tumoral comparable à l’approche peptidique que j’ai développée, grâce aux modèles cellulaires et murins disponibles au laboratoire / Hepatocellular carcinoma (HCC) is a very bad prognostic cancer, with few therapeutic options. The development of new therapeutic strategies is an emergency. In previous studies, our team showed that overexpression of Frizzled-7 receptor (FZD7) in HCC plays a role in the control of cancer phenotype. The aim of my thesis was to develop a strategy to inhibit the FZD7 signal and study the potential antitumor effect of such an inhibition. I used a small-peptide approach to inhibit the binding between FZD7 and its cytoplasmic adaptator, Dishevelled, and showed that the inhibition of FZD7 signal display antitumor effects in vitro on HCC cell lines, as well as in vivo, in a murine transgenic model of HCC. I explored the molecular mechanisms of this antitumor effect on a HCC cell line. Thanks to this small-peptide approach, I validated the inhibition of FZD7 signal as a target for HCC therapy. We also developed a two-hybrid high throughput screening with IMAXIO (Lyon) to identify chemicals able to inhibit the FZD/Dvl interaction and we identified 8 compounds. Prospects of this study are to test the potential anti-tumor effect of these compounds on HCC cell lines and HCC in vivo models.

Durant le développement myocardique, les cardiomyocytes s'allongent et se connectent entre eux grâce à une structure spécialisée, le disque intercalaire. Cette organisation des cardiomyocytes est essentielle pour le couplage mécanique et la conduction électrique. Un des éléments responsables de l'insuffisance cardiaque est la perturbation de ces sites de contact intercellulaire. Actuellement, aucun facteur n'est connu pour coordonner l'organisation polarisée des cardiomyocytes. Ici, nous présentons une augmentation importante de Pdzrn3 dans des cardiomyopathies hypertrophiques humaines et dans des myocardes murins, corrélée à une perte de l'élongation polarisée des cardiomyocytes. De plus la délétion spécifique intramyocardique de l'expression de Pdzrn3, dans un modèle murin, protège de la survenue d'une insuffisance cardiaque secondaire à une cardiomyopathie hypertrophique. Nos résultats révèlent une nouvelle voie de signalisation qui contrôle un programme génétique essentiel pour le développement myocardique, le maintien de la géométrie et de la fonction contractile des cardiomyocytes. Cette voie de signalisation implique PDZRN3 et cette molécule constitue une cible thérapeutique potentielle pour la protection de l’insuffisance cardiaque chez l’homme. / During heart maturation, individual cardiomyocytes stretch out and connect some with the others via their extremities by intercalated disk protein complexes. This planar and directionnel organization of the myocyte sis crucial for the machanical coupling and the anisotropic conduction of the electric signal in the heart. One of the hallmarks of heart failure concerns alterations in the contact sites between cardiomyocytes. Yet no factors on its own is known to coordinate cardiomyocyte polarized organization. Here we reported enhanced levels of Pdzrn3 in the diseased hypertrophic human and mouse myocardium, correlated with the loss of cardiomyocyte polarized elongation. Furthermore, mouse cardiac Pdzrn3 deficiency protected against heart failure in a mouse model of hypertrophic cardiomyopathy. Our results reveal a novel signaling that controls a genetic program essential for heart maturation and for maintain of cardiomyocyte overall geometry and contractile function and implicates PDZRN3 as a potential therapeutic target for human heart failure protection.

A conclusão do seqüênciamento do genoma de múltiplos vertebrados trouxe um importante desafio para entender e predizer função, particularmente para seqüências não-codificantes, a partir de seqüências primarias de DNA. A hipótese de que a conservação evolutiva prediz seqüências funcionais é comumente aceita, inclusive para seqüências envolvidas na regulação da transcrição gênica, mesmo que a conservação de seqüências tenha gerado imperfeitas predições de enhancers (acentuadores) funcionais. O colágeno tipo XVIII é um componente da maioria das membranas basais; mutações no gene COL18A1 levam a síndrome de Knobloch, uma doença autossômica recessiva caracterizada por degeneração vitreoretiniana e macular e encefalocele occipital. O COL18A1 tem 43 exons que transcrevem três isoformas a partir de dois promotores diferentes. As três isoformas apresentam um complexo modelo de expressão tecido-específico, incluindo expressão em rim, pulmão, cérebro e retina. Os níveis de expressão do COL18A1 são considerados clinicamente importantes na vasculogenese, e em predisposição para o hepatocarcinoma e diabetes tipo 2. Dessa forma, a identificação de regiões regulatórias fornecerá indícios sobre a regulação da expressão do COL18A1 em estados normal e patogênico. Além disso, a endostatina e o FRZC18 são fragmentos proteolíticos do colágeno XVIII envolvidos na sinalização Wnt. No entanto, o papel in vivo do FRZC18 ainda não foi estudado. Uma profunda investigação do papel deste domínio na via de sinalização Wnt é indubitavelmente necessária, bem como a compreensão da regulação do COL18A1 pela via de Wnt. Empregamos um sistema eficiente de teste em zebrafish para analisar o potencial funcional de elementos enhancers na regulação transcricional do COL18A1. Identificamos quatro elementos enhancers que controlam a transcrição consistente com o COL18A1 endógeno de zebrafish, em tecidos incluindo retina, rim, vasos sanguíneos, intestino, cartilagem e fígado. Apesar dos algoritmos utilizados não tenham detectado conservação em seqüências não-codificantes de humanos à teleósteos no lócus do COL18A1 estudado, as seqüências humanas funcionaram apropriadamente em zebrafish transgênicos. Adicionais análises computacionais post hoc revelaram similaridade entre seqüência humana e de zebrafish dentro ou próximo das quatro regiões enhancers. Testamos funcionalmente o FRZC18 com superexpressão de seu RNAm em embriões de zebrafish. Este experimento resultou em embriões com fenótipos que assemelharam à mutantes da via não-canônica de Wnt (slb e ppt). Este resultado aponta o FRZC18 como um antagonista da via de sinalização não-canônica de Wnt, possivelmente por interação com Wnt11 e Wnt5. Dissecamos o promotor 1 do COL18A1, o qual mostrou características de genes housekeeping e similaridades com o promotor 2 do COL18A1. Também mostramos possível ligação de TCF/LEF aos promotores do COL18A1. A via de Wnt levou à redução de atividade dos promotores do COL18A1 e também redução dos níveis de expressão através de superexpressão de β-catenina. Este trabalho elucidou de forma geral, os elementos regulatórios em cis do COL18A1 e melhor caracterizou o seu papel na via de sinalização Wnt como um antagonista também da via não-canônica e como um alvo da via canônica. / The completion of multiple vertebrate genome sequences has presented an important challenge to understand and predict function from primary DNA sequence, particularly for noncoding sequence. It is commonly hypothesized that evolutionary conservation predicts functional DNA sequences, including those involved in regulating gene transcription, although sequence conservation has proven to be an imperfect predictor of enhancer function. Type XVIII collagen is a component of most basement membranes; mutations in the COL18A1 gene lead to Knobloch Syndrome, an autosomal recessive disease characterized by vitreoretinal and macular degeneration and occipital encephalocele. COL18A1 has 43 exons that transcribe three isoforms from two different promoters. The three isoforms display complex patterns of tissue-specific expression, including in kidney, lung, brain, and retina. Expression levels of COL18A1 are thought to be clinically important in vasculogenesis, and in predisposition to hepatocarcinoma and diabetes type 2. Therefore, identification of the regulatory regions will provide insight into normal and pathogenic regulation of COL18A1 expression. Furthermore, endostatin and FRZC18 are cleaved fragments from collagen XVIII that are involved in Wnt signaling, however in vivo role of FRZC18 has not been investigated yet in any model organism. Thus, a deeper investigation of FRZC18 role in Wnt signaling is indubitable necessary, as well as a comprehension of COL18A1 regulation by Wnt signaling. We have employed an efficient system of transgenesis in the zebrafish to functionally evaluate potential enhancer elements regulating COL18A1 transcription. We identified four enhancer elements that control transcription consistent with zebrafish endogenous COL18A1, in tissues including retina, kidney, blood vessels, gut, cartilage and liver. Although the algorithms we used did not detect noncoding conservation from human to teleosts at the COL18A1 locus, the human sequences functioned appropriately in zebrafish transgenics. Additional post hoc computational analysis revealed detectable sequence similarities between human and zebrafish in or near two of the four enhancer regions. We tested one of these zebrafish regions and confirmed orthologous enhancer activity. We functionally tested FRZC18 with its mRNA overexpression in zebrafish embryos. This experiment resulted in embryos with phenotype remaining slb and ppt, mutants of non-canonical wnt components. This result points FRZC18 as an antagonist of non-canonincal Wnt signaling possibly by interaction with Wnt11 and Wnt5. We dissected COL18A1 promoter 1 and it showed characteristics of a housekeeping gene and similarities with promoter 2 and we also showed possible TCF/LEF binding to COL18A1 promoters. Wnt signaling responded to downregulate promoter activity of COL18A1 and also decrease its expression by overexpression of s-catenin. This work broadly elucidated COL18A1 cis regulatory elements and better characterized its role in Wnt signaling as an antagonist of noncanonical and also as a target of canonial signaling.

A conclusão do seqüênciamento do genoma de múltiplos vertebrados trouxe um importante desafio para entender e predizer função, particularmente para seqüências não-codificantes, a partir de seqüências primarias de DNA. A hipótese de que a conservação evolutiva prediz seqüências funcionais é comumente aceita, inclusive para seqüências envolvidas na regulação da transcrição gênica, mesmo que a conservação de seqüências tenha gerado imperfeitas predições de enhancers (acentuadores) funcionais. O colágeno tipo XVIII é um componente da maioria das membranas basais; mutações no gene COL18A1 levam a síndrome de Knobloch, uma doença autossômica recessiva caracterizada por degeneração vitreoretiniana e macular e encefalocele occipital. O COL18A1 tem 43 exons que transcrevem três isoformas a partir de dois promotores diferentes. As três isoformas apresentam um complexo modelo de expressão tecido-específico, incluindo expressão em rim, pulmão, cérebro e retina. Os níveis de expressão do COL18A1 são considerados clinicamente importantes na vasculogenese, e em predisposição para o hepatocarcinoma e diabetes tipo 2. Dessa forma, a identificação de regiões regulatórias fornecerá indícios sobre a regulação da expressão do COL18A1 em estados normal e patogênico. Além disso, a endostatina e o FRZC18 são fragmentos proteolíticos do colágeno XVIII envolvidos na sinalização Wnt. No entanto, o papel in vivo do FRZC18 ainda não foi estudado. Uma profunda investigação do papel deste domínio na via de sinalização Wnt é indubitavelmente necessária, bem como a compreensão da regulação do COL18A1 pela via de Wnt. Empregamos um sistema eficiente de teste em zebrafish para analisar o potencial funcional de elementos enhancers na regulação transcricional do COL18A1. Identificamos quatro elementos enhancers que controlam a transcrição consistente com o COL18A1 endógeno de zebrafish, em tecidos incluindo retina, rim, vasos sanguíneos, intestino, cartilagem e fígado. Apesar dos algoritmos utilizados não tenham detectado conservação em seqüências não-codificantes de humanos à teleósteos no lócus do COL18A1 estudado, as seqüências humanas funcionaram apropriadamente em zebrafish transgênicos. Adicionais análises computacionais post hoc revelaram similaridade entre seqüência humana e de zebrafish dentro ou próximo das quatro regiões enhancers. Testamos funcionalmente o FRZC18 com superexpressão de seu RNAm em embriões de zebrafish. Este experimento resultou em embriões com fenótipos que assemelharam à mutantes da via não-canônica de Wnt (slb e ppt). Este resultado aponta o FRZC18 como um antagonista da via de sinalização não-canônica de Wnt, possivelmente por interação com Wnt11 e Wnt5. Dissecamos o promotor 1 do COL18A1, o qual mostrou características de genes housekeeping e similaridades com o promotor 2 do COL18A1. Também mostramos possível ligação de TCF/LEF aos promotores do COL18A1. A via de Wnt levou à redução de atividade dos promotores do COL18A1 e também redução dos níveis de expressão através de superexpressão de β-catenina. Este trabalho elucidou de forma geral, os elementos regulatórios em cis do COL18A1 e melhor caracterizou o seu papel na via de sinalização Wnt como um antagonista também da via não-canônica e como um alvo da via canônica. / The completion of multiple vertebrate genome sequences has presented an important challenge to understand and predict function from primary DNA sequence, particularly for noncoding sequence. It is commonly hypothesized that evolutionary conservation predicts functional DNA sequences, including those involved in regulating gene transcription, although sequence conservation has proven to be an imperfect predictor of enhancer function. Type XVIII collagen is a component of most basement membranes; mutations in the COL18A1 gene lead to Knobloch Syndrome, an autosomal recessive disease characterized by vitreoretinal and macular degeneration and occipital encephalocele. COL18A1 has 43 exons that transcribe three isoforms from two different promoters. The three isoforms display complex patterns of tissue-specific expression, including in kidney, lung, brain, and retina. Expression levels of COL18A1 are thought to be clinically important in vasculogenesis, and in predisposition to hepatocarcinoma and diabetes type 2. Therefore, identification of the regulatory regions will provide insight into normal and pathogenic regulation of COL18A1 expression. Furthermore, endostatin and FRZC18 are cleaved fragments from collagen XVIII that are involved in Wnt signaling, however in vivo role of FRZC18 has not been investigated yet in any model organism. Thus, a deeper investigation of FRZC18 role in Wnt signaling is indubitable necessary, as well as a comprehension of COL18A1 regulation by Wnt signaling. We have employed an efficient system of transgenesis in the zebrafish to functionally evaluate potential enhancer elements regulating COL18A1 transcription. We identified four enhancer elements that control transcription consistent with zebrafish endogenous COL18A1, in tissues including retina, kidney, blood vessels, gut, cartilage and liver. Although the algorithms we used did not detect noncoding conservation from human to teleosts at the COL18A1 locus, the human sequences functioned appropriately in zebrafish transgenics. Additional post hoc computational analysis revealed detectable sequence similarities between human and zebrafish in or near two of the four enhancer regions. We tested one of these zebrafish regions and confirmed orthologous enhancer activity. We functionally tested FRZC18 with its mRNA overexpression in zebrafish embryos. This experiment resulted in embryos with phenotype remaining slb and ppt, mutants of non-canonical wnt components. This result points FRZC18 as an antagonist of non-canonincal Wnt signaling possibly by interaction with Wnt11 and Wnt5. We dissected COL18A1 promoter 1 and it showed characteristics of a housekeeping gene and similarities with promoter 2 and we also showed possible TCF/LEF binding to COL18A1 promoters. Wnt signaling responded to downregulate promoter activity of COL18A1 and also decrease its expression by overexpression of s-catenin. This work broadly elucidated COL18A1 cis regulatory elements and better characterized its role in Wnt signaling as an antagonist of noncanonical and also as a target of canonial signaling.

Les macrophages ont un rôle majeur dans la régulation de l’inflammation. Ils sont capables de répondre rapidement aux signaux extérieurs en passant d’un état pro- à anti-inflammatoire (respectivement nommés macrophages M1 et M2). Certaines sous populations de macrophages M2 ont des propriétés angiogéniques. Parmi les voies de signalisation permettant aux macrophages de répondre aux signaux extérieurs on trouve les voies Wnt/Frizzled (Fzd). Elles reposent sur 10 récepteurs Fzd et 19 ligands Wnt. Il a été établi que les couples Wnt3a/Fzd1 et Wnt5a/Fzd5 sont impliqués dans la réponse inflammatoire des macrophages. Récemment le rôle d’une voie Wnt/Flt1 a été mis en évidence dans la régulation de l’angiogenèse, par les macrophages, lors du développement rétinien. Notre objectif de recherche fut d’étudier le rôle de la signalisation induite par Fzd7 sur les propriétés angiogéniques des macrophages. Nous avons étudié l’angiogenèse pathologique par l’utilisation de plusieurs modèles murins d’inflammation (irritation cutané, ischémie du membre inférieur, infarctus du myocarde). Chez des souris déficientes en Fzd7 dans les macrophages, l’angiogenèse est plus importante durant la phase d’inflammation, comparées aux souris contrôles. Par immuno-histologie et cytométrie en flux nous avons démontré que cette augmentation du nombre de vaisseaux s’accompagne d’une population de macrophages M2 plus importante, sans modification du nombre total de macrophages. Ces résultats obtenus dans plusieurs contextes inflammatoires chez la souris suggèrent que Fzd7 serait impliqué dans le contrôle de l’angiogenèse via une régulation de la polarisation M1 versus M2 des macrophages. / Macrophages play a major role in regulating inflammation. They are able to respond quickly to external signals from a pro to anti-inflammatory state (respectively named M1 and M2 macrophages). Some subpopulations of M2 macrophages have angiogenic properties. The Wnt/Frizzled (Fzd) pathway is part of pathways allowing macrophages to respond to their environment. They are composed of 10 Fzd receptors and 19 Wnt ligands. It was established that Wnt3a/Fzd1 and Wnt5a/Fzd5 are involved in the inflammatory response of macrophages. Recently the role of a Wnt/Flt1 pathway has been highlighted in the regulation of angiogenesis, by macrophages, during retinal development. Our aim was to study the role of the Fzd7-induced signaling on macrophages angiogenic properties. We studied pathological angiogenesis by using several mouse models of inflammation (skin irritation, hindlimb ischemia, myocardial infarction). In mice with Fzd7-deleted macrophages, angiogenesis is greater during the inflammatory phase, compared with control mice. By immune-histochemistry and flow cytometry we demonstrated that the increase in the vessels number is associated with a greater M2 macrophages population, without change in the total number of macrophages. These results obtained in several inflammatory contexts in mice suggest that Fzd7 be involved in the control of angiogenesis through regulation of the M1 versus M2 polarization of macrophages.

L’angiogenèse physiologique est un processus clé du développement embryonnaire et chez l’adulte. Une anomalie de la formation des vaisseaux sanguins est à l’origine de nombreuses pathologies. Une meilleure compréhension des mécanismes de l’angiogenèse est un pré-requis essentiel à la mise au point de nouvelles stratégies thérapeutiques ayant pour objectif d’inhiber ou stimuler cette angiogenèse pour mieux traiter l’ensemble de ces pathologies. Au cours de ces dernières années, les voies de signalisation Wnt/Fzd sont apparues comme jouant un rôle fondamental dans le développement vasculaire. Au début de cette thèse, un premier projet nous a permis de montrer le rôle important de Fzd7 dans le contrôle de la perméabilité vasculaire, in vitro et in vivo, via la voie canonique et la régulation des complexes jonctionnels dépendantes de la VE-cadhérine. La deuxième partie de ce travail s’est focalisé sur le rôle de Fzd7 dans la formation des vaisseaux. Nous avons mis en évidence que Fzd7 contrôle la vascularisation post-natale de la rétine chez la souris. La voie de signalisation Fzd7/DVL/β-caténine régule le sprouting et la prolifération des cellules endothéliale (CE) via l’activation de la voie Notch, tandis que la voie de signalisation de Fzd7 contrôle la migration des CE via la régulation de MMP2/9 indépendamment de la voie Notch. Enfin, la troisième partie de cette thèse a eu pour objectif d’étudier l’implication de Fzd7 sur l’angiogenèse pathologique. Nos résultats préliminaires indiquent que Fzd7 participe aux phases de vaso-oblitération et de néovascularisation dans un modèle de rétinopathie induite par l’oxygène chez la souris, suggérant que Fzd7 pourrait être une nouvelle cible dans le traitement des rétinopathies. / Physiological angiogenesis is a key process in embryonic development but also in adult. Abnormal formation of blood vessels is the cause of many diseases. A better understanding of the mechanisms of angiogenesis is an essential prerequisite for the development of new therapeutic strategies aimed to inhibit or stimulate angiogenesis to better address these pathologies. In recent years, the Wnt/Fzd signaling pathways appeared to play a key role in vascular development. At the beginning of this study, a first project allowed us to show the important role of Fzd7 in controlling vascular permeability in vitro and in vivo, through the canonical pathway and the regulation of VE-cadherin junctional complexes. The second and main part of this work focused on the role of Fzd7 in the formation of blood vessels. We have demonstrated that Fzd7 controls postnatal vascularization of mice retina. The signaling pathway Fzd7/DVL/β-catenin regulates the sprouting and proliferation of endothelial cells (EC) through activation of Notch signaling, but also controls EC migration through MMP2/9 but independently of the Notch pathway. Finally, the third part of this work aimed to study the involvement of Fzd7 on pathological angiogenesis. Our preliminary data indicate that Fzd7 regulates vaso-obliteration and neovascularization in a mice model of oxygen-induced retinopathy suggesting that Fzd7 could be a new target for the treatment of retinopathy.

Glypican-3 (GPC3) belongs to a family of cell surface proteoglycans. GPC3 regulates the activity of several morphogens and growth factors that play critical roles during development. Disrupting the function of GPC3 leads to disease, including the overgrowth disease Simpson Golabi Behmel Syndrome (SGBS) and Cancer. Previous work has shown that GPC3 is over expressed in Hepatocellular Carcinoma (HCC), and that HCC proliferation is stimulated through GPC3 mediated activation of the Wnt signaling pathway. Glypicans are known to regulate Wnt signaling in a variety of model organisms including Drosophila and mouse. This work investigates the hypothesis that GPC3 stimulates Wnt signaling by facilitating/stabilizing the interaction between Wnt and its receptor Frizzled (Fzd). Consistent with this hypothesis, we found that GPC3 is able to bind both Wnt and Fzd. The binding of GPC3 to Fzd is mediated by the GPC3 glycosaminoglycan chains and by the cysteine rich domain of Fzd.