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Obesity is a major health problem that has reached epidemic proportions worldwide. Therapeutic intervention for obesity has proven extremely challenging. Obesity is a complex trait involving the interaction of genes involved in fundamental aspects of weight maintenance, exposure to an environment characterised by an over-abundance of food and sedentary life-style choices with limited physical activity (Poskitt 2009). A marked shift in diet has occurred worldwide (Popkin 2001) with greater saturated fat intake, reduced intake of complex carbohydrates and dietary fibre, and reduced fruit and vegetable intake (WHO 2003). In terms of pharmacological strategies, the current anti-obesity drugs on the market are primarily concerned with reducing appetite or fat absorption in the gut. However, serious side effects have been documented with some of these drugs, including an increased rate of cardiovascular events with Sibutramine use (Curfman et al., 2010). In 2007, the Scottish Medicines Consortium removed Rimonabant from use in the NHS Scotland due to risks of adverse psychiatric events (Burch et al., 2009). However, despite these setbacks, there have been considerable advancements in the treatment of obesity, achieved by combining pharmacological treatments with diet, exercise, behavioral approaches and surgery (gastric band surgery and liposuction). However, the prevalence of obesity continues to increase inexorably, particularly in the Asia Pacific region (Gill 2006; Low et al., 2009), and thus further advancements in obesity treatment are needed, ideally avoiding invasive procedures such as surgery.

Increased angiogenesis is an important characteristic of remodeling in asthmatic airways which stems from the imbalance between pro-angiogenic and anti-angiogenic factors. Surprisingly, the factors regulating this process in allergic asthma are poorly defined. The focus of this thesis is to investigate the effect of Semaphorin 3E (Sema3E) on angiogenesis events within the airways of murine model of allergic asthma. The role of Sema3E in asthma angiogenesis was tested in wild type and Sema3e-/- mice exposed to House Dust Mite (HDM) and monitored for changes in blood vessels number in the lungs. In addition, the potential of Sema3E, in reversing features of allergen inflammation, was tested in mouse model. In both cases immunohistochemistry and immunofluorescence staining of lung tissues used to assess the changes in the level of angiogenesis. Moreover, the expression of pro- and anti-angiogenic factors in total lung homogenate was assessed by ELISA and Real-Time PCR. The results showed that WT and Sema3e-/- mice both developed the HDM induced allergic asthma phenotype, but, the lung sections of HDM exposed Sema3e-/- mice had enhanced number of blood vessels compared to WT mice. The enhanced angiogenesis in Sema3-/- mice was coupled with increased level of angiogenesis driving factors VEGF and it receptor VEGFR-2. However, in WT mice the level of soluble VEGFR-1 secretion increased significantly which inhibited VEGF / VEGFR-2 binding. Besides, Sema3E treatment reduced the level of angiogenesis and inhibited HDM-induced secretion of VEGF and the expression of its receptor VEGFR-2 while increased the level of soluble VEGFR-1. Analyzing the ratio of VEGF / soluble VEGFR-1 revealed that in the presence of Sema3E in both models, soluble VEGFR-1 is the dominant factor which has an inhibitory role on angiogenic effect of VEGF. Taken together, this study provided the first evidence that Sema3E can modulate angiogenesis in allergic asthmatic airways. / February 2016

Le rôle important joué par les protéases dans plusieurs processus physiologiques (reproduction, cicatrisation et régénération tissulaire) nexclut pas leurs contributions parfois primordiales dans divers pathologies, telles que la croissance tumorale et les métrorragies dysfonctionnelles. Lors de ce travail, nous nous sommes intéressés à létude des protéases et de langiogenèse au niveau de deux aspects distincts de la reproduction : 1) Une situation physiologique quest la placentation par le biais dun modèle murin ; 2) Une situation pathologique chez les femmes souffrant de métrorragies dysfonctionnelles suite à lutilisation dun système intra-utérin à libération de progestatif comme moyen de contraception

Le développement dune placentation hémochoriale requiert une invasion trophoblastique de lendomètre maternel jusquà ses couches profondes ainsi quune érosion des vaisseaux maternels jusquau niveau du tiers supérieur du myomètre. Les cellules trophoblastiques envahissent et modifient les vaisseaux utérins pour permettre une augmentation du flux sanguin vers lunité foeto-placentaire. Dautre part, lembryon qui simplante va non seulement se connecter aux vaisseaux maternels mais aussi générer des vaisseaux de novo. Le processus dangiogenèse endométriale au niveau du site dimplantation est modulé spatio-temporellement par un dialogue complexe entre des facteurs endocrines, paracrines et autocrines. Parmi ces facteurs, lhCG, sécrétée précocement par le trophoblaste, va tenir un rôle de choix. Outre son rôle lutéotrophique bien décrit, lhCG promeut linvasion trophoblastique et présente une action clé sur la tolérance maternelle de lembryon. Nos travaux ont mis en évidence un rôle direct de lhCG sur langiogène endométriale ainsi quun rôle indirect via une boucle paracrine par activation du récepteur hCG/LH à la surface des cellules endothéliales et épithéliales de lendomètre et une stimulation de lexpression du VEGF dans les cellules épithéliales sous linfluence de lhCG. Grâce à de nombreux modèles dangiogenèse in vitro, in vivo et ex-vivo, nous avons décrypté la signalisation cellulaire mise en uvre lors de la liaison de lhCG à son récepteur. De plus, nos travaux démontrent un rôle artériogène inattendu de cette hormone. Ceci revêt toute son importance dans létude de leffet angiogène de lhCG lié à des processus physiologiques comme langiogenèse au niveau du site dimplantation, pathologiques comme leffet tumorigène de lhCG sécrété en fortes quantités lors de pathologies trophoblastiques.

Forward genetic screens in the mouse are contributing significantly to our understanding of basic mammalian development and human disease. In one such screen, our laboratory has identified the novel mouse gumby mutant, which affects the development of the neural and vascular systems. Here, I describe the characterization of the gumby mutant phenotype and the identification of its causative mutation in a novel, vertebrate-specific gene, which is one of the genes deleted in patients affected by Cri du Chat Syndrome that exhibit mental retardation and craniofacial deficits. Expression and phenotypic analyses revealed a requirement for the gumby gene in the facial nerve axon guidance and angiogenesis. Lately, it has become evident that many common mechanisms and molecules operate during neural and vascular development. My results suggest that the gumby gene is an attractive candidate for regulating both processes and its analysis in the future may help us understand how the navigational mechanisms for both systems are intertwined. My studies show that gumby is a cytoplasmic protein that is present in many embryonic and adult tissues. In yeast-two-hybrid assays gumby interacts with a member of the highly conserved Wnt pathway - Dishevelled 2 (Dvl2). In both Dvl2-/- and gumby homozygotes, the level of the cardiac neural crest cell marker Plexin2A is decreased. The three branches of the Wnt pathway have been shown to regulate a wide range of events during embryogenesis and adult homeostasis, and subsequently have been implicated in multiple human pathologies. Taken together my data suggest that gumby may be required for Wnt signaling in angiogenesis and/or facial nerve guidance. Given that Wnt signaling has been shown to play key roles in axon guidance, gumby and its roles in Wnt signaling may also contribute to the mental retardation seen in patients with Cri du Chat Syndrome. Thus, further analyses of molecular and biologic roles of gumby will provide important avenues for understanding the cell biology of human disease.

Stroke is the leading cause of long term disability and the third leading cause of death in the United States. Perlecan plays a significant role in brain development by sequestering and delivering growth factors to developing neuronal precursor cells in a neurovascular niche. Previous results demonstrated that perlecan proteolysis results in the cleavage of perlecan’s most C-terminal domain five (DV) in the post-ischemic brain. As post-stroke angiogenesis is an important step in post-stroke brain repair, I focused on the mechanism of DV’s role in brain angiogenesis in vitro. I first demonstrated that DV significantly increased brain endothelial (BE) cell migration, proliferation and tube-like formation suggesting DV is a pro-angiogenic factor for BE cells. I next investigated VEGF secretion from BE cells in the presence of DV. DV significantly increased VEGF secretion into the cell media, which was both dose and time dependent. Using quantitative real-time PCR, DV induced a maximal nine-fold increase in VEGF expression, compared to control, indicating DV is an upstream regulator of VEGF transcription. DV treated cells show an increase in phosphorylation of ERK-(1/2) that could be blocked by the pharmacological inhibitor U0126. This inhibitor could also block DV’s effect on VEGF mRNA expression and secretion indicating ERK is involved with DV’s effect on VEGF regulation. Optical sensor binding assays confirmed that DV binds to the α5β1 integrin with a Kd of 160nM, and cells treated with DV showed a visual representation of integrin α5β1-DV colocalization. Furthermore, shRNA-mediated knockdown of integrin α5 blocked DV’s effect on VEGF mRNA expression, indicating integrin α5 is involved with DV’s regulation of VEGF expression. In conclusion, these results demonstrate that DV has an unexpected proangiogenic effect in brain angiogenesis. This occurs via a previously unreported interaction between DV and the α5β1 integrin, resulting in the activation of the ERK, eIF4A and HIF1α signaling pathway and an ultimate increase in VEGF mRNA expression and VEGF secretion. As DV is generated post-stroke, these results suggest a novel mechanism by which brain tissue recovery following ischemia is influenced by processed fragments from the extracellular matrix.

Angiogenesis, the formation of new blood vessels from pre-existing vasculature, is important in post-injury scar formation but its role in scar-free regeneration remains relatively unexplored. This study investigates vascular regeneration during tail regeneration in the leopard gecko (Eublepharis macularius). It is hypothesized that blood vessel regeneration follows a conserved sequence of events similar to physiological angiogenesis. To test this hypothesis the onset and pattern of expression of common vascular and angiogenic proteins (von Willebrand factor, α-smooth muscle actin, vascular endothelial growth factor, thrombospondin-1 and cluster differentiation 36) was investigated. The effect of the anti-angiogenic peptide ABT-510 on tail regeneration was also explored by documenting changes in vascular morphology and histology of regenerate tails. Results show that the proteins of interest are expressed in a conserved sequence consistent with physiological angiogenesis. ABT-510 did not consistently prevent tail regeneration, but did have some small-scale effects. These results provide the basis for further investigations into the importance of angiogenesis during multi-tissue regeneration.