Patients on peritoneal dialysis (PD) are reliant on the peritoneum to provide a semi-permeable barrier to allow for dialysis (solute clearance), salt and water removal (ultrafiltration). PD patients are at risk of developing peritoneal fibrosis and angiogenesis which can lead to a decline in peritoneal membrane function. Specifically, PD patients develop increased solute transport and decreased osmotic conductance leading to ultrafiltration failure. Peritoneal angiogenesis is the leading factor that results in augmented peritoneal membrane solute transport which is associated with worse outcomes – increased risk of mortality and PD technique failure. Transforming growth factor beta (TGFB) is one of the primary cytokines involved in inducing epithelial to mesenchymal transition (EMT) and fibrosis. We hypothesize that PD leads to injury of the epithelial lining of the peritoneum – the mesothelial cells. These cells undergo a transition process and transitioned mesothelium are a source for angiogenic and fibrogenic growth factors. Matrix Metalloproteinase (MMP) 9 is an angiogeneic factor and has been observed to correlate with increased expression of vascular endothelial growth factor (VEGF). MMP9 has the ability to cleave and activate membrane bound factors such as E-cadherin and b-catenin respectively. There is substantial evidence that the canonical WNT/b-catenin pathway is active during fibrosis, and angiogenesis in different biological contexts. Thus, we investigated the role of MMP9 and WNT signaling in peritoneal angiogenesis. Limited evidence exists describing the role of noncanonical WNT signaling but some reports suggest that non-canonical WNT signaling inhibits WNT/b-catenin signaling. Non-canonical WNT5A has differential effects based on receptor context and has been shown to block WNT/b-catenin signaling in the presence of Receptor Tyrosine Kinase Like Orphan Receptor 2 (Ror2). The overall hypothesis of this PhD thesis is that MMP9 and WNT signaling play a key role in inducing peritoneal angiogenesis and are associated with changes in peritoneal membrane function. We expect WNT5A and Ror2 to protect against peritoneal membrane injury. From the overnight effluent of stable PD patients, we cultured mesothelial cells and assayed these for expression of MMP and WNT related genes. MMP9 and WNT1 gene expression were observed to be strongly correlated with peritoneal membrane solute transport in patients on PD. WNT2 mRNA was also positively correlated with peritoneal solute transport. We overexpressed MMP9 in the mouse peritoneum to demonstrate its role in angiogenesis and confirmed these findings using MMP9 -/- mice. In addition to this, we have shown a novel mechanism by which MMP9 induces angiogenesis by E-cadherin cleavage and b-catenin mediated signaling. The observed cross-talk between MMP9 and b-catenin prompted investigation of the activation of canonical WNT/b-catenin signaling in development of peritoneal membrane injury. In an experimental model of TGFB induced pertioneal injury, we confirmed the activation of WNT/b-catenin signaling. In addition to this we, we blocked the WNT pathway and observed that WNT/b-catenin signaling is required to induce peritoneal angiogenesis. WNT5A mRNA was downregulated during TGFB induced injury suggesting a more protective role. Furthermore, several studies have demonstrated its ability to antagonize the WNT/b-catenin signaling pathway. We demonstrated that WNT5A protected against angiogenesis by blocking the canonical WNT pathway. WNT5A is thought to antagonize the WNT/b-catenin signaling pathway by signaling through receptor Ror2. In cell culture, we overexpressed TGFB and blocked Ror2. This resulted in elevated levels of VEGF and fibronectin suggesting that Ror2 is involved in mediating protection. Therefore, Ror2 possesses the ability to regulate VEGF and may be a potential candidate by which WNT5A mediates its protective effects. In conclusion, our findings identified MMP9 and WNT1 as potential biomarkers of increased peritoneal solute transport in patients that are on PD. We have also found a novel mechanism by which MMP9 interacts with b-catenin to induce peritoneal angiogenesis and have provided a first look at WNT/b-catenin signaling in peritoneal angiogenesis. Lastly, we have shown WNT5A to protect against peritoneal angiogenesis. Taken together, our findings are not only significant to the realm of PD research but hold wide applicability to research in the biomedical sciences. / Thesis / Doctor of Philosophy (PhD)

Les maladies cardiovasculaires sont la première cause de mortalité dans le monde. Une meilleure compréhension des mécanismes physiopathologiques est nécessaire. Dans ce travail de thèse nous nous sommes intéressés à deux pathologies cardiovasculaires : l’athérosclérose et l’insuffisance cardiaque. Récemment, nous avons identifié le récepteur LRP1 et la protéine adaptatrice ShcA comme étant deux protéines impliquées dans deux de ces pathologies cardiovasculaires. Nousavons montré que ShcA joue un rôle protecteur dans l’insuffisance cardiaque. Chez les souris déficientes en ShcA au niveau cardiaque, nous observons une cardiomyopathie caractérisée par une dilatation du ventricule gauche associée à une perte de la contractilité. Nous avons montré que ShcA est essentiel à l’organisation des sarcomères et ceci très tôt durant l’embryogenèse. Dans une deuxième partie nous avons montré qu’en l’absence de PPARgamma, LRP1 était nécessaire à la calcification vasculaire en activant la voie prochondrogénique de Wnt5a. Nous avons montré que PPARgamma protège de la calcification vasculaire en induisant l’expression de Sfrp2 qui agit comme un antagoniste de Wnt5a. / Cardiovascular disease is the number one cause of death worldwide. A better understanding of the pathophysiological mechanisms is necessary. In this thesis we are focused on two cardiovascular diseases: atherosclerosis and heart failure. Recently, we identified the LRP1 receptor and the adapter protein ShcA as two proteins involved in two of these cardiovascular diseases. We have shown that ShcA exerts a protective role against heart failure. Mutant mice lacking ShcA in the heart exhibit a dilated cardiomyopathy with reduced cardiac contractility. Myocyte ultrastructure analysis shows that Shc A is essential to maintain sarcomeric intégrity in early embryonic heart development. in last part we have shown vascular calcification in the absence of PPARgamma requires expression of LRP1 in vascular smooth muscle cells. LRP1 promotes a Wnt5a-dependent prochondrogenic pathway. We show that PPARgamma protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2 (Sfrp2, wich functions as a Wnt5a antagonist.

No description available.

The AER has a vital role in directing embryonic limb development. Several models have been developed that attempt to explain how the AER directs limb development, but none of them are fully supported by existing data. I provide evidence that FGFs secreted from the AER induce a gradient of Wnt5a. I also demonstrate that limb mesenchyme grows toward increasing concentrations of Wnt5a. We hypothesize that the changing shape of the AER is critical for patterning the limb along the proximal to distal axis. To better understand the pathway through which Wnt5a elicits its effects, we have performed various genetic studies. We demonstrate that Wnt5a does not signal via the Wnt/β-catenin pathway. However, we show that Wnt5a mutants share many common defects with Vangl2 mutants suggesting that Wnt5a signals through the Wnt/planar cell polarity (PCP) pathway.

Le développement du tractus reproducteur femelle est issu de la coordination minutieuse de nombreuses voies de signalisation régulant les processus de prolifération, différenciation et d’apoptose cellulaire durant l’embryogenèse. Les voies Wnt et Hippo se démarquent à cet égard. L’activation de la voie Wnt, via des ligands spécifiques, participe à la stabilisation et l’augmentation de l’activité transcriptionnelle du coactivateur de transcription β-catenin. La voie Hippo, pour sa part, ne possède aucun de ligand spécifique. L’inactivation de la voie Hippo (via les kinases Lats1 et Lats2) entraine la stabilisation des coactivateurs de la transcription YAP/TAZ et l’augmentation de leur activité transcriptionnelle. Plusieurs évidences suggèrent notamment la possibilité de redondance fonctionnelle entre certains ligands de la voie Wnt, dont Wnt4 et Wnt5a, dans le développement du tractus reproducteur femelle. Cette avenue demeure toutefois peu étudiée. L’implication de la voie Hippo n’a pas été rapportée dans le développement du tractus reproducteur femelle. Toutefois, les nombreuses interactions rapportées dans la littérature entre les deux voies suggèrent un rôle méconnu de la voie Hippo. L’objectif de ce projet était donc d’élucider les rôles de Wnt4, Wnt5a, Lats1 et Lats2 dans le mésenchyme de Müller et le développement de l’utérus. Les résultats de notre première étude ont confirmé la fonction partiellement redondante de Wnt4 et Wnt5a dans le développement de l’utérus. Notre modèle est notamment caractérisé par des anomalies développementales ainsi qu’une perte de fonction utérine associée à des anomalies de décidualisation in vivo et une diminution de la viabilité des concepti. Les résultats de notre seconde étude ont confirmé les rôles redondants de Lats1 et Lats2 dans le maintien de la multipotentialité des cellules mésenchymateuses müllériennes. Une différenciation hâtive des cellules mésenchymateuses müllériennes en myofibroblastes via, entre autres, l’expression du gène cible Ctgf, a été observée. Nos résultats additionnels n’ont pu mettre en évidence une interaction potentielle entre les voies Wnt et Hippo pouvant expliquer l’apparition des phénotypes. Ces deux études permettent de confirmer certains rôles connus et d’établir de nouveaux rôles de ces voies dans le développement des canaux de Müller. Ils pourront aussi établir les fondements de modèles permettant l’étude de différentes pathologies utérines et l’identification de cibles thérapeutiques. / The development of the female reproductive tract arises from the coordination of numerous signaling pathways regulating processes such as proliferation, differentiation and apoptosis during embryogenesis. The Wnt and Hippo pathways are known to be involved in these processes. Wnt pathway activation, via its specific ligands, results in the stabilisation and increased transcriptional activity of β-catenin. The Hippo pathway does not possess any specific ligands. In contrast to Wnt, inactivation of the Hippo pathway (via Lats1 and Lats2 kinases) is required for the stabilization and increased activity of the transcriptional coactivators YAP and TAZ. The Wnt pathway is known to be involved in the development of the female reproductive tract. Further evidence also suggests the possibility of functional redundancy amongst certain WNT ligands such as Wnt4 and Wnt5a. The Hippo pathway is not known to be implicated in the development of the female reproductive tract. However, numerous interactions have been reported between both pathways, suggesting a possible unknown role of Hippo in that context. The objective of this project was to elucidate the roles of Wnt4, Wnt5a, Lats1 and Lats2 in the Müllerian mesenchyme and the development of the uterus. Results from our first study confirmed the partially redundant roles of Wnt4 and Wnt5a in the development of the uterus. Our model was notably characterized by developmental abnormalities and loss of uterine functions resulting in in vivo decidualization defects and loss of conceptus viability. Results from our second study confirmed the redundant roles of Lats1 and Lats2 in the maintenance of Müllerian mesenchymal cell multipotency. We observed premature differentiation of Müllerian mesenchymal cells into myofibroblasts in absence of both Lats1 and Lats2. These changes were in part due to the increased expression of the target gene Ctgf. Our additional results could not demonstrate any potential interactions between the Wnt and Hippo pathways that could explain the phenotypic changes. In conclusion, our studies confirmed and further discovered novel roles of these pathways in the development of the Müllerian ducts. These models could also lead to better understanding of the pathophysiology of certain uterine diseases and the discovery of potential therapeutic approaches.

No description available.