>Magister Scientiae - MSc / Cancer is the most common leading cause of death worldwide. Glioblastoma and breast cancer are the most aggressive solid tumour. The survival rate of these tumours depends on their ability to progress and spread. These cancers use their high proliferative capabilities for survival, increasing their malignancies. Glioblastoma is considered the most aggressive tumour initiated in the brain, whereas breast cancer is the most common metastatic cancer in the brain, both types of cancer are known as high infiltrated cancer and their invasiveness due to their capability to release factors that can alter the neighbouring cells to facilitate their progression.

The building blocks of human tissues are cells. The cells interact and respond to the characteristics of their local microenvironment. The cellular microenvironment is formed by three main components, the extracellular matrix, neighbouring cells and signalling molecules. Particularly, the extracellular matrix and neighbouring cells impose boundary conditions that limits the cell volume and cell spreading. However, these characteristics are often not present in traditional in vitro models, where cells experience a stiff and vast environment.   An approach to improve in vitro models is to use hydrogels, soft and highly hydrated polymers. Through chemical modifications, polymers naturally found in the extracellular matrix can be functionalized to form crosslinked hydrogels. Moreover, these functionalities can also be used to prepare micropatterns, micrometre sized cell adhesive areas on the hydrogels. These micropatterns guide the cell shape and permit the study of the cell response to these changes in shape, which has been observed in e.g. endothelial cells from various origins.   Taken all together, the aim of this work was to develop a hydrogel-based cell culture scaffold that permits the control of the spatial adhesion of brain endothelial cells in order to study the morphological effects on these cells and contribute to the understanding of the function of brain endothelial cells in health and disease.   This thesis demonstrates the functionalization of hyaluronic acid, a naturally occurring extracellular matrix polymer, to prepare photocrosslinkable hydrogels. Furthermore, through photolithography, micropatterns of cell adhesive peptides were prepared on these hydrogels. Brain microvascular endothelial cells, a highly specialized type of endothelial cells, adhered to the micropatterns, and the effect on their alignment depending on the micropatterned sized was studied. Furthermore, changes in their alignment were also observed when exposed to different glucose concentration.

No description available.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

Due to the high metabolic demand and low capacity for energy storage of the brain, neurons are vitally reliant on a constant oxygen supply. Under chronic mild hypoxic conditions (10% oxygen), angiogenesis is induced in the brain in an attempt to restore tissue oxygen tension to normal levels. In brain hypoxia, vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis; however, the role of its homolog placental growth factor (PlGF) is unknown. Using PlGF knockout (PlGF-/-) mice exposed to whole body hypoxia (10% oxygen) for 7, 14 and 21-days, we show that PlGF-/- animals exhibit a delay in the angiogenic response of the brain to hypoxia. PlGF-/- microvessels had a significant increase in fibrinogen accumulation and extravasation, which correlated with disruption of the tight-junction protein claudin-5. These vessels displayed large lumens, were surrounded by reactive astrocytes, lacked mural cell coverage and endothelial VEGF expression, and regressed after 21 days of hypoxia. The lack of PlGF, in combination with reduced VEGF expression levels observed in the brain of PlGF-/- animals during the first 5 days of hypoxia, is likely the cause of the delayed angiogenic response and the prothrombotic phenotype of these mice. In vitro studies conducted to analyze mechanisms involved in the impaired angiogenic phenotype and enhanced astrocytic reactivity to hypoxia of PlGF-/- animals indicated that: i) PlGF-/- mouse brain endothelial cells exhibit alterations in intracellular signaling pathways associated with sprouting (ERK1/2) and vessel branching morphogenesis (GSK-3β) and ii) PlGF-/- astrocytes overexpress VEGF receptor-2 (VEGFR-2) which through activation of the ERK1/2 signaling pathway leads to a more proliferative astrocytic phenotype. These astrocytes were more resistant to oxygen and glucose deprivation (OGD) than PlGF+/+ astrocytes, a characteristic that was shown to be independent of the classical antiapoptotic VEGFR-2-dependent PI3K/Akt pathway. The findings presented in this thesis demonstrated a critical role of PlGF in vascular remodeling in the hypoxic brain.

No description available.

La barrière hémato-encéphalique (BHE) régule le transport des molécules et des cellules du compartiment sanguin vers le système nerveux central. Pour assurer cette fonction, l’endothélium microvasculaire cérébral bénéficie d’un système particulier d’enzymes, de pompes d’efflux et de jonctions cellulaires spécialisées, qui ensemble contrôlent scrupuleusement le passage des molécules plasmatiques et des cellules circulantes. La VE-cadhérine est une molécule d’adhérence qui occupe une position unique dans l’organisation des jonctions endothéliales et le maintien de l’intégrité vasculaire. Cependant, même si le rôle de la VE-cadhérine est décrit comme fondamental au cours du développement du réseau vasculaire, sa participation dans l’intégrité de la BHE nécessite d’être explorée plus en détail. Le glioblastome, la tumeur cérébrale la plus agressive et la plus létale, est associée à une vascularisation hautement perméable. En outre, ces tumeurs renferment une sous-population de cellules souches gliomateuses (CSG) dérivant d’une fraction de cellules transformées à caractère souche, qui joueraient un rôle dans l’initiation et la progression tumorales, ainsi que dans la résistance aux thérapies conventionnelles. Plus particulièrement, ces cellules ont été retrouvées in situ en interaction directe avec les cellules endothéliales cérébrales. Néanmoins, l’implication des CSG dans la plasticité des cellules endothéliales cérébrales, et notamment la perméabilité vasculaire, n’a pas été étudiée. Au sein de notre équipe, nous avons démontré que les CSG sécrètent la Sémaphorine 3A (S3A), une molécule de répulsion caractérisée par une activité antiangiogénique et pro-perméabilité. La S3A induit une augmentation de la phosphorylation et de l’internalisation de la VE-cadhérine, conduisant à une perte de la fonction de barrière des cellules endothéliales cérébrales. Au niveau moléculaire, nous avons montré que Src, une tyrosine kinase, et Set, un inhibiteur naturel de PP2A, coopèrent pour inhiber l’activité phosphatase de PP2A en réponse à la S3A. Plus particulièrement, PP2A interagit avec la VE-cadhérine bloquant sa phosphorylation, son internalisation et l’ouverture de la barrière endothéliale. PP2A confère ainsi une stabilité à la VE-cadhérine, qui serait perturbée par la S3A produite localement par les CSG. Ce mécanisme pourrait jouer un rôle clé dans les défauts des vaisseaux irriguant les glioblastomes, et d’une manière générale dans la perméabilité vasculaire tumorale. L’ensemble de ces résultats nous permet de mieux caractériser les mécanismes moléculaires mis en jeu au cours de l’angiogenèse tumorale et d’envisager à long terme des molécules à visée thérapeutique, en ciblant par exemple la voie de signalisation activée par la S3A / The blood brain barrier (BBB) regulates the transport of molecules and cells from blood into the central nervous system. This implies that the cerebral microvascular endothelium has developed a particular system of enzymes, efflux pumps and specialized cell junctions, which together carefully control the passage of plasma molecules and circulating cells. Vascular endothelial (VE)-cadherin is an adhesion molecule that occupies a unique position in the organization of endothelial junctions and the maintenance of vascular integrity. In particular, phosphorylation and internalization of VE-cadherin destabilizes cell-cell contacts and increases permeability. However, though VE-cadherin is fundamental in the development of the vascular network, its participation in the integrity of the BBB needs to be further explored. Glioblastoma is the most aggressive and the most lethal brain tumor, which is characterized by a highly leaky vasculature. Furthermore, these tumors contain a subpopulation of glioma stem cells (GSC), which derive from a fraction of transformed stem cells. GSCs play a role in the tumor initiation, progression and resistance to conventional therapies. Notably, these cells were found in direct interaction with cerebral endothelial cells in situ. However, the involvement of GSCs in the plasticity of cerebral endothelial cells, including vascular permeability, has not been studied. Our team has demonstrated that GSCs secrete semaphorin 3A (S3A), a repulsive molecule characterized by anti-angiogenic and pro-permeability activity. S3A increased phosphorylation and internalization of VE-cadherin in cerebral endothelial cells, leading to a loss of barrier integrity. At the molecular level, Src, a tyrosine kinase, and Set, a natural inhibitor of PP2A, cooperate to inhibit the phosphatase activity of PP2A, in response to S3A. Specifically, we demonstrated that PP2A interacts with VE-cadherin at the basal level. This interaction blocks VE-cadherin phosphorylation and internalization and thereby prevents opening of the endothelial barrier. Thus, PP2A stabilizes VE-cadherin, and we further showed that this complex could be disrupted by S3A produced by GSCs. This mechanism could play a key role in the dysfunctions of the vessels supplying glioblastoma, and in tumor vascular permeability in general

<p>Angiogenesis is the formation of new blood vessels from the pre-existing blood vessels. Blood vessels are composed of endothelial cells and supporting musculature. Angiogenesis is regulated by numerous soluble ligands and by cell-matrix interactions. We have studied the molecular mechanisms in fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-A (VEGF-A)-induced angiogenesis using immortalized endothelial cell lines in different angiogenesis assays.</p><p>The role of the signaling protein H-Ras in FGF-2-induced <i>in vitro</i> angiogenesis was studied by expressing mutated versions of H-Ras in immortalized mouse brain endothelial cells using a tetracycline-regulated expression system. <i>In vitro</i> angiogenesis was analyzed as the ability of cells to invade a fibrin matrix and form branching structures in response to a combination of FGF-2 and tumor necrosis factor-α (TNF-α). Inhibition of H-Ras through the expression of dominant negative (S17N) H-Ras or pharmacological inactivation of H-Ras with a farnesyl transferase inhibitor, did not inhibit growth factor-induced invasion. In contrast, expression of constitutively active (G12V) H-Ras caused cells to adopt a transformed phenotype which inhibited invasive growth and cells formed solid tumors when injected in nude mice. These studies suggest that H-Ras activity is not required for differentiation but its activity must be tightly regulated as aberrant activity impairs endothelial cell differentiation.</p><p>In order to screen for both known and novel genes that regulate angiogenesis we used large scale microarray analysis. In VEGF-A-stimulated telomerase immortalized human microvascular endothelial cells undergoing invasive growth in fibrin gels, or forming cord-like structures on collagen, we identified several genes that were differentially expressed. Some of these are known to be important for endothelial cell functions and angiogenesis while others have no previous connection with endothelial cell function or were transcripts with no assigned function. Further analysis of these proteins will aid in elucidating the molecular mechanisms underlying endothelial cell differentiation. </p>