Glioblastoma multiforme (GBM) is the most deadly and common form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment is known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN comprised of either collagen Type 1 or HyStem-C®, human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBMECs), and LN229 (GBM) cells. A synergistic response between HUVECs and LN229 cells was observed in co-culture, including 10 – 16-fold increased cell proliferation, a decrease in the height of hydrogels of up to 68%, as well as elevated secretion of TGF-β and CXCL12 up to 2.6-fold from Day 8 to 14. These trends correlated with cell colocalization, indicating a chemotactic role for CXCL12 in enabling the migration of LN229 cells towards HUVECs in co-cultures. Von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP) in up to 40% of LN229 cells after 14 days in co-culture in collagen (2.2 mg/mL) and HyStem-C® gels. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® on EC networks, LN229 cell morphology and alignment, cytotoxicity, colocalization, and trans-differentiation. TMZ was observed to primarily affect LN229 cells, with treatment at high concentrations resulting in up to 2.3-fold reduced alignment as well as an increase in cell circularity. Cytotoxicity of up to 94% was also observed up to in LN229 monocultures, and was significantly higher in collagen (1.1 mg/mL) gels. Avastin® treatment resulted in changes to ECs. Network features were significantly reduced and EC cellular proliferation decreased up to 69% with Avastin® treatment. Significant increases in percentages of colocalized and GFAP+/vWF+ cells were also observed when treated with 8 µg/mL Avastin®. This suggests that chemotactic signaling may have been altered. TGF-β secretion was reduced in co-cultures when 150 µM TMZ or 8 µg/mL Avastin® were administered. / Doctor of Philosophy / Glioblastoma (GBM) is the most common and deadly form of brain cancer and is responsible for over 50% of adult brain tumors. A specific region within the GBM environment of particular interest is located near the vasculature, known as the perivascular niche (PVN). We have designed a 3D in vitro model of the PVN consisting of either collagen type 1 or HyStem-C®, a material made of primarily hyaluronic acid. Human umbilical vein endothelial cells (HUVECs), an immortalized cell line, or primary human brain microvascular endothelial cells (HBMECs) as well as LN229 (GBM) cells were used. A synergistic response was observed between HUVECs and LN229 cells in co-culture, including changes to the extracellular matrix, and signaling factor secretion. Further supporting this data, colocalization between LN229 cells and HUVECs was observed. Colocalization is a phenomenon where two cell types come into physical contact after one moves toward another. This indicated preferential migration, specifically in response to CXCL12. Endothelial cell marker von Willebrand factor (vWF) was co-expressed with glial fibrillary acidic protein (GFAP), commonly used to identify GBM cells. This percentage was increased in co-cultures with HBMECs, pointing to differences in the response of primary cells to immortalized cell lines. The expression of vWF indicates the early stages of trans-differentiation of LN229 cells to an endothelial cell phenotype. We then investigated the effect of chemotherapeutic drugs temozolomide (TMZ) and Avastin® in the PVN model. TMZ was observed to primarily affect LN229 cells, by reducing their alignment as well as causing cell death. Avastin® treatment resulted in changes to ECs. Networks and cell growth were significantly reduced after Avastin® treatment. When either TMZ or Avastin® was administered, the secretion of TGF-β, was reduced.

Chez les patients souffrant d'insuffisance rénale chronique, les calcifications vasculaires représentent la première cause de mortalité. Elles résultent de la trans-différenciation des cellules musculaires lisses (CMLs) en cellules de type ostéoblastique et/ou chondrocytaire, en réponse à des cytokines inflammatoires ou à une hyperphosphatémie. Les CMLs forment alors des cristaux par l'activité de la phosphatase alcaline non-spécifique du tissu (TNAP). A la lumière de résultats récents, nous avons émis l'hypothèse que la TNAP module la trans différenciation des CMLs. Nos objectifs étaient donc de déterminer l'effet de la TNAP dans la trans-différenciation des CMLs, et d'étudier les mécanismes impliqués dans son induction, avec un intérêt particulier pour les microRNAs. Nous avons observé que l'ajout de phosphatase alcaline purifiée ou la surexpression de TNAP stimule l'expression de marqueurs chondrocytaires en culture de CMLs et de cellules souches mésenchymateuses. De plus, l'inhibition de la TNAP bloque la maturation de chondrocytes primaires. Nous excluons un rôle des cristaux formés par la TNAP, puisque l'ajout de cristaux seuls ou associés à une matrice collagénique n'a pas reproduit les effets de la TNAP. Nous suspectons que la TNAP agit en hydrolysant le pyrophosphate inorganique (PPi). En effet, c'est la TNAP qui hydrolyse le PPi en culture de CMLs et de chondrocytes, et le PPi mime les effets de l'inhibition de TNAP en culture de chondrocytes. Enfin, nous rapportons le profil de microRNA des artères cultivées en conditions hyperphosphatémiques. Ces résultats pourraient être particulièrement importants dans le développement de nouvelles approches thérapeutiques / In patients with chronic kidney disease (CKD), vascular calcification represents the main cause of mortality. Vascular calcification results from the trans-differentiation of vascular smooth muscle cells (VSMCs) into cells similar to osteoblasts and/or chondrocytes, in response to inflammatory cytokines or hyperphosphatemia. Calcifying VSMCs form calcium phosphate crystals through the activity of tissue nonspecific alkaline phosphatase (TNAP). In light of recent findings, we hypothesized that TNAP also modulates VSMC trans-differentiation. Our objectives were therefore to determine the effect of TNAP activity on VSMC trans-differentiation, and secondly to investigate the molecular mechanisms involved in TNAP expression in aortas, with a particular interest in microRNAs. We first observed that addition of purified alkaline phosphatase or TNAP over-expression stimulates the expression of chondrocyte markers in culture of the mouse and rat VSMC lines, and of mesenchymal stem cells. Moreover, TNAP inhibition blocks the maturation of mouse primary chondrocytes and reduces mineralization. We exclude a role for crystals in TNAP effects, since addition of crystals alone or associated to a collagenous matrix fails to mimic TNAP effects. We rather suspect that TNAP acts through the hydrolysis of inorganic pyrophosphate (PPi). Indeed, PPi is hydrolyzed by TNAP in VSMCs and chondrocytes and addition of PPi mimics the effects of TNAP inhibition on chondrocyte maturation. Finally, we report microRNA signature of aortic explants treated under hyperphosphatemic conditions that induce vascular calcification. These results could be of particular importance in patients with CKD

Plusieurs études ont démontré que les agonistes du PPARgamma, dont la pioglitazone (PGZ), améliorent les paramètres métaboliques et histologiques de la stéatohépatite non-alcoolique (NASH) chez l'homme et la souris, et qu’ils ont des effets bénéfiques sur la fibrose hépatique chez le rat. Les mécanismes d’action sont mal connus. La NASH, caractérisée par de la stéatose, des lésions hépatocytaires, de l’inflammation et une fibrose variable, est considérée comme une complication hépatique du syndrome métabolique. L'obésité, un des facteurs de risque pour le développement de la NASH, est caractérisée par de faibles taux d'adiponectine sérique. Cette adipocytokine, dont l'expression génique est régulée par le PPARgamma, possède des propriétés anti-stéatosique et anti-fibrotique chez la souris. L'activité intracellulaire de l'adiponectine est médiée via ses récepteurs spécifiques qui activent la protéine kinase AMPK et/ou le PPARalpha. Une fois activée, l’AMPK induit les voies cataboliques de production d’énergie (telles que l'oxydation des acide gras) et inhibe les voies consommant de l’ATP (telles que la lipogenèse). L'activation du PPARalpha augmente l'oxydation des acides gras et inhibe la réponse inflammatoire. Le but de notre travail est d’évaluer l'implication de la voie PGZ–adiponectine–AMPK et/ou PPARalpha dans la prévention de la NASH et de la fibrose hépatique. Nous avons tout d’abord évalué l'effet de la PGZ sur la fibrose hépatique chez la souris. Nos observations montrent que, contrairement aux résultats observés chez le rat, la PGZ n’inhibe pas le développement de la fibrose hépatique chez la souris in vivo. Ces résultats ont été confirmés par des études sur les cellules stellaires hépatiques (HSCs), les cellules effectrices de la fibrose, in vitro. Dans une seconde étude, nous avons évalué l'impact de l’AMPK sur la fibrose hépatique in vivo et sur l’activation des HSCs in vitro. Nous avons constaté que l’AMPK jouait un rôle dans le contrôle de la trans-différentiation des HSCs in vitro mais pas dans le développement de la fibrose hépatique chez la souris in vivo. Finalement, nous avons évalué l'hypothèse que l'effet bénéfique de la PGZ sur la NASH résulte de la stimulation de l'AMPK et/ou du PPARalpha par l’adiponectine. Nos résultats ont montrés que cet effet de la PGZ était strictement dépendant de l’adiponectine mais ne semblait pas impliquer l'AMPK ni le PPARalpha. Nous avons également identifié SREBP-1c, régulant la lipogenèse de novo, comme cible thérapeutique potentielle pour le développement de la NASH. Les résultats obtenus dans le cadre de ce travail de thèse fournissent une meilleure compréhension de l’axe PPARgamma–adiponectine–AMPK dans le contrôle du développement de la NASH et de la fibrose hépatique chez la souris. / Several studies have demonstrated that peroxisome proliferator-activated receptor gamma (PPARg) agonists, such as pioglitazone (PGZ), improve metabolic parameters and histology of nonalcoholic steatohepatitis (NASH) development in humans and mice, and have beneficial effects on liver fibrosis in rats. NASH, characterized by steatosis, hepatocellular damage, inflammation and variable fibrosis, is recognised as the hepatic complication of the metabolic syndrome. Obesity, one of the risk factors for NASH development, is characterized by low serum adiponectin levels. This adipocytokine, of which gene expression is regulated by PPARg, demonstrates anti-steatotic and anti-fibrotic properties in mice. Intracellular activity of adiponectin is mediated through its specific receptors which activate AMP-activated protein kinase (AMPK) and PPARalpha. Once activated, AMPK switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis). Activation of PPARalpha increases fatty acid oxidation and reduces inflammatory reaction. The aim of the present work is to analyse the activation of the axis PGZ-adiponectin-AMPK and/or PPARalpha as a way to control NASH and hepatic fibrosis development. We first evaluated the effect of PGZ on hepatic fibrosis in mice. We observed that, by contrast with results in rats, PGZ did not prevent hepatic fibrosis development in vivo in mice. These results were confirmed by in vitro studies on the key effector cells of fibrogenesis, the hepatic stellate cells (HSCs). We then assessed the impact of AMPK on hepatic fibrosis in vivo and on HSC trans-differentiation/activation phenomenon in vitro. We found that AMPK played a role in the control of HSC trans-differentiation in vitro but was not implicated in the wound-healing fibrosis in vivo in mice. Finally, we tested the hypothesis that the beneficial effect of PGZ on steatohepatitis results from the adiponectin-dependent stimulation of AMPK and/or PPARalpha. We found that this preventive effect was clearly dependent of adiponectin but did not involve AMPK or PPARalpha activation. We have also identified SREBP-1c, implicated in the regulation of de novo lipogenesis, as a potential therapeutic target for the control of the development of NASH. The present thesis provides a better understanding of the axis PPARg–adiponectin–AMPK in the control of NASH and hepatic fibrosis development in mouse.

Der CCAAT enhancer binding protein beta (C/EBPβ) Transkriptionsfaktor reguliert die Differenzierung, Proliferation und Funktion vieler Zelltypen, einschließlich verschiedener Zellen des Immunsystems. Eine detaillierte molekulare Analyse des Mechanismus, wie C/EBPβ alternative Zellschicksale steuert, wurde jedoch bisher noch nicht unternommen. Es wurde gezeigt, dass die ektopische Expression von C/EBPβ in determinierten B- Vorläuferzellen diese zu inflammatorischen Makrophagen reprogrammieren kann. Wir haben dieses Reprogrammierungsystem verwendet, um die Strukturelemente in C/EBPβ, die für die Regulation der (Trans)Differenzierung durch C/EBPβ wichtig sind, zu untersuchen. Um die maßgeblichen C/EBPβ Proteinmodule für die Reprogrammierung zu bestimmen, wurden entweder C/EBPβ Wildtyp Isoformen oder Mutanten in primären murinen B-Vorläuferzellen ektopisch exprimiert. Die Analysen ergaben, dass die translational regulierten langen Isoformen LAP* and LAP, jedoch nicht die kurze Isoform LIP lymphoide Zellen zu myeloischen Zellen reprogrammieren können. Des weiteren haben wir gezeigt, dass die konservierten Regionen 2, 3 und 4 der C/EBPβ Transaktivierungsdomäne essentiell und ausreichend für die Konvertierung von B Zellen zu myeloischen Zellen sind. Die reprogrammierten myeloischen Zellen setzten sich aus einer heterogenen Population verschiedener myeloischer Zelltypen zusammen. Detaillierte Analysen von CD11b+ reprogrammierten Zellen zeigten, dass diskrete konservierte Regionen von C/EBPβ verschiedene pro- und anti-inflammatorische Gene und divergente Entwicklungsprogramme aktivierten. Des Weiteren führten nicht nur strukturelle C/EBPβ Mutanten sondern auch Puktmutationen an Stellen, die posttranslationalen Modifikationen (PTM) unterliegen, zu verschiedenen Reprogrammierungsergebnissen. Diese Daten zeigen, dass die C/EBPβ abhängige myeloische Diversifikation durch die Integration von strukturellen C/EBPβ Proteinmodulen und deren signalabhängigen PTMs erreicht wird. / The CCAAT enhancer binding protein beta (C/EBPβ) transcription factor regulates differentiation, proliferation, and functionality of many cell types, including various cells of the immune system. A detailed molecular understanding of how C/EBPβ directs alternative cell fates remains largely elusive. Ectopic expression of C/EBPβ has been previously shown to reprogram committed B cell progenitors into inflammatory macrophages. We took advantage of this reprogramming system in order to examine how C/EBPβ regulates (trans)differentiation. To determine which C/EBPβ protein modules are important for reprogramming, C/EBPβ wild type isoforms and mutants were ectopically expressed in primary mouse B cell progenitors. The data showed that the translationally regulated long isoforms LAP* and LAP, but not the N-terminally truncated isoform LIP can reprogram lymphoid cells into myeloid cells. Furthermore, we found that conserved regions 2,3 and 4 in the C/EBPβ protein transactivation domain are necessary and sufficient for B-to-myeloid cell conversion. Interestingly, the reprogrammed myeloid cells were found to represent a heterogeneous mixture of different myeloid cell types. Detailed analyses of the reprogrammed CD11b+ cells revealed that discrete conserved regions in C/EBPβ activated distinct pro- and anti-inflammatory genes and triggered divergent differentiation programs. Moreover, not only structural C/EBPβ mutants, but also post-translational modification (PTM) site mutations led to different reprogramming outcomes. These data suggest that C/EBPβ orchestrates myeloid diversification by integrating PTMs with structural plasticity as signal dependent adaptable modular properties to determine cell fate.