Angiogenesis and vascular permeability occur in physiological and pathological conditions. Angiogenesis denotes the process of blood vessel formation from preexisting quiescent vessels. Angiogenesis is initiated by proangiogenic factors, inducing endothelial cell sprouting, migration and anastomosis, followed by regression of the new vessels or maturation into a quiescent status. Vascular permeability is the process where blood vessels exchange nutrients, solutes and inflammatory cells with the surrounding tissue. Small molecules freely cross the endothelial wall, however macromolecules and cells leak out from the vasculature only after stimulation by certain factors, including VEGF. Angiogenesis and vascular permeability are tightly regulated physiological processes, but uncontrolled angiogenesis and excessive leakage lead to pathological conditions and the progression of several diseases. VEGF and its receptor VEGFR2 are critical players in angiogenesis and in vascular permeability. The binding of the ligand to the receptor is not the only event involved in the activation and regulation of the signaling cascade. Coreceptors, kinases, phosphatases, and other proteins involved in the trafficking of the complex modulate the signal amplitude and duration. VEGF/VEGFR2 complex combined with the coreceptor NRP1 has a strong pro-angiogenic action and a critical role in angiogenesis. Both VEGFR2 and NRP1 bind VEGF and can present VEGF in cis, when both VEGFR2 and NRP1 are expressed on the same endothelial cell or in trans, when NRP1 is expressed on an adjacent endothelial cell or another type of cell. Y949 and Y1212 are two of the main phosphorylation sites of VEGFR2 induced by VEGFA. The binding of phosphorylated Y949 to the SH2 domain of TSAd regulates vascular permeability leading to Src activation and adherens junction opening in vitro. Phospho-Y1212 is implicated in actin stress fiber remodeling via the adapter Nck, affecting the actin cytoskeleton and endothelial cell migration in vitro. Paladin is a vascular-enriched phosphatase-domain containing protein without reported phosphatase activity and is a negative regulator of insulin receptor and Toll-like receptor 9 signaling. In this thesis work, I have investigated the spatial dynamics of NRP1/VEGFR2 complex formation (in cis and in trans) for coordinating VEGF-mediated angiogenesis in physiological and in pathological conditions (Paper I). I have studied, in vivo, the role of VEGFR2 Y949 in vascular permeability and metastatic spread (Paper II) and the role of VEGFR2 Y1212 in angiogenic remodeling and vessel stability (Paper III). Furthermore, I have examined paladin’s role in regulating VEGF/VEGFR2 signaling and VE-cadherin junction stability, in angiogenic sprouting and vascular permeability (Paper IV). In conclusion, VEGF/VEGFR2 signaling is regulated by a multifactor system and each individual regulatory mechanism leads to a specific outcome in angiogenesis, vascular permeability and vessel stability.

Der menschliche Neokortex wird als Hauptsitz kognitiver Funktionen höherer Ordnung angesehen. Das Verständnis der neokortikalen Entwicklung anderer Säugetierarten ist von wesentlicher Bedeutung, um die menschliche Gehirnorganisation im Allgemeinen und neurologische Entwicklungsstörungen im Speziellen besser zu verstehen. In dieser Arbeit habe ich die Rolle des mit dem Mowat-Wilson-Syndrom assoziierten Transkriptionsfaktors Zeb2 in der neokortikalen Entwicklung der Maus untersucht. Ich habe nachgewiesen, dass Zeb2 die Adhäsion neugeborener kortikaler Neurone sowohl vor als auch nach der radialen Migration über zwei unabhängige molekulare Wege reguliert. Hierbei konnte ich zeigen, dass die Adhäsion im Vorfeld der radialen Migration über den molekularen Zeb2-Nrp1-Itgβ1- Weg reguliert wird. Zeb2 unterdrückt zell-intrinsisch die neuronale Adhäsion an die extrazelluläre Matrix und kontrolliert dadurch den Beginn der radialen Migration, die Dauer des multipolaren Stadiums sowie die Motilität multipolarer Neurone, ohne die radiale Migration selbst oder das spätere Zellschicksal innerhalb der kortikalen Schichten zu beeinflussen. Hierbei sind die apikalen Dendriten der Neurone normalerweise parallel zueinander und senkrecht zur Hirnhautoberfläche ausgerichtet. Ich habe gezeigt, dass die Ausrichtung der Neurone im Anschluss an ihre Migration von der Adhäsion der Zellen untereinander sowie zur extrazellulären Matrix abhängt und dieser Prozess unabhängig von der radialen Migration erfolgt. Zeb2 koordiniert das gesamt e Repertoire dieser postmigratorischen Adhäsion über den molekularen Zeb2-Cdh6-Itgβ1-Weg. Zusammenfassend zeigt diese Studie die Bedeutung der neuronalen Adhäsion während der neokortikalen Entwicklung auf und entschlüsselt die Regulationsmechanismen für die Initiierung der radialen Migration sowie für die postmigratorische Orientierung der Neurone der oberen kortikalen Schichten. / The human brain is a highly sophisticated biological structure and its formation is a highly orchestrated process. The human neocortex, in particular, is the main place of higher-order cognitive functions. Understanding the neocortical development of other mammalian species is essential for understanding brain organisation in common neurodevelopmental disorders in particular. Here I studied the role of Mowat-Wilson syndrome-associated transcription factor Zeb2 in mouse neocortical development. I have shown in this study that Zeb2 regulates adhesion of new born cortical neurons both before and after radial locomotion via two independent molecular pathways. I have shown that adhesion prior to radial locomotion is tightly regulated via Zeb2-Nrp1-Itgβ1 molecular pathway. Zeb2 cell-intrinsically suppresses adhesion of neurons to the extracellular matrix and therefore restricts the initiation of radial locomotion, multipolar stage duration and motility of multipolar neurons without affecting radial locomotion itself and layer cell fate acquisition. Once radial migration is finished neurons have to form apical dendrite and establish contact with the meningeal surface. Normally, apical dendrites of neurons are oriented parallel to each other and perpendicular to the meningeal surface. I have shown that postmigratory orientation of neurons is dependent on cell-to-cell and cell-to-extracellular matrix adhesion and occurs independently from radial migration. Zeb2 orchestrates the whole repertoire of adhesion of neurons completed radial migration via Zeb2-Cdh6-Itgβ1 molecular pathway. I have demonstrated that Cadherin 6 balance is crucial for establishment of postmigratory neuronal orientation under normal conditions. Taken together, this study has revealed the importance of neuronal adhesion during neocortical development and separated the regulation mechanisms for initiation of radial migration and postmigratory orientation of upper layer neurons.

La reconnaissance d’un antigène présenté par les cellules présentatrices d’antigène induit la prolifération et la différenciation des lymphocytes T naïfs en lymphocytes T effecteurs et mémoires. Cette reconnaissance se fait par l’interaction du récepteur des cellules T (TCR) des lymphocytes T et le complexe CMH-peptide présent à la surface des DC. Cependant, des signaux additionnels sont requis, une meilleure activation des lymphocytes T implique des corécepteurs présents à la surface de ces deux types cellulaires. Après l’élimination de l’antigène, la plupart des lymphocytes T effecteurs vont mourir. Une petite population de lymphocytes T va persister pour se différencier en lymphocytes T mémoires capables de protéger l’organisme contre une réinfection. Les signaux qui contrôlent le maintien des lymphocytes T mémoires sont encore mal compris. Pour comprendre le rôle de la molécule de costimulation 4-1BB dans le maintien des lymphocytes T CD8 mémoires, nous avons émis l’hypothèse que l’état de phosphorylation de la protéine adaptatrice TRAF1, qui se lie à 4-1BB, module le maintien des lymphocytes T CD8 mémoires. Ainsi, nous avons montré par des expériences de spectrométrie de masse que TRAF1 s’associe préférentiellement à TBK1 lorsqu’elle n’est pas phosphorylée. Nous avons aussi montré que la présence de TRAF1 est requise pour stabiliser TBK1 au récepteur 4-1BB après stimulation des lymphocytes T. Par ailleurs, les lymphocytes T CD8 OT-I TRAF1-/- reconstituées avec un mutant phospho-déficient de TRAF1 (S139A) et ensuite différenciées en lymphocytes T mémoires in vitro induisent une activation de la voie de signalisation NF-ĸB contrairement à ceux exprimant la forme phospho-mimétique de TRAF1 (S139D). Ces premières études démontrent l’importance de l’état de phosphorylation de TRAF1 en aval de 4-1BB dans les cellules T. Dans la seconde partie, nous avons évalué le rôle d’un autre corécepteur; la neuropiline 1, dans la maturation des DC. A cet effet, nous avons émis l’hypothèse que l’interaction de la neuropiline 1 et ses ligands contribuerait à la fonction des DC. Nous avons démontré que l’absence de la neuropiline 1 n’a pas d’effet sur la maturation au LPS des DC. Cependant, la présence du VEGF (un ligand de Nrp-1) inhibe la maturation des DC dérivées de la moelle osseuse. Notre étude a démontré que VEGF inhibe l’expression des molécules de costimulation, la sécrétion des cytokines pro inflammatoires et la signalisation TLR4 principalement les voies MAP Kinase et NF-ĸB. Contrairement aux résultats avec les cellules WT, VEGF n’est pas capable d’affecter la maturation, la sécrétion des cytokines et la signalisation TLR4 des DC Nrp1-Lyz où la neuropiline 1 est délétée. Ainsi, nos résultats ont démontré que VEGF inhibe la maturation des DC de façon Nrp1-dépendante. Enfin, l’analyse des molécules partenaires de la neuropiline 1 montre que Nrp1, VEGF et TLR4 se retrouvent dans le même complexe. Nos résultats démontrent que VEGF, en présence de la neuropiline 1 est capable d’interagir avec TLR4 pour inhiber la maturation des DC. Toutefois, en absence de la neuropiline1, VEGF n’est pas capable de recruter TLR4 pour réduire l’expression des molécules de costimulation. Ces études sur les corécepteurs pourraient être importantes dans l’élaboration de nouvelles approches vaccinales. / Antigen presentation by dendritic cells induces the proliferation and differentiation of naïve T lymphocytes into effector and memory T lymphocytes. This recognition is due to the interaction of the T cell receptor (TCR) with the cognate peptide-MHC complex presented on the surface of dendritic cells. However, additional signals are required from co-receptors on both cell types to ensure optimal T cell activation. Following the elimination of the antigen, most of the effector T cells will die with a small population of T cells remaining that will continue to differentiate into memory T cells which protect the organism against reinfection. The signals that control the maintenance of memory T cells are poorly understood. To dissect the role of the 4-1BB costimulatory molecules in the maintenance of CD8 memory T cells, we hypothesized that the phosphorylation state of the TRAF1 adaptor protein that binds to 4-1BB, modulates the maintenance of CD8 memory T lymphocytes. Thus, we have demonstrated by mass spectrometry that TRAF1 preferentially associates with TBK1 when it is not phosphorylated. We also have shown that the presence of TRAF1 is required to stabilize the interaction between TBK1 and 4-1BB after T cell activation. Furthermore, OT-I TRAF1-/- CD8 T cells reconstituted with a phospho-deficient TRAF1 mutant (S139A) and differentiated into memory CD8 T cells induced the activation of the NF-ĸB signaling pathway, in contrast to cells expressing a phospho-mimetic form of TRAF1 (S139D). Together, these results highlight the importance of the phosphorylation state of TRAF1 downstream of 4-1BB in T cells. In the second part, we evaluated the role of the neuropilin 1 coreceptor in the maturation of dendritic cells. To this end, we hypothesized that the interactions of neuropilin-1 with its ligands contribute to the function of dendritic cells. We have demonstrated that the absence of neuropilin-1 has no effect on the maturation of dendritic cells in the presence of LPS. However, the presence of VEGF (a neuropilin-1 ligand) inhibits the maturation of dendritic cells derived from the bone marrow. Our study further demonstrated that VEGF inhibits the expression of costimulatory molecules, the secretion of proinflammatory cytokines and TLR4 signaling pathways mainly MAP Kinase and NF-ĸB. Contrary to the results with wild-type cells, VEGF is not able to affect maturation, cytokine secretion and TLR4 signaling in NRP1-Lyz dendritic cells when neuropilin-1 is deleted. Thus, our results demonstrated that VEGF inhibits the maturation of dendritic cells in a NRP1-dependent manner. Finally, analysis of neuropilin 1 partners shows that NRP1, VEGF and TLR4 are found in the same complex. Our results show that VEGF, in the presence of neuropilin-1 is able to interact with TLR4 and inhibit the maturation of dendritic cells. However, in the absence of the neuropiline1, VEGF is not able to recruit TLR4 to reduce the expression of costimulatory molecules. These studies on coreceptors could be important in the development of novel vaccine therapies.

Various diseases, including cancer, stroke and heart attack, are associated with disruption of the vascular system. However, lack of a profound understanding of the transcription regulation during vascular development hinders the formation of effective molecular intervention strategies targeting angiogenesis. Here we describe an enhancer of Neuropilin1 (Nrp1) from the second intron of the gene that directs arterial and coronary endothelial cell-specific expression. Mice transgenic for either human or mouse sequences of the Nrp1in2 enhancers drove expression of the LacZ reporter gene specifically in the endothelial cells within the arterial compartment from early in development, while no expression was detected in veins. In addition, the hNrp1in2 enhancer directed expression to the endothelial cells in the developing coronary vasculature, with the initial expansion from around the sinus venosus at E11.5, and eventually contributed to the capillary, venous and arterial compartments of the coronary vessels but not the endocardium. This expression pattern is consistent with that reported in the Apelin-nlacZ line (Red-Horse et al., 2010), making the Nrp1 enhancer the first identified mammalian regulating enhancer of the coronary endothelial cell. Phylogenetic footprinting, and a tissue culture reporter assay suggested that this enhancer contains a 184bp minimal core region hNrp1in2peakA2 that recapitulates the expression profile of the full length enhancer. hNrp1in2peakA2 has conserved and in vitro validated recognition sites for Gata, Ets, and Fox. The validated Fox and Ets sites form a functional FOX:ETS motif, and the FOX:ETS motif is responsible for synergistic activation ofthe enhancer by FoxC2 and Etv2 in reporter assays. Mutation introduction to the functional Ets sites or compound ablation of the Gata and Fox site in hNrp1in2peakA2 result in total loss of vascular expression, in terms of both arterial and coronary expression. The Fox, Ets and Gata recognition sites may be sufficient to achieve arterial- and coronary- specific expression of the hNrp1in2peakA2.

Background: The focus of this study was to investigate how producing human bone marrow (hBM) derived mesenchymal stromal cell (MSC) extracellular vehicles (EVs) in a high purity isolation system would affect their established characterization criteria and address the validity of these methods of EV production. Additionally, we set out to functionally characterize the hBM-MSC-EVs for their identified immunomodulatory ability while also assessing the presence of novel MSC-EV marker NRP1 identified by our group to further affirm its validity as a functional MSC-EV identity marker. Methods: Each hBM-MSC-EV donor was cultured in a hollow-fiber bioreactor system in non-stimulating serum/xeno-free conditions for 25 days to produce EVs persistently under quiescent conditions to characterize the hBM-MSC-EVs in their native form. EVs were isolated by traditional PEG-based precipitation for preliminary characterization to monitor bioreactor production wherein they were characterized using multimodal tangential flow filtration coupled with fast protein liquid chromatograph (FPLC) size exclusion/high-affinity purifications to obtain the final highly purified EV sample. Additionally, functional analysis of their immunomodulatory ability, EVs and MSCs were incubated with activated peripheral blood mononuclear cells (PBMCs) as an in-vitro model to evaluate their potency. Results: The hBM-MSC-EVs produced from the bioreactor system showed consistent characterization in accordance with the MISEV2018 establish criteria. We were also able to demonstrate their functional ability by observing statistically significantly immunomodulatory ability of activated PBMCs equivalent to native MSC ability. We were also able to validate the present of NRP1 on all hBM-MSC-EV samples produced confirming its validity as a MSC-EV marker. Conclusion: The significance of the results obtained from this study confirms the production of MSC-EV using a bioreactor and high purity isolation for obtaining consistent MSC-EVs for downstream investigation. Additionally, we were able to demonstrate the significance of MSC-EVs on MSC signaling for immunomodulation by showing equivalent functional potency when tested in-vitro. These results contribute to further understanding the biological attributes of MSC-EVs and contribute to the validation of currently established characterization guidelines.