The cardiovascular system is the first functional organ to develop during embryogenesis. As the embryo reaches above a certain size, passive diffusion of gases and nutrients is no longer compatible with efficient growth. During embryogenesis, endothelial progenitor cells (angioblasts) are recruited from the primitive streak mesoderm and instructed to express vascular endothelial growth factor receptor-2 (VEGFR-2). This thesis examines the roles played by VEGFR-2 in the events through which a subpopulation of embryonic stem (ES) cells differentiate into endothelial cells and form the vasculature. We show that ES cells gene targeted for VEGFR-2 (flk1-/-) develop immature endothelial cells (ECs), precursors, when differentiated in vitro as embryoid bodies (EBs). The flk1-/- ECs are unresponsive to VEGF-stimulation and consistently fail to form vessels. However, when co-cultured with wild type ES cells in chimeric EBs, flk1-/- endothelial precursors are guided by wild type ECs to form transient, chimeric vascular structures. Use of lentivirus in an add-back approach allowed reconstitution of VEGFR-2 expression in flk1-/- ES cells, and rescue of vasculogenesis and sprouting angiogenesis. We propose that recruitment to the endothelial lineage is not dependent on VEGFR-2, although this receptor tyrosine kinase appears indispensible for EC integrity, survival and for differentation of endothelial precursors into mature ECs formating a stable vasculature. Neuropilin-1 (NRP1) and heparan sulfate proteoglycans (HSPGs) function as co-receptors for VEGFs. The co-receptors influence, qualitatively and quantitatively, the intracellular signal relayed by VEGFR-2 but it is unclear how. We examined the contribution of NRP1 to VEGFR-2 signaling in EB cultures, in zebrafish and in mice. Only NRP1-binding VEGFs were able to promote sprouting angiogenesis and formation of properly branched vascular tubes, supported by pericytes. Downstream of VEGFR-2/NRP1 activation, we identified recruitment of p38MAPK in signal transduction regulating sprouting angiogenesis.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

Newly established F1 hybrid Embryonic Stem cells allow the production of ES cell-derived animals at a high enough efficiency to directly make ES cell based genetics feasible. An F1 hybrid ES cell line, G4 was used to generate transgenic over-expressing cell lines. The consequence of the expression of a panel of transgenes was assessed directly from ES cell-derived embryos produced by the tetraploid complementation assay. The generation of ES cell-derived embryos/animals was very efficient. A sufficient number of mutants for initial phenotypic analyses was derived only a few weeks after the establishment of the cell lines. The genes used in the study had either angiogenic/vasculogenic, anti-angiogenic or unknown properties. Of these transgenic mouse lines VEGF-A and Flt-Fc were used to further elucidate the effects of altered VEGF signaling on cell fate decisions in embryonic development and ES differentiation in two experimental systems. A. Early but transient Flk-1 activation led to enhanced generation of blood progenitors, whereas continuous activation of Flk-1 abolished this effect and enhanced endothelial cell generation. Ex vivo analysis of cells derived from E7.5 embryos demonstrated that sFlt-1-mediated control of Flk-1 activity also impacted the fate of hematopoietic and endothelial cells. The Flt-1-Fc transgenic mouse model was used to alter Flk-1 activation in vivo and show the relevance of the in vitro observations. These results demonstrate that sFlt-1 regulates Flk-1 activation in an oxygen responsive manner. Inhibition of Flk-1 activation by sFlt-1 increases the specification of hemangioblasts to blood cells consistent with a VEGF-independent default mechanism. B. Ubiquitous over-expression of VEGF164 isoform led to E8.75 embryonic lethality. The primary cause of lethality was the failure to form an organized cardiovascular system, which was manifested in three ways: the absence of yolk sac blood vessels, the lack of embryonic-maternal circulation due to the failure of allantochorionic fusion and improper cardiac function. The described phenotypes suggest that VEGF does not inhibit embryonic or extra-embryonic mesoderm formation at gastrulation but perturbs the balance amongst the mesodermal components.

Vascularization of tissue-engineered substitutes is imperative for successful implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.

I developed a system to release multiple growth factors from PEGylated fibrin gels with varying profiles to induce vasculogenesis from embedded human MSCs. Zero-order release can be obtained by conjugating a growth factor with a homobifunctional, amine-reactive, PEG derivative. Growth factors can be entrapped during thrombin-mediated crosslinking and released rapidly. Growth factors with physical affinity for fibrinogen or fibrin can be sequestered within the matrix and released via degradation and/or disassociation. PDGF-BB was loaded via entrapment while TGF-β1 was sequestered through a combination of physical affinity and conjugation. The affinity of TGF-β1 and fibrinogen had never been previously examined or quantified. I aimed to determine the Ka and Kd between TGF-β1 and fibrinogen through a variety of assays. Binding ELISAs were developed for TGF-β1 and fibronectin, a protein associated with fibrin gels, and TGF-β1 and fibrinogen. However, background was high due to insufficient blocking agents. Other assays explored included western blots, surface plasmon resonance, and radiolabeled TGF-β1 with limited success. The affect of TGF-β1 on human MSC differentiation towards vascular cell phenotypes was examined both in 2D and fibrin gels embedded with MSCs. With exposure to TGF-β1, MSC proliferation was significantly inhibited in both 2D and within fibrin gels indicating that loaded TGF-β1 maintained bioactivity for at least 7 days. Gene expression of MSCs exposed to TGF-β1 demonstrated inhibited endothelial cell differentiation and stimulated smooth muscle cell differentiation. However, confocal and light microscopy indicated that endothelial cell differentiation is maintained with TGF-β1 loaded PEGylated fibrin gels. The system developed is highly modular and can be applied to other tissue engineering systems. Furthermore, other growth factors could be incorporated to promote vascular cell differentiation. / text

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.

Chez la souris, la néphrogenèse débute par l'apparition du blastème metanéphrogène à 9.5 dpc. Une transition mésenchymo-épithéliale, comportant 5 étapes, débute a 11.5 dpc et aboutit au rein mature, composé de 3 structures : glomérules, tubules, et capillaires glomérulaires. Les étapes initiales du développement rénal peuvent être récapitulées en culture ex vivo; toutefois, l'organogenèse terminale et la maturation rénale sont incomplètes, et les structures rénales obtenues ex vivo ne sont pas fonctionnelles. Une étude du développement vasculaire in vivo au cours du développement rénal montre une angiogenèse (cellules Pecam-1 positives) et une vasculogenèse (cellules VEGFR-1 positives) précoces, dès 10.5 dpc. Une analyse quantitative par qRT-PCR confirme le rôle de Hif1α et VEGF dans la vasculogenèse rénale. En outre, la voie PGC1α, inductrice de VEGF indépendante de HIF, est activée en conditions hypoxiques. Pour améliorer le développement vasculaire rénal ex vivo, nous proposons un modèle de culture avec micro-perfusion rénale. L'étude morphologique par immunofluorescence des reins après culture micro-perfusée montre une survie tissulaire normale (TUNEL), et une intégrité anatomique (Néphrine, Cytokératine, WT1), en particulier vasculaire (Pecam-1). Une perfusion de vivo-morpholinos WT1 aboutit à une perte d'expression de WT1, confirmant le caractère fonctionnel de notre modèle. En conclusion, nous montrons le rôle précoce de l'angiogenèse et de la vasculogenèse au cours du développement rénal ; nous identifions le rôle de PGC1α dans la vasculogenèse rénale en conditions hypoxiques, et nous proposons une nouvelle technique de culture rénale ex vivo. / In mice, nephrogenesis starts with the formation of the metanephric mesenchyme, at e9.5 dpc. A mesenchymal epithelial transition, consisting of 5 steps, starts at e11.5 dpc, and leads to a mature kidney, composed of 3 main structures: glomeruli, tubules, and capillaries. The initial steps of renal development can be recapitulated ex vivo; however, terminal organogenesis and maturation are impaired, and the explants are not functional. A study of vascular development in vivo during renal development shows that angiogenesis (Pecam-1 positive cells) and vasculogenesis (VEGF-R1 positive cells) occur early, at e10.5 dpc. A quantitative analysis, by qRT-PCR, shows that Hif1α and VEGF play a major role in renal vasculogenesis. Moreover, the PGC1α signaling pathway, a HIF independent VEGF inductor, is activated under hypoxic conditions. To improve ex vivo vascular development, we propose a novel culture technique, with micro-perfusion of the explant. A morphologic analysis of the kidneys obtained by micro-perfused cultures shows no apoptosis (TUNEL), a conserved parenchymal structure (Nephrin, Cytokeratin, WT1), and a proper vascular development (Pecam-1). A micro-perfusion of WT1 vivo-morpholinos leads to a decrease in WT1 expression, thus validating our model. In conclusion, we showed the early role of angiogenesis and vasculogenesis in renal development, we analyzed PGC1α role in hypoxic kidney cultures, and we proposed a novel kidney culture model.

L’Osteoprotégérine est une cytokine soluble qui joue un rôle clé dans le métabolisme osseux et est impliquée dans la réponse immunitaire et l’hématopoïèse. Elle est associée à la dysfonction endothéliale et semble intervenir dans l’angiogenèse. Cette cytokine constituerait en fait, un trait d’union entre le tissu osseux et vasculaire. Son rôle dans la formation de la matrice osseuse est aujourd’hui bien élucidé mais son implication dans la vascularisation reste à établir. L’OPG est rapidement libérée par l’endothélium dans des conditions inflammatoires et est donc en mesure d’intervenir dans le processus de revascularisation initié par les cellules progénitrices endothéliales (PECs). Au cours de cette étude, nous avons tenté de comprendre le rôle joué par cette cytokines dans la néovascularisation induite in vitro, par une sous population de PECs appelées ECFCs (endothelial colony-forming cells), et sur la formation des néovaisseaux in vivo.Nous avons montré qu’elle agit sur la « souchitude » des cellules CD34+, potentialise les propriétés proangiogènes des ECFCs in vitro, et participe au processus angiogénique in vivo. L’OPG agit sur les ECFCs via le syndécanne-1, inhibe leur adhésion à la matrice extracellulaire, favorise leur migration et leur tubulogenèse via la voie SDF-1/CXCR4, et potentialise leur adhésion à l'endothélium activé. Les effets observés sont corrélés à la libération du SDF-1, une activation des voies de signalisation ERK1/2, Akt et mTOR et à une activation de l’intégrine αVβ3. Par ailleurs, nous avons montré que l'OPG potentialise l’effet proangiogène du FGF-2 in vivo. Elle participe également au développement tumoral et à la dissémination des métastases, probablement via l'inhibition de l'apoptose des cellules tumorales, mais aussi par la promotion de l'angiogenèse tumorale. / Osteoprotegerin is a key regulator of bone metabolism involved in the immune response, hematopoiesis, and endothelial dysfunction. It seems to be implicated in angiogenesis and may represent a link between bone and vascular system. Although its role in bone is well recognized, its involvement in vasculature remains to be established. In inflammatory conditions, OPG is constitutively released by endothelial cells and smooth muscle cells, and therefore is able to participate in blood vessels formation induced by endothelial progenitor cells (EPCs). In this study we attempted to determine, in vitro the precise role of OPG in angiogenesis process induced by a subpopulation of EPCs called “endothelial colony-forming cells” (ECFCs), and on neovessel formation in vivo.We found that OPG causes phenotype changes of ECFCs via the activation of different molecular pathways targeting cell clonogenicity, differentiation, proliferation, migration and adhesion. Our results suggest that OPG may interact with ECFCs through its binding to syndecan-1, to induce an anti-adhesive effect and thereby promoting ECFCs migration through a SDF-1/CXCR4 dependant pathway and the ERK1/2, Akt and the mTOR pathways activation. OPG can intervene on the autocrine effect of ECFCs by inducing their adhesion to activated endothelium and their tubulogenesis, and potentiate their paracrine effects by inducing SDF-1 release. Alternatively, it can promote ECFCs survival, probably, in a αVβ3 integrin-dependent manner. In vivo, OPG potentiates FGF-2 proangiogenic effects and may participate in tumour growth, invasion and metastasis, possibly through inhibition of tumour cell apoptosis but also by promoting tumour angiogenesis.

In cardiovascular disease the repair response is insufficient to restore blood flow, leading to the death of muscle and loss of tissue function. Therefore, strategies to augment the endogenous cell response and its effects may help improve tissue recovery and function. In this study we explored the use of tissue-engineered collagen matrices for augmenting endogenous regenerative processes after myocardial infarction. Treatment with the sLeX-collagen matrix reduced inflammation and apoptosis and had a positive regenerative effect on the infarcted mouse heart, through improved vascular density and possibly enhanced cardiomyogenesis. Additionally, we investigated the effects of cryopreservation on generating circulating angiogenic cells (CACs) from peripheral blood mononuclear cells (PBMCs), as a potential source of stem cells that could be used in combination with our collagen scaffold. Our findings show that despite PBMCs experiencing phenotypic changes after cryopreservation, they may still be used to generate the same therapeutic CACs as freshly procured PBMCs.