Endothelial colony forming cells (ECFCs) are circulating cells able to differentiate into mature endothelial cells and replenish the endothelial lining at the sites of vascular damage. Their utilization for cell therapies aiming to restore healthy endothelial lining of blood vessels and stimulate neovascularization of ischemic tissues has been the object of intense investigation. The overall aim of this project was to investigate and develop novel approaches for the promotion of vasculogenesis and endothelisation of vascular grafts by ECFCs. First, protease-activated receptors (PARs) were investigated as potential targets to stimulate in ECFC-driven vasculogenesis and promote therapeutic revascularization. Our data showed that PAR-1 and PAR-2 are both expressed in ECFCs and functionally coupled to the ERK1/2 pathway. Specific stimulation of PAR-1, but not PAR-2, significantly inhibits in vitro tube formation by ECFCs, and this effect is due to the down-regulation of VEGFR-2. Although the role of PAR-2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade. Secondly, we investigated the use of human platelet lysate gel (hPLG) as an animal product-free and patient-specific tool to isolate, amplify, differentiate and deliver endothelial progenitor cells. This extracellular matrix (ECM) was able to support the proliferation of ECFCs in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Interestingly, the culture of ECFCs on hPLG led to the upregulation of several angiogenic genes, such as VEGFR-2 and PDGFR-β, and also induced the robust sprouting of existing vessels in an ex vivo model. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. Finally, the biomimetic and pro-angiogenic properties of human platelet lysate (hPL) were utilised to facilitate adhesion and proliferation of ECFCs on polymeric materials. hPL was shown to promote endothelisation of biomaterials, which can be utilised for tissue engineering purposes. Novel electrospun polymeric tubular scaffolds were developed and their surface properties enhanced using plasma treatment. These scaffolds exhibit increased adsorption of proteins from hPL, which acted as an interfacial layer to promote the adhesion and proliferation of ECFCs on their surface. Such findings demonstrate that the pro-angiogenic and pro-vasculogenic properties of platelet-derived factors can be transferred to scaffolds to stimulate endothelial growth on synthetic scaffolds for tissue engineering without the use of recombinant or animal products. In conclusion, we propose the use of ECFCs with platelet-derived products as an ideal synergy for the vascularization of tissue engineered constructs.

Les progéniteurs endothéliaux semblent offrir de nouvelles perspectives dans le traitement des pathologies ischémiques. Nos travaux portent sur l'étude des effets d'une transplantation d'une sous population homogène de progéniteurs endothéliaux dits tardifs, les ECFCs, sur un modèle d'occlusion de l'artère cérébrale moyenne (MCAO) transitoire chez le rat. 4x106 ECFCs cultivés à partir du sang de cordon humain ou 1ml de PBS ont été injectés en intraveineuse (IV) 24h après une MCAO d'une durée d'une heure chez les rats appartenant respectivement au groupe greffé et au groupe contrôle. On a pu mettre en évidence le passage des cellules greffées dans l'hémisphère cérébral ischémié par radiomarquage à l'oxinate d'indium 111 (111In) et marquage fluorescent au CM-Dil des ECFCs avant transplantation. Ceci a été confirmé par la visualisation d'ECFCs d'origine humaine en périphérie de la zone infarcie par marquages immunohistochimiques au MAB1281 et CD31. La transplantation d'ECFCs a augmenté significativement le taux de survie et a amélioré la récupération fonctionnelle des animaux. L'effet bénéfique observé est associé à une réduction du nombre de cellules apoptotiques ainsi qu'une augmentation de la densité capillaire et de la neurogenèse en périphérie de la zone lésée. Ces effets semblent corrélés à une surexpression en zone de pénombre de VEGF et IGF1 aux propriétés pro-angiogéniques et neurotrophiques, et à une diminution de l'expression d'un facteur pro-apoptotique proBDNF. De plus, nous avons montré que les ECFCs sont capables de sécréter des cytokines pro-angiogéniques. / Endothelial progenitor cells (EPCs) seem to be a promising option to treat patients with ischemic diseases. Here, we investigated the effects of late EPCs or Endothelial Colony-Forming Cells (ECFCs), a recently defined homogeneous subtype of EPCs, in a rat model of transient middle cerebral artery occlusion (MCAO). Either vehicle or 4.106 ECFCs, isolated from human cord blood, were intravenously injected 24h after 1 hour of MCAO in rats assigned to control and transplanted groups respectively. 111In-oxine-labeled ECFCs specifically homed to ischemic hemisphere and CM-Dil prelabeled ECFCs preferentially settled in the inner boundary of the core area of transplanted animals. The presence of human cells in rat brain sections was detected by immunohistochemical staining (MAB1281, CD31). We demonstrated that ECFCs injected 24h after MCAO improved functional recovery and survival rate. Beneficial effect was associated with an increase in growth factors expression in homogenates from ischemic area (VEGF, IGF-1, proBDNF) and may be related to the secretion by ECFCs of soluble factors that could affect apoptosis, vascular growth and neurogenesis. These findings raise perspectives for the use of ECFCs as a well-characterized cell therapy product for optimal therapeutic outcome after stroke.