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.
|Date of Award||26 Apr 2017|
|Supervisor||Giordano Pula (Supervisor) & Paul De Bank (Supervisor)|