Exploring the Interactions between Mesenchymal Stem Cells and Endothelial Cells in Engineered Perivascular Niches.

Abstract

Therapeutic approaches to induce neovascularization comprise cell-based therapies using endothelial cells (ECs) co-cultured with stromal cells to form long lasting functional blood vessels. This dissertation investigates the role of cross-talk between ECs and stromal cells in governing the processes of neovascularization in fibrin matrices, representative of the wound healing environment in vivo. Special attention was paid to a specific molecular interaction between the alpha 6 beta 1 integrin adhesion receptor on bone marrow derived stromal cells (BMSCs) and EC-deposited laminin. Prior studies have shown that co-delivery of ECs and stromal cells supports the formation of robust blood vessel networks. To validate this phenomenon in vitro, we utilized an established three dimensional microfluidic device as a model system. ECs suspended within fibrin gels patterned in the device adjacent to stromal cells executed a morphogenetic process akin to vasculogenesis, forming a primitive vascular plexus and maturing into a robust capillary network with hollow well-defined lumens. Both BMSCs and fibroblasts associated with the ECs like pericytes, but promoted capillary morphogenesis with distinct kinetics. Furthermore, biochemical assays revealed that the perivascular association of BMSCs required their alpha 6 beta 1 integrin receptor, presumably to mediate an interaction with EC-deposited laminin. To further investigate the alpha 6 beta1 integrin-laminin interactions, we used an in vitro model of angiogenesis in which ECs coated on microcarrier beads were co-cultured with BMSCs within a fibrin matrix. Using RNA interference, we demonstrated that α6 integrin inhibition in BMSCs reduced capillary sprouting, and blocked their ability to associate with nascent blood vessels. Furthermore, we demonstrated that the BMSCs with attenuated alpha 6 integrin expression levels proliferate at a significantly lower rate relative to either control cells expressing non-targeting shRNA or wild type BMSCs. Despite the addition of cells to compensate for this deficit in proliferation, deficient sprouting persists. These data collectively underscore the importance of understanding integrin-mediated interactions between EC-deposited basement membranes and supporting stromal cells during the complex process of vessel formation. The results presented in this dissertation could have significant implications for both physiological and pathological neovascularization.