The Effects of Endothelial Cell Contractility and Extracellular Matrix Density on Capillary Morphogenesis and Maintenance in 3D Biomaterials

Angiogenesis refers to new blood vessel formation from preexisting vasculature into previously avascular tissue. Identifying the mechanisms regulating angiogenesis in pathologic conditions such as cancer and heart disease is crucial to develop successful therapies. The dependence of angiogenesis on characteristic properties of these conditions, such as alterations in tissue stiffness due to changes in the composition of the extracellular matrix (ECM), the types of cells contributing to vessel formation and providing pro-angiogenic factors, as well as their contractile machinery, may shed light on potential therapeutic strategies.;Prior studies have established that increasing ECM density inhibits capillary morphogenesis in vitro, and that addition of human mesenchymal stem cells partially rescues a healthy angiogenic phenotype, leading us to investigate if these effects can be recapitulated in vivo. By quantifying vessel numbers, perfusion, thickness, maturity, and perivascular collagen deposition within an in vivo angiogenesis model, we showed that changing ECM density inhibits capillary morphogenesis in vivo consistent with in vitro observations.;Seeking to elucidate more complex nuances of the angiogenesis process related to ECM's physical resistance to endothelial cell-generated contractile forces, we sought to create a mechanically tunable biosynthetic scaffold based on PEGylated fibrinogen. Despite promise with other cell types, the ability of this material to support vessel formation was limited. Nevertheless, we were able to explore the hypothesis that ECM density may regulate angiogenesis via a mechanism involving actin-mediated cell-generated forces. We utilized an in vitro model of angiogenesis in which endothelial cells (ECs) coated on microcarrier beads are distributed within fibrin ECM and are supported by a fibroblast monolayer. Fibrin gel density variations, and a library of pharmacological agents that inhibit actin cytoskeleton-generated forces, were used to prove the necessity of EC-generated tractional forces in angiogenesis. Finally, we used spatio-temporal image correlation spectroscopy to quantify EC-mediated deformation of individual ECM elements during capillary sprouting, while manipulating the ability of ECs to generate traction using genetic mutants of the small GTPase RhoA. The combination of experimental techniques and materials enabled this dissertation to elucidate the interdependence of capillary morphogenesis, ECM mechanical properties, and cell contractile function both in vitro and in vivo.