Microenvironmental Control of Capillary Morphogenesis

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Vascularization is of critical importance to the treatment of numerous pathologies and the success of tissue engineering. Many cell-based strategies to promote therapeutic vascularization have been explored in both pre-clinical models and clinical trials. However, approaches involving delivery of a single cell type to ischemic sites either by intravenous or intramuscular injection have shown little efficacy in clinical trials, particularly for the treatment of critical limb ischemia. By contrast, a common approach to vascularize engineered tissues involves co-encapsulation of endothelial cells (or their progenitors) combined with supportive stromal cells in a hydrogel-based biomaterial intended to mimic the extracellular matrix (ECM). However, the choices of stromal cells and materials have varied widely across studies. Our long-term goal is to mechanistically understand how these elements of the microenvironment influence the quantity, functional quality, and stability of the resulting vasculature. Using a combination of in vitro and in vivo models, we have shown the rate of formation of nascent vasculature is regulated by both the biophysical properties of the ECM and the identity of the supporting stromal cells. Furthermore, we have demonstrated that stromal cell identity critically regulates the functional qualities of the microvasculature formed within fibrin hydrogels, both in vitro and in vivo, with multipotent bone marrow stromal cells (BMSCs) inducing more stable, less permeable capillaries compared to fibroblasts from a range of different tissues. Our data suggest stromal cells of different origins differentially remodel and stiffen the ECM to influence the rate of vascular morphogenesis, leading to our hypothesis that vessels which form quickly are of poor quality, while those which form more slowly show superior functionality, maturation, and persistence. In Aim 1, a combination of approaches will be used in 3D fibrin-based co- culture models to evaluate the impact of stromal cell identity on vascular morphogenesis rate, ECM mechanics (global and local), and permeability. In Aim 2, an engineered hydrogel material will be used to alter the relationships between stromal cell identity, ECM mechanics, vascularization rate, and vascular permeability. Finally, Aim 3 will examine the effects of stromal cell identity and morphogenesis rates on the quantity and quality of neovasculature in multiple in vivo models. Successful completion of these aims will expand the mechanistic insights attained during the prior funding periods to better understand the roles of the microenvironment in vascular morphogenesis, and thereby enhance efforts to create functional vasculature suitable for regenerative medicine and revascularization applications.