In vitro and in vivo evaluation of endothelial cells in microvascular networks

The utility of promising biomedical applications, such as tissue engineering and cell-based gene therapy, is currently limited by inadequate transport from the microvasculature. Success of these applications depends, in part, upon improved understanding of incorporation of implanted endothelial cells (ECs). Towards this understanding, the work presented in this thesis focuses on evaluating the effects of the presence of non-endothelial supporting cells, endothelial cell origin, extracellular matrix (ECM) support, the mechanical environment, and implant location on microvascular network (MVN) formation and EC incorporation into vessels. Supporting cells. When mixed with fibroblasts within Matrigel, BAEC show improved MVN formation in vitro, increased survival in vitro and in vivo, prolonged hGH delivery, and increased vascular incorporation, as compared to BAEC alone, which remain round and show rapidly decreasing hGH levels. Cell source. Adult human bone marrow-derived endothelial cells (BOEC) show superior MVN and lumen formation relative to three vessel-derived endothelial cell lines. Although hypothesized to have a survival advantage because of their large proliferative capacity, BOEC did not show a survival advantage. Mechanical environment. ECs in a stiff ECM environment (mechanically constrained or high collagen concentration) form large, thin-walled lumen structures, elongate as single cells, and show actin stress fibers. ECs in a more malleable environment (floating gels or low collagen concentration) form in vivo-like, elongated, multi-cellular structures with smaller, thicker-walled lumens and punctate actin staining. These behaviors can be modulated by use of cells that exert larger (BOEC) or smaller (HUVEC) tractional forces, implying that the balance between cellular force generation and matrix stiffness modulates capillary morphogenesis. ECM support. Salmon fibrin was evaluated as an alternative ECM support by evaluating sprouting of human umbilical vein ECs (HUVEC) on dextran beads suspended in either salmon or human fibrin. Salmon fibrin shows an increase in the number of sprouts per bead relative to human fibrin, while average sprout lengths is similar. Further, degradation is decreased in salmon fibrin relative to human fibrin in the absence of a protease inhibitor. Implant location. Low base-line vessel density seen in subcutaneous dorsal implants in nude mice can be improved by using the abdominal location in SCID mice