Three-dimensional (3-D) control of microvessel formation is critical for regeneration medicine and tissue engineering. Formation and maintenance of organ functions require deep and extensive microvessel networks that can supply O2 and nutrients to tissue-forming cells. In this study, we examined the effects of hypoxia on 3-D network formation using an in vitro model system in which the effects of hypoxia were isolated from those of other factors affecting network formation, such as growth factors. When we quantified network formation by endothelial cells (ECs) cultured on collagen gel under hypoxic (5% O2) and normoxic (21% O2) conditions, we found that hypoxia caused ECs to penetrate into the underlying collagen gel and to form 3-D, capillary-like networks. We also examined the detailed 3-D morphology of the networks using confocal laser-scanning microscopy. The networks promoted by hypoxia were more extensive and penetrated more deeply into the underlying collagen gel than did those formed under normoxic conditions. Our results demonstrate that hypoxia can induce 3-D network formation by ECs in vitro in the absence of other factors. In examining the mechanism by which hypoxia induces network formation, we found that hypoxia promotes expression of many EC genes, causes EC to secrete network-inducing factors, and increases the sensitivity of ECs to growth factors.
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