Our long-term goal is to understand the cellular mechanisms that regulate vesicle trafficking during endothelial tube formation. Our objective is to define critical genes involved in the formation of the apical membrane and the coordination between vesicle trafficking and the remodeling of cellular junctions during endothelial tube formation. Our principle hypothesis is that endothelial tube formation is tightly coupled to adherence and tight junction remodeling and that these apical junctions direct vesicle trafficking during formation of the apical membrane in vitro and in vivo. In support of this hypothesis, we have identified a number of proteins involved in cellular junctions and vesicle trafficking and demonstrated that these genes are required for endothelial tube formation in human umbilical vein endothelial cells in culture and during embryonic angiogenesis in zebrafish. We have chosen to work with zebrafish because of the combined advantages of a well-defined genome that relates to the human genome, the ability to efficiently knockdown genes and produce transgenic animals, and the optical clarity of the embryo that allows a detailed examination of tube formation on the single cell level in vivo. Complementary to this approach, we follow the same processes biochemically in vitro. We provide preliminary evidence suggesting a close coupling of vesicle trafficking and apical junctional remodeling. Our team has extensive experience with the generation of transgenic zebrafish using transposon-mediated transgenesis techniques, the injection of antisense morpholino oligonucleotides, and the examination of protein-protein interactions in vitro. We have taken an innovative and comparative approach to this problem by combining in vitro and in vivo experiments. We use transgenic zebrafish and human endothelial cells that express fluorescently tagged proteins to answer fundamental cell biological questions regarding endothelial tube formation. At the end of the granting period, we will have defined novel mechanisms that function during endothelial tube formation. This understanding is critical to the development of new therapeutic strategies to modulate of endothelial tube formation and diameter. This ability would have direct implications for the treatment of heart disease, ischemia, and cancer.
The creation of tubes by endothelial cells during angiogenesis and vasculogenesis is a critical step in the formation of functional blood vessels. Acquiring a more detailed understanding of the cellular and molecular pathways guiding this process has direct implications for the development of therapeutic interventions for the treatment of heart disease, ischemia, and cancer.
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