This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. VEGFR-3 signaling is essential for normal embryonic angiogenesis and lymphatic development. The magnitude of this signaling pathway is tightly associated with the abundance of VEGFR-3 at the cell surface. Although the abundance of cell surface VEGFR-3 is thought to be controlled by VEGF-C-induced endocytosis and degradation, very little is known about the endocytic machinery that governs VEGFR-3 internalization. Epsin is an emerging major endocytic adaptor protein that mediates activated receptor endocytosis. We recently showed that reduced expression of epsins 1 and 2 increased VEGFR-3 signaling in lymphatic endothelial cells (LEC), suggesting a potential regulatory role. To probe in vivo function of epsin 1 and 2 in VEGFR-3 signaling, we engineered endothelial cell-specific, tamoxifen-inducible epsin double knockout mice (EC-iDKO) by crossing epsin 1 floxed and epsin 2 KO mice with VEcad-Cre-ERT2 mice expressing tamoxifen-inducible Cre recombinase in endothelial cells (EC). In our preliminary studies, EC-iDKO mice exhibited increased lymphangiogenesis with enhanced lymphatic vessel density, dilated vessel lumen, and increased vessel sprouting. We hypothesize that epsin-mediated endocytosis of VEGFR-3 is crucial for lymphangiogenesis under physiological and pathological conditions. To test this hypothesis, we will first determine how endothelial-cell epsins regulate embryonic and postnatal lymphangiogenesis. We will utilize EC-iDKO mice to explore lymphatic development at various embryonic and postnatal stages under both physiological and pathological conditions. Moreover, we seek to define the molecular mechanisms by which endothelial-cell epsins regulate VEGFR-3 signaling. We plan to test the endocytic fate of VEGFR-3 in the absence of epsin and the interaction of epsin with VEGFR-3 using biochemical and cell morphological approaches, to determine the role of epsin in VEGFR-3 endocytosis and degradation. These studies should provide a unique perspective into the regulation of lymphangiogenesis, and contribute to the application of endocytic components to new therapeutic strategies for a number of lymphatic diseases and cancer.
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