EPSIN-REGULATED VEGFR-3 SIGNALING IN LYMPHANGIOGENESIS

淋巴管生成中 EPSIN 调节的 VEGFR-3 信号传导

• 批准号: 8364975
• 负责人: HONG CHEN
• 金额: $ 32.08万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8364975
• 关键词: Adaptor Signaling Protein; Biochemical; Biology; Blood Vessels; Cell surface; Cells; Development; Embryo; Endocytosis; Endothelial Cells; Engineering; Exhibits; Funding; Grant; Interdisciplinary Study; Knockout Mice; Lymphangiogenesis; Lymphatic; Lymphatic Diseases; Lymphatic Endothelial Cells; Lymphatic vessel; Mediating; Molecular; Mus; National Center for Research Resources; Physiological; Principal Investigator; Regulation; Research; Research Infrastructure; Resources; Role; Signal Pathway; Signal Transduction; Source; Staging; Tamoxifen; Testing; United States National Institutes of Health; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-3; angiogenesis; cost; density; epsin; epsin 1; in vivo; lymphatic cancer; novel therapeutics; postnatal; receptor; recombinase

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 VEGFR-3信号传导对于正常胚胎血管生成和淋巴管发育至关重要。该信号通路的大小与细胞表面VEGFR-3的丰度密切相关。尽管细胞表面VEGFR-3的丰度被认为是由VEGF-C诱导的内吞作用和降解控制的，但对支配VEGFR-3内化的内吞机制知之甚少。Epsin是一种新出现的介导激活受体内吞作用的主要内吞衔接蛋白。我们最近发现，epsins 1和2的表达减少增加了淋巴管内皮细胞（LEC）中的VEGFR-3信号，这表明了潜在的调节作用。为了探测epsin 1和2在VEGFR-3信号传导中的体内功能，我们通过将epsin 1 floxed和epsin 2 KO小鼠与在内皮细胞（EC）中表达他莫昔芬诱导型Cre重组酶的VEcad-Cre-ERT 2小鼠杂交来工程化内皮细胞特异性的他莫昔芬诱导型epsin双敲除小鼠（EC-iDKO）。在我们的初步研究中，EC-iDKO小鼠表现出淋巴管生成增加，淋巴管密度增加，血管管腔扩张，血管出芽增加。我们推测，在生理和病理条件下，epsin介导的VEGFR-3内吞作用对淋巴管生成至关重要。为了验证这一假设，我们将首先确定内皮细胞胰蛋白酶如何调节胚胎和产后淋巴管生成。我们将利用EC-iDKO小鼠在生理和病理条件下探索不同胚胎和出生后阶段的淋巴发育。此外，我们试图确定内皮细胞epsins调节VEGFR-3信号转导的分子机制。我们计划使用生物化学和细胞形态学方法测试在不存在epsin的情况下VEGFR-3的内吞命运以及epsin与VEGFR-3的相互作用，以确定epsin在VEGFR-3内吞和降解中的作用。这些研究应该提供一个独特的视角到淋巴管生成的调节，并有助于应用内吞成分的新的治疗策略，一些淋巴疾病和癌症。