Regulation of endothelial cell invasion, migration and cell junction plasticity

项目摘要 我实验室的主要研究方向之一是内皮屏障的调节， 内皮细胞迁移。这些过程对血管系统的生理功能至关重要， 在人类疾病中经常失调。在理解信号方面已经取得了很大的进展， 调节内皮屏障和细胞迁移。然而，刺激内皮细胞迁移， 血管生成是一种高度局部化和短暂的事件。定义本地和时间组件的角色 由于目前工具的局限性，血管生成信号传导的研究一直具有挑战性。此外，时空 这些刺激对内皮屏障的调节还知之甚少。我们的工作重点是 确定迁移信号的位置和持续时间如何指导内皮细胞的侵袭和迁移 通过细胞外基质，以及它们如何影响内皮屏障的组织和渗透性。 内皮屏障在粘附连接（AJs）水平上受到控制，粘附连接是细胞-细胞粘附结构， 由跨膜蛋白VE-钙粘蛋白介导。磷酸化介导的信号转导调节 结构和渗透性。在我们最近的研究中，我们描述了酪氨酸激酶Src及其受体的双重作用。 VE-钙粘蛋白的磷酸化在调节内皮通透性中的作用。我们的研究结果表明，Src- 介导的磷酸化诱导仍然保持其屏障功能的动态AJs的形成。这表明 一种调节AJ可塑性的机制，该机制不会损害屏障渗透性， 内皮细胞迁移在平行的研究中，我们剖析了Src调控的细胞降解的机制， 细胞外基质的内皮细胞，并发现了一种新的细胞骨架成分，介导 基质降解的podosomes的形成。本报告所建议的研究将继续以我们以往的研究为基础， 研究结果和重点是解剖如何磷酸化VE钙粘蛋白和血管生成信号的血管 生长因子受体2（VEGFR 2）、鞘氨醇-1-磷酸受体1（S1 PR 1）和Src调节 AJs的可塑性以及内皮细胞的侵袭和迁移。我们将使用新颖的光遗传学工具 这将使我们能够通过精确的空间和时间控制来询问这些过程。我们将使用 工程化的光调节VEGFR 2，S1 PR 1和Src，以确定局部和时间的影响， 控制血管生成信号和解剖机制，介导AJs的调节和 三维环境中的内皮细胞。我们的长期目标是定义控制 血管生成过程中内皮细胞的迁移和内皮屏障功能。

Project Summary One of the main research directions of my laboratory focuses on regulation of the endothelial barrier and endothelial cell migration. These processes are critical for physiological function of vascular system and they are often dysregulated in human diseases. A lot of progress has been made in understanding signaling that regulates endothelial barrier and cell migration. However, stimulation of endothelial cell migration during angiogenesis is a highly localized and transient event. Defining the role of the local and temporal components of angiogenic signaling has been challenging due to limitations of current tools. Furthermore, spatiotemporal regulation of the endothelial barrier by these stimuli has been poorly understood. Our proposed work will focus on determining how the location and duration of migratory signals direct endothelial cell invasion and migration through extracellular matrix, and how they affect the organization and permeability of the endothelial barrier. The endothelial barrier is controlled at the level of adherens junctions (AJs), cell-cell adhesion structures mediated by the transmembrane protein VE-cadherin. Phosphorylation-mediated signaling regulates the structure and permeability of AJs. In our recent studies, we described a dual role of tyrosine kinase Src and its phosphorylation of VE-cadherin in regulation of endothelial permeability. Our results demonstrated that Src- mediated phosphorylation induces formation of dynamic AJs that still retain their barrier function. This suggests a mechanism for the regulation of AJ plasticity that does not compromise barrier permeability during endothelial cell migration. In parallel studies, we dissected a mechanism of Src-regulated degradation of the extracellular matrix by the endothelial cell and discovered a novel cytoskeletal component that mediates formation of matrix-degrading podosomes. The studies proposed here will continue to build on our previous findings and focus on dissecting how phosphorylation of VE-cadherin and angiogenic signaling by Vascular Growth Factor Receptor 2 (VEGFR2), Sphingosine-1-phosphate Receptor 1 (S1PR1), and Src regulate plasticity of AJs as well as invasion and migration of endothelial cells. We will employ novel optogenetic tools that will allow us to interrogate these processes with precise spatial and temporal control. We will use engineered light-regulated VEGFR2, S1PR1, and Src to determine the effects of locally and temporally controlled angiogenic signals and dissect mechanisms that mediate regulation of AJs and migration of endothelial cells in three dimensional environment. Our long-term goal is to define the processes that control migration of endothelial cells and endothelial barrier function during angiogenesis.