Robo4 and heparan sulfate proteoglycan in angiogenesis

 描述(申请人提供)：我们研究的长期目标是了解轴突引导分子Robo4信号转导和硫酸乙酰肝素(HS)蛋白多糖在血管系统中的作用，并获得新的知识来指导基于Robo4和HS的人类疾病新疗法的开发。我们最近的研究发现，SLIT3与Robo4相互作用，在体外促进内皮细胞(EC)功能和体内新生血管形成，并证明EC-HS通过作为SLIT3-Robo4信号的共同受体促进横隔膜发育的血管生成。这些研究记录了体内证据，证明EC-HS是横隔膜发育血管生成所必需的，并阐明了EC-HS在Robo4信号转导中的积极调节作用。在我们正在进行的研究中，我们发现了Robo4和Syndecan-1(SDC-1)，这是一种HS蛋白多糖，通过在EC表面形成SDC-1-SLIT3-Robo4三元复合体而发挥协同递增作用，两者都从EC表面结构性地脱落，这种脱落降低了细胞表面Robo4和SDC-1的水平，并减弱了SLIT3诱导的EC迁移。这些新的发现揭示了Robo4信号中基于脱落的调节机制，并导致了我们的第一个假设，即Robo4和SDC-1的胞外结构域脱落都对Robo4信号进行负面调节。同时，我们的新研究还产生并检测了EC特异的Ext1基因敲除(Ext1ECKO)小鼠，在这些小鼠中EC-HS的表达完全取消。Ext1ECKO小鼠表现出严重的发育血管生成缺陷，并具有胚胎致死性，表明EC-HS在多个器官的发育血管生成中是必不可少的。表型分析发现，EC-Ext1消融干扰EC功能，包括顶端细胞出芽和丝状足突形成，以及壁细胞(MC)募集，分别复制了血管内皮生长因子(VEGF)和血小板衍生生长因子-B(PDGF-B)缺乏的小鼠的血管发育缺陷，导致我们的第二个假设，EC-HS促进VEGF-和PDGF-B信号转导，本质上调节发育血管生成。为了有力地检验我们的新假设，我们将追求三个具体目标。目的1通过鉴定Robo4和SDC-1脱落体(S)并评估Robo4和SDC-1脱落区的改变是否影响Robo4信号转导，阐明Robo4和SDC-1胞外结构域脱落对Robo4信号在血管生成中的调节作用(S)。目的2通过鉴定Ext1ECKO小鼠的血管缺陷并评估相关的EC-HS和MC功能，确定EC-HS在发育中血管生成中的重要作用及其潜在的细胞机制。AIM 3将破译EC-HS调控发育血管生成的分子信号，即血管内皮生长因子-B和PDGF-B信号。拟议的研究将使用新的和已建立的遗传、细胞和生化方法，并结合体外和体内血管生成模型。这些系列研究有望阐明Robo4-和SDC-1胞外结构域脱落作为Robo4信号在血管生成中的一种新的调节机制，以确定EC-HS对于发育的血管生成是必不可少的，并揭示EC-HS促进VEGF和PDGF-B信号转导作为EC-HS在发育中基本的促血管生成功能的分子机制。

 DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the roles of axon guidance molecule Robo4 signaling and heparan sulfate (HS) proteoglycans in vasculature and gain new knowledge to guide development of Robo4- and HS-based novel therapeutics for human diseases. Our recent studies uncovered that Slit3 interacts with Robo4 to promote endothelial cell (EC) functions in vitro and neovascularization in vivo, and demonstrated that EC-HS promotes developmental angiogenesis in diaphragm by functioning as a co- receptor for Slit3-Robo4 signaling. These studies documented in vivo evidence that EC-HS is required for developmental angiogenesis in diaphragm and elucidated a positive regulatory role of EC-HS in Robo4 signaling. In our ongoing studies, we uncovered that Robo4 and syndecan-1 (SDC-1), the HS proteoglycan that functions as a co-preceptor by forming SDC-1-Slit3-Robo4 ternary complex on the EC surface, both are constitutively shed from the EC surface, and the shedding reduces cell surface levels of Robo4 and SDC-1 and attenuates Slit3-induced EC migration. These new findings revealed a shedding-based regulatory mechanism in Robo4 signaling and led to our first hypothesis that ectodomain shedding of Robo4 and SDC-1 both negatively regulates Robo4 signaling. In parallel, our new studies also generated and examined the EC- specific Ext1 knockout (Ext1ECKO) mice in which the expression of EC-HS is completely abolished. The Ext1ECKO mice exhibit profound and severe developmental angiogenesis defects and are embryonic lethal, revealing that EC-HS is essentially required for developmental angiogenesis in multiple organs. Phenotype characterization uncovered that the EC-Ext1 ablation disturbs EC functions, including tip cell sprouting and filopodia formation, and mural cell (MC) recruitment, phenocopying the vascular development defects displayed in mice that are deficient in vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B), respectively, leading to our second hypothesis that EC-HS facilitates both VEGF- and PDGF-B signaling to essentially modulate developmental angiogenesis. To vigorously test our new hypotheses we will pursue three Specific Aims. Aim 1 will delineate the regulatory roles of Robo4- and SDC- 1 ectodomain shedding on Robo4 signaling in angiogenesis by identifying Robo4- and SDC-1 sheddaes(s) and assessing if alteration of Robo4- and SDC-1 shedding affects Robo4 signaling. Aim 2 will determine the essential role of EC-HS in developmental angiogenesis and the underlying cellular mechanisms by characterizing vascular defects in the Ext1ECKO mice and assessing related EC- and MC functions. Aim 3 will decipher the molecular signaling, i.e., VEGF- and PDGF-B signaling by which EC-HS modulates developmental angiogenesis. The proposed studies will use both novel and established genetic, cellular and biochemical approaches in conjunction with in vitro and in vivo angiogenesis models. These serial investigations are anticipated to elucidate Robo4- and SDC-1 ectodomain shedding as a novel regulatory mechanism of Robo4 signaling in angiogenesis, to establish that EC-HS is essentially required for developmental angiogenesis, and to reveal that EC-HS facilitates both VEGF and PDGF-B signaling as the molecular mechanisms underlying the essential pro-angiogenesis function of EC-HS in development.