Netrins in vascular and Stem cell guidance

描述（由申请人提供）：Netrin蛋白家族首先被鉴定为生长轴突的有吸引力的指导线索。我们最近发表的数据表明，它们在血管引导中具有相应的作用。有三个主要结果。首先，Netrin-1、-2和-4诱导多种内皮细胞系中的迁移、增殖和管形成。第二，在斑马鱼胚胎发生期间敲低netrinla抑制了副索血管（帕拉vessel，PAV）的形成，其最近被证明对于形成斑马鱼淋巴系统是关键的。第三，外源性Netrins在两种鼠疾病模型中加速缺血组织的再血管化和再灌注。因此，使用斑马鱼和哺乳动物系统、功能丧失和功能获得策略以及体内和体外测定，我们表明Netrins刺激血管生成。我们最近的初步数据显示，其他Netrin途径基因（netrin 2、dec、uncSb）也是斑马鱼PAV形成所需的; UncSb缺失和内皮特异性条件突变小鼠在胎盘血管生成中具有特定缺陷; Netrin还可以影响培养物中的造血干细胞和间充质干细胞（HSC和MSC）。在本修订提案中，我们将在这些成果的基础上再接再厉。利用PI和co-Pi在细胞培养、斑马鱼和小鼠系统方面的专业知识，我们将同时追求三个具体目标。每一个目标都涉及Netrin功能的单独作用，使我们能够在血管生成的不同方面测试共同和不同的机制。第一个目标是确定Netrin和Netrin受体在斑马鱼PAV形成中的作用。第二个目标将定义UncSb小鼠的胎盘血管缺陷，并解决潜在的发育机制。第三个目标将确定Netrin对培养中的HSC和MSC的影响，并进一步测试Netrin是否可以在体内指导这些干细胞的归巢，提供外源Netrin可以促进治疗模型中血管重建的潜在机制。与公共卫生的相关性。血管系统的形成对正常发育至关重要，并且血管重塑在对损伤或疾病的反应期间至关重要。这些研究将为Netrin信号传导在血管发育和内皮细胞生物学中的作用提供基本的见解，并对其治疗潜力提供机制性见解。这些结果可能对理解淋巴系统和胎盘的发育以及缺血性损伤的治疗具有特别的意义。

DESCRIPTION (provided by applicant): The Netrin family of proteins were first identified as attractive guidance cues for growing axons. Our recently-published data show they have a corresponding role in vascular guidance. There were three main results. First, Netrin-1, -2 and -4 induce migration, proliferation, and tube formation in multiple endothelial cell lines. Second, knockdown of netrinla during zebrafish embryogenesis inhibits the formation of the para- chordal vessel (PAV), which has recently been shown to be critical for forming the zebrafish lymphatic sys- tem. Third, exogenous Netrins accelerate revascularization and reperfusion of ischemic tissue in two murine disease models. Thus, using zebrafish and mammalian systems, loss-of-function and gain-of-function strat- egies, and in vivo and in vitro assays, we showed that Netrins stimulate angiogenesis. Our more recent preliminary data show that other Netrin-pathway genes (netrin2, dec, uncSb) are also required for zebrafish PAV formation; that UncSb null and endothelial-specific conditional mutant mice have a specific defect in placental angiogenesis; and that Netrins can also influence hematopoietic and mesen- chymal stem cells (HSCs and MSCs) in culture. In this revised proposal we will build on these results. Exploiting the PI and co-Pi's expertise with cell culture, zebrafish, and mouse systems, we will pursue three specific aims in parallel. Each aim addresses a separate role of Netrin function, allowing us to test for both common and divergent mechanisms during differ- ent aspects of angiogenesis. The first aim will define the role of Netrins and Netrin receptors in zebrafish PAV formation. The second aim will define the placental vascular defect in UncSb mice and address the underlying developmental mechanisms. The third aim will define the effects of Netrins on HSCs and MSCs in culture, and further test whether Netrin can direct the homing of these stem cells in vivo, providing a potential mechanism by which exogenous Netrins can promote revascularization in therapeutic models. Relevance to public health. Formation of the vascular system is critical to normal development, and vas- cular remodeling is critical during responses to injury or disease. These studies will provide fundamental in- sight into the role of Netrin signaling in vascular development and endothelial cell biology, and mechanistic insight into its therapeutic potential. The results may have particular relevance to understanding develop- ment of the lymphatic system and the placenta, and to therapies for ischemic injury.