Netrins in vascular and Stem cell guidance

描述（由申请人提供）：Netrin蛋白家族最初被确定为轴突生长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。主要有三个结果。首先，Netrin-1、-2和-4诱导多种内皮细胞系的迁移、增殖和管状形成。其次，在斑马鱼胚胎发生过程中，神经原的敲除抑制了脊索旁血管（PAV）的形成，而PAV最近被证明对斑马鱼淋巴系统的形成至关重要。第三，外源性netrin在两种小鼠疾病模型中加速缺血组织的血运重建和再灌注。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，以及体内和体外实验，我们表明，网蛋白刺激血管生成。我们最近的初步数据表明，斑马鱼PAV的形成也需要其他netrin通路基因（netrin2, dec, uncSb）；UncSb缺失和内皮特异性条件突变小鼠在胎盘血管生成中存在特异性缺陷；Netrins还可以影响培养的造血干细胞和间充质干细胞（hsc和MSCs）。在这项修订后的建议中，我们将以这些成果为基础。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，我们将并行追求三个具体目标。每个目标都解决了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。第一个目标是确定网蛋白和网蛋白受体在斑马鱼PAV形成中的作用。第二个目的是明确UncSb小鼠胎盘血管缺损并探讨其潜在的发育机制。第三个目标是确定Netrin对培养的造血干细胞和间质干细胞的影响，并进一步测试Netrin是否可以指导这些干细胞在体内的归巢，从而为外源性Netrin促进治疗模型中的血管重建提供潜在的机制。与公共卫生有关。血管系统的形成对正常发育至关重要，在对损伤或疾病的反应中，血管重塑是至关重要的。这些研究将对Netrin信号在血管发育和内皮细胞生物学中的作用以及其治疗潜力的机制提供基本的见解。该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相关性。

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.