GENETIC ANALYSIS OF EPHRIN-EPH SIGNALING IN ANGIOGENESIS

描述（来自申请的逐字）：跨膜配体ephrinB 2 在两种组织中均由动脉内皮细胞特异性表达，而不是静脉内皮细胞。 胚胎和成人，而它的受体EphB 4相反地由静脉表达 而不是动脉。配体和受体都是胚胎发育所必需的 心血管发育本提案的目的是了解 更确切地说，ephrinB 2-EphB 4信号传导在血管生成中的功能。使用 敲除和转基因中的条件性功能丧失和获得操作 在小鼠中，我们将测试假设，即双向信号之间 由这种配体-受体对介导的动脉和静脉对于 血管生成，并询问这种信号传导的细胞功能是否 主要是吸引人的或令人厌恶的。在《特定目标I》中， ephrinB 2特异性地存在于内皮细胞和内皮细胞中，以确定 它的基本功能是否确实在循环系统中发挥作用。 这些研究将得到实验的补充， ephrinB 2敲除表型在循环系统内。时间上 还将进行ephrinB 2的受控的泛内皮敲除， 确定该基因是否也需要在血管生成的后期阶段。在 具体目标II，我们将使用两种血管的体外胚状体试验 形成和体内胚胎嵌合体， EphrinB 2或EphB 4的纯合ES细胞突变体，以区分 这些基因在早期心血管发育中的主要需求是 心脏、外周脉管系统或两者。在第三阶段，我们将 进行ephrinB 2的组成性和/或条件性泛内皮表达 和EphB 4转基因，以确定动脉和静脉特异性 这些基因的表达对于它们的正常功能是必需的 在循环系统中。在具体目标IV中，我们将创建 可用于同时区分动脉的“双指标”小鼠 和静脉使用遗传编码的光化学标记。这些老鼠将 用于体内和体外实验，以进一步研究 动脉和静脉之间的细胞间相互作用以及 ephrinB 2-EphB 4信号转导介导这些相互作用。机制研究 ephrinB 2-EphB 4信号传导在发育中的作用与 为我们了解其在成人血管生成中的功能提供了信息， 抑制或促进血管生成的新治疗策略 临床环境，如癌症和心脏病，通过药理学 操纵这些动脉和静脉特异性信号分子。

DESCRIPTION (Verbatim from the application): The transmembrane ligand ephrinB2 is specifically expressed by arterial but not venous endothelial cells in both embryos and adults, whereas its receptor EphB4 is conversely expressed by veins but not arteries. Both the ligand and receptor are essential for embryonic cardiovascular development. The objective of this proposal is to understand more precisely the function of ephrinB2-EphB4 signaling in angiogenesis. Using conditional loss-and gain-of-function manipulations in knockout and transgenic mice, we will test the hypothesis that bi-directional signaling between arteries and veins mediated by this ligand-receptor pair is essential for angiogenesis, and ask whether the cellular function of this signaling is primarily attractive or repulsive. In Specific Aim I, we will knock out ephrinB2 specifically within endothelial and endocardial cells, to determine whether its essential function is indeed exerted within the circulatory system. These studies will be complemented by experiments to selectively rescue the ephrinB2 knockout phenotype within the circulatory system. Temporally controlled, pan-endothelial knockout of ephrinB2 will also be performed, to determine whether the gene is also required at later stages of angiogenesis. In Specific Aim II, we will use both in vitro embryoid body assays of blood vessel formation and in vivo embryo chimeras in conjunction with heterozygous and homozygous ES cells mutant for ephrinB2 or EphB4 to distinguish whether the primary requirement for these genes in early cardiovascular development is in the heart, the peripheral vasculature, or both. In Specific Aim III we will perform constitutive and/or conditional pan-endothelial expression of ephrinB2 and EphB4 transgenes to determine whether the arterial- and venous-specific expression, respectively, of these genes is essential for their proper function in the circulatory system. In Specific Aim IV we will create lines of "dual-indicator" mice that can be used to simultaneously distinguish arteries and veins using genetically encoded photochemical markers. These mice will be used in both in vivo and in vitro experiments to further study the role of cell-cell interactions between arteries and veins and the role of ephrinB2-EphB4 signaling in mediating these interactions. Mechanistic studies of the role of ephrinB2-EphB4 signaling in development are highly like to inform our understanding of its function in adult angiogenesis, and may suggest new therapeutic strategies for the inhibition or promotion of angiogenesis in clinical settings such as cancer and heart disease, via pharmacological manipulation of these artery- and vein-specific signaling molecules.