GENETIC ANALYSIS OF EPHRIN-EPH SIGNALING IN ANGIOGENESIS

描述(逐字摘自申请书)：跨膜配体ewitinB2 都是由动脉内皮细胞特异性表达，而不是静脉内皮细胞表达 胚胎和成人，而其受体EphB4则相反地通过静脉表达 但不是动脉。配体和受体都是胚胎发育所必需的。 心血管发育。这项建议的目标是理解 更准确地说，ewitinB2-EphB4信号在血管生成中的作用。vbl.使用 基因敲除和转基因中的条件性功能丧失和功能获得操纵 小鼠，我们将测试假设之间的双向信号 由这种配体-受体对介导的动脉和静脉是 血管生成，并询问该信号的细胞功能是否 主要是吸引人的或令人厌恶的。在特定的目标一，我们将击倒 血管内皮细胞和心内膜细胞特异性表达的ewitinB2 它的基本功能是否确实在循环系统内发挥作用。 这些研究将得到实验的补充，有选择地拯救 循环系统中的ewitinB2基因敲除表型。暂时地 还将进行受控的、泛内皮细胞基因敲除ewitinB2，以 确定在血管生成的后期阶段是否也需要该基因。在……里面 具体目标二，我们将使用两种体外血管胚体分析方法 杂合子与体内胚胎嵌合体的形成和 EphB4基因突变的纯合子ES细胞能否区分 这些基因在早期心血管发育中的主要需求是 心脏，外周血管，或两者兼而有之。在第三个具体目标中，我们将 进行结构性和/或条件性泛内皮细胞表达ePhrinB2 和EphB4转基因来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不可少的。 在循环系统中。在特定的目标四中，我们将创建 可同时区分动脉的“双指示器”小鼠 和静脉使用基因编码的光化学标记。这些老鼠将会是 在体内和体外实验中使用，以进一步研究 动脉和静脉之间的细胞-细胞相互作用及血管内皮生长因子的作用 EphB4信号转导信号调节这些相互作用。机械学研究 EphB4信号在发育中的作用 使我们了解它在成人血管生成中的作用，并可能提示 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的信号分子。

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.