CNS angiogenesis and blood-brain barrier regulation by the Wnt inhibitor Apcdd1.

描述(由申请人提供)：血管生成与内皮细胞在不同器官中排列血管的特定属性的获取的协调对于它们的正常功能是必不可少的。这一原理的一个重要例子是在脑内皮细胞中发现的，即血脑屏障(BBB)，它通过在内皮细胞之间形成高阻力的紧密连接来限制分子在细胞旁扩散进入大脑。尽管它对中枢神经系统(CNS)的功能很重要，但调节血管生成和这一屏障的发展的机制仍不清楚。我们先前已经证实，在中枢神经系统血管生成和屏障形成过程中，Wnt/β-catenin信号在脑内活跃，而在肝内皮细胞中不活跃。此外，这一途径对中枢神经系统血管生成和内皮细胞获得某些屏障特性是必不可少的。此外，Wnt/β-catenin信号的下游效应分子Apcdd1在血管生成后的中枢神经系统内皮细胞中高表达，此时内皮细胞具有BBB特性。Apcdd1选择性表达于中枢神经系统，但不表达于外周内皮细胞。Apccd1在出生后20天(P20)一直存在于中枢神经系统内皮细胞中，但在成年中枢神经系统血管中，当血管生成完成和血脑屏障完全形成时，Apccd1就消失了。该蛋白定位于细胞膜和分泌途径中，当在接受Wnt信号的细胞中过表达时，它以细胞自主的方式抑制Wnt/？-catenin信号的激活。我们认为Apcdd1抑制中枢神经系统内皮细胞的Wnt/？-catenin信号转导，从而使细胞成熟并获得BBB特性。在这项研究中，我们将研究Apcdd1在中枢神经系统血管生成和血脑屏障形成中的作用。我们将首先检查Apcdd1是否与Wnt配体(例如Wnt7a/7b)相互作用，从而在内皮细胞中诱导屏障属性。然后，我们将测试Apcdd1是否在体外诱导内皮细胞血管生成和屏障特性的各个方面是必要和充分的。我们已经用基因打靶的方法产生了Apcdd1基因敲除小鼠，并以可诱导的方式在中枢神经系统内皮细胞中过表达Apcdd1。这些小鼠将使我们能够在体内测试Apcdd1对中枢血管生成和血脑屏障形成的需求和充分性。了解中枢神经系统血管生成和血脑屏障的发展，将有助于阐明中枢神经系统内皮细胞紧密连接形成的机制，以及与中枢神经系统血管生成异常和血脑屏障破坏相关的病理性中枢神经系统疾病的病因，并有助于开发新的治疗方法来调节这些过程。

DESCRIPTION (provided by applicant): The coordination of angiogenesis with the acquisition of specific properties of endothelial cells that line the blood vessels in distinct organs is essenial for their proper function. An important example of this principle is found in the brain endothelium, namely the blood-brain barrier (BBB), that restricts paracellular diffusion of molecules into the brain by forming high resistance tight junctions between endothelial cells. Despite its importance for the central nervous system (CNS) function, the mechanisms that regulate angiogenesis and development of this barrier remain poorly characterized. We have previously identified that Wnt/¿-catenin signaling is active in brain but not liver endothelium during CNS angiogenesis and barrier formation. Moreover, this pathway is essential for CNS angiogenesis and acquisition of some barrier properties by endothelial cells. In addition, Apcdd1, a downstream effector of Wnt/¿-catenin signaling, is highly expressed in CNS endothelial cells after angiogenesis when endothelial cells acquire BBB properties. Apcdd1 is selectively expressed in CNS, but not, peripheral endothelial cells. Apccd1 is present in CNS endothelium until postnatal day 20 (P20), but it is extinguished in the adult CNS blood vessels when angiogenesis is complete and BBB is fully formed. The protein is localized within the plasma membrane and the secretory pathway and it inhibits the activation of Wnt/¿-catenin signaling in a cell-autonomous manner when overexpressed in cells that receive Wnt signaling. We propose that Apcdd1 inhibits Wnt/¿-catenin signaling in CNS endothelium to allow cells to mature and acquire BBB properties. In this proposal, we will investigate the role of Apcdd1 in CNS angiogenesis and BBB formation. We will first examine if Apcdd1 interacts with Wnt ligands (e.g. Wnt7a/7b) that induce barrier properties in endothelial cells. Then we will test if Apcdd1 is necessary and sufficient to induce various aspects of angiogenesis and barrier properties in endothelial cells in vitro. We have generated Apcdd1 knockout mice using gene targeting methods and mice that overexpress Apcdd1 in CNS endothelial cells in an inducible manner. These mice will allow us to test the requirement and sufficiency of Apcdd1 for CNS angiogenesis and BBB formation in vivo. Understanding the development of CNS angiogenesis and BBB will shed light on the mechanisms of tight junction formation within CNS endothelial cells, the etiology of pathological CNS conditions associated with abnormal CNS angiogenesis and BBB breakdown, and help to develop novel therapeutic approaches to regulate these processes.