Regulation of blood-brain barrier formation and function by EHDs

 描述（由申请人提供）：血脑屏障（BBB）是用于描述中枢神经系统（CNS）血管化血管的独特性质的术语。这一屏障对于维持脑内稳态（允许适当的神经元功能）和保护CNS免受损伤和疾病都至关重要。中风、多发性硬化症、水肿和脑创伤的许多症状是由于伴随原发性损伤的血脑屏障的破坏。此外，BBB为所有神经系统疾病的治疗提供了障碍，因为它极大地阻碍了药物递送至CNS。因此，了解调节BBB功能的机制可以鉴定调节BBB的新靶点，以修复BBB并开发绕过BBB用于药物递送的方法。血脑屏障的许多特性表现在构成血管壁的内皮细胞内，这使得它们能够限制血液和大脑之间的分子，离子和细胞的运动。一个重要的屏障特性是CNS内皮细胞以极低的速率进行胞吞作用，从而限制了跨细胞渗透性。虽然这些是内皮细胞的特性，但研究表明它们是由与CNS微环境的相互作用诱导的。特别是，我们已经确定，中枢神经系统周细胞是重要的，以诱导血脑屏障的性质，在内皮细胞在发展过程中，他们这样做，通过抑制转胞吞通过内皮细胞。在初步的实验中，我们已经确定，特定的EHD家族成员是丰富的外周'漏'血管，如肝和肺，经历了大量的转胞吞，相比之下，中枢神经系统血管，经历非常低的量的转胞吞。我们已经进一步证明，周细胞的遗传丢失、中风和神经炎症都导致CNS血管中EHD基因的上调，这与BBB渗漏相关。我们已经使用遗传小鼠模型来证明EHD 4的过表达导致体内BBB的渗漏，并且外周内皮细胞中的EHD 1和EHD 4的条件性敲除导致较少的外周血管渗漏。因此，我们的目的是测试的假设：周细胞通过抑制CNS内皮细胞中的EHD的表达来调节BBB，并且EHD的上调是神经系统疾病期间BBB功能障碍的重要组成部分。我们已经开发了在内皮细胞中可逆过表达EHD-GFP融合蛋白的转基因小鼠模型，以及从内皮细胞中删除EHD的条件性敲除模型。使用这些模型，我们将解决以下问题：如何上调EHDs导致BBB功能障碍？疾病期间BBB功能障碍是否需要EHD？什么导致EHD在BBB上调？阐明这些问题将使我们能够确定EHD在调节BBB中的作用，并确定它们是否是调节BBB以治疗神经系统疾病和帮助药物递送至CNS的靶点。

 DESCRIPTION (provided by applicant): The blood-brain barrier (BBB) is a term used to describe the unique properties of the blood vessels that vascularize the central nervous system (CNS). This barrier is crucial both to maintain brain homeostasis, which allows for proper neuronal function, and to protect the CNS from injury and disease. Many of the symptoms of stroke, multiple sclerosis, edema, and brain traumas are due to a breakdown of the BBB that accompanies the primary insult. Furthermore, the BBB provides an obstacle for the treatment of all neurological diseases as it greatly impedes drug delivery to the CNS. Therefore, understanding the mechanisms that regulate BBB function may identify novel targets to modulate the BBB, both to repair the BBB and to develop methods to bypass the BBB for drug delivery. Many of the properties of the BBB are manifested within the endothelial cells that make up the walls of the blood vessels, which allows them to restrict the movement of molecules, ions and cells between the blood and the brain. One important barrier property is that the CNS endothelial cells undergo transcytosis at extremely low rates, thus limiting the transcellular permeability. Although these are properties of the endothelial cells, studies have revealed that they are induced by interactions with the CNS microenvironment. In particular we have identified that CNS pericytes are important to induce BBB properties in endothelial cells during development, and they do so by inhibiting transcytosis through the endothelial cells. In preliminary experiments, we have identified that specific EHD family members are enriched in peripheral `leaky' vessels such as the liver and lung which undergo lots of transcytosis, compared to CNS vessels which undergo very low amounts of transcytosis. We have further demonstrated that genetic loss of pericytes, stroke and neuroinflammation all lead to up-regulation of EHD genes in CNS blood vessels that correlates with BBB leakage. We have used genetic mouse models to demonstrate that over-expression of EHD4 leads to leakage of the BBB in vivo, and conditional knockout of EHD1 and EHD4 in peripheral endothelial cells leads to less peripheral vascular leakage. Therefore we aim to test the hypothesis that: Pericytes regulate the BBB by inhibiting the expression of EHDs in CNS endothelial cells, and that up-regulation of EHDs is an important component of BBB dysfunction during neurological disease. We have developed transgenic mouse models to reversibly over-express EHD- GFP fusion proteins in endothelial cells, as well as conditional knockout models to delete EHDs from endothelial cells. Using these models we will address the following questions: How does up-regulation of EHDs lead to BBB dysfunction? Are EHDs required for BBB dysfunction during diseases? What leads to EHD upregulation at the BBB? Answering these questions will enable us to determine the role of EHDs in regulating the BBB and determine if they are a target to modulate the BBB to treat neurological diseases and aid drug delivery to the CNS.