A Vascularized Micro-Organ platform for the study of Brain-BBB-Blood interaction

项目总结 长期以来，对血脑屏障(BBB)的研究忽视了这一界面的血液成分， 几乎只关注神经血管单位(NVU)的细胞。我们所要达到的FOA的目标 回应旨在改变这一点： FOA的目的是通过重新定义血液为基础的科学的一个新领域的发展 作为血-脑界面组成部分的神经血管单位。这将促进人的发展-- 基于神经血管-血液模型确定诊断和调节血脑的靶点 接口…“ NVU由内皮细胞、周细胞和星形胶质细胞以及复杂的基底膜组成， 它们共同作用，严重限制了分子从血液进入脑实质的自由运动。 在发育过程中，BBB EC对本地信号作出响应，形成紧密连接，并具有非常低的 细胞穿透。这样做的副作用是潜在的治疗药物进入大脑的途径也 妥协了。在这项提案中，我们将建立在我们成熟的人类血管微器官(VMO)的基础上 平台创造了一种新型的血脑接口模型VMO-B。在这个模型中，人的网络 微血管与代表动脉和静脉的微流体通道吻合，并被诱导为血脑屏障 Wnt信号的表型。血管由周细胞包被，并由星形胶质细胞足突接触。 重要的是，我们将通过血管运行一种血液替代品-VMO血液-它将模拟 血液，包括蛋白质和脂肪含量。然后我们将使用VMO-B来研究BBB的过程 亨廷顿氏病发病机制的解体。我们已经有初步数据表明 突变型HTT蛋白在EC中的表达可导致血脑屏障缺陷。我们将调查血液和血液之间的串音 NVU的细胞，以及mHTT在每种细胞类型中的表达如何影响细胞间的通讯和屏障 功能。在R61阶段，我们将追求三个目标：目标1建立一个稳定的MPS血脑屏障模型 微血管学；目标2将血液流动纳入BBB微流体模型；以及，目标3表征关键 血脑交界处的转运体。在R33阶段，我们将使用此平台来检查 HD病理中的血-脑界面通过另外两个目标：Aim 4检验假设 在EC中mHTT的表达破坏了跨血脑屏障的运输，导致神经微环境的改变； 和，目标5测试假设，即mHTT的表达破坏了血液中多种细胞之间的相互作用- 大脑接口。该项目的完成不仅将揭示亨廷顿氏病的神经病理学 疾病，但也将产生一个理想的平台，适合药物开发和研究血液的作用- 阿尔茨海默病、多发性硬化症、帕金森氏症等多种神经系统疾病的脑界面 疾病、中风、CADASIL和创伤性脑损伤。

PROJECT SUMMARY For too long, studies of the Blood-Brain Barrier (BBB) have ignored the blood component of this interface, focusing almost exclusively on the cells of the Neurovascular Unit (NVU). The goal of the FOA to which we are responding aims to change this: “The intent of this FOA is to stimulate the development of a new field of blood-based science by re-defining the neurovascular unit as a component of the blood-brain interface. This will facilitate development of human- based neurovascular-blood models to identify targets for diagnostics and regulation of the blood-brain interface…” The NVU is comprised of endothelial cells (EC), pericytes and astrocytes, and a complex basement membrane, which work together to severely limit the free movement of molecules from the blood into the brain parenchyma. In response to local signals during development BBB EC develop tight junctions and have very low rates of transcytosis. The side-effect of this is that access of potentially therapeutic drugs into the brain is also compromised. In this proposal we will build on our well-established human Vascularized Micro-Organ (VMO) platform to create a novel blood-brain interface model, the VMO-B. In this model a network of human microvessels anastomoses to microfluidic channels representing an artery and a vein and are induced to a BBB phenotype by Wnt signaling. The vessels are invested by pericytes and contacted by astrocyte foot-processes. Importantly, we will run a blood substitute – VMOBlood – through the vessels that will mimic the composition of blood, including protein and lipid content. We will then use the VMO-B to investigate the process of BBB breakdown in the pathogenesis of Huntington’s disease. We already have preliminary data suggesting that expression of mutant HTT protein in EC causes BBB deficits. We will investigate crosstalk between blood and the cells of the NVU, and how expression of mHTT in each cell type affects cell-cell communication and barrier function. In the R61 phase we will pursue three aims: Aim 1 Develop a stable MPS BBB model with perfused microvasculature; Aim 2 Incorporate flow of blood into BBB microfluidic model; and, Aim 3 Characterize key transporters at the blood-brain interface. In the R33 phase we will use this platform to examine the role of the blood-brain interface in the pathology of HD through an additional two aims: Aim 4 Test the hypothesis that expression of mHTT in EC disrupts transport across the BBB leading to changes in the neural micro-environment; and, Aim 5 Test the hypothesis that expression of mHTT disrupts multiple cell-to-cell interactions at the blood- brain interface. Completion of this project will not only shed light on the neuropathology of Huntington’s disease, but will also yield a platform ideally suited to drug development and investigating the role of the blood- brain interface in numerous neurological diseases including Alzheimer’s disease, Multiple Sclerosis, Parkinson’s disease, stroke, CADASIL, and traumatic brain injury.