The role of VWF and platelets in BBB alterations associated with hypoxia

描述（由申请人提供）：缺氧后再氧合通常被用作研究缺血/再灌注相关病理的模型，因为后者存在于包括中风在内的几种疾病状态中。在动物模型中，缺氧后再氧合已被证明会导致紧密连接蛋白异常、血脑屏障细胞旁通透性增加和水肿，但导致这些病理的完整机制尚不清楚。血管性血变因子（VWF）是一种仅由内皮细胞和巨核细胞合成的糖蛋白。虽然已知VWF在脑内皮细胞（CECs）中大量表达，但对于VWF在内皮生物学中的作用，特别是在应激条件下对血脑屏障的调节中所知甚少。我们所获得的数据表明，在广义缺氧/再氧化模型中，vwf缺陷小鼠的血脑屏障通透性明显低于野生型。VWF是血小板在内皮活化过程中的主要配体，我们收集的初步数据支持血小板在缺氧暴露生存中的作用。血小板在止血和炎症中的作用最为人所知，血小板与内皮和暴露的内皮下层结合的主要配体是VWF。血小板可以激活内皮细胞，但也可以被发炎的内皮细胞激活。有数据支持暴露于缺氧后血小板数量和功能的改变。然而，研究得出了相互矛盾的结果，这给该领域的进步留下了很大的空间。有趣的是，在人脑内皮细胞培养中，血小板作为细胞旁通透性增加的增强剂的作用已被记录，最近的文献表明血小板在涉及血脑屏障的各种中枢神经系统疾病中发挥作用。VWF和血小板在血脑屏障生物学中的作用尚未得到充分研究，因此我们的总体目标是确定VWF和血小板在应激条件下促进血脑屏障通透性的程度。为了做到这一点，我们将使用一个广义常压缺氧然后再氧合的模型。在我们的第一个目标中，我们建议研究血浆vs.内皮下VWF在与广泛性缺氧/再氧化相关的血脑屏障通透性增加中的作用。我们还建议确定与野生型相比，vwf缺陷小鼠在缺氧/再氧化后保持血脑屏障完整性的程度，从而避免认知能力下降。其次，我们将确定血小板与VWF和内皮相互作用导致血脑屏障通透性增加的程度。我们建议使用敲除小鼠技术、血脑屏障渗透性测定、行为神经科学测试、共聚焦显微镜、血小板耗尽研究和紧密连接蛋白测定来实现这些目标。本提案中概述的研究旨在进一步了解脑微血管表达的糖蛋白如何与造血谱系的细胞类型相互作用以影响缺氧时的血脑屏障，因此与NINDS的任务直接相关，特别是神经环境分裂。

DESCRIPTION (provided by applicant): Hypoxia followed by reoxygenation is commonly used as a model to investigate pathology associated with ischemia/reperfusion as the latter condition is present in several disease states including stroke. In animal models, hypoxia followed by reoxygenation has been shown to cause tight junction protein abnormalities, increased BBB paracellular permeability and edema however, a complete mechanism leading to these pathologies is still undefined. von Willebrand Factor (VWF) is a glycoprotein that is only synthesized by endothelial cells and megakaryocytes. While it is known that VWF is expressed abundantly by cerebral endothelial cells (CECs), very little is known about the role of VWF in endothelial biology, particularly, in regulation of the BBB under stressful conditions. Data we have generated demonstrate that VWF-deficient mice have significantly less BBB permeability than wild type in a model of generalized hypoxia/reoxygenation. VWF is a major ligand for platelets during endothelial activation and preliminary data we have gathered support a role for platelets in survival of exposure to hypoxia. Platelets are best known for their role in hemostasis and inflammation and the primary ligand for platelet binding to the endothelium and exposed subendothelium is VWF. Platelets can activate endothelium but are also activated by inflamed endothelium. Data supporting alteration of platelet number and function after exposure to hypoxia have been documented. However, studies have yielded conflicting results leaving much room for advancements in this field. Intriguingly, a role for platelets as potentiators of increased paracellular permeability in human brain endothelial cell cultures has been documented and recent literature suggests a role for platelets in various CNS diseases involving the BBB. The role of VWF and platelets in BBB biology is understudied and warrants further investigation, thus our overall objective is to determine the extent VWF and platelets promote BBB permeability under stressful conditions. To accomplish this, we will use a model of generalized normobaric hypoxia followed by reoxygenation. In our first aim, we propose to examine the role of plasma vs. subendothelial VWF in increased BBB permeability associated with generalized hypoxia/reoxygenation. We also propose to determine the extent that VWF-deficient mice are saved from cognitive decline as they have preserved BBB integrity after hypoxia/reoxygenation when compared to wild-type. Secondly, we will determine the extent that platelet interactions with VWF and the endothelium lead to increased BBB permeability. We propose to accomplish these aims using knock out mouse technology, BBB permeability assays, behavioral neuroscience tests, confocal microscopy, platelet-depletion studies and tight junction protein assays. The studies outlined in this proposal aim to further our understanding of how a glycoprotein expressed by brain microvasculature interacts with a cell type of hematopoietic lineage to influence the BBB during hypoxia and thus are directly relevant to the mission of NINDS, specifically the neural environment division. PUBLIC HEALTH RELEVANCE: Aberrant blood brain barrier (BBB) permeability is a common pathology in many diseases of the central nervous system (CNS). The studies outlined in this proposal are original in that we propose to investigate the role of two non-CNS components in BBB alterations during hypoxia. These studies will further our knowledge of cerebral endothelial cell biology while potentially finding new therapeutic targets for neurological disorders involving the BBB.