Endothelial Beta 1-integrins in Cerebral Vascular Barrier Integrity

内皮β1-整合素在脑血管屏障完整性中的作用

基本信息

批准号：
10269018
负责人：
Gregory J Del Zoppo
金额：
$ 53.63万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10269018
关键词：
Acute Adherens Junction Adhesions Adhesives Affect Astrocytes Basal lamina Behavior Biochemical Blood Blood - brain barrier anatomy Blood Vessels Brain Edema Cell Culture Techniques Cell Line Cell Survival Cells Cerebrovascular system Chronic Complex Corpus striatum structure Disease Edema Endothelial Cells Endothelium Event Extravasation F-Actin Feasibility Studies Generations Glues Goals Hour Image Immunoglobulin G In Vitro Injections Injury Integrin Signaling Pathway Integrins Interruption Intervention Ischemia Ischemic Brain Injury Ischemic Stroke Knock-out Liquid substance Magnetic Resonance Imaging Middle Cerebral Artery Occlusion Modeling Molecular Conformation Mus Myosin Light Chain Kinase Myosin Light Chains Outcome PECAM1 gene Pathway interactions Permeability Phosphorylation Prevention Primary Cell Cultures Process Proteins Proteolysis Research Research Project Grants Rho-associated kinase Role Signal Pathway Signal Transduction Stroke Structure Testing Tight Junctions Time Tissues acute stroke adhesion receptor brain endothelial cell cadherin 5 cerebral microvasculature cerebrovascular clinically relevant experimental study improved outcome in vitro Model in vivo in vivo Model inhibitor/antagonist mortality neurovascular nonhuman primate novel preservation prevent response scaffold tissue injury treatment strategy vasogenic edema

项目摘要

Ischemic stroke acutely targets the cerebral microvasculature. Within hours following proximal MCA occlusion, the microvessel endothelial permeability barrier opens causing “vasogenic” edema in the corpus striatum. This process is not fully understood and has no treatment currently. Cerebral microvessel structure and signaling appears central to these acute changes. Interfering with the established adhesion of endothelial cell β1- integrins to the underlying matrix acutely increases T2 but not ADC by 14T MRI within 6 hours, and acutely increases permeability in vitro, alters tight junction (TJ) expression, and alters F-actin conformation, without loss of endothelial cell viability. However, inhibition of myosin light chain (MLC) phosphorylation, within 6 hours in vitro, prevents the permeability increase when established β1-integrin–matrix adhesion is disrupted. Hence, modulating endothelial β1-integrin signaling acutely could preserve the barrier and decrease striatal edema. The hypotheses to be tested in this proposal state that i) interactions of microvessel endothelial cells with their basal lamina matrix proteins through β1-integrin adhesion receptors are a major structural and signaling determinant of blood-brain barrier behavior, ii) focal ischemia disrupts stable β1-integrin–matrix interactions, iii) disruption of these interactions increases microvessel permeability, and iv) this acutely increased permeability and edema can be prevented by inhibiting β1-integrin signaling. The three Specific Aims will demonstrate that: 1) rapid modulation of the MLC and associated β1-integrin signaling can prevent the acute permeability increase caused by interference with β1-integrin–matrix adhesion, 2) experimental ischemia disrupts endothelial cell β1-integrin–matrix adhesion, induces endothelial signaling that increases permeability, and this can be prevented, and 3) focal ischemia, through tissue injury, decreases β1-integrin expression or matrix adhesion, and increases endothelial permeability, which can be prevented. A central role for endothelial cell β1-integrin–matrix adhesion as the determinant of acute “vasogenic” edema in focal ischemia is conceptually novel and testable. With high-quality primary cerebral endothelial cell cultures from wild type and conditional endothelial β1-integrin knockout constructs to define β1-integrin signaling events (Specific Aim 1), in vitro models to quantify the effects of ischemia on endothelial cell β1-integrin signaling (Specific Aim 2), and real- time assessment of acute edema formation in murine stereotaxic injection and MCA occlusion models using real-time 14T MRI to guide acute assessments of permeability and its prevention with signaling inhibitors (Specific Aims 1 and 3), this Project will demonstrate that β1-integrin–matrix adhesion is pivotal to edema prevention. These very feasible studies are a new direction in stroke research that will substantially further our understanding of acute blood-brain barrier behavior under focal ischemia. Understanding β1-integrin signaling mechanisms is also likely to identify novel specific targets to preserve or selectively change endothelial barrier function in other neurovascular disorders, with the aim to improve outcome.

缺血性中风以大脑微血管为目标，在近端 MCA 闭塞后数小时内，微血管内皮通透性屏障打开，导致纹状体出现“血管源性”水肿。目前尚未完全了解该过程，也没有治疗方法。似乎是这些急性变化的核心，干扰内皮细胞 β1- 已建立的粘附。通过 14T MRI，整合素与底层基质在 6 小时内急剧增加 T2，但不增加 ADC，并且急剧增加增加体外通透性，改变紧密连接 (TJ) 表达，并改变 F-肌动蛋白构象，而无需内皮细胞活力丧失，但在 6 小时内抑制肌球蛋白轻链 (MLC) 磷酸化。在体外，当已建立的 β1-整合素-基质粘附被破坏时，可防止通透性增加。剧烈调节内皮β1-整合素信号传导可以保护屏障并减少纹状体水肿。本提案中要测试的假设指出，i) 微血管内皮细胞与其自身的相互作用层基质蛋白通过β1-整合素粘附基底受体是主要的结构和信号传导血脑屏障行为的决定因素，ii) 局灶性缺血破坏稳定的 β1-整合素-基质相互作用，iii) 这些相互作用的破坏会增加微血管的通透性，并且 iv) 这种急剧增加的通透性抑制 β1-整合素信号传导可以预防水肿，这三个具体目标将得到证明。认为：1) 快速调节 MLC 和相关的 β1-整合素信号传导可以预防急性通透性由于干扰 β1-整合素-基质粘附而引起的增加，2) 实验性缺血破坏内皮细胞β1-整合素-基质粘附，诱导内皮信号传导，增加通透性，这可以预防，并且 3) 通过组织损伤，局灶性缺血会降低 β1-整合素表达或基质粘附，并增加内皮细胞的通透性，这是可以预防的。 β1-整合素-基质粘附作为局部缺血中急性“血管源性”水肿的决定因素在概念上是新颖且可测试的野生型和条件型原代脑内皮细胞培养物。内皮 β1-整合素敲除构建体用于定义 β1-整合素信号传导事件（具体目标 1），体外量化缺血对内皮细胞 β1-整合素信号传导影响的模型（具体目标 2），以及真实的小鼠立体定位注射和 MCA 闭塞模型中急性水肿形成的时间评估实时 14T MRI 指导渗透性的急性评估及其信号抑制剂的预防（具体目标 1 和 3），该项目将证明 β1-整合素-基质粘附对于水肿至关重要这些非常可行的研究是中风研究的新方向，将大大推进我们的研究。了解局灶性缺血下的急性血脑屏障行为。机制还可能确定新的特定靶点来保护或选择性改变内皮屏障在其他神经血管疾病中发挥作用，旨在改善结果。