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：
10118345
负责人：
Gregory J Del Zoppo
金额：
$ 54.77万
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/10118345
关键词：
Acute Adherens Junction Adhesions Adhesives Affect Astrocytes Basal lamina Behavior Biochemical Blood Blood - brain barrier anatomy Blood Vessels Brain Brain Edema Cell Culture Techniques Cell Line Cell Survival Cells 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.

缺血性中风敏锐地靶向大脑微举行。近端MCA闭塞后的几个小时内，微血管内皮通透性屏障在纹状体中引起“血管生成”水肿的打开。这过程尚未完全理解，目前尚无治疗。脑微血管结构和信号传导这些急性变化似乎是核心。干扰内皮细胞β1-建立的依从性在基础矩阵上的整合素急性地增加T2，但在6小时内不增加14T MRI，并且急性增加体外渗透性，改变紧密连接（TJ）的表达，并改变F-肌动蛋白构象，而无需内皮细胞活力的丧失。但是，在6小时内抑制肌球蛋白轻链（MLC）磷酸化在体外，当建立的β1-整合素 - 蛋白胶粘剂被破坏时，可以防止渗透率增加。因此，调节内皮β1-整合蛋白信号传导可急性保留屏障并降低纹状体水肿。在此提案中要测试的假设，即i）微血管内皮细胞与它们的相互作用通过β1-整合蛋白粘附受体碱性层层基质蛋白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 - 马trix相互作用，iii）这些相互作用的破坏增加了微血管的渗透性，iv）这种急性渗透性急剧提高可以通过抑制β1-整合蛋白信号传导来预防水肿。三个特定目标将证明 1）MLC和相关的β1-整合蛋白信号的快速调节可以防止急性渗透率由干扰β1-整合蛋白 - 玛trix粘合剂引起的增加，2）实验缺血破坏内皮细胞β1-整合蛋白 - 雄性粘合剂，诱导内皮信号，增加渗透性，这可以预防，3）局灶性缺血，通过组织损伤下降β1-整合蛋白表达或基质粘附并增加内皮渗透性，可以预防。内皮细胞的中心作用 β1-整合蛋白 - 玛蛋白粘合剂是局灶性缺血中急性“加血管”水肿的决定因素是概念上的新颖且可测试。具有高质量的原发性大脑内皮细胞培养物，来自野生型和条件内皮β1-整合蛋白基因敲除构建体以定义β1-整合蛋白信号事件（特定AIM 1），体外量化缺血对内皮细胞β1-整合蛋白信号传导（特定AIM 2）的影响的模型使用鼠立体定位注射和MCA遮挡模型中急性水肿形成的时间评估实时14T MRI指导渗透率的急性评估及其预防信号抑制剂（具体目的1和3），该项目将证明β1-整合素 - 玛trix粘合剂与水肿至关重要预防。这些非常可行的研究是中风研究的新方向，将大大进一步了解β1-整合蛋白信号传导机制还可能识别新的特定目标以保存或选择性更改内皮屏障在其他神经血管疾病中发挥作用，目的是改善预后。