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

• 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
• 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

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.

缺血性中风急性靶向脑微血管。近端MCA闭塞后数小时内， 微血管内皮通透性屏障打开，引起纹状体中的“血管源性”水肿。这 该过程尚未完全了解，目前也没有治疗方法。脑微血管结构与信号转导 似乎是这些急剧变化的核心。干扰内皮细胞β1- 在6小时内，通过14 T MRI，整合素与底层基质的结合急剧增加T2，但不增加ADC， 增加体外渗透性，改变紧密连接（TJ）表达，并改变F-肌动蛋白构象， 内皮细胞活力丧失。然而，肌球蛋白轻链（MLC）磷酸化的抑制，在6小时内， 在体外，当建立的β1-整联蛋白-基质粘附被破坏时，可防止渗透性增加。因此，我们认为， 急性调节内皮β1-整合素信号可以保护屏障并减轻纹状体水肿。 本提案中待检验的假设表明：i）微血管内皮细胞与其 基底层基质蛋白通过β1-整合素粘附受体是一种主要的结构和信号转导途径， 血脑屏障行为的决定因素，ii）局灶性缺血破坏稳定的β1-整联蛋白-基质相互作用，iii） 这些相互作用的破坏增加了微血管的渗透性，和iv）这种急剧增加的渗透性 并且可以通过抑制β1-整联蛋白信号传导来预防水肿。三个具体目标将表明 结论：1）MLC和相关β1-整合素信号的快速调节可以防止急性通透性 干扰β1-整合素-基质粘附引起的增加，2）实验性缺血破坏 内皮细胞β1-整合素-基质粘附，诱导内皮细胞信号传导，增加通透性， 3）通过组织损伤，局灶性缺血减少β1-整合素表达或基质 粘附，并增加内皮渗透性，这是可以预防的。内皮细胞的核心作用 β1-整合素-基质粘附作为局灶性缺血急性“血管源性”水肿的决定因素， 新颖且可测试。使用来自野生型和条件性的高质量原代脑内皮细胞培养物 内皮β1-整联蛋白敲除构建体，以定义β1-整联蛋白信号传导事件（特异性目的1），体外 模型来量化缺血对内皮细胞β1-整联蛋白信号传导的影响（特异性目标2），以及真实的- 在小鼠立体定位注射和MCA闭塞模型中使用 实时14 T 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），本项目将证明β1-整合素-基质粘附是水肿的关键 预防这些非常可行的研究是中风研究的一个新方向，将大大促进我们的研究。 了解局灶性缺血下急性血脑屏障的行为。了解β1-整合素信号传导 机制也有可能确定新的特定目标，以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，目的是改善结果。