Regulation of CNS leukocyte extravasation

CNS白细胞外渗的调节

基本信息

批准号：
10163922
负责人：
Joel S Pachter
金额：
$ 36.72万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-06-15 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10163922
关键词：
3-Dimensional Address Adherens Junction Adhesions Alzheimer&apos s Disease Appearance Atherosclerosis Binding Biology Blood Blood - brain barrier anatomy Blood Circulation Blood Proteins Blood Vessels Brain Cells Cerebrum Clinical Disease Ectopic Expression Endothelial Cells Endothelium Engineering Epithelial Cells Experimental Autoimmune Encephalomyelitis Experimental Models Extravasation Fostering Future Imaging technology Immune Immunophenotyping In Situ In Vitro Inflammation Inflammatory Invaded Leukocytes Link Mediating Membrane Meningitis Messenger RNA Metabolic Methods Microscopy Modality Modeling Molecular Movement Multiple Sclerosis Mus Neoplasm Metastasis Nervous System Trauma Neuraxis Neurons Neurosciences Pathogenesis Pathogenicity Patients Phase Phenotype Physiological Process Property Proteins RNA Regulation Relapse Reporting Resolution Role Series Signal Transduction Site Source Spinal Cord Technology Testing Therapeutic Tight Junctions Time Tissues United States National Institutes of Health Work cancer cell cell motility effective therapy exosome extracellular vesicles fluorescence imaging in vivo insight knock-down microvesicles migration nanosized neoplastic cell neuroAIDS neuroinflammation neurovascular injury novel novel therapeutic intervention novel therapeutics peptidomimetics potential biomarker pre-clinical repaired stem cells tissue repair vesicular release

项目摘要

While the entry of leukocytes into the central nervous system (CNS) is fundamental to the pathogenesis of many neuroinflammatory conditions, mechanisms regulating the last and most critical step of this process, transendothelial migration (TEM), remain obscure. But two recent series of findings from separate fields could reveal novel and vital clues to how and where TEM takes place. One, ectopic expression of tight junction (TJ) proteins has been described on circulating leukocytes in patients with relapses of the neuroinflammatory disease multiple sclerosis (MS). Elevated expression of TJ proteins by leukocytes in inflammatory conditions, as well as by stem and malignant cells, might endow these cells with heightened ability to cross tissue barriers – possibly by enabling transient interactions with endothelial TJ proteins via a ‘zipper’ mechanism. Two, nano- sized extracellular vesicles (EVs), e.g., exosomes and microvesicles, bearing junctional proteins and shed by endothelial cells have been reported. EVs are elevated in blood during inflammation (including MS), shuttle protein and RNA between cells, and interact with various immune cells to alter their adhesion and migration properties. These collective findings could suggest EVs transfer TJ proteins to leukocytes and serve as the missing links functionally connecting TJ proteins on leukocytes, TEM and neuroinflammation. Specifically, by transferring TJ cargo from endothelial cells to leukocytes in a juxtacrine manner, EVs might enable adherent leukocytes to transiently engage corresponding vascular TJ proteins and, thereby, foster TEM across the highly restrictive endothelium of the blood-brain barrier (BBB). Using state-of-the-art technologies, we will focus on leukocytes displaying claudin 5 (CLN-5) – a major TJ protein of the BBB – and test the following hypothesis: Leukocytes exploit CLN-5 and endothelial-derived EVs via novel interactions to extravasate across the BBB during neuroinflammation. Aim 1 will use high-resolution 3D fluorescence imaging and FACS to detect CLN-5+-leukocytes and identify their immunophenotypes and activation states at various times in the blood and different CNS regions of mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. Aim 2, will use novel endothelial conditional, eGFP-CLN-5 mice or CLN-5 knockdown mice to determine if leukocytes express CLN-5 endogenously or acquire it exogenously from endothelial cells. Aim 3, will use 3D fluorescence imaging/FACS to analyze transfer of CLN-5 cargo (protein and/or mRNA) from endothelial-derived EVs to leukocytes, and a novel CLN-5 peptidomimetic, Pep5, to establish if this action is CLN-5-dependent. Aim 4, will use a recognized in vitro BBB model and Pep5 to examine the functional role(s) of leukocyte CLN-5 and EVs in promoting TEM. These studies will be the first systematical and methodical approach to address the relationship between leukocyte TJ proteins and EVs, and their concerted role in neuroinflammation. As similar processes might generically operate in other instances of cell extravasation, these studies should reveal common pathogenic mechanisms and highlight new, therapeutic approaches that target EVs. !

虽然白细胞进入中枢神经系统（CNS）是对的发病机理的基础许多神经炎症条件，调节此过程中最后和最关键步骤的机制，跨内皮迁移（TEM）保持晦涩难懂。揭示新的和重要的线索，并在哪里发生TEM。已经描述了蛋白质有关神经炎症复发的患者循环白细胞的描述疾病多发性硬化症（MS）。以及通过茎和恶性细胞，可能会使细胞具有越过的越过组织屏障的能力 - 可能通过“拉链”机构与内皮TJ蛋白启用交易。大小的细胞外囊泡（EV），例如外泌体和微泡，带有连接蛋白，并由电动汽车在发炎期间（包括MS），班车升高细胞之间的蛋白质和RNA，并与各种免疫细胞相互作用以改变粘附和迁移这些集体发现的属性表明，电动汽车将TJ蛋白转移到白细胞中，并用作缺少链接在白细胞，TEM和神经炎症上的TJ蛋白将TJ货物从近去的货物转移到白细胞，EVS可能会粘附白细胞瞬时接合相关的血管TJ蛋白，从而促进您的tem 血脑屏障（BBB）的高度限制性内皮。在表现出Claudin 5（CLN-5）的白细胞上 - BBB的主要TJ蛋白，并检验以下假设：白细胞通过新颖的相互作用来利用CLN-5和内皮衍生的EV，以遍布您在神经炎症期间的BBB AIM 1。 Cln-5+ - 白细胞，并在血液中的不同时间识别其毫米型和激活状态具有实验性自身免疫性脑脊髓炎（EAE）的小鼠的不同CNS区域，AIM 2女士的模型，将使用新颖的内皮结构内源性表达CLN-5或从内皮细胞外源获取。成像/FACS分析CLN-5货物（蛋白质和/或mRNA）从内皮衍生的EV转移到白细胞和一种新型的CLN-5肽型PEP5，以确定这是否依赖于Cln-5 使用公认的体外BBB模型和PEP5检查白细胞CLN-5和EV的功能作用促进TEM。白细胞TJ蛋白质和电动汽车之间的关系，以及在神经炎症中的一致作用过程可能在其他细胞渗出的其他实例中起作用，这些研究应揭示常见的致病机制并突出了新的，治疗方法的目标电动汽车。呢