Mechanisms for matrix-dependent BBB dysfunction

基质依赖性血脑屏障功能障碍的机制

• 批准号: 10609845
• 负责人: Richard Scott Beard
• 金额: $ 33.02万
• 依托单位: BOISE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609845
• 关键词: AKT2 gene; Acute; Adhesions; Animal Model; Basement membrane; Blood - brain barrier anatomy; Blood Vessels; Blood brain barrier dysfunction; Cell Adhesion Inhibition; Cell Communication; Cell Separation; Cells; Central Nervous System; Central Nervous System Diseases; Collagen Type IV; Cues; Data; Dementia; Demyelinating Diseases; Development; Disease; Disease Progression; Down-Regulation; Encephalopathies; Endothelial Cells; Endothelium; Experimental Autoimmune Encephalomyelitis; Experimental Models; Extracellular Matrix; FOXO1A gene; Functional disorder; Future; Gene Transfer; Genetic Transcription; Goals; Hemorrhage; Histopathology; Homeostasis; Impairment; In Vitro; Individual; Inflammation; Inflammation Mediators; Inflammatory; Injury; Integrins; Interleukin-1 beta; Intervention; Knock-in Mouse; Knockout Mice; Knowledge; Laboratories; Lesion; Leucine; Liquid substance; Maintenance; Mediating; Metastatic malignant neoplasm to brain; Modeling; Molecular; Multiple Sclerosis; Nerve Degeneration; Neuronal Dysfunction; Pathogenicity; Pathologic; Pathway interactions; Persons; Phase; Physiological; Protein Isoforms; Proteins; Proteoglycan; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Repression; Research; Role; Signal Pathway; Signal Transduction; Stroke; Techniques; Testing; Therapeutic; Tight Junctions; Tissues; Transgenic Mice; Translating; Traumatic Brain Injury; United States; Up-Regulation; Work; autocrine; biglycan; blood-brain barrier function; cost; decorin; experimental study; expression vector; gene repression; in vivo; innovation; insight; integrin-linked kinase; mouse model; multiple sclerosis patient; neuroinflammation; new therapeutic target; novel; overexpression; pharmacologic; preservation; prevent; screening; seal; solute; surface coating; tool

PROJECT SUMMARY Over the past decade, the importance of the extracellular matrix (ECM) composition surrounding the endothelial cells (ECs) of the blood-brain barrier (BBB) has been increasingly recognized not only in barrier development and maintenance, but also in dysfunction. The tight junction protein claudin-5 (CLDN5) is critical for sealing paracellular pores between ECs to prevent the passage of fluids, solutes, and cells, thereby forming the de facto BBB. Major gaps in knowledge include what ECM alterations occur during inflammatory injuries and how they contribute to barrier disruption, plus how ECM-EC interactions regulate CLDN5. Our long-term goal is to elucidate the endothelial-specific signaling pathways responsible for BBB dysfunction during inflammation. The overall objective of this proposal is to define the role of ECM-mediated dysregulation of CLDN5-dependent BBB function during inflammation. Emphasis is placed on a novel role for the isoform-specific function of AKT2 in maintaining maximal CLDN5 expression during homeostasis, plus a proinflammatory role of two small leucine- rich proteoglycans (SLRPs), decorin and biglycan, in CLDN5 downregulation during neuroinflammation. The central hypothesis is that inflammation triggers a release of endothelial-derived SLRPs which act in an autocrine fashion to interfere with constitutive ECM-dependent regulation of CLDN5, contributing to BBB dysfunction. This hypothesis was derived from preliminary findings generated in the applicant’s laboratory. The rationale for the proposed research is that a better understanding of ECM pathobiology will translate into increased insights of the pathogenic role of BBB dysfunction in a multitude of inflammation-associated diseases in the central nervous system (CNS) which collectively account for the suffering of approximately 9 million people in the United States alone and bear a cost burden of 300 billion dollars annually. Guided by robust preliminary data, this hypothesis will be tested by two specific aims: 1) Determine the role of endothelial-derived SLRPs in BBB dysfunction during inflammation; and 2) Define the role of impaired β1-ILK-AKT2 signaling in ECM-dependent BBB dysfunction. The approach will be multifaceted, combining in vivo physiological analyses in a relevant animal model with comparable ex vivo and in vitro experiments using primary ECs isolated from CNS tissue. Innovative experimental models include transgenic mice with inducible BBB-specific SLRP deficiency or AKT2 overexpression; new molecular tools such as Tet-On constructs for gene transfer of decorin, biglycan, ILK, and AKT2; targeted screening of pharmacologic agents; and state-of-the-art histopathology techniques to detect inflammatory-mediated ECM alterations likely to only be present surrounding a small percentage of CNS microvessels (perivenular inflammatory lesions). The proposed research is significant, as data derived from these studies will not only establish novel concepts in ECM-dependent regulation of the endothelium but will also provide new mechanistic insights into the pathophysiology of BBB dysfunction with the potential to provide a basis for the development of new therapeutictargets.

项目总结 在过去的十年中，围绕内皮细胞的细胞外基质(ECM)成分的重要性 血脑屏障(BBB)的细胞(ECs)不仅在屏障的发育过程中得到越来越多的认识 和维持性，但也有功能障碍。紧密连接蛋白claudin-5(CLDN5)对封闭 内皮细胞之间的细胞旁小孔，以防止流体、溶质和细胞通过，从而形成血管内皮细胞。 事实上的血脑屏障。知识方面的主要差距包括炎性损伤期间发生了哪些ECM改变以及如何 它们有助于屏障的破坏，以及ECM-EC相互作用如何调节CLDN5。我们的长期目标是 阐明炎症过程中导致血脑屏障功能障碍的内皮细胞特异性信号通路。 这项提案的总体目标是确定ECM介导的CLDN5依赖的失调的作用 血脑屏障在炎症过程中发挥作用。重点放在了AKT2的异构体特异性功能的新作用上 在动态平衡期间维持CLDN5的最大表达，以及两个小亮氨酸的促炎作用- 在神经炎症过程中，CLDN5中丰富的蛋白多糖(SLRP)、核心蛋白聚糖和大聚糖下调表达。这个 中心假说是炎症触发内皮源性SLRPs的释放，这种SLRPs以自分泌的方式发挥作用 干扰细胞外基质对CLDN5的结构性调节，导致血脑屏障功能障碍。这 假设是从申请人的实验室产生的初步结果得出的。该计划的基本原理 拟议的研究是，对ECM病理生物学的更好理解将转化为对 血脑屏障功能障碍在中枢神经系统多种炎症相关疾病中的致病作用 这一系统(CNS)总共造成了美国约900万人的痛苦 仅此一项，每年就要承担3000亿美元的成本负担。在稳健的初步数据的指导下，这一假设 将通过两个特定的目标进行测试：1)确定内皮来源的SLRPs在血脑屏障功能障碍中的作用 2)明确受损的β1-ILK-AKT2信号在细胞外基质依赖性血脑屏障功能障碍中的作用。 该方法将是多方面的，将相关动物模型的体内生理分析与 使用从中枢神经系统组织中分离的原代内皮细胞进行体外和体外可比实验。创新型 实验模型包括带有可诱导的BBB特异性SLRP缺陷或AKT2的转基因小鼠 超量表达；新的分子工具，如用于核心蛋白，双聚糖，ilk， 和AKT2；有针对性的药物筛选；以及最先进的组织病理学技术 检测炎症介导的ECM改变可能只出现在一小部分周围 中枢神经系统微血管(小静脉周围炎性病变)。拟议的研究具有重要意义，因为数据是从 这些研究不仅将在ECM依赖的内皮调节方面建立新的概念，而且 也将为血脑屏障功能障碍的病理生理学提供新的机械学见解，有可能 为开发新的治疗靶点提供依据。