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: 10206277
• 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/10206277
• 关键词: AKT2 gene; Acute; Adhesions; Animal Model; Basement membrane; Blood; Blood - brain barrier anatomy; Blood Vessels; Cell Adhesion; Cell Communication; Cells; 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; Neuraxis; Neuronal Dysfunction; Pathogenicity; Pathologic; Pathway interactions; Pharmacology; Phase; Physiological; Protein Isoforms; Proteins; Proteoglycan; Proto-Oncogene Proteins c-akt; Regulation; Reporting; Repression; Research; Role; Signal Pathway; Signal Transduction; Stroke; Surface; Techniques; Testing; Tetanus Helper Peptide; Therapeutic; Tight Junctions; Tissues; Transgenic Mice; Translating; Traumatic Brain Injury; United States; Up-Regulation; Ursidae Family; Work; autocrine; biglycan; blood-brain barrier function; brain cell; cost; decorin; experimental study; expression vector; in vivo; innovation; insight; integrin-linked kinase; mouse model; multiple sclerosis patient; neuroinflammation; new therapeutic target; novel; overexpression; preservation; prevent; screening; seal; solute; 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.

项目摘要 在过去的十年中，内皮细胞周围的细胞外基质（ECM）组成的重要性， 血脑屏障（BBB）的内皮细胞（EC）不仅在屏障发育中， 和维护，但也在功能障碍。紧密连接蛋白claudin-5（CLDN 5）对于密封至关重要 内皮细胞之间的细胞旁孔，以防止液体，溶质和细胞的通过，从而形成细胞外基质。 事实上的BBB。知识的主要差距包括在炎症损伤期间ECM发生了什么变化以及如何发生变化。 它们有助于屏障破坏，以及ECM-EC相互作用如何调节CLDN 5。我们的长期目标是 阐明炎症期间负责BBB功能障碍的内皮特异性信号通路。 本提案的总体目标是确定ECM介导的CLDN 5依赖性细胞因子调节异常的作用。 炎症期间BBB功能。重点放在AKT 2亚型特异性功能的新作用上 在体内平衡过程中维持最大CLDN 5表达，加上两个小亮氨酸的促炎作用， 丰富的蛋白聚糖（SLRP）、核心蛋白聚糖和双糖蛋白聚糖在神经炎症期间CLDN 5下调中的作用。的 中心假设是炎症触发内皮源性SLRP的释放， 以干扰CLDN 5的组成型ECM依赖性调节，从而导致BBB功能障碍。这 假设是从申请人实验室的初步发现中得出的。的理由 拟议的研究是，更好地了解ECM病理生物学将转化为更多的见解， 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 系统（CNS），这些系统共同造成了美国约900万人的痛苦 每年承担3000亿美元的成本负担。根据可靠的初步数据，这一假设 将通过两个具体目的进行测试：1）确定内皮源性SLRP在BBB功能障碍中的作用， 2）确定受损的β1-ILK-AKT 2信号传导在ECM依赖性BBB功能障碍中的作用。 该方法将是多方面的，结合相关动物模型中的体内生理分析， 使用从CNS组织分离的原代EC的可比较的离体和体外实验。创新 实验模型包括具有可诱导的BBB特异性SLRP缺陷或AKT 2 过表达;新的分子工具，例如用于核心蛋白聚糖、双糖链蛋白聚糖、ILK， 和AKT 2;药理学药物的靶向筛选;以及最先进的组织病理学技术， 检测炎症介导的ECM改变，可能仅存在于一小部分周围， CNS微血管（小静脉周围炎性病变）。这项研究是有意义的，因为数据来自 从这些研究中，不仅将建立ECM依赖性内皮调节的新概念， 还将为BBB功能障碍的病理生理学提供新的机制见解， 为开发新的治疗靶点提供了基础。