Mechanisms for matrix-dependent BBB dysfunction

基质依赖性血脑屏障功能障碍的机制

基本信息

批准号：
10397156
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
项目类别：
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/10397156
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 Persons 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

项目摘要

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.

项目概要在过去的十年中，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（EC）不仅在屏障发育中得到越来越多的认识紧密连接蛋白claudin-5 (CLDN5) 对于密封至关重要。 EC 之间的细胞旁孔可阻止液体、溶质和细胞的通过，从而形成 de 事实上 BBB 的主要知识空白包括炎症损伤期间 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 CLDN5。阐明炎症期间导致 BBB 功能障碍的内皮特异性信号传导途径。该提案的总体目标是定义 ECM 介导的 CLDN5 依赖性失调的作用炎症期间的 BBB 功能被重点放在 AKT2 异构体特异性功能的新作用上。在稳态期间维持最大 CLDN5 表达，加上两个小亮氨酸的促炎作用富含蛋白聚糖 (SLRP)、核心蛋白聚糖和双糖链蛋白聚糖在神经炎症期间 CLDN5 下调中的作用。中心假设是炎症触发内皮源性 SLRP 的释放，该 SLRP 在自分泌中发挥作用时尚干扰 CLDN5 的组成性 ECM 依赖性调节，导致 BBB 功能障碍。假设源自申请人实验室的初步发现。拟议的研究是，更好地了解 ECM 病理学将转化为更多的见解 BBB 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），它共同造成了美国约 900 万人的痛苦根据可靠的初步数据，这一假设每年会造成 3000 亿美元的成本负担。将通过两个具体目标进行测试：1）确定内皮源性 SLRP 在 BBB 功能障碍中的作用炎症；2) 定义受损的 β1-ILK-AKT2 信号在 ECM 依赖性 BBB 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 CNS 组织中分离的原代 EC 进行可比的离体和体外实验。实验模型包括具有诱导性 BBB 特异性 SLRP 缺陷或 AKT2 的转基因小鼠过度表达；新的分子工具，例如用于核心蛋白聚糖、双糖链蛋白聚糖、ILK 基因转移的 Tet-On 构建体， 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可能仅存在于一小部分周围的炎症介导的 ECM 改变中枢神经系统微血管（静脉周围炎症病变）根据数据得出的结果具有重要意义。这些研究不仅将建立 ECM 依赖性内皮调节的新概念，而且还将为 BBB 功能障碍的病理生理学提供新的机制见解，并有可能为开发新的治疗靶点提供基础。