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Endothelial laminin in blood brain barrier regulation

内皮层粘连蛋白在血脑屏障调节中的作用

基本信息

批准号：
10588183
负责人：
Yao Yao
金额：
$ 37.46万
依托单位：
UNIVERSITY OF SOUTH FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-07-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10588183
关键词：
Ablation Adenylate Cyclase Affect Astrocytes Basement membrane Biochemical Biological Assay Biological Process Biology Blood - brain barrier anatomy Brain Cell physiology Cells Cerebral hemisphere hemorrhage Compensation Cre lox recombination system Cyclic AMP Cyclic AMP-Dependent Protein Kinases Deposition Development Endothelial Cells Endothelium Extracellular Matrix Proteins Generations Hematopoietic In Vitro Innovative Therapy Integrins Knock-out Knockout Mice Laboratories Laminin Maintenance Mediating Molecular Molecular Target Molecular Weight Mus National Heart, Lung, and Blood Institute Pathology Pathway interactions Pericytes Phenotype Physiological Protein Isoforms Proteins Regulation Research Role Sagittaria Second Messenger Systems Signal Pathway Signal Transduction Signaling Molecule Structural defect Structure Testing Tight Junctions Tracer Transgenic Mice Vesicle adenylyl cyclase 2 blood treatment blood-brain barrier disruption blood-brain barrier permeabilization brain endothelial cell cadherin 5 cerebrovascular gain of function in vivo inhibitor innovation integrin-linked kinase knock-down laminin S laminin alpha5 loss of function mutant nervous system disorder novel overexpression protein expression tool transcriptome sequencing transcytosis

项目摘要

Project Summary/Abstract The long-term objective of this application is to fully understand the biology of endothelial laminin in cerebrovascular development and function in physiological conditions. This is in line with NHLBI’s overarching objective to understand normal biological function. This proposal aims to investigate the biological function of endothelial laminin in blood brain barrier (BBB) integrity under homeostatic conditions, and explore its underlying molecular mechanism. In Aim 1, whether loss of endothelial laminin affects BBB formation in early development or BBB maintenance at later stage will be investigated using an innovative endothelial laminin knockout mouse line (EKO) generated in our laboratory. The extent of BBB disruption will be determined using fluorescent tracers of various molecular weights. Next, the underlying molecular mechanism (paracellular and/or transcellular transport) will be explored at biochemical, ultrastructural, and functional levels both in vitro and in vivo. Since hematopoietic cell-derived laminin is also ablated in these mutants, we will further investigate if BBB breakdown in EKO mice is due to loss of laminin in endothelial cells or hematopoietic cells using the VE-Cadherin-CreERT line. In Aim 2, the hypothesis that endothelial laminin regulates BBB integrity via adenylyl cyclase-2 (AC2) will be tested. First, whether and how exactly endothelial laminin regulates AC2 expression in brain microvascular endothelial cells will be examined in vitro and in vivo. Next, the function of AC2 in BBB permeability and paracellular/transcellular transport will be investigated using both loss-of-function and gain-of-function approaches. Third, whether the effect of AC2 on BBB integrity relies on its adenylyl cyclase activity (generation of cAMP) will be investigated using AC2 inhibitors and/or activators. If the answer is yes, which cAMP-downstream effector/signaling pathway (PKA versus Epac) mediates AC2’s BBB-regulating function will be explored using PKA- and/or Epac-specific inhibitors and activators. Successful completion of this proposal will elucidate the biological function of endothelial laminin in BBB integrity and its underlying molecular mechanism; uncover a previously unrecognized role of AC2 in transcellular transport (transcytosis); and identify endothelial laminin and AC2 as novel molecular targets in BBB regulation, which will promote the development of innovative treatments for BBB disruption. In addition, this proposal will also generate innovative research materials (e.g. transgenic mouse line and lentiviral constructs) useful in the field of laminin/basement membrane, which is understudied due to its intrinsic complexity and lack of research tools.

项目总结/摘要本申请的长期目标是充分了解内皮层粘连蛋白的生物学，生理条件下脑血管的发育和功能。这与NHLBI的总体目标是了解正常的生物功能。本提案旨在调查内皮层粘连蛋白在稳态血脑屏障完整性中的生物学功能条件下，并探讨其潜在的分子机制。在目标1中，是否内皮细胞的损失层粘连蛋白在早期发育中影响BBB的形成或在后期阶段维持BBB将是可能的。研究使用创新的内皮层粘连蛋白敲除小鼠系（EKO），在我们的研究中产生实验室BBB破坏的程度将使用各种荧光示踪剂来确定。分子量其次，潜在的分子机制（细胞旁和/或跨细胞的运输）将在体外和体内的生化、超微结构和功能水平上进行探索。由于造血细胞源性层粘连蛋白在这些突变体中也被消融，我们将进一步研究， EKO小鼠中的BBB破坏是由于内皮细胞或造血细胞中的层粘连蛋白的损失， VE-Cadherin-CreERT系。在目的2中，内皮层粘连蛋白调节血脑屏障完整性的假设通过腺苷酸环化酶-2（AC 2）进行检测。首先，内皮层粘连蛋白是否以及如何调节将在体外和体内检查脑微血管内皮细胞中的AC 2表达。接下来 AC 2在血脑屏障通透性和细胞旁/跨细胞转运中的功能将使用功能丧失和功能获得两种方法。第三，AC 2对血脑屏障完整性的影响依赖于其腺苷酸环化酶活性（cAMP的产生），将使用AC 2抑制剂进行研究和/或活化剂。如果答案是肯定的，那么cAMP下游效应物/信号通路（PKA）相对于Epac）介导的AC 2的BBB调节功能将使用PKA和/或Epac特异性抑制剂和活化剂。成功完成这一提案将阐明生物功能的内皮层粘连蛋白在血脑屏障的完整性及其潜在的分子机制;揭示了一个以前 AC 2在跨细胞转运（胞吞作用）中未被认识的作用;并鉴定内皮层粘连蛋白和 AC 2作为血脑屏障调控的新分子靶点，将促进创新的治疗BBB破坏。此外，这项提案还将产生创新的研究材料 (e.g.转基因小鼠系和慢病毒构建体）用于层粘连蛋白/基底膜领域，由于其内在的复杂性和缺乏研究工具，其研究不足。