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

• 批准号: 10558332
• 负责人: Yao Yao
• 金额: $ 37.38万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10558332
• 关键词: Adenylate Cyclase; Affect; Astrocytes; Basement membrane; Biochemical; Biological Assay; Biological Process; Biology; Blood - brain barrier anatomy; Brain; Cell physiology; Cells; Cre lox recombination system; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Deposition; Development; Endothelial Cells; Endothelium; Extracellular Matrix; Financial compensation; Generations; Hematopoietic; In Vitro; Innovative Therapy; Integrins; Knock-out; Knockout Mice; Laboratories; Laminin; Lead; 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; base; 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

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.

项目概要/摘要 该应用的长期目标是充分了解内皮层粘连蛋白的生物学特性 生理条件下脑血管的发育和功能。这符合 NHLBI 的规定 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下血脑屏障（BBB）完整性中的生物学功能 条件，并探讨其潜在的分子机制。在目标 1 中，内皮细胞是否丧失 层粘连蛋白影响发育早期BBB的形成或后期BBB的维持 使用我们的创新型内皮层粘连蛋白敲除小鼠系 (EKO) 进行了研究 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。接下来，潜在的分子机制（细胞旁和/或跨细胞） 转运）将在体外和体内的生物化学、超微结构和功能水平上进行探索。 由于造血细胞来源的层粘连蛋白在这些突变体中也被消除，我们将进一步研究是否 EKO 小鼠的血脑屏障崩溃是由于内皮细胞或造血细胞中层粘连蛋白的丢失 VE-钙粘蛋白-CreERT 线。在目标 2 中，内皮层粘连蛋白调节 BBB 完整性的假设 将通过腺苷酸环化酶-2 (AC2) 进行测试。一、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 AC2 的表达。接下来， AC2 在 BBB 通透性和细胞旁/跨细胞转运中的功能将使用以下方法进行研究 功能丧失和功能获得方法。三、AC2对BBB完整性是否有影响 依赖于其腺苷酸环化酶活性（cAMP 的产生）将使用 AC2 抑制剂进行研究 和/或活化剂。如果答案是肯定的，则哪个 cAMP-下游效应器/信号通路（PKA 与 Epac）介导 AC2 的 BBB 调节功能将使用 PKA 和/或 Epac 特异性进行探索 抑制剂和激活剂。该提案的成功完成将阐明 内皮层粘连蛋白对血脑屏障完整性的影响及其分子机制；揭开之前的 AC2 在跨细胞转运（转胞吞作用）中的作用未被认识；并鉴定内皮层粘连蛋白和 AC2作为BBB调控的新型分子靶点，将促进创新药物的发展 BBB 破坏的治疗。此外，该提案还将产生创新的研究材料 （例如转基因小鼠系和慢病毒构建体）可用于层粘连蛋白/基底膜领域， 由于其内在的复杂性和缺乏研究工具，该问题尚未得到充分研究。