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: 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
• 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

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.

项目摘要/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 脑血管发育和生理条件下的功能。这与NHLBI的 理解正常生物学功能的总体目标。该建议旨在调查 血液脑屏障（BBB）完整性下的内皮层粘连蛋白的生物学功能 条件，并探索其基本的分子机制。在AIM 1中，是否失去内皮 层粘连蛋白会影响早期开发或后期BBB维护的BBB形成 使用创新的内皮层粘连蛋白基因敲除小鼠系（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确定 分子量。接下来，基础分子机理（细胞细胞和/或跨细胞 运输）将在体外和体内探索生化，超微结构和功能水平。 由于造血细胞衍生的层粘连蛋白在这些突变体中也被消融，我们将进一步研究是否是否 EKO小鼠中的BBB分解是由于内皮细胞或造血细胞中层粘连蛋白的损失所致 VE-cadherin-Creert系列。在AIM 2中，内皮层粘连蛋白调节BBB完整性的假设 通过腺苷酸环化酶2（AC2）将测试。首先，是否以及如何准确调节内皮层粘连蛋白 将在体外和体内检查脑微血管内皮细胞中的AC2表达。接下来， AC2在BBB通透性和细胞细胞/跨细胞运输中的功能将使用 功能丧失和功能收益的方法。第三，AC2对BBB完整性的影响是否 将使用AC2抑制剂研究其依赖其腺苷循环活性（cAMP的产生） 和/或激活剂。如果答案是肯定的，哪个营地效应器/信号通路（PKA） 将使用PKA-和/或EPAC特异性探索EPAC）介导AC2的BBB调节功能 抑制剂和激活剂。该提案的成功完成将阐明 BBB完整性及其潜在的分子机制中的内皮层粘连蛋白；发现以前的一个 AC2在跨细胞运输（跨胞菌病）中的未识别作用；并确定内皮层粘连蛋白和 AC2作为BBB调节中的新分子靶标，这将促进创新的发展 BBB中断的治疗方法。此外，该建议还将生成创新的研究材料 （例如，转基因小鼠系和慢病毒构建体）在层粘连蛋白/地下膜的领域有用， 由于其内在的复杂性和缺乏研究工具而被理解。