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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：
10164853
负责人：
Yao Yao
金额：
$ 37.75万
依托单位：
UNIVERSITY OF GEORGIA
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 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10164853
关键词：
Adenylate Cyclase Affect Astrocytes Basement membrane Biochemical Biological Assay Biological Process Biology Blood - brain barrier anatomy Brain Cell physiology Cells 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/antagonist innovation integrin-linked kinase knock-down laminin S laminin alpha5 loss of function mutant nervous system disorder novel overexpression protein expression recombinase-mediated cassette exchange 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/文摘