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Molecular Tools to Study FLVCR2-mediated Heme Transport in Brain Angiogenesis

研究 FLVCR2 介导的脑血管生成中血红素转运的分子工具

基本信息

批准号：
10539852
负责人：
Thomas Darmody Arnold
金额：
$ 46.38万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10539852
关键词：
Address Affect Affinity Air Antibodies Binding Biological Assay Blood Blood - brain barrier anatomy Blood Vessels Brain Brain Ischemia Brain Vascular Malformation CRISPR/Cas technology Cell Culture System Cell Culture Techniques Cell membrane Cells Cellular biology Central Nervous System Diseases Cerebrovascular Disorders Communities Complex Cryoelectron Microscopy Data Data Collection Development Disease Drug Delivery Systems Endothelial Cells Foundations Functional disorder Future Genes Genetic Genetic Diseases Genetically Engineered Mouse Genotype Goals Growth Heme Human Hydranencephaly Hydrocephalus Immunoglobulin Fragments In Vitro Ions Ischemia Knowledge Link Lipids Maps Mediating Membrane Proteins Methods Modeling Molecular Movement Mus Mutagenesis Mutate Mutation Neuraxis Nutrient Online Mendelian Inheritance In Man Pathology Pharmaceutical Preparations Phenotype Preparation Property Publishing Recombinants Reporter Research Resolution Role Sampling Structural Models Structure System Testing Therapeutic Vascular Diseases Vascular Endothelial Cell Water Work angiogenesis base behavior in vitro brain endothelial cell experimental study heme a innovation knock-down loss of function mutation mind control nanodisk particle reconstitution tool trafficking uptake

项目摘要

PROJECT ABSTRACT The blood-brain barrier (BBB) is a term used to describe the unique properties of the blood vessels that vascularize the central nervous system (CNS) and restrict the movement of molecules, ions and cells between the blood and the brain. BBB dysfunction contributes to various CNS diseases and greatly impedes drug delivery. Despite its importance, little progress has been made in manipulating the BBB due to a major gap in our understanding, and a lack of tools to interrogate, constituents of the BBB at a molecular and cellular level. Our recent work and preliminary data begin to characterize a role for FLVCR2, a plasma membrane heme transporter, in brain vascular development and disease. Flvcr2 is specifically expressed in BBB endothelial cells and its mutation in humans causes Proliferative Vasculopathy and Hydranencephaly Hydrocephalus PVHH - a rare genetic condition characterized by defective brain angiogenesis leading to ischemia and ventriculomegaly. Our work links BBB heme transport to brain angiogenesis, and uncovers a central role for heme in these pathologies. However, the mechanisms by which FLVCR2 transports heme and controls brain angiogenesis are unknown, and we currently lack the tools to probe these mechanisms at a molecular level. In this proposal we will address this void through two specific Aims: In Aim 1, we will determine the structure of FLVCR2 using cryo-electron microscopy (cryoEM), so as to provide a model to generate structure-based functional hypotheses. In Aim 2, using CRISPR-Cas9 technology combined with innovative heme reporters and angiogenesis assays well-known to our lab, we will develop cell-based angiogenesis assays to allow rapid and efficient testing of structure-based hypotheses. Our preliminary data include cryoEM analysis of recombinant FLVCR2 and complex formation with FLVCR2-specific antibodies (Fabs); and the manipulation of Flvcr2 expression in cell culture angiogenesis assays utilizing mouse brain endothelial cells. These data demonstrate that we are well-poised for structural determination and the development of an adaptable cell-based assay for future structure/function studies on FLVCR2, and its role in BBB heme transport and CNS disease.

项目摘要