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

• 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
• 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

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 血脑屏障（BBB）是用于描述血管的独特性质的术语， 使中枢神经系统（CNS）血管化并限制分子、离子和细胞在中枢神经系统（CNS）之间的运动。 血液和大脑。血脑屏障功能障碍导致各种中枢神经系统疾病，并极大地阻碍药物递送。 尽管它的重要性，但由于我们的研究中存在重大差距，在操纵BBB方面几乎没有取得进展。 在分子和细胞水平上理解和询问BBB的组成的工具的缺乏。我们 最近的工作和初步数据开始表征FLVCR 2（一种质膜血红素）的作用 转运蛋白，在脑血管发育和疾病中。Flvcr 2在BBB内皮细胞中特异性表达 它在人类中的突变会导致脑血管病和无脑积水PVHH-a 一种罕见的遗传病，特征是脑血管生成缺陷，导致局部缺血和脑室扩大。 我们的工作将BBB血红素转运与脑血管生成联系起来，并揭示了血红素在这些血管生成中的核心作用。 病理学然而，FLVCR2转运血红素和控制脑血管生成的机制是 我们目前还不知道，我们目前缺乏在分子水平上探测这些机制的工具。在本提案中，我们 我将通过两个具体的目标来解决这个问题：在目标1中，我们将使用以下方法确定FLVCR 2的结构： 冷冻电子显微镜（cryoEM），从而提供一个模型，以产生基于结构的功能假说。 在目标2中，使用CRISPR-Cas9技术结合创新的血红素报告基因和血管生成测定， 众所周知，我们的实验室，我们将开发基于细胞的血管生成测定，以允许快速和有效的测试， 基于结构的假设我们的初步数据包括重组FLVCR 2和复合物的cryoEM分析。 用FLVCR 2特异性抗体（Fab）形成;以及在细胞培养物中操纵Flvcr 2表达 利用小鼠脑内皮细胞的血管生成测定。这些数据表明，我们已经做好准备， 结构测定和开发用于未来结构/功能的适应性细胞测定 FLVCR2及其在BBB血红素转运和CNS疾病中的作用。