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

项目摘要 血脑屏障(BBB)是一个用来描述血管的独特性质的术语，这些血管 使中枢神经系统(CNS)血管化，限制分子、离子和细胞之间的运动 血液和大脑。血脑屏障功能障碍是多种中枢神经系统疾病的致病因素，严重阻碍药物的转运。 尽管它很重要，但由于我们的主要差距，在操纵血脑屏障方面进展甚微 在分子和细胞水平上缺乏对血脑屏障成分的了解，以及缺乏审问工具。我们的 最近的工作和初步数据开始表征FLVCR2的作用，一种质膜血红素 转运蛋白，在脑血管发育和疾病中的作用。Flvcr2在血脑屏障内皮细胞中特异表达 在人类中，它的突变会导致增生性血管病和积水无脑积水PVHH-a 罕见的遗传性疾病，其特征是脑血管生成缺陷，导致脑缺血和脑室增大。 我们的工作将血脑屏障血红素运输与脑血管生成联系起来，并揭示了血红素在这些过程中的核心作用。 病理学。然而，FLVCR2转运血红素和控制脑血管生成的机制是 未知，我们目前缺乏在分子水平上探索这些机制的工具。在这份提案中，我们 我们将通过两个具体目标来解决这一空白：在目标1中，我们将使用 低温电子显微镜(CREYO-EM)，以便提供一个模型来生成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和血管生成分析 为我们的实验室所熟知，我们将开发基于细胞的血管生成测试，以允许快速和有效地测试 基于结构的假设。我们的初步数据包括重组FLVCR2和复合体的低温电子显微镜分析 FLVCR2特异性抗体的形成和FlvCR2在细胞培养中表达的调控 利用小鼠脑内皮细胞进行血管生成检测。这些数据表明，我们已经做好了准备 未来结构/功能的结构确定和适应性细胞分析方法的发展 FLVCR2及其在血脑屏障血红素转运和中枢神经系统疾病中作用的研究。

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.