Wnt signaling-mediated control of blood-retinal barrier

Wnt信号介导的血视网膜屏障控制

基本信息

批准号：
9918370
负责人：
JING CHEN
金额：
$ 44.25万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-30 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9918370
关键词：
Affect Binding Biological Assay Blindness Blood - brain barrier anatomy Blood Vessels Blood capillaries Blood-Retinal Barrier Carrier Proteins Caveolae Cell Culture Techniques Cell Nucleus Cells Clathrin Complement Data Development Drug Delivery Systems Electron Microscope Endocytosis Endothelial Cells Endothelium Exhibits Exocytosis Exudate Eye Eye diseases Familial exudative vitreoretinopathy Family Fluorescence Fundus Genes Genetic Transcription Goals Human In Vitro LDL-Receptor Related Protein 1 Lasers Lead Ligands Link Low Density Lipoprotein Receptor Luciferases Measures Mediating Membrane Transport Proteins Methods Microscopy Molecular Molecular Weight Mus Mutation Neuraxis Norrie&apos s disease Nuclear Osmotic Pressure Pathologic Pathway interactions Prevention Public Health Regulation Reporter Retina Retinal Edemas Role TCF Transcription Factor Testing Tight Junctions Transcriptional Regulation Transportation Vascular Diseases Vascular Endothelium Vascular Permeabilities Vesicle WNT Signaling Pathway Work angiogenesis beta catenin caveolin 1 design drug development improved in vivo inhibitor/antagonist interstitial lipoprotein receptor related protein 5 loss of function macula microscopic imaging mouse model novel novel therapeutic intervention novel therapeutics overexpression public health relevance receptor retina blood vessel structure retinal angiogenesis transcytosis

项目摘要

Project Summary Breakdown of the inner endothelial blood-retinal barrier (BRB) is a major cause of retinal edema and resultant vision loss in vascular eye diseases. In addition to paracellular transport though the tight junctions between endothelial cells (ECs), transportation of substance, particularly large molecules, also occurs through transcellular vesicles (transcytosis) across ECs. Normal healthy blood vessels in the central nervous system, including retinas, display low rates of transcytosis essential for maintaining blood-brain barrier (BBB) and inner BRB. Yet molecular mechanisms governing retinal EC transcytosis are poorly understood, which considerably limits our ability to manipulate inner BRB to treat vascular eye disease. In this project we identified that Wnt signaling pathway, a pathway fundamentally important for angiogenesis and vascular integrity control, may act as a critical negative regulator of EC transcytosis to maintain inner BRB. Mutations in the inter-related Wnt signaling pathway involving the ligand Norrin, and the receptor Frizzled4 and co-receptor low density lipoprotein receptor-related protein 5 (LRP5) are linked with development of familial exudative vitreoretinopathy (FEVR) and Norrie disease, both with inner endothelial BRB breakdown. Activation of canonical Wnt signaling involves stabilization of β-catenin, which then translocates to the nucleus to bind nuclear T-cell factor /lymphoid enhancer factor (TCF/LEF) to influence target genes. Our preliminary data show that loss of Wnt signaling in mouse models of FEVR and Norrie disease (Lrp5-/- and Norriny/- mice) significantly increased EC transcytosis, with decreased levels of MFSD2a (major facilitator super family domain containing 2a), a membrane transporter protein that suppresses EC transcytosis. We hypothesize that Wnt signaling is essential for maintaining a low transcytosis rate in retinal EC critical for inner BRB integrity, through Mfsd2a-dependent caveola vesicle modulation. We will test this hypothesis with three aims. In Aim 1 we will determine whether Wnt signaling controls EC transcytosis by measuring vascular permeability of both small and large molecules in Lrp5-/- and Norriny/- retinas, as well as in EC culture using transcytosis assay with Wnt modulation. In Aim 2 we will determine whether Wnt signaling regulates EC transcytosis through transcriptional regulation of MFSD2a, a transcytotic inhibitor, using a combination of ex vivo and in vitro approaches. In Aim 3, we will delineate whether Wnt signaling and MFSD2a control MFSD2a-assisted caveolar vesicle formation, transportation, and exocytosis. This proposed work will uncover novel fundamental molecular mechanisms governing EC transcytosis and inner BRB integrity, which is of significance in aiding development of improved strategies to manipulate inner BRB. Findings from this work will also be highly relevant for modulation of BBB in the central nervous system, as well as development of drug delivery methods through BBB and inner BRB.

项目概要内皮血视网膜屏障（BRB）的破坏是视网膜水肿的主要原因血管性眼病导致的视力丧失除了通过细胞间紧密连接进行的细胞运输外。内皮细胞（EC）物质的运输，特别是大分子，也通过跨细胞囊泡（转胞吞作用）穿过中枢神经系统中的正常健康血管，包括视网膜，显示出维持血脑屏障（BBB）和内部细胞所必需的低转胞吞作用率然而，人们对控制视网膜 EC 转胞吞作用的分子机制知之甚少，这在很大程度上限制了我们操纵内部 BRB 治疗血管性眼病的能力。在这个项目中，我们发现了 Wnt。信号通路是对血管生成和血管完整性控制至关重要的通路，可能会发挥作用作为 EC 转胞吞作用的关键负调节因子，以维持相互关联的 Wnt 中的内部 BRB 突变。信号通路涉及配体 Norrin、受体 Frizzled4 和辅助受体低密度脂蛋白受体相关蛋白 5 (LRP5) 与家族性渗出性玻璃体视网膜病变的发生有关 (FEVR) 和 Norrie 病，均与经典 Wnt 信号传导的内皮 BRB 破坏有关。涉及 β-连环蛋白的稳定，然后转位到细胞核以结合核 T 细胞因子/淋巴细胞我们的初步数据表明，增强子因子 (TCF/LEF) 会影响靶基因。 FEVR 和 Norrie 病小鼠模型（Lrp5-/- 和 Norriny/- 小鼠）显着增加 EC 转胞吞作用， MFSD2a（含有 2a 的主要促进子超家族结构域）水平降低，这是一种膜抑制 EC 转胞吞作用的转运蛋白我们认为 Wnt 信号传导对于其至关重要。通过 Mfsd2a 依赖性维持视网膜 EC 中的低转胞吞率，这对内部 BRB 完整性至关重要在目标 1 中，我们将通过三个目标来检验这一假设。 Wnt 信号通过测量小分子和大分子的血管通透性来控制 EC 转胞吞作用在 Lrp5-/- 和 Norriny/- 视网膜中，以及在 EC 培养中使用 Wnt 调制的转胞吞测定。我们将确定 Wnt 信号传导是否通过转录调节来调节 EC 转胞吞作用 MFSD2a 是一种转胞吞抑制剂，在目标 3 中，我们将结合使用离体和体外方法。描绘 Wnt 信号传导和 MFSD2a 是否控制 MFSD2a 辅助的小凹囊泡形成，这项拟议的工作将揭示新的基本分子机制。控制 EC 转胞吞作用和内部 BRB 完整性，这对于帮助改进改进的开发具有重要意义这项工作的结果也与 BBB 的调节高度相关。中枢神经系统，以及开发通过 BBB 和内部 BRB 的药物输送方法。