Wnt signaling-mediated control of blood-retinal barrier

Wnt信号介导的血视网膜屏障控制

• 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
• 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' 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

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.

项目摘要 内皮血红视网膜屏障（BRB）的崩溃是残留水肿的主要原因和导致的原因 血管疾病的视力丧失。除了细胞细胞运输外， 内皮细胞（EC），物质的转运，尤其是大分子，也发生 ECS的跨细胞蔬菜（跨胞菌病）。中枢神经系统中正常健康的血管， 包括视网膜，表现出较低的转胞菌病率对于维持血脑屏障（BBB）和内部至关重要 brb。然而，掌管视网膜EC转胞病的分子机制知之甚少，这相当多 限制了我们操纵内BRB治疗血管眼病的能力。在这个项目中，我们确定了Wnt 信号通路是一种对血管生成和血管完整性控制至关重要的途径，可能会起作用 作为维持内部BRB的EC转胞细胞增多症的关键负调节剂。相互关联的Wnt中的突变 涉及配体诺林的信号通路，受体毛躁4和受体低密度 脂蛋白受体相关蛋白5（LRP5）与家族正宗玻璃体蛋白病的发展有关 （FEVR）和Norrie病，均具有内皮内皮BRB分解。典型Wnt信号的激活 涉及β-catenin的稳定，然后将其转移到核与核T细胞因子 /淋巴管结合 增强子因子（TCF/LEF）影响靶基因。我们的初步数据表明，Wnt信号传导丢失 FEVR和Norrie疾病的小鼠模型（LRP5 - / - 和Norriny/ - 小鼠）显着增加了EC跨胞菌病 MFSD2A的水平提高（主要促进剂超级家族域2a），膜 抑制EC转身的转运蛋白。我们假设Wnt信号对于 通过MFSD2A依赖性保持残留的EC的残留EC的跨经细胞增多率较低 小窝囊泡调制。我们将以三个目标检验这一假设。在AIM 1中，我们将确定是否 Wnt信号通过测量小分子的血管渗透性来控制EC转胞胞菌病 在LRP5 - / - 和Norriny/ - 视网膜以及使用Wnt调制的转胞胞菌病测定法中以及EC培养中。在目标2中 我们将确定Wnt信号传导是否通过转录调节调节EC的转胞膜 MFSD2A是一种转胞细胞抑制剂，使用了离体和体外方法的组合。在AIM 3中，我们将 描述Wnt信号传导和MFSD2A是否控制MFSD2A辅助的Caveolar蔬菜形成， 运输和胞吐作用。这项提出的工作将发现新颖的基本分子机制 管理EC跨介症和内部BRB完整性，这对于有助于改善的发展具有重要意义 操纵内BRB的策略。这项工作的发现也将与BBB的调制高度相关 在中枢神经系统中，以及通过BBB和内BRB开发药物输送方法。