Roundabout4 signaling in endothelial cells

内皮细胞中的 Roundabout4 信号传导

• 批准号: 8017385
• 负责人: Ramani Ramchandran
• 金额: $ 37.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-16 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8017385
• 关键词: Address; Age related macular degeneration; Antisense Oligonucleotides; Appearance; Area; Biochemical; Biological Assay; Blood Vessels; Candidate Disease Gene; Cell Line; Cell Surface Receptors; Cell surface; Cells; Clinical; Complex; Cues; Cytoplasmic Tail; Diabetic Retinopathy; Disease; Drug Delivery Systems; Drug Design; Endothelial Cells; Extravasation; Family; Filopodia; Goals; Growth; Guanosine Triphosphate; Health; Homeostasis; Immigration; Immunoprecipitation; In Vitro; Injection of therapeutic agent; Intracellular Signaling Proteins; Laboratories; Ligands; Mediating; Migration Assay; Molecular; Pharmaceutical Preparations; Serum; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Small Interfering RNA; Testing; Tumor Angiogenesis; Western Blotting; Zebrafish; angiogenesis; axon guidance; base; cell motility; imaging modality; in vivo; interest; member; migration; protein complex; research study; response; rho GTP-Binding Proteins; selective expression; therapeutic development; tumor; tumor growth; vascular bed

DESCRIPTION (provided by applicant): Over the past four decades, endothelial cells lining the vessel wall have become an intense subject of clinical interest since ease of accessibility allows for effective drug targeting. Dysregulation of vessel growth is associated with wide range of disease states such as diabetic retinopathy, wet form of age related macular degeneration, and tumor growth. Tumor growth is angiogenesis dependent. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. The goal of this laboratory is to identify and study candidate genes that are selectively upregulated in tumor vasculature. Robo4 is an excellent target since it is expressed selectively in tumor vessels and is critical for directional migration of endothelial cells. Further, the cell surface expression of Robo4 allows easy access to potential drugs. Importantly, specific signaling complex assembly on Robo4, if identified to be responsible for directional migration can also be targeted. Previously, we have shown that robo4 is essential for angiogenesis in vivo in zebra fish and activates Rho GTPases in endothelial cells in vitro. Preliminary results suggest that: (A) Robo1 and Robo4 interact and share common signaling molecules at both the cell surface and intracellular level; (B) A complex of proteins including IRSp53, Mena, Cdc42-GTP, and Vilse together assembles in endothelial cells in response to Robo4; (C) Members of the Sry-related high-mobility-group (Sox) family interact with Robo4's cytoplasmic tail. We hypothesize that: "Endothelial cell surface receptors Robo1 and Robo4 co-operatively interact with Slit2 to direct cell migration via a specific complex assembly of intracellular signaling proteins (Cdc42-GTP, Vilse, IRSp53, Mena, Sox)." To test this hypothesis in aim 1, we will investigate whether Robo1 and Robo4 co- operatively interact with Slit2 and direct signaling complex assembly of Vilse and Mena in endothelial cells using a combination of molecular, biochemical and imaging methods. In aim 2, we will identify the mechanism of assembly of IRSp53, Mena, Cdc42-GTP, Vilse and Sox with Robo1 and Robo4's cytoplasmic tail in a signaling complex responsible for directing endothelial cell migration using a combination of in vitro biochemical and in vivo functional assays in zebra fish. We anticipate that by accomplishing aims 1 and 2, we will understand how Robo1 and Robo4 dictate the endothelial tip cell to navigate through complex milieu by unraveling the similarities between the two molecules in triggering signaling in endothelial cells, the order in which the different signaling molecules assemble on Robo1 and Robo4's cytoplasmic tail, the cues they respond to, and the integration of different signaling components to form filopodia. Each mechanistic step is a putative target and drugs targeting these steps will benefit diseases characterized by excessive angiogenesis and vascular leakage. PUBLIC HEALTH RELEVANCE: Tumor growth is angiogenesis dependent. We are interested in identifying vascular targets preferably cell surface molecules that are differentially expressed in tumor versus normal endothelial cells. This proposal studies one such target namely Robo4. Robo4 is highly expressed in tumor vessels. Tumor vessels are leaky, and the vascular bed often has a chaotic appearance. The first step in tumor angiogenesis is endothelial cell activation and directional migration of endothelial cells towards the tumor. Therefore, understanding the mechanisms of directional migration is critical for development of therapeutics that can selectively target tumor endothelial cells from taking this first step. This proposal will identify the mechanisms utilized by Robos in mediating directional migration in endothelial cells. We propose two aims to investigate mechanisms used by Robos to mediate directional migration. Accomplishing both aims will determine how Robo1 and Robo4 mechanistically assemble a signaling complex inside endothelial cells in response to Slit2 ligand, thereby coordinating directional migration of endothelial cells. This study will identify intracellular molecules common to the Robo1 and Robo4 signaling apparatus that serve as targets for drug design benefiting conditions associated with deregulated endothelial cell migration such as those associated with tumor growth.

描述（由申请人提供）：在过去的四十年中，血管壁内衬的内皮细胞已成为临床关注的热点，因为易于接近允许有效的药物靶向。血管生长失调与多种疾病状态有关，例如糖尿病性视网膜病、湿性年龄相关性黄斑变性和肿瘤生长。肿瘤生长依赖于血管生成。肿瘤血管生成的第一步是内皮细胞激活和内皮细胞向肿瘤的定向迁移。因此，了解定向迁移的机制对于开发能够选择性地靶向肿瘤内皮细胞迈出第一步的治疗方法至关重要。该实验室的目标是识别和研究肿瘤血管系统中选择性上调的候选基因。 Robo4 是一个极好的靶点，因为它在肿瘤血管中选择性表达，并且对于内皮细胞的定向迁移至关重要。此外，Robo4 的细胞表面表达可以轻松获得潜在的药物。重要的是，如果确定 Robo4 上的特定信号复合体组装负责定向迁移，也可以成为目标。此前，我们已经证明 robo4 对于斑马鱼体内血管生成至关重要，并在体外激活内皮细胞中的 Rho GTPases。初步结果表明：（A）Robo1和Robo4在细胞表面和细胞内水平相互作用并共享共同的信号分子； (B) 包括 IRSp53、Mena、Cdc42-GTP 和 Vilse 在内的蛋白质复合物在内皮细胞中组装在一起，以响应 Robo4； (C) Sry 相关高迁移率族 (Sox) 家族的成员与 Robo4 的细胞质尾部相互作用。我们假设：“内皮细胞表面受体 Robo1 和 Robo4 与 Slit2 协同相互作用，通过细胞内信号蛋白（Cdc42-GTP、Vilse、IRSp53、Mena、Sox）的特定复杂组装来指导细胞迁​​移。”为了检验目标 1 中的这一假设，我们将结合分子、生化和成像方法，研究 Robo1 和 Robo4 是否与 Slit2 协同相互作用，并直接在内皮细胞中组装 Vilse 和 Mena 信号复合物。在目标 2 中，我们将结合体外生化和体内功能，确定 IRSp53、Mena、Cdc42-GTP、Vilse 和 Sox 与 Robo1 和 Robo4 细胞质尾部在负责指导内皮细胞迁移的信号复合物中的组装机制。斑马鱼的检测。我们预计，通过实现目标 1 和 2，我们将了解 Robo1 和 Robo4 如何通过揭示两个分子在触发内皮细胞信号传导方面的相似性（不同信号分子的顺序）来指示内皮尖端细胞在复杂的环境中导航。组装在 Robo1 和 Robo4 的细胞质尾部，它们响应的线索以及不同信号成分的整合形成丝状伪足。每个机械步骤都是一个假定的目标，针对这些步骤的药物将有益于以过度血管生成和血管渗漏为特征的疾病。公共卫生相关性：肿瘤生长依赖于血管生成。我们感兴趣的是识别血管靶标，优选是在肿瘤与正常内皮细胞中差异表达的细胞表面分子。该提案研究了一个这样的目标，即 Robo4。 Robo4 在肿瘤血管中高表达。肿瘤血管有渗漏，血管床常常呈现混乱的外观。肿瘤血管生成的第一步是内皮细胞激活和内皮细胞向肿瘤的定向迁移。因此，了解定向迁移的机制对于开发能够选择性地靶向肿瘤内皮细胞迈出第一步的治疗方法至关重要。该提案将确定 Robos 介导内皮细胞定向迁移的机制。我们提出了两个目标来研究机器人用于调解定向迁移的机制。实现这两个目标将决定 Robo1 和 Robo4 如何在内皮细胞内机械地组装信号复合物以响应 Slit2 配体，从而协调内皮细胞的定向迁移。这项研究将鉴定 Robo1 和 Robo4 信号装置共有的细胞内分子，这些分子可作为药物设计的靶标，有益于与内皮细胞迁移失调相关的疾病，例如与肿瘤生长相关的疾病。