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，围墙壁的内皮细胞已成为临床感兴趣的强烈主题，因为易于访问性允许有效的药物靶向。血管生长的失调与广泛的疾病状态有关，例如糖尿病性视网膜病，与年龄相关的黄斑变性和肿瘤生长有关。肿瘤生长取决于血管生成。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可以选择性地靶向肿瘤内皮细胞的治疗剂至关重要。该实验室的目的是鉴定和研究在肿瘤脉管系统中有选择上调的候选基因。 ROBO4是一个极好的靶标，因为它在肿瘤血管中选择性表达，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取潜在药物。重要的是，Robo4上的特定信号传导复合物组件，如果确定为负责方向迁移，也可以针对。以前，我们已经证明ROBO4对于斑马鱼中的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上相互作用并共享共同的信号分子； （b）一种蛋白质的复合物，包括IRSP53，MENA，CDC42-GTP和VILSE响应Robo4，在内皮细胞中聚集在一起； （c）与SRY相关的高动力组（SOX）家族的成员与Robo4的细胞质尾巴相互作用。我们假设：“内皮细胞表面受体ROBO1和ROBO4合作与SLIT2相互作用，通过特定的细胞内信号蛋白（CDC42-GTP，VILSE，IRSP53，IRSP53，MENA，MENA，SOX）的特定复合物组装引导细胞迁移。”为了在AIM 1中检验这一假设，我们将研究Robo1和Robo4是否与SLIT2共同相互作用，并使用分子，生化和成像方法的组合使用内皮细胞中VILSE和MENA的直接信号传导复合物组装。在AIM 2中，我们将通过Robo1和Robo4和Robo4的细胞质尾巴来确定IRSP53，MENA，CDC42-GTP，VILSE和SOX的组装机制，这些信号传导复合体负责使用体外生物化学和Zebra in Vivo功能分析的组合来指导内皮细胞迁移。 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形成丝状。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使以过度血管生成和血管泄漏为特征的疾病受益。公共卫生相关性：肿瘤生长取决于血管生成。我们有兴趣确定在肿瘤与正常内皮细胞中差异表达的细胞表面分子优选的血管靶标。该建议研究一个这样的目标，即robo4。 ROBO4在肿瘤血管中高度表达。肿瘤血管漏水，血管床通常具有混乱的外观。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可以选择性地靶向肿瘤内皮细胞的治疗剂至关重要。该建议将确定机器人在介导内皮细胞中定向迁移中使用的机制。我们提出了两个目的，以研究机器人用来介导定向迁移的机制。完成两个目标将确定Robo1和Robo4如何在响应SLIT2配体的情况下机械地组装内皮细胞内部的信号传导复合物，从而协调内皮细胞的定向迁移。这项研究将确定ROBO1和ROBO4信号传导设备常见的细胞内分子，这些分子是药物设计的靶标，使与失调的内皮细胞迁移相关的疾病（例如与肿瘤生长相关的疾病）。