Investigation of endomucin as a novel regulator of angiogenesis

内粘蛋白作为血管生成的新型调节剂的研究

• 批准号: 10456724
• 负责人: Patricia Ann D'Amore
• 金额: $ 50.05万
• 依托单位: SCHEPENS EYE RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10456724
• 关键词: Adult; Age related macular degeneration; Antigens; Binding; Binding Proteins; Biochemical; Biological; Blood Vessels; Blood capillaries; Capillary Endothelial Cell; Cells; Characteristics; Choroidal Neovascularization; Chronic; Combined Modality Therapy; Data; Development; Dimerization; Disease; Endocytosis; Endothelial Cells; Endothelium; Extracellular Domain; Glycocalyx; Glycoproteins; Growth; Human; Immune Sera; Immunoprecipitation; In Vitro; Investigation; KDR gene; Knock-out; Lasers; Lead; Ligand Binding; Ligands; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Methods; Modeling; Molecular; Monoclonal Antibodies; Mus; Mutagenesis; N-Glycosylation Site; Ocular Pathology; Pathologic; Pathologic Neovascularization; Pathology; Patients; Permeability; Pinocytosis; Process; Reagent; Receptor Activation; Receptor Signaling; Regulation; Reporting; Retina; Role; Route; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Structure; Surface; Tamoxifen; Testing; Tube; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular Permeabilities; Vascularization; Work; angiogenesis; bevacizumab; cadherin 5; cell motility; endothelial-specific sialomucin; genetic approach; glycosylation; in vivo; inhibitor; insight; knock-down; migration; mouse model; mutant; neovascularization; new growth; novel; prevent; vascular factor

Project Summary We have identified endomucin (EMCN), a component of the endothelial cell (EC) glycocalyx, to be a novel regulator of VEGFR2 signaling. siRNA-mediated knockdown of EMCN in human retinal capillary EC blocks VEGF-induced angiogenic functions (proliferation, migration, and tube formation) in vitro and neovascularization in vivo. Our data indicate that EMCN is necessary for VEGF-stimulated VEGFR2 internalization. We hypothesize that EMCN regulates VEGF-induced VEGFR2 endocytosis, and thus VEGF signaling in EC. We propose: (i) To use a genetic approach to examine the role of EMCN in developmental angiogenesis and pathologic neovascularization as well as in adult vascular stability. We have generated mice with floxed EMCN that will be bred with Rosa-Cre to assess the effect of total EMCN knockout, and with tamoxifen-inducible VE-cadherin to examine EC-specific knockout. (ii) To elucidate the molecular mechanism through which EMCN regulates VEGFR2 internalization and signaling, cell biological and biochemical methods will be employed to determine how EMCN functions in VEGF-VEGFR2 endocytosis, to elucidate the structural characteristics of EMCN necessary for its role in VEGFR2 endocytosis, and to identify EMCN-VEGFR2 binding proteins that may be involved in VEGFR2 internalization. (iii) To develop a monoclonal antibody that interferes with the association between EMCN and VEGFR2, the glycosylated extracellular domain of EMCN will be used as an antigen. Our preliminary data using truncation mutants of EMCN indicate that the extracellular domain of EMCN is necessary for its effect on VEGFR2 signaling. Antisera will be screened on the basis of its effects on VEGF-induced EC migration and VEGFR2 internalization. Antisera, that we have shown to interfere with the effect of EMCN on VEGF-induced migration and VEGFR2 internalization, will be tested for its the ability to block pathologic VEGF-induced permeability and angiogenesis in vivo – alone, compared to aflibercept (VEGF trap), or as a combination therapy with aflibercept. VEGF neutralization is the primary mode of treatment for a number of ocular pathologies that involve neovascularization and vessel permeability. While remarkably successful, there is a significant proportion of patients who appear unresponsive to anti-VEGF therapy. In addition, a number of non-vascular cells in the retina express VEGFR2, and are thus vulnerable to chronic neutralization of local VEGF, with implications for neurotrophic and survival functions. Results of these studies will provide new information about the role of EMCN in vascular development, vascular integrity, and pathologic vessel growth; will reveal novel insights into the regulation of VEGF-stimulated VEGFR2 signaling; and, will test EMCN as a unique endothelial cell-specific target for blocking abnormal VEGF-induced angiogenesis and vascular permeability. !

项目摘要 我们已经确定内核素（EMCN）是内皮细胞（EC）糖脂的成分，是一种新颖 VEGFR2信号的调节剂。 siRNA介导的EMCN在人类残留毛细管EC中的敲低 VEGF诱导的血管生成功能（增殖，迁移和管形成）体外， 体内新血管化。我们的数据表明EMCN对于VEGF刺激的VEGFR2是必需的 内部化。我们假设EMCN调节VEGF诱导的VEGFR2内吞作用，从而调节VEGF 在EC中发出信号。我们建议：（i）使用一种遗传方法来检查EMCN在发育中的作用 血管生成和病理新生血管形成以及成人血管稳定性。我们已经产生了小鼠 用Floxed Emcn与Rosa-Cre一起繁殖，以评估总EMCN敲除的影响，并与 他莫昔芬可诱导的VE-钙粘着蛋白检查EC特异性敲除。 （ii）阐明分子机制 EMCN调节VEGFR2内在化和信号传导，细胞生物学和生化方法 将使用EMCN在VEGF-VEGFR2内吞作用中的功能，以阐明结构 EMCN在VEGFR2内吞作用中所必需的特征，并鉴定EMCN-VEGFR2结合 可能参与VEGFR2内在化的蛋白质。 （iii）开发一种干扰的单克隆抗体 与EMCN和VEGFR2之间的关联，将使用EMCN的糖基化细胞外结构域 作为抗原。我们使用EMCN的截断突变体的初步数据表明， EMCN对于其对VEGFR2信号传导的影响是必要的。 Antisera将根据其对 VEGF诱导的EC迁移和VEGFR2内在化。 Antisera，我们已经证明了 EMCN对VEGF诱导的迁移和VEGFR2内在化的影响，将测试其能力 与Aflibercept（VEGF）相比 陷阱），或作为与Aflibercept的组合疗法。 VEGF神经元化是A的主要治疗方式 涉及新血管形成和血管渗透性的眼科病理数量。虽然非常明显 成功的是，似乎对抗VEGF治疗无反应的患者有很大一部分。在 此外，视网膜Express VEGFR2中的许多非血管细胞，因此容易受到慢性的影响 局部VEGF的谈判，对神经营养和生存功能有影响。这些研究的结果 将提供有关EMCN在血管发育，血管完整性和 病理血管生长；将揭示对VEGF刺激的VEGFR2信号传导调节的新见解； 并且，将测试EMCN作为阻断异常VEGF诱导的独特内皮细胞特异性靶标 血管生成和血管渗透性。 呢