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. !

项目概要 我们已经确定内粘蛋白（EMCN）是内皮细胞（EC）糖萼的一种成分，是一种新型的 VEGFR2 信号传导的调节因子。 siRNA介导的人视网膜毛细血管EC块中EMCN的敲低 VEGF 诱导的体外血管生成功能（增殖、迁移和管形成） 体内新血管形成。我们的数据表明 EMCN 对于 VEGF 刺激的 VEGFR2 是必需的 内化。我们假设 EMCN 调节 VEGF 诱导的 VEGFR2 内吞作用，从而调节 VEGF EC 中的信号传导。我们建议： (i) 使用遗传学方法来检查 EMCN 在发育中的作用 血管生成和病理性新血管形成以及成人血管稳定性。我们已经生成了老鼠 与 floxed EMCN 一起与 Rosa-Cre 一起培育，以评估完全 EMCN 敲除的效果，并与 他莫昔芬诱导的 VE-钙粘蛋白用于检查 EC 特异性敲除。 (ii) 阐明分子机制 EMCN 通过其调节 VEGFR2 内化和信号传导、细胞生物学和生化方法 将用于确定 EMCN 在 VEGF-VEGFR2 内吞作用中的功能，以阐明结构 EMCN 的特征对于其在 VEGFR2 内吞作用中的作用是必要的，并鉴定 EMCN-VEGFR2 结合 可能参与 VEGFR2 内化的蛋白质。 (iii) 开发干扰单克隆抗体 由于EMCN和VEGFR2之间的关联，将使用EMCN的糖基化胞外结构域 作为抗原。我们使用 EMCN 截短突变体的初步数据表明， EMCN 因其对 VEGFR2 信号传导的影响而必不可少。抗血清将根据其对人体的影响进行筛选 VEGF 诱导的 EC 迁移和 VEGFR2 内化。抗血清，我们已证明它可以干扰 EMCN 对 VEGF 诱导的迁移和 VEGFR2 内化的影响，将测试其能力 与阿柏西普相比，单独阻断体内病理性 VEGF 诱导的通透性和血管生成（VEGF trap），或与阿柏西普联合治疗。 VEGF 中和是治疗该病的主要方式 许多涉及新血管形成和血管通透性的眼部病变。虽然引人注目 尽管取得了成功，但仍有相当一部分患者对抗 VEGF 治疗没有反应。在 此外，视网膜中的许多非血管细胞表达 VEGFR2，因此容易受到慢性疾病的影响。 局部 VEGF 的中和，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、血管完整性和血管发育中的作用的新信息 病理性血管生长；将揭示有关 VEGF 刺激的 VEGFR2 信号传导调节的新见解； 并且，将测试 EMCN 作为独特的内皮细胞特异性靶点，以​​阻断异常 VEGF 诱导的 血管生成和血管通透性。 ！