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 我们已经鉴定出内皮细胞(EC)糖萼的一种成分--内粘蛋白(EMCN)是一种新的 VEGFR2信号调节因子。SiRNA介导的EMCN在人视网膜毛细血管内皮细胞中的敲除 血管内皮生长因子诱导的血管生成功能(增殖、迁移和管状形成) 体内新生血管的形成。我们的数据表明EMCN是血管内皮生长因子刺激的VEGFR2所必需的 内部化。我们假设EMCN调节血管内皮生长因子诱导的VEGFR2内吞，从而调节血管内皮细胞生长因子 EC中的信令。我们建议：(I)使用遗传学方法来研究EMCN在发育过程中的作用 血管生成和病理性新生血管以及成人血管稳定性。我们已经培育了小鼠 使用将与ROSA-CRE一起繁殖的花序EMCN来评估完全敲除EMCN的效果，以及使用 他莫昔芬诱导的VE-钙粘附素用于检测EC特异性基因敲除。(Ii)阐明分子机制 EMCN通过其调节VEGFR2的内化和信号、细胞生物学和生化方法 将被用来确定EMCN在VEGF-VEGFR2内吞作用中的作用，以阐明其结构 EMCN在VEGFR2内吞作用中所必需的特性及其与VEGFR2结合的鉴定 可能参与VEGFR2内化的蛋白质。(Iii)研制一种可干扰 随着EMCN和VEGFR2之间的关联，EMCN的糖基化胞外区将被使用 作为一种抗原。我们使用EMCN截断突变体的初步数据表明， EMCN对VEGFR2信号通路的影响是必需的。将根据抗血清的作用对其进行筛选 血管内皮生长因子诱导的内皮细胞迁移和血管内皮细胞生长因子受体2内化。抗血清，我们已经证明了它可以干扰 EMCN对血管内皮生长因子诱导的迁移和VEGFR2内化的影响，将被测试其能力 阻断病理性血管内皮生长因子诱导的通透性和血管生成--单独与阿普利赛特(血管内皮生长因子)比较 Trap)，或作为与afLibercept的联合治疗。血管内皮生长因子中和是治疗非小细胞肺癌的主要模式 涉及新生血管和血管通透性的眼部病变的数量。虽然值得注意的是 在成功的情况下，有相当比例的患者对抗血管内皮生长因子治疗似乎没有反应。在……里面 此外，视网膜中的一些非血管细胞表达VEGFR2，因此容易受到慢性疾病的影响 局部血管内皮生长因子的中和，对神经营养和生存功能的影响。这些研究的结果 将提供有关EMCN在血管发育、血管完整性和 病理性血管生长；将揭示对血管内皮生长因子刺激的VEGFR2信号调节的新见解； 并且，将测试EMCN作为一个独特的内皮细胞特异性靶点来阻断异常的血管内皮生长因子诱导 血管生成和血管通透性。 好了！