Investigation of endomucin as a novel regulator of angiogenesis

项目摘要 我们已经鉴定了内皮细胞（EC）糖萼的一种成分--内粘蛋白（EMCN）， VEGFR 2信号转导的调节因子。siRNA介导的人视网膜毛细血管EC阻滞中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VEGFR 2 内化我们假设EMCN调节VEGF诱导的VEGFR 2内吞作用，从而调节VEGF 信号在EC我们建议：（i）使用遗传学方法来研究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。我们已经培养出了 用将与Rosa-Cre一起繁殖的floxed EMCN来评估完全EMCN敲除的效果， 他莫昔芬诱导的VE-钙粘蛋白来检查EC特异性敲除。(ii)为了阐明 EMCN通过其调节VEGFR 2内化和信号传导，细胞生物学和生物化学方法 将用于确定EMCN在VEGF-VEGFR 2内吞作用中的功能，以阐明EMCN的结构， EMCN在VEGFR 2内吞作用中所必需的特征，并鉴定EMCN-VEGFR 2结合 可能参与VEGFR 2内化的蛋白质。(iii)开发一种单克隆抗体， 由于EMCN和VEGFR 2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是其对VEGFR 2信号传导的作用所必需的。抗血清将根据其对以下方面的影响进行筛选： VEGF诱导的EC迁移和VEGFR 2内化。抗血清，我们已经证明可以干扰 EMCN对VEGF诱导的迁移和VEGFR 2内化的影响，将测试其 阻断病理性VEGF诱导的渗透性和体内血管生成-单独与aflibercept（VEGF）相比 trap），或作为与阿柏西普的组合疗法。VEGF中和是治疗血管内皮生长因子缺乏的主要方式。 涉及新血管形成和血管渗透性的多种眼部病理。虽然值得注意的是 成功的，有显着比例的患者出现对抗VEGF治疗无反应。在 此外，视网膜中的许多非血管细胞表达VEGFR 2，因此易受慢性炎症的影响。 中和局部VEGF，暗示神经营养和存活功能。这些研究结果 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VEGF刺激的VEGFR 2信号调节的新见解; 并将测试EMCN作为一种独特的内皮细胞特异性靶点， 血管生成和血管通透性。 !

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. !