The Endothelial Glycocalyx: Its Structure and Function and as a Mechanotransducer

描述(申请人提供)：血管内皮细胞(ECs)的管腔表面覆盖着一层膜结合的大分子的糖基催化剂，这些大分子包括硫酸盐蛋白多糖、透明质酸、唾液酸、糖球蛋白和血浆蛋白，它们附着在这个表面基质上。血管内皮糖帽层(EGL)为血管系统提供了一种多功能涂层，在动脉粥样硬化和糖尿病等疾病状态下会降解。由于与常规电子显微镜相关的脱水伪影，即使是像层的厚度这样的基本特征也没有得到牢固的确定。然而，体内和体外研究表明，硫酸乙酰肝素蛋白多糖在流体剪切应力下介导内皮重塑(细胞伸长和排列)，并与透明质酸一起控制重要的机械转导事件，如流体剪切诱导的一氧化氮产生刺激，但参与这些特征反应的核心蛋白尚不清楚。为了解决这些对我们理解血管在健康和疾病中的功能至关重要的基本问题，我们将在拟议的研究中进行以下研究：为了阐明内皮细胞糖帽层(EGL)的结构，我们将首次将冷冻透射电子显微镜(Cryo-TEM)与共聚焦显微镜相结合，以确定其厚度和组织。为了确定在流体剪切力作用下介导EC重塑和机械转导的蛋白多糖核心蛋白，我们将使用糖胺多聚糖(GAG)降解酶、RNA干扰技术和体外黏附阻断氨基酸序列以及体内敲除动物来解构这些过程。为了实施生物工程研究基金(BRG)中的项目，我们组织了一个具有生物工程核心专业知识的研究团队，包括体外剪切实验(Tarbell)和体内剪切实验(FU)，并辅之以显微镜和分子生物学(SPEAM)方面的专业知识。 公共卫生相关性：这项研究对公共健康很重要，因为内皮糖衣层(EGL)为血管系统提供了一种多功能涂层，在动脉粥样硬化和糖尿病等疾病状态下，血管系统会降解。EGL的降解导致血管调节功能障碍，例如，血流丧失导致强大的血管扩张剂一氧化氮的刺激。如果要开发重建或加强EGL的方法，则需要了解EGL的结构以及参与机械转导/重塑的核心蛋白和糖胺聚糖。重建的EGL将恢复关键的血管调节功能，从而对抗疾病。本申请中提出的基础性工作对于作为本项目长期目标的一部分的翻译工作至关重要。

DESCRIPTION (provided by applicant): The luminal surfaces of endothelial cells (ECs) that line our vasculature are coated with a glycocalyx of membrane-bound macromolecules comprised of sulfated proteoglycans, hyaluronic acid, sialic acids, glycocproteins and plasma proteins that adhere to this surface matrix. The endothelial glycocalyx layer (EGL) provides a multifunctional coating to the vasculature that is degraded in disease states such as atherosclerosis and diabetes. Because of dehydration artifacts associated with conventional electron microscopy, even such rudimentary characteristics as the thickness of the layer have not been firmly established. In vivo and in vitro studies have, however, shown that heparan sulfate proteoglycans mediate endothelial remodeling (cell elongation and alignment) in response to fluid shear stress and along with hyaluronic acid control vital mechanotransduction events such as fluid shear-induced stimulation of nitric oxide production, but the core proteins that are involved in these characteristic responses are not known. To address these fundamental questions that are crucial for our understanding of vascular function in health and disease, we will pursue the following studies in the proposed research: To elucidate the structure of the endothelial glycocalyx layer (EGL) we will apply, for the first time, cryo-transmission electron microscopy (cryo-TEM) in conjunction with confocal microscopy to determine its thickness and organization. To determine the proteoglycan core proteins that mediate EC remodeling and mechanotransduction in response to fluid shear stress we will use glycosaminoglycan (GAG) degrading enzymes, RNA interference technology and adhesion blocking amino acid sequences in vitro and knockout animals in vivo to deconstruct these processes. To carry out the projects in this Bioengineering Research Grant (BRG), we have organized a research team with core expertise in bioengineering including: in vitro shear experiments (Tarbell), and in vivo shear experiments (Fu) that is complemented by expertise in microscopy and molecular biology (Spray). PUBLIC HEALTH RELEVANCE: The research is important to public health because the endothelial glycocalyx layer (EGL) provides a multifunctional coating to the vasculature that is degraded in disease states such as atherosclerosis and diabetes. Degradation of the EGL leads to vasoregulatory dysfunction through, for example, loss of blood flow-induced stimulation of the potent vasodilator, nitric oxide. Knowledge of EGL structure and the core proteins and glycosaminoglycans involved in mechanotransduction/remodeling will be required if methods are to be developed to re-constitute or reinforce the EGL. A re-constituted EGL will restore critical vasoregulatory functions, thus combating disease. The foundational work proposed in this application is essential for translational work that will follow as part of the long range goals of this project.