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

内皮糖萼：其结构和功能以及作为机械传感器

基本信息

批准号：
8247713
负责人：
JOHN M TARBELL
金额：
$ 62.1万
依托单位：
CITY COLLEGE OF NEW YORK
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-15 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8247713
关键词：
Address Adhesions Amino Acid Sequence Aorta Atherosclerosis Binding Biomedical Engineering Blood Vessels Blood flow Caveolae Cell Line Cells Characteristics Chondroitinases Complement Confocal Microscopy Core Protein Cytoskeleton Dehydration Diabetes Mellitus Disease Electron Microscopy Endothelial Cells Enzymes Epoprostenol Event Focal Adhesions Functional disorder Glycocalyx Glycosaminoglycans Glypican Goals Gold Health Heparan Sulfate Proteoglycan Heparin Lyase Heparitin Sulfate Hyaluronic Acid Hyaluronidase Hypertension In Vitro Inorganic Sulfates Knowledge Label Liquid substance Mediating Membrane Methods Microscopy Molecular Biology Morphologic artifacts Mus Neuraminidase Nitric Oxide Plasma Proteins Process Production Prostaglandins I Proteins Proteoglycan Public Health RNA Interference Research Research Project Grants Series Sialic Acids Signaling Molecule Structure Surface Technology Thick Time Transmission Electron Microscopy Unspecified or Sulfate Ion Sulfates Vascular Diseases Vasodilator Agents Work combat in vivo insight knockout animal macromolecule proteoglycan core protein public health relevance research study response shear stress small hairpin RNA syndecan

项目摘要

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.

描述（由申请人提供）：我们的脉管系统的内皮细胞（EC）的腔表面被膜结合的大分子的糖蛋白覆盖，由硫化蛋白聚糖，透明质酸，唾液酸，唾液酸，唾液酸，乳糖蛋白和葡萄球菌蛋白组成，这些蛋白质和质量蛋白与此表面配饰。内皮糖脂层（EGL）为脉管系统提供了多功能涂层，该层在动脉粥样硬化和糖尿病等疾病状态下降解。由于与常规电子显微镜相关的脱水伪像，即使是层的厚度等基本特征也尚未被牢固确定。然而，体内和体外研究表明，乙par硫酸盐蛋白聚糖蛋白聚糖可以介导内皮重塑（细胞伸长和比对），以响应液体剪切应力，以及透明质酸控制的重要机械传导事件，例如流体剪切诱导的氧化蛋白，但核心的蛋白质涉及这些特征，这些蛋白质与这些特征相关的特征，这些蛋白质涉及氧化物的蛋白质的特征，这些蛋白质涉及这些特征。为了解决这些对我们对健康和疾病中血管功能的理解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：为了阐明我们将首次适用内皮性糖脂层（EGL）的结构（EGL），第一次使用Cryo-trans-Trans-Trans-Trans-Trans-Trans-Trans-Trans-Trans-Trans-Trans-Trans-Transerifection（Cryo-Tem）在Conjunciption中以浓度的厚度和孔隙cococal microsction来确定其厚度及其厚度的厚度。为了确定介导EC重塑和机械转移的蛋白酶核心蛋白，以响应液体剪切应力，我们将使用糖胺聚糖（GAG）降解酶，RNA干扰技术和粘附技术阻止氨基酸氨基酸序列在体外和敲除动物中以训练这些过程来构造这些过程。为了在这项生物工程研究补助金（BRG）中执行这些项目，我们组织了一个具有生物工程核心知识的研究团队，包括：体外剪切实验（Tarbell）和体内剪切实验（FU），该实验（FU）在显微镜和分子生物学方面的专业知识得到了补充。公共卫生相关性：这项研究对公共卫生很重要，因为内皮糖脂层（EGL）为脉管系统提供了多功能涂层，该涂层在疾病状态下降解，例如动脉粥样硬化和糖尿病。 EGL的降解会导致血管调节功能障碍，例如，血流诱导的有效血管扩张剂，一氧化氮的刺激丧失。如果要开发重新建立或加强EGL的方法，则需要了解与机械转导/重塑有关的EGL结构以及涉及机械转导/重塑的糖胺聚糖的知识。重新构成的EGL将恢复关键的血管调节功能，从而对抗疾病。本申请中提出的基本工作对于转化工作至关重要，这将是该项目远程目标的一部分。