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

描述（由申请人提供）：我们的血管系统中的内皮细胞（EC）的管腔表面包被有膜结合大分子的糖萼，该糖萼由硫酸化蛋白聚糖、透明质酸、唾液酸、糖蛋白和血浆蛋白组成，粘附在该表面基质上。内皮糖萼层（EGL）为血管系统提供多功能涂层，其在诸如动脉粥样硬化和糖尿病的疾病状态下降解。由于与常规电子显微镜相关的脱水伪影，甚至层的厚度这样的基本特征也没有被牢固地建立。然而，体内和体外研究表明，硫酸乙酰肝素蛋白聚糖介导内皮重塑（细胞伸长和排列），以响应流体剪切应力，并沿着与透明质酸控制重要的机械转导事件，如流体剪切诱导的刺激一氧化氮的产生，但参与这些特征性反应的核心蛋白是未知的。为了解决这些对于我们理解健康和疾病中的血管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：为了阐明内皮糖萼层（EGL）的结构，我们将首次应用冷冻透射电子显微镜（cryo-TEM）结合共聚焦显微镜来确定其厚度和组织。为了确定介导EC重塑和机械转导的蛋白聚糖核心蛋白，以响应流体剪切应力，我们将使用糖胺聚糖（GAG）降解酶，RNA干扰技术和粘附阻断氨基酸序列在体外和敲除动物在体内解构这些过程。 为了开展这项生物工程研究资助（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），并辅以显微镜和分子生物学（Spray）的专业知识。

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).