Biophysics of HDL Dysfunction

HDL 功能障碍的生物物理学

基本信息

批准号：
8511809
负责人：
Kirkwood Arthur Pritchard
金额：
$ 62.81万
依托单位：
MEDICAL COLLEGE OF WISCONSIN
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-16 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8511809
关键词：
ATP binding cassette transporter 1 ATP-Binding Cassette Transporters Affect Affinity Anti-Inflammatory Agents Anti-inflammatory Antioxidants Apolipoprotein A-I Atherosclerosis Automation Binding Biological Assay Biophysics Biosensor Blinded Cardiovascular Diseases Carrier Proteins Cells Cholesterol Cholesterol Esters Cholesterol Homeostasis Clinical Clinical Research Complex Cultured Cells Development Diagnosis Diagnostic Diagnostic tests Effectiveness Enzymes Esterification Event Family Foundations Functional disorder Genetic Engineering Goals Healthcare Heart Diseases High Density Lipoprotein Cholesterol High Density Lipoproteins Hospitals Human In Vitro Individual Inflammation Inflammatory Interferometry Knowledge Label Laboratories Lecithin Lipids Lipoprotein Binding Lipoproteins Liver Low Density Lipoprotein oxidation Manuals Measurement Measures Mediating Metabolism Modeling Modification Molecular Mus Myocardial Infarction Oxidative Stress Pathway interactions Patients Peroxidases Pharmaceutical Preparations Phospholipids Physiological Platelet Activating Factor Play Property Protective Agents Proteins Publishing Reactive Oxygen Species Reporting Research Risk Role SR-B proteins Sampling Series Source Stroke Techniques Testing Time Transferase Validation Vascular Diseases Xanthine Oxidase aryldialkylphosphatase base case control cost design experience glutathione peroxidase heart disease risk high density lipoprotein-1 high density lipoprotein-2 high density lipoprotein-3 improved in vitro Assay in vitro Bioassay indexing insight novel oxidation oxidized lipid prevent public health relevance receptor reconstitution research study reverse cholesterol transport scavenger receptor uptake

项目摘要

DESCRIPTION (provided by applicant): The oxidation status of HDL plays an important role in determining how this lipoprotein prevents or promotes atherosclerosis. Since patients with normal levels of HDL experience atherogenic events (ie., stroke, myocardial infarction), HDL function itself may be a stronger indicator of cardiovascular disease than the actual levels of HDL cholesterol. Bioassays of HDL function are complex, time-consuming, technician- and technique-dependent and difficult to reproduce between labs. Thus, highly efficient assays of HDL function are desperately needed. In this application, we hypothesize that the biophysics of HDL interactions with biomolecules the mediate HDL-dependent cholesterol metabolism represent a novel source of physiological information that can be used to assess the functional state of HDL. Using biolayer interferometry (BLI), a new label-free technique for measuring biomolecular interactions, we will develop a series of novel assays of HDL "function" to determine if and the extent to which oxidation impacts on the ability of HDL to interact with 1) anti- and pro-inflammatory enzymes (paraoxonase [PON1], platelet activating factor acetylhydrolase [PAF-AH], myeloperoxidase [MPO] and xanthine oxidase [XO]) to prevent LDL oxidation; 2) or ATP binding cassette (ABC) transporter 1 (ABCA1)/ABCG1, lecithin cholesteryl acyl transferase (LCAT), cholesteryl ester transfer protein (CETP) and scavenger receptor class B1 (SR-B1) to mediate HDL-cholesterol release, esterification, transfer and uptake, respectively. The mechanisms by which HDL is anti-inflammatory or participates in reverse cholesterol transport is directly dependent on the ability of HDL to bind these biomolecules. As such, any oxidation-induced changes in HDL binding affinity for these biomolecules should provide a sensitive index of HDL functionality. In Aim 1, we propose to measure the extent to which oxidized lipids and proteins in reconstituted HDL (r-HDL) impair HDL function using established in vitro bioassays of cholesterol release, esterification, transfer and uptake. In Aim 2, we will ue BLI assays to determine how oxidized lipids and proteins in r-HDL alter binding rates and affinity for the proteins and enzymes that mediate HDL's ability to inhibit LDL oxidation or promote specific steps in the reverse cholesterol transport pathway. In Aim 3, we propose to verify and validate that BLI assays by 1) correlating BLI assays with bioassays of HDL function in established murine models of vascular disease and 2) by using BLI assays to predict which patients have clinically-diagnosed atherosclerosis in case-controlled, blinded human studies. Successful completion of these studies will lay the foundation for the development of a new clinical assay for determining HDL functionality. Validation of these new assays will allow us to identify which patients have dysfunctional HDL in a time frame and for a cost that is compatible with clinical reference laboratories. Development of these novel assays will make it possible, for the first time, to perform large clinical studies to fully test the idea that dysfunctional HDL is better indicator of atherosclerotic risk.

描述（由申请人提供）：HDL 的氧化状态在决定这种脂蛋白如何预防或促进动脉粥样硬化方面起着重要作用。由于HDL水平正常的患者会经历动脉粥样硬化事件（即中风、心肌梗塞），因此HDL功能本身可能是比HDL胆固醇的实际水平更强的心血管疾病指标。 HDL 功能的生物测定复杂、耗时、依赖技术人员和技术，并且难以在实验室之间重现。因此，迫切需要对 HDL 功能进行高效测定。在此应用中，我们假设 HDL 与生物分子相互作用的生物物理学介导 HDL 依赖性胆固醇代谢代表了一种新的生理信息来源，可用于评估 HDL 的功能状态。使用生物层干涉测量法 (BLI)（一种用于测量生物分子相互作用的新型无标记技术），我们将开发一系列 HDL“功能”的新颖检测方法，以确定氧化是否影响 HDL 与 1 相互作用的能力以及影响程度。 ) 抗炎和促炎酶（对氧磷酶 [PON1]、血小板活化因子乙酰水解酶 [PAF-AH]、髓过氧化物酶 [MPO] 和黄嘌呤氧化酶[XO]）防止LDL氧化； 2) 或 ATP 结合盒 (ABC) 转运蛋白 1 (ABCA1)/ABCG1、卵磷脂胆固醇酰基转移酶 (LCAT)、胆固醇酯转移蛋白 (CETP) 和 B1 类清道夫受体 (SR-B1) 介导 HDL-胆固醇释放、酯化，分别为转移和吸收。 HDL 抗炎或参与反向胆固醇转运的机制直接取决于 HDL 结合这些生物分子的能力。因此，任何氧化诱导的 HDL 对这些生物分子的结合亲和力的变化都应提供 HDL 功能的敏感指数。在目标 1 中，我们建议使用已建立的胆固醇释放、酯化、转移和摄取体外生物测定法来测量重构 HDL (r-HDL) 中氧化脂质和蛋白质损害 HDL 功能的程度。在目标 2 中，我们将使用 BLI 测定来确定 r-HDL 中氧化的脂质和蛋白质如何改变蛋白质和酶的结合率和亲和力，从而介导 HDL 抑制 LDL 氧化或促进反向胆固醇转运途径中特定步骤的能力。在目标 3 中，我们建议通过以下方式验证 BLI 测定：1）将 BLI 测定与已建立的血管疾病小鼠模型中 HDL 功能的生物测定相关联；2）使用 BLI 测定来预测哪些患者患有临床诊断的动脉粥样硬化：受控、盲法人类研究。这些研究的成功完成将为开发确定 HDL 功能的新临床检测方法奠定基础。这些新检测方法的验证将使我们能够在一定的时间范围内识别出哪些患者患有 HDL 功能障碍，并且费用与临床参考实验室相一致。这些新检测方法的开发将首次使大型临床研究成为可能，以充分检验功能失调的 HDL 是动脉粥样硬化风险更好指标的观点。