Biophysics of HDL Dysfunction

HDL 功能障碍的生物物理学

基本信息

批准号：
8620821
负责人：
Kirkwood Arthur Pritchard
金额：
$ 1.89万
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/8620821
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 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的氧化状态在确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水平正常的患者经历了动脉粥样硬化事件（即中风，心肌梗死），因此HDL功能本身可能比实际水平的HDL胆固醇水平更强。 HDL功能的生物测定很复杂，耗时，技术人员和技术依赖性且在实验室之间难以再现。因此，迫切需要高效的HDL功能测定法。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的HDL依赖性胆固醇代谢代表了可用于评估HDL功能状态的新型生理信息来源。 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],髓过氧化物酶[MPO]和黄嘌呤氧化酶[XO]），以防止LDL氧化； 2）或ATP结合盒（ABC）转运蛋白1（ABCA1）/ABCG1，卵磷脂胆固醇酰基酰基转移酶（LCAT），胆固醇酯转移蛋白（CETP）和Scavenger受体B1类（SR-B1）（SR-B1），以介导HDL-胆固醇释放，酯化酯化，Uptersification and Transserification and Transsericate and Transfice complate and Transfice complate and Trissity complate and Trissity complate and tistity complate and complate and tistity。 HDL具有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，对这些生物分子的HDL结合亲和力的任何氧化诱导的变化都应提供HDL功能的敏感指数。在AIM 1中，我们建议使用建立的胆固醇释放，酯化，转移和吸收的体外生物测定在重构HDL（R-HDL）中氧化的脂质和蛋白质的程度。在AIM 2中，我们将使用BLI测定法确定R-HDL中的氧化脂质和蛋白质如何改变结合速率，并对蛋白质和酶的亲和力介导HDL抑制LDL氧化能力或在反向胆固醇转运途径中促进特定步骤。在AIM 3中，我们建议通过1）在已建立的血管疾病的鼠模型中验证和验证BLI分析与HDL功能的生物测定法相关联，以及2）使用BLI分析来预测哪些患者在病例控制的，盲人的人类研究中临床诊断的动脉粥样硬化。这些研究的成功完成将为开发确定HDL功能的新临床测定奠定基础。对这些新测定的验证将使我们能够在时间范围内确定哪些患者患有功能失调的HDL，并且与临床参考实验室兼容的成本。这些新颖的测定法的开发将使得第一次进行大型临床研究，以充分测试功能失调的HDL可以更好地指示动脉粥样硬化风险。