Biophysics of HDL Dysfunction

描述(申请人提供)：高密度脂蛋白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。由于高密度脂蛋白水平正常的患者会经历动脉粥样硬化事件(如中风、心肌梗死)，高密度脂蛋白功能本身可能比高密度脂蛋白胆固醇的实际水平更能反映心血管疾病。高密度脂蛋白功能的生物测定是复杂的、耗时的、依赖于技术人员和技术的，并且很难在实验室之间复制。因此，迫切需要高密度脂蛋白功能的高效检测。在这一应用中，我们假设高密度脂蛋白与生物分子相互作用的生物物理学代表了一种新的生理信息来源，可用于评估高密度脂蛋白的功能状态。利用生物层干涉计量学(BLI)这一新的无标记生物分子相互作用技术，我们将开发一系列新的高密度脂蛋白“功能”的分析，以确定氧化是否以及在多大程度上影响高密度脂蛋白与1)抗炎和促炎酶(对氧磷酶[PON1]、血小板激活因子乙酰水解酶[PAF-AH]、髓过氧化酶[MPO]和黄嘌呤氧化酶[XO])相互作用的能力，以防止低密度脂蛋白的氧化；2)或三磷酸腺苷结合盒(ABC)转运体1(ABCA1/Abcg1)、卵磷脂胆固醇酰基转移酶(LCAT)、胆固醇酯转移蛋白(CETP)和清道夫受体B1(SR-B1)分别介导高密度脂蛋白的释放、酯化、转运和摄取。高密度脂蛋白抗炎或参与胆固醇反向转运的机制直接依赖于高密度脂蛋白与这些生物分子结合的能力。因此，氧化引起的这些生物分子的高密度脂蛋白结合亲和力的任何变化都应该提供高密度脂蛋白功能的敏感指标。在目标1中，我们建议使用建立的胆固醇释放、酯化、转移和摄取的体外生物测定来测量重组高密度脂蛋白(r-HDL)中氧化的脂类和蛋白质对高密度脂蛋白功能的损害程度。在目标2中，我们将使用BLI分析来确定r-高密度脂蛋白中氧化的脂类和蛋白质如何改变蛋白质和酶的结合率和亲和力，这些蛋白质和酶介导高密度脂蛋白抑制低密度脂蛋白氧化或促进反向胆固醇运输途径中的特定步骤的能力。在目标3中，我们建议通过1)在已建立的血管疾病小鼠模型中将BLI检测与高密度脂蛋白功能的生物检测相关联，以及2)在病例对照、盲法人类研究中使用BLI检测来预测哪些患者临床诊断为动脉粥样硬化来验证和确认BLI检测。这些研究的成功完成将为开发一种新的临床测定高密度脂蛋白功能的方法奠定基础。对这些新检测方法的验证将使我们能够在一个时间框架内确定哪些患者具有功能障碍的高密度脂蛋白，并且成本与临床参考实验室兼容。这些新的分析方法的开发将首次使进行大规模临床研究成为可能，以充分测试功能失调的高密度脂蛋白是动脉粥样硬化风险的更好指示器这一想法。

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.