PRESERVATION OF HDL FUNCTION DURING EARLY ATHEROGENESIS

早期动脉粥样硬化过程中 HDL 功能的保留

• 批准号: 6389799
• 负责人: JOHN K BIELICKI
• 金额: $ 18.12万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6389799
• 关键词: apolipoproteins; atherosclerosis; blood lipoprotein metabolism; cholesterol; crosslink; cysteine; disease /disorder onset; electrospray ionization mass spectrometry; enzyme activity; enzyme inhibitors; enzyme structure; gas chromatography mass spectrometry; high density lipoproteins; high performance liquid chromatography; human tissue; hydrogen peroxide; lipid peroxides; low density lipoprotein; oxidation; oxidative stress; phosphatidylcholine sterol acyltransferase; phospholipids; protein sequence; site directed mutagenesis

The minimal oxidation of low density lipoproteins (LDL) in the artery wall is thought to initiate the atherogenic process by contributing to macrophage foam cell formation. High density lipoproteins (HDL) promote the efflux of excess cholesterol from macrophage foam cells thus reversing the atherosclerotic process. The enzyme lecithin cholesterol acyltransferase (LCAT) esterifies cholesterol on HDL and thus facilitates the net efflux of cholesterol from foam cells. As a result, LCAT plays an important protective function in reversing atheromatous lesions. Recent studies suggest that HDL may serve a beneficial function by accepting oxidized lipids from minimally oxidized LDL thus inhibiting early atherogenesis. However, lipophilic oxidation products have been found to produce a dramatic inhibition of LCAT activity suggesting the transfer of oxidized lipids from minimally oxidized LDL to HDL may impair HDL function and exacerbate developing atherosclerotic lesions. The aims of this proposal are to 1) identify the oxidized lipids that inhibit LCAT activity, 2) elucidate the underlying mechanism of LCAT impairment, and 3) establish whether the HDL-associated enzyme, paraoxonase (PON), can protect LCAT activity from specific molecular species of lipid peroxides. Preliminary results establish that physiological concentrations of phospholipid hydroperoxides (PL-OOH) are potent inhibitors of LCAT activity and that PON may play a protective role. A major goal of the present proposal will be to define natural boundaries wherein HDL can accept and degrade PL-OOH without sacrificing LCAT activity. It is hypothesized that the first step in HDL degrading PL-OOH involves the transfer of PL-OOH from LDL to HDL; if allowed to accumulate in HDL, PL-OOH directly inactivate the LCAT enzyme. A sensitive HPLC equipped with an on-line, post-column chemiluminescence detection system will be used to examine the transfer of PL-OOH from LDL to HDL. State-of-the-art Electrospray Mass Spectroscopy and protein sequencing techniques will be employed to identify the oxidation products forming specific amino acid adducts directly involved in LCAT inactivation. Site directed mutagenesis will be performed to genetically engineer an active LCAT enzyme resistant to the inhibitory effects of PL-OOH; thus, the underlying mechanism of LCAT impairment will be definitively established. Purified preparations PL-OOH, LCAT and PON enzymes will be used to define the capacity of HDL to accept/degrade PL-OOH without sacrificing LCAT activity. The proposed studies will greatly advance our understanding of the atherogenic process by defining deleterious effects of oxidized lipids on HDL cholesterol transport. Moreover, the research will uncover novel mechanisms for preserving HDL/LCAT function that can be utilized therapeutically to fight the onset of cardiovascular disease.

人们认为，动脉壁中低密度脂蛋白（LDL）的最小氧化被认为可以通过造成巨噬细胞泡沫细胞的形成来引发动脉粥样硬化过程。 高密度脂蛋白（HDL）从巨噬细胞泡沫细胞中促进过量胆固醇的外流，从而逆转了动脉粥样硬化过程。酶卵磷脂胆固醇酰基转移酶（LCAT）在HDL上酯化胆固醇，因此促进了泡沫细胞中胆固醇的净外排。 结果，LCAT在逆转动脉瘤病变时起着重要的保护功能。最近的研究表明，HDL可以通过接受最小氧化的LDL的氧化脂质来发挥有益的功能，从而抑制早期动脉粥样硬化。 然而，已经发现亲脂性氧化产物会产生对LCAT活性的显着抑制作用，这表明将氧化脂质从最小氧化的LDL转移到HDL可能会损害HDL功能并加剧产生动脉粥样硬化病变的HDL功能。 该提案的目的是1）确定抑制LCAT活性的氧化脂质，2）阐明LCAT损伤的潜在机制，以及3）确定HDL相关酶，副氧蛋白酶（PON）是否可以保护LCAT活性免受LIPIDEXIDES的特定分子的脂质分子的保护。 初步结果表明，磷脂氢过氧化物（PL-OOH）的生理浓度是LCAT活性的有效抑制剂，并且PON可能起保护作用。 本提案的一个主要目标是定义自然界限，其中HDL可以在不牺牲LCAT活动的情况下接受并降低PL-OOH。假设HDL降解PL-OOH的第一步涉及将PL-OOH从LDL转移到HDL。如果允许在HDL中积聚，则PL-OOH直接使LCAT酶失活。 配备了在线，柱后化学发光检测系统的敏感HPLC将使用PL-OOH从LDL到HDL的转移。 将采用最先进的电喷雾质谱和蛋白质测序技术来识别直接参与LCAT失活的特定氨基酸加合物的氧化产物。 将进行定位的诱变，以遗传来设计一种活性LCAT酶，可抵抗PL-OOH的抑制作用。因此，将确定LCAT损伤的潜在机制。 纯化的制剂PL-OOH，LCAT和PON酶将用于定义HDL接受/降解PL-OOH的能力，而无需牺牲LCAT活性。 提出的研究将通过定义氧化脂质对HDL胆固醇转运的有害影响，从而大大提高我们对动脉粥样硬化过程的理解。此外，该研究将发现可保留HDL/LCAT功能的新型机制，该机制可以在治疗上用于抵抗心血管疾病的发作。