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: 2909313
• 负责人: JOHN K BIELICKI
• 金额: $ 17.95万
• 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/2909313
• 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.

动脉壁中低密度脂蛋白（LDL）的最小氧化被认为通过促进巨噬细胞泡沫细胞的形成而引发动脉粥样硬化过程。 高密度脂蛋白（HDL）促进过量胆固醇从巨噬细胞泡沫细胞中流出，从而逆转动脉粥样硬化过程。卵磷脂胆固醇酰基转移酶 (LCAT) 酯化 HDL 上的胆固醇，从而促进胆固醇从泡沫细胞中净流出。 因此，LCAT 在逆转动脉粥样硬化病变方面发挥着重要的保护功能。最近的研究表明，HDL 可能通过接受最低限度氧化的 LDL 中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形成。 然而，已发现亲脂性氧化产物对 LCAT 活性产生显着抑制，表明氧化脂质从最低限度氧化的 LDL 转移到 HDL 可能会损害 HDL 功能并加剧动脉粥样硬化病变的发展。 该提案的目的是 1) 识别抑制 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 相关酶对氧磷酶 (PON) 是否可以保护 LCAT 活性免受特定影响脂质过氧化物的分子种类。 初步结果表明，生理浓度的磷脂氢过氧化物 (PL-OOH) 是 LCAT 活性的有效抑制剂，而 PON 可能发挥保护作用。 本提案的主要目标是定义 HDL 可以接受和降解 PL-OOH 而不牺牲 LCAT 活性的自然边界。据推测，HDL 降解 PL-OOH 的第一步涉及 PL-OOH 从 LDL 转移到 HDL；如果允许在 HDL 中积累，PL-OOH 会直接使 LCAT 酶失活。 配备在线柱后化学发光检测系统的灵敏 HPLC 将用于检查 PL-OOH 从 LDL 到 HDL 的转移。 最先进的电喷雾质谱和蛋白质测序技术将用于鉴定形成直接参与 LCAT 失活的特定氨基酸加合物的氧化产物。 将进行定点诱变，以基因工程设计出一种能够抵抗 PL-OOH 抑制作用的活性 LCAT 酶；因此，LCAT损伤的根本机制将被明确建立。 纯化制剂 PL-OOH、LCAT 和 PON 酶将用于确定 HDL 接受/降解 PL-OOH 而不牺牲 LCAT 活性的能力。 拟议的研究将通过定义氧化脂质对高密度脂蛋白胆固醇转运的有害影响，极大地增进我们对动脉粥样硬化过程的理解。此外，该研究还将揭示保留 HDL/LCAT 功能的新机制，可用于治疗心血管疾病的发作。