PRESERVATION OF HDL FUNCTION DURING EARLY ATHEROGENESIS

动脉壁中低密度脂蛋白(LDL)的轻微氧化被认为通过促进巨噬细胞泡沫细胞的形成而启动了动脉粥样硬化的形成过程。高密度脂蛋白(高密度脂蛋白)促进过多的胆固醇从巨噬细胞泡沫细胞外流，从而逆转动脉粥样硬化的过程。卵磷脂胆固醇酰基转移酶(LCAT)将胆固醇酯化到高密度脂蛋白上，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性病变中起着重要的保护作用。最近的研究表明，高密度脂蛋白可能通过接受氧化程度最低的低密度脂蛋白的氧化脂质而发挥有益的作用，从而抑制早期动脉粥样硬化的形成。然而，亲脂性氧化产物被发现对LCAT活性产生显著的抑制作用，这表明氧化的脂质从氧化程度最低的低密度脂蛋白转移到高密度脂蛋白可能会损害高密度脂蛋白的功能，并加剧动脉粥样硬化病变的发展。这一建议的目的是：1)确定抑制LCAT活性的氧化脂质；2)阐明LCAT损伤的潜在机制；3)确定高密度脂蛋白相关酶对氧磷酶(PON)是否能保护LCAT活性不受特定分子种类过氧化脂质的影响。初步结果表明，生理浓度的磷脂过氧化氢(PL-OHO)是LCAT活性的有效抑制物，PON可能起到保护作用。本提案的一个主要目标将是定义自然边界，其中高密度脂蛋白可以在不牺牲LCAT活性的情况下接受和降解PL-OOH。假设高密度脂蛋白降解磷脂酰胆碱的第一步涉及将磷脂酰胆碱从低密度脂蛋白转移到高密度脂蛋白；如果允许在高密度脂蛋白中积累，磷脂酰胆碱酯酶直接失活LCAT酶。配有在线柱后化学发光检测系统的灵敏高效液相将被用来检测PL-OOH从低密度脂蛋白到高密度脂蛋白的转移。将使用最先进的电喷雾质谱学和蛋白质测序技术来鉴定形成直接参与LCAT失活的特定氨基酸加合物的氧化产物。我们将进行定点突变，从基因工程的角度设计一种活性的LCAT酶，以抵抗PL-OOH的抑制作用，从而明确LCAT损伤的潜在机制。纯化的制剂PL-OOH、LCAT和PON酶将被用来确定高密度脂蛋白在不牺牲LCAT活性的情况下接受/降解PL-OOH的能力。通过定义氧化脂质对高密度脂蛋白运输的有害影响，建议的研究将极大地促进我们对动脉粥样硬化形成过程的理解。此外，这项研究将揭示保存高密度脂蛋白/低密度脂蛋白胆固醇功能的新机制，这些机制可以在治疗上用于对抗心血管疾病的发作。

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.