PRESERVATION OF HDL FUNCTION DURING EARLY ATHEROGENESIS

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来启动动脉粥样硬化过程。高密度脂蛋白（HDL）促进巨噬细胞泡沫细胞多余胆固醇的外排，从而逆转动脉粥样硬化过程。卵磷脂胆固醇酰基转移酶（LCAT）将胆固醇在高密度脂蛋白上酯化，从而促进胆固醇从泡沫细胞的净流出。因此，LCAT对逆转动脉粥样硬化病变具有重要的保护作用。最近的研究表明，HDL可能通过接受来自最低限度氧化LDL的氧化脂质而发挥有益的功能，从而抑制早期动脉粥样硬化的发生。然而，已经发现亲脂氧化产物对LCAT活性产生显著的抑制作用，这表明氧化脂质从最低限度氧化的LDL向HDL的转移可能损害HDL的功能并加剧正在发生的动脉粥样硬化病变。本研究的目的是：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hdl相关酶对氧氧化酶（PON）是否可以保护LCAT活性免受特定分子种类的脂质过氧化物的影响。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，PON可能发挥保护作用。本提案的一个主要目标是定义HDL可以在不牺牲LCAT活性的情况下接受和降解PL-OOH的自然边界。据推测，HDL降解PL-OOH的第一步涉及PL-OOH从LDL向HDL的转移；如果允许在HDL中积累，PL-OOH直接使LCAT酶失活。配备在线柱后化学发光检测系统的灵敏高效液相色谱将用于检测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被用来鉴定氧化产物形成特定的氨基酸加合物直接参与LCAT失活。位点定向诱变将用于基因工程，使活性LCAT酶抵抗PL-OOH的抑制作用；从而明确LCAT损伤的潜在机制。纯化的PL-OOH、LCAT和PON酶将用于确定HDL在不牺牲LCAT活性的情况下接受/降解PL-OOH的能力。提出的研究将通过定义氧化脂质对高密度脂蛋白胆固醇运输的有害影响，极大地促进我们对动脉粥样硬化过程的理解。此外，该研究将揭示保持HDL/LCAT功能的新机制，可用于治疗心血管疾病的发作。

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.