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形成而启动致动脉粥样硬化过程。 高密度脂蛋白（HDL）促进过量胆固醇从巨噬细胞泡沫细胞流出，从而逆转动脉粥样硬化过程。酶卵磷脂胆固醇酰基转移酶（LCAT）使胆固醇在HDL上变性，从而促进胆固醇从泡沫细胞的净流出。 因此，LCAT在逆转动脉粥样硬化病变中起重要的保护作用。最近的研究表明，HDL可能通过接受来自最低氧化LDL的氧化脂质从而抑制早期动脉粥样硬化形成而发挥有益的功能。 然而，已经发现亲脂性氧化产物产生对LCAT活性的显著抑制，表明氧化脂质从最低限度氧化的LDL转移至HDL可能损害HDL功能并加剧发展中的动脉粥样硬化病变。 本提案的目的是：1）鉴定抑制LCAT活性的氧化脂质，2）阐明LCAT损伤的潜在机制，3）确定HDL相关酶对氧磷酶（PON）是否可以保护LCAT活性免受脂质过氧化物特定分子种类的影响。 初步结果表明，生理浓度的磷脂过氧化氢（PL-OOH）是LCAT活性的有效抑制剂，PON可能起保护作用。 本提案的主要目标是定义HDL可以接受和降解PL-00 H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直接破坏LCAT酶。 一个灵敏的HPLC配备了在线，柱后化学发光检测系统将用于检查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鉴定形成直接参与LCAT灭活的特定氨基酸加合物的氧化产物。 将进行定点诱变，以遗传工程改造对PL-OOH抑制作用具有抗性的活性LCAT酶;因此，将明确确立LCAT损伤的潜在机制。 将使用纯化制剂PL-0 OH、LCAT和PON酶来确定HDL接受/降解PL-0 OH而不牺牲LCAT活性的能力。 通过确定氧化脂质对高密度脂蛋白胆固醇转运的有害作用，拟议的研究将大大推进我们对致动脉粥样硬化过程的理解。此外，该研究还将揭示保护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.