ENDOTHELIAL ACTIVATION BY AN ISOPROSTANE PHOSPHOLIPID

这项建议的总体目标是合成一种新的生物活性环氧异前列腺素磷脂，并将其用于生物学研究，该磷脂可能在动脉粥样硬化病变的发展中发挥重要作用。1-棕榈酰基-2-(5，6-环氧基异前列烷E_2)-甘油-3-磷胆碱，质量为828.5，存在于低密度脂蛋白中，在动脉粥样硬化病变中增加。PEIPC是MM-低密度脂蛋白的五个828.5异构体之一，初步研究表明，与其他相对不活跃的异构体相比，它在体外能强烈激活内皮细胞与单核细胞结合。拟议的研究将检验PEIPC通过与PAF样受体相互作用来激活内皮细胞的假设，并将表征与该受体的结合。我们对这种质量的分子的大部分初步研究都是用混合异构体完成的。828.5的异构体在高密度脂蛋白中高度积累(与其他生物活性磷脂相比)，可能是因为高密度脂蛋白酶比其他生物活性磷脂更不容易降解828.5的异构体。在初步研究中，我们已经证明，高密度脂蛋白，其他人显示，对内皮激活的保护作用较弱，含有增加的828.5的水平。高密度脂蛋白已被证明与至少一个清道夫受体结合。威茨图姆研究小组的研究表明，另一种抗原性类似的生物活性磷脂L-棕榈酰基-2-(氧代戊酰基)-sn-甘油-3-磷胆碱也与巨噬细胞清道夫受体结合。我们将检验这样一种假设，即在内皮细胞中，PEIPC与主要的清道夫受体LOX-L结合，阻止其与高密度脂蛋白的正常相互作用。最后，有证据表明，一些氧化的磷脂可能与蛋白质结合，并以这种加合形式与细胞相互作用。PEIPC的结构表明，在生理或病理生理条件下，它可能与蛋白质结合。这种加合磷脂可能比游离生物活性磷脂更稳定。我们将检验PEIPC与蛋白质和多肽形成加合物的假设；我们将使用这些加合物进行有限的研究。进行这些研究的关键是获得可靠的PEIPC供应和获得放射性标记分子的能力。这项提案是由一位在合成含有环氧化物分子方面具有丰富经验的化学家、一位在测试脂质内皮相互作用方面经验丰富的细胞生物学家和一位在这两个领域都有经验的合作研究员提交的联合意见书。综上所述，这项跨学科的建议将提供有关新型环氧异前列烷磷脂PEIPC的受体相互作用的重要信息。

The overall goal of this proposal is to synthesize and use for biologic studies a novel bioactive epoxy isoprostane phospholipid that may play an important role the development of atherosclerotic lesions. 1-Palmitoyl-2- (5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC), mass 828.5, is present in minimally oxidized LDL (MM-LDL) and increased in atherosclerotic lesions. PEIPC is one of five 828.5 isomers in MM-LDL and in preliminary studies causes a strong activation (compared to the other relatively inactive isomers) of endothelial cell to bind monocytes in vitro. The proposed studies will test the hypothesis that PEIPC activates endothelial cells by interacting with a PAF-like receptor and will characterize binding to this receptor. Most of our preliminary studies of molecules of this mass have been done with mixed isomers. Isomers of 828.5 were shown to highly accumulate in HDL (compared to other bioactive phospholipids) probably because HDL enzymes less easily degrade isomers of 828.5 than other bioactive phospholipids. In preliminary studies we have demonstrated that HDL, shown by others to be less protective against endothelial activation, contained increased levels of 828.5. HDL has been shown to bind at least one of the scavenger receptors. Studies by the Witztum group have shown that l-Palmitoyl-2- (oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), another bioactive phospholipid with antigenic similarity to PEIPC, also binds to macrophage scavenger receptors. We will test the hypothesis that, in endothelial cells, PEIPC binds to the major scavenger receptor LOX-l blocking its normal interaction with HDL. Finally there is evidence that some oxidized phospholipids may bind to proteins and interact with cells in this adducted form. The structure of PEIPC suggests that it may bind to proteins under physiological or pathophysiological conditions. Such adducted phospholipids may be more stable than free bioactive phospholipids. We will test the hypothesis that PEIPC forms adduct with proteins and peptides; and we will perform limited studies using these adducts. Key to carrying out these studies is the availability of a reliable supply of PEIPC and the ability to obtain radiolabeled molecules. This proposal is a combined submission from a chemist with extensive experience in the synthesis of epoxide containing molecules, a cell biologist experienced with testing lipid endothelial interactions and a coinvestigator experienced in both areas. In summary, this interdisciplinary proposal will provide important information about the receptor interactions of the novel epoxy isoprostane phospholipid PEIPC.