ENDOTHELIAL ACTIVATION BY AN ISOPROSTANE PHOSPHOLIPID

本提案的总体目标是合成并用于生物学研究一种新的生物活性环氧异前列腺烷磷脂，它可能在动脉粥样硬化病变的发展中起重要作用。1-棕榈酰-2-(5,6-环氧异前列腺素E2)- san -甘油-3-磷酸胆碱（PEIPC），质量828.5，存在于最低氧化LDL （MM-LDL）中，在动脉粥样硬化病变中增加。PEIPC是MM-LDL的五种828.5异构体之一，在初步研究中，与其他相对不活跃的异构体相比，PEIPC在体外可引起内皮细胞结合单核细胞的强烈激活。拟议的研究将验证PEIPC通过与paf样受体相互作用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进行的。与其他生物活性磷脂相比，828.5的异构体在高密度脂蛋白中高度积累，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（HDL）的828.5水平升高，而其他研究显示HDL对内皮细胞活化的保护作用较弱。高密度脂蛋白已被证明与至少一种清道夫受体结合。Witztum小组的研究表明，l-棕榈酰-2- (oxovaleroyl)- n-甘油-3-磷酸胆碱（POVPC）是另一种与PEIPC具有抗原相似性的生物活性磷脂，也能与巨噬细胞清道夫受体结合。我们将验证在内皮细胞中，PEIPC与主要清清剂受体lox - 1结合，阻断其与HDL的正常相互作用的假设。最后，有证据表明，一些氧化磷脂可能与蛋白质结合，并以这种内合形式与细胞相互作用。PEIPC的结构表明它可能在生理或病理生理条件下与蛋白质结合。这种内合的磷脂可能比游离的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。开展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。该提案是由一位在含环氧化物分子合成方面经验丰富的化学家、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共同研究者共同提交的。总之，这一跨学科的建议将提供关于新型环氧异前列腺烷磷脂PEIPC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.