ENDOTHELIAL ACTIVATION BY AN ISOPROSTANE PHOSPHOLIPID

本提案的总体目标是合成一种新型生物活性环氧异前列烷磷脂，并将其用于生物学研究，这种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-2-（5，6-环氧异前列烷E2）-sn-甘油-3-磷酸胆碱（PEIPC），质量828.5，存在于最低限度氧化的LDL（MM-LDL）中，并在动脉粥样硬化病变中增加。PEIPC是MM-LDL中的五种828.5异构体之一，并且在初步研究中引起内皮细胞的强烈活化（与其他相对无活性的异构体相比）以在体外结合单核细胞。拟议的研究将测试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初步研究大多是用混合异构体进行的。显示828.5的异构体在HDL中高度积累（与其他生物活性磷脂相比），这可能是因为HDL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，HDL，其他人显示对内皮激活的保护作用较小，含有828.5的水平增加。HDL已显示结合至少一种清道夫受体。 Witztum小组的研究表明，与PEIPC具有抗原相似性的另一种生物活性磷脂1-棕榈酰-2-（氧代戊酰基）-sn-甘油-3-磷酸胆碱（POVPC）也与巨噬细胞清道夫受体结合。我们将检验以下假设：在内皮细胞中，PEIPC结合主要的清道夫受体LOX-1，阻断其与HDL的正常相互作用。最后，有证据表明，一些氧化磷脂可以结合蛋白质，并与这种加合物形式的细胞相互作用。PEIPC的结构表明，它可以在生理或病理生理条件下与蛋白质结合。这样的加合磷脂可以比游离生物活性磷脂更稳定。我们将测试PEIPC与蛋白质和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PEIPC的可靠供应的可用性和获得放射性标记分子的能力。本提案是由一位在含环氧化物分子合成方面具有丰富经验的化学家、一位在检测脂质内皮相互作用方面具有丰富经验的细胞生物学家和一位在这两个领域都具有丰富经验的合作研究者提交的合并提交材料。总之，这一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.