Endothelial activation by an isoprostane phospholipid

描述（申请人提供）：1-棕榈酰-2-(5,6-环氧异prostanoyl E)- cn -甘油-3-磷脂酰胆碱（PEIPC）正在成为血管细胞功能的主要调节剂。在内皮细胞中，它已被证明可以增加炎症，促进凝血反应，调节连接通透性和增加氧化应激。在巨噬细胞中，它已被证明可以调节树突状细胞的分化。本研究的目的是合成足够数量的最活跃的非对映体，以深入了解PEIPC的作用机制。在前人的研究中，我们合成了一个与天然PEIPC核磁共振一致的PEIPC异构体。在目标1中，我们现在将合成消旋异构体，并确定在调节内皮细胞反应的两个异构体中最活跃的。我们已经观察到PEIPC可以与至少20种内皮细胞蛋白共价结合。在目标2中，我们将确定与蛋白质相互作用的PEIPC功能（可能是烯酮或环氧化物）。我们将首先检查与氨基酸官能团的相互作用，然后检查PEIPC最活跃的官能团与肽的相互作用。利用结合PEIPC的两种模型蛋白VEGFR2和H-ras，我们将确定与PEIPC或其游离脂肪酸结合的氨基酸序列。使用电喷雾电离串联质谱法，这些特定的脂质-蛋白相互作用产生的独特碎片谱将被鉴定用于发现PEIPC的其他内皮细胞蛋白靶点。了解了PEIPC与模型蛋白共价结合的机制后，我们将合成PEIPC类似物并测试它们对PEIPC作用的影响。我们之前已经确定，VEGFR2的激活是OxPAPC和PEIPC介导的ERK和SREBP激活所必需的。我们还确定了H-Ras的失活对于导致单核细胞结合的β 1整合素的激活是必要的。拮抗剂对这些反应的影响将被确定。总的来说，这些研究将确定PEIPC与控制内皮细胞功能的选定蛋白质相互作用的化学性质，并将验证PEIPC的共价结合激活控制内皮细胞炎症反应的途径的假设。公共卫生相关性：氧化磷脂在动脉粥样硬化病变和其他慢性炎症部位积聚，已被证明是内皮细胞炎症和促凝反应的重要调节剂，有助于动脉粥样硬化。这些研究将深入了解一种特殊的氧化磷脂（PEIPC）是如何引起这种激活的，并将开发类似物来抑制激活。因此，这些研究可能为动脉粥样硬化和其他慢性炎症疾病提供新的药物靶点。

DESCRIPTION (provided by applicant): 1-Palmitoyl-2-(5,6-epoxyisoprostanoyl E )-sn-glycero-3-phosphatidylcholine (PEIPC) is emerging as a major regulator of vascular cell function. In endothelial cells it has been demonstrated to increase inflammation, procoagulant responses, to regulate junction permeability and to increase oxidative stress. In macrophages it has been demonstrated to regulate dendritic cell differentiation. The goal of the proposed studies is to synthesize sufficient quantities of the most active diastereomer in order to gain insight into the mechanism of action of PEIPC. In previous studies we synthesized one isomer of PEIPC consistent with the NMR of the natural PEIPC. In Aim 1 we will now synthesize the diasteromer and identify the most active of the two isomers in regulating endothelial cell responses. We have observed that PEIPC can covalently bind to at least 20 endothelial cell proteins. In Aim 2 we will identify the PEIPC functionality (likely the enone or the epoxide) that interacts with proteins. We will begin by examining the interaction with functional groups of amino acids, then examine the interaction of the most active functional group of PEIPC with peptides. Using two model proteins that bind PEIPC, VEGFR2 and H-ras, we will determine the amino acid sequence involved in binding of PEIPC or its free fatty acid. Using electrospray ionization-tandem mass spectrometry, unique fragmentation spectra produced by these specific lipid-protein interactions will be identified for use in discovering additional endothelial cell protein targets of PEIPC. Armed with this knowledge of the mechanism of the covalent binding of PEIPC to model proteins, we will synthesize analogues of PEIPC and test their effects on PEIPC action. We have previously determined that activation of VEGFR2 is required for the OxPAPC and PEIPC mediated activation of ERK and SREBP. We have also determined that inactivation of H-Ras is necessary for the activation of beta one integrins that lead to monocyte binding. Effects of antagonists on these responses will be determined. Overall these studies will define the chemistry of the PEIPC interaction with selected proteins that control endothelial cell function and will test the hypothesis that covalent binding of PEIPC activates pathways that control the endothelial cell inflammatory response. PUBLIC HEALTH RELEVANCE: Oxidized phospholipids, which accumulate in atherosclerotic lesions and other chronic inflammatory sites, have been shown to be important regulators of endothelial cell inflammatory and pro-coagulant responses that contribute to atherosclerosis. These studies will gain insight into how a particular oxidized phospholipid, PEIPC, causes this activation and will develop analogues to inhibit activation. These studies thus may provide a new drug target for atherosclerosis and other chronic inflammatory diseases.