Endothelial activation by an isoprostane phospholipid

描述(申请人提供)：1-棕榈酰基-2-(5，6-环氧基异前列腺素E)-sn-甘油-3-磷脂酰胆碱(PEIPC)是一种重要的血管细胞功能调节剂。在内皮细胞中，它已被证明可以增加炎症、促凝血反应、调节连接通透性和增加氧化应激。在巨噬细胞中，它已被证明调节树突状细胞的分化。拟议研究的目标是合成足够数量的最具活性的非对映异构体，以深入了解PEIPC的作用机理。在以往的研究中，我们合成了一个与天然PEIPC的核磁共振谱一致的PEIPC异构体。在目标1中，我们现在将合成非对映异构体，并确定两个异构体中在调节内皮细胞反应方面最活跃的一个。我们观察到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)。我们将首先研究与氨基酸官能团的相互作用，然后研究PEIPC最活跃的官能团与多肽的相互作用。使用两个结合PEIPC的模型蛋白VEGFR2和H-ras，我们将确定与PEIPC或其游离脂肪酸结合有关的氨基酸序列。利用电喷雾电离-串联质谱仪，这些特定的脂质-蛋白质相互作用产生的独特的裂解光谱将被识别出来，用于发现PEIPC的其他内皮细胞蛋白质靶标。在了解了PEIPC与模型蛋白的共价结合机制后，我们将合成PEIPC的类似物，并测试它们对PEIPC作用的影响。我们先前已经确定，在OxPAPC和PEIPC介导的ERK和SREBP的激活中，VEGFR2的激活是必需的。我们还确定了H-RAS的失活对于导致单核细胞结合的β1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。总体而言，这些研究将定义PEIPC与控制内皮细胞功能的选定蛋白质相互作用的化学，并将检验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：氧化磷脂聚集在动脉粥样硬化病变和其他慢性炎症部位，已被证明是导致动脉粥样硬化的内皮细胞炎症和促凝血反应的重要调节因素。这些研究将深入了解一种特定的氧化磷脂PEIPC是如何导致这种激活的，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性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.