Endothelial Activation By An Isoprostane Phospholipid

描述（由申请方提供）：这些研究的目的是完成1-棕榈酰-2-（5，6-环氧异前列烷E2）-甘油基-3-磷酰胆碱（PEIPC）的合成，鉴定PEIPC与蛋白质相互作用的机制，并检验PEIPC与其受体的共价结合是其强效炎症活性的原因这一假设。我们已经表明，PEIPC的异构体有效地激活内皮细胞合成趋化因子IL-8和MCP-1，并表达含有纤连蛋白的单核细胞结合分子CS-1。支持体内炎症作用，已证明PEIPC在来自喂食高脂肪饮食的动物的脂蛋白中、在动脉粥样硬化病变中、在垂死细胞中和在用细胞因子处理的细胞中积累。PEIPC是一种难以从脂质混合物中分离出足够量用于实验的分子。因此，我们开发了PEIPC的5，6环氧异构体的合成方法。我们已经完成了合成该分子的环氧异前列烷基团的最复杂的方面。拟议的研究将通过将异前列烷与市售溶血磷脂酰胆碱连接来完成5，6环氧异构体的全合成。在目前的资助期间，我们已经做出了重要的观察，即PEIPC在中性pH下可以共价连接到许多细胞膜蛋白，其中之一可能是其受体。我们将通过a）确定其与候选氨基酸（例如硫醇、胺和胍）反应的产物的结构B）鉴定PEIPC结合的所选蛋白质的肽序列来鉴定PEIPC的反应性官能度。我们将制备几种结构相似的PEIPC类似物，其中反应性官能团被结构相似但反应性较低的基团取代，以保持与受体的合理结合而不与其反应。这些化合物将测试它们刺激内皮炎症活性和抑制PEIPC激活内皮细胞合成趋化因子的能力（MCP 1和IL-8）并诱导单核细胞结合分子纤连蛋白在内皮细胞表面上的沉积。这些研究的成功完成将为PEIPC（一种有效的炎症介质）的作用机制提供深入了解，并将提供一种潜在的治疗方法，以限制这种脂质在动脉粥样硬化和其他炎症性疾病中的炎症作用。

DESCRIPTION (provided by applicant): The goal of these studies is to complete synthesis of 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-snglycero-3-phosphoryl choline (PEIPC), identify the mechanism by which PEIPC interacts with proteins and to test the hypothesis that covalent binding of PEIPC to its receptor is responsible for its potent inflammatory activity. We have shown that isomers of PEIPC potently activate endothelial cells to synthesize chemokines IL-8 and MCP-1 and express the monocyte binding molecule CS-1 containing fibronectin. Supporting an inflammatory role in vivo, PEIPC has been demonstrated to accumulate in lipoproteins from animals fed a high fat diet, in atheroscierotic lesions, in dying cells and in cells treated with cytokines. PEIPC is a difficult molecule to isolate from lipid mixtures in sufficient quantity for experimentation. We have therefore developed methods for the synthesis of the 5,6 epoxy isomer of PEIPC. We have completed the most complex aspect of the synthesis of the epoxyisoprostane group of this molecule. The proposed studies will complete total synthesis of 5,6 epoxy isomer by linking the isoprostane to commercially available lysophosphatidylcholine. During the current grant period we have made the important observation that PEIPC at neutral pH can covalently link to a number of cell membrane proteins, one of which is likely its receptor. We will identify the reactive functionality of PEIPC by a) determining the structure of the products of its reaction with candidate amino acids (e.g. thiol, amine and guanidine) b) identifying the peptide sequence of selected proteins to which PEIPC binds. We will prepare several close structural analogues of PEIPC in which the reactive functional groups are replaced by structurally similar but less reactive groups in order to maintain reasonable binding to the receptor without reacting with it. These compounds will be tested for their ability to stimulate endothelial inflammatory activities and inhibit PEIPC activation of endothelial cells to synthesize chemokines (MCP1 and IL-8) and to induce deposition of the monocyte binding molecule fibronectin on the endothelial cell surface. The successful completion of these studies will provide insight into the mechanisms of action of PEIPC a potent inflammatory mediator and will provide a potential therapeutic approach to limit the inflammatory effects of this lipid in atherooscierosis and other inflammatory diseases.