Endothelial Activation By An Isoprostane Phospholipid

描述（由申请人提供）：这些研究的目标是完成1-棕榈酰-2-(5,6-环氧异前列腺素E2)-snglycero-3- phospylcholine （PEIPC）的合成，确定PEIPC与蛋白质相互作用的机制，并验证PEIPC与其受体的共价结合是其有效炎症活性的原因。我们已经证明，PEIPC的异构体能有效地激活内皮细胞合成趋化因子IL-8和MCP-1，并表达含有纤维连接蛋白的单核细胞结合分子CS-1。PEIPC在体内支持炎症作用，已被证明在高脂肪饮食动物的脂蛋白、动脉粥样硬化病变、垂死细胞和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数量用于实验的分子。因此，我们开发了PEIPC的5,6环氧异构体的合成方法。我们已经完成了这个分子环氧异前列腺烷基团合成的最复杂的部分。拟议的研究将完成5,6环氧异构体的全合成，通过连接异前列腺素和市售的溶血磷脂酰胆碱。在目前的资助期间，我们已经进行了重要的观察，即中性pH下的PEIPC可以与许多细胞膜蛋白共价连接，其中一个可能是它的受体。我们将通过a)确定PEIPC与候选氨基酸（如硫醇、胺和胍）反应产物的结构，b)确定PEIPC结合的选定蛋白质的肽序列来确定PEIPC的活性功能。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受体发生反应。这些化合物将被测试其刺激内皮炎症活动和抑制PEIPC激活内皮细胞合成趋化因子（MCP1和IL-8）和诱导单核细胞结合分子纤维连接蛋白沉积在内皮细胞表面的能力。这些研究的成功完成将提供对PEIPC的作用机制的深入了解，PEIPC是一种有效的炎症介质，并将提供一种潜在的治疗方法来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.