Endothelial Activation By An Isoprostane Phospholipid

描述(申请人提供)：这些研究的目标是完成1-棕榈酰基-2-(5，6-环氧异前列腺素E2)-甘油-3-磷酰胆碱(PEIPC)的合成，确定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有效炎症活性的原因的假说。我们已经证明，PEIPC的异构体可以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支持体内的炎症作用，已被证明在高脂饲料喂养的动物的脂蛋白、动脉粥样硬化病变、濒死细胞和经细胞因子处理的细胞中积累。PEIPC是一种很难从脂质混合物中分离出足够数量用于实验的分子。因此，我们开发了合成PEIPC的5，6环氧异构体的方法。我们已经完成了这个分子中环氧异前列烷基团合成的最复杂的方面。拟议的研究将通过将异前列腺素与商业上可获得的溶血磷脂酰胆碱连接起来，完成5，6环氧异构体的全合成。在目前的授权期内，我们已经做了一个重要的观察，在中性pH下PEIPC可以与许多细胞膜蛋白共价连接，其中之一可能是它的受体。我们将通过a)确定其与候选氨基酸(如硫醇、胺和胍)反应产物的结构来鉴定PEIPC的反应功能；b)鉴定与PEIPC结合的选定蛋白质的肽序列。我们将制备几个PEIPC的紧密结构类似物，其中反应性官能团被结构相似但活性较低的基团取代，以保持与受体的合理结合而不与其反应。这些化合物将被测试其刺激内皮炎性活动和抑制内皮细胞的PEIPC激活、合成趋化因子(MCP1和IL-8)以及诱导单核细胞结合分子纤维连接蛋白在内皮细胞表面沉积的能力。这些研究的成功完成将有助于深入了解PEIPC的作用机制，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效应。

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.