Regulation of Endothelial Cell Inflammatory Responses

这些研究的目的是确定特定的磷脂氧化产物1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine(PEIPC)和1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine(POVPC)作为内皮细胞炎症反应调节因子的重要性。这些磷脂已经被证明通过非经典的信号转导途径增加单核细胞-内皮细胞和抑制中性粒细胞-内皮细胞的相互作用。这些磷脂在体内的重要性是通过它们在动脉粥样硬化病变和其他慢性炎症部位的积聚来表明的。我们已经确定了这些生物活性脂类改变内皮细胞功能的候选信号转导途径，并获得了多个受体参与其对IL-8和MCP-1转录和单核细胞结合的影响的证据。这项提议的一个主要目标是完成对这些受体和信号转导途径的识别。在目标1中，我们将测试PEIPC与受体复合体结合的假设，该受体复合体由GPI锚定蛋白和修饰形式的Toll4组成，它改变了肺泡功能。假设这些腔隙变化会导致Src和/或 SREBP。结果，LEF和/或SREBP被移位到细胞核，导致IL-8转录激活。我们发现cAMP及其下游效应因子R-RAS是激活内皮细胞与单核细胞结合的重要信号。在目标2中，我们将表达已知和孤立的脂结合G蛋白偶联受体，以确定介导单核细胞结合的PEIPC受体。已识别的受体将通过腺病毒载体在小鼠的主动脉中过表达，并确定它们对脂肪条纹形成的影响。我们还将使用定量形态学和免疫组织化学方法来确定动脉粥样硬化中所提出的信号转导途径是否发生了变化。在目标3中，我们将检验这一假设 炎症部位磷脂氧化产物的聚集阻止了中性粒细胞的进入。在这些研究中，我们将使用细胞培养和细菌性眼内炎的小鼠模型。为了测试氧化磷脂积累的作用，我们将过度和低表达PON-2，一种降解这些氧化磷脂的酶。拟议的研究将确定POVPC和PEIPC作为动脉粥样硬化和其他慢性炎症过程的介质的重要性。

The goal of these studies is to determine the importance of specific phospholipid oxidation products, 1- palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC) and 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC) as regulators of endothelial cell inflammatory responses. These phospholipids have been shown to increase monocyte-endothelial and inhibit neutrophil-endothelial interactions by non-classical signal transduction pathways. The in vivo importance of these phospholipids is suggested by their accumulation in atherosclerotic lesions and at other sites of chronic inflammation. We have identified candidate signal transduction pathways by which these bioactive lipids alter endothelial cell function and have obtained evidence for the involvement of more than one receptor in their effects on transcription of IL-8 and MCP-1 and monocyte binding. A major goal of this proposal is to complete identification of these receptors and signal transduction pathways. In Aim 1 we will test the hypothesis that PEIPC binds to a receptor complex, composed of a GPI anchored protein and a modified form of Toll 4, which alters caveolar function. These caveolar changes are hypothesized to result in activation of Src and/or SREBP. As a result LEF and/or SREBP are translocated into the nucleus resulting in activation of IL-8 transcription. We have identified cAMP and its downstream effector R-Ras as important signals activating endothelial cells to bind monocytes. In Aim 2 we will express both known and orphan lipid-binding G-protein coupled receptors to identify the PEIPC receptor mediating monocyte binding. The identified receptors will be overexpressed in mouse aorta, using adenovirai transfection, and their effect on fatty streak formation determined. We will also determine, using quantitative morphology and immunohistochemistry, if the proposed signal transduction pathways are altered in atherosclerosis. In Aim 3 we will test the hypothesis that the accumulation of phosholipid oxidiation products at sites of inflammation blocks neutrophil entry. For these studies we will use cell culture and also a mouse model involving bacterial endopthalmitis. To test the role of oxidized phospholipid accumulation, we will over and underexpress PON-2, an enzyme that hydrolyzes these oxidized phospholipids. The proposed studies will determine the importance of POVPC and PEIPC as mediators of atherosclerosis and other chronic inflammatory processes.