Convergence of the Cox-2 and 5-Lipoxygenase Pathways

描述（由申请人提供）：在炎症和其他生理和病理生理过程中形成的类二十烷酸脂质介质家族包括公认的花生四烯酸、白三烯、脂氧素和花生四烯酸的羟基和环氧衍生物。这些短寿命的脂质类物质调节炎症事件的发生和消退以及免疫学处理中的关键步骤。前列腺素和白三烯分别通过环氧合酶-2（考克斯-2）或5-脂氧合酶（5-LOX）对花生四烯酸进行初始氧化后，通过不同的途径进行生物合成。非常出乎意料地，我们发现5-LOX和考克斯-2酶可以串联作用，导致以前未识别的类二十烷酸的生物合成：AA通过5-LOX和考克斯-2的连续氧化产生不稳定的二内过氧化物，其重排为两种新的半缩酮类二十烷酸，我们命名为半缩酮（HK）D2和HKE 2.在初步实验中，我们已经显示了HKD 2和HKE 2在活化的人白细胞的混合物中的生物合成以及它们在刺激内皮细胞小管形成中的生物活性，这两个关键发现暗示了半缩酮作为炎症中的新型脂质autacoids。在具体目标1中，我们将检验半缩酮的生物合成受内源性5-HETE的w-3类似物调节的假设，以及NSAID和考克斯-2特异性药物在低于前列腺素形成的剂量下影响半缩酮形成的假设，这表明低剂量NSAID的治疗应用可以影响半缩酮水平，同时避免前列腺素形成和相关的胃肠道或心血管副作用。我们还将通过全化学合成制备HKD 2和HKE 2，以全面分析其生物效应。在具体目标2中，我们将检验半缩酮是通过用表达5-LOX的白细胞提供5-HETE底物（例如，嗜中性粒细胞、嗜酸性粒细胞或嗜碱性粒细胞）对在活化的巨噬细胞或内皮细胞中表达的考克斯-2。这些研究将通过在人类动脉粥样硬化病变（典型炎症性疾病）的不同阶段和两种自发炎症/消退的动物模型中对半缩酮进行量化来补充。在特定目标3中，我们将确定半缩酮作为内皮细胞小管形成、单核细胞粘附于内皮细胞以及参与炎症反应的受体和转录因子的活化的调节剂的作用和机制。这些实验一起被设计为将5-LOX/考克斯-2衍生的半缩酮定义为在炎症过程的调节中具有功能作用的新型脂质自体素。现有的抗炎药物的新的治疗应用迫在眉睫。

DESCRIPTION (provided by applicant): The families of eicosanoid lipid mediators formed during inflammation and other physiological and pathophysiological processes include the well-established prostaglandins, leukotrienes, lipoxins, and hydroxy- and epoxy-derivatives of arachidonic acid. These short-lived lipid autacoids regulate crucial steps in the onset and resolution as well as immunological processing of an inflammatory event. Prostaglandins and leukotrienes are biosynthesized via separate pathways following the initial oxygenation of arachidonic acid by either cyclooxygenase-2 (COX-2) or 5-lipoxygenase (5-LOX), respectively. Quite unexpectedly, we discovered that the 5-LOX and COX-2 enzymes could be acting in tandem resulting in the biosynthesis of previously unrecognized eicosanoids: Consecutive oxygenation of AA by 5-LOX and COX-2 gives an unstable di- endoperoxide that rearranges to two novel hemiketal eicosanoids that we named hemiketal (HK) D2 and HKE2. In preliminary experiments we have shown biosynthesis of HKD2 and HKE2 in mixtures of activated human leukocytes and their biological activity in stimulating endothelial cell tubulogenesis, two key findings that implicate the hemiketals as novel lipid autacoids in inflammation. In Specific Aim 1 we will test the hypothesis that biosynthesis of the hemiketals is regulated by endogenous w-3 analogs of 5-HETE, and that NSAIDs and COX-2 specific drugs affect hemiketal formation at lower doses than prostaglandin formation, implicating that therapeutic application of low-dose NSAIDs could affect hemiketal levels while sparing prostaglandin formation and the associated gastrointestinal or cardiovascular side-effects. We will also prepare HKD2 and HKE2 by total chemical synthesis for comprehensive analysis of their biological effects. In Specific Aim 2 we will test the hypothesis that the hemiketals are formed through transcellular biosynthesis with a 5-LOX expressing leukocyte providing the 5-HETE substrate (e.g., a neutrophil, eosinophil, or basophil) for COX-2 expressed in an activated macrophage or endothelial cell. These studies will be complemented by the quantification of hemiketals in different stages of human atherosclerotic lesions as a prototypical inflammatory disease and in two animal models of spontaneous inflammation/resolution. In Specific Aim 3 we will determine the role and mechanism of hemiketals as regulators of endothelial cell tubulogenesis, monocyte adhesion to endothelial cells, and activation of receptors and transcription factors involved in the inflammatory response. Together these experiments are designed to define the 5-LOX/COX-2 derived hemiketals as novel lipid autacoids with a functional role in the regulation of the inflammatory process. Novel therapeutic applications of available anti- inflammatory medications are imminent.