Convergence of the Cox-2 and 5-Lipoxygenase Pathways

描述(申请人提供)：在炎症和其他生理和病理生理过程中形成的二十烷基类脂质介体家族包括公认的前列腺素、白三烯、脂素和花生四烯酸的羟基和环氧衍生物。这些短暂的类脂类化合物调节炎症事件的发生和消退以及免疫过程中的关键步骤。前列腺素和白三烯分别通过环氧合酶-2(COX-2)或5-脂氧合酶(5-LOX)氧化花生四烯酸后通过不同的途径合成。出乎意料的是，我们发现5-LOX和COX-2酶可能是同时作用的，导致了以前未知的二十烷类化合物的生物合成：5-LOX和COX-2连续氧化AA产生不稳定的双内酯，它重排成两个新的半酮醛二十烷类化合物，我们将其命名为半酮醛(HK)D2和HKE2。在初步实验中，我们显示了HKD2和HKE2在激活的人白细胞混合物中的生物合成及其刺激内皮细胞小管生成的生物活性，这两个关键发现表明半酮类化合物是一种新的炎症中的类脂类化合物。在特定的目标1中，我们将检验这样的假设，即半缩酮的生物合成受内源性5-HETE的w-3类似物调控，并且非类固醇抗炎药和环氧合酶-2特异性药物影响半酮合成的剂量低于前列腺素的形成，这意味着治疗应用低剂量的非类固醇抗炎药可以影响半酮的水平，同时避免前列腺素的形成和相关的胃肠道或心血管副作用。我们亦会以全化学合成的方法制备HKD2和HKE2，以全面分析它们的生物效应。在特定目标2中，我们将测试半酮是通过跨细胞生物合成形成的，表达5-LOX的白细胞为激活的巨噬细胞或内皮细胞表达的COX-2提供5-HETE底物(例如，中性粒细胞、嗜酸性粒细胞或嗜碱性粒细胞)。作为一种典型的炎症性疾病，在人类动脉粥样硬化病变的不同阶段和在两种自发炎症/消退的动物模型中，半酮类药物的定量将补充这些研究。在具体目标3中，我们将确定半酮类化合物作为内皮细胞小管形成、单核细胞与内皮细胞黏附以及参与炎症反应的受体和转录因子的激活的调节因子的作用和机制。总之，这些实验旨在定义5-LOX/COX-2衍生的半酮类化合物为新的类脂类化合物，具有调节炎症过程的功能。现有抗炎药物的新治疗应用迫在眉睫。

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.