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脂质介质家族包括公认的前列腺素、白三烯、脂毒素以及花生四烯酸的羟基和环氧衍生物。这些短暂的脂质类自身调节炎症事件的发生和解决以及免疫处理的关键步骤。前列腺素和白三烯分别在花生四烯酸被环氧化酶-2 （COX-2）或5-脂氧化酶（5-LOX）初始氧化后通过不同的途径进行生物合成。出乎意料的是，我们发现5-LOX和COX-2酶可以串联作用，导致以前未被识别的二十烷类化合物的生物合成：5-LOX和COX-2连续氧化AA产生不稳定的二内过氧化物，重新排列成两种新的半晶状二十烷类化合物，我们将其命名为半晶状（HK） D2和HKE2。在初步实验中，我们已经证明了HKD2和HKE2在活化的人类白细胞混合物中的生物合成，以及它们在刺激内皮细胞小管形成方面的生物活性，这两个关键发现暗示了半酮类物质在炎症中是一种新的脂质类药物。在Specific Aim 1中，我们将验证半酮类药物的生物合成是由内源性5-HETE的w-3类似物调节的假设，以及非甾体抗炎药和COX-2特异性药物在低于前列腺素形成的剂量下影响半细胞形成的假设，这意味着低剂量非甾体抗炎药的治疗应用可以影响半细胞水平，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。我们亦会采用全化学合成方法制备HKD2及HKE2，以全面分析其生物效应。在特异性目标2中，我们将验证半酮是通过表达5-LOX的白细胞提供5-HETE底物（例如，中性粒细胞，嗜酸性粒细胞或嗜碱性粒细胞）的跨细胞生物合成形成的，用于活化的巨噬细胞或内皮细胞中表达的COX-2。这些研究将通过在人类动脉粥样硬化病变不同阶段（作为一种典型炎症疾病）和两种自发炎症/消退动物模型中半酮的定量来补充。在Specific Aim 3中，我们将确定半酮类药物作为内皮细胞小管形成、单核细胞粘附内皮细胞以及参与炎症反应的受体和转录因子激活的调节剂的作用和机制。这些实验旨在将5-LOX/COX-2衍生的半酮类化合物定义为在炎症过程调节中具有功能作用的新型脂质类药物。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.