Convergence of the Cox-2 and 5-Lipoxygenase Pathways

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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.