Principal investigator/Program Director: (Last, first, middle): Serhan, Charles N. Abstract of Research Plan Inflammation has emerged as a key component in many prevalent human diseases including heart attacks, atherosclerosis, Alzheimer' s, and cancer, as well as those such as arthritis, already well known to be inflammatory diseases.* Therefore, it is important to establish the contribution of lipid mediators (LM) to inflammation as well as its natural resolution. Although LM were widely held to be solely pro- inflammatory or ' bad guys', evidence first obtained via this MERIT (1-39) and now many labs worldwide 1-35 indicates that specific arachidonate-derived lipoxygenase (LOX) products, i.e. lipoxins (LX), their aspirin triggered (AT) epimers, and designer stable analogs, are potent anti-inflammatory and pro-resolving agonists. Recently, we found that transgenic animals (mouse and rabbit) overexpressing key human enzyme and receptors in the LX pathway give reduced inflammatory responses. Employing new mediator-lipidomic techniques that we've introduced permitted identification of several previously unknown LM and novel pathways from essential n-3 fatty acids. These new LM termed resolvins (RV), neuroprotectins (NP), and docosatrienes (DT) are generated during the resolution phase of inflammation indicating, for the first time, that resolution is an active process. In work in progress, these novel LM and their aspirin triggered (AT) epimers actively down-regulate leukocytes and inflammation. Since these are the first results to link n-3 fatty acids, known from many clinical studies to have beneficial effects of importance in many diseases _'36, to the biosynthesis of novel potent LM, the following hypothesis is proposed: The newly uncovered resolvins, neuroprotectins, and docosatrienes are generated in vivo from essential n-3 fatty acids (e.g. DHA and EPA) by novel lipoxygenase-catalyzed pathways and serve both protective and anti-inflammatory roles that regulate the potentially deleterious side effects of host defense. To test this, we will focus on the following aims: 1. Novel fat-1 and human LXA 4 receptor (ALXR) Transgenic (TG) mice inflammatory phenotype. We prepared new TG mice overexpressing ALXR, and our collaborators (see letters) recently prepared fat-1 TG mice with enhanced endogenous n-3 biosynthesis 2. To determine whether the novel protective LM (RVD, NPD and DT) are made from n-3 in vivo and indeed causally responsible for anti-inflammation, we shall test their formation and actions in 3 separate models a) acute colitis inflammation, b) dermal air pouch and c) second organ injury following ischemia-reperfusion. A second set will determine the formation of AT forms of RV and DT in fat-1 transgenic mice given aspirin before and during challenge. 2. Elucidate transcellular biosynthesis of RV, NPD, and DT during innate cellular-neural interactions. In work in progress, human T helper cells in co-incubations with monocytes produce DT and novel related compounds; their biosynthesis will be established in this'proposed period. Also, a series will focus on transcellular biosynthesis between neural cells, rich in DHA, and leukocytes to establish the key pathways for production and their novel protective actions relevant to neuropathic pain and inflammation. 3. Identify intracellular protective signaling pathways activated by Resolvin, NPD, and novel DT. To achieve this, we've set-up new techniques and instrumentation for differential proteomics to assess specific andor unique intracellular signaling pathways initiated by these novel agonists. Here, we will systematically identify intracellular pathways activated by each novel LM class with leukocytes and recombinant receptor expressing cells. A second series will determine whether these LM share intracellular protein signaling. 4. Determine the role(s) of novel microbial and human modified (LOX) in resolvin, neuroprotectin, and docosatriene generation from DHA. In work in progress, we found that a single LOX produces both RVD and NPD 1. Here, we shall establish stereochemistry and catalytic parameters (pH optima, substrate preference) for a new soluble microbial 15-LOX we've identified (see letters). A second series will test the role of LOX modification (i.e. phosphorylation) on substrate utilization with the human leukocyte recombinant 5-LOX. Results will establish the new pathways and enzymatic mechanisms. 5. Human translation requires swift methods for RV, NP and DT identification. Throughout these studies, focus will be directed to new selective, sensitive, and simple techniques (i.e., ELISA) for pathways monitoring these. The long term goals are to l) elucidate the formation and actions of host protective and endogenous anti- inflammatory LM, 2) determine their relevance in inflammatory diseases, pain and relationship to dietary n-3 fatty acids and 3) assess whether these novel pathways are suitable for new therapeutic approaches. * Time Magazine cover, February 23, 2004. PHS 2590 (Rev. 05/01) Page 2 ========================================Section End===========================================
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