Atherosclerosis is a chronic inflammatory disease of the vasculature that often progresses to debilitating or fatal myocardial and stroke events. Macrophages are key players in progression of this disorder, interacting with lymphocytes and smooth muscle cells. 12/15-Lipoxygenase (12/15-LO) and 5-Lipoxygenase (5-LO) are present in subpopulations of macrophages and can oxygenate accumulating lipids to form hydroperoxides and leukotrienes, which have a variety of potent pro-inflammatory actions. Data generated during the past cycle of this grant established strong evidence for a pro-atherogenic role of 12/15-LO in three distinct mouse models and provided initial insight into the mechanisms involved. Recent data from other labs have shown the presence of 5-LO in atherosclerotic lesions and implicated this gene as a major atherosclerosis susceptibility gene in mice. The overall goal of this proposal is to establish the role and mechanisms for lipoxygenases in atherosclerosis. A central hypothesis is that subpopulations of 12/15-LO and 5-LO expressing macrophages can contribute to atherogenesis via specific pro-inflammatory gene signaling networks.
In Specific Aim 1, the expression and roles of 12/15-LO and 5-LO in atherosclerosis will be investigated. Considerable controversy now exists as to the true expression pattern of these enzymes throughout lesion development in humans and mice. We shall investigate expression patterns of 12/15-LO and 5-LO in atherosclerosis prone mice using immunohistochemistry and in specific macrophage populations using laser capture microdissecfion. The role of 5-LO in atherogenesis throughout the lifetime of mice on apoE and LDL-R genetic backgrounds will be examined by en face lesion analysis and potential additive or synergistic actions with 12/15-LO explored. Preliminary data in atherosclerotic models and microarray studies have indicated that lipoxygenase inhibition via gene disruption influences expression of several important cytokine and inflammatory mediators, which may offer an explanation for the roles of lipoxygenases in atherogenesis.
In Specific Aim 2, we will employ a microarray approach to examine the alterations in gene expression in both 12/15-LO and 5-LO deficient macrophages compared to C57BL/6 wildtype controls. Candidate gene changes in expression will be verified and functional links to lipoxygenase/cytokine signaling explored.
In Specific Aim 3, a small interfering RNA (siRNA) gene silencing approach will be instituted to block macrophage lipoxygenase expression. Overall, these studies will illuminate the importance of lipoxygenase pathways in macrophages in relation to atherosclerotic disease.
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