Lipoxygenases in Atherosclerosis

描述（由申请人提供）：动脉粥样硬化是一种慢性血管炎症性疾病，常发展为衰弱或致命的心肌和中风事件。巨噬细胞与淋巴细胞和平滑肌细胞相互作用，在这种疾病的进展中起着关键作用。12/15-脂氧合酶（12/15-LO）和5-脂氧合酶（5-LO）存在于巨噬细胞亚群中，可以氧化积累的脂质形成氢过氧化物和白三烯，具有多种强效的促炎作用。在此拨款的上一个周期中产生的数据为在三种不同的小鼠模型中12/15-LO的促动脉粥样硬化作用提供了强有力的证据，并初步了解了所涉及的机制。最近来自其他实验室的数据表明，5-LO存在于动脉粥样硬化病变中，并暗示该基因是小鼠动脉粥样硬化的主要易感基因。本建议的总体目标是建立脂氧合酶在动脉粥样硬化中的作用和机制。一个中心假设是，表达12/15-LO和5-LO的巨噬细胞亚群可以通过特定的促炎基因信号网络促进动脉粥样硬化。在Specific Aim 1中，我们将研究12/15-LO和5-LO在动脉粥样硬化中的表达及其作用。对于这些酶在人类和小鼠病变发展过程中的真实表达模式，目前存在相当大的争议。我们将利用免疫组织化学方法研究动脉粥样硬化易感小鼠中12/15-LO和5-LO的表达模式，并利用激光捕获显微解剖技术研究特定巨噬细胞群体中的表达模式。5-LO在apoE和LDL-R遗传背景小鼠的整个生命周期中在动脉粥样硬化中的作用将通过表面病变分析来检验，并探索其与12/15-LO的潜在相加或协同作用。动脉粥样硬化模型和微阵列研究的初步数据表明，通过基因破坏抑制脂氧合酶会影响几种重要细胞因子和炎症介质的表达，这可能为脂氧合酶在动脉粥样硬化中的作用提供了解释。在特异性目标2中，我们将采用微阵列方法检测与C57BL/6野生型对照相比，12/15-LO和5-LO缺陷巨噬细胞中基因表达的变化。候选基因的表达变化将得到验证，并探索与脂氧合酶/细胞因子信号传导的功能联系。在Specific Aim 3中，将建立小干扰RNA （siRNA）基因沉默方法来阻断巨噬细胞脂氧合酶的表达。总之，这些研究将阐明巨噬细胞中脂氧合酶途径在动脉粥样硬化疾病中的重要性。

DESCRIPTION (provided by applicant): 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.