Formation of Omega 3-Derived Electrophiles During Inflammation

描述(申请人提供)：动脉粥样硬化占所有心血管疾病死亡的四分之三，动脉粥样硬化血栓疾病预计将在2020年成为全球主要的死亡原因，这是一个主要的健康负担。Omega-3(w-3)多不饱和脂肪酸具有强大的抗炎作用和有益心血管的作用。虽然这些特性得到了广泛的认可，但其潜在的机制在很大程度上仍然不清楚。巨噬细胞激活过程中形成的脂类的亲电图谱显示，在高达200 nM的细胞内浓度下，酮基衍生的w-3脂肪酸omega-3衍生的脂肪酸存在。这些物种的形成依赖于COX-2，并给予阿司匹林增加这些w-3亲电脂肪酸氧基衍生物(EFOX)的水平。最近，EFOX已被证明在人类巨噬细胞、中性粒细胞和受炎症影响的动物组织中形成。此外，已发现EFOX可形成谷胱甘肽和蛋白质加合物，并通过激活Nrf2途径、抑制细胞因子表达和降低诱导型一氧化氮合酶水平来调节炎症途径。这一提议的假设是，EFOX是在炎症和动脉粥样硬化过程中形成的具有抗动脉粥样硬化特性的生物相关分子，通过信号通路的亲电调节转导w-3脂肪酸和阿司匹林的有益效果。为了解决这一假设，我们建议用化学方法产生分析工具(即氚和标记的标准)，以帮助对EFOX进行严格的生物学鉴定和定量。这些标准将用于分析巨噬细胞的生物合成途径，并评估相关的动脉粥样硬化小鼠模型的体内形成。这些物种及其目标的生化和信号特性将使用细胞模型进行分析。最后，将在小鼠动脉粥样硬化模型上测试EFOX的药理作用。在完成时，我们预计已经确定了不同的EFOX异构体物种及其在体内的相关性和形成。此外，我们希望对它们的亲电反应性、对第二阶段基因表达的调控，特别是Nrf2/Keap1途径以及它们的PPARg激活有一个清楚的了解。预计这项建议将导致更好地了解w-3脂肪酸的影响，并改进减少动脉粥样硬化及其有害影响的药理学方法。 公共卫生相关性：在美国，动脉粥样硬化每年导致50多万人死亡。欧米茄-3多不饱和脂肪酸通常存在于鱼油中，具有强大的抗炎作用和有益心血管的作用，尽管其作用机制在很大程度上仍不清楚。我们最近发现，在炎症过程中，人的巨噬细胞和中性粒细胞会形成一种名为EFOX的新型抗炎脂质。我们建议描述这些物种的生化功能，以及它们在动脉粥样硬化中的作用和水平。预计这些结果不仅将增加我们对这种疾病的了解，还将使我们能够更好地设计治疗该疾病的药理学方法。

DESCRIPTION (provided by applicant): Atherosclerosis accounts for three fourth of all deaths from cardiovascular disease and atherothrombotic diseases are projected to be the leading cause of death worldwide in 2020, representing a major health burden. Omega-3 (w-3) polyunsaturated fatty acids exert potent anti-inflammatory actions and beneficial cardiovascular effects. Although these properties are widely recognized, the underlying mechanisms remain largely unknown. An electrophilic mapping of lipids formed during macrophage activation showed that keto-derived w-3 fatty acids omega 3 derived-fatty acids were present at intracellular concentrations up to 200 nM. The formation of these species was dependent on COX-2, and administered aspirin increased the levels of these w-3 electrophilic fatty acid oxo-derivatives (EFOX). Recently, EFOX have been shown to be formed in human macrophages, neutrophils and animal tissues subjected to inflammation. Additionally, EFOX have been found to form glutathione and protein adducts and to modulate the inflammatory pathways through activation of Nrf2 pathway, inhibition of cytokine expression and reduction of inducible nitric oxide synthase levels. The hypothesis of this proposal is that EFOX are biologically relevant molecules with anti-atherogenic properties formed during inflammation and atherosclerosis that transduce the beneficial effects of w-3 fatty acids and aspirin through the electrophilic regulation of signaling pathways. To address this hypothesis we propose to chemically generate analytical tools (i.e. deuterated and labeled standards) to aid in the rigorous biological identification and quantitation of EFOX. These standards will be used for the analysis of biosynthetic pathways in macrophages and to assess the in vivo formation in a relevant murine model of atherosclerosis. The biochemical and signaling properties of these species and its targets will be analyzed using cellular models. Finally, the pharmacological effects of EFOX will be tested on a murine atherosclerosis model. At completion, we expect to have determined the different EFOX isomeric species and their in vivo relevance and formation. In addition, we expect to have gained a clear understanding of their electrophilic reactivities, modulation of phase 2 gene expression, in particular the Nrf2/KEAP1 pathway and their PPARg activation. It is expected that this proposal will lead to a better understanding of w-3 fatty acid effects and to improved pharmacological approaches to decrease atherosclerosis and its detrimental effects. PUBLIC HEALTH RELEVANCE: Atherosclerosis is responsible for over 500,000 deaths per year in the United States. Omega-3 polyunsaturated fatty acids, commonly found in fish oil, exert potent anti-inflammatory actions and beneficial cardiovascular effects although the operative mechanisms remain largely unknown. We recently discovered the formation of novel anti-inflammatory lipids called EFOX by human macrophages and neutrophils during inflammation. We propose to characterize the biochemical functionalities of these species and their effects and levels during atherosclerosis. It is expected that the results will not only increase our understanding of this disease, but also allow for a better design of pharmacological approaches for its treatment.