一类新型LDL磷脂氧化产物在动脉粥样硬化中的作用机制研究

Cardiovascular diseases (CVDs) caused by atherosclerosis are among the leading causes for mortality and morbidity in industrialized countries while China,as a developing country, has even higher mortality rate of CVD than USA and Japan. The molecular mechanisms of atherosclerosis appear to be multifactual and remain to be clearly defined; mounting evidence suggests that chronic inflammatory and oxidative stress play an important role. Atherosclerotic lesion formation resulted from interactions among different cell types including macrophages, endothelial cells, smooth muscle cells, and platelets within the vessel wall. Recent studies have demonstrated that oxidized phospholipids on LDL have deep impact on atherosclerosis. Macrophages are one of the major cell types found in the atherosclerotic plaque and acculating data suggest that macrophges play a critical role in every stage of athersclerosis.The early lesion formation appears to be resulted from phagocytosis of oxidized LDL by macrophage. However, the exact components in oxidized LDL that may be responsible for mediating early lesion formation have not been clearly elucidated. We have identified a novel class of oxidized phospholipid in oxidized LDL in vitro and in human atherosclerotic plaques. These free radical oxidation products were termed deoxy-A2/J2-IsoP-PC due to the presence of a cyclopentenone moiety, the similar structural motif found in cyclooxygenase (COX)-derived prostaglandins, 15-deoxy-PGJ2 (15d-PGJ2). These electrophilic compounds are known to have profound biological activities by covalent modification of critical cystein residues in proteins through Michael addition or Schiff base formation with lysines. We chemically synthesized a representative compound 15d-PGJ2-PC for this novel class of oxidized phospholipids and our preliminary experiments showed that 15d-PGJ2-PC can active Nrf2 and PPARs including PPARα, PPARδ and PPARγ, while inhibition of NFκB, suggesting that 15d-PGJ2-PC may regulate inflammatory response and oxidative stress.Interestingly, these compounds can induce a new phenotype of polarized macrophage, Mox (induced by oxPAPC). Our lipidomic and metabolomic studies demonstrated that this novel Mox phenotype had distinct metabolic features from M1 and M2 phenotypes including arachidonic acid pathways and glycolysis. Based on these preliminary data, we hypothesize that these novel phospholipid oxidation products may modulate atherosclerotic process by interacting with macrophages in response to inflammation and oxidative stress. In this project, we will utilize biochemistry, synthetic chemistry, lipidomics, metabolomics, and Click chemistry-based proteomics to study the roles of deoxy-A2/J2-PC in macrophages and atherosclerosis animal model. We contend that successful completion of this project may shed light into the novel molecular mechanisms of atherosclerosis and provide a potential therapeutic strategy for the prevention and treatment of atherosclerosis.

动脉粥样硬化是心脑血管病的主要致病原因之一，是由血管壁中的多种细胞相互作用引起组织损伤、斑块形成的一种慢性疾病，炎症和氧化应激对其发生发展极其重要。巨噬细胞是动脉粥样硬化斑块的主要成分，具有高度的可塑性，小鼠斑块中主要检测到M1、M2、Mox 三种亚型。近年来许多的证据表明LDL磷脂氧化产物作用于巨噬细胞并影响动脉粥样硬化，相关机制尚待进一步阐明。利用质谱我们在体外和体内实验中鉴定了一类新型的磷脂氧化产物，我们合成了其中的一个代表化合物，它不仅可以诱导Mox，还可以通过激活PPAR和Nrf2，抑制NFκB的活性，参与炎症和氧化应激。因此我们认为这类磷脂氧化产物可能是影响动脉粥样硬化过程炎症和氧化应激的重要因子。在本项目中，我们将进一步利用生物化学、化学合成、现代质谱技术、组学手段、细胞和动物模型等，研究其对动脉粥样硬化的作用以及分子机制，该项目可能对动脉粥样硬化的预防和治疗提供新思路。

动脉粥样硬化是心脑血管疾病的主要致病原因，大量的研究表明炎症和氧化应激在动脉粥样硬化发生发展过程中发挥极其重要的作用。既往的证据显示低密度脂蛋白LDL中含多不饱和脂肪酸的脂质在氧化应激条件下发生过氧化，这些脂氧化产物可能作用于血管壁中的巨噬细胞、内皮细胞、平滑肌细胞等参与动脉粥样硬化，然而相关分子机制尚待进一步阐明。在本项目中，我们取得了一系列重要的成果：（1）利用基于质谱技术的代谢组学和脂质组学，发现了脂质过氧化是心血管疾病（CVD）病人血浆脂质的重要特征 ( Lu et al, Redox Biol, 2018）, 其磷脂和胆固醇脂氧化产物在心肌梗塞病人血浆中均显著上升（Lu et al, Redox Biol. 2018; Guo et al, Redox Biol. 2019）; （2）利用合成化学和质谱等手段，我们鉴定了一类新型的带环戊烯酮结构的磷脂氧化产物，进一步利用生化、细胞和动物实验发现这类新型氧化产物不仅可以诱导巨噬细胞Mox表型，还可以激活 PPAR 和 Nrf2，抑制NFκB的活性，具有抗炎和抗氧化活性（Lu et al, J. Biol. Chem., 2017)，该研究首次明确了这类新型氧化产物在动脉硬化中的作用机制; （3）胆固醇脂过氧化物通过LXRa-IDOL-LDLR 通路抑制巨噬细胞和肝细胞胆固醇的摄入，从而升高血液中胆固醇的水平（Guo et al， Redox Biol., 2019），首次揭示了这类氧化胆固醇脂参与动脉粥样硬化的新机制; （4）线粒体乙醛脱氢酶2(ALDH2)主要代谢乙醛和脂质过氧化产生的活性醛类，40%的东亚人携带一个SNP (rs671), 除喝酒脸红以外CVD的风险大大增加，但其机制不明，我们发现ALDH2通过与LDLR和AMPK的相互作用影响泡沫细胞的形成：AMPK磷酸化ALDH2 SNP 使其从线粒体转移到细胞核，抑制调控溶酶体pH的重要蛋白ATP6V0e2的表达，减弱溶酶体对胆固醇脂的水解，从而促进泡沫细胞的形成 (Zhong et al, J. Clin. Invest., 2019)。项目的基础和临床意义: CVD病人血浆脂质组特征可能成为CVD检测的新型生物标志物; 进一步加深了我们对脂质过氧化影响动脉粥样硬化发生发展分子机制的理解，为动脉粥样硬化和CVD的预防和治疗提供了新思路。