IRGM/Irgm1 facilitates macrophage apoptosis through ROS generation and MAPK signal transduction: Irgm1(+/-) mice display increases atherosclerotic plaque stability.
IRGM/Irgm1 facilitates macrophage apoptosis through ROS generation and MAPK signal transduction: Irgm1(+/-) mice display increases atherosclerotic plaque stability.
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IRGM/Irgm1 通过 ROS 生成和 MAPK 信号转导促进巨噬细胞凋亡：Irgm( /-) 小鼠显示增加动脉粥样硬化斑块稳定性
作者：
Fang S;Sun S;Cai H;Zou X;Wang S;Hao X;Wan X;Tian J;Li Z;He Z;Huang W;Liang C;Zhang Z;Yang L;Tian J;Yu B;Sun B
理论基础：动脉粥样硬化斑块破裂(PR)是大多数急性心血管事件和死亡的病理基础和罪魁祸首。鉴于巨噬细胞凋亡和自噬在影响斑块稳定性中的复杂而重要的作用，一个重要的悬而未决的问题包括免疫相关的GTPase家族M蛋白(IRGM)及其小鼠同源基因IRGM1是否以及如何影响巨噬细胞的存活和动脉粥样硬化斑块的稳定性。方法：为探讨ST段抬高型心肌梗死(STEMI)患者血清IRGM水平与斑块形态的关系，将85例STEMI患者按OCT图像分析分为斑块破裂组(PR组)和非PR组(非PR组)，并对其血清IRGM水平进行定量。接下来，我们在ApoE-/-背景下设计了Irgm1缺陷小鼠(Irgm1+/-)和骨髓中Irgm1缺乏的嵌合体小鼠，然后给它们喂高脂饮食16周。病理染色检测小鼠的坏死斑核心、中性脂与胆固醇结晶的比值以及胶原纤维含量，以表征斑块的稳定性。免疫荧光、免疫组织化学染色和免疫印迹法检测斑块内巨噬细胞的凋亡情况。在体外，用ox-LDL刺激THP-1和RAW264.7细胞，模拟体内环境，用特异性siRNA(敲除)或IRGM质粒(敲入)改变IRGM/IRGM1的表达。观察IRGM/Irgm1对ox-LDL诱导的巨噬细胞自噬和凋亡的影响。此外，我们还引入了JNK/p38/ERK信号通路的抑制剂来验证Irgm1调控RAW264.7细胞凋亡的具体机制。结果：PR患者血清IRGM水平显著高于非PR患者和健康志愿者，可作为PR的有效预测指标。在高脂饮食中，Irgm1缺陷小鼠的坏死斑核心减少，中性脂肪和胆固醇结晶比率增加，胶原纤维含量增加。此外，在Irgm1基因缺陷小鼠的斑块中，巨噬细胞的凋亡受到抑制。在体外，IRGM/Irgm1缺陷可迅速抑制OX-LDL诱导的巨噬细胞自噬，并在晚期抑制OX-LDL诱导的巨噬细胞凋亡。此外，IRGM/Irgm1缺乏抑制巨噬细胞产生的活性氧(ROS)，而ROS的去除有效地抑制了IRGM过表达诱导的巨噬细胞凋亡。我们进一步证明Irgm1可以通过调节MAPK信号通路中JNK/p38/ERK的磷酸化来影响巨噬细胞的凋亡。结论：血清IRGM可能与STEMI患者PR过程有关，IRGM/Irgm1缺陷增加了斑块的稳定性。此外，IRGM/Irgm1缺陷通过抑制ROS的产生和MAPK信号转导来抑制巨噬细胞的凋亡。总体而言，这些结果表明靶向IRGM可能是预防和治疗PR引起的急性心血管死亡的一种新的治疗策略。
Rationale: Atherosclerosis plaque rupture (PR) is the pathological basis and chief culprit of most acute cardiovascular events and death. Given the complex and important role of macrophage apoptosis and autophagy in affecting plaque stability, an important unanswered question include is whether, and how, immunity-related GTPase family M protein (IRGM) and its mouse orthologue IRGM1 affect macrophage survival and atherosclerotic plaque stability. Methods: To investigate whether serum IRGM of ST-segment elevation myocardial infarction (STEMI) patients is related to plaque morphology, we divided 85 STEMI patients into those with and without plaque rupture (PR and non-PR, respectively) based on OCT image analysis, and quantified the patients' serum IRGM levels. Next, we engineered Irgm1 deficient mice (Irgm1+/-) and chimera mice with Irgm1 deficiency in the bone marrow on an ApoE-/- background, which were then fed a high-fat diet for 16 weeks. Pathological staining was used to detect necrotic plaque cores, ratios of neutral lipids and cholesterol crystal, as well as collagen fiber contents in these mice to characterize plaque stability. In addition, immunofluorescence, immunohistochemical staining and western blot were used to detect the apoptosis of macrophages in the plaques. In vitro, THP-1 and RAW264.7 cells were stimulated with ox-LDL to mimic the in vivo environment, and IRGM/IRGM1 expression were modified by specific siRNA (knockdown) or IRGM plasmid (knocked-in). The effect of IRGM/Irgm1 on autophagy and apoptosis of macrophages induced by ox-LDL was then evaluated. In addition, we introduced inhibitors of the JNK/p38/ERK signaling pathway to verify the specific mechanism by which Irgm1 regulates RAW264.7 cell apoptosis. Results: The serum IRGM levels of PR patients is significantly higher than that of non-PR patients and healthy volunteers, which may be an effective predictor of PR. On a high-fat diet, Irgm1-deficient mice exhibit reduced necrotic plaque cores, as well as neutral lipid and cholesterol crystal ratios, with increased collagen fiber content. Additionally, macrophage apoptosis is inhibited in the plaques of Irgm1-deficient mice. In vitro, IRGM/Irgm1 deficiency rapidly inhibits ox-LDL-induced macrophage autophagy while inhibiting ox-LDL-induced macrophage apoptosis in late stages. Additionally, IRGM/Irgm1 deficiency suppresses reactive oxygen species (ROS) production in macrophages, while removal of ROS effectively inhibits macrophage apoptosis induced by IRGM overexpression. We further show that Irgm1 can affect macrophage apoptosis by regulating JNK/p38/ERK phosphorylation in the MAPK signaling pathway. Conclusions: Serum IRGM may be related to the process of PR in STEMI patients, and IRGM/Irgm1 deficiency increases plaque stability. In addition, IRGM/Irgm1 deficiency suppresses macrophage apoptosis by inhibiting ROS generation and MAPK signaling transduction. Cumulatively, these results suggest that targeting IRGM may represent a new treatment strategy for the prevention and treatment of acute cardiovascular deaths caused by PR.
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