目标:在晚期动脉粥样硬化病变中,巨噬细胞死亡导致坏死核心形成和斑块易损性。亲环蛋白D(CypD)是一种线粒体特异性亲环蛋白,参与器官缺血再灌注后的细胞死亡过程。然而,CypD在动脉粥样硬化中的作用,特别是在坏死核形成中,是未知的。因此,本实验旨在阐明CypD在坏死核形成中的作用。
方法:为了阐明CypD的具体作用,由小鼠中的ppif编码,产生载脂蛋白-E/CypD-双敲除(Apo-/-Ppif-/-)小鼠。这些小鼠被喂食含有0.15%胆固醇的高脂肪饮食24周以加速动脉粥样硬化病变的发展。
结果:CypD的缺失减少了坏死核的大小,与对照Apoe-/-小鼠相比,巨噬细胞凋亡减少。在RAW264.7细胞中,siRNA介导的CypD敲低减弱了内质网应激诱导剂thapsigargin诱导的细胞色素c从线粒体释放到细胞质。此外,坏死,由TNF-α和caspase抑制剂诱导,通过敲低CypD而减弱。CypD缺失降低了外周血白细胞中的Ly-6Chigh炎性单核细胞和主动脉中Il1b的mRNA表达。相比之下,siRNA介导的CypD敲低在LPS和IFN-γ处理的RAW264.7细胞中不显著降低Il1b和Ccl2mRNA表达,这表明体内炎症的抑制可能是由于动脉粥样硬化病变中细胞死亡的减少,而不是CypD缺失对巨噬细胞的直接作用。
结论:这些结果表明CypD在晚期动脉粥样硬化病变中诱导巨噬细胞死亡并介导坏死核心形成。CypD可能成为治疗动脉粥样硬化性血管疾病的新靶点。
OBJECTIVE: In advanced atherosclerotic lesions, macrophage deaths result in necrotic core formation and plaque vulnerability. Cyclophilin D (CypD) is a mitochondria-specific cyclophilin involved in the process of cell death after organ ischemia-reperfusion. However, the role of CypD in atherosclerosis, especially in necrotic core formation, is unknown. Therefore, this experiment aims to clarify the role of CypD in necrotic core formation.
METHODS: To clarify the specific role of CypD, encoded by Ppif in mice, apolipoprotein-E/CypD-double knockout (Apoe-/-Ppif-/-) mice were generated. These mice were fed a high-fat diet containing 0.15 % cholesterol for 24 weeks to accelerate atherosclerotic lesion development.
RESULTS: Deletion of CypD decreased the necrotic core size, accompanied by a reduction of macrophage apoptosis compared to control Apoe-/- mice. In RAW264.7 cells, siRNA-mediated knockdown of CypD attenuated the release of cytochrome c from the mitochondria to the cytosol induced by endoplasmic reticulum stress inducer thapsigargin. In addition, necroptosis, induced by TNF-α and caspase inhibitor, was attenuated by knockdown of CypD. Ly-6Chigh inflammatory monocytes in peripheral blood leukocytes and mRNA expression of Il1b in the aorta were decreased by deletion of CypD. In contrast, siRNA-mediated knockdown of CypD did not significantly decrease Il1b nor Ccl2 mRNA expression in RAW264.7 cells treated with LPS and IFN-γ, suggesting that inhibition of inflammation in vivo is likely due to decreased cell death in the atherosclerotic lesions rather than a direct action of CypD deletion on the macrophage.
CONCLUSIONS: These results indicate that CypD induces macrophage death and mediates necrotic core formation in advanced atherosclerotic lesions. CypD could be a novel therapeutic target for treating atherosclerotic vascular diseases.