OBJECTIVES During the advancement of atherosclerosis, plaque cellularity is governed by the influx of monocyte-derived macrophages and their turnover via apoptotic and nonapoptotic forms of cell death. Previous reports have demonstrated that programmed necrosis, or necroptosis, of plaque macrophages contribute to necrotic core formation. Knockdown or inhibition of the necrosome components RIPK1 (receptor-interacting protein kinase 1) and RIPK3 (receptor-interacting protein kinase 3) slow atherogenesis, and activation of the terminal step of necroptosis, MLKL (mixed lineage kinase domain-like protein), has been demonstrated in advanced human atherosclerotic plaques. However, whether MLKL directly contributes to lesion development and necrotic core formation has not been investigated. Approaches and Results: MLKL expression was knocked down in atherogenic Apoe-knockout mice via the administration of antisense oligonucleotides. During atherogenesis, Mlkl knockdown decreased both programmed cell death and the necrotic core in the plaque. However, total lesion area remained unchanged. Furthermore, treatment with the MLKL antisense oligonucleotide unexpectedly reduced circulating cholesterol levels compared with control antisense oligonucleotide but increased the accumulation of lipids within the plaque and in vitro in macrophage foam cells. MLKL colocalized with the late endosome and multivesicular bodies in peritoneal macrophages incubated with atherogenic lipoproteins. Transfection with MLKL antisense oligonucleotide increased lipid localization with the multivesicular bodies, suggesting that upon Mlkl knockdown, lipid trafficking becomes defective leading to enhanced lipid accumulation in macrophages. CONCLUSIONS These studies confirm the requirement for MLKL as the executioner of necroptosis, and as such a significant contributor to the necrotic core during atherogenesis. We also identified a previously unknown role for MLKL in regulating endosomal trafficking to facilitate lipid handling in macrophages during atherogenesis.

中文翻译：

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MLKL（混合谱系激酶结构域类似蛋白）的丢失减少了坏死的核心，但增加了动脉粥样硬化中巨噬细胞脂质的积累。

目的在动脉粥样硬化的进展过程中，斑块的细胞性受单核细胞衍生的巨噬细胞的流入及其通过凋亡和非凋亡形式的细胞死亡的转换的支配。先前的报道表明，斑块巨噬细胞的程序性坏死或坏死性坏死有助于坏死核心的形成。敲低或抑制坏死因子RIPK1（受体相互作用蛋白激酶1）和RIPK3（受体相互作用蛋白激酶3）可减缓动脉粥样硬化的形成，并激活坏死病终末期激活MLKL（混合谱系激酶结构域样蛋白）。已在晚期人类动脉粥样硬化斑块中得到证实。然而，尚未研究MLKL是否直接有助于病变发展和坏死核心形成。方法和结果：通过给予反义寡核苷酸，在致动脉粥样硬化的Apoe基因敲除小鼠中敲低了MLKL的表达。在动脉粥样硬化发生过程中，Mlk1敲低可减少程序性细胞死亡和斑块中的坏死核心。但是，总病变面积保持不变。此外，与对照反义寡核苷酸相比，用MLKL反义寡核苷酸处理意外地降低了循环胆固醇水平，但增加了巨噬细胞泡沫细胞中斑块内和体外脂质的积累。MLKL在与致动脉粥样硬化脂蛋白孵育的腹膜巨噬细胞中与晚期内体和多囊泡体共定位。MLKL反义寡核苷酸转染可增加脂质在多囊泡体中的定位，这表明在敲除Mlk1后，脂质运输变得有缺陷，导致脂质在巨噬细胞中积累增加。结论这些研究证实了MLKL作为坏死性死刑执行者的要求，并因此是动脉粥样硬化形成过程中坏死核心的重要贡献者。我们还确定了MLKL在调节内体运输以促进动脉粥样硬化形成过程中巨噬细胞脂质处理方面的未知作用。