NYU Cardiovascular Research Center, The Leon H. Charney Division of Cardiology, Department of Medicine, New York University Grossman School of Medicine (M. Schlegel, M. Sharma, E.J.B., A.A.C.N., Y.C., M.S.A., E.M.C., G.J.K., C.v.S., J.G., R.F., C.A.N., L.C.S., D.P., E.A.F., K.J.M.).
Department of Anesthesiology and Intensive Care, Technical University of Munich, School of Medicine, Germany (M. Schlegel).
Circ Res. 2021 Aug 20;129(5):530-546. doi: 10.1161/CIRCRESAHA.121.319313. Epub 2021 Jul 22.
Rationale: Therapeutic efforts to decrease atherosclerotic cardiovascular disease risk have focused largely on reducing atherogenic lipoproteins, yet lipid-lowering therapies alone are insufficient to fully regress plaque burden. We postulate that arterial repair requires resolution of a maladaptive immune response and that targeting factors that hinder inflammation resolution will facilitate plaque regression. Objective: The guidance molecule Ntn1 (netrin-1) is secreted by macrophages in atherosclerotic plaques, where it sustains inflammation by enhancing macrophage survival and blocking macrophage emigration. We tested whether silencing Ntn1 in advanced atherosclerosis could resolve arterial inflammation and regress plaques. Methods and Results: To temporally silence Ntn1 in myeloid cells, we generated genetically modified mice in which Ntn1 could be selectively deleted in monocytes and macrophages using a tamoxifen-induced CX3CR1-driven cre recombinase (Ntn1fl/flCx3cr1creERT2+) and littermate control mice (Ntn1fl/flCx3cr1WT). Mice were fed Western diet in the setting of hepatic PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression to render them atherosclerotic and then treated with tamoxifen to initiate deletion of myeloid Ntn1 (MøΔNtn1) or not in controls (MøWT). Morphometric analyses performed 4 weeks later showed that myeloid Ntn1 silencing reduced plaque burden in the aorta (−50%) and plaque complexity in the aortic root. Monocyte-macrophage tracing experiments revealed lower monocyte recruitment, macrophage retention, and proliferation in MøΔNtn1 compared with MøWT plaques, indicating a restructuring of monocyte-macrophage dynamics in the artery wall upon Ntn1 silencing. Single-cell RNA sequencing of aortic immune cells before and after Ntn1 silencing revealed upregulation of gene pathways involved in macrophage phagocytosis and migration, including the Ccr7 chemokine receptor signaling pathway required for macrophage emigration from plaques and atherosclerosis regression. Additionally, plaques from MøΔNtn1 mice showed hallmarks of inflammation resolution, including higher levels of proresolving macrophages, IL (interleukin)-10, and efferocytosis, as compared to plaques from MøWT mice. Conclusion: Our data show that targeting Ntn1 in advanced atherosclerosis ameliorates atherosclerotic inflammation and promotes plaque regression.
治疗动脉粥样硬化性心血管疾病的努力主要集中在降低致动脉粥样硬化脂蛋白上，但单独使用降脂疗法不足以完全消退斑块负担。我们推测，动脉修复需要解决适应性免疫反应，而针对阻碍炎症消退的因素将有助于斑块消退。目的：指导分子 Ntn1（神经纤毛蛋白 1）由动脉粥样硬化斑块中的巨噬细胞分泌，通过增强巨噬细胞存活和阻止巨噬细胞迁移来维持炎症。我们测试了在晚期动脉粥样硬化中沉默 Ntn1 是否可以缓解动脉炎症并消退斑块。方法和结果：为了在髓样细胞中暂时沉默 Ntn1，我们利用可通过他莫昔芬诱导的 CX3CR1 驱动的 cre 重组酶（Ntn1fl/flCx3cr1creERT2+）在单核细胞和巨噬细胞中选择性删除 Ntn1 的基因修饰小鼠和同窝对照小鼠（Ntn1fl/flCx3cr1WT）生成了遗传修饰的小鼠。将小鼠喂食西方饮食，并在肝脏中过表达 PCSK9（前蛋白转化酶枯草溶菌素 9），使它们发生动脉粥样硬化，然后用他莫昔芬处理以启动髓样细胞 Ntn1 的缺失（MøΔNtn1）或在对照中不缺失（MøWT）。4 周后进行形态计量学分析显示，髓样细胞 Ntn1 沉默减少了主动脉中的斑块负担（-50%）和主动脉根部的斑块复杂性。单核细胞-巨噬细胞示踪实验显示，与 MøWT 斑块相比，MøΔNtn1 斑块中的单核细胞募集、巨噬细胞保留和增殖减少，表明 Ntn1 沉默后动脉壁中单核细胞-巨噬细胞动力学发生重构。在沉默 Ntn1 前后对主动脉免疫细胞进行单细胞 RNA 测序显示，参与巨噬细胞吞噬和迁移的基因途径上调，包括 Ccr7 趋化因子受体信号通路，该通路对于巨噬细胞从斑块和动脉粥样硬化中迁移和消退是必需的。此外，与 MøWT 斑块相比，MøΔNtn1 小鼠的斑块表现出炎症消退的特征，包括更高水平的促修复巨噬细胞、白细胞介素（IL）-10 和吞噬作用。结论：我们的数据表明，在晚期动脉粥样硬化中靶向 Ntn1 可改善动脉粥样硬化炎症并促进斑块消退。
