Department of Medicine, Marc and Ruti Bell Vascular Biology and Disease Program, Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, NY 10016, USA.
Arterioscler Thromb Vasc Biol. 2013 Jun;33(6):1180-8. doi: 10.1161/ATVBAHA.112.301008. Epub 2013 Apr 18.
Hypoxia is intimately linked to atherosclerosis and has become recognized as a primary impetus of inflammation. We recently demonstrated that the neuroimmune guidance cue netrin-1 (Ntn1) inhibits macrophage emigration from atherosclerotic plaques, thereby fostering chronic inflammation. However, the mechanisms governing netrin-1 expression in atherosclerosis are not well understood. In this study, we investigate the role of hypoxia in regulating expression of netrin-1 and its receptor uncoordinated-5-B receptor (Unc5b) in plaque macrophages and its functional consequences on these immune cells.
We show by immunostaining that netrin-1 and Unc5b are expressed in macrophages in hypoxia-rich regions of human and mouse plaques. In vitro, Ntn1 and Unc5b mRNA are upregulated in macrophages treated with oxidized low-density lipoprotein or inducers of oxidative stress (CoCl2, dimethyloxalylglycine, 1% O2). These responses are abrogated by inhibiting hypoxia-inducible transcription factor (HIF)-1α, indicating a causal role for this transcription factor in regulating Ntn1 and Unc5b expression in macrophages. Indeed, using promoter-luciferase reporter genes, we show that Ntn1- and Unc5b-promoter activities are induced by oxidized low-density lipoprotein and require HIF-1α. Correspondingly, J774 macrophages overexpressing active HIF-1α show increased netrin-1 and Unc5b expression and reduced migratory capacity compared with control cells, which was restored by blocking the effects of netrin-1. Finally, we show that netrin-1 protects macrophages from apoptosis under hypoxic conditions in a HIF-1α-dependent manner.
These findings provide a molecular mechanism by which netrin-1 and its receptor Unc5b are expressed in atherosclerotic plaques and implicate hypoxia and HIF-1α-induced netrin-1/Unc5b in sustaining inflammation by inhibiting the emigration and promoting the survival of lesional macrophages.
缺氧与动脉粥样硬化密切相关，已被认为是炎症的主要诱因。我们最近证明，神经免疫导向分子轴突导向因子 1（Ntn1）抑制了巨噬细胞从动脉粥样硬化斑块中的迁出，从而促进了慢性炎症。然而，调节动脉粥样硬化中轴突导向因子 1 表达的机制尚不清楚。在这项研究中，我们研究了缺氧在调节斑块巨噬细胞中轴突导向因子 1 及其受体 UNC5B 表达中的作用及其对这些免疫细胞的功能后果。
我们通过免疫染色显示，轴突导向因子 1 和 UNC5B 在富含人源和鼠源斑块缺氧区的巨噬细胞中表达。在体外，用氧化型低密度脂蛋白或氧化应激诱导剂（CoCl2、二甲草酰甘氨酸、1%O2）处理巨噬细胞后，Ntn1 和 UNC5B mRNA 上调。这些反应被抑制缺氧诱导转录因子（HIF）-1α 的抑制剂所阻断，表明该转录因子在调节巨噬细胞中 Ntn1 和 UNC5B 的表达中起因果作用。事实上，我们使用启动子-荧光素酶报告基因显示，氧化型低密度脂蛋白诱导 Ntn1 和 UNC5B 启动子活性，并需要 HIF-1α。相应地，与对照细胞相比，过表达活性 HIF-1α 的 J774 巨噬细胞表现出更高的轴突导向因子 1 和 UNC5B 表达和降低的迁移能力，这一能力通过阻断轴突导向因子 1 的作用得到恢复。最后，我们发现轴突导向因子 1 在缺氧条件下以依赖 HIF-1α 的方式保护巨噬细胞免于凋亡。
这些发现提供了一种分子机制，解释了轴突导向因子 1 和其受体 UNC5B 在动脉粥样硬化斑块中的表达，并表明缺氧和 HIF-1α 诱导的轴突导向因子 1/UNC5B 通过抑制迁移和促进病变巨噬细胞的存活来维持炎症。
