Division of Vascular Medicine, Medical Clinic and Policlinic IV, University Hospital Munich, Munich, Germany.
Helmholtz Center Munich, Institute of Developmental Genetics, Germany.
Atherosclerosis. 2014 Jul;235(1):213-22. doi: 10.1016/j.atherosclerosis.2014.04.020. Epub 2014 May 10.
Deletion of inducible nitric oxide synthase (iNOS) in apolipoprotein E knockout mice was shown to mitigate the extent of arteriosclerosis. Oxidized low density lipoprotein (oxLDL) inhibits macrophage migration and traps foam cells, possibly through a mechanism involving oxidative stress. Here, we addressed whether a reduction of iNOS-mediated oxidative stress remobilizes macrophage-derived foam cells and may reverse plaque formation.
Migration of RAW264.7 cells and bone marrow cells was quantified using a modified Boyden chamber. iNOS expression, phalloidin staining, focal adhesion kinase phosphorylation, lipid peroxides, nitric oxide (NO) and reactive oxygen species (ROS) production were assessed.
oxLDL treatment significantly reduced cell migration compared to unstimulated cells (p < 0.05). This migratory arrest was reversed by co-incubation with a pharmacologic iNOS inhibitor 1400 W (p < 0.05) and iNOS-siRNA (p > 0.05). Furthermore, apoE/iNOS double knockout macrophages do not show migratory arrest in response to oxLDL uptake, compared to apoE knockout controls (p > 0.05). We documented significantly increased iNOS expression following oxLDL treatment and downregulation using 1400 W and small inhibitory RNA (siRNA). iNOS inhibition was associated with a reduction in NO and peroxynitrite (ONOO-)- and increased superoxide generation. Trolox treatment of RAW264.7 cells restored migration indicating that peroxynitrite mediated lipid peroxide formation is involved in the signaling pathway mediating cell arrest..
Here, we provide pharmacologic and genetic evidence that oxLDL induced iNOS expression inhibits macrophage-derived foam cell migration. Therefore, reduction of peroxynitrite and possibly lipid hydroperoxide levels in plaques represents a valuable therapeutic approach to reverse migratory arrest of macrophage-derived foam cells and to impair plaque formation.
研究表明，载脂蛋白 E 基因敲除小鼠中诱导型一氧化氮合酶（iNOS）的缺失可减轻动脉硬化的程度。氧化型低密度脂蛋白（oxLDL）可抑制巨噬细胞迁移并使泡沫细胞陷巢，其可能通过氧化应激机制发挥作用。在此，我们探讨了 iNOS 介导的氧化应激减少是否能使巨噬细胞源性泡沫细胞重新迁移，并可能逆转斑块形成。
采用改良 Boyden 室法定量检测 RAW264.7 细胞和骨髓细胞的迁移。检测 iNOS 表达、鬼笔环肽染色、粘着斑激酶磷酸化、脂质过氧化物、一氧化氮（NO）和活性氧（ROS）的产生。
与未刺激细胞相比，oxLDL 处理显著降低细胞迁移（p<0.05）。这种迁移抑制可通过与药理学 iNOS 抑制剂 1400 W（p<0.05）和 iNOS-siRNA（p>0.05）共孵育而逆转。此外，与 apoE 基因敲除对照组相比，apoE/iNOS 双基因敲除巨噬细胞对 oxLDL 摄取没有发生迁移抑制（p>0.05）。我们发现 oxLDL 处理后 iNOS 表达显著增加，用 1400 W 和小干扰 RNA（siRNA）处理后表达下调。iNOS 抑制与 NO 和过氧亚硝酸盐（ONOO-）生成减少以及超氧化物生成增加有关。用 Trolox 处理 RAW264.7 细胞恢复了迁移，表明过氧亚硝酸盐介导的脂质过氧化物形成参与了介导细胞阻滞的信号通路。
本研究提供了药理学和遗传学证据，表明 oxLDL 诱导的 iNOS 表达抑制了巨噬细胞源性泡沫细胞的迁移。因此，减少斑块中过氧亚硝酸盐和可能的脂质氢过氧化物水平是逆转巨噬细胞源性泡沫细胞迁移抑制和损害斑块形成的一种有价值的治疗方法。
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