Division of Biological Chemistry, Department of Pharmaceutical Sciences, Showa University School of Pharmacy, Tokyo, Japan.
Division of Electron Microscopy, Showa University School of Medicine, Tokyo, Japan.
Front Immunol. 2019 Aug 9;10:1899. doi: 10.3389/fimmu.2019.01899. eCollection 2019.
The function of oxidatively modified low-density lipoprotein (oxLDL) in the progression of cardiovascular diseases has been extensively investigated and well-characterized with regards to the activation of multiple cellular responses in macrophages and endothelial cells. Although accumulated evidence has revealed the presence of neutrophils in vascular lesions, the effect of oxLDL on neutrophil function has not been properly investigated. In the present decade, neutrophil extracellular traps (NETs) gained immense attention not only as a primary response against pathogenic bacteria but also due to their pathological roles in tissue damage in various diseases, such as atherosclerosis and thrombosis. In this study, we investigated if oxLDL affects NET formation and if it is a risk factor for inflammatory reactions in endothelial cells. HL-60-derived neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA) for 30 min to induce NET formation, followed by incubation with 20 μg/mL native or oxidized LDL for additional 2 h. Culture media of the stimulated cells containing released NETs components were collected to evaluate NET formation by fluorometric quantitation of released DNA and detection of myeloperoxidase (MPO) by western blot analysis. NET formation of HL-60-derived neutrophils induced by PMA was significantly enhanced by additional incubation with oxLDL but not with native LDL. Treatment of HL-60-derived neutrophils with oxLDL alone in the absence of PMA did not induce NET formation. Furthermore, the culture media of HL-60-derived neutrophils after NET formation were then transferred to human aortic endothelial cell (HAECs) culture. Treatment of HAECs with the culture media containing NETs formed by HL-60-derived neutrophils increased the expression of metalloproteinase-1 protein in HAECs when HL-60-derived neutrophils were incubated with native LDL, and the expression was accelerated in the case of oxLDL. In addition, the culture media from NETs formed by HL-60-derived neutrophils caused the elongation of HAECs, which was immensely enhanced by coincubation with native LDL or oxLDL. These data suggest that oxLDL may act synergistically with neutrophils to form NETs and promote vascular endothelial inflammation.
氧化修饰的低密度脂蛋白（oxLDL）在心血管疾病进展中的作用已经得到了广泛的研究，并且在巨噬细胞和内皮细胞中，其对多种细胞反应的激活作用已经得到了很好的描述。尽管有大量证据表明中性粒细胞存在于血管病变中，但 oxLDL 对中性粒细胞功能的影响尚未得到适当的研究。在过去的十年中，中性粒细胞胞外诱捕网（NETs）不仅作为对抗病原菌的主要反应引起了广泛关注，而且由于它们在动脉粥样硬化和血栓形成等各种疾病中的组织损伤中的病理作用而受到关注。在这项研究中，我们研究了 oxLDL 是否影响 NET 形成，以及它是否是内皮细胞炎症反应的一个危险因素。HL-60 衍生的中性粒细胞用佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激 30 分钟以诱导 NET 形成，然后再与 20μg/ml 的天然或氧化 LDL 孵育 2 小时。刺激细胞的培养物介质中包含释放的 NET 成分，通过荧光定量法检测释放的 DNA 和用 Western blot 分析检测髓过氧化物酶（MPO）来评估 NET 形成。PMA 诱导的 HL-60 衍生的中性粒细胞的 NET 形成，通过与 oxLDL 进一步孵育而显著增强，但与天然 LDL 孵育则没有增强。单独用 oxLDL 处理 HL-60 衍生的中性粒细胞而不使用 PMA 则不会诱导 NET 形成。此外，将 NET 形成后 HL-60 衍生的中性粒细胞的培养物介质转移到人主动脉内皮细胞（HAECs）培养物中。用 HL-60 衍生的中性粒细胞形成的 NETs 的培养物介质处理 HAECs 时，当 HL-60 衍生的中性粒细胞与天然 LDL 孵育时，HAECs 中的金属蛋白酶-1 蛋白表达增加，而在 oxLDL 的情况下，表达加速。此外，来自 HL-60 衍生的中性粒细胞形成的 NETs 的培养物介质导致 HAECs 的伸长，当与天然 LDL 或 oxLDL 共孵育时，伸长幅度大大增加。这些数据表明，oxLDL 可能与中性粒细胞协同作用形成 NETs，并促进血管内皮炎症。
