Department of Cardiovascular, The First Hospital of Harbin, No. 151 Diduan Street, Daoli District, Harbin 150010, Heilongjiang, China.
Department of Pneumology, Qingdao Women and Children's Hospital, No. 6 Tongfu Road, Shibei District, Qingdao 266000, Shandong, China.
Biol Chem. 2020 Feb 25;401(3):367-376. doi: 10.1515/hsz-2019-0219.
The objective of this study was to reveal a novel mechanism underlying the progression of atherosclerosis (AS) associated with endothelial cells (ECs) and neutrophils. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) were used to observe the morphology and particle size of isolated exosomes. Western blotting was applied to examine exosomal markers, while the expression of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The production of inflammatory cytokines and reactive oxygen species (ROS) was determined by an enzyme-linked immunosorbent assay (ELISA) and a dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay. Circulating neutrophil extracellular traps (NETs) were represented by myeloperoxidase (MPO)-DNA complexes. NETs formation was assessed using immunofluorescence microscopy. Atherosclerotic lesion development was measured by Oil Red O (ORO) staining. In the results, MALAT1 expression was increased in exosomes extracted from oxidized low-density lipoprotein (ox-LDL)-treated human umbilical vein endothelial cells (HUVECs). When co-cultured with human neutrophils, exosomes derived from ox-LDL-treated HUVECs were revealed to promote NETs formation, which was mediated by exosomal MALAT1. Furthermore, ox-LDL-treated HUVECs-derived exosomes were demonstrated to trigger hyperlipidemia, inflammatory response and NETs release in a mouse model of AS. In conclusion, exosomal MALAT1 derived from ox-LDL-treated ECs initiated NETs formation, which in turn deteriorated AS.
本研究旨在揭示与内皮细胞（EC）和中性粒细胞相关的动脉粥样硬化（AS）进展的新机制。透射电子显微镜（TEM）和纳米颗粒跟踪分析（NTA）用于观察分离的外泌体的形态和粒径。Western blot 用于检测外泌体标志物，而转移相关肺腺癌转录物 1（MALAT1）的表达则通过定量实时聚合酶链反应（qRT-PCR）进行测量。通过酶联免疫吸附测定（ELISA）和二氯二氢荧光素二乙酸酯（DCFH-DA）测定来确定炎性细胞因子和活性氧（ROS）的产生。循环中性粒细胞细胞外陷阱（NETs）用髓过氧化物酶（MPO）-DNA 复合物表示。通过免疫荧光显微镜评估 NETs 形成。通过油红 O（ORO）染色测量动脉粥样硬化病变的发展。结果表明，氧化低密度脂蛋白（ox-LDL）处理的人脐静脉内皮细胞（HUVEC）中提取的外泌体中 MALAT1 表达增加。当与人中性粒细胞共培养时，发现来自 ox-LDL 处理的 HUVEC 的外泌体促进了 NETs 的形成，这是由外泌体 MALAT1 介导的。此外，ox-LDL 处理的 HUVEC 衍生的外泌体在 AS 小鼠模型中引发了高脂血症、炎症反应和 NETs 释放。总之，ox-LDL 处理的 EC 衍生的外泌体 MALAT1 引发了 NETs 的形成，进而使 AS 恶化。
