细胞间通讯对于几乎所有生理和病理过程都是至关重要的。内皮细胞（EC）来源的外泌体作为细胞间信息交流的介质，参与动脉粥样硬化的病理生理机制。然而，炎症内皮外泌体对巨噬细胞（Mϕ）功能的影响尚未明确定义。本研究旨在揭示肿瘤坏死因子-α（TNF-α）刺激的EC衍生外泌体（exo-T）对体外Mϕ的影响。使用透射电子显微镜（TEM）、纳米粒子跟踪分析（NTA）和西方印迹等技术鉴定了未经处理的EC外泌体（exo）和exo-T，我们观察到PKH67标记的exo/exo-T被Mϕ摄取。将Mϕ暴露于exo-T 24小时不仅使Mϕ偏向M1型亚型并加重脂质沉积，还促进了Mϕ凋亡，但没有明显影响Mϕ的迁移，如RT-qPCR、Dil-ox-LDL摄取实验、流式细胞术、伤口愈合实验和Transwell实验中检测到的。此外，exo/exo-T相关的microRNA-Seq揭示了104个显著差异表达的microRNA（DE-miRNAs）。通过基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路分析确定，DE-miRNAs的靶基因主要在代谢通路、MAPK信号通路等功能上富集。我们进一步通过免疫印迹实验证明，exo-T介入改善了MAPK/NF-κB相关蛋白的磷酸化。总之，本研究揭示了炎症内皮外泌体（TNF-α刺激EC衍生外泌体）作为功能中介物影响Mϕ功能，并且可能通过MAPK/NF-κB信号通路激活Mϕ。版权所有©2023年Lin, Huang, Yuan, Li, Lin, Zhu, Lin和Zhu.

Intercellular communication is essential for almost all physiological and pathological processes. Endothelial cell (EC)-derived exosomes, working as mediators for intercellular information exchange, are involved in the pathophysiological mechanisms of atherosclerosis. However, the effect of inflamed endothelial exosomes on the function of macrophages (Mϕ) is poorly defined. This study aims to unravel how exosomes derived from tumor necrosis factor-α (TNF-α)-stimulated ECs (exo-T) affect Mϕ in vitro.Exosomes derived from untreated ECs (exo) and exo-T were identified by using TEM, NTA, and western blot, and we observed that PKH67-labeled exo/exo-T were taken up by Mϕ. Exposure to exo-T for 24 h not only skewed Mϕ to the M1 subtype and exacerbated lipid deposition, but also promoted Mϕ apoptosis, while it did not significantly affect Mϕ migration, as detected by RT-qPCR, Dil-ox-LDL uptake assay, flow cytometry, wound healing assay, and transwell assay, respectively. In addition, exo/exo-T-related microRNA-Seq revealed 104 significantly differentially expressed microRNAs (DE-miRNAs). The target genes of DE-miRNAs were mainly enriched functionally in metabolic pathways, MAPK signaling pathway, etc., as determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. We further demonstrated by immunoblotting that exo-T intervention improves the phosphorylation of MAPK/NF-κB-related proteins.Collectively, this study reveals that inflamed endothelial exosomes (TNF-α-stimulated EC-derived exosomes) work as a functional mediator to affect Mϕ function and may activate Mϕ through MAPK/NF-κB signaling pathways.Copyright © 2023 Lin, Huang, Yuan, Li, Lin, Zhu, Lin and Zhu.