肝窦内皮细胞（LSEC）的病理性血管生成在代谢功能障碍相关脂肪性肝炎（MASH）诱导的肝纤维化的进展中起着至关重要的作用。间充质干细胞衍生的小细胞外囊泡（MSC-sEV）已显示出针对 MASH 的有希望的治疗潜力。本研究旨在探讨 MSC-sEV 对 LSEC 血管生成的影响并阐明潜在的分子机制。在肿瘤坏死因子-α (TNF-α) 处理的 LSEC 体外和蛋氨酸和胆碱缺乏饮食 (MCD) 诱导的 MASH 小鼠体内评估 MSC-sEV 对 LSEC 血管生成的影响。在此，我们发现 MSC-sEV 通过靶向 TNF-α 处理的 LSEC 和 MASH 小鼠中的血管生成标记物血管生成素 2 (Ang-2) 有效抑制 LSEC 血管生成。基因操作实验表明，MSC-sEV抑制LSEC血管生成的主要机制是通过调节核因子κB抑制剂α（IκBα）/核因子κB（NF-κB）/Ang-2通路。此外，质谱和免疫共沉淀 (Co-IP) 数据表明 MSC-sEV 将泛素特异性肽酶 9 X 连接 (USP9X) 蛋白递送至 LSEC，从而增强 IκBα 去泛素化和 NF-κB 激活，最终导致抑制 Ang-2 介导的 LSEC 血管生成。 USP9X 的敲低减弱了 MSC-sEV 对 Ang-2 表达、LSEC 血管生成和 MASH 进展的调节作用。总之，我们的研究结果表明，通过 MSC-sEV 传递的 USP9X 可以通过 IκBα/NF-κB/Ang-2 信号通路抑制 LSEC 血管生成并减轻 MASH 诱导的肝纤维化。版权所有 © 2024 作者。由爱思唯尔有限公司出版。保留所有权利。

Pathological angiogenesis of liver sinusoidal endothelial cells (LSEC) plays a crucial role in the progression of metabolic dysfunction-associated steatohepatitis (MASH)-induced liver fibrosis. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEV) have shown promising therapeutic potential against MASH. This study aimed to investigate the impact of MSC-sEV on LSEC angiogenesis and elucidate the underlying molecular mechanisms. The effects of MSC-sEV on LSEC angiogenesis were evaluated in Tumor Necrosis Factor- alpha (TNF-α)-treated LSECs in vitro and in Methionine and Choline Deficient Diet (MCD)-induced MASH mice in vivo. Herein, we found that MSC-sEV effectively suppressed LSEC angiogenesis by targeting the angiogenesis marker Angiogenin 2 (Ang-2) in both TNF-α-treated LSECs and MASH mice. Gene manipulation experiments revealed that the primary mechanism by which MSC-sEV inhibited LSEC angiogenesis was through the modulation of nuclear factor kappa B inhibitor alpha (IκBα) / nuclear factor kappa B (NF-κB) / Ang-2 pathway. Additionally, mass spectrometry and co-immunoprecipitation (Co-IP) data suggested that MSC-sEV delivered the ubiquitin specific peptidase 9 X-linked (USP9X) protein to LSECs, leading to enhanced IκBα deubiquitination and NF-κB activation, ultimately resulting in the inhibition of Ang-2-mediated LSEC angiogenesis. Knockdown of USP9X attenuated the regulatory effects of MSC-sEV on Ang-2 expression, LSEC angiogenesis, and the progression of MASH. In conclusion, our findings indicate that USP9X delivered via MSC-sEV can suppress LSEC angiogenesis and alleviate MASH-induced liver fibrosis through the IκBα/NF-κB/Ang-2 signaling pathway.Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.