Liu Libo, An Ziyu, Zhang Huan, Wan Xueqi, Zhao Xin, Yang Xueyao, Tian Jinfan, Song Xiantao
Department of Cardiology, Beijing Anzhen Hospital, Capital Medical University, No.2 Anzhen Road, Chaoyang District, 100029, Beijing, China.
Department of Cardiology, The Second Affiliated Hospital of Shandong First Medical University, No. 366 Taishan Street, Taishan District, 271000, Tai'an, China.
Cardiovasc Diabetol. 2025 Jan 29;24(1):48. doi: 10.1186/s12933-025-02603-0.
Bone marrow-derived mesenchymal stem cell-derived extracellular vesicles (BMSC-EVs) are widely used for therapeutic purposes in preclinical studies. However, their utility in treating diabetes-associated atherosclerosis remains largely unexplored. Here, we aimed to characterize BMSC-EV-mediated regulation of autophagy and macrophage polarization.
EVs were isolated from the supernatant of cultured BMSCs and characterized with transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. A diabetes-related atherosclerotic ApoE mouse model was established through feeding with a high-fat diet (HFD) and streptozotocin (STZ). Histopathological analyses were carried out using Oil Red O, H&E, and Masson staining of the aorta. TEM and immunohistochemistry (IHC) were applied to evaluate autophagy, and immunofluorescence (IF) was used to identify macrophage polarization. RAW264.7 macrophages were induced with oxidized low-density lipoprotein (ox-LDL) and high glucose (HG), co-cultured with BMSC-EVs, and analyzed for macrophage proliferation, migration, and foam cell formation. RAW264.7 cells were transduced with autophagy marker mRFP-GFP-LC3 lentivirus and analyzed with IF and western blotting.
Diabetic mice (DA group) had larger aortic plaque areas and lower collagen content than the HFD mice. BMSC-EV treatment significantly reduced blood glucose, LDL levels, and aortic plaque areas while increasing collagen content. BMSC-EV-treated aortas contained a higher number of autophagosomes/autolysosomes, with increased expression of LC3BII correlating with decreased P62 levels and a lower proportion of M1 macrophages. In vitro, BMSC-EVs inhibited proliferation, migration, and foam cell formation in ox-LDL and HG-induced activated RAW264.7 cells. These effects were reversed by the autophagy blocker bafilomycin A1. Consistent with the in vivo findings, BMSC-EVs elevated levels of the autophagy-related protein LC3BII/I and decreased P62 in ox-LDL and HG-induced RAW264.7 cells. These cells also expressed the M1 macrophage markers CD86 and iNOS, but showed reduced expression of the M2 marker Arg-1. Further, BMSC-EVs decreased AMPKα and mTOR phosphorylation levels, which were blocked by the AMPK inhibitor compound C.
BMSC-EVs attenuate diabetes-exacerbated atherosclerosis by inhibiting vascular macrophage proliferation, migration, and foam cell formation via AMPK/mTOR signaling-regulated autophagy and macrophage polarization. BMSC-EVs thus hold promise as therapeutic agents for atherosclerosis.
骨髓间充质干细胞衍生的细胞外囊泡（BMSC-EVs）在临床前研究中被广泛用于治疗目的。然而，它们在治疗糖尿病相关动脉粥样硬化方面的效用在很大程度上仍未得到探索。在此，我们旨在表征BMSC-EV介导的自噬调节和巨噬细胞极化。
从培养的BMSCs上清液中分离出细胞外囊泡，并通过透射电子显微镜（TEM）、纳米颗粒跟踪分析（NTA）和蛋白质印迹法进行表征。通过高脂饮食（HFD）和链脲佐菌素（STZ）建立糖尿病相关动脉粥样硬化ApoE小鼠模型。使用油红O、苏木精和伊红（H&E）以及主动脉的Masson染色进行组织病理学分析。应用TEM和免疫组织化学（IHC）评估自噬，并使用免疫荧光（IF）鉴定巨噬细胞极化。用氧化低密度脂蛋白（ox-LDL）和高糖（HG）诱导RAW264.7巨噬细胞，与BMSC-EVs共培养，并分析巨噬细胞增殖、迁移和泡沫细胞形成。用自噬标记物mRFP-GFP-LC3慢病毒转导RAW264.7细胞，并通过IF和蛋白质印迹法进行分析。
糖尿病小鼠（DA组）的主动脉斑块面积比HFD小鼠大，胶原蛋白含量比HFD小鼠低。BMSC-EV治疗显著降低血糖、低密度脂蛋白水平和主动脉斑块面积，同时增加胶原蛋白含量。BMSC-EV处理的主动脉中含有更多的自噬体/自溶酶体，LC3BII表达增加与P62水平降低以及M1巨噬细胞比例降低相关。在体外，BMSC-EVs抑制ox-LDL和HG诱导的活化RAW264.7细胞的增殖、迁移和泡沫细胞形成。自噬阻断剂巴弗洛霉素A1可逆转这些作用。与体内研究结果一致，BMSC-EVs提高了ox-LDL和HG诱导的RAW264.7细胞中自噬相关蛋白LC3BII/I的水平，并降低了P62水平。这些细胞还表达M1巨噬细胞标志物CD86和诱导型一氧化氮合酶（iNOS），但M2标志物精氨酸酶-1（Arg-1）的表达降低。此外，BMSC-EVs降低了AMPKα和mTOR的磷酸化水平，这被AMPK抑制剂化合物C阻断。
BMSC-EVs通过AMPK/mTOR信号调节的自噬和巨噬细胞极化抑制血管巨噬细胞增殖、迁移和泡沫细胞形成，从而减轻糖尿病加重的动脉粥样硬化。因此，BMSC-EVs有望成为动脉粥样硬化的治疗药物。
