Shaban Shadi Abdolrahman, Rezaie Jafar, Nejati Vahid
Department of Biology, Urmia University, Urmia, Post Box 165, 5756151818, Iran.
Solid Tumor Research Center, Cellular and Molecular Medicine Institute, Urmia University of Medical Sciences, Urmia, Iran.
Cardiovasc Toxicol. 2022 Jun;22(6):592-601. doi: 10.1007/s12012-022-09740-y. Epub 2022 Apr 19.
Exosomes from senescence cells play pivotal roles in endothelium dysfunction. We investigated the exosomal angiogenic cargo of endothelial cells (ECs) in a model of senescence in vitro. After inducing aging by HO, the expression of P53, P21, and P16 was investigated by western blotting, while the expression of FMR1, miR-21, and miR-126 were measured by real-time PCR (q-PCR). Oil Red O dye was used to stain cells. Acetylcholinesterase (AChE) assay, transmission electron microscopy (TEM), and western blotting characterized Exosomes. Exosomal miR-21, miR-126, matrix metallopeptidase-9 (MMP-9), and tumor necrosis factor- ɑ (TNF-ɑ) proteins were measured by Q-PCR and western blotting. A wound-healing assay was used to explore the effect of exosomes on ECs migration rate. The results showed that the expression of P53, P21, P16, FMR1, and miR-21 was increased in treated cells as compared with control cells (P < 0.05). In addition, the expression of miR-126 was decreased in treated cells (P < 0.05). The number of Oil Red O-positive-treated cells increased (P < 0.05). The AChE activity of exosomes from treated cells was increased (P < 0.05). In comparison with control cells, an increase in the expression levels of exosomal miR-21 and TNF-ɑ of treated cells coincided with a decrease in the expression levels of miR-126 and MMP-9 levels (P < 0.05). We found that the migration rate of ECs co-cultured with exosomes from treated cells was decreased (P < 0.05). The data indicate ECs under HO condition produce exosomes with distinct cargo that may be useful as a biomarker of age-related vascular disease.
衰老细胞来源的外泌体在内皮功能障碍中起关键作用。我们在体外衰老模型中研究了内皮细胞（ECs）外泌体的血管生成相关成分。通过过氧化氢（HO）诱导衰老后，采用蛋白质免疫印迹法检测P53、P21和P16的表达，同时采用实时定量聚合酶链反应（q-PCR）检测脆性X智力低下蛋白1（FMR1）、微小RNA-21（miR-21）和微小RNA-126（miR-126）的表达。用油红O染料对细胞进行染色。采用乙酰胆碱酯酶（AChE）活性检测、透射电子显微镜（TEM）观察及蛋白质免疫印迹法对外泌体进行鉴定。通过q-PCR和蛋白质免疫印迹法检测外泌体miR-21、miR-126、基质金属蛋白酶-9（MMP-9）和肿瘤坏死因子-α（TNF-α）蛋白水平。采用划痕实验探究外泌体对ECs迁移率的影响。结果显示，与对照细胞相比，处理后细胞中P53、P21、P16、FMR1和miR-21的表达增加（P<0.05）。此外，处理后细胞中miR-126的表达降低（P<0.05）。油红O阳性处理细胞的数量增加（P<0.05）。处理后细胞来源外泌体的AChE活性增加（P<0.05）。与对照细胞相比，处理后细胞外泌体miR-21和TNF-α表达水平升高，同时miR-126和MMP-9表达水平降低（P<0.05）。我们发现，与处理后细胞来源外泌体共培养的ECs迁移率降低（P<0.05）。数据表明，HO条件下的ECs产生具有不同成分的外泌体，这些外泌体可能作为与年龄相关血管疾病的生物标志物。
