Department of Pharmacology and Toxicology, Boonshoft School of Medicine, Wright State University, Dayton, OH 45435, USA.
Department of Biomedical Sciences, Joan C Edwards School of Medicine, Marshall University, Huntington, WV 25755, USA.
Exp Neurol. 2022 Dec;358:114211. doi: 10.1016/j.expneurol.2022.114211. Epub 2022 Aug 24.
We have previously demonstrated that endothelial progenitor cells (EPCs) provide beneficial effects on ischemic stroke by reducing oxidative stress, which could be through EPCs-released exosomes (EPC-EXs). EXs are emerging as a bioagent for mediating cell-cell communications via their carried microRNAs (miR). miR-210 is shown to provide a neuroprotection effect against ischemic stroke. Here, we aimed to determine whether the combination of EPC-EXs and miR-210 would provide an enhanced protective effect on neurons. The hypoxia and reoxygenation (H/R) model were applied to neurons to mimic the ischemic injury of neurons. EPCs were transfected with miR-210 mimic to elevate the level of miR-210 in cells and EPC-EXs (miR210-EPC-EXs). For functional studies, EPC-EXs were co-incubated with H/R-injured neurons, then the cell viability and reactive oxygen species (ROS) production were determined. The results showed 1) H/R induced apoptosis and ROS overproduction in neurons; 2) miR-210 mimic increased the level of miR-210 in both EPCs and EPC-EXs; 3) EPCs cultured in serum-free medium released more exosomes in comparison with cells grown in complete growth media, suggesting serum starving induce the release of EXs; 4) After transfection, EPCs grown in complete media had almost 50 times higher miR-210 level than EPCs had in serum-free media, while the EPCs-EXs isolated from the complete media has lower miR-210 expression than from the serum-free media in a time-dependent manner, suggesting the transfer of miR-210 through EXs; 5) After co-incubation, EPC-EXs and miR210-EPC-EXs were uptaken by neurons, and the miR-210 level in neurons was elevated by miR210-EPC-EXs; 6) miR210-EPC-EXs were more effective in promoting cell viability and decreasing apoptosis and ROS production than EPC-EXs. The present study demonstrated that EPCs-carried miR-210 could be released and transferred to neurons in a time-dependent manner and that miR-210 loading can enhance the protective effects of EPC-EXs on H/R-induced neuron apoptosis, oxidative stress, and decreased viability.
我们之前已经证明，内皮祖细胞（EPCs）通过减少氧化应激对缺血性中风产生有益影响，这可能是通过 EPCs 释放的外泌体（EPC-EXs）实现的。外泌体作为一种生物制剂，通过其携带的 microRNAs（miR）来介导细胞间通讯。miR-210 被证明对缺血性中风具有神经保护作用。在这里，我们旨在确定 EPC-EXs 和 miR-210 的组合是否会对神经元产生增强的保护作用。我们应用缺氧和复氧（H/R）模型模拟神经元的缺血性损伤，将 miR-210 模拟物转染到 EPCs 中，以提高细胞和 EPC-EXs（miR210-EPC-EXs）中的 miR-210 水平。在功能研究中，将 EPC-EXs 与 H/R 损伤的神经元共孵育，然后测定细胞活力和活性氧（ROS）的产生。结果表明：1）H/R 诱导神经元凋亡和 ROS 过度产生；2）miR-210 模拟物增加了 EPCs 和 EPC-EXs 中的 miR-210 水平；3）与在完全生长培养基中生长的细胞相比，在无血清培养基中培养的 EPCs 释放更多的外泌体，提示血清饥饿诱导外泌体的释放；4）转染后，在完全培养基中生长的 EPCs 的 miR-210 水平几乎比在无血清培养基中生长的 EPCs 高 50 倍，而从无血清培养基中分离的 EPC-EXs 的 miR-210 表达水平则随时间呈下降趋势，提示 miR-210 通过外泌体转移；5）共孵育后，EPC-EXs 和 miR210-EPC-EXs 被神经元摄取，神经元中的 miR-210 水平被 miR210-EPC-EXs 升高；6）miR210-EPC-EXs 比 EPC-EXs 更有效地促进细胞活力，减少细胞凋亡和 ROS 的产生。本研究表明，EPCs 携带的 miR-210 可以以时间依赖性的方式被释放并转移到神经元中，miR-210 负载可以增强 EPC-EXs 对 H/R 诱导的神经元凋亡、氧化应激和活力降低的保护作用。
