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Abstract：
BACKGROUND: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O2) to dissect the molecular mechanisms responsible for cardioprotection.
METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.
RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.
CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
METHODS: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385.
RESULTS: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs.
CONCLUSIONS: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases.
摘要：
背景：干细胞衍生的细胞外囊泡（EV）是一类具有优异生物相容性的新兴治疗剂，生物活性和促再生能力。基于EV的药物的潜在目标之一是心血管疾病（CVD）。在这项工作中，我们使用了在不同氧气浓度（21、5和3％O2）下培养的人诱导多能干细胞（hiPSCs;hiPPS-EV）衍生的EV来剖析负责心脏保护的分子机制。
方法：通过超滤结合尺寸排阻色谱（UFSEC）分离电动汽车，然后通过纳米粒子跟踪分析进行表征，原子力显微镜（AFM）和蛋白质印迹方法。液相色谱和串联质谱结合生物信息学分析用于鉴定各种氧气条件下的差异富集蛋白。我们直接比较了这些EV在心肌细胞（CM）损伤的氧-葡萄糖剥夺/复氧（OGD/R）模型中的心脏保护作用。利用先进的分子生物学，荧光显微镜,原子力谱和生物信息学技术，我们研究了参与细胞存活调节的细胞内信号通路，细胞凋亡和抗氧化反应。在用ML385抑制NRF2后的CM中评估EV对NRF2调节的信号传导的直接作用。
结果：我们证明，与其他测试的氧气条件（常氧；EV-N和低氧3％O2；EV-H3）衍生自5％O2的生理性缺氧（EV-H5）的hiPS-EV对受损CM的细胞保护功能增强。这是由于转移后受体细胞中Akt激酶的磷酸化率较高，调节AMPK活性和减少细胞凋亡。此外，我们提供了使用AFM测量的EV-H5治疗的CM的钙信号改善和持续收缩性的直接证据.机械上,我们的质谱和生物信息学分析显示,EV-H5中的差异富集蛋白与NRF2调节的抗氧化途径相关.在这方面,EV-H5在OGD/R时增加NRF2蛋白的核易位并增强其在CMs中的转录。相比之下,用ML385抑制NRF2消除了电动汽车对CMs的保护作用。
结论：在这项工作中，与EV-N和EV-H3相比,我们证明了EV-H5具有更好的心脏保护功能.这样的电动汽车在恢复应力CM的氧化还原平衡方面最有效，保持其收缩功能并防止细胞死亡。我们的数据支持生理性缺氧产生的hiPS-EV的潜在用途，作为具有再生特性的无细胞疗法，用于治疗心脏病。
方法：通过超滤结合尺寸排阻色谱（UFSEC）分离电动汽车，然后通过纳米粒子跟踪分析进行表征，原子力显微镜（AFM）和蛋白质印迹方法。液相色谱和串联质谱结合生物信息学分析用于鉴定各种氧气条件下的差异富集蛋白。我们直接比较了这些EV在心肌细胞（CM）损伤的氧-葡萄糖剥夺/复氧（OGD/R）模型中的心脏保护作用。利用先进的分子生物学，荧光显微镜,原子力谱和生物信息学技术，我们研究了参与细胞存活调节的细胞内信号通路，细胞凋亡和抗氧化反应。在用ML385抑制NRF2后的CM中评估EV对NRF2调节的信号传导的直接作用。
结果：我们证明，与其他测试的氧气条件（常氧；EV-N和低氧3％O2；EV-H3）衍生自5％O2的生理性缺氧（EV-H5）的hiPS-EV对受损CM的细胞保护功能增强。这是由于转移后受体细胞中Akt激酶的磷酸化率较高，调节AMPK活性和减少细胞凋亡。此外，我们提供了使用AFM测量的EV-H5治疗的CM的钙信号改善和持续收缩性的直接证据.机械上,我们的质谱和生物信息学分析显示,EV-H5中的差异富集蛋白与NRF2调节的抗氧化途径相关.在这方面,EV-H5在OGD/R时增加NRF2蛋白的核易位并增强其在CMs中的转录。相比之下,用ML385抑制NRF2消除了电动汽车对CMs的保护作用。
结论：在这项工作中，与EV-N和EV-H3相比,我们证明了EV-H5具有更好的心脏保护功能.这样的电动汽车在恢复应力CM的氧化还原平衡方面最有效，保持其收缩功能并防止细胞死亡。我们的数据支持生理性缺氧产生的hiPS-EV的潜在用途，作为具有再生特性的无细胞疗法，用于治疗心脏病。
