METHODS: EVs were isolated from normoxic and hypoxic cultured ASC conditioned culture medium. The miRNA expression profile was determined by miRseq, and EV markers were determined by Western blot and immunofluorescence staining. The uptake dynamics of fluorescently labelled EVs were monitored for 24 h. ASC-EVs\' pro-angiogenic effect was assessed by sprouting ex vivo rat aorta rings in left ventricular-decellularized extracellular matrix (LV dECM) hydrogel or basement membrane hydrogel (Geltrex®).
RESULTS: ASC-EVs augmented vascular network formation by aorta rings. The vascular network topology and stability were influenced in a hydrogel scaffold-dependent fashion. The ASC-EVs were enriched for several miRNA families/clusters, including Let-7 and miR-23/27/24. The miRNA-1290 was the highest enriched non-clustered miRNA, accounting for almost 20% of all reads in hypoxia EVs.
CONCLUSIONS: Our study revealed that ASC-EVs augment in vitro and ex vivo vascularisation, likely due to the enriched pro-angiogenic miRNAs in EVs, particularly miR-1290. Our results show promise for regenerative and revascularisation therapies based on ASC-EV-loaded ECM hydrogels.
方法:从常氧和低氧培养的ASC条件培养基中分离EV。通过miRseq测定miRNA表达谱,通过Westernblot和免疫荧光染色确定EV标志物。监测荧光标记的EVs的摄取动力学24小时。通过在左心室去细胞化的细胞外基质(LVdECM)水凝胶或基底膜水凝胶(Geltrex®)中发芽离体大鼠主动脉环来评估ASC-EVs的促血管生成作用。
结果:ASC-EV通过主动脉环增强血管网形成。以水凝胶支架依赖性方式影响血管网络拓扑和稳定性。ASC-EV富集了几个miRNA家族/簇,包括Let-7和miR-23/27/24。miRNA-1290是最高富集的非簇miRNA,占低氧电动汽车所有读数的近20%。
结论:我们的研究表明,ASC-EV在体外和离体血管形成中增加,可能是由于电动汽车中富集的促血管生成miRNA,特别是miR-1290。我们的结果显示了基于ASC-EV负载的ECM水凝胶的再生和血运重建疗法的前景。