Normoxia-Harvested hUC-MSC-Derived Exosomes Enhance Proangiogenic Activity Compared to Hypoxia-Derived Exosomes Via VEGFR2 Upregulation

Abstract Introduction Angiogenesis is essential for tissue regeneration and repair, a process largely mediated by the VEGFR2 signaling pathway, which is a key driver of endothelial cell proliferation and vessel formation. Mesenchymal stem cells (MSCs)-derived exosomes are known to promote angiogenesis under hypoxic conditions through their cargo of proteins and miRNAs. This study aimed to evaluate the proangiogenic potential of exosomes derived from human umbilical cord mesenchymal stem cells (hUC-MSCs) under normoxic (21% O₂) and hypoxic (1% O₂) conditions. Methods hUC-MSC–derived exosomes harvested under normoxic and hypoxic conditions were isolated and characterized in terms of size distribution, particle concentration, total protein content, and morphology. These exosomes under normoxic and hypoxic conditions were subsequently applied to human umbilical vein endothelial cells (HUVECs) at two concentrations (50 and 200 µg/mL) to evaluate their proangiogenic potential. The angiogenic activity was assessed using an in vitro Matrigel tube formation assay, and endothelial differentiation was quantified through image-based analysis of predefined angiogenesis parameters. Results Exosomes were isolated from human umbilical cord-derived MSCs and characterized with respect to size distribution, CD63 and CD9 surface protein content, and morphological imaging. Normoxic hUC-MSCs exhibited significantly higher VEGFR2 expression compared to hypoxia-treated cells ( P = 0.032). Exosomal protein yield was significantly higher under normoxia compared to hypoxia, the latter of which was evidenced by elevated HIF-1α expression. No significant dose-dependent differences (50 vs. 200 µg/mL) were observed in either condition. However, tube formation in the 50 µg/mL hypoxic group was significantly reduced compared with both normoxic groups, accompanied by a near absence of VEGFR2 expression in differentiating HUVECs. Conclusion hUC-MSCs-derived exosomes from normoxic conditions demonstrated superior in vitro angiogenic activity compared to those from hypoxia. The angiogenic performance of normoxic exosomes was associated with significantly higher VEGFR2 expression of hUC-MSCs, suggesting that normoxia may better preserve or promote the essential signaling pathways required for vessel formation. These findings suggest that low oxygen levels may reduce angiogenic protein content in exosomes, warranting further research to identify specific factors in hUC-MSCs exosomes under varying oxygen conditions.