Abstract:
Background Human umbilical cord mesenchymal stem cell-derived exosomes (HucMSC-Exo) have become a promising therapeutic tool in the field of regenerative medicine, but the exact mechanism of their action is still poorly understood.
Objective To explore whether HucMSC-Exo could drive neutrophil switching to N2 proangiogenic subtype.
Methods HucMSC-Exo was isolated and purified by ultracentrifugation, and the isolated exosomes were characterized by transmission electron microscopy, particle size analysis and western blot. Isolated HucMSC-Exo was co-cultured with neutrophils in vitro and the expression of phenotype-related molecules in neutrophils was detected using qRT-PCR. After co-culture with HucMSC-Exo, neutrophils were subjected to cell-cell co-culture system with vascular endothelial cells, and the effects of neutrophils on the proliferation, migration and invasion ability of vascular endothelial cells were observed by CCK8 assay, Edu assay, scratch assay and Transwell assay, respectively. Gene expression changes related to angiogenesis in neutrophils after receiving HucMSC-Exo stimulation were analyzed using qRT-PCR.
Results The extracts were shown in transmission electron microscopy as a round-like membrane-like structure with a diameter of approximately 100 nm, and were able to express the exosomal markers CD9 and Alix, indicating that the extracts were HucMSC-Exo. Compared to the PBS-treated group, treatment of neutrophils with HucMSC-Exo revealed increased expression of N2 markers, with Arg1 (P < 0.05), CD163 (P < 0.05) and CD206 (P< 0.05) by qRT-PCR. Following co-culture of HucMSC-Exo-stimulated neutrophils with vascular endothelial cells, the vascular endothelial cells became more proliferative, migratory, and invasive as observed by CCK8 assay, Edu assay, scratch assay, and Transwell assay, respectively. Compared with the PBS-treated group, neutrophils in HucMSC-Exo-treated group displayed elevated expression of pro-angiogenic factors BV8 (P < 0.05) and VEGFα (P < 0.05).
Conclusion HucMSC-Exo converts neutrophils to the N2 phenotype, and this N2 type of neutrophil promotes vascular endothelial cell proliferation, migration, and invasion.