HucMSC-Exo driving neutrophil to N2 promoting angiogenic phenotypic transformation

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 CCK-8 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 significantly increased expression of N2 markers, such as 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 CCK-8 assay, Edu assay, scratch assay, and Transwell assay, respectively (P < 0.05). 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.