PRMT1-rich exosomes derived from M2 macrophages as novel therapeutics for enhancing fracture healing.

PURPOSE: sM2 macrophage-derived exosomes (M2-Exo) have emerged as promising mediators of bone repair due to their anti-inflammatory and pro-regenerative properties. However, the molecular mechanisms underlying the interaction between M2-Exo and bone marrow mesenchymal stem cells (BMSCs) are awaiting clarification. Our previous study found that protein arginine methyltransferase 1 (PRMT1) in fracture-derived exosomes promotes bone healing. This study aimed to determine whether PRMT1-containing M2-Exo exerts an osteoprotective effect during fracture healing and elucidate its underlying mechanisms. METHODS: Following knockdown of PRMT1 in M2-Exo and then treating BMSCs, osteogenic differentiation ability of BMSCs was evaluated by qRT-PCR, western blot, Alizarin Red S, and Alkaline phosphatase (ALP). Immunoprecipitation was employed to clarify regulatory effect of PRMT1 on Src-associated in mitosis 68 kDa (Sam68) and its asymmetric dimethylarginine (ADMA) modification. RESULTS: PRMT1 was overexpressed in M2-Exo extracted from serum samples of fracture rats. M2-Exo delivered PRMT1 to BMSCs. PRMT1 knockdown in M2-Exo impaired osteogenic differentiation, as evidenced by suppressing mineral deposition and ALP activity (42.3 % and 60.8 % reduction vs. sh-NC; p < 0.01), with a decrease in expression of Bone Morphogenetic Protein 2 (BMP2) and RUNX Family Transcription Factor 2 (RUNX2) (74.9 % and 61.8 % decline vs. sh-NC; p < 0.01). PRMT1 interacted with Sam68, and its knockdown reduced ADMA modification and Sam68 protein expression. Sam68 overexpression partially rescued the osteogenic defects caused by PRMT1 knockdown, with increased mineralization level and ALP (44.3 % and 87.6 % increase vs. M2-Exo-sh-PRMT1 + oe-NC; p < 0.05), along with elevated BMP2 and RUNX2 expression (79.5 % and 98.8 % increase vs. M2-Exo-sh-PRMT1 + oe-NC; p < 0.01). CONCLUSION: PRMT1 released by M2-Exo is delivered to BMSCs, which enhances the osteogenic differentiation by catalyzing Sam68 ADMA. These findings provide a mechanistic and preclinical rationale for the potential use of M2-Exo to improve bone healing.