The Effects of M2 Macrophages-Derived Exosomes on Urethral Fibrosis and Stricture in Scar Formation.

Background: Macrophages are highly plastic cells, and macrophage-derived exosomes (M-Exos) have been implicated in inflammation-related pathophysiologies, such as tissue injury and fibrosis repair. This study aimed to investigate the possible effects of M-Exos on the initiation and development of urethral fibrosis and stricture after injury, and to elucidate the underlying mechanisms. Methods: In this study, we used time-tracking immunofluorescence staining for M1 and M2 macrophage markers to characterize sequential properties in the site of injured urethra. Next, we harvested these exosomes from different macrophages to co-culture with fibroblasts to further confirm the role of exosome-mediated M2 macrophage-fibroblast communication. Then, high-throughput micro-RNA (miRNA) sequencing was performed to detect the candidate exosomal miRNA and its target gene. Furthermore, fibroblasts were transfected with mRFP-GFP-LC3 plasmid to detect the autophagy role of SIRT1 in the urethral fibroblasts fibrogenesis. Results: Here we found that M2-polarized macrophages triggered and dominated the fibrotic scene, purified exosomes from M2 macrophages exacerbated urethral fibroblast fibrogenesis, and the inhibition of exosome secretion abolished fibroblast fibrogenesis. Moreover, miR-34a-5p, which is highly enriched and packaged within M2-Exos, can be transferred from M2 macrophages into urethral fibroblasts, resulting in deterioration of proliferation and fibrogenesis. Mechanistically, M2-Exos miR-34a-5p could directly interact with the 3'-UTR of SIRT1, thereby suppressing SIRT1 expression in fibroblasts, leading to the blockage of autophagosome-lysosome fusion to impair urethral fibroblast autophagy flux and further exacerbate fibrogenesis. More importantly, repression of miR-34a-5p in M2-Exos mitigated-urethral strictures in rats with damaged urethra. Conclusion: M2 macrophage-derived exosomes miR-34a-5p could aggravate the development of urethral fibrosis and stricture by blocking autophagosome-lysosome fusion in urethral fibroblasts and further accelerating fibrogenesis by directly targeting SIRT1, suggesting that M2-Exo miR-34a-5p and SIRT1 could serve as promising therapeutic targets for urethral stricture.