Profibrotic macrophage-derived CXCL4 promotes pericyte-to-myofibroblast transition after spinal cord injury

Introduction Spinal cord injury (SCI) induces fibrotic scarring that impairs axonal regeneration. Pericytes contribute to scar formation via pericyte-to-myofibroblast transition (PMT), yet the mechanisms underlying PMT in SCI remain unclear. Although CXCL4, a pleiotropic chemokine, is implicated in various fibrotic disorders, its role in driving PMT post-SCI remains unexplored. Objectives To investigate whether CXCL4 drives PMT after SCI, elucidate its mechanisms, and assess its therapeutic potential. Methods scRNA-seq characterized cell-type dynamics and profibrotic signals in injured mouse spinal cords. In vitro , primary pericytes were exposed to exogenous CXCL4 or co-cultured with Spp1 + Fn1 + macrophages. PMT was evaluated by RT-qPCR, Western blot, immunofluorescence, and flow cytometry. PI3K/Akt inhibition or CXCR3 knockdown dissected signaling pathways. In vivo , intrathecal injections of a CXCL4-neutralizing antibody or PI3K inhibitor were administered post-injury. Pericyte differentiation and fibrotic remodeling were assessed via immunostaining, Masson's trichrome staining, and gene expression profiling. Axonal regeneration and motor function were evaluated using CST tracing, serotonergic fiber labeling, Basso Mouse Scale scoring, and footprint analysis. Results PMT occurred post SCI, with Pdgfrβ + Acta2 + pericytes acting as major contributors. Spp1 + Fn1 + macrophage subpopulation was identified as the main source of CXCL4, transcriptionally regulated by MAFB. CXCL4 levels were significantly upregulated post-injury, while pericytes in the lesion expressed its receptor, CXCR3. In vitro , exogenous CXCL4 induced PMT in pericytes via PI3K/Akt signaling. Co-culture experiments confirmed that Spp1 + Fn1 + macrophages promoted pericyte transition through the CXCL4/CXCR3 axis. In vivo , blocking CXCL4 or PI3K suppressed PMT, reduced fibrotic scarring, enhanced axonal regeneration, and improved locomotor function in SCI mice. Conclusion Profibrotic macrophage-derived CXCL4 activates CXCR3/PI3K/Akt signaling in pericytes, driving their transition into scar-forming myofibroblasts after SCI. Blocking this axis mitigates fibrosis and enhances axonal regeneration and motor recovery. The translational potential of this article This study indicates that targeting the CXCL4-driven pericyte-to-myofibroblast transition to reduce fibrotic scar formation may provide an effective therapeutic strategy for enhancing axonal regeneration and functional recovery after SCI.