M2 macrophages promote heterotopic ossification through MSX2 binding to LEF1-mediated endothelial-mesenchymal...

Heterotopic ossification (HO) is a pathological condition characterized by dysregulated regenerative processes in skeletal muscle, resulting in the formation of mature bone within ectopic sites. Accumulating studies indicate that M2 macrophages facilitate endothelial-mesenchymal transition (EndMT), which contributes centrally to HO pathogenesis. This study explored the mechanism of M2 macrophage involvement in EndMT-mediated HO and analyzed the underlying molecular pathways.

Clinical samples of immature, mature, and autologous cancellous bones were collected to analyze macrophage infiltration and the cellular origin of HO. Using the GEO and GeneCards databases, MSX2 was identified as a candidate regulatory factor. An endothelial-macrophage co-culture system was established to investigate the specific molecular mechanisms by which macrophages affect EndMT by modulating MSX2 expression. Achilles tendon incision surgery was performed in C57BL/6 mice to simulate trauma-induced HO. Histological examination, immunohistochemistry, immunofluorescence, and ELISA were performed to verify the role of macrophages in influencing HO progression via MSX2. The STRING database was used to predict LEF1 as a downstream target gene of MSX2. Regulatory interactions between MSX2 and LEF1 were examined using dual-luciferase reporter assays, western blotting, and quantitative real-time PCR.

Compared to autologous cancellous bone, there was an increase in M2 macrophage infiltration in HO tissues, accompanied by the transformation of endothelial cells at the injury site into mesenchymal stem cells. The degree of HO positively correlated with elevated levels of BMP-2, SP, Act A, TGF-β, OSM, and NT-3 in the serum. In vitro experiments demonstrated that under co-culture conditions, M2 macrophages induced mouse aortic endothelial cells (MAOECs) to exhibit elevated expression of mesenchymal markers (N-cadherin, vimentin) and mesenchymal stem cell markers (CD44, CD90), while reducing levels of endothelial markers (E-cadherin, occludin). Moreover, M2 macrophage-driven EndMT activation further promoted the upregulation of chondrogenic (Sox9, SP7) and osteogenic (OPN, OCN, Runx2) markers. However, MSX2 depletion rescued M2 macrophage-induced EndMT activation. In vivo, MSX2 inhibition or macrophage depletion reduced the osteogenic capacity in mice and decreased HO formation, whereas injection of a lentivirus overexpressing MSX2 promoted HO formation. Mechanistically, MSX2 directly binds to the LEF1 promoter to enhance its transcriptional activity, thereby activating Wnt signaling and maintaining EndMT.

Our findings suggest that M2 macrophages regulate EndMT-driven osteogenesis through MSX2/LEF1/Wnt axis, providing new insights into future therapeutic strategies for HO.