Identification of novel macrophages and bone morphogenetic protein signals causing ectopic calcification and impairing muscle regeneration

Ectopic calcification is an abnormal regenerative response occurring in various tissues following injury, surgery, or genetic mutations. However, its underlying mechanisms remain unclear. By comparing three macrophage depletion methods using clodronate liposome, Csf1r neutralizing antibody, and macrophage-specific <i>Csf1r</i> gene deletion (<i>Csf1r cKO</i>), we found that F4/80(+)Csf1r(-) macrophages were specifically increased in calcified muscles in notexin-injected mice and BaCl₂-injected <i>Csf1r cKO</i> mice. Mechanistically, bone morphogenetic protein (BMP) signaling was found to contribute to ectopic calcification. Reanalysis of public single-cell sequencing data and lineage-tracing analysis using <i>Cdh5</i> ^{<i>creERT2</i>} mice revealed that endothelial-to-mesenchymal transition (EndoMT) is also a key contributor to ectopic calcification, as evidenced by the high expression of EndoMT-related markers in mesenchymal progenitor cells. Notably, the administration of BMP inhibitors reduced calcification and promoted muscle regeneration. Thus, F4/80(+)Csf1r(-) macrophages and BMP signals represent promising therapeutic targets for preventing ectopic calcifications triggered by trauma, burns, infections, or surgical interventions.