Descendants of hypertrophic chondrocytes promote angiogenesis by secreting THBS4 during bone growth and injury repair

Hypertrophic chondrocytes (HCs) could transform into osteoblastic lineage cells while the pathophysiological implications of HC transformation remain largely unknown. Here, we generated a mouse line utilizing Col10a1-Cre to induce DTA expression to genetically ablate HCs and their descendants. Col10a1-Cre; R26^DTA/+ mice displayed dwarf phenotype, abnormal spongy bone, and significantly delayed drill-hole injuries healing, suggesting an indispensable role of HC lineage extension in bone growth and injury repair. Intriguingly, single-cell RNA sequencing analysis revealed the most significant loss of a cell cluster expressing multiple angiogenic factors (Pro-Angiogenic Descendants of HCs, PADs) among cells derived from Col10a1-Cre; R26^DTA/+ and control femurs. In silico analysis of cell-cell communication supported Thrombospondin 4 (THBS4) as a specific angiogenic factor mediating the crosstalk between PADs and vascular endothelial cells. Concordantly, analyses using immunostaining combined with tissue clearing revealed that PADs physically contacted with endothelial cells, whereas Col10a1-Cre; R26^DTA/+ mice showed defective metaphyseal and cortical vessel formation and post-injury angiogenesis along with a significant loss of THBS4. Moreover, in vitro assays showed that supplying THBS4 was sufficient to promote proliferation and tube formation of endothelial cells and rescue defective angiogenesis of Col10a1-Cre; R26^DTA/+ metatarsal explants. Collectively, these findings demonstrate a critical role of PADs in bone growth and injury repair by secreting THBS4 to regulate angiogenesis.