摘要While hypoxic signaling has been shown to play a role in many cellular processes,its role in metabolism-linked extracellular matrix(ECM)organization and downstream processes of cell fate after musculoskeletal injury remains to be determined.Heterotopic ossification(HO)is a debilitating condition where abnormal bone formation occurs within extra-skeletal tissues.Hypoxia and hypoxia-inducible factor 1α(HIF-1α)activation have been shown to promote HO.However,the underlying molecular mechanisms by which the HIF-1α pathway in mesenchymal progenitor cells(MPCs)contributes to pathologic bone formation remain to be elucidated.Here,we used a proven mouse injury-induced HO model to investigate the role of HIF-1α on aberrant cell fate.Using single-cell RNA sequencing(scRNA-seq)and spatial transcriptomics analyses of the HO site,we found that collagen ECM organization is the most highly up-regulated biological process in MPCs.Zeugopod mesenchymal cell-specific deletion of Hif1α(Hoxa11-CreERT2;Hif1afl/fl)significantly mitigated HO in vivo.ScRNA-seq analysis of these Hoxa11-CreERT2;Hif1afl/flmice identified the PLOD2/LOX pathway for collagen cross-linking as downstream of the HIF-1α regulation of HO.Importantly,our scRNA-seq data and mechanistic studies further uncovered that glucose metabolism in MPCs is most highly impacted by HIF-1α deletion.From a translational aspect,a pan-LOX inhibitor significantly decreased HO.A newly screened compound revealed that the inhibition of PLOD2 activity in MPCs significantly decreased osteogenic differentiation and glycolytic metabolism.This suggests that the HIF-1α/PLOD2/LOX axis linked to metabolism regulates HO-forming MPC fate.These results suggest that the HIF-1α/PLOD2/LOX pathway represents a promising strategy to mitigate HO formation.