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-CreER^T2; Hif1a^fl/fl) significantly mitigated HO in vivo. ScRNA-seq analysis of these Hoxa11-CreER^T2; Hif1a^fl/fl mice 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.

虽然缺氧信号已被证明在许多细胞过程中发挥作用，但其在代谢相关的细胞外基质 (ECM) 组织和肌肉骨骼损伤后细胞命运的下游过程中的作用仍有待确定。异位骨化 (HO) 是一种使人衰弱的疾病，其中骨骼外组织内发生异常骨形成。缺氧和缺氧诱导因子 1α (HIF-1α) 激活已被证明可促进 HO。然而，间充质祖细胞（MPC）中的 HIF-1α 通路促进病理性骨形成的潜在分子机制仍有待阐明。在这里，我们使用经过验证的小鼠损伤诱导的 HO 模型来研究 HIF-1α 对异常细胞命运的作用。利用单细胞 RNA 测序 (scRNA-seq) 和 HO 位点的空间转录组学分析，我们发现胶原蛋白 ECM 组织是 MPC 中上调程度最高的生物过程。 Zeugopod间充质细胞特异性删除Hif1α （ Hoxa11-CreER ^T2 ； Hif1a ^fl/fl ）可显着减轻体内H2O。这些Hoxa11-CreER ^T2的 ScRNA-seq 分析； Hif1a ^fl/fl小鼠发现胶原蛋白交联的 PLOD2/LOX 通路是 HIF-1α 调节 H2O 的下游。重要的是，我们的 scRNA-seq 数据和机制研究进一步发现，MPC 中的葡萄糖代谢受 HIF-1α 缺失的影响最大。从翻译方面来看，pan-LOX 抑制剂显着降低了 HO。 一种新筛选的化合物表明，抑制 MPC 中的 PLOD2 活性可显着降低成骨分化和糖酵解代谢。这表明与代谢相关的 HIF-1α/PLOD2/LOX 轴调节 HO 形成的 MPC 命运。这些结果表明 HIF-1α/PLOD2/LOX 途径是减轻 HO 形成的有前途的策略。