Single-cell RNA sequencing analysis reveals the evolution and regulatory features of specialized endothelial cell subsets in non-traumatic osteonecrosis of the femoral head.

Non-traumatic osteonecrosis of the femoral head (NONFH) is a disabling disease mainly caused by glucocorticoid use and alcohol abuse, characterized by local impairment of blood supply and limited bone repair; however, the composition and regulatory mechanisms of specialized endothelial cells (ECs) in its microenvironment remain unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to identify four EC subsets, including type L, type H, type R, and arterial ECs. Type H and type R ECs were primarily enriched in cell adhesion and angiogenesis pathways and represented key subsets that promote bone repair in NONFH. Their abundance increased during the mid-stage of NONFH but declined in the late stage. Stromal cells in the femoral head interacted with type H and type R ECs through multiple ligand–receptor axes, maintaining their function. Further screening identified PDK4 as a critical regulator of type H and type R EC abundance. PDK4 not only promotes endothelial fatty acid oxidation but also participates in iron homeostasis and angiogenesis. In vitro experiments showed that PDK4 knockdown significantly impaired the angiogenic phenotype of ECs, which was associated with suppressed fatty acid oxidation, highlighting PDK4’s role in sustaining the activity and function of type H and type R ECs under disease conditions. Peripheral blood transcriptome analysis revealed that the dynamic expression pattern of PDK4 across NONFH stages was consistent with the single-cell results, suggesting potential value for stage-specific diagnosis. By integrating single-cell and peripheral blood transcriptome analyses with in vitro experiments and metabolomic validation, this study elucidates the dynamic changes of specialized ECs and the PDK4-mediated metabolic regulatory mechanisms during NONFH progression. These findings provide new insights into vascular repair and potential therapeutic strategies for NONFH.