Fei Chengshuo, Chen Yanlin, Tan Ruiqian, Yang Xinxing, Wu Guanda, Li Chenglong, Shi Jiawei, Le Shiyong, Yang Wenjie, Xu Jiajia, Wang Liang, Zhang Zhongmin
Division of Spine Surgery, Department of Orthopedics, Nanfang Hospital, Southern Medical University, Guangzhou, 510515, China.
Division of Spine Surgery, Department of Orthopaedics, The Third Affiliated Hospital, Southern Medical University, Guangzhou, 510630, China.
Biomark Res. 2025 Feb 25;13(1):33. doi: 10.1186/s40364-025-00746-6.
Ligamentum flavum hypertrophy (LFH) is a primary contributor to lumbar spinal stenosis. However, a thorough understanding of the cellular and molecular mechanisms driving LFH fibrotic progression remains incomplete.
Single-cell RNA sequencing (scRNA-seq) was performed to construct the single-cell map of human ligamentum flavum (LF) samples. An integrated multi-omics approach, encompassing scRNA-seq, bulk RNA sequencing (bulk RNA-seq), and Mendelian randomization (MR), was applied to conduct comprehensive functional analysis. Clinical tissue specimens and animal models were employed to further confirm the multi-omics findings.
ScRNA-seq provided a single-cell level view of the fibrotic microenvironment in LF, revealing significantly increased proportions of fibroblasts, myofibroblasts, and macrophages in LFH. Using transmission electron microscopy, single-cell gene set scoring, and MR analysis, ferroptosis was identified as a critical risk factor and pathway within LFH. Subcluster analysis of fibroblasts revealed functional heterogeneity among distinct subpopulations, highlighting the functional characteristics and the metabolic dynamics of fibroblast with a high ferroptosis score (High Ferro-score FB). The quantification of gene expression at single-cell level revealed that ferroptosis increased along with fibrosis in LFH specimens, a finding further validated in both human and mice tissue sections. Consistently, bulk RNA-seq confirmed increased proportions of fibroblasts and macrophages in LFH specimens, underscoring a strong correlation between these cell types through Spearman correlation analysis. Notably, subcluster analysis of the mononuclear phagocytes identified a specific subset of SPP1 macrophages (SPP1 Mac) enriched in LFH, which exhibited activation of fibrosis and ferroptosis-related metabolic pathways. Cell-cell communication analysis highlighted that SPP1 Mac exhibited the strongest outgoing and incoming interactions among mononuclear phagocytes in the LFH microenvironment. Ligand-receptor analysis further revealed that the SPP1-CD44 axis could serve as a key mediator regulating the activity of High Ferro-score FB. Multiplex immunofluorescence confirmed substantial Collagen I deposition and reduced Ferritin Light Chain expression in regions with SPP1-CD44 co-localization in LFH specimens.
Our findings indicated that SPP1 Mac may contribute to LFH fibrosis by regulating ferroptosis in High Ferro-score FB through the SPP1-CD44 axis. This study enhances our understanding of the cellular and molecular mechanisms underlying LFH progression, potentially improving early diagnostic strategies and identifying new therapeutic targets.
黄韧带肥厚（LFH）是腰椎管狭窄的主要原因。然而，对驱动LFH纤维化进展的细胞和分子机制的全面了解仍不完整。
进行单细胞RNA测序（scRNA-seq）以构建人黄韧带（LF）样本的单细胞图谱。应用包括scRNA-seq、批量RNA测序（bulk RNA-seq）和孟德尔随机化（MR）的综合多组学方法进行全面的功能分析。使用临床组织标本和动物模型进一步证实多组学研究结果。
scRNA-seq提供了LF纤维化微环境的单细胞水平视图，显示LFH中成纤维细胞、肌成纤维细胞和巨噬细胞的比例显著增加。通过透射电子显微镜、单细胞基因集评分和MR分析，铁死亡被确定为LFH中的一个关键危险因素和途径。成纤维细胞的亚群分析揭示了不同亚群之间的功能异质性，突出了高铁死亡评分的成纤维细胞（High Ferro-score FB）的功能特征和代谢动态。单细胞水平的基因表达定量显示，LFH标本中的铁死亡随着纤维化增加，这一发现在人和小鼠组织切片中得到进一步验证。同样，批量RNA-seq证实LFH标本中成纤维细胞和巨噬细胞的比例增加，通过Spearman相关性分析强调了这些细胞类型之间的强相关性。值得注意的是，单核吞噬细胞的亚群分析确定了LFH中富集的SPP1巨噬细胞（SPP1 Mac）的一个特定亚群，其表现出纤维化和铁死亡相关代谢途径的激活。细胞间通讯分析强调，SPP1 Mac在LFH微环境中的单核吞噬细胞中表现出最强的传出和传入相互作用。配体-受体分析进一步揭示，SPP1-CD44轴可能是调节High Ferro-score FB活性的关键介质。多重免疫荧光证实LFH标本中SPP1-CD44共定位区域的I型胶原蛋白大量沉积，铁蛋白轻链表达减少。
我们的研究结果表明，SPP1 Mac可能通过SPP1-CD44轴调节High Ferro-score FB中的铁死亡，从而促进LFH纤维化。这项研究增强了我们对LFH进展潜在的细胞和分子机制的理解，可能改善早期诊断策略并确定新的治疗靶点。
