Nakajima Hideaki, Johnson William E B, Kamitani Mikiko, Watanabe Shuji, Honjoh Kazuya, Kubota Arisa, Matsumine Akihiko
Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Yoshida-gun, Fukui 910-1193, Japan.
Chester Medical School, University of Chester, Chester CH1 4BJ, United Kingdom.
JBMR Plus. 2025 Feb 2;9(4):ziaf021. doi: 10.1093/jbmrpl/ziaf021. eCollection 2025 Apr.
Ossification of the spinal ligament (OSL), including ossification of the posterior longitudinal ligament and ossification of the ligamentum flavum (OLF), is a multifactorial disease that includes genetic predisposition. The association between the rate of ossification in the spinal canal and the severity of myelopathy symptoms is well known, but the degree of progression varies widely among patients. Although many candidate genes and biomarkers have been reported, there are no definitive and quantitative conclusions to date, probably because of low reproducibility due to individual differences. In this study, we focused on exosomes secreted by ossified spinal ligament cells. Exosomes are crucial for intercellular communication during development and progression of disease. In a co-culture study of non-OLF cells with OLF cells, there was increased osteogenic differentiation, including Runx2 and Wnt3a expression, with use of exosome-penetrating filters (1.2 μm) compared to exosome-non-penetrating filters (0.03 μm). Dose-dependent increases in alkaline phosphatase activity and mineral deposition were observed in non-OLF cells treated with OLF-derived exosomes. These results support the hypothesis that OLF-derived exosomes are involved in regulation of osteogenic differentiation. In comparative proteomics analysis, 32 factors were increased and 40 were decreased in OLF-derived exosomes compared to non-OLF-derived exosomes. Molecular network analysis of these 72 factors indicated 10 significant pathways, including the matrix metalloproteinase (MMP) signaling, mTOR signaling, Wnt signaling and VDR-associated pathways. Among the upregulated exosomal membrane proteins in OLF samples, COL IV, FMNL3, mTORC2, and PIP4K showed increased expression with greater ossification, suggesting they may serve as biomarkers of disease activity and therapeutic targets. These factors are involved in the PI3K/Akt/mTOR signaling pathway, and particularly mTOR is known to regulate osteogenic and chondrogenic differentiation. In contrast, fatty acid-binding protein 5, several KRT family proteins, S100A8, SERPINB3, and transglutaminase, were significantly downregulated in OLF-derived exosomes. These findings provide novel insights into the molecular mechanisms underlying OSL pathogenesis.
脊柱韧带骨化（OSL），包括后纵韧带骨化和黄韧带骨化（OLF），是一种具有遗传易感性的多因素疾病。椎管内骨化速率与脊髓病症状严重程度之间的关联已为人所知，但患者之间的进展程度差异很大。尽管已报道了许多候选基因和生物标志物，但迄今为止尚无明确和定量的结论，这可能是由于个体差异导致的低重复性。在本研究中，我们重点关注骨化脊柱韧带细胞分泌的外泌体。外泌体在疾病的发生和发展过程中的细胞间通讯中起着至关重要的作用。在非OLF细胞与OLF细胞的共培养研究中，与非外泌体穿透过滤器（0.03μm）相比，使用外泌体穿透过滤器（1.2μm）时，包括Runx2和Wnt3a表达在内的成骨分化增加。在用OLF衍生的外泌体处理的非OLF细胞中观察到碱性磷酸酶活性和矿物质沉积呈剂量依赖性增加。这些结果支持以下假设：OLF衍生的外泌体参与成骨分化的调节。在比较蛋白质组学分析中，与非OLF衍生的外泌体相比，OLF衍生的外泌体中有32种因子增加，40种因子减少。对这72种因子的分子网络分析表明有10条重要途径，包括基质金属蛋白酶（MMP）信号通路、mTOR信号通路、Wnt信号通路和VDR相关通路。在OLF样本中上调的外泌体膜蛋白中，COL IV、FMNL3、mTORC2和PIP4K随着骨化程度的增加而表达增加，表明它们可能作为疾病活动的生物标志物和治疗靶点。这些因子参与PI3K/Akt/mTOR信号通路，特别是mTOR已知可调节成骨和软骨分化。相反，脂肪酸结合蛋白5、几种KRT家族蛋白、S100A8、SERPINB3和转谷氨酰胺酶在OLF衍生的外泌体中显著下调。这些发现为OSL发病机制的分子机制提供了新的见解。
