Tsuchiya Maho, Ohashi Yoshihisa, Kodera Yoshio, Satoh Masashi, Matsui Takashi, Fukushima Kensuke, Iwase Dai, Aikawa Jun, Mukai Manabu, Inoue Gen, Takaso Masashi, Uchida Kentaro
Department of Orthopaedic Surgery, Kitasato University School of Medicine, 1-15-1 Minami-ku, Kitasato, Sagamihara 252-0374, Kanagawa, Japan.
Department of Physics, School of Science, Kitasato University, 1-15-1 Kitasato, Minami-ku, Sagamihara 252-0373, Kanagawa, Japan.
Biomedicines. 2023 Nov 14;11(11):3047. doi: 10.3390/biomedicines11113047.
Recent studies utilizing single-cell analysis have unveiled the presence of various fibroblast (Fb) subsets within the synovium under inflammatory conditions in osteoarthritis (OA), distinguishing them from those in rheumatoid arthritis (RA). Moreover, it has been reported that pain in knee OA patients is linked to specific fibroblast subsets. Single-cell expression profiling methods offer an incredibly detailed view of the molecular states of individual cells. However, one limitation of these methods is that they require the destruction of cells during the analysis process, rendering it impossible to directly assess cell function. In our study, we employ flow cytometric analysis, utilizing cell surface markers CD39 and CD55, in an attempt to isolate fibroblast subsets and investigate their relationship with OA pathology. Synovial tissues were obtained from 25 knee OA (KOA) patients. Of these, six samples were analyzed by RNA-seq ( = 3) and LC/MS analysis ( = 3). All 25 samples were analyzed to estimate the proportion of Fb (CD45-CD31-CD90+) subset by flow cytometry. The proportion of Fb subsets (CD39+CD55- and CD39-CD55+) and their association with osteoarthritis pathology were evaluated. CD39+CD55- Fb highly expressed myogenic markers such as CNN1, IGFBP7, MYH11, and TPM1 compared to CD39-CD55+ Fb. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of upregulated differentially expressed genes (DEGs) in CD39+CD55- Fb identified the Apelin pathway and cGMP-PKC-signaling pathway as possibly contributing to pain. LC/MS analysis indicated that proteins encoded by myogenic marker genes, including CNN1, IGFBP7, and MYH11, were also significantly higher than in CD39-CD55+ Fb. CD39-CD55+ Fb highly expressed PRG4 genes and proteins. Upregulated DEGs were enriched for pathways associated with proinflammatory states ('RA', 'TNF signaling pathway', 'IL-17 signaling pathway'). The proportion of CD39+CD55- Fb in synovium significantly correlated with both resting and active pain levels in knee OA (KOA) patients (resting pain, ρ = 0.513, = 0.009; active pain, ρ = 0.483, = 0.015). There was no correlation between joint space width (JSW) and the proportion of CD39+CD55- Fb. In contrast, there was no correlation between the proportion of CD39-CD55+ Fb and resting pain, active pain, or JSW. In conclusion, CD39+CD55- cells exhibit a myofibroblast phenotype, and its proportion is associated with KOA pain. Our study sheds light on the potential significance of CD39+CD55- synovial fibroblasts in osteoarthritis, their myofibroblast-like phenotype, and their association with joint pain. These findings provide a foundation for further research into the mechanisms underlying fibrosis, the impact of altered gene expression on osteoarthritic joints, and potential therapeutic strategies.
最近利用单细胞分析的研究揭示了骨关节炎(OA)炎症条件下滑膜内存在各种成纤维细胞(Fb)亚群,将它们与类风湿性关节炎(RA)中的成纤维细胞区分开来。此外,有报道称膝关节OA患者的疼痛与特定的成纤维细胞亚群有关。单细胞表达谱分析方法提供了单个细胞分子状态的极其详细的视图。然而,这些方法的一个局限性是它们在分析过程中需要破坏细胞,从而无法直接评估细胞功能。在我们的研究中,我们采用流式细胞术分析,利用细胞表面标志物CD39和CD55,试图分离成纤维细胞亚群并研究它们与OA病理的关系。从25名膝关节OA(KOA)患者中获取滑膜组织。其中,6个样本通过RNA测序(n = 3)和液相色谱/质谱分析(n = 3)进行分析。通过流式细胞术分析所有25个样本,以估计Fb(CD45-CD31-CD90+)亚群的比例。评估Fb亚群(CD39+CD55-和CD39-CD55+)的比例及其与骨关节炎病理的关联。与CD39-CD55+ Fb相比,CD39+CD55- Fb高表达肌源性标志物,如CNN1、IGFBP7、MYH11和TPM1。对CD39+CD55- Fb中上调的差异表达基因(DEG)进行京都基因与基因组百科全书(KEGG)分析,确定阿片肽途径和cGMP-PKC信号通路可能与疼痛有关。液相色谱/质谱分析表明,包括CNN1、IGFBP7和MYH11在内的肌源性标志物基因编码的蛋白质也显著高于CD39-CD55+ Fb。CD39-CD55+ Fb高表达PRG4基因和蛋白质。上调的DEG在与促炎状态相关的途径(“RA”、“TNF信号通路”、“IL-17信号通路”)中富集。滑膜中CD39+CD55- Fb的比例与膝关节OA(KOA)患者的静息痛和活动痛水平均显著相关(静息痛,ρ = 0.513,P = 0.009;活动痛,ρ = 0.483,P = 0.015)。关节间隙宽度(JSW)与CD39+CD55- Fb的比例之间无相关性。相反,CD39-CD55+ Fb的比例与静息痛、活动痛或JSW之间无相关性。总之,CD39+CD55-细胞表现出肌成纤维细胞表型,其比例与KOA疼痛相关。我们的研究揭示了CD39+CD55-滑膜成纤维细胞在骨关节炎中的潜在意义、它们的肌成纤维细胞样表型以及它们与关节疼痛的关联。这些发现为进一步研究纤维化的潜在机制、基因表达改变对骨关节炎关节的影响以及潜在治疗策略提供了基础。