CD39+CD55− Fb Subset Exhibits Myofibroblast-Like Phenotype and Is Associated with Pain in Osteoarthritis of the Knee

Abstract

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 (n = 3) and LC/MS analysis (n = 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, p = 0.009; active pain, ρ = 0.483, p = 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.

Shotgun Proteomics Links Proteoglycan-4+ Extracellular Vesicles to Cognitive Protection in Amyotrophic Lateral Sclerosis

“…By drawing parallels to the established interaction between PRG-4 and CD44, a transmembrane receptor found on the surface of EVs [ 33 ], we speculate that a similar mode of interaction could occur between PRG-4 and GPI-anchored receptors. Notably, EVs have been shown to express CD55, a GPI-anchored protein [ 34 ] to which PRG-4 could potentially bind, as demonstrated elsewhere [ 35 ]. Among all the identified proteins, PRG-4 showed the highest fold change and it was the only one associated with a clinical outcome.…”

Fibrocyte Phenotype of ENTPD1+CD55+ Cells and Its Association with Pain in Osteoarthritic Synovium

“…The PECAM1-CD45-CD55+ENTPD1-Thy-1+ cells, referred to as ENTPD1+CD55+ cells, and PECAM1-CD45-CD55+ENTPD1-Thy-1+ cells, referred to as ENTPD1−CD55+ cells, were individually sorted using a cell sorter (MA900, SONY, Tokyo, Japan). Gating criteria were established using control samples and single-stained samples [12].…”

“…Eluted peptides were analyzed using a Q Exactive for data-independent acquisition (DIA)-MS. MS1 spectra were obtained at 17,500 resolutions in the range of 350-770 m/z, and MS2 spectra were collected at >200 m/z at 35,000 resolutions. DIA were performed using a previously described protocol [12]. DIA files were assessed using the DIA-NN software (v.1.8.1, https://github.com/vdemichev/DiaNN, accessed on 12 March 2024) with fixed modifications of carbamidomethyl and variable modifications of Met oxidation.…”

“…These cells play a crucial role by forming a critical interface with the joint space and expressing high levels of lubricin, a glycoprotein, as well as hyaluronic acid, both of which are essential for joint lubrication and cartilage health [8,9]. However, in the context of OA, abnormal fibroblastic cells with a fibrotic or inflammatory phenotype become prominent, contributing significantly to the pathology of the joint disorder [11][12][13].…”