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.

Keywords: CD39; fibroblast subset; knee osteoarthritis; pain.

Figure 1

Flow cytometric analysis of CD39+ and CD55+ fibroblast obtained from knee osteoarthritis patients. (A) CD45− gate in synovial cells. X-axis, forward scattering (FSC); y-axis, CD45. (B) CD31−CD90+ gate in CD45-negative gate. X-axis, CD31; y-axis, CD90 (C) Dot plot analysis of the fibroblast population. X-axis, CD39; y-axis, CD55.

Figure 2

Key driver and pathway analysis of upregulated genes in CD39+CD55− cells. (A) Common upregulated genes in CD39+CD55− cells compared to CD39−CD55+ cells among three patients were determined using a Venn diagram. (B) Key driver gene analysis of common upregulated genes in CD39+CD55− cells. Blue dots indicate the gene selected to estimate the key driver gene. Red dots indicate key driver genes. *¹ MYH11 transcript variant SM1A, *² MYH11 transcript variant SM2A (C) KEGG analysis of the common upregulated genes in CD39+CD55− cells.

Figure 3

Key driver and pathway analysis of upregulated genes in CD39−CD55+cells. (A) Common upregulated genes in CD39−CD55+ cells compared to CD39+CD55− cells among three patients were determined using a Venn diagram. (B) Key driver gene analysis of the common upregulated genes in CD39−CD55+ cells. Blue dots indicate the gene selected to estimate the key driver gene. Red dots indicate key driver genes. *¹ PDPN transcript variant 1, *² PDPN transcript variant 2, *³ NRP2 transcript variant 2, *⁴ NRP2 transcript variant 5 (C) KEGG analysis of the common upregulated genes in CD39−CD55+ cells.

Figure 4

Differently expressed proteins between CD39+CD55− and CD39−CD55+. Differently expressed proteins between CD39+CD55− and CD39−CD55+ were shown using a volcano plot. Green dot. Blue dots indicate the gene selected to estimate the key driver gene. Red dots indicate key driver genes. Statistically significant values (p < 0.05) are depicted as green circle, not significant values as black spots.

Figure 5

Correlation between the proportion of synovial CD39+CD55− and CD39−CD55+ cells and radiographic joint space width and pain level in knee osteoarthritis patients. Scatter plots showing correlations. Correlation between the proportion of CD39+CD55− fibroblasts and (A) resting pain evaluated using a visual analog scale (VAS), (B) active pain evaluated using a VAS, and (C) radiographic joint space width (JSW). Correlation between the proportion of CD39−CD55+ fibroblasts and (D) resting pain evaluated using VAS, (E) active pain evaluated using a VAS, and (F) JSW. Statistical analysis was performed using Spearman’s correlation coefficient, as indicated by the ρ values. p < 0.05 indicates statistical significance.