Characterization of exosomes derived from synovial fluid
Exosomes derived from synovial fluid (SF-exo) were first extracted and characterized by transmission electron microscopy (TEM) and nanoparticle-tracking analysis. As shown in Fig. 1A-B, the morphology of SF-exo was typical cup-shaped and the average diameter of the vesicles is around 150 nm (Fig. 1B), which is consistent with international standard of exosomes[13]. It proves that the extracted small particles are exosomes.
Proteomic profiling of exosomes derived from synovial fluid
The dynamic profiles of SF-exo proteins between OA patients and healthy controls were detected using iTRAQ quantitative proteomics, and a total of 432 proteins were identified, with a labelling efficiency of 95.22% (Supplementary table 1). Volcano plots further illustrated the differentially expressed proteins (DEPs) in SF-exo, with 20 proteins up-regulated and 5 proteins down-regulated in OA group (Fig. 2A-B). DEPs were annotated using GO and KEGG databases (Fig. 2C-F). In biological process terms, DEPs were significantly enriched in immunity-related process terms, such as complement activation, humoral immune response mediated by circulating immunoglobulin, and immunoglobulin mediated immune response. In cellular component terms, DEPs were enriched in many extracellular terms, extracellular space, immunoglobulin complex, and extracellular exosome. In molecular function terms, DEPs were enriched in antigen binding, immunoglobulin receptor binding, and C5a anaphylatoxin chemotactic receptor binding. In addition, KEGG pathway analysis demonstrated that DEPs were enriched in many immune-related diseases pathways, such as Rheumatoid arthritis, Asthma, Systemic lupus erythematosus. Collectively, the above results indicated that DEPs in SF-exo were highly correlated with inflammatory regulation, and some of them may be therapeutic target or biomarker of OA.
Comparative proteomic analyses of synovial fluid and exosomes derived from synovial fluid
One synovial fluid (SF) dataset from OA patients and healthy control (PXD023708) was downloaded from a publicly available database. A total of 377 DEPs were identified in this dataset, of which135 proteins were up-regulated and 242 were down-regulated in SF from OA patients. Venn analysis of DEPs revealed overlapping expression patterns between the SF and SF-exo groups, with five DEPs overlapping includingC3, C4B, APOM, MMP3, and DPYSL2 (Fig. 3A). PPI network analysis was performed on these 5 overlapping DEPs with STRING database. We found that C3, C4B and APOM formed a tight interaction network (Fig. 3B). The functions of the overlapping proteins were determined using KEGG and GO analysis (Fig. 3C-F). Of the overlapping proteins, DPYSL2 was involved in axon guidance, which was a key stage of formation of neuronal network. MMP3 were involved in some inflammatory reaction, such as IL-17 signaling pathway, TNF signaling pathway and Rheumatoid arthritis pathway. C3 and C4B were important molecules in complement and coagulation cascades. APOM, an apolipoprotein, was enriched in terms of antioxidant activity and lipid transporter activity. In addition, the broad range of non-overlapping features between the synovial fluid and exosomes groups might imply certain selective effects from synovial fluid and exosomes on proteins.
Validation of biomarkers for OA
Based on the above data, we selected MMP3, DPYSL2, C3, C4B and APOM to identify their potential as OA biomarker. ROC analyses based on these 5 proteins were performed. These 5 proteins of synovial fluid in ROC analysis exhibited high AUC values (AUC, 72.650-84.188), which could be used to differentiate OA and healthy controls (Fig. 4A). Interestingly, these 5 proteins in SF-exo were able to significantly distinguished OA from healthy people (Fig. 4B). Then, the levels of biomarkers in the synovial fluid of OA patients and healthy controls were determined using ELISA. We found that the levels of C3, C4B, APOM, and MMP3 were significantly higher in the OA group than in the normal group, while the levels of DPYSL2 were significantly lower, which is consistent with the expression of PXD023708 data (Fig. 4C-D). In conclusion, we found SF-exo maybe a novel selection for early OA detection.