Tear-derived exosomal biomarkers of Graves' ophthalmopathy

Abstract

Graves' ophthalmopathy (GO), the most frequent extrathyroidal manifestation of Graves' disease (GD), can lead to a significant decline in the quality of life in patients. Exosomes, which contain proteins, lipids and DNA, play important roles in the pathological processes of various diseases. However, their roles in Graves' ophthalmopathy are still unclear. We aimed to isolate exosomes and analyze the different exosomal proteins. Tear fluids were collected from twenty-four GO patients, twenty-four GD patients and sixteen control subjects. The numbers of tear exosomes were assayed using nanoparticle tracking analysis. A Luminex 200 kit and ELISA kit were used to confirm the different cytokine concentrations in serum. Extraocular muscle from GO patients and controls was extracted, and western blotting was used to assay the levels of Caspase-3 and complement C4A. Our study demonstrated that the number of tear exosomes differ from GD patients and control. The expression levels of cytokines, including IL-1 and IL-18, were significantly increased in the tear exosomes and serum from GO patients compared with GD patients and controls. The levels of the exosomal proteins Caspase-3, complement C4A and APOA-IV were significantly increased in GO patients compared to GD patients and controls. Orbital fibroblasts from GO patients showed significantly higher levels of Caspase-3 and complement C4A than those from controls. The levels of serum APOA-IV in GO patients were significantly higher than those in GD patients and controls. Specific proteins showed elevated expression in tear exosomes from GO patients, indicating that they may play important roles in GO pathogenesis.

Keywords: APOA-IV; Caspase-3; Graves’ ophthalmopathy; biomarker; exosome; tear.

Figure 1

Workflow and preparation before LC−MS/MS analysis. (A) Experimental workflow of this study. (B) Representative nanoparticle tracking analysis of tear exosomes. (C) Representative transmission electron microscopy images of tear exosomes. The scale bar on the left represents 50 nm, and the scale bar on the right represents 200 nm. (D) Expression of CD9 and CD63 in exosomes of active GO patients, GD patients and healthy controls. (E) Exosomal protein presented in SDS-PAGE with Coomassie Brilliant Blue.

Figure 2

Pairwise comparison of the differentially expressed proteins in exosomes among the three groups. (A, E, I) Venn diagram comparing the number of identified proteins between GO and GD, GD and controls, GO and controls. (B, F, J) Volcano plots showing the differentially expressed proteins in exosomes between the GO group and the GD group, between the GD group and the healthy control group, and between the GO group and the healthy control group. A fold change of 1.5 and a p < 0.05 were used as the cutoff points. Upregulated proteins are shown in red, and downregulated proteins are shown in green. (C, G, K) Clustering heatmaps of differentially expressed proteins in exosomes from the three groups as described above. (D, H, L) List of the top 5 upregulated and downregulated proteins among the differentially expressed proteins from the three groups described above.

Figure 3

Gene Ontology and KEGG analyses of differentially expressed proteins from the GO group and the GD group. (A-C) Gene Ontology annotation (A), Gene Ontology enrichment (B), and KEGG enrichment (C) analyses of differentially expressed proteins in exosomes from the GO group and the GD group.

Figure 4

Gene Ontology and KEGG analyses of differentially expressed proteins from the GD group and the healthy control group. (A-C) GO annotation (A), GO enrichment (B), and KEGG enrichment (C) analysis of differentially expressed proteins in exosomes from the GD group and the healthy control group.

Figure 5

Gene Ontology and KEGG analyses of differentially expressed proteins from the GO group and the healthy control group. (A-C) Gene Ontology annotation (A), Gene Ontology enrichment (B), and KEGG enrichment (C) analyses of differentially expressed proteins in exosomes from the GD group and healthy controls.

Figure 6

Validation of differentially expressed proteins in exosomes from the GO patients, GD patients and controls. (A) Proteins in exosomes from the GO group and the GD group that were significantly varied in the proteomics analysis are shown in columns. (B) Inflammatory cytokines such as IL-1 and IL-18 among three groups. (C) Representative images and protein expression levels of Caspase-3, Complement C4A and GAPDH in western blots in GO patients and controls. (D) Apolipoprotein A-IV concentrations were measured by ELISA in the GO group, the GD group and the healthy control group. * P<0.05, ** P<0.01, *** P<0.001.