High-Throughput Proteomics and Immunohistochemistry of Orbital Connective Tissue in Graves' Orbitopathy.

Background: High-throughput proteomics (HTP) helps identify characteristic signaling networks specific to certain diseases, thus offering a range of potential applications in basic research and precision medicine. Immunohistochemistry can uncover local orbital immunity.

Methods: Thirty-one orbital connective tissue samples from patients with Graves' Orbitopathy, GO (11 with clinically severe and 12 with mild GO), and from eight control subjects were examined using HPT. We focused on the intersection of differentially expressed proteins (DEPs) that differ between severe GO and both mild GO and control. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes were used to analyze DEPs. Immunohistochemical verification was performed using 11 severe and 7 mild GO orbital tissues. Immunostaining was quantitatively analyzed using reliable and automated software that ensures objective, operator-independent evaluation.

Results: A total of 4,579 proteins were identified: 847, 790, and 208, DEPs were registered when comparing mild GO vs. control, severe GO vs control, and severe versus mild GO, respectively (all p<0.05). Using a cut-off threshold of fold change ≥ 2.0 during screening, five significantly up-regulated DEPs (Latent-transforming growth factor β-binding protein 2, Lysyl oxidase homolog, Ras-interacting protein 1, Integrin beta 3, Coagulation factor XII) overlapped between severe GO and both mild GO and control; while 163 up-regulated DEPs were involved in various biological processes (angiogenesis, inflammation, tissue remodeling and fibrosis). Subsequent immunohistochemical tissue validation confirmed the HTP findings.

Conclusions: For the first time, combined proteomics and immunohistochemistry detected upregulated orbital tissue proteins potentially implicated in GO progression.