A proteomic profile of synoviocyte lesions microdissected from formalin-fixed paraffin-embedded synovial tissues of rheumatoid arthritis

Junji Hayashi, Makoto Kihara, Harubumi Kato, Toshihide Nishimura

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation of the synovial joints. Early intervention followed by early diagnosis can result in disease remission; however, both early stage diagnosis and provision of effective treatment have been impeded by the heterogeneity of RA, which details of pathological mechanism are unclear. Regardless of numerous investigations of RA by means of genomic and proteomic approaches, proteins interplaying in RA synovial tissues that contain various types of synoviocytes, are not yet sufficiently understood. Hence we have conducted an HPLC/mass spectrometry-based exploratory proteomic analysis focusing on synoviocyte lesions laser-microdissected (LMD) from formalin-fixed paraffin-embedded (FFPE) synovial tissues (RA, n = 15; OA, n = 5), where those of Osteoarthritis (OA) were used as the control. A total of 508 proteins were identified from the RA and OA groups. With the semi-quantitative comparisons, the spectral index (SpI), log2 protein ratio (R SC ) based on spectral counting, and statistical G-test, 98 proteins were found to be significant (pair-wise p < 0.05) to the RA synovial tissues. These include stromelysin-1 (MMP3), proteins S100-A8 and S100-A9, plastin-2, galectin-3, calreticulin, cathepsin Z, HLA-A, HLA-DRB1, ferritin, neutrophil defensin 1, CD14, MMP9 etc. Our results confirmed the involvement of known RA biomarkers such as stromelysin-1 (MMP3) and proteins S100-A8 and S100-A9, and also that of leukocyte antigens such as HLA-DRB1. Network analyses of protein-protein interaction for those proteins significant to RA revealed a dominant participation of ribosome pathway (p = 5.91 × 10(-45)), and, interestingly, the associations of the p53 signaling (p = 2.34 × 10(-5)). An involvement of proteins including CD14, S100-A8/S100-A9 seems to suggest an activation of the NF-kB/MAPK signaling pathway. Our strategy of laser-microdissected FFPE-tissue proteomic analysis in Rheumatoid Arthritis thus demonstrated its technical feasibility in profiling proteins expressed in synovial tissues, which may play important roles in the RA pathogenesis.