AB1323\u2005OSTEOPHYTOSIS PROCESS SUGGEST INVOLVEMENT OF MAST CELLS, REVEALED BY WHOLE TRANSCRIPTOME ANALYSIS OF OSTEOPHYTES FROM KNEE OSTEOARTHRITIS PATIENTS

Abstract

Background: Cartilage-loss is a central pathology of osteoarthritis while synovitis, sub-chondral bone sclerosis and osteophytes (OFs) formation are counted as integral phenomenon. Osteophytosis is a process of OFs formation, in which mesenchymal stem cells from periosteum and synovium, along with a number of cytokines, growth factors and biochemical factors gives out bony out-growth at margin of the affected joint. Although frequently associated, OFs are one of the less focused areas in OA research, particularly from the molecular mechanism point of view, involved in their formation. Also, it is a good ambiguity if OFs is a pathological condition linked with altered biochemical milieu or a functional adaptation for the joint instability. Objectives: Accounting its importance in clinical OA and the obscure biochemical background, this study strives for a deeper comprehension of molecular mechanism and active pathways, involved in OF formation Methods: We performed whole transcriptome analysis of OFs, collected from six knee OA patients during knee replacement surgery. Non-OF tissue from tibial plateau of the same patient was used as a control. After RNA isolation, RNA-seq was performed using SOLiD 5500W platform and fragment sequencing chemistry. For differential gene expression analysis, non- normalized raw counts were used for the EdgeR package. To find differential transcriptome between OF and control, we used group-wise comparisons where negative binomial fitting was followed by exact test. Finally, RT-PCR of significantly expressed genes was run for trascriptome validation. Results: After cluster analysis, total 597 genes were significantly expressed between OFs and controls out of which, 323 genes were seen up-regulated (LogFC≥ 2) while 274 were down-regulated (LogFC≤ -2). Further, 88 genes showed marked increase (P<0.01); here CPA3, Selectin E, MS4A2, PLA2G2A and CSN1S1 were prominent. Similarly, 23 genes were with a noteworthy down-regulation (P<0.01) and APOB, CADM2, TNEFF2, GNAZ and GABRA2 remained as first five. GO analysis of the best extracted genes indicated significant involvement of ECM proteoglycan, GABA A receptor pathway (Reactome 2016 - homo sapiens_R-HSA-3000178 and R-HSA-977441, respectively), osteoblast and TNF-α signalling pathways (WikiPathways 2016 -homo sapiens WP322 and homo sapiens WP231 respectively). Finally, tracsriptome results were validated using RT-PCR of significantly expressed genes (CPA3, MMP-1, 3 and 13). Conclusion: Significant expression of CXCL9, 10, 11 and PGR4 indicate chemotaxis and immune cells deployment; outstanding CPA3 and MS4A2 indicate active involvement of mast cells. Accounting a previous knowledge that ‘mast cell products’ are linked with increased osteoblastic and osteoclastic activity and impose pathological changes in articular and peri-articular tissue, marked expressions of MC products or mediators here, enable us to propose an association between MCs and OF. Presence of E-selectin on endothelial cell surface is known to promote adhesion of neutrophils and further release of TNFα and IL1. Expression of PLA2G2A suggests increased prostaglandin synthesis and indicates activation of inflammatory cascade. In conclusion, this transcriptome analysis suggests a significant association of mast cells with osteophytosis. The genes involving chemotaxis, degranulation, cytokinin synthesis mark the initiation of OF. On the other hand, proteases metrixremodelling enzymes may indicate their growth. A host of different signalling molecules denotes involvement of canonical and non-cannonical pathways that warrants further investigations. Disclosure of Interests: None declared