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AB0101 SECRETOME ANALYSIS OF CHONDROCYTES AND SYNOVIAL FIBROBLASTS IN OSTEOARTHRITIS: MODULATION BY VIP

Background Osteoarhthritis (OA) is a chronic, degenerative and multifactorial disease, and the main cause of pain and dysfunction among elder people. It is characterized by a progressive loss of function of synovial joints (1). The role of chondrocytes in this pathology has been widely studied (2), but other joint cells are also involved, including the synovial fibroblast (SF) (3-5). During joint destruction, inflammatory and degradative mediators are released by joint cells and from the extracellular matrix (ECM), including fibronectin degradation fragments (Fn-fs) (3, 4, 6). On the other hand, vasoactive intestinal peptide (VIP) exerts anti-inflammatory and immunomodulatory actions in several autoimmune and inflammatory disorders, including OA (3, 5). The study of the mediators released by joint cells and their modulation by pro- and anti-inflammatory mediators would be useful for the design of novel therapies for OA treatment. Objectives To analyse the mediators released from co-cultures of OA chondrocytes and SF, and to elucidate the effect of Fn-fs and VIP on these cells. Methods Human articular chondrocytes (HAC) and SF from 4 OA patients were provided by the Rheumatology Service at Complejo Hospitalario Universitario A Coruña. Isolated cells were recovered and plated in SILAC DMEM-Flex lacking Arginine and Lysine. In the case of medium and heavy media, isotope-labeled L-lysine and L-arginine were used. When 100% of labeling was reached, cells were put in co-culture and incubated in serum-free medium with or without Fn-fs (10-8M) or Fn-fs + VIP (10-8M) for 48h. Cell secretomes were separated on a 10% SDS PAGE gel. Gels were stained with Coomassie blue and the resulting lanes were cut into slices and subjected to in-gel digestion. Extracted peptide mixtures were desalted and concentrated via NuTip, subjected to liquid chromatography, using a Tempo nano LC equipped with a Sun Collect MALDI Spotter, and analyzed by MALDI-TOF/TOF. Identification of peptides and proteins and relative quantification were performed using Protein Pilot software (Sciex). Results Cell secretomes were analysed in 4 OA patients in duplicate. Database search (UniprotKB/Swissprot) and Venn’s diagrams drawing tool (Venny 2.1.0) allowed us to identify 79 common proteins in the HACs-SF co-cultures. Among them, VIP was able to modulate 33 different proteins, significantly reducing 9 of them: CH3L1, PTX3, PGS2, MMP2, Complement C1R, Complement C3, TBA1, QSOX1, and CATB. These proteins include inflammatory and ECM proteins, proteases and complement system proteins among others, which play a main role in the OA pathogenesis. Conclusion VIP decreases inflammatory and degradative mediators in HAC-SF co-cultures, potentially slowing the progression of the disease and supporting its therapeutic role in osteoarthritis. References [1] Loeser RF, et al. Arthritis Rheum. 2012;64(6):1697-707. [2] Calamia V, et al. J Proteome Res. 2011;10(8):3701-11. [3] Pérez-García S, et al. Journal of molecular neuroscience : MN. 2014;52(1):18-27. [4] Pérez-García S, et al. Am J Pathol. 2016;186(9):2449-61. [5] Pérez-García S, et al. J Cell Mol Med. 2016;20(4):678-87. [6] Zack MD, et al. Arthritis Rheum. 2006;54(9):2912-22. Acknowledgement This work has been supported by Instituto de Salud Carlos III, Spain, cofinanced by FEDER, European Union: RETICS program, RIER (RD16/0012/0002 and RD16/0012/0008), and the projects PI17/00027 and PI16/2124. Disclosure of Interests Selene Pérez García: None declared, Valentina Calamia: None declared, Tamara Hermida Gómez: None declared, Mar Carrión: None declared, Raúl Villanueva Romero: None declared, Yasmina Juarranz: None declared, Francisco J. Blanco Consultant for: AbbVie, Bioiberica, BMS, GSK, Grünenthal, Janssen, Lilly, Pfizer, Regeneron, Roche, Sanofi, TRB Chemedica, and UCB, Rosa P. Gomariz: None declared