Background Fibroblast-like synoviocytes (FLS) are pivotal in inflammation and joint damage of rheumatoid arthritis (RA). These cells proliferate, become resistant to apoptosis, migrate and invade, contributing to perpetuate synovial inflammation and destruction of cartilage and bone. Current treatments of RA are focused against inflammatory factors and immune cells, however, a significant percentage of patients do not successfully respond. Combined treatments with drugs that control inflammation and with others that reverse the pathogenic phenotype of FLS could improve the prognosis of these patients. An unexplored area includes vitamin A and its metabolites. These compounds modulate differentiation, development, apoptosis and proliferation. Indeed, retinoids are being successfully used in the treatment of several cancers for their anti-proliferative and pro-differentiative actions. In addition, several studies have shown a notable reduction of cell migration and invasion in different cell types after treatment with all-trans retinoic acid (ATRA). However, it is not known if ATRA could modify the migratory and invasive ability of rheumatoid synoviocytes.
Objectives To analyse the effect of treatment with the retinoid all-trans retinoic acid in proliferation, migration and invasion of rheumatoid synoviocytes.
Methods FLS were obtained from 8 RA patients. Cellular proliferation was determined using the CellTiter-Glo luminescent viability assay (Promega). Migration was analysed by wound healing assay, using Ibidi inserts. Percentage of migrating cells was determined by Image J. Invasion was tested by the Boyden chamber method using inserts (Millicell) coated with Matrigel (BD Biosciences). Invasion was determined by quantification of Giemsa stained cells on the bottom side of the inserts under the microscope. Proteomic analysis was performed using LC-MALDI-TOF/TOF and 1-DE and 2-DE gels. MS and MS/MS data was searched against the UniProt/Swiss-Prot database of protein sequences.
Results We analysed the effect of ATRA in the spontaneous proliferation of FLS from 8 RA patients. ATRA treatment significantly reduced proliferation at 48, 72 and 96 h (p=0,005; p=0,002; p=0,04; respectively). Next, we analysed the effect of ATRA on RA FLS migration and invasion. Migration of RA FLS treated with ATRA for 96 h was reduced by 46% when compared with cells treated with vehicle control (p=0,002). In addition, RA FLS invasion was also impaired after ATRA treatment, as the invaded area was 31% lower than in controls (p=0,018). To elucidate the mechanisms underlying the effects of ATRA on RA FLS a proteomic analysis was performed. We compared the proteome of FLS from 5 RA patients treated with ATRA or with vehicle using LC-MALDI analysis. The differentially identified proteins in ATRA-treated FLS vs control FLS were Septin, Rho GTPase activating protein (ARHGAP1) and actin related protein 2 (ARP2). Validation experiments using 1-DE and 2-DE gels were performed.
Conclusions Overall these results reveal that retinoid treatment reduces the proliferative, migratory and invasive capacity of RA FLS, indicating that this treatment could decrease joint damage and loss of function in RA patients.
Acknowledgements Supported by grant PI14/01153 of the ISCIII (Spain), partially financed by FEDER.
Disclosure of Interest None declared
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