THU0064 Antibodies against carbamylated proteins from patients with rheumatoid arthritisactivate endothelial cells

A. Pecani, T. Colasanti, F.R. Spinelli, C. Barbati, F. Conti, G. Valesini, C. Alessandri
2018 THURSDAY, 14 JUNE 2018   unpublished
inflammation through regulation of cytokine, chemokine and metalloprotease expression. Thus, the study of the non-canonical pathway of Wnt5a in the aggressive phenotype of FLS and the inflammatory response may provide new therapeutic targets for the RA. Objectives: To analyse the involvement of Wnt5a in the proliferation, migration, invasion and inflammatory responses of FLS from RA patients. Methods: FLS were obtained from eight RA patients. Expression of Wnt5a was suppressed by siRNA
more » ... ion (Dharmacon). Cellular proliferation was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega). Migration was analysed by the wound-healing assay using Ibidi inserts. The occupation area was determined by Image J. Invasion was tested by quantifying Giemsa stained cells on the bottom side of Matrigel coated trans-membrane (Millipore). The expression of inflammatory mediators and metalloproteases was analysed by real-time PCR after stimulation with recombinant Wnt5a protein (rWnt5a), TNF and combined treatment. Results: We analysed the effect of the absence of Wnt5a and rWnt5a on the proliferation, migration and invasion of RA FLS. FLS lacking Wnt5a showed a significant migration decrease, 27.9% in basal condition and 16.9% after TNF stimulation, when compared with FLS transfected with siRNA control. Accordingly, migration of RA FLS treated with rWnt5a was increased by 48.5% when compared with cells treated with the vehicle. No change in migration was observed in TNF-stimulated FLS. Invasive capacity was reduced, reaching 71.4% of that observed in FLS transfected with siRNA control in basal conditions. In addition, invasion in FLS treated with rWnt5a was 35.7% higher than in controls. We also analysed the effect of rWnt5a on the inflammatory response of RA FLS. We found a significantly increase of IL6, IL8, CCL2, CXCL5, MMP9 and MMP13 basal expression and the TNF-induced expression of IL6, IL8, CXCL5 and MMP3. Nevertheless, the lack of Wnt5a or the addition of rWnt5a did not modify the spontaneous or the TNF-induced proliferation. Conclusions: These results indicate that Wnt5a contributes to the aggressive phenotype of FLS by potentiating their migration and invasion, and by stimulating the inflammatory response. Acknowledgements: Supported by grant of the ISCIII/PI14/01660/RETICS Program, RD16/0012/0014/cofinanced FEDER. Abstract THU0064 -Figure 1. Expression of addhesion molecules (A, B), activation of IRAK (C), NFkB (D) signalling and apoptosis (E) in EAhy 926.
doi:10.1136/annrheumdis-2018-eular.6217 fatcat:n7ghl2vsjbeavhkyx4q6g27jmu