Background/Purpose: Activin A and follistatin belong to an anti-inflammatory auto-regulatory cycle. Patients with rheumatoid arthritis (RA) have increased activin A levels in the synovial fluid and tissue. During inflammation, activin A is released systemically, then inducing its antagonist follistatin. This negative feedback is active in different cell types but not RA synovial fibroblasts (SF). Fibroblasts are known to interact with endothelial cells in inflamed tissues. Objective: Evaluation of the role of activin A and follistatin on RASF and endothelial cell interactions.

Methods: RA synovium was used for RASF isolation, HUVEC were commercially obtained. RASF and HUVEC were stimulated in mono- and coculture. Direct: RASF together with HUVEC; indirect: inserts with HUVEC separated by a membrane from RASF in the lower chamber. Stimuli: 15ng/ml activin A, 500ng/ml follistatin, 1ng/ml IL-1. Proteins were measured by ELISA. Proliferation was analyzed by BrdU assay. RASF were Calcein-AM stained for short-term cell adhesion assays in direct coculture. Cells were transferred to 24-well plates after 18h stimulation. After adhesion for 1h, non-adherent cells were removed by shaking 3x 5 min. Afterwards, cells were quantified. For the flow-adhesion assay, HUVEC were cultured on rat tail collagen coated capillary slides. HUVEC and RASF were stimulated for 4h with TNF, TNF+activin A or TNF+follistatin. After stimulation, 1×10^6 RASF were resuspended in 20ml medium. Two 1min videos were recorded at 18.4ml/h and 30.5ml/h using TNF-stimulated HUVEC or TNF-stimulated RASF on activin A or follistatin stimulated HUVEC.

Results: IL-1 induced activin A in RASF and HUVEC monoculture (8-fold, p< 0.01, 4-fold, p< 0.05) and direct and indirect coculture (5-fold, p< 0.05; 4-fold, p< 0.05). IL-1-induced activin A release was reduced by follistatin in HUVEC monoculture (12-fold, p< 0.01, n=5), direct and indirect coculture (10-fold, p< 0.01; 5-fold, p< 0.01) compared to IL-1 alone but not in RASF monoculture. IL-1-induced IL-6 release was reduced by activin A in HUVEC (42.6%, p< 0.05) and indirect coculture (31.8%, p< 0.05) but not in RASF monoculture and direct coculture. Follistatin did not alter IL-6 responses. IL-1 induced VEGF in RASF but not in HUVEC and was not altered by activin A. Proliferation of RASF, HUVEC or both was not altered by activin A or follistatin. Short-term adhesion showed no significant influence of activin A or follistatin (n=3). Flow adhesion assay showed reduced adherence / rolling of RASF on HUVEC stimulated with TNFα and activin A.

Conclusion: In direct and indirect coculture of HUVEC with RASF the effect on HUVEC is dominant leading to reduced IL-1-induced activin A release. However, the IL-1-induced IL-6 release in RASF or HUVECs was decreased by activin A in HUVEC monoculture and indirect coculture but not during cell-contact of both cell types. The direct interaction of RASF with HUVEC seems to prevent the reducing activin A effect on IL-6 release in HUVECs. Activin A seems to not to have an impact on short-term cell adhesion but first observations show that activin A has an influence on selectin-mediated adhesion under flow conditions.

« Back to ACR Convergence 2020

ACR Meeting Abstracts - https://acrabstracts.org/abstract/activin-a-and-follistatin-alter-endothelial-cell-and-rheumatoid-arthritis-synovial-fibroblast-adhesion-and-interaction/