0075: Impact of CXCL2 and IL-11 from Rheumatoid Arthritis Synovial Fibroblasts on Angiogenesis and Endothelial Cell Network Formation

Background/Purpose: In rheumatoid arthritis (RA), hypervascularization contributes to synovial inflammation. RA synovial fibroblasts (RASF) contribute to cartilage and bone erosion but also promote angiogenesis. Activated RASF secrete inflammatory, angiogenesis and matrix remodeling factors including CXCL2 and IL-11. While CXCL2 is primarily recognized for recruiting leukocytes to inflamed tissues, it also promotes angiogenesis. IL-11 is involved in bone resorption and fibrosis. Angiopoietin-2 (ANGPT2) is known to be dysregulated in pathological vessel formation. Therefore the role of CXCL2 and IL-11 in RASF-mediated altered angiogenesis was evaluated.

Methods: In the SCID mouse model of RA, healthy cartilage was subcutaneously co-implanted with RASF. RASF-mediated helix-like vessel (HLV) formation was evaluated after 3-45 days. Implants, RA and osteoarthritis (OA) synovium were stained for ANGPT2 and CXCL2. RASF were stimulated with 0.1 or 1ng/ml IL-1β once or repetitively and ELISA, realtime-PCR and RNAseq performed and supernatants used for 2D tube formation assays (TFA). TFA were performed by seeding HUVEC on Matrigel®. HUVEC were treated with supernatants or CXCL2 (25/50/100ng/ml) or 50ng/ml CXCL2 was combined with the CXCR2 antagonist SB225002 (0.015/0.03/0.06μM) or 10ng/ml IL-11 with/without the IL-11 inhibitor AF-218 (100/250/500ng/ml). To evaluate the role of VEGF, bevacizumab or ECM without VEGF with/without stimulants were used. After 4h, cell network area of TFA was measured using the Keyence BZ-X800© software.

Results: In SCID mice, RASF-specific HLV formation started at d3 and increased until d30. Compared to OA synovium, ANGPT2 was significantly upregulated in RA vessels and implants. RNAseq showed altered signaling pathways of angiogenesis. Repetitive RASF-stimulation significantly reduced IL-1β-induced IL-6, IL-11 and CXCL2 compared to RASF stimulated once (e.g. IL-6: 1^st: 5076±1730 vs 3^rd: 1890±758pg/ml, p< 0.0001; IL-11: 1^st: 451±205 vs 3^rd: 69±51pg/ml, p< 0.0001). Repetitive stimulation of HUVEC+15%RASF resulted in a significant IL-6 increase for subsequent stimulations (1^stvs.3^rd: p=0.02). RASF significantly reduced tube thickness (22.9µm SD=6.3 to 16.6µm SD=2.2) compared to HUVEC alone (p=0.014) but increased ANGPT2 release (4h: 5%, 48h: 14%). IL-1β-stimulated RASF supernatants (1^st vs 3^rd) reduced HUVEC network area to 78%, p=0.01 vs 77%, p=0.04 (100%: no supernatant-transfer control). CXCL2 decreased the network area (e.g. 50ng/ml: 71% of 100% control, p=0.02; 25ng/ml: 73%, p=0.04). SB225002 partially reversed the CXCL2 effect (0.015: 93%; 0.03: 87%; 0.06: 92%). IL-11 also disturbed the network area (63%, p=0.08) and AF-218 partially reversed the effect (e.g. 250ng/ml: 72%, 500: 90%). Bevacizumab prevented tube formation which could in part be restored by addition of ANGPT2. Addition of ANGPT2 to ECM had a similar effect.

Conclusion: RASF affect vascularization in vitro and in vivo. CXCL2, IL-11 and RASF-derived supernatants altered the HUVEC network. These findings suggest that CXCL2 as well as IL-11 produced by RASF may significantly contribute to the dysregulated angiogenesis involving ANGPT2 observed in RA.