Background/Purpose: In the inflamed synovium of RA patients, increased and altered angiogenesis is a pathological feature. Key players are chronically activated RA synovial fibroblasts (RASF), which promote synovial angiogenesis and matrix degradation. Canstatin is a matrix-derived anti-angiogenic collagen IV fragment that blocks the angiopoietin (ANGPT)/Tie2 pathway in endothelial cells (EC).

Objective: To analyse the effects of repetitively stimulated RASF and canstatin on vessel formation in the tube formation assay, the SCID-mouse model of RA and synovial tissue of RA patients with respect to ANGPT2 expression and RASF-EC interactions.

Methods: RASF were repetitively stimulated thrice every 24h with 0.05ng/ml IL-1ß. 2D tube formation assay was performed using HUVEC (+/- prestimulation with 0.2µg/ml canstatin for 20h) and 15%RASF seeded on Matrigel®. RASF/HUVEC were treated with 0.5µg/ml canstatin. Tube thickness and the area covered by the formed cellular network were measured. Supernatants were measured by ELISA. Cartilage was subcutaneously co-implanted with RASF alone or with 0.5µg/ml canstatin into SCID mice. Contralaterally, cartilage without RASF but with canstatin was implanted. Vessel formation and RASF-invasion were evaluated after 3-45 days.

Results: RASF or HUVEC stimulated once showed a significant IL-6 increase compared to unstimulated controls. Subsequent repetitive stimulation resulted in a significant IL-6 decrease compared to 1st stimulation in RASF (1st vs. 3rd: p< 0.0001) or HUVEC (1st vs. 3rd: p=0.041). The same effect was observed for IL-11 and CXCL2 in RASF (1st: vs. 3rd: p< 0.0001). In contrast, repetitive stimulation of HUVEC+15%RASF resulted in a significant IL-6 increase for each subsequent stimulation (1st vs. 3rd: p=0.02). Regarding tube formation, RASF significantly reduced tube thickness (p=0.01) and cell network area (p < 0.0001). RASF stimulated only once further reduced the network area (p=0.04), while repetitive stimulation significantly attenuated the proinflammatory effect (p=0.03). Stimulation of pre-treated HUVEC and unstimulated RASF with canstatin led to disturbed tube formation with reduced tube thickness (p=0.01). Co-culture of RASF with pre-treated HUVEC with canstatin further increased the RASF-mediated effect by reducing tube thickness (p < 0.001). In SCID mice, RASF-mediated helix-like vessel formation started at day 3. Number of helix-like vessels was significantly increased ipsilaterally compared to contralateral implants on day 3 and 30 (p=0.04). In contrast, number of helix-like vessels was significantly reduced ipsilaterally in implants containing canstatin on day 3 and 30 (p=0.03 both). In human RA synovium, the pathological vessel regulator ANGPT2 was significantly upregulated in vessels compared to OA tissue.

Conclusion: RASF-mediated effects on EC were detectable in the tube formation assay, since RASF and canstatin both specifically reduced tube thickness. RASF specifically altered neovascularization in SCID mice by promoting the formation of helix-like vessels. Human synovial tissue of RA patients showed significantly upregulated ANGPT2 expression compared to OA patients showing the effects on vessel formation in RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/chronic-inflammation-and-collagen-iv-fragment-canstatin-influence-rheumatoid-arthritis-synovial-fibroblast-and-endothelial-cell-interactions-in-vitro-and-in-vivo/