Background/Purpose: In the pathogenesis of rheumatoid arthritis (RA), neovascularization is increased in the activated and inflamed synovium. RA synovial fibroblasts (RASF) are key players in RA progression by degrading and invading into cartilage and altering synovial connective tissue. The consecutive matrix degradation results in a release of matrix proteins such as the angiogenic inhibitor canstatin (collagen IV α2), which, in turn, may lead to RASF activation and altered angiogenesis. The objective is the analysis of RASF- and canstatin-mediated effects on vessel formation in the SCID-mouse model for RA and in the tube formation assay with respect to protein expression and RASF-endothelial cell (EC) interactions.

Methods: RASF were isolated from RA synovium after joint replacement surgery. Human cartilage was subcutaneously co-implanted with RASF into SCID mice. Contralaterally, cartilage without RASF was implanted and vessel formation evaluated after 3-45 days (number of total vessels vs. helix-like vessels, vessel size). 2D tube formation assay was performed using HUVEC seeded (7,800 cells/well) on Matrigel® coating for 4h. 15% Calcein-AM stained RASF were added. RASF and HUVEC were treated with 0.2 or 0.5µg/ml canstatin. HUVEC were pre-treated with 0.2µg/ml canstatin for 20h. Quantification of tube formation was performed using 5 parameters: lumen circumference, tube thickness, numbers of lumen, tubes and branch points. Supernatants were collected to evaluate the vascular markers synthesis, e.g. angiopoietin-2 (ANGPT2).

Results: In the SCID mouse implants, vessel formation started at day 3. Helix-like vessels were detectable already at early time points of vessel formation in implants with RASF (d3-9) but not with healthy SF. On day 3, 60% of vessels were helix-like in implants after RASF implantation. RASF-mediated altered neovascularization was also observed in vitro. In the tube formation assay, RASF significantly reduced tube thickness from 22.9µm (SD=6.3) to 16.6µm (SD=2.2) (p=0.014) compared to HUVEC alone. Interestingly, fluorescence labelled RASF were located within the tubes. Additional stimulation of pre-treated HUVEC with 0.5 µg/ml canstatin resulted in disturbed tube formation with reduced tube thickness from 22.9 to 16.9µm (SD=4.4) (p=0.011) and in a 1.5-fold increase of ANGPT2. Co-culture of RASF with pre-treated HUVEC and addition of 0.5µg/ml canstatin further increased the RASF-mediated effect by reducing tube thickness from 22.9 to 14.6µm (SD=1.4) (p< 0.001), with an 1.6-fold increase of ANGPT2.

Conclusion: RASF specifically alter neovascularization in SCID mice by promoting the formation of helix-like vessels. RASF-mediated effects on EC were also detectable in the short-term tube formation assay, since RASF and canstatin both play a critical role in altering neovascularization by specifically reducing the tube thickness and increasing ANGPT2, a factor expressed at sites of vascular remodelling.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neovascularization-is-altered-by-rheumatoid-arthritis-synovial-fibroblasts-and-canstatin-in-vitro-and-in-vivo/