gms | German Medical Science

Introduction: The pathogenesis of rheumatoid arthritis (RA) is characterized by increased angiogenesis, which is predominantly located in the inflamed synovium. Key players in progression of RA are the synovial fibroblasts (RASF), which degrade and invade into cartilage and synovial connective tissue. The consecutive matrix degradation leads to the release of matrix fragments such as the angiogenic inhibitor collagen IV alpha 2 (COL4A2), which in turn may lead to RASF activation. Objective: Analysis and characterization of RASF-mediated altered vessel formation in the tube formation assay regarding gene and protein expression, and RASF-endothelial cell (EC) interactions.

Methods: RASF were isolated from RA synovium. 2D tube formation assay was performed with human umbilical vein endothelial cells (HUVEC) seeded (1x104 cells/well) on Matrigel^® coating for 4h. 15% RASF (1.500 cells) were added. RASF and HUVEC were treated with 0.2 or 0.5 µg/ml COL4A2. HUVEC were pre-treated with 0.2 µg/ml COL4A2 for 20 h. Quantification was performed using 5 parameters: lumen circumference, tube thickness, numbers of lumen, tubes, branch points. RNA extraction from tubes was done after dispase digestion for 20min at 37°C. Healthy human cartilage was subcutaneously co-implanted with RASF into SCID mice. Contralaterally, healthy cartilage without RASF was implanted and vessel formation evaluated after 3–45 days.

Results: Altered angiogenesis showing helix-like vessels started at day 3 in RASF-containing SCID mouse implants compared to controls without RASF or normal SF. RASF co-culture with HUVEC influenced tube formation by arranging into tubes and significantly reducing tube thickness from 22.9 µm (SD=6.3) to 16.6 µm (SD=2.2) (p=0.014) compared to HUVEC alone in vitro. Additional treatment of pre-treated HUVEC with 0.5 µg/ml COL4A2 also resulted in significantly reduced tube thickness from 22.9 µm to 16.9 µm (SD=4.4) (p=0.011). Combination of RASF and treatment with 0.5 µg/ml COL4A2 of pre-treated HUVEC significantly increased the RASF-mediated effect (14. 6µm, SD=1.4, p<0.001).

Conclusion: Angiogenesis is specifically altered by RASF in vivo in SCID mice, since helix-like vessels were only detectable using RASF but not after osteoarthritis or healthy SF implantation. RASF-mediated alterations of tubes were also observed in the tube-formation assay in vitro. RASFand COL4A2 both play a critical role in altering angiogenesis in vitro by specifically altering tube thickness.