AB0737 SYNOVIAL FIBROBLASTS FROM PATIENTS WITH RHEUMATOID ARTHRITIS AFFECT ENDOTHELIAL CELLS VIA CXCL2
Authors: F. Lötfering, C. Heck, D. Kürsammer, K. Frommer, M. Arnold-Gräf, S. Rehart, U. Müller-Ladner, E. Neumann
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> In the pathogenesis of rheumatoid arthritis (RA), neovascularization is an important factor by contributing to increase the inflammation. In previous studies, RA synovial fibroblasts (RASF) have shown to exert a proangiogenic effect on angiogenesis. The chemokine CXCL2 is secreted e.g. by macrophages, mast cells, neutrophil granulocytes and also RASF. CXCL2 attracts, among others, lymphocytes to migrate to the site of inflammation. Apart from this proinflammatory effect, other studies have shown an enhancing effect of CXCL2 on angiogenesis, e.g. in neoplastic diseases. Thus, CXCL2 may play a role in the RASF-mediated effect on angiogenesis. <h3>Objectives:</h3> To analyze the CXCL2-mediated effect of RASF on vessel formation in a 2D tube formation assay. <h3>Methods:</h3> 2D tube formation assays using Human Umbilical Vein Endothelial Cells (HUVEC) seeded on Matrigel® were performed to investigate CXCL2-mediated effects on angiogenesis. The tube formation assays were evaluated by measuring the area covered by tubes after four hours of incubation. In a first setup, the effect of RASF on HUVEC-mediated tube formation was investigated by adding RASF to the tube formation assays. In a second setup, HUVEC were treated with 25/50/100 ng/ml CXCL2. Subsequently, in the third setup, 0.015/0.03/0.06/0.125 μM of CXCR2 antagonist SB225002 were added to HUVEC treated with 50 ng/ml CXCL2, to confirm that the effect was mediated by CXCL2. The SB225002 stock was dissolved in dimethyl sulfoxide (DMSO). <h3>Results:</h3> RASF significantly reduced tube thickness from 22.9 µm (<i>SD = 6.3</i>) to 16.6 µm (<i>SD = 2.2</i>) (p = 0.014) in the tube formation assay compared to HUVEC alone. The fluorescence-labelled RASF were located within the tubes. All CXCL2 concentrations added to the tube formation assay showed a reducing effect on tube formation with the strongest effect with 50 ng/ml (70.4 %, p=0.02), followed by 25 ng/ml (73.24 %, p=0.04) and 100 ng/ml CXCL2 (74.7 %, p=0.08). The addition of SB225002 using different concentrations could partly inhibit the network-reducing effect of CXCL2 (0.015 µM 91.5 %; 0.03 µM 86.9 %; 0.06 µM 92 %). Only at the highest concentration of 0.125 µM SB225002, a proapoptotic effect on HUVEC was observed, consequently, this concentration was not further analyzed. A DMSO solvent control added according to 0.015 and 0.03 µM of SB225002 did not alter the tube network area. <h3>Conclusion:</h3> Both RASF and CXCL2 had a significant decreasing effect on the tube network in 2D tube formation assays. Thus, the observed effect suggests an angiogenesis-modulating effect mediated by RASF-released CXCL2. The addition of the CXCR2 antagonist SB225002 confirmed the CXCL2-mediated effect by partly inhibiting the reduction of the tube network. <h3>REFERENCES:</h3> <b>NIL.</b> <h3>Acknowledgements:</h3> <b>NIL.</b> <h3>Disclosure of Interests:</h3> <b>None declared.</b>