Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis

Abstract

The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor165 (VEGF165) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF165 production. In addition, sFasL inhibited VEGF165-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF165-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF165-producing cells but also by blocking VEGF165-induced migration of endothelial cells, independent of Fas-mediated apoptosis.

Figure 1

Inverse correlation between soluble Fas ligand (sFasL) and vascular endothelial growth factor (VEGF) levels in patients with rheumatoid arthritis (RA). (a) Concentrations of sFasL in synovial fluid of patients with RA (n = 29) and osteoarthritis (OA, n = 30). (b) Correlation of VEGF₁₆₅ concentration with sFasL level in the sera of patients with RA. (c) Correlation between VEGF₁₆₅ and sFasL levels in the synovial fluid of patients with RA.

Figure 2

Effect of soluble Fas ligand (sFasL) on vascular endothelial growth factor (VEGF)₁₆₅ production by synovial fibroblasts. (a) Fibroblast-like synoviocytes (FLSs) were cultured in triplicate for 24 hours with medium alone and transforming growth factor (TGF)-β (10 ng/ml) in the presence of various concentrations of sFasL (10 to 100 ng/ml). The amount of VEGF₁₆₅ in the culture supernatants was determined by ELISA. Data are the mean ± standard deviation (SD) of three independent experiments in triplicate. *P < 0.05; ^†P < 0.01 versus the cells stimulated with medium alone or TGF-β in the absence of sFasL. (b) The rheumatoid arthritis FLSs (RAFLS; n = 7) or osteoarthritis FLSs (OAFLS; n = 5) were treated with increasing concentrations of sFasL (1 to 100 ng/ml) in the absence or presence of 1% FCS for 24 hours. The viability of FLS was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are expressed as the mean ± SD. *P < 0.01 versus OAFLS.

Figure 3

Soluble Fas ligand (sFasL) does not affect the survival, proliferation and tube formation of endothelial cells (ECs). (a,b) No effect of sFasL on the survival (a) and proliferation (b) of ECs. The ECs were incubated for 24 hours in DMEM supplemented with 1% FCS, and were treated with various concentrations of sFasL (1 to 100 ng/ml) in the absence or presence of vascular endothelial growth factor (VEGF)₁₆₅ (10 ng/ml). The viability and proliferation of ECs were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and [³H]-thymidine incorporation assays, respectively. Data are expressed as the mean ± standard deviation (SD) of three independent experiments. (c) No effect of sFasL on tube formation by ECs. The ECs were plated on Matrigel matrices with VEGF₁₆₅ (20 ng/ml) in the presence of various concentrations (10 to 1,000 ng/ml) of sFasL or soluble CD40 ligand (sCD40L) for 48 hours. The total length of the tube network was calculated using Image-Pro Plus software. Data are representative of three independent experiments with similar results, presented as the mean ± SD.

Figure 4

Soluble Fas ligand (sFasL) strongly inhibits migration and chemotaxis of endothelial cells (ECs). (a) Inhibition of wounding migration of ECs by sFasL. Confluent ECs were wounded with the tip of a micropipette, and incubated further in M199 containing 1% FCS with vascular endothelial growth factor (VEGF)₁₆₅ (20 ng/ml) in the absence or presence of sFasL (50 ng/ml). After 12 hours, the cells migrating beyond the reference line were photographed (at ×50 magnification) and counted. A representative of three independent experiments is shown. (b) sFasL suppresses the chemotaxis of ECs induced by VEGF₁₆₅. The ECs were placed in the upper wells of the chemotaxis chamber with various concentrations (0.1 to 100 ng/ml) of soluble CD40 ligand (sCD40L) or sFasL in the presence of VEGF (10 ng/ml) added in the lower wells. The number of cells that had migrated to the lower surface of the membrane was counted after hematoxylin and eosin staining. Data are representative of three independent experiments, presented as the mean ± standard deviation. ^†P < 0.01 versus the VEGF₁₆₅-stimulated cells without sFasL.

Figure 5

Inhibition of vascular endothelial growth factor (VEGF)₁₆₅-induced phsospho-Akt (pAkt) activity by soluble Fas ligand (sFasL). Endothelial cells (ECs) were incubated with various concentrations of sFasL (1 to 50 ng/ml) in the presence of VEGF₁₆₅ (20 ng/ml) for 10 minutes. The pAkt and phospho-extracellular signal-regulated kinase (pERK) expression levels in the ECs were determined by western blot analysis. The data are a representative result of three independent experiments, and are expressed as the mean (± standard deviation) optical density ratio [pAkt/β-actin]. *P < 0.05 versus the VEGF₁₆₅-stimulated cells without sFasL.

Figure 6

Effect of soluble Fas ligand (sFasL) on neovascularization in vivo. Matrigel containing vascular endothelial growth factor (VEGF)₁₆₅ and heparin were injected subcutaneously with or without sFasL (100 ng/ml) into the abdomen of C57BL/6 mice. After 14 days, the mice were sacrificed and the Matrigel plugs were excised and fixed. (a) Representative Matrigel plugs containing PBS (none), VEGF₁₆₅ (500 ng/ml), or VEGF₁₆₅ (500 ng/ml) plus sFasL (100 ng/ml). (b) Quantification of new vessel formation via measurements of the hemoglobin (Hb) content within the Matrigel is shown. Eight mice were used for each group. Data represent the mean ± standard deviation, and similar results were obtained with two different experiments. *P < 0.001 versus the Hb content of the Matrigel containing VEGF₁₆₅. (c) Representative photograph of the gels shown in cross-section and stained with hematoxylin and eosin. Original magnification (×100).