Leptin-induced migration and angiogenesis in rheumatoid arthritis is mediated by reactive oxygen species

Abstract

Rheumatoid arthritis (RA) is a progressive autoimmune disease affecting the joints. In this study, we investigated the role of the pro-angiogenic factor leptin in regulating reactive oxygen species (ROS) to promote cell migration and angiogenesis in RA. We showed that leptin triggered RA fibroblast-like synoviocyte (FLS) migration by increased ROS expression. Additionally, leptin enhanced human umbilical vein endothelial cell (HUVEC) tube formation in a ROS/hypoxia-inducible factor-1α-dependent manner, accompanied by increased production of vascular endothelial growth factor and interleukin (IL)-6. We also revealed that antagonists of tumor necrosis factor, IL-6 and IL-1β down-regulated ROS production of RA FLS induced by leptin, which subsequently attenuated RA FLS migration and HUVEC tube formation. These findings demonstrated that leptin might play an important role in RA FLS migration and HUVEC angiogenesis.

Keywords: angiogenesis; leptin; migration; reactive oxygen species; rheumatoid arthritis.

Figure 1

Leptin induced RA FLS migration and HUVEC tube formation. (A) RA FLSs isolated from RA patients were stimulated with or without leptin (100 ng·mL⁻¹) for 24 h. Cell migration was measured by using the scratch assay and Transwell chambers. Representative photographs of control and leptin‐treated cells at 0 and 24 h are shown (n = 6). (B) RA FLSs were treated with or without leptin (100 ng·mL⁻¹) for 24 h. CM was then collected and applied to HUVEC cultures after addition of these cells to the Matrigel. The number of HUVEC tubes formed was determined by microscopy (n = 6). (C) RA FLSs were stimulated with or without leptin (100 ng·mL⁻¹) for 24 h. The level of VEGF and IL‐6 in the supernatant was determined by ELISA (n = 8). All experiments were repeated three times. Data represent the mean ± SEM (Wilcoxon's signed‐rank test; *P < 0.05, **P < 0.01).

Figure 2

ROS generation was involved in leptin‐stimulated RA FLS migration and HUVEC tube formation. (A) RA FLSs were labelled with DCFHDA (5 μm) after being incubated with leptin (100 ng·mL⁻¹) for 1 h. The fluorescent intensity of ROS was measured by flow cytometry and immunofluorescence (n = 6). (B,C) RA FLSs were pretreated with NAC (5 mm) or DPI (5 μm) for 1 h, and then stimulated with leptin (100 ng·mL⁻¹) for 24 h. Cell migration was examined with Transwell chambers. Matrigel assay was performed to test HUVEC tube formation (n = 6). (D,E) The levels of VEGF and IL‐6 in the supernatant were measured by ELISA (n = 8). All experiments were repeated three times. Data represent the mean ± SEM (Wilcoxon's signed‐rank test; *P < 0.05, **P < 0.01).

Figure 3

The ROS/HIF‐1α pathway participated in leptin‐induced HUVEC tube formation. (A) RA FLSs were pretreated with NAC and DPI for 1 h and then stimulated with leptin (100 ng·mL⁻¹) for 4 h. HIF‐1α mRNA expression of RA FLSs was determined by real‐time PCR. GAPDH was used as a control in real‐time PCR. FACS was used to detect HIF‐1α protein level (n = 6). (B) RA FLSs were preincubated with 10 μm 2‐methoxyestradiol (a HIF‐1α inhibitor) for 1 h and then stimulated with leptin (100 ng·mL⁻¹) for 24 h. The levels of VEGF and IL‐6 in the supernatant were examined by ELISA (n = 8). All experiments were repeated three times. Data represent the mean ± SEM (one‐way ANOVA; **P < 0.01, ***P < 0.001).

Figure 4

ROS production was decreased by antagonists of TNF, IL‐6 and IL‐1β. RA FLSs were incubated with anti‐TNFR2 (0.25 μg·mL⁻¹), anti‐IL‐6R (0.5 μg·mL⁻¹) and anti‐IL‐1βR (0.5 μg·mL⁻¹) for 2 h and then cultured with leptin (100 ng·mL⁻¹) for 1 h. ROS production was measured by flow cytometry (A) and immunofluorescence (B) (n = 6). All experiments were repeated three times. Data represent the mean ± SEM (one‐way ANOVA; **P < 0.01).

Figure 5

RA FLS migration and HUVEC tube formation were attenuated by antagonists of TNF, IL‐6 and IL‐1β. RA FLSs were pretreated with anti‐TNFR2 (0.25 μg·mL⁻¹), anti‐IL‐6R (0.5 μg·mL⁻¹) and anti‐IL‐1βR (0.5 μg·mL⁻¹) for 2 h before stimulated with leptin (100 ng·mL⁻¹) for 24 h. (A) RA FLS migration was tested by Transwell chambers (n = 6). (B) A Matrigel assay was performed to measure HUVEC tube formation by microscopy (n = 6). All experiments were repeated three times. Data represent the mean ± SEM (one‐way ANOVA; *P < 0.05, ***P < 0.001).