Results

Effect of adiponectin on the production of proinflammatory mediators in rheumatoid arthritis fibroblast-like synoviocytes
To evaluate whether adiponectin contributes significantly to the inflammation of joints in RA patients, the level of proinflammatory mediators in response to IL-1β or adiponectin was compared in FLSs from patients with RA (Figure 1). The RA synovial cells were treated with 1 or 10 μg/mL adiponectin. For comparison, the cells were treated with 0.1 or 1 ng/mL IL-1β. Adiponectin stimulated the production of IL-6 and IL-8 in RA FLSs, but the production of these proteins was very low in comparison with that induced by IL-1β in these cells. IL-1β at 1 ng/mL increased the production of IL-6 and IL-8 about fivefold and ninefold, respectively, compared with adiponectin at 10 μg/mL. In particular, 1 ng/mL IL-1β increased the level of PGE2 about 40-fold more than IL-1β at 0.1 ng/mL and adiponectin at 10 μg/mL. Consistent with the protein levels, the mRNA levels of the respective genes were also increased by the evaluation of real-time PCR (data not shown). This result suggests that adiponectin may not greatly induce the production of proinflammatory mediators such as IL-6, IL-8, and PGE2 in FLSs like IL-1β does.
Figure 1  Comparative effect of adiponectin and interleukin (IL)-1β on the production of IL-6, IL-8, and prostaglandin E2 (PGE2) in rheumatoid arthritis fibroblast-like synoviocytes. IL-1β at 1 ng/mL stimulates the production of (a) IL-6, (b) IL-8, and (c) PGE2 more than 10 μg/mL adiponectin. (d) The expression of cycloxygenase-2 (COX-2) increased only in the presence of IL-1β in proportion to the level of PGE2. The data shown are representative of three independent experiments, and similar results were obtained from all three. Values are expressed as mean ± standard error of the mean. * P < 0.05, ** P < 0.01 versus nontreated group. ns, not significant.

Effect of adiponectin on the production of vascular endothelial growth factor and matrix metalloproteinases in rheumatoid arthritis fibroblast-like synoviocytes
Next, to evaluate whether adiponectin stimulates the production of VEGF and MMPs for angiogenesis of pannus and joint destruction in RA FLSs, VEGF and MMP production was evaluated in the supernatants of cell cultures treated with adiponectin or IL-1β (Figure 2). Both adiponectin and IL-1β strongly stimulated the production of VEGF, MMP-1, and MMP-13 in RA FLSs. However, the expressions of MMP-2 and MMP-9 were not increased by either adiponectin or IL-1β at the mRNA or protein level. Consistent with the mRNA levels (data not shown), the protein levels of VEGF, MMP-1, and MMP-13 elevated by treatment with 10 μg/mL adiponectin were similar to those after treatment with 1 ng/mL IL-1β. The difference between adiponectin and IL-1β was not statistically significant, suggesting that adiponetin may have a role in the production of VEGF and MMPs like IL-1β does.
Figure 2  Effect of adiponectin on the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1, and MMP-13 in rheumatoid arthritis fibroblast-like synoviocytes. Adiponectin stimulates the expression of (a) VEGF and (b) MMP-1 and MMP-13 at a level comparable to 1 ng/mL interleukin-1-beta (IL-1β). The data shown are representative of three independent experiments, and similar results were obtained from all three. Values are expressed as mean ± standard error of the mean. * P < 0.05, ** P < 0.01 versus nontreated group.

Correlation of matrix metalloproteinase levels with adiponectin in the joint fluid of patients
To evaluate whether the in vitro effect of adiponectin on the expression of VEGF, MMP-1, and MMP-13 is associated with the expression of these genes in the joint fluid of RA patients, we measured the levels of adiponectin, VEGF, MMP-1, and MMP-13 in the joint fluid of RA or OA patients (Figure 3). As reported previously [8,9], the level of adiponectin was significantly elevated in the joint fluid of RA patients compared with that of OA patients (Figure 3a). The MMP-1 level was also higher in RA patients than in OA patients (Figure 3b). However, the level of MMP-13 and VEGF was not significantly different between RA and OA patients (Figure 3c, d). Next, we checked whether adiponectin levels correlated with VEGF, MMP-1, and MMP-13 levels in the joint fluid from RA or OA patients (Figure 4). However, the level of adiponectin did not positively correlate with MMP levels in the joint fluid of either RA or OA patients. MMP levels in joint fluid might be affected by other factors like proinflammatory cytokines and hypoxia or by unknown factors. However, the adiponectin levels significantly and positively correlated with VEGF levels in RA joint fluid, but not OA joint fluid. This result suggests that adipopectin may play a role in inducing the expression of VEGF in RA FLSs, leading to angiogenesis and the formation of pannus in RA patients.
Figure 3  The levels of adiponectin, vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1, and MMP-13 in the joint fluid of patients with rheumatoid arthritis (RA) or osteoarthritis (OA). The joint fluid levels of (a) adiponectin and (c) MMP-1 were significantly higher in RA patients than in OA patients, whereas (b) VEGF and (d) MMP-13 were not statistically different between RA and OA patients. Joint fluid samples from 30 patients in each group were used for the analysis. Values are expressed as mean ± standard error of the mean. * P < 0.05, *** P < 0.001 versus OA patient group.
Figure 4  Correlation of adiponectin with vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-1, or MMP-13 in the joint fluid from rheumatoid arthritis (RA) or osteoarthritis (OA) patients. The level of adiponectin was positively correlated with the level of VEGF in the joint fluid of (a) RA patients, but not (b) OA patients. The level of adiponectin did not correlate with the levels of MMP-1 and MMP-13 in either RA or OA patients.

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