Adiponectin may contribute to synovitis and joint destruction in rheumatoid arthritis by stimulating vascular endothelial growth factor, matrix metalloproteinase-1, and matrix metalloproteinase-13 expression in fibroblast-like synoviocytes more than proinflammatory mediators

Adipose tissue, once viewed as simply a storage and release depot for lipids, is now considered an endocrine tissue [1, 2] that secretes various substances (adipokines), including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, leptin, adiponectin, resistin, visfatin, and omenetin [3, 4]. Among these adipokines, much attention has been paid to adiponectin's relationship with insulin sensitivity and glucose and lipid metabolism in the past 10 years. In addition, adiponectin is known to exhibit potent anti-inflammatory [5], atheroprotective [6], and antidiabetic [7] effects.

Recent findings suggest that adiponectin may be involved in the pathogenesis of rheumatoid arthritis (RA). Levels of adiponectin in synovial fluid and sera were elevated in patients with RA [8, 9]. Adiponectin also induces the production of proinflammatory cytokines, IL-6, matrix metalloproteinase (MMP)-1, and IL-8 from RA synovial fibroblasts in vitro [10, 11]. Thus, it was suggested that adiponectin can also exert significant proinflammatory and matrix-degrading effects. However, the role of adiponectin in the pathogenesis of RA is still controversial because of conflicting reports about its function [10, 12‐15]. In particular, adiponectin seems to play an anti-inflammatory role because it significantly inhibited IL-1β-stimulated synovial cell proliferation in collagen-induced arthritic mice, despite increased IL-6 expression [16]. In contrast, high-grade inflammation in RA patients was negatively correlated with circulating adiponectin concentrations [17]. Rather, it was suggested that circulating adiponectin may be involved in cardiovascular disease in RA patients. Although this contradiction was partly explained by the induction of tolerance to inflammatory stimuli by adiponectin [18], the pro- or anti-inflammatory effects of adiponectin on the pathogenesis of RA remain unknown.

With regard to adiponectin's proinflammatory effects, we wondered whether adiponectin might stimulate the production of vascular endothelial growth factor (VEGF) and MMPs as well as proinflammatory mediators like IL-1β and TNF-α do. In this study, we investigated the stimulatory effect of adiponectin on the production of IL-6, IL-8, prostaglandin E₂ (PGE₂), VEGF, and MMPs. In addition, the correlation between adiponectin and VEGF or MMPs was investigated by measuring the levels of these three proteins in the joint fluid of patients with RA or osteoarthritis (OA).

Adiponectin is known to play a significant role in the pathogenesis of RA, although whether adiponectin acts as an anti-inflammatory or proinflammatory mediator is controversial. Assuming that adiponectin plays a role in the pathogenesis of arthritis as a proinflammatory mediator, we tried to determine how significantly adiponectin contributes to joint inflammation and destruction compared with another proinflammatory cytokine, IL-1β. Thus, we evaluated the effect of adiponectin on the production of the proinflammatory mediators VEGF and MMPs and compared the result with that of IL-1β, one of the major proinflammatory cytokines in RA FLSs.

In this study, RA FLSs stimulated with 10 μg/mL adiponectin increased the production of IL-6, IL-8, and PGE₂. However, the level of these proteins was low in comparison with that of RA FLSs stimulated with 1 ng/mL IL-1β. Adiponectin also greatly increased the mRNA and protein levels of collagenases (MMP-1 and MMP-13) and VEGF. MMPs and VEGF were elevated to a level comparable to that of IL-1β in RA FLSs. Interestingly, the mRNA and protein levels of gelatinases (MMP-2 and MMP-9) were not stimulated by either adiponectin or IL-1β. These results are consistent with some, but not all, previous results. Ehling and colleagues [10] reported that adiponectin increased the expression of pro-MMP-1 and IL-6 in RA FLSs, but not pro-MMP-13. In addition, adiponectin stimulated the expression of MMP-9, but not MMP-2, in cultured murine chondrocytes [20].

Some questions about this effect may be raised. First, the human recombinant adiponectin protein used in these studies is a single nonglycosylated polypeptide chain produced in Escherichia coli with a molecular weight of 25.1 kDa and containing 231 amino acids. Thus, the adiponectin used in this system may not completely duplicate the effect of native glycosylated adiponectin under physiological conditions. However, the adiponectin monomer forms multimeric complexes under physiological conditions and include a low-molecular-weight trimeric form (75 to 90 kDa) made up of three monomers strongly bound together at the globular domain level, a medium-molecular-weight hexameric form, and a high-molecular-weight form (about 500 kDa) [21]. The biological activity of each form has not yet been established. Furthermore, a proteolytic cleavage product of adiponectin, known as globular adiponectin, circulates in human plasma [22]. The percentage of each form with respect to the total amount of adiponectin could vary as a function of the different pathophysiological conditions. Thus, the biological activities may differ according to the different isoforms of adiponectin. However, we are confident that this result may represent, at least in part, the effect of a physiological adiponectin. Second, the concentration of adiponectin used to treat cells may have been at a physiological concentration. However, the level of IL-1β (1 ng/mL) used in this study may not represent the physiological concentration given that its concentration in the joint fluid of patients with RA is about 200 pg/mL [23]. Thus, a comparison of in vitro effects of adiponectin and IL-1β may not represent the in vivo effect of these factors. Adiponectin may play a more important role in the stimulation of MMPs in FLSs than IL-1β does. Our study provides insight into an understanding of adiponectin's role in joint destruction and inflammation in RA patients. Third, the cells were stimulated with adiponectin for 24 hours. Since potent stimulatory factors such as IL-6 and IL-8 are induced by adiponectin soon after stimulation with this factor, the effects observed with adiponectin may be mediated by these proinflammatory cytokines. However, we evaluated the levels of IL-6 and IL-8 after 6 hours of stimulation with adiponectin or IL-1β. The production levels of IL-6 and IL-8 after treatment with adiponectin were about 400 and 200 pg/mL, respectively; such levels are too low to increase MMP expression in this system. In contrast, IL-1β stimulated IL-6 and IL-8 protein expression to levels of about 10 and 8 ng/mL, respectively. This result suggests that adiponectin-mediated proinflammatory cytokines IL-6 and IL-8 could not increase the production of MMPs (Figure 5a). Finally, a recombinant protein produced in an E. coli system may have endotoxin contamination, and the endotoxin contaminant could stimulate the production of MMPs and VEGF. It was reported that commercial recombinant adiponectin contained lipopolysacchride (LPS) at concentrations of up to 30 pg/μg of protein [18]. However, even at LPS concentrations of 1 μg/mL, the production of MMPs, IL-6, and IL-8 was only weakly stimulated and the levels of these proteins were much lower than after treatment with 10 μg/mL adiponentin (Figure 5b). Therefore, we believe that the increase in MMPs and VEGF in this system was due to the effect of adiponectin alone.

Next, we determined whether adiponectin affected the gene expression levels of the joint fluid in patients with arthritis in vitro by measuring the levels of adiponectin, MMPs, and VEGF in the joint fluid of patients with RA or OA, and we checked for a correlation between adiponectin and MMPs or VEGF. The level of adiponectin was not correlated with MMP levels in the joint fluid of either RA or OA patients. However, adiponectin was positively correlated with VEGF in the joint fluid of RA patients. Although the synovial adiponectin level was significantly higher in RA patients than in OA patients in our study (a result that is consistent with a previous report [13]), the elevated adiponectin may counteract the local inflammatory process since the adiponectin was negatively associated with local inflammatory factors in patients with RA. In addition, the level of adiponectin and VEGF in the joint fluids of RA or OA patients did not correlate with the level of C-reactive protein, suggesting that adiponectin may not be significantly involved in inflammation (data not shown). Furthermore, it was suggested that adiponectin may have a protective role in OA [14]. Nevertheless, serum adiponectin concentrations correlate with the severity of RA evaluated by the extent of joint destruction, indirectly suggesting that adiponectin may be involved in joint destruction by stimulating the production of MMPs [24]. In contrast, VEGF levels were positively correlated with adiponectin levels in RA joint fluid, but not in OA joint fluid, although a correlation does not necessarily imply a causal relationship given that the levels may be affected by various factors. Thus, we cannot explain why there are differences between the two groups, even though the VEGF level between the two groups was not significantly different. The relationship between adiponectin and VEGF has not been addressed previously. Inconsistent with our data, treatment with recombinant adiponectin in a mouse model of laser-induced choroidal neovascularization resulted in decreased levels of VEGF [25]. Also, adiponectin did not regulate VEGF release in human airway smooth muscle cells whereas leptin did stimulate VEGF release [26]. However, the concentration used in these treatments may not be the physiological concentrations. In our in vitro experiments, 1 to 10 μg/mL adiponectin was used to detect increased gene expression of various factors. At adiponectin concentrations of less than 1 μg/mL, the genes in RA FLSs were not activated. If its physiological concentration is taken into consideration, adiponectin should be used at a concentration of at least 1 μg/mL for in vitro experiments [13].

Pathological processes cannot be fully understood based on the change in expression of individual genes alone since various factors act in concert in the development of specific diseases in the body. Thus, systems biology can provide a novel conceptual framework for understanding a disease [27]. Joint inflammation in arthritic patients is a complex immune reaction that is affected by various factors. Thus, joint inflammation should be understood as the integrated results of several factors, such as immune cell types infiltrating the joint cavity, cytokines, adipokines, hypoxia, and so on. In our study, the in vitro effect of adiponectin on MMP production was not demonstrated in the joint fluid of RA patients by evaluating the correlation between MMPs and adiponectin. The expression of MMPs also seems to be differentially regulated by various factors, including hypoxia (unpublished data) and nuclear factor-kappa-B inhibitors [28, 29]. In addition, MMP levels may be more significantly affected by the stage of disease than other factors, such as cytokines. MMP-9 level was increased more in early stages of arthritis than in later stages [30]. Thus, the level of MMPs in the joint fluid of RA patients may not be affected by IL-1β or adiponectin alone. Rather, an integral effect of various unknown factors may impact the level of MMPs in joint fluid.

Factors that increase the expression of MMPs and VEGF have been suggested as potential therapeutic targets to delay or reduce the joint destruction that occurs in RA patients [31]. Based on the in vitro effect of adiponectin on the expression of MMPs and VEGF, adiponectin may be a potential target to block MMP or VEGF expression. However, further studies should be performed to better understand factors that control the expression of MMPs and VEGF in the joint fluid of RA patients. This knowledge may open new doors to treatment and prevent the pathological processes of RA.

We show for the first time that adiponectin increases the level of VEGF and MMPs in RA FLSs as much as IL-1β but causes much smaller increases in IL-6, IL-8, and COX-2 compared with IL-1β. Furthermore, the level of adiponectin in the joint fluid of RA patients, but not OA patients, positively correlated with the level of VEGF. Although these data suggest a role for adiponectin in the perpetuation of synovitis in RA, no conclusions can be drawn from these results with regard to the relationship between adiponectin and inflammatory arthritis. Additional mechanistic and longitudinal studies in humans in the future might help to correlate our findings with clinical features.