In the present study we show that visfatin/Nampt is produced by the three main tissues of the human OA joint but to a greater degree by synovium. Visfatin/Nampt is naturally produced in a dimeric conformation by OA tissues, which corresponds to its enzymatically active form. Moreover, Nampt enzymatic activity is involved in the proinflammatory effects of visfatin/Nampt on chondrocytes and osteoblasts.
The detection of visfatin/Nampt in OA tissues has been reported previously, showing that all OA human tissues expressed visfatin/Nampt (that is, cartilage, subchondral bone, synovium as well as infrapatellar fat pad), but the conformation was not assessed [36, 37]. Interestingly, Meier and colleagues detected visfatin/Nampt by immunohistochemistry within OA synovium, especially around vessels . Here, we describe the conformation of visfatin/Nampt produced by the joint and its enzymatic activity. We previously reported that cultured human OA chondrocytes express visfatin/Nampt in response to IL-1β . Here, we demonstrate that the three main tissues of the human OA joint (that is, cartilage, subchondral bone and synovial membrane) store and release visfatin/Nampt, which is more significantly produced by synovial membrane than cartilage and subchondral bone. This finding suggests a potential paracrine effect of visfatin/Nampt from synovium to the other tissular and cellular components of the joint. Similarly, other mediators such as TNFα or IL-1β are released by the action of OA-activated synovial membrane on adjacent cartilage and subchondral bone tissues . In agreement with Duan and colleagues, visfatin/Nampt was present in synovial fluid from OA patients .
Visfatin/Nampt is a unique proinflammatory adipokine because it displays both cytokinic and enzymatic activities, the latter requiring a dimerization of two 52 kDa monomers to organize the enzymatic active site capable of converting nicotinamide to nicotinamide mononucleotide [19, 39]. We therefore investigated whether these dual roles of visfatin/Nampt are linked and are involved in OA pathophysiologic features. Use of nondenaturing conditions revealed that visfatin/Nampt stored and released by OA human tissues is naturally dimerized, because we detected the 120 kDa form. This natural dimeric form has been detected in human serum and is also constitutively released by human hepatocytes [40, 41].
Interestingly, visfatin/Nampt is also released by infrapatellar fat pad: the role of such a release by this tissue needs to be further addressed considering the intracapsular but extrasynovial localization of this tissue .
Because visfatin/Nampt came predominantly from the synovium, we assessed and found that it had synovial enzymatic activity. We did not systematically find Nampt activity in other OA joint tissues, probably because of the lower amount of visfatin/Nampt stored in these tissues (data not shown). We were not able to measure the enzymatic activity in CM or synovial fluid because visfatin/Nampt is much more diluted there than in tissues. Furthermore, we could not discriminate which cell type (that is, fibroblastic synoviocytes or infiltrating mononuclear cells) showed Nampt activity in OA synovium. Given the increased synovial production of visfatin/Nampt in an enzymatically active form, we hypothesize that visfatin/Nampt present in the OA joint originates mainly from synovium and acts on adjacent cartilage and subchondral bone in a paracrine pathway, with its enzymatic activity involved in its cytokinic effect.
The effect of visfatin/Nampt on synovial fibroblasts from patients with rheumatoid arthritis has been extensively studied and is characterized by proinflammatory and prodegradative effects (that is, IL-6, IL-8, MCP-1 and MMP release) [16, 26, 27]. Here, we investigated the effects of visfatin/Nampt on chondrocytes and, for the first time, on osteoblasts. Our team had demonstrated that human OA chondrocytes stimulated by visfatin/Nampt could acquire a prodegradative and proinflammatory phenotype by increasing prostaglandin E2, MMP-3 and MMP-13  Here, we further characterize the visfatin/Nampt-induced proinflammatory phenotype of chondrocytes since visfatin/Nampt was also responsible for stimulation of a cytokine (IL-6) and chemokines (Kc, MCP-1), all mediators critical in the OA pathological process because they participate in cartilage extracellular matrix degradation and attraction of proinflammatory cells [2, 3]. Despite the ubiquitous proinflammatory effect of visfatin and considering the ubiquitous role of Nampt enzymatic activity, such effects remain selective because we did not find any change in expression of growth factors or hypertrophic differentiation markers. Busso and colleagues treated efficiently collagen-induced arthritic mice with the specific enzymatic inhibitor APO866 and found decreased expression of proinflammatory mediators (IL-1β, IL-6 and MCP-1) but not other mediators (IL-10, interferon-gamma, regulated upon activation normal T-cell expressed and presumably secreted [RANTES], and IL-12), which again illustrates the selective inhibitory effect of APO866 .
To determine the involvement of the enzymatic activity of visfatin/Nampt in these proinflammatory effects, we pretreated chondrocytes and osteoblasts with the inhibitor APO866 – which specifically antagonizes the enzymatic activity only if the visfatin/Nampt dimerizes. The induction of the proinflammatory cytokines was greatly decreased (up to 94% and 63% for IL-6 in chondrocytes and osteoblasts, respectively), which demonstrates that the proinflammatory effects of visfatin/Nampt on chondrocytes greatly depend on its enzymatic activity. In agreement, we previously reported a similar decrease in visfatin/Nampt-induced prostaglandin E2 release with APO866 treatment . The inhibitory role of APO866 seems to specifically antagonize the proinflammatory effects of recombinant visfatin/Nampt and is not due to depletion of NAD (resulting in slower cellular function) or to inhibition of endogenous intracellular visfatin/Nampt. Indeed, we stimulated chondrocytes with IL-1β (0.1 ng/ml) treated with APO866 (10 nM) and found no decrease in mRNA levels of IL-6, Kc or MCP-1 (data not shown) while IL-1β is known to induce intracellular visfatin/Nampt in chondrocytes . In other conditions and other cell types, extracellular visfatin/Nampt had similar effects because it induced proinflammatory signaling in human vascular smooth muscle cells through Nampt activity, which was blocked by APO866 treatment .
We investigated the effects of visfatin/Nampt on murine osteoblasts for the first time. Osteoblasts were sensitive to visfatin/Nampt because their stimulation induced the expression and production of the same proinflammatory cytokines as in the chondrocyte experiments (that is, IL-6, Kc and MCP-1). Interestingly, the induction of proinflammatory cytokines was much higher in osteoblasts than chondrocytes. As seen with chondrocytes, the use of APO866 on osteoblasts decreased the production of proinflammatory cytokines, which confirms the enzymatic effect of visfatin/Nampt on osteoblasts. Visfatin/Nampt has a proinflammatory effect on the three main cell types within the joint: chondrocytes, osteoblasts (depending in part on Nampt activity) and synoviocytes [16, 26, 27].