Abstract
IntroductionVisfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). With a homodimeric conformation, it exerts nicotinamide phosphoribosyltransferase (Nampt) enzymatic activity, essential for nicotinamide adenine dinucleotide biosynthesis. We examined the tissular origin and conformation of visfatin/Nampt in human OA joints and investigated the role of visfatin/Nampt in chondrocytes and osteoblasts by studying Nampt enzymatic activity.MethodsSynovium, cartilage and subchondral bone from human OA joints were used for protein extraction or incubated for 24 hours in serum-free media (conditioned media), and synovial fluid was obtained from OA patients. Visfatin/Nampt expression in tissular extracts and conditioned media was evaluated by western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Nampt activity was assessed in OA synovium by colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated with visfatin/Nampt and pretreated or not with APO866, a pharmacologic inhibitor of Nampt activity. The effect on cytokines, chemokines, growth factors and hypertrophic markers expression was examined by quantitative reverse transcriptase polymerase chain reaction and/or ELISA.ResultsIn tissular explants, conditioned media and synovial fluid, visfatin/Nampt was found as a homodimer, corresponding to the enzymatically active conformation. All human OA joint tissues released visfatin/Nampt (synovium: 628 ± 106 ng/g tissue; subchondral bone: 195 ± 26 ng/g tissue; cartilage: 152 ± 46 ng/g tissue), with significantly higher level for synovium (P <0.0005). Nampt activity was identified ex vivo in synovium. In vitro, visfatin/Nampt significantly induced the expression of interleukin 6, keratinocyte chemoattractant and monocyte chemoattractant protein 1 in chondrocytes and osteoblasts. APO866 decreased the mRNA and protein levels of these pro-inflammatory cytokines in the two cell types (up to 94% and 63% inhibition, respectively). Levels of growth factors (vascular endothelial growth factor, transforming growth factor β) and hypertrophic genes were unchanged with treatment.ConclusionVisfatin/Nampt is released by all human OA tissues in a dimeric enzymatically active conformation and mostly by the synovium, which displays Nampt activity. The Nampt activity of visfatin is involved in chondrocyte and osteoblast activation, so targeting this enzymatic activity to disrupt joint tissue interactions may be novel in OA therapy.
Highlights
Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA)
We previously showed that visfatin/nicotinamide phosphoribosyltransferase (Nampt) is produced by IL-1β-stimulated OA chondrocytes and may induce a prodegradative and proinflammatory phenotype of chondrocytes characterized by the induction of matrix metalloproteinase (MMP)-3 and MMP-13 and synthesis of prostaglandin E2 [17]
We aimed to address the expression, conformation and enzymatic properties of visfatin/Nampt in human OA joints, to decipher the proinflammatory role of this adipokine in two cell types involved in OA, and to connect the cytokinic and enzymatic effects of this adipokine enzyme, investigating whether these effects are mediated by Nampt activity
Summary
Visfatin is an adipokine that may be involved in intertissular joint communication in osteoarthritis (OA). In OA, synovium is inflamed and characterized by hypertrophic and hyperplasic synoviocytes and infiltrating mononuclear cells All of these cells produce interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor alpha (TNFα), major proinflammatory cytokines in OA [3,4]. Recent data support a direct communication between the subchondral bone and cartilage via a process of diffusion through vessels, microcracks and fissures [5] This diffusion permits the exchange of soluble products with the ability to modulate the activities of resident cells in these tissues [6,7].
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