Background Visfatin exerts a nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity, which is the rate-limiting stage in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. NAD is involved in many biological processes including redox reactions, sirtuins activity and TNFαsynthesis (1). We have previously demonstrated the induction of prostaglandin E2 release by visfatin-stimulated chondrocytes (2).
Objectives i) Analyze the production and the enzymatic active conformation of visfatin/Nampt in human joint with osteoarthritis (OA), ii) Investigate its pro-inflammatory role on chondrocytes and osteoblasts, iii) Determine the involvement of the Nampt activity in the activation of those two cell types.
Methods Human OA joint tissues (synovium, cartilage, subchondral bone) from patients undergoing total knee arthroplasty were frozen or incubated 24h in serum-free media. Visfatin/NAMPT conformation in OA synovial fluids, tissue media and extracts was evaluated by Immunoblot (IB). NAMPT/visfatin concentration in tissue media was measured by ELISA. NAMPT activity was assessed in OA synovium extracts using Cyclex colorimetric assay. Primary cultures of murine chondrocytes and osteoblasts were stimulated for 24h with recombinant visfatin/NAMPT (5 µg/mL) with or without APO866 (10nM), a pharmacologic inhibitor of NAMPT activity. mRNA and protein expression of IL-6, IL-8/Kc, MCP-1, VEGF TGFβ, Ihh, Runx2 and type X collagen was examined by qRT-PCR and ELISA.
Results Human OA synovium, cartilage and subchondral bone all released visfatin/NAMPT (628±106, 152±46, 195±26 ng/g tissue, respectively) with significantly higher level for the synovium (p<0.0005). In tissue conditioned media and synovial fluids visfatin/NAMPT was found as a homodimer, corresponding to the enzymatic active conformation. Moreover, NAMPT activity was measured in synovium (1.38E-3 ± 8E-4 OD min-1, n=3). Visfatin/NAMPT significantly induced IL-6, IL-8/Kc and MCP-1 mRNA and protein expression by chondrocytes and osteoblasts (n=6) (Table). Nampt activity inhibition by APO866 decreased pro-inflammatory cytokine expression at mRNA (up to 94 %) and protein level (up to 63 %) (Table). Growth factor (VEGF, TGFβ) and hypertrophic gene expression (Ihh, Runx2, type X collagen) was unchanged upon stimulation, suggesting selective properties of visfatin/NAMPT.
Conclusions Visfatin/NAMPT is released by all OA tissues under a dimeric enzymatically active conformation and mostly by the synovium which displays a detectable NAMPT activity. The NAMPT activity of visfatin is involved in chondrocytes and osteoblasts activation. Thus, targeting its enzymatic activity with a compound like APO866 (being currently tested in hematological malignancies) may be a new therapeutic approach of OA.
Wang et al., Nature Structural & Molecular Biology, 2006;
Gosset et al., Arthritis & Rheumatism, 2008
Acknowledgements Grant from the French Society of Rheumatology (SFR)
Disclosure of Interest None Declared