Expression and function of visfatin (Nampt), an adipokine-enzyme involved in inflammatory pathways of osteoarthritis

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.

Circulating levels of adiponectin, resistin, and visfatin after mud-bath therapy in patients with bilateral knee osteoarthritis

“…Visfatin has proinflammatory and immunomodulating functions (Sun et al 2013) and manifested a pro-degradative effect in cartilage by i n c r e a s i n g t h e s y n t h e s i s o f M M P -3 , M M P -1 3 , thrombospondin motifs (ADAMTS)-4, and ADAMTS-5 (Gosset et al 2008;Hu et al 2011;Laiguillon et al 2014).…”

The nuclear factor-erythroid 2-related factor/heme oxygenase-1 axis is critical for the inflammatory features of type 2 diabetes–associated osteoarthritis

“…Briefly, the cartilage explants were cut into small pieces (≃5 mm3), washed several times with PBS and incubated in DMEM (25 mM) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 4 mM glutamine for 24 h at 37°C. After incubation, the explants were frozen and protein extracts were obtained using previously established methods (56). Briefly, the tissues were ground in liquid nitrogen using a mortar and pestle, after which the proteins were extracted with lysis buffer and used for the western blot experiments.…”

“…Protein extraction and western blotting-Protein extracts from human cartilage explants were prepared using previously described methods (56). Murine chondrocytes were cultured in 12-well plates (4×10 4 cells/well) in duplicate and were treated as indicated.…”

The role of adipocytokines in the pathogenesis of knee joint osteoarthritis

“…In OA patients visfatin SF level positively correlated with the degradation markers of collagen, (CTX-II) and aggrecans (AGG1, AGG2) [64]. Recent studies show that most articular visfatin derives from synovium, and the 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 [65].…”