Background/Purpose: The role of cytokines produced by obese-derived adipose tissue, namely adipokines, in the pathophysiology of osteoarthritis (OA) is now well established. We recently suggested that one of them, visfatin, may play a role in OA by activating chondrocytes (1,2). Along with its cytokine effect, visfatin has an enzymatic activity called nicotinamide phosphoribosyltransferase (Nampt), which is the rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis from nicotinamide, an intracellular pathway involved in many biological processes including TNFα and IL-6 synthesis. Thus, we aimed to i) characterize the local site(s) of production of visfatin/Nampt by the human OA joint tissues ii) further investigate the role of its enzymatic activity in the expression of pro-inflammatory cytokines by chondrocytes and iii) determine whether visfatin/Nampt may also play a role in subchondral bone.

Methods: Human OA joint tissues (synovial membrane, cartilage, subchondral bone) from patients undergoing surgical knee replacement were incubated for 24h in serum-free media. Visfatin/Nampt release in media by the different tissues was evaluated using Western Blot and ELISA. Primary cultures of mouse chondrocytes and osteoblasts were stimulated with recombinant visfatin/Nampt (5µg/mL) for 24h. To determine the role of the enzyme activity, cells were pretreated or not 4h before visfatin/Nampt stimulation with APO866 (10nM), a pharmacologic inhibitor of Nampt activity (3). Effects of stimulation on IL-6, IL-8/Kc, IL-1β, MCP-1, VEGF and TGFβ expression, and on IL-6 and IL-8/Kc release were examined by quantitative RT-PCR and ELISA, respectively.

Results: All human OA joint tissues released visfatin/Nampt (synovium: 529 ± 356, cartilage: 237 ± 380, subchondral bone: 200 ± 104 ng/g tissue) with higher level for the synovium compared to cartilage (p<0.01). Visfatin/Nampt significantly induced IL-6, IL-8/Kc, IL-1β and MCP-1 expression by chondrocytes (n=6) and osteoblasts (n=5) (Table). Visfatin/Nampt increased the production of IL-6 and IL-8/Kc proteins by both cell types. Nampt activity inhibition by APO866 decreased pro-inflammatory cytokines expression at mRNA level (up to 97 % of inhibition) as well as at protein level (up to 63 % of inhibition) and was especially efficient in chondrocytes (Table). Effect of visfatin/Nampt was selective since VEGF and TGFβ were not modulated upon stimulation.

		Gene expression				Protein production
		IL-6	Kc/IL-8	IL-1β	MCP-1	IL-6	Kc/IL-8
Chondrocytes (n=6)	Fold induction after visfatin/Nampt stimulation (Mean ± SEM), p-value	10.6 ± 3 (p=0.015)	4.9 ± 1.3 (p=0.015)	878 ± 320 (p=0.015)	2.5 ± 0.4 (p=0.03)	5.8 ± 5 (p=0.016)	29.7 ± 33 (p=0.015)
	Mean % of inhibition by APO866	94 %	83 %	97 %	62 %	63 %	57%
Osteoblasts (n=5)	Fold induction after visfatin/Nampt stimulation (Mean ± SEM), p-value	296 ± 59 (p=0.03)	142 ± 64 (p=0.03)	396 ± 112 (p=0.03)	133 ± 83 (p=0.03)	51 ± 19 (p=0.05)	54 ± 23 (p=0.05)
	Mean % of inhibition by APO866	63 %	79 %	73 %	84 %	39 %	49%

Conclusion: Visfatin/Nampt is produced by the cartilage, the subchondral bone and mostly by the synovial membrane. We demonstrate that Nampt activity of visfatin plays a major role in chondrocytes and osteoblasts activation, suggesting that targeting the Nampt enzymatic activity with a compound like APO866 (being tested in hematological malignancies) may be a new therapeutic approach of OA.

References: (1)Gosset M. Arthritis Rheum 2008 (2) Jacques C. J Biol Chem 2012 (3) Busso N. PLoS One 2008

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/visfatinnampt-in-osteoarthritis-sites-of-production-in-human-joints-and-role-of-its-enzymatic-activity/