Investigating the role of Nicotinamide phosphoribosyltransferase (NAMPT) in cartilage catabolism.
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NAMPT (nicotinamide phosphoribosyltransferase) is a universally expressed protein elevated in the serum and synovial fluid of rheumatoid arthritis (RA) sufferers. NAMPT is a rate-limiting enzyme in the biosynthesis of (nicotinamide adenine dinucleotide) NAD+, an essential cellular coenzyme, and has also been shown to exert cytokine-like activities as a mediator of innate immunity. However little is currently known of the role of NAMPT in cartilage metabolism. In this thesis, the role and regulation of NAMPT was studied in a variety of model systems. Addition of exogenous (e)NAMPT to fibroblasts in vitro increased MMP-3 release, an effect attenuated by APO866. In vivo studies in a murine arthritis model showed APO866 treatment reduced arthritis index, with a slight reduction in synovial hyperplasia, inflammatory infiltrate and cartilage degradation. In vivo imaging with MMP activatable fluorescent probes showed a reduction in MMP activity in APO866-treated animals compared with placebo. Analyses of archived tissue indicated that APO866 reduced MMP gene expression in affected limbs in a time and dose dependent manner. In a cartilage explant culture system, APO866 significantly attenuated cytokine-mediated proteoglycan (PG) depletion and MMP-2 and -9 release. This was associated with a dose-dependent loss of cell viability. These effects were reversed by co-incubation with NAD+ metabolites NMN and NA, suggesting that cell survival was highly NAD+-dependent, and that cytokine-induced MMP expression could be NAMPT independent. Finally, in vitro studies in RA synovial fibroblasts revealed that intracellular (i)NAMPT is upregulated by cytokine stimulation, with concurrent upregulation of MMPs -1 and -3. The data emphasise the pleiotropic function of NAMPT in health and disease, and suggest a role for extracellular (but not intracellular) NAMPT in cartilage catabolism. Finally, NAMPT inhibition is highlighted as a promising therapeutic strategy for RA.
|Item Type:||Thesis (PhD)|
|Subjects:||R Medicine > R Medicine (General)|
|Last Modified:||19 Mar 2016 23:37|
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