Introduction: Synovial fibroblasts (SF) display a ‘hyperactive’ phenotype in patients with rheumatoid arthritis (RA). Nicotinamide adenine dinucleotide (NAD+) plays a role in cell metabolism, but may also be a key molecule in maintaining this ‘activated’ phenotype. NAD+ can be synthesised from precursor vitamin molecules, nicotinamide (Nam), nicotinic acid (NA) and Tryptophan (TRP); with their respective phosphoribosyl transferases (NAMPT, NAPRT, QAPRT) and Indoleamine (IDO) being the rate limiting enzymes involved in these pathways. NAMPT and IDO are known to be elevated in RA synovial tissue (ST). However, the expression and regulation of other NAD+ biosynthesis enzymes are unknown. Methods: RA, OA and normal ST were obtained from joints of patients undergoing surgery and expression of NAD+ biosynthesis enzymes were quantified using qPCR. Synovial fibroblasts were cultured and stimulated with 10ng/ml of TNF-α, IL-1β, OSM & IFN- and the expression of NAD+ biosynthesis enzymes were quantified using qPCR. Results: qPCR analyses showed that all NAD+ biosynthesis enzymes tested were constitutively expressed in synovial tissue ex vivo and in vitro, with the exception of NMN adenyltransferase (NMNAT)-3. NAMPT, IDO, QAPRT, NADSYN and NMNAT-2 were all upregulated in RA ST compared to normal tissue, however only NAMPT was significantly upregulated in RA compared to OA and normal, (NAMPT reached statistical significance when patients on anti-TNF therapy were excluded). Moreover, NAMPT was found to be upregulated in ST of young actively developing individuals, decreasing with age. Expression of NAD salvage enzymes, NAMPT and NMNAT-2 in ST correlated with each other and de novo NAD enzymes, IDO, QAPRT, NADSYN and NMNAT-2 were also correlated with each other in ST. NAMPT and IDO were both significantly upregulated in vitro following stimulation with OSM & IFN- but only NAMPT and NMNAT-2 were upregulated following stimulation with TNF-α & IL-1β. NAPRT expression was found to be low in RA ST and there was no upregulation following stimulation by OSM, IFN-, TNF-α & IL-1β. Conclusion: The data presented in this thesis emphasises NAMPT and IDO as a potential therapeutic target in rheumatoid arthritis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:701996 |
| Date | January 2016 |
| Creators | Moideen, Abdul Nazeer |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/97161/ |
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