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Title: Dissecting out the mechanisms of hypoxia-dependent responses in rheumatoid arthritis
Author: Muz, Barbara
ISNI:       0000 0004 2713 3049
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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Rheumatoid arthritis (RA) is a chronic inflammatory disease with a significant impact on patients’ quality of life. One of the well-described features in RA is hypoxia. It has been shown that both RA synovial fluid and synovial tissue are characterised by insufficient amount of oxygen. Abnormality in hypoxia-inducible factor (HIF) expression is a marker of decreased oxygen tension in RA and in other pathological conditions. HIFs, together with the proteins which regulate their stabilisation and transactivation, namely prolyl hydroxylase domain (PHD) enzymes and factor inhibiting HIF-1 (FIH-1), were the main focus of this thesis. The first aim of the study was to investigate the specific roles of HIF isoforms in RA fibroblast-like synoviocytes (FLS), which are the key cells in RA pathogenesis. Expression of HIF-1α, HIF-2α and a panel of genes involved in angiogenesis (ANGPTL-4, ephrin-A3, VEGF), glycolysis (GLUT-1, ENO-1), pH regulation (CA9) and apoptosis (BNIP-3) were significantly increased by hypoxia. Successful silencing of both HIFs using short interfering RNA (siRNA) was achieved in RA FLS, and was followed by examination of the HIF-dependence of the candidate genes. The majority of these genes were found to be HIF-1α- dependent, whereas only ANGPTL-4 and VEGF were regulated by both HIF-1α and HIF-2α. Subsequently, the mRNA and protein expression of PHDs and FIH-1 in RA FLS, their response to hypoxic conditions and their HIF-dependence were examined. Specific knockdown of the hydroxylases was performed and the effects on HIFs and genes of interest were assessed. The most significant changes were noticed after silencing PHD-2, which led to HIF-α stabilisation and upregulation of HIF-dependent genes. In addition, PHD-2 depletion increased a number of pro-angiogenic genes and also contributed to new tubule formation in a functional angiogenesis assay. Comparison of RA, osteoarthritis (OA) FLS and normal human dermal fibroblasts (NHDF) revealed that PHD-2 has similar functions in promoting neovascularisation in arthritic cells (RA and OA), but not in non-arthritic cells (NHDF). Finally, the last chapter describes preliminary data on the expression of the least investigated HIF-3α subunit and its negative splice variant, HIF-3α4, in RA FLS. This research contributes to a better understanding of the upstream and downstream regulation of HIF signalling in RA, showing that PHD-2 has a key role in regulating hypoxic responses via HIFs in RA FLS. Because this pathway controls the expression of so many genes relevant to the disease, it may be an important target for RA therapy. One of the most critical challenges would be to target the correct HIF/PHDs molecule in the right tissue/cell, ensuring that it will not affect angiogenesis in healthy tissues.
Supervisor: Paleolog, Ewa Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral