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.