Hypoxia regulated responses in the pathogenesis of rheumatoid arthritis

• Main Author: Larsen, Helene
• Other Authors: Feldmann, Marc ; Paleolog, Ewa
• Published: Imperial College London 2009
• Subjects: 616.7
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.513436

Increased angiogenesis in the synovium is a hallmark of rheumatoid arthritis (RA), and is thought to be driven by the presence of both local regions of reduced oxygen tension (hypoxia) and inflammatory cytokines in the synovium. RA fibroblast-like synoviocytes (FLS) are the main cell type at the proliferating and invading edge of the pannus, and are therefore likely to be exposed to the greatest degree of hypoxia. In culture, RA FLS can secrete a plethora of angiogenic factors in response to hypoxia and inflammatory cytokines, and these cells are thus potential regulators of synovial angiogenesis. Transcriptional control of many hypoxia-inducible genes, including several involved in angiogenesis, occurs via hypoxia-inducible factor (HIF)-1α and/or HIF-2α, powerful transcription factors known to be induced by hypoxia and cytokines. The regulation of HIFs by hypoxia and inflammatory cytokines (tumour necrosis factor α, interleukin-1β and transforming growth factor β) was studied in RA FLS. The main findings were that HIF-1α mRNA was downregulated in hypoxia (1% oxygen) possibly due to a simultaneous upregulation of a natural antisense to HIF-1α, aHIF. HIF-2α was also shown to be negatively regulated by hypoxia at the mRNA level but the mechanisms behind this were not uncovered in the present study. Cytokines induced HIF-1α mRNA, protein and activity but reduced levels of HIF-2α mRNA. The description of regulation of HIF-1α by cytokines such as tumour necrosis factor α, supports the concept that HIF transcription factors may represent a key convergence point in RA for hypoxia and cytokines. In addition to studying the regulation of HIFs by inflammatory cytokines and hypoxia, 84 angiogenic genes were investigated for their hypoxia-responsiveness in RA FLS by PCR array screening. Two genes, angiopoietin-like 4 (ANGPTL-4) and leptin, were strongly induced in response to hypoxia in RA FLS in comparison to cells grown in normoxia (21% oxygen). Small interfering RNA technology was used to transiently knock-down HIF- 1α and/or HIF-2α which demonstrated that the hypoxia-inducibility of both ANGPTL-4 and leptin was dependent on HIFs, but that ANGPTL-4 induction was mainly HIF-1α dependent, compared to leptin which seemed to be regulated predominantly by HIF-2α. The secretion of leptin was measured by ELISA, and the functional significance of RA FLS-derived leptin in angiogenesis was further investigated using ELISAs and an MTT assay. As leptin has been reported to be elevated in the serum and synovial fluid of RA patients, leptin may represent a future target for anti-angiogenic therapy in RA. The research described in this thesis aimed to gain a better understanding of how HIFs are regulated by hypoxia and cytokines in RA FLS, using FLS grown from total dissociated human RA synovial membranes, and hereby to understand what governs the production of angiogenic factors in the RA synovial milieu.