Hypoxia as a key regulator of angiogenesis and inflammation in rheumatoid arthritis: the role of HIF hydroxylases
Creators: name: Konisti, Sofia
Publisher: name: Imperial College London
Year: 2013

description: 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, together with increased infiltration of macrophages into the inflamed joints. Members of the Hypoxia-inducible factor (HIF) family play key roles in activating the transcription of hypoxia regulated genes. Regulation of HIFs, including the enzymes 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. This work aimed to shed more light on the influence of monocyte-to-macrophage differentiation on the Hypoxia/HIF axis as well as how PHDs are being regulated during the differentiation process and thus influence the HIF pathway. For this purpose I have used macrophages derived from a monocytic cell line namely THP-1, or from freshly isolated monocytes, derived from peripheral blood mononuclear cells. Monocytes were differentiated to either the classical (M1) or the alternative (M2) macrophages using granulocyte-macrophage colony stimulating factor or macrophage-colony stimulating factor respectively. THP-1 monocytes were differentiated to a M2-like phenotype using phorbol 12-myristate 13-acetate. Possible differences between the two macrophage phenotypes with regard to the Hypoxia/HIF pathway were also investigated in this study. One of the major findings was that macrophage differentiation leads to stabilisation of HIF-1α isoform even in normoxic (20 % O2) conditions. In particular, in PMA-treated THP-1 cells, the normoxic HIF-1α stabilisation was partly due to an increased HIF α gene transcription. Moreover, PHD-2 expression and enzymatic activity were also affected during the differentiation process, and were linked to reduced HIF hydroxylation and increased HIFα protein accumulation. However, expression of downstream HIF dependent angiogenic genes was not affected during the differentiation process, suggesting an additional level of control, mediated possibly by transcriptional inactivation of HIF via asparagine hydroxylation by FIH-1, or inhibition of the HIF α nuclear translocation machinery. Using the M1 and M2 polarised macrophages, there were no significant differences observed between the two phenotypes with regard to the HIF α isoform expression. In both phenotypes downstream gene expression was observed for Bcl2/adenovirus E1B 19d-interactin protein (BNIP3) and ephrinA3 (EPHRINA3), although in the M2 phenotype expression of both genes was significantly greater. Exposure of macrophages to the hypoxia mimetic dimethyloxaloyglycine (DMOG) stabilised both HIF α protein isoforms with no siginificant differences in expression levels, and led to transcription of the HIF α target genes BNIP3 and EphrinA3. Expresion of PHD-2 and PHD-3 were also increased in response to DMOG but more in the M2 macrophages. The effect of HIF stabilisation by DMOG was also studied in two different in vivo mouse models of arthritis; collagen induced arthritis (CIA) and antigen induced arthritis (AIA). HIF activation by DMOG was observed by imaging analysis, using a previously described transgenic mouse oxygen-dependent degradation domain (ODD)-luciferase reporter mice. It was demonstrated that DMOG activates the HIF pathway in vivo, and appears to exert a protective role in the setting of arthritis (reduced paw/knee swelling), possibly by promoting the expansion of the anti-inflammatory M2 macrophage population. Furthermore the effect of DMOG in anaemia of inflammation was also observed in the CIA mouse model. Arthritic mice showed decreased haematocrit levels, which were corrected back to normal levels after DMOG treatment. In conclusion my data contributes to a better understanding of the Hypoxia/HIF pathway in RA, and also suggest an additional possibly protective role of HIF possibly by shifting macrophages towards a less inflammatory phenotype, and also inhibiting anaemia of inflammation. These findings also set the basis for further research that will allow the development of new therapeutic strategies for the treatment of RA.