Background/Purpose: Angiogenesis is a hallmark of the pathogenesis of rheumatoid arthritis (RA) but also of other inflammatory processes, tissue regeneration and progressing tumors. In RA, the neovascular network is dysfunctional and fails to restore tissue oxygen homeostasis, so that the inflamed joint remains markedly hypoxic. The hypoxia-induced multifunctional and pro-inflammatory protein macrophage migration inhibitory factor (MIF) participates in the regulation of hypoxia-induced angiogenesis. At cellular level, hypoxia is detected by a mechanism that regulates the amount of the oxygen-sensitive α-subunits of the transcription factors, hypoxia-inducible factor (HIF)-1 and -2, which activate a gene program associated with angiogenesis and glycolysis. Here, we focus on the role of MIF and its mechanism of action in the process of hypoxia-induced angiogenesis and in the regulation of HIFs.

Methods: Therefore, we developed a specific knockdown of MIF and its receptor CD74 in Human Microvascular Endothelial Cell (HMEC) using lentiviral-based shRNA technology allowing us to analyse the role of MIF in hypoxia-induced angiogenesis. We investigated the adaption of these cells towards pathophysiologic hypoxic conditions (1% O₂) analysing protein-levels of HIF-1α and HIF-2α, HIF-target gene expression, angiogenesis and VEGFA release. To further identify the underlying mechanisms angiogenesis assay was performed with HMECs treated with anti-CD74-IgG, rhMIF and 4-IPP, a small molecule inhibitor of MIF.

Results: Lentiviral-mediated reduction of MIF in HMEC led to significantly decreased angiogenesis (p<0.01) which could be restored by adding extracellular rhMIF. Interestingly, reduction of MIF did not influence hypoxia-induced (i) protein-levels of HIF-1α and HIF-2α, respectively, and (ii) HIF-target gene expression of PGK1, GAPDH and VEGFA but (iii) induced secretion of pro-angiogenic VEGFA and IL8. Moreover, addition of 4-IPP also decreased angiogenic response of non-transduced HMECs but enhanced the hypoxia-induced HIF-target gene expression of PGK1 and VEGFA. Inhibiting MIF-signalling by the addition of extracellular anti-CD74-IgG also reduced angiogenic potential of HMEC (p<0.001), which was not restorable by the addition of extracellular rhMIF. Furthermore, lentiviral-mediated reduction of CD74 in HMEC also decreased angiogenesis (p<0.01) again without influencing HIF-target gene expression of PGK1, GAPDH and VEGFA.

Conclusion: