Background/Purpose: Angiogenesis is a prominent feature of the pathogenesis of RA. Although new blood vessels deliver oxygen to the augmented inflammatory cell mass, the neovascular network is dysfunctional and fails to restore tissue oxygen homeostasis, so that the inflamed joint remains markedly hypoxic. At a cellular level, hypoxia is detected by a mechanism that regulates the amount of hypoxia-inducible factor (HIF)-1α and -2α. After translocation to the nucleus, HIF-1α or HIF-2α subunit bind its partner HIF-1β to form a functional, heterodimeric transcription factor, HIF-1 and HIF-2, which activate a gene program associated with angiogenesis, glycolysis, and adaptation to pH. The distinct functions of HIF-1 and HIF-2 in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown.

Methods: For this purpose we investigated the adaption of Human Microvascular Endothelial Cell (HMEC)-1 to pathophysiologic hypoxic conditions (1% O₂). Angiogenesis assay and cytokine analyses were conducted to describe angiogenesis. Gene expression and ATP/ADP measurements were performed to visualize the bioenergetic switch under hypoxia. shRNA-technology enabled a specific knockdown of either HIF-1α or HIF-2α or the combination of HIF-1α and HIF-2α, in order to identify distinct functions of both transcription factors, which were assessed via microarray analysis.

Results: Hypoxia induced angiogenesis and a bioenergetic switch from oxidative phosphorylation towards glycolysis. Knockdown of either HIF-1α or HIF-2α leads to a reduced angiogenesis in vitro. Although HIF-1α and HIF-2α show overlapping functions during adaption of HMEC-1 towards hypoxia reduction of HIF-1α resulted in a diminished cellular energy supply due to a reduction in hypoxia-induced glycolytic enzyme gene expression thereby not altering pro-angiogenic factors VEGF, VEGF receptor 1 and 2, and IL8. In contrast, reduction of HIF-2α resulted in a reduction of hypoxia induced pro-angiogenic VEGF and IL8 gene and protein expression but induction of VEGFR1 without altering glycolytic enzyme gene expression. Finally, the results of HIF-1α / HIF-2α combined knockdown demonstrate that the lack of both factors significantly reduced cell survival as compared to the scramble control thereby diminishing both gene expression of pro angiogenic factors and glycolytic enzymes.

Conclusion: Our results indicate distinct but also overlapping functions of HIF-1α and HIF-2α in ECs which are of high interest regarding the intervention of tumour growth but also the chronicity of the inflammatory process in RA. Finally we clearly demonstrate a major impact of HIF-1α and HIF-2α on angiogenesis mediated either via the expression of pro-angiogenic factors in case of HIF-2α or via the glycolytic switch in case of HIF-1α. At least both are essential for endothelial cellular function. These findings open new possibilities for therapeutic approaches by targeting the specific hypoxia induced factors.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/disentangling-the-role-of-hypoxia-inducible-factor-1-and-2-in-the-adaption-process-of-human-microvascular-endothelial-cells-to-pathophysiological-hypoxia/