Background/Purpose: Hypoxia and angiogenesis are features of inflamed and injured tissues. The transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2 control cellular metabolic response to decreased oxygen tension thereby promoting angiogenesis with implications on the pathogenesis of rheumatoid arthritis (RA). Our studies aims to knockdown HIF-1α and HIF-2α in human microvascular endothelial cells (HMEC), respectively, in order to investigate resulting effects on angiogenesis and bioenergetics under hypoxic versus normoxic conditions.

Methods: Specific knockdown of HIF-1α or HIF-2α was achieved using lentiviral-based shRNA technology. Angiogenesis of transduced HMECs was studied by investigating both tubuli and node formation under hypoxia (<1% O₂) versus normoxia (~18% O₂). Expression of hypoxia driven genes involved in metabolic response to hypoxia (GAPDH, PGK, GLUT1) was quantified by realtime RT-PCR. Bioenergetic state of the cells was investigated via ADP/ATP measurements.

Results: Knockdown of HIF-1α led to a loss in the hypoxia induced node (p=0.007) and tubuli formation (p=0.09). HIF-2α knockdown also resulted in a significant loss of hypoxia induced formation of tubuli (p=0.04). Focussing on bioenergetics, we found hypoxia to significantly induce PGK (p=0.0004) and GAPDH (p=0.049) in control cells. Interestingly, HIF-1α knockdown – but not HIF-2α knockdown – resulted in a loss of hypoxic induction of PGKexpression.

In both HIF-1α (p=0.01) and HIF-2α (p=0.13) knockdown cells, hypoxia was still capable of inducing GAPDH, but the effect was much less pronounced in HIF-1α knockdown cells.

Hypoxia did not up-regulate GLUT1,neither in control nor in HIF-1α and HIF-2α knockdown cells, respectively.

We also found the ADP/ATP ratio to be similar in control and HIF-1α or HIF-2α knockdown cells under normoxia. Under hypoxic conditions, however, HIF-1α knockdown cells showed a significantly enhanced ADP/ATP ratio (p<0.05) – indicating that less ATP is available – compared to control cells. This was not the case in HIF-2α knockdown cells.

Conclusion:

HIF-1α and HIF-2α are both key regulators of angiogenesis. However, they do differ in their ability to regulate cellular energy metabolism. This leads us to conclude that HIF-2α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (1), whereas HIF-1α affects angiogenesis via effects on cellular energy metabolism.

These findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in RA.

(1) Hahne et al. Angiogenic potential of HMECs – analysis of two HIFa isoforms and their overlapping functions. 75. Annual Scientific Meeting of the American College of Rheumatology, Chicago, USA, 4-9 November 2011. Abstract in Arthritis & Rheumatism 2011;63(10)Supplement:S10

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-bioenergetic-role-of-hif-1-and-hif-2-during-angiogenesis-of-human-microvascular-endothelial-cells/