Background/Purpose: Hypoxia is a feature of inflamed tissues. The transcription factors Hypoxia inducible factor (HIF)-1 and (HIF)-2 regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of rheumatoid arthritis (RA). However, functional differences and similarities between these factors in regard to bionergetics have not been investigated so far. Our aim was to knockdown either HIF-1α or HIF-2α in human microvascular endothelial cells (HMEC) in order to investigate resulting effects on angiogenesis and bioenergetics under hypoxia vs. normoxia. In previous studies we showed that HIF-2α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors. In this study we focused on the regulation of bioenergetics by HIF-1α and HIF-2α, respectively. 

Methods:

Specific knockdown of either 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 hypoxic (<1% O2) versus normoxic (~18% O2) conditions.

Expression of genes involved in the metabolic response to hypoxia (GAPDH, PGK, GLUT1, LDHA) was quantified by realtime RT-PCR. The bioenergetic status of the cells was quantified via ATP/ADP measurements.

Results:

Knockdown of HIF-1α resulted in a loss of both hypoxia induced node (p<0.01) and tubuli formation (p=0.09). Also HIF-2α knockdown was followed by a significant reduction of hypoxia induced formation of tubuli (p<0.05).

Focusing on bioenergetic aspects, we found hypoxia to significantly induce the gene expression of PGK (p<0.001), LDHA (p<0.05) and GAPDH (p<0.05) in control cells. Interestingly, knockdowns of HIF-1α and HIF-2α, respectively, did not affect the hypoxic induction of PGK and LDHA expression.

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 considerably less pronounced in HIF-1α knockdown cells. Hypoxia did not significantly up-regulate GLUT1 (encoding the glucose transporter 1), neither in control nor in HIF-1α or HIF-2α knockdown cells.

However, the knockdown of HIF-2α resulted in significantly decreased expression levels of GLUT1 under hypoxia (p<0.01).

We also found the ATP/ADP ratio to be similar in control, HIF-1α knockdown and HIF-2α knockdown cells under normoxia. Under hypoxia, however, HIF-1α knockdown cells showed a significantly reduced ATP/ADP ratio (p<0.05) – indicating that less ATP is available – compared to HIF-2α knockdown cells.

Conclusion: HIF-1α and HIF-2α are both key regulators of angiogenesis. However, they do differ in their potency to regulate cellular energy metabolism. We show here HIF-1α to affect angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of GAPDH and the diminished ATP/ADP ratio. In contrast, HIF-2α has rather modest effects on cellular energy metabolism but has been shown to affect angiogenesis directly via regulating the synthesis of proangiogenic factors. These findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in RA.

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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-2/