Background Hypoxia and angiogenesis are features of inflamed and injured 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).
Objectives 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 hypoxic versus normoxic conditions.
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 hypoxia driven 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=0.007) and tubuli formation (p=0.09). Also HIF-2α knockdown was followed by a significant reduction of hypoxia induced formation of tubuli (p=0.04). Focussing on bioenergetic aspects, we found hypoxia to significantly induce PGK (p=0.0004), LDHA (p=0,039) and GAPDH (p=0.049) in control cells. Interestingly, knockdown 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=0,0039).
We also found the ATP/ADP ratio to be similar in control, HIF-1α knockdown and HIF-2α knockdown cells under normoxia. Under hypoxic conditions, 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.
Conclusions HIF-1α and HIF-2α are both key regulators of angiogenesis. However, they do differ in their potency to regulate cellular energy metabolism. This leads us to conclude that HIF-2α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas HIF-1α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of GAPDH and the diminished ATP/ADP ratio.
These findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in RA.
Disclosure of Interest None Declared
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