THU0088 The Bioenergetic Role of HIF-1 and HIF-2 During Angiogenesis of Human Microvascular Endothelial Cells
Authors: M. Hahne, C. Strehl, M. Jakstadt, P. Hoff, T. Gaber, G.-R. Burmester, F. Buttgereit, Frank Buttgereit
Journal: Annals of the Rheumatic Diseases (2013)

<h3>Background</h3> 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). <h3>Objectives</h3> 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. <h3>Methods</h3> 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 (&lt;1% O<sub>2</sub>) versus normoxic (~18% O<sub>2</sub>) conditions. Expression of hypoxia driven genes involved in the metabolic response to hypoxia <i>(GAPDH, PGK, GLUT1, LDHA)</i> was quantified by realtime RT-PCR. The bioenergetic status of the cells was quantified via ATP/ADP measurements. <h3>Results</h3> 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 <i>PGK</i> (p=0.0004), LDHA (p=0,039) and <i>GAPDH</i> (p=0.049) in control cells. Interestingly, knockdown of HIF-1α and HIF-2α, respectively, did not affect the hypoxic induction of <i>PGK</i> and <i>LDHA</i> 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 <i>GLUT1</i> (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 <i>GLUT1</i> 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&lt;0.05) – indicating that less ATP is available – compared to HIF-2α knockdown cells. <h3>Conclusions</h3> 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 <i>GAPDH</i> 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. <h3>Disclosure of Interest</h3> None Declared