THU0088 The Bioenergetic Role of HIF-1 and HIF-2 During Angiogenesis of Human Microvascular Endothelial Cells. (23rd January 2014)

• Record Type: Journal Article
• Authors: Hahne, M.; Strehl, C.; Jakstadt, M.; Hoff, P.; Gaber, T.; Burmester, G.-R.; Buttgereit, F.; Buttgereit, Frank
• Is Part Of: Annals of the rheumatic diseases. Volume 72:Supplement 3(2013)
• Page Start: A192
• Page End: A193
• Publication Date: 2014-01-23
• Subjects: Rheumatism -- Periodicals; 616.723005
• DOI: 10.1136/annrheumdis-2013-eular.616
• Languages: English
• ISSNs: 0003-4967

Abstract

Abstract : 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