Abstract 1676: Constitutive Activation of Hypoxia-Inducible Factor 1α (HIF-1α) C-Transactivation Domain is Essential for Optimal Transcription and Angiogenesis
Background: HIF-1α is a transcription factor that regulates genes involved in angiogenesis. In normoxia, degradation of HIF-1α is enabled by two hydroxylations at residues P402 and P564, while inactivation occurs through hydroxylation at residue N803 within its C-transactivation domain (C-TAD). For purposes of therapeutic angiogenesis, HIF-1α stabilization was previously achieved by introducing two point mutations at residues P402 and P564. We assessed the hypothesis that constitutive activation of HIF-1α in addition to its stabilization would result in greater HIF-1α transcriptional and angiogenic effects than mere stabilization of the molecule.
Methods and Results: We constructed a triple mutant HIF-1α (TM) plasmid, both stabilized and constitutively active, by combining mutations P402A, P564G and N803A. Transient co-transfections with hypoxia-responsive element-luciferase construct in human embryonic kidney cells revealed 2 and 2.9-fold increase in transcriptional activity of TM compared with P402A P564G double mutant and wild-type HIF-1α, respectively (p<0.01). In-vitro angiogenesis assay using transfected human umbilical vein endothelial cells showed that TM stimulated tube formation more greatly than both P402A P564G mutant and wild-type HIF-1α (13±1.2 tubes per microscopic field vs. 5±0.4 and 4.5±0.6 respectively, p<0.001). In order to examine the in-vivo angiogenic activity of the triple mutant, we induced hindlimb ischemia in mice by femoral artery ligation. Intramuscular delivery of adenoviruses expressing either the TM, wild-type HIF-1α (WT) or saline (C) (n=8–12/group) was performed on the day of surgery. Laser Doppler measurement at day 28 post-surgery demonstrated significantly enhanced blood flow ratio of ischemic/normal limb in the triple mutant treated group (C=31.4±2.1%; WT =35.6±2.9%; TM=51.8±2.5%; p<0.01). Capillary density, indicated by CD31 levels, was also greater for the triple mutant treated group (C=25.4±2.2, WT=38.9±3.8, TM=65.6±6.9, p<0.05).
Conclusions: We show that constitutive activation of the HIF-1α C-TAD is essential for optimal HIF-mediated transcription and angiogenic effects, and that the triple mutant HIF-1α could improve current therapeutic angiogenesis efforts.