Hypoxia-inducible factor as an angiogenic master switch

Abstract

Hypoxia-inducible factors (HIFs) regulate the transcription of genes that mediate the response to hypoxia. HIFs are constantly expressed and degraded under normoxia, but stabilized under hypoxia. HIFs have been widely studied in physiological and pathological conditions and have been shown to contribute to the pathogenesis of various vascular diseases. In clinical settings, the HIF pathway has been studied for its role in inhibiting carcinogenesis. HIFs might also play a protective role in the pathology of ischemic diseases. Clinical trials of therapeutic angiogenesis after the administration of a single growth factor have yielded unsatisfactory or controversial results, possibly because the coordinated activity of different HIF-induced factors is necessary to induce mature vessel formation. Thus, manipulation of HIF activity to simultaneously induce a spectrum of angiogenic factors offers a superior strategy for therapeutic angiogenesis. Because HIF-2α plays an essential role in vascular remodeling, manipulation of HIF-2α is a promising approach to the treatment of ischemic diseases caused by arterial obstruction, where insufficient development of collateral vessels impedes effective therapy. Eukaryotic initiation factor 3 subunit e (eIF3e)/INT6 interacts specifically with HIF-2α and induces the proteasome inhibitor-sensitive degradation of HIF-2α, independent of hypoxia and von Hippel-Lindau protein. Treatment with eIF3e/INT6 siRNA stabilizes HIF-2α activity even under normoxic conditions and induces the expression of several angiogenic factors, at levels sufficient to produce functional arteries and veins in vivo. We have demonstrated that administration of eIF3e/INT6 siRNA to ischemic limbs or cold-injured brains reduces ischemic damage in animal models. This review summarizes the current understanding of the relationship between HIFs and vascular diseases. We also discuss novel oxygen-independent regulatory proteins that bind HIF-α and the implications of a new method for therapeutic angiogenesis using HIF stabilizers.

Keywords: HIF-1α; HIF-2α; Int6/eIF3e; angiogenesis; collateral vessel; pVHL; peripheral arterial disease.

Figure 1

Schema illustrating the degradation of HIF-2α. Hypoxia-inducible factor 2 (HIF-2) activates gene transcription in response to hypoxia. Under normoxic conditions (blue arrows), HIF-2α is hydroxylated on proline residues 496 and 542 by a prolyl-hydroxylase domain (PHD) protein. Hydroxylation is required for binding of the von Hippel-Lindau protein (VHL), the recognition subunit of a ubiquitin protein ligase that targets HIF-2α for ubiquitination and proteasomal degradation. In addition, hydroxylation on asparagine residue 847 by factor-inhibiting HIF (FIH) blocks the binding of the co-activator p300. On the other hand, binding of eIF3e/Int6 to the Int6/eIF3e binding site (IBS) leads to the proteasomal degradation of HIF-2α, irrespective of hypoxia/normoxia. This mechanism of post-transcriptional regulation is specific for the HIF-2α subtype. Thus, inhibition of eIF3e/Int6 by siRNA leads to the accumulation of HIF-2α.

Figure 2

Feedback mechanisms regulating the expression of HIF-2α. In hypoxia, stabilized and dimerized HIF-2α recognizes hypoxia responsive elements (HREs) in its own promoter and in the eIF3e/Int6 promoter, resulting in the transcription of both genes as part of positive and negative feedback mechanisms, respectively. eIF3e/Int6 binds to and degrades newly synthesized HIF-2α, even under hypoxic conditions.

Figure 3

DNA microarray analysis of eIF3e/Int6 silencing in MCF-7 cells. DNA microarray analysis of MCF-7 cells transfected with eIF3e/Int6 silencing plasmids identified 378 upregulated genes and 244 downregulated genes, relative to expression in cells transfected with GAPDH.