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Symposium Reports |
1 Vascular Biology Program and Institute for Cell Engineering, Departments of Pediatrics, Medicine, Oncology and Radiation Oncology; and McKusick-Nathans Institute of Genetic Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Abstract
The regulation of tissue perfusion is a major mechanism by which oxygen homeostasis is maintained. Hypoxia-inducible factor 1 (HIF-1) is a transcriptional regulator that mediates adaptive responses to reduced partial pressure of O2 in all metazoan species. In mammals, HIF-1 promotes angiogenesis, arteriogenesis and vasculogenesis through the production of multiple angiogenic growth factors in ischaemic tissue and by cell-autonomous effects on endothelial cells and bone marrow-derived angiogenic cells. Administration of viral vectors encoding constitutively active forms of the HIF-1
subunit results in increased tissue perfusion in animal models of ischaemic cardiovascular disease.
(Received 17 May 2007;
accepted after revision 14 August 2007; first published online 24 August 2007)
Corresponding author G. L. Semenza: Broadway Research Building, Suite 671, 733 North Broadway, Baltimore, MD 21205, USA. Email: gsemenza{at}jhmi.edu
The regulation of tissue perfusion is a major mechanism by which oxygen homeostasis is maintained. During embryogenesis, this involves the de novo formation of blood vessels from angioblasts that coalesce to form the primary vascular network, a process that is referred to as vasculogenesis. Once the primary network is established, new blood vessels can branch off from existing vessels, a process that is referred to as angiogenesis. Finally, the initial vascular plexus, which consists of a network of vessels of similar luminal diameter, is remodelled to form an arterial system in which a smaller number of large-calibre conductance vessels give rise to progressively increasing numbers of smaller calibre vessels in a hierarchical organization extending from arteries to arterioles to capillaries. The remodelling of vessels to accept increased flow is referred to as arteriogenesis (Heil et al. 2006).
When cells are hypoxic owing to inadequate perfusion, transcription of genes encoding angiogenic growth factors is increased (Losordo & Dimmeler, 2004a). The protein products of these genes then bind to receptors on endothelial and vascular smooth muscle cells, and this stimulates the outgrowth of new capillaries, resulting in increased O2 delivery to the hypoxic cells, which extinguishes the stimulus for further angiogenesis, thereby closing the homeostatic loop. Several dozen angiogenic growth factors have been identified, including angiopoietin 1, angiopoietin 2, placental growth factor, platelet-derived growth factor B, stromal-derived factor 1 and vascular endothelial growth factor (VEGF). These factors also promote the recruitment of circulating angiogenic cells to sites of hypoxia/ischaemia, where they also participate in the angiogenic process, either by differentiating into endothelial cells (in the case of endothelial progenitor cells) or by producing angiogenic cytokines that activate endogenous vascular endothelial or smooth muscle cells (Kinnaird et al. 2004; Losordo & Dimmeler, 2004b; Ziegelhoeffer et al. 2004).
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional regulator that mediates adaptive responses to reduced partial pressure of O2
. Hypoxia-inducible factor 1 is a heterodimer of a constitutively expressed HIF-1β subunit and an O2-regulated HIF-1 subunit (Wang et al. 1995; Wang & Semenza, 1995). Levels of the HIF-1 subunit increase dramatically as O2 concentrations decrease below 6% O2, which corresponds to a 
of 
40 mmHg (Jiang et al. 1996). Under aerobic conditions, HIF-1 is hydroxylated on proline residue 402 and/or 564 by the prolyl hydroxylase domain protein PHD2, which uses O2 as a substrate (Ehrismann et al. 2007). Hydroxylated HIF-1 is bound by the von Hippel-Lindau tumour suppressor protein (VHL), which recruits a ubiquitin–ligase complex (Maxwell et al. 1999; Kaelin, 2005). Ubiquitinated HIF-1 is targeted for proteasomal degradation. Under hypoxic conditions, the hydroxylation reaction is inhibited and unhydroxylated HIF-1 accumulates, dimerizes with HIF-1β and binds to target DNA that contains the core sequence 5'-RCGTG-3' (where R = A or G; Semenza et al. 1996). Asparagine residue 803 in HIF-1 is hydroxylated by the dioxygenase FIH-1 (factor inhibiting HIF-1) under aerobic conditions (Lando et al. 2002a; Ehrismann et al. 2007). Asparaginyl hydroxylation prevents the interaction of HIF-1 with the coactivator protein p300 (Lando et al. 2002b). Under hypoxic conditions, interaction of unhydroxylated HIF-1 with p300/CBP renders HIF-1 competent to activate transcription. Thus, both the half-life and the specific activity of HIF-1 are determined by O2-dependent hydroxylation, which provides a direct mechanism for the transduction of changes in 
to the nucleus, leading to changes in gene transcription. Both gain-of-function and loss-of-function studies in cultured cells and animal models have demonstrated that HIF-1 is involved in the increased expression of angiopoietin 1, angiopoietin 2, placental growth factor, platelet-derived growth factor B, stromal-derived factor 1 and vascular endothelial growth factor in response to hypoxia or ischaemia (Kelly et al. 2003; Ceradini et al. 2004).
Hypoxia-inducible factor 1β can also dimerize with HIF-2, which shares sequence similarity with HIF-1 and is also subject to O2-dependent prolyl and asparaginyl hydroxylation. In contrast to the ubiquitous expression of HIF-1, HIF-2 is expressed in a more restricted subset of cell types, most notably within vascular endothelial cells. Compared with HIF-1:HIF-1β heterodimers, HIF-2:HIF-1β heterodimers transactivate an overlapping but distinct set of target genes (Elvidge et al. 2006).
Knockout mice lacking HIF-1 die at midgestation (embryonic day 10) with defects in cardiac development, red blood cell production and vascularization, indicating that all three components of the circulatory system require HIF-1 for normal development (Iyer et al. 1998; Ryan et al. 1998; Compernolle et al. 2003; Yoon et al. 2006). Mice lacking HIF-1β also die at midgestation with vascular defects (Maltepe et al. 1997). Mice with complete HIF-2 deficiency are born without vascular defects in certain genetic backgrounds (Scortegagna et al. 2003). Mice with conditional knockout of HIF-1 in endothelial cells are also viable and without major vascular defects (Tang et al. 2004). Endothelial cell-specific expression of a dominant negative form of HIF-2 that interferes with the activity of both HIF-1:HIF-1β and HIF-2:HIF-1β heterodimers resulted in embryonic lethality with cardiovascular defects, indicating that endothelial cell expression of both HIF-1 and HIF-2 contributes to normal cardiovascular development (Licht et al. 2006).
Cardiovascular disease is the leading cause of morbidity and mortality in the US population, accounting for 38.5% of all deaths. Atherosclerotic narrowing of coronary arteries results in myocardial ischaemia owing to reduced tissue perfusion. Among patients with critical (>70%) narrowing of one or more major coronary vessels, approximately two-thirds develop collateral vessels that supply blood from another major artery downstream of the site of stenosis. Patients with collaterals are more likely to survive a heart attack than patients without collaterals (Hansen, 1989). Analysis of a single nucleotide polymorphism in the HIF1A gene that alters the coding sequence for HIF-1 (changing proline at residue 582 to serine P582S) revealed that the frequency of the variant allele was fivefold higher among patients with critical stenosis of a coronary artery who lacked collaterals compared with patients having collaterals (Resar et al. 2005). These results suggest that variation at the HIF1A locus may be an important determinant of whether patients with coronary artery disease develop collaterals.
In addition to coronary artery disease, atherosclerosis can result in stenosis of major arteries in the leg. The incidence of lower limb amputations is 200 per million in the non-diabetic and 3900 per million among the diabetic population the U.S. (Most & Sinnock, 1983). In a rabbit model of limb ischaemia, the femoral artery was occluded by the intravascular placement of coils and the animals received an injection of replication-defective recombinant adenovirus AdLacZ, which encodes β-galactosidase (control for non-specific effects of adenoviral injection), or AdCA5, which encodes a constitutively active form of HIF-1. Analysis of the rabbits by arteriography 2 weeks later revealed significantly increased collateral vessel diameter and perfusion of the occluded limb in animals receiving AdCA5 compared with those receiving AdLacZ (Patel et al. 2005). The ratio of blood pressure in the occluded to the non-occluded limb was 0.94 ± 0.05 in the AdCA5-treated rabbits compared with 0.55 ± 0.05 in the AdLacZ-treated rabbits. Immunohistochemistry for the endothelial-specific cell surface receptor CD31 revealed that the ratio of capillaries to myocytes in the ischaemic limb of AdCA5-treated rabbits was significantly greater than in the AdLacZ-treated rabbits, demonstrating that AdCA5 had promoted angiogenesis. In addition, immunohistochemistry for smooth muscle -actin revealed a significant increase in the luminal area of conductance arteries with a diameter >100 µm. Both arteriography and histology demonstrated that there was no increase in the number of collateral vessels, but rather in the diameter of the vessels, which allowed them to accept increased blood flow, i.e. arteriogenesis (Patel et al. 2005).
Intramuscular injection of mice with adeno-associated virus vectors encoding VEGF or a stable form of mouse HIF-1 with proline-to-alanine mutations (mHIF-1) at the two residues that are subject to O2-dependent hydroxylation revealed that whereas VEGF induced proliferation of endothelial cells without the proper formation of capillaries, mHIF-1 induced well-demarcated sprouting capillaries with organized pericyte coverage (Pajusola et al. 2005). In addition, whereas VEGF induced increased vascular permeability, mHIF-1 did not. Finally, perfusion of the injected muscle was significantly increased by injection of mHIF-1 and was not further increased by coexpression of angiopoietin 1 or platelet-derived growth factor B (Pajusola et al. 2005).
The effectiveness of treatment with constitutively active forms of HIF-1 may reflect the fact that HIF-1 promotes vascular remodelling through multiple mechanisms. First, HIF-1 stimulates the production of multiple angiogenic growth factors in ischaemic tissue, including angiopoietin 1, angiopoietin 2, placental growth factor, platelet-derived growth factor B, stromal-derived factor 1 and vascular endothelial growth factor (Kelly et al. 2003). Second, HIF-1 has cell-autonomous effects in endothelial cells (Tang et al. 2004; Manalo et al. 2005; Calvani et al. 2006) and bone marrow-derived progenitors (Okuyama et al. 2006) that promote the homing of these cells to ischaemic tissue and their activation, especially when they are stimulated by angiogenic growth factors (Fig. 1).
A phase I clinical trial of an adenovirus encoding a chimeric HIF-1/VP-16 fusion protein (protein consisting of the basic helix-loop-hellix-PAS domain from HIF-1 fused to the transactivation domain of herpes simplex virus protein VP-16) in patients with critical limb ischaemia was recently reported (Rajagopalan et al. 2007), but many questions remain to be answered regarding the translation of these findings to the clinic. Can HIF-1 gene therapy overcome the impairment of angiogenesis and arteriogenesis that occurs with ageing? Are certain subgroups of patients (e.g. patients with the P582S genotype) more (or less) likely to respond to this therapy? Can the clinical response be improved by combining gene therapy with progenitor (e.g. autologous bone marrow) cell therapy? Owing to the difficulty in translating success in preclinical models to the clinic (Hirsch, 2006), it is imperative to extend these models from young healthy animals (as were used in all of the studies described above) to aged, atherosclerotic and diabetic animals that more accurately model the patients in whom such novel therapies must ultimately prove their efficacy.
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