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* From The Johns Hopkins University School of Medicine, Baltimore, MD.
Correspondence to: Gregg L. Semenza, MD, PhD, 733 North Broadway, Suite 671, Baltimore, MD 21205; e-mail: gsemenza{at}jhmi.edu
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subunit. HIF-1 has been implicated in the pathogenesis of pulmonary hypertension based on both experimental and clinical data. In a mouse model of pulmonary hypertension, hypoxia-induced increases in right ventricular mass, right ventricular pressure, and medial wall thickness of pulmonary arterioles were impaired in mice that were heterozygous for a null allele at the locus encoding HIF-1 compared to wild-type littermates. Electrophysiologic analyses revealed that the hypoxia-induced hypertrophy and depolarization of pulmonary arterial smooth muscle cells from wild-type mice was significantly impaired in heterozygotes. In clinical studies, immunohistochemical analyses of plexiform lesions within the lungs of patients with severe pulmonary hypertension revealed dramatic overexpression of HIF-1 within proliferating endothelial cells. These cells also expressed vascular endothelial growth factor (VEGF), which is the product of a known HIF-1 target gene, indicating that autocrine VEGF-VEGF receptor signaling may contribution to the pathogenesis of plexiform lesions. These studies implicate HIF-1 in pathophysiologic alterations of both smooth muscle and endothelial cell biology in patients with pulmonary hypertension.
Key Words: gene expression • hypoxia • pulmonary hypertension
Hypoxia-inducible factor (HIF)-1 is a transcription factor that functions as a master regulator of oxygen homeostasis. HIF-1 has been shown to regulate the expression of dozens of target genes, the protein products of which play important roles in angiogenesis, erythropoiesis, energy metabolism, and cell survival.1 HIF-1 binds to hypoxia response elements that contain the core nucleotide sequence 5'-(A/G)CGTG-3'.2 Binding of HIF-1 to target genes leads to the recruitment of the coactivators,34 which interact with the transcription initiation complex containing RNA polymerase II and participate in histone acetylation that is required for transcription to occur.5
HIF-1 is a heterodimer composed of an oxygen-regulated HIF-1 subunit and a constitutively expressed HIF-1ß subunit.67 The HIF-1 subunit is subjected to posttranslational modification at proline residues 402 and 564 by a group of prolyl hydroxylases that utilize molecular O2 and the tricyclic acid cycle intermediate 2-oxoglutarate (-ketoglutarate) as substrates.89 Hydroxylation of Pro-402 and Pro-564 is required for the binding of the von Hippel-Lindau protein, which is the recognition component of an E3 ubiquitin-protein ligase that targets HIF-1 for degradation by the 26S proteasome.910111213141516 The HIF-1 prolyl hydroxylases have a relatively high Km for O2 such that physiologic reductions in O2 concentration result in increased levels of HIF-1.17 Asparagine 803 is also subjected to O2-dependent hydroxylation by the factor inhibiting HIF-1, which blocks the interaction of HIF-1 with the coactivators p300/cyclic adenosine monophosphate response element-binding protein and the transcriptional activation of HIF-1 target genes.1819202122 When isolated perfused ferret lung preparations were ventilated with hypoxic gas mixtures, HIF-1 expression was induced throughout the lung in an O2 concentration-dependent and time-dependent manner.23 Similar results were obtained with various pulmonary cell types that were exposed to hypoxia ex vivo.23
In addition to the O2-dependent regulation of HIF-1 expression and activity, an O2-independent regulatory pathway has been identified through which a wide variety of cytokines and growth factors have been shown to induce the expression of HIF-1 and HIF-1 target genes.24 Included among these are epidermal growth factor, fibroblast growth factor 2, hepatocyte growth factor, insulin-like growth factor 1 and 2, interleukin-1ß, insulin, prostaglandin E2, transforming growth factor (TGF)-, TGF-ß, thrombin, and tumor necrosis factor-.25 These cytokines and growth factors induce HIF-1 expression via the activation of the mitogen-activated protein kinase and/or phosphatidylinositol 3-kinase signal transduction pathways.262728
To analyze the role of HIF-1 in mammalian development and physiology, the gene encoding the HIF-1 subunit was inactivated by homologous recombination in mouse embryonic stem cells. Mouse embryos that were homozygous for the null allele arrested in their development by E9.0 and died by E10.5 with cardiac malformations, vascular regression, and extensive cell death.2930 Mice that were heterozygous for the null allele developed normally and were indistinguishable from their wild-type littermates under normoxic conditions. However, when the mice were subjected to chronic hypoxia (3 weeks at 10% O2), the wild-type and heterozygous littermates showed significant differences. Wild-type mice developed right ventricular hypertrophy and elevated right ventricular pressures as a result of hypoxia-induced remodeling of the small pulmonary arterioles. In contrast, heterozygotes showed significantly less right ventricular hypertrophy and pulmonary hypertrophy.31 In addition, pulmonary morphometry revealed that in wild-type mice chronic hypoxia increased the number of completely muscularized pulmonary arterioles as well as their medial wall thickness. The hypoxia-induced muscularization of pulmonary arterioles that was observed in the wild-type mice was significantly blunted in the heterozygous littermates.31
The increase in medial wall thickness in response to hypoxia is due both to an increase in the number of pulmonary arterial smooth muscle cells (hyperplasia) and an increase in the volume of individual cells (hypertrophy). To analyze the latter, pulmonary arterial smooth muscle cells were isolated from wild-type or heterozygous mice and the capacitance of individual cells was determined as an electrophysiologic measure of cell volume. Pulmonary arterial smooth muscle cells from wild-type mice exposed to hypoxia showed a significant increase in capacitance compared to those from normoxic mice. In contrast, pulmonary arterial smooth muscle cells from heterozygous wild-type mice exposed to hypoxia showed no increase in capacitance.32
Another major electrophysiologic response observed in pulmonary arterial smooth muscle cells from wild-type mice exposed to chronic hypoxia is membrane depolarization. The loss of membrane potential is associated with a reduction in voltage-gated K+ channel (Kv) currents.33 In pulmonary arterial smooth muscle cells from heterozygous mice, hypoxia-induced changes in membrane potential and Kv currents were markedly impaired.32 Thus, partial HIF-1 deficiency has dramatic effects on pulmonary arterial tone and remodeling. These effects of partial HIF-1 deficiency may be cell-autonomous and/or may result from impaired HIF-1 activity in pulmonary artery endothelial cells, which produce paracrine regulators of smooth muscle cells. Among these are endothelin 1, which is the product of a gene that is regulated by HIF-1.
HIF-2 is a protein that is structurally similar to HIF-1, and can dimerize with HIF-1ß and regulate the expression of an overlapping set of target genes.343536 The expression of HIF-1 is ubiquitous, whereas HIF-2 is expressed in a more limited number of tissues that include, however, both the lung and vasculature. Mice that are heterozygous for a null allele at the locus encoding HIF-2 show a complete absence of hypoxia-induced pulmonary hypertension.37 Increased levels of endothelin-1 and plasma catecholamines were induced in wild-type mice subjected to hypoxia, whereas these responses were lost in mice partially deficient for HIF-2.
In addition to the hypoxia-induced changes in pulmonary arterial smooth muscle structure and function that were described above, a hallmark of lung pathology in severe pulmonary hypertension in humans is the presence of plexiform lesions in which the dysregulated proliferation of pulmonary arterial endothelial cells results in vascular occlusion. Immunohistochemical analysis revealed high levels of HIF-1 expression within actively proliferating endothelial cells of plexiform lesions.38 In situ hybridization revealed that these cells also expressed vascular endothelial growth factor (VEGF) messenger RNA, which is the product of a known HIF-1 target gene. These same endothelial cells express VEGF receptor 2, the cognate receptor for VEGF, and signaling via VEGF receptor 2 induces endothelial cell survival and proliferation, indicating the establishment of an autocrine-signaling pathway. These data suggest that plexiform lesions arise via a process of disordered angiogenesis resulting from the aberrant activation of the HIF-1-VEGF pathway in endothelial cells by an unknown signal. It is interesting that cancer is another disease state in which HIF-1 expression is dramatically increased, leading to increased VEGF production and paracrine activation of endothelial cell proliferation.1 In cancers, HIF-1 also participates in the activation of other autocrine-signaling pathways involving TGF-/endothelial growth factor receptor and IGF-2/IGF-1R, which promote cell survival and proliferation. Additional studies are required to determine the extent to which plexiform lesions share properties with neoplasms and the precise role of HIF-1 in their pathogenesis.
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. Mol Cell Biol 2000;20,402-415 in response to hypoxia: their stabilization and redox signal-induced interaction with CBP/p300. EMBO J 1999;18,1905-1914[CrossRef][ISI][Medline] chains activated by prolyl hydroxylation. EMBO J 2001;20,5197-5206[CrossRef][ISI][Medline] targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 2001;292,464-468 to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292,468-472 by the ubiquitin-proteasome pathway. J Biol Chem 1999;274,6519-6525 (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions: its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997;272,22642-22647 by the von Hippel-Lindau tumor suppressor protein. EMBO J 2000;19,4298-4309[CrossRef][ISI][Medline]/CBP recognition in the cellular hypoxic response. Proc Natl Acad Sci U S A 2002;99,5271-5276. Proc Natl Acad Sci U S A 2002;99,5367-5372 and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001;15,2675-2686 and insulin-like growth factor 2. Cancer Res 1999;59,3915-3918 (HIF-1) synthesis: a novel mechanism for HIF-1-mediated vascular endothelial growth factor expression. Mol Cell Biol 2001;21,3995-4004. Genes Dev 1998;12,149-162 is required for solid tumor formation and embryonic vascularization. EMBO J 1998;17,3005-3015[CrossRef][ISI][Medline]. J Clin Invest 1999;103,691-696[ISI][Medline] deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia. Am J Physiol 2001;281,L202-L208[ISI] (HIF-1) and HIF-2 in hypoxic gene regulation. Mol Cell Biol 2003;23,9361-9374 versus HIF-2 in regulation of the transcriptional response to hypoxia. Cancer Res 2003;63,6130-6134 protects mice against pulmonary hypertension and right ventricular dysfunction during prolonged hypoxia. J Clin Invest 2003;111,1519-1527[CrossRef][ISI][Medline]This article has been cited by other articles:
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  J. M. Kahn, L. Goitein, D. R. Ouellette, and R. S. Irwin Pharmaceutical Industry Sponsorship of Journal Supplements Chest, May 1, 2006; 129(5): 1387 - 1388. [Full Text] [PDF]
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