New insights into oxygen sensing at a cellular level
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《胸》
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke’s and Papworth Hospitals, Cambridge, UK
Correspondence to:
Professor E Chilvers
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Box 157, Level 5 Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK; erc24@cam.ac.uk
The oxygen sensing pathway offers a new set of therapeutic targets for conditions ranging from inflammatory lung disease to pulmonary hypertension
Keywords: hypoxia; oxygen sensing; hypoxia inducible factor-1 (HIF-1); prolyl hydroxylase domain containing enzymes (PHDs)
Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; FIH, factor inhibiting HIF; HIF, hypoxia inducible factor; HRE, hypoxic response element; PHD, prolyl hydroxylase domain containing enzyme
The ability of cells to detect and respond to a fall in oxygen tension is of fundamental importance for maintaining oxidative metabolism and tissue homeostasis. One of the challenges facing scientists working in this area has been that any proposed mechanism for oxygen sensing has to accommodate the very different tolerances of certain tissues to hypoxia and the extreme variation in the cellular responses observed. Hence, while skeletal muscle cells can recover function after 30 minutes of anoxia, the brain suffers irreparable damage after only 4–6 minutes of ischaemia.1 Moreover, while carotid body cells respond to changes in oxygen tension that barely register in non-chemosensory tissues (and do so within seconds),2 upregulation of erythropoietin synthesis in the interstitial peritubular cells is transcriptionally regulated and requires far more protracted periods of hypoxaemia.3 Despite such variances in oxygen sensitivity and response time, all cells appear capable of responding to hypoxia and the essential components of a universal oxygen sensing mechanism have at last begun to emerge. Moreover, from studies conducted in stroke and heart disease, it is apparent that therapeutic targeting of this novel pathway is set to transform our approach to pathology previously deemed intractable.
RESEARCH STUDIES
Initial clues into how cells respond to low oxygen came from studies undertaken in the early 1990s examining the hypoxic response element (HRE) of the erythropoietin (Epo) gene. This led to the identification of a transcriptional activator called hypoxia inducible factor (HIF).4 HIF is a heterodimer composed of HIF-1? (or aryl hydrocarbon receptor nuclear translocator, ARNT), which is constitutively expressed, and HIF-1 whose expression and transcriptional activity are tightly regulated by the ambient oxygen concentration.5 Once formed, this protein complex migrates to the cell nucleus and, together with the co-activator CBP/p300, binds to the HREs present on the promoter region of genes involved in regulating metabolic supply and demand.6 Examples of HIF regulated genes include those involved in regulating vascular tone (for example, iNOS and adrenomedullin), angiogenesis (for example, vascular endothelial growth factor, VEGF), cell metabolism (for example, lactate dehydrogenase A, the glucose transporter GLUT-1), and haemoglobin biosynthesis (for example, erythropoietin).7 These findings, together with the ubiquitous nature of HIF and the demonstration that HIF deficient animals show major defects in many core physiological responses to oxygen, have resulted in HIF being regarded as one of the master regulators of the cellular response to hypoxia.8
The next step in this quest was to define the mechanism responsible for hypoxic induction of HIF-1. Through a combined structural and genetic approach, it has now been possible to show that HIF-1 activity is regulated by enzymatic hydroxylation at specific prolyl and asparaginyl residues by a novel 2-oxoglutarate dependent class of dioxygenases.9 Critically, these enzymes have been shown to display an absolute requirement for oxygen in addition to iron (Fe2+) and ascorbate. These oxygen sensitive enzymes inhibit HIF activity in a complementary manner since the prolyl hydroxylase domain containing enzymes (PHDs) result in an interaction between HIF-1 and the von Hippel-Lindau protein which targets HIF-1 for proteosomal destruction,10 and factor inhibiting HIF (FIH) causes asparaginyl hydroxylation and blocks HIF association with CBP/p300.11,12 Hence, under normoxic conditions, HIF-1 levels remain low and this prevents the transcription of genes containing HRE promoters (fig 1).
Figure 1 Oxygen dependent inhibition of HIF-1. Under normoxic conditions the oxygen requiring PHD and FIH dioxygenases lead to prolyl and asparaginyl hydroxylation of HIF-1 which results in proteosomal destruction and inhibition of transcriptional activity. PHD, prolyl hydroxylase domain containing enzyme; FIH, factor inhibiting HIF; NAD, amine activation domain; CAD, carboxyl activation domain; pVHL, von Hippel-Lindau protein.
CLINICAL APPLICATIONS
How does this inform our understanding of the pathophysiology of lung disease and can it provide the basis of novel therapies? Mice homozygous for a null mutation in the HIF-113 or HIF-1? genes14 die at mid gestation with vascular defects primarily involving the embryonic and extraembryonic circulation, respectively. In contrast, HIF-1+/- mice develop normally and are indistinguishable from wild type littermates. However, when exposed to 10% oxygen for up to 6 weeks, the HIF-1+/- mice demonstrate reduced susceptibility to pulmonary hypertension, polycythemia, and right ventricular hypertrophy relative to their wild type littermates.15 Morphometric analysis showed that the chronically hypoxic HIF-1+/- mice have fewer completely muscularised pulmonary arterioles and the degree of muscularisation in such vessels is reduced compared with HIF-1 wild type mice. Thus, HIF-1 appears to play a major role in mediating pulmonary vascular remodelling in chronic hypoxia, and therapeutic manoeuvres that inhibit HIF-1 activity in the lung may slow the progression of hypoxia induced pulmonary hypertension.
Likewise, in many human cancers including primary lung tumours, increased levels of HIF-1 expression are associated with resistance to tissue hypoxia and enhanced tumour angiogenesis.16 This abnormality correlates with a reduced sensitivity to radiation and chemotherapy and disease progression.17 Moreover, in vitro work has shown that depleting tumour cells of HIF-1 renders them more radioresponsive and thus provides an opportunity to enhance the therapeutic potential of conventional treatment.18 In addition, by targeting bioreductive pro-drugs to hypoxic tumour sites, the potential for normal tissue damage can be reduced.
One of the real surprises in this area of research, however, has been the recent demonstration of the importance of oxygen sensing pathways in the function of inflammatory cells. Hence, in contrast to the effects of hypoxia observed in many other cell types, oxygen deprivation causes a profound but reversible delay in the rate of constitutive apoptosis in human neutrophils.19 Moreover, removing the capacity of neutrophils to respond to hypoxia using a myeloid targeted HIF-1 knock-out results in a substantial fall in the glycolytic capacity of these cells and, as a consequence, a profound impairment of myeloid cell adhesion migration and bacterial killing.20 As persistent accumulation of primed and activated neutrophils is a cardinal feature of a number of lung diseases, these data predict that inhibition of the HIF-1 pathway may promote the resolution of granulocyte inflammation.
One final example of the importance of this pathway relates to the potential to prevent ischaemia-reperfusion injury in the transplanted lung. Hence, in the heart, ischaemic pre-conditioning, which reduces dramatically the degree of myocardial damage observed following subsequent coronary artery occlusion, has been shown to relate to HIF dependent upregulation of erythropoietin.21 Indeed, this protein has the capacity to prevent myocardial cell apoptosis, and erythropoietin infusion is effective in reducing infarct size and improving outcome in stroke.22 Application of these principles to lung protection is bound to follow.
Despite such striking advances in our knowledge of how cells sense oxygen, much remains to be defined. In particular, it is likely that a quite separate pathway underlies the ability of acute hypoxia to induce membrane depolarisation and L-type calcium channel activity in excitable cells. These events trigger the acute contractile response of pulmonary artery resistance vessels to hypoxia and the firing of carotid body glomus cells. The current contenders in this area are the mitochondrial NAD(P)H oxidases, which are postulated to produce a diffusible redox mediator in response to hypoxia.
CONCLUSIONS
These studies show that, while cells display widely differing tolerances and responses to hypoxia, oxygen sensing by dioxygenases and their consequent effects on HIF dependent transcriptional events appear to be a property that is shared by most, if not all, cells. This pathway now offers a new set of therapeutic targets for an array of lung diseases ranging from inflammatory lung disease through to pulmonary hypertension.
ACKNOWLEDGEMENTS
The work in the authors’ laboratories is sup-ported by the Wellcome Trust, British Heart Foundation, National Asthma Campaign, MRC, and British Lung Foundation. SRW holds an MRC Clinical Research Training Fellowship and KKS holds a BHF Clinical Fellowship.
REFERENCES
1. Siesjo BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metab 1981;1:155–85.
2. Lahiri S, Rozanov C, Roy A, et al. Regulation of oxygen sensing in peripheral arterial chemoreceptors. Int J Biochem Cell Biol 2001;33:755–74.
3. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988;242:1412–5.
4. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene. Mol Cell Biol 1992;12:5447–54.
5. Wang GL, Jiang B-H, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1992;92:5510–4.
6. Wenger RH, Gassmann M. Oxygen(es) and the hypoxia-inducible factor-1. Biol Chem 1997;378:609–16.
7. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 2000;88:1474–80.
8. Wenger RH. Cellular adaption to hypoxia: O2-sensing protein hydroxilases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 2002;16:1151–62.
9. Epstein ACR, Gleadle JM, McNeill LA, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001;107:43–54.
10. Jaakkola P, Mole DR, Tian Y-M, et al. Targeting of HIF- to the von Huppel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72.
11. Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002;295:858–61.
12. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001;15:2675–86.
13. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 1998;17:3005–15.
14. Maltepe E, Schmidt JV, Baunoch D, et al. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 1997;386:403–7.
15. Yu AY, Shimoda LA, Iyer NV, et al. Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1-alpha. J Clin Invest 1999;103:691–6.
16. Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci USA 1997;94:8104–9.
17. Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in radiotherapy of oropharyngeal cancer. Cancer Res 2001;61:2911–6.
18. Unruh A, Ressel A, Mohamed HG, et al. The hypoxia-inducible factor-1 alpha is a negative factor for tumour therapy. Oncogene 2003;22:3213–20.
19. Mecklenburgh KI, Walmsley SR, Cowburn AS, et al. Involvement of a ferroprotein sensor in hypoxia-mediated inhibition of neutrophil apoptosis. Blood 2002;100:3008–11.
20. Cramer T, Yamanishi Y, Clausen BE, et al. HIF-1 is essential for myeloid cell-mediated inflammation. Cell 2003;112:645–57.
21. Cai Z, Manalo DJ, Wei G, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischaemia-reperfusion injury. Circulation 2003;108:79–85.
22. Ehreneich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med 2002;8:495–505.(S R Walmsley, K K K Shear)
Correspondence to:
Professor E Chilvers
Respiratory Medicine Division, Department of Medicine, University of Cambridge School of Clinical Medicine, Box 157, Level 5 Addenbrooke’s Hospital, Hills Road, Cambridge CB2 2QQ, UK; erc24@cam.ac.uk
The oxygen sensing pathway offers a new set of therapeutic targets for conditions ranging from inflammatory lung disease to pulmonary hypertension
Keywords: hypoxia; oxygen sensing; hypoxia inducible factor-1 (HIF-1); prolyl hydroxylase domain containing enzymes (PHDs)
Abbreviations: ARNT, aryl hydrocarbon receptor nuclear translocator; FIH, factor inhibiting HIF; HIF, hypoxia inducible factor; HRE, hypoxic response element; PHD, prolyl hydroxylase domain containing enzyme
The ability of cells to detect and respond to a fall in oxygen tension is of fundamental importance for maintaining oxidative metabolism and tissue homeostasis. One of the challenges facing scientists working in this area has been that any proposed mechanism for oxygen sensing has to accommodate the very different tolerances of certain tissues to hypoxia and the extreme variation in the cellular responses observed. Hence, while skeletal muscle cells can recover function after 30 minutes of anoxia, the brain suffers irreparable damage after only 4–6 minutes of ischaemia.1 Moreover, while carotid body cells respond to changes in oxygen tension that barely register in non-chemosensory tissues (and do so within seconds),2 upregulation of erythropoietin synthesis in the interstitial peritubular cells is transcriptionally regulated and requires far more protracted periods of hypoxaemia.3 Despite such variances in oxygen sensitivity and response time, all cells appear capable of responding to hypoxia and the essential components of a universal oxygen sensing mechanism have at last begun to emerge. Moreover, from studies conducted in stroke and heart disease, it is apparent that therapeutic targeting of this novel pathway is set to transform our approach to pathology previously deemed intractable.
RESEARCH STUDIES
Initial clues into how cells respond to low oxygen came from studies undertaken in the early 1990s examining the hypoxic response element (HRE) of the erythropoietin (Epo) gene. This led to the identification of a transcriptional activator called hypoxia inducible factor (HIF).4 HIF is a heterodimer composed of HIF-1? (or aryl hydrocarbon receptor nuclear translocator, ARNT), which is constitutively expressed, and HIF-1 whose expression and transcriptional activity are tightly regulated by the ambient oxygen concentration.5 Once formed, this protein complex migrates to the cell nucleus and, together with the co-activator CBP/p300, binds to the HREs present on the promoter region of genes involved in regulating metabolic supply and demand.6 Examples of HIF regulated genes include those involved in regulating vascular tone (for example, iNOS and adrenomedullin), angiogenesis (for example, vascular endothelial growth factor, VEGF), cell metabolism (for example, lactate dehydrogenase A, the glucose transporter GLUT-1), and haemoglobin biosynthesis (for example, erythropoietin).7 These findings, together with the ubiquitous nature of HIF and the demonstration that HIF deficient animals show major defects in many core physiological responses to oxygen, have resulted in HIF being regarded as one of the master regulators of the cellular response to hypoxia.8
The next step in this quest was to define the mechanism responsible for hypoxic induction of HIF-1. Through a combined structural and genetic approach, it has now been possible to show that HIF-1 activity is regulated by enzymatic hydroxylation at specific prolyl and asparaginyl residues by a novel 2-oxoglutarate dependent class of dioxygenases.9 Critically, these enzymes have been shown to display an absolute requirement for oxygen in addition to iron (Fe2+) and ascorbate. These oxygen sensitive enzymes inhibit HIF activity in a complementary manner since the prolyl hydroxylase domain containing enzymes (PHDs) result in an interaction between HIF-1 and the von Hippel-Lindau protein which targets HIF-1 for proteosomal destruction,10 and factor inhibiting HIF (FIH) causes asparaginyl hydroxylation and blocks HIF association with CBP/p300.11,12 Hence, under normoxic conditions, HIF-1 levels remain low and this prevents the transcription of genes containing HRE promoters (fig 1).
Figure 1 Oxygen dependent inhibition of HIF-1. Under normoxic conditions the oxygen requiring PHD and FIH dioxygenases lead to prolyl and asparaginyl hydroxylation of HIF-1 which results in proteosomal destruction and inhibition of transcriptional activity. PHD, prolyl hydroxylase domain containing enzyme; FIH, factor inhibiting HIF; NAD, amine activation domain; CAD, carboxyl activation domain; pVHL, von Hippel-Lindau protein.
CLINICAL APPLICATIONS
How does this inform our understanding of the pathophysiology of lung disease and can it provide the basis of novel therapies? Mice homozygous for a null mutation in the HIF-113 or HIF-1? genes14 die at mid gestation with vascular defects primarily involving the embryonic and extraembryonic circulation, respectively. In contrast, HIF-1+/- mice develop normally and are indistinguishable from wild type littermates. However, when exposed to 10% oxygen for up to 6 weeks, the HIF-1+/- mice demonstrate reduced susceptibility to pulmonary hypertension, polycythemia, and right ventricular hypertrophy relative to their wild type littermates.15 Morphometric analysis showed that the chronically hypoxic HIF-1+/- mice have fewer completely muscularised pulmonary arterioles and the degree of muscularisation in such vessels is reduced compared with HIF-1 wild type mice. Thus, HIF-1 appears to play a major role in mediating pulmonary vascular remodelling in chronic hypoxia, and therapeutic manoeuvres that inhibit HIF-1 activity in the lung may slow the progression of hypoxia induced pulmonary hypertension.
Likewise, in many human cancers including primary lung tumours, increased levels of HIF-1 expression are associated with resistance to tissue hypoxia and enhanced tumour angiogenesis.16 This abnormality correlates with a reduced sensitivity to radiation and chemotherapy and disease progression.17 Moreover, in vitro work has shown that depleting tumour cells of HIF-1 renders them more radioresponsive and thus provides an opportunity to enhance the therapeutic potential of conventional treatment.18 In addition, by targeting bioreductive pro-drugs to hypoxic tumour sites, the potential for normal tissue damage can be reduced.
One of the real surprises in this area of research, however, has been the recent demonstration of the importance of oxygen sensing pathways in the function of inflammatory cells. Hence, in contrast to the effects of hypoxia observed in many other cell types, oxygen deprivation causes a profound but reversible delay in the rate of constitutive apoptosis in human neutrophils.19 Moreover, removing the capacity of neutrophils to respond to hypoxia using a myeloid targeted HIF-1 knock-out results in a substantial fall in the glycolytic capacity of these cells and, as a consequence, a profound impairment of myeloid cell adhesion migration and bacterial killing.20 As persistent accumulation of primed and activated neutrophils is a cardinal feature of a number of lung diseases, these data predict that inhibition of the HIF-1 pathway may promote the resolution of granulocyte inflammation.
One final example of the importance of this pathway relates to the potential to prevent ischaemia-reperfusion injury in the transplanted lung. Hence, in the heart, ischaemic pre-conditioning, which reduces dramatically the degree of myocardial damage observed following subsequent coronary artery occlusion, has been shown to relate to HIF dependent upregulation of erythropoietin.21 Indeed, this protein has the capacity to prevent myocardial cell apoptosis, and erythropoietin infusion is effective in reducing infarct size and improving outcome in stroke.22 Application of these principles to lung protection is bound to follow.
Despite such striking advances in our knowledge of how cells sense oxygen, much remains to be defined. In particular, it is likely that a quite separate pathway underlies the ability of acute hypoxia to induce membrane depolarisation and L-type calcium channel activity in excitable cells. These events trigger the acute contractile response of pulmonary artery resistance vessels to hypoxia and the firing of carotid body glomus cells. The current contenders in this area are the mitochondrial NAD(P)H oxidases, which are postulated to produce a diffusible redox mediator in response to hypoxia.
CONCLUSIONS
These studies show that, while cells display widely differing tolerances and responses to hypoxia, oxygen sensing by dioxygenases and their consequent effects on HIF dependent transcriptional events appear to be a property that is shared by most, if not all, cells. This pathway now offers a new set of therapeutic targets for an array of lung diseases ranging from inflammatory lung disease through to pulmonary hypertension.
ACKNOWLEDGEMENTS
The work in the authors’ laboratories is sup-ported by the Wellcome Trust, British Heart Foundation, National Asthma Campaign, MRC, and British Lung Foundation. SRW holds an MRC Clinical Research Training Fellowship and KKS holds a BHF Clinical Fellowship.
REFERENCES
1. Siesjo BK. Cell damage in the brain: a speculative synthesis. J Cereb Blood Flow Metab 1981;1:155–85.
2. Lahiri S, Rozanov C, Roy A, et al. Regulation of oxygen sensing in peripheral arterial chemoreceptors. Int J Biochem Cell Biol 2001;33:755–74.
3. Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 1988;242:1412–5.
4. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene. Mol Cell Biol 1992;12:5447–54.
5. Wang GL, Jiang B-H, Rue EA, et al. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1992;92:5510–4.
6. Wenger RH, Gassmann M. Oxygen(es) and the hypoxia-inducible factor-1. Biol Chem 1997;378:609–16.
7. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 2000;88:1474–80.
8. Wenger RH. Cellular adaption to hypoxia: O2-sensing protein hydroxilases, hypoxia-inducible transcription factors, and O2-regulated gene expression. FASEB J 2002;16:1151–62.
9. Epstein ACR, Gleadle JM, McNeill LA, et al. C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 2001;107:43–54.
10. Jaakkola P, Mole DR, Tian Y-M, et al. Targeting of HIF- to the von Huppel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 2001;292:468–72.
11. Lando D, Peet DJ, Whelan DA, et al. Asparagine hydroxylation of the HIF transactivation domain a hypoxic switch. Science 2002;295:858–61.
12. Mahon PC, Hirota K, Semenza GL. FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev 2001;15:2675–86.
13. Ryan HE, Lo J, Johnson RS. HIF-1 alpha is required for solid tumor formation and embryonic vascularization. EMBO J 1998;17:3005–15.
14. Maltepe E, Schmidt JV, Baunoch D, et al. Abnormal angiogenesis and responses to glucose and oxygen deprivation in mice lacking the protein ARNT. Nature 1997;386:403–7.
15. Yu AY, Shimoda LA, Iyer NV, et al. Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1-alpha. J Clin Invest 1999;103:691–6.
16. Maxwell PH, Dachs GU, Gleadle JM, et al. Hypoxia-inducible factor-1 modulates gene expression in solid tumors and influences both angiogenesis and tumor growth. Proc Natl Acad Sci USA 1997;94:8104–9.
17. Aebersold DM, Burri P, Beer KT, et al. Expression of hypoxia-inducible factor-1alpha: a novel predictive and prognostic parameter in radiotherapy of oropharyngeal cancer. Cancer Res 2001;61:2911–6.
18. Unruh A, Ressel A, Mohamed HG, et al. The hypoxia-inducible factor-1 alpha is a negative factor for tumour therapy. Oncogene 2003;22:3213–20.
19. Mecklenburgh KI, Walmsley SR, Cowburn AS, et al. Involvement of a ferroprotein sensor in hypoxia-mediated inhibition of neutrophil apoptosis. Blood 2002;100:3008–11.
20. Cramer T, Yamanishi Y, Clausen BE, et al. HIF-1 is essential for myeloid cell-mediated inflammation. Cell 2003;112:645–57.
21. Cai Z, Manalo DJ, Wei G, et al. Hearts from rodents exposed to intermittent hypoxia or erythropoietin are protected against ischaemia-reperfusion injury. Circulation 2003;108:79–85.
22. Ehreneich H, Hasselblatt M, Dembowski C, et al. Erythropoietin therapy for acute stroke is both safe and beneficial. Mol Med 2002;8:495–505.(S R Walmsley, K K K Shear)