Publications by authors named "Christopher W Pugh"

58 Publications

Accelerometer-measured physical activity and functional behaviours among people on dialysis.

Clin Kidney J 2021 Mar 31;14(3):950-958. Epub 2020 Aug 31.

Oxford Kidney Unit, Oxford University Hospitals NHS Foundation Trust, Oxford, UK.

Background: The feasibility of wrist-worn accelerometers, and the patterns and determinants of physical activity, among people on dialysis are uncertain.

Methods: People on maintenance dialysis were fitted with a wrist-worn AxivityAX3 accelerometer. Subsets also wore a 14-day electrocardiograph patch (ZioPatchXT) and wearable cameras. Age-, sex- and season-matched UK Biobank control groups were derived for comparison.

Results: Median (interquartile range) accelerometer wear time for the 101 recruits was 12.5 (10.4-13.5) days, of which 73 participants (mean age 66.5 years) had excellent wear on both dialysis and non-dialysis days. Mean (standard error) overall physical activity levels were 15.5 (0.7) milligravity units (m), 14.8 (0.7) m on dialysis days versus 16.2 (0.8) m on non-dialysis days. This compared with 28.1 (0.5) m for apparently healthy controls, 23.4 (0.4) m for controls with prior cardiovascular disease (CVD) and/or diabetes mellitus and 22.9 (0.6) m for heart failure controls. Each day, we estimated that those on dialysis spent an average of about 1 hour (h/day) walking, 0.6 h/day engaging in moderate-intensity activity, 0.7 h/day on light tasks, 13.2 h/day sedentary and 8.6 h/day asleep. Older age and self-reported leg weakness were associated with decreased levels of physical activity, but the presence of prior CVD, arrhythmias and listing for transplantation were not.

Conclusions: Wrist-worn accelerometers are an acceptable and reliable method to measure physical activity in people on dialysis and may also be used to estimate functional behaviours. Among people on dialysis, who are broadly half as active as general population controls, age and leg weakness appear to be more important determinants of low activity levels than CVD.
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http://dx.doi.org/10.1093/ckj/sfaa045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986362PMC
March 2021

Recent advances in the biology of tumour hypoxia with relevance to diagnostic practice and tissue-based research.

J Pathol 2020 04 18;250(5):593-611. Epub 2020 Mar 18.

Nuffield Department of Medicine, University of Oxford, Oxford, UK.

In this review article, we examine the importance of low levels of oxygen (hypoxia) in cancer biology. We provide a brief description of how mammalian cells sense oxygen. The hypoxia-inducible factor (HIF) pathway is currently the best characterised oxygen-sensing system, but recent work has revealed that mammals also use an oxygen-sensing system found in plants to regulate the abundance of some proteins and peptides with an amino-terminal cysteine residue. We discuss how the HIF pathway is affected during the growth of solid tumours, which develop in microenvironments with gradients of oxygen availability. We then introduce the concept of 'pseudohypoxia', a state of constitutive, oxygen-independent HIF system activation that occurs due to oncogenic stimulation in a number of specific tumour types that are of immediate relevance to diagnostic histopathologists. We provide an overview of the different methods of quantifying tumour hypoxia, emphasising the importance of pre-analytic factors in interpreting the results of tissue-based studies. Finally, we review recent approaches to targeting hypoxia/HIF system activation for therapeutic benefit, the application of which may require knowledge of which hypoxia signalling components are being utilised by a given tumour. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
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http://dx.doi.org/10.1002/path.5402DOI Listing
April 2020

Marked and rapid effects of pharmacological HIF-2α antagonism on hypoxic ventilatory control.

J Clin Invest 2020 05;130(5):2237-2251

Target Discovery Institute and.

Hypoxia-inducible factor (HIF) is strikingly upregulated in many types of cancer, and there is great interest in applying inhibitors of HIF as anticancer therapeutics. The most advanced of these are small molecules that target the HIF-2 isoform through binding the PAS-B domain of HIF-2α. These molecules are undergoing clinical trials with promising results in renal and other cancers where HIF-2 is considered to be driving growth. Nevertheless, a central question remains as to whether such inhibitors affect physiological responses to hypoxia at relevant doses. Here, we show that pharmacological HIF-2α inhibition with PT2385, at doses similar to those reported to inhibit tumor growth, rapidly impaired ventilatory responses to hypoxia, abrogating both ventilatory acclimatization and carotid body cell proliferative responses to sustained hypoxia. Mice carrying a HIF-2α PAS-B S305M mutation that disrupts PT2385 binding, but not dimerization with HIF-1β, did not respond to PT2385, indicating that these effects are on-target. Furthermore, the finding of a hypomorphic ventilatory phenotype in untreated HIF-2α S305M mutant mice suggests a function for the HIF-2α PAS-B domain beyond heterodimerization with HIF-1β. Although PT2385 was well tolerated, the findings indicate the need for caution in patients who are dependent on hypoxic ventilatory drive.
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http://dx.doi.org/10.1172/JCI133194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190921PMC
May 2020

Structure-Activity Relationship and Crystallographic Studies on 4-Hydroxypyrimidine HIF Prolyl Hydroxylase Domain Inhibitors.

ChemMedChem 2020 02 3;15(3):270-273. Epub 2019 Dec 3.

Department of Chemistry, University of Oxford Chemistry Research Laboratory, 12 Mansfield Road, Oxford, OX1 3TA, UK.

The 2-oxoglutarate-dependent hypoxia inducible factor prolyl hydroxylases (PHDs) are targets for treatment of a variety of diseases including anaemia. One PHD inhibitor is approved for use for the treatment of renal anaemia and others are in late stage clinical trials. The number of reported templates for PHD inhibition is limited. We report structure-activity relationship and crystallographic studies on a promising class of 4-hydroxypyrimidine-containing PHD inhibitors.
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http://dx.doi.org/10.1002/cmdc.201900557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496690PMC
February 2020

PHD2 inactivation in Type I cells drives HIF-2α-dependent multilineage hyperplasia and the formation of paraganglioma-like carotid bodies.

J Physiol 2018 Jun 19. Epub 2018 Jun 19.

Target Discovery Institute, University of Oxford, Oxford, UK.

Key Points: The carotid body is a peripheral arterial chemoreceptor that regulates ventilation in response to both acute and sustained hypoxia. Type I cells in this organ respond to low oxygen both acutely by depolarization and dense core vesicle secretion and, over the longer term, via cellular proliferation and enhanced ventilatory responses. Using lineage analysis, the present study shows that the Type I cell lineage itself proliferates and expands in response to sustained hypoxia. Inactivation of HIF-2α in Type I cells impairs the ventilatory, proliferative and cell intrinsic (dense core vesicle) responses to hypoxia. Inactivation of PHD2 in Type I cells induces multilineage hyperplasia and ultrastructural changes in dense core vesicles to form paraganglioma-like carotid bodies. These changes, similar to those observed in hypoxia, are dependent on HIF-2α. Taken together, these findings demonstrate a key role for the PHD2-HIF-2α couple in Type I cells with respect to the oxygen sensing functions of the carotid body.

Abstract: The carotid body is a peripheral chemoreceptor that plays a central role in mammalian oxygen homeostasis. In response to sustained hypoxia, it manifests a rapid cellular proliferation and an associated increase in responsiveness to hypoxia. Understanding the cellular and molecular mechanisms underlying these processes is of interest both to specialized chemoreceptive functions of that organ and, potentially, to the general physiology and pathophysiology of cellular hypoxia. We have combined cell lineage tracing technology and conditionally inactivated alleles in recombinant mice to examine the role of components of the HIF hydroxylase pathway in specific cell types within the carotid body. We show that exposure to sustained hypoxia (10% oxygen) drives rapid expansion of the Type I, tyrosine hydroxylase expressing cell lineage, with little transdifferentiation to (or from) that lineage. Inactivation of a specific HIF isoform, HIF-2α, in the Type I cells was associated with a greatly reduced proliferation of Type I cells and hypoxic ventilatory responses, with ultrastructural evidence of an abnormality in the action of hypoxia on dense core secretory vesicles. We also show that inactivation of the principal HIF prolyl hydroxylase PHD2 within the Type I cell lineage is sufficient to cause multilineage expansion of the carotid body, with characteristics resembling paragangliomas. These morphological changes were dependent on the integrity of HIF-2α. These findings implicate specific components of the HIF hydroxylase pathway (PHD2 and HIF-2α) within Type I cells of the carotid body with respect to the oxygen sensing and adaptive functions of that organ.
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http://dx.doi.org/10.1113/JP275996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138294PMC
June 2018

Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials.

Chem Sci 2017 Nov 11;8(11):7651-7668. Epub 2017 Sep 11.

Chemistry Research Laboratory , Department of Chemistry , University of Oxford , Oxford OX1 3TA , UK . Email:

Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities representatives of other human 2OG oxygenase subfamilies. The 'clinical' PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIF-α oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell-based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N- and C-terminal ODDs; the latter differences correlate with the biophysical observations.
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http://dx.doi.org/10.1039/c7sc02103hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5802278PMC
November 2017

Fumarate Hydratase Deletion in Pancreatic β Cells Leads to Progressive Diabetes.

Cell Rep 2017 Sep;20(13):3135-3148

Radcliffe Department of Medicine, OCDEM, Churchill Hospital, University of Oxford, Oxford OX3 7LE, UK; Department of Physiology, Institute of Neuroscience and Physiology, University of Göteborg, 405 30 Göteborg, Sweden. Electronic address:

We explored the role of the Krebs cycle enzyme fumarate hydratase (FH) in glucose-stimulated insulin secretion (GSIS). Mice lacking Fh1 in pancreatic β cells (Fh1βKO mice) appear normal for 6-8 weeks but then develop progressive glucose intolerance and diabetes. Glucose tolerance is rescued by expression of mitochondrial or cytosolic FH but not by deletion of Hif1α or Nrf2. Progressive hyperglycemia in Fh1βKO mice led to dysregulated metabolism in β cells, a decrease in glucose-induced ATP production, electrical activity, cytoplasmic [Ca] elevation, and GSIS. Fh1 loss resulted in elevated intracellular fumarate, promoting succination of critical cysteines in GAPDH, GMPR, and PARK 7/DJ-1 and cytoplasmic acidification. Intracellular fumarate levels were increased in islets exposed to high glucose and in islets from human donors with type 2 diabetes (T2D). The impaired GSIS in islets from diabetic Fh1βKO mice was ameliorated after culture under normoglycemic conditions. These studies highlight the role of FH and dysregulated mitochondrial metabolism in T2D.
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http://dx.doi.org/10.1016/j.celrep.2017.08.093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5637167PMC
September 2017

Hypoxia and HIF pathway in cancer and the placenta.

Placenta 2017 Aug 16;56:8-13. Epub 2017 Mar 16.

Nuffield Department of Medicine, University of Oxford, United Kingdom. Electronic address:

In this review we note that the placenta and cancer both develop in microenvironments in which there are gradients of oxygen availability. Whilst fundamentally different in that placental development is organised and physiological whilst cancer is chaotic and pathological, there are similarities in their respective capacities to proliferate, invade adjacent tissues, generate a blood supply and avoid rejection by the immune system. We provide a brief description of the hypoxia-inducible factor (HIF) pathway and indicate the ways by which HIF activity can be regulated to achieve oxygen homeostasis. We then exemplify the potential role of the HIF pathway in contributing to those functions shared between the placenta and cancer through effects on cellular proliferation, cell death, angiogenesis, blood vessel co-option, vascular mimicry, cell adhesion molecules, secretion of matrix metalloproteinases, antigen presentation mechanisms and immunosuppressive factors. We advocate future studies to explore these similarities and differences in the hope of improving our understanding of both systems and hence treatments of placental disorders and cancer.
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http://dx.doi.org/10.1016/j.placenta.2017.03.010DOI Listing
August 2017

New horizons in hypoxia signaling pathways.

Exp Cell Res 2017 07 15;356(2):116-121. Epub 2017 Mar 15.

Target Discovery Institute, University of Oxford, OX3 7FZ, UK; Francis Crick Institute, Midland Road, London NW1 1AT, UK. Electronic address:

Investigation into the regulation of the erythropoietin gene by oxygen led to the discovery of a process of direct oxygen sensing that transduces many cellular and systemic responses to hypoxia. The oxygen-sensitive signal is generated through the catalytic action of a series of 2-oxoglutarate-dependent oxygenases that regulate the transcription factor hypoxia-inducible factor (HIF) by the post-translational hydroxylation of specific amino acid residues. Here we review the implications of the unforeseen complexity of the HIF transcriptional cascade for the physiology and pathophysiology of hypoxia, and consider the origins of post-translational hydroxylation as a signaling process.
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http://dx.doi.org/10.1016/j.yexcr.2017.03.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5653532PMC
July 2017

Structural basis for oxygen degradation domain selectivity of the HIF prolyl hydroxylases.

Nat Commun 2016 08 26;7:12673. Epub 2016 Aug 26.

Chemistry Research Laboratory, Department of Chemistry, Oxford Centre for Integrative Systems Biology, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK.

The response to hypoxia in animals involves the expression of multiple genes regulated by the αβ-hypoxia-inducible transcription factors (HIFs). The hypoxia-sensing mechanism involves oxygen limited hydroxylation of prolyl residues in the N- and C-terminal oxygen-dependent degradation domains (NODD and CODD) of HIFα isoforms, as catalysed by prolyl hydroxylases (PHD 1-3). Prolyl hydroxylation promotes binding of HIFα to the von Hippel-Lindau protein (VHL)-elongin B/C complex, thus signalling for proteosomal degradation of HIFα. We reveal that certain PHD2 variants linked to familial erythrocytosis and cancer are highly selective for CODD or NODD. Crystalline and solution state studies coupled to kinetic and cellular analyses reveal how wild-type and variant PHDs achieve ODD selectivity via different dynamic interactions involving loop and C-terminal regions. The results inform on how HIF target gene selectivity is achieved and will be of use in developing selective PHD inhibitors.
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http://dx.doi.org/10.1038/ncomms12673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007464PMC
August 2016

Tuning the Transcriptional Response to Hypoxia by Inhibiting Hypoxia-inducible Factor (HIF) Prolyl and Asparaginyl Hydroxylases.

J Biol Chem 2016 09 8;291(39):20661-73. Epub 2016 Aug 8.

From the Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA,

The hypoxia-inducible factor (HIF) system orchestrates cellular responses to hypoxia in animals. HIF is an α/β-heterodimeric transcription factor that regulates the expression of hundreds of genes in a tissue context-dependent manner. The major hypoxia-sensing component of the HIF system involves oxygen-dependent catalysis by the HIF hydroxylases; in humans there are three HIF prolyl hydroxylases (PHD1-3) and an asparaginyl hydroxylase (factor-inhibiting HIF (FIH)). PHD catalysis regulates HIFα levels, and FIH catalysis regulates HIF activity. How differences in HIFα hydroxylation status relate to variations in the induction of specific HIF target gene transcription is unknown. We report studies using small molecule HIF hydroxylase inhibitors that investigate the extent to which HIF target gene expression is induced by PHD or FIH inhibition. The results reveal substantial differences in the role of prolyl and asparaginyl hydroxylation in regulating hypoxia-responsive genes in cells. PHD inhibitors with different structural scaffolds behave similarly. Under the tested conditions, a broad-spectrum 2-oxoglutarate dioxygenase inhibitor is a better mimic of the overall transcriptional response to hypoxia than the selective PHD inhibitors, consistent with an important role for FIH in the hypoxic transcriptional response. Indeed, combined application of selective PHD and FIH inhibitors resulted in the transcriptional induction of a subset of genes not fully responsive to PHD inhibition alone. Thus, for the therapeutic regulation of HIF target genes, it is important to consider both PHD and FIH activity, and in the case of some sets of target genes, simultaneous inhibition of the PHDs and FIH catalysis may be preferable.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034057PMC
http://dx.doi.org/10.1074/jbc.M116.749291DOI Listing
September 2016

Modulation of the Hypoxic Response.

Adv Exp Med Biol 2016 ;903:259-71

Department of Renal Medicine, University of Oxford, Oxford, UK.

Hypoxia stimulates a variety of adaptive responses, many mediated via the hypoxia inducible factors (HIF) family of transcriptional complexes. The balance of HIF-1, -2 and -3 controls a variety of genes, directly up-regulating transcription of genes involved in erythropoiesis, angiogenesis, vasomotor tone, metabolic pathways and processes related to cell multiplication and survival, and indirectly reducing the transcription of genes with other effects. HIF transcription factors are heterodimers consisting of an oxygen-regulated alpha chain bound to the constitutive aryl hydrocarbon receptor nuclear translocator. Under circumstances where oxygen is abundant the activity of the alpha chain is blocked by the actions of members of a family of oxygen-, iron- and oxoglutarate-dependent dioxygenase enzymes. Hydroxylation of two critical prolyl residues by the HIF prolyl hydroxylases (PHD1-3) leads to recognition by the von Hippel-Lindau E3 ubiquitin ligase complex, polyubiquitylation of the alpha chain and its consequent destruction by the proteasome. Hydroxylation of an asparaginyl residue by Factor Inhibiting HIF prevents any surviving HIF alpha chains from recruiting p300-CBP proteins, important for maximal transcriptional activation. Under conditions of acute hypoxia enzyme activity is suppressed, the HIF alpha chains are allowed to exist in their active form and target gene transcription is enhanced. In sustained hypoxia, adaptive responses mediated by the HIF pathway reduce oxygen demand and increase oxygen supply and thus ultimately down-regulate the pathway. However, a number of other processes also modulate HIF signalling and the balance between HIF-1 and HIF-2 actions. These include the generation of antisense HIF-1 and micro RNAs, up-regulation of HIF-3 alpha, antagonism of the HIF-p300 interaction by CITED2, increased PHD2 and PHD3 levels and effects on the pool of ankyrins within the cell which compete with HIF for the action of FIH. Additionally, effects on intermediary metabolism, reactive oxygen species, iron availability, nitric oxide levels and redox status within the cell may modulate HIF activity. Together, these effects lead to a reduction in the magnitude of the HIF response even if oxygenation is not restored and are predicted to alter the responsiveness of the system when oxygenation is restored.
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http://dx.doi.org/10.1007/978-1-4899-7678-9_18DOI Listing
June 2017

Regulation of ventilatory sensitivity and carotid body proliferation in hypoxia by the PHD2/HIF-2 pathway.

J Physiol 2016 Mar 6;594(5):1179-95. Epub 2015 Oct 6.

Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford, OX3 7BN, UK.

Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia-inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF-1α and HIF-2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen-treated Phd2(f/f);Rosa26(+/CreERT2) mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min(-1) g(-1) in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF-2α, but not HIF-1α. Furthermore, inducible inactivation of HIF-2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min(-1) g(-1) in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine-positive cells mm(-2) in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF-2α enzyme-substrate couple in modulating ventilatory sensitivity to hypoxia.
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http://dx.doi.org/10.1113/JP271050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771794PMC
March 2016

Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

PLoS One 2015 6;10(7):e0132004. Epub 2015 Jul 6.

Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom.

As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132004PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492579PMC
April 2016

The relationship between symptoms and blood pressure during maintenance hemodialysis.

Hemodial Int 2015 Oct 7;19(4):543-52. Epub 2015 May 7.

Medical Statistics, Centre for Statistics in Medicine, Oxford, UK.

Intradialytic hypotension (IDH) is a detrimental complication of maintenance hemodialysis, but how it is defined and reported varies widely in the literature. European Best Practice Guideline and Kidney Disease Outcomes Quality Initiative guidelines require symptoms and a mitigating intervention to fulfill the diagnosis, but morbidity and mortality outcomes are largely based on blood pressure alone. Furthermore, little is known about the incidence of asymptomatic hypotension, which may be an important cause of hypoperfusion injury and impaired outcome. Seventy-seven patients were studied over 456 dialysis sessions. Blood pressure was measured at 15-minute intervals throughout the session and compared with post-dialysis symptom questionnaire results using mixed modeling to adjust for repeated measures in the same patient. The frequency of asymptomatic hypotension was estimated by logistic regression using a variety of commonly cited blood pressure metrics that describe IDH. In 113 sessions (25%) where symptoms were recorded on the questionnaire, these appear not to have been reported to dialysis staff. When symptoms were reported (293 sessions [64%]), an intervention invariably followed. Dizziness and cramp were strongly associated with changes in systolic blood pressure (SBP), but not diastolic blood pressure. Nausea occurred more frequently in younger patients but was not associated with falls in blood pressure. Thresholds that maximized the probability of an intervention rather than a session remaining asymptomatic were SBP <100 mmHg or a 20% reduction in SBP from baseline. The probability of SBP falling to <100 mmHg in an asymptomatic session was 0.23. Symptoms are frequently not reported by patients who are hypotensive during hemodialysis, which leads to an underestimation of IDH if symptom-based definitions are used. A revised definition of IDH excluding patient-reported symptoms would be in line with literature reporting morbidity and mortality outcomes and include sessions in which potentially detrimental asymptomatic hypotension occurs.
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http://dx.doi.org/10.1111/hdi.12306DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682453PMC
October 2015

Hypoxia-inducible factor 2α regulates key neutrophil functions in humans, mice, and zebrafish.

Blood 2014 Jan 6;123(3):366-76. Epub 2013 Nov 6.

Department of Infection and Immunity and.

Neutrophil lifespan and function are regulated by hypoxia via components of the hypoxia inducible factor (HIF)/von Hippel Lindau/hydroxylase pathway, including specific roles for HIF-1α and prolyl hydroxylase-3. HIF-2α has both distinct and overlapping biological roles with HIF-1α and has not previously been studied in the context of neutrophil biology. We investigated the role of HIF-2α in regulating key neutrophil functions. Human and murine peripheral blood neutrophils expressed HIF-2α, with expression up-regulated by acute and chronic inflammatory stimuli and in disease-associated inflammatory neutrophil. HIF2A gain-of-function mutations resulted in a reduction in neutrophil apoptosis both ex vivo, through the study of patient cells, and in vivo in a zebrafish tail injury model. In contrast, HIF-2α-deficient murine inflammatory neutrophils displayed increased sensitivity to nitrosative stress induced apoptosis ex vivo and increased neutrophil apoptosis in vivo, resulting in a reduction in neutrophilic inflammation and reduced tissue injury. Expression of HIF-2α was temporally dissociated from HIF-1α in vivo and predominated in the resolution phase of inflammation. These data support a critical and selective role for HIF-2α in persistence of neutrophilic inflammation and provide a platform to dissect the therapeutic utility of targeting HIF-2α in chronic inflammatory diseases.
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http://dx.doi.org/10.1182/blood-2013-05-500207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894493PMC
January 2014

Carotid body hyperplasia and enhanced ventilatory responses to hypoxia in mice with heterozygous deficiency of PHD2.

J Physiol 2013 Jul 20;591(14):3565-77. Epub 2013 May 20.

Wellcome Trust Centre for Human Genetics, Roosevelt Drive, University of Oxford, Oxford OX3 7BN, UK.

Oxygen-dependent prolyl hydroxylation of hypoxia-inducible factor (HIF) by a set of closely related prolyl hydroxylase domain enzymes (PHD1, 2 and 3) regulates a range of transcriptional responses to hypoxia. This raises important questions about the role of these oxygen-sensing enzymes in integrative physiology. We investigated the effect of both genetic deficiency and pharmacological inhibition on the change in ventilation in response to acute hypoxic stimulation in mice. Mice exposed to chronic hypoxia for 7 days manifest an exaggerated hypoxic ventilatory response (HVR) (10.8 ± 0.3 versus 4.1 ± 0.7 ml min(-1) g(-1) in controls; P < 0.01). HVR was similarly exaggerated in PHD2(+/-) animals compared to littermate controls (8.4 ± 0.7 versus 5.0 ± 0.8 ml min(-1) g(-1); P < 0.01). Carotid body volume increased (0.0025 ± 0.00017 in PHD2(+/-) animals versus 0.0015 ± 0.00019 mm(3) in controls; P < 0.01). In contrast, HVR in PHD1(-/-) and PHD3(-/-) mice was similar to littermate controls. Acute exposure to a small molecule PHD inhibitor (PHI) (2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetic acid) did not mimic the ventilatory response to hypoxia. Further, 7 day administration of the PHI induced only modest increases in HVR and carotid body cell proliferation, despite marked stimulation of erythropoiesis. This was in contrast with chronic hypoxia, which elicited both exaggerated HVR and cellular proliferation. The findings demonstrate that PHD enzymes modulate ventilatory sensitivity to hypoxia and identify PHD2 as the most important enzyme in this response. They also reveal differences between genetic inactivation of PHDs, responses to hypoxia and responses to a pharmacological inhibitor, demonstrating the need for caution in predicting the effects of therapeutic modulation of the HIF hydroxylase system on different physiological responses.
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http://dx.doi.org/10.1113/jphysiol.2012.247254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731614PMC
July 2013

Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases.

ACS Chem Biol 2013 Jul 12;8(7):1488-96. Epub 2013 Jun 12.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford , Mansfield Road, Oxford, OX1 3TA, United Kingdom.

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.
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http://dx.doi.org/10.1021/cb400088qDOI Listing
July 2013

Roles of individual prolyl-4-hydroxylase isoforms in the first 24 hours following transient focal cerebral ischaemia: insights from genetically modified mice.

J Physiol 2012 Aug 21;590(16):4079-91. Epub 2012 May 21.

Nuffield Department of Medicine, University of Oxford, John Radcliffe Hospital, Headley Way, Headington, Oxford, OX3 9DU, UK.

This study investigated the function of each of the hypoxia inducible factor (HIF) prolyl-4-hydroxylase enzymes (PHD1–3) in the first 24 h following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed. Male, 8- to 12-week old PHD1−/−, PHD2+/− and PHD3−/− mice and their wild-type (WT) littermate were subjected to 45 min of middle cerebral artery occlusion (MCAO). During the experiments, regional cerebral blood flow (rCBF) was recorded by laser Doppler flowmetry. Behaviour was assessed at both 2 h and 24 h after reperfusion with a common neuroscore. Infarct volumes, blood–brain barrier (BBB) disruption, cerebral vascular density, apoptosis, reactive oxygen species (ROS), HIF1α, and glycogen levels were then determined using histological and immunohistochemical techniques. When compared to their WT littermates, PHD2+/− mice had significantly increased cerebral microvascular density and more effective restoration of CBF upon reperfusion. PHD2+/− mice showed significantly better functional outcomes and higher activity rates at both 2 h and 24 h after MCAO, associated with significant fewer apoptotic cells in the penumbra and less BBB disruption; PHD3−/− mice had impaired rCBF upon early reperfusion but comparable functional outcomes; PHD1−/− mice did not show any significant changes following the MCAO. Production of ROS, HIF1α staining and glycogen content in the brain were not different in any comparison. Life-long genetic inhibition of PHD enzymes produces different effects on outcome in the first 24 h after transient cerebral ischaemia. These need to be considered in optimizing therapeutic effects of PHD inhibitors, particularly when isoform specific inhibitors become available.
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http://dx.doi.org/10.1113/jphysiol.2012.232884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476649PMC
August 2012

The impact of renin-angiotensin-aldosterone system inhibitors on Type 1 and Type 2 diabetic patients with and without early diabetic nephropathy.

Kidney Int 2012 Apr 21;81(7):674-83. Epub 2011 Dec 21.

Department of Primary Care Health Sciences, University of Oxford, Oxford, UK.

Renin-angiotensin-aldosterone system inhibitors prevent the progression of kidney disease in patients with diabetic nephropathy, and we studied how that benefit varies by the type of diabetes and baseline urinary albumin. We pooled data from 49 randomized controlled trials in a meta-analysis using the ratio of endpoint urinary albumin levels in those treated compared to those untreated with renin-angiotensin-aldosterone system inhibitors in both fixed- and random-effects models. The urinary albumin excretion for treated microalbuminuric patients with Type 1 diabetes was on average 60% lower at the end of the trial compared with patients not treated with renin-angiotensin-aldosterone system inhibitors using the fixed-effects model and 67% lower using the random-effects model. There was no significant effect of treatment in patients with normal albumin excretion. For normoalbuminuric patients with Type 2 diabetes, urinary albumin excretion was on average 12% lower after treatment using the fixed-effects model compared to 21% lower using the random-effects model. For microalbuminuric patients, urinary albumin excretion was on average 23% lower using the fixed-effects model and 27% lower using the random-effects model. Thus, renin-angiotensin-aldosterone system inhibition reduced urinary albumin excretion for Type 1 diabetic patients with micro-, but not those with normoalbuminuria. Treatment reduced urinary albumin excretion for Type 2 diabetic patients with and without microalbuminuria.
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http://dx.doi.org/10.1038/ki.2011.413DOI Listing
April 2012

Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status.

J Pathol 2011 Sep 1;225(1):4-11. Epub 2011 Jun 1.

Molecular and Population Genetics Laboratory, Wellcome Trust Centre for Human Genetics, Oxford, UK.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.
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http://dx.doi.org/10.1002/path.2932DOI Listing
September 2011

High-resolution genome-wide mapping of HIF-binding sites by ChIP-seq.

Blood 2011 Jun 29;117(23):e207-17. Epub 2011 Mar 29.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford, UK.

Hypoxia-inducible factor (HIF) regulates the major transcriptional cascade central to the response of all mammalian cells to alterations in oxygen tension. Expression arrays indicate that many hundreds of genes are regulated by this pathway, controlling diverse processes that in turn orchestrate both oxygen delivery and utilization. However, the extent to which HIF exerts direct versus indirect control over gene expression together with the factors dictating the range of HIF-regulated genes remains unclear. Using chromatin immunoprecipitation linked to high throughput sequencing, we identify HIF-binding sites across the genome, independently of gene architecture. Using gene set enrichment analysis, we demonstrate robust associations with the regulation of gene expression by HIF, indicating that these sites operate over long genomic intervals. Analysis of HIF-binding motifs demonstrates sequence preferences outside of the core RCGTG-binding motif but does not reveal any additional absolute sequence requirements. Across the entire genome, only a small proportion of these potential binding sites are bound by HIF, although occupancy of potential sites was enhanced approximately 20-fold at normoxic DNAse1 hypersensitivity sites (irrespective of distance from promoters), suggesting that epigenetic regulation of chromatin may have an important role in defining the response to hypoxia.
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http://dx.doi.org/10.1182/blood-2010-10-314427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3374576PMC
June 2011

Cardiopulmonary function in two human disorders of the hypoxia-inducible factor (HIF) pathway: von Hippel-Lindau disease and HIF-2alpha gain-of-function mutation.

FASEB J 2011 Jun 9;25(6):2001-11. Epub 2011 Mar 9.

Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Rd., Oxford, OX1 3PT, UK.

The hypoxia-inducible factors (HIFs; isoforms HIF-1α, HIF-2α, HIF-3α) mediate many responses to hypoxia. Their regulation is principally by oxygen-dependent degradation, which is initiated by hydroxylation of specific proline residues followed by binding of von Hippel-Lindau (VHL) protein. Chuvash polycythemia is a disorder with elevated HIF. It arises through germline homozygosity for hypomorphic VHL alleles and has a phenotype of hematological, cardiopulmonary, and metabolic abnormalities. This study explores the phenotype of two other HIF pathway diseases: classic VHL disease and HIF-2α gain-of-function mutation. No cardiopulmonary abnormalities were detected in classic VHL disease. HIF-2α gain-of-function mutations were associated with pulmonary hypertension, increased cardiac output, increased heart rate, and increased pulmonary ventilation relative to metabolism. Comparison of the HIF-2α gain-of-function responses with data from studies of Chuvash polycythemia suggested that other aspects of the Chuvash phenotype were diminished or absent. In classic VHL disease, patients are germline heterozygous for mutations in VHL, and the present results suggest that a single wild-type allele for VHL is sufficient to maintain normal cardiopulmonary function. The HIF-2α gain-of-function phenotype may be more limited than the Chuvash phenotype either because HIF-1α is not elevated in the former condition, or because other HIF-independent functions of VHL are perturbed in Chuvash polycythemia.
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http://dx.doi.org/10.1096/fj.10-177378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3159892PMC
June 2011

Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors.

J Biol Chem 2011 Apr 18;286(15):13041-51. Epub 2011 Feb 18.

Nuffield Department of Clinical Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.

Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.
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http://dx.doi.org/10.1074/jbc.M110.211110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075650PMC
April 2011

Prolyl hydroxylase 3 (PHD3) is essential for hypoxic regulation of neutrophilic inflammation in humans and mice.

J Clin Invest 2011 Mar 7;121(3):1053-63. Epub 2011 Feb 7.

Academic Unit of Respiratory Medicine, The Medical School, University of Sheffield, Sheffield, United Kingdom.

The regulation of neutrophil lifespan by induction of apoptosis is critical for maintaining an effective host response and preventing excessive inflammation. The hypoxia-inducible factor (HIF) oxygen-sensing pathway has a major effect on the susceptibility of neutrophils to apoptosis, with a marked delay in cell death observed under hypoxic conditions. HIF expression and transcriptional activity are regulated by the oxygen-sensitive prolyl hydroxylases (PHD1-3), but the role of PHDs in neutrophil survival is unclear. We examined PHD expression in human neutrophils and found that PHD3 was strongly induced in response to hypoxia and inflammatory stimuli in vitro and in vivo. Using neutrophils from mice deficient in Phd3, we demonstrated a unique role for Phd3 in prolonging neutrophil survival during hypoxia, distinct from other hypoxia-associated changes in neutrophil function and metabolic activity. Moreover, this selective defect in neutrophil survival occurred in the presence of preserved HIF transcriptional activity but was associated with upregulation of the proapoptotic mediator Siva1 and loss of its binding target Bcl-xL. In vivo, using an acute lung injury model, we observed increased levels of neutrophil apoptosis and clearance in Phd3-deficient mice compared with WT controls. We also observed reduced neutrophilic inflammation in an acute mouse model of colitis. These data support what we believe to be a novel function for PHD3 in regulating neutrophil survival in hypoxia and may enable the development of new therapeutics for inflammatory disease.
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http://dx.doi.org/10.1172/JCI43273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3049381PMC
March 2011

Expression profiling in progressive stages of fumarate-hydratase deficiency: the contribution of metabolic changes to tumorigenesis.

Cancer Res 2010 Nov 26;70(22):9153-65. Epub 2010 Oct 26.

Department of Cardiovascular Medicine, University of Oxford, John Radcliffe Hospital, Headington, United Kingdom.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by mutations in the Krebs cycle enzyme fumarate hydratase (FH). It has been proposed that "pseudohypoxic" stabilization of hypoxia-inducible factor-α (HIF-α) by fumarate accumulation contributes to tumorigenesis in HLRCC. We hypothesized that an additional direct consequence of FH deficiency is the establishment of a biosynthetic milieu. To investigate this hypothesis, we isolated primary mouse embryonic fibroblast (MEF) lines from Fh1-deficient mice. As predicted, these MEFs upregulated Hif-1α and HIF target genes directly as a result of FH deficiency. In addition, detailed metabolic assessment of these MEFs confirmed their dependence on glycolysis, and an elevated rate of lactate efflux, associated with the upregulation of glycolytic enzymes known to be associated with tumorigenesis. Correspondingly, Fh1-deficient benign murine renal cysts and an advanced human HLRCC-related renal cell carcinoma manifested a prominent and progressive increase in the expression of HIF-α target genes and in genes known to be relevant to tumorigenesis and metastasis. In accord with our hypothesis, in a variety of different FH-deficient tissues, including a novel murine model of Fh1-deficient smooth muscle, we show a striking and progressive upregulation of a tumorigenic metabolic profile, as manifested by increased PKM2 and LDHA protein. Based on the models assessed herein, we infer that that FH deficiency compels cells to adopt an early, reversible, and progressive protumorigenic metabolic milieu that is reminiscent of that driving the Warburg effect. Targets identified in these novel and diverse FH-deficient models represent excellent potential candidates for further mechanistic investigation and therapeutic metabolic manipulation in tumors.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-1949DOI Listing
November 2010

Regulation of type II transmembrane serine proteinase TMPRSS6 by hypoxia-inducible factors: new link between hypoxia signaling and iron homeostasis.

J Biol Chem 2011 Feb 21;286(6):4090-7. Epub 2010 Oct 21.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom.

Hepcidin is a liver-derived hormone with a key role in iron homeostasis. In addition to iron, it is regulated by inflammation and hypoxia, although mechanisms of hypoxic regulation remain unclear. In hepatocytes, hepcidin is induced by bone morphogenetic proteins (BMPs) through a receptor complex requiring hemojuvelin (HJV) as a co-receptor. Type II transmembrane serine proteinase (TMPRSS6) antagonizes hepcidin induction by BMPs by cleaving HJV from the cell membrane. Inactivating mutations in TMPRSS6 lead to elevated hepcidin levels and consequent iron deficiency anemia. Here we demonstrate that TMPRSS6 is up-regulated in hepatic cell lines by hypoxia and by other activators of hypoxia-inducible factor (HIF). We show that TMPRSS6 expression is regulated by both HIF-1α and HIF-2α. This HIF-dependent up-regulation of TMPRSS6 increases membrane HJV shedding and decreases hepcidin promoter responsiveness to BMP signaling in hepatocytes. Our results reveal a potential role for TMPRSS6 in hepcidin regulation by hypoxia and provide a new molecular link between oxygen sensing and iron homeostasis.
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http://dx.doi.org/10.1074/jbc.M110.173096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3039360PMC
February 2011

Dysregulation of hypoxia pathways in fumarate hydratase-deficient cells is independent of defective mitochondrial metabolism.

Hum Mol Genet 2010 Oct 21;19(19):3844-51. Epub 2010 Jul 21.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK.

Mutations in the gene encoding the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer in affected individuals. FH-associated neoplasia is characterized by defective mitochondrial function and by upregulation of transcriptional pathways mediated by hypoxia-inducible factor (HIF), although whether and by what means these processes are linked has been disputed. We analysed the HIF pathway in Fh1-/- mouse embryonic fibroblasts (MEFs), in FH-defective neoplastic tissues and in Fh1-/- MEFs re-expressing either wild-type or an extra-mitochondrial restricted form of FH. These experiments demonstrated that upregulation of HIF-1alpha occurs as a direct consequence of FH inactivation. Fh1-/- cells accumulated intracellular fumarate and manifested severe impairment of HIF prolyl but not asparaginyl hydroxylation which was corrected by provision of exogenous 2-oxoglutarate (2-OG). Re-expression of the extra-mitochondrial form of FH in Fh1-/- cells was sufficient to reduce intracellular fumarate and to correct dysregulation of the HIF pathway completely, even in cells that remained profoundly defective in mitochondrial energy metabolism. The findings indicate that upregulation of HIF-1alpha arises from competitive inhibition of the 2-OG-dependent HIF hydroxylases by fumarate and not from disruption of mitochondrial energy metabolism.
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http://dx.doi.org/10.1093/hmg/ddq305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2935862PMC
October 2010

Mutation analysis of hypoxia-inducible factors HIF1A and HIF2A in renal cell carcinoma.

Anticancer Res 2009 Nov;29(11):4337-43

Department of Medical and Molecular Genetics, University of Birmingham, Institute of Biomedical Research, Edgbaston, Birmingham, B15 2TT, UK.

Background: Inactivation of the Von Hippel-Lindau (VHL) tumour suppressor gene leading to overexpression of hypoxia-inducible transcription factors (HIF)-1alpha and -2alpha is a critical event in the pathogenesis of most clear cell renal cell carcinomas (RCC). HIF-1alpha and HIF-2alpha share significant homology and regulate overlapping repertoires of hypoxia-inducible target genes but may have differing effects on RCC cell growth. Loss of HIF-1alpha expression has been described in RCC cell lines and primary tumours. Whether mutations in the alpha-subunits of HIF-1alpha and HIF-2alpha contribute to renal tumourigenesis was investigated here.

Materials And Methods: Mutation analysis of the complete coding sequence of HIF-1alpha and HIF-2alpha was carried out in primary RCC (n=40).

Results: The analysis revealed a somatic HIF1A missense substitution, p.Val116Glu, in a single RCC. Functional studies demonstrated that p.Val116Glu impaired HIF-1alpha transcriptional activity. Genotyping of HIF1A variants p.Pro582Ser and p.Ala588Thr demonstrated no significant differences between RCC patients and controls.

Conclusion: The detection of a loss-of-function HIF1A mutation in a primary RCC is consistent with HIF-1 and HIF-2 having different roles in renal tumourigenesis, However, somatic mutations of HIF1A are not frequently implicated in the pathogenesis of RCC.
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November 2009

Hypoxia, hypoxia-inducible factors (HIF), HIF hydroxylases and oxygen sensing.

Cell Mol Life Sci 2009 Nov 12;66(22):3539-54. Epub 2009 Sep 12.

Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Headington, Oxford, OX3 7BN, UK.

This article outlines the need for a homeostatic response to alterations in cellular oxygenation. It describes work on erythropoietin control that led to the discovery of the hypoxia-inducible transcription factor (HIF-1) and the parallel recognition that this system was responsive to a widespread oxygen-sensing mechanism. Subsequently, multiple HIF isoforms have been shown to have overlapping but non-redundant functions, controlling expression of genes involved in diverse processes such as angiogenesis, vascular tone, metal transport, glycolysis, mitochondrial function, cell growth and survival. The major role of prolyl and asparaginyl hydroxylation in regulating HIFs is described, as well as the identification of PHD1-3 and FIH as the oxygen-sensing enzymes responsible for these hydroxylations. Current understanding of other processes that modulate overall HIF activity, including influences from other signalling mechanisms such as kinases and nitric oxide levels, and the existence of a variety of feedback loops are outlined. The effects of some mutations in this pathway are documented as is knowledge of other substrates for these enzymes. The importance of PHD1-3 and FIH, and the large family of 2-oxoglutarate and iron(II)-dependent dioxygenases of which they are a part, in biology and medicine are discussed.
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http://dx.doi.org/10.1007/s00018-009-0147-7DOI Listing
November 2009