Publications by authors named "Tammie Bishop"

21 Publications

  • Page 1 of 1

Hypoxic and pharmacological activation of HIF inhibits SARS-CoV-2 infection of lung epithelial cells.

Cell Rep 2021 04 5;35(3):109020. Epub 2021 Apr 5.

Nuffield Department of Medicine, University of Oxford, Oxford, UK; Chinese Academy of Medical Sciences (CAMS) Oxford Institute (COI), University of Oxford, Oxford, UK. Electronic address:

COVID-19, caused by the novel coronavirus SARS-CoV-2, is a global health issue with more than 2 million fatalities to date. Viral replication is shaped by the cellular microenvironment, and one important factor to consider is oxygen tension, in which hypoxia inducible factor (HIF) regulates transcriptional responses to hypoxia. SARS-CoV-2 primarily infects cells of the respiratory tract, entering via its spike glycoprotein binding to angiotensin-converting enzyme 2 (ACE2). We demonstrate that hypoxia and the HIF prolyl hydroxylase inhibitor Roxadustat reduce ACE2 expression and inhibit SARS-CoV-2 entry and replication in lung epithelial cells via an HIF-1α-dependent pathway. Hypoxia and Roxadustat inhibit SARS-CoV-2 RNA replication, showing that post-entry steps in the viral life cycle are oxygen sensitive. This study highlights the importance of HIF signaling in regulating multiple aspects of SARS-CoV-2 infection and raises the potential use of HIF prolyl hydroxylase inhibitors in the prevention or treatment of COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020087PMC
April 2021

Silent hypoxaemia in COVID-19 patients.

J Physiol 2021 02 4;599(4):1057-1065. Epub 2021 Jan 4.

Division of Pulmonary, Critical Care, & Sleep Medicine, Department of Medicine, University of California, San Diego, CA, USA.

The clinical presentation of COVID-19 due to infection with SARS-CoV-2 is highly variable with the majority of patients having mild symptoms while others develop severe respiratory failure. The reason for this variability is unclear but is in critical need of investigation. Some COVID-19 patients have been labelled with 'happy hypoxia', in which patient complaints of dyspnoea and observable signs of respiratory distress are reported to be absent. Based on ongoing debate, we highlight key respiratory and neurological components that could underlie variation in the presentation of silent hypoxaemia and define priorities for subsequent investigation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/JP280769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902403PMC
February 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI133194DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190921PMC
May 2020

Genetic basis of oxygen sensing in the carotid body: HIF2α and an isoform switch in cytochrome c oxidase subunit 4.

Sci Signal 2020 01 21;13(615). Epub 2020 Jan 21.

Target Discovery Institute, University of Oxford, Oxford OX3 7FZ, UK.

The mechanistic basis of the marked oxygen sensitivity of glomus cells in the carotid body has long puzzled physiologists. In this issue of , Moreno-Domínguez show the critical importance of high levels of hypoxia-inducible factor, HIF2α/EPAS1, and the nuclear-encoded mitochondrial cytochrome c oxidase subunit, COX4I2, in glomus cell sensitivity to hypoxia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scisignal.aba1302DOI Listing
January 2020

Systemic silencing of PHD2 causes reversible immune regulatory dysfunction.

J Clin Invest 2019 06 4;129(9):3640-3656. Epub 2019 Jun 4.

Nuffield Department of Medicine Research Building, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Physiological effects of cellular hypoxia are sensed by prolyl hydroxylase (PHD) enzymes which regulate HIFs. Genetic interventions on HIF/PHD pathways reveal multiple phenotypes that extend the known biology of hypoxia. Recent studies unexpectedly implicate HIF in aspects of multiple immune and inflammatory pathways. However such studies are often limited by systemic lethal effects and/or use tissue-specific recombination systems, which are inherently irreversible, un-physiologically restricted and difficult to time. To study these processes better we developed recombinant mice which express tetracycline-regulated shRNAs broadly targeting the main components of the HIF/PHD pathway, permitting timed bi-directional intervention. We have shown that stabilization of HIF levels in adult mice through PHD2 enzyme silencing by RNA interference, or inducible recombination of floxed alleles, results in multi-lineage leukocytosis and features of autoimmunity. This phenotype was rapidly normalized on re-establishment of the hypoxia-sensing machinery when shRNA expression was discontinued. In both situations these effects were mediated principally through the Hif2a isoform. Assessment of cells bearing regulatory T cell markers from these mice revealed defective function and pro-inflammatory effects in vivo. We believe our findings have shown a new role for the PHD2/Hif2a couple in the reversible regulation of T cell and immune activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI124099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715380PMC
June 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/JP275996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138294PMC
June 2018

Hypoxia-inducible factor 1-alpha does not regulate osteoclastogenesis but enhances bone resorption activity via prolyl-4-hydroxylase 2.

J Pathol 2017 07 29;242(3):322-333. Epub 2017 May 29.

Nuffield Department of Orthopaedics Rheumatology & Musculoskeletal Sciences, University of Oxford, Oxford, UK.

Osteogenic-angiogenic coupling is promoted by the hypoxia-inducible factor 1-alpha (HIF-1α) transcription factor, provoking interest in HIF activation as a therapeutic strategy to improve osteoblast mineralization and treat pathological osteolysis. However, HIF also enhances the bone-resorbing activity of mature osteoclasts. It is therefore essential to determine the full effect(s) of HIF on both the formation and the bone-resorbing function of osteoclasts in order to understand how they might respond to such a strategy. Expression of HIF-1α mRNA and protein increased during osteoclast differentiation from CD14+ monocytic precursors, additionally inducing expression of the HIF-regulated glycolytic enzymes. However, HIF-1α siRNA only moderately affected osteoclast differentiation, accelerating fusion of precursor cells. HIF induction by inhibition of the regulatory prolyl-4-hydroxylase (PHD) enzymes reduced osteoclastogenesis, but was confirmed to enhance bone resorption by mature osteoclasts. Phd2 murine osteoclasts also exhibited enhanced bone resorption, associated with increased expression of resorption-associated Acp5, in comparison with wild-type cells from littermate controls. Phd3 bone marrow precursors displayed accelerated early fusion, mirroring results with HIF-1α siRNA. In vivo, Phd2 and Phd3 mice exhibited reduced trabecular bone mass, associated with reduced mineralization by Phd2 osteoblasts. These data indicate that HIF predominantly functions as a regulator of osteoclast-mediated bone resorption, with little effect on osteoclast differentiation. Inhibition of HIF might therefore represent an alternative strategy to treat diseases characterized by pathological levels of osteolysis. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/path.4906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518186PMC
July 2017

Hyperplasia and hypertrophy of pulmonary neuroepithelial bodies, presumed airway hypoxia sensors, in hypoxia-inducible factor prolyl hydroxylase-deficient mice.

Hypoxia (Auckl) 2016 12;4:69-80. Epub 2016 Apr 12.

Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

Pulmonary neuroepithelial bodies (NEBs), presumed polymodal airway sensors, consist of innervated clusters of amine (serotonin) and peptide-producing cells. While NEB responses to acute hypoxia are mediated by a membrane-bound O sensor complex, responses to sustained and/or chronic hypoxia involve a prolyl hydroxylase (PHD)-hypoxia-inducible factor-dependent mechanism. We have previously reported hyperplasia of NEBs in the lungs of -/- mice associated with enhanced serotonin secretion. Here we use a novel multilabel immunofluorescence method to assess NEB distribution, frequency, and size, together with the number and size of NEB cell nuclei, and to colocalize multiple cytoplasmic and nuclear epitopes in the lungs of -/-, +/-, and -/- mice and compare them with wild-type controls. To define the mechanisms of NEB cell hyperplasia, we used antibodies against Mash1 and Prox1 (neurogenic genes involved in NEB cell differentiation/maturation), hypoxia-inducible factor-1alpha, and the cell proliferation marker Ki67. Morphometric analysis of (% total lung area) immunostaining for synaptophysin (% synaptophysin), a cytoplasmic marker of NEB cells, was significantly increased in -/- and -/- mice compared to wild-type mice. In addition, NEB size and the number and size of NEB nuclei were also significantly increased, indicating that deficiency of s is associated with striking hyperplasia and hypertrophy of NEBs. In +/- mice, while mean % synaptophysin was comparable to wild-type controls, the NEB size was moderately increased, suggesting an effect even in heterozygotes. NEBs in all -deficient mice showed increased expression of Mash1, Prox1, Ki67, and hypoxia-inducible factor-1alpha, in keeping with enhanced differentiation from precursor cells and a minor component of cell proliferation. Since the loss of PHD activity mimics chronic hypoxia, our data provide critical information on the potential role of PHDs in the pathobiology and mechanisms of NEB cell hyperplasia that is relevant to a number of pediatric lung disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2147/HP.S103957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5085281PMC
April 2016

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/JP271050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4771794PMC
March 2016

Augmented 5-HT Secretion in Pulmonary Neuroepithelial Bodies from PHD1 Null Mice.

Adv Exp Med Biol 2015 ;860:309-13

Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children and Department of Laboratory Medicine and Pathobiology, University of Toronto, 555 University Avenue, M5G1X8, Toronto, ON, Canada.

Sustained exposure to low oxygen concentration leads to profound changes in gene expression to restore oxygen homeostasis. Hypoxia-inducible factors (HIFs) comprise a group of transcription factors which accumulate under hypoxia and contribute to the complex changes in gene expression. Under normoxic conditions HIFs are degraded by prolyl-hydroxylases (PHD), however during hypoxia this degradation is inhibited causing HIF accumulation and subsequent changes in gene expression. Pulmonary neuroepithelial bodies (NEB) are innervated serotonin (5-HT)-producing cells distributed throughout the airway epithelium. These putative O(2) sensors are hypothesized to contribute to the ventilatory response to hypoxia. NEB dysfunction has been implicated in several paediatric lung diseases including neuroendocrine cell hyperplasia of infancy and sudden infant death syndrome, both characterized by a marked NEB hyperplasia with unknown functional significance. We have previously reported striking NEB hyperplasia in PHD1(-/-) mice making these mice a potential model to study the role of NEBs in paediatric lung diseases. Here we report in vitro studies on 5-HT release from NEB using this model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-3-319-18440-1_35DOI Listing
January 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0132004PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492579PMC
April 2016

HIF hydroxylase pathways in cardiovascular physiology and medicine.

Circ Res 2015 Jun;117(1):65-79

From the Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

Hypoxia inducible factors (HIFs) are α/β heterodimeric transcription factors that direct multiple cellular and systemic responses in response to changes in oxygen availability. The oxygen sensitive signal is generated by a series of iron and 2-oxoglutarate-dependent dioxygenases that catalyze post-translational hydroxylation of specific prolyl and asparaginyl residues in HIFα subunits and thereby promote their destruction and inactivation in the presence of oxygen. In hypoxia, these processes are suppressed allowing HIF to activate a massive transcriptional cascade. Elucidation of these pathways has opened several new fields of cardiovascular research. Here, we review the role of HIF hydroxylase pathways in cardiac development and in cardiovascular control. We also consider the current status, opportunities, and challenges of therapeutic modulation of HIF hydroxylases in the therapy of cardiovascular disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.117.305109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4501273PMC
June 2015

Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives.

Hypoxia (Auckl) 2014 5;2:197-213. Epub 2014 Dec 5.

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

By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of "oxygen-sensing" 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2147/HP.S47598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045067PMC
December 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2012.247254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731614PMC
July 2013

Hyperplasia of pulmonary neuroepithelial bodies (NEB) in lungs of prolyl hydroxylase -1(PHD-1) deficient mice.

Adv Exp Med Biol 2012 ;758:149-55

Department of The Paediatric Laboratory Medicine Research Institute, University of Toronto, Toronto, ON, Canada.

Pulmonary NEB, widely distributed within the airway mucosa of mammalian lungs, are presumed hypoxia sensitive airway O(2) sensors responding to changes in airway gas concentration. NEB cell hyperplasia has been reported after exposure to chronic hypoxia and in a variety of paediatric and adult lung disorders. Prolyl hydroxylases (PHD 1-3) regulate the stability of hypoxia-inducible factors (HIF's) in an O(2)-dependent manner and function as intrinsic oxygen sensors. To determine a possible role of PHD-1in NEB cells we have quantitated NEB's in lungs of neonatal (P2) and adult (2 months) PHD-1-deficient mice and compared them to wild type (WT) control mice. Lung tissues fixed in formalin and embedded in paraffin were processed for immunoperoxidase method and frozen sections for multilabel immunoflourescence using antibodies for NEB markers synaptophysin, synaptic vesicle protein 2 and the peptide CGRP. The frequency and size of NEB in lungs of PHD-1 deficient neonatal mice (P2) and at 2 months was increased significantly compared to WT controls (p < 0.01). The present data suggests an important role for PHD enzymes in NEB cell biology deserving further studies. Since the PHD-1 deficient mouse appears to be the first animal model showing NEB cell hyperplasia it may be useful for studies of NEB physiology and pathobiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-94-007-4584-1_21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389447PMC
April 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1113/jphysiol.2012.232884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3476649PMC
August 2012

Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.

Gastroenterology 2010 Mar 8;138(3):1143-54.e1-2. Epub 2009 Oct 8.

Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Germany.

Background & Aims: Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage.

Methods: Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo.

Results: PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism.

Conclusions: Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2009.09.057DOI Listing
March 2010

Abnormal sympathoadrenal development and systemic hypotension in PHD3-/- mice.

Mol Cell Biol 2008 May 10;28(10):3386-400. Epub 2008 Mar 10.

The Henry Wellcome Building for Molecular Physiology, University of Oxford, Headington Campus, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.

Cell culture studies have implicated the oxygen-sensitive hypoxia-inducible factor (HIF) prolyl hydroxylase PHD3 in the regulation of neuronal apoptosis. To better understand this function in vivo, we have created PHD3(-/-) mice and analyzed the neuronal phenotype. Reduced apoptosis in superior cervical ganglion (SCG) neurons cultured from PHD3(-/-) mice is associated with an increase in the number of cells in the SCG, as well as in the adrenal medulla and carotid body. Genetic analysis by intercrossing PHD3(-/-) mice with HIF-1a(+/-) and HIF-2a(+/-) mice demonstrated an interaction with HIF-2alpha but not HIF-1alpha, supporting the nonredundant involvement of a PHD3-HIF-2alpha pathway in the regulation of sympathoadrenal development. Despite the increased number of cells, the sympathoadrenal system appeared hypofunctional in PHD3(-/-) mice, with reduced target tissue innervation, adrenal medullary secretory capacity, sympathoadrenal responses, and systemic blood pressure. These observations suggest that the role of PHD3 in sympathoadrenal development extends beyond simple control of cell survival and organ mass, with functional PHD3 being required for proper anatomical and physiological integrity of the system. Perturbation of this interface between developmental and adaptive signaling by hypoxic, metabolic, or other stresses could have important effects on key sympathoadrenal functions, such as blood pressure regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.02041-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423159PMC
May 2008

Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism.

Nat Genet 2008 Feb 6;40(2):170-80. Epub 2008 Jan 6.

The Center for Transgene Technology and Gene Therapy, Katholieke Universiteit (K.U.) Leuven, Leuven, B-3000, Belgium.

HIF prolyl hydroxylases (PHD1-3) are oxygen sensors that regulate the stability of the hypoxia-inducible factors (HIFs) in an oxygen-dependent manner. Here, we show that loss of Phd1 lowers oxygen consumption in skeletal muscle by reprogramming glucose metabolism from oxidative to more anaerobic ATP production through activation of a Pparalpha pathway. This metabolic adaptation to oxygen conservation impairs oxidative muscle performance in healthy conditions, but it provides acute protection of myofibers against lethal ischemia. Hypoxia tolerance is not due to HIF-dependent angiogenesis, erythropoiesis or vasodilation, but rather to reduced generation of oxidative stress, which allows Phd1-deficient myofibers to preserve mitochondrial respiration. Hypoxia tolerance relies primarily on Hif-2alpha and was not observed in heterozygous Phd2-deficient or homozygous Phd3-deficient mice. Of medical importance, conditional knockdown of Phd1 also rapidly induces hypoxia tolerance. These findings delineate a new role of Phd1 in hypoxia tolerance and offer new treatment perspectives for disorders characterized by oxidative stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ng.2007.62DOI Listing
February 2008

Genetic analysis of pathways regulated by the von Hippel-Lindau tumor suppressor in Caenorhabditis elegans.

PLoS Biol 2004 Oct 7;2(10):e289. Epub 2004 Sep 7.

The Henry Wellcome Building of Genomic Medicine, University of Oxford, Oxford, United Kingdom.

The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated alpha subunits of hypoxia-inducible factor (HIF). Although studies of VHL-defective renal carcinoma cells suggest the existence of other VHL tumor suppressor pathways, dysregulation of the HIF transcriptional cascade has extensive effects that make it difficult to distinguish whether, and to what extent, observed abnormalities in these cells represent effects on pathways that are distinct from HIF. Here, we report on a genetic analysis of HIF-dependent and -independent effects of VHL inactivation by studying gene expression patterns in Caenorhabditis elegans. We show tight conservation of the HIF-1/VHL-1/EGL-9 hydroxylase pathway. However, persisting differential gene expression in hif-1 versus hif-1; vhl-1 double mutant worms clearly distinguished HIF-1-independent effects of VHL-1 inactivation. Genomic clustering, predicted functional similarities, and a common pattern of dysregulation in both vhl-1 worms and a set of mutants (dpy-18, let-268, gon-1, mig-17, and unc-6), with different defects in extracellular matrix formation, suggest that dysregulation of these genes reflects a discrete HIF-1-independent function of VHL-1 that is connected with extracellular matrix function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.0020289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC515368PMC
October 2004

Primary causes of decreased mitochondrial oxygen consumption during metabolic depression in snail cells.

Am J Physiol Regul Integr Comp Physiol 2002 Feb;282(2):R372-82

Medical Research Council, Dunn Human Nutrition Unit, Cambridge CB2 2XY, United Kingdom.

Cells isolated from the hepatopancreas of estivating snails (Helix aspersa) have strongly depressed mitochondrial respiration compared with controls. Mitochondrial respiration was divided into substrate oxidation (which produces the mitochondrial membrane potential) and ATP turnover and proton leak (which consume it). The activity of substrate oxidation (and probably ATP turnover) decreased, whereas the activity of proton leak remained constant in estivation. These primary changes resulted in a lower mitochondrial membrane potential in hepatopancreas cells from estivating compared with active snails, leading to secondary decreases in respiration to drive ATP turnover and proton leak. The respiration to drive ATP turnover and proton leak decreased in proportion to the overall decrease in mitochondrial respiration, so that the amount of ATP turned over per O2 consumed remained relatively constant and aerobic efficiency was maintained in this hypometabolic state. At least 75% of the total response of mitochondrial respiration to estivation was caused by primary changes in the kinetics of substrate oxidation, with only 25% or less of the response occurring through primary effects on ATP turnover.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpregu.00401.2001DOI Listing
February 2002