Tammie Bishop

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

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 Pparα 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-2α 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.

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

ABSTRACT 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-2α but not HIF-1α, supporting the nonredundant involvement of a PHD3-HIF-2α 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.

Genetic Analysis of Pathways Regulated by the von Hippel-Lindau Tumor Suppressor in Caenorhabditis elegans

The von Hippel-Lindau (VHL) tumor suppressor functions as a ubiquitin ligase that mediates proteolytic inactivation of hydroxylated α 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.

Loss or Silencing of the PHD1 Prolyl Hydroxylase Protects Livers of Mice Against Ischemia/Reperfusion Injury

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.

HIF Hydroxylase Pathways in Cardiovascular Physiology and Medicine

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.

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

•  Arterial hypoxaemia leads to a rapid increase in ventilation. If the hypoxaemia is sustained, a further increase in ventilation develops over hours to days in a process termed ventilatory acclimatisation. •  Studies in transgenic mice implicate the hypoxia‐inducible factor (HIF) pathway in the latter process. •  The aim of this study was to investigate the role of HIF prolyl hydroxylase (PHD) enzymes in ventilatory acclimatisation. •  We find that PHD2+/−, but not PHD1−/− or PHD3−/−, mice mimic chronic hypoxia in exhibiting exaggerated ventilatory responses to acute hypoxia. This was associated with carotid body overgrowth. However, use of a PHD inhibitor (PHI) induced both hypoxic ventilatory sensitivity and carotid body proliferation only marginally despite strongly inducing erythropoiesis. •  Taken together, these findings implicate HIF/PHD2 in ventilatory control and carotid body biology but highlight the difficulty of translation from genetic models to pharmacological intervention.

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

•  Cerebral ischaemia results in the activation of multiple pathways that can independently lead to neuronal death. Agents targeting a number of processes at one time are likely to be translated into stroke therapy. •  Hypoxia‐inducible factor (HIF) is a transcription complex which responds to changes in oxygen. HIF levels are tightly regulated by a group of prolyl hydroxylases (PHDs). •  In this study, we investigated the function of each of the HIF‐PHDs in the first 24 hours following transient focal cerebral ischaemia by using mice with each isoform genetically suppressed. •  We found that the PHD1−/−, PHD2+/−, PHD3−/− mice had different outcomes after inducing ischaemia. In particular, the PHD2+/− mice had an improved rCBF response post‐reperfusion with better behavioural scores. The PHD3−/− mice have worse rCBF but no behavioural change. •  The information gained enhances understanding of the biological processes involved and informs strategies for therapeutic targeting of the PHD enzymes.

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

Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation. Oxygen‐sensitive signals are transduced in animal cells by post‐translational hydroxylation of transcription factors termed hypoxia‐inducible factors (HIFs). Mice heterozygous for the principal ‘oxygen‐sensing’ HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia. To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF‐1α and HIF‐2α. Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF‐2α. Inducible inactivation of HIF‐2α, but not HIF‐1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation. These findings demonstrate a key role for PHD2 and HIF‐2α in ventilatory control and carotid body biology.

Structure and Function of Mitochondria in Hepatopancreas Cells from Metabolically Depressed Snails

Mitochondria in cells isolated from the hepatopancreas of aestivating land snails (Helix aspersa) consume oxygen at 30% of the active control rate. The aim of this study was to investigate whether the lower respiration rate is caused by a decrease in the density of mitochondria or by intrinsic changes in the mitochondria. Mitochondria occupied 2% of cellular volume, and the mitochondrial inner membrane surface density was 17 μm−1, in cells from active snails. These values were not different in cells from aestivating snails. The mitochondrial protein and mitochondrial phospholipid contents of cells were also similar. There was little difference in the phospholipid fatty acyl composition of mitochondria isolated from metabolically depressed or active snails, except for arachidonic acid, which was 18% higher in mitochondria from aestivating snails. However, the activities of citrate synthase and cytochrome c oxidase in mitochondria isolated from aestivating snails were 68% and 63% of control, respectively. Thus the lower mitochondrial respiration rate in hepatopancreas cells from aestivating snails was not caused by differences in mitochondrial volume or surface density but was associated with intrinsic changes in the mitochondria.

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

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.