Reduced Oxygen Availability Research Articles

Oxygen Sensing and Signaling

Oxygen is an indispensable substrate for many biochemical reactions in plants, including energy metabolism (respiration). Despite its importance, plants lack an active transport mechanism to distribute oxygen to all cells. Therefore, steep oxygen gradients occur within most plant tissues, which can be exacerbated by environmental perturbations that further reduce oxygen availability. Plants possess various responses to cope with spatial and temporal variations in oxygen availability, many of which involve metabolic adaptations to deal with energy crises induced by low oxygen. Responses are induced gradually when oxygen concentrations decrease and are rapidly reversed upon reoxygenation. A direct effect of the oxygen level can be observed in the stability, and thus activity, of various transcription factors that control the expression of hypoxia-induced genes. Additional signaling pathways are activated by the impact of oxygen deficiency on mitochondrial and chloroplast functioning. Here, we describe the molecular components of the oxygen-sensing pathway.

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.

Therapies targeting haematopoietic stem cell niches

To maintain haematopoiesis, haematopoietic stem cells (HSCs) require the support of a specific microenvironment (stem cell niche). Specific interactions within these niches anchor HSCs and regulate HSC quiescence, proliferation or differentiation. An understanding of HSC niche composition has now led to improved strategies for HSC harvesting for transplantations. It is now understood that specialised BM macrophages regulate HSC niches. Indeed BM macrophages act as a conduit sensing and responding to inflammation and cytokines in a way that can affect HSC retention and activation. GCSF administration directly affects these BM macrophages causing the down-regulation of CXCL12 and VCAM-1, two key proteins expressed by mesenchymal progenitor cells to retain HSCs within the bone marrow. G-CSF administration also leads to a reduction of oxygen availability in the bone marrow, and this hypoxia further contributes to HSC mobilisation. These discoveries have enabled the development of drugs targeting these niche interactions such as CXCR4 inhibitors and prolylhydoxylase inhibitors to further boost HSC mobilisation for transplantation, particularly in patients for autologous transplantations, as prior successive rounds of radiotherapy and chemotherapy compromise subsequent HSC mobilisation and transplantation. Finally, recent research suggests that leukaemia initiating cells also reside in protective niches and therapeutic disruption of these niches sensitises them to chemotherapy.

Morg1 heterozygous deficiency ameliorates hypoxia-induced acute renal injury.

Acute kidney injury is a common complication of critically ill patients and may occur as a result of various factors and coexisting previous illnesses. Some pathophysiological responses seen in critical illness can be similar to the human physiological response to extreme environmental challenges, such as hypoxia from reduced oxygen availability at high altitudes (systemic hypoxia). Due to oxygen deficiency, mammalian cells activate the transcriptional factor hypoxia-inducible factor (HIF); its degradation is regulated by prolyl hydroxylase 3 (PHD3) in interaction with the scaffold protein MAPK organizer 1 (Morg1). While homozygous Morg1(-/-) mice are embryonically lethal, the kidneys of heterozygous Morg1(+/-) mice reveal elevated HIF protein levels and increased serum erythropoietin compared with wild-type Morg1(+/+) mice. In this study, we exposed wild-type and Morg1(+/-) mice to 10% oxygen in a hypoxic chamber for 3 days. This reduced oxygen concentration leads to a deterioration of renal function, an increase in renal inflammation, and significantly more tubular damage and apoptosis in the kidneys of wild-type (Morg1(+/+)) mice. In sharp contrast, Morg1(+/-) kidneys were protected against systemic hypoxia. They show significantly less renal lesions, reduced or no inflammation, and less tubular damage and apoptosis. Thus short-term systemic and subsequently renal hypoxia which may occur in many patients in the intensive care unit induces in wild-type mice renal injury, which is ameliorated by Morg1 deficiency. Our findings suggest that therapeutical manipulation of Morg1 may be an interesting novel target to prevent hypoxia-associated renal damage.

The effects of smoking on the eye: the role of the ophthalmic nurse

Cigarette smoking is an important factor in the pathogenesis and progression of several diseases of the eye, including age-related macular degeneration, cataracts and Graves’ ophthalmopathy, and also reduces the efficacy of therapy and surgical treatment. Nicotine and reactive oxygen species contribute to vasoconstriction, reduced oxygen availability and chronic inflammation. The awareness among eye care professionals of the effects of smoking on the eye is adequate, but many do not participate actively in identifying smokers and providing them with advice and support to quit. A barrier to quitting smoking in patients with eye diseases is their lack of knowledge that smoking can be harmful to their eyesight. More awareness is needed to increase the education process and to engage with patients to advise and support them to quit.

Capillary transit time heterogeneity and flow-metabolism coupling after traumatic brain injury.

Most patients who die after traumatic brain injury (TBI) show evidence of ischemic brain damage. Nevertheless, it has proven difficult to demonstrate cerebral ischemia in TBI patients. After TBI, both global and localized changes in cerebral blood flow (CBF) are observed, depending on the extent of diffuse brain swelling and the size and location of contusions and hematoma. These changes vary considerably over time, with most TBI patients showing reduced CBF during the first 12 hours after injury, then hyperperfusion, and in some patients vasospasms before CBF eventually normalizes. This apparent neurovascular uncoupling has been ascribed to mitochondrial dysfunction, hindered oxygen diffusion into tissue, or microthrombosis. Capillary compression by astrocytic endfeet swelling is observed in biopsies acquired from TBI patients. In animal models, elevated intracranial pressure compresses capillaries, causing redistribution of capillary flows into patterns argued to cause functional shunting of oxygenated blood through the capillary bed. We used a biophysical model of oxygen transport in tissue to examine how capillary flow disturbances may contribute to the profound changes in CBF after TBI. The analysis suggests that elevated capillary transit time heterogeneity can cause critical reductions in oxygen availability in the absence of ‘classic' ischemia. We discuss diagnostic and therapeutic consequences of these predictions.

High altitude may alter oxygen availability and renal metabolism in diabetics as measured by hyperpolarized [1-13C]pyruvate magnetic resonance imaging

The kidneys account for about 10% of the whole body oxygen consumption, whereas only 0.5% of the total body mass. It is known that intrarenal hypoxia is present in several diseases associated with development of kidney disease, including diabetes, and when renal blood flow is unaffected. The importance of deranged oxygen metabolism is further supported by deterioration of kidney function in patients with diabetes living at high altitude. Thus, we argue that reduced oxygen availability alters renal energy metabolism. Here, we introduce a novel magnetic resonance imaging (MRI) approach to monitor metabolic changes associated with diabetes and oxygen availability. Streptozotocin diabetic and control rats were given reduced, normal, or increased inspired oxygen in order to alter tissue oxygenation. The effects on kidney oxygen metabolism were studied using hyperpolarized [1-(13)C]pyruvate MRI. Reduced inspired oxygen did not alter renal metabolism in the control group. Reduced oxygen availability in the diabetic kidney altered energy metabolism by increasing lactate and alanine formation by 23% and 34%, respectively, whereas the bicarbonate flux was unchanged. Thus, the increased prevalence and severity of nephropathy in patients with diabetes at high altitudes may originate from the increased sensitivity toward inspired oxygen. This increased lactate production shifts the metabolic routs toward hypoxic pathways.

How wasting is saving: weight loss at altitude might result from an evolutionary adaptation.

At extreme altitude (>5,000 – 5,500 m), sustained hypoxia threatens human function and survival, and is associated with marked involuntary weight loss (cachexia). This seems to be a coordinated response: appetite and protein synthesis are suppressed, and muscle catabolism promoted. We hypothesise that, rather than simply being pathophysiological dysregulation, this cachexia is protective. Ketone bodies, synthesised during relative starvation, protect tissues such as the brain from reduced oxygen availability by mechanisms including the reduced generation of reactive oxygen species, improved mitochondrial efficiency and activation of the ATP-sensitive potassium (KATP) channel. Amino acids released from skeletal muscle also protect cells from hypoxia, and may interact synergistically with ketones to offer added protection. We thus propose that weight loss in hypoxia is an adaptive response: the amino acids and ketone bodies made available act not only as metabolic substrates, but as metabolic modulators, protecting cells from the hypoxic challenge.

OP0295 Hypoxic Dysregulation of Energy Metabolism in the Inflamed Arthritic Joint

BackgroundHypoxia in the inflamed joint is a powerful trigger of synovial fibroblasts and endothelial cell (EC) activation, proliferation and survival. The metabolic turnover of the inflamed synovium outpaces the dysfunctional...

Membrane transporters mediating root signalling and adaptive responses to oxygen deprivation and soil flooding

This review provides a comprehensive assessment of a previously unexplored topic: elucidating the role that plasma- and organelle-based membrane transporters play in plant-adaptive responses to flooding. We show that energy availability and metabolic shifts under hypoxia and anoxia are critical in regulating membrane-transport activity. We illustrate the high tissue and time dependence of this regulation, reveal the molecular identity of transporters involved and discuss the modes of their regulation. We show that both reduced oxygen availability and accumulation of transition metals in flooded roots result in a reduction in the cytosolic K(+) pool, ultimately determining the cell's fate and transition to programmed cell death (PCD). This process can be strongly affected by hypoxia-induced changes in the amino acid pool profile and, specifically, ϒ-amino butyric acid (GABA) accumulation. It is suggested that GABA plays an important regulatory role, allowing plants to proceed with H2 O2 signalling to activate a cascade of genes that mediate plant adaptation to flooding while at the same time, preventing the cell from entering a 'suicide program'. We conclude that progress in crop breeding for flooding tolerance can only be achieved by pyramiding the numerous physiological traits that confer efficient energy maintenance, cytosolic ion homeostasis, and reactive oxygen species (ROS) control and detoxification.

Hyperpolarized 13C MRS reveals hypoxia accelerates pseudo hypoxia in the diabetic kidney (890.8)

In diabetic patients, reduced renal oxygen availability is associated with a higher risk of diabetic nephropathy (DN) [1]. Recently, dynamic nuclear polarization hyperpolarized [1‐13C]pyruvate has shown an increased lactate conversion (lactate dehydrogenase activity) in the diabetic kidney, as a consequence of increased nicotinamide adenine dinucleotide (NADH) substrate generated by glucose‐mediated elevated flux through the polyol pathway, while the pyruvate dehydrogenase activity (PDH) is unaltered (pseudo hypoxia) [2]. In this study, we evaluated the renal oxygenation level and energy metabolism in diabetes‐suffering rats in response to altered oxygen availability, using blood oxygen level dependent (BOLD) MRI and hyperpolarized [1‐13C]pyruvate MRI, in response to hypoxic, normoxic and hyperoxic conditions. Specifically, we investigated the enzymatic activity of lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and pyruvate dehydrogenase (PDH). We show that reduced oxygen availability in the diabetic kidney alters the energy metabolism of pyruvate by increasing the lactate and alanine formation from pyruvate despite intact oxidative phosphorylation.Grant Funding Source: Supported by The Simon Spies Foundation, The Danish Kidney Foundation, Helen and Ejnar Bjørnows Foun

Acute hypoxia produces a superoxide burst in cells

Oxygen is a key molecule for cell metabolism. Eukaryotic cells sense the reduction in oxygen availability (hypoxia) and trigger a series of cellular and systemic responses to adapt to hypoxia, including the optimization of oxygen consumption. Many of these responses are mediated by a genetic program induced by the hypoxia-inducible transcription factors (HIFs), regulated by a family of prolyl hydroxylases (PHD or EGLN) that use oxygen as a substrate producing HIF hydroxylation. In parallel to these oxygen sensors modulating gene expression within hours, acute modulation of protein function in response to hypoxia is known to occur within minutes. Free radicals acting as second messengers, and oxidative posttranslational modifications, have been implied in both groups of responses. Localization and speciation of the paradoxical increase in reactive oxygen species production in hypoxia remain debatable. We have observed that several cell types respond to acute hypoxia with a transient increase in superoxide production for about 10min, probably originating in the mitochondria. This may explain in part the apparently divergent results found by various groups that have not taken into account the time frame of hypoxic ROS production. We propose that this acute and transient hypoxia-induced superoxide burst may be translated into oxidative signals contributing to hypoxic adaptation and preconditioning.

Transcriptional divergence of the duplicated hypoxia-inducible factor alpha genes in zebrafish

Oxygen availability has been a major force in shaping the physiological evolution of animals. Under reduced oxygen availability (hypoxia) major changes in gene expression are mediated by hypoxia-inducible factors (HIF alphas). Tetrapods have three hif alpha genes, whereas zebrafish (Danio rerio) and other cyprinids have six due to a teleost lineage-specific genome duplication. We studied the transcriptional divergence of the six teleost-specific hif alphas by inspecting the tissue-specific transcription patterns in adult zebrafish and by monitoring the early developmental transcription of normoxia- and hypoxia-grown zebrafish embryos. Overall we observed the highest hif alpha mRNA levels in tissues that are important for hypoxic survival, including the brain, gill and heart. Of the paralogs that have not previously received attention (hif alpha-1A, hif alpha-2B and hif alpha-3B) especially the hif alpha-2B transcription levels suggest functional relevance. The hif alpha-1A/B paralogs that have considerable coding sequence divergence displayed more overall transcriptional divergence than the hif alpha-2A/B paralog pair. The hif alpha-2A/B paralogs that are similarly conserved in coding sequence had a divergent transcription pattern during early development. When zebrafish grown in modest hypoxia were compared to normoxia grown fish, only hif alpha-3A transcription was significantly altered. These results suggest that, in zebrafish, the evolutionary retention of each hif alpha paralog pair has involved unique patterns of coding sequence divergence, adult tissue-specific transcriptional divergence or developmental transcriptional divergence.

Exometabolome analysis reveals hypoxia at the up-scaling of a Saccharomyces cerevisiae high-cell density fed-batch biopharmaceutical process.

BackgroundScale-up to industrial production level of a fermentation process occurs after optimization at small scale, a critical transition for successful technology transfer and commercialization of a product of interest. At the large scale a number of important bioprocess engineering problems arise that should be taken into account to match the values obtained at the small scale and achieve the highest productivity and quality possible. However, the changes of the host strain’s physiological and metabolic behavior in response to the scale transition are still not clear.ResultsHeterogeneity in substrate and oxygen distribution is an inherent factor at industrial scale (10,000 L) which affects the success of process up-scaling. To counteract these detrimental effects, changes in dissolved oxygen and pressure set points and addition of diluents were applied to 10,000 L scale to enable a successful process scale-up. A comprehensive semi-quantitative and time-dependent analysis of the exometabolome was performed to understand the impact of the scale-up on the metabolic/physiological behavior of the host microorganism. Intermediates from central carbon catabolism and mevalonate/ergosterol synthesis pathways were found to accumulate in both the 10 L and 10,000 L scale cultures in a time-dependent manner. Moreover, excreted metabolites analysis revealed that hypoxic conditions prevailed at the 10,000 L scale. The specific product yield increased at the 10,000 L scale, in spite of metabolic stress and catabolic-anabolic uncoupling unveiled by the decrease in biomass yield on consumed oxygen.ConclusionsAn optimized S. cerevisiae fermentation process was successfully scaled-up to an industrial scale bioreactor. The oxygen uptake rate (OUR) and overall growth profiles were matched between scales. The major remaining differences between scales were wet cell weight and culture apparent viscosity. The metabolic and physiological behavior of the host microorganism at the 10,000 L scale was investigated with exometabolomics, indicating that reduced oxygen availability affected oxidative phosphorylation cascading into down- and up-stream pathways producing overflow metabolism. Our study revealed striking metabolic and physiological changes in response to hypoxia exerted by industrial bioprocess up-scaling.

Imaging tumor hypoxia: Blood-borne delivery of imaging agents is fundamentally different in hypoxia subtypes

Hypoxic tissue subvolumes are a hallmark feature of solid malignant tumors, relevant for cancer therapy and patient outcome because they increase both the intrinsic aggressiveness of tumor cells and their resistance to several commonly used anticancer strategies. Pathogenetic mechanisms leading to hypoxia are diverse, may coexist within the same tumor and are commonly grouped according to the duration of their effects. Chronic hypoxia is mainly caused by diffusion limitations resulting from enlarged intercapillary distances and adverse diffusion geometries and — to a lesser extent — by hypoxemia, compromised perfusion or long-lasting microregional flow stops. Conversely, acute hypoxia preferentially results from transient disruptions in perfusion. While each of these features of the tumor microenvironment can contribute to a critical reduction of oxygen availability, the delivery of imaging agents (as well as nutrients and anticancer agents) may be compromised or remain unaffected. Thus, a critical appraisal of the effects of the various mechanisms leading to hypoxia with regard to the blood-borne delivery of imaging agents is necessary to judge their ability to correctly represent the hypoxic phenotype of solid malignancies.

Extracellular superoxide dismutase suppresses hypoxia-inducible factor-1α in pancreatic cancer

AbstractHypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that governs cellular responses to reduced oxygen availability by mediating crucial homeostatic processes and is a major survival determinant for tumor cells growing in a low-oxygen environment. Clinically, HIF-1α seems to be important in pancreatic cancer, as HIF-1α correlates with metastatic status of the tumor. Extracellular superoxide dismutase (EcSOD) inhibits pancreatic cancer cell growth by scavenging nonmitochondrial superoxide. We hypothesized that EcSOD overexpression leads to changes in the O2−/H2O2 balance modulating the redox status affecting signal transduction pathways. Both transient and stable overexpression of EcSOD suppressed the hypoxic accumulation of HIF-1α in human pancreatic cancer cells. This suppression of HIF-1α had a strong inverse correlation with levels of EcSOD protein. Coexpression of the hydrogen peroxide-removing protein glutathione peroxidase did not prevent the EcSOD-induced suppression of HIF-1α, suggesting that the degradation of HIF-1α observed with high EcSOD overexpression is possibly due to a low steady-state level of superoxide. Hypoxic induction of vascular endothelial growth factor (VEGF) was also suppressed with increased EcSOD. Intratumoral injections of an adenoviral vector containing the EcSOD gene into preestablished pancreatic tumors suppressed both VEGF levels and tumor growth. These results demonstrate that the transcription factor HIF-1α and its important gene target VEGF can be modulated by the antioxidant enzyme EcSOD.

Capacity and hypoxic response of subcutaneous adipose tissue blood flow in humans.

The blood flow capacity in subcutaneous adipose tissue in humans remains largely unknown, and therefore the aim of this study was to determine the physiological range of blood flow in this tissue. The subcutaneous adipose tissue blood flow (ATBF) was measured in 9 healthy young men by positron emission tomography using radiowater tracer. Subcutaneous ATBF was determined in regions adjacent to knee extensors at rest and during dynamic knee extensor exercise, and with 2 physiological perturbations: while breathing moderate systemic hypoxic air (14% O2) at rest and during exercise, and during intra-femoral artery infusion of high-dose adenosine infusion. ATBF was 1.3±0.6ml·100g(-1)·min(-1) at rest and increased with exercise (8.0±3.0ml·100g(-1)·min(-1), P<0.001) and adenosine infusion (10.5±4.9ml·100g(-1)·min(-1), P=0.001), but not when breathing moderate systemic hypoxic air (1.5±0.4ml·100g(-1)·min(-1)). ATBF was similar during exercise and adenosine infusion, but vascular conductance was lower during adenosine infusion. Finally, ATBF during exercise in moderate systemic hypoxia was reduced (6.3±2.2ml·100g(-1)·min(-1)) compared to normoxic exercise (P=0.004). The vasodilatation capacity of human subcutaneous adipose blood flow appears to be comparable to, or even higher, than that induced by moderate intensity exercise. Furthermore, the reduced blood flow response in subcutaneous adipose tissue during systemic hypoxia is likely to contribute, in part, to the redistribution of blood flow to exercising muscle in a condition of reduced oxygen availability.

Continuous Hypoxic Culturing of Human Embryonic Stem Cells Enhances SSEA-3 and MYC Levels

Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state.

Smoking and skin ageing: how aesthetic nurses can identify and prevent damage

Whether cigarette smoke has direct contact with individuals or enters through their blood supply, smoking has a significant impact on the skin, leaving it dry, wrinkled and discoloured. While carbon monoxide reduces oxygen availability, carcinogens damage DNA and disrupt cell turnover. Free radicals also disturb the chemical structure of collagen, reducing its flexibility, and nicotine suppresses the immune system, leading to poor wound healing and increased risk of infection. Aesthetic nurses are in a prime position to counsel patients about their smoking habits and provide support to those who wish to quit. This may improve the efficacy of any treatments provided. Patients should be offered advice on ways to give up smoking and directed toward health services and websites that may help them.

Non-destructive sampling procedure for biochemical or gene expression studies on post-harvest physiological deterioration of cassava roots

Cassava (Manihot esculenta Crantz) roots spoil 2–3 days after harvest due to post-harvest physiological deterioration (PPD), which had remained an unsolved problem until recent reports of genetic variation for tolerance to it. PPD is a genetically active, oxidative process triggered when the harvested roots are separated from their mother plant. The short shelf life of harvested roots results in large losses and high transport and marketing costs. Recent reports on positive genetic variation for tolerance to PPD will facilitate breeding for extended shelf life of the roots and a better understanding of the biochemical and genetic events leading to PPD. However, PPD scoring is difficult and prone to large experimental errors. It is often the case that roots from the same plant can have 0 and 100% PPD score due to injuries during the harvest process, variation in dry matter content and, most likely, other variables yet to be identified. Therefore, sampling a root for biochemical or genetic studies and measuring PPD in a different root, is not a reliable approach. A device has been developed and tested for the possibility of extracting a core of root parenchyma, filling the space with melted paraffin (to reduce oxygen availability), and then one or two weeks later visually quantifying PPD in the same root. Sampling the roots did not have any significant effect on PPD suggesting that the protocol can be used for biochemical composition and gene expression studies related to the causes of PPD and the possibilities of developing tolerance to it.