Hypoxia activates IKK-NF-κB and the immune response in <em>Drosophila melanogaster</em>

Hypoxia, or low oxygen availability, is an important physiological and pathological stimulus for multicellular organisms. Molecularly, hypoxia activates a transcriptional programme directed at restoration of oxygen homoeostasis and cellular survival. In mammalian cells, hypoxia not only activates the HIF (hypoxia-inducible factor) family, but also additional transcription factors such as NF-κB (nuclear factor κB). Here we show that hypoxia activates the IKK–NF-κB [IκB (inhibitor of nuclear factor κB)–NF-κB] pathway and the immune response in Drosophila melanogaster. We show that NF-κB activation is required for organism survival in hypoxia. Finally, we identify a role for the tumour suppressor Cyld, as a negative regulator of NF-κB in response to hypoxia in Drosophila. The results indicate that hypoxia activation of the IKK–NF-κB pathway and the immune response is an important and evolutionary conserved response

Hypoxia Upregulates Estrogen Receptor β in Pulmonary Artery Endothelial Cells in a HIF-1α-Dependent Manner

17β-Estradiol (E2) attenuates hypoxia-induced pulmonary hypertension (HPH) through estrogen receptor (ER)-dependent effects, including inhibition of hypoxia-induced endothelial cell proliferation; however, the mechanisms responsible for this remain unknown. We hypothesized that the protective effects of E2 in HPH are mediated through hypoxia-inducible factor 1α (HIF-1α)-dependent increases in ERβ expression. Sprague-Dawley rats and ERα or ERβ knockout mice were exposed to hypobaric hypoxia for 2-3 weeks. The effects of hypoxia were also studied in primary rat or human pulmonary artery endothelial cells (PAECs). Hypoxia increased expression of ERβ, but not ERα, in lungs from HPH rats as well as in rat and human PAECs. ERβ mRNA time dependently increased in PAECs exposed to hypoxia. Normoxic HIF-1α/HIF-2α stabilization increased PAEC ERβ, whereas HIF-1α knockdown decreased ERβ abundance in hypoxic PAECs. In turn, ERβ knockdown in hypoxic PAECs increased HIF-2α expression, suggesting a hypoxia-sensitive feedback mechanism. ERβ knockdown in hypoxic PAECs also decreased expression of the HIF inhibitor prolyl hydroxylase 2 (PHD2), whereas ERβ activation increased PHD2 and decreased both HIF-1α and HIF-2α, suggesting that ERβ regulates the PHD2/HIF-1α/HIF-2α axis during hypoxia. Whereas hypoxic wild-type or ERα knockout mice treated with E2 demonstrated less pulmonary vascular remodeling and decreased HIF-1α after hypoxia compared with untreated hypoxic mice, ERβ knockout mice exhibited increased HIF-2α and an attenuated response to E2 during hypoxia. Taken together, our results demonstrate a novel and potentially therapeutically targetable mechanism whereby hypoxia, via HIF-1α, increases ERβ expression and the E2-ERβ axis targets PHD2, HIF-1α, and HIF-2α to attenuate HPH development

The paradox between resistance to hypoxia and liability to hypoxic damage in hyperglycemic peripheral nerves. Evidence for glycolysis involvement

Isolated ventral and dorsal rat spinal roots incubated in normal (2.5 mM) or high glucose (25 mM) concentrations or in high concentrations of other hexoses were exposed transiently to hypoxia (30 min) in a solution of low buffering power. Compound nerve action potentials, extracellular direct current potentials, and interstitial pH were continuously recorded before, during, and after hypoxia. Ventral roots incubated in 25 mM D-glucose showed resistance to hypoxia. Dorsal roots, on the other hand, revealed electrophysiological damage by hyperglycemic hypoxia as indicated by a lack of posthypoxic recovery. In both types of spinal roots, interstitial acidification was most pronounced during hyperglycemic hypoxia. The changes in the sensitivity to hypoxia induced by high concentrations of D-glucose were imitated by high concentrations of D-mannose. In contrast, D-galactose, L-glucose, D-fructose, and L-fucose did not have such effects. Resistance to hypoxia, hypoxia-generated interstitial acidification, and hypoxia-induced electrophysiological damage were absent after pharmacological inhibition of nerve glycolysis with iodoacetate. These observations indicate 1) that enhanced anaerobic glycolysis produces resistance to hypoxia in hyperglycemic peripheral nerves and 2) that acidification may impair the function of peripheral axons when anaerobic glycolysis proceeds in a tissue with reduced buffering power

Divergent mutational processes distinguish hypoxic and normoxic tumours.

Many primary tumours have low levels of molecular oxygen (hypoxia), and hypoxic tumours respond poorly to therapy. Pan-cancer molecular hallmarks of tumour hypoxia remain poorly understood, with limited comprehension of its associations with specific mutational processes, non-coding driver genes and evolutionary features. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2658 cancers across 38 tumour types, we quantify hypoxia in 1188 tumours spanning 27 cancer types. Elevated hypoxia associates with increased mutational load across cancer types, irrespective of underlying mutational class. The proportion of mutations attributed to several mutational signatures of unknown aetiology directly associates with the level of hypoxia, suggesting underlying mutational processes for these signatures. At the gene level, driver mutations in TP53, MYC and PTEN are enriched in hypoxic tumours, and mutations in PTEN interact with hypoxia to direct tumour evolutionary trajectories. Overall, hypoxia plays a critical role in shaping the genomic and evolutionary landscapes of cancer

Inhibition of vascular endothelial growth factor with a sequence-specific hypoxia response element antagonist

Vascular endothelial growth factor (VEGF) and its receptors have been implicated as key factors in tumor angiogenesis that are up-regulated by hypoxia. We evaluated the effects of DNA-binding small molecules on hypoxia-inducible transcription of VEGF. A synthetic pyrrole-imidazole polyamide designed to bind the hypoxia response element (HRE) was found to disrupt hypoxia-inducible factor (HIF) binding to HIRE. In cultured HeLa cells, this resulted in a reduction of VEGF mRNA and secreted protein levels. The observed effects were polyamide-specific and dose-dependent. Analysis of genome-wide effects of the HIRE-specific polyamide revealed that a number of hypoxia-inducible genes were down-regulated. Pathway-based regulation of hypoxia-inducible gene expression with DNA-binding small molecules may represent a new approach for targeting angiogenesis

Vascular changes in the rat brain during chronic hypoxia in the presence and absence of hypercapnia.

Changes in brain vascularity in adult rats during adaptation to chronic normobaric hypoxia with or without elevated CO(2) were morphometrically investigated. Immunohistochemistry with anti-rat endothelial cell antigen (RECA-1) antibody was carried out for the vascular analysis. After the rats were subjected to hypoxia for 2 to 8 weeks (wks)(10 percent O(2) in N(2)), the total area of blood vessels was measured in 6 brain regions. After 2 wks of hypoxia, the blood vessel area was found to be significantly increased in the frontal cortex, striatum, hippocampus, thalamus, cerebellum, and medulla oblongata, by 44% , 96% , 65% , 50% , 102% and 97% , respectively. The ratio of large vessels with an area &#62; 500 micro m(2) was also increased in all brain regions. Hypoxic adaptation in brain vascularity did not change during 8 wks of hypoxia, and the hypoxia-induced levels measured in the vasculature returned to control levels 2 wks after the termination of hypoxia in areas of the brain other than the cortex and thalamus. In addition, hypoxia-induced changes in terms of the total vascular area and vessel size distribution were significantly inhibited by the elevation in CO(2), whereas chronic hypercapnia without hypoxia had no effect on brain vascularity. These findings suggested that adaptations in brain vascularity in response to hypoxia are rapidly induced, and there are regional differences in the reversibility of such vascular changes. Carbon dioxide is a potent suppressor of hypoxia-induced vascular changes, and may play an important role in vascular remodeling during the process of adaptation to chronic hypoxia.</p

The Influence of Body Size and Hemoglobin Multiplicity on Critical Oxygen Threshold in Red Drum (Sciaenops ocellatus)

Hypoxia is common in marine environments and fishes use a suite of cardiorespiratory adjustments to defend aerobic metabolism, including reducing standard metabolic rate (SMR), the minimum metabolic rate needed to sustain life at a specified temperature, or increasing hemoglobin (Hb)-O2 affinity. Nonetheless, hypoxia can constrain oxygen transport whereby fish cannot accommodate standard metabolic rate; a point known as critical oxygen tension (Pcrit). Currently, it is unclear how life history traits may impact Pcrit, but available data on red drum (Sciaenops ocellatus) suggest that its SMR decreases with size, and its transcriptome contains multiple Hb-α and Hb-β subunits. Therefore we sought to explore the influence of body size and acclimation to hypoxia. Critical oxygen tension (Pcrit) was measured for fish over a 2500-fold range in mass (0.26 - 686 g) and surprisingly showed an increase (Pcrit = 3.15 logM + 16.19; R2 = 0.44) despite decreasing SMR. Two groups of S. ocellatus (90.96 ± 5.00 g ranging from 69.7 g to 141.9 g) were also subjected to either normoxia ( > 95% P_(O_2 )) or hypoxia (30%±5% P_(O_2 )) treatment for two weeks. Only fish subjected to hypoxia treatment showed a statistically significant decrease in Pcrit after the treatment. Acclimation had no impact on gill surface area, diffusion distance or relative ventricular mass, but mRNA expression levels of the major Hb-α subunit switched from Hbα-3.1 in the normoxia group to Hbα-3.2 in the hypoxia treatment group and expression levels of Hbα-2, Hbα-3.2 and Hbβ-3.1 showed a statistically significant increase in the hypoxia treatment group. Decrease in P50 and thus an increase in Hb-O2 binding affinity was observed for fish subjected to hypoxia treatment. Taken together these data indicate that hypoxia tolerance is affected by both developmental stage and hypoxia acclimation.Integrative Biolog

Progressive Hypoxia-on-a-chip: An In Vitro Oxygen Gradient Model for Capturing the Effects of Hypoxia on Primary Hepatocytes in Health and Disease

Oxygen is vital to the function of all tissues including the liver and lack of oxygen, that is, hypoxia can result in both acute and chronic injuries to the liver in vivo and ex vivo. Furthermore, a permanent oxygen gradient is naturally present along the liver sinusoid, which plays a role in the metabolic zonation and the pathophysiology of liver diseases. Accordingly, here, we introduce an in vitro microfluidic platform capable of actively creating a series of oxygen concentrations on a single continuous microtissue, ranging from normoxia to severe hypoxia. This range approximately captures both the physiologically relevant oxygen gradient generated from the portal vein to the central vein in the liver, and the severe hypoxia occurring in ischemia and liver diseases. Primary rat hepatocytes cultured in this microfluidic platform were exposed to an oxygen gradient of 0.3–6.9%. The establishment of an ascending hypoxia gradient in hepatocytes was confirmed in response to the decreasing oxygen supply. The hepatocyte viability in this platform decreased to approximately 80% along the hypoxia gradient. Simultaneously, a progressive increase in accumulation of reactive oxygen species and expression of hypoxia‐inducible factor 1α was observed with increasing hypoxia. These results demonstrate the induction of distinct metabolic and genetic responses in hepatocytes upon exposure to an oxygen (/hypoxia) gradient. This progressive hypoxia‐on‐a‐chip platform can be used to study the role of oxygen and hypoxia‐associated molecules in modeling healthy and injured liver tissues. Its use can be further expanded to the study of other hypoxic tissues such as tumors as well as the investigation of drug toxicity and efficacy under oxygen‐limited conditions

Intrapulmonary shunting and pulmonary gas exchange during normoxic and hypoxic exercise in healthy humans

Exercise-induced intrapulmonary arteriovenous shunting, as detected by saline contrast echocardiography, has been demonstrated in healthy humans. We have previously suggested that increases in both pulmonary pressures and blood flow associated with exercise are responsible for opening these intrapulmonary arteriovenous pathways. In the present study, we hypothesized that, although cardiac output and pulmonary pressures would be higher in hypoxia, the potent pulmonary vasoconstrictor effect of hypoxia would actually attenuate exercise-induced intrapulmonary shunting. Using saline contrast echocardiography, we examined nine healthy men during incremental (65W + 30 W/2 min) cycle exercise to exhaustion in normoxia and hypoxia (fraction of inspired O-2 = 0.12). Contrast injections were made into a peripheral vein at rest and during exercise and recovery (3-5 min postexercise) with pulmonary gas exchange measured simultaneously. At rest, no subject demonstrated intrapulmonary shunting in normoxia [ arterial P-O2 (Pa-O2) = 98 +/- 10 Torr], whereas in hypoxia (Pa-O2 = 47 +/- 5 Torr), intrapulmonary shunting developed in 3/9 subjects. During exercise, similar to 90% (8/9) of the subjects shunted during normoxia, whereas all subjects shunted during hypoxia. Four of the nine subjects shunted at a lower workload in hypoxia. Furthermore, all subjects continued to shunt at 3 min, and five subjects shunted at 5 min postexercise in hypoxia. Hypoxia has acute effects by inducing intrapulmonary arteriovenous shunt pathways at rest and during exercise and has longterm effects by maintaining patency of these vessels during recovery. Whether oxygen tension specifically regulates these novel pathways or opens them indirectly via effects on the conventional pulmonary vasculature remains unclear