Glycolysis determines dichotomous regulation of T cell subsets in hypoxia

Hypoxia occurs in many pathological conditions, including chronic inflammation and tumors, and is considered to be an inhibitor of T cell function. However, robust T cell responses occur at many hypoxic inflammatory sites, suggesting that functions of some subsets are stimulated under low oxygen conditions. Here, we investigated how hypoxic conditions influence human T cell functions and found that, in contrast to naive and central memory T cells (T_N and T_CM), hypoxia enhances the proliferation, viability, and cytotoxic action of effector memory T cells (T_EM). Enhanced TEM expansion in hypoxia corresponded to high hypoxia-inducible factor 1α (HIF1α) expression and glycolytic activity compared with that observed in T_N and T_CM. We determined that the glycolytic enzyme GAPDH negatively regulates HIF1A expression by binding to adenylate-uridylate–rich elements in the 3′-UTR region of HIF1A mRNA in glycolytically inactive T_N and T_CM. Conversely, active glycolysis with decreased GAPDH availability in T_EM resulted in elevated HIF1α expression. Furthermore, GAPDH overexpression reduced HIF1α expression and impaired proliferation and survival of T cells in hypoxia, indicating that high glycolytic metabolism drives increases in HIF1α to enhance T_EM function during hypoxia. This work demonstrates that glycolytic metabolism regulates the translation of HIF1A to determine T cell responses to hypoxia and implicates GAPDH as a potential mechanism for controlling T cell function in peripheral tissue.     

Endothelially Derived Nitric Oxide Affects the Severity of Early Acetaminophen-induced Hepatic Injury in Mice

Objectives: The precise mechanism of hepatocellular toxicity following acetaminophen (APAP) poisoning remains unclear. Nitric oxide is implicated in APAP toxicity as an inflammatory signaling molecule and as a precursor to the free radical peroxynitrate. The effects of inducible nitric oxide synthase (iNOS)‐derived NO in APAP toxicity are known; however, the role of endothelial nitric oxide synthase (eNOS)‐derived NO is unknown. The authors sought to evaluate the effect of eNOS‐derived NO during APAP toxicity. Methods: C57BL6/J mice deficient in eNOS (eNOS KO) or iNOS (iNOS KO) and wild‐type mice (WT) were treated with 300 mg/kg APAP. Alanine aminotransferase levels and plasma nitrate and nitrite levels were measured. Hypoxia inducible factor (HIF)‐1α and Glucose Transporter 1 (Glut‐1) levels were determined by Western blot. Results: Alanine aminotransferase levels were significantly elevated in all treated animals. Alanine aminotransferase levels were significantly lower in eNOS KO and iNOS KO than in treated WT animals. Plasma nitrate/nitrite levels were significantly higher in WT animals than in iNOS KO and eNOS KO animals. HIF‐1α expression was increased in WT mice and decreased in iNOS KO mice. Glut‐1 is a downstream, indirect marker of HIF function. Glut‐1 expression was increased in WT and eNOS KO mice. Conclusions: Deficiency of either iNOS or eNOS results in decreased NO production and is associated with reduced hepatocellular injury following APAP poisoning. HIF‐1α and Glut‐1 levels are increased following APAP poisoning, implying that HIF‐1α is functional during the pathogenic response to APAP poisoning.     

Influence of long‐term oxygen therapy on heart rate and QT time‐series in hypoxic patients with chronic obstructive pulmonary disease

Background: There is increasing interest in the cardiovascular pathology independently associated with chronic obstructive pulmonary disease (COPD). We examined the influence of long‐term oxygen therapy (LTOT) on heart rate (RR) and QT time‐series in COPD. Methods: Ten hypoxic stable COPD patients underwent Holter ECG monitoring for 24 h and physical activity/energy expenditure monitoring for 5 days before and after LTOT. Variability of RR and QT time‐series was quantified using standard statistics and their structural (correlation/scaling) properties were assessed using multifractal analysis. Pre‐ and post‐LTOT cardiac/activity parameters were compared to examine the influence of oxygen therapy and circadian variation. Results: PaO₂ increased (P = 0·0004) whilst PaCO₂ was unchanged (P = 0·56) following LTOT. Activity/energy expenditure estimates were also unchanged following LTOT (P = 0·64–0·99), but RR variability was increased during the morning (P < 0·05) and night (P < 0·1, trend only). Multifractality of RR and QT time‐series was not significantly changed following LTOT, although QT multifractality showed some time‐dependent fluctuations. Trends in RR and QT time‐series over 24‐h were similar pre‐ and post‐LTOT, indicating a generally normal circadian response. Conclusions: An increase in HRV following LTOT (but notably in the absence of altered activity levels) provides tentative evidence that LTOT has a direct effect on heart rate control in COPD. This beneficial influence was expressed mainly during the morning, and the relevance of this diurnal variation in response requires further investigation. It was also confirmed that both RR and (to a lesser degree) QT time‐series in COPD have a multifractal structure, and this is not affected appreciably by LTOT.     

Hypoxia-inducible factor-1 confers resistance to the glycolytic inhibitor 2-deoxy-D-glucose

Hypoxic regions within solid tumors harbor cells that are resistant to standard chemotherapy and radiotherapy. Because oxygen is required to produce ATP by oxidative phosphorylation, under hypoxia, cells rely more on glycolysis to generate ATP and are thereby sensitive to 2-deoxy-d-glucose (2-DG), an inhibitor of this pathway. Universally, cells respond to lowered oxygen tension by increasing the amount of glycolytic enzymes and glucose transporters via the well-characterized hypoxia-inducible factor-1 (HIF). To evaluate the effects of HIF on 2-DG sensitivity, the following three models were used: (a) cells treated with oligomycin to block mitochondrial function in the presence (HIF(+)) or absence (HIF(-)) of hypoxia, (b) cells treated with small interfering RNA specific for HIF-1alpha and control cells cultured under hypoxia, and (c) a mutant cell line unable to initiate the HIF response and its parental HIF(+) counterpart under hypoxic conditions. In all three models, HIF increased resistance to 2-DG and other glycolytic inhibitors but not to other chemotherapeutic agents. Additionally, HIF reduced the effects of 2-DG on glycolysis (as measured by ATP and lactate assays). Because HIF increases glycolytic enzymes, it follows that greater amounts of 2-DG would be required to inhibit glycolysis, thereby leading to increased resistance to it under hypoxia. Indeed, hexokinase, aldolase, and lactate dehydrogenase were found to be increased as a function of HIF under the hypoxic conditions and cell types we used; however, phosphoglucose isomerase was not. Although both hexokinase and phosphoglucose isomerase are known to interact with 2-DG, our findings of increased levels of hexokinase more likely implicate this enzyme in the mechanism of HIF-mediated resistance to 2-DG. Moreover, because 2-DG is now in phase I clinical trials, our results suggest that glycolytic inhibitors may be more effective clinically when combined with agents that inhibit HIF.     

DNA methyltransferase inhibition restores erythropoietin production in fibrotic murine kidneys

Renal erythropoietin-producing cells (REPCs) remain in the kidneys of patients with chronic kidney disease, but these cells do not produce sufficient erythropoietin in response to hypoxic stimuli. Treatment with HIF stabilizers rescues erythropoietin production in these cells, but the mechanisms underlying the decreased response of REPCs in fibrotic kidneys to anemic stimulation remain elusive. Here, we show that fibroblast-like FOXD1⁺ progenitor-derived kidney pericytes, which are characterized by the expression of α1 type I collagen and PDGFRβ, produce erythropoietin through HIF2α regulation but that production is repressed when these cells differentiate into myofibroblasts. DNA methyltransferases and erythropoietin hypermethylation are upregulated in myofibroblasts. Exposure of myofibroblasts to nanomolar concentrations of the demethylating agent 5-azacytidine increased basal expression and hypoxic induction of erythropoietin. Mechanistically, the profibrotic factor TGF-β1 induced hypermethylation and repression of erythropoietin in pericytes; these effects were prevented by 5-azacytidine treatment. These findings shed light on the molecular mechanisms underlying erythropoietin repression in kidney myofibroblasts and demonstrate that clinically relevant, nontoxic doses of 5-azacytidine can restore erythropoietin production and ameliorate anemia in the setting of kidney fibrosis in mice.     

Functional diversification of three delta‐class glutathione S‐transferases involved in development and detoxification in Tribolium castaneum

Glutathione S‐transferases (GSTs) are members of a multifunctional enzyme superfamily. Forty‐one GSTs have been identified in Tribolium castaneum; however, none of the 41 GSTs has been functionally characterized. Here, three delta‐class GSTs, TcGSTd1, TcGSTd2 and TcGSTd3, of T. castaneum were successfully cloned and expressed in Escherichia coli. All of the studied GSTs catalysed the conjugation of reduced glutathione with 1‐chloro‐2,4‐dinitrobenzene. Insecticide treatment showed that the expression levels of TcGSTd3 and TcGSTd2 were significantly increased after exposure to phoxim and lambda‐cyhalothrin, whereas TcGSTd1 was slightly upregulated only in response to phoxim. A disc diffusion assay showed that overexpression of TcGSTD3, but not TcGSTD1 or TcGSTD2, in E. coli increased resistance to paraquat‐induced oxidative stress. RNA interference knockdown of TcGSTd1 caused metamorphosis deficiencies and reduced fecundity by regulating insulin/target‐of‐rapamycin signalling pathway‐mediated ecdysteroid biosynthesis, and knockdown of TcGSTd3 led to reduced fertility and a decreased hatch rate of the offspring, probably caused by the reduced antioxidative activity in the reproductive organs. These results indicate that TcGSTd3 and TcGSTd2 may play vital roles in cellular detoxification, whereas TcGSTd1 may play essential roles in normal development of T. castaneum. These delta‐class GSTs in T. castaneum have obtained different functions during the evolution.     

Regulation of lubricin/superficial zone protein by Wnt signalling in bovine synoviocytes

Lubricin, homologous to superficial zone protein (SZP), functions as a boundary lubricant in articular cartilage and plays an essential role in the maintenance of joint function and homeostasis. Wnt signalling plays a key role in joint development, including synovial joint formation, and several Wnt proteins are expressed in the synovium and articular cartilage in arthritis. The aim of this study was to determine the role of Wnt signalling on SZP accumulation in synoviocytes. Isolated synoviocytes from bovine knee joints were cultured with Wnt proteins (Wnt‐3a and Wnt‐5a) and antagonists or agonists of the Wnt–β‐catenin pathway or Wnt–Ca²⁺ pathway in serum‐free chemically defined medium. SZP accumulation in the culture medium was determined by enzyme‐linked immunosorbent assay. Wnt‐3a suppressed SZP accumulation via a Wnt–β‐catenin‐dependent pathway. In contrast, Wnt‐5a stimulated SZP accumulation via a β‐catenin independent pathway. The present investigation provides novel insights into the role of the Wnt signalling pathways in SZP accumulation in synoviocytes and their roles in the homeostasis of normal joints. Copyright © 2013 John Wiley & Sons, Ltd.     

Acute physiological and perceptual responses to high‐load resistance exercise in hypoxia

This study assessed whether hypoxia during high‐load resistance exercise could enhance the acute physiological responses related to muscular development. Twelve trained men performed exercise in three conditions: normoxia (fraction of inspired oxygen [F_IO₂] = 21%), moderate‐level hypoxia (F_IO₂ = 16%) and high‐level hypoxia (F_IO₂ = 13%). Exercise comprised high‐load squats and deadlifts (5 × 5 using 80% of 1‐repetition maximum with 180‐s rest). Muscle oxygenation and activation were monitored during exercise. Metabolic stress was estimated via capillary blood sampling. Perceived fatigue and soreness were also quantified following exercise. While the hypoxic conditions appeared to affect muscle oxygenation, significant differences between conditions were only noted for maximal deoxyhaemoglobin in the deadlift (P = 0·009). Blood lactate concentration increased from 1·1 to 1·2 mmol l^?1 at baseline to 9·5–9·8 mmol l^?1 after squats and 10·4–10·5 mmol l^?1 after deadlifts (P≤0·001), although there were no between‐condition differences. Perceived fatigue and muscle soreness were significantly elevated immediately and at 24 h following exercise, respectively, by similar magnitudes in all conditions (P≤0·001). Muscle activation did not differ between conditions. While metabolic stress is thought to moderate muscle activation and subsequent muscular development during hypoxic resistance training, it is not augmented during traditional high‐load exercise. This may be explained by the low number of repetitions performed and the long interset rest periods employed during this training. These findings suggest that high‐load resistance training might not benefit from additional hypoxia as has been shown for low‐ and moderate‐load training.     

Effects of haemoconcentration and haemodilution on acute hypoxia-induced pulmonary hypertension and changes in vascular compliance of isolated rabbit lungs

Objective: Erythrocytes influence the magnitude of hypoxia-induced pulmonary artery pressure increase. It is, however, unknown to what extent haemoconcentration and haemodilution affect this response and whether intrapulmonary blood volume (and thus vessel dimensions) alters the magnitude of pressure increase. Furthermore, it is unclear whether the haemodilution/haemoconcentration-dependent pressure increase is flow-related, via flow-dependent changes in vasomotor tone or rheologic effects, or can also be observed under no-flow conditions. Design: Experimental study in isolated rabbit lungs (n = 12) perfused with autologous blood at constant flow (100 ml/min) and ventilated with 5 % carbon dioxide in air. Setting: Laboratory for experimental studies. Interventions: Haemoconcentration (centrifugation) and haemodilution (Krebs-Henseleit/albumin) were carried out, resulting in haematocrits between 50 % and 0 %. During hypoxic ventilation, inspiratory oxygen fraction was reduced from 0.20 to 0.03. Measurements and results: Under constant flow conditions, haemodilution (from a Hct of 34–36 % to 0–1 %) decreased hypoxic pulmonary artery pressure response to one-third (from 10.8 ± 2.3 cmH₂O to 3.1 ± 1.0 cmH₂O, P < 0.05), while haemoconcentration did not affect the magnitude of hypoxic response (10.5 ± 2.0 cmH₂O). For all haematocrit values an increase in pulmonary blood volume (by 5 ml) decreased the magnitude of pressure response. Hypoxia-induced changes in static vascular filling pressure (double occlusion pressure) and vascular compliance were used to assess the strength of hypoxic vasoconstriction under static conditions. Neither haemoconcentration nor haemodilution altered hypoxia-induced changes in either variable. Conclusions: The magnitude of the acute hypoxic pressure response is not altered by haemoconcentration, but significantly reduced by haemodilution. In contrast, neither haemoconcentration nor haemodilution influenced hypoxia-induced changes in static vascular filling pressure and compliance. This suggests that the degree of hypoxic pulmonary vasoconstriction is not affected under static conditions and that the red blood cell-dependence of the magnitude of hypoxic pressure response is based on flow-related mechanisms. Received: 19 October 1999 Final revision received: 4 April 2000 Accepted: 28 April 2000     

Hypoxic preconditioning of myoblasts implanted in a tissue engineering chamber significantly increases local angiogenesis via upregulation of myoblast vascular endothelial growth factor‐A expression and downregulation of miRNA‐1, miRNA‐206 and angiopoietin‐1

Vascularization is a major hurdle for growing three‐dimensional tissue engineered constructs. This study investigated the mechanisms involved in hypoxic preconditioning of primary rat myoblasts in vitro and their influence on local angiogenesis postimplantation. Primary rat myoblast cultures were exposed to 90 min hypoxia at <1% oxygen followed by normoxia for 24 h. Real time (RT) polymerase chain reaction evaluation indicated that 90 min hypoxia resulted in significant downregulation of miR‐1 and miR‐206 (p < 0.05) and angiopoietin‐1 (p < 0.05) with upregulation of vascular endothelial growth factor‐A (VEGF‐A; p < 0.05). The miR‐1 and angiopoietin‐1 responses remained significantly downregulated after a 24 h rest phase. In addition, direct inhibition of miR‐206 in L6 myoblasts caused a significant increase in VEGF‐A expression (p < 0.05), further establishing that changes in VEGF‐A expression are influenced by miR‐206. Of the myogenic genes examined, MyoD was significantly upregulated, only after 24 h rest (p < 0.05). Preconditioned or control myoblasts were implanted with Matrigel? into isolated bilateral tissue engineering chambers incorporating a flow‐through epigastric vascular pedicle in severe combined immunodeficiency mice and the chamber tissue harvested 14 days later. Chambers implanted with preconditioned myoblasts had a significantly increased percentage volume of blood vessels (p = 0.0325) compared with chambers implanted with control myoblasts. Hypoxic preconditioned myoblasts promote vascularization of constructs via VEGF upregulation and downregulation of angiopoietin‐1, miR‐1 and miR‐206. The relatively simple strategy of hypoxic preconditioning of implanted cells ‐ including non‐stem cell types – has broad, future applications in tissue engineering of skeletal muscle and other tissues, as a technique to significantly increase implant site angiogenesis.     

Evaluating the feasibility of utilizing the small molecule phenamil as a novel biofactor for bone regenerative engineering

Osteoblast cell adhesion and differentiation on biomaterials are important achievements necessary for implants to be useful in bone regenerative engineering. Recombinant bone morphogenetic proteins (BMPs) have been shown to be important for these processes; however, there are many challenges associated with the widespread use of these proteins. A recent report demonstrated that the small molecule phenamil, a diuretic derivative, was able to induce osteoblast differentiation and mineralization in vitro via the canonical BMP signalling cascade (Park et al., 2009). In this study, the feasibility of using phenamil as a novel biofactor in conjunction with a biodegradable poly(lactide‐co‐glycolide acid) (PLAGA) polymeric scaffold for engineering bone tissue was evaluated. The in vitro cellular behaviour of osteoblast‐like MC3T3‐E1 cells cultured on PLAGA scaffolds in the presence of phenamil at 10 μM were characterized with regard to initial cell adhesion, proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. The results demonstrate that phenamil supported cell proliferation, promoted ALP activity and facilitated matrix mineralization of osteoblast‐like MC3T3‐E1 cells. Moreover, in this study, we found that phenamil promoted integrin‐mediated cell adhesion on PLAGA scaffolds. It was also shown that phenamil encapsulated within porous, microsphere PLAGA scaffolds retained its osteogenic activity upon release. Based on these findings, the small molecule phenamil has the potential to serve as a novel biofactor for the repair and regeneration of bone tissues. Copyright © 2012 John Wiley & Sons, Ltd.     

Knockdown of mitogen‐activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos

Arthropod‐borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK‐ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti‐MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3–4 days after morpholino feeding. Further, anti‐MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.     

Hyperosmolarity and hypoxia induce chondrogenesis of adipose-derived stem cells in a collagen type 2 hydrogel

Apart from soluble growth factors, various other biophysicochemical cues are known to promote chondrogenesis. Under physiological conditions, cartilage in the joint comprises a hyperosmotic and hypoxic environment. Therefore, in this study, we examined the inductive effects of hyperosmotic and/or hypoxic conditions on adipose stem cells (ASCs) and compared them with conventional TGFβ1‐induction. After encapsulation in collagen type II hydrogels and specific induction, ASCs were assessed for viability, proliferation, morphology and chondrogenic differentiation potential. Viability was similar under all conditions, with low proliferative activity. After 4 days, hypoxia and/or hyperosmolarity did not affect round cell morphology, while cells were mainly stretched in the TGFβ1‐induced group. At 21 days, the TGFß1‐treated group had aggregated into a cell nodule. Hyperosmolarity mimicked this aggregation to a lesser extent, whereas cells under hypoxia stretched out after 21 days, with a combined effect in the hypoxic/hyperosmotic group. Both individual and combined hyperosmotic and/or hypoxic conditions significantly upregulated SOX5, SOX9, COMP and Link‐p gene expression compared with the non‐induced group, and to similar levels as the TGFβ1‐induced group. GAG synthesis in both hydrogel and medium was increased under hypoxic conditions, whereas hyperosmolarity decreased GAG formation in the hydrogels, but increased GAG formation in the medium. We conclude that in a joint mimicking the three‐dimensional (3D) micro‐environment, a combination of hyperosmolarity and hypoxia is able to induce chondrogenesis to the same extent as TGFβ1. This might lead to an interesting alternative when considering short‐term triggering in a one‐step surgical procedure for the treatment of cartilaginous defects. Copyright © 2011 John Wiley & Sons, Ltd.