Reduced Oxygen Tension Research Articles

Methylsulfonylmethane relieves cobalt chloride-induced hypoxic toxicity in C2C12 myoblasts

AimsIn biology and medicine, hypoxia refers to reduced oxygen tension or oxygen starvation resulting from various environmental or pathological conditions. Prolonged hypoxia may lead to an imbalance in protein production and a loss of muscle mass in animals. The physiological response to hypoxia includes oxidative stress-induced activation of complex cell-signaling networks such as hypoxia-inducible factor (HIF), phosphoinositide 3-kinase (PI3K), and Janus kinase/signal transducer and activator of transcription (JAK-STAT). Methylsulfonylmethane (MSM) is a natural sulfur compound that regulates HIF-1α expression and provides cytoprotection from oxidative stress. In this study, we explored the anti-hypoxic activity and cytoprotective effect of MSM in cobalt chloride (CoCl2)-induced hypoxic C2C12 mouse myoblast culture. Materials and methodsWe used western blotting, real time PCR, flow cytometry for molecular signaling studies and we also used MTT assay and ChIP assay along with comet assay for cellular processes. Key findingsMSM prevented the CoCl2 induced cytotoxicity. Molecular markers of hypoxia, induced by CoCl2, were normalized or reduced by MSM, which also inhibited the effect of CoCl2-induced JAK2/STAT5b/Cyclin D1 and PI3K/AKT signaling. CoCl2-induced oxidative stress results in activation of the NRF2/HO-1-mediated cell survival pathway and inhibition of DNA repair, both of which were prevented by MSM. SignificanceWe suggest MSM can be considered as a candidate drug for reducing the effects of hypoxia in both animals and humans.

Soluble P-Selectin and von Willebrand Factor Rise in Healthy Volunteers Following Non-exertional Ascent to High Altitude.

Reduced oxygen tensions experienced at high altitudes are thought to predispose to thrombosis, yet there are few studies linking hypoxia, platelet activation, and thrombosis. Reports of platelet phenotypes in hypoxia are inconsistent, perhaps due to differing degrees of hypoxia experienced and the duration of exposure. This study aimed to investigate the relationship between soluble P-selectin, a marker of platelet activation, and von Willebrand factor (vWF) on exposure to hypoxia. We measured plasma concentrations of P-selectin and vWF in sixteen healthy volunteers before, during and after the APEX 2 expedition. APEX 2 consisted of a non-exertional ascent to 5,200 m, followed by 7 consecutive days at high altitude. We showed that high altitude significantly increased mean plasma P-selectin and vWF compared to pre-expedition levels. Both plasma marker levels returned to baseline post-expedition. We found a strong positive correlation between vWF and P-selectin, but no association between P-selectin and platelet count. Our results are consistent with previous work showing evidence of platelet activation at high altitude and demonstrate that the rise in P-selectin is not simply due to an increase in platelet count. As vWF and P-selectin could be derived from either platelets or endothelial cells, further work assessing more specific markers of endothelial activation is proposed to provide insight into the source of these potential pro-thrombotic biomarkers at altitude.

Lycopene Supplementation to Serum-Free Maturation Medium Improves In Vitro Bovine Embryo Development and Quality and Modulates Embryonic Transcriptomic Profile.

Bovine embryos are typically cultured at reduced oxygen tension to lower the impact of oxidative stress on embryo development. However, oocyte in vitro maturation (IVM) is performed at atmospheric oxygen tension since low oxygen during maturation has a negative impact on oocyte developmental competence. Lycopene, a carotenoid, acts as a powerful antioxidant and may protect the oocyte against oxidative stress during maturation at atmospheric oxygen conditions. Here, we assessed the effect of adding 0.2 μM lycopene (antioxidant), 5 μM menadione (pro-oxidant), and their combination on the generation of reactive oxygen species (ROS) in matured oocytes and the subsequent development, quality, and transcriptome of the blastocysts in a bovine in vitro model. ROS fluorescent intensity in matured oocytes was significantly lower in the lycopene group, and the resulting embryos showed a significantly higher blastocyst rate on day 8 and a lower apoptotic cell ratio than all other groups. Transcriptomic analysis disclosed a total of 296 differentially expressed genes (Benjamini–Hochberg-adjusted p < 0.05 and ≥ 1-log2-fold change) between the lycopene and control groups, where pathways associated with cellular function, metabolism, DNA repair, and anti-apoptosis were upregulated in the lycopene group. Lycopene supplementation to serum-free maturation medium neutralized excess ROS during maturation, enhanced blastocyst development and quality, and modulated the transcriptomic landscape.

Hypoxia-Conditioned Mesenchymal Stem Cells in Tissue Regeneration Application.

Mesenchymal stem cells (MSCs) have been demonstrated as promising cell sources for tissue regeneration due to their capability of self-regeneration, differentiation, and immunomodulation. MSCs also exert extensive paracrine effects through release of trophic factors and extracellular vesicles (EVs). However, despite extended exploration of MSCs in preclinical studies, the results are far from satisfactory due to the poor engraftment and low level of survival after implantation. Hypoxia preconditioning has been proposed as an engineering approach to improve the therapeutic potential of MSCs. During in vitro culture, hypoxic conditions can promote MSC proliferation, survival, and migration through various cellular responses to the reduction of oxygen tension. The multilineage differentiation potential of MSCs is altered under hypoxia, with consistent reports of enhanced chondrogenesis. Hypoxia also stimulates the paracrine activities of MSCs and increases the production of secretome both in terms of soluble factors as well as EVs. The secretome from hypoxia-preconditioned MSCs play important roles in promoting cell proliferation and migration, enhancing angiogenesis while inhibiting apoptosis and inflammation. In this review, we summarize current knowledge of hypoxia-induced changes in MSCs and discuss the application of hypoxia-preconditioned MSCs as well as hypoxic secretome in different kinds of disease models. Impact statement Mesenchymal stem cells (MSCs) have been applied in numerous cell-based and secretome-based therapies for tissue regeneration. Hypoxic conditions enhance the function of MSCs by increasing proliferation, survival, homing, differentiation, and paracrine activities. A timely up-to-date comprehensive overview of the effect of low oxygen tension to MSC, with emphasis on the influence and molecular mechanism of hypoxia preconditioning toward MSC's functionality is provided, including the therapeutic use of hypoxia-preconditioned MSC as well as hypoxic secretome in various prove-of-concept disease models. This knowledge would contribute to future engineering of MSC culture conditions for improved translational application.

Engagement of vascular early response genes typifies mild cognitive impairment.

Molecular responses in the brains of persons with mild cognitive impairment (MCI), the earliest transitional state between normal aging and early Alzheimer's disease (AD), are poorly understood. We examined AD-related neuropathology and transcriptome changes in the neocortex of individuals with MCI relative to controls and temporal responses to the mild hypoxia in mouse brains. Subsets of vascular early response to hypoxia genes were upregulated in MCI prior to the buildup of AD neuropathology. Early activation of pro-angiogenic hypoxia-inducible factor signaling in response to mild hypoxia was detected in mouse brains similar to those that were altered in MCI. Protracted responses to hypoxia were characterized by activation of phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt)-the mammalian target of rapamycin (mTOR) pathways in brain microvessel isolates. These findings suggest that cerebrovascular remodeling is an important antecedent to the development of dementia and a component of the homeostatic response to reduced oxygen tension in aging prior to the onset of AD.

High altitude simulation testing in patients with congenital diaphragmatic hernia

Aim of studyCongenital Diaphragmatic Hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension. Many patients receive care in specialty centers requiring air travel upon discharge and for subsequent follow-up care. Premature infants can experience significant hypoxia in flight, but this has not been studied in the CDH population. This report describes our center's experience with simulated altitude testing among CDH patients. MethodsIn a single center retrospective cohort study, CDH patients who underwent a High Altitude Simulation Test (HAST) from 2006 to 2019 were analyzed. HAST simulates increased altitude by reducing oxygen tension to an FIO2 of 0.15. Patients were tested only when flight was anticipated. Patients requiring oxygen were challenged on their baseline requirement. To pass, patients had to maintain oxygen saturation > 90%, and 94% if diagnosed with pulmonary hypertension. Supplemental oxygen was titrated as needed to achieve this goal. ResultsOf twenty patients tested, only six (30%) passed on their first attempt. Ten (50%) eventually passed, after an average of 3.2 additional attempts over 1.8 years. No patient passed initially who utilized ECMO support, diaphragmatic agenesis, or had elevated right ventricular pressure on echocardiogram. All patients achieved the targeted SpO2 with supplemental oxygen. ConclusionCDH patients experience hypoxia when exposed to the simulated hypobaric nature of air travel and therefore may become hypoxic in flight, requiring oxygen supplementation. Disease severity seems to correlate with risk of in-flight hypoxia. This data suggests that CDH patients should be screened to assess their need for supplemental oxygen to ensure safe air travel. Level of evidenceLevel 4 case series.

Role of hydrogen sulfide in the regulation of respiration, blood flow and bile secretory function of the liver

In acute experiments on laboratory rats, intra-portal administration of L-cysteine (20 mg/kg), the precursor of hydrogen sulfide synthesis, stimulated oxygen consumption of liver by 38.6% and reduced oxygen tension by 37.1%. Activation of tissue respiration occurred due to the strengthening of oxygen-dependent synthetic processes in liver, in particular those associated with mitochondrial enzyme-catalysed bile acid biosynthesis through the acidic pathway. The concentrations of taurocholic acid and mixtures of taurodeoxycholic and taurohenodeoxycholic acids increased by 10.3 and 17.9%, respectively, compared to the initial levels. In addition, the level of free cholesterol was decreased by 33.9% and esterification processes were intensified, as indicated by an increase in the concentration of esterified cholesterol by 22.6% in the bile of rats. The latter was to some extent confirmed by a decrease in the level of free bile acids (by 15.8%) involved in the biosynthesis of cholesterol esters and intensification of tissue respiration in the liver. L-cysteine dilated intrahepatic vessels, resulting in a significant decrease of the systemic blood pressure and blood pressure in the portal vein by 17.6 and 24.5%, respectively. L-cysteine increased the rate of local blood flow in the liver and blood supply by 28.2 and 24.4%, respectively. Blockade of cystathionine-γ-lyase by DL-propargylglycine (11 mg/kg) significantly inhibited the L-cysteine-induced tissue respiration and bile acid biosynthesis in the liver. Administration of DL-propargylglycine resulted in constriction of blood vessels of the liver and, as a consequence, to an increased blood pressure and a decreased blood flow rate in tissue. Our data point to an involvement of hydrogen sulfide in the regulation of liver tissue respiration and bile secretory function.

Extracellular Vesicle Transmission of Chemoresistance to Ovarian Cancer Cells Is Associated with Hypoxia-Induced Expression of Glycolytic Pathway Proteins, and Prediction of Epithelial Ovarian Cancer Disease Recurrence.

Simple SummaryOvarian cancer is one of the most lethal cancers affecting women worldwide. Its high mortality rate is often attributed to the non-specific nature of early symptoms of the disease. Developing a better understanding of the disease progression and identifying clinically useful biomarkers that aid in clinical management are requisite to reducing the mortality rate of ovarian cancer. Reduced oxygen tension (i.e., hypoxia) is not only a characteristic of solid tumors but may also enhance the metastatic capacity of tumors by inducing the release of tumor growth promoting factors. Recently, it has been proposed that small tumor-derived extracellular vesicles (sEVs) facilitate cancer progression. In this study, we established that sEVs produced under low oxygen tension induce a metabolic switch in ovarian cancer cells associated with changes in glycolytic pathway proteins that promote resistance to carboplatin. Significantly, we identified a suite of sEV-associated glycolysis pathway proteins that are present in patients with ovarian cancer that can predict disease recurrence with over 90% accuracy.Hypoxia is a key regulator of cancer progression and chemoresistance. Ambiguity remains about how cancer cells adapt to hypoxic microenvironments and transfer oncogenic factors to surrounding cells. In this study, we determined the effects of hypoxia on the bioactivity of sEVs in a panel of ovarian cancer (OvCar) cell lines. The data obtained demonstrate a varying degree of platinum resistance induced in OvCar cells when exposed to low oxygen tension (1% oxygen). Using quantitative mass spectrometry (Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra, SWATH) and targeted multiple reaction monitoring (MRM), we identified a suite of proteins associated with glycolysis that change under hypoxic conditions in cells and sEVs. Interestingly, we identified a differential response to hypoxia in the OvCar cell lines and their secreted sEVs, highlighting the cells’ heterogeneity. Proteins are involved in metabolic reprogramming such as glycolysis, including putative hexokinase (HK), UDP-glucuronosyltransferase 1–6 (UD16), and 6-phosphogluconolactonase (6 PGL), and their presence correlates with the induction of platinum resistance. Furthermore, when normoxic cells were exposed to sEVs from hypoxic cells, platinum-resistance increased significantly (p < 0.05). Altered chemoresistance was associated with changes in glycolysis and fatty acid synthesis. Finally, sEVs isolated from a clinical cohort (n = 31) were also found to be enriched in glycolysis-pathway proteins, especially in patients with recurrent disease. These data support the hypothesis that hypoxia induces changes in sEVs composition and bioactivity that confers carboplatin resistance on target cells. Furthermore, we propose that the expression of sEV-associated glycolysis-pathway proteins is predictive of ovarian cancer recurrence and is of clinical utility in disease management.

HIF1\u03b1-dependent induction of the mitochondrial chaperone TRAP1 regulates bioenergetic adaptations to hypoxia

The mitochondrial paralog of the Hsp90 chaperone family TRAP1 is often induced in tumors, but the mechanisms controlling its expression, as well as its physiological functions remain poorly understood. Here, we find that TRAP1 is highly expressed in the early stages of Zebrafish development, and its ablation delays embryogenesis while increasing mitochondrial respiration of fish larvae. TRAP1 expression is enhanced by hypoxic conditions both in developing embryos and in cancer models of Zebrafish and mammals. The TRAP1 promoter contains evolutionary conserved hypoxic responsive elements, and HIF1α stabilization increases TRAP1 levels. TRAP1 inhibition by selective compounds or by genetic knock-out maintains a high level of respiration in Zebrafish embryos after exposure to hypoxia. Our data identify TRAP1 as a primary regulator of mitochondrial bioenergetics in highly proliferating cells following reduction in oxygen tension and HIF1α stabilization.

Rat in vitro spermatogenesis promoted by chemical supplementations and oxygen-tension control

In vitro spermatogenesis (IVS) using air–liquid interphase organ culture method is possible with mouse testis tissues. The same method, however, has been hardly applicable to animals other than mice, only producing no or limited progression of spermatogenesis. In the present study, we challenged IVS of rats with modifications of culture medium, by supplementing chemical substances, including hormones, antioxidants, and lysophospholipids. In addition, reducing oxygen tension by placing tissues in an incubator of lower oxygen concentration and/or applying silicone cover ceiling on top of the tissue were effective for improving the spermatogenic efficiency. Through these modifications of the culture condition, rat spermatogenesis up to round spermatids was maintained over 70 days in the cultured tissue. Present results demonstrated a significant progress in rat IVS, revealing conditions commonly favorable for mice and rats as well as finding rat-specific optimizations. This is an important step towards successful IVS in many animal species, including humans.

A Rapid Shift from Chronic Hyperoxia to Normoxia Induces Systemic Anaphylaxis via Transient Receptor Potential Ankyrin 1 Channels on Mast Cells.

Extensive activation of mast cells is the major switch that triggers systemic anaphylaxis, resulting in the subsequent release of anaphylactic mediators into circulation. We previously demonstrated that rapid changes in oxygen tension lead to mast cell degranulation, and the released tryptase triggers retinal angiogenesis in a murine oxygen-induced retinopathy model. However, whether a rapid shift from hyperoxia to normoxia (relative hypoxic stress) is a risk factor for systemic anaphylaxis remains unknown. In this study, we demonstrated that the relative hypoxia stress induces systemic mast cell activation via transient receptor potential ankyrin 1 (TRPA1) channels, which immediately leads to hypothermia and increased vascular permeability in adult mice. Although mast cell-deficient or TRPA1-deficient mice did not exhibit anaphylactic symptoms following a rapid sift to normoxia, preinjection with bone marrow-derived cultured mast cells (BMCMCs) derived from wild-type TRPA1-expressing mice restored anaphylactic responses. In addition, we found that the rapid reductions in oxygen tension in a culture atmosphere triggered the degranulation of BMCMCs derived from wild-type TRPA1-expressing mice but not that of BMCMCs derived from TRPA1-deficient mice. In human LAD2 mast cells, the relative hypoxic stress led to the degranulation, which was suppressed by the addition of a TRPA1 inhibitor. Gradual reductions from hyperoxia to normoxia led to no anaphylactic symptoms. Our results demonstrated that TRPA1-triggered mast cell degranulation is a novel pathway that induces anaphylactic shock without Ag-Ab reactions. These findings introduce a potential role for oxygen in inducing mast cell-dependent anaphylaxis and highlight the need to reconsider chronic pure oxygen therapy for anoxic diseases.

Abstract 17038: Influence of Cardiac Workload on Vascular Pyridine Nucleotide Redox State

Introduction: Myocardial perfusion is enhanced during periods of elevated cardiomyocyte oxygen consumption. The coupling between blood flow and myocardial oxygen demand is mediated by redox-dependent signals, yet the influence of cardiac workload on arterial myocyte pyridine nucleotide redox status is unknown. Hypothesis: Increased cardiac workload acutely alters intramyocardial arterial myocyte [NADH] i :[NAD + ] i . Methods and Results: The genetically-encoded fluorescent biosensor Peredox-mCherry was used to monitor real-time changes in intracellular [NADH] i :[NAD] i in isolated arterial myocytes. Treatment of cells expressing Peredox-mCherry with a range of [lactate] o :[pyruvate] o resulted in significant changes in green (Ex,Em: 400, 510 nm): red (Ex,Em: 585,615 nm) fluorescence ratio (1.903 ± 0.010, 10 mM lactate, 1.072 ± 0.073, 10 mM pyruvate), affording accurate quantification of arterial myocyte [NADH] i :[NAD] i . Increased arterial myocyte [NADH] i :[NAD] i was observed in response to reductions in oxygen tension from 5% (0.0021 ± 0.0001) to 1% (0.0050 ± 0.0001; P&lt;0.05). [NADH] i :[NAD] i was not altered by direct stimulation of arterial myocytes with the beta-adrenergic agonist isoproterenol (1 μM; P = .0630). Furthermore, biosensor-expressing arterial myocytes in co-culture with human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) recapitulated the effects of elevated [lactate] o :[pyruvate] o and reduced oxygen tension, exhibiting frequency-dependent increases in [NADH] i :[NAD] I following electrical pacing (e.g., ~3.3 fold increase in 3 Hz vs. unpaced; P&lt;0.05). Consistent with these results, intramyocardial coronary arteries in mice acutely treated with dobutamine (10 mg/kg, i.p.) to drive high cardiac work exhibited significantly greater lactate:pyruvate ratio, used as a surrogate for [NADH] i :[NAD] i , compared with those in hearts treated with vehicle (3.17 ± 0.23 vs. 2.21 ± 0.11; P&lt;0.05). Conclusions: Our results indicate that acute increases in cardiac workload promote the elevation of [NADH] i :[NAD] i in intramyocardial arterial myocytes.

An evaluation of dermal microcirculatory occlusion under repeated mechanical loads: Implication of lymphatic impairment in pressure ulcers

Pressure ulcers are caused by prolonged mechanical loads deforming the underlying soft tissues. However, the mechanical loads for microcirculatory occlusion are unknown. The present study was designed to characterize the simultaneous response of microvascular and lymphatic structures under repeated mechanical loading. The effects of two distinct loading/unloading cycles involving (a) incremental pressures 30, 60, and 90mmHg and (b) three repeated cycles of 30mmHg were evaluated on a cohort of able-bodied volunteers. Microvascular response involved the monitoring of transcutaneous gas tensions, while dermal lymphatic activity was estimated from near-infrared imaging. Responses were compared during each load and recovery cycle. Changes in microvascular response were dependent on the load magnitudes, with 30mmHg resulting in a reduction in oxygen tension only, while 90mmHg affected both oxygen and carbon dioxide values in most cases (54%). By contrast, lymphatics revealed near total occlusion at 30mmHg. Although there were intersubject differences, temporal trends consistently revealed partial or full impairment under load, with recovery during off-loading. The pressure required to cause microcirculatory occlusion differed between individuals, with lymphatic impairment occurring at a lower pressure to that of microvascular vessels. This highlights the need for personalized care strategies and regular off-loading of vulnerable tissues.

The mutational impact of culturing human pluripotent and adult stem cells

Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.

Proteomics Analysis Reveals that Warburg Effect along with Modification in Lipid Metabolism Improves In Vitro Embryo Development under Low Oxygen.

The molecular mechanism regulating embryo development under reduced oxygen tension remains elusive. This study aimed to identify the molecular mechanism impacting embryo development under low oxygen conditions. Buffalo embryos were cultured under 5% or 20% oxygen and were evaluated according to their morphological parameters related to embryo development. The protein profiles of these embryos were compared using iTRAQ-based quantitative proteomics. Physiological O2 (5%) significantly promoted blastocyst yield, hatching rate, embryo quality and cell count as compared to atmospheric O2 (20%). The embryos in the 5% O2 group had an improved hatching rate of cryopreserved blastocysts post-warming (p < 0.05). Comparative proteome profiles of hatched blastocysts cultured under 5% vs. 20% O2 levels identified 43 differentially expressed proteins (DEPs). Functional analysis indicated that DEPs were mainly associated with glycolysis, fatty acid degradation, inositol phosphate metabolism and terpenoid backbone synthesis. Our results suggest that embryos under physiological oxygen had greater developmental potential due to the pronounced Warburg Effect (aerobic glycolysis). Moreover, our proteomic data suggested that higher lipid degradation, an elevated cholesterol level and a higher unsaturated to saturated fatty acid ratio might be involved in the better cryo-survival ability reported in embryos cultured under low oxygen. These data provide new information on the early embryo protein repertoire and general molecular mechanisms of embryo development under varying oxygen levels.

Importance of Osmolarity and Oxygen Tension for Cartilage Tissue Engineering.

For cartilage repair in vivo or evaluation of new therapeutic approaches in vitro, the generation of functional cartilage tissue is of crucial importance and can only be achieved if the phenotype of the chondrocytes is preserved. Three-dimensional (3D) cell culture is broadly used for this purpose. However, adapting culture parameters like the oxygen tension or the osmolarity to their physiological values is often omitted. Indeed, articular cartilage is an avascular tissue subjected to reduced oxygen tension and presenting and increased osmolarity compared with most other tissues. In this study, we aimed at evaluating the effect of a physiological oxygen tension (3% instead of 21%) and physiological osmolarity (430 vs. 330 mOsm in nonadjusted DMEM) and the combination of both on the cell proliferation, matrix production, and the phenotype of porcine chondrocytes in a scaffold-free 3D culture system. We observed that a physiological osmolarity had no effect on cell proliferation and matrix production but positively influences the chondrocyte phenotype. A physiological oxygen level prevented cell proliferation but resulted in an increased matrix content/million cells and had a positive influence on the chondrocyte phenotype as well. The strongest benefit was reached with the combination of both physiological osmolarity and oxygen levels; with these conditions, type I collagen expression became undetectable. In addition, at 3% O2 the chondrocytes-matrix constructs were found to more closely resemble native cartilage regarding the matrix-to-cell ratio. In conclusion, this study clearly demonstrates the benefit of using physiological oxygen tension and osmolarity in cartilage tissue engineering with the combination of both showing the strongest benefit on the chondrocyte phenotype.

Abstract C122: NUC-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro

Abstract Background: Cordycepin or 3′-deoxyadenosine inhibits the growth of cancer cells by several possible mechanisms. The ProTide NUC-7738, a phosphoramidate transformation of cordycepin, is designed to overcome cancer resistance mechanisms through direct release of 3’-deoxyadenosine monophosphate in cells. We hypothesized that it might activate AMP-activated protein kinase (AMPK), the key cellular energy sensor, and thus disrupt metabolic homeostasis in cancer cells. Clear cell renal cell carcinoma (ccRCC) is characterized by lipid accumulation due to dysregulation of the genes fundamental in metabolic pathways and so this disease might be a promising target for metabolic treatment. Phosphorylation of AMPK (pAMPK) is associated with downregulation of mTOR signalling, highlighting the potential of NUC-7738 to regulate this important cancer pathway. Methods: The expression of pAMPK and AMPK in ccRCC from 293 patients was analyzed by immunofluorescence, images were captured digitally and were analyzed using QuPath software. The effect of NUC-7738 on the growth and confluence of nine renal cancer cell lines was assessed using SRB assay and Celigo scanner, under both 0.5% oxygen and normoxic conditions. Western blotting was used to assess changes in the ratio of pAMPK:AMPK caused by NUC-7738 and results read using LiCor Odyssey. Effect of NUC-7738 on AMPK activation in ex vivo ccRCC tissue slices was analyzed using immunofluorescence and QuPath software. Results: Whereas AMPK was widely expressed in ccRCC, pAMPK was focal and very heterogeneous. Cell lines by contrast strongly expressed pAMPK, but this was reduced when culture conditions were altered to be more physiologically appropriate through reduction of oxygen tension and lowering glucose levels. NUC-7738 inhibited the growth of renal cancer cell lines under both hypoxic and normoxic conditions, and increased pAMPK levels were noted after 1 hour, 6 hours, 24 hours, and 48 hours treatment, with inhibition of mTOR activity predominantly observed after 48 hours. NUC-7738 also increased pAMPK levels in ex vivo ccRCC tissue slices. Conclusion: Activation of AMPK was generally low in both primary ccRCC tissue and cell lines grown under physiologically appropriate conditions. NUC-7738 caused activation of AMPK in ex vivo ccRCC tissue slices and in cell lines, and demonstrated efficacy against cell lines in both normoxic and hypoxic conditions. Renal cancer tissue typically has low expression of pAMPK, raising the prospect that AMPK modulation may offer a therapeutic option for ccRCC. These results suggest that inhibition of the mTOR pathway may be one of the anti-cancer mechanisms through which NUC-7738 exerts its activity. Citation Format: Mary Kudsy, Mustafa Elshani, Sarah Puthur, In Hwa Um, Grant D. Stewart, Maeve Rahilly, Alex Chapman, Michelle Myers, David J. Harrison. NUC-7738, a novel ProTide modification of 3’-deoxyadenosine, activates AMPK and kills renal cancer cells in vitro [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C122. doi:10.1158/1535-7163.TARG-19-C122

High-throughput assessment of hemoglobin polymer in single red blood cells from sickle cell patients under controlled oxygen tension

Sickle cell disease (SCD) is caused by a variant hemoglobin molecule that polymerizes inside red blood cells (RBCs) in reduced oxygen tension. Treatment development has been slow for this typically severe disease, but there is current optimism for curative gene transfer strategies to induce expression of fetal hemoglobin or other nonsickling hemoglobin isoforms. All SCD morbidity and mortality arise directly or indirectly from polymer formation in individual RBCs. Identifying patients at highest risk of complications and treatment candidates with the greatest curative potential therefore requires determining the amount of polymer in individual RBCs under controlled oxygen. Here, we report a semiquantitative measurement of hemoglobin polymer in single RBCs as a function of oxygen. The method takes advantage of the reduced oxygen affinity of hemoglobin polymer to infer polymer content for thousands of RBCs from their overall oxygen saturation. The method enables approaches for SCD treatment development and precision medicine.

Natural Tolerance to Ischemia and Hypoxemia in Diving Mammals: A Review.

Reperfusion injury follows ischemia/reperfusion events occurring during myocardial infarction, stroke, embolism, and other peripheral vascular diseases. Decreased blood flow and reduced oxygen tension during ischemic episodes activate cellular pathways that upregulate pro-inflammatory signaling and promote oxidant generation. Reperfusion after ischemia recruits inflammatory cells to the vascular wall, further exacerbating oxidant production and ultimately resulting in cell death, tissue injury, and organ dysfunction. Diving mammals tolerate repetitive episodes of peripheral ischemia/reperfusion as part of the cardiovascular adjustments supporting long duration dives. These adjustments allow marine mammals to optimize the use of their body oxygen stores while diving but can result in selectively reduced perfusion to peripheral tissues. Remarkably, diving mammals show no apparent detrimental effects associated with these ischemia/reperfusion events. Here, we review the current knowledge regarding the strategies marine mammals use to suppress inflammation and cope with oxidant generation potentially derived from diving-induced ischemia/reperfusion.

Blood flow restricted resistance exercise and reductions in oxygen tension attenuate mitochondrial H2 O2 emission rates in human skeletal muscle.

Blood flow restricted resistance exercise (BFR-RE) is capable of inducing comparable adaptations to traditional resistance exercise (RE), despite a lower total exercise volume. It has been suggested that an increase in reactive oxygen species (ROS) production may be involved in this response; however, oxygen partial pressure ( ) is reduced during BFR-RE, and the influence of on mitochondrial redox balance remains poorly understood. In human skeletal muscle tissue, we demonstrate that both maximal and submaximal mitochondrial ROS emission rates are acutely decreased 2h following BFR-RE, but not RE, occurring along with a reduction in tissue oxygenation during BFR-RE. We further suggest that is involved in this response because an in vitro analysis revealed that reducing dramatically decreased mitochondrial ROS emissions and electron leak to ROS. Altogether, these data indicate that mitochondrial ROS emission rates are attenuated following BFR-RE, and such a response is likely influenced by reductions in . Low-load blood flow restricted resistance exercise (BFR-RE) training has been proposed to induce comparable adaptations to traditional resistance exercise (RE) training, however, the acute signalling events remain unknown. Although a suggested mechanism of BFR-RE is an increase in reactive oxygen species (ROS) production, oxygen partial pressure ( ) is reduced during BFR-RE, and the influence of O2 tension on mitochondrial redox balance remains ambiguous. We therefore aimed to determine whether skeletal muscle mitochondrial bioenergetics were altered following an acute bout of BFR-RE or RE, and to further examine the role of in this response. Accordingly, muscle biopsies were obtained from 10 males at rest and 2h after performing three sets of single-leg squats (RE or BFR-RE) to failure at 30% one-repetition maximum. We determined that mitochondrial respiratory capacity and ADP sensitivity were not altered in response to RE or BFR-RE. Although maximal (succinate) and submaximal (non-saturating ADP) mitochondrial ROS emission rates were unchanged following RE, BFR-RE attenuated these responses by ∼30% compared to pre-exercise, occurring along with a reduction in skeletal muscle tissue oxygenation during BFR-RE (P<0.01 vs. RE). In a separate cohort of participants, evaluation of mitochondrial bioenergetics in vitro revealed that mild O2 restriction (50µm) dramatically attenuated maximal (∼4-fold) and submaximal (∼50-fold) mitochondrial ROS emission rates and the fraction of electron leak to ROS compared to room air (200µm). Combined, these data demonstrate that mitochondrial ROS emissions are attenuated following BFR-RE, a response which may be mediated by a reduction in skeletal muscle .