Ambient Oxygen Tension Research Articles

Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology.

Abstract 4811: Oxygen tension - dependent differences in cancer cell kinome

Abstract Current approaches to preclinical cancer research often fail to consider the impact of ambient oxygen (O2; ~21%) on cancer cells. This is also true for hypoxia studies that typically involves cancer cells previously grown in ambient O2 before subsequent transfer to hypoxia. However, the tumor microenvironment is characterized by significantly lower O2 levels. We have previously demonstrated the impact of ambient O2 on stem cell populations, signaling pathways and resistance to therapy. We developed an experimental approach that allows us to collect and process tumor tissues from transgenic mammary tumor mouse models and ovarian cancer patients under physioxia (3% O2), such that they are never exposed to ambient O2. In this study, our goal was to explore oxygen-dependent signaling pathway alterations and to determine how these pathways are influenced by targeted drugs in the context of physioxia or ambient air (AA). Our studies revealed increased basal phosphorylation levels of EGFR (Y1068) in the tumor cells in AA, relative to physioxia. However, downstream signaling effectors AKT and ERK showed higher phosphorylation levels under physioxia, compared to AA, suggesting that their activation is independent of EGFR signaling. These findings correlate with the decreased sensitivity of the tumor cells under physioxia to target drugs lapatinib and alpelisib. We then sought to examine basal and target drug induced kinome changes in tumor cells under physioxia and AA via Multiplexed Inhibitor Beads (MIBs) kinome assay. This assay revealed significant differences in the kinome of the tumor cells under physioxia compared to AA. Although direct comparison between vehicle and lapatinib treated cells in physioxia and ambient air showed very minimal changes, pairwise comparison between lapatinib treated physioxia cells and vehicle treated AA cells revealed an increase in the activity of PDGFRB in lapatinib treated physioxia cells. Similarly, a receptor tyrosine kinase (RTK) array and western blotting showed increased basal and lapatinib induced phosphorylation of PDGFRB (Y751) under physioxia. Next, we determined the potential role of PDGFRB in downstream signaling pathway activation of AKT and ERK and resistance to lapatinib. We found that sunitinib, a multitarget RTK inhibitor with high affinity for PDGFR effectively decreased PGDFRB activity under physioxia, with a concurrent decrease in the phosphorylation of AKT. Moreover, tumor cells under physioxia were more sensitive to sunitinib treatment, relative to ambient air. Furthermore, a combination of lapatinib and sunitinib rendered tumor cells under physioxia more sensitive to treatment than with lapatinib alone. These findings suggest that ambient and physioxic oxygen tensions differentially impact cancer relevant signaling pathways. Therefore, it may be necessary to carry out preclinical cancer studies in the context of physiologically relevant oxygen tensions to aid translatability. Citation Format: Adedeji K. Adebayo, Brijesh Kumar, Christopher Davis, Steven P. Angus, Harikrishna Nakshatri. Oxygen tension - dependent differences in cancer cell kinome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4811.

Evaluating the Impact of Physiologically Relevant Oxygen Tensions on Drug Metabolism in 3D Hepatocyte Cultures in Paper Scaffolds.

Oxygen is an essential regulator of cellular function and phenotype. Despite its importance, the incorporation of physiologically relevant oxygen tensions is often overlooked in experimental setups. Ambient oxygen tensions (pO2 ∼152 mmHg) are significantly higher than those in the alveolar-capillary barrier of the lung, which is the most oxygen-rich interface in the body (pO2 ∼104 mmHg). The discrepancy between standard culture practices and physiologically relevant oxygen tensions is more pronounced when considering the hepatocyte-lined sinusoids of the liver, whose pO2 values range from 65 mm Hg in the periportal region to 30 mm Hg in the perivenous region. Our previous work highlights the need to transition from standard culture conditions to more physiologically relevant microenvironments when predicting hepatocyte responses to drug candidates or potential toxins. This protocol details an experimental pipeline for quantifying differences in transcript levels, protein levels, and activity of the cytochrome P450 1A (CYP1A) enzyme family in hepatocytes maintained in a three-dimensional environment at ambient and physiologically relevant oxygen tensions. We quantify changes in transcript with qRT-PCR, protein expression with western blots, and activity with the ethoxyresorufin-O-deethylase (EROD) assay. This approach can be adapted to any drug-metabolizing enzyme. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Preparing tissue-like environments to evaluate HepG2 cells in paper-based cell culture platform at physiological oxygen levels Basic Protocol 2: Evaluating CYP1A activity of hepatocytes grown in the paper scaffolds using the EROD assay Basic Protocol 3: Evaluating CYP1A transcript levels of hepatocytes grown in the paper scaffolds using RT-qPCR Basic Protocol 4: Evaluating CYP1A protein levels of hepatocytes grown in the paper scaffolds using western blotting.

Characterizing the hypoxic performance of a fish using a new metric: PAAS-50.

The hypoxic constraint on peak oxygen uptake (ṀO2,peak) was characterized in rainbow trout over a range of ambient oxygen tensions with different testing protocols and statistical models. The best-fit model was selected using both statistical criteria (R2 and AIC) and the model's prediction of three anchor points for hypoxic performance: critical PO2(Pcrit), maximum ṀO2and a new metric, the minimum PO2that supports 50% of absolute aerobic scope (PAAS-50). The best-fitting model was curvilinear using five strategically selected PO2values. This model predicted PAAS-50 as 70 mmHg (coefficient of variation, CV=9%) for rainbow trout. Thus, while a five-point hypoxic performance curve can characterize the limiting effects of hypoxia in fish, as envisaged by Fry over 75 years ago, PAAS-50 is a promising metric to compare hypoxic constraints on performance in a standardized manner both within and across fish species.

Hypoxia ameliorates maternal diet-induced insulin resistance during pregnancy while having a detrimental effect on the placenta.

Maternal overweight/obesity contributes significantly to the development of gestational diabetes, which causes risks to both mother and fetus and is increasing sharply in prevalence worldwide. Since hypoxia reprograms energy metabolism and can alleviate weight gain, adiposity, insulin resistance (IR), and dyslipidemia, we set out to study the potential of sustained reduced ambient oxygen tension (15% O2) during pregnancy for alleviating the detrimental effects of diet‐induced IR in C57Bl/6N mice, taking normal chow‐fed and normoxia (21% O2) groups as controls. Our data show that hypoxic intervention reduced maternal weight gain, adiposity, and adipose tissue inflammation, and ameliorated maternal glucose metabolism and IR during gestation in diet‐induced IR relative to normoxia. Where diet‐induced IR reduced maternal hemoglobin and increased serum erythropoietin levels, hypoxic intervention compensated for these changes. Diet‐induced IR reduced fetal growth in normoxia, and even more in hypoxia. Hypoxic intervention reduced liver weight gain during pregnancy in the dams with diet‐induced IR, maternal liver weight being positively associated with embryo number. In case of diet‐induced IR, the hypoxic intervention compromised placental energy metabolism and vascularization and increased end‐pregnancy placental necrosis. Altogether, these data show that although hypoxic intervention mediates several beneficial effects on maternal metabolism, the combination of it with diet‐induced IR is even more detrimental to the placental and fetal outcome than diet‐induced IR alone.

Induction of in vitro Metabolic Zonation in Primary Hepatocytes Requires Both Near-Physiological Oxygen Concentration and Flux.

Pre-clinical drug screening is an important step in assessing the metabolic effects and hepatic toxicity of new pharmaceutical compounds. However, due to the complexity of the liver microarchitecture, simplified in vitro models do not adequately reflect in vivo situations. Especially spatial heterogeneity, known as metabolic zonation, is often lost due to limitations introduced by typical culture conditions. By culturing primary rat hepatocytes in varied ambient oxygen levels on either gas-permeable or non-permeable culture plates, we highlight the importance of biomimetic oxygen supply for the targeted induction of zonation-like phenotypes. Resulting cellular profiles illustrate the effect of pericellular oxygen concentration and consumption rates on hepatic functionality in terms of zone-specific metabolism and β-catenin signaling. We show that modulation of ambient oxygen tension can partially induce metabolic zonation in vitro when considering high supply rates, leading to in vivo-like drug metabolism. However, when oxygen supply is limited, similar modulation instead triggers an ischemic reprogramming, resembling metabolic profiles of hepatocellular carcinoma and increasing susceptibility toward drug-induced injury. Application of this knowledge will allow for the development of more accurate drug screening models to better identify adverse effects in hepatic drug metabolism.

Decreased Ambient Oxygen Tension Alters the Expression of Endothelin-1, iNOS and cGMP in Rat Alveolar Macrophages.

Background: Hypoxia plays an important role in the vascular tone of pulmonary circulation via the vasculature and parenchymal tissue. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, plays a role in inflammation in mononuclear cells. Nitric oxide synthase (NOS), which generates nitric oxide (NO)/cyclic 3', 5'-monophosphate (cGMP), is coexpressed with ET-1 in many cell types. The aim of this study was to assess whether hypoxia induces the production of ET-1 and associated expression of NOS, NO/cGMP and chemokines in rat alveolar macrophages (AMs).Methods: NR8383 cells were cultured under hypoxic (1% oxygen) conditions for 0, 2, 4, 8 and 12 hours. Levels of ET-1, inducible NOS (iNOS), phosphorylated iNOS (p-iNOS), nitrite/nitrate (NOx), cGMP and monocyte chemoattractant protein-1 (MCP-1) were measured.Results: ET-1, p-iNOS, NOx, and cGMP increased significantly in AMs after 4 hours of hypoxia (p < 0.05). ET-1 and MCP-1 mRNA increased after 8 hours (p < 0.05). The protein expression of ET-1, MCP-1, and p-iNOS increased in a time-dependent manner, while iNOS expression decreased with time.Conclusions: The changes in ET-1, p-iNOS, and the NO/cGMP pathway in AMs may help elucidate the mechanisms in the hypoxic lung. Understanding changes in the endothelin axis in hypoxic AMs is a crucial first step to unravel its role in pulmonary circulation.

FIH permits NAA10 to catalyze the oxygen-dependent lysyl-acetylation of HIF-1α

The N-terminal acetyltransferase A (NatA) complex, which is composed of NAA10 and NAA15, catalyzes N-terminal acetylation of many proteins in a co-translational manner. Structurally, the catalytic subunit NAA10 was believed to have no activity toward an internal lysine residue because the gate of its catalytic pocket is too narrow. However, several studies have demonstrated that the monomeric NAA10 can acetylate the internal lysine residues of several substrates including hypoxia-inducible factor 1α (HIF-1α). How NAA10 acetylates lysine residues has been an unsolved question. We here found that human FIH (factor inhibiting HIF) hydroxylates human NAA10 at W38 oxygen-dependently and this permits NAA10 to express the lysyl-acetyltransferase activity. The hydroxylated W38 forms a new hydrogen-bond with A67 and widens the gate at the catalytic pocket, which allows the entrance of a lysine residue to the site. Since the FIH-dependent hydroxylation of NAA10 occurs oxygen-dependently, NAA10 acetylates HIF-1α under normoxia but does not under hypoxia. Consequently, the acetylation promotes the pVHL binding to HIF-1α, and in turn HIF-1α is destructed via the ubiquitin-proteasome system. This study provides a novel oxygen-sensing process that determines the substrate specificity of NAA10 depending on an ambient oxygen tension.

DIPG-67. HYPOXIA-INDUCIBLE FACTORS REGULATE DIFFUSE INTRINSIC PONTINE GLIOMA GROWTH IN NORMOXIC CULTURE

Diffuse intrinsic pontine glioma (DIPG) are incurable tumors and the leading cause of pediatric brain tumor deaths. They exhibit low blood perfusion and regions of necrosis, indicative of a low-oxygen environment that supports activation of hypoxia-inducible factors (HIF) that are associated with increased proliferation, invasion, and therapy resistance. However, previous reports suggest that HIF2-alpha slows growth in some glioma models. We therefore sought to test the hypothesis that HIFs regulate DIPG growth. We cultured the human DIPG tumors SU-DIPG-IV, VUMC-DIPG-X, and SU-DIPG-XIII at ambient oxygen tension and 5% carbon dioxide. We measured protein expression by Western blot and growth by trypan blue exclusion or tetrazolium reduction following exposure to the hypoxia-mimetic (HM) compounds, cobalt (II) chloride or deferoxamine, or selective HIF inhibitors. All three DIPG cultures retained stable expression of HIF1-alpha and HIF2-alpha protein at ambient oxygen tension, unchanged by HM treatment. Selective inhibition of HIF2-alpha by TC-S 7009 increased apparent growth, whereas selective inhibition of HIF1-alpha by CAY10585 did not. We conclude that hypoxia-independent HIF expression unchanged by either HM treatment or HIF inhibition suggests impaired HIF degradation, in which hypoxia-induced activation of HIF target genes more likely depends on transcriptional co-activators rather than blocked proteasomal degradation. In both ambient and hypoxic conditions, HIF2-alpha activity may oppose DIPG growth. Future experiments will investigate whether the effects of HIF2-alpha inhibition on tumor growth can be explained by enhanced HIF1-alpha activity through desequestration of common binding partners, or through direct action of HIF2-alpha on previously reported apoptotic pathways.

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

Propensity to calcification as a pathway to renal hypoxia in chronic kidney disease and in hypertension.

P 076 - The importance of culturing primary cells under physiological conditions: proliferation, senescence, pluripotency

Mesenchymal stem cells (MSCs), such as human dental pulp stem cells (hDPSCs), are currently a source for cell therapy. However, cell therapy protocols require 10–400 million cells per treatment, and consequently, they need to be expanded in vitro before implantation, with the inconvenience that MSCs undergo senescence following a certain number of cell expansion passages, loosing their stem cell qualities. Ambient oxygen tension (21% pO2) is normally used for in vitro culture, but physiological levels in vivo range between 3% and 6% pO2. We previously demonstrated that hDPSC proliferation rate is significantly lowered at 21% pO2 due to enhanced oxidative stress, which led to the activation of p38/p21/NRF-2 pathway, upregulating antioxidant defenses. Moreover, long-term in vitro culture of hDPSCs at 21% pO2 caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and lysil oxidase expression, which is mediated by p16INK4a pathway. This was accompanied by a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress- associated premature senescence. For all these reasons, we show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.

Role of p16INK4a and BMI-1 in oxidative stress-induced premature senescence in human dental pulp stem cells

Human dental pulp stem cells (hDPSCs) are a source for cell therapy. Before implantation, an in vitro expansion step is necessary, with the inconvenience that hDPSCs undergo senescence following a certain number of passages, loosing their stemness properties. Long-term in vitro culture of hDPSCs at 21% (ambient oxygen tension) compared with 3–6% oxygen tension (physiological oxygen tension) caused an oxidative stress-related premature senescence, as evidenced by increased β-galactosidase activity and increased lysil oxidase expression, which is mediated by p16INK4a pathway. Furthermore, hDPSCs cultured at 21% oxygen tension underwent a downregulation of OCT4, SOX2, KLF4 and c-MYC factors, which was recued by BMI-1 silencing. Thus, p16INK4a and BMI-1 might play a role in the oxidative stress-associated premature senescence. We show that it is important for clinical applications to culture cells at physiological pO2 to retain their stemness characteristics and to delay senescence.

Suppressors of Superoxide-H2O2 Production at Site IQ of Mitochondrial Complex I Protect against Stem Cell Hyperplasia and Ischemia-Reperfusion Injury

Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, "S1QELs"). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine invivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQis involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

Abstract: Reducing Ambient Oxygen Tension Optimizes the Fabrication and Maturation of Pre-Vascularized Tissue Engineered Flaps

INTRODUCTION: Oxygen is a potent modulator of cell function and wound repair in vivo. Hypoxia can enhance the production of specific extracellular matrix components and increase angiogenesis through the hypoxia-inducible factor-1 pathway. However, in vivo, very few cells within the body experience ambient (21%) oxygen tension. Thus, for clinically relevant tissue-engineered pre-vascularized skin flaps, hypoxic conditions can be exploited for promoting angiogenesis. We sought to identify the ideal oxygen tension in which to fabricate our novel, pre-vascularized tissue constructs containing a vascularized 1 mm diameter microchannel lined with human cells. MATERIALS AND METHODS: Vascular networks were fabricated by sacrificing Pluronic F127 macrofibers in type I collagen with encapsulated human foreskin fibroblasts (HFF1) and human placental pericytes (HPPL) at a density of 1 x 10^6 cells/mL, respectively. Twenty-four hours following fiber sacrifice, 5 x 10^6 cells/mL of human aortic smooth muscle cells (HASMC) and 5 x 10^6 cells/mL of human umbilical vein endothelial cells (HUVEC) were seeded sequentially into the patent luminal space. Subsequently, 48 hours after fiber sacrifice, 1 x 10^6 cells/mL of human epidermal keratinocytes (HEK) were topically seeded onto scaffolds. Scaffolds were incubated at 1.5%, 5.0%, or 20.0% oxygen, underwent daily media changes, and were analyzed after 7 and 14 days in culture. RESULTS: Macrochannels were successfully lined with HUVEC and HASMC, generating anatomically appropriate neointimal and neomedial layers by as early as day 7. The most robust cellular linings were seen in constructs incubated in 5.0% oxygen. Immunohistochemical analysis revealed CD31+ HUVEC along the luminal surface of the macrochannel, and α-SMA expressing HASMC in the subendothelial plane. Furthermore, proliferation of HFF1 was evident as early as 7 days after seeding. HEK proliferated leading to the formation of a stratified epidermal layer along the construct surface and fibroblast specific-1-expressing fibroblasts within the “neo dermis.” CONCLUSION: Hypoxic conditions promote increased angiogenesis and vascular stability in our tissue engineered, pre-vascularized skin flaps without detrimental effects on other flap cellular constituents. With a built-in vascular network, vital epidermal (HEK) and dermal (HFF1, collagen) components, these full-thickness, tissue engineered skin scaffolds hold tremendous promise as a platform to aid in evaluating cellular responses to changing oxygen concentrations in parallel to generating tissue-engineered flaps of clinically relevant sizes. DISCLOSURE/FINANCIAL SUPPORT:The authors have no financial interests in this manuscript.

Impact of Oxygen Levels on Human Hematopoietic Stem and Progenitor Cell Expansion.

Oxygen levels are an important variable during the in vitro culture of stem cells. There has been increasing interest in the use of low oxygen to maximize proliferation and, in some cases, effect differentiation of stem cell populations. It is generally assumed that the defined pO2 in the incubator reflects the pO2 to which the stem cells are being exposed. However, we demonstrate that the pO2 experienced by cells in static culture can change dramatically during the course of culture as cell numbers increase and as the oxygen utilization by cells exceeds the diffusion of oxygen through the media. Dynamic culture (whereby the cell culture plate is in constant motion) largely eliminates this effect, and a combination of low ambient oxygen and dynamic culture results in a fourfold increase in reconstituting capacity of human hematopoietic stem cells compared with those cultured in static culture at ambient oxygen tension. Cells cultured dynamically at 5% oxygen exhibited the best expansion: 30-fold increase by flow cytometry, 120-fold increase by colony assay, and 11% of human CD45 engraftment in the bone marrow of NOD/SCID mice. To our knowledge, this is the first study to compare individual and combined effects of oxygen and static or dynamic culture on hematopoietic ex vivo expansion. Understanding and controlling the effective oxygen tension experienced by cells may be important in clinical stem cell expansion systems, and these results may have relevance to the interpretation of low oxygen culture studies.

Hif-3α Fine-tunes Extracellular Matrix Production in Nucleus Pulposus Cell under Hypoxia

Introduction Nucleus pulposus (NP), the gelatinous tissue core in the intervertebral disc (IVD), is enriched with extracellular matrix type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1). Dysregulation of extracellular matrix proteins in the NP is tightly associated with disc degeneration, ultimately leading to a loss of mechanical function in motion segments.1 Expression of SOX9, the major chondrogenic transcription factor for COL2A1 and AGC1 gene activation, was evidenced in the normal and degenerated human NP, but weak in the annulus fibrosus (AF) region.2 Considering the virtually avascular characteristic of the NP, NP cells are regulated within a hypoxic microenvironment. Among the family of hypoxia inducible factor-α subunits (HIF-α), HIF-1α and HIF-2α (EPAS1) have been well illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the NP.3–5 However, the understanding of HIF-3α, the dominant negative regulator of HIF-1α,6 in IVD is limited. Here, we hypothesized that HIF-3α may regulate the SOX9-dependent transcription of the extracellular matrix genes in NP cells under hypoxia. We aimed at characterizing the sub-cellular expression patterns of HIF-1α and HIF-3α in mouse IVD tissues and human NP cells cultured under different oxygen tensions. Moreover, we tested the modulatory effects of HIF-1α/HIF-3α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods All animal and human works were approved by local ethical committee. IVD were harvested from wild-type C57BL/6N mice at 3 and 6 months-old. Lumbar IVD of scoliosis patients were collected under informed consent. Cells were harvested by digestion with 0.5 × of TrpLE Express ( Gibco) 37°C, 30 minute and 0.25mg/ml Collagenase II ( Gibco) 37°C, 90 minute in DMEM-HG ( Gibco). Human NP cells were expanded in DMEM-HG supplemented with 10% fetal bovine serum. Subsequently, human NP cells were subjected to ambient oxygen tension (21% O2) or intermediate hypoxia (5% O2) or extreme hypoxia (1% O2) for 72 hours prior to fixation or RNA collection. Immunohistochemistry staining of Hif-1α and Hif-3α was studied with mouse IVD paraffin sections and different sub-cellular locations of HIF-1α, HIF-3α and SOX9 expression were identified by immunofluorescence in human NP cells cultured under different oxygen tensions. To determine transcriptional activity of Col2a1 and Agc1, luciferase-based promoter assays on specific Col2a1 or Agc1 cis-acting elements with the overexpression for each of the HIF-α subunits and/or Sox9 in ATDC5 cells were performed. Results Hif-1α and Hif-3α expression pattern was identified in mouse NP, AF and endplate (EP) tissue. Expression of HIF-1α and SOX9 was detected in the nuclei of human NP cells independent of different oxygen tensions. Strikingly, induction of HIF-3α expression in nuclei was observed in human NP cells cultured under hypoxia (5% and 1% O2) versus normoxia (21% O2). Additionally, AGC1 gene expression was reduced in human NP cells cultured under hypoxia in comparison with normoxia. Furthermore, luciferase-based promoter assay showed that Hif-3α overexpression inhibited the Sox9–dependent transcriptional up-regulation of Col2a1 and Agc1. Conclusion Our findings illustrated that induction of HIF-3α in human NP cell nuclei was evidenced in hypoxia versus normoxia. We further demonstrated that Hif-3α has a function in inhibiting Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of HIF-3α in fine-tuning the extracellular matrix protein production in NP cells under hypoxia. Detailed molecular mechanism of HIF-3α in regulating human NP cell and matrix homeostasis under hypoxia warrants further investigations. References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol 2010;222(1):23–32 Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55(2):91–97 Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res 2002;308(3):401–407 Risbud MV, Guttapalli A, Stokes DG, et al. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 2006;98(1):152–159 Agrawal A, Gajghate S, Smith H, et al. Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 2008;58(12):3798–3808 Dengler VL, Galbraith MD, Espinosa JM. Transcriptional regulation by hypoxia inducible factors. Crit Rev Biochem Mol Biol 2014;49(1):1–15

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.

Oxidative stress under ambient and physiological oxygen tension in tissue culture.

Oxygen (O2) levels range from 2-9% in vivo. However, cell culture experiments are performed at atmospheric O2 levels (21%). Oxidative stress due to generation of reactive oxygen species (ROS) in cells cultured at higher than physiological levels is implicated in multitude of deleterious effects including DNA damage, genomic instability and senescence. In addition, oxidative stress activates redox sensitive transcription factors related to inflammatory signaling and apoptotic signaling. Furthermore, several chromatin-modifying enzymes are affected by ROS, potentially impacting epigenetic regulation of gene expression. While primary cells are cultured at lower O2 levels due to their inability to grow at higher O2, the immortalized cells, which display no such apparent growth difficulties, are typically cultured at 21% O2. This review will provide an overview of issues associated with increased oxygen levels in in vitro cell culture and point out the benefits of using lower levels of oxygen tension even for immortalized cells.

Paradoxical anaerobism in desert pupfish.

In order to estimate metabolic demands of desert pupfish for conservation purposes, we measured oxygen consumption in fish acclimated to the ecologically relevant temperatures of 28 or 33°C. For these experiments, we used fish derived from a refuge population of Devils Hole pupfish (Cyprinodon diabolis). Measurement of routine oxygen consumption (V̇O2,routine) revealed some 33°C-acclimated fish (10% of 295 assayed fish) periodically exhibited periods of no measurable oxygen consumption despite available ambient oxygen tensions that were above the critical PO2. We call this phenomenon paradoxical anaerobism. The longest observed continuous bout with no oxygen consumption was 149 min, although typical bouts were much shorter. Fish maintained normal posture and ventilation rate (>230 ventilations per minute) during paradoxical anaerobism. Fish rarely demonstrated a compensatory increase in oxygen use following a period of paradoxical anaerobism. In contrast, only one out of 262 sampled fish acclimated at 28°C spontaneously demonstrated paradoxical anaerobism. Muscle lactate concentration was not elevated during periods of paradoxical anaerobism. However, the amount of ethanol released by the 33°C-acclimated fish was 7.3 times greater than that released by the 28°C acclimation group, suggesting ethanol may be used as an alternative end product of anaerobic metabolism. Exposure to exogenous ethanol, in concentrations as low as 0.1%, produced periods of paradoxical anaerobism even in 28°C-acclimated fish.

PP41 - Oxygen concentration in oxidative stress and replicative senescence in dental pulp stem cells

The in vitro culture is routinely carried out under ambient oxygen tension. However, in vivo human Dental Pulp Stem Cells (hDPSC) are not exposed to such hyperoxic conditions. Dental pulp tissue physiological oxygen tension ranges between values of 3% to 6% O2. In vitro cellular senescence refers to both replicative and premature senescence. Replicative senescence is associated with a progressive increase in the expression of p16INK4a. Bmi-1 is a supresor of p14ARF and p16INK4a. Premature or accelerated senescence can be induced by ROS accumulation. It has been demonstrated that pluripotency can be induced by introducing 2 factors: Sox2 and Oct4. The aim of this study was to investigate the involvement of oxidative stress induced by culture under 21% oxygen, in the maintenance of pluripotency and in the mechanisms of chronological and premature aging of hDPSC. hDPSC cultured under 21% O2 showed dihydrorhodamine levels significantly higher, and their mitocondrial membrane potential was lower in comparison to hDPSC cultured under 21% O2. Bmi-1 expression increases with passages, but this expression is higher in hDPSC cultured under 21% O2 in comparison to 3% O2 since the beginning. At early passages, Sox2 and Oct4 are overexpressed when culture under 3% O2 compared to 21% O2, and its expression decreases with passages. p14ARF, p16INK4a expression and beta-Galactosidase activity remain very low in hDPSC cultured under 3% O2, whereas under 21% O2 they are significatively augmented. p21 expression increases with passages under both 3% or 21% O2 culture conditions. Because hDPSC expressed p14ARF and p16INK4a but not Bmi-1 or p21, we conclude that in vitro premature senescence under 21% O2 may be associated with oxidative stress and not with Bmi-1 overexpression. hDPSC culture under 3% O2 mantains its stemness and reduces premature senescence. Supported by SAF2010-19498; SAF2013-44663-R; ISCIII2012-RED-43-029; PROMETEOII2014/056; RS2012-609; CM1001 and FRAILOMIC-HEALTH.2012.2.1.1-2. The study has been co-financed by FEDER funds from the European Union.