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Oxygen sensing and signaling: Impact on the regulation of physiologically important genes
Abstract
A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. It is likely that cells from a wide variety of tissues share a common mechanism of oxygen sensing and signal transduction leading to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). Besides hypoxia, transition metals (Co2+, Ni2+ and Mn2+) and iron chelation also promote activation of HIF-1. Induction of HIF-1 by hypoxia is blocked by the heme ligands carbon monoxide and nitric oxide. There is growing, albeit indirect, evidence that the oxygen sensor is a flavoheme protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. The activation of HIF-1 by hypoxia depends upon signaling-dependent rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1beta (ARNT), which then translocates to the nucleus and impacts on the transcription of genes whose cis-acting elements contain cognate hypoxia response elements.
... Activated PKG opens the mitoK ATP channel, which results in increased ROS production followed by activation of other kinases. In this system, PKG is the terminal cytosolic component of the terminal signaling pathway transmitting the cardioprotective signal from the cytosol to the inner mitochondrial membrane through the protein kinase C (PKC)-dependent pathway (Wang and Semenza, 1993;Zhu and Bunn, 1999;Stroka et al., 2001;Semenza, 2002;Bruick, 2003;Butow and Avadhani, 2004;Murphy, 2004). In addition, mitochondria participate in cell-to-cell interactions and systemic regulation (Zhu and Bunn, 1999;Chandel and Schumacker, 2000;Nicholls and Budd, 2000;Nishimura et al., 2001;Brunk and Terman, 2002;Butow and Avadhani, 2004;Kuznetsov et al., 2004;Michiels, 2004;Felty and Roy, 2005;MacDonald et al., 2005;Lukyanova et al., 2008aLukyanova et al., , 2011. ...
... In this system, PKG is the terminal cytosolic component of the terminal signaling pathway transmitting the cardioprotective signal from the cytosol to the inner mitochondrial membrane through the protein kinase C (PKC)-dependent pathway (Wang and Semenza, 1993;Zhu and Bunn, 1999;Stroka et al., 2001;Semenza, 2002;Bruick, 2003;Butow and Avadhani, 2004;Murphy, 2004). In addition, mitochondria participate in cell-to-cell interactions and systemic regulation (Zhu and Bunn, 1999;Chandel and Schumacker, 2000;Nicholls and Budd, 2000;Nishimura et al., 2001;Brunk and Terman, 2002;Butow and Avadhani, 2004;Kuznetsov et al., 2004;Michiels, 2004;Felty and Roy, 2005;MacDonald et al., 2005;Lukyanova et al., 2008aLukyanova et al., , 2011. Mitochondria are the major regulators of the oxygen homeostasis in the body. ...
... At the systemic level, mitochondria determine the concentration gradient for oxygen delivered from the environment to the cell and represent the final step of interaction with molecular oxygen (Brunk and Terman, 2002;Lukyanova et al., 2008aLukyanova et al., , 2011. Due to this function, which determines both viability and vital activity of aerobic organisms, evolution has created very sophisticated physiological systems for oxygen delivery to mitochondria and maintenance of optimum oxygenation in cells (act of breathing; pulmonary system of oxygen transportation; cardiovascular circulatory system; blood mass-transfer system; red cells; and hemoglobin) (Zhu and Bunn, 1999;Nicholls and Budd, 2000;Prabhakar and Overholt, 2000;Di Lisa and Ziegler, 2001;Peers and Kemp, 2001;Brunk and Terman, 2002;Voos and Rotgers, 2002;Da Silva et al., 2003;Duchen, 2004;Kuznetsov et al., 2004;Michiels, 2004;Devin and Rigoulet, 2007). ...
The article is focused on the role of the cell bioenergetic apparatus, mitochondria, involved in development of immediate and delayed molecular mechanisms for adaptation to hypoxic stress in brain cortex. Hypoxia induces reprogramming of respiratory chain function and switching from oxidation of NAD-related substrates (complex I) to succinate oxidation (complex II). Transient, reversible, compensatory activation of respiratory chain complex II is a major mechanism of immediate adaptation to hypoxia necessary for 1) succinate-related energy synthesis in the conditions of oxygen deficiency and formation of urgent resistance in the body; 2) succinate-related stabilization of HIF-1α and initiation of its transcriptional activity related with formation of long-term adaptation; 3) succinate-related activation of the succinate-specific receptor, GPR91.This mechanism participates in at least four critical regulatory functions: 1) sensor function related with changes in kinetic properties of complex I and complex II in response to a gradual decrease in ambient oxygen concentration; this function is designed for selection of the most efficient pathway for energy substrate oxidation in hypoxia; 2) compensatory function focused on formation of immediate adaptive responses to hypoxia and hypoxic resistance of the body; 3) transcriptional function focused on activated synthesis of HIF-1 and the genes providing long-term adaptation to low рО2; 4) receptor function, which reflects participation of mitochondria in the intercellular signaling system via the succinate-dependent receptor, GPR91. Inallcases, the desired result is achieved by activation of the succinate-dependent oxidation pathway, which allows considering succinate as a signaling molecule. Patterns of mitochondria-controlled activation of GPR-91- and HIF-1-dependent reaction were considered, and a possibility of their participation in cellular-intercellular-systemic interactions in hypoxia and adaptation was proved.
... 23 These reactive oxygen and nitrogen radicals are produced after oxygen supply interruption and/or restoration. 3,24 The major intracellular sources of O 2 À are the electron transport chain in the mitochondria, the nicotinamide adenine dinucleotide dihydrophosphate (NADPH) oxidase, cytochrome P450 reductase system in the cellular plasma membrane, 1,2 and the xanthine oxidase 25 and NO synthase (NOS) 26,27 enzyme systems (Figure 1). ...
... 28 Nitric oxide, also known as endothelium-derived relaxing factor, is a key biological messenger that is synthesized endogenously during conversion of arginine to citrulline in a process that required molecular oxygen and NADPH with tetrahydrobiopterin (H 4 B) acting as a cofactor. Bioregulatory NO is generated by 3 isoforms of NOS enzymes, 26,27 namely, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Although the former 2 are calcium dependent for their regulation, iNOS is insensitive to endogenous calcium, likely due to its tight noncovalent interaction with calmodulin (CaM) and Ca 2þ . ...
Over the past several years, there has been increasing recognition that pathogenesis of adhesion development includes significant contributions of hypoxia induced at the site of surgery, the resulting oxidative stress, and the subsequent free radical production. Mitochondrial dysfunction generated by surgically induced tissue hypoxia and inflammation can lead to the production of reactive oxygen and nitrogen species as well as antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase which when optimal have the potential to abrogate mitochondrial dysfunction and oxidative stress, preventing the cascade of events leading to the development of adhesions in injured peritoneum. There is a significant cross talk between the several processes leading to whether or not adhesions would eventually develop. Several of these processes present avenues for the development of measures that can help in abrogating adhesion formation or reformation after intraabdominal surgery.
... An NAD(P)H oxidase with low affinity for oxygen and high affinity for cyanide is believed to act as one of the sensors for oxygen tension in the carotid body, controlling the rate of ventilation [2]. The function of oxygen sensing is apparently shared by several proteins, including a nonmitochondrial cytochrome b558, a mitochondrial protein, and possibly a third heme protein [105,209]. ...
... A similar group of proteins was suggested to be involved as oxygen sensors in the regulation of erythropoietin production in human hepatoma cells [209]. A microsomal NADH oxidase was implicated as an oxygen sensor in bovine pulmonary and coronary arteries, where changes in oxygen tension regulate vascular relaxation through changes in O2 •-production and cGMP formation [198]. ...
The paper outlines natural sources, mechanism of production of radicals in live organisms and consequences of their effect on various biological substrates. All aerobic organisms produce reactive oxygen species physiologically by plant photosynthesis, analogically by mitochondrial respiration, photosensitizing reactions, eicosanoids production pathways,
and during host defence. The four-electron reduction of oxygen occurs within the mitochondrial electron transport system of all aerobically respiring cells. Massive production of radicals in live systems may occur as a response to the presence of compounds of transient metals that are reduced by accepting one electron, which initiates in biological systems a vicious circle of oxidation and cyclic production of radicals of various substrates. The oxidised substrates themselves are potentially toxic to live organisms and their structure is modified which alters their functional properties.
Described are regulated and unregulated reactions that produce reactive oxygen species formation and the role of transit
metals in catalysis of these reactions either in the form of ions or complexes within metalloproteins. Discussed is the fate
of those products, their physiological significance and potential oxidative damage to the body components, such as fats,
proteins, carbohydrates and nucleic acids. In conclusion we describe antioxidative defense reactions catalyzed by metalloenzymes. Description of effective protection of organisms against reactive oxygen species in concurrence with other oxidoreduction systems shows that the most effective are proteins with enzymatic activity that contain the transit
metal as a cofactor.
... According to modern views, the mitochondrial respiratory chain is involved in various intracellular signaling programs and performs the role of a signaling transformative metabolic system that activates the functional response and the physiological reactions of the organism to a variety of impacts, and above all, to hypoxia [99][100][101][102][103][104][105][106][107][108][109][110][111]. As noted above, succinate-dependent signaling pathways induced by adaptive factors GPR91, VEGF, and HIF1-α play a special role in this process [24][25][26][27]30,31,[35][36][37]. ...
The effect of a single one-hour exposure to three modes of hypobaric hypoxia (HBH) differed in the content of O2 in inhaled air (FiO2—14%, 10%, 8%) in the development of mitochondrial-dependent adaptive processes in the myocardium was studied in vivo. The following parameters have been examined: (a) an urgent reaction of catalytic subunits of mitochondrial enzymes (NDUFV2, SDHA, Cyt b, COX2, ATP5A) in the myocardium as an indicator of the state of the respiratory chain electron transport function; (b) an urgent activation of signaling pathways dependent on GPR91, HIF-1α and VEGF, allowing us to assess their role in the formation of urgent mechanisms of adaptation to hypoxia in the myocardium; (c) changes in the ultrastructure of three subpopulations of myocardial mitochondria under these conditions. The studies were conducted on two rat phenotypes: rats with low resistance (LR) and high resistance (HR) to hypoxia. The adaptive and compensatory role of the mitochondrial complex II (MC II) in maintaining the electron transport and energy function of the myocardium in a wide range of reduced O2 concentrations in the initial period of hypoxic exposure has been established. The features of urgent reciprocal regulatory interaction of NAD- and FAD-dependent oxidation pathways in myocardial mitochondria under these conditions have been revealed. The data indicating the participation of GPR91, HIF-1a and VEGF in this process have been obtained. The ultrastructure of the mitochondrial subpopulations in the myocardium of LR and HR rats differed in normoxic conditions and reacted differently to hypoxia of varying severity. The parameters studied together are highly informative indicators of the quality of cardiac activity and metabolic biomarkers of urgent adaptation in various hypoxic conditions.
... Nickel, cobalt and iron chelators mimic hypoxia and can induce the expression of NDRG1 by inducing and stabilising HIF-1 [47][48][49][50]. Nickel and cobalt can substitute Fe 2+ in enzymes acting in HIF-1 expression or stabilisation pathways, and iron chelators can prevent the binding of oxygen to haeme groups or degradation of HIF-1a and HIF-2a [49,51,52]. Interestingly, NDRG1 might interact with metals directly: a 30-amino acid peptide containing the C-terminal repeats of NDRG1 (TRSRSHTSEG) 3 binds nickel [53,54], copper [55], zinc [56], manganese and cobalt [57]. ...
N‐myc downstream regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot‐Marie‐Tooth neuropathy. The 43‐kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C‐terminal region with three 10‐residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full‐length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small‐angle X‐ray scattering and CD spectroscopy indicated a variable conformation for the N‐ and C‐terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C‐terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1.
... Cellular responses to ischemia have largely been defined by studies focusing on hypoxia-inducible factor (HIF)-1 [92,101,122]. However, Yan and colleagues reported the role of Egr-1 in the regulation of ischemic stress-related gene pool [113]. ...
Despite improved treatment options myocardial infarction (MI) is still a leading cause of mortality and morbidity worldwide. Remote ischemic preconditioning (RIPC) is a mechanistic process that reduces myocardial infarction size and protects against ischemia reperfusion (I/R) injury. The zinc finger transcription factor early growth response-1 (Egr-1) is integral to the biological response to I/R, as its upregulation mediates the increased expression of inflammatory and prothrombotic processes. We aimed to determine the association and/or role of Egr-1 expression with the molecular mechanisms controlling the cardioprotective effects of RIPC. This study used H9C2 cells in vitro and a rat model of cardiac ischemia reperfusion (I/R) injury. We silenced Egr-1 with DNAzyme (ED5) in vitro and in vivo, before three cycles of RIPC consisting of alternating 5 min hypoxia and normoxia in cells or hind-limb ligation and release in the rat, followed by hypoxic challenge in vitro and I/R injury in vivo. Post-procedure, ED5 administration led to a significant increase in infarct size compared to controls (65.90 ± 2.38% vs. 41.00 ± 2.83%, p < 0.0001) following administration prior to RIPC in vivo, concurrent with decreased plasma IL-6 levels (118.30 ± 4.30 pg/ml vs. 130.50 ± 1.29 pg/ml, p < 0.05), downregulation of the cardioprotective JAK–STAT pathway, and elevated myocardial endothelial dysfunction. In vitro, ED5 administration abrogated IL-6 mRNA expression in H9C2 cells subjected to RIPC (0.95 ± 0.20 vs. 6.08 ± 1.40-fold relative to the control group, p < 0.05), resulting in increase in apoptosis (4.76 ± 0.70% vs. 2.23 ± 0.34%, p < 0.05) and loss of mitochondrial membrane potential (0.57 ± 0.11% vs. 1.0 ± 0.14%-fold relative to control, p < 0.05) in recipient cells receiving preconditioned media from the DNAzyme treated donor cells. This study suggests that Egr-1 functions as a master regulator of remote preconditioning inducing a protective effect against myocardial I/R injury through IL-6-dependent JAK–STAT signaling.
... HIF increases triglycerides storage and fatty acids synthesis. It also suppresses carnitine palmitoyltransferase 1 and acyl-CoA synthase long-chain family member 1, which facilitate fatty acid import and oxidation, respectively resulting in blocking of fatty acids oxidation, in mitochondria (Zhu and Bunn 1999;Semenza 2010;Huang et al. 2014). ...
Pheochromocytomas are rare tumors originating in the adrenal medulla. They may be sporadic or in the context of a hereditary syndrome. A considerable number of pheochromocytomas carry germline or somatic gene mutations, which are inherited in the autosomal dominant way. All patients should undergo genetic testing. Symptoms are due to catecholamines over production or to a mass effect. Diagnosis is confirmed by raised plasma or urine metanephrines or normetanephrines. Radiology assists in the tumor location and any local invasion or metastasis. All the patients should have preoperative preparation with α-blockers and/or other medications to control hypertension, arrhythmia, and volume expansion. Surgery is the definitive treatment. Follow up should be life-long.
... The hypoxic mechanism of CoCl 2 involves the replacement of Fe 2+ by Co 2+ in hemoglobin, changing hemoglobin to a deoxidized state. In this state, the cells appear hypoxic in a normoxic environment (71). The hypoxic environment induced by CoCl 2 is widely used due to of the advantages of simple use and easy, precise control of treatment conditions. ...
The unique hypoxic inflammatory microenvironment observed in the spinal cord following spinal cord injury (SCI) limits the survival and efficacy of transplanted bone mesenchymal stem cells (BMSCs). The aim of the present study was to determine whether hypoxic preconditioning (HP) increased the therapeutic effects of BMSC on SCI. BMSCs were pretreated with cobalt chloride (CoCl2) in vitro, and the proliferative apoptotic and migratory abilities of these hypoxic BMSCs (H‑BMSCs) were assessed. BMSCs and H‑BMSCs derived from green fluorescent protein (GFP) rats were transplanted into SCI rats in vivo. The neurological function, histopathology, inflammation, and number and migration of transplanted cells were examined. HP significantly enhanced BMSC migration (increased hypoxia inducible factor 1α and C‑X‑C motif chemokine receptor 4 expression) and tolerance to apoptotic conditions (decreased caspase‑3 and increased B‑cell lymphoma 2 expression) in vitro. In vivo, H‑BMSC transplantation significantly improved neurological function, decreased spinal cord damage and suppressed the inflammatory response associated with microglial activation. The number of GFP‑positive cells in the SCI core and peripheral region of H‑BMSC animals was increased compared with that in those of BMSC animals, suggesting that HP may increase the survival and migratory abilities of BMSCs and highlights their therapeutic potential for SCI.
... While there were consistent increases in VEGFA and SLC2A1 gene expression between the two cell types, in BT474 cells HIF1A expression showed a slight but significant increase, with little change in MCF-10A cells. Because activation of HIF-1 depends in part on posttranscriptional stabilization of HIF-1α (Zhu and Bunn, 1999), its near-absence in mRNA cell samples after 24 h of hypoxic conditioning is unsurprising (Kallio et al., 1997), and the upregulation of downstream reporter genes VEGFA, SLC2A1 and BNIP3 further supports this interpretation. Overall, the 24-h restrictive oxygen conditioning revealed key hypoxiadependent genes were upregulated within the hypoxia device. ...
Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.
... C'est par l'étude des phénomènes moléculaires régissant cette spécificité du gène de l'Epo à l'hypoxie que les mécanismes cellulaires de sensibilité à l'oxygène sont mieux compris aujourd'hui. Car, il est connu que la production d'Epo est aussi stimulée par des ions métalliques comme le cobalt, ou le nickel (Zhu 1999). Il a ainsi été montré que l'amplification de l'expression de l'ARNm de l'Epo et la production de la protéine étaient dose dépendante avec des concentrations croissantes de CoC12 et NiC12 de façon similaire à ce qui était observé avec des degrés croissants d'hypoxie (Fandrey 1993, Goldberg 1988. ...
L'évaluation du risque parasitaire passe par le développement de stratégies d'analyses efficaces, sensibles et spécifiques qui peuvent permettre non seulement de détecter le parasite, mais aussi d'estimer sa viabilité faute de pouvoir disposer d'un outil fiable pour déterminer son pouvoir infectieux. Parmi les trois protozoaires les plus fréquemment retrouvés au niveau mondial, Giardia est présent sous la forme de kyste dans l'environnement. En fonction de ces éléments, cette étude a pour objectif de développer des outils de biologie moléculaire pour réaliser la détection des kystes de Giardia et l'évaluation de leur viabilité. La première partie de ce travail a permis de définir une méthode d'amplification de l' ADN par semi-nested PCR analytique et de l' ARN par RT semi-nested PCR analytique de kystes de Giardia purifiés à partir de selles de malades, en partant de trois gènes cibles et spécifiques de Giardia: l'HSP 70,1' ADHE et la Giardine. Aucun fragment amplifié n'a été obtenu avec les amorces HSP 70, des produits d'amplification ont été observés à la suite d'une semi-nested PCR avec le gène de l' ADHE, et pour la dernière cible, la Giardine, des résultats positifs sont enregistrés aussi bien en PCR qu'en RT-PCR. Pour parvenir à ces résultats, un protocole d'extraction pour chaque acide nucléique (ADN et ARN), des conditions optimales d'amplification pour chaque cible (HSP, ADHE et Giardine), ainsi qu'un traitement de l'extrait ARN par une DNase pour s'affranchir de l'amplification exclusive de cet acide nucléique ont été successivement sélectionnés. Dans nos conditions d'expérimentation, le seuil de détection de la semi-nested PCR est fixé pour la Giardine à 6 kystes et de la RT semi-nested PCR à 3 kystes. En partant de ces outils d'analyses, des études de survies en fonction de la température et d'impactd'un choc thermique sur la morphologie et l'expression des kystes de Giardia ont été réalisées. Les résultats d'estimation de la viabilité par RT-PCR analytique ont également été comparés à ceux obtenus en microscopie optique après marquage des kystes par des colorants vitaux. Globalement, quelles que soient les conditions d'incubation (température et temps), nos expérimentations ont démontré la persistance de l' ADN et de l' ARN par PCR et RT -PCR respectivement. L'estimation de la viabilité déterminée par RT-PCR n'est pas cohérente avec celle issue de l'observation au microscope optique en épi fluorescence qui permet d'enregistrer une baisse de la viabilité d'autant plus importante que la température est éloignée de l'intervalle 4-20?C et que le temps d'incubation est important. D'après ces résultats, la disponibilité de l'ADN et de l'ARN ne semble pas être diminuée par le stockage des kystes de Giardia aux différentes températures, probablement grâce à la persistance des acides nucléiques et/ou l'absence de modification dans le métabolisme de l' ARNm. Dans une seconde partie du travail, à partir des acides nucléiques des mêmes parasites, les conditions d'amplification de l' ADN et de l' ARN, respectivement en PCR et en RT-PCR quantitatives, ont été définies en prenant toujours pour cible la séquence de la Giardine. A l'issue de l'optimisation des conditions d'amplification, le protocole de PCR quantitative est validé sur la base d'une reproductibilité supérieure à 95 %, d'un rendement estimé à 88% et d'un seuil de détection fixé à 6 kystes de Giardia. Dans le cas de la RT-PCR quantitative, le traitement enzymatique par la DNase ne modifie ni le rendement (94 %) ni la sensibilité de la détection. Il permet d'assurer, avec une variabilité inférieure à 5 %,l'amplification exclusive de l' ARN à partir de 3 kystes de Giardia. En utilisant la méthodologie Jle PCR et RT-PCR quantitatives développée et l'observation au microscope optique en épifluorescence, les mêmes études de survies en fonction de la température et de l'impact d'un choc thermique sur la morphologie et l'expression des kystes de Giardia ont été réalisées. Les résultats de l'estimation de la viabilité ont également été comparés successivement à ceux obtenus en microscopie optique. Cette fois, la persistance de l' ADN est vérifiée plus précisément par des valeurs d'abattement proche de zéro et quelles que soient les conditions d'expérimentation. Concernant l'ARNm, les résultats de RT -PCR permettent dans certaines conditions d'enregistrer de faibles abattements, alors qu'à l'issue de l'examen en microscopie optique il est possible de vérifier l'absence de kystes viables. Nos expérimentations ont démontré qu'il était possible d'amplifier de façon sensible et spécifique le génome de Giardia (ARN et ADN) aussi bien par PCR analytique que par PCR quantitative. En revanche pour l'évaluation de la viabilité des kystes de Giardia, seule la PCR quantitative fournit les informations nécessaires pour apprécier le pouvoir infectieux.
... The mechanism of its hypoxia simulation is the replacement of Fe 2+ by Co 2+ in hemoglobin, changing hemoglobin to a deoxidized state. In this state, the cells "feel" hypoxic in a normoxic environment [57]. It is easy to control the experimental conditions with CoCl 2 in vitro, though there are some limitations. ...
Objective
Special hypoxic and hypertonic microenvironment in intervertebral discs (IVDs) decreases the treatment effect of cell transplantation. We investigated the hypothesis that hypoxic preconditioning (HP) could improve the therapeutic effect of bone mesenchymal stem cells (BMSCs) to IVD degeneration.
Methods
BMSCs from green fluorescent protein-transgenic rats were pretreated with cobalt chloride (CoCl2, 100 μM, 24 h) for hypoxic conditions in vitro. Apoptosis (related pathways of caspase-3 and bcl-2) and migration (related pathways of HIF-1α and CXCR4) were detected in BMSCs. In vivo, BMSCs and HP BMSCs (H-BMSCs) were injected into the rat model of IVD degeneration. The IVD height, survival, migration, and differentiation of transplanted BMSCs and matrix protein expression (collagen II, aggrecan, and MMP-13) were tested.
Results
H-BMSCs could extensively decrease IVD degeneration by increasing IVD height and collagen II and aggrecan expressions when compared with BMSCs. Significantly, more GFP-positive BMSCs were observed in the nucleus pulposus and annulus fibrosus regions of IVD. HP could significantly decrease BMSC apoptosis (activating bcl-2 and inhibiting caspase-3) and improve BMSC migration (increasing HIF-1α and CXCR4) in vitro.
Conclusion
HP could significantly enhance the capacity of BMSCs to repair DDD by increasing the survival and migration of implanted cells and increasing matrix protein expression.
... This allows for a greater range of measurements to be performed that cannot be conducted in situ. However, the validity of these measurements is limited if the samples are liable to degradation, when removed from their immediate surroundings and if the measurement itself is influenced through interaction with external parameters (e.g., oxygen levels 15,24 ). Conversely, online monitoring allows real-time quantification of analytes of interest. ...
Cell and gene therapies (CGTs) are examples of future therapeutics that can be used to cure or alleviate the symptoms of disease, by repairing damaged tissue or reprogramming defective genetic information. However, despite the recent advancements in clinical trial outcomes, the path to wide-scale adoption of CGTs remains challenging, such that the emergence of a “blockbuster” therapy has so far proved elusive. Manufacturing solutions for these therapies require the application of scalable and replicable cell manufacturing techniques, which differ markedly from the existing pharmaceutical incumbent. Attempts to adopt this pharmaceutical model for CGT manufacture have largely proved unsuccessful. The most significant challenges facing CGT manufacturing are process analytical testing and quality control. These procedures would greatly benefit from improved sensory technologies that allow direct measurement of critical quality attributes, such as pH, oxygen, lactate and glucose. In turn, this would make manufacturing more robust, replicable and standardized. In this review, the present-day state and prospects of CGT manufacturing are discussed. In particular, the authors highlight the role of fluorescent optical sensors, focusing on their strengths and weaknesses, for CGT manufacture. The review concludes by discussing how the integration of CGT manufacture and fluorescent optical sensors could augment future bioprocessing approaches.
... iNOS is an isoform of nitric oxide synthase, which catalyzes the formation of citrulline and NO from L-arginine, NADPH, and O 2 , with the formation of N-hydroxy-L-arginine as an intermediate (23). NO is an important radical also produced during hypoxia (24,25). Interestingly, treatment with hypoxia led to a striking increase in iNOS levels and activity in myometrial cells but did not affect leiomyoma cells (Fig. 2). ...
Objective:
To determine the effects of attenuating oxidative stress with the use of dichloroacetate (DCA) on the expression of key redox enzymes myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) as well as on apoptosis.
Design:
Prospective experimental study.
Setting:
University medical center.
Patient(s):
Cells established from myometrium and uterine fibroid from the same patients.
Intervention(s):
Cells were exposed to normal (20% O2) or hypoxic (2% O2) conditions for 24 hours with or without DCA (20 μg/mL), a metabolic modulator that shifts anaerobic to aerobic metabolism.
Main outcome measure(s):
Nitrate/nitrite (iNOS activity indicator), iNOS, Bcl-2/Bax ratio, MPO, and caspase-3 activities and levels were determined by means of Greiss assay, real-time reverse-transcription polymerase chain reaction, and ELISA. Data were analyzed with the use of SPSS by means of one-way analysis of variance with Tukey post hoc analysis and independent t tests.
Result(s):
MPO, iNOS, and nitrate/nitrite expression were higher in leiomyoma than in myometrial cells, and they were further enhanced by hypoxia in myometrial cells. Treatment with the use of DCA decreased MPO, iNOS, and nitrate/nitrite levels and negated the effect of hypoxia in both types of cells. Leiomyoma cells showed less apoptosis, as indicated by both caspase-3 activity and the Bcl-2/Bax ratio, than myometrial cells. Hypoxia further decreased apoptosis in myometrial cells with no further effect on leiomyoma cells. Treatment with DCA resulted in increased apoptosis in both types of cells, even in the presence of hypoxia.
Conclusion(s):
Shifting anaerobic to aerobic metabolism with the use of DCA resulted in an increase in apoptosis in leiomyoma cells and protected myometrial cells from the acquisition of the leiomyoma-like phenotype.
... The identity of the cellular oxygen sensor is not known, but a favin-heme protein residing in the plasma membrane has been proposed (392). This heme protein functions as an nicotinamide adenine dinucleotide phosphate reduced oxidase, transferring electrons through favin and heme to oxygen and generating superoxide. ...
... A prerequisite for many hypoxia-mediated responses is the basic-helix-loop-helix-PAS (Per-Arnt-Sim) family member transcription factors, hypoxia-inducible transcription factor (HIF)-1␣ [5] and -2␣ [6,7], that heterodimerize with the aryl hydrocarbon nuclear translocator protein (ARNT or HIF-1) [4,5] and then bind to the hypoxia response element (core consensus sequence: 5Ј-CGTG-3Ј) in responsive genes. Although the identity of the oxygen sensor upstream of the HIF transcription factors remains elusive [8], the molecular mechanisms of HIF activation by hypoxia are becoming clearer and include protein stabilization [9,10], thiol-redox regulation [11,12], and interaction with CBP/p300 [12,13]. Interestingly, agents besides hypoxia have been shown to activate HIF under normoxic conditions, including a variety of growth factors [14][15][16], v-SRC [17], phosphorylation by mitogen-activated protein kinase [18], as well as transition metals such as cobalt chloride and iron-chelating agents [19]. ...
Transcription factors orchestrate the development of extraembryonic tissues. Because placental hypoxia likely plays an important role in both normal and abnormal placentation, we have been investigating the hypoxia-inducible transcription factors (HIFs) in the human placenta. In this report, we focus on the placentas from women with preeclampsia. Because the placenta is a large, heterogeneous organ, we employed a systematic and unbiased approach to placental sampling, and our results are based on the analyses of eight biopsy sites per placenta. We observed no significant differences in HIF-1α or -2α mRNA expression between normal term and preeclamptic placentas. Nor was HIF protein expression significantly different, with the notable exception of HIF-2α, which, on average, was increased by 1.7-fold in the preeclamptic placentas (P < 0.03 vs. normal term placentas). Considering all 48 paired placental biopsy sites (eight sites each for six normal term and six preeclamptic placentas), HIF-2α protein levels in the preeclamptic placentas exceeded those in the normal term placentas in 39, or 81%, of the paired sites (P < 0.0013). The HIF-2α immunoreactivity was mainly located in the nuclei of the syncytiotrophoblast and fetoplacental vascular endothelium in the preeclamptic villous placenta. To control for the earlier gestational age of the preeclamptic placentas, an additional group of placentas from preterm deliveries without preeclampsia were also evaluated. The HIF protein expression was comparable in these preterm specimens and the normal term placentas. We conclude that protein expression of HIF-2α, but not of HIF-1α or -1β, is selectively increased in the preeclamptic placenta. The molecular mechanism(s) of this abnormality as well as the genes affected downstream are currently under investigation. To our knowledge, this is the first report of abnormal HIF-2α expression in human disease other than cancer.
... At the tissue level, the availability of oxygen is dependent on the distance from the oxygen-supplying blood vessels. Although the diffusion distance of oxygen in vivo is estimated to be 100-200 μm, a pO 2 of almost zero has been recorded in tissues 100 μm away from the nourishing blood vessels [5]. Therefore, a cellular/tissue oxygen-sensing mechanism is needed to assist tissue adaptation to nature/pathology-induced variations in oxygen availability. ...
Human periodontitis is a chronic inflammatory disease induced by opportunistic Gram-negative anaerobic bacteria at the tooth-supporting apparatus. Within the gingivitis-affected sulcus or periodontal pocket, the resident anaerobic bacteria interact with the host inflammatory reactions leading to a lower oxygen or hypoxic environment. A cellular/tissue oxygen-sensing mechanism and its appropriate regulation are needed to assist tissue adaptation to natural/pathology-induced variations in oxygen availability. In this chapter, we reviewed the biological relevance of hypoxia in periodontal/oral cellular development, epithelial barrier function, periodontal inflammation, and immunity. The role of hypoxia-inducible factor-1α in pathogen-host cross talk and alveolar bone homeostasis was also discussed. The naturally occurring pathophysiological process of hypoxia appeared to entail fundamental relevance for periodontal defense and regeneration.
... ROS-mediated stabilization of HIF-1α (-2α) The first models for HIF-1α stabilization under hypoxia postulated that a Fe 2+ /heme-containing, NADPH oxidase-like cytochrome targeted HIF-1α for degradation through ROS-dependent oxidation. [130,131] Therefore, it would follow that a decrease in O 2 should inhibit ROS generation and stabilize HIF-1α. Contrary to expectations, HIF-1α stabilization under hypoxia was intact in primary cells isolated from NADPH oxidase (NOX)-2 knockout mice. ...
Hypoxia (low O2) is an essential microenvironmental driver of phenotypic diversity in human solid cancers. Hypoxic cancer cells hijack evolutionarily conserved, O2- sensitive pathways eliciting molecular adaptations that impact responses to radiotherapy, tumor recurrence and patient survival. In this review, we summarize the radiobiological, genetic, epigenetic and metabolic mechanisms orchestrating oncogenic responses to hypoxia. In addition, we outline emerging hypoxia- targeting strategies that hold promise for individualized cancer therapy in the context of radiotherapy and drug delivery
... This observation might be of relevance in vivo, since hypoxia is frequently observed in tumoural tissue [10,11]. Tumor cells, as well as healthy cells, adapt to hypoxia by various appropriate physiologic responses, for example, by altered expression of genes that switch from oxidative to glycolytic metabolism [29,30]. These cellular responses are caused by the induction of the hypoxia-inducible factor (HIF) protein complex due to hypoxia. ...
Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro , although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo . This might be detrimental for the appropriate host immune response towards relevant pathogens.
... 22,23 NO is also able to conjugate with heme group of molecules such as soluble guanylyl cyclase (sGC) which is a classical downstream signal molecule of NO pathway. 24,25 After binding with NO, the heme maintains the 'oxy' conformation in hypoxia environment and the downstream pathway would not be activated, which deactivates transport proteins. 26 In this research, a biodegradable nanomedicine based on BNN6/DOX co-loaded 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 4 remarkable gas/drug effect on reversing MDR via NO-sensitized DOX efficacy enhancement ( Fig. 1). ...
Multidrug resistance (MDR) is responsible for the relatively low effectiveness of chemotherapeutics. Herein, a nitric oxide (NO) gas-enhanced chemosensitization strategy is proposed to overcome MDR by constructing a biodegradable nanomedicine formula based on BNN6/DOX co-loaded mPEG-PLGA. On the one hand, the nanomedicine is featured with high biocompatibility due to the high density of PEG and biodegradable PLGA. On the other hand, the nanoformula exhibits excellent stability under physiological conditions but stimuli-responsive decomposition of BNN6 for NO gas release upon ultraviolet-visible irradiation. More importantly, after triggering NO release, gas molecules would be generated to break the nanoparticle shell and lead to the release of doxorubicin. Furthermore, NO was demonstrated to reverse the MDR of tumor cells and enhance the chemosensitization for doxorubicin therapy.
... The possible explanation of HIF-1α activation involves an iron-containing flavoheme protein which can sense the hypoxia. Ni 2+ can activate a signaling cascade leading to HIF-1α stabilization by substituting for iron in this sensor [68,69]. Subsequently, HIF-1α will promote fibrogenesis via several different mechanisms [70]. ...
Numerous analyses including in vivo and in vitro experiments have demonstrated that inhalation exposure of NiONPs can result in pulmonary fibrosis. However, the potential mechanisms of this pathological process remain elusive. Here, we investigate the role of HIF-1α and TGF-ß1 in NiONPs-induced pulmonary fibrosis with a focus on the interplay of the above two proteins. In vivo, male Sprague&Dawley rats were exposed to NiONPs and pulmonary fibrosis was demonstrated using H&E staining and immunochemistry of αSMA. In vitro, NiONPs contributed to cell proliferation and increased expressions of collagen-1 and αSMA in human fetal lung fibroblasts. Both HIF-1α and TGF-ß1 were upregulated by NiONPs treatment. Inhibition of HIF-1α reduced TGF-ß1 expression and downregulation of TGF-ß1 reduced HIF-1α protein level. Mechanism investigation revealed that TGF-ß1 affects nuclear translocation activity of HIF-1α. Taken together, these finding provide evidence that HIF-1α and TGF-ß1 act in synergy to foster NiONPs-induced pulmonary fibrosis, and the cross talk between them is a pivotal mechanism of pulmonary fibrosis.
... It is of note that AhR ligands induce oxidative stress via increased H 2 O 2 production in an AhR-dependent fashion [190]. Increased levels of ROS have been shown to promote the production of molecular O 2 resulting in destabilization of HIF [191]. As to how the AhR and hypoxia modulate the melatoninergic pathways, including the melatonin receptors, will be important to determine, given the inhibitory effects of melatonin on ROS activation of the AhR and its inhibition of HIF1 in GBM [165]. ...
Abstract
Primary glioma, as well as secondary metastases, provide significant treatment challenges. An understanding of the biological underpinnings of glioma is likely to provide new pharmaceutical targets that will improve patient survival. Here, we look at the role that the kynurenine pathways and associated tyrptophan catabolites (TRYCATs) play in glioma, linking this to changes in oxidative and nitrosative stress (O&NS), immuneinflammatory activity, the aryl hydrocarbon receptor (AhR), and the melatoninergic pathways. It is suggested that the interactions of O&NS and the immune-inflammatory processes in glioma contribute to the induction of the TRYCATs via the kynurenine activation of the AhR, leading to increased indoleamine 2,3-dioxygenase, which deprives tryptophan for the necessary serotonin that is required as a precursor for the melatoninergic pathways. A diverse array of data pertaining to glioma can be linked to these pathways, including changes in miRNAs, epigenetic processes, estrogen receptors, 14-3-3, chromosome 4q35, neurotrophins, tristetraprolin and the N-acetylserotonin (NAS)/melatonin ratio. As many of these factors directly or indirectly act on the melatoninergic pathways, including variations in the NAS/melatonin ratio, it is suggested that the melatoninergic pathways may act as a hub that co-ordinate the multitude of changes occurring in glioma. Consequently, the melatoninergic pathways may be a significant pharmaceutical target for the treatment of this still very poorly managed condition.
... It is of note that AhR ligands induce oxidative stress via increased H2O2 production in an AhR-dependent fashion [190]. Increased levels of ROS have been shown promote the production of molecular O2 resulting in destabilization of HIF [191]. As to how the AhR and hypoxia modulate the melatoninergic pathways, including the melatonin receptors, will be important to determine, given the inhibitory effects of melatonin on ROS activation of the AhR and its inhibition of HIF1 in GBM [165]. ...
Primary glioma, as well as secondary metastases, provide significant treatment challenges. An understanding of the biological underpinnings of glioma is likely to provide new pharmaceutical targets that will improve patient survival. Here, we look at the role that the kynrenine pathways and associated tyrptophan catabolites (TRYCATs) play in glioma, linking this to changes in oxidative and nitrosative stress (O&NS), immune-inflammatory activity, the aryl hydrocarbon receptor (AhR), and the melatoninergic pathways. It is suggested that the interactions of O&NS and the immune-inflammatory processes in glioma contribute to the induction of the TRYCATs via the kynurenine activation of the AhR, leading to increased indoleamine 2,3-dioxygenase, which deprives tryptophan for the necessary serotonin that is required as a precursor for the melatoninergic pathways. A diverse array of data pertaining to glioma can be linked to these pathways, including changes in miRNAs, epigenetic processes, estrogen receptors, 14-3-3, chromosome 4q35, neurotrophins, tristetraprolin and the N-acetylserotonin (NAS)/melatonin ratio. As many of these factors directly or indirectly act on the melatoninergic pathways, including variations in the NAS/melatonin ratio, it is suggested that the melatoninergic pathways may act as a hub that co-ordinate the multitude of changes occurring in glioma. Consequently, the melatoninergic pathways may be a significant pharmaceutical target for the treatment of this still very poorly managed condition.
... Expression of these angiogenic factors is likely to be induced by the hypoxic microenvironment present in the avascular tumor tissue (2). Significant evidence indicates that the VEGF gene is under the control of HIF-1␣, which itself is strongly induced by hypoxia (3)(4)(5). Furthermore, vascular growth may be regulated by complex interactions of extracellular matrix molecules, proteolytic enzymes, and cell adhesion molecules. The inhibition of angiogenesis is considered one of the most promising strategies leading to the development of novel antineoplastic therapies (6). ...
Tumor vascularization is the rate-limiting step for the progression of cancer. Differential steps of tumor-induced angiogenesis were studied by a novel in vitro confrontation culture of avascular multicellular prostate tumor spheroids and embryoid bodies grown from pluripotent embryonic stem (ES) cells. Vascularization in embryoid bodies started on day 5 of cell culture and was paralleled by down-regulation of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF). In parallel, a dissipation of gradients in the pericellular oxygen pressure was observed as measured by O2-sensitive microelectrodes. After 24–48 h of confrontation culture, cells positive for platelet endothelial cell adhesion molecule (PECAM-1) became visible in the contact region between the embryoid body and the tumor spheroid and sprouted within the confrontation cultures during subsequent days. Tumor-induced angiogenesis resulted in growth stimulation of tumor spheroids, disappearance of central necrosis and a reduction of the pericellular oxygen pressure. Furthermore, tumor vascularization resulted in elevated levels of HIF-1α, VEGF, heat shock protein 27 (HSP27), and P-glycoprotein. Tumor-induced angiogenesis may augment the oxygen consumption in tumors resulting in an increased expression of hypoxia-related, proangiogenic genes as well as of HSP27 and P-glycoprotein, which are involved in a multidrug resistance phenotype.—Wartenberg, M., Dönmez, F., Ling, F. C., Acker, H., Hescheler, J., Sauer, H. Tumor-induced angiogenesis studied in confrontation cultures of multicellular tumor spheroids and embryoid bodies grown from pluripotent embryonic stem cells.
Coronavirus disease 2019 (COVID-19) is a global pandemic affecting 185 countries and >18.8 million patients worldwide as of July 2020. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-2) and majorly manifesting as a hypoxic condition in the affected patients. It has shown to multiply rapidly among patients showing other debilities, majorly hypertension, and diabetes mellitus. Periodontitis is defined as a multifactorial disease predominantly governed by microbial dysbiosis and having risk factors as the presence of systemic pathology, decline in immunity, and increased stress. Keeping these factors as a plausible threat for periodontitis, there can be a postulation made that periodontitis can act as a probable risk factor for COVID-19. Limited resources and novelty of the disease hold back any confirmation of the co-relation.
Silica-coated superparamagnetic iron nanoparticles (SiMAGs) are an exciting biomedical technology capable of targeted delivery of cell-based therapeutics and disease diagnosis. However, in order to realise their full clinical potential, their intracellular fate must be determined. The analytical techniques of super-resolution fluorescence microscopy, particle counting flow cytometry and pH-sensitive nanosensors were applied to elucidate mechanisms of intracellular SiMAG processing in human mesenchymal stem cell (hMSCs). Super-resolution microscopy showed SiMAG fluorescently-tagged nanoparticles are endocytosed and co-localised within lysosomes. When exposed to simulated lysosomal conditions SiMAGs were solubilised and exhibited diminishing fluorescence emission over 7 days. The in vitro intracellular metabolism of SiMAGs was monitored in hMSCs using flow cytometry and co-localised pH-sensitive nanosensors. A decrease in SiMAG fluorescence emission, which corresponded to a decrease in lysosomal pH was observed, mirroring ex vivo observations, suggesting SiMAG lysosomal exposure degrades fluorescent silica-coatings and iron cores. These findings indicate although there is a significant decrease in intracellular SiMAG loading, sufficient particles remain internalised (>50%) to render SiMAG treated cells amenable to long-term magnetic cell manipulation. Our analytical approach provides important insights into the understanding of the intracellular fate of SiMAG processing, which could be readily applied to other particle therapeutics, to advance their clinical translation.
Background
Acupuncture is a traditional method that has been widely used in various fields of medicine with therapeutic effect. However, evidence of effectiveness to support the application of electroacupuncture (EA) during the process of ischaemia is scarce.
Objectives
To investigate dynamic changes in hypoxia-inducible factor (HIF)−1α expression as well as its association with neurological status in rats subjected to acute ischaemic stroke and EA intervention.
Methods
Forty adult male rats were randomly divided into three groups that received sham surgery (Control group, n=10) or underwent middle cerebral artery occlusion and EA (MCAO+EA group, n=15) or minimal acupuncture as a control treatment (MCAO+MA group, n=15). The rats in the MCAO+EA and MCAO+MA groups received EA or acupuncture without any electrical current, respectively, during 90 min of ischaemia. Rats in the Control group received the same surgical procedure but without MCAO. EA involved electrical stimulation of needles inserted into the quadriceps at 50 Hz frequency and 3 mA current intensity. Neurological status was evaluated on postoperative day 1, and cerebral infarction volume (IV) and HIF-1α expression 24 hours later.
Results
Neurological scores were improved and cerebral IV was decreased in the MCAO+EA group compared to the MCAO+MA group (both p<0.05). Moreover, HIF-1α expression was higher in the MCAO+EA group versus the MCAO+MA group (p<0.05).
Conclusions
EA enhanced recovery of neurological function, decreased cerebral IV and increased HIF-1α expression in ischaemic rats. Further research is needed to determine whether EA is effective for stroke treatment through the stimulation of muscle contraction.
Reactive oxygen and nitrogen species (RONS), once viewed solely as toxic by-products of aerobic metabolism and components of leukocyte bactericidal defenses, have since gained recognition as important intra- and intercellular signalling molecules in both normal as well as pathological conditions. Reactive oxygen and nitrogen species are formed in cells during oxidant stress, in reperfusion injury, and in response to several growth factors and cytokines (120). Indeed, these species may represent a novel class of second or third messengers. Like other 2nd messenger systems (e.g. Ca2+, cAMP), reactive oxygen and nitrogen species are ideal signalling molecules. They are rapidly formed and highly labile, which may serve to restrict them to specific cellular compartments and improve target specificity while reducing unwanted, non-specific effects. Most importantly, the biochemical modifications induced by modest levels of reactive nitrogen and oxygen species are largely reversible (119).
Oxygen (O-2) sensing in blood and regulation of microvascular tone appear to involve hemoglobin (Hb) conformational changes resulting from O-2 desaturation. This observation has prompted the thought that Hb functions as both an O-2 sensor and regulator of microvasular blood flow to meet local tissue oxygen demand. The mechanism(s) by which this is accomplished has recently been the subject of increasing debate. Three primary hypotheses are described within the literature and include release of adenosine 5'-triphosphate by red blood cells (RBCs), release of S-nitrosylated molecules from RBCs originally bound to beta93 cysteine residues of oxyHb, and nitrite conversion and storage of nitric oxide by Hb at the site of ferric (Fe3+) and ferrous (Fe2+) Hb. Within extravascular cells, the global regulator of oxygen homeostasis is hypoxia-inducible factor-1 (HIF-1). This transcriptional factor is tightly regulated by O-2 and cellular redox-sensitive mechanisms. HIF-1 activation is responsible for the up-regulation of proteins, which increase O-2 supply. We believe that there are important and yet unexplored mechanisms by which RBCs can directly or indirectly communicate via redox intermediates with extravascular sites as part of the global O-2 sensing mechanism.
Hypoxia is a typical hallmark of various diseases, including cancer, ischemic diseases, and stroke. It is also associated with the disease progression. Therefore, it is critical to develop an effective strategy to target the hypoxic region for diagnosis and treatment. In this review, we summarize recent progress in the development of hypoxia-responsive systems for imaging, sensing and therapy. Two types of hypoxia-sensitive systems, the hypoxia inducible factor-1 based systems and bioreductive molecule based systems, were reviewed with comments on their advantages and limitations. Future opportunities and challenges are also discussed in the end.
Numerous analyses including in vivo and in vitro experiments have demonstrated that inhalation exposure of NiONPs can result in pulmonary fibrosis. However, the potential mechanisms of this pathological process remain elusive. Here, we investigate the role of HIF-1 alpha and TGF-beta 1 in NiONPs-induced pulmonary fibrosis with a focus on the interplay of the above two proteins. In vivo, male Sprague&Dawley rats were exposed to NiONPs and pulmonary fibrosis was demonstrated using H&E staining and immunochemistry of alpha SMA. In vitro, NiONPs contributed to cell proliferation and increased expressions of collagen-1 and alpha SMA in human fetal lung fibroblasts. Both HIF-1 alpha and TGF-beta 1 were upregulated by NiONPs treatment. Inhibition of HIF-la reduced TGF-beta 1 expression and downregulation of TGF-beta 1 reduced HIF-1 alpha protein level. Mechanism investigation revealed that TGF-beta 1 affects nuclear translocation activity of HIF-1 alpha. Taken together, these finding provide evidence that HIF-1 alpha and TGF-61 act in synergy to foster NiONPs-induced pulmonary fibrosis, and the cross talk between them is a pivotal mechanism of pulmonary fibrosis.
OBJECTIVE: To investigate the effects of hypoxia on the proliferation of human breast cancer MCF-7 cells and the cellular expression of hypoxia inducible factor-1α, stromal cell derived factor-1 and CXC chemokine receptor 4.METHODS: Human breast cancer MCF-7 cells with exponential growth in routine culture were exposed to 50, 100, 150 and 200 μmol/L CoCl2 to mimic hypoxic conditions, At 12, 24 and 48 h, the cells were collected for MTT assay, RT-PCR for HIF-1α, SDF-1 and CXCR4 mRNAs expression, Western bolt for HIF-1α and CXCR4 proteins expression, and the expression of SDF-1 in MCF-7 cells exposed to hypoxia (by CoCl2 150 μmol/L) at different time points were detected by immunofluorescence. RESULTS: Compared with the cells without CoCl2 treatment, a significant growth inhibition which increased with CoCl2 concentration and exposure time was observed, the inhibition rate of 50, 100 and 150 μmol/L with CoCl2 for 24 h was (54.62±0.07)%, (65.21±0.03)%, (80.15±0.01)% and (94.51±0.01)%, respectively. With 150 μmol/L CoCl2 for 12, 24 and 48 h, the inhibition rate were (70.83±0.03)%, (80.15±0.01)% and (89.27±0.03)%, respectively. RT-PCR showed that over-expression of HIF-1α, SDF-1 and CXCR4 mRNAs and western boltshowed that over-expression of HIF-1α and CXCR4 proteins, which could be measured under hypoxia induced by CoCl2. The immunofluorescence showed that the expression of SDF-1 protein increased with time extension after the cells stimulated by 150 μmol/L CoCl2. CONCLUSION: Hypoxia mimiced by CoCl2 exposure significantly inhibits the proliferation of MCF-7 cells, and at the non-toxic doses, CoCl2 dose-and time-dependently increases the expression of HIF-1α, SDF-1 and CXCR4.
Objective: To explore the effects and probable mechanism of hypoxia preconditioning on the proliferation, apoptosis and necrosis of bone marrow derived mesenchymal stem cells (BMSCs). Methods: BMSCs were cultured by whole bone marrow adherence method. The characteristics of BMSCs were identified by inducing adipogenic and osteogenic differentiation and surface markers (CD29, CD90, CD45) with flow cytometry (FCM). Cultured BMSCs were divided into the following groups: normal culture group, normal transfection negative group, normal hypoxia inducible factor-1α (HIF-1α) siRNA group, hypoxia culture group, hypoxia transfection negative group and hypoxia HIF-1α siRNA group. MTT assay was used to detect the proliferation rate of cells. FCM was appliedto detect the apoptosis and lactic acid dehydrogenase (LDH) activity in supernatant was determined to evaluate cell necrosis. Theprotein and mRNA expressions of HIF-1α, glucose transporter (GLUT-1) and VEGF of BMSCs were detected by Western blot and real-time PCR. Results: Cultured BMSCs had the capacities for adipogenic and osteogenic differentiation, and highly expressed CD29 and CD90, and negative for CD45. Hypoxia culture group had higher proliferation rate and lower LDH activity than normal culture groups (P<0. 05), but there was no significant difference of apoptosis among the above groups (P>0. 05). The protein and mRNA levels of HIF-1α, GLUT-1 and vascular endothelial growth factor (VEGF) of BMSCs in hypoxia culture group were significantly higher than that in normal group. After silencing HIF-1α, the protein and mRNA levels of HIF-1α, GLUT-1 and VEGF of BMSCs in hypoxia HIF-1α siRNA group were lower than that in hypoxia culture groups. Besides, in hypoxia HIF-1α siRNA group, the proliferation rate was lower and the LDH activity was higher than that in hypoxia culture group (P<0. 05). Conclusions: Hypoxia preconditioning promoted the proliferation ability and inhibited the necrosis of BMSCs in vitro, which were attributable in part to the up-regulation of GLUT-1 and VEGF by HIF-1α activation after hypoxia preconditioning.
The concept of a "critmeter" within the kidney establishes the role of the kidney not only in regulating ECF volume and RBC mass, but in integrating these two volumes to generate the hematocrit. As the "normal" hematocrit is not a random number, but one that maximizes tissue oxygen delivery, it follows that the hematocrit should be regulated. It is hypothesized that the "critmeter" is a functional unit established by nephron heterogeneity in renal blood flow and in the reabsorption of sodium under physiological conditions. The kidney can uniquely translate a measure of plasma volume into a tissue oxygen pressure signal by the effects of sodium reabsorption on renal energy utilization and oxygen consumption. The RBC mass and plasma volumes are integrated at the level of the tissue partial oxygen pressure by the balance of oxygen consumption required for sodium reabsorption and oxygen delivery to the proximal tubule. This balance is modulated by the renin-angiotensin system in that Ang II affects these variables disproportionately. Clinical and experimental evidence supports the interaction of the reninangiotensin system with the production of erythropoietin. In terms of blood volume regulation, the effects of the renin-angiotensin system on erythropoietin production allow for the efferent signal of volume regulation to more closely reflect the components of the afferent signals (Figure 4). Examples of resetting or dysfunction of the "critmeter" are the functional deficiency of erythropoietin in some forms of chronic renal failure and the excessive production in some cases of post-transplant erythrocytosis.
Reactive oxygen species and reactive nitrogen species, also known as free radicals, are products of normal cellular metabolism. They are species, capable of independent existence, which contains one or more unpaired electrons. That usually gives a degree of chemically reactivity to the free radical with the capacity of attacking biological structures. Reactive oxygen species include superoxide, hydroxyl and hydrogen peroxide among others, and the most relevant reactive nitrogen species are nitric oxide and peroxynitrite. The sources of Reactive Species are mitochondria, enzymes like NADPH oxidase and xanthine oxidase, and the reactions with ion transition metals like iron and cupper. Free Radicals can act as physiological and pathophysiological agents, depending on their balance, and if the production is acute and reversible or chronic. The physiological functions include process like cellular signaling and immune response, and the pathological effects are as promoters and continuers of diverse diseases like cancer, cardiovascular diseases, inflammation and multi organ failure in the critical ill patient. The antioxidant system consist of enzymes such as catalase, glutathione and superoxide dismutase, and scavengers like vitamins and flavonoids. This chapter provides a comprehensive review of the basis for the biochemical and physiological mechanisms involving reactive species.
The mitochondrial respiratory chain participates in performance of the signal transforming system which activates metabolic compensatory processes and the related functional response to both single and repeated, long-term exposure to hypoxia. The suppressed function of mitochondrial enzyme complex I (MEC I) and the alternative activation of mitochondrial enzyme complex II (MEC II) comprise a signaling regulatory mechanism which contributes to the formation of tissue-specific and general resistance of the body to different types of hypoxia. This process is associated with recovery of the electron transport function in the major respiratory chain and changes in the kinetic properties of respiratory chain enzymes. The degree of MEC I inhibition and MEC II activation in rat brain in the first 2 hours after a hypoxic exposure correlates with pronouncement of adaptation to hypoxia. Activation of the mechanism is necessary for stabilization and accumulation of hypoxia inducible factor-1 (HIF-1). The mechanisms underlying regulatory and signaling roles of mitochondrial enzymes and of the tricarbonic acid cycle substrates, succinate and a-ketoglutarate, in the modulation of HIF-1 activity are discussed in this chapter. It was shown that while formation of adaptive signs occurs in the hypoxic period, the irritating effect of hypoxia may result in overstrain of the system and failure of adaptive bioenergetic mechanisms. Advantages of intermittent normobaric hypoxia training over other hypoxic regimens in formation of resistance to hypoxia are due to the modulating and normalizing effect of reoxygenation periods on irritating effects of hypoxia. These reoxygenation periods provide more efficient energy regulation in this case. We have also demonstrated a possibility for improving the efficiency of hypoxic therapy by using combinations of hypoxic training with succinate-containing drugs. It was proven that succinate is essential for formation of urgent and long-term adaptive responses in the period of hypoxic training. This finding opens new prospects for improvement of the efficiency and optimization of hypoxic therapy by using the combinations of hypoxia and succinate-containing drugs.
Angiogenesis refers to the formation of new capillary network arising from existing blood or lymph vessels. It has been known for years that hypoxia regulates both physiological and pathological angiogenesis, in which hypoxia inducible factor 1 (HIF-1) plays a critical role in the regulation of angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin 1 and 2, placental growth factor, and platelet-derived growth factor B, etc. This chapter provides an updated overview focusing on the pro-angiogenesis effects of intermittent hypoxia in cardiac and skeletal muscles. Evidence from animal and human studies has clearly indicated that intermittent hypoxia is an effective inducer of angiogenesis in both cardiac and skeletal muscles. The angiogenic changes are likely mediated by the signaling pathway that involves HIF-1α and VEGF, which is activated by either hypobaric or normobaric intermittent hypoxia. Other clinically relevant forms of intermittent hypoxia/ischemia that are resulted from heavy muscular exercise, peripheral vascular diseases, or repetitive coronary occlusions could also lead to similar adaptive changes in myocardium and skeletal muscles. Further studies are warranted to develop a well controlled and validated regimen of intermittent hypoxia, which may be considered as a non-invasive approach to induce angiogenesis that could be instrumental in the treatment of various diseases with the primary pathological conditions related to tissue hypoxia.
Systemic lupus erythematosus (SLE) is a complex autoimmune multiorgan disease. In SLE, the disruption of reactive intermediate homeostasis may lead to a break in immune tolerance, increased tissue damage, and altered enzyme function. This chapter reviews the latest evidence from both animal and human studies on the role of reactive intermediates (RIs) in the pathogenesis of SLE. RIs are short-lived molecules produced by normal cellular metabolism and aid in a multitude of physiological and pathological processes. Nitrogen-based reactive intermediates are known as reactive nitrogen intermediates (RNI), while those that are oxygen-based are known as reactive oxygen intermediates (ROIs). While iNOS activity can suppress parasitemia or tumor growth, its overexpression in the setting of lupus disease activity appears to lead to organ damage and an altered immune response. No study to date has used selective iNOS inhibitors to explore the pathogenic potential of iNOS in humans with lupus. However, several studies have demonstrated elevated markers of NO production in lupus patients compared to controls and a significant correlation between markers of systemic NO production and lupus disease activity. Conflicting results from murine studies using pharmacologic or genetic manipulation of NOS highlight the complex biology of reactive intermediates in lupus. Further studies should be directed toward investigating potential sources of RIs other than iNOS, defining the key reactive species that are dominant in lupus, and addressing how the interplay between RNIs and ROIs may lead to a break in tolerance and enhanced aggressiveness of lupus disease activity.
Anemia is a common complication of CKD, particularly among patients whose GFR is less than 30–40 mL/min. The most important factor contributing to this anemia is the relative deficiency of the glycoprotein hormone erythropoietin. Erythropoietin is produced in the kidneys by cortical peritubular interstitial fibroblasts. The production of erythropoietin by the kidneys declines as functioning renal mass declines. Iron deficiency is also a common factor contributing to anemia in CKD. Iron metabolism is normally tightly regulated by hepcidin. Abnormalities in hepcidin that reduce iron availability for erythropoiesis induced by inflammation contribute to anemia in CKD patients. Erythrocytosis is a much less common occurrence in patients with CKD, and is mostly seen in patients with polycystic kidney disease or following kidney transplantation. Defective platelet function with a bleeding tendency is well-known to occur in patients with advanced CKD and uremia. At what CKD stage platelet dysfunction becomes clinically relevant is not known. Therapies aimed at improving this acquired platelet dysfunction include initiation of dialysis, desmopressin (DDAVP), cryoprecipitate, improvement in anemia, and estrogens.
Chronic kidney disease (CKD) often leads to renal anaemia, due to gradual reduction of erythropoietin-producing renal cells. About 15–30% of geriatric cats develop CKD, with renal disease being the primary cause of death of older cats. Of those cats with CKD, up to 65% in later-stage CKD will develop renal anaemia. Recognising and treating anaemia of renal disease is an important part of CKD therapy in both dogs and cats. The use of erythropoiesis-stimulating agents (ESAs) is standard-of-care in humans and becoming more used in veterinary medicine. Darbepoetin alfa (DA) has been shown to be effective in the treatment of renal anaemia.
To compare the effect of shifting anaerobic to aerobic metabolism on key regulators of oxidative stress, including extracellular superoxide dismutase (SOD3), inducible nitric oxide synthase (iNOS), and its product, nitric oxide (NO), as well as mitochondrial potential (Δψm) and apoptosis in fibroblasts established from normal peritoneum and adhesion tissues.
Prospective, experimental study.
University medical center.
Fibroblasts established from normal peritoneum and adhesion tissues from the same patients.
Treatment with dichloroacetate (0, 20, 40, and 80 μg/mL, 24 hours) MAIN OUTCOME MEASURE(S): The expression of SOD3, iNOS, and NO levels were measured utilizing real-time reverse transcription-polymerase chain reaction and Greiss assay. The Δψm was evaluated by the JC-1 Mitochondrial Membrane Potential Assay. Apoptosis was determined by caspase-3 activity and TUNEL assays. Data were analyzed using SPSS 19.0. Mixed model repeated-measures analysis of variance was used with a Bonferroni correction. Significant interactions were analyzed with independent sample t tests.
Dichloroacetate increased apoptosis, SOD3 messenger RNA, iNOS messenger RNA, and NO levels in fibroblasts from peritoneum and adhesions. There was enhanced Δψm adhesion as compared with normal peritoneal fibroblasts. Creating oxidative stress by exposure by hypoxia markedly increased Δψm in fibroblasts from normal peritoneum to levels observed in adhesions; dichloroacetate protected against the effects of hypoxia.
Anaerobic metabolism and oxidative stress are associated with the development of the adhesion phenotype, which manifests decreased apoptosis. Dichloroacetate induces adhesion fibroblasts to undergo apoptosis via modulation of redox homeostasis. These findings may provide targets for therapeutic treatment for reduction of profibrotic disorders, including postoperative adhesions.
Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Oxygen as an essential element is critical for energy production and existence of all organisms on earth. However, there are potentially damaging effects also associated with it leading to production of oxygen centered free radicals. These radicals are highly reactive and can cause damage to various biomolecules. Presence of a physiological antioxidant defense system keeps these free radicals in check. Any imbalance in the levels of free radicals or reactive oxygen species (ROS) leads to oxidative stress in the body and may culminate in various patho-physiological conditions. In this chapter we have systematically provided details about the sources of free radicals, their types, their biomarkers and physiological significance.
Aluminum has been repeatedly implicated in the etiopathology of several human neurodegenerative disorders including Alzheimer's disease. Due largely to the extensive euchromatization of normal brain cell neuronal nuclei and their high intrinsic rate of transcription, one prime target for aluminum appears to be within the nucleic acid compartments of the central nervous system. Generally, the high positive charge density of aluminum (Z2/r = 17.65) acts as a pervasive repressor and demodulator of neural activity. This chapter will review our current knowledge concerning aluminum as a genotoxic 'dementing' cation, particularly as it applies to neural-specific gene transcription and transcription factors in the human brain.
Paragangliome sind meist benigne Tumoren, die aus paraganglionären Chemorezeptoren, hauptsächlich der Kopf-Hals-Region, hervorgehen. Diese Tumoren bilden nur in seltenen Fällen Metastasen aus, führen jedoch häufig zu Symptomen wie Schwerhörigkeit, Tinnitus und Gesichtsnervparalyse. Kürzlich wurde gezeigt, dass in familiären Fällen von Paragangliomen 3 Gene, die für Untereinheiten des Atmungskettenproteins Succinat-Dehydrogenase (SDH) kodieren, von Mutationen betroffen sein können. Das Gen, welches für die Untereinheit SDHD kodiert, ist hierbei in bis zu 50% dieser Fälle mutiert. Der Nachweis eines LOH („loss of heterozygosity“, Verlust der Heterozygotie) in Tumorproben betroffener Patienten machte SDHD zum ersten bekannten Tumorsuppressor-Gen, welches für ein mitochondrielles Protein kodiert. In diesem Artikel werden die aktuellen Forschungsergebnisse auf dem Gebiet der Paragangliome zusammengefasst.
Abstract
Paragangliomas of the head and neck region are usually benign tumors that develop from chemoreceptors of paraganglionic origin in the majority of patients. These receptors play an important role in sensing and regulation of the blood CO2 level. Genetic alterations in the mitochondrial enzyme complex II (SDH), which is involved in respiratory chain and citric acid cycle reactions, have been shown to lead to sporadic as well as familial cases of these tumors. The gene encoding the subunit SDHD shows mutations in up to 50% of these cases. In addition, loss of heterozygosity (LOH) was demonstrated in these tumor samples and has been shown to be connected with oncogenesis of paragangliomas. Thus, SDHD is the first known tumor suppressor gene encoding a mitochondrial protein. In this article we summarize the current state of knowledge concerning the development of paragangliomas.
Hypoxia-inducible factor 1 (HIF-1) activates erythropoietin gene transcription in Hep3B cells subjected to hypoxia. HIF-1 activity is also induced by hypoxia in non-erythropoietin-producing cells, suggesting a more general regulatory role. We now report that RNAs encoding the glycolytic enzymes aldolase A (ALDA), phosphoglycerate kinase 1 (PGK1), and pyruvate kinase M were induced by exposure of Hep3B or HeLa cells to inducers of HIF-1 (1% O2, cobalt chloride, or desferrioxamine), whereas cycloheximide blocked induction of glycolytic RNAs and HIF-1 activity. Oligonucleotides from the ALDA, PGK1, enolase 1, lactate dehydrogenase A, and phosphofructokinase L (PFKL) genes, containing sequences similar to the HIF-1 binding site in the erythropoietin enhancer, specifically bound HIF-1 present in crude nuclear extracts or affinity-purified preparations. Sequences from the ALDA, PFKL, and PGK1 genes containing HIF-1 binding sites mediated hypoxia-inducible transcription in transient expression assays. These results support the role of HIF-1 as a mediator of adaptive responses to hypoxia that underlie cellular and systemic oxygen homeostasis.
Cardiomyocytes suppress contraction and O2 consumption during hypoxia. Cytochrome oxidase undergoes a decrease in V
max during hypoxia, which could alter mitochondrial redox and increase generation of reactive oxygen species (ROS). We therefore
tested whether ROS generated by mitochondria act as second messengers in the signaling pathway linking the detection of O2 with the functional response. Contracting cardiomyocytes were superfused under controlled O2 conditions while fluorescence imaging of 2,7-dichlorofluorescein (DCF) was used to assess ROS generation. Compared with normoxia
(PO2 ∼ 107 torr, 15% O2), graded increases in DCF fluorescence were seen during hypoxia, with responses at PO2 = 7 torr > 20 torr > 35 torr. The antioxidants 2-mercaptopropionyl glycine and 1,10-phenanthroline attenuated these increases
and abolished the inhibition of contraction. Superfusion of normoxic cells with H2O2 (25 μm) for >60 min mimicked the effects of hypoxia by eliciting decreases in contraction that were reversible after washout of
H2O2. To test the role of cytochrome oxidase, sodium azide (0.75–2 μm) was added during normoxia to reduce theV
max of the enzyme. Azide produced graded increases in ROS signaling, accompanied by graded decreases in contraction that were
reversible. These results demonstrate that mitochondria respond to graded hypoxia by increasing the generation of ROS and
suggest that cytochrome oxidase may contribute to this O2 sensing.
1. Treatment of rats with small doses of CoCl2 decreases liver 5-aminolaevulinate synthase (EC 2.3.1.37) activity and impairs incorporation of 5-amino[14C]laevulinate into liver haem. Salts of other metals (cadmium, nickel, manganese and zinc) are all relatively inactive. 2. The dose-response curves obtained for both these effects closely mirror the accumulation in the liver of a compound that is labelled by 5-amino[14C]laevulinate and is unextractable by acetone/HCl. 3. Incorporation of 5-amino[14C]laevulinate into unextractable compound is also obtained in vitro by incubating liver homogenates with label in the presence of cobalt:isotope-dilution experiments show that the radioactivity passes through pools of porphobilinogen and protoporphyrin, but not of haem. 4. The unextractable compound is not covalently bound to protein and possesses the same extraction and spectral properties as authentic cobalt protoporphyrin. 5. It is concluded (a) that cobalt protoporphyrin is readily formed not only in vitro, but also in vivo, and (b) that its formation accounts for the impaired incorporation of 5-aminolaevulinate into haem and may also be responsible for the action of cobalt on 5-aminolaevulinate synthase.
The cofactor requirements of macrophage nitric oxide (NO.) synthase suggest involvement of an NADPH-dependent flavoprotein. This prompted us to test the effect of the flavoprotein inhibitors diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodoniumdiphenyl (ID) on the NO. synthases of macrophages and endothelium. DPI, DTI, and ID completely inhibited NO. synthesis by mouse macrophages, their lysates, and partially purified macrophage NO. synthase. Inhibition of NO. synthase by these agents was potent (IC50's 50-150 nM), irreversible, dependent on time and temperature, and independent of enzyme catalysis. The inhibition by DPI was blocked by NADPH, NADP+, or 2'5'-ADP, but not by NADH. Likewise, FAD or FMN, but not riboflavin or adenosine 5-diphosphoribose, protected NO. synthase from inhibition by DPI. Neither NADPH nor FAD reacted with DPI. Once NO. synthase was inhibited by DPI, neither NADPH nor FAD could restore its activity. DPI also inhibited acetylcholine-induced relaxation of norepinephrine-preconstricted rabbit aortic rings (IC50 300 nM). Inhibition of acetylcholine-induced relaxation persisted for at least 2 h after DPI was washed out. In contrast, DPI had no effect on norepinephrine-induced vasoconstriction itself nor on vasorelaxation induced by the NO.-generating agent sodium nitroprusside. These results suggest that NO. synthesis in both macrophages and endothelial cells depends on an NADPH-utilizing flavoprotein. As a new class of NO. synthase inhibitors, DPI and its analogs are likely to prove useful in analyzing the physiologic and pathophysiologic roles of NO(.).
The rat carotid body tissue reveals a photometrically measurable haem signal with absorbance maxima at 560 nm, 518 nm and 425 nm, suggesting the presence of a b-type cytochrome; this was confirmed by pyridine haemochrome and CO spectra. The quantity of cytochrome b was estimated to be 310 pmol.mg of protein-1. This haem is capable of H2O2 formation, which can be inhibited by 10 microM-diphenyliodonium (DPI). The hypoxia-induced increase in nervous chemoreceptor discharge and the reduction of FAD and NAD(P)+ were also inhibited by DPI (10 microM). These results suggest that an oxidase such as the NAD(P)H oxidase of neutrophils may act as a pO2 sensor protein in the rat carotid body, probably inducing the pO2 chemoreceptor process by H2O2 formation.
We demonstrate, by means of immunohistochemistry, that type I cells of human, guinea pig, and rat carotid bodies react with antisera raised against the subunits p22phox, gp91phox, p47phox, and p67phox of the NAD(P)H oxidase isolated from human neutrophil granulocytes. The findings support previous photometric studies that indicate that carotid body type I cells possess a putative oxygen sensor protein that is similar to the neutrophil NAD(P)H oxidase and consists of a hydrogen peroxide generating low-potential cytochrome b558 with cofactors regulating the electron transfer to oxygen.
Diphenylene iodonium chloride suppresses the cobaltous chloride-induced expression of erythropoietin by Hep3B cells to about
50% at a concentration of 30 nM. At that concentration, it has no effect on the response to low oxygen. The related compound
iodonium diphenyl chloride acts similarly but is a much less effective inhibitor.
If, as reported, diphenylene iodonium chloride is a specific inhibitor of cytochrome b, it follows that the response to CoCl2 is dependent on that enzyme but the response to hypoxia is not.
The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.
The ability to adapt successfully to periods of relative hypoxia is crucial to the survival of all higher life forms. Several genes have previously been identified which are up-regulated in response to hypoxia; these include the genes encoding erythropoietin (Epo), platelet-derived growth factor B chain, endothelin, interleukin-1 alpha, ornithine decarboxylase, and vascular endothelial growth factor (VEGF). However, the molecular mechanisms by which hypoxia is sensed remain enigmatic. In addition, it is unknown whether the genes mentioned share a common oxygen-sensing signal transduction pathway. In this report we demonstrate multiple similarities between the oxygen-sensing mechanisms regulating the expression of VEGF and Epo. The expression of both mRNAs is significantly up-regulated by hypoxia and cobalt chloride (CoCl2), and the half-life of both mRNAs is markedly prolonged by cycloheximide. In addition, hypoxic induction of both Epo and VEGF is inhibited by carbon monoxide. As part of our investigation into the signal transduction pathway responsible for the hypoxia and cobalt induction of these genes, we discovered that the expression of members of the jun and fos protooncogene families is also up-regulated early after exposure to either of these stimuli. These findings provide support for the hypothesis that the mechanism(s) by which hypoxia is sensed at a molecular level may be highly conserved and tightly regulated.
Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia- inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.
The regulation of erythropoietin (Epo) production was investigated by competitive polymerase chain reaction, a highly sensitive and accurate means of measuring Epo mRNA levels. Co-amplification of the test sample with added mutant Epo cDNA template corrects for variability in the efficiency of amplification. Epo mRNA levels were determined in tissues of normal rats and in animals with varying degrees of anemia. Reduction of the hematocrit level from 0.40 to 0.15–0.20 resulted in a 300-fold increase in kidney Epo mRNA, which comprised 80% of the total Epo mRNA versus 20% from the liver. In contrast, very low levels detected in lung and spleen were not significantly increased by anemia. The human hepatoma cell line, Hep3B, secretes high levels of Epo in response to hypoxia. This regulation is, to a large extent, transcriptional. When Hep3B cells were incubated in the presence of decreasing O2 tension from 160 to 7 mm Hg, there was a monotonic increase in Epo mRNA to 50 to 100 times the normoxic level. Hyperoxia did not suppress basal expression. When cells were incubated at a PO2 of 7 mm Hg, induction of Epo mRNA was first noted at 30 minutes and was maximal at 5 to 6 hours. After Epo mRNA was boosted by a 4-hour hypoxic incubation, cells were then exposed to normoxia, which shut off further transcription of the Epo gene. The decay of Epo mRNA levels closely followed first order kinetics with a half-life of 2 hours, an effective measurement of message stability.
Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild- type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia- activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.
This review focuses on the molecular stratagems utilized by bacteria, yeast, and mammals in their adaptation to hypoxia. Among this broad range of organisms, changes in oxygen tension appear to be sensed by heme proteins, with subsequent transfer of electrons along a signal transduction pathway which may depend on reactive oxygen species. These heme-based sensors are generally two-domain proteins. Some are hemokinases, while others are flavohemoproteins [flavohemoglobins and NAD(P)H oxidases]. Hypoxia-dependent kinase activation of transcription factors in nitrogen-fixing bacteria bears a striking analogy to the phosphorylation of hypoxia inducible factor-1 (HIF-1) in mammalian cells. Moreover, redox chemistry appears to play a critical role both in the trans-activation of oxygen-responsive genes in unicellular organisms as well as in the activation of HIF-1. In yeast and bacteria, regulatory operons coordinate expression of genes responsible for adaptive responses to hypoxia and hyperoxia. Similarly, in mammals, combinatorial interactions of HIF-1 with other identified transcription factors are required for the hypoxic induction of physiologically important genes.
Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.
The hypoxia-inducible factor 1 complex (HIF-1) is involved in the transcriptional activation of several genes, like erythropoietin
and vascular endothelial growth factor, that are responsive to the lack of oxygen. The HIF-1 complex is composed of two b-HLH
proteins: HIF-1β, that is constitutively expressed, and HIF-1α, that is present only in hypoxic cells. The HIF-1α subunit
is continuously synthesized and degraded by the ubiquitin-proteasome under oxic conditions. Hypoxia, transition metals, iron chelators, and several antioxidants stabilize the HIF-1α protein, allowing the
formation of the transcriptionally active HIF-1 complex. The mechanisms of oxygen sensing and the pathways leading to HIF-1α
stabilization are unclear. Because the involvement of a heme protein oxygen sensor has been postulated, we tested the heme
sensor hypothesis by using a luciferase-expressing cell line (B-1), that is highly responsive to hypoxia. Exposure of B-1
cells to carbon monoxide and heme synthesis inhibitors failed to show any effect on the hypoxia responsiveness of these cells,
suggesting that heme proteins are not involved in hypoxia sensing. Measurement of iron in recombinantly expressed HIF-1α protein
revealed that this protein binds ironin vivo. Iron binding was localized to a 129-amino acid peptide between sequences 529 and 658 of the HIF-1α protein. Although the
exact structure of the iron center has not been yet defined, a 2:1 metal/protein molar ratio suggests a di-iron center, probably
similar to the one found in hemerythrin. This finding is compatible with a model where redox reaction may occur directly in the iron center of the HIF-1α subunit, affecting its survival inoxic conditions.
The metal specificity of the iron-protoporphyrin chelating enzyme from rat liver was studied. The conversion of protoporphyrin plus Fe2+ to heme was demonstrated spectrophotometrically. At high concentrations Fe2+ inhibited, probably through reaction with the sulfhydryl groups. Co2+ behaved very similar to Fe2+, and isotope dilution experiments indicated that Co2+ was utilized via the same enzymic mechanism as was Fe2+, 54Mn2+ in low concentrations was incorporated into protoporphyrin in trace amounts, even though Mn2+ was an inhibitor of the enzyme. Mn2+ apparently did not attack the sulfhydryl groups but did inhibit non-competitively with respect to Fe2+. Sn2+ was neither utilized by the enzyme nor inhibitory, while all other metals tested (Mg2+, Ca2+, Ni2+, Cd2+, Pb2+, Cu2+, Mn2+, Zn2+, Hg2+) were inhibitory.
Erythrocytosis was induced in Fischer rats by intrarenal (ir) injection of nickel subsulfide (Ni3S2) in dosages ranging from 0.6 to 10 mg per rat. Measurements of blood packed cell volume (PCV) became increased by one month after ir injection of Ni3S2; reached maximum values at approximately two months; and gradually returned to control values by seven months. The duration and magnitude of erythrocytosis were related to the dosage of Ni3S2. Increases in hemoglobin concentrations and erythrocyte counts (RBC) in Ni3S2-treated rats were consistently proportional to blood PCV values. Blood hemoglobin concentration was 24.9 +/- 1.2 g per dl at two months after bilateral ir injection of Ni3S2 (10 MG PER RAT), VERSUs 15.8 +/- 0.5 g per dl in saline-injected controls (P less than 0.001). No significant changes occurred in leukocyte or platelet counts of Ni3S2-treated rats. Autopsy of rats killed two months after ir injection of Ni3S2 showed marked erythroid hyperplasia of bone marrow and fibrotic needle tracts in renal parenchyma with localized deposits of particles of Ni3S2. In contrast to the erythrocytosis induced by ir injection of Ni3S2, administration of Ni3S2 by intramuscular (im) injection (10 mg per rat) had no significant effect upon blood PCV, RBC or hemoglobin values or upon morphology of bone marrow.
In rats, injection of nickel sulfide (5 mg) into each pole of one kidney, unlike intramuscular administration, elicits a plethoric condition a few weeks later. The resulting hematologic changes (increased hematocrit, hemoglobin, erythrocytes and circulating erythrocyte mass with normal plasma volume) indicate that the plethoric condition is due to polycythemia, which is not associated with alterations in the 2,3-diphosphoglyceric acid content of erythrocytes. Removal of the treated kidney, following the development of the polycythemia, as well as the tumor growth and expansion in the renal parenchyma, reverse the plethoric condition, suggesting that the erythropoietic changes derive from nickel-induced renal lesions. Further studies are required to elucidate the nature and mechanisms of the cellular alterations.
1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of delta psi m with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta psi m closely followed changes in PO2 with time. 5. The rate of rise of [Ca2+]i in response to anoxia is strongly temperature sensitive. The rate of depolarization of delta psi m with anoxia similarly increased at least two- to fivefold on warming from 22 to 36 degrees C. The change with FCCP was not significantly altered by temperature. 6. These data show that the mitochondrial membrane potential changes over a physiological range of PO2 values in type I cells. This contrasts with the behaviour in dissociated chromaffin cells and sensory neurons, in which no change was measurable until the PO2 fell close to zero.(ABSTRACT TRUNCATED AT 400 WORDS)
K+ currents recorded from adult rabbit chemoreceptor cells are reversibly inhibited on lowering the pO2 in the bathing solution. Bath application of a hypoxic TTX-containing solution revealed that inhibition of K+ current by low pO2 proceeds faster than TTX inhibition of Na+ currents, the apparent t1/2 being 3.68 and 7.14 s, respectively. Addition of carbon monoxide to the hypoxic gas mixture used to equilibrate the bathing solution reversed the inhibition of K+ currents by approx. 70%.
The expression of the nitrogen-fixation genes of Rhizobium meliloti is controlled by oxygen. These genes are induced when the free oxygen concentration is reduced to microaerobic levels. Two regulator proteins, FixL and FixJ, initiate the oxygen-response cascade, and the genes that encode them have been cloned. The fixL product seems to be a transmembrane sensor that modulates the activity of the fixJ product, a cytoplasmic regulator. FixL and FixJ are homologous to a family of bacterial two-component regulators, for which the mode of signal transduction is phosphorylation. We report here the purification of both FixJ and a soluble truncated FixL (FixL*), overproduced from a single plasmid construct. FixL* catalyses its own phosphorylation and the transfer of the gamma-phosphate of ATP to Fix J. The resulting FixJ-phosphate linkage is sensitive to base, as are the aspartyl phosphates of homologous systems. Visible spectra of purified FixL* show that it is an oxygen-binding haemoprotein. We propose that FixL senses oxygen through its haem moiety and transduces this signal by controlling the phosphorylation of FixJ.
Type I cells of the carotid body are known to participate in the detection of O2 tension in arterial blood but the primary chemotransduction mechanisms are not well understood. Here we report the existence in excised membrane patches of type I cells of a single K+ channel type modulated by changes in PO2. Open probability of the O2-sensitive K+ channel reversibly decreased by at least 50% on exposure to hypoxia but single-channel conductance (approximately 20 pS) was unaltered. In the range between 70 and 150 mmHg (1 mmHg = 133 Pa) the decrease of single-channel open probability was proportional to the PO2 measured in the vicinity of the membrane patch. The inhibition of K+ channel activity by low PO2 was independent of the presence of non-hydrolyzable guanine triphosphate analogues at the internal face of the membrane. The results indicate that the O2 sensor of type I cells is in the plasma membrane and suggest that environmental O2 interacts directly with the K+ channels.
The ionic currents of carotid body type I cells and their possible involvement in the detection of oxygen tension (Po2) in arterial blood are unknown. The electrical properties of these cells were studied with the whole-cell patch clamp technique, and the hypothesis that ionic conductances can be altered by changes in PO2 was tested. The results show that type I cells have voltage-dependent sodium, calcium, and potassium channels. Sodium and calcium currents were unaffected by a decrease in PO2 from 150 to 10 millimeters of mercury, whereas, with the same experimental protocol, potassium currents were reversibly reduced by 25 to 50 percent. The effect of hypoxia was independent of internal adenosine triphosphate and calcium. Thus, ionic conductances, and particularly the O2-sensitive potassium current, play a key role in the transduction mechanism of arterial chemoreceptors.
Erythropoietin (Epo), the hormone that stimulates red blood cell production, is synthesized in the kidney and liver in response
to hypoxia. The human hepatoma cell line Hep3B regulates its production of Epo in a physiologic manner. Either hypoxia or
cobalt chloride markedly increases expression of Epo mRNA as well as production of biologically active and immunologically
distinct Epo protein. New protein synthesis is required before the induction of increased levels of hypoxia- or cobalt-induced
Epo mRNA. Hypoxia, cobalt chloride, and nickel chloride appear to stimulate Epo production through a common pathway. The inhibition
of Epo production at low partial pressures of oxygen by carbon monoxide provides evidence that a heme protein is integrally
involved in the oxygen-sensing mechanism. This hypothesis is further supported by the finding that when heme synthesis is
blocked, hypoxia-, cobalt-, and nickel-induced Epo production are all markedly inhibited. A model is proposed in which a ligand-dependent
conformational change in a heme protein accounts for the mechanism by which hypoxia as well as cobalt and nickel stimulate
the production of Epo.
An unsolved issue for the arterial chemoreceptors is the mechanism by which hypoxia and other natural stimuli lead to an increase of activity in the carotid sinus nerve. According to the 'metabolic hypothesis', the hypoxic activation of the carotid body (CB) is mediated by a decrease of the ATP levels in the type I cells, which then release a neurotransmitter capable of exciting the sensory nerve endings. Using an in vitro preparation of cat CB, we report that ATP levels in the CB do in fact decrease when the organs are exposed to moderate, short lasting hypoxia (5 min 20% O2). Additionally, we found that decreases in ATP levels induced by 2-deoxyglucose (2 mM) or sodium cyanide (0.1 mM) are closely correlated with dopamine release from type I cells and electrical activity in the carotid sinus nerve elicited by these agents. The possible cause-effect relationship of these events is discussed.
Cobalt protoporphyrin generated from 5-amino[4-14C]laevulinate by homogenates or primary cultures of chick embryo liver exposed to CoCl2 was found to be radioactivity unextractable by acid/acetone, when extra protein was added. The activity of ferrochelatase was required for formation of cobalt protoporphyrin since inhibition of ferrochelatase with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (in the presence of cycloheximide) inhibited formation of cobalt protoporphyrin and resulted in accumulation of protoporphyrin. Cobalt protoporphyrin was detected spectrophotometrically in hepatocyte cultures exposed to the combination of 2-allyl-2-isopropylacetamide and CoCl2: (1) as the pyridine haemochrome of the protein pellet remaining after acid-acetone extraction of the cells, or (2) as the material extracted from the protein pellet with acetic acid-pyridine-chloroform. The amount of cobalt protoporphyrin increased with increasing CoCl2 concentration as cellular haem declined. The decrease in haem was about equal to the amount of cobalt protoporphyrin that accumulated. 2-Allyl-2-isopropylacetamide and polychlorinated biphenyls were both powerful inducers of 5-aminolaevulinate synthase. The former led to protoporphyrin accumulation, whereas with the latter, uroporphyrin accumulated, probably due to a concomitant decrease in activity of uroporphyrinogen decarboxylase. The decrease in activity of 5-aminolaevulinate synthase produced by administration of CoCl2 was greater after treatment with 2-allyl-2-isopropylacetamide than after treatment with allylisopropylacetamide and 3,4,3',4'-tetrachlorobiphenyl. We conclude: (a) that cobalt protoporphyrin is readily formed in cultured hepatocytes, and (b) that its formation accounts for the action of cobalt on 5-aminolaevulinate synthase.
The effect on carotid chemoreceptor afferents of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation that does not affect energy conservation, was studied in 20 cats that were anesthetized, paralyzed, and artificially ventilated. Responses of single or a few chemoreceptor afferents to changes in arterial O2 tension (PaO2) at constant arterial CO2 tension were recorded. In addition, responses to nicotine, cyanide, and antimycin A or carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP) were tested in normoxia. Oligomycin (50-500 microgram) was administered by close intra-arterial injection, and the same tests were repeated at timed intervals. Initially, oligomycin caused vigorous stimulation of carotid chemoreceptor activity. Subsequently, although the afferent fibers were still active and could be vigorously stimulated by nicotine, they no longer responded to changes in PaO2 or to doses of cyanide, antimycin A, or FCCP. These results separate stimulation of chemoreceptor afferents by hypoxia and metabolic inhibitors and uncouplers from that by nicotine and suggest that intact oxidative phosphorylation, required for maintenance of the intracellular high-energy phosphate levels, forms the basis of O2 chemoreception in the carotid body.
Hypoxia-inducible factor 1 (HIF-1) is found in mammalian cells cultured under reduced O2 tension and is necessary for transcriptional activation mediated by the erythropoietin gene enhancer in hypoxic cells. We show that both HIF-1 subunits are basic-helix-loop-helix proteins containing a PAS domain, defined by its presence in the Drosophila Per and Sim proteins and in the mammalian ARNT and AHR proteins. HIF-1 alpha is most closely related to Sim. HIF-1 beta is a series of ARNT gene products, which can thus heterodimerize with either HIF-1 alpha or AHR. HIF-1 alpha and HIF-1 beta (ARNT) RNA and protein levels were induced in cells exposed to 1% O2 and decayed rapidly upon return of the cells to 20% O2, consistent with the role of HIF-1 as a mediator of transcriptional responses to hypoxia.
Recent work has indicated that oxygen-sensing mechanism(s) resembling those controlling erythropoietin production operate in many non-erythropoietin-producing cells. To pursue the implication that such a system might control other genes, we studied oxygen-regulated expression of mRNAs for vascular endothelial growth factor, platelet-derived growth factor (PDGF) A and B chains, placental growth factor (PLGF), and transforming growth factor in four different cell lines and compared the characteristics with those of erythropoietin regulation. Oxygen-regulated expression was demonstrated for each gene in at least one cell type. However, the response to hypoxia (1% oxygen) varied markedly, ranging from a 13-fold increase (PDGF-B in Hep G2 cells) to a 2-fold decrease (PLGF in the trophoblastic line BeWo). For each gene/cell combination, both the magnitude and direction of the response to hypoxia were mimicked by exposure to cobaltous ions or two different iron-chelating agents, desferrioxamine and hydroxypyridinones. These similarities with established characteristics of erythropoietin regulation indicate that a similar mechanism of oxygen sensing is operating on a variety of vascular growth factors, and they suggest that chelatable iron is closely involved in the mechanism.
High carbon monoxide (CO) gas tensions (> 500 Torr) at normoxic PO2 (125-140 Torr) stimulates carotid chemosensory discharge in the perfused carotid body (CB) in the absence but not in the presence of light. According to a metabolic hypothesis of O2 chemoreception, the increased chemosensory discharge should correspond to a photoreversible decrease of O2 consumption, unlike a non-respiratory hypothesis. We tested the respiratory vs. non-respiratory hypotheses of O2 chemoreception in the cat CB by measuring the effect of high CO. Experiments were conducted using CBs perfused and superfused in vitro with high CO in normoxic, normocapnic cell-free CO2-HCO3- buffer solution at 37 degrees C. Simultaneous measurements of the rate of O2 disappearance with recessed PO2 microelectrodes and chemosensory discharge were made after flow interruption with and without CO in the perfusate. The control O2 disappearance rate without CO was -3.66 +/- 0.43 (S.E.) Torr/s (100 measurements in 12 cat CBs). In the dark, high CO reduced the O2 disappearance rate to -2.35 +/- 0.33 Torr/s, or 64.2 +/- 9.0% of control (P < 0.005, 34 measurements). High CO was excitatory in the dark, with an increase in baseline neural discharge from 129.2 +/- 47.0 to 399.3 +/- 49.1 impulses per s (P < 0.0001), and maximum discharge rate of 659 +/- 76 impulses/s (N.S. compared to control) during flow interruption. During perfusion with high CO in the light, there were no significant differences in baseline neural discharge or in the maximum neural discharge after flow interruption, and little effect on O2 metabolism (88.8 +/- 11.5% of control, N.S., 29 measurements).(ABSTRACT TRUNCATED AT 250 WORDS)
Vascular endothelial growth factor (VEGF) is a specific endothelial cell mitogen with potent angiogenic properties. In tumors, VEGF has been localized to the most necrotic and ischemic areas of the tissues, suggesting that local hypoxia is a potent inducer of VEGF production. Initial experiments in vitro confirmed the stimulatory effect of hypoxia on VEGF expression. The extent of this response and the mechanisms involved in oxygen sensing are poorly characterized.
Confluent monolayers of malignant cell lines or primary cultures of fibroblast or endothelial cells were exposed to hypoxia or incubated with either cobalt chloride, a stimulator of erythropoietin gene expression, or sodium azide, an inhibitor of oxydative phosphorylation. VEGF expression was analyzed by Northern blot or RNase protection assays. The expression VEGF in vivo was studied in animals subjected to hypobaric hypoxia or functional anemia.
Hypoxia greatly stimulated VEGF expression in tumor cell lines and primary fibroblast cultures. Endothelial cells, that expressed very low constitutive levels of VEGF, were resistant to hypoxic stimulation. RNase protection analysis showed that hypoxia primarily stimulated the induction of smaller and medium VEGF isoforms, i.e., the same ones expressed under normal conditions. The stimulatory effect of hypoxia on VEGF could be reproduced in vitro by cobalt chloride but not with sodium azide. In vivo, both hypoxia and anemia were found to be potent inducers of VEGF expression in several organs including heart, brain, liver, kidney, and muscle. As in vitro, cobalt was also found to be a potent stimulator of VEGF in vivo.
Hypoxia is a potent inducer of VEGF expression in malignant as well as normal cultured cells. It is also a stimulator of VEGF expression in vivo. The VEGF gene appears to respond to hypoxia like the erythropoietin gene, and the mechanism of oxygen sensing probably is mediated by a heme-containing protein.
We have previously demonstrated that an exposure of rat cardiomyocytes to anoxia induces the expression of mRNAs coding for vascular endothelial growth factor (VEGF). The action of anoxia was mimicked by Co, Ni and Mn. The actions of Co and of anoxia were not additive and did not involve AP-1 binding sites. Experiments using actinomycin D and cycloheximide indicated that VEGF mRNA levels in cardiac cells are regulated both at transcriptional and post transcriptional levels. It is concluded that an oxygen sensing mechanism is present in cardiac cells and controls the expression of VEGF mRNAs. It may be important for the neovascularization of ischemic myocardium.
Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.
FixL's are chimeric heme protein kinases from symbiotic nitrogen-fixing Rhizobia. We have overexpressed three FixL variants in Escherichia coli. Bradyrhizobium japonicum FixL, a soluble dimeric protein, is the first full-length FixL to be purified. The other two proteins are soluble truncations of Rhizobium meliloti FixL, which is a membrane protein. One contains both heme and kinase domains and is dimeric; the other has only the heme domain and is monomeric. We find that all the FixL's bind oxygen and carbon monoxide non-cooperatively, with very low affinities due entirely to slow association rates. FixL P50's for oxygen are 17-76 mmHg. FixL's may sense nitric oxide and carbon monoxide in addition to oxygen, especially at the low oxygen pressures encountered in vivo. Autoxidation rates are about 50 times faster than that of sperm whale myoglobin. The carbon monoxide affinity of FixL's is about 300 times lower than that of myoglobin, resulting in the unusually low values of 7.5-17 for the partition constant, M = P50(O2)/P50(CO), between carbon monoxide and oxygen. Met-FixL's have their Soret absorption maximum at 395 nm instead of the typical 408 nm and a steep hydroxymet transition at pH > or = 9.3; these properties indicate a pentacoordinated high-spin ferric heme and suggest a sterically hindered hydrophobic heme pocket lacking a distal (E7) histidine. FixL is the first member of a new class of heme proteins, the heme-based sensors, distinct from the oxygen carriers and electron transporters. We expect that some of the novel properties of FixL will be characteristic of the class.
The hypoxia-induced increase of spectrophotometrically measured light absorption at 560 nm, considered as reduced cytochrome b, in HepG2 cells is diminished after exposure to cobalt chloride (50 or 100 microM) for 18-36 h. The redox state of cytochrome c and cytochrome aa3, however, remains stable, indicating a particular affinity of cytochrome b for cobalt. Erythropoietin production of HepG2 cells increases after application of cobalt chloride, whereas H2O2 production, as measured by the dihydrorhodamine technique, decreases. It is concluded that cobalt stimulates a signal cascade with cytochrome b as receptor and H2O2 as second messenger for regulating erythropoietin production.
Carbon monoxide was shown to be competitive with O2 in oxygen sensing by perfused carotid bodies isolated from cats, afferent electrical activity increasing with either decreasing O2 or increasing CO. The CO-induced increase in afferent activity was fully reversed by bright light. At submaximal light intensities the extent of reversal, after correcting to equal light intensity of light quanta at each wavelength, was maximal for light of 432 +/- 2 and 590 +/- 2 nm, with a ratio (432/590) of approximately 6. This spectrum is characteristic of the CO compound of mitochondrial cytochrome a3. The photo-reversible inhibition of oxygen sensing activity by CO accounts for at least 80% of the oxygen chemosensory activity of the carotid body.
Diphenylene iodonium (DPI), an inhibitor of NAD(P)H oxidase, blocks hypoxic excitation of the carotid body. We used the whole-cell patch-clamp technique to investigate the actions of DPI on ionic currents in isolated type I carotid body cells. DPI (10 microM) caused reversible blockade of K+ and Ca2+ currents in these cells, indicating that DPI is a non-selective ion channel blocker. Since hypoxic excitation of the carotid body is dependent on Ca2+ influx into type I cells, our observation that DPI blocks Ca2+ currents in type I cells can account for the ability of this compound to inhibit hypoxic excitation of the intact organ.
Hypoxia-inducible factor 1 (HIF-1) activates erythropoietin gene transcription in Hep3B cells subjected to hypoxia. HIF-1 activity is also induced by hypoxia in non-erythropoietin-producing cells, suggesting a more general regulatory role. We now report that RNAs encoding the glycolytic enzymes aldolase A (ALDA), phosphoglycerate kinase 1 (PGK1), and pyruvate kinase M were induced by exposure of Hep3B or HeLa cells to inducers of HIF-1 (1% O2, cobalt chloride, or desferrioxamine), whereas cycloheximide blocked induction of glycolytic RNAs and HIF-1 activity. Oligonucleotides from the ALDA, PGK1, enolase 1, lactate dehydrogenase A, and phosphofructokinase L (PFKL) genes, containing sequences similar to the HIF-1 binding site in the erythropoietin enhancer, specifically bound HIF-1 present in crude nuclear extracts or affinity-purified preparations. Sequences from the ALDA, PFKL, and PGK1 genes containing HIF-1 binding sites mediated hypoxia-inducible transcription in transient expression assays. These results support the role of HIF-1 as a mediator of adaptive responses to hypoxia that underlie cellular and systemic oxygen homeostasis.
Oxygen sensors in the body induce various cell activities to avoid any mismatch between oxygen demand and oxygen supply and to maintain an optimal level of oxygen partial pressure (PO2) in various organs. Oxygen sensing seems to be a well conserved process among procaryontic and eucaryontic cells. The molecular mechanism of oxygen sensing is unknown, but it has been suggested that a hemeprotein is involved that does not participate in the mitochondrial energy production. As examplified on the carotid body and on erythropoietin producing HepG2 cells, a cytochrome b was described for the NAD(P)H oxidase of neutrophiles might be an attractive candidate for this hemeprotein. It is hypothesised that hydrogen peroxide (H2O2) produced by this cytochrome b in direct correlation with cellular PO2, serves as a second messenger to regulate potassium channels or gene expression. One might forsee, that this new concept of oxygen sensing could have an impact on all processes in physiology and pathophysiology which are dealing with reactive oxygen intermediates.
Erythropoietin (EPO) gene transcription is activated in kidney cells in vivo and in Hep3B cells exposed to hypoxia or cobalt chloride. Hypoxia-inducible factor 1 (HIF-1) is a nuclear factor that binds to the hypoxia-inducible enhancer of the EPO gene at a site that is required for transcriptional activation. HIF-1 DNA-binding activity is induced by hypoxia or cobalt chloride treatment of Hep3B cells. We report that treatment of Hep3B cells with desferrioxamine (DFX) induced HIF-1 activity and EPO RNA expression with kinetics similar to the induction of HIF-1 by hypoxia or cobalt chloride. Induction by each of these stimuli was inhibited by cycloheximide, indicating a requirement for de novo protein synthesis. DFX appears to induce HIF-1 by chelating iron as induction was inhibited by coadministration of ferrous ammonium sulfate. DFX administration to mice transiently increased EPO RNA levels in the kidney. As previously shown for hypoxia and cobalt treatment, DFX also induced HIF-1 activity in non-EPO-producing cells, suggesting the existence of a common hypoxia signal-transduction pathway leading to HIF-1 induction in different cell types.