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Sample records for 2-nitroimidazole hypoxic cell

  1. Optical isomers of a new 2-nitroimidazole nucleoside analog (PR-350 series): Radiosensitization efficiency and toxicity

    SciTech Connect

    Oya, Natsuo; Sasai, Keisuke; Shibata, Toru

    1995-08-30

    A new 2-nitroimidazole nucleoside radiosensitizer, PR-350 (1-[1{prime},3{prime},4{prime}-trihydroxy-2{prime}-butoxy]-methyl-2-nitroimidazole), has been reported to be as efficient as and less toxic than etanidazole. This compound is racemic, and it was recently optically resolved into two isomers, PR-68 (2{prime}R,3{prime}S type) and PR-69 (2{prime}S,3{prime}R type). The other two isomers, PR-28 (2{prime}S,3{prime}S type) and PR-44 (2{prime}R,3{prime}R type), were asymmetrically synthesized. In the present study, we investigated the properties, sensitizing activity, and toxicity of PR-350 and the four optical isomers in comparison with those of other 2-nitroimidazole hypoxic cell radiosensitizers, etanidazole, KU-2285, KIN-804, and RP-170. Because PR-350 and PR-28 can be industrially synthesized, we evaluated whether either of these two drugs are suitable for further investigation. In vivo radiosensitizing activity differed among the four optical isomers, which appeared to be due, at least in part, to differences in lipophilicity. Although PR-28 was the least toxic, its low sensitization efficiency does not warrant clinical trials. Among the PR compounds, PR-68 appears to be most efficient, but optical resolution of PR-68 from PR-350 is expensive, and asymmetrical synthesis of PR-68 is not established. Therefore, PR-350 seems to be most suitable for further investigation among the PR-350 series compounds, considering its higher efficiency compared with PR-28 and PR-44, and established synthesis. 28 refs., 7 figs., 1 tab.

  2. Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent “Pimonidazole” in Hypoxia

    PubMed Central

    Yoshioka, Takeshi; Feng, Fei; Zhao, Songji; Higashino, Kenichi; Numata, Yoshito; Kuge, Yuji

    2016-01-01

    Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH. PMID:27580239

  3. Imaging Mass Spectrometry Revealed the Accumulation Characteristics of the 2-Nitroimidazole-Based Agent "Pimonidazole" in Hypoxia.

    PubMed

    Masaki, Yukiko; Shimizu, Yoichi; Yoshioka, Takeshi; Feng, Fei; Zhao, Songji; Higashino, Kenichi; Numata, Yoshito; Kuge, Yuji

    2016-01-01

    Hypoxia, or low oxygen concentration, is a key factor promoting tumor progression and angiogenesis and resistance of cancer to radiotherapy and chemotherapy. 2-Nitroimidazole-based agents have been widely used in pathological and nuclear medicine examinations to detect hypoxic regions in tumors; in particular, pimonidazole is used for histochemical staining of hypoxic regions. It is considered to accumulate in hypoxic cells via covalent binding with macromolecules or by forming reductive metabolites after reduction of its nitro group. However, the detailed mechanism of its accumulation remains unknown. In this study, we investigated the accumulation mechanism of pimonidazole in hypoxic tumor tissues in a mouse model by mass spectrometric analyses including imaging mass spectrometry (IMS). Pimonidazole and its reductive metabolites were observed in the tumor tissues. However, their locations in the tumor sections were not similar to the positively stained areas in pimonidazole-immunohistochemistry, an area considered hypoxic. The glutathione conjugate of reduced pimonidazole, a low-molecular-weight metabolite of pimonidazole, was found in tumor tissues by LC-MS analysis, and our IMS study determined that the intratumor localization of the glutathione conjugate was consistent with the area positively immunostained for pimonidazole. We also found complementary localization of the glutathione conjugate and reduced glutathione (GSH), implying that formation of the glutathione conjugate occurred in the tumor tissue. These results suggest that in hypoxic tumor cells, pimonidazole is reduced at its nitro group, followed by conjugation with GSH.

  4. Clinical perspectives for the use of new hypoxic cell sensitizers

    SciTech Connect

    Brown, J.M.

    1982-09-01

    Experience with high pressure oxygen in combination with radiotherapy has shown that, for some tumors at least, the presence of hypoxic cells is a limiting factor in the ability to cure these tumors even with conventional daily fractionation. This suggests that hypoxic cell radiosensitizers, of which misonidazole (MISO) is the prototype drug, may play a role in improving the cure-rate of some tumors when combined with daily fractionation. Even for those tumors for which no improvement is seen when combined with daily fractionation, it is likely that there will be an important role for these sensitizers by using them in combination with regimens of only a few dose fractions. Because of the limiting side effects of neuropathy, a less toxic radiosensitizer than MISO is required to gain the full clinical benefit of these drugs. A possible way of achieving this is to reduce the lipid solubility (lipophilicity) of the compounds while still retaining their electron-affinity. This reduces the concentration of drug in the neural tissues (brain, peripheral nerves) without affecting the tumor concentration. However, if the lipophilicity is too low, the drugs are unable to enter the hypoxic cells and hence lose their radiosensitivity efficiency. It would appear that a lipophilicity given by an octanol:water partition coefficient of approximately 0.04 is optimum (cf. MISO = 0.43) with the 2-nitroimidazole amide SR-2508 the best in this series. Tumor levels of this drug of at least 7-8 times those obtained with MISO should be attainable clinically for no increase in neurotoxicity. Another property of electron-affinic sensitizers shows clinical promise. This is their ability to preferentially sensitize tumors compared to normal tissues to the cytotoxic action of several chemotherapeutic agents.

  5. Marking hypoxic cells for complement and cytotoxic T lymphocyte-mediated lysis: using pimonidazole.

    PubMed Central

    Chou, S. C.; Flood, P. M.; Raleigh, J. A.

    1996-01-01

    Artificial antigens are created when 2-nitroimidazoles bind to hypoxic cells. These antigens have been used in the immunodetection of tumour hypoxia but they might also serve to stimulate immune lysis of hypoxic tumour cells by complement- and cell-mediated processes. In order to test this hypothesis, lymphocytes isolated from the spleens of C3H/HeN mice that had been immunised with pimonidazole-labelled 3152-PRO cells were subcultured and tested for their ability to lyse chromium-51 loaded, pimonidazole-labelled 3152-PRO cells in an in vitro assay. In a parallel study, commercially available, rabbit complement was tested for its ability to lyse pimonidazole-labelled V79-4 cells in the presence of monoclonal antibodies which recognise protein adducts of reductively activated pimonidazole. Complement-mediated cell lysis was measured by means of an MTT assay. Complement-mediated and cell-mediated lysis was observed at pimonidazole concentrations which, in themselves, do not produce cell killing. PMID:8763883

  6. Studies of methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804) 1: effect on free radical scavenging system in mice bearing Ehrlich ascites carcinoma.

    PubMed

    Abu-Zeid, M; Hori, H; Nagasawa, H; Uto, Y; Inayama, S

    2000-02-01

    Methyl 2-nitroimidazole-1-acetohydroxamate (KIN-804) is a 2-nitroimidazole derivative containing a hydroxamate side chain designed to enhance the radiosensitization response of hypoxic cells. The possible sensitization of tumor tissue by KIN-804 can be evaluated through investigation of the levels of the free radical scavengers; namely, glutathione (GSH) and its complex enzyme system including glutathione reductase (GR) and glutathione peroxidase (GSH-Px), as well as glucose-6-phosphate dehydrogenase (G-6-PD). Female albino mice were inoculated with Ehrlich carcinoma in the thigh. Administration of KIN-804 (i.p. 80 mg/kg body weight) was carried out 20 min before localized irradiation of 10 Gy. The data revealed that KIN-804 administration, followed or not by gamma irradiation, resulted in a significant decrease in GSH content in tumor tissues associated with inhibition in GR and G-6-PD activities. Blood GSH-Px was enhanced in tumor inoculated mice and the administration of KIN-804 returned it to the normal value. These changes were more noticeable in tumor bearing mice exposed to both KIN-804 and irradiation.

  7. 2-Nitroimidazole-ruthenium polypyridyl complex as a new conjugate for cancer treatment and visualization.

    PubMed

    Mazuryk, Olga; Maciuszek, Monika; Stochel, Grażyna; Suzenet, Franck; Brindell, Małgorzata

    2014-05-01

    A novel long-lifetime highly luminescent ruthenium polypyridyl complex containing 2-nitroimidazole moiety [Ru(dip)2(bpy-2-nitroIm)]Cl2 (dip=4,7-diphenyl-1,10-phenanthroline, bpy-2-nitroIm=4-[3-(2-nitro-1H-imidazol-1-yl)propyl]-2,2'-bipyridine) has been designed cancer treatment and imaging. The luminescence properties of the synthesized compound strongly depend on the oxygen concentration. Under oxygen-free conditions quantum yield of luminescence and the average lifetime of emission were found to be 0.034 and 1.9 μs, respectively, which is ca. three times higher in comparison to values obtained in air-equilibrated solution. The binding properties of the investigated ruthenium complex to human serum albumin have been studied and the apparent binding constant for the formation of the protein-ruthenium adduct was determined to be 1.1×10(5)M(-1). The quantum yield and the average lifetime of emission are greatly enhanced upon binding of ruthenium compound to the protein. The DNA binding studies revealed two distinguished binding modes which lead to a decrease in luminescence intensity of ruthenium complex up to 60% for [DNA]/[Ru]<2, and enhancement of emission for [DNA]/[Ru]>80. Preliminary biological studies confirmed fast and efficient accumulation of the ruthenium complex inside cells. Furthermore, the ruthenium complex was found to be relatively cytotoxic with LD50 of 12 and 13 μM for A549 and CT26 cell lines, respectively, under normoxic conditions. The retention and cellular uptake of ruthenium complex is enhanced under hypoxic conditions and its LD50 decreases to 8 μM for A549 cell line.

  8. Lipid Accumulation in Hypoxic Tissue Culture Cells

    PubMed Central

    Gordon, Gerald B.; Barcza, Maureen A.; Bush, Marilyn E.

    1977-01-01

    Lipid droplets have long been recognized by light microscopy to accumulate in hypoxic cells both in vivo and in vitro. In the present tissue culture experiments, correlative electron microscopic observations and lipid analyses were performed to determine the nature and significance of lipid accumulation in hypoxia. Strain L mouse fibroblasts were grown in suspension culture, both aerobically and under severe oxygen restriction obtained by gassing cultures daily with an 8% CO2-92% nitrogen mixture. After 48 hours, hypoxic cells showed an increase in total lipid/protein ratio of 42% over control cells. Most of this increase was accounted for by an elevation in the level of cellular triglyceride from 12.3 ± 0.9 μg/mg cell protein in aerobic cultures to 41.9 ± 0.7 in the hypoxic cultures, an increase of 240%. Levels of cellular free fatty acids (FFA) were 96% higher in the hypoxic cultures. No significant changes in the levels of cellular phospholipid or cholesterol were noted. Electron microscopic examination revealed the accumulation of homogeneous cytoplasmic droplets. The hypoxic changes were reversible upon transferring the cultures to aerobic atmospheres with disappearance of the lipid. These experiments indicate that hypoxic injury initially results in triglyceride and FFA accumulation from an inability to oxidize fatty acids taken up from the media and not from autophagic processes, as described in other types of cell injury associated with the sequestration of membranous residues and intracellular cholesterol and phospholipid accumulation. ImagesFigure 3Figure 4Figure 5Figure 6Figure 7Figure 1Figure 2 PMID:196505

  9. Evaluation of nitrobenzimidazoles as hypoxic cell radiosensitizers

    SciTech Connect

    Wright, J.; Frank, L.R.; Bush, D.; Harrison, G.H.

    1983-07-01

    Radiobiological and pharmacokinetic assays were performed to determine the potential of 2-nitrobenzimidazole (NBI) as a hypoxic cell radiosensitizing agent. As judged by comparing survival curve slopes of Serratia marcescens irradiated under aerated and hypoxic conditions, the NBI enhancement ratio (ER) at 2 mM concentration was 2.4 +/- 0.2, compared with an oxygen enhancement ratio of 3.3 +/- 0.3. 2,5-Dinitrobenzimidazole (DNBI) was investigated in vitro; its ER was 3.0 +/- 0.3 at 4 mM concentration. Very poor tissue penetration of DNBI precluded further testing in vivo. Acute toxic signs appeared in C3H/HeJ mice following ip injection of NBI at 100 mg/kg. These would be partly attributable to the stress caused by the high pH of the injection vehicle. The LD50 was estimated to be 125-150 mg/kg. Mammary adenocarcinoma tumors grown in the flanks of these mice exhibited maximum NBI levels at 5 min postinjection (ip). Peak tumor radiosensitization occurred in the interval between 5 and 10 min postinjection. The ER for tumor regrowth delay was 2.1 +/- 0.3 following 50 mg/kg injected into mice 5 min before irradiation. Functional evaluation up to 40 days after treatment revealed no evidence of neurological deficit.

  10. Characterization and properties of monoammine nitroimidazole complexes of platinum (PtCl sub 2 (NH sub 3 )(NO sub 2 Im)). Crystal and molecular structure of cis-Amminedichloro(1-((((2-hydroxyethyl)amino)carbonyl)methyl)-2-nitroimidazole)platinum(II)

    SciTech Connect

    Rochon, F.D.; Pichang Kong; Melanson, R. ); Skov, K.A. ); Farrell, N. )

    1991-11-27

    The characterization of monoammine(nitroimidazole)platinum(II) complexes of structure (PtCl{sub 2}(NH{sub 3})(NO{sub 2}Im)) (NO{sub 2}Im = 1-((((2-hydroxyethyl)amino)carbonyl)methyl)-2-nitroimidazole, Etanidazole (I), 1-(2-nitro-1-imidazolyl)-3-methoxy2-propanol, Misonidazole (II), and 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, Metronidazole (III)) is reported. Both is cis and trans isomers may be isolated for II and III. The crystal structure of cis-amminedichloro(1-((((2-hydroxyethyl)amino)carbonyl)methyl)-2-nitroimidazole)platinum(II) has been determined by X-ray diffraction. The crystals are orthorhombic, space group Pnab with cell dimensions a = 14.867 (7) {angstrom}, b = 9.915 (5) {angstrom}, c = 19.015 (9) {angstrom}, and Z = 8. The structure was refined to R = 0.062 and R{sub w} = 0.052. Platinum has the expected square-planar coordination. The Pt-Cl bond trans to the nitroimidazole ligand is shorter (2.269 (3) {angstrom}) than normal. The dihedral angle between the platinum plane and the imidazole ring is 111{degree}, while the nitro group makes an angle of 31{degree} with the imidazole ring plane. Electrochemistry and {sup 195}Pt NMR data are also reported. The relevance of the chemical properties to their biological properties as radiosensitizers and hypoxic cytotoxins is discussed.

  11. Stem cells, telomerase regulation and the hypoxic state.

    PubMed

    Mathews, Juanita; Davy, Philip M C; Gardner, Lauren H; Allsopp, Richard C

    2016-01-01

    The cellular response to a hypoxic environment is regulated by hypoxia inducible factors. Hypoxia inducible factor 1 alpha (Hif1alpha) in particular, is tightly regulated by the hypoxic environment in most cells, and plays an important role in regulating the stress response of cells to hypoxia. Interestingly, substantial observations are now emerging that point to an important role for Hif1alpha in stem cells, including embryonic stem cells, neuronal stem cells and hematopoietic stem cells. Notably, Hif1alpha has been shown to enhance self renewal of stem cells, mediate a shift to glycolytic metabolism, and promote telomerase expression.

  12. Hypoxic preconditioning enhances neural stem cell transplantation therapy after intracerebral hemorrhage in mice.

    PubMed

    Wakai, Takuma; Narasimhan, Purnima; Sakata, Hiroyuki; Wang, Eric; Yoshioka, Hideyuki; Kinouchi, Hiroyuki; Chan, Pak H

    2016-12-01

    Previous studies have shown that intraparenchymal transplantation of neural stem cells ameliorates neurological deficits in animals with intracerebral hemorrhage. However, hemoglobin in the host brain environment causes massive grafted cell death and reduces the effectiveness of this approach. Several studies have shown that preconditioning induced by sublethal hypoxia can markedly improve the tolerance of treated subjects to more severe insults. Therefore, we investigated whether hypoxic preconditioning enhances neural stem cell resilience to the hemorrhagic stroke environment and improves therapeutic effects in mice. To assess whether hypoxic preconditioning enhances neural stem cell survival when exposed to hemoglobin, neural stem cells were exposed to 5% hypoxia for 24 hours before exposure to hemoglobin. To study the effectiveness of hypoxic preconditioning on grafted-neural stem cell recovery, neural stem cells subjected to hypoxic preconditioning were grafted into the parenchyma 3 days after intracerebral hemorrhage. Hypoxic preconditioning significantly enhanced viability of the neural stem cells exposed to hemoglobin and increased grafted-cell survival in the intracerebral hemorrhage brain. Hypoxic preconditioning also increased neural stem cell secretion of vascular endothelial growth factor. Finally, transplanted neural stem cells with hypoxic preconditioning exhibited enhanced tissue-protective capability that accelerated behavioral recovery. Our results suggest that hypoxic preconditioning in neural stem cells improves efficacy of stem cell therapy for intracerebral hemorrhage.

  13. Comparison of hypoxic cell radiosensitizers, KIN-804, KIN-844, KIN-806 and TX-1877, on brain and liver metabolizing capacities in mice bearing Ehrlich ascites carcinoma.

    PubMed

    Abou-Bedair, Farid Ahmed; Hori, Hitoshi; Nagasawa, Hideko; Uto, Yoshihiro; Abu-Zeid, Medhat; Inayama, Seiichi

    2002-05-01

    The biochemical effects of the 2-nitroimidazole hypoxic cell radiosensitizers KIN-804, KIN-806, and their analogues KIN-844 and TX-1877 on brain acetylcholinesterase (AChE) and hepatic free radical scavenging systems, such as reduced glutathione (GSH) and glucose-6-phosphate dehydrogenase (G-6-PDH) levels, and hepatic antioxidants, such as superoxide dismutase (SOD) and catalase, were evaluated in Ehrlich ascites carcinoma (EAC)-bearing Swiss albino mice. The assay of brain AChE revealed nonsignificant changes with all drugs examined. To evaluate the hepatic metabolic capacity, groups of mice were divided into control, EAC-inoculated, 10-Gy local gamma-irradiated, and KIN-804, KIN-844, KIN-806, or TX-1877 (50 mg/kg body weight, i.p.) groups, and gamma-irradiation was combined with each drug. EAC inoculation markedly suppressed GSH, G-6-PDH, SOD, and catalase levels. On the other hand, treatment with gamma-irradiation significantly enhanced them. The treatment of EAC-bearing mice with each drug alone in the absence of gamma-irradiation revealed that KIN-806 and its derivative TX-1877 showed antitumor activity through their significant recovery of GSH and SOD levels, respectively, in the EAC-bearing mice group. Similarly, the combined treatment of EAC-bearing mice with gamma-irradiation with each of the drugs tested showed that KIN-806 and TX-1877 significantly increased GSH and SOD, and to a lesser extent G-6-PDH and catalase levels. On the other hand, KIN-804 and KIN-844 had only a nonsignificant effect on all parameters examined. In conclusion, these data reveal that the administration of KIN-806 and TX-1877 with or without subsequent gamma-irradiation, resulted in significant recovery of GSH and SOD activities that were inhibited by EAC inoculation.

  14. Catabolic pathway for 2-nitroimidazole involves a novel nitrohydrolase that also confers drug resistance.

    PubMed

    Qu, Yi; Spain, Jim C

    2011-04-01

    Antibiotic resistance in pathogens can be mediated by catabolic enzymes thought to originate from soil bacteria, but the physiological functions and evolutionary origins of the enzymes in natural ecosystems are poorly understood. 2-Nitroimidazole (2NI) is a natural antibiotic and an analogue of the synthetic nitroimidazoles used for treatment of tuberculosis, Chagas' disease and cancer. Mycobacterium sp. JS330 was isolated from soil based on its ability to use 2NI as a sole growth substrate. The initial step in the degradation pathway is the hydrolytic denitration of 2NI to produce imidazol-2-one and nitrite. The amino acid sequence of 2NI nitrohydrolase is highly divergent from those of biochemically characterized enzymes, and it confers drug resistance when it is heterologously expressed in Escherichia coli. The unusual enzymatic reaction seems likely to determine the flux of nitroimidazole in natural ecosystems and also represents the discovery of a previously unreported drug resistance mechanism in soil before its identification in clinical situations.

  15. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

    SciTech Connect

    Shima, Haruko; Takubo, Keiyo; Iwasaki, Hiroko; Yoshihara, Hiroki; Gomei, Yumiko; Hosokawa, Kentaro; Arai, Fumio; Takahashi, Takao; Suda, Toshio

    2009-01-16

    Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2R{gamma}{sup null} (NOG) mice. Hypoxic culture (1% O{sub 2}) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34{sup +}CD38{sup -} cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

  16. Hypoxic culture conditions enhance the generation of regulatory T cells

    PubMed Central

    Neildez-Nguyen, Thi My Anh; Bigot, Jérémy; Da Rocha, Sylvie; Corre, Guillaume; Boisgerault, Florence; Paldi, Andràs; Galy, Anne

    2015-01-01

    The generation of large amounts of induced CD4+ CD25+ Foxp3+ regulatory T (iTreg) cells is of great interest for several immunotherapy applications, therefore a better understanding of signals controlling iTreg cell differentiation and expansion is required. There is evidence that oxidative metabolism may regulate several key signalling pathways in T cells. This prompted us to investigate the effects of oxygenation on iTreg cell generation by comparing the effects of atmospheric (21%) or of low (5%) O2 concentrations on the phenotype of bead-stimulated murine splenic CD4+ T cells from Foxp3-KI-GFP T-cell receptor transgenic mice. The production of intracellular reactive oxygen species was shown to play a major role in the generation of iTreg cells, a process characterized by increased levels of Sirt1, PTEN and Glut1 on the committed cells, independently of the level of oxygenation. The suppressive function of iTreg cells generated either in atmospheric or low oxygen levels was equivalent. However, greater yields of iTreg cells were obtained under low oxygenation, resulting from a higher proliferative rate of the committed Treg cells and higher levels of Foxp3, suggesting a better stability of the differentiation process. Higher expression of Glut1 detected on iTreg cells generated under hypoxic culture conditions provides a likely explanation for the enhanced proliferation of these cells as compared to those cultured under ambient oxygen. Such results have important implications for understanding Treg cell homeostasis and developing in vitro protocols for the generation of Treg cells from naive T lymphocytes. PMID:25243909

  17. Hypoxic-Preconditioned Bone Marrow Stem Cell Medium Significantly Improves Outcome After Retinal Ischemia in Rats

    PubMed Central

    Roth, Steven; Dreixler, John C.; Mathew, Biji; Balyasnikova, Irina; Mann, Jacob R.; Boddapati, Venkat; Xue, Lai; Lesniak, Maciej S.

    2016-01-01

    Purpose We have previously demonstrated the protective effect of bone marrow stem cell (BMSC)-conditioned medium in retinal ischemic injury. We hypothesized here that hypoxic preconditioning of stem cells significantly enhances the neuroprotective effect of the conditioned medium and thereby augments the protective effect in ischemic retina. Methods Rats were subjected to retinal ischemia by increasing intraocular pressure to 130 to 135 mm Hg for 55 minutes. Hypoxic-preconditioned, hypoxic unconditioned, or normoxic medium was injected into the vitreous 24 hours after ischemia ended. Recovery was assessed 7 days after injections by comparing electroretinography measurements, histologic examination, and apoptosis (TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay). To compare proteins secreted into the medium in the groups and the effect of hypoxic exposure, we used rat cytokine arrays. Results Eyes injected with hypoxic BMSC–conditioned medium 24 hours after ischemia demonstrated significantly enhanced return of retinal function, decreased retinal ganglion cell layer loss, and attenuated apoptosis compared to those administered normoxic or hypoxic unconditioned medium. Hypoxic-preconditioned medium had 21 significantly increased protein levels compared to normoxic medium. Conclusions The medium from hypoxic-preconditioned BMSCs robustly restored retinal function and prevented cell loss after ischemia when injected 24 hours after ischemia. The protective effect was even more pronounced than in our previous studies of normoxic conditioned medium. Prosurvival signals triggered by the secretome may play a role in this neuroprotective effect. PMID:27367588

  18. Hypoxic cell sensitizers and heavy charged-particle radiations.

    PubMed Central

    Chapman, J. D.; Urtasun, R. C.; Blakely, E. A.; Smith, K. C.; Tobias, C. A.

    1978-01-01

    Stationary-phase populations of Chinese hamster V-79 cells were irradiated with 250 kV X-rays and the Bragg peaks (spread to a width of 4 cm) of energetic He-, C-, Ne-, and A-ion beams produced at the 184-inch cyclotron and BEVALAC at Lawrence Berkeley Laboratory. Survival curves were generated with each radiation for cells suspended in air-saturated and nitrogen-saturated medium with and without sensitizer present. The oxygen enhancement ratios (OERs) measured for X-rays with 1mM metronidazole and 0.5 mM misonidazole were 2.0 and 1.6 respectively. The OERs without sensitizer for He-, C-, Ne-, and A-ion Bragg peaks were 2.4, 1.7, 1.6 and 1.4 respectively. For each type of radiation tested the presence of hypoxic-cell sensitizers resulted in an additional reduction in the measured OERs, indicating that these drugs should be of benefit in the radiotherapy planned with these and other high LET radiations. PMID:277223

  19. Phenotypic heterogeneity of disseminated tumour cells is preset by primary tumour hypoxic microenvironments.

    PubMed

    Fluegen, Georg; Avivar-Valderas, Alvaro; Wang, Yarong; Padgen, Michael R; Williams, James K; Nobre, Ana Rita; Calvo, Veronica; Cheung, Julie F; Bravo-Cordero, Jose Javier; Entenberg, David; Castracane, James; Verkhusha, Vladislav; Keely, Patricia J; Condeelis, John; Aguirre-Ghiso, Julio A

    2017-02-01

    Hypoxia is a poor-prognosis microenvironmental hallmark of solid tumours, but it is unclear how it influences the fate of disseminated tumour cells (DTCs) in target organs. Here we report that hypoxic HNSCC and breast primary tumour microenvironments displayed upregulation of key dormancy (NR2F1, DEC2, p27) and hypoxia (GLUT1, HIF1α) genes. Analysis of solitary DTCs in PDX and transgenic mice revealed that post-hypoxic DTCs were frequently NR2F1(hi)/DEC2(hi)/p27(hi)/TGFβ2(hi) and dormant. NR2F1 and HIF1α were required for p27 induction in post-hypoxic dormant DTCs, but these DTCs did not display GLUT1(hi) expression. Post-hypoxic DTCs evaded chemotherapy and, unlike ER(-) breast cancer cells, post-hypoxic ER(+) breast cancer cells were more prone to enter NR2F1-dependent dormancy. We propose that primary tumour hypoxic microenvironments give rise to a subpopulation of dormant DTCs that evade therapy. These post-hypoxic dormant DTCs may be the source of disease relapse and poor prognosis associated with hypoxia.

  20. Acute Hypoxic Stress Affects Migration Machinery of Tissue O2-Adapted Adipose Stromal Cells

    PubMed Central

    Lobanova, Margarita V.; Andreeva, Elena R.

    2016-01-01

    The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O2 tension in an MSC niche in vivo is about 4–7%. However, most in vitro studies of MSC functional activity are performed at 20% O2. Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O2, 24 h) on adipose tissue-derived MSC motility at tissue-related O2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1, and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury. PMID:28115943

  1. Will hyperthermia conquer the elusive hypoxic cell? Implications of heat effects on tumor and normal-tissue microcirculation.

    PubMed

    Dewhirst, M W; Ozimek, E J; Gross, J; Cetas, T C

    1980-12-01

    In tumor radiobiology, the hypoxic cell has become especially important because of recent evidence of acute hypoxic regions within experimental tumors. The impact of hyperthermia on the development of acute hypoxia is discussed. Two experiments for studying hyperthermia and hypoxia in tumors are presented. Knowledge of the microenvironment of tumor cells will be necessary to understand and improve tumor control.

  2. Cytosine Deaminase/5-Fluorocytosine Exposure Induces Bystander and Radiosensitization Effects in Hypoxic Glioblastoma Cells in vitro

    SciTech Connect

    Chen, Jennifer K.; Hu, Lily J.; Wang Dongfang; Lamborn, Kathleen R.; Deen, Dennis F. . E-mail: dennisdeen@juno.com

    2007-04-01

    Purpose: Treatment of glioblastoma (GBM) is limited by therapeutic ratio; therefore, successful therapy must be specifically cytotoxic to cancer cells. Hypoxic cells are ubiquitous in GBM, and resistant to radiation and chemotherapy, and, thus, are logical targets for gene therapy. In this study, we investigated whether cytosine deaminase (CD)/5-fluorocytosine (5-FC) enzyme/prodrug treatment induced a bystander effect (BE) and/or radiosensitization in hypoxic GBM cells. Methods and Materials: We stably transfected cells with a gene construct consisting of the SV40 minimal promoter, nine copies of a hypoxia-responsive element, and the yeast CD gene. During hypoxia, a hypoxia-responsive element regulates expression of the CD gene and facilitates the conversion of 5-FC to 5-fluorouracil, a highly toxic antimetabolite. We used colony-forming efficiency (CFE) and immunofluorescence assays to assess for BE in co-cultures of CD-expressing clone cells and parent, pNeo- or green fluorescent protein-stably transfected GBM cells. We also investigated the radiosensitivity of CD clone cells treated with 5-FC under hypoxic conditions, and we used flow cytometry to investigate treatment-induced cell cycle changes. Results: Both a large BE and radiosensitization occurred in GBM cells under hypoxic conditions. The magnitude of the BE depended on the number of transfected cells producing CD, the functionality of the CD, the administered concentration of 5-FC, and the sensitivity of cell type to 5-fluorouracil. Conclusion: Hypoxia-inducible CD/5-FC therapy in combination with radiation therapy shows both a pronounced BE and a radiosensitizing effect under hypoxic conditions.

  3. Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

    SciTech Connect

    Sprong, Debbie; Janssen, Hilde L.; Vens, Conchita; Begg, Adrian C. . E-mail: a.begg@nki.nl

    2006-02-01

    Purpose: To determine the role of DNA repair in hypoxic radioresistance. Methods and Materials: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. Results: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. Conclusion: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.

  4. Inhibition of hypoxia-induced miR-155 radiosensitizes hypoxic lung cancer cells.

    PubMed

    Babar, Imran A; Czochor, Jennifer; Steinmetz, Allison; Weidhaas, Joanne B; Glazer, Peter M; Slack, Frank J

    2011-11-15

    miR-155 is a prominent microRNA (miRNA) that regulates genes involved in immunity and cancer-related pathways. miR-155 is overexpressed in lung cancer, which correlates with poor patient prognosis. It is unclear how miR-155 becomes increased in lung cancers and how this increase contributes to reduced patient survival. Here, we show that hypoxic conditions induce miR-155 expression in lung cancer cells and trigger a corresponding decrease in a validated target, FOXO3A. Furthermore, we find that increased levels of miR-155 radioprotects lung cancer cells, while inhibition of miR-155 radiosensitizes these cells. Moreover, we reveal a therapeutically important link between miR-155 expression, hypoxia, and irradiation by demonstrating that anti-miR-155 molecules also sensitize hypoxic lung cancer cells to irradiation. Our study helps explain how miR-155 becomes elevated in lung cancers, which contain extensive hypoxic microenvironments, and demonstrates that inhibition of miR-155 may have important therapeutic potential as a means to radiosensitize hypoxic lung cancer cells.

  5. Pharmacokinetics of SPECT radiopharmaceuticals for imaging hypoxic tissues.

    PubMed

    Wiebe, L I; Stypinski, D

    1996-09-01

    Although hypoxia has been known for decades to play an important role in the outcome of radiotherapy in oncology, and inspite of the contribution of hypoxia to a myriad of pathologies that involve vascular disease, the selective imaging of hypoxic tissue has attained prominence only within the past decade. Contemporary research in the hypoxia imaging field is based largely on radiosensitizer research of the 1960's and 1970's. Early sensitizer research identified a family of nitro-organic compounds, the N-1 substituted 2-nitroimidazoles as candidate drugs. The early champion, and still the reference standard for therapeutic radiosensitization of hypoxic tumor cells is misonidazole (MISO). Its peripheral neurotoxicity led to failure in clinical studies, but its biological, biophysical and biochemical properties have been investigated in detail and serve as a basis for further design, not only of sensitizers, but of diagnostic radiopharmaceuticals for imaging tissue hypoxia. Pharmacokinetic characterization of radiopharmaceuticals, specifically radiopharmaceuticals for imaging tissue hypoxia, has not been a central theme in their development. The advent of PET, through which quantitative determinations first became possible, opened the field for both descriptive and analytical radiopharmacokinetic studies. In SPECT, however, this approach is still undergoing refinement. This paper addresses some of the underlying issues in radiopharmaceutical pharmacokinetics. There is a paucity of published radiopharmacokinetic data for SPECT hypoxia imaging agents. Consequently, the pharmacokinetic issues for MISO are presented as a basis for development of pharmacokinetics for the chemically-related imaging agents. Properties of an hypoxia marker are described from a pharmacokinetic viewpoint, a theoretical model for descriptive pharmacokinetics is introduced and finally, recent pharmacokinetic studies from our laboratory are described.

  6. T-Cell Activation under Hypoxic Conditions Enhances IFN-γ Secretion

    PubMed Central

    Roman, Jessica; Rangasamy, Tirumalai; Guo, Jia; Sugunan, Siva; Meednu, Nida; Packirisamy, Gopinath; Shimoda, Larissa A.; Golding, Amit; Semenza, Gregg; Georas, Steve N.

    2010-01-01

    Secondary lymphoid organs and peripheral tissues are characterized by hypoxic microenvironments, both in the steady state and during inflammation. Although hypoxia regulates T-cell metabolism and survival, very little is known about whether or how hypoxia influences T-cell activation. We stimulated mouse CD4+ T cells in vitro with antibodies directed against the T-cell receptor (CD3) and CD28 under normoxic (20% O2) and hypoxic (1% O2) conditions. Here we report that stimulation under hypoxic conditions augments the secretion of effector CD4+ T-cell cytokines, especially IFN-γ. The enhancing effects of hypoxia on IFN-γ secretion were independent of mouse strain, and were also unaffected using CD4+ T cells from mice lacking one copy of the gene encoding hypoxia-inducible factor-1α. Using T cells from IFN-γ receptor–deficient mice and promoter reporter studies in transiently transfected Jurkat T cells, we found that the enhancing effects of hypoxia on IFN-γ expression were not due to effects on IFN-γ consumption or proximal promoter activity. In contrast, deletion of the transcription factor, nuclear erythroid 2 p45–related factor 2 attenuated the enhancing effect of hypoxia on IFN-γ secretion and other cytokines. We conclude that hypoxia is a previously underappreciated modulator of effector cytokine secretion in CD4+ T cells. PMID:19372249

  7. Quantitative measurement of redox potential in hypoxic cells using SERS nanosensors

    NASA Astrophysics Data System (ADS)

    Jiang, Jing; Auchinvole, Craig; Fisher, Kate; Campbell, Colin J.

    2014-09-01

    Hypoxia is considered to be a reductive disorder of cells that is caused either by a lack of oxygen or by the dysregulation of metabolic pathways and is thought to play a role in the pathology of diseases including stroke and cancer. One aspect of hypoxia that remains poorly investigated is the dysregulation of cellular redox potential and its role in controlling biological pathway activation. Since there is currently no way of quantitatively measuring the intracellular redox potential of hypoxic cells, this provided us with the motivation to develop optical nanosensors whose Surface-Enhanced Raman (SER) spectrum provides a quantitative measure of redox potential in hypoxic cells. Our nanosensors are made from organic reporter molecules that show oxidation-state-dependent changes in the Raman spectrum and are chemically adsorbed onto gold nanoshells. These nanosensors can be taken up by cells, and by collecting the SER spectrum we can calculate the localised intracellular redox potential from single hypoxic cells in a non-invasive, reversible way.Hypoxia is considered to be a reductive disorder of cells that is caused either by a lack of oxygen or by the dysregulation of metabolic pathways and is thought to play a role in the pathology of diseases including stroke and cancer. One aspect of hypoxia that remains poorly investigated is the dysregulation of cellular redox potential and its role in controlling biological pathway activation. Since there is currently no way of quantitatively measuring the intracellular redox potential of hypoxic cells, this provided us with the motivation to develop optical nanosensors whose Surface-Enhanced Raman (SER) spectrum provides a quantitative measure of redox potential in hypoxic cells. Our nanosensors are made from organic reporter molecules that show oxidation-state-dependent changes in the Raman spectrum and are chemically adsorbed onto gold nanoshells. These nanosensors can be taken up by cells, and by collecting the SER

  8. Stereotactic Ablative Radiotherapy Should Be Combined With a Hypoxic Cell Radiosensitizer

    SciTech Connect

    Brown, J. Martin; Diehn, Maximilian; Loo, Billy W.

    2010-10-01

    Purpose: To evaluate the effect of tumor hypoxia on the expected level of cell killing by regimens of stereotactic ablative radiotherapy (SABR) and to determine the extent to which the negative effect of hypoxia could be prevented using a clinically available hypoxic cell radiosensitizer. Results and Discussion: We have calculated the expected level of tumor cell killing from regimens of SABR, both with and without the assumption that 20% of the tumor cells are hypoxic, using the standard linear quadratic model and the universal survival curve modification. We compare the results obtained with our own clinical data for lung tumors of different sizes and with published data from other studies. We also have calculated the expected effect on cell survival of adding the hypoxic cell sensitizer etanidazole at clinically achievable drug concentrations. Modeling tumor cell killing with any of the currently used regimens of SABR produces results that are inconsistent with the majority of clinical findings if tumor hypoxia is not considered. However, with the assumption of tumor hypoxia, the expected level of cell killing is consistent with clinical data. For only some of the smallest tumors are the clinical data consistent with no tumor hypoxia, but there could be other reasons for the sensitivity of these tumors. The addition of etanidazole at clinically achievable tumor concentrations produces a large increase in the expected level of tumor cell killing from the large radiation doses used in SABR. Conclusions: The presence of tumor hypoxia is a major negative factor in limiting the curability of tumors by SABR at radiation doses that are tolerable to surrounding normal tissues. However, this negative effect of hypoxia could be overcome by the addition of clinically tolerable doses of the hypoxic cell radiosensitizer etanidazole.

  9. Bone marrow long label-retaining cells reside in the sinusoidal hypoxic niche

    SciTech Connect

    Kubota, Yoshiaki; Takubo, Keiyo; Suda, Toshio

    2008-02-08

    In response to changing signals, quiescent hematopoietic stem cells (HSCs) can be induced to an activated cycling state and provide multi-lineage hematopoietic cells to the whole body via blood vessels. However, the precise localization of quiescent HSCs in bone marrow microenvironment is not fully characterized. Here, we performed whole-mount immunostaining of bone marrow and found that BrdU label-retaining cells (LRCs) definitively reside in the sinusoidal hypoxic zone distant from the 'vascular niche'. Although LRCs expressed very low level of a well-known HSC marker, c-kit in normal circumstances, myeloablation by 5-FU treatment caused LRCs to abundantly express c-kit and proliferate actively. These results demonstrate that bone marrow LRCs reside in the sinusoidal hypoxic niche, and function as a regenerative cell pool of HSCs.

  10. Downregulation of Metabolic Activity Increases Cell Survival Under Hypoxic Conditions: Potential Applications for Tissue Engineering

    PubMed Central

    Kim, Jaehyun; Andersson, Karl-Erik; Jackson, John D.; Lee, Sang Jin; Atala, Anthony

    2014-01-01

    A major challenge to the success of cell-based implants for tissue regeneration is an insufficient supply of oxygen before host vasculature is integrated into the implants, resulting in premature cell death and dysfunction. Whereas increasing oxygenation to the implants has been a major focus in the field, our strategy is aimed at lowering oxygen consumption by downregulating cellular metabolism of cell-based implants. Adenosine, which is a purine nucleoside that functions as an energy transferring molecule, has been reported to increase under hypoxia, resulting in reducing the adenosine triphosphate (ATP) demands of the Na+/K+ ATPase. In the present study, we investigated whether adenosine could be used to downregulate cellular metabolism to achieve prolonged survival under hypoxic conditions. Murine myoblasts (C2C12) lacking a self-survival mechanism were treated with adenosine under 0.1% hypoxic stress. The cells, cultured in the presence of 5 mM adenosine, maintained their viability under hypoxia, and regained their normal growth and function of forming myotubes when transferred to normoxic conditions at day 11 without further supply of adenosine, whereas nontreated cells failed to survive. An increase in adenosine concentrations shortened the onset of reproliferation after transfer to normoxic conditions. This increase correlated with an increase in metabolic downregulation during the early phase of hypoxia. A higher intracellular ATP level was observed in adenosine-treated cells throughout the duration of hypoxia. This strategy of increasing cell survival under hypoxic conditions through downregulating cellular metabolism may be utilized for cell-based tissue regeneration applications as well as protecting tissues against hypoxic injuries. PMID:24524875

  11. Lethal or protective effects of prolonged treatment with hypoxic cell sensitizers

    SciTech Connect

    Edgren, M.R.

    1995-12-31

    AK-2123 [N-(2-methoxyethyl)-2-(3-nitro-1-triazolyl)acetamide] is a hypoxic cell radiosensitizer which is currently being tested in several oncology clinics and which has a lower toxicity than misonidazole (MISO) in vivo. The positive experiences reported recently certainly warrant further clinical evaluations. The experimental observations reported so far need further experimental studies to clarify the sensitization mechanism, especially as recent intratumoral strategies used in the clinical administration of the sensitizers can result in a large local concentration of the drug that may persist for a prolonged period of time between and after radiation exposures. Model experiments in vitro using V79 cells were performed with AK-2123 under these conditions. Misonidazole (MISO) and metronidazole (METRO), well known hypoxic cell radiosensitizers, were used for comparison of the effects. Clonogenic survival and induction and repair of DNA damage were used as end-points.

  12. Chromosomal variability of human mesenchymal stem cells cultured under hypoxic conditions

    PubMed Central

    Ueyama, Hanae; Horibe, Tomohisa; Hinotsu, Shiro; Tanaka, Tomoaki; Inoue, Takeomi; Urushihara, Hisashi; Kitagawa, Akira; Kawakami, Koji

    2012-01-01

    Abstract Bone marrow derived human mesenchymal stem cells (hMSCs) have attracted great interest from both bench and clinical researchers because of their pluripotency and ease of expansion ex vivo. However, these cells do finally reach a senescent stage and lose their multipotent potential. Proliferation of these cells is limited up to the time of their senescence, which limits their supply, and they may accumulate chromosomal changes through ex vivo culturing. The safe, rapid expansion of hMSCs is critical for their clinical application. Chromosomal aberration is known as one of the hallmarks of human cancer, and therefore it is important to understand the chromosomal stability and variability of ex vivo expanded hMSCs before they are used widely in clinical applications. In this study, we examined the effects of culturing under ambient (20%) or physiologic (5%) O2 concentrations on the rate of cell proliferation and on the spontaneous transformation of hMSCs in primary culture and after expansion, because it has been reported that culturing under hypoxic conditions accelerates the propagation of hMSCs. Bone marrow samples were collected from 40 patients involved in clinical research. We found that hypoxic conditions promote cell proliferation more favourably than normoxic conditions. Chromosomal aberrations, including structural instability or aneuploidy, were detected in significantly earlier passages under hypoxic conditions than under normoxic culture conditions, suggesting that amplification of hMSCs in a low-oxygen environment facilitated chromosomal instability. Furthermore, smoothed hazard-function modelling of chromosomal aberrations showed increased hazard after the fourth passage under both sets of culture conditions, and showed a tendency to increase the detection rate of primary karyotypic abnormalities among donors aged 60 years and over. In conclusion, we propose that the continuous monitoring of hMSCs will be required before they are used in

  13. Autocrine production of IL-11 mediates tumorigenicity in hypoxic cancer cells

    PubMed Central

    Onnis, Barbara; Fer, Nicole; Rapisarda, Annamaria; Perez, Victor S.; Melillo, Giovanni

    2013-01-01

    IL-11 and its receptor, IL-11Ra, are expressed in human cancers; however, the functional role of IL-11 in tumor progression is not known. We found that IL11 is a hypoxia-inducible, VHL-regulated gene in human cancer cells and that expression of IL11 mRNA was dependent, at least in part, on HIF-1. A cooperative interaction between HIF-1 and AP-1 mediated transcriptional activation of the IL11 promoter. Additionally, we found that human cancer cells expressed a functional IL-11Ra subunit, which triggered signal transduction either by exogenous recombinant human IL-11 or by autocrine production of IL-11 in cells cultured under hypoxic conditions. Silencing of IL11 dramatically abrogated the ability of hypoxia to increase anchorage-independent growth and significantly reduced tumor growth in xenograft models. Notably, these results were phenocopied by partial knockdown of STAT1 in a human prostate cancer cell line (PC3), suggesting that this pathway may play an important role in mediating the effects of IL-11 under hypoxic conditions. In conclusion, these results identify IL11 as an oxygen- and VHL-regulated gene and provide evidence of a pathway “hijacked” by hypoxic cancer cells that may contribute to tumor progression. PMID:23549086

  14. Erdosteine protects rat testis tissue from hypoxic injury by reducing apoptotic cell death.

    PubMed

    Guven, A; Ickin, M; Uzun, O; Bakar, C; Balbay, E Gulec; Balbay, O

    2014-02-01

    The purpose of this study was to examine the effects of hypobaric hypoxia on testis morphology and the effects of erdosteine on testis tissue. Caspase-3 and hypoxia-inducible factor 1α expressions were detected by immunohistochemistry. Adult male Wistar rats were placed in a hypobaric hypoxic chamber. Rats in the erdosteine group were exposed to the same conditions and treated orally with erdosteine (20 mg kg(-1) daily) at the same time from the first day of hypoxic exposure for 2 weeks. The normoxia group was evaluated as the control. The hypoxia group showed decreased height of spermatogenic epithelium in some seminiferous tubules, vacuolisation in spermatogenic epithelial cells, deterioration and gaps in the basal membrane and an increase in blood vessels in the interstitial area. The erdosteine group showed amelioration of both epithelial cell vacuolisation and basal membrane deterioration. Numbers of hypoxia-inducible factor 1α-immunostained Sertoli and Leydig cells were significantly higher in the hypoxia group than in the erdosteine group. The number of seminiferous tubules with caspase-3-immunostained germ cells was highest in the hypoxia group and decreased in the erdosteine and normoxia groups respectively. Based on these observations, erdosteine protects testis tissue from hypoxic injury by reducing apoptotic cell death.

  15. Metformin increases PDH and suppresses HIF-1α under hypoxic conditions and induces cell death in oral squamous cell carcinoma

    PubMed Central

    Guimarães, Talita Antunes; Farias, Lucyana Conceição; Santos, Eliane Sobrinho; de Carvalho Fraga, Carlos Alberto; Orsini, Lissur Azevedo; de Freitas Teles, Leandro; Feltenberger, John David; de Jesus, Sabrina Ferreira; de Souza, Marcela Gonçalves; Sousa Santos, Sérgio Henrique; de Paula, Alfredo Maurício Batista

    2016-01-01

    Background Metformin is a biguanide, belonging to the oral hypoglycemic agents and is a widely used in the treatment of type 2 diabetes. Evidence indicate that Metformin inhibits cell proliferation in several human cancers and inhibits the Warburg phenomenon in tumor cells. Results Low PDH levels were observed in OSCC, and Metformin promotes an increase in PDH levels in hypoxic conditions. Metformin also reduced HIF-1α mRNA and protein levels. Metformin demonstrated antiproliferative effects, inhibited migration, increased the number of apoptotic cells and increased the transcription of caspase 3. Objective The present study aims to explore the effects of Metformin in hypoxic conditions. Specifically, we focused on pyruvate dehydrogenase (PDH), (hypoxia-inducible factor 1α) HIF-1α levels and the oral squamous cell carcinoma (OSCC) cell phenotype. Additionally, we also investigated a theoretical consequence of Metformin treatment. Methods PDH levels in patients with OSCC and oral dysplasia were evaluated. Metformin was administered in vitro to test the effect of Metformin under hypoxic conditions. The results were complemented by Bioinformatics analyses. Conclusions In conclusion, our current findings show that Metformin reduces HIF-1α gene expression and increases PDH expression. Metformin inhibits cell proliferation and migration in the OSCC cell line model. Additionally, Metformin enhances the number of apoptotic cells and caspase 3 levels. Interestingly enough, Metformin did not increase the mutant p53 levels under hypoxic conditions. PMID:27474170

  16. Galectin-3 Up-Regulation in Hypoxic and Nutrient Deprived Microenvironments Promotes Cell Survival

    PubMed Central

    Ikemori, Rafael Yamashita; Machado, Camila Maria Longo; Furuzawa, Karina Mie; Nonogaki, Suely; Osinaga, Eduardo; Umezawa, Kazuo; de Carvalho, Marcelo Alex; Verinaud, Liana; Chammas, Roger

    2014-01-01

    Galectin-3 (gal-3) is a β-galactoside binding protein related to many tumoral aspects, e.g. angiogenesis, cell growth and motility and resistance to cell death. Evidence has shown its upregulation upon hypoxia, a common feature in solid tumors such as glioblastoma multiformes (GBM). This tumor presents a unique feature described as pseudopalisading cells, which accumulate large amounts of gal-3. Tumor cells far from hypoxic/nutrient deprived areas express little, if any gal-3. Here, we have shown that the hybrid glioma cell line, NG97ht, recapitulates GBM growth forming gal-3 positive pseudopalisades even when cells are grafted subcutaneously in nude mice. In vitro experiments were performed exposing these cells to conditions mimicking tumor areas that display oxygen and nutrient deprivation. Results indicated that gal-3 transcription under hypoxic conditions requires previous protein synthesis and is triggered in a HIF-1α and NF-κB dependent manner. In addition, a significant proportion of cells die only when exposed simultaneously to hypoxia and nutrient deprivation and demonstrate ROS induction. Inhibition of gal-3 expression using siRNA led to protein knockdown followed by a 1.7–2.2 fold increase in cell death. Similar results were also found in a human GBM cell line, T98G. In vivo, U87MG gal-3 knockdown cells inoculated subcutaneously in nude mice demonstrated decreased tumor growth and increased time for tumor engraftment. These results indicate that gal-3 protected cells from cell death under hypoxia and nutrient deprivation in vitro and that gal-3 is a key factor in tumor growth and engraftment in hypoxic and nutrient-deprived microenvironments. Overexpression of gal-3, thus, is part of an adaptive program leading to tumor cell survival under these stressing conditions. PMID:25369297

  17. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells

    PubMed Central

    Fu, Shibo; Tar, Moses Tarndie; Melman, Arnold; Davies, Kelvin Paul

    2014-01-01

    Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM “relaxant” pathways; excessive activation of these pathways results in priapism.—Fu, S., Tar, M. T., Melman, A., Davies, K. P. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells. PMID:24803544

  18. Phytoglobins Improve Hypoxic Root Growth by Alleviating Apical Meristem Cell Death1[OPEN

    PubMed Central

    Stasolla, Claudio

    2016-01-01

    Hypoxic root growth in maize (Zea mays) is influenced by the expression of phytoglobins (ZmPgbs). Relative to the wild type, suppression of ZmPgb1.1 or ZmPgb1.2 inhibits the growth of roots exposed to 4% oxygen, causing structural abnormalities in the root apical meristems. These effects were accompanied by increasing levels of reactive oxygen species (ROS), possibly through the transcriptional induction of four Respiratory Burst Oxidase Homologs. TUNEL-positive nuclei in meristematic cells indicated the involvement of programmed cell death (PCD) in the process. These cells also accumulated nitric oxide and stained heavily for ethylene biosynthetic transcripts. A sharp increase in the expression level of several 1-aminocyclopropane synthase (ZmAcs2, ZmAcs6, and ZmAcs7), 1-aminocyclopropane oxidase (Aco15, Aco20, Aco31, and Aco35), and ethylene-responsive (ZmErf2 and ZmEbf1) genes was observed in hypoxic ZmPgb-suppressing roots, which overproduced ethylene. Inhibiting ROS synthesis with diphenyleneiodonium or ethylene perception with 1-methylcyclopropene suppressed PCD, increased BAX inhibitor-1, an effective attenuator of the death programs in eukaryotes, and restored root growth. Hypoxic roots overexpressing ZmPgbs had the lowest level of ethylene and showed a reduction in ROS staining and TUNEL-positive nuclei in the meristematic cells. These roots retained functional meristems and exhibited the highest growth performance when subjected to hypoxic conditions. Collectively, these results suggest a novel function of Pgbs in protecting root apical meristems from hypoxia-induced PCD through mechanisms initiated by nitric oxide and mediated by ethylene via ROS. PMID:27702845

  19. Macrophages are recruited to hypoxic tumor areas and acquire a Pro-Angiogenic M2-Polarized phenotype via hypoxic cancer cell derived cytokines Oncostatin M and Eotaxin

    PubMed Central

    Tripathi, Chakrapani; Tewari, Brij Nath; Kanchan, Ranjana Kumari; Baghel, Khemraj Singh; Nautiyal, Naveen; Shrivastava, Richa; Kaur, Harbeer; Bhatt, Madan Lal Bramha; Bhadauria, Smrati

    2014-01-01

    TAMs, a unique and distinct M2-skewed myeloid population of tumor stroma, exhibiting pro-tumor functions is fast emerging as a potential target for anti-cancer immunotherapy. Macrophage-recruitment and M2-polarization represent key TAMs-related phenomenon that are amenable to therapeutic intervention. However successful translation of these approaches into effective therapeutic regimen requires better characterization of tumor-microenvironment derived signals that regulate macrophage recruitment and their polarization. Owing to hypoxic milieu being a persistent feature of tumor-microenvironment and a major contributor to malignancy and treatment resistance, the current study was planned with an aim to decipher tumor cell responses to hypoxia vis-a-vis macrophage homing and phenotype switching. Here, we show that hypoxia-primed cancer cells chemoattract and polarize macrophages to pro-angiogenic M2-polarized subtype via Eotaxin and Oncostatin M. Concordantly, hypoxic regions of human breast-cancer specimen exhibited elevated Eotaxin and Oncostatin M levels with concurrently elevated M2-macrophage content. Blockade of Eotaxin/Oncostatin M not only prevented hypoxic breast-cancer cells from recruiting and polarizing macrophages towards an M2-polarized phenotype and retarded tumor progression in 4T1/BALB/c-syngenic-mice-model of breast-cancer but also enhanced the efficacy of anti-angiogenic Bevacizumab. The findings established these two cytokines as novel targets for devising effective anticancer therapy particularly for tumors that are refractory or develop resistance to anti-angiogenic therapeutics. PMID:25051364

  20. Periostin Promotes Neural Stem Cell Proliferation and Differentiation following Hypoxic-Ischemic Injury.

    PubMed

    Ma, Si-Min; Chen, Long-Xia; Lin, Yi-Feng; Yan, Hu; Lv, Jing-Wen; Xiong, Man; Li, Jin; Cheng, Guo-Qiang; Yang, Yi; Qiu, Zi-Long; Zhou, Wen-Hao

    2015-01-01

    Neural stem cell (NSC) proliferation and differentiation are required to replace neurons damaged or lost after hypoxic-ischemic events and recover brain function. Periostin (POSTN), a novel matricellular protein, plays pivotal roles in the survival, migration, and regeneration of various cell types, but its function in NSCs of neonatal rodent brain is still unknown. The purpose of this study was to investigate the role of POSTN in NSCs following hypoxia-ischemia (HI). We found that POSTN mRNA levels significantly increased in differentiating NSCs. The proliferation and differentiation of NSCs in the hippocampus is compromised in POSTN knockout mice. Moreover, NSC proliferation and differentiation into neurons and astrocytes significantly increased in cultured NSCs treated with recombinant POSTN. Consistently, injection of POSTN into neonatal hypoxic-ischemic rat brains stimulated NSC proliferation and differentiation in the subventricular and subgranular zones after 7 and 14 days of brain injury. Lastly, POSTN treatment significantly improved the spatial learning deficits of rats subjected to HI. These results suggest that POSTN significantly enhances NSC proliferation and differentiation after HI, and provides new insights into therapeutic strategies for the treatment of hypoxic-ischemic encephalopathy.

  1. [SOS response of DNA repair and genetic cell instability under hypoxic conditions].

    PubMed

    Vasil'eva, S V; Strel'tsova, D A

    2011-01-01

    The SOS DNA repair pathway is induced in E. coli as a multifunctional cell response to a wide variety of signals: UV, X or gamma-irradiation, mitomycin C or nalidixic acid treatment, thymine starvation, etc. Triggering of the system can be used as a general and early sign of DNA damage. Additionally, the SOS-response is known to be an "error-prone" DNA repair pathway and one of the sources of genetic instability. Hypoxic conditions are established to be the major factor of genetic instability as well. In this paper we for the first time studied the SOS DNA repair response under hypoxic conditions induced by the well known aerobic SOS-inducers. The SOS DNA repair response was examined as a reaction of E. coli PQ37 [sfiA::lacZ] cells to UVC, NO-donating agents and 4NQO. Here we provide evidence that those agents were able to induce the SOS DNA repair response in E. coli at anaerobic growth conditions. The process does not depend on the transcriptional activity of the universal protein of E. col anaerobic growth Fnr [4Fe-4S]2+ or can not be referred to as an indicator of genetic instability in hypoxic conditions.

  2. A comparative immunological analysis of CoCl2 treated cells with in vitro hypoxic exposure.

    PubMed

    Shweta; Mishra, K P; Chanda, S; Singh, S B; Ganju, L

    2015-02-01

    The hypoxic preconditioning of mammalian cells has been shown to have beneficial effects against hypoxic injuries. However, very little information is available on the comparative analysis of immunological responses to hypoxic and hypoxia mimetic exposure. Therefore, in the present study, mouse peritoneal macrophages and splenocytes were subjected to hypoxia exposure (0.5 % O2) and hypoxia mimetic Cobalt chloride (CoCl2) treatment to evaluate their effect on immune response and delineate the underlying signaling mechanisms. The results obtained indicated that super oxide generation increased while TLR4 expression and cell surface markers like CD25, CD40 and CD69 were suppressed in both the treatments as compared to normoxia. Cobalt chloride treatment increased NF-κB expression, nitric oxide (NO) and iNOS expression, cytokines TNF-α and IL-6 as compared to hypoxia exposure. Our study showed that CoCl2 stabilizes HIF-1α to create hypoxia like conditions but it mainly influences the inflammatory response via NF-κB signaling pathway by skewing the production of proinflammatory molecules like TNF-α, IL-6 and NO.

  3. Hypoxic stellate cells of pancreatic cancer stroma regulate extracellular matrix fiber organization and cancer cell motility.

    PubMed

    Sada, Masafumi; Ohuchida, Kenoki; Horioka, Kohei; Okumura, Takashi; Moriyama, Taiki; Miyasaka, Yoshihiro; Ohtsuka, Takao; Mizumoto, Kazuhiro; Oda, Yoshinao; Nakamura, Masafumi

    2016-03-28

    Desmoplasia and hypoxia in pancreatic cancer mutually affect each other and create a tumor-supportive microenvironment. Here, we show that microenvironment remodeling by hypoxic pancreatic stellate cells (PSCs) promotes cancer cell motility through alteration of extracellular matrix (ECM) fiber architecture. Three-dimensional (3-D) matrices derived from PSCs under hypoxia exhibited highly organized parallel-patterned matrix fibers compared with 3-D matrices derived from PSCs under normoxia, and promoted cancer cell motility by inducing directional migration of cancer cells due to the parallel fiber architecture. Microarray analysis revealed that procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in PSCs was the gene that potentially regulates ECM fiber architecture under hypoxia. Stromal PLOD2 expression in surgical specimens of pancreatic cancer was confirmed by immunohistochemistry. RNA interference-mediated knockdown of PLOD2 in PSCs blocked parallel fiber architecture of 3-D matrices, leading to decreased directional migration of cancer cells within the matrices. In conclusion, these findings indicate that hypoxia-induced PLOD2 expression in PSCs creates a permissive microenvironment for migration of cancer cells through architectural regulation of stromal ECM in pancreatic cancer.

  4. The Antiproliferative and Colony-suppressive Activities of STAT3 Inhibitors in Human Cancer Cells Is Compromised Under Hypoxic Conditions.

    PubMed

    Tian, Jilai; Xiao, Hui; Wu, Ruohan; Cao, Yang; Li, Chenglong; Xu, Ronald; Pierson, Christopher R; Finlay, Jonathan L; Yang, Fang; Gu, Ning; Lin, Jiayuh

    2017-02-01

    Constitutive activation of signal transducer and activator of transcription 3 (STAT3) has been indicated as a novel cancer drug target, since it plays an important role in diverse oncogenic processes including survival, cell proliferation and migration. Emerging STAT3 inhibitors have demonstrated efficacy in cancer cells and animal tumor models. It is well known that most solid tumors are characterized by hypoxia, but it is not clear if hypoxic conditions affect activity of STAT3 inhibitors. To examine this, two STAT3 inhibitors were tested to investigate their inhibitory efficacy in cancer cells grown under hypoxic conditions compared with those without hypoxia. Cell proliferation, colony formation and western blot assays were performed to examine the differences in the cell viability, proliferation and proteins in the STAT3 pathway. Under hypoxic conditions, the half-maximal inhibitory concentration values for both STAT3 inhibitors were increased compared to normoxic conditions in human pancreatic cancer, medulloblastoma and sarcoma cell lines. In addition, the ability of both STAT3 inhibitors to inhibit colony formation in pancreatic cancer, medulloblastoma and sarcoma cell lines was reduced under hypoxic conditions when compared to cells under normoxic conditions. Furthermore, there was an increase in phosphorylated STAT3 levels in cancer cells under hypoxic conditions, suggesting this may be one of the mechanisms of resistance. In summary, the results presented here provide a novel finding of STAT3 inhibitor activity under hypoxic conditions and indicate that under such low oxygen conditions, the anticancer efficacy of STAT3 inhibitors was indeed hampered. These results highlight the need to develop new therapeutic strategies to overcome the resistance of cancer cells to STAT3 inhibitors under hypoxic conditions.

  5. Hypoxic preconditioning of human cardiosphere-derived cell sheets enhances cellular functions via activation of the PI3K/Akt/mTOR/HIF-1α pathway

    PubMed Central

    Tanaka, Yuya; Hosoyama, Tohru; Mikamo, Akihito; Kurazumi, Hiroshi; Nishimoto, Arata; Ueno, Koji; Shirasawa, Bungo; Hamano, Kimikazu

    2017-01-01

    Cell sheet technology is a promising therapeutic strategy for the treatment of ischemic diseases such as myocardial infarction. We recently developed a novel protocol, termed “hypoxic preconditioning,” capable of augmenting the therapeutic efficacy of cell sheets. Following this protocol, the pro-angiogenic and anti-fibrotic activity of cell sheets were enhanced by brief incubation of cell sheets under hypoxic culture conditions. However, the precise molecular mechanism underlying the hypoxic preconditioning of cell sheets is unclear. In the present study, we examined signal transducers in cell sheets to identify those responsive to hypoxic preconditioning, using cardiosphere-derived cell (CDC) sheets. We initially tested whether sheet-like structures were suitable for hypoxic preconditioning by comparing them with individual cells. Hypoxic preconditioning was more effective in sheeted cells than in individual cells. Expression of hypoxia inducible factor-1α (HIF-1α) and mammalian target of rapamycin (mTOR) were induced upon hypoxic preconditioning of cell sheets, as was the phosphoinositide 3-kinase (PI3K)/Akt pathway. In addition, hypoxic preconditioning increased phosphorylation of epidermal growth factor receptor (EGFR) and heat shock protein 60 (HSP60) in CDC sheets. Our findings provide novel insights into the utility of hypoxic preconditioning in cell sheet-based technologies for the treatment of ischemic diseases. PMID:28337294

  6. Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine.

    PubMed

    Muscari, Claudio; Giordano, Emanuele; Bonafè, Francesca; Govoni, Marco; Pasini, Alice; Guarnieri, Carlo

    2013-08-29

    The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.

  7. Cyctotoxicities of mitomycin C and x rays to aerobic and hypoxic cells in vitro

    SciTech Connect

    Rockwell, S.

    1982-01-01

    Aerobic and hypoxic EMT6 mouse mammary tumor cells in exponential growth in vitro were used to study cell survival after treatment with radiation (250k V X rays) and mitomycin C in various combinations. The cytotoxicities of the two agents were found to be additive as judged by comparing dose-response curves for each agent alone with survival curves after combination therapy and by isobologram analysis. The cytotoxicities resulting from combination treatments were found to be independent of the sequence of the treatments or the interval between treatments.

  8. Radiation-induced changes in nucleoid halo diameteres of aerobic and hypoxic SF-126 human brain tumor cells

    SciTech Connect

    Wang, J.; Basu, H.S.; Hu, L.; Feuerstein, B.G.; Deen, D.F.

    1995-02-01

    Nucleoid halo diameters were measured to assay changes in DNA supercoiling in human brain tumor cell line SF-126 after irradiation under aerobic or hypoxic conditions. In unirradiated aerobic cells, a typical propidium iodide titration curve showed that with increasing concentrations of propodium iodide, the halo diameter increased and then decreased with the unwinding and subsequent rewinding of DNA supercoils. In irradiated cells, the rewinding of DNA supercoils was inhibited, resulting in an increased halo diameter, in a radiation dose-dependent manner. To produce equal increases in halo diameter required about a threefold higher radiation dose in hypoxic cells than in aerobic cells. Quantitatively similiar differences in the radiation sensitivities of hypoxic and aerobic cells were demonstrated by a colony-forming efficiency assay. These findings suggest that the nucleoid halo assay may be used as a rapid measure of the inherent radiation sensitivity of human tumors. 22 refs., 5 figs.

  9. Hypoxic Preconditioning Increases Survival and Pro-Angiogenic Capacity of Human Cord Blood Mesenchymal Stromal Cells In Vitro

    PubMed Central

    Bader, Andreas Matthäus; Klose, Kristin; Bieback, Karen; Korinth, Dirk; Schneider, Maria; Seifert, Martina; Choi, Yeong-Hoon; Kurtz, Andreas; Falk, Volkmar; Stamm, Christof

    2015-01-01

    Hypoxic preconditioning was shown to improve the therapeutic efficacy of bone marrow-derived multipotent mesenchymal stromal cells (MSCs) upon transplantation in ischemic tissue. Given the interest in clinical applications of umbilical cord blood-derived MSCs, we developed a specific hypoxic preconditioning protocol and investigated its anti-apoptotic and pro-angiogenic effects on cord blood MSCs undergoing simulated ischemia in vitro by subjecting them to hypoxia and nutrient deprivation with or without preceding hypoxic preconditioning. Cell number, metabolic activity, surface marker expression, chromosomal stability, apoptosis (caspases-3/7 activity) and necrosis were determined, and phosphorylation, mRNA expression and protein secretion of selected apoptosis and angiogenesis-regulating factors were quantified. Then, human umbilical vein endothelial cells (HUVEC) were subjected to simulated ischemia in co-culture with hypoxically preconditioned or naïve cord blood MSCs, and HUVEC proliferation was measured. Migration, proliferation and nitric oxide production of HUVECs were determined in presence of cord blood MSC-conditioned medium. Cord blood MSCs proved least sensitive to simulated ischemia when they were preconditioned for 24 h, while their basic behavior, immunophenotype and karyotype in culture remained unchanged. Here, “post-ischemic” cell number and metabolic activity were enhanced and caspase-3/7 activity and lactate dehydrogenase release were reduced as compared to non-preconditioned cells. Phosphorylation of AKT and BAD, mRNA expression of BCL-XL, BAG1 and VEGF, and VEGF protein secretion were higher in preconditioned cells. Hypoxically preconditioned cord blood MSCs enhanced HUVEC proliferation and migration, while nitric oxide production remained unchanged. We conclude that hypoxic preconditioning protects cord blood MSCs by activation of anti-apoptotic signaling mechanisms and enhances their angiogenic potential. Hence, hypoxic preconditioning

  10. Radiosensitization of EMT6 cells by four platinum complexes

    SciTech Connect

    Teicher, B.A.; Rockwell, S.; Lee, J.B.

    1985-05-01

    The compounds described here are dichloro complexes of bivalent platinum with one or two potentially radiosensitizing ligands. The radiosensitization of oxygenated and hypoxic exponentially growing EMT6 cells in vitro was measured. The dose modifying factors obtained with 200 ..mu..M and 400 ..mu..M trans-bis(2-nitroimidazole)dichloroplatinum II (NIPt) in hypoxic cells were 1.5 and 2.1, respectively. For trans-bis(2-amino-5-nitrothiazole)dichloroplatinum II (Plant) under the same conditions, the dose modifying factor was 1.5 at 200 ..mu..M and 1.8 at 400 ..mu..M. Neither compound sensitized oxygenated cells when tested similar protocols. Unlike the trans complexes (1,2-diamino-4-nitrobenzene)dichloroplatinum II (Plato) was cytotoxic toward the hypoxic cells in the absence of X rays. The time course of cytotoxicity for 100 ..mu..M Plato in exponentially growing cells showed rapid killing of hypoxic cells, and much less toxicity toward oxygenated cells. In radiosensitization studies, dose modifying factors of 1.6 and 2.0 were found with 200 ..mu..M and 400 ..mu..M Plato in hypoxic cells. The compound did not sensitize aerobic cells. The well-known platinum complex cis-dipyridinedichloroplatinum II (PyPt) represents a cis-platinum heterocyclic aromatic complex that does not have a nitro-functionality. The dose modifying factor obtained with 400 ..mu..M PyPt in hypoxic cells was 1.7. On a molar basis, the nitro-functional platinum complexes appear to be more effective as hypoxic cell radiosensitizers than the corresponding free ligands.

  11. Isolation and characterization of 2-nitroimidazole produced by Streptomyces species as an inhibitor of both carbonic anhydrase and shell formation in the barnacle Balanus amphitrite.

    PubMed

    Fukushima, Mari; Ozaki, Noriaki; Ikeda, Hiroyuki; Furihata, Keiko; Hayakawa, Yoichi; Sakuda, Shohei; Nagasawa, Hiromichi

    2002-03-01

    Carbonic anhydrase is thought to be involved in the process of calcium carbonate deposition in calcified tissues of many organisms. Barnacles form hard calcified shells for protection against predation, and represent a class of marine-fouling animals. In order to inhibit barnacle growth by inhibiting shell formation, we searched for carbonic anhydrase inhibitors from microbial secondary metabolites. A simple assay for assessing carbonic-anhydrase-inhibiting activity was developed. Screening of many microorganisms isolated from soil with this assay resulted in a microbial strain that produced a carbonic anhydrase inhibitor. This strain was identified as Streptomyces eurocidicus mf294. The inhibitor was isolated through 4 purification steps and identified as 2-nitroimidazole on the basis of spectroscopic data. 2-Nitroimidazole inhibited barnacle carbonic anhydrase dose-dependently and complete inhibition was reached at the concentration of 1 x 10(-5) M. 2-Nitroimidazole did not affect settlement or metamorphosis of barnacle larvae, but inhibited shell formation at concentrations higher than 1 x 10(-4) M. These findings strongly support the idea that carbonic anhydrase is involved in calcification.

  12. Opiorphin is a master regulator of the hypoxic response in corporal smooth muscle cells.

    PubMed

    Fu, Shibo; Tar, Moses Tarndie; Melman, Arnold; Davies, Kelvin Paul

    2014-08-01

    Men with sickle cell disease (SCD) risk developing priapism. Recognizing that SCD is a disease of hypoxia, we investigated the effect of hypoxia on gene expression in corporal smooth muscle (CSM) cells. Rat CSM cells in vitro were treated with CoCl2 or low oxygen tension to mimic hypoxia. Hypoxic conditions increased expression of genes previously associated with priapism in animal models. Variable coding sequence a1 (Vcsa1; the rat opiorphin homologue, sialorphin), hypoxia-inducible factor 1a (Hif-1a), and A2B adenosine receptor (a2br) were increased by 10-, 4-, and 6-fold, respectively, by treatment with CoCl2, whereas low oxygen tension caused increases in expression of 3-, 4-, and 1.5-fold, respectively. Sialorphin-treated CSM cells increased expression of Hif-1a and a2br by 4-fold, and vcsa1-siRNA treatment reduced expression by ∼50%. Using a Hif-1a inhibitor, we demonstrated up-regulation of a2br by sialorphin is dependent on Hif-1a, and knockdown of vcsa1 expression with vcsa1-siRNA demonstrated that hypoxic-up-regulation of Hif-1a is dependent on vcsa1. In CSM from a SCD mouse, there was 15-fold up-regulation of opiorphin at a life stage prior to priapism. We conclude that in CSM, opiorphins are master regulators of the hypoxic response. Opiorphin up-regulation in response to SCD-associated hypoxia activates CSM "relaxant" pathways; excessive activation of these pathways results in priapism.

  13. Therapeutic effect of human umbilical cord mesenchymal stem cells on neonatal rat hypoxic-ischemic encephalopathy.

    PubMed

    Zhang, Xinhua; Zhang, Qinfen; Li, Wei; Nie, Dekang; Chen, Weiwei; Xu, Chunxiang; Yi, Xin; Shi, Jinhong; Tian, Meiling; Qin, Jianbing; Jin, Guohua; Tu, Wenjuan

    2014-01-01

    The therapeutic potential of umbilical cord blood mesenchymal stem cells has been studied in several diseases. However, the possibility that human umbilical cord Wharton's jelly-derived mesenchymal stem cells (hUCMSCs) can be used to treat neonatal hypoxic-ischemic encephalopathy (HIE) has not yet been investigated. This study focuses on the potential therapeutic effect of hUCMSC transplantation in a rat model of HIE. Dermal fibroblasts served as cell controls. HIE was induced in neonatal rats aged 7 days. hUCMSCs labeled with Dil were then transplanted into the models 24 hr or 72 hr post-HIE through the peritoneal cavity or the jugular vein. Behavioral testing revealed that hUCMSC transplantation but not the dermal fibroblast improved significantly the locomotor function vs. vehicle controls. Animals receiving cell grafts 24 hr after surgery showed a more significant improvement than at 72 hr. More hUCMSCs homed to the ischemic frontal cortex following intravenous administration than after intraperitoneal injection. Differentiation of engrafted cells into neurons was observed in and around the infarct region. Gliosis in ischemic regions was significantly reduced after hUCMSC transplantation. Administration of ganglioside (GM1) enhanced the behavioral recovery on the base of hUCMSC treatment. These results demonstrate that intravenous transplantation of hUCMSCs at an early stage after HIE can improve the behavior of hypoxic-ischemic rats and decrease gliosis. Ganglioside treatment further enhanced the recovery of neurological function following hUCMSC transplantation.

  14. System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions.

    PubMed

    Kagawa, Yuki; Miyahara, Hirotaka; Ota, Yuri; Tsuneda, Satoshi

    2016-01-01

    Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three-dimensional (3-D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3-D culture conditions, contributing crucial data for an efficient 3-D culture system design.

  15. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells

    PubMed Central

    Kusuma, Sravanti; Zhao, Stephen; Gerecht, Sharon

    2012-01-01

    Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony-forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC-derived ECM is abrogated in response to TGFβ signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O2 tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O2 tension. ECM production from all ECs is found to be regulated by hypoxia-inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.—Kusuma, S., Zhao, S., Gerecht, S. The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells. PMID:22919069

  16. Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

    SciTech Connect

    Ren Hongying; Cai Huiguo; Han Zhongchao; Yang Renchi; Zhao, Qinjun; Cao Ying; Li Jing; Zhou Cixiang; Liao Lianming; Jia Mingyue; Zhao Qian; Chen Guoqiang . E-mail: chengq@shsmu.edu.cn; Zhao, R.C. |. E-mail: chunhuaz@public.tpt.tj.cn

    2006-08-18

    Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O{sub 2}, bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G{sub 2}/S/M phase cells increased evidently under 8% O{sub 2} condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O{sub 2} condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl{sub 2}) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.

  17. Experimental study on the cryopreservation of LLC-PK1 epithelial cells with hypoxic UW solution.

    PubMed

    Wan, Chidan; Wang, Chunyou; Liu, Tao; Wang, Hongbo; Yang, Zhiyong

    2007-08-01

    The effects of oxygen partial pressure on cryopreservation of the cells with organ preservation solution were explored. Hypoxic UW solution was made by purging the UW solution with argon. The pig proximal tubule epithelial cells (LLC-PK1 cells) were cryopreserved in hypoxic UW solution (Ar-UW group) or standard UW solution (UW group) at 4 degrees C for 48 h. Trypan blue staining and LDH detection were performed to evaluate the injury of the cells. The results showed that the oxygen partial pressure in Ar-UW group was significantly declined from 242+/-6 mmHg to 83+/-10 mmHg. After cryopreservation at 4 degrees C for 48 h, LDH leakage rate and Trypan blue-stained rate in Ar-UW group were (11.3+/-3.4)% and (10.5+/-4.7)%, respectively, which were significantly lower than in UW group [(49.5+/-6.9)% and (47.6+/-9.3)% respectively, both P<0.01]. It was concluded that lower oxygen partial pressure of UW solution was more beneficial to the cryopreservation of LLC.

  18. SENP1 desensitizes hypoxic ovarian cancer cells to cisplatin by up-regulating HIF-1α

    PubMed Central

    Ao, Qilin; Su, Wenjing; Guo, Shuang; Cai, Lei; Huang, Lei

    2015-01-01

    Hypoxia-inducible factor 1 alpha (HIF-1α) is closely related to chemoresistance of ovarian cancers. Although it is reported that HIF-1α can be regulated by Sentrin/SUMO-specific protease 1 (SENP1), the effects of SENP1 on HIF-1α is still controversial. In this study, we identified that SENP1 positively regulated the expression of HIF-1α by deSUMOylation and weakened the sensitivity of hypoxic ovarian cancer cells to cisplatin. These results indicate that SENP1 is a positive regulator of HIF-1α and plays a negative role in ovarian cancer chemotherapy. PMID:26548925

  19. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment.

    PubMed

    Li, Xianchao; Hou, Wensheng; Wu, Xiaoying; Jiang, Wei; Chen, Haiyan; Xiao, Nong; Zhou, Ping

    2014-02-01

    Bone marrow mesenchymal stem cell transplantation is an effective treatment for neonatal hypoxic-ischemic brain damage. However, the in vivo transplantation effects are poor and their survival, colonization and differentiation efficiencies are relatively low. Red or near-infrared light from 600-1,000 nm promotes cellular migration and prevents apoptosis. Thus, we hypothesized that the combination of red light with bone marrow mesenchymal stem cell transplantation would be effective for the treatment of hypoxic-ischemic brain damage. In this study, the migration and colonization of cultured bone marrow mesenchymal stem cells on primary neurons after oxygen-glucose deprivation were detected using Transwell assay. The results showed that, after a 40-hour irradiation under red light-emitting diodes at 660 nm and 60 mW/cm(2), an increasing number of green fluorescence-labeled bone marrow mesenchymal stem cells migrated towards hypoxic-ischemic damaged primary neurons. Meanwhile, neonatal rats with hypoxic-ischemic brain damage were given an intraperitoneal injection of 1 × 10(6) bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm(2) for 7 successive days. Shuttle box test results showed that, after phototherapy and bone marrow mesenchymal stem cell transplantation, the active avoidance response rate of hypoxic-ischemic brain damage rats was significantly increased, which was higher than that after bone marrow mesenchymal stem cell transplantation alone. Experimental findings indicate that 660 nm red light emitting diode irradiation promotes the migration of bone marrow mesenchymal stem cells, thereby enhancing the contribution of cell transplantation in the treatment of hypoxic-ischemic brain damage.

  20. Mineralization of bone-related SaOS-2 cells under physiological hypoxic conditions.

    PubMed

    Müller, Werner E G; Schröder, Heinz C; Tolba, Emad; Diehl-Seifert, Bärbel; Wang, Xiaohong

    2016-01-01

    Inorganic polyphosphate (polyP) is a physiological energy-rich polymer with multiple phosphoric anhydride bonds. In cells such as bone-forming osteoblasts, glycolysis is the main pathway generating metabolic energy in the form of ATP. In the present study, we show that, under hypoxic culture conditions, the growth/viability of osteoblast-like SaOS-2 cells is not impaired. The addition of polyP to those cells, administered as amorphous calcium polyP nanoparticles (aCa-polyP-NP; approximate size 100 nm), significantly increased the proliferation of the cells. In the presence of polyP, the cells produce significant levels of lactate, the end product of anaerobic glycolysis. Under those conditions, an eight-fold increase in the steady-state level of the membrane-associated carbonic anhydrase IX is found, as well as a six-fold induction of the hypoxia-inducible factor 1. Consequently, biomineral formation onto the SaOS-2 cells decreases under low oxygen tension. If the polyP nanoparticles are added to the cells, the degree of mineralization is enhanced. These changes had been measured also in human mesenchymal stem cells. The assumption that the bicarbonate, generated by the carbonic anhydrase in the presence of polyP under low oxygen, is deposited as a constituent of the bioseeds formed during initial hydroxyapatite formation is corroborated by the identification of carbon besides of calcium, oxygen and phosphorus in the initial biomineral deposit onto the cells using the sensitive technology of high-resolution energy dispersive spectrometry mapping. Based on these data, we conclude that polyP is required for the supply of metabolic energy during bone mineral formation under physiological, hypoxic conditions, acting as a 'metabolic fuel' for the cells to grow.

  1. Selective toxicity of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide toward hypoxic mammalian cells

    SciTech Connect

    Rauth, A.M.; Mohindra, J.K.

    1981-12-01

    The chemotherapeutic agent 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) is used in the treatment of malignant melanoma where response rates of 15 to 30% have been reported. Some current interest exists in combining DTIC chemotherapy with localized high-dose (800 rads)-per-fraction radiotherapy in the treatment of unresectable metastatic melanoma. The present work investigates the radiosensitizing and chemotherapeutic properties of DTIC in an in vitro system using Chinese hamster ovary or HeLa cells and in vivo, using the KHT transplantable murine tumor. No evidence of a radiosensitizing effect of DTIC was found towards hypoxic or aerobic cells either in vitro or in vivo. In vitro, high drug concentrations (1 mg/ml) were approximately 5 times more effective in killing hypoxic Chinese hamster ovary or HeLa cells than in killing aerobic cells over exposure times of 0 to 12 hr. The degree of toxicity was drug dose and temperature dependent but was not highly dependent on cell number or cell type. In vivo plasma levels of DTIC were measured with high-pressure liquid chromatography after i.p. injection of drug into C3H mice. At the highest drug doses tested, near the 50% lethal dose in mice for DTIC (0.5 mg/g), the drug was toxic to both aerobic and hypoxic tumor cells with some evidence of increased toxicity towards hypoxic cells. The present work suggests that DTIC may be more efficiently activated under hypoxic conditions as compared to aerobic conditions. The increased toxicity of DTIC under hypoxic versus aerobic conditions may prove to be a feature of this drug that can be exploited in its clinical use and in the design of new analogs of DTIC.

  2. Effect of nitroimidazole sensitizers on in vitro glycolytic metabolism of hypoxic squamous cell carcinoma.

    PubMed

    Minn, H; Clavo, A C; Fisher, S J; Wahl, R L

    2000-01-01

    Two nitroimidazole compounds, misonidazole (MISO) and nimorazole (NIMO), were evaluated for their potential to modify uptake of [5,6-3H] 2-fluoro-2-deoxy-D-glucose (3H-FDG) in the human squamous carcinoma cell line UT-SCC-5 exposed to increasing levels of hypoxia. UT-SCC-5 cells were incubated with 0-10 mM of MISO or NIMO under normal or reduced oxygen concentrations of 20%, 1.5%, or 0% with 5% CO2 for 6 h, after which 74 KBq of 3H-FDG was added in media for 1 h. In the presence of normal concentrations of O2, both sensitizers increased 3H-FDG uptake by up to 178% (MISO) or 84% (NIMO) when compared with untreated cells. In anoxia, MISO decreased 3H-FDG uptake to 35% of that of control whereas NIMO-treated cells showed a respective decrease in tracer uptake to 62%. Clonogenic assays clearly indicated that MISO was toxic and NIMO moderately toxic for hypoxic cells, whereas both sensitizers exerted only a very modest effect on survival of fully oxygenated cells. Our findings indicate that nitroimidazole treatment consistently increases 3H-FDG uptake into UT-SCC-5 cells under normal oxygen concentrations. In hypoxia, the observed decrease in tracer uptake is dependent on both the level of ambient oxygen and drug concentration and may reflect both direct toxicity and inhibition of glycolysis. The observations may be useful for further applications of 18F-FDG positron emission tomography (PET) to monitor effects of hypoxic cell radiosensitizers on tumor metabolism in vivo.

  3. Hypoxic tumor-derived microvesicles negatively regulate NK cell function by a mechanism involving TGF-β and miR23a transfer

    PubMed Central

    Berchem, Guy; Noman, Muhammad Zaeem; Bosseler, Manon; Paggetti, Jerome; Baconnais, Sonia; Le cam, Eric; Nanbakhsh, Arash; Moussay, Etienne; Mami-Chouaib, Fathia; Janji, Bassam; Chouaib, Salem

    2016-01-01

    ABSTRACT Tumor-derived microvesicles (TD-MVs) are key mediators which are shed by cancer cells and can sensitize neighboring cells in the tumor microenvironment. TD-MVs are extracellular vesicles composed of exosomes and MVs and promote cancer invasion and metastasis. Intratumoral hypoxia is an integral component of all solid tumors. The relationship between hypoxic tumor-shed MVs and NK-mediated cytotoxicity remains unknown. In this paper, we reported that MVs derived from hypoxic tumor cells qualitatively differ from those derived from normoxic tumor cells. Using multiple tumor models, we showed that hypoxic MVs inhibit more NK cell function as compared to normoxic MVs. Hypoxic TD-MVs package two immunosuppressive factors involved in the impairment of natural killer (NK) cell cytotoxicity against different tumor cells in vitro and in vivo. We showed that following their uptake by NK cells, hypoxic TD-MVs transfer TGF-β1 to NK cells, decreasing the cell surface expression of the activating receptor NKG2D, thereby inhibiting NK cell function. MicroRNA profiling revealed the presence of high levels of miR-210 and miR-23a in hypoxic TD-MVs. We demonstrated that miR-23a in hypoxic TD-MVs operates as an additional immunomosuppressive factor, since it directly targets the expression of CD107a in NK cells. To our knowledge, this is the first study to show that hypoxic tumor cells by secreting MVs can educate NK cells and decrease their antitumor immune response. This study highlights the existence of a novel mechanism of immune suppression mediated by hypoxic TD-MVs and further improves our understanding of the immunosuppressive mechanisms prevailing in the hypoxic tumor microenvironment. PMID:27141372

  4. Important role of PLC-γ1 in hypoxic increase in intracellular calcium in pulmonary arterial smooth muscle cells.

    PubMed

    Yadav, Vishal R; Song, Tengyao; Joseph, Leroy; Mei, Lin; Zheng, Yun-Min; Wang, Yong-Xiao

    2013-02-01

    An increase in intracellular calcium concentration ([Ca(2+)](i)) in pulmonary arterial smooth muscle cells (PASMCs) induces hypoxic cellular responses in the lungs; however, the underlying molecular mechanisms remain incompletely understood. We report, for the first time, that acute hypoxia significantly enhances phospholipase C (PLC) activity in mouse resistance pulmonary arteries (PAs), but not in mesenteric arteries. Western blot analysis and immunofluorescence staining reveal the expression of PLC-γ1 protein in PAs and PASMCs, respectively. The activity of PLC-γ1 is also augmented in PASMCs following hypoxia. Lentiviral shRNA-mediated gene knockdown of mitochondrial complex III Rieske iron-sulfur protein (RISP) to inhibit reactive oxygen species (ROS) production prevents hypoxia from increasing PLC-γ1 activity in PASMCs. Myxothiazol, a mitochondrial complex III inhibitor, reduces the hypoxic response as well. The PLC inhibitor U73122, but not its inactive analog U73433, attenuates the hypoxic vasoconstriction in PAs and hypoxic increase in [Ca(2+)](i) in PASMCs. PLC-γ1 knockdown suppresses its protein expression and the hypoxic increase in [Ca(2+)](i). Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP(3)) production, which is blocked by U73122. The IP(3) receptor (IP(3)R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca(2+)](i). PLC-γ1 knockdown or U73122 reduces H(2)O(2)-induced increase in [Ca(2+)](i) in PASMCs and contraction in PAs. 2-APB and xestospongin-C produce similar inhibitory effects. In conclusion, our findings provide novel evidence that hypoxia activates PLC-γ1 by increasing RISP-dependent mitochondrial ROS production in the complex III, which causes IP(3) production, IP(3)R opening, and Ca(2+) release, playing an important role in hypoxic Ca(2+) and contractile responses in PASMCs.

  5. Polysialic acid sustains cancer cell survival and migratory capacity in a hypoxic environment

    PubMed Central

    Elkashef, Sara M.; Allison, Simon J.; Sadiq, Maria; Basheer, Haneen A.; Ribeiro Morais, Goreti; Loadman, Paul M.; Pors, Klaus; Falconer, Robert A.

    2016-01-01

    Polysialic acid (polySia) is a unique carbohydrate polymer expressed on the surface of NCAM (neuronal cell adhesion molecule) in a number of cancers where it modulates cell-cell and cell-matrix adhesion, migration, invasion and metastasis and is strongly associated with poor clinical prognosis. We have carried out the first investigation into the effect of polySia expression on the behaviour of cancer cells in hypoxia, a key source of chemoresistance in tumours. The role of polysialylation and associated tumour cell migration and cell adhesion were studied in hypoxia, along with effects on cell survival and the potential role of HIF-1. Our findings provide the first evidence that polySia expression sustains migratory capacity and is associated with tumour cell survival in hypoxia. Initial mechanistic studies indicate a potential role for HIF-1 in sustaining polySia-mediated migratory capacity, but not cell survival. These data add to the growing body of evidence pointing to a crucial role for the polysialyltransferases (polySTs) in neuroendocrine tumour progression and provide the first evidence to suggest that polySia is associated with an aggressive phenotype in tumour hypoxia. These results have significant potential implications for polyST inhibition as an anti-metastatic therapeutic strategy and for targeting hypoxic cancer cells. PMID:27611649

  6. Relaxing the formation of hypoxic bottom water with sediment microbial fuel cells.

    PubMed

    Touch, Narong; Hibino, Tadashi; Morimoto, Yuki; Kinjo, Nobutaka

    2017-02-08

    The method of improving bottom water environment using industrial wastes to suppress diffusion substances from bottom sediment has recently captured the attention of many researchers. In this study, wastewater discharge-derived sediment was used to examine an alternative approach involving the use of sediment microbial fuel cells (SMFCs) in relaxing the formation of hypoxic bottom water, and removing reduced substances from sediment. Concentrations of dissolved oxygen (DO) and other ions were measured in overlying water and sediment pore water with and without the application of SMFCs. The results suggest that SMFCs can markedly reduce hydrogen sulfide and manganese ion concentrations in overlying water, and decrease the depletions of redox potential and DO concentration. In addition, SMFCs can dissolve ferric compounds in the sediment and thereby release the ferric ion available to fix phosphate in the sediment. Our results indicate that SMFCs can be used as an alternative method to relax the formation of hypoxic bottom water and to remove reduced substances from the sediment, thus improving the quality of both water and sediment environments.

  7. Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles

    PubMed Central

    Agrawal, Rahul; Dale, Tina P.; Al-Zubaidi, Mohammed A.; Benny Malgulwar, Prit; Forsyth, Nicholas R.; Kulshreshtha, Ritu

    2016-01-01

    MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology. PMID:27783707

  8. Influence of the hypoxic cell sensitizer misonidazole on the proliferation of well-oxygenated cells in vitro during prolonged exposure.

    PubMed Central

    Deys, B. F.; Stap, J.

    1979-01-01

    Analysis of time-lapse cinematographic film permitted the construction of pedigrees from 88 well oxygenated cells of a mouse osteosarcoma (MOS). These cells have been chronically treated with various concentrations of the hypoxic cell sensitizer misonidazole (MIS) over periods of up to 96 h. At concentrations of 0.5 and 7 mM there is a 2--3 h increase in cell-cycle time. Concentrations of 2 mM show an intermitotic time delay of 7.6--10.3 h. At 4 mM cells divided only once. With increasing drug concentration there was an increase in the number of abnormal mitoses. These results were compared with cloning efficiency (PE) experiments. PE at 0.5 mM is 80%, at 1 mM 40 and at 2 mM is reduced to 4%. Cells treated with 2mM MIS over a period of 28.6 h resume their normal cycle when the drug is washed from the culture. This may indicate that DNA is not a major target for MIS. It is concluded that this hypoxic cell sensitizer is also toxic for MOS cells in well oxygenated conditions. PMID:292453

  9. Preliminary research on 1-(4-bromo-2-nitroimidazol-1-yl)-3-[(18)F]fluoropropan-2-ol as a novel brain hypoxia PET tracer in a rodent model of stroke.

    PubMed

    Nieto, Elena; Delgado, Mercedes; Sobrado, Mónica; de Ceballos, María L; Alajarín, Ramón; García-García, Luis; Kelly, James; Lizasoain, Ignacio; Pozo, Miguel A; Álvarez-Builla, Julio

    2015-08-28

    The synthesis of the new radiotracer precursor 4-Br-NITTP and the radiolabeling of the new tracer 1-(4-bromo-2-nitroimidazol-1-yl)-3-[(18)F]fluoropropan-2-ol (4-Br-[(18)F]FMISO) is reported. The cyclic voltammetry behaviour, neuronal cell toxicity, transport through the brain endothelial cell monolayer, in vivo PET imaging and preliminary calculations of the tracer uptake for a rodent model of stroke were studied for the new compound and the results were compared to those obtained with [(18)F]FMISO, the current gold standard PET hypoxia tracer. The new PET brain hypoxia tracer is more easily reduced, has higher CLogP than [(18)F]FMISO and it diffuses more rapidly through brain endothelial cells. The new compound is non-toxic to neuronal cells and it allows the in vivo mapping of stroke in mice with higher sensitivity. 4-Br-[(18)F]FMISO is a good candidate for further development in ischemic stroke.

  10. Hepatocytes Determine the Hypoxic Microenvironment and Radiosensitivity of Colorectal Cancer Cells Through Production of Nitric Oxide That Targets Mitochondrial Respiration

    SciTech Connect

    Jiang, Heng; Verovski, Valeri N.; Leonard, Wim; Law, Ka Lun; Vermeersch, Marieke; Storme, Guy; Van den Berge, Dirk; Gevaert, Thierry; Sermeus, Alexandra; De Ridder, Mark

    2013-03-01

    Purpose: To determine whether host hepatocytes may reverse hypoxic radioresistance through nitric oxide (NO)-induced oxygen sparing, in a model relevant to colorectal cancer (CRC) liver metastases. Methods and Materials: Hepatocytes and a panel of CRC cells were incubated in a tissue-mimetic coculture system with diffusion-limited oxygenation, and oxygen levels were monitored by an oxygen-sensing fluorescence probe. To activate endogenous NO production, cocultures were exposed to a cytokine mixture, and the expression of inducible nitric oxide synthase was analyzed by reverse transcription–polymerase chain reaction, Western blotting, and NO/nitrite production. The mitochondrial targets of NO were examined by enzymatic activity. To assess hypoxic radioresponse, cocultures were irradiated and reseeded for colonies. Results: Resting hepatocytes consumed 10-40 times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116, and SW480 CRC cells, and thus seemed to be the major effectors of hypoxic conditioning. As a result, hepatocytes caused uniform radioprotection of tumor cells at a 1:1 ratio. Conversely, NO-producing hepatocytes radiosensitized all CRC cell lines more than 1.5-fold, similar to the effect of selective mitochondrial inhibitors. The radiosensitizing effect was associated with a respiratory self-arrest of hepatocytes at the level of aconitase and complex II, which resulted in profound reoxygenation of tumor cells through oxygen sparing. Nitric oxide–producing hepatocytes were at least 10 times more active than NO-producing macrophages to reverse hypoxia-induced radioresistance. Conclusions: Hepatocytes were the major determinants of the hypoxic microenvironment and radioresponse of CRC cells in our model of metabolic hypoxia. We provide evidence that reoxygenation and radiosensitization of hypoxic CRC cells can be achieved through oxygen sparing induced by endogenous NO production in host hepatocytes.

  11. Lung tumorigenic response of strain A mice exposed to hypoxic cell sensitizers alone and in combination with gamma-radiation

    SciTech Connect

    Mian, T.A.; Theiss, J.C.; Grdina, D.J.

    1983-01-01

    The influence of metronidazole, misonidazole, and desmethylmisonidazole on the induction of lung adenomas in the strain A mouse was examined. Two dose levels of the hypoxic cell sensitizers, 0.2 and 0.6 mg/g, were used either alone or in combination with 900 rads of gamma-radiation in a fractionated dose schedule of twice a week for 3 weeks. In the groups of mice which received hypoxic cell sensitizers only, the prevalence and the mean number of lung tumors per mouse were somewhat increased (p less than 0.10) in the group receiving the higher dose (0.6 mg/g) of misonidazole but was not significantly different from results for the control animals in the other two sensitizer groups. The combination of hypoxic cell sensitizer and radiation did not show any significant enhancement of lung tumor response when compared with the group which received radiation only. The dose of radiation used in this study significantly enhanced lung tumor formation in mice when compared with that in the control group. Thus, under the experimental exposure conditions used in this investigation, which were somewhat similar to the exposure conditions occurring in clinical treatment, each of the hypoxic cell sensitizers tested failed to sensitize significantly the mice to the carcinogenic effects of gamma-radiation.

  12. A redox-silent analogue of tocotrienol inhibits hypoxic adaptation of lung cancer cells.

    PubMed

    Kashiwagi, Korehito; Harada, Kayono; Yano, Yoshihisa; Kumadaki, Itsumaro; Hagiwara, Kiyokazu; Takebayashi, Jun; Kido, Wakiko; Virgona, Nantiga; Yano, Tomohiro

    2008-01-25

    We have previously reported that a redox-silent analogue of alpha-tocotrienol (T3), 6-O-carboxypropyl-alpha-tocotrienol (T3E) shows more potential anti-carcinogenic property than T3 in a lung cancer cell (A549 cell). However, the mechanisms by which T3E exerts its potential anti-carcinogenic effect is still unclear. As tumor malignancy is associated with hypoxia adaptation, in this study, we examined whether T3E could suppress survival and invasion in A549 cells under hypoxia. Hypoxia treatment drastically-induced activation of the protein tyrosine kinase, Src, and its regulated signaling required for hypoxia adaptation of A549 tumor cells. The survival and invasion capacity of the tumor cells under hypoxia was suppressed by T3E via the inactivation of Src. More specifically, T3E-dependent inhibition of Src-induced Akt activation contributed to suppression of cell survival under hypoxia, and the reduction of fibrinolytic factors such as plasminogen activator-1(PAI-1) via the decrease of hypoxia-inducible factor-2alpha by T3E led to inhibition of hypoxic invasion. Overall these results suggest that T3E suppresses hypoxia adaptation of A549 cells by the inhibition in hypoxia-induced activation of Src signaling.

  13. Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells

    PubMed Central

    Morgan, J. Brian; Liu, Yang; Coothankandaswamy, Veena; Mahdi, Fakhri; Jekabsons, Mika B.; Gerwick, William H.; Valeriote, Frederick A.; Zhou, Yu-Dong; Nagle, Dale G.

    2015-01-01

    The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells. PMID:25803180

  14. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    NASA Astrophysics Data System (ADS)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  15. Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology.

    PubMed

    Moreno, Marta; Fernández, Virginia; Monllau, Josep M; Borrell, Víctor; Lerin, Carles; de la Iglesia, Núria

    2015-08-11

    Neural stem cells (NSCs) reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs) that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA) of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT) and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state.

  16. Evaluation of the antineoplastic activity of gallic acid in oral squamous cell carcinoma under hypoxic conditions.

    PubMed

    Guimaraes, Talita A; Farias, Lucyana C; Fraga, Carlos A; Feltenberger, John D; Melo, Geraldo A; Coletta, Ricardo D; Souza Santos, Sergio H; de Paula, Alfredo M B; Guimaraes, Andre L

    2016-06-01

    The purpose of the current study was to develop and test a theoretical model that could explain the mechanism of action of gallic acid (GA) in the oral squamous cell carcinoma context for the first time. The theoretical model was developed using bioinformatics and interaction network analysis to evaluate the effect of GA on oral squamous cell carcinoma. In a second step to confirm theoretical results, migration, invasion, proliferation, and gene expression (Col1A1, E-cadherin, HIF-1α, and caspase-3) were performed under normoxic and hypoxic conditions. Our study indicated that treatment with GA resulted in the inhibition of cell proliferation, migration, and invasion in neoplastic cells. Observation of the molecular mechanism showed that GA upregulates E-cadherin expression and downregulates Col1A1 and HIF-1α expression, suggesting that GA might be a potential anticancer compound. In conclusion, the present study demonstrated that GA significantly reduces cell proliferation, invasion, and migration by increasing E-cadherin and repressing Col1A1.

  17. Human bone marrow stem cells cultured under hypoxic conditions present altered characteristics and enhanced in vivo tissue regeneration.

    PubMed

    Lee, Jung-Seok; Park, Jung-Chul; Kim, Tae-Wan; Jung, Byung-Joo; Lee, Youngseok; Shim, Eun-Kyung; Park, Soyon; Choi, Eun-Young; Cho, Kyoo-Sung; Kim, Chang-Sung

    2015-09-01

    Human bone marrow mesenchymal stem cells (hBMSCs) were isolated from bone marrow of the vertebral body. The hBMSCs were cultured under either hypoxic (1% O2) or normoxic (21% O2; control) conditions and the characteristics as mesenchymal stem cells were compared. Results revealed that hypoxia reduced proliferative potential and colony-forming efficiency of hBMSCs, and significantly enhanced osteogenic and chondrogenic differentiation. The hBMSCs enhanced the regenerative potential of bone in vivo. In vitro synthesis of soluble and insoluble collagen was significantly increased in the hypoxic condition. In vivo collagen tissue regeneration was also enhanced under the hypoxic condition, with concomitant increased expressions of various subtypes of collagen and lysyl-oxidase family mRNA. MicroRNA assays revealed that miR-155-5p, which negatively regulates HIF-1α, was significantly highly expressed. These observations demonstrate that hBMSCs obtained from human vertebrae exhibit altered characteristics under hypoxic conditions, and each factor contributing to hBMSC-mediated tissue healing should be evaluated with the goal of allowing their clinical application.

  18. SIRT1 is required for mitochondrial biogenesis reprogramming in hypoxic human pulmonary arteriolar smooth muscle cells.

    PubMed

    Li, Pengyun; Liu, Yan; Burns, Nana; Zhao, Ke-Seng; Song, Rui

    2017-03-22

    Although recent studies have reported that mitochondria are putative oxygen sensors underlying hypoxic pulmonary vasoconstriction, little is known concerning the sirtuin 1 (SIRT1)-mediated mitochondrial biogenesis regulatory program in pulmonary arteriolar smooth muscle cells (PASMCs) during hypoxia/reoxygenation (H/R). We investigated the epigenetic regulatory mechanism of mitochondrial biogenesis and function in human PASMCs during H/R. Human PASMCs were exposed to hypoxia of 24-48 h and reoxygenation of 24-48 h. The expression of SIRT1 was reduced in a time-dependent manner. Mitochondrial transcription factor A (TFAM) expression was increased during hypoxia and decreased during reoxygenation, while the release of TFAM was increased in a time-dependent manner. Lentiviral overexpression of SIRT1 preserved SIRT3 deacetylase activity in human PASMCs exposed to H/R. Knockdown of PGC-1α suppressed the effect of SIRT1 on SIRT3 activity. Knockdown of SIRT3 abrogated SIRT1-mediated deacetylation of cyclophilin D (CyPD). Notably, knockdown of SIRT3 or PGC-1α suppressed the incremental effect of SIRT1 on mitochondrial TFAM, mitochondrial DNA (mtDNA) content and cellular ATP levels. Importantly, polydatin restored SIRT1 levels in human PASMCs exposed to H/R. Knockdown of SIRT1 suppressed the effect of polydatin on mitochondrial TFAM, mtDNA content and cellular ATP levels. In conclusion, SIRT1 expression is decreased in human PASMCs during H/R. TFAM expression in mitochondria is reduced and the release of TFAM is increased by H/R. PGC-1α/SIRT3/CyPD mediates the protective effect of SIRT1 on expression and release of TFAM and mitochondrial biogenesis and function. Polydatin improves mitochondrial biogenesis and function by enhancing SIRT1 expression in hypoxic human PASMCs.

  19. Hypoxic culture conditions induce increased metabolic rate and collagen gene expression in ACL-derived cells.

    PubMed

    Kowalski, Tomasz J; Leong, Natalie L; Dar, Ayelet; Wu, Ling; Kabir, Nima; Khan, Adam Z; Eliasberg, Claire D; Pedron, Andrew; Karayan, Ashant; Lee, Siyoung; Di Pauli von Treuheim, Theodor; Jiacheng, Jin; Wu, Ben M; Evseenko, Denis; McAllister, David R; Petrigliano, Frank A

    2016-06-01

    There has been substantial effort directed toward the application of bone marrow and adipose-derived mesenchymal stromal cells (MSCs) in the regeneration of musculoskeletal tissue. Recently, resident tissue-specific stem cells have been described in a variety of mesenchymal structures including ligament, tendon, muscle, cartilage, and bone. In the current study, we systematically characterize three novel anterior cruciate ligament (ACL)-derived cell populations with the potential for ligament regeneration: ligament-forming fibroblasts (LFF: CD146(neg) , CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ), ligament perivascular cells (LPC: CD146(pos) CD34(neg) CD44(pos) , CD31(neg) , CD45(neg) ) and ligament interstitial cells (LIC: CD34(pos) CD146(neg) , CD44(pos) , CD31(neg) , CD45(neg) )-and describe their proliferative and differentiation potential, collagen gene expression and metabolism in both normoxic and hypoxic environments, and their trophic potential in vitro. All three groups of cells (LIC, LPC, and LFF) isolated from adult human ACL exhibited progenitor cell characteristics with regard to proliferation and differentiation potential in vitro. Culture in low oxygen tension enhanced the collagen I and III gene expression in LICs (by 2.8- and 3.3-fold, respectively) and LFFs (by 3- and 3.5-fold, respectively) and increased oxygen consumption rate and extracellular acidification rate in LICs (by 4- and 3.5-fold, respectively), LFFs (by 5.5- and 3-fold, respectively), LPCs (by 10- and 4.5-fold, respectively) as compared to normal oxygen concentration. In summary, this study demonstrates for the first time the presence of three novel progenitor cell populations in the adult ACL that demonstrate robust proliferative and matrix synthetic capacity; these cells may play a role in local ligament regeneration, and consequently represent a potential cell source for ligament engineering applications. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc

  20. Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways.

    PubMed

    Hamasu, T; Inanami, O; Tsujitani, M; Yokoyama, K; Takahashi, E; Kashiwakura, I; Kuwabara, M

    2005-05-01

    To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28-33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18-20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.

  1. Collagen density regulates xenobiotic and hypoxic response of mammary epithelial cells.

    PubMed

    Curran, Colleen S; Carrillo, Esteban R; Ponik, Suzanne M; Keely, Patricia J

    2015-01-01

    Breast density, where collagen I is the dominant component, is a significant breast cancer risk factor. Cell surface integrins interact with collagen, activate focal adhesion kinase (FAK), and downstream cell signals associated with xenobiotics (AhR, ARNT) and hypoxia (HIF-1α, ARNT). We examined if mammary cells cultured in high density (HD) or low density (LD) collagen gels affected xenobiotic or hypoxic responses. ARNT production was significantly reduced by HD culture and in response to a FAK inhibitor. Consistent with a decrease in ARNT, AhR and HIF-1α reporter activation and VEGF production was lower in HD compared to LD. However, P450 production was enhanced in HD and induced by AhR and HIF-1α agonists, possibly in response to increased NF-κB activaton. Thus, collagen density differentially regulates downstream cell signals of AhR and HIF-1α by modulating the activity of FAK, the release of NF-κB transcriptional factors, and the levels of ARNT.

  2. Collagen density regulates xenobiotic and hypoxic response of mammary epithelial cells

    PubMed Central

    Curran, Colleen S.; Carrillo, Esteban R.; Ponik, Suzanne M.; Keely, Patricia J.

    2014-01-01

    Breast density, where collagen I is the dominant component, is a significant breast cancer risk factor. Cell surface integrins interact with collagen, activate focal adhesion kinase (FAK), and downstream cell signals associated with xenobiotics (AhR, ARNT) and hypoxia (HIF-1α, ARNT). We examined if mammary cells cultured in high density (HD) or low density (LD) collagen gels affected xenobiotic or hypoxic responses. ARNT production was significantly reduced by HD culture and in response to a FAK inhibitor. Consistent with a decrease in ARNT, AhR and HIF-1α reporter activation and VEGF production was lower in HD compared to LD. However, P450 production was enhanced in HD and induced by AhR and HIF-1α agonists, possibly in response to increased NF-kB activaton. Thus, collagen density differentially regulates downstream cell signals of AhR and HIF-1α by modulating the activity of FAK, the release of NF-kB transcriptional factors, and the levels of ARNT. PMID:25481308

  3. Hypoxia-induced angiotensin II by the lactate-chymase-dependent mechanism mediates radioresistance of hypoxic tumor cells

    PubMed Central

    Xie, Guozhu; Liu, Ying; Yao, Qiwei; Zheng, Rong; Zhang, Lanfang; Lin, Jie; Guo, Zhaoze; Du, Shasha; Ren, Chen; Yuan, Quan; Yuan, Yawei

    2017-01-01

    The renin-angiotensin system (RAS) is a principal determinant of arterial blood pressure and fluid and electrolyte balance. RAS component dysregulation was recently found in some malignancies and correlated with poor patient outcomes. However, the exact mechanism of local RAS activation in tumors is still unclear. Here, we find that the local angiotensin II predominantly exists in the hypoxic regions of tumor formed by nasopharyngeal carcinoma CNE2 cells and breast cancer MDA-MB-231 cells, where these tumor cells autocrinely produce angiotensin II by a chymase-dependent rather than an angiotensin converting enzyme-dependent mechanism. We further demonstrate in nasopharyngeal carcinoma CNE2 and 5–8F cells that this chymase-dependent effect is mediated by increased levels of lactate, a by-product of glycolytic metabolism. Finally, we show that the enhanced angiotensin II plays an important role in the intracellular accumulation of HIF-1α of hypoxic nasopharyngeal carcinoma cells and mediates the radiation-resistant phenotype of these nasopharyngeal carcinoma cells. Thus, our findings reveal the critical role of hypoxia in producing local angiotensin II by a lactate-chymase-dependent mechanism and highlight the importance of local angiotensin II in regulating radioresistance of hypoxic tumor cells. PMID:28205588

  4. Mild hypothermia combined with neural stem cell transplantation for hypoxic-ischemic encephalopathy: neuroprotective effects of combined therapy

    PubMed Central

    Wang, Lin; Jiang, Feng; Li, Qifeng; He, Xiaoguang; Ma, Jie

    2014-01-01

    Neural stem cell transplantation is a useful treatment for ischemic stroke, but apoptosis often occurs in the hypoxic-ischemic environment of the brain after cell transplantation. In this study, we determined if mild hypothermia (27–28°C) can increase the survival rate of neural stem cells (1.0 × 105/μL) transplanted into neonatal mice with hypoxic-ischemic encephalopathy. Long-term effects on neurological functioning of the mice were also examined. After mild hypothermia combined with neural stem cell transplantation, we observed decreased expression levels of inflammatory factor nuclear factor-kappa B and apoptotic factor caspase-3, reduced cerebral infarct volumes, increased survival rate of transplanted cells, and marked improvements in neurological function. Thus, the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation are superior to those of monotherapy. Moreover, our findings suggest that the neuroprotective effects of mild hypothermia combined with neural stem cell transplantation on hypoxic-ischemic encephalopathy are achieved by anti-inflammatory and anti-apoptotic mechanisms. PMID:25422635

  5. Stromal cell-derived factor-1-directed bone marrow mesenchymal stem cell migration in response to inflammatory and/or hypoxic stimuli

    PubMed Central

    Yu, Yang; Wu, Rui-Xin; Gao, Li-Na; Xia, Yu; Tang, Hao-Ning; Chen, Fa-Ming

    2016-01-01

    ABSTRACT Directing cell trafficking toward a target site of interest is critical for advancing stem cell therapy in clinical theranostic applications. In this study, we investigated the effects of inflammatory and/or hypoxic stimuli on the migration of bone marrow mesenchymal stem cells (BMMSCs) during in vitro culture and after in vivo implantation. Using tablet scratch experiments and observations from a transwell system, we found that both inflammatory and hypoxic stimuli significantly enhanced cell migration. However, the combination of inflammatory and hypoxic stimuli did not result in a synergistic effect. The presence of stromal cell-derived factor-1 (SDF-1) significantly enhanced cell migration irrespective of the incubation conditions, and these positive effects could be blocked by treatment with AMD3100. Based on a time course experiment, we found that preconditioning cells with either inflammatory or hypoxic stimuli for 24 h or with both stimuli for 12 h led to high levels of chemokine receptor type 4 (CXCR4) expression. In vivo studies further demonstrated that pretreatment of BMMSCs with inflammatory and/or hypoxic stimuli resulted in an increased number of systemically injected cells migrating toward skin injuries, and local SDF-1 administration significantly increased cell migration. These findings suggest that in vitro control of either inflammatory or hypoxic stimuli has significant potential to enhance SDF-1-directed BMMSC migration via the upregulation of CXCR4 expression. Although combining the stimuli did not necessarily lead to a synergistic effect, the potential to reduce the dose and time required for cell preconditioning indicates that combinations of various strategies warrant further exploration. PMID:26745021

  6. Ultrasound mediated delivery of oxygen and LLL12 loaded stimuli responsive microdroplets for the treatment of hypoxic cancer cells.

    PubMed

    Xu, Jinshun; Yuan, Shuai; Tian, Jilai; Martin, Kyle A; Song, Jinhua; Li, Chenglong; Wang, Zhigang; Lin, Jiayuh; Si, Ting; Xu, Ronald X

    2017-03-21

    LLL12 exhibits high specificity for inhibiting STAT3 phosphorylation and dimerization, and inducing apoptosis to constitutively activated STAT3 cancer cells without cytotoxicity to normal cells with dormant STAT3. However, clinical deployment of LLL12 in cancer treatment is hindered by its low bioavailability and hypoxia-induced resistance. To overcome these limitations, we encapsulate both oxygen and LLL12 in stimuli responsive microdroplets (SRMs) by a gas-driven coaxial flow focusing (CFF) process for ultrasound mediated treatment of hypoxic cancer cells. Our benchtop experiments demonstrate that the CFF process is able to produce SRMs with uniform size distribution, large oxygen loading capacity, high LLL12 encapsulation efficiency, well protection of bioactivity, and steadily long shelf time. The in vitro therapeutic studies in pancreatic cancer cells (PANC-1 and CAPAN-1) demonstrate the immediate release of oxygen and LLL12 in exposure to therapeutic ultrasound pulses as well as the improved anticancer effects under hypoxic conditions. The findings suggest that the proposed oxygen and LLL12 loaded SRMs provide a promising drug delivery strategy for more effective treatment of hypoxic cancer cells.

  7. Ultrasound mediated delivery of oxygen and LLL12 loaded stimuli responsive microdroplets for the treatment of hypoxic cancer cells

    PubMed Central

    Xu, Jinshun; Yuan, Shuai; Tian, Jilai; Martin, Kyle A.; Song, Jinhua; Li, Chenglong; Wang, Zhigang; Lin, Jiayuh; Si, Ting; Xu, Ronald X.

    2017-01-01

    LLL12 exhibits high specificity for inhibiting STAT3 phosphorylation and dimerization, and inducing apoptosis to constitutively activated STAT3 cancer cells without cytotoxicity to normal cells with dormant STAT3. However, clinical deployment of LLL12 in cancer treatment is hindered by its low bioavailability and hypoxia-induced resistance. To overcome these limitations, we encapsulate both oxygen and LLL12 in stimuli responsive microdroplets (SRMs) by a gas-driven coaxial flow focusing (CFF) process for ultrasound mediated treatment of hypoxic cancer cells. Our benchtop experiments demonstrate that the CFF process is able to produce SRMs with uniform size distribution, large oxygen loading capacity, high LLL12 encapsulation efficiency, well protection of bioactivity, and steadily long shelf time. The in vitro therapeutic studies in pancreatic cancer cells (PANC-1 and CAPAN-1) demonstrate the immediate release of oxygen and LLL12 in exposure to therapeutic ultrasound pulses as well as the improved anticancer effects under hypoxic conditions. The findings suggest that the proposed oxygen and LLL12 loaded SRMs provide a promising drug delivery strategy for more effective treatment of hypoxic cancer cells. PMID:28322306

  8. Hypoxic preconditioning enhances the therapeutic potential of the secretome from cultured human mesenchymal stem cells in experimental traumatic brain injury.

    PubMed

    Chang, Ching-Ping; Chio, Chung-Ching; Cheong, Chong-Un; Chao, Chien-Ming; Cheng, Bor-Chieh; Lin, Mao-Tsun

    2013-02-01

    Bone-marrow-derived human MSCs (mesenchymal stem cells) support repair when administered to animals with TBI (traumatic brain injury) in large part through secreted trophic factors. We directly tested the ability of the culture medium (or secretome) collected from human MSCs under normoxic or hypoxic conditions to protect neurons in a rat model of TBI. Concentrated conditioned medium from cultured human MSCs or control medium was infused through the tail vein of rats subjected to TBI. We have demonstrated that MSCs cultured in hypoxia were superior to those cultured in normoxia in inducing expression of both HGF (hepatocyte growth factor) and VEGF (vascular endothelial growth factor) in the cultured medium. We showed further that rats treated with the secretome from both normoxic- and hypoxic-preconditioned MSCs performed significantly better than the controls in both motor and cognitive functional test. Subsequent post-mortem evaluation of brain damage at the 4-day time point confirmed that both normoxic- and hypoxic-preconditioned MSC secretome-treated rats had significantly greater numbers of newly forming neurons, but significantly less than the controls in brain damaged volume and apoptosis. The TBI rats treated with hypoxic-preconditioned MSC secretome performed significantly better in both motor and cognitive function tests and neurogenesis, and had significantly less brain damage than the TBI rats treated with the normoxic-preconditioned MSC secretome. Collectively, these findings suggest that MSCs secrete bioactive factors, including HGF and VEGF, that stimulate neurogenesis and improve outcomes of TBI in a rat model. Hypoxic preconditioning enhances the secretion of these bioactive factors from the MSCs and the therapeutic potential of the cultured MSC secretome in experimental TBI.

  9. Respiration of mammalian cells at low concentrations of oxygen: I. Effect of hypoxic-cell radiosensitizing drugs.

    PubMed Central

    Koch, C. J.; Biaglow, J. E.

    1978-01-01

    Drugs which sensitize hypoxic mammalian cells to radiation damage in vitro can also affect the cellular respiration rate. This phenomenon was studied in detail to determine whether the changes in oxygen consumption occur at low oxygen concentrations and under optimal nutritional conditions. We have found that cells in tissue culture can undergo adaptive changes in respiration (electron flow) which make them insensitive to the effects of radiosensitizing drugs and even respiration uncouplers such as dinitrophenol, and the inhibitors rotenone and cyanide. At low cell densities, where nutrient depletion in the medium would be negligible, the drugs have reduced effects, particularly at low oxygen concentrations (below 40 mmHg oxygen partial pressure). Parallel cytotoxicity and growht inhibition studies indicate that most drugs are unlikely to have substantial effect on respiration at non-cytotoxic levels. PMID:277219

  10. Thiopental Inhibits Global Protein Synthesis by Repression of Eukaryotic Elongation Factor 2 and Protects from Hypoxic Neuronal Cell Death

    PubMed Central

    Schwer, Christian I.; Lehane, Cornelius; Guelzow, Timo; Zenker, Simone; Strosing, Karl M.; Spassov, Sashko; Erxleben, Anika; Heimrich, Bernd; Buerkle, Hartmut; Humar, Matjaz

    2013-01-01

    Ischemic and traumatic brain injury is associated with increased risk for death and disability. The inhibition of penumbral tissue damage has been recognized as a target for therapeutic intervention, because cellular injury evolves progressively upon ATP-depletion and loss of ion homeostasis. In patients, thiopental is used to treat refractory intracranial hypertension by reducing intracranial pressure and cerebral metabolic demands; however, therapeutic benefits of thiopental-treatment are controversially discussed. In the present study we identified fundamental neuroprotective molecular mechanisms mediated by thiopental. Here we show that thiopental inhibits global protein synthesis, which preserves the intracellular energy metabolite content in oxygen-deprived human neuronal SK-N-SH cells or primary mouse cortical neurons and thus ameliorates hypoxic cell damage. Sensitivity to hypoxic damage was restored by pharmacologic repression of eukaryotic elongation factor 2 kinase. Translational inhibition was mediated by calcium influx, activation of the AMP-activated protein kinase, and inhibitory phosphorylation of eukaryotic elongation factor 2. Our results explain the reduction of cerebral metabolic demands during thiopental treatment. Cycloheximide also protected neurons from hypoxic cell death, indicating that translational inhibitors may generally reduce secondary brain injury. In conclusion our study demonstrates that therapeutic inhibition of global protein synthesis protects neurons from hypoxic damage by preserving energy balance in oxygen-deprived cells. Molecular evidence for thiopental-mediated neuroprotection favours a positive clinical evaluation of barbiturate treatment. The chemical structure of thiopental could represent a pharmacologically relevant scaffold for the development of new organ-protective compounds to ameliorate tissue damage when oxygen availability is limited. PMID:24167567

  11. Investigation of the modifying effects of vitamin A and hypoxic cell sensitizers in radiation carcinogenesis in mice

    SciTech Connect

    Mian, T.A.

    1982-01-01

    The effect of vitamin A (retinyl acetate) and three hypoxic cell sensitizers (metronidazole, misonidazole and desmethylmisonidazole) on lung tumor development in strain A mice exposed to radiation was assessed. In experiments involving vitamin A, two groups of mice were fed a low vitamin A diet (< 100 IU/100g diet) while the two other groups were fed a high vitamin A diet (800 IU/100 g diet). After two weeks one group maintained on the high vitamin A diet and one group maintained on the low vitamin A diet were given an acute dose of 500 rad of gamma radiation to the thoracic region. Mice were killed, their lungs were removed and the number of surface adenomas were counted. There was a significant increase in the number of mice bearing lung tumors and the mean number of lung tumors per mouse in the irradiated group maintained on the high vitamin A diet at 40 weeks post irradiation as compared to the irradiated group maintained on a low vitamin A diet. In the other experiment two dose levels of the hypoxic cell sensitizers, 0.2 mg/g and 0.6 mg/g, were used either alone or in combination with 900 rad of gamma radiation in a fractionated dose schedule of twice a week for three weeks. In the groups of mice which received hypoxic cell sensitizers only, the prevalence and the mean number of lung tumors per mouse were somewhat increased in the higher dose group (0.6 mg/g) of misonidazole but was not significantly different from the control animals in the other two sensitizer groups. The combination of hypoxic cell sensitizer and radiation did not show any significant enhancement of lung tumor response when compared with the group which received radiation only. The dose of radiation used in this study significantly enhanced lung tumor formation in mice when compared with the control group.

  12. Neural stem cells: properties and therapeutic potentials for hypoxic-ischemic brain injury in newborn infants.

    PubMed

    Lee, Il-Shin; Jung, Kwangsoo; Kim, Miri; Park, Kook In

    2010-12-01

    Neural stem cells (NSCs) are defined by their ability to self-renew, to differentiate into cells of all glial and neuronal lineages throughout the neuraxis, and to populate developing or degenerating central nervous system (CNS) regions. The recognition that NSCs propagated in culture could be reimplanted into the mammalian brain, where they might integrate appropriately throughout the mammalian CNS and stably express foreign genes, has unveiled a new role for neural transplantation and gene therapy and a possible strategy for addressing the CNS manifestations of diseases that hitherto had been refractory to intervention. An intriguing phenomenon with possible therapeutic potentials has begun to emerge from our observations of the behavior of NSCs in animal models of neonatal hypoxic-ischemic (HI) brain injury. During phases of active neurodegeneration, factors seem to be transiently elaborated to which NSCs may respond by migrating to degenerating regions and differentiating specifically towards replacement of dying neural cells. NSCs may attempt to repopulate and reconstitute ablated regions. These 'repair mechanisms' may actually reflect the reexpression of basic developmental principles that may be harnessed for therapeutic ends. In addition, NSCs may serve as vehicles for gene delivery and appear capable of simultaneous neural cell replacement and gene therapy (e.g. with factors that might enhance neuronal differentiation, neurites outgrowth, proper connectivity, and/or neuroprotection). When combined with certain synthetic biomaterials, NSCs may be even more effective in 'engineering' the damaged CNS towards reconstitution. We have also cultured human NSCs or progenitors as neurospheres which were derived from fetal cadavers at 13 weeks of gestation, and transplanted them into HI-injured immature brains to investigate their therapeutic potentials in this type of model.

  13. Mesenchymal stem cells protect neurons against hypoxic-ischemic injury via inhibiting parthanatos, necroptosis, and apoptosis, but not autophagy.

    PubMed

    Kong, Deyan; Zhu, Juehua; Liu, Qian; Jiang, Yongjun; Xu, Lily; Luo, Ning; Zhao, Zhenqiang; Zhai, Qijin; Zhang, Hao; Zhu, Mingyue; Liu, Xinfeng

    2017-03-01

    Cellular therapy with mesenchymal stem cells (MSCs) protects cortical neurons against hypoxic-ischemic injury of stroke. Although sorts of efforts have been made to confirm the neuroprotective effect of MSCs on neurons against hypoxic-ischemic injury, the mechanism is until now far away from clear. Here in this study, oxygen-glucose deprivation (OGD)-injured neuron model was applied to mimic the neuronal hypoxic-ischemic injury in vitro. Co-culturing with MSCs in a transwell co-culture system, the OGD injured neurons were rescued by 75.0 %. Further data demonstrated that co-culturing with MSCs protected the cortical neurons from the OGD-induced parthanatos by alleviating apoptosis-inducing factor (AIF) nuclear translocation; attenuated the neuronal necroptosis by down-regulating the expression of the two essential kinases in necroptosis, receptor interacting protein kinase1 (RIP1) and 3 (RIP3); rescued the neurons from apoptosis by deactivating caspase-3; whilst performed no significant influence on OGD-induced neuronal autophagy, according to its failed regulation on Beclin1. In conclusion, MSCs potentially protect the cortical neurons from OGD-injury in vitro, through rescuing neurons from the cell death of parthanatos, necroptosis, and apoptosis, but not autophagy, which could provide some evidence to the mechanism explanation on stem cell treatment for ischemic stroke.

  14. Therapeutic effects of human umbilical cord mesenchymal stem cells transplantation on hypoxic ischemic encephalopathy

    PubMed Central

    Xie, Bingchuan; Gu, Ping; Wang, Wenting; Dong, Ci; Zhang, Lina; Zhang, Jun; Liu, Huimiao; Qiu, Fucheng; Han, Rui; Zhang, Zhenqing; Yan, Baoyong

    2016-01-01

    Objective: Human umbilical cord mesenchymal stem cells (hUC-MSCs) hold substantial promise for the treatment of ischemic neurological disease, but few clinical data are currently available about its therapeutic effects in hypoxic ischemic encephalopathy (HIE). This study is to evaluate the effects of hUC-MSCs transplantation on patients with HIE. Methods A total 22 patients with HIEwere randomly divided into hUC-MSCs transplantation group (n = 12) and control group (n = 10). After isolation, hUC-MSCs were cultured for 3 to 5 passages in vitro and then intravenously administered to HIE patients in the transplantation group, while the control group received routine treatment only. The outcomes of HIE patients were evaluated at designated time points by clinical assessment scales, including NIHSS, Barthel Index, MMSE, HAMA24, HAMD14 and UPDRS. Results: hUC-MSCs were identified by morphological analysis and flow cytometry assays before clinic transplantation. No significant differences of demographic characteristics were observed between the two groups of subjects. Compared to the control group, hUC-MSCs transplantation markedly improved the outcomes of HIE patients leading to better recovery of neurological function, cognition ability, emotional reaction and extrapyramidal function. No significant adverse effects were found in subjects with hUC-MSCs transplantation during a 180-day follow-up period. Conclusion: These data suggest that hUC-MSCs therapy markedly improves the outcomes of patients with HIE, which is potential for the routine treatment of ischemic neurological disease. PMID:27508046

  15. In vivo click reaction between Tc-99m-labeled azadibenzocyclooctyne-MAMA and 2-nitroimidazole-azide for tumor hypoxia targeting.

    PubMed

    Sun, Wenjing; Chu, Taiwei

    2015-10-15

    The bioactivity of nitroimidazole in Tc-99m-labeled 2-nitroimidazole, a traditional solid tumor hypoxia-imaging agent for single photon emission computed tomography (SPECT), is reduced by the presence of large ligand and metallic radionuclide, exhibiting lower tumor-to-nontumor ratios. In an effort to solve this general problem, a pretargeting strategy based on click chemistry (strain-promoted cyclooctyne-azide cycloaddition) was applied. The functional click synthons were synthesized as pretargeting components: an azide group linked to 2-nitroimidazole (2NIM-Az) serves for tumor hypoxia-targeting and azadibenzocyclooctyne conjugated with monoamine monoamide dithiol ligand (AM) functions as radiolabeling and binding group to azides in vivo. 2NIM-triazole-MAMA was obtained from in vitro click reaction with a reaction rate constant of 0.98M(-1)s(-1). AM and 2NIM-triazole-MAMA were radiolabeled with Tc-99m. The hypoxia-pretargeting biodistribution was studied in Kunming mice bearing S180 tumor; (99m)Tc-AM and (99m)Tc-triazole-2NIM were used as blank control and conventional control. Compared to the control groups, the pretargeting experiment exhibits the best radio-uptake and retention in tumor, with higher tumor-to-muscle and tumor-to-blood ratios (up to 8.55 and 1.44 at 8h post-(99m)Tc-complex-injection, respectively). To some extent, the pretargeting strategy protects the bioactivity of nitroimidazole and therefore provides an innovative approach for the development of tumor hypoxia-SPECT imaging agents.

  16. The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells.

    PubMed

    Herwartz, Christine; Castillo-Juárez, Paola; Schröder, Linda; Barron, Blanca L; Steger, Gertrud

    2015-01-01

    Hypoxia activates the expression of proangiogenic and survival promoting factors as well as proinflammatory cytokines that support tissue inflammation. Hypoxia and inflammation are associated with tumor progression. The identification of the factors participating in the hypoxia associated inflammation is essential to develop strategies to control tumor hypoxia. The transcription factor ZNF395 was found to be overexpressed in various tumors including glioblastomas particularly in the network of a hypoxic response pointing to a functional role of ZNF395. On the other hand, ZNF395 was suggested to have tumor suppressor activities which may rely on its repression of proinflammatory factors. To address these conflictive observations, we investigated the role of ZNF395 in the expression of proinflammatory cytokines in the astrocytoma cell line U87-MG under hypoxia. We show that ZNF395 is a target gene of the hypoxia inducible factor HIF-1α. By gene expression analysis, RT-PCR and ELISA, we demonstrated that the siRNA-mediated suppression of ZNF395 impairs the hypoxic induction of IL-1β, IL-6, IL-8, and LIF in U87-MG cells. At ambient oxygen concentrations, ZNF395 had no enhancing effect, indicating that this transcriptional activation by ZNF395 is restricted to hypoxic conditions. Our results suggest that ZNF395 contributes to hypoxia associated inflammation by superactivating proinflammatory cytokines.

  17. The Transcription Factor ZNF395 Is Required for the Maximal Hypoxic Induction of Proinflammatory Cytokines in U87-MG Cells

    PubMed Central

    Herwartz, Christine; Castillo-Juárez, Paola; Schröder, Linda; Barron, Blanca L.; Steger, Gertrud

    2015-01-01

    Hypoxia activates the expression of proangiogenic and survival promoting factors as well as proinflammatory cytokines that support tissue inflammation. Hypoxia and inflammation are associated with tumor progression. The identification of the factors participating in the hypoxia associated inflammation is essential to develop strategies to control tumor hypoxia. The transcription factor ZNF395 was found to be overexpressed in various tumors including glioblastomas particularly in the network of a hypoxic response pointing to a functional role of ZNF395. On the other hand, ZNF395 was suggested to have tumor suppressor activities which may rely on its repression of proinflammatory factors. To address these conflictive observations, we investigated the role of ZNF395 in the expression of proinflammatory cytokines in the astrocytoma cell line U87-MG under hypoxia. We show that ZNF395 is a target gene of the hypoxia inducible factor HIF-1α. By gene expression analysis, RT-PCR and ELISA, we demonstrated that the siRNA-mediated suppression of ZNF395 impairs the hypoxic induction of IL-1β, IL-6, IL-8, and LIF in U87-MG cells. At ambient oxygen concentrations, ZNF395 had no enhancing effect, indicating that this transcriptional activation by ZNF395 is restricted to hypoxic conditions. Our results suggest that ZNF395 contributes to hypoxia associated inflammation by superactivating proinflammatory cytokines. PMID:26229239

  18. The dietary flavonoid kaempferol effectively inhibits HIF-1 activity and hepatoma cancer cell viability under hypoxic conditions

    SciTech Connect

    Mylonis, Ilias; Lakka, Achillia; Tsakalof, Andreas; Simos, George

    2010-07-16

    Research highlights: {yields} Kaempferol inhibits HIF-1 activity in hepatocarcinoma cells; {yields} Kaempferol causes cytoplasmic mislocalization of HIF-1{alpha} by impairing the MAPK pathway. {yields} Viability of hepatocarcinoma cells under hypoxia is reduced by kaempferol. -- Abstract: Hepatocellular carcinoma (HCC) is characterized by high mortality rates and resistance to conventional treatment. HCC tumors usually develop local hypoxia, which stimulates proliferation of cancer cells and renders them resilient to chemotherapy. Adaptation of tumor cells to the hypoxic conditions depends on the hypoxia-inducible factor 1 (HIF-1). Over-expression of its regulated HIF-1{alpha} subunit, an important target of anti-cancer therapy, is observed in many cancers including HCC and is associated with severity of tumor growth and poor patient prognosis. In this report we investigate the effect of the dietary flavonoid kaempferol on activity, expression levels and localization of HIF-1{alpha} as well as viability of human hepatoma (Huh7) cancer cells. Treatment of Huh7 cells with kaempferol under hypoxic conditions (1% oxygen) effectively inhibited HIF-1 activity in a dose-dependent manner (IC{sub 50} = 5.16 {mu}M). The mechanism of this inhibition did not involve suppression of HIF-1{alpha} protein levels but rather its mislocalization into the cytoplasm due to inactivation of p44/42 MAPK by kaempferol (IC{sub 50} = 4.75 {mu}M). Exposure of Huh7 cells to 10 {mu}{Mu} kaempferol caused significant reduction of their viability, which was remarkably more evident under hypoxic conditions. In conclusion, kaempferol, a non-toxic natural food component, inhibits both MAPK and HIF-1 activity at physiologically relevant concentrations (5-10 {mu}M) and suppresses hepatocarcinoma cell survival more efficiently under hypoxia. It has, therefore, potential as a therapeutic or chemopreventive anti-HCC agent.

  19. CDKN2B Regulates TGFβ Signaling and Smooth Muscle Cell Investment of Hypoxic Neovessels

    PubMed Central

    Nanda, Vivek; Downing, Kelly P.; Ye, Jianqin; Xiao, Sophia; Kojima, Yoko; Spin, Joshua M.; DiRenzo, Daniel; Nead, Kevin T.; Connolly, Andrew J; Dandona, Sonny; Perisic, Ljubica; Hedin, Ulf; Maegdefessel, Lars; Dalman, Jessie; Guo, Liang; Zhao, XiaoQing; Kolodgie, Frank D.; Virmani, Renu; Davis, Harry R.; Leeper, Nicholas J.

    2015-01-01

    Rationale Genetic variation at the chromosome 9p21 cardiovascular risk locus has been associated with peripheral artery disease (PAD), but its mechanism remains unknown. Objective To determine whether this association is secondary to an increase in atherosclerosis, or is the result of a separate angiogenesis-related mechanism. Methods and Results Quantitative evaluation of human vascular samples revealed that carriers of the 9p21 risk allele possess a significantly higher burden of immature intraplaque microvessels than carriers of the ancestral allele, irrespective of lesion size or patient comorbidity. To determine whether aberrant angiogenesis also occurs under non-atherosclerotic conditions, we performed femoral artery ligation surgery in mice lacking the 9p21 candidate gene, Cdkn2b. These animals developed advanced hind-limb ischemia and digital auto-amputation, secondary to a defect in the capacity of the Cdkn2b-deficient smooth muscle cell (SMC) to support the developing neovessel. Microarray studies identified impaired TGFβ signaling in cultured CDKN2B-deficient cells, as well as TGFβ1 upregulation in the vasculature of 9p21 risk allele carriers. Molecular signaling studies indicated that loss of CDKN2B impairs the expression of the inhibitory factor, SMAD-7, which promotes downstream TGFβ activation. Ultimately, this manifests in the upregulation of a poorly studied effector molecule, TGFβ1-induced-1, which is a TGFβ-‘rheostat’ known to have antagonistic effects on the EC and SMC. Dual knockdown studies confirmed the reversibility of the proposed mechanism, in vitro. Conclusions These results suggest that loss of CDKN2B may not only promote cardiovascular disease through the development of atherosclerosis, but may also impair TGFβ signaling and hypoxic neovessel maturation. PMID:26596284

  20. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    PubMed

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing.

  1. Cancer stem-like cells can be induced through dedifferentiation under hypoxic conditions in glioma, hepatoma and lung cancer

    PubMed Central

    Wang, Pan; Wan, Wen-wu; Xiong, Shuang-Long; Feng, Hua; Wu, Nan

    2017-01-01

    Traditional studies have shown that transcription factors, including SOX-2, OCT-4, KLF-4, Nanog and Lin-28A, contribute to the dedifferentiation and reprogramming process in normal tissues. Hypoxia is a physiological phenomenon that exists in tumors and promotes the expression of SOX-2, OCT-4, KLF-4, Nanog and Lin-28A. Therefore, an interesting question is whether hypoxia as a stimulating factor promotes the process of dedifferentiation and induces the formation of cancer stem-like cells. Studies have shown that OCT-4 and Nanog overexpression induced the formation of cancer stem cell-like cells through dedifferentiation and enhanced malignancy in lung adenocarcinoma, and reprogramming SOX-2 in pancreatic cancer cells also promoted the dedifferentiation process. Therefore, we investigated this phenomenon in glioma, lung cancer and hepatoma cells and found that the transcription factors mentioned above were highly expressed under hypoxic conditions and induced the formation of spheres, which exhibited asymmetric division and cell cycle arrest. The dedifferentiation process induced by hypoxia highlights a new pattern of cancer development and recurrence, demonstrating that all kinds of cancer cells and the hypoxic microenvironment should be taken into consideration when developing tumor therapies. PMID:28179999

  2. Patient-specific modeling and analysis of dynamic behavior of individual sickle red blood cells under hypoxic conditions

    NASA Astrophysics Data System (ADS)

    Li, Xuejin; Du, E.; Li, Zhen; Tang, Yu-Hang; Lu, Lu; Dao, Ming; Karniadakis, George

    2015-11-01

    Sickle cell anemia is an inherited blood disorder exhibiting heterogeneous morphology and abnormal dynamics under hypoxic conditions. We developed a time-dependent cell model that is able to simulate the dynamic processes of repeated sickling and unsickling of red blood cells (RBCs) under physiological conditions. By using the kinetic cell model with parameters derived from patient-specific data, we present a mesoscopic computational study of the dynamic behavior of individual sickle RBCs flowing in a microfluidic channel with multiple microgates. We investigate how individual sickle RBCs behave differently from healthy ones in channel flow, and analyze the alteration of cellular behavior and response to single-cell capillary obstruction induced by cell rheologic rigidification and morphological change due to cell sickling under hypoxic conditions. We also simulate the flow dynamics of sickle RBCs treated with hydroxyurea (HU) and quantify the relative enhancement of hemodynamic performance of HU. This work was supported by the National Institutes of Health (NIH) Grant U01HL114476.

  3. Factors associated with the preincubation effect of hypoxic cell sensitizers in vitro and their possible implications in chemosensitization

    SciTech Connect

    Roizin-Towle, L.; Biaglow, J.E.; Meltzer, H.L.; Varnes, M.E.

    1984-06-01

    The enhancement of melphalan toxicity was observed by preincubation of V-79-379A cells in spinner culture with multiple doses of misonidazole (miso) or SR-2508 under hypoxic conditions. Chemosensitization was shown to be a function of sensitizer concentration and duration of exposure to the alkylating agent. Cells preincubated with miso not only had lower levels of nonprotein thiols, but also were shown to have altered levels of intracellular calcium and a lower threshold to oxidative stress as measured by toxicity to cysteamine or H/sub 2/O/sub 2/. Preincubated cells, hypoxic cells, and cells receiving moderate hyperthermia (42.5/sup 0/C for 3 hr) all showed increased sensitivity to either cysteamine or H/sub 2/O/sub 2/. The increased killing of preincubated cells by cysteamine was shown to be similar to that of H/sub 2/O/sub 2/, and the dramatic reduction of cysteamine toxicity by catalase indicated H/sub 2/O/sub 2/ was the major reaction associated with this effect. These results indicate that preincubated cells exhibit a variety of biological effects that may significantly influence their response to further treatment with drugs or radiation, especially where peroxidative and free radical mechanisms are involved.

  4. Combination of nicotinamide and hyperthermia to eliminate radioresistant chronically and acutely hypoxic tumor cells

    SciTech Connect

    Horsman, M.R.; Chaplin, D.J.; Overgaard, J. )

    1990-12-01

    The interaction among nicotinamide, radiation, and heat was studied in vivo using a C3H mouse mammary carcinoma grown in the feet of CDF1 mice. Response following local tumor treatment was assessed by tumor control and regrowth delay. Nicotinamide (1000 mg/kg i.p.) produced maximal radiosensitization when injected 30 min to 2 h before irradiation (enhancement ratios (ERs), 1.2-1.5). Radiation damage was also increased by heating tumors (43.5 degrees C for 60 min) 4 h after irradiation (ERs = 1.6-2.6). This combined radiation and heat treatment was enhanced by nicotinamide but the effect depended on the assay procedure, such that although a significant increase was observed with the tumor control assay, only a slight increase was seen using regrowth delay as the end point. The development of moist desquamation in normal feet was used to estimate skin damage after irradiation. Nicotinamide and heat both resulted in a small yet significant increase in skin damage (ERs less than 1.2 and 1.1, respectively). A combined treatment resulted in a greater ER of 1.7, but when compared to the tumor response it still gave a therapeutic gain. A histological fluorescent staining technique was used to assess functional tumor vasculature at two periods in time separated by 20 min. Under normal conditions 7.7% of the vessels in this tumor were functional at one time but not the other. This value was reduced to 2.8% after nicotinamide administration. Since these fluctuations in blood flow can result in acute hypoxia we conclude that while heat eliminates chronically hypoxic tumor cells, nicotinamide probably removes the presence of acute hypoxia.

  5. Se-methylselenocysteine sensitizes hypoxic tumor cells to irinotecan by targeting hypoxia-inducible factor 1α

    PubMed Central

    Chintala, Sreenivasulu; Tóth, Károly; Cao, Shousong; Durrani, Farukh A.; Vaughan, Mary M.; Jensen, Randy L.; Rustum, Youcef M.

    2010-01-01

    Purpose Hypoxic tumor cells overexpressing hypoxia-inducible factor 1alpha (HIF-1α) are generally resistant to chemo/radiotherapy. We have reported that Se-methylselenocysteine (MSC) therapeutically enhances the efficacy and selectivity of irinotecan against human tumor xenografts. The aim of this study was to delineate the mechanism responsible for the observed efficacy targeting on HIF-1α and its transcriptionally regulated genes VEGF and CAIX. Methods We investigated the mechanism of HIF-1α inhibition by MSC and its critical role in the therapeutic outcome by generating HIF-1α stable knockdown (KD) human head and neck squamous cell carcinoma, FaDu by transfecting HIF-1α short hairpin RNA. Results While cytotoxic efficacy in combination with methylselenic acid (MSA) with SN-38 (active metabolites of MSC and irinotecan) could not be confirmed in vitro against normoxic tumor cells, the hypoxic tumor cells were more sensitive to the combination. Reduction in HIF-1α either by MSA or shRNA knockdown resulted in significant increase in cytotoxicity of SN38 in vitro against hypoxic, but not the normoxic tumor cells. Similarly, in vivo, either MSC in combination with irinotecan treatment of parental xenografts or HIF-1α KD tumors treated with irinotecan alone resulted in comparable therapeutic response and increase in the long-term survival of mice bearing FaDu xenografts. Conclusions Our results show that HIF-1α is a critical target for MSC and its inhibition was associated with enhanced antitumor activity of irinotecan. Inhibition of HIF-1α appeared to be mediated through stabilization of PHD2, 3 and downregulation of ROS by MSC. Thus, our findings support the development of MSC as a HIF-1α inhibitor in combination chemotherapy. PMID:20066420

  6. Downregulation of miR-210 expression inhibits proliferation, induces apoptosis and enhances radiosensitivity in hypoxic human hepatoma cells in vitro

    SciTech Connect

    Yang, Wei; Sun, Ting; Cao, Jianping; Liu, Fenju; Tian, Ye; Zhu, Wei

    2012-05-01

    Hypoxia is a common feature of solid tumors and an important contributor to tumor radioresistance. miR-210 is the most consistently and robustly induced microRNA under hypoxia in different types of tumor cells and normal cells. In the present study, to explore the feasibility of miR-210 as an effective therapeutic target, lentiviral-mediated anti-sense miR-210 gene transfer technique was employed to downregulate miR-210 expression in hypoxic human hepatoma SMMC-7721, HepG2 and HuH7 cells, and phenotypic changes of which were analyzed. Hypoxia led to an increased hypoxia inducible factor-1{alpha} (HIF-1{alpha}) and miR-210 expression and cell arrest in the G{sub 0}/G{sub 1} phase in all cell lines. miR-210 downregulation significantly suppressed cell viability, induced cell arrest in the G{sub 0}/G{sub 1} phase, increased apoptotic rate and enhanced radiosensitivity in hypoxic human hepatoma cells. Moreover, apoptosis-inducing factor, mitochondrion-associated, 3 (AIFM3) was identified as a direct target gene of miR-210. AIFM3 downregulation by siRNA attenuated radiation induced apoptosis in miR-210 downregulated hypoxic human hepatoma cells. Taken together, these data suggest that miR-210 might be a potential therapeutic target and specific inhibition of miR-210 expression in combination with radiotherapy might be expected to exert strong anti-tumor effect on hypoxic human hepatoma cells. -- Highlights: Black-Right-Pointing-Pointer miR-210 downregulation radiosensitized hypoxic hepatoma. Black-Right-Pointing-Pointer AIFM3 was identified as a direct target gene of miR-210. Black-Right-Pointing-Pointer miR-210 might be a therapeutic target to hypoxic hepatoma.

  7. Transcriptional Profiling of Hypoxic Neural Stem Cells Identifies Calcineurin-NFATc4 Signaling as a Major Regulator of Neural Stem Cell Biology

    PubMed Central

    Moreno, Marta; Fernández, Virginia; Monllau, Josep M.; Borrell, Víctor; Lerin, Carles; de la Iglesia, Núria

    2015-01-01

    Summary Neural stem cells (NSCs) reside in a hypoxic microenvironment within the brain. However, the crucial transcription factors (TFs) that regulate NSC biology under physiologic hypoxia are poorly understood. Here we have performed gene set enrichment analysis (GSEA) of microarray datasets from hypoxic versus normoxic NSCs with the aim of identifying pathways and TFs that are activated under oxygen concentrations mimicking normal brain tissue microenvironment. Integration of TF target (TFT) and pathway enrichment analysis identified the calcium-regulated TF NFATc4 as a major candidate to regulate hypoxic NSC functions. Nfatc4 expression was coordinately upregulated by top hypoxia-activated TFs, while NFATc4 target genes were enriched in hypoxic NSCs. Loss-of-function analyses further revealed that the calcineurin-NFATc4 signaling axis acts as a major regulator of NSC self-renewal and proliferation in vitro and in vivo by promoting the expression of TFs, including Id2, that contribute to the maintenance of the NSC state. PMID:26235896

  8. Oxidative Stress Promotes Doxorubicin-Induced Pgp and BCRP Expression in Colon Cancer Cells Under Hypoxic Conditions.

    PubMed

    Pinzón-Daza, Martha L; Cuellar-Saenz, Yenith; Nualart, Francisco; Ondo-Mendez, Alejandro; Del Riesgo, Lilia; Castillo-Rivera, Fabio; Garzón, Ruth

    2017-01-20

    P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) are ATP binding cassette (ABC) transporters that are overexpressed in different drug-resistant cancer cell lines. In this study, we investigated whether doxorubicin promotes Pgp and/or BCRP expression to induce drug resistance in colon cancer cells under hypoxic conditions. We analyzed HIF-1α activity via ELISA, Pgp, and BCRP expression by qRT-PCR and the relationship between doxorubicin uptake and ABC transporter expression via confocal microscopy in HT-29WT and HT-29 doxorubicin-resistant colon cancer cells (HT-29DxR). These cells were treated with doxorubicin and/or CoCl2 (chemical hypoxia), and reactive oxygen species inductors. We found that the combination of chemically induced hypoxia and doxorubicin promoted Pgp mRNA expression within 24 h in HT-29WT and HT-29DxR cells. Both doxorubicin and CoCl2 alone or in combination induced Pgp and BCRP expression, as demonstrated via confocal microscopy in each of the above two cell lines. Thus, we surmised that Pgp and BCRP expression may result from synergistic effects exerted by the combination of doxorubicin-induced ROS production and HIF-1α activity under hypoxic conditions. However, HIF-1α activity disruption via the administration of E3330, an APE-1 inhibitor, downregulated Pgp expression and increased doxorubicin delivery to HT-29 cells, where it served as a substrate for Pgp, indicating the existence of an indirect relationship between Pgp expression and doxorubicin accumulation. Thus, we concluded that Pgp and BCRP expression can be regulated via cross-talk between doxorubicin and hypoxia, promoting drug resistance in HT-29 WT, and HT-29DxR cells and that this process may be ROS dependent. J. Cell. Biochem. 9999: 1-11, 2017. © 2017 Wiley Periodicals, Inc.

  9. Tanshinone IIA pretreatment renders free flaps against hypoxic injury through activating Wnt signaling and upregulating stem cell-related biomarkers.

    PubMed

    Xu, Zihan; Zhang, Zhenxin; Wu, Lijun; Sun, Yaowen; Guo, Yadong; Qin, Gaoping; Mu, Shengzhi; Fan, Ronghui; Wang, Benfeng; Gao, Wenjie

    2014-10-09

    Partial or total flap necrosis after flap transplantation is sometimes clinically encountered in reconstructive surgery, often as a result of a period of hypoxia that exceeds the tolerance of the flap tissue. In this study, we determine whether tanshinone IIA (TSA) pretreatment can protect flap tissue against hypoxic injury and improve its viability. Primary epithelial cells isolated from the dorsal skin of mice were pretreated with TSA for two weeks. Cell counting kit-8 and Trypan Blue assays were carried out to examine the proliferation of TSA-pretreated cells after exposure to cobalt chloride. Then, Polymerase chain reaction and Western blot analysis were used to determine the expression of β-catenin, GSK-3β, SOX2, and OCT4 in TSA-treated cells. In vivo, after mice were pretreated with TSA for two weeks, a reproducible ischemic flap model was implemented, and the area of surviving tissue in the transplanted flaps was measured. Immunohistochemistry was also conducted to examine the related biomarkers mentioned above. Results show that epidermal cells, pretreated with TSA, showed enhanced resistance to hypoxia. Activation of the Wnt signaling pathway in TSA-pretreated cells was characterized by the upregulation of β-catenin and the downregulation of GSK-3β. The expression of SOX2 and OCT4 controlled by Wnt signaling were also found higher in TSA pretreated epithelial cells. In the reproducible ischaemic flap model, pretreatment with TSA enhanced resistance to hypoxia and increased the area of surviving tissue in transplanted flaps. The expression of Wnt signaling pathway components, stem-cell related biomarkers, and CD34, which are involved in the regeneration of blood vessels, was also upregulated in TSA-pretreated flap tissue. The results show that TSA pretreatment protects free flaps against hypoxic injury and increases the area of surviving tissue by activating Wnt signaling and upregulating stem cell-related biomarkers.

  10. Azobenzene-caged sulforhodamine dyes: a novel class of ‘turn-on’ reactive probes for hypoxic tumor cell imaging

    NASA Astrophysics Data System (ADS)

    Chevalier, Arnaud; Piao, Wen; Hanaoka, Kenjiro; Nagano, Tetsuo; Renard, Pierre-Yves; Romieu, Anthony

    2015-12-01

    New sulforhodamine-based fluorescent ‘turn-on’ probes have been developed for the direct imaging of cellular hypoxia. Rapid access to this novel class of water-soluble ‘azobenzene-caged’ fluorophores was made possible through an easily-implementable azo-coupling reaction between a fluorescent primary arylamine derived from a sulforhodamine 101 scaffold (named SR101-NaphtNH 2 ) and a tertiary aniline whose N-substituents are neutral, cationic, or zwitterionic. The detection mechanism is based on the bioreductive cleavage of the azo bond that restores strong far-red fluorescence (emission maximum at 625 nm) by regenerating the original sulforhodamine SR101-NaphtNH 2 . This valuable fluorogenic response was obtained for the three ‘smart’ probes studied in this work, as shown by an in vitro assay using rat liver microsomes placed under aerobic and then under hypoxic conditions. Most importantly, the probe namely SR101-NaphtNH 2 -Hyp-diMe was successfully applied for imaging the hypoxic status of tumor cells (A549 cells).

  11. Stem cells decreased neuronal cell death after hypoxic stress in primary fetal rat neurons in vitro.

    PubMed

    Sakai, Tetsuro; Xu, Yan

    2012-01-01

    To explore stem cell-mediated neuronal protection through extracellular signaling pathways by transplanted stem cells, we sought to identify potential candidate molecules responsible for neuronal protection using an in vitro coculture system. Primary fetal rat hippocampal neurons underwent hypoxia (≤1% oxygen) for 96 h nad then were returned to a normoxic condition. The study group then received rat umbilical cord matrix-derived stem cells, while the control group received fresh media only. The experimental group showed decreased neuronal apoptosis compared to the control group [44.5 ± 1.6% vs. 71.0 ± 4.2% (mean ± SD, p = 0.0005) on day 5] and higher neuronal survival (4.9 ± 1.2 cells/100× field vs. 2.2 ± 0.3, p = 0.02 on day 5). Among 90 proteins evaluated using a protein array, stem cell coculture media showed increased protein secretion of TIMP-1 (5.61-fold), TIMP-2 (4.88), CNTF-Rα (3.42), activin A (2.20), fractalkine (2.04), CCR4 (2.02), and decreased secretion in MIP-2 (0.30-fold), AMPK α1 (0.43), TROY (0.48), and TIMP-3 (0.50). This study demonstrated that coculturing stem cells with primary neurons in vitro decreased neuronal cell death after hypoxia with significantly altered protein secretion. The results suggest that stem cells may offer neuronal protection through extracellular signaling.

  12. The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16INK4A mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent

    PubMed Central

    Ito, Akira; Aoyama, Tomoki; Yoshizawa, Makoto; Nagai, Momoko; Tajino, Junichi; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Kuroki, Hiroshi

    2015-01-01

    A hypoxic environment is thought to be important for the maintenance of stemness and suppressing cell senescence, in stem cells. Therefore, a hypoxic condition is induced during cell expansion and/or induction of intended differentiation. However, the induction of these conditions requires a specially equipped hypoxia chamber and expensive gas mixtures, which are expensive and space-consuming. Owing to these restrictions, appropriate hypoxic conditions cannot be provided during cell transportation, which is increasingly required for regenerative medicine. Hence, a simple and economical culture system is required. The purpose of this study was to investigate the effects of short-term hypoxic conditions on human mesenchymal stem cell (MSC) proliferation, viability, and senescence, utilizing the CulturePal system (CulturePal-Zero and CulturePal-Five), a novel and simple hypoxic culture system with a built-in deoxidizing agent. The O2 concentration in the CulturePal-Zero was observed to reduce to <0.1% within 1 h, and to 5% within 24h in the CulturePal-Five system. Cell proliferation under these hypoxic conditions showed a sharp increase at 5% O2 concentration, and no noticeable cell death was observed even at severe hypoxic conditions (<0.1% O2) up to 72h. The p16INK4A (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16INK4A expression altered proportionately to the O2 concentration. These results indicated that the short-term hypoxic condition, at an approximate O2 concentration of 5%, would be suitable for promoting cell proliferation and repressing cell senescence, without aggravating the MSC viability. Therefore, the CulturePal systems may be suitable for providing an appropriate hypoxic condition in stem cell research and transportation. PMID:26195892

  13. Lecithin-Bound Iodine Prevents Disruption of Tight Junctions of Retinal Pigment Epithelial Cells under Hypoxic Stress

    PubMed Central

    Sugimoto, Masahiko; Kondo, Mineo

    2016-01-01

    Aim. We investigated whether lecithin-bound iodine (LBI) can protect the integrity of tight junctions of retinal pigment epithelial cells from hypoxia. Method. Cultured human retinal pigment epithelial (ARPE-19) cells were pretreated with LBI. To mimic hypoxic conditions, cells were incubated with CoCl2. We compared the integrity of the tight junctions (TJs) of control to cells with either LBI alone, CoCl2 alone, or LBI + CoCl2. The levels of cytokines in the conditioned media were also determined. Results. Significant decrease in the zonula occludens-1 (ZO-1) intensity in the CoCl2 group compared to the control (5787.7 ± 4126.4 in CoCl2 group versus 29244.6 ± 2981.2 in control; average ± standard deviation). But the decrease was not significant in the LBI + CoCl2 (27189.0 ± 11231.1). The levels of monocyte chemoattractant protein-1 (MCP-1) and Chemokine (C-C Motif) Ligand 11 (CCL-11) were significantly higher in the CoCl2 than in the control (340.8 ± 43.3 versus 279.7 ± 68.3 pg/mL for MCP-1, and 15.2 ± 12.9 versus 12.5 ± 6.1 pg/mL for CCL-11. With LBI pretreatment, the levels of both cytokines were decreased to 182.6 ± 23.8 (MCP-1) and 5.46 ± 1.9 pg/mL for CCL-11). Blockade of MCP-1 or CCL-11 also shows similar result representing TJ protection from hypoxic stress. Conclusions. LBI results in a protective action from hypoxia. PMID:27340563

  14. Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage.

    PubMed

    Feng, Zhichun; Liu, Jing; Ju, Rong

    2013-05-05

    Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats (7 days old) subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2'-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2'-deoxyuridine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.

  15. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells.

    PubMed

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPK α blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPK α in hypoxic SW620 cells, implying cross-talk between ERK and AMPK α . Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1 α and Akt/mTOR and the activation of AMPK α and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPK α in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPK α and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells.

  16. N-myc Downstream-Regulated Gene 1 (NDRG1) mediates pomegranate juice protection from apoptosis in hypoxic BeWo cells but not in primary human trophoblasts

    PubMed Central

    Chen, Baosheng; Zaveri, Parul G.; Longtine, Mark S.; Nelson, D. Michael

    2015-01-01

    Introduction N-Myc downstream-regulated gene 1 (NDRG1) expression is increased in placentas of human pregnancies with intrauterine growth restriction and in hypoxic cultured primary trophoblasts. We previously showed that elevated NDRG1 decreases trophoblast apoptosis induced by hypoxia. Separately, we found that pomegranate juice (PJ) decreases cell death induced by hypoxia in trophoblasts. Here, we test the hypothesis that PJ protects trophoblasts from hypoxia-induced apoptosis by modulating NDRG1 expression. Methods Quantitative rtPCR was used to investigate the effects of PJ treatment on mRNA levels of 22 candidate genes involved in apoptosis, oxidative stress, and differentiation in trophoblasts. Western blotting and immunofluorescence were used to analyze NDRG1 protein levels. siRNA-mediated NDRG1 knockdown was used to investigate the role of NDRG1 in response to PJ in hypoxic BeWo choriocarcinoma cells and hypoxic cultured primary human trophoblasts. Results The mRNA levels of eight genes were altered, with NDRG1 showing the largest response to PJ and thus, we pursued the role of NDRG1 here. PJ significantly increased NDRG1 protein expression in primary trophoblasts and in BeWo cells. Knockdown of NDRG1 in hypoxic BeWo cells in the presence of PJ yielded increased apoptosis. In contrast, knockdown of NDRG1 in hypoxic primary trophoblasts in the presence of PJ did not increase apoptosis. Discussion We conclude that the PJ-mediated decrease in cell death in hypoxia is partially mediated by NDRG1 in BeWo cells but not in primary trophoblasts. The disparate effects of NDRG1 between BeWo cells and primary trophoblasts indicate caution is required when extrapolating from results obtained with cell lines to primary trophoblasts. PMID:26028238

  17. Activation of AMP-Activated Protein Kinase α and Extracelluar Signal-Regulated Kinase Mediates CB-PIC-Induced Apoptosis in Hypoxic SW620 Colorectal Cancer Cells

    PubMed Central

    Cho, Sung-Yun; Lee, Hyo-Jeong; Lee, Hyo-Jung; Jung, Deok-Beom; Kim, Hyunseok; Sohn, Eun Jung; Kim, Bonglee; Jung, Ji Hoon; Kwon, Byoung-Mog; Kim, Sung-Hoon

    2013-01-01

    Here, antitumor mechanism of cinnamaldehyde derivative CB-PIC was elucidated in human SW620 colon cancer cells. CB-PIC significantly exerted cytotoxicity, increased sub-G1 accumulation, and cleaved PARP with apoptotic features, while it enhanced the phosphorylation of AMPK alpha and ACC as well as activated the ERK in hypoxic SW620 cells. Furthermore, CB-PIC suppressed the expression of HIF1 alpha, Akt, and mTOR and activated the AMPK phosphorylation in hypoxic SW620 cells. Conversely, silencing of AMPKα blocked PARP cleavage and ERK activation induced by CB-PIC, while ERK inhibitor PD 98059 attenuated the phosphorylation of AMPKα in hypoxic SW620 cells, implying cross-talk between ERK and AMPKα. Furthermore, cotreatment of CB-PIC and metformin enhanced the inhibition of HIF1α and Akt/mTOR and the activation of AMPKα and pACC in hypoxic SW620 cells. In addition, CB-PIC suppressed the growth of SW620 cells inoculated in BALB/c athymic nude mice, and immunohistochemistry revealed that CB-PIC treatment attenuated the expression of Ki-67, CD34, and CAIX and increased the expression of pAMPKα in CB-PIC-treated group. Interestingly, CP-PIC showed better antitumor activity in SW620 colon cancer cells under hypoxia than under normoxia, since it may be applied to chemoresistance. Overall, our findings suggest that activation of AMPKα and ERK mediates CB-PIC-induced apoptosis in hypoxic SW620 colon cancer cells. PMID:23589723

  18. Targeted Identification of Sialoglycoproteins in Hypoxic Endothelial Cells and Validation in Zebrafish Reveal Roles for Proteins in Angiogenesis

    PubMed Central

    Delcourt, Nicolas; Quevedo, Celia; Nonne, Christelle; Fons, Pierre; O'Brien, Donogh; Loyaux, Denis; Diez, Maria; Autelitano, François; Guillemot, Jean-Claude; Ferrara, Pascual; Muriana, Arantza; Callol, Carlos; Hérault, Jean-Pascal; Herbert, Jean-Marc; Favre, Gilles; Bono, Françoise

    2015-01-01

    The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis. PMID:25384978

  19. A 3D engineered tumour for spatial snap-shot analysis of cell metabolism and phenotype in hypoxic gradients

    PubMed Central

    Rodenhizer, Darren; Gaude, Edoardo; Cojocari, Dan; Mahadevan, Radhakrishnan; Frezza, Christian; Wouters, Bradly G.; McGuigan, Alison P.

    2016-01-01

    The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional (3D) tumours. Here, we describe an engineered model to assemble 3D tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snap-shot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia. PMID:26595121

  20. Hypoxic Tumor Cell Modulates Its Microenvironment to Enhance Angiogenic and Metastatic Potential by Secretion of Proteins and Exosomes*

    PubMed Central

    Park, Jung Eun; Tan, Hon Sen; Datta, Arnab; Lai, Ruenn Chai; Zhang, Huoming; Meng, Wei; Lim, Sai Kiang; Sze, Siu Kwan

    2010-01-01

    Under hypoxia, tumor cells produce a secretion that modulates their microenvironment to facilitate tumor angiogenesis and metastasis. Here, we observed that hypoxic or reoxygenated A431 carcinoma cells exhibited enhanced angiogenic and metastatic potential such as reduced cell-cell and cell-extracellular matrix adhesion, increased invasiveness, and production of a secretion with increased chorioallantoic membrane angiogenic activity. Consistent with these observations, quantitative proteomics revealed that under hypoxia the tumor cells secreted proteins involved in angiogenesis, focal adhesion, extracellular matrix-receptor interaction, and immune cell recruitment. Unexpectedly, the secreted proteins were predominantly cytoplasmic and membrane proteins. Ultracentrifugation at 100,000 × g precipitated 54% of the secreted proteins and enriched for many exosome-associated proteins such as the tetraspanins and Alix and also proteins with the potential to facilitate angiogenesis and metastasis. Two tetraspanins, CD9 and CD81, co-immunoprecipitated. Together, these data suggested that tumor cells secrete proteins and exosomes with the potential to modulate their microenvironment and facilitate angiogenesis and metastasis. PMID:20124223

  1. A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients

    NASA Astrophysics Data System (ADS)

    Rodenhizer, Darren; Gaude, Edoardo; Cojocari, Dan; Mahadevan, Radhakrishnan; Frezza, Christian; Wouters, Bradly G.; McGuigan, Alison P.

    2016-02-01

    The profound metabolic reprogramming that occurs in cancer cells has been investigated primarily in two-dimensional cell cultures, which fail to recapitulate spatial aspects of cell-to-cell interactions as well as tissue gradients present in three-dimensional tumours. Here, we describe an engineered model to assemble three-dimensional tumours by rolling a scaffold-tumour composite strip. By unrolling the strip, the model can be rapidly disassembled for snapshot analysis, allowing spatial mapping of cell metabolism in concert with cell phenotype. We also show that the establishment of oxygen gradients within samples that are shaped by oxygen-dependent signalling pathways, as well as the consequential variations in cell growth, response to hypoxic gradients extending from normoxia to severe hypoxia, and therapy responsiveness, are consistent with those of tumours in vivo. Moreover, by using liquid chromatography tandem mass spectrometry, we mapped cellular metabolism and identified spatially defined metabolic signatures of cancer cells to reveal both known and novel metabolic responses to hypoxia.

  2. Selective Intracellular Delivery of Recombinant Arginine Deiminase (ADI) Using pH-Sensitive Cell Penetrating Peptides To Overcome ADI Resistance in Hypoxic Breast Cancer Cells.

    PubMed

    Yeh, Tzyy-Harn; Chen, Yun-Ru; Chen, Szu-Ying; Shen, Wei-Chiang; Ann, David K; Zaro, Jennica L; Shen, Li-Jiuan

    2016-01-04

    Arginine depletion strategies, such as pegylated recombinant arginine deiminase (ADI-PEG20), offer a promising anticancer treatment. Many tumor cells have suppressed expression of a key enzyme, argininosuccinate synthetase 1 (ASS1), which converts citrulline to arginine. These tumor cells become arginine auxotrophic, as they can no longer synthesize endogenous arginine intracellularly from citrulline, and are therefore sensitive to arginine depletion therapy. However, since ADI-PEG20 only depletes extracellular arginine due to low internalization, ASS1-expressing cells are not susceptible to treatment since they can synthesize arginine intracellularly. Recent studies have found that several factors influence ASS1 expression. In this study, we evaluated the effect of hypoxia, frequently encountered in many solid tumors, on ASS1 expression and its relationship to ADI-resistance in human MDA-MB-231 breast cancer cells. It was found that MDA-MB-231 cells developed ADI resistance in hypoxic conditions with increased ASS1 expression. To restore ADI sensitivity as well as achieve tumor-selective delivery under hypoxia, we constructed a pH-sensitive cell penetrating peptide (CPP)-based delivery system to carry ADI inside cells to deplete both intra- and extracellular arginine. The delivery system was designed to activate the CPP-mediated internalization only at the mildly acidic pH (6.5-7) associated with the microenvironment of hypoxic tumors, thus achieving better selectivity toward tumor cells. The pH sensitivity of the CPP HBHAc was controlled by recombinant fusion to a histidine-glutamine (HE) oligopeptide, generating HBHAc-HE-ADI. The tumor distribution of HBHAc-HE-ADI was comparable to ADI-PEG20 in a mouse xenograft model of human breast cancer cells in vivo. In addition, HBHAc-HE-ADI showed increased in vitro cellular uptake in cells incubated in a mildly acidic pH (hypoxic conditions) compared to normal pH (normoxic conditions), which correlated with p

  3. Dedifferentiated Fat Cells as a Novel Source for Cell Therapy to Target Neonatal Hypoxic-Ischemic Encephalopathy.

    PubMed

    Mikrogeorgiou, Alkisti; Sato, Yoshiaki; Kondo, Taiki; Hattori, Tetsuo; Sugiyama, Yuichiro; Ito, Miharu; Saito, Akiko; Nakanishi, Keiko; Tsuji, Masahiro; Kazama, Tomohiko; Kano, Koichiro; Matsumoto, Taro; Hayakawa, Masahiro

    2017-03-09

    Neonatal hypoxic-ischemic (HI) encephalopathy (HIE) remains a major cause of mortality and persistent neurological disabilities in affected individuals. At present, hypothermia is considered to be the only applicable treatment option, although growing evidence suggests that cell-based therapy might achieve better outcomes. Dedifferentiated fat (DFAT) cells are derived from mature adipocytes via a dedifferentiation strategy called ceiling culture. Their abundance and ready availability might make them an ideal therapeutic tool for the treatment of HIE. In the present study, we aimed to determine whether the outcome of HIE can be improved by DFAT cell treatment. HI injury was achieved by ligating the left common carotid artery in 7-day-old rat pups, followed by 1-h exposure to 8% O2. Subsequently, the severity of damage was assessed by diffusion-weighted magnetic resonance imaging to assign animals to equivalent groups. 24 h after hypoxia, DFAT cells were injected at 105 cells/pup into the right external jugular vein. To evaluate brain damage in the acute phase, a group of animals was sacrificed 48 h after the insult, and paraffin sections of the brain were stained to assess several acute injury markers. In the chronic phase, the behavioral outcome was measured by performing a series of behavioral tests. From the 24th day of age, the sensorimotor function was examined by evaluating the initial forepaw placement on a cylinder wall and the latency to falling from a rotarod treadmill. The cognitive function was tested with the novel object recognition (NOR) test. In vitro conditioned medium (CM) prepared from cultured DFAT cells was added at various concentrations to neuronal cell cultures, which were then exposed to oxygen-glucose deprivation (OGD). The number of cells that stained positive for the apoptosis marker active caspase-3 decreased by 73 and 52% in the hippocampus and temporal cortex areas of the brain, respectively, in the DFAT-treated pups. Similarly, the

  4. Interval hypoxic training.

    PubMed

    Bernardi, L

    2001-01-01

    Interval hypoxic training (IHT) is a technique developed in the former Soviet Union, that consists of repeated exposures to 5-7 minutes of steady or progressive hypoxia, interrupted by equal periods of recovery. It has been proposed for training in sports, to acclimatize to high altitude, and to treat a variety of clinical conditions, spanning from coronary heart disease to Cesarean delivery. Some of these results may originate by the different effects of continuous vs. intermittent hypoxia (IH), which can be obtained by manipulating the repetition rate, the duration and the intensity of the hypoxic stimulus. The present article will attempt to examine some of the effects of IH, and, whenever possible, compare them to those of typical IHT. IH can modify oxygen transport and energy utilization, alter respiratory and blood pressure control mechanisms, induce permanent modifications in the cardiovascular system. IHT increases the hypoxic ventilatory response, increase red blood cell count and increase aerobic capacity. Some of these effects might be potentially beneficial in specific physiologic or pathologic conditions. At this stage, this technique appears interesting for its possible applications, but still largely to be explored for its mechanisms, potentials and limitations.

  5. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells

    PubMed Central

    Armitage, Emily G.; Kotze, Helen L.; Allwood, J. William; Dunn, Warwick B.; Goodacre, Royston; Williams, Kaye J.

    2015-01-01

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments. PMID:26508589

  6. Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells.

    PubMed

    Armitage, Emily G; Kotze, Helen L; Allwood, J William; Dunn, Warwick B; Goodacre, Royston; Williams, Kaye J

    2015-10-28

    Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments.

  7. Mammea E/BB, an isoprenylated dihydroxycoumarin protonophore that potently uncouples mitochondrial electron transport, disrupts hypoxic signaling in tumor cells.

    PubMed

    Du, Lin; Mahdi, Fakhri; Jekabsons, Mika B; Nagle, Dale G; Zhou, Yu-Dong

    2010-11-29

    The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC(50) values of 0.96 and 0.89 μM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 μM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.

  8. Mammea E/BB, An Isoprenylated Dihydroxycoumarin Protonophore that Potently Uncouples Mitochondrial Electron Transport Disrupts Hypoxic Signaling in Tumor Cells

    PubMed Central

    Du, Lin; Mahdi, Fakhri; Jekabsons, Mika B.; Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC50 values of 0.96 and 0.89 µM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 µM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlay their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines. PMID:20929261

  9. Hypoxic radiosensitization: adored and ignored.

    PubMed

    Overgaard, Jens

    2007-09-10

    Since observations from the beginning of the last century, it has become well established that solid tumors may contain oxygen-deficient hypoxic areas and that cells in such areas may cause tumors to become radioresistant. Identifying hypoxic cells in human tumors has improved by the help of new imaging and physiologic techniques, and a substantial amount of data indicates the presence of hypoxia in many types of human tumors, although with a considerable heterogeneity among individual tumors. Controlled clinical trials during the last 40 years have indicated that this source of radiation resistance can be eliminated or modified by normobaric or hyperbaric oxygen or by the use of nitroimidazoles as hypoxic radiation sensitizers. More recently, attention has been given to hypoxic cytotoxins, a group of drugs that selectively or preferably destroys cells in a hypoxic environment. An updated systematic review identified 10,108 patients in 86 randomized trials designed to modify tumor hypoxia in patients treated with curative attempted primary radiation therapy alone. Overall modification of tumor hypoxia significantly improved the effect of radiotherapy, with an odds ratio of 0.77 (95% CI, 0.71 to 0.86) for the outcome of locoregional control and with an associated significant overall survival benefit (odds ratio = 0.87; 95% CI, 0.80 to 0.95). No significant influence was found on the incidence of distant metastases or on the risk of radiation-related complications. Ample data exist to support a high level of evidence for the benefit of hypoxic modification. However, hypoxic modification still has no impact on general clinical practice.

  10. Improvement of neuronal cell survival by astrocyte-derived exosomes under hypoxic and ischemic conditions depends on prion protein.

    PubMed

    Guitart, Kathrin; Loers, Gabriele; Buck, Friedrich; Bork, Ute; Schachner, Melitta; Kleene, Ralf

    2016-06-01

    Prion protein (PrP) protects neural cells against oxidative stress, hypoxia, ischemia, and hypoglycemia. In the present study we confirm that cultured PrP-deficient neurons are more sensitive to oxidative stress than wild-type neurons and present the novel findings that wild-type, but not PrP-deficient astrocytes protect wild-type cerebellar neurons against oxidative stress and that exosomes released from stressed wild-type, but not from stressed PrP-deficient astrocytes reduce neuronal cell death induced by oxidative stress. We show that neuroprotection by exosomes of stressed astrocytes depends on exosomal PrP but not on neuronal PrP and that astrocyte-derived exosomal PrP enters into neurons, suggesting neuronal uptake of astrocyte-derived exosomes. Upon exposure of wild-type astrocytes to hypoxic or ischemic conditions PrP levels in exosomes were increased. By mass spectrometry and Western blot analysis, we detected increased levels of 37/67 kDa laminin receptor, apolipoprotein E and the ribosomal proteins S3 and P0, and decreased levels of clusterin/apolipoprotein J in exosomes from wild-type astrocytes exposed to oxygen/glucose deprivation relative to exosomes from astrocytes maintained under normoxic conditions. The levels of these proteins were not altered in exosomes from stressed PrP-deficient astrocytes relative to unstressed PrP-deficient astrocytes. These results indicate that PrP in astrocytes is a sensor for oxidative stress and mediates beneficial cellular responses, e.g. release of exosomes carrying PrP and other molecules, resulting in improved survival of neurons under hypoxic and ischemic conditions.

  11. Tumour necrosis factor-α suppresses the hypoxic response by NF-κB-dependent induction of inhibitory PAS domain protein in PC12 cells.

    PubMed

    Goryo, Kenji; Torii, Satoru; Yasumoto, Ken-Ichi; Sogawa, Kazuhiro

    2011-09-01

    Inflammation is often accompanied by hypoxia. However, crosstalk between signalling pathways activated by inflammation and signalling events that control adaptive response to hypoxia is not fully understood. Here we show that exposure to tumour necrosis factor-α (TNF-α) activates expression of the inhibitory PAS domain protein (IPAS) to suppress the hypoxic response caused by hypoxia-inducible factor (HIF)-1 and HIF-2 in rat pheochromocytoma PC12 cells but not in human hepatoma Hep3B cells. This induction of IPAS was dependent on the nuclear factor-κB (NF-κB) pathway and attenuated hypoxic induction of HIF-1 target genes such as tyrosine hydroxylase (TH) and vascular endothelial growth factor (VEGF). HIF-dependent reporter activity in hypoxia was also decreased following TNF-α treatment. Knockdown of IPAS mRNA by small interfering RNA (siRNA) restored the TNF-α-suppressed hypoxic response. These results indicate that TNF-α is a cell-type specific suppressor of HIFs and suggest a novel crosstalk between stimulation by inflammatory mediators and HIF-dependent hypoxic response.

  12. Serum- and stromal cell-free hypoxic generation of embryonic stem cell-derived hematopoietic cells in vitro, capable of multilineage repopulation of immunocompetent mice.

    PubMed

    Lesinski, Dietrich Armin; Heinz, Niels; Pilat-Carotta, Sandra; Rudolph, Cornelia; Jacobs, Roland; Schlegelberger, Brigitte; Klump, Hannes; Schiedlmeier, Bernhard

    2012-08-01

    Induced pluripotent stem cells (iPSCs) may become a promising source for the generation of patient-specific hematopoietic stem cells (HSCs) in vitro. A crucial prerequisite will be the availability of reliable protocols for the directed and efficient differentiation toward HSCs. So far, the most robust strategy for generating HSCs from pluripotent cells in vitro has been established in the mouse model involving ectopic expression of the human transcription factor HOXB4. However, most differentiation protocols include coculture on a xenogenic stroma cell line and the use of animal serum. Involvement of any of both would pose a major barrier to the translation of those protocols to human autologous iPSCs intended for clinical use. Therefore, we asked whether long-term repopulating HSCs can, in principle, be generated from embryonic stem cells without stroma cells or serum. Here, we showed that long-term multilineage engraftment could be accomplished in immunocompetent mice when HSCs were generated in serum-free medium without stroma cell support and when hypoxic conditions were used. Under those conditions, HOXB4(+) embryonic stem cell-derived hematopoietic stem and progenitor cells were immunophenotypically similar to definitive bone marrow resident E-SLAM(+) (CD150(+)CD48(-)CD45(+)CD201(+)) HSCs. Thus, our findings may ease the development of definitive, adult-type HSCs from pluripotent stem cells, entirely in vitro.

  13. Human bone marrow-derived mesenchymal stem cells display enhanced clonogenicity but impaired differentiation with hypoxic preconditioning.

    PubMed

    Boyette, Lisa B; Creasey, Olivia A; Guzik, Lynda; Lozito, Thomas; Tuan, Rocky S

    2014-02-01

    Stem cells are promising candidate cells for regenerative applications because they possess high proliferative capacity and the potential to differentiate into other cell types. Mesenchymal stem cells (MSCs) are easily sourced but do not retain their proliferative and multilineage differentiative capabilities after prolonged ex vivo propagation. We investigated the use of hypoxia as a preconditioning agent and in differentiating cultures to enhance MSC function. Culture in 5% ambient O(2) consistently enhanced clonogenic potential of primary MSCs from all donors tested. We determined that enhanced clonogenicity was attributable to increased proliferation, increased vascular endothelial growth factor secretion, and increased matrix turnover. Hypoxia did not impact the incidence of cell death. Application of hypoxia to osteogenic cultures resulted in enhanced total mineral deposition, although this effect was detected only in MSCs preconditioned in normoxic conditions. Osteogenesis-associated genes were upregulated in hypoxia, and alkaline phosphatase activity was enhanced. Adipogenic differentiation was inhibited by exposure to hypoxia during differentiation. Chondrogenesis in three-dimensional pellet cultures was inhibited by preconditioning with hypoxia. However, in cultures expanded under normoxia, hypoxia applied during subsequent pellet culture enhanced chondrogenesis. Whereas hypoxic preconditioning appears to be an excellent way to expand a highly clonogenic progenitor pool, our findings suggest that it may blunt the differentiation potential of MSCs, compromising their utility for regenerative tissue engineering. Exposure to hypoxia during differentiation (post-normoxic expansion), however, appears to result in a greater quantity of functional osteoblasts and chondrocytes and ultimately a larger quantity of high-quality differentiated tissue.

  14. Overexpression of HIF-1α in mesenchymal stem cells contributes to repairing hypoxic-ischemic brain damage in rats.

    PubMed

    Lin, Deju; Zhou, Liping; Wang, Biao; Liu, Lizhen; Cong, Li; Hu, Chuanqin; Ge, Tingting; Yu, Qin

    2017-01-01

    Preclinical researches on mesenchymal stem cells (MSCs) transplantation, which is used to treat hypoxic-ischemic (HI) brain damage, have received inspiring achievements. However, the insufficient migration of active cells to damaged tissues has limited their potential therapeutic effects. There are some evidences that hypoxia inducible factor-1 alpha (HIF-1α) promotes the viability and migration of the cells. Here, we aim to investigate whether overexpression of HIF-1α in MSCs could improve the viability and migration capacity of cells, and its therapeutic efficiency on HI brain damage. In the study, MSCs with HIF-1α overexpression was achieved by recombinant lentiviral vector and transplanted to the rats subsequent to HI. Our data indicated that overexpression of HIF-1α promoted the viability and migration of MSCs, HIF-1α overexpressed MSCs also had a stronger therapeutic efficiency on HI brain damaged treatment by mitigating the injury on behavioral and histological changes evoked by HI insults, accompanied with more MSCs migrating to cerebral damaged area. This study demonstrated that HIF-1α overexpression could increase the MSCs' therapeutic efficiency in HI and the promotion of the cells' directional migration to cerebral HI area by overexpression may be responsible for it, which showed that transplantation of MSCs with HIF-1α overexpression is an attractive therapeutic option to treat HI-induced brain injury in the future.

  15. Sustained radiosensitization of hypoxic glioma cells after oxygen pretreatment in an animal model of glioblastoma and in vitro models of tumor hypoxia.

    PubMed

    Clarke, Ryon H; Moosa, Shayan; Anzivino, Matthew; Wang, Yi; Floyd, Desiree Hunt; Purow, Benjamin W; Lee, Kevin S

    2014-01-01

    Glioblastoma multiforme (GBM) is the most common and lethal form of brain cancer and these tumors are highly resistant to chemo- and radiotherapy. Radioresistance is thought to result from a paucity of molecular oxygen in hypoxic tumor regions, resulting in reduced DNA damage and enhanced cellular defense mechanisms. Efforts to counteract tumor hypoxia during radiotherapy are limited by an attendant increase in the sensitivity of healthy brain tissue to radiation. However, the presence of heightened levels of molecular oxygen during radiotherapy, while conventionally deemed critical for adjuvant oxygen therapy to sensitize hypoxic tumor tissue, might not actually be necessary. We evaluated the concept that pre-treating tumor tissue by transiently elevating tissue oxygenation prior to radiation exposure could increase the efficacy of radiotherapy, even when radiotherapy is administered after the return of tumor tissue oxygen to hypoxic baseline levels. Using nude mice bearing intracranial U87-luciferase xenografts, and in vitro models of tumor hypoxia, the efficacy of oxygen pretreatment for producing radiosensitization was tested. Oxygen-induced radiosensitization of tumor tissue was observed in GBM xenografts, as seen by suppression of tumor growth and increased survival. Additionally, rodent and human glioma cells, and human glioma stem cells, exhibited prolonged enhanced vulnerability to radiation after oxygen pretreatment in vitro, even when radiation was delivered under hypoxic conditions. Over-expression of HIF-1α reduced this radiosensitization, indicating that this effect is mediated, in part, via a change in HIF-1-dependent mechanisms. Importantly, an identical duration of transient hyperoxic exposure does not sensitize normal human astrocytes to radiation in vitro. Taken together, these results indicate that briefly pre-treating tumors with elevated levels of oxygen prior to radiotherapy may represent a means for selectively targeting radiation

  16. Mitochondrial DNA damage and a hypoxic response are induced by CoCl2 in rat neuronal PC12 cells

    PubMed Central

    Wang, Guichun; Hazra, Tapas K.; Mitra, Sankar; Lee, Heung-Man; Englander, Ella W.

    2000-01-01

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl2, mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 µM CoCl2. In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1α (HIF-1α), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage. PMID:10773083

  17. Mitochondrial DNA damage and a hypoxic response are induced by CoCl(2) in rat neuronal PC12 cells.

    PubMed

    Wang, G; Hazra, T K; Mitra, S; Lee, H M; Englander, E W

    2000-05-15

    Generation of reactive oxygen species (ROS) and activation of a transcriptional program that mimics the hypoxic response have been documented in cultured cells in the presence of cobalt chloride. We found that in the presence of hypoxia-mimicking concentrations of CoCl(2), mitochondrial but not nuclear DNA damage is induced in rat neuronal, PC12 cells. To our knowledge, this is the first documentation of induction of mitochondrial DNA (mtDNA) damage under these conditions. Likewise, we provide the first evidence for elevation of MYH, the mammalian homolog of the Escherichia coli MutY DNA glycosylase, in mammalian cells. Recently, the human MYH was implicated in repair of oxidative DNA damage and shown to carry a mitochondrial localization sequence. Here, an induction of mtDNA damage and a time-dependent increase in the MYH level were detected with exposure of cells to 100 microM CoCl(2). In addition, the levels of proteins involved in cellular responses to hypoxia, ROS and nuclear DNA damage; hypoxia-inducible factor 1alpha(HIF-1alpha), p53, p21 and PCNA were also modulated temporally. Earlier studies suggested that the mtDNA is a primary target for oxidative damage. Our findings extend these observations and suggest that activation of DNA repair processes is associated with the presence of mtDNA damage.

  18. Orexins protect neuronal cell cultures against hypoxic stress: an involvement of Akt signaling.

    PubMed

    Sokołowska, Paulina; Urbańska, Anna; Biegańska, Kaja; Wagner, Waldemar; Ciszewski, Wojciech; Namiecińska, Magdalena; Zawilska, Jolanta B

    2014-01-01

    Orexins A and B are peptides produced mainly by hypothalamic neurons that project to numerous brain structures. We have previously demonstrated that rat cortical neurons express both types of orexin receptors, and their activation by orexins initiates different intracellular signals. The present study aimed to determine the effect of orexins on the Akt kinase activation in the rat neuronal cultures and the significance of that response in neurons subjected to hypoxic stress. We report the first evidence that orexins A and B stimulated Akt in cortical neurons in a concentration- and time-dependent manner. Orexin B more potently than orexin A increased Akt phosphorylation, but the maximal effect of both peptides on the kinase activation was very similar. Next, cultured cortical neurons were challenged with cobalt chloride, an inducer of reactive oxygen species and hypoxia-mediated signaling pathways. Under conditions of chemical hypoxia, orexins potently increased neuronal viability and protected cortical neurons against oxidative stress. Our results also indicate that Akt kinase plays an important role in the pro-survival effects of orexins in neurons, which implies a possible mechanism of the orexin-induced neuroprotection.

  19. Digoxin downregulates NDRG1 and VEGF through the inhibition of HIF-1α under hypoxic conditions in human lung adenocarcinoma A549 cells.

    PubMed

    Wei, Dong; Peng, Jing-Jing; Gao, Hui; Li, Hua; Li, Dong; Tan, Yong; Zhang, Tao

    2013-04-02

    Digoxin, an inhibitor of Na+/K+ ATPase, has been used in the treatment of heart-related diseases (such as congestive heart failure and atrial arrhythmia) for decades. Recently, it was reported that digoxin is also an effective HIF-1α inhibitor. We investigated whether digoxin could suppress tumor cell growth through HIF-1α in non-small cell lung cancer cells (A549 cells) under hypoxic conditions. An MTT assay was used to measure cell viability. RT-PCR and western blotting were performed to analyze the mRNA and protein expression of VEGF, NDRG1, and HIF-1α. HIF-1α nuclear translocation was then determined by EMSA. Digoxin was found to inhibit the proliferation of A549 cells under hypoxic conditions. Our results showed that hypoxia led to the upregulation of VEGF, NDRG1, and HIF-1α both at the mRNA and protein levels. We also found that the hypoxia-induced overexpression of VEGF, NDRG1, and HIF-1α was suppressed by digoxin in a concentration-dependent manner. As expected, our EMSA results demonstrated that under hypoxic conditions HIF-1α nuclear translocation was also markedly reduced by digoxin in a concentration-dependent manner. Our results suggest that digoxin downregulated hypoxia-induced overexpression of VEGF and NDRG1 at the transcriptional level probably through the inhibition of HIF-1α synthesis in A549 cells.

  20. Insights into the neuroprotective mechanisms of 2-iminobiotin employing an in-vitro model of hypoxic-ischemic cell injury.

    PubMed

    Zitta, Karina; Peeters-Scholte, Cacha; Sommer, Lena; Parczany, Kerstin; Steinfath, Markus; Albrecht, Martin

    2016-12-05

    Several animal models have been used to simulate cerebral hypoxia-ischemia and suggested neuroprotective effects of the biotin analogue 2-iminobiotin (2-IB). The aims of this study were to employ a human in-vitro hypoxia model to confirm protective effects of 2-IB on neuronal cells, determine the optimal neuroprotective concentrations of 2-IB and scrutinize underlying cellular effects of 2-IB. Neuronal IMR-32 cells were exposed to hypoxia employing an enzymatic hypoxia system and were thereafter incubated with various concentrations of 2-IB (10 to 300ng/ml). Cell damage, metabolic activity and generation of reactive oxygen species were quantified using colorimetric/fluorometric lactate dehydrogenase (LDH), tetrazolium-based (MTS) and reactive oxygen species assays. Proteome profiling arrays were performed to evaluate the regulation of cell stress protein expression by hypoxia and 2-IB. Seven hours of hypoxia led to morphological changes in IMR-32 cultures, increased neuronal cell damage (P<0.001), reduction of metabolic activity (P<0.01) and enhanced reactive oxygen species production (P<0.05). Post-hypoxic application of 2-IB (30ng/ml) attenuated hypoxia-induced LDH release (P<0.05) and increased metabolic activity of IMR-32 cells (P<0.05), while reactive oxygen species production was only by trend decreased. Array-based protein expression profiling revealed that 2-IB attenuated the expression of several hypoxia-induced cell stress-associated proteins by more than 25% (pp38α, HIF2α, ADAMTS1, pHSP27, PON2, PON3 and p27). Hypoxia-induced neuronal cell damage can be simulated using the described in-vitro model. 2-IB inhibits hypoxia-mediated neurotoxicity most efficiently at 30ng/ml and the underlying mechanisms involve a downregulation of stress-associated protein expression. Our results suggest 2-IB as a potential drug for the treatment of perinatal hypoxia-ischemia.

  1. [The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

    PubMed

    Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

    2014-01-01

    It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

  2. Physiological and hypoxic oxygen concentration differentially regulates human c-Kit+ cardiac stem cell proliferation and migration.

    PubMed

    Bellio, Michael A; Rodrigues, Claudia O; Landin, Ana Marie; Hatzistergos, Konstantinos E; Kuznetsov, Jeffim; Florea, Victoria; Valasaki, Krystalenia; Khan, Aisha; Hare, Joshua M; Schulman, Ivonne Hernandez

    2016-12-01

    Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O2). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O2 concentrations. Physiological O2 levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of β-galactosidase activity (-4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O2 reduces senescence and promotes quiescence. Furthermore, physiological O2 levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O2 migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O2 concentration on CSC biology and has important implications for refining stem cell therapies.

  3. Hypoxic stress triggers a programmed cell death pathway to induce vascular cavity formation in Pisum sativum roots.

    PubMed

    Sarkar, Purbasha; Gladish, Daniel K

    2012-12-01

    Flooding at warm temperatures induces hypoxic stress in Pisum sativum seedling roots. In response, some undifferentiated cells in the primary root vascular cylinder start degenerating and form a longitudinal vascular cavity. Changes in cellular morphology and cell wall ultrastructure detected previously in the late stages of cavity formation suggest possible involvement of programmed cell death (PCD). In this study, cytological events occurring in the early stages of cavity formation were investigated. Systematic DNA fragmentation, a feature of many PCD pathways, was detected in the cavity-forming roots after 3 h of flooding in situ by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay and in isolated total DNA by gel electrophoresis. High molecular weight DNA fragments of about 20-30 kb were detected by pulse-field gel electrophoresis, but no low-molecular weight internucleosomal DNA fragments were detected by conventional gel electrophoresis. Release of mitochondrial cytochrome c protein into the cytosol, an integral part of mitochondria-dependent PCD pathways, was detected in the cavity-forming roots within 2 h of flooding by fluorescence microscopy of immunolabeled cytochrome c in situ and in isolated mitochondrial and cytosolic protein fractions by western blotting. DNA fragmentation and cytochrome c release remained confined to the undifferentiated cells in center of the root vascular cylinders, even after 24 h of flooding, while outer vascular cylinder cells and cortical cells maintained cellular integrity and normal activity. These findings confirm that hypoxia-induced vascular cavity formation in P. sativum roots involves PCD, and provides a chronological model of cytological events involved in this rare and understudied PCD system.

  4. Astroglial Activation by an Enriched Environment after Transplantation of Mesenchymal Stem Cells Enhances Angiogenesis after Hypoxic-Ischemic Brain Injury

    PubMed Central

    Cho, Sung-Rae; Suh, Hwal; Yu, Ji Hea; Kim, Hyongbum (Henry); Seo, Jung Hwa; Seo, Cheong Hoon

    2016-01-01

    Transplantation of mesenchymal stem cells (MSCs) has paracrine effects; however, the effects are known to be largely limited. Here we investigated the combination effects of cell transplantation and enriched environment (EE) in a model of hypoxic-ischemic brain injury. Brain damage was induced in seven-day-old mice by unilateral carotid artery ligation and exposure to hypoxia (8% O2 for 90 min). At six weeks of age, the mice were randomly assigned to four groups: phosphate-buffered saline (PBS)-control (CON), PBS-EE, MSC-CON, and MSC-EE. Rotarod and grip strength tests were performed to evaluate neurobehavioral functions. Histologic evaluations were also performed to confirm the extent of astrocyte activation and endogenous angiogenesis. An array-based multiplex ELISA and Western blot were used to identify growth factors in vivo and in vitro. Two weeks after treatment, levels of astrocyte density and angiogenic factors were increased in MSC-EE mice, but glial scarring was not increased. Eight weeks after treatment, angiogenesis was increased, and behavioral outcomes were synergistically improved in the MSC-EE group. Astrocytes co-cultured with MSCs expressed higher levels of angiogenic factors than astrocytes cultured alone. The mechanisms of this synergistic effect included enhanced repair processes, such as increased endogenous angiogenesis and upregulation of angiogenic factors released from activated astrocytes. PMID:27649153

  5. A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia.

    PubMed

    Bouquet, Fanny; Ousset, Marielle; Biard, Denis; Fallone, Frédérique; Dauvillier, Stéphanie; Frit, Philippe; Salles, Bernard; Muller, Catherine

    2011-06-01

    DNA-dependent protein kinase (DNA-PK) is involved in DNA double-strand break (DSB) signalling and repair. We report that DNA-PK is activated by mild hypoxia conditions (0.1-1% O₂) as shown by (1) its autophosphorylation on Ser2056, and (2) its mobilisation from a soluble nucleoplasmic compartment to a less extractable nuclear fraction. The recruitment of DNA-PK was not followed by activation and recruitment of the XRCC4-DNA-ligase-IV complex, suggesting that DSBs are not responsible for activation of DNA-PK. To unravel the mechanism of DNA-PK activation, we show that exposure of cells to trichostatin A, a histone deacetylase inhibitor, leads to DNA-PK autophosphorylation and relocalisation to DNA. Histone acetylation (mainly H3K14) is increased in hypoxic cells and treatment with anacardic acid, an inhibitor of histone acetyl transferase, prevented both histone modifications and DNA-PK activation in hypoxic conditions. Importantly, in using either silenced DNA-PK cells or cells exposed to a specific DNA-PK inhibitor (NU7026), we demonstrated that hypoxic DNA-PK activation positively regulates the key transcription factor HIF-1 and one subsequent target gene, GLUT1. Our results show that hypoxia initiates chromatin modification and consequently DNA-PK activation, which positively regulate cellular oxygen-sensing and oxygen-signalling pathways.

  6. Final report on the United States phase I clinical trial of the hypoxic cell radiosensitizer, misonidazole (Ro-07-0582; NSC No. 261037

    SciTech Connect

    Phillips, T.L.; Wasserman, T.H.; Johnson, R.J.; Levin, V.A.; VanRaalte, G.

    1981-10-15

    The hypoxic cell sensitizer misonidazole began phase I evaluation in the United States in July 1977. One hundred two patients received 104 individual courses of drug. Drug was administered from once to five times per week over time spans from one to six weeks. The individual doses ranged 1 to 5 g/m. The major toxicity noted was neurologic; 49% of evaluable courses showed peripheral neuropathy, and 9% of evaluable courses showed central nervous system effects and/or ototoxicity. In addition, 48 of 102 patients exhibited some degree of nausea and vomiting. The concomitant administration of dexamethasone and phenytoin sodium appeared to lower the incidence of neuropathy. Observations of efficacy were made comparatively in five patients who had multiple lesions treated with and without misonidazole. All five showed increased response in the lesions treated with misonidazole. It is concluded that misonidazole is a reasonably safe and potentially effective hypoxic cell sensitizer whose dose-limiting toxicity is neurologic.

  7. [The effect of the gas hypoxic mixture GHM-8 on the capacity of the stromal clonogenic cells (CFU-F) in rat bone marrow for postradiation recovery].

    PubMed

    Konopliannikov, A G; Waĭnson, A A; Kolesnikova, A I; Zaĭtsev, V A; Kal'sina, S Sh; Lepekhina, L A

    1993-01-01

    The effect of gas hypoxic mixture, containing 8% of O2 (GHM-8), on the ability of cell precursors of haemopoietic stroma (which form colonies (clones) of fibroblasts (CFU-F) in a culture, and are present in the bone marrow of adult rats) to repair potentially lethal and sublethal radiation damages has been investigated. The recovery of CFU-F from potentially lethal damages, that was studied after their delayed survival in a culture following irradiation of animals, proceeds at nearly the same rate in cells irradiated both in the air and in hypoxic conditions (GHM-8). Fractionated irradiation reduces the radioprotective effect of GHM-8 for CFU-F, particularly for the radioresistant subpopulation; the ability of CFU-F to recover from sublethal radiation damages decreases.

  8. Cold preservation of endothelial cells in sucrose-based solution (SbS) and University of Wisconsin (UW) solutions: comparison of normoxic or hypoxic storage.

    PubMed

    Hawkins, M; Sales, K M; Dijk, S; Fuller, B

    2005-01-01

    Cold preservation of endothelial cells was studied, comparing primary endothelial cells (human umbilical vein endothelial cells - HUVECs) and a continuously growing cell line (ECV304 cells). Viability at the end of 24h cold preservation was measured by dye exclusion, whilst metabolism was assessed by Alamar blue conversion. Two preservation solutions were studied (UW solution) and sucrose-based (SbS) in both cell types. The response was similar in both cell types to preservation under normoxic conditions (with percentage dye exclusion maintained at about 80 percent in both preservation solutions) whereas under hypoxic conditions ECV304 were more sensitive to preservation in UW solution (dye exclusion reduced to 43.5+/-1.4 percent versus 73.6+/-14 percent (P<0.01). Metabolism assessed by Alamar blue conversion after cold preservation and rewarming was similar in both ECV304 and HUVECs after storage under normoxic conditions in UW solution, but in both cell types, metabolism was higher in SbS (P<0.05 and p<0.01) than in UW solution. Under hypoxic conditions, both cell types showed similar recovery of metabolism after storage in either UW or SbS. If the cells (in this case ECV304 under aerobic conditions) were stored for 24h and then allowed to rewarm in either of the respective preservation solutions (UW or SbS for 1h) before the Alamar blue test, metabolism was higher (p less than 0.01) in those exposed to SbS. UW solution and SbS provide similar protection for endothelial cells under hypoxic conditions, but SbS has some advantages under normoxic storage or if the cells experience variable temperatures in the presence of residual preservation solution at the end of cold preservation period.

  9. Remarkable photocytotoxicity in hypoxic HeLa cells by a dipyridophenazine copper(II) Schiff base thiolate.

    PubMed

    Lahiri, Debojyoti; Majumdar, Ritankar; Mallick, Dibyendu; Goswami, Tridib K; Dighe, Rajan R; Chakravarty, Akhil R

    2011-08-01

    Copper(II) complexes [Cu(satp)(L)] (1-3) of a Schiff base thiolate (salicylidene-2-aminothiophenol, H2satp) and phenanthroline bases (L), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 3), were prepared, characterized and their anaerobic DNA photocleavage activity and hypoxic photocytotoxicity studied. The redox active complexes show the Cu(II)-Cu(I) couple near -0.5 V for 1 and near 0.0 V vs. SCE (saturated calomel electrode) for 2 and 3. The one-electron paramagnetic complexes (~1.85 μB) are avid DNA binders giving Kb values within 1.0×10(5)-8.0×10(5) M(-1). Thermal melting and viscosity data along with molecular docking calculations suggest DNA groove and/or partial intercalative binding of the complexes. The complexes show anaerobic DNA cleavage activity in red light under argon via type-I pathway, while DNA photocleavage in air proceeds via hydroxyl radical pathway. The DFT (density functional theory) calculations reveal a thyil radical pathway for the anaerobic DNA photocleavage activity and suggest the possibility of generation of a transient copper(I) species due to bond breakage between the copper and sulfur to generate the thyil radical. An oxidation of the copper(I) species is likely by oxygen in an aerobic medium or by the buffer medium in an anaerobic condition. Complex 3 exhibits significant photocytotoxicity in HeLa cells (IC50=8.3(±1.0) μM) in visible light, while showing lower dark toxicity (IC50=17.2(±1.0) μM). A significant reduction in the dark toxicity is observed under hypoxic cellular conditions (IC50=30.0(±1.0) μM in dark), while retaining its photocytotoxicity (IC50=8.0(±1.0) μM).

  10. Brusatol inhibits HIF-1 signaling pathway and suppresses glucose uptake under hypoxic conditions in HCT116 cells

    PubMed Central

    Lu, Yapeng; Wang, Bo; Shi, Qian; Wang, Xueting; Wang, Dang; Zhu, Li

    2016-01-01

    Hypoxia-inducible factor-1 (HIF-1) is an important transcription factor that induces adaptive responses upon low oxygen conditions in human cancers and triggers off a poor prognostic outcome of conventional treatments. In this study, we discovered for the first time that brusatol (BRU), a quassinoid extracted from Brucea Esters, has the capability to inhibit HIF-1 signaling pathway. We found that BRU concentration-dependently down-regulated HIF-1α protein levels under hypoxia or CoCl2-induced mimic hypoxia in HCT116 cells without causing significant cytotoxicity. Besides, the transactivation activity of HIF-1 was suppressed by BRU under hypoxic conditions, as well as the expression of HIF-1 target genes, including VEGF, GLUT1, HK2 and LDHA. In addition, BRU can also decrease glucose consumption under hypoxia through inhibition of HIF-1 signaling pathway. Further studies revealed that the inhibitory effect of BRU on HIF-1 signaling pathway might be attributed to promoting degradation of HIF-1α. Interestingly, intracellular reactive oxygen species (ROS) levels and mitochondrial ROS level were both decreased by BRU treatment, indicating the involvment of mitochondrial ROS regulation in the action of BRU. Taken together, these results provided clear evidence for BRU-mediated HIF-1α regulation and suggested its therapeutic potential in colon tumors. PMID:27982118

  11. Hypoxic Preconditioning Enhances the Benefit of Cardiac Progenitor-Cell Therapy for Treatment of Myocardial Infarction by Inducing CXCR4 Expression

    PubMed Central

    Tang, Yao Liang; Zhu, Wuqiang; Cheng, Min; Chen, Lijuan; Zhang, John; Sun, Tao; Kishore, Raj; Phillips, M. Ian; Losordo, Douglas W.; Qin, Gangjian

    2009-01-01

    Myocardial infarction (MI) rapidly depletes the endogenous cardiac progenitor-cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac-cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal-cell–derived factor 1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone-marrow—derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine MI model. We found that hypoxic preconditioning increased CXCR4 expression in cardiosphere-derived, Lin−/c-kit+ progenitor (CLK) cells and markedly augmented CLK-cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced MI, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the SDF-1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor-cell—based regenerative therapy. PMID:19407239

  12. Identification of miRNAs Involved in the Protective Effect of Sevoflurane Preconditioning Against Hypoxic Injury in PC12 Cells.

    PubMed

    Sun, Yingying; Li, Yuanhai; Liu, Lei; Wang, Yiqiao; Xia, Yingjing; Zhang, Lingli; Ji, Xuewu

    2015-11-01

    The mechanism of sevoflurane preconditioning-induced neuroprotection is poorly understood. This study was aimed at identifying microRNAs (miRNAs) involved in the protective effect of sevoflurane preconditioning against hypoxic injury using the miRCURYTM LNA Array. The screened differentially expressed miRNAs were further validated using qRT-PCR. Finally, after transfection of miRNA (miR-101a or miR-34b) mimics or inhibitor, MTT and flow cytometry assays were used to evaluate cell survival and apoptosis in sevoflurane preconditioning. qRT-PCR confirmed the changes in expression of differentially expressed miRNAs that were screened by the microarray: down-regulation of rno-miR-101a, rno-miR-106b, and rno-miR-294 and up-regulation of rno-miR-883, rno-miR-16, and rno-miR-34b. MiR-101a and miR-34b were the most differentially expressed miRNAs. Sevoflurane preconditioning-inhibited apoptosis and preconditioning-enhanced cell viability of PC12 cells were significantly attenuated by transfection of miR-101a mimetic or miR-34b inhibitors, but were significantly enhanced by transfection of miR-34b mimetic. Therefore, a number of miRNAs, including miR-101a and miR-34b, might play important roles in the neuroprotection induced by sevoflurane preconditioning. Such miRNAs might provide novel targets for preventive and therapeutic strategies against cerebral ischemia-reperfusion injury.

  13. Influence of high-altitude hypoxic environments on the survival of cochlear hair cells and spiral ganglion neurons in rats

    PubMed Central

    Fan, Dongyan; Ren, Hailong; Danzeng, Dunzhu; Li, Haonan; Wang, Ping

    2016-01-01

    The aim of the present study was to observe the histological changes in the peripheral auditory system in rats at different time-points after relocating from low altitude to high altitude (3,600 m). The general physical condition of the rats was observed and cochlear tissue samples were obtained every month. The morphology and survival of the cochlear hair cells (HCs) were observed using cochlear surface preparation at 1, 30, 90, 120, 150 and 180 days after moving to the plateau area. Changes in spiral ganglion neurons (SGNs) were detected at different time-points using immunofluorescence technology on frozen sections. No obvious morphological changes were observed in the cochlear HCs within 1–3 months of the rats moving to the plateau area, and there was little loss of outer HCs (OHCs) at 3 months. Cell swelling, dislocation and loss of cochlear OHCs were apparent at 4 months, and the losses of cochlear OHCs and inner HCs (IHCs) were 54 and 39%, respectively at 6 months. The loss of SGNs was observed at 3 months, and there was a loss of 28–35% of SGNs during 3–6 months. Thus, a high-altitude hypoxic environment influenced the cochlear HCs in rats after moving to the plateau area in a time-dependent manner. The damage to SGNs occurred earlier than the HCs, although SGN damage was not aggravated with time. Furthermore, compared with cochlear HCs, cochlear SGNs were identified to be markedly more sensitive to hypoxia, and exerted an adaptive mechanism to protect neurons from hypoxia. PMID:28101341

  14. Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

    PubMed Central

    Chang, Woochul; Kim, Ran; Park, Sang In; Jung, Yu Jin; Ham, Onju; Lee, Jihyun; Kim, Ji Hyeong; Oh, Sekyung; Lee, Min Young; Kim, Jongmin; Park, Moon-Seo; Chung, Yong-An; Hwang, Ki-Chul; Maeng, Lee-So

    2015-01-01

    The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects. PMID:26062554

  15. uPAR induces epithelial–mesenchymal transition in hypoxic breast cancer cells

    PubMed Central

    Lester, Robin D.; Jo, Minji; Montel, Valérie; Takimoto, Shinako; Gonias, Steven L.

    2007-01-01

    Hypoxia activates genetic programs that facilitate cell survival; however, in cancer, it may promote invasion and metastasis. In this study, we show that breast cancer cells cultured in 1.0% O2 demonstrate changes consistent with epithelial–mesenchymal transition (EMT). Snail translocates to the nucleus, and E-cadherin is lost from plasma membranes. Vimentin expression, cell migration, Matrigel invasion, and collagen remodeling are increased. Hypoxia-induced EMT is accompanied by increased expression of the urokinase-type plasminogen activator receptor (uPAR) and activation of cell signaling factors downstream of uPAR, including Akt and Rac1. Glycogen synthase kinase-3β is phosphorylated, and Snail expression is increased. Hypoxia-induced EMT is blocked by uPAR gene silencing and mimicked by uPAR overexpression in normoxia. Antagonizing Rac1 or phosphatidylinositol 3-kinase also inhibits development of cellular properties associated with EMT in hypoxia. Breast cancer cells implanted on chick chorioallantoic membranes and treated with CoCl2, to model hypoxia, demonstrate increased dissemination. We conclude that in hypoxia, uPAR activates diverse cell signaling pathways that cooperatively induce EMT and may promote cancer metastasis. PMID:17664334

  16. FoxO proteins mediate hypoxic induction of connective tissue growth factor in endothelial cells.

    PubMed

    Samarin, Jana; Wessel, Julia; Cicha, Iwona; Kroening, Sven; Warnecke, Christina; Goppelt-Struebe, Margarete

    2010-02-12

    Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

  17. DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

    PubMed Central

    Kaluzová, Milota; Kaluz, Stefan; Lerman, Michael I.; Stanbridge, Eric J.

    2004-01-01

    We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53+/+ but not the isogenic p53−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented. PMID:15199132

  18. Neuroprotective effects of oligodendrocyte progenitor cell transplantation in premature rat brain following hypoxic-ischemic injury.

    PubMed

    Chen, Long-Xia; Ma, Si-Min; Zhang, Peng; Fan, Zi-Chuan; Xiong, Man; Cheng, Guo-Qiang; Yang, Yi; Qiu, Zi-Long; Zhou, Wen-Hao; Li, Jin

    2015-01-01

    Periventricular leukomalacia (PVL) is a common ischemic brain injury in premature infants for which there is no effective treatment. The objective of this study was to determine whether transplanted mouse oligodendrocyte progenitor cells (OPCs) have neuroprotective effects in a rat model of PVL. Hypoxia-ischemia (HI) was induced in 3-day-old rat pups by left carotid artery ligation, followed by exposure to 6% oxygen for 2.5 h. Animals were assigned to OPC transplantation or sham control groups and injected with OPCs or PBS, respectively, and sacrificed up to 6 weeks later for immunohistochemical analysis to investigate the survival and differentiation of transplanted OPCs. Apoptosis was evaluated by double immunolabeling of brain sections for caspase-3 and neuronal nuclei (NeuN), while proliferation was assessed using a combination of anti-Nestin and -bromodeoxyuridine antibodies. The expression of brain-derived neurotrophic factor (BDNF) and Bcl-2 was examined 7 days after OPC transplantation. The Morris water maze was used to test spatial learning and memory. The results showed that transplanted OPCs survived and formed a myelin sheath, and stimulated BDNF and Bcl-2 expression and the proliferation of neural stem cells (NSC), while inhibiting HI-induced neuronal apoptosis relative to control animals. Moreover, deficits in spatial learning and memory resulting from HI were improved by OPC transplantation. These results demonstrate an important neuroprotective role for OPCs that can potentially be exploited in cell-based therapeutic approaches to minimize HI-induced brain injury.

  19. Immunosuppression by hypoxic cell radiosensitizers: a phenomenon of potential clinical importance

    SciTech Connect

    Rockwell, S.; Kapp, D.S.

    1982-06-01

    The nitroimidazoles metronidazole, misonidazol, and desmethyl misonidazole are currently undergoing clinical trials as possible adjuncts to radiotherapy. Ongoing clinical trials are evaluating the effectiveness of these agents and also documenting the pharmacokinetics and toxicities of radiosensitizing doses of these drugs in man. A variety of toxic effects have been noted in man, including anorexia, nausea and vomiting, peripheral neuropathy, central nervous system symptoms, ototoxicity, allergy, and fear. Laboratory studies have also suggested that these agents have potential to be mutagenic, carcinogenic, and teratogenic. In the editorial presented, the author attempts to draw attention to an additional toxic effect of nitroimidazoles - the inhibition of cell-mediated immune responses. (JMT)

  20. Radiation dose fractionation studies with hypoxic cell radiosensitizers using a murine tumor. [X-ray; mice

    SciTech Connect

    Hill, R.P.

    1982-03-01

    The ability of five nitroimidazoles, metronidazole (MET), misonidazole (MISO), desmethymisonidazole (DMM), SR 2508 and SR 2555, to sensitize the KHT sarcoma to radiation treatment has been compared for drug doses in the range 0-1.5 g/Kg. Single radiation doses or two different daily fractionation schedules (4 fractions of 5 Gy each or 7 fraction of 3 Gy each) were used; the tumor cell survival was determined using either an in vivo or in vitro colony assay. Each radiation (100 kVp X rays at 11 Gy/min) treatment was given locally, 60-70 min (MET) or 30-40 min (other drugs) after either intraperitoneal (MET, MISO, DMM) or intraveous (SR 2508, SR 2555) injection of the drugs; these times have been shown to be optimum for this tumor. For the single doses and both fractionation schedules the tumor cell survival, following the irradiation treatment, declined as the drug dose increased in the range 0 to 0.75 g/Kg for all the drugs, but above this dose level a plateau was reached and the amount of sensitization remained essentially constant. In this plateau region the reduction in survival achieved was similar for single doses and 5 Gy fraction but was less for 3 Gy fractions, indicating that sensitization was smaller for the smaller dose fractions. For the 4 x 5 Gy fractionation schedule the plateau level of survival was lowest for MISO, DMM and SR 2508, slightly higher for SR 2555 and much higher for MET. For the 3 Gy fractions SR 2508 appeared slightly less effective than MISO and DMM.

  1. Novel PI3K and mTOR Inhibitor NVP-BEZ235 Radiosensitizes Breast Cancer Cell Lines under Normoxic and Hypoxic Conditions

    PubMed Central

    Kuger, Sebastian; Cörek, Emre; Polat, Bülent; Kämmerer, Ulrike; Flentje, Michael; Djuzenova, Cholpon S.

    2014-01-01

    In the present study, we assessed, if the novel dual phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor NVP-BEZ235 radiosensitizes triple negative (TN) MDA-MB-231 and estrogen receptor (ER) positive MCF-7 cells to ionizing radiation under various oxygen conditions, simulating different microenvironments as occurring in the majority of breast cancers (BCs). Irradiation (IR) of BC cells cultivated in hypoxic conditions revealed increased radioresistance compared to normoxic controls. Treatment with NVP-BEZ235 completely circumvented this hypoxia-induced effects and radiosensitized normoxic, reoxygenated, and hypoxic cells to similar extents. Furthermore, NVP-BEZ235 treatment suppressed HIF-1α expression and PI3K/mTOR signaling, induced autophagy, and caused protracted DNA damage repair in both cell lines in all tested oxygen conditions. Moreover, after incubation with NVP-BEZ235, MCF-7 cells revealed depletion of phospho-AKT and considerable signs of apoptosis, which were significantly enhanced by radiation. Our findings clearly demonstrate that NVP-BEZ235 has a clinical relevant potential as a radiosensitizer in BC treatment. PMID:24678241

  2. Laurenditerpenol, a New Diterpene from the Tropical Marine Alga Laurencia intricata Potently Inhibits HIF-1 Mediated Hypoxic Signaling in Breast Tumor Cells

    PubMed Central

    Mohammed, Kaleem A.; Hossain, Chowdhury Faiz; Zhang, Lei; Bruick, Richard K.; Zhou, Yu-Dong; Nagle, Dale G.

    2010-01-01

    The degree of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) promotes tumor cell adaptation and survival under hypoxic conditions. Therefore, specific HIF-1 inhibitors represent an important new class of potential tumor-selective therapeutic agents. A T47D human breast tumor cell-based reporter assay was used to examine extracts of plants and marine organisms for inhibitors of HIF-1 activation. Bioassay-guided fractionation of the lipid extract of the red alga Laurencia intricata yielded a structurally novel diterpene laurenditerpenol (1). The structure of 1 was determined spectroscopically. The relative configurations of the substituents of each ring system were assigned based on NOESY correlations. The absolute configurations of positions C-1 was determined by the modified Mosher ester procedure (directly in NMR tubes). Compound 1 potently inhibited hypoxia-activated HIF-1 (IC50: 0.4 μM) and hypoxia-induced VEGF (a potent angiogenic factor) in T47D cells. Compound 1 selectively inhibits HIF-1 activation by hypoxia but not iron chelator induced activation. Further, 1 suppresses tumor cell survival under hypoxic conditions without affecting normoxic cell growth. Compound 1 inhibits HIF-1 by blocking the induction of the oxygen-regulated HIF-1α protein. Mitochondrial respiration studies revealed that 1 suppresses oxygen consumption. PMID:15620241

  3. HIF2α/EFEMP1 cascade mediates hypoxic effects on breast cancer stem cell hierarchy

    PubMed Central

    Hong, In-Sun; Nam, Jeong-Seok

    2016-01-01

    Breast cancer stem cells (BCSCs) have been shown to contribute to tumor growth, metastasis, and recurrence. They are also markedly resistant to conventional cancer treatments, such as chemotherapy and radiation. Recent studies have suggested that hypoxia is one of the prominent micro-environmental factors that increase the self-renewal ability of BCSCs, partially by enhancing CSC phenotypes. Thus, the identification and development of new therapeutic approaches based on targeting the hypoxia-dependent responses in BCSCs is urgent. Through various in vitro studies, we found that hypoxia specifically up-regulates BCSC sphere formation and a subset of CD44+/CD24−/low CSCs. Hypoxia inducible factors 2α (HIF2α) depletion suppressed CSC-like phenotypes and CSC-mediated drug resistance in breast cancer. Furthermore, the stimulatory effects of hypoxia-induced HIF2α on BCSC sphere formation were successfully attenuated by epidermal growth factor-containing fibulin-like extracellular matrix protein 1 (EFEMP1) knockdown. Taken together, these data suggest that HIF2α mediates hypoxia-induced cancer growth/metastasis and that EFEMP1 is a downstream effector of hypoxia-induced HIF2α during breast tumorigenesis. PMID:27270657

  4. Hypoxic pre-conditioning increases the infiltration of endothelial cells into scaffolds for dermal regeneration pre-seeded with mesenchymal stem cells.

    PubMed

    Fierro, Fernando A; O'Neal, Adam J; Beegle, Julie R; Chávez, Myra N; Peavy, Thomas R; Isseroff, Roslyn R; Egaña, José T

    2015-01-01

    Many therapies using mesenchymal stem cells (MSC) rely on their ability to produce and release paracrine signals with chemotactic and pro-angiogenic activity. These characteristics, however, are mostly studied under standard in vitro culture conditions. In contrast, various novel cell-based therapies imply pre-seeding MSC into bio-artificial scaffolds. Here we describe human bone marrow-derived MSC seeded in Integra matrices, a common type of scaffold for dermal regeneration (SDR). We show and measured the distribution of MSC within the SDR, where cells clearly establish physical interactions with the scaffold, exhibiting constant metabolic activity for at least 15 days. In the SDR, MSC secrete VEGF and SDF-1α and induce transwell migration of CD34(+) hematopoietic/endothelial progenitor cells, which is inhibited in the presence of a CXCR4/SDF-1α antagonist. MSC in SDR respond to hypoxia by altering levels of angiogenic signals such as Angiogenin, Serpin-1, uPA, and IL-8. Finally, we show that MSC-containing SDR that have been pre-incubated in hypoxia show higher infiltration of endothelial cells after implantation into immune deficient mice. Our data show that MSC are fully functional ex vivo when implanted into SDR. In addition, our results strongly support the notion of hypoxic pre-conditioning MSC-containing SDR, in order to promote angiogenesis in the wounds.

  5. A Novel Microfluidic Platform for High-Resolution Imaging of a Three-Dimensional Cell Culture under a Controlled Hypoxic Environment

    PubMed Central

    Funamoto, Kenichi; Zervantonakis, Ioannis K.; Liu, Yuchun; Ochs, Christopher J.; Kim, Choong

    2014-01-01

    Low oxygen tensions experienced in various pathological and physiological conditions are a major stimulus for angiogenesis. Hypoxic conditions play a critical role in regulating cellular behaviour including migration, proliferation and differentiation. This study introduces the use of a microfluidic device that allows for the control of oxygen tension for the study of different three-dimensional (3D) cell cultures for various applications. The device has a central 3D gel region acting as an external cellular matrix, flanked by media channels. On each side, there is a peripheral gas channel through which suitable gas mixtures are supplied to establish a uniform oxygen concentration or gradient within the device. The effects of various parameters, such as gas and media flow rates, device thickness, and diffusion coefficients of oxygen were examined using numerical simulations to determine the characteristics of the microfluidic device. A polycarbonate (PC) film with a low oxygen diffusion coefficient was embedded in the device in proximity above the channels to prevent oxygen diffusion from the incubator environment into the Polydimethylsiloxane (PDMS) device. The oxygen tension in the device was then validated experimentally using a ruthenium-coated (Ru-coated) oxygen-sensing glass cover slip which confirmed the establishment of low uniform oxygen tensions (< 3%) or an oxygen gradient across the gel region. To demonstrate the utility of the microfluidic device for cellular experiments under hypoxic conditions, migratory studies of MDA-MB-231 human breast cancer cells were performed. The microfluidic device allowed for imaging cellular migration with high-resolution, exhibiting an enhanced migration in hypoxia in comparison to normoxia. This microfluidic device presents itself as a promising platform for the investigation of cellular behaviour in a 3D gel scaffold under varying hypoxic conditions. PMID:23023115

  6. Comparative Study on the Protective Effects of Salidroside and Hypoxic Preconditioning for Attenuating Anoxia-Induced Apoptosis in Pheochromocytoma (PC12) Cells

    PubMed Central

    Hu, Yao; Lv, Xiumei; Zhang, Jing; Meng, Xianli

    2016-01-01

    Background Hypoxia is an important sign that can result from body injuries or a special condition such as being at a high altitude or deep water diving. In the current studies, hypoxic preconditioning (HPC) plays a key role in reducing hypoxia-induced apoptosis. We aimed to study the pharmacologic preconditioning effects of salidroside versus those of HPC in hypoxia-/anoxia-induced apoptosis in PC12 cells (pheochromocytoma). Material/Methods PC12 cells were treated by different experimental conditions: control condition, hypoxia condition, HPC condition, low-/middle-/high-dose condition of salidroside, cyclosporine A (CsA), and oratractyloside (ATR). The cell viability, lactate dehydrogenase (LDH) activity, apoptosis, mitochondrial membrane potential (MMP), intracellular Ca2+, caspase-3 activity, and expression of Bcl-2 were detected in PC12 cells after the hypoxia treatment. Salidroside, extracted from the traditional Chinese herb Rhodiola rosea L, plays an essential role in reducing hypoxia-induced apoptosis in PC12 cells by the mitochondrial pathway. Results Salidroside decreased the apoptosis and increased the viability of hypoxia-induced PC12 cells more effectively than HPC Moreover, salidroside markedly stabilized MMP and intracellular Ca2+, reduced or inhibited LDH and caspase-3 activity, and up-regulated Bcl-2; CsA and ATR showed corresponding function. Conclusions Salidroside administration restrains apoptosis induced by hypoxia in PC12 cells. The protective effects are mediated by preservation of mitochondrial integrity and MMP to inhibit the excessive Ca2+ influx and caspase-3 activity and to promote the Bcl-2 expression, providing a potential clinical and effective therapeutic mechanism to reduce deaths from ischemic or hypoxic injury. PMID:27794583

  7. NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress

    PubMed Central

    Alonso, Judith; Galán, María; Martí-Pàmies, Ingrid; Romero, José María; Camacho, Mercedes; Rodríguez, Cristina; Martínez-González, José

    2016-01-01

    Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells. PMID:27654514

  8. NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress.

    PubMed

    Alonso, Judith; Galán, María; Martí-Pàmies, Ingrid; Romero, José María; Camacho, Mercedes; Rodríguez, Cristina; Martínez-González, José

    2016-09-22

    Vascular cell survival is compromised under pathological conditions such as abdominal aortic aneurysm (AAA). We have previously shown that the nuclear receptor NOR-1 is involved in the survival response of vascular cells to hypoxia. Here, we identify the anti-apoptotic protein cIAP2 as a downstream effector of NOR-1. NOR-1 and cIAP2 were up-regulated in human AAA samples, colocalizing in vascular smooth muscle cells (VSMC). While NOR-1 silencing reduced cIAP2 expression in vascular cells, lentiviral over-expression of this receptor increased cIAP2 mRNA and protein levels. The transcriptional regulation of the human cIAP2 promoter was analyzed in cells over-expressing NOR-1 by luciferase reporter assays, electrophoretic mobility shift analysis and chromatin immunoprecipitation, identifying a NGFI-B site (NBRE-358/-351) essential for NOR-1 responsiveness. NOR-1 and cIAP2 were up-regulated by hypoxia and by a hypoxia mimetic showing a similar time-dependent pattern. Deletion and site-directed mutagenesis studies show that NOR-1 mediates the hypoxia-induced cIAP2 expression. While NOR-1 over-expression up-regulated cIAP2 and limited VSMC apoptosis induced by hypoxic stress, cIAP2 silencing partially prevented this NOR-1 pro-survival effect. These results indicate that cIAP2 is a target of NOR-1, and suggest that this anti-apoptotic protein is involved in the survival response to hypoxic stress mediated by NOR-1 in vascular cells.

  9. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

  10. Patterns and Levels of Hypoxia in Head and Neck Squamous Cell Carcinomas and Their Relationship to Patient Outcome

    SciTech Connect

    Evans, Sydney M. V. Du, Kevin L.; Chalian, Ara A.; Mick, Rosemarie; Zhang, Paul J.; Hahn, Stephen M.; Quon, Harry; Lustig, Robert; Weinstein, Gregory S.; Koch, Cameron J.

    2007-11-15

    Purpose: EF5, a 2-nitroimidazole hypoxia marker, was used to study the presence, levels, and prognostic significance of hypoxia in primary head and neck squamous cell tumors. Methods and Materials: Twenty-two patients with newly diagnosed squamous cell carcinoma of the oral cavity, oropharynx, or larynx with at least 2 years of clinical follow-up were included in this study. Quantitative analyses of EF5 immunofluorescence was carried out, and these data were compared with patient outcome. Results: EF5 immunostaining showed substantial intra- and intertumoral hypoxic heterogeneity. The majority of cells in all tumors were well oxygenated. Three patterns of EF5 binding in cells were identified using criteria based on the cellular region that was stained (peripheral or central) and the relationship of binding to necrosis. We tested the association between EF5-binding levels with event-free and overall survival irrespective of the pattern of cellular binding or treatment regimen. Patients with tumors containing EF5-binding regions corresponding to severe hypoxia ({<=}0.1% oxygen) had a shorter event-free survival time than patients with pO{sub 2} values greater than 0.1% (p = 0.032). Nodal status was also predictive for outcome. Conclusions: These data illustrate the potential utility of EF5 binding based on quantitative immunohistochemistry of tissue pO{sub 2} and provide support for the development of noninvasive hypoxia positron emission tomographic studies with fluorine 18-labeled EF5.

  11. Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture.

    PubMed

    Zeng, Rui; Chen, Yu-Cheng; Zeng, Zhi; Liu, Rui; Qiang, Ou; Jiang, Xiao-Fei; Liu, Xiao-Xia; Li, Xian; Wang, Hao-Yu

    2008-03-01

    Aim We studied the role of mini-TyrRS and mini-TrpRS in angiogenesis by using small interfering RNA-mediated mini-TyrRS/mini-TrpRS knockout in hypoxic culture of human umbilical vein endothelial cells. Methods SiRNA was used as the main method to inhibited the gene function. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction and western blotting. The angiogenic activity in vitro was evaluated by transwell migration assay and Matrigel-induced capillary tube formation in hypoxic culture. Cell proliferation was determined by crystal violet staining. Results The results showed that levels of the mini-TyrRS/mini-TrpRS gene and protein in mock transfection group and negative control group were higher, but noticeably decreased in experimental group. However, no significant difference was detected between mock transfection group and negative control group, but there was a statistically significant difference compared with experimental group. For mini-TyrRS-siRNA group, the cell migration, tube formation and the rate of cell proliferation were respectively inhibited by (47.4, 56.3, 65.4, 73.7%), (60.5, 69.1, 75.9, 83.6%) and (40.4, 56.2, 61.2, 68.0%). For mini-TrpRS-siRNA, were respectively increased by (18.0, 33.8, 45.1, 56.4%), (18.3, 31.2, 40.3, 45.7%) and (8.4, 26.4, 38.2, 46.6%). Conclusion These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in matrigel models in hypoxic culture, raising the possibility that mini-TyrRS stimulates a common downstream signaling event. Thus, naturally occurring fragments of two proteins involved in translation, TyrRS and TrpRS, have opposing activity on endothelial cell angiogenesis in the matrigel assays. The opposing activities of the two tRNA synthetases suggest tight regulation of the balance between pro- and anti-angiogenic stimuli.

  12. The long non-coding RNA – HIF1A-AS2 facilitates the maintenance of mesenchymal glioblastoma stem-like cells in hypoxic niches

    PubMed Central

    Mineo, Marco; Ricklefs, Franz; Rooj, Arun K.; Lyons, Shawn M.; Ivanov, Pavel; Ansari, Khairul I.; Nakano, Ichiro; Chiocca, E. Antonio; Godlewski, Jakub; Bronisz, Agnieszka

    2016-01-01

    Long-non-coding RNAs (lncRNAs) have an undefined role in the pathobiology of glioblastoma multiforme (GBM). These tumors are genetically and phenotypically heterogeneous with transcriptome subtype-specific GBM stem-like cells (GSCs) that adapt to the brain tumor microenvironment, including hypoxic niches. We identified hypoxia inducible factor 1 alpha-antisense RNA 2 (HIF1A-AS2) as a subtype-specific hypoxia inducible lncRNA, up-regulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal and hypoxia-dependent molecular reprogramming. Amongst the HIF1A-AS2 interactome, IGF2BP2 and DHX9 were identified as direct partners. This association was needed for maintenance of expression of their target gene, HMGA1. Down-regulation of HIF1A-AS2 led to delayed growth of mesenchymal GSC tumors, survival benefits, and impaired expression of HMGA1 in vivo. Our data demonstrate that HIF1A-AS2 contributes to GSCs’ speciation and adaptation to hypoxia within the tumor microenvironment, acting directly through its interactome/targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context. PMID:27264189

  13. The Archipelago Ubiquitin Ligase Subunit Acts in Target Tissue to Restrict Tracheal Terminal Cell Branching and Hypoxic-Induced Gene Expression

    PubMed Central

    Mortimer, Nathan T.; Moberg, Kenneth H.

    2013-01-01

    The Drosophila melanogaster gene archipelago (ago) encodes the F-box/WD-repeat protein substrate specificity factor for an SCF (Skp/Cullin/F-box)-type polyubiquitin ligase that inhibits tumor-like growth by targeting proteins for degradation by the proteasome. The Ago protein is expressed widely in the fly embryo and larva and promotes degradation of pro-proliferative proteins in mitotically active cells. However the requirement for Ago in post-mitotic developmental processes remains largely unexplored. Here we show that Ago is an antagonist of the physiologic response to low oxygen (hypoxia). Reducing Ago activity in larval muscle cells elicits enhanced branching of nearby tracheal terminal cells in normoxia. This tracheogenic phenotype shows a genetic dependence on sima, which encodes the HIF-1α subunit of the hypoxia-inducible transcription factor dHIF and its target the FGF ligand branchless (bnl), and is enhanced by depletion of the Drosophila Von Hippel Lindau (dVHL) factor, which is a subunit of an oxygen-dependent ubiquitin ligase that degrades Sima/HIF-1α protein in metazoan cells. Genetic reduction of ago results in constitutive expression of some hypoxia-inducible genes in normoxia, increases the sensitivity of others to mild hypoxic stimulus, and enhances the ability of adult flies to recover from hypoxic stupor. As a molecular correlate to these genetic data, we find that Ago physically associates with Sima and restricts Sima levels in vivo. Collectively, these findings identify Ago as a required element of a circuit that suppresses the tracheogenic activity of larval muscle cells by antagonizing the Sima-mediated transcriptional response to hypoxia. PMID:23459416

  14. Kinetics of a putative hypoxic tissue marker, Technetium-99m-nitroimidazole (BMS181321), in normoxic, hypoxic, ischemic and stunned myocardium

    SciTech Connect

    Kusuoka, Hideo; Hashimoto, Katsuji; Fukuchi, Kazuki

    1994-08-01

    This study focused on the kinetics of the newly developed {sup 99m}TTc-nitroimidazole, propyleneamine oxime-1,2-nitroimidazole (BMS181321) in the different setting of myocardial perfusion states and oxygenation levels, and compared the kinetics of BMS181321 with those of other technetium analogues. The kinetics of BMS181321 were evaluated in isolated perfused rat hearts. Technetium-99m-hexamethyl propyleneamine oxime (HMPAO) and a non-nitroimidazole-containing analogue of BMS 181321 (6-methyl propyleneamine oxime; PAO-6-Me) were used to compare their kinetics with those of BMS181321. BMS181321 cleared quickly from normoxic hearts and the retention in the myocardium 10 min after injection was 0.84% {plus_minus} 0.04% ID/g wet wt (mean {plus_minus} s.e.m.). In contrast, BMS181321 was retained after reperfusion when it was injected before ischemia; the uptake in the myocardium 10 min after reperfusion was significantly greater than in controls (23.9% {plus_minus} 3.9%ID/g wt, p<0.05). These results indicate that {sup 99m}Tc-BMS181321 is well trapped in ischemic myocardium and moderately trapped in hypoxic myocardium, but washed out quickly in stunned myocardium. The residence time influences the amount retained. 14 refs., 7 figs., 1 tab.

  15. Preparation of Nucleosides Derived from 2-Nitroimidazole and d-Arabinose, d-Ribose, and d-Galactose by the Vorbrüggen Method and Their Conversion to Potential Precursors for Tracers To Image Hypoxia

    PubMed Central

    2011-01-01

    2-Nitroimidazole was silylated using hexaethyldisilazane and then reacted with 1-O-acetyl derivatives of d-arabinose, d-ribose, and d-galactose in acetonitrile at mild temperatures (−20 °C to rt), catalyzed by triethylsilyl triflate (Vorbrüggen conditions). The α-anomer was formed in the former case and the β-anomers in the latter two cases (highly) selectively. When d-arabinose and d-ribose were silylated with tert-butyldiphenylsilyl chloride in pyridine at the hydroxyl groups at C-5 and acetylated at the other ones in a one-pot reaction, mixtures of anomeric 1-O-acetyl derivatives were obtained. These were coupled by the Vorbrüggen method and then deblocked at C-5 and tosylated to give precursors for tracers to image hypoxia in four steps without using Hg(CN)2 necessary for other methods. The Vorbrüggen conditions enable a shorter route to azomycin nucleoside analogues than the previous coupling procedures. PMID:21905640

  16. Modulation of prostaglandin biosynthesis in murine mammary adenocarcinoma tumor cells

    SciTech Connect

    Shalinsky, D.R.

    1988-01-01

    In efforts to exploit the differential oxygen levels within the subcompartments of solid neoplasms, this project has focused on modulating prostaglandin (PG) biosynthesis under aerobic and hypoxic conditions. Mammary adenocarcinoma tumor cells (Line 4526), either intact or sonicated, were incubated with either 2.0 uM {sup 14}C-arachidonic acid (AA) or 20.0 uM {sup 14}C-PGH{sub 2}, respectively. Following metabolism, products were extracted, separated by thin layer chromatography and analyzed by radiochromatographic scan. PGE{sub 2} was predominantly formed with minimal amounts of PGF{sub 2a} or PGD{sub 2}. Indomethacin and ibuprofen inhibited the PGE{sub 2} formation from AA with an IC{sub 50} value of 6.3 {times} 10{sup {minus}8} and 9.6 {times} 10{sup {minus}5}M, respectively. Suspended cells in glass vials were made hypoxic by flushing with N{sub 2} for varying time intervals to study AA metabolism. A time-dependent inhibition of PG biosynthesis was observed under hypoxia, and by 30 min, the PGE{sub 2} synthesis was reduced by 50% which was further inhibited by indomethacin. Misonidazole, a 2-nitroimidazole analogue, partially reversed the inhibition of PGE{sub 2} synthesis under hypoxia by 49% at 100 uM. However, misonidazole did not affect PG biosynthesis under aerobic conditions. The stimulation of PGE{sub 2} biosynthesis by misonidazole under hypoxia was blocked by indomethacin, suggesting that misonidazole can not act independently of the cyclooxygenase.

  17. Estrogen receptor α is a novel target of the Von Hippel-Lindau protein and is responsible for the proliferation of VHL-deficient cells under hypoxic conditions

    PubMed Central

    Jung, Youn-Sang; Lee, Su-Jin; Yoon, Min-Ho; Ha, Nam Chul; Park, Bum-Joon

    2012-01-01

    The Von Hippel-Lindau gene (VHL) is frequently deleted or mutated in human renal cell carcinoma (RCC) at the early stage. According to the well-established theory, pVHL acts as a tumor suppressor through its E3 ligase activity, which targets hypoxia-inducing factor-1α (HIF-1α). However, the elevated expression of HIF-1α did not promote cell proliferation, indicating that there would be another target, which could promote cell proliferation at the early cancer stage of RCC. In this study, we show that estrogen receptor-α (ER-α) is a novel proteasomal degradation target of the pVHL E3 ligase. Indeed, the overexpression of VHL suppresses exo- and endogenous ER-α expression, whereas si-pVHL can increase ER-α expression. The negative regulation of pVHL on ER-α expression is achieved by its E3 ligase activity. Thus, pVHL can promote the ER-α ubiquitinylation. In addition, we revealed that ER-α and HIF-1α are competitive substrates of pVHL. Thus, under normal conditions, ER-α overexpression can increase the transcription factor activity of HIF-1α. Under the hypoxic condition, where HIF-1α is not a suitable target of pVHL, ER-α is more rapidly degraded by pVHL. However, in VHL-deficient cells, the expression of ER-α and HIF-1α is retained, so that the hypoxic condition did not suppress cell proliferation obviously compared with cells that are expressing pVHL. Thus, blocking of ER-α using its inhibitor could suppress the proliferation of VHL-deficient cells as effectively as hypoxia-induced growth suppression. Considering our results, blocking of ER-α signaling in VHL-deficient cancer cells would be beneficial for cancer suppression. Indeed, we showed the anti-proliferative effect of Faslodex in VHL-deficient cells. PMID:23159849

  18. BDNF Pretreatment of Human Embryonic-Derived Neural Stem Cells Improves Cell Survival and Functional Recovery After Transplantation in Hypoxic-Ischemic Stroke.

    PubMed

    Rosenblum, Sahar; Smith, Tenille N; Wang, Nancy; Chua, Joshua Y; Westbroek, Erick; Wang, Kendrick; Guzman, Raphael

    2015-01-01

    Intra-arterial neural stem cell (NSC) therapy has the potential to improve long-term outcomes after stroke. Here we evaluate if pretreatment of NSCs with brain-derived neurotrophic factor (BDNF) prior to transplantation improves cell engraftment and functional recovery following hypoxic-ischemic (HI) stroke. Human embryonic-derived NSCs with or without BDNF pretreatment (1 h, 100 ng/ml) were transplanted 3 days after HI stroke. Functional recovery was assessed using the horizontal ladder test. Cell engraftment was evaluated using bioluminescence imaging (BLI) and histological counts of SC121(+) cells. Fluoro-Jade C (FJC) and NeuN stains were used to evaluate neuroprotection. The effect of BDNF on NSCs was analyzed using a migration assay, immunocytochemistry, Luminex proteomic assay, and RT-qPCR.BLI analysis demonstrated significantly higher photon flux in the BDNF-treated NSC group compared to untreated NSC (p = 0.049) and control groups (p = 0.0021) at 1 week after transplantation. Immunohistochemistry confirmed increased transplanted cell survival in the cortex (p = 0.0126) and hippocampus (p = 0.0098) of animals injected with BDNF-treated NSCs compared to untreated NSCs. Behavioral testing revealed that the BDNF-treated NSC group demonstrated increased sensorimotor recovery compared to the untreated NSC and control groups (p < 0.001) over the 1-month period (p < 0.001) following transplantation. A significant improvement in performance was found in the BDNF-treated NSC group compared to the control group at 14, 21, and 28 (p < 0.05) days after transplantation. The cortex and hippocampus of the BDNF-treated NSC group had significantly more SC121(+) NSCs (p = 0.0125, p = 0.0098), fewer FJC(+) neurons (p = 0.0370, p = 0.0285), and a higher percentage of NeuN(+) expression (p = 0.0354) in the cortex compared to the untreated NSC group. BDNF treatment of NSCs resulted in significantly greater migration to SDF-1, secretion of M-CSF, VEGF, and expression of CXCR4

  19. Proof of Concept to Isolate and Culture Primary Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-hold Diving

    DTIC Science & Technology

    2012-09-30

    Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-hold Diving...To isolate and culture primary muscle cells from the swimming muscles of northern elephant seals . OBJECTIVES Objective 1. To test the...Proof of Concept to Isolate and Culture Primary Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under

  20. Hypoxia in Tumor Angiogenesis and Metastasis: Evaluation of VEGF and MMP Over-expression and Down-Regulation of HIF-1alpha with RNAi in Hypoxic Tumor Cells

    NASA Astrophysics Data System (ADS)

    Shah, Shruti

    Background: As tumor mass grows beyond a few millimeters in diameter, the angiogenic "switch" is turned on leading to recruitment of blood vessels from surrounding artery and veins. However, the tumor mass is poorly perfused and there are pockets of hypoxia or lower oxygen concentrations relative to normal tissue. Hypoxia-inducing factor-1a (HIF-1a), a transcription factor, is activated when the oxygen concentration is low. Upon activation of HIF-1a, a number of other genes also turn on that allows the tumor to become more aggressive and resistant to therapy. Purpose: The main objectives of this study were to evaluate the effect of hypoxia-induced HIF-1a followed by over-expression of angiogenic and metastatic markers in tumor cells and down-regulation of HIF-1a using nanoparticle-delivered RNA interference therapy. Methods: Human ovarian (SKOV3) and breast (MDA-MB-231) adenocarcinoma cells were incubated under normoxic and hypoxic conditions. Following hypoxia treatment of the cells, HIF-1α, vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP-2), and MMP-9 expression was analyzed qualitatively and quantitatively. For intracellular delivery of HIF-1a gene silencing small interfering RNA (siRNA), type B gelatin nanoparticles were fabricated using the solvent displacement method and the surface was modified with poly(ethylene glycol) (PEG, Mol. wt. 2kDa). Cellular uptake and distribution of the nanoparticles was observed with Cy3-siRNA loaded, FITC-conjugated gelatin nanoparticles. Cytotoxicity of the nanoparticle formulations was evaluated in both the cell lines. siRNA was transfected in the gelatin nanoparticles under hypoxic conditions. Total cellular protein and RNA were extracted for analysis of HIF1a, VEGF, MMP-2 and MMP-9 expression. Results: MDA-MB-231 and SKOV3 cells show increased expression of HIF1a under hypoxic conditions compared to baseline levels at normoxic conditions. ELISA and western blots of VEGF, MMP-2 and MMP-9 appear to

  1. The anti-necrosis role of hypoxic preconditioning after acute anoxia is mediated by aldose reductase and sorbitol pathway in PC12 cells.

    PubMed

    Wu, Li-Ying; Ma, Zi-Min; Fan, Xue-Lai; Zhao, Tong; Liu, Zhao-Hui; Huang, Xin; Li, Ming-Ming; Xiong, Lei; Zhang, Kuan; Zhu, Ling-Ling; Fan, Ming

    2010-07-01

    It has been demonstrated that hypoxic preconditioning (HP) enhances the survival ability of the organism against the subsequent acute anoxia (AA). However, it is not yet clear whether necrosis induced by AA can be prevented by HP, and what are the underlying mechanisms. In this study, we examined the effect of HP (10% O(2), 48 h) on necrosis induced by AA (0% O(2), 24 h) in PC12 cells. We found that HP delayed the regulatory volume decrease and reduced cell swelling after 24 h of exposure to AA. Since aldose reductase (AR) is involved in cell volume regulation, we detected AR mRNA expression with reverse transcription-polymerase chain reaction (RT-PCR) techniques. The AR mRNA level was dramatically elevated by HP. Furthermore, an HP-induced decrease in cell injury was reversed by berberine chloride (BB), the inhibitor of AR. In addition, sorbitol synthesized from glucose catalyzed by AR is directly related to cell volume regulation. Subsequently, we tested sorbitol content in the cytoplasm. HP clearly elevated sorbitol content, while BB inhibited the elevation induced by HP. Further study showed that a strong inhibitor of sorbitol permease, quinidine, completely reversed the protection induced by HP after AA. These data provide evidence that HP prevents necrosis induced by AA and is mediated by AR and sorbitol pathway.

  2. Human Umbilical Cord Blood CD34-Positive Cells as Predictors of the Incidence and Short-Term Outcome of Neonatal Hypoxic-Ischemic Encephalopathy: A Pilot Study

    PubMed Central

    Nasr Eldin, Mohamed Hassan; Amer, Hanaa A.; Abdelhamid, Adel E.; El Houssinie, Moustafa; Ibrahim, Abir

    2017-01-01

    Background and Purpose Neonatal hypoxic-ischemic encephalopathy (HIE) is one of the leading causes of neurological handicap in developing countries. Human umbilical cord blood (hUCB) CD34-positive (CD34+) stem cells exhibit the potential for neural repair. We tested the hypothesis that hUCB CD34+ stem cells and other cell types [leukocytes and nucleated red blood cells (NRBCs)] that are up-regulated during the acute stage of perinatal asphyxia (PA) could play a role in the early prediction of the occurrence, severity, and mortality of HIE. Methods This case-control pilot study investigated consecutive neonates exposed to PA. The hUCB CD34+ cell count in mononuclear layers was assayed using a flow cytometer. Twenty full-term neonates with PA and 25 healthy neonates were enrolled in the study. Results The absolute CD34+ cell count (p=0.02) and the relative CD34+ cell count (CD34+%) (p<0.001) in hUCB were higher in the HIE patients (n=20) than the healthy controls. The hUCB absolute CD34+ cell count (p=0.04), CD34+% (p<0.01), and Hobel risk scores (p=0.04) were higher in patients with moderate-to-severe HIE (n=9) than in those with mild HIE (n=11). The absolute CD34+ cell count was strongly correlated with CD34+% (p<0.001), Hobel risk score (p=0.04), total leukocyte count (TLC) (p<0.001), and NRBC count (p=0.01). CD34+% was correlated with TLC (p=0.02). Conclusions hUCB CD34+ cells can be used to predict the occurrence, severity, and mortality of neonatal HIE after PA. PMID:28079317

  3. Hyperbaric oxygenation promotes neural stem cell proliferation and protects the learning and memory ability in neonatal hypoxic-ischemic brain damage.

    PubMed

    Wei, Lixia; Wang, Jinshen; Cao, Yuntao; Ren, Qing; Zhao, Lili; Li, Xingang; Wang, Jiwen

    2015-01-01

    The aim of our study was to evaluate whether hyperbaric oxygenation (HBO) was an effective therapy for neonatal hypoxic ischemic brain damage (HIBD). Seven-day-old rat pups were divided into 3 groups: sham, hypoxia-ischemia (HI) control and HI-HBO group. HBO was administered for HI rats daily. The pathologic changes in brain tissues were observed by hematoxylin-eosin (H-E) staining. The immunohistochemical staining was applied to detect the Nestin and 5-bromo-2-deoxyuridine (BrdU) positive cells in hippocampal dentate gyrus region. The learning and memory function of rats was examined by Morris water maze. The HI rats showed obvious pathologic changes accompanied by levels decreasing and disorder arrangement of pyramidal cells, glial cells proliferation in postoperative, and nerve nuclei broken, while pathologic changes of rats in sham group was approximate to that in the HI + HBO group that was opposite to the HI group. Compared with the sham group, the Nestin and BrdU positive cells in HBO + HI group at different time points increased significantly (P < 0.01). Learning and memory function of rats in HI group was poor compared with the sham/HI + HBO group (P < 0.01), while that in HI + HBO group was approximate to that in sham group (P > 0.05). HBO treatment improved the learning and memory ability of the HI rats. HBO therapy may be effective for neonatal HIBD treatment.

  4. Avian embryos in hypoxic environments.

    PubMed

    León-Velarde, F; Monge-C, C

    2004-08-12

    Avian embryos at high altitude do not benefit of the maternal protection against hypoxia as in mammals. Nevertheless, avian embryos are known to hatch successfully at altitudes between 4,000 and 6,500 m. This review considers some of the processes that bring about the outstanding modifications in the pressure differences between the environment and mitochondria of avian embryos in hypoxic environments. Among species, some maintain normal levels of oxygen consumption ( VO2) have a high oxygen carrying capacity, lower the air cell-arterial pressure difference ( PAO2 - PaO2 ) with a constant pH. Other species decrease VO2, increase only slightly the oxygen carrying capacity, have a higher PAO2 - PaO2 difference than sea-level embryos and lower the PCO2 and pH. High altitude embryos, and those exposed to hypoxia have an accelerated decline of erythrocyte ATP levels during development and an earlier stimulation of 2,3-BPG synthesis. A higher Bohr effect may ensure high tissue PO2 in the presence of the high-affinity hemoglobin. Independently of the strategy used, they serve together to promote suitable rates of development and successful hatching of high altitude birds in hypoxic environments.

  5. Correlation between the expression of divalent metal transporter 1 and the content of hypoxia-inducible factor-1 in hypoxic HepG2 cells

    PubMed Central

    Li, Zhu; Lai, Zhang; Ya, Ke; Fang, Du; Ho, Yung Wing; Lei, Yang; Ming, Qian Zhong

    2008-01-01

    Abstract Transferrin and transferrin receptor are two key proteins of iron metabolism that have been identified to be hypoxia-inducible genes. Divalent metal transporter 1 (DMT1) is also a key transporter of iron under physiological conditions. In addition, in the 5′ regulatory region of human DMT1 (between −412 and −570), there are two motifs (CCAAAGTGCTGGG) that are similar to hypoxia-inducible factor-1 (HIF-1) binding sites. It was therefore speculated that DMT1 might also be a hypoxia-inducible gene. We investigated the effects of hypoxia and hypoxia/re-oxygenation on the expression of DMT1 and the content of HIF-1alpha in HepG2 cells. As we expected, a very similar tendency in the responses of the expression of HIF-1α, DMT1+IRE (iron response element) and DMT1−IRE proteins to chemical (CoCl2) or physical hypoxia was observed. A highly significant correlation was found between the expression of DMT1 proteins and the contents of HIF-1 in hypoxic cells. After the cells were exposed to hypoxia and subsequent normoxia, no HIF-1α could be detected and a significant decrease in DMT1+IRE expression (P<0.05), but not in DMT1−IRE protein (versus the hypoxia group), was observed. The findings implied that the HIF-1 pathway might have a role in the regulation of DMT1+IRE expression during hypoxia. PMID:18419598

  6. Radio-sensitization effect of an mTOR inhibitor, temsirolimus, on lung adenocarcinoma A549 cells under normoxic and hypoxic conditions

    PubMed Central

    Ushijima, Hiroki; Suzuki, Yoshiyuki; Oike, Takahiro; Komachi, Mayumi; Yoshimoto, Yuya; Ando, Ken; Okonogi, Noriyuki; Sato, Hiro; Noda, Shin-ei; Saito, Jun-ichi; Nakano, Takashi

    2015-01-01

    The mammalian target of rapamycin (mTOR) correlates with cell survival under hypoxia and regulates hypoxia-inducible factor-1α (HIF-1α), a key protein in hypoxia-related events. However, the role of mTOR in radio-resistance has not been fully investigated. Therefore, the effect of mTOR on the radio-resistance of cancer cells under hypoxia was evaluated using the mTOR inhibitor temsirolimus. Clonogenic survival was examined in the A549 human lung adenocarcinoma cell line under normoxia or hypoxia, with or without temsirolimus. An oxygen enhancement ratio (OER) was calculated using the D10 values, the doses giving 10% survival. Western blotting was performed to investigate the effect of temsirolimus on mTOR and the HIF-1α pathway under normoxia and hypoxia. A549 cells showed a radio-resistance of 5.1 and 14.2 Gy, as indicated by D10 values under normoxia and hypoxia, respectively; the OER was 2.8. The cell survival rates under hypoxia and with temsirolimus remarkably decreased compared with those under normoxia. The D10 values of the cells under normoxia and hypoxia were 4.8 and 5.4 Gy, respectively (OER = 1.1). mTOR expression was suppressed by temsirolimus under both normoxia and hypoxia. HIF-1α expression decreased under hypoxia in the presence of temsirolimus. These results suggest that temsirolimus can overcome the radio-resistance induced by hypoxia. When the fact that mTOR acts upstream of HIF-1α is considered, our data suggest that the restoration of radiation sensitivity by temsirolimus under hypoxia may be associated with the suppression of the HIF-1α pathway. Temsirolimus could therefore be used as a hypoxic cell radio-sensitizer. PMID:25887043

  7. 3,3'-Diindolylmethane inhibits VEGF expression through the HIF-1α and NF-κB pathways in human retinal pigment epithelial cells under chemical hypoxic conditions.

    PubMed

    Park, Hongzoo; Lee, Dae-Sung; Yim, Mi-Jin; Choi, Yung Hyun; Park, Saegwang; Seo, Su-Kil; Choi, Jung Sik; Jang, Won Hee; Yea, Sung Su; Park, Won Sun; Lee, Chang-Min; Jung, Won-Kyo; Choi, Il-Whan

    2015-07-01

    Oxidative stress in the retinal pigment epithelium (RPE) can lead to the pathological causes of age-related macular degeneration (AMD). Hypoxia induces oxidative damage in retinal pigment epithelial cells (RPE cells). In this study, we investigated the capacity of 3,3'-diindolylmethane (DIM) to reduce the expression of vascular endothelial growth factor (VEGF) under hypoxic conditions, as well as the molecular mechanisms involved. Human RPE cells (ARPE-19 cells) were treated with cobalt chloride (CoCl2, 200 µM) and/or DIM (10 and 20 µM). The production of VEGF was measured by enzyme-linked immunosorbent assay. The translocation of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor-κB (NF-κB) was determined by western blot analysis. The binding activity of HIF-1α and NF-κB was analyzed by electrophoretic mobility shift assay. The phosphorylation levels of mitogen-activated protein kinases (MAPKs) were measured by western blot analysis. The levels of mitochondrial reactive oxygen species (ROS) were detected by fluorescence microplate assay. The results revealed that DIM significantly attenuated the CoCl2-induced expression of VEGF in the ARPE-19 cells. The CoCl2-induced translocation and activation of HIF-1α and NF-κB were also attenuated by treatment with DIM. In addition, DIM inhibited the CoCl2-induced activation of p38 MAPK in the ARPE-19 cells. Pre-treatment with YCG063, a mitochondrial ROS inhibitor, led to the downregulation of the CoCl2-induced production of VEGF by suppressing HIF-1α and NF-κB activity. Taken together, the findings of our study demonstrate that DIM inhibits the CoCl2-induced production of VEGF by suppressing mitochondrial ROS production, thus attenuating the activation of HIF-1α and p38 MAPK/NF-κB.

  8. DNA damage following combination of radiation with the bioreductive drug AQ4N: possible selective toxicity to oxic and hypoxic tumour cells.

    PubMed Central

    Hejmadi, M. V.; McKeown, S. R.; Friery, O. P.; McIntyre, I. A.; Patterson, L. H.; Hirst, D. G.

    1996-01-01

    AQ4N (1,4-bis-([2-(dimethylamino-N- oxide)ethyl]amino)5,8-dihydroxyanthracene-9,10-dione) is a novel bioreductive agent that can be reduced to a stable, DNA-affinic compound, AQ4. The alkaline comet assay was used to evaluate DNA damage induced by AQ4N and radiation. Cells prepared from freshly excised T50/80 murine tumours were shown to have the ability to reduce AQ4N to a DNA-damaging agent; this had disappeared within 24 h of excision. When T50/80 tumours implanted in BDF mice were exposed to radiation in vivo a considerable amount of DNA damage was present in tumours excised immediately. Minimal levels of DNA damage were detectable in tumours excised after 2-5 h. AQ4N given 30 min before radiation had no appreciable influence on this effect and AQ4N alone caused only a small amount of damage. When AQ4N and radiation were combined an increasing number of damaged cells were seen in tumours excised 24-96 h after irradiation. This was interpreted as evidence of the continued presence of AQ4, or AQ4-induced damage, which was formed in cells hypoxic at the time of administration of AQ4N. AQ4, a potent topoisomerase II inhibitor, would be capable of damaging cells recruited into the cell cycle following radiation damage to the well-oxygenated cells of the tumour. The kinetics of the expression of the DNA damage is consistent with this hypothesis and shows that AQ4 has persistent activity in vivo. PMID:8595165

  9. Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte

    PubMed Central

    Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

    2016-01-01

    Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway. PMID:26766745

  10. Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte.

    PubMed

    Gu, Yan; He, Mulan; Zhou, Xiaoqin; Liu, Jinngjing; Hou, Nali; Bin, Tan; Zhang, Yun; Li, Tingyu; Chen, Jie

    2016-01-14

    Mesenchymal stem cell (MSC) transplantation reduces the neurological impairment caused by hypoxic-ischemic brain damage (HIBD) via immunomodulation. In the current study, we found that MSC transplantation improved learning and memory function and enhanced long-term potentiation in neonatal rats subjected to HIBD and the amount of IL-6 released from MSCs was far greater than that of other cytokines. However, the neuroprotective effect of MSCs infected with siIL-6-transduced recombinant lentivirus (siIL-6 MSCs) was significantly weakened in the behavioural tests and electrophysiological analysis. Meanwhile, the hippocampal IL-6 levels were decreased following siIL-6 MSC transplantation. In vitro, the levels of IL-6 release and the levels of IL-6R and STAT3 expression were increased in both primary neurons and astrocytes subjected to oxygen and glucose deprivation (OGD) following MSCs co-culture. The anti-apoptotic protein Bcl-2 was upregulated and the pro-apoptotic protein Bax was downregulated in OGD-injured astrocytes co-cultured with MSCs. However, the siIL-6 MSCs suppressed ratio of Bcl-2/Bax in the injured astrocytes and induced apoptosis number of the injured astrocytes. Taken together, these data suggest that the neuroprotective effect of MSC transplantation in neonatal HIBD rats is partly mediated by IL-6 to enhance anti-apoptosis of injured astrocytes via the IL-6/STAT3 signaling pathway.

  11. Vasoreparative dysfunction of CD34+ cells in diabetic individuals involves hypoxic desensitization and impaired autocrine/paracrine mechanisms.

    PubMed

    Jarajapu, Yagna P R; Hazra, Sugata; Segal, Mark; Li Calzi, Sergio; LiCalzi, Sergio; Jadhao, Chandra; Jhadao, Chandra; Qian, Kevin; Mitter, Sayak K; Raizada, Mohan K; Boulton, Michael E; Grant, Maria B

    2014-01-01

    We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34+ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

  12. Opiorphin-dependent up-regulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice

    PubMed Central

    Fu, Shibo; Davies, Kelvin P.

    2015-01-01

    The precise molecular mechanisms underlying priapism associated with sickle cell disease remain to be defined. However, there is increasing evidence that up-regulated activity of the opiorphin and adenosine pathways in corporal tissue, resulting in heighted relaxation of smooth muscle, play an important role in development of priapism. A key enzyme in the adenosine pathway is CD73, an ecto-5-prime-nucleotidase (5-prime-ribonucleotide phosphohydrolase; EC 3.1.3.5) which catalyzes the conversion of adenosine mononucleotides to adenosine. In the present study we investigated how sickle cell disease and hypoxia regulate the interplay between opiorphin and CD73. In the corpora of sickle cell mice we observed significantly elevated expression of both the mouse opiorphin homologue mSmr3a (14-fold) and CD73 (2.2-fold) relative to non-sickle cell controls at a life-stage prior to the exhibition of priapism. Sickle cell disease has a pronounced hypoxic component, therefore we determined if CD73 was also modulated in in vitro corporal smooth muscle (CSM) models of hypoxia. Hypoxia significantly increased CD73 protein and mRNA expression by 1.5-fold and 2-fold, respectively. We previously demonstrated that expression of another component of the adenosine signaling pathway, the adensosine 2B receptor, can be regulated by sialorphin (the rat opiorphin homolologue), and we demonstrate that sialorphin also regulates CD73 expression in a dose and time dependent fashion. Using siRNA to knock-down sialorphin mRNA expression in CSM cells in vitro, we demonstrate that the hypoxic up-regulation of CD73 is dependent on the up-regulation of sialorphin. Overall our data provides further evidence to support a role for opiorphin in CSM in regulating the cellular response regulating response to hypoxia or sickle cell disease by activating smooth muscle relaxant pathways. PMID:25833166

  13. Opiorphin-dependent upregulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice.

    PubMed

    Fu, S; Davies, K P

    2015-07-01

    The precise molecular mechanisms underlying priapism associated with sickle cell disease remain to be defined. However, there is increasing evidence that upregulated activity of the opiorphin and adenosine pathways in corporal tissue, resulting in heighted relaxation of smooth muscle, have an important role in development of priapism. A key enzyme in the adenosine pathway is CD73, an ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5) which catalyzes the conversion of adenosine mononucleotides to adenosine. In the present study we investigated how sickle cell disease and hypoxia regulate the interplay between opiorphin and CD73. In the corpora of sickle cell mice we observed significantly elevated expression of both the mouse opiorphin homolog mSmr3a (14-fold) and CD73 (2.2-fold) relative to non-sickle cell controls at a life stage before the exhibition of priapism. Sickle cell disease has a pronounced hypoxic component, therefore we determined if CD73 was also modulated in in vitro corporal smooth muscle (CSM) models of hypoxia. Hypoxia significantly increased CD73 protein and mRNA expression by 1.5-fold and 2-fold, respectively. We previously demonstrated that expression of another component of the adenosine signaling pathway, the adensosine 2B receptor, can be regulated by sialorphin (the rat opiorphin homolologue), and we demonstrate that sialorphin also regulates CD73 expression in a dose- and time-dependent fashion. Using siRNA to knockdown sialorphin mRNA expression in CSM cells in vitro, we demonstrate that the hypoxic upregulation of CD73 is dependent on the upregulation of sialorphin. Overall, our data provide further evidence to support a role for opiorphin in CSM in regulating the cellular response to hypoxia or sickle cell disease by activating smooth muscle relaxant pathways.

  14. Lysyl oxidase mediates hypoxic control of metastasis.

    PubMed

    Erler, Janine T; Giaccia, Amato J

    2006-11-01

    Hypoxic cancer cells pose a great challenge to the oncologist because they are especially aggressive, metastatic, and resistant to therapy. Recently, we showed that elevation of the extracellular matrix protein lysyl oxidase (LOX) correlates with metastatic disease and is essential for hypoxia-induced metastasis. In an orthotopic rodent model of breast cancer, a small-molecule or antibody inhibitor of LOX abolished metastasis, offering preclinical validation of this enzyme as a therapeutic target.

  15. Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2014-02-15

    To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

  16. Sphingosine Kinase-1 Involves the Inhibitory Action of HIF-1α by Chlorogenic Acid in Hypoxic DU145 Cells

    PubMed Central

    Lee, Myoung-Sun; Lee, Seon-Ok; Kim, Kyu-Ri; Lee, Hyo-Jeong

    2017-01-01

    Hypoxia enhances cancer development in a solid tumor. Hypoxia-inducible factor-1 α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor that regulates tumor. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, anti-apoptosis and cell proliferation as well as imparts resistance to cancer treatment. In this study, we assessed Crataegus Pinnatifida Bunge var. typical Schneider ethanol extract (CPE) for its anti-cancer effects in hypoxia-induced DU145 human prostate cancer cell line. CPE decreased the abundance of HIF-1α and sphingosine kinase-1 (SPHK-1) in hypoxia-induced prostate cancer DU145 cells. CPE decreased HIF-1α and SPHK-1 as well as SPHK-1 activity. Chlorogenic acid (CA) is one of four major compounds of CPE. Compared to CPE, CA significantly decreased the expression of HIF-1α and SPHK-1 as well as SPHK-1 activity in hypoxia-induced DU145 cells. Furthermore, CA decreased phosphorylation AKT and GSK-3β, which are associated with HIF-1α stabilization and affected SPHK-1 in a concentration-dependent manner. We confirmed the mechanism of CA-induced inhibition of HIF-1α by SPHK-1 signaling pathway using SPHK-1 siRNA and SPHK inhibitor (SKI). CA decreased the secretion and cellular expression of VEGF, thus inhibiting hypoxia-induced angiogenesis. Treatment of DU145cells with SPHK1 siRNA and CA for 48 h decreased cancer cell growth, and the inhibitory action of SPHK siRNA and CA on cell growth was confirmed by decrease in the abundance of Proliferating cell nuclear antigen (PCNA). PMID:28165392

  17. Prenatal Hypoxic-Ischemic Insult Changes the Distribution and Number of NADPH-Diaphorase Cells in the Cerebellum

    PubMed Central

    Savignon, Tiago; Costa, Everton; Tenorio, Frank; Manhães, Alex C.; Barradas, Penha C.

    2012-01-01

    Astrogliosis, oligodendroglial death and motor deficits have been observed in the offspring of female rats that had their uterine arteries clamped at the 18th gestational day. Since nitric oxide has important roles in several inflammatory and developmental events, here we evaluated NADPH-diaphorase (NADPH-d) distribution in the cerebellum of rats submitted to this hypoxia-ischemia (HI) model. At postnatal (P) day 9, Purkinje cells of SHAM and non-manipulated (NM) animals showed NADPH-d+ labeling both in the cell body and dendritic arborization in folia 1 to 8, while HI animals presented a weaker labeling in both cellular structures. NADPH-d+ labeling in the molecular (ML), and in both the external and internal granular layer, was unaffected by HI at this age. At P23, labeling in Purkinje cells was absent in all three groups. Ectopic NADPH-d+ cells in the ML of folia 1 to 4 and folium 10 were present exclusively in HI animals. This labeling pattern was maintained up to P90 in folium 10. In the cerebellar white matter (WM), at P9 and P23, microglial (ED1+) NADPH-d+ cells, were observed in all groups. At P23, only HI animals presented NADPH-d labeling in the cell body and processes of reactive astrocytes (GFAP+). At P9 and P23, the number of NADPH-d+ cells in the WM was higher in HI animals than in SHAM and NM ones. At P45 and at P90 no NADPH-d+ cells were observed in the WM of the three groups. Our results indicate that HI insults lead to long-lasting alterations in nitric oxide synthase expression in the cerebellum. Such alterations in cerebellar differentiation might explain, at least in part, the motor deficits that are commonly observed in this model. PMID:22540005

  18. T Helper Cell Activation and Expansion Is Sensitive to Glutaminase Inhibition under Both Hypoxic and Normoxic Conditions

    PubMed Central

    Sener, Zeynep; Cederkvist, Fritjof H.; Volchenkov, Roman; Holen, Halvor L.; Skålhegg, Bjørn S.

    2016-01-01

    Immune responses often take place where nutrients and O2 availability are limited. This has an impact on T cell metabolism and influences activation and effector functions. T cell proliferation and expansion are associated with increased consumption of glutamine which is needed in a number of metabolic pathways and regulate various physiological processes. The first step in endogenous glutamine metabolism is reversible and is regulated by glutaminase (GLS1 and GLS2) and glutamine synthase (GLUL). There are two isoforms of GLS1, Kidney type glutaminase (KGA) and Glutaminase C (GAC). The aim of this study is to investigate the expression, localization and role of GLS1 and GLUL in naïve and activated human CD4+ T cells stimulated through the CD3 and CD28 receptors under normoxia and hypoxia. In proliferating cells, GAC was upregulated and KGA was downregulated, and both enzymes were located to the mitochondria irrespective of O2 levels. By contrast GLUL is localized to the cytoplasm and was upregulated under hypoxia. Proliferation was dependent on glutamine consumption, as glutamine deprivation and GLS1 inhibition decreased proliferation and expression of CD25 and CD226, regardless of O2 availability. Again irrespective of O2, GLS1 inhibition decreased the proportion of CCR6 and CXCR3 expressing CD4+ T cells as well as cytokine production. We propose that systemic Th cell activation and expansion might be dependent on glutamine but not O2 availability. PMID:27467144

  19. Regulation of HIF-1-Alpha, miR-200, and Markers of Cancer Stem Cells by CDF Under Hypoxic Condition

    DTIC Science & Technology

    2012-04-01

    Ali,S., Kong,D., Banerjee,S., Ahmad,A., Li,Y., Azmi,A.S., Miele ,L. and Sarkar,F.H. Over-expression of FoxM1 leads to epithelial-mesenchymal...Ahmad,A., Banerjee,S., Azmi,A.S., Miele ,L. and Sarkar,F.H. Notch-1 induces epithelial-mesenchymal transition consistent with cancer stem cell

  20. A hypoxic signature marks tumors formed by disseminated tumor cells in the BALB-neuT mammary cancer model

    PubMed Central

    Msaki, Aichi; Pastò, Anna; Curtarello, Matteo; Arigoni, Maddalena; Barutello, Giuseppina; Calogero, Raffaele Adolfo; Macagno, Marco; Cavallo, Federica

    2016-01-01

    Metastasis is the final stage of cancer progression. Some evidence indicates that tumor cell dissemination occurs early in the natural history of cancer progression. Disseminated tumor cells (DTC) have been described in the bone marrow (BM) of cancer patients as well as in experimental models, where they correlate with later development of metastasis. However, little is known about the tumorigenic features of DTC obtained at different time points along tumor progression. Here, we found that early DTC isolated from BM of 15-17 week-old Her2/neu transgenic (BALB-neuT) mice were not tumorigenic in immunodeficient mice. In contrast, DTC-derived tumors were easily detectable when late DTC obtained from 19-22 week-old BALB-neuT mice were injected. Angiogenesis, which contributes to regulate tumor dormancy, appeared dispensable to reactivate late DTC, although it accelerated growth of secondary DTC tumors. Compared with parental mammary tumors, gene expression profiling disclosed a distinctive transcriptional signature of late DTC tumors which was enriched for hypoxia-related transcripts and was maintained in ex-vivo cell culture. Altogether, these findings highlight a different tumorigenic potential of early and late DTC in the BALB-neuT model and describe a HIF-1α-related transcriptional signature in DTC tumors, which may render DTC angiogenesis-competent, when placed in a favourable environment. PMID:27105499

  1. Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells

    PubMed Central

    Babcock, Jennifer; Herrera, Alberto; Coricor, George; Karch, Christopher; Liu, Alexander H.; Rivera-Gines, Aida; Ko, Jane L.

    2017-01-01

    Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO) to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR) and hypoxia inducible factor-1α (HIF-1α). The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D) out of four potential HIF response elements of the hKOR gene (HIFA–D) synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing) produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing), suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation. PMID:28117678

  2. Phase I study of the combination of two hypoxic cell radiosensitizers, Ro 03-8799 and SR-2508: toxicity and pharmacokinetics

    SciTech Connect

    Newman, H.F.; Bleehen, N.M.; Workman, P.

    1986-07-01

    The hypoxic cell radiosensitizer Ro 03-8799 produces acute central nervous system toxicity which limits repeated doses of the drug to 0.75 g/m/sup 2/, but peripheral neuropathy does not occur. SR-2508 causes no acute effects at doses greater than 3.0 g/m/sup 2/, but causes peripheral neuropathy at cumulative doses of 30 g/m/sup 2/. By combining maximum tolerated doses of each agent, it may be possible to increase efficacy, but not toxicity. Escalating single doses of Ro 03-8799 and SR-2508 were administered to 10 patients. The drugs were infused together in 50 ml of 0.9% saline over 10 min, beginning at 0.5 g/m/sup 2/ of each agent, and proceeding to a fixed dose of 0.75 g/m/sup 2/ Ro 03-8799 with 0.5, 1.0, 2.0, and 3.0 g/m/sup 2/ SR-2508. Four patients experienced the expected acute syndrome related to Ro 03-8799, but the incidence was not increased by escalating doses of SR-2508, and no peripheral neuropathy was seen. Plasma and urine pharmacokinetic studies showed that no drug interaction occurred. Six patients have been given a 9-dose regime over a 3 week period, using 0.75 g/m/sup 2/ Ro 03-8799 and escalating doses of 0.5, 1.0, and 1.5 g/m2 SR-2508. All exhibited the expected acute side effects related to Ro 03-8799, but with no increase at the higher doses of SR-2508. No other toxicity was seen. Plasma pharmacokinetics performed at the beginning and end of the schedule were similar. Biopsies were taken from six superficial tumors following combined radiosensitizer administration. Mean tumor concentrations over the 30 min following the end of infusion were 30 and 72 micrograms/g for Ro 03-8799 and SR-2508, respectively. These values would be expected to translate into an approximate single dose sensitizer enhancement ratio of 1.5 to 1.6, offering a significant gain over the enhancement possible with the drugs given alone.

  3. Hypoxic cell radiosensitizers in the treatment of high grade gliomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole)

    SciTech Connect

    Newman, H.F.; Bleehen, N.M.; Ward, R.; Workman, P.

    1988-09-01

    The hypoxic cell radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole) have been evaluated for their simultaneous penetration into human brain tumors and surrounding normal tissue. Thirteen patients received a dose of 1 g of each agent, infused over a 10 minute period during neurosurgery. Samples of glioma (20), brain (10) and cerebrospinal fluid (1) were obtained at a mean time (+/- SD) of 31 +/- 18 min from the end of infusion. A 24 hr plasma time course was measured in six patients. Nitroimidazole concentrations were determined by HPLC. For a mean dose of 0.55 g/m2 of each agent, the mean tumor concentrations (+/- SD) were 17.0 +/- 12.0 micrograms/g for Ro 03-8799 and 13.5 +/- 10.9 micrograms/g for SR 2508. The tumor/plasma ratios were 279 +/- 230% and 47 +/- 34% respectively. For adjacent 'normal' brain tissue, the radiosensitizer concentrations were 29.9 +/- 13.1 micrograms/g for Ro 03-8799, and 4.0 +/- 1.7 micrograms/g for SR 2508, and the brain/plasma ratios were 430 +/- 29% and 14 +/- 8% respectively. There was a significant trend towards increasing accumulation of both agents with time, in both tumor and normal brain. Concentrations in cerebrospinal fluid were very low. Plasma pharmacokinetics for Ro 03-8799 were similar to previous experience, but for SR 2508 the terminal half-life was greater in this series by a factor of 1.3. The results confirm that Ro 03-8799 is distributed widely in the central nervous system, and demonstrate that SR 2508 can achieve high tumor concentrations when the blood-brain barrier is compromised. The concentrations achieved with the combination are indicative of a significant advantage over metronidazole, misonidazole, or either agent alone, and normalized to the therapeutic dose of 0.75 g/m2 plus 2.0 g/m2 SR 2508 are consistent with those giving additive sensitization in an in vivo mouse tumor model.

  4. Hypoxic viscosity and diabetic retinopathy.

    PubMed Central

    Rimmer, T; Fleming, J; Kohner, E M

    1990-01-01

    Diabetic and sickle retinopathy have features in common--for example, venous dilatation, microaneurysms, and capillary closure preceding neovascularisation. Bearing in mind that haemoglobin in poorly controlled diabetes is abnormal and that extremely low oxygen tensions (known to cause sickling) exist in the healthy cat retina, we wished to explore the possibility that diabetic blood, like that of sickle cell disease, may become more viscous when deoxygenated. To do this we measured whole blood viscosity, under oxygenated and deoxygenated conditions, of 23 normal persons, 23 diabetic patients without retinopathy, and 34 diabetic patients with retinopathy. The shear rate used was 230 s-1, which is similar to that thought to prevail in the major retinal veins. The viscosity of blood from normal persons, corrected for packed cell volume, did not change significantly on deoxygenation: mean 4.54 (SD 0.38) cps, versus, 4.57 (0.39) paired t test, p = 0.66. Similarly the blood from diabetics without retinopathy showed no change: 4.42 (0.45) versus 4.42 (0.30), p = 0.98; whereas the blood from patients with retinopathy changed from 4.82 (0.48) to 4.95 (0.63), p = 0.027. The hypoxic viscosity ratio (deoxygenated divided by oxygenated viscosity) correlated with total serum cholesterol (r = 0.44, p = 0.018) but not with HbA1, serum glucose, triglycerides, or age. A disproportionate increase in venous viscosity relative to arterial viscosity would lead to increased intraluminal and transmural pressure and therefore exacerbate leakage across capillary walls. PMID:2378855

  5. Hypoxic Episodes in Bronchopulmonary Dysplasia

    PubMed Central

    Martin, Richard J.; Di Fiore, Juliann M.; Walsh, Michele C.

    2015-01-01

    Hypoxic episodes are troublesome components of bronchopulmonary dysplasia in preterm infants. Immature respiratory control appears to be the major contributor, typically superimposed upon abnormal respiratory function. As a result, relatively short respiratory pauses may precipitate desaturation and accompanying bradycardia. As this population is predisposed to pulmonary hypertension, it is likely that pulmonary vasoconstriction may also play a role in hypoxic episodes. The natural history of intermittent hypoxic episodes has been well characterized in the preterm population at risk for BPD. However, the consequences of these episodes are less clear. Proposed associations of intermittent hypoxia include retinopathy of prematurity, sleep disordered breathing, and neurodevelopmental delay. Future study should address whether these associations are causal relationships. PMID:26593081

  6. Hypoxia-inducible factor-1β (HIF-1β) is upregulated in a HIF-1α-dependent manner in 518A2 human melanoma cells under hypoxic conditions

    SciTech Connect

    Mandl, Markus Kapeller, Barbara; Lieber, Roman; Macfelda, Karin

    2013-04-26

    Highlights: •HIF-1β is a hypoxia-responsive protein in 518A2 human melanoma cells. •HIF-1β is upregulated in a HIF-1α-dependent manner under hypoxic conditions. •HIF-1β is not elevated due to heterodimerization with HIF-1α per se. •HIF-1β inducibility has a biological relevance as judged in Het-CAM model. -- Abstract: Solid tumors include hypoxic areas due to excessive cell proliferation. Adaptation to low oxygen levels is mediated by the hypoxia-inducible factor (HIF) pathway promoting invasion, metastasis, metabolic alterations, chemo-resistance and angiogenesis. The transcription factor HIF-1, the major player within this pathway consists of HIF-1α and HIF-1β. The alpha subunit is continuously degraded under normoxia and becomes stabilized under reduced oxygen supply. In contrast, HIF-1β is generally regarded as constitutively expressed and being present in excess within the cell. However, there is evidence that the expression of this subunit is more complex. The aim of this study was to investigate the role of HIF-1β in human melanoma cells. Among a panel of five different cell lines, in 518A2 cells exposed to the hypoxia-mimetic cobalt chloride HIF-1β was rapidly elevated on protein level. Knockdown experiments performed under cobalt chloride-exposure and hypoxia revealed that this effect was mediated by HIF-1α. The non-canonical relationship between these subunits was further confirmed by pharmacologic inhibition of HIF-1α and by expression of a dominant-negative HIF mutant. Overexpression of HIF-1α showed a time delay in HIF-1β induction, thus arguing for HIF-1β de novo synthesis rather than protein stabilization by heterodimerization. A Hen’s egg test-chorioallantoic membrane model of angiogenesis and invasion indicated a local expression of HIF-1β and implies a biological relevance of these findings. In summary, this study demonstrates the HIF-1α-dependent regulation of HIF-1β under hypoxic conditions for the first time. The

  7. Tirapazamine: hypoxic cytotoxicity and interaction with radiation as assessed by the micronucleus assay.

    PubMed Central

    Shibata, T.; Shibamoto, Y.; Sasai, K.; Oya, N.; Murata, R.; Takagi, T.; Hiraoka, M.; Takahashi, M.; Abe, M.

    1996-01-01

    We investigated the cytotoxicity and the interaction with low-dose radiation (1-4Gy) of tirapazamine by the in vitro cytokinesis-block micronucleus (MN) assay. Murine SCCVII and human melanoma (G-361) cells were treated with tirapazamine under aerobic or hypoxic conditions for 1 h and the MN frequency was determined using cytochalasin-B. The cells were also treated with or without tirapazamine or KU-2285 (hypoxic cell sensitiser) under hypoxic conditions and irradiated with or without reaeration of the cell suspensions. A dose-dependent increase of MN frequency was observed by tirapazamine treatment and the hypoxic toxicity ratio was about 130 for SCCVII and 37 for G-361. The radiation dose-response curves of MN frequency suggested that the interaction of tirapazamine with irradiation appeared to be essentially additive in both cell lines. In contrast, the dose-response curve became steeper by KU-2285 treatment. Combined effects of tirapazamine and irradiation on the hypoxic cells were much higher than the radiation effect on aerobic cells at low doses, while the effects of KU-2285 did not exceed that of aerobic irradiation. In conclusion, tirapazamine appeared to be superior to hypoxic radiosensitisers at clinically relevant doses, not because of aerobic radiosensitisation but because of its potent hypoxic cytotoxicity additive to radiation effect. PMID:8763848

  8. Chronic nicotine blunts hypoxic sensitivity in perinatal rat adrenal chromaffin cells via upregulation of KATP channels: role of alpha7 nicotinic acetylcholine receptor and hypoxia-inducible factor-2alpha.

    PubMed

    Buttigieg, Josef; Brown, Stephen; Holloway, Alison C; Nurse, Colin A

    2009-06-03

    Fetal nicotine exposure blunts hypoxia-induced catecholamine secretion from neonatal adrenomedullary chromaffin cells (AMCs), providing a link between maternal smoking, abnormal arousal responses, and risk of sudden infant death syndrome. Here, we show that the mechanism is attributable to upregulation of K(ATP) channels via stimulation of alpha7 nicotinic ACh receptors (AChRs). These K(ATP) channels open during hypoxia, thereby suppressing membrane excitability. After in utero exposure to chronic nicotine, neonatal AMCs show a blunted hypoxic sensitivity as determined by inhibition of outward K(+) current, membrane depolarization, rise in cytosolic Ca(2+), and catecholamine secretion. However, hypoxic sensitivity could be unmasked in nicotine-exposed AMCs when glibenclamide, a blocker of K(ATP) channels, was present. Both K(ATP) current density and K(ATP) channel subunit (Kir 6.2) expression were significantly enhanced in nicotine-exposed cells relative to controls. The entire sequence could be reproduced in culture by exposing neonatal rat AMCs or immortalized fetal chromaffin (MAH) cells to nicotine for approximately 1 week, and was prevented by coincubation with selective blockers of alpha7 nicotinic AChRs. Additionally, coincubation with inhibitors of protein kinase C and CaM kinase, but not protein kinase A, prevented the effects of chronic nicotine in vitro. Interestingly, chronic nicotine failed to blunt hypoxia-evoked responses in MAH cells bearing short hairpin knockdown (>90%) of the transcription factor, hypoxia-inducible factor-2alpha (HIF-2alpha), suggesting involvement of the HIF pathway. The therapeutic potential of K(ATP) channel blockers was validated in experiments in which hypoxia-induced neonatal mortality in nicotine-exposed pups was significantly reduced after pretreatment with glibenclamide.

  9. Effects of various acute hypoxic conditions on the hemorheological response during exercise and recovery1.

    PubMed

    Moon, Hwang-Woon; Shin, Se-Hyun; Lee, Chul-Hyun; Park, Hun-Young; Sunoo, Sub; Nam, Sang-Seok

    2016-10-05

    Even though exercise hemorheology at hypoxic condition has been considered as a good tool to understand clinical hemorheology, there have been limited studies reported. Previous researches showed that hemorheological variables are closely correlated with oxygen delivery capacity during exercise. The present study investigated hypoxic responses including RBC deformability and aggregation, metabolic parameters and complete blood cell counts at various hypoxic conditions during cycling exercise and recovery. Eleven Korean healthy male subjects performed submaximal bike exercise at sea level (20.9% O2) and under various hypoxic conditions (16.5% O2, 14.5% O2, 12.8% O2, and 11.2% O2) in a random order. The submaximal bike exercise intensity of the subjects was 70% maximum heart rate at sea level. All variables were measured at rest, during exercise and recovery 30-minute, respectively. As oxygen partial pressure decreased, arterial blood oxygen saturation decreased but oxygen uptake did not change much. Heart rate and lactate concentration during exercise increased when oxygen partial pressure is less than or equal to 14.5% O2 condition. Red blood cell (RBC) counts, hemoglobin counts, and hematocrit level were not apparently altered with hypoxic conditions. RBC deformability showed significant alterations at 11.2% O2 conditions compared with other hypoxic conditions during exercise or recovery, except at 10 minutes recovery. However, decreases in oxygen partial pressure did not affect red blood cell aggregation. Therefore, we conclude that alterations in RBC deformability may reduce aerobic capabilities at hypoxic condition.

  10. Phase I/II Trial of Sequential Chemoradiotherapy Using a Novel Hypoxic Cell Radiosensitizer, Doranidazole (PR-350), in Patients With Locally Advanced Non-Small-Cell Lung Cancer (WJTOG-0002)

    SciTech Connect

    Nishimura, Yasumasa Nakagawa, Kazuhiko; Takeda, Koji; Tanaka, Masahiro; Segawa, Yoshihiko; Tsujino, Kayoko; Negoro, Shunichi; Fuwa, Nobukazu; Hida, Toyoaki; Kawahara, Masaaki; Katakami, Nobuyuki; Hirokawa, Keiko; Yamamoto, Nobuyuki; Fukuoka, Masahiro; Ariyoshi, Yutaka

    2007-11-01

    Purpose: This Phase I/II trial was conducted to assess the efficacy and safety of PR-350, a novel hypoxic cell radiosensitizer, when administered with thoracic radiation therapy (RT) after induction chemotherapy (CT) for locally advanced non-small-cell lung cancer (NSCLC). Methods and Materials: Two cycles of cisplatin (80 mg/m{sup 2}) and paclitaxel (180 mg/m{sup 2}), or carboplatin (AUC = 6) and paclitaxel (200 mg/m{sup 2}) were given before RT of 60 Gy in 30 fractions. In the Phase I portion, the starting dosage of PR-350 was 10 daily administrations (2000 mg/m{sup 2}) in combination with RT, and this number was increased in increments of 10 for successive groups to 30 doses. Results: In total, 37 patients were enrolled. In Phase I (n = 20), PR-350 could be administered 30 times with concurrent thoracic RT. Thus, in Phase II (n = 17), PR-350 was administered 30 times. The major toxicity was radiation pneumonitis, with Grade 3 or more pneumonitis noted in 6 patients (16%) including 2 with treatment-related deaths. However, no Grade 3 or more esophageal toxicity was noted, and only Grade 1 peripheral neuropathy was noted in 9 patients (24%). For all 37 patients, the median survival time (MST) and the 2-year survival rate were 15.9 months and 24%, respectively. For 18 patients receiving 21 to 30 doses of PR-350, the MST and 2-year survival rate were 20.9 months and 33%, respectively. Conclusions: Thoracic RT combined with 30 daily administrations of PR-350 after induction CT was well tolerated and promising for locally advanced NSCLC.

  11. Adaptation of iron requirement to hypoxic conditions at high altitude.

    PubMed

    Gassmann, Max; Muckenthaler, Martina U

    2015-12-15

    Adequate acclimatization time to enable adjustment to hypoxic conditions is one of the most important aspects for mountaineers ascending to high altitude. Accordingly, most reviews emphasize mechanisms that cope with reduced oxygen supply. However, during sojourns to high altitude adjustment to elevated iron demand is equally critical. Thus in this review we focus on the interaction between oxygen and iron homeostasis. We review the role of iron 1) in the oxygen sensing process and erythropoietin (Epo) synthesis, 2) in gene expression control mediated by the hypoxia-inducible factor-2 (HIF-2), and 3) as an oxygen carrier in hemoglobin, myoglobin, and cytochromes. The blood hormone Epo that is abundantly expressed by the kidney under hypoxic conditions stimulates erythropoiesis in the bone marrow, a process requiring high iron levels. To ensure that sufficient iron is provided, Epo-controlled erythroferrone that is expressed in erythroid precursor cells acts in the liver to reduce expression of the iron hormone hepcidin. Consequently, suppression of hepcidin allows for elevated iron release from storage organs and enhanced absorption of dietary iron by enterocytes. As recently observed in sojourners at high altitude, however, iron uptake may be hampered by reduced appetite and gastrointestinal bleeding. Reduced iron availability, as observed in a hypoxic mountaineer, enhances hypoxia-induced pulmonary hypertension and may contribute to other hypoxia-related diseases. Overall, adequate systemic iron availability is an important prerequisite to adjust to high-altitude hypoxia and may have additional implications for disease-related hypoxic conditions.

  12. Marine Natural Products as Inhibitors of Hypoxic Signaling in Tumors

    PubMed Central

    Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    Marine natural products have become a major source of new chemical entities in the discovery of potential anticancer agents that potently suppress various antitumor molecular targets. As a consequence of insufficient vascularization, hypoxic regions form within rapidly growing solid tumor masses. Specific alterations of gene expression in these hypoxic tumor cells help facilitate the survival and metastatic spread of solid tumors. The transcriptional response to cellular hypoxia is primarily mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1) that regulates the expression of more than 100 genes involved in cellular adaptation and survival under hypoxic stress. Clinical studies in cancer patients indicate that HIF-1 activation is directly correlated with advanced disease stages and treatment resistance. HIF-1 has emerged as an important tumor-selective molecular target for anticancer drug discovery. As a result, natural product-based inhibitors of HIF-1 activation have been identified from plants and microorganisms. Recently, structurally unique natural products from marine sponges, crinoids, and algae have been identified as HIF-1 activation inhibitors. The US National Cancer Institute’s Open Repository of marine invertebrate and algae extracts has proven to be a valuable source of natural product HIF-1 inhibitors. Among the active compounds identified, certain marine natural products have also been shown to suppress the hypoxic induction of HIF-1 target genes such as vascular endothelial growth factor (VEGF). Some of these marine HIF-1 inhibitors act by interfering with the generation of mitochondrial signaling molecules in hypoxic cells. However, the precise mechanisms of action for many newly identified marine natural product HIF-1 inhibitors remain unresolved. PMID:20622986

  13. HIF-α/MIF and NF-κB/IL-6 axes contribute to the recruitment of CD11b+Gr-1+ myeloid cells in hypoxic microenvironment of HNSCC.

    PubMed

    Zhu, Guiquan; Tang, Yaling; Geng, Ning; Zheng, Min; Jiang, Jian; Li, Ling; Li, Kaide; Lei, Zhengge; Chen, Wei; Fan, Yunlong; Ma, Xiangrui; Li, Longjiang; Wang, Xiaoyi; Liang, Xinhua

    2014-02-01

    CD11b+Gr-1+ myeloid cells have gained much attention due to their roles in tumor immunity suppression as well as promotion of angiogenesis, invasion, and metastases. However, the mechanisms by which CD11b+Gr-1+ myeloid cells recruit to the tumor site have not been well clarified. In the present study, we showed that hypoxia could stimulate the migration of CD11b+Gr-1+ myeloid cells through increased production of macrophage migration inhibitory factor (MIF) and interleukin-6 (IL-6) by head and neck squamous cell carcinoma (HNSCC) cells. Hypoxia-inducible factor-1α (HIF-1α)- and HIF-2α-dependent MIF regulated chemotaxis, differentiation, and pro-angiogenic function of CD11b+Gr-1+ myeloid cells through binding to CD74/CXCR2, and CD74/CXCR4 complexes, and then activating p38/mitogen-activated protein kinase (MAPK) and phosphatidylinositide 3-kinases (PI3K)/AKT signaling pathways. Knockdown (KD) of HIF-1α and HIF-2α in HNSCC cells decreased MIF level but failed to inhibit the CD11b+Gr-1+ myeloid cell migration, because HIF-1α/2α KD enhanced nuclear factor κB (NF-κB) activity that increased IL-6 secretion. Simultaneously blocking NF-κB and HIF-1α/HIF-2α had better inhibitory effect on CD11b+Gr-1+ myeloid cell recruitment in the hypoxic zone than individually silencing HIF-1α/2α or NF-κB. In conclusion, the interaction between HIF-α/MIF and NF-κB/IL-6 axes plays an important role in the hypoxia-induced accumulation of CD11b+Gr-1+ myeloid cells and tumor growth in HNSCC.

  14. Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy

    PubMed Central

    Dixon, Brandon J.; Reis, Cesar; Ho, Wing Mann; Tang, Jiping; Zhang, John H.

    2015-01-01

    Neonatal hypoxic ischemic encephalopathy (HIE) is a devastating disease that primarily causes neuronal and white matter injury and is among the leading cause of death among infants. Currently there are no well-established treatments; thus, it is important to understand the pathophysiology of the disease and elucidate complications that are creating a gap between basic science and clinical translation. In the development of neuroprotective strategies and translation of experimental results in HIE, there are many limitations and challenges to master based on an appropriate study design, drug delivery properties, dosage, and use in neonates. We will identify understudied targets after HIE, as well as neuroprotective molecules that bring hope to future treatments such as melatonin, topiramate, xenon, interferon-beta, stem cell transplantation. This review will also discuss some of the most recent trials being conducted in the clinical setting and evaluate what directions are needed in the future. PMID:26389893

  15. Intranasal delivery of cell-penetrating anti-NF-κB peptides (Tat-NBD) alleviates infection-sensitized hypoxic-ischemic brain injury.

    PubMed

    Yang, Dianer; Sun, Yu-Yo; Lin, Xiaoyi; Baumann, Jessica M; Dunn, R Scott; Lindquist, Diana M; Kuan, Chia-Yi

    2013-09-01

    Perinatal infection aggravates neonatal hypoxic-ischemic (HI) brain injury and may interfere with therapeutic hypothermia. While the NF-κB signaling pathway has been implicated in microglia activation in infection-sensitized HI, the current therapeutic strategies rely on systemic intervention, which could impair neonatal immunity and increase the risk of severe infection. To devise a brain-targeted anti-NF-κB strategy, we examined the effects of intranasal delivery of tat-NBD peptides in two animal models of neonatal infection-sensitized HI. Kinetic experiments showed that tat-NBD peptides entered the olfactory bulbs rapidly (10-30 min) and peaked in the cerebral cortex around 60 min after intranasal application in P7 rats. Further, intranasal delivery of 1.4 mg/kg tat-NBD, which is only 7% of the intravenous dose in past studies, markedly attenuated NF-κB signaling, microglia activation, and brain damage triggered by HI with 4 or 72 h pre-exposure to the bacterial endotoxin lipopolysaccharide (LPS). In contrast, intranasal delivery of mutant tat-NBD peptides or systemic application of minocycline failed to block LPS-sensitized HI injury. Yet, intranasal delivery of up to 5.6 mg/kg tat-NBD peptides immediately after pure-HI insult showed little protection, likely due to its rapid clearance from the brain and inability to inhibit parenchymal plasminogen activators. Together, these results suggest a novel therapy of infection-sensitized HI brain injury in newborns.

  16. Hypoxic radiosensitizers: substituted styryl derivatives.

    PubMed

    Nudelman, A; Falb, E; Odesa, Y; Shmueli-Broide, N

    1994-10-01

    A number of novel styryl epoxides, N-substituted-styryl-ethanolamines, N-mono and N,N'-bis-(2-hydroxyethyl)-cinnamamides--analogues to the known radiosensitizers RSU-1069, pimonidazole and etanidazole--display selective hypoxic radiosensitizing activity. The styryl group, especially when substituted by electron withdrawing groups, was found to be bioisosteric to the nitroimidazolyl functionality. The most active derivative 2-(2'-nitrophenyl)ethen-1-yl-oxirane 8a displayed a sensitizer enhancement ratio (SER) of 5 relative to misonidazole.

  17. The anatomy of a hypoxic operator in Saccharomyces cerevisiae.

    PubMed Central

    Deckert, J; Torres, A M; Hwang, S M; Kastaniotis, A J; Zitomer, R S

    1998-01-01

    Aerobic repression of the hypoxic genes of Saccharomyces cerevisiae is mediated by the DNA-binding protein Rox1 and the Tup1/Ssn6 general repression complex. To determine the DNA sequence requirements for repression, we carried out a mutational analysis of the consensus Rox1-binding site and an analysis of the arrangement of the Rox1 sites into operators in the hypoxic ANB1 gene. We found that single base pair substitutions in the consensus sequence resulted in lower affinities for Rox1, and the decreased affinity of Rox1 for mutant sites correlated with the ability of these sites to repress expression of the hypoxic ANB1 gene. In addition, there was a general but not complete correlation between the strength of repression of a given hypoxic gene and the compliance of the Rox1 sites in that gene to the consensus sequence. An analysis of the ANB1 operators revealed that the two Rox1 sites within an operator acted synergistically in vivo, but that Rox1 did not bind cooperatively in vitro, suggesting the presence of a higher order repression complex in the cell. In addition, the spacing or helical phasing of the Rox1 sites was not important in repression. The differential repression by the two operators of the ANB1 gene was found to be due partly to the location of the operators and partly to the sequences between the two Rox1-binding sites in each. Finally, while Rox1 repression requires the Tup1/Ssn6 general repression complex and this complex has been proposed to require the aminoterminal regions of histones H3 and H4 for full repression of a number of genes, we found that these regions were dispensable for ANB1 repression and the repression of two other hypoxic genes. PMID:9832521

  18. Marine Compound Xyloketal B Reduces Neonatal Hypoxic-Ischemic Brain Injury

    PubMed Central

    Xiao, Ai-Jiao; Chen, Wenliang; Xu, Baofeng; Liu, Rui; Turlova, Ekaterina; Barszczyk, Andrew; Sun, Christopher Lf; Liu, Ling; Deurloo, Marielle; Wang, Guan-Lei; Feng, Zhong-Ping; Sun, Hong-Shuo

    2014-01-01

    Neonatal hypoxic-ischemic encephalopathy causes neurodegeneration and brain injury, leading to sensorimotor dysfunction. Xyloketal B is a novel marine compound isolated from a mangrove fungus Xylaria species (no. 2508) with unique antioxidant effects. In this study, we investigated the effects and mechanism of xyloketal B on oxygen-glucose deprivation-induced neuronal cell death in mouse primary cortical culture and on hypoxic-ischemic brain injury in neonatal mice in vivo. We found that xyloketal B reduced anoxia-induced neuronal cell death in vitro, as well as infarct volume in neonatal hypoxic-ischemic brain injury model in vivo. Furthermore, xyloketal B improved functional behavioral recovery of the animals following hypoxic-ischemic insult. In addition, xyloketal B significantly decreased calcium entry, reduced the number of TUNEL-positive cells, reduced the levels of cleaved caspase-3 and Bax proteins, and increased the level of Bcl-2 protein after the hypoxic-ischemic injury. Our findings indicate that xyloketal B is effective in models of hypoxia-ischemia and thus has potential as a treatment for hypoxic-ischemic brain injury. PMID:25546517

  19. Hyperbaric oxygen suppresses hypoxic-ischemic brain damage in newborn rats.

    PubMed

    Zhu, Min; Lu, Mengru; Li, Qing-Jie; Zhang, Zhuo; Wu, Zheng-Zheng; Li, Jie; Qian, Lai; Xu, Yun; Wang, Zhong-Yuan

    2015-01-01

    The optimal therapeutic time-window and protective mechanism of hyperbaric oxygen in hypoxic-ischemic brain damage remain unclear. This study aimed to determine the neuroprotective effects of hyperbaric oxygen. Following hypoxic-ischemic brain damage modeling in neonatal rats, hyperbaric oxygen was administered at 6, 24, 48, and 72 hours and 1 week after hypoxia, respectively, once daily for 1 week. Fourteen days after hypoxic-ischemic brain damage, cell density and apoptosis rate, number of Fas-L+, caspase-8+, and caspase-3+ neuronal cells, levels of nitric oxide, malondialdehyde, and superoxide dismutase in hippocampus were examined. Morris water maze test was conducted 28 days after insult. Significant improvements were found in cell density, rate of apoptosis, oxidative stress markers, FasL, and caspases in rats treated with hyperbaric oxygen within 72 hours compared to hypoxic-ischemic injury. Similarly, time-dependent behavioral amelioration was observed in pups treated with hyperbaric oxygen. Our findings suggest that hyperbaric oxygen protects against hypoxic-ischemic brain damage by inhibiting oxidative stress and FasL-induced apoptosis, and optimal therapeutic time window is within 72 hours after hypoxic-ischemic brain damage.

  20. Hypoxic preconditioning protects photoreceptors against light damage independently of hypoxia inducible transcription factors in rods.

    PubMed

    Kast, Brigitte; Schori, Christian; Grimm, Christian

    2016-05-01

    Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors.

  1. Differential roles of prostaglandin E-type receptors in activation of hypoxia-inducible factor 1 by prostaglandin E1 in vascular-derived cells under non-hypoxic conditions

    PubMed Central

    Suzuki, Kengo; Nishi, Kenichiro; Takabuchi, Satoshi; Kai, Shinichi; Matsuyama, Tomonori; Kurosawa, Shin; Adachi, Takehiko; Maruyama, Takayuki; Fukuda, Kazuhiko

    2013-01-01

    Prostaglandin E1 (PGE1), known pharmaceutically as alprostadil, has vasodilatory properties and is used widely in various clinical settings. In addition to acute vasodilatory properties, PGE1 may exert beneficial effects by altering protein expression of vascular cells. PGE1 is reported to be a potent stimulator of angiogenesis via upregulation of VEGF expression, which is under the control of the transcription factor hypoxia-inducible factor 1 (HIF-1). However, the molecular mechanisms behind the phenomenon are largely unknown. In the present study, we investigated the mechanism by which PGE1 induces HIF-1 activation and VEGF gene expression in human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs), both vascular-derived cells. HUVECs and HASMCs were treated with PGE1 at clinically relevant concentrations under 20% O2 conditions and HIF-1 protein expression was investigated. Expression of HIF- 1α protein and the HIF-1-downstream genes were low under 20% O2 conditions and increased in response to PGE1 treatment in both HUVECs and HASMCs in a dose- and time-dependent manner under 20% O2 conditions as comparable to exposure to 1% O2 conditions. Studies using EP-receptor-specific agonists and antagonists revealed that EP1 and EP3 are critical to PGE1-induced HIF-1 activation. In vitro vascular permeability assays using HUVECs indicated that PGE1 increased vascular permeability in HUVECs. Thus, we demonstrate that PGE1 induces HIF- 1α protein expression and HIF-1 activation under non-hypoxic conditions and also provide evidence that the activity of multiple signal transduction pathways downstream of EP1 and EP3 receptors is required for HIF-1 activation. PMID:24349900

  2. Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

    SciTech Connect

    Hong, Kyung-Soo; Park, Jun-Ik; Kim, Mi-Ju; Kim, Hak-Bong; Lee, Jae-Won; Dao, Trong Tuan; Oh, Won Keun; Kang, Chi-Dug; Kim, Sun-Hee

    2012-03-01

    SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning. -- Graphical abstract: Polyphenols mimicked hypoxic preconditioning by up-regulating HIF-1α and SIRT1 and down-regulating c-Myc, PHD2, and β-catenin. HepG2 cells were pretreated with the indicated doses of myricetin (MYR; A), quercetin (QUR; B), or piceatannol (PIC; C) for 4 h and then exposed to hypoxia for 4 h. Levels of HIF-1α, SIRT1, c-Myc, β-catenin, and PHD2 were determined by western blot analysis. The data are representative of three individual experiments. Highlights: ► SIRT1 expression is increased in hypoxia

  3. VHL: Cullin-g the hypoxic response.

    PubMed

    Kershaw, Nadia J; Babon, Jeffrey J

    2015-03-03

    Hypoxia inducible factor (HIF)-mediated response to hypoxic conditions is turned off by VHL-mediated ubiquitination of HIFα. To achieve this, VHL binds HIFα and recruits it to a specific E3 ubiquitin ligase complex, a Cullin-RING-ligase. In this issue of Structure, Nguyen et al. provide a structural view of how VHL engages the ligase in order to inhibit the hypoxic response.

  4. Expression and hypoxic regulation of hif1alpha and hif2alpha during early blood and endothelial cell differentiation in chick.

    PubMed

    Ota, Kanako; Nagai, Hiroki; Sheng, Guojun

    2007-08-01

    HIF1 and HIF2 are major mediators for hypoxia sensing and response. Their roles in early differentiation of two key cell types involved in oxygen supply in amniotes, the primitive blood cells and endothelial cells, are unclear. We show that, in pre-circulation avian embryos, hif1alpha and hif2alpha are expressed in embryonic and extraembryonic tissues, respectively. hif2alpha, first identified as epas1, is not present in endothelial cells at any pre-circulation stage under either normoxia or hypoxia conditions. Differentiating blood cells express low levels of hif2alpha under normoxia, but show a strong and rapid upregulation under hypoxia. Blood cell differentiation, however, is not affected under either hypoxia or hyperoxia conditions.

  5. Adapting radiotherapy to hypoxic tumours

    NASA Astrophysics Data System (ADS)

    Malinen, Eirik; Søvik, Åste; Hristov, Dimitre; Bruland, Øyvind S.; Rune Olsen, Dag

    2006-10-01

    In the current work, the concepts of biologically adapted radiotherapy of hypoxic tumours in a framework encompassing functional tumour imaging, tumour control predictions, inverse treatment planning and intensity modulated radiotherapy (IMRT) were presented. Dynamic contrast enhanced magnetic resonance imaging (DCEMRI) of a spontaneous sarcoma in the nasal region of a dog was employed. The tracer concentration in the tumour was assumed related to the oxygen tension and compared to Eppendorf histograph measurements. Based on the pO2-related images derived from the MR analysis, the tumour was divided into four compartments by a segmentation procedure. DICOM structure sets for IMRT planning could be derived thereof. In order to display the possible advantages of non-uniform tumour doses, dose redistribution among the four tumour compartments was introduced. The dose redistribution was constrained by keeping the average dose to the tumour equal to a conventional target dose. The compartmental doses yielding optimum tumour control probability (TCP) were used as input in an inverse planning system, where the planning basis was the pO2-related tumour images from the MR analysis. Uniform (conventional) and non-uniform IMRT plans were scored both physically and biologically. The consequences of random and systematic errors in the compartmental images were evaluated. The normalized frequency distributions of the tracer concentration and the pO2 Eppendorf measurements were not significantly different. 28% of the tumour had, according to the MR analysis, pO2 values of less than 5 mm Hg. The optimum TCP following a non-uniform dose prescription was about four times higher than that following a uniform dose prescription. The non-uniform IMRT dose distribution resulting from the inverse planning gave a three times higher TCP than that of the uniform distribution. The TCP and the dose-based plan quality depended on IMRT parameters defined in the inverse planning procedure (fields

  6. Learning and cognitive deficits in hypoxic neonatal rats intensified by BAX mediated apoptosis: protective role of glucose, oxygen, and epinephrine.

    PubMed

    Raveendran, Anju Thoppil; Skaria, Paulose Cheramadatikudiyil

    2013-02-01

    Hypoxic brain injury during neonatal development can lead to neuronal damage and produce learning and cognitive impairments. TOPRO-3 staining was used to visualize cell loss and real-time polymerase chain reaction (PCR) analysis of BAX mRNA was used to evaluate the level of apoptosis in the cerebral cortex, cerebellum, brain stem, and striatum of hypoxic neonatal rats and hypoxic rats resuscitated with glucose, oxygen, and epinephrine. The long-term effects of neonatal hypoxic insult on cognition and behavior were studied using Morris water maze experiment on 1-month-old rats exposed to neonatal hypoxia. In hypoxic neonatal rats, a significant cell loss (p < .001) within the brain regions was observed in TOPRO-3 staining and BAX mRNA expression was significantly upregulated (p < .001). Immediate resuscitation of hypoxic neonates with glucose, alone and along with oxygen, significantly downregulated (p < .001) BAX mRNA expression. The BAX expression in epinephrine resuscitated and 100% oxygen resuscitated groups were found to be upregulated in the brain regions. In water maze experiment, 1-month-old rats exposed to neonatal hypoxia spent lesser time in the platform quadrant (p < .001) and showed longer escape latency (p < .001) highlighting the learning and cognitive deficits. Our study revealed the effect of glucose resuscitation alone and along with oxygenation in ameliorating the spatial memory and learning deficits induced by neonatal hypoxic insult mediated brain cell loss.

  7. The Galvanotactic Migration of Keratinocytes is Enhanced by Hypoxic Preconditioning

    PubMed Central

    Guo, Xiaowei; Jiang, Xupin; Ren, Xi; Sun, Huanbo; Zhang, Dongxia; Zhang, Qiong; Zhang, Jiaping; Huang, Yuesheng

    2015-01-01

    The endogenous electric field (EF)-directed migration of keratinocytes (galvanotaxis) into wounds is an essential step in wound re-epithelialization. Hypoxia, which occurs immediately after injury, acts as an early stimulus to initiate the healing process; however, the mechanisms for this effect, remain elusive. We show here that the galvanotactic migration of keratinocytes was enhanced by hypoxia preconditioning as a result of the increased directionality rather than the increased motility of keratinocytes. This enhancement was both oxygen tension- and preconditioning time-dependent, with the maximum effects achieved using 2% O2 preconditioning for 6 hours. Hypoxic preconditioning (2% O2, 6 hours) decreased the threshold voltage of galvanotaxis to < 25 mV/mm, whereas this value was between 25 and 50 mV/mm in the normal culture control. In a scratch-wound monolayer assay in which the applied EF was in the default healing direction, hypoxic preconditioning accelerated healing by 1.38-fold compared with the control conditions. Scavenging of the induced ROS by N-acetylcysteine (NAC) abolished the enhanced galvanotaxis and the accelerated healing by hypoxic preconditioning. Our data demonstrate a novel and unsuspected role of hypoxia in supporting keratinocyte galvanotaxis. Enhancing the galvanotactic response of cells might therefore be a clinically attractive approach to induce improved wound healing. PMID:25988491

  8. Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment.

    PubMed

    Song, Yu-Jiao; Zhang, Shan-Shan; Guo, Xian-Ling; Sun, Kai; Han, Zhi-Peng; Li, Rong; Zhao, Qiu-Dong; Deng, Wei-Jie; Xie, Xu-Qin; Zhang, Jian-Wei; Wu, Meng-Chao; Wei, Li-Xin

    2013-10-01

    Liver cancer stem cells (LCSCs) can drive and maintain hepatocellular carcinoma (HCC) growth, metastasis, and recurrence. Therefore, they are potentially responsible for the poor prognosis of HCC. Oxygen and nutrient deficiencies are common characteristics of the tumor microenvironment. However, how LCSCs adapt to oxygen- and nutrient-deprived conditions is unclear. Here, we used immunofluorescent staining and flow cytometry analysis to show that CD133+ cells were significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7. Sorted CD133+ cells showed higher survival, less apoptosis, and possess higher clonogenic ability under H/S compared to the CD133- population. Under H/S, electron microscopy revealed more advanced autophagic vesicles in CD133+ cells. Additionally, CD133+ cells had higher autophagy levels as measured by both RT-qPCR and Western blotting. CD133+ cells had more accumulated GFP-LC3 puncta, which can be detected by fluorescence microscopy. The autophagic inhibitor chloroquine (CQ) significantly increased apoptosis and decreased the clonogenic capacity of CD133+ cells under H/S. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+ cells. However, CQ significantly impaired this process. Therefore, autophagy is essential for LCSCs maintenance. CD133+ cells were also found to have a higher tumor-forming ability in vivo, which could be inhibited by CQ administration. Collectively, our results indicate that the involvement of autophagy in maintenance of CD133+ LCSCs under the oxygen- and nutrient-deprived conditions that are typical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may make LCSCs more sensitive to the tumor microenvironment and be useful in improving anti-cancer treatments.

  9. Effects of chloride channel blockers on hypoxic injury in rat proximal tubules.

    PubMed

    Reeves, W B

    1997-05-01

    These studies examined the pathways and consequences of chloride uptake into proximal tubule cells during in vitro hypoxia. The chloride channel blocker diphenylamine-2-carboxylate (DPC) markedly reduced the degree of hypoxia-induced membrane damage as measured by the release of lactate dehydrogenase (LDH). DPC reduced the release of LDH from hypoxic tubules from 38 +/- 2.7% to 16 +/- 1.7% after 30 minutes of hypoxia (P < 0.001, N = 16) and also reduced 36Cl- uptake by hypoxic tubules. The reduction in LDH release was not associated with better preservation of cell ATP content or with protection against hypoxia-induced DNA damage. Other Cl- channel blockers, such as niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 2-[(2-cyclopentyl-6,7-dichloro-2,3-dihyrdo-2-methyl-1-oxo-1H-in den-5-yl)oxy] acetic acid (IAA-94) provided even greater protection than DPC and were as effective as 2 mM glycine. The Cl- channel blockers appear to act late in the course of hypoxic injury since DNA damage, an early manifestation of injury, is not prevented by the blockers and since addition of the Cl- channel blocker after the hypoxic injury has begun reduces further membrane damage. These results support the conclusion that transport through Cl- channels contributes to hypoxic cell injury in proximal tubular cells.

  10. Regulation of c-jun expression during hypoxic and low-glucose stress.

    PubMed Central

    Ausserer, W A; Bourrat-Floeck, B; Green, C J; Laderoute, K R; Sutherland, R M

    1994-01-01

    Hypoxic stress in tumor cells has been implicated in malignant progression and in the development of therapeutic resistance. We have investigated the effects of acute hypoxic exposure on regulation of the proto-oncogene c-jun in SiHa cells, a human squamous carcinoma cell line. Hypoxic exposure produced increased levels of c-jun mRNA resulting from both message stabilization and transcriptional activation. A superinduction of c-jun message resulted during simultaneous oxygen and glucose deprivation, with several characteristics of an induction mediated by oxidative-stress pathways. This superinduction was blocked by preincubation of cells with the glutathione precursor N-acetyl cysteine or with phorbol 12-myristate 13-acetate, which indicates redox control of c-jun expression and probable involvement of protein kinase C. By gel retardation assay, no increase in AP-1 DNA binding activity was found to be concomitant with the transcriptional activation of c-jun. A lack of increased DNA binding was observed for the consensus AP-1 sequence and for the two AP-1 sequence variants found within the c-Jun promoter. Additionally, hypoxic and low-glucose stress produced no activation of stably transfected AP-1 reporter sequences. Taken together, these results indicate that the transcriptional activation of c-jun during hypoxic and low-glucose stress involves redox control and is unlikely to be mediated by AP-1 recognition elements within the c-jun promoter. Images PMID:8035787

  11. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

    PubMed Central

    Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke. PMID:26881424

  12. Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells.

    PubMed

    Song, Haoming; Cheng, Youjun; Bi, Gang; Zhu, Yihui; Jun, Wei; Ma, Wenlin; Wu, Huimin

    2016-01-01

    Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.

  13. Microfluidic Platform Generates Oxygen Landscapes for Localized Hypoxic Activation

    PubMed Central

    Rexius, Megan L.; Mauleon, Gerardo; Malik, Asrar B.; Rehman, Jalees; Eddington, David T.

    2014-01-01

    An open-well microfluidic platform generates an oxygen landscape using gas-perfused networks which diffuse across a membrane. The device enables real-time analysis of cellular and tissue responses to oxygen tension to define how cells adapt to heterogeneous oxygen conditions found in the physiological setting. We demonstrate that localized hypoxic activation of cells elicited specific metabolic and gene responses in human microvascular endothelial cells and bone marrow-derived mesenchymal stem cells. A robust demonstration of the compatibility of the device with standard laboratory techniques demonstrates the wide utility of the method. This platform is ideally suited to study real-time cell responses and cell-cell interactions within physiologically relevant oxygen landscapes. PMID:25315003

  14. Microfluidic platform generates oxygen landscapes for localized hypoxic activation.

    PubMed

    Rexius-Hall, Megan L; Mauleon, Gerardo; Malik, Asrar B; Rehman, Jalees; Eddington, David T

    2014-12-21

    An open-well microfluidic platform generates an oxygen landscape using gas-perfused networks which diffuse across a membrane. The device enables real-time analysis of cellular and tissue responses to oxygen tension to define how cells adapt to heterogeneous oxygen conditions found in the physiological setting. We demonstrate that localized hypoxic activation of cells elicited specific metabolic and gene responses in human microvascular endothelial cells and bone marrow-derived mesenchymal stem cells. A robust demonstration of the compatibility of the device with standard laboratory techniques demonstrates the wide utility of the method. This platform is ideally suited to study real-time cell responses and cell-cell interactions within physiologically relevant oxygen landscapes.

  15. The lethal interaction of x ray and penicillin induced lesions following x-irradiation of Escherichia coli B/r in the presence of hypoxic cell sensitizers

    SciTech Connect

    Gillies, N.E.; Obioha, F.I.

    1982-03-01

    When Escherichia coli B/r were x-irradiated under anoxia in the presence of different electron-affinic sensitizers and then incubated in broth containing penicillin (at a concentration that did not kill unirradiated cells) additional killing of the bacteria occurred provided the sensitizers were of relatively high lipophilicity. The overall effect was to increase the efficiency of these sensitizers. It is concluded that sensitizer-dependent latent radiation lesions(s) are produced in membrane components of the cell envelope that interact with damage caused by penicillin in the peptidoglycan layer and this causes the additional lethality.

  16. TCN, an AKT inhibitor, exhibits potent antitumor activity and enhances radiosensitivity in hypoxic esophageal squamous cell carcinoma in vitro and in vivo

    PubMed Central

    Guo, Qing; He, Jia; Shen, Feng; Zhang, Wei; Yang, Xi; Zhang, Chi; Zhang, Qu; Huang, Jun-Xing; Wu, Zheng-Dong; Sun, Xin-Chen; Dai, Sheng-Bin

    2017-01-01

    The aim of the present study was to investigate the radiosensitization effect of triciribine (TCN) on human esophageal squamous cell carcinoma (ESCC) in normoxia or hypoxia and its mechanism. The cytotoxicity and radiosensitization mechanism of TCN were investigated by Cell Counting Kit 8, clonogenic assay, flow cytometry, western blotting (WB) and immunofluorescence staining of phospho-histone H2A.X, Ser139 (γ-H2AX) in ESCC in vitro, while the protein expression levels of AKT, phosphorylated (p)-AKT, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) were evaluated by WB in vivo. The cytotoxicity of TCN was dose dependent. Upon exposure to TCN, ESCC cells in hypoxia treated with 4-Gy radiotherapy exhibited an evidently higher apoptotic rate than cells subjected to other treatments. TCN could significantly inhibit the protein expression of p-AKT, HIF-1α and VEGF in vitro and in vivo. The present results suggested that TCN can effectively inhibit AKT, p-AKT, HIF-1α and VEGF, thus conferring radiosensitivity to ESCC in vitro and vivo. TCN is considered as an adjuvant in radiotherapy of ESCC in clinical application. PMID:28356983

  17. A lab-on-a-chip for hypoxic patch clamp measurements combined with optical tweezers and spectroscopy- first investigations of single biological cells.

    PubMed

    Alrifaiy, Ahmed; Borg, Johan; Lindahl, Olof A; Ramser, Kerstin

    2015-04-18

    The response and the reaction of the brain system to hypoxia is a vital research subject that requires special instrumentation. With this research subject in focus, a new multifunctional lab-on-a-chip (LOC) system with control over the oxygen content for studies on biological cells was developed. The chip was designed to incorporate the patch clamp technique, optical tweezers and absorption spectroscopy. The performance of the LOC was tested by a series of experiments. The oxygen content within the channels of the LOC was monitored by an oxygen sensor and verified by simultaneously studying the oxygenation state of chicken red blood cells (RBCs) with absorption spectra. The chicken RBCs were manipulated optically and steered in three dimensions towards a patch-clamp micropipette in a closed microfluidic channel. The oxygen level within the channels could be changed from a normoxic value of 18% O 2 to an anoxic value of 0.0-0.5% O 2. A time series of 3 experiments were performed, showing that the spectral transfer from the oxygenated to the deoxygenated state occurred after about 227 ± 1 s and a fully developed deoxygenated spectrum was observed after 298 ± 1 s, a mean value of 3 experiments. The tightness of the chamber to oxygen diffusion was verified by stopping the flow into the channel system while continuously recording absorption spectra showing an unchanged deoxygenated state during 5400 ± 2 s. A transfer of the oxygenated absorption spectra was achieved after 426 ± 1 s when exposing the cell to normoxic buffer. This showed the long time viability of the investigated cells. Successful patching and sealing were established on a trapped RBC and the whole-cell access (Ra) and membrane (Rm) resistances were measured to be 5.033 ± 0.412 M Ω and 889.7 ± 1.74 M Ω respectively.

  18. Effect of Hypoxic Stress–Activated Polo-like Kinase 3 on Corneal Epithelial Wound Healing

    PubMed Central

    Lu, Jiawei; Wang, Ling; Dai, Wei

    2010-01-01

    Purpose. Hypoxia/reoxygenation conditions can generate oxidative stresses resulting in the suppression of cell proliferation and the delay of corneal epithelial wound healing. The purpose of this study was to investigate the cellular mechanism involving the role of the stress-responsive Polo-like kinase 3 (Plk3) in hypoxic stress–induced delay of corneal epithelial wound healing. Methods. Plk3 activities were determined by immunochemistry and immunocomplex kinase assay approaches. Corneal epithelial wound healing was evaluated by a whole-eye organ culture model and by scratch-induced wound closure assay. Corneal epithelial layer was removed by using a corneal rust-ring-remover in wild-type and Plk3−/− mice. Wound healing was analyzed using a confocal imaging system. Cell growth was measured by MTT assays. Results. The effect of hypoxic stress on early stages of corneal epithelial wound healing was compared with other oxidative stresses, including UV, CoCl2, and H2O2 treatments. Hypoxic stress–induced delay of corneal epithelial wound healing was further evaluated in human corneal epithelial cells and in the corneas of wild-type and Plk3 knockout (Plk3−/−) mice. Hypoxic stress–induced Plk3 activation resulted in growth attenuation and delay of wound healing. Further evidence demonstrated that the increase in Plk3 activity in constitutively active Plk3-expressed cells significantly enhanced stress-induced delay of wound healing. In contrast, hypoxic stress–induced delay of wound healing was markedly diminished in the corneas of Plk3 deficient Plk3−/− mice. Conclusions. These results provide for the first time important evidence that Plk3 plays a significant role in hypoxic stress–induced attenuation of cell growth and delay of corneal epithelial wound healing. PMID:20505196

  19. Adrenocorticotropic Hormone and PI3K/Akt Inhibition Reduce eNOS Phosphorylation and Increase Cortisol Biosynthesis in Long-Term Hypoxic Ovine Fetal Adrenal Cortical Cells.

    PubMed

    Newby, Elizabeth A; Kaushal, Kanchan M; Myers, Dean A; Ducsay, Charles A

    2015-08-01

    This study was designed to determine the role of the MEK/ERK1/2 and PI3K/Akt pathways in cortisol production and endothelial nitric oxide synthase (eNOS) phosphorylation (peNOS) in the ovine fetal adrenal in response to long-term hypoxia (LTH). Pregnant ewes were maintained at high altitude (3820 m) for the last 100 days of gestation (dGa). At 138 to 142 dGa, fetal adrenal cortical cells (FACs) were collected from LTH and age-matched normoxic fetuses. Cortisol production and peNOS were measured in response to pretreatment with the MEK/ERK1/2 pathway inhibitor UO126 (UO) and adrenocorticotropic hormone (ACTH) stimulation. UO126 reduced ACTH-stimulated cortisol in both normoxic and LTH FACs. UO126 alone or in combination with ACTH reduced peNOS in the normoxic group, while ACTH alone or ACTH + UO inhibited peNOS in LTH FACs. Additionally, cortisol was measured in response to pretreatment with UO and treatment with 22R-hydroxycholesterol (22R-OHC) or water-soluble cholesterol (WSC) with and without ACTH stimulation. UO126 had no effect on 22R-OHC-treated cells, but reduced cortisol in cells treated with WSC and/or ACTH. Cortisol and peNOS were also measured in response to pretreatment with PI3K/Akt pathway inhibitor Wortmannin (WT) and ACTH stimulation. Wortmannin further increased cortisol under ACTH-stimulated conditions and, like ACTH, reduced peNOS in LTH but not normoxic FACs. Together, these data suggest that in LTH FACs MEK/ERK1/2 does not regulate peNOS but that UO acts downstream from eNOS, possibly at cholesterol transport, to affect cortisol production in LTH FACs, while the PI3K/Akt pathway, along with ACTH, regulates peNOS and plays a role in the fetal adaptation to LTH in FACs.

  20. Sirtuin 6 protects the heart from hypoxic damage

    SciTech Connect

    Maksin-Matveev, Anna; Kanfi, Yariv; Hochhauser, Edith; Isak, Ahuva; Cohen, Haim Y.; Shainberg, Asher

    2015-01-01

    Sirtuin 6 (SIRT6) is a protein associated with prolonged life expectancy. We investigated whether life extension is associated with cardioprotection against hypoxia. The proposed study is to develop approaches to reduce hypoxic damage through the use of the sirtuin pathway and to elucidate the mechanism involved. For that purpose we subjected cardiomyocytes from transgenic mice (TG) with over-expression of SIRT6, to hypoxic stress in cell cultures. We hypothesized that cardiomyocytes from transgenic mice subjected to prolonged hypoxia may release survival factors or fewer damage markers to protect them from hypoxic stress compared with wild type (WT) mice. Lactate dehydrogenase (LDH) and creatine kinase (CK) released to the medium and propidium iodide (PI) binding, were markedly decreased following hypoxia in TG cardiomyocytes. The protective mechanism of SIRT6 over-expression includes the activation of pAMPKα pathway, the increased protein level of B-cell lymphoma 2 (Bcl2), the inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), the decrease of reactive oxygen species (ROS) and the reduction in the protein level of phospho-protein kinase B (pAkt) during hypoxia. Together, all these processes impede the necrosis/apoptosis pathways leading to the improved survival of cardiomyocytes following hypoxia, which might explain life extension. - Highlights: • Sirtuin 6 is a protein associated with prolonged life expectancy. • Over-expression of sirtuin 6 protects cardiocytes from hypoxia and oxidative stress. • Over-expression of sirtuin 6 activates the pAMPKα pathway and the Bcl2 expression. • Over-expression of sirtuin 6 decreases ROS formation and pAkt level during hypoxia. • These pathways protect cardiocytes from hypoxia and might explain lifespan extension.

  1. First Autologous Cell Therapy of Cerebral Palsy Caused by Hypoxic-Ischemic Brain Damage in a Child after Cardiac Arrest—Individual Treatment with Cord Blood

    PubMed Central

    Jensen, A.; Hamelmann, E.

    2013-01-01

    Each year, thousands of children incur brain damage that results in lifelong sequelae. Therefore, based on experimental evidence, we explored the therapeutic potential of human cord blood, known to contain stem cells, to examine the functional neuroregeneration in a child with cerebral palsy after cardiac arrest. The boy, whose cord blood was stored at birth, was 2.5 years old and normally developed when global ischemic brain damage occurred resulting in a persistent vegetative state. Nine weeks later, he received autologous cord blood (91.7 mL, cryopreserved, 5.75 × 10e8 mononuclear cells) intravenously. Active rehabilitation (physio- and ergotherapy) was provided daily, follow-up at 2, 5, 12, 24, 30, and 40 months. At 2-months follow-up the boy's motor control improved, spastic paresis was largely reduced, and eyesight was recovered, as did the electroencephalogram. He smiled when played with, was able to sit and to speak simple words. At 40 months, independent eating, walking in gait trainer, crawling, and moving from prone position to free sitting were possible, and there was significantly improved receptive and expressive speech competence (four-word sentences, 200 words). This remarkable functional neuroregeneration is difficult to explain by intense active rehabilitation alone and suggests that autologous cord blood transplantation may be an additional and causative treatment of pediatric cerebral palsy after brain damage. PMID:23762741

  2. In vivo profiling of hypoxic gene expression in gliomas using the hypoxia marker EF5 and laser-capture microdissection

    PubMed Central

    Marotta, Diane; Karar, Jayashree; Jenkins, W. Timothy; Kumanova, Monika; Jenkins, Kevin W.; Tobias, John W.; Baldwin, Donald; Hatzigeorgiou, Artemis; Alexiou, Panagiotis; Evans, Sydney M.; Alarcon, Rodolfo; Maity, Amit; Koch, Cameron; Koumenis, Constantinos

    2010-01-01

    Hypoxia is a key determinant of tumor aggressiveness, yet little is known regarding hypoxic global gene regulation in vivo. We have employed the hypoxia marker EF5 coupled with laser capture microdissection to isolate RNA from viable hypoxic and normoxic regions of 9L experimental gliomas. Through microarray analysis, we have identified several mRNAs (including the HIF targets Vegf, Glut-1 and Hsp27) with increased levels under hypoxia compared to normoxia both in vitro and in vivo. However, we also found striking differences between the global in vitro and in vivo hypoxic mRNA profiles. Intriguingly, the mRNA levels of a substantial number of immunomodulatory and DNA repair proteins including CXCL9, CD3D and RAD51 were found to be downregulated in hypoxic areas in vivo, consistent with a pro-tumorigenic role of hypoxia in solid tumors. Immunohistochemical staining verified increased HSP27 and decreased RAD51 protein levels in hypoxic vs. normoxic tumor regions. Moreover, CD8+ T cells which are recruited to tumors upon stimulation by CXCL9 and CXCL10, were largely excluded from viable hypoxic areas in vivo. This is the first study to analyze the influence of hypoxia on mRNA levels in vivo and can be readily adapted to obtain a comprehensive picture of hypoxic regulation of gene expression and its influence on biological functions in solid tumors. PMID:21266355

  3. Initiation of premature senescence by Bcl-2 in hypoxic condition.

    PubMed

    Wang, Wei; Wang, Desheng; Li, Hong

    2014-01-01

    Senescence, a state of cell cycle arrest, has been regarded as an intrinsic barrier to malignance. Although being repressed in most immortal tumors, the genetic program of senescence can be reactivated by critical regulators, including the apoptosis regulator Bcl-2. We showed here that hypoxic condition resulted in an irreversible senescence-like phenotype with increased expression of Bcl-2 in mouse melanoma B16 cells. In CoCl2-simulating hypoxic condition, characteristic morphological alterations and increased activity of senescence-associated β-galactosidase (SA-β-gal) can be detected with high level of Bcl-2, which was confirmed by western blot and co-staining of SA-β-gal and Bcl-2 by immunocytochemistry. Accordingly, Bcl-2 silence by specific siRNA ahead of hypoxia treatment interrupted the senescent development. Moreover Bcl-2 overexpression led to early onset of senescence. We propose that Bcl-2 is required to initiate and maintain the senescent phenotype. In addition, p53 and p16 were not involved in hypoxia-induced senescence according to the expression levels during senescent process. These results suggest that when encountering harmful stress (hypoxia), melanoma cells overexpress Bcl-2 and turn to senescence, a permanent cell-cycle arrest, for prolonged survival.

  4. Phosphorylation of the pyruvate dehydrogenase complex precedes HIF-1-mediated effects and pyruvate dehydrogenase kinase 1 upregulation during the first hours of hypoxic treatment in hepatocellular carcinoma cells

    PubMed Central

    Zimmer, Andreas David; Walbrecq, Geoffroy; Kozar, Ines; Behrmann, Iris; Haan, Claude

    2016-01-01

    The pyruvate dehydrogenase complex (PDC) is an important gatekeeper enzyme connecting glycolysis to the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, it has a strong impact on the glycolytic flux as well as the metabolic phenotype of a cell. PDC activity is regulated via reversible phosphorylation of three serine residues on the pyruvate dehydrogenase (PDH) E1α subunit. Phosphorylation of any of these residues by the PDH kinases (PDKs) leads to a strong decrease in PDC activity. Under hypoxia, the inactivation of the PDC has been described to be dependent on the hypoxia-inducible factor 1 (HIF-1)-induced PDK1 protein upregulation. In this study, we show in two hepatocellular carcinoma cell lines (HepG2 and JHH-4) that, during the adaptation to hypoxia, PDH is already phosphorylated at time points preceding HIF-1-mediated transcriptional events and PDK1 protein upregulation. Using siRNAs and small molecule inhibitor approaches, we show that this inactivation of PDC is independent of HIF-1α expression but that the PDKs need to be expressed and active. Furthermore, we show that reactive oxygen species might be important for the induction of this PDH phosphorylation since it correlates with the appearance of an altered redox state in the mitochondria and is also inducible by H2O2 treatment under normoxic conditions. Overall, these results show that neither HIF-1 expression nor PDK1 upregulation is necessary for the phosphorylation of PDH during the first hours of the adaptation to hypoxia. PMID:27800515

  5. Synthesis and Evaluation of a CBZ-AAN-Dox Prodrug and its in vitro Effects on SiHa Cervical Cancer Cells Under Hypoxic Conditions.

    PubMed

    Chen, Hongyuan; Liu, Xiao; Clayman, Eric S; Shao, Fangyuan; Xiao, Manshan; Tian, Xuyan; Fu, Wuyu; Zhang, Caiyun; Ruan, Bibo; Zhou, Pengjun; Liu, Zhong; Wang, Yifei; Rui, Wen

    2015-10-01

    Although doxorubicin (Dox) is widely used in clinical treatment for solid tumors, it causes many side-effects such as heart and kidney damage, bone marrow suppression, and drug resistance. Legumain is a lysosomal protease that is elevated and associated with an invasive and metastatic phenotype in a number of solid tumors. In this study, we designed and synthesized a Dox prodrug, N-benzyloxycarbonyl-Ala-Ala-Asn-Doxorubicin (CBZ-AAN-Dox), with 94% purity. Single substrate kinetic assays demonstrated hLegumain-specific enzymatic cleavage and activation of the prodrug in vitro, and this enzymatic cleavage of the prodrug substrate was more sensitive in acidic conditions, releasing more than 70% of Dox after 24 h. Treatment of tumor cells with our prodrug demonstrated a much higher IC50 value, significantly enhanced uptake of the prodrug, and considerably less cellular toxicity compared to Dox treatment alone. Our study presents a novel prodrug, CBZ-AAN-Dox, to potentially increase both the safety and efficacy of clinical treatment of tumors by exploiting the tumor's innate expression of legumain.

  6. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction

    PubMed Central

    Tabeling, Christoph; Yu, Hanpo; Wang, Liming; Ranke, Hannes; Goldenberg, Neil M.; Zabini, Diana; Noe, Elena; Krauszman, Adrienn; Gutbier, Birgitt; Yin, Jun; Schaefer, Michael; Arenz, Christoph; Hocke, Andreas C.; Suttorp, Norbert; Proia, Richard L.; Witzenrath, Martin; Kuebler, Wolfgang M.

    2015-01-01

    Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca2+ mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca2+ mobilization. Ca2+ mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction. PMID:25829545

  7. Vascular endothelial growth factor: an attractive target in the treatment of hypoxic/ischemic brain injury

    PubMed Central

    Guo, Hui; Zhou, Hui; Lu, Jie; Qu, Yi; Yu, Dan; Tong, Yu

    2016-01-01

    Cerebral hypoxia or ischemia results in cell death and cerebral edema, as well as other cellular reactions such as angiogenesis and the reestablishment of functional microvasculature to promote recovery from brain injury. Vascular endothelial growth factor is expressed in the central nervous system after hypoxic/ischemic brain injury, and is involved in the process of brain repair via the regulation of angiogenesis, neurogenesis, neurite outgrowth, and cerebral edema, which all require vascular endothelial growth factor signaling. In this review, we focus on the role of the vascular endothelial growth factor signaling pathway in the response to hypoxic/ischemic brain injury, and discuss potential therapeutic interventions. PMID:26981109

  8. Hypoxic vasodilatation in isolated, perfused guinea-pig heart: an analysis of the underlying mechanisms.

    PubMed Central

    von Beckerath, N; Cyrys, S; Dischner, A; Daut, J

    1991-01-01

    measured in the steady state was linear. In the presence of 10 microM-NNA coronary resistance was increased more than twofold at low perfusion rates; at perfusion rates between 4 and 10 ml min-1 coronary resistance decreased progressively. This change in the pressure-flow relationship may be responsible for the alterations in the time course of hypoxic vasodilatation induced by NNA. 7. In order to test whether changes in energy metabolism in coronary smooth muscle cells were responsible for hypoxic vasodilatation we blocked glycolysis by replacing the glucose in the perfusate with deoxyglucose (DOG).(ABSTRACT TRUNCATED AT 400 WORDS) Images Fig. 1 PMID:1798031

  9. Combining hypoxic methods for peak performance.

    PubMed

    Millet, Gregoire P; Roels, B; Schmitt, L; Woorons, X; Richalet, J P

    2010-01-01

    New methods and devices for pursuing performance enhancement through altitude training were developed in Scandinavia and the USA in the early 1990s. At present, several forms of hypoxic training and/or altitude exposure exist: traditional 'live high-train high' (LHTH), contemporary 'live high-train low' (LHTL), intermittent hypoxic exposure during rest (IHE) and intermittent hypoxic exposure during continuous session (IHT). Although substantial differences exist between these methods of hypoxic training and/or exposure, all have the same goal: to induce an improvement in athletic performance at sea level. They are also used for preparation for competition at altitude and/or for the acclimatization of mountaineers. The underlying mechanisms behind the effects of hypoxic training are widely debated. Although the popular view is that altitude training may lead to an increase in haematological capacity, this may not be the main, or the only, factor involved in the improvement of performance. Other central (such as ventilatory, haemodynamic or neural adaptation) or peripheral (such as muscle buffering capacity or economy) factors play an important role. LHTL was shown to be an efficient method. The optimal altitude for living high has been defined as being 2200-2500 m to provide an optimal erythropoietic effect and up to 3100 m for non-haematological parameters. The optimal duration at altitude appears to be 4 weeks for inducing accelerated erythropoiesis whereas <3 weeks (i.e. 18 days) are long enough for beneficial changes in economy, muscle buffering capacity, the hypoxic ventilatory response or Na(+)/K(+)-ATPase activity. One critical point is the daily dose of altitude. A natural altitude of 2500 m for 20-22 h/day (in fact, travelling down to the valley only for training) appears sufficient to increase erythropoiesis and improve sea-level performance. 'Longer is better' as regards haematological changes since additional benefits have been shown as hypoxic exposure

  10. Honey dilution impact on in vitro wound healing: Normoxic and hypoxic condition.

    PubMed

    Chaudhary, Amrita; Bag, Swarnendu; Barui, Ananya; Banerjee, Provas; Chatterjee, Jyotirmoy

    2015-01-01

    Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2 ), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell-cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription-polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E-cadherin, cytoskeletal protein F-actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real-time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition.

  11. Plasticity in the Neonatal Brain following Hypoxic-Ischaemic Injury

    PubMed Central

    Rocha-Ferreira, Eridan

    2016-01-01

    Hypoxic-ischaemic damage to the developing brain is a leading cause of child death, with high mortality and morbidity, including cerebral palsy, epilepsy, and cognitive disabilities. The developmental stage of the brain and the severity of the insult influence the selective regional vulnerability and the subsequent clinical manifestations. The increased susceptibility to hypoxia-ischaemia (HI) of periventricular white matter in preterm infants predisposes the immature brain to motor, cognitive, and sensory deficits, with cognitive impairment associated with earlier gestational age. In term infants HI causes selective damage to sensorimotor cortex, basal ganglia, thalamus, and brain stem. Even though the immature brain is more malleable to external stimuli compared to the adult one, a hypoxic-ischaemic event to the neonate interrupts the shaping of central motor pathways and can affect normal developmental plasticity through altering neurotransmission, changes in cellular signalling, neural connectivity and function, wrong targeted innervation, and interruption of developmental apoptosis. Models of neonatal HI demonstrate three morphologically different types of cell death, that is, apoptosis, necrosis, and autophagy, which crosstalk and can exist as a continuum in the same cell. In the present review we discuss the mechanisms of HI injury to the immature brain and the way they affect plasticity. PMID:27047695

  12. Magneto-aerotactic bacteria deliver drug-containing nanoliposomes to tumour hypoxic regions

    NASA Astrophysics Data System (ADS)

    Felfoul, Ouajdi; Mohammadi, Mahmood; Taherkhani, Samira; de Lanauze, Dominic; Zhong Xu, Yong; Loghin, Dumitru; Essa, Sherief; Jancik, Sylwia; Houle, Daniel; Lafleur, Michel; Gaboury, Louis; Tabrizian, Maryam; Kaou, Neila; Atkin, Michael; Vuong, Té; Batist, Gerald; Beauchemin, Nicole; Radzioch, Danuta; Martel, Sylvain

    2016-11-01

    Oxygen-depleted hypoxic regions in the tumour are generally resistant to therapies. Although nanocarriers have been used to deliver drugs, the targeting ratios have been very low. Here, we show that the magneto-aerotactic migration behaviour of magnetotactic bacteria, Magnetococcus marinus strain MC-1 (ref. 4), can be used to transport drug-loaded nanoliposomes into hypoxic regions of the tumour. In their natural environment, MC-1 cells, each containing a chain of magnetic iron-oxide nanocrystals, tend to swim along local magnetic field lines and towards low oxygen concentrations based on a two-state aerotactic sensing system. We show that when MC-1 cells bearing covalently bound drug-containing nanoliposomes were injected near the tumour in severe combined immunodeficient beige mice and magnetically guided, up to 55% of MC-1 cells penetrated into hypoxic regions of HCT116 colorectal xenografts. Approximately 70 drug-loaded nanoliposomes were attached to each MC-1 cell. Our results suggest that harnessing swarms of microorganisms exhibiting magneto-aerotactic behaviour can significantly improve the therapeutic index of various nanocarriers in tumour hypoxic regions.

  13. Phosphorylation of carbonic anhydrase IX controls its ability to mediate extracellular acidification in hypoxic tumors.

    PubMed

    Ditte, Peter; Dequiedt, Franck; Svastova, Eliska; Hulikova, Alzbeta; Ohradanova-Repic, Anna; Zatovicova, Miriam; Csaderova, Lucia; Kopacek, Juraj; Supuran, Claudiu T; Pastorekova, Silvia; Pastorek, Jaromir

    2011-12-15

    In the hypoxic regions of a tumor, carbonic anhydrase IX (CA IX) is an important transmembrane component of the pH regulatory machinery that participates in bicarbonate transport. Because tumor pH has implications for growth, invasion, and therapy, determining the basis for the contributions of CA IX to the hypoxic tumor microenvironment could lead to new fundamental and practical insights. Here, we report that Thr443 phosphorylation at the intracellular domain of CA IX by protein kinase A (PKA) is critical for its activation in hypoxic cells, with the fullest activity of CA IX also requiring dephosphorylation of Ser448. PKA is activated by cAMP, which is elevated by hypoxia, and we found that attenuating PKA in cells disrupted CA IX-mediated extracellular acidification. Moreover, following hypoxia induction, CA IX colocalized with the sodium-bicarbonate cotransporter and other PKA substrates in the leading edge membranes of migrating tumor cells, in support of the concept that bicarbonate metabolism is spatially regulated at cell surface sites with high local ion transport and pH control. Using chimeric CA IX proteins containing heterologous catalytic domains derived from related CA enzymes, we showed that CA IX activity was modulated chiefly by the intracellular domain where Thr443 is located. Our findings indicate that CA IX is a pivotal mediator of the hypoxia-cAMP-PKA axis, which regulates pH in the hypoxic tumor microenvironment.

  14. pH-responsive hybrid quantum dots for targeting hypoxic tumor siRNA delivery.

    PubMed

    Zhu, HongYan; Zhang, ShengYu; Ling, Yong; Meng, GuoLiang; Yang, Yu; Zhang, Wei

    2015-12-28

    Hypoxia is a characteristic of cancer and plays a key role in tumorigenesis, angiogenesis and resistance to cancer therapies. SiRNA treatment is effective against hypoxic tumors by gene silencing. However, siRNA delivery to the hypoxic regions of solid tumors still presents a challenge due to the distance from blood vessels and the increased presence of efflux transporters. Therefore, tumor therapies would be improved through the immediate development of an effective siRNA delivery system to hypoxic regions. To this end, we synthesized a system to deliver HIF-1α siRNA into hypoxic tumor cells. The system consists of a functional shell composed of 2-deoxyglucose (DG)-polyethylene glycol (PEG) connected with the compound of lipoic acid, lysine and 9-poly-d-arginine (LA-Lys-9R) by a hydrazone bond and a core of CdTe quantum dots (QDs). The molecular structure of DG-PEG-LA-Lys-9R was confirmed by liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) spectroscopy, Fourier transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The multifunctional CdTe QDs measured approximately 200 nm and showed excellent biocompatibility, perfect siRNA binding capability and enhanced hypoxic tumor targeting. Importantly, the system described here is pH-responsive with a hydrazone bond; therefore, it avoids GLUT1 receptor-mediated endocytic recycling, resulting in irreversible delivery of the siRNA. We used Western blots to confirm the superior gene silencing efficiency induced by the DG-PEG-LA-Lys-9R with hydrazone modified CdTe QDs. Here, we demonstrate high efficacy of the siRNA tumor delivery system using in vitro and in vivo experiments. In addition, these studies demonstrate that pH-responsive hybrid quantum dots show improved antitumor efficacy with decreased organ toxicity, indicating a promising siRNA delivery system for hypoxic cancer therapy.

  15. Effects of chronic normobaric hypoxic and hypercapnic exposure in rats: Prevention of experimental chronic mountain sickness by hypercapnia

    NASA Astrophysics Data System (ADS)

    Lincoln, B.; Bonkovsky, H. L.; Ou, Lo-Chang

    1987-09-01

    A syndrome of experimental chronic mountain sickness can be produced in the Hilltop strain of Sprague-Dawley rats by chronic hypobaric hypoxic exposure. This syndrome is characterized by polycythemia, plasma hemoglobinemia, pulmonary hypertension and right ventricular hypertrophy with eventual failure and death. It has generally been assumed that these changes are caused by chronic hypoxemia, not by hypobaric exposure per se. We have now confirmed this directly by showing that chronic normobaric hypoxic exposure (10.5% O2) produces similar hematologic and hemodynamic changes. Further, the addition of hypercapnic exposure to the hypoxic exposure blunted or prevented the effects of the hypoxic exposure probably by stimulating respiration, thus increasing the rate of oxygen delivery to the cells. Changes in the rate-controlling enzymes of hepatic heme metabolism, 5-aminolevulinate synthase and heme oxygenase, and in cytochrome(s) P-450, the major hepatic hemoprotein(s), were also measured in hypoxic and hypercapnic rats. Hypoxia decreased 5-aminolevulinate synthase and increased cytochrome(s) P-450, probably by increasing the size of a “regulatory” heme pool within hepatocytes. These changes were also prevented by the addition of hypercapnic to hypoxic exposure.

  16. Facilitation of human osteoblast apoptosis by sulindac and indomethacin under hypoxic injury.

    PubMed

    Liu, Cheng; Tsai, An-Ly; Chen, Yen-Chu; Fan, Shih-Chen; Huang, Chun-Hsien; Wu, Chia-Ching; Chang, Chih-Han

    2012-01-01

    Hypoxic-ischemia injury occurs after trauma causes consequential bone necrosis. Non-steroid anti-inflammatory drugs (NSAIDs) are frequently used in orthopedic clinics for pain relief. However, the underlying mechanism and outcome for usage of NSAIDs is poorly understood. To investigate the damage and loss of osteoblast function in hypoxia, two hypoxia mimetics, cobalt chloride (CoCl(2)) and desferrioxamine (DFO), were used to create an in vitro hypoxic microenvironment. The cell damage was observed by decreases of cell viability and increases in cyclooxygenase-2 and cleaved poly(ADP-ribose) polymerase (PARP). Cell apoptosis was confirmed by WST-1 cytotoxic assays and flow cytometry. The functional expression of osteoblast in alkaline phosphatase (ALP) activity was significantly decreased by CoCl(2) and inhibited when treated with DFO. To simulate the use of NSAID after hypoxic injury, four types of anti-inflammatory drugs, sulindac sulfide (SUL), indomethacin (IND), aspirin (Asp), and sodium salicylate (NaS), were applied to osteoblasts after 1 h of hypoxia mimetic treatment. SUL and IND further enhanced cell death after hypoxia. ALP activity was totally abolished in hypoxic osteoblasts under IND treatment. Facilitation of osteoblast apoptosis occurred regardless of IND dosage under hypoxic conditions. To investigate osteoblast in vivo, local hypoxia was created by fracture of tibia and then treated the injured mice with IND by oral feeding. IND-induced osteoblast apoptosis was confirmed by positive staining of TUNEL assay in fractured mice. Significant delay of fracture healing in bone tissue was also observed with the treatment of IND. These results provide information pertaining to choosing appropriate anti-inflammatory drugs for orthopedic patients.

  17. Improved tolerance of acute severe hypoxic stress in chronic hypoxic diaphragm is nitric oxide-dependent.

    PubMed

    Lewis, Philip; McMorrow, Clodagh; Bradford, Aidan; O'Halloran, Ken D

    2015-09-01

    The effects of chronic hypoxia (CH) on respiratory muscle performance have hardly been investigated, despite clinical relevance. Results from recent studies are indicative of unique adaptive strategies in hypoxic diaphragm. Respiratory muscle tolerance of acute severe hypoxic stress was examined in normoxic and CH diaphragm in the presence and absence of a nitric oxide (NO) synthase inhibitor. We tested the hypothesis that improved tolerance of severe hypoxic stress in CH diaphragm is NO-dependent. Wistar rats were exposed to normoxia (sea-level, n = 6) or CH (ambient pressure = 380 mmHg, n = 6) for 6 weeks. Diaphragm muscle functional properties were determined ex vivo under severe hypoxic conditions (gassed with 95%N2/5% CO2) with and without 1 mM L-N(G)-nitroarginine (L-NNA, nNOS inhibitor). Fatigue tolerance, but not force, was significantly improved in CH diaphragm (p = 0.008). CH exposure did not affect diaphragm muscle fibre oxidative capacity determined from cluster analysis of area-density plots of muscle fibre succinate dehydrogenase activity. Acute NOS inhibition reduced diaphragm peak tetanic force (p = 0.018), irrespective of gas treatment, and completely reversed improved fatigue tolerance of the CH diaphragm. We conclude that CH exposure improves fatigue tolerance during acute severe hypoxic stress in an NO-dependent manner, independent of muscle fibre oxidative capacity.

  18. Molecular chaperones and hypoxic-ischemic encephalopathy

    PubMed Central

    Hua, Cong; Ju, Wei-na; Jin, Hang; Sun, Xin; Zhao, Gang

    2017-01-01

    Hypoxic-ischemic encephalopathy (HIE) is a disease that occurs when the brain is subjected to hypoxia, resulting in neuronal death and neurological deficits, with a poor prognosis. The mechanisms underlying hypoxic-ischemic brain injury include excitatory amino acid release, cellular proteolysis, reactive oxygen species generation, nitric oxide synthesis, and inflammation. The molecular and cellular changes in HIE include protein misfolding, aggregation, and destruction of organelles. The apoptotic pathways activated by ischemia and hypoxia include the mitochondrial pathway, the extrinsic Fas receptor pathway, and the endoplasmic reticulum stress-induced pathway. Numerous treatments for hypoxic-ischemic brain injury caused by HIE have been developed over the last half century. Hypothermia, xenon gas treatment, the use of melatonin and erythropoietin, and hypoxic-ischemic preconditioning have proven effective in HIE patients. Molecular chaperones are proteins ubiquitously present in both prokaryotes and eukaryotes. A large number of molecular chaperones are induced after brain ischemia and hypoxia, among which the heat shock proteins are the most important. Heat shock proteins not only maintain protein homeostasis; they also exert anti-apoptotic effects. Heat shock proteins maintain protein homeostasis by helping to transport proteins to their target destinations, assisting in the proper folding of newly synthesized polypeptides, regulating the degradation of misfolded proteins, inhibiting the aggregation of proteins, and by controlling the refolding of misfolded proteins. In addition, heat shock proteins exert anti-apoptotic effects by interacting with various signaling pathways to block the activation of downstream effectors in numerous apoptotic pathways, including the intrinsic pathway, the endoplasmic reticulum-stress mediated pathway and the extrinsic Fas receptor pathway. Molecular chaperones play a key role in neuroprotection in HIE. In this review, we

  19. Hypoxic Ischemic Encephalopathy in the Term Infant

    PubMed Central

    Fatemi, Ali; Wilson, Mary Ann; Johnston, Michael V.

    2010-01-01

    Synopsis Hypoxia-ischemia in the perinatal period is an important cause of cerebral palsy and associated disabilities in children. There has been significant research progress in hypoxic-ischemic encephalopathy over the last two decades and many new molecular mechanisms have been identified. Despite all these advances, therapeutic interventions are still limited. In this review paper, we discuss a number of molecular pathways involved in hypoxia-ischemia, and potential therapeutic targets. PMID:19944838

  20. [Radiation protection using a gaseous hypoxic mixture in oncological practice].

    PubMed

    Strelkov, R B; Chizhov, A Ia; Il'ina, A I; Kuznetsova, L E; Pines, E V

    1979-01-01

    Studies on volunteers have shown that the gas hypoxic mixture containing 10% of oxygen and 90% of nitrogen (GHM-10) renders a protective action on the genetic apparatus of human skin cells but provides no protection of the peripheral blood leucocytes, which show the identical character of metabolic processes as neoplastic cells. Under clinically performed distant x-ray therapy for breast cancer the inhaling of GHM-10 was found to render the antiradiation protective action on different normal tissues (skin, subcellular connective tissue, muscle tissue, mammary gland tissue), but it fails to protect the tumor tissue and regional lymph nodes involved. The clinical observations were supported by pathomorphological examination of the operation material.

  1. A voxel-based multiscale model to simulate the radiation response of hypoxic tumors

    SciTech Connect

    Espinoza, I.; Peschke, P.; Karger, C. P.

    2015-01-15

    Purpose: In radiotherapy, it is important to predict the response of tumors to irradiation prior to the treatment. This is especially important for hypoxic tumors, which are known to be highly radioresistant. Mathematical modeling based on the dose distribution, biological parameters, and medical images may help to improve this prediction and to optimize the treatment plan. Methods: A voxel-based multiscale tumor response model for simulating the radiation response of hypoxic tumors was developed. It considers viable and dead tumor cells, capillary and normal cells, as well as the most relevant biological processes such as (i) proliferation of tumor cells, (ii) hypoxia-induced angiogenesis, (iii) spatial exchange of cells leading to tumor growth, (iv) oxygen-dependent cell survival after irradiation, (v) resorption of dead cells, and (vi) spatial exchange of cells leading to tumor shrinkage. Oxygenation is described on a microscopic scale using a previously published tumor oxygenation model, which calculates the oxygen distribution for each voxel using the vascular fraction as the most important input parameter. To demonstrate the capabilities of the model, the dependence of the oxygen distribution on tumor growth and radiation-induced shrinkage is investigated. In addition, the impact of three different reoxygenation processes is compared and tumor control probability (TCP) curves for a squamous cells carcinoma of the head and neck (HNSSC) are simulated under normoxic and hypoxic conditions. Results: The model describes the spatiotemporal behavior of the tumor on three different scales: (i) on the macroscopic scale, it describes tumor growth and shrinkage during radiation treatment, (ii) on a mesoscopic scale, it provides the cell density and vascular fraction for each voxel, and (iii) on the microscopic scale, the oxygen distribution may be obtained in terms of oxygen histograms. With increasing tumor size, the simulated tumors develop a hypoxic core. Within the

  2. Hypoxic remodelling of Ca{sup 2+} stores does not alter human cardiac myofibroblast invasion

    SciTech Connect

    Riches, K.; Hettiarachchi, N.T.; Porter, K.E.; Peers, C.

    2010-12-17

    Research highlights: {yields} Bradykinin promotes migration and proliferation of myofibroblasts. {yields} Such activity is Ca{sup 2+}-dependent and occurs under hypoxic conditions. {yields} Hypoxia increased myofibroblast Ca{sup 2+} stores but not influx evoked by bradykinin. {yields} Myofibroblast migration and proliferation was unaffected by hypoxia. -- Abstract: Cardiac fibroblasts are the most abundant cell type in the heart, and play a key role in the maintenance and repair of the myocardium following damage such as myocardial infarction by transforming into a cardiac myofibroblast (CMF) phenotype. Repair occurs through controlled proliferation and migration, which are Ca{sup 2+} dependent processes, and often requires the cells to operate within a hypoxic environment. Angiotensin converting enzyme (ACE) inhibitors reduce infarct size through the promotion of bradykinin (BK) stability. Although CMF express BK receptors, their activity under the reduced O{sub 2} conditions that occur following infarct are entirely unexplored. Using Fura-2 microfluorimetry on primary human CMF, we found that hypoxia significantly increased the mobilisation of Ca{sup 2+} from intracellular stores in response to BK whilst capacitative Ca{sup 2+} entry (CCE) remained unchanged. The enhanced store mobilisation was due to a striking increase in CMF intracellular Ca{sup 2+}-store content under hypoxic conditions. However, BK-induced CMF migration or proliferation was not affected following hypoxic exposure, suggesting that Ca{sup 2+} influx rather than mobilisation is of primary importance in CMF migration and proliferation.

  3. Gold nanoparticles enhance anti-tumor effect of radiotherapy to hypoxic tumor

    PubMed Central

    Kim, Mi Sun; Lee, Eun-Jung; Kim, Jae-Won; Chung, Ui Seok; Koh, Won-Gun; Keum, Ki Chang; Koom, Woong Sub

    2016-01-01

    Purpose Hypoxia can impair the therapeutic efficacy of radiotherapy (RT). Therefore, a new strategy is necessary for enhancing the response to RT. In this study, we investigated whether the combination of nanoparticles and RT is effective in eliminating the radioresistance of hypoxic tumors. Materials and Methods Gold nanoparticles (GNPs) consisting of a silica core with a gold shell were used. CT26 colon cancer mouse model was developed to study whether the combination of RT and GNPs reduced hypoxia-induced radioresistance. Hypoxia inducible factor-1α (HIF-1α) was used as a hypoxia marker. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining were conducted to evaluate cell death. Results Hypoxic tumor cells had an impaired response to RT. GNPs combined with RT enhanced anti-tumor effect in hypoxic tumor compared with RT alone. The combination of GNPs and RT decreased tumor cell viability compare to RT alone in vitro. Under hypoxia, tumors treated with GNPs + RT showed a higher response than that shown by tumors treated with RT alone. When a reactive oxygen species (ROS) scavenger was added, the enhanced antitumor effect of GNPs + RT was diminished. Conclusion In the present study, hypoxic tumors treated with GNPs + RT showed favorable responses, which might be attributable to the ROS production induced by GNPs + RT. Taken together, GNPs combined with RT seems to be potential modality for enhancing the response to RT in hypoxic tumors. PMID:27730800

  4. Possible role of lysophosphatidic acid in rat model of hypoxic pulmonary vascular remodeling

    PubMed Central

    2014-01-01

    Abstract Pulmonary hypertension is characterized by cellular and structural changes in the vascular wall of pulmonary arteries. We hypothesized that lysophosphatidic acid (LPA), a bioactive lipid, is implicated in this vascular remodeling in a rat model of hypoxic pulmonary hypertension. Exposure of Wistar rats to 10% O2 for 3 weeks induced an increase in the mean serum levels of LPA, to 40.9 (log-detransformed standard deviations: 23.4–71.7) μM versus 21.6 (11.0–42.3) μM in a matched control animal group (P = 0.037). We also observed perivascular LPA immunohistochemical staining in lungs of hypoxic rats colocalized with the secreted lysophospholipase D autotaxin (ATX). Moreover, ATX colocalized with mast cell tryptase, suggesting implication of these cells in perivascular LPA production. Hypoxic rat lungs expressed more ATX transcripts (2.4-fold) and more transcripts of proteins implicated in cell migration: β2 integrin (1.74-fold), intracellular adhesion molecule 1 (ICAM-1; 1.84-fold), and αM integrin (2.70-fold). Serum from the hypoxic group of animals had significantly higher chemoattractant properties toward rat primary lung fibroblasts, and this increase in cell migration could be prevented by the LPA receptor 1 and 3 antagonists. LPA also increased adhesive properties of human pulmonary artery endothelial cells as well as those of human peripheral blood mononuclear cells, via the activation of LPA receptor 1 or 3 followed by the stimulation of gene expression of ICAM-1, β-1, E-selectin, and vascular cell adhesion molecule integrins. In conclusion, chronic hypoxia increases circulating and tissue levels of LPA, which might induce fibroblast migration and recruitment of mononuclear cells in pulmonary vasculature, both of which contribute to pulmonary vascular remodeling. PMID:25621161

  5. Impact of perinatal systemic hypoxic-ischemic injury on the brain of male offspring rats: an improved model of neonatal hypoxic-ischemic encephalopathy in early preterm newborns.

    PubMed

    Huang, Yuejun; Lai, Huihong; Xu, Hongwu; Wu, Weizhao; Lai, Xiulan; Ho, Guyu; Chen, Yunbin; Ma, Lian

    2013-01-01

    In this study, we attempted to design a model using Sprague-Dawley rats to better reproduce perinatal systemic hypoxic-ischemic encephalopathy (HIE) in early preterm newborns. On day 21 of gestation, the uterus of pregnant rats were exposed and the blood supply to the fetuses of neonatal HIE groups were thoroughly abscised by hemostatic clamp for 5, 10 or 15 min. Thereafter, fetuses were moved from the uterus and manually stimulated to initiate breathing in an incubator at 37 °C for 1 hr in air. We showed that survival rates of offspring rats were decreased with longer hypoxic time. TUNEL staining showed that apoptotic cells were significant increased in the brains of offspring rats from the 10 min and 15 min HIE groups as compared to the offspring rats in the control group at postnatal day (PND) 1, but there was no statistical difference between the offspring rats in the 5 min HIE and control groups. The perinatal hypoxic treatment resulted in decreased neurons and increased cleaved caspase-3 protein levels in the offspring rats from all HIE groups at PND 1. Platform crossing times and the percentage of the time spent in the target quadrant of Morris Water Maze test were significantly reduced in the offspring rats of all HIE groups at PND 30, which were associated with decreased brain-derived neurotrophic factor levels and neuronal cells in the hippocampus of offspring rats at PND 35. These data demonstrated that perinatal ischemic injury led to the death of neuronal cells and long-lasting impairment of memory. This model reproduced hypoxic ischemic encephalopathy in early preterm newborns and may be appropriate for investigating therapeutic interventions.

  6. Simulating hypoxia-induced acidic environment in cancer cells facilitates mobilization and redox-cycling of genomic copper by daidzein leading to pro-oxidant cell death: implications for the sensitization of resistant hypoxic cancer cells to therapeutic challenges.

    PubMed

    Ullah, Mohammad F; Ahmad, Aamir; Bhat, Showket H; Khan, Husain Y; Zubair, Haseeb; Sarkar, Fazlul H; Hadi, Sheikh M

    2016-04-01

    This study was conducted to investigate the mechanism of action involved in the anti-cancer activity of daidzein and identification of cancer specific micro-environment as therapeutic target of this secondary metabolite derived from soy. Our data indicated that daidzein induces cellular DNA breakage, anti-proliferative effects and apoptosis in a concentration-dependent manner. We demonstrated that such a daidzein-induced anti-cancer action involves a copper-dependant pathway in which endogenous copper is mobilized by daidzein and redox-cycled to generate reactive oxygen species which act as an upstream signal leading to pro-oxidant cell death. Further in the context of hypoxia being a resistant factor against standard therapies and that an effect secondary to hypoxia is the intracellular acidification, we show that the anticancer activity of daidzein is modulated positively in acidic pH but copper-specific chelator is still able to inhibit daidzein activity. Moreover, an experimental setup of hypoxia mimic (cobalt chloride) revealed an enhanced sensitivity of cancer cells to the cytotoxic effects of daidzein which was neutralized in the presence of neocuproine. The findings support a paradigm shift from the conventional antioxidant property of dietary isoflavones to molecules capable of initiating a pro-oxidant signaling mediated by reactive oxygen species. Further, the clinical relevance of such an action mechanism in cancer chemoprevention is also proposed. This study identified endogenous copper as a molecular target and acidic pH as a modulating factor for the therapeutic activity of daidzein against cancer. The evidence presented highlights the potential of dietary agents as adjuvants to standard therapeutic regimens.

  7. Time Domains of the Hypoxic Ventilatory Response and Their Molecular Basis

    PubMed Central

    Pamenter, Matthew E.; Powell, Frank L.

    2016-01-01

    Ventilatory responses to hypoxia vary widely depending on the pattern and length of hypoxic exposure. Acute, prolonged, or intermittent hypoxic episodes can increase or decrease breathing for seconds to years, both during the hypoxic stimulus, and also after its removal. These myriad effects are the result of a complicated web of molecular interactions that underlie plasticity in the respiratory control reflex circuits and ultimately control the physiology of breathing in hypoxia. Since the time domains of the physiological hypoxic ventilatory response (HVR) were identified, considerable research effort has gone toward elucidating the underlying molecular mechanisms that mediate these varied responses. This research has begun to describe complicated and plastic interactions in the relay circuits between the peripheral chemoreceptors and the ventilatory control circuits within the central nervous system. Intriguingly, many of these molecular pathways seem to share key components between the different time domains, suggesting that varied physiological HVRs are the result of specific modifications to overlapping pathways. This review highlights what has been discovered regarding the cell and molecular level control of the time domains of the HVR, and highlights key areas where further research is required. Understanding the molecular control of ventilation in hypoxia has important implications for basic physiology and is emerging as an important component of several clinical fields. PMID:27347896

  8. [Autonomic regulation at emotional stress under hypoxic conditions in the elderly with physiological and accelerated aging: effect of hypoxic training].

    PubMed

    Os'mak, E D; Asanov, É O

    2014-01-01

    The effect of hypoxic training on autonomic regulation in psycho-emotional stress conditions in hypoxic conditions in older people with physiological (25 people) and accelerated (28 people) aging respiratory system. It is shown that hypoxic training leads to an increase in vagal activity indicators (HF) and reduced simpatovagal index (LF/HF), have a normalizing effect on the autonomic balance during stress loads in older people with different types of aging respiratory system.

  9. Erythropoietin modulates the neural control of hypoxic ventilation.

    PubMed

    Gassmann, Max; Soliz, Jorge

    2009-11-01

    Numerous factors involved in general homeostasis are able to modulate ventilation. Classically, this comprises several kind of molecules, including neurotransmitters and steroids that are necessary for fine tuning ventilation under different conditions such as sleep, exercise, and acclimatization to high altitude. Recently, however, we have found that erythropoietin (Epo), the main regulator of red blood cell production, influences both central (brainstem) and peripheral (carotid bodies) respiratory centers when the organism is exposed to hypoxic conditions. Here, we summarize the effect of Epo on the respiratory control in mammals and highlight the potential implication of Epo in the ventilatory acclimatization to high altitude, as well as in the several respiratory sickness and syndromes occurring at low and high altitude.

  10. The effects of sugar phosphates in reducing hif-1? Under hypoxic conditions.

    PubMed

    Tucci, Michelle; Perrett, Wesley; Scott, Victoria; Wilson, Gerri; Black, David; Benghuzzi, Hamed

    2012-01-01

    Preliminary research has shown evidence of the presence of connective tissue growth factor CTGF in the tenosynovium of patients with carpal tunnel syndrome, a finding which supports the fibrotic pathophysiological progression of the disease. Connective tissue growth factor (CTGF) expression is regulated through the actions of two distinct molecular signals; transforming growth factor-beta (TGF-ß) and hypoxia inducible factor-1 alpha (HIF-1a). Mannose-6 phosphate is a natural inhibitor of TGF-ß and can also is converted to fructose 6 phosphate which we have shown is capable of reducing HIF-1 a. The goal of this experiment was to determine if mannose 6 phosphate is capable of reducing HIF-1a reducing both components of the CTGF pathway. Fibroblast cells were subjected to 5µM or 50µM concentration of either fructose 1,6 diphosphate (F6P) or mannose 6 phosphate (M6P) for a period of 24 hours in either ambient or hypoxic conditions. After the incubation period, cell viability, cell damage, morphology, and concentration of HIF-1a were determined. Cell numbers were reduced by approximately 50% in hypoxic conditions compared with ambient control. Intracellular glutathione concentration was increased significantly under hypoxic conditions compared with control. The concentration of reduced glutathione in both F6P and M6P were similar to ambient air values indicating a protection against oxidative stress. Hypoxia inducible factor-1 alpha was increased in cells under hypoxic conditions compared to base line levels in normoxic treated cells. Interestingly, only 5µM F6P was able to reduce HIF-1a back toward control normoxic values. The data suggest that the HIF-1a pathway leading to fibrosis is not the primary pathway for reductions in CTGF following MP6 treatments. This information is important in terms of developing compounds which decrease adhesion while not decreasing cell viability or impairing cellular function.

  11. Germination under Extreme Hypobaric and Hypoxic Environment

    NASA Astrophysics Data System (ADS)

    Hashimoto, Hirofumi

    Is the agriculture on Mars without a pressured greenhouse dome possible? In order to inves-tigate a possibility of plant cultivation for the space agriculture on Mars, germination rate for six species of plant, Jute, Chrysanthemum, Komatsuna, Cucumber, Okra, and Eggplant under extreme hypobaric and hypoxic condition was measured. Oxygen partial pressure was 1kPa which was equal to 1/100 of normal earth atmosphere. Seeds of Jute and Cucumber were able to germinate in six species. In the case of Jute, germination rate under the oxygen partial pressure of 1kPa was very high, 70

  12. Extreme Hypoxic Conditions Induce Selective Molecular Responses and Metabolic Reset in Detached Apple Fruit

    PubMed Central

    Cukrov, Dubravka; Zermiani, Monica; Brizzolara, Stefano; Cestaro, Alessandro; Licausi, Francesco; Luchinat, Claudio; Santucci, Claudio; Tenori, Leonardo; Van Veen, Hans; Zuccolo, Andrea; Ruperti, Benedetto; Tonutti, Pietro

    2016-01-01

    The ripening physiology of detached fruit is altered by low oxygen conditions with profound effects on quality parameters. To study hypoxia-related processes and regulatory mechanisms, apple (Malus domestica, cv Granny Smith) fruit, harvested at commercial ripening, were kept at 1°C under normoxic (control) and hypoxic (0.4 and 0.8 kPa oxygen) conditions for up to 60 days. NMR analyses of cortex tissue identified eight metabolites showing significantly different accumulations between samples, with ethanol and alanine displaying the most pronounced difference between hypoxic and normoxic treatments. A rapid up-regulation of alcohol dehydrogenase and pyruvate-related metabolism (lactate dehydrogenase, pyruvate decarboxylase, alanine aminotransferase) gene expression was detected under both hypoxic conditions with a more pronounced effect induced by the lowest (0.4 kPa) oxygen concentration. Both hypoxic conditions negatively affected ACC synthase and ACC oxidase transcript accumulation. Analysis of RNA-seq data of samples collected after 24 days of hypoxic treatment identified more than 1000 genes differentially expressed when comparing 0.4 vs. 0.8 kPa oxygen concentration samples. Genes involved in cell-wall, minor and major CHO, amino acid and secondary metabolisms, fermentation and glycolysis as well as genes involved in transport, defense responses, and oxidation-reduction appeared to be selectively affected by treatments. The lowest oxygen concentration induced a higher expression of transcription factors belonging to AUX/IAA, WRKY, HB, Zinc-finger families, while MADS box family genes were more expressed when apples were kept under 0.8 kPa oxygen. Out of the eight group VII ERF members present in apple genome, two genes showed a rapid up-regulation under hypoxia, and western blot analysis showed that apple MdRAP2.12 proteins were differentially accumulated in normoxic and hypoxic samples, with the highest level reached under 0.4 kPa oxygen. These data suggest

  13. Visualizing the antivascular effect of bortezomib on the hypoxic tumor microenvironment.

    PubMed

    Sun, Xiaorong; Ackerstaff, Ellen; He, Fuqiu; Xing, Ligang; Hsiao, Hung Tsung; Koutcher, Jason A; Ling, C Clifton; Li, Gloria C

    2015-10-27

    Bortezomib, a novel proteasome inhibitor, has been approved for treating multiple myeloma and mantle cell lymphoma and studied pre-clinically and clinically for solid tumors. Preferential cytotoxicity of bortezomib was found toward hypoxic tumor cells and endothelial cells in vitro. The purpose of this study is to investigate the role of a pretreatment hypoxic tumor microenvironment on the effects of bortezomib in vitro and ex vivo, and explore the feasibility of dynamic contrast enhanced magnetic resonance imaging (DCE MRI) to noninvasively evaluate the biological effects of bortezomib. It was shown in vitro by Western blot, flow cytometry, and ELISA that bortezomib accumulated HIF-1α in non-functional forms and blocks its hypoxia response in human colorectal cancer cell lines. Ex vivo experiments were performed with fluorescent immunohistochemical staining techniques using multiple endogenous and exogenous markers to identify hypoxia (pimonidazole, HRE-TKeGFP), blood flow/permeability (Hoechst 33342), micro-vessels (CD31 and SMA), apoptosis (cleaved caspase 3) and hypoxia response (CA9). After bortezomib administration, overall apoptosis index was significantly increased and blood perfusion was dramatically decreased in tumor xenografts. More importantly, apoptosis signals were found preferentially located in moderate and severe pretreatment hypoxic regions in both tumor and endothelial cells. Meanwhile, DCE MRI examinations showed that the tumor blood flow and permeability decreased significantly after bortezomib administration. The present study revealed that bortezomib reduces tumor hypoxia response and blood perfusion, thus, presenting antivascular properties. It will be important to determine the hypoxic/perfusion status pre- and during treatment at further translational studies.

  14. Synaptic NMDA Receptors Mediate Hypoxic Excitotoxic Death

    PubMed Central

    Wroge, Christine M.; Hogins, Joshua; Eisenman, Larry; Mennerick, Steven

    2012-01-01

    Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDA receptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDAR hypothesis posits that synaptic NMDARs are neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. In part, the extrasynaptic hypothesis is built on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a non-substrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase (GPT), which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate. PMID:22573696

  15. Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas.

    PubMed

    Duque-Correa, María A; Kühl, Anja A; Rodriguez, Paulo C; Zedler, Ulrike; Schommer-Leitner, Sandra; Rao, Martin; Weiner, January; Hurwitz, Robert; Qualls, Joseph E; Kosmiadi, George A; Murray, Peter J; Kaufmann, Stefan H E; Reece, Stephen T

    2014-09-23

    Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.

  16. Carbohydrate Supplementation Influences Serum Cytokines after Exercise under Hypoxic Conditions

    PubMed Central

    Caris, Aline Venticinque; Da Silva, Edgar Tavares; Dos Santos, Samile Amorim; Lira, Fabio Santos; Oyama, Lila Missae; Tufik, Sergio; Dos Santos, Ronaldo Vagner Thomatieli

    2016-01-01

    Introduction: Exercise performed at the hypoxia equivalent of an altitude of 4200 m is associated with elevated inflammatory mediators and changes in the Th1/Th2 response. By contrast, supplementation with carbohydrates has an anti-inflammatory effect when exercise is performed under normoxic conditions. The objective of this study was to evaluate the effect of carbohydrate supplementation on cytokines and cellular damage markers after exercise under hypoxic conditions at a simulated altitude of 4200 m. Methods: Seven adult male volunteers who exercised for 60 min at an intensity of 50% VO2Peak were randomly evaluated under three distinct conditions; normoxia, hypoxia and hypoxia + carbohydrate supplementation. Blood samples were collected at rest, at the end of exercise and after 60 min of recovery. To evaluate hypoxia + carbohydrate supplementation, volunteers received a solution of 6% carbohydrate (maltodextrin) or a placebo (strawberry-flavored Crystal Light®; Kraft Foods, Northfield, IL, USA) every 20 min during exercise and recovery. Statistical analyses comprised analysis of variance, with a one-way ANOVA followed by the Tukey post hoc test with a significance level of p < 0.05. Results: Under normoxic and hypoxic conditions, there was a significant increase in the concentration of IL-6 after exercise and after recovery compared to at rest (p < 0.05), while in the hypoxia + carbohydrate group, there was a significant increase in the concentration of IL-6 and TNF-α after exercise compared to at rest (p < 0.05). Furthermore, under this condition, TNF-α, IL-2 and the balance of IL-2/IL-4 were increased after recovery compared to at rest (p < 0.05). Conclusion: We conclude that carbohydrate supplementation modified the IL-6 and TNF-α serum concentrations and shifted the IL-2/IL-4 balance towards Th1 in response without glycemic, glutaminemia and cell damage effects. PMID:27827949

  17. Ginkgolides protect primary cortical neurons from potassium cyanide-induced hypoxic injury.

    PubMed

    Zhu, Li; Xu, You Jia; Du, Fang; Qian, Zhong Ming

    2007-06-01

    In this study, we investigated the effects of ginkgolides (Gins A, B, C and J), the main constituent of the non-flavone fraction of EGb 761, on hypoxic injury induced by potassium cyanide (KCN) in primary cortical neurons. The neurons were pretreated with or without ginkgolides for 24 h before incubation with KCN for 4 h. Cell viability was then determined by a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyletrazolium bromide] assay and lactate dehydrogenase (LDH) release from neurons into the medium was measured. The morphological changes of neurons were observed under inverse microscopy and electron microscopy. The results demonstrated that KCN (0.05 mmol/l) significantly decreased cell viability and increased LDH release (P < 0.05 versus the control). The characteristic changes of neuronal morphology induced by KCN were observed. However, pretreatment of neurons with 37.5 microg/ml of ginkgolides (ginkgolides + KCN group) led to a significant increase in cell viability, a decrease in LDH release (P < 0.05 versus the KCN group) and a remarkable improvement in cellular morphology in hypoxic neurons compared with the KCN group. The data suggested that ginkgolides have a significant role to protect the primary cortical neurons from hypoxic injury induced by KCN.

  18. [The function of the oxytocin-synthesizing system of the hypothalamus in rats with diabetes mellitus undergoing hypoxic training].

    PubMed

    Kolesnyk, Iu M; Abramov, A V; Trzhetsyns'kyĭ, S D; Hancheva, O V

    1999-01-01

    The state of hypothalamic oxytocin-synthesizing system in Wistar rats were investigating. The morphometric measurements and immunocytochemical detection of oxytocin-containing cells was used for determining of the functional state of supraoptic nucleus, anterior and posterior-medialis magnocellular subdivisions of paraventricular nucleus. It was established intermittent hypoxic training exert positive influence on rats with experimental diabetes mellitus. This effects depending on increasing synthesis and secretion of hypothalamic oxytocin. Intermittent hypoxic training elevate contents of immunoreactive oxytocin without changing morphometric characteristics in neurons of supraoptic and paraventricular nuclei and median eminence of hypothalamus. In comparison oxytocin contents in these neurons elevade less significance in diabetic rats, but it was observed increasing of nucleolus volume in hypothalamic oxytocin-synthesizing neurons. Intermittent hypoxic training of diabetic rats stimulate more significance elevating oxytocin contents in hypothalamic neurons and median eminence that evidence high level activity of hypothalamic oxytocin-synthesizing system.

  19. Cellular prion protein directly interacts with and enhances lactate dehydrogenase expression under hypoxic conditions.

    PubMed

    Ramljak, Sanja; Schmitz, Matthias; Zafar, Saima; Wrede, Arne; Schenkel, Sara; Asif, Abdul R; Carimalo, Julie; Doeppner, Thorsten R; Schulz-Schaeffer, Walter J; Weise, Jens; Zerr, Inga

    2015-09-01

    Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified, a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion protein knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the former group. Likewise, cerebral ischemia significantly increases LDH levels in WT vs. Prnp(0/0) mice with accompanying smaller lesions in the WT group. HEK293 cells overexpressing PrP(c) show significantly higher LDH expression/activity following 90-min of hypoxia as compared to control cells. Moreover, a cytoplasmic co-localization of LDH and PrP(c) was recorded under both normoxic and hypoxic conditions. Interestingly, an expression of monocarboxylate transporter 1, responsible for cellular lactate uptake, increases with PrP(c)-overexpression under normoxic conditions. Our data suggest LDH as a direct PrP(c) interactor with possible physiological relevance under low oxygen conditions.

  20. Detection and specific targeting of hypoxic regions within solid tumors: current preclinical and clinical strategies.

    PubMed

    Bache, M; Kappler, M; Said, H M; Staab, A; Vordermark, D

    2008-01-01

    Poor oxygenation of solid tumors is a major indicator of adverse prognosis after standard treatment, e.g. radiotherapy. This observation founded on intratumoral pO(2) electrode measurements has been supported more recently by studies of injected hypoxia markers (pimonidazole, EF5) or hypoxia-related proteins (hypoxia-inducible factor-1alpha, carbonic anhydrase IX) detected immunohistochemically. Alternative approaches include imaging of tumor hypoxia by nuclear medicine studies and the measurement of hypoxia-related proteins (osteopontin) in patient plasma. Low oxygen levels as found in tumors are rarely observed in normal tissues. The presence of hypoxic tumor cells is therefore regarded not only as an adverse prognostic factor but as an opportunity for tumor-specific treatment. Classic approaches to normalize tumor oxygenation involve the breathing of modified gas mixtures and pharmacologic modification of blood flow as in the "accelerated radiotherapy, carbogen, nicotinamide" (ARCON) scheme. Specific killing of hypoxic tumor cells can potentially be achieved by hypoxia-selective cytotoxins (model substance tirapazamine), which has shown promise in head and neck cancer. Direct targeting of hypoxia-related molecules such as hypoxia-inducible factor-1alpha, the central regulator of the hypoxic response in tumor cells, is an attractive approach currently tested in preclinical models. For clinical applications, the appropriate combination of hypoxia detection for patient selection with a hypoxia-specific treatment is essential. A therapeutic benefit has been suggested for the selection of patients by plasma osteopontin level and treatment with the hypoxic radiosensitizer nimorazole in addition to radiotherapy, for selection by F-misonidazole positron-emission tomography (PET) and treatment with tirapazamine in addition to chemoradiation and for selection by pimonidazole immunohistochemistry and ARCON treatment, all in head and neck cancer.

  1. Kill-painting of hypoxic tumours in charged particle therapy

    PubMed Central

    Tinganelli, Walter; Durante, Marco; Hirayama, Ryoichi; Krämer, Michael; Maier, Andreas; Kraft-Weyrather, Wilma; Furusawa, Yoshiya; Friedrich, Thomas; Scifoni, Emanuele

    2015-01-01

    Solid tumours often present regions with severe oxygen deprivation (hypoxia), which are resistant to both chemotherapy and radiotherapy. Increased radiosensitivity as a function of the oxygen concentration is well described for X-rays. It has also been demonstrated that radioresistance in anoxia is reduced using high-LET radiation rather than conventional X-rays. However, the dependence of the oxygen enhancement ratio (OER) on radiation quality in the regions of intermediate oxygen concentrations, those normally found in tumours, had never been measured and biophysical models were based on extrapolations. Here we present a complete survival dataset of mammalian cells exposed to different ions in oxygen concentration ranging from normoxia (21%) to anoxia (0%). The data were used to generate a model of the dependence of the OER on oxygen concentration and particle energy. The model was implemented in the ion beam treatment planning system to prescribe uniform cell killing across volumes with heterogeneous radiosensitivity. The adaptive treatment plans have been validated in two different accelerator facilities, using a biological phantom where cells can be irradiated simultaneously at three different oxygen concentrations. We thus realized a hypoxia-adapted treatment plan, which will be used for painting by voxel of hypoxic tumours visualized by functional imaging. PMID:26596243

  2. Natural Compounds Regulate Glycolysis in Hypoxic Tumor Microenvironment

    PubMed Central

    Gao, Jian-Li; Chen, Ying-Ge

    2015-01-01

    In the early twentieth century, Otto Heinrich Warburg described an elevated rate of glycolysis occurring in cancer cells, even in the presence of atmospheric oxygen (the Warburg effect). Recently it became a therapeutically interesting strategy and is considered as an emerging hallmark of cancer. Hypoxia inducible factor-1 (HIF-1) is one of the key transcription factors that play major roles in tumor glycolysis and could directly trigger Warburg effect. Thus, how to inhibit HIF-1-depended Warburg effect to assist the cancer therapy is becoming a hot issue in cancer research. In fact, HIF-1 upregulates the glucose transporters (GLUT) and induces the expression of glycolytic enzymes, such as hexokinase, pyruvate kinase, and lactate dehydrogenase. So small molecules of natural origin used as GLUT, hexokinase, or pyruvate kinase isoform M2 inhibitors could represent a major challenge in the field of cancer treatment. These compounds aim to suppress tumor hypoxia induced glycolysis process to suppress the cell energy metabolism or enhance the susceptibility of tumor cells to radio- and chemotherapy. In this review, we highlight the role of natural compounds in regulating tumor glycolysis, with a main focus on the glycolysis under hypoxic tumor microenvironment. PMID:25685782

  3. Post-hypoxic Myoclonus: Current Concepts, Neurophysiology, and Treatment

    PubMed Central

    Gupta, Harsh V.; Caviness, John N.

    2016-01-01

    Background Myoclonus may occur after hypoxia. In 1963, Lance and Adams described persistent myoclonus with other features after hypoxia. However, myoclonus occurring immediately after hypoxia may demonstrate different syndromic features from classic Lance–Adams syndrome (LAS). The aim of this review is to provide up-to-date information about the spectrum of myoclonus occurring after hypoxia with emphasis on neurophysiological features. Methods A literature search was performed on PubMed database from 1960 to 2015. The following search terms were used: “myoclonus,” “post anoxic myoclonus,” “post hypoxic myoclonus,” and “Lance Adams syndrome.” The articles describing clinical features, neurophysiology, management, and prognosis of post-hypoxic myoclonus cases were included for review. Results Several reports in the literature were separated clinically into “acute post-hypoxic myoclonus,” which occurred within hours of severe hypoxia, and “chronic post-hypoxic myoclonus,” which occurred with some recovery of mental status as the LAS. Acute post-hypoxic myoclonus was generalized in the setting of coma. Chronic post-hypoxic myoclonus presented as multifocal cortical action myoclonus that was significantly disabling. There was overlap of neurophysiological findings for these two syndromes but also different features. Treatment options for these two distinct clinical–neurophysiologic post-hypoxic myoclonus syndromes were approached differently. Discussion The review of clinical and neurophysiological findings suggests that myoclonus after hypoxia manifests in one or a combination of distinct syndromes: acute and/or chronic myoclonus. The mechanism of post-hypoxic myoclonus may arise either from cortical and/or subcortical structures. More research is needed to clarify mechanisms and treatment of post-hypoxic myoclonus. PMID:27708982

  4. Actualities on molecular pathogenesis and repairing processes of cerebral damage in perinatal hypoxic-ischemic encephalopathy.

    PubMed

    Distefano, Giuseppe; Praticò, Andrea D

    2010-09-16

    Hypoxic-ischemic encephalopathy (HIE) is the most important cause of cerebral damage and long-term neurological sequelae in the perinatal period both in term and preterm infant. Hypoxic-ischemic (H-I) injuries develop in two phases: the ischemic phase, dominated by necrotic processes, and the reperfusion phase, dominated by apoptotic processes extending beyond ischemic areas. Due to selective ischemic vulnerability, cerebral damage affects gray matter in term newborns and white matter in preterm newborns with the typical neuropathological aspects of laminar cortical necrosis in the former and periventricular leukomalacia in the latter. This article summarises the principal physiopathological and biochemical processes leading to necrosis and/or apoptosis of neuronal and glial cells and reports recent insights into some endogenous and exogenous cellular and molecular mechanisms aimed at repairing H-I cerebral damage.

  5. Actualities on molecular pathogenesis and repairing processes of cerebral damage in perinatal hypoxic-ischemic encephalopathy

    PubMed Central

    2010-01-01

    Hypoxic-ischemic encephalopathy (HIE) is the most important cause of cerebral damage and long-term neurological sequelae in the perinatal period both in term and preterm infant. Hypoxic-ischemic (H-I) injuries develop in two phases: the ischemic phase, dominated by necrotic processes, and the reperfusion phase, dominated by apoptotic processes extending beyond ischemic areas. Due to selective ischemic vulnerability, cerebral damage affects gray matter in term newborns and white matter in preterm newborns with the typical neuropathological aspects of laminar cortical necrosis in the former and periventricular leukomalacia in the latter. This article summarises the principal physiopathological and biochemical processes leading to necrosis and/or apoptosis of neuronal and glial cells and reports recent insights into some endogenous and exogenous cellular and molecular mechanisms aimed at repairing H-I cerebral damage. PMID:20846380

  6. Hypoxic Stress Facilitates Acute Activation and Chronic Down-Regulation of Fanconi Anemia Proteins

    PubMed Central

    Scanlon, Susan E.; Glazer, Peter M.

    2014-01-01

    Hypoxia induces genomic instability through replication stress and dysregulation of vital DNA repair pathways. The Fanconi anemia (FA) proteins, FANCD2 and FANCI, are key members of a DNA repair pathway that responds to replicative stress, suggesting that they undergo regulation by hypoxic conditions. Here acute hypoxic stress activates the FA pathway via ubiquitination of FANCD2 and FANCI in an ATR-dependent manner. In addition, the presence of an intact FA pathway is required for preventing hypoxia-induced DNA damage measurable by the comet assay, limiting the accumulation of γH2AX (a marker of DNA damage or stalled replication), and protecting cells from hypoxia-induced apoptosis. Furthermore, prolonged hypoxia induces transcriptional repression of FANCD2 in a manner analogous to the hypoxic down-regulation of BRCA1 and RAD51. Thus, hypoxia-induced FA pathway activation plays a key role in maintaining genome integrity and cell survival, while FA protein down-regulation with prolonged hypoxia contributes to genomic instability. PMID:24688021

  7. Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition

    PubMed Central

    Xu, Xuehu; An, Xiuli; Wang, Shu

    2016-01-01

    The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. PMID:27965712

  8. Overexpression of mitochondrial uncoupling protein 1 (UCP1) induces a hypoxic response in Nicotiana tabacum leaves

    PubMed Central

    Barreto, Pedro; Okura, Vagner; Pena, Izabella A.; Maia, Renato; Maia, Ivan G.; Arruda, Paulo

    2016-01-01

    Mitochondrial uncoupling protein 1 (UCP1) decreases reactive oxygen species production under stress conditions by uncoupling the electrochemical gradient from ATP synthesis. This study combined transcriptome profiling with experimentally induced hypoxia to mechanistically dissect the impact of Arabidopsis thaliana UCP1 (AtUCP1) overexpression in tobacco. Transcriptomic analysis of AtUCP1-overexpressing (P07) and wild-type (WT) plants was carried out using RNA sequencing. Metabolite and carbohydrate profiling of hypoxia-treated plants was performed using 1H-nuclear magnetic resonance spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection. The transcriptome of P07 plants revealed a broad induction of stress-responsive genes that were not strictly related to the mitochondrial antioxidant machinery, suggesting that overexpression of AtUCP1 imposes a strong stress response within the cell. In addition, transcripts that mapped into carbon fixation and energy expenditure pathways were broadly altered. It was found that metabolite markers of hypoxic adaptation, such as alanine and tricarboxylic acid intermediates, accumulated in P07 plants under control conditions at similar rates to WT plants under hypoxia. These findings indicate that constitutive overexpression of AtUCP1 induces a hypoxic response. The metabolites that accumulated in P07 plants are believed to be important in signalling for an improvement in carbon assimilation and induction of a hypoxic response. Under these conditions, mitochondrial ATP production is less necessary and fermentative glycolysis becomes critical to meet cell energy demands. In this scenario, the more flexible energy metabolism along with an intrinsically activated hypoxic response make these plants better adapted to face several biotic and abiotic stresses. PMID:26494730

  9. Joint immobilization induced hypoxic and inflammatory conditions in rat knee joints.

    PubMed

    Yabe, Yutaka; Hagiwara, Yoshihiro; Suda, Hideaki; Ando, Akira; Onoda, Yoshito; Tsuchiya, Masahiro; Hatori, Kouki; Itoi, Eiji

    2013-01-01

    The purpose of this study was to examine the hypoxic and inflammatory conditions after immobilization in the joint capsule of rat knees. The unilateral knee joints of adult male rats were immobilized with an internal fixator (Im group) for 1 day, 3 days, and 1, 2, 4, 8, and 16 weeks. Sham-operated animals had holes drilled in the femur and tibia and screws inserted without a plate (control group). The number of cells and blood vessels in the capsule were histologically examined. The hypoxic condition in the capsule was histologically examined with a Hypoxyprobe™-1. The gene expressions related to the hypoxic (hypoxia inducible factor-1α, vascular endothelial growth factor, and fibroblast growth factor 2) and inflammatory conditions [interleukin-6 (IL-6), IL-1α, IL-1β, tumor necrosis factor-α, and tumor necrosis factor-β] were evaluated by quantitative reverse transcription polymerase chain reaction. The number of cells was unchanged at 1 day in the two groups; however, the number significantly increased at 3 days in the Im group. The number of blood vessels in the Im group gradually decreased. Strong immunostaining of Hypoxyprobe™-1 around the blood vessels was observed in the Im group. The gene expressions of hypoxia inducible factor-1α and fibroblast growth factor 2 were significantly higher in the Im group compared with those in the control group. The gene expressions of IL-6, IL-1α, IL-1β, and tumor necrosis factor-β were significantly higher in the Im group compared with those in the control group. These data indicated that joint immobilization induced hypoxic and inflammatory conditions in the joint capsule, which might be an initiating factor for joint contracture.

  10. [Follow-up of newborns with hypoxic-ischaemic encephalopathy].

    PubMed

    Martínez-Biarge, M; Blanco, D; García-Alix, A; Salas, S

    2014-07-01

    Hypothermia treatment for newborn infants with hypoxic-ischemic encephalopathy reduces the number of neonates who die or have permanent neurological deficits. Although this therapy is now standard of care, neonatal hypoxic-ischaemic encephalopathy still has a significant impact on the child's neurodevelopment and quality of life. Infants with hypoxic-ischaemic encephalopathy should be enrolled in multidisciplinary follow-up programs in order to detect impairments, to initiate early intervention, and to provide counselling and support for families. This article describes the main neurodevelopmental outcomes after term neonatal hypoxic-ischaemic encephalopathy. We offer recommendations for follow-up based on the infant's clinical condition and other prognostic indicators, mainly neonatal neuroimaging. Other aspects, such as palliative care and medico-legal issues, are also briefly discussed.

  11. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

    PubMed Central

    Cox, Thomas R.; Schoof, Erwin M.; Gartland, Alison; Erler, Janine T.; Linding, Rune

    2015-01-01

    The cancer secretome includes all of the macromolecules secreted by cells into their microenvironment. Cancer cell secretomes are significantly different to that of normal cells reflecting the changes that normal cells have undergone during their transition to malignancy. More importantly, cancer secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397 and the complete proteomic, bioinformatic and biological analyses are reported in Cox et al. (2015) [1]. PMID:26649326

  12. Thalamic mediation of hypoxic respiratory depression in lambs.

    PubMed

    Koos, Brian J; Rajaee, Arezoo; Ibe, Basil; Guerra, Catalina; Kruger, Lawrence

    2016-04-01

    Immaturity of respiratory controllers in preterm infants dispose to recurrent apnea and oxygen deprivation. Accompanying reductions in brain oxygen tensions evoke respiratory depression, potentially exacerbating hypoxemia. Central respiratory depression during moderate hypoxia is revealed in the ventilatory decline following initial augmentation. This study determined whether the thalamic parafascicular nuclear (Pf) complex involved in adult nociception and sensorimotor regulation (Bentivoglio M, Balerecia G, Kruger L. Prog Brain Res 87: 53-80, 1991) also becomes a postnatal controller of hypoxic ventilatory decline. Respiratory responses to moderate isocapnic hypoxia were studied in conscious lambs. Hypoxic ventilatory decline was compared with peak augmentation. Pf and/or adjacent thalamic structures were destroyed by the neuron-specific toxin ibotenic acid (IB). IB lesions involving the thalamic Pf abolished hypoxic ventilatory decline. Lesions of adjacent thalamic nuclei that spared Pf and control injections of vehicle failed to blunt hypoxic respiratory depression. Our findings reveal that the thalamic Pf region is a critical controller of hypoxic ventilatory depression and thus a key target for exploring molecular concomitants of forebrain pathways regulating hypoxic ventilatory depression in early development.

  13. NDRG1 deficiency attenuates fetal growth and the intrauterine response to hypoxic injury.

    PubMed

    Larkin, Jacob; Chen, Baosheng; Shi, Xiao-Hua; Mishima, Takuya; Kokame, Koichi; Barak, Yaacov; Sadovsky, Yoel

    2014-03-01

    Intrauterine mammalian development depends on the preservation of placental function. The expression of the protein N-myc downstream-regulated gene 1 (NDRG1) is increased in placentas of human pregnancies affected by fetal growth restriction and in hypoxic primary human trophoblasts, where NDRG1 attenuates cell injury. We sought to assess the function of placental NDRG1 in vivo and tested the hypothesis that NDRG1 deficiency in the mouse embryo impairs placental function and consequently intrauterine growth. We found that Ndrg1 knock-out embryos were growth restricted in comparison to wild-type or heterozygous counterparts. Furthermore, hypoxia reduced the survival of female, but not male, knock-out embryos. Ndrg1 deletion caused significant alterations in placental gene expression, with a marked reduction in transcription of several lipoproteins in the placental labyrinth. These transcriptional changes were associated with reduced fetal:maternal serum cholesterol ratio exclusively in hypoxic female embryos. Collectively, our findings indicate that NDRG1 promotes fetal growth and regulates the metabolic response to intrauterine hypoxic injury in a sexually dichotomous manner.

  14. Reactive Nitrogen Species in Mitochondria and Their Implications in Plant Energy Status and Hypoxic Stress Tolerance

    PubMed Central

    Gupta, Kapuganti Jagadis; Igamberdiev, Abir U.

    2016-01-01

    Hypoxic and anoxic conditions result in the energy crisis that leads to cell damage. Since mitochondria are the primary organelles for energy production, the support of these organelles in a functional state is an important task during oxygen deprivation. Plant mitochondria adapted the strategy to survive under hypoxia by keeping electron transport operative even without oxygen via the use of nitrite as a terminal electrons acceptor. The process of nitrite reduction to nitric oxide (NO) in the mitochondrial electron transport chain recycles NADH and leads to a limited rate of ATP production. The produced ATP alongside with the ATP generated by fermentation supports the processes of transcription and translation required for hypoxic survival and recovery of plants. Non-symbiotic hemoglobins (called phytoglobins in plants) scavenge NO and thus contribute to regeneration of NAD+ and nitrate required for the operation of anaerobic energy metabolism. This overall operation represents an important strategy of biochemical adaptation that results in the improvement of energy status and thereby in protection of plants in the conditions of hypoxic stress. PMID:27047533

  15. Enhancement of hypoxic liver damage by ethanol. Involvement of xanthine oxidase and the role of glycolysis.

    PubMed

    Younes, M; Strubelt, O

    1987-09-15

    Using isolated hemoglobin-free perfused rat livers we investigated the hepatotoxic effects of hypoxia, ethanol or the combination of both. Hypoxia only (90 min) led to a weak toxicity as evidenced by the efflux of the enzymes glutamate-pyruvate-transaminase (GPT) and sorbitol dehydrogenase (SDH). This toxic effect was slightly higher in livers treated with ethanol (3 g/l) under normoxic conditions. Ethanol added under hypoxic conditions, however, showed a strong hepatotoxic effect. Under hypoxic conditions, lactate + pyruvate production was increased fivefold over control, indicating that glycolysis was more effectively undergone as main source of energy. Addition of ethanol suppressed this effect, indicating that ethanol inhibited glycolysis. These results indicate that ethanol potentiates hypoxic liver damage by inhibiting the main metabolic pathway yielding ATP under low oxygen tension resulting in a severe energy deficit. Allopurinol (100 mg/l) inhibited the toxic effects seen with ethanol + hypoxia. Also, the inhibitory action of ethanol on glycolysis was antagonized. Our results are consistent with the following model: hypoxia converts NAD-dependent xanthine dehydrogenase (XD) into the oxygen-dependent xanthine oxidase (XO). Due to hypoxia and ethanol, purine metabolites and acetaldehyde accumulate and are metabolized via XO. This process leads to the production of oxygen radicals which most probably mediate both the inhibition of glycolysis and the direct toxic effects towards liver cells.

  16. Hypoxic pulmonary vasoconstriction, carotid body function and erythropoietin production in adult rats perinatally exposed to hyperoxia

    PubMed Central

    Prieto-Lloret, Jesus; Ramirez, Maria; Olea, Elena; Moral-Sanz, Javier; Cogolludo, Angel; Castañeda, Javier; Yubero, Sara; Agapito, Teresa; Gomez-Niño, Angela; Rocher, Asuncion; Rigual, Ricardo; Obeso, Ana; Perez-Vizcaino, Francisco; González, Constancio

    2015-01-01

    Adult mammalians possess three cell systems that are activated by acute bodily hypoxia: pulmonary artery smooth muscle cells (PASMC), carotid body chemoreceptor cells (CBCC) and erythropoietin (EPO)-producing cells. In rats, chronic perinatal hyperoxia causes permanent carotid body (CB) atrophy and functional alterations of surviving CBCC. There are no studies on PASMC or EPO-producing cells. Our aim is to define possible long-lasting functional changes in PASMC or EPO-producing cells (measured as EPO plasma levels) and, further, to analyse CBCC functional alterations. We used 3- to 4-month-old rats born and reared in a normal atmosphere or exposed to perinatal hyperoxia (55–60% O2 for the last 5–6 days of pregnancy and 4 weeks after birth). Perinatal hyperoxia causes an almost complete loss of hypoxic pulmonary vasoconstriction (HPV), which was correlated with lung oxidative status in early postnatal life and prevented by antioxidant supplementation in the diet. O2-sensitivity of K+ currents in the PASMC of hyperoxic animals is normal, indicating that their inhibition is not sufficient to trigger HPV. Perinatal hyperoxia also abrogated responses elicited by hypoxia on catecholamine and cAMP metabolism in the CB. An increase in EPO plasma levels elicited by hypoxia was identical in hyperoxic and control animals, implying a normal functioning of EPO-producing cells. The loss of HPV observed in adult rats and caused by perinatal hyperoxia, comparable to oxygen therapy in premature infants, might represent a previously unrecognized complication of such a medical intervention capable of aggravating medical conditions such as regional pneumonias, atelectases or general anaesthesia in adult life. Key points Adult animals that have been perinatally exposed to oxygen-rich atmospheres (hyperoxia), recalling those used for oxygen therapy in infants, exhibit a loss of hypoxic pulmonary vasoconstriction, whereas vasoconstriction elicited by depolarizing agents is

  17. Pulmonary hypoxic vasoconstriction: how strong? How fast?

    NASA Technical Reports Server (NTRS)

    Sheehan, D. W.; Klocke, R. A.; Farhi, L. E.

    1992-01-01

    We have developed a minimally invasive technique for studying regional blood flow in conscious sheep, bypassing the complications of open-chest surgery, flow probes and tracer infusion. We quantitate regional perfusion continuously on the basis of regional clearance of methane (methane is produced in the sheep rumen, enters the circulation and is eliminated nearly completely (greater than 95%) in the lung). Tracheal intubation with a dual-lumen catheter isolates the gas exchange of the right apical lobe (RAL; less than 15% of the lung) from that of the remainder of the lung, which serves as a control (CL). We measure RAL and CL methane elimination by entraining expirates in constant flows, sampled continuously for methane. Results obtained with this technique and from regional oxygen uptake are in excellent agreement. We have found that hypoxic vasoconstriction is far more potent and stable during eucapnic hypoxia than during hypocapnic hypoxia. The time course of the vasoconstriction suggests that many of the data in the literature may have been obtained prior to steady state.

  18. Hypoxic Hepatitis: A Review and Clinical Update

    PubMed Central

    Waseem, Najeff; Chen, Po-Hung

    2016-01-01

    Abstract Hypoxic hepatitis (HH), also known as ischemic hepatitis or shock liver, is characterized by a massive, rapid rise in serum aminotransferases resulting from reduced oxygen delivery to the liver. The most common predisposing condition is cardiac failure, followed by circulatory failure as occurs in septic shock and respiratory failure. HH does, however, occur in the absence of a documented hypotensive event or shock state in 50% of patients. In intensive care units, the incidence of HH is near 2.5%, but has been reported as high as 10% in some studies. The pathophysiology is multifactorial, but often involves hepatic congestion from right heart failure along with reduced hepatic blood flow, total body hypoxemia, reduced oxygen uptake by hepatocytes or reperfusion injury following ischemia. The diagnosis is primarily clinical, and typically does not require liver biopsy. The definitive treatment of HH involves correction of the underlying disease state, but successful management includes monitoring for the potential complications such as hypoglycemia, hyperglycemia, hyperammonemia and hepatopulmonary syndrome. Prognosis of HH remains poor, especially for cases in which there was a delay in diagnosis. The in-hospital mortality rate is >50%, and the most frequent cause of death is the predisposing condition and not the liver injury itself. PMID:27777895

  19. Mass Law Predicts Hyperbolic Hypoxic Ventilatory Response

    NASA Astrophysics Data System (ADS)

    Severinghaus, John W.

    The hyperbolic hypoxic ventilatory response vs PaO2, HVRp, is interpreted as relecting a mass hyperbolic relationship of cytochrome PcO2 to cytochrome potential Ec, offset 32 torr by the constant diffusion gradient between arterial blood and cytochrome in CB at its constant metabolic rate dot VO_2 . Ec is taken to be a linear function of redox reduction and CB ventilatory drive. As Ec rises in hypoxia, the absolute potentials of each step in the citric acid cycle rises equally while the potential drop across each step remains constant because flux rate remains constant. A hypothetic HVRs ( dot VE vs SaO2) response curve computed from these assumptions is strikingly non linear. A hypothetic HVRp calculated from an assumed linear HVRs cannot be fit to the observed hyperbolic increase of ventilation in response to isocapnic hypoxia at PO2 less than 40 torr. The incompatibility of these results suggest that in future studies HVRs will not be found to be linear, especially below 80% SaO2 and HVRp will fail to be accurately hyperbolic.

  20. Bifurcate effects of glucose on caspase-independent cell death during hypoxia

    SciTech Connect

    Aki, Toshihiko; Nara, Akina; Funakoshi, Takeshi; Uemura, Koichi

    2010-06-04

    We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

  1. Advancing hypoxic training in team sports: from intermittent hypoxic training to repeated sprint training in hypoxia.

    PubMed

    Faiss, Raphaël; Girard, Olivier; Millet, Grégoire P

    2013-12-01

    Over the past two decades, intermittent hypoxic training (IHT), that is, a method where athletes live at or near sea level but train under hypoxic conditions, has gained unprecedented popularity. By adding the stress of hypoxia during 'aerobic' or 'anaerobic' interval training, it is believed that IHT would potentiate greater performance improvements compared to similar training at sea level. A thorough analysis of studies including IHT, however, leads to strikingly poor benefits for sea-level performance improvement, compared to the same training method performed in normoxia. Despite the positive molecular adaptations observed after various IHT modalities, the characteristics of optimal training stimulus in hypoxia are still unclear and their functional translation in terms of whole-body performance enhancement is minimal. To overcome some of the inherent limitations of IHT (lower training stimulus due to hypoxia), recent studies have successfully investigated a new training method based on the repetition of short (<30 s) 'all-out' sprints with incomplete recoveries in hypoxia, the so-called repeated sprint training in hypoxia (RSH). The aims of the present review are therefore threefold: first, to summarise the main mechanisms for interval training and repeated sprint training in normoxia. Second, to critically analyse the results of the studies involving high-intensity exercises performed in hypoxia for sea-level performance enhancement by differentiating IHT and RSH. Third, to discuss the potential mechanisms underpinning the effectiveness of those methods, and their inherent limitations, along with the new research avenues surrounding this topic.

  2. Elevation of Circulating miR-210-3p in High-Altitude Hypoxic Environment

    PubMed Central

    Yan, Yan; Wang, Cheng; Zhou, Wanqing; Shi, Yonghui; Guo, Pengtao; Liu, Yuxiu; Wang, Junjun; Zhang, Chen-Yu; Zhang, Chunni

    2016-01-01

    Background: The induction of miR-210-3p, a master hypoxamir, is a consistent feature of the hypoxic response in both normal and malignant cells. However, whether miR-210-3p acts as a circulating factor in response to a hypoxic environment remains unknown. The current study aimed to examine the effect of a high-altitude hypoxic environment on circulating miR-210-3p. Methods: We examined and compared the levels of miR-210-3p using TaqMan-based qRT-PCR in both peripheral blood cells and plasma from 84 ethnic Chinese Tibetans residing at 3560 m, 46 newly arrived migrant Han Chinese (Tibet Han) and 82 Han Chinese residing at 8.9 m (Nanjing Han). Furthermore, we analyzed the correlations of miR-210-3p with hematological indices. Results: The relative concentrations of miR-210-3p to internal reference U6 in blood cells were significantly higher in the Tibet Han group (1.01 ± 0.11, P < 0.001) and in the Tibetan group (1.17 ± 0.09, P < 0.001) than in the Nanjing Han group (0.51 ± 0.04). The absolute concentrations of plasma miR-210-3p were also markedly elevated in the Tibet Han group (503.54 ± 42.95 fmol/L, P = 0.004) and in the Tibetan group (557.78 ± 39.84 fmol/L, P < 0.001) compared to the Nanjing Han group (358.39 ± 16.16 fmol/L). However, in both blood cells and plasma, miR-210-3p levels were not significantly different between the Tibet Han group and the Tibetan group (P = 0.280, P = 0.620, respectively). Plasma miR-210-3p concentrations were positively correlated with miR-210-3p levels in blood cells (r = 0.192, P = 0.005). Furthermore, miR-210-3p levels in both blood cells and plasma showed strong positive correlations with red blood cell counts and hemoglobin and hematocrit values. Conclusion: These data demonstrated, for the first time, that miR-210-3p might act as a circulating factor in response to hypoxic environments and could be associated with human adaptation to life at high altitudes. PMID:27014085

  3. SILAC-based proteomic analysis reveals that salidroside antagonizes cobalt chloride-induced hypoxic effects by restoring the tricarboxylic acid cycle in cardiomyocytes.

    PubMed

    Xu, Zhong-Wei; Chen, Xi; Jin, Xiao-Han; Meng, Xiang-Yan; Zhou, Xin; Fan, Feng-Xu; Mao, Shi-Yun; Wang, Yue; Zhang, Wen-Cheng; Shan, Na-Na; Li, Yu-Ming; Xu, Rui-Cheng

    2016-01-01

    Hypoxic status alters the energy metabolism and induces cell injury in cardiomyocytes, and it further triggers the occurrence and development of cardiovascular diseases. Our previous studies have shown that salidroside (SAL) exhibits anti-hypoxic activity. However, the mechanisms remain obscure. In the present study, we successfully screened 92 different expression proteins in CoCl2-induced hypoxic conditions, 106 different expression proteins in the SAL-mediated anti-hypoxic group were compared with the hypoxic group using quantitative proteomics strategy, respectively. We confirmed that SAL showed a positive protective function involving the acetyl-CoA metabolic, tricarboxylic acid (TCA) cycle using bioinformatics analysis. We also demonstrated that SAL plays a critical role in restoring the TCA cycle and in protecting cardiomyocytes from oxidative injury via up-regulation expressions of PDHE1-B, ACO2, SUCLG1, SUCLG2 and down-regulation of MDH2. SAL also inhibited H9c2 cell apoptosis by inhibiting the activation of pro-apoptotic molecules caspase 3 and caspase 9 as well as activation of the anti-apoptotic molecular Bcl-2. Additionally, SAL also improved mitochondrial membrane potential (ΔΨm), reduced reactive oxygen species (ROS) and intercellular Ca(2+) concentration ([Ca(2+)]i) accumulation and inhibited the excessive consumption of ATP in H9c2 cells.

  4. The different mechanisms of hypoxic acclimatization and adaptation in Lizard Phrynocephalus vlangalii living on Qinghai-Tibet Plateau.

    PubMed

    He, Jianzheng; Xiu, Minghui; Tang, Xiaolong; Yue, Feng; Wang, Ningbo; Yang, Shaobin; Chen, Qiang

    2013-03-01

    Phrynocephalus vlangalii is a species of lizard endemic in China, which lives on Qinghai-Tibet Plateau ranging from 2000 to 4600 m above sea level. In this study, P. vlangalii were collected from low altitude (2750 m) and high altitude (4564 m). The lizards from low altitude were acclimatized in simulated hypoxic chamber (equivalent to 4600 m) for 7, 15, and 30 days. The hematological parameters, heart weight, myocardial capillary density, and myocardial enzyme activities were examined. The results showed that acclimatization to hypoxia significantly increased hemoglobin concentration ([Hb]), hematocrit (Hct), heart weight (HW), heart weight to body mass (HW/BM), lactate dehydrogenase (LDH) activity, but markedly decreased mean corpuscular hemoglobin concentration (MCHC) and succinate dehydrogenase (SDH) activity. Red blood cell (RBC) count, body mass (BM), myocardial capillary density did not change markedly during hypoxic acclimatization. On the other hand, [Hb], Hct, MCHC, HW/BM, myocardium capillary density, and SDH activity of P. vlangalii from high altitude were remarkably higher than those from low-altitude; however, LDH activity of high-altitude P. vlangalii was lower than that of low-altitude lizards. There was no significant difference in HW or BM between populations of high-altitude and low-altitude. Based on the present data, we suggest that P. vlangalii has special anatomical, physiological, and biochemical accommodate mechanisms to live in hypoxic environment, and the regulative mechanisms are different between hypoxic acclimatization and adaptation.

  5. In vitro Ischemia Suppresses Hypoxic Induction of Hypoxia Inducible Factor-1α by Inhibition of Synthesis and Not Enhanced Degradation

    PubMed Central

    Karuppagounder, Saravanan S.; Basso, Manuela; Sleiman, Sama F.; Ma, Thong C.; Speer, Rachel E.; Smirnova, Natalya A.; Gazaryan, Irina G.; Ratan, Rajiv R.

    2015-01-01

    Hypoxia Inducible Factor (HIF) mediates a broad, conserved adaptive response to hypoxia, and the HIF pathway is a potential therapeutic target in cerebral ischemia. In this study, we investigated the mechanism by which in vitro ischemia (oxygen-glucose deprivation, OGD) affects canonical hypoxic HIF-1α stabilization. We validated the use of a reporter containing the oxygen dependent degradation domain of HIF-1α fused to firefly luciferase (ODD-luc) to quantitatively monitor distinct biochemical events leading to hypoxic HIF-1α expression or stabilization in a human neuroblastoma cell line (SH-SY5Y). When OGD was imposed following a 2 hour hypoxic stabilization of ODD-luc, the levels of the reporter were reduced, consistent with prior models proposing that OGD enhances HIF prolyl hydroxylase (PHD) activity. Surprisingly, PHD inhibitors and proteasome inhibitors do not stabilize ODD-luc in OGD. Further, OGD does not affect the half-life of ODD-luc protein following hypoxia, suggesting that OGD abrogates hypoxic HIF-1α induction by reducing HIF-1α synthesis rather than by enhancing its degradation. We observed ATP depletion under OGD versus hypoxia, and propose that ATP depletion enhances translational suppression, overcoming the selective synthesis of HIF concurrent with global decreases in protein synthesis in hypoxia. Taken together, these findings biochemically characterize a practical reporter for monitoring HIF-1α levels and support a novel model for HIF regulation in an in vitro model of human ischemia. PMID:23456821

  6. [Mechanism of protective effects of tumor necrosis factor receptor associated protein 1 on hypoxic cardiomyocytes of rats].

    PubMed

    Xiang, F; Zhang, D X; Ma, S Y; Huang, Y S

    2016-12-20

    Objective: To investigate the mechanism of protective effects of tumor necrosis factor receptor associated protein 1 (TRAP1) on hypoxic cardiomyocytes of rats. Methods: Primary cultured cardiomyocytes were obtained from neonatal Sprague-Dawley rats (aged 1 to 3 days) and then used in the following experiments. (1) Cells were divided into group TRAP1 and control group according to the random number table (the same grouping method below), and then the total protein of cells was extracted. Total protein of cells in group TRAP1 was added with mouse anti-rat TRAP1 monoclonal antibody, while that in control group was added with the same type of IgG from mouse. Co-immunoprecipitation and protein mass spectrography analysis were used to determine the possible proteins interacted with TRAP1. (2) Cells were divided into normoxia blank control group (NBC), normoxia+ TRAP1 interference control group (NTIC), normoxia+ TRAP1 interference group (NTI), normoxia+ TRAP1 over-expression control group (NTOC), and normoxia+ TRAP1 over-expression group (NTO), with 1 well in each group. Cells in group NBC were routinely cultured, while cells in the latter four groups were respectively added with TRAP1 RNA interference empty virus vector, TRAP1 RNA interference adenovirus vector, TRAP1 over-expression empty virus vector, and TRAP1 over-expression adenovirus vector. Another batch of cells were divided into group NBC, hypoxic blank control group (HBC), hypoxic+ TRAP1 interference control group (HTIC), hypoxic+ TRAP1 interference group (HTI), hypoxic+ TRAP1 over-expression control group (HTOC), and hypoxic+ TRAP1 over-expression group (HTO), with 1 well in each group. Cells in hypoxic groups were under hypoxic condition for 6 hours after being treated as those in the corresponding normoxia groups, respectively. The mRNA expression of cytochrome c oxidase subunit Ⅱ (COXⅡ) of cells in each group was detected by real time fluorescent quantitive reverse transcription polymerase chain

  7. Psychological strain: examining the effect of hypoxic bedrest and confinement.

    PubMed

    Stavrou, Nektarios A M; McDonnell, Adam C; Eiken, Ola; Mekjavic, Igor B

    2015-02-01

    The aim was to assess the effect of a 10-day exposure to the environmental stressors anticipated in future lunar habitats on indices of psychological strain. In addition to the reduced gravity of the Moon, future habitats on the Moon will likely maintain a hypobaric hypoxic environment. The hypobaric environment will eliminate the need for long decompression profiles prior to each extra-vehicular activity. We investigated the indices of psychological strain during three 10-day conditions, designed to assess the separate and combined effects of inactivity/unloading and normobaric hypoxia on several physiological systems. Eleven male participants underwent three 10-day campaigns in a randomised manner: 1) normobaric normoxic bed rest (NBR), 2) normobaric hypoxic bed rest (HBR) and 3) normobaric hypoxic ambulatory confinement (HAMB). The most negative psychological profile appeared on day 10 of the HBR and HAMB (hypoxic) conditions. Concomitantly, a decrease in positive emotions was observed from baseline to day 10 of the HBR and NBR conditions. Thus, confinement in a hypoxic environment seems to exert a negative effect on an individual's psychological mood.

  8. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    PubMed

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response.

  9. Hypoxic Pulmonary Vasoconstriction: From Molecular Mechanisms to Medicine.

    PubMed

    Dunham-Snary, Kimberly J; Wu, Danchen; Sykes, Edward A; Thakrar, Amar; Parlow, Leah R G; Mewburn, Jeffrey D; Parlow, Joel L; Archer, Stephen L

    2017-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism that is intrinsic to the pulmonary vasculature. Intrapulmonary arteries constrict in response to alveolar hypoxia, diverting blood to better-oxygenated lung segments, thereby optimizing ventilation/perfusion matching and systemic oxygen delivery. In response to alveolar hypoxia, a mitochondrial sensor dynamically changes reactive oxygen species and redox couples in pulmonary artery smooth muscle cells (PASMC). This inhibits potassium channels, depolarizes PASMC, activates voltage-gated calcium channels, and increases cytosolic calcium, causing vasoconstriction. Sustained hypoxia activates rho kinase, reinforcing vasoconstriction, and hypoxia-inducible factor (HIF)-1α, leading to adverse pulmonary vascular remodeling and pulmonary hypertension (PH). In the nonventilated fetal lung, HPV diverts blood to the systemic vasculature. After birth, HPV commonly occurs as a localized homeostatic response to focal pneumonia or atelectasis, which optimizes systemic Po2 without altering pulmonary artery pressure (PAP). In single-lung anesthesia, HPV reduces blood flow to the nonventilated lung, thereby facilitating thoracic surgery. At altitude, global hypoxia causes diffuse HPV, increases PAP, and initiates PH. Exaggerated or heterogeneous HPV contributes to high-altitude pulmonary edema. Conversely, impaired HPV, whether due to disease (eg, COPD, sepsis) or vasodilator drugs, promotes systemic hypoxemia. Genetic and epigenetic abnormalities of this oxygen-sensing pathway can trigger normoxic activation of HIF-1α and can promote abnormal metabolism and cell proliferation. The resulting pseudohypoxic state underlies the Warburg metabolic shift and contributes to the neoplasia-like phenotype of PH. HPV and oxygen sensing are important in human health and disease.

  10. Melatonin Suppresses Toll Like Receptor 4-Dependent Caspase-3 Signaling Activation Coupled with Reduced Production of Proinflammatory Mediators in Hypoxic Microglia

    PubMed Central

    Yao, Linli; Lu, Pengfei; Ling, Eng-Ang

    2016-01-01

    Microglia activation and associated inflammatory response play pivotal roles in the pathogenesis of different neurodegenerative diseases including neonatal hypoxic brain injury. Here we show that caspase3 expression was upregulated in activated microglia after hypoxic exposure, and remarkably, the cell viability remained unaffected alluding to the possibility of a non-apoptotic role of caspase3 in activated microglia. Chemical inhibition of caspase3 suppressed microglia activation as evident by an obvious reduction in expression of proinflammatory mediators and NF-κB signaling activation. Hypoxia induced caspase3 activation was TLR4 dependent as supported by the fact that caspase3 activation was hindered in cells with TLR4 knockdown. Interestingly, melatonin treatment significantly suppressed caspase3 activation. More importantly, melatonin also inhibited the increase in TLR4 protein and mRNA expression in hypoxic microglia. Inhibition of TLR4 expression by melatonin was also found in microglia of postnatal rats subjected to hypoxic exposure. Taken together, it is concluded that melatonin could inhibit TLR4 expression in hypoxic microglia followed by suppression of caspase3 activation leading to decrease in production of proinflammatory mediators. PMID:27812200

  11. Small Molecule Inhibition of microRNA-210 Reprograms an Oncogenic Hypoxic Circuit.

    PubMed

    Costales, Matthew G; Haga, Christopher L; Velagapudi, Sai Pradeep; Childs-Disney, Jessica L; Phinney, Donald G; Disney, Matthew D

    2017-03-08

    A hypoxic state is critical to the metastatic and invasive characteristics of cancer. Numerous pathways play critical roles in cancer maintenance, many of which include noncoding RNAs such as microRNA (miR)-210 that regulates hypoxia inducible factors (HIFs). Herein, we describe the identification of a small molecule named Targapremir-210 that binds to the Dicer site of the miR-210 hairpin precursor. This interaction inhibits production of the mature miRNA, derepresses glycerol-3-phosphate dehydrogenase 1-like enzyme (GPD1L), a hypoxia-associated protein negatively regulated by miR-210, decreases HIF-1α, and triggers apoptosis of triple negative breast cancer cells only under hypoxic conditions. Further, Targapremir-210 inhibits tumorigenesis in a mouse xenograft model of hypoxic triple negative breast cancer. Many factors govern molecular recognition of biological targets by small molecules. For protein, chemoproteomics and activity-based protein profiling are invaluable tools to study small molecule target engagement and selectivity in cells. Such approaches are lacking for RNA, leaving a void in the understanding of its druggability. We applied Chemical Cross-Linking and Isolation by Pull Down (Chem-CLIP) to study the cellular selectivity and the on- and off-targets of Targapremir-210. Targapremir-210 selectively recognizes the miR-210 precursor and can differentially recognize RNAs in cells that have the same target motif but have different expression levels, revealing this important feature for selectively drugging RNAs for the first time. These studies show that small molecules can be rapidly designed to selectively target RNAs and affect cellular responses to environmental conditions, resulting in favorable benefits against cancer. Further, they help define rules for identifying druggable targets in the transcriptome.

  12. Enhanced non-eupneic breathing following hypoxic, hypercapnic or hypoxic-hypercapnic gas challenges in conscious mice.

    PubMed

    Getsy, Paulina M; Davis, Jesse; Coffee, Gregory A; May, Walter J; Palmer, Lisa A; Strohl, Kingman P; Lewis, Stephen J

    2014-12-01

    C57BL6 mice display non-eupneic breathing and spontaneous apneas during wakefulness and sleep as well as markedly disordered breathing following cessation of a hypoxic challenge. We examined whether (1) C57BL6 mice display marked non-eupneic breathing following hypercapnic or hypoxic-hypercapnic challenges, and (2) compared the post-hypoxia changes in non-eupneic breathing of C57BL6 mice to those of B6AF1 (57BL6 dam × A/J sire) and Swiss-Webster mice, which display different ventilatory responses than C57BL6 mice. C57BL6 mice displayed marked increases in respiratory frequency and non-eupneic breathing upon return to room-air after hypoxic (10% O2, 90% N2), hypercapnic (5% CO2, 21% O2 and 74% N2) and hypoxic-hypercapnic (10% O2, 5% CO2 and 85% N2) challenges. B6AF1 mice displayed less tachypnea and reduced non-eupneic breathing post-hypoxia, whereas Swiss-Webster mice displayed robust tachypnea with minimal increases in non-eupneic breathing post-hypoxia. These studies demonstrate that non-eupneic breathing increases after physiologically-relevant hypoxic-hypercapnic challenge in C57BL6 mice and suggest that further studies with these and B6AF1 and Swiss-Webster mice will help define the genetics of non-eupneic breathing.

  13. Enhanced non-eupneic breathing following hypoxic, hypercapnic or hypoxic-hypercapnic gas challenges in conscious mice

    PubMed Central

    Getsy, Paulina M.; Davis, Jesse; Coffee, Gregory A.; May, Walter J.; Palmer, Lisa A.; Strohl, Kingman P.; Lewis, Stephen J.

    2014-01-01

    C57BL6 mice display non-eupneic breathing and spontaneous apneas during wakefulness and sleep as well as markedly disordered breathing following cessation of a hypoxic challenge. We examined whether (1) C57BL6 mice display marked non-eupneic breathing following hypercapnic or hypoxic-hypercapnic challenges, and (2) compared the post-hypoxia changes in non-eupneic breathing of C57BL6 mice to those of B6AF1 (57BL6 dam × A/J sire) and Swiss-Webster mice, which display different ventilatory responses than C57BL6 mice. C57BL6 mice displayed marked increases in respiratory frequency and non-eupneic breathing upon return to room-air after hypoxic (10% O2, 90% N2), hypercapnic (5% CO2, 21% O2, 74% N2) and hypoxic-hypercapnic (10% O2, 5% CO2, 85% N2) challenges. B6AF1 mice displayed less tachypnea and reduced non-eupneic breathing post-hypoxia, whereas Swiss-Webster mice displayed robust tachypnea with minimal increases in non-eupneic breathing post-hypoxia. These studies demonstrate that non-eupneic breathing increases after physiologically-relevant hypoxic-hypercapnic challenge in C57BL6 mice and suggest that further studies with these and B6AF1 and Swiss-Webster mice will help define the genetics of non-eupneic breathing. PMID:25242462

  14. Increase of cellular hypoxic tolerance by erythromycin and other antibiotics.

    PubMed

    Huber, R; Kasischke, K; Ludolph, A C; Riepe, M W

    1999-05-14

    Antibiotics are used extensively, but in addition to their anti-infectious effects some inhibit cellular energy metabolism. We investigated hypoxic tolerance following in vivo pretreatment with erythromycin and kanamycin, or in vitro pretreatment with ampicillin. Recovery of the CA1 population spike amplitude in hippocampal slices upon 15 min hypoxia improved time-dependently following single i.p. in vivo pretreatment with erythromycin (maximum at 6 h: recovery 90+/-7% (mean s.d.) vs 30% in untreated controls; p<0.01). The hypoxia-induced increase in NADH was smaller in slices that recovered from hypoxia. We conclude that antibiotics increase cellular hypoxic tolerance to a varying extent. Use of antibiotics in experimental studies may, therefore, distort conclusions about hypoxic sensitivity and confounding mechanisms. In contrast, antibiotics may provide an effective strategy to induce chemical preconditioning in humans.

  15. Hypoxic pulmonary hypertension in chronic lung diseases: novel vasoconstrictor pathways.

    PubMed

    Rowan, Simon C; Keane, Michael P; Gaine, Seán; McLoughlin, Paul

    2016-03-01

    Pulmonary hypertension is a well recognised complication of chronic hypoxic lung diseases, which are among the most common causes of death and disability worldwide. Development of pulmonary hypertension independently predicts reduced life expectancy. In chronic obstructive pulmonary disease, long-term oxygen therapy ameliorates pulmonary hypertension and greatly improves survival, although the correction of alveolar hypoxia and pulmonary hypertension is only partial. Advances in understanding of the regulation of vascular smooth muscle tone show that chronic vasoconstriction plays a more important part in the pathogenesis of hypoxic pulmonary hypertension than previously thought, and that structural vascular changes contribute less. Trials of existing vasodilators show that pulmonary hypertension can be ameliorated and systemic oxygen delivery improved in carefully selected patients, although systemic hypotensive effects limit the doses used. Vasoconstrictor pathways that are selective for the pulmonary circulation can be blocked to reduce hypoxic pulmonary hypertension without causing systemic hypotension, and thus provide potential targets for novel therapeutic strategies.

  16. The metal-responsive transcription factor-1 contributes to HIF-1 activation during hypoxic stress

    SciTech Connect

    Murphy, Brian J. . E-mail: brian.murphy@sri.com; Sato, Barbara G.; Dalton, Timothy P.; Laderoute, Keith R.

    2005-11-25

    Hypoxia-inducible factor-1 (HIF-1), the major transcriptional regulator of the mammalian cellular response to low oxygen (hypoxia), is embedded within a complex network of signaling pathways. We have been investigating the importance of another stress-responsive transcription factor, MTF-1, for the adaptation of cells to hypoxia. This article reports that MTF-1 plays a central role in hypoxic cells by contributing to HIF-1 activity. Loss of MTF-1 in transformed Mtf1 null mouse embryonic fibroblasts (MEFs) results in an attenuation of nuclear HIF-1{alpha} protein accumulation, HIF-1 transcriptional activity, and expression of an established HIF-1 target gene, glucose transporter-1 (Glut1). Mtf1 null (Mtf1 KO) MEFs also have constitutively higher levels of both glutathione (GSH) and the rate-limiting enzyme involved in GSH synthesis-glutamate cysteine ligase catalytic subunit-than wild type cells. The altered cellular redox state arising from increased GSH may perturb oxygen-sensing mechanisms in hypoxic Mtf1 KO cells and decrease the accumulation of HIF-1{alpha} protein. Together, these novel findings define a role for MTF-1 in the regulation of HIF-1 activity.

  17. Advancing hypoxic training in team sports: from intermittent hypoxic training to repeated sprint training in hypoxia

    PubMed Central

    Faiss, Raphaël; Girard, Olivier; Millet, Grégoire P

    2013-01-01

    Over the past two decades, intermittent hypoxic training (IHT), that is, a method where athletes live at or near sea level but train under hypoxic conditions, has gained unprecedented popularity. By adding the stress of hypoxia during ‘aerobic’ or ‘anaerobic’ interval training, it is believed that IHT would potentiate greater performance improvements compared to similar training at sea level. A thorough analysis of studies including IHT, however, leads to strikingly poor benefits for sea-level performance improvement, compared to the same training method performed in normoxia. Despite the positive molecular adaptations observed after various IHT modalities, the characteristics of optimal training stimulus in hypoxia are still unclear and their functional translation in terms of whole-body performance enhancement is minimal. To overcome some of the inherent limitations of IHT (lower training stimulus due to hypoxia), recent studies have successfully investigated a new training method based on the repetition of short (<30 s) ‘all-out’ sprints with incomplete recoveries in hypoxia, the so-called repeated sprint training in hypoxia (RSH). The aims of the present review are therefore threefold: first, to summarise the main mechanisms for interval training and repeated sprint training in normoxia. Second, to critically analyse the results of the studies involving high-intensity exercises performed in hypoxia for sea-level performance enhancement by differentiating IHT and RSH. Third, to discuss the potential mechanisms underpinning the effectiveness of those methods, and their inherent limitations, along with the new research avenues surrounding this topic. PMID:24282207

  18. Peptidylarginine Deiminases as Drug Targets in Neonatal Hypoxic-Ischemic Encephalopathy.

    PubMed

    Lange, Sigrun

    2016-01-01

    Oxygen deprivation and infection are major causes of perinatal brain injury leading to cerebral palsy and other neurological disabilities. The identification of novel key factors mediating white and gray matter damage are crucial to allow better understanding of the specific contribution of different cell types to the injury processes and pathways for clinical intervention. Recent studies in the Rice-Vannucci mouse model of neonatal hypoxic ischemia (HI) have highlighted novel roles for calcium-regulated peptidylarginine deiminases (PADs) and demonstrated neuroprotective effects of pharmacological PAD inhibition following HI and synergistic infection mimicked by lipopolysaccharide stimulation.

  19. Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise.

    PubMed

    Casey, Darren P; Madery, Brandon D; Pike, Tasha L; Eisenach, John H; Dietz, Niki M; Joyner, Michael J; Wilkins, Brad W

    2009-10-01

    We tested the hypothesis that adenosine contributes to augmented skeletal muscle vasodilation during hypoxic exercise. In separate protocols, subjects performed incremental rhythmic forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n = 8), subjects received an intra-arterial administration of saline (control) and aminophylline (adenosine receptor antagonist). In protocol 2 (n = 10), subjects received intra-arterial phentolamine (alpha-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml x min(-1).100 mmHg(-1)) was calculated from forearm blood flow (in ml/min) and blood pressure (in mmHg). In protocol 1, the change in FVC (DeltaFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 +/- 29 and 314 +/- 34 ml x min(-1) x 100 mmHg(-1) (10% and 20%, respectively). Aminophylline administration did not affect DeltaFVC during hypoxic exercise at 10% (190 +/- 29 ml x min(-1)x100 mmHg(-1), P = 0.4) or 20% (287 +/- 48 ml x min(-1) x 100 mmHg(-1), P = 0.3). In protocol 2, DeltaFVC due to hypoxic exercise with phentolamine infusion was 313 +/- 30 and 453 +/- 41 ml x min(-1) x 100 mmHg(-1) (10% and 20% respectively). DeltaFVC was similar at 10% (352 +/- 39 ml min(-1) x 100 mmHg(-1), P = 0.8) and 20% (528 +/- 45 ml x min(-1) x 100 mmHg(-1), P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, DeltaFVC to exogenous adenosine was reduced by aminophylline administration in both protocols (P < 0.05 for both). These observations suggest that adenosine receptor activation is not obligatory for the augmented hyperemia during hypoxic exercise in humans.

  20. Exercise-induced oxidative stress and hypoxic exercise recovery.

    PubMed

    Ballmann, Christopher; McGinnis, Graham; Peters, Bridget; Slivka, Dustin; Cuddy, John; Hailes, Walter; Dumke, Charles; Ruby, Brent; Quindry, John

    2014-04-01

    Hypoxia due to altitude diminishes performance and alters exercise oxidative stress responses. While oxidative stress and exercise are well studied, the independent impact of hypoxia on exercise recovery remains unknown. Accordingly, we investigated hypoxic recovery effects on post-exercise oxidative stress. Physically active males (n = 12) performed normoxic cycle ergometer exercise consisting of ten high:low intensity intervals, 20 min at moderate intensity, and 6 h recovery at 975 m (normoxic) or simulated 5,000 m (hypoxic chamber) in a randomized counter-balanced cross-over design. Oxygen saturation was monitored via finger pulse oximetry. Blood plasma obtained pre- (Pre), post- (Post), 2 h post- (2Hr), 4 h post- (4Hr), and 6 h (6Hr) post-exercise was assayed for Ferric Reducing Ability of Plasma (FRAP), Trolox Equivalent Antioxidant Capacity (TEAC), Lipid Hydroperoxides (LOOH), and Protein Carbonyls (PC). Biopsies from the vastus lateralis obtained Pre and 6Hr were analyzed by real-time PCR quantify expression of Heme oxygenase 1 (HMOX1), Superoxide Dismutase 2 (SOD2), and Nuclear factor (euthyroid-derived2)-like factor (NFE2L2). PCs were not altered between trials, but a time effect (13 % Post-2Hr increase, p = 0.044) indicated exercise-induced blood oxidative stress. Plasma LOOH revealed only a time effect (p = 0.041), including a 120 % Post-4Hr increase. TEAC values were elevated in normoxic recovery versus hypoxic recovery. FRAP values were higher 6Hr (p = 0.045) in normoxic versus hypoxic recovery. Exercise elevated gene expression of NFE2L2 (20 % increase, p = 0.001) and SOD2 (42 % increase, p = 0.003), but hypoxic recovery abolished this response. Data indicate that recovery in a hypoxic environment, independent of exercise, may alter exercise adaptations to oxidative stress and metabolism.

  1. Application of altitude/hypoxic training by elite athletes.

    PubMed

    Wilber, Randall L

    2007-09-01

    At the Olympic level, differences in performance are typically less than 0.5%. This helps explain why many contemporary elite endurance athletes in summer and winter sport incorporate some form of altitude/hypoxic training within their year-round training plan, believing that it will provide the "competitive edge" to succeed at the Olympic level. The purpose of this paper is to describe the practical application of altitude/hypoxic training as used by elite athletes. Within the general framework of the paper, both anecdotal and scientific evidence will be presented relative to the efficacy of several contemporary altitude/hypoxic training models and devices currently used by Olympic-level athletes for the purpose of legally enhancing performance. These include the three primary altitude/hypoxic training models: 1) live high+train high (LH+TH), 2) live high+train low (LH+TL), and 3) live low+train high (LL+TH). The LH+TL model will be examined in detail and will include its various modifications: natural/terrestrial altitude, simulated altitude via nitrogen dilution or oxygen filtration, and hypobaric normoxia via supplemental oxygen. A somewhat opposite approach to LH+TL is the altitude/hypoxic training strategy of LL+TH, and data regarding its efficacy will be presented. Recently, several of these altitude/hypoxic training strategies and devices underwent critical review by the World Anti-Doping Agency (WADA) for the purpose of potentially banning them as illegal performance-enhancing substances/methods. This paper will conclude with an update on the most recent statement from WADA regarding the use of simulated altitude devices.

  2. Microbial mineralization of dichloroethene and vinyl chloride under hypoxic conditions

    USGS Publications Warehouse

    Bradley, Paul M.; Chapelle, Francis H.

    2011-01-01

    Mineralization of 14C-radiolabled vinyl chloride ([1,2-14C] VC) and cis-dichloroethene ([1,2-14C] cis-DCE) under hypoxic (initial dissolved oxygen (DO) concentrations about 0.1 mg/L) and nominally anoxic (DO minimum detection limit = 0.01 mg/L) was examined in chloroethene-exposed sediments from two groundwater and two surface water sites. The results show significant VC and dichloroethene (DCE) mineralization under hypoxic conditions. All the sample treatments exhibited pseudo-first-order kinetics for DCE and VC mineralization over an extended range of substrate concentrations. First-order rates for VC mineralization were approximately 1 to 2 orders of magnitude higher in hypoxic groundwater sediment treatments and at least three times higher in hypoxic surface water sediment treatments than in the respective anoxic treatments. For VC, oxygen-linked processes accounted for 65 to 85% of mineralization at DO concentrations below 0.1 mg/L, and 14CO2 was the only degradation product observed in VC treatments under hypoxic conditions. Because the lower detection limit for DO concentrations measured in the field is typically 0.1 to 0.5 mg/L, these results indicate that oxygen-linked VC and DCE biodegradation can be significant under field conditions that appear anoxic. Furthermore, because rates of VC mineralization exceeded rates of DCE mineralization under hypoxic conditions, DCE accumulation without concomitant accumulation of VC may not be evidence of a DCE degradative “stall” in chloroethene plumes. Significantly, mineralization of VC above the level that could reasonably be attributed to residual DO contamination was also observed in several nominally anoxic (DO minimum detection limit = 0.01 mg/L) microcosm treatments.

  3. Hypoxic Preconditioning Combined with Microbubble-Mediated Ultrasound Effect on MSCs Promote SDF-1/CXCR4 Expression and its Migration Ability: An In Vitro Study.

    PubMed

    Li, Lu; Wu, Shengzheng; Li, Peijing; Zhuo, Lisha; Gao, Yunhua; Xu, Yali

    2015-12-01

    Our objective is to investigate the promoting effect of hypoxic preconditioning combined with microbubble (MB)-mediated ultrasound (US) on the SDF-1/CXCR4 expression and the migration ability of mesenchymal stem cells (MSCs). Based on the uniform design, the parameters of MB-mediated US, such as the total treatment time (T), acoustic intensity (Q), and the dosage of MBs, were optimized firstly. The results were assessed by regression analysis. Using the optimum irradiation parameters, the concentration of SDF-1 in the supernatant, the expression levels of membrane CXCR4, and the cell viability of hypoxic MSCs or normoxic MSCs were compared. The in vitro transwell migration assay was performed as well. The best combination of parameters for more SDF-1 secretion and less MSCs death was T = 30 s, A = 0.6 W/cm(2), and MB = 10(6)/ml. After 24 h of hypoxic preconditioning, the expression of SDF-1 and surface CXCR4 was increased in the hypoxic MSC group as compared to the normoxic MSC group (P < 0.05). On the basis of that, MB-mediated US could further upregulate the expression of SDF-1/CXCR4 with the optimum parameters (P < 0.05), while the cell viability was only decreased by about 9-10 % compared to the untreated groups. The number of successfully migrated cells was also the largest in the hypoxic preconditioning combined with MB-mediated US group than all the other groups. The results obtained indicate the combination of hypoxic preconditioning, and MB-mediated US can upregulate the SDF-1/CXCR4 expression and improve the migration ability in MSCs.

  4. Hypoxic Pulmonary Vasoconstriction in Humans: Tale or Myth

    PubMed Central

    Hussain, A.; Suleiman, M.S.; George, S.J.; Loubani, M.; Morice, A.

    2017-01-01

    Hypoxic Pulmonary vasoconstriction (HPV) describes the physiological adaptive process of lungs to preserves systemic oxygenation. It has clinical implications in the development of pulmonary hypertension which impacts on outcomes of patients undergoing cardiothoracic surgery. This review examines both acute and chronic hypoxic vasoconstriction focusing on the distinct clinical implications and highlights the role of calcium and mitochondria in acute versus the role of reactive oxygen species and Rho GTPases in chronic HPV. Furthermore it identifies gaps of knowledge and need for further research in humans to clearly define this phenomenon and the underlying mechanism. PMID:28217180

  5. Chemical composition of rainbow trout urine following acute hypoxic stress

    USGS Publications Warehouse

    Hunn, Joseph B.

    1969-01-01

    Rainbow trout (Salmo gairdnerii) were anesthetized with MS-222, catheterized, and introduced into urine collecting chambers. Twenty-four hours after introduction, a 4-hour accumulation of urine was collected to serve as the control. Water flow to the chambers was then discontinued for 30 minutes during which the oxygen content of the water exiting in the chamber dropped from 4.9 to 2.8 mg/l. Following this hypoxic stress fresh water was restored and accumulated urine samples were taken for analysis at 1, 4, and 20 hours post-hypoxic stress. Rainbow trout excrete abnormally high concentrations of Na, K, Mg, Cl, and inorganic PO4 following hypoxia.

  6. A DNA-binding Molecule Targeting the Adaptive Hypoxic Response in Multiple Myeloma has Potent Anti-tumor Activity

    PubMed Central

    Mysore, Veena S.; Szablowski, Jerzy; Dervan, Peter B.; Frost, Patrick J.

    2016-01-01

    Multiple myeloma (MM) is incurable and invariably becomes resistant to chemotherapy. Although the mechanisms remain unclear, hypoxic conditions in the bone marrow have been implicated in contributing to MM progression, angiogenesis, and resistance to chemotherapy. These effects occur via adaptive cellular responses mediated by hypoxia-inducible transcription factors (HIFs), and targeting HIFs can have anti-cancer effects in both solid and hematological malignancies. Here, it was found that in most myeloma cell lines tested, HIF1α, but not HIF2α expression was oxygen dependent and this could be explained by the differential expression of the regulatory prolyl-hydroxylase isoforms. The anti-MM effects of a sequence-specific DNA-binding pyrrole-imidazole polyamide (HIF-PA), that disrupts the HIF heterodimer from binding to its cognate DNA sequences, were also investigated. HIF-PA is cell permeable, localizes to the nuclei, and binds specific regions of DNA with an affinity comparable to that of HIF transcription factors. Most of the MM cells were resistant to hypoxia-mediated apoptosis, and HIF-PA treatment could overcome this resistance in vitro. Using xenograft models, it was determined that HIF-PA significantly decreased tumor volume and increased hypoxic and apoptotic regions within solid tumor nodules and the growth of myeloma cells engrafted in the bone marrow. This provides a rationale for targeting the adaptive cellular hypoxic response of the O2-dependent activation of HIFα using polyamides. PMID:26801054

  7. Time-Course Analysis of Gene Expression During the Saccharomyces cerevisiae Hypoxic Response

    PubMed Central

    Bendjilali, Nasrine; MacLeon, Samuel; Kalra, Gurmannat; Willis, Stephen D.; Hossian, A. K. M. Nawshad; Avery, Erica; Wojtowicz, Olivia; Hickman, Mark J.

    2016-01-01

    Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new regulated genes. Reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that the lysine methyltransferase EFM6 and the recombinase DMC1, both conserved in humans, are indeed oxygen-responsive. Looking more broadly, oxygen-regulated genes participate in expected processes like respiration and lipid metabolism, but also in unexpected processes like amino acid and vitamin metabolism. Using principle component analysis, we discovered that the hypoxic response largely occurs during the first 2 hr and then a new steady-state expression state is achieved. Moreover, we show that the oxygen-dependent genes are not part of the previously described environmental stress response (ESR) consisting of genes that respond to diverse types of stress. While hypoxia appears to cause a transient stress, the hypoxic response is mostly characterized by a transition to a new state of gene expression. In summary, our results reveal that hypoxia causes widespread and complex changes in gene expression to prepare the cell to function with little or no oxygen. PMID:27883312

  8. Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

    PubMed Central

    Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

    2014-01-01

    Background: Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. Methods: C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). Results and conclusions: The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions. PMID:24966910

  9. Time-Course Analysis of Gene Expression During the Saccharomyces cerevisiae Hypoxic Response.

    PubMed

    Bendjilali, Nasrine; MacLeon, Samuel; Kalra, Gurmannat; Willis, Stephen D; Hossian, A K M Nawshad; Avery, Erica; Wojtowicz, Olivia; Hickman, Mark J

    2017-01-05

    Many cells experience hypoxia, or low oxygen, and respond by dramatically altering gene expression. In the yeast Saccharomyces cerevisiae, genes that respond are required for many oxygen-dependent cellular processes, such as respiration, biosynthesis, and redox regulation. To more fully characterize the global response to hypoxia, we exposed yeast to hypoxic conditions, extracted RNA at different times, and performed RNA sequencing (RNA-seq) analysis. Time-course statistical analysis revealed hundreds of genes that changed expression by up to 550-fold. The genes responded with varying kinetics suggesting that multiple regulatory pathways are involved. We identified most known oxygen-regulated genes and also uncovered new regulated genes. Reverse transcription-quantitative PCR (RT-qPCR) analysis confirmed that the lysine methyltransferase EFM6 and the recombinase DMC1, both conserved in humans, are indeed oxygen-responsive. Looking more broadly, oxygen-regulated genes participate in expected processes like respiration and lipid metabolism, but also in unexpected processes like amino acid and vitamin metabolism. Using principle component analysis, we discovered that the hypoxic response largely occurs during the first 2 hr and then a new steady-state expression state is achieved. Moreover, we show that the oxygen-dependent genes are not part of the previously described environmental stress response (ESR) consisting of genes that respond to diverse types of stress. While hypoxia appears to cause a transient stress, the hypoxic response is mostly characterized by a transition to a new state of gene expression. In summary, our results reveal that hypoxia causes widespread and complex changes in gene expression to prepare the cell to function with little or no oxygen.

  10. Satellite-based empirical models linking river plume dynamics with hypoxic area andvolume

    EPA Science Inventory

    Satellite-based empirical models explaining hypoxic area and volume variation were developed for the seasonally hypoxic (O2 < 2 mg L−1) northern Gulf of Mexico adjacent to the Mississippi River. Annual variations in midsummer hypoxic area and ...

  11. Blunted Hypoxic Pulmonary Vasoconstriction in Experimental Neonatal Chronic Lung Disease

    PubMed Central

    Rey-Parra, Gloria Juliana; Archer, Stephen L.; Bland, Richard D.; Albertine, Kurt H.; Carlton, David P.; Cho, Soo-Chul; Kirby, Beth; Haromy, Al; Eaton, Farah; Wu, Xichen; Thébaud, Bernard

    2008-01-01

    Rationale: Neonatal chronic lung disease (CLD), caused by prolonged mechanical ventilation (MV) with O2-rich gas, is the most common cause of long-term hospitalization and recurrent respiratory illness in extremely premature infants. Recurrent episodes of hypoxemia and associated ventilator adjustments often lead to worsening CLD. The mechanism that causes these hypoxemic episodes is unknown. Hypoxic pulmonary vasoconstriction (HPV), which is partially controlled by O2-sensitive voltage-gated potassium (Kv) channels, is an important adaptive response to local hypoxia that helps to match perfusion and ventilation in the lung. Objectives: To test the hypothesis that chronic lung injury (CLI) impairs HPV. Methods: We studied preterm lambs that had MV with O2-rich gas for 3 weeks and newborn rats that breathed 95%-O2 for 2 weeks, both of which resulted in airspace enlargement and pulmonary vascular changes consistent with CLD. Measurements and Main Results: HPV was attenuated in preterm lambs with CLI after 2 weeks of MV and in newborn rats with CLI after 2 weeks of hyperoxia. HPV and constriction to the Kv1.x-specific inhibitor, correolide, were preferentially blunted in excised distal pulmonary arteries (dPAs) from hyperoxic rats, whose dPAs exhibited decreased Kv1.5 and Kv2.1 mRNA and K+ current. Intrapulmonary gene transfer of Kv1.5, encoding the ion channel that is thought to trigger HPV, increased O2-sensitive K+ current in cultured smooth muscle cells from rat dPAs, and restored HPV in hyperoxic rats. Conclusions: Reduced expression/activity of O2-sensitive Kv channels in dPAs contributes to blunted HPV observed in neonatal CLD. PMID:18511704

  12. Microglia-derived proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta induce Purkinje neuronal apoptosis via their receptors in hypoxic neonatal rat brain.

    PubMed

    Kaur, Charanjit; Sivakumar, Viswanathan; Zou, Zhirong; Ling, Eng-Ang

    2014-01-01

    The developing cerebellum is extremely vulnerable to hypoxia which can damage the Purkinje neurons. We hypothesized that this might be mediated by tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) derived from activated microglia as in other brain areas. One-day-old rats were subjected to hypoxia following, which the expression changes of various proteins in the cerebellum including hypoxia inducible factor-1α, TNF-α, IL-1β, TNF-R1 and IL-1R1 were analyzed. Following hypoxic exposure, TNF-α and IL-1β immunoexpression in microglia was enhanced coupled by that of TNF-R1 and IL-1R1 in the Purkinje neurons. Along with this, hypoxic microglia in vitro showed enhanced release of TNF-α and IL-1β whose receptor expression was concomitantly increased in the Purkinje neurons. In addition, nitric oxide (NO) level was significantly increased in the cerebellum and cultured microglia subjected to hypoxic exposure. Moreover, cultured Purkinje neurons treated with conditioned medium derived from hypoxic microglia underwent apoptosis but the incidence was significantly reduced when the cells were treated with the same medium that was neutralized with TNF-α/IL-1β antibody. We conclude that hypoxic microglia in the neonatal cerebellum produce increased amounts of NO, TNF-α and IL-1β which when acting via their respective receptors could induce Purkinje neuron death.

  13. Proof of Concept to Isolate and Culture Primary Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-Hold Diving

    DTIC Science & Technology

    2014-09-30

    be resolvable with the addition both antibiotics and antimycotics to the transfer and culturing medias. With those changes and our anticipated...transfer cells exclusively in F10 (with antibiotics /antimycotics) for the next field trip. February 2013 With newly-weaned pups available to...of f-10 based growth media + 0.5% lipid (prewarmed to 37oC) 22. Plate cells on matrigel- coated plates for initial growth phase. 23. Check on

  14. Nitrous oxide emissions from the Gulf of Mexico Hypoxic Zone

    EPA Science Inventory

    The production of nitrous oxide (N2O), a potent greenhouse gas, in hypoxic coastal zones remains poorly characterized due to a lack of data, though large nitrogen inputs and deoxygenation typical of these systems create the potential for large N2O emissions. We report the first N...

  15. Notification: Evaluate the Gulf of Mexico Hypoxic Zone Reduction

    EPA Pesticide Factsheets

    Project #OPE-FY13-0012, January 30, 2013. The Office of Inspector General (OIG) is starting preliminary research on the U.S. Environmental Protection Agency’s (EPA’s) and states’ efforts to reduce the Gulf of Mexico hypoxic zone.

  16. Population differences in response to hypoxic stress in Atlantic salmon.

    PubMed

    Côte, J; Roussel, J-M; Le Cam, S; Bal, G; Evanno, G

    2012-12-01

    Understanding whether populations can adapt to new environmental conditions is a major issue in conservation and evolutionary biology. Aquatic organisms are increasingly exposed to environmental changes linked with human activities in river catchments. For instance, the clogging of bottom substratum by fine sediments is observed in many rivers and usually leads to a decrease in dissolved oxygen concentrations in gravel beds. Such hypoxic stress can alter the development and even be lethal for Atlantic salmon (Salmo salar) embryos that spend their early life into gravel beds. In this study, we used a common garden experiment to compare the responses to hypoxic stress of four genetically differentiated and environmentally contrasted populations. We used factorial crossing designs to measure additive genetic variation of early life-history traits in each population. Embryos were reared under normoxic and hypoxic conditions, and we measured their survival, incubation time and length at the end of embryonic development. Under hypoxic conditions, embryos had a lower survival and hatched later than in normoxic conditions. We found different hypoxia reaction norms among populations, but almost no population effect in both treatments. We also detected significant sire × treatment interactions in most populations and a tendency for heritability values to be lower under stressful conditions. Overall, these results reveal a high degree of phenotypic plasticity in salmon populations that nevertheless differ in their adaptive potential to hypoxia given the distinct reaction norms observed between and within populations.

  17. Chemoreceptor stimulation interferes with regional hypoxic pulmonary vasoconstriction.

    PubMed

    Chapleau, M W; Wilson, L B; Gregory, T J; Levitzky, M G

    1988-02-01

    Hypoxemia interferes with the diversion of blood flow away from hypoxic regions of the lung, possibly through activation of the arterial chemoreceptor reflex. The purpose of this study was to determine if selective stimulation of carotid chemoreceptors reduces the diversion of flow (hypoxic vasoconstriction) when normal systemic oxygen levels are present. Chloralose anesthetized dogs were paralyzed and each lung was separately ventilated via a dual-lumen endobronchial tube. Left pulmonary artery (QL) and main pulmonary artery (QT) blood flows were measured with electromagnetic flow probes. Chemoreceptors were stimulated by perfusion of the carotid sinuses with hypoxic, hypercapnic blood. QL/QT averaged 46 +/- 4, 29 +/- 2, and 36 +/- 4% during bilateral O2 ventilation (control), left lung N2 ventilation, and left lung N2 plus chemoreceptor stimulation in dogs treated with the cyclo-oxygenase inhibitor meclofenamate. After vagotomy, QL/QT averaged 45 +/- 4, 27 +/- 3, and 28 +/- 2% during the same conditions. QL/QT decreased significantly from control (P less than 0.05) during left lung N2 alone but did not decrease during left lung N2 plus chemoreceptor stimulation in dogs with intact vagi. In contrast, QL/QT decreased significantly both before and during chemoreceptor stimulation in vagotomized dogs. The same responses were observed in dogs not treated with meclofenamate. These results indicate that selective stimulation of arterial chemoreceptors can interfere with regional hypoxic vasoconstriction and suggest that the vagus nerves may mediate this effect.

  18. MODULATION OF HYPOXIC PULMONARY VASOCONSTRICTION BY ERYTHROCYTIC NITRIC OXIDE

    EPA Science Inventory

    Abstract
    American Heart Association 2001

    Modulation of Hypoxic Pulmonary Vasoconstriction by Erythrocytic NO
    McMahon TJ1, Gow AJ1, Huang YCT4, Stamler JS1,2,3
    Departments of Medicine1 and Biochemistry2, and Howard Hughes Medical Institute3,
    Duke University Med...

  19. Physiological Responses to Two Hypoxic Conditioning Strategies in Healthy Subjects

    PubMed Central

    Chacaroun, Samarmar; Borowik, Anna; Morrison, Shawnda A.; Baillieul, Sébastien; Flore, Patrice; Doutreleau, Stéphane; Verges, Samuel

    2017-01-01

    Objective: Hypoxic exposure can be used as a therapeutic tool by inducing various cardiovascular, neuromuscular, and metabolic adaptations. Hypoxic conditioning strategies have been evaluated in patients with chronic diseases using either sustained (SH) or intermittent (IH) hypoxic sessions. Whether hypoxic conditioning via SH or IH may induce different physiological responses remains to be elucidated. Methods: Fourteen healthy active subjects (7 females, age 25 ± 8 years, body mass index 21.5 ± 2.5 kg·m−2) performed two interventions in a single blind, randomized cross-over design, starting with either 3 x SH (48 h apart), or 3 x IH (48 h apart), separated by a 2 week washout period. SH sessions consisted of breathing a gas mixture with reduced inspiratory oxygen fraction (FiO2), continuously adjusted to reach arterial oxygen saturations (SpO2) of 70–80% for 1 h. IH sessions consisted of 5 min with reduced FiO2 (SpO2 = 70–80%), followed by 3-min normoxia, repeated seven times. During the first (S1) and third (S3) sessions of each hypoxic intervention, cardiorespiratory parameters, and muscle and pre-frontal cortex oxygenation (near infrared spectroscopy) were assessed continuously. Results: Minute ventilation increased significantly during IH sessions (+2 ± 2 L·min−1) while heart rate increased during both SH (+11 ± 4 bpm) and IH (+13 ± 5 bpm) sessions. Arterial blood pressure increased during all hypoxic sessions, although baseline normoxic systolic blood pressure was reduced from S1 to S3 in IH only (−8 ± 11 mmHg). Muscle oxygenation decreased significantly during S3 but not S1, for both hypoxic interventions (S3: SH −6 ± 5%, IH −3 ± 4%); pre-frontal oxygenation decreased in S1 and S3, and to a greater extent in SH vs. IH (−13 ± 3% vs. −6 ± 6%). Heart rate variability indices indicated a significantly larger increase in sympathetic activity in SH vs. IH (lower SDNN, PNN50, and RMSSD values in SH). From S1 to S3, further reduction in

  20. NF-kappaB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1alpha.

    PubMed

    Rius, Jordi; Guma, Monica; Schachtrup, Christian; Akassoglou, Katerina; Zinkernagel, Annelies S; Nizet, Victor; Johnson, Randall S; Haddad, Gabriel G; Karin, Michael

    2008-06-05

    The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose alpha subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1alpha, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-kappaB plays a central role. NF-kappaB activation is controlled by IkappaB kinases (IKK), mainly IKK-beta, needed for phosphorylation-induced degradation of IkappaB inhibitors in response to infection and inflammation. IKK-beta is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-kappaB and HIF-1alpha has proven elusive. Using in vitro systems, it was reported that HIF-1alpha activates NF-kappaB, that NF-kappaB controls HIF-1alpha transcription and that HIF-1alpha activation may be concurrent with inhibition of NF-kappaB. Here we show, with the use of mice lacking IKK-beta in different cell types, that NF-kappaB is a critical transcriptional activator of HIF-1alpha and that basal NF-kappaB activity is required for HIF-1alpha protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-beta deficiency results in defective induction of HIF-1alpha target genes including vascular endothelial growth factor. IKK-beta is also essential for HIF-1alpha accumulation in macrophages experiencing a bacterial infection. Hence, IKK-beta is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

  1. Effects of regional phentolamine on hypoxic vasodilatation in healthy humans.

    PubMed

    Weisbrod, C J; Minson, C T; Joyner, M J; Halliwill, J R

    2001-12-01

    1. Limb vascular beds exhibit a graded dilatation in response to hypoxia despite increased sympathetic vasoconstrictor nerve activity. We investigated the extent to which sympathetic vasoconstriction can mask hypoxic vasodilatation and assessed the relative contributions of beta-adrenergic and nitric oxide (NO) pathways to hypoxic vasodilatation. 2. We measured forearm blood flow responses (plethysmography) to isocapnic hypoxia (arterial saturation approximately 85%) in eight healthy men and women (18-26 years) after selective alpha-adrenergic blockade (phentolamine) of one forearm. Subsequently, we measured hypoxic responses after combined alpha- and beta-adrenergic blockade (phentolamine and propranolol) and after combined alpha- and beta-adrenergic blockade coupled with NO synthase inhibition (N(G)-monomethyl-L-arginine, L-NMMA). 3. Hypoxia increased forearm vascular conductance by 49.0 +/- 13.5% after phentolamine (compared to +16.8 +/- 7.0% in the control arm without phentolamine, P < 0.05). After addition of propranolol, the forearm vascular conductance response to hypoxia was reduced by approximately 50%, but dilatation was still present (+24.7 +/- 7.0%, P < 0.05 vs. normoxia). When L-NMMA was added, there was no further reduction in the forearm vascular conductance response to hypoxia (+28.2 +/- 4.0%, P < 0.05 vs. normoxia). 4. Thus, selective regional alpha-adrenergic blockade unmasked a greater hypoxic vasodilatation than occurs in the presence of functional sympathetic nervous system responses to hypoxia. Furthermore, approximately half of the hypoxic vasodilatation in the forearm appears to be mediated by beta-adrenergic receptor-mediated pathways. Finally, since considerable dilatation persists in the presence of both beta-adrenergic blockade and NO synthase inhibition, it is likely that an additional vasodilator mechanism is activated by hypoxia in humans.

  2. Effects of oxygen on intrinsic radiation sensitivity: A test of the relationship between aerobic and hypoxic linear-quadratic (LQ) model parameters

    SciTech Connect

    Carlson, David J.; Stewart, Robert D.; Semenenko, Vladimir A.

    2006-09-15

    The poor treatment prognosis for tumors with high levels of hypoxia is usually attributed to the decreased sensitivity of hypoxic cells to ionizing radiation. Mechanistic considerations suggest that linear quadratic (LQ) survival model radiosensitivity parameters for hypoxic (H) and aerobic (A) cells are related by {alpha}{sub H}={alpha}{sub A}/oxygen enhancement ratio (OER) and ({alpha}/{beta}){sub H}=OER({alpha}/{beta}){sub A}. The OER parameter may be interpreted as the ratio of the dose to the hypoxic cells to the dose to the aerobic cells required to produce the same number of DSBs per cell. The validity of these expressions is tested against survival data for mammalian cells irradiated in vitro with low- and high-LET radiation. Estimates of hypoxic and aerobic radiosensitivity parameters are derived from independent and simultaneous least-squares fits to the survival data. An external bootstrap procedure is used to test whether independent fits to the survival data give significantly better predictions than simultaneous fits to the aerobic and hypoxic data. For low-LET radiation, estimates of the OER derived from the in vitro data are between 2.3 and 3.3 for extreme levels of hypoxia. The estimated range for the OER is similar to the oxygen enhancement ratios reported in the literature for the initial yield of DSBs. The half-time for sublethal damage repair was found to be independent of oxygen concentration. Analysis of patient survival data for cervix cancer suggests an average OER less than or equal to 1.5, which corresponds to a pO{sub 2} of 5 mm Hg (0.66%) in the in vitro experiments. Because the OER derived from the cervix cancer data is averaged over cells at all oxygen levels, cells irradiated in vivo under extreme levels of hypoxia (<0.5 mm Hg) may have an OER substantially higher than 1.5. The reported analyses of in vitro data, as well as mechanistic considerations, provide strong support for the expressions relating hypoxic and aerobic

  3. Targeting the hypoxic fraction of tumours using hypoxia-activated prodrugs.

    PubMed

    Phillips, Roger M

    2016-03-01

    The presence of a microenvironment within most tumours containing regions of low oxygen tension or hypoxia has profound biological and therapeutic implications. Tumour hypoxia is known to promote the development of an aggressive phenotype, resistance to both chemotherapy and radiotherapy and is strongly associated with poor clinical outcome. Paradoxically, it is recognised as a high-priority target and one of the therapeutic strategies designed to eradicate hypoxic cells in tumours is a group of compounds known collectively as hypoxia-activated prodrugs (HAPs) or bioreductive drugs. These drugs are inactive prodrugs that require enzymatic activation (typically by 1 or 2 electron oxidoreductases) to generate cytotoxic species with selectivity for hypoxic cells being determined by (1) the ability of oxygen to either reverse or inhibit the activation process and (2) the presence of elevated expression of oxidoreductases in tumours. The concepts underpinning HAP development were established over 40 years ago and have been refined over the years to produce a new generation of HAPs that are under preclinical and clinical development. The purpose of this article is to describe current progress in the development of HAPs focusing on the mechanisms of action, preclinical properties and clinical progress of leading examples.

  4. Differential effects of hypoxic and hyperoxic stress-induced hypertrophy in cultured chick fetal cardiac myocytes.

    PubMed

    Greco, Allison A; Gomez, George

    2014-02-01

    The adult heart responds to contraction demands by hypertrophy, or enlargement, of cardiac myocytes. Adaptive hypertrophy can occur in response to hyperoxic conditions such as exercise, while pathological factors that result in hypoxia ultimately result in heart failure. The difference in the outcomes produced by pathologically versus physiologically induced hypertrophy suggests that the cellular signaling pathways or conditions of myocytes may be different at the cellular level. The structural and functional changes in myocytes resulting from hyperoxia (simulated using hydrogen peroxide) and hypoxia (using oxygen deprivation) were tested on fetal chick cardiac myocytes grown in vitro. Structural changes were measured using immunostaining for α-sarcomeric actin or MyoD, while functional changes were assessed using immunostaining for calcium/calmodulin-dependent kinase (CaMKII) and by measuring intracellular calcium fluxes using live cell fluorescence imaging. Both hypoxic and hyperoxic stress resulted in an upregulation of actin and MyoD expression. Similarly, voltage-gated channels governing myocyte depolarization and the regulation of CaMK were unchanged by hyperoxic or hypoxic conditions. However, the dynamic features of calcium fluxes elicited by caffeine or epinephrine were different in cells subjected to hypoxia versus hyperoxia, suggesting that these different conditions differentially affect components of ligand-activated signaling pathways that regulate calcium. Our results suggest that changes in signaling pathways, rather than structural organization, may mediate the different outcomes associated with hyperoxia-induced versus hypoxia-induced hypertrophy, and these changes are likely initiated at the cellular level.

  5. The long noncoding RNA landscape in hypoxic and inflammatory renal epithelial injury

    PubMed Central

    Zhang, Xuan; Xue, Chenyi; Zhang, Hanrui; Shashaty, Michael G. S.; Gosai, Sager J.; Meyer, Nuala; Grazioli, Alison; Hinkle, Christine; Caughey, Jennifer; Li, Wenjun; Susztak, Katalin; Gregory, Brian D.; Li, Mingyao; Reilly, Muredach P.

    2015-01-01

    Long noncoding RNAs (lncRNAs) are emerging as key species-specific regulators of cellular and disease processes. To identify potential lncRNAs relevant to acute and chronic renal epithelial injury, we performed unbiased whole transcriptome profiling of human proximal tubular epithelial cells (PTECs) in hypoxic and inflammatory conditions. RNA sequencing revealed that the protein-coding and noncoding transcriptomic landscape differed between hypoxia-stimulated and cytokine-stimulated human PTECs. Hypoxia- and inflammation-modulated lncRNAs were prioritized for focused followup according to their degree of induction by these stress stimuli, their expression in human kidney tissue, and whether exposure of human PTECs to plasma of critically ill sepsis patients with acute kidney injury modulated their expression. For three lncRNAs (MIR210HG, linc-ATP13A4-8, and linc-KIAA1737-2) that fulfilled our criteria, we validated their expression patterns, examined their loci for conservation and synteny, and defined their associated epigenetic marks. The lncRNA landscape characterized here provides insights into novel transcriptomic variations in the renal epithelial cell response to hypoxic and inflammatory stress. PMID:26400545

  6. Hypoxic induction of caspase-11/caspase-1/interleukin-1beta in brain microglia.

    PubMed

    Kim, Nam-Gon; Lee, Heasuk; Son, Eunyung; Kwon, Oh-Young; Park, Jae-Yong; Park, Jae-Hoon; Cho, Gyeong Jae; Choi, Wan Sung; Suk, Kyoungho

    2003-06-10

    Caspase-11 is an inducible protease that plays an important role in both inflammation and apoptosis. Inflammatory stimuli induce and activate caspase-11, which is required for the activation of caspase-1 or interleukin-1beta (IL-1beta) converting enzyme (ICE). Caspase-1 in turn mediates the maturation of proinflammatory cytokines such as IL-1beta, which is one of the crucial mediators of neurodegeneration in the central nervous system. Here, we report that hypoxic exposure of cultured brain microglia (BV-2 mouse microglia cells and rat primary microglial cultures) induces expression and activation of caspase-11, which is accompanied by activation of caspase-1 and secretion of mature IL-1beta and IL-18. Hypoxic induction of caspase-11 was observed in both mRNA and protein levels, and was mediated through p38 mitogen-activated protein kinase pathway. Transient global ischemia in rats also induced caspase-11 expression and IL-1beta production in hippocampus supporting our in vitro findings. Caspase-11-expressing cells in hippocampus were morphologically identified as microglia. Taken together, our results indicate that hypoxia induces a sequential event-caspase-11 induction, caspase-1 activation, and IL-1beta release-in brain microglia, and point out the importance of initial caspase-11 induction in hypoxia-induced inflammatory activation of microglia.

  7. Metabolism of exogenously applied fructose 1,6-bisphosphate in hypoxic vascular smooth muscle.

    PubMed

    Hardin, C D; Roberts, T M

    1994-12-01

    Exogenously administered fructose 1,6-bisphosphate reportedly protects ischemic or hypoxic tissue and facilitates metabolic recovery. The mechanism of action of exogenous fructose 1,6-bisphosphate has been an issue of considerable debate, since there is a lack of direct evidence that fructose 1,6-bisphosphate can cross the cell membrane and act as an intermediate in glycolysis. We synthesized [1,6-13C]fructose 1,6-bisphosphate and directly examined its cellular metabolism in hog carotid artery segments using 13C-nuclear magnetic resonance (NMR) spectroscopy. [1,6-13C]fructose 1,6-bisphosphate (2.1 mM) was metabolized by hog carotid artery during normoxia and hypoxia with a major metabolic product being [3-13C]lactate. The production of [3-13C]lactate was greater during hypoxia than during normoxia, indicating that fructose 1,6-bisphosphate metabolism responded to the energetic state of the tissue. We found that exogenously added fructose 1,6-bisphosphate at 2.1 mM did not significantly improve the ability of hypoxic hog carotid artery to maintain isometric force, whereas 20 mM fructose 1,6-bisphosphate did significantly, although modestly, improve isometric force maintenance. These results indicate that exogenously added fructose 1,6-bisphosphate is capable of entering cells and serving as a glycolytic intermediate.

  8. Role of Antioxidants in Neonatal Hypoxic-Ischemic Brain Injury: New Therapeutic Approaches.

    PubMed

    Arteaga, Olatz; Álvarez, Antonia; Revuelta, Miren; Santaolalla, Francisco; Urtasun, Andoni; Hilario, Enrique

    2017-01-28

    Hypoxic-ischemic brain damage is an alarming health and economic problem in spite of the advances in neonatal care. It can cause mortality or detrimental neurological disorders such as cerebral palsy, motor impairment and cognitive deficits in neonates. When hypoxia-ischemia occurs, a multi-faceted cascade of events starts out, which can eventually cause cell death. Lower levels of oxygen due to reduced blood supply increase the production of reactive oxygen species, which leads to oxidative stress, a higher concentration of free cytosolic calcium and impaired mitochondrial function, triggering the activation of apoptotic pathways, DNA fragmentation and cell death. The high incidence of this type of lesion in newborns can be partly attributed to the fact that the developing brain is particularly vulnerable to oxidative stress. Since antioxidants can safely interact with free radicals and terminate that chain reaction before vital molecules are damaged, exogenous antioxidant therapy may have the potential to diminish cellular damage caused by hypoxia-ischemia. In this review, we focus on the neuroprotective effects of antioxidant treatments against perinatal hypoxic-ischemic brain injury, in the light of the most recent advances.

  9. Proof of Concept to Isolate and Culture Primary Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-hold Diving

    DTIC Science & Technology

    2013-09-30

    To isolate and culture primary muscle cells from the swimming muscles of northern elephant seals. OBJECTIVES To develop a protocol for the...Muscle sampling Biopsy samples of approximately 50 mg will be collected with a 6-mm biopsy cannula (Depuy, Warsaw, Indiana) from the swimming (M... swimming muscles of northern elephant seals. Select myoblasts are identified in each image by red arrows. The myoblasts can be distinguished from

  10. HDAC6 maintains mitochondrial connectivity under hypoxic stress by suppressing MARCH5/MITOL dependent MFN2 degradation

    SciTech Connect

    Kim, Hak-June; Nagano, Yoshito; Choi, Su Jin; Park, Song Yi; Kim, Hongtae; Yao, Tso-Pang; Lee, Joo-Yong

    2015-09-04

    Mitochondria undergo fusion and fission in response to various metabolic stresses. Growing evidences have suggested that the morphological change of mitochondria by fusion and fission plays a critical role in protecting mitochondria from metabolic stresses. Here, we showed that hypoxia treatment could induce interaction between HDAC6 and MFN2, thus protecting mitochondrial connectivity. Mechanistically, we demonstrated that a mitochondrial ubiquitin ligase MARCH5/MITOL was responsible for hypoxia-induced MFN2 degradation in HDAC6 deficient cells. Notably, genetic abolition of HDAC6 in amyotrophic lateral sclerosis model mice showed MFN2 degradation with MARCH5 induction. Our results indicate that HDAC6 is a critical regulator of MFN2 degradation by MARCH5, thus protecting mitochondrial connectivity from hypoxic stress. - Highlights: • Hypoxic stress induces the interaction between HDAC6 and MFN2. • Hypoxic stress activates MARCH5 in HDAC6 deficient cells to degrade MFN2. • HDAC6 is required to maintain mitochondrial connectivity under hypoxia. • MARCH5 is increased and promotes the degradation of MFN2 in HDAC6 KO ALS mice.

  11. AMP-activated Protein Kinase Deficiency Blocks the Hypoxic Ventilatory Response and Thus Precipitates Hypoventilation and Apnea

    PubMed Central

    Mahmoud, Amira D.; Lewis, Sophronia; Juričić, Lara; Udoh, Utibe-Abasi; Hartmann, Sandy; Jansen, Maurits A.; Ogunbayo, Oluseye A.; Puggioni, Paolo; Holmes, Andrew P.; Kumar, Prem; Navarro-Dorado, Jorge; Foretz, Marc; Viollet, Benoit; Dutia, Mayank B.; Marshall, Ian

    2016-01-01

    Rationale: Modulation of breathing by hypoxia accommodates variations in oxygen demand and supply during, for example, sleep and ascent to altitude, but the precise molecular mechanisms of this phenomenon remain controversial. Among the genes influenced by natural selection in high-altitude populations is one for the adenosine monophosphate–activated protein kinase (AMPK) α1-catalytic subunit, which governs cell-autonomous adaptations during metabolic stress. Objectives: We investigated whether AMPK-α1 and/or AMPK-α2 are required for the hypoxic ventilatory response and the mechanism of ventilatory dysfunctions arising from AMPK deficiency. Methods: We used plethysmography, electrophysiology, functional magnetic resonance imaging, and immediate early gene (c-fos) expression to assess the hypoxic ventilatory response of mice with conditional deletion of the AMPK-α1 and/or AMPK-α2 genes in catecholaminergic cells, which compose the hypoxia-responsive respiratory network from carotid body to brainstem. Measurements and Main Results: AMPK-α1 and AMPK-α2 deletion virtually abolished the hypoxic ventilatory response, and ventilatory depression during hypoxia was exacerbated under anesthesia. Rather than hyperventilating, mice lacking AMPK-α1 and AMPK-α2 exhibited hypoventilation and apnea during hypoxia, with the primary precipitant being loss of AMPK-α1 expression. However, the carotid bodies of AMPK-knockout mice remained exquisitely sensitive to hypoxia, contrary to the view that the hypoxic ventilatory response is determined solely by increased carotid body afferent input to the brainstem. Regardless, functional magnetic resonance imaging and c-fos expression revealed reduced activation by hypoxia of well-defined dorsal and ventral brainstem nuclei. Conclusions: AMPK is required to coordinate the activation by hypoxia of brainstem respiratory networks, and deficiencies in AMPK expression precipitate hypoventilation and apnea, even when carotid body

  12. Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

    PubMed Central

    Wilde, Aimee D.; Snyder, Daniel J.; Putnam, Nicole E.; Valentino, Michael D.; Hammer, Neal D.; Lonergan, Zachery R.; Hinger, Scott A.; Aysanoa, Esar E.; Blanchard, Catlyn; Dunman, Paul M.; Wasserman, Gregory A.; Chen, John; Shopsin, Bo; Gilmore, Michael S.; Skaar, Eric P.; Cassat, James E.

    2015-01-01

    Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction. PMID:26684646

  13. Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide.

    PubMed

    Hemschemeier, Anja; Düner, Melis; Casero, David; Merchant, Sabeeha S; Winkler, Martin; Happe, Thomas

    2013-06-25

    Hemoglobins are recognized today as a diverse family of proteins present in all kingdoms of life and performing multiple reactions beyond O2 chemistry. The physiological roles of most hemoglobins remain elusive. Here, we show that a 2-on-2 ("truncated") hemoglobin, termed THB8, is required for hypoxic growth and the expression of anaerobic genes in Chlamydomonas reinhardtii. THB8 is 1 of 12 2-on-2 hemoglobins in this species. It belongs to a subclass within the 2-on-2 hemoglobin class I family whose members feature a remarkable variety of domain arrangements and lengths. Posttranscriptional silencing of the THB8 gene results in the mis-regulation of several genes and a growth defect under hypoxic conditions. The latter is intensified in the presence of an NO scavenger, which also impairs growth of wild-type cells. As recombinant THB8 furthermore reacts with NO, the results of this study indicate that THB8 is part of an NO-dependent signaling pathway.

  14. Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide

    PubMed Central

    Hemschemeier, Anja; Düner, Melis; Casero, David; Merchant, Sabeeha S.; Winkler, Martin; Happe, Thomas

    2013-01-01

    Hemoglobins are recognized today as a diverse family of proteins present in all kingdoms of life and performing multiple reactions beyond O2 chemistry. The physiological roles of most hemoglobins remain elusive. Here, we show that a 2-on-2 (“truncated”) hemoglobin, termed THB8, is required for hypoxic growth and the expression of anaerobic genes in Chlamydomonas reinhardtii. THB8 is 1 of 12 2-on-2 hemoglobins in this species. It belongs to a subclass within the 2-on-2 hemoglobin class I family whose members feature a remarkable variety of domain arrangements and lengths. Posttranscriptional silencing of the THB8 gene results in the mis-regulation of several genes and a growth defect under hypoxic conditions. The latter is intensified in the presence of an NO scavenger, which also impairs growth of wild-type cells. As recombinant THB8 furthermore reacts with NO, the results of this study indicate that THB8 is part of an NO-dependent signaling pathway. PMID:23754374

  15. Mechanisms and consequences of ATMIN repression in hypoxic conditions: roles for p53 and HIF-1

    PubMed Central

    Leszczynska, Katarzyna B.; Göttgens, Eva-Leonne; Biasoli, Deborah; Olcina, Monica M.; Ient, Jonathan; Anbalagan, Selvakumar; Bernhardt, Stephan; Giaccia, Amato J.; Hammond, Ester M.

    2016-01-01

    Hypoxia-induced replication stress is one of the most physiologically relevant signals known to activate ATM in tumors. Recently, the ATM interactor (ATMIN) was identified as critical for replication stress-induced activation of ATM in response to aphidicolin and hydroxyurea. This suggests an essential role for ATMIN in ATM regulation during hypoxia, which induces replication stress. However, ATMIN also has a role in base excision repair, a process that has been demonstrated to be repressed and less efficient in hypoxic conditions. Here, we demonstrate that ATMIN is dispensable for ATM activation in hypoxia and in contrast to ATM, does not affect cell survival and radiosensitivity in hypoxia. Instead, we show that in hypoxic conditions ATMIN expression is repressed. Repression of ATMIN in hypoxia is mediated by both p53 and HIF-1α in an oxygen dependent manner. The biological consequence of ATMIN repression in hypoxia is decreased expression of the target gene, DYNLL1. An expression signature associated with p53 activity was negatively correlated with DYNLL1 expression in patient samples further supporting the p53 dependent repression of DYNLL1. Together, these data demonstrate multiple mechanisms of ATMIN repression in hypoxia with consequences including impaired BER and down regulation of the ATMIN transcriptional target, DYNLL1. PMID:26875667

  16. The role of inflammation in hypoxic pulmonary hypertension: from cellular mechanisms to clinical phenotypes

    PubMed Central

    Poth, Jens M.; Fini, Mehdi A.; Olschewski, Andrea; El Kasmi, Karim C.; Stenmark, Kurt R.

    2014-01-01

    Hypoxic pulmonary hypertension (PH) comprises a heterogeneous group of diseases sharing the common feature of chronic hypoxia-induced pulmonary vascular remodeling. The disease is usually characterized by mild to moderate pulmonary vascular remodeling that is largely thought to be reversible compared with the progressive irreversible disease seen in World Health Organization (WHO) group I disease. However, in these patients, the presence of PH significantly worsens morbidity and mortality. In addition, a small subset of patients with hypoxic PH develop “out-of-proportion” severe pulmonary hypertension characterized by pulmonary vascular remodeling that is irreversible and similar to that in WHO group I disease. In all cases of hypoxia-related vascular remodeling and PH, inflammation, particularly persistent inflammation, is thought to play a role. This review focuses on the effects of hypoxia on pulmonary vascular cells and the signaling pathways involved in the initiation and perpetuation of vascular inflammation, especially as they relate to vascular remodeling and transition to chronic irreversible PH. We hypothesize that the combination of hypoxia and local tissue factors/cytokines (“second hit”) antagonizes tissue homeostatic cellular interactions between mesenchymal cells (fibroblasts and/or smooth muscle cells) and macrophages and arrests these cells in an epigenetically locked and permanently activated proremodeling and proinflammatory phenotype. This aberrant cellular cross-talk between mesenchymal cells and macrophages promotes transition to chronic nonresolving inflammation and vascular remodeling, perpetuating PH. A better understanding of these signaling pathways may lead to the development of specific therapeutic targets, as none are currently available for WHO group III disease. PMID:25416383

  17. Popularity of hypoxic training methods for endurance-based professional and amateur athletes.

    PubMed

    Álvarez-Herms, J; Julià-Sánchez, S; Hamlin, M J; Corbi, F; Pagès, T; Viscor, G

    2015-05-01

    Scientific debate continues into whether hypoxic training has any performance benefit for athletes, and although this type of training seems popular, to our knowledge little empirical evidence on its popularity with endurance-based athletes exists. To quantify the usage of hypoxic training in endurance-based athletes we asked 203 athletes (amateur = 108, professional = 95) to complete a 17-question survey during 2013-2014 season. Compared to amateurs, professional athletes were 4.5 times (3.0-6.8, odds ratio, 95% confidence limits) more likely to undertake hypoxic training. Live-high train-low was the most popular hypoxic training protocol for athletes (52% professional and 80% amateur) with live-high train-high also used (38% professional, 20% amateur). Compared to amateurs, professional athletes tended to use evidence-based hypoxic training methods, seek advice on hypoxic training from reliable sources and were generally more realistic about the potential performance gains as a result of hypoxic training. Almost one third (25-30%) of all athletes suffered illness during their hypoxic training. Compared to amateurs, professional athletes are more likely to undertake hypoxic training and tend to follow current scientific guidelines. Attenuation of the ill effects that occur during hypoxic training may be accomplished if athletes give more attention to monitoring stress and training levels.

  18. Regulation of HIF-1α signaling and chemoresistance in acute lymphocytic leukemia under hypoxic conditions of the bone marrow microenvironment

    PubMed Central

    Frolova, Olga; Samudio, Ismael; Benito, Juliana Maria; Jacamo, Rodrigo; Kornblau, Steven M.; Markovic, Ana; Schober, Wendy; Lu, Hongbo; Qiu, Yi Hua; Buglio, Daniela; McQueen, Teresa; Pierce, Sherry; Shpall, Elizabeth; Konoplev, Sergej; Thomas, Deborah; Kantarjian, Hagop; Lock, Richard; Andreeff, Michael; Konopleva, Marina

    2012-01-01

    Overcoming resistance to chemotherapy is the main therapeutic challenge in the treatment of acute lymphocytic leukemia (ALL). Interactions between leukemia cells and the microenvironment promote leukemia cell survival and confer resistance to chemotherapy. Hypoxia is an integral component of bone marrow (BM) microenvironment. Hypoxia-inducible factor-1α (HIF-1), a key regulator of the cellular response to hypoxia, regulates cell growth and metabolic adaptation to hypoxia. HIF-1α expression, analyzed by Reverse Phase Protein Arrays in 92 specimens from newly diagnosed patients with pre-B-ALL, had a negative prognostic impact on survival (p = 0.0025). Inhibition of HIF-1α expression by locked mRNA antagonist (LNA) promoted chemosensitivity under hypoxic conditions, while pharmacological or genetic stabilization of HIF-1α under normoxia inhibited cell growth and reduced apoptosis induction by chemotherapeutic agents. Co-culture of pre-B ALL or REH cells with BM-derived mesenchymal stem cells (MSC) under hypoxia resulted in further induction of HIF-1α protein and acquisition of the glycolytic phenotype, in part via stroma-induced AKT/mTOR signaling. mTOR blockade with everolimus reduced HIF-1α expression, diminished glucose uptake and glycolytic rate and partially restored the chemosensitivity of ALL cells under hypoxia/stroma co-cultures. Hence, mTOR inhibition or blockade of HIF-1α-mediated signaling may play an important role in chemosensitization of ALL cells under hypoxic conditions of the BM microenvironment. PMID:22785211

  19. Magnetic resonance imaging of hypoxic injury to the murine placenta.

    PubMed

    Tomlinson, Tracy M; Garbow, Joel R; Anderson, Jeff R; Engelbach, John A; Nelson, D Michael; Sadovsky, Yoel

    2010-02-01

    We assessed the use of magnetic resonance imaging (MRI) to define placental hypoxic injury associated with fetal growth restriction. On embryonic day 18.5 (E18.5) we utilized dynamic contrast-enhanced (DCE)-MRI on a 4.7-tesla small animal scanner to examine the uptake and distribution of gadolinium-based contrast agent. Quantitative DCE parameter analysis was performed for the placenta and fetal kidneys of three groups of pregnant C57BL/6 mice: 1) mice that were exposed to Fi(O(2)) = 12% between E15.5 and E18.5, 2) mice in normoxia with food restriction similar to the intake of hypoxic mice between E15.5 and E18.5, and 3) mice in normoxia that were fed ad libitum. After imaging, we assessed fetoplacental weight, placental histology, and gene expression. We found that dams exposed to hypoxia exhibited fetal growth restriction (weight reduction by 28% and 14%, respectively, P < 0.05) with an increased placental-to-fetal ratio. By using MRI-based assessment of placental contrast agent kinetics, referenced to maternal paraspinous muscle, we found decreased placental clearance of contrast media in hypoxic mice, compared with either control group (61%, P < 0.05). This was accompanied by diminished contrast accumulation in the hypoxic fetal kidneys (23%, P < 0.05), reflecting reduced transplacental gadolinium transport. These changes were associated with increased expression of placental Phlda2 and Gcm1 transcripts. Exposure to hypoxia near the end of mouse pregnancy reduces placental perfusion and clearance of contrast. MRI-based DCE imaging provides a novel tool for dynamic, in vivo assessment of placental function.

  20. Hypoxic cutaneous vasodilation is sustained during brief cold stress and is not affected by changes in CO2.

    PubMed

    Simmons, Grant H; Fieger, Sarah M; Minson, Christopher T; Halliwill, John R

    2010-04-01

    Hypoxia decreases core body temperature in animals and humans during cold exposure. In addition, hypoxia increases skin blood flow in thermoneutral conditions, but the impact of hypoxic vasodilation on vasoconstriction during cold exposure is unknown. In this study, skin blood flow was assessed using laser-Doppler flowmetry, and cutaneous vascular conductance (CVC) was calculated as red blood cell flux/mean arterial pressure and normalized to baseline (n = 7). Subjects were exposed to four different conditions in the steady state (normoxia and poikilocapnic, isocapnic, and hypercapnic hypoxia) and were cooled for 10 min using a water-perfused suit in each condition. CVC increased during all three hypoxic exposures (all P < 0.05 vs. baseline), and the magnitude of these steady-state responses was not affected by changes in end-tidal CO(2) levels. During poikilocapnic and hypercapnic hypoxia, cold exposure reduced CVC to the same levels observed during normoxic cooling (P > 0.05 vs. normoxia), whereas CVC remained elevated throughout cold exposure during isocapnic hypoxia (P < 0.05 vs. normoxia). The magnitude of vasoconstriction during cold stress was similar in all conditions (P > 0.05). Thus the magnitude of cutaneous vasodilation during steady-state hypoxia is not affected by CO(2) responses. In addition, the magnitude of reflex vasoconstriction is not altered by hypoxia, such that the upward shift in skin blood flow (hypoxic vasodilation) is maintained during whole body cooling.

  1. Hypoxic repression of CYP7A1 through a HIF-1α- and SHP-independent mechanism

    PubMed Central

    Moon, Yunwon; Park, Bongju; Park, Hyunsung

    2016-01-01

    Liver cells experience hypoxic stress when drug-metabolizing enzymes excessively consume O2 for hydroxylation. Hypoxic stress changes the transcription of several genes by activating a heterodimeric transcription factor called hypoxia-inducible factor-1α/β (HIF-1α/β). We found that hypoxic stress (0.1% O2) decreased the expression of cytochrome P450 7A1 (CYP7A1), a rate-limiting enzyme involved in bile acid biosynthesis. Chenodeoxycholic acid (CDCA), a major component of bile acids, represses CYP7A1 by activating a transcriptional repressor named small heterodimer partner (SHP). We observed that hypoxia decreased the levels of both CDCA and SHP, suggesting that hypoxia repressed CYP7A1 without inducing SHP. The finding that overexpression of HIF-1α increased the activity of the CYP7A1 promoter suggested that hypoxia decreased the expression of CYP7A1 in a HIF-1-independent manner. Thus, the results of this study suggested that hypoxia decreased the activity of CYP7A1 by limiting its substrate O2, and by decreasing the transcription of CYP7A1. [BMB Reports 2016; 49(3): 173-178] PMID:26521940

  2. Hypoxic regulation of lactate dehydrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements.

    PubMed

    Firth, J D; Ebert, B L; Ratcliffe, P J

    1995-09-08

    The oxygen-regulated control system responsible for the induction of erythropoietin (Epo) by hypoxia is present in most (if not all) cells and operates on other genes, including those involved in energy metabolism. To understand the organization of cis-acting sequences that are responsible for oxygen-regulated gene expression, we have studied the 5' flanking region of the mouse gene encoding the hypoxically inducible enzyme lactate dehydrogenase A (LDH). Deletional and mutational analysis of the function of mouse LDH-reporter fusion gene constructs in transient transfection assays defined three domains, between -41 and -84 base pairs upstream of the transcription initiation site, which were crucial for oxygen-regulated expression. The most important of these, although not capable of driving hypoxic induction in isolation, had the consensus of a hypoxia-inducible factor 1 (HIF-1) site, and cross-competed for the binding of HIF-1 with functionally active Epo and phosphoglycerate kinase-1 sequences. The second domain was positioned close to the HIF-1 site, in an analogous position to one of the critical regions in the Epo 3' hypoxic enhancer. The third domain had the motif of a cAMP response element (CRE). Activation of cAMP by forskolin had no effect on the level of LDH mRNA in normoxia, but produced a magnified response to hypoxia that was dependent upon the integrity of the CRE, indicating an interaction between inducible factors binding the HIF-1 and CRE sites.

  3. Hypoxic repression of CYP7A1 through a HIF-1α- and SHP-independent mechanism.

    PubMed

    Moon, Yunwon; Park, Bongju; Park, Hyunsung

    2016-03-01

    Liver cells experience hypoxic stress when drug-metabolizing enzymes excessively consume O2 for hydroxylation. Hypoxic stress changes the transcription of several genes by activating a heterodimeric transcription factor called hypoxia-inducible factor- 1α/β (HIF-1α/β). We found that hypoxic stress (0.1% O2) decreased the expression of cytochrome P450 7A1 (CYP7A1), a rate-limiting enzyme involved in bile acid biosynthesis. Chenodeoxycholic acid (CDCA), a major component of bile acids, represses CYP7A1 by activating a transcriptional repressor named small heterodimer partner (SHP). We observed that hypoxia decreased the levels of both CDCA and SHP, suggesting that hypoxia repressed CYP7A1 without inducing SHP. The finding that overexpression of HIF-1α increased the activity of the CYP7A1 promoter suggested that hypoxia decreased the expression of CYP7A1 in a HIF-1-independent manner. Thus, the results of this study suggested that hypoxia decreased the activity of CYP7A1 by limiting its substrate O2, and by decreasing the transcription of CYP7A1. [BMB Reports 2016; 49(3): 173-178].

  4. Attenuating Ischemic Disruption of K(+) Homeostasis in the Cortex of Hypoxic-Ischemic Neonatal Rats: DOR Activation vs. Acupuncture Treatment.

    PubMed

    Chao, Dongman; Wang, Qinyu; Balboni, Gianfranco; Ding, Guanghong; Xia, Ying

    2016-12-01

    Perinatal hypoxic-ischemic (HI) brain injury results in death or profound long-term neurologic disability in both children and adults. However, there is no effective pharmacological therapy due to a poor understanding of HI events, especially the initial triggers for hypoxic-ischemic injury such as disrupted ionic homeostasis and the lack of effective intervention strategy. In the present study, we showed that neonatal brains undergo a developmental increase in the disruption of K(+) homeostasis during simulated ischemia, oxygen-glucose deprivation (OGD) and neonatal HI cortex has a triple phasic response (earlier attenuation, later enhancement, and then recovery) of disrupted K(+) homeostasis to OGD. This response partially involves the activity of the δ-opioid receptor (DOR) since the earlier attenuation of ischemic disruption of K(+) homeostasis could be blocked by DOR antagonism, while the later enhancement was reversed by DOR activation. Similar to DOR activation, acupuncture, a strategy to promote DOR activity, could partially reverse the later enhanced ischemic disruption of K(+) homeostasis in the neonatal cortex. Since maintaining cellular K(+) homeostasis and inhibiting excessive K(+) fluxes in the early phase of hypoxic-ischemic insults may be of therapeutic benefit in the treatment of ischemic brain injury and related neurodegenerative conditions, and since many neurons and other cells can be rescued during the "window of opportunity" after HI insults, our first findings regarding the role of acupuncture and DOR in attenuating ischemic disruption of K(+) homeostasis in the neonatal HI brain suggest a potential intervention therapy in the treatment of neonatal brain injury, especially hypoxic-ischemic encephalopathy.

  5. Combined radiation-protective and radiation-sensitizing agents. II. Radiosensitivity of hypoxic or aerobic Chinese hamster fibroblasts in the presence of cysteamine and misonidazole: implications for the oxygen effect (with Appendix on calculation of dose-modifying factors. [/sup 60/Co

    SciTech Connect

    Koch, C.J.; Howell, R.L.

    1981-08-01

    Experiments have been done to test whether a hypoxic cell radiosensitizing agent (misonidazole) can be combined with a radioprotecting agent (cysteamine) to equalize partially the radiation response of hypoxic and aerobic mammalian cells in tissue culture. The results indicate that cysteamine will protect against the radiosensitization of a hypoxic cell sensitizing drug (2.5 mM misonidazole) at much lower concentration than it will protect against the radiosensitization of oxygen (350 ..mu..M). Thus the addition of a radiation-protective drug tends to cancel the drug benefit of the radiosensitizer and therefore increases the differential response of hypoxic and aerobic cells rather than equalizing this response. The data suggest that even in situations where tumor tissue absorbs far less radioprotective drug than normal tissue (e.g., WR 2721), one might expect difficulties with the simultaneous administration of radiosensitizing and radioprotecting drugs.

  6. Mitochondrial Akt Regulation of Hypoxic Tumor Reprogramming.

    PubMed

    Chae, Young Chan; Vaira, Valentina; Caino, M Cecilia; Tang, Hsin-Yao; Seo, Jae Ho; Kossenkov, Andrew V; Ottobrini, Luisa; Martelli, Cristina; Lucignani, Giovanni; Bertolini, Irene; Locatelli, Marco; Bryant, Kelly G; Ghosh, Jagadish C; Lisanti, Sofia; Ku, Bonsu; Bosari, Silvano; Languino, Lucia R; Speicher, David W; Altieri, Dario C

    2016-08-08

    Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism toward glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an "actionable" therapeutic target in cancer.

  7. Flows and hypoxic blackwater events in managed ephemeral river channels

    NASA Astrophysics Data System (ADS)

    Hladyz, Sally; Watkins, Susanne C.; Whitworth, Kerry L.; Baldwin, Darren S.

    2011-04-01

    SummaryAs pressure increases on the availability of water resources worldwide, especially in the face of climatic change, it is probable that the likelihood of streams undergoing at least some periods of drying will increase in arid and semi-arid regions. This has implications for the ongoing management of waterways in these areas. One area of concern is the potential occurrence of hypoxic blackwater events upon re-instatement of flows in creek and river channels following periods of drying. Hypoxic blackwater events are characterised by high levels of dissolved organic carbon (DOC), the metabolism of which results in low dissolved oxygen (DO) in the water column, which can cause fish and crustacean mortality. Therefore, understanding hypoxic blackwater events is important in order to reduce the potential for fish mortalities and other water quality impacts from both managed and natural flows. In this study, we set out to determine the factors that influenced the occurrence of a hypoxic blackwater event in the Edward-Wakool river system, in southern NSW, Australia during the previous austral summer (2008-2009). Standing stocks of plant litter, emergent macrophytes and river red gum saplings ( Eucalyptus camaldulensis Dehn.), as well as rates of litterfall, were determined in dry and inundated channels. A series of mesocosm experiments were undertaken to determine which carbon source was the greatest contributor to DOC and to DO depletion, and what loadings could result in hypoxia. These experiments were then used to create a simple algorithm relating carbon loading in a dry channel to DOC in the overlying water column following inundation. Results revealed that plant litter was the main contributor to water column DOC and to DO depletion. Litter loadings equal to or greater than 370 g m -2 were found to cause DO in a shallow (20 cm) water column at 20 °C to fall to zero within two days. This loading was approximately half of that found in dry channels in the

  8. [Biochemical and pharmacological mechanisms of different types of hypoxic preconditioning in cerebral ischemia in mice].

    PubMed

    Kulinskiĭ, V I; Gavrilina, T V; Minakina, L N; Kovtun, V Iu

    2006-01-01

    Different types of hypoxic preconditioning (hypoxic, circulatory, hemic and tissue hypoxia) increase the tolerance to complete global cerebral ischemia at early terms (hours). Biochemico-pharmacological analysis with the use of selective agonists and antagonists showed the importance of adenosine A1-receptors and K+(ATP)-channels in the mechanisms of the neuroprotective effect and natural tolerance. The general scheme of the investigated mechanisms of different types of hypoxic preconditioning has been proposed.

  9. Bax inhibiting peptide reduces apoptosis in neonatal rat hypoxic-ischemic brain damage

    PubMed Central

    Sun, Meng-Ya; Cui, Kai-Jie; Yu, Mao-Min; Zhang, Hui; Peng, Xiang-Li; Jiang, Hong

    2015-01-01

    Neonatal hypoxic ischemic encephalopathy (HIE) has been reported to induce apoptosis in neonates. We, therefore, analyzed the ability of Bax-inhibiting peptide (BIP) to provide neuroprotective effects during hypoxic-ischemic brain damage (HIBD). Seven-day-old wistar rat pups (n = 198) were randomly divided into a sham-operated group (Group S, n = 18), saline group (Group C, n = 90) and BIP group (Group B, n = 90). Pathological changes in the cerebral tissues of rat pups were analyzed using hematoxylin and eosin stain, TUNEL and Western blot. The expression of cytochrome c and caspase-3 was determined using western blot technique. Rat pups demonstrated neurobehavioral alteration in Groups C and B. TUNEL-positive cells in the left hippocampus were significantly increased in Group C and Group B after HIBD (P < 0.01) when compared with Group S. There was a marked reduction in TUNEL positive cells in subgroups B1 through B4 when compared with the respective subgroups C1 through C5. Compared with Group S, the expression of caspase-3 and cytochrome c was significantly increased in Groups C and B (P < 0.01). The difference in expression of caspase-3 and cytochrome c between subgroups B1 through B4 and C1 through C4 was significant (P < 0.01). In conclusions, the neuro-protective effect of BIP was due to a reduction of nerve cell apoptosis in our neonatal HIE rat model. We propose that BIP has potential as a neuro-protective drug in neonatal HIE cases. PMID:26823794

  10. Monte-Carlo model development for evaluation of current clinical target volume definition for heterogeneous and hypoxic glioblastoma.

    PubMed

    Moghaddasi, L; Bezak, E; Harriss-Phillips, W

    2016-05-07

    Clinical target volume (CTV) determination may be complex and subjective. In this work a microscopic-scale tumour model was developed to evaluate current CTV practices in glioblastoma multiforme (GBM) external radiotherapy. Previously, a Geant4 cell-based dosimetry model was developed to calculate the dose deposited in individual GBM cells. Microscopic extension probability (MEP) models were then developed using Matlab-2012a. The results of the cell-based dosimetry model and MEP models were combined to calculate survival fractions (SF) for CTV margins of 2.0 and 2.5 cm. In the current work, oxygenation and heterogeneous radiosensitivity profiles were incorporated into the GBM model. The genetic heterogeneity was modelled using a range of α/β values (linear-quadratic model parameters) associated with different GBM cell lines. These values were distributed among the cells randomly, taken from a Gaussian-weighted sample of α/β values. Cellular oxygen pressure was distributed randomly taken from a sample weighted to profiles obtained from literature. Three types of GBM models were analysed: homogeneous-normoxic, heterogeneous-normoxic, and heterogeneous-hypoxic. The SF in different regions of the tumour model and the effect of the CTV margin extension from 2.0-2.5 cm on SFs were investigated for three MEP models. The SF within the beam was increased by up to three and two orders of magnitude following incorporation of heterogeneous radiosensitivities and hypoxia, respectively, in the GBM model. However, the total SF was shown to be overdominated by the presence of tumour cells in the penumbra region and to a lesser extent by genetic heterogeneity and hypoxia. CTV extension by 0.5 cm reduced the SF by a maximum of 78.6  ±  3.3%, 78.5  ±  3.3%, and 77.7  ±  3.1% for homogeneous and heterogeneous-normoxic, and heterogeneous hypoxic GBMs, respectively. Monte-Carlo model was developed to quantitatively evaluate SF for genetically

  11. Monte-Carlo model development for evaluation of current clinical target volume definition for heterogeneous and hypoxic glioblastoma

    NASA Astrophysics Data System (ADS)

    Moghaddasi, L.; Bezak, E.; Harriss-Phillips, W.

    2016-05-01

    Clinical target volume (CTV) determination may be complex and subjective. In this work a microscopic-scale tumour model was developed to evaluate current CTV practices in glioblastoma multiforme (GBM) external radiotherapy. Previously, a Geant4 cell-based dosimetry model was developed to calculate the dose deposited in individual GBM cells. Microscopic extension probability (MEP) models were then developed using Matlab-2012a. The results of the cell-based dosimetry model and MEP models were combined to calculate survival fractions (SF) for CTV margins of 2.0 and 2.5 cm. In the current work, oxygenation and heterogeneous radiosensitivity profiles were incorporated into the GBM model. The genetic heterogeneity was modelled using a range of α/β values (linear-quadratic model parameters) associated with different GBM cell lines. These values were distributed among the cells randomly, taken from a Gaussian-weighted sample of α/β values. Cellular oxygen pressure was distributed randomly taken from a sample weighted to profiles obtained from literature. Three types of GBM models were analysed: homogeneous-normoxic, heterogeneous-normoxic, and heterogeneous-hypoxic. The SF in different regions of the tumour model and the effect of the CTV margin extension from 2.0-2.5 cm on SFs were investigated for three MEP models. The SF within the beam was increased by up to three and two orders of magnitude following incorporation of heterogeneous radiosensitivities and hypoxia, respectively, in the GBM model. However, the total SF was shown to be overdominated by the presence of tumour cells in the penumbra region and to a lesser extent by genetic heterogeneity and hypoxia. CTV extension by 0.5 cm reduced the SF by a maximum of 78.6  ±  3.3%, 78.5  ±  3.3%, and 77.7  ±  3.1% for homogeneous and heterogeneous-normoxic, and heterogeneous hypoxic GBMs, respectively. Monte-Carlo model was developed to quantitatively evaluate SF for genetically

  12. Definitive evidence for hypoxic cells influencing cure in cancer therapy.

    PubMed

    Bush, R S; Jenkin, R D; Allt, W E; Beale, F A; Bean, H; Dembo, A J; Pringle, J F

    1978-06-01

    From an analysis of 2803 patients with carcinoma of the cervix treated by radiation therapy, a 62% cure rate can be shown. In those patients with Stage IIb and III disease, a haemoglobin level during treatment of below 12 g% was associated with a significantly higher pelvic recurrence rate, and also lower cure rate, than for those with a haemoglobin level 12g% or more. A prospective study shows that the correction of anaemia is associated with a decreased pelvic recurrence rate and an increased cure rate consistent with tumour hypoxia being greater in anaemic patients than in those with a normal haemoglobin level. It is also consistent with the thesis that hypoxia controls the radiation local control rate in patients with advanced carcinoma of the cervix.

  13. Resveratrol alleviate hypoxic pulmonary hypertension via anti-inflammation and anti-oxidant pathways in rats

    PubMed Central

    Xu, Dunquan; Li, Yan; Zhang, Bo; Wang, Yanxia; Liu, Yi; Luo, Ying; Niu, Wen; Dong, Mingqing; Liu, Manling; Dong, Haiying; Zhao, Pengtao; Li, Zhichao

    2016-01-01

    Resveratrol, a plant-derived polyphenolic compound and a phytoestrogen, was shown to possess multiple protective effects including anti-inflammatory response and anti-oxidative stress. Hypoxic pulmonary hypertension (HPH) is a progressive disease characterized by sustained vascular resistance and marked pulmonary vascular remodeling. The exact mechanisms of HPH are still unclear, but inflammatory response and oxidative stress was demonstrated to participate in the progression of HPH. The present study was designed to investigate the effects of resveratrol on HPH development. Sprague-Dawley rats were challenged by hypoxia exposure for 28 days to mimic hypoxic pulmonary hypertension along with treating resveratrol (40 mg/kg/day). Hemodynamic and pulmonary pathomorphology data were then obtained, and the anti-proliferation effect of resveratrol was determined by in vitro assays. The anti-inflammation and anti-oxidative effects of resveratrol were investigated in vivo and in vitro. The present study showed that resveratrol treatment alleviated right ventricular systolic pressure and pulmonary arterial remodeling induced by hypoxia. In vitro experiments showed that resveratrol notably inhibited proliferation of pulmonary arterial smooth muscle cells in an ER-independent manner. Data showed that resveratrol administration inhibited HIF-1 α expression in vivo and in vitro, suppressed inflammatory cells infiltration around the pulmonary arteries, and decreased ROS production induced by hypoxia in PAMSCs. The inflammatory cytokines' mRNA levels of tumor necrosis factor α, interleukin 6, and interleukin 1β were all suppressed by resveratrol treatment. The in vitro assays showed that resveratrol inhibited the expression of HIF-1 α via suppressing the MAPK/ERK1 and PI3K/AKT pathways. The antioxidant axis of Nuclear factor erythroid-2 related factor 2/ Thioredoxin 1 (Nrf-2/Trx-1) was up-regulated both in lung tissues and in cultured PASMCs. In general, the current study

  14. Resveratrol alleviate hypoxic pulmonary hypertension via anti-inflammation and anti-oxidant pathways in rats.

    PubMed

    Xu, Dunquan; Li, Yan; Zhang, Bo; Wang, Yanxia; Liu, Yi; Luo, Ying; Niu, Wen; Dong, Mingqing; Liu, Manling; Dong, Haiying; Zhao, Pengtao; Li, Zhichao

    2016-01-01

    Resveratrol, a plant-derived polyphenolic compound and a phytoestrogen, was shown to possess multiple protective effects including anti-inflammatory response and anti-oxidative stress. Hypoxic pulmonary hypertension (HPH) is a progressive disease characterized by sustained vascular resistance and marked pulmonary vascular remodeling. The exact mechanisms of HPH are still unclear, but inflammatory response and oxidative stress was demonstrated to participate in the progression of HPH. The present study was designed to investigate the effects of resveratrol on HPH development. Sprague-Dawley rats were challenged by hypoxia exposure for 28 days to mimic hypoxic pulmonary hypertension along with treating resveratrol (40 mg/kg/day). Hemodynamic and pulmonary pathomorphology data were then obtained, and the anti-proliferation effect of resveratrol was determined by in vitro assays. The anti-inflammation and anti-oxidative effects of resveratrol were investigated in vivo and in vitro. The present study showed that resveratrol treatment alleviated right ventricular systolic pressure and pulmonary arterial remodeling induced by hypoxia. In vitro experiments showed that resveratrol notably inhibited proliferation of pulmonary arterial smooth muscle cells in an ER-independent manner. Data showed that resveratrol administration inhibited HIF-1 α expression in vivo and in vitro, suppressed inflammatory cells infiltration around the pulmonary arteries, and decreased ROS production induced by hypoxia in PAMSCs. The inflammatory cytokines' mRNA levels of tumor necrosis factor α, interleukin 6, and interleukin 1β were all suppressed by resveratrol treatment. The in vitro assays showed that resveratrol inhibited the expression of HIF-1 α via suppressing the MAPK/ERK1 and PI3K/AKT pathways. The antioxidant axis of Nuclear factor erythroid-2 related factor 2/ Thioredoxin 1 (Nrf-2/Trx-1) was up-regulated both in lung tissues and in cultured PASMCs. In general, the current study

  15. The effects of altitude/hypoxic training on oxygen delivery capacity of the blood and aerobic exercise capacity in elite athletes – a meta-analysis

    PubMed Central

    Park, Hun-young; Hwang, Hyejung; Park, Jonghoon; Lee, Seongno; Lim, Kiwon

    2016-01-01

    [Purpose] This study was designed as a meta-analysis of randomized controlled trials comparing effectiveness of altitude/hypoxic training (experimental) versus sea-level training (control) on oxygen delivery capacity of the blood and aerobic exercise capacity of elite athletes in Korea. [Methods] Databases (Research Information Service System, Korean studies Information Service System, National Assembly Library) were for randomized controlled trials comparing altitude/hypoxic training versus sea-level training in elite athletes. Studies published in Korea up to December 2015 were eligible for inclusion. Oxygen delivery capacity of the blood was quantified by red blood cell (RBC), hemoglobin (Hb), hematocrit (Hct), erythropoietin (EPO); and aerobic exercise capacity was quantified by maximal oxygen consumption (VO2max). RBC, Hb, Hct, VO2max represented heterogeneity and compared post-intervention between altitude/hypoxic training and sea-level training in elite athletes by a random effect model meta-analysis. EPO represented homogeneity and meta-analysis performed by a fixed effect model. Eight independent studies with 156 elite athletes (experimental: n = 82, control: n = 74) were included in the metaanalysis. [Results] RBC (4.499×105 cell/ul, 95 % CI: 2.469 to 6.529), Hb (5.447 g/dl, 95 % CI: 3.028 to 7.866), Hct (3.639 %, 95 % CI: 1.687 to 5.591), EPO (0.711 mU/mL, 95% CI: 0.282 to 1.140), VO2max (1.637 ml/kg/min, 95% CI: 0.599 to 1.400) showed significantly greater increase following altitude/hypoxic training, as compared with sea-level training. [Conclusion] For elite athletes in Korea, altitude/ hypoxic training appears more effective than sea-level training for improvement of oxygen delivery capacity of the blood and aerobic exercise capacity. PMID:27298808

  16. Quantifying Metabolic Heterogeneity in Head and Neck Tumors in Real Time: 2-DG Uptake Is Highest in Hypoxic Tumor Regions

    PubMed Central

    Nakajima, Erica C.; Laymon, Charles; Oborski, Matthew; Hou, Weizhou; Wang, Lin; Grandis, Jennifer R.; Ferris, Robert L.; Mountz, James M.; Van Houten, Bennett

    2014-01-01

    Purpose Intratumoral metabolic heterogeneity may increase the likelihood of treatment failure due to the presence of a subset of resistant tumor cells. Using a head and neck squamous cell carcinoma (HNSCC) xenograft model and a real-time fluorescence imaging approach, we tested the hypothesis that tumors are metabolically heterogeneous, and that tumor hypoxia alters patterns of glucose uptake within the tumor. Experimental Design Cal33 cells were grown as xenograft tumors (n = 16) in nude mice after identification of this cell line's metabolic response to hypoxia. Tumor uptake of fluorescent markers identifying hypoxia, glucose import, or vascularity was imaged simultaneously using fluorescent molecular tomography. The variability of intratumoral 2-deoxyglucose (IR800-2-DG) concentration was used to assess tumor metabolic heterogeneity, which was further investigated using immunohistochemistry for expression of key metabolic enzymes. HNSCC tumors in patients were assessed for intratumoral variability of 18F-fluorodeoxyglucose (18F-FDG) uptake in clinical PET scans. Results IR800-2-DG uptake in hypoxic regions of Cal33 tumors was 2.04 times higher compared to the whole tumor (p = 0.0001). IR800-2-DG uptake in tumors containing hypoxic regions was more heterogeneous as compared to tumors lacking a hypoxic signal. Immunohistochemistry staining for HIF-1α, carbonic anhydrase 9, and ATP synthase subunit 5β confirmed xenograft metabolic heterogeneity. We detected heterogeneous 18F-FDG uptake within patient HNSCC tumors, and the degree of heterogeneity varied amongst tumors. Conclusion Hypoxia is associated with increased intratumoral metabolic heterogeneity. 18F-FDG PET scans may be used to stratify patients according to the metabolic heterogeneity within their tumors, which could be an indicator of prognosis. PMID:25127378

  17. Hypoxic-preconditioning enhances the regenerative capacity of neural stem/progenitors in subventricular zone of newborn piglet brain.

    PubMed

    Ara, Jahan; De Montpellier, Sybille

    2013-09-01

    Perinatal hypoxia-ischemia (HI) results in brain injury, whereas mild hypoxic episodes result in preconditioning, which can significantly reduce the vulnerability of the brain to subsequent severe hypoxia-ischemia. Hypoxic-preconditioning (PC) has been shown to enhance cell survival and differentiation of progenitor cells in the central nervous system (CNS). The purpose of this study was to determine whether pretreatment with PC prior to HI stimulates subventricular zone (SVZ) proliferation and neurogenesis in newborn piglets. One-day-old piglets were subjected to PC (8% O2/92% N2) for 3h and 24h later were exposed to HI produced by combination of hypoxia (5% FiO2) for a pre-defined period of 30min and ischemia induced by a period of 10min of hypotension. Here we demonstrate that SVZ derived neural stem/progenitor cells (NSPs) from PC, HI and PC+HI piglets proliferated as neurospheres, expressed neural progenitor and neurodevelopmental markers, and that greater proportion of the spheres generated are multipotential. Neurosphere assay revealed that preconditioning pretreatment increased the number of NSP-derived neurospheres in SVZ following HI compared to normoxic and HI controls. NSPs from preconditioned SVZ generated twice as many neurons and astrocytes in vitro. Injections with 5-Bromo-2-deoxyuridine (BrdU) after PC revealed a robust proliferative response within the SVZ that continued for one week. PC also increased neurogenesis in vivo, doublecortin positive cells with migratory profiles were observed streaming from the SVZ to striatum and neocortex. These findings show that the induction of proliferation and neurogenesis by PC might be a positive adaptation for an efficient repair and plasticity in the event of a hypoxic-ischemic insult.

  18. 2-Chlorobenzoate biodegradation by recombinant Burkholderia cepacia under hypoxic conditions in a membrane bioreactor.

    PubMed

    Urgun-Demirtas, Meltem; Stark, Benjamin; Pagilla, Krishna

    2005-01-01

    The feasibility of applying bacterial hemoglobin technology to degrade 2-chlorobenzoate (2-CBA) through co-metabolism under hypoxic conditions in a membrane bioreactor (MBR) process has been studied in the laboratory. 2-chlorobenzoate removal and chloride release rates in the MBR system varied from 99 to 78% and 98 to 73%, respectively, depending on the operation conditions. Chemical oxygen demand (COD) removal efficiencies were more than 90% at food-to-microorganism ratios ranging from 0.32 to 0.62 g/g/d, and the observed yield was 0.13 to 0.20 g biomass/g COD. The bacterial cell-floc size-distribution analysis showed that there is a significant change in bacterial floc size due to high shear stress during operation of the MBR. To characterize growth kinetics of Burkholderia cepacia strain dinitrotoluene, a mathematical model that describes co-metabolic oxidation of 2-CBA in an MBR has been developed.

  19. Neuroinflammation and Neuroimmune Dysregulation after Acute Hypoxic-Ischemic Injury of Developing Brain

    PubMed Central

    Bhalala, Utpal S.; Koehler, Raymond C.; Kannan, Sujatha

    2015-01-01

    Hypoxic-ischemic (HI) injury to developing brain results from birth asphyxia in neonates and from cardiac arrest in infants and children. It is associated with varying degrees of neurologic sequelae, depending upon the severity and length of HI. Global HI triggers a series of cellular and biochemical pathways that lead to neuronal injury. One of the key cellular pathways of neuronal injury is inflammation. The inflammatory cascade comprises activation and migration of microglia – the so-called “brain macrophages,” infiltration of peripheral macrophages into the brain, and release of cytotoxic and proinflammatory cytokines. In this article, we review the inflammatory and immune mechanisms of secondary neuronal injury after global HI injury to developing brain. Specifically, we highlight the current literature on microglial activation in relation to neuronal injury, proinflammatory and anti-inflammatory/restorative pathways, the role of peripheral immune cells, and the potential use of immunomodulators as neuroprotective compounds. PMID:25642419

  20. Drosophila cyclin D/Cdk4 regulates mitochondrial biogenesis and aging and sensitizes animals to hypoxic stress

    PubMed Central

    Icreverzi, Amalia; Flor de la Cruz, Aida; Van Voorhies, Wayne A

    2012-01-01

    Drosophila cyclin D (CycD) is the single fly ortholog of the mammalian cyclin D1 and promotes both cell cycle progression and cellular growth. However, little is known about how CycD promotes cell growth. We show here that CycD/Cdk4 hyperactivity leads to increased mitochondrial biogenesis (mitobiogenesis), mitochondrial mass, NRF-1 activity (Tfam transcript levels) and metabolic activity in Drosophila, whereas loss of CycD/Cdk4 activity has the opposite effects. Surprisingly, both CycD/Cdk4 addition and loss of function increase mitochondrial superoxide production and decrease lifespan, indicating that an imbalance in mitobiogenesis may lead to oxidative stress and aging. In addition, we provide multiple lines of evidence indicating that CycD/Cdk4 activity affects the hypoxic status of cells and sensitizes animals to hypoxia. Both mitochondrial and hypoxia-related effects can be detected at global transcriptional level. We propose that mitobiogenesis and the hypoxic stress response have an antagonistic relationship, and that CycD/Cdk4 levels regulate mitobiogenesis contemporaneous to the cell cycle, such that only when cells are sufficiently oxygenated can they proliferate. PMID:22293404

  1. Cytoprotection against Hypoxic and/or MPP+ Injury: Effect of δ–Opioid Receptor Activation on Caspase 3

    PubMed Central

    Xu, Yuan; Zhi, Feng; Shao, Naiyuan; Wang, Rong; Yang, Yilin; Xia, Ying

    2016-01-01

    The pathological changes of Parkinson’s disease (PD) are, at least partially, associated with the dysregulation of PTEN-induced putative kinase 1 (PINK1) and caspase 3. Since hypoxic and neurotoxic insults are underlying causes of PD, and since δ-opioid receptor (DOR) is neuroprotective against hypoxic/ischemic insults, we sought to determine whether DOR activation could protect the cells from damage induced by hypoxia and/or MPP+ by regulating PINK1 and caspase 3 expressions. We exposed PC12 cells to either severe hypoxia (0.5%–1% O2) for 24–48 h or to MPP+ at different concentrations (0.5, 1, 2 mM) and then detected the levels of PINK1 and cleaved caspase 3. Both hypoxia and MPP+ reduced cell viability, progressively suppressed the expression of PINK1 and increased the cleaved caspase 3. DOR activation using UFP-512, effectively protected the cells from hypoxia and/or MPP+ induced injury, reversed the reduction in PINK1 protein and significantly attenuated the increase in the cleaved caspase 3. On the other hand, the application of DOR antagonist, naltrindole, greatly decreased cell viability and increased cleaved caspase 3. These findings suggest that DOR is cytoprotective against both hypoxia and MPP+ through the regulation of PINK1 and caspase 3 pathways. PMID:27517901

  2. Expressions of hypoxic stress sensor proteins after transient cerebral ischemia in mice.

    PubMed

    Shang, Jingwei; Liu, Ning; Tanaka, Nobuhito; Abe, Koji

    2012-03-01

    The role of hypoxia sensor proteins is important in responding and protecting cells against hypoxic/ischemic injury in brain. Seven in absentia homolog 1 (Siah1) regulates primarily the downstream sensor proteins factor inhibiting alpha subunit of hypoxia-inducible factor-1 (FIH) under normoxic conditions and prolyl hydroxylases domain 3 (PHD3) under hypoxic conditions. In the present study, we investigated the temporal and spatial changes of these hypoxia sensor proteins, Siah1, FIH, and PHD3, after 60 min of transient middle cerebral artery occlusion (tMCAO) up to 72 hr after reperfusion in ICR mice. Immunohistochemistry and Western blot analyses showed that Siah1 was quickly and strongly induced in neuronal cells of the ischemic penumbra, with a peak at 2 hr, and gradually returned toward the sham control (SC) level until 72 hr. In contrast, the expressions of FIH and PHD3 were strongly visualized in the SC brains, and significantly reduced in a time-dependent manner with reperfusion until 72 hr. In the ischemic core region, Siah1, FIH, and PHD3 showed a similar change of strong and progressive decrease until 72 hr. Double-immunofluorescence analyses showed a cytoplasmic localization of Siah1 and both cytoplasmic and nuclear localizations of FIH and PHD3 and that Siah1 plus FIH or PHD3 were well colocalized in same neuron at 2 hr after tMCAO. The present study suggests that hypoxia sensor proteins (Siah1, FIH, and PHD3) showed temporally and spatially different expressions after tMCAO, which could provide an effective neuroprotective reaction through their further downstream proteins after cerebral ischemia.

  3. Time domains of the hypoxic ventilatory response in ectothermic vertebrates.

    PubMed

    Porteus, Cosima; Hedrick, Michael S; Hicks, James W; Wang, Tobias; Milsom, William K

    2011-04-01

    Over a decade has passed since Powell et al. (Respir Physiol 112:123-134, 1998) described and defined the time domains of the hypoxic ventilatory response (HVR) in adult mammals. These time domains, however, have yet to receive much attention in other vertebrate groups. The initial, acute HVR of fish, amphibians and reptiles serves to minimize the imbalance between oxygen supply and demand. If the hypoxia is sustained, a suite of secondary adjustments occur giving rise to a more long-term balance (acclimatization) that allows the behaviors of normal life. These secondary responses can change over time as a function of the nature of the stimulus (the pattern and intensity of the hypoxic exposure). To add to the complexity of this process, hypoxia can also lead to metabolic suppression (the hypoxic metabolic response) and the magnitude of this is also time dependent. Unlike the original review of Powell et al. (Respir Physiol 112:123-134, 1998) that only considered the HVR in adult animals, we also consider relevant developmental time points where information is available. Finally, in amphibians and reptiles with incompletely divided hearts the magnitude of the ventilatory response will be modulated by hypoxia-induced changes in intra-cardiac shunting that also improve the match between O(2) supply and demand, and these too change in a time-dependent fashion. While the current literature on this topic is reviewed here, it is noted that this area has received little attention. We attempt to redefine time domains in a more 'holistic' fashion that better accommodates research on ectotherms. If we are to distinguish between the genetic, developmental and environmental influences underlying the various ventilatory responses to hypoxia, however, we must design future experiments with time domains in mind.

  4. Reflex gelastic-dacrystic seizures following hypoxic-ischaemic encephalopathy.

    PubMed

    Verma, Rajesh; Praharaj, Heramba Narayan

    2013-07-12

    Reflex or stimulus-sensitive epilepsies are uncommon epileptic syndromes triggered by exogenous-specific sensory stimulus or endogenous various mental activities. Gelastic-dacrystic seizures are rare epileptic manifestations characterised by ictal laughter and crying. Gelastic-dacrystic seizures are commonly caused by hypothalamic hamartoma but rarely described due to cortical dysplasia, lesions of frontal and temporal lobes, tumours and vascular malformations. We report a young woman who presented with somatosensory-evoked gelastic-dacrystic seizures. This patient had a positive history of perinatal insult substantiated by MRI findings. Hypoxic-ischaemic encephalopathy as the cause of gelastic-dacrystic seizures has not been reported so far in the literature.

  5. Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF

    PubMed Central

    Lee, Sae-Won; Jeong, Han-Kyul; Lee, Ji-Young; Yang, Jimin; Lee, Eun Ju; Kim, Su-Yeon; Youn, Seock-Won; Lee, Jaewon; Kim, Woo Jean; Kim, Kyu-Won; Lim, Jeong Mook; Park, Jong-Wan; Park, Young-Bae; Kim, Hyo-Soo

    2012-01-01

    Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed ‘hypoxic-priming’) efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation. PMID:22821840

  6. Targeting Hypoxia-inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment

    PubMed Central

    Nigim, Fares; Cavanaugh, Jill; Patel, Anoop P.; Curry, William T.; Esaki, Shin-ichi; Kasper, Ekkehard M.; Chi, Andrew S.; Louis, David N.; Martuza, Robert L.; Rabkin, Samuel D.; Wakimoto, Hiroaki

    2015-01-01

    Tissue hypoxia and necrosis represent pathophysiological and histological hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently >50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness, and is a suitable platform for studying disease biology and developing hypoxia-targeted agents. PMID:26083570

  7. Beta-receptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon.

    PubMed

    Gamperl, A K; Vijayan, M M; Pereira, C; Farrell, A P

    1998-02-01

    We examined the in vivo effect of acute hypoxemia on myocardial cell-surface (sarcolemmal) beta-adrenoreceptor density (Bmax) and binding affinity (KD) and on stress protein 70 (sp70) expression by exposing rainbow trout (Oncorhynchus mykiss; 2.1-2.7 kg) to hypoxic water (3 mg/l O2) at 15 degrees C for 6 h. This degree of hypoxia was the minimum O2 level that these trout could tolerate without losing equilibrium and struggling violently. Hypoxic exposure reduced arterial PO2 (PaO2) from 98 to 26 mmHg and arterial oxygen content (CaO2) from 10.8 to 7.4 vol/100 vol, but did not elevate epinephrine and norepinephrine levels above 10 and 30 nM, respectively. Despite the substantial reduction in blood oxygen status, the Bmax and KD of myocardial cell-surface beta-adrenoreceptors were unaffected by 6 h of hypoxic exposure. In addition, acute hypoxemia did not increase myocardial sp70 expression. The failure of short-term hypoxia to decrease trout myocardial beta-adrenoreceptor density clearly contrasts with the established hypoxia-mediated down-regulation shown for mammals. To further investigate the influence of low PO2 on salmonid myocardial beta-adrenoreceptors, binding studies were performed on the spongy (continuously exposed to deoxygenated venous blood) and compact (perfused by oxygenated blood supplied by the coronary artery) myocardia of chinook salmon. The spongy myocardium has adapted to its microenvironment of continuous low PO2 by having 14% more cell-surface beta-adrenoreceptors compared with the compact myocardium. There was no tissue-specific difference in KD and no evidence of sexual dimorphism in Bmax or KD. We conclude from our studies that the salmonid heart is well adapted for sustained performance under hypoxic conditions. We found that wild chinook salmon had 2.8 x more cell-surface beta-adrenoreceptors compared with hatchery-reared rainbow trout. This difference suggests a significant degree of plasticity exists for fish myocardial beta

  8. Ionic storm in hypoxic/ischemic stress: Can opioid receptors subside it?

    PubMed Central

    Chao, Dongman; Xia, Ying

    2010-01-01

    Neurons in the mammalian central nervous system are extremely vulnerable to oxygen deprivation and blood supply insufficiency. Indeed, hypoxic/ischemic stress triggers multiple pathophysiological changes in the brain, forming the basis of hypoxic/ischemic encephalopathy. One of the initial and crucial events induced by hypoxia/ischemia is the disruption of ionic homeostasis characterized by enhanced K+ efflux and Na+-, Ca2+- and Cl− influx, which causes neuronal injury or even death. Recent data from our laboratory and those of others have shown that activation of opioid receptors, particularly δ-opioid receptors (DOR), is neuroprotective against hypoxic/ischemic insult. This protective mechanism may be one of the key factors that determine neuronal survival under hypoxic/ischemic condition. An important aspect of the DOR-mediated neuroprotection is its action against hypoxic/ischemic disruption of ionic homeostasis. Specially, DOR signal inhibits Na+ influx through the membrane and reduces the increase in intracellular Ca2+, thus decreasing the excessive leakage of intracellular K+. Such protection is dependent on a PKC-dependent and PKA-independent signaling pathway. Furthermore, our novel exploration shows that DOR attenuates hypoxic/ischemic disruption of ionic homeostasis through the inhibitory regulation of Na+ channels. In this review, we will first update current information regarding the process and features of hypoxic/ischemic disruption of ionic homeostasis and then discuss the opioid-mediated regulation of ionic homeostasis, especially in hypoxic/ischemic condition, and the underlying mechanisms. PMID:20036308

  9. Least-cost control of agricultural nutrient contributions to the Gulf of Mexico hypoxic zone

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In 2007, the hypoxic zone in the Gulf of Mexico, measuring 20,720 km**2, was one of the two largest reported since measurement of the zone began in 1985. The extent of the hypoxic zone is related to nitrogen and phosphorous loadings originating on agricultural fields in the upper Midwest. This stud...

  10. Tetrahydrobiopterin in the Prevention of Hypertonia in Hypoxic Fetal Brain

    PubMed Central

    Vásquez-Vivar, Jeannette; Whitsett, Jennifer; Derrick, Matthew; Ji, Xinhai; Yu, Lei; Tan, Sidhartha

    2009-01-01

    Objective: Tetrahydrobiopterin (BH4) deficiency is one of the causes of dystonia at birth. We hypothesized that BH4 is a developmental factor determining vulnerability of the immature fetal brain to hypoxic-ischemic injury and subsequent motor deficits in newborns. Methods: Pregnant rabbits were subjected to 40-min uterine ischemia and fetal brains were investigated for global and focal changes in BH4. Newborn kits were assessed by neurobehavioral tests following vehicle and sepiapterin (BH4-analog) treatment of dams. Results: Naive fetal brains at 70% gestation (E22) were severely deficient for BH4 compared to maternal and other fetal tissues. BH4 concentration rapidly increased normally in the perinatal period with the highest concentrations found in the thalamus compared to basal ganglia, frontal, occipital, hippocampus and parietal cortex. Global sustained 40-min hypoxia-ischemia depleted BH4 in E22 thalamus and to a lesser extent in basal ganglia, but not in the frontal, occipital and parietal regions. Maternal supplementation prior to hypoxia-ischemia with sepiapterin increased BH4 in all brain regions and especially in the thalamus, but did not increase the intermediary metabolite, 7,8-BH2. Sepiapterin treatment also reduced incidence of severe motor deficits and perinatal death following E22 hypoxia-ischemia. Interpretation: We conclude that early developmental BH4 deficiency plays a critical role in hypoxic-ischemic brain injury. Increasing brain BH4 via maternal supplementation may be an effective strategy in preventing motor deficits from antenatal hypoxia-ischemia. PMID:19798726

  11. Chemoreceptor stimulation and hypoxic pulmonary vasoconstriction in conscious dogs.

    PubMed

    Levitzky, M G

    1979-07-01

    Dogs with electromagnetic flow probes implanted on their left (QL) and main (QT) pulmonary arteries, catheters in their left atria and external jugular veins, and chronic tracheostomies were trained to accept Carlens dual-lumen endotracheal tubes into their tracheostomies, thus allowing separate ventilation of the two lungs. Swan-Ganz catheters were inserted through the jugular vein catheters. Pneumotachographs measured air flow to each lung. During bilateral ventilation with room air or O2, QL was about 36% of QT. When the left lung was ventilated with N2 while the right remained on O2, PAO2 was above 90 mmHg and QL fell to about 25% of QT. When the left lung was ventilated with N2 and the right with room air, PAO2 fell below 40 mm Hg and QL increased to control levels. This increase in perfusion of the hypoxic lung during systemic hypoxemia was not seen in dogs after surgical deafferentation of the systemic arterial chemoreceptors, indicating that stimulation of the arterial chemoreceptors may interfere with the hypoxic pulmonary vasoconstriction.

  12. Regional hypoxic pulmonary vasoconstriction in dogs with asymptomatic dirofilariasis.

    PubMed

    Chapleau, M W; Fish, R E; Levitzky, M G

    1985-06-01

    The pulmonary hemodynamic response to unilateral alveolar hypoxia was investigated in pentobarbital-anesthetized dogs with mild heartworm (HW) disease and in dogs free of HW (HWF). Left lung nitrogen ventilation in HWF dogs resulted in a decrease in the fraction of the cardiac output (QT) perfusing the left lung (QL) from 0.37 +/- 0.03 (SEM) to 0.20 +/- 0.02 (P less than 0.01). In contrast, dogs with mild HW disease did not develop a significant decrease in QL/QT which decreased from 0.38 +/- 0.02 to 0.33 +/- 0.02. This attenuated pulmonary vascular response to regional alveolar hypoxia in dogs with HW disease was associated with a normal pulmonary arterial pressure (14.8 +/- 1.5 mm of Hg) that was not different from that seen in HWF dogs (15.8 +/- 1.7 mm of Hg). These results indicate that mild HW disease interferes with the ability of hypoxic pulmonary vasoconstriction to redistribute pulmonary blood flow away from hypoxic regions of the lung.

  13. The Effects of Acidic and Hypoxic Conditions on the Estuarine ...

    EPA Pesticide Factsheets

    The interactive and combined effects of coastal acidification and hypoxia on estuarine species is an increasing concern as these stressors change concomitantly. There is a need to understand how these environmental factors interact, as well as their effect on estuarine organisms. A method was developed for this research whereby four exposure treatments were created simultaneously: ambient, elevated pCO2, (~1300µatm, IPCC RCP 8.5 scenario), hypoxic (low dissolved oxygen, ~2 mg/L), and combined elevated pCO2 with low dissolved oxygen. An exposure with variant water quality parameters allows for the comparative study of organismal survival response to acidified and hypoxic conditions. The goal of this research is to determine acute species sensitivity, which is determined by survivability, to the combined effects of elevated pCO2 and hypoxia over a 5 day period, as well as possible differences in sensitivity between life-stages. Preliminary research on sheepshead minnow and mysid shrimp, indicates that mysid shrimp were tolerant of both elevated pCO2 and low DO exposure regardless of life-stage, whereas sheepshead minnows were more sensitive to the combined effects of acidification and hypoxia. This work is part of the first phase of the NECAH project, which is identifying species that are sensitive to the combined effects of acidification and hypoxia. The project describes the initial work on the first 2 species selected for testing and the final product will be

  14. Efferent inhibition of carotid body chemoreception in chronically hypoxic cats.

    PubMed

    Lahiri, S; Smatresk, N; Pokorski, M; Barnard, P; Mokashi, A

    1983-11-01

    The effects of chronic hypoxia on carotid chemoreceptor afferent activity before and after sectioning the carotid sinus nerves (CSN) were studied in cats exposed to 10% O2 for 21-49 days in a chamber at sea level. For comparison, chronically normoxic cats at sea level were also studied. The cats were anesthetized, paucifiber preparation for the measurement of carotid chemosensory activity from a small slip of CSN was made, and their steady-state responses to 4-5 levels of arterial pressure of O2 (PaO2) at a constant PaCO2 and to 3-4 levels of PaCO2 in hyperoxia were measured before and after sectioning the CSN. The chemosensory response to hypoxia in the cats with intact CSN after chronic exposure to hypoxia was not reduced relative to the cats that breathed room air at sea level. Sectioning the CSN significantly augmented the chemosensory responses to hypoxia in all the chronically hypoxic but not significantly in the normoxic cats. The responses to moderate hypercapnia during hyperoxia were not significantly changed by cutting the CSN in either group. We conclude that there is a significant CSN efferent inhibition of chemosensory activity due to chronic hypoxia in the cat. This implies that without the efferent inhibition the hypoxic chemosensitivity is increased by chronic hypoxia.

  15. Differential hypoxic cytotoxicity of bioreductive agents determined in vitro by the MTT assay

    SciTech Connect

    Stratford, I.J.; Stephens, M.A.

    1989-04-01

    We obtained good agreement between the MTT assay and conventional clonogenic assays regarding the concentration and contact time required to produce a given level of killing of Chinese hamster V79 cells treated in either air of N/sub 2/ with a range of bioreductive cytotoxic drugs. All agents chosen for these experiments represented classes of compounds known to be more toxic towards hypoxic cells than they are to aerobic cells. Namely a quinone, mitomycin C; a di-N-oxide, SR4233; and a number of nitro-heterocyclics including misonidazole. The MTT assay is carried out with V79 cells attached to the bottom of 1 cm glass wells within a 24 well plate. All procedures, that is drug exposure, cell growth, metabolism of MTT are then carried out in situ. To measure optical density we used an ELIZA plate reader modified to take 24-well plates. We propose that this method provides a simple, rapid procedure for evaluating the cytotoxicity of bioreductive drugs.

  16. Hypoxic environments as refuge against predatory fish in the Amazonian floodplains.

    PubMed

    Anjos, M B; De Oliveira, R R; Zuanon, J

    2008-02-01

    Several groups of Amazonian fishes exhibit behavioral, morphological and physiological characteristics that allow occupying hypoxic environments, despite the energetic costs of living in such harsh conditions. One of the supposed advantages of occupying hypoxic habitats would be a lower predation pressure resulting from a lower number of piscivorous fishes in those environments. We tested this hypothesis in an area of the Amazon River floodplain through gill net fishing in normoxic and hypoxic habitats. From the 103 species caught, 38 were classified as piscivores. We found no difference in the number of piscivorous species captured in hypoxic and normoxic habitats (chi2 = 0.23; p = 0.63; df = 1) but piscivorous individuals were more numerous in normoxic than in hypoxic sampling stations (chi2 = 104.4; p < 0.001; df = 1). This indicates that environments submitted to low oxygen conditions may in fact function as refuges against piscivorous fishes in the Amazonian floodplains.

  17. Hypoxic pulmonary vasodilation: a paradigm shift with a hydrogen sulfide mechanism

    PubMed Central

    Whitfield, Nathan L.; Bearden, Shawn E.; St. Leger, Judy; Nilson, Erika; Gao, Yan; Madden, Jane A.

    2010-01-01

    Hypoxic pulmonary vasoconstriction (HVC), an intrinsic and assumed ubiquitous response of mammalian pulmonary blood vessels, matches regional ventilation to perfusion via an unknown O2-sensing mechanism. Global pulmonary hypoxia experienced by individuals suffering from chronic obstructive pulmonary disease or numerous hypoventilation syndromes, including sleep apnea, often produces maladaptive pulmonary hypertension, but pulmonary hypertension is not observed in diving mammals, where profound hypoxia is routine. Here we examined the response of cow and sea lion pulmonary arteries (PA) to hypoxia and observed the expected HVC in the former and a unique hypoxic vasodilation in resistance vessels in the latter. We then used this disparate response to examine the O2-sensing mechanism. In both animals, exogenous H2S mimicked the vasoactive effects of hypoxia in isolated PA. H2S-synthesizing enzymes, cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfur transferase, were identified in lung tissue from both animals by one-dimensional Western blot analysis and immunohistochemistry. The relationship between H2S production/consumption and O2 was examined in real time by use of amperometric H2S and O2 sensors. H2S was produced by sea lion and cow lung homogenate in the absence of O2, but it was rapidly consumed when O2 was present. Furthermore, consumption of exogenous H2S by cow lung homogenate, PA smooth muscle cells, and heart mitochondria was O2 dependent and exhibited maximal sensitivity at physiologically relevant Po2 levels. These studies show that HVC is not an intrinsic property of PA and provide further evidence for O2-dependent H2S metabolism in O2 sensing. PMID:19889863

  18. Neuroprotective effects of electro acupuncture on hypoxic-ischemic encephalopathy in newborn rats Ass.

    PubMed

    Xu, Tao; Li, Wenjie; Liang, Yiqun; Yang, Zhonghua; Liu, Jingdong; Wang, Yejun; Su, Nailun

    2014-11-01

    Hypoxic-ischemic encephalopathy (HIE) is a common and potentially devastating condition in the neonate, associated with high mortality and morbidity. Effective treatment options are limited and therefore alternative therapies such as acupuncture are increasingly used. Previous studies have shown that electro acupuncture promoted proliferation of neural progenitor cell and increased expression of neurotrophic factor in HIE. However, effects of electro acupuncture on downstream signaling pathways have been rarely researched. So, in the present study, we aimed to evaluate the neuroprotective effects of electro acupuncture on HIE and to further investigate the role of GDNF family receptor member RET and its key downstream PI3-K/Akt pathway in the process. A rat HIE model was constructed by the left common carotid artery (LCCA) ligation method in combination with hypoxic treatment. Considering that Baihui (GV20), Dazhui (GV14), Quchi (LI11) and Yongquan (KI1) are commonly used in clinics for stroke treatment and are easy to locate, we chose the above four acupoints as the combination for electro acupuncture treatment which was performed once a day for different time periods. Hematoxylin-eosin (HE) staining and transmission electron microscopy results showed that electro acupuncture could ameliorate neurologic damage and alleviate the degenerative changes of ultra structure of cortical neurons in rats subjected to HIE. And the longer acupuncture treatment lasted, the better its therapeutic effect would be. This was accompanied by gradually increased expression of GDNF family receptor RET at the mRNA level and its downstream signaling Akt at the protein level in the ischemic cortex. These findings suggest that electro acupuncture shows neuroprotective effects in HIE, which at least in part is attributed to activation of PI3-K/Akt signaling pathway.

  19. Metabolite analysis of Mycobacterium species under aerobic and hypoxic conditions reveals common metabolic traits.

    PubMed

    Drapal, Margit; Wheeler, Paul R; Fraser, Paul D

    2016-08-01

    A metabolite profiling approach has been implemented to elucidate metabolic adaptation at set culture conditions in five Mycobacterium species (two fast- and three slow-growing) with the potential to act as model organisms for Mycobacterium tuberculosis (Mtb). Analysis has been performed over designated growth phases and under representative environments (nutrient and oxygen depletion) experienced by Mtb during infection. The procedure was useful in determining a range of metabolites (60-120 compounds) covering nucleotides, amino acids, organic acids, saccharides, fatty acids, glycerols, -esters, -phosphates and isoprenoids. Among these classes of compounds, key biomarker metabolites, which can act as indicators of pathway/process activity, were identified. In numerous cases, common metabolite traits were observed for all five species across the experimental conditions (e.g. uracil indicating DNA repair). Amino acid content, especially glutamic acid, highlighted the different properties between the fast- and slow-growing mycobacteria studied (e.g. nitrogen assimilation). The greatest similarities in metabolite composition between fast- and slow-growing mycobacteria were apparent under hypoxic conditions. A comparison to previously reported transcriptomic data revealed a strong correlation between changes in transcription and metabolite content. Collectively, these data validate the changes in the transcription at the metabolite level, suggesting transcription exists as one of the predominant modes of cellular regulation in Mycobacterium. Sectors with restricted correlation between metabolites and transcription (e.g. hypoxic cultivation) warrant further study to elucidate and exploit post-transcriptional modes of regulation. The strong correlation between the laboratory conditions used and data derived from in vivo conditions, indicate that the approach applied is a valuable addition to our understanding of cell regulation in these Mycobacterium species.

  20. Peptidylarginine deiminases: novel drug targets for prevention of neuronal damage following hypoxic ischemic insult (HI) in neonates.

    PubMed

    Lange, Sigrun; Rocha-Ferreira, Eridan; Thei, Laura; Mawjee, Priyanka; Bennett, Kate; Thompson, Paul R; Subramanian, Venkataraman; Nicholas, Anthony P; Peebles, Donald; Hristova, Mariya; Raivich, Gennadij

    2014-08-01

    Neonatal hypoxic ischaemic (HI) injury frequently causes neural impairment in surviving infants. Our knowledge of the underlying molecular mechanisms is still limited. Protein deimination is a post-translational modification caused by Ca(+2) -regulated peptidylarginine deiminases (PADs), a group of five isozymes that display tissue-specific expression and different preference for target proteins. Protein deimination results in altered protein conformation and function of target proteins, and is associated with neurodegenerative diseases, gene regulation and autoimmunity. In this study, we used the neonatal HI and HI/infection [lipopolysaccharide (LPS) stimulation] murine models to investigate changes in protein deimination. Brains showed increases in deiminated proteins, cell death, activated microglia and neuronal loss in affected brain areas at 48 h after hypoxic ischaemic insult. Upon treatment with the pan-PAD inhibitor Cl-amidine, a significant reduction was seen in microglial activation, cell death and infarct size compared with control saline or LPS-treated animals. Deimination of histone 3, a target protein of the PAD4 isozyme, was increased in hippocampus and cortex specifically upon LPS stimulation and markedly reduced following Cl-amidine treatment. Here, we demonstrate a novel role for PAD enzymes in neural impairment in neonatal HI Encephalopathy, highlighting their role as promising new candidates for drug-directed intervention in neurotrauma. Hypoxic Ischaemic Insult (HI) results in activation of peptidylarginine deiminases (PADs) because of calcium dysregulation. Target proteins undergo irreversible changes of protein bound arginine to citrulline, resulting in protein misfolding. Infection in synergy with HI causes up-regulation of TNFα, nuclear translocation of PAD4 and change in gene regulation as a result of histone deimination. Pharmacological PAD inhibition significantly reduced HI brain damage.

  1. [Effect of the intermittent hypoxic training on the functioning of peptidergic neurons of the paraventricular hypothalamic nucleus and brain stem neurons in rats].

    PubMed

    Abramov, A V

    1998-03-01

    Internittent hypoxic training (IHT) increased the quantity and secretory activity of peptidergic neurons of the paraventricular hypothalamic nucleus (PHN) and activated neurons of the dorsal motor nucleus of n.vagus. These structures seem to take part in realisation of the IHT activating effect on condition of the pancreatic delta-cells. The effect involves insulin-stimulating and insuloprotective effects realised via hypothalamic and neuro-conducting ways of regulation of the endocrine pancreas with a direct participation of hypothalamic neuropeptides.

  2. The influence of hypoxic physical activity on cfDNA as a new marker of vascular inflammation

    PubMed Central

    Zembron-Lacny, Agnieszka; Baldy-Chudzik, Katarzyna; Orysiak, Joanna; Sitkowski, Dariusz; Banach, Maciej

    2015-01-01

    The phenomenon of circulating cell-free DNA (cfDNA) is important for many biomedical disciplines including the field of exercise biochemistry and physiology. It is likely that cfDNA is released into the plasma by apoptosis of endothelial cells and circulating endothelial progenitor cells (EPCs), and/or by NETosis of immune cells induced by strenuous exercise. Increases of cfDNA are described to be a potential hallmark for the overtraining syndrome, and might be related to aseptic vascular inflammation in athletes. Yet, the relevance of systemic inflammation and cfDNA with endothelial dysfunction in athletes still remains unclear. In this review article, we provide a current overview of exercise-induced cfDNA release to the circulation with special emphasis on its relationship with apoptosis and NETosis and the effect of hypoxic physical activity on vascular inflammation in athletes. PMID:26788076

  3. Hypoxic Living and Exercise Training Alter Adipose Tissue Leptin/Leptin Receptor in Rats.

    PubMed

    Lu, Yingli; Feng, Lianshi; Xie, Minhao; Zhang, Li; Xu, Jianfang; He, Zihong; You, Tongjian

    2016-01-01

    Background: Hypobaric hypoxia results in weight loss in obese individuals, and exercise training is advocated for the treatment of obesity and its related metabolic dysfunctions. The purpose of this study was to investigate the effects of hypoxic living and exercise training on obesity and adipose tissue leptin/leptin receptor in dietary-induced obese rats. Methods: One hundred and thirty high-fat diet fed Sprague-Dawley rats were assigned into one of the following groups (n = 10 each): control, sedentary hypoxic living for 1-4 weeks (SH1, SH2, SH3, and SH4), living, and exercise training in normoxic conditions for 1-4 weeks (TN1, TN2, TN3, and TN4), and living and exercise training in hypoxic conditions for 1-4 weeks (TN1, TN2, TN3, and TN4). Epididymal adipose tissue expression levels of leptin and leptin receptor were determined Results: Compared to hypoxic living and living and exercise training in normoxic conditions, living and exercise training in hypoxic conditions for 3-4 weeks resulted in lower Lee index (P < 0.05-0.01), and higher expression of leptin and leptin receptor (P < 0.05-0.01) in adipose tissue. Conclusion: In a rodent model of altitude training, living, and exercise training in hypoxic conditions resulted in greater alterations in obesity and adipose tissue leptin/leptin receptor than hypoxic living alone and living and exercise training in normoxic conditions.

  4. Hypoxic Living and Exercise Training Alter Adipose Tissue Leptin/Leptin Receptor in Rats

    PubMed Central

    Lu, Yingli; Feng, Lianshi; Xie, Minhao; Zhang, Li; Xu, Jianfang; He, Zihong; You, Tongjian

    2016-01-01

    Background: Hypobaric hypoxia results in weight loss in obese individuals, and exercise training is advocated for the treatment of obesity and its related metabolic dysfunctions. The purpose of this study was to investigate the effects of hypoxic living and exercise training on obesity and adipose tissue leptin/leptin receptor in dietary-induced obese rats. Methods: One hundred and thirty high-fat diet fed Sprague-Dawley rats were assigned into one of the following groups (n = 10 each): control, sedentary hypoxic living for 1–4 weeks (SH1, SH2, SH3, and SH4), living, and exercise training in normoxic conditions for 1–4 weeks (TN1, TN2, TN3, and TN4), and living and exercise training in hypoxic conditions for 1–4 weeks (TN1, TN2, TN3, and TN4). Epididymal adipose tissue expression levels of leptin and leptin receptor were determined Results: Compared to hypoxic living and living and exercise training in normoxic conditions, living and exercise training in hypoxic conditions for 3–4 weeks resulted in lower Lee index (P < 0.05–0.01), and higher expression of leptin and leptin receptor (P < 0.05–0.01) in adipose tissue. Conclusion: In a rodent model of altitude training, living, and exercise training in hypoxic conditions resulted in greater alterations in obesity and adipose tissue leptin/leptin receptor than hypoxic living alone and living and exercise training in normoxic conditions. PMID:27932989

  5. Satellite-based empirical models linking river plume dynamics with hypoxic area and volume

    NASA Astrophysics Data System (ADS)

    Le, Chengfeng; Lehrter, John C.; Hu, Chuanmin; Obenour, Daniel R.

    2016-03-01

    Satellite-based empirical models explaining hypoxic area and volume variation were developed for the seasonally hypoxic (O2 < 2 mg L-1) northern Gulf of Mexico adjacent to the Mississippi River. Annual variations in midsummer hypoxic area and volume were related to Moderate Resolution Imaging Spectroradiometer-derived monthly estimates of river plume area (km2) and average, inner shelf chlorophyll a concentration (Chl a, mg m-3). River plume area in June was negatively related with midsummer hypoxic area (km2) and volume (km3), while July inner shelf Chl a was positively related to hypoxic area and volume. Multiple regression models using river plume area and Chl a as independent variables accounted for most of the variability in hypoxic area (R2 = 0.92) or volume (R2 = 0.89). These models explain more variation in hypoxic area than models using Mississippi River nutrient loads as independent variables. The results here also support a hypothesis that confinement of the river plume to the inner shelf is an important mechanism controlling hypoxia area and volume in this region.

  6. A scenario and forecast model for Gulf of Mexico hypoxic area and volume

    USGS Publications Warehouse

    Scavia, Donald; Evans, Mary Anne; Obenour, Daniel R.

    2013-01-01

    For almost three decades, the relative size of the hypoxic region on the Louisiana-Texas continental shelf has drawn scientific and policy attention. During that time, both simple and complex models have been used to explore hypoxia dynamics and to provide management guidance relating the size of the hypoxic zone to key drivers. Throughout much of that development, analyses had to accommodate an apparent change in hypoxic sensitivity to loads and often cull observations due to anomalous meteorological conditions. Here, we describe an adaptation of our earlier, simple biophysical model, calibrated to revised hypoxic area estimates and new hypoxic volume estimates through Bayesian estimation. This application eliminates the need to cull observations and provides revised hypoxic extent estimates with uncertainties, corresponding to different nutrient loading reduction scenarios. We compare guidance from this model application, suggesting an approximately 62% nutrient loading reduction is required to reduce Gulf hypoxia to the Action Plan goal of 5,000 km2, to that of previous applications. In addition, we describe for the first time, the corresponding response of hypoxic volume. We also analyze model results to test for increasing system sensitivity to hypoxia formation, but find no strong evidence of such change.

  7. Adenosine A1 and A3 receptors protect astrocytes from hypoxic damage.

    PubMed

    Björklund, Olga; Shang, Mingmei; Tonazzini, Ilaria; Daré, Elisabetta; Fredholm, Bertil B

    2008-10-31

    Brain levels of adenosine are elevated during hypoxia. Through effects on adenosine receptors (A(1), A(2A), A(2B) and A(3)) on astrocytes, adenosine can influence functions such as glutamate uptake, reactive gliosis, swelling, as well as release of neurotrophic and neurotoxic factors having an impact on the outcome of metabolic stress. We have studied the roles of these receptors in astrocytes by evaluating their susceptibility to damage induced by oxygen deprivation or exposure to the hypoxia mimic cobalt chloride (CoCl(2)). Hypoxia caused ATP breakdown and purine release, whereas CoCl(2) (0.8 mM) mainly reduced ATP by causing cell death in human D384 astrocytoma cells. Further experiments were conducted in primary astrocytes prepared from specific adenosine receptor knock-out (KO) and wild type (WT) mice. In WT cells purine release following CoCl(2) exposure was mainly due to nucleotide release, whereas hypoxia-induced intracellular ATP breakdown followed by nucleoside efflux. N-ethylcarboxamidoadenosine (NECA), an unselective adenosine receptor agonist, protected from cell death following hypoxia. Cytotoxicity was more pronounced in A(1)R KO astrocytes and tended to be higher in WT cells in the presence of the A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Genetic deletion of A(2A) receptor resulted in less prominent effects. A(3)R KO glial cells were more affected by hypoxia than WT cells. Accordingly, the A(3) receptor agonist 2-chloro-N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (CL-IB-MECA) reduced ATP depletion caused by hypoxic conditions. It also reduced apoptosis in human astroglioma D384 cells after oxygen deprivation. In conclusion, the data point to a cytoprotective role of adenosine mediated by both A(1) and A(3) receptors in primary mouse astrocytes.

  8. Switching off malignant mesothelioma: exploiting the hypoxic microenvironment

    PubMed Central

    Nabavi, Noushin; Bennewith, Kevin L.; Churg, Andrew; Wang, Yuzhuo; Collins, Colin C.; Mutti, Luciano

    2016-01-01

    Malignant mesotheliomas are aggressive, asbestos-related cancers with poor patient prognosis, typically arising in the mesothelial surfaces of tissues in pleural and peritoneal cavity. The relative unspecific symptoms of mesotheliomas, misdiagnoses, and lack of precise targeted therapies call for a more critical assessment of this disease. In the present review, we categorize commonly identified genomic aberrations of mesotheliomas into their canonical pathways and discuss targeting these pathways in the context of tumor hypoxia, a hallmark of cancer known to render solid tumors more resistant to radiation and most chemo-therapy. We then explore the concept that the intrinsic hypoxic microenvironment of mesotheliomas can be Achilles' heel for targeted, multimodal therapeutic intervention. PMID:28191281

  9. Atrial natriuretic factor in neonatal hypoxic-ischemic encephalopathy.

    PubMed

    Carbonell, X; Figueras, J; Salvia, M D; Esque, M T; Delgado, M P; Jimenez, R

    1993-01-01

    The influence of perinatal asphyxia in the secretion of atrial natriuretic factor (ANF) during the first 6 days of life, and its renal consequences are discussed. Comparison between 20 healthy term neonates and 19 with first--or second--degree hypoxic-ischemic encephalopathy (HIE) is made. Daily controls were performed on clinical and neurological examinations and administration of sodium and fluids. On the first and sixth days of life, 24 hours urine collection, natremia, natriuresis, fractionated excretion of sodium and creatinine clearance were determined. The ANF was performed at 1, 2, 3 and 6 days old, by R.I.A. The full term newborns with HIE showed a peak in ANF values on day two, as does the control group, thereafter maintaining higher levels, with a significant difference on day three and six. No correlation could be found between the ANF levels and the renal variables analyzed.

  10. Hypoxia inducible factors and the response to hypoxic stress

    PubMed Central

    Majmundar, Amar J.; Wong, Waihay J.; Simon, M. Celeste

    2011-01-01

    Oxygen (O2) is an essential nutrient that serves as a key substrate in cellular metabolism and bioenergetics. In a variety of physiological and pathological states, organisms encounter insufficient O2 availability, or hypoxia. In order to cope with this stress, evolutionarily conserved responses are engaged. In mammals, the primary transcriptional response to hypoxic stress is mediated by the Hypoxia-inducible factors (HIFs). While canonically regulated by prolyl hydroxylase domain-containing enzymes (PHDs), the HIFα subunits are intricately responsive to numerous other factors including Factor Inhibiting HIF-1α (FIH1), sirtuins, and metabolites. These transcription factors function in normal tissue homeostasis and impinge on critical aspects of disease progression and recovery. Insights from basic HIF biology are being translated into pharmaceuticals targeting the HIF pathway. PMID:20965423

  11. The hypoxia signaling pathway and hypoxic adaptation in fishes.

    PubMed

    Xiao, Wuhan

    2015-02-01

    The hypoxia signaling pathway is an evolutionarily conserved cellular signaling pathway present in animals ranging from Caenorhabditis elegans to mammals. The pathway is crucial for oxygen homeostasis maintenance. Hypoxia-inducible factors (HIF-1α and HIF-2α) are master regulators in the hypoxia signaling pathway. Oxygen concentrations vary a lot in the aquatic environment. To deal with this, fishes have adapted and developed varying strategies for living in hypoxic conditions. Investigations into the strategies and mechanisms of hypoxia adaptation in fishes will allow us to understand fish speciation and breed hypoxia-tolerant fish species/strains. This review summarizes the process of the hypoxia signaling pathway and its regulation, as well as the mechanism of hypoxia adaptation in fishes.

  12. First aid kit for hypoxic survival: sensors and strategies.

    PubMed

    López-Barneo, J; Nurse, C A; Nilsson, G E; Buck, L T; Gassmann, M; Bogdanova, A Yu

    2010-01-01

    Survival success under conditions of acute oxygen deprivation depends on efficiency of the central and peripheral chemoreception, optimization of oxygen extraction from the hypoxic environment and its delivery to the periphery, and adjustments of energy production and consumption. This article uses a comparative approach to assess the efficiency of adaptive strategies used by anoxia-tolerant and hypoxia-sensitive species to support survival during the first minutes to 1 h of oxygen deprivation. An aquatic environment is much more demanding in terms of diurnal and seasonal variations of the ambient oxygen availability from anoxia to hyperoxia than is an air environment. Therefore, fishes and aquatic turtles have developed a number of adaptive responses, which are lacking in most of the terrestrial mammals, to cope with these extreme conditions. These include efficient central and peripheral chemoreception, acute changes in respiratory rate and amplitude, and acute increase of the gas-exchange interface. A special set of adaptive mechanisms are engaged in reduction of the energy expenditure of the major oxygen-consuming organs: the brain and the heart. Both reduction of ATP consumption and a switch to alterative energy sources contribute to the maintenance of ATP and ion balance in hypoxia-tolerant animals. Hypoxia and hyperoxia are conditions favoring development of oxidative stress. Efficient protection from oxidation in anoxia-tolerant species includes reduction in the glutamate levels in the brain, stabilization of the mitochondrial function, and maintenance of nitric oxide production under conditions of oxygen deprivation. We give an overview of the current state of knowledge on some selected molecular and cellular acute adaptive mechanisms. These include the mechanisms of chemoreception in adult and neonatal mammals and in fishes, acute metabolic adaptive responses in the brain, and the role of nitrite in the preservation of heart function under hypoxic

  13. Role of Mitochondria in Neonatal Hypoxic-Ischemic Brain Injury

    PubMed Central

    Lu, Yujiao; Tucker, Donovan; Dong, Yan; Zhao, Ningjun; Zhuo, Xiaoying; Zhang, Quanguang

    2016-01-01

    Hypoxic-ischemia (HI) causes severe brain injury in neonates. It’s one of the leading causes to neonatal death and pediatric disability, resulting in devastating consequences, emotionally and economically, to their families. A series of events happens in this process, e.g. excitatory transmitter release, extracelluar Ca2+ influxing, mitochondrial dysfunction, energy failure, and neuron death. There are two forms of neuron death after HI insult: necrosis and apoptosis, apoptosis being the more prevalent form. Mitochondria handle a series of oxidative reactions, and yield energy for various cellular activities including the maintainance of membrane potential and preservation of intracellular ionic homeostasis. Therefore mitochondria play a critical role in neonatal neurodegeneration following HI, and mitochondrial dysfunction is the key point in neurodegenerative evolution. Because of this, exploring effective mitochondria-based clinical strategies is crucial. Today the only efficacious clinic treatment is hypothermia. However, due to its complex management, clinical complication and autoimmune decrease, its clinical application is limited. So far, many mitochondria-based strategies have been reported neuroprotective in animal models, which offers promise on neonatal therapy. However, since their clinical effectiveness are still unclear, plenty of studies need to be continued in the future. According to recent reports, two novel strategies have been proposed: methylene blue (MB) and melatonin. Although they are still in primary stage, the underlying mechanisms indicate promising clinical applications. Every neurological therapeutic strategy has its intrinsic deficit and limited efficacy, therefore in the long run, the perfect clinical therapy for hypoxic-ischemic neonatal brain injury will be based on the combination of multiple strategies. PMID:27441209

  14. Oxygen transport in conscious newborn dogs during hypoxic hypometabolism.

    PubMed

    Rohlicek, C V; Saiki, C; Matsuoka, T; Mortola, J P

    1998-03-01

    We questioned whether the decrease in O2 consumption (VO2) during hypoxia in newborns is a regulated response or reflects a limitation in O2 availability. Experiments were conducted on previously instrumented conscious newborn dogs. VO2 was measured at a warm ambient temperature (30 degrees C, n = 7) or in the cold (20 degrees C, n = 6), while the animals breathed air or were sequentially exposed to 15 min of fractional inspired O2 (FIO2): 21, 18, 15, 12, 10, 8, and 6%. In normoxia, VO2 averaged 15 +/- 1 (SE) and 25 +/- 1 ml . kg-1 . min-1 in warm and cold conditions, respectively. In the warm condition, hypometabolism (i.e., hypoxic VO2 < normoxic VO2) occurred at FIO2 hypoxic drop in VO2 in the newborn reflects a limitation in O2 availability. The results are compatible with the idea that the phenomenon is one of "regulated conformism" to hypoxia.

  15. Hypoxic repression of pyruvate dehydrogenase activity is necessary for metabolic reprogramming and growth of model tumours

    PubMed Central

    Golias, Tereza; Papandreou, Ioanna; Sun, Ramon; Kumar, Bhavna; Brown, Nicole V.; Swanson, Benjamin J.; Pai, Reetesh; Jaitin, Diego; Le, Quynh-Thu; Teknos, Theodoros N.; Denko, Nicholas C.

    2016-01-01

    Tumour cells fulfil the bioenergetic and biosynthetic needs of proliferation using the available environmental metabolites. Metabolic adaptation to hypoxia causes decreased mitochondrial function and increased lactate production. This work examines the biological importance of the hypoxia-inducible inhibitory phosphorylations on the pyruvate dehydrogenase E1α subunit. Pancreatic cancer cell lines were genetically manipulated to alter the net phosphorylation of PDH E1α through reduced kinase expression or enhanced phosphatase expression. The modified cells were tested for hypoxic changes in phosphorylated E1α, mitochondrial metabolism and growth as xenografted tumours. Even though there are four PDHK genes, PDHK1 is essential for inhibitory PDH phosphorylation of E1α at serine 232, is partially responsible for modification of serines 293 and 300, and these phosphorylations are necessary for model tumour growth. In order to determine the clinical relevance, a cohort of head and neck cancer patient biopsies was examined for phosphorylated E1α and expression of PDHK1. Patients with detectable 232 phosphorylation or expression of PDHK1 tend to have worse clinical outcome. These data show that PDHK1 activity is unique and non-redundant in the family of PHDK enzymes and a PDHK1 specific inhibitor would therefore have anti-cancer activity with reduced chance of side effects from inhibition of other PDHKs. PMID:27498883

  16. Marine bacterioplankton community turnover within seasonally hypoxic waters of a subtropical sound: Devil's Hole, Bermuda.

    PubMed

    Parsons, Rachel J; Nelson, Craig E; Carlson, Craig A; Denman, Carmen C; Andersson, Andreas J; Kledzik, Andrew L; Vergin, Kevin L; McNally, Sean P; Treusch, Alexander H; Giovannoni, Stephen J

    2015-10-01

    Understanding bacterioplankton community dynamics in coastal hypoxic environments is relevant to global biogeochemistry because coastal hypoxia is increasing worldwide. The temporal dynamics of bacterioplankton communities were analysed throughout the illuminated water column of Devil's Hole, Bermuda during the 6-week annual transition from a strongly stratified water column with suboxic and high-pCO2 bottom waters to a fully mixed and ventilated state during 2008. A suite of culture-independent methods provided a quantitative spatiotemporal characterization of bacterioplankton community changes, including both direct counts and rRNA gene sequencing. During stratification, the surface waters were dominated by the SAR11 clade of Alphaproteobacteria and the cyanobacterium Synechococcus. In the suboxic bottom waters, cells from the order Chlorobiales prevailed, with gene sequences indicating members of the genera Chlorobium and Prosthecochloris--anoxygenic photoautotrophs that utilize sulfide as a source of electrons for photosynthesis. Transitional zones of hypoxia also exhibited elevated levels of methane- and sulfur-oxidizing bacteria relative to the overlying waters. The abundance of both Thaumarcheota and Euryarcheota were elevated in the suboxic bottom waters (> 10(9) cells l(-1)). Following convective mixing, the entire water column returned to a community typical of oxygenated waters, with Euryarcheota only averaging 5% of cells, and Chlorobiales and Thaumarcheota absent.

  17. [Hemo- and neurodynamics of the human brain during exposure to moderate hypoxic hypoxia].

    PubMed

    Alekseev, D A; Zubarev, A F; Krupina, T N; Iarullin, Kh Kh; Kuznets, E I

    1984-01-01

    Synchronous electro- and rheoencephalography were used to study tolerance to moderate hypoxic hypoxia for 30 min at an altitude of 5000 m without additional oxygen supply. As test subject, men with autonomic-vascular dystonia (29-39 years old), 15 men over 40 (41-56 years old), and 16 essentially healthy controls (23-36 years old) were used. The aged volunteers (41-56 years old) did not differ from the controls with respect to their tolerance to hypoxic hypoxia. The men with early symptoms of hypertonic-type dystonia also showed high tolerance to hypoxic hypoxia. The subjects with hypotonic-type dystonia displayed lower tolerance.

  18. Immuno-modulator inter-alpha inhibitor proteins ameliorate complex auditory processing deficits in rats with neonatal hypoxic-ischemic brain injury.

    PubMed

    Threlkeld, Steven W; Lim, Yow-Pin; La Rue, Molly; Gaudet, Cynthia; Stonestreet, Barbara S

    2017-03-10

    Hypoxic-ischemic (HI) brain injury is recognized as a significant problem in the perinatal period, contributing to life-long language-learning and other cognitive impairments. Central auditory processing deficits are common in infants with hypoxic-ischemic encephalopathy and have been shown to predict language learning deficits in other at risk infant populations. Inter-alpha inhibitor proteins (IAIPs) are a family of structurally related plasma proteins that modulate the systemic inflammatory response to infection and have been shown to attenuate cell death and improve learning outcomes after neonatal brain injury in rats. Here, we show that systemic administration of IAIPs during the early HI injury cascade ameliorates complex auditory discrimination deficits as compared to untreated HI injured subjects, despite reductions in brain weight. These findings have significant clinical implications for improving central auditory processing deficits linked to language learning in neonates with HI related brain injury.

  19. Molecular mediators of hypoxic-ischemic injury and implications for epilepsy in the developing brain.

    PubMed

    Hossain, Mir Ahamed

    2005-09-01

    Perinatal hypoxia-ischemia (HI) is the most common cause of cerebral palsy, and an important consequence of perinatal HI is epilepsy. Epilepsy is a disorder in which the balance between cerebral excitability and inhibition is tipped toward uncontrolled excitability. Selected neuronal circuits as well as certain populations of glial cells die from the excitotoxicity triggered by HI. Excitotoxicity, a term referring to cell death caused by overstimulation of the excitatory glutamate neurotransmitter receptors, plays a critical role in brain injury caused by perinatal HI. Ample evidence suggests distinct differences between the immature and mature brain with respect to the pathology and consequences of hypoxic-ischemic brain injury. Thus, the intrinsic vulnerability of specific cell types and systems in the developing brain is particularly important in determining the final pattern of damage and functional disability caused by perinatal HI. These patterns of neuronal vulnerability are associated with clinical syndromes of neurologic disorders such as cerebral palsy, epilepsy, and seizures. Recent studies have uncovered important molecular and cellular aspects of hypoxic-ischemic brain injury. The cascade of biochemical and histopathological events initiated by HI can extend for days to weeks after the insult is triggered, which may provide a "therapeutic window" for intervening in the pathogenesis in the developing brain. Activation of apoptotic programs accounts for the majority of HI-induced pathophysiology in neonatal brain disorders. New experimental approaches to protecting brain tissue from the effects of neonatal HI include administration of neuronal growth factors and effective inhibition of the death effector pathways, such as caspase cascade, and their downstream targets, which execute apoptosis and/or induction of their regulatory cellular proteins. Our recent findings that a novel neuronal protein, neuronal pentraxin 1 (NP1), is induced following HI in

  20. Adenosine A1 receptors contribute to immune regulation after neonatal hypoxic ischemic brain injury.

    PubMed

    Winerdal, Max; Winerdal, Malin E; Wang, Ying-Qing; Fredholm, Bertil B; Winqvist, Ola; Ådén, Ulrika

    2016-03-01

    Neonatal brain hypoxic ischemia (HI) often results in long-term motor and cognitive impairments. Post-ischemic inflammation greatly effects outcome and adenosine receptor signaling modulates both HI and immune cell function. Here, we investigated the influence of adenosine A1 receptor deficiency (A1R(-/-)) on key immune cell populations in a neonatal brain HI model. Ten-day-old mice were subjected to HI. Functional outcome was assessed by open locomotion and beam walking test and infarction size evaluated. Flow cytometry was performed on brain-infiltrating cells, and semi-automated analysis of flow cytometric data was applied. A1R(-/-) mice displayed larger infarctions (+33%, p < 0.05) and performed worse in beam walking tests (44% more mistakes, p < 0.05) than wild-type (WT) mice. Myeloid cell activation after injury was enhanced in A1R(-/-) versus WT brains. Activated B lymphocytes expressing IL-10 infiltrated the brain after HI in WT, but were less activated and did not increase in relative frequency in A1R(-/-). Also, A1R(-/-) B lymphocytes expressed less IL-10 than their WT counterparts, the A1R antagonist DPCPX decreased IL-10 expression whereas the A1R agonist CPA increased it. CD4(+) T lymphocytes including FoxP3(+) T regulatory cells, were unaffected by genotype, whereas CD8(+) T lymphocyte responses were smaller in A1R(-/-) mice. Using PCA to characterize the immune profile, we could discriminate the A1R(-/-) and WT genotypes as well as sham operated from HI-subjected animals. We conclude that A1R signaling modulates IL-10 expression by immune cells, influences the activation of these cells in vivo, and affects outcome after HI.

  1. Sildenafil inhibits chronically hypoxic upregulation of canonical transient receptor potential expression in rat pulmonary arterial smooth muscle

    PubMed Central

    Lu, Wenju; Zhang, Dandan; Peng, Gongyong; Li, Bing; Zhong, Nanshan

    2010-01-01

    In pulmonary arterial smooth muscle cells (PASMCs), Ca2+ influx through store-operated Ca2+ channels thought to be composed of canonical transient receptor potential (TRPC) proteins is an important determinant of intracellular free calcium concentration ([Ca2+]i) and pulmonary vascular tone. Sildenafil, a type V phosphodiesterase inhibitor that increases cellular cGMP, is recently identified as a promising agent for treatment of pulmonary hypertension. We previously demonstrated that chronic hypoxia elevated basal [Ca2+]i in PASMCs due in large part to enhanced store-operated Ca2+ entry (SOCE); moreover, ex vivo exposure to prolonged hypoxia (4% O2 for 60 h) upregulated TRPC1 and TRPC6 expression in PASMCs. We examined the effect of sildenafil on basal [Ca2+]i, SOCE, and the expression of TRPC in PASMCs under prolonged hypoxia exposure. We also examined the effect of sildenafil on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle (PA) from rats that developed chronically hypoxic pulmonary hypertension (CHPH). Compared with vehicle control, treatment with sildenafil (300 nM) inhibited prolonged hypoxia induced increases of 1) basal [Ca2+]i, 2) SOCE, and 3) mRNA and protein expression of TRPC in PASMCs. Moreover, sildenafil (50 mg · kg−1 · day−1) inhibited mRNA and protein expression of TRPC1 and TRPC6 in PA from chronically hypoxic (10% O2 for 21 days) rats, which was associated with decreased right ventricular pressure and right ventricular hypertrophy. Furthermore, we found, in PASMCs exposed to prolonged hypoxia, that knockdown of TRPC1 or TRPC6 by their specific small interference RNA attenuated the hypoxic increases of SOCE and basal [Ca2+]i, suggesting a cause and effect link between increases of TRPC1 and TRPC6 expression and the hypoxic increases of SOCE and basal [Ca2+]i. These results suggest that sildenafil may alter basal [Ca2+]i in PASMCs by decreasing SOCE through downregulation of TRPC1 and TRPC6 expression, thereby contributing to

  2. Effect of Intermittent Hypoxic Training Followed by Intermittent Hypoxic Exposure on Aerobic Capacity of Long Distance Runners.

    PubMed

    Nakamoto, Fernanda P; Ivamoto, Rafael K; Andrade, Marilia Dos S; de Lira, Claudio A B; Silva, Bruno M; da Silva, Antonio C

    2016-06-01

    Effects of intermittent hypoxic training (IHT) are still controversial and detraining effects remain uninvestigated. Therefore, we investigated (a) whether IHT improves aerobic capacity; (b) whether aerobic detraining occurs post-IHT; and (c) whether intermittent hypoxic exposure (IHE) at rest reduces a possible aerobic detraining post-IHT. Twenty eight runners (21 men/7 women; 36 ± 2 years; maximal oxygen uptake [V[Combining Dot Above]O2max] 55.4 ± 1.3 ml·kg·min) participated in a single-blinded placebo-controlled trial. Twice a week, 1 group performed 6 weeks of IHT (n = 11), followed by 4 weeks of IHE (n = 11) at rest (IHT+IHE group). Another group performed 6 weeks of IHT (n = 10), followed by 4 weeks of normoxic exposure (NE, n = 9) at rest (IHT+NE group). A control group performed 6 weeks of normoxic training (NT, n = 7), followed by 4 weeks of NE (n = 6) at rest (NT+NE group). Hematological and submaximal/maximal aerobic measurements were conducted in normoxia at pretraining, posttraining, and postexposure. Hemoglobin concentration did not change, but lactate threshold and running economy improved in all groups at posttraining (p ≤ 0.05 vs. pretraining). Ventilatory threshold, respiratory compensation point, and V[Combining Dot Above]O2max increased after IHT (IHT+IHE group: 7.3, 5.4, and 9.2%, respectively; IHT+NE group: 10.7, 7.5, and 4.8%; p ≤ 0.05 vs. pretraining), but not after NT (-1.1, -1.0, and -3.8%; p > 0.05 vs. pretraining). Such IHT-induced adaptations were maintained at postexposure (p > 0.05 vs. postexposure). In conclusion, IHT induced further aerobic improvements than NT. These additional IHT adaptations were maintained for 4 weeks post-IHT, regardless of IHE.

  3. The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase.

    PubMed

    Cox, Thomas R; Rumney, Robin M H; Schoof, Erwin M; Perryman, Lara; Høye, Anette M; Agrawal, Ankita; Bird, Demelza; Latif, Norain Ab; Forrest, Hamish; Evans, Holly R; Huggins, Iain D; Lang, Georgina; Linding, Rune; Gartland, Alison; Erler, Janine T

    2015-06-04

    Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia. Tumour-secreted proteins play a crucial role in these interactions and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in patients with oestrogen-receptor negative breast cancer. Global quantitative analysis of the hypoxic secretome identified lysyl oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonize and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications.

  4. The hypoxic cancer secretome induces pre-metastatic bone lesions through lysyl oxidase

    PubMed Central

    Cox, Thomas R.; Rumney, Robin M.H.; Schoof, Erwin M.; Perryman, Lara; Høye, Anette M.; Agrawal, Ankita; Bird, Demelza; Latif, Norain Ab; Forrest, Hamish; Evans, Holly R.; Huggins, Iain D; Lang, Georgina; Linding, Rune

    2016-01-01

    Tumour metastasis is a complex process involving reciprocal interplay between cancer cells and host stroma at both primary and secondary sites, and is strongly influenced by microenvironmental factors such as hypoxia1. Tumour-secreted proteins play a crucial role in these interactions2–5 and present strategic therapeutic potential. Metastasis of breast cancer to the bone affects approximately 85% of patients with advanced disease and renders them largely untreatable6. Specifically, osteolytic bone lesions, where bone is destroyed, lead to debilitating skeletal complications and increased patient morbidity and mortality6,7. The molecular interactions governing the early events of osteolytic lesion formation are currently unclear. Here we show hypoxia to be specifically associated with bone relapse in ER-negative breast cancer patients. Global quantitative analysis of the hypoxic secretome identified Lysyl Oxidase (LOX) as significantly associated with bone-tropism and relapse. High expression of LOX in primary breast tumours or systemic delivery of LOX leads to osteolytic lesion formation whereas silencing or inhibition of LOX activity abrogates tumour-driven osteolytic lesion formation. We identify LOX as a novel regulator of NFATc1-driven osteoclastogenesis, independent of RANK Ligand, which disrupts normal bone homeostasis leading to the formation of focal pre-metastatic lesions. We show that these lesions subsequently provide a platform for circulating tumour cells to colonise and form bone metastases. Our study identifies a novel mechanism of regulation of bone homeostasis and metastasis, opening up opportunities for novel therapeutic intervention with important clinical implications. PMID:26017313

  5. Disturbed hypoxic responses as a pathogenic mechanism of diabetic foot ulcers.

    PubMed

    Catrina, Sergiu-Bogdan; Zheng, Xiaowei

    2016-01-01

    Diabetic foot ulceration (DFU) is a chronic complication of diabetes that is characterized by impaired wound healing in the lower extremities. DFU remains a major clinical challenge because of poor understanding of its pathogenic mechanisms. Impaired wound healing in diabetes is characterized by decreased angiogenesis, reduced bone marrow-derived endothelial progenitor cell (EPC) recruitment, and decreased fibroblast and keratinocyte proliferation and migration. Recently, increasing evidence has suggested that increased hypoxic conditions and impaired cellular responses to hypoxia are essential pathogenic factors of delayed wound healing in DFU. Hypoxia-inducible factor-1 (HIF-1, a heterodimer of HIF-1α and HIF-1β) is a master regulator of oxygen homeostasis that mediates the adaptive cellular responses to hypoxia by regulating the expression of genes involved in angiogenesis, metabolic changes, proliferation, migration, and cell survival. However, HIF-1 signalling is inhibited in diabetes as a result of hyperglycaemia-induced HIF-1α destabilization and functional repression. Increasing HIF-1α expression and activity using various approaches promotes angiogenesis, EPC recruitment, and granulation, thereby improving wound healing in experimental diabetes. The mechanisms underlying HIF-1α regulation in diabetes and the therapeutic strategies targeting HIF-1 signalling for the treatment of diabetic wounds are discussed in this review. Further investigations of the pathways involved in HIF-1α regulation in diabetes are required to advance our understanding of the mechanisms underlying impaired wound healing in diabetes and to provide a foundation for developing novel therapeutic approaches to treat DFU.

  6. Myostatin up-regulation is associated with the skeletal muscle response to hypoxic stimuli.

    PubMed

    Hayot, Maurice; Rodriguez, Julie; Vernus, Barbara; Carnac, Gilles; Jean, Elise; Allen, David; Goret, Lucie; Obert, Philippe; Candau, Robin; Bonnieu, Anne

    2011-01-30

    Myostatin and hypoxia signalling pathways are able to induce skeletal muscle atrophy, but whether a relationship between these two pathways exists is currently unknown. Here, we tested the hypothesis that a potential mechanism for hypoxia effect on skeletal muscle may be through regulation of myostatin. We reported an induction of myostatin expression in muscles of rats exposed to chronic hypoxia. Interestingly, we also demonstrated increased skeletal muscle myostatin protein expression in skeletal muscle of hypoxemic patients with severe chronic obstructive pulmonary disease (COPD). Parallel studies in human skeletal muscle cell cultures showed that induction of myostatin expression in myotubes treated with hypoxia-mimicking agent such as cobalt chloride (CoCl(2)) is associated with myotube atrophy. Furthermore, we demonstrated that inhibition of myostatin by means of genetic deletion of myostatin or treatment with blocking antimyostatin antibodies inhibits the CoCl(2)-induced atrophy in muscle cells. Finally, addition of recombinant myostatin restored the CoCl(2)-induced atrophy in myostatin deficient myotubes. These results strongly suggest that myostatin can play an essential role in the adaptation of skeletal muscle to hypoxic environment.

  7. Novel Genes Critical for Hypoxic Preconditioning in Zebrafish Are Regulators of Insulin and Glucose Metabolism

    PubMed Central

    Manchenkov, Tania; Pasillas, Martina P.; Haddad, Gabriel G.; Imam, Farhad B.

    2015-01-01

    Severe hypoxia is a common cause of major brain, heart, and kidney injury in adults, children, and newborns. However, mild hypoxia can be protective against later, more severe hypoxia exposure via “hypoxic preconditioning,” a phenomenon that is not yet fully understood. Accordingly, we have established and optimized an embryonic zebrafish model to study hypoxic preconditioning. Using a functional genomic approach, we used this zebrafish model to identify and validate five novel hypoxia-protective genes, including irs2, crtc3, and camk2g2, which have been previously implicated in metabolic regulation. These results extend our understanding of the mechanisms of hypoxic preconditioning and affirm the discovery potential of this novel vertebrate hypoxic stress model. PMID:25840431

  8. Ixr1p and the control of the Saccharomyces cerevisiae hypoxic response.

    PubMed

    Vizoso-Vázquez, Angel; Lamas-Maceiras, Mónica; Becerra, Manuel; González-Siso, M Isabel; Rodríguez-Belmonte, Esther; Cerdán, M Esperanza

    2012-04-01

    In Saccharomyces cerevisiae, adaptation to hypoxia/anaerobiosis requires the transcriptional induction or derepression of multiple genes organized in regulons controlled by specific transcriptional regulators. Ixr1p is a transcriptional regulatory factor that causes aerobic repression of several hypoxic genes (COX5B, TIR1, and HEM13) and also the activation of HEM13 during hypoxic growth. Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative Δixr1, grown in aerobic and hypoxic conditions, reveals differential regulation of genes related not only to the hypoxic and oxidative stress responses but also to the re-adaptation of catabolic and anabolic fluxes in response to oxygen limitation. The function of Ixr1p in the transcriptional regulation of genes from the sulfate assimilation pathway and other pathways producing α-keto acids is of biotechnological importance for industries based on yeast-derived fermentation products.

  9. The involvement of reactive oxygen species in hypoxic injury to rat liver.

    PubMed

    Younes, M; Strubelt, O

    1988-03-01

    Isolated perfused livers from fasted, but not from fed rats showed hepatotoxic responses when subjected to 30 min of hypoxia followed by 60 min of reoxygenation. Toxicity was evident by a release of glutamate-pyruvate-transaminase, lactate dehydrogenase and glutathione into the perfusate, by a depletion of hepatic glutathione and by an accumulation of calcium in the liver. This indicates, that the liver is resistant to hypoxic injury as long as glycogen is present to maintain anaerobic ATP-synthesis. This is substantiated by the fact that addition of fructose--but not glucose--to the medium resulted in a protection of the liver against hypoxic injury concomitant with its degradation to lactate + pyruvate. Superoxide dismutase, catalase, desferrioxamine and allopurinol prevented hypoxic liver injury suggesting a substantial role of reactive oxygen species formed via the xanthine oxidase reaction in mediating hypoxic liver injury.

  10. [Age, gender and individually-typological characteristics of reaction to acute hypoxic exposure].

    PubMed

    Krivoshchekov, S G; Balioz, N V; Nekipelova, N V; Kapilevich, L V

    2014-01-01

    Individual pequliarities of hypoxic resistance, assessed by the response of cardiorespiratory system to acute normobaric hypoxia (10% O2), were studied in healthy subjects. Age changes in dynamics of blood oxygen saturation after the acute hypoxia are shown at level of separate sites curve SpO2 (phases of a delay, decrease and lifting). It is established, that at children sensitivity to acute hypoxia above, than at teenagers, and at teenagers above, than at adults. Higher lability of mental processes, sympathetic activity, and personal anxiety are associated with choleric temperament. Cholerics are characterized by slower restoration of blood oxygen saturation after the acute hypoxia compared with sanguine persons that we consider an indication of less hypoxic tolerance of the first group. We have developed the complex algorithm, dynamics describing dependence oxygen saturation in various phases of the hypoxic test, which can be used as a universal method of an estimation hypoxic stability at different groups of the population.

  11. Scalloped hammerhead shark Sphyrna lewini, utilizes deep-water, hypoxic zone in the Gulf of California.

    PubMed

    Jorgensen, S J; Klimley, A P; Muhlia-Melo, A F

    2009-05-01

    A hammerhead shark Sphyrna lewini tracked for 74 days revealed an expansion of the range of vertical distribution for the species to include the extreme hypoxic environment of the oxygen minimum layer in the Gulf of California.

  12. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition

    NASA Astrophysics Data System (ADS)

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S.; Grzes, Katarzyna M.; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D.; Cantrell, Doreen A.; Rocha, Sonia; Ciulli, Alessio

    2016-11-01

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

  13. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition

    PubMed Central

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S.; Grzes, Katarzyna M.; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D.; Cantrell, Doreen A.; Rocha, Sonia; Ciulli, Alessio

    2016-01-01

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein–protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling. PMID:27811928

  14. Potent and selective chemical probe of hypoxic signalling downstream of HIF-α hydroxylation via VHL inhibition.

    PubMed

    Frost, Julianty; Galdeano, Carles; Soares, Pedro; Gadd, Morgan S; Grzes, Katarzyna M; Ellis, Lucy; Epemolu, Ola; Shimamura, Satoko; Bantscheff, Marcus; Grandi, Paola; Read, Kevin D; Cantrell, Doreen A; Rocha, Sonia; Ciulli, Alessio

    2016-11-04

    Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

  15. Modulation of the hypoxic sensory response of the carotid body by 5-hydroxytryptamine: role of the 5-HT2 receptor.

    PubMed

    Jacono, F J; Peng, Y-J; Kumar, G K; Prabhakar, N R

    2005-02-15

    Previous studies have shown that glomus cells of the carotid body express 5-hydroxytryptamine (5-HT). The aim of this study was to elucidate the role of 5-HT on the hypoxic sensory response (HSR) of the carotid body. Sensory activity was recorded from multi-fiber (n=16) and single-fiber (n=8) preparations of ex vivo carotid bodies harvested from anesthetized, adult rats. 5-HT (3 microM) had no significant effect on the magnitude or on the onset of the HSR. However, 5-HT consistently prolonged the time necessary for the sensory activity to return to baseline following the termination of the hypoxic challenge. Ketanserin (40 microM), a 5-HT2 receptor antagonist completely prevented 5-HT-induced prolongation of the HSR, whereas had no effect on the control HSR (onset, magnitude, and time for decay without 5-HT). Carotid bodies expressed 5-HT, but hypoxia did not facilitate 5-HT release. These observations suggest that 5-HT is not critical for the HSR of the rat carotid body, but it modulates the dynamics of the HSR via its action on 5-HT2 receptors.

  16. Antisense suppression of cucumber (Cucumis sativus L.) sucrose synthase 3 (CsSUS3) reduces hypoxic stress tolerance.

    PubMed

    Wang, Hongyun; Sui, Xiaolei; Guo, Jinju; Wang, Zhenyu; Cheng, Jintao; Ma, Si; Li, Xiang; Zhang, Zhenxian

    2014-03-01

    Sucrose synthase (SUS; EC 2.4.1.13) plays important roles in sugar metabolism and abiotic stress response. But the genes encoding SUS in cucumber (Cucumis sativus L.) have not been well studied. Here, we isolated four cucumber sucrose synthase genes (CsSUS). Among them, CsSUS3, which highly expressed in the roots, was chosen for further study. Immunolocalization and subcellular localization analysis indicated that CsSUS3 localized in the cytosol and the plasma membrane, and mainly existed in the companion cells of phloem in the roots. When suffering hypoxia stress from flooding, CsSUS3 expression and SUS activity in roots increased, especially in the lateral roots; moreover, the soluble SUS activity increased clearly, but the membrane fraction hardly changed. Compared with the wild-type cucumbers, the transgenic lines with antisense expression of CsSUS3 were more sensitive to flooding. After 6 d of flooding, the SUS activity, soluble sugar and uridine 5'-diphosphate glucose (UDPG) content and the ratio of ATP/ADP in the roots of transgenic plants were significantly lower than that in wild-type plants. Moreover, the transgenic lines grew more slowly with more yellow necrosis in the leaves. These findings suggested CsSUS3 participated in resisting hypoxic stress. Furthermore, the mechanism of CsSUS3 in resisting hypoxic stress was also discussed.

  17. Hypoxic mitophagy regulates mitochondrial quality and platelet activation and determines severity of I/R heart injury

    PubMed Central

    Zhang, Weilin; Ren, He; Xu, Chunling; Zhu, Chongzhuo; Wu, Hao; Liu, Dong; Wang, Jun; Liu, Lei; Li, Wei; Ma, Qi; Du, Lei; Zheng, Ming; Zhang, Chuanmao; Liu, Junling; Chen, Quan

    2016-01-01

    Mitochondrial dysfunction underlies many prevalent diseases including heart disease arising from acute ischemia/reperfusion (I/R) injury. Here, we demonstrate that mitophagy, which selectively removes damaged or unwanted mitochondria, regulated mitochondrial quality and quantity in vivo. Hypoxia induced extensive mitochondrial degradation in a FUNDC1-dependent manner in platelets, and this was blocked by in vivo administration of a cell-penetrating peptide encompassing the LIR motif of FUNDC1 only in wild-type mice. Genetic ablation of Fundc1 impaired mitochondrial quality and increased mitochondrial mass in platelets and rendered the platelets insensitive to hypoxia and the peptide. Moreover, hypoxic mitophagy in platelets protected the heart from worsening of I/R injury. This represents a new mechanism of the hypoxic preconditioning effect which reduces I/R injury. Our results demonstrate a critical role of mitophagy in mitochondrial quality control and platelet activation, and suggest that manipulation of mitophagy by hypoxia or pharmacological approaches may be a novel strategy for cardioprotection. DOI: http://dx.doi.org/10.7554/eLife.21407.001 PMID:27995894

  18. Sterol Regulatory Element Binding Protein (Srb1) Is Required for Hypoxic Adaptation and Virulence in the Dimorphic Fungus Histoplasma capsulatum

    PubMed Central

    DuBois, Juwen C.; Smulian, A. George

    2016-01-01

    The Histoplasma capsulatum sterol regulatory element binding protein (SREBP), Srb1 is a member of the basic helix-loop-helix (bHLH), leucine zipper DNA binding protein family of transcription factors that possess a unique tyrosine (Y) residue instead of an arginine (R) residue in the bHLH region. We have determined that Srb1 message levels increase in a time dependent manner during growth under oxygen deprivation (hypoxia). To further understand the role of Srb1 during infection and hypoxia, we silenced the gene encoding Srb1 using RNA interference (RNAi); characterized the resulting phenotype, determined its response to hypoxia, and its ability to cause disease within an infected host. Silencing of Srb1 resulted in a strain of H. capsulatum that is incapable of surviving in vitro hypoxia. We found that without complete Srb1 expression, H. capsulatum is killed by murine macrophages and avirulent in mice given a lethal dose of yeasts. Additionally, silencing Srb1 inhibited the hypoxic upregulation of other known H. capsulatum hypoxia-responsive genes (HRG), and genes that encode ergosterol biosynthetic enzymes. Consistent with these regulatory functions, Srb1 silenced H. capsulatum cells were hypersensitive to the antifungal azole drug itraconazole. These data support the theory that the H. capsulatum SREBP is critical for hypoxic adaptation and is required for H. capsulatum virulence. PMID:27711233

  19. Does amifostine reduce metabolic rate? Effect of the drug on gas exchange and acute ventilatory hypoxic response in humans.

    PubMed

    Pandit, Jaideep J; Allen, Caroline; Little, Evelyn; Formenti, Federico; Harris, Adrian L; Robbins, Peter A

    2015-04-16

    Amifostine is added to chemoradiation regimens in the treatment of many cancers on the basis that, by reducing the metabolic rate, it protects normal cells from toxic effects of therapy. We tested this hypothesis by measuring the metabolic rate (by gas exchange) over 255 min in 6 healthy subjects, at two doses (500 mg and 1000 mg) of amifostine infused over 15 min at the start of the protocol. We also assessed the ventilatory response to six 1 min exposures to isocapnic hypoxia mid-protocol. There was no change in metabolic rate with amifostine as measured by oxygen uptake (p = 0.113). However in carbon dioxide output and respiratory quotient, we detected a small decline over time in control and drug protocols, consistent with a gradual change from carbohydrate to fat metabolism over the course of the relatively long study protocol. A novel result was that amifostine (1000 mg) increased the mean ± SD acute hypoxic ventilatory response from 12.4 ± 5.1 L/min to 20.3 ± 11.9 L/min (p = 0.045). In conclusion, any cellular protective effects of amifostine are unlikely due to metabolic effects. The stimulatory effect on hypoxic ventilatory responses may be due to increased levels of hypoxia inducible factor, either peripherally in the carotid body, or centrally in the brain.

  20. [Modification of the radiation injury of hematopoiesis in rats using the gaseous hypoxic mixture GHM-10].

    PubMed

    Zhavoronkov, L P; Sklobovskaia, I E; Strelkov, R B; Chizhov, A Ia

    1986-01-01

    In experiments on Wistar rats it was shown that gas hypoxic mixture containing O2 (10%) and N2 (90%) had a radioprotective action with regard to the survival rate for 30 days and to the haemopoietic system status. The application of gas hypoxic mixture reduced the postirradiation cytopenia in the blood and lowered the degree of the bone marrow depletion by the 3d day following irradiation; DMF was 1.25 as determined by total bone marrow cellularity.

  1. Analysis of Hypoxic and Hypercapnic Ventilatory Response in Healthy Volunteers

    PubMed Central

    Lin, Ling; Sharifi, Husham; Rico, Tom; Andlauer, Olivier; Aran, Adi; Bloomrosen, Efrat; Faraco, Juliette; Fang, Han; Mignot, Emmanuel

    2017-01-01

    Introduction A previous study has suggested that the Human Leukocyte Antigen (HLA) allele DQB1*06:02 affects hypoxic ventilatory response (HVR) but not hypercapnic ventilatory response (HCVR) in an Asian population. The current study evaluated the relationship in Caucasians and Asians. In addition we assessed whether gender or polymorphisms in genes participating in the control of breathing affect HVR and HCVR. Methods A re-breathing system was used to measure HVR and HCVR in 551 young adults (56.8% Caucasians, 30% Asians). HLA-DQB1*06:02 and tagged polymorphisms and coding variants in genes participating in breathing (PHOX2B, GPR4 and TASK2/KCNK5) were analyzed. The associations between HVR/HCVR and HLA-DQB1*06:02, genetic polymorphisms, and gender were evaluated using ANOVA or frequentist association testing with SNPTEST. Results HVR and gender are strongly correlated. HCVR and gender are not. Mean HVR in women was 0.276±0.168 (liter/minute/%SpO2) compared to 0.429±0.266 (liter/minute/%SpO2) in men, p<0.001 (55.4% higher HVR in men). Women had lower baseline minute ventilation (8.08±2.36 l/m vs. 10.00±3.43l/m, p<0.001), higher SpO2 (98.0±1.3% vs. 96.6±1.7%, p<0.001), and lower EtCO2 (4.65±0.68% vs. 4.82±1.02%, p = 0.025). One hundred and two (18.5%) of the participants had HLA-DQB1*06:02. No association was seen between HLA-DQB1*06:02 and HVR or HCVR. Genetic analysis revealed point wise, uncorrected significant associations between two TASK2/KCNK5 variants (rs2815118 and rs150380866) and HCVR. Conclusions This is the largest study to date reporting the relationship between gender and HVR/ HCVR and the first study assessing the association between genetic polymorphisms in humans and HVR/HCVR. The data suggest that gender has a large effect on hypoxic breathing response. PMID:28045995

  2. Effects of intermittent hypoxic training on aerobic and anaerobic performance.

    PubMed

    Morton, James Peter; Cable, Nigel Tim

    The aim of the present study was to determine whether short-term intermittent hypoxic training would enhance sea level aerobic and anaerobic performance over and above that occurring with equivalent sea level training. Over a 4-week period, two groups of eight moderately trained team sports players performed 30 min of cycling exercise three times per week. One group trained in normobaric hypoxia at a simulated altitude of 2750 m (F(I)O2= 0.15), the other group trained in a laboratory under sea level conditions. Each training session consisted of ten 1-min bouts at 80% maximum workload maintained for 2 min (Wmax) during the incremental exercise test at sea level separated by 2-min active recovery at 50% Wmax. Training intensities were increased by 5% after six training sessions and by a further 5% (of original Wmax) after nine sessions. Pre-training assessments of VO(2max), power output at onset of 4 mM blood lactate accumulation (OBLA), Wmax and Wingate anaerobic performance were performed on a cycle ergometer at sea level and repeated 4-7 d following the training intervention. Following training there were significant increases (p < 0.01) in VO(2max) (7.2 vs. 8.0%), Wmax (15.5 vs. 17.8%), OBLA (11.1 vs. 11.9%), mean power (8.0 vs. 6.5%) and peak power (2.9 vs. 9.3%) in both the hypoxic and normoxic groups respectively. There were no significant differences between the increases in any of the above-mentioned performance parameters in either training environment (p > 0.05). In addition, neither haemoglobin concentration nor haematocrit were significantly changed in either group (p > 0.05). It is concluded that acute exposure of moderately trained subjects to normobaric hypoxia during a short-term training programme consisting of moderate- to high-intensity intermittent exercise has no enhanced effect on the degree of improvement in either aerobic or anaerobic performance. These data suggest that if there are any advantages to training in hypoxia for sea level

  3. Hemoglobin Effects on Nitric Oxide Mediated Hypoxic Vasodilation.

    PubMed

    Rong, Zimei; Cooper, Chris E

    2016-01-01

    The brain responds to hypoxia with an increase in cerebral blood flow (CBF). However, such an increase is generally believed to start only after the oxygen tension decreases to a certain threshold level. Although many mechanisms (different vasodilator and different generation and metabolism mechanisms of the vasodilator) have been proposed at the molecular level, none of them has gained universal acceptance. Nitric oxide (NO) has been proposed to play a central role in the regulation of oxygen supply since it is a vasodilator whose production and metabolism are both oxygen dependent. We have used a computational model that simulates blood flow and oxygen metabolism in the brain (BRAINSIGNALS) to test mechanism by which NO may elucidate hypoxic vasodilation. The first model proposed that NO was produced by the enzyme nitric oxide synthase (NOS) and metabolized by the mitochondrial enzyme cytochrome c oxidase (CCO). NO production declined with decreasing oxygen concentration given that oxygen is a substrate for nitric oxide synthase (NOS). However, this was balanced by NO metabolism by CCO, which also declined with decreasing oxygen concentration. However, the NOS effect was dominant; the resulting model profiles of hypoxic vasodilation only approximated the experimental curves when an unfeasibly low K m for oxygen for NOS was input into the model. We therefore modified the model such that NO generation was via the nitrite reductase activity of deoxyhemoglobin instead of NOS, whilst keeping the metabolism of NO by CCO the same. NO production increased with decreasing oxygen concentration, leading to an improved reproduction of the experimental CBF versus PaO2 curve. However, the threshold phenomenon was not perfectly reproduced. In this present work, we incorporated a wider variety of oxygen dependent and independent NO production and removal mechanisms. We found that the addition of NO removal via oxidation to nitrate mediated by oxyhemoglobin resulted in the

  4. Archaeal enrichment in the hypoxic zone in the northern Gulf of Mexico.

    PubMed

    Gillies, Lauren E; Thrash, J Cameron; deRada, Sergio; Rabalais, Nancy N; Mason, Olivia U

    2015-10-01

    Areas of low oxygen have spread exponentially over the past 40 years, and are cited as a key stressor on coastal ecosystems. The world's second largest coastal hypoxic (≤ 2 mg of O2 l(-1)) zone occurs annually in the northern Gulf of Mexico. The net effect of hypoxia is the diversion of energy flow away from higher trophic levels to microorganisms. This energy shunt is consequential to the overall productivity of hypoxic water masses and the ecosystem as a whole. In this study, water column samples were collected at 39 sites in the nGOM, 21 of which were hypoxic. Analysis of the microbial community along a hypoxic to oxic dissolved oxygen gradient revealed that the relative abundance (iTag) of Thaumarchaeota species 16S rRNA genes (> 40% of the microbial community in some hypoxic samples), the absolute abundance (quantitative polymerase chain reaction; qPCR) of Thaumarchaeota 16S rRNA genes and archaeal ammonia-monooxygenase gene copy number (qPCR) were significantly higher in hypoxic samples. Spatial interpolation of the microbial and chemical data revealed a continuous, shelfwide band of low dissolved oxygen waters that were dominated by Thaumarchaeota (and Euryarchaeota), amoA genes and high concentrations of phosphate in the nGOM, thus implicating physicochemical forcing on microbial abundance.

  5. Physiological biomarkers of hypoxic stress in red swamp crayfish Procambarus clarkii from field and laboratory experiments.

    PubMed

    Bonvillain, Christopher P; Rutherford, D Allen; Kelso, William E; Green, Christopher C

    2012-09-01

    The crayfish industry in Louisiana is the largest in the United States, with crayfish frequently harvested from waters that experience episodic or chronic hypoxia (dissolved oxygen [DO]≤ 2 mg/l). We examined physiological biomarkers (hemolymph lactate, glucose, and protein concentrations) of hypoxic stress in the red swamp crayfish Procambarus clarkii from chronically hypoxic natural habitats and laboratory hypoxia experiments. P. clarkii from normoxic and hypoxic areas in the Atchafalaya River Basin were sampled monthly from April to July 2010. Laboratory experiments subjected P. clarkii to severe hypoxia (1 mg/l DO), moderate hypoxia (2 mg/l DO), or normoxic conditions (control: DO>7.5 mg/l) for 12, 24, and 48 h. P. clarkii from normoxic and hypoxic natural habitats did not display significantly different hemolymph lactate or glucose concentrations; however, mean hemolymph protein concentration was significantly lower in crayfish from hypoxic areas. P. clarkii exposed to severe hypoxia in laboratory experiments had significantly higher hemolymph lactate and glucose concentrations for all three exposure times, whereas large differences in protein concentrations were not observed. These results suggest that elevated hemolymph lactate and glucose concentrations are responses to acute hypoxia in P. clarkii, while differences in protein concentrations are the result of chronic hypoxic exposure.