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Sample records for 2-nitroimidazole hypoxic cell

  1. Detection of hypoxic cells with the 2-nitroimidazole, EF5, correlates with early redox changes in rat brain after perinatal hypoxia-ischemia.

    PubMed

    Bergeron, M; Evans, S M; Sharp, F R; Koch, C J; Lord, E M; Ferriero, D M

    1999-01-01

    The hypoxia-dependent activation of nitroheterocyclic drugs by cellular nitroreductases leads to the formation of intracellular adducts between the drugs and cellular macromolecules. Because this covalent binding is maximal in the absence of oxygen, detection of bound adducts provides an assay for estimating the degree of cellular hypoxia in vivo. Using a pentafluorintated derivative of etanidazole called EF5, we studied the distribution of EF5 adducts in seven-day-old rats subjected to different treatments which decrease the level of oxygen in the brain. EF5 solution was administered intraperitoneally 30 min prior to each treatment. The effect of acute and chronic hypoxia on EF5 adduct formation (binding) was studied in the brain of newborn rats exposed to global hypoxia (8% O2 for 30, 90 or 150 min) and in the brain of chronically hypoxic rat pups with congenital cardiac defects (Wistar Kyoto). The effect of combined hypoxia-ischemia was investigated in rat pups subjected to right carotid coagulation and concurrent exposure to 8% O2 for 30, 90 or 150 min. Brains were frozen immediately at the end of each treatment. Using a Cy3-conjugated monoclonal mouse antibody (ELK3-51) raised against EF5 adducts, hypoxic cells within brain regions were visualized by fluorescence immunocytochemistry. Brains from controls or vehicle-injected animals showed no EF5 binding. Notably, brains from animals which were chronically hypoxemic as a result of congenital cardiac defects also showed no EF5 binding. A short exposure (30 min) to hypoxia or to combined hypoxia-ischemia resulted in increased background stain and few scattered cells with low-intensity immunostaining. Acute hypoxia exposure of at least 90-150 min, which in this age animal does not result in frank cellular damage, produced patchy areas of low- to moderate-intensity fluorescence scattered throughout the brain. In contrast, 90-150 min of hypoxia-ischemia was associated with intense immunofluorescence in the

  2. NLP-1: a DNA intercalating hypoxic cell radiosensitizer and cytotoxin

    SciTech Connect

    Panicucci, R.; Heal, R.; Laderoute, K.; Cowan, D.; McClelland, R.A.; Rauth, A.M.

    1989-04-01

    The 2-nitroimidazole linked phenanthridine, NLP-1 (5-(3-(2-nitro-1-imidazoyl)-propyl)-phenanthridinium bromide), was synthesized with the rationale of targeting the nitroimidazole to DNA via the phenanthridine ring. The drug is soluble in aqueous solution (greater than 25 mM) and stable at room temperature. It binds to DNA with a binding constant 1/30 that of ethidium bromide. At a concentration of 0.5 mM, NLP-1 is 8 times more toxic to hypoxic than aerobic cells at 37 degrees C. This concentration is 40 times less than the concentration of misonidazole, a non-intercalating 2-nitroimidazole, required for the same degree of hypoxic cell toxicity. The toxicity of NLP-1 is reduced at least 10-fold at 0 degrees C. Its ability to radiosensitize hypoxic cells is similar to misonidazole at 0 degrees C. Thus the putative targeting of the 2-nitroimidazole, NLP-1, to DNA, via its phenanthridine group, enhances its hypoxic toxicity, but not its radiosensitizing ability under the present test conditions. NLP-1 represents a lead compound for intercalating 2-nitroimidazoles with selective toxicity for hypoxic cells.

  3. Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

    PubMed

    Matthews, J; Adomat, H; Farrell, N; King, P; Koch, C; Lord, E; Palcic, B; Poulin, N; Sangulin, J; Skov, K

    1996-07-01

    The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy. PMID:8763880

  4. Immunocytochemical labelling of aerobic and hypoxic mammalian cells using a platinated derivative of EF5.

    PubMed Central

    Matthews, J.; Adomat, H.; Farrell, N.; King, P.; Koch, C.; Lord, E.; Palcic, B.; Poulin, N.; Sangulin, J.; Skov, K.

    1996-01-01

    The monoclonal antibody ELK3-51 was previously developed to detect adducts of the 2-nitroimidazole EF5. Direct immunofluorescence was used to detect adducts of EF5 or of a platinated derivative cis-[PtCl2(NH3)EF5] in SCCVII cells treated under aerobic or hypoxic conditions. Fluorescence measurements of these cells using both image and flow cytometric methods were compared, giving similar profiles. Platination significantly decreased immunofluorescence levels (approximately 4-fold less than EF5) after 3 h in hypoxia, but also increased levels after exposure in air (approximately 1.5 x) such that the hypoxic ratio decreased from approximately 50 to approximately 13. Platinated EF5 also showed significantly greater cytotoxicity than its parent in both aerobic and hypoxic cells. These results are consistent with targeting of EF5 to DNA, which was confirmed qualitatively by confocal microscopy. Images Figure 1 PMID:8763880

  5. 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. PMID:27580239

  6. 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.

  7. S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid: a model for potential bioreductively activated prodrugs for inhibitors of nitric oxide synthase (NOS) activity.

    PubMed

    Ulhaq, S; Naylor, M A; Chinje, E C; Threadgill, M D; Stratford, I J

    1997-01-01

    Treatment of 1,1-dimethylethyl S-(2-1,1-dimethylethoxycarbonylamino)-5-bromopentanoate with 1-potassio-2-nitroimidazole, followed by deprotection, afforded S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid, which was reduced to S-2-amino-5-(2-aminoimidazol-1-yl)pentanoic acid. This aminoimadazole inhibited rat brain nitric oxide synthase (NOS) activity 3.2 times more potently than did the nitro analogue. Thus S-2-amino-5-(2-nitroimidazol-1-yl)pentanoic acid is a potent prodrug which may be bioreductively activated to a NOS inhibitor in hypoxic solid tumours. PMID:9051114

  8. The Search for New Hypoxic Cell Radiosensitizers

    PubMed Central

    Mansfield, Carl M.; Kimler, Bruce F.; Cheng, C.C.; Abrahams, Iris L.; Podrebarac, Eugene G.; Wittek, Philip J.; Reddy, Eashwer K.

    1980-01-01

    A number of newly synthesized compounds whose chemical structure suggested possible or remotely possible ability to radiosensitize hypoxic mammalian cells were studied in an in-vitro system. Those compounds that were not excluded because of insolubility or extreme cytotoxicity were tested for radiosensitizing ability. The correlation between chemical structure and radiosensitizing ability will be used for the rational design of additional compounds with a high probability of being effective hypoxic cell radiosensitizers. It is hoped that this will contribute to attempts to improve the cure rate of patients with malignant tumors through the use of radiation therapy and hypoxic cell radiosensitizers. PMID:7401185

  9. Activation of radiosensitizers by hypoxic cells.

    PubMed Central

    Olive, P. L.; Durand, R. E.

    1978-01-01

    Hypoxic cells can metabolize nitroheterocyclic compounds to produce toxic intermediates capable of affecting the survival of neighbouring oxygenated cells. Mutagenesis experiments with E. coli WP-2 343 (deficient in nitroreductase) indicated that reduction of nitroheterocyclics outside bacteria causes killing and mutations within bacteria, presumably due to the transfer of the "active" specie (s). Using animal tissue slices to reduce nitrofurans, cultured L-929 cells incubated under aerobic conditions were far more sensitive to the toxic and DNA damaging effects of these drugs. Transfer of the active species also occurs in a tissue-like environment in multicell spheroids where the presence of a hypoxic central core served to convert the nitroheterocyclics to intermediates which also damaged the neighbouring oxygenated cells. PMID:354676

  10. DNA-Targeted 2-Nitroimidazoles: Studies of the Influence of the Phenanthridine-Linked Nitroimidazoles, 2-NLP-3 and 2-NLP-4, on DNA Damage Induced by Ionizing Radiation

    SciTech Connect

    Buchko, Garry W. ); Weinfeld, Michael

    2002-01-01

    The nitroimidazole-linked phenanthridines 2-NLP-3 (5-[3-(2-nitro-1-imidazoyl)-propyl]-phenanthridinium bromide) and 2-NLP-4 (5-[3-(2-nitro-1-imidazoyl)-butyl1]-phenanthridinium bromide) are composed of the radiosensitizer, 2-nitroimidazole, attached to the DNA intercalator phenanthridine via a 3- and 4-carbon linker, respectively. Previous in vitro assays show both compounds to be 10 - 100 times more efficient as hypoxic cell radiosensitizer, misonidazole[Cowan et al., Radiat. Res. 127, 81-89, 1991]. Here we have used a 32P postlabeling assay and 5'-end labeled oligonucleotide assay to compare the radiogenic DNA damage generated in the presence of 2-NLP-3, 2-NLP-4 compared to irradiation in the presence of misonidazole. This may account, at least in part, for the greater cellular radiosensitization shown by the nitroimidazole-linked phenanthridines over misonidazole.

  11. Dendritic cell reprogramming by the hypoxic environment.

    PubMed

    Bosco, Maria Carla; Varesio, Luigi

    2012-12-01

    Myeloid dendritic cells (DCs) are professional antigen-presenting cells central to the orchestration of innate and acquired immunity and the maintenance of self-tolerance. The local microenvironment contributes to the regulation of DC development and functions, and deregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. DC generation from monocytic precursors recruited at sites of inflammation, tissue damage, or neoplasia occurs under condition of low partial oxygen pressure (pO(2), hypoxia). We reviewed the literature addressing the phenotypic and functional changes triggered by hypoxia in monocyte-derived immature (i) and mature (m) DCs. The discussion will revolve around in vitro studies of gene expression profile, which give a comprehensive representation of the complexity of response of these cells to low pO(2). The gene expression pattern of hypoxic DC will be discussed to address the question of the relationship with a specific maturation stage. We will summarize data relative to the regulation of the chemotactic network, which points to a role for hypoxia in promoting a migratory phenotype in iDCs and a highly proinflammatory state in mDCs. Current knowledge of the strict regulatory control exerted by hypoxia on the expression of immune-related cell surface receptors will also be addressed, with a particular focus on a newly identified marker of hypoxic DCs endowed with proinflammatory properties. Furthermore, we discuss the literature on the transcription mechanisms underlying hypoxia-regulated gene expression in DCs, which support a major role for the HIF/HRE pathway. Finally, recent advances shedding light on the in vivo influence of the local hypoxic microenvironment on DCs infiltrating the inflamed joints of juvenile idiopathic arthritis patients are outlined. PMID:22901977

  12. Hypoxic cell turnover in different solid tumor lines

    SciTech Connect

    Ljungkvist, Anna S.E. . E-mail: a.ljungkvist@rther.umcn.nl; Bussink, Johan; Kaanders, Johannes H.A.M.; Rijken, Paulus F.J.W.; Begg, Adrian C.; Raleigh, James A.; Kogel, Albert J. van der

    2005-07-15

    Purpose: Most solid tumors contain hypoxic cells, and the amount of tumor hypoxia has been shown to have a negative impact on the outcome of radiotherapy. The efficacy of combined modality treatments depends both on the sequence and timing of the treatments. Hypoxic cell turnover in tumors may be important for optimal scheduling of combined modality treatments, especially when hypoxic cell targeting is involved. Methods and Materials: Previously we have shown that a double bioreductive hypoxic marker assay could be used to detect changes of tumor hypoxia in relation to the tumor vasculature after carbogen and hydralazine treatments. This assay was used in the current study to establish the turnover rate of hypoxic cells in three different tumor models. The first hypoxic marker, pimonidazole, was administered at variable times before tumor harvest, and the second hypoxic marker, CCI-103F, was injected at a fixed time before harvest. Hypoxic cell turnover was defined as loss of pimonidazole (first marker) relative to CCI-103F (second marker). Results: The half-life of hypoxic cell turnover was 17 h in the murine C38 colon carcinoma line, 23 h and 49 h in the human xenograft lines MEC82 and SCCNij3, respectively. Within 24 h, loss of pimonidazole-stained areas in C38 and MEC82 occurred concurrent with the appearance of pimonidazole positive cell debris in necrotic regions. In C38 and MEC82, most of the hypoxic cells had disappeared after 48 h, whereas in SCCNij3, viable cells that had been labeled with pimonidazole were still observed after 5 days. Conclusions: The present study demonstrates that the double hypoxia marker assay can be used to study changes in both the proportion of hypoxic tumor cells and their lifespan at the same time. The present study shows that large differences in hypoxic cell turnover rates may exist among tumor lines, with half-lives ranging from 17-49 h.

  13. The Effect of Hypoxic Preconditioning on Induced Schwann Cells under Hypoxic Conditions

    PubMed Central

    Chen, Ou; Wu, Miaomiao; Jiang, Liangfu

    2015-01-01

    Object Our objective was to explore the protective effects of hypoxic preconditioning on induced Schwann cells exposed to an environment with low concentrations of oxygen. It has been observed that hypoxic preconditioning of induced Schwann cells can promote axonal regeneration under low oxygen conditions. Method Rat bone marrow mesenchymal stem cells (MSCs) were differentiated into Schwann cells and divided into a normal oxygen control group, a hypoxia-preconditioning group and a hypoxia group. The ultrastructure of each of these groups of cells was observed by electron microscopy. In addition, flow cytometry was used to measure changes in mitochondrial membrane potential. Annexin V-FITC/PI staining was used to detect apoptosis, and Western blots were used to detect the expression of Bcl-2/Bax. Fluorescence microscopic observations of axonal growth in NG-108 cells under hypoxic conditions were also performed. Results The hypoxia-preconditioning group maintained mitochondrial cell membrane and crista integrity, and these cells exhibited less edema than the hypoxia group. In addition, the cells in the hypoxia-preconditioning group were found to be in early stages of apoptosis, whereas cells from the hypoxia group were in the later stages of apoptosis. The hypoxia-preconditioning group also had higher levels of Bcl-2/Bax expression and longer NG-108 cell axons than were observed in the hypoxia group. Conclusion Hypoxic preconditioning can improve the physiological state of Schwann cells in a severe hypoxia environment and improve the ability to promote neurite outgrowth. PMID:26509259

  14. Therapeutic attack of hypoxic cells of solid tumors: presidential address.

    PubMed

    Sartorelli, A C

    1988-02-15

    Hypoxic cells of solid tumors are relatively resistant to therapeutic assault. Studies have demonstrated that oxygen-deficient tumor cells exist in an environment conducive to reductive reactions making hypoxic cells particularly sensitive to bioreductive alkylating agents. Mitomycin C, the prototype bioreductive alkylating agent available for clinical use, is capable of preferentially killing oxygen-deficient cells both in vitro and in vivo. This phenomenon is at least in part the result of differences in the uptake and metabolism of mitomycin C by hypoxic and oxygenated tumor cells, with the ultimate critical lesion being the cross-linking of DNA by the mitomycin antibiotic. The combination of mitomycin C with X-irradiation, to attack hypoxic and oxygenated tumor cell populations, respectively, has led to enhanced antitumor effects in mice bearing solid tumor implants and in patients with cancer of the head and neck. More efficacious kill of hypoxic tumor cells may be possible by the use of dicoumarol in combination with mitomycin or by the use of the related antibiotic porfiromycin. The findings support the use of an agent with specificity for hypoxic tumor cells in potentially curative regimens for solid tumors. PMID:3123053

  15. Tracking hypoxic signaling within encapsulated cell aggregates.

    PubMed

    Skiles, Matthew L; Sahai, Suchit; Blanchette, James O

    2011-01-01

    , is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 10(10) pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion. Western blot analysis of encapsulated cells in 20% and 1% oxygen also

  16. Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

    PubMed Central

    Skiles, Matthew L.; Sahai, Suchit; Blanchette, James O.

    2011-01-01

    nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact β-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80° C as many 150 μL aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated

  17. Targeting tumor hypoxia with 2-nitroimidazole-indocyanine green dye conjugates

    PubMed Central

    Xu, Yan; Zanganeh, Saeid; Mohammad, Innus; Aguirre, Andres; Wang, Tianheng; Yang, Yi; Kuhn, Liisa; Smith, Michael B.

    2013-01-01

    Abstract. Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs, and fluorescence optical tomography has tremendous potential to provide clinically useful, functional information by identifying tumor hypoxia. The synthesis of a 2-nitroimidazole-indocyanine green conjugate using a piperazine linker (piperazine-2-nitroimidazole-ICG) capable of robust fluorescent imaging of tumor hypoxia is described. In vivo mouse tumor imaging studies were completed and demonstrate an improved imaging capability of the new dye relative to an earlier version of the dye that was synthesized with an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG). Mouse tumors located at imaging depths of 1.5 and 2.0 cm in a turbid medium were imaged at various time points after intravenous injection of the dyes. On average, the reconstructed maximum fluorescence concentration of the tumors injected with piperazine-2-nitroimidazole-ICG was twofold higher than that injected with ethanolamine-2-nitroimidazole-ICG within 3 h postinjection period and 1.6 to 1.7 times higher beyond 3 h postinjection. The untargeted bis-carboxylic acid ICG completely washed out after 3 h postinjection. Thus, the optimal window to assess tumor hypoxia is beyond 3 h postinjection. These findings were supported with fluorescence images of histological sections of tumor samples and an immunohistochemistry technique for identifying tumor hypoxia. PMID:23764695

  18. Targeting tumor hypoxia with 2-nitroimidazole-indocyanine green dye conjugates

    NASA Astrophysics Data System (ADS)

    Xu, Yan; Zanganeh, Saeid; Mohammad, Innus; Aguirre, Andres; Wang, Tianheng; Yang, Yi; Kuhn, Liisa; Smith, Michael B.; Zhu, Quing

    2013-06-01

    Tumor hypoxia is a major indicator of treatment resistance to chemotherapeutic drugs, and fluorescence optical tomography has tremendous potential to provide clinically useful, functional information by identifying tumor hypoxia. The synthesis of a 2-nitroimidazole-indocyanine green conjugate using a piperazine linker (piperazine-2-nitroimidazole-ICG) capable of robust fluorescent imaging of tumor hypoxia is described. In vivo mouse tumor imaging studies were completed and demonstrate an improved imaging capability of the new dye relative to an earlier version of the dye that was synthesized with an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG). Mouse tumors located at imaging depths of 1.5 and 2.0 cm in a turbid medium were imaged at various time points after intravenous injection of the dyes. On average, the reconstructed maximum fluorescence concentration of the tumors injected with piperazine-2-nitroimidazole-ICG was twofold higher than that injected with ethanolamine-2-nitroimidazole-ICG within 3 h postinjection period and 1.6 to 1.7 times higher beyond 3 h postinjection. The untargeted bis-carboxylic acid ICG completely washed out after 3 h postinjection. Thus, the optimal window to assess tumor hypoxia is beyond 3 h postinjection. These findings were supported with fluorescence images of histological sections of tumor samples and an immunohistochemistry technique for identifying tumor hypoxia.

  19. Development of a real-time imaging system for hypoxic cell apoptosis

    PubMed Central

    Kagiya, Go; Ogawa, Ryohei; Hyodo, Fuminori; Yamashita, Kei; Nakamura, Mizuki; Ishii, Ayumi; Sejimo, Yukihiko; Tominaga, Shintaro; Murata, Masaharu; Tanaka, Yoshikazu; Hatashita, Masanori

    2016-01-01

    Hypoxic regions within the tumor form due to imbalances between cell proliferation and angiogenesis; specifically, temporary closure or a reduced flow due to abnormal vasculature. They create environments where cancer cells acquire resistance to therapies. Therefore, the development of therapeutic approaches targeting the hypoxic cells is one of the most crucial challenges for cancer regression. Screening potential candidates for effective diagnostic modalities even under a hypoxic environment would be an important first step. In this study, we describe the development of a real-time imaging system to monitor hypoxic cell apoptosis for such screening. The imaging system is composed of a cyclic luciferase (luc) gene under the control of an improved hypoxic-responsive promoter. The cyclic luc gene product works as a caspase-3 (cas-3) monitor as it gains luc activity in response to cas-3 activation. The promoter composed of six hypoxic responsible elements and the CMV IE1 core promoter drives the effective expression of the cyclic luc gene in hypoxic conditions, enhancing hypoxic cell apoptosis visualization. We also confirmed real-time imaging of hypoxic cell apoptosis in the spheroid, which shares properties with the tumor. Thus, this constructed system could be a powerful tool for the development of effective anticancer diagnostic modalities. PMID:26966700

  20. Correlation between drug uptake and selective toxicity of porfiromycin to hypoxic EMT6 cells.

    PubMed

    Keyes, S R; Rockwell, S; Sartorelli, A C

    1987-11-01

    Mitomycin C and its methylated analogue porfiromycin (Por) have significant potential as adjuncts to regimens presently used for treating solid tumors because of their preferential toxicity to cells existing in an hypoxic environment. An understanding of the factors producing the differential activity of these drugs under aerobic and hypoxic conditions would facilitate the development of new agents of this class. Previous studies have focused on the enzymes that reductively activate the mitomycins and on the interaction of these drugs with DNA; none of these studies has fully explained the differences in cytotoxicity observed under hypoxic and aerobic conditions. The present investigation demonstrates that the rate of Por uptake is directly correlated with cytotoxicity under both aerobic and hypoxic conditions. Uptake of Por into hypoxic cells is more rapid than into aerobic cells at equal drug concentrations. Hypoxic cells also accumulate drug in concentrations well in excess of those in the extracellular medium; this is apparently a reflection of drug sequestration in these cells. This sequestration of Por, which affects the rate and extent of uptake in hypoxic cells, does not take place in aerobic cells. The failure of aerobic cells to sequester drug is evidenced by the very rapid efflux of Por from these cells upon removal of extracellular Por and by the fact that aerobic cells attain a state of equilibrium between the intracellular and extracellular drug concentrations. The findings demonstrate that differences in the uptake and retention of Por are associated with the preferential toxicity of Por to hypoxic cells. PMID:3664473

  1. 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.

  2. Some factors affecting the specific toxicity of misonidazole towards hypoxic mammalian cells.

    PubMed Central

    Stratford, I. J.; Gray, P.

    1978-01-01

    The toxic action of misonidazole towards hypoxic mammalian cells has been shown to be a function of serum concentration, with higher serum concentrations enhancing the toxic effect. Added thiols protect cells against misonidazole toxicity. In addition, the action of misonidazole on hypoxic cells labelled with 5-BUdR has been examined. Cells with incroported 5-BUdR are no more sensitive to misonidazole toxicity than are cells without label. PMID:277212

  3. Stem Cell Therapy for Neonatal Hypoxic-Ischemic Encephalopathy

    PubMed Central

    Gonzales-Portillo, Gabriel S.; Reyes, Stephanny; Aguirre, Daniela; Pabon, Mibel M.; Borlongan, Cesar V.

    2014-01-01

    Treatments for neonatal hypoxic-ischemic encephalopathy (HIE) have been limited. The aim of this paper is to offer translational research guidance on stem cell therapy for neonatal HIE by examining clinically relevant animal models, practical stem cell sources, safety and efficacy of endpoint assays, as well as a general understanding of modes of action of this cellular therapy. In order to do so, we discuss the clinical manifestations of HIE, highlighting its overlapping pathologies with stroke and providing insights on the potential of cell therapy currently investigated in stroke, for HIE. To this end, we draw guidance from recommendations outlined in stem cell therapeutics as an emerging paradigm for stroke or STEPS, which have been recently modified to Baby STEPS to cater for the “neonatal” symptoms of HIE. These guidelines recognized that neonatal HIE exhibit distinct disease symptoms from adult stroke in need of an innovative translational approach that facilitates the entry of cell therapy in the clinic. Finally, new information about recent clinical trials and insights into combination therapy are provided with the vision that stem cell therapy may benefit from available treatments, such as hypothermia, already being tested in children diagnosed with HIE. PMID:25161645

  4. Screening and identification of a specific peptide for targeting hypoxic hepatoma cells.

    PubMed

    Liu, Yiming; Xia, Xiangwen; Wang, Yong; Li, Xin; Zhou, Guofeng; Liang, Huiming; Feng, Gansheng; Zheng, Chuansheng

    2016-08-01

    The biological behaviors of residual hepatoma cells after transarterial embolization therapy, which exist in a hypoxic or even anaerobic tumor microenvironment, differ from the tumor cells under normoxic conditions. This study aimed to use a phage display peptide library for in vivo and in vitro screening to obtain a peptide which could specifically bind to hypoxic hepatoma cells, allowing further targeted diagnosis and treatment for liver cancer. In this study, hypoxic hepatoma cells HepG2 (targeted cells), and normal liver cells HL-7702 (control cells), were utilized to perform three rounds of in vitro screening using a phage-displayed 7-mer peptide library. In addition, hypoxic HepG2 were subcutaneously injected into nude mice to establish a hepatocarcinoma model, followed by performing three rounds of in vivo screening on the phages identified from the in vitro screening. The products from the screening were further identified using ELISA and immunofluorescence staining on cells and tissues. The results indicated that the P11 positive clone had the highest binding effect with hypoxic hepatoma cells. The sequence of the exogenous insert fragment of P11 positive clone was obtained by sequencing: GSTSFSK. The binding assay indicated that GSTSFSK could specifically bind to hypoxic hepatoma cells and hepatocarcinoma tissues. This 7-mer peptide has the potential to be developed as an useful molecular to the targeting diagnosis and treatment of residual hepatoma cells after transarterial chemoembolization. PMID:27381416

  5. Puerarin Induces Mitochondria-Dependent Apoptosis in Hypoxic Human Pulmonary Arterial Smooth Muscle Cells

    PubMed Central

    Chen, Chan; Chen, Chun; Wang, Zhiyi; Wang, Liangxing; Yang, Lehe; Ding, Minjiao; Ding, Cheng; Sun, Yu; Lin, Quan; Huang, Xiaoying; Du, Xiaohong; Zhao, Xiaowei; Wang, Chuangyi

    2012-01-01

    Background Pulmonary vascular medial hypertrophy in hypoxic pulmonary arterial hypertension (PAH) is caused in part by decreased apoptosis in pulmonary artery smooth muscle cells (PASMCs). Puerarin, an isoflavone purified from the Chinese medicinal herb kudzu, ameliorates chronic hypoxic PAH in animal models. Here we investigated the effects of puerarin on apoptosis of hypoxic human PASMCs (HPASMCs), and to determine the possible underlying mechanisms. Methodology/Principal Findings HPASMCs were cultured for 24 h in normoxia or hypoxia (5% O2) conditions with and without puerarin. Cell number and viability were determined with a hemacytometer or a cell counting kit. Apoptosis was detected with a TUNEL test, rhodamine-123 (R-123) fluorescence, a colorimetric assay, western blots, immunohistochemical staining and RT-PCR. Hypoxia inhibited mitochondria-dependent apoptosis and promoted HPASMC growth. In contrast, after puerarin (50 µM or more) intervention, cell growth was inhibited and apoptosis was observed. Puerarin-induced apoptosis in hypoxic HPASMCs was accompanied by reduced mitochondrial membrane potential, cytochrome c release from the mitochondria, caspase-9 activation, and Bcl-2 down-regulation with concurrent Bax up-regulation. Conclusions/Significance Puerarin promoted apoptosis in hypoxic HPASMCs by acting on the mitochondria-dependent pathway. These results suggest a new mechanism of puerarin relevant to the management of clinical hypoxic pulmonary hypertension. PMID:22457823

  6. Targeting tumor hypoxia: a third generation 2-nitroimidazole-indocyanine dye-conjugate with improved fluorescent yield.

    PubMed

    Zhou, Feifei; Zanganeh, Saeid; Mohammad, Innus; Dietz, Christopher; Abuteen, Akram; Smith, Michael B; Zhu, Quing

    2015-12-14

    Tumor hypoxia is associated with the rapid proliferation and growth of malignant tumors, and the ability to detect tumor hypoxia is important for predicting tumor response to anti-cancer treatments. We have developed a class of dye-conjugates that are related to indocyanine green (ICG, ) to target tumor hypoxia, based on in vivo infrared fluorescence imaging using nitroimidazole moieties linked to indocyanine fluorescent dyes. We previously reported that linking 2-nitroimidazole to an indocyanine dicarboxylic acid dye derivative () using an ethanolamine linker (ethanolamine-2-nitroimidazole-ICG, ), led to a dye-conjugate that gave promising results for targeting cancer hypoxia in vivo. Structural modification of the dye conjugate replaced the ethanolamine unit with a piperazineacetyl unit and led a second generation dye conjugate, piperzine-2-nitroimidazole-ICG (). This second generation dye-conjugate showed improved targeting of tumor hypoxia when compared with . Based on the hypothesis that molecules with more planar and rigid structures have a higher fluorescence yield, as they could release less absorbed energy through molecular vibration or collision, we have developed a new 2-nitroimidazole ICG conjugate, , with two carbon atoms less in the polyene linker. Dye-conjugate was prepared from our new dye (), and coupled to 2-nitroimidazole using a piperazine linker to produce this third-generation dye-conjugate. Spectral measurements showed that the absorption/emission wavelengths of 657/670 were shifted ∼100 nm from the second-generation hypoxia dye of 755/780 nm. Its fluorescence quantum yield was measured to be 0.467, which is about 5 times higher than that of (0.083). In vivo experiments were conducted with balb/c mice and showed more than twice the average in vivo fluorescence intensity in the tumor beyond two hours post retro-orbital injection as compared with . These initial results suggest that may significantly improve in vivo tumor hypoxia targeting

  7. Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression.

    PubMed

    Kagiya, Go; Ogawa, Ryohei; Choudhuri, Rajani; Cook, John A; Hatashita, Masanori; Tanaka, Yoshikazu; Koda, Kana; Yamashita, Kei; Kubo, Makoto; Kawakami, Fumitaka; Mitchell, James B

    2015-08-01

    The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication. PMID:26034980

  8. Characterization of radiation resistant hypoxic cell subpopulations in KHT sarcomas. (II). Cell sorting.

    PubMed Central

    Siemann, D. W.; Keng, P. C.

    1988-01-01

    Hypoxic cells in KHT sarcomas were characterized using fluorescence activated cell sorting based on the diffusion properties of the fluorochrome Hoechst 33342. Tumour-bearing female C3H/HeJ mice were injected i.v. with 10 micrograms g-1 Hoechst 33342 and the cells derived from the tumours sorted on the basis of their staining intensities. For each sorted fraction the DNA histogram was evaluated using FCM analysis. The results indicated that the bright and dim cells were not equally distributed about the cell cycle. For example, a greater proportion of S phase cells were in the bright subpopulations whereas the dim subpopulations contained an increased proportion of cells in G1. When the tumours were irradiated with a single dose of radiation prior to cell sorting, the dim cells survived preferentially. Dose response curves for the 20% most dim and 20% most bright cells, sorted on the basis of fluorescence intensity, then were determined. The survival curves of the dim and bright cells were found to have slopes similar to those of KHT cells irradiated in situ in dead animals or in vitro under fully oxic conditions, respectively. In addition, when KHT sarcoma-bearing mice were given a 2.5 mmol kg-1 dose of misonidazole (MISO) prior to irradiation and cell sorting, the dim subpopulation was sensitized whereas the bright subpopulation was not. These findings suggest that (i) compared to well-oxygenated areas, hypoxic regions of KHT tumours contain a smaller percentage of cells actively proliferating and (ii) Hoechst 33342 sorting may allow the detailed in situ evaluation of agents acting directly against hypoxic cells in solid tumours. PMID:3179180

  9. C-1027, a radiomimetic enediyne anticancer drug, preferentially targets hypoxic cells.

    PubMed

    Beerman, Terry A; Gawron, Loretta S; Shin, Seulkih; Shen, Ben; McHugh, Mary M

    2009-01-15

    The hypoxic nature of cells within solid tumors limits the efficacy of anticancer therapies such as ionizing radiation and conventional radiomimetics because their mechanisms require oxygen to induce lethal DNA breaks. For example, the conventional radiomimetic enediyne neocarzinostatin is 4-fold less cytotoxic to cells maintained in low oxygen (hypoxic) compared with normoxic conditions. By contrast, the enediyne C-1027 was nearly 3-fold more cytotoxic to hypoxic than to normoxic cells. Like other radiomimetics, C-1027 induced DNA breaks to a lesser extent in cell-free, or cellular hypoxic, compared with normoxic environments. However, the unique DNA interstrand cross-linking ability of C-1027 was markedly enhanced under the same hypoxic conditions that reduced its DNA break induction. Although the unique chemistry of C-1027 allows it to concurrently generate both DNA breaks and cross-links in normoxic cells, a low oxygen environment represses the former and promotes the latter. Thus, treatment with C-1027 offers a facile approach for overcoming the radioresistance associated with poorly oxygenated cells. PMID:19147573

  10. The Immune-Metabolic Basis of Effector Memory CD4+ T Cell Function under Hypoxic Conditions.

    PubMed

    Dimeloe, Sarah; Mehling, Matthias; Frick, Corina; Loeliger, Jordan; Bantug, Glenn R; Sauder, Ursula; Fischer, Marco; Belle, Réka; Develioglu, Leyla; Tay, Savaş; Langenkamp, Anja; Hess, Christoph

    2016-01-01

    Effector memory (EM) CD4(+) T cells recirculate between normoxic blood and hypoxic tissues to screen for cognate Ag. How mitochondria of these cells, shuttling between normoxia and hypoxia, maintain bioenergetic efficiency and stably uphold antiapoptotic features is unknown. In this study, we found that human EM CD4(+) T cells had greater spare respiratory capacity (SRC) than did naive counterparts, which was immediately accessed under hypoxia. Consequently, hypoxic EM cells maintained ATP levels, survived and migrated better than did hypoxic naive cells, and hypoxia did not impair their capacity to produce IFN-γ. EM CD4(+) T cells also had more abundant cytosolic GAPDH and increased glycolytic reserve. In contrast to SRC, glycolytic reserve was not tapped under hypoxic conditions, and, under hypoxia, glucose metabolism contributed similarly to ATP production in naive and EM cells. However, both under normoxic and hypoxic conditions, glucose was critical for EM CD4(+) T cell survival. Mechanistically, in the absence of glycolysis, mitochondrial membrane potential (ΔΨm) of EM cells declined and intrinsic apoptosis was triggered. Restoring pyruvate levels, the end product of glycolysis, preserved ΔΨm and prevented apoptosis. Furthermore, reconstitution of reactive oxygen species (ROS), whose production depends on ΔΨm, also rescued viability, whereas scavenging mitochondrial ROS exacerbated apoptosis. Rapid access of SRC in hypoxia, linked with built-in, oxygen-resistant glycolytic reserve that functionally insulates ΔΨm and mitochondrial ROS production from oxygen tension changes, provides an immune-metabolic basis supporting survival, migration, and function of EM CD4(+) T cells in normoxic and hypoxic conditions. PMID:26621861

  11. Keynote address: cellular reduction of nitroimidazole drugs: potential for selective chemotherapy and diagnosis of hypoxic cells.

    PubMed

    Chapman, J D; Lee, J; Meeker, B E

    1989-04-01

    Nitroimidazole drugs were initially developed as selective radiosensitizers of hypoxic cells and, consequently, as adjuvants to improve the local control probabilities of current radiotherapies. Misonidazole (MISO), the prototype radiosensitizing drug, was found in Phase I clinical studies to cause dose-limiting neurotoxicities (mainly peripheral neuropathies). MISO was also found to be cytotoxic in the absence of radiation and to covalently bind to cellular molecules, both processes demonstrating rates much higher in hypoxic compared with oxygenated cells. It is likely that neurotoxicity, cellular cytotoxicity and adduct formation results from reactions between reduction intermediates of MISO and cellular target molecules. Spin-offs from radiosensitizer research include the synthesis and characterization of more potent hypoxic cytotoxins and the exploitation of sensitizer-adducts as probes for measuring cellular and tissue oxygen levels. Current developments in hypoxic cell cytotoxin and hypoxic cell marker research are reviewed with specific examples from studies which characterize the cellular reduction of TF-MISO, (1-(2-nitro-1-imidazolyl)-3[2,2,2-trifluoroethoxy]-2-propanol). PMID:2649465

  12. Hypoxic stress up-regulates Kir2.1 expression and facilitates cell proliferation in brain capillary endothelial cells.

    PubMed

    Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao; Asai, Kiyofumi; Imaizumi, Yuji

    2016-08-01

    The blood-brain barrier (BBB) is mainly composed of brain capillary endothelial cells (BCECs), astrocytes and pericytes. Brain ischemia causes hypoxic encephalopathy and damages BBB. However, it remains still unclear how hypoxia affects BCECs. In the present study, t-BBEC117 cells, an immortalized bovine brain endothelial cell line, were cultured under hypoxic conditions at 4-5% oxygen for 72 h. This hypoxic stress caused hyperpolarization of resting membrane potential. Patch-clamp recordings revealed a marked increase in Ba(2+)-sensitive inward rectifier K(+) current in t-BBEC117 cells after hypoxic culture. Western blot and real-time PCR analyses showed that Kir2.1 expression was significantly up-regulated at protein level but not at mRNA level after the hypoxic culture. Ca(2+) imaging study revealed that the hypoxic stress enhanced store-operated Ca(2+) (SOC) entry, which was significantly reduced in the presence of 100 μM Ba(2+). On the other hand, the expression of SOC channels such as Orai1, Orai2, and transient receptor potential channels was not affected by hypoxic stress. MTT assay showed that the hypoxic stress significantly enhanced t-BBEC117 cell proliferation, which was inhibited by approximately 60% in the presence of 100 μM Ba(2+). We first show here that moderate cellular stress by cultivation under hypoxic conditions hyperpolarizes membrane potential via the up-regulation of functional Kir2.1 expression and presumably enhances Ca(2+) entry, resulting in the facilitation of BCEC proliferation. These findings suggest potential roles of Kir2.1 expression in functional changes of BCECs in BBB following ischemia. PMID:27235552

  13. 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.

  14. 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.

  15. Global Profiling of Metabolic Adaptation to Hypoxic Stress in Human Glioblastoma Cells

    PubMed Central

    Kucharzewska, Paulina; Christianson, Helena C.; Belting, Mattias

    2015-01-01

    Oncogenetic events and unique phenomena of the tumor microenvironment together induce adaptive metabolic responses that may offer new diagnostic tools and therapeutic targets of cancer. Hypoxia, or low oxygen tension, represents a well-established and universal feature of the tumor microenvironment and has been linked to increased tumor aggressiveness as well as resistance to conventional oncological treatments. Previous studies have provided important insights into hypoxia induced changes of the transcriptome and proteome; however, how this translates into changes at the metabolite level remains to be defined. Here, we have investigated dynamic, time-dependent effects of hypoxia on the cancer cell metabolome across all families of macromolecules, i.e., carbohydrate, protein, lipid and nucleic acid, in human glioblastoma cells. Using GC/MS and LC/MS/MS, 345 and 126 metabolites were identified and quantified in cells and corresponding media, respectively, at short (6 h), intermediate (24 h), and prolonged (48 h) incubation at normoxic or hypoxic (1% O2) conditions. In conjunction, we performed gene array studies with hypoxic and normoxic cells following short and prolonged incubation. We found that levels of several key metabolites varied with the duration of hypoxic stress. In some cases, metabolic changes corresponded with hypoxic regulation of key pathways at the transcriptional level. Our results provide new insights into the metabolic response of glioblastoma cells to hypoxia, which should stimulate further work aimed at targeting cancer cell adaptive mechanisms to microenvironmental stress. PMID:25633823

  16. Increased betulinic acid induced cytotoxicity and radiosensitivity in glioma cells under hypoxic conditions

    PubMed Central

    2011-01-01

    Background Betulinic acid (BA) is a novel antineoplastic agent under evaluation for tumor therapy. Because of the selective cytotoxic effects of BA in tumor cells (including gliomas), the combination of this agent with conservative therapies (such as radiotherapy and chemotherapy) may be useful. Previously, the combination of BA with irradiation under hypoxic conditions had never been studied. Methods In this study, the effects of 3 to 30 μM BA on cytotoxicity, migration, the protein expression of PARP, survivin and HIF-1α, as well as radiosensitivity under normoxic and hypoxic conditions were analyzed in the human malignant glioma cell lines U251MG and U343MG. Cytotoxicity and radiosensitivity were analyzed with clonogenic survival assays, migration was analyzed with Boyden chamber assays (or scratch assays) and protein expression was examined with Western blot analyses. Results Under normoxic conditions, a half maximal inhibitory concentration (IC50) of 23 μM was observed in U251MG cells and 24 μM was observed in U343MG cells. Under hypoxic conditions, 10 μM or 15 μM of BA showed a significantly increased cytotoxicity in U251MG cells (p = 0.004 and p = 0.01, respectively) and U343MG cells (p < 0.05 and p = 0.01, respectively). The combination of BA with radiotherapy resulted in an additive effect in the U343MG cell line under normoxic and hypoxic conditions. Weak radiation enhancement was observed in U251MG cell line after treatment with BA under normoxic conditions. Furthermore, under hypoxic conditions, the incubation with BA resulted in increased radiation enhancement. The enhancement factor, at an irradiation dose of 15 Gy after treatment with 10 or 15 μM BA, was 2.20 (p = 0.02) and 4.50 (p = 0.03), respectively. Incubation with BA led to decreased cell migration, cleavage of PARP and decreased expression levels of survivin in both cell lines. Additionally, BA treatment resulted in a reduction of HIF-1α protein under hypoxic conditions. Conclusion Our

  17. Modification of SR 2508 sensitization in hypoxic V79 cells by manipulation of glutathione levels

    SciTech Connect

    Phillips, T.L.; Mitchell, J.B.; DeGraff, W.G.; Russo, A.; Albright, N.; Rajpal, R.

    1989-05-01

    This series of experiments employed the hypoxic cell sensitizer SR 2508 in concentrations ranging from 0.1 to 10 mM and V-79 cells irradiated in air or made hypoxic in glass syringes, then irradiated with 15 MV X rays. Using a series of survival curves measured at the various concentrations, K curves relating sensitizer enhancement ratio (SER) to SR 2508 concentration were calculated with normal GSH levels or with depletion of GSH to 0% using 1 mM buthionine sulfoximine (BSO) or elevation to 200% of normal using 1 mM oxothiazolidine carboxylate (OTZ). Survival curves were fitted by computer, allowing calculation of standard errors for the SER values. The depletion of GSH by BSO sensitized hypoxic and aerated cells significantly and caused more than additive enhancement of SR 2508 sensitization in hypoxic cells. Elevation of GSH with OTZ protects cells irradiated in air or hypoxia and reduces the SER obtained with SR 2508. The results further support the importance of GSH levels in influencing sensitization by nitroimidazoles.

  18. Killing Hypoxic Cell Populations in a 3D Tumor Model with EtNBS-PDT

    PubMed Central

    Evans, Conor L.; Abu-Yousif, Adnan O.; Park, Yong Jin; Klein, Oliver J.; Celli, Jonathan P.; Rizvi, Imran; Zheng, Xiang; Hasan, Tayyaba

    2011-01-01

    An outstanding problem in cancer therapy is the battle against treatment-resistant disease. This is especially true for ovarian cancer, where the majority of patients eventually succumb to treatment-resistant metastatic carcinomatosis. Limited perfusion and diffusion, acidosis, and hypoxia play major roles in the development of resistance to the majority of front-line therapeutic regimens. To overcome these limitations and eliminate otherwise spared cancer cells, we utilized the cationic photosensitizer EtNBS to treat hypoxic regions deep inside in vitro 3D models of metastatic ovarian cancer. Unlike standard regimens that fail to penetrate beyond ∼150 µm, EtNBS was found to not only penetrate throughout the entirety of large (>200 µm) avascular nodules, but also concentrate into the nodules' acidic and hypoxic cores. Photodynamic therapy with EtNBS was observed to be highly effective against these hypoxic regions even at low therapeutic doses, and was capable of destroying both normoxic and hypoxic regions at higher treatment levels. Imaging studies utilizing multiphoton and confocal microscopies, as well as time-lapse optical coherence tomography (TL-OCT), revealed an inside-out pattern of cell death, with apoptosis being the primary mechanism of cell killing. Critically, EtNBS-based photodynamic therapy was found to be effective against the model tumor nodules even under severe hypoxia. The inherent ability of EtNBS photodynamic therapy to impart cytotoxicity across a wide range of tumoral oxygenation levels indicates its potential to eliminate treatment-resistant cell populations. PMID:21876751

  19. Cyclooxygenase-2 inhibition attenuates hypoxic cancer cells induced m2-polarization of macrophages.

    PubMed

    Dubey, P; Shrivastava, R; Tripathi, C; Jain, N K; Tewari, B N; Lone, M-U-D; Baghel, K S; Kumar, V; Misra, S; Bhadauria, S; Bhatt, M L B

    2014-01-01

    Tumor-associated macrophages (TAMs), represent a major subpopulation of tumor infiltrating immune cells. These alternatively activated M2-polarized macrophages are well known for their pro-tumor functions. Owing to their established role in potentiating tumor-neovasculogenesis and metastasis, TAMs have emerged as promising target for anti-cancer immunotherapy. One of the key TAMs related phenomenon that is amenable to therapeutic intervention is their phenotype switching into alternatively activated M2-polarized macrophages. Hindering macrophage polarization towards a pro-tumor M2 phenotype, or better still reprogramming the M2 like TAMs towards M1 subtype is being considered a beneficial anti-cancer strategy. Hypoxic tumor milieu has been proposed as one of the most plausible factor governing M2-polarization of macrophages. We recently demonstrated that hypoxic tumor cells imparted a pro—angiogenic M2 skewed phenotype to macrophages. Furthermore, sizeable body of data indicates for participation of cyclooxygenase-2 (COX-2) in macrophage polarization. Concordantly, inhibition of COX-2 is associated with impaired macrophage polarization. Prompted by this in the current study we decided to explore if inhibition of COX-2 activity via chemical inhibitors may prevent hypoxic cancer cell induced M2-polarization of macrophages. We observed that treatment with Flunixin meglumine, an established preferential inhibitor of COX-2 activity markedly inhibited hypoxic cancer cell induced of M2-polarization of macrophages thereby indicating for usage of COX-2 inhibition as possible anti-cancer treatment modality. PMID:25210855

  20. Targeting lactate-fueled respiration selectively kills hypoxic tumor cells in mice

    PubMed Central

    Sonveaux, Pierre; Végran, Frédérique; Schroeder, Thies; Wergin, Melanie C.; Verrax, Julien; Rabbani, Zahid N.; De Saedeleer, Christophe J.; Kennedy, Kelly M.; Diepart, Caroline; Jordan, Bénédicte F.; Kelley, Michael J.; Gallez, Bernard; Wahl, Miriam L.; Feron, Olivier; Dewhirst, Mark W.

    2008-01-01

    Tumors contain oxygenated and hypoxic regions, so the tumor cell population is heterogeneous. Hypoxic tumor cells primarily use glucose for glycolytic energy production and release lactic acid, creating a lactate gradient that mirrors the oxygen gradient in the tumor. By contrast, oxygenated tumor cells have been thought to primarily use glucose for oxidative energy production. Although lactate is generally considered a waste product, we now show that it is a prominent substrate that fuels the oxidative metabolism of oxygenated tumor cells. There is therefore a symbiosis in which glycolytic and oxidative tumor cells mutually regulate their access to energy metabolites. We identified monocarboxylate transporter 1 (MCT1) as the prominent path for lactate uptake by a human cervix squamous carcinoma cell line that preferentially utilized lactate for oxidative metabolism. Inhibiting MCT1 with α-cyano-4-hydroxycinnamate (CHC) or siRNA in these cells induced a switch from lactate-fueled respiration to glycolysis. A similar switch from lactate-fueled respiration to glycolysis by oxygenated tumor cells in both a mouse model of lung carcinoma and xenotransplanted human colorectal adenocarcinoma cells was observed after administration of CHC. This retarded tumor growth, as the hypoxic/glycolytic tumor cells died from glucose starvation, and rendered the remaining cells sensitive to irradiation. As MCT1 was found to be expressed by an array of primary human tumors, we suggest that MCT1 inhibition has clinical antitumor potential. PMID:19033663

  1. Cytotoxicity and DNA crosslinks produced by mitomycin analogs in aerobic and hypoxic EMT6 cells.

    PubMed

    Keyes, S R; Loomis, R; DiGiovanna, M P; Pritsos, C A; Rockwell, S; Sartorelli, A C

    1991-01-01

    Several mitomycin antibiotics were evaluated for their capacities to kill EMT6 tumor cells and to produce DNA crosslinks under conditions of oxygenation and hypoxia. The agents examined included mitomycin C, porfiromycin, and the 7-aminomethyl dithioacetal derivative of mitomycin C (BMY-43324), all of which caused greater kill of hypoxic cells than of their oxygenated counterparts; the N,N'-dimethylaminomethylene derivative of mitomycin C (BMY-25282), which was considerably more cytotoxic under oxygenated conditions than in hypoxia; and the N,N'-dimethylaminomethylene derivative of porfiromycin (BL-6783), which was equal in its toxicity to hypoxic and oxygenated cells. All of these agents produced DNA crosslinks in EMT6 cells, as measured by alkaline elution. The number of crosslinks required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation, suggesting that the crosslinking of DNA was a major lesion in the cytodestructive action of the mitomycins. PMID:1760250

  2. 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

  3. 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.

  4. 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.

  5. Loss of CSL Unlocks a Hypoxic Response and Enhanced Tumor Growth Potential in Breast Cancer Cells.

    PubMed

    Braune, Eike-Benjamin; Tsoi, Yat Long; Phoon, Yee Peng; Landor, Sebastian; Silva Cascales, Helena; Ramsköld, Daniel; Deng, Qiaolin; Lindqvist, Arne; Lian, Xiaojun; Sahlgren, Cecilia; Jin, Shao-Bo; Lendahl, Urban

    2016-05-10

    Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL, and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1α protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1,750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a role for CSL in tumorigenesis and regulation of the cellular hypoxic response. PMID:27066863

  6. Beneficial Effects of Hypoxic Preconditioning on Human Umbilical Cord Mesenchymal Stem Cells.

    PubMed

    Zhang, Li; Yang, Jing; Tian, Yan-Ming; Guo, Hui; Zhang, Yi

    2015-10-31

    As human umbilical cord mesenchymal stem cells (hUC-MSCs) transplanation may be promising in heart failure treatment, it is important to know whether hypoxic preconditioning (HP) promote hUC-MSCs proliferation and differentiation and protect them against chemical hypoxic damages. This study aimed to investigate the effects of HP on proliferation and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). The study also aimed to confirm our hypothesis that HP could promote hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells as well as effectively protecting hUC-MSCs and cardiomyocyte-like cells against chemical hypoxic damages. Isolated hUC-MSCs were cultured in hypoxia at 1%, 3% and 5% O₂ for 72 hours. 5-azacytidine (5-AZA) induced differentiation of hUC-MSCs to cardiomyocyte-like cells was determined by streptavidin-perosidase (SP) immunohistochemical staining and the content of troponin (TnI). Flow cytometry was used to measure cell cycle in hUC-MSCs and cardiomyocyte-like cells. The mitochondrial membrane potential (ΔΨ(m)) and mitochondrial Ca²⁺ concentration ([Ca²⁺](m)), were measured in hUC-MSCs and cardiomyocyte-like cells during chemical hypoxia induced by cobalt chloride (100 μmol/L). HP optimally promoted the proliferation of hUC-MSCs at 3% O₂ and enhanced the differentiation of hUC-MSCs to cardiomyocyte-like cells by 5-AZA in a concentration-dependent manner. The cell cycle distribution of cardiomyocyte-like cells, but not hUC-MSCs, was clearly changed by HP. Chemical hypoxic damage, decreased ΔΨ(m) and increased [Ca²⁺](m), were alleviated significantly in HP-treated cells compared with the normaxia-treated cells. The results demonstrate that HP promoted hUC-MSCs proliferation and differentiation to cardiomyocyte-like cells, and protected both cell types against chemical hypoxic damage. PMID:26536910

  7. Transcriptomic changes in human renal proximal tubular cells revealed under hypoxic conditions by RNA sequencing.

    PubMed

    Yu, Wenmin; Li, Yiping; Wang, Zhi; Liu, Lei; Liu, Jing; Ding, Fengan; Zhang, Xiaoyi; Cheng, Zhengyuan; Chen, Pingsheng; Dou, Jun

    2016-09-01

    Chronic hypoxia often occurs among patients with chronic kidney disease (CKD). Renal proximal tubular cells may be the primary target of a hypoxic insult. However, the underlying transcriptional mechanisms remain undefined. In this study, we revealed the global changes in gene expression in HK‑2 human renal proximal tubular cells under hypoxic and normoxic conditions. We analyzed the transcriptome of HK‑2 cells exposed to hypoxia for 24 h using RNA sequencing. A total of 279 differentially expressed genes was examined, as these genes could potentially explain the differences in HK‑2 cells between hypoxic and normoxic conditions. Moreover, 17 genes were validated by qPCR, and the results were highly concordant with the RNA seqencing results. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these differentially expressed genes. The upregulated genes appeared to be significantly enriched in the pathyway of extracellular matrix (ECM)-receptor interaction, and in paticular, the pathway of renal cell carcinoma was upregulated under hypoxic conditions. The downregulated genes were enriched in the signaling pathway related to antigen processing and presentation; however, the pathway of glutathione metabolism was downregulated. Our analysis revealed numerous novel transcripts and alternative splicing events. Simultaneously, we also identified a large number of single nucleotide polymorphisms, which will be a rich resource for future marker development. On the whole, our data indicate that transcriptome analysis provides valuable information for a more in depth understanding of the molecular mechanisms in CKD and renal cell carcinoma. PMID:27432315

  8. An Argonaute 2 Switch Regulates Circulating miR-210 to Coordinate Hypoxic Adaptation across Cells

    PubMed Central

    Hale, Andrew; Lee, Changjin; Annis, Sofia; Min, Pil-Ki; Pande, Reena; Creager, Mark A.; Julian, Colleen G.; Moore, Lorna G.; Mitsialis, S. Alex; Hwang, Sarah J.; Kourembanas, Stella; Chan, Stephen Y.

    2014-01-01

    Complex organisms may coordinate molecular responses to hypoxia by specialized avenues of communication across multiple tissues, but these mechanisms are poorly understood. Plasma-based, extracellular microRNAs have been described, yet, their regulation and biological functions in hypoxia remain enigmatic. We found a unique pattern of release of the hypoxia-inducible microRNA-210 (miR-210) from hypoxic and reoxygenated cells. This microRNA is also elevated in human plasma in physiologic and pathologic conditions of altered oxygen demand and delivery. Released miR-210 can be delivered to recipient cells, and its direct suppression of its direct target ISCU and mitochondrial metabolism is primarily evident in hypoxia. To regulate these hypoxia-specific actions, prolyl-hydroxylation of Argonaute 2 acts as a molecular switch that reciprocally modulates miR-210 release and intracellular activity in source cells as well as regulates intracellular activity in recipient cells after miR-210 delivery. Therefore, Argonaute 2-dependent control of released miR-210 represents a unique communication system that integrates the hypoxic response across anatomically distinct cells, preventing unnecessary activity of delivered miR-210 in normoxia while still preparing recipient tissues for incipient hypoxic stress and accelerating adaptation. PMID:24983771

  9. The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

    PubMed Central

    Fens, Marcel H.; Larkin, Sandra K.; Oronsky, Bryan; Scicinski, Jan; Morris, Claudia R.; Kuypers, Frans A.

    2014-01-01

    Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release. PMID:25007272

  10. Clinical results of hypoxic cell radiosensitisation from hyperbaric oxygen to accelerated radiotherapy, carbogen and nicotinamide.

    PubMed Central

    Saunders, M.; Dische, S.

    1996-01-01

    The 40-year history of hypoxic cell sensitisation can be traced from hyperbaric oxygen to the present clinical studies with carbogen, nicotinamide and accelerated radiotherapy. A meta-analysis by Overgaard (1995) included 10703 cases entered into 83 randomised controlled trials and showed an overall improvement in local tumour control of 4.6% (P = 0.00001) and in survival of 2.8% (P = 0.005). Hyperbaric oxygen gave a 6.6% (P = 0.003) improvement in local control and hypoxic cell sensitisers 3.9% (P = 0.04). Despite this, the only hypoxic cell-sensitising method in routine clinical use is the giving of nimorazole in supraglottic and pharyngeal carcinomas. Acute, as well as chronic hypoxia has been recognised and nicotinamide, the amide derivative of B3 is believed to prevent the former. Thus ARCON (accelerated radiotherapy, carbogen and nicotinamide) has been introduced in the clinic in an effort to overcome tumour proliferation, chronic and acute hypoxia, respectively. The success of future randomised controlled trials would be improved greatly if methods were available to measure the concentration of hypoxic cells in tumours before treatment and thus select those where benefit may be gained. The use of ARCON recognises that tumour cell proliferation is an important cause of failure in addition to hypoxia. However, intrinsic radiosensitivity may also need to be taken into account in the future. Clinical trials aim to improve the therapeutic ratio and thus the study of morbidity is as important as local tumour control. International collaboration is essential if randomised controlled trials are to be carried out within reasonable periods of time. PMID:8763896

  11. ZEB1 Promotes Invasion in Human Fetal Neural Stem Cells and Hypoxic Glioma Neurospheres.

    PubMed

    Kahlert, Ulf D; Suwala, Abigail K; Raabe, Eric H; Siebzehnrubl, Florian A; Suarez, Maria J; Orr, Brent A; Bar, Eli E; Maciaczyk, Jaroslaw; Eberhart, Charles G

    2015-11-01

    Diffuse spread through brain parenchyma and the presence of hypoxic foci rimmed by neoplastic cells are two cardinal features of glioblastoma, and low oxygen is thought to drive movement of malignant gliomas in the core of the lesions. Transcription factors associated with epithelial-to-mesenchymal transition (EMT) have been linked to this invasion, and we found that hypoxia increased in vitro invasion up to fourfold in glioblastoma neurosphere lines and induced the expression of ZEB1. Immunohistochemical assessment of 295 surgical specimens consisting of various types of pediatric and adult brain cancers showed that ZEB1 expression was significantly higher in infiltrative lesions than less invasive tumors such as pilocytic astrocytoma and ependymoma. ZEB1 protein was also present in human fetal periventricular stem and progenitor cells and ZEB1 inhibition impaired migration of in vitro propagated human neural stem cells. The induction of ZEB1 protein in hypoxic glioblastoma neurospheres could be partially blocked by the HIF1alpha inhibitor digoxin. Targeting ZEB1 blocked hypoxia-augmented invasion of glioblastoma cells in addition to slowing them in normoxia. These data support the role for ZEB1 in invasive and high-grade brain tumors and suggest its key role in promoting invasion in the hypoxic tumor core as well as in the periphery. PMID:25521330

  12. Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells.

    PubMed

    Shen, Jiangang; Lee, Waisin; Li, Yue; Lau, Chi Fai; Ng, Kwong Man; Fung, Man Lung; Liu, Ke Jian

    2008-10-01

    Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells. PMID:18717816

  13. Tirapazamine-induced DNA damage measured using the comet assay correlates with cytotoxicity towards hypoxic tumour cells in vitro.

    PubMed Central

    Siim, B. G.; van Zijl, P. L.; Brown, J. M.

    1996-01-01

    Tirapazamine (SR 4233), a bioreductive drug selectively toxic towards hypoxic cells, is presently in phase II clinical trials. Since it would not be expected that all tumours would respond equally to the drug, we are exploring ways of predicting the response of individual tumours. In this study we have tested whether the comet assay, which measures DNA damage in individual cells, can provide a simple, surrogate end point for cell killing by tirapazamine. We examined the relationship between the cytotoxicity of tirapazamine under hypoxic conditions and tirapazamine-induced DNA strand breaks in murine (SCCVII, EMT6, RIF-1) and human (HT1080, A549, HT29) tumour cell lines. These results were compared with the relationship between tirapazamine cytotoxicity and another measure of the ability of cells to metabolise tirapazamine; high-performance liquid chromatography (HPLC) analysis of tirapazamine loss or formation of the two electron reduction product SR 4317. The correlation between the hypoxic cytotoxic potency of tirapazamine and DNA damage was highly significant (r = 0.905, P = 0.013). A similar correlation was observed for hypoxic potency and tirapazamine loss (r = 0.812, P = 0.050), while the correlation between hypoxic potency and SR 4317 formation was not significant (r = 0.634, P = 0.171). The hypoxic cytotoxicity of tirapazamine in vitro can therefore be predicted by measuring tirapazamine-induced DNA damage using the comet assay. This approach holds promise for predicting the response of individual tumours to tirapazamine in the clinic. PMID:8611431

  14. G-CSF and hypoxic conditioning improve the proliferation, neural differentiation and migration of canine bone marrow mesenchymal stem cells

    PubMed Central

    Yu, Jing; Liu, Xing-Long; Cheng, Qi-Guang; Lu, Shan-Shan; Xu, Xiao-Quan; Zu, Qing-Quan; Liu, Sheng

    2016-01-01

    Transplantation using bone marrow mesenchymal stem cells (BMSCs) is emerging as a potential regenerative therapy after ischemic attacks in the brain. However, it has been questioned because very few transplanted BMSCs are detected homing to and survived in the ischemic region. Improving the cell viability and migration ability under the complex ischemic condition seems very important. The aim of our study is to identify whether hypoxic condition and granulocyte colony-stimulating factor (G-CSF) could improve the cell survival and migration ability of transplanted cells or hypoxic condition could promote BMSC's neural differentiation. BMSCs were treated under either normoxic (21% O2) or hypoxic (1% O2) (HP-BMSCs) conditions, no significant apoptosis was observed in hypoxic precondition (HP) group, our study confirmed that HP improves BMSCs proliferation and migration. Meanwhile, neural induction of BMSCs under hypoxic condition exhibited significant superior results than normoxic condition. Additionally, the addition of G-CSF in HP-BMSCs culture media promoted HP efficiency on BMSCs. These findings shed light on novel efficient strategy on the prosperity of BMSCs. Hypoxic preconditioning and cultured with G-CSF may become a promising therapeutics for cell-based therapy in the treatments of ischemia stroke. PMID:27588100

  15. Preferential kill of hypoxic EMT6 mammary tumor cells by the bioreductive alkylating agent porfiromycin.

    PubMed

    Sartorelli, A C; Belcourt, M F; Hodnick, W F; Keyes, S R; Pritsos, C A; Rockwell, S

    1995-01-01

    Hypoxic cells in solid tumors represent a therapeutically resistant population that limits the curability of many solid tumors by irradiation and by most chemotherapeutic agents. The oxygen deficit, however, creates an environment conducive to reductive processes; this results in a major exploitable difference between normal and neoplastic tissues. The mitomycin antibiotics can be reductively activated by a number of oxidoreductases, in a process required for the production of their therapeutic effects. Preferential activation of these drugs under hypoxia and greater toxicity to oxygen-deficient cells than to their oxygenated counterparts are obtained in most instances. The demonstration that mitomycin C and porfiromycin, used to kill the hypoxic fraction, in combination with irradiation, to eradicate the oxygenated portion of the tumor, produced enhanced cytodestructive effects on solid tumors in animals has led to the clinical evaluation of the mitomycins in combination with radiation therapy in patients with head and neck cancer. The findings from these clinical trials have demonstrated the value of directing a concerted therapeutic attack on the hypoxic fraction of solid tumors as an approach toward enhancing the curability of localized neoplasms by irradiation. PMID:7572339

  16. Down-Regulation of Rad51 and Decreased Homologous Recombination in Hypoxic Cancer Cells

    PubMed Central

    Bindra, Ranjit S.; Schaffer, Paul J.; Meng, Alice; Woo, Jennifer; Måseide, Kårstein; Roth, Matt E.; Lizardi, Paul; Hedley, David W.; Bristow, Robert G.; Glazer, Peter M.

    2004-01-01

    There is an emerging concept that acquired genetic instability in cancer cells can arise from the dysregulation of critical DNA repair pathways due to cell stresses such as inflammation and hypoxia. Here we report that hypoxia specifically down-regulates the expression of RAD51, a key mediator of homologous recombination in mammalian cells. Decreased levels of Rad51 were observed in multiple cancer cell types during hypoxic exposure and were not associated with the cell cycle profile or with expression of hypoxia-inducible factor. Analyses of RAD51 gene promoter activity, as well as mRNA and protein stability, indicate that the hypoxia-mediated regulation of this gene occurs via transcriptional repression. Decreased expression of Rad51 was also observed to persist in posthypoxic cells for as long as 48 h following reoxygenation. Correspondingly, we found reduced levels of homologous recombination in both hypoxic and posthypoxic cells, suggesting that the hypoxia-associated reduction in Rad51 expression has functional consequences for DNA repair. In addition, hypoxia-mediated down-regulation of Rad51 was confirmed in vivo via immunofluorescent image analysis of experimental tumors in mice. Based on these findings, we propose a novel mechanism of genetic instability in the tumor microenvironment mediated by hypoxia-induced suppression of the homologous recombination pathway in cancer cells. The aberrant regulation of Rad51 expression may also create heterogeneity in the DNA damage response among cells within tumors, with implications for the response to cancer therapies. PMID:15367671

  17. Molecular mechanisms mediating metastasis of hypoxic breast cancer cells.

    PubMed

    Semenza, Gregg L

    2012-09-01

    Breast cancers contain regions of intratumoral hypoxia in which reduced O(2) availability activates the hypoxia-inducible factors HIF-1 and HIF-2, which increase the transcription of genes encoding proteins that are required for many important steps in cancer progression. Recently, HIFs have been shown to play critical roles in the metastasis of breast cancer to the lungs through the transcriptional activation of genes encoding angiopoietin-like 4 and L1 cell adhesion molecule, which promote the extravasation of circulating cancer cells from the lung vasculature, and the lysyl oxidase family members LOX, LOXL2, and LOXL4, which promote invasion and metastatic niche formation. Digoxin, a drug that inhibits HIF-1 activity, blocks primary tumor growth, vascularization, invasion, and metastasis in ex vivo and in vivo assays. PMID:22921864

  18. Porfiromycin as a bioreductive alkylating agent with selective toxicity to hypoxic EMT6 tumor cells in vivo and in vitro.

    PubMed

    Keyes, S R; Rockwell, S; Sartorelli, A C

    1985-08-01

    Hypoxic cells may limit the curability of solid tumors by conventional chemotherapeutic agents and radiotherapy. Agents which are preferentially toxic to cells with low oxygen contents could therefore be useful as adjuncts to the regimens now used to treat these cancers. To date, the best agent of this type that we have tested is porfiromycin. Porfiromycin is similar to mitomycin C in its toxicity to hypoxic EMT6 cells in vitro but has much less toxicity than mitomycin C to well-oxygenated EMT6 cells. EMT6 cell sonicates reduce mitomycin C and porfiromycin to reactive electrophiles at similar rates under hypoxic conditions, a finding that correlates with cytotoxicity, whereas the rate of production of reactive species from both drugs is very slow under aerobic conditions. We also show that porfiromycin is capable of killing hypoxic radiation-resistant cells in solid EMT6 tumors. Appropriate regimens combining porfiromycin (which preferentially kills hypoxic cells) and radiation (which preferentially kills aerated cells) may therefore be especially efficacious for the treatment of solid tumors. PMID:3926306

  19. Hypoxic preconditioning of mesenchymal stromal cells induces metabolic changes, enhances survival, and promotes cell retention in vivo.

    PubMed

    Beegle, Julie; Lakatos, Kinga; Kalomoiris, Stefanos; Stewart, Heather; Isseroff, R Rivkah; Nolta, Jan A; Fierro, Fernando A

    2015-06-01

    Mesenchymal stem cells/multipotent stromal cells (MSCs) are promising therapeutics for a variety of conditions. However, after transplantation, cell retention remains extremely challenging. Given that many hypoxic signals are transitory and that the therapeutic administration of MSCs is typically into tissues that are normally hypoxic, we studied the effect of hypoxic preconditioning (HP) prior to new exposure to hypoxia. We show that preincubation for 2 days or more in 1% oxygen reduces serum deprivation-mediated cell death, as observed by higher cell numbers and lower incorporation of EthD-III and Annexin V. Consistently, HP-MSCs expressed significantly lower levels of cytochrome c and heme oxygenase 1 as compared to controls. Most importantly, HP-MSCs showed enhanced survival in vivo after intramuscular injection into immune deficient NOD/SCID-IL2Rgamma(-/-) mice. Interestingly, HP-MSCs consume glucose and secrete lactate at a slower rate than controls, possibly promoting cell survival, as glucose remains available to the cells for longer periods of time. In addition, we compared the metabolome of HP-MSCs to controls, before and after hypoxia and serum deprivation, and identified several possible mediators for HP-mediated cell survival. Overall, our findings suggest that preincubation of MSCs for 2 days or more in hypoxia induces metabolic changes that yield higher retention after transplantation. PMID:25702874

  20. Stretch-activated channels in pulmonary arterial smooth muscle cells from normoxic and chronically hypoxic rats.

    PubMed

    Ducret, Thomas; El Arrouchi, Jalila; Courtois, Arnaud; Quignard, Jean-François; Marthan, Roger; Savineau, Jean-Pierre

    2010-11-01

    Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment. PMID:21035852

  1. 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. PMID:26558344

  2. The Induction of Metformin Inhibitory Effects on Tumor Cell Growth in Hypoxic Condition.

    PubMed

    Safari, Zohreh; Safaralizadeh, Reza; Seyedzadeh, Mir Hadi; Valinezad Orang, Ayla; Zare, Ahad; Hosseinpour Feizi, Mohammad Ali; Kardar, Gholam Ali

    2015-12-01

    It is aimed to evaluate the actual anti-cancerous effects of metformin on cancer cells in hypoxic condition. Non-cancerous cells (HEK293) and cancer cells (MCF-7) were cultured in both hypoxia and normoxia conditions and treated with different concentrations of metformin. The proliferation, apoptosis, and necrosis rate were assessed using MTT test and Annexin V assay. The S6K1 phosphorylation was assessed using western blotting. Zymography was used to measure the activity of metalloproteinase-9 (MMP-9). Metformin treatment inhibited proliferation of cancer cells in the optimal concentration of 10 mM under hypoxia condition, while it showed no effects on non-cancerous cell viability. The statistical analysis of MTT assay indicated that the pro-apoptotic function of metformin for cancer cells under hypoxia condition compared to normoxia was significant with different metformin concentrations (p<0.01). However, the effect of metformin treatments for non-cancerous cells under hypoxia condition compared to normoxia was not significant. Western-blot analysis indicated a significant decrease in S6K1 phosphorylation in cancer cells under hypoxia condition (p<0.05). Nevertheless, there was no considerable difference between normoxia and hypoxia conditions in non-cancerous cells. MMP-9 zymography analysis revealed that the highest inhibition of MMP-9 activity was observed in hypoxia condition by 20mM of metformin concentration only in cancer cell. The results indicate that in hypoxia condition metformin exerts its anti-cancerous function by inhibiting proliferation and tumor progression and inducing cell apoptosis more effectively than normoxia condition. In line with cancer cell conditions, most importantly hypoxic condition, metformin can be considered as a potential anti-cancerous drug. PMID:26725558

  3. δ-Tocotrienol treatment is more effective against hypoxic tumor cells than normoxic cells: potential implications for cancer therapy.

    PubMed

    Shibata, Akira; Nakagawa, Kiyotaka; Tsuduki, Tsuyoshi; Miyazawa, Teruo

    2015-08-01

    Tocotrienols, unsaturated forms of vitamin E, inhibit the proliferation of a variety of cancer cells and suppress angiogenesis. However, the mechanisms underlying those effects on cancer cell growth remain unclear especially under hypoxic conditions. In this study, we demonstrated that δ-tocotrienol (δ-T3) could be used as a novel anticancer agent against human colorectal adenocarcinoma (DLD-1) cells under both normoxic and hypoxic conditions. δ-T3 inhibited the growth of DLD-1 cells in a dose-dependent fashion by inducing cell cycle arrest and apoptosis. This effect was more potent under hypoxic than normoxic conditions. The anticancer effect of δ-T3 was achieved by its up-regulation of cyclin-dependent kinase inhibitors (p21 and p27), the activation of caspases and the suppression of phosphorylation of protein kinase B (Akt) at Thr(308) and Ser(473). In in vivo studies, oral administration of rice bran tocotrienol (RBT3, mainly γ-T3) (10 mg/mouse/day) significantly inhibited tumor growth in nude mice. In tumor analyses, RBT3 activated p21, p27, caspase-3 and caspase-9 and decreased Akt phosphorylation. Furthermore, immunostaining revealed that RBT3 decreased the number of cells positive for CD31/platelet endothelial cell adhesion molecule-1 in microvessels in the tumor. Taken together, these data suggest that tocotrienols are potent antitumor agents capable of inducing apoptosis and inhibiting angiogenesis under both hypoxic and normoxic conditions. Tocotrienols could have significant therapeutic potential in the clinical treatment of tumors. PMID:25979648

  4. TAT-ODD-p53 enhances the radiosensitivity of hypoxic breast cancer cells by inhibiting Parkin-mediated mitophagy

    PubMed Central

    Du, Shasha; Ren, Chen; Wang, Yuxia; Yuan, Yawei

    2015-01-01

    Radiation therapy has an important role in the treatment of breast cancer. Dysfunction p53 and hypoxia are typical biological characteristics of breast cancer that constitute barriers to the efficacy of radiotherapy. Mitophagy plays a protective role in cellular homeostasis under hypoxic conditions, while mitophagy is inhibited by p53 in normal cells. We explored the effects of a p53 fusion protein, TAT-ODD-p53, on the radiosensitivity of hypoxic breast cancer cells both in vitro and in vivo, as well as investigating the related molecular mechanisms. We found that selective accumulation of TAT-ODD-p53 occurred under hypoxic conditions and significantly increased tumor cell radiosensitivity both in vitro and in vivo. Mitophagy had an important role in maintaining hypoxia-induced radioresistance. Mitophagy was inhibited by TAT-ODD-p53 and this inhibition was suppressed by over-expression of Parkin in hypoxic irradiated breast cancer cells. In addition, mitophagy was induced by deletion of p53, with this effect being weakened by Parkin knockdown at a low oxygen tension. By interacting with Parkin, p53 inhibited the translocation of Parkin to the mitochondria, disrupting the protective mitophagy process. These results suggest that TAT-ODD-p53 has a significant and preferential radiosensitizing effect on hypoxic breast cancer cells by inhibition of Parkin-mediated mitophagy. PMID:26025927

  5. Chemotherapeutic attack of hypoxic tumor cells by the bioreductive alkylating agent mitomycin C.

    PubMed

    Keyes, S R; Heimbrook, D C; Fracasso, P M; Rockwell, S; Sligar, S G; Sartorelli, A C

    1985-01-01

    Since the cure of solid tumors is limited by the presence of cells with low oxygen contents, we have approached the development of treatment regimens and of new drugs for these tumors by investigating agents which are preferentially bioactivated under hypoxia. Major emphasis has been directed at studying the mode of action of the mitomycin antibiotics, as bioreductive alkylating agents. Using primarily the EMT6 mouse mammary carcinoma as a solid tumor model, we have found that mitomycin C and porfiromycin are preferentially toxic to cells with low oxygen contents. The mitomycin analog BMY-25282 is more toxic to hypoxic cells than are mitomycin C and porfiromycin; however, unlike these antibiotics, BMY-25282 is preferentially toxic to well-oxygenated cells. With these three mitomycins, we have observed a correlation between cytotoxicity to hypoxic cells, the rate of generation of reactive products, and the redox potentials of the drugs. Investigations of the enzymes in EMT6 cells that could possibly activate mitomycin C have revealed that cytochrome P-450 and xanthine oxidase are not present in measurable quantities and therefore are not responsible for activation of mitomycin C. Activities representative of NADPH-cytochrome c reductase and DT-diaphorase are present in these neoplastic cells. Comparison of these enzymatic activities in EMT6, CHO, and V79 cells with the rate of generation of reactive products under hypoxia shows a direct correlation between these two parameters, but there is no quantitative correlation between these two parameters and the amount of cytotoxicity. Use of purified NADPH-cytochrome c reductase and inhibitors of this enzyme demonstrated that NADPH-cytochrome c reductase can activate mitomycin C, but that it is probably not the only enzyme participating in this bioactivation in EMT6 cells. The DT-diaphorase inhibitor dicoumarol was employed to show that this enzyme is not involved in the activation of mitomycin C to a cytotoxic agent

  6. 660 nm red light-enhanced bone marrow mesenchymal stem cell transplantation for hypoxic-ischemic brain damage treatment

    PubMed Central

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

    2014-01-01

    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/cm2, 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 × 106 bone marrow mesenchymal stem cells, followed by irradiation under red light-emitting diodes at 660 nm and 60 mW/cm2 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. PMID:25206807

  7. Bioactivation of mitomycin antibiotics by aerobic and hypoxic Chinese hamster ovary cells overexpressing DT-diaphorase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-06-28

    DT-Diaphorase catalyzes a two-electron reduction of mitomycin C (MC) and porfiromycin (POR) to reactive species. Many cell lines that overexpress DT-diaphorase and are sensitive to the mitomycins are protected from the aerobic cytotoxicity of these drugs by the DT-diaphorase inhibitor dicumarol. The cytoprotective properties of this relatively non-specific inhibitor, however, vanish under hypoxic conditions. To ascertain the role of DT-diaphorase in mitomycin bioactivation and cytotoxicity in living cells, a rat liver DT-diaphorase cDNA was transfected into Chinese hamster ovary cells. MC was equitoxic to the parental cells under oxygenated and hypoxic conditions. In contrast, POR was less toxic than MC to these cells under aerobic conditions, but significantly more toxic than MC under hypoxia. Two DT-diaphorase-transfected clones displayed increases in DT-diaphorase activity of 126- and 133-fold over parental cells. The activities of other oxidoreductases implicated in mitomycin bioreduction were unchanged. MC was more toxic to both DT-diaphorase-transfected lines than to parental cells; the toxicity of MC to the transfected lines was similar in air and hypoxia. POR was also more toxic to the DT-diaphorase-elevated clones than to parental cells under oxygenated conditions. Under hypoxia, however, the toxicity of POR to the transfected clones was unchanged from that of parental cells. The findings implicate DT-diaphorase in mitomycin bioactivation in living cells, but suggest that this enzyme does not contribute to the differential toxicity of MC or POR in air and hypoxia. PMID:8687482

  8. 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. PMID:26453899

  9. Critical Role of Tumor Microenvironment in Shaping NK Cell Functions: Implication of Hypoxic Stress.

    PubMed

    Hasmim, Meriem; Messai, Yosra; Ziani, Linda; Thiery, Jerome; Bouhris, Jean-Henri; Noman, Muhammad Zaeem; Chouaib, Salem

    2015-01-01

    Blurring the boundary between innate and adaptive immune system, natural killer (NK) cells, a key component of the innate immunity, are recognized as potent anticancer mediators. Extensive studies have been detailed on how NK cells get activated and recognize cancer cells. In contrast, few studies have been focused on how tumor microenvironment-mediated immunosubversion and immunoselection of tumor-resistant variants may impair NK cell function. Accumulating evidences indicate that several cell subsets (macrophages, myeloid-derived suppressive cells, T regulatory cells, dendritic cells, cancer-associated fibroblasts, and tumor cells), their secreted factors, as well as metabolic components (i.e., hypoxia) have immunosuppressive roles in the tumor microenvironment and are able to condition NK cells to become anergic. In this review, we will describe how NK cells react with different stromal cells in the tumor microenvironment. This will be followed by a discussion on the role of hypoxic stress in the regulation of NK cell functions. The aim of this review is to provide a better understanding of how the tumor microenvironment impairs NK cell functions, thereby limiting the use of NK cell-based therapy, and we will attempt to suggest more efficient tools to establish a more favorable tumor microenvironment to boost NK cell cytotoxicity and control tumor progression. PMID:26441986

  10. Critical Role of Tumor Microenvironment in Shaping NK Cell Functions: Implication of Hypoxic Stress

    PubMed Central

    Hasmim, Meriem; Messai, Yosra; Ziani, Linda; Thiery, Jerome; Bouhris, Jean-Henri; Noman, Muhammad Zaeem; Chouaib, Salem

    2015-01-01

    Blurring the boundary between innate and adaptive immune system, natural killer (NK) cells, a key component of the innate immunity, are recognized as potent anticancer mediators. Extensive studies have been detailed on how NK cells get activated and recognize cancer cells. In contrast, few studies have been focused on how tumor microenvironment-mediated immunosubversion and immunoselection of tumor-resistant variants may impair NK cell function. Accumulating evidences indicate that several cell subsets (macrophages, myeloid-derived suppressive cells, T regulatory cells, dendritic cells, cancer-associated fibroblasts, and tumor cells), their secreted factors, as well as metabolic components (i.e., hypoxia) have immunosuppressive roles in the tumor microenvironment and are able to condition NK cells to become anergic. In this review, we will describe how NK cells react with different stromal cells in the tumor microenvironment. This will be followed by a discussion on the role of hypoxic stress in the regulation of NK cell functions. The aim of this review is to provide a better understanding of how the tumor microenvironment impairs NK cell functions, thereby limiting the use of NK cell-based therapy, and we will attempt to suggest more efficient tools to establish a more favorable tumor microenvironment to boost NK cell cytotoxicity and control tumor progression. PMID:26441986

  11. Hypoxia-Responsive Polymersomes for Drug Delivery to Hypoxic Pancreatic Cancer Cells.

    PubMed

    Kulkarni, Prajakta; Haldar, Manas K; You, Seungyong; Choi, Yongki; Mallik, Sanku

    2016-08-01

    Hypoxia in tumors contributes to overall tumor progression by assisting in epithelial-to-mesenchymal transition, angiogenesis, and metastasis of cancer. In this study, we have synthesized a hypoxia-responsive, diblock copolymer poly(lactic acid)-azobenzene-poly(ethylene glycol), which self-assembles to form polymersomes in an aqueous medium. The polymersomes did not release any encapsulated contents for 50 min under normoxic conditions. However, under hypoxia, 90% of the encapsulated dye was released in 50 min. The polymersomes encapsulated the combination of anticancer drugs gemcitabine and erlotinib with entrapment efficiency of 40% and 28%, respectively. We used three-dimensional spheroid cultures of pancreatic cancer cells BxPC-3 to demonstrate hypoxia-mediated release of the drugs from the polymersomes. The vesicles were nontoxic. However, a significant decrease in cell viability was observed in hypoxic spheroidal cultures of BxPC-3 cells in the presence of drug encapsulated polymersomes. These polymersomes have potential for future applications in imaging and treatment of hypoxic tumors. PMID:27303825

  12. Increased calcium loading and inotropy without greater cell death in hypoxic rat cardiomyocytes.

    PubMed

    Kondo, R P; Apstein, C S; Eberli, F R; Tillotson, D L; Suter, T M

    1998-12-01

    To test whether contractile function in "hypoxic" myocytes treated with high glucose (19.5 mM) can be improved by increasing intracellular Ca2+ without accelerating cell contracture or death, we challenged metabolically inhibited, paced myocytes with high extracellular Ca2+ concentration ([Ca2+]o) and measured simultaneously cell shortening and intracellular Ca2+ concentration ([Ca2+]i). NaCN exposure at a physiological [Ca2+]o level (1.2 mM) caused a decline of contractile function to 58 +/- 8% of the pre-NaCN value (P < 0.001) but increased systolic and diastolic [Ca2+]i by 104 +/- 17 and 37 +/- 9% above baseline (P < 0.01), respectively. Consequent doubling of [Ca2+]o to 2.4 mM, in the presence of NaCN, immediately restored contractile function, and twitch amplitude after 18 min was 123 +/- 14% (P < 0.001) of baseline pre-NaCN values, whereas systolic [Ca2+]i increased further to 225 +/- 63% (P < 0.05) and diastolic [Ca2+]i to 73 +/- 16% above baseline (P < 0.01). This marked increase in [Ca2+]i had no deleterious effect on myocyte diastolic function or survival. These results suggest that if adequate metabolic substrate is provided, contractile function in metabolically inhibited, hypoxic myocytes can be restored by increasing [Ca2+]i without causing short-term cell injury. PMID:9843829

  13. 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.

  14. 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.

  15. Radiosensitization of EMT6 cells by four platinum complexes.

    PubMed

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

    1985-05-01

    The greatest research effort in producing radiation sensitizers has been directed toward organic compounds. However, platinum complexes also have radiosensitizing capabilities, perhaps because they bind to DNA. The compound 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 microM and 400 microM 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 microM and 1.8 at 400 microM. 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 microM 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 microM and 400 microM 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 microM 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. PMID:4039304

  16. SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells.

    PubMed

    Turkoglu, Sumeyye Aydogan; Kockar, Feray

    2016-01-01

    ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition. PMID:26299656

  17. 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

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

    PubMed

    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

  19. Hypoxic preconditioning increases the protective effect of bone marrow mesenchymal stem cells on spinal cord ischemia/reperfusion injury.

    PubMed

    Wang, Zhilin; Fang, Bo; Tan, Zhibin; Zhang, Dong; Ma, Hong

    2016-03-01

    Transplantation of bone marrow mesenchymal stem cells (BMSCs) protect against spinal cord ischemia/reperfusion injury (SCIRI). However, a large number of transplanted BMSCs often undergo apoptosis, which severely affects the treatment outcome. Previous studies have demonstrated that hypoxic preconditioning effectively increases the survival rate of BMSCs following transplantation, and increases their protective effect on injured tissues. However, there have been few reports regarding roles of hypoxic preconditioning in SCIRI. The present study isolated rat BMSCs and separately transplanted hypoxia‑ and non‑hypoxia‑preconditioned BMSCs into the spinal cord tissues of rats with SCIRI. The role of hypoxic preconditioning in the promotion of the protective effect of BMSCs on SCIRI was investigated using neurological function scores, Evans blue staining, hematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling. In addition, reverse transcription‑quantitative polymerase chain reaction and western blotting were used to detect the expression levels of hypoxia‑inducible factor 1α (HIF‑1α), and to investigate its possible underlying mechanism of action. The results indicated that hypoxic preconditioning effectively increased the protective effects of BMSCs on neurological function, blood spinal cord barrier and tissue damage following SCIRI, and inhibited apoptosis. Furthermore, hypoxic preconditioned BMSCs upregulated the expression of HIF‑1α in spinal cord tissues. Therefore, hypoxic preconditioning effectively increased the protective effect of BMSCs on SCIRI and may be associated with upregulation of the expression of HIF‑1α. Hypoxic preconditioning may serve as an effective means of increasing the protective effect of BMSCs on SCIRI. PMID:26783161

  20. 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. PMID:23204067

  1. Loss of Interleukin-21 Receptor Activation in Hypoxic Endothelial Cells Impairs Perfusion Recovery after Hindlimb Ischemia

    PubMed Central

    Wang, Tao; Cunningham, Alexis; Dokun, Ayotunde O; Hazarika, Surovi; Houston, Kevin; Chen, Lingdan; Lye, R. John; Spolski, Rosanne; Leonard, Warren J.; Annex, Brian H.

    2016-01-01

    Objective Surgical hindlimb ischemia (HLI) in mice has become a valuable preclinical model to study peripheral arterial disease (PAD). We previously identified that the different phenotypical outcomes following HLI across inbred mouse strains is related a region on the short arm of mouse chromosome 7. The gene coding the interleukin-21 receptor (IL-21R) lies at the peak of association in this region. Approach and Results With quantitative RT-PCR, we found that a mouse strain with a greater ability to up-regulate IL-21R following HLI had better perfusion recovery than a strain with no up-regulation after HLI. Immunofluorescent staining of ischemic hind-limb tissue showed IL-21R expression on endothelial cells (EC) from these C57BL/6 mice. An EC-enriched fraction isolated from ischemic hind-limb muscle showed higher Il-21R levels than an EC-enriched fraction from non-ischemic limbs. In-vitro, human umbilical vein EC (HUVEC) showed elevated IL-21R expression after hypoxia and serum starvation. Under these conditions, IL-21 treatment increased cell viability, decreased cell apoptosis, and augmented tube formation. In-vivo, either knockout Il21r or blocking IL-21 signaling by treating with IL-21R-Fc (fusion protein that blocks IL-21 binding to its receptor) in C57BL/6 mice resulted in less perfusion recovery after HLI. Both in-vitro and in-vivo modulation of the IL-21/IL-21R axis under hypoxic conditions resulted in increasedSTAT3 phosphorylation and a subsequent increase in the BCL-2/BAX ratio. Conclusion Our data indicate that IL-21R up-regulation and ligand activation in hypoxic endothelial cells may help perfusion recovery by limiting/preventing apoptosis and/or favoring cell survival and angiogenesis through the STAT3 pathway. PMID:25838422

  2. Synthesis and evaluation of two novel 2-nitroimidazole derivatives as potential PET radioligands for tumor imaging

    PubMed Central

    Zha, Zhihao; Zhu, Lin; Liu, Yajing; Du, Fenghua; Gan, Hongmei; Qiao, Jinpin; Kung, Hank F.

    2011-01-01

    Introduction Nitroimidazole (azomycin) derivatives labeled with radioisotopes have been developed as cancer imaging and radiotherapeutic agents based on the oncological hypoxic mechanism. By attaching nitroimidazole core with different functional groups, we synthesized new nitroimidazole derivatives, and evaluated their potentiality as tumor imaging agents. Methods Starting with commercially available 2-nitroimdazole, 2-fluoro-N-(2-(2-nitro-1H-imidazol-1-yl)ethyl)acetamide (NEFA, [19F]7) and 2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl 2-fluoroacetate (NEFT, [19F]8), as well as radiolabeling precursors - the bromo substituted analogs were quickly synthesized through a three-step synthetic pathway. The precursors were radiolabeled with [18F]F-/18-crown-6/KHCO3 in DMSO at 90 °C for 10 min followed by purification with an Oasis HLB cartridge. Biodistribution studies were carried out in EMT-6 tumor-bearing mice. The uptake (%ID/g) in tumors and normal tissues were measured at 30 min post injection. Liquid chromatography-electrospray ionization mass spectrometry (LC/MS) was used to distinguish metabolites from parent drugs in urine and plasma of rat injected with “cold” NEFA ([19F]7) and NEFT ([19F]8). Results Two radiotracers, [18F]NEFA ([18F]7) and [18F]NEFT ([18F]8), were prepared with average yields of 6-7% and 9-10% (no decay corrected). Radiochemical purity for both tracers was >95% as determined by HPLC. Biodistribution studies in EMT-6 tumor-bearing mice indicated that the tumor to blood and tumor to liver ratios of both [18F]7 (0.96, 0.98) and [18F]8 (0.61,1.10) at 30 min were higher than those observed for [18F]FMISO (1) (0.91, 0.59), a well-investigated azomycin type hypoxia radiotacer. LC/MS analysis demonstrated that fluoroacetate was the main in vivo metabolite for both NEFA ([19F]7) and NEFT ([19F]8). Conclusions In this research, two new fluorine-18 labeled 2-nitroimdazole derivatives, [18F]7 and [18F]8, both of which containing in vivo hydrolyzable

  3. 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.

  4. Magnesium sulfate protects oligodendrocyte lineage cells in a rat cell-culture model of hypoxic-ischemic injury.

    PubMed

    Itoh, Kanako; Maki, Takakuni; Shindo, Akihiro; Egawa, Naohiro; Liang, Anna C; Itoh, Naoki; Lo, Eng H; Lok, Josephine; Arai, Ken

    2016-05-01

    Hypoxic-ischemic (HI) brain injury in newborns results in serious damage. Magnesium sulfate has been clinically used as a cyto-protective agent against HI brain injury in newborns in some countries, including Japan. However, it is not clear how magnesium exerts this effect and how it acts on the individual types of cells within the newborn brain. In this study, we exposed cultured rat oligodendrocyte precursor cells to magnesium sulfate during the period when they differentiate into oligodendrocytes, and showed that magnesium-exposed oligodendrocytes exhibited more resistance to HI injury. Our data may support the use of magnesium sulfate in the clinical setting. PMID:26699082

  5. Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

    PubMed

    Germain, Todd; Ansari, Megan; Pappas, Dimitri

    2016-09-14

    Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. PMID:27566353

  6. Efficient Monte Carlo modelling of individual tumour cell propagation for hypoxic head and neck cancer

    NASA Astrophysics Data System (ADS)

    Tuckwell, W.; Bezak, E.; Yeoh, E.; Marcu, L.

    2008-09-01

    A Monte Carlo tumour model has been developed to simulate tumour cell propagation for head and neck squamous cell carcinoma. The model aims to eventually provide a radiobiological tool for radiation oncology clinicians to plan patient treatment schedules based on properties of the individual tumour. The inclusion of an oxygen distribution amongst the tumour cells enables the model to incorporate hypoxia and other associated parameters, which affect tumour growth. The object oriented program FORTRAN 95 has been used to create the model algorithm, with Monte Carlo methods being employed to randomly assign many of the cell parameters from probability distributions. Hypoxia has been implemented through random assignment of partial oxygen pressure values to individual cells during tumour growth, based on in vivo Eppendorf probe experimental data. The accumulation of up to 10 million virtual tumour cells in 15 min of computer running time has been achieved. The stem cell percentage and the degree of hypoxia are the parameters which most influence the final tumour growth rate. For a tumour with a doubling time of 40 days, the final stem cell percentage is approximately 1% of the total cell population. The effect of hypoxia on the tumour growth rate is significant. Using a hypoxia induced cell quiescence limit which affects 50% of cells with and oxygen levels less than 1 mm Hg, the tumour doubling time increases to over 200 days and the time of tumour growth for a clinically detectable tumour (109 cells) increases from 3 to 8 years. A biologically plausible Monte Carlo model of hypoxic head and neck squamous cell carcinoma tumour growth has been developed for real time assessment of the effects of multiple biological parameters which impact upon the response of the individual patient to fractionated radiotherapy.

  7. Efficient Monte Carlo modelling of individual tumour cell propagation for hypoxic head and neck cancer.

    PubMed

    Tuckwell, W; Bezak, E; Yeoh, E; Marcu, L

    2008-09-01

    A Monte Carlo tumour model has been developed to simulate tumour cell propagation for head and neck squamous cell carcinoma. The model aims to eventually provide a radiobiological tool for radiation oncology clinicians to plan patient treatment schedules based on properties of the individual tumour. The inclusion of an oxygen distribution amongst the tumour cells enables the model to incorporate hypoxia and other associated parameters, which affect tumour growth. The object oriented program FORTRAN 95 has been used to create the model algorithm, with Monte Carlo methods being employed to randomly assign many of the cell parameters from probability distributions. Hypoxia has been implemented through random assignment of partial oxygen pressure values to individual cells during tumour growth, based on in vivo Eppendorf probe experimental data. The accumulation of up to 10 million virtual tumour cells in 15 min of computer running time has been achieved. The stem cell percentage and the degree of hypoxia are the parameters which most influence the final tumour growth rate. For a tumour with a doubling time of 40 days, the final stem cell percentage is approximately 1% of the total cell population. The effect of hypoxia on the tumour growth rate is significant. Using a hypoxia induced cell quiescence limit which affects 50% of cells with and oxygen levels less than 1 mm Hg, the tumour doubling time increases to over 200 days and the time of tumour growth for a clinically detectable tumour (10(9) cells) increases from 3 to 8 years. A biologically plausible Monte Carlo model of hypoxic head and neck squamous cell carcinoma tumour growth has been developed for real time assessment of the effects of multiple biological parameters which impact upon the response of the individual patient to fractionated radiotherapy. PMID:18677039

  8. 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

  9. Hypoxic Culture Conditions as a Solution for Mesenchymal Stem Cell Based Regenerative Therapy

    PubMed Central

    Haque, Nazmul; Rahman, Mohammad Tariqur; Abu Kasim, Noor Hayaty; Alabsi, Aied Mohammed

    2013-01-01

    Cell-based regenerative therapies, based on in vitro propagation of stem cells, offer tremendous hope to many individuals suffering from degenerative diseases that were previously deemed untreatable. Due to the self-renewal capacity, multilineage potential, and immunosuppressive property, mesenchymal stem cells (MSCs) are considered as an attractive source of stem cells for regenerative therapies. However, poor growth kinetics, early senescence, and genetic instability during in vitro expansion and poor engraftment after transplantation are considered to be among the major disadvantages of MSC-based regenerative therapies. A number of complex inter- and intracellular interactive signaling systems control growth, multiplication, and differentiation of MSCs in their niche. Common laboratory conditions for stem cell culture involve ambient O2 concentration (20%) in contrast to their niche where they usually reside in 2–9% O2. Notably, O2 plays an important role in maintaining stem cell fate in terms of proliferation and differentiation, by regulating hypoxia-inducible factor-1 (HIF-1) mediated expression of different genes. This paper aims to describe and compare the role of normoxia (20% O2) and hypoxia (2–9% O2) on the biology of MSCs. Finally it is concluded that a hypoxic environment can greatly improve growth kinetics, genetic stability, and expression of chemokine receptors during in vitro expansion and eventually can increase efficiency of MSC-based regenerative therapies. PMID:24068884

  10. Exosomes Derived from Hypoxic Oral Squamous Cell Carcinoma Cells Deliver miR-21 to Normoxic Cells to Elicit a Prometastatic Phenotype.

    PubMed

    Li, Ling; Li, Chao; Wang, Shaoxin; Wang, Zhaohui; Jiang, Jian; Wang, Wei; Li, Xiaoxia; Chen, Jin; Liu, Kun; Li, Chunhua; Zhu, Guiquan

    2016-04-01

    Hypoxia is a common feature of solid tumors and is associated with aggressiveness and poor patient outcomes. Exosomes, initially considered to be cellular "garbage dumpsters," are now implicated in mediating interactions with the cellular environment. However, the mechanisms underlying the association between exosomes and hypoxia during cancer progression remain poorly understood. In this study, we found that exosomes derived from hypoxic oral squamous cell carcinoma (OSCC) cells increased the migration and invasion of OSCC cells in a HIF-1α and HIF-2α-dependent manner. Given that exosomes have been shown to transport miRNAs to alter cellular functions, we performed miRNA sequencing of normoxic and hypoxic OSCC-derived exosomes. Of the 108 miRNAs that were differentially expressed, miR-21 stood out as one of the most significantly upregulated miRNAs under hypoxic conditions. miR-21 depletion in hypoxic OSCC cells led to decreased miR-21 levels in exosomes and significantly reduced cell migration and invasion. Conversely, restoration of miR-21 expression in HIF-1α and HIF-2α-depleted exosomes rescued OSCC cell migration and invasion. Moreover, exosomal miR-21 markedly enhanced snail and vimentin expression, while significantly decreasing E-cadherin levels in OSCC cells, in vitro and in vivo Finally, circulating exosomal miR-21 levels were closely associated with HIF-1α/HIF-2α expression, T stage, and lymph node metastasis in patients with OSCC. In conclusion, our findings suggest that the hypoxic microenvironment may stimulate tumor cells to generate miR-21-rich exosomes that are delivered to normoxic cells to promote prometastatic behaviors and prompt further investigation into the therapeutic value of exosome inhibition for cancer treatment. Cancer Res; 76(7); 1770-80. ©2016 AACR. PMID:26992424

  11. 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

  12. 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. PMID:26849170

  13. Metabolic potentiation of the radiosensitization of hypoxic bacterial cells afforded by nitroaromatic compounds.

    PubMed

    Anderson, R F; Patel, K B

    1983-03-01

    Prolonged preirradiation incubation of nitroaromatic radiosensitizers with Escherichia coli cells has been found to increase the degree of radiosensitization of the cells in anoxia. Studies with E. coli strains which differ in their nitroreductase activity indicate that the increase in sensitization arises from the action of metabolites produced by the nitroreductase system of the cell. The metabolites alone appear to decrease the extrapolation number of irradiated hypoxic cells and when combined with the parent compound give a biphasic survival curve. The combination of misonidazole (1 mmole dm-3) and its metabolites (1 mmole dm-3) gave initial and final enhancement ratios of 2.4 and 1.4, respectively. The final enhancement ratio is that expected for 1 mmole dm-3 misonidazole alone, whereas the initial enhancement ratio indicates that the metabolites potentiate the action of misonidazole. The preirradiation incubation effect is removed by dithiothreitol at concentrations which do not affect the radiosensitization level of the nitroaromatic sensitizer. This result indicates that the active metabolite probably depletes a certain amount of the free-thiol compounds inside the cell which assist in the repair of radiation-induced damage. PMID:6344127

  14. 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

  15. 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. PMID:25952967

  16. Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells.

    PubMed

    Li, Mingchuan; Wang, Yong Xing; Luo, Yong; Zhao, Jiahui; Li, Qing; Zhang, Jiao; Jiang, Yongguang

    2016-07-01

    Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death. Hypoxia is an environmental stimulus that plays an important role in the development and cancer progression especially for solid tumors. The key regulator under hypoxic conditions is stabilized hypoxia-inducible factor (HIF)-1α. In the present study, immune-fluorescent staining, siRNAs, qRT-PC, immunoblotting, cell migration and invasion assays were carried out to test typical epithelial to mesenchymal transition under hypoxia and the key regulators of this process in PC3, a human prostate cancer cell line. Our data demonstrated that hypoxia induces diverse molecular, phenotypic and functional changes in prostate cancer cells that are consistent with EMT. We also showed that a cell signal factor such as HIF-1α, which might be stabilized under hypoxic environment, is involved in EMT and cancer cell invasive potency. The induced hypoxia could be blocked by HIF-1α gene silencing and reoxygenation of EMT in prostate cancer cells, hypoxia partially reversed accompanied by a process of mesenchymal-epithelial reverting transition (MErT). EMT might be induced by activation of HIF-1α-dependent cell signaling in hypoxic prostate cancer cells. PMID:27108616

  17. 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. PMID:25481308

  18. 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

  19. Autophagy mediates survival of pancreatic tumour-initiating cells in a hypoxic microenvironment.

    PubMed

    Rausch, Vanessa; Liu, Li; Apel, Anja; Rettig, Theresa; Gladkich, Jury; Labsch, Sabrina; Kallifatidis, Georgios; Kaczorowski, Adam; Groth, Ariane; Gross, Wolfgang; Gebhard, Martha M; Schemmer, Peter; Werner, Jens; Salnikov, Alexei V; Zentgraf, Hanswalter; Büchler, Markus W; Herr, Ingrid

    2012-07-01

    Involvement of dysregulated autophagy in cancer growth and progression has been shown in different tumour entities, including pancreatic ductal adenocarcinoma (PDA). PDA is an extremely aggressive tumour characterized by a small population of highly therapy-resistant cancer stem cells (CSCs) capable of self-renewal and migration. We examined whether autophagy might be involved in the survival of CSCs despite nutrition and oxygen deprivation typical for the hypoxic tumour microenvironment of PDA. Immunohistochemistry revealed that markers for hypoxia, CSCs and autophagy are co-expressed in patient-derived tissue of PDA. Hypoxia starvation (H/S) enhanced clonogenic survival and migration of established pancreatic cancer cells with stem-like properties (CSC(high)), while pancreatic tumour cells with fewer stem cell markers (CSC(low)) did not survive these conditions. Electron microscopy revealed more advanced autophagic vesicles in CSC(high) cells, which exhibited higher expression of autophagy-related genes under normoxic conditions and relative to CSC(low) cells, as found by RT-PCR and western blot analysis. LC3 was already fully converted to the active LC3-II form in both cell lines, as evaluated by western blot and detection of accumulated GFP-LC3 protein by fluorescence microscopy. H/S increased formation of autophagic and acid vesicles, as well as expression of autophagy-related genes, to a higher extent in CSC(high) cells. Modulation of autophagy by inhibitors and activators resensitized CSC(high) to apoptosis and diminished clonogenicity, spheroid formation, expression of CSC-related genes, migratory activity and tumourigenicity in mice. Our data suggest that enhanced autophagy levels may enable survival of CSC(high) cells under H/S. Interference with autophagy-activating or -inhibiting drugs disturbs the fine-tuned physiological balance of enhanced autophagy in CSC and switches survival signalling to suicide. PMID:22262369

  20. Quantitative Imaging of Hematopoietic Stem and Progenitor Cell localization and hypoxic status in the Bone Marrow microenvironment

    PubMed Central

    Nombela-Arrieta, César; Pivarnik, Gregory; Winkel, Beatrice; Canty, Kimberly J.; Harley, Brendan; Mahoney, John E.; Park, Shin-Young; Lu, Jiayun; Protopopov, Alexei; Silberstein, Leslie E.

    2014-01-01

    The existence of a hematopoietic stem cell niche as a spatially confined regulatory entity relies on the notion that hematopoietic stem and progenitor cells (HSPCs) are strategically positioned in unique bone marrow (BM) microenvironments with defined anatomical and functional features. Here, we employ a powerful imaging cytometry platform to perform a comprehensive quantitative analysis of HSPC distribution in BM cavities of femoral bones. We find that HSPCs preferentially localize in endosteal zones, where the majority closely interacts with sinusoidal and non-sinusoidal BM microvessels, which form a distinctive circulatory system. In situ tissue analysis reveals that HSPCs exhibit a hypoxic profile, defined by strong retention of pimonidazole and expression of HIF-1α, regardless of localization throughout the BM, adjacency to vascular structures or cell cycle status. These studies argue that the characteristic hypoxic state of HSPCs is not solely the result of a minimally oxygenated niche but may be partially regulated by cell-specific mechanisms. PMID:23624405

  1. 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

  2. Cytotoxic Activity of Anticancer Drugs on Hepatocellular Carcinoma Cells in Hypoxic-Hyponutritional Culture

    PubMed Central

    Li, Qiang; Zhu, Lin-Zhong; Yang, Ren-Jie; Zhu, Xu

    2014-01-01

    To investigate which anticancer drugs and combination of dual drugs could further promote the inhibition of cell growth in vitro against HCC cell line (HepG2) in the hypoxic and hyponutritional culture medium (HHCM) mimicked the different scenarios of transcatheter arterial chemoembolization (TACE). The cells of hepatocellular carcinoma (HCC) treated by TACE suffered various hypoxia and hyponutrition. The cells were treated for 2 hours, 4 hours, 6 hours, and 24 hours, respectively, using 10 drugs including epirubicin (EPI), cisplatin (DDP), mitomycin-C (MMC), oxaliplatin (OXA), hydroxycamptothecin (HCPT), 5-fluorouracil (5-FU), gemcitabine (GEM), docetaxel (DTX), thiotepa (TSPA), and pemetrexed disodium (PEM) in 4 concentrations of HHCM (5%, 10%, 25%, and 50%, respectively) mimicking the scenario of TACE and were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells treated with combinations of dual drugs for 24 hours were also tested. The sensitive drugs with inhibition rates more than 30% were EPI, MMC, HCPT, OXA, and PEM in 4 types of HHCMs. The sensitivity of the cells to treatment with drugs for 24 hours was significantly higher than the sensitivity of the cells to treatment with drugs for 2 hours in 5%, 10%, and 25% HHCM. The sensitivity of the combination of dual drugs was no more than the sensitivity of the single drug with higher sensitivity in 4 concentrations of HHCM. EPI, MMC, HCPT, OXA, and PEM exhibited cytotoxic activity against HepG2 cells in various hypoxia and hyponutrition states. Prolonging the time of exposure could increase the sensitivity of drug, and the combination of dual drugs cannot enhance the cytotoxic effect. PMID:25437582

  3. Cytotoxic activity of anticancer drugs on hepatocellular carcinoma cells in hypoxic-hyponutritional culture.

    PubMed

    Li, Qiang; Zhu, Lin-Zhong; Yang, Ren-Jie; Zhu, Xu

    2014-01-01

    To investigate which anticancer drugs and combination of dual drugs could further promote the inhibition of cell growth in vitro against HCC cell line (HepG2) in the hypoxic and hyponutritional culture medium (HHCM) mimicked the different scenarios of transcatheter arterial chemoembolization (TACE). The cells of hepatocellular carcinoma (HCC) treated by TACE suffered various hypoxia and hyponutrition. The cells were treated for 2 hours, 4 hours, 6 hours, and 24 hours, respectively, using 10 drugs including epirubicin (EPI), cisplatin (DDP), mitomycin-C (MMC), oxaliplatin (OXA), hydroxycamptothecin (HCPT), 5-fluorouracil (5-FU), gemcitabine (GEM), docetaxel (DTX), thiotepa (TSPA), and pemetrexed disodium (PEM) in 4 concentrations of HHCM (5%, 10%, 25%, and 50%, respectively) mimicking the scenario of TACE and were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells treated with combinations of dual drugs for 24 hours were also tested. The sensitive drugs with inhibition rates more than 30% were EPI, MMC, HCPT, OXA, and PEM in 4 types of HHCMs. The sensitivity of the cells to treatment with drugs for 24 hours was significantly higher than the sensitivity of the cells to treatment with drugs for 2 hours in 5%, 10%, and 25% HHCM. The sensitivity of the combination of dual drugs was no more than the sensitivity of the single drug with higher sensitivity in 4 concentrations of HHCM. EPI, MMC, HCPT, OXA, and PEM exhibited cytotoxic activity against HepG2 cells in various hypoxia and hyponutrition states. Prolonging the time of exposure could increase the sensitivity of drug, and the combination of dual drugs cannot enhance the cytotoxic effect. PMID:25437582

  4. Studies on the mechanism of the cytotoxic action of the mitomycin antibiotics in hypoxic and oxygenated EMT6 cells.

    PubMed

    Sartorelli, A C; Tomasz, M; Rockwell, S

    1993-01-01

    The mitomycin antibiotics, because of their preferential toxicities for hypoxic cells, have significant potential as adjuncts to ionizing radiation in the treatment of solid tumors. To gain information on the mechanism by which these agents exert their cytotoxicities to hypoxic and aerobic cells, the effects of MC, POR and several of their analogs were studied in EMT6 mammary carcinoma cells. The rate of uptake of POR by these cells was directly correlated with the cytotoxicity produced by this agent under both hypoxia and aeration. At equivalent concentrations, uptake of POR into hypoxic cells was more rapid than into aerobic cells. Hypoxic cells also accumulated the antibiotic in concentrations well in excess of that present in the extracellular medium, presumably as a result of reductive activation and covalent binding of POR to cellular structures. Such activation and binding occur to a much lesser degree in aerated cells, resulting in the rapid efflux of POR from these cells when the antibiotic is removed from the extracellular environment. To gain information on the reaction of POR with DNA, mono- and bis-adducts formed in EMT6 cells exposed to this agent were measured. Three major adducts were formed. Two were mono-adducts consisting of deoxyguanosine linked at its N2-position to the C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct in which POR was cross-linked to two deoxyguanosines at their N2-positions. More adducts were formed in hypoxia than in air, and more bis-adducts were present in hypoxic cells. Simultaneous exposure of cells to both POR and DIC reduced the total adduct level and a new unknown adduct was formed, primarily under hypoxia. Several mitomycins were evaluated for their capacity to kill EMT6 cells and to produce DNA cross-links in both hypoxia and aeration. The number of cross-links required to produce a given amount of cell kill was similar, regardless of the mitomycin employed or the degree of oxygenation. The findings

  5. Cripto regulates hematopoietic stem cells as a hypoxic-niche-related factor through cell surface receptor GRP78.

    PubMed

    Miharada, Kenichi; Karlsson, Göran; Rehn, Matilda; Rörby, Emma; Siva, Kavitha; Cammenga, Jörg; Karlsson, Stefan

    2011-10-01

    Hematopoietic stem cells (HSCs) are maintained in hypoxic niches in endosteal regions of bones. Here we demonstrate that Cripto and its receptor GRP78 are important regulators of HSCs in the niche. Flow cytometry analyses revealed two distinct subpopulations of CD34(-)KSL cells based on the expression of GRP78, and these populations showed different reconstitution potential in transplantation assays. GRP78(+)HSCs mainly reside in the endosteal area, are more hypoxic, and exhibit a lower mitochondrial potential, and their HSC capacity was maintained in vitro by Cripto through induction of higher glycolytic activity. Additionally, HIF-1α KO mice have decreased numbers of GRP78(+)HSCs and reduced expression of Cripto in the endosteal niche. Furthermore, blocking GRP78 induced a movement of HSCs from the endosteal to the central marrow area. These data suggest that Cripto/GRP78 signaling is an important pathway that regulates HSC quiescence and maintains HSCs in hypoxia as an intermediary of HIF-1α. PMID:21982233

  6. PIM Kinase Inhibitors Kill Hypoxic Tumor Cells by Reducing Nrf2 Signaling and Increasing Reactive Oxygen Species.

    PubMed

    Warfel, Noel A; Sainz, Alva G; Song, Jin H; Kraft, Andrew S

    2016-07-01

    Intratumoral hypoxia is a significant obstacle to the successful treatment of solid tumors, and it is highly correlated with metastasis, therapeutic resistance, and disease recurrence in cancer patients. As a result, there is an urgent need to develop effective therapies that target hypoxic cells within the tumor microenvironment. The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases represent a prosurvival pathway that is upregulated in response to hypoxia, in a HIF-1-independent manner. We demonstrate that pharmacologic or genetic inhibition of PIM kinases is significantly more toxic toward cancer cells in hypoxia as compared with normoxia. Xenograft studies confirm that PIM kinase inhibitors impede tumor growth and selectively kill hypoxic tumor cells in vivo Experiments show that PIM kinases enhance the ability of tumor cells to adapt to hypoxia-induced oxidative stress by increasing the nuclear localization and activity of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), which functions to increase the expression of antioxidant genes. Small molecule PIM kinase inhibitors prevent Nrf2 from accumulating in the nucleus, reducing the transcription of cytoprotective genes and leading to the build-up of intracellular reactive oxygen species (ROS) to toxic levels in hypoxic tumor cells. This toxic effect of PIM inhibitors can be successfully blocked by ROS scavengers, including N-acetyl cystine and superoxide dismutase. Thus, inhibition of PIM kinases has the potential to oppose hypoxia-mediated therapeutic resistance and induce cell death in the hypoxic tumor microenvironment. Mol Cancer Ther; 15(7); 1637-47. ©2016 AACR. PMID:27196781

  7. Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

    SciTech Connect

    Pilgaard, L.; Lund, P.; Duroux, M.; Lockstone, H.; Taylor, J.; Emmersen, J.; Fink, T.; Ragoussis, J.; Zachar, V.

    2009-07-01

    Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

  8. The radiomimetic enediyne, 20'-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance.

    PubMed

    Beerman, Terry A; Gawron, Loretta S; Shen, Ben; Kennedy, Daniel R

    2014-09-01

    The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027's induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20'-deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors. PMID:24986640

  9. The radiomimetic enediyne, 20′-deschloro-C-1027 induces inter-strand DNA crosslinks in hypoxic cells and overcomes cytotoxic radioresistance

    PubMed Central

    Beerman, Terry A.; Gawron, Loretta S.; Shen, Ben; Kennedy, Daniel R.

    2014-01-01

    The ability of the radiomimetic anti-tumor enediyne C-1027 to induce DNA inter-strand crosslinks (ICLs), in addition to the expected DNA strand breaks, is unique among traditional DNA targeted cancer therapies. Importantly, radiation therapy and most radiomimetic drugs have diminished effect in hypoxic environments due to decreased induction of DNA strand breaks, which is an oxygen requiring process. However, C-1027’s induction of ICLs is enhanced under hypoxia and it is actually more potent against hypoxic cells, overcoming this common tumor resistance mechanism. In this study, an analog of C-1027, 20′deschloro-C-1027 was examined for its ability to induce DNA ICLs under hypoxic conditions. Deschloro-induced ICLs were detected under hypoxic cell-free conditions, with a concomitant reduction in the induction of DNA strand breaks. In cells deschloro behaved similarly, inducing cellular ICLs under hypoxic conditions with a reduction in DNA breaks. The cytotoxicity of deschloro treatment was similar in normoxic and hypoxic cells, suggesting that the ICL induction allows deschloro to retain its cytotoxic activity under hypoxia. It appears that rational engineering of the C-1027 family of radiomimetics holds promise toward overcoming the radioresistance associated with the hypoxic environment associated with solid tumors. PMID:24986640

  10. Repetitive hypoxic preconditioning induces an immunosuppressed B cell phenotype during endogenous protection from stroke

    PubMed Central

    2014-01-01

    Background Repetitive hypoxic preconditioning (RHP) creates an anti-inflammatory phenotype that protects from stroke-induced injury for months after a 2-week treatment. The mechanisms underlying long-term tolerance are unknown, though one exposure to hypoxia significantly increased peripheral B cell representation. For this study, we sought to determine if RHP specifically recruited B cells into the protected ischemic hemisphere, and whether RHP could phenotypically alter B cells prior to stroke onset. Methods Adult, male SW/ND4 mice received RHP (nine exposures over 2 weeks; 8 to 11 % O2; 2 to 4 hours) or identical exposures to 21 % O2 as control. Two weeks following RHP, a 60-minute transient middle cerebral artery occlusion was induced. Standard techniques quantified CXCL13 mRNA and protein expression. Two days after stroke, leukocytes were isolated from brain tissue (70:30 discontinuous Percoll gradient) and profiled on a BD-FACS Aria flow cytometer. In a separate cohort without stroke, sorted splenic CD19+ B cells were isolated 2 weeks after RHP and analyzed on an Illumina MouseWG-6 V2 Bead Chip. Final gene pathways were determined using Ingenuity Pathway Analysis. Student’s t-test or one-way analysis of variance determined significance (P < 0.05). Results CXCL13, a B cell-specific chemokine, was upregulated in post-stroke cortical vessels of both groups. In the ischemic hemisphere, RHP increased B cell representation by attenuating the diapedesis of monocyte, macrophage, neutrophil and T cells, to quantities indistinguishable from the uninjured, contralateral hemisphere. Pre-stroke splenic B cells isolated from RHP-treated mice had >1,900 genes differentially expressed by microarray analysis. Genes related to B-T cell interactions, including antigen presentation, B cell differentiation and antibody production, were profoundly downregulated. Maturation and activation were arrested in a cohort of B cells from pre-stroke RHP-treated mice while

  11. HIF1α represses cell stress pathways to allow proliferation of hypoxic fetal cardiomyocytes

    PubMed Central

    Guimarães-Camboa, Nuno; Stowe, Jennifer; Aneas, Ivy; Sakabe, Noboru; Cattaneo, Paola; Henderson, Lindsay; Kilberg, Michael S.; Johnson, Randall S.; Chen, Ju; McCulloch, Andrew D.; Nobrega, Marcelo A.; Evans, Sylvia M.; Zambon, Alexander C.

    2015-01-01

    Summary Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with each other are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we have discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay between HIF1α, ATF4 and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions between major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia, and may have relevance to other diseases, including cancer. PMID:26028220

  12. Ultrasound-mediated destruction of oxygen and paclitaxel loaded lipid microbubbles for combination therapy in hypoxic ovarian cancer cells.

    PubMed

    Sun, Jiangchuan; Yin, Mingyue; Zhu, Shenyin; Liu, Li; Zhu, Yi; Wang, Zhigang; Xu, Ronald X; Chang, Shufang

    2016-01-01

    We synthesized oxygen and paclitaxel (PTX) loaded lipid microbubbles (OPLMBs) for ultrasound mediated combination therapy in hypoxic ovarian cancer cells. Our experiments successfully demonstrated that ultrasound induced OPLMBs destruction significantly enhanced the local oxygen release. We also demonstrated that OPLMBs in combination with ultrasound (300 kHz, 0.5 W/cm(2), 15s) yielded anti-proliferative activities of 52.8 ± 2.75% and cell apoptosis ratio of 35.25 ± 0.17% in hypoxic cells at 24h after the treatment, superior to other treatment groups such as PTX only and PTX-loaded MBs (PLMBs) with or without ultrasound mediation. RT-PCR and Western blot tests further confirmed the reduced expression of HIF-1α and MDR-1/P-gp after ultrasound mediation of OPLMBs. Our experiment suggests that ultrasound mediation of oxygen and drug-loaded MBs may be a useful method to overcome chemoresistance in the hypoxic ovarian cancer cells. PMID:26384914

  13. Embryonic stem cells conditioned medium enhances Wharton's jelly-derived mesenchymal stem cells expansion under hypoxic condition.

    PubMed

    Prasajak, Patcharee; Rattananinsruang, Piyaporn; Chotinantakul, Kamonnaree; Dechsukhum, Chavaboon; Leeanansaksiri, Wilairat

    2015-05-01

    Mesenchymal stem cells (MSCs) are accepted as a promising tool for therapeutic purposes. However, low proliferation and early senescence are still main obstacles of MSCs expansion for using as cell-based therapy. Thus, clinical scale of cell expansion is needed to obtain a large number of cells serving for further applications. In this study, we investigated the value of embryonic stem cells conditioned medium (ESCM) for in vitro expansion of Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) as compared to typical culture medium for MSCs, Dulbecco's modified Eagle's medium with 1.0 g/l glucose (DMEM-LG) supplemented with 10 % FBS, under hypoxic condition. The expanded cells from ESCM (ESCM-MSCs) and DMEM-LG (DMEM-MSCs) were characterized for both phenotype and biological activities including proliferation rate, population doubling time, cell cycle distribution and MSCs characteristics. ESCM and DMEM-LG could enhance WJ-MSCs proliferation as 204.66 ± 10.39 and 113.77 ± 7.89 fold increase at day 12, respectively. ESCM-MSCs could express pluripotency genes including Oct-4, Oct-3/4, Nanog, Klf-4, C-Myc and Sox-2 both in early and late passages whereas the downregulations of Oct-4 and Nanog were detected in late passage cells of DMEM-MSCs. The 2 cell populations also showed common MSCs characteristics including normal cell cycle, fibroblastic morphology, cell surface markers expressions (CD29(+), CD44(+), CD90(+), CD34(-), CD45(-)) and differentiation capacities into adipogenic, chondrogenic and osteogenic lineages. Moreover, our results revealed that ESCM exhibited as a rich source of several factors which are required for supportive WJ-MSCs proliferation. In conclusion, ESCM under hypoxic condition could accelerate WJ-MSCs expansion while maintaining their pluripotency properties. Our knowledge provide short term and cost-saving in WJ-MSCs expansion which has benefit to overcome insufficient cell numbers for clinical applications by reusing the

  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. Continuous Hypoxic Culturing of Human Embryonic Stem Cells Enhances SSEA-3 and MYC Levels

    PubMed Central

    Laiho, Asta; Rahkonen, Nelly; Emani, Maheswara Reddy; Viitala, Miro; Laurila, Kirsti; Sahla, Roosa; Lund, Riikka; Lähdesmäki, Harri; Jaakkola, Panu; Lahesmaa, Riitta

    2013-01-01

    Low oxygen tension (hypoxia) contributes critically to pluripotency of human embryonic stem cells (hESCs) by preventing spontaneous differentiation and supporting self-renewal. However, it is not well understood how hESCs respond to reduced oxygen availability and what are the molecular mechanisms maintaining pluripotency in these conditions. In this study we characterized the transcriptional and molecular responses of three hESC lines (H9, HS401 and HS360) on short (2 hours), intermediate (24 hours) and prolonged (7 days) exposure to low oxygen conditions (4% O2). In response to prolonged hypoxia the expression of pluripotency surface marker SSEA-3 was increased. Furthermore, the genome wide gene-expression analysis revealed that a substantial proportion (12%) of all hypoxia-regulated genes in hESCs, were directly linked to the mechanisms controlling pluripotency or differentiation. Moreover, transcription of MYC oncogene was induced in response to continuous hypoxia. At the protein level MYC was stabilized through phosphorylation already in response to a short hypoxic exposure. Total MYC protein levels remained elevated throughout all the time points studied. Further, MYC protein expression in hypoxia was affected by silencing HIF2α, but not HIF1α. Since MYC has a crucial role in regulating pluripotency we propose that induction of sustained MYC expression in hypoxia contributes to activation of transcriptional programs critical for hESC self-renewal and maintenance of enhanced pluripotent state. PMID:24236059

  16. Regulation of angiogenin expression and epithelial-mesenchymal transition by HIF-1α signaling in hypoxic retinal pigment epithelial cells.

    PubMed

    Lai, Kairan; Luo, Chenqi; Zhang, Xiaobo; Ye, Panpan; Zhang, Yidong; He, Jiliang; Yao, Ke

    2016-09-01

    Choroidal neovascularization (CNV) is a major cause of vision loss in many retinal diseases. Hypoxia is determined to be a key inducer of CNV and hypoxia-inducible factor-1 (HIF-1) is an important transcription factor. Epithelial-mesenchymal transition (EMT) and the synthesis of proangiogenic cytokines make great contributions to the development of CNV. In the present study, the role of HIF-1α signaling in the regulation of angiogenin (ANG) expression and EMT in hypoxic retinal pigment epithelial cells was investigated. A significant elevation expression of ANG expression level in a mouse model of laser-induced CNV was demonstrated. In a hypoxic model of ARPE-19, an increased expression level of ANG and induction of EMT accompanied with stabilization and nucleus translocation of HIF-1α. Blockage of HIF-1α signaling resulted in inhibition of high expression of ANG and EMT features. The direct interaction between HIF-1α and ANG promoter region was identified by ChIP-qPCR. The association of RNase 4 mRNA level with HIF-1α signaling was also clarified in APRE-19. Moreover, the exogenous ANG translocated into the nucleus, enhanced 45S rRNA transcription, promoted cell proliferation and tube formation in human retinal microvascular endothelial cells. In conclusion, the hypoxic conditions regulate the expression of ANG and EMT via an activation of HIF-1α signaling. It provides molecular evidence for potential therapy strategies of treating CNV. PMID:27259982

  17. Synthesis and Biological Evaluation of Iodoglucoazomycin (I-GAZ), an Azomycin-Glucose Adduct with Putative Applications in Diagnostic Imaging and Radiotherapy of Hypoxic Tumors.

    PubMed

    Kumar, Piyush; Elsaidi, Hassan R H; Zorniak, Bohdarianna; Laurens, Evelyn; Yang, Jennifer; Bacchu, Veena; Wang, Monica; Wiebe, Leonard I

    2016-08-01

    Iodoglucoazomycin (I-GAZ; N-(2-iodo-3-(6-O-glucosyl)propyl)-2-nitroimidazole), a non-glycosidic nitroimidazole-6-O-glucose adduct, was synthesized, radioiodinated, and evaluated as a substrate of glucose transporter 1 (GLUT1) for radiotheranostic (therapy+diagnostic) management of hypoxic tumors. Nucleophilic iodination of the nosylate synthon of I-GAZ followed by deprotection afforded I-GAZ in 74 % overall yield. I-GAZ was radioiodinated via 'exchange' labeling using [(123/131) I]iodide (50-70 % RCY) and then purified by Sep-Pak™ (>96 % RCP). [(131) I]I-GAZ was stable in 2 % ethanolic solution in sterile water for 14 days when stored at 5 °C. In cell culture, I-GAZ was found to be nontoxic to EMT-6 cells at concentrations <0.5 mm, and weakly radiosensitizing (SER 1.1 at 10 % survival of EMT-6 cells; 1.2 at 0.1 % survival in MCF-7 cells). The hypoxic/normoxic uptake ratio of [(123) I]I-GAZ in EMT-6 cells was 1.46 at 2 h, and under normoxic conditions the uptake of [(123) I]I-GAZ by EMT-6 cells was unaltered in the presence of 5 mm glucose. The biodistribution of [(131) I]I-GAZ in EMT-6 tumor-bearing Balb/c mice demonstrated rapid clearance from blood and extensive renal and hepatic excretion. Tumor/blood and tumor/muscle ratios reached ∼3 and 8, respectively, at 4 h post-injection. Regression analysis of the first order polynomial plots of the blood and tumor radioactivity concentrations supported a perfusion-excretion model with low hypoxia-dependent binding. [(131) I]I-GAZ was found to be stable in vivo, and did not deiodinate. PMID:27377671

  18. Melatonin promotes osteoblast differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions through activation of PKD/p38 pathways.

    PubMed

    Son, Jang-Ho; Cho, Yeong-Cheol; Sung, Iel-Yong; Kim, In-Ryoung; Park, Bong-Soo; Kim, Yong-Deok

    2014-11-01

    Osteoblastic differentiation and bone-forming capacity are known to be suppressed under hypoxic conditions. Melatonin has been shown to influence cell differentiation. A number of in vitro and in vivo studies have suggested that melatonin also has an anabolic effect on bone, by promoting osteoblastic differentiation. However, the precise mechanisms and the signaling pathways involved in this process, particularly under hypoxic conditions, are unknown. This study investigated whether melatonin could promote osteoblastic differentiation and mineralization of preosteoblastic MC3T3-E1 cells under hypoxic conditions. Additionally, we examined the molecular signaling pathways by which melatonin mediates this process. We found that melatonin is capable of promoting differentiation and mineralization of MC3T3-E1 cells cultured under hypoxic conditions. Melatonin upregulated ALP activity and mRNA levels of Alp, Osx, Col1, and Ocn in a time- and concentration-dependent manner. Alizarin red S staining showed that the mineralized matrix in hypoxic MC3T3-E1 cells formed in a manner that was dependent on melatonin concentration. Moreover, melatonin stimulated phosphorylation of p38 Mapk and Prkd1 in these MC3T3-E1 cells. We concluded that melatonin promotes osteoblastic differentiation of MC3T3-E1 cells under hypoxic conditions via the p38 Mapk and Prkd1 signaling pathways. PMID:25250639

  19. Loss of cell adhesion molecule CHL1 improves homeostatic adaptation and survival in hypoxic stress.

    PubMed

    Huang, X; Sun, J; Rong, W; Zhao, T; Li, D H; Ding, X; Wu, L Y; Wu, K; Schachner, M; Xiao, Z C; Zhu, L L; Fan, M

    2013-01-01

    Close homologue of L1 (CHL1) is a transmembrane cell adhesion molecule that is critical for brain development and for the maintenance of neural circuits in adults. Recent studies revealed that CHL1 has diverse roles and is involved in the regulation of recovery after spinal cord injury. CHL1 expression was downregulated in the cerebral cortex, hypothalamus, and brain stem after the induction of acute hypoxia (AH). In the current study, we sought to address the role of CHL1 in regulating homeostasis responses to hypoxia using CHL1-knockout (CHL1(-/-)) mice. We found that, compared with wild-type littermates, CHL1(-/-) mice showed a dramatically lower mortality rate and an augmented ventilatory response after they were subjected to AH. Immunofluorescence staining revealed that CHL1 was expressed in the carotid body (CB), the key oxygen sensor in rodents, and CHL1 expression level in the CB as assayed by western blot was decreased after hypoxic exposure. The number of glomus cells and the expression of tyrosine hydroxylase (a marker for glomus cells) in the CB of CHL1(-/-) mice appeared to be increased compared with CHL1(+/+) mice. In addition, in the ex vivo CB preparation, hypoxia induced a significantly greater afferent nerve discharge in CHL1(-/-) mice compared with CHL1(+/+) mice. Furthermore, the arterial blood pressure and plasma catecholamine levels of CHL1(-/-) mice were also significantly higher than those of CHL1(+/+) mice. Our findings first demonstrate that CHL1 is a novel intrinsic factor that is involved in CB function and in the ventilatory response to AH. PMID:23949217

  20. Modification of the metabolism and cytotoxicity of bioreductive alkylating agents by dicoumarol in aerobic and hypoxic murine tumor cells.

    PubMed

    Keyes, S R; Rockwell, S; Sartorelli, A C

    1989-06-15

    We have demonstrated previously that dicoumarol (DIC) increased the generation of reactive metabolites from mitomycin C (MC) in EMT6 cells under hypoxic conditions in vitro. This increased reaction rate was associated with an increased toxicity of MC to hypoxic EMT6 cells. In contrast, aerobic cells treated with DIC in vitro were protected from MC toxicity. We now demonstrate that DIC sensitizes EMT6 cells to two MC analogues, porfiromycin (POR) and the 7-N-dimethylaminomethylene analogue of mitomycin C (BMY-25282), in hypoxia and protects cells from these agents in air, despite the fact that POR is preferentially toxic to hypoxic cells and BMY-25282 is preferentially toxic to aerobic cells. In contrast, DIC increases menadione cytotoxicity in both air and hypoxia and has no effect on the cytotoxicity of Adriamycin. We have also shown previously that the preferential toxicity of POR to hypoxic cells is associated with an increased rate of drug uptake. In the present study, DIC had no measurable effect on the uptake of [3H]POR but increased the extent of efflux of this agent. MC-induced DNA cross-links, which have been proposed as the lesions responsible for the lethality of MC, are decreased by DIC in air and increased by DIC in hypoxia, in concert with the observed modifications of MC cytotoxicity by DIC. However, in aerobic cells treated with DIC and MC, the decrease in DNA interstrand cross-links is not directly associated with a decrease in cytotoxicity. L1210 cells, which have no measurable quinone reductase activity, demonstrate increased toxicity when treated with DIC and MC in hypoxia, as observed with EMT6 cells. Unlike EMT6 cells, however, L1210 cells are not protected by DIC from MC toxicity in air. Taken together, these findings suggest that DIC is altering the intracellular metabolism of MC and that quinone reductase or another, unidentified, enzyme sensitive to DIC may be involved in activating MC to a toxic product in aerobic EMT6 cells. PMID:2470504

  1. Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions.

    PubMed

    Marshall, R S; Paterson, M C; Rauth, A M

    1989-03-01

    Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells. PMID:2467684

  2. Deficient activation by a human cell strain leads to mitomycin resistance under aerobic but not hypoxic conditions.

    PubMed Central

    Marshall, R. S.; Paterson, M. C.; Rauth, A. M.

    1989-01-01

    Two non-transformed human skin fibroblast strains, GM38 and 3437T, were found to be more sensitive to the bioreductive alkylating agents mitomycin C (MMC) and porfiromycin (PM) under hypoxic compared to aerobic conditions. One of these strains, 3437T, was 6-7 times more resistant to these agents under aerobic exposure conditions, but was identical in sensitivity to the normal strain, GM38, under hypoxic conditions. Aerobic 3437T cells demonstrated no increased resistance to cisplatin compared to the normal strain, arguing against enhanced ability to repair DNA interstrand cross-links as the underlying explanation for the mitomycin resistance. The aerobic resistance of 3437T was not altered by dicumarol, an inhibitor of the enzyme DT-diaphorase which is believed to be involved in aerobic activation of MMC and PM. Dicumarol did increase the resistance of GM38, but not to the same level of resistance demonstrated by 3437T. These results suggest that the aerobic MMC and PM resistance of 3437T may arise, in part, from a deficiency in DT-diaphorase activity. The identical sensitivities under hypoxic conditions indicate that drug activation pathways operative in the absence of oxygen are similar in both the normal and 3437T cells. PMID:2467684

  3. 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

  4. Preclinical and Clinical Evidence of Mycobacterium tuberculosis Persistence in the Hypoxic Niche of Bone Marrow Mesenchymal Stem Cells after Therapy.

    PubMed

    Garhyan, Jaishree; Bhuyan, Seema; Pulu, Ista; Kalita, Deepjyoti; Das, Bikul; Bhatnagar, Rakesh

    2015-07-01

    Mycobacterium tuberculosis (MTB), the causative agent of pulmonary tuberculosis, is difficult to eliminate by antibiotic therapy. We recently identified CD271(+) bone marrow-mesenchymal stem cells (BM-MSCs) as a potential site of MTB persistence after therapy. Herein, we have characterized the potential hypoxic localization of the post-therapy MTB-infected CD271(+) BM-MSCs in both mice and human subjects. We first demonstrate that in a Cornell model of MTB persistence in mice, green fluorescent protein-labeled virulent MTB-strain H37Rv was localized to pimonidazole (an in vivo hypoxia marker) positive CD271(+) BM-MSCs after 90 days of isoniazid and pyrazinamide therapy that rendered animal's lung noninfectious. The recovered CD271(+) BM-MSCs from post-therapy mice, when injected into healthy mice, caused active tuberculosis infection in the animal's lung. Moreover, MTB infection significantly increased the hypoxic phenotype of CD271(+) BM-MSCs. Next, in human subjects, previously treated for pulmonary tuberculosis, the MTB-containing CD271(+) BM-MSCs exhibited high expression of hypoxia-inducible factor 1α and low expression of CD146, a hypoxia down-regulated cell surface marker of human BM-MSCs. These data collectively demonstrate the potential localization of MTB harboring CD271(+) BM-MSCs in the hypoxic niche, a critical microenvironmental factor that is well known to induce the MTB dormancy phenotype. PMID:26066709

  5. BrdU-positive cells in the neonatal mouse hippocampus following hypoxic-ischemic brain injury

    PubMed Central

    Bartley, John; Soltau, Thomas; Wimborne, Hereward; Kim, Sunjun; Martin-Studdard, Angeline; Hess, David; Hill, William; Waller, Jennifer; Carroll, James

    2005-01-01

    Background Mechanisms that affect recovery from fetal and neonatal hypoxic-ischemic (H-I) brain injury have not been fully elucidated. The incidence of intrapartum asphyxia is approximately 2.5%, but the occurrence of adverse clinical outcome is much lower. One of the factors which may account for this relatively good outcome is the process of neurogenesis, which has been described in adult animals. We used a neonatal mouse model to assess new cells in the hippocampus after H-I injury. Results Neonatal mice underwent permanent unilateral carotid ligation on the seventh postnatal day followed by exposure to 8% hypoxia for 75 minutes. The presence of new cells was determined by bromodeoxyuridine (BrdU) incorporation into cells with sacrifice of the animals at intervals. Brain sections were stained for BrdU in combination with neuronal, glial, endothelial and microglial stains. We found a significant increase in BrdU-positive cells in the neonatal mouse hippocampus in the injured area compared to the non-injured area, most prominent in the dentate gyrus (DG) (154.5 ± 59.6 v. 92.9 ± 32.7 at 3 days after injury; 68.9 ± 23.4 v. 52.4 ± 17.1 at 35 days after injury, p < 0.0011). Among the cells which showed differentiation, those which were stained as either microglial or endothelial cells showed a peak increase at three days after the injury in the DG, injured versus non-injured side (30.5 ± 17.8 v. 2.7 ± 2.6, p < 0.0002). As in the adult animal, neurogenesis was significantly increased in the DG with injury (15.0 ± 4.6 v. 5.2 ± 1.6 at 35 days after injury, p < 0.0002), and this increase was subsequent to the appearance of the other dividing cells. Numbers of new oligodendrocytes were significantly higher in the DG on the non-injured side (7.0 ± 24.2 v. 0.1 ± 0.3, p < 0.0002), suggesting that oligodendrocyte synthesis was reduced in the injured hippocampus. Conclusion These findings demonstrate that the neonatal animal responds to brain injury with neurogenesis

  6. 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.

  7. Cytotoxicity and DNA lesions produced by mitomycin C and porfiromycin in hypoxic and aerobic EMT6 and Chinese hamster ovary cells.

    PubMed

    Fracasso, P M; Sartorelli, A C

    1986-08-01

    Solid neoplasms may contain deficient or poorly functional vascular beds, a property that leads to the formation of hypoxic tumor cells, which form a therapeutically resistant cell population within the tumor that is difficult to eradicate by ionizing irradiation and most existing chemotherapeutic agents. As an approach to the therapeutic attack of hypoxic cells, we have measured the cytotoxicity and DNA lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin, two structurally similar antibiotics, in oxygen-deficient and aerobic cells. Mitomycin C and porfiromycin were preferentially cytotoxic to hypoxic EMT6 cells in culture, with porfiromycin producing a greater differential kill of hypoxic EMT6 cells relative to their oxygenated counterparts than did mitomycin C. Chinese hamster ovary cells were more resistant to these quinone antibiotics; although in this cell line, porfiromycin was significantly more cytotoxic to hypoxic cells than to aerobic cells, and the degree of oxygenation did not affect the toxicity of mitomycin C. Alkaline elution methodology was utilized to study the formation of DNA single-strand breaks and DNA interstrand cross-links produced by mitomycin C and porfiromycin in both EMT6 and Chinese hamster ovary cells. A negligible quantity of DNA single-strand breaks and DNA interstrand cross-links were produced in hypoxic and aerobic Chinese hamster ovary cells by exposure to mitomycin C or porfiromycin, a finding consistent with the considerably lower sensitivity of this cell line to these agents. In EMT6 tumor cells, no single-strand breaks appeared to be produced by these antitumor antibiotics under both hypoxic and aerobic conditions; however, a significant number of DNA interstrand cross-links were formed in this cell line following drug treatment, with substantially more DNA interstrand cross-linking being produced under hypoxic conditions. Mitomycin C and porfiromycin caused the same amount of cross-linking under

  8. Activity of C-7 substituted cyclic acetal derivatives of mitomycin C and porfiromycin against hypoxic and oxygenated EMT6 carcinoma cells in vitro and in vivo.

    PubMed

    Rockwell, S; Keyes, S R; Loomis, R; Kelley, M; Vyas, D M; Wong, H; Doyle, T W; Sartorelli, A C

    1991-06-01

    A series of cyclic acetal derivatives of mitomycin C (MC) and porfiromycin (POR) were tested for their ability to kill hypoxic and oxygenated EMT6 tumor cells. Amino methyl acetal and thioacetal substitutions at C-7 of MC and POR dramatically increased the cytotoxicity of the compounds to hypoxic EMT6 tumor cells in vitro but had little effect on the aerobic toxicities. In contrast, a methyl substitution at N1a markedly decreased the aerobic cytotoxicities of the compounds but did not alter the hypoxic cytotoxicities. The POR acetal, BMY-42355, had the largest differential between hypoxic and aerobic cytotoxicities yet observed among MC analogs. Preliminary studies in mice showed that BMY-42355 had good antineoplastic activity when used alone or in combination with radiation and was less toxic than POR; the therapeutic ratio of this compound in these initial studies was higher than those of either MC or POR. PMID:2049227

  9. Protection of Cardiomyocytes from Ischemic/Hypoxic Cell Death via Drbp1 and pMe2GlyDH in Cardio-specific ARC Transgenic Mice*

    PubMed Central

    Pyo, Jong-Ok; Nah, Jihoon; Kim, Hyo-Jin; Chang, Jae-Woong; Song, Young-Wha; Yang, Dong-Kwon; Jo, Dong-Gyu; Kim, Hyung-Ryong; Chae, Han-Jung; Chae, Soo-Wan; Hwang, Seung-Yong; Kim, Seung-Jun; Kim, Hyo-Joon; Cho, Chunghee; Oh, Chang-Gyu; Park, Woo Jin; Jung, Yong-Keun

    2008-01-01

    The ischemic death of cardiomyocytes is associated in heart disease and heart failure. However, the molecular mechanism underlying ischemic cell death is not well defined. To examine the function of apoptosis repressor with a caspase recruitment domain (ARC) in the ischemic/hypoxic damage of cardiomyocytes, we generated cardio-specific ARC transgenic mice using a mouse α-myosin heavy chain promoter. Compared with the control, the hearts of ARC transgenic mice showed a 3-fold overexpression of ARC. Langendoff preparation showed that the hearts isolated from ARC transgenic mice exhibited improved recovery of contractile performance during reperfusion. The cardiomyocytes cultured from neonatal ARC transgenic mice were significantly resistant to hypoxic cell death. Furthermore, the ARC C-terminal calcium-binding domain was as potent to protect cardiomyocytes from hypoxic cell death as ARC. Genome-wide RNA expression profiling uncovered a list of genes whose expression was changed (>2-fold) in ARC transgenic mice. Among them, expressional regulation of developmentally regulated RNA-binding protein 1 (Drbp1) or the dimethylglycine dehydrogenase precursor (pMe2GlyDH) affected hypoxic death of cardiomyocytes. These results suggest that ARC may protect cardiomyocytes from hypoxic cell death by regulating its downstream, Drbp1 and pMe2GlyDH, shedding new insights into the protection of heart from hypoxic damages. PMID:18782777

  10. 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. PMID:26612622

  11. 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.

  12. 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.

  13. 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.

  14. Exposure of yeast cells to anoxia induces transient oxidative stress. Implications for the induction of hypoxic genes.

    PubMed

    Dirmeier, Reinhard; O'Brien, Kristin M; Engle, Marcella; Dodd, Athena; Spears, Erick; Poyton, Robert O

    2002-09-20

    The mitochondrial respiratory chain is required for the induction of some yeast hypoxic nuclear genes. Because the respiratory chain produces reactive oxygen species (ROS), which can mediate intracellular signal cascades, we addressed the possibility that ROS are involved in hypoxic gene induction. Recent studies with mammalian cells have produced conflicting results concerning this question. These studies have relied almost exclusively on fluorescent dyes to measure ROS levels. Insofar as ROS are very reactive and inherently unstable, a more reliable method for measuring changes in their intracellular levels is to measure their damage (e.g. the accumulation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) in DNA, and oxidative protein carbonylation) or to measure the expression of an oxidative stress-induced gene, e.g. SOD1. Here we used these approaches as well as a fluorescent dye, carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA), to determine whether ROS levels change in yeast cells exposed to anoxia. These studies reveal that the level of mitochondrial and cytosolic protein carbonylation, the level of 8-OH-dG in mitochondrial and nuclear DNA, and the expression of SOD1 all increase transiently during a shift to anoxia. These studies also reveal that carboxy-H(2)-DCFDA is an unreliable reporter of ROS levels in yeast cells shifted to anoxia. By using two-dimensional electrophoresis and mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), we have found that specific proteins become carbonylated during a shift to anoxia and that some of these proteins are the same proteins that become carbonylated during peroxidative stress. The mitochondrial respiratory chain is responsible for much of this carbonylation. Together, these findings indicate that yeast cells exposed to anoxia experience transient oxidative stress and raise the possibility that this initiates the induction of hypoxic genes. PMID:12089150

  15. Correlation between radiosensitivity, percentage hypoxic cells and pO2 measurements in one rodent and two human tumor xenografts.

    PubMed

    Thomas, C D; Chavaudra, N; Martin, L; Guichard, M

    1994-07-01

    Computerized pO2 histography has been used to measure the intratumor pO2 in patients for the past few years, and there is now evidence that these tumors contain hypoxic cells. One of the major questions that remains to be answered is the relevance of such data to radiosensitivity. The present study looks for a correlation between intratumor pO2, the percentage of hypoxic cells in the tumor and the radiosensitization induced by carbogen and/or the oxygen carrier, perflubron emulsion. Two human tumor xenografts (HRT18, Na11+) and one rodent tumor (EMT6) were used. The radiosensitivity (clonogenic assay) and the oxygen tension (computerized pO2 histography) were measured. All experiments were performed under similar conditions. Carbogen increased tumor radiosensitivity; sensitization was greatest when 4 ml/kg perflubron emulsion was used in conjunction with carbogen. The pO2 distribution was shifted to higher pO2 values in the tumors whatever the treatment; the shift was greater for perflubron emulsion plus carbogen. The low pO2 values (< 0.4 kPa) were lost for the HRT18 cells. A correlation (EMT6, HRT18) or a link (Na11+) between the radiosensitization and the oxygen tension measurements was found for values below 1.07 or 1.33 kPa. A trend between the percentage of hypoxic cells and pO2 measurements was found taking into account pO2 measurements comprised between 0.27 and 0.67 kPa. PMID:8016297

  16. The PI3K/Akt/mTOR pathway mediates retinal progenitor cell survival under hypoxic and superoxide stress.

    PubMed

    Sanghera, Karan P; Mathalone, Nurit; Baigi, Ramteen; Panov, Elan; Wang, Dan; Zhao, Xu; Hsu, Howard; Wang, Hai; Tropepe, Vincent; Ward, Michael; Boyd, Shelley R

    2011-06-01

    Oxygen (O₂) tension has emerged as a major regulator of stem cell (SC) biology. Low O₂ concentrations that are toxic to mature cells can confer advantage to stem and early progenitors, while superoxide stress remains a constant threat in aerobic biology and may be partially avoided through sequestration of SCs in the relatively hypoxic stem or regenerative niche. Using primary retina-derived retinal progenitor cells (RPCs) and the R28 progenitor cell line in vitro, we show that RPCs are sensitive to hydrogen peroxide (H₂O₂) induced damage and resistant to moderate levels of low oxygen stress (1% O₂). Under hypoxic conditions, multipotent RPCs upregulate Epo receptors, and Epo, along with insulin, protects against both superoxide- and severe hypoxia- (0.25% O₂) induced apoptosis through activation of the canonical PI3K/Akt/mTOR pathway. This survival advantage is sensitive to inhibitors of PI3K and mTOR. We further demonstrate phosphorylation of the p70S6 ribosomal kinase, a downstream mediator of PI3K/Akt/mTOR and translational activator. Overall, these data confirm that RPCs are sensitive to superoxide stress and resistant to hypoxia and that this resistance is mediated in part by Epo. They further suggest that manipulation of RPCs ex vivo prior to ocular delivery, or the in vivo delivery of exogenous survival factors at the time of cell implantation, could enhance the success of regenerative therapies aimed to restore sight. PMID:21463685

  17. 1,2-Benzisothiazole Derivatives Bearing 4-, 5-, or 6-Alkyl/arylcarboxamide Moieties Inhibit Carbonic Anhydrase Isoform IX (CAIX) and Cell Proliferation under Hypoxic Conditions.

    PubMed

    Coviello, Vito; Marchi, Beatrice; Sartini, Stefania; Quattrini, Luca; Marini, Anna Maria; Simorini, Francesca; Taliani, Sabrina; Salerno, Silvia; Orlandi, Paola; Fioravanti, Anna; Desidero, Teresa Di; Vullo, Daniela; Da Settimo, Federico; Supuran, Claudiu T; Bocci, Guido; La Motta, Concettina

    2016-07-14

    Three novel series of 1,2-benzisothiazole derivatives have been developed as inhibitors of carbonic anhydrase isoform IX. Compounds 5c and 5j, tested in vitro on the human colon cell line HT-29, blocked the growth of cells cultured under chemically induced hypoxic conditions, displaying a specific activity against cancer cells characterized by CAIX up-regulation. Moreover, a synergistic activity of 5c with SN-38 (the active metabolite of irinotecan) and 5-fluorouracil on cell proliferation under hypoxic conditions was demonstrated. PMID:27305384

  18. Role of the ERK1/2 Signaling Pathway in Osteogenesis of Rat Tendon-Derived Stem Cells in Normoxic and Hypoxic Cultures

    PubMed Central

    Li, Pei; Xu, Yuan; Gan, Yibo; Song, Lei; Zhang, Chengmin; Wang, Liyuan; Zhou, Qiang

    2016-01-01

    Background: Ectopic ossification and increased vascularization are two common phenomena in the chronic tendinopathic tendon. The increased vascularization usually leads to an elevated local oxygen tension which is one of micro-environments that can influence differentiate status of stem cells. Objective: This study aimed to investigate the osteogenesis capacity of rat tendon-derived stem cells TDSCs (rTDSCs) in normoxic and hypoxic cultures, and to study the role of ERK1/2 signaling pathway in this process. Methods: rTDSCs were subjected to osteogenesis inductive culture in hypoxic (3% O2) and normoxic (20% O2) conditions. The inhibitor U0126 was added along with culture medium to determine the role of ERK1/2 signaling pathway. Cell viability, cell proliferation, alizarin red staining, alkaline phosphatase (AKP) activity, gene expression (ALP, osteocalcin, collagen I and RUNX2) and protein expression (p-ERK1/2 and RUNX2) of osteogenic-cultured rTSDCs were analyzed in this study. Results: Hypoxic and normoxic culture had no effects on cell viability of rTDSCs, whereas the proliferation potential of rTDSCs was significantly increased in hypoxic culture. The osteogenesis capacity of rTDSCs in normoxic culture was significantly promoted compared with hypoxic culture, which was reflected by an increased alizarin red staining intensity, an elevated ALP activity, and the up-regulated gene (ALP, osteocalcin, collagen I and RUNX2) or protein (RUNX2) expression of osteogenic makers. However, the osteogenesis capacity of rTDSCs in both hypoxic and normoxic cultures was attenuated by the inhibitor U0126. Conclusion: Normoxic culture promotes osteogenic differentiation of rTDSCs compared with the hypoxic culture, and the ERK1/2 signaling pathway is involved in this process. PMID:27499695

  19. 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.

  20. Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism.

    PubMed

    Park, Chi-Yeon; Choi, Seung-Cheol; Kim, Jong-Ho; Choi, Ji-Hyun; Joo, Hyung Joon; Hong, Soon Jun; Lim, Do-Sun

    2016-01-01

    Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31- human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31- CSCs(hTERT)), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31- CSCs(hTERT) sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31- CSCs(hTERT) were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31- CSCs(hTERT) conditioned medium (CM). Sca-1+/CD31- CSCs(hTERT) CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31- CSCs(hTERT) CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31- CSCs(hTERT) exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31- CSCs(hTERT) CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field. PMID:27231894

  1. Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

    PubMed Central

    Park, Chi-Yeon; Choi, Seung-Cheol; Kim, Jong-Ho; Choi, Ji-Hyun; Joo, Hyung Joon; Hong, Soon Jun; Lim, Do-Sun

    2016-01-01

    Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31− human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31− CSCshTERT), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31− CSCshTERT sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31− CSCshTERT were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31− CSCshTERT conditioned medium (CM). Sca-1+/CD31− CSCshTERT CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31− CSCshTERT CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31− CSCshTERT exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31− CSCshTERT CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field. PMID:27231894

  2. Vitamin D Prevents Endothelial Progenitor Cell Dysfunction Induced by Sera from Women with Preeclampsia or Conditioned Media from Hypoxic Placenta

    PubMed Central

    Myerski, Ashley C.; von Kaisenberg, Constantin S.; Grundmann, Magdalena; Hubel, Carl A.; von Versen-Höynck, Frauke

    2014-01-01

    Context Placenta-derived circulating factors contribute to the maternal endothelial dysfunction underlying preeclampsia. Endothelial colony forming cells (ECFC), a sub-population of endothelial progenitor cells (EPCs), are thought to be involved in vasculogenesis and endothelial repair. Low vitamin D concentrations are associated with an increased risk for preeclampsia. Objective We hypothesized that the function of human fetal ECFCs in culture would be suppressed by exposure to preeclampsia-related factors–preeclampsia serum or hypoxic placental conditioned medium– in a fashion reversed by vitamin D. Design, Setting, Patients ECFCs were isolated from cord blood of uncomplicated pregnancies and expanded in culture. Uncomplicated pregnancy villous placenta in explant culture were exposed to either 2% (hypoxic), 8% (normoxic) or 21% (hyperoxic) O2 for 48 h, after which the conditioned media (CM) was collected. Outcome Measures ECFC tubule formation (Matrigel assay) and migration were examined in the presence of either maternal serum from preeclampsia cases or uncomplicated pregnancy controls, or pooled CM, in the presence or absence of 1,25(OH)2 vitamin D3. Results 1,25(OH)2 vitamin D3 reversed the adverse effects of preeclampsia serum or CM from hypoxic placenta on ECFCs capillary-tube formation and migration. Silencing of VDR expression by VDR siRNA, VDR blockade, or VEGF pathway blockade reduced ECFC functional abilities. Effects of VDR or VEGF blockade were partially prevented by vitamin D. Conclusion Vitamin D promotes the capillary-like tubule formation and migration of ECFCs in culture, minimizing the negative effects of exposure to preeclampsia-related factors. Further evaluation of the role of vitamin D in ECFC regulation and preeclampsia is warranted. PMID:24887145

  3. Hypoxic Preconditioning Differentially Affects GABAergic and Glutamatergic Neuronal Cells in the Injured Cerebellum of the Neonatal Rat

    PubMed Central

    Patterson, Sean I.; Muñoz, Estela M.; Seltzer, Alicia M.

    2014-01-01

    In this study we examined cerebellar alterations in a neonatal rat model of hypoxic-ischemic brain injury with or without hypoxic preconditioning (Pc). Between postnatal days 7 and 15, the cerebellum is still undergoing intense cellular proliferation, differentiation and migration, dendritogenesis and synaptogenesis. The expression of glutamate decarboxylase 1 (GAD67) and the differentiation factor NeuroD1 were examined as markers of Purkinje and granule cells, respectively. We applied quantitative immunohistochemistry to sagittal cerebellar slices, and Western blot analysis of whole cerebella obtained from control (C) rats and rats submitted to Pc, hypoxia-ischemia (L) and a combination of both treatments (PcL). We found that either hypoxia-ischemia or Pc perturbed the granule cells in the posterior lobes, affecting their migration and final placement in the internal granular layer. These effects were partially attenuated when the Pc was delivered prior to the hypoxia-ischemia. Interestingly, whole nuclear NeuroD1 levels in Pc animals were comparable to those in the C rats. However, a subset of Purkinje cells that were severely affected by the hypoxic-ischemic insult—showing signs of neuronal distress at the levels of the nucleus, cytoplasm and dendritic arborization—were not protected by Pc. A monoclonal antibody specific for GAD67 revealed a three-band pattern in cytoplasmic extracts from whole P15 cerebella. A ∼110 kDa band, interpreted as a potential homodimer of a truncated form of GAD67, was reduced in Pc and L groups while its levels were close to the control animals in PcL rats. Additionally we demonstrated differential glial responses depending on the treatment, including astrogliosis in hypoxiated cerebella and a selective effect of hypoxia-ischemia on the vimentin-immunolabeled intermediate filaments of the Bergmann glia. Thus, while both glutamatergic and GABAergic cerebellar neurons are compromised by the hypoxic-ischemic insult, the former are

  4. Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells.

    PubMed

    Elanchezhian, R; Palsamy, P; Madson, C J; Mulhern, M L; Lynch, D W; Troia, A M; Usukura, J; Shinohara, T

    2012-01-01

    Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions. PMID:22513875

  5. Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

    PubMed Central

    Elanchezhian, R; Palsamy, P; Madson, C J; Mulhern, M L; Lynch, D W; Troia, A M; Usukura, J; Shinohara, T

    2012-01-01

    Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions. PMID:22513875

  6. NQDI-1, an inhibitor of ASK1 attenuates acute perinatal hypoxic-ischemic cerebral injury by modulating cell death

    PubMed Central

    HAO, HU; LI, SITAO; TANG, HUI; LIU, BINGQING; CAI, YAO; SHI, CONGCONG; XIAO, XIN

    2016-01-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed protein kinase, which regulates cell fate in numerous injury conditions. Therefore, ASK1 may be a promising novel therapeutic target for injury. However, the expression and distribution of ASK1 in the perinatal brain following hypoxia-ischemia (HI) remains to be elucidated. In the present study, western blotting and immunofluorescence were used to determine the expression and distribution of ASK1 and any associated downstream targets in the perinatal rat brain following HI. NQDI-1, a specific inhibitor of ASK1 was intracerebroventricularly injected following neonatal rats brain insult for neuroprotection. The results revealed an increased expression of ASK1 and this expression was localized to the neurons and astrocytes, compared with the sham controls. Additionally, it was demonstrated that the ASK1/c-Jun N-terminal kinases (JNK) pathway was involved in the brain damage following HI in neonatal rats. Notably, NQDI-1 significantly inhibited the in vivo expression levels of ASK1, phosphorylated (p-)JNK, p-c-Jun, p53 and caspase 3. Reduced acute hypoxic-ischemic cerebral injury and cell apoptosis was observed following the injection of NQDI-1. Collectively, NQDI-1 attenuated acute perinatal hypoxic-ischemic cerebral injury by inhibiting the expression of ASK1 and cell apoptosis. This may be a promising novel neuroprotective inhibitor for perinatal cerebra injury. PMID:27081917

  7. [Lycium barbarum polysaccharides enhances SIRT1 expression and decreases MMP-9 and HIF-1α expressions in hypoxic pulmonary vascular smooth muscle cells].

    PubMed

    Zhu, Yanni; Sun, Yuning; Guan, Wenxia; Yan, Yan; Zhang, Wei; Bai, Lugen; Kong, Haitao; Li, Fang

    2016-07-01

    Objective To investigate the expressions of silent mating type information regulation 2 homolog-1 (SIRT1), matrix metalloproteinase-9 (MMP-9) and hypoxia inducible factor 1α (HIF-1α) in hypoxic pulmonary vascular smooth muscle cells (PVSMCs) treated with Lycium barbarum polysaccharides (LBP). Methods PVSMCs were divided into 10 groups: normal oxygen (200 mL/L oxygen) cells, 2 μmol/mL LBP-treated normal oxygen cells, DMSO-treated normal oxygen cells, DMSO-treated hypoxic (100 mL/L oxygen) cells, (0.5, 1, 2) μmol/mL LBP-treated hypoxic cells; SIRT1 agonist (resveratrol or SRT-1720)-treated hypoxic cells, SIRT1 inhibitor EX-527-treated hypoxic cells. After 6, 12, 24 hours, the mRNA and protein expressions of SIRT1, MMP-9 and HIF-1α were measured by real-time quantitative PCR and Western blotting, respectively. In LBP-treated groups, the expressions of SIRT1, MMP-9 and HIF-1α mRNA and protein were detected 12 hours after LBP treatment. Results Under the condition of hypoxia, the expression levels of SIRT1 mRNA and protein in PVSMCs decreased, while MMP-9 and HIF-1α mRNA increased. Under hypoxia, SIRT1 expression was raised and MMP-9, HIF-1α were reduced by LBP treatment in a dose-dependent manner. Morever, resveratrol could inhibit the expression of MMP-9. Conclusion LBP can enhance the expression of SIRT1 and decrease the expression of MMP-9 and HIF-1α in hypoxic PVSMCs. PMID:27363270

  8. c-kit-Positive Cardiac Stem Cells Nested in Hypoxic Niches are Activated by Stem Cell Factor Reversing the Aging Myopathy

    PubMed Central

    Sanada, Fumihiro; Kim, Junghyun; Czarna, Anna; Chan, Noel Yan-Ki; Signore, Sergio; Ogórek, Barbara; Isobe, Kazuya; Wybieralska, Ewa; Borghetti, Giulia; Pesapane, Ada; Sorrentino, Andrea; Mangano, Emily; Cappetta, Donato; Mangiaracina, Chiara; Ricciardi, Mario; Cimini, Maria; Ifedigbo, Emeka; Perrella, Mark A.; Goichberg, Polina; Choi, Augustine; Kajstura, Jan; Hosoda, Toru; Rota, Marcello; Anversa, Piero; Leri, Annarosa

    2014-01-01

    Rationale Hypoxia favors stem cell quiescence, while normoxia is required for their activation; but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. Objective A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. Methods and Results Here we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot reenter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. Conclusions Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggests that a pool of functionally-competent CSCs persists in the senescent heart and this stem cell compartment can promote myocyte regeneration effectively, correcting partly the aging myopathy. PMID:24170267

  9. Tanshinone IIA Pretreatment Renders Free Flaps against Hypoxic Injury through Activating Wnt Signaling and Upregulating Stem Cell-Related Biomarkers

    PubMed Central

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

    2014-01-01

    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. PMID:25302618

  10. Modulation of p75 neurotrophin receptor under hypoxic conditions induces migration and invasion of C6 glioma cells.

    PubMed

    Wang, Ting-Chung; Luo, Sheng-Jie; Lin, Chun-Liang; Chang, Pey-Jium; Chen, Miao-Fen

    2015-01-01

    p75 neurotrophin receptor (p75NTR) has been reported to play important roles in various cancer types. However, the exact mechanism of tumorigenesis involving p75NTR is unknown. In this study, we investigated the relationship between the expression of p75NTR in malignant glioma and the impact on tumor cell migration and invasion. p75NTR and hypoxia-inducible factor-1α (HIF-1α) expression was down-regulated by short-hairpin RNA and up-regulated with expression vectors. By immunohistochemical staining and Western blot analysis, we found that p75NTR was expressed in both human and rat malignant gliomas. Knockdown of p75NTR increased the expression of vimentin, vascular endothelial growth factor, Matrix metalloproteinase 9, and TWIST, and enhanced the invasion and migration abilities assessed by transwell assay in the C6 tumor cells. Inverse expressions of p75NTR and HIF-1α were detected in glioma cell lines under hypoxic conditions, while increased HIF-1α significantly downregulated the expression of p75NTR, suggesting a HIF-1α-p75NTR-EMT pathway that may regulate glioma cells invasion and migration. Downregulation of p75NTR increased phosphorylation of Src, focal adhesion kinase (FAK) and paxillin. Knockdown of p75NTR also dysregulated β-catenin-mediated cell junctions, and up-regulated the expressions of fibronectin and L1CAM in the cell-cell junctions, thus suggesting that p75NTR knockdown contributed to a more aggressive migration phenotype via FAK signaling pathway. Our studies suggested that modulation of p75NTR under hypoxic condition could enhance C6 cells migration and invasion by induction of EMT, and activation of the FAK pathway. The HIF-1α-p75NTR-EMT axis may play a central role in glioma tumorigenesis. PMID:25527128

  11. 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. PMID:26358160

  12. 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).

  13. Developing a New Two-Step Protocol to Generate Functional Hepatocytes from Wharton's Jelly-Derived Mesenchymal Stem Cells under Hypoxic Condition

    PubMed Central

    Prasajak, Patcharee

    2013-01-01

    The shortage of donor livers and hepatocytes is a major limitation of liver transplantation. Thus, generation of hepatocyte-like cells may provide alternative choice for therapeutic applications. In this study, we developed a new method to establish hepatocytes from Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) cell lines named WJMSCs-SUT1 and WJMSCs-SUT2 under hypoxic condition. This new method could rapidly drive both WJ-MSCs cell lines into hepatic lineage within 18 days. The achievement of hepatogenic differentiation was confirmed by the characterization of both phenotypes and functions. More than 80% MSCs-derived hepatocyte-like cells (MSCDHCs) achieved functional hepatocytes including hepatic marker expressions both at gene and protein levels, glycogen storage, low-density lipoprotein uptake, urea production, and albumin secretion. This study highlights the establishment of new hepatogenic induction protocol under hypoxic condition in order to mimic hypoxic microenvironment in typical cell physiology. In conclusion, we present a simple, high-efficiency, and time saving protocol for the generation of functional hepatocyte-like cells from WJ-MSCs in hypoxic condition. The achievement of this method may overcome the limitation of donor hepatocytes and provides a new avenue for therapeutic value in cell-based therapy for life-threatening liver diseases, regenerative medicine, toxicity testing for pharmacological drug screening, and other medical related applications. PMID:23818908

  14. Effect of deficiencies in DNA repair on the toxicity of mitomycin C and porfiromycin to CHO cells under aerobic and hypoxic conditions.

    PubMed

    Hughes, C S; Irvin, C G; Rockwell, S

    1991-02-01

    A wild type Chinese hamster cell line (AA8) and three repair-deficient sublines of AA8 (EM9, UV4, and UV5) were used to study the nature of the cytotoxic lesions produced by the bioreductive alkylating agents mitomycin C and porfiromycin under aerobic and hypoxic conditions. The sensitivities of the repair-deficient sublines to the drugs varied markedly: EM9 was similar to AA8, whereas UV4 was exquisitely sensitive and UV5 was of intermediate sensitivity. Moreover, both the relative toxicities of the two drugs and the relative toxicities of each drug under aerobic and hypoxic conditions varied for the different cell lines. These data suggest that there are differences in the spectra of toxic lesions produced by mitomycin and porfiromycin and that there are differences in the lesions produced by these drugs under aerobic and hypoxic conditions. PMID:1899798

  15. Inhibition of protein synthesis by imexon reduces HIF-1alpha expression in normoxic and hypoxic pancreatic cancer cells.

    PubMed

    Samulitis, Betty K; Landowski, Terry H; Dorr, Robert T

    2009-02-01

    Hypoxia-inducing factor-1 alpha (HIF-1alpha), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O(2)) and hypoxic (1% O(2)) conditions. Western blot analyses of imexon-treated cells demonstrated that imexon reduces HIF-1alpha protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1alpha levels. Imexon did not reduce transcription of new HIF-1alpha mRNA, but did reduce the synthesis of new proteins, including HIF-1alpha, measured by (35)S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1alpha protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1alpha translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2alpha (eIF-2alpha) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug's cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1alpha, and this effect overcomes an increase in the stability of preformed HIF-1alpha due to proteasome inhibition by imexon. Because net HIF-1alpha levels are reduced by imexon, combination studies with other drugs affected by HIF-1alpha survival signaling are warranted. PMID:18607542

  16. Inhibition of protein synthesis by imexon reduces HIF-1α expression in normoxic and hypoxic pancreatic cancer cells

    PubMed Central

    Samulitis, Betty K.; Landowski, Terry H.; Dorr, Robert T.

    2008-01-01

    Summary Hypoxia-inducing factor-1 alpha (HIF-1α), is a major survival factor for tumor cells growing in a low oxygen environment. The anti-cancer agent imexon binds thiols and causes accumulation of reactive oxygen species (ROS) in pancreatic cancer cells. Unlike many cytotoxic agents, imexon is equi-cytotoxic in human MiaPaCa-2 and Panc-1 cells grown in normoxic (21% O2) and hypoxic (1% O2) conditions. Western blot analyses of imexon treated cells demonstrated that imexon reduces HIF-1α protein levels in both normoxic and hypoxic conditions in a time- and concentration-dependant fashion. Gemcitabine did not similarly affect HIF-1α levels. Imexon did not reduce transcription of new HIF-1α mRNA, but did reduce the synthesis of new proteins, including HIF-1α, measured by 35S methionine/cysteine (Met/Cys) incorporation. Concurrently, the half-life of existing HIF-1α protein was increased by imexon, in association with a marked inhibition of chymotryptic activity in the 20S proteasome. The inhibition of HIF-1α translation was not specific, rather it was part of a general decrease in protein translation caused by imexon. This inhibitory effect on translation did not involve phosphorylation of eukaryotic initiation factor-2α (eIF-2α) and was not closely correlated to cell growth inhibition by imexon, suggesting that mechanisms other than protein synthesis inhibition contribute to the drug’s cytotoxic effects. In summary, imexon blocks the translation of new proteins, including HIF-1α, and this effect overcomes an increase in the stability of preformed HIF-1α due to proteasome inhibition by imexon. Because net HIF-1α levels are reduced by imexon, combination studies with other drugs affected by HIF-1α survival signaling are warranted. PMID:18607542

  17. 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

  18. 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

  19. Addition of a hypoxic cell selective cytotoxic agent (mitomycin C or porfiromycin) to Fluosol-DA/carbogen/radiation.

    PubMed

    Holden, S A; Herman, T S; Teicher, B A

    1990-05-01

    In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O2/5% CO2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and

  20. PERK/eIF2α signaling protects therapy resistant hypoxic cells through induction of glutathione synthesis and protection against ROS.

    PubMed

    Rouschop, Kasper M; Dubois, Ludwig J; Keulers, Tom G; van den Beucken, Twan; Lambin, Philippe; Bussink, Johan; van der Kogel, Albert J; Koritzinsky, Marianne; Wouters, Bradly G

    2013-03-19

    Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers. PMID:23471998

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

    PubMed

    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. PMID:26595121

  3. 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

  4. 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

    2016-01-01

    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

  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. 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

  7. The current status of drug development of hypoxic cell radiosensitizers and their potential role in gynecologic oncology

    SciTech Connect

    Coleman, C.N.; Ballon, S.C.; Howes, A.E.; Martinez, A.; Halsey, J.; Hirst, V.K.

    1984-05-01

    Both laboratory and clinical data suggest that hypoxia contributes to the failure of radiotherapy to achieve local control of bulky gynecologic tumors. As part of a Phase I trial of hypoxic cell radiosensitizers, 19 women at Stanford University with advanced (n . 6) or recurrent (n . 13) pelvic neoplasms were treated with radiotherapy plus desmethylmisonidazole. Complete or partial response occurred in 42% of patients with some patients achieving local control for over 1 year. It is unknown if the sensitizer added to the results of radiotherapy alone. A Phase I trial of a theoretically superior sensitizer, SR-2508, is soon to begin. It is anticipated that the dose-limiting neurotoxicity seen with misonidazole and desmethylmisonidazole will either be eliminated or will occur at a much higher total dose of drug. Many patients with gynecologic tumors could potentially benefit from participation in the new drug trials.

  8. 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. GLIA 2016;64:896-910. PMID:26992135

  9. Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation? in vivo and in vitro studies

    PubMed Central

    Edmunds, Nicholas J; Moncada, Salvador; Marshall, Janice M

    2003-01-01

    Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O2 sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O2 for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O2 tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with l-NAME, but restored when baseline FVC is restored by infusion of NO donor. This ‘restored’ hypoxic response, like the control hypoxic response, is inhibited by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by l-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after l-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with l-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; l-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent Km of endothelial cytochrome oxidase for O2, allowing the endothelium to act as an O2 sensor, releasing adenosine in response to moderate falls in O2. PMID:12527738

  10. Serum- and Stromal Cell-Free Hypoxic Generation of Embryonic Stem Cell-Derived Hematopoietic Cells In Vitro, Capable of Multilineage Repopulation of Immunocompetent Mice

    PubMed Central

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

    2012-01-01

    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. PMID:23197864

  11. miR-210 and hypoxic microvesicles: Two critical components of hypoxia involved in the regulation of killer cells function.

    PubMed

    Noman, Muhammad Zaeem; Janji, Bassam; Berchem, Guy; Chouaib, Salem

    2016-09-28

    It has become clear that tumor stroma components are engaged in an active and complex molecular cross-talk that has serious implications for immunological recognition of tumor cells in shaping the microenvironment. Hypoxia which is a major component of tumor microenvironment influences the characteristics of neoplasia by favoring heterogeneity, invasiveness, metastatic potency and tumor progression. In this regard, an important mode of communication between carcinoma cells and immune cells may involve tumor-derived microvesicles, which are able to carry lipids, proteins, mRNAs and miRNAs. This review covers new evidence indicating that the efficacy of the cell-mediated cytotoxicity (CTLs and NK) may be dependent on hypoxia induced miRNA and microvesicles in the tumor microenvironment by inhibiting the efficacy of natural host anti-tumor immune response and improving the ability of tumors to avoid immunosurveillance. This emphasizes that hypoxic tumors actively develop additional mechanisms to suppress the sensing of the immunologic danger signals in order to survive and propagate without inciting anti-tumor immunity. PMID:26523672

  12. Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury

    PubMed Central

    Hsueh, Yuan-Yu; Chang, Ya-Ju; Huang, Chia-Wei; Handayani, Fitri; Chiang, Yi-Lun; Fan, Shih-Chen; Ho, Chien-Jung; Kuo, Yu-Min; Yang, Shang-Hsun; Chen, Yuh-Ling; Lin, Sheng-Che; Huang, Chao-Ching; Wu, Chia-Ching

    2015-01-01

    Perinatal cerebral hypoxic-ischemic (HI) injury damages the architecture of neurovascular units (NVUs) and results in neurological disorders. Here, we differentiated adipose-derived stem cells (ASCs) toward the progenitor of endothelial progenitor cells (EPCs) and neural precursor cells (NPCs) via microenvironmental induction and investigated the protective effect by transplanting ASCs, EPCs, NPCs, or a combination of EPCs and NPCs (E+N) into neonatal HI injured rat pups. The E+N combination produced significant reduction in brain damage and cell apoptosis and the most comprehensive restoration in NVUs regarding neuron number, normal astrocytes, and vessel density. Improvements in cognitive and motor functions were also achieved in injured rats with E+N therapy. Synergistic interactions to facilitate transmigration under in vitro hypoxic microenvironment were discovered with involvement of the neuropilin-1 (NRP1) signal in EPCs and the C-X-C chemokine receptor 4 (CXCR4) and fibroblast growth factor receptor 1 (FGFR1) signals in NPCs. Therefore, ASCs exhibit great potential for cell sources in endothelial and neural lineages to prevent brain from HI damage. PMID:26447335

  13. Synergy of endothelial and neural progenitor cells from adipose-derived stem cells to preserve neurovascular structures in rat hypoxic-ischemic brain injury.

    PubMed

    Hsueh, Yuan-Yu; Chang, Ya-Ju; Huang, Chia-Wei; Handayani, Fitri; Chiang, Yi-Lun; Fan, Shih-Chen; Ho, Chien-Jung; Kuo, Yu-Min; Yang, Shang-Hsun; Chen, Yuh-Ling; Lin, Sheng-Che; Huang, Chao-Ching; Wu, Chia-Ching

    2015-01-01

    Perinatal cerebral hypoxic-ischemic (HI) injury damages the architecture of neurovascular units (NVUs) and results in neurological disorders. Here, we differentiated adipose-derived stem cells (ASCs) toward the progenitor of endothelial progenitor cells (EPCs) and neural precursor cells (NPCs) via microenvironmental induction and investigated the protective effect by transplanting ASCs, EPCs, NPCs, or a combination of EPCs and NPCs (E+N) into neonatal HI injured rat pups. The E+N combination produced significant reduction in brain damage and cell apoptosis and the most comprehensive restoration in NVUs regarding neuron number, normal astrocytes, and vessel density. Improvements in cognitive and motor functions were also achieved in injured rats with E+N therapy. Synergistic interactions to facilitate transmigration under in vitro hypoxic microenvironment were discovered with involvement of the neuropilin-1 (NRP1) signal in EPCs and the C-X-C chemokine receptor 4 (CXCR4) and fibroblast growth factor receptor 1 (FGFR1) signals in NPCs. Therefore, ASCs exhibit great potential for cell sources in endothelial and neural lineages to prevent brain from HI damage. PMID:26447335

  14. Sustained Radiosensitization of Hypoxic Glioma Cells after Oxygen Pretreatment in an Animal Model of Glioblastoma and In Vitro Models of Tumor Hypoxia

    PubMed Central

    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

  15. Inhibition of Hypoxia Inducible Factor Alpha and Astrocyte-Elevated Gene-1 Mediates Cryptotanshinone Exerted Antitumor Activity in Hypoxic PC-3 Cells

    PubMed Central

    Lee, Hyo-Jeong; Jung, Deok-Beom; Sohn, Eun Jung; Kim, Hanna Hyun; Park, Moon Nyeo; Lew, Jae-Hwan; Lee, Seok Geun; Kim, Bonglee; Kim, Sung-Hoon

    2012-01-01

    Although cryptotanshinone (CT) was known to exert antitumor activity in several cancers, its molecular mechanism under hypoxia still remains unclear. Here, the roles of AEG-1 and HIF-1α in CT-induced antitumor activity were investigated in hypoxic PC-3 cells. CT exerted cytotoxicity against prostate cancer cells and suppressed HIF-1α accumulation and AEG-1 expression in hypoxic PC-3 cells. Also, AEG-1 was overexpressed in prostate cancer cells. Interestingly, HIF-1α siRNA transfection enhanced the cleavages of caspase-9,3, and PAPR and decreased expression of Bcl-2 and AEG1 induced by CT in hypoxic PC-3 cells. Of note, DMOG enhanced the stability of AEG-1 and HIF-1α during hypoxia. Additionally, CT significantly reduced cellular level of VEGF in PC-3 cells and disturbed tube formation of HUVECs. Consistently, ChIP assay revealed that CT inhibited the binding of HIF-1α to VEGF promoter. Furthermore, CT at 10 mg/kg suppressed the growth of PC-3 cells in BALB/c athymic nude mice by 46.4% compared to untreated control. Consistently, immunohistochemistry revealed decreased expression of Ki-67, CD34, VEGF, carbonic anhydrase IX, and AEG-1 indices in CT-treated group compared to untreated control. Overall, our findings suggest that CT exerts antitumor activity via inhibition of HIF-1α, AEG1, and VEGF as a potent chemotherapeutic agent. PMID:23243443

  16. Bcl-2 Regulates HIF-1α Protein Stabilization in Hypoxic Melanoma Cells via the Molecular Chaperone HSP90

    PubMed Central

    Trisciuoglio, Daniela; Gabellini, Chiara; Desideri, Marianna; Ziparo, Elio; Zupi, Gabriella; Del Bufalo, Donatella

    2010-01-01

    Background Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is a critical mediator of the cellular response to hypoxia. Enhanced levels of HIF-1α, the oxygen-regulated subunit of HIF-1, is often associated with increased tumour angiogenesis, metastasis, therapeutic resistance and poor prognosis. It is in this context that we previously demonstrated that under hypoxia, bcl-2 protein promotes HIF-1/Vascular Endothelial Growth Factor (VEGF)-mediated tumour angiogenesis. Methodology/Principal Findings By using human melanoma cell lines and their stable or transient derivative bcl-2 overexpressing cells, the current study identified HIF-1α protein stabilization as a key regulator for the induction of HIF-1 by bcl-2 under hypoxia. We also demonstrated that bcl-2-induced accumulation of HIF-1α protein during hypoxia was not due to an increased gene transcription or protein synthesis. In fact, it was related to a modulation of HIF-1α protein expression at a post-translational level, indeed its degradation rate was faster in the control lines than in bcl-2 transfectants. The bcl-2-induced HIF-1α stabilization in response to low oxygen tension conditions was achieved through the impairment of ubiquitin-dependent HIF-1α degradation involving the molecular chaperone HSP90, but it was not dependent on the prolyl hydroxylation of HIF-1α protein. We also showed that bcl-2, HIF-1α and HSP90 proteins form a tri-complex that may contribute to enhancing the stability of the HIF-1α protein in bcl-2 overexpressing clones under hypoxic conditions. Finally, by using genetic and pharmacological approaches we proved that HSP90 is involved in bcl-2-dependent stabilization of HIF-1α protein during hypoxia, and in particular the isoform HSP90β is the main player in this phenomenon. Conclusions/Significance We identified the stabilization of HIF-1α protein as a mechanism through which bcl-2 induces the activation of HIF-1 in hypoxic tumour cells involving the

  17. 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. PMID:22486732

  18. Pharmacokinetic/pharmacodynamic modeling identifies SN30000 and SN29751 as tirapazamine analogs with improved tissue penetration and hypoxic cell killing in tumors

    PubMed Central

    Hicks, Kevin O.; Siim, Bronwyn G.; Jaiswal, Jagdish K.; Pruijn, Frederik B.; Fraser, Annie M.; Patel, Rita; Hogg, Alison; Liyanage, H.D. Sarath; Jo Dorie, Mary; Brown, J. Martin; Denny, William. A.; Hay, Michael P.; Wilson, William R.

    2012-01-01

    Purpose Tirapazamine (TPZ) has attractive features for targeting hypoxic cells in tumors but limited clinical activity, in part because of poor extravascular penetration. Here we identify improved TPZ analogs by using a spatially resolved pharmacokinetic/pharmacodynamic (SR-PKPD) model that considers tissue penetration explicitly during lead optimization. Experimental design The SR-PKPD model was used to guide progression of 281 TPZ analogs through a hierarchical screen. For compounds exceeding hypoxic selectivity thresholds in single cell cultures, SR-PKPD model parameters (kinetics of bioreductive metabolism, clonogenic cell killing potency, diffusion coefficients in multicellular layers, plasma pharmacokinetics at well tolerated doses in mice) were measured to prioritize testing in xenograft models in combination with radiation. Results SR-PKPD-guided lead optimization identified SN29751 and SN30000 as the most promising hypoxic cytotoxins from two different structural subseries. Both were reduced to the corresponding 1-oxide selectively under hypoxia by HT29 cells, with an oxygen dependence quantitatively similar to that of TPZ. SN30000, in particular, showed higher hypoxic potency and selectivity than TPZ in tumor cell cultures and faster diffusion through HT29 and SiHa multicellular layers. Both compounds also provided superior plasma PK in mice and rats at equivalent toxicity. In agreement with SR-PKPD predictions, both were more active than TPZ with single dose or fractionated radiation against multiple human tumor xenografts. Conclusions SN30000 and SN29751 are improved TPZ analogs with potential for targeting tumor hypoxia in humans, and illustrate the utility of novel SR-PKPD modeling approaches for lead optimization during anticancer drug development. PMID:20732963

  19. 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. PMID:21576354

  20. Differential toxicity of mitomycin C and porfiromycin to aerobic and hypoxic Chinese hamster ovary cells overexpressing human NADPH:cytochrome c (P-450) reductase.

    PubMed

    Belcourt, M F; Hodnick, W F; Rockwell, S; Sartorelli, A C

    1996-01-01

    Purified NADPH:cytochrome c (P-450) reductase (FpT; NADPH-ferrihemoprotein oxidoreductase, EC 1.6.2.4) can reductively activate mitomycin antibiotics through a one-electron reduction to species that alkylate DNA. To assess the involvement of FpT in the intracellular activation of the mitomycins, transfectants overexpressing a human FpT cDNA were established from a Chinese hamster ovary cell line deficient in dihydrofolate reductase (CHO-K1/dhfr-). The parental cell line was equisensitive to the cytotoxic action of mitomycin C under oxygenated and hypoxic conditions. In contrast, porfiromycin was considerably less cytotoxic to wild-type parental cells than was mitomycin C in air and markedly more cytotoxic under hypoxia. Two FpT-transfected clones were selected that expressed 19- and 27-fold more FpT activity than the parental line. Levels of other oxidoreductases implicated in the activation of the mitomycins were unchanged. Significant increases in sensitivity to mitomycin C and porfiromycin in the two FpT-transfected clones were seen under both oxygenated and hypoxic conditions, with the increases in toxicity being greater under hypoxia than in air. These findings demonstrate that FpT can bioreductively activate the mitomycins in living cells and implicate FpT in the differential aerobic/hypoxic toxicity of the mitomycins. PMID:8552660

  1. 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.

  2. Amperometric sensing of HIF1α expressed in cancer cells and the effect of hypoxic mimicking agents.

    PubMed

    Hussain, Khalil K; Gurudatt, N G; Mir, Tanveer Ahmad; Shim, Yoon-Bo

    2016-09-15

    Hypoxia inducible factor 1 alpha (HIF1α) overexpression was detected in cancerous cells using an amperometric immunosensor with a nano-bioconjugate. The sensor probe was fabricated by covalently immobilizing the antibody (anti-HIF1α) onto a composite layer of functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA) formed on a layer of gold nanoparticles (AuNPs). A nano-bioconjugate with hydrazine and a secondary antibody of HIF1α (sec-Ab2) attached on AuNPs reveals the immunoreaction at the sensor probe through the catalytic reduction of H2O2 by hydrazine at -0.35V vs. Ag/AgCl. Morphology and performance of the sensor probe were characterized using FE-SEM, XPS, EIS, and cyclic voltammetry. The calibration plot at optimized experimental conditions shows a dynamic range of 25-350pM/mL with a detection limit of 5.35±0.02pM/mL. The reliability of the sensor was evaluated using non-cancerous Vero and cancerous MCF-7 cell lysates, where the HIF1α expression was compared with three cancerous cell lines MCF-7, PC-3, and A549. Furthermore, the sensor probe confirms the stable expression of HIF1α in the A549 lung cancer cells when exposing them to hypoxic mimicking agents Co, Ni, and Mn ions. Of these, Co ions show the highest stabilization effect on HIF1α followed by Ni and Mn ions, respectively. PMID:27132006

  3. Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells

    PubMed Central

    Neelam, Sudha; Brooks, Morgan M.

    2015-01-01

    Purpose The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. Methods HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3β (GSK-3β) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of β-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear β-catenin and its downstream effect on EMT marker proteins and VEGF expression. Results SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3β activity prompting a significant increase in the translocation of cytoplasmic β-catenin to the nucleus. The enhancement of nuclear β-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear β-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed

  4. Upregulation of miRNA3195 and miRNA374b Mediates the Anti-Angiogenic Properties of Melatonin in Hypoxic PC-3 Prostate Cancer Cells

    PubMed Central

    Sohn, Eun Jung; Won, Gunho; Lee, Jihyun; Lee, Sangyoon; Kim, Sung-hoon

    2015-01-01

    Recently microRNAs (miRNAs) have been attractive targets with their key roles in biological regulation through post-transcription to control mRNA stability and protein translation. Though melatonin was known as an anti-angiogenic agent, the underlying mechanism of melatonin in PC-3 prostate cancer cells under hypoxia still remains unclear. Thus, in the current study, we elucidated the important roles of miRNAs in melatonin-induced anti-angiogenic activity in hypoxic PC-3 cells. miRNA array revealed that 33 miRNAs (>2 folds) including miRNA3195 and miRNA 374b were significantly upregulated and 16 miRNAs were downregulated in melatonin-treated PC-3 cells under hypoxia compared to untreated control. Melatonin significantly attenuated the expression of hypoxia-inducible factor (HIF)-1 alpha, HIF-2 alpha and vascular endothelial growth factor (VEGF) at mRNA level in hypoxic PC-3 cells. Consistently, melatonin enhanced the expression of miRNA3195 and miRNA 374b in hypoxic PC-3 cells by qRT-PCR analysis. Of note, overexpression of miRNA3195 and miRNA374b mimics attenuated the mRNA levels of angiogenesis related genes such as HIF-1alpha, HIF-2 alpha and VEGF in PC-3 cells under hypoxia. Furthermore, overexpression of miRNA3195 and miRNA374b suppressed typical angiogenic protein VEGF at the protein level and VEGF production induced by melatonin, while antisense oligonucleotides against miRNA 3195 or miRNA 374b did not affect VEGF production induced by melatonin. Also, overexpression of miR3195 or miR374b reduced HIF-1 alpha immunofluorescent expression in hypoxic PC-3 compared to untreated control. Overall, our findings suggest that upregulation of miRNA3195 and miRNA374b mediates anti-angiogenic property induced by melatonin in hypoxic PC-3 cells. PMID:25553085

  5. Hypergravity and hypobaric hypoxic conditions promote endothelial cell and platelet activation.

    PubMed

    Rubenstein, David A; Yin, Wei

    2014-09-01

    Cardiovascular disease risk is heightened during exposure to altered gravity and/or altered barometric conditions. Previous work has suggested that this heightened cardiovascular risk is due to enhancements of endothelial cell inflammatory and/or thrombogenic responses. In recent work, the role of platelets on instigating or inhibiting endothelial cell responses associated with cardiovascular disease has been found to be dependent on both biochemical and biophysical factors. In this work, we aimed to determine how two biophysical forces, gravity and atmospheric pressure, alter endothelial cell and platelet functions and their interactions to instigate or inhibit cardiovascular disease responses. To address this aim, endothelial cells and platelets were subjected to a force 8 times greater than the normal gravitational force, for up to 30 minutes. In separate experiments, endothelial cells and platelets were subjected to 50% of normal atmospheric pressure. Endothelial cell and platelet responses, associated with cardiovascular diseases, were measured as a time course during exposure. In general, the exposure of endothelial cells to either hypergravity or hypobaric conditions enhanced cardiovascular disease responses. However, the presence of platelets generally inhibited endothelial cell responses. Platelet activation was, however, somewhat enhanced under both hypergravity and hypobaric conditions. Our data suggest that altered biophysical forces can modulate endothelial cell and platelet responses that are salient for cardiovascular disease progression. However, the interaction of these two cells tends to restrain the progression of the pro-cardiovascular disease responses. PMID:25211651

  6. Epithelial-mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions.

    PubMed

    Sueki, Akane; Matsuda, Kazuyuki; Iwashita, Chinami; Taira, Chiaki; Ishimine, Nau; Shigeto, Shohei; Kawasaki, Kenji; Sugano, Mitsutoshi; Yamamoto, Hiroshi; Honda, Takayuki

    2014-10-31

    Epithelial-mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O2) and hypoxic (2% O2) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis. PMID:25445593

  7. Study of the mechanism underlying the inhibitory effects of transglutaminase II on apoptosis in the osteosarcoma MG-63 cell line under hypoxic conditions

    PubMed Central

    WANG, GUOBIN; FU, LIMEI; CHEN, FANGMIN

    2015-01-01

    The aim of the present study was to investigate the association between the apoptosis phenomenon in the MG-63 osteosarcoma cell line, and transglutaminase II (TG2) expression. The relationship between the anti-apoptotic mechanism of TG2 and the expression of cytochrome c as well as caspase-3 under hypoxic conditions was also verified. A hypoxic culture of MG-63 cells was prepared. The hypoxia and TG2 siRNA hypoxia groups were established, and the cultures were incubated for 12 h under hypoxic conditions. TG2 activity, TG2 protein expression and its mRNA level were investigated. Cytochrome c and caspase-3 protein levels in the TG2 nucleus and cytoplasm were measured. The apoptotic rate was also monitored. The results showed that TG2 activity, TG2 protein expression and its mRNA level in the hypoxia group were significantly higher than those of the siRNA hypoxia group. The results showed statistically insignificant differences (P<0.05). By contrast, a comparison of the two groups in the cytoplasm yielded no statistically significant differences (P>0.05). Cytochrome c and caspase-3 protein levels in the hypoxia group were significantly higher than those of the TG2 siRNA hypoxia group. The results showed statistically significant differences (P<0.05). By contrast, the protein levels in the cytoplasm were significantly lower than those of the TG2 siRNA hypoxia group, with differences being statistically significant (P<0.05). The differences in apoptotic rates between the hypoxia and TG2 siRNA hypoxia groups were also statistically significant (P<0.05). Under hypoxic conditions, a high TG2 expression inhibited the apoptosis of the MG-63 osteosarcoma cell line. This effect was probably associated with its suppressive activity on the transportation of cytochrome c and caspase-3 from nucleus to cytoplasm. PMID:26788145

  8. Cell Death Conversion under Hypoxic Condition in Tumor Development and Therapy

    PubMed Central

    Qiu, Yu; Li, Peng; Ji, Chunyan

    2015-01-01

    Hypoxia, which is common during tumor progression, plays important roles in tumor biology. Failure in cell death in response to hypoxia contributes to progression and metastasis of tumors. On the one hand, the metabolic and oxidative stress following hypoxia could lead to cell death by triggering signal cascades, like LKB1/AMPK, PI3K/AKT/mTOR, and altering the levels of effective components, such as the Bcl-2 family, Atg and p62. On the other hand, hypoxia-induced autophagy can serve as a mechanism to turn over nutrients, so as to mitigate the adverse condition and then avoid cell death potentially. Due to the effective role of hypoxia, this review focuses on the crosstalk in cell death under hypoxia in tumor progression. Additionally, the illumination of cell death in hypoxia could shed light on the clinical applications of cell death targeted therapy. PMID:26512660

  9. Up-Regulation of ENO1 by HIF-1α in Retinal Pigment Epithelial Cells after Hypoxic Challenge Is Not Involved in the Regulation of VEGF Secretion

    PubMed Central

    Zheng, Feihui; Jang, Wai-Chi; Fung, Frederic K. C.; Lo, Amy C. Y.; Wong, Ian Y. H.

    2016-01-01

    Purpose Alpha-enolase (ENO1), a major glycolytic enzyme, is reported to be over-expressed in various cancer tissues. It has been demonstrated to be regulated by the Hypoxia-inducible factor 1-α (HIF-1α), a crucial transcriptional factor implicated in tumor progression and cancer angiogenesis. Choroidal neovascularization (CNV), which is a leading cause of severe vision loss caused by newly formed blood vessels in the choroid, is also engendered by hypoxic stress. In this report, we investigated the expression of ENO1 and the effects of its down-regulation upon cobalt (II) chloride-induced hypoxia in retinal pigment epithelial cells, identified as the primary source of ocular angiogenic factors. Methods HIF-1α-diminished retinal pigment epithelial cells were generated by small interfering RNA (siRNA) technology in ARPE-19 cells, a human retinal pigment epithelial cell line. Both normal and HIF-1α-diminished ARPE-19 cells were then subjected to hypoxic challenge using cobalt (II) chloride (CoCl2) or anaerobic chamber. The relation between ENO1 expression and vascular endothelial growth factor (VEGF) secretion by retinal pigment epithelial cells were examined. Protein levels of HIF-1α and ENO1 were analyzed using Western Blot, while VEGF secretion was essayed by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity after hypoxia was detected by Lactate Dehydrogenase (LDH) Assay. Results Upon 24 hr of CoCl2-induced hypoxia, the expression levels of ENO1 and VEGF were increased along with HIF-1α in ARPE-19 cells, both of which can in turn be down-regulated by HIF-1α siRNA application. However, knockdown of ENO1 alone or together with HIF-1α did not help suppress VEGF secretion in hypoxic ARPE-19 cells. Conclusion ENO1 was demonstrated to be up-regulated by HIF-1α in retinal pigment epithelial cells in response to hypoxia, without influencing VEGF secretion. PMID:26882120

  10. Hypoxic control of metastasis.

    PubMed

    Rankin, Erinn B; Giaccia, Amato J

    2016-04-01

    Metastatic disease is the leading cause of cancer-related deaths and involves critical interactions between tumor cells and the microenvironment. Hypoxia is a potent microenvironmental factor promoting metastatic progression. Clinically, hypoxia and the expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are associated with increased distant metastasis and poor survival in a variety of tumor types. Moreover, HIF signaling in malignant cells influences multiple steps within the metastatic cascade. Here we review research focused on elucidating the mechanisms by which the hypoxic tumor microenvironment promotes metastatic progression. These studies have identified potential biomarkers and therapeutic targets regulated by hypoxia that could be incorporated into strategies aimed at preventing and treating metastatic disease. PMID:27124451

  11. Hypoxic control of metastasis

    PubMed Central

    Rankin, Erinn B.; Giaccia, Amato J.

    2016-01-01

    Metastatic disease is the leading cause of cancer-related deaths and involves critical interactions between tumor cells and the microenvironment. Hypoxia is a potent microenvironmental factor promoting metastatic progression. Clinically, hypoxia and the expression of the hypoxia-inducible transcription factors HIF-1 and HIF-2 are associated with increased distant metastasis and poor survival in a variety of tumor types. Moreover, HIF signaling in malignant cells influences multiple steps within the metastatic cascade. Here we review research focused on elucidating the mechanisms by which the hypoxic tumor microenvironment promotes metastatic progression. These studies have identified potential biomarkers and therapeutic targets regulated by hypoxia that could be incorporated into strategies aimed at preventing and treating metastatic disease. PMID:27124451

  12. Multiparameter digitized video microscopy of toxic and hypoxic injury in single cells.

    PubMed Central

    Lemasters, J J; Gores, G J; Nieminen, A L; Dawson, T L; Wray, B E; Herman, B

    1990-01-01

    There is no clear picture of the critical events that lead to the transition from reversible to irreversible injury. Many studies have suggested that a rise in cytosolic free Ca2+ initiates plasma membrane bleb formation and a sequence of events that lead ultimately to cell death. In recent studies, we have measured changes in cytosolic free Ca2+, mitochondrial membrane potential, cytosolic pH, and cell surface blebbing in relation to the onset of irreversible injury and cell death following anoxic and toxic injury to single hepatocytes by using multiparameter digitized video microscopy (MDVM). MDVM is an emerging new technology that permits single living cells to be labeled with multiple probes whose fluorescence is responsive to specific cellular parameters of interest. Fluorescence images specific for each probe are collected over time, digitized, and stored. Image analysis and processing then permits quantitation of the spatial distribution of the various parameters with the single living cells. Our results indicate the following: The formation of plasma membrane blebs accompanies all types of injury in hepatocytes. Cell death is a rapid event initiated by rupture of a plasma membrane bleb, and it is coincident with the onset of irreversible injury. An increase of cytosolic free Ca2+ is not the stimulus for bleb formation or the final common pathway leading to cell death. A decrease of mitochondrial membrane potential precedes the loss of cell viability. Cytosolic pH falls by more than 1 pH unit during chemical hypoxia. This acidosis protects against the onset of cell death. Images FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 10. FIGURE 13. FIGURE 15. PMID:2190822

  13. 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

  14. Hypoxic conditions increases H₂S-induced ER stress in A2870 cells.

    PubMed

    Lencesova, Lubomira; Vlcek, Miroslav; Krizanova, Olga; Hudecova, Sona

    2016-03-01

    Hypoxia - a state of lower oxygen demand-is responsible for a higher aggressiveness of tumors and therefore a worse prognosis. During hypoxia, several metabolic pathways are re-organized, e.g., energetic metabolism, modulation of pH, and calcium transport. Calcium is an important second messenger that regulates variety of processes in the cell. Thus, aim of this work was to compare H2S modulation of the intracellular calcium transport systems in hypoxia and in cells grown in standard culture conditions. For all experiments, we used ovarian cancer cell line (A2780). H2S is a novel gasotransmitter, known to be involved in a modulation of several calcium transport systems, thus resulting in altered calcium signaling. Two models of hypoxia were used in our study-chemical (induced by dimethyloxallyl glycine) and 2 % O2 hypoxia, both combined with a treatment using a slow H2S donor GYY4137. In hypoxia, we observed rapid changes in cytosolic and reticular calcium levels compared to cells grown in standard culture conditions, and these changes were even more exagerrated when combined with the GYY4137. Changes in a calcium homeostasis result from IP3 receptor´s up-regulation and down-regulation of the SERCA 2, which leads to a development of the endoplasmic reticulum stress. Based on our results, we propose a higher vulnerability of calcium transport systems to H2S regulation under hypoxia. PMID:26868821

  15. Expression and significance of hypoxia-inducible factor-1α and MDR1/P-glycoprotein in laryngeal carcinoma tissue and hypoxic Hep-2 cells

    PubMed Central

    XIE, JIN; LI, DA-WEI; CHEN, XIN-WEI; WANG, FEI; DONG, PIN

    2013-01-01

    The present study aimed to evaluate the expression of hypoxia-inducible factor-1α (HIF-1α) and MDR1/P-glycoprotein (P-gp) in human laryngeal squamous cell carcinoma (LSCC) tissues, and also to investigate the regulation of MDR1 gene expression by HIF-1α in Hep-2 cells under hypoxic conditions. The expression of HIF-1α and MDR1/P-gp in human LSCC tissues was examined using immunohistochemistry. The HIF-1α and MDR1 gene expression in the Hep-2 cells was detected using real-time quantitative reverse transcription (QRT)-PCR and western blot analysis under normoxic and hypoxic conditions. In hypoxia, HIF-1α expression was inhibited by RNA interference. HIF-1α and MDR1/P-gp expression was high in the LSCC tissues and was associated with the clinical stage and lymph node metastasis (P<0.05). HIF-1α expression was positively correlated with MDR1/P-gp expression (P<0.01). In the Hep-2 cells, HIF-1α and MDR1/P-gp expression significantly increased in response to hypoxia. The inhibition of HIF-1α expression synergistically downregulated the expression of the MDR1 gene in hypoxic Hep-2 cells. HIF-1α expression is positively correlated with MDR1/P-gp expression in LSCC, and the two proteins may be able to serve as potential biomarkers for predicting the malignant progression and metastasis of LSCC. HIF-1α may be critical for the upregulation of MDR1 gene expression induced by hypoxia in Hep-2 cells. PMID:23946810

  16. 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)

  17. Targeted inhibition of survivin with YM155 promotes apoptosis of hypoxic human pulmonary arterial smooth muscle cells via the upregulation of voltage-dependent K+ channels

    PubMed Central

    ZHANG, SHUAI; LIU, BO; FAN, ZAIWEN; WANG, DONG; LIU, YING; LI, JIAN; WANG, NING; LIU, YI; ZHANG, BO

    2016-01-01

    Hypoxic pulmonary hypertension (PH) is a common disease characterized by a disturbance to the balance of apoptosis and cell proliferation in pulmonary artery smooth muscle cells (PASMCs). The anti-apoptotic protein, survivin, has been observed to be upregulated in pulmonary arteries (PAs) of chronic hypoxia-induced PH rats. The present study aimed to investigate the therapeutic potential of sepantronium bromide (YM155), a selective survivin inhibitor, on hypoxic human PASMCs and examine the potential underlying mechanisms. Cultured human PASMCs (HPASMCs) were randomly divided into the following groups: i) Normoxia (N); ii) normoxia + 100 nmol/l YM155 (NY100); iii) hypoxia (H); iv) hypoxia + 1 nmol/l YM155 (HY1); v) hypoxia + 10 nmol/l YM155 (HY10); and hypoxia + 100 nmol/l YM155 (HY100) groups. The cells were exposed to the different conditions for 24 h, according to the group. Cell viability was then determined using a Cell Counting Kit-8 assay, and apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. The expression levels of survivin were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry and Western blot analyses. The expression levels of the voltage-dependent K+ (Kv) channels, Kv1.5 and Kv2.1, were measured using RT-qPCR and Western blotting. Cell proliferation in the hypoxic PASMCs was significantly increased by hypoxia, however, apoptosis of the HPASMCs was suppressed, the expression of survivin were upregulated and the expression levels of Kv1.5 and Kv2.1 were downregulated. YM155 treatment ameliorated the hypoxia-induced increase in cell proliferation and expression of survivin in a concentration-dependent manner, increased apoptosis, and increased the expression levels of Kv1.5 and Kv2.1 (P<0.05). By contrast, YM155 treatment in normoxic HPASMCs had no significant effects on proliferation, apop-tosis, or the expression

  18. Targeted inhibition of survivin with YM155 promotes apoptosis of hypoxic human pulmonary arterial smooth muscle cells via the upregulation of voltage-dependent K⁺ channels.

    PubMed

    Zhang, Shuai; Liu, Bo; Fan, Zaiwen; Wang, Dong; Liu, Ying; Li, Jian; Wang, Ning; Liu, Yi; Zhang, Bo

    2016-04-01

    Hypoxic pulmonary hypertension (PH) is a common disease characterized by a disturbance to the balance of apoptosis and cell proliferation in pulmonary artery smooth muscle cells (PASMCs). The anti-apoptotic protein, survivin, has been observed to be upregulated in pulmonary arteries (PAs) of chronic hypoxia-induced PH rats. The present study aimed to investigate the therapeutic potential of sepantronium bromide (YM155), a selective survivin inhibitor, on hypoxic human PASMCs and examine the potential underlying mechanisms. Cultured human PASMCs (HPASMCs) were randomly divided into the following groups: i) Normoxia (N); ii) normoxia + 100 nmol/l YM155 (NY100); iii) hypoxia (H); iv) hypoxia + 1 nmol/l YM155 (HY1); v) hypoxia + 10 nmol/l YM155 (HY10); and hypoxia + 100 nmol/l YM155 (HY100) groups. The cells were exposed to the different conditions for 24 h, according to the group. Cell viability was then determined using a Cell Counting Kit‑8 assay, and apoptosis was detected using a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay. The expression levels of survivin were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunocytochemistry and Western blot analyses. The expression levels of the voltage-dependent K+ (Kv) channels, Kv1.5 and Kv2.1, were measured using RT-qPCR and Western blotting. Cell proliferation in the hypoxic PASMCs was significantly increased by hypoxia, however, apoptosis of the HPASMCs was suppressed, the expression of survivin were upregulated and the expression levels of Kv1.5 and Kv2.1 were downregulated. YM155 treatment ameliorated the hypoxia‑induced increase in cell proliferation and expression of survivin in a concentration‑dependent manner, increased apoptosis, and increased the expression levels of Kv1.5 and Kv2.1 (P<0.05). By contrast, YM155 treatment in normoxic HPASMCs had no significant effects on proliferation, apoptosis, or the expression

  19. Neutrophils Oppose Uterine Epithelial Carcinogenesis via Debridement of Hypoxic Tumor Cells.

    PubMed

    Blaisdell, Adam; Crequer, Amandine; Columbus, Devin; Daikoku, Takiko; Mittal, Khush; Dey, Sudhansu K; Erlebacher, Adrian

    2015-12-14

    Polymorphonuclear neutrophils (PMNs) are largely considered to foster cancer development despite wielding an arsenal of cytotoxic agents. Using a mouse model of PTEN-deficient uterine cancer, we describe a surprising inhibitory role for PMNs in epithelial carcinogenesis. By inducing tumor cell detachment from the basement membrane, PMNs impeded early-stage tumor growth and retarded malignant progression. Unexpectedly, PMN recruitment and tumor growth control occurred independently of lymphocytes and cellular senescence and instead ensued as part of the tumor's intrinsic inflammatory response to hypoxia. In humans, a PMN gene signature correlated with improved survival in several cancer subtypes, including PTEN-deficient uterine cancer. These findings provide insight into tumor-associated PMNs and reveal a context-specific capacity for PMNs to directly combat tumorigenesis. PMID:26678340

  20. Diffuse-type gastric cancer cells switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis in hypoxic tumor microenvironments.

    PubMed

    Kinoshita, Haruhito; Yashiro, Masakazu; Fukuoka, Tatsunari; Hasegawa, Tsuyoshi; Morisaki, Tamami; Kasashima, Hiroaki; Masuda, Go; Noda, Satoru; Hirakawa, Kosei

    2015-12-01

    Cancer-associated fibroblasts (CAFs) have been considered to play an important role for tumor progression of cancer. Solid tumors contain heterogeneous distribution of oxygen in their microenvironments. This study investigated the growth signaling of gastric cancer (GC) cells in focus on the interaction with CAFs and GC cells under normoxia and hypoxia. Four diffuse-type GC cell lines, two intestinal-type GC cell lines and three CAF cell lines were used. Cells were examined for expression of C-X-C chemokine receptor 4 (CXCR4), fibroblast growth factor receptor 2 (FGFR2) and stromal-derived factor 1 (SDF1) by RT-PCR, western blot, ELISA and immunohistochemical staining of xenografted tumors. GC cell proliferation was examined under hypoxia in the presence or absence of CAFs, a FGFR2 inhibitor, a CXCR4 inhibitor and HIF1α siRNA. Proliferation of diffuse-type GC cells, but not intestinal-type GC cells, was significantly increased by CAFs. CXCR4 expression by diffuse-type GC cells was significantly increased in hypoxia, while FGFR2 expression was decreased. CXCR4 expression was correlated with hypoxic microenvironment of xenografted tumor, but FGFR2 expression was not. FGFR2 inhibition significantly decreased the growth-stimulating activity of CAFs for diffuse-type GC cells in normoxia. In contrast, CXCR4 inhibition significantly decreased the growth-stimulating activity of CAFs in hypoxia. SDF1 production by CAFs was increased in hypoxia, while cancer cells did not produce SDF1. HIF1 siRNA significantly decreased both CXCR4 expression by diffuse-type GC cells and SDF1 production by CAFs. These findings suggest that diffuse-type GC cells might switch their driver pathways from FGFR2 signaling to SDF1/CXCR4 axis through HIF1 in hypoxic tumor microenvironments. PMID:26385890

  1. Autophagy regulates the apoptosis of bone marrow-derived mesenchymal stem cells under hypoxic condition via AMP-activated protein kinase/mammalian target of rapamycin pathway.

    PubMed

    Zhang, Zheng; Yang, Ming; Wang, Yabin; Wang, Le; Jin, Zhitao; Ding, Liping; Zhang, Lijuan; Zhang, Lina; Jiang, Wei; Gao, Guojie; Yang, Junke; Lu, Bingwei; Cao, Feng; Hu, Taohong

    2016-06-01

    Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been demonstrated as an ideal autologous stem cells source for cell-based therapy for myocardial infarction (MI). However, poor viability of donor stem cells after transplantation limits their therapeutic efficiency, whereas the underlying mechanism is still poorly understood. Autophagy, a highly conserved process of cellular degradation, is required for maintaining homeostasis and normal function. Here, we investigated the potential role of autophagy on apoptosis in BM-MSCs induced by hypoxic injury. BM-MSCs, isolated from male C57BL/6 mice, were subjected to hypoxia and serum deprivation (H/SD) injury for 6, 12, and 24 h, respectively. The autophagy state was regulated by 3-methyladenine (3MA) and rapamycin administration. Furthermore, compound C was administrated to inhibit AMPK. The apoptosis induced by H/SD was determined by TUNEL assays. Meanwhile, autophagy was measured by GFP-LC3 plasmids transfection and transmission electron microscope. Moreover, protein expressions were evaluated by Western blot assay. In the present study, we found that hypoxic stress increased autophagy and apoptosis in BM-MSCs time dependently. Meanwhile, hypoxia increased the activity of AMPK/mTOR signal pathway. Moreover, increased apoptosis in BM-MSCs under hypoxia was abolished by 3-MA, whereas was aggravated by rapamycin. Furthermore, the increased autophagy and apoptosis in BM-MSCs induced by hypoxia were abolished by AMPK inhibitor compound C. These data provide evidence that hypoxia induced AMPK/mTOR signal pathway activation which regulated the apoptosis and autophagy in BM-MSCs. Furthermore, the apoptosis of BM-MSCs under hypoxic condition was regulated by autophagy via AMPK/mTOR pathway. PMID:27005844

  2. Inactivation of hypoxia-induced YAP by statins overcomes hypoxic resistance tosorafenib in hepatocellular carcinoma cells

    PubMed Central

    Zhou, Tian-yi; Zhuang, Lin-han; Hu, Yan; Zhou, Yu-lu; Lin, Wen-kai; Wang, Dan-dan; Wan, Zi-qian; Chang, Lin-lin; Chen, Ying; Ying, Mei-dan; Chen, Zi-bo; Ye, Song; Lou, Jian-shu; He, Qiao-jun; Zhu, Hong; Yang, Bo

    2016-01-01

    Sorafenib is a multikinase inhibitor used as a first-line treatment for advanced hepatocellular carcinoma (HCC), but it has shown modest to low response rates. The characteristic tumour hypoxia of advanced HCC maybe a major factor underlying hypoxia-mediated treatment failure. Thus, it is urgent to elucidate the mechanisms of hypoxia-mediated sorafenib resistance in HCC. In this study, we found that hypoxia induced the nuclear translocation of Yes associate-Protein (YAP) and the subsequent transactivation of target genes that promote cell survival and escape apoptosis, thereby leading to sorafenib resistance. Statins, the inhibitors of hydroxymethylglutaryl-CoA reductase, could ameliorate hypoxia-induced nuclear translocation of YAP and suppress mRNA levels of YAP target genes both in vivo and in vitro. Combined treatment of statins with sorafenib greatly rescued the loss of anti-proliferative effects of sorafenib under hypoxia and improved the inhibitory effects on HepG2 xenograft tumour growth, accompanied by enhanced apoptosis as evidenced by the increased sub-G1 population and PARP cleavage. The expression levels of YAP and its target genes were highly correlated with poor prognosis and predicted a high risk of HCC patients. These findings collectively suggest that statins utilization maybe a promising new strategy to counteract hypoxia-mediated resistance to sorafenib in HCC patients. PMID:27476430

  3. Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

    SciTech Connect

    Noguchi, K.; Hattori, T.; Igarashi, T.; Ueno, K.; Satoh, T.; Kitagawa, H.; Hori, H.; Shibata, T.; Inayama, S.

    1985-08-19

    A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitromidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H/sub 2/O/sub 2/ as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H/sub 2/O/sub 2/ as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H/sub 2/O/sub 2/ as substrate. 26 references, 2 figures, 4 tables.

  4. Altered Stra13 and Dec2 circadian gene expression in hypoxic cells

    SciTech Connect

    Guillaumond, Fabienne; Lacoche, Samuel; Dulong, Sandrine; Grechez-Cassiau, Aline; Filipski, Elisabeth; Li, Xiao-Mei; Levi, Francis; Berra, Edurne; Delaunay, Franck; Teboul, Michele

    2008-05-16

    The circadian system regulates rhythmically most of the mammalian physiology in synchrony with the environmental light/dark cycle. Alteration of circadian clock gene expression has been associated with tumour progression but the molecular links between the two mechanisms remain poorly defined. Here we show that Stra13 and Dec2, two circadian transcriptional regulators which play a crucial role in cell proliferation and apoptosis are overexpressed and no longer rhythmic in serum shocked fibroblasts treated with CoCl{sub 2,} a substitute of hypoxia. This effect is associated with a loss of circadian expression of the clock genes Rev-erb{alpha} and Bmal1, and the clock-controlled gene Dbp. Consistently, cotransfection assays demonstrate that STRA13 and DEC2 both antagonize CLOCK:BMAL1 dependent transactivation of the Rev-erb{alpha} and Dbp promoters. Using a transplantable osteosarcoma tumour model, we show that hypoxia is associated with altered circadian expression of Stra13, Dec2, Rev-erb{alpha}, Bmal1 and Dbp in vivo. These observations collectively support the notion that overexpression of Stra13 and Dec2 links hypoxia signalling to altered circadian clock gene expression.

  5. 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

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

    PubMed Central

    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. PMID:26579521

  7. Enhancement of Anti-Hypoxic Activity and Differentiation of Cardiac Stem Cells by Supernatant Fluids from Cultured Macrophages that Phagocytized Dead Mesenchymal Stem Cells

    PubMed Central

    Liu, Liang; Jin, Xian; Zhou, Zhong’e; Shen, Chengxing

    2016-01-01

    Background: Most mesenchymal stem cells (MSCs) die shortly after transplantation into a myocardial infarcted area. Dead MSCs (dMSCs) are phagocytized by macrophages (pMΦ) in vivo and in vitro; however, the effects of pMΦ on cardiac stem cells (CSCs) remain unknown. Methods: MSCs, CSCs, and macrophages were obtained from bone marrow, hearts, and peritoneal cavity of mice, respectively. dMSCs were harvested after hypoxia for 24 h, and incubated with macrophages (2:1) for another 2 days with or without lipopolysaccharide (LPS, 50 ng/mL) and sorted by flow cytometry to obtain pMΦ. Viability and apoptosis of CSCs were respectively evaluated with the cell counting kit-8 (CCk-8) assay and Annexin V-PE/7-AAD staining at 0, 6, 12, and 24 h of culture with supernatant fluids from macrophages (MΦ), LPS-stimulated macrophages (LPS-pMΦ), pMΦ, and MSCs. GATA-4 and c-TnI expression was measured by flow cytometry on the seventh day. Expression of inflammation and growth factors was assessed by real-time polymerase chain reaction (RT-PCR) in MΦ, LPS-pMΦ, and pMΦ cells. Results: pMΦ expressed higher levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β)and lower levels of tumor necrosis factor-α(TNF-α)and IL-6 than LPS-pMΦ, higher levels of growth factors and of GATA-4 and c-TnI at the 7th day, which were similar to those in MSCs. CSCs cultured with supernatant fluids of pMΦ exhibited higher proliferative, anti-hypoxic, and differentiation activities. Conclusion: The supernatant fluids of macrophages that had phagocytized dead MSCs encouraged changes in phenotype and growth factor expression, enhanced proliferation, differentiation, and anti-hypoxic activity of CSCs, which is relevant to understanding the persistent therapeutic effect of MSCs after their massive demise upon transplantation in myocardial infarction. Furthermore, some miRNAs or proteins which were extracted from the supernatant fluids may give us a new insight into the treatment of

  8. Bidirectional signalling between EphA2 and ephrinA1 increases tubular cell attachment, laminin secretion and modulates erythropoietin expression after renal hypoxic injury.

    PubMed

    Rodriguez, Stéphane; Rudloff, Stefan; Koenig, Katrin Franziska; Karthik, Swapna; Hoogewijs, David; Huynh-Do, Uyen

    2016-08-01

    Acute kidney injury (AKI) is common in hospitalized patients and has a poor prognosis, the severity of AKI being linked to progression to chronic kidney disease. This stresses the need to search for protective mechanisms during the acute phase. We investigated kidney repair after hypoxic injury using a rat model of renal artery branch ligation, which led to an oxygen gradient vertical to the corticomedullary axis. Three distinct zones were observed: tubular necrosis, infarction border zone and preserved normal tissue. EphA2 is a receptor tyrosine kinase with pivotal roles in cell architecture, migration and survival, upon juxtacrine contact with its membrane-bound ligand EphrinA1. Following hypoxia, EphA2 was up-regulated in cortical and medullary tubular cells, while EphrinA1 was up-regulated in interstitial cells adjacent to peritubular capillaries. Moreover, erythropoietin (EPO) messenger RNA (mRNA) was strongly expressed in the border zone of infarcted kidney within the first 6 h. To gain more insight into the biological impact of EphA2 and EphrinA1 up-regulation, we activated the signalling pathways in vitro using recombinant EphrinA1/Fc or EphA2/Fc proteins. Stimulation of EphA2 forward signalling in the proximal tubular cell line HK2 increased cell attachment and laminin secretion at the baso-lateral side. Conversely, activation of reverse signalling through EphrinA1 expressed by Hep3B cells promoted EPO production at both the transcriptional and protein level. Strikingly, in co-culture experiments, juxtacrine contact between EphA2 expressing MDCK and EphrinA1 expressing Hep3B was sufficient to induce a significant up-regulation of EPO mRNA production in the latter cells, even in the absence of hypoxic conditions. The synergistic effects of EphA2 and hypoxia led to a 15-20-fold increase of EPO expression. Collectively, our results suggest an important role of EphA2/EphrinA1 signalling in kidney repair after hypoxic injury through stimulation of (i) tubular

  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. Hypoxic HepG2 cell adaptation decreases ATP synthase dimers and ATP production in inflated cristae by mitofilin down-regulation concomitant to MICOS clustering.

    PubMed

    Plecitá-Hlavatá, Lydie; Engstová, Hana; Alán, Lukáš; Špaček, Tomáš; Dlasková, Andrea; Smolková, Katarína; Špačková, Jitka; Tauber, Jan; Strádalová, Vendula; Malínský, Jan; Lessard, Mark; Bewersdorf, Joerg; Ježek, Petr

    2016-05-01

    The relationship of the inner mitochondrial membrane (IMM) cristae structure and intracristal space (ICS) to oxidative phosphorylation (oxphos) is not well understood. Mitofilin (subunit Mic60) of the mitochondrial contact site and cristae organizing system (MICOS) IMM complex is attached to the outer membrane (OMM) via the sorting and assembly machinery/topogenesis of mitochondrial outer membrane β-barrel proteins (SAM/TOB) complex and controls the shape of the cristae. ATP synthase dimers determine sharp cristae edges, whereas trimeric OPA1 tightens ICS outlets. Metabolism is altered during hypoxia, and we therefore studied cristae morphology in HepG2 cells adapted to 5% oxygen for 72 h. Three dimensional (3D), super-resolution biplane fluorescence photoactivation localization microscopy with Eos-conjugated, ICS-located lactamase-β indicated hypoxic ICS expansion with an unchanged OMM (visualized by Eos-mitochondrial fission protein-1). 3D direct stochastic optical reconstruction microscopy immunocytochemistry revealed foci of clustered mitofilin (but not MICOS subunit Mic19) in contrast to its even normoxic distribution. Mitofilin mRNA and protein decreased by ∼20%. ATP synthase dimers vs monomers and state-3/state-4 respiration ratios were lower during hypoxia. Electron microscopy confirmed ICS expansion (maximum in glycolytic cells), which was absent in reduced or OMM-detached cristae of OPA1- and mitofilin-silenced cells, respectively. Hypoxic adaptation is reported as rounding sharp cristae edges and expanding cristae width (ICS) by partial mitofilin/Mic60 down-regulation. Mitofilin-depleted MICOS detaches from SAM while remaining MICOS with mitofilin redistributes toward higher interdistances. This phenomenon causes partial oxphos dormancy in glycolytic cells via disruption of ATP synthase dimers.-Plecitá-Hlavatá, L., Engstová, H., Alán, L., Špaček, T., Dlasková, A., Smolková, K., Špačková, J., Tauber, J., Strádalová, V., Malínský, J

  11. 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

  12. The Long Non-coding RNA HIF1A-AS2 Facilitates the Maintenance of Mesenchymal Glioblastoma Stem-like Cells in Hypoxic Niches.

    PubMed

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

    2016-06-14

    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, upregulated in mesenchymal GSCs. Its deregulation affects GSC growth, self-renewal, and hypoxia-dependent molecular reprogramming. Among 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. Downregulation 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 and targets and indirectly by modulating responses to hypoxic stress depending on the subtype-specific genetic context. PMID:27264189

  13. Dichloroacetate should be considered with platinum-based chemotherapy in hypoxic tumors rather than as a single agent in advanced non-small cell lung cancer

    PubMed Central

    Garon, Edward B.; Christofk, Heather R.; Hosmer, Wylie; Britten, Carolyn D; Bahng, Agnes; Crabtree, Matthew J; Hong, Candice Sun; Kamranpour, Naeimeh; Pitts, Sharon; Kabbinavar, Fairooz; Patel, Cecil; von Euw, Erika; Black, Alexander; Michelakis, Evangelos D.; Dubinett, Steven M.; Slamon, Dennis J.

    2014-01-01

    Objectives Dichloroacetate (DCA) is a highly bioavailable small molecule that inhibits pyruvate dehydrogenase kinase, promoting glucose oxidation and reversing the glycolytic phenotype in preclinical cancer studies. We designed this open label phase II trial to determine the response rate, safety, and tolerability of oral DCA in patients with metastatic breast cancer and advanced stage NSCLC. Materials and Methods This trial was conducted with DCA 6.25 mg/kg orally twice daily in previously treated stage IIIB/IV non-small cell lung cancer (NSCLC) or stage IV breast cancer. Growth inhibition by DCA was also evaluated in a panel of 54 NSCLC cell lines with and without cytotoxic chemotherapeutics (cisplatin and docetaxel) in normoxic and hypoxic conditions. Results and Conclusions Under normoxic conditions in vitro, single agent IC50 was > 2 mM for all evaluated cell lines. Synergy with cisplatin was seen in some cell lines under hypoxic conditions. In the clinical trial, after seven patients were enrolled, the study was closed based on safety concerns. The only breast cancer patient had stable disease after 8 weeks, quickly followed by progression in the brain. Two patients withdrew consent within a week of enrollment. Two patients had disease progression prior to the first scheduled scans. Within one week of initiating DCA, one patient died suddenly of unknown cause, and one experienced a fatal pulmonary embolism. We conclude that patients with previously treated advanced NSCLC did not benefit from oral DCA. In the absence of a larger controlled trial, firm conclusions regarding the association between these adverse events and DCA are unclear. Further development of DCA should be in patients with longer life expectancy, in whom sustained therapeutic levels can be achieved, and potentially in combination with cisplatin. PMID:24442098

  14. 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.

  15. Factors influencing survival of mammalian cells exposed to hypothermia. VI. Effects of prehypothermic hypoxia followed by aerobic or hypoxic storage at various hypothermic temperatures.

    PubMed

    Kruuv, J; Lepock, J R

    1995-04-01

    The Arrhenius plot of inactivation (killing) rates of V-79 Chinese hamster cells exposed to hypothermia in air-equilibrated (aerobic) medium contains a break at about 8 degrees C, which corresponds to the minimum inactivation rate, implying that there are distinct hypothermic damage mechanisms above (range I, 8 to 25 degrees C) and below (range II, 0 to 8 degrees C) 8 degrees C. Prehypothermic hypoxia (PHH) for 75 min at room temperature sensitizes cells to subsequent aerobic hypothermia at both 5 and 10 degrees C (range II and I). However, PHH followed by severe hypoxia (0.03 microM oxygen in the medium) protected cells during 10 degrees C (range I) storage by increasing the shoulder, but not the slope, of the cell survival curve compared to the PHH plus 10 degrees C aerobic hypothermia case. On the other hand, PHH plus severe hypoxia during 5 degrees C storage (range II) protected cells by decreasing the slope, but not the shoulder, of the cell survival curve compared to the PHH plus 5 degrees C aerobic hypothermia case. Furthermore, PHH plus severe hypoxia during 5 degrees C storage was not significantly worse than aerobic storage without PHH at 5 degrees C. With or without severe hypoxia, 10 degrees C storage is preferable to 5 degrees C storage in this cell line. Extrapolated to organ storage, the results may imply that if warm ischemia (PHH) has occurred, subsequent hypoxic hypothermic perfusion storage may be preferable to aerobic hypothermic perfusion storage. PMID:7743821

  16. 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.

  17. 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

  18. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling.

    PubMed

    Huang, Chia-Wei; Huang, Chao-Ching; Chen, Yuh-Ling; Fan, Shih-Chen; Hsueh, Yuan-Yu; Ho, Chien-Jung; Wu, Chia-Ching

    2015-01-01

    Neonatal hypoxic-ischemic (HI) brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs) from adipose-derived stem cells (ASCs) and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1) and vascular endothelial growth factor receptor 2 (VEGFR2) was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment. PMID:26509169

  19. Shear Stress Induces Differentiation of Endothelial Lineage Cells to Protect Neonatal Brain from Hypoxic-Ischemic Injury through NRP1 and VEGFR2 Signaling

    PubMed Central

    Huang, Chia-Wei; Huang, Chao-Ching; Chen, Yuh-Ling; Fan, Shih-Chen; Hsueh, Yuan-Yu; Ho, Chien-Jung; Wu, Chia-Ching

    2015-01-01

    Neonatal hypoxic-ischemic (HI) brain injuries disrupt the integrity of neurovascular structure and lead to lifelong neurological deficit. The devastating damage can be ameliorated by preserving the endothelial network, but the source for therapeutic cells is limited. We aim to evaluate the beneficial effect of mechanical shear stress in the differentiation of endothelial lineage cells (ELCs) from adipose-derived stem cells (ASCs) and the possible intracellular signals to protect HI injury using cell-based therapy in the neonatal rats. The ASCs expressed early endothelial markers after biochemical stimulation of endothelial growth medium. The ELCs with full endothelial characteristics were accomplished after a subsequential shear stress application for 24 hours. When comparing the therapeutic potential of ASCs and ELCs, the ELCs treatment significantly reduced the infarction area and preserved neurovascular architecture in HI injured brain. The transplanted ELCs can migrate and engraft into the brain tissue, especially in vessels, where they promoted the angiogenesis. The activation of Akt by neuropilin 1 (NRP1) and vascular endothelial growth factor receptor 2 (VEGFR2) was important for ELC migration and following in vivo therapeutic outcomes. Therefore, the current study demonstrated importance of mechanical factor in stem cell differentiation and showed promising protection of brain from HI injury using ELCs treatment. PMID:26509169

  20. Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation.

    PubMed

    Park, Yoon Shin; Kim, Gyungah; Jin, Yoon Mi; Lee, Jee Young; Shin, Jong Wook; Jo, Inho

    2016-01-01

    We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. PMID:26655815

  1. Differential hypoxic regulation of the microRNA-146a/CXCR4 pathway in normal and leukemic monocytic cells: impact on response to chemotherapy

    PubMed Central

    Spinello, Isabella; Quaranta, Maria Teresa; Paolillo, Rosa; Pelosi, Elvira; Cerio, Anna Maria; Saulle, Ernestina; Coco, Francesco Lo; Testa, Ugo; Labbaye, Catherine

    2015-01-01

    High expression of the chemokine receptor 4, CXCR4, associated with a negative prognosis in acute myeloid leukemia, is related to hypoxia. Because CXCR4 expression is under the post-transcriptional control of microRNA-146a in normal and leukemic monocytic cells, we first investigated the impact of hypoxia on microRNA-146a and CXCR4 expression during monocytopoiesis and in acute monocytic leukemia. We then analyzed the effects of hypoxia on drug sensitivity of CXCR4-expressing leukemic cells. We found that microRNA-146a is a target of hypoxia-inducible factor-1α or -2α in relation to the stage of monocytopoiesis and the level of hypoxia, and demonstrated the regulation of the microRNA-146a/CXCR4 pathway by hypoxia in monocytes derived from CD34+ cells. Thus, in myeloid leukemic cell lines, hypoxia-mediated control of the microRNA-146a/CXCR4 pathway depends only on the capacity of hypoxia-inducible factor-1α to up-regulate microRNA-146a, which in turn decreases CXCR4 expression. However, at variance with normal monocytic cells and leukemic cell lines, in acute monocytic leukemia overexpressing CXCR4, hypoxia up-modulates microRNA-146a but fails to down-modulate CXCR4 expression. We then investigated the effect of hypoxia on the response of leukemic cells to chemotherapy alone or in combination with stromal-derived factor-1α. We found that hypoxia increases stromal-derived factor-1α-induced survival of leukemic cells by decreasing their sensitivity to anti-leukemic drugs. Altogether, our results demonstrate that hypoxia-mediated regulation of microRNA-146a, which controls CXCR4 expression in monocytic cells, is lost in acute monocytic leukemia, thus contributing to maintaining CXCR4 overexpression and protecting the cells from anti-leukemic drugs in the hypoxic bone marrow microenvironment. PMID:26045293

  2. Protective Effects of N-Acetyl-L-Cysteine in Human Oligodendrocyte Progenitor Cells and Restoration of Motor Function in Neonatal Rats with Hypoxic-Ischemic Encephalopathy

    PubMed Central

    Park, Dongsun; Shin, Kyungha; Choi, Ehn-Kyoung; Choi, Youngjin; Jang, Ja-Young; Kim, Jihyun; Jeong, Heon-Sang; Lee, Wooryoung; Lee, Yoon-Bok; Kim, Seung Up; Joo, Seong Soo; Kim, Yun-Bae

    2015-01-01

    Objective. Since oligodendrocyte progenitor cells (OPCs) are the target cells of neonatal hypoxic-ischemic encephalopathy (HIE), the present study was aimed at investigating the protective effects of N-acetyl-l-cysteine (NAC), a well-known antioxidant and precursor of glutathione, in OPCs as well as in neonatal rats. Methods. In in vitro study, protective effects of NAC on KCN cytotoxicity in F3.Olig2 OPCs were investigated via MTT assay and apoptotic signal analysis. In in vivo study, NAC was administered to rats with HIE induced by hypoxia-ischemia surgery at postnatal day 7, and their motor functions and white matter demyelination were analyzed. Results. NAC decreased KCN cytotoxicity in F3.Olig2 cells and especially suppressed apoptosis by regulating Bcl2 and p-ERK. Administration of NAC recovered motor functions such as the using ratio of forelimb contralateral to the injured brain, locomotor activity, and rotarod performance of neonatal HIE animals. It was also confirmed that NAC attenuated demyelination in the corpus callosum, a white matter region vulnerable to HIE. Conclusion. The results indicate that NAC exerts neuroprotective effects in vitro and in vivo by preserving OPCs, via regulation of antiapoptotic signaling, and that F3.Olig2 human OPCs could be a good tool for screening of candidates for demyelinating diseases. PMID:25918547

  3. Hypoxic Preconditioning Inhibits Hypoxia-induced Apoptosis of Cardiac Progenitor Cells via the PI3K/Akt-DNMT1-p53 Pathway.

    PubMed

    Xu, Rongfeng; Sun, Yuning; Chen, Zhongpu; Yao, Yuyu; Ma, Genshan

    2016-01-01

    Research has demonstrated that hypoxic preconditioning (HP) can enhance the survival and proliferation of cardiac progenitor cells (CPCs); however, the underlying mechanisms are not fully understood. Here, we report that HP of c-kit (+) CPCs inhibits p53 via the PI3K/Akt-DNMT1 pathway. First, CPCs were isolated from the hearts of C57BL/6 mice and further purified by magnetic-activated cell sorting. Next, these cells were cultured under either normoxia (H0) or HP for 6 hours (H6) followed by oxygen-serum deprivation for 24 hours (24h). Flow cytometric analysis and MTT assays revealed that hypoxia-preconditioned CPCs exhibited an increased survival rate. Western blot and quantitative real-time PCR assays showed that p53 was obviously inhibited, while DNMT1 and DNMT3β were both significantly up-regulated by HP. Bisulphite sequencing analysis indicated that DNMT1 and DNMT3β did not cause p53 promoter hypermethylation. A reporter gene assay and chromatin immunoprecipitation analysis further demonstrated that DNMT1 bound to the promoter locus of p53 in hypoxia-preconditioned CPCs. Together, these observations suggest that HP of CPCs could lead to p53 inhibition by up-regulating DNMT1 and DNMT3β, which does not result in p53 promoter hypermethylation, and that DNMT1 might directly repress p53, at least in part, by binding to the p53 promoter locus. PMID:27488808

  4. Hypoxic Preconditioning Inhibits Hypoxia-induced Apoptosis of Cardiac Progenitor Cells via the PI3K/Akt-DNMT1-p53 Pathway

    PubMed Central

    Xu, Rongfeng; Sun, Yuning; Chen, Zhongpu; Yao, Yuyu; Ma, Genshan

    2016-01-01

    Research has demonstrated that hypoxic preconditioning (HP) can enhance the survival and proliferation of cardiac progenitor cells (CPCs); however, the underlying mechanisms are not fully understood. Here, we report that HP of c-kit (+) CPCs inhibits p53 via the PI3K/Akt-DNMT1 pathway. First, CPCs were isolated from the hearts of C57BL/6 mice and further purified by magnetic-activated cell sorting. Next, these cells were cultured under either normoxia (H0) or HP for 6 hours (H6) followed by oxygen–serum deprivation for 24 hours (24h). Flow cytometric analysis and MTT assays revealed that hypoxia-preconditioned CPCs exhibited an increased survival rate. Western blot and quantitative real-time PCR assays showed that p53 was obviously inhibited, while DNMT1 and DNMT3β were both significantly up-regulated by HP. Bisulphite sequencing analysis indicated that DNMT1 and DNMT3β did not cause p53 promoter hypermethylation. A reporter gene assay and chromatin immunoprecipitation analysis further demonstrated that DNMT1 bound to the promoter locus of p53 in hypoxia-preconditioned CPCs. Together, these observations suggest that HP of CPCs could lead to p53 inhibition by up-regulating DNMT1 and DNMT3β, which does not result in p53 promoter hypermethylation, and that DNMT1 might directly repress p53, at least in part, by binding to the p53 promoter locus. PMID:27488808

  5. 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. PMID:15288603

  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. Combination treatment with hypoxia-activated prodrug evofosfamide (TH-302) and mTOR inhibitors results in enhanced antitumor efficacy in preclinical renal cell carcinoma models

    PubMed Central

    Sun, Jessica D; Ahluwalia, Dharmendra; Liu, Qian; Li, Wenwu; Wang, Yan; Meng, Fanying; Bhupathi, Deepthi; Matteucci, Mark D; Hart, Charles P

    2015-01-01

    Tumors often consist of hypoxic regions which are resistant to chemo- and radiotherapy. Evofosfamide (also known as TH-302), a 2-nitroimidazole triggered hypoxia-activated prodrug, preferentially releases the DNA cross-linker bromo-isophosphoramide mustard in hypoxic cells. The intracellular kinase mTOR plays a key role in multiple pathways which are important in cancer progression. Here we investigated the enhanced efficacy profile and possible mechanisms of evofosfamide in combination with mTOR inhibitor (mTORi) everolimus or temsirolimus in renal cell carcinoma (RCC) xenograft models. The antitumor activities of the mTORi everolimus or temsirolimus alone, evofosfamide alone, or the combination were investigated in the 786-O and Caki-1 RCC cells in vitro and in vivo xenograft models. Two schedules were tested in which evofosfamide was started on the same day as the mTORi or 1 week after. Combination mechanisms were investigated by measuring a panel of pharmacodynamic biomarkers by immunohistochemistry. Antitumor efficacy in both RCC xenograft models was enhanced by the combination of evofosfamide and mTORi. Evofosfamide reduced the increased hypoxia induced by mTORi. Combination treatment induced increased DNA damage, decreased cell proliferation, and decreased survivin. Addition of mTORi did not change evofosfamide-mediated cytotoxicity in 786-O or Caki-1 cells in vitro which might suggest cell non-autonomous effects, specifically increased tumor hypoxia, are important for the in vivo combination activity. Taken together, evofosfamide potentiates the antitumor efficacy of mTOR inhibitors and inhibits the increased tumor hypoxia caused by mTOR inhibition. These studies provide a translational rationale for combining evofosfamide with mTOR inhibitors in clinical studies. PMID:26328245

  8. Tonicity enhancer binding protein (TonEBP) and hypoxia-inducible factor (HIF) coordinate heat shock protein 70 (Hsp70) expression in hypoxic nucleus pulposus cells: role of Hsp70 in HIF-1α degradation.

    PubMed

    Gogate, Shilpa S; Fujita, Nobuyuki; Skubutyte, Renata; Shapiro, Irving M; Risbud, Makarand V

    2012-05-01

    The objective of our study was to examine the regulation of hypoxic expression of heat shock protein 70 (Hsp70) in nucleus pulposus cells and to determine if Hsp70 promoted hypoxia-inducible factor (HIF)-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss-of-function and gain-of-function experiments and mutational analysis of the Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of the Hsp70 promoter. In contrast, mutation of hypoxia response element (HRE) motifs coupled with loss-of-function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses the inductive effect of TonEBP on the Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through the proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP, and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis. PMID:22322648

  9. AMP-activated protein kinase (AMPK)–dependent and –independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells

    PubMed Central

    Tan, CD; Smolenski, RT; Harhun, MI; Patel, HK; Ahmed, SG; Wanisch, K; Yáñez-Muñoz, RJ; Baines, DL

    2012-01-01

    BACKGROUND AND PURPOSE Pulmonary transepithelial Na+ transport is reduced by hypoxia, but in the airway the regulatory mechanisms remain unclear. We investigated the role of AMPK and ROS in the hypoxic regulation of apical amiloride-sensitive Na+ channels and basolateral Na+K+ ATPase activity. EXPERIMENTAL APPROACH H441 human airway epithelial cells were used to examine the effects of hypoxia on Na+ transport, AMP : ATP ratio and AMPK activity. Lentiviral constructs were used to modify cellular AMPK abundance and activity; pharmacological agents were used to modify cellular ROS. KEY RESULTS AMPK was activated by exposure to 3% or 0.2% O2 for 60 min in cells grown in submerged culture or when fluid (0.1 mL·cm−2) was added to the apical surface of cells grown at the air–liquid interface. Only 0.2% O2 activated AMPK in cells grown at the air–liquid interface. AMPK activation was associated with elevation of cellular AMP : ATP ratio and activity of the upstream kinase LKB1. Hypoxia inhibited basolateral ouabain-sensitive Isc (Iouabain) and apical amiloride-sensitive Na+ conductance (GNa+). Modification of AMPK activity prevented the effect of hypoxia on Iouabain (Na+K+ ATPase) but not apical GNa+. Scavenging of superoxide and inhibition of NADPH oxidase prevented the effect of hypoxia on apical GNa+ (epithelial Na+ channels). CONCLUSIONS AND IMPLICATIONS Hypoxia activates AMPK-dependent and -independent pathways in airway epithelial cells. Importantly, these pathways differentially regulate apical Na+ channels and basolateral Na+K+ ATPase activity to decrease transepithelial Na+ transport. Luminal fluid potentiated the effect of hypoxia and activated AMPK, which could have important consequences in lung disease conditions. PMID:22509822

  10. Vasoreparative Dysfunction of CD34+ Cells in Diabetic Individuals Involves Hypoxic Desensitization and Impaired Autocrine/Paracrine Mechanisms

    PubMed Central

    Jarajapu, Yagna P. R.; Hazra, Sugata; Segal, Mark; LiCalzi, Sergio; 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. PMID:24713821

  11. Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

    PubMed

    Sweda, Romy; Phillips, Andre W; Marx, Joel; Johnston, Michael V; Wilson, Mary Ann; Fatemi, Ali

    2016-07-01

    Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration. PMID:27149035

  12. Effect of hyperbaric oxygenation on mitochondrial function of neuronal cells in the cortex of neonatal rats after hypoxic-ischemic brain damage

    PubMed Central

    Yang, L.; Hei, M.Y.; Dai, J.J.; Hu, N.; Xiang, X.Y.

    2016-01-01

    The timing and mechanisms of protection by hyperbaric oxygenation (HBO) in hypoxic-ischemic brain damage (HIBD) have only been partially elucidated. We monitored the effect of HBO on the mitochondrial function of neuronal cells in the cerebral cortex of neonatal rats after HIBD. Neonatal Sprague-Dawley rats (total of 360 of both genders) were randomly divided into normal control, HIBD, and HIBD+HBO groups. The HBO treatment began immediately after hypoxia-ischemia (HI) and continued once a day for 7 consecutive days. Animals were euthanized 0, 2, 4, 6, and 12 h post-HI to monitor the changes in mitochondrial membrane potential (ΔΨm) occurring soon after a single dose of HBO treatment, as well as 2, 3, 4, 5, 6, and 7 days post-HI to study ΔΨm changes after a series of HBO treatments. Fluctuations in ΔΨm were observed in the ipsilateral cortex in both HIBD and HIBD+HBO groups. Within 2 to 12 h after HI insult, the ΔΨm of the HIBD and HIBD+HBO groups recovered to some extent. A secondary drop in ΔΨm was observed in both groups during the 1-4 days post-HI period, but was more severe in the HIBD+HBO group. There was a secondary recovery of ΔΨm observed in the HIBD+HBO group, but not in the HIBD group, during the 5-7 days period after HI insult. HBO therapy may not lead to improvement of neural cell mitochondrial function in the cerebral cortex in the early stage post-HI, but may improve it in the sub-acute stage post-HI. PMID:27119428

  13. Effect of hyperbaric oxygenation on mitochondrial function of neuronal cells in the cortex of neonatal rats after hypoxic-ischemic brain damage.

    PubMed

    Yang, L; Hei, M Y; Dai, J J; Hu, N; Xiang, X Y

    2016-01-01

    The timing and mechanisms of protection by hyperbaric oxygenation (HBO) in hypoxic-ischemic brain damage (HIBD) have only been partially elucidated. We monitored the effect of HBO on the mitochondrial function of neuronal cells in the cerebral cortex of neonatal rats after HIBD. Neonatal Sprague-Dawley rats (total of 360 of both genders) were randomly divided into normal control, HIBD, and HIBD+HBO groups. The HBO treatment began immediately after hypoxia-ischemia (HI) and continued once a day for 7 consecutive days. Animals were euthanized 0, 2, 4, 6, and 12 h post-HI to monitor the changes in mitochondrial membrane potential (ΔΨm) occurring soon after a single dose of HBO treatment, as well as 2, 3, 4, 5, 6, and 7 days post-HI to study ΔΨm changes after a series of HBO treatments. Fluctuations in ΔΨm were observed in the ipsilateral cortex in both HIBD and HIBD+HBO groups. Within 2 to 12 h after HI insult, the ΔΨm of the HIBD and HIBD+HBO groups recovered to some extent. A secondary drop in ΔΨm was observed in both groups during the 1-4 days post-HI period, but was more severe in the HIBD+HBO group. There was a secondary recovery of ΔΨm observed in the HIBD+HBO group, but not in the HIBD group, during the 5-7 days period after HI insult. HBO therapy may not lead to improvement of neural cell mitochondrial function in the cerebral cortex in the early stage post-HI, but may improve it in the sub-acute stage post-HI. PMID:27119428

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

    PubMed

    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

  15. 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

  16. Hypoxia-inducible factor-1α mediates the toll-like receptor 4 signaling pathway leading to anti-tumor effects in human hepatocellular carcinoma cells under hypoxic conditions

    PubMed Central

    Zhang, Xiaoyu; Li, Shuchen; Li, Mingrong; Huang, Haiying; Li, Jingyuan; Zhou, Changwei

    2016-01-01

    Hypoxia-inducible factor-1α (HIF-1α) and toll-like receptor 4 (TLR4) are involved in numerous mechanisms of cancer biology, including cell proliferation and survival; however the interaction of the two factors under hypoxic conditions remains unclear. The present study investigated the in vitro mechanism that results in the suppression of tumor cell growth and cellular functions when HIF-1α is silenced. In the present study, the human hepatocellular carcinoma HepG2 cell line was transfected with short hairpin RNA (shRNA) against HIF-1α and cultured under hypoxic conditions (1% O2 for 24 h). The expression of HIF-1α and various growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2), were examined using quantitative polymerase chain reaction and immunoblotting. Tumor growth was measured using a Cell Counting Kit-8 assay and tumor activity was measured using tumor cell invasion and migration assays. Lipopolysaccharide and TAK-242 were used to activate and inhibit TLR4, respectively, to observe the role of TLR4 in the HIF-1α silenced tumor cells. The expression of TLR4 signaling pathway associates, including myeloid differentiation primary response gene 88 (MyD88), apoptosis signal-regulating kinase 1 (ASK1), p38 mitogen-activated protein kinases and HIF-1α, were analyzed by western blot assay. Under hypoxic conditions, silencing of HIF-1α expression suppressed tumor cell growth and regulated the expression of tumor growth-associated genes, including EGF, HGF, VEGF and FG2. Suppression of tumor cell invasion and migration was also observed in the HIF-1α silenced HepG2 cell line. In addition, TLR4 was identified to be involved in HIF-1α and MyD88 accumulation, and activation of ASK1 and p38 were demonstrated to be critical for TLR4-mediated HIF-1α pathway. In conclusion, silencing of HIF-1α expression may induce anti-tumor effects under hypoxic

  17. Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress.

    PubMed

    Hoffmann, Brian R; Wagner, Jordan R; Prisco, Anthony R; Janiak, Agnieszka; Greene, Andrew S

    2013-11-01

    Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration. PMID:24022223

  18. Vascular endothelial growth factor-A signaling in bone marrow-derived endothelial progenitor cells exposed to hypoxic stress

    PubMed Central

    Hoffmann, Brian R.; Wagner, Jordan R.; Prisco, Anthony R.; Janiak, Agnieszka

    2013-01-01

    Bone marrow-derived endothelial progenitor cells (BM-EPCs) are stimulated by vascular endothelial growth factor-A (VEGF-A) and other potent proangiogenic factors. During angiogenesis, an increase in VEGF-A expression stimulates BM-EPCs to enhance endothelial tube formation and contribute to an increase in microvessel density. Hypoxia is known to produce an enhanced angiogenic response and heightened levels of VEGF-A have been seen in oxygen deprived epithelial and endothelial cells, yet the pathways for VEGF-A signaling in BM-EPCs have not been described. This study explores the influence of hypoxia on VEGF-A signaling in rat BM-EPCs utilizing a novel proteomic strategy to directly identify interacting downstream components of the combined VEGF receptor(s) signaling pathways, gene expression analysis, and functional phenotyping. VEGF-A signaling network analysis following liquid chromatographic separation and tandem mass spectrometry revealed proteins related to inositol/calcium signaling, nitric oxide signaling, cell survival, cell migration, and inflammatory responses. Alterations in BM-EPC expression of common angiogenic genes and tube formation in response to VEGF-A during hypoxia were measured and combined with the proteomic analysis to enhance and support the signaling pathways detected. BM-EPC tube formation assays in response to VEGF-A exhibited little tube formation; however, a cell projection/migratory phenotype supported the signaling data. Additionally, a novel assay measuring BM-EPC incorporation into preformed endothelial cell tubes indicated a significant increase of incorporated BM-EPCs after pretreatment with VEGF-A during hypoxia. This study verifies known VEGF-A pathway components and reveals several unidentified mechanisms of VEGF-A signaling in BM-EPCs during hypoxia that may be important for migration to sites of vascular regeneration. PMID:24022223

  19. Understanding Hypoxic Drive and the Release of Hypoxic Vasoconstriction.

    PubMed

    Inkrott, Jon C

    2016-01-01

    Understanding the hypoxic drive and release of hypoxic vasoconstriction in the chronic obstructive pulmonary disease population can be somewhat confusing and misunderstood. Furthermore, the hypoxic drive theory is one in which there really is no scientific evidence to support and yet continues to prosper in every aspect of care in regard to the chronic lung patient, from prehospital all the way to intensive care unit and home care therapy. This subject review will hopefully enhance some understanding of what exactly goes on with these patients and the importance of providing oxygen when it is desperately needed. PMID:27393755

  20. [Hypoxic lung failure].

    PubMed

    David, S; Wiesner, O

    2016-04-01

    Hypoxic lung failure is among the major indications for patients' referral to intensive care units either for surveillance or if necessary therapy. There are a vast number of pathophysiological causes of lung failure and the optimal treatment highly depends on the underlying pathology; therefore, no standard algorithm exists. So-called acute respiratory distress syndrome (ARDS) represents a very severe manifestation of hypoxemic lung failure that is of particular relevance for intensivists and is therefore the focus of this review. In addition to fundamental pathophysiology of lung injury, the article also focuses on established and modern treatment strategies. Moreover, we will briefly highlight innovative concepts of ARDS treatment that might become relevant in the future. PMID:27084180

  1. 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.

  2. Involvement of gap junctions between smooth muscle cells in sustained hypoxic pulmonary vasoconstriction development: a potential role for 15-HETE and 20-HETE.

    PubMed

    Kizub, Igor V; Lakhkar, Anand; Dhagia, Vidhi; Joshi, Sachindra R; Jiang, Houli; Wolin, Michael S; Falck, John R; Koduru, Sreenivasulu Reddy; Errabelli, Ramu; Jacobs, Elizabeth R; Schwartzman, Michal L; Gupte, Sachin A

    2016-04-15

    In response to hypoxia, the pulmonary artery normally constricts to maintain optimal ventilation-perfusion matching in the lung, but chronic hypoxia leads to the development of pulmonary hypertension. The mechanisms of sustained hypoxic pulmonary vasoconstriction (HPV) remain unclear. The aim of this study was to determine the role of gap junctions (GJs) between smooth muscle cells (SMCs) in the sustained HPV development and involvement of arachidonic acid (AA) metabolites in GJ-mediated signaling. Vascular tone was measured in bovine intrapulmonary arteries (BIPAs) using isometric force measurement technique. Expression of contractile proteins was determined by Western blot. AA metabolites in the bath fluid were analyzed by mass spectrometry. Prolonged hypoxia elicited endothelium-independent sustained HPV in BIPAs. Inhibition of GJs by 18β-glycyrrhetinic acid (18β-GA) and heptanol, nonspecific blockers, and Gap-27, a specific blocker, decreased HPV in deendothelized BIPAs. The sustained HPV was not dependent on Ca(2+) entry but decreased by removal of Ca(2+) and by Rho-kinase inhibition with Y-27632. Furthermore, inhibition of GJs decreased smooth muscle myosin heavy chain (SM-MHC) expression and myosin light chain phosphorylation in BIPAs. Interestingly, inhibition of 15- and 20-hydroxyeicosatetraenoic acid (HETE) synthesis decreased HPV in deendothelized BIPAs. 15-HETE- and 20-HETE-stimulated constriction of BIPAs was inhibited by 18β-GA and Gap-27. Application of 15-HETE and 20-HETE to BIPAs increased SM-MHC expression, which was also suppressed by 18β-GA and by inhibitors of lipoxygenase and cytochrome P450 monooxygenases. More interestingly, 15,20-dihydroxyeicosatetraenoic acid and 20-OH-prostaglandin E2, novel derivatives of 20-HETE, were detected in tissue bath fluid and synthesis of these derivatives was almost completely abolished by 18β-GA. Taken together, our novel findings show that GJs between SMCs are involved in the sustained HPV in BIPAs, and

  3. Hypoxic apnea and gasping.

    PubMed Central

    Guntheroth, W G; Kawabori, I

    1975-01-01

    We have tested the hypothesis that severe lypoxia causes apnea, regardless of the arterial CO2 and pH, and that extreme hypoxia causes gasping. Acute experiments with airway occlusion and with low inspired oxygen (FIo2) were performed on anesthetized adult dogs and monkeys. Arterial oxygen saturation was recorded continuously with fiberoptic oximetry, and Pco2 by an electrode catheter. In addition, blood samples were obtained for Po2, Pco2, and pH. Apnea was induced regularly when the Pao2 fell below 10 torr, whether the Paco2 was high with asphyxia (63 torr) or low (26 torr) with low FIo2. Similarly, the Pao2 at apnea was the same whether the pH was 7.17 with asphyxic hypoxia or 7.46 with hypoxic hypoxia. Gasping occurred at even lower Pao2 (below 5 torr) after 1 or 2 min of apnea. Gasping promptly restored the Pao2 to levels of moderate hypoxia (over 30 torr) which permitted resumption of regular respiration, with gradual elimination of the gasping. Fetal monkeys at term were studied in a similar manner from the moment of cord clamping. Their blood gases with apnea were quite similar to adult values in the narrow range of Pao2 and the wide range of Paco2 and pH. In the fetus, gasping was less immediately effective in improving arterial oxygen, but more persistent than in the adult. Regular respirations would not develop in the absence of oxygen in either the fetus or adult animal. Images PMID:811688

  4. Caffeic acid phenethyl ester reduces the secretion of vascular endothelial growth factor through the inhibition of the ROS, PI3K and HIF-1α signaling pathways in human retinal pigment epithelial cells under hypoxic conditions.

    PubMed

    Paeng, Sung Hwa; Jung, Won-Kyo; Park, Won Sun; Lee, Dae-Sung; Kim, Gi-Young; Choi, Yung Hyun; Seo, Su-Kil; Jang, Won Hee; Choi, Jung Sik; Lee, Young-Min; Park, Saegwang; Choi, Il-Whan

    2015-05-01

    Choroidal neovascularization (CNV) can lead to progressive and severe visual loss. Vascular endothelial growth factor (VEGF) promotes the development of CNV. Caffeic acid phenethyl ester (CAPE), a biologically active component of the honeybee (Apis mellifera) propolis, has been demonstrated to have several interesting biological regulatory properties. The objective of this study was to determine whether treatment with CAPE results in the inhibition of the production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial cells (RPE cells) under hypoxic conditions and to explore the possible underlying mechanisms. An in vitro experimental model of hypoxia was used to mimic an ischemic microenvironment for the RPE cells. Human RPE cells (ARPE-19) were exposed to hypoxia with or without CAPE pre-treatment. ARPE-19 cells were used to investigate the pathway involved in the regulation of VEGF production under hypoxic conditions, based on western blot analysis, enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assay (EMSA). The amount of VEGF released from the hypoxia-exposed cells was significantly higher than that of the normoxic controls. Pre-treatment with CAPE suppressed the hypoxia-induced production of VEGF in the ARPE-19 cells, and this effect was inhibited through the attenuation of reactive oxygen species (ROS) production, and the inhibition of phosphoinositide 3-kinase (PI3K)/AKT and hypoxia-inducible factor-1α (HIF-1α) expression. These in vitro findings suggest that CAPE may prove to be a novel anti-angiogenic agent for the treatment of diseases associated with CNV. PMID:25738890

  5. 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

  6. Hypoxic adipocytes pattern early heterotopic bone formation.

    PubMed

    Olmsted-Davis, Elizabeth; Gannon, Francis H; Ozen, Mustafa; Ittmann, Michael M; Gugala, Zbigniew; Hipp, John A; Moran, Kevin M; Fouletier-Dilling, Christine M; Schumara-Martin, Shannon; Lindsey, Ronald W; Heggeness, Michael H; Brenner, Malcolm K; Davis, Alan R

    2007-02-01

    The factors contributing to heterotopic ossification, the formation of bone in abnormal soft-tissue locations, are beginning to emerge, but little is known about microenvironmental conditions promoting this often devastating disease. Using a murine model in which endochondral bone formation is triggered in muscle by bone morphogenetic protein 2 (BMP2), we studied changes near the site of injection of BMP2-expressing cells. As early as 24 hours later, brown adipocytes began accumulating in the lesional area. These cells stained positively for pimonidazole and therefore generated hypoxic stress within the target tissue, a prerequisite for the differentiation of stem cells to chondrocytes and subsequent heterotopic bone formation. We propose that aberrant expression of BMPs in soft tissue stimulates production of brown adipocytes, which drive the early steps of heterotopic endochondral ossification by lowering oxygen tension in adjacent tissue, creating the correct environment for chondrogenesis. Results in misty gray lean mutant mice not producing brown fat suggest that white adipocytes convert into fat-oxidizing cells when brown adipocytes are unavailable, providing a compensatory mechanism for generation of a hypoxic microenvironment. Manipulation of the transcriptional control of adipocyte fate in local soft-tissue environments may offer a means to prevent or treat development of bone in extraskeletal sites. PMID:17255330

  7. Hypoxic Pulmonary Vasoconstriction in Humans

    PubMed Central

    Loubani, Mahmoud; Morice, Alyn H.

    2013-01-01

    Hypoxic pulmonary vasoconstriction is the elegant theory put forward more than six decades ago to explain regional variations in perfusion within the lung in certain animal species in response to localised restrictions in oxygenation. Although considerable progress has been made to describe the phenomenon at the macroscopic level and explain it at the microscopic level, we are far from a universal agreement about the process in humans. This review attempts to highlight some of the important evidence bases of hypoxic pulmonary vasoconstriction in humans and the significant gaps in our knowledge that would need bridging. PMID:24024204

  8. Targeting hypoxic response for cancer therapy

    PubMed Central

    Paolicchi, Elisa; Gemignani, Federica; Krstic-Demonacos, Marija; Dedhar, Shoukat; Mutti, Luciano; Landi, Stefano

    2016-01-01

    Hypoxic tumor microenvironment (HTM) is considered to promote metabolic changes, oncogene activation and epithelial mesenchymal transition, and resistance to chemo- and radio-therapy, all of which are hallmarks of aggressive tumor behavior. Cancer cells within the HTM acquire phenotypic properties that allow them to overcome the lack of energy and nutrients supply within this niche. These phenotypic properties include activation of genes regulating glycolysis, glucose transport, acidosis regulators, angiogenesis, all of which are orchestrated through the activation of the transcription factor, HIF1A, which is an independent marker of poor prognosis. Moreover, during the adaptation to a HTM cancer cells undergo deep changes in mitochondrial functions such as “Warburg effect” and the “reverse Warburg effect”. This review aims to provide an overview of the characteristics of the HTM, with particular focus on novel therapeutic strategies currently in clinical trials, targeting the adaptive response to hypoxia of cancer cells. PMID:26859576

  9. Hypoxic Tumor Environments Exhibit Disrupted Collagen I Fibers and Low Macromolecular Transport

    PubMed Central

    Kakkad, Samata M.; Penet, Marie-France; Akhbardeh, Alireza; Pathak, Arvind P.; Solaiyappan, Meiyappan; Raman, Venu; Leibfritz, Dieter; Glunde, Kristine; Bhujwalla, Zaver M.

    2013-01-01

    Hypoxic tumor microenvironments result in an aggressive phenotype and resistance to therapy that lead to tumor progression, recurrence, and metastasis. While poor vascularization and the resultant inadequate drug delivery are known to contribute to drug resistance, the effect of hypoxia on molecular transport through the interstitium, and the role of the extracellular matrix (ECM) in mediating this transport are unexplored. The dense mesh of fibers present in the ECM can especially influence the movement of macromolecules. Collagen 1 (Col1) fibers form a key component of the ECM in breast cancers. Here we characterized the influence of hypoxia on macromolecular transport in tumors, and the role of Col1 fibers in mediating this transport using an MDA-MB-231 breast cancer xenograft model engineered to express red fluorescent protein under hypoxia. Magnetic resonance imaging of macromolecular transport was combined with second harmonic generation microscopy of Col1 fibers. Hypoxic tumor regions displayed significantly decreased Col1 fiber density and volume, as well as significantly lower macromolecular draining and pooling rates, than normoxic regions. Regions adjacent to severely hypoxic areas revealed higher deposition of Col1 fibers and increased macromolecular transport. These data suggest that Col1 fibers may facilitate macromolecular transport in tumors, and their reduction in hypoxic regions may reduce this transport. Decreased macromolecular transport in hypoxic regions may also contribute to poor drug delivery and tumor recurrence in hypoxic regions. High Col1 fiber density observed around hypoxic regions may facilitate the escape of aggressive cancer cells from hypoxic regions. PMID:24349142

  10. Hypoxic turtles keep their cool.

    PubMed

    Madsen, Jesper G; Wang, Tobias; Madsen, Peter T

    2015-01-01

    Several species of freshwater turtles spend the winter submerged in ice-covered lakes in a state of severe metabolic depression. It has been proposed that the hibernating turtles are comatose and entirely unresponsive, which raises the question of how they detect the arrival of spring and whether they respond to sensory information during hibernation. Using evoked potential studies in cold hypoxic turtles exposed to light and vibration, we show that hibernating turtles maintain neural responsiveness to light stimuli during prolonged hypoxia, while responsiveness to vibration is lost. This reveals a state of differential neural shutdown, in different sensory systems in the cold hypoxic turtle brain. In behavioral studies we show that turtles held for 14 days in hibernation increase locomotor activity in response to light or elevated temperatures, but not to vibration or increased oxygen. We conclude that hibernating freshwater turtles are not comatose, but remain vigilant during overwintering in cold hypoxia. PMID:27227001

  11. Hypoxic turtles keep their cool

    PubMed Central

    Madsen, Jesper G; Wang, Tobias; Madsen, Peter T

    2015-01-01

    Several species of freshwater turtles spend the winter submerged in ice-covered lakes in a state of severe metabolic depression. It has been proposed that the hibernating turtles are comatose and entirely unresponsive, which raises the question of how they detect the arrival of spring and whether they respond to sensory information during hibernation. Using evoked potential studies in cold hypoxic turtles exposed to light and vibration, we show that hibernating turtles maintain neural responsiveness to light stimuli during prolonged hypoxia, while responsiveness to vibration is lost. This reveals a state of differential neural shutdown, in different sensory systems in the cold hypoxic turtle brain. In behavioral studies we show that turtles held for 14 days in hibernation increase locomotor activity in response to light or elevated temperatures, but not to vibration or increased oxygen. We conclude that hibernating freshwater turtles are not comatose, but remain vigilant during overwintering in cold hypoxia.

  12. 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.

  13. Effects of sensitizers on cell respiration: II. The effects of hypoxic cell sensitizers on oxygen utilization in cellular and chemical models.

    PubMed Central

    Greenstock, C. L.; Biaglow, J. E.; Durand, R. E.

    1978-01-01

    The metabolic activity of nitroheterocyclic sensitizers could limit their usefulness in vivo. Biochemical mechanisms of drug metabolism, toxicity and effects on cell respiration have been studied in microsomes, and the kinetics of the simulated redox reactions determined by pulse radiolysis. Stimulated oxidation of coenzyme, glucose, ascorbate or glutathione substrate radicals by nitroheterocyclic sensitizers, with the concomitant appearance of the respective nitro radical anions, is observed. Under hypoxia, the nitro radical anions decay slowly by second order processes, forming reduced metabolites. In air, the nitro radical anions react with oxygen forming superoxide radical anions, peroxide and regenerating the drug. Nitro radical-anions also react with cytochrome-c indicating a possible interference with mitochondrial energy metabolism. PMID:277209

  14. 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. PMID:24709424

  15. HIF-α/MIF and NF-κB/IL-6 Axes Contribute to the Recruitment of CD11b+Gr-1+ Myeloid Cells in Hypoxic Microenvironment of HNSCC12

    PubMed Central

    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

    2014-01-01

    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. PMID:24709424

  16. 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

  17. Neuroprotective Strategies after Neonatal Hypoxic Ischemic Encephalopathy.

    PubMed

    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

  18. 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. PMID:26183475

  19. The hTERT Promoter Enhances the Antitumor Activity of an Oncolytic Adenovirus under a Hypoxic Microenvironment

    PubMed Central

    Hashimoto, Yuuri; Tazawa, Hiroshi; Teraishi, Fuminori; Kojima, Toru; Watanabe, Yuichi; Uno, Futoshi; Yano, Shuya; Urata, Yasuo; Kagawa, Shunsuke; Fujiwara, Toshiyoshi

    2012-01-01

    Hypoxia is a microenvironmental factor that contributes to the invasion, progression and metastasis of tumor cells. Hypoxic tumor cells often show more resistance to conventional chemoradiotherapy than normoxic tumor cells, suggesting the requirement of novel antitumor therapies to efficiently eliminate the hypoxic tumor cells. We previously generated a tumor-specific replication-competent oncolytic adenovirus (OBP-301: Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives viral E1 expression. Since the promoter activity of the hTERT gene has been shown to be upregulated by hypoxia, we hypothesized that, under hypoxic conditions, the antitumor effect of OBP-301 with the hTERT promoter would be more efficient than that of the wild-type adenovirus 5 (Ad5). In this study, we investigated the antitumor effects of OBP-301 and Ad5 against human cancer cells under a normoxic (20% oxygen) or a hypoxic (1% oxygen) condition. Hypoxic condition induced nuclear accumulation of the hypoxia-inducible factor-1α and upregulation of hTERT promoter activity in human cancer cells. The cytopathic activity of OBP-301 was significantly higher than that of Ad5 under hypoxic condition. Consistent with their cytopathic activity, the replication of OBP-301 was significantly higher than that of Ad5 under the hypoxic condition. OBP-301-mediated E1A was expressed within hypoxic areas of human xenograft tumors in mice. These results suggest that the cytopathic activity of OBP-301 against hypoxic tumor cells is mediated through hypoxia-mediated activation of the hTERT promoter. Regulation of oncolytic adenoviruses by the hTERT promoter is a promising antitumor strategy, not only for induction of tumor-specific oncolysis, but also for efficient elimination of hypoxic tumor cells. PMID:22720091

  20. Oral (po) dosing with RSU 1069 or RB 6145 maintains their potency as hypoxic cell radiosensitizers and cytotoxins but reduces systemic toxicity compared with parenteral (ip) administration in mice

    SciTech Connect

    Cole, S.; Stratford, I.J.; Bowler, J.; Nolan, J.; Wright, E.G.; Lorimore, S.A.; Adams, G.E. )

    1991-07-01

    RB 6145 is a pro-drug of the hypoxic cell radiosensitizer RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for C3H/He mice was 80 mg/kg (0.38 mmol/kg) ip but 320 mg/kg (1.5 mmol/kg) following po administration. The MTD values of RB 6145 were 350 mg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 toward bone marrow stem cells was also less after po administration than after ip administration; 0.1 mmol/kg ip RSU 1069 and 0.38 mmol/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. Oral administration of RSU 1069 resulted in lower spermatogenic toxicity. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Some nephrotoxicity was observed in half of the mice given the highest po dose of 1.5 mmol/kg of RSU 1069; this was not observed following the highest ip dose of drug. For RSU 1069 and RB 6145, administered by either route, the maximum hypoxic cell radiosensitization in murine KHT sarcomas, occurred when the drugs were given 45-60 min before 10 Gy of X rays. The degree of radiosensitization produced by a particular dose of either compound was largely independent of the route of administration. Preliminary pharmacokinetic studies, using 3H-RSU 1069, suggested that anti-tumor efficacy correlated with peak blood level of label and concentration in the tumor at the time of irradiation, which were not reduced by po compared with ip administration. Normal tissue toxicity tended to correlate with total exposure over time, which was reduced approximately two-fold by po administration. Oral administration of RSU 1069 or RB 6145, as well as being convenient, may give therapeutic benefit since dose-limiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was less affected.

  1. Mechanical properties and reactivity of vessels in isolated perfused lungs of chronically hypoxic rats.

    PubMed

    Emery, C J; Bee, D; Barer, G R

    1981-11-01

    1. Chronically hypoxic rats kept in 10% (v/v) O2 for 3--6 weeks, were compared with littermate control rats. Pulmonary vascular resistance, measured from the slope of the pressure-flow relationship in isolated lungs perfused with blood of normal packed cell volume was higher in chronically hypoxic than control rats even during normoxia. 2. Chronically hypoxic rats weighed less than control rats but their pulmonary vascular volume, measured with labelled albumin was similar to control rats. This, together with evidence that the number of precapillary vessels is not reduced, does not suggest a large reduction in the vascular bed in chronic hypoxia. 3. A greater vasodilator action of isoprenaline and adenosine in chronically hypoxic than control lungs suggested a higher normoxic vascular tone. This higher tone was not the sole cause of increased resistance in chronically hypoxic lungs, since maximal vasodilatation did not reduce resistance to control levels. The chief cause was probably encroachment of new muscle on the vascular lumen of small vessels. 4. Pulmonary arterial compliance was reduced in chronically hypoxic lungs. 5. Reactivity of vessels to ventilation hypoxia, over a wide range of oxygen tension, to angiotensin II (ANG II) and to adenosine 5'-triphosphate (ATP) was significantly greater in chronically hypoxic than control lungs, but thresholds to these stimuli were not reduced. PMID:7285503

  2. Hydrogen sulfide mediates hypoxic vasoconstriction through a production of mitochondrial ROS in trout gills.

    PubMed

    Skovgaard, Nini; Olson, Kenneth R

    2012-09-01

    Hypoxic pulmonary vasoconstriction (HPV) is an adaptive response that diverts pulmonary blood flow from poorly ventilated and hypoxic areas of the lung to more well-ventilated parts. This response is important for the local matching of blood perfusion to ventilation and improves pulmonary gas exchange efficiency. HPV is an ancient and highly conserved response, expressed in the respiratory organs of all vertebrates, including lungs of mammals, birds, and reptiles; amphibian skin; and fish gills. The mechanism underlying HPV and how cells sense low Po(2) remains elusive. In perfused trout gills (Oncorhynchus mykiss), acute hypoxia, as well as H(2)S, caused an initial and transient constriction of the vasculature. Inhibition of the enzymes cystathionine-β-synthase and cystathionine-γ-lyase, which blocks H(2)S production, abolished the hypoxic response. Individually blocking the four complexes in the electron transport chain abolished both the hypoxic and the H(2)S-mediated constriction. Glutathione, an antioxidant and scavenger of superoxide, attenuated the vasoconstriction in response to hypoxia and H(2)S. Furthermore, diethyldithiocarbamate, an inhibitor of superoxide dismutase, attenuated the hypoxic and H(2)S constriction. This strongly suggests that H(2)S mediates the hypoxic vasoconstriction in trout gills. H(2)S may stimulate the mitochondrial production of superoxide, which is then converted to hydrogen peroxide (H(2)O(2)). Thus, H(2)O(2) may act as the "downstream" signaling molecule in hypoxic vasoconstriction. PMID:22739350

  3. Hypoxic Conditioned Medium from Human Amniotic Fluid-Derived Mesenchymal Stem Cells Accelerates Skin Wound Healing through TGF-β/SMAD2 and PI3K/Akt Pathways

    PubMed Central

    Jun, Eun Kyoung; Zhang, Qiankun; Yoon, Byung Sun; Moon, Jai-Hee; Lee, Gilju; Park, Gyuman; Kang, Phil Jun; Lee, Jung Han; Kim, Areee; You, Seungkwon

    2014-01-01

    In a previous study, we isolated human amniotic fluid (AF)-derived mesenchymal stem cells (AF-MSCs) and utilized normoxic conditioned medium (AF-MSC-norCM) which has been shown to accelerate cutaneous wound healing. Because hypoxia enhances the wound healing function of mesenchymal stem cell-conditioned medium (MSC-CM), it is interesting to explore the mechanism responsible for the enhancement of wound healing function. In this work, hypoxia not only increased the proliferation of AF-MSCs but also maintained their constitutive characteristics (surface marker expression and differentiation potentials). Notably, more paracrine factors, VEGF and TGF-β1, were secreted into hypoxic conditioned medium from AF-MSCs (AF-MSC-hypoCM) compared to AF-MSC-norCM. Moreover, AF-MSC-hypoCM enhanced the proliferation and migration of human dermal fibroblasts in vitro, and wound closure in a skin injury model, as compared to AF-MSC-norCM. However, the enhancement of migration of fibroblasts accelerated by AF-MSC-hypoCM was inhibited by SB505124 and LY294002, inhibitors of TGF-β/SMAD2 and PI3K/AKT, suggesting that AF-MSC-hypoCM-enhanced wound healing is mediated by the activation of TGF-β/SMAD2 and PI3K/AKT. Therefore, AF-MSC-hypoCM enhances wound healing through the increase of hypoxia-induced paracrine factors via activation of TGF-β/SMAD2 and PI3K/AKT pathways. PMID:24398984

  4. Transforming growth factor-beta1 upregulation triggers pulmonary artery smooth muscle cell proliferation and apoptosis imbalance in rats with hypoxic pulmonary hypertension via the PTEN/AKT pathways.

    PubMed

    Liu, Yun; Cao, Yonggang; Sun, Shuyang; Zhu, Jinquan; Gao, Shan; Pang, Jie; Zhu, Daling; Sun, Zengxian

    2016-08-01

    Transforming growth factor-beta1 (TGFβ1) and Phosphatase and Tensin homolog deleted on chromosome ten (PTEN) are involved in the regulation of proliferation, differentiation, migration and apoptosis of various cell types. In previous studies, we have shown that TGFβ1 and PTEN play an important role in the progression of pulmonary vascular remodeling induced by pulmonary artery smooth muscle cells (PASMCs). However, the mechanisms involved in the activation of PASMCs between TGFβ1 and PTEN pathways remain unknown. We found that pulmonary vascular walls in hypoxic pulmonary arterial hypertension (PAH) rats were thicker than the vessels from normal rats in vivo. Substantially higher levels of TGFβ1 and significant loss of PTEN expression were observed in the lungs of PAH rats when compared with normoxia. Meanwhile, AKT, a downstream proliferative signaling protein of the PTEN antagonist PI3K, was markedly activated in the lungs of PAH rats. In vitro studies using PASMCs showed that TGFβ1 increased cell proliferation in PTEN-dependent manner. Moreover, we found that TGFβ1 enhanced cell survival, up-regulated the expression of Bcl-2 and procaspase-3, decreased the number of TUNEL-positive cells and caspase-3 expression in PASMCs under serum-deprived (SD) condition via PI3K/AKT pathway. The results further establish that TGFβ1 promoted PAH by decreasing PTEN expression and increasing PI3K/AKT activation in the lung. In conclusion, TGFβ1 mediated PTEN inactivation and resistance to apoptosis seems to be key mediators of lung vascular remodeling associated with PAH. These findings further clarify molecular mechanisms that support targeting PTEN/AKT signaling pathway to attenuate pathogenic derangements in PAH. PMID:27297622

  5. Fentanyl activates hypoxia-inducible factor 1 in neuronal SH-SY5Y cells and mice under non-hypoxic conditions in a μ-opioid receptor-dependent manner.

    PubMed

    Daijo, Hiroki; Kai, Shinichi; Tanaka, Tomoharu; Wakamatsu, Takuhiko; Kishimoto, Shun; Suzuki, Kengo; Harada, Hiroshi; Takabuchi, Satoshi; Adachi, Takehiko; Fukuda, Kazuhiko; Hirota, Kiichi

    2011-09-30

    Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia. PMID:21703258

  6. [Hypoxic training of high qualification sportsmen].

    PubMed

    Radziievs'kiĭ, P O; Radziievs'ka, M P

    2003-01-01

    Normobaric intermittent hypoxic training (IHT) is an effective method for improving of of the functional respiration system (FRS) state, increase of aerobic productivity, general and special capacity for work in sportsmen of high qualification. The advantage of the use of intermittent hypoxic training during the preparation of high qualification sportsmen is so that hypoxic hypoxia and hypoxia of load influence on the sportsman organism in different time (IHT is doing during the rest, out of the planned sport training without the influence on it). This is a difference with the hypoxic training in mountain conditions where two types of hypoxia influence on the sportsman organism simultaneously and permanently--hypoxia of load and hypoxic hypoxia, and additive destructive action of the both types of hypoxia may be revealed. High efficacy of intermittent hypoxic training in improving of all parts of sportsmen FRS is a result alternating of hypoxic influences and normoxic intervals between them during which the level of plastic processes remains increased, oxygen tension in arterial blood and tissues increases to normoxic values. After IHT course, the state of respiration organs improves, the respiration volume, part of alveolar ventilation in the minute volume of respiration, saturation of arterial blood by oxygen, haemoglobin content in blood--increase as well as increase economy and efficacy of oxygen regimes of organism, general and special (especially important) physical capacity for work increase too. PMID:12918261

  7. The neuronal control of hypoxic ventilation: erythropoietin and sexual dimorphism.

    PubMed

    Gassmann, Max; Tissot van Patot, Martha; Soliz, Jorge

    2009-10-01

    Using mice, we demonstrated that when oxygen supply is lowered, erythropoietin (Epo), the main regulator of red blood cell production, modulates the ventilatory response by interacting with central (brainstem) and peripheral (carotid bodies) respiratory centers. We showed that enhanced Epo levels in the brainstem increased the hypoxic ventilatory response, and that intracerebroventricular injection of an Epo antagonist (soluble Epo receptor) abolished the ventilatory acclimatization to hypoxia. More recently, we have found that the impact of Epo on ventilation occurs in a sex-dependent manner. Keeping in mind that women are less susceptible to several respiratory sicknesses and syndromes than men, we suggest that Epo plays a key role in sexually-dimorphic hypoxic ventilation. Accordingly, we foresee that Epo has a potential therapeutic use as treatment for hypoxia-associated ventilatory diseases. PMID:19845617

  8. Low-dose radiation suppresses Pokemon expression under hypoxic conditions.

    PubMed

    Kim, Seung-Whan; Yu, Kweon; Shin, Kee-Sun; Kwon, Kisang; Hwang, Tae-Sik; Kwon, O-Yu

    2014-01-01

    Our previous data demonstrated that CoCl2-induced hypoxia controls endoplasmic reticulum (ER) stress-associated and other intracellular factors. One of them, the transcription factor Pokemon, was differentially regulated by low-dose radiation (LDR). There are limited data regarding how this transcription factor is involved in expression of the unfolded protein response (UPR) under hypoxic conditions. The purpose of this study was to obtain clues on how Pokemon is involved in the UPR. Pokemon was selected as a differentially expressed gene under hypoxic conditions; however, its regulation was clearly repressed by LDR. It was also demonstrated that both expression of ER chaperones and ER stress sensors were affected by hypoxic conditions, and the same results were obtained when cells in which Pokemon was up- or down-regulated were used. The current state of UPR and LDR research associated with the Pokemon pathway offers an important opportunity to understand the oncogenesis, senescence, and differentiation of cells, as well as to facilitate introduction of new therapeutic radiopharmaceuticals. PMID:24772825

  9. Hypoxic-Ischemic Neonatal Encephalopathy: Animal Experiments for Neuroprotective Therapies

    PubMed Central

    Ikenoue, Tsuyomu

    2013-01-01

    Hypoxic-ischemic neonatal encephalopathy and ensuing brain damage is still an important problem in modern perinatal medicine. In this paper, we would like to share some of the results of our recent studies on neuroprotective therapies in animal experiments, as well as some literature reviews. From the basic animal studies, we have now obtained some possible candidates for therapeutic measures against hypoxic-ischemic neonatal encephalopathy. For example, they are hypothermia, rehabilitation, free radical scavenger, neurotrophic factors and growth factors, steroid, calcium channel blocker, vagal stimulation, some anti apoptotic agents, pre- and post conditioning, antioxidants, cell therapy with stem cells, modulators of K(+)-ATP channels, and so on. Whether combination of these therapies may be more beneficial than any single therapy needs to be clarified. Hypoxia-ischemia is a complicated condition, in which the cause, severity, and time-course are different in each case. Likewise, each fetus has its own inherent potentials such as adaptation, preconditioning-tolerance, and intolerance. Therefore, further extensive studies are required to establish an individualized strategy for neuroprotection against perinatal hypoxic-ischemic insult. PMID:23533962

  10. Carbonic Anhydrase 9 mRNA/microRNA34a Interplay in Hypoxic Human Mammospheres.

    PubMed

    De Carolis, Sabrina; Bertoni, Sara; Nati, Marina; D'Anello, Laura; Papi, Alessio; Tesei, Anna; Cricca, Monica; Bonafé, Massimiliano

    2016-07-01

    The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche. PMID:26553365

  11. [The Role of ABCG2 Protein in Maintenance of Viability and Proliferative Activity of Bone Marrow Mesenchymal Stem Cells Under Hypoxic Conditions].

    PubMed

    Poleshko, A G; Volotovski, I D

    2016-01-01

    It has been shown that hypoxia (5% 02) and fibroblast growth factor bFGF reduce the doubling time of bone marrow mesenchymal stem cells under their cultivation in vitro that indicates an increase in cell culture proliferation. It has been found out that low concentrations of O2 and factor bFGF added to the cell culture medium increase an expression of abcg2 gene and its gene protein, ABCG2 transport gene, in mesenchymal stem cells. These events potentiate the effects of hypoxia observed in mesenchymal stem cells. We revealed that blocking of ABCG2 protein functional activity led to increased generation of reactive oxygen species in mesenchymal stem cells. The effect of hypoxia and/or bFGF on protein profile of mesenchymal stem cells was studied. The results represented in this work together with previous data proved a link between ABCG2 protein expression, its activity and maintenance of viability and proliferative activity of mesenchymal stem cells cultivated under hypoxia. ABCG2 acts as protector. PMID:27192835

  12. 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

  13. 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

  14. Epigenetic regulation of hypoxic sensing disrupts cardiorespiratory homeostasis

    PubMed Central

    Nanduri, Jayasri; Makarenko, Vladislav; Reddy, Vaddi Damodara; Yuan, Guoxiang; Pawar, Anita; Wang, Ning; Khan, Shakil A.; Zhang, Xin; Kinsman, Brian; Peng, Ying-Jie; Kumar, Ganesh K.; Fox, Aaron P.; Godley, Lucy A.; Semenza, Gregg L.; Prabhakar, Nanduri R.

    2012-01-01

    Recurrent apnea with intermittent hypoxia is a major clinical problem in preterm infants. Recent studies, although limited, showed that adults who were born preterm exhibit increased incidence of sleep-disordered breathing and hypertension, suggesting that apnea of prematurity predisposes to autonomic dysfunction in adulthood. Here, we demonstrate that adult rats that were exposed to intermittent hypoxia as neonates exhibit exaggerated responses to hypoxia by the carotid body and adrenal chromaffin cells, which regulate cardio-respiratory function, resulting in irregular breathing with apneas and hypertension. The enhanced hypoxic sensitivity was associated with elevated oxidative stress, decreased expression of genes encoding antioxidant enzymes, and increased expression of pro-oxidant enzymes. Decreased expression of the Sod2 gene, which encodes the antioxidant enzyme superoxide dismutase 2, was associated with DNA hypermethylation of a single CpG dinucleotide close to the transcription start site. Treating neonatal rats with decitabine, an inhibitor of DNA methylation, during intermittent hypoxia exposure prevented oxidative stress, enhanced hypoxic sensitivity, and autonomic dysfunction. These findings implicate a hitherto uncharacterized role for DNA methylation in mediating neonatal programming of hypoxic sensitivity and the ensuing autonomic dysfunction in adulthood. PMID:22232674

  15. 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

  16. 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. PMID:26881424

  17. Oroxylin A regulates glucose metabolism in response to hypoxic stress with the involvement of Hypoxia-inducible factor-1 in human hepatoma HepG2 cells.

    PubMed

    Dai, Qinsheng; Yin, Qian; Wei, Libin; Zhou, Yuxin; Qiao, Chen; Guo, Yongjian; Wang, Xiaotang; Ma, Shiping; Lu, Na

    2016-08-01

    Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. In this study, we investigated the influence and signaling ways of oroxylin A affecting cancer cell energy metabolism under hypoxia. The data showed that oroxylin A remarkably reduced the generation of lactate and glucose uptake under hypoxia in HepG2 cells. Moreover, oroxylin A inhibited HIF-1α expression and its stability. The downstream targets (PDK1, LDHA, and HK II), as well as their mRNA levels were also suppressed by oroxylin A under hypoxia. The silencing or the overexpression of HIF-1α assays suggested that HIF-1α is required for metabolic effect of oroxylin A in HepG2 cells during hypoxia. Furthermore, oroxylin A could reduce the expression of complex III in mitochondrial respiratory chain, and then decrease the accumulation of ROS at moderate concentrations (0-50 µM) under hypoxia, which was benefit for its inhibition on glycolytic activity by decreasing ROS-mediated HIF-1 expression. Besides, oroxylin A didn't cause the loss of MMP under hypoxia and had no obvious effects on the expression of OXPHOS complexes, suggesting that oroxylin A did not affect mitochondrial mass at the moderate stress of oroxylin A. The suppressive effect of oroxylin A on glycolysis led to a significantly repress of ATP generation, for ATP generation mostly depends on glycolysis in HepG2 cells. This study revealed a new aspect of glucose metabolism regulation of oroxylin A under hypoxia, which may contribute to its new anticancer mechanism. © 2015 Wiley Periodicals, Inc. PMID:26259145

  18. 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.

  19. Differential regulation of pro- and antiapoptotic proteins in fish adipocytes during hypoxic conditions.

    PubMed

    Ekambaram, Padmini; Parasuraman, Parimala; Jayachandran, Tharani

    2016-06-01

    Worldwide, the frequencies and magnitudes of hypoxic events in estuarine waters have increased considerably over the past two decades. Fish populations are suitable indicators for the assessment of quality of aquatic ecosystems and often comprise a variety of adaptation systems by triggering oxidants, antioxidants and hypoxia-responsive signaling proteins. Signaling pathway may lead to cell survival or cell death which is fine-tuned by both positive and negative factors, which includes hypoxia-inducible factor-1α (HIF1α), heat-shock protein-70 (HSP70), phospho-c-Jun N-terminal kinase 1/2 (p-JNK1/2) and apoptosis signal-regulating kinase-1 (ASK1). In the present study, we attempt to determine stress-mediated signaling changes and molecular mechanism behind the cell survival by comparing adipocytes of fish from field hypoxic condition and laboratory-induced hypoxic condition (in vitro hypoxia). Comparison of field and laboratory studies in fish adipocytes showed differential expression of HIF1α, HSP70, p-JNK1/2 and ASK1 with altered oxidants and antioxidants. Further, the results also suggest that in vitro hypoxic conditions mimic field hypoxic conditions. Trends of hypoxia response were same in in vitro hypoxia of control adipocytes as in Ennore estuary, and hypoxia response was more pronounced in the test adipocytes under in vitro hypoxic condition. Results of the present work suggest that hypoxia is the major crusade of water pollutants affecting fish by differential regulation of pro- and antiapoptotic proteins probably through HSP70. This may play a vital role by providing cytoprotection in pollutant-induced stressed fish adipocytes substantiated by the in vitro hypoxic studies. PMID:26744268

  20. Hypoxic tumor cell death and modulation of endothelial adhesion molecules in the regression of granulocyte colony-stimulating factor-transduced tumors.

    PubMed Central

    Colombo, M. P.; Lombardi, L.; Melani, C.; Parenza, M.; Baroni, C.; Ruco, L.; Stoppacciaro, A.

    1996-01-01

    C-26 colon adenocarcinoma cells transduced with the granulocyte colony-stimulating factor (G-CSF) gene form large tumors when injected into sublethally irradiated mice. These tumors regress when leukocyte function is reconstituted. Electron microscopy and immunocytochemical analysis of regressing C-26/G-CSF nodules indicates that tumor destruction is due mainly to hypoxia resulting from the functional loss of tumor vasculature and is only marginally due to direct cytolysis. Desegregation of basal lamina, cell swelling, and loss of junctions characterized the vessels within regressing tumors. Tumor cells were necrotic or filled with lipid vacuoles regardless of the distance from nearby vessels. Damage of tumor vasculature was dependent on the infiltrating leukocytes and the cytotoxic cytokines they produced. Locally produced interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) induced vascular cellular adhesion molecule-1 (VCAM-1) and E-selectin on tumor vessels. Treatment with monoclonal antibodies to interferon-gamma (IFN-gamma) or TNF-alpha blocked tumor regression by inhibiting VCAM-1 and E-selectin expression on tumor-associated endothelial cells resulting in a reduced number of infiltrating leukocytes. Thus, C-26/G-CSF tumor regression presents features typical of hemorrhagic necrosis that occurs through the cytokines produced by infiltrating leukocytes in response to G-CSF. Images Figure 1 p477-a Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8579110

  1. The novel dual PI3K/mTOR inhibitor NVP-BGT226 displays cytotoxic activity in both normoxic and hypoxic hepatocarcinoma cells

    PubMed Central

    Martelli, Alberto M.; Zauli, Giorgio; Alameen, Ayman A.M.; Ultimo, Simona; Tabellini, Giovanna; McCubrey, James A.; Capitani, Silvano; Neri, Luca M.

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most common lethal human malignancies worldwide and its advanced status is frequently resistant to conventional chemotherapeutic agents and radiation. We evaluated the cytotoxic effect of the orally bioavailable dual PI3K/mTOR inhibitor, NVP-BGT226, on a panel of HCC cell lines, since hyperactivated PI3K/Akt/mTOR signaling pathway could represent a biomolecular target for Small Inhibitor Molecules in this neoplasia. We analyzed the drug activity in both normoxia and hypoxia conditions, the latter playing often a relevant role in the induction of chemoresistance and angiogenesis. In normoxia NVP-BGT226 caused cell cycle arrest in the G0/G1 phase of the cell cycle, induced apoptosis and autophagy at low concentrations. Interestingly the drug inactivated p-Akt and p-S6 at < 10 nM concentration. In hypoxia NVP-BGT226 maintained its cytotoxic efficacy at the same concentration as documented by MTT assays and Western blot analysis. Moreover, the drug showed in hypoxia inhibitory properties against angiogenesis by lowering the expression of the transcription factor HIF-1α and of VEGF. Our results indicate that NVP-BGT226 has a potent cytotoxic effect on HCC cell lines also in hypoxia condition, thus emerging as a potential candidate for cancer treatment in HCC targeted therapy. PMID:26003166

  2. 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

    2015-01-01

    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. PMID:25907197

  3. Changes in misonidazole binding with hypoxic fraction in mouse tumors

    SciTech Connect

    Hirst, D.G.; Hazlehurst, J.L.; Brown, J.M.

    1985-07-01

    Binding of misonidazole (MISO) or a derivative to hypoxic cells in tumors has been proposed as a method for identifying tumors, and measuring their level of hypoxia. The author has recently shown that the hypoxic fraction of tumor cells can be altered over a wide range in vivo by acutely changing the hematocrit of the host animal by transfusion. The present study is aimed to investigate the changes in binding by /sup 14/C MISO that accompanied this procedure. Tumor bearing mice were injected with /sup 14/C MISO, irradiated with a single dose of X rays (20 Gy) and their tumor excised and bisected. One half of each tumor was used to determine cell survival in vitro, the other was used for /sup 14/C scintillation counting. As previously described, tumor cell survival was dramatically increased in acutely anemic mice and this was accompanied by an increase in /sup 14/C MISO binding to the tumors. The relationship between clonogenic cell survival and binding was found to be linear on a log-log plot for each of the tumor lines studied, but the slopes of the lines were different in different tumor lines and generally steeper than the value of 1.0 expected for a 1:1 correspondence between cells binding radioactivity and radiobiological resistance.

  4. 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

  5. Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells

    PubMed Central

    Trisciuoglio, D; Gabellini, C; Desideri, M; Ragazzoni, Y; De Luca, T; Ziparo, E; Del Bufalo, D

    2011-01-01

    In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2. PMID:21233846

  6. Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells.

    PubMed

    Trisciuoglio, D; Gabellini, C; Desideri, M; Ragazzoni, Y; De Luca, T; Ziparo, E; Del Bufalo, D

    2011-06-01

    In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2. PMID:21233846

  7. 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

    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

  8. 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.

  9. Treadmill exercise ameliorates impairment of spatial learning ability through enhancing dopamine expression in hypoxic ischemia brain injury in neonatal rats.

    PubMed

    Park, Chang-Youl; Lee, Shin-Ho; Kim, Bo-Kyun; Shin, Mal-Soon; Kim, Chang-Ju; Kim, Hong

    2013-01-01

    Substantia nigra and striatum are vulnerable to hypoxic ischemia brain injury. Physical exercise promotes cell survival and functional recovery after brain injury. However, the effects of treadmill exercise on nigro-striatal dopaminergic neuronal loss induced by hypoxic ischemia brain injury in neonatal stage are largely unknown. We determined the effects of treadmill exercise on survival of dopamine neurons in the substantia nigra and dopaminergic fibers in the striatum after hypoxic ischemia brain injury. On postnatal 7 day, left common carotid artery of the neonatal rats ligated for two hours and the neonatal rats were exposed to hypoxia conditions for one hour. The rat pups in the exercise groups were forced to run on a motorized treadmill for 30 min once a day for 12 weeks, starting 22 days after induction of hypoxic ischemia brain injury. Spatial learning ability in rat pups was determined by Morris water maze test after last treadmill exercise. The viability of dopamine neurons in the substantia nigra and dopamine fibers in the striatum were analyzed using immunohistochemistry. In this study, hypoxic ischemia injury caused loss of dopamine neurons in the substantia nigra and dopaminergic fibers in the striatum. Induction of hypoxic ischemia deteriorated spatial learning ability. Treadmill exercise ameliorated nigro-striatal dopaminergic neuronal loss, resulting in the improvement of spatial learning ability. The present study suggests the possibility that treadmill exercise in early adolescent period may provide a useful strategy for the recovery after neonatal hypoxic ischemia brain injury. PMID:24278893

  10. Novel Roles for Protein Kinase Cδ-dependent Signaling Pathways in Acute Hypoxic Stress-induced Autophagy*S⃞

    PubMed Central

    Chen, Jo-Lin; Lin, Her H.; Kim, Kwang-Jin; Lin, Anning; Forman, Henry J.; Ann, David K.

    2008-01-01

    Macroautophagy, a tightly orchestrated intracellular process for bulk degradation of cytoplasmic proteins or organelles, is believed to be essential for cell survival or death in response to stress conditions. Recent observations indicate that autophagy is an adaptive response in cells subjected to prolonged hypoxia. However, the signaling mechanisms that activate autophagy under acute hypoxic stress are not clearly understood. In this study, we show that acute hypoxic stress by treatment with 1% O2 or desferroxamine, a hypoxia-mimetic agent, of cells renders a rapid induction of LC3-II level changes and green fluorescent protein-LC3 puncta accumulation, hallmarks of autophagic processing, and that this process involves protein kinase Cδ (PKCδ), and occurs prior to the induction of BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3). Interestingly, hypoxic stress leads to a rapid and transient activation of JNK in Pa-4 or mouse embryo fibroblast cells. Acute hypoxic stress-induced changes in LC3-II level and JNK activation are attenuated in Pa-4 cells by dominant negative PKCδKD or in mouse embryo fibroblast/PKCδ-null cells. Intriguingly, the requirement of PKCδ is not apparent for starvation-induced autophagy. The importance of PKCδ in hypoxic stress-induced adaptive responses is further supported by our findings that inhibition of PKCδ-facilitated autophagy by 3-methyladenine or Atg5 knock-out renders a greater prevalence of cell death following prolonged desferroxamine treatment, whereas PKCδ- or JNK1-deficient cells exhibit resistance to extended hypoxic exposure. These results uncover dual roles of PKCδ-dependent signaling in the cell fate determination upon hypoxic exposure. PMID:18836180

  11. CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction.

    PubMed

    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

    2015-03-31

    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. Ca(2+) 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 Ca(2+) mobilization. Ca(2+) 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

  12. 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

  13. Hypoxic Conditioning as a New Therapeutic Modality

    PubMed Central

    Verges, Samuel; Chacaroun, Samarmar; Godin-Ribuot, Diane; Baillieul, Sébastien

    2015-01-01

    Preconditioning refers to a procedure by which a single noxious stimulus below the threshold of damage is applied to the tissue in order to increase resistance to the same or even different noxious stimuli given above the threshold of damage. Hypoxic preconditioning relies on complex and active defenses that organisms have developed to counter the adverse consequences of oxygen deprivation. The protection it confers against ischemic attack for instance as well as the underlying biological mechanisms have been extensively investigated in animal models. Based on these data, hypoxic conditioning (consisting in recurrent exposure to hypoxia) has been suggested a potential non-pharmacological therapeutic intervention to enhance some physiological functions in individuals in whom acute or chronic pathological events are anticipated or existing. In addition to healthy subjects, some benefits have been reported in patients with cardiovascular and pulmonary diseases as well as in overweight and obese individuals. Hypoxic conditioning consisting in sessions of intermittent exposure to moderate hypoxia repeated over several weeks may induce hematological, vascular, metabolic, and neurological effects. This review addresses the existing evidence regarding the use of hypoxic conditioning as a potential therapeutic modality, and emphasizes on many remaining issues to clarify and future researches to be performed in the field. PMID:26157787

  14. 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

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

    PubMed

    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

  16. Performance of an active inspired hypoxic guard.

    PubMed

    Ghijselings, Idris E; De Cooman, Sofie; Carette, Rik; Peyton, Philip J; De Wolf, Andre M; Hendrickx, Jan F A

    2016-02-01

    Current hypoxic guards systems fail to maintain the inspired O2 concentration (FIO2) ≥ 21 % across the entire fresh gas flow (FGF) range when a second carrier gas is used (N2O or air). We examined the performance of the Maquet O2 Guard(®), a smart hypoxic guard that increases O2 delivery if an inspired hypoxic mixture is formed. After obtaining IRB approval and informed consent, 12 ASA I-II patients were enrolled. During anesthesia with sevoflurane in O2/air, the O2 Guard(®) was tested by administering O2/air at the following delivered hypoxic guard limits [expressed as (total FGF in L min(-1); FDO2 in %)] for 4 min each: [0.3;67], [0.4;50], [0.6;34], [0.8;25], [1.0;21], [1.2;21], [1.5;21], [2;21], [3;21], and [5;21]. The following data were collected: (1) time from FIO2 = 30 to 20 %; (2) time from FIO2 = 20 % to O2 Guard(®) activation; (3) time from O2 Guard(®) activation to FIO2 = 25 %; (4) FGF and FDO2 used by the O2 Guard. If SpO2 was <90 % for 10 s or longer at any time, the patient was excluded. Three patients were excluded for low SpO2. The incidence of FIO2 < 21 % was 100 % within the 1-2 L min(-1) FGF range. The O2 Guard(®) was activated within 20 s after FIO2 became 20 %, except in one patient where FIO2 oscillated between 20 and 21 %. FDO2 was increased to 60 % and FGF to 1 L min(-1) (the latter only if it was lower than 1 L min(-1) prior to activation of the O2 Guard). FIO2 increased to 25 % within 55 s after O2 Guard activation in all patients. The O2 Guard(®), an active inspired hypoxic guard, rapidly reverses and limits the duration of inspired hypoxic episodes when the delivered hypoxic guard fails to do so. PMID:25757405

  17. 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

  18. Monte Carlo radiotherapy simulations of accelerated repopulation and reoxygenation for hypoxic head and neck cancer

    PubMed Central

    Harriss-Phillips, W M; Bezak, E; Yeoh, E K

    2011-01-01

    Objective A temporal Monte Carlo tumour growth and radiotherapy effect model (HYP-RT) simulating hypoxia in head and neck cancer has been developed and used to analyse parameters influencing cell kill during conventionally fractionated radiotherapy. The model was designed to simulate individual cell division up to 108 cells, while incorporating radiobiological effects, including accelerated repopulation and reoxygenation during treatment. Method Reoxygenation of hypoxic tumours has been modelled using randomised increments of oxygen to tumour cells after each treatment fraction. The process of accelerated repopulation has been modelled by increasing the symmetrical stem cell division probability. Both phenomena were onset immediately or after a number of weeks of simulated treatment. Results The extra dose required to control (total cell kill) hypoxic vs oxic tumours was 15–25% (8–20 Gy for 5×2 Gy per week) depending on the timing of accelerated repopulation onset. Reoxygenation of hypoxic tumours resulted in resensitisation and reduction in total dose required by approximately 10%, depending on the time of onset. When modelled simultaneously, accelerated repopulation and reoxygenation affected cell kill in hypoxic tumours in a similar manner to when the phenomena were modelled individually; however, the degree was altered, with non-additive results. Simulation results were in good agreement with standard linear quadratic theory; however, differed for more complex comparisons where hypoxia, reoxygenation as well as accelerated repopulation effects were considered. Conclusion Simulations have quantitatively confirmed the need for patient individualisation in radiotherapy for hypoxic head and neck tumours, and have shown the benefits of modelling complex and dynamic processes using Monte Carlo methods. PMID:21933980

  19. 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

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

    PubMed

    Rocha-Ferreira, Eridan; Hristova, Mariya

    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

  1. 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

  2. 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. PMID:22037869

  3. Targeting constitutively-activated STAT3 in hypoxic ovarian cancer, using a novel STAT3 inhibitor

    PubMed Central

    McCann, Georgia A.; Naidu, Shan; Rath, Kellie S.; Bid, Hemant K.; Tierney, Brent J.; Suarez, Adrian; Varadharaj, Saradhadevi; Zhang, Jianying; Hideg, Kálmán; Houghton, Peter; Kuppusamy, Periannan; Cohn, David E.; Selvendiran, Karuppaiyah

    2014-01-01

    Tumor hypoxia, a feature of many solid tumors including ovarian cancer, is associated with resistance to therapies. We previously demonstrated that hypoxic exposure results in increased expression of phosphorylated signal transducer and activator of transcription 3 (pSTAT3). We hypothesized the activation of STAT3 could lead to chemotherapeutic resistance in ovarian cancer cells in hypoxic conditions. In this study, we demonstrate the level of pSTAT3 Tyr705 is increased in the hypoxic regions of human epithelial ovarian cancer (EOC) specimens, as determined by HIF-1α and CD-31 staining. In vitro mutagenesis studies proved that pSTAT3 Tyr705 is necessary for cell survival and proliferation under hypoxic conditions. In addition, we show that S1PR1, a regulator of STAT3 transcription via the JAK/STAT pathway, is highly expressed in hypoxic ovarian cancer cells (HOCCs). Knock down of S1PR1 in HOCCs reduced pSTAT3 Tyr705 levels and was associated with decreased cell survival. Treatment of HOCCs with the STAT3 inhibitor HO-3867 resulted in a rapid and dramatic decrease in pSTAT3 Tyr705 levels as a result of ubiquitin proteasome degradation. STAT3-target proteins Bcl-xL, cyclin D2 and VEGF showed similar decreases in HO-3867 treated cells. Taken together, these findings suggest that activation of STAT3 Tyr705 promotes cell survival and proliferation in HOCCs, and that S1PR1 is involved in the initiation of STAT3 activation. Targeting hypoxia-mediated STAT3 activation represents a therapeutic option for ovarian cancer and other solid tumors. PMID:25594014

  4. Study of capillary network directionality and irrigation of hypoxic tissue in an angiogenesis lattice model

    NASA Astrophysics Data System (ADS)

    Moglia, Belén; Guisoni, Nara; Albano, Ezequiel V.

    2013-12-01

    To shed light on the understanding of the angiogenesis process, we study a simplified lattice model for the capillary network formation between an existing blood vessel and an initially hypoxic tissue. We consider that the cells of the tissue surface can release growth factors that will diffuse, leading to the formation of new capillaries that ultimately arrive at the tissue. Additionally, we consider the local production of growth factors by the growing capillary network. We also propose the existence of an inhibition mechanism at the hypoxic surface, i.e., a fixed number of neighboring sites of an already irrigated site of the hypoxic tissue stop releasing growth factors due to the arrival of nutrients. Particularly, the goal of this work is to study the effect of the release of local growth factors and the inhibition mechanism on properties such as the directionality of the growing network and the irrigation of the hypoxic tissue. Therefore we propose the quantification of these two relevant features for angiogenesis modeling. We establish a relationship between the model behavior without the release of local growth factors in the presence of the inhibition mechanism and a normal angiogenesis process. In this situation, the model gives a directional capillary network and a good irrigation of the hypoxic tissue. On the other hand, for a large number of released local growth factors in the absence of the inhibition mechanism, the model could be appropriate for the description of tumor angiogenesis. In this case, the model provides a rather small directionality for the growing structure, with a worse degree of irrigation of the hypoxic tissue, as well as a more tortuous capillary network with many closed branches and loops.

  5. 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. PMID:26590349

  6. Hypoxic Ischemic Encephalopathy: Pathophysiology and Experimental Treatments

    PubMed Central

    Allen, Kimberly A.; Brandon, Debra H.

    2011-01-01

    Hypoxic ischemic encephalopathy (HIE) is a serious birth complication affecting full term infants: 40–60% of affected infants die by 2 years of age or have severe disabilities. The majority of the underlying pathologic events of HIE are a result of impaired cerebral blood flow and oxygen delivery to the brain with resulting primary and secondary energy failure. In the past, treatment options were limited to supportive medical therapy. Currently, several experimental treatments are being explored in neonates and animal models to ameliorate the effects of secondary energy failure. This review discusses the underlying pathophysiologic effects of a hypoxic-ischemic event and experimental treatment modalities being explored to manage infants with HIE. Further research is needed to better understand if the long-term impact of the experimental treatments and whether the combinations of experimental treatments can improve outcomes in infants with HIE. PMID:21927583

  7. 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. PMID:21882223

  8. 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.

  9. 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

  10. 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.

  11. Temporal Rac1 - HIF-1 crosstalk modulates hypoxic survival of aged neurons.

    PubMed

    Güntert, Tanja; Gassmann, Max; Ogunshola, Omolara O

    2016-07-01

    Neurodegenerative diseases are frequently associated with hypoxic conditions. During hypoxia the neuronal cytoskeleton is rapidly reorganized and such abnormalities are directly linked to adverse outcomes. Besides their roles as master regulators of the cytoskeleton, the Rho GTPases are also involved in cellular processes stimulated by hypoxic stress. We investigated the contribution of Rac1-mediated signaling to hypoxic responses of mature neurons using primary cortical cells cultured for 17 days in vitro. We show Rac1 is both upregulated and activated during hypoxia. Pharmacological inhibition of Rac1, but not RhoA, completely abrogated hypoxic HIF-1α stabilization and expression of the HIF-1 targets VEGF and GLUT1. Furthermore activity of JNK and GSK3β were also highly dependent on Rac1 activity and biphasic effects were observed after 6 and 24h of exposure. Notably, inhibition of either pathway suppressed HIF-1α accumulation. Although inhibition of Rac1 did not affect neuronal viability during acute exposure cell death was strongly induced after 24h revealing a time-dependent effect of Rac1 signaling on survival. Thus hypoxia-activated Rac1 is critical for neuronal HIF-1α stabilization and survival during oxygen deprivation via integration of complex signaling cascades. PMID:27018294

  12. 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

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

    PubMed

    Pamenter, Mathhew 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. © 2016 American Physiological Society. Compr Physiol 6:1345-1385, 2016. PMID:27347896

  14. Buyanghuanwu Tang therapy for neonatal rats with hypoxic ischemic encephalopathy

    PubMed Central

    Liu, Xiyao; Min, Yue; Gu, Weiwang; Wang, Yujue; Tian, Yuguang

    2015-01-01

    Background: Neonatal hypoxic-ischemic encephalopathy (HIE) is a clinical syndrome manifested by neurological symptoms in the first days of life in term infants. Purpose: To investigate the therapy effect of Buyanghuanwu Tang (BYHWT), a decoction with 7 herbal ingredients, on neonatal rats with hypoxic ischemic encephalopathy (HIE) and its mechanism. Methods: 50 3-week male Sprague-Dawley rats were divided into normal control group, model group, BYHWT 1d group, BYHWT 3d group and BYHWT 7d group, 10 rats in each group. The HIE model of was established in later 4 groups. The later 3 groups were treated with BYHWT for 1, 3 and 7 days, respectively, and the normal control group and model group were treated with PBS. The Morris water maze test and dynamic 18F-FDG-PET/CT imaging were performed. The changes of hippocampal tissue observed by histopathologic examination, and the expressions of JNK1/JNK2 and TNF-α protein were observed western blotting. Results: Compared with model group, the impaired performance on distance and latency parameters was mitigated in BYHWT 1d group, BYHWT 3d group and BYHWT 7d group (P < 0.01), the FDG uptake was decreased in BYHWT 3d group and BYHWT 7d group, the apoptotic cells and inflammatory cells were significantly decreased in BYHWT 3d group and BYHWT 7d group, and the expressions of JNK1/JNK2 and TNF-α protein were significantly decreased in BYHWT 7d group (P < 0.05). Conclusion: BYHWT can delay the HIE onset and preserve the motor function, primarily by regulating inflammation, apoptosis and inhibition by mediating JNK signaling. PMID:26770451

  15. Metabolic plasticity in CLL: adaptation to the hypoxic niche

    PubMed Central

    Koczula, K M; Ludwig, C; Hayden, R; Cronin, L; Pratt, G; Parry, H; Tennant, D; Drayson, M; Bunce, C M; Khanim, F L; Günther, U L

    2016-01-01

    Metabolic transformation in cancer is increasingly well understood. However, little is known about the metabolic responses of cancer cells that permit their survival in different microenvironments. We have used a nuclear magnetic resonance based approach to monitor metabolism in living primary chronic lymphoid leukemia (CLL) cells and to interrogate their real-time metabolic responses to hypoxia. Our studies demonstrate considerable metabolic plasticity in CLL cells. Despite being in oxygenated blood, circulating CLL cells are primed for hypoxia as measured by constitutively low level hypoxia-inducible factor (HIF-1α) activity and modest lactate production from glycolysis. Upon entry to hypoxia we observed rapid upregulation of metabolic rates. CLL cells that had adapted to hypoxia returned to the ‘primed' state when re-oxygenated and again showed the same adaptive response upon secondary exposure to hypoxia. We also observed HIF-1α independent differential utilization of pyruvate in oxygenated and hypoxic conditions. When oxygenated, CLL cells released pyruvate, but in hypoxia imported pyruvate to protect against hypoxia-associated oxidative stress. Finally, we identified a marked association of slower resting glucose and glutamine consumption, and lower alanine and lactate production with Binet A0 stage samples indicating that CLL may be divided into tumors with higher and lower metabolic states that reflect disease stage. PMID:26202928

  16. 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

  17. DW-MRI in assessment of the hypoxic fraction, interstitial fluid pressure, and metastatic propensity of melanoma xenografts

    PubMed Central

    2014-01-01

    Background Cancer patients with primary tumors showing extensive hypoxia and highly elevated interstitial fluid pressure (IFP) have poor prognosis. The potential of diffusion-weighted magnetic resonance imaging (DW-MRI) in assessing the hypoxic fraction, IFP, and metastatic propensity of tumors was investigated in this study. Methods A-07 and R-18 melanoma xenografts were used as general models of human cancer. DW-MRI was performed at 1.5 T, and maps of the apparent diffusion coefficient (ADC) were produced with in-house-made software developed in Matlab. Pimonidazole was used as a hypoxia marker. Tumor cell density and hypoxic fraction were assessed by quantitative analysis of histological sections. IFP was measured with a Millar catheter. Metastatic propensity was determined by examining tumor-bearing mice for pulmonary micrometastases post mortem. Results ADC decreased with increasing tumor cell density, independent of whether the A-07 and R-18 data were analyzed separately or together. In the A-07 line, ADC decreased with increasing hypoxic fraction and increasing IFP and was lower in metastatic than in nonmetastatic tumors, and in the R-18 line, ADC decreased with increasing hypoxic fraction. There was a strong inverse correlation between ADC and hypoxic fraction as well as between ADC and IFP across the two tumor lines, primarily because low ADC as well as high hypoxic fraction and high IFP were associated with high cell density. Conclusion Low ADC is a potentially useful biomarker of poor prognosis in cancer, since low ADC is mainly a consequence of high cell density, and high cell density may lead to increased hypoxia and interstitial hypertension and, therefore, increased microenvironment-associated metastasis. PMID:24528854

  18. Hypoxia-inducible Factor-dependent Production of Profibrotic Mediators by Hypoxic Hepatocytes

    PubMed Central

    Copple, Bryan L.; Bustamante, Juan J.; Welch, Timothy P.; Kim, Nam Deuk; Moon, Jeon-OK

    2011-01-01

    Background/Aims During the development of liver fibrosis, mediators are produced that stimulate cells in the liver to differentiate into myofibroblasts and to produce collagen. Recent studies demonstrated that the transcription factor, hypoxia-inducible factor-1α (HIF-1α), is critical for upregulation of profibrotic mediators, such as platelet-derived growth factor-A (PDGF-A), PDGF-B, and plasminogen activator inhibitor-1 (PAI-1) in the liver during the development of fibrosis. What remains unknown is the cell type-specific regulation of these genes by HIF-1α in liver cell types. Accordingly, the hypothesis was tested that HIF-1α is activated in hypoxic hepatocytes and regulates production of profibrotic mediators by these cells. Methods In this study, hepatocytes were isolated from the livers of control and HIF-1α or HIF-1β-Deficient mice and exposed to hypoxia. Results Exposure of primary mouse hepatocytes to 1% oxygen stimulated nuclear accumulation of HIF-1α and upregulated PAI-1, vascular endothelial cell growth factor, and the vasoactive peptides adrenomedullin-1 (ADM-1) and ADM-2. In contrast, levels of PDGF-A and PDGF-B mRNAs were unaffected in these cells by hypoxia. Exposure of HIF-1α-Deficient hepatocytes to 1% oxygen only partially prevented upregulation of these genes, suggesting that other hypoxia-regulated transcription factors, such as HIF-2α, may also regulate these genes. In support of this, HIF-2α was activated in hypoxic hepatocytes, and exposure of HIF-1β-Deficient hepatocytes to 1% oxygen completely prevented upregulation PAI-1, VEGF, and ADM-1, suggesting that HIF-2α may also contribute to upregulation of these genes in hypoxic hepatocytes. Conclusions Collectively, our results suggest that HIFs may be important regulators of profibrotic and vasoactive mediators by hypoxic hepatocytes. PMID:19302442

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

    PubMed

    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

  20. 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

  1. [STRESS AND INFARCT LIMITING EFFECTS OF EARLY HYPOXIC PRECONDITIONING].

    PubMed

    Lishmanov, Yu B; Maslov, L N; Sementsov, A S; Naryzhnaya, N V; Tsibulnikov, S Yu

    2015-09-01

    It was established that early hypoxic preconditioning is an adaptive state different from eustress and distress. Hypoxic preconditioning has the cross effects, increasing the tolerance of the heart to ischemia-reperfusion and providing antiulcerogenic effect during immobilization stress. PMID:26672158

  2. Prevention of experimental stroke by hypercapnic-hypoxic preconditioning.

    PubMed

    Yakushev, N N; Bespalov, A G; Kulikov, V P

    2008-09-01

    The effectiveness of hypercapnic hypoxic training in the prevention of acute disturbances in cerebral circulation was studied under experimental conditions. Hypercapnic hypoxic training was followed by a significant decrease in the severity of neurological deficit and locomotor and coordination disorders after cerebral ischemic injury. PMID:19240841

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

    PubMed Central

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

    2015-01-01

    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. PMID:26416246

  4. The role of hydrogen sulphide in the control of breathing in hypoxic zebrafish (Danio rerio)

    PubMed Central

    Porteus, Cosima S; Abdallah, Sara J; Pollack, Jacob; Kumai, Yusuke; Kwong, Raymond W M; Yew, Hong M; Milsom, William K; Perry, Steve F

    2014-01-01

    The current study investigated the role of hydrogen sulphide (H2S) in oxygen sensing, intracellular signalling and promotion of ventilatory responses to hypoxia in adult and larval zebrafish (Danio rerio). Both larval and adult zebrafish exhibited a dose-dependent increase in ventilation to sodium sulphide (Na2S), an H2S donor. In vertebrates, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) are enzymes that catalyse the endogenous production of H2S. In adult zebrafish, inhibition of both CBS and CSE with aminooxyacetate (AOA) and propargyl glycine (PPG) blunted or abolished the hypoxic hyperventilation, and the addition of Na2S to the water partially rescued the effects of inhibiting endogenous H2S production. In zebrafish larvae (4 days post-fertilization), gene knockdown of either CBS or CSE using morpholinos attenuated the hypoxic ventilatory response. Furthermore, the intracellular calcium concentration of isolated neuroepithelial cells (NECs), which are putative oxygen chemoreceptors, increased significantly when these cells were exposed to 50 μm Na2S, supporting a role for H2S in Ca2+-evoked neurotransmitter release in these cells. Finally, immunohistochemical labelling showed that NECs dissociated from adult gill contained CBS and CSE, whereas cutaneous NECs in larval zebrafish expressed only CSE. Taken together, these data show that H2S can be produced in the putative oxygen-sensing cells of zebrafish, the NECs, in which it appears to play a pivotal role in promoting the hypoxic ventilatory response. PMID:24756639

  5. Pharmacological Preconditioning of Mesenchymal Stem Cells with Trimetazidine (1-[2,3,4-Trimethoxybenzyl]piperazine) Protects Hypoxic Cells against Oxidative Stress and Enhances Recovery of Myocardial Function in Infarcted Heart through Bcl-2 Expression

    PubMed Central

    Wisel, Sheik; Khan, Mahmood; Kuppusamy, M. Lakshmi; Mohan, I. Krishna; Chacko, Simi M.; Rivera, Brian K.; Sun, Benjamin C.; Hideg, Kálmán; Kuppusamy, Periannan

    2009-01-01

    Stem cell transplantation is a possible therapeutic option to repair ischemic damage to the heart. However, it is faced with a number of challenges including the survival of the transplanted cells in the ischemic region. The present study was designed to use stem cells preconditioned with trimetazidine (1-[2,3,4-trimethoxybenzyl]piperazine; TMZ), a widely used anti-ischemic drug for treating angina in cardiac patients, to increase the rate of their survival after transplantation. Bone marrow-derived rat mesenchymal stem cells (MSCs) were subjected to a simulated host tissue environment by culturing them under hypoxia (2% O2) and using hydrogen peroxide (H2O2) to induce oxidative stress. MSCs were preconditioned with 10 μM TMZ for 6 h followed by treatment with 100 μM H2O2 for 1 h and characterized for their cellular viability and metabolic activity. The preconditioned cells showed a significant protection against H2O2-induced loss of cellular viability, membrane damage, and oxygen metabolism accompanied by a significant increase in HIF-1α, survivin, phosphorylated Akt (pAkt), and Bcl-2 protein levels and Bcl-2 gene expression. The therapeutic efficacy of the TMZ-preconditioned MSCs was evaluated in an in vivo rat model of myocardial infarction induced by permanent ligation of left anterior descending coronary artery. A significant increase in the recovery of myocardial function and up-regulation of pAkt and Bcl-2 levels were observed in hearts transplanted with TMZ-preconditioned cells. This study clearly demonstrated the potential benefits of pharmacological preconditioning of MSCs with TMZ for stem cell therapy for repairing myocardial ischemic damage. PMID:19218529

  6. Hypoxic guard systems do not prevent rapid hypoxic inspired mixture formation.

    PubMed

    De Cooman, Sofie; Schollaert, Caroline; Hendrickx, Jan F A; Peyton, Philip J; Van Zundert, Tom; De Wolf, Andre M

    2015-08-01

    Because a case report and theoretical mass balances suggested that hypoxic guard systems may not prevent the formation of hypoxic inspired mixtures (FIO2 ≤ 21 %) over the clinically used fresh gas flow (FGF) range, we measured FIO2 over a wide range of hypoxic guard limits for O2/N2O and O2/air mixtures. After IRB approval, 16 ASA I-II patients received sevoflurane in either O2/N2O (n = 8) or O2/air (n = 8) using a Zeus(®) anesthesia machine in the conventional mode. After using an 8 L/min FGF with FDO2 = 25% for 10 min, the following hypoxic guard limits were tested for 4 min each, expressed as [total FGF in L/min; FDO2 in %]: [0.3;85], [0.4;65], [0.5;50], [0.7;36], [0.85;30], [1.0;25], [1.25;25], [1.5;25], [2;25], [3;25], [5;25], and [8;25]. In between these [FGF;FDO2] combinations, 8 L/min FGF with 25% O2 was used for 4 min to return to the same baseline FIO2 (25%) before the start of the next combination. This sequence was studied once in each patient receiving O2/air (n = 8), but twice in each patient who received O2/N2O (n = 8) to examine the effect of decreasing N2O uptake over time, resulting in three groups: early O2/N2O, late O2/N2O, and O2/air group. The [FGF;FDO2]-FIO2 relationship was examined. The overall [FGF;FDO2]-FIO2 relationship in the three groups was similar. In all 1, 1.25, and 1.5 L/min FGF groups, FIO2 decreased below 21% in all but one patient; this occurred within 1 min in at least one patient. In the 0.7 L/min O2/air group and the 3 L/min late O2/N2O and O2/air groups, FIO2 decreased below 21% in one patient. Current hypoxic guard systems do not reliably prevent a hypoxic FIO2 with O2/N2O and O2/air mixtures, particularly between 0.7 and 3 L/min. PMID:25270987

  7. Extensive reproductive disruption, ovarian masculinization and aromatase suppression in Atlantic croaker in the northern Gulf of Mexico hypoxic zone

    PubMed Central

    Thomas, Peter; Rahman, Md. Saydur

    2012-01-01

    The long-term impacts on marine ecosystems of the recent dramatic worldwide increase in the incidence of coastal hypoxia are unknown. Here, we show widespread reproductive disruption in Atlantic croakers collected from hypoxic sites approximately 120 km apart in the extensive northern Gulf of Mexico continental shelf hypoxic zone. Gonadal growth and gamete production were impaired in croakers from hypoxic sites compared with fish from reference normoxic sites east of the Mississippi River Delta. Male germ cells were detected in approximately 19 per cent of croaker ovaries collected in the hypoxic region, but were absent in ovaries from normoxic sites. In addition, the sex ratio was skewed towards males at the hypoxic sites. The masculinization and other reproductive disruptions were associated with declines in neuroendocrine function, as well as ovarian and brain expression of aromatase (the enzyme that converts androgens to oestrogens). A similar incidence of ovarian masculinization and decline in ovarian aromatase expression were observed in croaker after chronic laboratory hypoxia exposure, indicating that ovarian masculinization is a specific hypoxia response and is due to decreased aromatase activity. The results suggest severe reproductive impairment can occur over large coastal regions in marine fish populations exposed to seasonal hypoxia, with potential long-term impacts on population abundance. PMID:21613294

  8. Acute Hypoxic Test in Patients with Prediabetes.

    PubMed

    Shatylo, Valerii B; Serebrovska, Tatiana V; Gavalko, Anna V; Egorov, Egor; Korkushko, Oleg V

    2016-06-01

    Shatylo, Valerii B., Tetiana V. Serebrovska, Anna V. Gavalko, Egor Egorov, and Oleg V. Korkushko. Acute hypoxic test in patients with prediabetes. High Alt Med Biol. 17:101-107, 2016.-Prediabetes is a state of impaired carbohydrate metabolism when not all of the symptoms required to label a person as diabetic are present, but blood glucose is higher than in healthy subjects. Recent evidence suggests that intermittent hypoxia training (IHT) might provide a cost-effective strategy for improving metabolic functioning. One of the most important aspects of the successful IHT application is individualized approach to hypoxic dose and regimen prescription. To establish the relationships between indices of carbohydrate metabolism and individual resistance to hypoxia, the acute hypoxic test (AHT, breathing gas mixture with 12% O2 during 20 minutes) was performed in 33 healthy volunteers (mean age, 63.0, range, 44-76; fasting plasma glucose (FPG) less than 5.6 mmol/L and 2 hours postoral glucose tolerance test (OGTT) glycemia less than 7.8 mmol/L) and 30 patients with impaired glucose metabolism (mean age, 65.5, range, 44-75; FPG from 5.6 to 6.9 mmol/L and 2 hours post-OGTT glycemia from 7.8 to 11 mmol/L). Negative correlation was found between the SaO2 level at 20th minute AHT and FPG (r = -0.83; p < 0.01) and insulin (r = -0.27; p < 0.05), as well as 2 hours post-OGTT glucose and insulin levels (r = -0.75 and -0.40, respectively). Longer recovery time and less effective functioning of respiratory and cardiovascular systems were also registered in patients with prediabetes showing that their cardiovascular resilience is impaired compared to normoglycemic controls. These patterns of relationship must be considered when assigning the individual modes of IHT. PMID:27213550

  9. Neuroimaging in Neonatal Hypoxic Ischemic Encephalopathy.

    PubMed

    Krishnan, Pradeep; Shroff, Manohar

    2016-09-01

    Magnetic resonance (MR) imaging is emerging as one of the most important tools in identifying the etiology of neonatal encephalopathy as well as in predicting long-term outcomes. This makes it imperative to have a broader understanding of normal myelination of the neonatal brain on MR imaging and to be familiar with the spectrum of imaging features in ischemic and non-ischemic neonatal encephalopathy. Hypoxic ischemic injury (HIE) is one of the most common causes of neonatal encephalopathy and imaging appearances are influenced by factors such as the stage of maturation of the neonatal brain and severity as well as duration of ischemic insult. Other common causes of neonatal encephalopathy include infectious diseases, congenital disorders and inborn errors of metabolism. PMID:26909496

  10. 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

  11. Renal kallikrein in chronic hypoxic rats.

    PubMed

    Chen, C F; Chen, L W; Chien, C T; Wu, M S; Tsai, T J

    1996-09-01

    1. We have studied the role of kallikrein (KK) in the maintenance of renal function in chronic hypoxic rats (high altitude; HA), compared with control rats kept at sea level (SL). Hypoxia was induced by placing female Wistar rats (198-290 g) in an altitude chamber (5500 m) 15 h/day for 4 weeks. Experiments were also conducted to study the interaction of KK with renal nerve activity and endothelin (ET), two parameters previously shown to be altered in this model. 2. It was found that renal cortex tissue KK activity (TKA) was not significantly different in 10 SL and 10 HA rats. However, the urinary KK activity (UKA) was reduced nearly to half (from 35.2 +/- 4.6 to 18.5 +/- 1.7 pkat/min) in HA rats after 4 weeks of chronic hypoxia. 3. Acute renal denervated diuresis was accompanied by a significant increase in UKA (from 9 +/- 2 to 14 +/- 2 pkat/min in HA and denervated HA rats, respectively; P < 0.05) in HA rats. Intrarenal arterial pretreatment of aprotinin reduced the denervated diuresis. 4. Endothelin (600 ng/kg per h) reduced urine flow, sodium and potassium excretion in the ipsilateral kidney in another 10 SL and 10 HA rats. The extent of the drop of these parameters was significantly less in HA rats. Urinary KK activity was correlated significantly with the measured renal functional parameters (r ranging from 0.472 to 0.612) in SL rats, but was insignificant in HA rats (r ranging from 0.032 to 0.192). 5. We have demonstrated that chronic exposure to hypoxia decreases urinary KK excretion and that KK is involved in acute renal denervated diuresis generated in these animals. The present study suggests that KK plays a partial role in the maintenance of renal function in chronic hypoxic rats. PMID:8911720

  12. Inhibition of TYRO3/Akt signaling participates in hypoxic injury in hippocampal neurons

    PubMed Central

    Zhu, Yan-zhen; Wang, Wei; Xian, Na; Wu, Bing

    2016-01-01

    In this study, we investigated the role of the TYRO3/Akt signaling pathway in hypoxic injury to hippocampal neurons. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that hypoxia inhibited the proliferation and viability of hippocampal neurons. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay demonstrated that hypoxia induced neuronal apoptosis in a time-dependent manner, with a greater number of apoptotic cells with longer hypoxic exposure. Immunofluorescence labeling revealed that hypoxia suppressed TYRO3 expression. Western blot assay showed that hypoxia decreased Akt phosphorylation levels in a time-dependent manner. Taken together, these findings suggest that hypoxia inhibits the proliferation of hippocampal neurons and promotes apoptosis, and that the inhibition of the TYRO3/Akt signaling pathway plays an important role in hypoxia-induced neuronal injury. PMID:27335558

  13. 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

  14. Notch1 is associated with the multidrug resistance of hypoxic osteosarcoma by regulating MRP1 gene expression.

    PubMed

    Li, C; Guo, D; Tang, B; Zhang, Y; Zhang, K; Nie, L

    2016-01-01

    Hypoxia and Notch signaling pathway are closely related and both participate in cell proliferation and drug resistance of tumors. However, the molecular mechanisms of hypoxia and Notch signaling pathway in cell proliferation and drug resistance of osteosarcoma (OS) remain unclear. In this study, to further evaluate the role of hypoxia and Notch1 on drug resistance of OS, we investigated the influence of inhibiting Notch1 pathway by Notch1 small interference RNA (siRNA) on human MG-63 OS cells in hypoxia. Our data showed that hypoxia promoted OS cell proliferation, induced the G0/G1-S-G2/M phase transition, and increased multidrug resistance of human OS cells. Western blot analysis suggested that hypoxia increased the expression of HIF-1α, Notch1, and multidrug resistance protein-1 (MRP1) in human OS cells. Notch1 siRNA inhibits proliferation and increases apoptosis of hypoxic OS cells. Finally, these hypoxic OS cells can be sensitized to multidrug treatment through inhibition of the Notch protein expression by siRNA. Repression of the Notch protein expression resulted in down-regulation of MRP1 protein. These data support the conclusion that Notch signaling is up-regulated in human OS cells under hypoxia and Notch1 may represent a viable target to overcome chemoresistant OS cells in a hypoxic niche by regulating MRP1 gene expression. PMID:27468877

  15. Hypoxic preconditioning: effect, mechanism and clinical implication (Part 1).

    PubMed

    Lu, Guo-wei; Shao, Guo

    2014-11-01

    Hypoxic preconditioning (HPC) refers to exposure of organisms, systems, organs, tissues or cells to moderate hypoxia/ischemia that is able to result in a resistance to subsequent severe hypoxia/ischemia in tissues and cells. The effects exerted by HPC are well documented. The original local in situ (LiHPC) is now broadened to remote ectopic organs-tissues (ReHPC) and extended crossly to cross pluripotential HPC(CpHPC) induced by a variety of stresses other than hypoxia/ischemia, including cancer, for example. We developed a unique animal model of repetitive autohypoxia in adult mice, and studied systematically on the effects and mechanisms of HPC on the model in our laboratory since the early 1960s. The tolerances to hypoxia and protection from injury increased significantly in this model. The adult mice behave like hypoxia-intolerant mammalian newborns and hypoxia-tolerant adult animals during their exposure to repetitive autohypoxia. The overall energy supply and demand decreased, the microorganization of the brain maintained and the spacial learning and memory ability improved but not impaired, the detrimental neurochemicals such as free radicals down-regulated and the beneficial neurochemicals such as adenosine(ADO) and antihypoxic gene(s)/factor(s) (AHGs/AHFs) up-regulated. Accordingly, we hypothesize that mechanisms for the tolerance/protective effects of HPC are fundamentally depending on energy saving and brain plasticity in particular. It is thought that these two major mechanisms are triggered by exposure to hypoxia/ischemia via oxygen sensing-transduction pathways and HIF-1 initiation cascades. We suggest that HPC is an intrinsic mechanism developed in biological evolution and is a novel potential strategy for fighting against hypoxia-ischemia and other stresses. Motivation of endogenous antihypoxic potential, activation of oxygen sensing--signal transduction systems and supplement of exogenous antihypoxic substances as well as development of HPC

  16. [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. PMID:24290154

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

    PubMed

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

    2015-12-01

    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

  18. 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 Res87: 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. PMID:26818057

  19. Individual variation in response to simulated hypoxic stress of rats.

    PubMed

    Ghosh, Dishari; Kumar, Rajesh; Pal, Karan

    2012-10-01

    With an aim to categorize the animals exposed to simulated hypobaric hypoxia and to evaluate the hormonal profile responsible for individual variation in response to hypoxic stress, degree of tolerance to hypobaric hypoxia was measured by exposing the animals to a simulated altitude of 10,668 m at 32 degrees C and animals were categorized as low and high tolerant groups based on their gasping time. The hormonal profiles of these groups were evaluated just after exposure to the test. The results showed a distinct individual difference in response to hypoxic tolerance test. There was a significant increase in plasma norepinephrine concentration in high tolerant group than low tolerant rats. After hypoxic tolerance test, total circulating corticosterone (CORT) level also increased but this was not significant in high tolerant rats as compared to low tolerant rats. Corticosteroid binding globulin (CBG) concentration differ significantly between high and low tolerant groups of rats resulting in significant changes in circulating free corticosterone that in turn may be responsible for individual differences in hypoxic gasping time. Significant differences were also observed in prolactin and testosterone levels of both the groups. The results established the method of differentiating the animals according their response to hypoxic tolerance test. These data indicate that multiple components rather than only plasma glucocorticoid of the stress response are providing a basis for individual differences in physiological responses to hypoxic stress. PMID:23214269

  20. 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

  1. Modulation of the LKB1-AMPK Signalling Pathway Underpins Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension.

    PubMed

    Evans, A Mark; Lewis, Sophronia A; Ogunbayo, Oluseye A; Moral-Sanz, Javier

    2015-01-01

    Perhaps the defining characteristic of pulmonary arteries is the process of hypoxic pulmonary vasoconstriction (HPV) which, under physiological conditions, supports ventilation-perfusion matching in the lung by diverting blood flow away from oxygen deprived areas of the lung to oxygen rich regions. However, when alveolar hypoxia is more widespread, either at altitude or with disease (e.g., cystic fibrosis), HPV may lead to hypoxic pulmonary hypertension. HPV is driven by the intrinsic response to hypoxia of pulmonary arterial smooth muscle and endothelial cells, which are acutely sensitive to relatively small changes in pO2 and have evolved to monitor oxygen supply and thus address ventilation-perfusion mismatch. There is now a consensus that the inhibition by hypoxia of mitochondrial oxidative phosphorylation represents a key step towards the induction of HPV, but the precise nature of the signalling pathway(s) engaged thereafter remains open to debate. We will consider the role of the AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1), an upstream kinase through which AMPK is intimately coupled to changes in oxygen supply via mitochondrial metabolism. A growing body of evidence, from our laboratory and others, suggests that modulation of the LKB1-AMPK signalling pathway underpins both hypoxic pulmonary vasoconstriction and the development of pulmonary hypertension. PMID:26303471

  2. 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

  3. Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling.

    PubMed

    Fediuk, Jena; Sikarwar, Anurag S; Nolette, Nora; Dakshinamurti, Shyamala

    2014-12-01

    In hypoxic pulmonary arterial (PA) myocytes, challenge with thromboxane mimetic U46619 induces marked actin polymerization and contraction, phenotypic features of persistent pulmonary hypertension of the newborn (PPHN). Rho GTPases regulate the actin cytoskeleton. We previously reported that U46619-induced actin polymerization in hypoxic PA myocytes occurs independently of the RhoA pathway and hypothesized involvement of the Cdc42 pathway. PA myocytes grown in normoxia or hypoxia for 72 h were stimulated with U46619, then analyzed for Rac/Cdc42 activation by affinity precipitation, phosphatidylinositide-3-kinase (PI3K) activity by phospho-Akt, phospho-p21-activated kinase (PAK) by immunoblot, and association of Cdc42 with neuronal Wiskott Aldrich Syndrome protein (N-WASp) by immunoprecipitation. The effect of Rac or PAK inhibition on filamentous actin was quantified by laser-scanning cytometry and by cytoskeletal fractionation; effects of actin-modifying agents were measured by isometric myography. Basal Cdc42 activity increased in hypoxia, whereas Rac activity decreased. U46619 challenge increased Cdc42 and Rac activity in hypoxic cells, independently of PI3K. Hypoxia increased phospho-PAK, unaltered by U46619. Association of Cdc42 with N-WASp decreased in hypoxia but increased after U46619 exposure. Hypoxia doubled filamentous-to-globular ratios of α- and γ-actin isoforms. Jasplakinolide stabilized γ-filaments, increasing force; cytochalasin D depolymerized all actin isoforms, decreasing force. Rac and PAK inhibition decreased filamentous actin in tissues although without decrease in force. Rho inhibition decreased myosin phosphorylation and force. Hypoxia induces actin polymerization in PA myocytes, particularly increasing filamentous α- and γ-actin, contributing to U46619-induced contraction. Hypoxic PA myocytes challenged with a thromboxane mimetic polymerize actin via the Cdc42 pathway, reflecting increased Cdc42 association with N-WASp. Mechanisms

  4. Myocardial kinetics of fluorine-18 misonidazole: A marker of hypoxic myocardium

    SciTech Connect

    Shelton, M.E.; Dence, C.S.; Hwang, D.R.; Welch, M.J.; Bergmann, S.R.

    1989-03-01

    Fluoromisonidazole, a member of a class of compounds referred to as ''hypoxic sensitizers,'' accumulates in hypoxic, viable tumor cells. We hypothesized that it might therefore accumulate also in ischemic, but non-necrotic myocardium potentially salvageable by interventional therapy. To evaluate the myocardial kinetics of (18F)fluoromisonidazole (FM), 20 isolated perfused rabbit hearts were used to characterize the uptake and binding of tracer under control conditions (n = 6), or with ischemia (flow 10% of control, n = 5), hypoxia without low flow (control flow rates with hypoxic medium, n = 5), or with reperfusion (n = 4). Myocardial retention of tracer detected externally with gamma scintillation probes after 20 min of constant (18F)FM infusion followed by 20 min of washout with nonradioactive buffer was 41 +/- 7% and 46 +/- 8% of peak activity in hearts subjected to ischemia or hypoxia, respectively, and significantly higher than in hearts subjected to either control perfusion or to ischemia followed by reperfusion (18 +/- 6 and 16 +/- 5% of peak activity, respectively, p less than 0.01). The biologic half-time of retained tracer was 40 hr in all hearts indicating essentially irreversible binding. Based on these findings, we measured uptake of (18F)FM using positron emission tomography in five dogs subjected to acute coronary occlusion. Five to thirteen millicuries of tracer were injected within 3 hr of occlusion. Within 30 min after administration of tracer, 18F accumulation in ischemic myocardium was greater than that observed in normal myocardium. The results indicate that (18F)FM accumulates in ischemic myocardium in relation to diminished tissue oxygen content and not simply because of diminished flow. Thus, this class of compounds may be potentially useful to help identify hypoxic myocardium.

  5. Hypoxic preconditioning with cobalt ameliorates hypobaric hypoxia induced pulmonary edema in rat.

    PubMed

    Shukla, Dhananjay; Saxena, Saurabh; Purushothaman, Jayamurthy; Shrivastava, Kalpana; Singh, Mrinalini; Shukla, Shirish; Malhotra, Vineet Kumar; Mustoori, Sairam; Bansal, Anju

    2011-04-10

    Exposure to high altitude results in hypobaric hypoxia which is considered as an acute physiological stress and often leads to high altitude maladies such as high altitude pulmonary edema (HAPE) and high altitude cerebral edema (HACE). The best way to prevent high altitude injuries is hypoxic preconditioning which has potential clinical usefulness and can be mimicked by cobalt chloride. Preconditioning with cobalt has been reported to provide protection in various tissues against ischemic injury. However, the effect of preconditioning with cobalt against high altitude induced pulmonary edema has not been investigated in vivo. Therefore, in the present study, rats pretreated with saline or cobalt (12.5mg/kg body weight) for 7days were exposed to hypobaric hypoxia of 9142m for 5h at 24°C. Formation of pulmonary edema was assessed by measuring transvascular leakage of sodium fluorescein dye and lung water content. Total protein content, albumin content, vascular endothelial growth factor (VEGF) and cytokine levels were measured in bronchoalveolar lavage fluid. Expression of HO-1, MT, NF-κB DNA binding activity and lung tissue pathology were evaluated to determine the effect of preconditioning on HAPE. Hypobaric hypoxia induced increase in transvascular leakage of sodium fluorescein dye, lung water content, lavage total protein, albumin, VEGF levels, pro-inflammatory cytokine levels, tissue expression of cell adhesion molecules and NF-κB DNA binding activity were reduced significantly after hypoxic preconditioning with cobalt. Expression of anti-inflammatory protein HO-1, MT, TGF-β and IL-6 were increased after hypoxic preconditioning. These data suggest that hypoxic preconditioning with cobalt has protective effect against HAPE. PMID:21296072

  6. FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions.

    PubMed

    Wu, Wenxian; Lin, Chunxia; Wu, Keng; Jiang, Lei; Wang, Xiaojing; Li, Wen; Zhuang, Haixia; Zhang, Xingliang; Chen, Hao; Li, Shupeng; Yang, Yue; Lu, Yue; Wang, Jingjing; Zhu, Runzhi; Zhang, Liangqing; Sui, Senfang; Tan, Ning; Zhao, Bin; Zhang, Jingjing; Li, Longxuan; Feng, Du

    2016-07-01

    In hypoxic cells, dysfunctional mitochondria are selectively removed by a specialized autophagic process called mitophagy. The ER-mitochondrial contact site (MAM) is essential for fission of mitochondria prior to engulfment, and the outer mitochondrial membrane protein FUNDC1 interacts with LC3 to recruit autophagosomes, but the mechanisms integrating these processes are poorly understood. Here, we describe a new pathway mediating mitochondrial fission and subsequent mitophagy under hypoxic conditions. FUNDC1 accumulates at the MAM by associating with the ER membrane protein calnexin. As mitophagy proceeds, FUNDC1/calnexin association attenuates and the exposed cytosolic loop of FUNDC1 interacts with DRP1 instead. DRP1 is thereby recruited to the MAM, and mitochondrial fission then occurs. Knockdown of FUNDC1, DRP1, or calnexin prevents fission and mitophagy under hypoxic conditions. Thus, FUNDC1 integrates mitochondrial fission and mitophagy at the interface of the MAM by working in concert with DRP1 and calnexin under hypoxic conditions in mammalian cells. PMID:27145933

  7. 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.

  8. The effects of hypoxic bradycardia and extracellular HCO3(-)/CO2 on hypoxic performance in the eel heart.

    PubMed

    Joyce, William; Simonsen, Maj; Gesser, Hans; Wang, Tobias

    2016-02-01

    During hypoxia, fishes exhibit a characteristic hypoxic bradycardia, the functional significance of which remains debated. Here, we investigated the hypothesis that hypoxic bradycardia primarily safeguards cardiac performance. In preparations from the European eel (Anguilla anguilla), a decrease in stimulation frequency from 40 to 15 beats min(-1), which replicates hypoxic bradycardia in vivo, vastly improved cardiac performance during hypoxia in vitro. As eels display dramatic shifts in extracellular HCO3(-)/CO2, we further investigated the effect this has upon hypoxic cardiac performance. Elevations from 10 mmol l(-1) HCO3(-)/1% CO2 to 40 mmol l(-1) HCO3(-)/4% CO2 had few effects on performance; however, further, but still physiologically relevant, increases to 70 mmol l(-1) HCO3(-)/7% CO2 compromised hypoxia tolerance. We revealed a four-way interaction between HCO3(-)/CO2, contraction frequency, hypoxia and performance over time, whereby the benefit of hypoxic bradycardia was most prolonged at 10 mmol l(-1) HCO3(-)/1% CO2. Together, our data suggest that hypoxic bradycardia greatly benefits cardiac performance, but its significance may be context specific. PMID:26596533

  9. Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling.

    PubMed

    Ko, Young San; Cho, Sung Jin; Park, Jinju; Choi, Yiseul; Lee, Jae-Seon; Youn, Hong-Duk; Kim, Woo Ho; Kim, Min A; Park, Jong-Wan; Lee, Byung Lan

    2016-09-01

    Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3β (GSK-3β) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3β (WT-GSK-3β) or kinase-dead mutant of GSK-3β (KD-GSK-3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3β activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3β transfectants than in WT-GSK-3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3β tumors than in WT-GSK-3β tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3β at the translational level. Our data suggest that GSK-3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway. PMID:27365055

  10. 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.

  11. HIF1A induces expression of the WASF3 metastasis-associated gene under hypoxic conditions.

    PubMed

    Ghoshal, Pushpankur; Teng, Yong; Lesoon, Leslie Ann; Cowell, John K

    2012-09-15

    The WASF3 (WAVE3) gene is an important mediator of cell motility, invasion and metastasis and is expressed at high levels in some advanced stage tumors. In our survey of breast cancer cells, we now demonstrate that exposure to hypoxic conditions increases WASF3 expression levels in MDA231, SKBR3 and MCF7 cells. The WASF3 promoter region contains HIF1A response elements (HRE). ChIP assays demonstrate that HIF1A binds to these HRE elements in the promoter region, and luciferase reporter assays using the WASF3 gene minimal promoter shows that hypoxia results in its upregulation. Phosphorylation of WASF3 is required for its ability to affect invasion and increased phosphoactivation of WASF3 is also seen in cells challenged with hypoxia. These cells also show increased motility in the scratch wound assay. Cells in which WASF3 has been knocked down show no response to hypoxia as expected, implicating the specificity of the hypoxic response to WASF3. Overall, these experiments demonstrate WASF3 is a HIF1A-regulated gene and suggests a mechanism to explain the observation of elevated expression of WASF3 in advanced stage tumors. PMID:22581642

  12. 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

  13. Chick embryos have the same pattern of hypoxic lower-brain activation as fetal mammals.

    PubMed

    Landry, Jeremy P; Hawkins, Connor; Lee, Aaron; Coté, Alexandra; Balaban, Evan; Pompeiano, Maria

    2016-01-01

    cFos expression (indicating a particular kind of neuronal activation) was examined in embryonic day (E) 18 chick embryos after exposure to 4 h of either normoxia (21% O2), modest hypoxia (15% O2), or medium hypoxia (10% O2). Eight regions of the brainstem and hypothalamus were surveyed, including seven previously shown to respond to hypoxia in late-gestation mammalian fetuses (Breen et al., 1997; Nitsos and Walker, 1999b). Hypoxia-related changes in chick embryo brain activation mirrored those found in fetal mammals with the exception of the medullary Raphe, which showed decreased hypoxic activation, compared with no change in mammals. This difference may be explained by the greater anapyrexic responses of chick embryos relative to mammalian fetuses. Activation in the A1/C1 region was examined in more detail to ascertain whether an O2-sensitive subpopulation of these cells containing heme oxygenase 2 (HMOX2) may drive hypoxic brain responses before the maturation of peripheral O2-sensing. HMOX2-positive and -negative catecholaminergic cells and interdigitating noncatecholaminergic HMOX2-positive cells all showed significant changes in cFos expression to hypoxia, with larger population responses seen in the catecholaminergic cells. Hypoxia-induced activation of lower-brain regions studied here was significantly better correlated with activation of the nucleus of the solitary tract (NTS) than with that of HMOX2-containing A1/C1 neurons. Together, these observations suggest that (1) the functional circuitry controlling prenatal brain responses to hypoxia is strongly conserved between birds and mammals, and (2) NTS neurons are a more dominant driving force for prenatal hypoxic cFos brain responses than O2-sensing A1/C1 neurons. PMID:25964066

  14. Oxygen-regulated isoforms of cytochrome c oxidase have differential effects on its nitric oxide production and on hypoxic signaling

    PubMed Central

    Castello, Pablo R.; Woo, Dong Kyun; Ball, Kerri; Wojcik, Jay; Liu, Laura; Poyton, Robert O.

    2008-01-01

    Recently, it has been reported that mitochondria possess a novel pathway for nitric oxide (NO) synthesis. This pathway is induced when cells experience hypoxia, is nitrite (NO2−)-dependent, is independent of NO synthases, and is catalyzed by cytochrome c oxidase (Cco). It has been proposed that this mitochondrially produced NO is a component of hypoxic signaling and the induction of nuclear hypoxic genes. In this study, we examine the NO2−-dependent NO production in yeast engineered to contain alternative isoforms, Va or Vb, of Cco subunit V. Previous studies have shown that these isoforms have differential effects on oxygen reduction by Cco, and that their genes (COX5a and COX5b, respectively) are inversely regulated by oxygen. Here, we find that the Vb isozyme has a higher turnover rate for NO production than the Va isozyme and that the Vb isozyme produces NO at much higher oxygen concentrations than the Va isozyme. We have also found that the hypoxic genes CYC7 and OLE1 are induced to higher levels in a strain carrying the Vb isozyme than in a strain carrying the Va isozyme. Together, these results demonstrate that the subunit V isoforms have differential effects on NO2−-dependent NO production by Cco and provide further support for a role of Cco in hypoxic signaling. These findings also suggest a positive feedback mechanism in which mitochondrially produced NO induces expression of COX5b, whose protein product then functions to enhance the ability of Cco to produce NO in hypoxic/anoxic cells. PMID:18388202

  15. Targeting radiosensitizers to DNA by attachment of an intercalating group: Nitroimidazole-linked phenanthridines

    SciTech Connect

    Cowan, D.S.; Panicucci, R.; McClelland, R.A.; Rauth, A.M. )

    1991-07-01

    The nitroimidazole-linked phenanthridine series of compounds (NLP-1, 2, and 3) were synthesized under the assumption that it should be possible to enhance the molar efficiency of 2-nitroimidazoles as hypoxic cell radiosensitizers and cytotoxins by targeting them to their likely site of action, DNA. The targeting group chosen was the phenanthridine moiety, the major component of the classical DNA intercalating compound, ethidium bromide. The sole difference between the compounds is the length of the hydrocarbon chain linking the nitroimidazole to the phenanthridine. The phenanthridine group with a three-carbon side chain, P-1, was also synthesized to allow studies on the effect of the targeting group by itself. The ability of the compounds to bind to DNA is inversely proportional to their linker chain length with binding constant values ranging from approximately 1 {times} 10(5) mol-1 for NLP-2 to 6 {times} 10(5) mol-1 for NLP-3. The NLP compounds show selective toxicity to hypoxic cells at 37 degrees C at external drug concentrations 10-40 times lower than would be required for untargeted 2-nitroimidazoles such as misonidazole in vitro. Toxicity to both hypoxic and aerobic cells is dependent on the linker chain: the shorter the chain, the greater the toxicity. In addition, the NLP compounds radiosensitize hypoxic cells at external drug concentrations as low as 0.05 mM with almost the full oxygen effect being observed at a concentration of 0.5 mM. These concentrations are 10-100 times lower than would be required for similar radiosensitization using misonidazole. Radiosensitizing ability is independent of linker chain length. The present compounds represent prototypes for further studies of the efficacy and mechanism of action of 2-nitroimidazoles targeted to DNA by linkage to an intercalating group.

  16. The effect of PCO2 on hypoxic pulmonary vasoconstriction.

    PubMed

    Noble, W H; Kay, J C; Fisher, J A

    1981-09-01

    Lung areas with a low V/Q ratio cause hypoxaemia. The low alveolar oxygen concentration may cause local hypoxic pulmonary vasoconstriction (HPV) which reduces perfusion, raises the V/Q ratio, and hence reduces the tendency to a low PaO2. By changing PCO2, the HPV response can be altered. We examined this relationship in anaesthetized dogs by using a tracheal divider to separate hypoxic (nitrogen ventilated) from oxygenated (100 per cent oxygen ventilated) lung. Relative perfusion was assessed from total 133Xe exhaled from each lung area after intravenous infusions. When PaCO2 was changed by changing ventilation, we found that an increasing PaCO2 increased HPV and also PaO2. At a PaCO2 of 3.3 kPa, HPV was abolished and PaO2 fell. We also changed PaCO2 by altering PICO2 to one or both lung areas while ventilation remained constant throughout the experiment. Again as PaCO2 increased, HPV and PaCO2 increased. When PaCO2 fell and end tidal carbon dioxide in the hypoxic lung (PETCO2) remained elevated by maintaining PICO2 in the hypoxic lung and removing CO2 from the oxygenated lung) HPV was maintained. Thus it is the alveolar concentration of CO2 in the hypoxic lung which is important in modifying HPV. We conclude that in this model a low PETCO2 (3.3 kPa) in hypoxic lung will reduce HPV, and will result in more severe hypoxaemia. This may have relevance in both anaesthetized and intensive care unit patients when a higher PaO2 may be obtained by increasing hypoxic lung PETCO2. The effect of PETCO2 on PaO2 will be influenced by other variables, but when hypoventilated or hypoxic exist, increasing PETCO2 may reinforce hypoxic pulmonary vasoconstriction and thus may increase PaO2. PMID:6793220

  17. Adaptation of the myoglobin knockout mouse to hypoxic stress.

    PubMed

    Schlieper, Georg; Kim, Jie-Hoon; Molojavyi, Andrei; Jacoby, Christoph; Laussmann, Tim; Flögel, Ulrich; Gödecke, Axel; Schrader, Jürgen

    2004-04-01

    Myoglobin knockout (myo-/-) mice were previously reported to show no obvious phenotype but revealed several compensatory mechanisms that include increases in cardiac capillary density, coronary flow, and hemoglobin. The aim of this study was to investigate whether severe hypoxic stress can exhaust these compensatory mechanisms and whether this can be monitored on the gene and protein level. Myo-/- and wild-type (WT) mice we e exposed to hypoxia (10% O(2)) fo 2 wk. Thereafter hemodynamic parameters were investigated by invasive measurement combined with magnetic resonance imaging. Cardiac gene and protein expression were analyzed using cDNA arrays and two-dimensional gel electrophoresis plus mass spectrometry, respectively. Hematocrit levels increased from 44% (WT) and 48% (myo-/-) to 72% in both groups. Similar to WT controls, hypoxic myo-/- animals maintained stable cardiovascular function (mean arterial blood pressure 82.4 mmHg, ejection fraction 72.5%). Cardiac gene expression of hypoxic myo-/- mice differed significantly from WT controls in 17 genes (e.g., keratinocyte lipid binding protein +202%, cytochrome c oxidase Vb +41%). Interestingly, hypoxia inducible factor-1alpha remained unchanged in both groups. Proteome analysis revealed reduced levels of heart fatty acid-binding protein and heat shock protein 27 both in hypoxic myo-/- and WT mice. Our data thus demonstrate that myo-/- mice do not decompensate du ing hypoxic st ess but a e surprisingly well adapted. Changes in ene gy metabolism of fatty acids may contribute to the robustness of myoglobin-deficient mice. PMID:14656764

  18. 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. PMID:25484354

  19. 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. PMID:24491563

  20. KLF5 mediates vascular remodeling via HIF-1α in hypoxic pulmonary hypertension.

    PubMed

    Li, Xiaochen; He, Yuanzhou; Xu, Yongjian; Huang, Xiaomin; Liu, Jin; Xie, Min; Liu, Xiansheng

    2016-02-15

    Hypoxic pulmonary hypertension (HPH) is characterized by active vasoconstriction and profound vascular remodeling. KLF5, a zinc-finger transcription factor, is involved in the excessive proliferation and apoptotic resistance phenotype associated with monocrotaline-induced pulmonary hypertension. However, the molecular mechanisms of KLF5-mediated pathogenesis of HPH are largely undefined. Adult male Sprague-Dawley rats were exposed to normoxia or hypoxia (10% O2) for 4 wk. Hypoxic rats developed pulmonary arterial remodeling and right ventricular hypertrophy with significantly increased right ventricular systolic pressure. The levels of KLF5 and hypoxia-inducible factor-1α (HIF-1α) were upregulated in distal pulmonary arterial smooth muscle from hypoxic rats. The knockdown of KLF5 via short-hairpin RNA attenuated chronic hypoxia-induced hemodynamic and histological changes in rats. The silencing of either KLF5 or HIF-1α prevented hypoxia-induced (5%) proliferation and migration and promoted apoptosis in human pulmonary artery smooth muscle cells. KLF5 was immunoprecipitated with HIF-1α under hypoxia and acted as an upstream regulator of HIF-1α. The cell cycle regulators cyclin B1 and cyclin D1 and apoptosis-related proteins including bax, bcl-2, survivin, caspase-3, and caspase-9, were involved in the regulation of KLF5/HIF-1α-mediated cell survival. This study demonstrated that KLF5 plays a crucial role in hypoxia-induced vascular remodeling in an HIF-1α-dependent manner and provided a better understanding of the pathogenesis of HPH. PMID:26702149

  1. Hypoxic Stress Induced by Hydralazine Leads to a Loss of Blood-Brain Barrier Integrity and an Increase in Efflux Transporter Activity

    PubMed Central

    Chatard, Morgane; Puech, Clémentine

    2016-01-01

    Understanding cellular and molecular mechanisms induced by hypoxic stress is crucial to reduce blood-brain barrier (BBB) disruption in some neurological diseases. Since the brain is a complex organ, it makes the interpretation of in vivo data difficult, so BBB studies are often investigated using in vitro models. However, the investigation of hypoxia in cellular pathways is complex with physical hypoxia because HIF-1α (factor induced by hypoxia) has a short half-life. We had set up an innovative and original method of induction of hypoxic stress by hydralazine that was more reproducible, which allowed us to study its impact on an in vitro BBB model. Our results showed that hydralazine, a mimetic agent of the hypoxia pathway, had the same effect as physical hypoxia, with few cytotoxicity effects on our cells. Hypoxic stress led to an increase of BBB permeability which corresponded to an opening of our BBB model. Study of tight junction proteins revealed that this hypoxic stress decreased ZO-1 but not occludin expression. In contrast, cells established a defence mechanism by increasing expression and activity of their efflux transporters (Pgp and MRP-1). This induction method of hypoxic stress by hydralazine is simple, reproducible, controllable and suitable to understand the cellular and molecular mechanisms involved by hypoxia on the BBB. PMID:27337093

  2. 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.

  3. 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

  4. Hypoxic tumor microenvironment: Opportunities to develop targeted therapies.

    PubMed

    Patel, Akhil; Sant, Shilpa

    2016-01-01

    In recent years, there has been great progress in the understanding of tumor biology and its surrounding microenvironment. Solid tumors create regions with low oxygen levels, generally termed as hypoxic regions. These hypoxic areas offer a tremendous opportunity to develop targeted therapies. Hypoxia is not a random by-product of the cellular milieu due to uncontrolled tumor growth; rather it is a constantly evolving participant in overall tumor growth and fate. This article reviews current trends and recent advances in drug therapies and delivery systems targeting hypoxia in the tumor microenvironment. In the first part, we give an account of important physicochemical changes and signaling pathways activated in the hypoxic microenvironment. This is then followed by various treatment strategies including hypoxia-sensitive signaling pathways and approaches to develop hypoxia-targeted drug delivery systems. PMID:27143654

  5. 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. PMID:26895650

  6. 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

  7. The role of classical transient receptor potential channels in the regulation of hypoxic pulmonary vasoconstriction.

    PubMed

    Fuchs, B; Dietrich, A; Gudermann, T; Kalwa, H; Grimminger, F; Weissmann, N

    2010-01-01

    Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism of the lung matching blood perfusion to ventilation during local alveolar hypoxia. HPV thus optimizes pulmonary gas exchange. In contrast chronic and generalized hypoxia leads to pulmonary vascular remodeling with subsequent pulmonary hypertension and right heart hypertrophy. Among other non-selective cation channels, the family of classical transient receptor potential channels (TRPC) has been shown to be expressed in pulmonary arterial smooth muscle cells. Among this family, TRPC6 is essential for the regulation of acute HPV in mice. Against this background, in this chapter we give an overview about the TRPC family and their role in HPV. PMID:20204731

  8. 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. PMID:24384982

  9. 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. PMID:19661449

  10. 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.

  11. 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.

  12. Modeling the Transcriptional Regulatory Network That Controls the Early Hypoxic Response in Candida albicans

    PubMed Central

    van het Hoog, Marco; Tebbji, Faiza; Beaurepaire, Cécile; Whiteway, Malcolm

    2014-01-01

    We determined the changes in transcriptional profiles that occur in the first hour following the transfer of Candida albicans to hypoxic growth conditions. The impressive speed of this response is not compatible with current models of fungal adaptation to hypoxia that depend on the depletion of sterol and heme. Functional analysis using Gene Set Enrichment Analysis (GSEA) identified the Sit4 phosphatase, Ccr4 mRNA deacetylase, and Sko1 transcription factor (TF) as potential regulators of the early hypoxic response. Cells mutated in these and other regulators exhibit a delay in their transcriptional responses to hypoxia. Promoter occupancy data for 29 TFs were combined with the transcriptional profiles of 3,111 in vivo target genes in a Network Component Analysis (NCA) to produce a model of the dynamic and highly interconnected TF network that controls this process. With data from the TF network obtained from a variety of sources, we generated an edge and node model that was capable of separating many of the hypoxia-upregulated and -downregulated genes. Upregulated genes are centered on Tye7, Upc2, and Mrr1, which are associated with many of the gene promoters that exhibit the strongest activations. The connectivity of the model illustrates the high redundancy of this response system and the challenges that lie in determining the individual contributions of specific TFs. Finally, treating cells with an inhibitor of the oxidative phosphorylation chain mimics most of the early hypoxic profile, which suggests that this response may be initiated by a drop in ATP production. PMID:24681685

  13. 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

  14. 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

  15. Can targeting glutamate receptors with long-term heat acclimation improve outcomes following hypoxic injury?

    PubMed Central

    Ely, Brett R; Brunt, Vienna E; Minson, Christopher T

    2015-01-01

    Long-term heat acclimation appears to improve tolerance to hypoxic insults in various tissues, including brain, providing a promising avenue to improve functional outcomes following cerebrovascular events. Glutamate discharge is implicated in dysfunction following hypoxic stress and thus, targeting glutamate receptors with heat acclimation could improve cognitive outcomes following hypoxic injury. PMID:27227003

  16. 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 ...

  17. Wnt-Responsive Cancer Stem Cells Are Located Close to Distorted Blood Vessels and Not in Hypoxic Regions in a p53-Null Mouse Model of Human Breast Cancer

    PubMed Central

    Landua, John D.; Bu, Wen; Wei, Wei; Li, Fuhai; Wong, Stephen T.C.; Dickinson, Mary E.; Rosen, Jeffrey M.; Lewis, Michael T.

    2014-01-01

    Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature. PMID:24797826

  18. Wnt-responsive cancer stem cells are located close to distorted blood vessels and not in hypoxic regions in a p53-null mouse model of human breast cancer.

    PubMed

    Vadakkan, Tegy J; Landua, John D; Bu, Wen; Wei, Wei; Li, Fuhai; Wong, Stephen T C; Dickinson, Mary E; Rosen, Jeffrey M; Lewis, Michael T; Zhang, Mei

    2014-07-01

    Cancer stem cells (CSCs, or tumor-initiating cells) may be responsible for tumor formation in many types of cancer, including breast cancer. Using high-resolution imaging techniques, we analyzed the relationship between a Wnt-responsive, CSC-enriched population and the tumor vasculature using p53-null mouse mammary tumors transduced with a lentiviral Wnt signaling reporter. Consistent with their localization in the normal mammary gland, Wnt-responsive cells in tumors were enriched in the basal/myoepithelial population and generally located in close proximity to blood vessels. The Wnt-responsive CSCs did not colocalize with the hypoxia-inducible factor 1α-positive cells in these p53-null basal-like tumors. Average vessel diameter and vessel tortuosity were increased in p53-null mouse tumors, as well as in a human tumor xenograft as compared with the normal mammary gland. The combined strategy of monitoring the fluorescently labeled CSCs and vasculature using high-resolution imaging techniques provides a unique opportunity to study the CSC and its surrounding vasculature. PMID:24797826

  19. Molecular profiling of cetuximab and bevacizumab treatment of colorectal tumours reveals perturbations in metabolic and hypoxic response pathways

    PubMed Central

    Ji, Hong; Simpson, Richard J.; Rigopoulos, Angela; Murone, Carmel; Fang, Catherine; Gong, Sylvia; O'Keefe, Graeme

    2015-01-01

    Angiogenesis and epidermal growth factor receptor (EGFR) inhibition has been shown to have anti-tumour efficacy, and enhance the therapeutic effects of cytotoxic chemotherapy in metastatic colorectal cancer. The interplay of signalling alterations and changes in metabolism and hypoxia in tumours following anti-VEGF and anti-EGFR treatment is not well understood. We aimed to explore the pharmacodynamics of cetuximab and bevacizumab treatment in human colon carcinoma tumour cells in vitro and xenograft models through proteomic profiling, molecular imaging of metabolism and hypoxia, and evaluation of therapy-induced changes in tumour cells and the tumour microenvironment. Both cetuximab and bevacizumab inhibited tumour growth in vivo, and this effect was associated with selectively perturbed glucose metabolism and reduced hypoxic volumes based on PET/MRI imaging. Global proteomic profiling of xenograft tumours (in presence of cetuximab, bevacizumab, and combination treatments) revealed alterations in proteins involved in glucose, lipid and fatty acid metabolism (e.g., GPD2, ATP5B, STAT3, FASN), as well as hypoxic regulators and vasculogenesis (e.g., ATP5B, THBS1, HSPG2). These findings correlated with western immunoblotting (xenograft lysates) and histological examination by immunohistochemistry. These results define important mechanistic insight into the dynamic changes in metabolic and hypoxic response pathways in colorectal tumours following treatment with cetuximab and bevacizumab, and highlight the ability of these therapies to selectively impact on tumour cells and extracellular microenvironment. PMID:26517691

  20. 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...

  1. Study for Germination Under Extreme Hypobaric and Hypoxic Condition

    NASA Astrophysics Data System (ADS)

    Hashimoto, H.

    2010-04-01

    In order to investigate a possibility of plant cultivation for the space agriculture on Mars, germination rate for six species of plant under extreme hypobaric and hypoxic condition was measured. As a result, seeds of Jute and Cucumber were able to germinate in six species.

  2. 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...

  3. The impact of hypofractionation on simultaneous dose-boosting to hypoxic tumor subvolumes

    SciTech Connect

    Ruggieri, Ruggero; Nahum, Alan E.

    2006-11-15

    In a previous study, the dependence of the therapeutic ratio on the number of fractions (n), including both acute and chronic hypoxia, was investigated for homogeneously irradiated tumors. The present study further develops the model to include simultaneous dose-boosting to the hypoxic tumour subvolumes. The acutely hypoxic (ah) tumor subvolume was partitioned into a large number (10{sup 2}-10{sup 3}) of oxygenation subvolumes, modelled through rectangular pO{sub 2}(t) waves all with the same frequency and fractional time spent below the hypoxic threshold, but with randomly distributed phases. Three quite different assumptions were considered for the effect of prolonged hypoxia on the radiosensitivity ({alpha}) of the chronically hypoxic (ch) clonogens, ranging from equal radiosensitivity to that of the ah-cells to an even greater radiosensitivity than that of the well-oxygenated (ox) cells. The linear-quadratic model, including tumor repopulation, intertumor {alpha}-heterogeneity, and dependence of the oxygen enhancement ratio on the dose per fraction, was adopted for tumor control probability (TCP) computation. To include a consideration of therapeutic ratio, lung irradiation was considered and the mean normalized total lung dose (NTD{sub L}) was used as a risk indicator. For those 1(fr/d){center_dot}5(d/w) schedules yielding 50% TCP with homogeneous irradiation (our reference benchmark), we estimated the gain in TCP and the corresponding NTD{sub L} from dose boosting only the ch-subvolume, both the ah- and the ch-subvolumes, or 50% of the pretreatment tumor volume without specific targeting to tumor hypoxia. For two of the three assumptions for the radiosensitivity of the ch-clonogens, dose-boosting the ch-subvolume was associated with a substantial gain in TCP, and with a trend including minima in NTD{sub L}, for severely hypofractionated schedules only, whereas when dose-boosting both the ah- and the ch-subvolumes a substantial gain in TCP was always obtained

  4. VEGF receptors mediate hypoxic remodeling of adult ovine carotid arteries.

    PubMed

    Adeoye, Olayemi O; Bouthors, Vincent; Hubbell, Margaret C; Williams, James M; Pearce, William J

    2014-10-01

    Recent studies suggest that VEGF contributes to hypoxic remodeling of arterial smooth muscle, although hypoxia produces only transient increases in VEGF that return to normoxic levels despite sustained changes in arterial structure and function. To explore how VEGF might contribute to long-term hypoxic vascular remodeling, this study explores the hypothesis that chronic hypoxia produces sustained increases in smooth muscle VEGF receptor density that mediate long-term vascular effects of hypoxia. Carotid arteries from adult sheep maintained at sea level or altitude (3,820 m) for 110 days were harvested and denuded of endothelium. VEGF levels were similar in chronically hypoxic and normoxic arteries, as determined by immunoblotting. In contrast, VEGF receptor levels were significantly increased by 107% (VEGF-R1) and 156% (VEGF-R2) in hypoxic compared with normoxic arteries. In arteries that were organ cultured 24 h with 3 nM VEGF, VEGF replicated effects of hypoxia on abundances of smooth muscle α actin (SMαA), myosin light chain kinase (MLCK), and MLC20 and the effects of hypoxia on colocalization of MLC20 with SMαA, as measured via confocal microscopy. VEGF did not replicate the effects of chronic hypoxia on colocalization of MLCK with SMαA or MLCK with MLC20, suggesting that VEGF's role in hypoxic remodeling is highly protein specific, particularly for contractile protein organization. VEGF effects in organ culture were inhibited by VEGF receptor blockers vatalinib (240 nM) and dasatinib (6.3 nM). These findings support the hypothesis that long-term upregulation of VEGF receptors help mediate sustained effects of hypoxia on the abundance and colocalization of contractile proteins in arterial smooth muscle. PMID:25038104

  5. Sex-dependent mitochondrial respiratory impairment and oxidative stress in a rat model of neonatal hypoxic-ischemic encephalopathy.

    PubMed

    Demarest, Tyler G; Schuh, Rosemary A; Waddell, Jaylyn; McKenna, Mary C; Fiskum, Gary

    2016-06-01

    Increased male susceptibility to long-term cognitive deficits is well described in clinical and experimental studies of neonatal hypoxic-ischemic encephalopathy. While cell death signaling pathways are known to be sexually dimorphic, a sex-dependent pathophysiological mechanism preceding the majority of secondary cell death has yet to be described. Mitochondrial dysfunction contributes to cell death following cerebral hypoxic-ischemia (HI). Several lines of evidence suggest that there are sex differences in the mitochondrial metabolism of adult mammals. Therefore, this study tested the hypothesis that brain mitochondrial respiratory impairment and associated oxidative stress is more severe in males than females following HI. Maximal brain mitochondrial respiration during oxidative phosphorylation was two-fold more impaired in males following HI. The endogenous antioxidant glutathione was 30% higher in the brain of sham females compared to males. Females also exhibited increased glutathione peroxidase (GPx) activity following HI injury. Conversely, males displayed a reduction in mitochondrial GPx4 protein levels and mitochondrial GPx activity. Moreover, a 3-4-fold increase in oxidative protein carbonylation was observed in the cortex, perirhinal cortex, and hippocampus of injured males, but not females. These data provide the first evidence for sex-dependent mitochondrial respiratory dysfunction and oxidative damage, which may contribute to the relative male susceptibility to adverse long-term outcomes following HI. Lower basal GSH levels, lower post-hypoxic mitochondrial glutathione peroxidase (mtGPx) activity, and mitochondrial glutathione peroxidase 4 (mtGPx4) protein levels may contribute to the susceptibility of the male brain to oxidative damage and mitochondrial dysfunction following neonatal hypoxic-ischemia (HI). Treatment of male pups with acetyl-L-carnitine (ALCAR) protects against the loss of mtGPx activity, mtGPx4 protein, and increases in protein

  6. Neurochemical correlates of cyanide-induced hypoxic neuronal damage in vitro.

    PubMed

    Sher, P K

    1988-02-01

    Neuronal cortical cell cultures obtained from fetal mice were subjected to an hypoxic insult produced by sodium cyanide (1 mM) for 24 h. Neurochemical assays were performed 13-14 days after plating on intact cells in situ to determine if there was a specific pattern of cellular dysfunction in addition to morphologic change. Ro5-4864-displaceable benzodiazepine (BDZ) binding and high-affinity [3H] beta-alanine uptake were not reduced when compared to control values. However, specific and clonazepam-displaceable BDZ binding (81 +/- 4% and 50 +/- 9% of control values, respectively), high-affinity [3H]GABA uptake (75 +/- 2%), and choline acetyltransferase activity (82 +/- 2%) were significantly lower. When the data were expressed in terms of protein content, high-affinity [3H] beta-alanine uptake was significantly increased in cyanide-exposed and magnesium-treated cultures (123 +/- 5% and 117 +/- 3%, respectively) as was R05-4864-displaceable BDZ binding (152 +/- 14%), consistent with stimulation of nonneuronal BDZ binding and increased glial neurotransmitter uptake. Moreover, pretreatment of the cultures with magnesium effectively prevented both the morphologic and neurochemical evidence of hypoxic injury. These data lend further support to the notion that the release of excitatory neurotransmitters may mediate neurotoxicity in developing brain. PMID:2834659

  7. Thromboxane receptor hyper-responsiveness in hypoxic pulmonary hypertension requires serine 324

    PubMed Central

    Santhosh, K T; Sikarwar, A S; Hinton, M; Chelikani, P; Dakshinamurti, S

    2014-01-01

    Background and Purpose Dysregulation of the thromboxane A2 (TP) receptor, resulting in agonist hypersensitivity and hyper-responsiveness, contributes to exaggerated vasoconstriction in the hypoxic pulmonary artery in neonatal persistent pulmonary hypertension. We previously reported that hypoxia inhibits TP receptor phosphorylation, causing desensitization. Hence, we examined the role of PKA-accessible serine residues in determining TP receptor affinity, using site-directed mutational analysis. Experimental Approach Vasoconstriction to a thromboxane mimetic and phosphorylation of TP receptor serine was examined in pulmonary arteries from neonatal swine with persistent pulmonary hypertension and controls. Effects of hypoxia were determined in porcine and human TP receptors. Human TPα serines at positions 324, 329 and 331 (C-terminal tail) were mutated to alanine and transiently expressed in HEK293T cells. Saturation binding and displacement kinetics of a TP antagonist and agonist were determined in porcine TP, wild-type human TPα and all TP mutants. Agonist-elicited calcium mobilization was determined for each TP mutant, in the presence of a PKA activator or inhibitor, and in hypoxic and normoxic conditions. Key Results The Ser324A mutant was insensitive to PKA activation and hypoxia, had a high affinity for agonist and increased agonist-induced calcium mobilization. Ser329A was no different from wild-type TP receptors. Ser331A was insensitive to hypoxia and PKA with a decreased agonist-mediated response. Conclusions and Implications In hypoxic pulmonary hypertension, loss of site-specific phosphorylation of the TP receptor causes agonist hyper-responsiveness. Ser324 is the primary residue phosphorylated by PKA, which regulates TP receptor-agonist interactions. Ser331 mutation confers loss of TP receptor-agonist interaction, regardless of PKA activity. PMID:24490858

  8. A model to simulate the oxygen distribution in hypoxic tumors for different vascular architectures

    SciTech Connect

    Espinoza, Ignacio; Peschke, Peter; Karger, Christian P.

    2013-08-15

    Purpose: As hypoxic cells are more resistant to photon radiation, it is desirable to obtain information about the oxygen distribution in tumors prior to the radiation treatment. Noninvasive techniques are currently not able to provide reliable oxygenation maps with sufficient spatial resolution; therefore mathematical models may help to simulate microvascular architectures and the resulting oxygen distributions in the surrounding tissue. Here, the authors present a new computer model, which uses the vascular fraction of tumor voxels, in principle measurable noninvasively in vivo, as input parameter for simulating realistic PO2 histograms in tumors, assuming certain 3D vascular architectures.Methods: Oxygen distributions were calculated by solving a reaction-diffusion equation in a reference volume using the particle strength exchange method. Different types of vessel architectures as well as different degrees of vascular heterogeneities are considered. Two types of acute hypoxia (ischemic and hypoxemic) occurring additionally to diffusion-limited (chronic) hypoxia were implemented as well.Results: No statistically significant differences were observed when comparing 2D- and 3D-vessel architectures (p > 0.79 in all cases) and highly heterogeneously distributed linear vessels show good agreement, when comparing with published experimental intervessel distance distributions and PO2 histograms. It could be shown that, if information about additional acute hypoxia is available, its contribution to the hypoxic fraction (HF) can be simulated as well. Increases of 128% and 168% in the HF were obtained when representative cases of ischemic and hypoxemic acute hypoxia, respectively, were considered in the simulations.Conclusions: The presented model is able to simulate realistic microscopic oxygen distributions in tumors assuming reasonable vessel architectures and using the vascular fraction as macroscopic input parameter. The model may be used to generate PO2 histograms

  9. BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

    PubMed

    Gabellini, Chiara; De Luca, Teresa; Trisciuoglio, Daniela; Desideri, Marianna; Di Martile, Marta; Passeri, Daniela; Candiloro, Antonio; Biffoni, Mauro; Rizzo, Maria Giulia; Orlandi, Augusto; Del Bufalo, Donatella

    2013-11-01

    Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis. PMID:23836782

  10. Hypoxia targeted carbon nanotubes as a sensitive contrast agent for photoacoustic imaging of tumors

    NASA Astrophysics Data System (ADS)

    Zanganeh, Saeid; Aguirre, Andres; Biswal, Nrusingh C.; Pavlik, Christopher; Smith, Michael B.; Alqasemi, Umar; Li, Hai; Zhu, Quing

    2011-03-01

    Development of new and efficient contrast agents is of fundamental importance to improve detection sensitivity of smaller lesions. Within the family of nanomaterials, carbon nanotubes (CNT) not only have emerged as a new alternative and efficient transporter and translocater of therapeutic molecules but also as a photoacoustic molecular imaging agent owing to its strong optical absorption in the near-infrared region. Drugs, Antibodies and nucleic acids could functionalize the CNT and prepare an appropriate system for delivering the cargos to cells and organs. In this work, we present a novel photoacoustic contrast agent which is based on a unique hypoxic marker in the near infrared region, 2-nitroimidazole -bis carboxylic acid derivative of Indocyanine Green conjugated to single walled carbon nanotube (SWCNT-2nitroimidazole-ICG). The 2-nitroimidazole-ICG has an absorption peak at 755 nm and an extinction coefficient of 20,5222 M-1cm-1. The conjugation of this marker with SWCNT shows more than 25 times enhancement of optical absorption of carbon nanotubes in the near infrared region. This new conjugate has been optically evaluated and shows promising results for high contrast photoacoustic imaging of deeply located tumors. The conjugate specifically targets tumor hypoxia, an important indicator of tumor metabolism and tumor therapeutic response. The detection sensitivity of the new contrast agent has been evaluated in-vitro cell lines and with in-vivo tumors in mice.

  11. Hypoxic acidemia, hyperviscosity, and maternal hypertension do not affect the umbilical arterial velocity waveform in fetal sheep.

    PubMed

    Morrow, R J; Adamson, S L; Bull, S B; Ritchie, J W

    1990-10-01

    The effect of hypoxic acidemia, hyperviscosity, and maternal hypertension on the umbilical arterial velocity waveform was studied in 23 chronically catheterized fetal sheep. Fetal hypoxic acidemia induced by lowering the maternal inspired oxygen concentration (n = 7) caused no change in the ratio of systolic/diastolic blood velocity even when fetal arterial pH was as low as 6.8. Fetal blood hyperviscosity (n = 7) induced by exchange transfusion with packed maternal blood cells increased placental vascular resistance by greater than or equal to 50% but had no significant effect on the systolic/diastolic ratio. Similarly, maternal hypertension induced by intravenous infusion of angiotensin II to the ewe (n = 9) did not affect the systolic/diastolic ratio despite a 50% increase in maternal arterial blood pressure. We conclude that umbilical arterial velocity waveform abnormalities observed in growth-restricted human fetuses are probably not a direct result of fetal hypoxemia or hyperviscosity or maternal hypertension. PMID:2220943

  12. An inducible autoregulatory loop between HIPK2 and Siah2 at the apex of the hypoxic response.

    PubMed

    Calzado, Marco A; de la Vega, Laureano; Möller, Andreas; Bowtell, David D L; Schmitz, M Lienhard

    2009-01-01

    Oxygen deprivation (hypoxia) results in reprogrammed gene expression patterns that induce multifaceted cellular responses. Here we identify a regulated interaction between the serine/threonine kinase HIPK2 and the ubiquitin E3 ligase Siah2 as a mechanism controlling the hypoxic response. Under normoxic conditions, several mechanisms ensure HIPK2 stability: only a fraction of HIPK2 is found in association with Siah2, whereas HIPK2-mediated phosphorylation of this E3 ligase at positions 26, 28 and 68 weakens mutual binding and destabilizes its phosphorylated interaction partner. Hypoxic conditions allow a markedly increased HIPK2/Siah2 interaction and result in efficient polyubiquitylation and proteasomal degradation of the kinase. Accordingly, hypoxia-induced HIPK2 elimination is markedly reduced in Siah2-deficient cells. As HIPK2 has an important role as a negative regulator of gene expression, its elimination from promoter-associated repressor complexes allows the induction of a substantial fraction of hypoxia-induced genes. PMID:19043406

  13. Evaluation of Nonpeptidic Ligand Conjugates for SPECT Imaging of Hypoxic and Carbonic Anhydrase IX-Expressing Cancers.

    PubMed

    Lv, Peng-Cheng; Putt, Karson S; Low, Philip S

    2016-07-20

    As tumors grow, vasculature is often deficient or malformed, resulting in many localized areas of hypoxia. Cells located in these hypoxic regions exhibit an altered gene expression pattern that can significantly alter resistance to conventional anticancer treatments such as ionizing radiation and chemotherapeutic drugs. A priori knowledge of the level of hypoxia within a tumor may better guide clinical care. In an effort to create a hypoxia specific imaging agent, a ligand for the tissue hypoxia marker, carbonic anhydrase IX (CA IX), was synthesized and used as a targeting ligand to deliver an attached (99m)Tc-chelating agent. Binding of the resulting conjugates to hypoxic cancer cells was first characterized in vitro. Whole animal imaging and biodistribution studies then were performed to determine tumor specificity in vivo. Several conjugates were found to bind selectively to CA IX expressing tumors in a receptor-dependent manner. We suggest that such conjugates could prove useful in identifying hypoxic cancers and/or quantitating the level of hypoxia within a tumor. PMID:27362480

  14. 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.

  15. Pentoxifylline Alleviates Perinatal Hypoxic-Ischemia-Induced Short-term Memory Impairment by Suppressing Apoptosis in the Hippocampus of Rat Pups

    PubMed Central

    2016-01-01

    Purpose: Perinatal hypoxic-ischemic brain damage is a major cause of acute mortality and chronic neurologic morbidity in infants and children. We investigated the effects of pentoxifylline, a methylxanthine derivative and type-4 phosphodiesterase inhibitor, on short-term memory and apoptotic neuronal cell death in the hippocampus following perinatal hypoxic-ischemia in newborn rats. Methods: We used a step-down avoidance task to evaluate short-term memory and 3ʹ-5ʹ-cyclic adenosine monophosphate (cAMP) assay to detect cAMP levels. We evaluated apoptosis using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for evidence of DNA fragmentation, immunohistochemistry for caspase-3 levels, and western blot for Bcl-2 and Bax. Results: Perinatal hypoxic-ischemic injury increased apoptotic cell death in the hippocampus, resulting in impaired short-term memory with decreased cAMP levels. Pentoxifylline treatment improved short-term memory by suppressing apoptotic cell death in the hippocampus with elevated cAMP levels. Conclusions: Pentoxifylline ameliorated perinatal hypoxic-ischemia in rat pups. This alleviating effect could be ascribed to the inhibition apoptosis due to increased cAMP production by pentoxifylline. PMID:27377942

  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. Prolonged hypothermia exposure diminishes neuroprotection for severe ischemic-hypoxic primary neurons.

    PubMed

    Gao, Xiao-Ya; Zhu, Shu-Zhen; Xiang, Wei; Huang, Kai-Bin; Hu, Ya-Fang; Gu, Yong; Pan, Su-Yue

    2016-04-01

    This study aimed to identify optimal mild hypothermic (MH) condition that would provide the best protection for neuronal cells undergoing severe ischemia and hypoxia. We also sought to determine if longer exposure to mild hypothermia would confer greater protection to severe ischemia and hypoxia in these cells. We designed a primary neuronal cell model for severe glucose and oxygen deprivation/reoxygenation (OGD/R) to simulate the hypoxic-ischemic condition of patients with severe stroke, trauma, or hypoxic-ischemic encephalopathy. We evaluated the viability of these neurons following 3 h of OGD/R and variable MH conditions including different temperatures and durations of OGD/R exposure. We further explored the effects of the optimal MH condition on several parts which are associated with mitochondrial apoptosis pathway: intracellular calcium, reactive oxygen species (ROS), and mitochondrial transmembrane potential (MTP). The results of this study showed that the apoptosis proportion (AP) and cell viability proportion (CVP) after OGD/R significantly varied depending on which MH condition cells were exposed to (p < 0.001). Further, our findings showed that prolonged MH reduced the neuroprotection to AP and CVP. We also determined that the optimal MH conditions (34 °C for 4.5 h) reduced intracellular calcium, ROS, and recovered MTP. These findings indicate that there is an optimal MH treatment strategy for severely hypoxia-ischemic neurons, prolonged duration might diminish the neuroprotection, and that MH treatment likely initiates neuroprotection by inhibiting the mitochondrial apoptosis pathway. PMID:26802735

  18. Influence of a high-altitude hypoxic environment on human plasma microRNA profiles.

    PubMed

    Yan, Yan; Shi, Yonghui; Wang, Cheng; Guo, Pengtao; Wang, Junjun; Zhang, Chen-Yu; Zhang, Chunni

    2015-01-01

    Circulating microRNAs (miRNAs) are promising disease biomarkers. However, the influence of high-altitude hypoxic environments on plasma miRNA profiles remains unknown. This study included a total of 509 plasma samples from 278 native Tibetans and 80 newly arrived migrant Han Chinese (Tibet Han) residing at 3560 m and 151 Han Chinese residing at 8.9 m (Nanjing Han). The levels of 754 miRNAs were initially determined using a TaqMan Low Density Array (TLDA) in two pooled samples from 50 Tibet Han and 50 Nanjing Han individuals. Some markedly altered miRNAs in Tibet Han were subsequently measured in all 509 plasma samples by individual qRT-PCR. Compared with the Nanjing Han, 172 miRNAs were differentially expressed in the Tibet Han (105 upregulated and 67 downregulated). The correlation coefficient for the two groups was 0.72. Several upregulated miRNAs were randomly selected for analysis by qRT-PCR, and the results were consistent with those identified by TLDA. These miRNAs were also significantly increased in the Tibetans compared with the Nanjing Han. Furthermore, these altered miRNAs showed strong positive correlations with red blood cell counts and hemoglobin values. These data are the first to provide clear evidence that a high-altitude hypoxic environment significantly affects human plasma miRNA profiles. PMID:26468998

  19. Influence of a high-altitude hypoxic environment on human plasma microRNA profiles

    PubMed Central

    Yan, Yan; Shi, Yonghui; Wang, Cheng; Guo, Pengtao; Wang, Junjun; Zhang, Chen-Yu; Zhang, Chunni

    2015-01-01

    Circulating microRNAs (miRNAs) are promising disease biomarkers. However, the influence of high-altitude hypoxic environments on plasma miRNA profiles remains unknown. This study included a total of 509 plasma samples from 278 native Tibetans and 80 newly arrived migrant Han Chinese (Tibet Han) residing at 3560 m and 151 Han Chinese residing at 8.9 m (Nanjing Han). The levels of 754 miRNAs were initially determined using a TaqMan Low Density Array (TLDA) in two pooled samples from 50 Tibet Han and 50 Nanjing Han individuals. Some markedly altered miRNAs in Tibet Han were subsequently measured in all 509 plasma samples by individual qRT-PCR. Compared with the Nanjing Han, 172 miRNAs were differentially expressed in the Tibet Han (105 upregulated and 67 downregulated). The correlation coefficient for the two groups was 0.72. Several upregulated miRNAs were randomly selected for analysis by qRT-PCR, and the results were consistent with those identified by TLDA. These miRNAs were also significantly increased in the Tibetans compared with the Nanjing Han. Furthermore, these altered miRNAs showed strong positive correlations with red blood cell counts and hemoglobin values. These data are the first to provide clear evidence that a high-altitude hypoxic environment significantly affects human plasma miRNA profiles. PMID:26468998

  20. Simple radiosensitizing of hypoxic tumor tissues by N2O/Br(-) mixture.

    PubMed

    Billik, P

    2015-07-01

    The radiosensitization model of hypoxic tumor tissues based on the N2O/Br(-) mixture is described. The well-documented radiolysis of water in the presence of N2O and Br(-) ions at a low concentration supports this model. An aqueous solution saturated with N2O gas during the radiolysis generates OH radicals in a large extent. In N2O/Br- media at pH<9, Br2 is formed. Br2 hydrolyzes in an aqueous solution to form a very reactive hypobromous (HOBr) acid. Such process is described by the following chemical reaction: H2O + Br(-) + N2O + ionizing radiation (IR) --> HOBr + OH(-). In vivo formed HOBr as a long-lived product with a high biological activity induces the hypoxic tumor cell damage via many unique mechanisms. A local application or inhalation of an N2O-O2 mixture before or during the radiotherapy to enhance the saturation of tissues with N2O is a key prerequisite. Since the extracellular concentration of Br(-) ions is very low (0.02-0.05 mM), an oral or local application of NaBr should be used to shift the extracellular concentration of Br(-) ions to the mM region. PMID:25812437

  1. 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

  2. Hypoxic and ischemic hypoxia exacerbate brain injury associated with metabolic encephalopathy in laboratory animals.

    PubMed Central

    Vexler, Z S; Ayus, J C; Roberts, T P; Fraser, C L; Kucharczyk, J; Arieff, A I

    1994-01-01

    Hypoxemia is a major comorbid factor for permanent brain damage in several metabolic encephalopathies. To determine whether hypoxia impairs brain adaptation to hyponatremia, worsening brain edema, we performed in vitro and in vivo studies in cats and rats with hyponatremia plus either ischemic or hypoxic hypoxia. Mortality with hypoxic hypoxia was 0%; with hyponatremia, 22%; and with hyponatremia+hypoxia, 100%. Hyponatremia in cats produced brain edema, with a compensatory decrease of brain sodium. Ischemic hypoxia also resulted in brain edema, but with elevation of brain sodium. However, when ischemic hypoxia was superimposed upon hyponatremia, there was elevation of brain sodium with further elevation of water. Outward sodium transport in cat cerebral cortex synaptosomes was measured via three major pathways through which brain osmolality can be decreased. After hyponatremia, sodium transport was significantly altered such that brain cell osmolality would decrease: 44% increase in Na(+)-K(+)-ATPase transport activity (ouabain inhibitable); 26% decrease in amiloride-sensitive sodium uptake. The change in veratridine-stimulated sodium uptake was not significant (P > 0.05). When ischemic hypoxia was superimposed upon hyponatremia, all of the cerebral adaptive changes induced by hyponatremia alone were eliminated. Thus, hypoxia combined with hyponatremia produces a major increase in brain edema and mortality, probably by eliminating the compensatory mechanisms of sodium transport initiated by hyponatremia that tend to minimize brain swelling. Images PMID:8282795

  3. 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

  4. Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection.

    PubMed

    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

    2015-12-01

    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

  5. 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

  6. The Hypoxic Testicle: Physiology and Pathophysiology

    PubMed Central

    Reyes, Juan G.; Farias, Jorge G.; Henríquez-Olavarrieta, Sebastián; Madrid, Eva; Parraga, Mario; Zepeda, Andrea B.; Moreno, Ricardo D.

    2012-01-01

    Mammalian spermatogenesis is a complex biological process occurring in the seminiferous tubules in the testis. This process represents a delicate balance between cell proliferation, differentiation, and apoptosis. In most mammals, the testicles are kept in the scrotum 2 to 7°C below body core temperature, and the spermatogenic process proceeds with a blood and oxygen supply that is fairly independent of changes in other vascular beds in the body. Despite this apparently well-controlled local environment, pathologies such as varicocele or testicular torsion and environmental exposure to low oxygen (hypoxia) can result in changes in blood flow, nutrients, and oxygen supply along with an increased local temperature that may induce adverse effects on Leydig cell function and spermatogenesis. These conditions may lead to male subfertility or infertility. Our literature analyses and our own results suggest that conditions such as germ cell apoptosis and DNA damage are common features in hypoxia and varicocele and testicular torsion. Furthermore, oxidative damage seems to be present in these conditions during the initiation stages of germ cell damage and apoptosis. Other mechanisms like membrane-bound metalloproteinases and phospholipase A2 activation could also be part of the pathophysiological consequences of testicular hypoxia. PMID:23056665

  7. [Progress on Hypoxic-ischemic Brain Damage Associated with CCR2 and CCL2].

    PubMed

    Luo, Yu-jia; Li, Ru-bo; Ma, Shi-yu; Lü, Meng-yan

    2016-02-01

    Hypoxic-ischemic brain damage (HIBD) is referred to a common type of cerebral damage, which is caused by injury, leading to shallow bleeding in the cortex with intact cerebral pia mater. In recent years, studies show that a various kinds of immune cells and immune cellular factors are involved in the occurrence of HIBD. CC chemokine receptor 2 (CCR2) is a representative of CC chemokine receptor, and is widely distributed in cerebral neuron, astrocyte, and microglial cells, and is the main chemo-tactic factor receptor in brain tissue. CC chemokine ligand 2 (CCL2) is a kind of basophilic protein and the ligand of CCR2, and plays an important role in inflammation. In order to provide evidence for correlational studies in HIBD, this review will introduce the biological characteristics of CCR2 and CCL2, and illustrate the relationship between the immunoreactivity and HIBD. PMID:27295859

  8. Perfusion of nonventilated lung: failure of hypoxic vasoconstriction

    SciTech Connect

    Sostman, H.D.; Neumann, R.D.; Gottschalk, A.; Greenspan, R.H.

    1983-07-01

    Alveolar hypoxia is a well established cause of regional vasoconstriction such that nonventilated segments are not perfused. The paradoxical situation of retained perfusion of nonventilated lung has seldom been discussed. Three clinical examples are illustrated. In each case coexistent chronic obstructive lung disease may have contributed to this unexpected finding by reducing pulmonary vascular capacity such that blood flow diversion from hypoxic segments was not possible.

  9. The Syndrome of Delayed Post-Hypoxic Leukoencephalopathy

    PubMed Central

    Shprecher, David; Mehta, Lahar

    2010-01-01

    Delayed post-hypoxic leukoencephalopathy (DPHL) is a demyelinating syndrome characterized by acute onset of neuropsychiatric symptoms days to weeks following apparent recovery from coma after a period of prolonged cerebral hypo-oxygenation. It is diagnosed, after excluding other potential causes of delirium, with a clinical history of carbon monoxide poisoning, narcotic overdose, myocardial infarction, or another global cerebral hypoxic event. The diagnosis can be supported by neuroimaging evidence of diffuse hemispheric demyelination sparing cerebellar and brainstem tracts, or by an elevated cerebrospinal fluid myelin basic protein. Standard or hyperbaric oxygen following CO poisoning may reduce the likelihood of DPHL or other neurologic sequelae. Bed rest and avoidance of stressful procedures for the first 10 days following any prolonged hypoxic event may also lower the risk. Gradual recovery over a 3 to 12 month period is common, but impaired attention or executive function, parkinsonism, or corticospinal tract signs can persist. Stimulants, amantadine or levodopa may be considered for lasting cognitive or parkinsonian symptoms. Anticipation and recognition of DPHL should lead to earlier and more appropriate utilization of health care services. PMID:20166270

  10. Cerebral blood flow autoregulation during intracranial hypertension in hypoxic lambs

    SciTech Connect

    Borel, C.O.; Backofen, J.E.; Koehler, R.C.; Jones, M.D. Jr.; Traystman, R.J. )

    1987-12-01

    The authors tested the hypothesis that hypoxic hypoxia interferes with cerebral blood flow (CBF) autoregulation when intracranial pressure (ICP) is elevated in pentobarbital-anesthetized lambs (3 to 9 days old). Cerebral perfusion pressure (CPP) was lowered stepwise from 73 to 23 mmHg in eight normoxic lambs and from 65 to 31 mmHg in eight other hypoxic lambs by ventricular infusion of artificial cerebrospinal fluid. In normoxic lambs, CBF measured by microspheres labeled with six different radioisotopes was not significantly changed over this range of CPP. In animals made hypoxic, base-line CBF was twice that of normoxic lambs. CBF was unchanged as CPP was reduced to 31 mmHg. Lower levels of CPP were not attained because a pressor response occurred with further elevations of ICP. No regional decrements in blood flow to cortical arterial watershed areas or to more caudal regions, such as cerebellum, brain stem, or thalamus, were detected with elevated ICP. Cerebral O{sub 2} uptake was similar in both groups and did not decrease when CPP was reduced. These results demonstrate that normoxic lambs have a considerable capacity for effective autoregulation of CBF when ICP is elevated. Moreover, cerebral vasodilation in response to a level of hypoxia approximating that normally seen prenatally does not abolish CBF autoregulation when ICP is elevated during the first postnatal week.

  11. 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

  12. Ventilatory effects of substance P-saporin lesions in the nucleus tractus solitarii of chronically hypoxic rats

    PubMed Central

    Fu, Zhenxing; Powell, Frank L.

    2011-01-01

    During ventilatory acclimatization to hypoxia (VAH), time-dependent increases in ventilation lower Pco2 levels, and this persists on return to normoxia. We hypothesized that plasticity in the caudal nucleus tractus solitarii (NTS) contributes to VAH, as the NTS receives the first synapse from the carotid body chemoreceptor afferents and also contains CO2-sensitive neurons. We lesioned cells in the caudal NTS containing the neurokinin-1 receptor by microinjecting the neurotoxin saporin conjugated to substance P and measured ventilatory responses in awake, unrestrained rats 18 days later. Lesions did not affect hypoxic or hypercapnic ventilatory responses in normoxic control rats, in contrast to published reports for similar lesions in other central chemosensitive areas. Also, lesions did not affect the hypercapnic ventilatory response in chronically hypoxic rats (inspired Po2 = 90 Torr for 7 days). These results suggest functional differences between central chemoreceptor sites. However, lesions significantly increased ventilation in normoxia or acute hypoxia in chronically hypoxic rats. Hence, chronic hypoxia increases an inhibitory effect of neurokinin-1 receptor neurons in the NTS on ventilatory drive, indicating that these neurons contribute to plasticity during chronic hypoxia, although such plasticity does not explain VAH. PMID:21593425

  13. Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

    PubMed Central

    Weissmann, Norbert; Dietrich, Alexander; Fuchs, Beate; Kalwa, Hermann; Ay, Mahmut; Dumitrascu, Rio; Olschewski, Andrea; Storch, Ursula; Mederos y Schnitzler, Michael; Ghofrani, Hossein Ardeschir; Schermuly, Ralph Theo; Pinkenburg, Olaf; Seeger, Werner; Grimminger, Friedrich; Gudermann, Thomas

    2006-01-01

    Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6−/− mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6−/− but not in WT mice. This effect was mirrored by a lack of hypoxia-induced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6−/− mice. In both WT and TRPC6−/− PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6−/− PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange. PMID:17142322

  14. Phosphorescence Monitoring of Hypoxic Microenvironment in Solid-Tumors to Evaluate Chemotherapeutic Effects Using the Hypoxia-Sensitive Iridium (III) Coordination Compound

    PubMed Central

    Shang, Jin; Ma, Jingwen; Wang, Rong; Deng, Lei; Guo, Youmin; Zhong, Fan; Bai, Mingfeng; Zhang, Shaojuan; Wu, Daocheng

    2015-01-01

    Objectives To utilize phosphorescence to monitor hypoxic microenvironment in solid-tumors and investigate cancer chemotherapeutic effects in vivo. Methods A hypoxia-sensitive probe named BTP was used to monitor hypoxic microenvironment in solid-tumors. The low-dose metronomic treatment with cisplatin was used in anti-angiogenetic chemotherapeutic programs. The phosphorescence properties of BTP were detected by a spectrofluorometer. BTP cytotoxicity utilized cell necrosis and apoptosis, which were evaluated by trypan blue dye exclusion and Hoechst33342 plus propidium iodide assays. Tumor-bearing mouse models of colon adenocarcinoma were used for tumor imaging in vivo. Monitoring of the hypoxic microenvironment in tumors was performed with a Maestro 2 fluorescence imaging system. Tumor tissues in each group were harvested regularly and treated with pathological hematoxylin and eosin and immunohistochemical staining to confirm imaging results. Results BTP did not feature obvious cytotoxicity for cells, and tumor growth in low-dose metronomic cisplatin treated mice was significantly inhibited by chemotherapy. Hypoxic levels significantly increased due to cisplatin, as proven by the expression level of related proteins. Phosphorescence intensity in the tumors of mice in the cisplatin group was stronger and showed higher contrast than that in tumors of saline treated mice. Conclusions We develop a useful phosphorescence method to evaluate the chemotherapeutic effects of cisplatin. The proposed method shows potential as a phosphorescence imaging approach for evaluating chemotherapeutic effects in vivo, especially anti-angiogenesis. PMID:25786221

  15. Patterning hypoxic multicellular spheroids in a 3D matrix - a promising method for anti-tumor drug screening.

    PubMed

    Ma, Jingyun; Zhang, Xu; Liu, Yang; Yu, Haibo; Liu, Lianqing; Shi, Yang; Li, Yanfeng; Qin, Jianhua

    2016-01-01

    3D multicellular spheroid models are of great value in the investigation of tumor biology and tumor responses to chemotherapy and radiation. To establish a mimicking tumor microenvironment in vitro, we developed a straightforward method by patterning hypoxic multicellular spheroids in a 3D matrix. The efficacy of this approach was evaluated by characterizing spheroid formation, invasive capability and phenotypic transition in aggressive human glioma cells. We observed enhanced cell proliferation, spheroid formation and invasive capability in U87 glioma cells transfected with hypoxia-inducible factors (HIFs) compared with non-treated cells. We also demonstrated that the overexpression of HIFs in hypoxic glioma cells may promote cell migration by epithelial-mesenchymal transition within the 3D matrix. Compared with conventional 3D culturing techniques, the simple operation, rapid prototyping, low cost and high throughput format of the micro-patterning method facilitates the characterization of cell proliferation, migration, phenotypic function and drug evaluation in physiologically relevant 3D microenvironments. This in vitro 3D system can recapitulate the physiologically relevant tumor microenvironment and is a promising method for 3D anti-tumor drug screening and the identification of novel targets for tumor invasion and angiogenesis. PMID:26647062

  16. 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

    2016-08-01

    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. PMID:27505672

  17. 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

  18. Blood-retinal barrier in hypoxic ischaemic conditions: basic concepts, clinical features and management.

    PubMed

    Kaur, C; Foulds, W S; Ling, E A

    2008-11-01

    The blood-retinal barrier (BRB) plays an important role in the homeostatic regulation of the microenvironment in the retina. It consists of inner and outer components, the inner BRB (iBRB) being formed by the tight junctions between neighbouring retinal capillary endothelial cells and the outer barrier (oBRB) by tight junctions between retinal pigment epithelial cells. Astrocytes, Müller cells and pericytes contribute to the proper functioning of the iBRB. In many clinically important conditions including diabetic retinopathy, ischaemic central retinal vein occlusion, and some respiratory diseases, retinal hypoxia results in a breakdown of the iBRB. Disruption of the iBRB associated with increased vascular permeability, results in vasogenic oedema and tissue damage, with consequent adverse effects upon vision. Factors such as enhanced production of vascular endothelial growth factor (VEGF), NO, oxidative stress and inflammation underlie the increased permeability of the iBRB and inhibition of these factors is beneficial. Experimental studies in our laboratory have shown melatonin to be a protective agent for the iBRB in hypoxic conditions. Although oBRB breakdown can occur in conditions such as accelerated hypertension and the toxaemia of pregnancy, both of which are associated with choroidal ischaemia and in age-related macular degeneration (ARMD), and is a feature of exudative (serous) retinal detachment, our studies have shown that the oBRB remains intact in hypoxic/ischaemic conditions. Clinically, anti-VEGF therapy has been shown to improve vision in diabetic maculopathy and in neovascular ARMD. The visual benefit in both conditions appears to arise from the restoration of BRB integrity with a reduction of retinal oedema. PMID:18940262

  19. Metabolic reprogramming and inflammation act in concert to control vascular remodeling in hypoxic pulmonary hypertension.

    PubMed

    Stenmark, Kurt R; Tuder, Rubin M; El Kasmi, Karim C

    2015-11-15

    Pulmonary hypertension (PH) is a complex, multifactorial syndrome that remains poorly understood despite decades of research. PH is characterized by profound pulmonary artery (PA) remodeling that includes significant fibro-proliferative and inflammatory changes of the PA adventitia. In line with the emerging concept that PH shares key features with cancer, recent work centers on the idea that PH results from a multistep process driven by reprogramming of gene-expression patterns that govern changes in cell metabolism, inflammation, and proliferation. Data demonstrate that in addition to PA endothelial cells and smooth muscle cells, adventitial fibroblasts from animals with experimental hypoxic PH and from humans with PH (hereafter, termed PH-Fibs) exhibit proinflammatory activation, increased proliferation, and apoptosis resistance, all in the context of metabolic reprogramming to aerobic glycolysis. PH-Fibs can also recruit, retain, and activate naïve macrophages (Mϕ) toward a proinflammatory/proremodeling phenotype through secretion of chemokines, cytokines, and glycolytic metabolites, among which IL-6 and lactate play key roles. Furthermore, these fibroblast-activated Mϕ (hereafter, termed FAMϕ) exhibit aerobic glycolysis together with high expression of arginase 1, Vegfa, and I1lb, all of which require hypoxia-inducible factor 1α and STAT3 signaling. Strikingly, in situ, the adventitial Mϕ phenotype in the remodeled PA closely resembles the Mϕ phenotype induced by fibroblasts in vitro (FAMϕ), suggesting that FAMϕ crosstalk involving metabolic and inflammatory signals is a critical, pathogenetic component of vascular remodeling. This review discusses metabolic and inflammatory changes in fibroblasts and Mϕ in PH with the goal of raising ideas about new interventions to abrogate remodeling in hypoxic forms of PH. PMID:25930027

  20. 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

  1. 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

  2. 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

    2016-05-01

    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

  3. [The hypoxic syndrome in children with cardiomyopathy].

    PubMed

    Yurieva, E A; Sukhorukov, V S; Vozdvijenskaia, E S; Novikova, N M; Kharabadze, M N; Yablonskaia, M I

    2015-02-01

    The children with inherited cardiopathy including hypersensitive (n = 85) and dilatation (n=10) cardiopathy as well as cardiopathy under Ehlers-Danlos Syndrome (n = 70) combined with different inherited heart disease were examined to establish signs of hematic and tissue hypoxia. The most typical signs turned out periodic decrease of blood pCO2 with increasing of content of lactate and pyruvate in blood and saliva, multiple caries of teeth and high rate of systemic hypoplasia of enamel of both temporary and permanent teeth. The study established decrease in blood of level of macro-ergic compounds (ATP, ADF AMP) with increasing of excretion calcium and phosphates with urine. The increase of rate of mutations of hypoxanthine guanine phosphoribosyltransferase in lymphocytes with increasing of content of uric acid in blood and/or in urine was detected. The study revealed increasing of processes of peroxide oxidation, alterations of morphology of cells of skeletal muscles (RRF) and accumulation ofcalcium, lipids and alteration of structure of mitochondria. PMID:26027255

  4. The Regenerative Potential of Female Skeletal Muscle upon Hypobaric Hypoxic Exposure

    PubMed Central

    Mancinelli, Rosa; Di Filippo, Ester S.; Verratti, Vittore; Fulle, Stefania; Toniolo, Luana; Reggiani, Carlo; Pietrangelo, Tiziana

    2016-01-01

    Aim: The aim of this study was to determine whether a 14-day trekking expeditions, in high altitude hypoxic environment, triggers redox disturbance at the level of satellite cells (adult stem cells) in young women. Methods: We collected muscle biopsies from Vastus Lateralis muscle for both single fiber analysis and satellite cells isolation. The samples collected before (PRE-Hypoxia) and after (POST-Hypoxia) the trekking in the Himalayas were compared. Satellite cells were investigated for oxidative stress (oxidant production, antioxidant enzyme activity, and lipid damage), mitochondrial potential variation, gene profile of HIF, and myogenic transcription factors (Pax7, MyoD, myogenin), and miRNA expression (miR-1, miR-133, miR-206). Results: The nuclear domain analysis showed a significant fusion and consequent reduction of the Pax7+ satellite cells in the single mature fibers. The POST-Hypoxia myoblasts obtained by two out of six volunteers showed high superoxide anion production and lipid peroxidation along with impaired dismutase and catalase and mitochondrial potential. The transcription profile and miRNA expression were different for oxidized and non-oxidized cells. Conclusions: The present study supports the phenomenon of hypobaric-hypoxia-induced oxidative stress and its role in the impairment of the regenerative capacity of satellite cells derived from the V. Lateralis muscle of young adult female subjects. PMID:27471475

  5. 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

  6. 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

  7. 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

  8. Cytoprotection against Hypoxic and/or MPP⁺ Injury: Effect of δ-Opioid Receptor Activation on Caspase 3.

    PubMed

    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

  9. Targeting the hypoxic response in bone tissue engineering: A balance between supply and consumption to improve bone regeneration.

    PubMed

    Stiers, Pieter-Jan; van Gastel, Nick; Carmeliet, Geert

    2016-09-01

    Bone tissue engineering is a promising therapeutic alternative for bone grafting of large skeletal defects. It generally comprises an ex vivo engineered combination of a carrier structure, stem/progenitor cells and growth factors. However, the success of these regenerative implants largely depends on how well implanted cells will adapt to the hostile and hypoxic host environment they encounter after implantation. In this review, we will discuss how hypoxia signalling may be used to improve bone regeneration in a tissue-engineered construct. First, hypoxia signalling induces angiogenesis which increases the survival of the implanted cells as well as stimulates bone formation. Second, hypoxia signalling has also angiogenesis-independent effects on mesenchymal cells in vitro, offering exciting new possibilities to improve tissue-engineered bone regeneration in vivo. In addition, studies in other fields have shown that benefits of modulating hypoxia signalling include enhanced cell survival, proliferation and differentiation, culminating in a more potent regenerative implant. Finally, the stimulation of endochondral bone formation as a physiological pathway to circumvent the harmful effects of hypoxia will be briefly touched upon. Thus, angiogenic dependent and independent processes may counteract the deleterious hypoxic effects and we will discuss several therapeutic strategies that may be combined to withstand the hypoxia upon implantation and improve bone regeneration. PMID:26768117

  10. 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

  11. Hypoxic regulation of glycosylation via the N-acetylglucosamine cycle

    PubMed Central

    Shirato, Ken; Nakajima, Kazuki; Korekane, Hiroaki; Takamatsu, Shinji; Gao, Congxiao; Angata, Takashi; Ohtsubo, Kazuaki; Taniguchi, Naoyuki

    2011-01-01

    Glucose is an energy substrate, as well as the primary source of nucleotide sugars, which are utilized as donor substrates in protein glycosylation. Appropriate glycosylation is necessary to maintain the stability of protein, and is also important in the localization and trafficking of proteins. The dysregulation of glycosylation results in the development of a variety of disorders, such as cancer, diabetes mellitus and emphysema. Glycosylation is kinetically regulated by dynamically changing the portfolio of glycosyltransferases, nucleotide sugars, and nucleotide sugar transporters, which together form a part of what is currently referred to as the ”Glycan cycle”. An excess or a deficiency in the expression of glycosyltransferases has been shown to alter the glycosylation pattern, which subsequently leads to the onset, progression and exacerbation of a number of diseases. Furthermore, alterations in intracellular nucleotide sugar levels can also modulate glycosylation patterns. It is observed that pathological hypoxic microenvironments frequently occur in solid cancers and inflammatory foci. Hypoxic conditions dramatically change gene expression profiles, by activating hypoxia-inducible factor-1, which mediates adaptive cellular responses. Hypoxia-induced glycosyltransferases and nucleotide suga