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-resistant hypoxic cancer cells, and could serve as a safe and effective adjuvant to radiation therapy for patients with GBM. PMID:25350400

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

PubMed

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

2015-01-01

Honey is known as a popular healing agent against tropical infections and wounds. However, the effects of honey dilutions on keratinocyte (HaCaT) wound healing under hypoxic condition is still not explored. In this study, we examined whether honey dilution have wound healing potential under hypoxic stress. The antioxidant potential and healing efficacy of honey dilution on in vitro wound of human epidermal keratinocyte (HaCaT cells) under hypoxia (3% O2 ), and normoxia is explored by nitro blue tetrazolium assay. The cell survival % quantified by MTT assay to select four honey dilutions like 10, 1, 0.1, and 0.01 v/v% and the changes in cellular function was observed microscopically. Further, the cell proliferation, migration, cell-cell adhesion, and relevant gene expression were studied by flow cytometry, migration/scratch assay, immunocytochemistry, and reverse transcription-polymerase chain reaction, respectively. The expression pattern of cardinal molecular features viz. E-cadherin, cytoskeletal protein F-actin, p63, and hypoxia marker Hif 1α were examined. Honey dilution in 0.1% v/v combat wound healing limitations in vitro under normoxia and hypoxia (3%). Its wound healing potential was quantified by immunocytochemistry and real-time PCR for the associated molecular features that were responsible for cell proliferation and migration. Our data showed that honey dilution can be effective in hypoxic wound healing. Additionally, it reduced superoxide generation and supplied favorable bioambience for cell proliferation, migration, and differentiation during hypoxic wound healing. These findings may reveal the importance of honey as an alternative and cost effective therapeutic natural product for wound healing in hypoxic condition. © 2015 by the Wound Healing Society.

Multiscale Model of Mycobacterium tuberculosis Infection Maps Metabolite and Gene Perturbations to Granuloma Sterilization Predictions

PubMed Central

Pienaar, Elsje; Matern, William M.; Linderman, Jennifer J.

2016-01-01

Granulomas are a hallmark of tuberculosis. Inside granulomas, the pathogen Mycobacterium tuberculosis may enter a metabolically inactive state that is less susceptible to antibiotics. Understanding M. tuberculosis metabolism within granulomas could contribute to reducing the lengthy treatment required for tuberculosis and provide additional targets for new drugs. Two key adaptations of M. tuberculosis are a nonreplicating phenotype and accumulation of lipid inclusions in response to hypoxic conditions. To explore how these adaptations influence granuloma-scale outcomes in vivo, we present a multiscale in silico model of granuloma formation in tuberculosis. The model comprises host immunity, M. tuberculosis metabolism, M. tuberculosis growth adaptation to hypoxia, and nutrient diffusion. We calibrated our model to in vivo data from nonhuman primates and rabbits and apply the model to predict M. tuberculosis population dynamics and heterogeneity within granulomas. We found that bacterial populations are highly dynamic throughout infection in response to changing oxygen levels and host immunity pressures. Our results indicate that a nonreplicating phenotype, but not lipid inclusion formation, is important for long-term M. tuberculosis survival in granulomas. We used virtual M. tuberculosis knockouts to predict the impact of both metabolic enzyme inhibitors and metabolic pathways exploited to overcome inhibition. Results indicate that knockouts whose growth rates are below ∼66% of the wild-type growth rate in a culture medium featuring lipid as the only carbon source are unable to sustain infections in granulomas. By mapping metabolite- and gene-scale perturbations to granuloma-scale outcomes and predicting mechanisms of sterilization, our method provides a powerful tool for hypothesis testing and guiding experimental searches for novel antituberculosis interventions. PMID:26975995

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

PubMed

Rosignoli, Carine; Thibaut de Ménonville, Séverine; Orfila, Danielle; Béal, Méline; Bertino, Béatrice; Aubert, Jérôme; Mercenier, Annick; Piwnica, David

2018-04-01

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Radiosensitization of Hypoxic Tumor Cells by Depletion of Intracellular Glutathione

NASA Astrophysics Data System (ADS)

Bump, Edward A.; Yu, Ning Y.; Brown, J. Martin

1982-08-01

Depletion of glutathione in Chinese hamster ovary cells in vitro by diethyl maleate resulted in enhancement of the effect of x-rays on cell survival under hypoxic conditions but not under oxygenated conditions. Hypoxic EMT6 tumor cells were similarly sensitized in vivo. The action of diethyl maleate is synergistic with the effect of the electron-affinic radiosensitizer misonidazole, suggesting that the effectiveness of misonidazole in cancer radiotherapy may be improved by combining it with drugs that deplete intracellular glutathione.

Radiosensitization of hypoxic tumor cells by depletion of intracellular glutathione

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bump, E.A.; Yu, N.Y.; Brown, J.M.

1982-08-06

Depletion of glutathione in Chinese hamster ovary cells in vitro by diethyl maleate resulted in enhancement of the effect of x-rays on cell survival under hypoxic conditions but not under oxygenated conditions. Hypoxic EMT6 tumor cells were similarly sensitized in vivo. The action of diethyl maleate is synergistic with the effect of the electron-affinic radiosensitizer misonidazole, suggesting that the effectiveness of misonidazole in cancer radiotherapy may be improved by combining it with drugs that deplete intracellular glutathione.

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. © 2016. Published by The Company of Biologists Ltd.

Radiosensitization of paclitaxel, etanidazole and paclitaxel+etanidazole nanoparticles on hypoxic human tumor cells in vitro.

PubMed

Jin, Cheng; Bai, Ling; Wu, Hong; Tian, Furong; Guo, Guozhen

2007-09-01

Paclitaxel and etanidazole are hypoxic radiosensitizers that exhibit cytotoxic action at different mechanisms. The poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles containing paclitaxel, etanidazole and paclitaxel+etanidazole were prepared by o/w and w/o/w emulsification-solvent evaporation method. The morphology of the nanoparticles was investigated by scanning electron microscope (SEM). The drug encapsulation efficiency (EE) and release profile in vitro were measured by high-performance liquid chromatography (HPLC). The cellular uptake of nanoparticles for the human breast carcinoma cells (MCF-7) and the human carcinoma cervicis cells (HeLa) was evaluated by transmission electronic microscopy and fluorescence microscopy. Cell viability was determined by the ability of single cell to form colonies in vitro. The prepared nanoparticles were spherical shape with size between 80 and 150 nm. The EE was higher for paclitaxel and lower for etanidazole. The drug release was controlled over time. The cellular uptake of nanoparticles was observed. Co-culture of the two tumor cell lines with drug-loaded nanoparticles demonstrated that released drug effectively sensitized hypoxic tumor cells to radiation. The radiosensitization of paclitaxel+etanidazole nanoparticles was more significant than that of single drug-loaded nanoparticles.

Oxidative Phosphorylation as a Target Space for Tuberculosis: Success, Caution, and Future Directions.

PubMed

Cook, Gregory M; Hards, Kiel; Dunn, Elyse; Heikal, Adam; Nakatani, Yoshio; Greening, Chris; Crick, Dean C; Fontes, Fabio L; Pethe, Kevin; Hasenoehrl, Erik; Berney, Michael

2017-06-01

The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, have inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis and the opportunities for drug discovery.

A limited innate immune response is induced by a replication-defective herpes simplex virus vector following delivery to the murine central nervous system

PubMed Central

Zeier, Zane; Aguilar, J Santiago; Lopez, Cecilia M; Devi-Rao, G B; Watson, Zachary L; Baker, Henry V; Wagner, Edward K; Bloom, David C

2010-01-01

Herpes simplex virus type 1 (HSV-1)–based vectors readily transduce neurons and have a large payload capacity, making them particularly amenable to gene therapy applications within the central nervous system (CNS). Because aspects of the host responses to HSV-1 vectors in the CNS are largely unknown, we compared the host response of a nonreplicating HSV-1 vector to that of a replication-competent HSV-1 virus using microarray analysis. In parallel, HSV-1 gene expression was tracked using HSV-specific oligonucleotide-based arrays in order to correlate viral gene expression with observed changes in host response. Microarray analysis was performed following stereotactic injection into the right hippocampal formation of mice with either a replication-competent HSV-1 or a nonreplicating recombinant of HSV-1, lacking the ICP4 gene (ICP4−). Genes that demonstrated a significant change (P < .001) in expression in response to the replicating HSV-1 outnumbered those that changed in response to mock or nonreplicating vector by approximately 3-fold. Pathway analysis revealed that both the replicating and nonreplicating vectors induced robust antigen presentation but only mild interferon, chemokine, and cytokine signaling responses. The ICP4− vector was restricted in several of the Toll-like receptor-signaling pathways, indicating reduced stimulation of the innate immune response. These array analyses suggest that although the nonreplicating vector induces detectable activation of immune response pathways, the number and magnitude of the induced response is dramatically restricted compared to the replicating vector, and with the exception of antigen presentation, host gene expression induced by the non-replicating vector largely resembles mock infection. PMID:20095947

Effects of neural stem cell media on hypoxic injury in rat hippocampal slice cultures.

PubMed

Lee, Na Mi; Chae, Soo Ahn; Lee, Hong Jun

2017-12-15

Neonatal hypoxic-ischemic brain injuries cause serious neurological sequelae, yet there is currently no effective treatment for them. We hypothesized that neurotrophic factors released into the medium by stem cells could supply hypoxia-damaged organotypic hippocampal slice cultures with regenerative abilities. We prepared organotypic slice cultures of the hippocampus of 7-day-old Sprague-Dawley rats based on the modified Stoppini method; slices were cultured for 14days in vitro using either Gahwiler's medium (G-medium) or stem cell-conditioned medium (S-medium) as culture medium. At day 14 in vitro, hippocampal slice cultures were exposed to 95% N 2 and 5% CO 2 for 3h to induce hypoxic damage, the extent of which was then measured using propidium iodide fluorescence and immunohistochemistry images. We performed dot blotting to estimate neurotrophic/growth factor levels in the G- and S-media. Organotypic hippocampal slices cultured using S-medium after hypoxic injury were significantly less damaged than those cultured using G-medium. GLUT1, NGF, GDNF, VEGF, GCSF, and IGF2 levels were higher in S-medium than in G-medium, whereas FGF1, HIF, and MCP3 levels were not significantly different between media. In conclusion, we found that stem cell-conditioned medium had a neuroprotective effect against hypoxic injury, and that, of the various neurotrophic factors in S-medium, NGF, GDNF, and VEGF can contribute to neuroprotection. Copyright © 2017 Elsevier B.V. All rights reserved.

A Hypoxia-Targeted Boron Neutron Capture Therapy Agent for the Treatment of Glioma.

PubMed

Luderer, Micah John; Muz, Barbara; de la Puente, Pilar; Chavalmane, Sanmathi; Kapoor, Vaishali; Marcelo, Raymundo; Biswas, Pratim; Thotala, Dinesh; Rogers, Buck; Azab, Abdel Kareem

2016-10-01

Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells. B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo. B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively. B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on hypoxic tumor metabolism. Further studies are warranted to evaluate B-381 and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma.

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

PubMed Central

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

2015-01-01

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

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

PubMed

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

2016-04-01

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

Stationary phase gene expression of Mycobacterium tuberculosis following a progressive nutrient depletion: a model for persistent organisms?,

PubMed Central

Hampshire, Tobias; Soneji, Shamit; Bacon, Joanna; James, Brian W.; Hinds, Jason; Laing, Ken; Stabler, Richard A; Marsh, Philip D.; Butcher, Philip D

2011-01-01

Summary The majority of individuals infected with TB develop a latent infection, in which organisms survive within the body while evading the host immune system. Such persistent bacilli are capable of surviving several months of combinatorial antibiotic treatment. Evidence suggests that stationary phase bacteria adapt to increase their tolerance to environmental stresses. We have developed a unique in vitro model of dormancy based on the characterization of a single, large volume fermenter culture of M. tuberculosis, as it adapts to stationary phase. Cells are maintained in controlled and defined aerobic conditions (50% dissolved oxygen tension), using probes that measure dissolved oxygen tension, temperature, and pH. Microarray analysis has been used in conjunction with viability and nutrient depletion assays to dissect differential gene expression. Following exponential phase growth the gradual depletion of glucose/glycerol resulted in a small population of survivors that were characterized for periods in excess of 100 days. Bacilli adapting to nutrient depletion displayed characteristics associated with persistence in vivo, including entry into a non-replicative state and the up-regulation of genes involved in β-oxidation of fatty acids and virulence. A reduced population of non-replicating bacilli went on to adapt sufficiently to re-initiate cellular division. PMID:15207492

Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response

PubMed Central

Grant, Sarah Schmidt; Hung, Deborah T.

2013-01-01

Certain bacterial pathogens are able to evade the host immune system and persist within the human host. The consequences of persistent bacterial infections potentially include increased morbidity and mortality from the infection itself as well as an increased risk of dissemination of disease. Eradication of persistent infections is difficult, often requiring prolonged or repeated courses of antibiotics. During persistent infections, a population or subpopulation of bacteria exists that is refractory to traditional antibiotics, possibly in a non-replicating or metabolically altered state. This review highlights the clinical significance of persistent infections and discusses different in vitro models used to investigate the altered physiology of bacteria during persistent infections. We specifically focus on recent work establishing increased protection against oxidative stress as a key element of the altered physiologic state across different in vitro models and pathogens. PMID:23563389

Effects of turpentine-induced inflammation on the hypoxic stimulation of intestinal Fe3+ absorption in mice.

PubMed Central

Raja, K. B.; Duane, P.; Peters, T. J.

1990-01-01

Chronic subcutaneous turpentine administration (weekly for 6 weeks) induced a mild normocytic anaemia in mice. In-vitro and in-vivo intestinal Fe3+ absorption parameters were, however, not significantly altered from values in saline-treated or untreated mice. Normal mice, when exposed to 3 days hypoxia demonstrated a 2-3-fold increase in iron absorption in vivo, mainly due to changes in the amount of iron transferred from the mucosa to the plasma and thence to the carcass. A 2-3-fold increase in Vmax was also observed in in-vitro uptake experiments using isolated duodenal fragments. In contrast, turpentine-treated animals, though demonstrating an enhanced in-vitro maximal uptake capacity, failed to elicit an adaptive response in vivo following hypoxic exposure. These findings suggest that a circulating (humoral) factor may be responsible for the inhibition in absorption in vivo in this turpentine-induced inflammatory model. PMID:2278822

Susceptibility Profiles of Amphotericin B and Posaconazole against Clinically Relevant Mucorales Species under Hypoxic Conditions

PubMed Central

Maurer, Elisabeth; Sparber, Manuela; Lackner, Michaela; Caramalho, Rita; Lass-Flörl, Cornelia

2014-01-01

The effect of hypoxic conditions on the in vitro efficacy of amphotericin B and posaconazole against Mucorales was evaluated by defining MICs with Etest and broth microdilution and identifying minimal fungicidal concentrations (MFCs). With Etest, oxygen-dependent changes were detected, while the MIC and the MFC determined with broth microdilution remained unaltered with reduced oxygen levels. The observed differences depended on the method used. PMID:25451049

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes

PubMed Central

Santhosh, KT; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, AJ; Dakshinamurti, S

2011-01-01

BACKGROUND AND PURPOSE Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor–mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS TP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. PMID:21385177

Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi*

PubMed Central

Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina

2017-01-01

Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types. PMID:27852749

A Hypoxia-Targeted Boron Neutron Capture Therapy Agent for the Treatment of Glioma

PubMed Central

Luderer, Micah John; Muz, Barbara; de la Puente, Pilar; Chavalmane, Sanmathi; Kapoor, Vaishali; Marcelo, Raymundo; Biswas, Pratim; Thotala, Dinesh; Rogers, Buck; Azab, Abdel Kareem

2016-01-01

Purpose Boron neutron capture therapy (BNCT) has the potential to become a viable cancer treatment modality, but its clinical translation has been limited by the poor tumor selectivity of agents. To address this unmet need, a boronated 2-nitroimidazole derivative (B-381) was synthesized and evaluated for its capability of targeting hypoxic glioma cells. Methods B-381 has been synthesized from a 1-step reaction. Using D54 and U87 glioma cell lines, the in vitro cytotoxicity and cellular accumulation of B-381 has been evaluated under normoxic and hypoxic conditions compared to L-boronophenylalanine (BPA). Furthermore, tumor retention of B-381 was evaluated in vivo. Results B-381 had low cytotoxicity in normal and cancer cells. Unlike BPA, B-381 illustrated preferential retention in hypoxic glioma cells compared to normoxic glioma cells and normal tissues in vitro. In vivo, B-381 illustrated significantly higher long-term tumor retention compared to BPA, with 9.5-fold and 6.5-fold higher boron levels at 24 and 48 h, respectively. Conclusions B-381 represents a new class of BNCT agents in which their selectivity to tumors is based on tumor hypoxic metabolism, and further studies are warranted to evaluate this compound and similar compounds as preclinical candidates for future BNCT clinical trials for the treatment of glioma. PMID:27401411

Enhancement of nitrosourea cytotoxicity by misonidazole in vitro: correlation with carbamoylating potential.

PubMed Central

Mulcahy, R. T.; Dembs, N. L.; Ublacker, G. A.

1984-01-01

We have investigated the relationships between nitrosourea structure and physicochemical properties and the ability of misonidazole (MISO) to potentiate nitrosourea cytotoxicity in an in vitro model system. EMT-6/Ro tumour cells were exposed in suspension to each of 9 different nitrosourea anti-tumour drugs under hypoxic and aerobic culture conditions. Additional cultures were similarly treated with nitrosoureas in the presence of 1.0 mM MISO. Seven of the 9 nitrosoureas did not demonstrate any selective toxicity toward aerobic or hypoxic cells. In contrast, chlorozotocin (CHLZ) was slightly more toxic toward hypoxic cells while Bis-OH CyNU more effectively killed aerobic cells. The addition of MISO to the drug treatment enhanced the effectiveness of all the nitrosoureas under hypoxic conditions, with the exception of CHLZ which was uninfluenced by MISO. The magnitude of the MISO dose enhancement factor (DEF, defined as the ratio of drug doses required to reduce cell survival to S = 10(-3) in 4 hours in the absence and presence of 1.0 mM MISO) for each combination was examined as a function of the relative carbamoylating or alkylating activity of the nitrosourea included in that combination. Such an analysis revealed a significant (P less than 0.05) positive correlation between relative carbamoylating potency and DEF. No significant (P greater than 0.20) relationship could be established for DEF and alkylating activity. PMID:6704305

FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state.

PubMed

Masaki, Yukiko; Shimizu, Yoichi; Yoshioka, Takeshi; Nishijima, Ken-Ichi; Zhao, Songji; Higashino, Kenichi; Numata, Yoshito; Tamaki, Nagara; Kuge, Yuji

2017-10-01

18 F-fluoromisonidazole (FMISO), a well-known PET imaging probe for diagnosis of hypoxia, is believed to accumulate in hypoxic cells via covalent binding with macromolecules after reduction of the nitro group. Previously, we showed the majority of 18 F-FMISO was incorporated into low-molecular-weight metabolites in hypoxic tumors, and the glutathione conjugate of reduced FMISO (amino-FMISO-GS) distributed in the tumor hypoxic regions as revealed by imaging mass spectrometry (IMS). The present study was conducted to clarify whether FMISO is metabolized to amino-FMISO-GS within tumor cells and how amino-FMISO-GS contributes to FMISO accumulation in hypoxic cells. We also evaluated the relationship between FMISO accumulation and the glutathione conjugation-related factors in the cells. Tumor cells (FaDu, LOVO, and T24) were treated with 18 F-FMISO and incubated under normoxic or hypoxic conditions for 4 h. The FMISO metabolites were analyzed with LC-ESI-MS. Several glutathione conjugation-related factors of tumor cells were evaluated in vitro. FaDu tumor-bearing mice were intravenously injected with 18 F-FMISO and the tumors were excised at 4 h post-injection. Autoradiography, IMS and histologic studies were performed. Amino-FMISO-GS was the main contributor to FMISO incorporated in hypoxic FaDu cells in vitro and in vivo. Total FMISO uptake levels and amino-FMISO-GS levels were highest in FaDu, followed by LOVO, and then T24 (total uptake: 0.851 ± 0.009 (FaDu), 0.617 ± 0.021 (LOVO) and 0.167 ± 0.006 (T24) % dose/mg protein; amino-FMISO-GS: 0.502 ± 0.035 (FaDu), 0.158 ± 0.013 (LOVO), and 0.007 ± 0.001 (T24) % dose/mg protein). The glutathione level of FaDu was significantly higher than those of LOVO and T24. The enzyme activity of glutathione-S-transferase catalyzing the glutathione conjugation reaction in FaDu was similar levels to that in LOVO, and was higher than that in T24. Quantitative RT-PCR analysis revealed that the expression levels of efflux transporters of the glutathione conjugate (multidrug resistance-associated protein 1) were lowest in FaDu, followed by LOVO, and then T24. FMISO accumulates in hypoxic cells through reductive metabolism followed by glutathione conjugation. We illustrated the possibility that increased production and decreased excretion of amino-FMISO-GS contribute to FMISO accumulation in tumor cells under hypoxic conditions.

Drug Discovery & Development: State-of-the-Art and Future Directions” on the topic of “Targets”

PubMed Central

Cook, Gregory M.; Hards, Kiel; Dunn, Elyse; Heikal, Adam; Nakatani, Yoshio; Greening, Chris; Crick, Dean C.; Fontes, Fabio L.; Pethe, Kevin; Hasenoehrl, Erik; Berney, Michael

2017-01-01

Chapter summary The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, has inspired scientists to seek out new targets for drug development. The Mycobacterium tuberculosis complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth. M. tuberculosis is an obligately aerobic heterotroph that depends on oxidative phosphorylation (OXPHOS) for growth and survival. However, several studies are redefining the metabolic breadth of the genus. Alternative electron donors and acceptors may provide the maintenance energy for the pathogen to maintain viability in hypoxic, nonreplicating states relevant to latent infection. This hidden metabolic flexibility may ultimately decrease the efficacy of drugs targeted against primary dehydrogenases and terminal oxidases. However, it may also open up opportunities to develop novel antimycobacterials targeting persister cells. In this review, we discuss the progress in understanding the role of energetic targets in mycobacterial physiology and pathogenesis, and the opportunities for drug discovery. PMID:28597820

Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

PubMed

Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

2011-07-01

Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

Susceptibility profiles of amphotericin B and posaconazole against clinically relevant mucorales species under hypoxic conditions.

PubMed

Maurer, Elisabeth; Binder, Ulrike; Sparber, Manuela; Lackner, Michaela; Caramalho, Rita; Lass-Flörl, Cornelia

2015-02-01

The effect of hypoxic conditions on the in vitro efficacy of amphotericin B and posaconazole against Mucorales was evaluated by defining MICs with Etest and broth microdilution and identifying minimal fungicidal concentrations (MFCs). With Etest, oxygen-dependent changes were detected, while the MIC and the MFC determined with broth microdilution remained unaltered with reduced oxygen levels. The observed differences depended on the method used. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Ultrasonic Estimation of Mechanical Properties of Pulmonary Arterial Wall Under Normoxic and Hypoxic Conditions

NASA Astrophysics Data System (ADS)

Waters, Kendall R.; Mukdadi, Osama M.

2005-04-01

Secondary pediatric pulmonary hypertension is a disease that could benefit from improved ultrasonic diagnostic techniques. We perform high-frequency in vitro ultrasound measurements (25 MHz to 100 MHz) on fresh and fixed pulmonary arterial walls excised from normoxic and hypoxic Long-Evans rat models. Estimates of the elastic stiffness coefficients are determined from measurements of the speed of sound. Preliminary results indicate that hypoxia leads to up to increase of 20 % in stiffening of the pulmonary arterial wall.

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

PubMed

Udartseva, Olga O; Lobanova, Margarita V; Andreeva, Elena R; Buravkov, Sergey V; Ogneva, Irina V; Buravkova, Ludmila B

2016-01-01

The ability of mesenchymal stromal (stem) cells (MSCs) to be mobilised from their local depot towards sites of injury and to participate in tissue repair makes these cells promising candidates for cell therapy. Physiological O 2 tension in an MSC niche in vivo is about 4-7%. However, most in vitro studies of MSC functional activity are performed at 20% O 2 . Therefore, this study focused on the effects of short-term hypoxic stress (0.1% O 2 , 24 h) on adipose tissue-derived MSC motility at tissue-related O 2 level. No significant changes in integrin expression were detected after short-term hypoxic stress. However, O 2 deprivation provoked vimentin disassembly and actin polymerisation and increased cell stiffness. In addition, hypoxic stress induced the downregulation of ACTR3, DSTN, MACF1, MID1, MYPT1, NCK1, ROCK1, TIAM1 , and WASF1 expression, the products of which are known to be involved in leading edge formation and cell translocation. These changes were accompanied by the attenuation of targeted and nontargeted migration of MSCs after short-term hypoxic exposure, as demonstrated in scratch and transwell migration assays. These results indicate that acute hypoxic stress can modulate MSC function in their native milieu, preventing their mobilisation from sites of injury.

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

PubMed Central

Lobanova, Margarita V.; Andreeva, Elena R.

2016-01-01

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

In vitro and in vivo efficacy of β-lactams against replicating and slowly growing/nonreplicating Mycobacterium tuberculosis.

PubMed

Solapure, Suresh; Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K G; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V

2013-06-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.

In Vitro and In Vivo Efficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

PubMed Central

Dinesh, Neela; Shandil, Radha; Ramachandran, Vasanthi; Sharma, Sreevalli; Bhattacharjee, Deepa; Ganguly, Samit; Reddy, Jitendar; Ahuja, Vijaykamal; Panduga, Vijender; Parab, Manish; Vishwas, K. G.; Kumar, Naveen; Balganesh, Meenakshi; Balasubramanian, V.

2013-01-01

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%TMIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model. PMID:23507276

Quantitative Proteomic Analysis of Replicative and Nonreplicative Forms Reveals Important Insights into Chromatin Biology of Trypanosoma cruzi.

PubMed

Leandro de Jesus, Teresa Cristina; Calderano, Simone Guedes; Vitorino, Francisca Nathalia de Luna; Llanos, Ricardo Pariona; Lopes, Mariana de Camargo; de Araújo, Christiane Bezerra; Thiemann, Otavio Henrique; Reis, Marcelo da Silva; Elias, Maria Carolina; Chagas da Cunha, Julia Pinheiro

2017-01-01

Chromatin associated proteins are key regulators of many important processes in the cell. Trypanosoma cruzi, a protozoa flagellate that causes Chagas disease, alternates between replicative and nonreplicative forms accompanied by a shift on global transcription levels and by changes in its chromatin architecture. Here, we investigated the T. cruzi chromatin proteome using three different protocols and compared it between replicative (epimastigote) and nonreplicative (trypomastigote) forms by high-resolution mass spectrometry. More than 2000 proteins were identified and quantified both in chromatin and nonchromatin extracts. Besides histones and other known nuclear proteins, trypanosomes chromatin also contains metabolic (mainly from carbohydrate pathway), cytoskeleton and many other proteins with unknown functions. Strikingly, the two parasite forms differ greatly regarding their chromatin-associated factors composition and amount. Although the nucleosome content is the same for both life forms (as seen by MNase digestion), the remaining proteins were much less detected in nonreplicative forms, suggesting that they have a naked chromatin. Proteins associated to DNA proliferation, such as PCNA, RPA, and DNA topoisomerases were exclusively found in the chromatin of replicative stages. On the other hand, the nonreplicative stages have an enrichment of a histone H2B variant. Furthermore, almost 20% of replicative stages chromatin-associated proteins are expressed in nonreplicative forms, but located at nonchromatin space. We identified different classes of proteins including phosphatases and a Ran-binding protein, that may shuttle between chromatin and nonchromatin space during differentiation. Seven proteins, including those with unknown functions, were selected for further validation. We confirmed their location in chromatin and their differential expression, using Western blotting assays and chromatin immunoprecipitation (ChIP). Our results indicate that the replicative state in trypanosomes involves an increase of chromatin associated proteins content. We discuss in details, the qualitative and quantitative implication of this chromatin set in trypanosome chromatin biology. Because trypanosomes are early-branching organisms, this data can boost our understanding of chromatin-associated processes in other cell types. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Argon protects against hypoxic-ischemic brain injury in neonatal rats through activation of nuclear factor (erythroid-derived 2)-like 2

PubMed Central

Zhao, Hailin; Mitchell, Sian; Ciechanowicz, Sarah; Savage, Sinead; Wang, Tianlong; Ji, Xunming; Ma, Daqing

2016-01-01

Perinatal hypoxic ischaemic encephalopathy (HIE) has a high mortality rate with neuropsychological impairment. This study investigated the neuroprotective effects of argon against neonatal hypoxic-ischaemic brain injury. In vitro cortical neuronal cell cultures derived from rat foetuses were subjected to an oxygen and glucose deprivation (OGD) challenge for 90 minutes and then exposed to 70% argon or nitrogen with 5% carbon dioxide and balanced with oxygen for 2 hours. In vivo, seven-day-old rats were subjected to unilateral common carotid artery ligation followed by hypoxic (8% oxygen balanced with nitrogen) insult for 90 minutes. They were exposed to 70% argon or nitrogen balanced with oxygen for 2 hours. In vitro, argon treatment of cortical neuronal cultures resulted in a significant increase of p-mTOR and Nuclear factor (erythroid-derived 2)-like 2(Nrf2) and protection against OGD challenge. Inhibition of m-TOR through Rapamycin or Nrf2 through siRNA abolished argon-mediated cyto-protection. In vivo, argon exposure significantly enhanced Nrf2 and its down-stream effector NAD(P)H Dehydrogenase, Quinone 1(NQO1) and superoxide dismutase 1(SOD1). Oxidative stress, neuroinflammation and neuronal cell death were significantly decreased and brain infarction was markedly reduced. Blocking PI-3K through wortmannin or ERK1/2 through U0126 attenuated argon-mediated neuroprotection. These data provide a new molecular mechanism for the potential application of argon as a neuroprotectant in HIE. PMID:27016422

Targeting tumor hypoxia: suppression of breast tumor growth and metastasis by novel carbonic anhydrase IX inhibitors.

PubMed

Lou, Yuanmei; McDonald, Paul C; Oloumi, Arusha; Chia, Stephen; Ostlund, Christina; Ahmadi, Ardalan; Kyle, Alastair; Auf dem Keller, Ulrich; Leung, Samuel; Huntsman, David; Clarke, Blaise; Sutherland, Brent W; Waterhouse, Dawn; Bally, Marcel; Roskelley, Calvin; Overall, Christopher M; Minchinton, Andrew; Pacchiano, Fabio; Carta, Fabrizio; Scozzafava, Andrea; Touisni, Nadia; Winum, Jean-Yves; Supuran, Claudiu T; Dedhar, Shoukat

2011-05-01

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDA-MB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4(Luc+) cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis.

Parkin modulates expression of HIF-1α and HIF-3α during hypoxia in gliobastoma-derived cell lines in vitro.

PubMed

Maugeri, Grazia; D'Amico, Agata Grazia; Reitano, Rita; Saccone, Salvatore; Federico, Concetta; Cavallaro, Sebastiano; D'Agata, Velia

2016-06-01

Mutation of the Parkin gene causes an autosomal recessive juvenile-onset form of Parkinson's disease. However, recently, it has been also linked to a wide variety of malignancies, including glioblastoma multiforme (GBM). In this pathology, Parkin exhibits a tumor suppressor role by mitigating the proliferation rate in both in vitro and in vivo models. However, Parkin involvement in the hypoxic process has not as yet been investigated. GBM is the most common and aggressive primary brain tumor in adults and is characterized by hypoxic areas. The low oxygen supply causes the expression of hypoxia-inducible factors (HIFs) leading to an accumulation of pro-angiogenic factors and tumoral invasiveness. We assess the relationship between Parkin and two HIFs expressed during hypoxic conditions, namely HIF-1α and HIF-3α. Our data show that Parkin is downregulated under hypoxia and that it interferes with HIF expression based on cellular oxygen tension. These results suggest a role for the involvement of Parkin in GBM, although further studies will be needed to understand the mechanism by which it modulates HIF-1α and HIF-3α expression.

Impaired gamete production and viability in Atlantic croaker collected throughout the 20,000 km(2) hypoxic region in the northern Gulf of Mexico.

PubMed

Thomas, Peter; Rahman, Md Saydur; Picha, Matthew E; Tan, Wenxian

2015-12-15

The long-term impacts of recent marked increases in the incidence and extent of hypoxia (dissolved oxygen <2 mg/L) in coastal regions worldwide on fisheries and ecosystems are unknown. Reproductive impairment was investigated in Atlantic croaker collected in 2010 from the extensive coastal hypoxic region in the northern Gulf of Mexico. Potential fecundity was significantly lower in croaker collected throughout the ~20,000 km(2) hypoxic region than in croaker from normoxic sites. In vitro bioassays of gamete viability showed reductions in oocyte maturation and sperm motility in croaker collected from the hypoxic sites in response to reproductive hormones which were accompanied by decreases in gonadal levels of membrane progestin receptor alpha, the receptor regulating these processes. The finding that environmental hypoxia exposure reduces oocyte viability in addition to decreasing oocyte production in croaker suggests that fecundity estimates need to be adjusted to account for the decrease in oocyte maturation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pulmonary vascular responses during acute and sustained respiratory alkalosis or acidosis in intact newborn piglets.

PubMed

Gordon, J B; Rehorst-Paea, L A; Hoffman, G M; Nelin, L D

1999-12-01

Acute alkalosis-induced pulmonary vasodilation and acidosis-induced pulmonary vasoconstriction have been well described, but responses were generally measured within 5-30 min of changing pH. In contrast, several in vitro studies have found that relatively brief periods of sustained alkalosis can enhance, and sustained acidosis can decrease, vascular reactivity. In this study of intact newborn piglets, effects of acute (20 min) and sustained (60-80 min) alkalosis or acidosis on baseline (35% O2) and hypoxic (12% O2) pulmonary vascular resistance (PVR) were compared with control piglets exposed only to eucapnia. Acute alkalosis decreased hypoxic PVR, but sustained alkalosis failed to attenuate either baseline PVR or the subsequent hypoxic response. Acute acidosis did not significantly increase hypoxic PVR, but sustained acidosis markedly increased both baseline PVR and the subsequent hypoxic response. Baseline PVR was similar in all piglets after resumption of eucapnic ventilation, but the final hypoxic response was greater in piglets previously exposed to alkalosis than in controls. Thus, hypoxic pulmonary vasoconstriction was not attenuated during sustained alkalosis, but was accentuated during sustained acidosis and after the resumption of eucapnia in alkalosis-treated piglets. Although extrapolation of data from normal piglets to infants and children with pulmonary hypertension must be done with caution, this study suggests that sustained alkalosis may be of limited efficacy in treating acute hypoxia-induced pulmonary hypertension and the risks of pulmonary hypertension must be considered when using ventilator strategies resulting in permissive hypercapnic acidosis.

Oxygen Generating Biomaterials Preserve Skeletal Muscle Homeostasis under Hypoxic and Ischemic Conditions

PubMed Central

Ward, Catherine L.; Corona, Benjamin T.; Yoo, James J.; Harrison, Benjamin S.; Christ, George J.

2013-01-01

Provision of supplemental oxygen to maintain soft tissue viability acutely following trauma in which vascularization has been compromised would be beneficial for limb and tissue salvage. For this application, an oxygen generating biomaterial that may be injected directly into the soft tissue could provide an unprecedented treatment in the acute trauma setting. The purpose of the current investigation was to determine if sodium percarbonate (SPO), an oxygen generating biomaterial, is capable of maintaining resting skeletal muscle homeostasis under otherwise hypoxic conditions. In the current studies, a biologically and physiologically compatible range of SPO (1–2 mg/mL) was shown to: 1) improve the maintenance of contractility and attenuate the accumulation of HIF1α, depletion of intramuscular glycogen, and oxidative stress (lipid peroxidation) that occurred following ∼30 minutes of hypoxia in primarily resting (duty cycle = 0.2 s train/120 s contraction interval <0.002) rat extensor digitorum longus (EDL) muscles in vitro (95% N2–5% CO2, 37°C); 2) attenuate elevations of rat EDL muscle resting tension that occurred during contractile fatigue testing (3 bouts of 25 100 Hz tetanic contractions; duty cycle = 0.2 s/2 s = 0.1) under oxygenated conditions in vitro (95% O2–5% CO2, 37°C); and 3) improve the maintenance of contractility (in vivo) and prevent glycogen depletion in rat tibialis anterior (TA) muscle in a hindlimb ischemia model (i.e., ligation of the iliac artery). Additionally, injection of a commercially available lipid oxygen-carrying compound or the components (sodium bicarbonate and hydrogen peroxide) of 1 mg/mL SPO did not improve EDL muscle contractility under hypoxic conditions in vitro. Collectively, these findings demonstrate that a biological and physiological concentration of SPO (1–2 mg/mL) injected directly into rat skeletal muscle (EDL or TA muscles) can partially preserve resting skeletal muscle homeostasis under hypoxic conditions. PMID:23991116

SU-F-T-685: Evaluation of Tumor Hypoxic Fraction Using Serial Volumetric Imaging During Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chvetsov, A

Purpose: To develop a tumor response model which could be uses to compute tumor hypoxic fraction using serial volumetric tumor imaging. This algorithm may be used for treatment response assessment and also for guidance of more expensive PET imaging of hypoxia. Methods: Previously developed two-level cell population tumor response model was modified to include a third cell level describing hypoxic and necrotic cells. This third level was considered constant value during radiotherapy treatment; therefore, inclusion additional parameter did not compromise stability of model fitting to imaging data. Fitting the model to serial volumetric imaging data was performed using a leastmore » squares objective function and simulated annealing algorithm. The problem of reconstruction of radiobiological parameters from serial imaging data was considered as inverse ill-posed problem described by the Fredholm integral equation of the first kind. Variational regularization was used to stabilize solutions. Results: To evaluate performance of the algorithm, we used a set of serial CT imaging data on tumor-volume for 14 head and neck cancer patients. The hypoxic fractions were reconstructed for each patient and the distribution of hypoxic fractions was compared to the distribution of initial hypoxic fractions previously measured using histograph. The measured and reconstructed from imaging data distributions of hypoxic fractions are in good agreement. The reconstructed distribution of cell surviving fraction was also in better agreement with in vitro data than previously obtained using the two-level cell population model. Conclusion: Our results indicate that it is possible to evaluate the initial hypoxic tumor fraction using serial volumetric imaging and a tumor response model. This algorithm can be used for treatment response assessment and guidance of more expensive PET imaging.« less

SU-E-T-146: Effects of Uncertainties of Radiation Sensitivity of Biological Modelling for Treatment Planning

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oita, M; Department of Life System, Institute of Technology and Science, Graduate School, The Tokushima University; Uto, Y

Purpose: The aim of this study was to evaluate the distribution of uncertainty of cell survival by radiation, and assesses the usefulness of stochastic biological model applying for gaussian distribution. Methods: For single cell experiments, exponentially growing cells were harvested from the standard cell culture dishes by trypsinization, and suspended in test tubes containing 1 ml of MEM(2x10{sup 6} cells/ml). The hypoxic cultures were treated with 95% N{sub 2}−5% CO{sub 2} gas for 30 minutes. In vitro radiosensitization was also measured in EMT6/KU single cells to add radiosensitizer under hypoxic conditions. X-ray irradiation was carried out by using an Xraymore » unit (Hitachi X-ray unit, model MBR-1505R3) with 0.5 mm Al/1.0 mm Cu filter, 150 kV, 4 Gy/min). In vitro assay, cells on the dish were irradiated with 1 Gy to 24 Gy, respectively. After irradiation, colony formation assays were performed. Variations of biological parameters were investigated at standard cell culture(n=16), hypoxic cell culture(n=45) and hypoxic cell culture(n=21) with radiosensitizers, respectively. The data were obtained by separate schedule to take account for the variation of radiation sensitivity of cell cycle. Results: At standard cell culture, hypoxic cell culture and hypoxic cell culture with radiosensitizers, median and standard deviation of alpha/beta ratio were 37.1±73.4 Gy, 9.8±23.7 Gy, 20.7±21.9 Gy, respectively. Average and standard deviation of D{sub 50} were 2.5±2.5 Gy, 6.1±2.2 Gy, 3.6±1.3 Gy, respectively. Conclusion: In this study, we have challenged to apply these uncertainties of parameters for the biological model. The variation of alpha values, beta values, D{sub 50} as well as cell culture might have highly affected by probability of cell death. Further research is in progress for precise prediction of the cell death as well as tumor control probability for treatment planning.« less

ORC1/CDC6 and MCM7 distinct associate with chromatin through Trypanosoma cruzi life cycle.

PubMed

Calderano, Simone; Godoy, Patricia; Soares, Daiane; Sant'Anna, Osvaldo Augusto; Schenkman, Sergio; Elias, M Carolina

2014-02-01

Trypanosoma cruzi alternates between replicative and non-replicative stages. We analyzed the expression of components of the pre-replication machinery TcORC1/CDC6 and TcMCM7 and their interaction with DNA in all T. cruzi stages. TcORC1/CDC6 remains in the nuclear space during all stages of the life cycle and interacts with DNA in the replicative stages; however, it does not bind to DNA in the non-replicative forms. Moreover, TcMCM7 is not present in the non-replicative stages. These data suggest that the lacking of DNA replication during the T. cruzi life cycle may be a consequence of the blocking of TcORC1/CDC6-DNA interaction and of the down regulation of the TcMCM7 expression. Copyright © 2014 Elsevier B.V. All rights reserved.

Short-term hypoxic preconditioning promotes prevascularization in 3D bioprinted bone constructs with stromal vascular fraction derived cells† †Electronic supplementary information (ESI) available: qPCR primers and Fig. S1. See DOI: 10.1039/c7ra04372d Click here for additional data file.

PubMed Central

Kuss, Mitchell A.; Harms, Robert; Wu, Shaohua; Wang, Ying; Untrauer, Jason B.; Carlson, Mark A.

2017-01-01

Reconstruction of complex, craniofacial bone defects often requires autogenous vascularized bone grafts, and still remains a challenge today. In order to address this issue, we isolated the stromal vascular fraction (SVF) from adipose tissues and maintained the phenotypes and the growth of endothelial lineage cells within SVF derived cells (SVFC) by incorporating an endothelial cell medium. We 3D bioprinted SVFC within our hydrogel bioinks and conditioned the constructs in either normoxia or hypoxia. We found that short-term hypoxic conditioning promoted vascularization-related gene expression, whereas long-term hypoxia impaired cell viability and vascularization. 3D bioprinted bone constructs composed of polycaprolactone/hydroxyapatite (PCL/HAp) and SVFC-laden hydrogel bioinks were then implanted into athymic mice, after conditioning in normoxic or short-term hypoxic environments, in order to determine the in vitro and in vivo vascularization and osteogenic differentiation of the constructs. Short-term hypoxic conditioning promoted microvessel formation in vitro and in vivo and promoted integration with existing host vasculature, but did not affect osteogenic differentiation of SVFC. These findings demonstrate the benefit of short-term hypoxia and the potential for utilization of SVFC and 3D bioprinting for generating prevascularized 3D bioprinted bone constructs. Furthermore, the ability to custom design complex anatomical shapes has promising applications for the regeneration of both large and small craniofacial bone defects. PMID:28670447

Role of aldehyde dehydrogenase in hypoxic vasodilator effects of nitrite in rats and humans

PubMed Central

Arif, Sayqa; Borgognone, Alessandra; Lin, Erica Lai-Sze; O'Sullivan, Aine G; Sharma, Vishal; Drury, Nigel E; Menon, Ashvini; Nightingale, Peter; Mascaro, Jorge; Bonser, Robert S; Horowitz, John D; Feelisch, Martin; Frenneaux, Michael P; Madhani, Melanie

2015-01-01

Background and Purpose Hypoxic conditions favour the reduction of nitrite to nitric oxide (NO) to elicit vasodilatation, but the mechanism(s) responsible for bioconversion remains ill defined. In the present study, we assess the role of aldehyde dehydrogenase 2 (ALDH2) in nitrite bioactivation under normoxia and hypoxia in the rat and human vasculature. Experimental Approach The role of ALDH2 in vascular responses to nitrite was studied using rat thoracic aorta and gluteal subcutaneous fat resistance vessels from patients with heart failure (HF; 16 patients) in vitro and by measurement of changes in forearm blood flow (FBF) during intra-arterial nitrite infusion (21 patients) in vivo. Specifically, we investigated the effects of (i) ALDH2 inhibition by cyanamide or propionaldehyde and the (ii) tolerance-independent inactivation of ALDH2 by glyceryl trinitrate (GTN) on the vasodilator activity of nitrite. In each setting, nitrite effects were measured via evaluation of the concentration–response relationship under normoxic and hypoxic conditions in the absence or presence of ALDH2 inhibitors. Key Results Both in rat aorta and human resistance vessels, dilatation to nitrite was diminished following ALDH2 inhibition, in particular under hypoxia. In humans there was a non-significant trend towards attenuation of nitrite-mediated increases in FBF. Conclusions and Implications In human and rat vascular tissue in vitro, hypoxic nitrite-mediated vasodilatation involves ALDH2. In patients with HF in vivo, the role of this enzyme in nitrite bioactivation is at the most, modest, suggesting the involvement of other more important mechanisms. PMID:25754766

Mapping of PARK2 and PACRG overlapping regulatory region reveals LD structure and functional variants in association with leprosy in unrelated indian population groups.

PubMed

Chopra, Rupali; Ali, Shafat; Srivastava, Amit K; Aggarwal, Shweta; Kumar, Bhupender; Manvati, Siddharth; Kalaiarasan, Ponnusamy; Jena, Mamta; Garg, Vijay K; Bhattacharya, Sambit N; Bamezai, Rameshwar N K

2013-01-01

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations.

The Mycobacterium tuberculosis MmpL11 Cell Wall Lipid Transporter Is Important for Biofilm Formation, Intracellular Growth, and Nonreplicating Persistence

PubMed Central

Wright, Catherine C.; Hsu, Fong Fu; Arnett, Eusondia; Dunaj, Jennifer L.; Davidson, Patrick M.; Pacheco, Sophia A.; Harriff, Melanie J.; Lewinsohn, David M.; Schlesinger, Larry S.

2017-01-01

ABSTRACT The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosis mmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity. PMID:28507063

Foamy Macrophages from Tuberculous Patients' Granulomas Constitute a Nutrient-Rich Reservoir for M. tuberculosis Persistence

PubMed Central

Poquet, Yannick; Levillain, Florence; Botanch, Catherine; Bardou, Fabienne; Daffé, Mamadou; Emile, Jean-François; Marchou, Bruno; Cardona, Pere-Joan; de Chastellier, Chantal; Altare, Frédéric

2008-01-01

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria. PMID:19002241

Mapping of PARK2 and PACRG Overlapping Regulatory Region Reveals LD Structure and Functional Variants in Association with Leprosy in Unrelated Indian Population Groups

PubMed Central

Chopra, Rupali; Aggarwal, Shweta; Kumar, Bhupender; Manvati, Siddharth; Kalaiarasan, Ponnusamy; Jena, Mamta; Garg, Vijay K.; Bhattacharya, Sambit N.; Bamezai, Rameshwar N. K.

2013-01-01

Leprosy is a chronic infectious disease caused by Mycobacterium Leprae, where the host genetic background plays an important role toward the disease pathogenesis. Various studies have identified a number of human genes in association with leprosy or its clinical forms. However, non-replication of results has hinted at the heterogeneity among associations between different population groups, which could be due to differently evolved LD structures and differential frequencies of SNPs within the studied regions of the genome. A need for systematic and saturated mapping of the associated regions with the disease is warranted to unravel the observed heterogeneity in different populations. Mapping of the PARK2 and PACRG gene regulatory region with 96 SNPs, with a resolution of 1 SNP per 1 Kb for PARK2 gene regulatory region in a North Indian population, showed an involvement of 11 SNPs in determining the susceptibility towards leprosy. The association was replicated in a geographically distinct and unrelated population from Orissa in eastern India. In vitro reporter assays revealed that the two significantly associated SNPs, located 63.8 kb upstream of PARK2 gene and represented in a single BIN of 8 SNPs, influenced the gene expression. A comparison of BINs between Indian and Vietnamese populations revealed differences in the BIN structures, explaining the heterogeneity and also the reason for non-replication of the associated genomic region in different populations. PMID:23861666

Hypoxic exosomes facilitate bladder tumor growth and development through transferring long non-coding RNA-UCA1.

PubMed

Xue, Mei; Chen, Wei; Xiang, An; Wang, Ruiqi; Chen, He; Pan, Jingjing; Pang, Huan; An, Hongli; Wang, Xiang; Hou, Huilian; Li, Xu

2017-08-25

To overcome the hostile hypoxic microenvironment of solid tumors, tumor cells secrete a large number of non-coding RNA-containing exosomes that facilitate tumor development and metastasis. However, the precise mechanisms of tumor cell-derived exosomes during hypoxia are unknown. Here, we aim to clarify whether hypoxia affects tumor growth and progression by transferring long non-coding RNA-urothelial cancer-associated 1 (lncRNA-UCA1) enriched exosomes secreted from bladder cancer cells. We used bladder cancer 5637 cells with high expression of lncRNA-UCA1 as exosome-generating cells and bladder cancer UMUC2 cells with low expression of lncRNA-UCA1 as recipient cells. Exosomes derived from 5637 cells cultured under normoxic or hypoxic conditions were isolated and identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting analysis. These exosomes were co-cultured with UMUC2 cells to evaluate cell proliferation, migration and invasion. We further investigated the roles of exosomal lncRNA-UCA1 derived from hypoxic 5637 cells by xenograft models. The availability of lncRNA-UCA1 in serum-derived exosomes as a biomarker for bladder cancer was also assessed. We found that hypoxic exosomes derived from 5637 cells promoted cell proliferation, migration and invasion, and hypoxic exosomal RNAs could be internalized by three bladder cancer cell lines. Importantly, lncRNA-UCA1 was secreted in hypoxic 5637 cell-derived exosomes. Compared with normoxic exosomes, hypoxic exosomes derived from 5637 cells showed the higher expression levels of lncRNA-UCA1. Moreover, Hypoxic exosomal lncRNA-UCA1 could promote tumor growth and progression though epithelial-mesenchymal transition, in vitro and in vivo. In addition, the expression levels of lncRNA-UCA1 in the human serum-derived exosomes of bladder cancer patients were higher than that in the healthy controls. Together, our results demonstrate that hypoxic bladder cancer cells remodel tumor microenvironment to facilitate tumor growth and development though secreting the oncogenic lncRNA-UCA1-enriched exosomes and exosomal lncRNA-UCA1 in human serum has the possibility as a diagnostic biomarker for bladder cancer.

Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

PubMed Central

Turapov, Obolbek; Waddell, Simon J.; Burke, Bernard; Glenn, Sarah; Sarybaeva, Asel A.; Tudo, Griselda; Labesse, Gilles; Young, Danielle I.; Young, Michael; Andrew, Peter W.; Butcher, Philip D.; Cohen-Gonsaud, Martin

2014-01-01

Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis. PMID:24590482

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines

PubMed Central

Gasper, David J.; Neldner, Brandon; Plisch, Erin H.; Rustom, Hani; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M.

2016-01-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract. PMID:27997610

Effective Respiratory CD8 T-Cell Immunity to Influenza Virus Induced by Intranasal Carbomer-Lecithin-Adjuvanted Non-replicating Vaccines.

PubMed

Gasper, David J; Neldner, Brandon; Plisch, Erin H; Rustom, Hani; Carrow, Emily; Imai, Hirotaka; Kawaoka, Yoshihiro; Suresh, M

2016-12-01

CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract.

Intratumoral oxygen gradients mediate sarcoma cell invasion

PubMed Central

Lewis, Daniel M.; Park, Kyung Min; Tang, Vitor; Xu, Yu; Pak, Koreana; Eisinger-Mathason, T. S. Karin; Simon, M. Celeste; Gerecht, Sharon

2016-01-01

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm3) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1–6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets. PMID:27486245

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Wei, E-mail: detachedy@yahoo.com.cn; Sun, Ting; Cao, Jianping

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 inmore » 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.« less

Intratumoral oxygen gradients mediate sarcoma cell invasion.

PubMed

Lewis, Daniel M; Park, Kyung Min; Tang, Vitor; Xu, Yu; Pak, Koreana; Eisinger-Mathason, T S Karin; Simon, M Celeste; Gerecht, Sharon

2016-08-16

Hypoxia is a critical factor in the progression and metastasis of many cancers, including soft tissue sarcomas. Frequently, oxygen (O2) gradients develop in tumors as they grow beyond their vascular supply, leading to heterogeneous areas of O2 depletion. Here, we report the impact of hypoxic O2 gradients on sarcoma cell invasion and migration. O2 gradient measurements showed that large sarcoma mouse tumors (>300 mm(3)) contain a severely hypoxic core [≤0.1% partial pressure of O2 (pO2)] whereas smaller tumors possessed hypoxic gradients throughout the tumor mass (0.1-6% pO2). To analyze tumor invasion, we used O2-controllable hydrogels to recreate the physiopathological O2 levels in vitro. Small tumor grafts encapsulated in the hydrogels revealed increased invasion that was both faster and extended over a longer distance in the hypoxic hydrogels compared with nonhypoxic hydrogels. To model the effect of the O2 gradient accurately, we examined individual sarcoma cells embedded in the O2-controllable hydrogel. We observed that hypoxic gradients guide sarcoma cell motility and matrix remodeling through hypoxia-inducible factor-1α (HIF-1α) activation. We further found that in the hypoxic gradient, individual cells migrate more quickly, across longer distances, and in the direction of increasing O2 tension. Treatment with minoxidil, an inhibitor of hypoxia-induced sarcoma metastasis, abrogated cell migration and matrix remodeling in the hypoxic gradient. Overall, we show that O2 acts as a 3D physicotactic agent during sarcoma tumor invasion and propose the O2-controllable hydrogels as a predictive system to study early stages of the metastatic process and therapeutic targets.

High targeted migration of human mesenchymal stem cells grown in hypoxia is associated with enhanced activation of RhoA

PubMed Central

2013-01-01

Introduction A feature which makes stem cells promising candidates for cell therapy is their ability to migrate effectively into damaged or diseased tissues. Recent reports demonstrated the increased motility of human mesenchymal stem cells (hMSC) grown under hypoxic conditions compared to normoxic cells. However, the directional migration of hMSC cultured in hypoxia has not been investigated. In this study we examined the in vitro transmembrane migration of hMSC permanently cultured in hypoxia in response to various cytokines. We also studied the involvement of RhoA, a molecule believed to play an essential role in the migration of MSC via reorganization of the cytoskeleton. Methods We compared the directional migration of human hMSCs grown permanently under normal (21%, normoxic) and low O2 (5%, hypoxic) conditions until passage 4 using an in vitro transmembrane migration assay. A series of 17 cytokines was used to induce chemotaxis. We also compared the level of GTP-bound RhoA in the cell extracts of calpeptin-activated hypoxic and normoxic hMSC. Results We found that hMSC cultured in hypoxia demonstrate markedly higher targeted migration activity compared to normoxic cells, particularly towards wound healing cytokines, including those found in ischemic and myocardial infarction. We also demonstrated for the first time that hMSC are dramatically more sensitive to activation of RhoA. Conclusions The results of this study indicate that high directional migration of hMSCs permanently grown in hypoxia is associated with the enhanced activation of RhoA. The enhanced migratory capacity of hypoxic hMSC would further suggest their potential advantages for clinical applications. PMID:23295150

Short- and long-term immunogenicity and protection induced by non-replicating smallpox vaccine candidates in mice and comparison with the traditional 1st generation vaccine.

PubMed

Ferrier-Rembert, Audrey; Drillien, Robert; Tournier, Jean-Nicolas; Garin, Daniel; Crance, Jean-Marc

2008-03-25

This study assessed three non-replicating smallpox vaccine candidates (modified vaccinia Ankara (MVA), NYVAC and HR) for their immunogenicity and ability to protect mice against an intranasal cowpox virus challenge and compared them with the traditional replicating vaccine. A single immunisation with the non-replicating vaccines induced a complete protection from death at short-term, but was not fully protective when mice were challenged 150 days post-vaccination with protection correlated with the specific neutralizing antibodies and CD4(+) T-cells responses. Prime-boost vaccination enabled effective long-term protection from death for mice vaccinated with MVA, but protection from disease and CD4(+) T-cell level were lower than the ones induced by the traditional vaccine over the long-term period. Further investigations are necessary with MVA to determine the optimal conditions of immunisation to induce at long-term immunogenicity and protection observed with the 1st generation smallpox vaccine.

Novel poly (ADP-ribose) polymerase inhibitor, AZD2281, enhances radiosensitivity of both normoxic and hypoxic esophageal squamous cancer cells.

PubMed

Zhan, L; Qin, Q; Lu, J; Liu, J; Zhu, H; Yang, X; Zhang, C; Xu, L; Liu, Z; Cai, J; Ma, J; Dai, S; Tao, G; Cheng, H; Sun, X

2016-04-01

Radiotherapy plays an important role in the treatment of esophageal squamous cell carcinoma (ESCC). However, the outcome of radiotherapy in ESCC remains unsatisfactory because esophageal squamous cancer cells, particularly those under hypoxic condition, exhibit radioresistance. The aim of this study was to determine whether or not AZD2281, a potent poly (ADP-ribose) polymerase (PARP) inhibitor, could enhance the radiation sensitivity of two ESCC cell lines, namely ECA109 and TE13. The radiosensitizing effect of AZD2281 was evaluated on the basis of cell death, clonogenic survival and tumor xenograft progression. AZD2281 alone was slightly toxic to ESCC cell lines. Apoptosis was increased and clonogenic survival was decreased in both cell lines when AZD2281 was combined with ionizing radiation (IR) under normoxic condition. AZD2281 enhanced IR-induced apoptosis to a more significant level under chronic hypoxic condition (0.2% O(2), 48 hour) than under normoxic condition. AZD2281 also slightly enhanced clonogenic cell death under chronic hypoxic condition compared with that under normoxic condition. This result could be associated with increased radiation-induced DNA double-strand breaks (DSB), decreased DSB repair and increased apoptosis of ESCC cells. Furthermore, homologous recombination (HR) protein Rad51 expression and focus formation were decreased in ESCC cells exposed to moderate chronic hypoxic condition (0.2% O(2), 48 hour); this result indicated that chronic hypoxic ESCC cells were HR deficient, possibly causing contextual synthetic lethality with PARP inhibitor in radiation sensitization. AZD2281 was also a radiation sensitizer in ESCC tumor xenograft models. Hence, in vitro and in vivo findings provide evidence that AZD2281 potently sensitizes ESCC cells to X-ray irradiation. The selective cell killing of HR-defective hypoxic cells contributes to radiosensitization by PARP inhibitor in ESCC cells under hypoxic condition. © 2015 International Society for Diseases of the Esophagus.

Contrasting hypoxic effects on breast cancer stem cell hierarchy is dependent on ER-α status.

PubMed

Harrison, Hannah; Rogerson, Lynsey; Gregson, Hannah J; Brennan, Keith R; Clarke, Robert B; Landberg, Göran

2013-02-15

Tumor hypoxia is often linked to decreased survival in patients with breast cancer and current therapeutic strategies aim to target the hypoxic response. One way in which this is done is by blocking hypoxia-induced angiogenesis. Antiangiogenic therapies show some therapeutic potential with increased disease-free survival, but these initial promising results are short lived and followed by tumor progression. We hypothesized that this may be due to altered cancer stem cell (CSC) activity resulting from increased tumor hypoxia. We studied the effects of hypoxia on CSC activity, using in vitro mammosphere and holoclone assays as well as in vivo limiting dilution experiments, in 13 patient-derived samples and four cell lines. There was a HIF-1α-dependent CSC increase in ER-α-positive cancers following hypoxic exposure, which was blocked by inhibition of estrogen and Notch signaling. A contrasting decrease in CSC was seen in ER-α-negative cancers. We next developed a xenograft model of cell lines and patient-derived samples to assess the hypoxic CSC response. Varying sizes of xenografts were collected and analyzed for HIF1-α expression and CSC. The same ER-α-dependent contrasting hypoxic-CSC response was seen validating the initial observation. These data suggest that ER-α-positive and negative breast cancer subtypes respond differently to hypoxia and, as a consequence, antiangiogenic therapies will not be suitable for both subgroups.

Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival

PubMed Central

Komatsu, Hirotake; Cook, Colin; Wang, Chia-Hao; Medrano, Leonard; Lin, Henry; Kandeel, Fouad; Tai, Yu-Chong; Mullen, Yoko

2017-01-01

Background Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency. Methodology The oxygen tension inside isolated human islets of different sizes was simulated under varying oxygen environments using a computational in silico model. In vitro human islet viability was also assessed after culturing in different oxygen conditions. Correlation between simulation data and experimentally measured islet viability was examined. Using these in vitro viability data of human islets, the effect of islet diameter and oxygen tension of the culture environment on islet viability was also analyzed using a logistic regression model. Principal findings Computational simulation clearly revealed the oxygen gradient inside the islet structure. We found that oxygen tension in the islet core was greatly lower (hypoxic) than that on the islet surface due to the oxygen consumption by the cells. The hypoxic core was expanded in the larger islets or in lower oxygen cultures. These findings were consistent with results from in vitro islet viability assays that measured central necrosis in the islet core, indicating that hypoxia is one of the major causes of central necrosis. The logistic regression analysis revealed a negative effect of large islet and low oxygen culture on islet survival. Conclusions/Significance Hypoxic core conditions, induced by the oxygen gradient inside islets, contribute to the development of central necrosis of human isolated islets. Supplying sufficient oxygen during culture could be an effective and reasonable method to maintain isolated islets viable. PMID:28832685

Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

PubMed

Ong, Huan Ting; Redmond, Sharon L; Marano, Robert J; Atlas, Marcus D; von Unge, Magnus; Aabel, Peder; Dilley, Rodney J

2017-03-15

Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CM ADSC ) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CM ADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CM ADSC . Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

A hypoxia- and {alpha}-fetoprotein-dependent oncolytic adenovirus exhibits specific killing of hepatocellular carcinomas.

PubMed

Kwon, Oh-Joon; Kim, Pyung-Hwan; Huyn, Steven; Wu, Lily; Kim, Minjung; Yun, Chae-Ok

2010-12-15

Oncolytic adenoviruses (Ad) constitute a new promising modality of cancer gene therapy that displays improved efficacy over nonreplicating Ads. We have previously shown that an E1B 19-kDa-deleted oncolytic Ad exhibits a strong cell-killing effect but lacks tumor selectivity. To achieve hepatoma-restricted cytotoxicity and enhance replication of Ad within the context of tumor microenvironment, we used a modified human α-fetoprotein (hAFP) promoter to control the replication of Ad with a hypoxia response element (HRE). We constructed Ad-HRE(6)/hAFPΔ19 and Ad-HRE(12)/hAFPΔ19 that incorporated either 6 or 12 copies of HRE upstream of promoter. The promoter activity and specificity to hepatoma were examined by luciferase assay and fluorescence-activated cell sorting analysis. In addition, the AFP expression- and hypoxia-dependent in vitro cytotoxicity of Ad-HRE(6)/hAFPΔ19 and Ad-HRE(12)/hAFPΔ19 was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cytopathic effect assay. In vivo tumoricidal activity on subcutaneous and liver orthotopic model was monitored by noninvasive molecular imaging. Ad-HRE(12)/hAFPΔ19 exhibited enhanced tumor selectivity and cell-killing activity when compared with Ad-hAFPΔ19. The tumoricidal activity of Ad-HRE(12)/hAFPΔ19 resulted in significant inhibition of tumor growth in both subcutaneous and orthotopic models. Histologic examination of the primary tumor after treatment confirmed accumulation of viral particles near hypoxic areas. Furthermore, Ad-HRE(12)/hAFPΔ19 did not cause severe inflammatory immune response and toxicity after systemic injection. The results presented here show the advantages of incorporating HREs into a hAFP promoter-driven oncolytic virus. This system is unique in that it acts in both a tissue-specific and tumor environment-selective manner. The greatly enhanced selectivity and tumoricidal activity of Ad-HRE(12)/hAFPΔ19 make it a promising therapeutic agent in the treatment of liver cancers. ©2010 AACR.

A Mn(II) complex of boradiazaindacene (BODIPY) loaded graphene oxide as both LED light and H2O2 enhanced anticancer agent.

PubMed

Xu, Xiao-Lei; Shao, Jian; Chen, Qiu-Yun; Li, Cheng-Hao; Kong, Meng-Yun; Fang, Fang; Ji, Ling; Boison, Daniel; Huang, Tao; Gao, Jing; Feng, Chang-Jian

2016-06-01

Cancer cells are more susceptible to H2O2 induced cell death than normal cells. H2O2-activatable and O2-evolving nanoparticles could be used as photodynamic therapy agents in hypoxic environments. In this report, a photo-active Mn(II) complex of boradiazaindacene derivatives (Mn1) was used as a dioxygen generator under irradiation with LED light in water. Moreover, the in vitro biological evaluation for Mn1 and its loaded graphene oxide (herein called Mn1@GO) on HepG-2 cells in normal and hypoxic conditions has been performed. In particular, Mn1@GO can react with H2O2 resulting active anticancer species, which show high inhibition on both HepG-2 cells and CoCl2-treated HepG-2 cells (hypoxic cancer cells). The mechanism of LED light enhanced anticancer activity for Mn1@GO on HepG-2 cells was discussed. Our results show that Mn(II) complexes of boradiazaindacene (BODIPY) derivatives loaded GO can be both LED light and H2O2-activated anticancer agents in hypoxic environments. Copyright © 2016 Elsevier Inc. All rights reserved.

Family A and B DNA Polymerases in Cancer: Opportunities for Therapeutic Interventions

PubMed Central

Shanbhag, Vinit; Sachdev, Shrikesh; Flores, Jacqueline A.; Modak, Mukund J.; Singh, Kamalendra

2018-01-01

DNA polymerases are essential for genome replication, DNA repair and translesion DNA synthesis (TLS). Broadly, these enzymes belong to two groups: replicative and non-replicative DNA polymerases. A considerable body of data suggests that both groups of DNA polymerases are associated with cancer. Many mutations in cancer cells are either the result of error-prone DNA synthesis by non-replicative polymerases, or the inability of replicative DNA polymerases to proofread mismatched nucleotides due to mutations in 3′-5′ exonuclease activity. Moreover, non-replicative, TLS-capable DNA polymerases can negatively impact cancer treatment by synthesizing DNA past lesions generated from treatments such as cisplatin, oxaliplatin, etoposide, bleomycin, and radiotherapy. Hence, the inhibition of DNA polymerases in tumor cells has the potential to enhance treatment outcomes. Here, we review the association of DNA polymerases in cancer from the A and B families, which participate in lesion bypass, and conduct gene replication. We also discuss possible therapeutic interventions that could be used to maneuver the role of these enzymes in tumorigenesis. PMID:29301327

Discovery and Optimization of Benzotriazine Di-N-Oxides Targeting Replicating and Non-replicating Mycobacterium tuberculosis

PubMed Central

Chopra, Sidharth; Koolpe, Gary A.; Tambo-ong, Arlyn A.; Matsuyama, Karen N.; Ryan, Kenneth J.; Tran, Tran B.; Doppalapudi, Rupa S.; Riccio, Edward S.; Iyer, Lalitha V.; Green, Carol E.; Wan, Baojie; Franzblau, Scott G.; Madrid, Peter B.

2012-01-01

Compounds bactericidal against both replicating and non-replicating Mtb may shorten the length of TB treatment regimens by eliminating infections more rapidly. Screening of a panel of antimicrobial and anticancer drug classes that are bioreduced into cytotoxic species revealed that 1,2,4-benzotriazine di-N-oxides (BTOs) are potently bactericidal against replicating and non-replicating Mtb. Medicinal chemistry optimization, guided by semi-empirical molecular orbital calculations, identified a new lead compound (20q) from this series with an MIC of 0.31 μg/mL against H37Rv and a cytotoxicity (CC50) against Vero cells of 25 μg/mL. 20q also had equivalent potency against a panel of single-drug resistant strains of Mtb and remarkably selective activity for Mtb over a panel of other pathogenic bacterial strains. 20q was also negative in a L5178Y MOLY assay, indicating low potential for genetic toxicity. These data along with measurements of the physiochemical properties and pharmacokinetic profile demonstrate that BTOs have the potential to be developed into a new class of antitubercular drugs. PMID:22691154

Germinal center hypoxia potentiates immunoglobulin class switch recombination

PubMed Central

Abbott, Robert K.; Thayer, Molly; Labuda, Jasmine; Silva, Murillo; Philbrook, Phaethon; Cain, Derek W.; Kojima, Hidefumi; Hatfield, Stephen; Sethumadhavan, Shalini; Ohta, Akio; Reinherz, Ellis L.; Kelsoe, Garnett; Sitkovsky, Michail

2016-01-01

Germinal centers (GCs) are anatomic sites where B cells undergo secondary diversification to produce high affinity, class switched antibodies. We hypothesized that proliferating B cells in GCs create a hypoxic microenvironment that governs their further differentiation. Using molecular markers, we found GCs to be predominantly hypoxic. Compared to normoxia (21% O2), hypoxic culture conditions (1% O2) in vitro accelerated class switching and plasma cell formation and enhanced expression of GL-7 on B and CD4+ T cells. Reversal of GC hypoxia in vivo by breathing 60% O2 during immunization resulted in reduced frequencies of GC B cells, T follicular helper (TFH) cells and plasmacytes, as well as lower expression of ICOS on TFH. Importantly, this reversal of GC hypoxia decreased antigen-specific serum IgG1 and reduced the frequency of IgG1+ B cells within the antigen specific GC. Taken together, these observations reveal a critical role for hypoxia in GC B cell differentiation. PMID:27798169

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

Impact of Hypoxia on Drug Resistance and Growth Characteristics of Mycobacterium tuberculosis Clinical Isolates.

PubMed

Liu, Zhonghua; Gao, Yulu; Yang, Hua; Bao, Haiyang; Qin, Lianhua; Zhu, Changtai; Chen, Yawen; Hu, Zhongyi

2016-01-01

Mycobacterium tuberculosis (MTB) is a specific aerobic bacterium, but can survive under hypoxic conditions, such as those in lung cheese necrosis, granulomas, or macrophages. It is not clear whether the drug sensitivity and growth characteristics of MTB under hypoxic conditions are different from those under aerobic conditions. In this study, we examined the drug resistance and growth characteristics of MTB clinical isolates by a large sample of in vitro drug susceptibility tests, using an automatic growth instrument. Under hypoxic conditions, variance in drug resistance was observed in nearly one-third of the MTB strains and was defined as MTB strains with changed drug sensitivity (MTB-CDS). Among these strains, resistance in a considerable proportion of clinical strains was significantly increased, and some strains emerged as multi-drug resistant. Growth test results revealed a high growth rate and large survival number in macrophages under hypoxia in MTB-CDS. According to the results of fluorescence quantitative PCR, the expression of some genes, including RegX3 (involving RIF resistance), Rv0194 (efflux pump gene), four genes related to transcription regulation (KstR, DosR, Rv0081 and WhiB3) and gene related to translation regulation (DATIN), were upregulated significantly under hypoxic conditions compared to that under aerobic conditions (p < 0.05). Thus, we concluded that some MTB clinical isolates can survive under hypoxic conditions and their resistance could change. As for poor clinical outcomes in patients, based on routine drug susceptibility testing, drug susceptibility tests for tuberculosis under hypoxic conditions should also be recommended. However, the detailed mechanisms of the effect of hypoxia on drug sensitivity and growth characteristics of MTB clinical isolates still requires further study.

Differential expression of IL-6/IL-6R and MAO-A regulates invasion/angiogenesis in breast cancer.

PubMed

Bharti, Rashmi; Dey, Goutam; Das, Anjan Kumar; Mandal, Mahitosh

2018-04-26

Monoamine oxidases (MAO) are mitochondrial enzymes functioning in oxidative metabolism of monoamines. The action of MAO-A has been typically described in neuro-pharmacological domains. Here, we have established a co-relation between IL-6/IL-6R and MAO-A and their regulation in hypoxia induced invasion/angiogenesis. We employed various in-vitro and in-vivo techniques and clinical samples. We studied a co-relation among MAO-A and IL-6/IL-6R and tumour angiogenesis/invasion in hypoxic environment in breast cancer model. Activation of IL-6/IL-6R and its downstream was found in hypoxic cancer cells. This elevation of IL-6/IL-6R caused sustained inhibition of MAO-A in hypoxic environment. Inhibition of IL-6R signalling or IL-6R siRNA increased MAO-A activity and inhibited tumour angiogenesis and invasion significantly in different models. Further, elevation of MAO-A with 5-azacytidine (5-Aza) modulated IL-6 mediated angiogenesis and invasive signatures including VEGF, MMPs and EMT in hypoxic breast cancer. High grade invasive ductal carcinoma (IDC) clinical specimen displayed elevated level of IL-6R and depleted MAO-A expression. Expression of VEGF and HIF-1α was unregulated and loss of E-Cadherin was observed in high grade IDC tissue specimen. Suppression of MAO-A by IL-6/IL-6R activation promotes tumour angiogenesis and invasion in hypoxic breast cancer environment.

Chloroquine inhibits autophagy and deteriorates the mitochondrial dysfunction and apoptosis in hypoxic rat neurons.

PubMed

Li, Peng; Hao, Lei; Guo, Yan-Yan; Yang, Guang-Lu; Mei, Hua; Li, Xiao-Hua; Zhai, Qiong-Xiang

2018-06-01

Mitochondrial dysfunction (MD) and apoptosis in the neurons are associated with neonatal hypoxic-ischemic (HI) encephalopathy (HIE). The present study was to explore the influence of autophagy on the induction of MD and apoptosis in the neurons in a neonatal HIE rats and in hypoxia-treated neurons in vitro. Ten-day-old HI rat pups were sacrificed for brain pathological examination and immunohistochemical analysis. The induction of autophagy, apoptosis and MD were also determined in the neurons under hypoxia, with or without autophagy inhibitor, chloroquine (CQ) treatment. HI treatment caused atrophy and apoptosis of neurons, with a significantly increased levels of apoptosis- and autophagy-associated proteins, such as cleaved caspase 3 and the B subunit of autophagy-related microtubule-associated protein 1 light chain 3 (LC3-B). in vitro experiments demonstrated that the hypoxia induced autophagy in neurons, as was inhibited by CQ. The hypoxia-induced cytochrome c release, cleaved caspase 3 and cleaved caspase 9 were aggravated by CQ. Moreover, there were higher levels of reactive oxygen species, more mitochondrial superoxide and less mitochondrial membrane potential in the CQ-treated neurons under hypoxia than in the neurons singularly under hypoxia. Apoptosis and autophagy were induced in HI neonatal rat neurons, autophagy inhibition deteriorates the hypoxia-induced neuron MD and apoptosis. It implies a neuroprotection of autophagy in the hypoxic-ischemic encephalopathy. Administration of autophagy inducer agents might be promising in HIE treatment. Copyright © 2018. Published by Elsevier Inc.

Hydrogen Sulfide Protects Renal Grafts Against Prolonged Cold Ischemia-Reperfusion Injury via Specific Mitochondrial Actions.

PubMed

Lobb, I; Jiang, J; Lian, D; Liu, W; Haig, A; Saha, M N; Torregrossa, R; Wood, M E; Whiteman, M; Sener, A

2017-02-01

Ischemia-reperfusion injury is unavoidably caused by loss and subsequent restoration of blood flow during organ procurement, and prolonged ischemia-reperfusion injury IRI results in increased rates of delayed graft function and early graft loss. The endogenously produced gasotransmitter, hydrogen sulfide (H 2 S), is a novel molecule that mitigates hypoxic tissue injury. The current study investigates the protective mitochondrial effects of H 2 S during in vivo cold storage and subsequent renal transplantation (RTx) and in vitro cold hypoxic renal injury. Donor allografts from Brown Norway rats treated with University of Wisconsin (UW) solution + H 2 S (150 μM NaSH) during prolonged (24-h) cold (4°C) storage exhibited significantly (p < 0.05) decreased acute necrotic/apoptotic injury and significantly (p < 0.05) improved function and recipient Lewis rat survival compared to UW solution alone. Treatment of rat kidney epithelial cells (NRK-52E) with the mitochondrial-targeted H 2 S donor, AP39, during in vitro cold hypoxic injury improved the protective capacity of H 2 S >1000-fold compared to similar levels of the nonspecific H 2 S donor, GYY4137 and also improved syngraft function and survival following prolonged cold storage compared to UW solution. H 2 S treatment mitigates cold IRI-associated renal injury via mitochondrial actions and could represent a novel therapeutic strategy to minimize the detrimental clinical outcomes of prolonged cold IRI during RTx. © 2016 The American Society of Transplantation and the American Society of Transplant Surgeons.

In vivo retinal and choroidal hypoxia imaging using a novel activatable hypoxia-selective near-infrared fluorescent probe.

PubMed

Fukuda, Shinichi; Okuda, Kensuke; Kishino, Genichiro; Hoshi, Sujin; Kawano, Itsuki; Fukuda, Masahiro; Yamashita, Toshiharu; Beheregaray, Simone; Nagano, Masumi; Ohneda, Osamu; Nagasawa, Hideko; Oshika, Tetsuro

2016-12-01

Retinal hypoxia plays a crucial role in ocular neovascular diseases, such as diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusion. Fluorescein angiography is useful for identifying the hypoxia extent by detecting non-perfusion areas or neovascularization, but its ability to detect early stages of hypoxia is limited. Recently, in vivo fluorescent probes for detecting hypoxia have been developed; however, these have not been extensively applied in ophthalmology. We evaluated whether a novel donor-excited photo-induced electron transfer (d-PeT) system based on an activatable hypoxia-selective near-infrared fluorescent (NIRF) probe (GPU-327) responds to both mild and severe hypoxia in various ocular ischemic diseases animal models. The ocular fundus examination offers unique opportunities for direct observation of the retina through the transparent cornea and lens. After injection of GPU-327 in various ocular hypoxic diseases of mouse and rabbit models, NIRF imaging in the ocular fundus can be performed noninvasively and easily by using commercially available fundus cameras. To investigate the safety of GPU-327, electroretinograms were also recorded after GPU-327 and PBS injection. Fluorescence of GPU-327 increased under mild hypoxic conditions in vitro. GPU-327 also yielded excellent signal-to-noise ratio without washing out in vivo experiments. By using near-infrared region, GPU-327 enables imaging of deeper ischemia, such as choroidal circulation. Additionally, from an electroretinogram, GPU-327 did not cause neurotoxicity. GPU-327 identified hypoxic area both in vivo and in vitro.

Chloronitroimidazoles as radiosensitizers of hypoxic cells in vitro.

PubMed

Wideł, M; Watras, J; Suwiński, J; Salwińska, E

1987-01-01

Some results of the first more complex studies in vitro on radio-sensitizing efficiency, cytotoxicity and reactivity with blood-thiols of a series of 4- or 5-nitroimidazoles substituted in the 5, 4 or 2 position with chlorine are presented. The derivatives of 4-nitroimidazole substituted in 5 position ("ortho" position) with Cl show higher radiosensitizing efficiency than one may expect from their reduction potential, E1/2. At the same time they are extremely toxic, especially for aerobic cells. It is considered that high biological activity of ortho-substituted 4-nitroimidazoles is connected with their considerable chemical reactivity towards thiols and suppression of those natural protective compounds in the cells. The corresponding 5-nitro isomers are about tenfold weaker sensitizers, and simultaneously much less cytotoxic, either in aerobic or in hypoxic conditions. The chloro-4(5)-nitroimidazoles nonsubstituted at N-1 and ionizable in aqueous solution are relatively weaker at the same time less toxic radiosensitizers. It is evaluated that potential application in radiotherapy may have those chloronitroimidazoles which show low aerobic cytotoxicity, moderate radiosensitizing ability and no reactivity towards thiols. On the basis of the study in vitro, we have selected such a compound: 1-methyl-2-chloro-4-nitroimidazole (P13) for screening in vivo.

Induction of dormancy in hypoxic human papillomavirus-positive cancer cells

PubMed Central

Hoppe-Seyler, Karin; Bossler, Felicitas; Lohrey, Claudia; Bulkescher, Julia; Rösl, Frank; Jansen, Lars; Mayer, Arnulf; Vaupel, Peter; Dürst, Matthias; Hoppe-Seyler, Felix

2017-01-01

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation. PMID:28115701

Proline oxidase promotes tumor cell survival in hypoxic tumor microenvironments

PubMed Central

Liu, Wei; Glunde, Kristine; Bhujwalla, Zaver M.; Raman, Venu; Sharma, Anit; Phang, James M.

2012-01-01

Proline is a readily released stress substrate that can be metabolized by proline oxidase (POX) to generate either reactive oxygen species to induce apoptosis or autophagy or ATP during times of nutrient stress. However, the contribution of proline metabolism to tumorigenesis in hypoxic microenvironments has not been explored. In this study, we investigated the different functions of POX under hypoxia and glucose depletion. We found that hypoxia induced POX expression in cancer cells in vitro and that POX upregulation co-localized with hypoxic tissues in vivo. In addition, the combination of hypoxia and low-glucose showed additive effects on POX expression. Similar to conditions of low glucose, hypoxia-mediated POX induction was dependent on AMP-activated protein kinase (AMPK) activation, but was independent of HIF-1α and HIF-2α. Under low-glucose and combined low-glucose and hypoxic conditions, proline catabolized by POX was used preferentially for ATP production, whereas under hypoxia, POX mediated autophagic signaling for survival by generating ROS. Although the specific mechanism was different for hypoxia and glucose deprivation, POX consistently contributed to tumor cell survival under these conditions. Together, our findings offer new insights into the metabolic reprogramming of tumor cells present within a hostile microenvironment and suggest that proline metabolism is a potential target for cancer therapeutics. PMID:22609800

Hypoxia-activated prodrug enhances therapeutic effect of sunitinib in melanoma

PubMed Central

Liu, Shujing; Tetzlaff, Michael T.; Wang, Tao; Chen, Xiang; Yang, Ruifeng; Kumar, Suresh M.; Vultur, Adina; Li, Pengxiang; Martin, James S.; Herlyn, Meenhard; Amaravadi, Ravi

2017-01-01

Angiogenesis is a critical step during tumor progression. Anti-angiogenic therapy has only provided modest benefits in delaying tumor progression despite its early promise in cancer treatment. It has been postulated that anti-angiogenic therapy may promote the emergence of a more aggressive cancer cell phenotype by generating increased tumor hypoxia—a well-recognized promoter of tumor progression. TH-302 is a 2-nitroimidazole triggered hypoxia-activated prodrug (HAP) which has been shown to selectively target the hypoxic tumor compartment and reduce tumor volume. Here, we show that melanoma cells grown under hypoxic conditions exhibit increased resistance to a wide variety of therapeutic agents in vitro and generate larger and more aggressive tumors in vivo than melanoma cells grown under normoxic conditions. However, hypoxic melanoma cells exhibit a pronounced sensitivity to TH-302 which is further enhanced by the addition of sunitinib. Short term sunitinib treatment fails to prolong the survival of melanoma bearing genetically engineered mice (Tyr::CreER; BRafCA;Ptenlox/lox) but increases tumor hypoxia. Long term TH-302 alone modestly prolongs the overall survival of melanoma bearing mice. Combination therapy of TH-302 with sunitinib further increases the survival of treated mice. These studies provide a translational rationale for combining hypoxic tumor cell targeted therapies with anti-angiogenics for treatment of melanoma. PMID:29383148

Induction of dormancy in hypoxic human papillomavirus-positive cancer cells.

PubMed

Hoppe-Seyler, Karin; Bossler, Felicitas; Lohrey, Claudia; Bulkescher, Julia; Rösl, Frank; Jansen, Lars; Mayer, Arnulf; Vaupel, Peter; Dürst, Matthias; Hoppe-Seyler, Felix

2017-02-07

Oncogenic human papillomaviruses (HPVs) are closely linked to major human malignancies, including cervical and head and neck cancers. It is widely assumed that HPV-positive cancer cells are under selection pressure to continuously express the viral E6/E7 oncogenes, that their intracellular p53 levels are reconstituted on E6/E7 repression, and that E6/E7 inhibition phenotypically results in cellular senescence. Here we show that hypoxic conditions, as are often found in subregions of cervical and head and neck cancers, enable HPV-positive cancer cells to escape from these regulatory principles: E6/E7 is efficiently repressed, yet, p53 levels do not increase. Moreover, E6/E7 repression under hypoxia does not result in cellular senescence, owing to hypoxia-associated impaired mechanistic target of rapamycin (mTOR) signaling via the inhibitory REDD1/TSC2 axis. Instead, a reversible growth arrest is induced that can be overcome by reoxygenation. Impairment of mTOR signaling also interfered with the senescence response of hypoxic HPV-positive cancer cells toward prosenescent chemotherapy in vitro. Collectively, these findings indicate that hypoxic HPV-positive cancer cells can induce a reversible state of dormancy, with decreased viral antigen synthesis and increased therapeutic resistance, and may serve as reservoirs for tumor recurrence on reoxygenation.

2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study.

PubMed

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

2017-01-01

Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy (HIE), but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB) superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C), and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml). Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS) was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml) reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho Rad17. In summary, addition of 2-IB during hypothermia is able to attenuate hypoxia-induced neuronal cell damage in vitro . Combination treatment of hypothermia with 2-IB could be a promising strategy to reduce hypoxia-induced neuronal cell damage and should be considered in further animal and clinical studies.

Hypoxia promotes proliferation of human myogenic satellite cells: a potential benefactor in tissue engineering of skeletal muscle.

PubMed

Koning, Merel; Werker, Paul M N; van Luyn, Marja J A; Harmsen, Martin C

2011-07-01

Facial paralysis is a physically, psychologically, and socially disabling condition. Innovative treatment strategies based on regenerative medicine, in particular tissue engineering of skeletal muscle, are promising for treatment of patients with facial paralysis. The natural source for tissue-engineered muscle would be muscle stem cells, that is, human satellite cells (SC). In vivo, SC respond to hypoxic, ischemic muscle damage by activation, proliferation, differentiation to myotubes, and maturation to muscle fibers, while maintaining their reserve pool of SC. Therefore, our hypothesis is that hypoxia improves proliferation and differentiation of SC. During tissue engineering, a three-dimensional construct, or implanting SC in vivo, SC will encounter hypoxic environments. Thus, we set out to test our hypothesis on SC in vitro. During the first five passages, hypoxically cultured SC proliferated faster than their counterparts under normoxia. Moreover, also at higher passages, a switch from normoxia to hypoxia enhanced proliferation of SC. Hypoxia did not affect the expression of SC markers desmin and NCAM. However, the average surface expression per cell of NCAM was downregulated by hypoxia, and it also downregulated the gene expression of NCAM. The gene expression of the myogenic transcription factors PAX7, MYF5, and MYOD was upregulated by hypoxia. Moreover, gene expression of structural proteins α-sarcomeric actin, and myosins MYL1 and MYL3 was upregulated by hypoxia during differentiation. This indicates that hypoxia promotes a promyogenic shift in SC. Finally, Pax7 expression was not influenced by hypoxia and maintained in a subset of mononucleated cells, whereas these cells were devoid of structural muscle proteins. This suggests that during myogenesis in vitro, at least part of the SC adopt a quiescent, that is, reserve cells, phenotype. In conclusion, tissue engineering under hypoxic conditions would seem favorable in terms of myogenic proliferation, while maintaining the quiescent SC pool.

Neurotrophic and neuroprotective potential of human limbus-derived mesenchymal stromal cells.

PubMed

Liang, Chang-Min; Weng, Shao-Ju; Tsai, Tung-Han; Li, I-Hsun; Lu, Pin-Hui; Ma, Kuo-Hsing; Tai, Ming-Cheng; Chen, Jiann-Torng; Cheng, Cheng-Yi; Huang, Yuahn-Sieh

2014-10-01

The purpose of this study was to examine neurotrophic and neuroprotective effects of limbus stroma-derived mesenchymal stromal cells (L-MSCs) on cortical neurons in vitro and in vivo. Cultured L-MSCs were characterized by flow cytometry and immunofluorescence through the use of specific MSC marker antibodies. Conditioned media were collected from normoxia- and hypoxia-treated L-MSCs to assess neurotrophic effects. Neuroprotective potentials were evaluated through the use of in vitro hypoxic cortical neuron culture and in vivo rat focal cerebral ischemia models. Neuronal morphology was confirmed by immunofluorescence with the use of anti-MAP2 antibody. Post-ischemic infarct volume and motor behavior were assayed by means of triphenyltetrazolium chloride staining and open-field testing, respectively. Human growth antibody arrays and enzyme-linked immunoassays were used to analyze trophic/growth factors contained in conditioned media. Isolated human L-MSCs highly expressed CD29, CD90 and CD105 but not CD34 and CD45. Mesenchymal lineage cell surface expression pattern and differentiation capacity were identical to MSCs derived form human bone marrow and adipose tissue. The L-MSC normoxic and hypoxic conditioned media both promoted neurite outgrowth in cultured cortical neurons. Hypoxic conditioned medium showed superior neurotrophic function and neuroprotective potential with reduced ischemic brain injury and improved functional recovery in rat focal cerebral ischemia models. Human growth factor arrays and enzyme-linked immunoassays measurements showed neuroprotective and growth-associated cytokines (vascular endothelial growth factor [VEGF], VEGFR3, brain-derived neurotrophic factor, insulin-like growth factor -2 and hepatocyte growth factor) contained in conditioned media. Hypoxic exposure caused VEGF and brain-derived neurotrophic factor upregulation, possibly contributing to neurotrophic and neuroprotective effects. L-MSCs can secrete various neurotrophic factors stimulating neurite outgrowth and protecting neurons against brain ischemic injury through paracrine mechanism. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

PubMed

Segretti, Natanael Dante; Simões, Cristina Kortstee; Corrêa, Michelle Fidelis; Felli, Veni Maria Andres; Miyata, Marcelo; Cho, Sang Hyun; Franzblau, Scott Gary; Fernandes, João Paulo Dos Santos

2016-07-01

Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile. Copyright © 2016 Elsevier Ltd. All rights reserved.

Do hypoxia/normoxia culturing conditions change the neuroregulatory profile of Wharton Jelly mesenchymal stem cell secretome?

PubMed

Teixeira, Fábio G; Panchalingam, Krishna M; Anjo, Sandra Isabel; Manadas, Bruno; Pereira, Ricardo; Sousa, Nuno; Salgado, António J; Behie, Leo A

2015-07-24

The use of human umbilical cord Wharton Jelly-derived mesenchymal stem cells (hWJ-MSCs) has been considered a new potential source for future safe applications in regenerative medicine. Indeed, the application of hWJ-MSCs into different animal models of disease, including those from the central nervous system, has shown remarkable therapeutic benefits mostly associated with their secretome. Conventionally, hWJ-MSCs are cultured and characterized under normoxic conditions (21 % oxygen tension), although the oxygen levels within tissues are typically much lower (hypoxic) than these standard culture conditions. Therefore, oxygen tension represents an important environmental factor that may affect the performance of mesenchymal stem cells in vivo. However, the impact of hypoxic conditions on distinct mesenchymal stem cell characteristics, such as the secretome, still remains unclear. In the present study, we have examined the effects of normoxic (21 % O2) and hypoxic (5 % O2) conditions on the hWJ-MSC secretome. Subsequently, we address the impact of the distinct secretome in the neuronal cell survival and differentiation of human neural progenitor cells. The present data indicate that the hWJ-MSC secretome collected from normoxic and hypoxic conditions displayed similar effects in supporting neuronal differentiation of human neural progenitor cells in vitro. However, proteomic analysis revealed that the use of hypoxic preconditioning led to the upregulation of several proteins within the hWJ-MSC secretome. Our results suggest that the optimization of parameters such as hypoxia may lead to the development of strategies that enhance the therapeutic effects of the secretome for future regenerative medicine studies and applications.

Hypoxia-Activated Prodrug TH-302 Targets Hypoxic Bone Marrow Niches in Preclinical Leukemia Models.

PubMed

Benito, Juliana; Ramirez, Marc S; Millward, Niki Zacharias; Velez, Juliana; Harutyunyan, Karine G; Lu, Hongbo; Shi, Yue-Xi; Matre, Polina; Jacamo, Rodrigo; Ma, Helen; Konoplev, Sergej; McQueen, Teresa; Volgin, Andrei; Protopopova, Marina; Mu, Hong; Lee, Jaehyuk; Bhattacharya, Pratip K; Marszalek, Joseph R; Davis, R Eric; Bankson, James A; Cortes, Jorge E; Hart, Charles P; Andreeff, Michael; Konopleva, Marina

2016-04-01

To characterize the prevalence of hypoxia in the leukemic bone marrow, its association with metabolic and transcriptional changes in the leukemic blasts and the utility of hypoxia-activated prodrug TH-302 in leukemia models. Hyperpolarized magnetic resonance spectroscopy was utilized to interrogate the pyruvate metabolism of the bone marrow in the murine acute myeloid leukemia (AML) model. Nanostring technology was used to evaluate a gene set defining a hypoxia signature in leukemic blasts and normal donors. The efficacy of the hypoxia-activated prodrug TH-302 was examined in the in vitro and in vivo leukemia models. Metabolic imaging has demonstrated increased glycolysis in the femur of leukemic mice compared with healthy control mice, suggesting metabolic reprogramming of hypoxic bone marrow niches. Primary leukemic blasts in samples from AML patients overexpressed genes defining a "hypoxia index" compared with samples from normal donors. TH-302 depleted hypoxic cells, prolonged survival of xenograft leukemia models, and reduced the leukemia stem cell pool in vivo In the aggressive FLT3/ITD MOLM-13 model, combination of TH-302 with tyrosine kinase inhibitor sorafenib had greater antileukemia effects than either drug alone. Importantly, residual leukemic bone marrow cells in a syngeneic AML model remain hypoxic after chemotherapy. In turn, administration of TH-302 following chemotherapy treatment to mice with residual disease prolonged survival, suggesting that this approach may be suitable for eliminating chemotherapy-resistant leukemia cells. These findings implicate a pathogenic role of hypoxia in leukemia maintenance and chemoresistance and demonstrate the feasibility of targeting hypoxic cells by hypoxia cytotoxins. ©2015 American Association for Cancer Research.

MTA1 and MTA3 Regulate HIF1a Expression in Hypoxia-Treated Human Trophoblast Cell Line HTR8/Svneo

PubMed Central

Wang, Kai; Chen, Ying; Ferguson, Susan D.; Leach, Richard E.

2015-01-01

Hypoxia plays an important role in placental trophoblast differentiation and function during early pregnancy. Hypoxia-inducible factor 1 alpha (HIF1a) is known to regulate cellular adaption to hypoxic conditions. However, our current understanding of the role of HIF1a in trophoblast physiology is far from complete. Metastasis Associated Protein 1 and 3 (MTA1 and MTA3) are components of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a chromatin remodeling complex, and are highly expressed in term placental trophoblasts. However, the role of MTA1 and MTA3 in the hypoxic placental environment of early pregnancy is unknown. In the present study, we examined the association among MTA1, MTA3 and HIF1a expression under hypoxic conditions in trophoblasts both in vivo and in vitro. We first investigated the localization of MTA1 and MTA3 with HIF1a expression in the placental trophoblast of 1st trimester placenta via immunohistochemistry. Our data reveals that under physiologically hypoxic environment, MTA1 and MTA3 along with HIF1a are highly expressed by villous trophoblasts. Next, we investigated the effect of hypoxia on these genes in vitro using the first trimester-derived HTR8/SVneo cell line and observed up-regulation of MTA1 and MTA3 as well as HIF1a protein following hypoxia treatment. To investigate the direct effect of MTA1 and MTA3 upon HIF1a, we over-expressed MTA1 and MTA3 genes in HTR8/SVneo cells respectively and examined protein levels of HIF1a via Western blot as well as HIF1a target gene expression using a luciferase assay driven by a hypoxia-response element promoter (HRE-luciferase). We found that over-expressions of MTA1 and MTA3 up-regulate both HIF1a protein level and HRE-luciferase activity under hypoxic condition. In summary, both MTA1 and MTA3 are induced by hypoxia and up-regulate HIF1a expression and HIF1a target gene expression in trophoblasts. These data suggest that MTA1 and MTA3 play critical roles in trophoblast function and differentiation during early pregnancy. PMID:25705708

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).

Selective reversible inhibition of autophagy in hypoxic breast cancer cells promotes pulmonary metastasis

PubMed Central

Dower, Christopher M.; Bhat, Neema; Wang, Edward W.; Wang, Hong-Gang

2016-01-01

Autophagy influences how cancer cells respond to nutrient deprivation and hypoxic stress, two hallmarks of the tumor microenvironment (TME). In this study, we explored the impact of autophagy on the pathophysiology of breast cancer cells, using a novel hypoxia-dependent, reversible dominant negative strategy to regulate autophagy at the cellular level within the TME. Suppression of autophagy via hypoxia-induced expression of the kinase-dead unc-51 like autophagy activating kinase (ULK1) mutant K46N increased lung metastases in MDA-MB-231 xenograft mouse models. Consistent with this effect, expressing a dominant-negative mutant of ULK1 or ATG4b or a ULK1-targeting shRNA facilitated cell migration in vitro. Functional proteomic and transcriptome analysis revealed that loss of hypoxia-regulated autophagy promotes metastasis via induction of the fibronectin integrin signaling axis. Indeed, loss of ULK1 function increased fibronectin deposition in the hypoxic TME. Together, our results indicated that hypoxia-regulated autophagy suppresses metastasis in breast cancer by preventing tumor fibrosis. These results also suggest cautions in the development of autophagy-based strategies for cancer treatment. PMID:28115361

Oxygen nanobubbles revert hypoxia by methylation programming.

PubMed

Bhandari, Pushpak N; Cui, Yi; Elzey, Bennett D; Goergen, Craig J; Long, Christopher M; Irudayaraj, Joseph

2017-08-24

Targeting the hypoxic tumor microenvironment has a broad impact in cancer epigenetics and therapeutics. Oxygen encapsulated nanosize carboxymethyl cellulosic nanobubbles were developed for mitigating the hypoxic regions of tumors to weaken the hypoxia-driven pathways and inhibit tumor growth. We show that 5-methylcytosine (5mC) hypomethylation in hypoxic regions of a tumor can be reverted to enhance cancer treatment by epigenetic regulation, using oxygen nanobubbles in the sub-100 nm size range, both, in vitro and in vivo. Oxygen nanobubbles were effective in significantly delaying tumor progression and improving survival rates in mice models. Further, significant hypermethylation was observed in promoter DNA region of BRCA1 due to oxygen nanobubble (ONB) treatment. The nanobubbles can also reprogram several hypoxia associated and tumor suppressor genes such as MAT2A and PDK-1, in addition to serving as an ultrasound contrast agent. Our approach to develop nanosized oxygen encapsulated bubbles as an ultrasound contrast agent for methylation reversal is expected to have a significant impact in epigenetic programming and to serve as an adjuvant to cancer treatment.

Post-natal hypoxic activity of the central respiratory command is improved in transgenic mice overexpressing Epo in the brain.

PubMed

Caravagna, Céline; Kinkead, Richard; Soliz, Jorge

2014-08-15

Previous studies indicated that erythropoietin modulates central respiratory command in mice. Specifically, a one-hour incubation of the brainstems with erythropoietin attenuates hypoxia-induced central respiratory depression. Here, using transgenic mice constitutively overexpressing erythropoietin specifically in the brain (Tg21), we investigated the effect of chronic erythropoietin stimulation on central respiratory command activity during post-natal development. In vitro brainstem-spinal cord preparations from mice at 0 (P0) or 3 days of age (P3) were used to record the fictive inspiratory activity from the C4 ventral root. Our results show that erythropoietin already stimulates the hypoxic burst frequency at P0, and at P3, erythropoietin effectively stimulates the hypoxic burst frequency and amplitude. Because the maturation of the central respiratory command in mice is characterized by a decrease in the burst frequency with age, our results also suggest that erythropoietin accelerates the maturation of the newborn respiratory network and its response to hypoxia. Copyright © 2014 Elsevier B.V. All rights reserved.

Hypoxic ventilatory response in Tac1-/- neonatal mice following exposure to opioids.

PubMed

Berner, J; Shvarev, Y; Zimmer, A; Wickstrom, R

2012-12-01

Morphine is the dominating analgetic drug used in neonates, but opioid-induced respiratory depression limits its therapeutic use. In this study, we examined acute morphine effects on respiration during intermittent hypoxia in newborn Tac1 gene knockout mice (Tac1-/-) lacking substance P and neurokinin A. In vivo, plethysmography revealed a blunted hypoxic ventilatory response (HVR) in Tac1-/- mice. Morphine (10 mg/kg) depressed the HVR in wild-type animals through an effect on respiratory frequency, whereas it increased tidal volumes in Tac1-/- during hypoxia, resulting in increased minute ventilation. Apneas were reduced during the first hypoxic episode in both morphine-exposed groups, but were restored subsequently in Tac1-/- mice. Morphine did not affect ventilation or apnea prevalence during baseline conditions. In vitro, morphine (50 nM) had no impact on anoxic response of brain stem preparations of either strain. In contrast, it suppressed the inspiratory rhythm during normoxia and potentiated development of posthypoxic neuronal arrest, especially in Tac1-/-. Thus this phenotype has a higher sensitivity to the depressive effects of morphine on inspiratory rhythm generation, but morphine does not modify the reactivity to oxygen deprivation. In conclusion, although Tac1-/- mice are similar to wild-type animals during normoxia, they differed by displaying a reversed pattern with an improved HVR during intermittent hypoxia both in vivo and in vitro. These data suggest that opioids and the substance P-ergic system interact in the HVR, and that reducing the activity in the tachykinin system may alter the respiratory effects of opioid treatment in newborns.

Neuroprotective effect of lidocaine: is there clinical potential?

PubMed Central

Leng, Tiandong; Gao, Xiuren; Dilger, James P; Lin, Jun

2016-01-01

Local anesthetic lidocaine has been shown to be protective in animal models of focal and global ischemia as well as in in vitro hypoxic models. Lidocaine has been tested in patients for its potential protective effect on postoperative cognitive dysfunction. This mini-review summarizes the laboratory and clinical evidences and discusses its clinical applications as neuroprotective agent. PMID:27186318

Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: Influence of oxygen and salicylic acid concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zitta, Karina; Meybohm, Patrick; Bein, Berthold

In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10 {mu}M SA on CaCo-2 cells grownmore » under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen. -- Highlights: Black-Right-Pointing-Pointer Effects of salicylic acid on colon carcinoma cells grown under normoxic and hypoxic conditions Black-Right-Pointing-Pointer Salicylic acid increases caspase-3/7 activity and hydrogen peroxide release under normoxia Black-Right-Pointing-Pointer Salicylic acid decreases pro-survival erk-1/2 phosphorylation under normoxia Black-Right-Pointing-Pointer Salicylic acid does not influence any of the investigated parameters under hypoxia.« less

Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

PubMed

Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

2013-01-01

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

Differential Requirements for T Cells in Viruslike Particle- and Rotavirus-Induced Protective Immunity▿

PubMed Central

Blutt, Sarah E.; Warfield, Kelly L.; Estes, Mary K.; Conner, Margaret E.

2008-01-01

Correlates of protection from rotavirus infection are controversial. We compared the roles of B and T lymphocytes in protective immunity induced either by intranasally administered nonreplicating viruslike particles or inactivated virus or by orally administered murine rotavirus. We found that protection induced by nonreplicating vaccines requires CD4+ T cells and CD40/CD40L. In contrast, T cells were not required for short-term protective immunity induced by infection, but both T-cell-dependent and -independent mechanisms contributed to long-term maintenance of protection. Our findings indicate that more than one marker of protective immunity exists and that these markers depend on the vaccine that is administered. PMID:18184712

Design, Synthesis, and Characterization of N-Oxide-Containing Heterocycles with in Vivo Sterilizing Antitubercular Activity

PubMed Central

2017-01-01

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the infectious disease responsible for the highest number of deaths worldwide. Herein, 22 new N-oxide-containing compounds were synthesized followed by in vitro and in vivo evaluation of their antitubercular potential against Mtb. Compound 8 was found to be the most promising compound, with MIC90 values of 1.10 and 6.62 μM against active and nonreplicating Mtb, respectively. Additionally, we carried out in vivo experiments to confirm the safety and efficacy of compound 8; the compound was found to be orally bioavailable and highly effective, leading to a reduction of Mtb to undetectable levels in a mouse model of infection. Microarray-based initial studies on the mechanism of action suggest that compound 8 blocks translation. Altogether, these results indicate that benzofuroxan derivative 8 is a promising lead compound for the development of a novel chemical class of antitubercular drugs. PMID:28968083

Tricyclic [1,2,4]triazine 1,4-dioxides as hypoxia selective cytotoxins.

PubMed

Hay, Michael P; Hicks, Kevin O; Pchalek, Karin; Lee, Ho H; Blaser, Adrian; Pruijn, Frederik B; Anderson, Robert F; Shinde, Sujata S; Wilson, William R; Denny, William A

2008-11-13

A series of novel tricyclic triazine-di- N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen-, and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUC req) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics.

Tricyclic [1,2,4]Triazine 1,4-Dioxides As Hypoxia Selective Cytotoxins

PubMed Central

Hay, Michael P.; Hicks, Kevin O.; Pchalek, Karin; Lee, Ho H.; Blaser, Adrian; Pruijn, Frederik B.; Anderson, Robert F.; Shinde, Sujata S.; Wilson, William R.; Denny, William A.

2009-01-01

A series of novel tricyclic triazine-di-N-oxides (TTOs) related to tirapazamine have been designed and prepared. A wide range of structural arrangements with cycloalkyl, oxygen- and nitrogen-containing saturated rings fused to the triazine core, coupled with various side chains linked to either hemisphere, resulted in TTO analogues that displayed hypoxia-selective cytotoxicity in vitro. Optimal rates of hypoxic metabolism and tissue diffusion coefficients were achieved with fused cycloalkyl rings in combination with both the 3-aminoalkyl or 3-alkyl substituents linked to weakly basic soluble amines. The selection was further refined using pharmacokinetic/pharmacodynamic model predictions of the in vivo hypoxic potency (AUCreq) and selectivity (HCD) with 12 TTO analogues predicted to be active in vivo, subject to the achievement of adequate plasma pharmacokinetics. PMID:18847185

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

PubMed Central

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

2014-01-01

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

Molecular and functional evaluation of a novel HIF inhibitor, benzopyranyl 1,2,3-triazole compound

PubMed Central

Park, Kyunghye; Lee, Hye Eun; Lee, Sun Hee; Lee, Doohyun; Lee, Taeho; Lee, You Mie

2017-01-01

Hypoxia occurs in a variety of pathological events, including the formation of solid tumors. Hypoxia-inducible factor (HIF)-1α is stabilized under hypoxic conditions and is a key molecule in tumor growth and angiogenesis. Seeking to develop novel cancer therapeutics, we investigated small molecules from our in-house chemical libraries to target HIF-1α. We employed a dual-luciferase assay that uses a luciferase (Luc) reporter vector harboring five copies of hypoxia-responsive element (HRE) in the promoter. Under hypoxic conditions that increased Luc reporter activity by four-fold, we screened 144 different compounds, nine of which showed 30–50% inhibition of hypoxia-induced Luc reporter activity. Among these, “Compound 12, a benzopyranyl 1,2,3-triazole” was the most efficient at inhibiting the expression of HIF-1α under hypoxic conditions, reducing its expression by 80%. Under hypoxic conditions, the half maximal IC50 of the compound was 24 nM in HEK-293 human embryonic kidney cells, and 2 nM in A549 human lung carcinoma cells. Under hypoxic conditions, Compound 12 increased hydroxylated HIF-1α levels and HIF-1α ubiquitination, and also dose-dependently decreased HIF-1α target gene expression as well as vascular endothelial growth factor (VEGF) secretion. Furthermore, this compound inhibited VEGF-induced in vitro angiogenesis in human umbilical vein endothelial cells (HUVECs), and in vivo, it inhibited chick chorioallantoic membrane angiogenesis. In allogaft assays, cotreatment with Compound 12 and gefitinib significantly inhibited tumor growth and angiogenesis. Compound 12 can be a novel inhibitor of HIF-1α by accelerating its degradation, and shows much potential as an anti-cancer agent through its ability to suppress tumor growth and angiogenesis. PMID:27999195

Polymeric Nano-Encapsulation of Curcumin Enhances its Anti-Cancer Activity in Breast (MDA-MB231) and Lung (A549) Cancer Cells Through Reduction in Expression of HIF-1α and Nuclear p65 (Rel A).

PubMed

Khan, Mohammed N; Haggag, Yusuf A; Lane, Majella E; McCarron, Paul A; Tambuwala, Murtaza M

2018-02-14

The anti-cancer potential of curcumin, a natural NFκβ inhibitor, has been reported extensively in breast, lung and other cancers. In vitro and in vivo studies indicate that the therapeutic efficacy of curcumin is enhanced when formulated in a nanoparticulate carrier. However, the mechanism of action of curcumin at the molecular level in the hypoxic tumour micro-environment is not fully understood. Hence, the aim of our study was to investigate the mechanism of action of curcumin formulated as nanoparticles in in vitro models of breast and lung cancer under an hypoxic microenvironment. Biodegradable poly(lactic-co-glycolic acid) PLGA nanoparticles (NP), loaded with curcumin (cur-PLGA-NP), were fabricated using a solvent evaporation technique to overcome solubility issues and to facilitate intracellular curcumin delivery. Cytotoxicity of free curcumin and cur-PLGA-NP was evaluated in MDA-MB-231 and A549 cell lines using migration, invasion and colony formation assays. All treatments were performed under an hypoxic micro-environment and whole cell lysates from controls and test groups were used to determine the expression of HIF-1α and p65 levels using ELISA assays. A ten-fold increase in solubility, three-fold increase in anti-cancer activity and a significant reduction in the levels of cellular HIF-1α and nuclear p65 (Rel A) were observed for cur-PLGA-NP, when compared to free curcumin. Our findings indicate that curcumin can effectively lower the elevated levels of HIF-1α and nuclear p65 (Rel A) in breast and lung cancer cells under an hypoxic tumour micro-environment when delivered in nanoparticulate form. This applied means of colloidal delivery could explain the improved anti-cancer efficacy of curcumin and has further potential applications in enhancing the activity of anti-cancer agents of low solubility. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Irradiation at 636 nm positively affects diabetic wounded and hypoxic cells in vitro.

PubMed

Sekhejane, Palesa R; Houreld, Nicolette N; Abrahamse, Heidi

2011-08-01

This study investigated the effect of low-intensity laser irradiation (LILI) on pro-inflammatory cytokines involved in wound healing processes in diabetes and hypoxia. Diabetes is associated with impaired wound healing and a prolonged inflammatory phase. Pro-inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6 are elevated in diabetes. LILI has been reported to accelerate wound healing and decrease inflammatory cytokines. A human skin fibroblast cell line (WS1) was used in vitro. Cells were exposed to various insults, namely, wounding, and a diabetic or hypoxic environment. Experimental cells were exposed to an energy density of 5  J/cm(2) using a continuous wave 636-nm diode laser at an average power of 95  mW, an illuminated area of 9.05  cm(2), and an irradiance of 11 mW/cm(2) (irradiation time, 476  sec). The effect of laser irradiation on cytokine expression was examined at 1 or 24  h post-irradiation. Cellular morphology, viability, proliferation, and cytokine expression (IL-1β, IL-6, and TNF-α) were investigated. Translocation of nuclear factor-kappa B (NF-κB) was also determined. There was a higher rate of migration in irradiated wounded cultures, and irradiated hypoxic cells showed an improvement in cellular morphology. All cell models showed an increase in proliferation. Normal wounded cells showed a decrease in apoptosis, TNF-α, and IL-1β. Diabetic wounded cells showed an increase in viability and a decrease in apoptosis and IL-1β, whereas hypoxic cells showed an increase in viability and IL-6, and a decrease in apoptosis and TNF-α. NF-κB was translocated into the nucleus post-irradiation. Phototherapy resulted in hastened wound closure, increased proliferation, and normalization of cellular function. The decrease in the different pro-inflammatory cytokines and NF-κB translocation was model and time dependent. Overall, laser irradiation resulted in a reduction in inflammatory cytokines and directed cells into the cell survival pathway.

Fertilizer Response Curves for Commercial Southern Forest Species Defined with an Un-Replicated Experimental Design.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Coleman, Mark; Aubrey, Doug; Coyle, David, R.

2005-11-01

There has been recent interest in use of non-replicated regression experimental designs in forestry, as the need for replication in experimental design is burdensome on limited research budgets. We wanted to determine the interacting effects of soil moisture and nutrient availability on the production of various southeastern forest trees (two clones of Populus deltoides, open pollinated Platanus occidentalis, Liquidambar styraciflua and Pinus taeda). Additionally, we required an understanding of the fertilizer response curve. To accomplish both objectives we developed a composite design that includes a core ANOVA approach to consider treatment interactions, with the addition of non-replicated regression plots receivingmore » a range of fertilizer levels for the primary irrigation treatment.« less

Development and characterization of a human three-dimensional chondrosarcoma culture for in vitro drug testing.

PubMed

Voissiere, Aurélien; Jouberton, Elodie; Maubert, Elise; Degoul, Françoise; Peyrode, Caroline; Chezal, Jean-Michel; Miot-Noirault, Élisabeth

2017-01-01

It has been suggested that chemoresistance of chondrosarcoma (CHS), the cartilage tumor, is caused by the phenotypic microenvironmental features of the tumor tissue, mainly the chondrogenic extracellular matrix (ECM), and hypoxia. We developed and characterized a multicellular tumor spheroid (MCTS) of human chondrosarcoma HEMC-SS cells to gain insight into tumor cell biology and drug response. At Day 7, HEMC-SS spheroids exhibited a homogeneous distribution of proliferative Ki-67 positive cells, whereas in larger spheroids (Day 14 and Day 20), proliferation was mainly localized in the periphery. In the core of larger spheroids, apoptotic cells were evidenced by TUNEL assay, and hypoxia by pimonidazole staining. Interestingly, VEGF excretion, evidenced by ELISA on culture media, was detectable from Day 14 spheroids, and increased as the spheroids grew in size. HEMC-SS spheroids synthesized a chondrogenic extracellular matrix rich in glycosaminoglycans and type-2 collagen. Finally, we investigated the sensitivity of Day 7 and Day 14 chondrosarcoma MCTS to hypoxia-activated prodrug TH-302 and doxorubicin compared with their 2D counterparts. As expected, TH-302 exhibited higher cytotoxic activity on larger hypoxic spheroids (Day 14) than on non-hypoxic spheroids (Day 7), with multicellular resistance index (MCRI) values of 7.7 and 9.1 respectively. For doxorubicin, the larger-sized spheroids exhibited higher drug resistance (MCRI of 5.0 for Day 7 and 18.3 for Day 14 spheroids), possibly due to impeded drug penetration into the deep layer of spheroids, evidenced by its auto-fluorescence property. We have developed a model of human chondrosarcoma MCTS that combines an ECM rich in glycosaminoglycans with a high hypoxic core associated with VEGF excretion. This model could offer a more predictive in vitro chondrosarcoma system for screening drugs targeting tumor cells and their microenvironment.

Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function.

PubMed

Damous, Luciana Lamarão; Nakamuta, Juliana Sanajotti; Soares, José Maria; Maciel, Gustavo Arantes Rosa; Simões, Ricardo Dos Santos; Montero, Edna Frasson de Souza; Krieger, José Eduardo; Baracat, Edmund Chada

2014-03-20

Cryopreservation of the ovarian tissue has shown promising results. However, there remain controversial issues such as the short half-life of grafts. In this aspect, there are some evidences that preconditioning the ovarian tissue before transplantation is beneficial. To determine the effect of hypoxic preconditioning in vitro on ovarian tissue prior to transplantation. Eighteen female adult Wistar rats, were sorted into three experimental groups. Ovaries were maintained in DMEM low glucose serum free at 37°C with 5% CO2, at atmospheric oxigen concentration (normoxia) or 1% O2 (hypoxia) for 16 hours. Oxigen concentration was determined by injection of nitrogen in the incubator. Animals submitted to ovarian transplantation immediately after oophorectomy were the Control Group (C). After this, the ovaries were implanted in the retroperitoneum with nonabsorbable suture and animals evaluated for thirty days after transplantation. Beginning on postoperative (PO) day 11, a daily collection of vaginal smear was carried out. Analyses comprised morphological, morphometric (counting ovarian follicles and corpora lutea) and immunohistochemistry for cleaved caspase-3 (apoptosis). In normoxia and control groups all animals recovered their estrous cycles, while in the hypoxia group, two animals did not ovulate but, among those which did, resumption took longer than in the other groups (p < 0.05). The number of ovarian follicles and corpora lutea decreased significantly in the hypoxia group when compared to the other two groups (p < 0.001) and apoptosis was increased in the few ovarian follicles which remained viable (p < 0.001). The hypoxic preconditioning in vitro was not beneficial to the graft and worsened their viability, compromising its functionality or delaying the return of this.

Females transplanted with ovaries subjected to hypoxic preconditioning show impair of ovarian function

PubMed Central

2014-01-01

Background Cryopreservation of the ovarian tissue has shown promising results. However, there remain controversial issues such as the short half-life of grafts. In this aspect, there are some evidences that preconditioning the ovarian tissue before transplantation is beneficial. Objective To determine the effect of hypoxic preconditioning in vitro on ovarian tissue prior to transplantation. Methods Eighteen female adult Wistar rats, were sorted into three experimental groups. Ovaries were maintained in DMEM low glucose serum free at 37°C with 5% CO2, at atmospheric oxigen concentration (normoxia) or 1% O2 (hypoxia) for 16 hours. Oxigen concentration was determined by injection of nitrogen in the incubator. Animals submitted to ovarian transplantation immediately after oophorectomy were the Control Group (C). After this, the ovaries were implanted in the retroperitoneum with nonabsorbable suture and animals evaluated for thirty days after transplantation. Beginning on postoperative (PO) day 11, a daily collection of vaginal smear was carried out. Analyses comprised morphological, morphometric (counting ovarian follicles and corpora lutea) and immunohistochemistry for cleaved caspase-3 (apoptosis). Results In normoxia and control groups all animals recovered their estrous cycles, while in the hypoxia group, two animals did not ovulate but, among those which did, resumption took longer than in the other groups (p < 0.05). The number of ovarian follicles and corpora lutea decreased significantly in the hypoxia group when compared to the other two groups (p < 0.001) and apoptosis was increased in the few ovarian follicles which remained viable (p < 0.001). Conclusion The hypoxic preconditioning in vitro was not beneficial to the graft and worsened their viability, compromising its functionality or delaying the return of this. PMID:24655551

The expression of Argonaute2 and related microRNA biogenesis proteins in normal and hypoxic trophoblasts.

PubMed

Donker, Rogier B; Mouillet, Jean-François; Nelson, D Michael; Sadovsky, Yoel

2007-04-01

Endogenous microRNAs (miRNAs) post-transcriptionally regulate mRNA and protein expression during tissue development and function. Whereas adaptation to environmental insults are tightly regulated in human tissues, the role of miRNAs and miRNA biogenesis proteins in this context is inadequately explored. We sought to analyse the expression of the key RNAi enzyme Argonaute2 (Ago2) and other miRNA biogenesis proteins in human trophoblasts during differentiation and in hypoxic environment. Using an in vitro analysis of primary term human trophoblasts, we identified the expression of the core miRNA biogenesis proteins in human villous trophoblasts, with expression levels unaffected by cellular differentiation. We found that the miRNA biosynthetic pathway was functional and produced miRNAs, with miR-93 up-regulated and miR-424 down-regulated in hypoxic environment. In contrast, hypoxia did not alter the expression of key miRNA machinery proteins. The pivotal miRNA processing enzyme Ago2, along with its interacting protein DP103, were expressed in normal placentas as well as in placentas from pregnancies complicated by placental hypoperfusion that resulted in fetal growth restriction. Ago2 and DP103 co-immunoprecipitated, and did not limit trophoblast response to hypoxic stress. We concluded that the core miRNA machinery proteins are expressed and functional in human trophoblasts. The influence of hypoxia on the expression of a subset of placental miRNA species is unlikely to reflect altered expression of key miRNA biogenesis proteins.

Hypoxic Three-Dimensional Scaffold-Free Aggregate Cultivation of Mesenchymal Stem Cells in a Stirred Tank Reactor.

PubMed

Egger, Dominik; Schwedhelm, Ivo; Hansmann, Jan; Kasper, Cornelia

2017-05-23

Extensive expansion of mesenchymal stem cells (MSCs) for cell-based therapies remains challenging since long-term cultivation and excessive passaging in two-dimensional conditions result in a loss of essential stem cell properties. Indeed, low survival rate of cells, alteration of surface marker profiles, and reduced differentiation capacity are observed after in vitro expansion and reduce therapeutic success in clinical studies. Remarkably, cultivation of MSCs in three-dimensional aggregates preserve stem cell properties. Hence, the large scale formation and cultivation of MSC aggregates is highly desirable. Besides other effects, MSCs cultivated under hypoxic conditions are known to display increased proliferation and genetic stability. Therefore, in this study we demonstrate cultivation of adipose derived human MSC aggregates in a stirred tank reactor under hypoxic conditions. Although aggregates were exposed to comparatively high average shear stress of 0.2 Pa as estimated by computational fluid dynamics, MSCs displayed a viability of 78-86% and maintained their surface marker profile and differentiation potential after cultivation. We postulate that cultivation of 3D MSC aggregates in stirred tank reactors is valuable for large-scale production of MSCs or their secreted compounds after further optimization of cultivation parameters.

VEGF production and signaling in Müller glia are critical to modulating vascular function and neuronal integrity in diabetic retinopathy and hypoxic retinal vascular diseases.

PubMed

Le, Yun-Zheng

2017-10-01

Müller glia (MG) are major retinal supporting cells that participate in retinal metabolism, function, maintenance, and protection. During the pathogenesis of diabetic retinopathy (DR), a neurovascular disease and a leading cause of blindness, MG modulate vascular function and neuronal integrity by regulating the production of angiogenic and trophic factors. In this article, I will (1) briefly summarize our work on delineating the role and mechanism of MG-modulated vascular function through the production of vascular endothelial growth factor (VEGF) and on investigating VEGF signaling-mediated MG viability and neural protection in diabetic animal models, (2) explore the relationship among VEGF and neurotrophins in protecting Müller cells in in vitro models of diabetes and hypoxia and its potential implication to neuroprotection in DR and hypoxic retinal diseases, and (3) discuss the relevance of our work to the effectiveness and safety of long-term anti-VEGF therapies, a widely used strategy to combat DR, diabetic macular edema, neovascular age-related macular degeneration, retinopathy of prematurity, and other hypoxic retinal vascular disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genistein inhibition of OGD-induced brain neuron death correlates with its modulation of apoptosis, voltage-gated potassium and sodium currents and glutamate signal pathway.

PubMed

Ma, Xue-Ling; Zhang, Feng; Wang, Yu-Xiang; He, Cong-Cong; Tian, Kun; Wang, Hong-Gang; An, Di; Heng, Bin; Liu, Yan-Qiang

2016-07-25

In the present study, we established an in vitro model of hypoxic-ischemia via exposing primary neurons of newborn rats to oxygen-glucose deprivation (OGD) and observing the effects of genistein, a soybean isoflavone, on hypoxic-ischemic neuron viability, apoptosis, voltage-activated potassium (Kv) and sodium (Nav) currents, and glutamate receptor subunits. The results indicated that OGD exposure reduced the viability and increased the apoptosis of brain neurons. Meanwhile, OGD exposure caused changes in the current-voltage curves and current amplitude values of voltage-activated potassium and sodium currents; OGD exposure also decreased GluR2 expression and increased NR2 expression. However, genistein at least partially reversed the effects caused by OGD. The results suggest that hypoxic-ischemia-caused neuronal apoptosis/death is related to an increase in K(+) efflux, a decrease in Na(+) influx, a down-regulation of GluR2, and an up-regulation of NR2. Genistein may exert some neuroprotective effects via the modulation of Kv and Nav currents and the glutamate signal pathway, mediated by GluR2 and NR2. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

CCL11 promotes migration and proliferation of mouse neural progenitor cells.

PubMed

Wang, Feifei; Baba, Nobuyasu; Shen, Yuan; Yamashita, Tatsuyuki; Tsuru, Emi; Tsuda, Masayuki; Maeda, Nagamasa; Sagara, Yusuke

2017-02-07

Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period, resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (NPCs) migrate through the parenchyma and home in to injury sites in the rodent brain, the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury. Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse NPCs. The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion, which was extended to the cortical and striatal areas. NPCs migrated toward an injured area, where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of NPCs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist, SB297006. Our data implicate an important effect of CCL11 for mouse NPCs. The effective activation of NPCs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury.

Targeted temperature management in neurological intensive care unit

PubMed Central

Muengtaweepongsa, Sombat; Srivilaithon, Winchana

2017-01-01

Targeted temperature management (TTM) shows the most promising neuroprotective therapy against hypoxic/ischemic encephalopathy (HIE). In addition, TTM is also useful for treatment of elevated intracranial pressure (ICP). HIE and elevated ICP are common catastrophic conditions in patients admitted in Neurologic intensive care unit (ICU). The most common cause of HIE is cardiac arrest. Randomized control trials demonstrate clinical benefits of TTM in patients with post-cardiac arrest. Although clinical benefit of ICP control by TTM in some specific critical condition, for an example in traumatic brain injury, is still controversial, efficacy of ICP control by TTM is confirmed by both in vivo and in vitro studies. Several methods of TTM have been reported in the literature. TTM can apply to various clinical conditions associated with hypoxic/ischemic brain injury and elevated ICP in Neurologic ICU. PMID:28706860

Oxygen and Cell Fate Decisions

DTIC Science & Technology

2009-05-27

mesenchymal stem cells may still be able to commit to chondro- genic differentiation under hypoxic conditions in vivo. Surprisingly, Malladi et al. have...are induced to undergo differentiation in vitro at 2% O2 ( Malladi et al. 2006). Nonetheless, targeted deletion of HIF-1α in adipose-derived adult... Malladi et al. 2007). These dis- crepancies could potentially be appreciated from at least two perspectives. (1) Chondrogenic dif- ferentiation is

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever.

PubMed

Schäfer, Birgit; Holzer, Georg W; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A; Kreil, Thomas R; Barrett, P Noel; Falkner, Falko G

2011-01-01

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.

Glycoprotein from street rabies virus BD06 induces early and robust immune responses when expressed from a non-replicative adenovirus recombinant.

PubMed

Wang, Shuchao; Sun, Chenglong; Zhang, Shoufeng; Zhang, Xiaozhuo; Liu, Ye; Wang, Ying; Zhang, Fei; Wu, Xianfu; Hu, Rongliang

2015-09-01

The rabies virus (RABV) glycoprotein (G) is responsible for inducing neutralizing antibodies against rabies virus. Development of recombinant vaccines using the G genes from attenuated strains rather than street viruses is a regular practice. In contrast to this scenario, we generated three human adenovirus type 5 recombinants using the G genes from the vaccine strains SRV9 and Flury-LEP, and the street RABV strain BD06 (nrAd5-SRV9-G, nrAd5-Flury-LEP-G, and nrAd5-BD06-G). These recombinants were non-replicative, but could grow up to ~10(8) TCID50/ml in helper HEK293AD cells. Expression of the G protein was verified by immunostaining, quantitative PCR and cytometry. Animal experiments revealed that immunization with nrAd5-BD06-G can induce a higher seroconversion rate, a higher neutralizing antibody level, and a longer survival time after rabies virus challenge in mice when compared with the other two recombinants. Moreover, the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) was significantly higher in mice immunized with nrAd5-BD06-G, which might also contribute to the increased protection. These results show that the use of street RABV G for non-replicative systems may be an alternative for developing effective recombinant rabies vaccines.

Radioresistance in murine solid tumors induced by interleukin-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Braunschweiger, P.G.; Basrur, V.; Santos, O.

1996-02-01

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1{alpha} on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1{alpha} on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 h after administration of IL-1{alpha} (0.5 {mu}g/mouse, ip) and characterized by increases in D{sub 0}, D{sub q}, {alpha}/{Beta} and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1{alpha}more » had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1{alpha} was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1{alpha} had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D{sub 0}, {alpha}/{Beta} and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1{alpha} under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1{alpha} in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1{alpha} may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 may play an important role in the activity of IL-1{alpha} both in vivo and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different. 32 refs., 6 figs., 1 tab.« less

PRMT5 as a druggable target for glioblastoma therapy.

PubMed

Banasavadi-Siddegowda, Yeshavanth Kumar; Welker, Alessandra M; An, Min; Yang, Xiaozhi; Zhou, Wei; Shi, Guqin; Imitola, Jaime; Li, Chenglong; Hsu, Sigmund; Wang, Jiang; Phelps, Mitch; Zhang, Jianying; Beattie, Christine E; Baiocchi, Robert; Kaur, Balveen

2018-05-18

In spite of standard multimodal therapy consisting of surgical resection followed by radiation and concurrent chemotherapy, prognosis for glioblastoma (GBM) patients remains poor. The identification of both differentiated and undifferentiated "stem cell like" populations in the tumor highlights the significance of finding novel targets that affect the heterogeneous tumor cell population. Protein arginine methyltransferase 5 (PRMT5) is one such candidate gene whose nuclear expression correlates with poor survival and has been reported to be required for survival of differentiated GBM cells and self-renewal of undifferentiated GBM cells. In the current study we screened the specificity and efficacy of 4 novel PRMT5 inhibitors in the treatment of GBM. Efficacies of these inhibitors were screened using an in vitro GBM neurosphere model and an in vivo intracranial zebrafish model of glioma. Standard molecular biology methods were employed to investigate changes in cell cycle, growth, and senescence. In vitro and in vivo studies revealed that among the 4 PRMT5 inhibitors, treatment of GBM cells with compound 5 (CMP5) mirrored the effects of PRMT5 knockdown wherein it led to apoptosis of differentiated GBM cells and drove undifferentiated primary patient derived GBM cells into a nonreplicative senescent state. In vivo antitumor efficacy combined with the specificity of CMP5 underscores the importance of developing it for translation.

In Vitro Alterations Do Not Reflect a Requirement for Host Cell Cycle Progression during Plasmodium Liver Stage Infection

PubMed Central

Hanson, Kirsten K.; March, Sandra; Ng, Shengyong; Bhatia, Sangeeta N.

2014-01-01

Prior to invading nonreplicative erythrocytes, Plasmodium parasites undergo their first obligate step in the mammalian host inside hepatocytes, where each sporozoite replicates to generate thousands of merozoites. While normally quiescent, hepatocytes retain proliferative capacity and can readily reenter the cell cycle in response to diverse stimuli. Many intracellular pathogens, including protozoan parasites, manipulate the cell cycle progression of their host cells for their own benefit, but it is not known whether the hepatocyte cell cycle plays a role during Plasmodium liver stage infection. Here, we show that Plasmodium parasites can be observed in mitotic hepatoma cells throughout liver stage development, where they initially reduce the likelihood of mitosis and ultimately lead to significant acquisition of a binucleate phenotype. However, hepatoma cells pharmacologically arrested in S phase still support robust and complete Plasmodium liver stage development, which thus does not require cell cycle progression in the infected cell in vitro. Furthermore, murine hepatocytes remain quiescent throughout in vivo infection with either Plasmodium berghei or Plasmodium yoelii, as do Plasmodium falciparum-infected primary human hepatocytes, demonstrating that the rapid and prodigious growth of liver stage parasites is accomplished independent of host hepatocyte cell cycle progression during natural infection. PMID:25416236

The diacylglycerol acyltransferase Rv3371 of Mycobacterium tuberculosis is required for growth arrest and involved in stress-induced cell wall alterations.

PubMed

Rastogi, Shivangi; Singh, Amit Kumar; Chandra, Gyan; Kushwaha, Pragati; Pant, Garima; Singh, Kavita; Mitra, Kalyan; Sashidhara, Koneni V; Krishnan, Manju Y

2017-05-01

Triacylglycerol (TAG) is important to mycobacteria both as cell envelope component and energy reservoir. Mycobacterium tuberculosis (Mtb) genome encodes at least 15 putative TAG synthase (tgs)s. We report that one of these genes, Rv3371, specific to pathogenic mycobacteria, when expressed in M. smegmatis leads to modifications in colony morphotype, bacterial architecture, cell surface properties and elevated TAG levels. Rv3371 was found to largely localize in the cell membrane. The Rv3371 promoter is minimally active during exponential growth in vitro, however, is up-regulated under stationary phase, hypoxia, nutrient starvation, nitrosative stress, low iron, in IFN-γ activated macrophages and infected mice. The low iron-induced expression of Rv3371 is likely due to the de-repression by Rv1404, which is probably activated by ideR. An Rv3371 deletion mutant of Mtb showed impaired non-replicating persistence in vitro and altered sensitivity to anti-mycobacterial drugs. In low iron medium, the Rv3371 deletion mutant showed reduced formation of TAG containing extracellular vesicles. Therefore Rv3371 is likely involved in Mtb growth arrest and cell wall alterations during persistence. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hypoxia-induced long non-coding RNA Malat1 is dispensable for renal ischemia/reperfusion-injury.

PubMed

Kölling, Malte; Genschel, Celina; Kaucsar, Tamas; Hübner, Anika; Rong, Song; Schmitt, Roland; Sörensen-Zender, Inga; Haddad, George; Kistler, Andreas; Seeger, Harald; Kielstein, Jan T; Fliser, Danilo; Haller, Hermann; Wüthrich, Rudolf; Zörnig, Martin; Thum, Thomas; Lorenzen, Johan

2018-02-21

Renal ischemia-reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Non-coding RNAs are crucially involved in its pathophysiology. We identified hypoxia-induced long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) to be upregulated in renal I/R injury. We here elucidated the functional role of Malat1 in vitro and its potential contribution to kidney injury in vivo. Malat1 was upregulated in kidney biopsies and plasma of patients with AKI, in murine hypoxic kidney tissue as well as in cultured and ex vivo sorted hypoxic endothelial cells and tubular epithelial cells. Malat1 was transcriptionally activated by hypoxia-inducible factor 1-α. In vitro, Malat1 inhibition reduced proliferation and the number of endothelial cells in the S-phase of the cell cycle. In vivo, Malat1 knockout and wildtype mice showed similar degrees of outer medullary tubular epithelial injury, proliferation, capillary rarefaction, inflammation and fibrosis, survival and kidney function. Small-RNA sequencing and whole genome expression analysis revealed only minor changes between ischemic Malat1 knockout and wildtype mice. Contrary to previous studies, which suggested a prominent role of Malat1 in the induction of disease, we did not confirm an in vivo role of Malat1 concerning renal I/R-injury.

Melatonin reduces hypoxic-ischaemic (HI) induced autophagy and apoptosis: An in vivo and in vitro investigation in experimental models of neonatal HI brain injury.

PubMed

Hu, Yingying; Wang, Zhouguang; Liu, Yanlong; Pan, Shulin; Zhang, Hao; Fang, Mingchu; Jiang, Huai; Yin, Jiayu; Zou, Shuangshuang; Li, Zhenmao; Zhang, Hongyu; Lin, Zhenlang; Xiao, Jian

2017-07-13

Melatonin has neuroprotective effects in many diseases, including neonatal hypoxic-ischaemic (HI) brain injury. The purpose of this study was to evaluate the neuroprotective effects of melatonin both in vivo and in vitro and associated molecular mechanisms behind these effects. Postnatal day 7 male and female rat pups were subjected to unilateral HI, melatonin was injected intraperitoneally 1h before HI and an additional six doses were administered at 24h intervals. The pups were sacrificed at 24h and 7 d after HI. Pre-treatment with melatonin significantly reduced brain damage at 7 d after HI, with 15mg/kg melatonin achieving over 30% recovery in tissue loss compared to vehicle-treated animals. Autophagy and apoptotic cell death as indicated by autophagy associated proteins, cleaved caspase 3 and Tunel staining, was significantly inhibited after melatonin treatment in vivo as well as in PC12 cells. Melatonin treatment also significantly increased the GAP43 in the cortex. In conclusion, melatonin treatment reduced neonatal rat brain injury after HI, and this appeared to be related to inhibiting autophagy as well as reducing apoptotic cell death. Copyright © 2017 Elsevier B.V. All rights reserved.

HYPOXIA-ACTIVATED PRO-DRUG TH-302 EXHIBITS POTENT TUMOUR SUPPRESSIVE ACTIVITY AND COOPERATES WITH CHEMOTHERAPY AGAINST OSTEOSARCOMA

PubMed Central

Liapis, Vasilios; Labrinidis, Agatha; Zinonos, Irene; Hay, Shelley; Ponomarev, Vladimir; Panagopoulos, Vasilios; DeNichilo, Mark; Ingman, Wendy; Atkins, Gerald J.; Findlay, David M.; Zannettino, Andrew CW.; Evdokiou, Andreas

2015-01-01

Tumour hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumour hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumours. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS. PMID:25444931

Hypoxia-activated pro-drug TH-302 exhibits potent tumor suppressive activity and cooperates with chemotherapy against osteosarcoma.

PubMed

Liapis, Vasilios; Labrinidis, Agatha; Zinonos, Irene; Hay, Shelley; Ponomarev, Vladimir; Panagopoulos, Vasilios; DeNichilo, Mark; Ingman, Wendy; Atkins, Gerald J; Findlay, David M; Zannettino, Andrew C W; Evdokiou, Andreas

2015-02-01

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS. Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.

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

PubMed

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

2014-08-01

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

Extracellular Vesicles from Hypoxic Adipocytes and Obese Subjects Reduce Insulin‐Stimulated Glucose Uptake

PubMed Central

Mleczko, Justyna; Ortega, Francisco J.; Falcon‐Perez, Juan Manuel; Wabitsch, Martin; Fernandez‐Real, Jose Manuel

2018-01-01

Scope We investigate the effects of extracellular vesicles (EVs) obtained from in vitro adipocyte cell models and from obese subjects on glucose transport and insulin responsiveness. Methods and results EVs are isolated from the culture supernatant of adipocytes cultured under normoxia, hypoxia (1% oxygen), or exposed to macrophage conditioned media (15% v/v). EVs are isolated from the plasma of lean individuals and subjects with obesity. Cultured adipocytes are incubated with EVs and activation of insulin signalling cascades and insulin‐stimulated glucose transport are measured. EVs released from hypoxic adipocytes impair insulin‐stimulated 2‐deoxyglucose uptake and reduce insulin mediated phosphorylation of AKT. Insulin‐mediated phosphorylation of extracellular regulated kinases (ERK1/2) is not affected. EVs from individuals with obesity decrease insulin stimulated 2‐deoxyglucose uptake in adipocytes (p = 0.0159). Conclusion EVs released by stressed adipocytes impair insulin action in neighboring adipocytes. PMID:29292863

Treatment of hypoxic-ischemic encephalopathy in mouse by transplantation of embryonic stem cell-derived cells.

PubMed

Ma, Jie; Wang, Yu; Yang, Jianhua; Yang, Min; Chang, Keun-A; Zhang, Linhua; Jiang, Feng; Li, Yi; Zhang, Zhonggong; Heo, Chaejeong; Suh, Yoo-Hun

2007-07-01

A 7-day-old hypoxic-ischemic encephalopathy (HIE) mouse model was used to study the effect of transplantation of embryonic stem (ES) cell-derived cells on the HIE. After the inducement in vitro, the ES cell-derived cells expressed Nestin and MAP-2, rather than GFAP mRNA. After transplantation, ES cell-derived cells can survive, migrate into the injury site, and specifically differentiate into neurons, showing improvement of the learning ability and memory of the HIE mouse at 8 months post-transplantation. The non-grafted HIE mouse brain showed typical pathological changes in the hippocampus and cerebral cortex, where the number of neurons was reduced, while in the cell graft group, number of the neurons increased in the same regions. Although further study is necessary to elucidate the precise mechanisms responsible for this functional recovery, we believe that ES cells have advantages for use as a donor source in HIE.

X-ray Radiation-Controlled NO-Release for On-Demand Depth-Independent Hypoxic Radiosensitization.

PubMed

Fan, Wenpei; Bu, Wenbo; Zhang, Zhen; Shen, Bo; Zhang, Hui; He, Qianjun; Ni, Dalong; Cui, Zhaowen; Zhao, Kuaile; Bu, Jiwen; Du, Jiulin; Liu, Jianan; Shi, Jianlin

2015-11-16

Multifunctional stimuli-responsive nanotheranostic systems are highly desirable for realizing simultaneous biomedical imaging and on-demand therapy with minimized adverse effects. Herein, we present the construction of an intelligent X-ray-controlled NO-releasing upconversion nanotheranostic system (termed as PEG-USMSs-SNO) by engineering UCNPs with S-nitrosothiol (R-SNO)-grafted mesoporous silica. The PEG-USMSs-SNO is designed to respond sensitively to X-ray radiation for breaking down the S-N bond of SNO to release NO, which leads to X-ray dose-controlled NO release for on-demand hypoxic radiosensitization besides upconversion luminescent imaging through UCNPs in vitro and in vivo. Thanks to the high live-body permeability of X-ray, our developed PEG-USMSs-SNO may provide a new technique for achieving depth-independent controlled NO release and positioned radiotherapy enhancement against deep-seated solid tumors. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis.

PubMed

Bozec, Aline; Hannemann, Nicole

2016-06-03

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes.

Protein kinase G confers survival advantage to Mycobacterium tuberculosis during latency-like conditions.

PubMed

Khan, Mehak Zahoor; Bhaskar, Ashima; Upadhyay, Sandeep; Kumari, Pooja; Rajmani, Raju S; Jain, Preeti; Singh, Amit; Kumar, Dhiraj; Bhavesh, Neel Sarovar; Nandicoori, Vinay Kumar

2017-09-29

Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis , required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Energetics of Respiration and Oxidative Phosphorylation in Mycobacteria.

PubMed

Cook, Gregory M; Hards, Kiel; Vilchèze, Catherine; Hartman, Travis; Berney, Michael

2014-06-01

Mycobacteria inhabit a wide range of intracellular and extracellular environments. Many of these environments are highly dynamic and therefore mycobacteria are faced with the constant challenge of redirecting their metabolic activity to be commensurate with either replicative growth or a non-replicative quiescence. A fundamental feature in this adaptation is the ability of mycobacteria to respire, regenerate reducing equivalents and generate ATP via oxidative phosphorylation. Mycobacteria harbor multiple primary dehydrogenases to fuel the electron transport chain and two terminal respiratory oxidases, an aa3 -type cytochrome c oxidase and cytochrome bd-type menaquinol oxidase, are present for dioxygen reduction coupled to the generation of a protonmotive force. Hypoxia leads to the downregulation of key respiratory complexes, but the molecular mechanisms regulating this expression are unknown. Despite being obligate aerobes, mycobacteria have the ability to metabolize in the absence of oxygen and a number of reductases are present to facilitate the turnover of reducing equivalents under these conditions (e.g. nitrate reductase, succinate dehydrogenase/fumarate reductase). Hydrogenases and ferredoxins are also present in the genomes of mycobacteria suggesting the ability of these bacteria to adapt to an anaerobic-type of metabolism in the absence of oxygen. ATP synthesis by the membrane-bound F1FO-ATP synthase is essential for growing and non-growing mycobacteria and the enzyme is able to function over a wide range of protonmotive force values (aerobic to hypoxic). The discovery of lead compounds that target respiration and oxidative phosphorylation in Mycobacterium tuberculosis highlights the importance of this area for the generation of new front line drugs to combat tuberculosis.

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

PubMed

Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

2015-03-11

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Absolute hypoxic exercise training enhances in vitro thrombin generation by increasing procoagulant platelet-derived microparticles under high shear stress in sedentary men.

PubMed

Chen, Yu-Wen; Chen, Yi-Ching; Wang, Jong-Shyan

2013-05-01

HS (high shear) stress associated with artery stenosis facilitates TG (thrombin generation) by increasing the release of procoagulant PDMPs (platelet-derived microparticles). Physical exercise and hypoxia may paradoxically modulate vascular thrombotic risks. The aim of the present study was to investigate how exercise training with/without hypoxia affected TG mediated by PDMPs under physio-pathological shear flows. A total of 75 sedentary males were randomly divided into five groups (n=15 in each group): 21% O2 [NC (normoxic control)] or 15% O2 [HC (hypoxic control)] at rest or were trained at 50% of peak work rate under 21% O2 [NT (normoxic training)] or 15% O2 [HAT (hypoxic-absolute training)], or 50% of HR (heart rate) reserve under 15% O2 [HRT (hypoxic-relative training)] for 30 min/day, 5 days/week for 4 weeks. The PDMP characteristics and dynamic TG were measured by flow cytometry and thrombinography respectively. Before the intervention, strenuous exercise markedly increased the PDMP count (14.8%) and TG rate (19.5%) in PDMP-rich plasma at 100 dynes/cm2 of shear stress (P<0.05). After the interventions, both NT and HRT significantly attenuated the enhancement of HS-induced PDMPs (4.7 and 4.9%) and TG rate (3.8 and 3.0%) (P<0.05) by severe exercise. Conversely, HAT notably promoted the PDMP count (37.3%) and TG rate (38.9%) induced by HS (P<0.05), concurrent with increasing plasma TF (tissue factor) and coagulation factor V levels at rest or following exercise. We conclude that both HRT and NT depress similarly HS-mediated TG during exercise, but HAT accelerates the prothrombotic response to vigorous exercise. These findings provide new insights into how exercise training under a hypoxic condition influences the risk of thrombosis associated with stenotic arteries.

Post-hypoxic constriction of retinal arterioles is impaired during nitric oxide and cyclo-oxygenase inhibition and in diabetic patients without retinopathy.

PubMed

Petersen, Line; Bek, Toke

2017-10-01

Occlusion of retinal vessels leads to retinal ischaemia and hypoxia, which induces vasodilatation in adjacent retinal areas in order to normalize retinal oxygenation. Previous studies have shown that NO and COX products are involved in hypoxia-induced dilatation of retinal arterioles in vitro and in vivo, and that this response is disturbed in patients with diabetes mellitus. However, it is unknown to what extent post-hypoxic recovery of the diameter of retinal arterioles depends on NO and COX products in normal persons and in diabetic patients. The Dynamic Vessel Analyzer (DVA) was used to study the post-hypoxic diameter changes of larger retinal vessels in 20 normal persons, 20 diabetic patients without diabetic retinopathy, and in 18 patients with diabetic maculopathy before and after inhibition of the synthesis of nitric oxide and COX products. In normal persons, the arterioles had re-constricted (p > 0.99) 2 minutes after termination of hypoxia in the absence of antagonists, but not after treatment with L-NMMA and diclofenac (p < 0.01 for all comparisons). In diabetic patients without retinopathy, the arterioles showed no diameter changes after termination of hypoxia during any of the interventions. In patients with diabetic maculopathy hypoxia had not dilated retinal arterioles (p > 0.1 for all comparisons) to allow the study of re-constriction. In all groups, the dilatation of venules remained significantly increased during the post-hypoxic observation period, both in the absence and in the presence of L-NMMA and diclofenac.Post-hypoxic constriction of retinal arterioles depends on NO and COX products, and is impaired in diabetic patients before the development of retinopathy. This disturbance may contribute to the development of diabetic retinopathy, and should be the target of future interventional studies aimed at preventing and treating the disease.ClinicalTrials.gov identifier: NCT01689090.

DT-13 attenuates human lung cancer metastasis via regulating NMIIA activity under hypoxia condition.

PubMed

Wei, Xiao-Hui; Lin, Sen-Sen; Liu, Yang; Zhao, Ren-Ping; Khan, Ghulam Jilany; Du, Hong-Zhi; Mao, Ting-Ting; Yu, Bo-Yang; Li, Rui-Ming; Yuan, Sheng-Tao; Sun, Li

2016-08-01

Cancer metastasis plays a major role in tumor deterioration. Metastatic processes are known to be regulated by hypoxic microenvironment and non-muscle myosin IIA (NMIIA). DT-13, a bioactive saponin monomer isolated from Ophiopogon japonicus, has been reported to inhibit various cancer metastasis, but whether NMIIA is involved in the anti-metastatic activity of DT-13 under hypoxia remains to be determined. Thus, this study aims to clarify the role of DT-13 in regulating 95D cell metastasis under hypoxic microenvironment and to further investigate whether NMIIA is involved in the anti-metastatic mechanism of DT-13. We found that DT-13 significantly inhibited 95D cells metastasis in vitro and in vivo. Furthermore, hypoxia significantly inhibited the expression of NMIIA and redistributed NMIIA to the cell periphery, whereas DT-13 reversed the hypoxic effects by upregulating the expression of NMIIA. Moreover, DT-13 treatment redistributed NMIIA to the nuclear periphery and reduced the formation of F-actin in 95D cells. In addition, we found that the Raf-ERK1/2 signaling pathway is involved in regulation of NMIIA by DT-13. Collectively, these findings support NMIIA as a target of DT-13 to prevent lung cancer metastasis.

Characterization and In Vivo Testing of Mesenchymal Stem Cells Derived from Human Embryonic Stem Cells

PubMed Central

Gruenloh, William; Kambal, Amal; Sondergaard, Claus; McGee, Jeannine; Nacey, Catherine; Kalomoiris, Stefanos; Pepper, Karen; Olson, Scott; Fierro, Fernando

2011-01-01

Mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of tissues through homing to injured areas, especially to hypoxic, apoptotic, or inflamed areas and releasing factors that hasten endogenous repair. In some cases genetic engineering of the MSC is desired, since they are excellent delivery vehicles. We have derived MSCs from the human embryonic stem cell (hESC) line H9 (H9-MSCs). They expressed CD105, CD90, CD73, and CD146, and lacked expression of CD45, CD34, CD14, CD31, and HLA-DR, the hESC pluripotency markers SSEA-4 and Tra-1-81, and the hESC early differentiation marker SSEA-1. Marrow-derived MSCs showed a similar phenotype. H9-MSCs did not form teratoma in our initial studies, whereas the parent H9 line did so robustly. H9-MSCs differentiated into bone, cartilage, and adipocytes in vitro, and displayed increased migration under hypoxic conditions. Finally, using a hindlimb ischemia model, H9-MSCs were shown to home to the hypoxic muscle, but not the contralateral limb, by 48 h after IV injection. In summary, we have defined methods for differentiation of hESCs into MSCs and have defined their characteristics and in vivo migratory properties. PMID:21275830

Neuroprotective effects of scutellarin against hypoxic-ischemic-induced cerebral injury via augmentation of antioxidant defense capacity.

PubMed

Guo, Hong; Hu, Li-Min; Wang, Shao-Xia; Wang, Yu-Lin; Shi, Fang; Li, Hui; Liu, Yang; Kang, Li-Yuan; Gao, Xiu-Mei

2011-12-31

An increasing number of studies has indicated that hypoxic-ischemic-induced cerebral injury is partly mediated via oxidative stress. Recent researches have focused on searching for drug and herbal manipulations to protect against hypoxic-ischemic-induced oxidative cell damage. Scutellarin is a flavonoid derived from the Erigeron breviscapus (vant.) and has been reported to exhibit neuroprotective properties. However, its precise mechanism, particularly its antioxidation mechanism, remains elusive. In the present study, we investigated the neuroprotective effects of scutellarin on middle cerebral artery occlusion (MCAO)-induced brain damage in rats, and oxygen-glucose deprivation (OGD)-induced toxicity in primary culture of rat cortical neurons. In vivo, intraperitoneal injections of scutellarin (20 and 60 mg/kg) improved the neurological score and diminished the percentage of brain infarct volume. At the same time, scutellarin significantly increased superoxide dismutase (SOD), catalase (CAT) activities and glutathione (GSH) level in ischemic brain tissues, enhancing endogenous antioxidant activity. Moreover, pretreatment of scutellarin (25, 50 and 100 μM) protected neurons against lethal stimuli, decreased the percentage of apoptotic cells and inhibited reactive oxygen species (ROS) generation in OGD-induced primary cortical neurons in vitro. These results suggest that the preventive and therapeutic potential of scutellarin in cerebral injury patients is, at least in part, ascribed to augmentation of cellular antioxidant defense capacity.

The Effects of Pleiotrophin in Proliferative Diabetic Retinopathy

PubMed Central

Zhu, Xuemei; Bai, Yujing; Yu, Wenzhen; Pan, Chungting; Jin, Enzhong; Song, Dan; Xu, Qiong; Yao, Yuou; Huang, Lvzhen; Tao, Yong; Li, Xiaoxin; Zhao, Mingwei

2015-01-01

Pleiotrophin (PTN), a secreted, multifunctional cytokine, is involved in angiogenic, fibrotic and neurodegenerative diseases. However, little is known about its effects on diabetic retinopathy, a neurovascular disease. To investigate the role of PTN in proliferative diabetic retinopathy (PDR), PTN concentration in the vitreous was evaluated in PDR patients and non-diabetic controls. PTN expression was observed in epiretinal membranes from patients. PTN knockdown was performed using small interfering (si)RNA, and the effects on retinal pigment epithelium (RPE) cells and human umbilical vascular endothelia cells (HUVECs) were observed in vitro under hyperglycemic and hypoxic conditions. Cell attachment, proliferation, migration, tube formation, cell cycle, apoptosis, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation, and VEGF levels were studied. The vitreous PTN concentration in PDR patients was higher than that in non-diabetic controls, and PTN was highly expressed in the fibrovascular membranes of PDR patients. Under hyperglycemic and hypoxic conditions, PTN knockdown reduced cell attachment, proliferation, migration, and tube formation and induced cell cycle arrest and apoptosis in vitro. Mechanically, PTN depletion decreased ERK 1/2 phosphorylation. Recombinant PTN up regulated the concentration of VEGF in vitro, which can be attenuated by the ERK 1/2 inhibitor. Taken together, our results implied that elevated PTN in PDR patients might participate in the critical processes of the development of PDR, most likely playing roles in angiogenesis and proliferation, possibly by activating the ERK 1/2 pathway and regulating VEGF secretion. These findings provide new insight into the roles of PTN in PDR and suggest that PTN may become a new target for therapeutic intervention in PDR. PMID:25617851

The effects of pleiotrophin in proliferative diabetic retinopathy.

PubMed

Zhu, Xuemei; Bai, Yujing; Yu, Wenzhen; Pan, Chungting; Jin, Enzhong; Song, Dan; Xu, Qiong; Yao, Yuou; Huang, Lvzhen; Tao, Yong; Li, Xiaoxin; Zhao, Mingwei

2015-01-01

Pleiotrophin (PTN), a secreted, multifunctional cytokine, is involved in angiogenic, fibrotic and neurodegenerative diseases. However, little is known about its effects on diabetic retinopathy, a neurovascular disease. To investigate the role of PTN in proliferative diabetic retinopathy (PDR), PTN concentration in the vitreous was evaluated in PDR patients and non-diabetic controls. PTN expression was observed in epiretinal membranes from patients. PTN knockdown was performed using small interfering (si)RNA, and the effects on retinal pigment epithelium (RPE) cells and human umbilical vascular endothelia cells (HUVECs) were observed in vitro under hyperglycemic and hypoxic conditions. Cell attachment, proliferation, migration, tube formation, cell cycle, apoptosis, extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation, and VEGF levels were studied. The vitreous PTN concentration in PDR patients was higher than that in non-diabetic controls, and PTN was highly expressed in the fibrovascular membranes of PDR patients. Under hyperglycemic and hypoxic conditions, PTN knockdown reduced cell attachment, proliferation, migration, and tube formation and induced cell cycle arrest and apoptosis in vitro. Mechanically, PTN depletion decreased ERK 1/2 phosphorylation. Recombinant PTN up regulated the concentration of VEGF in vitro, which can be attenuated by the ERK 1/2 inhibitor. Taken together, our results implied that elevated PTN in PDR patients might participate in the critical processes of the development of PDR, most likely playing roles in angiogenesis and proliferation, possibly by activating the ERK 1/2 pathway and regulating VEGF secretion. These findings provide new insight into the roles of PTN in PDR and suggest that PTN may become a new target for therapeutic intervention in PDR.

Human skin-derived fibroblasts used as a 'Trojan horse' for drug delivery.

PubMed

Coccè, V; Vitale, A; Colombo, S; Bonomi, A; Sisto, F; Ciusani, E; Alessandri, G; Parati, E; Brambilla, P; Brambilla, M; La Porta, C A; Pessina, A

2016-06-01

Drug toxicity currently represents the main challenge of tumour chemotherapy. Our group recently developed a new method for drug delivery inspired by the 'Trojan Horse' concept. Human mesenchymal stem cells (hMSCs) have been shown to play the role of new 'horses' in delivering anti-tumour agents, without involving any genetic manipulation. As human stromal dermal fibroblasts (hSDFs) represent an interesting alternative to hMSCs, being easy to isolate, they could be an ideal candidate for this kind of procedure. To investigate whether hSDFs can take up and deliver paclitaxel (PTX) in sufficient concentrations to inhibit a very aggressive melanoma tumour (IgR39) in vitro. hSDFs were primed with high doses of PTX, and then the effect of drug delivery on IgR39 melanoma proliferation in vitro was evaluated using several assays (antiproliferation, transwell cocultures, rosette assays and colony growth assays). Furthermore, the cell cycle and PTX uptake/release mechanism of hSDFs were studied both under both normal and hypoxic conditions. hSDFs incorporated PTX and then released it with unaffected pharmacological activity, inhibiting human IgR39 melanoma growth in vitro. The hypoxic conditions did not induce changes in cell cycle pattern and the uptake-release mechanism with PTX was not affected. hSDFs can be used as a Trojan horse, as the released drug was functionally active. These results indicated that these cells could be used for clinical treatment as the drug was released into the cellular environment and the primed cells underwent apoptosis. © 2016 British Association of Dermatologists.

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

Efficiently estimating salmon escapement uncertainty using systematically sampled data

USGS Publications Warehouse

Reynolds, Joel H.; Woody, Carol Ann; Gove, Nancy E.; Fair, Lowell F.

2007-01-01

Fish escapement is generally monitored using nonreplicated systematic sampling designs (e.g., via visual counts from towers or hydroacoustic counts). These sampling designs support a variety of methods for estimating the variance of the total escapement. Unfortunately, all the methods give biased results, with the magnitude of the bias being determined by the underlying process patterns. Fish escapement commonly exhibits positive autocorrelation and nonlinear patterns, such as diurnal and seasonal patterns. For these patterns, poor choice of variance estimator can needlessly increase the uncertainty managers have to deal with in sustaining fish populations. We illustrate the effect of sampling design and variance estimator choice on variance estimates of total escapement for anadromous salmonids from systematic samples of fish passage. Using simulated tower counts of sockeye salmon Oncorhynchus nerka escapement on the Kvichak River, Alaska, five variance estimators for nonreplicated systematic samples were compared to determine the least biased. Using the least biased variance estimator, four confidence interval estimators were compared for expected coverage and mean interval width. Finally, five systematic sampling designs were compared to determine the design giving the smallest average variance estimate for total annual escapement. For nonreplicated systematic samples of fish escapement, all variance estimators were positively biased. Compared to the other estimators, the least biased estimator reduced bias by, on average, from 12% to 98%. All confidence intervals gave effectively identical results. Replicated systematic sampling designs consistently provided the smallest average estimated variance among those compared.

Pre-Clinical Efficacy and Safety of Experimental Vaccines Based on Non-Replicating Vaccinia Vectors against Yellow Fever

PubMed Central

Schäfer, Birgit; Holzer, Georg W.; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A.; Kreil, Thomas R.; Barrett, P. Noel; Falkner, Falko G.

2011-01-01

Background Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. Methodology/Principal Findings A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 105 TCID50. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. Conclusions/Significance The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice. PMID:21931732

Neuroprotective Effects of Glutamate Antagonists and Extracellular Acidity

NASA Astrophysics Data System (ADS)

Kaku, David A.; Giffard, Rona G.; Choi, Dennis W.

1993-06-01

Glutamate antagonists protect neurons from hypoxic injury both in vivo and in vitro, but in vitro studies have not been done under the acidic conditions typical of hypoxia-ischemia in vivo. Consistent with glutamate receptor antagonism, extracellular acidity reduced neuronal death in murine cortical cultures that were deprived of oxygen and glucose. Under these acid conditions, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isox-azolepropionate-kainate antagonists further reduced neuronal death, such that some neurons tolerated prolonged oxygen and glucose deprivation almost as well as did astrocytes. Neuroprotection induced by this combination exceeded that induced by glutamate antagonists alone, suggesting that extracellular acidity has beneficial effects beyond the attenuation of ionotropic glutamate receptor activation.

Extract from Mimosa pigra attenuates chronic experimental pulmonary hypertension.

PubMed

Rakotomalala, G; Agard, C; Tonnerre, P; Tesse, A; Derbré, S; Michalet, S; Hamzaoui, J; Rio, M; Cario-Toumaniantz, C; Richomme, P; Charreau, B; Loirand, G; Pacaud, P

2013-06-21

Different parts of Mimosa pigra (MPG) are used in traditional medicine in Madagascar, tropical Africa, South America and Indonesia for various troubles including cardiovascular disorders. To investigate the mechanisms underlying the vascular effects of MPG by assessing in vitro its antioxidant and anti-inflammatory properties, and its vascular relaxing effects, and in vivo, its action on hypoxic pulmonary hypertension (PAH) in rats. The antioxidant activity of MPG leaf hydromethanolic extract was determined by using both the 1,1-diphenyl-2-picrylhydrazyl radical scavenging and the oxygen radical absorbance capacity in vitro assays. Anti-inflammatory properties were assayed on TNFα-induced VCAM-1 expression in endothelial cells. The vasorelaxant effect of MPG extract was studied on rat arterial rings pre-contracted with phenylephrine (1μM) in the presence or absence of the endothelium. In vivo MPG extract effects were analyzed in chronic hypoxic PAH, obtained by housing male Wistar rats, orally treated or not with MPG extract (400mg/kg/d), in a hypobaric chamber for 21 days. MPG leaf extract had antioxidant and anti-inflammatory properties. It induced endothelium-dependent, NO-mediated relaxation of rat aorta and pulmonary artery. In vivo, chronic MPG treatment reduced hypoxic PAH in rat by decreasing by 22.3% the pulmonary arterial pressure and by 20.0% and 23.9% the pulmonary artery and cardiac remodelling, respectively. This effect was associated with a restoration of endothelium function and a 2.3-fold increase in endothelial NO synthase phosphorylation. MPG leaf hydromethanolic extract contained tryptophan and flavonoids, including quercetin glycosides. Both compounds also efficiently limit hypoxia-induced PAH. Our results show endothelial protective action of MPG leaf hydromethanolic extract which is likely to be due to its antioxidant action. MPG successfully attenuated the development of PAH, thus demonstrating the protective effect of MPG on cardiovascular diseases. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Hypoxic-preconditioning enhances the regenerative capacity of neural stem/progenitors in subventricular zone of newborn piglet brain.

PubMed

Ara, Jahan; De Montpellier, Sybille

2013-09-01

Perinatal hypoxia-ischemia (HI) results in brain injury, whereas mild hypoxic episodes result in preconditioning, which can significantly reduce the vulnerability of the brain to subsequent severe hypoxia-ischemia. Hypoxic-preconditioning (PC) has been shown to enhance cell survival and differentiation of progenitor cells in the central nervous system (CNS). The purpose of this study was to determine whether pretreatment with PC prior to HI stimulates subventricular zone (SVZ) proliferation and neurogenesis in newborn piglets. One-day-old piglets were subjected to PC (8% O2/92% N2) for 3h and 24h later were exposed to HI produced by combination of hypoxia (5% FiO2) for a pre-defined period of 30min and ischemia induced by a period of 10min of hypotension. Here we demonstrate that SVZ derived neural stem/progenitor cells (NSPs) from PC, HI and PC+HI piglets proliferated as neurospheres, expressed neural progenitor and neurodevelopmental markers, and that greater proportion of the spheres generated are multipotential. Neurosphere assay revealed that preconditioning pretreatment increased the number of NSP-derived neurospheres in SVZ following HI compared to normoxic and HI controls. NSPs from preconditioned SVZ generated twice as many neurons and astrocytes in vitro. Injections with 5-Bromo-2-deoxyuridine (BrdU) after PC revealed a robust proliferative response within the SVZ that continued for one week. PC also increased neurogenesis in vivo, doublecortin positive cells with migratory profiles were observed streaming from the SVZ to striatum and neocortex. These findings show that the induction of proliferation and neurogenesis by PC might be a positive adaptation for an efficient repair and plasticity in the event of a hypoxic-ischemic insult. Copyright © 2013 Elsevier B.V. All rights reserved.

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

Differential patterns of injury to the proximal tubule of renal cortical slices following in vitro exposure to mercuric chloride, potassium dichromate, or hypoxic conditions.

PubMed

Ruegg, C E; Gandolfi, A J; Nagle, R B; Brendel, K

1987-09-15

The innate susceptibility of renal cell types to these agents was investigated using precision-cut rabbit renal cortical slices made perpendicular to the cortical-papillary axis. Slices were incubated in DME/F12 medium containing 10 microM, 100 microM, or 1 mM concentrations of either metal for 12 hr or in Krebs-Hepes buffer gassed with nitrogen (100%) for 0.75 to 5 hr of hypoxic exposure. To simulate postischemic reperfusion, some slices were transferred to vessels gassed with oxygen after an initial hypoxic period. Mercuric chloride (100 microM) exposure resulted in damage to the straight regions of proximal tubules by 12 hr leaving convoluted regions unaffected. Hypoxia (2.25 hr) and potassium dichromate (100 microM for 12 hr) both caused injury to the convoluted proximal tubules without affecting straight proximal tubular regions. Mercury concentrations of 10 microM and 1 mM had no effect or injured all cell types within the slice, respectively. Similar results were observed for hypoxic periods less than 1.5 hr or greater than 3 hr of exposure. Potassium dichromate had no measurable affect at 10 microM, but at 1 mM focal lesions were observed after 4 hr of exposure, and by 12 hr all cell types within the slice were affected. Intracellular potassium content normalized to DNA correlated well, but always preceded the pathological lesions observed. These results demonstrate that injury to specific regions of the proximal tubule by these agents relates to an innate susceptibility of the intoxicated cell type independent of physiologic feedback or blood delivery patterns proposed as mechanisms of selective injury from in vivo studies.

Could Killing Bacterial Subpopulations Hit Tuberculosis out of the Park?

PubMed

Baranowski, Catherine; Rubin, Eric J

2016-07-14

One hurdle to treating tuberculosis could be that it is so difficult to kill nonreplicating subpopulations of the causative pathogens. This work describes two new cephalosporin derivatives that specifically target this population of Mycobacterium tuberculosis.

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography.

PubMed

Bunker, Richard D; Mandal, Kalyaneswar; Bashiri, Ghader; Chaston, Jessica J; Pentelute, Bradley L; Lott, J Shaun; Kent, Stephen B H; Baker, Edward N

2015-04-07

Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L- and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L- and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.

Helicase promotes replication re-initiation from an RNA transcript.

PubMed

Sun, Bo; Singh, Anupam; Sultana, Shemaila; Inman, James T; Patel, Smita S; Wang, Michelle D

2018-06-13

To ensure accurate DNA replication, a replisome must effectively overcome numerous obstacles on its DNA substrate. After encountering an obstacle, a progressing replisome often aborts DNA synthesis but continues to unwind. However, little is known about how DNA synthesis is resumed downstream of an obstacle. Here, we examine the consequences of a non-replicating replisome collision with a co-directional RNA polymerase (RNAP). Using single-molecule and ensemble methods, we find that T7 helicase interacts strongly with a non-replicating T7 DNA polymerase (DNAP) at a replication fork. As the helicase advances, the associated DNAP also moves forward. The presence of the DNAP increases both helicase's processivity and unwinding rate. We show that such a DNAP, together with its helicase, is indeed able to actively disrupt a stalled transcription elongation complex, and then initiates replication using the RNA transcript as a primer. These observations exhibit T7 helicase's novel role in replication re-initiation.

Large-scale production of foot-and-mouth disease virus (serotype Asia1) VLP vaccine in Escherichia coli and protection potency evaluation in cattle.

PubMed

Xiao, Yan; Chen, Hong-Ying; Wang, Yuzhou; Yin, Bo; Lv, Chaochao; Mo, Xiaobing; Yan, He; Xuan, Yajie; Huang, Yuxin; Pang, Wenqiang; Li, Xiangdong; Yuan, Y Adam; Tian, Kegong

2016-07-02

Foot-and-mouth disease (FMD) is an acute, highly contagious disease that infects cloven-hoofed animals. Vaccination is an effective means of preventing and controlling FMD. Compared to conventional inactivated FMDV vaccines, the format of FMDV virus-like particles (VLPs) as a non-replicating particulate vaccine candidate is a promising alternative. In this study, we have developed a co-expression system in E. coli, which drove the expression of FMDV capsid proteins (VP0, VP1, and VP3) in tandem by a single plasmid. The co-expressed FMDV capsid proteins (VP0, VP1, and VP3) were produced in large scale by fermentation at 10 L scale and the chromatographic purified capsid proteins were auto-assembled as VLPs in vitro. Cattle vaccinated with a single dose of the subunit vaccine, comprising in vitro assembled FMDV VLP and adjuvant, developed FMDV-specific antibody response (ELISA antibodies and neutralizing antibodies) with the persistent period of 6 months. Moreover, cattle vaccinated with the subunit vaccine showed the high protection potency with the 50 % bovine protective dose (PD50) reaching 11.75 PD50 per dose. Our data strongly suggest that in vitro assembled recombinant FMDV VLPs produced from E. coli could function as a potent FMDV vaccine candidate against FMDV Asia1 infection. Furthermore, the robust protein expression and purification approaches described here could lead to the development of industrial level large-scale production of E. coli-based VLPs against FMDV infections with different serotypes.

The Methods and Mechanisms to Differentiate Endothelial-Like Cells and Smooth Muscle Cells from Mesenchymal Stem Cells for Vascularization in Vaginal Reconstruction.

PubMed

Zhang, Hua; Zhang, Jingkun; Huang, Xianghua; Li, Yanan

2018-06-01

Endothelial cells and smooth muscle cells (SMCs) are important aspects of vascularization in vaginal reconstruction. Research has confirmed that mesenchymal stem cells could differentiate into endothelial-like cells and SMCs. But the methods were more complicated and the mechanism was unknown. In the current study, we induced the bone mesenchymal stem cells (BMSCs) to differentiate into endothelial-like cells and SMCs in vitro by differentiation medium and investigated the effect of Wnt/β-catenin signaling on the differentiation process of BMSCs. Results showed that the hypoxic environment combined with VEGF and bFGF could induce increased expression of endothelial-like cells markers VEGFR1, VEGFR2, and vWF. The SMCs derived from BMSCs induced by TGF-β1 and PDGF-AB significantly expressed SMC markers SMMHC11 and α-SMA. The data also showed that activation of Wnt/β-catenin signaling could promote the differentiation of BMSCs into endothelial-like cells and SMCs. Thus, we established endothelial-like cells and SMCs in vitro by more simple methods, presented the important role of hypoxic environment on the differentiation of BMSCs into endothelial-like cells, and confirmed that the Wnt/β-catenin signaling pathway has a positive impact on the differentiation of BMSCs into endothelial-like cells and SMCs. This is important for vascular reconstruction.

Use of estetrol with other steroids for attenuation of neonatal hypoxic-ischemic brain injury: to combine or not to combine?

PubMed

Tskitishvili, Ekaterine; Pequeux, Christel; Munaut, Carine; Viellevoye, Renaud; Nisolle, Michelle; Noël, Agnes; Foidart, Jean-Michel

2016-06-07

Estetrol (E4), estradiol (E2) and progesterone (P4) have important antioxidative and neuroprotective effects in neuronal system. We aimed to study the consequence of combined steroid therapy in neonatal hypoxic-ischemic encephalopathy (HIE). In vitro the effect of E4 combined with other steroids on oxidative stress and the cell viability in primary hippocampal cultures was evaluated by lactate dehydrogenase and cell survival assays. In vivo neuroprotective and therapeutic efficacy of E4 combined with other steroids was studied in HIE model of immature rats. The rat pups rectal temperature, body and brain weights were evaluated.The hippocampus and the cortex were investigated by histo/immunohistochemistry: intact cell number counting, expressions of markers for early gray matter lose, neuro- and angiogenesis were studied. Glial fibrillary acidic protein was evaluated by ELISA in blood samples. In vitro E4 and combinations of high doses of E4 with P4 and/or E2 significantly diminished the LDH activity and upregulated the cell survival.In vivopretreatment or treatment by different combinations of E4 with other steroids had unalike effects on body and brain weight, neuro- and angiogenesis, and GFAP expression in blood. The combined use of E4 with other steroids has no benefit over the single use of E4.

DNA Ligase C1 Mediates the LigD-Independent Nonhomologous End-Joining Pathway of Mycobacterium smegmatis

PubMed Central

Bhattarai, Hitesh; Gupta, Richa

2014-01-01

Nonhomologous end joining (NHEJ) is a recently described bacterial DNA double-strand break (DSB) repair pathway that has been best characterized for mycobacteria. NHEJ can religate transformed linear plasmids, repair ionizing radiation (IR)-induced DSBs in nonreplicating cells, and seal I-SceI-induced chromosomal DSBs. The core components of the mycobacterial NHEJ machinery are the DNA end binding protein Ku and the polyfunctional DNA ligase LigD. LigD has three autonomous enzymatic modules: ATP-dependent DNA ligase (LIG), DNA/RNA polymerase (POL), and 3′ phosphoesterase (PE). Although genetic ablation of ku or ligD abolishes NHEJ and sensitizes nonreplicating cells to ionizing radiation, selective ablation of the ligase activity of LigD in vivo only mildly impairs NHEJ of linearized plasmids, indicating that an additional DNA ligase can support NHEJ. Additionally, the in vivo role of the POL and PE domains in NHEJ is unclear. Here we define a LigD ligase-independent NHEJ pathway in Mycobacterium smegmatis that requires the ATP-dependent DNA ligase LigC1 and the POL domain of LigD. Mycobacterium tuberculosis LigC can also support this backup NHEJ pathway. We also demonstrate that, although dispensable for efficient plasmid NHEJ, the activities of the POL and PE domains are required for repair of IR-induced DSBs in nonreplicating cells. These findings define the genetic requirements for a LigD-independent NHEJ pathway in mycobacteria and demonstrate that all enzymatic functions of the LigD protein participate in NHEJ in vivo. PMID:24957619

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

PubMed Central

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

2017-01-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

Chondroprotective effect of zinc oxide nanoparticles in conjunction with hypoxia on bovine cartilage-matrix synthesis.

PubMed

Mirza, Eraj Humayun; Pan-Pan, Chong; Wan Ibrahim, Wan Mohd Azhar Bin; Djordjevic, Ivan; Pingguan-Murphy, Belinda

2015-11-01

Articular cartilage is a tissue specifically adapted to a specific niche with a low oxygen tension (hypoxia), and the presence of such conditions is a key factor in regulating growth and survival of chondrocytes. Zinc deficiency has been linked to cartilage-related disease, and presence of Zinc is known to provide antibacterial benefits, which makes its inclusion attractive in an in vitro system to reduce infection. Inclusion of 1% zinc oxide nanoparticles (ZnONP) in poly octanediol citrate (POC) polymer cultured in hypoxia has not been well determined. In this study we investigated the effects of ZnONP on chondrocyte proliferation and matrix synthesis cultured under normoxia (21% O2 ) and hypoxia (5% O2 ). We report an upregulation of chondrocyte proliferation and sulfated glycosaminoglycan (S-GAG) in hypoxic culture. Results demonstrate a synergistic effect of oxygen concentration and 1% ZnONP in up-regulation of anabolic gene expression (Type II collagen and aggrecan), and a down regulation of catabolic (MMP-13) gene expression. Furthermore, production of transcription factor hypoxia-inducible factor 1A (HIF-1A) in response to hypoxic condition to regulate chondrocyte survival under hypoxia is not affected by the presence of 1% ZnONP. Presence of 1% ZnONP appears to act to preserve homeostasis of cartilage in its hypoxic environment. © 2015 Wiley Periodicals, Inc.

Hypoxia/hepatoma dual specific suicide gene expression plasmid delivery using bio-reducible polymer for hepatocellular carcinoma therapy.

PubMed

Kim, Hyun Ah; Nam, Kihoon; Lee, Minhyung; Kim, Sung Wan

2013-10-10

Gene therapy is suggested as a promising alternative strategy of hepatocellular carcinoma (HCC, also called hepatoma) therapy. To achieve a successful and safe gene therapy, tight regulation of gene expression is required to minimize side-effects in normal tissues. In this study, we developed a novel hypoxia and hepatoma dual specific gene expression vector. The constructed vectors were transfected into various cell lines using bio-reducible polymer, PAM-ABP. First, pAFPS-Luc or pAFPL-Luc vector was constructed with the alpha-fectoprotein (AFP) promoter and enhancer for hepatoma tissue specific gene expression. Then, pEpo-AFPL-Luc was constructed by insertion of the erythropoietin (Epo) enhancer for hypoxic cancer specific gene expression. In vitro transfection assay showed that pEpo-AFPL-Luc transfected hepatoma cell increased gene expression under hypoxic condition. To confirm the therapeutic effect of dual specific vector, herpes simplex virus thymidine kinase (HSV-TK) gene was introduced for cancer cell killing. The pEpo-AFPL-TK was transfected into hepatoma cell lines in the presence of ganciclovir (GCV) pro-drug. Caspase-3/7, MTT and TUNEL assays elucidated that pEpo-AFPL-TK transfected cells showed significant increasing of death rate in hypoxic hepatoma cells compared to controls. Therefore, the hypoxia/hepatoma dual specific gene expression vector with the Epo enhancer and AFP promoter may be useful for hepatoma specific gene therapy. © 2013.

To establish a pharmacological experimental platform for the study of cardiac hypoxia using the microelectrode array.

PubMed

Yeung, Chi-Kong; Sommerhage, Frank; Wrobel, Günter; Law, Jessica Ka-Yan; Offenhäusser, Andreas; Rudd, John Anthony; Ingebrandt, Sven; Chan, Mansun

2009-01-01

Simultaneous recording of electrical potentials from multiple cells may be useful for physiological and pharmacological research. The present study aimed to establish an in vitro cardiac hypoxia experimental platform on the microelectrode array (MEA). Embryonic rat cardiac myocytes were cultured on the MEAs. Following >or=90 min of hypoxia, changes in lactate production (mM), pH, beat frequency (beats per min, bpm), extracellular action potential (exAP) amplitude, and propagation velocity between the normoxic and hypoxic cells were compared. Under hypoxia, the beat frequency of cells increased and peaked at around 42.5 min (08.1+/-3.2 bpm). The exAP amplitude reduced as soon as the cells were exposed to the hypoxic medium, and this reduction increased significantly after approximately 20 min of hypoxia. The propagation velocity of the hypoxic cells was significantly lower than that of the control throughout the entire 90+ min of hypoxia. The rate of depolarisation and Na(+) signal gradually reduced over time, and these changes had a direct effect on the exAP duration. The extracellular electrophysiological measurements allow a partial reconstruction of the signal shape and time course of the underlying hypoxia-associated physiological changes. The present study showed that the cardiac myocyte-integrated MEA may be used as an experimental platform for the pharmacological studies of cardiovascular diseases in the future.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

PubMed

Hu, Yae; Yan, Ruhong; Zhang, Ce; Zhou, Zhichao; Liu, Meng; Wang, Can; Zhang, Hong; Dong, Liang; Zhou, Tiantian; Wu, Yi; Dong, Ningzheng; Wu, Qingyu

2018-04-12

Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia. © 2018 American Heart Association, Inc.

Enhanced Prostate Cancer Gene Transfer and Therapy Using a Novel Serotype Chimera Cancer Terminator Virus (Ad.5/3-CTV)

PubMed Central

AZAB, BELAL M.; DASH, RUPESH; DAS, SWADESH K.; BHUTIA, SUJIT K.; SARKAR, SIDDIK; SHEN, XUE-NING; QUINN, BRIDGET A.; DENT, PAUL; DMITRIEV, IGOR P.; WANG, XIANG-YANG; CURIEL, DAVID T.; PELLECCHIA, MAURIZIO; REED, JOHN C.; SARKAR, DEVANAND; FISHER, PAUL B.

2015-01-01

Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting. PMID:23868767

Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

PubMed Central

Bozec, Aline; Hannemann, Nicole

2016-01-01

Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes. PMID:27284940

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

PubMed Central

Huang, Ying; Zitta, Karina; Bein, Berthold; Steinfath, Markus; Albrecht, Martin

2013-01-01

SUMMARY Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs. PMID:24046359

Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

PubMed

Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

2017-04-01

RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Development of a metabolically active, non-replicating sporozoite vaccine to prevent Plasmodium falciparum malaria.

PubMed

Hoffman, Stephen L; Billingsley, Peter F; James, Eric; Richman, Adam; Loyevsky, Mark; Li, Tao; Chakravarty, Sumana; Gunasekera, Anusha; Chattopadhyay, Rana; Li, Minglin; Stafford, Richard; Ahumada, Adriana; Epstein, Judith E; Sedegah, Martha; Reyes, Sharina; Richie, Thomas L; Lyke, Kirsten E; Edelman, Robert; Laurens, Matthew B; Plowe, Christopher V; Sim, B Kim Lee

2010-01-01

Immunization of volunteers by the bite of mosquitoes carrying radiation-attenuated Plasmodium falciparum sporozoites protects greater than 90% of such volunteers against malaria, if adequate numbers of immunizing biting sessions and sporozoite-infected mosquitoes are used. Nonetheless, until recently it was considered impossible to develop, license and commercialize a live, whole parasite P. falciparum sporozoite (PfSPZ) vaccine. In 2003 Sanaria scientists reappraised the potential impact of a metabolically active, non-replicating PfSPZ vaccine, and outlined the challenges to producing such a vaccine. Six years later, significant progress has been made in overcoming these challenges. This progress has enabled the manufacture and release of multiple clinical lots of a 1(st) generation metabolically active, non-replicating PfSPZ vaccine, the Sanaria PfSPZ Vaccine, submission of a successful Investigational New Drug application to the US Food and Drug Administration, and initiation of safety, immunogenicity and protective efficacy studies in volunteers in MD, US. Efforts are now focused on how best to achieve submission of a successful Biologics License Application and introduce the vaccine to the primary target population of African children in the shortest possible period of time. This will require implementation of a systematic, efficient clinical development plan. Short term challenges include optimizing the (1) efficiency and scale up of the manufacturing process and quality control assays, (2) dosage regimen and method of administration, (3) potency of the vaccine, and (4) logistics of delivering the vaccine to those who need it most, and finalizing the methods for vaccine stabilization and attenuation. A medium term goal is to design and build a facility for manufacturing highly potent and stable vaccine for pivotal Phase 3 studies and commercial launch.

Synthesis and structure-activity relationships of varied ether linker analogues of the antitubercular drug (6S)-2-nitro-6-{[4-(trifluoromethoxy)benzyl]oxy}-6,7-dihydro-5h-imidazo[2,1-b][1,3]oxazine (PA-824).

PubMed

Thompson, Andrew M; Sutherland, Hamish S; Palmer, Brian D; Kmentova, Iveta; Blaser, Adrian; Franzblau, Scott G; Wan, Baojie; Wang, Yuehong; Ma, Zhenkun; Denny, William A

2011-10-13

New analogues of antitubercular drug PA-824 were synthesized, featuring alternative side chain ether linkers of varying size and flexibility, seeking drug candidates with enhanced metabolic stability and high efficacy. Both α-methyl substitution and removal of the benzylic methylene were broadly tolerated in vitro, with a biaryl example of the latter class exhibiting an 8-fold better efficacy than the parent drug in a mouse model of acute Mycobacterium tuberculosis infection and negligible fragmentation to an alcohol metabolite in liver microsomes. Extended linkers (notably propenyloxy, propynyloxy, and pentynyloxy) provided greater potencies against replicating M. tb (monoaryl analogues), with propynyl ethers being most effective under anaerobic (nonreplicating) conditions (mono/biaryl analogues). For benzyloxybenzyl and biaryl derivatives, aerobic activity was maximal with the original (OCH(2)) linker. One propynyloxy-linked compound displayed an 89-fold higher efficacy than the parent drug in the acute model, and it was slightly superior to antitubercular drug OPC-67683 in a chronic infection model.

Latent Tuberculosis Infection: Myths, Models, and Molecular Mechanisms

PubMed Central

Dutta, Noton K.

2014-01-01

SUMMARY The aim of this review is to present the current state of knowledge on human latent tuberculosis infection (LTBI) based on clinical studies and observations, as well as experimental in vitro and animal models. Several key terms are defined, including “latency,” “persistence,” “dormancy,” and “antibiotic tolerance.” Dogmas prevalent in the field are critically examined based on available clinical and experimental data, including the long-held beliefs that infection is either latent or active, that LTBI represents a small population of nonreplicating, “dormant” bacilli, and that caseous granulomas are the haven for LTBI. The role of host factors, such as CD4+ and CD8+ T cells, T regulatory cells, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ), in controlling TB infection is discussed. We also highlight microbial regulatory and metabolic pathways implicated in bacillary growth restriction and antibiotic tolerance under various physiologically relevant conditions. Finally, we pose several clinically important questions, which remain unanswered and will serve to stimulate future research on LTBI. PMID:25184558

Recombination within the nonstructural genes of the parvovirus minute virus of mice (MVM) generates functional levels of wild-type NS1, which can be detected in the absence of selective pressure following transfection of nonreplicating plasmids.

PubMed

Pearson, J L; Pintel, D J

2000-03-30

Recombination within the coding region of the nonstructural genes of minute virus of mice (MVM), which generates functional levels of wild-type NS1, was observed in the absence of selective pressure following cotransfection of nonreplicating plasmids. P38 activity was used as a measure of recombinant NS1 production, which, together with direct detection of recombinant-generated products by RT-PCR, allowed an estimation of recombination efficiency. In addition, we show that very low levels of wild-type NS1 were able to significantly transactivate P38. Given that recombination following cotransfection can generate NS1 at these levels, our observations have implications for the study of parvoviral genetics, the construction of recombinant parvoviral vectors for gene therapy applications, and perhaps other systems using cotransfection of plasmids that share homologous sequences. Copyright 2000 Academic Press.

Novel Imidazoline Antimicrobial Scaffold That Inhibits DNA Replication with Activity against Mycobacteria and Drug Resistant Gram-Positive Cocci

PubMed Central

2015-01-01

Bacterial antimicrobial resistance is an escalating public health threat, yet the current antimicrobial pipeline remains alarmingly depleted, making the development of new antimicrobials an urgent need. Here, we identify a novel, potent, imidazoline antimicrobial compound, SKI-356313, with bactericidal activity against Mycobacterium tuberculosis and Gram-positive cocci, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA). SKI-356313 is active in murine models of Streptococcus pneumoniae and MRSA infection and is potently bactericidal for both replicating and nonreplicating M. tuberculosis. Using a combination of genetics, whole genome sequencing, and a novel target ID approach using real time imaging of core macromolecular biosynthesis, we show that SKI-356313 inhibits DNA replication and displaces the replisome from the bacterial nucleoid. These results identify a new antimicrobial scaffold with a novel mechanism of action and potential therapeutic utility against nonreplicating M. tuberculosis and antibiotic resistant Gram-positive cocci. PMID:25222597

Enhanced differentiation of neural stem cells to neurons and promotion of neurite outgrowth by oxygen-glucose deprivation.

PubMed

Wang, Qin; Yang, Lin; Wang, Yaping

2015-06-01

Stroke has become the leading cause of mortality worldwide. Hypoxic or ischemic insults are crucial factors mediating the neural damage in the brain tissue of stroke patients. Neural stem cells (NSCs) have been recognized as a promising tool for the treatment of ischemic stroke and other neurodegenerative diseases due to their inducible pluripotency. In this study, we aim to mimick the cerebral hypoxic-ischemic injury in vitro using oxygen-glucose deprivation (OGD) strategy, and evaluate the effects of OGD on the NSC's neural differentiation, as well as the differentiated neurite outgrowth. Our data showed that NSCs under the short-term 2h OGD treatment are able to maintain cell viability and the capability to form neurospheres. Importantly, this moderate OGD treatment promotes NSC differentiation to neurons and enhances the performance of the mature neuronal networks, accompanying increased neurite outgrowth of differentiated neurons. However, long-term 6h and 8h OGD exposures in NSCs lead to decreased cell survival, reduced differentiation and diminished NSC-derived neurite outgrowth. The expressions of neuron-specific microtubule-associated protein 2 (MAP-2) and growth associated protein 43 (GAP-43) are increased by short-term OGD treatments but suppressed by long-term OGD. Overall, our results demonstrate that short-term OGD exposure in vitro induces differentiation of NSCs while maintaining their proliferation and survival, providing valuable insights of adopting NSC-based therapy for ischemic stroke and other neurodegenerative disorders. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemoattractant signaling between tumor cells and macrophages regulates cancer cell migration, metastasis and neovascularization.

PubMed

Green, Chad E; Liu, Tiffany; Montel, Valerie; Hsiao, Gene; Lester, Robin D; Subramaniam, Shankar; Gonias, Steven L; Klemke, Richard L

2009-08-21

Tumor-associated macrophages are known to influence cancer progression by modulation of immune function, angiogenesis, and cell metastasis, however, little is known about the chemokine signaling networks that regulate this process. Utilizing CT26 colon cancer cells and RAW 264.7 macrophages as a model cellular system, we demonstrate that treatment of CT26 cells with RAW 264.7 conditioned medium induces cell migration, invasion and metastasis. Inflammatory gene microarray analysis indicated CT26-stimulated RAW 264.7 macrophages upregulate SDF-1alpha and VEGF, and that these cytokines contribute to CT26 migration in vitro. RAW 264.7 macrophages also showed a robust chemotactic response towards CT26-derived chemokines. In particular, microarray analysis and functional testing revealed CSF-1 as the major chemoattractant for RAW 264.7 macrophages. Interestingly, in the chick CAM model of cancer progression, RAW 264.7 macrophages localized specifically to the tumor periphery where they were found to increase CT26 tumor growth, microvascular density, vascular disruption, and lung metastasis, suggesting these cells home to actively invading areas of the tumor, but not the hypoxic core of the tumor mass. In support of these findings, hypoxic conditions down regulated CSF-1 production in several tumor cell lines and decreased RAW 264.7 macrophage migration in vitro. Together our findings suggest a model where normoxic tumor cells release CSF-1 to recruit macrophages to the tumor periphery where they secrete motility and angiogenic factors that facilitate tumor cell invasion and metastasis.

Antihelminthic benzimidazoles are novel HIF activators that prevent oxidative neuronal death via binding to tubulin.

PubMed

Aleyasin, Hossein; Karuppagounder, Saravanan S; Kumar, Amit; Sleiman, Sama; Basso, Manuela; Ma, Thong; Siddiq, Ambreena; Chinta, Shankar J; Brochier, Camille; Langley, Brett; Haskew-Layton, Renee; Bane, Susan L; Riggins, Gregory J; Gazaryan, Irina; Starkov, Anatoly A; Andersen, Julie K; Ratan, Rajiv R

2015-01-10

Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21(cip1/waf1), in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke.

Effect of radiation on cell proliferation and tumor hypoxia in HPV-positive head and neck cancer in vivo models.

PubMed

Sørensen, Brita Singers; Busk, Morten; Horsman, Michael R; Alsner, Jan; Overgaard, Jens; Kyle, Alastair H; Minchinton, Andrew I

2014-11-01

Human papilloma virus-associated head and neck squamous cell carcinomas (HNSCC) represent a distinct subgroup of HNSCC characterized by a favorable prognosis and a distinct molecular biology. There is a range of unresolved questions regarding the different biology and clinical outcome of HPV-positive HNSCC. The purpose of the present project was to obtain insight into the biology of treatment responsiveness of HPV-related HNSCC. Tumor xenografts were established from HPV-negative (FaDuDD,) and HPV-positive (UD2 and UMSCC47) HNSCC cell lines. Tumors were treated with 10 Gy or 20 Gy and the effect on the tumor microenvironment was studied at different time points after treatment. Cryosections were imaged for cell proliferation, hypoxia, vessel density and vessel perfusion. In the HPV-positive tumor models the levels of cell proliferation decreased significantly following irradiation. This was not seen in the HPV-negative model (FaDuDD). Furthermore, it was found that the tumor hypoxic fraction decreased over time after treatment in irradiated HPV-positive tumors and not in the HPV-negative tumors. The radiosensitivity previously observed in vitro could be applied in vivo in respect to a radiation-induced decrease in proliferating cells. A decreasing hypoxic fraction following irradiation in the HPV-positive tumors could explain the lack of benefit from hypoxic modifiers observed in patients. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Systemic Hypoxia Changes the Organ-Specific Distribution of Vascular Endothelial Growth Factor and Its Receptors

NASA Astrophysics Data System (ADS)

Marti, Hugo H.; Risau, Werner

1998-12-01

Vascular endothelial growth factor (VEGF) plays a key role in physiological blood vessel formation and pathological angiogenesis such as tumor growth and ischemic diseases. Hypoxia is a potent inducer of VEGF in vitro. Here we demonstrate that VEGF is induced in vivo by exposing mice to systemic hypoxia. VEGF induction was highest in brain, but also occurred in kidney, testis, lung, heart, and liver. In situ hybridization analysis revealed that a distinct subset of cells within a given organ, such as glial cells and neurons in brain, tubular cells in kidney, and Sertoli cells in testis, responded to the hypoxic stimulus with an increase in VEGF expression. Surprisingly, however, other cells at sites of constitutive VEGF expression in normal adult tissues, such as epithelial cells in the choroid plexus and kidney glomeruli, decreased VEGF expression in response to the hypoxic stimulus. Furthermore, in addition to VEGF itself, expression of VEGF receptor-1 (VEGFR-1), but not VEGFR-2, was induced by hypoxia in endothelial cells of lung, heart, brain, kidney, and liver. VEGF itself was never found to be up-regulated in endothelial cells under hypoxic conditions, consistent with its paracrine action during normoxia. Our results show that the response to hypoxia in vivo is differentially regulated at the level of specific cell types or layers in certain organs. In these tissues, up- or down-regulation of VEGF and VEGFR-1 during hypoxia may influence their oxygenation after angiogenesis or modulate vascular permeability.

In vitro-in vivo activity relationship of substituted benzimidazole cell division inhibitors with activity against Mycobacteria tuberculosis

PubMed Central

Knudson, Susan E.; Kumar, Kunal; Awasthi, Divya; Ojima, Iwao; Slayden, Richard A.

2014-01-01

Structure based drug design was used to develop a compound library of novel 2,5,6- and 2,5,7-trisubstituted benzimidazoles. Three structural analogs, SB-P1G10, SB-P8B2 and SB-P3G2 were selected from this library based on previous studies for advanced study. In vitro studies revealed that SB-P8B2 and SB-P3G2 had sigmoidal kill-curves while in contrast SB-P1G10 showed a narrow zonal susceptibility. The in vitro studies also demonstrated that exposure to SB-P8B2 or SB-P3G2 was bactericidal, while SB-P1G10 treatment never resulted in complete killing. The dose curves for the three compounds against clinical isolates were comparable to their respective dose curves in the laboratory strain of M. tuberculosis. SB-P8B2 and SB-P3G2 exhibited antibacterial activity against non-replicating bacilli under low oxygen conditions. SB-P3G2 and SB-P1G10 were assessed in acute short-term animal models of tuberculosis, which showed that SB-P3G2 treatment demonstrated activity against M. tuberculosis. Together, these studies reveal an in vitro- in vivo relationship of the 2,5,6-trisubstituted benzimidazoles that serves as a criterion for advancing this class of cell division inhibitors into more resource intensive in vivo efficacy models such as the long-term murine model of tuberculosis and Pre-IND PK/PD studies. Specifically, these studies are the first demonstration of efficacy and an in vitro–in vivo activity relationship for 2,5,6-trisubstituted benzimidazoles. The in vivo activity presented in this manuscript substantiates this class of cell division inhibitors as having potency and efficacy against M. tuberculosis. PMID:24746463

In vitro and in vivo activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis.

PubMed

Upton, A M; Cho, S; Yang, T J; Kim, Y; Wang, Y; Lu, Y; Wang, B; Xu, J; Mdluli, K; Ma, Z; Franzblau, S G

2015-01-01

Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

In Vitro and In Vivo Activities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis

PubMed Central

Cho, S.; Yang, T. J.; Kim, Y.; Wang, Y.; Lu, Y.; Wang, B.; Xu, J.; Mdluli, K.; Ma, Z.; Franzblau, S. G.

2014-01-01

Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10−7. In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole. PMID:25331696

Magnolol suppresses hypoxia-induced angiogenesis via inhibition of HIF-1α/VEGF signaling pathway in human bladder cancer cells.

PubMed

Chen, Meng-Chuan; Lee, Chi-Feng; Huang, Wen-Hsin; Chou, Tz-Chong

2013-05-01

The hypoxic environment in tumors is an important factor causing tumor angiogenesis by activating the key transcription factor, hypoxia-inducible factors-1α (HIF-1α). Magnolol isolated from Magnolia officinalis has been reported to exhibit an anticancer activity via elevation of apoptosis. However, whether magnolol inhibits tumor angiogenesis remains unknown. In the present study, we demonstrated that magnolol significantly inhibited angiogenesis in vitro and in vivo evidenced by the attenuation of hypoxia and vascular endothelial growth factor (VEGF)-induced tube formation of human umbilical vascular endothelial cells, vasculature generation in chicken chorioallantoic membrane and Matrigel plug. In hypoxic human bladder cancer cells (T24), treatment with magnolol inhibited hypoxia-stimulated H2O2 formation, HIF-1α induction including mRNA, protein expression, and transcriptional activity as well as VEGF secretion. Additionally, the enhanced degradation of HIF-1α protein via enhancing prolyl hydroxylase activity and the decreased newly-synthesized HIF-1α protein in hypoxic T24 cells may involve the reduction of HIF-1α protein accumulation by magnolol. Interestingly, magnolol also acts as a VEGFR2 antagonist, and subsequently attenuates the down-stream AKT/mTOR/p70S6K/4E-BP-1 kinase activation both in hypoxic T24 cells and tumor tissues. As expected, administration of magnolol greatly attenuated tumor growth, angiogenesis and the protein expression of HIF-1α, VEGF, CD31, a marker of endothelial cells, and carbonic anhydrase IX, an endogenous marker for hypoxia, in the T24 xenograft mouse model. Collectively, these findings strongly indicate that the anti-agngiogenic activity of magnolol is, at least in part, mediated by suppressing HIF-1α/VEGF-dependent pathways, and suggest that magnolol may be a potential drug for human bladder cancer therapy. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro

PubMed Central

Zhou, Liping; Zhang, Xiaowei; Zhou, Panpan; Li, Xue; Xu, Xuejing; Shi, Qing; Li, Dong; Ju, Xiuli

2017-01-01

Successfully expanding hematopoietic stem cells (HSCs) is advantageous for clinical HSC transplantation. The present study investigated the influence of testosterone on the proliferation, antigen phenotype and expression of hematopoiesis-related genes in umbilical cord blood-derived cluster of differentiation (CD)34+ cells under normoxic or hypoxia conditions. Cord blood (CB) CD34+ cells were separated using magnetic activated cell sorting. A cytokine cocktail and feeder cells were used to stimulate the expansion of CD34+ cells under normoxic (20% O2) and hypoxic (1% O2) conditions for 7 days and testosterone was added accordingly. Cells were identified using flow cytometry and reconstruction capacity was determined using a colony-forming unit (CFU) assay. The effects of oxygen concentration and testosterone on the expression of hematopoietic-related genes, including homeobox (HOX)A9, HOXB2, HOXB4, HOXC4 and BMI-1, were measured using reverse transcription-quantitative polymerase chain reaction. The results indicated that the number of CFUs and total cells in the testosterone group increased under normoxic and hypoxic conditions compared with the corresponding control groups. Furthermore, the presence of testosterone increased the number of CFU-erythroid colonies. In liquid culture, the growth of CD34+ cells was rapid under normoxic conditions compared with under hypoxic conditions, however CD34+ cells were maintained in an undifferentiated state under hypoxic conditions. The addition of testosterone under hypoxia promoted the differentiation of CD34+ cells into CD34+CD38+CD71+ erythroid progenitor cells. Furthermore, it was determined that the expression of hematopoietic-related genes was significantly increased (P<0.05) in the hypoxia testosterone group compared with the other groups. Therefore, the results of the current study indicate that a combination of hypoxia and testosterone may be a promising cultivation condition for HSC/hemopoietic progenitor cell expansion ex vivo. PMID:29067121

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro.

PubMed

Zhou, Liping; Zhang, Xiaowei; Zhou, Panpan; Li, Xue; Xu, Xuejing; Shi, Qing; Li, Dong; Ju, Xiuli

2017-11-01

Successfully expanding hematopoietic stem cells (HSCs) is advantageous for clinical HSC transplantation. The present study investigated the influence of testosterone on the proliferation, antigen phenotype and expression of hematopoiesis-related genes in umbilical cord blood-derived cluster of differentiation (CD)34 + cells under normoxic or hypoxia conditions. Cord blood (CB) CD34 + cells were separated using magnetic activated cell sorting. A cytokine cocktail and feeder cells were used to stimulate the expansion of CD34 + cells under normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions for 7 days and testosterone was added accordingly. Cells were identified using flow cytometry and reconstruction capacity was determined using a colony-forming unit (CFU) assay. The effects of oxygen concentration and testosterone on the expression of hematopoietic-related genes, including homeobox (HOX)A9, HOXB2, HOXB4, HOXC4 and BMI-1, were measured using reverse transcription-quantitative polymerase chain reaction. The results indicated that the number of CFUs and total cells in the testosterone group increased under normoxic and hypoxic conditions compared with the corresponding control groups. Furthermore, the presence of testosterone increased the number of CFU-erythroid colonies. In liquid culture, the growth of CD34 + cells was rapid under normoxic conditions compared with under hypoxic conditions, however CD34 + cells were maintained in an undifferentiated state under hypoxic conditions. The addition of testosterone under hypoxia promoted the differentiation of CD34 + cells into CD34 + CD38 + CD71 + erythroid progenitor cells. Furthermore, it was determined that the expression of hematopoietic-related genes was significantly increased (P<0.05) in the hypoxia testosterone group compared with the other groups. Therefore, the results of the current study indicate that a combination of hypoxia and testosterone may be a promising cultivation condition for HSC/hemopoietic progenitor cell expansion ex vivo .

Combining antiangiogenic therapy with adoptive cell immunotherapy exerts better antitumor effects in non-small cell lung cancer models.

PubMed

Shi, Shujing; Wang, Rui; Chen, Yitian; Song, Haizhu; Chen, Longbang; Huang, Guichun

2013-01-01

Cytokine-induced killer cells (CIK cells) are a heterogeneous subset of ex-vivo expanded T lymphocytes which are characterized with a MHC-unrestricted tumor-killing activity and a mixed T-NK phenotype. Adoptive CIK cells transfer, one of the adoptive immunotherapy represents a promising nontoxic anticancer therapy. However, in clinical studies, the therapeutic activity of adoptive CIK cells transfer is not as efficient as anticipated. Possible explanations are that abnormal tumor vasculature and hypoxic tumor microenvironment could impede the infiltration and efficacy of lymphocytes. We hypothesized that antiangiogenesis therapy could improve the antitumor activity of CIK cells by normalizing tumor vasculature and modulating hypoxic tumor microenvironment. We combined recombinant human endostatin (rh-endostatin) and CIK cells in the treatment of lung carcinoma murine models. Intravital microscopy, dynamic contrast enhanced magnetic resonance imaging, immunohistochemistry, and flow cytometry were used to investigate the tumor vasculature and hypoxic microenvironment as well as the infiltration of immune cells. Our results indicated that rh-endostatin synergized with adoptive CIK cells transfer to inhibit the growth of lung carcinoma. We found that rh-endostatin normalized tumor vasculature and reduced hypoxic area in the tumor microenvironment. Hypoxia significantly inhibited the proliferation, cytotoxicity and migration of CIK cells in vitro and impeded the homing of CIK cells into tumor parenchyma ex vivo. Furthermore, we found that treatment with rh-endostatin significantly increased the homing of CIK cells and decreased the accumulation of suppressive immune cells in the tumor tissue. In addition, combination therapy produced higher level of tumor-infiltration lymphocytes compared with other treatments. Our results demonstrate that rh-endostatin improves the therapeutic effect of adoptive CIK cells therapy against lung carcinomas and unmask the mechanisms of the synergistic antitumor efficacy, providing a new rationale for combining antiangiogenesis therapy with immunotherapy in the treatment of lung cancer.

Comprehensive evaluation of immunomodulation by moderate hypoxia in S. agalactiae vaccinated Nile tilapia.

PubMed

Gallage, Sanchala; Katagiri, Takayuki; Endo, Masato; Maita, Masashi

2017-07-01

Streptococcus agalactiae is a major bacterial pathogen in tilapia aquaculture. Vaccines are known to provide protection but S. agalactiae clearance in tilapia can be reduced by marginal environmental conditions. Therefore, the purpose of this study is to examine S. agalactiae clearance in vaccinated Nile tilapia under moderate hypoxic (55± 5% DO) and normoxic (85 ± 5%DO) conditions. Fish were acclimatized to either moderate hypoxia or normoxia and immunized with formalin-inactivated S. agalactiae. Fish were experimentally challenged with S. agalactiae at 30 days post-vaccination. Serum antibody titer was significantly higher in vaccinated fish kept under normoxic condition compared to the moderate hypoxic condition at fifteen and thirty days post-vaccination. The cumulative mortality following challenge was significantly reduced in vaccinated fish kept under normoxic condition compared to those in moderate hypoxic condition reflecting that pre-challenge antibody titer may correlate with survival of fish. Blood and tissue pathogen burden detection of S. agalactiae studies revealed that culturable S. agalactiae cells could not be detected in the blood of normoxic vaccinated fish at all the sampling points. In contrast, fish vaccinated in moderate hypoxic condition had considerable number of culturable S. agalactiae cells in their blood up to 5 days following challenge. Phagocytosis and intracellular reactive oxygen species (ROS) production were lowered by moderate hypoxia in vitro. Furthermore, presence of specific antibodies and higher specific antibody level in the serum increased phagocytosis, ROS production and lowered intracellular survival of S. agalactiae in head kidney leukocytes. Overall this study has highlighted that S. agalactiae clearance in vaccinated Nile tilapia is modulated by moderate hypoxia. One of the possible explanations for this might be less efficient phagocytic activities due to low oxygen availability and lower specific antibody production in vaccinated fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Transepithelial Ion Transport is Suppressed in Hypoxic Sinonasal Epithelium

PubMed Central

Blount, Angela; Zhang, Shaoyan; Chestnut, Michael; Hixon, Brian; Skinner, Daniel; Sorscher, Eric J.; Woodworth, Bradford A.

2011-01-01

Objectives/Hypothesis Sinonasal respiratory epithelial mucociliary clearance (MCC) is dependent on the transepithelial transport of ions such as Cl−. The objectives of the present study were to investigate the role of oxygen restriction in 1) Cl− transport across primary sinonasal epithelial monolayers, 2) expression of the apical Cl− channels CFTR and TMEM16A, and 3) the pathogenesis of chronic rhinosinusitis (CRS). Study Design In vitro investigation. Methods Murine nasal septal epithelial (MNSE, wild type) and human sinonasal epithelial (HSNE) cultures were incubated under hypoxic conditions (1% O2, 5% CO2). Cultures were mounted in Ussing chambers for ion transport measurements. CFTR and TMEM16A expression were measured using quantitative RT-PCR. Results The change in short-circuit current (ΔISC (µA/cm2) attributable to CFTR (forskolin-stimulated) was significantly decreased due to a 12 hour hypoxia exposure in both MNSE (13.55+/− 0.46 vs. 19.23+/−0.18) and HSNE (19.55+/−0.56 vs. 25.49+/−1.48 (control); p<0.05. TMEM16A (UTP-stimulated transport) was inhibited by 48 hours of hypoxic exposure in MNSE (15.92+/−2.87 vs. 51.44+/−3.71(control) p<0.05] and by 12 hours of hypoxic exposure in HSNE (16.75+/−0.68 vs. 24.15+/−1.35 (control). Quantitative RT-PCR (reported as relative mRNA levels+/−S.D.) demonstrated significant reductions in both CFTR and TMEM16A mRNA expression in MNSE and HSNE due to airway epithelial hypoxia. Conclusions Sinonasal epithelial CFTR and TMEM16A-mediated Cl− transport and mRNA expression were robustly decreased in an oxygen restricted environment. The findings in the present study indicate persistent hypoxia may lead to acquired defects in sinonasal Cl− transport in a fashion likely to confer mucociliary dysfunction in CRS. Level of Evidence 1b PMID:22024847

Tissue factor transcription driven by Egr-1 is a critical mechanism of murine pulmonary fibrin deposition in hypoxia

PubMed Central

Yan, Shi-Fang; Zou, Yu Shan; Gao, Yun; Zhai, Chao; Mackman, Nigel; Lee, Stephen L.; Milbrandt, Jeffrey; Pinsky, David; Kisiel, Walter; Stern, David

1998-01-01

Local hypoxemia and stasis trigger thrombosis. We have demonstrated previously that in a murine model of normobaric hypoxia pulmonary fibrin deposition is a result of expression of tissue factor, especially in oxygen-deprived mononuclear phagocytes (MPs). We now show that transcription factor early-growth-response gene product (Egr-1) is rapidly activated in hypoxia, both in vitro and in vivo, and is responsible for transcription and expression of tissue factor in hypoxic lung. MPs and HeLa cells subjected to hypoxia (pO2 ≈13 torr) had increased levels of tissue factor transcripts (≈18-fold) and an increased rate of transcription (≈15-fold), based on nuclear run-on analysis. Gel-shift analysis of nuclear extracts from hypoxic MPs and HeLa cells demonstrated increased DNA-binding activity at the serum response region (SRR; −111/+14 bp) of the tissue factor promoter at Egr-1 motifs. Using 32P-labeled Egr consensus oligonucleotide, we observed induction of DNA-binding activity in nuclear extracts from hypoxic lung and HeLa cells because of activation of Egr-1, by means of supershift analysis. Transient transfection of HeLa cells with chimeric plasmids containing wild-type or mutant SRR from the tissue factor promoter showed that intact Sp1 sites are necessary for basal promoter activity, whereas the integrity of Egr-1 sites was required for hypoxia-enhanced expression. A central role for Egr-1 in hypoxia-mediated tissue factor expression was confirmed by experiments with homozygous Egr-1 null mice; wild-type mice subjected to oxygen deprivation expressed tissue factor and showed fibrin deposition, but hypoxic homozygous Egr-1 null mice displayed neither tissue factor nor fibrin. These data delineate a novel biology for hypoxia-induced fibrin deposition, in which oxygen deprivation-induced activation of Egr-1, resulting in expression of tissue factor, has an unexpected and central role. PMID:9653181

Combining Antiangiogenic Therapy with Adoptive Cell Immunotherapy Exerts Better Antitumor Effects in Non-Small Cell Lung Cancer Models

PubMed Central

Shi, Shujing; Wang, Rui; Chen, Yitian; Song, Haizhu; Chen, Longbang; Huang, Guichun

2013-01-01

Introduction Cytokine-induced killer cells (CIK cells) are a heterogeneous subset of ex-vivo expanded T lymphocytes which are characterized with a MHC-unrestricted tumor-killing activity and a mixed T-NK phenotype. Adoptive CIK cells transfer, one of the adoptive immunotherapy represents a promising nontoxic anticancer therapy. However, in clinical studies, the therapeutic activity of adoptive CIK cells transfer is not as efficient as anticipated. Possible explanations are that abnormal tumor vasculature and hypoxic tumor microenvironment could impede the infiltration and efficacy of lymphocytes. We hypothesized that antiangiogenesis therapy could improve the antitumor activity of CIK cells by normalizing tumor vasculature and modulating hypoxic tumor microenvironment. Methods We combined recombinant human endostatin (rh-endostatin) and CIK cells in the treatment of lung carcinoma murine models. Intravital microscopy, dynamic contrast enhanced magnetic resonance imaging, immunohistochemistry, and flow cytometry were used to investigate the tumor vasculature and hypoxic microenvironment as well as the infiltration of immune cells. Results Our results indicated that rh-endostatin synergized with adoptive CIK cells transfer to inhibit the growth of lung carcinoma. We found that rh-endostatin normalized tumor vasculature and reduced hypoxic area in the tumor microenvironment. Hypoxia significantly inhibited the proliferation, cytotoxicity and migration of CIK cells in vitro and impeded the homing of CIK cells into tumor parenchyma ex vivo. Furthermore, we found that treatment with rh-endostatin significantly increased the homing of CIK cells and decreased the accumulation of suppressive immune cells in the tumor tissue. In addition, combination therapy produced higher level of tumor-infiltration lymphocytes compared with other treatments. Conclusions Our results demonstrate that rh-endostatin improves the therapeutic effect of adoptive CIK cells therapy against lung carcinomas and unmask the mechanisms of the synergistic antitumor efficacy, providing a new rationale for combining antiangiogenesis therapy with immunotherapy in the treatment of lung cancer. PMID:23799045

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cowan, D.S.; Panicucci, R.; McClelland, R.A.

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 ofmore » 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.« less

Enhanced prostate cancer gene transfer and therapy using a novel serotype chimera cancer terminator virus (Ad.5/3-CTV).

PubMed

Azab, Belal M; Dash, Rupesh; Das, Swadesh K; Bhutia, Sujit K; Sarkar, Siddik; Shen, Xue-Ning; Quinn, Bridget A; Dent, Paul; Dmitriev, Igor P; Wang, Xiang-Yang; Curiel, David T; Pellecchia, Maurizio; Reed, John C; Sarkar, Devanand; Fisher, Paul B

2014-01-01

Few options are available for treating patients with advanced prostate cancer (PC). As PC is a slow growing disease and accessible by ultrasound, gene therapy could provide a viable option for this neoplasm. Conditionally replication-competent adenoviruses (CRCAs) represent potentially useful reagents for treating PC. We previously constructed a CRCA, cancer terminator virus (CTV), which showed efficacy both in vitro and in vivo for PC. The CTV was generated on a serotype 5-background (Ad.5-CTV) with infectivity depending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequently reduced in many tumor types, including PCs thereby limiting effective Ad-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV) was created by replacing the Ad.5 fiber knob with the Ad.3 fiber knob thereby facilitating infection in a CAR-independent manner. We evaluated Ad.5/3-CTV in comparison with Ad.5-CTV in low CAR human PC cells, demonstrating higher efficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV potently suppressed in vivo tumor growth in a nude mouse xenograft model and in a spontaneously induced PC that develops in Hi-myc transgenic mice. Considering the significant responses in a Phase I clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers, Ad.5/3-CTV may exert improved therapeutic benefit in a clinical setting. © 2013 Wiley Periodicals, Inc.

Copper-boosting compounds: a novel concept for antimycobacterial drug discovery.

PubMed

Speer, Alexander; Shrestha, Tej B; Bossmann, Stefan H; Basaraba, Randall J; Harber, Gregory J; Michalek, Suzanne M; Niederweis, Michael; Kutsch, Olaf; Wolschendorf, Frank

2013-02-01

We and others recently identified copper resistance as important for virulence of Mycobacterium tuberculosis. Here, we introduce a high-throughput screening assay for agents that induce a copper hypersensitivity phenotype in M. tuberculosis and demonstrate that such copper-boosting compounds are effective against replicating and nonreplicating M. tuberculosis strains.

Evaluating Great Lakes bald eagle nesting habitat with Bayesian inference

Treesearch

Teryl G. Grubb; William W. Bowerman; Allen J. Bath; John P. Giesy; D. V. Chip Weseloh

2003-01-01

Bayesian inference facilitated structured interpretation of a nonreplicated, experience-based survey of potential nesting habitat for bald eagles (Haliaeetus leucocephalus) along the five Great Lakes shorelines. We developed a pattern recognition (PATREC) model of our aerial search image with six habitat attributes: (a) tree cover, (b) proximity and...

Dexamethasone treatment differentially alters viral shedding and the antibody and acute phase protein response after multivalent respiratory vaccination in beef steers

USDA-ARS?s Scientific Manuscript database

Our objective was to examine immunosuppression induced by dexamethasone (DEX) administration in cattle upon immunological responses to a multivalent respiratory vaccine containing replicating and non-replicating agents. Steers ( n = 32; 209 +/- 8 kg) seronegative to infectious bovine rhinotracheitis...

On the cross-cultural trail, searching for (non)-replication.

PubMed

Weiner, Bernard

2015-08-01

In this response to the manuscript by Pilati, attribution theory as applied to motivation is first reviewed. Then shortcomings of Pilati's method and analysis are pointed out. In addition, issues concerning the significance of lack of cross-cultural replication are discussed. © 2015 International Union of Psychological Science.

Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.

PubMed

Sanna, K; Rofstad, E K

1994-07-15

Rodent cell lines can develop resistance to doxorubicin and methotrexate during hypoxic stress. This has so far not been observed in human tumor cell lines. The purpose of our communication is to show that doxorubicin and methotrexate resistance can also develop in human melanoma cells during exposure to hypoxia. Four cell lines (BEX-c, COX-c, SAX-c, WIX-c) have been studied. Cells were exposed to hypoxia (O2 concentration < 10 ppm) for 24 hr prior to reoxygenation. Doxorubicin and methotrexate cell survival curves were determined immediately after as well as 18 and 42 hr after reoxygenation. The 4 cell lines were relatively sensitive to doxorubicin without hypoxia pre-treatment, and all developed resistance during exposure to hypoxia. Hypoxic stress also induced methotrexate resistance in BEX-c and SAX-c but not in COX-c and WIX-c. BEX-c and SAX-c were sensitive to methotrexate without hypoxia pre-treatment, whereas COX-c and WIX-c were resistant initially. Hypoxia-induced drug resistance was present immediately after reoxygenation and tended to decrease with time but remained statistically significant even 42 hr after reoxygenation.

2H,3H-decafluoropentane-based nanodroplets: new perspectives for oxygen delivery to hypoxic cutaneous tissues.

PubMed

Prato, Mauro; Magnetto, Chiara; Jose, Jithin; Khadjavi, Amina; Cavallo, Federica; Quaglino, Elena; Panariti, Alice; Rivolta, Ilaria; Benintende, Emilio; Varetto, Gianfranco; Argenziano, Monica; Troia, Adriano; Cavalli, Roberta; Guiot, Caterina

2015-01-01

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues.

2H,3H-Decafluoropentane-Based Nanodroplets: New Perspectives for Oxygen Delivery to Hypoxic Cutaneous Tissues

PubMed Central

Jose, Jithin; Khadjavi, Amina; Cavallo, Federica; Quaglino, Elena; Panariti, Alice; Rivolta, Ilaria; Benintende, Emilio; Varetto, Gianfranco; Argenziano, Monica; Troia, Adriano; Cavalli, Roberta; Guiot, Caterina

2015-01-01

Perfluoropentane (PFP)-based oxygen-loaded nanobubbles (OLNBs) were previously proposed as adjuvant therapeutic tools for pathologies of different etiology sharing hypoxia as a common feature, including cancer, infection, and autoimmunity. Here we introduce a new platform of oxygen nanocarriers, based on 2H,3H-decafluoropentane (DFP) as core fluorocarbon. These new nanocarriers have been named oxygen-loaded nanodroplets (OLNDs) since DFP is liquid at body temperature, unlike gaseous PFP. Dextran-shelled OLNDs, available either in liquid or gel formulations, display spherical morphology, ~600 nm diameters, anionic charge, good oxygen carrying capacity, and no toxic effects on human keratinocytes after cell internalization. In vitro OLNDs result more effective in releasing oxygen to hypoxic environments than former OLNBs, as demonstrated by analysis through oxymetry. In vivo, OLNDs effectively enhance oxy-hemoglobin levels, as emerged from investigation by photoacoustic imaging. Interestingly, ultrasound (US) treatment further improves transdermal oxygen release from OLNDs. Taken together, these data suggest that US-activated, DFP-based OLNDs might be innovative, suitable and cost-effective devices to topically treat hypoxia-associated pathologies of the cutaneous tissues. PMID:25781463

Effect of propofol on hypoxia re-oxygenation induced neuronal cell damage in vitro*.

PubMed

Huang, Y; Zitta, K; Bein, B; Scholz, J; Steinfath, M; Albrecht, M

2013-01-01

Propofol may protect neuronal cells from hypoxia re-oxygenation injury, possibly via an antioxidant actions under hypoxic conditions. This study investigated the molecular effects of propofol on hypoxia-induced cell damage using a neuronal cell line. Cultured human IMR-32 cells were exposed to propofol (30 μm) and biochemical and molecular approaches were used to assess cellular effects. Propofol significantly reduced hypoxia-mediated increases in lactate dehydrogenase, a marker of cell damage (mean (SD) for normoxia: 0.39 (0.07) a.u.; hypoxia: 0.78 (0.21) a.u.; hypoxia+propofol: 0.44 (0.17) a.u.; normoxia vs hypoxia, p<0.05; hypoxia vs hypoxia+propofol, p<0.05), reactive oxygen species and hydrogen peroxide. Propofol also diminished the morphological signs of cell damage. Increased amounts of catalase, which degrades hydrogen peroxide, were detected under hypoxic conditions. Propofol decreased the amount of catalase produced, but increased its enzymatic activity. Propofol protects neuronal cells from hypoxia re-oxygenation injury, possibly via a combined direct antioxidant effect along with induced cellular antioxidant mechanisms. Anaesthesia © 2012 The Association of Anaesthetists of Great Britain and Ireland.

Effects of human recombinant erythropoietin on differentiation and distribution of erythroid progenitor cells on murine medullary and splenic erythropoiesis during hypoxia and post-hypoxia.

PubMed

Mide, S M; Huygens, P; Bozzini, C E; Fernandez Pol, J A

2001-01-01

Hemopoietic cells, the extracellular matrix, growth factors and the microenvironment are involved in the regulation of hemopoiesis. Although the regulation of erythropoiesis is well understood at the cellular level in vivo and in vitro, the role of hemopoietic sites of erythroid progenitors production has not been well defined in both steady state conditions and in stress erythropoiesis. In this study we examined the qualitative erythroid differentiation and quantitative changes of the erythroid progenitors in different erythropoietic organs during erythropoiesis of stress in a hypoxia-induced polycythemia and post-hypoxic changes in a mice model. Chronic intermittent exposure to hypobaric hypoxia induced polycythemia in mice and the post-hypoxic period was characterized by total suppression of erythropoiesis. The number and distribution in hemopoietic sites of Immature Erythroid Burst (BFU-EI), Mature Erythroid Burst (BFU-EM) and Erythroid Colony Forming Units (CFU-E) was evaluated in bone marrow and spleen of hypoxic and post-hypoxic mice after removal from the chamber. The number of BFU-EI and CFU-E, was evaluated in both femoral bone marrow and spleen of ex-hypoxic polycythemic mice, at two times intervals after the end of hypoxia. We found that in both bone marrow and spleen, the kinetics of the CFU-E pool was characterized by a sharp fall from above normal to lower than normal levels. BFU-EM increased from normal to higher than normal levels. These results have been correlated with both erythropoietin (EPO) and the erythropoietic activity. The results show that EPO levels largely control both the differentiation and the amplification of the CFU-E pool and they suggest that EPO may acts as a "survival factor" at the CFU-E level and/or increase the flow of cells from BFU-E to CFU-E. After the termination of the period of hypoxia and during post-hypoxia there was a reduction in EPO production which subsequently caused a depletion of the CFU-E population, indicating that the size of the CFU-E pool is EPO-dependent. After the injection of 1U of recombinant human erythropoietin (rHuEPO) the size of that pool was increased and the pool of BFU-EI was decreased. It is noteworthy that our studies show that the spleen functions as a large reservoir of erythroid precursors for hypoxia-induced stress erythropoiesis.

[Effects of hypoxic acclimatization on myocardial sarcoplasmic reticulum ATPase and 45Ca2+ uptake in rats].

PubMed

Long, Chao-liang; Zhang, Yan-fang; Yin, Zhao-yun; Wang, Hai

2005-08-01

To study the effect of acute hypoxia and hypoxic acclimatization on myocardial function of rats. Eighteen male Wistar rats were randomly divided into three groups: normoxic control, acute hypoxia and intermittent hypoxic acclimatization group (n=6). After being exposed to hypoxia (8000 m) for 4 h before and after intermittent hypoxic acclimatization (3000 m and 5000 m, 14 d respectively, 4 h/d), the rats were decapitated and then myocardial sarcoplasmic reticulum (SR) were derived from cardiac muscles. Activities of Na+, K(+)-ATPase, Ca2+, Mg2(+)-ATPase in SR, phosphorylation of phospholamban (PLB) and the ability of 45Ca2+ uptake in SR were observed in all these three groups. 1) Hypoxia had no effects on the activity of Na+, K(+)-ATPase in rats myocardial SR of rats. 2) Compared with normoxic control rats, the activity of Ca2+, Mg2(+)-ATPase in myocardial SR of rats after acute hypoxia was reduced significantly (P<0.01). After intermittent hypoxic acclimatization, its activity increased significantly as compared with that of acute hypoxic rats (P<0.01). 3) The phosphorylation of PLB in acute hypoxic rats was reduced significantly compared with normoxic control rats. After intermittent hypoxic acclimatization, its phosphorylation was increased significantly compared with that of acute hypoxic rats. It suggests that hypoxic acclimatization could alleviate the inhibition of calcium pump. 4) The ability of 45Ca2+ uptake of SR in acute hypoxic rats was decreased significantly. After hypoxic acclimatization, its ability was strengthened significantly. These results suggest that the increased function of myocardial SR calcium pump, the strengthened phosphorylation of PLB to alleviate the inhibition of calcium pump and the increased function of Ca2+ transport in SR are the mechanisms of hypoxic acclimatization protecting cardiac functions from injury induced by severe hypoxia.

Persisting mild hypothermia suppresses hypoxia-inducible factor-1alpha protein synthesis and hypoxia-inducible factor-1-mediated gene expression.

PubMed

Tanaka, Tomoharu; Wakamatsu, Takuhiko; Daijo, Hiroki; Oda, Seiko; Kai, Shinichi; Adachi, Takehiko; Kizaka-Kondoh, Shinae; Fukuda, Kazuhiko; Hirota, Kiichi

2010-03-01

The transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in regulating gene expression in response to hypoxia-ischemia. Ischemia causes the tissue not only to be hypoxic but also to be hypothermic because of the hypoperfusion under certain circumstances. On the other hand, the induced hypothermia is one of the most common therapeutic modalities to extend tolerance to hypoxia. Although hypoxia elicits a variety of cellular and systemic responses at different organizational levels in the body, little is known about how hypoxia-induced responses are affected by low temperature. We examined the influence of mild hypothermic conditions (28-32 degrees C) on HIF-1 in both in vitro and in vivo settings. In vitro experiments adopting cultured cells elucidated that hypoxia-induced HIF-1 activation was resistant to 4-h exposure to the low temperature. In contrast, exposure to the low temperature as long as 24 h suppressed HIF-1 activation and the subsequent upregulation of HIF-1 target genes such as VEGF or GLUT-1. HIF-1alpha protein stability in the cell was not affected by hypothermic treatment. Furthermore, intracellular ATP content was reduced under 1% O(2) conditions but was not largely affected by hypothermic treatment. The evidence indicates that reduction of oxygen consumption is not largely involved in suppression of HIF-1. In addition, we demonstrated that HIF-1 DNA-binding activity and HIF-1-dependent gene expressions induced under 10% O(2) atmosphere in mouse brain were not influenced by treatment under 3-h hypothermic temperature but were inhibited under 5-h treatment. On the other hand, we indicated that warming ischemic legs of mice for 24 h preserved HIF-1 activity. In this report we describe for the first time that persisting low temperature significantly reduced HIF-1alpha neosynthesis under hypoxic conditions, leading to a decrease in gene expression for adaptation to hypoxia in both in vitro and in vivo settings.

Human Adipose-Derived Mesenchymal Stem Cells Respond to Short-Term Hypoxia by Secreting Factors Beneficial for Human Islets In Vitro and Potentiate Antidiabetic Effect In Vivo

PubMed Central

Schive, Simen W.; Mirlashari, Mohammad Reza; Hasvold, Grete; Wang, Mengyu; Josefsen, Dag; Gullestad, Hans Petter; Korsgren, Olle; Foss, Aksel; Kvalheim, Gunnar; Scholz, Hanne

2017-01-01

Adipose-derived mesenchymal stem cells (ASCs) release factors beneficial for islets in vitro and protect against hyperglycemia in rodent models of diabetes. Oxygen tension has been shown to induce metabolic changes and alter ASCs’ release of soluble factors. The effects of hypoxia on the antidiabetic properties of ASCs have not been explored. To investigate this, we incubated human ASCs for 48 h in 21% (normoxia) or 1% O2 (hypoxia) and compared viability, cell growth, surface markers, differentiation capability, and soluble factors in the conditioned media (CM). Human islets were exposed to CM from ASCs incubated in either normoxia or hypoxia, and islet function and apoptosis after culture with or without proinflammatory cytokines were measured. To test hypoxic preconditioned ASCs’ islet protective effects in vivo, ASCs were incubated for 48 h in normoxia or hypoxia before being injected into Balb/c Rag 1–/– immunodeficient mice with streptozotocin-induced insulitis. Progression of diabetes and insulin content of pancreas were measured. We found that incubation in hypoxia was well tolerated by ASCs and that levels of VEGF-A, FGF-2, and bNGF were elevated in CM from ASCs incubated in hypoxia compared to normoxia, while levels of HGF, IL-8, and CXCL1 were reduced. CM from ASCs incubated in hypoxia significantly improved human islet function and reduced apoptosis after culture, and reduced cytokine-induced apoptosis. In our mouse model, pancreas insulin content was higher in both groups receiving ASCs compared to control, but the mice receiving preconditioned ASCs had lower random and fasting blood glucose, as well as improved oral glucose tolerance compared to untreated mice. In conclusion, our in vitro results indicate that the islet protective potential of ASCs improves in hypoxia, and we give insight into factors involved in this. Finally we show that hypoxic preconditioning potentiates ASCs’ antidiabetic effect in vivo. PMID:28713640

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage.

PubMed

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J; Francis, Richard O; Roach, Robert C; Dzieciatkowska, Monika; Rogers, Stephen C; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T; Thomas, Tiffany A; Hansen, Kirk C; Spitalnik, Steven L; Xia, Yang; Zimring, James C; Hod, Eldad A; D'Alessandro, Angelo

2018-02-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1-7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13 C 1 -aspartate or 13 C 5 -adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and - preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. Copyright© 2018 Ferrata Storti Foundation.

Enhancement of hypoxia-activated prodrug TH-302 anti-tumor activity by Chk1 inhibition.

PubMed

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

2015-05-21

The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and selectively under hypoxic conditions releases the DNA cross-linker bromo-isophosphoramide mustard (Br-IPM). Here, we have explored the effect of Chk1 inhibition on TH-302-mediated pharmacological activities. We employed in vitro cell viability, DNA damage, cellular signaling assays and the in vivo HT29 human tumor xenograft model to study the effect of Chk1inhibition on TH-302 antitumor activities. TH-302 cytotoxicity is greatly enhanced by Chk1 inhibition in p53-deficient but not in p53-proficient human cancer cell lines. Chk1 inhibitors reduced TH-302-induced cell cycle arrest via blocking TH-302-induced decrease of phosphorylation of histone H3 and increasing Cdc2-Y15 phosphorylation. Employing the single-cell gel electrophoresis (comet) assay, we observed a potentiation of the TH-302 dependent tail moment. TH-302 induced γH2AX and apoptosis were also increased upon the addition of Chk1 inhibitor. Potentiation of TH-302 cytotoxicity by Chk1 inhibitor was only observed in cell lines proficient in, but not deficient in homology-directed DNA repair. We also show that combination treatment led to lowering of Rad51 expression levels as compared to either agent alone. In vivo data demonstrate that Chk1 inhibitor enhances TH-302 anti-tumor activity in p53 mutant HT-29 human tumor xenografts, supporting the hypothesis that these in vitro results can translate to enhanced in vivo efficacy of the combination. TH-302-mediated in vitro and in vivo anti-tumor activities were greatly enhanced by the addition of Chk1 inhibitors. The preclinical data presented in this study support a new approach for the treatment of p53-deficient hypoxic cancers by combining Chk1 inhibitors with the hypoxia-activated prodrug TH-302.

Hypoxia modulates the purine salvage pathway and decreases red blood cell and supernatant levels of hypoxanthine during refrigerated storage

PubMed Central

Nemkov, Travis; Sun, Kaiqi; Reisz, Julie A.; Song, Anren; Yoshida, Tatsuro; Dunham, Andrew; Wither, Matthew J.; Francis, Richard O.; Roach, Robert C.; Dzieciatkowska, Monika; Rogers, Stephen C.; Doctor, Allan; Kriebardis, Anastasios; Antonelou, Marianna; Papassideri, Issidora; Young, Carolyn T.; Thomas, Tiffany A.; Hansen, Kirk C.; Spitalnik, Steven L.; Xia, Yang; Zimring, James C.; Hod, Eldad A.; D’Alessandro, Angelo

2018-01-01

Hypoxanthine catabolism in vivo is potentially dangerous as it fuels production of urate and, most importantly, hydrogen peroxide. However, it is unclear whether accumulation of intracellular and supernatant hypoxanthine in stored red blood cell units is clinically relevant for transfused recipients. Leukoreduced red blood cells from glucose-6-phosphate dehydrogenase-normal or -deficient human volunteers were stored in AS-3 under normoxic, hyperoxic, or hypoxic conditions (with oxygen saturation ranging from <3% to >95%). Red blood cells from healthy human volunteers were also collected at sea level or after 1–7 days at high altitude (>5000 m). Finally, C57BL/6J mouse red blood cells were incubated in vitro with 13C1-aspartate or 13C5-adenosine under normoxic or hypoxic conditions, with or without deoxycoformycin, a purine deaminase inhibitor. Metabolomics analyses were performed on human and mouse red blood cells stored for up to 42 or 14 days, respectively, and correlated with 24 h post-transfusion red blood cell recovery. Hypoxanthine increased in stored red blood cell units as a function of oxygen levels. Stored red blood cells from human glucose-6-phosphate dehydrogenase-deficient donors had higher levels of deaminated purines. Hypoxia in vitro and in vivo decreased purine oxidation and enhanced purine salvage reactions in human and mouse red blood cells, which was partly explained by decreased adenosine monophosphate deaminase activity. In addition, hypoxanthine levels negatively correlated with post-transfusion red blood cell recovery in mice and – preliminarily albeit significantly - in humans. In conclusion, hypoxanthine is an in vitro metabolic marker of the red blood cell storage lesion that negatively correlates with post-transfusion recovery in vivo. Storage-dependent hypoxanthine accumulation is ameliorated by hypoxia-induced decreases in purine deamination reaction rates. PMID:29079593

HIF-2α dictates the susceptibility of pancreatic cancer cells to TRAIL by regulating survivin expression

PubMed Central

Harashima, Nanae; Takenaga, Keizo; Akimoto, Miho; Harada, Mamoru

2017-01-01

Cancer cells develop resistance to therapy by adapting to hypoxic microenvironments, and hypoxia-inducible factors (HIFs) play crucial roles in this process. We investigated the roles of HIF-1α and HIF-2α in cancer cell death induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) using human pancreatic cancer cell lines. siRNA-mediated knockdown of HIF-2α, but not HIF-1α, increased susceptibility of two pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL in vitro under normoxic and hypoxic conditions. The enhanced sensitivity to TRAIL was also observed in vivo. This in vitro increased TRAIL sensitivity was observed in other three pancreatic cancer cell lines. An array assay of apoptosis-related proteins showed that knockdown of HIF-2α decreased survivin expression. Additionally, survivin promoter activity was decreased in HIF-2α knockdown Panc-1 cells and HIF-2α bound to the hypoxia-responsive element in the survivin promoter region. Conversely, forced expression of the survivin gene in HIF-2α shRNA-expressing Panc-1 cells increased resistance to TRAIL. In a xenograft mouse model, the survivin suppressant YM155 sensitized Panc-1 cells to TRAIL. Collectively, our results indicate that HIF-2α dictates the susceptibility of human pancreatic cancer cell lines, Panc-1 and AsPC-1, to TRAIL by regulating survivin expression transcriptionally, and that survivin could be a promising target to augment the therapeutic efficacy of death receptor-targeting anti-cancer therapy. PMID:28476028

Protective Effects of Myricetin on Acute Hypoxia-Induced Exercise Intolerance and Mitochondrial Impairments in Rats

PubMed Central

Zou, Dan; Liu, Peng; Chen, Ka; Xie, Qi; Liang, Xinyu; Bai, Qian; Zhou, Qicheng; Liu, Kai; Zhang, Ting; Zhu, Jundong; Mi, Mantian

2015-01-01

Purpose Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro. Methods Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM). The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA). mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence. Results Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regluators, in addition, AMP-activated protein kinase(AMPK) plays a crucial role in this process. Conclusions Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis. PMID:25919288

HIF-2α-ILK Is Involved in Mesenchymal Stromal Cell Angiogenesis in Multiple Myeloma Under Hypoxic Conditions

PubMed Central

Zhang, Xiaoying; Xu, Yinhui; Liu, Hongbo; Zhao, Pan; Chen, Yafang; Yue, Zhijie; Zhang, Zhiqing; Wang, Xiaofang

2018-01-01

Mesenchymal stromal cells are proven to be likely induce the angiogenic response in multiple myeloma and thus represent an enticing target for antiangiogenesis therapies for multiple myeloma. Substantial evidence indicates that angiogenesis in multiple myeloma is complex and involves direct production of angiogenic cytokines by abnormal plasma cells and these B-cell neoplasia generated pathophysiology change within the microenvironment. In this study, we demonstrated that mesenchymal stromal cells cultured with U266/Lp-1 under hypoxic conditions resulted in an increased α-smooth muscle actin expression and high productive levels of both hypoxia-inducible factor-2α and integrin-linked kinase proteins. Moreover, inhibition of hypoxia-inducible factor-2α by Small interfering RNA (siRNA) in mesenchymal stromal cells decreased the protein levels of both α-smooth muscle actin and integrin-linked kinase after mesenchymal stromal cells cultured with U266 under hypoxic conditions. We further demonstrated that transfection of integrin-linked kinase-siRNA reduced the protein level of α-smooth muscle actin and attenuated angiogenesis in vitro by decreasing the attachment of Q-dot labeled cells and secretion of angiogenic factors. In conclusion, our research showed that mesenchymal stromal cells cultured with myeloma cells under hypoxia participated in the angiogenesis of multiple myeloma, which is regulated by the hypoxia-inducible factor-2α-integrin-linked kinase pathway. Thus, targeting integrin-linked kinase may represent an effective strategy to block hypoxia-inducible factor-2α-induced angiogenesis in the treatment of multiple myeloma. PMID:29656700

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

PubMed Central

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

2015-01-01

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

Effects of hypoxia on osteogenic differentiation of mesenchymal stromal cells used as a cell therapy for avascular necrosis of the femoral head.

PubMed

Ciapetti, Gabriela; Granchi, Donatella; Fotia, Caterina; Savarino, Lucia; Dallari, Dante; Del Piccolo, Nicola; Donati, Davide Maria; Baldini, Nicola

2016-09-01

Avascular necrosis of the femoral head (AVN) occurs as common result of various conditions or develops as a primary entity, with a high freqency in young adults. Because of its tendency toward osteoarthritis requiring total hip arthroplasty, alternative treatments are being advocated, including cell therapy with mesenchymal stromal cells (MSCs). Because osteonecrotic bone is a severely hypoxic tissue, with a 1-3% oxygen tension, the survival and function of multipotent cells is questionable. In this study, the proliferative, immunophenotypic and osteogenic properties of bone marrow (BM)-derived MSCs from a clinical series of patients with AVN were evaluated under in vitro conditions mimicking the hypoxic milieu of AVN to verify the rationale for cell therapy. MSCs retrieved from the iliac crest (BM-MSC) were isolated, expanded and induced to osteogenic differentiation under a 2% pO2 atmosphere (hypoxia) in comparison with the standard 21% pO2 (normoxia) that is routinely used in cell culture assays. Both proliferation and colony-forming ability were significantly enhanced in hypoxia-exposed BM-MSCs compared with BM-MSCs under normoxia. The expression of bone-related genes, including alkaline phosphatase, Type I collagen, and osteocalcin was significantly increased under hypoxia. Moreover, mineral deposition after osteogenic induction was not hampered, but in some cases even enhanced under low oxygen tension. These findings support autologous cell therapy as an effective treatment to stimulate bone healing in the hypoxic microenvironment of AVN. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Astrocyte - neuron lactate shuttle may boost more ATP supply to the neuron under hypoxic conditions - in silico study supported by in vitro expression data

PubMed Central

2011-01-01

Background Neuro-glial interactions are important for normal functioning of the brain as well as brain energy metabolism. There are two major working models - in the classical view, both neurons and astrocytes can utilize glucose as the energy source through oxidative metabolism, whereas in the astrocyte-neuron lactate shuttle hypothesis (ANLSH) it is the astrocyte which can consume glucose through anaerobic glycolysis to pyruvate and then to lactate, and this lactate is secreted to the extracellular space to be taken up by the neuron for further oxidative degradation. Results In this computational study, we have included hypoxia-induced genetic regulation of these enzymes and transporters, and analyzed whether the ANLSH model can provide an advantage to either cell type in terms of supplying the energy demand. We have based this module on our own experimental analysis of hypoxia-dependent regulation of transcription of key metabolic enzymes. Using this experimentation-supported in silico modeling, we show that under both normoxic and hypoxic conditions in a given time period ANLSH model does indeed provide the neuron with more ATP than in the classical view. Conclusions Although the ANLSH is energetically more favorable for the neuron, it is not the case for the astrocyte in the long term. Considering the fact that astrocytes are more resilient to hypoxia, we would propose that there is likely a switch between the two models, based on the energy demand of the neuron, so as to maintain the survival of the neuron under hypoxic or glucose-and-oxygen-deprived conditions. PMID:21995951

Antihelminthic Benzimidazoles Are Novel HIF Activators That Prevent Oxidative Neuronal Death via Binding to Tubulin

PubMed Central

Aleyasin, Hossein; Karuppagounder, Saravanan S.; Kumar, Amit; Sleiman, Sama; Basso, Manuela; Ma, Thong; Siddiq, Ambreena; Chinta, Shankar J.; Brochier, Camille; Langley, Brett; Haskew-Layton, Renee; Bane, Susan L.; Riggins, Gregory J.; Gazaryan, Irina; Starkov, Anatoly A.; Andersen, Julie K.

2015-01-01

Abstract Aims: Pharmacological activation of the adaptive response to hypoxia is a therapeutic strategy of growing interest for neurological conditions, including stroke, Huntington's disease, and Parkinson's disease. We screened a drug library with known safety in humans using a hippocampal neuroblast line expressing a reporter of hypoxia-inducible factor (HIF)-dependent transcription. Results: Our screen identified more than 40 compounds with the ability to induce hypoxia response element-driven luciferase activity as well or better than deferoxamine, a canonical activator of hypoxic adaptation. Among the chemical entities identified, the antihelminthic benzimidazoles represented one pharmacophore that appeared multiple times in our screen. Secondary assays confirmed that antihelminthics stabilized the transcriptional activator HIF-1α and induced expression of a known HIF target gene, p21cip1/waf1, in post-mitotic cortical neurons. The on-target effect of these agents in stimulating hypoxic signaling was binding to free tubulin. Moreover, antihelminthic benzimidazoles also abrogated oxidative stress-induced death in vitro, and this on-target effect also involves binding to free tubulin. Innovation and Conclusions: These studies demonstrate that tubulin-binding drugs can activate a component of the hypoxic adaptive response, specifically the stabilization of HIF-1α and its downstream targets. Tubulin-binding drugs, including antihelminthic benzimidazoles, also abrogate oxidative neuronal death in primary neurons. Given their safety in humans and known ability to penetrate into the central nervous system, antihelminthic benzimidazoles may be considered viable candidates for treating diseases associated with oxidative neuronal death, including stroke. Antioxid. Redox Signal. 22, 121–134. PMID:24766300

Placental growth factor expression is reversed by antivascular endothelial growth factor therapy under hypoxic conditions.

PubMed

Zhou, Ai-Yi; Bai, Yu-Jing; Zhao, Min; Yu, Wen-Zhen; Huang, Lv-Zhen; Li, Xiao-Xin

2014-08-01

Clinical trials have revealed that the antivascular endothelial growth factor (VEGF) therapies are effective in retinopathy of prematurity (ROP). But the low level of VEGF was necessary as a survival signal in healthy conditions, and endogenous placental growth factor (PIGF) is redundant for development. The purpose of this study was to elucidate the PIGF expression under hypoxia as well as the influence of anti-VEGF therapy on PIGF. CoCl2-induced hypoxic human umbilical vein endothelial cells (HUVECs) were used for an in vitro study, and oxygen-induced retinopathy (OIR) mice models were used for an in vivo study. The expression patterns of PIGF under hypoxic conditions and the influence of anti-VEGF therapy on PIGF were evaluated by quantitative reverse transcription-polymerase chain reaction (RTPCR). The retinal avascular areas and neovascularization (NV) areas of anti-VEGF, anti-PIGF and combination treatments were calculated. Retina PIGF concentration was evaluated by ELISA after treatment. The vasoactive effects of exogenous PIGF on HUVECs were investigated by proliferation and migration studies. PIGF mRNA expression was reduced by hypoxia in OIR mice, in HUVECs under hypoxia and anti-VEGF treatment. However, PIGF expression was reversed by anti-VEGF therapy in the OIR model and in HUVECs under hypoxia. Exogenous PIGF significantly inhibited HUVECs proliferation and migration under normal conditions, but it stimulated cell proliferation and migration under hypoxia. Anti-PIGF treatment was effective for neovascular tufts in OIR mice (P<0.05). The finding that PIGF expression is iatrogenically up-regulated by anti-VEGF therapy provides a consideration to combine it with anti-PIGF therapy.

Astrocyte-neuron lactate shuttle may boost more ATP supply to the neuron under hypoxic conditions--in silico study supported by in vitro expression data.

PubMed

Genc, Seda; Kurnaz, Isil A; Ozilgen, Mustafa

2011-10-13

Neuro-glial interactions are important for normal functioning of the brain as well as brain energy metabolism. There are two major working models--in the classical view, both neurons and astrocytes can utilize glucose as the energy source through oxidative metabolism, whereas in the astrocyte-neuron lactate shuttle hypothesis (ANLSH) it is the astrocyte which can consume glucose through anaerobic glycolysis to pyruvate and then to lactate, and this lactate is secreted to the extracellular space to be taken up by the neuron for further oxidative degradation. In this computational study, we have included hypoxia-induced genetic regulation of these enzymes and transporters, and analyzed whether the ANLSH model can provide an advantage to either cell type in terms of supplying the energy demand. We have based this module on our own experimental analysis of hypoxia-dependent regulation of transcription of key metabolic enzymes. Using this experimentation-supported in silico modeling, we show that under both normoxic and hypoxic conditions in a given time period ANLSH model does indeed provide the neuron with more ATP than in the classical view. Although the ANLSH is energetically more favorable for the neuron, it is not the case for the astrocyte in the long term. Considering the fact that astrocytes are more resilient to hypoxia, we would propose that there is likely a switch between the two models, based on the energy demand of the neuron, so as to maintain the survival of the neuron under hypoxic or glucose-and-oxygen-deprived conditions.

Fabrication and characterization of UV-emitting nanoparticles as novel radiation sensitizers targeting hypoxic tumor cells

NASA Astrophysics Data System (ADS)

Squillante, Michael R.; Jüstel, Thomas; Anderson, R. Rox; Brecher, Charles; Chartier, Daniel; Christian, James F.; Cicchetti, Nicholas; Espinoza, Sara; McAdams, Daniel R.; Müller, Matthias; Tornifoglio, Brooke; Wang, Yimin; Purschke, Martin

2018-06-01

Radiation therapy is one of the primary therapeutic techniques for treating cancer, administered to nearly two-thirds of all cancer patients. Although largely effective in killing cancer cells, radiation therapy, like other forms of cancer treatment, has difficulty dealing with hypoxic regions within solid tumors. The incomplete killing of cancer cells can lead to recurrence and relapse. The research presented here is investigating the enhancement of the efficacy of radiation therapy by using scintillating nanoparticles that emit UV photons. UV photons, with wavelengths between 230 nm and 280 nm, are able to inactivate cells due to their direct interaction with DNA, causing a variety of forms of damage. UV-emitting nanoparticles will enhance the treatment in two ways: first by generating UV photons in the immediate vicinity of cancer cells, leading to direct and oxygen-independent DNA damage, and second by down-converting the applied higher energy X-rays into softer X-rays and particles that are more efficiently absorbed in the targeted tumor region. The end result will be nanoparticles with a higher efficacy in the treatment of hypoxic cells in the tumor, filling an important, unmet clinical need. Our preliminary experiments show an increase in cell death using scintillating LuPO4:Pr nanoparticles over that achieved by the primary radiation alone. This work describes the fabrication of the nanoparticles, their physical characterization, and the spectroscopic characterization of the UV emission. The work also presents in vitro results that demonstrate an enhanced efficacy of cell killing with x-rays and a low unspecific toxicity of the nanoparticles.

Improvement of mesh recolonization in abdominal wall reconstruction with adipose vs. bone marrow mesenchymal stem cells in a rodent model.

PubMed

van Steenberghe, M; Schubert, T; Guiot, Y; Goebbels, R M; Gianello, P

2017-08-01

Reconstruction of muscle defects remains a challenge. Our work assessed the potential of an engineered construct made of a human acellular collagen matrix (HACM) seeded with porcine mesenchymal stem cells (MSCs) to reconstruct abdominal wall muscle defects in a rodent model. This study compared 2 sources of MSCs (bone-marrow, BMSCs, and adipose, ASCs) in vitro and in vivo for parietal defect reconstruction. Cellular viability and growth factor release (VEGF, FGF-Beta, HGF, IGF-1, TGF-Beta) were investigated under normoxic/hypoxic culture conditions. Processed and recellularized HACMs were mechanically assessed. The construct was tested in vivo in full thickness abdominal wall defect treated with HACM alone vs. HACM+ASCs or BMSCs (n=14). Tissue remodeling was studied at day 30 for neo-angiogenesis and muscular reconstruction. A significantly lower secretion of IGF was observed with ASCs vs. BMSCs under hypoxic conditions (-97.6%, p<0.005) whereas significantly higher VEGF/FGF secretions were found with ASCs (+92%, p<0.001 and +72%, p<0.05, respectively). Processing and recellularization did not impair the mechanical properties of the HACM. In vivo, angiogenesis and muscle healing were significantly improved by the HACM+ASCs in comparison to BMSCs (p<0.05) at day 30. A composite graft made of an HACM seeded with ASCs can improve muscle repair by specific growth factor release in hypoxic conditions and by in vivo remodeling (neo-angiogenesis/graft integration) while maintaining mechanical properties. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of hypoxia-ischemia and MK-801 treatment on the binding of a phencyclidine analogue in the developing rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Silverstein, F.S.; McDonald, J.W. III; Bommarito, M.

1990-02-01

The phencyclidine analogue ({sup 3}H)(1-(2-thienyl)cyclohexyl)piperidine ({sup 3}H-TCP) binds to the ion channel associated with the N-methyl-D-aspartate receptor channel complex. In vitro autoradiography indicates that the distribution of {sup 3}H-TCP binding in brain closely parallels that of ({sup 3}H)glutamate binding to the N-methyl-D-aspartate receptor. In nine 7-day-old rats, an acute focal hypoxic-ischemic insult produced by unilateral carotid artery ligation and subsequent exposure to 8% oxygen acutely reduced {sup 3}H-TCP binding ipsilateral to the ligation by 30% in the CA1, by 27% in the CA3, by 26% in the dentate gyrus, and by 17% in the striatum compared with values from themore » contralateral hemisphere. In 10 littermates that received 1 mg/kg of the neuroprotective noncompetitive N-methyl-D-aspartate antagonist MK-801 immediately before hypoxic exposure, the regional distribution of {sup 3}H-TCP binding in hypoxic-ischemic brain was relatively preserved and there were no interhemispheric asymmetries in {sup 3}H-TCP binding densities. In addition, in three unoperated rats decapitated 24 hours after MK-801 treatment, {sup 3}H-TCP binding was reduced by 15-35%; similar bilateral suppression of {sup 3}H-TCP binding was detected in MK-801-treated ligates. Our data indicate that {sup 3}H-TCP autoradiography can be used to assay the efficacy of neuroprotective agents in this experimental model of perinatal stroke.« less

Guinea Pig Oxygen-Sensing and Carotid Body Functional Properties

PubMed Central

Gonzalez-Obeso, Elvira; Docio, Inmaculada; Olea, Elena; Cogolludo, Angel; Obeso, Ana; Rocher, Asuncion; Gomez-Niño, Angela

2017-01-01

Mammals have developed different mechanisms to maintain oxygen supply to cells in response to hypoxia. One of those mechanisms, the carotid body (CB) chemoreceptors, is able to detect physiological hypoxia and generate homeostatic reflex responses, mainly ventilatory and cardiovascular. It has been reported that guinea pigs, originally from the Andes, have a reduced ventilatory response to hypoxia compared to other mammals, implying that CB are not completely functional, which has been related to genetically/epigenetically determined poor hypoxia-driven CB reflex. This study was performed to check the guinea pig CB response to hypoxia compared to the well-known rat hypoxic response. These experiments have explored ventilatory parameters breathing different gases mixtures, cardiovascular responses to acute hypoxia, in vitro CB response to hypoxia and other stimuli and isolated guinea pig chemoreceptor cells properties. Our findings show that guinea pigs are hypotensive and have lower arterial pO2 than rats, probably related to a low sympathetic tone and high hemoglobin affinity. Those characteristics could represent a higher tolerance to hypoxic environment than other rodents. We also find that although CB are hypo-functional not showing chronic hypoxia sensitization, a small percentage of isolated carotid body chemoreceptor cells contain tyrosine hydroxylase enzyme and voltage-dependent K+ currents and therefore can be depolarized. However hypoxia does not modify intracellular Ca2+ levels or catecholamine secretion. Guinea pigs are able to hyperventilate only in response to intense acute hypoxic stimulus, but hypercapnic response is similar to rats. Whether other brain areas are also activated by hypoxia in guinea pigs remains to be studied. PMID:28533756

Guinea Pig Oxygen-Sensing and Carotid Body Functional Properties.

PubMed

Gonzalez-Obeso, Elvira; Docio, Inmaculada; Olea, Elena; Cogolludo, Angel; Obeso, Ana; Rocher, Asuncion; Gomez-Niño, Angela

2017-01-01

Mammals have developed different mechanisms to maintain oxygen supply to cells in response to hypoxia. One of those mechanisms, the carotid body (CB) chemoreceptors, is able to detect physiological hypoxia and generate homeostatic reflex responses, mainly ventilatory and cardiovascular. It has been reported that guinea pigs, originally from the Andes, have a reduced ventilatory response to hypoxia compared to other mammals, implying that CB are not completely functional, which has been related to genetically/epigenetically determined poor hypoxia-driven CB reflex. This study was performed to check the guinea pig CB response to hypoxia compared to the well-known rat hypoxic response. These experiments have explored ventilatory parameters breathing different gases mixtures, cardiovascular responses to acute hypoxia, in vitro CB response to hypoxia and other stimuli and isolated guinea pig chemoreceptor cells properties. Our findings show that guinea pigs are hypotensive and have lower arterial pO 2 than rats, probably related to a low sympathetic tone and high hemoglobin affinity. Those characteristics could represent a higher tolerance to hypoxic environment than other rodents. We also find that although CB are hypo-functional not showing chronic hypoxia sensitization, a small percentage of isolated carotid body chemoreceptor cells contain tyrosine hydroxylase enzyme and voltage-dependent K + currents and therefore can be depolarized. However hypoxia does not modify intracellular Ca 2+ levels or catecholamine secretion. Guinea pigs are able to hyperventilate only in response to intense acute hypoxic stimulus, but hypercapnic response is similar to rats. Whether other brain areas are also activated by hypoxia in guinea pigs remains to be studied.

Sulfonamide derivative targeting carbonic anhydrase IX as a nuclear imaging probe for colorectal cancer detection in vivo

PubMed Central

Guan, Siao-Syun; Cheng, Chun-Chia; Ho, Ai-Sheng; Wang, Chia-Chi; Luo, Tsai-Yueh; Liao, Tse-Zung; Chang, Jungshan; Wu, Cheng-Tien; Liu, Shing-Hwa

2015-01-01

Hypoxic microenvironment is a common situation in solid tumors. Carbonic anhydrase IX (CA9) is one of the reliable cellular biomarkers of hypoxia. The role of CA9 in colorectal cancer (CRC) remains to be clarified. CA9 inhibitor such as sulfonamides is known to block CA9 activation and reduce tumor growth consequently. Here, we aimed to investigate the CA9 expression in serum and tumor from different stages of CRC patients and utilize sulfonamide derivative with indium-111 labeling as a probe for CRC nuclear imaging detection in vivo. The serum CA9 was correlated with the tumor CA9 levels in different stages of CRC patients. Hypoxia increased cell viability and CA9 expression in colorectal cancer HCT-15 cells. Sulfonamide derivative 5-(2-aminoethyl)thiophene-2-sulfonamide (ATS) could bind with CA9 in vitro under hypoxia. Moreover, tumor tissues in HCT-15-induced xenograft mice possessed higher hypoxic fluorescence signal as compared with other organs. We also found that the radioisotope signal of indium-111 labeled ATS, which was utilized for CRC detection in HCT-15-induced xenograft mice, was markedly enhanced in tumors as compared with non-ATS control. Taken together, these findings suggest that CA9 is a potential hypoxic CRC biomarker and measurement of serum CA9 can be as a potential tool for diagnosing CA9 expressions in CRC clinical practice. The radioisotope-labeled sulfonamide derivative (ATS) may be useful to apply in CRC patients for nuclear medicine imaging. PMID:26447758

Hypoxia-Targeting Drug Evofosfamide (TH-302) Enhances Sunitinib Activity in Neuroblastoma Xenograft Models.

PubMed

Kumar, Sushil; Sun, Jessica D; Zhang, Libo; Mokhtari, Reza Bayat; Wu, Bing; Meng, Fanying; Liu, Qian; Bhupathi, Deepthi; Wang, Yan; Yeger, Herman; Hart, Charles; Baruchel, Sylvain

2018-05-23

Antiangiogenic therapy has shown promising results in preclinical and clinical trials. However, tumor cells acquire resistance to this therapy by gaining ability to survive and proliferate under hypoxia induced by antiangiogenic therapy. Combining antiangiogenic therapy with hypoxia-activated prodrugs can overcome this limitation. Here, we have tested the combination of antiangiogenic drug sunitinib in combination with hypoxia-activated prodrug evofosfamide in neuroblastoma. In vitro, neuroblastoma cell line SK-N-BE(2) was 40-folds sensitive to evofosfamide under hypoxia compared to normoxia. In IV metastatic model, evofosfamide significantly increased mice survival compared to the vehicle (P=.02). In SK-N-BE(2) subcutaneous xenograft model, we tested two different treatment regimens using 30 mg/kg sunitinib and 50 mg/kg evofosfamide. Here, sunitinib therapy when started along with evofosfamide treatment showed higher efficacy compared to single agents in subcutaneous SK-N-BE(2) xenograft model, whereas sunitinib when started 7 days after evofosfamide treatment did not have any advantage compared to treatment with either single agent. Immunofluorescence of tumor sections revealed higher number of apoptotic cells and hypoxic areas compared to either single agent when both treatments were started together. Treatment with 80 mg/kg sunitinib with 50 mg/kg evofosfamide was significantly superior to single agents in both xenograft and metastatic models. This study confirms the preclinical efficacy of sunitinib and evofosfamide in murine models of aggressive neuroblastoma. Sunitinib enhances the efficacy of evofosfamide by increasing hypoxic areas, and evofosfamide targets hypoxic tumor cells. Consequently, each drug enhances the activity of the other. Copyright © 2018. Published by Elsevier Inc.

Estimation of genetic parameters and their sampling variances of quantitative traits in the type 2 modified augmented design

USDA-ARS?s Scientific Manuscript database

We proposed a method to estimate the error variance among non-replicated genotypes, thus to estimate the genetic parameters by using replicated controls. We derived formulas to estimate sampling variances of the genetic parameters. Computer simulation indicated that the proposed methods of estimatin...

Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells

PubMed Central

Sedlik, C.; Saron, M.-F.; Sarraseca, J.; Casal, I.; Leclerc, C.

1997-01-01

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+ T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+ class I-restricted cytotoxic activity persisted in vivo for at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells. PMID:9207121

Nonreplicating Influenza A Virus Vaccines Confer Broad Protection against Lethal Challenge

PubMed Central

Baz, Mariana; Boonnak, Kobporn; Paskel, Myeisha; Santos, Celia; Powell, Timothy; Townsend, Alain

2015-01-01

ABSTRACT New vaccine technologies are being investigated for their ability to elicit broadly cross-protective immunity against a range of influenza viruses. We compared the efficacies of two intranasally delivered nonreplicating influenza virus vaccines (H1 and H5 S-FLU) that are based on the suppression of the hemagglutinin signal sequence, with the corresponding H1N1 and H5N1 cold-adapted (ca) live attenuated influenza virus vaccines in mice and ferrets. Administration of two doses of H1 or H5 S-FLU vaccines protected mice and ferrets from lethal challenge with homologous, heterologous, and heterosubtypic influenza viruses, and two doses of S-FLU and ca vaccines yielded comparable effects. Importantly, when ferrets immunized with one dose of H1 S-FLU or ca vaccine were challenged with the homologous H1N1 virus, the challenge virus failed to transmit to naive ferrets by the airborne route. S-FLU technology can be rapidly applied to any emerging influenza virus, and the promising preclinical data support further evaluation in humans. PMID:26489862

Exosomes Secreted under Hypoxia Enhance Invasiveness and Stemness of Prostate Cancer Cells by Targeting Adherens Junction Molecules

PubMed Central

Ramteke, Anand; Ting, Harold; Agarwal, Chapla; Mateen, Samiha; Somasagara, Ranganathan; Hussain, Anowar; Graner, Michael; Frederick, Barbara; Agarwal, Rajesh; Deep, Gagan

2015-01-01

Hypoxic conditions in prostate cancer (PCA) are associated with poor prognosis; however, precise mechanism/s through which hypoxia promotes malignant phenotype remains unclear. Here, we analyzed the role of exosomes from hypoxic PCA cells in enhancing the invasiveness and stemness of naïve PCA cells, as well as in promoting cancer-associated fibroblast (CAF) phenotype in prostate stromal cells (PrSC). Human PCA LNCaP and PC3 cells were exposed to hypoxic (1% O2) or normoxic (21% O2) conditions, and exosomes secreted under hypoxic (ExoHypoxic) and normoxic (ExoNormoxic) conditions were isolated from conditioned media. Nanoparticle tracking analysis revealed that ExoHypoxic have smaller average size as compared to ExoNormoxic. Immunoblotting results showed a higher level of tetraspanins (CD63 and CD81), heat shock proteins (HSP90 and HSP70) and Annexin II in ExoHypoxic compared to ExoNormoxic. Co-culturing with ExoHypoxic increased the invasiveness and motility of naïve LNCaP and PC3 cells, respectively. ExoHypoxic also promoted prostasphere formation by both LNCaP and PC3 cells, and enhanced α-SMA (a CAF biomarker) expression in PrSC. Compared to ExoNormoxic, ExoHypoxic showed higher metalloproteinases activity and increased level of diverse signaling molecules (TGF-β2, TNF1α, IL6, TSG101, Akt, ILK1, and β-catenin). Furthermore, proteome analysis revealed a higher number of proteins in ExoHypoxic (160 proteins) compared to ExoNormoxic (62 proteins), primarily associated with the remodeling of epithelial adherens junction pathway. Importantly, ExoHypoxic targeted the expression of adherens junction proteins in naïve PC3 cells. These findings suggest that ExoHypoxic are loaded with unique proteins that could enhance invasiveness, stemness and induce microenvironment changes; thereby, promoting PCA aggressiveness. PMID:24347249

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 ...

Immunogenicity, Protective Efficacy, and Non-Replicative Status of the HSV-2 Vaccine Candidate HSV529 in Mice and Guinea Pigs

PubMed Central

Bernard, Marie-Clotilde; Barban, Véronique; Pradezynski, Fabrine; de Montfort, Aymeric; Ryall, Robert; Caillet, Catherine; Londono-Hayes, Patricia

2015-01-01

HSV-2 vaccine is needed to prevent genital disease, latent infection, and virus transmission. A replication-deficient mutant virus (dl5-29) has demonstrated promising efficacy in animal models of genital herpes. However, the immunogenicity, protective efficacy, and non-replicative status of the highly purified clinical vaccine candidate (HSV529) derived from dl5-29 have not been evaluated. Humoral and cellular immune responses were measured in mice and guinea pigs immunized with HSV529. Protection against acute and recurrent genital herpes, mortality, latent infection, and viral shedding after vaginal HSV-2 infection was determined in mice or in naïve and HSV-1 seropositive guinea pigs. HSV529 replication and pathogenicity were investigated in three sensitive models of virus replication: severe combined immunodeficient (SCID/Beige) mice inoculated by the intramuscular route, suckling mice inoculated by the intracranial route, and vaginally-inoculated guinea pigs. HSV529 immunization induced HSV-2-neutralizing antibody production in mice and guinea pigs. In mice, it induced production of specific HSV-2 antibodies and splenocytes secreting IFNγ or IL-5. Immunization effectively prevented HSV-2 infection in all three animal models by reducing mortality, acute genital disease severity and frequency, and viral shedding. It also reduced ganglionic viral latency and recurrent disease in naïve and HSV-1 seropositive guinea pigs. HSV529 replication/propagation was not detected in the muscles of SCID/Beige mice, in the brains of suckling mice, or in vaginal secretions of inoculated guinea pigs. These results confirm the non-replicative status, as well as its immunogenicity and efficacy in mice and guinea pigs, including HSV-1 seropositive guinea pigs. In mice, HSV529 produced Th1/Th2 characteristic immune response thought to be necessary for an effective vaccine. These results further support the clinical investigation of HSV529 in human subjects as a prophylactic vaccine. PMID:25837802

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis

PubMed Central

Armstrong, Richard M.; Adams, Katherine L.; Zilisch, Joseph E.; Bretl, Daniel J.; Sato, Hiromi; Anderson, David M.

2016-01-01

ABSTRACT Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis. IMPORTANCE Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis. This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism. PMID:27002134

Measles virus induces persistent infection by autoregulation of viral replication.

PubMed

Doi, Tomomitsu; Kwon, Hyun-Jeong; Honda, Tomoyuki; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

2016-11-24

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

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

PubMed

Os'mak, E D; Asanov, É O

2014-01-01

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

[3H]-nitrendipine binding in membranes obtained from hypoxic and reoxygenated heart.

PubMed

Matucci, R; Bennardini, F; Sciammarella, M L; Baccaro, C; Stendardi, I; Franconi, F; Giotti, A

1987-04-01

We compared the binding properties of [3H]-nitrendipine in heart membranes from normal guinea-pig heart and from hypoxic or hypoxic and reoxygenated heart. The [3H]-nitrendipine binds a single class of high capacity (Bmax 667.2 +/- 105.2) with high affinity (KD 0.14 +/- 0.02) binding sites. By contrast, in membranes of hypoxic and reoxygenated heart the Bmax decreases significantly while it remains unaffected during hypoxia. Xanthinoxidase activity is increased in hypoxic-reoxygenated hearts.

Protection Against Dengue Virus by Non-Replicating and Live Attenuated Vaccines Used Together in a Prime Boost Vaccination Strategy

DTIC Science & Technology

2010-01-01

vaccines primed rhesus maca - ques for an immune response to a tetravalent live attenuated virus (TLAV) vaccine. An initial experiment was performed in 16...and 4 and no measurable increase for DENV 1. These two experiments clearly demonstrated that rhesus maca - ques could be successfully immunized and

Basamid® G for weed control in forest tree nurseries

Treesearch

Adam Muckenfuss; Bill Isaacs

2006-01-01

Basamid® G is a granular soil fumigant containing dazomet, which has activity on weeds, insects, nematodes, and diseases. Basamid® G was compared to methyl bromide:chloropicrin and was equally effective as a weed control material in forest tree nurseries. Pine and hardwood plantings were treated with both materials in replicated and nonreplicated trials...

Communicating to Policymakers: What to Do with Ambiguous Results from Three Impact Studies of the Same Education Program

ERIC Educational Resources Information Center

Hartry, Ardice

2013-01-01

This paper considers one challenge from recent emphasis on rigorous research for decision-making in education: the interpretation of non-replicated findings. Under Clinton and Bush, the federal government called for and funded highly rigorous research. The "gold-standard" for research was randomized-control trials (RCTs), which…

Clinical and diagnostic features of delayed hypoxic leukoencephalopathy.

PubMed

Shprecher, David R; Flanigan, Kevin M; Smith, A Gordon; Smith, Shawn M; Schenkenberg, Thomas; Steffens, John

2008-01-01

Delayed hypoxic leukoencephalopathy is an underrecognized syndrome of delayed demyelination, which is important to consider when delayed onset of neuropsychiatric symptoms follows a hypoxic event. The authors describe clinical and diagnostic features of three such cases, review the pathophysiology of delayed hypoxic leukoencephalopathy, and discuss features which may help distinguish it from toxic leukoencephalopathy.

Neuroprotective Role of Exogenous Brain-Derived Neurotrophic Factor in Hypoxia-Hypoglycemia-Induced Hippocampal Neuron Injury via Regulating Trkb/MiR134 Signaling.

PubMed

Huang, Weidong; Meng, Facai; Cao, Jie; Liu, Xiaobin; Zhang, Jie; Li, Min

2017-05-01

Hypoxic-ischemic brain injury is an important cause of neonatal mortality and morbidity. Brain-derived neurotrophic factor (BDNF) has been reported to play a neuroprotective role in hypoxic-ischemic brain injury; however, the specific effects and mechanism of BDNF on hypoxic-hypoglycemic hippocampal neuron injury remains unknown. The current study investigated the action of BDNF in regulating cerebral hypoxic-ischemic injury by simulating hippocampal neuron ischemia and hypoxia. We found that BDNF, p-Trkb, and miR-134 expression levels decreased, and that exogenous BDNF increased survival and reduced apoptosis in hypoxic-hypoglycemic hippocampal neurons. The results also show that BDNF inhibits MiR-134 expression by activating the TrkB pathway. Transfection with TrkB siRNA and pre-miR-134 abrogated the neuroprotective role of BDNF in hypoxic-hypoglycemic hippocampal neurons. Our results suggest that exogenous BDNF alleviates hypoxic-ischemic brain injury through the Trkb/MiR-134 pathway. These findings may help to identify a potential therapeutic agent for the treatment of hypoxic-ischemic brain injury.

Cobalt-Doped Brushite Cement: Preparation, Characterization, and In Vitro Interaction with Osteosarcoma Cells

NASA Astrophysics Data System (ADS)

Cummings, Haley; Han, Weiguo; Vahabzadeh, Sahar; Elsawa, Sherine F.

2017-08-01

Brushite cement (BrC) is being widely used in bone and dental tissue engineering application because of its significant biocompatibility, bioresorbability, and moldability. Here, we have reported the effects of cobalt (Co) and its concentration on physical and biological properties of BrC. Our results show that Co addition stabilizes the tricalcium phosphate structure and decreases the amount of BrC phase in the final product. The in vitro interaction of samples with osteosarcoma MG-63 cells proved the cytocompatibility of all compositions in both normoxic and hypoxic conditions. Although the cell viability increased under hypoxia, the change was insignificant compared with normoxic conditions. Our data show that Co addition reduced hypoxia inducible factor-1α and glioma-associated oncogene family zinc finger 2 expression in MG-63, suggesting Co may provide the benefit of reducing the effects of hypoxia on gene expression in the osteosarcoma cell line.

Stress Erythropoiesis Model Systems.

PubMed

Bennett, Laura F; Liao, Chang; Paulson, Robert F

2018-01-01

Bone marrow steady-state erythropoiesis maintains erythroid homeostasis throughout life. This process constantly generates new erythrocytes to replace the senescent erythrocytes that are removed by macrophages in the spleen. In contrast, anemic or hypoxic stress induces a physiological response designed to increase oxygen delivery to the tissues. Stress erythropoiesis is a key component of this response. It is best understood in mice where it is extramedullary occurring in the adult spleen and liver and in the fetal liver during development. Stress erythropoiesis utilizes progenitor cells and signals that are distinct from bone marrow steady-state erythropoiesis. Because of that observation many genes may play a role in stress erythropoiesis despite having no effect on steady-state erythropoiesis. In this chapter, we will discuss in vivo and in vitro techniques to study stress erythropoiesis in mice and how the in vitro culture system can be extended to study human stress erythropoiesis.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

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

Apoptosis, energy metabolism, and fraction of radiobiologically hypoxic cells: a study of human melanoma multicellular spheroids.

PubMed

Rofstad, E K; Eide, K; Skøyum, R; Hystad, M E; Lyng, H

1996-09-01

The magnitude of the fraction of radiobiologically hypoxic cells in tumours is generally believed to reflect the efficiency of the vascular network. Theoretical studies have suggested that the hypoxic fraction might also be influenced by biological properties of the tumour cells. Quantitative experimental results of cell energy metabolism, hypoxia- induced apoptosis, and radiobiological hypoxia are reported here. Human melanoma multicellular spheroids (BEX-c and WIX-c) were used as tumour models to avoid confounding effects of the vascular network. Radiobiological studies showed that the fractions of hypoxic cells in 1000-microM spheroids were 32 +/- 12% (BEX-c) and 2.5 +/- 1.1% (WIX-c). The spheroid hypoxic volume fractions (28 +/- 6% (BEX-c) and 1.4 +/- 7% (WIX-c)), calculated from the rate of oxygen consumption per cell, the cell packing density, and the thickness of the viable rim, were similar to the fractions of radiobiologically hypoxic cells. Large differences between tumours in fraction of hypoxic cells are therefore not necessarily a result of differences in the efficiency of the vascular network. Studies of monolayer cell cultures, performed to identify the biological properties of the BEX-c and WIX-c cells leading to this large difference in fraction of hypoxic cells, gave the following results: (1) WIX-c showed lower cell surviving fractions after exposure to hypoxia than BEX-c, (2) WIX-c showed higher glucose uptake and lactate release rates than BEX-c both under aerobic and hypoxic conditions, and (3) hypoxia induced apoptosis in WIX-c but not in BEX-c. These observations suggested that the difference between BEX-c and WIX-c spheroids in fraction of hypoxic cells resulted partly from differences in cell energy metabolism and partly from a difference in capacity to retain viability under hypoxic stress. The induction of apoptosis by hypoxia was identified as a phenomenon which has an important influence on the magnitude of the fraction of radiobiologically hypoxic cells in multicellular spheroids.

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

PubMed

Lerman, Oren Z; Galiano, Robert D; Armour, Mary; Levine, Jamie P; Gurtner, Geoffrey C

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O(2)), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 +/- 1.3 pg/ml versus 34.8 +/- 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients.

Hypoxia Potentiates the Radiation-Sensitizing Effect of Olaparib in Human Non-Small Cell Lung Cancer Xenografts by Contextual Synthetic Lethality

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yanyan; Verbiest, Tom; Devery, Aoife M.

Purpose: Poly(ADP-ribose) polymerase (PARP) inhibitors potentiate radiation therapy in preclinical models of human non-small cell lung cancer (NSCLC) and other types of cancer. However, the mechanisms underlying radiosensitization in vivo are incompletely understood. Herein, we investigated the impact of hypoxia on radiosensitization by the PARP inhibitor olaparib in human NSCLC xenograft models. Methods and Materials: NSCLC Calu-6 and Calu-3 cells were irradiated in the presence of olaparib or vehicle under normoxic (21% O{sub 2}) or hypoxic (1% O{sub 2}) conditions. In vitro radiosensitivity was assessed by clonogenic survival assay and γH2AX foci assay. Established Calu-6 and Calu-3 subcutaneous xenografts were treated with olaparib (50 mg/kg, dailymore » for 3 days), radiation (10 Gy), or both. Tumors (n=3/group) were collected 24 or 72 hours after the first treatment. Immunohistochemistry was performed to assess hypoxia (carbonic anhydrase IX [CA9]), vessels (CD31), DNA double strand breaks (DSB) (γH2AX), and apoptosis (cleaved caspase 3 [CC3]). The remaining xenografts (n=6/group) were monitored for tumor growth. Results: In vitro, olaparib showed a greater radiation-sensitizing effect in Calu-3 and Calu-6 cells in hypoxic conditions (1% O{sub 2}). In vivo, Calu-3 tumors were well-oxygenated, whereas Calu-6 tumors had extensive regions of hypoxia associated with down-regulation of the homologous recombination protein RAD51. Olaparib treatment increased unrepaired DNA DSB (P<.001) and apoptosis (P<.001) in hypoxic cells of Calu-6 tumors following radiation, whereas it had no significant effect on radiation-induced DNA damage response in nonhypoxic cells of Calu-6 tumors or in the tumor cells of well-oxygenated Calu-3 tumors. Consequently, olaparib significantly increased radiation-induced growth inhibition in Calu-6 tumors (P<.001) but not in Calu-3 tumors. Conclusions: Our data suggest that hypoxia potentiates the radiation-sensitizing effects of olaparib by contextual synthetic killing, and that tumor hypoxia may be a potential biomarker for selecting patients who may get the greatest benefit from the addition of olaparib to radiation therapy.« less

Cellular Dysfunction in the Diabetic Fibroblast

PubMed Central

Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P < 0.001) and was not significantly stimulated by hypoxia (1% O2), whereas wild-type fibroblast migration was up-regulated nearly twofold in hypoxic conditions (P < 0.05). Diabetic fibroblasts produced twice the amount of pro-matrix metalloproteinase-9 as normal fibroblasts, as measured by both gelatin zymography and enzyme-linked immunosorbent assay (P < 0.05). Adult diabetic fibroblasts exhibited a sevenfold impairment in vascular endothelial growth factor (VEGF) production (4.5 ± 1.3 pg/ml versus 34.8 ± 3.3 pg/ml, P < 0.001) compared to wild-type fibroblasts. Moreover, wild-type fibroblast production of VEGF increased threefold in response to hypoxia, whereas diabetic fibroblast production of VEGF was not up-regulated in hypoxic conditions (P < 0.001). To address the question whether these differences resulted from chronic hyperglycemia or absence of the leptin receptor, fibroblasts were harvested from newborn db/db mice before the onset of diabetes (4 to 5 weeks old). These fibroblasts showed no impairments in VEGF production under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients. PMID:12507913

Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures.

PubMed

Roy, A; Krzykwa, E; Lemieux, R; Néron, S

2001-12-01

Several normal human cells, such as hematopoietic stem cells, dendritic cells, and B cells, can be cultured in vitro in defined optimal conditions. Several ex vivo culture systems require the use of feeder cells to support the growth of target cells. In such systems, proliferation of feeder cells has to be stopped, so that they can be used as nonreplicating viable support cells. Because feeder cells need to provide one or few active signals, it is important to maintain them in an metabolically active state, allowing continued expression of specific ligands or cytokines. Mitomycin C and gamma-irradiation treatments are commonly used to prepare nonproliferating feeder cells and are usually considered to be equivalent. Normal human B lymphocytes can be expanded in vitro in the presence of feeder cells expressing the CD40 ligand CD154. Here we compared the ability of gamma-irradiation- and mitomycin C-treated feeder cells to support the expansion of normal human B lymphocytes. The results indicate that expansion of B cells during a long-term culture was 100 times more potent using gamma-irradiated feeder cells compared to mitomycin C-treated cells. This difference could be related to a significant reduction in both cellular metabolism and level of CD154 expression observed in mitomycin C-treated feeder cells, but not in gamma-irradiated cells nor in control untreated cells. These results indicate that mitomycin C-treated feeder cells are metabolically altered, and consequently less efficient at maintaining cell expansion in the long-term cell culture system used.

Apoptin towards safe and efficient anticancer therapies.

PubMed

Backendorf, Claude; Noteborn, Mathieu H M

2014-01-01

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent.Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

Hypoxia promotes production of neural crest cells in the embryonic head.

PubMed

Scully, Deirdre; Keane, Eleanor; Batt, Emily; Karunakaran, Priyadarssini; Higgins, Debra F; Itasaki, Nobue

2016-05-15

Hypoxia is encountered in either pathological or physiological conditions, the latter of which is seen in amniote embryos prior to the commencement of a functional blood circulation. During the hypoxic stage, a large number of neural crest cells arise from the head neural tube by epithelial-to-mesenchymal transition (EMT). As EMT-like cancer dissemination can be promoted by hypoxia, we investigated whether hypoxia contributes to embryonic EMT. Using chick embryos, we show that the hypoxic cellular response, mediated by hypoxia-inducible factor (HIF)-1α, is required to produce a sufficient number of neural crest cells. Among the genes that are involved in neural crest cell development, some genes are more sensitive to hypoxia than others, demonstrating that the effect of hypoxia is gene specific. Once blood circulation becomes fully functional, the embryonic head no longer produces neural crest cells in vivo, despite the capability to do so in a hypoxia-mimicking condition in vitro, suggesting that the oxygen supply helps to stop emigration of neural crest cells in the head. These results highlight the importance of hypoxia in normal embryonic development. © 2016. Published by The Company of Biologists Ltd.

Gab1 Is Modulated by Chronic Hypoxia in Children with Cyanotic Congenital Heart Defect and Its Overexpression Reduces Apoptosis in Rat Neonatal Cardiomyocytes

PubMed Central

Cherif, Myriam; Nakaoka, Yoshikazu; Angelini, Gianni D.; Ghorbel, Mohamed T.

2015-01-01

Gab1 (Grb2 associated binding protein 1) is a member of the scaffolding/docking proteins (Gab1, Gab2, and Gab3). It is required for fibroblast cell survival and maintaining cardiac function. Very little is known about human Gab1 expression in response to chronic hypoxia. The present study examined the hypothesis that hypoxia regulates Gab1 expression in human paediatric myocardium and cultured rat cardiomyocytes. Here we showed that Gab1 is expressed in myocardial tissue in acyanotic and cyanotic children with congenital heart defects. Gab1 protein was upregulated in cyanotic compared to acyanotic hearts suggesting that Gab1 upregulation is a component of the survival program initiated by hypoxia in cyanotic children. The expression of other Gab1 interacting partners was not affected by hypoxia and Gab1 regulation. Additionally, using an in vitro model, we demonstrated that overexpressing Gab1 in neonatal cardiomyocytes, under hypoxic condition, resulted in the reduction of apoptosis suggesting a role for this protein in cardiomyocyte survival. Altogether, our data provide strong evidence that Gab1 is important for heart cell survival following hypoxic stress. PMID:26090437

An ultrasonically powered implantable micro-oxygen generator (IMOG).

PubMed

Maleki, Teimour; Cao, Ning; Song, Seung Hyun; Kao, Chinghai; Ko, Song-Chu Arthur; Ziaie, Babak

2011-11-01

In this paper, we present an ultrasonically powered implantable micro-oxygen generator (IMOG) that is capable of in situ tumor oxygenation through water electrolysis. Such active mode of oxygen generation is not affected by increased interstitial pressure or abnormal blood vessels that typically limit the systemic delivery of oxygen to hypoxic regions of solid tumors. Wireless ultrasonic powering (2.15 MHz) was employed to increase the penetration depth and eliminate the directional sensitivity associated with magnetic methods. In addition, ultrasonic powering allowed for further reduction in the total size of the implant by eliminating the need for a large area inductor. IMOG has an overall dimension of 1.2 mm × 1.3 mm × 8 mm, small enough to be implanted using a hypodermic needle or a trocar. In vitro and ex vivo experiments showed that IMOG is capable of generating more than 150 μA which, in turn, can create 0.525 μL/min of oxygen through electrolytic disassociation. In vivo experiments in a well-known hypoxic pancreatic tumor models (1 cm (3) in size) also verified adequate in situ tumor oxygenation in less than 10 min.

PTP1B controls non-mitochondrial oxygen consumption by regulating RNF213 to promote tumour survival during hypoxia.

PubMed

Banh, Robert S; Iorio, Caterina; Marcotte, Richard; Xu, Yang; Cojocari, Dan; Rahman, Anas Abdel; Pawling, Judy; Zhang, Wei; Sinha, Ankit; Rose, Christopher M; Isasa, Marta; Zhang, Shuang; Wu, Ronald; Virtanen, Carl; Hitomi, Toshiaki; Habu, Toshiyuki; Sidhu, Sachdev S; Koizumi, Akio; Wilkins, Sarah E; Kislinger, Thomas; Gygi, Steven P; Schofield, Christopher J; Dennis, James W; Wouters, Bradly G; Neel, Benjamin G

2016-07-01

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

Cavitation-enhanced extravasation for drug delivery.

PubMed

Arvanitis, Costas D; Bazan-Peregrino, Miriam; Rifai, Bassel; Seymour, Leonard W; Coussios, Constantin C

2011-11-01

A flow-through tissue-mimicking phantom composed of a biocompatible hydro-gel with embedded tumour cells was used to assess and optimize the role of ultrasound-induced cavitation on the extravasation of a macromolecular compound from a channel mimicking vessel in the gel, namely a non-replicating luciferase-expressing adenovirus (Ad-Luc). Using a 500 KHz therapeutic ultrasound transducer confocally aligned with a focussed passive cavitation detector, different exposure conditions and burst mode timings were selected by performing time and frequency domain analysis of passively recorded acoustic emissions, in the absence and in the presence of ultrasound contrast agents acting as cavitation nuclei. In the presence of Sonovue, maximum ultraharmonic emissions were detected for peak rarefactional pressures of 360 kPa, and maximum broadband emissions occurred at 1250 kPa. The energy of the recorded acoustic emissions was used to optimise the pulse repetition frequency and duty cycle in order to maximize either ultraharmonic or broadband emissions while keeping the acoustic energy delivered to the focus constant. Cell viability measurements indicated that none of the insonation conditions investigated induces cell death in the absence of a therapeutic agent (i.e. virus). Phase contrast images of the tissue-mimicking phantom showed that short range vessel disruption can occur when ultra-harmonic emissions (nf0/2) are maximised whereas formation of a micro-channel perpendicular to the flow can be obtained in the presence of broadband acoustic emissions. Following Ad-Luc delivery, luciferase expression measurements showed that a 60-fold increase in its bioavailability can be achieved when broadband noise emissions are present during insonation, even for modest contrast agent concentrations. The findings of the present study suggest that drug delivery systems based on acoustic cavitation may help enhance the extravasation of anticancer agents, thus increasing their penetration distance to hypoxic regions and poorly vascularised tumour regions. Copyright © 2011 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Embryonic Stem Cell-Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic-Ischemic Mouse Brain.

PubMed

Hawkins, Kate E; Corcelli, Michelangelo; Dowding, Kate; Ranzoni, Anna M; Vlahova, Filipa; Hau, Kwan-Leong; Hunjan, Avina; Peebles, Donald; Gressens, Pierre; Hagberg, Henrik; de Coppi, Paolo; Hristova, Mariya; Guillot, Pascale V

2018-05-01

Human mesenchymal stem cells (MSCs) have huge potential for regenerative medicine. In particular, the use of pluripotent stem cell-derived mesenchymal stem cells (PSC-MSCs) overcomes the hurdle of replicative senescence associated with the in vitro expansion of primary cells and has increased therapeutic benefits in comparison to the use of various adult sources of MSCs in a wide range of animal disease models. On the other hand, fetal MSCs exhibit faster growth kinetics and possess longer telomeres and a wider differentiation potential than adult MSCs. Here, for the first time, we compare the therapeutic potential of PSC-MSCs (ES-MSCs from embryonic stem cells) to fetal MSCs (AF-MSCs from the amniotic fluid), demonstrating that ES-MSCs have a superior neuroprotective potential over AF-MSCs in the mouse brain following hypoxia-ischemia. Further, we demonstrate that nuclear factor (NF)-κB-stimulated interleukin (IL)-13 production contributes to an increased in vitro anti-inflammatory potential of ES-MSC-conditioned medium (CM) over AF-MSC-CM, thus suggesting a potential mechanism for this observation. Moreover, we show that induced pluripotent stem cell-derived MSCs (iMSCs) exhibit many similarities to ES-MSCs, including enhanced NF-κB signaling and IL-13 production in comparison to AF-MSCs. Future studies should assess whether iMSCs also exhibit similar neuroprotective potential to ES-MSCs, thus presenting a potential strategy to overcome the ethical issues associated with the use of embryonic stem cells and providing a potential source of cells for autologous use against neonatal hypoxic-ischemic encephalopathy in humans. Stem Cells Translational Medicine 2018;7:439-449. © 2018 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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

PubMed

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

2016-12-01

Cardiac stem cells (CSCs) are being evaluated for their efficacy in the treatment of heart failure. However, numerous factors impair the exogenously delivered cells' regenerative capabilities. Hypoxia is one stress that contributes to inadequate tissue repair. Here, we tested the hypothesis that hypoxia impairs cell proliferation, survival, and migration of human CSCs relative to physiological and room air oxygen concentrations. Human endomyocardial biopsy-derived CSCs were isolated, selected for c-Kit expression, and expanded in vitro at room air (21% O 2 ). To assess the effect on proliferation, survival, and migration, CSCs were transferred to physiological (5%) or hypoxic (0.5%) O 2 concentrations. Physiological O 2 levels increased proliferation (P < 0.05) but did not affect survival of CSCs. Although similar growth rates were observed in room air and hypoxia, a significant reduction of β-galactosidase activity (-4,203 fluorescent units, P < 0.05), p16 protein expression (0.58-fold, P < 0.001), and mitochondrial content (0.18-fold, P < 0.001) in hypoxia suggests that transition from high (21%) to low (0.5%) O 2 reduces senescence and promotes quiescence. Furthermore, physiological O 2 levels increased migration (P < 0.05) compared with room air and hypoxia, and treatment with mesenchymal stem cell-conditioned media rescued CSC migration under hypoxia to levels comparable to physiological O 2 migration (2-fold, P < 0.05 relative to CSC media control). Our finding that physiological O 2 concentration is optimal for in vitro parameters of CSC biology suggests that standard room air may diminish cell regenerative potential. This study provides novel insights into the modulatory effects of O 2 concentration on CSC biology and has important implications for refining stem cell therapies. Copyright © 2016 the American Physiological Society.

Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy.

PubMed

Fernández-López, David; Martínez-Orgado, José; Casanova, Ignacio; Bonet, Bartolomé; Leza, Juan Carlos; Lorenzo, Pedro; Moro, Maria Angeles; Lizasoain, Ignacio

2005-06-30

To analyze whether exposure to oxygen-glucose deprivation (OGD) of immature rat brain slices might reproduce the main pathophysiologic events leading to neuronal death in neonatal hypoxic-ischemic encephalopathy (NHIE), 500 microm-thick brain slices were obtained from 7-day-old Wistar rats, and incubated in oxygenated physiological solution. In OGD group, oxygen and glucose were removed from the medium for 10-30 min (n = 25); then, slices were re-incubated in normal medium. In control group the medium composition remained unchanged (CG, n = 30). Medium samples were obtained every 30 min for 3 h. To analyze neuronal damage, slices were stained with Nissl and CA1 area of hippocampus and cortex were observed under microscopy. In addition, neuronal death was quantified as LDH released to the medium determined by spectrophotometry. Additionally, medium glutamate (Glu) levels were determined by HPLC and those of TNFalpha by ELISA, whereas inducible nitric oxide synthase expression was determined by Western blot performed on slices homogenate. Optimal OGD time was established in 20 min. After OGD, a significant decrease in the number of neurones in hippocampus and cortex was observed. LDH release was maximal at 30 min, when it was five-fold greater than in CG. Furthermore, medium Glu concentrations were 200 times greater than CG levels at the end of OGD period. A linear relationship between Glu and LDH release was demonstrated. Finally, 3 h after OGD a significant induction of iNOS as well as an increase in TNFalpha release were observed. In conclusion, OGD appears as a feasible and reproducible in vitro model, leading to a neuronal damage, which is physiopathologically similar to that found in NHIE.

n-3 Polyunsaturated Fatty Acids Reduce Neonatal Hypoxic/Ischemic Brain Injury by Promoting Phosphatidylserine Formation and Akt Signaling.

PubMed

Zhang, Wenting; Liu, Jia; Hu, Xiaoming; Li, Peiying; Leak, Rehana K; Gao, Yanqin; Chen, Jun

2015-10-01

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate neonatal hypoxic/ischemic (H/I) brain damage, but the underlying mechanisms are not fully understood. This study tested the hypothesis that n-3 PUFAs enhance Akt-dependent prosurvival signaling by promoting the biosynthesis of phosphatidylserine in neuronal cell membranes. Dietary n-3 PUFA supplementation was initiated on the second day of pregnancy in dams. H/I was induced in 7-day-old rat pups by ipsilateral common carotid artery occlusion followed by hypoxia (8% oxygen for 2.5 hours). Neurological outcomes, brain tissue loss, cell death, and the activation of signaling events were assessed after H/I. The effects of n-3 PUFAs (docosahexaenoic acid and eicosapentaenoic acid) on oxygen-glucose deprivation-induced cell death and the underlying mechanism of protection were also examined in primary cortical neuron cultures. n-3 PUFAs reduced brain tissue loss at 7 days after H/I and improved neurological outcomes, whereas inhibition of PI3K/Akt signaling by LY294002 partially abrogated this neuroprotective effect. Docosahexaenoic acid/eicosapentaenoic acid also prevented ischemic neuronal death through the Akt prosurvival pathway in vitro. Furthermore, docosahexaenoic acid/eicosapentaenoic acid increased the production of phosphatidylserine, the major membrane-bound phospholipids, after ischemia both in vitro and in vivo. A reduction in membrane phosphatidylserine by shRNA-mediated knockdown of phosphatidylserine synthetase-1 attenuated Akt activation and neuronal survival after docosahexaenoic acid/eicosapentaenoic acid treatment in the oxygen-glucose deprivation model. n-3 PUFAs robustly protect against H/I-induced brain damage in neonates by activating Akt prosurvival pathway in compromised neurons. In addition, n-3 PUFAs promote the formation of membrane phosphatidylserine, thereby promoting Akt activity and improving cellular survival. © 2015 American Heart Association, Inc.

Targeted identification of sialoglycoproteins in hypoxic endothelial cells and validation in zebrafish reveal roles for proteins in angiogenesis.

PubMed

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

2015-02-06

The formation of new vessels in the tumor, termed angiogenesis, is essential for primary tumor growth and facilitates tumor invasion and metastasis. Hypoxia has been described as one trigger of angiogenesis. Indeed, hypoxia, which is characterized by areas of low oxygen levels, is a hallmark of solid tumors arising from an imbalance between oxygen delivery and consumption. Hypoxic conditions have profound effects on the different components of the tumoral environment. For example, hypoxia is able to activate endothelial cells, leading to angiogenesis but also thereby initiating a cascade of reactions involving neutrophils, smooth muscle cells, and fibroblasts. In addition, hypoxia directly regulates the expression of many genes for which the role and the importance in the tumoral environment remain to be completely elucidated. In this study, we used a method to selectively label sialoglycoproteins to identify new membrane and secreted proteins involved in the adaptative process of endothelial cells by mass spectrometry-based proteomics. We used an in vitro assay under hypoxic condition to observe an increase of protein expression or modifications of glycosylation. Then the function of the identified proteins was assessed in a vasculogenesis assay in vivo by using a morpholino strategy in zebrafish. First, our approach was validated by the identification of sialoglycoproteins such as CD105, neuropilin-1, and CLEC14A, which have already been described as playing key roles in angiogenesis. Second, we identified several new proteins regulated by hypoxia and demonstrated for the first time the pivotal role of GLUT-1, TMEM16F, and SDF4 in angiogenesis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Low oxygen tension reverses antineoplastic effect of iron chelator deferasirox in human glioblastoma cells.

PubMed

Legendre, Claire; Avril, Sylvie; Guillet, Catherine; Garcion, Emmanuel

2016-02-01

Overcoming resistance to treatment is an essential issue in many cancers including glioblastoma (GBM), the deadliest primary tumor of the central nervous system. As dependence on iron is a key feature of tumor cells, using chelators to reduce iron represents an opportunity to improve conventional GBM therapies. The aim of the present study was, therefore, to investigate the cytostatic and cytotoxic impact of the new iron chelator deferasirox (DFX) on human GBM cells in well-defined clinical situations represented by radiation therapy and mild-hypoxia. Under experimental normoxic condition (21% O2), deferasirox (DFX) used at 10 μM for 3 days reduced proliferation, led cell cycle arrest in S and G2-M phases and induced cytotoxicity and apoptosis in U251 and U87 GBM cells. The abolition of the antineoplastic DFX effects when cells were co-treated with ferric ammonium sulfate supports the hypothesis that its effects result from its ability to chelate iron. As radiotherapy is the main treatment for GBM, the combination of DFX and X-ray beam irradiation was also investigated. Irradiation at a dose of 16 Gy repressed proliferation, cytotoxicity and apoptosis, but only in U251 cells, while no synergy with DFX was observed in either cell line. Importantly, when the same experiment was conducted in mild-hypoxic conditions (3% O2), the antiproliferative and cytotoxic effects of DFX were abolished, and its ability to deplete iron was also impaired. Taken together, these in vitro results could raise the question of the benefit of using iron chelators in their native forms under the hypoxic conditions often encountered in solid tumors such as GBM. Developing new chemistry or a new drug delivery system that would keep DFX active in hypoxic cells may be the next step toward their application.

Conversion of Stationary to Invasive Tumor Initiating Cells (TICs): Role of Hypoxia in Membrane Type 1-Matrix Metalloproteinase (MT1-MMP) Trafficking

PubMed Central

Li, Jian; Zucker, Stanley; Pulkoski-Gross, Ashleigh; Kuscu, Cem; Karaayvaz, Mihriban; Ju, Jingfang; Yao, Herui; Song, Erwei; Cao, Jian

2012-01-01

Emerging evidence has implicated the role of tumor initiating cells (TICs) in the process of cancer metastasis. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. In this report, we employed less invasive breast cancer TICs, SK-3rd, that displays CD44high/CD24low with high mammosphere-forming and tumorigenic capacities, to investigate the mechanism by which stationary TICs are converted to invasive TICs. Invasive ability of SK-3rd TICs was markedly enhanced when the cells were cultured under hypoxic conditions. Given the role of membrane type 1-matrix metalloproteinase (MT1-MMP) in cancer invasion/metastasis, we explored a possible involvement of MT1-MMP in hypoxia-induced TIC invasion. Silencing of MT1-MMP by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. Under hypoxic conditions, MT1-MMP redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MT1-MMP. Interestingly, cell surface MT1-MMP gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MT1-MMP in cytoplasmic storage pools under normoxic conditions, by demonstrating a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MT1-MMP expression in SK-3rd TICs. These observations suggest that MT1-MMP is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis. PMID:22679501

Hypothermia reduces VEGF-165 expression, but not osteogenic differentiation of human adipose stem cells under hypoxia

PubMed Central

Bakker, Astrid D.; Hogervorst, Jolanda M. A.; Nolte, Peter A.; Klein-Nulend, Jenneke

2017-01-01

Cryotherapy is successfully used in the clinic to reduce pain and inflammation after musculoskeletal damage, and might prevent secondary tissue damage under the prevalent hypoxic conditions. Whether cryotherapy reduces mesenchymal stem cell (MSC) number and differentiation under hypoxic conditions, causing impaired callus formation is unknown. We aimed to determine whether hypothermia modulates proliferation, apoptosis, nitric oxide production, VEGF gene and protein expression, and osteogenic/chondrogenic differentiation of human MSCs under hypoxia. Human adipose MSCs were cultured under hypoxia (37°C, 1% O2), hypothermia and hypoxia (30°C, 1% O2), or control conditions (37°C, 20% O2). Total DNA, protein, nitric oxide production, alkaline phosphatase activity, gene expression, and VEGF protein concentration were measured up to day 8. Hypoxia enhanced KI67 expression at day 4. The combination of hypothermia and hypoxia further enhanced KI67 gene expression compared to hypoxia alone, but was unable to prevent the 1.2-fold reduction in DNA amount caused by hypoxia at day 4. Addition of hypothermia to hypoxic cells did not alter the effect of hypoxia alone on BAX-to-BCL-2 ratio, alkaline phosphatase activity, gene expression of SOX9, COL1, or osteocalcin, or nitric oxide production. Hypothermia decreased the stimulating effect of hypoxia on VEGF-165 gene expression by 6-fold at day 4 and by 2-fold at day 8. Hypothermia also decreased VEGF protein expression under hypoxia by 2.9-fold at day 8. In conclusion, hypothermia decreased VEGF-165 gene and protein expression, but did not affect differentiation, or apoptosis of MSCs cultured under hypoxia. These in vitro results implicate that hypothermia treatment in vivo, applied to alleviate pain and inflammation, is not likely to harm early stages of callus formation. PMID:28166273

Enhancement of human neural stem cell self-renewal in 3D hypoxic culture.

PubMed

Ghourichaee, Sasan Sharee; Powell, Elizabeth M; Leach, Jennie B

2017-05-01

The pathology of neurological disorders is associated with the loss of neuronal and glial cells that results in functional impairments. Human neural stem cells (hNSCs), due to their self-renewing and multipotent characteristics, possess enormous tissue-specific regenerative potential. However, the efficacy of clinical applications is restricted due to the lack of standardized in vitro cell production methods with the capability of generating hNSC populations with well-defined cellular compositions. At any point, a population of hNSCs may include undifferentiated stem cells, intermediate and terminally differentiated progenies, and dead cells. Due to the plasticity of hNSCs, environmental cues play crucial roles in determining the cellular composition of hNSC cultures over time. Here, we investigated the independent and synergistic effect of three important environmental factors (i.e., culture dimensionality, oxygen concentration, and growth factors) on the survival, renewal potential, and differentiation of hNSCs. Our experimental design included two dimensional (2D) versus three dimensional (3D) cultures and normoxic (21% O 2 ) versus hypoxic (3% O 2 ) conditions in the presence and absence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2). Additionally, we discuss the feasibility of mathematical models that predict hNSC growth and differentiation under these culture conditions by adopting a negative feedback regulatory term. Our results indicate that the synergistic effect of culture dimensionality and hypoxic oxygen concentration in the presence of growth factors enhances the proliferation of viable, undifferentiated hNSCs. Moreover, the same synergistic effect in the absence of growth factors promotes the differentiation of hNSCs. Biotechnol. Bioeng. 2017;114: 1096-1106. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Ultrasensitive near-infrared fluorescence-enhanced probe for in vivo nitroreductase imaging.

PubMed

Li, Yuhao; Sun, Yun; Li, Jiachang; Su, Qianqian; Yuan, Wei; Dai, Yu; Han, Chunmiao; Wang, Qiuhong; Feng, Wei; Li, Fuyou

2015-05-20

Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.

Unsaturation of Very-Long-Chain Ceramides Protects Plant from Hypoxia-Induced Damages by Modulating Ethylene Signaling in Arabidopsis

PubMed Central

Yu, Lu-Jun; Huang, Li; Chen, Liang; Wang, Feng-Zhu; Xia, Fan-Nv; Zhu, Tian-Ren; Wu, Jian-Xin; Yin, Jian; Liao, Bin; Shi, Jianxin; Zhang, Jian-Hua; Aharoni, Asaph; Yao, Nan; Shu, Wensheng; Xiao, Shi

2015-01-01

Lipid remodeling is crucial for hypoxic tolerance in animals, whilst little is known about the hypoxia-induced lipid dynamics in plants. Here we performed a mass spectrometry-based analysis to survey the lipid profiles of Arabidopsis rosettes under various hypoxic conditions. We observed that hypoxia caused a significant increase in total amounts of phosphatidylserine, phosphatidic acid and oxidized lipids, but a decrease in phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Particularly, significant gains in the polyunsaturated species of PC, PE and phosphatidylinositol, and losses in their saturated and mono-unsaturated species were evident during hypoxia. Moreover, hypoxia led to a remarkable elevation of ceramides and hydroxyceramides. Disruption of ceramide synthases LOH1, LOH2 and LOH3 enhanced plant sensitivity to dark submergence, but displayed more resistance to submergence under light than wild type. Consistently, levels of unsaturated very-long-chain (VLC) ceramide species (22:1, 24:1 and 26:1) predominantly declined in the loh1, loh2 and loh3 mutants under dark submergence. In contrast, significant reduction of VLC ceramides in the loh1-1 loh3-1 knockdown double mutant and lacking of VLC unsaturated ceramides in the ads2 mutants impaired plant tolerance to both dark and light submergences. Evidence that C24:1-ceramide interacted with recombinant CTR1 protein and inhibited its kinase activity in vitro, enhanced ER-to-nucleus translocation of EIN2-GFP and stabilization of EIN3-GFP in vivo, suggests a role of ceramides in modulating CTR1-mediated ethylene signaling. The dark submergence-sensitive phenotypes of loh mutants were rescued by a ctr1-1 mutation. Thus, our findings demonstrate that unsaturation of VLC ceramides is a protective strategy for hypoxic tolerance in Arabidopsis. PMID:25822663

Sulphonylurea drugs reduce hypoxic damage in the isolated perfused rat kidney.

PubMed

Engbersen, R; Moons, M M; Wouterse, A C; Dijkman, H B; Kramers, C; Smits, P; Russel, F G

2000-08-01

Sulphonylurea drugs have been shown to protect against hypoxic damage in isolated proximal tubules of the kidney. In the present study we investigated whether these drugs can protect against hypoxic damage in a whole kidney preparation. Tolbutamide (200 microM) and glibenclamide (10 microM) were applied to the isolated perfused rat kidney prior to changing the gassing from oxygen to nitrogen for 30 min. Hypoxic perfusions resulted in an increased fractional excretion of glucose (FE % glucose 14.3+/-1.5 for hypoxic perfusions vs 4.9+/-1.6 for normoxic perfusions, mean +/- s.e. mean, P<0.05), which could be completely restored by 200 microM tolbutamide (5.7+/-0.4 for tolbutamide vs 14.3+/-1.5 for untreated hypoxic kidneys, P<0.01). Furthermore, tolbutamide reduced the total amount of LDH excreted in the urine (220+/-100 mU for tolbutamide vs. 1220+/-160 mU for untreated hypoxic kidneys, P<0.01). Comparable results were obtained with glibenclamide (10 microM). In agreement with the effect on functional parameters, ultrastructural analysis of proximal tubules showed increased brush border preservation in tolbutamide treated kidneys compared to untreated hypoxic kidneys. We conclude that glibenclamide and tolbutamide are both able to reduce hypoxic damage to proximal tubules in the isolated perfused rat kidney when applied in the appropriate concentrations.

A scenario and forecast model for Gulf of Mexico hypoxic area and volume

USGS Publications Warehouse

Scavia, Donald; Evans, Mary Anne; Obenour, Daniel R.

2013-01-01

For almost three decades, the relative size of the hypoxic region on the Louisiana-Texas continental shelf has drawn scientific and policy attention. During that time, both simple and complex models have been used to explore hypoxia dynamics and to provide management guidance relating the size of the hypoxic zone to key drivers. Throughout much of that development, analyses had to accommodate an apparent change in hypoxic sensitivity to loads and often cull observations due to anomalous meteorological conditions. Here, we describe an adaptation of our earlier, simple biophysical model, calibrated to revised hypoxic area estimates and new hypoxic volume estimates through Bayesian estimation. This application eliminates the need to cull observations and provides revised hypoxic extent estimates with uncertainties, corresponding to different nutrient loading reduction scenarios. We compare guidance from this model application, suggesting an approximately 62% nutrient loading reduction is required to reduce Gulf hypoxia to the Action Plan goal of 5,000 km2, to that of previous applications. In addition, we describe for the first time, the corresponding response of hypoxic volume. We also analyze model results to test for increasing system sensitivity to hypoxia formation, but find no strong evidence of such change.

Renal effects of metallothionein induction by zinc in vitro and in vivo.

PubMed

Schanz, Moritz; Schaaf, Lea; Dippon, Juergen; Biegger, Dagmar; Fritz, Peter; Alscher, Mark Dominik; Kimmel, Martin

2017-03-16

Metallothionein (MTT) is an endogenous antioxidant that can be induced by both zinc (Zn) and ischemia. In kidneys, increased MTT expression exerts a putative protective role in diabetes and hypoxia. Our goal was to further investigate the behavior of MTT under the influence of Zn and hypoxia in vitro and in vivo. MTT expression was measured in vitro in cell cultures of proximal tubular cells (LCC-PK1) by immune-histochemistry and real-time PCR after incubation with increasing concentrations of Zn under hypoxic and non-hypoxic conditions. In addition, in vivo studies were carried out in 54 patients to study MTT induction through Zn. This is a sub-study of a prospective, randomized, double-blind trial on prevention of contrast-media-induced nephropathy using Placebo, Zn and N-Acetylcysteine. Blood samples were obtained before and after 2 days p.o. treatment with or without Zn (60 mg). ELISA-based MTT level measurements were done to evaluate the effects of Zn administration. For in vivo analysis, we considered the ratio of MTT to baseline MTT (MTT 1 /MTT 0 ) and the ratio of eGFR (eGFR 1 /eGFR 0 ), correspondingly. In vitro quantitative immuno-histochemical analysis (IHC) and real-time PCR showed that at increasing levels of Zn (5, 10, and 15 μg/ml) led to a progressive increase of MTTs: Median (IQR) expression of IHC also increased progressively from 0.10 (0.09-0.12), 0.15 (0.12-0.18), 0.25 (0.25-0.27), 0.59 (0.48-0.70) (p < 0.0001). Median (IQR) expression of PCR: 0.59 (0.51-1.72), 1.62 (1.38-4.70), 3.58 (3.06-10.42) and 10.81 (9.24-31.47) (p < 0.0001). In contrast, hypoxia did not change MTT-levels in vitro (p > 0.05). In vivo no significant differences (p = 0.96) occurred in MTT-levels after 2 days of Zn administration compared with no Zn intake. Nevertheless, there was a significant correlation between MTT (MTT 1 /MTT 0 ) and eGFR (eGFR 1 /eGFR 0 ) in case of Zn administration (rho = -0.49; 95%-CI: -0.78 to -0.03; p = 0.04). We found that Zn did induce MTTs in vitro, whereas hypoxia had no significant impact. In contrast, no significant increase of MTTs was detected after in vivo administration of Zn. However, there was a significant negative correlation between MTT and eGFR in vivo in case of Zn administration, this could indicate a protective role of MTTs in a setting of reduced kidney function, which is possibly influenced by Zn. ClinicalTrials.gov Identifier: NCT00399256 . Retrospectively registered 11/13/2006.

Asymptotic Properties of Induced Maximum Likelihood Estimates of Nonlinear Models for Item Response Variables: The Finite-Generic-Item-Pool Case.

ERIC Educational Resources Information Center

Jones, Douglas H.

The progress of modern mental test theory depends very much on the techniques of maximum likelihood estimation, and many popular applications make use of likelihoods induced by logistic item response models. While, in reality, item responses are nonreplicate within a single examinee and the logistic models are only ideal, practitioners make…

Intracellular Survival of Staphylococcus aureus in Endothelial Cells: A Matter of Growth or Persistence

PubMed Central

Rollin, Guillaume; Tan, Xin; Tros, Fabiola; Dupuis, Marion; Nassif, Xavier; Charbit, Alain; Coureuil, Mathieu

2017-01-01

The Gram-positive human pathogen Staphylococcus aureus is a leading cause of severe bacterial infections. Recent studies have shown that various cell types could readily internalize S. aureus and infected cells have been proposed to serve as vehicle for the systemic dissemination of the pathogen. Here we focused on the intracellular behavior of the Community-Associated Methicillin-Resistant S. aureus strain USA300. Supporting earlier observations, we found that wild-type S. aureus strain USA300 persisted for longer period within endothelial cells than within macrophages and that a mutant displaying the small colony variant phenotype (ΔhemDBL) had increased intracellular persistence. Time-lapse microscopy revealed that initial persistence of wild-type bacteria in endothelial cells corresponded to distinct single cell events, ranging from active intracellular bacterial proliferation, leading to cell lysis, to non-replicating bacterial persistence even 1 week after infection. In sharp contrast, ΔhemDBL mutant bacteria were essentially non-replicating up to 10 days after infection. These findings suggest that internalization of S. aureus in endothelial cells triggers its persistence and support the notion that endothelial cells might constitute an intracellular persistence niche responsible for reported relapse of infection after antibiotic therapy. PMID:28769913

Replication of type 5 adenovirus promotes middle ear infection by Streptococcus pneumoniae in the chinchilla model of otitis media

PubMed Central

Murrah, Kyle A.; Turner, Roberta L.; Pang, Bing; Perez, Antonia C.; Reimche, Jennifer L.; King, Lauren B.; Wren, John; Gandhi, Uma; Swords, W. Edward; Ornelles, David A.

2015-01-01

Adenoviral infection is a major risk factor for otitis media. We hypothesized that adenovirus promotes bacterial ascension into the middle ear through the disruption of normal function in the Eustachian tubes due to inflammation-induced changes. An intranasal infection model of the chinchilla was used to test the ability of type 5 adenovirus to promote middle ear infection by Streptococcus pneumoniae. The hyperinflammatory adenovirus mutant dl327 and the nonreplicating adenovirus mutant H5wt300ΔpTP were used to test the role of inflammation and viral replication, respectively, in promotion of pneumococcal middle ear infection. Precedent infection with adenovirus resulted in a significantly greater incidence of middle ear disease by S. pneumoniae as compared to nonadenovirus infected animals. Infection with the adenovirus mutant dl327 induced a comparable degree of bacterial ascension into the middle ear as did infection with the wild-type virus. By contrast, infection with the nonreplicating adenovirus mutant H5wt300ΔpTP resulted in less extensive middle ear infection compared to the wild-type adenovirus. We conclude that viral replication is necessary for adenoviral-induced pneumococcal middle ear disease. PMID:25251686

Inhibitors Selective for Mycobacterial Versus Human Proteasomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, G.; Li, D; Sorio de Carvalho, L

Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-onemore » compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.« less

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

2016-08-05

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. Copyright © 2016 Elsevier Inc. All rights reserved.

Hypoxic pretreatment protects against neuronal damage of the rat hippocampus induced by severe hypoxia.

PubMed

Gorgias, N; Maidatsi, P; Tsolaki, M; Alvanou, A; Kiriazis, G; Kaidoglou, K; Giala, M

1996-04-01

The present study investigates whether under conditions of successive hypoxic exposures pretreatment with mild (15% O(2)) or moderate (10% O(2)) hypoxia, protects hippocampal neurones against damage induced by severe (3% O(2)) hypoxia. The ultrastructural findings were also correlated with regional superoxide dismutase (SOD) activity changes. In unpretreated rats severe hypoxia induced ultrastructural changes consistent with the aspects of delayed neuronal death (DND). However, in preexposed animals hippocampal damage was attenuated in an inversely proportional way with the severity of the hypoxic pretreatment. The ultrastructural hypoxic tolerance findings were also closely related to increased regional SOD activity levels. Thus the activation of the endogenous antioxidant defense by hypoxic preconditioning, protects against hippocampal damage induced by severe hypoxia. The eventual contribution of increased endogenous adenosine and/or reduced excitotoxicity to induce hypoxic tolerance is discussed.

Sulphonylurea drugs reduce hypoxic damage in the isolated perfused rat kidney

PubMed Central

Engbersen, Richard; Moons, Miek M; Wouterse, Alfons C; Dijkman, Henry B; Kramers, Cees; Smits, Paul; Russel, Frans G M

2000-01-01

Sulphonylurea drugs have been shown to protect against hypoxic damage in isolated proximal tubules of the kidney. In the present study we investigated whether these drugs can protect against hypoxic damage in a whole kidney preparation. Tolbutamide (200 μM) and glibenclamide (10 μM) were applied to the isolated perfused rat kidney prior to changing the gassing from oxygen to nitrogen for 30 min. Hypoxic perfusions resulted in an increased fractional excretion of glucose (FE % glucose 14.3±1.5 for hypoxic perfusions vs 4.9±1.6 for normoxic perfusions, mean±s.e.mean, P<0.05), which could be completely restored by 200 μM tolbutamide (5.7±0.4 for tolbutamide vs 14.3±1.5 for untreated hypoxic kidneys, P<0.01). Furthermore, tolbutamide reduced the total amount of LDH excreted in the urine (220±100 mU for tolbutamide vs 1220±160 mU for untreated hypoxic kidneys, P<0.01). Comparable results were obtained with glibenclamide (10 μM). In agreement with the effect on functional parameters, ultrastructural analysis of proximal tubules showed increased brush border preservation in tolbutamide treated kidneys compared to untreated hypoxic kidneys. We conclude that glibenclamide and tolbutamide are both able to reduce hypoxic damage to proximal tubules in the isolated perfused rat kidney when applied in the appropriate concentrations. PMID:10928974

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

PubMed Central

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

2016-01-01

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

A model study of the response of hypoxia to upwelling-favorable wind on the northern Gulf of Mexico shelf

NASA Astrophysics Data System (ADS)

Feng, Yang; Fennel, Katja; Jackson, George A.; DiMarco, Steven F.; Hetland, Robert D.

2014-03-01

The hypoxic region in the northern Gulf of Mexico, one of the largest man-made hypoxic zones in the world, has received extensive scientific study and management interest. A previous statistical study has concluded that in addition to anthropogenic nitrogen loading, the observed hypoxic extent is correlated to the duration of upwelling favorable (westerly) wind without elucidating the underlying mechanism. In this study, we use a three-dimensional, coupled hydrological-biogeochemical model to mechanistically examine how variations of the hypoxic area are related to the duration of upwelling-favorable wind. We performed scenario experiments with different durations of upwelling-favorable wind using realistic winds from summer 2002 (when upwelling-favorable winds were present only for about 1 month) and summer 2009 (when upwelling-favorable conditions started early and persisted for about 2 months). While the maximum simulated hypoxic area is approximately 15,000 km2 in both cases, the evolutions of the hypoxic area and the dates when its maximum extent are reached are different. With an early start of persistently upwelling-favorable wind in 2009, the hypoxic area reached its maximum in early summer and decreased afterwards. By contrast, the hypoxic area was small in early summer of 2002 and peaked during the short period of upwelling-favorable wind in late summer. The model revealed that the wind influences the evolution of the hypoxic area by changing the vertical and horizontal distributions of the low salinity, high chlorophyll water on the shelf.

Construction of Large-Volume Tissue Mimics with 3D Functional Vascular Networks

PubMed Central

Kang, Tae-Yun; Hong, Jung Min; Jung, Jin Woo; Kang, Hyun-Wook; Cho, Dong-Woo

2016-01-01

We used indirect stereolithography (SL) to form inner-layered fluidic networks in a porous scaffold by introducing a hydrogel barrier on the luminal surface, then seeded the networks separately with human umbilical vein endothelial cells and human lung fibroblasts to form a tissue mimic containing vascular networks. The artificial vascular networks provided channels for oxygen transport, thus reducing the hypoxic volume and preventing cell death. The endothelium of the vascular networks significantly retarded the occlusion of channels during whole-blood circulation. The tissue mimics have the potential to be used as an in vitro platform to examine the physiologic and pathologic phenomena through vascular architecture. PMID:27228079

Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

PubMed

Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

2016-03-01

Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls. Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

Hypoxia-activated prodrug TH-302 decreased survival rate of canine lymphoma cells under hypoxic condition.

PubMed

Yamazaki, Hiroki; Lai, Yu-Chang; Tateno, Morihiro; Setoguchi, Asuka; Goto-Koshino, Yuko; Endo, Yasuyuki; Nakaichi, Munekazu; Tsujimoto, Hajime; Miura, Naoki

2017-01-01

We tested the hypotheses that hypoxic stimulation enhances growth potentials of canine lymphoma cells by activating hypoxia-inducible factor 1α (HIF-1α), and that the hypoxia-activated prodrug (TH-302) inhibits growth potentials in the cells. We investigated how hypoxic culture affects the growth rate, chemoresistance, and invasiveness of canine lymphoma cells and doxorubicin (DOX)-resistant lymphoma cells, and influences of TH-302 on survival rate of the cells under hypoxic conditions. Our results demonstrated that hypoxic culture upregulated the expression of HIF-1α and its target genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter G2 (ABCG2), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and survivin, and enhanced the growth rate, DOX resistance, and invasiveness of the cells. Additionally, TH-302 decreased the survival rate of the cells under hypoxic condition. Our studies suggest that hypoxic stimulation may advance the tumorigenicity of canine lymphoma cells, favoring malignant transformation. Therefore, the data presented may contribute to the development of TH-302-based hypoxia-targeting therapies for canine lymphoma.

Tumorigenicity of hypoxic respiring cancer cells revealed by a hypoxia–cell cycle dual reporter

PubMed Central

Le, Anne; Stine, Zachary E.; Nguyen, Christopher; Afzal, Junaid; Sun, Peng; Hamaker, Max; Siegel, Nicholas M.; Gouw, Arvin M.; Kang, Byung-hak; Yu, Shu-Han; Cochran, Rory L.; Sailor, Kurt A.; Song, Hongjun; Dang, Chi V.

2014-01-01

Although aerobic glycolysis provides an advantage in the hypoxic tumor microenvironment, some cancer cells can also respire via oxidative phosphorylation. These respiring (“non-Warburg”) cells were previously thought not to play a key role in tumorigenesis and thus fell from favor in the literature. We sought to determine whether subpopulations of hypoxic cancer cells have different metabolic phenotypes and gene-expression profiles that could influence tumorigenicity and therapeutic response, and we therefore developed a dual fluorescent protein reporter, HypoxCR, that detects hypoxic [hypoxia-inducible factor (HIF) active] and/or cycling cells. Using HEK293T cells as a model, we identified four distinct hypoxic cell populations by flow cytometry. The non-HIF/noncycling cell population expressed a unique set of genes involved in mitochondrial function. Relative to the other subpopulations, these hypoxic “non-Warburg” cells had highest oxygen consumption rates and mitochondrial capacity consistent with increased mitochondrial respiration. We found that these respiring cells were unexpectedly tumorigenic, suggesting that continued respiration under limiting oxygen conditions may be required for tumorigenicity. PMID:25114222

Effect of the Discharge Water which Mixed Sewage Disposal Water with Seawater Desalting Treated Sewage for Bottom Sediment and Hypoxic Water Mass

NASA Astrophysics Data System (ADS)

Watanabe, Ryoichi; Yamasaki, Koreyoshi; Minagawa, Tomoko; Iyooka, Hiroki; Kitano, Yoshinori

For every time in summer season, hypoxic water mass has formed at the inner part of Hakata Bay. Field observation study has carried out at the inner part of Hakata Bay since 2004 with the particular aim of tracking the movement of hypoxic water mass. Hypoxic water masses form the end of June to September on this area because the consumption of oxygen in bottom water layers exceeds the re-supply of oxygen from the atmosphere. Under such hypoxic conditions, the seawater desalination plant has begun to use in 2005. After seawater desalination plant operation starting, hypoxic water mass tends to improve. In this research, the authors show the following result. After seawater desalination plant has begun to operate, the hypoxia around the mixed discharge water outlet tends to be improved.

Antioxidants prevent depression of the acute hypoxic ventilatory response by subanaesthetic halothane in men

PubMed Central

Teppema, Luc J; Nieuwenhuijs, Diederik; Sarton, Elise; Romberg, Raymonda; Olievier, Cees N; Ward, Denham S; Dahan, Albert

2002-01-01

We studied the effect of the antioxidants (AOX) ascorbic acid (2 g, I.V.) and α-tocopherol (200 mg, P.O.) on the depressant effect of subanaesthetic doses of halothane (0.11 % end-tidal concentration) on the acute isocapnic hypoxic ventilatory response (AHR), i.e. the ventilatory response upon inhalation of a hypoxic gas mixture for 3 min (leading to a haemoglobin saturation of 82 ± 1.8 %) in healthy male volunteers. In the first set of protocols, two groups of eight subjects each underwent a control hypoxic study, a halothane hypoxic study and finally a halothane hypoxic study after pretreatment with AOX (study 1) or placebo (study 2). Halothane reduced the AHR by more than 50 %, from 0.79 ± 0.31 to 0.36 ± 0.14 l min−1 %−1 in study 1 and from 0.79 ± 0.40 to 0.36 ± 0.19 l min−1 %−1 in study 2, P < 0.01 for both. Pretreatment with AOX prevented this depressant effect of halothane in the subjects of study 1 (AHR returning to 0.77 ± 0.32 l min−1 %−1, n.s. from control), whereas placebo (study 2) had no effect (AHR remaining depressed at 0.36 ± 0.27 l min−1 %−1, P < 0.01 from control). In a second set of protocols, two separate groups of eight subjects each underwent a control hypoxic study, a sham halothane hypoxic study and finally a sham halothane hypoxic study after pretreatment with AOX (study 3) or placebo (study 4). In studies 3 and 4, sham halothane did not modify the control hypoxic response, nor did AOX (study 3) or placebo (study 4). The 95 % confidence intervals for the ratio of hypoxic sensitivities, (AOX + halothane):halothane in study 1 and (AOX - sham halothane):sham halothane in study 3, were [1.7, 2.6] and [1.0, 1.2], respectively. Because the antioxidants prevented the reduction of the acute hypoxic response by halothane, we suggest that this depressant effect may be caused by reactive species produced by a reductive metabolism of halothane during hypoxia or that a change in redox state of carotid body cells by the antioxidants prevented or changed the binding of halothane to its effect site. Our findings may also suggest that reactive species have an inhibiting effect on the acute hypoxic ventilatory response. PMID:12411535

Age specific effect of MK-801 on hypoxic body temperature regulation in rats.

PubMed

Baig, Mirza Shafiulla; Joseph, Vincent

2008-02-01

Hypoxic exposure produces a consistent decrease of rectal temperature (Tb), which is recognized as a potent protective response. While some of the neural mechanisms underlying this response have recently been described, it remains poorly known how these mechanisms evolve during post-natal development. We recently reported that in rat pups NMDA glutamate receptor limits Tb drop upon hypoxic exposure, an effect that has not been reported by others in adult rats. Accordingly, we tested the hypothesis that the implication of NMDA receptors on temperature control during hypoxic exposure evolves during development. To this aim, we evaluated the hypoxic (30 min - 12% O(2)) responses of Tb, metabolic rate, and ventilation in rats after injection of vehicle, or the NMDA receptor antagonist MK-801, at different ages (post-natal days 4, 10, 20 and 2-3 month-old - P4, P10, P20 and P60). MK-801 amplified the magnitude of the hypoxic-induced Tb drop in P4, P10 and P20 rats, but this effect was not apparent in adults. In P20 rats MK-801 tripled the hypoxic induced Tb drop, which was 0.5 degrees C in control and 1.4 degrees C in treated rats (p<0.0001). This effect was specific to temperature regulation, and was not accompanied by similar changes of other recorded parameters. MK-801 induced a significant decrease of the hypoxic ventilatory response in adults only. We conclude that NMDA glutamate receptor acts as a counter-regulatory factor that limits the hypoxic-induced drop of rectal temperature during post-natal development in rats.

Assessment of the Emerging Biocruise Threat

DTIC Science & Technology

2000-08-01

navigation and guidance system is located in the front; and the fuel and warhead are typically located in the midbody .66 (See Figure 1.) Guidance of a...unitary or submunitions Undetermined Undetermined MUPSOW South Africa Air & ground Conventional/ unitary or submunitions 200+ 2002 Storm Shadow ...replicating agents, bacteria or viruses, or nonreplicating materials, toxins or physiologically active proteins or peptides, that can be produced by living

Differential responsiveness in VEGF receptor subtypes to hypoxic stress in various tissues of plateau animals.

PubMed

Xie, Hui-Chun; Li, Jin-Gang; He, Jian-Ping

2017-05-04

With hypoxic stress, hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) are elevated and their responses are altered in skeletal muscles of plateau animals [China Qinghai-Tibetan plateau pikas (Ochotona curzoniae)] as compared with control animals [normal lowland Sprague-Dawley (SD) rats]. The results indicate that HIF-1alpha and VEGF are engaged in physiological functions under hypoxic environment. The purpose of the current study was to examine the protein levels of VEGF receptor subtypes (VEGFRs: VEGFR-1, VEGFR-2 and VEGFR-3) in the end organs, namely skeletal muscle, heart and lung in response to hypoxic stress. ELISA and Western blot analysis were employed to determine HIF-1alpha and the protein expression of VEGFRs in control animals and plateau pikas. We further blocked HIF-1alpha signal to determine if HIF-1alpha regulates alternations in VEGFRs in those tissues. We hypothesized that responsiveness of VEGFRs in the major end organs of plateau animals is differential with insult of hypoxic stress and is modulated by low oxygen sensitive HIF-1alpha. Our results show that hypoxic stress induced by exposure of lower O(2) for 6 h significantly increased the levels of VEGFR-2 in skeletal muscle, heart and lung and the increases were amplified in plateau pikas. Our results also demonstrate that hypoxic stress enhanced VEGFR-3 in lungs of plateau animals. Nonetheless, no significant alternations in VEGFR-1 were observed in those tissues with hypoxic stress. Moreover, we observed decreases of VEGFR-2 in skeletal muscle, heart and lung; and decreases of VEGFR-3 in lung following HIF-1alpha inhibition. Overall, our findings suggest that in plateau animals 1) responsiveness of VEGFRs is different under hypoxic environment; 2) amplified VEGFR-2 response appears in skeletal muscle, heart and lung, and enhanced VEGFR-3 response is mainly observed in lung; 3) HIF-1alpha plays a regulatory role in the levels of VEGFRs. Our results provide the underlying cellular and molecular mechanisms responsible for hypoxic environment in plateau animals, having an impact on research of physiological and ecological adaptive responses to acute or chronic hypoxic stress in humans who living at high attitude and who live at a normal sea level but suffer from hypoxic disorders.

Nitric oxide-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased intensity of hypoxic exercise

PubMed Central

Curry, Timothy B.; Wilkins, Brad W.; Joyner, Michael J.

2011-01-01

Hypoxic vasodilation in skeletal muscle at rest is known to include β-adrenergic receptor-stimulated nitric oxide (NO) release. We previously reported that the augmented skeletal muscle vasodilation during mild hypoxic forearm exercise includes β-adrenergic mechanisms. However, it is unclear whether a β-adrenergic receptor-stimulated NO component exists during hypoxic exercise. We hypothesized that NO-mediated vasodilation becomes independent of β-adrenergic receptor activation with increased exercise intensity during hypoxic exercise. Ten subjects (7 men, 3 women; 23 ± 1 yr) breathed hypoxic gas to titrate arterial O2 saturation to 80% while remaining normocapnic. Subjects performed two consecutive bouts of incremental rhythmic forearm exercise (10% and 20% of maximum) with local administration (via a brachial artery catheter) of propranolol (β-adrenergic receptor inhibition) alone and with the combination of propranolol and nitric oxide synthase inhibition [NG-monomethyl-l-arginine (l-NMMA)] under normoxic and hypoxic conditions. Forearm blood flow (FBF, ml/min; Doppler ultrasound) and blood pressure [mean arterial pressure (MAP), mmHg; brachial artery catheter] were assessed, and forearm vascular conductance (FVC, ml·min−1·100 mmHg−1) was calculated (FBF/MAP). During propranolol alone, the rise in FVC (Δ from normoxic baseline) due to hypoxic exercise was 217 ± 29 and 415 ± 41 ml·min−1·100 mmHg−1 (10% and 20% of maximum, respectively). Combined propranolol-l-NMMA infusion during hypoxic exercise attenuated ΔFVC at 20% (352 ± 44 ml·min−1·100 mmHg−1; P < 0.001) but not at 10% (202 ± 28 ml·min−1·100 mmHg−1; P = 0.08) of maximum compared with propranolol alone. These data, when integrated with earlier findings, demonstrate that NO contributes to the compensatory vasodilation during mild and moderate hypoxic exercise; a β-adrenergic receptor-stimulated NO component exists during low-intensity hypoxic exercise. However, the source of the NO becomes less dependent on β-adrenergic mechanisms as exercise intensity increases. PMID:21193565

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamamura, Hideto; Suzuki, Yoshiaki; Yamamura, Hisao

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{sup 2+}-sensitive inward rectifier K{sup +} 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 butmore » not at mRNA level after the hypoxic culture. Ca{sup 2+} imaging study revealed that the hypoxic stress enhanced store-operated Ca{sup 2+} (SOC) entry, which was significantly reduced in the presence of 100 μM Ba{sup 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{sup 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{sup 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. -- Highlights: •Hypoxic culture of brain endothelial cells (BEC) caused membrane hyperpolarization. •This hyperpolarization was due to the increased expression of Kir2.1 channels. •Hypoxia enhanced store-operated Ca{sup 2+} (SOC) entry via Kir2.1 up-regulation. •Expression levels of putative SOC channels were not affected by hypoxia. •Kir2.1 up-regulation is responsible for hypoxia-enhanced BEC proliferation.« less

Using the endocannabinoid system as a neuroprotective strategy in perinatal hypoxic-ischemic brain injury

PubMed Central

Lara-Celador, I.; Goñi-de-Cerio, F.; Alvarez, Antonia; Hilario, Enrique

2013-01-01

One of the most important causes of brain injury in the neonatal period is a perinatal hypoxic-ischemic event. This devastating condition can lead to long-term neurological deficits or even death. After hypoxic-ischemic brain injury, a variety of specific cellular mechanisms are set in motion, triggering cell damage and finally producing cell death. Effective therapeutic treatments against this phenomenon are still unavailable because of complex molecular mechanisms underlying hypoxic-ischemic brain injury. After a thorough understanding of the mechanism underlying neural plasticity following hypoxic-ischemic brain injury, various neuroprotective therapies have been developed for alleviating brain injury and improving long-term outcomes. Among them, the endocannabinoid system emerges as a natural system of neuroprotection. The endocannabinoid system modulates a wide range of physiological processes in mammals and has demonstrated neuroprotective effects in different paradigms of acute brain injury, acting as a natural neuroprotectant. The aim of this review is to study the use of different therapies to induce long-term therapeutic effects after hypoxic-ischemic brain injury, and analyze the important role of the endocannabinoid system as a new neuroprotective strategy against perinatal hypoxic-ischemic brain injury. PMID:25206720

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.

[Effect of progesterone on the expression of GLUT in the brain following hypoxic-ischemia in newborn rats].

PubMed

Li, Dong-Liang; Han, Hua

2008-08-01

To investigate the expression of GLUT1 and GLUT3 in the hippocampus after cerebral hypoxic-ischemia (HI) in newborn rats and the effect of progesterone (PROG) on them. Forty newborn SD rats were randomly divided into four groups: normal group, sham-operated group, hypoxic-ischemic group and progesterone group. Model of hypoxic-ischemia encephalopathy (HIE) was established in the 7-day-old newborn SD rats. Immunohistochemical method was applied to detect the expression of GLUT1 and GLUT3 in hippocampus. GLUT1 and GLUT3 were slightly seen in normal and sham operation group, there was no obviously difference between the two groups (P > 0.05). The expression of GLUT1 and GLUT3 in hypoxic-ischemia group were all higher than that in sham operated group (P < 0.05). Not only the expression of GLUT in progesterone group were significantly higher than that in sham operated group (P < 0.01), but also than that in hypoxic-ischemia group (P < 0.05). PROG could increase the tolerance of neuron to hypoxic-ischemia with maintaining the energy supply in the brain by up-regulating GLUT expression.

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations.

PubMed

Saito, Kohta; Warrier, Thulasi; Somersan-Karakaya, Selin; Kaminski, Lina; Mi, Jianjie; Jiang, Xiuju; Park, Suna; Shigyo, Kristi; Gold, Ben; Roberts, Julia; Weber, Elaina; Jacobs, William R; Nathan, Carl F

2017-06-13

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB , which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2.

PubMed

Lee, Jung-Seok; Kim, Seul Ki; Jung, Byung-Joo; Choi, Seong-Bok; Choi, Eun-Young; Kim, Chang-Sung

2018-04-01

This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs) expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2) treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia) and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis), and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Elevated levels of hypoxia-inducible microRNA-210 in pre-eclampsia: new insights into molecular mechanisms for the disease

PubMed Central

Zhang, Yi; Fei, Mingyu; Xue, Geng; Zhou, Qi; Jia, Yin; Li, Li; Xin, Hong; Sun, Shuhan

2012-01-01

Abstract Pre-eclampsia is a leading cause of maternal and foetal morbidity and mortality worldwide. Insufficient uteroplacental oxygenation is believed to be responsible for the disease. However, what molecular events involve in hypoxic responses and how they affect placental development remain unclear. Recently, miRNAs have emerged as a new class of molecules in response to hypoxia. We show here that the expression of microRNA-210 (mir-210) is up-regulated in patients with pre-eclampsia, as well as in trophoblast cells cultured under hypoxic conditions. Ectopic expression of mir-210 inhibited the migration and invasion capability of trophoblast cells. Ephrin-A3 and Homeobox-A9, which related with cell migration and vascular remodelling, were then experimentally validated as the functional targets of mir-210 both in vivo and in vitro. Using luciferase reporter, chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) experiments, we finally identified a new transcriptional mechanism that the overexpression of mir-210 under hypoxia was regulated by NF-κB transcriptional factor p50, apart from the well-known HIF 1α. Taken together, our study implicates an important role for mir-210 in the molecular mechanism of pre-eclampsia. PMID:21388517

HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

PubMed

Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

2017-04-01

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Role of substance P in neutral endopeptidase modulation of hypoxic response of the carotid body.

PubMed

Kumar, G K; Yu, R K; Overholt, J L; Prabhakar, N R

2000-01-01

Carotid body expresses neutral endopeptidase (NEP)-like enzyme activity and phosphoramidon, an inhibitor of NEP augments sensory response of the carotid body to hypoxia (Kumar et al., 1990). NEP hydrolyzes substance P (SP) and methionine enkephalin (Met-ENK) in the nervous system. In the present study, we determined whether NEP hydrolyzes Met-ENK and SP in the carotid body and whether these peptides contribute to the phosphoramidon-induced potentiation of the sensory response to hypoxia. Experiments were performed on carotid bodies excised from anaesthetized adult cats. HPLC analysis showed that both SP and Met-ENK were hydrolyzed by the carotid body. Phosphoramidon (400 microM) markedly inhibited SP (approximately 90%) but had only marginal effect on Met-ENK hydrolysis (approximately 15%). Sensory responses of the carotid body in vitro to hypoxia (pO2, 68 +/- 6 mmHg) and SP (10 nmoles) were potentiated by phosphoramidon by approximately 80% and approximately 275% respectively (p < 0.01). SP-receptor antagonist abolished phosphoramidon-induced potentiation of the sensory response to hypoxia as well as to SP. These results demonstrate that SP is a preferred substrate for NEP in the carotid body and SP plays a major role in the potentiation of the hypoxic response of the carotid body by phosphoramidon.

Glucocorticoid-induced pancreatic-hepatic trans-differentiation in a human cell line in vitro.

PubMed

Fairhall, Emma A; Leitch, Alistair C; Lakey, Anne F; Probert, Philip M E; Richardson, Gabriella; De Santis, Carol; Wright, Matthew C

2018-05-22

The rodent pancreatic AR42J-B13 (B-13) cell line differentiates into non-replicative hepatocyte-like cells in response to glucocorticoid mediated via the glucocorticoid receptor (GR). The aims of this study were to identify a human cell line that responds similarly and investigate the mechanisms underpinning any alteration in differentiation. Exposing the human pancreatic adenocarcinoma (HPAC) cell line to 1-10 µM concentrations of dexamethasone (DEX) resulted an inhibition of proliferation, suppressed carcinoembryonic antigen expression, limited expression of pancreatic acinar and hepatic gene expression and significant induction of the constitutively-expressed hepatic CYP3A5 mRNA transcript. These changes were associated with a pulse of genomic DNA methylation and suppressed notch signalling activity. HPAC cells expressed high levels of GR transcript in contrast to other nuclear receptors - such as the glucocorticoid-activated pregnane X receptor (PXR) - and GR transcriptional function was activated by DEX in HPAC cells. Expression of selected hepatocyte transcripts in response to DEX was blocked by co-treatment with the GR antagonist RU486. These data indicate that the HPAC response to glucocorticoid exposure includes an inhibition in proliferation, alterations in notch signalling and a limited change in the expression of genes associated with an acinar and hepatic phenotype. This is the first demonstration of a human cell responding to similarly to the rodent B-13 cell regarding formation of hepatocyte-like cells in response to glucocorticoid. Identifying and modulating the ablating factor(s) may enhance the hepatocyte-like forming capacity of HPAC cells after exposure to glucocorticoid and generate an unlimited in vitro supply of human hepatocytes for toxicology studies and a variety of clinical applications. Copyright © 2018 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Recombinant Hepatitis E virus like particles can function as RNA nanocarriers.

PubMed

Panda, Subrat Kumar; Kapur, Neeraj; Paliwal, Daizy; Durgapal, Hemlata

2015-06-24

Assembled virus-like particles (VLPs) without genetic material, with structure similar to infectious virions, have been successfully used as vaccines. We earlier described in vitro assembly, characterisation and tissue specific receptor dependent Clathrin mediated entry of empty HEV VLPs, produced from Escherichia coli expressed HEV capsid protein (pORF2). Similar VLP's have been described as a potential candidate vaccine (Hecolin) against HEV. We have attempted to use such recombinant assembled Hepatitis E virus (HEV) VLPs as a carrier for heterologous RNA with protein coding sequence fused in-frame with HEV 5' region (containing cap and encapsidation signal) and investigated, if the relevant protein could be expressed and elicit an immune response in vivo. In vitro transcribed red fluorescent protein (RFP)/Hepatitis B virus surface antigen (HBsAg) RNA, fused to 5'-HEV sequence with cap and encapsidation signal (1-249 nt), was packaged into the recombinant HEV-VLPs and incubated with five different cell lines (Huh7, A549, Vero, HeLa and SiHa). The pORF2-VLPs could specifically transfer exogenous coding RNA into Huh7 and A549 cells. In vivo, Balb/c mice were immunized (intramuscular injections) with 100 µg pORF2-VLP encapsidated with 5'-methyl-G-HEV (1-249 nt)-HBsAg RNA, blood samples were collected and screened by ELISA for anti-pORF2 and anti-HBsAg antibodies. Humoral immune response could be elicited in Balb/c mice against both HEV capsid protein and cargo RNA encoded HBsAg protein. These findings suggest that other than being a possible vaccine, HEV pORF2-VLPs can be used as a promising non-replicative tissue specific gene delivery system.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

PubMed Central

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C.; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-01-01

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned. PMID:28877647

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

PubMed

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-12-01

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

The Influence of Changes in Tumor Hypoxia on Dose-Painting Treatment Plans Based on {sup 18}F-FMISO Positron Emission Tomography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin Zhixiong; Mechalakos, James; Nehmeh, Sadek

2008-03-15

Purpose: To evaluate how changes in tumor hypoxia, according to serial fluorine-18-labeled fluoro-misonidazole ({sup 18}F-FMISO) positron emission tomography (PET) imaging, affect the efficacy of intensity-modulated radiotherapy (IMRT) dose painting. Methods and Materials: Seven patients with head and neck cancers were imaged twice with FMISO PET, separated by 3 days, before radiotherapy. Intensity-modulated radiotherapy plans were designed, on the basis of the first FMISO scan, to deliver a boost dose of 14 Gy to the hypoxic volume, in addition to the 70-Gy prescription dose. The same plans were then applied to hypoxic volumes from the second FMISO scan, and the efficacymore » of dose painting evaluated by assessing coverage of the hypoxic volumes using D{sub max}, D{sub min}, D{sub mean}, D{sub 95}, and equivalent uniform dose (EUD). Results: Similar hypoxic volumes were observed in the serial scans for 3 patients but dissimilar ones for the other 4. There was reduced coverage of hypoxic volumes of the second FMISO scan relative to that of the first scan (e.g., the average EUD decreased from 87 Gy to 80 Gy). The decrease was dependent on the similarity of the hypoxic volumes of the two scans (e.g., the average EUD decrease was approximately 4 Gy for patients with similar hypoxic volumes and approximately 12 Gy for patients with dissimilar ones). Conclusions: The changes in spatial distribution of tumor hypoxia, as detected in serial FMISO PET imaging, compromised the coverage of hypoxic tumor volumes achievable by dose-painting IMRT. However, dose painting always increased the EUD of the hypoxic volumes.« less

Hyperadditive Ventilatory Response Arising from Interaction between the Carotid Chemoreflex and the Muscle Mechanoreflex in Healthy Humans.

PubMed

Silva, Talita M; Aranda, Liliane C; Paula-Ribeiro, Marcelle; Oliveira, Diogo M; Medeiros, Wladimir Musetti; Vianna, Lauro C; Nery, Luiz E; Silva, Bruno M

2018-03-22

Physical exercise potentiates the carotid chemoreflex control of ventilation (VE). Hyperadditive neural interactions may partially mediate the potentiation. However, some neural interactions remain incompletely explored. As the potentiation occurs even during low-intensity exercise, we tested the hypothesis that the carotid chemoreflex and the muscle mechanoreflex could interact in a hyperadditive fashion. Fourteen young healthy subjects inhaled, randomly, in separate visits, 12% O 2 to stimulate the carotid chemoreflex, and 21% O 2 as control. A rebreathing circuit maintained isocapnia. During gases administration, subjects either remained at rest (i.e., normoxic and hypoxic rest) or the muscle mechanoreflex was stimulated, via passive knee movement (i.e., normoxic and hypoxic movement). Surface muscle electrical activity did not increase during the passive movement, confirming the absence of active contractions. Hypoxic rest and normoxic movement similarly increased VE [change (mean {plus minus} SEM) = 1.24 {plus minus} 0.72 vs. 0.73 {plus minus} 0.43 L/min, respectively; P = 0.46], but hypoxic rest only increased tidal volume (Vt) and normoxic movement only increased breathing frequency (BF). Hypoxic movement induced greater VE and mean inspiratory flow (Vt/Ti) increase than the sum of hypoxic rest and normoxic movement isolated responses (VE change: hypoxic movement = 3.72 {plus minus} 0.81 vs. sum = 1.96 {plus minus} 0.83 L/min, P = 0.01; Vt/Ti change: hypoxic movement = 0.13 {plus minus} 0.03 vs. sum = 0.06 {plus minus} 0.03 L/s, P = 0.02). Moreover, hypoxic movement increased both Vt and BF. Collectively, the results indicate the carotid chemoreflex and the muscle mechanoreflex interacted mediating a hyperadditive ventilatory response in healthy humans.

Preclinical testing of an Atr inhibitor demonstrates improved response to standard therapies for esophageal cancer.

PubMed

Leszczynska, Katarzyna B; Dobrynin, Greg; Leslie, Rhea E; Ient, Jonathan; Boumelha, Adam J; Senra, Joana M; Hawkins, Maria A; Maughan, Tim; Mukherjee, Somnath; Hammond, Ester M

2016-11-01

Esophageal cancer has a persistently low 5-year survival rate and has recently been classified as a cancer of unmet need by Cancer Research UK. Consequently, new approaches to therapy are urgently required. Here, we tested the hypothesis that an ATR inhibitor, VX-970, used in combination with standard therapies for esophageal cancer could improve treatment outcome. Using esophageal cancer cell lines we evaluated the efficacy of combining VX-970 with cisplatin and carboplatin in vitro and with radiation in vitro and in vivo. Radiation experiments were also carried out in hypoxic conditions to mimic the tumor microenvironment. Combining VX-970 with cisplatin, carboplatin and radiation increased tumor cell kill in vitro. A significant tumor growth delay was observed when VX-970 was combined with radiotherapy in vivo. VX-970 is an effective chemo/radiosensitizer which could be readily integrated in the current treatment paradigm to improve the treatment response in esophageal cancer and we plan to test it prospectively in the forthcoming phase I dose escalation safety study combining the ATR inhibitor VX-970 with chemoradiotherapy in esophageal cancer (EudraCT number: 2015-003965-27). Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ashok, Ajay; Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288; Kanwar, Jagat Rakesh

Survivin, as an anti-apoptotic protein and a cell cycle regulator, is recently gaining importance for its regenerative potential in salvaging injured hypoxic cells of vital organs such as heart. Different strategies are being employed to upregulate survivin expression in dying hypoxic cardiomyocytes. We investigated the cardioprotective potential of a cell permeable survivin mutant protein SurR9C84A, for the management of hypoxia mediated cardiomyocyte apoptosis, in a novel and clinically relevant model employing primary human cardiomyocytes (HCM). The aim of this research work was to study the efficacy and mechanism of SurR9C84A facilitated cardioprotection and regeneration in hypoxic HCM. To mimic hypoxicmore » microenvironment in vitro, well characterized HCM were treated with 100 µm (48 h) cobalt chloride to induce hypoxia. Hypoxia induced (HI) HCM were further treated with SurR9C84A (1 µg/mL) in order to analyse its cardioprotective efficacy. Confocal microscopy showed rapid internalization of SurR9C84A and scanning electron microscopy revealed the reinstatement of cytoskeleton projections in HI HCM. SurR9C84A treatment increased cell viability, reduced cell death via, apoptosis (Annexin-V assay), and downregulated free cardiac troponin T and MMP-9 expression. SurR9C84A also upregulated the expression of proliferation markers (PCNA and Ki-67) and downregulated mitochondrial depolarization and ROS levels thereby, impeding cell death. Human Apoptosis Array further revealed that SurR9C84A downregulated expression of pro-apoptotic markers and augmented expression of HSPs and HTRA2/Omi. SurR9C84A treatment led to enhanced levels of survivin, VEGF, PI3K and pAkt. SurR9C84A proved non-toxic to normoxic HCM, as validated through unaltered cell proliferation and other marker levels. Its pre-treatment exhibited lesser susceptibility to hypoxia/damage. SurR9C84A holds a promising clinical potential for human cardiomyocyte survival and proliferation following hypoxic injury. - Highlights: • Protection/regeneration of dying myocardium post myocardial infarction is important. • Downregulation of survivin induces apoptosis in hypoxic human cardiomyocytes (HCM). • Bio-replenishment with SurR9-C84A reinstates HCM survival, recovery and growth. • SurR9-C84A targets mitochondrial depolarization, fcTnT and ROS generation in HCM. • SurR9-C84A upregulates survivin, PCNA, PI3K/Akt pathway, VEGF and HSP levels. • SurR9-C84A holds promise as a treatment and preventive agent to replenish survivin.« less

Effects of Hypoxia on the Phylogenetic Composition and Species Distribution of Protists in a Subtropical Harbor.

PubMed

Rocke, Emma; Jing, Hongmei; Xia, Xiaomin; Liu, Hongbin

2016-07-01

Tolo Harbor, a subtropical semi-enclosed coastal water body, is surrounded by an expanding urban community, which contributes to large concentrations of nutrient runoff, leading to algal blooms and localized hypoxic episodes. Present knowledge of protist distributions in subtropical waters during hypoxic conditions is very limited. In this study, therefore, we combined parallel 454 pyrosequencing technology and denaturing gradient gel electrophoresis (DGGE) fingerprint analyses to reveal the protist community shifts before, during, and after a 2-week hypoxic episode during the summer of 2011. Hierarchical clustering for DGGE demonstrated similar grouping of hypoxic samples separately from oxic samples. Dissolved oxygen (DO) concentration and dissolved inorganic nitrogen:phosphate (DIN:PO4) concentrations significantly affected OTU distribution in 454 sequenced samples, and a shift toward a ciliate and marine alveolate clade II (MALV II) species composition occurred as waters shifted from oxic to hypoxic. These results suggest that protist community shifts toward heterotrophic and parasitic tendencies as well as decreased diversity and richness in response to hypoxic outbreaks.

[Effect of argon and nitrogen on the peritoneal macrophages in mice and their resistance to the UV damaging effect in vitro].

PubMed

Galchuk, S V; Turovetskiĭ, V B; Andreev, A I; Buravkova, L B

2001-01-01

Explored were effects of argon and nitrogen on intracellular pH in peritoneal macrophages in mice and resistance of cellular membranes to the UV damaging effect in vitro. Blasting argon or nitrogen along the surface of cell cultures in airtight chamber for 20 minutes was shown to decrease 5-folds the oxygen content of solution as compared with initial level with culture pH unchanged. Ten-minute blasting argon or nitrogen through the incubation chamber slightly elevates intracellular pH in macrophages. The standard cell incubation conditions recovered following approximately 60 minutes in hypoxic atmosphere, the ability of macrophages to build up fluorescein was degraded and they increased intracellular pH no matter the indifferent gas yet more marked in case of nitrogen in use. It was demonstrated that the normobaric gas environment with oxygen partly replaced by nitrogen or argon protects plasmatic membranes of cells from UV-induced damage.

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 cell attachment, (ii) secretion of basal membrane proteins and (iii) EPO production. These findings could thus pave the way to new therapeutic approaches.

Blockade of the swelling-induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo.

PubMed

Wong, Raymond; Abussaud, Ahmed; Leung, Joseph Wh; Xu, Bao-Feng; Li, Fei-Ya; Huang, Sammen; Chen, Nai-Hong; Wang, Guan-Lei; Feng, Zhong-Ping; Sun, Hong-Shuo

2018-05-01

Activation of swelling-induced Cl - current (I Cl,swell ) during neonatal hypoxia-ischemia (HI) may induce brain damage. Hypoxic-ischemic brain injury causes chronic neurological morbidity in neonates as well as acute mortality. In this study, we investigated the role of I Cl,swell in hypoxic-ischemic brain injury using a selective blocker, 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl) oxybutyric acid (DCPIB). In primary cultured cortical neurons perfusion of a 30% hypotonic solution activated I Cl,swell , which was completely blocked by the application of DCPIB (10 μmol/L). The role of I Cl,swell in neonatal hypoxic-ischemic brain injury in vivo was evaluated in a modified neonatal hypoxic-ischemic brain injury model. Before receiving the ischemic insult, the mouse pups were injected with DCPIB (10 mg/kg, ip). We found that pretreatment with DCPIB significantly reduced the brain damage assessed using TTC staining, Nissl staining and whole brain imaging, and improved the sensorimotor and vestibular recovery outcomes evaluated in neurobehavioural tests (i.e. geotaxis reflex, and cliff avoidance reflex). These results show that DCPIB has neuroprotective effects on neonatal hypoxic-ischemic brain injury, and that the I Cl,swell may serve as a therapeutic target for treatment of hypoxic-ischemic encephalopathy.

Effect of acute hypoxic shock on the rat brain morphology and tripeptidyl peptidase I activity.

PubMed

Petrova, Emilia B; Dimitrova, Mashenka B; Ivanov, Ivaylo P; Pavlova, Velichka G; Dimitrova, Stella G; Kadiysky, Dimitar S

2016-06-01

Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress. Copyright © 2016 Elsevier GmbH. All rights reserved.

Ethyl pyruvate inhibits hypoxic pulmonary vasoconstriction and attenuates pulmonary artery cytokine expression

PubMed Central

Tsai, Ben M.; Lahm, Tim; Morrell, Eric D.; Crisostomo, Paul R.; Markel, Troy; Wang, Meijing; Meldrum, Daniel R.

2009-01-01

Hypoxic pulmonary vasoconstriction is a common consequence of acute lung injury and may be mediated by increased local production of proinflammatory cytokines. Ethyl pyruvate is a novel anti-inflammatory agent that has been shown to downregulate proinflammatory genes following hemorrhagic shock; however, its effects on hypoxic pulmonary vasoconstriction are unknown. We hypothesized that ethyl pyruvate would inhibit hypoxic pulmonary vasoconstriction and downregulate pulmonary artery cytokine expression during hypoxia. To study this, isometric force displacement was measured in isolated rat pulmonary artery rings (n=8/group) during hypoxia (95% N2/5% CO2) with or without prior ethyl pyruvate (10 mM) treatment. Following 60 minutes of hypoxia, pulmonary artery rings were analyzed for TNF-α and IL-1 mRNA via RT-PCR. Ethyl pyruvate inhibited hypoxic pulmonary artery contraction (4.49±2.32% vs. 88.80±5.68% hypoxia alone) and attenuated the hypoxic upregulation of pulmonary artery TNF and IL-1 mRNA (p<0.05). These data indicate that: 1) hypoxia increases pulmonary artery vasoconstriction and proinflammatory cytokine gene expression; 2) ethyl pyruvate decreases hypoxic pulmonary vasoconstriction and downregulates hypoxia-induced pulmonary artery proinflammatory cytokine gene expression; and 3) ethyl pyruvate may represent a novel therapeutic adjunct in the treatment of acute lung injury. PMID:17574585

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hong, Kyung-Soo; Research Center for Ischemic Tissue regeneration, Pusan National University School of Medicine, Yangsan; Park, Jun-Ik

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-cateninmore » 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-exposed or hypoxic preconditioned cells. ► SIRT1 deacetylates c-Myc and β-catenin ► HIF-1α is up-regulated by down-regulation of c-Myc and β-catenin expression. ► Polyphenolic SIRT1 activators mimics hypoxic preconditioning.« less

Three-dimensional cultured glioma cell lines

NASA Technical Reports Server (NTRS)

Gonda, Steve R. (Inventor); Marley, Garry M. (Inventor)

1991-01-01

Three-dimensional glioma spheroids were produced in vitro with size and histological differentiation previously unattained. The spheroids were grown in liquid media suspension in a Johnson Space Center (JSC) Rotating Wall Bioreactor without using support matrices such as microcarrier beads. Spheroid volumes of greater than 3.5 cu mm and diameters of 2.5 mm were achieved with a viable external layer or rim of proliferating cells, a transitional layer beneath the external layer with histological differentiation, and a degenerative central region with a hypoxic necrotic core. Cell debris was evident in the degenerative central region. The necrotics centers of some of the spheroids had hyaline droplets. Granular bodies were detected predominantly in the necrotic center.

Rapid Growth of Uropathogenic Escherichia coli during Human Urinary Tract Infection.

PubMed

Forsyth, Valerie S; Armbruster, Chelsie E; Smith, Sara N; Pirani, Ali; Springman, A Cody; Walters, Matthew S; Nielubowicz, Greta R; Himpsl, Stephanie D; Snitkin, Evan S; Mobley, Harry L T

2018-03-06

Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo , matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization. Copyright © 2018 Forsyth et al.

Detection of hypoxic fractions in murine tumors by comet assay: Comparison with other techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Q.; Kavanagh, M.C.; Newcombe, D.

1995-12-01

The alkaline comet assay was used to detect the hypoxic fractions of murine tumors. A total of four tumor types were tested using needle aspiration biopsies taken immediately after a radiation dose of 15 Gy. Initial studies confirmed that the normalized tail moment, a parameter reflecting single-strand DNA breaks induced by the radiation, was linearly related to radiation dose. Further, it was shown that for a mixed population (1:1) of cells irradiated under air-breathing or hypoxic conditions, the histogram of normal tail moment values obtained from analyzing 400 cells in the population had a double peak which, when fitted withmore » two Gaussian distributions, gave a good estimate of the proportion of the two subpopulations. For the four tumor types, the means of the calculated hypoxic fractions from four or five individual tumors were 0.15 {+-} 0.04 for B16F1, 0.08 {+-} 0.04 for KHT-LP1, 0.17 {+-} 0.04 for RIF-1 and 0.04 {+-} 0.01 for SCCVII. Analysis of variance showed that the hypoxic fraction in KHT-LP1 tumors is significantly lower than those of the other three tumors (P = 0.026) but that there is no significant difference in hypoxic fraction between B16F1, RIF-1 and SCCVII tumors (P = 0.574). Results from multiple samples taken from each of five RIF-1 tumors showed that the intertumor heterogeneity of hypoxic fractions was greater than that within the same tumor. The mean hypoxic fraction obtained using the comet assay for the four tumor types was compared with the hypoxic fraction determined by the clonogenic assay, or median pO{sub 2} values, or [{sup 3}H]misonidazole binding in the same tumor types. The values of hypoxic fraction obtained with the comet assay were two to four times lower than those measured by the paired survival method. Preliminary results obtained with a dose of 5 Gy were consistent with those obtained using 15 Gy. These results suggest the further development of the comet assay for clinical studies. 21 refs., 7 figs., 5 tabs.« less

Priming of the Cells: Hypoxic Preconditioning for Stem Cell Therapy.

PubMed

Wei, Zheng Z; Zhu, Yan-Bing; Zhang, James Y; McCrary, Myles R; Wang, Song; Zhang, Yong-Bo; Yu, Shan-Ping; Wei, Ling

2017-10-05

Stem cell-based therapies are promising in regenerative medicine for protecting and repairing damaged brain tissues after injury or in the context of chronic diseases. Hypoxia can induce physiological and pathological responses. A hypoxic insult might act as a double-edged sword, it induces cell death and brain damage, but on the other hand, sublethal hypoxia can trigger an adaptation response called hypoxic preconditioning or hypoxic tolerance that is of immense importance for the survival of cells and tissues. This review was based on articles published in PubMed databases up to August 16, 2017, with the following keywords: "stem cells," "hypoxic preconditioning," "ischemic preconditioning," and "cell transplantation." Original articles and critical reviews on the topics were selected. Hypoxic preconditioning has been investigated as a primary endogenous protective mechanism and possible treatment against ischemic injuries. Many cellular and molecular mechanisms underlying the protective effects of hypoxic preconditioning have been identified. In cell transplantation therapy, hypoxic pretreatment of stem cells and neural progenitors markedly increases the survival and regenerative capabilities of these cells in the host environment, leading to enhanced therapeutic effects in various disease models. Regenerative treatments can mobilize endogenous stem cells for neurogenesis and angiogenesis in the adult brain. Furthermore, transplantation of stem cells/neural progenitors achieves therapeutic benefits via cell replacement and/or increased trophic support. Combinatorial approaches of cell-based therapy with additional strategies such as neuroprotective protocols, anti-inflammatory treatment, and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the recent progress regarding cell types and applications in regenerative medicine as well as future applications.

Regulation of breathing and body temperature of a burrowing rodent during hypoxic-hypercapnia.

PubMed

Barros, Renata C H; Abe, Augusto S; Cárnio, Evelin C; Branco, Luiz G S

2004-05-01

Burrowing mammals usually have low respiratory sensitivity to hypoxia and hypercapnia. However, the interaction between ventilation (V), metabolism and body temperature (Tb) during hypoxic-hypercapnia has never been addressed. We tested the hypothesis that Clyomys bishopi, a burrowing rodent of the Brazilian cerrado, shows a small ventilatory response to hypoxic-hypercapnia, accompanied by a marked drop in Tb and metabolism. V, Tb and O(2) consumption (V?O(2)) of C. bishopi were measured during exposure to air, hypoxia (10% and 7% O(2)), hypercapnia (3% and 5% CO(2)) and hypoxic-hypercapnia (10% O(2)+ 3% CO(2)). Hypoxia of 7% but not 10%, caused a significant increase in V, and a significant drop in Tb. Both hypoxic levels decreased V?O(2) and 7% O(2) significantly increased V/V?O(2). Hypercapnia of 5%, but not 3%, elicited a significant increase in V, although no significant change in Tb, V?O(2) or V/V?O(2) was detected. A combination of 10% O(2) and 3% CO(2) had minor effects on V and Tb, while V?O(2) decreased and V/V?O(2) tended to increase. We conclude that C. bishopi has a low sensitivity not only to hypoxia and hypercapnia, but also to hypoxic-hypercapnia, manifested by a biphasic ventilatory response, a drop in metabolism and a tendency to increase V/V?O(2). The effect of hypoxic-hypercapnia was the summation of the hypoxia and hypercapnia effects, with respiratory responses tending to have hypercapnic patterns while metabolic responses, hypoxic patterns.

Hypoxic events and concomitant factors in preterm infants on non-invasive ventilation.

PubMed

Fathabadi, Omid Sadeghi; Gale, Timothy; Wheeler, Kevin; Plottier, Gemma; Owen, Louise S; Olivier, J C; Dargaville, Peter A

2017-04-01

Automated control of inspired oxygen for newborn infants is an emerging technology, currently limited by reliance on a single input signal (oxygen saturation, SpO 2 ). This is while other signals that may herald the onset of hypoxic events or identify spurious hypoxia are not usually utilised. We wished to assess the frequency of apnoea, loss of circuit pressure and/or motion artefact in proximity to hypoxic events in preterm infants on non-invasive ventilation. Hypoxic events (SpO 2  < 80 %) were identified using a previously acquired dataset obtained from preterm infants receiving non-invasive ventilation. Events with concomitant apnoea, loss of circuit pressure or oximetry motion artefact were annotated, and the frequency of each of these factors was determined. The effect of duration and timing of apnoea on the characteristics of the associated hypoxic events was studied. Among 1224 hypoxic events, 555 (45 %) were accompanied by apnoea, 31 (2.5 %) by loss of circuit pressure and 696 (57 %) by motion artefact, while for 224 (18 %) there were no concomitant factors identified. Respiratory pauses of longer duration (>15 s) preceding hypoxic events, were associated with a relatively slow decline in SpO 2 and more prolonged hypoxia compared to shorter pauses. Hypoxic events are frequently accompanied by respiratory pauses and/or motion artefact. Real-time monitoring and input of respiratory waveform may thus improve the function of automated oxygen controllers, allowing pre-emptive responses to respiratory pauses. Furthermore, use of motion-resistant oximeters and plethysmographic waveform assessment procedures will help to optimise feedback control of inspired oxygen delivery.

Knockdown of miR-210 decreases hypoxic glioma stem cells stemness and radioresistance.

PubMed

Yang, Wei; Wei, Jing; Guo, Tiantian; Shen, Yueming; Liu, Fenju

2014-08-01

Glioma contains abundant hypoxic regions which provide niches to promote the maintenance and expansion of glioma stem cells (GSCs), which are resistant to conventional therapies and responsible for recurrence. Given the fact that miR-210 plays a vital role in cellular adaption to hypoxia and in stem cell survival and stemness maintenance, strategies correcting the aberrantly expressed miR-210 might open up a new therapeutic avenue to hypoxia GSCs. In the present study, to explore the possibility of miR-210 as an effective therapeutic target to hypoxic GSCs, we employed a lentiviral-mediated anti-sense miR-210 gene transfer technique to knockdown miR-210 expression and analyze phenotypic changes in hypoxic U87s and SHG44s cells. We found that hypoxia led to an increased HIF-2α mRNA expression and miR-210 expression in GSCs. Knockdown of miR-210 decreased neurosphere formation capacity, stem cell marker expression and cell viability, and induced differentiation and G0/G1 arrest in hypoxic GSCs by partially rescued Myc antagonist (MNT) protein expression. Knockdown of MNT could reverse the gene expression changes and the growth inhibition resulting from knockdown of miR-210 in hypoxic GSCs. Moreover, knockdown of miR-210 led to increased apoptotic rate and Caspase-3/7 activity and decreased invasive capacity, reactive oxygen species (ROS) and lactate production and radioresistance in hypoxic GSCs. These findings suggest that miR-210 might be a potential therapeutic target to eliminate GSCs located in hypoxic niches. Copyright © 2014 Elsevier Inc. All rights reserved.

[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. Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression: functional analysis of p62 and autophagy in hypoxic neuronal cells.

PubMed

Tanabe, Fumito; Yone, Kazunori; Kawabata, Naoya; Sakakima, Harutoshi; Matsuda, Fumiyo; Ishidou, Yasuhiro; Maeda, Shingo; Abematsu, Masahiko; Komiya, Setsuro; Setoguchi, Takao

2011-12-01

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients.

Co-delivery of siRNA and hypericin into cancer cells by hyaluronic acid modified PLGA-PEI nanoparticles.

PubMed

Li, Yanan; Zhang, Junling; Wang, Buhai; Shen, Yan; Ouahab, Ammar

2016-01-01

Malignant tumors cause more death because of the resistance of the hypoxic cancer cell toward radiotherapy. Targeting for hypoxic cancer area and gene silencing to overcome the hypoxia are two kinds of important therapeutic strategies for treating tumors. In order to explore the combined effects of gene therapy and hypericin (Hy) on tumor cells, hypoxia-inducible factor 1 alpha (HIF-1α) small interfering ribonucleic acid (siRNA) was transfected into the hypoxic human nasopharyngeal carcinoma (CNE2) cells using Hy-encapsulated nanocomplexes (Hy-HPP NPs) as a carrier which would achieve dual targeting to the tumor necrosis area. NPs were prepared by emulsion-diffusion-evaporation method. Formulations were evaluated by conducting in vitro physicochemical studies, electrophoresis, in vivo study, and biochemical studies. Hy-loaded nanoparticles with a mean size of around 160 nm was able to enhance the accumulation in the tumors by enhanced permeability and retention effect. The electrophoresis confirmed the good stability of siRNA/Hy-HPP NPs in the presence of phosphate-buffered saline (pH 7.4), competitive heparin, and RNase. The results of transfection showed that the uptake of siRNA was significantly increased up to 50% in CNE2 cells. The level of the HIF-1α with Hy-encapsulated nanocomplexes was significantly reduced to 30% in the transfected CNE2 cells. In vivo studies, the carrier exhibited higher intensity at the tumor tissue cells and higher affinity toward the necrotic tumor tissue. Results demonstrated that Hy-HPP NPs could significantly enhance the tranfection efficiency of siRNA, suggesting Hy-encapsulated nanoparticle as an efficient gene carrier. The co-delivery of HIF-1α siRNA (siHIF-1α) and Hy could efficiently decrease the level of HIF-1α and increase the affinity toward necrotic tissues. Hence, this is a promising strategy for further application in radiotherapy.

Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation.

PubMed

Castro-Prego, Raquel; Lamas-Maceiras, Mónica; Soengas, Pilar; Carneiro, Isabel; González-Siso, Isabel; Cerdán, M Esperanza

2009-12-14

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1-Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides -557 to -376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

Archaeal enrichment in the hypoxic zone in the northern Gulf of Mexico.

PubMed

Gillies, Lauren E; Thrash, J Cameron; deRada, Sergio; Rabalais, Nancy N; Mason, Olivia U

2015-10-01

Areas of low oxygen have spread exponentially over the past 40 years, and are cited as a key stressor on coastal ecosystems. The world's second largest coastal hypoxic (≤ 2 mg of O2 l(-1)) zone occurs annually in the northern Gulf of Mexico. The net effect of hypoxia is the diversion of energy flow away from higher trophic levels to microorganisms. This energy shunt is consequential to the overall productivity of hypoxic water masses and the ecosystem as a whole. In this study, water column samples were collected at 39 sites in the nGOM, 21 of which were hypoxic. Analysis of the microbial community along a hypoxic to oxic dissolved oxygen gradient revealed that the relative abundance (iTag) of Thaumarchaeota species 16S rRNA genes (> 40% of the microbial community in some hypoxic samples), the absolute abundance (quantitative polymerase chain reaction; qPCR) of Thaumarchaeota 16S rRNA genes and archaeal ammonia-monooxygenase gene copy number (qPCR) were significantly higher in hypoxic samples. Spatial interpolation of the microbial and chemical data revealed a continuous, shelfwide band of low dissolved oxygen waters that were dominated by Thaumarchaeota (and Euryarchaeota), amoA genes and high concentrations of phosphate in the nGOM, thus implicating physicochemical forcing on microbial abundance. © 2015 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

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

PubMed

Kast, Brigitte; Schori, Christian; Grimm, Christian

2016-05-01

Hypoxic preconditioning protects photoreceptors against light-induced degeneration preserving retinal morphology and function. Although hypoxia inducible transcription factors 1 and 2 (HIF1, HIF2) are the main regulators of the hypoxic response, photoreceptor protection does not depend on HIF1 in rods. Here we used rod-specific Hif2a single and Hif1a;Hif2a double knockout mice to investigate the potential involvement of HIF2 in rods for protection after hypoxic preconditioning. To identify potential HIF2 target genes in rods we determined the retinal transcriptome of hypoxic control and rod-specific Hif2a knockouts by RNA sequencing. We show that rods do not need HIF2 for hypoxia-induced increased survival after light exposure. The transcriptomic analysis revealed a number of genes that are potentially regulated by HIF2 in rods; among those were Htra1, Timp3 and Hmox1, candidates that are interesting due to their connection to human degenerative diseases of the retina. We conclude that neither HIF1 nor HIF2 are required in photoreceptors for protection by hypoxic preconditioning. We hypothesize that HIF transcription factors may be needed in other cells to produce protective factors acting in a paracrine fashion on photoreceptor cells. Alternatively, hypoxic preconditioning induces a rod-intrinsic response that is independent of HIF transcription factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

Least-cost control of agricultural nutrient contributions to the Gulf of Mexico hypoxic zone

USDA-ARS?s Scientific Manuscript database

In 2007, the hypoxic zone in the Gulf of Mexico, measuring 20,720 km**2, was one of the two largest reported since measurement of the zone began in 1985. The extent of the hypoxic zone is related to nitrogen and phosphorous loadings originating on agricultural fields in the upper Midwest. This stud...

[Effect of mexamine on the resistance of dogs to acute hypoxic hypoxia].

PubMed

Vasin, M V; Antipov, V V; Davydov, B I; Suvorov, N N

1975-01-01

As demonstrated in experiments staged on dogs mexamine hydrochloride, used in a dose of 20 mg/kg by the intraperiotoneal route 1.5 hours before the onset of acute hypoxic hypoxia increases the resistance of the organism to oxigen deficiency. Mexamine is capable of significantly intensity hypothermy in dogs during acute hypoxic hypoxia.

Efficacy of radiosensitizing doped titania nanoparticles under hypoxia and preparation of an embolic microparticle

PubMed Central

Morrison, Rachel A; Rybak-Smith, Malgorzata J; Thompson, James M; Thiebaut, Bénédicte; Hill, Mark A; Townley, Helen E

2017-01-01

The aim of this study was to develop a manufacturing protocol for large-scale production of doped titania radiosensitizing nanoparticles (NPs) to establish their activity under hypoxia and to produce a multimodal radiosensitizing embolic particle for cancer treatment. We have previously shown that radiosensitizing NPs can be synthesized from titania doped with rare earth elements, especially gadolinium. To translate this technology to the clinic, a crucial step is to find a suitable, scalable, high-throughput method. Herein, we have described the use of flame spray pyrolysis (FSP) to generate NPs from titanium and gadolinium precursors to produce titania NPs doped with 5 at% gadolinium. The NPs were fully characterized, and their capacity to act as radiosensitizers was confirmed by clonogenic assays. The integrity of the NPs in vitro was also ascertained due to the potentially adverse effects of free gadolinium in the body. The activity of the NPs was then studied under hypoxia since this is often a barrier to effective radiotherapy. In vitro radiosensitization experiments were performed with both the hypoxia mimetics deferoxamine and cobalt chloride and also under true hypoxia (oxygen concentration of 0.2%). It was shown that the radiosensitizing NPs were able to cause a significant increase in cell death even after irradiation under hypoxic conditions such as those found in tumors. Subsequently, the synthesized NPs were used to modify polystyrene embolization microparticles. The NPs were sintered to the surface of the microparticles by heating at 230°C for 15 minutes. This resulted in a good coverage of the surface and to generate embolization particles that were shown to be radiosensitizing. Such multimodal particles could therefore result in occlusion of the tumor blood vessels in conjunction with localized reactive oxygen species generation, even under hypoxic conditions such as those found in the center of tumors. PMID:28572729

Efficacy of radiosensitizing doped titania nanoparticles under hypoxia and preparation of an embolic microparticle.

PubMed

Morrison, Rachel A; Rybak-Smith, Malgorzata J; Thompson, James M; Thiebaut, Bénédicte; Hill, Mark A; Townley, Helen E

2017-01-01

The aim of this study was to develop a manufacturing protocol for large-scale production of doped titania radiosensitizing nanoparticles (NPs) to establish their activity under hypoxia and to produce a multimodal radiosensitizing embolic particle for cancer treatment. We have previously shown that radiosensitizing NPs can be synthesized from titania doped with rare earth elements, especially gadolinium. To translate this technology to the clinic, a crucial step is to find a suitable, scalable, high-throughput method. Herein, we have described the use of flame spray pyrolysis (FSP) to generate NPs from titanium and gadolinium precursors to produce titania NPs doped with 5 at% gadolinium. The NPs were fully characterized, and their capacity to act as radiosensitizers was confirmed by clonogenic assays. The integrity of the NPs in vitro was also ascertained due to the potentially adverse effects of free gadolinium in the body. The activity of the NPs was then studied under hypoxia since this is often a barrier to effective radiotherapy. In vitro radiosensitization experiments were performed with both the hypoxia mimetics deferoxamine and cobalt chloride and also under true hypoxia (oxygen concentration of 0.2%). It was shown that the radiosensitizing NPs were able to cause a significant increase in cell death even after irradiation under hypoxic conditions such as those found in tumors. Subsequently, the synthesized NPs were used to modify polystyrene embolization microparticles. The NPs were sintered to the surface of the microparticles by heating at 230°C for 15 minutes. This resulted in a good coverage of the surface and to generate embolization particles that were shown to be radiosensitizing. Such multimodal particles could therefore result in occlusion of the tumor blood vessels in conjunction with localized reactive oxygen species generation, even under hypoxic conditions such as those found in the center of tumors.

Energy metabolism in human melanoma cells under hypoxic and acidic conditions in vitro.

PubMed

Skøyum, R; Eide, K; Berg, K; Rofstad, E K

1997-01-01

The response to treatment and the malignant progression of tumours are influenced by the ability of the tumour cells to withstand severe energy deprivation during prolonged exposure to hypoxia at normal or low extracellular pH (pHe). The objective of the present work was to demonstrate intertumour heterogeneity under conditions of microenvironment-induced energy deprivation and to investigate whether the heterogeneity can be attributed to differences in the capacity of the tumour cells to generate energy in an oxygen-deficient microenvironment. Cultures of four human melanoma cell lines (BEX-c, COX-c, SAX-c, WIX-c) were exposed to hypoxia in vitro at pHe 7.4, 7.0 or 6.6 for times up to 31 h by using the steel-chamber method. High-performance liquid chromatography was used to assess adenylate energy charge as a function of exposure time. Cellular rates of glucose uptake and lactate release were determined by using standard enzymatic test kits. The adenylate energy charge decreased with time under hypoxia in all cell lines. The decrease was most pronounced shortly after the treatment had been initiated and then tapered off. BEX-c and SAX-c showed a significantly higher adenylate energy charge under hypoxic conditions than did COX-c and WIX-c whether the pHe was 7.4, 7.0 or 6.6, showing that tumours can differ in the ability to avoid energy deprivation during microenvironmental stress. There was no correlation between the adenylate energy charge and the rates of glucose uptake and lactate release. Intertumour heterogeneity in the ability to withstand energy deprivation in an oxygen-deficient microenvironment cannot therefore be attributed mainly to differences in the capacity of the tumour cells to generate energy by anaerobic metabolism. The data presented here suggest that the heterogeneity is rather caused by differences in the capacity of the tumour cells to reduce the rate of energy consumption when exposed to hypoxia.

Energy metabolism in human melanoma cells under hypoxic and acidic conditions in vitro.

PubMed Central

Skøyum, R.; Eide, K.; Berg, K.; Rofstad, E. K.

1997-01-01

The response to treatment and the malignant progression of tumours are influenced by the ability of the tumour cells to withstand severe energy deprivation during prolonged exposure to hypoxia at normal or low extracellular pH (pHe). The objective of the present work was to demonstrate intertumour heterogeneity under conditions of microenvironment-induced energy deprivation and to investigate whether the heterogeneity can be attributed to differences in the capacity of the tumour cells to generate energy in an oxygen-deficient microenvironment. Cultures of four human melanoma cell lines (BEX-c, COX-c, SAX-c, WIX-c) were exposed to hypoxia in vitro at pHe 7.4, 7.0 or 6.6 for times up to 31 h by using the steel-chamber method. High-performance liquid chromatography was used to assess adenylate energy charge as a function of exposure time. Cellular rates of glucose uptake and lactate release were determined by using standard enzymatic test kits. The adenylate energy charge decreased with time under hypoxia in all cell lines. The decrease was most pronounced shortly after the treatment had been initiated and then tapered off. BEX-c and SAX-c showed a significantly higher adenylate energy charge under hypoxic conditions than did COX-c and WIX-c whether the pHe was 7.4, 7.0 or 6.6, showing that tumours can differ in the ability to avoid energy deprivation during microenvironmental stress. There was no correlation between the adenylate energy charge and the rates of glucose uptake and lactate release. Intertumour heterogeneity in the ability to withstand energy deprivation in an oxygen-deficient microenvironment cannot therefore be attributed mainly to differences in the capacity of the tumour cells to generate energy by anaerobic metabolism. The data presented here suggest that the heterogeneity is rather caused by differences in the capacity of the tumour cells to reduce the rate of energy consumption when exposed to hypoxia. PMID:9275017

Preferential action of arsenic trioxide in solid-tumor microenvironment enhances radiation therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Griffin, Robert J.; Williams, Brent W.; Park, Heon Joo

2005-04-01

Purpose: To investigate the effect of arsenic trioxide, Trisenox (TNX), on primary cultures of endothelial cells and tumor tissue under varying pH and pO{sub 2} environments and the effects of combined TNX and radiation therapy on experimental tumors. Methods and Materials: Human dermal microvascular endothelial cells were cultured in vitro and exposed to TNX under various combinations of aerobic, hypoxic, neutral, or acidic conditions, and levels of activated JNK MAP kinase were assessed by Western blotting. FSaII fibrosarcoma cells grown in the hind limb of female C3H mice were used to study the effect of TNX on tumor blood perfusionmore » and oxygenation. The tumor-growth delay after a single or fractionated irradiation with or without TNX treatment was assessed. Results: A single intraperitoneal injection of 8 mg/kg TNX reduced the blood perfusion in FSaII tumors by 53% at 2 hours after injection. To increase the oxygenation of the tumor vasculature during TNX treatment, some animals were allowed to breathe carbogen (95% O{sub 2}/5% CO{sub 2}). Carbogen breathing alone for 2 hours reduced tumor perfusion by 33%. When carbogen breathing was begun immediately after TNX injection, no further reduction occurred in tumor blood perfusion at 2 hours after injection. In vitro, TNX exposure increased activity JNK MAP kinase preferentially in endothelial cells cultured in an acidic or hypoxic environment. In vivo, the median oxygenation in FSaII tumors measured at 3 or 5 days after TNX injection was found to be significantly elevated compared with control tumors. Subsequently, radiation-induced tumor-growth delay was synergistically increased when radiation and TNX injection were fractionated at 3-day or 5-day intervals. Conclusions: Trisenox has novel vascular-damaging properties, preferentially against endothelium in regions of low pH or pO{sub 2}, which leads to tumor cell death and enhancement of the response of tumors to radiotherapy.« less

Treatment of Neuroblastoma with an Engineered “Obligate” Anaerobic Salmonella typhimurium Strain YB1

PubMed Central

Ning, Bo-Tao; Yu, Bin; Chan, Shing; Chan, Jian-liang; Huang, Jian-Dong; Chan, Godfrey Chi-Fung

2017-01-01

Purpose Neuroblastoma is an embryonic solid tumor derived from the progenitors of the sympathetic nervous system. More than half of the patients developed metastatic disease at the time of initial diagnosis and had poor outcome with current therapeutic approaches. In recent years, some obligate and facultative anaerobic bacteria were reported to target the hypoxic and necrotic region of solid tumor models and caused tumor regression. We recently successfully constructed an “obligate” anaerobic Salmonella strain YB1 that was applied in breast cancer nude mice model by us. Here, we report the application of YB1 in neuroblastoma treatment. Methods The anti-cancer effect and side-effects of YB1 was examined in both in vitro and in vivo experiment. Previous established orthotopic neuroblastoma SCID/beige murine model using SK-NLP/luciferase cell line was adopted. Results In vitro, YB1 induced apoptosis for up to 31.4% of the neuroblastoma cells under anaerobic condition, three times more than that under aerobic condition (10.9%). The expression of both Toll like Receptor 4 and 5 (TLR4 and TLR5) in cancer cells were significantly up-regulated (p<0.05, p<0.01 respectively) after the treatment of YB1 under anaerobic condition. In mouse model, YB1 preferentially accumulated inside the core of the tumors, rather than in normal tissues as our previous reported. This is suggestive of the hypoxic nature of tumor core. Tumor growth was significantly retarded in YB1 treatment group (n=6, P<0.01). Furthermore, there was no long-term organ damage noted in all the organs examined including heart, lung, liver, spleen and brain in the YB1 treated mice. Conclusion The genetic modified Salmonella strain YB1 is a promising anti-tumor strategy against the tumor bulk for neuroblastoma. Future study can be extended to other common cancer types to verify the relative efficacy on different neoplastic cells. PMID:28775780

Priming of the Cells: Hypoxic Preconditioning for Stem Cell Therapy

PubMed Central

Wei, Zheng Z; Zhu, Yan-Bing; Zhang, James Y; McCrary, Myles R; Wang, Song; Zhang, Yong-Bo; Yu, Shan-Ping; Wei, Ling

2017-01-01

Objective: Stem cell-based therapies are promising in regenerative medicine for protecting and repairing damaged brain tissues after injury or in the context of chronic diseases. Hypoxia can induce physiological and pathological responses. A hypoxic insult might act as a double-edged sword, it induces cell death and brain damage, but on the other hand, sublethal hypoxia can trigger an adaptation response called hypoxic preconditioning or hypoxic tolerance that is of immense importance for the survival of cells and tissues. Data Sources: This review was based on articles published in PubMed databases up to August 16, 2017, with the following keywords: “stem cells,” “hypoxic preconditioning,” “ischemic preconditioning,” and “cell transplantation.” Study Selection: Original articles and critical reviews on the topics were selected. Results: Hypoxic preconditioning has been investigated as a primary endogenous protective mechanism and possible treatment against ischemic injuries. Many cellular and molecular mechanisms underlying the protective effects of hypoxic preconditioning have been identified. Conclusions: In cell transplantation therapy, hypoxic pretreatment of stem cells and neural progenitors markedly increases the survival and regenerative capabilities of these cells in the host environment, leading to enhanced therapeutic effects in various disease models. Regenerative treatments can mobilize endogenous stem cells for neurogenesis and angiogenesis in the adult brain. Furthermore, transplantation of stem cells/neural progenitors achieves therapeutic benefits via cell replacement and/or increased trophic support. Combinatorial approaches of cell-based therapy with additional strategies such as neuroprotective protocols, anti-inflammatory treatment, and rehabilitation therapy can significantly improve therapeutic benefits. In this review, we will discuss the recent progress regarding cell types and applications in regenerative medicine as well as future applications. PMID:28937044

Spatial differences of cellular origins and in vivo hypoxia modify contractile properties of pulmonary artery smooth muscle cells: lessons for arterial tissue engineering.

PubMed

Hall, S M; Soueid, A; Smith, T; Brown, R A; Haworth, S G; Mudera, V

2007-01-01

Tissue engineering of functional arteries is challenging. Within the pulmonary artery wall, smooth muscle cells (PASMCs) have site-specific developmental and functional phenotypes, reflecting differing contractile roles. The force generated by PASMCs isolated from the inner 25% and outer 50% of the media of intrapulmonary elastic arteries from five normal and eight chronically hypoxic (hypertensive) 14 day-old piglets was quantified in a three-dimensional (3D) collagen construct, using a culture force monitor. Outer medial PASMCs from normal piglets exerted more force (528 +/- 50 dynes) than those of hypoxic piglets (177 +/- 42 dynes; p < 0.01). Force generation by inner medial PASMCs from normal and hypoxic piglets was similar (349 +/- 35 and 239 +/- 60 dynes). In response to agonist (thromboxane) stimulation, all PASMCs from normal and hypoxic piglets contracted, but the increase in force generated by outer and inner hypoxic PASMCs (ranges 13-72 and 14-56 dynes) was less than by normal PASMCs (ranges 27-154 and 34-159 dynes, respectively; p < 0.05 for both). All hypoxic PASMCs were unresponsive to antagonist (sodium nitroprusside) stimulation, all normal PASMCs relaxed (range - 87 to - 494 dynes). Myosin heavy chain expression by both hypoxic PASMC phenotypes was less than normal (p < 0.05 for both), as was the activity of focal adhesion kinase, regulating contraction, in hypoxic inner PASMCs (p < 0.01). Chronic hypoxia resulted in the development of abnormal PASMC phenotypes, which in collagen constructs exhibited a reduction in contractile force and reactivity to agonists. Characterization of the mechanical response of spatially distinct cells and modification of their behaviour by hypoxia is critical for successful tissue engineering of major blood vessels.

Accumulation of p62 in degenerated spinal cord under chronic mechanical compression

PubMed Central

Tanabe, Fumito; Yone, Kazunori; Kawabata, Naoya; Sakakima, Harutoshi; Matsuda, Fumiyo; Ishidou, Yasuhiro; Maeda, Shingo; Abematsu, Masahiko; Komiya, Setsuro

2011-01-01

Intracellular accumulation of altered proteins, including p62 and ubiquitinated proteins, is the basis of most neurodegenerative disorders. The relationship among the accumulation of altered proteins, autophagy, and spinal cord dysfunction by cervical spondylotic myelopathy has not been clarified. We examined the expression of p62 and autophagy markers in the chronically compressed spinal cord of tiptoe-walking Yoshimura mice. In addition, we examined the expression and roles of p62 and autophagy in hypoxic neuronal cells. Western blot analysis showed the accumulation of p62, ubiquitinated proteins, and microtubule-associated protein 1 light chain 3 (LC3), an autophagic marker, in the compressed spinal cord. Immunohistochemical examinations showed that p62 accumulated in neurons, axons, astrocytes, and oligodendrocytes. Electron microscopy showed the expression of autophagy markers, including autolysosomes and autophagic vesicles, in the compressed spinal cord. These findings suggest the presence of p62 and autophagy in the degenerated compressed spinal cord. Hypoxic stress increased the expression of p62, ubiquitinated proteins, and LC3-II in neuronal cells. In addition, LC3 turnover assay and GFP-LC3 cleavage assay showed that hypoxic stress increased autophagy flux in neuronal cells. These findings suggest that hypoxic stress induces accumulation of p62 and autophagy in neuronal cells. The forced expression of p62 decreased the number of neuronal cells under hypoxic stress. These findings suggest that p62 accumulation under hypoxic stress promotes neuronal cell death. Treatment with 3-methyladenine, an autophagy inhibitor decreased the number of neuronal cells, whereas lithium chloride, an autophagy inducer increased the number of cells under hypoxic stress. These findings suggest that autophagy promotes neuronal cell survival under hypoxic stress. Our findings suggest that pharmacological inducers of autophagy may be useful for treating cervical spondylotic myelopathy patients. PMID:22082874

Responses to GABA(A) receptor activation are altered in NTS neurons isolated from chronic hypoxic rats.

PubMed

Tolstykh, Gleb; Belugin, Sergei; Mifflin, Steve

2004-04-23

The inhibitory amino acid GABA is released within the nucleus of the solitary tract (NTS) during hypoxia and modulates the respiratory response to hypoxia. To determine if responses of NTS neurons to activation of GABA(A) receptors are altered following exposure to chronic hypoxia, GABA(A) receptor-evoked whole cell currents were measured in enzymatically dispersed NTS neurons from normoxic and chronic hypoxic rats. Chronic hypoxic rats were exposed to 10% O(2) for 9-12 days. Membrane capacitance was the same in neurons from normoxic (6.9+/-0.5 pF, n=16) and hypoxic (6.3+/-0.5 pF, n=15) rats. The EC(50) for peak GABA-evoked current density was significantly greater in neurons from hypoxic (21.7+/-2.2 microM) compared to normoxic rats (12.2+/-0.9 microM) (p<0.001). Peak and 5-s adapted GABA currents evoked by 1, 3 and 10 microM were greater in neurons from normoxic compared to hypoxic rats (p<0.05) whereas peak and 5-s adapted responses to 30 and 100 microM GABA were not different comparing normoxic to hypoxic rats. Desensitization of GABA(A)-evoked currents was observed at concentrations greater than 3 microM and, measured as the ratio of the current 5 s after the onset of 100 microM GABA application to the peak GABA current, was the same in neurons from normoxic (0.37+/-0.03) and hypoxic rats (0.33+/-0.04). Reduced sensitivity to GABA(A) receptor-evoked inhibition in chronic hypoxia could influence chemoreceptor afferent integration by NTS neurons.

Curved reformat of the paediatric brain MRI into a 'flat-earth map' - standardised method for demonstrating cortical surface atrophy resulting from hypoxic-ischaemic encephalopathy.

PubMed

Simpson, Ewan; Andronikou, Savvas; Vedajallam, Schadie; Chacko, Anith; Thai, Ngoc Jade

2016-09-01

Hypoxic-ischaemic encephalopathy is optimally imaged with brain MRI in the neonatal period. However neuroimaging is often also performed later in childhood (e.g., when parents seek compensation in cases of alleged birth asphyxia). We describe a standardised technique for creating two curved reconstructions of the cortical surface to show the characteristic surface changes of hypoxic-ischaemic encephalopathy in children imaged after the neonatal period. The technique was applied for 10 cases of hypoxic-ischaemic encephalopathy and also for age-matched healthy children to assess the visibility of characteristic features of hypoxic-ischaemic encephalopathy. In the abnormal brains, fissural or sulcal widening was seen in all cases and ulegyria was identifiable in 7/10. These images could be used as a visual aid for communicating MRI findings to clinicians and other interested parties.

Serine protease activity contributes to control of Mycobacterium tuberculosis in hypoxic lung granulomas in mice

PubMed Central

Reece, Stephen T.; Loddenkemper, Christoph; Askew, David J.; Zedler, Ulrike; Schommer-Leitner, Sandra; Stein, Maik; Mir, Fayaz Ahmad; Dorhoi, Anca; Mollenkopf, Hans-Joachim; Silverman, Gary A.; Kaufmann, Stefan H.E.

2010-01-01

The hallmark of human Mycobacterium tuberculosis infection is the presence of lung granulomas. Lung granulomas can have different phenotypes, with caseous necrosis and hypoxia present within these structures during active tuberculosis. Production of NO by the inducible host enzyme NOS2 is a key antimycobacterial defense mechanism that requires oxygen as a substrate; it is therefore likely to perform inefficiently in hypoxic regions of granulomas in which M. tuberculosis persists. Here we have used Nos2–/– mice to investigate host-protective mechanisms within hypoxic granulomas and identified a role for host serine proteases in hypoxic granulomas in determining outcome of disease. Nos2–/– mice reproduced human-like granulomas in the lung when infected with M. tuberculosis in the ear dermis. The granulomas were hypoxic and contained large amounts of the serine protease cathepsin G and clade B serine protease inhibitors (serpins). Extrinsic inhibition of serine protease activity in vivo resulted in distorted granuloma structure, extensive hypoxia, and increased bacterial growth in this model. These data suggest that serine protease activity acts as a protective mechanism within hypoxic regions of lung granulomas and present a potential new strategy for the treatment of tuberculosis. PMID:20679732

The Hog1 Mitogen-Activated Protein Kinase Mediates a Hypoxic Response in Saccharomyces cerevisiae

PubMed Central

Hickman, Mark J.; Spatt, Dan; Winston, Fred

2011-01-01

We have studied hypoxic induction of transcription by studying the seripauperin (PAU) genes of Saccharomyces cerevisiae. Previous studies showed that PAU induction requires the depletion of heme and is dependent upon the transcription factor Upc2. We have now identified additional factors required for PAU induction during hypoxia, including Hog1, a mitogen-activated protein kinase (MAPK) whose signaling pathway originates at the membrane. Our results have led to a model in which heme and ergosterol depletion alters membrane fluidity, thereby activating Hog1 for hypoxic induction. Hypoxic activation of Hog1 is distinct from its previously characterized response to osmotic stress, as the two conditions cause different transcriptional consequences. Furthermore, Hog1-dependent hypoxic activation is independent of the S. cerevisiae general stress response. In addition to Hog1, specific components of the SAGA coactivator complex, including Spt20 and Sgf73, are also required for PAU induction. Interestingly, the mammalian ortholog of Spt20, p38IP, has been previously shown to interact with the mammalian ortholog of Hog1, p38. Taken together, our results have uncovered a previously unknown hypoxic-response pathway that may be conserved throughout eukaryotes. PMID:21467572

Medicinal electronomics bricolage design of hypoxia-targeting antineoplastic drugs and invention of boron tracedrugs as innovative future-architectural drugs.

PubMed

Hori, Hitoshi; Uto, Yoshihiro; Nakata, Eiji

2010-09-01

We describe herein for the first time our medicinal electronomics bricolage design of hypoxia-targeting antineoplastic drugs and boron tracedrugs as newly emerging drug classes. A new area of antineoplastic drugs and treatments has recently focused on neoplastic cells of the tumor environment/microenvironment involving accessory cells. This tumor hypoxic environment is now considered as a major factor that influences not only the response to antineoplastic therapies but also the potential for malignant progression and metastasis. We review our medicinal electronomics bricolage design of hypoxia-targeting drugs, antiangiogenic hypoxic cell radiosensitizers, sugar-hybrid hypoxic cell radiosensitizers, and hypoxia-targeting 10B delivery agents, in which we design drug candidates based on their electronic structures obtained by molecular orbital calculations, not based solely on pharmacophore development. These drugs include an antiangiogenic hypoxic cell radiosensitizer TX-2036, a sugar-hybrid hypoxic cell radiosensitizer TX-2244, new hypoxia-targeting indoleamine 2,3-dioxygenase (IDO) inhibitors, and a hypoxia-targeting BNCT agent, BSH (sodium borocaptate-10B)-hypoxic cytotoxin tirapazamine (TPZ) hybrid drug TX-2100. We then discuss the concept of boron tracedrugs as a new drug class having broad potential in many areas.

A Low Protein Diet Increases the Hypoxic Tolerance in Drosophila

PubMed Central

Vigne, Paul; Frelin, Christian

2006-01-01

Dietary restriction is well known to increase the life span of a variety of organisms from yeast to mammals, but the relationships between nutrition and the hypoxic tolerance have not yet been considered. Hypoxia is a major cause of cell death in myocardial infarction and stroke. Here we forced hypoxia-related death by exposing one-day-old male Drosophila to chronic hypoxia (5% O2) and analysed their survival. Chronic hypoxia reduced the average life span from 33.6 days to 6.3 days when flies were fed on a rich diet. A demographic analysis indicated that chronic hypoxia increased the slope of the mortality trajectory and not the short-term risk of death. Dietary restriction produced by food dilution, by yeast restriction, or by amino acid restriction partially reversed the deleterious action of hypoxia. It increased the life span of hypoxic flies up to seven days, which represented about 25% of the life time of an hypoxic fly. Maximum survival of hypoxic flies required only dietary sucrose, and it was insensitive to drugs such as rapamycin and resveratrol, which increase longevity of normoxic animals. The results thus uncover a new link between protein nutrition, nutrient signalling, and resistance to hypoxic stresses. PMID:17183686

CD44 Interacts with HIF-2α to Modulate the Hypoxic Phenotype of Perinecrotic and Perivascular Glioma Cells.

PubMed

Johansson, Elinn; Grassi, Elisa S; Pantazopoulou, Vasiliki; Tong, Bei; Lindgren, David; Berg, Tracy J; Pietras, Elin J; Axelson, Håkan; Pietras, Alexander

2017-08-15

Hypoxia-inducible factors enhance glioma stemness, and glioma stem cells have an amplified hypoxic response despite residing within a perivascular niche. Still, little is known about differential HIF regulation in stem versus bulk glioma cells. We show that the intracellular domain of stem cell marker CD44 (CD44ICD) is released at hypoxia, binds HIF-2α (but not HIF-1α), enhances HIF target gene activation, and is required for hypoxia-induced stemness in glioma. In a glioma mouse model, CD44 was restricted to hypoxic and perivascular tumor regions, and in human glioma, a hypoxia signature correlated with CD44. The CD44ICD was sufficient to induce hypoxic signaling at perivascular oxygen tensions, and blocking CD44 cleavage decreased HIF-2α stabilization in CD44-expressing cells. Our data indicate that the stem cell marker CD44 modulates the hypoxic response of glioma cells and that the pseudo-hypoxic phenotype of stem-like glioma cells is achieved by stabilization of HIF-2α through interaction with CD44, independently of oxygen. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Panicucci, R.; Heal, R.; Laderoute, K.

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 ismore » 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.« less

PI3K/Akt contributes to increased expression of Toll-like receptor 4 in macrophages exposed to hypoxic stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, So Young; Jeong, Eunshil; Joung, Sun Myung

2012-03-16

Highlights: Black-Right-Pointing-Pointer Hypoxic stress-induced TLR4 expression is mediated by PI3K/Akt in macrophages. Black-Right-Pointing-Pointer PI3K/Akt regulated HIF-1 activation leading to TLR4 expression. Black-Right-Pointing-Pointer p38 mitogen-activated protein kinase was not involved in TLR4 expression by hypoxic stress. Black-Right-Pointing-Pointer Sulforaphane suppressed hypoxia-mediated TLR4 expression by inhibiting PI3K/Akt. -- Abstract: Toll-like receptors (TLRs) play critical roles in triggering immune and inflammatory responses by detecting invading microbial pathogens and endogenous danger signals. Increased expression of TLR4 is implicated in aggravated inflammatory symptoms in ischemic tissue injury and chronic diseases. Results from our previous study showed that TLR4 expression was upregulated by hypoxic stress mediated bymore » hypoxia-inducible factor-1 (HIF-1) at a transcriptional level in macrophages. In this study, we further investigated the upstream signaling pathway that contributed to the increase of TLR4 expression by hypoxic stress. Either treatment with pharmacological inhibitors of PI3K and Akt or knockdown of Akt expression by siRNA blocked the increase of TLR4 mRNA and protein levels in macrophages exposed to hypoxia and CoCl{sub 2}. Phosphorylation of Akt by hypoxic stress preceded nuclear accumulation of HIF-1{alpha}. A PI3K inhibitor (LY294002) attenuated CoCl{sub 2}-induced nuclear accumulation and transcriptional activation of HIF-1{alpha}. In addition, HIF-1{alpha}-mediated upregulation of TLR4 expression was blocked by LY294002. Furthermore, sulforaphane suppressed hypoxia- and CoCl{sub 2}-induced upregulation of TLR4 mRNA and protein by inhibiting PI3K/Akt activation and the subsequent nuclear accumulation and transcriptional activation of HIF-1{alpha}. However, p38 was not involved in HIF-1{alpha} activation and TLR4 expression induced by hypoxic stress in macrophages. Collectively, our results demonstrate that PI3K/Akt contributes to hypoxic stress-induced TLR4 expression at least partly through the regulation of HIF-1 activation. These reveal a novel mechanism for regulation of TLR4 expression upon hypoxic stress and provide a therapeutic target for chronic diseases related to hypoxic stress.« less

Regulation of monocarboxylate transporter MCT1 expression by p53 mediates inward and outward lactate fluxes in tumors.

PubMed

Boidot, Romain; Végran, Frédérique; Meulle, Aline; Le Breton, Aude; Dessy, Chantal; Sonveaux, Pierre; Lizard-Nacol, Sarab; Feron, Olivier

2012-02-15

The monocarboxylate transporter (MCT) family member MCT1 can transport lactate into and out of tumor cells. Whereas most oxidative cancer cells import lactate through MCT1 to fuel mitochondrial respiration, the role of MCT1 in glycolysis-derived lactate efflux remains less clear. In this study, we identified a direct link between p53 function and MCT1 expression. Under hypoxic conditions, p53 loss promoted MCT1 expression and lactate export produced by elevated glycolytic flux, both in vitro and in vivo. p53 interacted directly with the MCT1 gene promoter and altered MCT1 mRNA stabilization. In hypoxic p53(-/-) tumor cells, NF-κB further supported expression of MCT1 to elevate its levels. Following glucose deprivation, upregulated MCT1 in p53(-/-) cells promoted lactate import and favored cell proliferation by fuelling mitochondrial respiration. We also found that MCT1 expression was increased in human breast tumors harboring p53 mutations and coincident features of hypoxia, with higher MCT1 levels associated with poorer clinical outcomes. Together, our findings identify MCT1 as a target for p53 repression and they suggest that MCT1 elevation in p53-deficient tumors allows them to adapt to metabolic needs by facilitating lactate export or import depending on the glucose availability.

Contrasting dynamic responses in vivo of the Bcl-xL and Bim erythropoietic survival pathways

PubMed Central

Koulnis, Miroslav; Porpiglia, Ermelinda; Porpiglia, P. Alberto; Liu, Ying; Hallstrom, Kelly; Hidalgo, Daniel

2012-01-01

Survival signaling by the erythropoietin (Epo) receptor (EpoR) is essential for erythropoiesis and for its acceleration in hypoxic stress. Several apparently redundant EpoR survival pathways were identified in vitro, raising the possibility of their functional specialization in vivo. Here we used mouse models of acute and chronic stress, including a hypoxic environment and β-thalassemia, to identify two markedly different response dynamics for two erythroblast survival pathways in vivo. Induction of the antiapoptotic protein Bcl-xL is rapid but transient, while suppression of the proapoptotic protein Bim is slower but persistent. Similar to sensory adaptation, however, the Bcl-xL pathway “resets,” allowing it to respond afresh to acute stress superimposed on a chronic stress stimulus. Using “knock-in” mouse models expressing mutant EpoRs, we found that adaptation in the Bcl-xL response occurs because of adaptation of its upstream regulator Stat5, both requiring the EpoR distal cytoplasmic domain. We conclude that survival pathways show previously unsuspected functional specialization for the acute and chronic phases of the stress response. Bcl-xL induction provides a “stop-gap” in acute stress, until slower but permanent pathways are activated. Furthermore, pathologic elevation of Bcl-xL may be the result of impaired adaptation, with implications for myeloproliferative disease mechanisms. PMID:22086418

Hypoxia-inducible vascular endothelial growth factor gene therapy using the oxygen-dependent degradation domain in myocardial ischemia.

PubMed

Kim, Hyun Ah; Lim, Soyeon; Moon, Hyung-Ho; Kim, Sung Wan; Hwang, Ki-Chul; Lee, Minhyung; Kim, Sun Hwa; Choi, Donghoon

2010-10-01

A hypoxia-inducible VEGF expression system with the oxygen-dependent degradation (ODD) domain was constructed and tested to be used in gene therapy for ischemic myocardial disease. Luciferase and VEGF expression vector systems were constructed with or without the ODD domain: pEpo-SV-Luc (or pEpo-SV-VEGF) and pEpo-SV-Luc-ODD (or pEpo-SV-VEGF-ODD). In vitro gene expression efficiency of each vector type was evaluated in HEK 293 cells under both hypoxic and normoxic conditions. The amount of VEGF protein was estimated by ELISA. The VEGF expression vectors with or without the ODD domain were injected into ischemic rat myocardium. Fibrosis, neovascularization, and cardiomyocyte apoptosis were assessed using Masson's trichrome staining, α-smooth muscle actin (α-SMA) immunostaining, and the TUNEL assay, respectively. The plasmid vectors containing ODD significantly improved the expression level of VEGF protein in hypoxic conditions. The enhancement of VEGF protein production was attributed to increased protein stability due to oxygen deficiency. In a rat model of myocardial ischemia, the pEpo-SV-VEGF-ODD group exhibited less myocardial fibrosis, higher microvessel density, and less cardiomyocyte apoptosis compared to the control groups (saline and pEpo-SV-VEGF treatments). An ODD-mediated VEGF expression system that facilitates VEGF-production under hypoxia may be useful in the treatment of ischemic heart disease.

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

PubMed

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

2016-01-14

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

Defined xenogeneic-free and hypoxic environment provides superior conditions for long-term expansion of human adipose-derived stem cells.

PubMed

Yang, Sufang; Pilgaard, Linda; Chase, Lucas G; Boucher, Shayne; Vemuri, Mohan C; Fink, Trine; Zachar, Vladimir

2012-08-01

Development and implementation of therapeutic protocols based on stem cells or tissue-engineered products relies on methods that enable the production of substantial numbers of cells while complying with stringent quality and safety demands. In the current study, we aimed to assess the benefits of maintaining cultures of adipose-derived stem cells (ASCs) in a defined culture system devoid of xenogeneic components (xeno-free) and hypoxia over a 49-day growth period. Our data provide evidence that conditions involving StemPro mesenchymal stem cells serum-free medium (SFM) Xeno-Free and hypoxia (5% oxygen concentration) in the culture atmosphere provide a superior proliferation rate compared to a standard growth environment comprised of alpha-modified Eagle medium (A-MEM) supplemented with fetal calf serum (FCS) and ambient air (20% oxygen concentration) or that of A-MEM supplemented with FCS and hypoxia. Furthermore, a flow cytometric analysis and in vitro differentiation assays confirmed the immunophenotype stability and maintained multipotency of ASCs when expanded under xeno-free conditions and hypoxia. In conclusion, our data demonstrate that growth conditions utilizing a xeno-free and hypoxic environment not only provide an improved environment for the expansion of ASCs, but also set the stage as a culture system with the potential broad spectrum utility for regenerative medicine and tissue engineering applications.

Monocarboxylate transporter-dependent mechanism confers resistance to oxygen- and glucose-deprivation injury in astrocyte-neuron co-cultures.

PubMed

Gao, Chen; Zhou, Liya; Zhu, Wenxia; Wang, Hongyun; Wang, Ruijuan; He, Yunfei; Li, Zhiyun

2015-05-06

Hypoxic and low-glucose stressors contribute to neuronal death in many brain diseases. Astrocytes are anatomically well-positioned to shield neurons from hypoxic injury. During hypoxia/ischemia, lactate released from astrocytes is taken up by neurons and stored for energy. This process is mediated by monocarboxylate transporters (MCTs) in the central nervous system. In the present study, we investigated the ability of astrocytes to protect neurons from oxygen- and glucose-deprivation (OGD) injury via an MCT-dependent mechanism in vitro. Primary cultures of neurons, astrocytes, and astrocytes-neurons derived from rat hippocampus were subjected to OGD, MCT inhibition with small interfering (si)RNA. Cell survival and expression of MCT4, MCT2, glial fibrillary acidic protein, and neuronal nuclear antigen were evaluated. OGD significantly increased cell death in neuronal cultures and up-regulated MCT4 expression in astrocyte cultures, but no increased cell death was observed in neuron-astrocyte co-cultures or astrocyte cultures. However, neuronal cell death in co-cultures was increased by exposure to MCT4- or MCT2-specific siRNA, and this effect was attenuated by the addition of lactate into the extracellular medium of neuronal cultures prior to OGD. These findings demonstrate that resistance to OGD injury in astrocyte-neuron co-cultures occurs via an MCT-dependent mechanism. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss.

PubMed

Zahoor, Muhammad; Westhrin, Marita; Aass, Kristin Roseth; Moen, Siv Helen; Misund, Kristine; Psonka-Antonczyk, Katarzyna Maria; Giliberto, Mariaserena; Buene, Glenn; Sundan, Anders; Waage, Anders; Sponaas, Anne-Marit; Standal, Therese

2017-12-26

Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α-dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients.

Hypoxia promotes IL-32 expression in myeloma cells, and high expression is associated with poor survival and bone loss

PubMed Central

Zahoor, Muhammad; Aass, Kristin Roseth; Moen, Siv Helen; Misund, Kristine; Psonka-Antonczyk, Katarzyna Maria; Giliberto, Mariaserena; Buene, Glenn; Sundan, Anders; Waage, Anders; Sponaas, Anne-Marit

2017-01-01

Multiple myeloma (MM) is a hematologic cancer characterized by expansion of malignant plasma cells in the bone marrow. Most patients develop an osteolytic bone disease, largely caused by increased osteoclastogenesis. The myeloma bone marrow is hypoxic, and hypoxia may contribute to MM disease progression, including bone loss. Here we identified interleukin-32 (IL-32) as a novel inflammatory cytokine expressed by a subset of primary MM cells and MM cell lines. We found that high IL-32 gene expression in plasma cells correlated with inferior survival in MM and that IL-32 gene expression was higher in patients with bone disease compared with those without. IL-32 was secreted from MM cells in extracellular vesicles (EVs), and those EVs, as well as recombinant human IL-32, promoted osteoclast differentiation both in vitro and in vivo. The osteoclast-promoting activity of the EVs was IL-32 dependent. Hypoxia increased plasma-cell IL-32 messenger RNA and protein levels in a hypoxia-inducible factor 1α–dependent manner, and high expression of IL-32 was associated with a hypoxic signature in patient samples, suggesting that hypoxia may promote expression of IL-32 in MM cells. Taken together, our results indicate that targeting IL-32 might be beneficial in the treatment of MM bone disease in a subset of patients. PMID:29296919

Creatine and creatine pyruvate reduce hypoxia-induced effects on phrenic nerve activity in the juvenile mouse respiratory system.

PubMed

Scheer, Monika; Bischoff, Anna M; Kruzliak, Peter; Opatrilova, Radka; Bovell, Douglas; Büsselberg, Dietrich

2016-08-01

Adequate concentrations of ATP are required to preserve physiological cell functions and protect tissue from hypoxic damage. Decreased oxygen concentration results in ATP synthesis relying increasingly on the presence of phosphocreatine. The lack of ATP through hypoxic insult to neurons that generate or regulate respiratory function, would lead to the cessation of breathing (apnea). It is not clear whether creatine plays a role in maintaining respiratory phrenic nerve (PN) activity during hypoxic challenge. The aim of the study was to test the effects of exogenously applied creatine or creatine pyruvate in maintaining PN induced respiratory rhythm against the deleterious effects of severe hypoxic insult using Working Heart-Brainstem (WHB) preparations of juvenile Swiss type mice. WHB's were perfused with control perfusate or perfusate containing either creatine [100μM] or creatine pyruvate [100μM] prior to hypoxic challenge and PN activity recorded throughout. Results showed that severe hypoxic challenge resulted in an initial transient increase in PN activity, followed by a reduction in that activity leading to respiratory apnea. The results demonstrated that perfusing the WHB preparation with creatine or creatine pyruvate, significantly reduced the onset of apnea compared to control conditions, with creatine pyruvate being the more effective substance. Overall, creatine and creatine pyruvate each produced time-dependent degrees of protection against severe hypoxic-induced disturbances of PN activity. The underlying protective mechanisms are unknown and need further investigations. Copyright © 2016 Elsevier Inc. All rights reserved.

Cardiovascular development in embryos of the American alligator Alligator mississippiensis: effects of chronic and acute hypoxia.

PubMed

Crossley, Dane A; Altimiras, Jordi

2005-01-01

Chronic hypoxic incubation is a common tool used to address the plasticity of morphological and physiological characteristics during vertebrate development. In this study chronic hypoxic incubation of embryonic American alligators resulted in both morphological (mass) and physiological changes. During normoxic incubation embryonic mass, liver mass and heart mass increased throughout the period of study, while yolk mass fell. Chronic hypoxia (10%O2) resulted in a reduced embryonic mass at 80% and 90% of incubation. This reduction in embryonic mass was accompanied by a relative enlargement of the heart at 80% and 90% of incubation, while relative embryonic liver mass was similar to the normoxic group. Normoxic incubated alligators maintained a constant heart rate during the period of study, while mean arterial pressure rose continuously. Both levels of hypoxic incubation (15% and 10%O2) resulted in a lower mean arterial pressure at 90% of incubation, while heart rate was lower in the 10%O2 group only. Acute (5 min) exposure to 10%O2 in the normoxic group resulted in a biphasic response, with a normotensive bradycardia occurring during the period of exposure and a hypertensive tachycardic response occurring during recovery. The embryos incubated under hypoxia also showed a blunted response to acute hypoxic stress. In conclusion, the main responses elicited by chronic hypoxic incubation, namely, cardiac enlargement, blunted hypoxic response and systemic vasodilation, may provide chronically hypoxic embryos with a new physiological repertoire for responding to hypoxia.

Hypoxia enhances the interaction between pancreatic stellate cells and cancer cells via increased secretion of connective tissue growth factor.

PubMed

Eguchi, Daiki; Ikenaga, Naoki; Ohuchida, Kenoki; Kozono, Shingo; Cui, Lin; Fujiwara, Kenji; Fujino, Minoru; Ohtsuka, Takao; Mizumoto, Kazuhiro; Tanaka, Masao

2013-05-01

Pancreatic cancer (PC), a hypovascular tumor, thrives under hypoxic conditions. Pancreatic stellate cells (PSCs) promote PC progression by secreting soluble factors, but their functions in hypoxia are poorly understood. This study aimed to clarify the effects of hypoxic conditions on the interaction between PC cells and PSCs. We isolated human PSCs from fresh pancreatic ductal adenocarcinomas and analyzed functional differences in PSCs between normoxia (21% O2) and hypoxia (1% O2), including expression of various factors related to tumor-stromal interactions. We particularly analyzed effects on PC invasiveness of an overexpressed molecule-connective tissue growth factor (CTGF)-in PSCs under hypoxic conditions, using RNA interference techniques. Conditioned media from hypoxic PSCs enhanced PC cell invasiveness more intensely than that from normoxic PSCs (P < 0.01). When co-cultured with PSCs, PC cell invasion was more enhanced under hypoxia than under normoxia (P < 0.05). Among various soluble factors, which were related to invasiveness, CTGF was one of the overexpressed molecules in hypoxic PSCs. A higher level of CTGF expression was also found in supernatant of hypoxic PSCs than in supernatant of normoxic PSCs. PC cell invasiveness was reduced by CTGF knockdown in hypoxic PSCs co-cultured with PC cells (P < 0.05). Hypoxia induces PSCs' secretion of CTGF, leading to enhancement of PC invasiveness. CTGF derived from hypoxia-stimulated PSCs may be a new therapeutic target for pancreatic cancer. Copyright © 2013 Elsevier Inc. All rights reserved.

Determination of concentration and molar absorptivity of hypochlorous acid and hypobromous acid species by hydrogen peroxide titration

NASA Astrophysics Data System (ADS)

Uehara, H.; Arakaki, T.

2017-12-01

Hypochlorous acid and hypobromous acid (abbreviated as "HypoX acids") are the main ingredients of bleaching and bactericides. The HypoX acids change their chemical forms depending on environmental factors such as pH and various chemical reactions. For example, it has been reported that hypobromite ion in water changes to carcinogenic bromate by photochemical reaction with ultraviolet light. In this study, concentrations of HypoX acids were determined by UV-VIS absorbance measurement utilizing the fact that HypoX acids react with hydrogen peroxide and do not co-exist in the solution. The method for determining the concentration by titration with hydrogen peroxide can be carried out simpler and more efficiently than the DPD method or the current titration method generally used for chlorine concentration measurement. Molar absorptivity between 250 - 500 nm of HypoX acids, including their conjugate base species, was determined by solving theoretical acid-base formula including molar fraction of each chemical species at various pHs. Molar absorptivity of OCl- and OBr- between 250 - 500 nm was determined based on the concentrations obtained from titration with hydrogen peroxide and absorbance at pH > 10, where OCl- and OBr- dominate. Furthermore, the HypoX acids solutions were irradiated with a solar simulator, and the photolysis rate constants were obtained. Based on those values, the half-lives were calculated and the behavior of HypoX acids in the environment was elucidated.

Metabolic adaptation to chronic hypoxia in cardiac mitochondria.

PubMed

Heather, Lisa C; Cole, Mark A; Tan, Jun-Jie; Ambrose, Lucy J A; Pope, Simon; Abd-Jamil, Amira H; Carter, Emma E; Dodd, Michael S; Yeoh, Kar Kheng; Schofield, Christopher J; Clarke, Kieran

2012-05-01

Chronic hypoxia decreases cardiomyocyte respiration, yet the mitochondrial mechanisms remain largely unknown. We investigated the mitochondrial metabolic pathways and enzymes that were decreased following in vivo hypoxia, and questioned whether hypoxic adaptation was protective for the mitochondria. Wistar rats were housed in hypoxia (7 days acclimatisation and 14 days at 11% oxygen), while control rats were housed in normoxia. Chronic exposure to physiological hypoxia increased haematocrit and cardiac vascular endothelial growth factor, in the absence of weight loss and changes in cardiac mass. In both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria isolated from hypoxic hearts, state 3 respiration rates with fatty acid were decreased by 17-18%, and with pyruvate were decreased by 29-15%, respectively. State 3 respiration rates with electron transport chain (ETC) substrates were decreased only in hypoxic SSM, not in hypoxic IFM. SSM from hypoxic hearts had decreased activities of ETC complexes I, II and IV, which were associated with decreased reactive oxygen species generation and protection against mitochondrial permeability transition pore (MPTP) opening. In contrast, IFM from hypoxic hearts had decreased activity of the Krebs cycle enzyme, aconitase, which did not modify ROS production or MPTP opening. In conclusion, cardiac mitochondrial respiration was decreased following chronic hypoxia, associated with downregulation of different pathways in the two mitochondrial populations, determined by their subcellular location. Hypoxic adaptation was not deleterious for the mitochondria, in fact, SSM acquired increased protection against oxidative damage under the oxygen-limited conditions.

[Adaptation to hypoxia and hyperoxia improves physical endurance: the role of reactive oxygen species and redox-signaling].

PubMed

Sazontova, T G; Glazachev, O S; Bolotova, A V; Dudnik, E N; Striapko, N V; Bedareva, I V; Anchishkina, N A; Arkhipenko, Iu V

2012-06-01

We have conducted theoretical foundation, experimental analysis and a pilot study of a new method of adaptation to hypoxia and hyperoxia in the prevention of hypoxic and stress-induced disorders and improving the body's tolerance to physical stress. It has been shown in the experimental part that a combination of physical exercise with adaptation to hypoxia-hyperoxia significantly increased tolerance to acute physical load (APL) and its active phase. Analysis of lipid peroxidation processes, antioxidant enzymes and HSPs showed that short-term training for physical exercise by itself compensates the stressor, but not the hypoxic component of the APL, the combination of training with adaptation to hypoxia-hyperoxia completely normalizes the stressor and hypoxic components of APL. The pilot study has been performed to evaluate the effectiveness of hypoxic-hyperoxic training course in qualified young athletes with over-training syndrome. After completing the course of hypoxia-hyperoxia adaptation, 14 sessions, accompanied by light mode sports training, the athletes set the normalization of autonomic balance, increased resistance to acute hypoxia in hypoxic test, increased physical performance--increased PWC170, maximal oxygen consumption (VO2max) parameters, their relative values to body mass, diminished shift of rate pressure product in the load. Thus, we confirmed experimental findings that hypoxic-hyperoxic training optimizes hypoxic (increased athletes resistance to proper hypoxia) and stress (myocardium economy in acute physical stress testing) components in systemic adaptation and restoration of athletes' with over-training syndrome.

Visualization of the Charcoal Agar Resazurin Assay for Semi-quantitative, Medium-throughput Enumeration of Mycobacteria.

PubMed

Gold, Ben; Roberts, Julia; Ling, Yan; Lopez Quezada, Landys; Glasheen, Jou; Ballinger, Elaine; Somersan-Karakaya, Selin; Warrier, Thulasi; Nathan, Carl

2016-12-14

There is an urgent need to discover and progress anti-infectives that shorten the duration of tuberculosis (TB) treatment. Mycobacterium tuberculosis, the etiological agent of TB, is refractory to rapid and lasting chemotherapy due to the presence of bacilli exhibiting phenotypic drug resistance. The charcoal agar resazurin assay (CARA) was developed as a tool to characterize active molecules discovered by high-throughput screening campaigns against replicating and non-replicating M. tuberculosis. Inclusion of activated charcoal in bacteriologic agar medium helps mitigate the impact of compound carry-over, and eliminates the requirement to pre-dilute cells prior to spotting on CARA microplates. After a 7-10 day incubation period at 37 °C, the reduction of resazurin by mycobacterial microcolonies growing on the surface of CARA microplate wells permits semi-quantitative assessment of bacterial numbers via fluorometry. The CARA detects approximately a 2-3 log10 difference in bacterial numbers and predicts a minimal bactericidal concentration leading to ≥99% bacterial kill (MBC≥99). The CARA helps determine whether a molecule is active on bacilli that are replicating, non-replicating, or both. Pilot experiments using the CARA facilitate the identification of which concentration of test agent and time of compound exposure require further evaluation by colony forming unit (CFU) assays. In addition, the CARA can predict if replicating actives are bactericidal or bacteriostatic.

Clinics in diagnostic imaging (153). Severe hypoxic ischaemic brain injury.

PubMed Central

Chua, Wynne; Lim, Boon Keat; Lim, Tchoyoson Choie Cheio

2014-01-01

A 58-year-old Indian woman presented with asystole after an episode of haemetemesis, with a patient downtime of 20 mins. After initial resuscitation efforts, computed tomography of the brain, obtained to evaluate neurological injury, demonstrated evidence of severe hypoxic ischaemic brain injury. The imaging features of hypoxic ischaemic brain injury and the potential pitfalls with regard to image interpretation are herein discussed. PMID:25091891

Bemithyl potentiates the antioxidant effect of intermittent hypoxic training.

PubMed

Zarubina, I V; Nurmanbetova, F N; Shabanov, P D

2005-08-01

The rats were adapted to hypoxic hypoxia by intermittent training in a flow pressure chamber for 3 days. The course of bemithyl treatment (25 mg/kg intraperitoneally, 3 days) started immediately after the 1st day of training. Bemithyl potentiated the adaptive metabolic changes in rat brain induced by repeated hypoxic hypoxia, increased the individual resistance to hypoxia, and produced a long-lasting effect.

Cardiorespiratory physiological phenotypic plasticity in developing air-breathing anabantid fishes (Betta splendens and Trichopodus trichopterus).

PubMed

Mendez-Sanchez, Jose F; Burggren, Warren W

2017-08-01

Developmental plasticity of cardiorespiratory physiology in response to chronic hypoxia is poorly understood in larval fishes, especially larval air-breathing fishes, which eventually in their development can at least partially "escape" hypoxia through air breathing. Whether the development air breathing makes these larval fishes less or more developmentally plastic than strictly water breathing larval fishes remains unknown. Consequently, developmental plasticity of cardiorespiratory physiology was determined in two air-breathing anabantid fishes ( Betta splendens and Trichopodus trichopterus ). Larvae of both species experienced an hypoxic exposure that mimicked their natural environmental conditions, namely chronic nocturnal hypoxia (12 h at 17 kPa or 14 kPa), with a daily return to diurnal normoxia. Chronic hypoxic exposures were made from hatching through 35 days postfertilization, and opercular and heart rates measured as development progressed. Opercular and heart rates in normoxia were not affected by chronic nocturnal hypoxic. However, routine oxygen consumption M˙O2 (~4  μ mol·O 2 /g per hour in normoxia in larval Betta ) was significantly elevated by chronic nocturnal hypoxia at 17 kPa but not by more severe (14 kPa) nocturnal hypoxia. Routine M˙O2 in Trichopodus (6-7  μ mol·O 2 /g per hour), significantly higher than in Betta , was unaffected by either level of chronic hypoxia. P Crit , the PO 2 at which M˙O2 decreases as ambient PO 2 falls, was measured at 35 dpf, and decreased with increasing chronic hypoxia in Betta , indicating a large, relatively plastic hypoxic tolerance. However, in contrast, P Crit in Trichopodus increased as rearing conditions grew more hypoxic, suggesting that hypoxic acclimation led to lowered hypoxic resistance. Species-specific differences in larval physiological developmental plasticity thus emerge between the relatively closely related Betta and Trichopodus Hypoxic rearing increased hypoxic tolerance in Betta , which inhabits temporary ponds with nocturnal hypoxia. Trichopodus , inhabiting more permanent oxygenated bodies of water, showed few responses to hypoxia, reflecting a lower degree of developmental phenotypic plasticity. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

PubMed

Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

2015-10-05

Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hypoxic Episodes in Bronchopulmonary Dysplasia

PubMed Central

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

2015-01-01

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

Rifamycin action on RNA polymerase in antibiotic-tolerant Mycobacterium tuberculosis results in differentially detectable populations

PubMed Central

Saito, Kohta; Warrier, Thulasi; Somersan-Karakaya, Selin; Kaminski, Lina; Mi, Jianjie; Jiang, Xiuju; Park, Suna; Shigyo, Kristi; Gold, Ben; Roberts, Julia; Weber, Elaina; Jacobs, William R.; Nathan, Carl F.

2017-01-01

Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such “differentially detectable” (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin. PMID:28559332

Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

PubMed

Sedlik, C; Dridi, A; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J; Casal, J I; Leclerc, C

1999-04-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration.

The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

PubMed Central

Gao, Wei; Kim, Jin-Yong; Anderson, Jeffrey R.; Akopian, Tatos; Hong, Seungpyo; Jin, Ying-Yu; Kandror, Olga; Kim, Jong-Woo; Lee, In-Ae; Lee, Sun-Young; McAlpine, James B.; Mulugeta, Surafel; Sunoqrot, Suhair; Wang, Yuehong; Yang, Seung-Hwan; Yoon, Tae-Mi; Goldberg, Alfred L.; Pauli, Guido F.; Cho, Sanghyun

2014-01-01

Drug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition of Mycobacterium tuberculosis viability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity against M. tuberculosis in vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains of M. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition of M. tuberculosis growth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistant M. tuberculosis strains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target against M. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development. PMID:25421483

Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

PubMed Central

Sedlik, C.; Dridi, A.; Deriaud, E.; Saron, M. F.; Rueda, P.; Sarraseca, J.; Casal, J. I.; Leclerc, C.

1999-01-01

We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+ T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120

Targeting hypoxic microenvironment of pancreatic xenografts with the hypoxia-activated prodrug TH-302.

PubMed

Lohse, Ines; Rasowski, Joanna; Cao, Pinjiang; Pintilie, Melania; Do, Trevor; Tsao, Ming-Sound; Hill, Richard P; Hedley, David W

2016-06-07

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX).The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche.

Response of the RIF-1 tumor in vitro and in C3H/Km mice to x-radiation (cell survival, regrowth delay, and tumor control), chemotherapeutic agents, and activated macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brown, J.M.; Twentyman, P.R.; Zamvil, S.S.

1980-03-01

The radiation response of logarithmic growth phase and fed plateau phase RIF-1 cells in vitro was found to be characterized by D/sub 0/ values of 110 and 133 rads and extrapolation numbs of 36 and 28, respectively. The response of the tumor in vivo to X-irradiation in nonanesthetized mice showed a dependence on the tumor implantation site. In the leg muscle, the response indicated that most cells were at an intermediate level of oxygenation, whereas in the subcutaneous tissue of the flank, the response of the tumor indicated that it had a small fraction of hypoxic cells of maximum radioresistance.more » Misonidazole radiosensitized the leg-implanted tumor as measured both by cell survival and regrowth delay. The tumor was relatively insensitive to a single dose of 1,3-bis(2-chloroethyl)-1-nitrosourea, sensitive to a single dose of cis-platinum, and highly sensitive to a single dose of cyclophosphamide.« less

Portable bioreactor for perfusion and electrical stimulation of engineered cardiac tissue.

PubMed

Tandon, Nina; Taubman, Alanna; Cimetta, Elisa; Saccenti, Laetitia; Vunjak-Novakovic, Gordana

2013-01-01

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Although bioreactors have facilitated the engineering of cardiac patches of clinically relevant size in vitro, a major drawback remains the transportation of the engineered tissues from a production facility to a medical operation facility while maintaining tissue viability and preventing contamination. Furthermore, after implantation, most of the cells are endangered by hypoxic conditions that exist before vascular flow is established. We developed a portable device that provides the perfusion and electrical stimulation necessary to engineer cardiac tissue in vitro, and to transport it to the site where it will be implantated. The micropump-powered perfusion apparatus may additionally function as an extracorporeal active pumping system providing nutrients and oxygen supply to the graft post-implantation. Such a system, through perfusion of oxygenated media and bioactive molecules (e.g. growth factors), could transiently support the tissue construct until it connects to the host vasculature and heart muscle, after which it could be taken away or let biodegrade.

In vitro therapeutic effect of PDT combined with VEGF-A gene therapy

NASA Astrophysics Data System (ADS)

Lecaros, Rumwald Leo G.; Huang, Leaf; Hsu, Yih-Chih

2014-02-01

Vascular endothelial growth factor A (VEGF-A), commonly known as VEGF, is one of the primary factors that affect tumor angiogenesis. It was found to be expressed in cancer cell lines including oral squamous cell carcinoma. Photodynamic therapy (PDT) is a novel therapeutic modality to treat cancer by using a photosensitizer which is activated by a light source to produce reactive oxygen species and mediates oxygen-independent hypoxic conditions to tumor. Another emerging treatment to cure cancer is the use of interference RNA (e.g. siRNA) to silence a specific mRNA sequence. VEGF-A was found to be expressed in oral squamous cell carcinoma and overexpressed after 24 hour post-PDT by Western blot analysis. Cell viability was found to decrease at 25 nM of transfected VEGF-A siRNA. In vitro combined therapy of PDT and VEGF-A siRNA showed better response as compared with PDT and gene therapy alone. The results suggest that PDT combined with targeted gene therapy has a potential mean to achieve better therapeutic outcome.

Hypoxia reduces testosterone synthesis in mouse Leydig cells by inhibiting NRF1-activated StAR expression

PubMed Central

Zou, Zhiran; Wang, Dan; Lu, Yapeng; Dong, Zhangji; Zhu, Li

2017-01-01

Male fertility disorders play a key role in half of all infertility cases. Reduction in testosterone induced by hypoxia might cause diseases in reproductive system and other organs. Hypoxic exposure caused a significant decrease of NRF1. Software analysis reported that the promoter region of steroidogenic acute regulatory protein (StAR) contained NRF1 binding sites, indicating NRF1 promoted testicular steroidogenesis. The purpose of this study is to determine NRF1 is involved in testosterone synthesis; and under hypoxia, the decrease of testosterone synthesis is caused by lower expression of NRF1. We designed both in vivo and in vitro experiments. Under hypoxia, the expressions of NRF1 in Leydig cells and testosterone level were significantly decreased both in vivo and in vitro. Overexpression and interference NRF1 could induced StAR and testosterone increased and decreased respectively. ChIP results confirmed the binding of NRF1 to StAR promoter region. In conclusion, decline of NRF1 expression downregulated the level of StAR, which ultimately resulted in a reduction in testosterone synthesis. PMID:28146428

Reoxygenation in the RIF-1 tumor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dorie, M.J.; Kallman, R.F.

1984-05-01

The proportion of hypoxic cells in the RIF-1 tumor was examined for 13 days following a 15 Gy conditioning dose. The paired survival curve technique indicated that 100% of the surviving cells were hypoxic immediately following this treatment. However, within 1 hour, only about 50% remained hypoxic; this proportion continued to drop to about 10% but did not reach the pretreatment level of 1.1% for the duration of the study.

An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors.

PubMed

Szablowski, Jerzy O; Raskatov, Jevgenij A; Dervan, Peter B

2016-04-01

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1: has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1: in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1: distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1: show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608-17. ©2015 AACR. ©2015 American Association for Cancer Research.

Hypothermia therapy for newborns with hypoxic ischemic encephalopathy.

PubMed

Silveira, Rita C; Procianoy, Renato S

2015-01-01

Therapeutic hypothermia reduces cerebral injury and improves the neurological outcome secondary to hypoxic ischemic encephalopathy in newborns. It has been indicated for asphyxiated full-term or near-term newborn infants with clinical signs of hypoxic-ischemic encephalopathy (HIE). A search was performed for articles on therapeutic hypothermia in newborns with perinatal asphyxia in PubMed; the authors chose those considered most significant. There are two therapeutic hypothermia methods: selective head cooling and total body cooling. The target body temperature is 34.5 °C for selective head cooling and 33.5 °C for total body cooling. Temperatures lower than 32 °C are less neuroprotective, and temperatures below 30 °C are very dangerous, with severe complications. Therapeutic hypothermia must start within the first 6h after birth, as studies have shown that this represents the therapeutic window for the hypoxic-ischemic event. Therapy must be maintained for 72 h, with very strict control of the newborn's body temperature. It has been shown that therapeutic hypothermia is effective in reducing neurologic impairment, especially in full-term or near-term newborns with moderate hypoxic-ischemic encephalopathy. Therapeutic hypothermia is a neuroprotective technique indicated for newborn infants with perinatal asphyxia and hypoxic-ischemic encephalopathy. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

PubMed

Uddin, Md Imam; Evans, Stephanie M; Craft, Jason R; Capozzi, Megan E; McCollum, Gary W; Yang, Rong; Marnett, Lawrence J; Uddin, Md Jashim; Jayagopal, Ashwath; Penn, John S

2016-08-05

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

Hypoxia interface behavior of the ctenophore Mnemiopsis leidyi

NASA Astrophysics Data System (ADS)

Gentry, L. A.; Moss, A.

2016-02-01

The ctenophore Mnemiopsis leidyi is most widely known as a destructive invasive species of the Black and Caspian seas. Ctenophores are also found endemically in oceans worldwide, where their low oxygen tolerances allow many of them to use hypoxic zones to escape predation and hunt disabled prey. Ctenophores have also been observed in the wild and laboratory conditions associating with the interface of hypoxic and normoxic waters, allowing them to feed on the organisms found there. In order to test the ability of M. leidyi to find oxygen interfaces, a 10 cm diameter X 60 cm tall cylindrical tank was designed with a sharp oxycline (<1cm) in the middle, stabilized by a 1 ppt salinity difference. The hypoxic water was produced by nitrogen bubbling prior to the experiment. We found that animals introduced to the tank under oxycline conditions increased their time spent at the interface by over nearly five-fold versus those in a fully normoxic or hypoxic water. These preliminary results would indicate that M. leidyi preferentially associates with the interfaces of hypoxic zones. As human effects such as overfishing, nutrient enrichment of coastal waters, and invasive introduction continue to rise, the complex interactions of these animals and hypoxic zones will become increasingly important to planktonic and pelagic ecosystems worldwide.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy

PubMed Central

Uddin, Md. Imam; Evans, Stephanie M.; Craft, Jason R.; Capozzi, Megan E.; McCollum, Gary W.; Yang, Rong; Marnett, Lawrence J.; Uddin, Md. Jashim; Jayagopal, Ashwath; Penn, John S.

2016-01-01

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs. PMID:27491345

Neurotransmitters and neuromodulators controlling the hypoxic respiratory response in anaesthetized cats.

PubMed

Richter, D W; Schmidt-Garcon, P; Pierrefiche, O; Bischoff, A M; Lalley, P M

1999-01-15

1. The contributions of neurotransmitters and neuromodulators to the responses of the respiratory network to acute hypoxia were analysed in anaesthetized cats. 2. Samples of extracellular fluid were collected at 1-1.5 min time intervals by microdialysis in the medullary region of ventral respiratory group neurones and analysed for their content of glutamate, gamma-aminobutyric acid (GABA), serotonin and adenosine by high performance liquid chromatography. Phrenic nerve activity was correlated with these measurements. 3. Levels of glutamate and GABA increased transiently during early periods of hypoxia, coinciding with augmented phrenic nerve activity and then fell below control during central apnoea. Serotonin and adenosine increased slowly and steadily with onset of hypoxic depression of phrenic nerve activity. 4. The possibility that serotonin contributes to hypoxic respiratory depression was tested by microinjecting the 5-HT-1A receptor agonist 8-OH-DPAT into the medullary region that is important for rhythmogenesis. Hypoxic activation of respiratory neurones and phrenic nerve activity were suppressed. Microinjections of NAN-190, a 5-HT-1A receptor blocker, enhanced hypoxic augmentation resulting in apneustic prolongation of inspiratory bursts. 5. The results reveal a temporal sequence in the release of neurotransmitters and neuromodulators and suggest a specific role for each of them in the sequential development of hypoxic respiratory disturbances.

Hypoxia induces triglycerides accumulation in prostate cancer cells and extracellular vesicles supporting growth and invasiveness following reoxygenation

PubMed Central

Kumar, Rahul; Dhar, Deepanshi; Agarwal, Chapla; Bergman, Bryan; Graner, Michael; Maroni, Paul; Singh, Rana P.; Agarwal, Rajesh; Deep, Gagan

2015-01-01

Hypoxia is an independent prognostic indicator of poor outcome in several malignancies. However, precise mechanism through which hypoxia promotes disease aggressiveness is still unclear. Here, we report that under hypoxia (1% O2), human prostate cancer (PCA) cells, and extracellular vesicles (EVs) released by these cells, are significantly enriched in triglycerides due to the activation of lipogenesis-related enzymes and signaling molecules. This is likely a survival response to hypoxic stress as accumulated lipids could support growth following reoxygenation. Consistent with this, significantly higher proliferation was observed in hypoxic PCA cells following reoxygenation associated with rapid use of accumulated lipids. Importantly, lipid utilization inhibition by CPT1 inhibitor etomoxir and shRNA-mediated CPT1-knockdown significantly compromised hypoxic PCA cell proliferation following reoxygenation. Furthermore, COX2 inhibitor celecoxib strongly reduced growth and invasiveness following hypoxic PCA cells reoxygenation, and inhibited invasiveness induced by hypoxic PCA EVs. This establishes a role for COX2 enzymatic products in the enhanced PCA growth and invasiveness. Importantly, concentration and loading of EVs secreted by PCA cells were significantly compromised under delipidized serum condition and by lipogenesis inhibitors (fatostatin and silibinin). Overall, present study highlights the biological significance of lipid accumulation in hypoxic PCA cells and its therapeutic relevance in PCA. PMID:26087400

Role of Kv7 channels in responses of the pulmonary circulation to hypoxia.

PubMed

Sedivy, Vojtech; Joshi, Shreena; Ghaly, Youssef; Mizera, Roman; Zaloudikova, Marie; Brennan, Sean; Novotna, Jana; Herget, Jan; Gurney, Alison M

2015-01-01

Hypoxic pulmonary vasoconstriction (HPV) is a beneficial mechanism that diverts blood from hypoxic alveoli to better ventilated areas of the lung, but breathing hypoxic air causes the pulmonary circulation to become hypertensive. Responses to airway hypoxia are associated with depolarization of smooth muscle cells in the pulmonary arteries and reduced activity of K(+) channels. As Kv7 channels have been proposed to play a key role in regulating the smooth muscle membrane potential, we investigated their involvement in the development of HPV and hypoxia-induced pulmonary hypertension. Vascular effects of the selective Kv7 blocker, linopirdine, and Kv7 activator, flupirtine, were investigated in isolated, saline-perfused lungs from rats maintained for 3-5 days in an isobaric hypoxic chamber (FiO2 = 0.1) or room air. Linopirdine increased vascular resistance in lungs from normoxic, but not hypoxic rats. This effect was associated with reduced mRNA expression of the Kv7.4 channel α-subunit in hypoxic arteries, whereas Kv7.1 and Kv7.5 were unaffected. Flupirtine had no effect in normoxic lungs but reduced vascular resistance in hypoxic lungs. Moreover, oral dosing with flupirtine (30 mg/kg/day) prevented short-term in vivo hypoxia from increasing pulmonary vascular resistance and sensitizing the arteries to acute hypoxia. These findings suggest a protective role for Kv7.4 channels in the pulmonary circulation, limiting its reactivity to pressor agents and preventing hypoxia-induced pulmonary hypertension. They also provide further support for the therapeutic potential of Kv7 activators in pulmonary vascular disease. Copyright © 2015 the American Physiological Society.

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

PubMed Central

Yadav, Vishal R.; Song, Tengyao; Joseph, Leroy; Mei, Lin; Zheng, Yun-Min

2013-01-01

An increase in intracellular calcium concentration ([Ca2+]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 [Ca2+]i in PASMCs. PLC-γ1 knockdown suppresses its protein expression and the hypoxic increase in [Ca2+]i. Hypoxia remarkably increases inositol 1,4,5-trisphosphate (IP3) production, which is blocked by U73122. The IP3 receptor (IP3R) antagonist 2-aminoethoxydiphenyl borate (2-APB) or xestospongin-C inhibits the hypoxic increase in [Ca2+]i. PLC-γ1 knockdown or U73122 reduces H2O2-induced increase in [Ca2+]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 IP3 production, IP3R opening, and Ca2+ release, playing an important role in hypoxic Ca2+ and contractile responses in PASMCs. PMID:23204067

Hypoxia monitoring activities within the FP7 EU-project HYPOX: diverse approaches to understand a complex phenomenon

NASA Astrophysics Data System (ADS)

Janssen, F.; Waldmann, C.; Boetius, A.

2012-04-01

Hypoxic conditions in aquatic systems and the occurrence of 'dead zones' increase worldwide due to man-made eutrophication and global warming with consequences for biodiversity, ecosystem functions and services such as fisheries, aquaculture and tourism. Monitoring of hypoxia and its consequences has to (1) account for the appropriate temporal and spatial scales, (2) separate anthropogenic from natural drivers and long-term trends from natural variations, (3) assess ecosystem response, (4) use modeling tools for generalization and prediction, and (5) share data and obtained knowledge. In 2009 the EU FP7 project HYPOX (www.hypox.net) started out as a pioneering attempt to improve and integrate hypoxia observation capacities addressing these requirements. Target ecosystems selected for HYPOX cover a broad range of settings (e.g., hydrography, oxygenation status, biological activity, anthropogenic impact) and differ in their sensitivity towards change. Semi-enclosed basins with permanent anoxia (Black Sea, Baltic Sea), are included as well as seasonally or locally hypoxic land-locked systems (fjords, lagoons, lakes) and open ocean systems with high sensitivity to global warming (North Atlantic - Arctic transition). Adopted monitoring approaches involve autonomous, cabled, and shipboard instruments and include static and profiling moorings, benthic observatories, drifters, as well as classical CTD surveys. In order to improve observatory performance, project activities encompass developments of oxygen sensors as well as calibration procedures and technologies to reduce biofouling. Modeling and data assimilation are used to synthesize findings, to obtain an in-depth understanding of hypoxia causes and consequences, and to improve forecasting capacities. For integration of the collected information into a global oxygen observing system, results are disseminated through the HYPOX portal following GEOSS data sharing principles. This presentation will give an overview of the scientific approach of HYPOX and highlight some key results comprising findings from individual ecosystems and indentified general patterns. The driving forces that lead to hypoxia are assessed as well as consequences of oxygen depletion for aquatic life and biogeochemical processes.

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 that ripe apple tissues finely and specifically modulate sensing and regulatory mechanisms in response to different hypoxic stress conditions. PMID:26909091

More than meets the eye: infant presenting with hypoxic ischaemic encephalopathy.

PubMed

Sen, Kuntal; Agarwal, Rajkumar

2018-04-05

We report a newborn infant who presented with poor Apgar scores and umbilical artery acidosis leading to the diagnosis of hypoxic ischaemic encephalopathy. During the course of the infant's hospitalisation, subsequent workup revealed an underlying genetic cause that masqueraded as hypoxic ischaemic encephalopathy. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Nonreplication of an Association of Apolipoprotein E2 With Sinistrality

PubMed Central

Piper, Brian J.; Yasen, Alia L.; Taylor, Amy E.; Ruiz, Jonatan R.; Gaynor, J. William; Dayger, Catherine A.; Gonzalez-Gross, Marcela; Kwon, Oh D.; Nilsson, Lars-Göran; Day, Ian N. M.; Raber, Jacob; Miller, Jeremy K.

2013-01-01

A recent report found that left-handed adolescents were over three-fold more likely to have an Apolipoprotein (APOE) ε2 allele. This study was unable to replicate this association in young-adults (N=166). A meta-analysis of nine other datasets (N = 360 to 7,559, Power > 0.999) including that of National Alzheimer’s Coordinating Center also failed to find an over-representation of ε2 among left-handers indicating that this earlier outcome was most likely a statistical artifact. PMID:22721421

Nitric oxide contributes to the augmented vasodilatation during hypoxic exercise

PubMed Central

Casey, Darren P; Madery, Brandon D; Curry, Timothy B; Eisenach, John H; Wilkins, Brad W; Joyner, Michael J

2010-01-01

We tested the hypotheses that (1) nitric oxide (NO) contributes to augmented skeletal muscle vasodilatation during hypoxic exercise and (2) the combined inhibition of NO production and adenosine receptor activation would attenuate the augmented vasodilatation during hypoxic exercise more than NO inhibition alone. In separate protocols subjects performed forearm exercise (10% and 20% of maximum) during normoxia and normocapnic hypoxia (80% arterial O2 saturation). In protocol 1 (n= 12), subjects received intra-arterial administration of saline (control) and the NO synthase inhibitor NG-monomethyl-l-arginine (l-NMMA). In protocol 2 (n= 10), subjects received intra-arterial saline (control) and combined l-NMMA–aminophylline (adenosine receptor antagonist) administration. Forearm vascular conductance (FVC; ml min−1 (100 mmHg)−1) was calculated from forearm blood flow (ml min−1) and blood pressure (mmHg). In protocol 1, the change in FVC (Δ from normoxic baseline) due to hypoxia under resting conditions and during hypoxic exercise was substantially lower with l-NMMA administration compared to saline (control; P < 0.01). In protocol 2, administration of combined l-NMMA–aminophylline reduced the ΔFVC due to hypoxic exercise compared to saline (control; P < 0.01). However, the relative reduction in ΔFVC compared to the respective control (saline) conditions was similar between l-NMMA only (protocol 1) and combined l-NMMA–aminophylline (protocol 2) at 10% (−17.5 ± 3.7 vs.−21.4 ± 5.2%; P= 0.28) and 20% (−13.4 ± 3.5 vs.−18.8 ± 4.5%; P= 0.18) hypoxic exercise. These findings suggest that NO contributes to the augmented vasodilatation observed during hypoxic exercise independent of adenosine. PMID:19948661

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.

Expression of MUC17 Is Regulated by HIF1α-Mediated Hypoxic Responses and Requires a Methylation-Free Hypoxia Responsible Element in Pancreatic Cancer

PubMed Central

Kitamoto, Sho; Yokoyama, Seiya; Higashi, Michiyo; Yamada, Norishige; Matsubara, Shyuichiro; Takao, Sonshin; Batra, Surinder K.; Yonezawa, Suguru

2012-01-01

MUC17 is a type 1 membrane-bound glycoprotein that is mainly expressed in the digestive tract. Recent studies have demonstrated that the aberrant overexpression of MUC17 is correlated with the malignant potential of pancreatic ductal adenocarcinomas (PDACs); however, the exact regulatory mechanism of MUC17 expression has yet to be identified. Here, we provide the first report of the MUC17 regulatory mechanism under hypoxia, an essential feature of the tumor microenvironment and a driving force of cancer progression. Our data revealed that MUC17 was significantly induced by hypoxic stimulation through a hypoxia-inducible factor 1α (HIF1α)-dependent pathway in some pancreatic cancer cells (e.g., AsPC1), whereas other pancreatic cancer cells (e.g., BxPC3) exhibited little response to hypoxia. Interestingly, these low-responsive cells have highly methylated CpG motifs within the hypoxia responsive element (HRE, 5′-RCGTG-3′), a binding site for HIF1α. Thus, we investigated the demethylation effects of CpG at HRE on the hypoxic induction of MUC17. Treatment of low-responsive cells with 5-aza-2′-deoxycytidine followed by additional hypoxic incubation resulted in the restoration of hypoxic MUC17 induction. Furthermore, DNA methylation of HRE in pancreatic tissues from patients with PDACs showed higher hypomethylation status as compared to those from non-cancerous tissues, and hypomethylation was also correlated with MUC17 mRNA expression. Taken together, these findings suggested that the HIF1α-mediated hypoxic signal pathway contributes to MUC17 expression, and DNA methylation of HRE could be a determinant of the hypoxic inducibility of MUC17 in pancreatic cancer cells. PMID:22970168

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.

Inhibition of Siah2 Ubiquitin Ligase by Vitamin K3 Attenuates Chronic Myeloid Leukemia Chemo-Resistance in Hypoxic Microenvironment.

PubMed

Huang, Jixian; Lu, Ziyuan; Xiao, Yajuan; He, Bolin; Pan, Chengyun; Zhou, Xuan; Xu, Na; Liu, Xiaoli

2018-02-05

BACKGROUND A hypoxic microenvironment is associated with resistance to tyrosine kinase inhibitors (TKIs) and a poor prognosis in chronic myeloid leukemia (CML). The E3 ubiquitin ligase Siah2 plays a vital role in the regulation of hypoxia response, as well as in leukemogenesis. However, the role of Siah2 in CML resistance is unclear, and it is unknown whether vitaminK3 (a Siah2 inhibitor) can improve the chemo-sensitivity of CML cells in a hypoxic microenvironment. MATERIAL AND METHODS The expression of Siah2 was detected in CML patients (CML-CP and CML-BC), K562 cells, and K562-imatinib-resistant cells (K562-R cells). We measured the expression of PHD3, HIF-1α, and VEGF in both cell lines under normoxia and hypoxic conditions, and the degree of leukemic sensitivity to imatinib and VitaminK3 were evaluated. RESULTS Siah2 was overexpressed in CML-BC patients (n=9) as compared to CML-CP patients (n=13). Similarly, K562-imatinib-resistant cells (K562-R cells) showed a significantly higher expression of Siah2 as compared to K562 cells in a hypoxic microenvironment. Compared to normoxia, under hypoxic conditions, both cell lines had lower PHD3, higher HIF-1α, and higher VEGF expression. Additionally, Vitamin K3 (an inhibitor of Siah2) reversed these changes and promoted a higher degree of leukemic sensitivity to imatinib. CONCLUSIONS Our findings indicate that the Siah2-PHD3- HIF-1α-VEGF axis is an important hypoxic signaling pathway in a leukemic microenvironment. An inhibitor of Siah2, combined with TKIs, might be a promising therapy for relapsing and refractory CML patients.

Hypoxic survival strategies in two fishes: extreme anoxia tolerance in the North European crucian carp and natural hypoxic preconditioning in a coral-reef shark.

PubMed

Nilsson, Göran E; Renshaw, Gillian M C

2004-08-01

Especially in aquatic habitats, hypoxia can be an important evolutionary driving force resulting in both convergent and divergent physiological strategies for hypoxic survival. Examining adaptations to anoxic/hypoxic survival in hypoxia-tolerant animals may offer fresh ideas for the treatment of hypoxia-related diseases. Here, we summarise our present knowledge of two fishes that have evolved to survive hypoxia under very different circumstances. The crucian carp (Carassius carassius) is of particular interest because of its extreme anoxia tolerance. During the long North European winter, it survives for months in completely oxygen-deprived freshwater habitats. The crucian carp also tolerates a few days of anoxia at room temperature and, unlike anoxia-tolerant freshwater turtles, it is still physically active in anoxia. Moreover, the crucian carp does not appear to reduce neuronal ion permeability during anoxia and may primarily rely on more subtle neuromodulatory mechanisms for anoxic metabolic depression. The epaulette shark (Hemiscyllium ocellatum) is a tropical marine vertebrate. It lives on shallow reef platforms that repeatedly become cut off from the ocean during periods of low tides. During nocturnal low tides, the water [O(2)] can fall by 80% due to respiration of the coral and associated organisms. Since the tides become lower and lower over a period of a few days, the hypoxic exposure during subsequent low tides will become progressively longer and more severe. Thus, this shark is under a natural hypoxic preconditioning regimen. Interestingly, hypoxic preconditioning lowers its metabolic rate and its critical P(O(2)). Moreover, repeated anoxia appears to stimulate metabolic depression in an adenosine-dependent way.

Prediction of Susceptibility to Acute Mountain Sickness Using Hypoxia-Induced Intrapulmonary Arteriovenous Shunt and Intracardiac Shunt Fractions

DTIC Science & Technology

2013-10-01

echocardiography to determine bubble/shunt scores. We will also use nuclear medicine imaging to determine shunt fractions following acute exposures to... echocardiography while breathing hypoxic gas mixtures. – TASK COMPLETED. For Task #1.3 “Quantify shunt during hypoxic exposure with SPECT CT – PFO...subjects.” 19 PFO+ subjects have completed saline contrast echocardiography while breathing hypoxic gas mixtures for 30 min. One PFO+ subject that had

Prediction of Susceptibility to Acute Mountain Sickness Using Hypoxia-Induced Intrapulmonary Arteriovenous Shunt and Intracardiac Shunt Fractions

DTIC Science & Technology

2012-10-31

intrapulmonary and intracardiac shunt using saline contrast echocardiography to determine bubble/shunt scores. We will also use nuclear medicine imaging to...subjects have completed saline contrast echocardiography while breathing hypoxic gas mixtures. For Task #2 “10 hr hypoxic exposure and AMS... echocardiography while breathing an FIO2=0.14, will be susceptible or resistant to developing AMS after 10 hr hypoxic exposure. For Task #3 “Hypoxia

OUTCOMES in CHILDHOOD FOLLOWING THERAPEUTIC HYPOTHERMIA for NEONATAL HYPOXIC-ISCHEMIC ENCEPHALOPATHY (HIE)

PubMed Central

Natarajan, Girija; Pappas, Athina; Shankaran, Seetha

2017-01-01

In this chapter we review the childhood outcomes of neonates with birth depression and/or hypoxic-ischemic encephalopathy. The outcomes of these children prior to the era of hypothermia for neuroprotection will first be summarized, followed by discussion of results from randomized controlled trials of therapeutic hypothermia for neonatal hypoxic ischemic encephalopathy. The predictors of outcome in childhood following neonatal HIE using clinical and imaging biomarkers following hypothermia therapy will be described. PMID:27863707

Neuroprotection Against Hypoxic/Ischemic Injury: δ-Opioid Receptors and BDNF-TrkB Pathway.

PubMed

Sheng, Shiying; Huang, Jingzhong; Ren, Yi; Zhi, Feng; Tian, Xuansong; Wen, Guoqiang; Ding, Guanghong; Xia, Terry C; Hua, Fei; Xia, Ying

2018-05-11

The delta-opioid receptor (DOR) is one of three classic opioid receptors in the opioid system. It was traditionally thought to be primarily involved in modulating the transmission of messages along pain signaling pathway. Although there were scattered studies on its other neural functions, inconsistent results and contradicting conclusions were found in past literatures, especially in terms of DOR's role in a hypoxic/ischemic brain. Taking inspiration from the finding that the turtle brain exhibits a higher DOR density and greater tolerance to hypoxic/ischemic insult than the mammalian brain, we clarified DOR's specific role in the brain against hypoxic/ischemic injury and reconciled previous controversies in this aspect. Our serial studies have strongly demonstrated that DOR is a unique neuroprotector against hypoxic/ischemic injury in the brain, which has been well confirmed in current research. Moreover, mechanistic studies have shown that during acute phases of hypoxic/ischemic stress, DOR protects the neurons mainly by the stabilization of ionic homeostasis, inhibition of excitatory transmitter release, and attenuation of disrupted neuronal transmission. During prolonged hypoxia/ischemia, however, DOR neuroprotection involves a variety of signaling pathways. More recently, our data suggest that DOR may display its neuroprotective role via the BDNF-TrkB pathway. This review concisely summarizes the progress in this field. © 2018 The Author(s). Published by S. Karger AG, Basel.

Phylogenetic composition and distribution of picoeukaryotes in the hypoxic northwestern coast of the Gulf of Mexico

PubMed Central

Rocke, Emma; Jing, Hongmei; Liu, Hongbin

2013-01-01

Coastal marine hypoxic, or low-oxygen, episodes are an increasing worldwide phenomenon, but its effect on the microbial community is virtually unknown by far. In this study, the community structure and phylogeny of picoeukaryotes in the Gulf of Mexico, which are exposed to severe hypoxia in these areas was explored through a clone library approach. Both oxic surface waters and suboxic bottom waters were collected in August 2010 from three representative stations on the inner Louisiana shelf near the Atchafalaya and Mississippi River plumes. The bottom waters of the two more western stations were much more hypoxic in comparison to those of the station closest to the Mississippi River plume, which were only moderately hypoxic. A phylogenetic analysis of a total 175 sequences, generated from six 18S rDNA clone libraries, demonstrated a clear dominance of parasitic dinoflagellates from Marine alveolate clades I and II in all hypoxic waters as well as in the surface layer at the more western station closest to the Atchafalaya River plume. Species diversity was significantly higher at the most hypoxic sites, and many novel species were present among the dinoflagellate and stramenopile clades. We concluded that hypoxia in the Gulf of Mexico causes a significant shift in picoeukaryote communities, and that hypoxia may cause a shift in microbial food webs from grazing to parasitism. PMID:23281331

Population pharmacokinetics of phenobarbital in infants with neonatal encephalopathy treated with therapeutic hypothermia.

PubMed

Shellhaas, Renée A; Ng, Chee M; Dillon, Christina H; Barks, John D E; Bhatt-Mehta, Varsha

2013-02-01

Phenobarbital is the first-line treatment for neonatal seizures. Many neonates with hypoxic ischemic encephalopathy are treated with therapeutic hypothermia, and about 40% have clinical seizures. Little is known about the pharmacokinetics of phenobarbital in infants with hypoxic ischemic encephalopathy who undergo therapeutic hypothermia. The objective of this study was to determine the effect of therapeutic hypothermia on phenobarbital pharmacokinetics, taking into account maturational changes. Level 3 neonatal ICU. Infants with hypoxic ischemic encephalopathy and suspected seizures, all treated with phenobarbital. Some of these infants also received treatment with therapeutic hypothermia. None. A retrospective cohort study of 39 infants with hypoxic ischemic encephalopathy treated with phenobarbital (20 were treated with therapeutic hypothermia and 19 were not). Data on phenobarbital plasma concentrations were collected in 39 subjects with hypoxic ischemic encephalopathy with or without therapeutic hypothermia. Using nonlinear mixed-effects modeling, population pharmacokinetics of phenobarbital were developed with a total of 164 plasma concentrations. A one-compartment model best described the pharmacokinetics. The clearance of phenobarbital was linearly related to body weight and matured with increasing age with a maturation half-life of 22.1 days. Therapeutic hypothermia did not influence the pharmacokinetic parameters of phenobarbital. Therapeutic hypothermia does not influence the clearance of phenobarbital after accounting for weight and age. Standard phenobarbital dosing is appropriate for the initial treatment of seizures in neonates with hypoxic ischemic encephalopathy treated with therapeutic hypothermia.

Redistribution of pulmonary blood flow during unilateral hypoxia in prone and supine dogs

NASA Technical Reports Server (NTRS)

Mann, C. M.; Domino, K. B.; Walther, S. M.; Glenny, R. W.; Polissar, N. L.; Hlastala, M. P.

1998-01-01

We used fluorescent-labeled microspheres in pentobarbital-anesthetized dogs to study the effects of unilateral alveolar hypoxia on the pulmonary blood flow distribution. The left lung was ventilated with inspired O2 fraction of 1.0, 0.09, or 0.03 in random order; the right lung was ventilated with inspired O2 fraction of 1.0. The lungs were removed, cleared of blood, dried at total lung capacity, then cubed to obtain approximately 1,500 small pieces of lung ( approximately 1.7 cm3). The coefficient of variation of flow increased (P < 0.001) in the hypoxic lung but was unchanged in the hyperoxic lung. Most (70-80%) variance in flow in the hyperoxic lung was attributable to structure, in contrast to only 30-40% of the variance in flow in the hypoxic lung (P < 0.001). When adjusted for the change in total flow to each lung, 90-95% of the variance in the hyperoxic lung was attributable to structure compared with 70-80% in the hypoxic lung (P < 0.001). The hilar-to-peripheral gradient, adjusted for change in total flow, decreased in the hypoxic lung (P = 0.005) but did not change in the hyperoxic lung. We conclude that hypoxic vasoconstriction alters the regional distribution of flow in the hypoxic, but not in the hyperoxic, lung.

Expression of Clock genes in the pineal glands of newborn rats with hypoxic-ischemic encephalopathy☆

PubMed Central

Sun, Bin; Feng, Xing; Ding, Xin; Bao, Li; Li, Yongfu; He, Jun; Jin, Meifang

2012-01-01

Clock genes are involved in circadian rhythm regulation, and surviving newborns with hypoxic-ischemic encephalopathy may present with sleep-wake cycle reversal. This study aimed to determine the expression of the clock genes Clock and Bmal1, in the pineal gland of rats with hypoxic-ischemic brain damage. Results showed that levels of Clock mRNA were not significantly changed within 48 hours after cerebral hypoxia and ischemia. Expression levels of CLOCK and BMAL1 protein were significantly higher after 48 hours. The levels of Bmal1 mRNA reached a peak at 36 hours, but were significantly reduced at 48 hours. Experimental findings indicate that Clock and Bmal1 genes were indeed expressed in the pineal glands of neonatal rats. At the initial stage (within 36 hours) of hypoxic-ischemic brain damage, only slight changes in the expression levels of these two genes were detected, followed by significant changes at 36–48 hours. These changes may be associated with circadian rhythm disorder induced by hypoxic-ischemic brain damage. PMID:25538743

Celecoxib enhances radiosensitivity of hypoxic glioblastoma cells through endoplasmic reticulum stress

PubMed Central

Suzuki, Kenshi; Gerelchuluun, Ariungerel; Hong, Zhengshan; Sun, Lue; Zenkoh, Junko; Moritake, Takashi; Tsuboi, Koji

2013-01-01

Background Refractoriness of glioblastoma multiforme (GBM) largely depends on its radioresistance. We investigated the radiosensitizing effects of celecoxib on GBM cell lines under both normoxic and hypoxic conditions. Methods Two human GBM cell lines, U87MG and U251MG, and a mouse GBM cell line, GL261, were treated with celecoxib or γ-irradiation either alone or in combination under normoxic and hypoxic conditions. Radiosensitizing effects were analyzed by clonogenic survival assays and cell growth assays and by assessing apoptosis and autophagy. Expression of apoptosis-, autophagy-, and endoplasmic reticulum (ER) stress–related genes was analyzed by immunoblotting. Results Celecoxib significantly enhanced the radiosensitivity of GBM cells under both normoxic and hypoxic conditions. In addition, combined treatment with celecoxib and γ-irradiation induced marked autophagy, particularly in hypoxic cells. The mechanism underlying the radiosensitizing effect of celecoxib was determined to be ER stress loading on GBM cells. Conclusion Celecoxib enhances the radiosensitivity of GBM cells by a mechanism that is different from cyclooxygenase-2 inhibition. Our results indicate that celecoxib may be a promising radiosensitizing drug for clinical use in patients with GBM. PMID:23658321

The effects of ROS in prostatic stromal cells under hypoxic environment.

PubMed

Ren, Hailin; Li, Xiaona; Cheng, Guojun; Li, Ning; Hou, Zhi; Suo, Jiming; Wang, Jian; Za, Xi

2015-06-01

The objective of this study is to explore the effects of reactive oxygen species (ROS) under hypoxic environment in prostatic stromal cells (PSC). To detect the expression of ROS in PSC and the tissues of benign prostatic hyperplasia (BPH) by flow cytometry; under hypoxic conditions, to observe the changes of cells growth and ROS in PSC; quantitative PCR was used to detect hypoxia inducible factor-1α (HIF-1α), androgen receptors (AR), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) in PSC; After edaravone intervening, to examine the changes of cells growth, ROS, HIF-1α, AR, VEGF, and IL-8 under hypoxic conditions. The expression of ROS in tissues and cells which under hypoxic condition was significantly increased. 3% O2 promoted the proliferation. The HIF-1α, AR, VEGF, and IL-8 were upregulated under 3% O2. After edaravone intervening, ROS significantly decreased, HIF-1α and VEGF were downregulated, and cell proliferation declined. Hypoxia stimulates the generation of ROS, and the ROS may play a key role in BPH.

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

NASA Astrophysics Data System (ADS)

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

2016-11-01

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

Targeting hypoxic microenvironment of pancreatic xenografts with the hypoxia-activated prodrug TH-302

PubMed Central

Lohse, Ines; Rasowski, Joanna; Cao, Pinjiang; Pintilie, Melania; Do, Trevor; Tsao, Ming-Sound; Hill, Richard P.; Hedley, David W.

2016-01-01

Previous reports have suggested that the hypoxic microenvironment provides a niche that supports tumor stem cells, and that this might explain clinical observations linking hypoxia to metastasis. To test this, we examined the effects of a hypoxia-activated prodrug, TH-302, on the tumor-initiating cell (TIC) frequency of patient-derived pancreatic xenografts (PDX). The frequencies of TIC, measured by limiting dilution assay, varied widely in 11 PDX models, and were correlated with rapid growth but not with the levels of hypoxia. Treatment with either TH-302 or ionizing radiation (IR), to target hypoxic and well-oxygenated regions, respectively, reduced TIC frequency, and the combination of TH-302 and IR was much more effective in all models tested. The combination was also more effective than TH-302 or IR alone controlling tumor growth, particularly treating the more rapidly-growing/hypoxic models. These findings support the clinical utility of hypoxia targeting in combination with radiotherapy to treat pancreatic cancers, but do not provide strong evidence for a hypoxic stem cell niche. PMID:27248663

Hydroethidine: a fluorescent redox probe for locating hypoxic cells in spheroids and murine tumours.

PubMed

Olive, P L

1989-09-01

The fluorescent redox probe hydroethidine was accumulated and metabolised about five times faster in aerobic than in hypoxic mammalian cells. Patterns of fluorescence in Chinese hamster V79 spheroids also indicated that internal hypoxic cells were less able to metabolise the drug; toxicity was observed in cells only when cell fluorescence exceeded about 500 times background. In medium equilibrated with air or nitrogen, cell accumulation of the stain was rapid, and began to plateau after 30 min; loss of ethidium was initially rapid, with a slower component after 30 min, and transfer of the metabolite ethidium between stained and unstained cells was observed after 2 h co-incubation. Sorting cells from irradiated spheroids on the basis of ethidium fluorescence provided good separation of aerobic radiosensitive and hypoxic radioresistant cells, although separation using the perfusion probe, Hoechst 33342, was superior. Similar experiments with the murine SCCVII squamous cell carcinoma suggested that hydroethidine might be a useful indirect stain for locating hypoxic cells in experimental tumours when used in combination with a perfusion probe such as Hoechst 33342.

Effects of acute hypoxic exposure on oxygen affinity of human red blood cells.

PubMed

Chowdhury, Aniket; Dasgupta, Raktim

2017-01-20

Adaptation of red blood cells subjected to acute hypoxia, crucial for managing high altitude syndrome and pulmonary diseases, has been investigated. For this, red blood cells were exposed to the acute hypoxic condition by purging nitrogen over increasing time periods from 15 to 60 min and thereafter equilibrated with atmospheric oxygen for 10 min. Raman spectra of these red blood cells were then recorded and analyzed to look for changes in the level of oxygenation compared to unexposed cells. A decreasing oxygen affinity for the cells was observed with increasing time of exposure to the hypoxic condition. This change in oxygen affinity for the red blood cells may result from metabolic adjustment of the cells under the hypoxic condition to promote increased concentration of intracellular 2, 3-diphosphoglycerate.

Indices of Psychological Strain During Hypoxis Bedrest

NASA Astrophysics Data System (ADS)

Stavrou, Nektarios A.; McDonnell, Adam C.; Eiken, Ola; Mekjavic, Igor B.

2013-02-01

Much attention has been devoted to the physiological changes that occur during bed rest. However, there has been a lack of focus on the psychological aspects per se. We investigated indices of psychological strain during three 10-d interventions, designed to assess the combined effects of inactivity/unloading and normobaric hypoxia on several physiological systems. Eleven male participants underwent three 10-d campaigns in a randomized manner: 1) normobaric hypoxic ambulatory confinement (HAMB), 2) normobaric hypoxic bed rest (HBR) and 3) normoxic bed rest (NBR). The most negative psychological profile appeared on BR10 of HBR and HAmb conditions (hypoxic conditions). Concomitantly a decrease in positive emotions was observed from BR-2 to BR10. Bed rest and exposure to hypoxic environments seems to exert a negative effect on person’s psychological mood.

Hypoxia-driven mechanism of vemurafenib resistance in melanoma

PubMed Central

Qin, Yong; Roszik, Jason; Chattopadhyay, Chandrani; Hashimoto, Yuuri; Liu, Chengwen; Cooper, Zachary A.; Wargo, Jennifer A.; Hwu, Patrick; Ekmekcioglu, Suhendan; Grimm, Elizabeth A.

2016-01-01

Melanoma is molecularly and structurally heterogeneous, with some tumor cells existing under hypoxic conditions. Our cell growth assays showed that under controlled hypoxic conditions, BRAF(V600E) melanoma cells rapidly became resistant to vemurafenib. By employing both a three-dimensional (3D) spheroid model and a two-dimensional (2D) hypoxic culture system to model hypoxia in vivo, we identified upregulation of HGF/MET signaling as a major mechanism associated with vemurafenib resistance as compared to 2D standard tissue culture in ambient air. We further confirmed that the upregulation of HGF/MET signaling was evident in drug-resistant melanoma patient tissues and mouse xenografts. Pharmacologic inhibition of the c-Met/Akt pathway restored the sensitivity of melanoma spheroids or 2D hypoxic cultures to vemurafenib. PMID:27458138

Different response to hypoxia of adipose-derived multipotent cells from obese subjects with and without metabolic syndrome

PubMed Central

Moreno-Indias, Isabel; Coín-Aragüez, Leticia; Lhamyani, Said; Alcaide Torres, Juan; Fernández-Veledo, Sonia; Vendrell, Joan; Camargo, Antonio; El Bekay, Rajaa; Tinahones, Francisco José

2017-01-01

Background/Objectives Multiple studies suggest that hypoxia, together with inflammation, could be one of the phenomena involved in the onset and progression of obesity-related insulin resistance. In addition, dysfunction of adipose tissue in obese subjects with metabolic syndrome is associated with decreased angiogenesis. However, some subjects with a high body mass index do not develop metabolic abnormalities associated with obesity. The aim of the current study was to examine the neovascular properties of visceral adipose tissue-derived multipotent mesenchymal cells subjected to hypoxia (hypox-visASCs) from normal-weight subjects (Nw) and obese patients with metabolic syndrome (MS) and without metabolic syndrome (NonMS). Methods This was a 2-year study to enroll subjects who underwent bariatric surgery or cholecystectomy. Eight patients who underwent either bariatric surgery or cholecystectomy (27 patients) participated in the study. Visceral adipose tissue samples from Nw, MS and NonMS subjects were processed by enzymatic digestion. VisASCs cultured under hypoxic conditions were characterized by tubule formation assay, ELISA, flow cytometry, migration rate, and qRT-PCR, and the effects of visASCs-conditioned medium on survival and endothelial cell tubule formation were evaluated. Results Hypox-visASCs from NonMS subjects showed a greater capacity for tubule formation than hypox-visASCs from Nw and MS subjects. The lower percentage of CD140b+/CD44+ and CD140b+/CD184+ cells observed in hypox-visASCs from NonMS subjects compared to MS subjects was accompanied not only by a lower migration rate from the chemotactic effects of stromal cell derived factor 1α, but also by lower levels of NOX5 mRNA expression. While the levels of monocyte chemoattractant protein 1 mRNA expressed by hypox-visASCs correlated positively with the body mass index and waist circumference of the subjects, the concentration of vascular endothelial growth factor present in hypox-visASC-conditioned culture medium decreased significantly with increasing plasma glucose. The survival rate and tubules formed by endothelial cells cultured in hypox-visASC-conditioned medium decreased significantly with increasing homeostasis model assessment to quantify insulin resistance. Conclusions Our results suggest that hypox-visASCs from NonMS subjects could promote healthy adipose tissue expansion, while hypox-visASCs from MS subjects appear to contribute to the decreased angiogenic potential and increased inflammation underlying adipose tissue dysfunction in obesity. Our results emphasize the importance of taking into account not only the BMI but also the metabolic profile of the subjects during the implementation of ASCs-based therapy to promote neovascularization. PMID:29166648

Preparation and characterization of dextran nanobubbles for oxygen delivery.

PubMed

Cavalli, R; Bisazza, A; Giustetto, P; Civra, A; Lembo, D; Trotta, G; Guiot, C; Trotta, M

2009-11-03

Dextran nanobubbles were prepared with a dextran shell and a perfluoropentan core in which oxygen was stored. To increase the stability polyvinylpirrolidone was also added to the formulation as stabilizing agent. Rhodamine B was used as fluorescent marker to obtain fluorescent nanobubbles. The nanobubble formulations showed sizes of about 500nm, a negative surface charge and a good capacity of loading oxygen, no hemolytic activity or toxic effect on cell lines. The fluorescent labelled nanobubbles could be internalized in Vero cells. Oxygen-filled nanobubbles were able to release oxygen in different hypoxic solutions at different time after their preparation in in vitro experiments. The oxygen release kinetics could be enhanced after nanobubble insonation with ultrasound at 2.5MHz. The oxygen-filled nanobubble formulations might be proposed for therapeutic applications in various diseases.

Outcomes in childhood following therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy (HIE).

PubMed

Natarajan, Girija; Pappas, Athina; Shankaran, Seetha

2016-12-01

In this article, we review the childhood outcomes of neonates with birth depression and/or hypoxic-ischemic encephalopathy. The outcomes of these children prior to the era of hypothermia for neuroprotection will first be summarized, followed by discussion of results from randomized controlled trials of therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy. The predictors of outcome in childhood following neonatal HIE using clinical and imaging biomarkers following hypothermia therapy will be described. Copyright © 2016 Elsevier Inc. All rights reserved.

Pretreatment with magnesium sulfate attenuates white matter damage by preventing cell death of developing oligodendrocytes.

PubMed

Seyama, Takahiro; Kamei, Yoshimasa; Iriyama, Takayuki; Imada, Shinya; Ichinose, Mari; Toshimitsu, Masatake; Fujii, Tomoyuki; Asou, Hiroaki

2018-04-01

Antenatal maternal administration of magnesium sulfate (MgSO 4 ) reduces cerebral palsy in preterm infants. However, it remains controversial as to whether it also reduces occurrence of white matter damage, or periventricular leukomalacia. We assessed the effect of MgSO 4 against white matter damage induced by hypoxic-ischemic insult using a neonatal rat model and culture of premyelinating oligodendrocytes (pre-OL). Rat pups at postnatal day (P) 6 were administered either MgSO 4 or vehicle intraperitoneally before hypoxic-ischemic insult (unilateral ligation of the carotid artery followed by 6% oxygen for 1 h). The population of oligodendrocyte (OL) markers and CD-68-positive microglia at P11, and TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL)-positive cells at P8 were evaluated in pericallosal white matter. Primary cultures of mouse pre-OL were subjected to oxygen glucose deprivation condition, and the lactate dehydrogenase release from culture cells was evaluated to assess cell viability. Pretreatment with MgSO 4 attenuated the loss of OL markers, such as myelin basic protein and Olig2, in ipsilateral pericallosal white matter and decreased the number of CD-68-positive microglia and TUNEL-positive cells in vivo. Pretreatment with MgSO 4 also inhibited lactate dehydrogenase release from pre-OL induced by oxygen glucose deprivation in vitro. Pretreatment with MgSO 4 attenuates white matter damage by preventing cell death of pre-OL. © 2018 Japan Society of Obstetrics and Gynecology.

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

PubMed

Poupard, Nicolas; Badarou, Pamela; Fasani, Fabienne; Groult, Hugo; Bridiau, Nicolas; Sannier, Frédéric; Bordenave-Juchereau, Stéphanie; Kieda, Claudine; Piot, Jean-Marie; Grillon, Catherine; Fruitier-Arnaudin, Ingrid; Maugard, Thierry

2017-05-09

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC 50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a Matrigel TM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Polyacetylene glycoside attenuates ischemic kidney injury by co-inhibiting inflammation, mitochondria dysfunction and lipotoxicity.

PubMed

Zhou, Yijie; Du, Dan; Liu, Shuyun; Zhao, Meng; Yuan, Yujia; Li, Lan; Chen, Younan; Lu, Yanrong; Cheng, Jingqiu; Liu, Jingping

2018-07-01

Ischemic acute kidney injury (AKI) is a serious clinical problem and no efficient therapeutics is available in clinic now. Natural polyacetylene glycosides (PGAs) had shown antioxidant and anti-inflammatory properties, but their effects on kidney injury have not been evaluated. This study aimed to investigate the protective effect of PGA on ischemic kidney injury in renal tubular epithelial cells (TECs) and mice. Hypoxic HK-2 cells and renal ischemia/reperfusion injury (IRI) mice were treated with PGA from Coreopsis tinctoria, and the cell viability, renal function, apoptosis, inflammation, mitochondrial injury, lipids metabolism were analyzed. In vitro results showed that PGA improved cell viability and reduced oxidative stress, pro-apoptotic/pro-inflammatory factors expression and NFκB activation in TECs under hypoxia/reperfusion (H/R). Moreover, PGA reduced mitochondria oxidative stress and improved ATP production, ΔΨm and mitochondria biogenesis, and inhibited lipids uptake, biosynthesis and accumulation in hypoxic TECs. In vivo, PGA significantly attenuated kidney injury and reduced blood urea nitrogen (BUN), serum creatinine (CREA) and urinary albumin (Alb), and increased creatinine clearance (CC) in IRI mice. PGA also decreased cell apoptosis, mitochondria oxidative stress, inflammatory response and lipid droplets accumulation, and promoted ATP generation in kidney of IRI mice. Our results proved that PGA ameliorated ischemic kidney injury via synergic anti-inflammation, mitochondria protection and anti-lipotoxicity actions, and it might be a promising multi-target therapy for ischemic AKI. Copyright © 2018. Published by Elsevier Inc.

* Hypoxia for Mesenchymal Stem Cell Expansion and Differentiation: The Best Way for Enhancing TGFß-Induced Chondrogenesis and Preventing Calcifications in Alginate Beads.

PubMed

Henrionnet, Christel; Liang, Gai; Roeder, Emilie; Dossot, Manuel; Wang, Hui; Magdalou, Jacques; Gillet, Pierre; Pinzano, Astrid

2017-09-01

We examined the respective influence of a sequential or a continuous hypoxia during expansion and transforming growth factor beta 1-driven chondrogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differentiation was performed within alginate beads, a classical tool for the implantation of MSCs within the joint. The standard normoxic 2D (expansion) and 3D (differentiation) MSCs cultures served as reference. To determine the quality of chondrogenesis, we analyzed typical markers such as type II and X collagens, SOX9, COMP, versican, and aggrecan mRNAs using polymerase chain reaction and we assessed the production of type II collagen and hypoxia-inducible factor (HIF)-1α by histological stainings. We simultaneously assessed the expression of osteogenic mRNAs (Alkaline Phosphatase, RUNX2, and Osteocalcin) and the presence of micro-calcifications by Alizarin red and Raman spectroscopy. Chondrogenic differentiation is clearly improved by hypoxia in 3D. Best results were obtained when the entire process, that is, 2D expansion and 3D differentiation, was performed under continuous 5% hypoxic condition. In addition, no calcification (hydroxyapatite, proved by RAMAN) was observed after 2D hypoxic expansion even in the case of a normoxic differentiation, in contrast with controls. Finally, a better chondrogenic differentiation of human MSCs is achieved when a reduced oxygen tension is applied during both expansion and differentiation times, avoiding in vitro osteogenic commitment of cells and subsequently the calcification deposition.

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

PubMed Central

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

2016-01-01

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

RBE and OER within the spread-out Bragg peak for proton beam therapy: in vitro study at the Proton Medical Research Center at the University of Tsukuba

PubMed Central

Kanemoto, Ayae; Hirayama, Ryoichi; Moritake, Takashi; Furusawa, Yoshiya; Sun, Lue; Sakae, Takeji; Kuno, Akihiro; Terunuma, Toshiyuki; Yasuoka, Kiyoshi; Mori, Yutaro; Tsuboi, Koji; Sakurai, Hideyuki

2014-01-01

There are few reports on the biological homogeneity within the spread-out Bragg peak (SOBP) of proton beams. Therefore, to evaluate the relative biological effectiveness (RBE) and the oxygen enhancement ratio (OER), human salivary gland tumor (HSG) cells were irradiated at the plateau position (position A) and three different positions within a 6-cm-wide SOBP (position B, 26 mm proximal to the middle; position C, middle; position D, 26 mm distal to the middle) using 155-MeV/n proton beams under both normoxic and hypoxic conditions at the Proton Medical Research Center, University of Tsukuba, Japan. The RBE to the plateau region (RBEplateau) and the OER value were calculated from the doses corresponding to 10% survival data. Under the normoxic condition, the RBEplateau was 1.00, 0.99 and 1.09 for positions B, C and D, respectively. Under the hypoxic condition, the RBEplateau was 1.10, 1.06 and 1.12 for positions B, C and D, respectively. The OER was 2.84, 2.60, 2.63 and 2.76 for positions A, B, C and D, respectively. There were no significant differences in either the RBEplateau or the OER between these three positions within the SOBP. In conclusion, biological homogeneity need not necessarily be taken into account for treatment planning for proton beam therapy at the University of Tsukuba. PMID:24876271

STAT3 precedes HIF1α transcriptional responses to oxygen and oxygen and glucose deprivation in human brain pericytes.

PubMed

Carlsson, Robert; Özen, Ilknur; Barbariga, Marco; Gaceb, Abderahim; Roth, Michaela; Paul, Gesine

2018-01-01

Brain pericytes are important to maintain vascular integrity of the neurovascular unit under both physiological and ischemic conditions. Ischemic stroke is known to induce an inflammatory and hypoxic response due to the lack of oxygen and glucose in the brain tissue. How this early response to ischemia is molecularly regulated in pericytes is largely unknown and may be of importance for future therapeutic targets. Here we evaluate the transcriptional responses in in vitro cultured human brain pericytes after oxygen and/or glucose deprivation. Hypoxia has been widely known to stabilise the transcription factor hypoxia inducible factor 1-alpha (HIF1α) and mediate the induction of hypoxic transcriptional programs after ischemia. However, we find that the transcription factors Jun Proto-Oncogene (c-JUN), Nuclear Factor Of Kappa Light Polypeptide Gene Enhancer In B-Cells (NFκB) and signal transducer and activator of transcription 3 (STAT3) bind genes regulated after 2hours (hs) of omitted glucose and oxygen before HIF1α. Potent HIF1α responses require 6hs of hypoxia to substantiate transcriptional regulation comparable to either c-JUN or STAT3. Phosphorylated STAT3 protein is at its highest after 5 min of oxygen and glucose (OGD) deprivation, whereas maximum HIF1α stabilisation requires 120 min. We show that STAT3 regulates angiogenic and metabolic pathways before HIF1α, suggesting that HIF1α is not the initiating trans-acting factor in the response of pericytes to ischemia.

ATR Kinase Inhibition Protects Non-cycling Cells from the Lethal Effects of DNA Damage and Transcription Stress*

PubMed Central

Kemp, Michael G.; Sancar, Aziz

2016-01-01

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase that maintains genome stability and halts cell cycle phase transitions in response to DNA lesions that block DNA polymerase movement. These DNA replication-associated features of ATR function have led to the emergence of ATR kinase inhibitors as potential adjuvants for DNA-damaging cancer chemotherapeutics. However, whether ATR affects the genotoxic stress response in non-replicating, non-cycling cells is currently unknown. We therefore used chemical inhibition of ATR kinase activity to examine the role of ATR in quiescent human cells. Although ATR inhibition had no obvious effects on the viability of non-cycling cells, inhibition of ATR partially protected non-replicating cells from the lethal effects of UV and UV mimetics. Analyses of various DNA damage response signaling pathways demonstrated that ATR inhibition reduced the activation of apoptotic signaling by these agents in non-cycling cells. The pro-apoptosis/cell death function of ATR is likely due to transcription stress because the lethal effects of compounds that block RNA polymerase movement were reduced in the presence of an ATR inhibitor. These results therefore suggest that whereas DNA polymerase stalling at DNA lesions activates ATR to protect cell viability and prevent apoptosis, the stalling of RNA polymerases instead activates ATR to induce an apoptotic form of cell death in non-cycling cells. These results have important implications regarding the use of ATR inhibitors in cancer chemotherapy regimens. PMID:26940878

The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer

DOE PAGES

Xu, Weidong; Neill, Thomas; Yang, Yuefeng; ...

2014-12-11

In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β- catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle tomore » establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Finally, based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.« less

A nonreplicating subunit vaccine protects mice against lethal Ebola virus challenge

PubMed Central

Phoolcharoen, Waranyoo; Dye, John M.; Kilbourne, Jacquelyn; Piensook, Khanrat; Pratt, William D.; Arntzen, Charles J.; Chen, Qiang; Mason, Hugh S.; Herbst-Kralovetz, Melissa M.

2011-01-01

Ebola hemorrhagic fever is an acute and often deadly disease caused by Ebola virus (EBOV). The possible intentional use of this virus against human populations has led to design of vaccines that could be incorporated into a national stockpile for biological threat reduction. We have evaluated the immunogenicity and efficacy of an EBOV vaccine candidate in which the viral surface glycoprotein is biomanufactured as a fusion to a monoclonal antibody that recognizes an epitope in glycoprotein, resulting in the production of Ebola immune complexes (EICs). Although antigen–antibody immune complexes are known to be efficiently processed and presented to immune effector cells, we found that codelivery of the EIC with Toll-like receptor agonists elicited a more robust antibody response in mice than did EIC alone. Among the compounds tested, polyinosinic:polycytidylic acid (PIC, a Toll-like receptor 3 agonist) was highly effective as an adjuvant agent. After vaccinating mice with EIC plus PIC, 80% of the animals were protected against a lethal challenge with live EBOV (30,000 LD50 of mouse adapted virus). Surviving animals showed a mixed Th1/Th2 response to the antigen, suggesting this may be important for protection. Survival after vaccination with EIC plus PIC was statistically equivalent to that achieved with an alternative viral vector vaccine candidate reported in the literature. Because nonreplicating subunit vaccines offer the possibility of formulation for cost-effective, long-term storage in biothreat reduction repositories, EIC is an attractive option for public health defense measures. PMID:22143779

Reduced O2 concentration during CAM development--its effect on angiogenesis and gene expression in the broiler embryo CAM.

PubMed

Druyan, S; Levi, E

2012-01-01

Hypoxia during embryogenesis may induce changes in the development of some physiological regulatory systems, thereby causing permanent phenotypic changes in the embryo. Various levels of hypoxia at different time points during embryogenesis were found to affect both anatomical and physiological morphogenesis. These changes and adaptations depended on the timing, intensity, and duration of the hypoxic exposure and, moreover, were regulated by differential expression of developmentally important genes, mostly expressed in a stage- and time-dependent manner. Eggs incubated in a 17%-oxygen atmosphere for 12 h/d from E5 through E12 exhibited a clear and significant increase in the vascular area of the chorioallantoic membrane (CAM); an increase that was already significant within 12 h after the end of the 1st hypoxic exposures (E6). We used the combination of the genes, β-actin, RPLP0 and HPRT as a reference for gene expression profiling, in studying the expression levels of hypoxia-inducible factor 1-alpha (HIF1α), vascular endothelial growth factor alpha-2 (VEGF α 2), vascular endothelial growth factor receptor 2 (KDR), matrix metalloproteinase-2 (MMP2), and fibroblast growth factor 2 (FGF2), under normal and hypoxic conditions. In general, expression of all five investigated genes throughout the embryonic day of development had similar patterns of hypoxia-induced alterations. In E5.5 embryos, expression of HIF1α, MMP2, VEGFα2, and KDR was significantly higher in hypoxic embryos than in controls. In E6 embryos expression of HIF1α, VEGFα2, and FGF2 was significantly higher in hypoxic embryos than in controls. From E6.5 onward expression levels of the examined genes did not show any differences between hypoxic and control embryos. It can be concluded that in this experimental model, exposing broiler embryos to 17% O(2) from E5 to E7 induced significant angiogenesis, as expressed by the above genes. Further studies to examine whether this early exposure to hypoxic condition affects the chick's ability to withstand a post-hatch hypoxic environment is still required. Copyright © 2012 Elsevier B.V. All rights reserved.

Transduction of hematopoietic stem cells to stimulate RNA interference against feline infectious peritonitis.

PubMed

Anis, Eman A; Dhar, Madhu; Legendre, Alfred M; Wilkes, Rebecca P

2017-06-01

Objectives The goals of the study were: (1) to develop and evaluate non-replicating lentivirus vectors coding for feline coronavirus (FCoV)-specific micro (mi)RNA as a potential antiviral therapy for feline infectious peritonitis (FIP); (2) to assess the feasibility of transducing hematopoietic stem cells (HSCs) with ex vivo introduction of the miRNA-expressing lentivirus vector; and (3) to assess the ability of the expressed miRNA to inhibit FCoV replication in HSCs in vitro. Methods HSCs were obtained from feline bone marrow and replicated in vitro. Three lentiviruses were constructed, each expressing a different anti-FCoV miRNA. HSCs were stably transduced with the miRNA-expressing lentivirus vector that produced the most effective viral inhibition in a feline cell line. The effectiveness of the transduction and the expression of anti-FCoV miRNA were tested by infecting the HSCs with two different strains of FCoV. The inhibition of coronavirus replication was determined by relative quantification of the inhibition of intracellular viral genomic RNA synthesis using real-time, reverse-transcription PCR. The assessment of virus replication inhibition was determined via titration of extracellular virus using the TCID 50 assay. Results Inhibition of FCoV was most significant in feline cells expressing miRNA-L2 that targeted the viral leader sequence, 48 h postinfection. miRNA-L2 expression in stably transduced HSCs resulted in 90% and 92% reductions in FIPV WSU 79-1146 genomic RNA synthesis and extracellular virus production, respectively, as well as 74% and 80% reduction in FECV WSU 79-1683 genomic RNA synthesis and extracellular virus production, respectively, as compared with an infected negative control sample producing non-targeting miRNA. Conclusions and relevance These preliminary results show that genetic modification of HSCs for constitutive production of anti-coronavirus miRNA will reduce FCoV replication.

Overexpression of IGF-I receptor in HeLa cells enhances in vivo radioresponse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaneko, Haruna; Yu, Dong; Miura, Masahiko

2007-11-30

Insulin-like growth factor I receptor (IGF-IR) is a transmembrane receptor tyrosine kinase whose activation strongly promotes cell growth and survival. We previously reported that IGF-IR activity confers intrinsic radioresistance in mouse embryo fibroblasts in vitro. However, it is still unclear whether tumor cells overexpressing IGF-IR exhibit radioresistance in vivo. For this purpose, we established HeLa cells that overexpress IGF-IR (HeLa-R), subcutaneously transplanted these cells into nude mice, and examined radioresponse in the resulting solid tumors. HeLa-R cells exhibited typical in vitro phenotypes generally observed in IGF-IR-overexpressing cells, as well as significant intrinsic radioresistance in vitro compared with parent cells. Asmore » expected, the transplanted HeLa-R tumors grew at a remarkably higher rate than parent tumors. Histological analysis revealed that HeLa-R tumors expressed more VEGF and had a higher density of tumor vessels. Unexpectedly, a marked growth delay was observed in HeLa-R tumors following 10 Gy of X-irradiation. Immunostaining of HeLa-R tumors for the hypoxia marker pimonidazole revealed a significantly lower level of hypoxic cells. Moreover, clamp hypoxia significantly increased radioresistance in HeLa-R tumors. Tumor microenvironments in vivo generated by the IGF-IR expression thus could be a major factor in determining the tumor radioresponse in vivo.« less

Organotypic three-dimensional assays based on human leiomyoma–derived matrices

PubMed Central

Dourado, Mauricio Rocha; Sundquist, Elias; Apu, Ehsanul Hoque; Alahuhta, Ilkka; Tuomainen, Katja; Vasara, Jenni; Al-Samadi, Ahmed

2018-01-01

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro. Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel®. However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel®. Additionally, Myogel can replace Matrigel® in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays. This article is part of the discussion meeting issue ‘Extracellular vesicles and the tumour microenvironment’. PMID:29158312

Organotypic three-dimensional assays based on human leiomyoma-derived matrices.

PubMed

Salo, Tuula; Dourado, Mauricio Rocha; Sundquist, Elias; Apu, Ehsanul Hoque; Alahuhta, Ilkka; Tuomainen, Katja; Vasara, Jenni; Al-Samadi, Ahmed

2018-01-05

Alongside cancer cells, tumours exhibit a complex stroma containing a repertoire of cells, matrix molecules and soluble factors that actively crosstalk between each other. Recognition of this multifaceted concept of the tumour microenvironment (TME) calls for authentic TME mimetics to study cancer in vitro Traditionally, tumourigenesis has been investigated in non-human, three-dimensional rat type I collagen containing organotypic discs or by means of mouse sarcoma-derived gel, such as Matrigel ® However, the molecular compositions of these simplified assays do not properly simulate human TME. Here, we review the main properties and benefits of using human leiomyoma discs and their matrix Myogel for in vitro assays. Myoma discs are practical for investigating the invasion of cancer cells, as are cocultures of cancer and stromal cells in a stiff, hypoxic TME mimetic. Myoma discs contain soluble factors and matrix molecules commonly present in neoplastic stroma. In Transwell, IncuCyte, spheroid and sandwich assays, cancer cells move faster and form larger colonies in Myogel than in Matrigel ® Additionally, Myogel can replace Matrigel ® in hanging-drop and tube-formation assays. Myogel also suits three-dimensional drug testing and extracellular vesicle interactions. To conclude, we describe the application of our myoma-derived matrices in 3D in vitro cancer assays.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'. © 2017 The Authors.

Designing a Microfluidic Device with Integrated Ratiometric Oxygen Sensors for the Long-Term Control and Monitoring of Chronic and Cyclic Hypoxia

PubMed Central

Grist, Samantha M.; Schmok, Jonathan C.; Liu, Meng-Chi (Andy); Chrostowski, Lukas; Cheung, Karen C.

2015-01-01

Control of oxygen over cell cultures in vitro is a topic of considerable interest, as chronic and cyclic hypoxia can alter cell behaviour. Both static and transient hypoxic levels have been found to affect tumour cell behaviour; it is potentially valuable to include these effects in early, in vitro stages of drug screening. A barrier to their inclusion is that rates of transient hypoxia can be a few cycles/hour, which is difficult to reproduce in traditional in vitro cell culture environments due to long diffusion distances from control gases to the cells. We use a gas-permeable three-layer microfluidic device to achieve spatial and temporal oxygen control with biologically-relevant switching times. We measure the oxygen profiles with integrated, ratiometric optical oxygen sensors, demonstrate sensor and system stability over multi-day experiments, and characterize a pre-bleaching process to improve sensor stability. We show, with both finite-element modelling and experimental data, excellent control over the oxygen levels by the device, independent of fluid flow rate and oxygenation for the operating flow regime. We measure equilibration times of approximately 10 min, generate complex, time-varying oxygen profiles, and study the effects of oxygenated media flow rates on the measured oxygen levels. This device could form a useful tool for future long-term studies of cell behaviour under hypoxia. PMID:26287202

Cell sheet engineering using the stromal vascular fraction of adipose tissue as a vascularization strategy.

PubMed

Costa, Marina; Cerqueira, Mariana T; Santos, Tírcia C; Sampaio-Marques, Belém; Ludovico, Paula; Marques, Alexandra P; Pirraco, Rogério P; Reis, Rui L

2017-06-01

Current vascularization strategies for Tissue Engineering constructs, in particular cell sheet-based, are limited by time-consuming and expensive endothelial cell isolation and/or by the complexity of using extrinsic growth factors. Herein, we propose an alternative strategy using angiogenic cell sheets (CS) obtained from the stromal vascular fraction (SVF) of adipose tissue that can be incorporated into more complex constructs. Cells from the SVF were cultured in normoxic and hypoxic conditions for up to 8days in the absence of extrinsic growth factors. Immunocytochemistry against CD31 and CD146 revealed spontaneous organization in capillary-like structures, more complex after hypoxic conditioning. Inhibition of HIF-1α pathway hindered capillary-like structure formation in SVF cells cultured in hypoxia, suggesting a role of HIF-1α. Moreover, hypoxic SVF cells showed a trend for increased secretion of angiogenic factors, which was reflected in increased network formation by endothelial cells cultured on matrigel using that conditioned medium. In vivo implantation of SVF CS in a mouse hind limb ischemia model revealed that hypoxia-conditioned CS led to improved restoration of blood flow. Both in vitro and in vivo data suggest that SVF CS can be used as simple and cost-efficient tools to promote functional vascularization of TE constructs. Neovascularization after implantation is a major obstacle for producing clinically viable cell sheet-based tissue engineered constructs. Strategies using endothelial cells and extrinsic angiogenic growth factors are expensive and time consuming and may raise concerns of tumorigenicity. In this manuscript, we describe a simplified approach using angiogenic cell sheets fabricated from the stromal vascular fraction of adipose tissue. The strong angiogenic behavior of these cell sheets, achieved without the use of external growth factors, was further stimulated by low oxygen culture. When implanted in an in vivo model of hind limb ischemia, the angiogenic cell sheets contributed to blood flux recovery. These cell sheets can therefore be used as a straightforward tool to increase the neovascularization of cell sheet-based thick constructs. Copyright © 2017. Published by Elsevier Ltd.

The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1{alpha} targeted gene expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyake, Kotaro, E-mail: hif.panc@gmail.com; Nishioka, Masanori; Imura, Satoru

Tumor hypoxia has been considered to be a potential therapeutic target, because hypoxia is a common feature of solid tumors and is associated with their malignant phenotype. In the present study, we investigated the antitumor effect of a novel hypoxic cytotoxin, 3-[2-hydroxyethyl(methyl)amino]-2-quinoxalinecarbonitrile 1,4-dioxide (TX-2098) in inhibiting the expression of hypoxia inducible factor-1{alpha} (HIF-1{alpha}), and consequently vascular endothelial cell growth factor (VEGF) expression in pancreatic cancer. The antitumor effects of TX-2098 under hypoxia were tested against various human pancreatic cancer cell lines using WST-8 assay. VEGF protein induced pancreatic cancer was determined on cell-free supernatant by ELISA. Moreover, nude mice bearingmore » subcutaneously (s.c.) or orthotopically implanted human SUIT-2 were treated with TX-2098. Tumor volume, survival and expression of HIF-1 and associated molecules were evaluated in treatment versus control groups. In vitro, TX-2098 inhibited the proliferation of various pancreatic cancer cell lines. In s.c model, tumors from nude mice injected with pancreatic cancer cells and treated with TX-2098 showed significant reductions in volume (P < 0.01 versus control). Quantitative real-time reverse transcription-PCR analysis revealed that TX-2098 significantly inhibited mRNA expression of the HIF-1 associated molecules, VEGF, glucose transporter 1 and Aldolase A (P < 0.01 versus control). These treatments also prolong the survival in orthotopic models. These results suggest that the effect of TX-2098 in pancreatic cancer might be correlated with the expression of VEGF and HIF-1 targeted molecules. -- Highlights: Black-Right-Pointing-Pointer We designed and synthesized novel hypoxic cytoxin, TX-2098. Black-Right-Pointing-Pointer TX-2098 inhibited the proliferation of human pancreatic cancer cells than TPZ. Black-Right-Pointing-Pointer TX-2098 reduced VEGF protein level than TPZ. Black-Right-Pointing-Pointer TX-2098 inhibited mRNA expression of VEGF, GLUT1 and Aldolase A, not HIF-1{alpha}. Black-Right-Pointing-Pointer TX-2098 improved the survival in orthotopic SUIT-2 xenograft model.« less

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

PubMed Central

Christou, Helen; Reslan, Ossama M.; Mam, Virak; Tanbe, Alain F.; Vitali, Sally H.; Touma, Marlin; Arons, Elena; Mitsialis, S. Alex; Kourembanas, Stella

2012-01-01

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O2) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH4Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH4Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH. PMID:22287610

[Elucidating the molecular mechanism of prostate cancer progression under chronic hypoxia and development of the novel therapeutic approach].

PubMed

Nomura, Takeo; Yamasaki, Mutsushi; Mimata, Hiromitsu

2014-12-01

Cancer cells encounter a hypoxic microenvironment during tumor growth and progression. In addition, androgen-deprivation therapy against prostate cancer can develop secondary to a hypoxic condition caused by drastic blood supply reduction because androgen drives angiogenic inducers including vascular endothelial growth factor (VEGF) and inhibits angiogenesis inhibitor prostatic pigment epithelium-derived factor (PEDF). Extreme hypoxic conditions are not suitable for cancer survival, however, cancer cells soon adapt to a hypoxic environment and survive. We established a prostate cancer cell line cultured under chronic hypoxia and analyzed a castration-resistant phenotype. Here, the Vav3 was identified as a key oncogenic molecule associated with castration-resistance under chronic hypoxia. We analyzed the functions of Vav3 and Vav3-mediated signaling to establish a novel therapeutic target for castration-resistant prostate cancer.

Disparate roles of zinc in chemical hypoxia-induced neuronal death

PubMed Central

Kim, Sujeong; Seo, Jung-Woo; Oh, Shin Bi; Kim, So Hee; Kim, Inki; Suh, Nayoung; Lee, Joo-Yong

2015-01-01

Accumulating evidence has provided a causative role of zinc (Zn2+) in neuronal death following ischemic brain injury. Using a hypoxia model of primary cultured cortical neurons with hypoxia-inducing chemicals, cobalt chloride (1 mM CoCl2), deferoxamine (3 mM DFX), and sodium azide (2 mM NaN3), we evaluated whether Zn2+ is involved in hypoxic neuronal death. The hypoxic chemicals rapidly elicited intracellular Zn2+ release/accumulation in viable neurons. The immediate addition of the Zn2+ chelator, CaEDTA or N,N,N’N’-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), prevented the intracellular Zn2+ load and CoCl2-induced neuronal death, but neither 3 hour later Zn2+ chelation nor a non-Zn2+ chelator ZnEDTA (1 mM) demonstrated any effects. However, neither CaEDTA nor TPEN rescued neurons from cell death following DFX- or NaN3-induced hypoxia, whereas ZnEDTA rendered them resistant to the hypoxic injury. Instead, the immediate supplementation of Zn2+ rescued DFX- and NaN3-induced neuronal death. The iron supplementation also afforded neuroprotection against DFX-induced hypoxic injury. Thus, although intracellular Zn2+ release/accumulation is common during chemical hypoxia, Zn2+ might differently influence the subsequent fate of neurons; it appears to play a neurotoxic or neuroprotective role depending on the hypoxic chemical used. These results also suggest that different hypoxic chemicals may induce neuronal death via distinct mechanisms. PMID:25667569

Disparate roles of zinc in chemical hypoxia-induced neuronal death.

PubMed

Kim, Sujeong; Seo, Jung-Woo; Oh, Shin Bi; Kim, So Hee; Kim, Inki; Suh, Nayoung; Lee, Joo-Yong

2015-01-01

Accumulating evidence has provided a causative role of zinc (Zn(2+)) in neuronal death following ischemic brain injury. Using a hypoxia model of primary cultured cortical neurons with hypoxia-inducing chemicals, cobalt chloride (1 mM CoCl2), deferoxamine (3 mM DFX), and sodium azide (2 mM NaN3), we evaluated whether Zn(2+) is involved in hypoxic neuronal death. The hypoxic chemicals rapidly elicited intracellular Zn(2+) release/accumulation in viable neurons. The immediate addition of the Zn(2+) chelator, CaEDTA or N,N,N'N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), prevented the intracellular Zn(2+) load and CoCl2-induced neuronal death, but neither 3 hour later Zn(2+) chelation nor a non-Zn(2+) chelator ZnEDTA (1 mM) demonstrated any effects. However, neither CaEDTA nor TPEN rescued neurons from cell death following DFX- or NaN3-induced hypoxia, whereas ZnEDTA rendered them resistant to the hypoxic injury. Instead, the immediate supplementation of Zn(2+) rescued DFX- and NaN3-induced neuronal death. The iron supplementation also afforded neuroprotection against DFX-induced hypoxic injury. Thus, although intracellular Zn(2+) release/accumulation is common during chemical hypoxia, Zn(2+) might differently influence the subsequent fate of neurons; it appears to play a neurotoxic or neuroprotective role depending on the hypoxic chemical used. These results also suggest that different hypoxic chemicals may induce neuronal death via distinct mechanisms.

In vitro evaluation of the monoclonal antibody 64Cu-IgG M75 against human carbonic anhydrase IX and its in vivo imaging.

PubMed

Čepa, Adam; Ráliš, Jan; Král, Vlastimil; Paurová, Monika; Kučka, Jan; Humajová, Jana; Lázníček, Milan; Lebeda, Ondřej

2018-03-01

Specific oncology diagnostics requires new types of the selective radiopharmaceuticals, particularly those suitable for the molecular PET imaging. The aim of this work is to present a new, specific PET-immunodiagnostic radiopharmaceutical based on the monoclonal antibody IgG M75 targeting human carbonic anhydrase IX labelled with 64 Cu (T ½ = 12.70h) and its in vitro and in vivo evaluation. The antibody IgG M75 was conjugated with a non-commercial copper-specific chelator "phosphinate" and then labelled with the positron emitter 64 Cu. Stability of the labelled conjugated was tested in human serum. The immunoreactivity of the labelled conjugate was evaluated in vitro on a suitable cell cultures of the colorectal carcinoma (HT-29) and its imaging properties were estimated in vivo on a mouse model with inoculated colorectal carcinoma HT-29 imaged on a µPET/CT. The tested radioimmunoconjugate was obtained in a specific activity of 0.25-0.5 MBq/µg. In vitro uptake experiments revealed specific binding to the HT-29 cells (45 ± 2.8% of the total added activity) and the measured K D value was found to be 9.2nM. Imaging clearly demonstrated significant uptake of the labelled monoclonal antibody in the tumour at 18h post administration. The radioimmunoconjugate 64 Cu-PS-IgG M75 seems to be a suitable candidate for PET diagnostics of hypoxic tumours expressing human carbonic anhydrase IX. Copyright © 2017 Elsevier Ltd. All rights reserved.

Enhancing Contents of γ-Aminobutyric Acid (GABA) and Other Micronutrients in Dehulled Rice during Germination under Normoxic and Hypoxic Conditions.

PubMed

Ding, Junzhou; Yang, Tewu; Feng, Hao; Dong, Mengyi; Slavin, Margaret; Xiong, Shanbai; Zhao, Siming

2016-02-10

Biofortification of staple grains with high contents of essential micronutrients is an important strategy to overcome micronutrient malnutrition. However, few attempts have targeted at γ-aminobutyric acid (GABA), a functional nutrient for aging populations. In this study, two rice cultivars, Heinuo and Xianhui 207, were used to investigate changes in GABA and other nutritional compounds of dehulled rice after germination under normoxic and hypoxic conditions. Forty-one metabolites were identified in both cultivars treated by normoxic germination, whereas the germinated dehulled rice of Heinuo and Xianhui 207 under hypoxic treatment had 43 and 41 metabolites identified, respectively. GABA increased in dehulled rice after germination, especially under hypoxia. Meanwhile, a number of other health-beneficial and/or flavor-related compounds such as lysine and d-mannose increased after the hypoxic treatment. The accumulation of GABA exhibited genotype-specific modes in both normoxic and hypoxic treatments. With regard to GABA production, Xianhui 207 was more responsive to the germination process than Heinuo, whereas Heinuo was more responsive to hypoxia than Xianhui 207. This study provides a promising approach to biofortify dehulled rice with increased GABA and other nutrients through metabolomic-based regulation.

Transcriptome analysis demonstrates that long noncoding RNA is involved in the hypoxic response in Larimichthys crocea.

PubMed

Liu, Wei; Liu, Xiaoxu; Wu, Changwen; Jiang, Lihua

2018-06-15

The large yellow croaker (Larimichthys crocea) has low hypoxia tolerance compared with other fish species, and the mRNA levels of hypoxia-inducible factor (HIF)-1α in its brain do not change markedly under hypoxic conditions. In this study, we investigated noncoding transcription in the hypoxic response mechanism of L. crocea. We generated a catalog of long noncoding RNAs (lncRNAs) from the brain of L. crocea individuals under hypoxic stress, investigated lncRNA expression patterns, and analyzed the HIF signaling pathway by RNA sequencing. Prolyl hydroxylase domain 2 (PHD2) expression significantly increased after 6 and 12 h of hypoxia, and a lncRNA (Linc_06633.1) was found in the upstream, antisense region of PHD2. Linc_06633.1 may be an important regulator that promotes PDH2 expression under hypoxia in L. crocea, and we constructed a regulatory profile of L. crocea under hypoxic conditions. To the best of our knowledge, it is the first study that has been conducted on hypoxia signaling pathway regulation by lncRNAs in L. crocea and elucidates the role played by lncRNAs in the regulation of the hypoxia stress response in teleost fish.

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Xiangjun; Yao, Qisheng, E-mail: yymcyqs@126.com; Sun, Xinbo

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treatedmore » with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates fibrosis after hypoxia-and LPS-induced epithelial cells injury. • Slit2 ameliorates inflammation and fibrosis after hypoxia-and LPS-induced renal epithelial cells injury via TLR4/NF-κB.« less

Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302.

PubMed

Wojtkowiak, Jonathan W; Cornnell, Heather C; Matsumoto, Shingo; Saito, Keita; Takakusagi, Yoichi; Dutta, Prasanta; Kim, Munju; Zhang, Xiaomeng; Leos, Rafael; Bailey, Kate M; Martinez, Gary; Lloyd, Mark C; Weber, Craig; Mitchell, James B; Lynch, Ronald M; Baker, Amanda F; Gatenby, Robert A; Rejniak, Katarzyna A; Hart, Charles; Krishna, Murali C; Gillies, Robert J

2015-01-01

Hypoxic niches in solid tumors harbor therapy-resistant cells. Hypoxia-activated prodrugs (HAPs) have been designed to overcome this resistance and, to date, have begun to show clinical efficacy. However, clinical HAPs activity could be improved. In this study, we sought to identify non-pharmacological methods to acutely exacerbate tumor hypoxia to increase TH-302 activity in pancreatic ductal adenocarcinoma (PDAC) tumor models. Three human PDAC cell lines with varying sensitivity to TH-302 (Hs766t > MiaPaCa-2 > SU.86.86) were used to establish PDAC xenograft models. PDAC cells were metabolically profiled in vitro and in vivo using the Seahorse XF system and hyperpolarized (13)C pyruvate MRI, respectively, in addition to quantitative immunohistochemistry. The effect of exogenous pyruvate on tumor oxygenation was determined using electroparamagnetic resonance (EPR) oxygen imaging. Hs766t and MiaPaCa-2 cells exhibited a glycolytic phenotype in comparison to TH-302 resistant line SU.86.86. Supporting this observation is a higher lactate/pyruvate ratio in Hs766t and MiaPaCa xenografts as observed during hyperpolarized pyruvate MRI studies in vivo. Coincidentally, response to exogenous pyruvate both in vitro (Seahorse oxygen consumption) and in vivo (EPR oxygen imaging) was greatest in Hs766t and MiaPaCa models, possibly due to a higher mitochondrial reserve capacity. Changes in oxygen consumption and in vivo hypoxic status to pyruvate were limited in the SU.86.86 model. Combination therapy of pyruvate plus TH-302 in vivo significantly decreased tumor growth and increased survival in the MiaPaCa model and improved survival in Hs766t tumors. Using metabolic profiling, functional imaging, and computational modeling, we show improved TH-302 activity by transiently increasing tumor hypoxia metabolically with exogenous pyruvate. Additionally, this work identified a set of biomarkers that may be used clinically to predict which tumors will be most responsive to pyruvate + TH-302 combination therapy. The results of this study support the concept that acute increases in tumor hypoxia can be beneficial for improving the clinical efficacy of HAPs and can positively impact the future treatment of PDAC and other cancers.

Understanding the hypoxic niche of multiple myeloma: therapeutic implications and contributions of mouse models

PubMed Central

Hu, Jinsong; Van Valckenborgh, Els; Menu, Eline; De Bruyne, Elke; Vanderkerken, Karin

2012-01-01

Multiple myeloma (MM) is the second most common hematological malignancy and is characterized by the clonal expansion of plasma cells in the bone marrow. Recently, hypoxia has received increased interest in the context of MM, in both basic and translational research. In this review, we describe the discovery of the hypoxic niche in MM and how it can be targeted therapeutically. We also discuss mouse models that closely mimic human MM, highlighting those that allow preclinical research into new therapies that exploit the hypoxic niche in MM. PMID:23115205

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

DTIC Science & Technology

2013-09-30

Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-hold Diving...Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-hold Diving 5a...two day period in September, 2012. The first major huddle to the study was to determine the effect of the overnight shipping of the viability of

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

DTIC Science & Technology

2014-09-30

that are too small have less effective results with mechanical trituration that follows digestion). 5. Move dish and sample into the cell culture...Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-Hold Diving...Muscle Cells from Northern Elephant Seals to Study the Mechanisms that Maintain Aerobic Metabolism Under the Hypoxic Conditions of Breath-Hold Diving 5a

Novel Models to Study Effect of High-Altitude Hypoxic Exposure and Placental Insufficiency on Fetal Oxygen Metabolism and Congenital Heart Defects

DTIC Science & Technology

2017-10-01

equivalent to O2 in air at altitudes from 25,000-4,000 ft elevation. ODDluc activity is measures in the fetal tissues as an index of hypoxic stress ...inspired O2. This corresponds to elevations of 25,000-7000 feet. The hypoxic stress placed on the embryo organs (heart, liver, brain) in a normal pregnancy...embryo is particularly vulnerable to reductions in the supply of O2 coming from the mother. 3) The combined stress of placental insufficiency plus

Efficacy of passive hypothermia and adverse events during transport of asphyxiated newborns according to the severity of hypoxic-ischemic encephalopathy.

PubMed

Carreras, Nuria; Alsina, Miguel; Alarcon, Ana; Arca-Díaz, Gemma; Agut, Thais; García-Alix, Alfredo

To determine if the efficacy of passive hypothermia and adverse events during transport are related to the severity of neonatal hypoxic-ischemic encephalopathy. This was a retrospective study of 67 infants with hypoxic-ischemic encephalopathy, born between April 2009 and December 2013, who were transferred for therapeutic hypothermia and cooled during transport. Fifty-six newborns (84%) were transferred without external sources of heat and 11 (16%) needed an external heat source. The mean temperature at departure was 34.4±1.4°C and mean transfer time was 3.3±2.0h. Mean age at arrival was 5.6±2.5h. Temperature at arrival was between 33 and 35°C in 41 (61%) infants, between 35°C and 36.5°C in 15 (22%) and <33°C in 11 (16%). Infants with severe hypoxic-ischemic encephalopathy had greater risk of having an admission temperature<33°C (OR: 4.5; 95% CI: 1.1-19.3). The severity of hypoxic-ischemic encephalopathy and the umbilical artery pH were independent risk factors for a low temperature on admission (p<0.05). Adverse events during transfer, mainly hypotension and bleeding from the endotracheal tube, occurred in 14 infants (21%), with no differences between infants with moderate or severe hypoxic-ischemic encephalopathy. The risk of overcooling during transport is greater in newborns with severe hypoxic-ischemic encephalopathy and those with more severe acidosis at birth. The most common adverse events during transport are related to physiological deterioration and bleeding from the endotracheal tube. This observation provides useful information to identify those asphyxiated infants who require closer clinical surveillance during transport. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

Hypoxia-preconditioned mesenchymal stem cells ameliorate ischemia/reperfusion-induced lung injury.

PubMed

Liu, Yung-Yang; Chiang, Chi-Huei; Hung, Shih-Chieh; Chian, Chih-Feng; Tsai, Chen-Liang; Chen, Wei-Chih; Zhang, Haibo

2017-01-01

Hypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels. I/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined. I/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism. Hypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

Hypoxic Preconditioning Results in Increased Motility and Improved Therapeutic Potential of Human Mesenchymal Stem Cells

PubMed Central

Rosová, Ivana; Dao, Mo; Capoccia, Ben; Link, Daniel; Nolta, Jan A.

2010-01-01

Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential. PMID:18511601

Adenosine receptor antagonist and augmented vasodilation during hypoxic exercise

PubMed Central

Madery, Brandon D.; Pike, Tasha L.; Eisenach, John H.; Dietz, Niki M.; Joyner, Michael J.; Wilkins, Brad W.

2009-01-01

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 (α-adrenoceptor antagonist) and combined phentolamine and aminophylline administration. Forearm vascular conductance (FVC; in ml·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 (ΔFVC; change from normoxic baseline) during hypoxic exercise with saline was 172 ± 29 and 314 ± 34 ml·min−1·100 mmHg−1 (10% and 20%, respectively). Aminophylline administration did not affect ΔFVC during hypoxic exercise at 10% (190 ± 29 ml·min−1·100 mmHg−1, P = 0.4) or 20% (287 ± 48 ml·min−1·100 mmHg−1, P = 0.3). In protocol 2, ΔFVC due to hypoxic exercise with phentolamine infusion was 313 ± 30 and 453 ± 41 ml·min−1·100 mmHg−1 (10% and 20% respectively). ΔFVC was similar at 10% (352 ± 39 ml·min−1·100 mmHg−1, P = 0.8) and 20% (528 ± 45 ml·min−1·100 mmHg−1, P = 0.2) hypoxic exercise with combined phentolamine and aminophylline. In contrast, ΔFVC 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

Enhanced cell survival and paracrine effects of mesenchymal stem cells overexpressing hepatocyte growth factor promote cardioprotection in myocardial infarction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Liyan; Liu, Xiaolin; Zhang, Yuelin

Poor cell survival post transplantation compromises the therapeutic benefits of mesenchymal stem cells (MSCs) in myocardial infarction (MI). Hepatocyte growth factor (HGF) is an important cytokine for angiogenesis, anti-inflammation and anti-apoptosis. This study aimed to evaluate the cardioprotective effects of MSCs overexpressing HGF in a mouse model of MI. The apoptosis of umbilical cord-derived MSCs (UC-MSCs) and HGF-UC-MSCs under normoxic and hypoxic conditions was detected. The conditioned medium (CdM) of UC-MSCs and HGF-UC-MSCs under a hypoxic condition was harvested and its protective effect on neonatal cardiomyocytes (NCMs) exposed to a hypoxic challenge was examined. UC-MSCs and HGF-UC-MSCs were transplanted intomore » the peri-infarct region in mice following MI and heart function assessed 4 weeks post transplantation. The apoptosis of HGF-UC-MSCs under hypoxic conditions was markedly decreased compared with that of UC-MSCs. NCMs treated with HGF-UC-MSC hypoxic CdM (HGF-UC-MSCs-hy-CdM) exhibited less cell apoptosis in response to hypoxic challenge than those treated with UC-MSC hypoxic CdM (UC-MSCs-hy-CdM). HGF-UC-MSCs-hy-CdM released the inhibited p-Akt and lowered the enhanced ratio of Bax/Bcl-2 induced by hypoxia in the NCMs. HGF-UC-MSCs-hy-CdM expressed higher levels of HGF, EGF, bFGF and VEGF than UC-MSCs-hy-CdM. Transplantation of HGF-UC-MSCs or UC-MSCs greatly improved heart function in the mouse model of MI. Compared with UC-MSCs, transplantation of HGF-UC-MSCs was associated with less cardiomyocyte apoptosis, enhanced angiogenesis and increased proliferation of cardiomyocytes. This study may provide a novel therapeutic strategy for MSC-based therapy in cardiovascular disease.« less

Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat.

PubMed

Li, Guohu; Xiao, Yuhui; Estrella, Jaymie L; Ducsay, Charles A; Gilbert, Raymond D; Zhang, Lubo

2003-07-01

Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the susceptibility of adult heart to ischemia-reperfusion (I-R) injury. Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) groups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R(10)) or 25 minutes of ischemia and 3 hours of reperfusion (I-R(25)). Prenatal hypoxia did not change basal left ventricular (LV) function. I-R(10) produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R(25) caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R(25)-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. There was a significant decrease in LV heat shock protein 70 and endothelial nitric oxide synthase levels in hypoxic hearts. Prenatal hypoxia did not change beta(1)-adrenoreceptor levels but significantly increased beta(2)-adrenoreceptor in the left ventricle. In addition, it increased G(s)alpha but decreased G(i)alpha. Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in beta(2)-adrenoreceptor and the G(s)alpha/G(i)alpha ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.

Hypoxia-preconditioned mesenchymal stem cells ameliorate ischemia/reperfusion-induced lung injury

PubMed Central

Chiang, Chi-Huei; Hung, Shih-Chieh; Chian, Chih-Feng; Tsai, Chen-Liang; Chen, Wei-Chih; Zhang, Haibo

2017-01-01

Background Hypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels. Methods I/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined. Results I/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism. Conclusions Hypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis. PMID:29117205

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, K

Purpose: Most prostate cancers are slow-growing diseases but normally require much higher doses (80Gy) with conventional fractionation radiotherapy, comparing to other more aggressive cancers. This study is to disclose the radiobiological basis of this discrepancy by proposing the concept of prostate cancer stem cells (CSCs) and examining their specific irradiation responses. Methods: There are overwhelming evidences that CSC may keep their stemness, e.g. the competency of cell differentiation, in hypoxic microenvironments and hence become radiation resistive, though the probability is tiny for aggressiveness cancers. Tumor hypoxia used to be considered as an independent reason for poor treatment outcomes, and recentmore » evidences showed that even prostate cancers were also hypoxic though they are very slow-growing. In addition, to achieve comparable outcomes to other much more aggressive cancers, much higher doses (rather than lower doses) are always needed for prostate cancers, regardless of its non-aggressiveness. All these abnormal facts can only be possibly interpreted by the irradiation responses characteristics of prostate CSCs. Results: Both normal cancer cells (NCCs) and CSCs exiting in tumors, in which NCCs are mainly for symptoms whereas killing all CSCs achieves disease-free. Since prostate cancers are slow-growing, the hypoxia in prostate cancers cannot possibly from NCCs, thus it is caused by hypoxic CSCs. However, single hypoxic cell cannot be imaged due to limitation of imaging techniques, unless a large group of hypoxic cells exist together, thus most of CSCs in prostate cancers are virtually hypoxic, i.e. not in working mode because CSCs in proliferating mode have to be normoxic, and this explains why prostate cancers are unaggressive. Conclusion: The fractional dose in conventional radiotherapy (∼2Gy) could only kill NCCs and CSCs in proliferating modes, whereas most CSCs survived fractional treatments since they were hypoxic, thus to eliminate all CSCs and achieve disease-free, much more fractionations are needed.« less

Development of a Metabolically Active, Non-Replicating Sporozoite Vaccine to Prevent Plasmodium falciparum Malaria

DTIC Science & Technology

2009-10-01

6 Minglin Li,2 Richard Stafford,1·2 Adriana Ahumada,l.2 Judith E. Epstein, 3 Martha Sedegah,3 Sharina Reyes, 3 Thomas L. Ric hie,3 Kirsten E. Lyke,4...200S; 4:567- 7S. 65. Kumar KA , Baxter P, Tarun AS, Kappc SH, Nusscnzwcig V. Conserved protective mechanisms in radiation and genetically attenuated...uis3{-) and uis4{-) Plasmodium sporozoitcs. PLoS One 2009; 4:44SO . 66. Purcell LA, Wong KA , Yanow SK, Lee M, Spithill TW, Rodriguez A. Chemically

Cost of areal reduction of gulf hypoxia through agricultural practice.

PubMed

Whittaker, Gerald; Barnhart, Bradley L; Srinivasan, Raghavan; Arnold, Jeffrey G

2015-02-01

A major share of the area of hypoxic growth in the Northern Gulf of Mexico has been attributed to nutrient run-off from agricultural fields, but no estimate is available for the cost of reducing Gulf hypoxic area using agricultural conservation practices. We apply the Soil and Water Assessment Tool using observed daily weather to simulate the reduction in nitrogen loading in the Upper Mississippi River Basin (UMRB) that would result from enrolling all row crop acreage in the Conservation Reserve Program (CRP). Nitrogen loadings at the outlet of the UMRB are used to predict Gulf hypoxic area, and net cash farm rent is used as the price for participation in the CRP. Over the course of the 42 year simulation, direct CRP costs total more than $388 billion, and the Inter-Governmental Task Force goal of hypoxic area less than 5000 square kilometers is met in only two years. Published by Elsevier B.V.

Hemin offers neuroprotection through inducing exogenous neuroglobin in focal cerebral hypoxic-ischemia in rats

PubMed Central

Song, Xue; Xu, Rui; Xie, Fei; Zhu, Haiyuan; Zhu, Ji; Wang, Xin

2014-01-01

Objective: To investigate the inducible effect of hemin on exogenous neuroglobin (Ngb) in focal cerebral hypoxic-ischemia in rats. Methods: 125 healthy SD rats were randomly divided into five groups: sham-operation control group, operation group, hemin treatment group, exogenous Ngb treatment group, and hemin and exogenous Ngb joint treatment group. Twenty-four hours after focal cerebral hypoxic-ischemia, Ngb expression was evaluated by immunocytochemistry, RT-PCR, and western blot analyses, while the brain water content and infarct volume were examined. Results: Immunocytochemistry, RT-PCR, and western blot analyses showed more pronounced Ngb expression in the hemin and exogenous Ngb joint operation group than in the hemin or exogenous Ngb individual treatment groups, thus producing significant differences in brain water content and infarct volume (p < 0.05). Conclusions: Hemin may be beneficial in protecting against focal cerebral hypoxic-ischemia through inducing the expression of exogenous Ngb. PMID:24966924

Effects of posture on blood flow diversion by hypoxic pulmonary vasoconstriction in dogs

NASA Technical Reports Server (NTRS)

Walther, S. M.; Domino, K. B.; Hlastala, M. P.

1998-01-01

We used differential excretion of sulphur hexafluoride from the left and right lung to measure blood flow diversion by hypoxic pulmonary vasoconstriction (HPV) in the prone and supine positions in dogs (n = 9). Gas exchange was assessed using the multiple inert gas elimination technique. Blood flow diversion from the hypoxic (3% oxygen) left lung was mean 70.7 (SD 11.2)% in the supine compared with 57.0 (12.1)% in the prone position (P < 0.02). The supine position was associated with increased perfusion to low VA/Q regions (P < 0.05). The increased flow diversion with hypoxia in the supine position was associated with more ventilation to high VA/Q regions (P < 0.05). We conclude that flow diversion by hypoxic pulmonary vasoconstriction is greater in the supine position. This effect could contribute to the variable response in gas exchange with positioning in patients with ARDS.

Selective Infection of Antigen-Specific B Lymphocytes by Salmonella Mediates Bacterial Survival and Systemic Spreading of Infection

PubMed Central

de Wit, Jelle; Martinoli, Chiara; Zagato, Elena; Janssen, Hans; Jorritsma, Tineke; Bar-Ephraïm, Yotam E.; Rescigno, Maria; Neefjes, Jacques; van Ham, S. Marieke

2012-01-01

Background The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer’s Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading. Methodology/Principal Findings Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood. Conclusions/Significance This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection. PMID:23209805

Measurement of Lactate Content and Amide Proton Transfer Values in the Basal Ganglia of a Neonatal Piglet Hypoxic-Ischemic Brain Injury Model Using MRI.

PubMed

Zheng, Y; Wang, X-M

2017-04-01

As amide proton transfer imaging is sensitive to protein content and intracellular pH, it has been widely used in the nervous system, including brain tumors and stroke. This work aimed to measure the lactate content and amide proton transfer values in the basal ganglia of a neonatal piglet hypoxic-ischemic brain injury model by using MR spectroscopy and amide proton transfer imaging. From 58 healthy neonatal piglets (3-5 days after birth; weight, 1-1.5 kg) selected initially, 9 piglets remained in the control group and 43 piglets, in the hypoxic-ischemic brain injury group. Single-section amide proton transfer imaging was performed at the coronal level of the basal ganglia. Amide proton transfer values of the bilateral basal ganglia were measured in all piglets. The ROI of MR spectroscopy imaging was the right basal ganglia, and the postprocessing was completed with LCModel software. After hypoxic-ischemic insult, the amide proton transfer values immediately decreased, and at 0-2 hours, they remained at their lowest level. Thereafter, they gradually increased and finally exceeded those of the control group at 48-72 hours. After hypoxic-ischemic insult, the lactate content increased immediately, was maximal at 2-6 hours, and then gradually decreased to the level of the control group. The amide proton transfer values were negatively correlated with lactate content ( r = -0.79, P < .05). This observation suggests that after hypoxic-ischemic insult, the recovery of pH was faster than that of lactate homeostasis. © 2017 by American Journal of Neuroradiology.

Predicting hypoxaemia during flights in children with cystic fibrosis

PubMed Central

Buchdahl, R; Babiker, A; Bush, A; Cramer, D

2001-01-01

BACKGROUND—We have previously suggested that it is possible to predict oxygen desaturation during flight in children with cystic fibrosis and chronic lung disease by non-invasive measurement of oxygen saturation following inhalation of 15% oxygen—the pre-flight hypoxic challenge. This study reports on the results of measurements over 5years.
METHODS—The study comprised a pre-flight hypoxic challenge measuring oxygen saturation by finger tip pulse oximetry (SpO2) during tidal breathing of 15% oxygen in nitrogen and spirometric testing 1 month before the flight followed by SpO2 measurements during intercontinental flights to and from holidays abroad with children in wake and sleep states.
RESULTS—Pre-flight tests were completed on 87 children with cystic fibrosis. Desaturation of <90% occurred in 10 children at some stage during the flight, three of whom received supplementary oxygen. Using a cut off SpO2 of 90%, the pre-flight hypoxic challenge correctly predicted desaturation in only two of these children. The sensitivity and specificity of the pre-flight hypoxic challenge were 20% and 99%, respectively, compared with 70% and 96% for spirometric tests (using a cut off for forced expiratory volume in 1 second (FEV1) of <50% predicted). Overall, pre-flight spirometric tests were a better predictor of desaturation during flight with the area under the Receiver Operating Characteristic (ROC) curve of 0.89 compared with 0.73 for the hypoxic challenge test.
CONCLUSIONS—In this group of subjects pre-flight spirometric testing was a better predictor of desaturation during flight than the pre-flight hypoxic challenge.

 PMID:11641514

When Is Embryonic Arrest Broken in Turtle Eggs?

PubMed

Williamson, Sean A; Evans, Roger G; Reina, Richard D

Turtle embryos enter a state of arrested development in the oviduct, allowing the mother greater flexibility in her reproductive schedule. Development recommences once eggs transition from the hypoxic oviduct to the normoxic nest. Significant mortality can occur if turtle eggs are moved between 12 h and 20 d after oviposition, and this is linked to the recommencement of embryonic development. To better understand the timing of developmental arrest and to determine how movement-induced mortality might be avoided, we determined the latency (i.e., time elapsed since oviposition) to recommencement of development following oviposition by exposing the eggs of green turtles (Chelonia mydas) to hypoxia (oxygen tension <8 mmHg) for 3 d, commencing 30 min to 48 h after oviposition. Embryonic development-including development of the characteristic opaque white spot on the eggshell-was halted by hypoxic incubation. When the delay before hypoxic incubation was 12 h or less, hatching success did not differ from a control group. If the hypoxic treatment began after 16 h or more in normoxia, then all embryos died. Thus, by returning eggs to a hypoxic environment before they have broken from arrest (i.e., within 12 h of oviposition), it is possible to extend embryonic arrest for at least 3 d, with no apparent detriment to hatching success. Therefore, hypoxic incubation may provide a new approach for avoidance of movement-induced mortality when conservation or research efforts require the relocation of eggs. Our findings also suggest that movement-induced mortality may have constrained the evolution of viviparity in turtles.

Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate

PubMed Central

Yin, Chengqian; He, Dan; Chen, Shuyang; Tan, Xiaoling; Sang, Nianli

2016-01-01

Molecular oxygen is the final electron acceptor in cellular metabolism but cancer cells often become adaptive to hypoxia, which promotes resistance to chemotherapy and radiation. The reduction of endogenous glycolytic pyruvate to lactate is known as an adaptive strategy for hypoxic cells. Whether exogenous pyruvate is required for hypoxic cell proliferation by either serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxic and ρ0 cells defective in electron transfer chain, we show that exogenous pyruvate is required to sustain proliferation of both cancer and non-cancer cells that cannot utilize oxygen. Particularly, we show that absence of pyruvate led to glycolysis inhibition and AMPK activation along with decreased NAD+ levels in ρ0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and suppresses AMPK activation. Knockdown of lactate dehydrogenase significantly inhibits the rescuing effects of exogenous pyruvate. In contrast, none of pyruvate-derived metabolites tested (including acetyl-CoA, α-ketoglutarate, succinate and alanine) can replace pyruvate in supporting ρ0 cell proliferation. Knockdown of pyruvate carboxylase, pyruvate dehydrogenase and citrate synthase do not impair exogenous pyruvate to rescue ρ0 cells. Importantly, we show that exogenous pyruvate relieves ATP insufficiency and mTOR inhibition and promotes proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. Taken together, our data support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate. The pyruvate cycle may be targeted as a new therapy of hypoxic cancers. PMID:27374086

Exogenous pyruvate facilitates cancer cell adaptation to hypoxia by serving as an oxygen surrogate.

PubMed

Yin, Chengqian; He, Dan; Chen, Shuyang; Tan, Xiaoling; Sang, Nianli

2016-07-26

Molecular oxygen is the final electron acceptor in cellular metabolism but cancer cells often become adaptive to hypoxia, which promotes resistance to chemotherapy and radiation. The reduction of endogenous glycolytic pyruvate to lactate is known as an adaptive strategy for hypoxic cells. Whether exogenous pyruvate is required for hypoxic cell proliferation by either serving as an electron acceptor or a biosynthetic substrate remains unclear. By using both hypoxic and ρ0 cells defective in electron transfer chain, we show that exogenous pyruvate is required to sustain proliferation of both cancer and non-cancer cells that cannot utilize oxygen. Particularly, we show that absence of pyruvate led to glycolysis inhibition and AMPK activation along with decreased NAD+ levels in ρ0 cells; and exogenous pyruvate increases lactate yield, elevates NAD+/NADH ratio and suppresses AMPK activation. Knockdown of lactate dehydrogenase significantly inhibits the rescuing effects of exogenous pyruvate. In contrast, none of pyruvate-derived metabolites tested (including acetyl-CoA, α-ketoglutarate, succinate and alanine) can replace pyruvate in supporting ρ0 cell proliferation. Knockdown of pyruvate carboxylase, pyruvate dehydrogenase and citrate synthase do not impair exogenous pyruvate to rescue ρ0 cells. Importantly, we show that exogenous pyruvate relieves ATP insufficiency and mTOR inhibition and promotes proliferation of hypoxic cells, and that well-oxygenated cells release pyruvate, providing a potential in vivo source of pyruvate. Taken together, our data support a novel pyruvate cycle model in which oxygenated cells release pyruvate for hypoxic cells as an oxygen surrogate. The pyruvate cycle may be targeted as a new therapy of hypoxic cancers.

Developmental amnesia associated with early hypoxic-ischaemic injury.

PubMed

Gadian, D G; Aicardi, J; Watkins, K E; Porter, D A; Mishkin, M; Vargha-Khadem, F

2000-03-01

We recently reported on three young patients with severe impairments of episodic memory resulting from brain injury sustained early in life. These findings have led us to hypothesize that such impairments might be a previously unrecognized consequence of perinatal hypoxic-ischaemic injury. Neuropsychological and quantitative magnetic resonance investigations were carried out on five young patients, all of whom had suffered hypoxic-ischaemic episodes at or shortly after birth. All five patients showed severe impairments of episodic memory (memory for events), with relative preservation of semantic memory (memory for facts). However, none had any of the major neurological deficits that are typically associated with hypoxic-ischaemic injury, and all attended mainstream schools. Quantitative magnetic resonance investigations revealed severe bilateral hippocampal atrophy in all cases. As a group, the patients also showed bilateral reductions in grey matter in the regions of the putamen and the ventral part of the thalamus. On the basis of their clinical histories and the pattern of magnetic resonance findings, we attribute the patients' pathology and associated memory impairments primarily to hypoxic-ischaemic episodes sustained very early in life. We suggest that the degree of hypoxia-ischaemia was sufficient to produce selective damage to particularly vulnerable regions of the brain, notably the hippocampi, but was not sufficient to result in the more severe neurological and cognitive deficits that can follow hypoxic-ischaemic injury. The impairments in episodic memory may be difficult to recognize, particularly in early childhood, but this developmental amnesia can have debilitating consequences, both at home and at school, and may preclude independent life in adulthood.

Effects of hypoxic exposure during feeding on SDA and postprandial cardiovascular physiology in the Atlantic cod, Gadus morhua.

PubMed

Behrens, Jane W; Axelsson, Michael; Neuenfeldt, Stefan; Seth, Henrik

2012-01-01

Some Atlantic cod in the Bornholm Basin undertake vertical foraging migrations into severely hypoxic bottom water. Hypoxic conditions can reduce the postprandial increase in gastrointestinal blood flow (GBF). This could subsequently postpone or reduce the postprandial increase in oxygen consumption (MO(2)), i.e. the SDA, leading to a disturbed digestion. Additionally, a restricted oxygen uptake could result in an oxygen debt that needs to be compensated for upon return to normoxic waters and this may also affect the ability to process the food. Long-term cardio-respiratory measurements were made on fed G. morhua in order to understand how the cardio-respiratory system of feeding fish respond to a period of hypoxia and a subsequent return to normoxia. These were exposed to 35% water oxygen saturation for 90 minutes, equivalent to the time and oxygen level cod voluntarily endure when searching for food in the Bornholm Basin. We found that i) gastric and intestinal blood flows, cardiac output and MO(2) increased after feeding, ii) gastric and intestinal blood flows were spared in hypoxia, and iii) there were no indications of an oxygen debt at the end of the hypoxic period. The magnitude and time course of the measured variables are similar to values obtained from fish not exposed to the hypoxic period. In conclusion, when cod in the field search for and ingest prey under moderate hypoxic conditions they appear to stay within safe limits of oxygen availability as we saw no indications of an oxygen debt, or negative influence on digestive capacity, when simulating field observations.

Early Macrophage Recruitment and Alternative Activation Are Critical for the Later Development of Hypoxia-induced Pulmonary Hypertension

PubMed Central

Vergadi, Eleni; Chang, Mun Seog; Lee, Changjin; Liang, Olin; Liu, Xianlan; Fernandez-Gonzalez, Angeles; Mitsialis, S. Alex; Kourembanas, Stella

2011-01-01

Background Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, is protective in HPH. Methods and Results We generated bitransgenic mice that overexpress human HO-1 under doxycycline (dox) control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of dox resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of Found in Inflammatory Zone-1, Arginase-1 and Chitinase-3-like-3. A brief, two-day pulse of dox delayed but did not prevent the peak of hypoxic inflammation, and could not protect from HPH. In contrast, a seven-day dox treatment sustained high HO-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage IL-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted proliferation of pulmonary artery smooth muscle cells while treatment with carbon monoxide, a HO-1 enzymatic product, abrogated this effect. Conclusions Early recruitment and alternative activation of macrophages in hypoxic lungs is critical for the later development of HPH. HO-1 may confer protection from HPH by effectively modifing macrophage activation state in hypoxia. PMID:21518986

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

Hypoxic pulmonary vasoconstriction does not affect hydrostatic pulmonary edema formation.

PubMed

Cheney, F W; Bishop, M J; Eisenstein, B L; Artman, L D

1987-02-01

We studied the effects of regional hypoxic pulmonary vasoconstriction (HPV) on lobar flow diversion in the presence of hydrostatic pulmonary edema. Ten anesthetized dogs with the left lower lobe (LLL) suspended in a net for continuous weighing were ventilated with a bronchial divider so the LLL could be ventilated with either 100% O2 or a hypoxic gas mixture (90% N2-5% CO2-5% O2). A balloon was inflated in the left atrium until hydrostatic pulmonary edema occurred, as evidenced by a continuous increase in LLL weight. Left lower lobe flow (QLLL) was measured by electromagnetic flow meter and cardiac output (QT) by thermal dilution. At a left atrial pressure of 30 +/- 5 mmHg, ventilation of the LLL with the hypoxic gas mixture caused QLLL/QT to decrease from 17 +/- 4 to 11 +/- 3% (P less than 0.05), pulmonary arterial pressure to increase from 35 +/- 5 to 37 +/- 6 mmHg (P less than 0.05), and no significant change in rate of LLL weight gain. Gravimetric confirmation of our results was provided by experiments in four animals where the LLL was ventilated with an hypoxic gas mixture for 2 h while the right lung was ventilated with 100% O2. In these animals there was no difference in bloodless lung water between the LLL and right lower lobe. We conclude that in the presence of left atrial pressures high enough to cause hydrostatic pulmonary edema, HPV causes significant flow diversion from an hypoxic lobe but the decrease in flow does not affect edema formation.

Indomethacin reduces short-circuit current and oxygen consumption in normal and chronically hypoxic rat colon.

PubMed

Saraví, Fernando D; Cincunegui, Liliana M; Saldeña, Teobaldo A; Carra, Graciela E; Ibáñez, Jorge E; Grzona, Esteban

2006-09-01

Chronic hypobaric hypoxia is a physiological environmental stressor. While its effects on most major organ systems have been extensively studied, few works have addressed hypoxia-induced changes in intestinal transport. The effects of cyclooxygenase blockade with indomethacin on short-circuit current (Isc) and oxygen consumption (QO2) of the distal colonic epithelium of control rats and rats submitted to hypoxia for 10 days at 0.52 atm were studied. Isolated mucosae were mounted in an Ussing chamber modified for measuring QO2 while preserving transepithelial vectorial transport. Amiloride was added to the mucosal hemichamber to block a sodium component of Isc present in hypoxic rats. In this condition, basal Isc did not differ between the hypoxic and the control group, but QO2 was higher in the former. Indomethacin (30 micromol/L) reduced Isc to the same extent in both groups, but QO2 reduction was larger in the hypoxic group. Pharmacological blockade of chloride secretion and a low-chloride solution abolished the indomethacin-induced reductions of Isc in both groups, and the reduction of QO2 in controls, and attenuated but did not suppress the QO2 reduction in the hypoxic group. Linear regression analysis of QO2 changes versus Isc changes yielded a significant correlation for both groups, with regression lines with the same slope, but a higher position in bypoxic animals. Results suggest that spontaneously releasedprostaglandins are equally important for maintaining colonic chloride secretion in hypoxic as in normoxic rats, but that, in the former, indomethacin has an additional effect on QO2 which is unrelated to ion transport.

Options for managing hypoxic blackwater events in river systems: a review.

PubMed

Kerr, Janice L; Baldwin, Darren S; Whitworth, Kerry L

2013-01-15

Blackwater events are characterised by a high concentration of dissolved organic carbon in the water column. They occur naturally in lowland rivers with forested floodplains and bring a variety of benefits to both aquatic and floodplain biota. However, particularly when accompanied by high temperatures, respiration of the organic carbon may cause blackwater to become hypoxic. This may lead to a range of lethal and sub-lethal effects on the aquatic biota. We review the current scientific knowledge concerning the management of blackwater and hypoxia, and examine how this knowledge may be applied to the management of hypoxic blackwater events in lowland river systems. A range of management options, which aim to either prevent the development of hypoxic blackwater or to reintroduce oxygen into deoxygenated waters, are reported. Mitigation options that may be applicable to lowland river systems include manipulating the season and magnitude of floods in regulated rivers, increasing roughness in flow paths, establishing oxygenated refugia for aquatic biota and introducing hydraulic structures that promote turbulence and re-aeration. With climatic changes trending towards a scenario where extreme events leading to the development of hypoxic blackwater are more probable, it is now vital to validate and optimise management options on local and regional scales and work towards closing knowledge gaps. With judicious management of regulated rivers, it is possible to minimise the impacts of hypoxic flows while preserving the benefits brought to floodplain and river ecosystems by seasonal flooding and carbon exchange. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Flight assessment in patients with respiratory disease: hypoxic challenge testing vs. predictive equations.

PubMed

Martin, S E; Bradley, J M; Buick, J B; Bradbury, I; Elborn, J S

2007-06-01

Predictive equations have been proposed as a simpler alternative to hypoxic challenge testing (HCT) for determining the risk of in-flight hypoxia. To assess agreement between hypoxic challenge testing (HCT) and predictive equations for assessment of in-flight hypoxia. Retrospective study. Patients with chronic obstructive pulmonary disease (COPD) (n = 15), interstitial lung disease (ILD) (n = 15) and cystic fibrosis (CF) (n = 15) were studied. Spirometry was recorded prior to hypoxic inhalation and oxygen saturations (SpO2) were recorded before, after and during hypoxic inhalation. Blood gases were analysed before and after hypoxic inhalation and when SpO2 = 85%. An HCT was performed using the Ventimask method. The PaO2 at altitude was estimated for each group using four published predictive equations, which use values of PaO2 (ground) and lung function measurements to predict altitude PaO2. Results were interpreted using the BTS recommendations for prescription of in-flight oxygen post HCT. The Stuart Maxwell test of overall homogeneity was used to assess agreement between HCT results and each of the predictive equations. Ground PaO2 was significantly greater in patients with CF than either ILD or COPD (p < 0.05). PaO2 in all three groups significantly decreased following HCT. With the exception of equation 3, significantly fewer patients in each group would require in-flight O2 if prescription was based on HCT, compared to predictive equations (p < 0.05). Predictive equations considerably overestimate the need for in-flight O2, compared to HCT.

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

PubMed

Bache, Matthias; Zschornak, Martin P; Passin, Sarina; Kessler, Jacqueline; Wichmann, Henri; Kappler, Matthias; Paschke, Reinhard; Kaluđerović, Goran N; Kommera, Harish; Taubert, Helge; Vordermark, Dirk

2011-09-09

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. 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. 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. Our results suggest that BA is capable of improving the effects of tumor therapy in human malignant glioma cells, particularly under hypoxic conditions. Further investigations are necessary to characterize its potential as a radiosensitizer.

Argon: Systematic Review on Neuro- and Organoprotective Properties of an “Inert” Gas

PubMed Central

Höllig, Anke; Schug, Anita; Fahlenkamp, Astrid V.; Rossaint, Rolf; Coburn, Mark

2014-01-01

Argon belongs to the group of noble gases, which are regarded as chemically inert. Astonishingly some of these gases exert biological properties and during the last decades more and more reports demonstrated neuroprotective and organoprotective effects. Recent studies predominately use in vivo or in vitro models for ischemic pathologies to investigate the effect of argon treatment. Promising data has been published concerning pathologies like cerebral ischemia, traumatic brain injury and hypoxic ischemic encephalopathy. However, models applied and administration of the therapeutic gas vary. Here we provide a systematic review to summarize the available data on argon’s neuro- and organoprotective effects and discuss its possible mechanism of action. We aim to provide a summary to allow further studies with a more homogeneous setting to investigate possible clinical applications of argon. PMID:25310646

Variable responses of small and large human hepatocytes to hypoxia and hypoxia/reoxygenation (H–R)

PubMed Central

Bhogal, Ricky H.; Weston, Christopher J.; Curbishley, Stuart M.; Bhatt, Anand N.; Adams, David H.; Afford, Simon C.

2011-01-01

Hypoxia and hypoxia–reoxygenation (H–R) regulate human hepatocyte cell death by mediating the accumulation of reactive oxygen species (ROS). Hepatocytes within the liver are organised into peri-portal (PP) and peri-venous (PV) subpopulations. PP and PV hepatocytes differ in size and function. We investigated whether PP and PV human hepatocytes exhibit differential susceptibility to hypoxic stress. Isolated hepatocytes were used in an in vitro model of hypoxia and H–R. ROS production and cell death were assessed using flow cytometry. PV, and not PP hepatocytes, accumulate intracellular ROS in a mitochondrial dependent manner during hypoxia and H–R. This increased ROS regulates hepatocyte apoptosis and necrosis via a mitochondrial pathway. These findings have implications on the understanding of liver injury and application of potential therapeutic strategies. PMID:21356211

Acute morphine effects on respiratory activity in mice with target deletion of the tachykinin 1 gene (Tac1-/-).

PubMed

Shvarev, Yuri; Berner, Jonas; Bilkei-Gorzo, Andras; Lagercrantz, Hugo; Wickström, Ronny

2010-01-01

Search for physiological mechanisms which could antagonize the opioid-induced respiratory depression is of important clinical value. In this study, we investigated the acute effects of morphine on respiratory activity in genetically modified newborn (P2) mice with target deletion of the (Tac1 -/-) gene lacking substance P (SP) and neurokinin A (NKA). In vivo, as shown with whole-body flow barometric plethysmography technique, morphine induced significantly attenuated minute ventilation during intermittent hypoxia in control animals. In contrast, knockout mice revealed significant increase in minute ventilation. In vitro, in brainstem preparation, knockout mice demonstrated greater changes in burst frequency during intermittent anoxia challenge. The data suggest that hereditary deficiency in tachykinins, SP and NKA results in more robust hypoxic response in newborn Tac1-/- mice during respiratory depression induced by morphine.

Notification: Evaluate the Gulf of Mexico Hypoxic Zone Reduction

EPA Pesticide Factsheets

Project #OPE-FY13-0012, January 30, 2013. The Office of Inspector General (OIG) is starting preliminary research on the U.S. Environmental Protection Agency’s (EPA’s) and states’ efforts to reduce the Gulf of Mexico hypoxic zone.

Hypobaric hypoxic cerebral insults: the neurological consequences of going higher.

PubMed

Maa, Edward H

2010-01-01

As increasing numbers of people live, work, and play at high altitudes, awareness of the neurological consequences of hypobaric hypoxic environments becomes paramount. Despite volumes of studies examining the pathophysiology of altitude sickness, the underlying mechanisms of the spectrum of altitude related illnesses is still elusive. High altitude headache, acute mountain sickness, high altitude cerebral edema and other neurological presentations including sleep disturbances and seizures at high altitude are reviewed. As our knowledge advances in the field of altitude physiology, the clinical and research techniques developed may help our understanding of hypoxic brain injury in general.

Global distribution of naturally occurring marine hypoxia on continental margins

NASA Astrophysics Data System (ADS)

Helly, John J.; Levin, Lisa A.

2004-09-01

Hypoxia in the ocean influences biogeochemical cycling of elements, the distribution of marine species and the economic well being of many coastal countries. Previous delineations of hypoxic environments focus on those in enclosed seas where hypoxia may be exacerbated by anthropogenically induced eutrophication. Permanently hypoxic water masses in the open ocean, referred to as oxygen minimum zones, impinge on a much larger seafloor surface area along continental margins of the eastern Pacific, Indian and western Atlantic Oceans. We provide the first global quantification of naturally hypoxic continental margin floor by determining upper and lower oxygen minimum zone depth boundaries from hydrographic data and computing the area between the isobaths using seafloor topography. This approach reveals that there are over one million km 2 of permanently hypoxic shelf and bathyal sea floor, where dissolved oxygen is <0.5 ml l -1; over half (59%) occurs in the northern Indian Ocean. We also document strong variation in the intensity, vertical position and thickness of the OMZ as a function of latitude in the eastern Pacific Ocean and as a function of longitude in the northern Indian Ocean. Seafloor OMZs are regions of low biodiversity and are inhospitable to most commercially valuable marine resources, but support a fascinating array of protozoan and metazoan adaptations to hypoxic conditions.

GABA is not elevated during neuroprotective neuronal depression in the hypoxic epaulette shark (Hemiscyllium ocellatum).

PubMed

Mulvey, Jamin M; Renshaw, Gillian M C

2009-02-01

Prolonged hypoxic exposure results in cell failure, glutamate excitotoxicity and apoptosis in the brain. The epaulette shark can withstand prolonged hypoxic exposure without brain injury, while maintaining normal function and activity at tropical temperatures. We examined whether the inhibitory neurotransmitter GABA was involved in hypoxia tolerance and neuroprotection during hypoxic preconditioning. Sharks were exposed to either cyclic hypoxic preconditioning or normoxic conditions. Whole brain GABA concentration was determined using high performance liquid chromatography; GABA distribution in neuronal structures was localised with immunohistochemistry and quantified. While the overall brain level of GABA was not significantly different, there was a significant heterogeneous change in GABA distribution. GABA immunoreactivity was elevated in key motor and sensory nuclei from preconditioned animals, including the nucleus motorius nervi vagi and the cerebellar crest (p<0.001), corresponding to areas of previously reported neuronal hypometabolism. Since the neuroprotection in all other hypoxia and anoxia tolerant species examined so far relies in part on significant elevations in GABA and the phylogenetically older epaulette shark does not, it is reasonable to assume that further research in this unique animal model may yield clues to new key modulators of neuroprotection. Understanding such mechanisms may facilitate the development of therapeutic interventions in the treatment of transient ischaemic attacks, strokes and traumatic brain injury.

Hypoxic Preconditioning Promotes the Bioactivities of Mesenchymal Stem Cells via the HIF-1α-GRP78-Akt Axis.

PubMed

Lee, Jun Hee; Yoon, Yeo Min; Lee, Sang Hun

2017-06-21

Mesenchymal stem cells (MSC) are ideal materials for stem cell-based therapy. As MSCs reside in hypoxic microenvironments (low oxygen tension of 1% to 7%), several studies have focused on the beneficial effects of hypoxic preconditioning on MSC survival; however, the mechanisms underlying such effects remain unclear. This study aimed to uncover the potential mechanism involving 78-kDa glucose-regulated protein (GRP78) to explain the enhanced MSC bioactivity and survival in hindlimb ischemia. Under hypoxia (2% O₂), the expression of GRP78 was significantly increased via hypoxia-inducible factor (HIF)-1α. Hypoxia-induced GRP78 promoted the proliferation and migration potential of MSCs through the HIF-1α-GRP78-Akt signal axis. In a murine hind-limb ischemia model, hypoxic preconditioning enhanced the survival and proliferation of transplanted MSCs through suppression of the cell death signal pathway and augmentation of angiogenic cytokine secretion. These effects were regulated by GRP78. Our findings indicate that hypoxic preconditioning promotes survival, proliferation, and angiogenic cytokine secretion of MSCs via the HIF-1α-GRP78-Akt signal pathway, suggesting that hypoxia-preconditioned MSCs might provide a therapeutic strategy for MSC-based therapies and that GRP78 represents a potential target for the development of functional MSCs.

Modeling the Relative Importance of Nutrient and Carbon Loads, Boundary Fluxes, and Sediment Fluxes on Gulf of Mexico Hypoxia.

PubMed

Feist, Timothy J; Pauer, James J; Melendez, Wilson; Lehrter, John C; DePetro, Phillip A; Rygwelski, Kenneth R; Ko, Dong S; Kreis, Russell G

2016-08-16

The Louisiana continental shelf in the northern Gulf of Mexico experiences bottom water hypoxia in the summer. In this study, we applied a biogeochemical model that simulates dissolved oxygen concentrations on the shelf in response to varying riverine nutrient and organic carbon loads, boundary fluxes, and sediment fluxes. Five-year model simulations demonstrated that midsummer hypoxic areas were most sensitive to riverine nutrient loads and sediment oxygen demand from settled organic carbon. Hypoxic area predictions were also sensitive to nutrient and organic carbon fluxes from lateral boundaries. The predicted hypoxic area decreased with decreases in nutrient loads, but the extent of change was influenced by the method used to estimate model boundary concentrations. We demonstrated that modeling efforts to predict changes in hypoxic area on the continental shelf in relationship to changes in nutrients should include representative boundary nutrient and organic carbon concentrations and functions for estimating sediment oxygen demand that are linked to settled organic carbon derived from water-column primary production. On the basis of our model analyses using the most representative boundary concentrations, nutrient loads would need to be reduced by 69% to achieve the Gulf of Mexico Nutrient Task Force Action Plan target hypoxic area of 5000 km(2).

Metabolic and hypoxic adaptation to anti-angiogenic therapy: a target for induced essentiality

PubMed Central

McIntyre, Alan; Harris, Adrian L

2015-01-01

Anti-angiogenic therapy has increased the progression-free survival of many cancer patients but has had little effect on overall survival, even in colon cancer (average 6–8 weeks) due to resistance. The current licensed targeted therapies all inhibit VEGF signalling (Table1). Many mechanisms of resistance to anti-VEGF therapy have been identified that enable cancers to bypass the angiogenic blockade. In addition, over the last decade, there has been increasing evidence for the role that the hypoxic and metabolic responses play in tumour adaptation to anti-angiogenic therapy. The hypoxic tumour response, through the transcription factor hypoxia-inducible factors (HIFs), induces major gene expression, metabolic and phenotypic changes, including increased invasion and metastasis. Pre-clinical studies combining anti-angiogenics with inhibitors of tumour hypoxic and metabolic adaptation have shown great promise, and combination clinical trials have been instigated. Understanding individual patient response and the response timing, given the opposing effects of vascular normalisation versus reduced perfusion seen with anti-angiogenics, provides a further hurdle in the paradigm of personalised therapeutic intervention. Additional approaches for targeting the hypoxic tumour microenvironment are being investigated in pre-clinical and clinical studies that have potential for producing synthetic lethality in combination with anti-angiogenic therapy as a future therapeutic strategy. PMID:25700172

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans

PubMed Central

Sun, C-L; Kim, E; Crowder, C M

2014-01-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2–3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death. PMID:24317200

Delayed innocent bystander cell death following hypoxia in Caenorhabditis elegans.

PubMed

Sun, C-L; Kim, E; Crowder, C M

2014-04-01

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2-3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

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

The effect of resuscitation in 100% oxygen on brain injury in a newborn rat model of severe hypoxic-ischaemic encephalopathy.

PubMed

Smit, Elisa; Liu, Xun; Gill, Hannah; Jary, Sally; Wood, Thomas; Thoresen, Marianne

2015-11-01

Infants with birth asphyxia frequently require resuscitation. Current guidance is to start newborn resuscitation in 21% oxygen. However, infants with severe hypoxia-ischaemia may require prolonged resuscitation with oxygen. To date, no study has looked at the effect of resuscitation in 100% oxygen following a severe hypoxic-ischaemic insult. Postnatal day 7 Wistar rats underwent a severe hypoxic-ischaemic insult (modified Vannucci unilateral brain injury model) followed by immediate resuscitation in either 21% or 100% oxygen for 30 min. Seven days following the insult, negative geotaxis testing was performed in survivors, and the brains were harvested. Relative ipsilateral cortical and hippocampal area loss was assessed histologically. Total area loss in the affected hemisphere and area loss within the hippocampus did not significantly differ between the two groups. The same results were seen for short-term neurological assessment. No difference was seen in weight gain between pups resuscitated in 21% and 100% oxygen. Resuscitation in 100% oxygen does not cause a deleterious effect on brain injury following a severe hypoxic-ischaemic insult in a rat model of hypoxia-ischaemia. Further work investigating the effects of resuscitation in 100% oxygen is warranted, especially for newborn infants with severe hypoxic-ischaemic encephalopathy. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

[The effect of 5-HD on expression of PKC-alpha in rats of chronic hypoxic pulmonary hypertension].

PubMed

Shu, Ying; Li, Qiu; Li, Yun-lei; Zhang, Li-ping; Chen, Cheng-shui

2011-08-01

To investigate the effect of mito chondrial K(ATP) channels (mitoK(ATP)) inhibitor 5-hydroxydecanoate(5-HD) on chronic hypoxic pulmonary artery hypertension (CHPAH) rats and its underlying mechanisms. Forty-eight male SD rats were equally divided into 4 groups randomly (n=12): normal group, hypoxia group, hypoxia + 5-HD group, hypoxia + Diazoxide group. Except the first group, the other three groups were put into hypoxic [O2 (10.0% +/- 0.3%] and nonrmobaric chamber for four weeks to establish chronic hypoxic model and received different interference. When the interference completed, right heart catheter was used to detect the mean pulmonary arterial pressure (mPAP) of each rat and PKC-alpha mRNA expression in pulmonary arteries was detected by reverse transcription-polymerase chain reaction (RT-PCR) and protein expression by Western blot. (mPAP was much higher in hypoxia group than that in normal group (P < 0.01) while in hypoxia + 5-HD group and hypoxia + diazoxide were decreased significantly compared to hypoxia group (P < 0.01). (2) The protein and mRNA levels of PKC-alpha in the hypoxic group were higher than those in normal group (P < 0.05). 5-HD plays a protective role on CHPAH. The mechanism of its effect may be attributed to inhibiting MitoK(ATP).

Accelerated acidosis in response to variable fetal heart rate decelerations in chronically hypoxic ovine fetuses.

PubMed

Amaya, Kevin E; Matushewski, Brad; Durosier, L Daniel; Frasch, Martin G; Richardson, Bryan S; Ross, Michael G

2016-02-01

Due to limitations of technology, clinicians are typically unable to determine if human fetuses are normoxic or moderately, chronically hypoxic. Risk factors for chronic hypoxia include fetal growth restriction, which is associated with an increased incidence of oligohydramnios and thus a risk for umbilical cord occlusion (UCO) and variable fetal heart rate (FHR) decelerations. At delivery, fetal growth restriction infants (<3rd percentile) have nearly twice the incidence of low Apgar scores and umbilical pH <7.0. Despite the risks of oligohydramnios and intermittent UCO, there is little understanding of the acid/base responses rates of chronically hypoxic fetuses to variable FHR decelerations as might occur during human labor. We sought to compare the increase in base deficit (BD) among chronically hypoxic as compared to normoxic ovine fetuses in response to simulated mild, moderate, and severe variable FHR decelerations. Near-term ovine fetuses were chronically prepared with brachial artery catheters and an inflatable umbilical cuff occluder. Following a recovery period, normoxic (n = 9) and spontaneously hypoxic (n = 5) fetuses were identified (arterial O2 saturation ≤55%). Both animal groups underwent graded, 1-minute occlusions every 2.5 minutes with 1 hour of mild (∼30 beats/min [bpm] decrease from baseline), 1 hour of moderate (∼60 bpm decrease from baseline), and up to 2 hours of severe (∼90 bpm decrease from baseline) variable FHR decelerations until fetal arterial pH reached 7.00, when occlusions were stopped. Repetitive UCO resulted in development of acidosis (pH <7.0) in both groups. Hypoxic and normoxic fetuses demonstrated similar BD increases in response to both mild (0.39, interquartile range [IQR] 0.28-0.45 vs 0.26, IQR 0.01-0.30 mEq/L/10 min, P = .25) and severe (1.97, IQR 1.50-2.43 vs 1.51, IQR 0.97-2.45 mEq/L/10 min, P = .63) variable decelerations. However, moderate variable decelerations increased BD in hypoxic fetuses at 2.5 times the rate of normoxic fetuses (0.97, IQR 0.52-1.72 vs 0.39, IQR 0.23-0.47 mEq/L/10 min, P = .03). During the recovery period, hypoxic fetuses cleared BD slower than normoxic fetuses (0.08 ± 0.02 vs 0.12 ± 0.03 mEq/L/min, P = .02). In comparison to normoxic fetuses, hypoxic fetuses can more rapidly progress to significant metabolic acidosis in response to moderate FHR variable decelerations, and more slowly recover with in utero resuscitation, likely a consequence of impaired placental function and fetal physiologic responses. Copyright © 2016 Elsevier Inc. All rights reserved.

NYVAC vector modified by C7L viral gene insertion improves T cell immune responses and effectiveness against leishmaniasis.

PubMed

Sánchez-Sampedro, L; Mejías-Pérez, E; S Sorzano, Carlos Óscar; Nájera, J L; Esteban, M

2016-07-15

The NYVAC poxvirus vector is used as vaccine candidate for HIV and other diseases, although there is only limited experimental information on its immunogenicity and effectiveness for use against human pathogens. Here we defined the selective advantage of NYVAC vectors in a mouse model by comparing the immune responses and protection induced by vectors that express the LACK (Leishmania-activated C-kinase antigen), alone or with insertion of the viral host range gene C7L that allows the virus to replicate in human cells. Using DNA prime/virus boost protocols, we show that replication-competent NYVAC-LACK that expresses C7L (NYVAC-LACK-C7L) induced higher-magnitude polyfunctional CD8(+) and CD4(+) primary adaptive and effector memory T cell responses (IFNγ, TNFα, IL-2, CD107a) to LACK antigen than non-replicating NYVAC-LACK. Compared to NYVAC-LACK, the NYVAC-LACK-C7L-induced CD8(+) T cell population also showed higher proliferation when stimulated with LACK antigen. After a challenge by subcutaneous Leishmania major metacyclic promastigotes, NYVAC-LACK-C7L-vaccinated mouse groups showed greater protection than the NYVAC-LACK-vaccinated group. Our results indicate that the type and potency of immune responses induced by LACK-expressing NYVAC vectors is improved by insertion of the C7L gene, and that a replication-competent vector as a vaccine renders greater protection against a human pathogen than a non-replicating vector. Copyright © 2016 Elsevier B.V. All rights reserved.

Mitochondria As Sources and Targets of Methane.

PubMed

Mészáros, András Tamás; Szilágyi, Ágnes Lilla; Juhász, László; Tuboly, Eszter; Érces, Dániel; Varga, Gabriella; Hartmann, Petra

2017-01-01

This review summarizes the current knowledge on the role of mitochondria in the context of hypoxic cell biology, while providing evidence of how these mechanisms are modulated by methane (CH 4 ). Recent studies have unambiguously confirmed CH 4 bioactivity in various in vitro and in vivo experimental models and established the possibility that CH 4 can affect many aspects of mitochondrial physiology. To date, no specific binding of CH 4 to any enzymes or receptors have been reported, and it is probable that many of its effects are related to physico-chemical properties of the non-polar molecule. (i) Mitochondria themselves can be sources of endogenous CH 4 generation under oxido-reductive stress conditions; chemical inhibition of the mitochondrial electron transport chain with site-specific inhibitors leads to increased formation of CH 4 in eukaryote cells, in plants, and in animals. (ii) Conventionally believed as physiologically inert, studies cited in this review demonstrate that exogenous CH 4 modulates key events of inflammation. The anti-apoptotic effects of exogenously administered CH 4 are also recognized, and these properties also suggest that CH 4 -mediated intracellular signaling is closely associated with mitochondria. (iii) Mitochondrial substrate oxidation is coupled with the reduction of molecular oxygen, thus providing energy for cellular metabolism. Interestingly, recent in vivo studies have shown improved basal respiration and modulated mitochondrial oxidative phosphorylation by exogenous CH 4 . Overall, these data suggest that CH 4 liberation and effectiveness in eukaryotes are both linked to hypoxic events and redox regulation and support the notion that CH 4 has therapeutic roles in mammalian pathophysiologies.

Mitochondria As Sources and Targets of Methane

PubMed Central

Mészáros, András Tamás; Szilágyi, Ágnes Lilla; Juhász, László; Tuboly, Eszter; Érces, Dániel; Varga, Gabriella; Hartmann, Petra

2017-01-01

This review summarizes the current knowledge on the role of mitochondria in the context of hypoxic cell biology, while providing evidence of how these mechanisms are modulated by methane (CH4). Recent studies have unambiguously confirmed CH4 bioactivity in various in vitro and in vivo experimental models and established the possibility that CH4 can affect many aspects of mitochondrial physiology. To date, no specific binding of CH4 to any enzymes or receptors have been reported, and it is probable that many of its effects are related to physico-chemical properties of the non-polar molecule. (i) Mitochondria themselves can be sources of endogenous CH4 generation under oxido-reductive stress conditions; chemical inhibition of the mitochondrial electron transport chain with site-specific inhibitors leads to increased formation of CH4 in eukaryote cells, in plants, and in animals. (ii) Conventionally believed as physiologically inert, studies cited in this review demonstrate that exogenous CH4 modulates key events of inflammation. The anti-apoptotic effects of exogenously administered CH4 are also recognized, and these properties also suggest that CH4-mediated intracellular signaling is closely associated with mitochondria. (iii) Mitochondrial substrate oxidation is coupled with the reduction of molecular oxygen, thus providing energy for cellular metabolism. Interestingly, recent in vivo studies have shown improved basal respiration and modulated mitochondrial oxidative phosphorylation by exogenous CH4. Overall, these data suggest that CH4 liberation and effectiveness in eukaryotes are both linked to hypoxic events and redox regulation and support the notion that CH4 has therapeutic roles in mammalian pathophysiologies. PMID:29181377

Photophysical properties and photocytotoxicity of free and liposome-entrapped diazepinoporphyrazines on LNCaP cells under normoxic and hypoxic conditions.

PubMed

Wieczorek, Ewelina; Mlynarczyk, Dariusz T; Kucinska, Malgorzata; Dlugaszewska, Jolanta; Piskorz, Jaroslaw; Popenda, Lukasz; Szczolko, Wojciech; Jurga, Stefan; Murias, Marek; Mielcarek, Jadwiga; Goslinski, Tomasz

2018-04-25

5,7-Diaryl-substituted symmetrical diazepinoporphyrazine and tribenzodiazepinoporphyrazine were synthesized and characterized using UV-Vis, MS MALDI, and various NMR techniques. The expected photosensitizing potentials of these porphyrazines were evaluated by measuring their abilities to generate singlet oxygen in organic solvents and by comparing them with that of the recently obtained dendrimeric G1-type diazepinoporhyrazine. Absorbance and fluorescence measurements were performed to study the aggregation properties of the novel macrocycles. The photocytotoxicity of tribenzodiazepinoporphyrazine towards LNCaP cells in its free form and after its incorporation into liposomes was examined using MTT assay under normoxic and hypoxic conditions. It is interesting that all tested liposome formulations maintained their phototoxic activity in hypoxia. Also, tribenzodiazepinoporphyrazine incorporated into liposomes revealed better photocytotoxic effect (IC 50 values of 0.600 ± 0.357 μM and 0.378 ± 0.002 μM) than its free form (IC 50 values of 3.135 ± 0.156 μM). Following the in vitro experiments, the most promising liposomal formulation containing l-α-phosphatidyl-DL-glycerol for tribenzodiazepinoporphyrazine was found. Moreover, tribenzodiazepinoporphyrazine incorporated into liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) revealed moderate phototoxicity at 5 × 10 -5  μM for antibacterial photodynamic therapy. It was established that an irradiation of planktonic bacterial strains significantly reduced CFUs of Staphylococcus aureus ATCC 25923 in comparison to tribenzodiazepinoporphyrazine containing l-α-phosphatidyl-DL-glycerol liposomes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.