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Sample records for comparing hypoxia-induced gene

  1. Screening of hypoxia-inducible genes in sporadic ALS.

    LENUS (Irish Health Repository)

    Cronin, Simon

    2008-10-01

    Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

  2. A prognostic profile of hypoxia-induced genes for localised high-grade soft tissue sarcoma

    DEFF Research Database (Denmark)

    Aggerholm-Pedersen, Ninna; Sørensen, Brita Singers; Overgaard, Jens

    2016-01-01

    sarcoma (STS). METHODS: The hypoxia-induced gene quantification was performed by real-time quantitative PCR (RT-qPCR) of formalin-fixed, paraffin-embedded tissue samples. The gene expression cut-points were determined in a test cohort of 55 STS patients and used to allocate each patient into a more......BACKGROUND: For decades, tumour hypoxia has been pursued as a cancer treatment target. However, prognostic and predictive biomarkers are essential for the use of this target in the clinic. This study investigates the prognostic value of a hypoxia-induced gene profile in localised soft tissue...

  3. Translational control is a major contributor to hypoxia induced gene expression

    International Nuclear Information System (INIS)

    Beucken, Twan van den; Magagnin, Michael G.; Jutten, Barry; Seigneuric, Renaud; Lambin, Philippe; Koritzinsky, Marianne; Wouters, Bradly G.

    2011-01-01

    Background and purpose: Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. Material and methods: Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia ( 2 ). Changes in transcription and translation were assessed using affymetrix microarray technology. Results: Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2-4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. Conclusions: Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.

  4. Hypoxia-Inducible Regulation of a Prodrug-Activating Enzyme for Tumor-Specific Gene Therapy

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    Toru Shibata

    2002-01-01

    Full Text Available Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

  5. Identification of crucial microRNAs and genes in hypoxia-induced human lung adenocarcinoma cells

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    Geng Y

    2016-07-01

    Full Text Available Ying Geng,1,* Lili Deng,2,* Dongju Su,1 Jinling Xiao,1 Dongjie Ge,3 Yongxia Bao,1 Hui Jing4 1Department of Respiratory, 2Department of Oncology, The Second Affiliated Hospital of Harbin Medical University, 3Department of Respiratory, The First Hospital of Harbin, 4Department of Emergency, The Second Affiliated Hospital of Harbin Medical University Harbin, Heilongjiang, People’s Republic of China *These authors contributed equally to this work Background: Variations of microRNA (miRNA expression profile in hypoxic lung cancer cells have not been studied so far. Therefore, using miRNA microarray technology, this study aimed to study the miRNA expression profile and investigate the potential crucial miRNAs and their target genes in hypoxia-induced human lung adenocarcinoma cells.Materials and methods: Based on miRNA microarray, miRNA expression profiling of hypoxia-induced lung adenocarcinoma A549 cells was obtained. After identification of differentially expressed miRNAs (DE-miRNAs in hypoxic cells, target genes of DE-miRNAs were predicted, and functional enrichment analysis of targets was conducted. Furthermore, the expression levels of DE-miRNAs and their target genes were validated by real-time quantitative polymerase chain reaction. In addition, using miRNA mimics, the effect of overexpressed DE-miRNAs on A549 cell behaviors (cell proliferation, cell cycle, and apoptosis was evaluated.Results: In total, 14 DE-miRNAs (nine upregulated miRNAs and five downregulated miRNAs were identified in hypoxic cells, compared with normoxic cells. Target genes of both upregulated and downregulated miRNAs were enriched in the functions such as chromatin modification, and pathways such as Wnt signaling pathway and transforming growth factor (TGF-β signaling pathway. The expression levels of several miRNAs and their target genes were confirmed, including hsa-miR-301b/FOXF2, hsa-miR-148b-3p/WNT10B, hsa-miR-769-5p/(SMAD2, ARID1A, and hsa-miR-622. Among them

  6. A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

    Energy Technology Data Exchange (ETDEWEB)

    Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

    2004-11-01

    Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

  7. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

    Science.gov (United States)

    Carlin, Sean; Pugachev, Andrei; Sun, Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C Clifton; Humm, John L

    2009-10-01

    To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the

  8. In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

    International Nuclear Information System (INIS)

    Carlin, Sean; Pugachev, Andrei; Sun Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C. Clifton; Humm, John L.

    2009-01-01

    Purpose: To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer 18 F-fluoromisonidazole ( 18 F-FMISO). Methods: Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe 124 I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil ( 124 I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between 124 I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe 18 F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with 124 I-FIAU (3 h before sacrifice) and 18 F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between 18 F-FMISO and 124 I-FIAU on a pixel-by-pixel basis was performed. Results: Correlation coefficients between 124 I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between 18 F-FMISO and 124 I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. Conclusions: We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of

  9. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

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    Hiroyasu Kamei

    2008-08-01

    Full Text Available Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

  10. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella

    NARCIS (Netherlands)

    Mengesha, Asferd; Dubois, Ludwig; Lambin, Philippe; Landuyt, Willy; Chiu, Roland K; Wouters, Bradly G; Theys, Jan

    To increase the potential of attenuated Salmonella as gene delivery vectors for cancer treatment, we developed a hypoxia-inducible promoter system to limit gene expression specifically to the tumor. This approach is envisaged to not only increase tumor specificity, but also to target those cells

  11. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

    Science.gov (United States)

    Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

    2001-11-01

    Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

  12. Hypoxia-inducible factor directs POMC gene to mediate hypothalamic glucose sensing and energy balance regulation.

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    Hai Zhang

    2011-07-01

    Full Text Available Hypoxia-inducible factor (HIF is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance.

  13. Hypoxia-Inducible Factor Directs POMC Gene to Mediate Hypothalamic Glucose Sensing and Energy Balance Regulation

    Science.gov (United States)

    Zhang, Hai; Zhang, Guo; Gonzalez, Frank J.; Park, Sung-min; Cai, Dongsheng

    2011-01-01

    Hypoxia-inducible factor (HIF) is a nuclear transcription factor that responds to environmental and pathological hypoxia to induce metabolic adaptation, vascular growth, and cell survival. Here we found that HIF subunits and HIF2α in particular were normally expressed in the mediobasal hypothalamus of mice. Hypothalamic HIF was up-regulated by glucose to mediate the feeding control of hypothalamic glucose sensing. Two underlying molecular pathways were identified, including suppression of PHDs by glucose metabolites to prevent HIF2α degradation and the recruitment of AMPK and mTOR/S6K to regulate HIF2α protein synthesis. HIF activation was found to directly control the transcription of POMC gene. Genetic approach was then employed to develop conditional knockout mice with HIF inhibition in POMC neurons, revealing that HIF loss-of-function in POMC neurons impaired hypothalamic glucose sensing and caused energy imbalance to promote obesity development. The metabolic effects of HIF in hypothalamic POMC neurons were independent of leptin signaling or pituitary ACTH pathway. Hypothalamic gene delivery of HIF counteracted overeating and obesity under conditions of nutritional excess. In conclusion, HIF controls hypothalamic POMC gene to direct the central nutrient sensing in regulation of energy and body weight balance. PMID:21814490

  14. Anti-vascular agent Combretastatin A-4-P modulates Hypoxia Inducible Factor-1 and gene expression

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    Currie Margaret J

    2006-12-01

    Full Text Available Abstract Background A functional vascular network is essential for the survival, growth and spread of solid tumours, making blood vessels a key target for therapeutic strategies. Combretastatin A-4 phosphate (CA-4-P is a tubulin-depolymerising agent in Phase II clinical trials as a vascular disrupting agent. Not much is known of the molecular effect of CA-4-P under tumour conditions. The tumour microenvironment differs markedly from that in normal tissue, specifically with respect to oxygenation (hypoxia. Gene regulation under tumour conditions is governed by hypoxia inducible factor 1 (HIF-1, controlling angiogenic and metastatic pathways. Methods We investigated the effect of CA-4-P on factors of the upstream and downstream signalling pathway of HIF-1 in vitro. Results CA-4-P treatment under hypoxia tended to reduce HIF-1 accumulation in a concentration-dependent manner, an effect which was more prominent in endothelial cells than in cancer cell lines. Conversely, CA-4-P increased HIF-1 accumulation under aerobic conditions in vitro. At these concentrations of CA-4-P under aerobic conditions, nuclear factor κB was activated via the small GTPase RhoA, and expression of the HIF-1 downstream angiogenic effector gene, vascular endothelial growth factor (VEGF-A, was increased. Conclusion Our findings advance the understanding of signal transduction pathways involved in the actions of the anti-vascular agent CA-4-P.

  15. Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation☆

    OpenAIRE

    Cui, Hong; Han, Weijuan; Yang, Lijun; Chang, Yanzhong

    2013-01-01

    Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor 1α, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage. There is little evidence of direct regulatory effects of hypoxia-inducible factor 1α on oligodendrocyte lineage gene-1. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxy...

  16. Hypoxia-induced decrease of UCP3 gene expression in rat heart parallels metabolic gene switching but fails to affect mitochondrial respiratory coupling.

    Science.gov (United States)

    Essop, M Faadiel; Razeghi, Peter; McLeod, Chris; Young, Martin E; Taegtmeyer, Heinrich; Sack, Michael N

    2004-02-06

    Mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) are postulated to contribute to antioxidant defense, nutrient partitioning, and energy efficiency in the heart. To distinguish isotype function in response to metabolic stress we measured cardiac mitochondrial function and cardiac UCP gene expression following chronic hypobaric hypoxia. Isolated mitochondrial O(2) consumption and ATP synthesis rate were reduced but respiratory coupling was unchanged compared to normoxic groups. Concurrently, left ventricular UCP3 mRNA levels were significantly decreased with hypoxia (pheart as opposed to uncoupling of mitochondria. Moreover, the divergent hypoxia-induced regulation of UCP2 and UCP3 supports distinct mitochondrial regulatory functions of these inner mitochondrial membrane proteins in the heart in response to metabolic stress.

  17. Altered expression of hypoxia-inducible factor-1α (HIF-1α and its regulatory genes in gastric cancer tissues.

    Directory of Open Access Journals (Sweden)

    Jihan Wang

    Full Text Available Tissue hypoxia induces reprogramming of cell metabolism and may result in normal cell transformation and cancer progression. Hypoxia-inducible factor 1-alpha (HIF-1α, the key transcription factor, plays an important role in gastric cancer development and progression. This study aimed to investigate the underlying regulatory signaling pathway in gastric cancer using gastric cancer tissue specimens. The integration of gene expression profile and transcriptional regulatory element database (TRED was pursued to identify HIF-1α ↔ NFκB1 → BRCA1 → STAT3 ← STAT1 gene pathways and their regulated genes. The data showed that there were 82 differentially expressed genes that could be regulated by these five transcription factors in gastric cancer tissues and these genes formed 95 regulation modes, among which seven genes (MMP1, TIMP1, TLR2, FCGR3A, IRF1, FAS, and TFF3 were hub molecules that are regulated at least by two of these five transcription factors simultaneously and were associated with hypoxia, inflammation, and immune disorder. Real-Time PCR and western blot showed increasing of HIF-1α in mRNA and protein levels as well as TIMP1, TFF3 in mRNA levels in gastric cancer tissues. The data are the first study to demonstrate HIF-1α-regulated transcription factors and their corresponding network genes in gastric cancer. Further study with a larger sample size and more functional experiments is needed to confirm these data and then translate into clinical biomarker discovery and treatment strategy for gastric cancer.

  18. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: insight into the cytotoxicity and antiangiogenesis

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    Yang T

    2016-12-01

    Full Text Available Tieshan Yang,1 Qian Yao,1 Fei Cao,1 Qianqian Liu,1 Binlei Liu,2 Xiu-Hong Wang1 1Laboratory for Biomedical Photonics, Institute of Laser Engineering, Beijing University of Technology, 2Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Abstract: Hypoxia-inducible factor-1 (HIF-1 is a transcription factor that is activated upon exposure to hypoxic stress. It modulates a number of cellular responses including proliferation, apoptosis, angiogenesis, and metabolism by activating a panel of target genes in response to hypoxia. The HIF-1 level is often upregulated in the hypoxic microenvironment of solid tumors, which contributes to cancer treatment failure. Here we report that silver nanoparticles (AgNPs, which are widely used as an antimicrobial agent, are an effective inhibitor of HIF-1. AgNPs inhibited the activation of a HIF-dependent reporter construct after the cells were exposed to hypoxic conditions or treated with cobalt chloride, a hypoxia mimetic agent. The AgNPs also interfered with the accumulation of HIF-1α protein and the induction of the endogenous HIF target genes, VEGF-A and GLUT1. Since both HIF-1 and vascular endothelial growth factor-A play an important role in angiogenesis, AgNPs also inhibited angiogenesis in vitro. Our data reveal a new mechanism of how AgNPs act on cellular function, that is, they disrupt HIF signaling pathway. This finding provides a novel insight into how AgNPs can inhibit cancer cell growth and angiogenesis. Keywords: silver nanoparticles (AgNPs, hypoxia-inducible factor, transcriptional activity, vascular endothelial growth factor-A, angiogenesis

  19. Silver nanoparticles inhibit the function of hypoxia-inducible factor-1 and target genes: insight into the cytotoxicity and antiangiogenesis.

    Science.gov (United States)

    Yang, Tieshan; Yao, Qian; Cao, Fei; Liu, Qianqian; Liu, Binlei; Wang, Xiu-Hong

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that is activated upon exposure to hypoxic stress. It modulates a number of cellular responses including proliferation, apoptosis, angiogenesis, and metabolism by activating a panel of target genes in response to hypoxia. The HIF-1 level is often upregulated in the hypoxic microenvironment of solid tumors, which contributes to cancer treatment failure. Here we report that silver nanoparticles (AgNPs), which are widely used as an antimicrobial agent, are an effective inhibitor of HIF-1. AgNPs inhibited the activation of a HIF-dependent reporter construct after the cells were exposed to hypoxic conditions or treated with cobalt chloride, a hypoxia mimetic agent. The AgNPs also interfered with the accumulation of HIF-1α protein and the induction of the endogenous HIF target genes, VEGF-A and GLUT1. Since both HIF-1 and vascular endothelial growth factor-A play an important role in angiogenesis, AgNPs also inhibited angiogenesis in vitro. Our data reveal a new mechanism of how AgNPs act on cellular function, that is, they disrupt HIF signaling pathway. This finding provides a novel insight into how AgNPs can inhibit cancer cell growth and angiogenesis.

  20. Duplication and Diversification of the Hypoxia-Inducible IGFBP-1 Gene in Zebrafish

    DEFF Research Database (Denmark)

    Kamei, Hiroyasu; Lu, Ling; Jiao, Shuang

    2008-01-01

    Background: Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenabilit...

  1. Polymorphisms in the hypoxia-inducible factor 1 alpha gene in Mexican patients with preeclampsia: A case-control study

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    Nava-Salazar Sonia

    2011-03-01

    Full Text Available Abstract Background Although the etiology of preeclampsia is still unclear, recent work suggests that changes in circulating angiogenic factors play a key role in its pathogenesis. In the trophoblast of women with preeclampsia, hypoxia-inducible factor 1 alpha (HIF-1α is over-expressed, and induces the expression of non-angiogenic factors and inhibitors of trophoblast differentiation. This observation prompted the study of HIF-1α and its relation to preeclampsia. It has been described that the C1772T (P582S and G1790A (A588T polymorphisms of the HIF1A gene have significantly greater transcriptional activity, correlated with an increased expression of their proteins, than the wild-type sequence. In this work, we studied whether either or both HIF1A variants contribute to preeclampsia susceptibility. Results Genomic DNA was isolated from 150 preeclamptic and 105 healthy pregnant women. Exon 12 of the HIF1A gene was amplified by PCR, and the genotypes of HIF1A were determined by DNA sequencing. In preeclamptic women and controls, the frequencies of the T allele for C1772T were 4.3 vs. 4.8%, and the frequencies of the A allele for G1790A were 0.0 vs. 0.5%, respectively. No significant differences were found between groups. Conclusion The frequency of the C1772T and G1790A polymorphisms of the HIF1A gene is very low, and neither polymorphism is associated with the development of preeclampsia in the Mexican population.

  2. Hypoxia-independent upregulation of placental hypoxia inducible factor-1α gene expression contributes to the pathogenesis of preeclampsia.

    Science.gov (United States)

    Iriyama, Takayuki; Wang, Wei; Parchim, Nicholas F; Song, Anren; Blackwell, Sean C; Sibai, Baha M; Kellems, Rodney E; Xia, Yang

    2015-06-01

    Accumulation of hypoxia inducible factor-1α (HIF-1α) is commonly an acute and beneficial response to hypoxia, whereas chronically elevated HIF-1α is associated with multiple disease conditions, including preeclampsia, a serious hypertensive disease of pregnancy. However, the molecular basis underlying the persistent elevation of placental HIF-1α in preeclampsia and its role in the pathogenesis of preeclampsia are poorly understood. Here we report that Hif-1α mRNA and HIF-1α protein were elevated in the placentas of pregnant mice infused with angiotensin II type I receptor agonistic autoantibody, a pathogenic factor in preeclampsia. Knockdown of placental Hif-1α mRNA by specific siRNA significantly attenuated hallmark features of preeclampsia induced by angiotensin II type I receptor agonistic autoantibody in pregnant mice, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and elevated maternal circulating soluble fms-like tyrosine kinase-1 levels. Next, we discovered that Hif-1α mRNA levels and HIF-1α protein levels were induced in an independent preeclampsia model with infusion of the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT). SiRNA knockdown experiments also demonstrated that elevated HIF-1α contributed to LIGHT-induced preeclampsia features. Translational studies with human placentas showed that angiotensin II type I receptor agonistic autoantibody or LIGHT is capable of inducing HIF-1α in a hypoxia-independent manner. Moreover, increased HIF-1α was found to be responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and production of soluble fms-like tyrosine kinase-1 in human villous explants. Overall, we demonstrated that hypoxia-independent stimulation of HIF-1α gene expression in the placenta is a common pathogenic mechanism promoting disease progression. Our findings reveal new insight to preeclampsia and highlight

  3. Hypoxia-inducible factors - regulation, role and comparative aspects in tumourigenesis

    DEFF Research Database (Denmark)

    Hansen, A E; Kristensen, A T; Law, I

    2011-01-01

    important prognostic information and may help identify potential hypoxia circumventing and targeting strategies. This review summarizes current knowledge on HIF regulation and function in tumour cells and discusses the aspects of using companion animals as comparative spontaneous cancer models. Spontaneous...... tumours in companion animals hold a great research potential for the evaluation and understanding of tumour hypoxia and in the development of hypoxia-targeting therapeutics....

  4. Mipu1, a novel direct target gene, is involved in hypoxia inducible factor 1-mediated cytoprotection.

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    Kangkai Wang

    Full Text Available Mipu1 (myocardial ischemic preconditioning up-regulated protein 1, recently identified in our lab, is a novel zinc-finger transcription factor which is up-regulated during ischemic preconditioning. However, it is not clear what transcription factor contributes to its inducible expression. In the present study, we reported that HIF-1 regulates the inducible expression of Mipu1 which is involved in the cytoprotection of HIF-1α against oxidative stress by inhibiting Bax expression. Our results showed that the inducible expression of Mipu1 was associated with the expression and activation of transcription factor HIF-1 as indicated by cobalt chloride (CoCl2 treatment, HIF-1α overexpression and knockdown assays. EMSA and luciferase reporter gene assays showed that HIF-1α bound to the hypoxia response element (HRE within Mipu1 promoter region and promoted its transcription. Moreover, our results revealed that Mipu1 inhibited the expression of Bax, an important pro-apoptosis protein associated with the intrinsic pathway of apoptosis, elevating the cytoprotection of HIF-1 against hydrogen peroxide (H2O2-mediated injury in H9C2 cells. Our findings implied that Bax may be a potential target gene of transcription factor Mipu1, and provided a novel insight for understanding the cytoprotection of HIF-1 and new clues for further elucidating the mechanisms by which Mipu1 protects cell against pathological stress.

  5. Improving heterologous protein secretion at aerobic conditions by activating hypoxia-induced genes in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Liu, Lifang; Zhang, Yiming; Liu, Zihe

    2015-01-01

    Oxygen is important for normal aerobic metabolism, as well as for protein production where it is needed for oxidative protein folding. However, several studies have reported that anaerobic conditions seem to be more favorable in terms of recombinant protein production. We were interested in incre......Oxygen is important for normal aerobic metabolism, as well as for protein production where it is needed for oxidative protein folding. However, several studies have reported that anaerobic conditions seem to be more favorable in terms of recombinant protein production. We were interested...... in increasing recombinant protein production under aerobic conditions so we focused on Rox1p regulation. Rox1p is a transcriptional regulator, which in oxidative conditions represses genes induced in hypoxia. We deleted ROX1 and studied the effects on the production of recombinant proteins in Saccharomyces...

  6. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

    International Nuclear Information System (INIS)

    Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.; Hales, Charles A.

    2006-01-01

    The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension

  7. Chenodeoxycholic Acid Reduces Hypoxia Inducible Factor-1α Protein and Its Target Genes.

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    Yunwon Moon

    Full Text Available This study evaluated HIF-1α inhibitors under different hypoxic conditions, physiological hypoxia (5% O2 and severe hypoxia (0.1% O2. We found that chenodeoxy cholic acid (CDCA reduced the amount of HIF-1α protein only under physiological hypoxia but not under severe hypoxia without decreasing its mRNA level. By using a proteasome inhibitor MG132 and a translation inhibitor cyclohexamide, we showed that CDCA reduced HIF-1α protein by decreasing its translation but not by enhancing its degradation. The following findings indicated that farnesoid X receptor (FXR, a CDCA receptor and its target gene, Small heterodimer partner (SHP are not involved in this effect of CDCA. Distinctly from CDCA, MG132 prevented SHP and an exogenous FXR agonist, GW4064 from reducing HIF-1α protein. Furthermore a FXR antagonist, guggulsterone failed to prevent CDCA from decreasing HIF-1α protein. Furthermore, guggulsterone by itself reduced HIF-1α protein even in the presence of MG132. These findings suggested that CDCA and guggulsterone reduced the translation of HIF-1α in a mechanism which FXR and SHP are not involved. This study reveals novel therapeutic functions of traditional nontoxic drugs, CDCA and guggulsterone, as inhibitors of HIF-1α protein.

  8. BAY 87-2243, a highly potent and selective inhibitor of hypoxia-induced gene activation has antitumor activities by inhibition of mitochondrial complex I

    International Nuclear Information System (INIS)

    Ellinghaus, Peter; Heisler, Iring; Unterschemmann, Kerstin; Haerter, Michael; Beck, Hartmut; Greschat, Susanne; Ehrmann, Alexander; Summer, Holger; Flamme, Ingo; Oehme, Felix; Thierauch, Karlheinz; Michels, Martin; Hess-Stumpp, Holger; Ziegelbauer, Karl

    2013-01-01

    The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel–Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors

  9. Association of hypoxia inducible factor-1 alpha gene polymorphism with both type 1 and type 2 diabetes in a Caucasian (Hungarian sample

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    Panczel Pal

    2009-08-01

    Full Text Available Abstract Background Hypoxia inducible factor-1 alpha (HIF-1α is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465 of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890 was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929. Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740 Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls, the genotypes were grouped as T absent (CC and T present (CT and TT. Genotype-wise analysis showed a significant increase of T present group in controls (24.0% as compared to patients (16.8%, P = 0.008. This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020, and also in type 2 (17.6%, P = 0.032 diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3% than in the clinical sample (8.7%, P = 0.002 with similar results in type 1 (7.8%, P = 0.010 and type 2 (9.1%, P = 0.011 diabetes. The odds ratio for diabetes (either type 1 or 2 was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect

  10. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    International Nuclear Information System (INIS)

    Taylor, Cormac T.; Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T.; Ryan, Silke

    2014-01-01

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  11. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Taylor, Cormac T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T. [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland); Ryan, Silke, E-mail: silke.ryan@ucd.ie [School of Medicine and Medical Science, The Conway Institute, University College Dublin (Ireland); Pulmonary and Sleep Disorders Unit, St. Vincent’s University Hospital, Dublin (Ireland)

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  12. Hypoxic induction of the regulator of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor pathway.

    Directory of Open Access Journals (Sweden)

    Sam W Z Olechnowicz

    Full Text Available The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2 in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4 protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered.

  13. Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway

    Science.gov (United States)

    Olechnowicz, Sam W. Z.; Fedele, Anthony O.; Peet, Daniel J.

    2012-01-01

    The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered. PMID:22970249

  14. Hypoxia-inducible factor-1α regulates chemotactic migration of pancreatic ductal adenocarcinoma cells through directly transactivating the CX3CR1 gene.

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    Tiansuo Zhao

    Full Text Available CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF-1α expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1α bound to the hypoxia-response element (HRE; 5'-A/GCGTG-3' of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1α significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1α may regulate cancer cell migration through CX3CR1. The HIF-1α/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC.

  15. Development of a cell-based reporter assay for screening of inhibitors of hypoxia-inducible factor 2-induced gene expression.

    Science.gov (United States)

    Woldemichael, Girma M; Vasselli, James R; Gardella, Roberta S; McKee, Tawnya C; Linehan, W Marston; McMahon, James B

    2006-09-01

    Reporter cell lines have been developed for the identification of inhibitors of gene expression enhanced by hypoxia-inducible factor 2, which has been implicated as a transcription factor involved in the tumorigenesis of clear cell renal carcinoma. Stably transformed reporter clones of the human renal clear cell carcinoma cell line 786-O were generated by transfection or retroviral infection. Luciferase reporter expression in the vectors used was driven by either the natural human vascular endothelial growth factor (VEGF) promoter-enhancer or by the VEGF and the human endothelial nitric oxide synthase enhancers modulating minimal human cytomegalovirus promoter. Utility of the generated reporter cell lines was validated by introducing the von Hippel-Lindau protein complex and testing for reporter inducibility by hypoxia. The dynamic range in reporter activity under hypoxic stress was found to be at least 30- to 40-fold, with a signal-to-noise ratio of 60:1. Properties of the cell lines such as tolerance to up to 3% DMSO, signal stability with multiple in vitro passages, and utility in both 96- and 384-well plate formats indicated their suitability for use in a high-throughput screen. In addition, the potential use of these reporter lines in the evaluation of high-throughput screening hits in vivo in various mice models has been demonstrated.

  16. Hypoxia induces telomerase reverse transcriptase (TERT gene expression in non-tumor fish tissues in vivo: the marine medaka (Oryzias melastigma model

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    Mok Helen OL

    2006-09-01

    Full Text Available Abstract Background Current understanding on the relationships between hypoxia, hypoxia-inducible factor-1 (HIF-1 and telomerase reverse transcriptase (TERT gene expression are largely based on in vitro studies in human cancer cells. Although several reports demonstrated HIF-1- mediated upregulation of the human TERT gene under hypoxia, conflicting findings have also been reported. Thus far, it remains uncertain whether these findings can be directly extrapolated to non-tumor tissues in other whole animal systems in vivo. While fish often encounter environmental hypoxia, the in vivo regulation of TERT by hypoxia in non-neoplastic tissues of fish remains virtually unknown. Results The adult marine medaka (Oryzias melastigma was employed as a model fish in this study. We have cloned and characterized a 3261-bp full-length TERT cDNA, omTERT, which encodes a protein of 1086 amino acids. It contains all of the functional motifs that are conserved in other vertebrate TERTs. Motif E is the most highly conserved showing 90.9–100% overall identity among the fish TERTs and 63.6% overall identity among vertebrates. Analysis of the 5'-flanking sequence of the omTERT gene identified two HRE (hypoxia-responsive element; nt. – 283 and – 892 cores. Overexpression of the HIF-1α induced omTERT promoter activity as demonstrated using transient transfection assays. The omTERT gene is ubiquitously expressed in fish under normoxia, albeit at varying levels, where highest expression was observed in gonads and the lowest in liver. In vivo expression of omTERT was significantly upregulated in testis and liver in response to hypoxia (at 96 h and 48 h, respectively, where concomitant induction of the omHIF-1α and erythropoietin (omEpo genes was also observed. In situ hybridization analysis showed that hypoxic induction of omTERT mRNA was clearly evident in hepatocytes in the caudal region of liver and in spermatogonia-containing cysts in testis. Conclusion This

  17. Myeloid cell leukemia-1 (Mc1-1 is a candidate target gene of hypoxia-inducible factor-1 (HIF-1 in the testis

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    Palladino Michael A

    2012-12-01

    Full Text Available Abstract Background Spermatic cord torsion can lead to testis ischemia (I and subsequent ischemia-reperfusion (I/R causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1 transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5’-RCGTG-3’ identified myeloid cell leukemia-1 (Mcl-1 as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1. Methods The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA and electrophoretic mobility shift assays (EMSA. MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP analysis. Results The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter. Conclusions The results

  18. Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

    Directory of Open Access Journals (Sweden)

    Mahendran Botlagunta

    2011-03-01

    Full Text Available DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

  19. Reactive oxygen species-generating mitochondrial DNA mutation up-regulates hypoxia-inducible factor-1alpha gene transcription via phosphatidylinositol 3-kinase-Akt/protein kinase C/histone deacetylase pathway.

    Science.gov (United States)

    Koshikawa, Nobuko; Hayashi, Jun-Ichi; Nakagawara, Akira; Takenaga, Keizo

    2009-11-27

    Lewis lung carcinoma-derived high metastatic A11 cells constitutively overexpress hypoxia-inducible factor (HIF)-1alpha mRNA compared with low metastatic P29 cells. Because A11 cells exclusively possess a G13997A mutation in the mitochondrial NADH dehydrogenase subunit 6 (ND6) gene, we addressed here a causal relationship between the ND6 mutation and the activation of HIF-1alpha transcription, and we investigated the potential mechanism. Using trans-mitochondrial cybrids between A11 and P29 cells, we found that the ND6 mutation was directly involved in HIF-1alpha mRNA overexpression. Stimulation of HIF-1alpha transcription by the ND6 mutation was mediated by overproduction of reactive oxygen species (ROS) and subsequent activation of phosphatidylinositol 3-kinase (PI3K)-Akt and protein kinase C (PKC) signaling pathways. The up-regulation of HIF-1alpha transcription was abolished by mithramycin A, an Sp1 inhibitor, but luciferase reporter and chromatin immunoprecipitation assays indicated that Sp1 was necessary but not sufficient for HIF-1alpha mRNA overexpression in A11 cells. On the other hand, trichostatin A, a histone deacetylase (HDAC) inhibitor, markedly suppressed HIF-1alpha transcription in A11 cells. In accordance with this, HDAC activity was high in A11 cells but low in P29 cells and in A11 cells treated with the ROS scavenger ebselene, the PI3K inhibitor LY294002, and the PKC inhibitor Ro31-8220. These results suggest that the ROS-generating ND6 mutation increases HIF-1alpha transcription via the PI3K-Akt/PKC/HDAC pathway, leading to HIF-1alpha protein accumulation in hypoxic tumor cells.

  20. Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia-inducible Factor-2 Alpha, is Regulated by von Hippel-Lindau Gene Mutation Status.

    Science.gov (United States)

    Messai, Yosra; Gad, Sophie; Noman, Muhammad Zaeem; Le Teuff, Gwenael; Couve, Sophie; Janji, Bassam; Kammerer, Solenne Florence; Rioux-Leclerc, Nathalie; Hasmim, Meriem; Ferlicot, Sophie; Baud, Véronique; Mejean, Arnaud; Mole, David Robert; Richard, Stéphane; Eggermont, Alexander M M; Albiges, Laurence; Mami-Chouaib, Fathia; Escudier, Bernard; Chouaib, Salem

    2016-10-01

    Clear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel-Lindau (VHL) gene. To elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. A series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon-intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. Fisher's exact test was used for VHL mutation and Kruskal-Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal-Wallis test was used; otherwise Cochran-Mantel-Haenzsel test was used. We also used the Fligner-Policello test to compare two medians when the distributions had different dispersions. We demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the human PD-L1 proximal promoter in 786-O cells. Our work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. We investigated the relationship between von Hippel-Lindau mutations and programmed death-ligand 1 expression. We

  1. The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1α targeted gene expression.

    Science.gov (United States)

    Miyake, Kotaro; Nishioka, Masanori; Imura, Satoru; Batmunkh, Erdenebulgan; Uto, Yoshihiro; Nagasawa, Hideko; Hori, Hitoshi; Shimada, Mitsuo

    2012-08-01

    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-1α (HIF-1α), 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 bearing 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

    Science.gov (United States)

    Leszczynska, Katarzyna B.; Foskolou, Iosifina P.; Abraham, Aswin G.; Anbalagan, Selvakumar; Tellier, Céline; Haider, Syed; Span, Paul N.; O’Neill, Eric E.; Buffa, Francesca M.; Hammond, Ester M.

    2015-01-01

    Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage–induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain–containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors. PMID:25961455

  3. 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

    Energy Technology Data Exchange (ETDEWEB)

    Miyake, Kotaro, E-mail: hif.panc@gmail.com [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Nishioka, Masanori; Imura, Satoru; Batmunkh, Erdenebulgan [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Uto, Yoshihiro [Department of Biological Science and Technology, Institute of Socio Technosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Nagasawa, Hideko [Laboratory of Pharmaceutical and Medicinal Chemistry, Gifu Pharmaceutical University, Gifu 501-1196 (Japan); Hori, Hitoshi [Department of Biological Science and Technology, Institute of Socio Technosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan); Shimada, Mitsuo [Department of Surgery, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503 (Japan)

    2012-08-01

    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 bearing 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

  4. The novel hypoxic cytotoxin, TX-2098 has antitumor effect in pancreatic cancer; possible mechanism through inhibiting VEGF and hypoxia inducible factor-1α targeted gene expression

    International Nuclear Information System (INIS)

    Miyake, Kotaro; Nishioka, Masanori; Imura, Satoru; Batmunkh, Erdenebulgan; Uto, Yoshihiro; Nagasawa, Hideko; Hori, Hitoshi; Shimada, Mitsuo

    2012-01-01

    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-1α (HIF-1α), 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 bearing 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: ► We designed and synthesized novel hypoxic cytoxin, TX-2098. ► TX-2098 inhibited the proliferation of human pancreatic cancer cells than TPZ. ► TX-2098 reduced VEGF protein level than TPZ. ► TX-2098 inhibited mRNA expression of VEGF, GLUT1 and Aldolase A, not HIF-1α. ► TX-2098 improved the survival in

  5. Baltic salmon (Salmo salar) yolk-sac fry mortality is associated with disturbances in the function of hypoxia-inducible transcription factor (HIF-1α) and consecutive gene expression

    International Nuclear Information System (INIS)

    Vuori, Kristiina A.M.; Soitamo, Arto; Vuorinen, Pekka J.; Nikinmaa, Mikko

    2004-01-01

    Baltic salmon (Salmo salar) suffer from abnormally high yolk-sac fry mortality designated as M74-syndrome. In 1990s, 25-80% of salmon females, which ascended rivers to spawn, produced yolk-sac fry suffering from the syndrome. Symptoms of M74-affected fry include neurological disturbances, impaired vascular development and abnormal haemorrhages. The latter symptoms are observed in mammalian embryos if the function of hypoxia inducible transcription factor (HIF-1α), its dimerization partner aryl hydrocarbon nuclear translocator (ARNT) or target gene vascular endothelial growth factor (VEGF) is disturbed. To study the possible involvement of HIF-1α and its target gene VEGF in the development of the syndrome, we collected healthy and M74-affected wild Baltic salmon yolk-sac fry and analyzed HIF-1α mRNA and protein expression, HIF-1α DNA-binding, target gene VEGF protein expression, and blood vessel density in both groups at different stages of yolk-sac fry development. In addition, since Baltic salmon females contain organochlorine contaminants, which have been suggested to be the cause of M74 syndrome via the aryl hydrocarbon receptor (AhR)-dependent gene expression pathway, we studied AhR protein expression, AhR DNA-binding and target gene CYP1A protein expression. Since the parents of both healthy and M74-affected wild fry will have experienced the organochlorine load from the Baltic Sea, hatchery-reared fry were included in the studies as an additional control. The results show that the vascular defects observed in fry suffering from M74 are associated with reduced DNA-binding activity of HIF-1α and subsequent downregulation of its target gene vascular endothelial growth factor (VEGF). In addition, also AhR function is decreased in diseased fry making it unlikely that symptoms of M74-affected fry would be caused by an upregulation of xenobiotically induced AhR-dependent gene expression pathway

  6. Dexamethasone impairs hypoxia-inducible factor-1 function

    International Nuclear Information System (INIS)

    Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of α- and β-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1α levels in the cytosol of HepG2 cells, while nuclear HIF-1α levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients

  7. Up-regulation of hypoxia-inducible factor (HIF)-1α and HIF-target genes in cortical neurons by the novel multifunctional iron chelator anti-Alzheimer drug, M30.

    Science.gov (United States)

    Avramovich-Tirosh, Y; Bar-Am, O; Amit, T; Youdim, M B H; Weinreb, O

    2010-06-01

    Based on a multimodal drug design paradigm, we have synthesized a multifunctional non-toxic, brain permeable iron chelator, M30, possessing the neuroprotective propargylamine moiety of the anti-Parkinsonian drug, rasagiline (Azilect) and antioxidant-iron chelator moiety of an 8-hydroxyquinoline derivative of our iron chelator, VK28. M30 was recently found to confer potential neuroprotective effects in vitro and in various preclinical neurodegenerative models and regulate the levels and processing of the Alzheimer's amyloid precursor protein and its toxic amyloidogenic derivative, Abeta. Here, we show that M30 activates the hypoxia-inducible factor (HIF)-1alpha signaling pathway, thus promoting HIF-1alpha mRNA and protein expression levels, as well as increasing transcription of HIF-1alpha-dependent genes, including vascular endothelial growth factor, erythropoietin, enolase-1, p21 and tyrosine hydroxylase in rat primary cortical cells. In addition, M30 also increased the expression levels of the transcripts of brain derived neurotrophic factor (BDNF) and growth-associated protein-43 (GAP-43). Regarding aspects of relevance to Alzheimer's disease (AD), western blotting analysis of glycogen synthase kinase- 3beta (GSK-3beta) signaling pathway revealed that M30 enhanced the levels of phospho-AKT (Ser473) and phospho- GSK-3beta (Ser9) and attenuated Tau phosphorylation. M30 was also shown to protect cultured cortical neurons against Abeta(25-35) toxicity. All these multimodal pharmacological activities of M30 might be beneficial for its potent efficacy in the prevention and treatment of neurodegenerative conditions, such as Parkinson's disease and AD in which oxidative stress and iron-mediated toxicity are involved.

  8. Hypoxia-inducible bidirectional shRNA expression vector delivery using PEI/chitosan-TBA copolymers for colorectal Cancer gene therapy.

    Science.gov (United States)

    Javan, Bita; Atyabi, Fatemeh; Shahbazi, Majid

    2018-04-12

    This investigation was conducted to construct a hypoxia/colorectal dual-specific bidirectional short hairpin RNA (shRNA) expression vector and to transfect it into the colon cancer cell line HT-29 with PEI/chitosan-TBA nanoparticles for the simultaneous knock down of β-catenin and Bcl-2 under hypoxia. To construct a pRNA-bipHRE-CEA vector, the carcinoma embryonic antigen (CEA) promoter designed in two directions and the vascular endothelial growth factor (VEGF) enhancer were inserted between two promoters for hypoxic cancer specific gene expression. To confirm the therapeutic effect of the dual-specific vector, β-catenin and Bcl-2 shRNAs were inserted downstream of each promoter. The physicochemical properties, the cytotoxicity, and the transfection efficiency of these PEI/chitosan-TBA nanoparticles were investigated. In addition, the antitumor effects of the designed vector on the expression of β-catenin and Bcl-2, cell cycle distribution, and apoptosis were investigated in vitro. The silencing effect of the hypoxia-response shRNA expression vector was relatively low (18%-25%) under normoxia, whereas it was significantly increased to approximately 50%-60% in the HT-29 cell line. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis due to gene silencing under hypoxia. Furthermore, MTS assay, fluorescence microscopy images, and flow cytometry analyses confirmed that the PEI/chitosan-TBA blend system provided effective transfection with low cytotoxicity. This novel hypoxia-responsive shRNA expression vector may be useful for RNA interference (RNAi)-based cancer gene therapy in hypoxic colorectal tumors. Moreover, the PEI/chitosan-TBA copolymer might be a promising gene carrier for use in gene transfer in vivo. Copyright © 2017. Published by Elsevier Inc.

  9. Progress toward overcoming hypoxia-induced resistance to solid tumor therapy

    International Nuclear Information System (INIS)

    Karakashev, Sergey V; Reginato, Mauricio J

    2015-01-01

    Hypoxic tumors are associated with poor clinical outcome for multiple types of human cancer. This may be due, in part, to hypoxic cancer cells being resistant to anticancer therapy, including radiation therapy, chemotherapy, and targeted therapy. Hypoxia inducible factor 1, a major regulator of cellular response to hypoxia, regulates the expression of genes that are involved in multiple aspects of cancer biology, including cell survival, proliferation, metabolism, invasion, and angiogenesis. Here, we review multiple pathways regulated by hypoxia/hypoxia inducible factor 1 in cancer cells and discuss the latest advancements in overcoming hypoxia-mediated tumor resistance

  10. Effects of 12 metal ions on iron regulatory protein 1 (IRP-1) and hypoxia-inducible factor-1 alpha (HIF-1α) and HIF-regulated genes

    International Nuclear Information System (INIS)

    Li Qin; Chen Haobin; Huang Xi; Costa, Max

    2006-01-01

    Several metal ions that are carcinogenic affect cellular iron homeostasis by competing with iron transporters or iron-regulated enzymes. Some metal ions can mimic a hypoxia response in cells under normal oxygen tension, and induce expression of HIF-1α-regulated genes. This study investigated whether 12 metal ions altered iron homeostasis in human lung carcinoma A549 cells as measured by an activation of IRP-1 and ferritin level. We also studied hypoxia signaling by measuring HIF-1α protein levels, hypoxia response element (HRE)-driven luciferase reporter activity, and Cap43 protein level (an HIF-1α responsive gene). Our results show the following: (i) Ni(II), Co(II), V(V), Mn(II), and to a lesser extent As(III) and Cu(II) activated the binding of IRP-1 to IRE after 24 h, while the other metal ions had no effect; (ii) 10 of 12 metal ions induced HIF-1α protein but to strikingly different degrees. Two of these metal ions, Al(III) and Cd(II), did not induce HIF-1α protein; however, as indicated below, only Ni(II), Co (II), and to lesser extent Mn(II) and V(V) activated HIF-1α-dependent transcription. The combined effects of both [Ni(II) + As(III)] and [Ni(II) + Cr(VI)] on HIF-1α protein were synergistic; (iii) Addition of Fe(II) with Ni(II), Co(II), and Cr(VI) attenuated the induction of HIF-1α after 4 h treatment; (iv) Ni(II), Co(II), and Mn(II) significantly decrease ferritin level after 24 h exposure; (v) Ni(II), Co(II), V(V), and Mn(II) activated HRE reporter gene after 20 h treatment; (vi) Ni(II), Co(II), V(V), and Mn(II) increased the HIF-1-dependent Cap43 protein level after 24 h treatment. In conclusion, only Ni (II), Co (II), and to a lesser extent Mn(II) and V(V) significantly stabilized HIF-1α protein, activated IRP, decreased the levels of ferritin, induced the transcription of HIF-dependent reporter, and increased the expression of Cap43 protein levels (HIF-dependent gene). The mechanism for the significant stabilization and elevation of HIF-1

  11. Unraveling the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the adaption process of human microvascular endothelial cells (HMEC-1) to hypoxia: Redundant HIF-dependent regulation of macrophage migration inhibitory factor.

    Science.gov (United States)

    Hahne, Martin; Schumann, Peggy; Mursell, Mathias; Strehl, Cindy; Hoff, Paula; Buttgereit, Frank; Gaber, Timo

    2018-03-01

    Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O 2 ) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Hypoxia-inducible factor-1α plays roles in Epstein-Barr virus's natural life cycle and tumorigenesis by inducing lytic infection through direct binding to the immediate-early BZLF1 gene promoter.

    Directory of Open Access Journals (Sweden)

    Richard J Kraus

    2017-06-01

    Full Text Available When confronted with poor oxygenation, cells adapt by activating survival signaling pathways, including the oxygen-sensitive transcriptional regulators called hypoxia-inducible factor alphas (HIF-αs. We report here that HIF-1α also regulates the life cycle of Epstein-Barr virus (EBV. Incubation of EBV-positive gastric carcinoma AGS-Akata and SNU-719 and Burkitt lymphoma Sal and KemIII cell lines with a prolyl hydroxylase inhibitor, L-mimosine or deferoxamine, or the NEDDylation inhibitor MLN4924 promoted rapid and sustained accumulation of both HIF-1α and lytic EBV antigens. ShRNA knockdown of HIF-1α significantly reduced deferoxamine-mediated lytic reactivation. HIF-1α directly bound the promoter of the EBV primary latent-lytic switch BZLF1 gene, Zp, activating transcription via a consensus hypoxia-response element (HRE located at nt -83 through -76 relative to the transcription initiation site. HIF-1α did not activate transcription from the other EBV immediate-early gene, BRLF1. Importantly, expression of HIF-1α induced EBV lytic-gene expression in cells harboring wild-type EBV, but not in cells infected with variants containing base-pair substitution mutations within this HRE. Human oral keratinocyte (NOK and gingival epithelial (hGET cells induced to differentiate by incubation with either methyl cellulose or growth in organotypic culture accumulated both HIF-1α and Blimp-1α, another cellular factor implicated in lytic reactivation. HIF-1α activity also accumulated along with Blimp-1α during B-cell differentiation into plasma cells. Furthermore, most BZLF1-expressing cells observed in lymphomas induced by EBV in NSG mice with a humanized immune system were located distal to blood vessels in hypoxic regions of the tumors. Thus, we conclude that HIF-1α plays central roles in both EBV's natural life cycle and EBV-associated tumorigenesis. We propose that drugs that induce HIF-1α protein accumulation are good candidates for

  13. Hypoxia-inducible factor signalling mechanisms in the central nervous system.

    Science.gov (United States)

    Corcoran, A; O'Connor, J J

    2013-08-01

    In the CNS, neurones are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased, neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma and neurodegenerative diseases. Cellular responses to oxygen deprivation are complex and result in activation of short- and long-term mechanisms to conserve energy and protect cells. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute effects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, pre-synaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurones to hypoxia is the activation of transcription factors such as hypoxia-inducible factor. The activation of hypoxia-inducible factor is governed by a family of dioxygenases called hypoxia-inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia-inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia-inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia-inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

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

    Directory of Open Access Journals (Sweden)

    Hyo-Jeong Lee

    2012-01-01

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

  15. Association of Hypoxia-Inducible Factor-2 Alpha Gene Polymorphisms with the Risk of Hepatitis B Virus-Related Liver Disease in Guangxi Chinese: A Case-Control Study.

    Directory of Open Access Journals (Sweden)

    Liling Huang

    Full Text Available Hypoxia-inducible factor-2 alpha (HIF-2a plays a major role in the progression of disease, although the role of HIF-2α gene polymorphisms in hepatitis B virus (HBV-related diseases remains elusive. The aim of this study is to determine whether HIF-2a rs13419896 and rs6715787 single-nucleotide polymorphisms (SNPs are associated with susceptibility to chronic hepatitis B (CHB, liver cirrhosis (LC, or hepatocellular carcinoma (HCC.A case-control study of 107 patients with CHB, 83 patients with LC, 234 patients with HCC, and 224 healthy control subjects was carried out, and the HIF-2a rs13419896 and rs6715787 SNPs were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP.No significant differences were observed in the genotype or allele frequency of two HIF-2a SNPs between the cases and controls (all p>0.05. However, in subgroup analysis by gender, the HIF-2a rs13419896 GA and AA genotypes were significantly associated with a risk of CHB (odds ratio [OR] = 3.565, 95% confidence interval [CI] = 1.123-11.314, p = 0.031 and OR = 12.506, 95% CI = 1.329-117.716, p = 0.027 in females, and the A allele of rs13419896 was associated with a risk of CHB (OR = 2.624, 95% CI = 1.244-5.537, p = 0.011 and LC (OR = 2.351, 95% CI = 1.002-5.518, p = 0.050 in females. The rs6715787 CG genotype polymorphism may contribute to a reduced risk of LC in the Guangxi Zhuang Chinese population (OR = 0.152, 95% CI = 0.028-0.807, p = 0.027, as determined via subgroup analysis by ethnicity. Moreover, binary logistic regression analyses that were adjusted by drinking status indicated that the AA genotype of rs13419896 may contribute to an increased risk of LC in the non-alcohol-drinking population (OR = 3.124, 95% CI = 1.091-8.947, p = 0.034. In haplotype analysis, GG haplotype was significantly associated with a reduced risk of LC (OR = 0.601, 95% CI = 0.419-0.862, p = 0.005.The HIF-2a rs13419896 polymorphism is associated with an increased

  16. Does Acute Normobaric Hypoxia Induce Anapyrexia in Adult Humans?

    Science.gov (United States)

    Seo, Yongsuk; Gerhart, Hayden D; Vaughan, Jeremiah; Kim, Jung-Hyun; Glickman, Ellen L

    2017-06-01

    Seo, Yongsuk, Hayden D. Gerhart, Jeremiah Vaughan, Jung-Hyun Kim, and Ellen L. Glickman. Does acute normobaric hypoxia induce anapyrexia in adult humans? High Alt Med Biol. 18:185-190, 2017.-Exposure to hypoxia is known to induce a reduction in core body temperature as a protective mechanism, which has been shown in both animals and humans. The purpose of this study was to test if acute exposure to normobaric hypoxia (NH) induces anapyrexia in adult humans in association with decreased peripheral oxygen saturation (SpO 2 ). Ten healthy male subjects were seated in atmospheres of normobaric normoxia 21% (NN21), NH 17% (NH17), and 13% (NH13) O 2 for 60 minutes in a counterbalanced manner. Rectal temperature (Tre) was continuously monitored together with the quantification of metabolic heat production (MHP) and body heat storage (S). Baseline physiological measurements showed no differences between the three conditions. SpO 2 was significantly decreased in NH17 and NH13 compared with NN21 (p ≤ 0.001). Tre decreased following 60 minutes of resting in all conditions, but, independent of the conditions, showed no association between Tre and levels of hypoxic SpO 2 . There was also no significant difference in either MHP or S between conditions. The present results showed no evidence of hypoxia-induced anapyrexia in adult humans during 1 hour of resting after exposure to NH either at 13% or 17% O 2 .

  17. Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors.

    Science.gov (United States)

    Aprelikova, Olga; Chandramouli, Gadisetti V R; Wood, Matthew; Vasselli, James R; Riss, Joseph; Maranchie, Jodi K; Linehan, W Marston; Barrett, J Carl

    2004-06-01

    Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Copyright 2004 Wiley-Liss, Inc.

  18. Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension.

    Science.gov (United States)

    Maston, Levi D; Jones, David T; Giermakowska, Wieslawa; Howard, Tamara A; Cannon, Judy L; Wang, Wei; Wei, Yongyi; Xuan, Weimin; Resta, Thomas C; Gonzalez Bosc, Laura V

    2017-05-01

    Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4 + T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4 + T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1 -/- , lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4 + , CD8 + , or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1 -/- mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4 + but not CD8 + T cells restored the hypertensive phenotype in RAG1 -/- mice. Interestingly, RAG1 -/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4 + cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension. Copyright © 2017 the American Physiological Society.

  19. Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

    DEFF Research Database (Denmark)

    Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

    Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer  Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc.1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... activates transcription of numerous genes associated with angiogenesis, ATP-metabolism, cell-proliferation, glycolysis and apoptosis. HIF-1α is over expressed in many malignant tumors and is reported to play an important role in tumor invasion and progression. The aim of this Ph.D. project is to investigate...... the predictive and prognostic value of HIF-1α in colorectal cancer.Materials and MethodsThe project is divided into 3 substudies:1. Biological and methodological aspects. The expression of HIF-1α measured by immunohistochemistry in paraffin embedded tissue is related to single nucleotide polymorphism (SNP...

  20. Hypoxia Inducible Factors and Hypertension: Lessons from Sleep Apnea Syndrome

    Science.gov (United States)

    Nanduri, Jayasri; Peng, Ying-Jie; Yuan, Guoxiang; Kumar, Ganesh K.; Prabhakar, Nanduri R.

    2015-01-01

    Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity is a major contributor to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2- mediated transcription. Dysregulation of HIF-1- and HIF-2- mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive species (ROS) generation in the chemosensory reflex which is central for developing hypertension. PMID:25772710

  1. Reactive oxygen species (ROS) and the heat stress response of Daphnia pulex: ROS-mediated activation of hypoxia-inducible factor 1 (HIF-1) and heat shock factor 1 (HSF-1) and the clustered expression of stress genes.

    Science.gov (United States)

    Klumpen, Eva; Hoffschröer, Nadine; Zeis, Bettina; Gigengack, Ulrike; Dohmen, Elias; Paul, Rüdiger J

    2017-01-01

    Heat stress in ectotherms involves direct (e.g. protein damage) and/or indirect effects (temperature-induced hypoxia and ROS formation), which cause activation of the transcription factors (TF) heat shock factor 1 (HSF-1) and/or hypoxia-inducible factor 1 (HIF-1). The present study focused on the links between stress (ROS) signals, nuclear (n) and cytoplasmic (c) HSF-1/HIF-1 levels, and stress gene expression on mRNA and protein levels (e.g. heat-shock protein 90, HSP90) upon acute heat and ROS (H 2 O 2 ) stress. Acute heat stress (30°C) evoked fluctuations in ROS level. Different feeding regimens, which affected the glutathione (GSH) level, allowed altering the frequency of ROS fluctuations. Other data showed fluctuation frequency to depend also on ROS production rate. The heat-induced slow or fast ROS fluctuations (at high or low GSH levels) evoked slow or fast fluctuations in the levels of nHIF-1α, nHSF-1 and gene products (mRNAs and protein), albeit after different time delays. Time delays to ROS fluctuations were, for example,shorter for nHIF-1α than for nHSF-1 fluctuations, and nHIF-1α fluctuations preceded and nHSF-1 fluctuations followed fluctuations in HSP90 mRNA level. Cytoplasmic TF levels either changed little (cHIF-1α) or showed a steady increase (cHSF-1). Applying acute H 2 O 2 stress (at 20°C) revealed effects on nHIF-1α and mRNA levels, but no significant effects on nHSF-1 level. Transcriptome data additionally showed coordinated fluctuations of mRNA levels upon acute heat stress, involving mRNAs for HSPs and other stress proteins, with all corresponding genes carrying DNA binding motifs for HIF-1 and HSF-1. This study provided evidence for promoting effects of ROS and HIF-1 on early haemoglobin, HIF-1α and HSP90 mRNA expressions upon heat or ROS stress. The increasing cHSF-1 level likely affected nHSF-1 level and later HSP90 mRNA expression. Heat stress evoked ROS fluctuations, with this stress signal forwarded via nHIF-1 and nHSF-1

  2. Hypoxia-induced dysfunction of rat diaphragm: role of peroxynitrite.

    NARCIS (Netherlands)

    Zhu, X.; Heunks, L.M.A.; Versteeg, E.M.M.; Heijden, E. van der; Ennen, L.; Kuppevelt, A.H.M.S.M. van; Vina, J.; Dekhuijzen, P.N.R.

    2005-01-01

    Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm

  3. HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

    Directory of Open Access Journals (Sweden)

    Chen-Ye Shi

    2013-08-01

    Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

  4. Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1α.

    Science.gov (United States)

    Yang, Yeon Ju; Na, Hwi Jung; Suh, Michelle J; Ban, Myung Jin; Byeon, Hyung Kwon; Kim, Won Shik; Kim, Jae Wook; Choi, Eun Chang; Kwon, Hyeong Ju; Chang, Jae Won; Koh, Yoon Woo

    2015-11-01

    Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1α (HIF-1α) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1α-induced invasive characteristics. Cells were cultured under controlled hypoxic environments (1% O₂) or normoxic conditions. The effect of hypoxia on HIF-1α, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1α and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1α, tissue analysis was done. Hypoxia induces HIF-1α expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1α via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1α with RNA interference suppressed hypoxia-induced HIF-1α and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1α. These were confirmed in the orthotopic FTC model. Hypoxia induced HIF-1α, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

  5. Hypoxia-inducible factor-1 alpha has a key role in hypoxic preconditioning.

    Science.gov (United States)

    Taie, Satoshi; Ono, Junichiro; Iwanaga, Yasuyuki; Tomita, Shuhei; Asaga, Takehiko; Chujo, Kosuke; Ueki, Masaaki

    2009-08-01

    Sublethal hypoxia induces tolerance to subsequent hypoxic insults in a process known as hypoxic preconditioning (HP). Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key transcription protein involved in the mechanism of HP. In this study, we investigated the effects of HP on tissue oxygenation and expression of HIF-1 alpha gene targets in the brain using neural cell-specific HIF-1 alpha-deficient mice. The animals were exposed to 8% oxygen for 3 hours. Twenty-four hours later, the oxygen partial pressure (pO(2)) of brain tissue and gene expression were measured during hypoxia. HP improved the pO(2) of brain tissue during subsequent hypoxia with upregulated inducible nitric oxide synthase in wild-type mice, whereas HP had no detectable effect in the mutant mice. Our results indicate that the protective effects of HP may be partially mediated by improving tissue oxygenation via HIF-1 alpha and inducible nitric oxide synthase.

  6. HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

    International Nuclear Information System (INIS)

    Liu, Junye; Zhang, Jing; Wang, Xiaowu; Li, Yan; Chen, Yongbin; Li, Kangchu; Zhang, Jian; Yao, Libo; Guo, Guozhen

    2010-01-01

    Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

  7. Increased hypoxia-inducible factor-1α in striated muscle of tumor-bearing mice.

    Science.gov (United States)

    Devine, Raymond D; Bicer, Sabahattin; Reiser, Peter J; Wold, Loren E

    2017-06-01

    Cancer cachexia is a progressive wasting disease resulting in significant effects on the quality of life and high mortality. Most studies on cancer cachexia have focused on skeletal muscle; however, the heart is now recognized as a major site of cachexia-related effects. To elucidate possible mechanisms, a proteomic study was performed on the left ventricles of colon-26 (C26) adenocarcinoma tumor-bearing mice. The results revealed several changes in proteins involved in metabolism. An integrated pathway analysis of the results revealed a common mediator in hypoxia-inducible factor-1α (HIF-1α). Work by other laboratories has shown that extensive metabolic restructuring in the C26 mouse model causes changes in gene expression that may be affected directly by HIF-1α, such as glucose metabolic genes. M-mode echocardiography showed progressive decline in heart function by day 19 , exhibited by significantly decreased ejection fraction and fractional shortening, along with posterior wall thickness. Using Western blot analysis, we confirmed that HIF-1α is significantly upregulated in the heart, whereas there were no changes in its regulatory proteins, prolyl hydroxylase domain-containing protein 2 (PHD2) and von Hippel-Lindau protein (VHL). PHD2 requires both oxygen and iron as cofactors for the hydroxylation of HIF-1α, marking it for ubiquination via VHL and subsequent destruction by the proteasome complex. We examined venous blood gas values in the tumor-bearing mice and found significantly lower oxygen concentration compared with control animals in the third week after tumor inoculation. We also examined select skeletal muscles to determine whether they are similarly affected. In the diaphragm, extensor digitorum longus, and soleus, we found significantly increased HIF-1α in tumor-bearing mice, indicating a hypoxic response, not only in the heart, but also in skeletal muscle. These results indicate that HIF-1α may contribute, in part, to the metabolic changes

  8. Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains▿ †

    OpenAIRE

    Bellot, Grégory; Garcia-Medina, Raquel; Gounon, Pierre; Chiche, Johanna; Roux, Danièle; Pouysségur, Jacques; Mazure, Nathalie M.

    2009-01-01

    While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy,...

  9. Structural and functional analysis of coral Hypoxia Inducible Factor.

    Science.gov (United States)

    Zoccola, Didier; Morain, Jonas; Pagès, Gilles; Caminiti-Segonds, Natacha; Giuliano, Sandy; Tambutté, Sylvie; Allemand, Denis

    2017-01-01

    Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O2 concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1) is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O2 concentrations of the coral model Stylophora pistillata. In silico analysis showed that homologs of HIF-1 α (SpiHIF-1α) and HIF-1β (SpiHIF-1β) exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE) sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD) relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O2 concentration between Cnidarians and Mammals whose ancestors diverged about 1,200-1,500 million years ago.

  10. Structural and functional analysis of coral Hypoxia Inducible Factor.

    Directory of Open Access Journals (Sweden)

    Didier Zoccola

    Full Text Available Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O2 concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1 is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O2 concentrations of the coral model Stylophora pistillata. In silico analysis showed that homologs of HIF-1 α (SpiHIF-1α and HIF-1β (SpiHIF-1β exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O2 concentration between Cnidarians and Mammals whose ancestors diverged about 1,200-1,500 million years ago.

  11. Saururus cernuus lignans-Potent small molecule inhibitors of hypoxia-inducible factor-1

    International Nuclear Information System (INIS)

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou Yudong

    2005-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B 1 , manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC 50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors

  12. Hypoxia-inducible transcription factor-1α promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

    International Nuclear Information System (INIS)

    Luo, FengMing; Liu, XiaoJing; Yan, NaiHong; Li, ShuangQing; Cao, GuiQun; Cheng, QingYing; Xia, QingJie; Wang, HongJing

    2006-01-01

    Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a 'master' gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression. A549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Knocking down expression of HIF-1α inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. During hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis

  13. Cinnamic aldehyde suppresses hypoxia-induced angiogenesis via inhibition of hypoxia-inducible factor-1α expression during tumor progression.

    Science.gov (United States)

    Bae, Woom-Yee; Choi, Jae-Sun; Kim, Ja-Eun; Jeong, Joo-Won

    2015-11-01

    During tumor progression, hypoxia-inducible factor 1 (HIF-1) plays a critical role in tumor angiogenesis and tumor growth by regulating the transcription of several genes in response to a hypoxic environment and changes in growth factors. This study was designed to investigate the effects of cinnamic aldehyde (CA) on tumor growth and angiogenesis and the mechanisms underlying CA's anti-angiogenic activities. We found that CA administration inhibits tumor growth and blocks tumor angiogenesis in BALB/c mice. In addition, CA treatment decreased HIF-1α protein expression and vascular endothelial growth factor (VEGF) expression in mouse tumors and Renca cells exposed to hypoxia in vitro. Interestingly, CA treatment did not affect the stability of von Hippel-Lindau protein (pVHL)-associated HIF-1α and CA attenuated the activation of mammalian target of rapamycin (mTOR) pathway. Collectively, these findings strongly indicate that the anti-angiogenic activity of CA is, at least in part, regulated by the mTOR pathway-mediated suppression of HIF-1α protein expression and these findings suggest that CA may be a potential drug for human cancer therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Hypoxia-induced retinopathy model in adult zebrafish

    DEFF Research Database (Denmark)

    Cao, Ziquan; Jensen, Lasse D.; Rouhi, Pegah

    2010-01-01

    Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available....... In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia......-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs....

  15. Hypoxia-induced angiogenesis is increased by the controlled release of deferoxiamine from gelatin hydrogels.

    Science.gov (United States)

    Saito, Takashi; Tabata, Yasuhiko

    2014-08-01

    The objective of this study is to design biodegradable hydrogels for the controlled release of deferoxiamine (DFO) and evaluate their biological activity. When the DFO was added to human umbilical vein endothelial cells cultured in 5.0% O2, the level of hypoxia-inducible factor-1α and vascular endothelial growth factor significantly increased compared with that without DFO. The expression of angiogenesis-related genes was accordingly increased by the DFO addition. An aqueous solution of mixed gelatin and DFO was freeze-dried, and dehydrothermally treated at 140°C for 24h to prepare a gelatin hydrogel incorporating DFO. In the release test with phosphate-buffered saline solution (PBS) at 37°C, an initial DFO release of 60% was observed, followed by no release. When placed in PBS containing collagenase, the hydrogel was enzymatically degraded with time, and consequently released DFO in a degradation-dependent manner. After the hydrogel incorporating DFO was injected intramuscularly into a mouse model of hind limb ischemia, the number of new blood vessels formed was significantly higher than that with free DFO and DFO-free hydrogel. It is concluded that the DFO-containing hydrogel shows promising for inducing angiogenesis locally. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  16. Hypoxia-Induced Signaling Promotes Prostate Cancer Progression: Exosomes Role as Messenger of Hypoxic Response in Tumor Microenvironment

    Science.gov (United States)

    Deep, Gagan; Panigrahi, Gati K.

    2017-01-01

    Prostate cancer (PCA) is the leading malignancy in men and the second leading cause of cancer-related deaths. Hypoxia (low O2 condition) is considered an early event in prostate carcinogenesis associated with an aggressive phenotype. In fact, clinically, hypoxia and hypoxia-related biomarkers are associated with treatment failure and disease progression. Hypoxia-inducible factor 1 (HIF-1) is the key factor that is activated under hypoxia, and mediates adaptation of cells to hypoxic conditions through regulating the expression of genes associated with angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, survival, proliferation, metabolism, stemness, hormone-refractory progression, and therapeutic resistance. Besides HIF-1, several other signaling pathways including PI3K/Akt/mTOR, NADPH oxidase (NOX), Wnt/β-catenin, and Hedgehog are activated in cancer cells under hypoxic conditions, and also contribute in hypoxia-induced biological effects in HIF-1-dependent and -independent manners. Hypoxic cancer cells cause extensive changes in the tumor microenvironment both local and distant, and recent studies have provided ample evidence supporting the crucial role of nanosized vesicles “exosomes” in mediating hypoxia-induced tumor microenvironment remodeling. Exosomes’ role has been reported in hypoxia-induced angiogenesis, stemness, activation of cancer-associated fibroblasts (CAFs), and EMT. Together, existing literature suggests that hypoxia plays a predominant role in PCA growth and progression, and PCA could be effectively prevented and treated via targeting hypoxia/hypoxia-related signaling pathways. PMID:27279239

  17. MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

    Science.gov (United States)

    Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

    2015-02-01

    The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

  18. Role of hypoxia-inducible factor in diabetic myocardial hypertrophy ...

    African Journals Online (AJOL)

    Purpose: This study was carried out to investigate the role of hypoxia-inducible factor (HIF) in diabetic cardiomyopathy in vitro. Methods: Hypoxia was induced chemically in H9C2 cells (cardiac hypertrophy model), and the cells were treated with phenylephrine (PE), deferoxamine (DFO), PE + DFO, and HIF-1α siRNA under ...

  19. Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

    NARCIS (Netherlands)

    Leszczynska, K.B.; Foskolou, I.P.; Abraham, A.G.; Anbalagan, S.; Tellier, C.; Haider, S.; Span, P.N.; O'Neill, E.E.; Buffa, F.M.; Hammond, E.M.

    2015-01-01

    Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent

  20. Role of Hypoxia-inducible factor-1 and its target genes in human lung adenocarcinoma cells after photon- versus carbon ion irradiation; Expression HIF-1-abhaengiger Gene in humanen Lungenadenokarzinom (A549)-Zellen und deren Regulation nach Photonen- und Schwerionenbestrahlung

    Energy Technology Data Exchange (ETDEWEB)

    Bill, Verena Maria

    2013-11-26

    Exposed to hypoxia tumor cells are notably resistant to photon irradiation. The hypoxiainducible transcription factor 1α (HIF-1α) seems to play a fundamental role in this resistance, while its role after heavy-ion beam remains unknown. The intention of this study was to determine how A549-cells (non-small-cell lung carcinoma) react in different oxygenation states after irradiation with photons or heavy ions, particularly in regards to their expression of HIF-1 target genes. Resistance of hypoxic A549 cells after photon irradiation was documented by cellular and clonogenic survival. In contrast, cellular survival after heavy-ion irradiation in hypoxic cells was not elevated to normoxic cells. Among the oxygen dependent regulation of HIF-1 target genes, gene expression analyses showed an increased expression of GLUT-1, LDH-A, PDK-1 and VEGF after photon irradiation but not after heavy-ion irradiation after 48 hours in normoxic cells. As expected, CDKN1A as inhibitor of cell cycle progression showed higher expression after both radiation forms; interestingly CDKN1A was also in an oxygen dependent manner lightly upregulated. In western blot analyses we demonstrated a significant increase of HIF-1 and GLUT-1 caused by hypoxia, but only a tendency of increased protein level in hypoxia after photon irradiation and no changes after heavy-ion irradiation. Significantly higher protein level of secreted VEGF-A could be measured 72 hours after photon irradiation in normoxic cells by ELISA analyses. Controversially discussed, I could not detect an association between HIF-1 and SCF or Trx-1 in A549-cells in this study. Whereas Trx-1-expression was neither influenced by changed oxygen partial pressure nor irradiation, I could show increased SCF mRNA by quantitative Real Time-PCR and secreted protein level by ELISA after photon irradiation independent of oxygen state. In summary, this study showed that HIF-1 and its target genes (GLUT-1, LDHA; PDK, VEGF) and also SCF was

  1. Glutamatergic neurotransmission modulates hypoxia-induced hyperventilation but not anapyrexia

    Directory of Open Access Journals (Sweden)

    Paula P.M. de

    2004-01-01

    Full Text Available The interaction between pulmonary ventilation (V E and body temperature (Tb is essential for O2 delivery to match metabolic rate under varying states of metabolic demand. Hypoxia causes hyperventilation and anapyrexia (a regulated drop in Tb, but the neurotransmitters responsible for this interaction are not well known. Since L-glutamate is released centrally in response to peripheral chemoreceptor stimulation and glutamatergic receptors are spread in the central nervous system we tested the hypothesis that central L-glutamate mediates the ventilatory and thermal responses to hypoxia. We measured V E and Tb in 40 adult male Wistar rats (270 to 300 g before and after intracerebroventricular injection of kynurenic acid (KYN, an ionotropic glutamatergic receptor antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamatergic receptor antagonist or vehicle (saline, followed by a 1-h period of hypoxia (7% inspired O2 or normoxia (humidified room air. Under normoxia, KYN (N = 5 or MCPG (N = 8 treatment did not affect V E or Tb compared to saline (N = 6. KYN and MCPG injection caused a decrease in hypoxia-induced hyperventilation (595 ± 49 for KYN, N = 7 and 525 ± 84 ml kg-1 min-1 for MCPG, N = 6; P < 0.05 but did not affect anapyrexia (35.3 ± 0.2 for KYN and 34.7 ± 0.4ºC for MCPG compared to saline (912 ± 110 ml kg-1 min-1 and 34.8 ± 0.2ºC, N = 8. We conclude that glutamatergic receptors are involved in hypoxic hyperventilation but do not affect anapyrexia, indicating that L-glutamate is not a common mediator of this interaction.

  2. Genome-wide identification of hypoxia-inducible factor-1 and -2 binding sites in hypoxic human macrophages alternatively activated by IL-10.

    Science.gov (United States)

    Tausendschön, Michaela; Rehli, Michael; Dehne, Nathalie; Schmidl, Christian; Döring, Claudia; Hansmann, Martin-Leo; Brüne, Bernhard

    2015-01-01

    Macrophages (MΦ) often accumulate in hypoxic areas, where they significantly influence disease progression. Anti-inflammatory cytokines, such as IL-10, generate alternatively activated macrophages that support tumor growth. To understand how alternative activation affects the transcriptional profile of hypoxic macrophages, we globally mapped binding sites of hypoxia-inducible factor (HIF)-1α and HIF-2α in primary human monocyte-derived macrophages prestimulated with IL-10. 713 HIF-1 and 795 HIF-2 binding sites were identified under hypoxia. Pretreatment with IL-10 altered the binding pattern, with 120 new HIF-1 and 188 new HIF-2 binding sites emerging. HIF-1 binding was most prominent in promoters, while HIF-2 binding was more abundant in enhancer regions. Comparison of ChIP-seq data obtained in other cells revealed a highly cell type specific binding of HIF. In MΦ HIF binding occurred preferentially in already active enhancers or promoters. To assess the roles of HIF on gene expression, primary human macrophages were treated with siRNA against HIF-1α or HIF-2α, followed by genome-wide gene expression analysis. Comparing mRNA expression to the HIF binding profile revealed a significant enrichment of hypoxia-inducible genes previously identified by ChIP-seq. Analysis of gene expression under hypoxia alone and hypoxia/IL-10 showed the enhanced induction of a set of genes including PLOD2 and SLC2A3, while another group including KDM3A and ADM remained unaffected or was reduced by IL-10. Taken together IL-10 influences the DNA binding pattern of HIF and the level of gene induction. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

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    Peng Zhang

    2014-03-01

    Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

  4. Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

    DEFF Research Database (Denmark)

    Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

    2010-01-01

    Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer  Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc.1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... Hospital, Vejle, Denmark4Institute of Regional Health Services Research, University of Southern Denmark, Odense DenmarkBackgroundPrognostic and predictive markers are needed for individualizing the treatment of colorectal cancer. Hypoxia-inducible factor 1α (HIF-1α) is a transcription-inducing factor which...... the predictive and prognostic value of HIF-1α in colorectal cancer.Materials and MethodsThe project is divided into 3 substudies:1. Biological and methodological aspects. The expression of HIF-1α measured by immunohistochemistry in paraffin embedded tissue is related to single nucleotide polymorphism (SNP...

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

    Science.gov (United States)

    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.

  6. Notch-1 mediates hypoxia-induced angiogenesis in rheumatoid arthritis.

    Science.gov (United States)

    Gao, Wei; Sweeney, Catherine; Connolly, Mary; Kennedy, Aisling; Ng, Chin Teck; McCormick, Jennifer; Veale, Douglas J; Fearon, Ursula

    2012-07-01

    To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis. The expression and regulation of Notch-1, its ligand delta-like protein 4 (DLL-4) and downstream signaling components (hairy-related transcription factor 1 [HRT-1], HRT-2), and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions (1-3%) were assessed in synovial tissue specimens from patients with inflammatory arthritis and controls and in human dermal microvascular endothelial cells (HDMECs) by immunohistology, dual immunofluorescence staining (Notch-1/factor VIII), Western blotting, and real-time polymerase chain reaction. In vivo synovial tissue oxygen levels (tissue PO2) were measured under direct visualization at arthroscopy. HDMEC activation under hypoxic conditions in the presence of Notch-1 small interfering RNA (siRNA), the γ-secretase inhibitor DAPT, or dimethyloxalylglycine (DMOG) was assessed by Matrigel tube formation assay, migration assay, invasion assay, and matrix metalloproteinase 2 (MMP-2)/MMP-9 zymography. Expression of Notch-1, its ligand DLL-4, and HRT-1 was demonstrated in synovial tissue, with the strongest expression localized to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual immunofluorescence staining. Notch-1 intracellular domain (NICD) expression was significantly higher in synovial tissue from patients with tissue PO2 of PO2 of >20 mm Hg (>3% O2). Exposure of HDMECs to 3% hypoxia induced HIF-1α and NICD protein expression and DLL-4, HRT-1, and HRT-2 messenger RNA expression. DMOG directly induced NICD expression, while Notch-1 siRNA inhibited hypoxia-induced HIF-1α expression, suggesting that Notch-1/HIF-1α signaling is bidirectional. Finally, 3% hypoxia-induced angiogenesis, endothelial cell migration, endothelial cell invasion, and proMMP-2 and proMMP-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT. Our

  7. Intermittent hypoxia induces hyperlipidemia in lean mice.

    Science.gov (United States)

    Li, Jianguo; Thorne, Laura N; Punjabi, Naresh M; Sun, Cheuk-Kwan; Schwartz, Alan R; Smith, Philip L; Marino, Rafael L; Rodriguez, Annabelle; Hubbard, Walter C; O'Donnell, Christopher P; Polotsky, Vsevolod Y

    2005-09-30

    Obstructive sleep apnea, a syndrome leading to recurrent intermittent hypoxia (IH), has been associated previously with hypercholesterolemia, independent of underlying obesity. We examined the effects of experimentally induced IH on serum lipid levels and pathways of lipid metabolism in the absence and presence of obesity. Lean C57BL/6J mice and leptin-deficient obese C57BL/6J-Lep(ob) mice were exposed to IH for five days to determine changes in serum lipid profile, liver lipid content, and expression of key hepatic genes of lipid metabolism. In lean mice, exposure to IH increased fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, phospholipids (PLs), and triglycerides (TGs), as well as liver TG content. These changes were not observed in obese mice, which had hyperlipidemia and fatty liver at baseline. In lean mice, IH increased sterol regulatory element binding protein 1 (SREBP-1) levels in the liver, increased mRNA and protein levels of stearoyl-coenzyme A desaturase 1 (SCD-1), an important gene of TG and PL biosynthesis controlled by SREBP-1, and increased monounsaturated fatty acid content in serum, which indicated augmented SCD-1 activity. In addition, in lean mice, IH decreased protein levels of scavenger receptor B1, regulating uptake of cholesterol esters and HDL by the liver. We conclude that exposure to IH for five days increases serum cholesterol and PL levels, upregulates pathways of TG and PL biosynthesis, and inhibits pathways of cholesterol uptake in the liver in the lean state but does not exacerbate the pre-existing hyperlipidemia and metabolic disturbances in leptin-deficient obesity.

  8. Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion

    NARCIS (Netherlands)

    Mattijsen, F.; Georgiadi, A.; Andasarie, T.; Szalowska, E.; Zota, A.; Krones-Herzig, A.; Kersten, A.H.

    2014-01-01

    Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a

  9. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

  10. Hypoxia-Inducible Factor 1 Is an Inductor of Transcription Factor Activating Protein 2 Epsilon Expression during Chondrogenic Differentiation

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    Stephan Niebler

    2015-01-01

    Full Text Available The transcription factor AP-2ε (activating enhancer-binding protein epsilon is expressed in cartilage of humans and mice. However, knowledge about regulatory mechanisms influencing AP-2ε expression is limited. Using quantitative real time PCR, we detected a significant increase in AP-2ε mRNA expression comparing initial and late stages of chondrogenic differentiation processes in vitro and in vivo. Interestingly, in these samples the expression pattern of the prominent hypoxia marker gene angiopoietin-like 4 (Angptl4 strongly correlated with that of AP-2ε suggesting that hypoxia might represent an external regulator of AP-2ε expression in mammals. In order to show this, experiments directly targeting the activity of hypoxia-inducible factor-1 (HIF1, the complex mediating responses to oxygen deprivation, were performed. While the HIF1-activating compounds 2,2′-dipyridyl and desferrioxamine resulted in significantly enhanced mRNA concentration of AP-2ε, siRNA against HIF1α led to a significantly reduced expression rate of AP-2ε. Additionally, we detected a significant upregulation of the AP-2ε mRNA level after oxygen deprivation. In sum, these different experimental approaches revealed a novel role for the HIF1 complex in the regulation of the AP-2ε gene in cartilaginous cells and underlined the important role of hypoxia as an important external regulatory stimulus during chondrogenic differentiation modulating the expression of downstream transcription factors.

  11. Role of hypoxia and hypoxia inducible factor in physiological and pathological conditions

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    Mozhgan Jahani

    2017-11-01

    Full Text Available Introduction: Organisms are exposed to oxygen deprivation (Hypoxia in various physiological and pathological conditions. There are different conserve evolutionary responses to counterview with this stress that primary transcriptional response to stress related to hypoxia is interceded by hypoxia-inducible factor (HIF-1 in mammals. This factor can regulate different genes that have essential roles in adaptation to this condition. In this review, the role of this factor in physiological and pathological conditions under hypoxic condition has been evaluated after examining structural features and regulation characteristics of HIF-1. Methods: First, articles related to the keywords of hypoxia and HIF-1 (from 1991-2016 were searched from valid databases such as Springer Link, Google Scholar, PubMed and Science direct. Then, the articles correlated with hypoxia, HIF-1 and their roles in physiological and pathological conditions (120 articles were searched and just 64 articles were selected for this study. Result: According to studies, there are different genes in cells and organs that can be regulated by HIF-1. Activation of genes expression by this protein occurs through its linkage to cis-acting of 50 base pair hypoxia response element (HRE region located in their promotor and enhancer. Depending on circumstances, activation of these genes can be beneficial or harmful. Conclusion: Activation of different genes in hypoxia by HIF-1 has different effects on physiological and pathological conditions. Therefore, HIF-1, as a hypoxia-inducible factor in hypoxic conditions, plays an essential role in the adaptation of cells and organs to changes related to the presence of oxygen.

  12. Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

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    Adriana M Zimnicka

    Full Text Available Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX, a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2 also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC. In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

  13. Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

    International Nuclear Information System (INIS)

    Ragnum, Harald Bull; Røe, Kathrine; Holm, Ruth; Vlatkovic, Ljiljana; Nesland, Jahn Marthin; Aarnes, Eva-Katrine; Ree, Anne Hansen; Flatmark, Kjersti; Seierstad, Therese; Lilleby, Wolfgang; Lyng, Heidi

    2013-01-01

    Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to

  14. Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Ragnum, Harald Bull [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Røe, Kathrine [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Division of Medicine, Department of Oncology, Akershus University Hospital, Lørenskog (Norway); Holm, Ruth; Vlatkovic, Ljiljana [Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Nesland, Jahn Marthin [Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Medical Faculty, University of Oslo, Oslo (Norway); Aarnes, Eva-Katrine [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Ree, Anne Hansen [Division of Medicine, Department of Oncology, Akershus University Hospital, Lørenskog (Norway); Medical Faculty, University of Oslo, Oslo (Norway); Flatmark, Kjersti [Department of Tumor Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Department of Gastrointestinal Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Seierstad, Therese [Department of Radiology and Nuclear Medicine, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Faculty of Health Sciences, Buskerud University College, Drammen (Norway); Lilleby, Wolfgang [Department of Oncology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Lyng, Heidi, E-mail: heidi.lyng@rr-research.no [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway)

    2013-11-15

    Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to

  15. Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

    Directory of Open Access Journals (Sweden)

    Lv Y

    2015-07-01

    Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

  16. Down-regulation of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor by HEXIM1 attenuates myocardial angiogenesis in hypoxic mice.

    Science.gov (United States)

    Yoshikawa, Noritada; Shimizu, Noriaki; Ojima, Hidenori; Kobayashi, Hiroshi; Hosono, Osamu; Tanaka, Hirotoshi

    2014-10-24

    Pulmonary hypertension (PH) sustains elevation of pulmonary vascular resistance and ultimately leads to right ventricular (RV) hypertrophy and failure and death. Recently, proangiogenic factors hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) have been known to promote left ventricular myocardial angiogenesis and lead to cardiac hypertrophy, and this would be involved in RV hypertrophy of PH patients. Previously, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced cardiomyocyte hypertrophy and hypertrophic genes expression, and that cardiomyocyte-specific HEXIM1 transgenic mice ameliorates RV hypertrophy in hypoxia-induced PH model. Given these results, here we analyzed the effect of HEXIM1 on the expression of HIF-1α and VEGF and on myocardial angiogenesis of RV in PH. We revealed that overexpression of HEXIM1 prevented hypoxia-induced expression of HIF-1α protein and its target genes including VEGF in the cultured cardiac myocytes and fibroblasts, and that cardiomyocyte-specific HEXIM1 transgenic mice repressed RV myocardial angiogenesis in hypoxia-induced PH model. Thus, we conclude that HEXIM1 could prevent RV hypertrophy, at least in part, via suppression of myocardial angiogenesis through down-regulation of HIF-1α and VEGF in the myocardium under hypoxic condition. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

    International Nuclear Information System (INIS)

    Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

    2003-01-01

    Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

  18. Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

    Directory of Open Access Journals (Sweden)

    Shahrooz Ghaderi

    2018-03-01

    Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

  19. Hypoxia Inducible Factor-1α (HIF-1 α and its Role in Tumour Progression to Malignancy

    Directory of Open Access Journals (Sweden)

    Gaurav Mrinal Sharma

    2008-07-01

    Full Text Available Hypoxia is a condition in which an area of the body or a tissue is deprived of sufficient supply of oxygen. The lack of nutrients in a hypoxic tissue generally causes apoptosis but some cells are able to adapt to this hypoxic environment and resist apoptosis. This adaptation occurs as a result of gene activation. Hypoxia is a characteristic feature of many cancers and is the stimulus for overexpression of HIF-1α - a basic loop-helix PAS protein family subunit of HIF, which allows the cell to adapt and survive in hostile environment. The presence of hypoxia and HIF-1α is correlated with an increased risk of metastasis and techniques that can inhibit hypoxia inducible factor may be instrumental in finding a cure for cancer.

  20. Suppression of the expression of hypoxia-inducible factor-1α by RNA interference alleviates hypoxia-induced pulmonary hypertension in adult rats.

    Science.gov (United States)

    Li, Ying; Shi, Bo; Huang, Liping; Wang, Xin; Yu, Xiaona; Guo, Baosheng; Ren, Weidong

    2016-12-01

    Hypoxia-inducible factor-1α (HIF-1α) has been implicated in the pathogenesis of hypoxic pulmonary hypertension (PH). However, the potential clinical value of HIF-1α as a therapeutic target in the treatment of PH has not yet been evaluated. In this study, an animal model of hypoxia-induced PH was established by exposing adult rats to 10% O2 for 3 weeks, and the effects of the lentivirus-mediated delivery of HIF-1α short hairpin RNA (shRNA) by intratracheal instillation prior to exposure to hypoxia on the manifestations of hypoxia-induced PH were assessed. The successful delivery of HIF-1α shRNA into the pulmonary arteries effectively suppressed the hypoxia-induced upregulation of HIF-1α, accompanied by the prominent attenuation the symptoms associated with hypoxia-induced PH, including the elevation of pulmonary arterial pressure, hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs), as well as the muscularization of pulmonary arterioles. In addition, the knockdown of HIF-1α in cultured rat primary PASMCs significantly inhibited the hypoxia-induced acceleration of the cell cycle and the proliferation of the PASMCs, suggesting that HIF-1α may be a direct mediator of PASMC hyperplasia in hypoxia-induced PH. In conclusion, this study demonstrates the potent suppressive effects of HIF-1α shRNA on hypoxia-induced PH and PASMC hyperplasia, providing evidence for the potential application of HIF-1α shRNA in the treatment of hypoxic PH.

  1. Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

    Science.gov (United States)

    Lee, Dae-Hee; Lee, Yong J

    2008-10-01

    Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

  2. Hypoxic Culture Promotes Dopaminergic-Neuronal Differentiation of Nasal Olfactory Mucosa Mesenchymal Stem Cells via Upregulation of Hypoxia-Inducible Factor-1α.

    Science.gov (United States)

    Zhuo, Yi; Wang, Lei; Ge, Lite; Li, Xuan; Duan, Da; Teng, Xiaohua; Jiang, Miao; Liu, Kai; Yuan, Ting; Wu, Pei; Wang, Hao; Deng, Yujia; Xie, Huali; Chen, Ping; Xia, Ying; Lu, Ming

    2017-08-01

    Olfactory mucosa mesenchymal stem cells (OM-MSCs) display significant clonogenic activity and may be easily propagated for Parkinson's disease therapies. Methods of inducing OM-MSCs to differentiate into dopaminergic (DAergic) neurons using olfactory ensheathing cells (OECs) are thus an attractive topic of research. We designed a hypoxic induction protocol to generate DAergic neurons from OM-MSCs using a physiological oxygen (O 2 ) level of 3% and OEC-conditioned medium (OCM; HI group). The normal induction (NI) group was cultured in O 2 at ambient air level (21%). The role of hypoxia-inducible factor-1α (HIF-1α) in the differentiation of OM-MSCs under hypoxia was investigated by treating cells with an HIF-1α inhibitor before induction (HIR group). The proportions of β-tubulin- and tyrosine hydroxylase (TH)-positive cells were significantly increased in the HI group compared with the NI and HIR groups, as shown by immunocytochemistry and Western blotting. Furthermore, the level of dopamine was significantly increased in the HI group. A slow outward potassium current was recorded in differentiated cells after 21 d of induction using whole-cell voltage-clamp tests. A hypoxic environment thus promotes OM-MSCs to differentiate into DAergic neurons by increasing the expression of HIF-1α and by activating downstream target gene TH. This study indicated that OCM under hypoxic conditions could significantly upregulate key transcriptional factors involved in the development of DAergic neurons from OM-MSCs, mediated by HIF-1α. Hypoxia promotes DAergic neuronal differentiation of OM-MSCs, and HIF-1α may play an important role in hypoxia-inducible pathways during DAergic lineage specification and differentiation in vitro.

  3. Effect of oxygen on cardiac differentiation in mouse iPS cells: role of hypoxia inducible factor-1 and Wnt/beta-catenin signaling.

    Directory of Open Access Journals (Sweden)

    Tanya L Medley

    Full Text Available BACKGROUND: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes. OBJECTIVE: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha and the canonical Wnt pathway in this process. METHODS: Embryoid bodies (EBs derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture. RESULTS: At 14 days of differentiation, 59 ± 2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway. CONCLUSION: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis.

  4. Characterization and functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in tilapia.

    Science.gov (United States)

    Li, Hong Lian; Gu, Xiao Hui; Li, Bi Jun; Chen, Xiao; Lin, Hao Ran; Xia, Jun Hong

    2017-01-01

    Hypoxia is a major cause of fish morbidity and mortality in the aquatic environment. Hypoxia-inducible factors are very important modulators in the transcriptional response to hypoxic stress. In this study, we characterized and conducted functional analysis of hypoxia-inducible factor HIF1α and its inhibitor HIF1αn in Nile tilapia (Oreochromis niloticus). By cloning and Sanger sequencing, we obtained the full length cDNA sequences for HIF1α (2686bp) and HIF1αn (1308bp), respectively. The CDS of HIF1α includes 15 exons encoding 768 amino acid residues and the CDS of HIF1αn contains 8 exons encoding 354 amino acid residues. The complete CDS sequences of HIF1α and HIF1αn cloned from tilapia shared very high homology with known genes from other fishes. HIF1α show differentiated expression in different tissues (brain, heart, gill, spleen, liver) and at different hypoxia exposure times (6h, 12h, 24h). HIF1αn expression level under hypoxia is generally increased (6h, 12h, 24h) and shows extremely highly upregulation in brain tissue under hypoxia. A functional determination site analysis in the protein sequences between fish and land animals identified 21 amino acid sites in HIF1α and 2 sites in HIF1αn as significantly associated sites (α = 0.05). Phylogenetic tree-based positive selection analysis suggested 22 sites in HIF1α as positively selected sites with a p-value of at least 95% for fish lineages compared to the land animals. Our study could be important for clarifying the mechanism of fish adaptation to aquatic hypoxia environment.

  5. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Do, Ji Yeon; Choi, Young Keun [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kook, Hyun [Department of Pharmacology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, Brain Science & Engineering Institute, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Lee, In-Kyu [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Division of Endocrinology and Metabolism, Department of Internal Medicine, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Dong Ho, E-mail: sarasate2222@gmail.com [Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  6. Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

    Science.gov (United States)

    Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

    2012-08-01

    Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

  7. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    International Nuclear Information System (INIS)

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-01-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O 2 ). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice

  8. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

    International Nuclear Information System (INIS)

    Park, Choa; Lee, YoungJoo

    2014-01-01

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression

  9. Overexpression of ERβ is sufficient to inhibit hypoxia-inducible factor-1 transactivation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Choa; Lee, YoungJoo, E-mail: yjlee@sejong.ac.kr

    2014-07-18

    Highlights: • We examined the effect of ERβ specific ligand on HIF-1 inhibition. • DPN down-regulates the ARNT protein levels in PC3 cells. • DPN did not show additional effect in ERβ transfected MCF-7 cells. • Our study shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression. - Abstract: Estrogen receptor (ER) β is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERβ suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERβ specific ligand on HIF-1 inhibition in ERβ positive PC3 cells and ERβ transfected MCF-7 cells. ERβ specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERβ specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERβ transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERβ was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERβ significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERβ. This result shows that unliganded ERβ is sufficient to inhibit HIF-1 in systems of overexpression.

  10. Hypoxia-induced metastasis model in embryonic zebrafish

    DEFF Research Database (Denmark)

    Rouhi, Pegah; Jensen, Lasse D.; Cao, Ziquan

    2010-01-01

    Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring...... of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent Di......I-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average...

  11. Retinal neuroprotection by hypoxic preconditioning is independent of hypoxia-inducible factor-1 alpha expression in photoreceptors.

    Science.gov (United States)

    Thiersch, Markus; Lange, Christina; Joly, Sandrine; Heynen, Severin; Le, Yun Zheng; Samardzija, Marijana; Grimm, Christian

    2009-06-01

    Hypoxic preconditioning stabilizes hypoxia-inducible factor (HIF) 1 alpha in the retina and protects photoreceptors against light-induced cell death. HIF-1 alpha is one of the major transcription factors responding to low oxygen tension and can differentially regulate a large number of target genes. To analyse whether photoreceptor-specific expression of HIF-1 alpha is essential to protect photoreceptors by hypoxic preconditioning, we knocked down expression of HIF-1 alpha specifically in photoreceptor cells, using the cyclization recombinase (Cre)-lox system. The Cre-mediated knockdown caused a 20-fold reduced expression of Hif-1 alpha in the photoreceptor cell layer. In the total retina, RNA expression was reduced by 65%, and hypoxic preconditioning led to only a small increase in HIF-1 alpha protein levels. Accordingly, HIF-1 target gene expression after hypoxia was significantly diminished. Retinas of Hif-1 alpha knockdown animals did not show any pathological alterations, and tolerated hypoxic exposure in a comparable way to wild-type retinas. Importantly, the strong neuroprotective effect of hypoxic preconditioning against light-induced photoreceptor degeneration persisted in knockdown mice, suggesting that hypoxia-mediated survival of light exposure does not depend on an autocrine action of HIF-1 alpha in photoreceptor cells. Hypoxia-mediated stabilization of HIF-2 alpha and phosphorylation of signal transducer and activator of transcription 3 (STAT 3) were not affected in the retinas of Hif-1 alpha knockdown mice. Thus, these factors are candidates for regulating the resistance of photoreceptors to light damage after hypoxic preconditioning, along with several potentially neuroprotective genes that were similarly induced in hypoxic knockdown and control mice.

  12. Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms.

    Science.gov (United States)

    Stenmark, Kurt R; Fagan, Karen A; Frid, Maria G

    2006-09-29

    Chronic hypoxic exposure induces changes in the structure of pulmonary arteries, as well as in the biochemical and functional phenotypes of each of the vascular cell types, from the hilum of the lung to the most peripheral vessels in the alveolar wall. The magnitude and the specific profile of the changes depend on the species, sex, and the developmental stage at which the exposure to hypoxia occurred. Further, hypoxia-induced changes are site specific, such that the remodeling process in the large vessels differs from that in the smallest vessels. The cellular and molecular mechanisms vary and depend on the cellular composition of vessels at particular sites along the longitudinal axis of the pulmonary vasculature, as well as on local environmental factors. Each of the resident vascular cell types (ie, endothelial, smooth muscle, adventitial fibroblast) undergo site- and time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors, and each resident cell type plays a specific role in the overall remodeling response. In addition, hypoxic exposure induces an inflammatory response within the vessel wall, and the recruited circulating progenitor cells contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation. The possibility exists that the lung or lung vessels also contain resident progenitor cells that participate in the remodeling process. Thus the hypoxia-induced remodeling of the pulmonary circulation is a highly complex process where numerous interactive events must be taken into account as we search for newer, more effective therapeutic interventions. This review provides perspectives on each of the aforementioned areas.

  13. [Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

    Science.gov (United States)

    Liu, Fang; Wuren, Tana; Ma, Lan; Yang, Ying-Zhong; Ge, Ri-Li

    2011-12-25

    In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

  14. Gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 signaling in pregnant rats.

    Science.gov (United States)

    Zhou, Jianjun; Xiao, Daliao; Hu, Yali; Wang, Zhiqun; Paradis, Alexandra; Mata-Greenwood, Eugenia; Zhang, Lubo

    2013-09-01

    Preeclampsia is a life-threatening pregnancy disorder. However, its pathogenesis remains unclear. We tested the hypothesis that gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 (ET-1) signaling. Time-dated pregnant and nonpregnant rats were divided into normoxic and hypoxic (10.5% O2 from the gestational day 6-21) groups. Chronic hypoxia had no significant effect on blood pressure or proteinuria in nonpregnant rats but significantly increased blood pressure on day 12 (systolic blood pressure, 111.7 ± 6.1 versus 138.5 ± 3.5 mm Hg; P=0.004) and day 20 (systolic blood pressure, 103.4 ± 4.6 versus 125.1 ± 6.1 mm Hg; P=0.02) in pregnant rats and urine protein (μg/μL)/creatinine (nmol/μL) ratio on day 20 (0.10 ± 0.01 versus 0.20 ± 0.04; P=0.04), as compared with the normoxic control group. This was accompanied with asymmetrical fetal growth restriction. Hypoxia resulted in impaired trophoblast invasion and uteroplacental vascular remodeling. In addition, plasma ET-1 levels, as well as the abundance of prepro-ET-1 mRNA, ET-1 type A receptor and angiotensin II type 1 receptor protein in the kidney and placenta were significantly increased in the chronic hypoxic group, as compared with the control animals. Treatment with the ET-1 type A receptor antagonist, BQ123, during the course of hypoxia exposure significantly attenuated the hypoxia-induced hypertension and other preeclampsia-like features. The results demonstrate that chronic hypoxia during gestation induces preeclamptic symptoms in pregnant rats via heightened ET-1 and ET-1 type A receptor-mediated signaling, providing a molecular mechanism linking gestational hypoxia and increased risk of preeclampsia.

  15. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    International Nuclear Information System (INIS)

    Sorensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Horsman, Michael R.

    2007-01-01

    Background: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. Methods: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu DD ]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24 h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. Results: In both SiHa and FaDu DD cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu DD cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu DD . Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu DD cells. Conclusion: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers

  16. Thiamine deficiency activates hypoxia inducible factor-1α to facilitate pro-apoptotic responses in mouse primary astrocytes.

    Directory of Open Access Journals (Sweden)

    Kristy Zera

    Full Text Available Thiamine is an essential enzyme cofactor required for proper metabolic function and maintenance of metabolism and energy production in the brain. In developed countries, thiamine deficiency (TD is most often manifested following chronic alcohol consumption leading to impaired mitochondrial function, oxidative stress, inflammation and excitotoxicity. These biochemical lesions result in apoptotic cell death in both neurons and astrocytes. Comparable histological injuries in patients with hypoxia/ischemia and TD have been described in the thalamus and mammillary bodies, suggesting a congruency between the cellular responses to these stresses. Consistent with hypoxia/ischemia, TD stabilizes and activates Hypoxia Inducible Factor-1α (HIF-1α under physiological oxygen levels. However, the role of TD-induced HIF-1α in neurological injury is currently unknown. Using Western blot analysis and RT-PCR, we have demonstrated that TD induces HIF-1α expression and activity in primary mouse astrocytes. We observed a time-dependent increase in mRNA and protein expression of the pro-apoptotic and pro-inflammatory HIF-1α target genes MCP1, BNIP3, Nix and Noxa during TD. We also observed apoptotic cell death in TD as demonstrated by PI/Annexin V staining, TUNEL assay, and Cell Death ELISA. Pharmacological inhibition of HIF-1α activity using YC1 and thiamine repletion both reduced expression of pro-apoptotic HIF-1α target genes and apoptotic cell death in TD. These results demonstrate that induction of HIF-1α mediated transcriptional up-regulation of pro-apoptotic/inflammatory signaling contributes to astrocyte cell death during thiamine deficiency.

  17. Absence of the inflammasome adaptor ASC reduces hypoxia-induced pulmonary hypertension in mice.

    Science.gov (United States)

    Cero, Fadila Telarevic; Hillestad, Vigdis; Sjaastad, Ivar; Yndestad, Arne; Aukrust, Pål; Ranheim, Trine; Lunde, Ida Gjervold; Olsen, Maria Belland; Lien, Egil; Zhang, Lili; Haugstad, Solveig Bjærum; Løberg, Else Marit; Christensen, Geir; Larsen, Karl-Otto; Skjønsberg, Ole Henning

    2015-08-15

    Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1β, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC(-/-), and NLRP3(-/-) mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC(-/-) mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P right ventricular remodeling. RVSP of NLRP3(-/-) mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1β in WT and NLRP3(-/-) mice, this response was absent in ASC(-/-) mice. Moreover, ASC(-/-) mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension. Copyright © 2015 the American Physiological Society.

  18. Hypoxia-Inducible Histone Lysine Demethylases: Impact on the Aging Process and Age-Related Diseases

    Science.gov (United States)

    Salminen, Antero; Kaarniranta, Kai; Kauppinen, Anu

    2016-01-01

    Hypoxia is an environmental stress at high altitude and underground conditions but it is also present in many chronic age-related diseases, where blood flow into tissues is impaired. The oxygen-sensing system stimulates gene expression protecting tissues against hypoxic insults. Hypoxia stabilizes the expression of hypoxia-inducible transcription factor-1α (HIF-1α), which controls the expression of hundreds of survival genes related to e.g. enhanced energy metabolism and autophagy. Moreover, many stress-related signaling mechanisms, such as oxidative stress and energy metabolic disturbances, as well as the signaling cascades via ceramide, mTOR, NF-κB, and TGF-β pathways, can also induce the expression of HIF-1α protein to facilitate cell survival in normoxia. Hypoxia is linked to prominent epigenetic changes in chromatin landscape. Screening studies have indicated that the stabilization of HIF-1α increases the expression of distinct histone lysine demethylases (KDM). HIF-1α stimulates the expression of KDM3A, KDM4B, KDM4C, and KDM6B, which enhance gene transcription by demethylating H3K9 and H3K27 sites (repressive epigenetic marks). In addition, HIF-1α induces the expression of KDM2B and KDM5B, which repress transcription by demethylating H3K4me2,3 sites (activating marks). Hypoxia-inducible KDMs support locally the gene transcription induced by HIF-1α, although they can also control genome-wide chromatin landscape, especially KDMs which demethylate H3K9 and H3K27 sites. These epigenetic marks have important role in the control of heterochromatin segments and 3D folding of chromosomes, as well as the genetic loci regulating cell type commitment, proliferation, and cellular senescence, e.g. the INK4 box. A chronic stimulation of HIF-1α can provoke tissue fibrosis and cellular senescence, which both are increasingly present with aging and age-related diseases. We will review the regulation of HIF-1α-dependent induction of KDMs and clarify their role in

  19. Hypoxia Inducible Factor 1 (HIF1) Activation in U87 Glioma Cells Involves a Decrease in Reactive Oxygen Species Production and Protein Kinase C Activity

    Science.gov (United States)

    1998-06-29

    Curcumin DFX Desferrioxamine DNA Deoxyribonucleic Acid DPI Diphenyliodinium DPPD Diphenylphenylenediamine DTH Dithionite EMSA Electrophoretic mobility shift... neuroprotective effects (Fern et al., 1996, Morishita et al., 1 1997). The identification of a hypoxia inducible transcription factor known as HIF-1 (Semenza...derived EPO in the eNS neuroprotective response to hypoxia. Cloning of the human and murine EPO gene, the availability of a convenient EPa producing

  20. URG11 mediates hypoxia-induced epithelial-to-mesenchymal transition by modulation of E-cadherin and β-catenin

    International Nuclear Information System (INIS)

    Du, Rui; Huang, Chen; Bi, Qian; Zhai, Ying; Xia, Lin; Liu, Jie; Sun, Shiren; Fan, Daiming

    2010-01-01

    Upregulated gene 11 (URG11), recently identified as a new HBx-upregulated gene that may activate β-catenin and Wnt signaling, was found to be upregulated in a human tubule cell line under low oxygen. Here, we investigated the potential role of URG11 in hypoxia-induced renal tubular epithelial-to-mesenchymal (EMT). Overexpression of URG11 in a human proximal tubule cell line (HK2) promoted a mesenchymal phenotype accompanied by reduced expression of the epithelial marker E-cadherin and increased expression of the mesenchymal markers vimentin and α-SMA, while URG11 knockdown by siRNA effectively reversed hypoxia-induced EMT. URG11 promoted the expression of β-catenin and increased its nuclear accumulation under normoxic conditions through transactivation of the β-catenin promoter. This in turn upregulated β-catenin/T-cell factor (TCF) and its downstream effector genes, vimentin, and α-SMA. In vivo, strong expression of URG11 was observed in the tubular epithelia of 5/6-nephrectomized rats, and a Western blot analysis demonstrated a close correlation between HIF-1α and URG11 protein levels. Altogether, our results indicate that URG11 mediates hypoxia-induced EMT through the suppression of E-cadherin and the activation of the β-catenin/TCF pathway.

  1. Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina.

    Science.gov (United States)

    Tan, Sih Min; Deliyanti, Devy; Figgett, William A; Talia, Dean M; de Haan, Judy B; Wilkinson-Berka, Jennifer L

    2015-07-01

    Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 μM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6-18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Hypoxia Induced Factor in Chronic Kidney Disease: Friend or Foe?

    Science.gov (United States)

    Li, Weiying; Zhao, Yuliang; Fu, Ping

    2017-01-01

    Many studies have shown evidence that erythropoiesis-stimulating agents (ESAs), as a classic treatment for chronic kidney disease (CKD)-related anemia, have several disadvantages and may trigger various adverse events with long-term use. The hypoxia-induced factor (HIF) pathway has been intensively investigated in kidney disease, especially in CKD, as research has shown that HIF-mediated erythropoiesis might work as a potential therapeutic strategy for managing CKD-related anemia. Development of prolyl hydroxylase domain inhibitors (PHIs), as an effective HIF activator, is a valuable step toward finding a replacement for ESAs, which showed an effective erythropoiesis through a comprehensive and physiological approach by promoting erythropoietin production, increasing iron bioavailability and improving chronic inflammatory status. Heretofore no adverse events or obvious off-target effects have been reported in clinical trials of PHIs. Nevertheless, a cautious inspection with extended follow-up period is warranted to validate the safety of prolonged HIF elevation, especially considering its ambiguous role in fibrogenesis and inflammation responses and possible risks in accelerating vascular calcification and tumorigenesis. A weighed dosing strategy might be the key to circumvent the unexpected side-effect brought by pleotropic effects of HIF elevation and achieve a selective augmentation of HIF-mediated signaling pathway. New studies with longer follow-up period and adequate analysis about the risks for proinflammation, vascular calcification and tumorigenesis are needed to ensure the drugs are safe for long-term use before being widely accepted in daily clinical practice.

  3. Development of 1-aryl-3-furanyl/thienyl-imidazopyridine templates for inhibitors against hypoxia inducible factor (HIF)-1 transcriptional activity.

    Science.gov (United States)

    Fuse, Shinichiro; Ohuchi, Toshiaki; Asawa, Yasunobu; Sato, Shinichi; Nakamura, Hiroyuki

    2016-12-15

    1,3-Disubstituted-imidazopyridines were designed for developing inhibitors against HIF-1 transcriptional activity. Designed compounds were rapidly synthesized from a key aromatic scaffold via microwave-assisted Suzuki-Miyaura coupling/CH direct arylation sequence. Evaluation of ability to inhibit the hypoxia induced transcriptional activity of HIF-1 revealed that the compound 2i and 3a retained the same level of the inhibitory activity comparing with that of known inhibitor, YC-1 (1). Identified, readily accessible 1-aryl-3-furanyl/thienyl-imidazopyridine templates should be useful for future drug development. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Flavonoids-induced accumulation of hypoxia-inducible factor (HIF)-1alpha/2alpha is mediated through chelation of iron.

    Science.gov (United States)

    Park, Sung-Soo; Bae, Insoo; Lee, Yong J

    2008-04-15

    Hypoxia-inducible factor-1 alpha (HIF-1alpha) is the regulatory subunit of the heterodimeric transcription factor HIF-1 that is the key regulator of cellular response to low oxygen tension. Under normoxic conditions, HIF-1alpha is continuously degraded by the ubiquitin-proteasome pathway through pVHL (von Hippel-Lindau tumor suppressor protein). Under hypoxic conditions, HIF-1alpha is stabilized and induces the transcription of HIF-1 target genes. Quercetin, a flavonoid with anti-oxidant, anti-inflammatory, and kinase modulating properties, has been found to induce HIF-1alpha accumulation and VEGF secretion in normoxia. In this study, the molecular mechanisms of quercetin-mediated HIF-1alpha accumulation were investigated. Previous studies have shown that, in addition to being induced by hypoxia, HIF-1alpha can be induced through the phosphatidylinositol 3-kinase (PI3K)/Akt and p53 signaling pathways. But our study revealed, through p53 mutant-type as well as p53 null cell lines, that neither the PI3K/Akt nor the p53 signaling pathway is required for quercetin-induced HIF-1alpha accumulation. And we observed that HIF-1alpha accumulated by quercetin is not ubiquitinated and the interaction of HIF-1alpha with pVHL is reduced, compared with HIF-1alpha accumulated by the proteasome inhibitor MG132. The use of quercetin's analogues showed that only quercetin and galangin induce HIF-1/2alpha accumulation and this effect is completely reversed by additional iron ions. This is because quercetin and galangin are able to chelate cellular iron ions that are cofactors of HIF-1/2alpha proline hydroxylase (PHD). These data suggest that quercetin inhibits the ubiquitination of HIF-1/2alpha in normoxia by hindering PHD through chelating iron ions.

  5. Proteomic Alterations in Response to Hypoxia Inducible Factor 2α in Normoxic Neuroblastoma Cells.

    Science.gov (United States)

    Cimmino, Flora; Pezone, Lucia; Avitabile, Marianna; Persano, Luca; Vitale, Monica; Sassi, Mauro; Bresolin, Silvia; Serafin, Valentina; Zambrano, Nicola; Scaloni, Andrea; Basso, Giuseppe; Iolascon, Achille; Capasso, Mario

    2016-10-07

    Hypoxia inducible factor (HIF)-2α protein expression in solid tumors promotes stem-like phenotype in cancer stem cells and increases tumorigenic potential in nonstem cancer cells. Recently, we have shown that HIF-1/2α gene expression is correlated to neuroblastoma (NB) poor survival and to undifferentiated tumor state; HIF-2α protein was demonstrated to enhance aggressive features of the disease. In this study, we used proteomic experiments on NB cells to investigate HIF-2α downstream-regulated proteins or pathways with the aim of providing novel therapeutic targets or bad prognosis markers. We verified that pathways mostly altered by HIF-2α perturbation are involved in tumor progression. In particular, HIF-2α induces alteration of central metabolism and splicing control pathways. Simultaneously, WNT, RAS/MAPK, and PI3K/AKT activity or expression are affected and may impact the sensitivity and the intensity of HIF-2α-regulated pathways. Furthermore, genes coding the identified HIF-2α-related markers built a signature able to stratify NB patients with unfavorable outcome. Taken together, our findings underline the relevance of dissecting the downstream effects of a poor survival marker in developing targeted therapy and improving patient stratification. Future prospective studies are needed to translate the use of these data into the clinical practice.

  6. Radiolabeled Probes Targeting Hypoxia-Inducible Factor-1-Active Tumor Microenvironments

    Directory of Open Access Journals (Sweden)

    Masashi Ueda

    2014-01-01

    Full Text Available Because tumor cells grow rapidly and randomly, hypoxic regions arise from the lack of oxygen supply in solid tumors. Hypoxic regions in tumors are known to be resistant to chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1 expressed in hypoxic regions regulates the expression of genes related to tumor growth, angiogenesis, metastasis, and therapy resistance. Thus, imaging of HIF-1-active regions in tumors is of great interest. HIF-1 activity is regulated by the expression and degradation of its α subunit (HIF-1α, which is degraded in the proteasome under normoxic conditions, but escapes degradation under hypoxic conditions, allowing it to activate transcription of HIF-1-target genes. Therefore, to image HIF-1-active regions, HIF-1-dependent reporter systems and injectable probes that are degraded in a manner similar to HIF-1α have been recently developed and used in preclinical studies. However, no probe currently used in clinical practice directly assesses HIF-1 activity. Whether the accumulation of 18F-FDG or 18F-FMISO can be utilized as an index of HIF-1 activity has been investigated in clinical studies. In this review, the current status of HIF-1 imaging in preclinical and clinical studies is discussed.

  7. Hypoxia-Inducible Factors: Mediators of Cancer Progression; Prognostic and Therapeutic Targets in Soft Tissue Sarcomas

    International Nuclear Information System (INIS)

    Sadri, Navid; Zhang, Paul J.

    2013-01-01

    Soft-tissue sarcomas remain aggressive tumors that result in death in greater than a third of patients due to either loco-regional recurrence or distant metastasis. Surgical resection remains the main choice of treatment for soft tissue sarcomas with pre- and/or post-operational radiation and neoadjuvant chemotherapy employed in more advanced stage disease. However, in recent decades, there has been little progress in the average five-year survival for the majority of patients with high-grade soft tissue sarcomas, highlighting the need for improved targeted therapeutic agents. Clinical and preclinical studies demonstrate that tumor hypoxia and up-regulation of hypoxia-inducible factors (HIFs) is associated with decreased survival, increased metastasis, and resistance to therapy in soft tissue sarcomas. HIF-mediated gene expression regulates many critical aspects of tumor biology, including cell survival, metabolic programming, angiogenesis, metastasis, and therapy resistance. In this review, we discuss HIFs and HIF-mediated genes as potential prognostic markers and therapeutic targets in sarcomas. Many pharmacological agents targeting hypoxia-related pathways are in development that may hold therapeutic potential for treating both primary and metastatic sarcomas that demonstrate increased HIF expression

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

    Science.gov (United States)

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

    2016-07-01

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

  9. Epigenetic control of hypoxia inducible factor-1α-dependent expression of placental growth factor in hypoxic conditions.

    Science.gov (United States)

    Tudisco, Laura; Della Ragione, Floriana; Tarallo, Valeria; Apicella, Ivana; D'Esposito, Maurizio; Matarazzo, Maria Rosaria; De Falco, Sandro

    2014-04-01

    Hypoxia plays a crucial role in the angiogenic switch, modulating a large set of genes mainly through the activation of hypoxia-inducible factor (HIF) transcriptional complex. Endothelial cells play a central role in new vessels formation and express placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, mainly involved in pathological angiogenesis. Despite several observations suggest a hypoxia-mediated positive modulation of PlGF, the molecular mechanism governing this regulation has not been fully elucidated. We decided to investigate if epigenetic modifications are involved in hypoxia-induced PlGF expression. We report that PlGF expression was induced in cultured human and mouse endothelial cells exposed to hypoxia (1% O 2), although DNA methylation at the Plgf CpG-island remains unchanged. Remarkably, robust hyperacetylation of histones H3 and H4 was observed in the second intron of Plgf, where hypoxia responsive elements (HREs), never described before, are located. HIF-1α, but not HIF-2α, binds to identified HREs. Noteworthy, only HIF-1α silencing fully inhibited PlGF upregulation. These results formally demonstrate a direct involvement of HIF-1α in the upregulation of PlGF expression in hypoxia through chromatin remodeling of HREs sites. Therefore, PlGF may be considered one of the putative targets of anti-HIF therapeutic applications.

  10. Hypoxia-induced oxidative base modifications in the VEGF hypoxia-response element are associated with transcriptionally active nucleosomes.

    Science.gov (United States)

    Ruchko, Mykhaylo V; Gorodnya, Olena M; Pastukh, Viktor M; Swiger, Brad M; Middleton, Natavia S; Wilson, Glenn L; Gillespie, Mark N

    2009-02-01

    Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxia-response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (K.A. Ziel et al., FASEB J. 19, 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multinucleosomes to transcriptionally active mono- and dinucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono- and dinucleosome fractions. The histone deacetylase inhibitor trichostatin A caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mononucleosomes and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.

  11. Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

    Science.gov (United States)

    Pawlus, Matthew R; Hu, Cheng-Jun

    2013-09-01

    Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

    International Nuclear Information System (INIS)

    Tse, Anna Chung-Kwan; Li, Jing-Woei; Chan, Ting-Fung; Wu, Rudolf Shiu-Sun; Lai, Keng-Po

    2015-01-01

    Highlights: • We demonstrate hypoxia induced miR-210 in ovarian follicular cells. • We show anti-apoptotic roles of miR-210 in ovarian follicular cells under hypoxia. • Apoptotic genes (DLC1, SLK, TNFRSF10B, RBM25, and USP7) are target of miR-210. • MiR-210 is vital for ovarian follicular cells proliferation in response to hypoxia. - Abstract: Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of mi

  13. Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

    Energy Technology Data Exchange (ETDEWEB)

    Tse, Anna Chung-Kwan [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Li, Jing-Woei; Chan, Ting-Fung [School of Life Sciences, Hong Kong Bioinformatics Centre, The Chinese University of Hong Kong, Hong Kong SAR (China); Wu, Rudolf Shiu-Sun [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Lai, Keng-Po, E-mail: balllai@hku.hk [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China)

    2015-08-15

    Highlights: • We demonstrate hypoxia induced miR-210 in ovarian follicular cells. • We show anti-apoptotic roles of miR-210 in ovarian follicular cells under hypoxia. • Apoptotic genes (DLC1, SLK, TNFRSF10B, RBM25, and USP7) are target of miR-210. • MiR-210 is vital for ovarian follicular cells proliferation in response to hypoxia. - Abstract: Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of mi

  14. Hypoxia inducible factor-1 alpha stabilization for regenerative therapy in traumatic brain injury

    Directory of Open Access Journals (Sweden)

    Mushfiquddin Khan

    2017-01-01

    Full Text Available Mild traumatic brain injury (TBI, also called concussion, initiates sequelae leading to motor deficits, cognitive impairments and subtly compromised neurobehaviors. While the acute phase of TBI is associated with neuroinflammation and nitroxidative burst, the chronic phase shows a lack of stimulation of the neurorepair process and regeneration. The deficiency of nitric oxide (NO, the consequent disturbed NO metabolome, and imbalanced mechanisms of S-nitrosylation are implicated in blocking the mechanisms of neurorepair processes and functional recovery in the both phases. Hypoxia inducible factor-1 alpha (HIF-1α, a master regulator of hypoxia/ischemia, stimulates the process of neurorepair and thus aids in functional recovery after brain trauma. The activity of HIF-1α is regulated by NO via the mechanism of S-nitrosylation of HIF-1α. S-nitrosylation is dynamically regulated by NO metabolites such as S-nitrosoglutathione (GSNO and peroxynitrite. GSNO stabilizes, and peroxynitrite destabilizes HIF-1α. Exogenously administered GSNO was found not only to stabilize HIF-1α and to induce HIF-1α-dependent genes but also to stimulate the regeneration process and to aid in functional recovery in TBI animals.

  15. Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies

    Science.gov (United States)

    Zimna, Agnieszka; Kurpisz, Maciej

    2015-01-01

    The cardiovascular system ensures the delivery of oxygen and nutrients to all cells, tissues, and organs. Under extended exposure to reduced oxygen levels, cells are able to survive through the transcriptional activation of a series of genes that participate in angiogenesis, glucose metabolism, and cell proliferation. The oxygen-sensitive transcriptional activator HIF-1 (hypoxia-inducible factor-1) is a key transcriptional mediator of the response to hypoxic conditions. The HIF-1 pathway was found to be a master regulator of angiogenesis. Whether the process is physiological or pathological, HIF-1 seems to participate in vasculature formation by synergistic correlations with other proangiogenic factors such as VEGF (vascular endothelial growth factor), PlGF (placental growth factor), or angiopoietins. Considering the important contributions of HIF-1 in angiogenesis and vasculogenesis, it should be considered a promising target for treating ischaemic diseases or cancer. In this review, we discuss the roles of HIF-1 in both physiological/pathophysiological angiogenesis and potential strategies for clinical therapy. PMID:26146622

  16. Cellular Oxygen Sensing: Crystal Structure of Hypoxia-Inducible Factor Prolyl Hydroxylase (PHD2)

    Energy Technology Data Exchange (ETDEWEB)

    McDonough,M.; Li, V.; Flashman, E.; Chowdhury, R.; Mohr, C.; Lienard, B.; Zondlo, J.; Oldham, N.; Clifton, I.; et al.

    2006-01-01

    Cellular and physiological responses to changes in dioxygen levels in metazoans are mediated via the posttranslational oxidation of hypoxia-inducible transcription factor (HIF). Hydroxylation of conserved prolyl residues in the HIF-{alpha} subunit, catalyzed by HIF prolyl-hydroxylases (PHDs), signals for its proteasomal degradation. The requirement of the PHDs for dioxygen links changes in dioxygen levels with the transcriptional regulation of the gene array that enables the cellular response to chronic hypoxia; the PHDs thus act as an oxygen-sensing component of the HIF system, and their inhibition mimics the hypoxic response. We describe crystal structures of the catalytic domain of human PHD2, an important prolyl-4-hydroxylase in the human hypoxic response in normal cells, in complex with Fe(II) and an inhibitor to 1.7 Angstroms resolution. PHD2 crystallizes as a homotrimer and contains a double-stranded {beta}-helix core fold common to the Fe(II) and 2-oxoglutarate-dependant dioxygenase family, the residues of which are well conserved in the three human PHD enzymes (PHD 1-3). The structure provides insights into the hypoxic response, helps to rationalize a clinically observed mutation leading to familial erythrocytosis, and will aid in the design of PHD selective inhibitors for the treatment of anemia and ischemic disease.

  17. Diacetoxyscirpenol as a new anticancer agent to target hypoxia-inducible factor 1

    Science.gov (United States)

    Choi, Yong-Joon; Shin, Hyun-Woo; Chun, Yang-Sook; Leutou, Alain Simplice; Son, Byeng Wha; Park, Jong-Wan

    2016-01-01

    Hypoxia activates hypoxia-inducible factor 1, which promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. Thus, HIF-1 is one of the most compelling targets for treating cancers. The aim of this study was to find a small molecule that inhibits HIF-1 under hypoxia in cancer cells. 7,280 compounds in a chemical library were tested in a cancer cell line expressing luciferase HIF-dependently. Through three rounds of screening, we finally picked up a compound that originates from a marine bacterium parasitizing red alga. The antibiotic potently inhibited HIF-1 expression and its transcriptional activity in cancer cells exposed to hypoxia. Through two-step fractionation, diacetoxyscirpenol was purified and identified as a HIF-inhibiting ingredient. Mechanistically, diacetoxyscirpenol inhibits the synthesis of HIF-1α protein and also interferes with the dimerization of HIF-1α and ARNT. It attenuates HIF-mediated gene expression in cancer cells exposed to hypoxia, and by doing so reduces tumorigenic and angiogenic potentials of cancer cells. More importantly, diacetoxyscirpenol retarded tumor growth in mice, and reduced HIF-1α expression and vascular formation in the tumors. Overall, diacetoxyscirpenol is considered a potential drug deregulating the HIF-1 signaling pathway, and it could be beneficially employed for treating malignant tumors with hypoxic microenvironment. PMID:27613833

  18. Cancer drug troglitazone stimulates the growth and response of renal cells to hypoxia inducible factors

    Energy Technology Data Exchange (ETDEWEB)

    Taub, Mary, E-mail: biochtau@buffalo.edu

    2016-03-11

    Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10{sup −5} M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia. - Highlights: • Troglitazone and rosiglitazone stimulate renal proximal tubule cell growth. • Troglitazone and linolenic acid stimulate growth via PPARϒ. • Linolenic acid stimulates growth in the presence of fatty acid free serum albumin. • Rosiglitazone stimulates transcription by a HRE luciferase construct.

  19. Cancer drug troglitazone stimulates the growth and response of renal cells to hypoxia inducible factors

    International Nuclear Information System (INIS)

    Taub, Mary

    2016-01-01

    Troglitazone has been used to suppress the growth of a number of tumors through apoptosis and autophagy. However, previous in vitro studies have employed very high concentrations of troglitazone (≥10"−"5 M) in order to elicit growth inhibitory effects. In this report, when employing lower concentrations of troglitazone in defined medium, troglitazone was observed to stimulate the growth of primary renal proximal tubule (RPT) cells. Rosiglitazone, like troglitazone, is a thiazolidinedione (TZD) that is known to activate Peroxisome Proliferator Activated Receptor Υ (PPARΥ). Notably, rosiglitazone also stimulates RPT cell growth, as does Υ-linolenic acids, another PPARΥ agonist. The PPARΥ antagonist GW9662 inhibited the growth stimulatory effect of troglitazone. In addition, troglitazone stimulated transcription by a PPAR Response Element/Luciferase construct. These results are consistent with the involvement of PPARΥ as a mediator of the growth stimulatory effect of troglitazone. In a number of tumor cells, the expression of hypoxia inducible factor (HIF) is increased, promoting the expression of HIF inducible genes, and vascularization. Troglitazone was observed to stimulate transcription by a HIF/luciferase construct. These observations indicate that troglitazone not only promotes growth, also the survival of RPT cells under conditions of hypoxia. - Highlights: • Troglitazone and rosiglitazone stimulate renal proximal tubule cell growth. • Troglitazone and linolenic acid stimulate growth via PPARϒ. • Linolenic acid stimulates growth in the presence of fatty acid free serum albumin. • Rosiglitazone stimulates transcription by a HRE luciferase construct.

  20. Hypoxia Induces a Metabolic Shift and Enhances the Stemness and Expansion of Cochlear Spiral Ganglion Stem/Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Hsin-Chien Chen

    2015-01-01

    Full Text Available Previously, we demonstrated that hypoxia (1% O2 enhances stemness markers and expands the cell numbers of cochlear stem/progenitor cells (SPCs. In this study, we further investigated the long-term effect of hypoxia on stemness and the bioenergetic status of cochlear spiral ganglion SPCs cultured at low oxygen tensions. Spiral ganglion SPCs were obtained from postnatal day 1 CBA/CaJ mouse pups. The measurement of oxygen consumption rate, extracellular acidification rate (ECAR, and intracellular adenosine triphosphate levels corresponding to 20% and 5% oxygen concentrations was determined using a Seahorse XF extracellular flux analyzer. After low oxygen tension cultivation for 21 days, the mean size of the hypoxia-expanded neurospheres was significantly increased at 5% O2; this correlated with high-level expression of hypoxia-inducible factor-1 alpha (Hif-1α, proliferating cell nuclear antigen (PCNA, cyclin D1, Abcg2, nestin, and Nanog proteins but downregulated expression of p27 compared to that in a normoxic condition. Low oxygen tension cultivation tended to increase the side population fraction, with a significant difference found at 5% O2 compared to that at 20% O2. In addition, hypoxia induced a metabolic energy shift of SPCs toward higher basal ECARs and higher maximum mitochondrial respiratory capacity but lower proton leak than under normoxia, where the SPC metabolism was switched toward glycolysis in long-term hypoxic cultivation.

  1. The histone demethylases JMJD1A and JMJD2B are transcriptional targets of hypoxia-inducible factor HIF

    DEFF Research Database (Denmark)

    Beyer, Sophie; Kristensen, Malene Maag; Jensen, Kim Steen

    2008-01-01

    of these modifications is exerted by histone methyltransferases and the recently discovered histone demethylases. Here we show that the hypoxia-inducible factor HIF-1a binds to specific recognition sites in the genes encoding the jumonji family histone demethylases JMJD1A and JMJD2B and induces their expression....... Accordingly, hypoxic cells express elevated levels of JMJD1A and JMJD2B mRNA and protein. Furthermore, we find increased expression of JMJD1A and JMJD2B in renal cancer cells that have lost the von Hippel Lindau tumor suppressor protein VHL and therefore display a deregulated expression of HIF. Studies...... on ectopically expressed JMJD1A and JMJD2B indicate that both proteins retain their histone lysine demethylase activity in hypoxia and thereby might impact the hypoxic gene expression program....

  2. COGNATE: comparative gene annotation characterizer.

    Science.gov (United States)

    Wilbrandt, Jeanne; Misof, Bernhard; Niehuis, Oliver

    2017-07-17

    The comparison of gene and genome structures across species has the potential to reveal major trends of genome evolution. However, such a comparative approach is currently hampered by a lack of standardization (e.g., Elliott TA, Gregory TR, Philos Trans Royal Soc B: Biol Sci 370:20140331, 2015). For example, testing the hypothesis that the total amount of coding sequences is a reliable measure of potential proteome diversity (Wang M, Kurland CG, Caetano-Anollés G, PNAS 108:11954, 2011) requires the application of standardized definitions of coding sequence and genes to create both comparable and comprehensive data sets and corresponding summary statistics. However, such standard definitions either do not exist or are not consistently applied. These circumstances call for a standard at the descriptive level using a minimum of parameters as well as an undeviating use of standardized terms, and for software that infers the required data under these strict definitions. The acquisition of a comprehensive, descriptive, and standardized set of parameters and summary statistics for genome publications and further analyses can thus greatly benefit from the availability of an easy to use standard tool. We developed a new open-source command-line tool, COGNATE (Comparative Gene Annotation Characterizer), which uses a given genome assembly and its annotation of protein-coding genes for a detailed description of the respective gene and genome structure parameters. Additionally, we revised the standard definitions of gene and genome structures and provide the definitions used by COGNATE as a working draft suggestion for further reference. Complete parameter lists and summary statistics are inferred using this set of definitions to allow down-stream analyses and to provide an overview of the genome and gene repertoire characteristics. COGNATE is written in Perl and freely available at the ZFMK homepage ( https://www.zfmk.de/en/COGNATE ) and on github ( https

  3. Hypoxia-inducible factor-dependent production of profibrotic mediators by hypoxic hepatocytes.

    Science.gov (United States)

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

    2009-08-01

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

  4. MicroRNA-145 Aggravates Hypoxia-Induced Injury by Targeting Rac1 in H9c2 Cells.

    Science.gov (United States)

    Wang, Ximing; Zhang, Yanxia; Wang, Hongshan; Zhao, Genshang; Fa, Xianen

    2017-01-01

    Myocardial infarction (MI) is a leading cause of morbidity and mortality. Here, we sought to explore the potential role and underlying mechanism of miR-145 in MI. H9c2 cells were cultured under persistent hypoxia to simulate MI. The hypoxia-induced injury was assessed on the basis of cell viability, migration, invasion and apoptosis. The expression of miR-145 was evaluated by qRT-PCR and the influence of aberrantly expressed miR-145 on H9c2 cells under hypoxia was also estimated. Utilizing bioinformatics methods, the target genes of miR-145 were verified by luciferase reporter assay. Then, effects of abnormally expressed target gene on miR-145 silenced H9c2 cells were assessed. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways were detected by Western blot analysis. Hypoxia remarkably lowered viability, migration and invasion but promoted cell apoptosis. Meantime, the miR-145 level was up-regulated in H9c2 cells under hypoxia. Following experiments suggested that hypoxia-induced injury was exacerbated by miR-145 overexpression while was alleviated by miR-145 silence. Rac1 was predicted and further validated to be a target gene of miR-145. The influence of miR-145 silencing on H9c2 cells under hypoxia could be reversed by down-regulation of Rac1. Additionally, the phosphorylation levels of PI3K, AKT, MAPK and ERK were all elevated in miR-145 silenced cells and these alterations were reversed by down-regulation of Rac1. miR-145 silencing could protect H9c2 cells against hypoxia-induced injury by targeting Rac1, in which PI3K/AKT and MAPK/ERK pathways might be involved. © 2017 The Author(s). Published by S. Karger AG, Basel.

  5. Effects of cadmium on hypoxia-induced expression of hemoglobin and erythropoietin in larval sheepshead minnow, Cyprinodon variegatus

    Energy Technology Data Exchange (ETDEWEB)

    Dangre, A.J.; Manning, S. [Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564 (United States); Brouwer, M., E-mail: marius.brouwer@usm.edu [Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564 (United States)

    2010-08-15

    Hypoxia and toxic metals are two common stressors found in the estuarine environment. To date little information is available on the combined effects of these stressors on early larval development in fish. We investigated the effect of cadmium and hypoxia exposure alone as well in combination on larval Cyprinodon variegatus. The LC{sub 10} for cadmium was determined to be 0.3 ppm in a 96 h acute exposure. This concentration was used in all studies. Cadmium in larvae increased significantly with exposure time (1, 3, 5 and 7 days post-hatch). The increase was proportional to body weight and not affected by hypoxia. Cadmium responsive genes were identified by suppression subtractive hybridization (SSH) in Cyprinodonvariegatus larvae after exposure to cadmium for 1, 3, 5 and 7 days. We obtained over 700 sequences from the cadmium cDNA library. Blast search of ESTs suggested that cadmium modulates multiple physiological processes. Pertinent to this study, cadmium was found to down-regulate both embryonic {alpha} and {beta} globin, which are expressed in erythrocytes generated during the first, or primitive, wave of erythropoiesis in teleosts. Hemoglobin (Hb) and erythropoietin (Epo) (the hormone that promotes red blood cell production) are known hypoxia-inducible genes. To explore the possibility that cadmium might offset the hypoxia-induced expression of Hb and Epo, we investigated the expression of both genes following hypoxia, cadmium and combined exposures for 1, 3, 5 and 7 days post-hatch. Since Epo had not yet been identified in C. variegatus we first successfully cloned a partial coding sequence of the C. variegatus hormone. Subsequent studies revealed that expression levels of Hb and Epo remained unchanged in the normoxic controls during the time course of the study. Hypoxia increased Epo expression relative to normoxic controls, on days 3, 5 and 7, while cadmium in hypoxia inhibited the increase. Only the changes on days 5 and 7 were statistically significant

  6. Rapid detection of hypoxia-inducible factor-1-active tumours: pretargeted imaging with a protein degrading in a mechanism similar to hypoxia-inducible factor-1{alpha}

    Energy Technology Data Exchange (ETDEWEB)

    Ueda, Masashi [Kyoto University, Radioisotopes Research Laboratory, Kyoto University Hospital, Faculty of Medicine, Kyoto (Japan); Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kudo, Takashi; Konishi, Hiroaki; Miyano, Azusa; Ono, Masahiro; Saji, Hideo [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Kuge, Yuji [Kyoto University, Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto (Japan); Hokkaido University, Central Institute of Isotope Science, Sapporo (Japan); Mukai, Takahiro [Kyushu University, Department of Biomolecular Recognition Chemistry, Graduate School of Pharmaceutical Sciences, Fukuoka (Japan); Tanaka, Shotaro; Kizaka-Kondoh, Shinae; Hiraoka, Masahiro [Kyoto University, Department of Radiation Oncology and Image-applied Therapy, Graduate School of Medicine, Kyoto (Japan)

    2010-08-15

    Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumour progression. For the imaging of HIF-1-active tumours, we previously developed a protein, POS, which is effectively delivered to and selectively stabilized in HIF-1-active cells, and a radioiodinated biotin derivative, (3-{sup 123}I-iodobenzoyl)norbiotinamide ({sup 123}I-IBB), which can bind to the streptavidin moiety of POS. In this study, we aimed to investigate the feasibility of the pretargeting method using POS and {sup 123}I-IBB for rapid imaging of HIF-1-active tumours. Tumour-implanted mice were pretargeted with POS. After 24 h, {sup 125}I-IBB was administered and subsequently, the biodistribution of radioactivity was investigated at several time points. In vivo planar imaging, comparison between {sup 125}I-IBB accumulation and HIF-1 transcriptional activity, and autoradiography were performed at 6 h after the administration of {sup 125}I-IBB. The same sections that were used in autoradiographic analysis were subjected to HIF-1{alpha} immunohistochemistry. {sup 125}I-IBB accumulation was observed in tumours of mice pretargeted with POS (1.6%ID/g at 6 h). This result is comparable to the data derived from {sup 125}I-IBB-conjugated POS-treated mice (1.4%ID/g at 24 h). In vivo planar imaging provided clear tumour images. The tumoral accumulation of {sup 125}I-IBB significantly correlated with HIF-1-dependent luciferase bioluminescence (R=0.84, p<0.01). The intratumoral distribution of {sup 125}I-IBB was heterogeneous and was significantly correlated with HIF-1{alpha}-positive regions (R=0.58, p<0.0001). POS pretargeting with {sup 123}I-IBB is a useful technique in the rapid imaging and detection of HIF-1-active regions in tumours. (orig.)

  7. The redox protein thioredoxin-1 (Trx-1) increases hypoxia-inducible factor 1alpha protein expression: Trx-1 overexpression results in increased vascular endothelial growth factor production and enhanced tumor angiogenesis.

    Science.gov (United States)

    Welsh, Sarah J; Bellamy, William T; Briehl, Margaret M; Powis, Garth

    2002-09-01

    Hypoxia-inducible factor 1 (HIF-1), a heterodimer of HIF-1alpha and HIF-1beta subunits, is a transcriptional activator central to the cellular response to low oxygen that includes metabolic adaptation, angiogenesis, metastasis, and inhibited apoptosis. Thioredoxin-1 (Trx-1) is a small redox protein overexpressed in a number of human primary tumors. We have examined the effects of Trx-1 on HIF activity and the activation of downstream genes. Stable transfection of human breast carcinoma MCF-7 cells with human Trx-1 caused a significant increase in HIF-1alpha protein levels under both normoxic (20% oxygen) and hypoxic (1% oxygen) conditions. Trx-1 increased hypoxia-induced HIF-1 transactivation activity measured using a luciferase reporter under the control of the hypoxia response element. Changes in HIF-1alpha mRNA levels did not account for the changes observed at the protein level, and HIF-1beta protein levels did not change. Trx-1 transfection also caused a significant increase in the protein products of hypoxia-responsive genes, including vascular endothelial growth factor (VEGF) and nitric oxide synthase 2 in a number of different cell lines (MCF-7 human breast and HT29 human colon carcinomas and WEHI7.2 mouse lymphoma cells) under both normoxic and hypoxic conditions. The pattern of expression of the different isoforms of VEGF was not changed by Trx-1. Transfection of a redox-inactive Trx-1 (C32S/C35S) markedly decreased levels of HIF-1alpha protein, HIF-1 transactivating activity, and VEGF protein in MCF-7 cells compared with empty vector controls. In vivo studies using WEHI7.2 cells transfected with Trx-1 showed significantly increased tumor VEGF and angiogenesis. The results suggest that Trx-1 increases HIF-1alpha protein levels in cancer cells and increases VEGF production and tumor angiogenesis.

  8. The Effects of Portulaca oleracea on Hypoxia-Induced Pulmonary Edema in Mice.

    Science.gov (United States)

    Yue, Tan; Xiaosa, Wen; Ruirui, Qi; Wencai, Shi; Hailiang, Xin; Min, Li

    2015-03-01

    Portulaca oleracea L. (PO) is known as "a vegetable for long life" due to its antioxidant, anti-inflammatory, and other pharmacological activities. However, the protective activity of the ethanol extract of PO (EEPO) against hypoxia-induced pulmonary edema has not been fully investigated. In this study, we exposed mice to a simulated altitude of 7000 meters for 0, 3, 6, 9, and 12 h to observe changes in the water content and transvascular leakage of the mouse lung. It was found that transvascular leakage increased to the maximum in the mouse lung after 6 h exposure to hypobaric hypoxia. Prophylactic administration of EEPO before hypoxic exposure markedly reduced the transvascular leakage and oxidative stress, and inhibited the upregulation of NF-kB in the mouse lung, as compared with the control group. In addition, EEPO significantly reduced the levels of proinflammatory cytokines and cell adhesion molecules in the lungs of mice, as compared with the hypoxia group. Our results show that EEPO can reduce initial transvascular leakage and pulmonary edema under hypobaric hypoxia conditions.

  9. Hypoxia Inducible Factor 1α Promotes Endogenous Adaptive Response in Rat Model of Chronic Cerebral Hypoperfusion

    Directory of Open Access Journals (Sweden)

    Ying Yang

    2017-01-01

    Full Text Available Hypoxia inducible factor 1α (HIF-1α, a pivotal regulator of gene expression in response to hypoxia and ischemia, is now considered to regulate both pro-survival and pro-death responses depending on the duration and severity of the stress. We previously showed that chronic global cerebral hypoperfusion (CCH triggered long-lasting accumulation of HIF-1α protein in the hippocampus of rats. However, the role of the stabilized HIF-1α in CCH is obscure. Here, we knock down endogenous HIF-1α to determine whether and how HIF-1α affects the disease processes and phenotypes of CCH. Lentivirus expressing HIF-1α small hairpin RNA was injected into the bilateral hippocampus and bilateral ventricles to knock down HIF-1α gene expression in the hippocampus and other brain areas. Permanent bilateral common carotid artery occlusions, known as 2-vessel occlusions (2VOs, were used to induce CCH in rats. Angiogenesis, oxidative stress, histopathological changes of the brain, and cognitive function were tested. Knockdown of HIF-1α prior to 2VO significantly exacerbates the impairment of learning and memory after four weeks of CCH. Mechanically, reduced cerebral angiogenesis, increased oxidative damage, and increased density of astrocytes and microglia in the cortex and some subregions of hippocampus are also shown after four weeks of CCH. Furthermore, HIF-1α knockdown also disrupts upregulation of regulated downstream genes. Our findings suggest that HIF-1α-protects the brain from oxidative stress and inflammation response in the disease process of CCH. Accumulated HIF-1α during CCH mediates endogenous adaptive processes to defend against more severe hypoperfusion injury of the brain, which may provide a therapeutic benefit.

  10. Regulation of glucose transporter protein-1 and vascular endothelial growth factor by hypoxia inducible factor 1α under hypoxic conditions in Hep-2 human cells.

    Science.gov (United States)

    Xu, Ou; Li, Xiaoming; Qu, Yongtao; Liu, Shuang; An, Jie; Wang, Maoxin; Sun, Qingjia; Zhang, Wen; Lu, Xiuying; Pi, Lihong; Zhang, Min; Shen, Yupeng

    2012-12-01

    The present study evaluated the regulation of glucose transporter protein-1 (Glut-1) and vascular endothelial growth factor (VEGF) by hypoxia inducible factor 1α (HIF-1α) under hypoxic conditions in Hep-2 human cells to explore the feasibility of these three genes as tumor markers. Hep-2 cells were cultured under hypoxic and normoxic conditions for 6, 12, 24, 36 and 48 h. The proliferation of Hep-2 cells was evaluated using an MTT assay. The protein and mRNA expression levels of HIF-1α, Glut-1 and VEGF were detected using the S-P immunocytochemical method, western blotting and reverse transcription polymerase chain reaction (RT-PCR). The results revealed that the expression levels of HIF-1α, Glut-1 and VEGF protein in Hep-2 cells were significantly elevated under hypoxic conditions compared with those under normoxic conditions over 36 h. Under hypoxic conditions, mRNA levels of HIF-1α were stable, while mRNA levels of Glut-1 and VEGF changed over time. In conclusion, Glut-1 and VEGF were upregulated by HIF-1α under hypoxic conditions in a time-dependent manner in Hep-2 cells and their co-expression serves as a tumor marker.

  11. Yak response to high-altitude hypoxic stress by altering mRNA expression and DNA methylation of hypoxia-inducible factors.

    Science.gov (United States)

    Xiong, Xianrong; Fu, Mei; Lan, Daoliang; Li, Jian; Zi, Xiangdong; Zhong, Jincheng

    2015-01-01

    Hypoxia-inducible factors (HIFs) are oxygen-dependent transcriptional activators, which play crucial roles in tumor angiogenesis and mammalian development, and regulate the transcription of genes involved in oxygen homeostasis in response to hypoxia. However, information on HIF-1α and HIF-2α in yak (Bos grunniens) is scarce. The complete coding region of yak HIF-2α was cloned, its mRNA expression in several tissues were determined, and the expression levels were compared with those of closely related low-altitude cattle (Bos taurus), and the methylation status of promoter regions were analyzed to better understand the roles of HIF-1α and HIF-2α in domesticated yak. The yak HIF-2α cDNA was cloned and sequenced in the present work reveals the evolutionary conservation through multiple sequence alignment, although 15 bases changed, resulting in 8 amino acid substitutions in the translated proteins in cattle. The tissue-specific expression results showed that HIF-1α is ubiquitously expressed, whereas HIF-2α expression is limited to endothelial tissues (kidney, heart, lung, spleen, and liver) and blood in yak. Both HIF-1α and HIF-2α expressions were higher in yak tissues than in cattle. The HIF-1α expression level is much higher in yak than cattle in these organs, except for the lung (P hypoxic stress response mechanism and may assist current medical research to understand hypoxia-related diseases.

  12. Hypoxia-induced retinal neovascularization in zebrafish embryos: a potential model of retinopathy of prematurity.

    Science.gov (United States)

    Wu, Yu-Ching; Chang, Chao-Yuan; Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.

  13. Comparative and Experimental Studies on the Genes Altered by Chronic Hypoxia in Human Brain Microendothelial Cells

    Directory of Open Access Journals (Sweden)

    Eugenia Mata-Greenwood

    2017-05-01

    Full Text Available Background : Hypoxia inducible factor 1 alpha (HIF1A is a master regulator of acute hypoxia; however, with chronic hypoxia, HIF1A levels return to the normoxic levels. Importantly, the genes that are involved in the cell survival and viability under chronic hypoxia are not known. Therefore, we tested the hypothesis that chronic hypoxia leads to the upregulation of a core group of genes with associated changes in the promoter DNA methylation that mediates the cell survival under hypoxia.Results : We examined the effect of chronic hypoxia (3 days; 0.5% oxygen on human brain micro endothelial cells (HBMEC viability and apoptosis. Hypoxia caused a significant reduction in cell viability and an increase in apoptosis. Next, we examined chronic hypoxia associated changes in transcriptome and genome-wide promoter methylation. The data obtained was compared with 16 other microarray studies on chronic hypoxia. Nine genes were altered in response to chronic hypoxia in all 17 studies. Interestingly, HIF1A was not altered with chronic hypoxia in any of the studies. Furthermore, we compared our data to three other studies that identified HIF-responsive genes by various approaches. Only two genes were found to be HIF dependent. We silenced each of these 9 genes using CRISPR/Cas9 system. Downregulation of EGLN3 significantly increased the cell death under chronic hypoxia, whereas downregulation of ERO1L, ENO2, adrenomedullin, and spag4 reduced the cell death under hypoxia.Conclusions : We provide a core group of genes that regulates cellular acclimatization under chronic hypoxic stress, and most of them are HIF independent.

  14. TMEM45A is essential for hypoxia-induced chemoresistance in breast and liver cancer cells

    International Nuclear Information System (INIS)

    Flamant, Lionel; Roegiers, Edith; Pierre, Michael; Hayez, Aurélie; Sterpin, Christiane; De Backer, Olivier; Arnould, Thierry; Poumay, Yves; Michiels, Carine

    2012-01-01

    Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance

  15. Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

    Science.gov (United States)

    Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

    2017-04-01

    To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

  16. A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

    DEFF Research Database (Denmark)

    Greenberger, Lee M; Horak, Ivan D; Filpula, David

    2008-01-01

    Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth facto...

  17. Long-term divergent tidal flat benthic community recovery following hypoxia-induced mortality

    NARCIS (Netherlands)

    Colen, van C.; Montserrat, F.; Vincx, M.; Herman, P.M.J.; Ysebaert, T.; Degraer, S.

    2010-01-01

    Macrobenthos recovery after hypoxia-induced mass mortality was assessed in an estuarine tidal mudflat during 3 years. During the first 2 years, a Pearson-Rosenberg type of community recovery took place along with the improving bottom water oxygen conditions. After 3 months, spionid polychaetes

  18. Antioxidant Mechanism of Rutin on Hypoxia-Induced Pulmonary Arterial Cell Proliferation

    Directory of Open Access Journals (Sweden)

    Qian Li

    2014-11-01

    Full Text Available Reactive oxygen species (ROS are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4 in pulmonary artery endothelial cells (PAECs. Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α. Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC, a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

  19. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

    NARCIS (Netherlands)

    Weijer, Ruud; Broekgaarden, Mans; Krekorian, Massis; Alles, Lindy K.; van Wijk, Albert C.; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C.; van Gulik, Thomas M.; Storm, Gert; Heger, Michal

    2016-01-01

    Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of

  20. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

    NARCIS (Netherlands)

    Weijer, R.; Broekgaarden, M.; Krekorian, M.; Alles, L.K.; van Wijk, A.C; Mackaaij, C.; Verheij, J.; van der Wal, A.C.; van Gullik, T.M.; Storm, Gerrit; Heger, M.

    2016-01-01

    Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression

  1. Antioxidant mechanism of Rutin on hypoxia-induced pulmonary arterial cell proliferation.

    Science.gov (United States)

    Li, Qian; Qiu, Yanli; Mao, Min; Lv, Jinying; Zhang, Lixin; Li, Shuzhen; Li, Xia; Zheng, Xiaodong

    2014-11-18

    Reactive oxygen species (ROS) are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC) proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4) in pulmonary artery endothelial cells (PAECs). Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α). Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC), a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

  2. Effect of hypoxia-inducible factor 1-alpha (HIF-1α) on proliferation ...

    African Journals Online (AJOL)

    Jane

    2011-07-25

    Jul 25, 2011 ... Full Length Research Paper. Effect of hypoxia-inducible factor 1-alpha ... 1Department of Neurosurgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025,. China. 2Department of Neurosurgery, Chaoyang Hospital, Huainan, Anhui, China. 3Department of Neurosurgery ...

  3. Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy.

    Science.gov (United States)

    Weijer, Ruud; Broekgaarden, Mans; Krekorian, Massis; Alles, Lindy K; van Wijk, Albert C; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C; van Gulik, Thomas M; Storm, Gert; Heger, Michal

    2016-01-19

    Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of HIF-1-associated proteins in human perihilar cholangiocarcinomas, (2) investigate the role of HIF-1 in PDT-treated human perihilar cholangiocarcinoma cells, and (3) determine whether HIF-1 inhibition reduces survival signaling and enhances PDT efficacy. Increased expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was confirmed in human perihilar cholangiocarcinomas. PDT with liposome-delivered zinc phthalocyanine caused HIF-1α stabilization in SK-ChA-1 cells and increased transcription of HIF-1α downstream genes. Acriflavine was taken up by SK-ChA-1 cells and translocated to the nucleus under hypoxic conditions. Importantly, pretreatment of SK-ChA-1 cells with acriflavine enhanced PDT efficacy via inhibition of HIF-1 and topoisomerases I and II. The expression of VEGF, CD105, CD31/Ki-67, and GLUT-1 was determined by immunohistochemistry in human perihilar cholangiocarcinomas. In addition, the response of human perihilar cholangiocarcinoma (SK-ChA-1) cells to PDT with liposome-delivered zinc phthalocyanine was investigated under both normoxic and hypoxic conditions. Acriflavine, a HIF-1α/HIF-1β dimerization inhibitor and a potential dual topoisomerase I/II inhibitor, was evaluated for its adjuvant effect on PDT efficacy. HIF-1, which is activated in human hilar cholangiocarcinomas, contributes to tumor cell survival following PDT in vitro. Combining PDT with acriflavine pretreatment improves PDT efficacy in cultured cells and therefore warrants further preclinical validation for therapy-recalcitrant perihilar cholangiocarcinomas.

  4. Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development.

    Science.gov (United States)

    Tsuji, Kenji; Kitamura, Shinji; Makino, Hirofumi

    2014-04-25

    The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly. In this study, we examined whether HIF regulates kidney development. We harvested kidneys from day 13 rat embryos (E13Ks) and cultured the organs under normoxic (20% O2/5% CO2) or hypoxic (5% O2/5% CO2) conditions. We evaluated the kidneys based on morphology and gene expression. E13Ks cultured under hypoxic conditions had significantly more ureteric bud (UB) branching than the E13Ks cultured under normoxic conditions. In addition, the mRNA levels of GDNF and GDNF receptor (GFR-α1), increased under hypoxic conditions in E13Ks. When we cultured E13Ks with the HIF-1α inhibitor digoxin or with siRNA targeting HIF-1α under hypoxic conditions, we did not observe increased UB branching. In addition, the expression of GDNF and GFR-α1 was inhibited under hypoxic conditions when the kidneys were treated with siRNA targeting HIF-1α. We also elucidated that hypoxia inhibited UB cell apoptosis and promoted the expression of FGF7 mRNA levels in metanephric mesenchymal (MM) cells in vitro. These findings suggest that hypoxic condition has important roles in inducing branching morphogenesis during kidney development. Hypoxia might mediate branching morphogenesis via not only GDNF/Ret but also FGF signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

  5. Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor.

    Science.gov (United States)

    Kwong, Raymond W M; Kumai, Yusuke; Tzaneva, Velislava; Azzi, Estelle; Hochhold, Nina; Robertson, Cayleih; Pelster, Bernd; Perry, Steve F

    2016-12-15

    The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P O 2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca 2+ levels and Ca 2+ uptake. Ca 2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca 2+ channel (ecac), but not plasma membrane Ca 2+ -ATPase (pmca2) or Na + /Ca 2+ -exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca 2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca 2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca 2+ balance during hypoxia, the results demonstrated that the reduction of Ca 2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca 2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression. © 2016. Published by The Company of Biologists Ltd.

  6. Chronic intermittent hypoxia induces cardiac inflammation and dysfunction in a rat obstructive sleep apnea model.

    Science.gov (United States)

    Wei, Qin; Bian, Yeping; Yu, Fuchao; Zhang, Qiang; Zhang, Guanghao; Li, Yang; Song, Songsong; Ren, Xiaomei; Tong, Jiayi

    2016-11-01

    Chronic intermittent hypoxia is considered to play an important role in cardiovascular pathogenesis during the development of obstructive sleep apnea (OSA). We used a well-described OSA rat model induced with simultaneous intermittent hypoxia. Male Sprague Dawley rats were individually placed into plexiglass chambers with air pressure and components were electronically controlled. The rats were exposed to intermittent hypoxia 8 hours daily for 5 weeks. The changes of cardiac structure and function were examined by ultrasound. The cardiac pathology, apoptosis, and fibrosis were analyzed by H&E staining, TUNNEL assay, and picosirius staining, respectively. The expression of inflammation and fibrosis marker genes was analyzed by quantitative real-time PCR and Western blot. Chronic intermittent hypoxia/low pressure resulted in significant increase of left ventricular internal diameters (LVIDs), end-systolic volume (ESV), end-diastolic volume (EDV), and blood lactate level and marked reduction in ejection fraction and fractional shortening. Chronic intermittent hypoxia increased TUNNEL-positive myocytes, disrupted normal arrangement of cardiac fibers, and increased Sirius stained collagen fibers. The expression levels of hypoxia induced factor (HIF)-1α, NF-kB, IL-6, and matrix metallopeptidase 2 (MMP-2) were significantly increased in the heart of rats exposed to chronic intermittent hypoxia. In conclusion, the left ventricular function was adversely affected by chronic intermittent hypoxia, which is associated with increased expression of HIF-1α and NF-kB signaling molecules and development of cardiac inflammation, apoptosis and fibrosis. © 2016 by the Journal of Biomedical Research. All rights reserved.

  7. Hypoxia-inducible factor 1 regulates heat and cold pain sensitivity and persistence.

    Science.gov (United States)

    Kanngiesser, Maike; Mair, Norbert; Lim, Hee-Young; Zschiebsch, Katja; Blees, Johanna; Häussler, Annett; Brüne, Bernhard; Ferreiròs, Nerea; Kress, Michaela; Tegeder, Irmgard

    2014-06-01

    The present study assessed the functions of the transcription factor hypoxia-inducible factor (HIF) in sensory neurons in models of acute, inflammatory, ischemic, and neuropathic pain. The alpha subunit, HIF1α, was specifically deleted in neurons of the dorsal root ganglia by mating HIF1α(fl/fl) mice with SNScre mice. SNS-HIF1α(-/-) mice were more sensitive to noxious heat and cold pain stimulation than were HIF1α(fl/fl) control mice. They also showed heightened first-phase nociceptive responses in the formalin and capsaicin tests with increased numbers of cFos-positive neurons in the dorsal horn, and intensified hyperalgesia in early phases after paw inflammation and hind limb ischemia/reperfusion. The behavioral cold and heat pain hypersensitivity was explained by increased calcium fluxes after transient receptor potential channel activation in primary sensory neurons of SNS-HIF1α(-/-) mice and lowered electrical activation thresholds of sensory fibers. SNS-HIF1α(-/-) mice however, developed less neuropathic pain after sciatic nerve injury, which was associated with an abrogation of HIF1-mediated gene up-regulation. The results suggest that HIF1α is protective in terms of acute heat and cold pain but in case of ongoing activation in injured neurons, it may promote the development of neuropathic pain. The duality of HIF1 in pain regulation may have an impact on the side effects of drugs targeting HIF1, which are being developed, for example, as anticancer agents. Specifically, in patients with cancer neuropathy, however, temporary HIF1 inhibition might provide a welcome combination of growth and pain reduction.

  8. Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

    International Nuclear Information System (INIS)

    Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

    2010-01-01

    Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas

  9. Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

    Directory of Open Access Journals (Sweden)

    Dirksen Uta

    2010-07-01

    Full Text Available Abstract Background Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor. Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. Methods HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. Results 17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Conclusions Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

  10. Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

    Science.gov (United States)

    Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

    2010-07-16

    Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

  11. Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

    Directory of Open Access Journals (Sweden)

    Jennifer Adamski

    Full Text Available Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1. In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target

  12. LncRNA TUG1 serves an important role in hypoxia-induced myocardial cell injury by regulating the miR-145-5p-Binp3 axis

    Science.gov (United States)

    Wu, Zhongwei; Zhao, Shengji; Li, Chunfu; Liu, Chaoquan

    2018-01-01

    The aim of the present study was to investigate the function of long non-coding RNA TUG1 in hypoxia-induced myocardial cell injury and to explore the potential molecular mechanisms. The cardiomyocyte cell line H9c2 was cultured under hypoxic and normoxic conditions. TUG1 expression under hypoxic conditions was then detected. The effects of TUG1 overexpression on viability, apoptosis, migration and invasion were assayed. In addition, the microRNA (miR)-145-5p expression was detected. Following H9c2 cell transfection with miR-145-5p mimics, the H9c2 cell viability, apoptosis, migration and invasion were also detected. Additionally, the target gene of miR-145-5p was assayed by Luciferase reporter assay. The protein expressions of Wnt-3a, Wnt5a, and β-catenin in H9c2 cells under hypoxic conditions were also determined. The results revealed that hypoxia induced injury in H9c2 cells, including inhibiting cell viability, migration and invasion, and promoting cell apoptosis. Overexpression of TUG1 aggravated hypoxia-induced injury in H9c2 cells. In addition, miR-145-5p was negatively regulated by TUG1, and TUG1 overexpression aggravated hypoxia-induced injury via the downregulation of miR-145-5p. Furthermore, B-cell lymphoma 2 interacting protein 3 (Bnip3) was a target of miR-145-5p, and overexpression of Bnip3 aggravated hypoxia-induced cell injury by activating Wnt/β-catenin signaling pathways in H9c2 cells. In conclusion, overexpression of TUG1 aggravated hypoxia-induced injury in cardiomyocytes by regulating the miR-145-5p-Binp3 axis. Activation of the Wnt/β-catenin signaling pathway may be a key mechanism to mediate the role of TUG1 in regulating hypoxia-induced myocardial injury. TUG1 may be an effective diagnostic marker and therapeutic target for myocardial ischemia. PMID:29207102

  13. Paeoniflorin prevents hypoxia-induced epithelial–mesenchymal transition in human breast cancer cells

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    Zhou Z

    2016-04-01

    Full Text Available Zhenyu Zhou,1,* Shunchang Wang,1,* Caijuan Song,2 Zhuang Hu11Department of Thyroid and Breast, Huaihe Hospital, Henan University, Kaifeng, 2Department of Immunization Program, Zhengzhou Center for Disease Control and Prevention, Zhengzhou, People’s Republic of China*These authors contributed equally to this workAbstract: Paeoniflorin (PF is a monoterpene glycoside extracted from the root of Paeonia lactiflora Pall. Previous studies have demonstrated that PF inhibits the growth, invasion, and metastasis of tumors in vivo and in vitro. However, the effect of PF on hypoxia-induced epithelial–mesenchymal transition (EMT in breast cancer cells remains unknown. Therefore, the objective of this study was to investigate the effect of PF on hypoxia-induced EMT in breast cancer cells, as well as characterize the underlying mechanism. The results presented in this study demonstrate that PF blocks the migration and invasion of breast cancer cells by repressing EMT under hypoxic conditions. PF also significantly attenuated the hypoxia-induced increase in HIF-1α level. Furthermore, PF prevented hypoxia-induced expression of phosphorylated PI3K and Akt in MDA-MB-231 cells. In conclusion, PF prevented hypoxia-induced EMT in breast cancer cells by inhibiting HIF-1α expression via modulation of PI3K/Akt signaling pathway. This finding provides evidence that PF can serve as a therapeutic agent for the treatment of breast cancer.Keywords: paeoniflorin, breast cancer, hypoxia, epithelial–mesenchymal transition, PI3K/Akt signaling pathway

  14. Overexpression of Hypoxia-Inducible Factor-1α Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

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    Pei Wang

    2013-09-01

    Full Text Available Background/Aims: The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells. Methods: The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER. The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1, zonular occludens-1 (ZO-1, occludin, and myosin light chain kinase (MLCK in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR. Results: Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression. Conclusion: We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.

  15. Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogenesis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts.

    Science.gov (United States)

    Rapisarda, Annamaria; Zalek, Jessica; Hollingshead, Melinda; Braunschweig, Till; Uranchimeg, Badarch; Bonomi, Carrie A; Borgel, Suzanne D; Carter, John P; Hewitt, Stephen M; Shoemaker, Robert H; Melillo, Giovanni

    2004-10-01

    We have previously shown that topotecan, a topoisomerase I poison, inhibits hypoxia-inducible factor (HIF)-1alpha protein accumulation by a DNA damage-independent mechanism. Here, we report that daily administration of topotecan inhibits HIF-1alpha protein expression in U251-HRE glioblastoma xenografts. Concomitant with HIF-1alpha inhibition, topotecan caused a significant tumor growth inhibition associated with a marked decrease of angiogenesis and expression of HIF-1 target genes in tumor tissue. These results provide a compelling rationale for testing topotecan in clinical trials to target HIF-1 in cancer patients.

  16. Hypoxia inducible factor 3α plays a critical role in alveolarization and distal epithelial cell differentiation during mouse lung development.

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    Yadi Huang

    Full Text Available Lung development occurs under relative hypoxia and the most important oxygen-sensitive response pathway is driven by Hypoxia Inducible Factors (HIF. HIFs are heterodimeric transcription factors of an oxygen-sensitive subunit, HIFα, and a constitutively expressed subunit, HIF1β. HIF1α and HIF2α, encoded by two separate genes, contribute to the activation of hypoxia inducible genes. A third HIFα gene, HIF3α, is subject to alternative promoter usage and splicing, leading to three major isoforms, HIF3α, NEPAS and IPAS. HIF3α gene products add to the complexity of the hypoxia response as they function as dominant negative inhibitors (IPAS or weak transcriptional activators (HIF3α/NEPAS. Previously, we and others have shown the importance of the Hif1α and Hif2α factors in lung development, and here we investigated the role of Hif3α during pulmonary development. Therefore, HIF3α was conditionally expressed in airway epithelial cells during gestation and although HIF3α transgenic mice were born alive and appeared normal, their lungs showed clear abnormalities, including a post-pseudoglandular branching defect and a decreased number of alveoli. The HIF3α expressing lungs displayed reduced numbers of Clara cells, alveolar epithelial type I and type II cells. As a result of HIF3α expression, the level of Hif2α was reduced, but that of Hif1α was not affected. Two regulatory genes, Rarβ, involved in alveologenesis, and Foxp2, a transcriptional repressor of the Clara cell specific Ccsp gene, were significantly upregulated in the HIF3α expressing lungs. In addition, aberrant basal cells were observed distally as determined by the expression of Sox2 and p63. We show that Hif3α binds a conserved HRE site in the Sox2 promoter and weakly transactivated a reporter construct containing the Sox2 promoter region. Moreover, Hif3α affected the expression of genes not typically involved in the hypoxia response, providing evidence for a novel

  17. MiR-142 modulates human pancreatic cancer proliferation and invasion by targeting hypoxia-inducible factor 1 (HIF-1α in the tumor microenvironments

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    Yebin Lu

    2017-02-01

    Full Text Available MicroRNAs regulate most protein-coding genes, including genes important in cancer and other diseases. In this study, we demonstrated that the expression of miR-142 could be significantly suppressed in pancreatic cancer specimens and cell lines compared to their adjacent tissues and normal pancreatic cells. Growth and invasion of PANC-1 and SW1990 cells were attenuated by overexpression of miR-142 in vitro. With the help of bioinformatics analysis, hypoxia-inducible factor 1 (HIF-1α was identified to be a direct target of miR-142, and a luciferase reporter experiment confirmed this discovery. Overexpression of miR-142 decreases protein expression of HIF-1α. In the hypoxic microenvironment, HIF-1α was up-regulated while miR-142 was down-regulated. The invaded cells significantly increased in the hypoxic microenvironment compared to the normoxic microenvironment. The hypoxia treatment induced cells’ proliferation, and invasion could be inhibited by miR-142 overexpression or HIF-1α inhibition. Moreover, expression of epithelial-mesenchymal transition (EMT markers, Vimentin, VEGF-C and E-cad, was altered under hypoxia conditions and regulated by miR-142/HIF-1α. Above all, these findings provided insights on the functional mechanism of miR-142, suggesting that the miR-142/HIF-1α axis may interfere with the proliferative and invasive properties of pancreatic cancer cells, and indicated that miR-142 could be a potential therapeutic target for pancreatic cancer.

  18. Impact of interleukin-6 on hypoxia-induced pulmonary hypertension and lung inflammation in mice

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    Izziki Mohamed

    2009-01-01

    Full Text Available Abstract Background Inflammation may contribute to the pathogenesis of various forms of pulmonary hypertension (PH. Recent studies in patients with idiopathic PH or PH associated with underlying diseases suggest a role for interleukin-6 (IL-6. Methods To determine whether endogenous IL-6 contributes to mediate hypoxic PH and lung inflammation, we studied IL-6-deficient (IL-6-/- and wild-type (IL-6+/+ mice exposed to hypoxia for 2 weeks. Results Right ventricular systolic pressure, right ventricle hypertrophy, and the number and media thickness of muscular pulmonary vessels were decreased in IL-6-/- mice compared to wild-type controls after 2 weeks' hypoxia, although the pressure response to acute hypoxia was similar in IL-6+/+ and IL-6-/- mice. Hypoxia exposure of IL-6+/+ mice led to marked increases in IL-6 mRNA and protein levels within the first week, with positive IL-6 immunostaining in the pulmonary vessel walls. Lung IL-6 receptor and gp 130 (the IL-6 signal transducer mRNA levels increased after 1 and 2 weeks' hypoxia. In vitro studies of cultured human pulmonary-artery smooth-muscle-cells (PA-SMCs and microvascular endothelial cells revealed prominent synthesis of IL-6 by PA-SMCs, with further stimulation by hypoxia. IL-6 also markedly stimulated PA-SMC migration without affecting proliferation. Hypoxic IL-6-/- mice showed less inflammatory cell recruitment in the lungs, compared to hypoxic wild-type mice, as assessed by lung protein levels and immunostaining for the specific macrophage marker F4/80, with no difference in lung expression of adhesion molecules or cytokines. Conclusion These data suggest that IL-6 may be actively involved in hypoxia-induced lung inflammation and pulmonary vascular remodeling in mice.

  19. Late gestational intermittent hypoxia induces metabolic and epigenetic changes in male adult offspring mice.

    Science.gov (United States)

    Khalyfa, Abdelnaby; Cortese, Rene; Qiao, Zhuanhong; Ye, Honggang; Bao, Riyue; Andrade, Jorge; Gozal, David

    2017-04-15

    Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia, a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis and metabolic function in offspring. Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue. Fetal perturbations by OSA during pregnancy impose long-term detrimental effects manifesting as metabolic dysfunction in adult male offspring. Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG-IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time-pregnant female C57BL/6 mice were exposed to LG-IH or room air (LG-RA) during days 13-18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP-chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG-IH male but not female offspring. LG-IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro-inflammatory macrophages emerged in VWAT along with 1520 DMRs (P < 0.0001), associated with 693 genes

  20. Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

    Directory of Open Access Journals (Sweden)

    Min Yu

    Full Text Available Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

  1. Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension

    International Nuclear Information System (INIS)

    Satwiko, Muhammad Gahan; Ikeda, Koji; Nakayama, Kazuhiko; Yagi, Keiko; Hocher, Berthold; Hirata, Ken-ichi; Emoto, Noriaki

    2015-01-01

    Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH. - Highlights: • Role of endothelin-1 in pulmonary arterial hypertension (PAH) was investigated. • The endothelin-1 transgenic (ETTG) and wild type (WT) mice were analyzed. • ETTG mice spontaneously developed PAH under normoxia conditions. • SU5416 further aggravated PAH in ETTG mice. • Enhanced endothelin system activity could be a causative factor in

  2. Targeted activation of endothelin-1 exacerbates hypoxia-induced pulmonary hypertension

    Energy Technology Data Exchange (ETDEWEB)

    Satwiko, Muhammad Gahan [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Ikeda, Koji [Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Nakayama, Kazuhiko [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Yagi, Keiko [Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan); Hocher, Berthold [Institute for Nutritional Science, University of Potsdam, Potsdam (Germany); Hirata, Ken-ichi [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Emoto, Noriaki, E-mail: emoto@med.kobe-u.ac.jp [Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe (Japan); Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe (Japan)

    2015-09-25

    Pulmonary arterial hypertension (PAH) is a fatal disease that eventually results in right heart failure and death. Current pharmacologic therapies for PAH are limited, and there are no drugs that could completely cure PAH. Enhanced activity of endothelin system has been implicated in PAH severity and endothelin receptor antagonists have been used clinically to treat PAH. However, there is limited experimental evidence on the direct role of enhanced endothelin system activity in PAH. Here, we investigated the correlation between endothelin-1 (ET-1) and PAH using ET-1 transgenic (ETTG) mice. Exposure to chronic hypoxia increased right ventricular pressure and pulmonary arterial wall thickness in ETTG mice compared to those in wild type mice. Of note, ETTG mice exhibited modest but significant increase in right ventricular pressure and vessel wall thickness relative to wild type mice even under normoxic conditions. To induce severe PAH, we administered SU5416, a vascular endothelial growth factor receptor inhibitor, combined with exposure to chronic hypoxia. Treatment with SU5416 modestly aggravated hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial vessel wall thickening in ETTG mice in association with increased interleukin-6 expression in blood vessels. However, there was no sign of obliterative endothelial cell proliferation and plexiform lesion formation in the lungs. These results demonstrated that enhanced endothelin system activity could be a causative factor in the development of PAH and provided rationale for the inhibition of endothelin system to treat PAH. - Highlights: • Role of endothelin-1 in pulmonary arterial hypertension (PAH) was investigated. • The endothelin-1 transgenic (ETTG) and wild type (WT) mice were analyzed. • ETTG mice spontaneously developed PAH under normoxia conditions. • SU5416 further aggravated PAH in ETTG mice. • Enhanced endothelin system activity could be a causative factor in

  3. Effect of Hypoxia-Inducible Factor 1α on Early Healing in Extraction Sockets

    Directory of Open Access Journals (Sweden)

    Hyun-Chang Lim

    2018-01-01

    Full Text Available The aim of the present study was to investigate the effect of hypoxia-inducible factor 1α (HIF1A on the early healing (4 weeks of extraction sockets exhibiting partial loss of the labial bone. Two extraction sockets of the maxillary incisors from each of six dogs were assigned to two treatment modalities: deproteinized bovine bone mineral (i with 10% collagen (DBBM-C soaked with HIF1A and covered by a collagen membrane (CM (HIF group or (ii treated with DBBM-C only and covered by a CM (control group. Microcomputed tomography revealed some degree of collapse of the labial contour. The totally augmented volume and new bone volume did not differ significantly between two groups (P>0.05. The histological analysis revealed that the apical area of the socket was mostly filled with newly formed bone, while there was less newly formed bone in the coronal area and incomplete cortex formation. The histomorphometric analysis revealed that the area of newly formed bone was significantly larger in the HIF group than the control group (12.16±3.04 versus 9.48±2.01 mm2, P<0.05, while there was no significant intergroup difference in the total augmented area. In conclusion, even though DBBM-C soaked with HIF1A enhanced histomorphometric bone formation, this intervention did not demonstrate superiority in preventing ridge shrinkage compared to DBBM-C alone. Clinical relevance of these findings should be further studied.

  4. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression.

    Science.gov (United States)

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-08-06

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

  5. Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

    International Nuclear Information System (INIS)

    Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

    2009-01-01

    Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed

  6. Effects of YC-1 on hypoxia-inducible factor 1 alpha in hypoxic human bladder transitional carcinoma cell line T24 cells.

    Science.gov (United States)

    Li, Yangle; Zhao, Xiaokun; Tang, Huiting; Zhong, Zhaohui; Zhang, Lei; Xu, Ran; Li, Songchao; Wang, Yi

    2012-01-01

    It was the aim of this study to explore the effects of 3-(5'-hydroxymethyl-2'-furyl)-l-benzyl indazole (YC-1) on transcription activity, cell proliferation and apoptosis of hypoxic human bladder transitional carcinoma cells (BTCC), mediated by hypoxia-inducible factor 1α (HIF-1α). BTCC cell line T24 cells were incubated under normoxic or hypoxic conditions, adding different doses of YC-1. The protein expression of HIF-1α and HIF-1α-mediated genes was detected by Western blotting. RT-PCR was used to detect HIF-1α mRNA expression. Cell proliferation, apoptosis and migration activity were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry and transwell migration assay. The cells were pretreated by two ERK/p38 MAPK pathway-specific inhibitors, PD98059 or SB203580, and then incubated with YC-1 treatment under hypoxic condition. HIF-1α protein expression was detected by Western blotting. Hypoxic T24 cells expressed a higher level of HIF-1α, vascular endothelial growth factor, matrix metalloproteinases-2, B-cell lymphoma/leukemia-2 protein and HIF-1α mRNA compared with normoxic controls, in which the above-mentioned expression was downregulated by YC-1 in a dose-dependent manner. Cell proliferation and migration activity were inhibited while apoptosis was induced by YC-1 under hypoxic condition. Moreover, YC-1-downregulated HIF-1α expression was reversed by PD98059 and SB203580, respectively. YC-1 inhibits HIF-1α and HIF-1α-mediated gene expression, cell proliferation and migration activity and induces apoptosis in hypoxic BTCC. The ERK/p38 MAPK pathway may be involved in YC-1-mediated inhibition of HIF-1α. Copyright © 2011 S. Karger AG, Basel.

  7. The effects of doxycycline and micronized purified flavonoid fraction on human vein wall remodeling are not hypoxia-inducible factor pathway-dependent.

    Science.gov (United States)

    Lim, Chung Sim; Kiriakidis, Serafim; Paleolog, Ewa M; Davies, Alun H

    2012-10-01

    Doxycycline and micronized purified flavonoid fraction (MPFF) modulate vein wall remodeling that may be associated with hypoxia in varicose veins (VVs), vein graft stenosis, and deep venous thrombosis. We recently reported that in vitro exposure of non-VV (NVVs) and VVs to hypoxic conditions activates the hypoxia-inducible factor (HIF) pathway. This study investigated the in vitro effects of doxycycline and MPFF on the HIF pathway in hypoxic NVVs and VVs. Six NVVs and six VVs obtained from surgery were used to prepare vein organ cultures, which were exposed to hypoxia (1% O(2)), with and without MPFF (10(-5) mol/L) or doxycycline (5 μg/mL) for 16 hours. The veins were analyzed for HIF-1α, HIF-2α, and their target gene expression, with real-time polymerase chain reaction and Western blot. The differences between gene expressions were tested with one-way analysis of variance with repeated measures, followed by the Dunnett test for multiple comparisons. P factor, B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3, prolyl hydroxylase domain-2, and prolyl hydroxylase domain-3, was not significantly altered in NVVs and VVs exposed to hypoxia and treated with doxycycline or MPFF compared with those untreated. Doxycycline and MPFF at a concentration corresponding to a therapeutic dose do not alter the activation of the HIF pathway in NVV and VV organ cultures exposed to hypoxia. Our findings suggest vein wall remodeling actions in NVVs and VVs are likely not HIF-dependent. Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

  8. Multi-Vitamin B Supplementation Reverses Hypoxia-Induced Tau Hyperphosphorylation and Improves Memory Function in Adult Mice.

    Science.gov (United States)

    Yu, Lixia; Chen, Yuan; Wang, Weiguang; Xiao, Zhonghai; Hong, Yan

    2016-08-04

    Hypobaric hypoxia (HH) leads to reduced oxygen delivery to brain. It could trigger cognitive dysfunction and increase the risk of dementia including Alzheimer's disease (AD). The present study was undertaken in order to examine whether B vitamins (B6, B12, folate, and choline) could exert protective effects on hypoxia-induced memory deficit and AD related molecular events in mice. Adult male Kunming mice were assigned to five groups: normoxic control, hypoxic model (HH), hypoxia+vitamin B6/B12/folate (HB), hypoxia+choline (HC), hypoxia+vitamin B6/B12/folate+choline (HBC). Mice in the hypoxia, HB, HC, and HBC groups were exposed to hypobaric hypoxia for 8 h/day for 28 days in a decompression chamber mimicking 5500 meters of high altitude. Spatial and passive memories were assessed by radial arm and step-through passive test, respectively. Levels of tau and glycogen synthase kinase (GSK)-3β phosphorylation were detected by western blot. Homocysteine (Hcy) concentrations were determined using enzymatic cycling assay. Mice in the HH group exhibited significant spatial working and passive memory impairment, increased tau phosphorylation at Thr181, Ser262, Ser202/Thr205, and Ser396 in the cortex and hippocampus, and elevated Hcy levels compared with controls. Concomitantly, the levels of Ser9-phosphorylated GSK-3β were significantly decreased in brain after hypoxic treatment. Supplementations of vitamin B6/B12/folate+choline could significantly ameliorate the hypoxia-induced memory deficits, observably decreased Hcy concentrations in serum, and markedly attenuated tau hyperphosphorylation at multiple AD-related sites through upregulating inhibitory Ser9-phosphorylated GSK-3β. Our finding give further insight into combined neuroprotective effects of vitamin B6, B12, folate, and choline on brain against hypoxia.

  9. Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

    Directory of Open Access Journals (Sweden)

    Kim Chan

    2007-10-01

    Full Text Available Abstract Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5. RGC-5 cells were cultured in a closed hypoxic chamber (5% O2 with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38 and nuclear factor-kappa B (NF-κB were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF, a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia.

  10. Agmatine protects retinal ganglion cells from hypoxia-induced apoptosis in transformed rat retinal ganglion cell line

    Science.gov (United States)

    Hong, Samin; Lee, Jong Eun; Kim, Chan Yun; Seong, Gong Je

    2007-01-01

    Background Agmatine is an endogenous polyamine formed by the decarboxylation of L-arginine. We investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells (RGC-5). RGC-5 cells were cultured in a closed hypoxic chamber (5% O2) with or without agmatine. Cell viability was determined by lactate dehydrogenase (LDH) assay and apoptosis was examined by annexin V and caspase-3 assays. Expression and phosphorylation of mitogen-activated protein kinases (MAPKs; JNK, ERK p44/42, and p38) and nuclear factor-kappa B (NF-κB) were investigated by Western immunoblot analysis. The effects of agmatine were compared to those of brain-derived neurotrophic factor (BDNF), a well-known protective neurotrophin for retinal ganglion cells. Results After 48 hours of hypoxic culture, the LDH assay showed 52.3% cell loss, which was reduced to 25.6% and 30.1% when agmatine and BDNF were administered, respectively. This observed cell loss was due to apoptotic cell death, as established by annexin V and caspase-3 assays. Although total expression of MAPKs and NF-κB was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. Agmatine reduced phosphorylation of JNK and NF-κB, while BDNF suppressed phosphorylation of ERK and p38. Conclusion Our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in RGC-5 cells and that its effects may act through the JNK and NF-κB signaling pathways. Our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia. PMID:17908330

  11. Role of hypoxia-inducible factor-α in hepatitis-B-virus X protein-mediated MDR1 activation

    International Nuclear Information System (INIS)

    Han, Hyo-Kyung; Han, Chang Yeob; Cheon, Eun-Pa; Lee, Jaewon; Kang, Keon Wook

    2007-01-01

    The transition from chemotherapy-responsive cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multi-drug resistance 1 (MDR1). We found that hepatitis-B-virus X protein (HBx) increases the transcriptional activity and protein level of MDR1 in a hepatoma cell line, H4IIE. In addition, HBx overexpression made H4IIE cells more resistant to verapamil-uptake. HBx stabilized hypoxia-inducible factor-1α (HIF-1α) and induced the nuclear translocation of C/EBPβ. Reporter gene analyses showed that HBx increased the reporter activity in the cells transfected with the reporter containing MDR1 gene promoter. Moreover, the luciferase reporter gene activity was significantly inhibited by HIF-1α siRNA but not by overexpression of C/EBP dominant negative mutant. These results imply that HBx increases the MDR1 transporter activity through the transcriptional activation of the MDR1 gene with HIF-1α activation, and suggest HIF-1α for the therapeutic target of HBV-mediated chemoresistance

  12. Induction of gastrin expression in gastrointestinal cells by hypoxia or cobalt is independent of hypoxia-inducible factor (HIF).

    Science.gov (United States)

    Xiao, Lin; Kovac, Suzana; Chang, Mike; Shulkes, Arthur; Baldwin, Graham S; Patel, Oneel

    2012-07-01

    Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

  13. Overexpression of hypoxia-inducible factor prolyl- hydoxylase ...

    African Journals Online (AJOL)

    Jane

    2011-08-08

    Aug 8, 2011 ... which is regulated by HIF prolyl-dydoxylase -mediated degradation. Taken together, our results ..... Chromatin immunoprecipitation analysis of gene ... phosphatidylinositol 3-kinase/Akt pathway. Endocrinology, 148(5):.

  14. Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway.

    Science.gov (United States)

    Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng; Qiao, Yong

    2016-01-01

    Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury. © 2015 by the Society for Experimental Biology and Medicine.

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

    Science.gov (United States)

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

    2016-01-08

    We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (-3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5'-deletions from -3040 to -1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides -2715 to -2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Hypoxia-inducible factor-2α-dependent hypoxic induction of Wnt10b expression in adipogenic cells.

    Science.gov (United States)

    Park, Young-Kwon; Park, Bongju; Lee, Seongyeol; Choi, Kang; Moon, Yunwon; Park, Hyunsung

    2013-09-06

    Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

  17. Hypoxic Stress Upregulates the Expression of Slc38a1 in Brown Adipocytes via Hypoxia-Inducible Factor-1α.

    Science.gov (United States)

    Horie, Tetsuhiro; Fukasawa, Kazuya; Iezaki, Takashi; Park, Gyujin; Onishi, Yuki; Ozaki, Kakeru; Kanayama, Takashi; Hiraiwa, Manami; Kitaguchi, Yuka; Kaneda, Katsuyuki; Hinoi, Eiichi

    2018-01-01

    The availability of amino acid in the brown adipose tissue (BAT) has been shown to be altered under various conditions; however, little is known about the possible expression and pivotal role of amino acid transporters in BAT under physiological and pathological conditions. The present study comprehensively investigated whether amino acid transporters are regulated by obesogenic conditions in BAT in vivo. Moreover, we investigated the mechanism underlying the regulation of the expression of amino acid transporters by various stressors in brown adipocytes in vitro. The expression of solute carrier family 38 member 1 (Slc38a1; gene encoding sodium-coupled neutral amino acid transporter 1) was preferentially upregulated in the BAT of both genetic and acquired obesity mice in vivo. Moreover, the expression of Slc38a1 was induced by hypoxic stress through hypoxia-inducible factor-1α, which is a master transcription factor of the adaptive response to hypoxic stress, in brown adipocytes in vitro. These results indicate that Slc38a1 is an obesity-associated gene in BAT and a hypoxia-responsive gene in brown adipocytes. © 2017 S. Karger AG, Basel.

  18. Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of); Park, Jong-Wan, E-mail: parkjw@snu.ac.kr [Department of Pharmacology, Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 110-799 (Korea, Republic of)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HIF-1{alpha} is expressed PRMT5-dependently in hypoxic cancer cells. Black-Right-Pointing-Pointer The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. Black-Right-Pointing-Pointer The de novo synthesis of HIF-1{alpha} depends on PRMT5. Black-Right-Pointing-Pointer PRMT5 is involved in the HIF-1{alpha} translation initiated by 5 Prime UTR of HIF-1{alpha} mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1-8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1{alpha} in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1{alpha} protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1{alpha} transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1{alpha} translation initiated by the 5 Prime UTR of HIF-1{alpha} mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  19. Protein arginine methyltransferase 5 is an essential component of the hypoxia-inducible factor 1 signaling pathway

    International Nuclear Information System (INIS)

    Lim, Ji-Hong; Choi, Yong-Joon; Cho, Chung-Hyun; Park, Jong-Wan

    2012-01-01

    Highlights: ► HIF-1α is expressed PRMT5-dependently in hypoxic cancer cells. ► The HIF-1 regulation of hypoxia-induced genes is attenuated in PRMT5-knocked-down cells. ► The de novo synthesis of HIF-1α depends on PRMT5. ► PRMT5 is involved in the HIF-1α translation initiated by 5′ UTR of HIF-1α mRNA. -- Abstract: Protein arginine methyltransferase 5 (PRMT5) is an enzyme that transfers one or two methyl groups to the arginine residues of histones or non-histone proteins, and that plays critical roles in cellular processes as diverse as receptor signaling and gene expression. Furthermore, PRMT5 is highly expressed in tumors, where it may be associated with tumor growth. Although much research has been conducted on PRMT5, little is known regarding its role in adaption to hypoxia. As hypoxia-inducible factor 1 (HIF-1) is a key player in hypoxic response, we examined the possible involvement of PRMT5 in the HIF-1 signaling pathway. Of the siRNAs targeting PRMT1–8, only PRMT5 siRNA attenuated the hypoxic induction of HIF-1α in A549 cells, and this result was reproducible in all three cancer cell lines examined. PRMT5 knock-down also repressed the promoter activities and the transcript levels of HIF-1-governed genes. Mechanistically, de novo synthesis of HIF-1α protein was reduced in PRMT5-knocked-down A549 cells, and this was rescued by PRMT5 restoration. In contrast, HIF-1α transcription, RNA processing, and protein stability were unaffected by PRMT5 knock-down. Furthermore, PRMT5 was found to be essential for the HIF-1α translation initiated by the 5′ UTR of HIF-1α mRNA. Given our results and previous reports, we believe that PRMT5 probably promotes tumor growth by stimulating cell proliferation and by participating in the construction of a tumor-favorable microenvironment via HIF-1 activation.

  20. Hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium

    Science.gov (United States)

    Polosukhin, Vasiliy V; Cates, Justin M; Lawson, William E; Milstone, Aaron P; Matafonov, Anton G; Massion, Pierre P; Lee, Jae Woo; Randell, Scott H; Blackwell, Timothy S

    2018-01-01

    Goblet cell hyperplasia is a common feature of chronic obstructive pulmonary disease (COPD) airways, but the mechanisms that underlie this epithelial remodelling in COPD are not understood. Based on our previous finding of hypoxia-inducible factor-1α (HIF-1α) nuclear localization in large airways from patients with COPD, we investigated whether hypoxia-inducible signalling could influence the development of goblet cell hyperplasia. We evaluated large airway samples obtained from 18 lifelong non-smokers and 13 former smokers without COPD, and 45 former smokers with COPD. In these specimens, HIF-1α nuclear staining occurred almost exclusively in COPD patients in areas of airway remodelling. In COPD patients, 93.2 ± 3.9% (range 65 – 100%) of goblet cells were HIF-1α positive in areas of goblet cell hyperplasia, whereas nuclear HIF-1α was not detected in individuals without COPD or in normal-appearing pseudostratified epithelium from COPD patients. To determine the direct effects of hypoxia-inducible signalling on epithelial cell differentiation in vitro, human bronchial epithelial cells (HBECs) were grown in air-liquid interface cultures under hypoxia (1% O2) or following treatment with a selective HIF-1α stabilizer, (2R)-[(4-biphenylylsulphonyl)amino]-N-hydroxy-3-phenyl-propionamide (BiPS). HBECs grown in hypoxia or with BiPS treatment were characterized by HIF-1α activation, carbonic anhydrase IX expression, mucus-producing cell hyperplasia and increased expression of MUC5AC. Analysis of signal transduction pathways in cells with HIF-1α activation showed increased ERK1/2 phosphorylation without activation of epidermal growth factor receptor, Ras, PI3K-Akt or STAT6. These data indicate an important effect of hypoxia-inducible signalling on airway epithelial cell differentiation and identify a new potential target to limit mucus production in COPD. PMID:21557221

  1. Decay Accelerating Factor (CD55) Protects Neuronal Cells from Chemical Hypoxia-Induced Injury

    Science.gov (United States)

    2010-04-09

    Pavlakovic G, Isom GE: Dopaminergic neurotoxicity of cyanide: neurochemical, histological and behavioral characterization. Toxicol Appl Pharmacol...provided the original work is properly cited. ResearchDecay accelerating factor (CD55) protects neuronal cells from chemical hypoxia-induced injury...deposition of C3a/C5a and membrane attack complex (MAC or C5b-9) production. The present study investigates the ability of DAF to protect primary cultured

  2. A New Hypoxia Inducible Factor-2 Inhibitory Pyrrolinone Alkaloid from Roots and Stems of Piper sarmentosum

    OpenAIRE

    BOKESCH, Heidi Rose; GARDELLA, Roberta Scott; RABE, Daniel Christopher; BOTTARO, Donald Paul; LINEHAN, William Marston; MCMAHON, James Brislin; MCKEE, Tawnya Carlene

    2011-01-01

    A new trimethoxycinnamoyl-2-pyrrolinone alkaloid, langkamide (1), along with the known compounds piplartine (2) and 3,4,5-trimethoxycinnamic acid (3) were isolated from the roots and stems of the shrub Piper sarmentosum Roxb. The structures were established by spectroscopic analyses and comparison of their spectral data with values reported in the literature. The compounds were tested for their ability to modulate hypoxia inducible factor-2 (HIF-2) transcription activity and all three showed ...

  3. PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells.

    Science.gov (United States)

    Gozal, Evelyne; Metz, Cynthia J; Dematteis, Maurice; Sachleben, Leroy R; Schurr, Avital; Rane, Madhavi J

    2017-09-05

    Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Stimulating retinal blood vessel protection with hypoxia-inducible factor stabilization: identification of novel small-molecule hydrazones to inhibit hypoxia-inducible factor prolyl hydroxylase (an American Ophthalmological Society thesis).

    Science.gov (United States)

    Sears, Jonathan E; Hoppe, George

    2013-09-01

    To discover novel small molecules that inhibit hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD), a key enzyme that regulates the posttranslational stability and hence activity of HIF. NIH3T3 cell line stably transfected with firefly luciferase under a HIF-1-inducible promoter was used to screen a Chembridge library of 34,000 small molecules of molecular weight 250 to 550 Da. Positive hits were considered at 4.5-fold higher luminescence than control. Selected compounds were validated in vitro. The most effective dose was then used to treat mice expressing firefly luciferase fused to the oxygen-dependent degradation domain (lucODD) in order to determine the location of the receptor for systemic treatment with small-molecule HIF PHD inhibitors. Twenty-three novel small molecules were discovered, the majority of which were hydrazones and hydrazines. Of the 23 compounds, each had different selectivity for expression of erythropoietin or vascular endothelial growth factor, two angiogenic, HIF-regulated gene products. In addition, each showed different selectivity for hepatocytes or kidney, or both or neither, when injected intraperitoneally in an in vivo reporter gene assay. The discovery of multiple small molecules that inhibit HIF PHD identifies new reagents to develop strategies to prevent the degradation of HIF by its selective PHD. These molecules are novel hypoxia mimetics that may provide new strategies to protect retinovasculature from hyperoxia.

  5. Genome-Wide Comparative Gene Family Classification

    Science.gov (United States)

    Frech, Christian; Chen, Nansheng

    2010-01-01

    Correct classification of genes into gene families is important for understanding gene function and evolution. Although gene families of many species have been resolved both computationally and experimentally with high accuracy, gene family classification in most newly sequenced genomes has not been done with the same high standard. This project has been designed to develop a strategy to effectively and accurately classify gene families across genomes. We first examine and compare the performance of computer programs developed for automated gene family classification. We demonstrate that some programs, including the hierarchical average-linkage clustering algorithm MC-UPGMA and the popular Markov clustering algorithm TRIBE-MCL, can reconstruct manual curation of gene families accurately. However, their performance is highly sensitive to parameter setting, i.e. different gene families require different program parameters for correct resolution. To circumvent the problem of parameterization, we have developed a comparative strategy for gene family classification. This strategy takes advantage of existing curated gene families of reference species to find suitable parameters for classifying genes in related genomes. To demonstrate the effectiveness of this novel strategy, we use TRIBE-MCL to classify chemosensory and ABC transporter gene families in C. elegans and its four sister species. We conclude that fully automated programs can establish biologically accurate gene families if parameterized accordingly. Comparative gene family classification finds optimal parameters automatically, thus allowing rapid insights into gene families of newly sequenced species. PMID:20976221

  6. Increased tumour ascorbate is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in human colorectal cancer

    Directory of Open Access Journals (Sweden)

    Caroline eKuiper

    2014-02-01

    Full Text Available Ascorbate is a co-factor for the hydroxylases that regulate the transcription factor hypoxia-inducible factor (HIF-1, which provides cancer cells with a metabolic and survival advantage in the hypoxic environment of solid tumors. However, whether ascorbate affects tumor development is a highly debated issue. We aimed to determine whether tumor ascorbate was associated with HIF-1 activation and patient disease-free survival. In this study we undertook a retrospective observational analysis of tissue-banked tumor and paired normal tissue from 49 colorectal cancer patients, measuring ascorbate levels, HIF-1α and its downstream gene products BNIP3 and VEGF. Patient survival was monitored for the first six years after surgery. We found that ascorbate levels were lower in tumor tissue compared to normal tissue (p< 0.001 but overall levels varied considerably. HIF-1α, VEGF and BNIP3 were elevated in tumor samples (p< 0.01. There was an inverse relationship between tumor ascorbate content and HIF-1 pathway activation (p=0.002 and tumor size (p=0.018. Higher tumor ascorbate content was associated with significantly improved disease-free survival in the first 6 years after surgery (p=0.006, with 141 - 1,094 additional disease free days. This was independent of tumor grade and stage. Survival advantage was associated with the amount of ascorbate in the tumor, but not with the amount in adjacent normal tissue. Our results demonstrate that higher tumor ascorbate content is associated decreased HIF-1 activation, most likely due to the co-factor activity of ascorbate for the regulatory HIF hydroxylases. Our findings support the need for future studies to determine whether raising tumor ascorbate is possible with clinical intervention and whether this results in modification of hydroxylase-dependent pathways in the tumor.

  7. Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Transcription

    Science.gov (United States)

    Chandel, N. S.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T.

    1998-09-01

    Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ 0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in ρ 0 cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

  8. Andrographolide inhibits hypoxia-inducible factor-1 through phosphatidylinositol 3-kinase/AKT pathway and suppresses breast cancer growth

    Directory of Open Access Journals (Sweden)

    Li J

    2015-02-01

    Full Text Available Jie Li,1 Chao Zhang,1 Hongchuan Jiang,1 Jiao Cheng21Department of General Surgery, 2Department of Gynaecology and Obstetrics, Beijing Chao-Yang Hospital, Beijing, People’s Republic of ChinaAbstract: Hypoxia-inducible factor-1 (HIF-1 is a master regulator of the transcriptional response to hypoxia. HIF-1α is one of the most compelling anticancer targets. Andrographolide (Andro was newly identified to inhibit HIF-1 in T47D cells (a half maximal effective concentration [EC50] of 1.03×10-7 mol/L, by a dual-luciferase reporter assay. It suppressed HIF-1α protein and gene accumulation, which was dependent on the inhibition of upstream phosphatidylinositol 3-kinase (PI3K/AKT pathway. It also abrogated the expression of HIF-1 target vascular endothelial growth factor (VEGF gene and protein. Further, Andro inhibited T47D and MDA-MB-231 cell proliferation and colony formation. In addition, it exhibited significant in vivo efficacy and antitumor potential against the MDA-MB-231 xenograft in nude mice. In conclusion, these results highlighted the potential effects of Andro, which inhibits HIF-1, and hence may be developed as an antitumor agent for breast cancer therapy in future.Keywords: Andrographolide (Andro, HIF-1α, inhibit, breast cancer, hypoxia, PI3k/AKT/mTOR pathway

  9. Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

    Science.gov (United States)

    Chang, Wei-Lun; Chang, Yi-Cheng; Lin, Kuan-Ting; Li, Han-Ru; Pai, Chih-Yu; Chen, Jen-Hao; Su, Yi-Hsien

    2017-08-15

    Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus , hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development. © 2017. Published by The Company of Biologists Ltd.

  10. Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Shigeo Saito

    2015-06-01

    Full Text Available Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia, cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches. Reactive oxygen species (ROS have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16Ink4a, which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs. During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs. ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

  11. Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells.

    Science.gov (United States)

    Jeon, Daun; Park, Heon Joo; Kim, Hong Seok

    2018-01-01

    Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial-mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Steroid receptor coactivator-3 regulates glucose metabolism in bladder cancer cells through coactivation of hypoxia inducible factor 1α.

    Science.gov (United States)

    Zhao, Wei; Chang, Cunjie; Cui, Yangyan; Zhao, Xiaozhi; Yang, Jun; Shen, Lan; Zhou, Ji; Hou, Zhibo; Zhang, Zhen; Ye, Changxiao; Hasenmayer, Donald; Perkins, Robert; Huang, Xiaojing; Yao, Xin; Yu, Like; Huang, Ruimin; Zhang, Dianzheng; Guo, Hongqian; Yan, Jun

    2014-04-18

    Cancer cell proliferation is a metabolically demanding process, requiring high glycolysis, which is known as "Warburg effect," to support anabolic growth. Steroid receptor coactivator-3 (SRC-3), a steroid receptor coactivator, is overexpressed and/or amplified in multiple cancer types, including non-steroid targeted cancers, such as urinary bladder cancer (UBC). However, whether SRC-3 regulates the metabolic reprogramming for cancer cell growth is unknown. Here, we reported that overexpression of SRC-3 accelerated UBC cell growth, accompanied by the increased expression of genes involved in glycolysis. Knockdown of SRC-3 reduced the UBC cell glycolytic rate under hypoxia, decreased tumor growth in nude mice, with reduction of proliferating cell nuclear antigen and lactate dehydrogenase expression levels. We further revealed that SRC-3 could interact with hypoxia inducible factor 1α (HIF1α), which is a key transcription factor required for glycolysis, and coactivate its transcriptional activity. SRC-3 was recruited to the promoters of HIF1α-target genes, such as glut1 and pgk1. The positive correlation of expression levels between SRC-3 and Glut1 proteins was demonstrated in human UBC patient samples. Inhibition of glycolysis through targeting HK2 or LDHA decelerated SRC-3 overexpression-induced cell growth. In summary, overexpression of SRC-3 promoted glycolysis in bladder cancer cells through HIF1α to facilitate tumorigenesis, which may be an intriguing drug target for bladder cancer therapy.

  13. Hypoxia induces cancer-associated cAMP/PKA signalling through HIF-mediated transcriptional control of adenylyl cyclases VI and VII.

    Science.gov (United States)

    Simko, Veronika; Iuliano, Filippo; Sevcikova, Andrea; Labudova, Martina; Barathova, Monika; Radvak, Peter; Pastorekova, Silvia; Pastorek, Jaromir; Csaderova, Lucia

    2017-08-31

    Hypoxia is a phenomenon often arising in solid tumours, linked to aggressive malignancy, bad prognosis and resistance to therapy. Hypoxia-inducible factor-1 has been identified as a key mediator of cell and tissue adaptation to hypoxic conditions through transcriptional activation of many genes involved in glucose metabolism and other cancer-related processes, such as angiogenesis, cell survival and cell invasion. Cyclic adenosine 3'5'-monophosphate is one of the most ancient and evolutionarily conserved signalling molecules and the cAMP/PKA signalling pathway plays an important role in cellular adaptation to hypoxia. We have investigated possible new mechanisms behind hypoxic activation of the cAMP/PKA pathway. For the first time, we have shown that hypoxia induces transcriptional up-regulation of the system of adenylyl cyclases, enzymes responsible for cAMP production, in a panel of carcinoma cell lines of various origin. Our data prove functional relevance of the hypoxic increase of adenylyl cyclases VI and VII at least partially mediated by HIF-1 transcription factor. We have identified adenylyl cyclase VI and VII isoforms as mediators of cellular response to hypoxia, which led to the elevation of cAMP levels and enhanced PKA activity, with an impact on cell migration and pH regulation.

  14. Menadione and ethacrynic acid inhibit the hypoxia-inducible factor (HIF) pathway by disrupting HIF-1α interaction with p300.

    Science.gov (United States)

    Na, Yu-Ran; Han, Ki-Cheol; Park, Hyunsung; Yang, Eun Gyeong

    2013-05-17

    Hypoxia is a general characteristic of most solid malignancies and intimately related to neoplastic diseases and cancer progression. Homeostatic response to hypoxia is primarily mediated by hypoxia inducible factor (HIF)-1α that elicits transcriptional activity through recruitment of the CREB binding protein (CBP)/p300 coactivator. Targeted blockade of HIF-1α binding to CBP/p300 would thus constitute a novel approach for cancer treatment by suppressing tumor angiogenesis and metastasis. Here, we identified inhibitors against the interaction between HIF-1α and p300 by a fluorescence polarization-based assay employing a fluorescently-labeled peptide containing the C-terminal activation domain of HIF-1α. Two small molecule inhibitors, menadione (MD) and ethacrynic acid (EA), were found to decrease expression of luciferase under the control of hypoxia-responsive elements in hypoxic cells as well as to efficiently block the interaction between the full-length HIF-1α and p300. While these compounds did not alter the expression level of HIF-1α, they down-regulated expression of a HIF-1α target vascular endothelial growth factor (VEGF) gene. Considering hypoxia-induced VEGF expression leading to highly aggressive tumor growth, MD and EA may provide new scaffolds for development of tumor therapeutic reagents as well as tools for a better understanding of HIF-1α-mediated hypoxic regulation. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lin, Wan-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Lii, Chong-Kuei; Chen, Haw-Wen

    2018-03-01

    Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation. © 2017 Wiley Periodicals, Inc.

  16. Hypoxia induces adipogenic differentitation of myoblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

    2010-09-03

    Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

  17. Hypoxia inducible BHLHB2 is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma

    International Nuclear Information System (INIS)

    Wang, Weibin; Reiser-Erkan, Carolin; Michalski, Christoph W.; Raggi, Matthias C.; Quan, Liao; Yupei, Zhao; Friess, Helmut; Erkan, Mert; Kleeff, Joerg

    2010-01-01

    weak/absent nuclear BHLHB2 staining had significantly worse median survival compared to those with strong staining (13 months vs. 27 months, p = 0.03). In a multivariable analysis, BHLHB2 staining was an independent prognostic factor (Hazard-Ratio = 2.348, 95% CI = 1.250-4.411, p = 0.008). Conclusions: Hypoxia-inducible BHLHB2 expression is a novel independent prognostic marker in pancreatic cancer patients and indicates increased chemosensitivity towards gemcitabine.

  18. Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression

    International Nuclear Information System (INIS)

    Simiantonaki, Nektaria; Taxeidis, Marios; Jayasinghe, Caren; Kurzik-Dumke, Ursula; Kirkpatrick, Charles James

    2008-01-01

    Hypoxia-inducible factor 1 alpha (HIF-1α) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1α during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas. Immunohistochemistry and Western blot is used to analyse HIF-1α expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1α inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry. In normal mucosa, HPP and TA-LGD HIF-1α was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1α were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1α overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1α expression was strongly accumulated in perinecrotic regions. In these cases HIF-1α activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1α was also seen in periiflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1α expression and nuclear translocation. We conclude that HIF-1α expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic

  19. Notch1 is required for hypoxia-induced proliferation, invasion and chemoresistance of T-cell acute lymphoblastic leukemia cells

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    Zou Jie

    2013-01-01

    Full Text Available Abstract Background Notch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL. Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL. Methods T-ALL cell lines (Jurkat, Sup-T1 transfected with HIF-1α or Notch1 small interference RNA (siRNA were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot. Results Hypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2 and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression. Conclusions Notch1 signalling is required for hypoxia/HIF-1α-induced proliferation

  20. The Zinc Finger of Prolyl Hydroxylase Domain Protein 2 Is Essential for Efficient Hydroxylation of Hypoxia-Inducible Factor α.

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    Arsenault, Patrick R; Song, Daisheng; Chung, Yu Jin; Khurana, Tejvir S; Lee, Frank S

    2016-09-15

    Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  1. Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification.

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    Mokas, Sophie; Larivière, Richard; Lamalice, Laurent; Gobeil, Stéphane; Cornfield, David N; Agharazii, Mohsen; Richard, Darren E

    2016-09-01

    Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

  2. Detection of the onset of ischemia and carcinogenesis by hypoxia-inducible transcription factor-based in vivo bioluminescence imaging.

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    Tetsuya Kadonosono

    Full Text Available An animal model for the early detection of common fatal diseases such as ischemic diseases and cancer is desirable for the development of new drugs and treatment strategies. Hypoxia-inducible factor 1 (HIF-1 is a transcription factor that regulates oxygen homeostasis and plays key roles in a number of diseases, including cancer. Here, we established transgenic (Tg mice that carry HRE/ODD-luciferase (HOL gene, which generates bioluminescence in an HIF-1-dependent manner and was successfully used in this study to monitor HIF-1 activity in ischemic tissues. To monitor carcinogenesis in vivo, we mated HOL mice with rasH2 Tg mice, which are highly sensitive to carcinogens and are used for short-term carcinogenicity assessments. After rasH2-HOL Tg mice were treated with N-methyl-N-nitrosourea, bioluminescence was detected noninvasively as early as 9 weeks in tissues that contained papillomas and malignant lesions. These results suggest that the Tg mouse lines we established hold significant potential for monitoring the early onset of both ischemia and carcinogenesis and that these lines will be useful for screening chemicals for carcinogenic potential.

  3. A3 Adenosine Receptors Modulate Hypoxia-inducible Factor-1a Expression in Human A375 Melanoma Cells

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    Stefania Merighi

    2005-10-01

    Full Text Available Hypoxia-inducible factor-1 (HIF-1 is a key regulator of genes crucial to many aspects of cancer biology. The purine nucleoside, adenosine, accumulates within many tissues under hypoxic conditions, including that of tumors. Because the levels of both HIF-1 and adenosine are elevated within the hypoxic environment of solid tumors, we investigated whether adenosine may regulate HIF-1. Here we show that, under hypoxic conditions (< 2% 02, adenosine upregulates HIF-1α protein expression in a dose-dependent and timedependent manner, exclusively through the A3 receptor subtype. The response to adenosine was generated at the cell surface because the inhibition of A3 receptor expression, by using small interfering RNA, abolished nucleoside effects. A3 receptor stimulation in hypoxia also increases angiopoietin-2 (Ang-2 protein accumulation through the induction of HIF-1α. In particular, we found that A3 receptor stimulation activates p44/p42 and p38 mitogen-activated protein kinases, which are required for A3-induced increase of HIF-1a and Ang-2. Collectively, these results suggest a cooperation between hypoxic and adenosine signals that ultimately may lead to the increase in HIF-1-mediated effects in cancer cells.

  4. Transient ureteral obstruction prevents against kidney ischemia/reperfusion injury via hypoxia-inducible factor (HIF-2α activation.

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    Shun Zhang

    Full Text Available Although the protective effect of transient ureteral obstruction (UO prior to ischemia on subsequent renal ischemia/reperfusion (I/R injury has been documented, the underlying molecular mechanism remains to be understood. We showed in the current study that 24 h of UO led to renal tubular hypoxia in the ipsilateral kidney in mice, with the accumulation of hypoxia-inducible factor (HIF-2α, which lasted for a week after the release of UO. To address the functions of HIF-2α in UO-mediated protection of renal IRI, we utilized the Mx-Cre/loxP recombination system to knock out target genes. Inactivation of HIF-2α, but not HIF-1α blunted the renal protective effects of UO, as demonstrated by much higher serum creatinine level and severer histological damage. UO failed to prevent postischemic neutrophil infiltration and apoptosis induction in HIF-2α knockout mice, which also diminished the postobstructive up-regulation of the protective molecule, heat shock protein (HSP-27. The renal protective effects of UO were associated with the improvement of the postischemic recovery of intra-renal microvascular blood flow, which was also dependent on the activation of HIF-2α. Our results demonstrated that UO protected the kidney via activation of HIF-2α, which reduced tubular damages via preservation of adequate renal microvascular perfusion after ischemia. Thus, preconditional HIF-2α activation might serve as a novel therapeutic strategy for the treatment of ischemic acute renal failure.

  5. Hypoxia-induced hypothermia mediated by GABA in the rostral parapyramidal area of the medulla oblongata.

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    Osaka, T

    2014-05-16

    Hypoxia evokes a regulated decrease in the body core temperature (Tc) in a variety of animals. The neuronal mechanisms of this response include, at least in part, glutamatergic activation in the lateral preoptic area (LPO) of the hypothalamus. As the sympathetic premotor neurons in the medulla oblongata constitute a cardinal relay station in the descending neuronal pathway from the hypothalamus for thermoregulation, their inhibition can also be critically involved in the mechanisms of the hypoxia-induced hypothermia. Here, I examined the hypothesis that hypoxia-induced hypothermia is mediated by glutamate-responsive neurons in the LPO that activate GABAergic transmission in the rostral raphe pallidus (rRPa) and neighboring parapyramidal region (PPy) of the medulla oblongata in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. Unilateral microinjection of GABA (15nmol) into the rRPa and PPy regions elicited a prompt increase in tail skin temperature (Ts) and decreases in Tc, oxygen consumption rate (VO2), and heart rate. Next, when the GABAA receptor blocker bicuculline methiodide (bicuculline methiodide (BMI), 10pmol) alone was microinjected into the rRPa, it elicited unexpected contradictory responses: simultaneous increases in Ts, VO2 and heart rate and a decrease in Tc. Then, when BMI was microinjected bilaterally into the PPy, no direct effect on Ts was seen; and thermogenic and tachycardic responses were slight. However, pretreatment of the PPy with BMI, but not vehicle saline, greatly attenuated the hypothermic responses evoked by hypoxic (10%O2-90%N2, 5min) ventilation or bilateral microinjections of glutamate (5nmol, each side) into the LPO. The results suggest that hypoxia-induced hypothermia was mediated, at least in part, by the activation of GABAA receptors in the PPy. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

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    Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

    2015-01-01

    Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

  7. Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

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    Shan-Shan Liu

    2013-12-01

    Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

  8. of Hypoxia-Inducible Factor-1α Activity by the Fusion of High-Resolution SPECT and Morphological Imaging Tests

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    Hirofumi Fujii

    2012-01-01

    Full Text Available Purpose. We aimed to clearly visualize heterogeneous distribution of hypoxia-inducible factor 1α (HIF activity in tumor tissues in vivo. Methods. We synthesized of 125I-IPOS, a 125I labeled chimeric protein probe, that would visualize HIF activity. The biodistribution of 125I-IPOS in FM3A tumor-bearing mice was evaluated. Then, the intratumoral localization of this probe was observed by autoradiography, and it was compared with histopathological findings. The distribution of 125I-IPOS in tumors was imaged by a small animal SPECT/CT scanner. The obtained in vivo SPECT-CT fusion images were compared with ex vivo images of excised tumors. Fusion imaging with MRI was also examined. Results. 125I-IPOS well accumulated in FM3A tumors. The intratumoral distribution of 125I-IPOS by autoradiography was quite heterogeneous, and it partially overlapped with that of pimonidazole. High-resolution SPECT-CT fusion images successfully demonstrated the heterogeneity of 125I-IPOS distribution inside tumors. SPECT-MRI fusion images could give more detailed information about the intratumoral distribution of 125I-IPOS. Conclusion. High-resolution SPECT images successfully demonstrated heterogeneous intratumoral distribution of 125I-IPOS. SPECT-CT fusion images, more favorably SPECT-MRI fusion images, would be useful to understand the features of heterogeneous intratumoral expression of HIF activity in vivo.

  9. Decreased extracellular adenosine levels lead to loss of hypoxia-induced neuroprotection after repeated episodes of exposure to hypoxia.

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    Mei Cui

    Full Text Available Achieving a prolonged neuroprotective state following transient ischemic attacks (TIAs is likely to effectively reduce the brain damage and neurological dysfunction associated with recurrent stroke. HPC is a phenomenon in which advanced exposure to mild hypoxia reduces the stroke volume produced by a subsequent TIA. However, this neuroprotection is not long-lasting, with the effects reaching a peak after 3 days. Therefore, in this study, we investigated the use of multiple episodes of hypoxic exposure at different time intervals to induce longer-term protection in a mouse stroke model. C57BL/6 mice were subjected to different hypoxic preconditioning protocols: a single episode of HPC or five identical episodes at intervals of 3 days (E3d HPC or 6 days (E6d HPC. Three days after the last hypoxic exposure, temporary middle cerebral artery occlusion (MCAO was induced. The effects of these HPC protocols on hypoxia-inducible factor (HIF regulated gene mRNA expression were measured by quantitative PCR. Changes in extracellular adenosine concentrations, known to exert neuroprotective effects, were also measured using in vivo microdialysis and high pressure liquid chromatography (HPLC. Neuroprotection was provided by E6d HPC but not E3d HPC. HIF-regulated target gene expression increased significantly following all HPC protocols. However, E3d HPC significantly decreased extracellular adenosine and reduced cerebral blood flow in the ischemic region with upregulated expression of the adenosine transporter, equilibrative nucleoside transporter 1 (ENT1. An ENT1 inhibitor, propentofylline increased the cerebral blood flow and re-established neuroprotection in E3d HPC. Adenosine receptor specific antagonists showed that adenosine mainly through A1 receptor mediates HPC induced neuroprotection. Our data indicate that cooperation of HIF-regulated genes and extracellular adenosine is necessary for HPC-induced neuroprotection.

  10. Apigenin ameliorates hypertension-induced cardiac hypertrophy and down-regulates cardiac hypoxia inducible factor-lα in rats.

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    Zhu, Zeng-Yan; Gao, Tian; Huang, Yan; Xue, Jie; Xie, Mei-Lin

    2016-04-01

    Apigenin is a natural flavonoid compound that can inhibit hypoxia-inducible factor (HIF)-1α expression in cultured tumor cells under hypoxic conditions. Hypertension-induced cardiac hypertrophy is always accompanied by abnormal myocardial glucolipid metabolism due to an increase of HIF-1α. However, whether or not apigenin may ameliorate the cardiac hypertrophy and abnormal myocardial glucolipid metabolism remains unknown. This study aimed to examine the effects of apigenin. Rats with cardiac hypertrophy induced by renovascular hypertension were treated with apigenin 50-100 mg kg(-1) (the doses can be achieved by pharmacological or dietary supplementation for an adult person) by gavage for 4 weeks. The results showed that after treatment with apigenin, the blood pressure, heart weight, heart weight index, cardiomyocyte cross-sectional area, serum angiotensin II, and serum and myocardial free fatty acids were reduced. It is important to note that apigenin decreased the expression level of myocardial HIF-1α protein. Moreover, apigenin simultaneously increased the expression levels of myocardial peroxisome proliferator-activated receptor (PPAR) α, carnitine palmitoyltransferase (CPT)-1, and pyruvate dehydrogenase kinase (PDK)-4 proteins and decreased the expression levels of myocardial PPARγ, glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter (GLUT)-4 proteins. These findings demonstrated that apigenin could improve hypertensive cardiac hypertrophy and abnormal myocardial glucolipid metabolism in rats, and its mechanisms might be associated with the down-regulation of myocardial HIF-1α expression and, subsequently increasing the expressions of myocardial PPARα and its target genes CPT-1 and PDK-4, and decreasing the expressions of myocardial PPARγ and its target genes GPAT and GLUT-4.

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

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

    2011-09-30

    Hypoxia-inducible factor 1 (HIF-1) is the main transcription factor responsible for hypoxia-induced gene expression. Perioperative drugs including anesthetics have been reported to affect HIF-1 activity. However, the effect of fentanyl on HIF-1 activity is not well documented. In this study, we investigated the effect of fentanyl and other opioids on HIF-1 activity in human SH-SY5Y neuroblastoma cells, hepatoma Hep3B cells, lung adenocarcinoma A549 cells and mice. Cells were exposed to fentanyl, and HIF-1 protein expression was examined by Western blot analysis using anti-HIF-1α and β antibodies. HIF-1-dependent gene expression was investigated by semi-quantitative real-time reverse transcriptase (RT)-PCR (qRT-PCR) and luciferase assay. Furthermore, fentanyl was administered intraperitoneally and HIF-1-dependent gene expression was investigated by qRT-PCR in the brains and kidneys of mice. A 10-μM concentration of fentanyl and other opioids, including 1 μM morphine and 4 μM remifentanil, induced HIF-1α protein expression and HIF-1 target gene expression in an opioid receptor-dependent manner in SH-SY5Y cells with activity peaking at 24h. Fentanyl did not augment HIF-1α expression during hypoxia-induced induction. HIF-1α stabilization assays and experiments with cycloheximide revealed that fentanyl increased translation from HIF-1α mRNA but did not stabilize the HIF-1α protein. Furthermore, fentanyl induced HIF-1 target gene expression in the brains of mice but not in their kidneys in a naloxone-sensitive manner. In this report, we describe for the first time that fentanyl, both in vitro and in vivo, induces HIF-1 activation under non-hypoxic conditions, leading to increases in expression of genes associated with adaptation to hypoxia. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Recombinant adeno-associated virus-delivered hypoxia-inducible stanniocalcin-1 expression effectively inhibits hypoxia-induced cell apoptosis in cardiomyocytes.

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    Shi, Xin; Wang, Jianzhong; Qin, Yan

    2014-12-01

    Ischemia/hypoxia-induced oxidative stress is detrimental for the survival of cardiomyocytes and cardiac function. Stanniocalcin-1 (STC-1), a glycoprotein, has been found to play an inhibitory role in the production of reactive oxygen species (ROS). Here, we speculated that the overexpression of STC-1 might alleviate oxidative damage in cardiomyocytes under conditions of hypoxia. To control the expression of STC-1 in hypoxia, we constructed a recombinant adeno-associated virus (AAV) carrying the hypoxia-responsive element (HRE) to mediate hypoxia induction. Cardiomyocytes were infected with AAV-HRE-STC-1 and cultured in normoxic or hypoxic conditions, and STC-1 overexpression was only detected in hypoxic cultured cardiomyocytes by using quantitative real-time polymerase chain reaction and Western blot analysis. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, AAV-HRE-STC-1 infection was shown to significantly enhance cell survival under hypoxia. Hypoxia-induced cell apoptosis was inhibited by AAV-HRE-STC-1 infection by using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide apoptosis assay. Moreover, the proapoptotic protein Caspase-3 and anti-apoptotic protein Bcl-2, which were dysregulated by hypoxia, were reversed by AAV-HRE-STC-1 infection. AAV-HRE-STC-1-mediated STC-1 overexpression markedly inhibited ROS production in cardiomyocytes cultured under hypoxic conditions. AAV-HRE-STC-1 infection significantly upregulated uncoupled protein 3 (UCP3), whereas silencing of UCP3 blocked the inhibitory effect of AAV-HRE-STC-1 on ROS production. In contrast, AAV-HRE-STC-1 infection had no effect on UCP2, and knockdown of UCP2 did not block the inhibitory effect of AAV-HRE-STC-1 on ROS production in the cardiomyocytes cultured under hypoxic conditions. Taken together, STC1 activates antioxidant pathway in cardiomyocytes through the induction of UCP3, implying that AAV-HRE-STC-1 has potential in the treatment of ischemic

  13. Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives

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    Bishop, Tammie; Ratcliffe, Peter J

    2014-01-01

    By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. PMID:27774477

  14. Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

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    Hyeong-Jun Han

    Full Text Available Peptidyl prolyl isomerase (PIN1 regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF-1α in human colon cancer (HCT116 cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

  15. Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

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    Li Chen

    2015-01-01

    Full Text Available Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2/Bcl-2 associated X protein (Bax in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases.

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

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

    2015-10-28

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

  17. Senp1 drives hypoxia-induced polycythemia via GATA1 and Bcl-xL in subjects with Monge's disease.

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    Azad, Priti; Zhao, Huiwen W; Cabrales, Pedro J; Ronen, Roy; Zhou, Dan; Poulsen, Orit; Appenzeller, Otto; Hsiao, Yu Hsin; Bafna, Vineet; Haddad, Gabriel G

    2016-11-14

    In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. © 2016 Azad et al.

  18. Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

    Directory of Open Access Journals (Sweden)

    Seeley TW

    2017-03-01

    Full Text Available Todd W Seeley, Mark D Sternlicht, Stephen J Klaus, Thomas B Neff, David Y Liu Therapeutics R&D, FibroGen, Inc., San Francisco, CA, USA Abstract: The effects of pharmacological hypoxia-inducible factor (HIF stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF, using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs, FG-4497 or roxadustat (FG-4592. In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors. Keywords: cancer progression, erythropoiesis, hypoxia-inducible factor, hypoxia-inducible factor prolyl hydroxylase inhibitors, vascular endothelial growth factor, MMTV-Neu breast cancer model

  19. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

    Directory of Open Access Journals (Sweden)

    Mun Chiang Chan

    Full Text Available As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs. Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs. Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4 induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

  20. Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer- and metastasis-initiating cells

    Science.gov (United States)

    Mimeault, Murielle; Batra, Surinder K

    2013-01-01

    Accumulating lines of experimental evidence have revealed that hypoxia-inducible factors, HIF-1α and HIF-2α, are key regulators of the adaptation of cancer- and metastasis-initiating cells and their differentiated progenies to oxygen and nutrient deprivation during cancer progression under normoxic and hypoxic conditions. Particularly, the sustained stimulation of epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), stem cell factor (SCF) receptor KIT, transforming growth factor-β receptors (TGF-βRs) and Notch and their downstream signalling elements such as phosphatidylinositol 3′-kinase (PI3K)/Akt/molecular target of rapamycin (mTOR) may lead to an enhanced activity of HIFs. Moreover, the up-regulation of HIFs in cancer cells may also occur in the hypoxic intratumoral regions formed within primary and secondary neoplasms as well as in leukaemic cells and metastatic prostate and breast cancer cells homing in the hypoxic endosteal niche of bone marrow. The activated HIFs may induce the expression of numerous gene products such as induced pluripotency-associated transcription factors (Oct-3/4, Nanog and Sox-2), glycolysis- and epithelial-mesenchymal transition (EMT) programme-associated molecules, including CXC chemokine receptor 4 (CXCR4), snail and twist, microRNAs and angiogenic factors such as vascular endothelial growth factor (VEGF). These gene products in turn can play critical roles for high self-renewal ability, survival, altered energy metabolism, invasion and metastases of cancer cells, angiogenic switch and treatment resistance. Consequently, the targeting of HIF signalling network and altered metabolic pathways represents new promising strategies to eradicate the total mass of cancer cells and improve the efficacy of current therapies against aggressive and metastatic cancers and prevent disease relapse. PMID:23301832

  1. Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor -1α in hepatocellular carcinoma

    International Nuclear Information System (INIS)

    Zhang, Lin; Feng, Xiaobin; Dong, Jiahong; Qian, Cheng; Huang, Gang; Li, Xiaowu; Zhang, Yujun; Jiang, Yan; Shen, Junjie; Liu, Jia; Wang, Qingliang; Zhu, Jin

    2013-01-01

    High invasion and metastasis are the primary factors causing poor prognosis of patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. In this study, we investigate the molecular mechanism by which hypoxia promotes HCC invasion and metastasis through inducing epithelial-mesenchymal transition (EMT). The expression of EMT markers was analyzed by immunohistochemistry. Effect of hypoxia on induction of EMT and ability of cell migration and invasion were performed. Luciferase reporter system was used for evaluation of Snail regulation by hypoxia-inducible factor -1α (HIF-1α). We found that overexpression of HIF-1α was observed in HCC liver tissues and was related to poor prognosis of HCC patients. HIF-1α expression profile was correlated with the expression levels of SNAI1, E-cadherin, N-cadherin and Vimentin. Hypoxia was able to induce EMT and enhance ability of invasion and migration in HCC cells. The same phenomena were also observed in CoCl2-treated cells. The shRNA-mediated HIF-1α suppression abrogated CoCl2-induced EMT and reduced ability of migration and invasion in HCC cells. Luciferase assay showed that HIF-1α transcriptional regulated the expression of SNAI1 based on two hypoxia response elements (HREs) in SNAI1 promoter. We demonstrated that hypoxia-stabilized HIF1α promoted EMT through increasing SNAI1 transcription in HCC cells. This data provided a potential therapeutic target for HCC treatment

  2. Furan- and Thiophene-2-Carbonyl Amino Acid Derivatives Activate Hypoxia-Inducible Factor via Inhibition of Factor Inhibiting Hypoxia-Inducible Factor-1

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    Shin-ichi Kawaguchi

    2018-04-01

    Full Text Available Induction of a series of anti-hypoxic proteins protects cells during exposure to hypoxic conditions. Hypoxia-inducible factor-α (HIF-α is a major transcription factor that orchestrates this protective effect. To activate HIF exogenously, without exposing cells to hypoxic conditions, many small-molecule inhibitors targeting prolyl hydroxylase domain-containing protein have been developed. In addition, suppression of factor inhibiting HIF-1 (FIH-1 has also been shown to have the potential to activate HIF-α. However, few small-molecule inhibitors of FIH-1 have been developed. In this study, we synthesized a series of furan- and thiophene-2-carbonyl amino acid derivatives having the potential to inhibit FIH-1. The inhibitory activities of these compounds were evaluated in SK-N-BE(2c cells by measuring HIF response element (HRE promoter activity. Several furan- and thiophene-2-carbonyl amino acid derivatives inhibited FIH-1 based on correlations among the docking score of the FIH-1 active site, the chemical structure of the compounds, and biological HIF-α/HRE transcriptional activity.

  3. Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

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    Richard Rosen

    2015-01-01

    Full Text Available Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

  4. The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and β in the tobacco hornworm, Manduca sexta.

    Science.gov (United States)

    Lundquist, Taylor A; Kittilson, Jeffrey D; Ahsan, Rubina; Greenlee, Kendra J

    2017-12-12

    As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-β dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Inhibition of phospholipaseD2 increases hypoxia-induced human colon cancer cell apoptosis through inactivating of the PI3K/AKT signaling pathway.

    Science.gov (United States)

    Liu, Maoxi; Fu, Zhongxue; Wu, Xingye; Du, Kunli; Zhang, Shouru; Zeng, Li

    2016-05-01

    Hypoxia is a common feature of solid tumor, and is a direct stress that triggers apoptosis in many human cell types. As one of solid cancer, hypoxia exists in the whole course of colon cancer occurrence and progression. Our previous studies shown that hypoxia induce high expression of phospholipase D2 (PLD2) and survivin in colon cancer cells. However, the correlation between PLD2 and survivin in hypoxic colon cancer cells remains unknown. In this study, we observed significantly elevated PLD2 and survivin expression levels in colon cancer tissues and cells. This is a positive correlation between of them, and co-expression of PLD2 and survivin has a positive correlation with the clinicpatholic features including tumor size, TNM stage, and lymph node metastasis. We also found that hypoxia induced the activity of PLD increased significant mainly caused by PLD2 in colon cancer cells. However, inhibition the activity of PLD2 induced by hypoxia promotes the apoptosis of human colon cancer cells, as well as decreased the expression of apoptosis markers including survivin and bcl2. Moreover, the pharmacological inhibition of PI3K/AKT supported the hypothesis that promotes the apoptosis of hypoxic colon cancer cells by PLD2 activity inhibition may through inactivation of the PI3K/AKT signaling pathway. Furthermore, interference the PLD2 gene expression leaded to the apoptosis of hypoxic colon cancer cells increased and also decreased the expression level of survivin and bcl2 may through inactivation of PI3K/AKT signaling pathway. These results indicated that PLD2 play antiapoptotic role in colon cancer under hypoxic conditions, inhibition of the activity, or interference of PLD2 gene expression will benefit for the treatment of colon cancer patients.

  6. Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

    2015-04-03

    Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

  7. Regulation of hypoxia-inducible factor-1α (HIF-1α expression by interleukin-1β (IL-1 β, insulin-like growth factors I (IGF-I and II (IGF-II in human osteoarthritic chondrocytes

    Directory of Open Access Journals (Sweden)

    Angelica Rossi Sartori-Cintra

    2012-01-01

    Full Text Available OBJECTIVE: Hypoxia-inducible factor 1 alpha regulates genes related to cellular survival under hypoxia. This factor is present in osteroarthritic chondrocytes, and cytokines, such as interleukin-1 beta, participate in the pathogenesis of osteoarthritis, thereby increasing the activities of proteolytic enzymes, such as matrix metalloproteinases, and accelerating cartilage destruction. We hypothesize that Hypoxia Inducible Factor-1 alpha (HIF-1α can regulate cytokines (catabolic action and/or growth factors (anabolic action in osteoarthritis. The purpose of this study was to investigate the modulation of HIF-1α in human osteoarthritic chondrocytes by interleukin-1 beta (IL-1β and insulin-like growth factors I (IGF-I and II (IGF-II and to determine the involvement of the phosphatidylinositol-3kinase (PI-3K pathway in this process. METHODS: Human osteroarthritic chondrocytes were stimulated with IL-1β, IGF-I and IGF-II and LY294002, a specific inhibitor of PI-3K. Nuclear protein levels and gene expression were analyzed by western blot and quantitative reverse transcription-polymerase chain reaction analyses, respectively. RESULTS: HIF-1α expression was upregulated by IL-1β at the protein level but not at the gene level. IGF-I treatment resulted in increases in both the protein and mRNA levels of HIF-1α , whereas IGF-II had no effect on its expression. However, all of these stimuli exploited the PI-3K pathway. CONCLUSION: IL-1β upregulated the levels of HIF-1α protein post-transcriptionally, whereas IGF-I increased HIF-1α at the transcript level. In contrast, IGF-II did not affect the protein or gene expression levels of HIF-1α . Furthermore, all of the tested stimuli exploited the PI-3K pathway to some degree. Based on these findings, we are able to suggest that Hypoxia inducible Factor-1 exhibits protective activity in chondrocytes during osteoarthritis.

  8. Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast

    NARCIS (Netherlands)

    Kuijper, Arno; Groep, P. van der; Wall, E. van der; Diest, P.J. van

    2005-01-01

    INTRODUCTION Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance

  9. IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression.

    Science.gov (United States)

    Hasan, Arif Ul; Kittikulsuth, Wararat; Yamaguchi, Fuminori; Musarrat Ansary, Tuba; Rahman, Asadur; Shibayama, Yuki; Nakano, Daisuke; Hitomi, Hirofumi; Tokuda, Masaaki; Nishiyama, Akira

    2017-09-15

    Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl 2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl 2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, β-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis

    NARCIS (Netherlands)

    Sluimer, Judith C.; Gasc, Jean-Marie; van Wanroij, Job L.; Kisters, Natasja; Groeneweg, Mathijs; Sollewijn Gelpke, Maarten D.; Cleutjens, Jack P.; van den Akker, Luc H.; Corvol, Pierre; Wouters, Bradly G.; Daemen, Mat J.; Bijnens, Ann-Pascale J.

    2008-01-01

    We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of

  11. Hypoxia-Inducible Lipid Droplet-Associated Is Not a Direct Physiological Regulator of Lipolysis in Adipose Tissue

    DEFF Research Database (Denmark)

    Dijk, Wieneke; Mattijssen, Frits; de la Rosa Rodriguez, Montserrat

    2017-01-01

    Triglycerides are stored in specialized organelles called lipid droplets. Numerous proteins have been shown to be physically associated with lipid droplets and govern their function. Previously, the protein hypoxia-inducible lipid droplet-associated (HILPDA) was localized to lipid droplets and wa...

  12. Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Veronica Kalhori

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1 is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3, Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.

  13. The β3-Integrin Binding Protein β3-Endonexin Is a Novel Negative Regulator of Hypoxia-Inducible Factor-1

    Science.gov (United States)

    Kračun, Damir; Rieß, Florian; Kanchev, Ivan; Gawaz, Meinrad

    2014-01-01

    Abstract Aims: Integrins are multifunctional heterodimeric adhesion receptors that mediate the attachment between a cell and the extracellular matrix or other surrounding cells. In endothelial cells, integrins can modulate cell migration and motility. In particular, β3-integrin is expressed in angiogenic vessels. Signal transduction by β3-integrins requires the recruitment of intracellular signaling molecules. β3-endonexin is a highly spliced molecule that has been identified as a β3-integrin binding protein. β3-endonexin isoforms are expressed in endothelial cells and have been suggested to act as shuttle proteins between the membrane and the nucleus. However, their functional role in angiogenesis is unclear. In this study, we investigated whether β3-endonexin isoforms are involved in endothelial angiogenic processes under hypoxia. Results: The overexpression of β3-endonexin isoforms decreased endothelial proliferation and tube formation under hypoxia, while the depletion of β3-endonexin by RNAi promoted angiogenic responses in vitro and in vivo. In hypoxia, β3-endonexin accumulated in the nucleus, and prevention of this response by depletion of β3-endonexin increased hypoxic activation and induction of the hypoxia-inducible factor (HIF)-1 and its target genes VEGF and PAI-1. β3-endonexin diminished nuclear factor kappa B (NFκB) activation and decreased NFκB binding to the HIF-1α promoter under hypoxia, subsequently diminishing NFκB-dependent transcription of HIF-1α under hypoxia. Innovation: Our results indicate for the first time that the overexpression of β3-endonexin can decrease hypoxic induction and activation of HIF-1α and can prevent hypoxic endothelial proliferation and angiogenic responses. Conclusion: β3-endonexin can act as a novel anti-angiogenic factor specifically in the response to hypoxia due to its negative impact on the activation of HIF-1. Antioxid. Redox Signal. 20, 1964–1976. PMID:24386901

  14. The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells.

    Science.gov (United States)

    Binó, Lucia; Kučera, Jan; Štefková, Kateřina; Švihálková Šindlerová, Lenka; Lánová, Martina; Kudová, Jana; Kubala, Lukáš; Pacherník, Jiří

    2016-01-25

    Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

    Science.gov (United States)

    Asghar, Muhammad Yasir; Bergelin, Nina; Jaakkola, Panu; Törnquist, Kid

    2013-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1. PMID:23824493

  16. TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

    Directory of Open Access Journals (Sweden)

    Tien-Ling Liao

    2014-09-01

    Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

  17. Hypoxia-inducible factor-2α (HIF-2α), but not HIF-1α, is essential for hypoxic induction of class III β-tubulin expression in human glioblastoma cells.

    Science.gov (United States)

    Bordji, Karim; Grandval, Alexandra; Cuhna-Alves, Leilane; Lechapt-Zalcman, Emmanuèle; Bernaudin, Myriam

    2014-12-01

    Glioblastoma multiforme (GBM) is the deadliest form of primary brain cancer. Several reports have indicated aberrant levels of βIII-tubulin (βIII-t) in human GBM. βIII-t overexpression was linked to increasing malignancy in glial tumors and described to determine the onset of resistance to chemotherapy. Furthermore, a linkage was suggested between the induction of βIII-t expression and hypoxia, a hallmark of GBM. We investigated the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the regulation of the βIII-t gene (TUBB3) in GBM cells cultured in either normoxia or hypoxia. We report for the first time that HIF-2α, but not HIF-1α, is involved in hypoxia-induced βIII-t expression in GBM cells. By gene-reporter experiments and site-directed mutagenesis, we found that two overlapping hypoxia response elements located in the 3' UTR of the gene were involved in the activation of TUBB3. This occurred through an enhanced binding of HIF-2α to the 3' region, as revealed by an electrophoretic mobility shift assay. Conversely, the promoter of TUBB3 was shown to be inactive. In addition, we observed that HIF-1α exhibits a repressive effect on βIII-t expression in cells cultured in normoxia. These results show that both HIF-α isoforms have opposing effects on βIII-t expression in GBM cells. Finally, we observed that hypoxia-induced βIII-t expression is well correlated with the kinetics of HIF-2α protein stabilization. The evidence for a direct linkage between HIF-2α and increased expression of βIII-t by hypoxia suggests that an anti-HIF-2α strategy (i.e. by downregulating βIII-t) could be of potential interest for improving the treatment of GBM. © 2014 FEBS.

  18. Thin Air Resulting in High Pressure: Mountain Sickness and Hypoxia-Induced Pulmonary Hypertension

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    Richter, Manuel; Tello, Khodr; Sommer, Natascha; Gall, Henning; Ghofrani, Hossein Ardeschir

    2017-01-01

    With rising altitude the partial pressure of oxygen falls. This phenomenon leads to hypobaric hypoxia at high altitude. Since more than 140 million people permanently live at heights above 2500 m and more than 35 million travel to these heights each year, understanding the mechanisms resulting in acute or chronic maladaptation of the human body to these circumstances is crucial. This review summarizes current knowledge of the body's acute response to these circumstances, possible complications and their treatment, and health care issues resulting from long-term exposure to high altitude. It furthermore describes the characteristic mechanisms of adaptation to life in hypobaric hypoxia expressed by the three major ethnic groups permanently dwelling at high altitude. We additionally summarize current knowledge regarding possible treatment options for hypoxia-induced pulmonary hypertension by reviewing in vitro, rodent, and human studies in this area of research. PMID:28522921

  19. Recent Advances in Developing Inhibitors for Hypoxia-Inducible Factor Prolyl Hydroxylases and Their Therapeutic Implications

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    So Yeon Kim

    2015-11-01

    Full Text Available Hypoxia-inducible factor (HIF prolyl hydroxylases (PHDs are members of the 2-oxoglutarate dependent non-heme iron dioxygenases. Due to their physiological roles in regulation of HIF-1α stability, many efforts have been focused on searching for selective PHD inhibitors to control HIF-1α levels for therapeutic applications. In this review, we first describe the structure of PHD2 as a molecular basis for structure-based drug design (SBDD and various experimental methods developed for measuring PHD activity. We further discuss the current status of the development of PHD inhibitors enabled by combining SBDD approaches with high-throughput screening. Finally, we highlight the clinical implications of small molecule PHD inhibitors.

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

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    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 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.

  1. The role of hypoxia-inducible factor-2 in digestive system cancers.

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    Zhao, J; Du, F; Shen, G; Zheng, F; Xu, B

    2015-01-15

    Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies.

  2. Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.

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    Maybin, Jacqueline A; Murray, Alison A; Saunders, Philippa T K; Hirani, Nikhil; Carmeliet, Peter; Critchley, Hilary O D

    2018-01-23

    Heavy menstrual bleeding (HMB) is common and debilitating, and often requires surgery due to hormonal side effects from medical therapies. Here we show that transient, physiological hypoxia occurs in the menstrual endometrium to stabilise hypoxia inducible factor 1 (HIF-1) and drive repair of the denuded surface. We report that women with HMB have decreased endometrial HIF-1α during menstruation and prolonged menstrual bleeding. In a mouse model of simulated menses, physiological endometrial hypoxia occurs during bleeding. Maintenance of mice under hyperoxia during menses decreases HIF-1α induction and delays endometrial repair. The same effects are observed upon genetic or pharmacological reduction of endometrial HIF-1α. Conversely, artificial induction of hypoxia by pharmacological stabilisation of HIF-1α rescues the delayed endometrial repair in hypoxia-deficient mice. These data reveal a role for HIF-1 in the endometrium and suggest its pharmacological stabilisation during menses offers an effective, non-hormonal treatment for women with HMB.

  3. Hypoxia targeted bifunctional suicide gene expression enhances radiotherapy in vitro and in vivo

    International Nuclear Information System (INIS)

    Sun, Xiaorong; Xing, Ligang; Deng, Xuelong; Hsiao, Hung Tsung; Manami, Akiko; Koutcher, Jason A.; Clifton Ling, C.; Li, Gloria C.

    2012-01-01

    Purpose: To investigate whether hypoxia targeted bifunctional suicide gene expression-cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with 5-FC treatments can enhance radiotherapy. Materials and methods: Stable transfectants of R3327-AT cells were established which express a triple-fusion-gene: CD, UPRT and monomoric DsRed (mDsRed) controlled by a hypoxia inducible promoter. Hypoxia-induced expression/function of CDUPRTmDsRed was verified by western blot, flow cytometry, fluorescent microscopy, and cytotoxicity assay of 5-FU and 5-FC. Tumor-bearing mice were treated with 5-FC and local radiation. Tumor volume was monitored and compared with those treated with 5-FC or radiation alone. In addition, the CDUPRTmDsRed distribution in hypoxic regions of tumor sections was visualized with fluorescent microscopy. Results: Hypoxic induction of CDUPRTmDsRed protein correlated with increased sensitivity to 5-FC and 5-FU. Significant radiosensitization effects were detected after 5-FC treatments under hypoxic conditions. In the tumor xenografts, the distribution of CDUPRTmDsRed expression visualized with fluorescence microscopy was co-localized with the hypoxia marker pimonidazole positive staining cells. Furthermore, administration of 5-FC to mice in combination with local irradiation resulted in significant tumor regression, as in comparison with 5-FC or radiation treatments alone. Conclusions: Our data suggest that the hypoxia-inducible CDUPRT/5-FC gene therapy strategy has the ability to specifically target hypoxic cancer cells and significantly improve the tumor control in combination with radiotherapy.

  4. Protective effects of myricetin on acute hypoxia-induced exercise intolerance and mitochondrial impairments in rats.

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    Dan Zou

    Full Text Available 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.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.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 regulators, in addition, AMP-activated protein kinase(AMPK plays a crucial role in this process.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.

  5. Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.

    Science.gov (United States)

    Drager, Luciano F; Yao, Qiaoling; Hernandez, Karen L; Shin, Mi-Kyung; Bevans-Fonti, Shannon; Gay, Jason; Sussan, Thomas E; Jun, Jonathan C; Myers, Allen C; Olivecrona, Gunilla; Schwartz, Alan R; Halberg, Nils; Scherer, Philipp E; Semenza, Gregg L; Powell, David R; Polotsky, Vsevolod Y

    2013-07-15

    Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

  6. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334 ameliorates murine colitis

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    Gupta R

    2014-01-01

    Full Text Available Ram Gupta,1 Anita R Chaudhary,2 Binita N Shah,1 Avinash V Jadhav,3 Shitalkumar P Zambad,1 Ramesh Chandra Gupta,4 Shailesh Deshpande,4 Vijay Chauthaiwale,4 Chaitanya Dutt4 1Department of Pharmacology, 2Cellular and Molecular Biology, 3Preclinical Safety Evaluation, 4Discovery, Torrent Research Centre, Torrent Pharmaceuticals Ltd, Gandhinagar, Gujarat, India Background and aim: Mucosal healing in inflammatory bowel disease (IBD can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor

  7. Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

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    Kumar Anil

    2010-10-01

    Full Text Available Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6 plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1% or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM, revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.

  8. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy.

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    Zhang, Jiankai; He, Zhangyou; Xiao, Wenjian; Na, Qingqing; Wu, Tianxiu; Su, Kaixin; Cui, Xiaojun

    2016-01-01

    Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-x03BA;B p65 and phosphorylated NF-x03BA;B p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-x03BA;B were activated by BAG3 overexpression, and the NF-x03BA;B inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-x03BA;B signaling pathway in hypoxia-injured cardiomyocytes. © 2016 The Author(s) Published by S. Karger AG, Basel.

  9. Overexpression of BAG3 Attenuates Hypoxia-Induced Cardiomyocyte Apoptosis by Inducing Autophagy

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    Jiankai Zhang

    2016-07-01

    Full Text Available Background: Hypoxia is a well-known factor in the promotion of apoptosis, which contributes to the development of numerous cardiac diseases, such as heart failure and myocardial infarction. Inhibiting apoptosis is an important therapeutic strategy for the treatment of related heart diseases caused by ischemia/hypoxic injury. Previous studies have demonstrated that BAG3 plays an important role in cardiomyocyte apoptosis and survival. However, the role of BAG3 in hypoxia-induced cardiomyocyte apoptosis remains to be clarified. Here, we demonstrate that BAG3 is induced by hypoxia stimuli in cultured cardiomyocytes. Methods: BAG3 expression level was measured in H9c2 cells treated with hypoxia for 48 h. Cell proliferation and apoptosis were tested using MTT assay and Annexin V FITC-PI staining assay, respectively. The mRNA or protein expression level of BAG3, LC3-I, LC3-II, Atg5, NF-κB p65 and phosphorylated NF-κB p65 were assessed by qRT-PCR and western blot assay, respectively. Resluts: Overexpression of BAG3 inhibited cell apoptosis and promoted proliferation in hypoxia-injured H9c2 cells. Furthermore, autophagy and NF-κB were activated by BAG3 overexpression, and the NF-κB inhibitor PDTC could inhibit the activation of autophagy induced by BAG3 overexpression. In addition, the autophagy inhibitor 3-MA partly impeded the inhibitory effect of BAG3 on hypoxia-induced cardiomyocyte apoptosis. Conclusion: these results suggested that overexpression of BAG3 promoted cell proliferation and inhibited apoptosis by activating autophagy though the NF-κB signaling pathway in hypoxia-injured cardiomyocytes.

  10. Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxia-inducible factor-1alpha expression via poly(ADP-ribose) polymerase-1.

    Science.gov (United States)

    Pan, Rong; Chen, Chen; Liu, Wen-Lan; Liu, Ke-Jian

    2013-07-01

    Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study tests the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-h hypoxic treatment. Although 3-h hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner. Exposure of astrocytes to hypoxia for 3 h remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably, HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. © 2013 John Wiley & Sons Ltd.

  11. A comparative gene expression database for invertebrates

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    Ormestad Mattias

    2011-08-01

    Full Text Available Abstract Background As whole genome and transcriptome sequencing gets cheaper and faster, a great number of 'exotic' animal models are emerging, rapidly adding valuable data to the ever-expanding Evo-Devo field. All these new organisms serve as a fantastic resource for the research community, but the sheer amount of data, some published, some not, makes detailed comparison of gene expression patterns very difficult to summarize - a problem sometimes even noticeable within a single lab. The need to merge existing data with new information in an organized manner that is publicly available to the research community is now more necessary than ever. Description In order to offer a homogenous way of storing and handling gene expression patterns from a variety of organisms, we have developed the first web-based comparative gene expression database for invertebrates that allows species-specific as well as cross-species gene expression comparisons. The database can be queried by gene name, developmental stage and/or expression domains. Conclusions This database provides a unique tool for the Evo-Devo research community that allows the retrieval, analysis and comparison of gene expression patterns within or among species. In addition, this database enables a quick identification of putative syn-expression groups that can be used to initiate, among other things, gene regulatory network (GRN projects.

  12. Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α.

    Science.gov (United States)

    Nagaya, Yoshiaki; Aoyama, Mineyoshi; Tamura, Tetsuya; Kakita, Hiroki; Kato, Shin; Hida, Hideki; Saitoh, Shinji; Asai, Kiyofumi

    2014-12-01

    Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  13. Exercise attenuates intermittent hypoxia-induced cardiac fibrosis associated with sodium-hydrogen exchanger-1 in rats

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    Tsung-I Chen

    2016-10-01

    Full Text Available Purpose: To investigate the role of sodium–hydrogen exchanger-1 (NHE-1 and exercise training on intermittent hypoxia-induced cardiac fibrosis in obstructive sleep apnea (OSA, using an animal model mimicking the intermittent hypoxia of OSA. Methods: Eight-week-old male Sprague–Dawley rats were randomly assigned to control (CON, intermittent hypoxia (IH, exercise (EXE or IH combined with exercise (IHEXE groups. These groups were randomly assigned to subgroups receiving either a vehicle or the NHE-1 inhibitor cariporide. The EXE and IHEXE rats underwent exercise training on an animal treadmill for 10 weeks (5 days/week, 60 minutes/day, 24–30 m/minute, 2–10% grade. The IH and IHEXE rats were exposed to 14 days of IH (30 seconds of hypoxia - nadir of 2-6% O2 - followed by 45 seconds of normoxia for 8 hours/day. At the end of 10 weeks, rats were sacrificed and then hearts were removed to determine the myocardial levels of fibrosis index, oxidative stress, antioxidant capacity and NHE-1 activation. Results: Compared to the CON rats, IH induced higher cardiac fibrosis, lower myocardial catalase and superoxidative dismutase activities, higher myocardial lipid and protein peroxidation and higher NHE-1 activation (p < 0.05 for each, which were all abolished by cariporide. Compared to the IH rats, lower cardiac fibrosis, higher myocardial antioxidant capacity, lower myocardial lipid and protein peroxidation and lower NHE-1 activation were found in the IHEXE rats (p < 0.05 for each. Conclusion: IH-induced cardiac fibrosis was associated with NHE-1 hyperactivity. However, exercise training and cariporide exerted an inhibitory effect to prevent myocardial NHE-1 hyperactivity, which contributed to reduced IH-induced cardiac fibrosis. Therefore, NHE-1 plays a critical role in the effect of exercise on IH-induced increased cardiac fibrosis.

  14. 3-Bromopyruvate reverses hypoxia-induced pulmonary arterial hypertension through inhibiting glycolysis: In vitro and in vivo studies.

    Science.gov (United States)

    Chen, Fangzheng; Wang, Heng; Lai, Jiadan; Cai, Shujing; Yuan, Linbo

    2018-05-04

    Pulmonary arterial smooth muscle cell (PASMC) proliferation is vital to pulmonary vascular remodeling in pulmonary arterial hypertension (PAH) pathogenesis, and inhibiting PASMC metabolism could serve as a new possible therapy to reverse the process. 3-Bromopyruvate (3-BrPA) is an effective glycolysis inhibitor with its effect in PAH remains unclear. Our study aims to assess the therapeutic effect of 3-BrPA in PAH rats and investigate the possible mechanism of 3-BrPA in PASMC proliferation and apoptosis. 27 healthy SD rats were grouped and treated with hypoxia/normoxia and administration of 3-BrPA/physiological saline. Mean pulmonary artery pressure (mPAP) and cardiac output (CO) were measured and pulmonary vascular resistance (PVR) was calculated. Right ventricular hypertrophy index (RVHI) was calculated to evaluate the right ventricular hypertrophy degree. The percentage of medial wall area (WA%) and medial wall thickness (WT%) were measured by image analysis. PASMCs groups received hypoxia/normoxia treatments and 3-BrPA/physiological saline. PASMC proliferation and migration were respectively detected by CCK-8 and cell wound scratch assay. Hexokinase II (HK-2) expression and lactate level were respectively measured by Western Blotting and lactate test kit to detect glycolysis. mPAP, PVR, PVHI, WA% and WT% in rats increased after the hypoxia treatment, but were lower compared to rats received 3-BrPA in hypoxia environment. HK-2 expression, lactate concentration, OD value and scratch areas in PASMCs increased after the hypoxia treatment, but were decreased after the administration of 3-BrPA. 3-BrPA can inhibit PASMC proliferation and migration by inhibiting glycolysis, and is effective in reversing the vascular remodeling in hypoxia-induced PAH rats. Copyright © 2017. Published by Elsevier B.V.

  15. Role of hypoxia inducing factor-1β in alcohol-induced autophagy, steatosis and liver injury in mice.

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    Hong-Min Ni

    Full Text Available Chronic alcohol causes liver hypoxia and steatosis, which eventually develops into alcoholic liver disease (ALD. While it has been known that alcohol consumption activates hepatic hypoxia inducing factor-1α (HIF-1α, conflicting results regarding the role of HIF-1α in alcohol-induced liver injury and steatosis in mice have been reported. In the present study, we aimed to use hepatocyte-specific HIF-1β knockout mice to eliminate the possible compensatory effects of the single knockout of the 1α subunit of HIF to study the role of HIFs in ALD. C57BL/6 wild type mice were treated with acute ethanol to mimic human binge drinking. Matched wild-type and hepatocyte specific HIF-1β knockout mice were also subjected to a recently established Gao-binge alcohol model to mimic chronic plus binge conditions, which is quite common in human alcoholics. We found that acute alcohol treatment increased BNIP3 and BNIP3L/NIX expression in primary cultured hepatocytes and in mouse livers, suggesting that HIF may be activated in these models. We further found that hepatocyte-specific HIF-1β knockout mice developed less steatosis and liver injury following the Gao-binge model or acute ethanol treatment compared with their matched wild type mice. Mechanistically, protection against Gao-binge treatment-induced steatosis and liver injury was likely associated with increased FoxO3a activation and subsequent induction of autophagy in hepatocyte-specific HIF-1β knockout mice.

  16. Kinetic Investigations of the Role of Factor Inhibiting Hypoxia-inducible Factor (FIH) as an Oxygen Sensor.

    Science.gov (United States)

    Tarhonskaya, Hanna; Hardy, Adam P; Howe, Emily A; Loik, Nikita D; Kramer, Holger B; McCullagh, James S O; Schofield, Christopher J; Flashman, Emily

    2015-08-07

    The hypoxia-inducible factor (HIF) hydroxylases regulate hypoxia sensing in animals. In humans, they comprise three prolyl hydroxylases (PHD1-3 or EGLN1-3) and factor inhibiting HIF (FIH). FIH is an asparaginyl hydroxylase catalyzing post-translational modification of HIF-α, resulting in reduction of HIF-mediated transcription. Like the PHDs, FIH is proposed to have a hypoxia-sensing role in cells, enabling responses to changes in cellular O2 availability. PHD2, the most important human PHD isoform, is proposed to be biochemically/kinetically suited as a hypoxia sensor due to its relatively high sensitivity to changes in O2 concentration and slow reaction with O2. To ascertain whether these parameters are conserved among the HIF hydroxylases, we compared the reactions of FIH and PHD2 with O2. Consistent with previous reports, we found lower Km(app)(O2) values for FIH than for PHD2 with all HIF-derived substrates. Under pre-steady-state conditions, the O2-initiated FIH reaction is significantly faster than that of PHD2. We then investigated the kinetics with respect to O2 of the FIH reaction with ankyrin repeat domain (ARD) substrates. FIH has lower Km(app)(O2) values for the tested ARDs than HIF-α substrates, and pre-steady-state O2-initiated reactions were faster with ARDs than with HIF-α substrates. The results correlate with cellular studies showing that FIH is active at lower O2 concentrations than the PHDs and suggest that competition between HIF-α and ARDs for FIH is likely to be biologically relevant, particularly in hypoxic conditions. The overall results are consistent with the proposal that the kinetic properties of individual oxygenases reflect their biological capacity to act as hypoxia sensors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Neuroprotective effect of electroacupuncture and upregulation of hypoxia-inducible factor-1α during acute ischaemic stroke in rats.

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    Li, Ce; Zhang, Tingting; Yu, Kewei; Xie, Hongyu; Bai, Yulong; Zhang, Li; Wu, Yi; Wang, Nianhong

    2017-10-01

    Acupuncture is a traditional method that has been widely used in various fields of medicine with therapeutic effect. However, evidence of effectiveness to support the application of electroacupuncture (EA) during the process of ischaemia is scarce. To investigate dynamic changes in hypoxia-inducible factor (HIF)-1α expression as well as its association with neurological status in rats subjected to acute ischaemic stroke and EA intervention. Forty adult male rats were randomly divided into three groups that received sham surgery (Control group, n=10) or underwent middle cerebral artery occlusion and EA (MCAO+EA group, n=15) or minimal acupuncture as a control treatment (MCAO+MA group, n=15). The rats in the MCAO+EA and MCAO+MA groups received EA or acupuncture without any electrical current, respectively, during 90 min of ischaemia. Rats in the Control group received the same surgical procedure but without MCAO. EA involved electrical stimulation of needles inserted into the quadriceps at 50 Hz frequency and 3 mA current intensity. Neurological status was evaluated on postoperative day 1, and cerebral infarction volume (IV) and HIF-1α expression 24 hours later. Neurological scores were improved and cerebral IV was decreased in the MCAO+EA group compared to the MCAO+MA group (both p<0.05). Moreover, HIF-1α expression was higher in the MCAO+EA group versus the MCAO+MA group (p<0.05). EA enhanced recovery of neurological function, decreased cerebral IV and increased HIF-1α expression in ischaemic rats. Further research is needed to determine whether EA is effective for stroke treatment through the stimulation of muscle contraction. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

  18. Fibroblast-Specific Deletion of Hypoxia Inducible Factor-1 Critically Impairs Murine Cutaneous Neovascularization and Wound Healing.

    Science.gov (United States)

    Duscher, Dominik; Maan, Zeshaan N; Whittam, Alexander J; Sorkin, Michael; Hu, Michael S; Walmsley, Graham G; Baker, Hutton; Fischer, Lauren H; Januszyk, Michael; Wong, Victor W; Gurtner, Geoffrey C

    2015-11-01

    Diabetes and aging are known risk factors for impaired neovascularization in response to ischemic insult, resulting in chronic wounds, and poor outcomes following myocardial infarction and cerebrovascular injury. Hypoxia-inducible factor (HIF)-1α, has been identified as a critical regulator of the response to ischemic injury and is dysfunctional in diabetic and elderly patients. To better understand the role of this master hypoxia regulator within cutaneous tissue, the authors generated and evaluated a fibroblast-specific HIF-1α knockout mouse model. The authors generated floxed HIF-1 mice (HIF-1) by introducing loxP sites around exon 1 of the HIF-1 allele in C57BL/6J mice. Fibroblast-restricted HIF-1α knockout (FbKO) mice were generated by breeding our HIF-1 with tamoxifen-inducible Col1a2-Cre mice (Col1a2-CreER). HIF-1α knockout was evaluated on a DNA, RNA, and protein level. Knockout and wild-type mice were subjected to ischemic flap and wound healing models, and CD31 immunohistochemistry was performed to assess vascularity of healed wounds. Quantitative real-time polymerase chain reaction of FbKO skin demonstrated significantly reduced Hif1 and Vegfa expression compared with wild-type. This finding was confirmed at the protein level (p wound closure and vascularity (p wound healing, reduced wound vascularity, and significant impairment in the ischemic neovascular response. These findings provide new insight into the importance of cell-specific responses to hypoxia during cutaneous neovascularization.

  19. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    International Nuclear Information System (INIS)

    Lee, Hae-June; Yoon, Changhwan; Park, Do Joong; Kim, Yeo-Jung; Schmidt, Benjamin; Lee, Yoon-Jin; Tap, William D.; Eisinger-Mathason, T.S. Karin; Choy, Edwin; Kirsch, David G.; Simon, M. Celeste

    2015-01-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm 3 within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm 3 for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature

  20. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hae-June [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Yoon, Changhwan [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Park, Do Joong [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Department of Surgery, Seoul National University Bundang Hospital, Sungnam (Korea, Republic of); Kim, Yeo-Jung [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Schmidt, Benjamin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Lee, Yoon-Jin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Tap, William D. [Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Eisinger-Mathason, T.S. Karin [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Choy, Edwin [Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Kirsch, David G. [Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (United States); Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina (United States); Simon, M. Celeste [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Howard Hughes Medical Institute (United States); and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  1. Comparative gene expression between two yeast species

    Directory of Open Access Journals (Sweden)

    Guan Yuanfang

    2013-01-01

    Full Text Available Abstract Background Comparative genomics brings insight into sequence evolution, but even more may be learned by coupling sequence analyses with experimental tests of gene function and regulation. However, the reliability of such comparisons is often limited by biased sampling of expression conditions and incomplete knowledge of gene functions across species. To address these challenges, we previously systematically generated expression profiles in Saccharomyces bayanus to maximize functional coverage as compared to an existing Saccharomyces cerevisiae data repository. Results In this paper, we take advantage of these two data repositories to compare patterns of ortholog expression in a wide variety of conditions. First, we developed a scalable metric for expression divergence that enabled us to detect a significant correlation between sequence and expression conservation on the global level, which previous smaller-scale expression studies failed to detect. Despite this global conservation trend, between-species gene expression neighborhoods were less well-conserved than within-species comparisons across different environmental perturbations, and approximately 4% of orthologs exhibited a significant change in co-expression partners. Furthermore, our analysis of matched perturbations collected in both species (such as diauxic shift and cell cycle synchrony demonstrated that approximately a quarter of orthologs exhibit condition-specific expression pattern differences. Conclusions Taken together, these analyses provide a global view of gene expression patterns between two species, both in terms of the conditions and timing of a gene's expression as well as co-expression partners. Our results provide testable hypotheses that will direct future experiments to determine how these changes may be specified in the genome.

  2. Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide

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    Li Zheng

    2010-10-01

    Full Text Available Abstract Background Hypoxia-inducible factor-1α (HIF-1α, a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α. Results Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide. Conclusions The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional

  3. [Effects of intermittent hypoxic exposure on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha and erythropoietin levels].

    Science.gov (United States)

    Zhang, Cheng-yan; Zhang, Ji-xin; Lü, Xiao-tao; Li, Bao-yu

    2009-10-01

    To investigate the effects of intermittent hypoxic exposure and normoxic convalescence on the parameter of erythrocyte and serum hypoxia inducible factor-1 alpha (HIF-1alpha) and erythropoietin (EPO) levels. Rat models of intermittent hypoxic exposure were established, combined with the clinical research on volunteers experiencing the intermittent plateau work. Blood samples for red blood cell (RBC) counts, hemoglobin (Hb) and hematocrit (HCT) were collected, serum HIF-1alpha and EPO levels were measured using enzyme linked immunosorbent assay. RBC counts, Hb concentration and HCT were significantly higher than the normoxic group (P hypoxic exposure can enhance serum hypoxia inducible factor-1 alpha and erythropointin levels and the generation of red blood cells, which leads to an increase in hemoglobin concentration and hematocrit. The results have changed with the hypoxic exposure period prolonged. Normoxic convalescence after intermittent hypoxic exposure can make the related indexes reduced, and contribute to the organism recovery.

  4. Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation.

    Science.gov (United States)

    Dale, Erica A; Fields, Daryl P; Devinney, Michael J; Mitchell, Gordon S

    2017-01-01

    Phrenic long-term facilitation (pLTF) is a form of hypoxia-induced spinal respiratory motor plasticity that requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, tropomyosin receptor kinase B (TrkB). Since the cellular location of relevant TrkB receptors is not known, we utilized intrapleural siRNA injections to selectively knock down TrkB receptor protein within phrenic motor neurons. TrkB receptors within phrenic motor neurons are necessary for BDNF-dependent acute intermittent hypoxia-induced pLTF, demonstrating that phrenic motor neurons are a critical site of respiratory motor plasticity. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Adenosine concentration in the porcine coronary artery wall and A2A receptor involvement in hypoxia-induced vasodilatation.

    Science.gov (United States)

    Frøbert, Ole; Haink, Gesine; Simonsen, Ulf; Gravholt, Claus H; Levin, Max; Deussen, Andreas

    2006-01-15

    We tested whether hypoxia-induced coronary artery dilatation could be mediated by an increase in adenosine concentration within the coronary artery wall or by an increase in adenosine sensitivity. Porcine left anterior descendent coronary arteries, precontracted with prostaglandin F(2alpha) (10(-5) M), were mounted in a pressure myograph and microdialysis catheters were inserted into the tunica media. Dialysate adenosine concentrations were analysed by HPLC. Glucose, lactate and pyruvate were measured by an automated spectrophotometric kinetic enzymatic analyser. The exchange fraction of [(14)C]adenosine over the microdialysis membrane increased from 0.32 +/- 0.02 to 0.46 +/- 0.02 (n = 4, P lactate/pyruvate ratio was significantly increased in hypoxic arteries but did not correlate with adenosine concentration. We conclude that hypoxia-induced coronary artery dilatation is not mediated by increased adenosine produced within the artery wall but might be facilitated by increased adenosine sensitivity at the A(2A) receptor level.

  6. Doxycycline Impairs Mitochondrial Function and Protects Human Glioma Cells from Hypoxia-Induced Cell Death: Implications of Using Tet-Inducible Systems.

    Science.gov (United States)

    Luger, Anna-Luisa; Sauer, Benedikt; Lorenz, Nadja I; Engel, Anna L; Braun, Yannick; Voss, Martin; Harter, Patrick N; Steinbach, Joachim P; Ronellenfitsch, Michael W

    2018-05-17

    Inducible gene expression is an important tool in molecular biology research to study protein function. Most frequently, the antibiotic doxycycline is used for regulation of so-called tetracycline (Tet)-inducible systems. In contrast to stable gene overexpression, these systems allow investigation of acute and reversible effects of cellular protein induction. Recent reports have already called for caution when using Tet-inducible systems as the employed antibiotics can disturb mitochondrial function and alter cellular metabolism by interfering with mitochondrial translation. Reprogramming of energy metabolism has lately been recognized as an important emerging hallmark of cancer and is a central focus of cancer research. Therefore, the scope of this study was to systematically analyze dose-dependent metabolic effects of doxycycline on a panel of glioma cell lines with concomitant monitoring of gene expression from Tet-inducible systems. We report that doxycycline doses commonly used with inducible expression systems (0.01⁻1 µg/mL) substantially alter cellular metabolism: Mitochondrial protein synthesis was inhibited accompanied by reduced oxygen and increased glucose consumption. Furthermore, doxycycline protected human glioma cells from hypoxia-induced cell death. An impairment of cell growth was only detectable with higher doxycycline doses (10 µg/mL). Our findings describe settings where doxycycline exerts effects on eukaryotic cellular metabolism, limiting the employment of Tet-inducible systems.

  7. The clinical impact of hypoxia-regulated gene expression in loco-regional gastroesophageal cancer

    DEFF Research Database (Denmark)

    Winther, M.; Alsner, J.; Tramm, T.

    2015-01-01

    Purpose/Objective: In a former study (1), the hypoxia gene expression classifier, developed in head and neck squamous cell carcinomas, was applied in 89 patients with loco-regional gastroesophageal cancer (GC). Analysis of the 15 genes was indicative of hypoxia being more profound in esophagus...... and display greater heterogeneity compared to AC. However, previous indications that the hypoxia classifier might hold prognostic significance in ESCC patients could not be confirmed. Ongoing work includes in vitro studies of esophageal cancer cell lines in order to identify alternative hypoxia induced genes...... and to further explore the prognostic value of hypoxia in patients with loco-regional gastroesophageal cancer. (Figure Presented)....

  8. KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

    International Nuclear Information System (INIS)

    Oommen, Deepu; Prise, Kevin M.

    2012-01-01

    Highlights: ► KNK437, a benzylidene lactam compound, is a novel radiosensitizer. ► KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1α under hypoxia. ► KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1α. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1α in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1α levels in KNK437-treated cells. This suggested that the absence of HIF-1α in hypoxic cells was not due to the enhanced protein degradation. HIF-1α is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1α mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1α levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

  9. Fucoidan Suppresses Hypoxia-Induced Lymphangiogenesis and Lymphatic Metastasis in Mouse Hepatocarcinoma

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    Hongming Teng

    2015-06-01

    Full Text Available Metastasis, the greatest clinical challenge associated with cancer, is closely connected to multiple biological processes, including invasion and adhesion. The hypoxic environment in tumors is an important factor that causes tumor metastasis by activating HIF-1α. Fucoidan, extracted from brown algae, is a sulfated polysaccharide and, as a novel marine biological material, has been used to treat various disorders in China, Korea, Japan and other countries. In the present study, we demonstrated that fucoidan derived from Undaria pinnatifida sporophylls significantly inhibits the hypoxia-induced expression, nuclear translocation and activity of HIF-1α, the synthesis and secretion of VEGF-C and HGF, cell invasion and lymphatic metastasis in a mouse hepatocarcinoma Hca-F cell line. Fucoidan also suppressed lymphangiogenesis in vitro and in vivo. In addition, accompanied by a reduction in the HIF-1α nuclear translocation and activity, fucoidan significantly reduced the levels of p-PI3K, p-Akt, p-mTOR, p-ERK, NF-κB, MMP-2 and MMP-9, but increased TIMP-1 levels. These results indicate strongly that the anti-metastasis and anti-lymphangiogenesis activities of fucoidan are mediated by suppressing HIF-1α/VEGF-C, which attenuates the PI3K/Akt/mTOR signaling pathways.

  10. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

    International Nuclear Information System (INIS)

    Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun

    2006-01-01

    Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Δp85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways

  11. hypoxia-inducible factors activate CD133 promoter through ETS family transcription factors.

    Directory of Open Access Journals (Sweden)

    Shunsuke Ohnishi

    Full Text Available CD133 is a cellular surface protein that has been reported to be a cancer stem cell marker, and thus it is considered to be a potential target for cancer treatment. However, the mechanism regulating CD133 expression is not yet understood. In this study, we analyzed the activity of five putative promoters (P1-P5 of CD133 in human embryonic kidney (HEK 293 cells and colon cancer cell line WiDr, and found that the activity of promoters, particularly of P5, is elevated by overexpression of hypoxia-inducible factors (HIF-1α and HIF-2α. Deletion and mutation analysis identified one of the two E-twenty six (ETS binding sites (EBSs in the P5 region as being essential for its promoter activity induced by HIF-1α and HIF-2α. In addition, a chromatin imunoprecipitation assay demonstrated that HIF-1α and HIF-2α bind to the proximal P5 promoter at the EBSs. The immunoprecipitation assay showed that HIF-1α physically interacts with Elk1; however, HIF-2α did not bind to Elk1 or ETS1. Furthermore, knockdown of both HIF-1α and HIF-2α resulted in a reduction of CD133 expression in WiDr. Taken together, our results revealed that HIF-1α and HIF-2α activate CD133 promoter through ETS proteins.

  12. Hypoxia-Inducible Factor and Its Role in the Management of Anemia in Chronic Kidney Disease

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    Joshua M. Kaplan

    2018-01-01

    Full Text Available Hypoxia-inducible factor (HIF plays a crucial role in the response to hypoxia at the cellular, tissue, and organism level. New agents under development to pharmacologically manipulate HIF may provide new and exciting possibilities in the treatment of anemia of chronic kidney disease (CKD as well as in multiple other disease states involving ischemia–reperfusion injury. This article provides an overview of recent studies describing current standards of care for patients with anemia in CKD and associated clinical issues, and those supporting the clinical potential for targeting HIF stabilization with HIF prolyl-hydroxylase inhibitors (HIF-PHI in these patients. Additionally, articles reporting the clinical potential for HIF-PHIs in ‘other’ putative therapeutic areas, the tissue and intracellular distribution of HIF- and prolyl-hydroxylase domain (PHD isoforms, and HIF isoforms targeted by the different PHDs, were identified. There is increasing uncertainty regarding the optimal treatment for anemia of CKD with poorer outcomes associated with treatment to higher hemoglobin targets, and the increasing use of iron and consequent risk of iron imbalance. Attainment and maintenance of more physiologic erythropoietin levels associated with HIF stabilization may improve the management of patients resistant to treatment with erythropoiesis-stimulating agents and improve outcomes at higher hemoglobin targets.

  13. Interleukin-17 limits hypoxia-inducible factor 1α and development of hypoxic granulomas during tuberculosis.

    Science.gov (United States)

    Domingo-Gonzalez, Racquel; Das, Shibali; Griffiths, Kristin L; Ahmed, Mushtaq; Bambouskova, Monika; Gopal, Radha; Gondi, Suhas; Muñoz-Torrico, Marcela; Salazar-Lezama, Miguel A; Cruz-Lagunas, Alfredo; Jiménez-Álvarez, Luis; Ramirez-Martinez, Gustavo; Espinosa-Soto, Ramón; Sultana, Tamanna; Lyons-Weiler, James; Reinhart, Todd A; Arcos, Jesus; de la Luz Garcia-Hernandez, Maria; Mastrangelo, Michael A; Al-Hammadi, Noor; Townsend, Reid; Balada-Llasat, Joan-Miquel; Torrelles, Jordi B; Kaplan, Gilla; Horne, William; Kolls, Jay K; Artyomov, Maxim N; Rangel-Moreno, Javier; Zúñiga, Joaquín; Khader, Shabaana A

    2017-10-05

    Mycobacterium tuberculosis (Mtb) is a global health threat, compounded by the emergence of drug-resistant strains. A hallmark of pulmonary tuberculosis (TB) is the formation of hypoxic necrotic granulomas, which upon disintegration, release infectious Mtb. Furthermore, hypoxic necrotic granulomas are associated with increased disease severity and provide a niche for drug-resistant Mtb. However, the host immune responses that promote the development of hypoxic TB granulomas are not well described. Using a necrotic Mtb mouse model, we show that loss of Mtb virulence factors, such as phenolic glycolipids, decreases the production of the proinflammatory cytokine IL-17 (also referred to as IL-17A). IL-17 production negatively regulates the development of hypoxic TB granulomas by limiting the expression of the transcription factor hypoxia-inducible factor 1α (HIF1α). In human TB patients, HIF1α mRNA expression is increased. Through genotyping and association analyses in human samples, we identified a link between the single nucleotide polymorphism rs2275913 in the IL-17 promoter (-197G/G), which is associated with decreased IL-17 production upon stimulation with Mtb cell wall. Together, our data highlight a potentially novel role for IL-17 in limiting the development of hypoxic necrotic granulomas and reducing disease severity in TB.

  14. Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

    Science.gov (United States)

    McNamee, Eóin N.; Johnson, Darlynn Korns; Homann, Dirk

    2014-01-01

    Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies. PMID:22961658

  15. Cell physiology regulation by hypoxia inducible factor-1: Targeting oxygen-related nanomachineries of hypoxic cells.

    Science.gov (United States)

    Eskandani, Morteza; Vandghanooni, Somayeh; Barar, Jaleh; Nazemiyeh, Hossein; Omidi, Yadollah

    2017-06-01

    Any dysfunctionality in maintaining the oxygen homeostasis by mammalian cells may elicit hypoxia/anoxia, which results in inescapable oxidative stress and possible subsequent detrimental impacts on certain cells/tissues with high demands to oxygen molecules. The ischemic damage in turn can trigger initiation of a number of diseases including organs ischemia, metabolic disorders, inflammatory diseases, different types of malignancies, and alteration in wound healing process. Thus, full comprehension of molecular mechanism(s) and cellular physiology of the oxygen homeostasis is the cornerstone of the mammalian cells metabolism, energetic pathways and health and disease conditions. An imbalance in oxygen content within the cellular microenvironment activates a cascade of molecular events that are often compensated, otherwise pathologic condition occurs through a complexed network of biomolecules. Hypoxia inducible factor-1 (HIF-1) plays a key transcriptional role in the adaptation of cell physiology in relation with the oxygen content within a cell. In this current study, we provide a comprehensive review on the molecular mechanisms of oxygen sensing and homeostasis and the impacts of HIF-1 in hypoxic/anoxic conditions. Moreover, different molecular and biochemical responses of the cells to the surrounding environment are discussed in details. Finally, modern technological approaches for targeting the hypoxia related proteins are articulated. Copyright © 2017. Published by Elsevier B.V.

  16. Hypoxia-inducible factor 1–mediated characteristic features of cancer cells for tumor radioresistance

    International Nuclear Information System (INIS)

    Harada, Hiroshi

    2016-01-01

    Tumor hypoxia has been attracting increasing attention in the fields of radiation biology and oncology since Thomlinson and Gray detected hypoxic cells in malignant solid tumors and showed that they exert a negative impact on the outcome of radiation therapy. This unfavorable influence has, at least partly, been attributed to cancer cells acquiring a radioresistant phenotype through the activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). On the other hand, accumulating evidence has recently revealed that, even though HIF-1 is recognized as an important regulator of cellular adaptive responses to hypoxia, it may not become active and induce tumor radioresistance under hypoxic conditions only. The mechanisms by which HIF-1 is activated in cancer cells not only under hypoxic conditions, but also under normoxic conditions, through cancer-specific genetic alterations and the resultant imbalance in intermediate metabolites have been summarized herein. The relevance of the HIF-1–mediated characteristic features of cancer cells, such as the production of antioxidants through reprogramming of the glucose metabolic pathway and cell cycle regulation, for tumor radioresistance has also been reviewed

  17. cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

    Directory of Open Access Journals (Sweden)

    Lorena Olivares-González

    Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

  18. Simulating sleep apnea by exposure to intermittent hypoxia induces inflammation in the lung and liver.

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    da Rosa, Darlan Pase; Forgiarini, Luiz Felipe; Baronio, Diego; Feijó, Cristiano Andrade; Martinez, Dênis; Marroni, Norma Possa

    2012-01-01

    Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH). IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n = 6) or a simulated IH (SIH) (n = 6) for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF-α), inducible NO synthase (iNOS), vascular endothelial growth factor (VEGF), and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

  19. Simulating Sleep Apnea by Exposure to Intermittent Hypoxia Induces Inflammation in the Lung and Liver

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    Darlan Pase da Rosa

    2012-01-01

    Full Text Available Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH. IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n=6 or a simulated IH (SIH (n=6 for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α, nuclear factor kappa B (NF-κB, and tumor necrosis factor (TNF-α, inducible NO synthase (iNOS, vascular endothelial growth factor (VEGF, and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

  20. Hypoxia-Inducible Factor-1 as a Therapeutic Target in Endometrial Cancer Management

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    Laura M. S. Seeber

    2010-01-01

    Full Text Available In the Western world, endometrial cancer (EC is the most common malignant tumor of the female genital tract. Solid tumors like EC outgrow their vasculature resulting in hypoxia. Tumor hypoxia is important because it renders an aggressive phenotype and leads to radio- and chemo-therapy resistance. Hypoxia-inducible factor-1 (HIF-1 plays an essential role in the adaptive cellular response to hypoxia and is associated with poor clinical outcome in EC. Therefore, HIF-1 could be an attractive therapeutic target. Selective HIF-1 inhibitors have not been identified. A number of nonselective inhibitors which target signaling pathways upstream or downstream HIF-1 are known to decrease HIF-1 protein levels. In clinical trials for the treatment of advanced and/or recurrent EC are the topoisomerase I inhibitor Topotecan, mTOR-inhibitor Rapamycin, and angiogenesis inhibitor Bevacizumab. Preliminary data shows encouraging results for these agents. Further work is needed to identify selective HIF-1 inhibitors and to translate these into clinical trials.

  1. Low sodium intake does not impair renal compensation of hypoxia-induced respiratory alkalosis.

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    Höhne, Claudia; Boemke, Willehad; Schleyer, Nora; Francis, Roland C; Krebs, Martin O; Kaczmarczyk, Gabriele

    2002-05-01

    Acute hypoxia causes hyperventilation and respiratory alkalosis, often combined with increased diuresis and sodium, potassium, and bicarbonate excretion. With a low sodium intake, the excretion of the anion bicarbonate may be limited by the lower excretion rate of the cation sodium through activated sodium-retaining mechanisms. This study investigates whether the short-term renal compensation of hypoxia-induced respiratory alkalosis is impaired by a low sodium intake. Nine conscious, tracheotomized dogs were studied twice either on a low-sodium (LS = 0.5 mmol sodium x kg body wt-1 x day-1) or high-sodium (HS = 7.5 mmol sodium x kg body wt-1 x day-1) diet. The dogs breathed spontaneously via a ventilator circuit during the experiments: first hour, normoxia (inspiratory oxygen fraction = 0.21); second to fourth hour, hypoxia (inspiratory oxygen fraction = 0.1). During hypoxia (arterial PO2 34.4 +/- 2.1 Torr), plasma pH increased from 7.37 +/- 0.01 to 7.48 +/- 0.01 (P respiratory alkalosis was not impaired by a low sodium intake. The increased sodium excretion during hypoxia seems to be combined with a decrease in plasma aldosterone and angiotensin II in LS as well as in HS dogs. Other factors, e.g., increased mean arterial blood pressure, minute ventilation, and renal blood flow, may have contributed.

  2. Leukemia kidney infiltration can cause secondary polycythemia by activating hypoxia-inducible factor (HIF) pathway.

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    Osumi, Tomoo; Awazu, Midori; Fujimura, Eriko; Yamazaki, Fumito; Hashiguchi, Akinori; Shimada, Hiroyuki

    2013-06-01

    Secondary polycythemia with increased production of erythropoietin (EPO) is known to occur in kidney diseases such as hydronephrosis and cystic disease, but the mechanism remains unclear. We report an 18-year-old female with isolated renal relapse of acute lymphoblastic leukemia accompanied by polycythemia. At the relapse, she presented with bilateral nephromegaly, mild renal dysfunction, and erythrocytosis with increased serum EPO levels up to 52.1 mIU/mL (9.1-32.8). Renal biopsy demonstrated diffuse lymphoblastic infiltration. The expression of hypoxia-inducible factor (HIF)-1α, which is undetectable in normal kidney, was observed in the renal tubule epithelium compressed by lymphoblastic cells. These findings suggest that erythrocytosis was caused by renal ischemia due to leukemic infiltration. Polycythemia probably became apparent because of the lack of leukemic involvement of the bone marrow. With chemotherapy, the serum EPO level rapidly decreased to normal range accompanied by the normalization of kidney size and function. Renal leukemic infiltration may enhance EPO production, although not recognized in the majority of cases because of bone marrow involvement. Our case has clarified the mechanism of previously reported polycythemia associated with renal diseases as renal ischemia. Furthermore, we have added renal ischemia resulting from tumor infiltration to the list of causes of secondary polycythemia.

  3. Hypoxia Inducible Factor Signaling and Experimental Persistent Pulmonary Hypertension of the Newborn: A Therapeutic Opportunity

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    Stephen eWedgwood

    2015-03-01

    Full Text Available BACKGROUND: Mitochondrial reactive oxygen species levels and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB activity are increased in a lamb model of persistent pulmonary hypertension of the newborn (PPHN. These events can trigger hypoxia inducible factor (HIF signaling in response to hypoxia, which has been shown to contribute to pulmonary vascular remodeling in rodent models of pulmonary hypertension. However the role of HIF signaling in chronic intrauterine pulmonary hypertension is not well understood.AIM: To determine if HIF signaling is increased in the lamb model of PPHN, and to identify the underlying mechanisms. RESULTS: PPHN was induced in lambs by antenatal ligation of the ductus arteriosus at 128 days gestation. After 9 days, lungs and pulmonary artery smooth muscle cells (PASMC were isolated from control and PPHN lambs. HIF-1α expression was increased in PPHN lungs and HIF activity was increased in PPHN PASMC relative to controls. Hypoxia increased HIF activity to a greater degree in PPHN vs. control PASMC. Control PASMC were exposed to cyclic stretch at 1Hz and 15% elongation for 24h, as an in vitro model of vascular stress. Stretch increased HIF activity, which was attenuated by inhibition of mitochondrial complex III and NFκB.CONCLUSION: Increased HIF signaling in PPHN is triggered by stretch, via mechanisms involving mitochondrial ROS and NFκB. Hypoxia substantially amplifies HIF activity in PPHN vascular cells. Targeting these signaling molecules may attenuate and reverse pulmonary vascular remodeling associated with PPHN.

  4. The histone deacetylase inhibitor, Vorinostat, represses hypoxia inducible factor 1 alpha expression through translational inhibition.

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    Darren M Hutt

    Full Text Available Hypoxia inducible factor 1α (HIF-1α is a master regulator of tumor angiogenesis being one of the major targets for cancer therapy. Previous studies have shown that Histone Deacetylase Inhibitors (HDACi block tumor angiogenesis through the inhibition of HIF-1α expression. As such, Vorinostat (Suberoylanilide Hydroxamic Acid/SAHA and Romidepsin, two HDACis, were recently approved by the Food and Drug Administration (FDA for the treatment of cutaneous T cell lymphoma. Although HDACis have been shown to affect HIF-1α expression by modulating its interactions with the Hsp70/Hsp90 chaperone axis or its acetylation status, the molecular mechanisms by which HDACis inhibit HIF-1α expression need to be further characterized. Here, we report that the FDA-approved HDACi Vorinostat/SAHA inhibits HIF-1α expression in liver cancer-derived cell lines, by a new mechanism independent of p53, prolyl-hydroxylases, autophagy and proteasome degradation. We found that SAHA or silencing of HDAC9 mechanism of action is due to inhibition of HIF-1α translation, which in turn, is mediated by the eukaryotic translation initiation factor--eIF3G. We also highlighted that HIF-1α translation is dramatically inhibited when SAHA is combined with eIF3H silencing. Taken together, we show that HDAC activity regulates HIF-1α translation, with HDACis such as SAHA representing a potential novel approach for the treatment of hepatocellular carcinoma.

  5. Negative regulation of hypoxia-inducible genes by the von Hippel-Lindau protein.

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    Iliopoulos, O; Levy, A P; Jiang, C; Kaelin, W G; Goldberg, M A

    1996-01-01

    Inactivation of the von Hippel-Lindau protein (pVHL) has been implicated in the pathogenesis of renal carcinomas and central nervous system hemangioblastomas. These are highly vascular tumors which overproduce angiogenic peptides such as vascular endothelial growth factor/vascular permeability factor (VEGF/VPF). Renal carcinoma cells lacking wild-type pVHL were found to produce mRNAs encoding VEGF/VPF, the glucose transporter GLUT1, and the platelet-derived growth factor B chain under both no...

  6. Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

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    Hagen Thilo

    2011-04-01

    Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

  7. Correlation of hypoxia inducible factor-1α and vascular endothelium growth factor in rat myocardium during aerobic and anaerobic exercise

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    Rostika Flora

    2012-08-01

    Full Text Available Background: Exercise increases the need for oxygen to generate ATP through oxidative phosphorylation. If the high energy demand during exercise is not balanced by sufficient oxygen supply, hypoxia occurs in skeletal muscle tissue leading to upregulation of hypoxia inducible factor-1α (HIF-1α. The activity of HIF-1α increases the expression of various genes in order to reduce the metabolic dependence on oxygen and to increase oxygen supply to the tissue, e.g., VEGF which plays a role in angiogenesis. In myocardium, it is unclear whether exercise leads to hypoxia and whether HIF-1α and VEGF play a role in the mechanism of hypoxic adaptation. This study aimed to investigate the correlation of HIF-1α and VEGF in heart muscle tissue of rats during aerobic and anaerobic exercise.Methods: A rat treadmill was used with a specific exercise program for 1, 3, 7 and 10 days. The concentrations of HIF-1α and VEGF were measured the myocardium.Results: Both, HIF-1α protein and VEGF were increased (p < 0.05 in the groups with aerobic and anaerobic exercise. Concentrations of HIF-1α were highest on the first day of activity, being higher in the anaerobic than in the aerobic group (156.8 ± 33.1 vs. 116.03 ± 5.66. Likewise, the highest concentration of VEGF in the group with anaerobic exercise occurred on the first day (36.37 ± 2:35, while in the aerobic group, VEGF concentration was highest on day 3 (40.66 ± 1.73. The correlation between the myocardial tissue consentrations of HIF-1α and VEGF is moderate (r = 0.59 in the aerobic group and strong in the anaerobic group (r = 0.69.Conclusion: Aerobic and anaerobic exercise increase HIF-1α and VEGF concentrations in rat myocardium in specific patterns. The anaerobic condition triggers vascularization stronger and obviously earlier than aerobic exercise. (Med J Indones. 2012;21:133-40Keywords: Exercise, HIF-1α, myocardium, VEGF

  8. Expression and function of hypoxia inducible factor-1 alpha in human melanoma under non-hypoxic conditions

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    Joshi Sandeep S

    2009-11-01

    Full Text Available Abstract Background Hypoxia inducible factor-1 alpha (HIF-1α protein is rapidly degraded under normoxic conditions. When oxygen tensions fall HIF-1α protein stabilizes and transactivates genes involved in adaptation to hypoxic conditions. We have examined the normoxic expression of HIF-1α RNA and protein in normal human melanocytes and a series of human melanoma cell lines isolated from radial growth phase (RGP, vertical growth phase (VGP and metastatic (MET melanomas. Results HIF-1α mRNA and protein was increased in RGP vs melanocytes, VGP vs RGP and MET vs VGP melanoma cell lines. We also detected expression of a HIF-1α mRNA splice variant that lacks part of the oxygen-dependent regulation domain in WM1366 and WM9 melanoma cells. Over-expression of HIF-1α and its splice variant in the RGP cell line SbCl2 resulted in a small increase in soft agar colony formation and a large increase in matrigel invasion relative to control transfected cells. Knockdown of HIF-1α expression by siRNA in the MET WM9 melanoma cell line resulted in a large decrease in both soft agar colony formation and matrigel invasion relative to cells treated with non-specific siRNA. There is a high level of ERK1/2 phosphorylation in WM9 cells, indicating an activated Ras-Raf-MEK-ERK1/2 MAPK pathway. Treatment of WM9 cells with 30 μM U0126 MEK inhibitor, decreased ERK1/2 phosphorylation and resulted in a decrease in HIF-1α expression. However, a 24 h treatment with 10 μM U0126 totally eliminated Erk1/2 phosphorylation, but did not change HIF-1alpha levels. Furthermore, siRNA knockdown of MEK siRNA did not change HIF-1alpha levels. Conclusion We speculate that metabolic products of U0126 decrease HIF-1alpha expression through "off target" effects. Overall our data suggest that increased HIF-1α expression under normoxic conditions contributes to some of the malignant phenotypes exhibited by human melanoma cells. The expanded role of HIF-1α in melanoma biology increases

  9. miR-138 protects cardiomyocytes from hypoxia-induced apoptosis via MLK3/JNK/c-jun pathway

    Energy Technology Data Exchange (ETDEWEB)

    He, Siyi; Liu, Peng; Jian, Zhao; Li, Jingwei; Zhu, Yun; Feng, Zezhou; Xiao, Yingbin, E-mail: xiaoyb@vip.sina.com

    2013-11-29

    Highlights: •First time to find miR-138 is up-regulated in hypoxic cardiomyocytes. •First time to find miR-138 targets MLK3 and regulates JNK/c-jun pathway. •Rare myocardial biopsy of patients with CHD were collected. •Both silence and overexpression of miR-138 were implemented. •Various methods were used to detect cell function. -- Abstract: Cardiomyocytes experience a series of complex endogenous regulatory mechanisms against apoptosis induced by chronic hypoxia. MicroRNAs are a class of endogenous small non-coding RNAs that regulate cellular pathophysiological processes. Recently, microRNA-138 (miR-138) has been found related to hypoxia, and beneficial for cell proliferation. Therefore, we intend to study the role of miR-138 in hypoxic cardiomyocytes and the main mechanism. Myocardial samples of patients with congenital heart disease (CHD) were collected to test miR-138 expression. Agomir or antagomir of miR-138 was transfected into H9C2 cells to investigate its effect on cell apoptosis. Higher miR-138 expression was observed in patients with cyanotic CHD, and its expression gradually increased with prolonged hypoxia time in H9C2 cells. Using MTT and LDH assays, cell growth was significantly greater in the agomir group than in the negative control (NC) group, while antagomir decreased cell survival. Dual luciferase reporter gene and Western-blot results confirmed MLK3 was a direct target of miR-138. It was found that miR-138 attenuated hypoxia-induced apoptosis using TUNEL, Hoechst staining and Annexin V-PE/7-AAD flow cytometry analysis. We further detected expression of apoptosis-related proteins. In the agomir group, the level of pro-apoptotic proteins such as cleaved-caspase-3, cleaved-PARP and Bad significantly reduced, while Bcl-2 and Bcl-2/Bax ratio increased. Opposite changes were observed in the antagomir group. Downstream targets of MLK3, JNK and c-jun, were also suppressed by miR-138. Our study demonstrates that up-regulation of miR-138 plays

  10. Hypoxia Inducible Factor-2 Alpha and Prolinhydroxylase 2 Polymorphisms in Patients with Acute Respiratory Distress Syndrome (ARDS

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    Annika Dötsch

    2017-06-01

    Full Text Available Hypoxia-inducible-factor-2α (HIF-2α and HIF-2 degrading prolyl-hydroxylases (PHD are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS. Specifically, functionally active genetic variants of HIF-2α (single nucleotide polymorphism (SNP [ch2:46441523(hg18] and PHD2 (C/T; SNP rs516651 and T/C; SNP rs480902 are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs’ prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2α or PHD2 genes are (1 common, and (2 independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay and HIF-2α-polymorphism (restriction digest + agarose-gel visualization, and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2α CC-carriers (CG: 0.38%; GG: 0%, 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%; CC: 78.8%, and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%; CC: 52.4%. PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09–10.22; p = 0.034 within 30-days, whereas the other SNPs had no significant impact (p = ns. The homozygous HIF-2α GG-genotype was not present in our Caucasian ARDS cohort; however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS.

  11. Comparative gene expression of intestinal metabolizing enzymes.

    Science.gov (United States)

    Shin, Ho-Chul; Kim, Hye-Ryoung; Cho, Hee-Jung; Yi, Hee; Cho, Soo-Min; Lee, Dong-Goo; Abd El-Aty, A M; Kim, Jin-Suk; Sun, Duxin; Amidon, Gordon L

    2009-11-01

    The purpose of this study was to compare the expression profiles of drug-metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S-transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S-transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug-metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs.

  12. Contractile Activity Is Necessary to Trigger Intermittent Hypobaric Hypoxia-Induced Fiber Size and Vascular Adaptations in Skeletal Muscle

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    David Rizo-Roca

    2018-05-01

    Full Text Available Altitude training has become increasingly popular in recent decades. Its central and peripheral effects are well-described; however, few studies have analyzed the effects of intermittent hypobaric hypoxia (IHH alone on skeletal muscle morphofunctionality. Here, we studied the effects of IHH on different myofiber morphofunctional parameters, investigating whether contractile activity is required to elicit hypoxia-induced adaptations in trained rats. Eighteen male Sprague-Dawley rats were trained 1 month and then divided into three groups: (1 rats in normobaria (trained normobaric inactive, TNI; (2 rats subjected daily to a 4-h exposure to hypobaric hypoxia equivalent to 4,000 m (trained hypobaric inactive, THI; and (3 rats subjected daily to a 4-h exposure to hypobaric hypoxia just before performing light exercise (trained hypobaric active, THA. After 2 weeks, the tibialis anterior muscle (TA was excised. Muscle cross-sections were stained for: (1 succinate dehydrogenase to identify oxidative metabolism; (2 myosin-ATPase to identify slow- and fast-twitch fibers; and (3 endothelial-ATPase to stain capillaries. Fibers were classified as slow oxidative (SO, fast oxidative glycolytic (FOG, fast intermediate glycolytic (FIG or fast glycolytic (FG and the following parameters were measured: fiber cross-sectional area (FCSA, number of capillaries per fiber (NCF, NCF per 1,000 μm2 of FCSA (CCA, fiber and capillary density (FD and CD, and the ratio between CD and FD (C/F. THI rats did not exhibit significant changes in most of the parameters, while THA animals showed reduced fiber size. Compared to TNI rats, FOG fibers from the lateral/medial fields, as well as FIG and FG fibers from the lateral region, had smaller FCSA in THA rats. Moreover, THA rats had increased NCF in FG fibers from all fields, in medial and posterior FIG fibers and in posterior FOG fibers. All fiber types from the three analyzed regions (except the posterior FG fibers displayed a

  13. Factors regulated by interferon gamma and hypoxia-inducible factor 1A contribute to responses that protect mice from Coccidioides immitis infection

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    Woelk Christopher H

    2012-09-01

    Full Text Available Abstract Background Coccidioidomycosis results from airborne infections caused by either Coccidioides immitis or C. posadasii. Both are pathogenic fungi that live in desert soil in the New World and can infect normal hosts, but most infections are self-limited. Disseminated infections occur in approximately 5% of cases and may prove fatal. Mouse models of the disease have identified strains that are resistant (e.g. DBA/2 or susceptible (e.g. C57BL/6 to these pathogens. However, the genetic and immunological basis for this difference has not been fully characterized. Results Microarray technology was used to identify genes that were differentially expressed in lung tissue between resistant DBA/2 and sensitive C57BL/6 mice after infection with C. immitis. Differentially expressed genes were mapped onto biological pathways, gene ontologies, and protein interaction networks, which revealed that innate immune responses mediated by Type II interferon (i.e., IFNG and the signal transducer and activator of transcription 1 (STAT1 contribute to the resistant phenotype. In addition, upregulation of hypoxia inducible factor 1A (HIF1A, possibly as part of a larger inflammatory response mediated by tumor necrosis factor alpha (TNFA, may also contribute to resistance. Microarray gene expression was confirmed by real-time quantitative PCR for a subset of 12 genes, which revealed that IFNG HIF1A and TNFA, among others, were significantly differentially expressed between the two strains at day 14 post-infection. Conclusion These results confirm the finding that DBA/2 mice express more Type II interferon and interferon stimulated genes than genetically susceptible strains and suggest that differential expression of HIF1A may also play a role in protection.

  14. Disrupting Hypoxia-Induced Bicarbonate Transport Acidifies Tumor Cells and Suppresses Tumor Growth.

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    McIntyre, Alan; Hulikova, Alzbeta; Ledaki, Ioanna; Snell, Cameron; Singleton, Dean; Steers, Graham; Seden, Peter; Jones, Dylan; Bridges, Esther; Wigfield, Simon; Li, Ji-Liang; Russell, Angela; Swietach, Pawel; Harris, Adrian L

    2016-07-01

    Tumor hypoxia is associated clinically with therapeutic resistance and poor patient outcomes. One feature of tumor hypoxia is activated expression of carbonic anhydrase IX (CA9), a regulator of pH and tumor growth. In this study, we investigated the hypothesis that impeding the reuptake of bicarbonate produced extracellularly by CA9 could exacerbate the intracellular acidity produced by hypoxic conditions, perhaps compromising cell growth and viability as a result. In 8 of 10 cancer cell lines, we found that hypoxia induced the expression of at least one bicarbonate transporter. The most robust and frequent inductions were of the sodium-driven bicarbonate transporters SLC4A4 and SLC4A9, which rely upon both HIF1α and HIF2α activity for their expression. In cancer cell spheroids, SLC4A4 or SLC4A9 disruption by either genetic or pharmaceutical approaches acidified intracellular pH and reduced cell growth. Furthermore, treatment of spheroids with S0859, a small-molecule inhibitor of sodium-driven bicarbonate transporters, increased apoptosis in the cell lines tested. Finally, RNAi-mediated attenuation of SLC4A9 increased apoptosis in MDA-MB-231 breast cancer spheroids and dramatically reduced growth of MDA-MB-231 breast tumors or U87 gliomas in murine xenografts. Our findings suggest that disrupting pH homeostasis by blocking bicarbonate import might broadly relieve the common resistance of hypoxic tumors to anticancer therapy. Cancer Res; 76(13); 3744-55. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Expression of hypoxia-inducible factor-1α and erythropoietin at corneal neovascularization in rats

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    Ji-Min Wang

    2014-12-01

    Full Text Available AIM: To describe the expression of hypoxia-inducible factor-1α(HIF-1αand erythropoietin(EPOin rats' corneal and evaluate its potential effect on corneal neovascularization(CNVgrowth. METHODS: The young SD rats(3mowas chosen and randomly divided into 2 groups, which were experimental group and normal control group. CNV model was established by alkali burn, and the length and area of CNV was observed everyday after operation by slit lamp. After that, the expression of HIF-1α and EPO was measured by SABC and RT-PCR methods at 1, 3, 5, 7, and 14d after alkali burn. The data was analyzed by SPSS 20.0. RESULTS: The area of CNV was increasing at 1, 3, 5, 7, and 14d after alkali burn, and the peak point appear at 7d. The growth speed was decreased after 14d. SABC method told us that no HIF-1α and very tiny amount EPO was detected at normal rats' corneal. The expression of the two factors increased at 1d after alkali burn in corneal epithelium and endoderm. The results of RT-PCR showed that a few amounts of HIF-1α and EPO mRNA were detected at normal group. The expression of the two factors was increased at 3d after alkali burn, and the peak value was found at 7d, however, it was decreased at 14d. Statistical difference was found at different time(PCONCLUSION: HIF-1α and EPO is closely related to CNV.

  16. Hypoxia-inducible factor 1α mediates neuroprotection of hypoxic postconditioning against global cerebral ischemia.

    Science.gov (United States)

    Zhu, Tingna; Zhan, Lixuan; Liang, Donghai; Hu, Jiaoyue; Lu, Zhiwei; Zhu, Xinyong; Sun, Weiwen; Liu, Liu; Xu, En

    2014-10-01

    Hypoxia administered after transient global cerebral ischemia (tGCI) has been shown to induce neuroprotection in adult rats, but the underlying mechanisms for this protection are unclear. Here, we tested the hypothesis that hypoxic postconditioning (HPC) induces neuroprotection through upregulation of hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF), and that this involves phosphatidylinositol-3-kinase (PI3K), p38 mitogen-activated protein kinase (p38 MAPK), and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) pathways. The expression of HIF-1α, VEGF, and cleaved caspase-9 were determined by immunohistochemistry and Western blot. As pharmacologic interventions, the HIF-1α inhibitor 2-methoxyestradiol (2ME2), PI3K inhibitor LY294002, p38 MAPK inhibitor SB203580, and MEK inhibitor U0126 were administered before HPC or after tGCI. We found that HPC maintained the higher expression of HIF-1α and VEGF and decreased cleaved caspase-9 levels in CA1 after tGCI. These effects were reversed by 2ME2 administered before HPC, and the neuroprotection of HPC was abolished. LY294002 and SB203580 decreased the expression of HIF-1α and VEGF after HPC, whereas U0126 increased HIF-1α and VEGF after tGCI. These findings suggested that HIF-1α exerts neuroprotection induced by HPC against tGCI through VEGF upregulation and cleaved caspase-9 downregulation, and that the PI3K, p38 MAPK, and MEK pathways are involved in the regulation of HIF-1α and VEGF.

  17. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    Science.gov (United States)

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  18. Potent inhibition of tumoral hypoxia-inducible factor 1α by albendazole

    International Nuclear Information System (INIS)

    Pourgholami, Mohammad H; Cai, Zhao Y; Badar, Samina; Wangoo, Kiran; Poruchynsky, Marianne S; Morris, David L

    2010-01-01

    Emerging reports suggest resistance, increased tumor invasiveness and metastasis arising from treatment with drugs targeting vascular endothelial growth factor (VEGF). It is believed that increased tumoral hypoxia plays a prominent role in the development of these phenomena. Inhibition of tumoral hypoxia inducible factor (HIF-1α) is thus becoming an increasingly attractive therapeutic target in the treatment of cancer. We hypothesized that the anti-VEGF effect of albendazole (ABZ) could be mediated through inhibition of tumoral HIF-1α. In vitro, the effects of ABZ on HIF-1α levels in human ovarian cancer cells (OVCAR-3) were investigated using hypoxic chamber or desferrioxamine (DFO) induced-hypoxia. In vivo, the effects of ABZ (150 mg/kg, i.p., single dose) on the tumor levels of HIF-1α and VEGF protein and mRNA were investigated by western blotting, RT-PCR and real time-PCR. In vitro, ABZ inhibited cellular HIF-1α protein accumulation resulting from placement of cells under hypoxic chamber or exposure to DFO. In vivo, tumors excised from vehicle treated mice showed high levels of both HIF-1α and VEGF. Whereas, tumoral HIF-1α and VEGF protein levels were highly suppressed in ABZ treated mice. Tumoral VEGFmRNA (but not HIF-1αmRNA) was also found to be highly suppressed by ABZ. These results demonstrate for the first time the effects of an acute dose of ABZ in profoundly suppressing both HIF-1α and VEGF within the tumor. This dual inhibition may provide additional value in inhibiting angiogenesis and be at least partially effective in inhibiting tumoral HIF-1α surge, tumor invasiveness and metastasis

  19. Disparate roles of zinc in chemical hypoxia-induced neuronal death

    Directory of Open Access Journals (Sweden)

    Sujeong eKim

    2015-01-01

    Full Text Available 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.

  20. Disparate roles of zinc in chemical hypoxia-induced neuronal death.

    Science.gov (United States)

    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.

  1. Radiosensitive effect of hypoxia-inducible factor 1α inhibitor YC-1 on hypoxic glioma SHG44 cell line

    International Nuclear Information System (INIS)

    Guo Xinwei; Lu Xueguan; Tong Liumei; Zong Tianzhou; Chen Liesong

    2011-01-01

    Objective: To investigate the radiosensitive effect of hypoxia-inducible factor 1α (HIF-1α) inhibitor YC-1 on hypoxic glioma SHG44 cell line and its related mechanism. Methods: Glioma SHG44 cell line was cultured in normoxic (20% O 2 ), continuous hypoxia (1% O 2 ) for 12 h and 24 h, continuous hypoxia plus YC-1 was performed for 12 h and 24 h, respectively. The expression of HIF-1α was assessed by Western blot. The radiosensitivity was evaluated by the survival curve, and the sublethal damage repair (SLDR) ability was measured by dose-fraction experiment. Results: HIF-1α protein levels of glioma SHG44 cells were significantly increased after hypoxic cultures for 12 h and 24 h than those of the corresponding cells cultured in normoxic, while the radiosensitivity was lower. The OER (oxygen-enhancement ratio) of SHG44 cells in hypoxia for 12 h and 24 h were 1.22 and 1.37, respectively. By the further statistical analysis it was found that SLDR ability of glioma SHG44 was increased at hypoxia, and when irradiation was carried one at the interval of 8, 10, 12 h it was statistically significant (P<0.05). HIF-1α protein levels of glioma SHG44 cells cultured in hypoxia plus YC-1 for 12 h and 24 h were decreased significantly compared to the corresponding cells cultured in hypoxia only, while the radiosensitivity was significantly increased. the EF (enhancement factor) of YC-1 for glioma SHG44 cells at hypoxia for 12 h and 24 h was 1.27. By the further statistical analysis it was also found that SLDR ability was decreased significantly for hypoxic SHG44 cells which was co-cultured with YC-1, and at the interval of 8, 10, 12 h irradiation was statistically significant (P<0.05). Conclusion: YC-1 can increase the radiosensitivity of hypoxic glioma SHG44 cell line, and its mechanism is related to SLDR inhibited by YC-1. (authors)

  2. [Altered expressions of alkane monooxygenase and hypoxia inducible factor-1α expression in lung tissue of rat hypoxic pulmonary hypertension].

    Science.gov (United States)

    Deng, Hua-jun; Yuan, Ya-dong

    2013-10-29

    To explore the altered expressions of alkane monooxygenase (AlkB) and hypoxia-inducible factor-1α (HIF-1α) in a rat model of hypoxic pulmonary arterial hypertension. Twenty Wistar rats were divided randomly into normal control and hypoxia groups after 1-week adaptive feeding. Hypoxia group was raised in a homemade organic glass tank with a 24-h continuous supply of air and nitrogen atmospheric mixed gas. And the oxygen concentration of (10.0 ± 0.5)% was controlled by oxygen monitoring control system. The control group was maintained in room air. Both groups stayed in the same room with the same diet. After 8 weeks, the level of mean pulmonary pressure (mPAP) was measured by right-heart catheterization, right ventricular hypertrophy index (RVHI) calculated by the ratio of right ventricle to left ventricle plus septum and hypoxic pulmonary vascular remodeling (HPSR) observed under microscope. And the levels of AlkB and HIF-1α mRNA and protein in lungs were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. At 8 weeks post-hypoxia, compared with the control group [11.0 ± 0.7 mm Hg (1 mm Hg = 0.133 kPa), 0.210 ± 0.035], the levels of mPAP and RVHI in hypoxia group (33.3 ± 1.3 mm Hg, 0.448 ± 0.013) increased significantly (both P < 0.05), the expressions of AlkB mRNA and protein in pulmonary tissue decreased significantly (0.338 ± 0.085 vs 0.688 ± 0.020, P < 0.01) (0.483 ± 0.052 vs 0.204 ± 0.010, P < 0.01), and the expressions of HIF-1α mRNA and protein increased significantly (0.790 ± 0.161 vs 0.422 ± 0.096, P < 0.01) (0.893 ± 0.080 vs 0.346 ± 0.008, P < 0.01). The down-regulation of AlkB in lung tissue may increase the activity of HIF-1 to participate in the occurrence and development of pulmonary hypertension.

  3. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

    2012-10-15

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

  4. Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer.

    Science.gov (United States)

    Bouquerel, P; Gstalder, C; Müller, D; Laurent, J; Brizuela, L; Sabbadini, R A; Malavaud, B; Pyronnet, S; Martineau, Y; Ader, I; Cuvillier, O

    2016-03-14

    The sphingosine kinase-1/sphingosine 1-phosphate (SphK1/S1P) signaling pathway has been reported to modulate the expression of the canonical transcription factor hypoxia-inducible HIF-1α in multiple cell lineages. HIF-2α is also frequently overexpressed in solid tumors but its role has been mostly studied in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, where HIF-2α has been established as a driver of a more aggressive disease. In this study, the role of SphK1/S1P signaling with regard to HIF-2α was investigated in various cancer cell models including ccRCC cells. Under hypoxic conditions or in ccRCC lacking a functional von Hippel-Lindau (VHL) gene and expressing high levels of HIF-2α, SphK1 activity controls HIF-2α expression and transcriptional activity through a phospholipase D (PLD)-driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, downregulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Taking advantage of a monoclonal antibody neutralizing extracellular S1P, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 or the S1P receptor 1 (S1P1) is silenced. Here, we report the first evidence that the SphK1/S1P signaling pathway regulates the transcription factor hypoxia-inducible HIF-2α in diverse cancer cell lineages notably ccRCC, where HIF-2α has been established as a driver of a more aggressive disease. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

  5. Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

    International Nuclear Information System (INIS)

    Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

    2012-01-01

    Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl 2 -induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical

  6. Comparative Genomic Analysis of Soybean Flowering Genes

    Science.gov (United States)

    Jung, Chol-Hee; Wong, Chui E.; Singh, Mohan B.; Bhalla, Prem L.

    2012-01-01

    Flowering is an important agronomic trait that determines crop yield. Soybean is a major oilseed legume crop used for human and animal feed. Legumes have unique vegetative and floral complexities. Our understanding of the molecular basis of flower initiation and development in legumes is limited. Here, we address this by using a computational approach to examine flowering regulatory genes in the soybean genome in comparison to the most studied model plant, Arabidopsis. For this comparison, a genome-wide analysis of orthologue groups was performed, followed by an in silico gene expression analysis of the identified soybean flowering genes. Phylogenetic analyses of the gene families highlighted the evolutionary relationships among these candidates. Our study identified key flowering genes in soybean and indicates that the vernalisation and the ambient-temperature pathways seem to be the most variant in soybean. A comparison of the orthologue groups containing flowering genes indicated that, on average, each Arabidopsis flowering gene has 2-3 orthologous copies in soybean. Our analysis highlighted that the CDF3, VRN1, SVP, AP3 and PIF3 genes are paralogue-rich genes in soybean. Furthermore, the genome mapping of the soybean flowering genes showed that these genes are scattered randomly across the genome. A paralogue comparison indicated that the soybean genes comprising the largest orthologue group are clustered in a 1.4 Mb region on chromosome 16 of soybean. Furthermore, a comparison with the undomesticated soybean (Glycine soja) revealed that there are hundreds of SNPs that are associated with putative soybean flowering genes and that there are structural variants that may affect the genes of the light-signalling and ambient-temperature pathways in soybean. Our study provides a framework for the soybean flowering pathway and insights into the relationship and evolution of flowering genes between a short-day soybean and the long-day plant, Arabidopsis. PMID:22679494

  7. A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4

    Energy Technology Data Exchange (ETDEWEB)

    Ando, Hitoshi [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Department of Neurosurgery, Fukushima Medical University School of Medicine, Fukushima (Japan); Natsume, Atsushi, E-mail: anatsume@med.nagoya-u.ac.jp [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Iwami, Kenichiro; Ohka, Fumiharu [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan); Kuchimaru, Takahiro; Kizaka-Kondoh, Shinae [Department of Biomolecular Engineering, Tokyo Institute of Technology Graduate School of Bioscience and Biotechnology, Yokohama (Japan); Ito, Kengo [National Center for Geriatrics and Gerontology, Aichi (Japan); Saito, Kiyoshi [Department of Neurosurgery, Fukushima Medical University School of Medicine, Fukushima (Japan); Sugita, Sachi; Hoshino, Tsuneyoshi [MICRON Inc.Medical Facilities Support Department, Aichi (Japan); Wakabayashi, Toshihiko [Department of Neurosurgery, Nagoya University School of Medicine, Nagoya (Japan)

    2013-03-29

    Highlights: ► HIF-3α4 is silenced by DNA methylation in meningiomas. ► Induction of HIF-3α4 impaired angiogenesis in meningiomas. ► Induction of HIF-3α4 impaired proliferation and oxygen-dependent metabolism. -- Abstract: Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas.

  8. Enzyme 15-lipoxygenase 1 promotes hypoxia-inducible factor 1α turnover and reduces vascular endothelial growth factor expression: implications for angiogenesis

    International Nuclear Information System (INIS)

    Zhong, Hua; Wang, Ruoxiang; Kelavkar, Uddhav; Wang, Christopher Y; Simons, Jonathan

    2014-01-01

    Hypoxia-inducible factor 1α (HIF-1α) is the regulatory subunit of the heterodimeric HIF-1 that plays a critical role in transcriptional regulation of genes in angiogenesis and hypoxic adaptation, while fatty acid metabolism mediated by lipoxygenases has been implicated in a variety of pathogeneses, including cancers. In this study, we report that 15-lipoxygenase 1 (15-LO1), a key member of the lipoxygenase family, promotes HIF-1α ubiquitination and degradation. Altering the level of 15-LO1 yields inverse changes in HIF-1α and HIF-1 transcriptional activity, under both normoxia and hypoxia, and even in CoCl 2 -treated cells where HIF-1α has been artificially elevated. The antagonistic effect of 15-LO1 is mediated by the Pro 564 /hydroxylation/26S proteasome system, while both the enzymatic activity and the intracellular membrane-binding function of 15-LO1 appear to contribute to HIF-1α suppression. Our findings provide a novel mechanism for HIF-1α regulation, in which oxygen-dependent HIF-1 activity is modulated by an oxygen-insensitive lipid metabolic enzyme

  9. Hypoxia inducible factor 1-alpha (HIF-1 alpha is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

    Directory of Open Access Journals (Sweden)

    Elisa Conde

    Full Text Available Acute tubular necrosis (ATN caused by ischemia/reperfusion (I/R during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α, using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

  10. Silencing of osteopontin promotes the radiosensitivity of breast cancer cells by reducing the expression of hypoxia inducible factor 1 and vascular endothelial growth factor

    Institute of Scientific and Technical Information of China (English)

    YANG Li; ZHAO Wei; ZUO Wen-shu; WEI Ling; SONG Xian-rang; WANG Xing-wu; ZHENG Gang; ZHENG Mei-zhu

    2012-01-01

    Background Osteopontin (OPN) is a secreted phosphoglycoprotein (SSP) that is overexpressed in a variety of tumors and was regarded as a molecular marker of tumors.In this study,we intended to demonstrate the role of OPN in human breast cancer cell line MDA-MB-231.Methods Recombinant plasmid expressing small interfering RNA (siRNA) specific to OPN mRNA was transfected into MDA-MB-231 cells to generate the stable transfected cell line MDA-MB-343,and the empty plasmid tansfected cells (MDA-MB-neg) or wildtype MDA-MB-231 cells were used as control cells respectively.Expression of OPN,hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) proteins was analyzed by Western blotting analysis.The radiosensitivity of cells was determined by detecting cell apoptosis,cell proliferation and cell senescence.Results HIF-1 and VEGF proteins in MDA-MB-343 cells were significantly downregulated upon the efficient knockdown of OPN expression under either hypoxia or normoxia environment.Moreover,expression of OPN protein was upregualted upon hypoxic culture.Stable OPN-silencing also decreased cell invasion,increased cell apoptosis and cell senescence,as well as reduced clonogenic survival,resulting in increase radiation tolerance.Conclusions Suppression of OPN gene expression can enhance radiosensitivity and affect cell apoptosis in breast cancer cells.OPN seems to be an attractive target for the improvement of radiotherapy.

  11. Hypoxia-inducible factor 1 alpha is a poor prognostic factor and potential therapeutic target in malignant peripheral nerve sheath tumor.

    Directory of Open Access Journals (Sweden)

    Suguru Fukushima

    Full Text Available Malignant peripheral nerve sheath tumor (MPNST is a rare soft tissue sarcoma with poor prognosis. Hypoxia-inducible factor 1 (HIF-1 plays a crucial role in the cellular response to hypoxia and regulates the expression of multiple genes involved in tumor progression in various cancers. However, the importance of the expression of HIF-1α in MPNSTs is unclear.The expression of HIF-1α was examined immunohistochemically in 82 MPNST specimens. Cell culture assays of human MPNST cells under normoxic and hypoxic conditions were used to evaluate the impact of anti-HIF-1α-specific siRNA inhibition on cell survival. A screening kit was employed to identify small molecules that inhibited HIF-1α.The nuclear expression of HIF-1α was positive in 75.6% of MPNST samples (62/82 cases. Positivity for HIF-1α was a significant poor prognostic factor both in univariate (P = 0.048 and multivariate (P ≤ 0.0001 analyses. HIF-1α knockdown abrogated MPNST cell growth, inducing apoptosis. Finally, chetomin, an inhibitor of HIF-1α, effectively inhibited the growth of MPNST cells and induced their apoptosis.Inhibition of HIF-1α signaling is a potential treatment option for MPNSTs.

  12. Hypoxia-Inducible Factor-1α: A Potential Factor for the Enhancement of Osseointegration between Dental Implants and Tissue-Engineered Bone

    Directory of Open Access Journals (Sweden)

    Duohong Zou

    2011-07-01

    Full Text Available Introduction: Tissue-engineered bones are widely utilized to protect healthy tissue, reduce pain, and increase the success rate of dental implants. one of the most challenging obstacles lies in obtaining effective os-seointegration between dental implants and tissue-engineered structures. Deficiencies in vascularization, osteogenic factors, oxygen, and other nutrients inside the tissue-engineered bone during the early stages following implantation all inhibit effective osseointe-gration. Oxygen is required for aerobic metabolism in bone and blood vessel tissues, but oxygen levels inside tissue-engineered bone are not suf-ficient for cell proliferation. HIF-1α is a pivotal regulator of hypoxic and ischemic vascular responses, driving transcriptional activation of hundreds of genes involved in vascular reactivity, angiogenesis, arteriogenesis, and osteogenesis.The hypothesis: Hypoxia-Inducible Factor-1α seems a potential factor for the enhancement of osseointegration between dental implants and tissue-engineered bone.Evaluation of the hypothesis: Enhancement of HIF-1α protein expression is recognized as the most promising approach for angiogenesis, because it can induce multiple angiogenic targets in a coordinated manner. Therefore, it will be a novel potential therapeutic methods targeting HIF-1α expression to enhance osseointegration be-tween dental implants and tissue-engineered bone.

  13. Radiation protective effect of hypoxia-inducible factor-1α (HIF-1α) on human oral squamous cell carcinoma cell lines

    International Nuclear Information System (INIS)

    Hosokawa, Y.; Okumura, K.; Terashima, S.; Sakakura, Y.

    2012-01-01

    We examined the effects of 5-Gy radiation on the expression of hypoxia-inducible factor-1α (HIF-1α) and the radiosensitivity of five human oral squamous cell carcinoma (OSCC) cell lines (SAS, Ca9-22, TT, BSC-OF and IS-FOM). In all of the cell lines, HIF-1α was expressed in mRNA, and radiation had no influence on gene transcription. The number of apoptotic cells increased 72 h after irradiation in cell lines SAS, Ca9-22 and TT cells, indicating low transcriptional levels of HIF-1α, and the levels of non-cleaved caspase-3, an executioner of apoptosis, and non-cleaved poly (adenosine diphosphate-ribose) polymerase (PARP), a marker of DNA damage early in apoptosis, decreased simultaneously. Conversely, radiation failed to induce apoptosis or to decrease expression of non-cleaved caspase-3 and PARP in cell-lines BSC-OF and IS-FOM cells that expressed high levels of HIF-1α. BSC-OF and IS-FOM cells exhibited high migratory capacity. When CoCl 2 was present in the medium, HIF-1α expression increased along with the survival of Ca9-22 cells after radiation exposure. These results suggest that OSCC cells expressing high levels of HIF-1α are resistant to radiation. HIF-1α can be used to control the short term radiosensitivity of cells. (authors)

  14. A hypoxia-inducible factor (HIF)-3α splicing variant, HIF-3α4 impairs angiogenesis in hypervascular malignant meningiomas with epigenetically silenced HIF-3α4

    International Nuclear Information System (INIS)

    Ando, Hitoshi; Natsume, Atsushi; Iwami, Kenichiro; Ohka, Fumiharu; Kuchimaru, Takahiro; Kizaka-Kondoh, Shinae; Ito, Kengo; Saito, Kiyoshi; Sugita, Sachi; Hoshino, Tsuneyoshi; Wakabayashi, Toshihiko

    2013-01-01

    Highlights: ► HIF-3α4 is silenced by DNA methylation in meningiomas. ► Induction of HIF-3α4 impaired angiogenesis in meningiomas. ► Induction of HIF-3α4 impaired proliferation and oxygen-dependent metabolism. -- Abstract: Hypoxia inducible factor is a dominant regulator of adaptive cellular responses to hypoxia and controls the expression of a large number of genes regulating angiogenesis as well as metabolism, cell survival, apoptosis, and other cellular functions in an oxygen level-dependent manner. When a neoplasm is able to induce angiogenesis, tumor progression occurs more rapidly because of the nutrients provided by the neovasculature. Meningioma is one of the most hypervascular brain tumors, making anti-angiogenic therapy an attractive novel therapy for these tumors. HIF-3α has been conventionally regarded as a dominant-negative regulator of HIF-1α, and although alternative HIF-3α splicing variants are extensively reported, their specific functions have not yet been determined. In this study, we found that the transcription of HIF-3α4 was silenced by the promoter DNA methylation in meningiomas, and inducible HIF-3α4 impaired angiogenesis, proliferation, and metabolism/oxidation in hypervascular meningiomas. Thus, HIF-3α4 could be a potential molecular target in meningiomas

  15. Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxia-induced apoptosis of retinal ganglion cells.

    Science.gov (United States)

    Yuan, Jing; Yu, Jian-Xiong

    2016-05-01

    Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

  16. Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

    Science.gov (United States)

    Cao, Bo; Chai, Chunxiang; Zhao, Sishun

    2015-12-01

    Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

  17. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

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    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  18. [Research progress of mechanism of hypoxia-inducible factor-1α signaling pathway in condylar cartilage growth and remodeling].

    Science.gov (United States)

    Gaoli, Xu; Lili, Wu; Zhiwu, Wu; Zhiyuan, Gu

    2016-12-01

    The condylar cartilage was adapted to hypoxic conditions in vivo. However, condylar cartilage cells exposed in normoxia in vitro affect the chondrocyte phenotype and cartilage matrix formation. This condition also resulted in great difficulty in chondrocyte research. Culturing chondrocyte should be simulated in in vivo hypoxia environment as much as possible. The hypoxia-inducible factor-1α (HIF-1α) demonstrates an important transcription factor of adaptive response to hypoxic conditions. HIF-1α also plays an active role in maintaining homeostasis and function of chondrocytes. This review summarized current knowledge of the HIF-1α structure, signaling pathway, and mechanism of HIF-1α in the condylar cartilage repair.

  19. 2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study

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    Karina Zitta

    2018-01-01

    Full Text Available 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

  20. Phrenic motor neuron TrkB expression is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

    OpenAIRE

    Dale, Erica A.; Fields, Daryl P.; Devinney, Michael J.; Mitchell, Gordon S.

    2016-01-01

    Phrenic long-term facilitation (pLTF) is a form of hypoxia-induced spinal respiratory motor plasticity that requires new synthesis of brain derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, tropomyosin receptor kinase B (TrkB). Since the cellular location of relevant TrkB receptors is not known, we utilized intrapleural siRNA injections to selectively knock down TrkB receptor protein within phrenic motor neurons. TrkB receptors within phrenic motor neurons are n...

  1. Changes in Hypoxia-Inducible Factor-1 (HIF-1) and Regulatory Prolyl Hydroxylase (PHD) Enzymes Following Hypoxic-Ischemic Injury in the Neonatal Rat.

    Science.gov (United States)

    Chu, Hannah X; Jones, Nicole M

    2016-03-01

    Hypoxia leads to activation of many cellular adaptive processes which are regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 consists of HIF-1α and HIF-1ß subunits and levels of HIF-1α protein are regulated by HIF prolyl-hydroxylase enzymes (PHD1, 2, 3). The aim of the current study was to investigate the expression of HIF-1α and PHDs at various time points after hypoxia-ischemia (HI), using a neonatal rat model of HI brain injury. Sprague-Dawley rat pups (postnatal day 7) were anaesthetized and underwent right carotid artery occlusion and were then exposed to 6 % oxygen for 2.5 h at 37 °C. HI injured animals demonstrated a significant reduction in the size of the ipsilateral hemisphere, compared to sham controls. Protein analysis using western blotting and enzyme-linked immunosorbent assay showed that 24 h after HI, there was a significant increase in PHD3 protein and an increase of HIF-1α compared to controls. At the 72 h time point, there was a reduction in PHD3 protein, which appeared to relate to cellular loss. There were no changes in PHD1 or PHD2 protein levels after HI when compared to age-matched controls. Further studies are necessary to establish roles for the HIF-1 regulatory enzyme PHD3 in brain injury processes.

  2. The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

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    Mehtap Kilic Eren

    Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

  3. SurR9C84A protects and recovers human cardiomyocytes from hypoxia induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Ashok, Ajay [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Department of Pathology, Case Western Reserve University, 2103 Cornell Rd. WRB 5128, Cleveland, OH 44106-7288 (United States); Kanwar, Jagat Rakesh [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia); Krishnan, Uma Maheswari [Centre for Nanotechnology & Advanced Biomaterials (CeNTAB), School of Chemical & Biotechnology (SCBT), SASTRA University, Thanjavur 613401 (India); Kanwar, Rupinder Kaur, E-mail: rupinder.kanwar@deakin.edu.au [Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Faculty of Health, Centre for Molecular and Medical Research - C-MMR, Deakin University, Waurn Ponds, Victoria 3216 (Australia)

    2017-01-01

    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 hypoxic 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

  4. Developmental Expression and Hypoxic Induction of Hypoxia Inducible Transcription Factors in the Zebrafish.

    Science.gov (United States)

    Köblitz, Louise; Fiechtner, Birgit; Baus, Katharina; Lussnig, Rebecca; Pelster, Bernd

    2015-01-01

    The hypoxia inducible transcription factor (HIF) has been shown to coordinate the hypoxic response of vertebrates and is expressed in three different isoforms, HIF-1α, HIF-2α and HIF-3α. Knock down of either Hif-1α or Hif-2α in mice results in lethality in embryonic or perinatal stages, suggesting that this transcription factor is not only controlling the hypoxic response, but is also involved in developmental phenomena. In the translucent zebrafish embryo the performance of the cardiovascular system is not essential for early development, therefore this study was designed to analyze the expression of the three Hif-isoforms during zebrafish development and to test the hypoxic inducibility of these transcription factors. To complement the existing zfHif-1α antibody we expressed the whole zfHif-2α protein and used it for immunization and antibody generation. Similarly, fragments of the zfHif-3α protein were used for immunization and generation of a zfHif-3α specific antibody. To demonstrate presence of the Hif-isoforms during development [between 1 day post fertilization (1 dpf) and 9 dpf] affinity-purified antibodies were used. Hif-1α protein was present under normoxic conditions in all developmental stages, but no significant differences between the different developmental stages could be detected. Hif-2α was also present from 1 dpf onwards, but in post hatching stages (between 5 and 9 dpf) the expression level was significantly higher than prior to hatching. Similarly, Hif-3α was expressed from 1 dpf onwards, and the expression level significantly increased until 5 dpf, suggesting that Hif-2α and Hif-3α play a particular role in early development. Hypoxic exposure (oxygen partial pressure = 5 kPa) in turn caused a significant increase in the level of Hif-1α protein even at 1 dpf and in later stages, while neither Hif-2α nor Hif-3α protein level were affected. In these early developmental stages Hif-1α therefore appears to be more important for

  5. Prognostic role of hypoxia-inducible factor-1 alpha expression in osteosarcoma: a meta-analysis

    Directory of Open Access Journals (Sweden)

    Ren HY

    2016-03-01

    Full Text Available Hai-Yong Ren,1 Yin-Hua Zhang,1,2 Heng-Yuan Li,1 Tao Xie,1 Ling-Ling Sun,1 Ting Zhu,1 Sheng-Dong Wang,1 Zhao-Ming Ye1 1Department of Orthopaedics, Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, 2The First Department of Orthopaedics, Hospital of Zhejiang General Corps of Armed Police Forces, Jiaxing, People’s Republic of China Background: Hypoxia-inducible factor-1α (HIF-1α plays an important role in tumor progression and metastasis. A number of studies have investigated the association of HIF-1α with prognosis and clinicopathological characteristics of osteosarcoma but yielded inconsistent results.  Method: Systematic computerized searches were performed in PubMed, Embase, and Web of Science databases for relevant original articles. The pooled hazard ratios (HRs and odds ratios (ORs with corresponding confidence intervals (CIs were calculated to assess the prognostic value of HIF-1α expression. The standard mean difference was used to analyze the continuous variable.  Results: Finally, nine studies comprising 486 patients were subjected to final analysis. Protein expression level of HIF-1α was found to be significantly related to overall survival (HR =3.0; 95% CI: 1.46–6.15, disease-free survival (HR =2.23; 95% CI: 1.26–3.92, pathologic grade (OR =21.33; 95% CI: 4.60–98.88, tumor stage (OR =10.29; 95% CI: 3.55–29.82, chemotherapy response (OR =9.68; 95% CI: 1.87–50.18, metastasis (OR =5.06; 95% CI: 2.87–8.92, and microvessel density (standard mean difference =2.83; 95% CI: 2.28–3.39.  Conclusion: This meta-analysis revealed that overexpression of HIF-1α is a predictive factor of poor outcomes for osteosarcoma. HIF-1α appeared to play an important role in prognostic evaluation and may be a potential target in antitumoral therapy. Keywords: HIF-1α, osteosarcoma, prognosis, meta-analysis

  6. The role of metformin and resveratrol in the prevention of hypoxia-inducible factor 1α accumulation and fibrosis in hypoxic adipose tissue.

    Science.gov (United States)

    Li, Xiaole; Li, Jia; Wang, Lulu; Li, Aiyun; Qiu, Zhixia; Qi, Lian-Wen; Kou, Junping; Liu, Kang; Liu, Baolin; Huang, Fang

    2016-06-01

    Hypoxic activation of hypoxia-inducible factor 1α (HIF-1α) and fibrosis in adipose tissue contribute to adipose dysfunction. This study was designed to investigate the effects of metformin and resveratrol on the regulation of HIF-1α and fibrosis in hypoxic adipose tissue. Mice were fed a high-fat diet to induce hypoxia and fibrosis in adipose tissue; adipose tissue incubated in vitro in 1% O2 showed a similar change. The effects of metformin and resveratrol on hypoxia, HIF-1α accumulation, endoplasmic reticulum stress and gene expressions of extracellular matrix components and pro-inflammatory cytokines were examined. Oral administration of metformin or resveratrol prevented hypoxia and reduced HIF-1α accumulation with dephosphorylation of inositol-requiring enzyme 1α and eukaryotic initiation factor 2α, indicative of suppression of hypoxic HIF-1α activation and endoplasmic reticulum stress. Metformin and resveratrol down-regulated gene expressions of Col3α, Col6α, elastin and lysyl oxidase and thereby reduced collagen deposition in adipose tissue. The increased gene expressions of TNF-α, IL-6, monocyte chemoattractant protein 1 and F4/80 were also down-regulated by metformin and resveratrol. Metformin and resveratrol had similar effects in adipose tissue exposed to 1% O2 . Metformin reduced ATP production and prevented the reduction in oxygen tension in 3T3-L1 cells, suggesting that it prevented hypoxia by limiting oxygen consumption, whereas resveratrol reduced HIF-1α accumulation by promoting its proteasomal degradation via the regulation of AMPK/SIRT1. Hypoxia and fibrosis are early causes of adipose dysfunction in obesity. Both metformin and resveratrol effectively inhibited HIF-1α activation-induced fibrosis and inflammation in adipose tissue, although by different mechanisms. © 2016 The British Pharmacological Society.

  7. Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions.

    Science.gov (United States)

    Xia, Tian; Cheng, Hao; Zhu, Yu

    2014-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in tumor cell adaption to hypoxia by inducing the transcription of numerous genes. The role of HIF-1α in malignant retinoblastoma remains unclear. We analyzed the role of HIF-1α in WERI-Rb-1 retinoblastoma cells under hypoxic conditions. CoCl2 (125 mmol/L) was added to the culture media to mimic hypoxia. HIF-1α was silenced using siRNA. Gene and protein expression were measured by semi-quantitative RT-PCR and Western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Cell proliferation, adhesion and invasion were assayed using MTT, Transwell invasion, and cell adhesion assays respectively. Hypoxia significantly upregulated HIF-1α protein expression and the HIF-1α target genes VEGF, GLUT1, and Survivin mRNA. HIF-1α mRNA expression was not affected by hypoxia. Transfection of the siRNA expression plasmid pRNAT-CMV3.2/Neo-HIF-1α silenced HIF-1α by approximately 80% in hypoxic WERI-Rb-1 cells. The knockdown of HIF-1α under hypoxic conditions downregulated VEGF, GLUT1, and Survivin mRNA. It also inhibited proliferation, promoted apoptosis, induced the G0/G1 phase cell cycle arrest, and reduced the adhesion and invasion of WERI-Rb-1 cells. HIF-1α plays a major role in the survival and aggressive phenotype of retinoblastoma cells under hypoxic conditions. Targeting HIF-1α may be a promising therapeutic strategy for human malignant retinoblastoma.

  8. Reverse translation of phase I biomarker findings links the activity of angiotensin-(1–7) to repression of hypoxia inducible factor-1α in vascular sarcomas

    International Nuclear Information System (INIS)

    Petty, W Jeffrey; Aklilu, Mebea; Varela, Victor A; Lovato, James; Savage, Paul D; Miller, Antonius A

    2012-01-01

    In a phase I study of angiotensin-(1–7) [Ang-(1–7)], clinical benefit was associated with reduction in plasma placental growth factor (PlGF) concentrations. The current study examines Ang-(1–7) induced changes in biomarkers according to cancer type and investigates mechanisms of action engaged in vitro. Plasma biomarkers were measured prior to Ang-(1–7) administration as well as 1, 2, 3, 4, and 6 hours after treatment. Tests for interaction were performed to determine the impact of cancer type on angiogenic hormone levels. If a positive interaction was detected, treatment-induced biomarker changes for individual cancer types were assessed. To investigate mechanisms of action, in vitro growth assays were performed using a murine endothelioma cell line (EOMA). PCR arrays were performed to identify and statistically validate genes that were altered by Ang-(1–7) treatment in these cells. Tests for interaction controlled for dose cohort and clinical response indicated a significant impact of cancer type on post-treatment VEGF and PlGF levels. Following treatment, PlGF levels decreased over time in patients with sarcoma (P = .007). Treatment of EOMA cells with increasing doses of Ang-(1–7) led to significant growth suppression at doses as low as 100 nM. PCR arrays identified 18 genes that appeared to have altered expression after Ang-(1–7) treatment. Replicate analyses confirmed significant changes in 8 genes including reduction in PlGF (P = .04) and hypoxia inducible factor 1α (HIF-1α) expression (P < .001). Ang-(1–7) has clinical and pre-clinical activity for vascular sarcomas that is linked to reduced HIF-1α and PlGF expression

  9. Prolonged fasting activates hypoxia inducible factors-1α, -2α and -3α in a tissue-specific manner in northern elephant seal pups.

    Science.gov (United States)

    Soñanez-Organis, José G; Vázquez-Medina, José P; Crocker, Daniel E; Ortiz, Rudy M

    2013-09-10

    Hypoxia inducible factors (HIFs) are important regulators of energy homeostasis and cellular adaptation to low oxygen conditions. Northern elephant seals are naturally adapted to prolonged periods (1-2 months) of food deprivation (fasting) which result in metabolic changes that may activate HIF-1. However, the effects of prolonged fasting on HIFs are not well defined. We obtained the full-length cDNAs of HIF-1α and HIF-2α, and partial cDNA of HIF-3α in northern elephant seal pups. We also measured mRNA and nuclear protein content of HIF-1α, -2α, -3α in muscle and adipose during prolonged fasting (1, 3, 5 & 7 weeks), along with mRNA expression of HIF-mediated genes, LDH and VEGF. HIF-1α, -2α and -3α are 2595, 2852 and 1842 bp and encode proteins of 823, 864 and 586 amino acid residues with conserved domains needed for their function (bHLH and PAS) and regulation (ODD and TAD). HIF-1α and -2α mRNA expression increased 3- to 5-fold after 7 weeks of fasting in adipose and muscle, whereas HIF-3α increased 5-fold after 7 weeks of fasting in adipose. HIF-2α protein expression was detected in nuclear fractions from adipose and muscle, increasing approximately 2-fold, respectively with fasting. Expression of VEGF increased 3-fold after 7 weeks in adipose and muscle, whereas LDH mRNA expression increased 12-fold after 7 weeks in adipose. While the 3 HIFα genes are expressed in muscle and adipose, only HIF-2α protein was detectable in the nucleus suggesting that HIF-2α may contribute more significantly in the up-regulation of genes involved in the metabolic adaptation during fasting in the elephant seal. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Andrographolide inhibits hypoxia-induced HIF-1α-driven endothelin 1 secretion by activating Nrf2/HO-1 and promoting the expression of prolyl hydroxylases 2/3 in human endothelial cells.

    Science.gov (United States)

    Lin, Hung-Chih; Su, Shih-Li; Lu, Chia-Yang; Lin, Ai-Hsuan; Lin, Wan-Chun; Liu, Chin-San; Yang, Ya-Chen; Wang, Hsiu-Miao; Lii, Chong-Kuei; Chen, Haw-Wen

    2017-03-01

    Andrographolide, the main bioactive component of the medicinal plant Andrographis paniculata, has been shown to possess potent anti-inflammatory activity. Endothelin 1 (ET-1), a potent vasoconstrictor peptide produced by vascular endothelial cells, displays proinflammatory property. Hypoxia-inducible factor 1α (HIF-1α), the regulatory member of the transcription factor heterodimer HIF-1α/β, is one of the most important molecules that responds to hypoxia. Changes in cellular HIF-1α protein level are the result of altered gene transcription and protein stability, with the latter being dependent on prolyl hydroxylases (PHDs). In this study, inhibition of pro-inflammatory ET-1 expression and changes of HIF-1α gene transcription and protein stability under hypoxia by andrographolide in EA.hy926 endothelial-like cells were investigated. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2. We found that hypoxia stimulated the production of reactive oxygen species (ROS), the expression of HIF-1α mRNA and protein, and the expression and secretion of ET-1. These effects, however, were attenuated by co-exposure to andrographolide, bilirubin, and RuCO. Silencing Nrf2 and heme oxygenase 1 (HO-1) reversed the inhibitory effects of andrographolide on hypxoia-induced HIF-1α mRNA and protein expression. Moreover, andrographolide increased the expression of prolyl hydroxylases (PHD) 2/3, which hydroxylate HIF-1α and promotes HIF-1α proteasome degradation, with an increase in HIF-1α hydroxylation was noted under hypoxia. Inhibition of p38 MAPK abrogated the hypoxia-induced increases in HIF-1α mRNA and protein expression as well as ET-1 mRNA expression and secretion. Taken together, these results suggest that andrographolide suppresses hypoxia-induced pro-inflammatory ET-1 expression by activating Nrf2/HO-1, inhibiting p38 MAPK signaling, and promoting PHD2/3 expression. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 918-930, 2017. © 2016 Wiley

  11. Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival

    Directory of Open Access Journals (Sweden)

    Joffrey ePelletier

    2012-02-01

    Full Text Available The hypoxia-inducible factor 1 (HIF-1, in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1, were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these hypoxia-preconditioned cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO2 acts as an alarm that prepares the cells to face subsequent nutrient depletion and to survive.

  12. Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose.

    Science.gov (United States)

    Ikeda, Ryuji; Tabata, Sho; Tajitsu, Yusuke; Nishizawa, Yukihiko; Minami, Kentaro; Furukawa, Tatsuhiko; Yamamoto, Masatatsu; Shinsato, Yoshinari; Akiyama, Shin-Ichi; Yamada, Katsushi; Takeda, Yasuo

    2013-09-01

    The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

  13. Hypoxia regulates the expression of the neuromedin B receptor through a mechanism dependent on hypoxia-inducible factor-1α.

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    Hyun-Joo Park

    Full Text Available The neuromedin B receptor (NMB-R, a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α. We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

  14. Hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage.

    Science.gov (United States)

    Frid, Maria G; Brunetti, Jacqueline A; Burke, Danielle L; Carpenter, Todd C; Davie, Neil J; Reeves, John T; Roedersheimer, Mark T; van Rooijen, Nico; Stenmark, Kurt R

    2006-02-01

    Vascular remodeling in chronic hypoxic pulmonary hypertension includes marked fibroproliferative changes in the pulmonary artery (PA) adventitia. Although resident PA fibroblasts have long been considered the primary contributors to these processes, we tested the hypothesis that hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage, termed fibrocytes. Using two neonatal animal models (rats and calves) of chronic hypoxic pulmonary hypertension, we demonstrated a dramatic perivascular accumulation of mononuclear cells of a monocyte/macrophage lineage (expressing CD45, CD11b, CD14, CD68, ED1, ED2). Many of these cells produced type I collagen, expressed alpha-smooth muscle actin, and proliferated, thus exhibiting mesenchymal cell characteristics attributed to fibrocytes. The blood-borne origin of these cells was confirmed in experiments wherein circulating monocytes/macrophages of chronically hypoxic rats were in vivo-labeled with DiI fluorochrome via liposome delivery and subsequently identified in the remodeled pulmonary, but not systemic, arterial adventitia. The DiI-labeled cells that appeared in the vessel wall expressed monocyte/macrophage markers and procollagen. Selective depletion of this monocytic cell population, using either clodronate-liposomes or gadolinium chloride, prevented pulmonary adventitial remodeling (ie, production of collagen, fibronectin, and tenascin-C and accumulation of myofibroblasts). We conclude that circulating mesenchymal precursors of a monocyte/macrophage lineage, including fibrocytes, are essential contributors to hypoxia-induced pulmonary vascular remodeling.

  15. Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

    Energy Technology Data Exchange (ETDEWEB)

    Pelletier, Joffrey; Bellot, Grégory [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France); Gounon, Pierre; Lacas-Gervais, Sandra [Centre Commun de Microscopie Appliquée, University of Nice-Sophia Antipolis, Nice (France); Pouysségur, Jacques; Mazure, Nathalie M., E-mail: mazure@unice.fr [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France)

    2012-02-28

    The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO{sub 2} acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

  16. Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

    International Nuclear Information System (INIS)

    Pelletier, Joffrey; Bellot, Grégory; Gounon, Pierre; Lacas-Gervais, Sandra; Pouysségur, Jacques; Mazure, Nathalie M.

    2012-01-01

    The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO 2 acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

  17. [Characteristics of sublingual vein and expressions of vascular endothelial growth factor and hypoxia-inducible factor 1alpha proteins in sublingual tissues of Beagle dogs with portal hypertension].

    Science.gov (United States)

    Li, Bai-yu; Wang, Li-na; Yue, Xiao-qiang; Li, Bai

    2009-05-01

    To observe sublingual vein characteristics and the expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1alpha (HIF-1alpha) proteins in sublingual tissues of Beagle dogs with cirrhotic portal hypertension. Twelve Beagle dogs were randomly divided into normal control group and cirrhotic portal hypertension group. There were 6 dogs in each group. A canine model of cirrhosis portal hypertension was established by injecting dimethylnitrosamine (DMN) into portal vein once a week for 7 weeks. The characteristics of sublingual vein were observed. Portal venous pressure was measured by using bioelectric recording techniques. The expressions of VEGF and HIF-1alpha proteins in sublingual vein were detected by immunohistochemical method. The shape and color of sublingual vein in beagle dogs in the cirrhotic portal hypertension group changed obviously as compared with the normal control group. Immunohistochemical results showed that there were almost no expressions of VEGF and HIF-1alpha proteins in sublingual tissues in the normal control group; however, the expressions of VEGF and HIF-1alpha proteins in sublingual tissues in the cirrhotic portal hypertension group significantly increased. Changes of portal pressure may lead to the formation of the abnormal sublingual vein by increasing the expressions of VEGF and HIF-1alpha proteins in sublingual tissues in Beagle dogs with portal hypertension.

  18. The expression of vascular endothelial growth factor is affected by hypoxia inducible factor-1α in peritoneum of endometriosis mice treated with genistein

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    Sylvi Wafda Nur Amellia

    2016-09-01

    Full Text Available This study aimed to investigate whether the genistein is able to decrease the expressions of vascular endothelial growth factor-A (VEGF-A and hypoxia inducible factor-1α (HIF-1α in mouse model of endometriosis. Forty female mice (Mus musculus were divided into eight groups (n = 5 each, including the control (untreated group, endometriosis group, and endometriosis groups treated with various doses of genistein (50; 100; 200; 300; 400; and 500 mg/day. VEGF-A and HIF-1α analyses were performed by immunohistochemistry. We found significant increases in the VEGF-A and HIF-α expressions in endometriosis group compared to the control group. The increased expressions of VEGF-A and HIF-1α were significantly (p < 0.05 attenuated by the administration of all doses of genistein. In conclusion, in mouse model of endometriosis, genistein potentially inhibits the increase in angiogenesis in peritoneal tissue. Therefore, this result may provide a novel anti-angiogenic treatment strategy for the therapy of endometriosis.

  19. Ginsenoside Rg3 enhances radiosensitization of hypoxic oesophageal cancer cell lines through vascular endothelial growth factor and hypoxia inducible factor 1α.

    Science.gov (United States)

    Ge, Xiaolin; Zhen, Fuxi; Yang, Baixia; Yang, Xi; Cai, Jing; Zhang, Chi; Zhang, Sheng; Cao, Yuandong; Ma, Jianxin; Cheng, Hongyan; Sun, Xinchen

    2014-06-01

    To determine if the pretreatment of hypoxic human oesophageal carcinoma cell lines (EC109, TE1 and KYSE170) with ginsenoside Rg3 (Rg3) increases their radiosensitivity to X-rays. The growth inhibitory effect of different Rg3 concentrations was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. Radiation sensitivity was measured using a clone formation assay and flow cytometry was used to measure the effects of Rg3 on radiation-induced apoptosis. Western blot analysis was used to measure the effects of Rg3 on the levels of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF). Rg3 inhibited EC109, TE1 and KYSE170 cell growth in a dose- and time-dependent manner. Pretreatment with 10 µmol/ml Rg3 increased EC109, TE1 and KYSE170 radiosensitivity. Rg3 plus radiation significantly increased the apoptosis rate compared with radiation alone. Rg3 also decreased VEGF and HIF-1α protein levels in EC109 cells in a dose-dependent manner. The combination of Rg3 and radiation increased the fragmentation of double-stranded DNA. Rg3 enhanced the radiosensitivity of human oesophageal carcinoma cell lines cultured under hypoxic conditions possibly by downregulating VEGF and HIF-1α protein levels. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

  20. Thymidine phosphorylase and hypoxia-inducible factor 1-α expression in clinical stage II/III rectal cancer: association with response to neoadjuvant chemoradiation therapy and prognosis.

    Science.gov (United States)

    Lin, Shuhan; Lai, Hao; Qin, Yuzhou; Chen, Jiansi; Lin, Yuan

    2015-01-01

    The aim of this study was to determine whether pretreatment status of thymidine phosphorylase (TP), and hypoxia-inducible factor alpha (HIF-1α) could predict pathologic response to neoadjuvant chemoradiation therapy with oxaliplatin and capecitabine (XELOXART) and outcomes for clinical stage II/III rectal cancer patients. A total of 180 patients diagnosed with clinical stage II/III rectal cancer received XELOXART. The status of TP, and HIF-1α were determined in pretreatment biopsies by immunohistochemistry (IHC). Tumor response was assessed in resected regimens using the tumor regression grade system and TNM staging system. 5-year disease free survival (DFS) and 5-year overall survival (OS) were evaluated with the Kaplan-Meier method and were compared by the log-rank test. Over expression of TP and low expression of HIF-1α were associated with pathologic response to XELOXART and better outcomes (DFS and OS) in clinical stage II/III rectal cancer patients (P rectal cancer received XELOXART. Additional well-designed, large sample, multicenter, prospective studies are needed to confirm the result of this study.

  1. Senp1 drives hypoxia-induced polycythemia via GATA1 and Bcl-xL in subjects with Monge’s disease

    Science.gov (United States)

    Azad, Priti; Zhao, Huiwen W.; Ronen, Roy; Zhou, Dan; Poulsen, Orit; Hsiao, Yu Hsin; Bafna, Vineet

    2016-01-01

    In this study, because excessive polycythemia is a predominant trait in some high-altitude dwellers (chronic mountain sickness [CMS] or Monge’s disease) but not others living at the same altitude in the Andes, we took advantage of this human experiment of nature and used a combination of induced pluripotent stem cell technology, genomics, and molecular biology in this unique population to understand the molecular basis for hypoxia-induced excessive polycythemia. As compared with sea-level controls and non-CMS subjects who responded to hypoxia by increasing their RBCs modestly or not at all, respectively, CMS cells increased theirs remarkably (up to 60-fold). Although there was a switch from fetal to adult HgbA0 in all populations and a concomitant shift in oxygen binding, we found that CMS cells matured faster and had a higher efficiency and proliferative potential than non-CMS cells. We also established that SENP1 plays a critical role in the differential erythropoietic response of CMS and non-CMS subjects: we can convert the CMS phenotype into that of non-CMS and vice versa by altering SENP1 levels. We also demonstrated that GATA1 is an essential downstream target of SENP1 and that the differential expression and response of GATA1 and Bcl-xL are a key mechanism underlying CMS pathology. PMID:27821551

  2. Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

    Science.gov (United States)

    Asai, Yoichiro; Yamada, Tetsuya; Tsukita, Sohei; Takahashi, Kei; Maekawa, Masamitsu; Honma, Midori; Ikeda, Masanori; Murakami, Keigo; Munakata, Yuichiro; Shirai, Yuta; Kodama, Shinjiro; Sugisawa, Takashi; Chiba, Yumiko; Kondo, Yasuteru; Kaneko, Keizo; Uno, Kenji; Sawada, Shojiro; Imai, Junta; Nakamura, Yasuhiro; Yamaguchi, Hiroaki; Tanaka, Kozo; Sasano, Hironobu; Mano, Nariyasu; Ueno, Yoshiyuki; Shimosegawa, Tooru; Katagiri, Hideki

    2017-05-01

    Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic

  3. Comparative GO: a web application for comparative gene ontology and gene ontology-based gene selection in bacteria.

    Directory of Open Access Journals (Sweden)

    Mario Fruzangohar

    Full Text Available The primary means of classifying new functions for genes and proteins relies on Gene Ontology (GO, which defines genes/proteins using a controlled vocabulary in terms of their Molecular Function, Biological Process and Cellular Component. The challenge is to present this information to researchers to compare and discover patterns in multiple datasets using visually comprehensible and user-friendly statistical reports. Importantly, while there are many GO resources available for eukaryotes, there are none suitable for simultaneous, graphical and statistical comparison between multiple datasets. In addition, none of them supports comprehensive resources for bacteria. By using Streptococcus pneumoniae as a model, we identified and collected GO resources including genes, proteins, taxonomy and GO relationships from NCBI, UniProt and GO organisations. Then, we designed database tables in PostgreSQL database server and developed a Java application to extract data from source files and loaded into database automatically. We developed a PHP web application based on Model-View-Control architecture, used a specific data structure as well as current and novel algorithms to estimate GO graphs parameters. We designed different navigation and visualization methods on the graphs and integrated these into graphical reports. This tool is particularly significant when comparing GO groups between multiple samples (including those of pathogenic bacteria from different sources simultaneously. Comparing GO protein distribution among up- or down-regulated genes from different samples can improve understanding of biological pathways, and mechanism(s of infection. It can also aid in the discovery of genes associated with specific function(s for investigation as a novel vaccine or therapeutic targets.http://turing.ersa.edu.au/BacteriaGO.

  4. Comparative genomics on Norrie disease gene.

    Science.gov (United States)

    Katoh, Masuko; Katoh, Masaru

    2005-05-01

    DAND1 (NBL1), DAND2 (CKTSF1B1 or GREM1 or GREMLIN), DAND3 (CKTSF1B2 or GREM2 or PRDC), DAND4 (CER1), DAND5 (CKTSF1B3 or GREM3 or DANTE), MUC2, MUC5AC, MUC5B, MUC6, MUC19, WISP1, WISP2, WISP3, VWF, NOV and Norrie disease (NDP or NORRIN) genes encode proteins with cysteine knot domain. Cysteine-knot superfamily proteins regulate ligand-receptor interactions for a variety of signaling pathways implicated in embryogenesis, homeostasis, and carcinogenesis. Although Ndp is unrelated to Wnt family members, Ndp is claimed to function as a ligand for Fzd4. Here, we identified and characterized rat Ndp, cow Ndp, chicken ndp and zebrafish ndp genes by using bioinformatics. Rat Ndp gene, consisting of three exons, was located within AC105563.4 genome sequence. Cow Ndp and chicken ndp complete CDS were derived from CB467544.1 EST and BX932859.2 cDNA, respectively. Zebrafish ndp gene was located within BX572627.5 genome sequence. Rat Ndp (131 aa) was a secreted protein with C-terminal cysteine knot-like (CTCK) domain. Rat Ndp showed 100, 96.9, 95.4, 87.8 and 66.4 total-amino-acid identity with mouse Ndp, cow Ndp, human NDP, chicken ndp and zebrafish ndp, respectively. Exon-intron structure of mammalian Ndp orthologs was well conserved. FOXA2, CUTL1 (CCAAT displacement protein), LMO2, CEBPA (C/EBPalpha)-binding sites and triple POU2F1 (OCT1)-binding sites were conserved among promoters of mammalian Ndp orthologs.

  5. Hypoxia-induced autophagy is inhibited by PADI4 knockdown, which promotes apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

    Science.gov (United States)

    Fan, Tingting; Zhang, Changsong; Zong, Ming; Fan, Lieying

    2018-01-01

    Impaired apoptosis of rheumatoid arthritis (RA)-fibroblast-like synoviocytes (FLS) is pivotal in the process of RA. Peptidyl arginine deiminase type IV (PADI4) is associated with autoantibody regulation via histone citrullination in RA. The present study aimed to investigate the role of PADI4 in the apoptosis of RA-FLS. FLS were isolated from patients with RA and a rat model. The effects of PADI4 on RA-FLS were investigated in vitro and in vivo. Hypoxia-induced autophagy was induced by 1% O2 and was detected by immunohistochemical and immunofluorescence analysis; in addition, apoptosis was detected by flow cytometry. RA-FLS obtained from RA rat model exhibited significant proliferation under severe hypoxia conditions. Hypoxia also significantly induced autophagy and elevated the expression of PADI4. Subsequently, short hairpin RNA-mediated PADI4 knockdown was demonstrated to significantly inhibit hypoxia-induced autophagy and promote apoptosis in RA-FLS. The results of these in vitro and in vivo studies suggested that PADI4 may be closely associated with hypoxia-induced autophagy, and the inhibition of hypoxia-induced autophagy by PADI4 knockdown may contribute to an increase in the apoptosis of RA-FLS. PMID:29393388

  6. Hypoxia-inducible factor-1α expression requires PI 3-kinase activity and correlates with Akt1 phosphorylation in invasive breast carcinomas

    NARCIS (Netherlands)

    Gort, E.H.; Groot, A.J.; Derks van de Ven, T.L.P.; Groep, P. van der; Verlaan, I.; Laar, T. van; Diest, P.J. van; Wall, E. van der; Shvarts, A.

    2006-01-01

    Hypoxia-inducible factor-1 alpha (HIF-1a) is the regulatory subunit of the heterodimeric transcription factor HIF-1 and the key factor in cellular response to low oxygen tension. Expression of HIF-1a protein is associated with poor patient survival and therapy resistance in many types of solid

  7. [Effects of hypoxia inducible factor-1α on P311 and its influence on the migration of murine epidermal stem cells].

    Science.gov (United States)

    Xu, Z D; Li, H S; Wang, S; He, W F; Wu, J; Luo, G X

    2017-05-20

    Objective: To explore the effects of hypoxia inducible factor-1α (HIF-1α) on P311 and its influence on the migration of murine epidermal stem cells (ESCs) under hypoxia in vitro. Methods: Two kinds of murine ESCs were isolated and obtained from 15 neonatal wild-type C57BL/6J mice and 5 congeneric source P311 gene knock-out mice, respectively. The first passage of cells were used in the following experiments after morphologic observation and detection of expression of cell surface markers CD71 and CD49f with flow cytometer. (1) After cell scratch assay, according to the random number table (the same dividing method below), ESCs of P311 gene knock-out mice were divided into normoxia group (cells were cultured with complete medium in normoxic carbon dioxide incubator, and the subsequent normoxic treatments were the same) and hypoxia group (cells were cultured in hypoxic carbon dioxide incubator containing 1% oxygen, and the subsequent hypoxic treatments were the same), with 12 inserts in each group. ESCs of wild-type mice were divided into normoxia group, pure hypoxia group, dimethyl sulfoxide (DMSO) control group (2 μL DMSO solvent was added for 1 h of normoxia treatment before hypoxia treatment), HIF-1α inhibitor group (cells were treated with 11 μmol/L HIF-1 inhibitor of 2 μL under normoxia condition for 1 h before hypoxia treatment), HIF-1α stabilizer group (the cells were treated with 2 μmol/L FG-4592 of 2 μL under normoxia condition for 1 h before hypoxia treatment), with 12 inserts in each group. Three inserts of each time point in each group were adopted respectively to measure the residual width of scratch under inverted phase contrast microscope at post scratch hour (PSH) 0 (immediately), 12, 24, and 48. (2) After hypoxia treatment, the protein level of HIF-1α in ESCs of wild-type mice was detected by Western blotting at post hypoxia hour (PHH) 0, 12, 24, and 48. (3) ESCs of wild-type mice were divided into pure hypoxia group, DMSO control group

  8. Hypoxia-inducible factor-1α, vascular endothelial growth factor, inducible nitric oxide synthase, and endothelin-1 expression correlates with angiogenesis in congenital heart disease

    Directory of Open Access Journals (Sweden)

    Hsin-Ling Yin

    2016-07-01

    Full Text Available In Taiwan, the average prevalence of congenital heart disease (CHD is 13.08/1000 live births. Most children with CHD die before the age of 5 years; therefore, identifying treatment methods to extend the life of CHD patients is an important issue in clinical practice. The objective of this study is to evaluate the roles of hypoxia-inducible factor-1α (HIF-1α, vascular endothelial growth factor (VEGF, inducible nitric oxide synthase (iNOS, endothelin-1 (ET-1, and CD34 in CHD autopsy cases in comparison with autopsy cases without CHD. The study included 19 autopsy cases, which were divided into the following four groups: acyanotic CHD (n = 11, cyanotic CHD (n = 3, CHD associated with chromosomal abnormalities (n = 3, and complex CHD (n = 2. Heart specimens obtained from 10 autopsy cases without CHD were included as controls. Our results indicated that high percentages of HIF-1α (100%, VEGF (89.5%, iNOS (78.9%, and ET-1 (84.2% expressions were observed in CHD autopsy cases and this was found to be significant. HIF-1α induced by hypoxia could play a potential role in relating downstream gene expressions in CHD patients. Upregulation of VEGF by HIF-1α could play an important role in triggering angiogenesis to protect myocardial cell survival in a hypoxic microenvironment. Therefore, HIF-1α could be a significant prognosis marker in CHD and be a prospective candidate in the development of target therapy in cardiovascular diseases.

  9. CD147 subunit of lactate/H+ symporters MCT1 and hypoxia-inducible MCT4 is critical for energetics and growth of glycolytic tumors.

    Science.gov (United States)

    Le Floch, Renaud; Chiche, Johanna; Marchiq, Ibtissam; Naiken, Tanesha; Naïken, Tanesha; Ilc, Karine; Ilk, Karine; Murray, Clare M; Critchlow, Susan E; Roux, Danièle; Simon, Marie-Pierre; Pouysségur, Jacques

    2011-10-04

    Malignant tumors exhibit increased dependence on glycolysis, resulting in abundant export of lactic acid, a hypothesized key step in tumorigenesis. Lactic acid is mainly transported by two H(+)/lactate symporters, MCT1/MCT4, that require the ancillary protein CD147/Basigin for their functionality. First, we showed that blocking MCT1/2 in Ras-transformed fibroblasts with AR-C155858 suppressed lactate export, glycolysis, and tumor growth, whereas ectopic expression of MCT4 in these cells conferred resistance to MCT1/2 inhibition and reestablished tumorigenicty. A mutant-derivative, deficient in respiration (res(-)) and exclusively relying on glycolysis for energy, displayed low tumorigenicity. These res(-) cells could develop resistance to MCT1/2 inhibition and became highly tumorigenic by reactivating their endogenous mct4 gene, highlighting that MCT4, the hypoxia-inducible and tumor-associated lactate/H(+) symporter, drives tumorigenicity. Second, in the human colon adenocarcinoma cell line (LS174T), we showed that combined silencing of MCT1/MCT4 via inducible shRNA, or silencing of CD147/Basigin alone, significantly reduced glycolytic flux and tumor growth. However, both silencing approaches, which reduced tumor growth, displayed a low level of CD147/Basigin, a multifunctional protumoral protein. To gain insight into CD147/Basigin function, we designed experiments, via zinc finger nuclease-mediated mct4 and basigin knockouts, to uncouple MCTs from Basigin expression. Inhibition of MCT1 in MCT4-null, Basigin(high) cells suppressed tumor growth. Conversely, in Basigin-null cells, in which MCT activity had been maintained, tumorigenicity was not affected. Collectively, these findings highlight that the major protumoral action of CD147/Basigin is to control the energetics of glycolytic tumors via MCT1/MCT4 activity and that blocking lactic acid export provides an efficient anticancer strategy.

  10. The zebrafish miR-125c is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations and embryogenesis.

    Science.gov (United States)

    He, Yan; Huang, Chun-Xiao; Chen, Nan; Wu, Meng; Huang, Yan; Liu, Hong; Tang, Rong; Wang, Wei-Min; Wang, Huan-Ling

    2017-09-26

    Hypoxia is a unique environmental stress. Hypoxia inducible factor-lα (HIF-lα) is a major transcriptional regulator of cellular adaptations to hypoxic stress. MicroRNAs (miRNAs) as posttranscriptional gene expression regulators occupy a crucial role in cell survival under low-oxygen environment. Previous evidences suggested that miR-125c is involved in hypoxia adaptation, but its precise biological roles and the regulatory mechanism underlying hypoxic responses remain unknown. The present study showed that zebrafish miR-125c is upregulated by hypoxia in a Hif-lα-mediated manner in vitro and in vivo . Dual-luciferase assay revealed that cdc25a is a novel target of miR-125c. An inverse correlation between miR-125c and cdc25a was further confirmed in vivo , suggesting miR-125c as a crucial physiological inhibitor of cdc25a which responds to cellular hypoxia. Overexpression of miR-125c suppressed cell proliferation, led to cell cycle arrest at the G1 phase in ZF4 cells and induced apoptotic responses during embryo development. More importantly, miR-125c overexpression resulted in severe malformation and reduction of motility during zebrafish embryonic development. Taken together, we conclude that miR-125c plays a pivotal role in cellular adaptations to hypoxic stress at least in part through the Hif-1α/miR-125c/cdc25a signaling and has great impact on zebrafish early embryonic development.

  11. The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations.

    Science.gov (United States)

    Huang, Chun-Xiao; Chen, Nan; Wu, Xin-Jie; Huang, Cui-Hong; He, Yan; Tang, Rong; Wang, Wei-Min; Wang, Huan-Ling

    2015-12-01

    Hypoxia, a unique and essential environmental stress, evokes highly coordinated cellular responses, and hypoxia-inducible factor (HIF) 1 in the hypoxia signaling pathway, an evolutionarily conserved cellular signaling pathway, acts as a master regulator of the transcriptional response to hypoxic stress. MicroRNAs (miRNAs), a major class of posttranscriptional gene expression regulators, also play pivotal roles in orchestrating hypoxia-mediated cellular adaptations. Here, global miRNA expression profiling and quantitative real-time PCR indicated that the up-regulation of the miR-462/miR-731 cluster in zebrafish larvae is induced by hypoxia. It was further validated that miR-462 and miR-731 are up-regulated in a Hif-1α-mediated manner under hypoxia and specifically target ddx5 and ppm1da, respectively. Overexpression of miR-462 and miR-731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis. In situ detection revealed that the miR-462/miR-731 cluster is highly expressed in a consistent and ubiquitous manner throughout the early developmental stages. Additionally, the transcripts become restricted to the notochord, pharyngeal arch, liver, and gut regions from postfertilization d 3 to 5. These data highlight a previously unidentified role of the miR-462/miR-731 cluster as a crucial signaling mediator for hypoxia-mediated cellular adaptations and provide some insights into the potential function of the cluster during embryonic development. © FASEB.

  12. MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner.

    Science.gov (United States)

    Joshi, Shweta; Singh, Alok R; Durden, Donald L

    2014-08-15

    Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner*

    Science.gov (United States)

    Joshi, Shweta; Singh, Alok R.; Durden, Donald L.

    2014-01-01

    Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α–HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. PMID:24982421

  14. Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

    Science.gov (United States)

    Oh, H; Kwak, J-S; Yang, S; Gong, M-K; Kim, J-H; Rhee, J; Kim, S K; Kim, H-E; Ryu, J-H; Chun, J-S

    2015-12-01

    Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Hypoxia-Inducible Factor-1α (HIF-1α) Expression on Endothelial Cells in Juvenile Nasopharyngeal Angiofibroma: A Review of 70 cases and Tissue Microarray Analysis.

    Science.gov (United States)

    Song, Xiaole; Yang, Chenhe; Zhang, Huankang; Wang, Jingjing; Sun, Xicai; Hu, Li; Liu, Zhuofu; Wang, Dehui

    2018-06-01

    To examine the expression of hypoxia-inducible factor-1α (HIF-1α) and its related molecules (cellular repressor of E1A-stimulated genes [CREG], osteopontin [OPN], proto-oncogene tyrosine-protein kinase Src [c-Src], and vascular endothelial growth factor [VEGF]) in juvenile nasopharyngeal angiofibroma (JNA) and explore the correlation between clinical prognosis and HIF-1α expression. The study performed a retrospective review of the clinical records of patients with JNA treated between 2003 and 2007. Specimens were analyzed by immunohistochemistry for HIF-1α, CREG, OPN, c-Src, and VEGF expression, and microvessel density (MVD) was assessed by tissue microarray. The correlation between expression levels and clinicopathological features including age, tumor stage, intraoperative blood loss, and recurrence was analyzed. HIF-1α, CREG, OPN, c-Src, and VEGF were upregulated in endothelial cells (ECs) of patients with JNA, and strong correlations in the expression of these molecules were observed. HIF-1α expression was higher in young patients ( P = .032) and in recurrent cases ( P = .01). Survival analysis showed that low HIF-1α levels in ECs predicted longer time to recurrence (log rank test P = .006). Receiver operating characteristic curve analysis showed that HIF-1α was a prognostic factor for recurrence (area under the curve = 0.690, P = .019). No correlation was found between the expression of molecules and Radkowski stage or intraoperative blood loss. In cases of JNA treated surgically, HIF-1α expression in ECs is a useful prognostic factor for tumor recurrence.

  16. Hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha recruits bone marrow-derived cells to the murine pulmonary vasculature.

    Directory of Open Access Journals (Sweden)

    Daniel J Angelini

    2010-06-01

    Full Text Available Pulmonary hypertension (PH is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo.We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP(+ transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+ BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+ cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+ and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs. The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner.These results demonstrate HIMF-dependent recruitment of BMD

  17. Insights into the therapeutic potential of hypoxia-inducible factor-1α small interfering RNA in malignant melanoma delivered via folate-decorated cationic liposomes

    Directory of Open Access Journals (Sweden)

    Chen Z

    2016-03-01

    Full Text Available Zhongjian Chen,1,* Tianpeng Zhang,2,* Baojian Wu,2 Xingwang Zhang2 1Department of Pharmaceutics, Shanghai Dermatology Hospital, 2Division of Pharmaceutics, College of Pharmacy, Jinan University, Gangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: Malignant melanoma (MM represents the most dangerous form of skin cancer, and its incidence is expected to rise in the coming time. However, therapy for MM is limited by low topical drug concentration and multidrug resistance. This article aimed to develop folate-decorated cationic liposomes (fc-LPs for hypoxia-inducible factor-1α (HIF-1α small interfering (siRNA delivery, and to evaluate the potential of such siRNA/liposome complexes in MM therapy. HIF-1α siRNA-loaded fc-LPs (siRNA-fc-LPs were prepared by a film hydration method followed by siRNA incubation. Folate decoration of liposomes was achieved by incorporation of folate/oleic acid-diacylated oligochitosans. The resulting siRNA-fc-LPs were 95.3 nm in size with a ζ potential of 2.41 mV. The liposomal vectors exhibited excellent loading capacity and protective effect toward siRNA. The in vitro cell transfection efficiency was almost parallel to the commercially available Lipofectamine™ 2000. Moreover, the anti-melanoma activity of HIF-1α siRNA was significantly enhanced through fc-LPs. Western blot analysis and apoptosis test demonstrated that siRNA-fc-LPs substantially reduced the production of HIF-1α-associated protein and induced the apoptosis of hypoxia-tolerant melanoma cells. Our designed liposomal vectors might be applicable as siRNA delivery vehicle to systemically or topically treat MM. Keywords: malignant melanoma, HIF-1α siRNA, chitosan, cationic liposomes, gene therapy

  18. Expression of hypoxia-inducible factor-1α and hepatocyte growth factor in development of fibrosis in the transplanted kidney

    DEFF Research Database (Denmark)

    Kellenberger, Terese; Marcussen, Niels; Nyengaard, Jens Randel

    2014-01-01

    Late renal graft loss is associated with interstitial fibrosis. Hypoxia-inducible factor-1α (HIF-1α) is thought to facilitate fibrosis through interaction with TGF-β1, while hepatocyte growth factor (HGF) may act antifibrotic in the kidney allograft. The aim of this study was to investigate...... transplantation, but an inverse significant correlation between the HGF expression and the fibrosis score 1 year after transplantation was shown. Even when adjusting for human leucocyte antigen mismatches, there was a significant relationship between fibrosis and HGF expression. Graft survival...... was not significantly correlated to HIF-1α or HGF at 1 year, although the trend was towards better graft survival with high HGF. HGF may have antifibrotic effects in human renal transplants. (Central.Denmark.Region.Committee number: 1-10-72-318-13)....

  19. Hypoxic stress up-regulates the expression of Toll-like receptor 4 in macrophages via hypoxia-inducible factor.

    Science.gov (United States)

    Kim, So Young; Choi, Yong Jun; Joung, Sun Myung; Lee, Byung Ho; Jung, Yi-Sook; Lee, Joo Young

    2010-04-01

    Toll-like receptors (TLRs) are germline-encoded innate immune receptors that recognize invading micro-organisms and induce immune and inflammatory responses. Deregulation of TLRs is known to be closely linked to various immune disorders and inflammatory diseases. Cells at sites of inflammation are exposed to hypoxic stress, which further aggravates inflammatory processes. We have examined if hypoxic stress modulates the TLR activity of macrophages. Hypoxia and CoCl(2) (a hypoxia mimetic) enhanced the expression of TLR4 messenger RNA and protein in macrophages (RAW264.7 cells), whereas the messenger RNA of other TLRs was not increased. To determine the underlying mechanism, we investigated the role of hypoxia-inducible factor 1 (HIF-1) in the regulation of TLR4 expression. Knockdown of HIF-1alpha expression by small interfering RNA inhibited hypoxia-induced and CoCl(2)-induced TLR4 expression in macrophages, while over-expression of HIF-1alpha potentiated TLR4 expression. Chromatin immunoprecipitation assays revealed that HIF-1alpha binds to the TLR4 promoter region under hypoxic conditions. In addition, deletion or mutation of a putative HIF-1-binding motif in the TLR4 promoter greatly attenuated HIF-1alpha-induced TLR4 promoter reporter expression. Up-regulation of TLR4 expression by hypoxic stress enhanced the response of macrophages to lipopolysaccharide, resulting in increased expression of cyclooxygenase-2, interleukin-6, regulated on activation normal T cell expressed and secreted, and interferon-inducible protein-10. These results demonstrate that TLR4 expression in macrophages is up-regulated via HIF-1 in response to hypoxic stress, suggesting that hypoxic stress at sites of inflammation enhances susceptibility to subsequent infection and inflammatory signals by up-regulating TLR4.

  20. Hypoxia-induced glucose-6-phosphate dehydrogenase overexpression and -activation in pulmonary artery smooth muscle cells: implication in pulmonary hypertension

    Science.gov (United States)

    Chettimada, Sukrutha; Gupte, Rakhee; Rawat, Dhwajbahadur; Gebb, Sarah A.; McMurtry, Ivan F.

    2014-01-01

    Severe pulmonary hypertension is a debilitating disease with an alarmingly low 5-yr life expectancy. Hypoxia, one of the causes of pulmonary hypertension, elicits constriction and remodeling of the pulmonary arteries. We now know that pulmonary arterial remodeling is a consequence of hyperplasia and hypertrophy of pulmonary artery smooth muscle (PASM), endothelial, myofibroblast, and stem cells. However, our knowledge about the mechanisms that cause these cells to proliferate and hypertrophy in response to hypoxic stimuli is still incomplete, and, hence, the treatment for severe pulmonary arterial hypertension is inadequate. Here we demonstrate that the activity and expression of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, are increased in hypoxic PASM cells and in lungs of chronic hypoxic rats. G6PD overexpression and -activation is stimulated by H2O2. Increased G6PD activity contributes to PASM cell proliferation by increasing Sp1 and hypoxia-inducible factor 1α (HIF-1α), which directs the cells to synthesize less contractile (myocardin and SM22α) and more proliferative (cyclin A and phospho-histone H3) proteins. G6PD inhibition with dehydroepiandrosterone increased myocardin expression in remodeled pulmonary arteries of moderate and severe pulmonary hypertensive rats. These observations suggest that altered glucose metabolism and G6PD overactivation play a key role in switching the PASM cells from the contractile to synthetic phenotype by increasing Sp1 and HIF-1α, which suppresses myocardin, a key cofactor that maintains smooth muscle cell in contractile state, and increasing hypoxia-induced PASM cell growth, and hence contribute to pulmonary arterial remodeling and pathogenesis of pulmonary hypertension. PMID:25480333

  1. Comparative genome analysis of PHB gene family reveals deep evolutionary origins and diverse gene function.

    Science.gov (United States)

    Di, Chao; Xu, Wenying; Su, Zhen; Yuan, Joshua S

    2010-10-07

    PHB (Prohibitin) gene family is involved in a variety of functions important for different biological processes. PHB genes are ubiquitously present in divergent species from prokaryotes to eukaryotes. Human PHB genes have been found to be associated with various diseases. Recent studies by our group and others have shown diverse function of PHB genes in plants for development, senescence, defence, and others. Despite the importance of the PHB gene family, no comprehensive gene family analysis has been carried to evaluate the relatedness of PHB genes across different species. In order to better guide the gene function analysis and understand the evolution of the PHB gene family, we therefore carried out the comparative genome analysis of the PHB genes across different kingdoms. The relatedness, motif distribution, and intron/exon distribution all indicated that PHB genes is a relatively conserved gene family. The PHB genes can be classified into 5 classes and each class have a very deep evolutionary origin. The PHB genes within the class maintained the same motif patterns during the evolution. With Arabidopsis as the model species, we found that PHB gene intron/exon structure and domains are also conserved during the evolution. Despite being a conserved gene family, various gene duplication events led to the expansion of the PHB genes. Both segmental and tandem gene duplication were involved in Arabidopsis PHB gene family expansion. However, segmental duplication is predominant in Arabidopsis. Moreover, most of the duplicated genes experienced neofunctionalization. The results highlighted that PHB genes might be involved in important functions so that the duplicated genes are under the evolutionary pressure to derive new function. PHB gene family is a conserved gene family and accounts for diverse but important biological functions based on the similar molecular mechanisms. The highly diverse biological function indicated that more research needs to be carried out

  2. Induction of erythropoiesis by hypoxia-inducible factor prolyl hydroxylase inhibitors without promotion of tumor initiation, progression, or metastasis in a VEGF-sensitive model of spontaneous breast cancer

    Science.gov (United States)

    Seeley, Todd W; Sternlicht, Mark D; Klaus, Stephen J; Neff, Thomas B; Liu, David Y

    2017-01-01

    The effects of pharmacological hypoxia-inducible factor (HIF) stabilization were investigated in the MMTV-Neundl-YD5 (NeuYD) mouse model of breast cancer. This study first confirmed the sensitivity of this model to increased vascular endothelial growth factor (VEGF), using bigenic NeuYD;MMTV-VEGF-25 mice. Tumor initiation was dramatically accelerated in bigenic animals. Bigenic tumors were also more aggressive, with shortened doubling times and increased lung metastasis as compared to NeuYD controls. In separate studies, NeuYD mice were treated three times weekly from 7 weeks of age until study end with two different HIF prolyl hydroxylase inhibitors (HIF-PHIs), FG-4497 or roxadustat (FG-4592). In NeuYD mice, HIF-PHI treatments elevated erythropoiesis markers, but no differences were detected in tumor onset or the phenotypes of established tumors. PMID:28331872

  3. Loss of hypoxia-inducible factor 2 alpha in the lung alveolar epithelium of mice leads to enhanced eosinophilic inflammation in cobalt-induced lung injury.

    Science.gov (United States)

    Proper, Steven P; Saini, Yogesh; Greenwood, Krista K; Bramble, Lori A; Downing, Nathaniel J; Harkema, Jack R; Lapres, John J

    2014-02-01

    Hard metal lung disease (HMLD) is an occupational lung disease specific to inhalation of cobalt-containing particles whose mechanism is largely unknown. Cobalt is a known hypoxia mimic and stabilizer of the alpha subunits of hypoxia-inducible factors (HIFs). Previous work revealed that though HIF1α contrib utes to cobalt toxicity in vitro, loss of HIF1α in the alveolar epithelial cells does not provide in vivo protection from cobalt-induced lung inflammation. HIF1α and HIF2α show unique tissue expression profiles, and HIF2α is known to be the predominant HIF mRNA isoform in the adult lung. Thus, if HIF2α activation by cobalt contributes to pathophysiology of HMLD, we hypothesized that loss of HIF2α in lung epithelium would provide protection from cobalt-induced inflammation. Mice with HIF2α-deficiency in Club and alveolar type II epithelial cells (ATIIs) (HIF2α(Δ/Δ)) were exposed to cobalt (60 µg/day) or saline using a subacute occupational exposure model. Bronchoalveolar lavage cellularity, cytokines, qRT-PCR, and histopathology were analyzed. Results show that loss of HIF2α leads to enhanced eosinophilic inflammation and increased goblet cell metaplasia. Additionally, control mice demonstrated a mild recovery from cobalt-induced lung injury compared with HIF2α(Δ/Δ) mice, suggesting a role for epithelial HIF2α in repair mechanisms. The expression of important cytokines, such as interleukin (IL)-5 and IL-10, displayed significant differences following cobalt exposure when HIF2α(Δ/Δ) and control mice were compared. In summary, our data suggest that although loss of HIF2α does not afford protection from cobalt-induced lung inflammation, epithelial HIF2α signaling does play an important role in modulating the inflammatory and repair response in the lung.

  4. Effects of 17β-estradiol and 2-methoxyestradiol on the oxidative stress-hypoxia inducible factor-1 pathway in hypoxic pulmonary hypertensive rats.

    Science.gov (United States)

    Wang, Li; Zheng, Quan; Yuan, Yadong; Li, Yanpeng; Gong, Xiaowei

    2017-05-01

    The present study aimed to investigate the effects of 17β-estradiol (E2) and 2-methoxyestradiol (2ME) on the oxidative stress-hypoxia inducible factor-1 (OS-HIF-1) pathway in hypoxic pulmonary hypertensive rats. Female Sprague-Dawley rats were divided randomly into 4 groups, as follows: i) Control (Group A); ii) ovariectomy (OVX) + hypoxia (Group B); iii) OVX + hypoxia + E2 injection (Group C); and iv) 2ME injection (Group D). The rats were maintained under hypoxic conditions for 8 weeks, and mean pulmonary artery pressure (mPAP) and pulmonary arteriole morphology were measured. The reactive oxygen species, superoxide dismutase (SOD), manganese superoxide dismutase (MnSOD), and copper-zinc superoxide dismutase (Cu/ZnSOD) levels in serum were also measured. MnSOD and HIF-1α expression levels in lung tissue were determined by western blotting and reverse transcription-quantitative polymerase chain reaction. The mPAP and arterial remodeling index were significantly elevated following chronic hypoxia exposure; however, experimental data revealed a reduced response in E2 and 2ME intervention rats. Compared with Group A, Group B had significantly elevated oxidative stress levels, as illustrated by increased serum ROS levels, decreased serum SOD and MnSOD levels and decreased MnSOD mRNA and protein expression levels in lung tissue. Furthermore, HIF-1α mRNA and protein expression in Group B was significantly elevated compared with Group A. E2 and 2ME intervention significantly attenuated the aforementioned parameter changes, suggesting that E2 and 2ME partially ameliorate hypoxic pulmonary hypertension. The underlying mechanism of this may be associated with the increase in MnSOD activity and expression and reduction in ROS level, which reduces the levels of transcription and translation of HIF-1α.

  5. The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α protein levels in endothelial cells under hypoxia.

    Directory of Open Access Journals (Sweden)

    Stefan K Alig

    Full Text Available The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia.SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin. SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132 returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS formation, as measured by oxidation of H2-DCF and DHE fluorescence.SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

  6. Hypoxia induces a phenotypic switch of fibroblasts to myofibroblasts through a MMP-2/TIMP mediated pathway: Implications for venous neointimal hyperplasia in hemodialysis access

    Science.gov (United States)

    Misra, Sanjay; Fu, Alex A.; Misra, Khamal D.; Shergill, Uday M.; Leof, Edward B; Mukhopadhyay, Debabrata

    2010-01-01

    Purpose Hemodialysis grafts fail because of venous neointimal hyperplasia formation caused by adventitial fibroblasts which have become myofibroblasts (α-smooth muscle actin positive cells) and migrate to the neointima. There is increased expression of hypoxia inducible factor-1 alpha (HIF-1α in venous neointimal hyperplasia formation in experimental animal model and clinical samples. We hypothesized that under hypoxic stimulus (HIF-1α fibroblasts will convert to myofibroblasts through a matrix metalloproteinase-2 (MMP-2) mediated pathway. Materials and methods Murine AKR-2B fibroblasts were made hypoxic or normoxic for 24, 48, and 72 hours. Protein expression for HIF-1α, α-smooth muscle actin, MMP-2, MMP-9, TIMP-1, and TIMP-2 was performed to determine the kinetic changes of these proteins. Immunostaining for α-smooth muscle actin, collagen, and fibronectin was performed. Results At all time points, there was significantly increased expression of HIF-1α in the hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was significantly increased expression α-smooth muscle actin at all time points which peaked by 48 hours in hypoxic fibroblasts when compared to normoxic fibroblasts (P<0.05). There was a significant increase in the expression of active MMP-2 by 48-72 hours and a significant increase in tissue inhibitor of metalloproteinase-1 (TIMP-1) by 48-72 hours by hypoxic fibroblasts (P<0.05). By 72 hours, there was significant increase in TIMP-2 expression (P<0.05). Immunohistochemical analysis demonstrated increased expression for α-smooth muscle actin, collagen, and fibronectin as the length of hypoxia increased. Conclusions Under hypoxia, fibroblasts will convert to myofibroblasts through a MMP-2 mediated pathway which may provide insight into the mechanism of venous neointimal hyperplasia. PMID:20434368

  7. Intermittent Hypoxia Is Associated With High Hypoxia Inducible Factor-1α but Not High Vascular Endothelial Growth Factor Cell Expression in Tumors of Cutaneous Melanoma Patients

    Directory of Open Access Journals (Sweden)

    Isaac Almendros

    2018-04-01

    Full Text Available Epidemiological associations linking between obstructive sleep apnea and poorer solid malignant tumor outcomes have recently emerged. Putative pathways proposed to explain that these associations have included enhanced hypoxia inducible factor (HIF-1α and vascular endothelial growth factor (VEGF cell expression in the tumor and altered immune functions via intermittent hypoxia (IH. Here, we examined relationships between HIF-1α and VEGF expression and nocturnal IH in cutaneous melanoma (CM tumor samples. Prospectively recruited patients with CM tumor samples were included and underwent overnight polygraphy. General clinical features, apnea–hypopnea index (AHI, desaturation index (DI4%, and CM characteristics were recorded. Histochemical assessments of VEGF and HIF-1α were performed, and the percentage of positive cells (0, <25, 25–50, 51–75, >75% was blindly tabulated for VEGF expression, and as 0, 0–5.9, 6.0–10.0, >10.0% for HIF-1α expression, respectively. Cases with HIF-1α expression >6% (high expression were compared with those <6%, and VEGF expression >75% of cells was compared with those with <75%. 376 patients were included. High expression of VEGF and HIF-1α were seen in 88.8 and 4.2% of samples, respectively. High expression of VEGF was only associated with increasing age. However, high expression of HIF-1α was significantly associated with age, Breslow index, AHI, and DI4%. Logistic regression showed that DI4% [OR 1.03 (95% CI: 1.01–1.06] and Breslow index [OR 1.28 (95% CI: 1.18–1.46], but not AHI, remained independently associated with the presence of high HIF-1α expression. Thus, IH emerges as an independent risk factor for higher HIF-1α expression in CM tumors and is inferentially linked to worse clinical CM prognostic indicators.

  8. A comparative analysis of soft computing techniques for gene prediction.

    Science.gov (United States)

    Goel, Neelam; Singh, Shailendra; Aseri, Trilok Chand

    2013-07-01

    The rapid growth of genomic sequence data for both human and nonhuman species has made analyzing these sequences, especially predicting genes in them, very important and is currently the focus of many research efforts. Beside its scientific interest in the molecular biology and genomics community, gene prediction is of considerable importance in human health and medicine. A variety of gene prediction techniques have been developed for eukaryotes over the past few years. This article reviews and analyzes the application of certain soft computing techniques in gene prediction. First, the problem of gene prediction and its challenges are described. These are followed by different soft computing techniques along with their application to gene prediction. In addition, a comparative analysis of different soft computing techniques for gene prediction is given. Finally some limitations of the current research activities and future research directions are provided. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. RNA Sequencing Reveals that Kaposi Sarcoma-Associated Herpesvirus Infection Mimics Hypoxia Gene Expression Signature

    Science.gov (United States)

    Viollet, Coralie; Davis, David A.; Tekeste, Shewit S.; Reczko, Martin; Pezzella, Francesco; Ragoussis, Jiannis

    2017-01-01

    Kaposi sarcoma-associated herpesvirus (KSHV) causes several tumors and hyperproliferative disorders. Hypoxia and hypoxia-inducible factors (HIFs) activate latent and lytic KSHV genes, and several KSHV proteins increase the cellular levels of HIF. Here, we used RNA sequencing, qRT-PCR, Taqman assays, and pathway analysis to explore the miRNA and mRNA response of uninfected and KSHV-infected cells to hypoxia, to compare this with the genetic changes seen in chronic latent KSHV infection, and to explore the degree to which hypoxia and KSHV infection interact in modulating mRNA and miRNA expression. We found that the gene expression signatures for KSHV infection and hypoxia have a 34% overlap. Moreover, there were considerable similarities between the genes up-regulated by hypoxia in uninfected (SLK) and in KSHV-infected (SLKK) cells. hsa-miR-210, a HIF-target known to have pro-angiogenic and anti-apoptotic properties, was significantly up-regulated by both KSHV infection and hypoxia using Taqman assays. Interestingly, expression of KSHV-encoded miRNAs was not affected by hypoxia. These results demonstrate that KSHV harnesses a part of the hypoxic cellular response and that a substantial portion of hypoxia-induced changes in cellular gene expression are induced by KSHV infection. Therefore, targeting hypoxic pathways may be a useful way to develop therapeutic strategies for KSHV-related diseases. PMID:28046107

  10. Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

    International Nuclear Information System (INIS)

    Eom, Hyun-Jeong; Ahn, Jeong-Min; Kim, Younghun; Choi, Jinhee

    2013-01-01

    In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO 3 in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO 3 did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO 3 . These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO 3 . • HIF-1 and PMK-1 were needed for AgNPs- and AgNO 3 -induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO 3 did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal amount of silver mass contained

  11. Hypoxia inducible factor-1 (HIF-1)–flavin containing monooxygenase-2 (FMO-2) signaling acts in silver nanoparticles and silver ion toxicity in the nematode, Caenorhabditis elegans

    Energy Technology Data Exchange (ETDEWEB)

    Eom, Hyun-Jeong; Ahn, Jeong-Min [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of); Kim, Younghun [Department of Chemical Engineering, Kwangwoon University, 447-1, Wolgye-dong, Nowon-gu, Seoul 139-701 (Korea, Republic of); Choi, Jinhee, E-mail: jinhchoi@uos.ac.kr [School of Environmental Engineering and Graduate School of Energy and Environmental System Engineering, University of Seoul, 90 Jeonnong-dong, Dongdaemun-gu, Seoul 130-743 (Korea, Republic of)

    2013-07-15

    In the present study, nanotoxicity mechanism associated with silver nanoparticles (AgNPs) exposure was investigated on the nematode, Caenorhabditis elegans focusing on the hypoxia response pathway. In order to test whether AgNPs-induced hypoxia inducible factor-1 (HIF-1) activation was due to hypoxia or to oxidative stress, depletion of dissolved oxygen (DO) in the test media and a rescue effect using an antioxidant were investigated, respectively. The results suggested that oxidative stress was involved in activation of the HIF-1 pathway. We then investigated the toxicological implications of HIF-1 activation by examining the HIF-1 mediated transcriptional response. Of the genes tested, increased expression of the flavin containing monooxygenase-2 (FMO-2) gene was found to be the most significant as induced by AgNPs exposure. We found that AgNPs exposure induced FMO-2 activation in a HIF-1 and p38 MAPK PMK-1 dependent manner, and oxidative stress was involved in it. We conducted all experiments to include comparison of AgNPs and AgNO{sub 3} in order to evaluate whether any observed toxicity was due to dissolution or particle specific. The AgNPs and AgNO{sub 3} did not produce any qualitative differences in terms of exerting toxicity in the pathways observed in this study, however, considering equal amount of silver mass, in every endpoint tested the AgNPs were found to be more toxic than AgNO{sub 3}. These results suggest that Ag nanotoxicity is dependent not only on dissolution of Ag ion but also on particle specific effects and HIF-1–FMO-2 pathway seems to be involved in it. - Highlights: • HIF-1 signaling was investigated in C. elegans exposed to AgNPs and AgNO{sub 3}. • HIF-1 and PMK-1 were needed for AgNPs- and AgNO{sub 3}-induced fmo-2 gene expression. • PMK-1–HIF-1–FMO-2 pathway was dependent on oxidative stress. • AgNPs and AgNO{sub 3} did not produce any qualitative differences in HIF-1 signaling. • AgNPs were more toxic than an equal

  12. Correlation of Hypoxia-Inducible Factor 1α with Angiogenesis in Liver Tumors After Transcatheter Arterial Embolization in an Animal Model

    International Nuclear Information System (INIS)

    Liang Bin; Zheng Chuansheng; Feng, Gan-Sheng; Wu Hanping; Wang Yong; Zhao Hui; Qian Jun; Liang Huimin

    2010-01-01

    This study sought to determine the expression of hypoxia-inducible factor 1α (HIF-1α) and its relation to angiogenesis in liver tumors after transcatheter arterial embolization (TAE) in an animal model. A total of 20 New Zealand White rabbits were implanted with VX2 tumor in liver. TAE-treated group animals (n = 10) received TAE with polyvinyl alcohol particles. Control group animals (n = 10) received sham embolization with distilled water. Six hours or 3 days after TAE, animals were humanely killed, and tumor samples were collected. Immunohistochemical staining was performed to evaluate HIF-1α and vascular endothelial growth factor (VEGF) protein expression and microvessel density (MVD). Real-time polymerase chain reaction was performed to examine VEGF mRNA levels. The levels of HIF-1α protein were significantly higher in TAE-treated tumors than those in the control tumors (P = 0.001). HIF-1α protein was expressed in viable tumor cells that were located predominantly at the periphery of necrotic tumor regions. The levels of VEGF protein and mRNA, and mean MVD were significantly increased in TAE-treated tumors compared with the control tumors (P = 0.001, 0.000, and 0.001, respectively). HIF-1α protein level was significantly correlated with VEGF mRNA (r = 0.612, P = 0.004) and protein (r = 0.554, P = 0.011), and MVD (r = 0.683, P = 0.001). We conclude that HIF-1α is overexpressed in VX2 tumors treated with TAE as a result of intratumoral hypoxia generated by the procedure and involved in activation of the TAE-associated tumor angiogenesis. HIF-1α might represent a promising therapeutic target for antiangiogenesis in combination with TAE against liver tumors.

  13. Hypoxia-inducible factor-1α and Wnt/β-catenin signaling pathways promote the invasion of hypoxic gastric cancer cells.

    Science.gov (United States)

    Liu, Hong-Lan; Liu, Dang; Ding, Guang-Rong; Liao, Peng-Fei; Zhang, Jun-Wen

    2015-09-01

    The present study aimed to examine the association between hypoxia-inducible factor (HIF)-1α and the Wnt/β-catenin signaling pathway in a hypoxic environment. The study also aimed to explore the possible mechanisms underlying the invasion of hypoxic gastric cancer cells in vitro and in vivo. The pcDNA™ 6.2‑GW/EmGFP‑miR‑β‑catenin plasmid was transfected into SGC‑7901 gastric cancer cells, resulting in cells with stable suppression of β‑catenin expression. The biological characteristics of the control, liposome, negative control, β‑catenin knockdown, hypoxia and hypoxia β‑catenin knockdown groups were tested using an invasion assay. The differences in the invasive capacity of the control, negative control and liposome groups were not statistically significant. However, the hypoxia group demonstrated a significantly enhanced invasive capacity, as compared with that in the control group (Phypoxic and control cells was high alongside increased HIF‑1α, β‑catenin, uPA and MMP‑7 levels according to western blot and immunohistochemical analyses, while growth and protein levels of tumors from hypoxic β‑catenin knockdown cells were significantly lower and those of β‑catenin knockdown cells were lowest. In conclusion, these results suggested that HIF‑1α activation was able to regulate the Wnt/β‑catenin pathway, and that HIF‑1α may be controlled by the Wnt/β‑catenin pathway. A potential mechanism underlying SGC‑7901 tumorigenicity is the activation of the Wnt/β‑catenin signaling pathway, which activates uPA and MMP‑7 expression and contributes to the enhanced invasion of hypoxic cancer cells.

  14. Sestrin2 induced by hypoxia inducible factor1 alpha protects the blood-brain barrier via inhibiting VEGF after severe hypoxic-ischemic injury in neonatal rats.

    Science.gov (United States)

    Shi, Xudan; Doycheva, Desislava Met; Xu, Liang; Tang, Jiping; Yan, Min; Zhang, John H

    2016-11-01

    Hypoxic ischemic (HI) encephalopathy remains the leading cause of perinatal brain injury resulting in long term disabilities. Stabilization of blood brain barrier (BBB) after HI is an important target, therefore, in this study we aim to determine the role of sestrin2, a stress inducible protein which is elevated after various insults, on BBB stabilization after moderate and severe HI injuries. Rat pups underwent common carotid artery ligation followed by either 150min (severe model) or 100min (moderate model) of hypoxia. 1h post HI, rats were intranasally administered with recombinant human sestrin2 (rh-sestrin2) and sacrificed for infarct area, brain water content, righting reflex and geotaxis reflex. Sestrin2 was silenced using siRNA and an activator/inhibitor of hypoxia inducible factor1α (HIF1α) was used to examine their roles on BBB permeability. Rats subjected to severe HI exhibited larger infarct area and higher sestrin2 expression compared to rats in the moderate HI group. rh-sestrin2 attenuated brain infarct and edema, while silencing sestrin2 reversed these protective effects after severe HI. HIF1α induced sestrin2 activation in severe HI but not in moderate HI groups. A HIF1a agonist was shown to increase permeability of the BBB via vascular endothelial growth factor (VEGF) after moderate HI. However, after severe HI, HIF1α activated both VEGF and sestrin2. But HIF1α dependent sestrin2 activation was the predominant pathway after severe HI which inhibited VEGF and attenuated BBB permeability. rh-sestrin2 attenuated BBB permeability via upregulation of endogenous sestrin2 which was induced by HIF1α after severe HI. However, HIF1α's effects as a prodeath or prosurvival signal were influenced by the severity of HI injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Transplantation of Rat Mesenchymal Stem Cells Overexpressing Hypoxia-Inducible Factor 2α Improves Blood Perfusion and Arteriogenesis in a Rat Hindlimb Ischemia Model

    Directory of Open Access Journals (Sweden)

    Weifeng Lu

    2017-01-01

    Full Text Available Mesenchymal stem cells (MSCs have been increasingly tested in cell-based therapy to treat numerous diseases. Genetic modification to improve MSC behavior may enhance posttransplantation outcome. This study aims to test the potential therapeutic benefits of rat bone marrow MSCs overexpressing hypoxia-inducible factor 2α (rMSCsHIF-2α in a rat hindlimb ischemia model. PBS, rMSCs, or rMSCsHIF-2α were injected into rat ischemic hindlimb. Compared with the injection of PBS or rMSCs, transplantation of rMSCsHIF-2α significantly improved blood perfusion, increased the number of vessel branches in the muscle of the ischemic hindlimb, and improved the foot mobility of the ischemic hindlimb (all P<0.05. rMSCHIF-2α transplantation also markedly increased the expression of proangiogenic factors VEGF, bFGF, and SDF1 and Notch signaling proteins including DII4, NICD, Hey1, and Hes1, whereas it reduced the expression of proapoptotic factor Bax in the muscle of the ischemic hindlimb. Overexpression of HIF-2α did not affect rMSC stemness and proliferation under normoxia but significantly increased rMSC migration and tube formation in matrigel under hypoxia (all P<0.05. RMSCsHIF-2α stimulated endothelial cell invasion under hypoxia significantly (P<0.05. Genetic modification of rMSCs via overexpression of HIF-2α improves posttransplantation outcomes in a rat hindlimb ischemia model possibly by stimulating proangiogenic growth factors and cytokines.

  16. Angiogenesis and expression of vascular endothelial growth factor, tumour necrosis factor-α and hypoxia inducible factor-1α in canine renal cell carcinoma.

    Science.gov (United States)

    Yhee, J Y; Yu, C H; Kim, J H; Im, K S; Kim, N H; Brodersen, B W; Doster, A R; Sur, J-H

    2012-01-01

    The aim of the present study was to determine the distribution and characteristics of microvessels in various histological types of canine renal cell carcinoma (RCC). The study compared microvessel density (MVD) and distribution of blood vessels according to histological type and evaluated the presence of angiogenesis-related proteins. Nine archival samples of canine RCC were studied. MVD was calculated as the mean number of blood vessels per mm(2). The diameter of blood vessels was calculated by determining either the length of the long axis of blood vessels (diameter(max)) or the mean distance from the centre of each blood vessel to the tunica adventia (diameter(mean)). A significant difference in MVD was evident between RCCs and normal kidneys (46.6 ± 28.0 versus 8.4 ± 2.2 microvessels/mm(2)). Diameter(max) in canine RCCs (34.1 ± 14.7 μm) was also significantly different from normal canine kidney (23.2 ± 3.4 μm). Vascular endothelial growth factor (VEGF) was expressed by tumour cells and vascular endothelial cells and tumour necrosis factor (TNF)-α expression was observed in vascular endothelial cells in both neoplastic and normal kidney. Although VEGF is involved in angiogenesis and correlates with tumour stage of development, no correlation was found between VEGF expression and MVD. Tumour-associated macrophages expressing TNF-α and hypoxia inducible factor 1α were identified in peritumoural tissue and may play an important role in angiogenesis. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Effects of stem cell factor on hypoxia-inducible factor 1 alpha accumulation in human acute myeloid leukaemia and LAD2 mast cells.

    Directory of Open Access Journals (Sweden)

    Bernhard F Gibbs

    Full Text Available Stem cell factor (SCF is a hematopoietic growth factor that exerts its activity by signalling through the tyrosine kinase receptor known as Kit or CD117. SCF-Kit signalling is crucial for the survival, proliferation and differentiation of hematopoietic cells of myeloid lineage. Furthermore, since myeloid leukaemia cells express the Kit receptor, SCF may play an important role in myeloid leukaemia progression too. However, the mechanisms of this pathophysiological effect remain unclear. Recent evidence shows that SCF triggers accumulation of the inducible alpha subunit of hypoxia-inducible factor 1 (HIF-1 in hematopoietic cells--a transcription complex that plays a pivotal role in cellular adaptation to low oxygen availability. However, it is unknown how SCF impacts on HIF-1α accumulation in human myeloid leukaemia and mast cells. Here we show that SCF induces HIF-1α accumulation in THP-1 human myeloid leukaemia cells but not in LAD2 mast cells. We demonstrated that LAD2 cells have a more robust glutathione (GSH-dependent antioxidative system compared to THP-1 cells and are therefore protected against the actions of ROS generated in an SCF-dependent manner. BSO-induced GSH depletion led to a significant decrease in HIF-1α prolyl hydroxylase (PHD activity in THP-1 cells and to near attenuation of it in LAD2 cells. In THP-1 cells, SCF-induced HIF-1α accumulation is controlled via ERK, PI3 kinase/PKC-δ/mTOR-dependent and to a certain extent by redox-dependent mechanisms. These results demonstrate for the first time an important cross-talk of signalling pathways associated with HIF-1 activation--an important stage of the myeloid leukaemia cell life cycle.

  18. Enhanced response to radiotherapy in tumours deficient in the function of hypoxia-inducible factor-1

    International Nuclear Information System (INIS)

    Williams, Kaye J.; Telfer, Brian A.; Xenaki, Dia; Sheridan, Mary R.; Desbaillets, Isabelle; Peters, Hans J.W.; Honess, Davina; Harris, Adrian L.; Dachs, Gabi U.; Kogel, Albert van der; Stratford, Ian J.

    2005-01-01

    Background and purpose: To test the hypothesis that deficiency in expression of the transcription factor, HIF-1, renders tumours more radioresponsive than HIF-1 proficient tumours. Patients and methods: Tumours comprising mouse hepatoma cells lacking HIF-1β (and thereby HIF-1 function) were grown in nude mice and radiation-induced growth delay compared with that seen for wild-type tumours and tumours derived from HIF-1β negative cells where HIF-1 function had been restored. Results: The xenografts that lack HIF-1 activity take longer to establish their growth and are more radioresponsive than both parental xenografts and those with restored HIF-1 function. Pre-treatment of the HIF-1 deficient xenografts with the hypoxic radiosensitizer misonidazole, had little effect on radioresponse. In contrast this treatment radiosensitized the parental xenografts. In spite of this, no difference in oxygenation status was found between the tumour types as measured by Eppendorf O 2 -electrodes and by binding of the hypoxic cell marker NITP. Admixing wild type and HIF-1 deficient cells in the same tumour at ratios of 1 in 10 and 1 in 100 restores the growth of the mixed tumours to that of a 100% HIF-1 proficient cell population. However, when comparing the effects of radiation on the mixed tumours, radioresponsiveness is maintained in those tumours containing the high proportion of HIF-1 deficient cells. Conclusions: The differences in radioresponse do not correlate with tumour oxygenation, suggesting that the hypoxic cells within the HIF-1 deficient tumours do not contribute to the outcome of radiotherapy. Thus, hypoxia impacts on tumour radioresponsiveness not simply because of the physio-chemical mechanism of oxygen with radiation-induced radicals causing damage 'fixation', but also because hypoxia/HIF-1 promotes expression of genes that allow tumour cells to survive under these adverse conditions. Further, the results from the cell mixing experiments uncouple the growth

  19. A comparative study of three different gene expression analysis methods.

    Science.gov (United States)

    Choe, Jae Young; Han, Hyung Soo; Lee, Seon Duk; Lee, Hanna; Lee, Dong Eun; Ahn, Jae Yun; Ryoo, Hyun Wook; Seo, Kang Suk; Kim, Jong Kun

    2017-12-04

    TNF-α regulates immune cells and acts as an endogenous pyrogen. Reverse transcription polymerase chain reaction (RT-PCR) is one of the most commonly used methods for gene expression analysis. Among the alternatives to PCR, loop-mediated isothermal amplification (LAMP) shows good potential in terms of specificity and sensitivity. However, few studies have compared RT-PCR and LAMP for human gene expression analysis. Therefore, in the present study, we compared one-step RT-PCR, two-step RT-LAMP and one-step RT-LAMP for human gene expression analysis. We compared three gene expression analysis methods using the human TNF-α gene as a biomarker from peripheral blood cells. Total RNA from the three selected febrile patients were subjected to the three different methods of gene expression analysis. In the comparison of three gene expression analysis methods, the detection limit of both one-step RT-PCR and one-step RT-LAMP were the same, while that of two-step RT-LAMP was inferior. One-step RT-LAMP takes less time, and the experimental result is easy to determine. One-step RT-LAMP is a potentially useful and complementary tool that is fast and reasonably sensitive. In addition, one-step RT-LAMP could be useful in environments lacking specialized equipment or expertise.

  20. Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

    DEFF Research Database (Denmark)

    Sørensen, Brita Singers; Alsner, Jan; Overgaard, Jens

    2007-01-01

    BACKGROUND: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression...

  1. Hypoxia-Inducible Factor-1α in carcinogenesis and progression of breast cancer

    NARCIS (Netherlands)

    Bos, R.

    2004-01-01

    This thesis is primarily focused on the previously hardly explored role of HIF-1 in breast cancer. HIF-1 is a transcription factor induced by hypoxia, but also by some oncogenes, tumor suppressor genes and growth factors. Activated HIF-1 can induce angiogenesis, glycolysis, erythropoiesis, and other

  2. Multiple roles of hypoxia in ovarian function: roles of hypoxia-inducible factor-related and -unrelated signals during the luteal phase

    OpenAIRE

    Nishimura, Ryo; Okuda, Kiyoshi

    2015-01-01

    There is increasing interest in the role of oxygen conditions in the microenvironment of organs because of the discovery of a hypoxia-specific transcription factor, namely hypoxia-inducible factor (HIF) 1. Ovarian function has several phases that change day by day, including ovulation, follicular growth and corpus luteum formation and regression. These phases are regulated by many factors, including pituitary hormones and local hormones, such as steroids, peptides and cytokines, as well as ox...

  3. Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

    Science.gov (United States)

    Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K; Jain, Anil K; Ramteke, Anand M; Serkova, Natalie J; Agarwal, Chapla; Agarwal, Rajesh

    2017-03-01

    Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O 2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELM?) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

    OpenAIRE

    Angelini, Daniel J.; Su, Qingning; Kolosova, Irina A.; Fan, Chunling; Skinner, John T.; Yamaji-Kegan, Kazuyo; Collector, Michael; Sharkis, Saul J.; Johns, Roger A.

    2010-01-01

    Background Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM?) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling ...

  5. Scaffold filling, contig fusion and comparative gene order inference

    Directory of Open Access Journals (Sweden)

    Rounsley Steve

    2010-06-01

    Full Text Available Abstract Background There has been a trend in increasing the phylogenetic scope of genome sequencing without finishing the sequence of the genome. Increasing numbers of genomes are being published in scaffold or contig form. Rearrangement algorithms, however, including gene order-based phylogenetic tools, require whole genome data on gene order or syntenic block order. How then can we use rearrangement algorithms to compare genomes available in scaffold form only? Can the comparative evidence predict the location of unsequenced genes? Results Our method involves optimally filling in genes missing from the scaffolds, while incorporating the augmented scaffolds directly into the rearrangement algorithms as if they were chromosomes. This is accomplished by an exact, polynomial-time algorithm. We then correct for the number of extra fusion/fission operations required to make scaffolds comparable to full assemblies. We model the relationship between the ratio of missing genes actually absent from the genome versus merely unsequenced ones, on one hand, and the increase of genomic distance after scaffold filling, on the other. We estimate the parameters of this model through simulations and by comparing the angiosperm genomes Ricinus communis and Vitis vinifera. Conclusions The algorithm solves the comparison of genomes with 18,300 genes, including 4500 missing from one genome, in less than a minute on a MacBook, putting virtually all genomes within range of the method.

  6. Scaffold filling, contig fusion and comparative gene order inference.

    Science.gov (United States)

    Muñoz, Adriana; Zheng, Chunfang; Zhu, Qian; Albert, Victor A; Rounsley, Steve; Sankoff, David

    2010-06-04

    There has been a trend in increasing the phylogenetic scope of genome sequencing without finishing the sequence of the genome. Increasing numbers of genomes are being published in scaffold or contig form. Rearrangement algorithms, however, including gene order-based phylogenetic tools, require whole genome data on gene order or syntenic block order. How then can we use rearrangement algorithms to compare genomes available in scaffold form only? Can the comparative evidence predict the location of unsequenced genes? Our method involves optimally filling in genes missing from the scaffolds, while incorporating the augmented scaffolds directly into the rearrangement algorithms as if they were chromosomes. This is accomplished by an exact, polynomial-time algorithm. We then correct for the number of extra fusion/fission operations required to make scaffolds comparable to full assemblies. We model the relationship between the ratio of missing genes actually absent from the genome versus merely unsequenced ones, on one hand, and the increase of genomic distance after scaffold filling, on the other. We estimate the parameters of this model through simulations and by comparing the angiosperm genomes Ricinus communis and Vitis vinifera. The algorithm solves the comparison of genomes with 18,300 genes, including 4500 missing from one genome, in less than a minute on a MacBook, putting virtually all genomes within range of the method.

  7. SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

    International Nuclear Information System (INIS)

    Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

    2012-01-01

    Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

  8. A New Pharmacological Agent (AKB-4924) Stabilizes Hypoxia Inducible Factor (HIF) and Increases Skin Innate Defenses Against Bacterial Infection

    Science.gov (United States)

    Okumura, Cheryl Y.M.; Hollands, Andrew; Tran, Dan N.; Olson, Joshua; Dahesh, Samira; von Köckritz-Blickwede, Maren; Thienphrapa, Wdee; Corle, Courtney; Jeung, Seung Nam; Kotsakis, Anna; Shalwitz, Robert A.; Johnson, Randall S.; Nizet, Victor

    2013-01-01

    Hypoxia inducible factor-1 (HIF-1) is a transcription factor that is a major regulator of energy homeostasis and cellular adaptation to low oxygen stress. HIF-1 is also activated in response to bacterial pathogens and supports the innate immune response of both phagocytes and keratinocytes. In this work, we show that a new pharmacological compound AKB-4924 (Akebia Therapeutics) increases HIF-1α levels and enhances the antibacterial activity of phagocytes and keratinocytes against both methicillin-sensitive and -resistant strains of Staphylococcus aureus in vitro. AKB-4924 is also effective in stimulating the killing capacity of keratinocytes against the important opportunistic skin pathogens Pseudomonas aeruginosa and Acinitobacter baumanii. The effect of AKB-4924 is mediated through the activity of host cells, as the compound exerts no direct antimicrobial activity. Administered locally as a single agent, AKB-4924 limits S. aureus proliferation and lesion formation in a mouse skin abscess model. This approach to pharmacologically boost the innate immune response via HIF-1 stabilization may serve as a useful adjunctive treatment for antibiotic-resistant bacterial infections. PMID:22371073

  9. Ethanol extract of Portulaca oleracea L. protects against hypoxia-induced neuro damage through modulating endogenous erythropoietin expression.

    Science.gov (United States)

    Wanyin, Wang; Liwei, Dong; Lin, Jia; Hailiang, Xin; Changquan, Ling; Min, Li

    2012-04-01

    In addition to its role in erythropoiesis, erythropoietin is also appreciated for its neuroprotective effects, and it has been suggested for treatment of some ischemic-hypoxic neurovascular diseases. The protective effects of endogenous erythropoietin in the brain give rise to the hypothesis that modulating erythropoietin expression might be a better way for treatment of ischemia-hypoxia neurovascular diseases. We have found that ethanol extract of Portulaca oleracea L. (EEPO) could increase erythropoietin expression in hypoxic mouse brain in our previous study. The present study is to investigate whether EEPO exerts its neuroprotective effects against hypoxia injury through regulating endogenous erythropoietin expression. The results demonstrated that EEPO decreased the serum neuron specific enolase level in hypoxia mice and the activity of caspase-3 in neuron, increased the neuron viability and attenuated the pathological damages caused by the hypoxia condition. Importantly, we also found that EEPO stimulated the endogenous erythropoietin expression at both mRNA and protein levels. Using the conditioned medium containing soluble erythropoietin receptor, we found that the neuroprotective effects of EEPO were dependent, at least partly, on erythropoietin expression. Although EEPO did not affect transcription of hypoxia inducible factor-1α (HIF-1α), it did stabilize expression of HIF-1α. It is concluded that EEPO has neuroprotective effects against hypoxia injury, which is at least partly through stimulating endogenous erythropoietin expression by stabilizing HIF-1α. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Hypoxia-Induced neonatal seizures diminish silent synapses and long-term potentiation in hippocampal CA1 neurons

    Science.gov (United States)

    Zhou, Chengwen; Bell, Jocelyn J. Lippman; Sun, Hongyu; Jensen, Frances E.

    2012-01-01

    Neonatal seizures can lead to epilepsy and long-term cognitive deficits in adulthood. Using a rodent model of the most common form of human neonatal seizures, hypoxia-induced seizures (HS), we aimed to determine whether these seizures modify long-term potentiation (LTP) and “silent” N-methyl-D-aspartate receptor (NMDAR)-only synapses in hippocampal CA1. At 48-72 hours (hrs) post-HS, electrophysiology and immunofluorescent confocal microscopy revealed a significant decrease in the incidence of silent synapses, and an increase in amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at the synapses. Coincident with this decrease in silent synapses, there was an attenuation of LTP elicited by either tetanic stimulation of Schaffer collaterals or a pairing protocol, and persistent attenuation of LTP in slices removed in later adulthood after P10 HS. Furthermore, post-seizure treatment in vivo with the AMPAR antagonist 2,3-dihydroxy-6-nitro-7-sulfonyl-benzo[f]quinoxaline (NBQX) protected against the HS-induced depletion of silent synapses and preserved LTP. Thus, this study demonstrates a novel mechanism by which early-life seizures could impair synaptic plasticity, suggesting a potential target for therapeutic strategies to prevent long-term cognitive deficits. PMID:22171027

  11. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    International Nuclear Information System (INIS)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-01

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  12. Hypoxia-inducible factor-1α upregulation in microglia following hypoxia protects against ischemia-induced cerebral infarction.

    Science.gov (United States)

    Huang, Tao; Huang, Weiyi; Zhang, Zhiqiang; Yu, Lei; Xie, Caijun; Zhu, Dongan; Peng, Zizhuang; Chen, Jiehan

    2014-10-01

    Activated microglia were considered to be the toxic inflammatory mediators that induce neuron degeneration after brain ischemia. Hypoxia can enhance the expression of hypoxia-inducible factor-1α (HIF-1α) in microglia and cause microglial activation. However, intermittent hypoxia has been reported recently to be capable of protecting the body from myocardial ischemia. We established a high-altitude environment as the hypoxic condition in this study. The hypoxic condition displayed a neuroprotective effect after brain ischemia, and mice exposed to this condition presented better neurological performance and smaller infarct size. At the same time, a high level of HIF-1α, low level of isoform of nitric oxide synthase, and a reduction in microglial activation were also seen in ischemic focus of hypoxic mice. However, this neuroprotective effect could be blocked by 2-methoxyestradiol, the HIF-1α inhibitor. Our finding suggested that HIF-1α expression was involved in microglial activation in vitro and was regulated by oxygen supply. The microglia were inactivated by re-exposure to hypoxia, which might be due to overexpression of HIF-1α. These results indicated that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain ischemia. This mechanism possibly lies in microglial inactivation through regulation of the expression of HIF-1α.

  13. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor--1 alpha protein in hypoxic conditions.

    Science.gov (United States)

    Wang, Ronghai; Zhang, Ping; Li, Jinhang; Guan, Hongzai; Shi, Guangjun

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG-HIF-1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Neuroprotection by hypoxic preconditioning involves upregulation of hypoxia-inducible factor-1 in a prenatal model of acute hypoxia.

    Science.gov (United States)

    Giusti, Sebastián; Fiszer de Plazas, Sara

    2012-02-01

    The molecular pathways underlying the neuroprotective effects of preconditioning are promising, potentially drugable targets to promote cell survival. However, these pathways are complex and are not yet fully understood. In this study we have established a paradigm of hypoxic preconditioning based on a chick embryo model of normobaric acute hypoxia previously developed by our group. With this model, we analyzed the role of hypoxia-inducible factor-1α (HIF-1α) stabilization during preconditioning in HIF-1 signaling after the hypoxic injury and in the development of a neuroprotective effect against the insult. To this end, we used a pharmacological approach, based on the in vivo administration of positive (Fe(2+), ascorbate) and negative (CoCl(2)) modulators of the activity of HIF-prolyl hydroxylases (PHDs), the main regulators of HIF-1. We have found that preconditioning has a reinforcing effect on HIF-1 accumulation during the subsequent hypoxic injury. In addition, we have also demonstrated that HIF-1 induction during hypoxic preconditioning is necessary to obtain an enhancement in HIF-1 accumulation and to develop a tolerance against a subsequent hypoxic injury. We provide in vivo evidence that administration of Fe(2+) and ascorbate modulates HIF accumulation, suggesting that PHDs might be targets for neuroprotection in the CNS. Copyright © 2011 Wiley Periodicals, Inc.

  15. Targeting Hypoxia-Inducible Factor 1α in a New Orthotopic Model of Glioblastoma Recapitulating the Hypoxic Tumor Microenvironment.

    Science.gov (United States)

    Nigim, Fares; Cavanaugh, Jill; Patel, Anoop P; Curry, William T; Esaki, Shin-ichi; Kasper, Ekkehard M; Chi, Andrew S; Louis, David N; Martuza, Robert L; Rabkin, Samuel D; Wakimoto, Hiroaki

    2015-07-01

    Tissue hypoxia and necrosis represent pathophysiologic and histologic hallmarks of glioblastoma (GBM). Although hypoxia inducible factor 1α (HIF-1α) plays crucial roles in the malignant phenotypes of GBM, developing HIF-1α-targeted agents has been hampered by the lack of a suitable preclinical model that recapitulates the complex biology of clinical GBM. We present a new GBM model, MGG123, which was established from a recurrent human GBM. Orthotopic xenografting of stem-like MGG123 cells reproducibly generated lethal tumors that were characterized by foci of palisading necrosis, hypervascularity, and robust stem cell marker expression. Perinecrotic neoplastic cells distinctively express HIF-1α and are proliferative in both xenografts and the patient tissue. The xenografts contain scattered hypoxic foci that were consistently greater than 50 μm distant from blood vessels, indicating intratumoral heterogeneity of oxygenation. Hypoxia enhanced HIF-1α expression in cultured MGG123 cells, which was abrogated by the HIF-1α inhibitors digoxin or ouabain. In vivo, treatment of orthotopic MGG123 xenografts with digoxin decreased HIF-1α expression, vascular endothelial growth factor mRNA levels, and CD34-positive vasculature within the tumors, and extended survival of mice bearing the aggressive MGG123 GBM. This preclinical tumor model faithfully recapitulates the GBM-relevant hypoxic microenvironment and stemness and is a suitable platform for studying disease biology and developing hypoxia-targeted agents.

  16. Ubiquitination is absolutely required for the degradation of hypoxia-inducible factor - 1 alpha protein in hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ronghai [Department of Urology, Linzi District People' s Hospital, Zibo, 255400 (China); Zhang, Ping, E-mail: zpskx001@163.com [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Li, Jinhang [Department of Gynecology, Qingdao Municipal Hospital, Qingdao, 266011 (China); Guan, Hongzai [Laboratory Department, School of Medicine, Qingdao University, Qingdao, 266071 (China); Shi, Guangjun, E-mail: qdmhshigj@yahoo.com [Department of Hepatobiliary Surgery, Qingdao Municipal Hospital, Qingdao, 266071 (China)

    2016-01-29

    The hypoxia-inducible factor (HIF) is recognized as the master regulator of hypoxia response. HIF-α subunits expression are tightly regulated. In this study, our data show that ts20 cells still expressed detectable E1 protein even at 39.5° C for 12 h, and complete depletion of E1 protein expression at 39.5° C by siRNA enhanced HIF-1α and P53 protein expression. Further inhibition of E1 at 39.5 °C by siRNA, or E1 inhibitor Ube1-41 completely blocked HIF-1α degradation. Moreover, immunoprecipitations of co-transfection of HA-ubiquitin and FLAG–HIF–1α plasmids directly confirmed the involvement of ubiquitin in the hypoxic degradation of HIF-1α. Additionally, hypoxic HIF-1 α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization. Taken together, our data suggest that constitutive HIF-1α protein degradation in hypoxia is absolutely ubiquitination-dependent, and unidentified E3 ligase may exist for this degradation pathway. - Highlights: • HIF-1α protein is constitutively degraded in hypoxic conditions. • Requirement of ubiquitination for HIF-1α degradation in hypoxia. • Hypoxic HIF-1α degradation is independent of HAF, RACK1, sumoylation or nuclear/cytoplasmic localization.

  17. The retinoblastoma protein regulates hypoxia-inducible genetic programs, tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells

    Science.gov (United States)

    Labrecque, Mark P.; Takhar, Mandeep K.; Nason, Rebecca; Santacruz, Stephanie; Tam, Kevin J.; Massah, Shabnam; Haegert, Anne; Bell, Robert H.; Altamirano-Dimas, Manuel; Collins, Colin C.; Lee, Frank J.S.; Prefontaine, Gratien G.; Cox, Michael E.; Beischlag, Timothy V.

    2016-01-01

    Loss of tumor suppressor proteins, such as the retinoblastoma protein (Rb), results in tumor progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumor that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role Rb plays in mediating hypoxia-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22Rv1 human prostate cancer cells. DNA microarray analysis revealed that loss of Rb in conjunction with hypoxia leads to aberrant expression of hypoxia-regulated genetic programs that increase cell invasion and promote neuroendocrine differentiation. For the first time, we have established a direct link between hypoxic tumor environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumors to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. PMID:27015368

  18. Study on radiation regulation of hypoxia inducible factor-1α expression and its correlation with hepatoma radiosensitivity

    International Nuclear Information System (INIS)

    Jin Wensen; Kong Zhaolu; Shen Zhifen; Tong Shungao; Ji Huajun; Jin Yizun

    2008-01-01

    Objective: To study the regulation of hypoxia inducible factor-1α (HIF-1α) expression in hepatoma cells after irradiation and the expression of HIF-1α effect on the radiosensitivity of heptoma cells. Methods: HepG2 cells were pretreated by Cobalt chloride (COCl 2 ), a chemical hypoxia agent, to induce and stabilize the expression of HIF-1α, and then exposed to different γ-irradiation doses. Clonogenic assay was used to evaluate HepG2 cell survival fraction (SF) after irradiation under normoxia and chemical hypoxia. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot assay (Western blot) were utilized to detect the changes of intracellular HIF-1α on the level of transcripation and translation. Results: Cell survival level was elevated by chemical hypoxia and there was a statistical difference between chemical hypoxic group and normoxic group. The ratios of SF(SF co /SF o 2 )on two different conditions were increased with irradiation doses. Meanwhile, the irradiation induced up-regulation of HIF-1α in dose-dependent manner. The expression of HIF-1α was correlated with HepG2 cell survival level to some extent. Conclusions: Irradiation could up-regulate the level of HIF-1α expression in HepG2 cells under chemical hypoxic condition. The cells survival level might be influenced by the changes in HIF-1α expression. (authors)

  19. RNAi Knockdown of Hypoxia-Inducible Factor-1α Decreased the Proliferation, Migration, and Invasion of Hypoxic Hepatocellular Carcinoma Cells.

    Science.gov (United States)

    Chen, ChengShi; Liu, Rong; Wang, JianHua; Yan, ZhiPing; Qian, Sheng; Zhang, Wei

    2015-04-01

    The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.

  20. Relationship between serum hypoxia-inducible factor-1α and carotid plaque in patients with type 2 diabetes mellitus

    International Nuclear Information System (INIS)

    E Weiqin; Shi Bimin

    2012-01-01

    Objective: To observe the changes of type 2 diabetes mellitus (T2DM) patients' serum hypoxia inducible factor-1α (HIF-1α), and evaluate the relationship between serum HIF-1α and vascular lesions of atherosclerosis. Methods: The serum level of HIF-1α in 32 T2DM with carotid plaques (T2DM+CP group), 24 T2DM without macrovascular complications (T2DM group), and 24 controls was studied with ELISA method. Results: The serum HIF-1α level in T2DM with and without carotid plaque group was significantly higher than that in the controls (all P<0.01). Furthermore, among T2DM, the level of HIF-1α was higher in patients with carotid plaque than that without carotid plaque (P<0.05) . The serum HIF-1α was positively correlated with fasting plasma glucose, HbAlc and HOMA-IR (all P<0.05). Multiple linear stepwise regression analysis showed that HbAlc was the independent determinants of HIF-1α. Conclusion: High level of serum HIF-1α in T2DM patients concerns with blood glucose and insulin resistance, which plays an important role in the development of macrovascular complications. (authors)

  1. Neutralizing S1P inhibits intratumoral hypoxia, induces vascular remodelling and sensitizes to chemotherapy in prostate cancer

    Science.gov (United States)

    Ader, Isabelle; Golzio, Muriel; Andrieu, Guillaume; Zalvidea, Santiago; Richard, Sylvain; Sabbadini, Roger A.; Malavaud, Bernard; Cuvillier, Olivier

    2015-01-01

    Hypoxia promotes neovascularization, increased tumor growth, and therapeutic resistance. The transcription factor, hypoxia-inducible factor 1α (HIF-1α), has been reported as the master driver of adaptation to hypoxia. We previously identified the sphingosine kinase 1/sphingosine 1-phosphate (SphK1/S1P) pathway as a new modulator of HIF-1α under hypoxia. Taking advantage of a monoclonal antibody neutralizing extracellular S1P (sphingomab), we report that inhibition of S1P extracellular signaling blocks HIF-1α accumulation and activity in several cancer cell models exposed to hypoxia. In an orthotopic xenograft model of prostate cancer, we show that sphingomab reduces hypoxia and modifies vessel architecture within 5 days of treatment, leading to increased intratumoral blood perfusion. Supporting the notion that a transient vascular normalization of tumor vessels is the mechanism by which sphingomab exerts its effects, we demonstrate that administration of the antibody for 5 days before chemotherapy is more effective at local tumor control and metastatic dissemination than any other treatment scheduling. These findings validate sphingomab as a potential new normalization agent that could contribute to successful sensitization of hypoxic tumors to chemotherapy. PMID:25915662

  2. Human monocytes undergo functional re-programming during sepsis mediated by hypoxia-inducible factor-1α.

    Science.gov (United States)

    Shalova, Irina N; Lim, Jyue Yuan; Chittezhath, Manesh; Zinkernagel, Annelies S; Beasley, Federico; Hernández-Jiménez, Enrique; Toledano, Victor; Cubillos-Zapata, Carolina; Rapisarda, Annamaria; Chen, Jinmiao; Duan, Kaibo; Yang, Henry; Poidinger, Michael; Melillo, Giovanni; Nizet, Victor; Arnalich, Francisco; López-Collazo, Eduardo; Biswas, Subhra K

    2015-03-17

    Sepsis is characterized by a dysregulated inflammatory response to infection. Despite studies in mice, the cellular and molecular basis of human sepsis remains unclear and effective therapies are lacking. Blood monocytes serve as the first line of host defense and are equipped to recognize and respond to infection by triggering an immune-inflammatory response. However, the response of these cells in human sepsis and their contribution to sepsis pathogenesis is poorly understood. To investigate this, we performed a transcriptomic, functional, and mechanistic analysis of blood monocytes from patients during sepsis and after recovery. Our results revealed the functional plasticity of monocytes during human sepsis, wherein they transited from a pro-inflammatory to an immunosuppressive phenotype, while enhancing protective functions like phagocytosis, anti-microbial activity, and tissue remodeling. Mechanistically, hypoxia inducible factor-1α (HIF1α) mediated this functional re-programming of monocytes, revealing a potential mechanism for their therapeutic targeting to regulate human sepsis. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. 17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

    Science.gov (United States)

    Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

    2010-03-01

    The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

  4. Hypoxia-inducible factors regulate pluripotency factor expression by ZNF217- and ALKBH5-mediated modulation of RNA methylation in breast cancer cells.

    Science.gov (United States)

    Zhang, Chuanzhao; Zhi, Wanqing Iris; Lu, Haiquan; Samanta, Debangshu; Chen, Ivan; Gabrielson, Edward; Semenza, Gregg L

    2016-10-04

    Exposure of breast cancer cells to hypoxia increases the percentage of breast cancer stem cells (BCSCs), which are required for tumor initiation and metastasis, and this response is dependent on the activity of hypoxia-inducible factors (HIFs). We previously reported that exposure of breast cancer cells to hypoxia induces the ALKBH5-mediated demethylation of N6-methyladenosine (m6A) in NANOG mRNA leading to increased expression of NANOG, which is a pluripotency factor that promotes BCSC specification. Here we report that exposure of breast cancer cells to hypoxia also induces ZNF217-dependent inhibition of m6A methylation of mRNAs encoding NANOG and KLF4, which is another pluripotency factor that mediates BCSC specification. Although hypoxia induced the BCSC phenotype in all breast-cancer cell lines analyzed, it did so through variable induction of pluripotency factors and ALKBH5 or ZNF217. However, in every breast cancer line, the hypoxic induction of pluripotency factor and ALKBH5 or ZNF217 expression was HIF-dependent. Immunohistochemistry revealed that expression of HIF-1α and ALKBH5 was concordant in all human breast cancer biopsies analyzed. ALKBH5 knockdown in MDA-MB-231 breast cancer cells significantly decreased metastasis from breast to lungs in immunodeficient mice. Thus, HIFs stimulate pluripotency factor expression and BCSC specification by negative regulation of RNA methylation.

  5. Muscle-specific expression of hypoxia-inducible factor in human skeletal muscle

    DEFF Research Database (Denmark)

    Mounier, Rémi; Pedersen, Bente Klarlund; Plomgaard, Peter

    2010-01-01

    fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies...... from triceps brachii (characterized by a high proportion of type II fibres), from soleus (characterized by a high proportion of type I fibres) and from vastus lateralis (characterized by an equal proportion of type I and II fibres). The hypothesis was that type I muscle fibres would have lower HIF-1......alpha protein level. Interestingly, none of the HIF-1alpha target genes, like the most studied angiogenic factor involved in muscle angiogenesis, vascular endothelial growth factor (VEGF), exhibited a muscle fibre-specific-related mRNA expression at rest in normoxia. However, soleus presented...

  6. Hypoxia regulates the expression and localization of CCAAT/enhancer binding protein α by hypoxia inducible factor-1α in bladder transitional carcinoma cells.

    Science.gov (United States)

    Xue, Mei; Li, Xu; Chen, Wei

    2015-08-01

    Hypoxia inducible factor-1α (HIF-1α) is overexpressed in various types of solid tumor in humans, including bladder cancer. HIF-1α regulates the expression of a series of genes, which are involved in cell proliferation, differentiation, apoptosis, angiogenesis, migration and invasion and represents a potential therapeutic target for the treatment of human cancer. Despite extensive investigation of the effects of HIF-1α in the progression and metastasis of bladder cancer, the possible regulatory mechanisms underlying the effects of HIF-1α on bladder cancer cell proliferation and differentiation remain to be elucidated. It has been suggested that the transcription factor CCAAT/enhancer binding protein α (C/EBPα) acts as a tumor suppressor in several types of cancer cell, which are involved in regulating cell differentiation, proliferation and apoptosis. The present study confirmed that, in bladder cancer cells, the expression and localization of C/EBPα was regulated by hypoxia through an HIF-1α -dependent mechanism, which may be significant in bladder cancer cell proliferation and differentiation. The 5637 and T24 bladder cancer cell lines were incubated under normoxic and hypoxic conditions. The expression levels of HIF-1α and C/EBPα were detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunofluorescence analysis. The results revealed that, under hypoxic conditions, the protein expression levels of HIF-1α were markedly upregulated, but the mRNA levels were not altered. However, the mRNA and protein levels of C/EBPα were significantly reduced. The present study further analyzed the subcellular localization of C/EBPα, which was markedly decreased in the nuclei under hypoxic conditions. Following HIF-1α small interference RNA silencing of HIF-1α, downregulation of C/EBPα was prevented in the bladder cancer cells cultured under hypoxic conditions. In addition, groups of cells treated with 3-(5'-hydroxymethyl

  7. Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner