Transcription profiling of mouse macrophage response to hypoxia, part 2
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ABSTRACT: We hypothesize that the culture media collected from macrophages exposed to intermittent hypoxia will induce a greater pro-inflammatory gene profile in naïve cultured macrophages than will culture media collected from macrophages exposed to sustained hypoxia. We will evaluate gene expression using microarray analysis of RNA collected from RAW 264.7 macrophages cultured for 24 hours in DMEM media obtained from 1) cells cultured with intermittent hypoxia (2 minute cycles: 90 seconds at 40 Torr and 30 seconds at 8 Torr), 2) media exposed to intermittent hypoxia, 3) cells cultured with sustained hypoxia (8 Torr), 4) media exposed to sustained hypoxia and 4) standard tissue culture conditions (fresh DMEM media; reference).
Project description:We hypothesize that cultured macrophages directly exposed intermittent hypoxia will have a greater change in expression in genes related to inflammatory response than macrophages exposed to sustained hypoxia. We will evaluate gene expression using microarray analysis of RNA collected from RAW 264.7 macrophages cultured under the following environmental conditions: 1) 4 hours of intermittent hypoxia (2 minute cycles: 90 seconds at 40 Torr and 30 seconds at 8 Torr), 2) 4 hours of sustained hypoxia (8 Torr), and 3 ) standard tissue culture conditions (141 Torr; reference).
Project description:Transcriptional responses to hypoxia were compared between a hypoxia tolerant fish (tidepool sculpin; Oligocottus maculosus) and a hypoxia intolerant fish (silverspotted sculpin; Blepsias cirrhosus). To determine if, and how, transcriptional plasticity is associated with differences in hypoxia tolerance, each species was subjected to a hypoxic time-course and liver was sampled at normoxia, 3hr, 8hr, 24hr, 48hr and 72hr of hypoxia. The hypoxic level for each species was scaled to the species' own tolerance level (relative exposure) in order to elicit similar tissue level hypoxia between the two sculpin species. Each species was also subjected to a single environmental O2 tension (absolute exposure) and sampled at 24 hrs of hypoxia.
Project description:Lactic acidosis and hypoxia are two prominent tumor microenvironmental stresses that are both known to exert important influences on gene expression and phenotypes of cancer cells. But very little is known about the cross-talk and interaction between these two stresses. We performed gene expression analysis of MCF7 cells exposed to lactic acidosis, hypoxia and combined lactic acidosis and hypoxia. We found the hypoxia response elicited under hypoxia was mostly abolished upon simultaneous exposure to lactic acidosis. The repression effects are due to loss of HIF-1α protein synthesis under lactic acidosis. In addition, we showed lactic acidosis strongly synergizes with hypoxia to activate the unfold protein response (UPR) and inflammation response which are highly similar to amino acid deprivation responses (AAR). The statistical factor analysis of hypoxia and lactic acidosis responses indicated that ATF4 locus, an important activator in the UPR/AAR pathway, is amplified in subsets of breast tumors and cancer cell lines. Varying ATF4 levels dramatically affect the ability to survive the post-stress recovery from hypoxia and lactic acidosis and may suggest its selection of ATF4 amplification in human cancers. These data suggest that lactic acidosis interacts with hypoxia by both inhibiting the canonical hypoxia response and while activating the UPR and inflammation response. Gain of ATF4 locus may offer survival advantages to allow successful adaptation to frequent fluctuations of oxygen and acidity in tumor microenvironment. Collectively, our studies have provided linkage between the short-term transcriptional responses to the long term selection of the DNA copy number alterations (CNAs) under tumor microenvironmental stresses. RNAs from MCF7 cells exposed to control condition (ambient air ~21% O2, no lactate and neutral pH), lactic acidosis (ambient air, 10 mM Lactate and pH 6.7), hypoxia (1% pO2, no lactate and neutral pH) and the combined lactic acidosis and hypoxia (1% pO2, 10 mM Lactate and pH 6.7) condition for 24 hours were extracted by miRVana kits (Ambion) and hybridized to Affymetrix Human genome 133A 2.0 arrays with standard protocol.
Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells
Project description:Here we used microarrays to characterize changes in global gene expression in the hepatopancreas of Pacific white shrimp, Litopenaeus vannamei, exposed to short term (4 h) hypoxia (H) or hypercapnic hypoxia (HH) or long term (24 h) H or HH, compared to animals in air-saturated water (normoxia). The transcriptomes of crustaceans exposed to low O2 and high CO2 contained both shared and treatment-specific signature genes (q M-bM-^IM-$ 0.01, FC M-bM-^IM-% 1.5), with shifts characteristic of metabolic depression rather than anaerobic metabolism. Down-regulated signature genes dominated the transcript profile in three of the four treatments (H 4 h, H 24 h, 4 h HH); many of these genes were involved in amino acid or RNA metabolism or in translation, including several tRNA synthetases. Unique patterns of gene expression such as increased lipid metabolism and hemocyanin synthesis (H 24 h) and initiation of apoptosis (24 h HH) were tied to specific treatments. This work contributes insight to the effects that human perturbations might have on estuarine organisms, and the importance of examining the impacts of environmentally relevant combinations of hypoxia and hypercapnia on estuarine populations. L. vannamei were exposed for 4 or 24 hours to one of the following conditions: normoxia, hypoxia or hypercapnic hypoxia. Hepatopancreas tissue from individual animals was dissected, total RNA extracted, labelled and hybridized to oligonucleotide microarrays with probes for 21,864 L. vannamei unigenes. Treatments were repeated until a total of 7 biological replicates was obtained for each time:treatment combination, except for the 24 h normoxia group, represented by 6 replicates.
Project description:Aspergillus fumigatus was cultured in a chemostat for 12.5 hours, and switched to hypoxia (0.2% oxygen). Samples were collected at the beginning of the experiment, before the switch to hypoxia, and 2, 6, 12 and 24 hours after the switch. RNA was extracted and microarrays performed to compare each time point to the time the experiment was switched. There are 3 biological replicates and 2 technical replicates.
Project description:Uncontrolled accumulation of pulmonary artery smooth muscle cells (PASMC) to the distal pulmonary arterioles (PAs) is one of the major characteristics of pulmonary hypertension (PH). Cellular senescence contributes to aging and lung diseases associated with PH and links to PH progression. However, the mechanism by which cellular senescence controls vascular remodeling in PH is not fully understood. The levels of senescence marker, p16INK4A and senescence-associated β-galactosidase (SA-β-gal) activity are higher in PA endothelial cells (ECs) isolated from idiopathic pulmonary arterial hypertension (IPAH) patients compared to those from healthy individuals. Hypoxia-induced accumulation of α-smooth muscle actin (αSMA)-positive cells to the PAs is attenuated in p16fl/fl-Cdh5(PAC)-CreERT2 (p16iΔEC) mice after tamoxifen induction. We have reported that endothelial TWIST1 mediates hypoxia-induced vascular remodeling by increasing platelet-derived growth factor (PDGFB) expression. Transcriptomic analyses of IPAH patient or hypoxia-induced mouse lung ECs reveal the alteration of senescence-related gene expression and their interaction with TWIST1. Knockdown of p16INK4A attenuates the expression of PDGFB and TWIST1 in IPAH patient PAECs or hypoxia-treated mouse lungs and suppresses accumulation of αSMA–positive cells to the supplemented ECs in the gel implanted on the mouse lungs. Hypoxia-treated mouse lung EC-derived exosomes stimulate DNA synthesis and migration of PASMCs in vitro and in the gel implanted on the mouse lungs, while p16iΔEC mouse lung EC-derived exosomes inhibit the effects. These results suggest that endothelial senescence controls αSMA–positive cell proliferation and migration in PH through TWIST1-PDGFB signaling.
Project description:To delineate the role of hypoxia in esophageal epithelial biology, we carried out gene array experiments using a non-transformed immortalized diploid human esophageal cell line, EPC2-hTERT (Mol Cancer Res. 2003;1:729-38). Unlike cancer cell lines, EPC2-hTERT has no genetic alterations at early passages that may affect the cellular response to hypoxia. Experiment Overall Design: EPC2-hTERT cells were exposed to moderate (1% O2) hypoxia in experiment 1 (Exp1) or severe (0.2% O2) hypoxia in experiment 2 (Exp2). Normoxia (21% O2) served as a control in both experiments.
Project description:Human Mammalian Epithelial Cells (HMEC) were exposed to different environmental stresses, including hypoxia, lactic acidosis, the combination of hypoxia and lactic acidosis, lactosis , as well as acidosis. We used microarrays to examine the genomic programs of cells incubated under different microenvironments. Experiment Overall Design: HMEC cells were exposed to different environmental stresses and RNAs were extracted and put on Affymetrix microarrays. We gathered RNAs from cells grown in regular media (control), lactic acidosis, hypoxia, the combinatino of lactic acidosis and hypoxia, lactosis, as well as acidosis.
Project description:Comprehensive analysis of coding and non -coding transcriptome using ribo-depleted total RNA-seq and poly A selected RNA-seq of MCF-7 cells grown in hypoxia and normoxia. Breast cancer cell line (MCF-7) is cultured in normoxic condition (21% O2) and hypoxic condition (1%O2) for 24 hours. Expression of HIF-1alpha and/or HIF-2alpha subunits was suppressed using siRNAs in hypoxic MCF-7 cells. Total RNA was isolated from both hypoxia and normoxia conditions were subjected for ribosomal depleted stand specific RNA-seq and poly A selected RNA-seq