Incubation In Hypoxia Research Articles

Oxygenation by Intravascular Photosynthesis Reduces Kidney Damage During ex Vivo Preservation.

Several clinical issues are associated with reduced oxygen delivery to tissues due to impaired vascular perfusion; moreover, organs procured for transplantation are subjected to severe hypoxia during preservation. Consequently, alternative tissue oxygenation is an active field in biomedical research where several innovative approaches have been recently proposed. Among these, intravascular photosynthesis represents a promising approach as it relies on the intrinsic capacity of certain microorganisms to produce oxygen upon illumination. In this context, this work aims at the development of photosynthetic perfusable solutions that could be applied to preserve organs for transplantation purposes. Our findings demonstrate that a biocompatible physiological solution containing the photosynthetic microalgae Chlamydomonas reinhardtii can fulfill the metabolic oxygen demand of rat kidney slices in vitro. Furthermore, intravascular administration of this solution does not induce tissue damage in the rat kidneys. Moreover, kidney slices obtained from these algae-perfused organs exhibited significantly improved preservation after 24 h of incubation in hypoxia while exposed to light, resulting in reduced tissue damage and enhanced metabolic status. Overall, the results presented here contribute to the development of alternative strategies for tissue oxygenation, supporting the use of perfusable photosynthetic solutions for organ preservation in transplantation.

THE ROLE OF ICAM1 IN TUMOR ASSOCIATED MACROPHAGES IN GLIOBLASTOMA TUMORIGENESIS

Abstract Glioblastoma (GBM) is a fatal brain cancer in adults with ineffective treatment methods. Cell adhesion molecules (CAMs) are proteins that are expressed on the surface of cells and enable them to interact with one another and the surrounding microenvironment. Intracellular adhesion molecule 1 (ICAM-1) is a cell adhesion molecule expressed by various cell types. Preliminary data showed that ICAM-1 is associated with poorer overall and progression free survival in patients and ICAM-1 expression levels increase in recurrent tumors. TAMs enhance GBM tumor growth. The aim of this study was to determine if ICAM-1 expression on TAMs contributes to GBM tumorigenesis, specifically within the hypoxic tumor microenvironment. We hypothesized that ICAM1 facilitates TAM migration, proliferation, cell adhesion and phagocytosis, thus enhancing tumor growth. We found that upon incubation of human and mouse primary macrophages in hypoxia, ICAM-1 expression levels increased and were further exasperated upon culturing with tumor cell-conditioned medium. The migration, cell adhesion and phagocytosis of ICAM-1 deficient macrophages was lower than wild type macrophages, and effects were further exasperated upon culturing with tumor conditioned media and incubation in hypoxia. Intracranial injection of tumor cells into ICAM-1 deficient and wild type mice revealed that ICAM1 deficient mice survived longer with lower overall tumor volume than wild type mice, with survival and volume differences being rescued upon ICAM1 bone marrow reconstitution. In conclusion, the expression of ICAM1 in TAMs likely promotes GBM tumorigenesis and hypoxia further enhances the expression of ICAM-1 in macrophages which is associated with more aggressive GBM.

Transcriptomic analysis of preovipositional embryonic arrest in a nonsquamate reptile (Chelonia mydas).

After gastrulation, oviductal hypoxia maintains turtle embryos in an arrested state prior to oviposition. Subsequent exposure to atmospheric oxygen upon oviposition initiates recommencement of embryonic development. Arrest can be artificially extended for several days after oviposition by incubation of the egg under hypoxic conditions, with development recommencing in an apparently normal fashion after subsequent exposure to normoxia. To examine the transcriptomic events associated with embryonic arrest in green sea turtles (Chelonia mydas), RNA‐sequencing analysis was performed on embryos from freshly laid eggs and eggs incubated in either normoxia (oxygen tension ~159 mmHg) or hypoxia (<8 mmHg) for 36 h after oviposition (n = 5 per group). The patterns of gene expression differed markedly among the three experimental groups. Normal embryonic development in normoxia was associated with upregulation of genes involved in DNA replication, the cell cycle, and mitosis, but these genes were commonly downregulated after incubation in hypoxia. Many target genes of hypoxia inducible factors, including the gene encoding insulin‐like growth factor binding protein 1 (igfbp1), were downregulated by normoxic incubation but upregulated by incubation in hypoxia. Notably, some of the transcriptomic effects of hypoxia in green turtle embryos resembled those reported to be associated with hypoxia‐induced embryonic arrest in diverse taxa, including the nematode Caenorhabditis elegans and zebrafish (Danio rerio). Hypoxia‐induced preovipositional embryonic arrest appears to be a unique adaptation of turtles. However, our findings accord with the proposition that the mechanisms underlying hypoxia‐induced embryonic arrest per se are highly conserved across diverse taxa.

The HIFα-Stabilizing Drug Roxadustat Increases the Number of Renal Epo-Producing Sca-1+ Cells

Inhibition of the prolyl-4-hydroxylase domain (PHD) enzymes, leading to the stabilization of hypoxia-inducible factor (HIF) α as well as to the stimulation of erythropoietin (Epo) synthesis, is the functional mechanism of the new anti-anemia drug roxadustat. Little is known about the effects of roxadustat on the Epo-producing cell pool. To gain further insights into the function of PHD inhibitors, we characterized the abundance of mesenchymal stem cell (MSC)-like cells after roxadustat treatment of mice. The number of Sca-1+ mesenchymal cells following roxadustat treatment increased exclusively in the kidneys. Isolated Sca-1+ cells demonstrated typical features of MSC-like cells, including adherence to tissue culture plates, trilineage differentiation potential, and expression of MSC markers. Kidney-derived Sca-1+ MSC-like cells were cultured for up to 21 days. Within the first few days in culture, cells stabilized HIF-1α and HIF-2α and temporarily increased Epo production upon incubation in hypoxia. In summary, we have identified a Sca-1+ MSC-like cell population that is involved in renal Epo production and might contribute to the strong anti-anemic effect of the PHD inhibitor roxadustat.

Revision of the Regioselectivity of the Beirut Reaction of Monosubstituted Benzofuroxans with Benzoylacetonitrile. 6-Substituted quinoxaline-2-carbonitrile 1,4- dioxides: Structural Characterization and Estimation of Anticancer Activity and Hypoxia Selectivity.

Quinoxaline 1,4-dioxides have a broad range of biological activity that causes a growing interest in their derivatives for drug discovery. Recent studies demonstrated that quinoxaline 1,4- dioxides have a promising anticancer activity and good hypoxia-selectivity. The preparation, isolation, structure characterization, and screening for anticancer activity of the first representatives of 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides have been described. A series of 7- and 6-halogeno-3-phenylquinoxaline-2-carbonitrile 1,4-dioxides was synthesized by the Beirut reaction. The cytotoxicity was assessed by MTT test (72 h incubation) in normoxia (21% O2) and hypoxia (1% O2) conditions. We found that during the Beirut reaction between a benzofuroxan bearing an electron withdrawing group and benzoylacetonitrile in the presence of triethylamine, in addition to well-known 7-substituted quinoxaline-2-carbonitrile 1,4-dioxides 7-11a, the 6-isomers 7-11b are formed. Moreover, the yield of the 6- isomers increased with the increase in the electron-withdrawing character of the substituent. For benzofuroxans with CO2Me and CF3 groups, 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides 10-11b were the major products. Despite similarities in physicochemical and spectroscopic properties, the obtained isomers exhibit considerable differences in their anticancer activity and hypoxia selectivity. Substituents and their electronic effects play a key role in the formation of 7- and 6-substituted quinoxaline-2-carbonitrile 1,4-dioxides in the Beirut reaction and in the cytotoxicity properties of the obtained isomers.

Hypoxia weakens mussel attachment by interrupting DOPA cross-linking during adhesive plaque curing.

Marine mussels (Mytilus spp.) attach to a wide variety of surfaces underwater using a network of byssal threads, each tipped with a protein-based adhesive plaque that uses the surrounding seawater environment as a curing agent. Plaques undergo environmental post-processing, requiring a basic seawater pH be maintained for up to 8 days for the adhesive to strengthen completely. Given the sensitivity of plaques to local pH conditions long after deposition, we investigated the effect of other aspects of the seawater environment that are known to vary in nearshore habitats on plaque curing. The effect of seawater temperature, salinity and dissolved oxygen concentration were investigated using tensile testing, atomic force microscopy and amino acid compositional analysis. High temperature (30°C) and hyposalinity (1 PSU) had no effect on adhesion strength, while incubation in hypoxia (0.9 mg l-1) caused plaques to have a mottled coloration and prematurely peel from substrates, leading to a 51% decrease in adhesion strength. AFM imaging of the plaque cuticle found that plaques cured in hypoxia had regions of lower stiffness throughout, indicative of reductions in DOPA cross-linking between adhesive proteins. A better understanding of the dynamics of plaque curing could aid in the design of better synthetic adhesives, particularly in medicine where adhesion must take place within wet body cavities.

Severe hypoxia selects hematopoietic progenitors with stem cell potential from primary Myelodysplastic syndrome bone marrow cell cultures.

Myelodysplastic Syndromes (MDS) are clonal neoplasms where stem/progenitor cells endowed with self-renewal and capable of perpetuating the disease have been demonstrated. It is known that oxygen tension plays a key role in driving normal hematopoiesis and that hematopoietic stem cells are maintained in hypoxic areas of the bone marrow (BM). Hypoxia could also regulate leukemic/dysplastic hematopoiesis. We evaluated the stem cell potential of MDS cells derived from the BM of 39 MDS patients and selected under severe hypoxia. MDS cells rescued from hypoxia-incubated cultures were subjected to stem and progenitor cell assays in vitro, as well as to hematopoietic reconstitution assay in NOD-SCID mice. Incubation in severe hypoxia of cells explanted from MDS patients selected a cell subset endowed with stem cell potential, as determined in vitro. This occurred only from the BM of patients classified as IPSS low/INT-1 risk. Transplantation into NOD-SCID mice confirmed using an in vivo model that severe hypoxia selects a cell subset endowed with stem cell potential from bone marrow mononuclear cells (BMMC). derived from patients belonging to the IPSS low/int-1 risk group. Data here reported show that cells endowed with stem cell potential and capable of adapting to hypoxia and escaping hypoxia-induced apoptosis exist within MDS cell populations.

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

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

Periods of cardiovascular susceptibility to hypoxia in embryonic american alligators (Alligator mississippiensis).

During embryonic development, environmental perturbations can affect organisms' developing phenotype, a process known as developmental plasticity. Resulting phenotypic changes can occur during discrete, critical windows of development. Critical windows are periods when developing embryos are most susceptible to these perturbations. We have previously documented that hypoxia reduces embryo size and increases relative heart mass in American alligator, and this study identified critical windows when hypoxia altered morphological, cardiovascular function and cardiac gene expression of alligator embryos. We hypothesized that incubation in hypoxia (10% O2) would increase relative cardiac size due to cardiac enlargement rather than suppression of somatic growth. We exposed alligator embryos to hypoxia during discrete incubation periods to target windows where the embryonic phenotype is altered. Hypoxia affected heart growth between 20 and 40% of embryonic incubation, whereas somatic growth was affected between 70 and 90% of incubation. Arterial pressure was depressed by hypoxic exposure during 50-70% of incubation, whereas heart rate was depressed in embryos exposed to hypoxia during a period spanning 70-90% of incubation. Expression of Vegf and PdgfB was increased in certain hypoxia-exposed embryo treatment groups, and hypoxia toward the end of incubation altered β-adrenergic tone for arterial pressure and heart rate. It is well known that hypoxia exposure can alter embryonic development, and in the present study, we have identified brief, discrete windows that alter the morphology, cardiovascular physiology, and gene expression in embryonic American alligator.

Preconditioning of Human Adipose-derived Stromal/Stem Cells: Evaluation of Short-term Preincubation Regimens to Enhance their Regenerative Potential

Objective: Stem cell-based therapy seems to be a promising option to support regeneration after organ failure and tissue injury. The transplantation of MSCs requires cells with a maximum regenerative capacity. Therefore, the research on new strategies to improve the release of regenerative factors of MSCs is urgently needed. Methods: Human adipose-derived stromal/stem cells (ASC) were isolated from lipoaspirates, characterized, and cultured. Cells were either cultured under standard conditions or preconditioned by incubation in a hypoxic environment (0.5% O2) or in normoxia in the presence of recombinant human tumor necrosis factor-α (TNFα) or recombinant human epidermal growth factor (EGF) for 48 hours. First, seven selected regeneration promoting factors were evaluated by qPCR analysis. Afterwards the secretome of ASCs was estimated using a commercially available protein array for 507 proteins. Results: PCR analysis showed a differential induction of ASCs` gene expression by the three pretreatments. Whereas ASCs in hypoxia showed a significant mRNA induction of VEGF, FGF-7, and IGF-II, the other pretreatments induced no significant change in VEGF expression. The gene expression of HB-EGF and M-CSF was significantly induced in hypoxia and by incubation with TNFα, but not EGF. Angiopoietin-like 1 mRNA was not significantly induced by all three preconditioning regimens. Evaluation by the protein array revealed that from the 507 proteins investigated 21.9% were found to be more than five-fold increased after incubation in hypoxia (111 of 507 proteins). Preincubation with EGF resulted in an upregulation of 32.3% (164/507), whereas TNFα upregulated 28.8% of all proteins evaluated (146/507). Conclusion: The findings indicate that all three preconditioning regimens induced a wide variety of proteins. However, short-term pretreatment with EGF induced the highest quantity of proteins, and, therefore, appears to be the best preconditioning regime for cell therapeutic approaches.

Abstract P5-08-09: Synergistic cytotoxicity of digoxin and 5-fluorouracil in doxorubicin-resistant breast cancer cell lines

Abstract BACKGROUND: Metastatic breast cancer is the leading cause of cancer death for women in the US. Cytotoxic chemotherapy remains one of the most important systemic treatment options for breast cancer patients. To enhance the tumor response to chemotherapy, attention has been focused on agents that reverse multidrug resistance (MDR) and increase the sensitivity of tumor cells to chemical drugs. Although many reversal drugs have been identified in vitro, their clinical application has been limited due to their own toxicity. It was reported that 5 years after mastectomy, the recurrence rate of breast cancer among patients on cardiac glycoside therapy, namely digoxin or digitoxin, was almost 10 times lower as compared with those who were not on cardiac glycoside therapy. Digoxin has also been shown to inhibit hypoxia-inducible factors (HIF) and block lung metastasis in a breast cancer model. Interestingly, digoxin was demonstrated to stimulate cell death in various cell lines including breast cancer. Therefore the aim of this study was to examine the reversal effect of combined digoxin and 5-fluorouracil (5-FU) on MDR resistance in human breast cell lines MCF-7 and MDA-MB-231 in vitro, as well as its mechanism of action. METHODS: Wild-type and doxorubicin-resistant ER-positive (MCF-7 and MCF-7/DoxR) and triple negative (MDA-MB-231 and MDA-231/DoxR) breast cancer lines were used for this study. We have previously shown that both doxorubicin-resistant cell lines are also cross-resistant to 5-FU. Cell viability of both resistant cancer cell lines to 5-FU, digoxin, and both 5-FU with digoxin were compared to control at 96 hours of incubation both in normoxia and hypoxia using MTT assay. Western blot was used to quantify the level of HIF-1α and p-glycoprotein (P-gP). RESULTS: In both doxorubicin-resistant cell lines, sub-IC50 concentration of digoxin together with 5-FU significantly decreased the cell viabilities at 96 hours compared to single agent digoxin and 5-FU. Under both normoxic and hypoxic conditions, the same synergistic cytotoxic effects were demonstrated. Western blot revealed that HIF-1α and P-gP were decreased in both doxorubicin-resistant breast cancer cells treated with the digoxin and 5-FU combination compared to single agent digoxin or 5-FU. CONCLUSION: The combination of digoxin and 5-FU demonstrates a synergistic cytotoxic effect in doxorubicin-resistant breast cancer cell lines that is maintained under hypoxic conditions. This is, at least in part, via the inhibitory effects on both HIF-1α and P-gP. The combination of digoxin and 5-FU could be an effective clinical treatment strategy to overcome MDR in breast cancer. A phase II clinical trial is ongoing to test this hypothesis in patients with doxorubicin-resistant metastatic breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-09.

Cardiovascular plasticity during hypoxic development in snapping turtle and alligator embryos

The cardiovascular (CV) system is the first operational organ system, and reptiles represent evolutionary intermediates between water‐breathing vertebrates and birds and mammals. Reptile eggs are subject to environmental stressors during development, including hypoxia, and we have shown that hypoxia impacts reptilian CV function and maturation. Using Snapping turtles and American alligators, we investigated in ovo species differences following chronic incubation in hypoxia (10% O2). Hypoxia alters tonic regulation of baseline CV variables, causing hypotension in both species and relative cardiac enlargement. Assessment of CV reflexes induced via acute hypoxia or pharmacological stimuli indicate reflexes can be plastic and blunted by chronic hypoxic incubation. Alligator embryos possess adrenergic tone originating from circulating catecholamines only, and cholinergic tone is absent until near hatching. Turtle embryos display adrenergic and cholinergic tone at 70% of development, and can utilize autonomic tone to control heart rate over the last 30% of incubation. Our data indicate that maturation of CV function exhibits phenotypic plasticity in response to hypoxic incubation in reptile species, and that turtle embryos display a more ‘advanced’ developmental program than alligator embryos. NSF CAREER IBN IOS‐0845741 to DAC

Functional and Transcriptional Induction of Aquaporin-1 Gene by Hypoxia; Analysis of Promoter and Role of Hif-1α

Aquaporin-1 (AQP1) is a water channel that is highly expressed in tissues with rapid O2 transport. It has been reported that this protein contributes to gas permeation (CO2, NO and O2) through the plasma membrane. We show that hypoxia increases Aqp1 mRNA and protein levels in tissues, namely mouse brain and lung, and in cultured cells, the 9L glioma cell line. Stopped-flow light-scattering experiments confirmed an increase in the water permeability of 9L cells exposed to hypoxia, supporting the view that hypoxic Aqp1 up-regulation has a functional role. To investigate the molecular mechanisms underlying this regulatory process, transcriptional regulation was studied by transient transfections of mouse endothelial cells with a 1297 bp 5′ proximal Aqp1 promoter-luciferase construct. Incubation in hypoxia produced a dose- and time-dependent induction of luciferase activity that was also obtained after treatments with hypoxia mimetics (DMOG and CoCl2) and by overexpressing stabilized mutated forms of HIF-1α. Single mutations or full deletions of the three putative HIF binding domains present in the Aqp1 promoter partially reduced its responsiveness to hypoxia, and transfection with Hif-1α siRNA decreased the in vitro hypoxia induction of Aqp1 mRNA and protein levels. Our results indicate that HIF-1α participates in the hypoxic induction of AQP1. However, we also demonstrate that the activation of Aqp1 promoter by hypoxia is complex and multifactorial and suggest that besides HIF-1α other transcription factors might contribute to this regulatory process. These data provide a conceptual framework to support future research on the involvement of AQP1 in a range of pathophysiological conditions, including edema, tumor growth, and respiratory diseases.

Respiratory mechanics in 1-day old chicken hatchlings and effects of prenatal hypoxia

This study examined the static and dynamic properties of the respiratory system in avian hatchlings, and the effects of incubation in hypoxia (15% O(2)) on these variables. In 1-day old chicken (Gallus gallus) hatchlings killed by an anesthetic overdose the static compliance of the respiratory system (C(rs)) was measured from the pressure-volume curve, constructed by step changes in lung volume. The dynamic compliance, C(rs)(dyn), resistance, R(rs), and time constant, τ(rs), were measured during mechanical ventilation at rates up to 90 cpm. The results indicated that (1) static C(rs) in hatchlings is several folds higher than in neonatal mammals of similar size, (2) during mechanical ventilation the respiratory system becomes hyperinflated and much stiffer; at 65 cpm (which is the respiratory frequency of hatchlings spontaneously breathing at rest) C(rs)(dyn) was about one tenth of the static value, (3) after prenatal hypoxia static C(rs), C(rs)(dyn), R(rs) and τ(rs) were similar to controls; only the magnitude of the hyperinflation was slightly decreased. It is concluded that in avian hatchlings (a) despite the large respiratory volume of the air sacs, expiration can occur passively because the hyperinflation greatly decreases C(rs)(dyn) and shortens τ(rs), and (b) prenatal hypoxia of the level tested has no major effects on the mechanical properties of the respiratory system.

Abstract 3106: Translational regulation of hypoxia-inducible factor-1α and 2α by mutant oncogenes in colon cancer

Abstract Background: KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1α and 2α, and mTOR pathways can regulate the translation of HIF proteins. We previously reported that HIF-1α promotes the growth of SW480 colon cancer cells but HIF-2α appears to restrain growth. We sought to determine the roles of KRAS, BRAF, and mTOR in the regulation of these two HIF isoforms. Methods: Colon cancer cell lines DLD-1 and HCT116 that carry mutant KRAS, HT29 and Colo205 that carry mutant BRAF and Caco2 that does not have mutation in either KRAS or BRAF gene were utilized. The cells were treated with siRNA directed against K-ras or BRAF or a non-specific control. Mutant K-ras and BRAF were lentivirally introduced in Caco2 cells. Following incubation in normoxia or hypoxia, HIF-1α and 2α expression were analyzed by western blotting and QRT-PCR. DKO-3 and HKe-3 cells are DLD-1 and HCT116 cells, respectively, in which the mutant KRAS gene is disrupted by homologous recombination. HIF-1α protein synthesis was measured by its accumulation during treatment with the proteasome inhibitor MG132. To analyze signaling pathways downstream of K-ras, the MEK inhibitor PD98059, PI3K inhibitor LY294002, and mTOR inhibitor Rapamycin were utilized. Results: Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1α, whereas mutant BRAF enhanced both HIF-1α and 2α. Knockout of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of HIF-1α and the rate of HIF-1α protein synthesis. In contrast, knockdown of mutant BRAF in HT29 cells impaired the translation of HIF-2α. In DLD-1 and HCT116 cells, LY294002 suppressed the hypoxic induction of HIF-1α, but not HIF-2α. PD98059 strongly inhibited the hypoxic induction of HIF-1α as well as HIF-2α in HT29 and Colo205 cells. p70 S6 kinase, which regulates global translation and can be phosphorylated by mTOR, was inactivated in hypoxia. Inhibition of mTOR by rapamycin minimally suppressed HIF-1α induction but not HIF-2α. Similarly, knockdown of Raptor and Rictor, which are components of mTORC1 and mTORC2, respectively, only partially suppressed HIF induction. Conclusion: Mutant KRAS enhances the hypoxic induction of HIF-1α by regulating its translation through PI3K pathways. However, mutant BRAF can enhance HIF-2α translation by activating MAPK pathways. The mTOR pathway does not appear to be an important regulator of HIF-1α or 2α translation in colon cancer cells. Mutant oncogenes appear to interact with hypoxia to enhance HIF expression in an mTOR-independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3106.

Morphological changes in the ductus arteriosus in response to development in hypoxia and hyperoxia

The ductus arteriosus (DA) is an oxygen sensitive fetal blood vessel that allows for a right‐to‐left shunt of right ventricular outflow away from the lungs. Previously we have shown that the maturation of the oxygen induced contraction of the DA can be altered by incubation under varying O2 levels. The objective of this study was to characterize the influence of development in hypoxia or hyperoxia on the morphological changes occurring in the chicken DA during hatching. White leghorn chicken eggs were incubated in normoxia (21% O2), hypoxia (15% O2), and hyperoxia (30% O2) for the entire incubation period. The DA was removed from day 18, internally pipped, and externally pipped embryos and hatchlings for histological examination. The major finding was a significant effect of incubation oxygen levels on the DA lumen diameter during hatching. During hatching, the DA lumen diameter from hyperoxic incubated embryos was significantly smaller than that from control embryos. Incubation in hypoxia resulted in a decrease in the closure of the DA in comparison to the control embryos. A similar pattern was observed in the overall length of the right and left DA. The timing of morphological closure of the O2‐sensitive DA is accelerated by in hyperoxia and delayed in hypoxia. This work was funded by the American Heart Association South Central Affiliate and the National Science Foundation.

Growth of the chicken embryo: Implications of egg size

Among avian species with large differences in egg size, changes in eggshell conductance and incubation time permit the water loss necessary for embryonic development. To what extent this happens for different-size eggs within a species is much less known. Chicken eggs with fresh egg weight (Wegg) either large (L,∼66g) or small (S,∼51g) were incubated at 38°C and 60% humidity; their yolk and albumen scaled almost in proportion to Wegg. Eggshell gas conductance scaled to 0.77 of Wegg, as it occurs inter-specifically, while external pipping and hatching occurred at similar times in S and L. Hence, L lost less water during incubation than S, and embryos of L were over-hydrated and those of S were dehydrated. The absolute values of embryo's weight, growth rate, oxygen consumption and the weight of the chorioallantoic membrane were similar between S and L during the first half of incubation, and greater in L in the second half. Incubation in hypoxia reduced growth rate in both sets and maintained the difference in growth trajectories between S and L. The energetic cost of growth and tissue maintenance did not differ significantly. It is concluded that,10+9 among chicken eggs of different sizes, 1) the growth rate of the embryo relates to the size of its egg, probably genetically and because of differences in water content, 2) eggshell conductance contributes, but incubation time does not, to the requirements for water loss. Therefore, the egg water balance during incubation may be the physiological constraint that limits the maximal variability in egg size compatible with embryonic survival.

Role of the Oxygen-Dependent Degradation Domain in a Hypoxia-Inducible Gene Expression System in Vascular Endothelial Growth Factor Gene Therapy

STUDY DESIGN.: An in vitro neural hypoxia model and rat spinal cord injury (SCI) model were used to assess the regulation effect of a reporter or therapeutic gene expression by an oxygen-dependent degradation (ODD) domain in a hypoxia-inducible gene expression system with or without the erythropoietin (EPO) enhancer. OBJECTIVE.: To increase vascular endothelial growth factor (VEGF) gene expression in SCI lesions but avoid unwanted overexpression of VEGF in normal sites, we developed a hypoxia-inducible gene expression system consisting of the EPO enhancer upstream of the SV promoter and an ODD domain C-terminally fused to VEGF. SUMMARY OF BACKGROUND DATA.: ODD domain plays a major role in the degradation of hypoxia-inducible factor 1alpha and has been used in a hypoxia-specific gene expression system as a post-translational regulatory factor. METHODS.: The hypoxia-inducible luciferase or VEGF plasmid was constructed using the EPO enhancer combined with or without the ODD domain. The constructed plasmid was transfected into mouse Neuro 2a (N2a) neuroblastoma cells by Lipofectamine 2000, followed by a 24-hour incubation in hypoxia or normoxia. For in vivo analysis, the naked plasmid DNA was directly injected into the injured rat spinal cord. The gene expression was evaluated by luciferase activity assay, enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, and immunofluorescence staining. RESULTS.: The EPO enhancer/ODD domain-combined hypoxia-inducible gene expression system clearly increased the expression of the reporter luciferase gene and therapeutic VEGF gene specifically under hypoxic conditions and SCI, and quickly downregulated protein expression to a very low level after reoxygenation. CONCLUSION.: These results strongly suggest the potential applicability of this EPO enhancer/ODD domain-based hypoxia-inducible gene expression system in the development of a safer and more effective VEGF gene therapy for SCI.

Hypoxia Inducible Factor 1á (HIF-1á) Regulates CD20 Expression in Lymphoma Cells: Possible Implications for Rituximab Based Therapy in Diffuse Large B Cell Lymphoma (DLBCL).

Abstract 1698Poster Board I-724 BackgroundHIF-1á is a transcription factor that regulates gene expression in response to decreases in cellular oxygenation (hypoxia). Genes activated by HIF are involved in glycolysis, glucose transport, angiogenesis, cell proliferation, cell migration, cell metabolism, and cell survival. Many of these genes confer protection against the consequences of oxygen deprivation while others enhance resistance to chemotherapy or radiotherapy. Clinically, evidence of elevated HIF-1á protein correlates with poor prognosis in lung, breast, colorectal, brain, pancreatic, ovarian, renal and bladder cancers. Our prior data found that constitutive expression of HIF-1á is enhanced in a set of established lymphoma cell lines (Evens et al, BJH, 2008), underscoring the potential impact of HIF in lymphoma. We also found that HIF-1á was stabilized in lymphoma specimens from patients with follicular lymphoma and DLBCL. These observations highlight a potential importance of HIF in tumor progression, but the full significance of HIF in lymphoma is not yet known. We recently reported that in 153 patients treated with either CHOP or R-CHOP, HIF-1á expression correlated with improved outcome only in patients who were treated with R-CHOP, and was a significant independent prognostic factor in the R-treated patients (Evens et al, JCO, in press, 2009). Based on these clinical observations and other data that showed density of CD20 expression correlated with outcome in DLBCL treated with R-CHOP, we hypothesized that there may be an important biological interaction between CD20 and HIF or downstream genes regulated by HIF. MethodsWe therefore investigated the relationship between HIF-1á, hypoxia, and CD20 expression in the DLBCL cell line SUDHL4. SUDHL4 cells were treated for 4 hours in normoxic and hypoxic conditions (with and without cobalt chloride, a hypoxia mimetic), or in hypoxia chambers (1.5% oxygen). Western blotting was used to measure HIF-1á expression and CD20 expression, while flow cytometry was used to quantify CD20 density. To on CD20 expression, we further investigate the contribution of HIF-1á downregulated HIF-1á with the targeted anti-HIF-1á small molecule inhibitor, PX-478, then subsequently measured CD20 expression by Western blotting. ResultsSUDHL4 cells were incubated with and without cobalt chloride for 24 hours and CD20 expression was measured. Compared with normoxic controls, a 2-fold increase (1.69-2.36) in CD20 expression was observed (by flow cytometric analysis) in the SUDHL4 cells incubated with cobalt chloride. SUDHL4 cells were next pre-incubated with PX-478 for 4 hours at increasing concentrations from 5-25 μM followed by 20 hour incubation in normoxia (20% oxygen) or hypoxia (1.5% oxygen). We found expected dose-dependent decreases in HIF-1á levels. Furthermore, we demonstrated a concomitant decrease in the level of CD20 expression in cells incubated under hypoxic conditions with increasing concentrations of PX-478 (see Figure below). Interestingly, much less alteration of CD20 was noted in cells incubated under normoxic conditions. ConclusionsThese data demonstrate for the first time that HIF-1á may regulate CD20 expression in lymphoma. These data also provide a potential explanation for the clinical observation that patients with DLBCL with increased HIF-1á expression have superior outcomes/survival when treated with R-CHOP, but not with CHOP. These findings may have implications for our understanding of the biology and treatment of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interactive effects of temperature and hypoxia on heart rate and oxygen consumption of the 3-day old chicken embryo

In the chick embryo at day 3, gas exchange occurs by diffusion and oxygen consumption ( V̇ O 2 ) does not depend on the cardiovascular convection of O 2. Whether or not this is the case in hypoxia is not known and represents the aim of the study. The heart of chicken embryos at 72 h (stage HH18) was filmed through a window of the eggshell by a camera attached to a microscope. Stroke volume was estimated from the changes in heart silhouette between systole and diastole. V̇ O 2 was measured by a closed system methodology. In normoxia, a decrease in temperature ( T) from 39 to 31 °C had parallel depressant effects on V̇ O 2 and HR. At 39 °C, a progressive decrease in O 2 lowered V̇ O 2 ; HR was maintained until the O 2 threshold of ∼ 15%. In severe hypoxia (4% O 2) V̇ O 2 and HR were, respectively, ∼ 12% and ∼ 62% of normoxia. At 32 °C the hypoxic threshold for HR was significantly lower. During constant hypoxia (7% O 2) V̇ O 2 did not respond to T, while the HR response was preserved. Stroke volume changed little with changes in T or O 2, except at 6 and 4% O 2, when it decreased by ∼ 20 and 30%. In embryos growth-retarded because of incubation in chronic hypoxia, V̇ O 2 and HR responses to T and hypoxia were similar to those of normal embryos. We conclude that in the early embryo during hypoxia cardiovascular O 2 convection is not responsible for the drop in V̇ O 2 . The generalised hypometabolic response, in combination with the extremely small cardiac V̇ O 2 , probably explains the minor effects of hypoxia on cardiac activity.