Hypoxia up-regulates expression of Eph receptors and ephrins in mouse skin.

Eph receptor tyrosine kinases and their ligands (ephrins) are key players during the development of the embryonic vasculature; however, their role and regulation in adult angiogenesis remain to be defined. Both receptors and ligands have been shown to be up-regulated in a variety of tumors. To address the hypothesis that hypoxia is an important regulator of Ephs/ephrins expression, we developed a mouse skin flap model of hypoxia. We demonstrate that our model truly represents segmental skin hypoxia by applying four independent methods: continuous measurement of partial cutaneous oxygen tension, monitoring of tissue lactate/pyruvate ratio, time course of hypoxia-inducible factor-1alpha (HIF-1alpha) induction, and localization of stabilized HIF-1alpha by immunofluorescence in the hypoxic skin flap. Our experiments indicate that hypoxia up-regulates not only HIF-1alpha and vascular endothelial growth factor (VEGF) expression, but also Ephs and ephrins of both A and B subclasses in the skin. In addition, we show that in Hep3B and PC-3 cells, the hypoxia-induced up-regulation of Ephs and ephrins is abrogated by small interfering RNA-mediated down-regulation of HIF-1alpha. These novel findings shed light on the role of this versatile receptor/ligand family in adult angiogenesis. Furthermore, our model offers considerable potential for analyzing distinct mechanisms of neovascularization in gene-targeted mice.

Changes in mitochondrial enzymatic activities of monocytes during prolonged hypobaric hypoxia and influence of antioxidants: A randomized controlled study

OBJECTIVES Exposure to high altitudes is associated with oxidative cellular damage due to the increased level of reactive oxygen and nitrogen species and altered activity of antioxidant systems. Subjects were submitted to prolonged hypoxia, to evaluate changes in mitochondrial enzyme activities of monocytes and their attenuation by supplementation with antioxidants. METHODS Twelve subjects were randomly assigned to receive antioxidant supplements or placebo prior to and during an expedition to Pik Lenin (7145 m). Monocytes were isolated from blood samples to determine the activity of mitochondrial enzymes cytochrome c oxidase and citrate synthase at 490 m (baseline) and at the altitudes of 3550 m, 4590 m, and 5530 m. RESULTS An increase in citrate synthase activity at all altitudes levels was observed. Hypoxia induced an increase in the activity of cytochrome c oxidase only at 4590 m. Neither citrate synthase activity nor cytochrome c oxidase activity differed between the subjects receiving antioxidant supplements and those receiving placebo. CONCLUSIONS Hypoxia leads to an increase in citrate synthase activity of monocyte mitochondria as a marker of mitochondrial mass, which is not modified by antioxidant supplementation. The increase in mitochondrial mass may represent a compensatory mechanism to preserve oxidative phosphorylation of monocytes at high altitudes.

Interplay between receptor tyrosine kinases and hypoxia signaling in cancer.

Deregulated signaling via receptor tyrosine kinase (RTK) pathways is prevalent in numerous types of human cancers and is commonly correlated with worst prognosis, resistance to various treatment modalities and increased mortality. Likewise, hypoxic tumors are often manifested by aggressive mode of growth and progression following an adaptive genetic reprogramming with consequent transcriptional activation of genes encoding proteins, which support tumor survival under low oxygen-related conditions. Consequently, both the hypoxia-inducible factor (HIF) system, which is the major mediator of hypoxia-related signaling, and numerous RTK systems are considered critical molecular targets in current cancer therapy. It is now evident that there is an intricate molecular crosstalk between RTKs and hypoxia-related signaling in the sense that hypoxia can activate expression of particular RTKs and/or their corresponding ligands, while some RTK systems have been shown to trigger activation of the HIF machinery. Moreover, signaling regulation of some RTK systems under hypoxic conditions has also been documented to take place in a HIF-independent manner. With this review we aim at overviewing the most current observations on that topic and highlight the importance of the potential co-drugging the HIF system along with particular relevant RTKs for better tumor growth control.

Hypoxia-induced transcriptional regulation through chromatin modifications and NC2 function

In response to tumor hypoxia, specific genes that promote angiogenesis, proliferation, and survival are induced. Globally, I find that hypoxia induces a mixed pattern of histone modifications that are typically associated with either transcriptional activation or repression. Furthermore, I find that selective activation of hypoxia-inducible genes occurs simultaneously with widespread repression of transcription. I analyzed histone modifications at the core promoters of hypoxia-repressed and -activated genes and find that distinct patterns of histone modifications correlate with transcriptional activity. Additionally, I discovered that trimethylated H3-K4, a modification generally associated with transcriptional activation, is induced at both hypoxia-activated and repressed genes, suggesting a novel pattern of histone modifications induced during hypoxia. ^ In order to determine the mechanism of hypoxia-induced widespread repression of transcription, I focused my studies on negative cofactor 2 (NC2). Previously, we found that hypoxia-induced repression of the alpha-fetoprotein (AFP) gene occurs during preinitiation complex (PIC) assembly, and I find that NC2, an inhibitor of PIC assembly, is induced during hypoxia. Moreover, I find that the beta subunit of NC2 is essential for hypoxia-mediated repression of AFP, as well as the widespread repression of transcription observed during hypoxia. Previous data in Drosophila and S. cerevisiae indicate that NC2 functions as either an activator or a repressor of transcription. The mechanism of NC2-mediated activation remains unclear; although, Drosophila NC2 function correlates with specific core promoter elements. I tested if NC2 activates transcription in mammalian cells using this core promoter-specific model as a guide. Utilizing site-specific mutagenesis, I find that NC2 function in mammalian cells is not dependent upon specific core promoter elements; however, I do find that mammalian NC2 does function in a gene-specific manner as either an activator or repressor of transcription during hypoxia. Furthermore, I find that binding of the alpha subunit of NC2 specifically correlates with NC2-mediated transcriptional activation. NC2α and NC2β are both required for NC2-mediated transcriptional activation; whereas, NC2β alone is required for hypoxia-induced transcriptional repression. Together, these data indicate that hypoxia mediates changes in gene expression through both chromatin modifications and NC2 function. ^

Gene expression and physiological changes of the Antarctic krill Euphausia superba under different hypoxia intensities

Antarctic krill (Euphausia superba) from South Georgia comprise one of the most northern and abundant krill stocks. South Georgia waters are undergoing rapid warming, as a result of climate change, which in turn could alter the oxygen concentration of the water. We investigated gene expression in Antarctic krill related to aerobic metabolism, antioxidant defence, and heat-shock response under severe (2.5% O2 saturation or 0.6 kPa) and threshold (20% O2 saturation or 4 kPa) hypoxia exposure compared to in situ levels (normoxic; 100% O2 saturation or 21 kPa). Biochemical metabolic and oxidative stress indicators complemented the genic expression analysis to detect in vivo signs of stress during the hypoxia treatments. Expression levels of the genes citrate synthase (CS), mitochondrial manganese superoxide dismutase (SODMn-m) and one heat-shock protein isoform (E) were higher in euphausiids incubated 6 h at 20% O2 saturation than in animals exposed to control (normoxic) conditions. All biochemical antioxidant defence parameters remained unchanged among treatments. Levels of lipid peroxidation were raised after 6 h of severe hypoxia. Overall, short-term exposure to hypoxia altered mitochondrial metabolic and antioxidant capacity, but did not induce anaerobic metabolism. Antarctic krill are swarming organisms and may experience short periods of hypoxia when present in dense swarms. A future, warmer Southern ocean, where oxygen saturation levels are decreased, may result in smaller, less dense swarms as they act to avoid greater levels of hypoxia.

Response of three krill species to hypoxia and warming: An experimental approach to oxygen minimum zones expansion in coastal ecosystems

To understand the adaptation of euphausiid (krill) species to oxygen minimum zones (OMZ), respiratory response and stress experiments combining hypoxia/reoxygenation exposure with warming were conducted. Experimental krill species were obtained from the Antarctic (South Georgia area), the Humboldt Current system (HCS, Chilean coast), and the Northern California Current system (NCCS, Oregon). Euphausia mucronata from the HCS shows oxyconforming or oxygen partial pressure (pO2)-dependent respiration below 80% air saturation (18 kPa). Normoxic subsurface oxygenation in winter posed a "high oxygen stress" for this species. The NCCS krill, Euphausia pacifica, and the Antarctic krill, Euphausia superba maintain respiration rates constant down to low critical pO2 values of 6 kPa (30% air saturation) and 11 kPa (55% air saturation), respectively. Antarctic krill had the lowest antioxidant enzyme activities, but the highest concentrations of the molecular antioxidant glutathione (GSH) and was not affected by 6 h exposure to moderate hypoxia. Temperate krill species had higher SOD (superoxide dismutase) values in winter than in summer, which relate to higher winter metabolic rate (E. pacifica). In all species, antioxidant enzyme activities remained constant during hypoxic exposure at habitat temperature. Warming by 7°C above habitat temperature in summer increased SOD activities and GSH levels in E. mucronata (HCS), but no oxidative damage occurred. In winter, when the NCCS is well mixed and the OMZ is deeper, +4°C of warming combined with hypoxia represents a lethal condition for E. pacifica. In summer, when the OMZ expands upwards (100 m subsurface), antioxidant defences counteracted hypoxia and reoxygenation effects in E. pacifica, but only at mildly elevated temperature (+2°C). In this season, experimental warming by +4°C reduced antioxidant activities and the hypoxia combination again caused mortality of exposed specimens. We conclude that a climate change scenario combining warming and hypoxia represents a serious threat to E. pacifica and, as a consequence, NCCS food webs.

Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-l-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 μM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 μM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 μM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1α or HIF-1α-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1α to a DNA-binding form.

Characterization of the α1B-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle

We previously demonstrated that α1B-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O2 exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that α1BAR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb α1BAR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The α1BAR 5′ flanking region was sequenced to −2,460 (relative to ATG +1). Transient transfection experiments identify the minimal promoter region between −270 and −143 and sequence between −270 and −248 that are required for transcription of the α1BAR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT1MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately −160 in aorta SMC. Sequence between −837 and −273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the cis-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the α1BAR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the α1BAR gene also were identified that may confer the differential hypoxic increase in α1BAR gene transcription in aorta, but not in vena cava SMC.

Use of intrinsic modes in biology: Examples of indicial response of pulmonary blood pressure to ± step hypoxia

Recently, a new method to analyze biological nonstationary stochastic variables has been presented. The method is especially suitable to analyze the variation of one biological variable with respect to changes of another variable. Here, it is illustrated by the change of the pulmonary blood pressure in response to a step change of oxygen concentration in the gas that an animal breathes. The pressure signal is resolved into the sum of a set of oscillatory intrinsic mode functions, which have zero “local mean,” and a final nonoscillatory mode. With this device, we obtain a set of “mean trends,” each of which represents a “mean” in a definitive sense, and together they represent the mean trend systematically with different degrees of oscillatory content. Correspondingly, the oscillatory content of the signal about any mean trend can be represented by a set of partial sums of intrinsic mode functions. When the concept of “indicial response function” is used to describe the change of one variable in response to a step change of another variable, we now have a set of indicial response functions of the mean trends and another set of indicial response functions to describe the energy or intensity of oscillations about each mean trend. Each of these can be represented by an analytic function whose coefficients can be determined by a least-squares curve-fitting procedure. In this way, experimental results are stated sharply by analytic functions.

An essential role for p300/CBP in the cellular response to hypoxia

p300 and CBP are homologous transcription adapters targeted by the E1A oncoprotein. They participate in numerous biological processes, including cell cycle arrest, differentiation, and transcription activation. p300 and/or CBP (p300/CBP) also coactivate CREB. How they participate in these processes is not yet known. In a search for specific p300 binding proteins, we have cloned the intact cDNA for HIF-1α. This transcription factor mediates hypoxic induction of genes encoding certain glycolytic enzymes, erythropoietin (Epo), and vascular endothelial growth factor. Hypoxic conditions lead to the formation of a DNA binding complex containing both HIF-1α and p300/CBP. Hypoxia-induced transcription from the Epo promoter was specifically enhanced by ectopic p300 and inhibited by E1A binding to p300/CBP. Hypoxia-induced VEGF and Epo mRNA synthesis were similarly inhibited by E1A. Hence, p300/CBP–HIF complexes participate in the induction of hypoxia-responsive genes, including one (vascular endothelial growth factor) that plays a major role in tumor angiogenesis. Paradoxically, these data, to our knowledge for the first time, suggest that p300/CBP are active in both transformation suppression and tumor development.

Identification of a Human VPF/VEGF 3′ Untranslated Region Mediating Hypoxia-induced mRNA Stability

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3′ untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3′ untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3′ untranslated region and distinct mRNA-binding proteins in human tumor cells.