Hypoxia-responsive Gene Expression Research Articles

Hypoxia responsive gene expression is mediated by various subsets of transcription factors and miRNAs that are determined by the actual oxygen availability

• Reduced oxygen availability is not only associated with flooding, but occurs also during growth and development. It is largely unknown how hypoxia is perceived and what signaling cascade is involved in activating adaptive responses. • We analysed the expression of over 1900 transcription factors (TFs) and 180 microRNA primary transcripts (pri-miRNAs) in Arabidopsis roots exposed to different hypoxic conditions by means of quantitative PCR. We also analysed the promoters of genes induced by hypoxia with respect to over-represented DNA elements that can act as potential TF binding sites and their in vivo interaction was verified. • We identified various subsets of TFs that responded differentially through time and in an oxygen concentration-dependent manner. The regulatory potential of selected TFs and their predicted DNA binding elements was validated. Although the expression of pri-miRNAs was differentially regulated under hypoxia, only one corresponding mature miRNA changed accordingly. Putative target transcripts of the miRNAs were not significantly affected. • Our results show that the regulation of hypoxia-induced genes is controlled via simultaneous interaction of various combinations of TFs. Under anoxic conditions, an additional set of TFs is induced. Regulation of gene expression via miRNAs appears to play a minor role during hypoxia.

Splenectomy improves survival by increasing arterial blood supply in a rat model of reduced-size liver

Prevention of acute portal hyperperfusion in small-for-size livers by inflow modulation results in beneficial postoperative outcome. The objective of this study was to unravel the underlying mechanism, emphasizing the intimate relationship between portal venous (PV) and hepatic arterial (HA) blood flow (BF). Rats underwent partial hepatectomy (pHx), splenectomy before pHx or splenectomy and ligation of the A. hepatica before pHx. Portal venous blood flow (PVBF), hepatic arterial blood flow (HABF), and tissue pO₂ were assessed during stepwise resection from 30% to 90%. Hepatic regeneration and hypoxia-responsive gene expression were analyzed in each group after nonlethal 85% pHx. 90% pHx caused a fourfold rise in PVBF, a slight decrease in HABF with a 50% reduction in pO₂, and high mortality. Splenectomy before pHx reduced the PVBF and caused a rise in HABF with doubling in tissue pO₂. An attenuation of hypoxia-responsive gene expression turned into enhanced hepatocellular regeneration and improved survival. A. hepatica ligation abolished the beneficial effect of splenectomy on tissue oxygenation, proliferation, and outcome. In conclusion, the beneficial effect of splenectomy in small-for-size livers can be attributed to a rise in HABF with sufficient oxygen supply rather than to a reduced portal venous hyperperfusion to the remnant liver.

Oxygen Sensing and Osteogenesis

Osteogenesis and angiogenesis are tightly coupled during bone formation and repair. Blood vessels not only carry oxygen and nutrients to the developing bone, but also play an active role in bone formation and remodeling by mediating the interaction between osteoblasts, osteocytes, osteoclasts, and vascular cells at a variety of levels. Tissue hypoxia is believed to be a major stimulus for angiogenesis by activating hypoxia-inducible factor alpha (HIFalpha) pathway, which is a central regulator of hypoxia adaptation in vertebrates. HIFalpha remains inactive under normoxic conditions through pVHL-mediated polyubiquitination and proteasomal degradation. Activation of the HIFalpha pathway by hypoxia triggers hypoxia-responsive gene expression, such as vascular endothelial growth factor (Vegf), which plays a critical role in angiogenesis, endochondral bone formation, and bone repair following fracture. Recent work from our laboratory has shown that osteoblasts use the HIFalpha pathway to sense reduced oxygen tension and transmit signals that impinge on angiogenic and osteogenic gene programs during bone formation. Using a genetic approach, we have demonstrated that overexpression of HIFalpha in mouse osteoblasts through disruption of Vhl results in profound increases in angiogenesis and osteogenesis, which appear to be mediated by cell nonautonomous mechanisms involving VEGF. These studies suggest that VEGF exerts many of its actions on bone indirectly by stimulation of angiogenesis. Whether or to what extent this angiogenic factor functions independent of endothelial cells remains to be determined.

926. Recombinant AAV Vectors Pseudotyped with Serotype-1 Capsid Mediates Early Onset of Gene Expression in Mouse Hearts

Top of pageAbstract Adeno-associated virus serotype 2 (AAV2) has been used most frequently as vector in experiments on cardiac gene therapy. We have used this vector to deliver vascular endothelial growth factor (VEGF) gene controlled by the hypoxia response element (HRE) and a cardiac myosin light chain 2v promoter. We achieved cardiac-specific and hypoxia-responsive gene expression that induced neovascular formation in ischemic murine hearts and improved cardiac function. However, gene expression mediated by AAV2 vector in hearts was delayed and did not reach the maximum level until about 4 weeks after intramyocardial injection. Irreversible myocardial injury could have occurred during this period of delay. Because hypoxia inducible factor 1 (HIF-1) is induced immediately at the onset of hypoxia, earlier onset of the HRE regulated gene expression should be more effective in inducing therapeutic angiogenesis. Recently, studies on AAV with different serotypes have shown that they differ in their transduction efficiency with different tissues. To identify a serotype that can efficiently mediate early expression of transgene in the myocardium, we packaged into serotypes 1 to 5 capsids, AAV vector carrying CMV promoter driving the mouse erythropoietin (Epo) gene. The packaged vectors were injected into murine hearts intramyocardially. Gene expression was studied by real-time RT-PCR. The kinetics of Epo expression was studied by measuring the hematocrit after AAVEpo gene transduction at different time points. We found that AAV1, 4 and 5 mediated earlier and higher gene expression than AAV2. Gene expression mediated by AAV1 increased faster than AAV4 and 5, and became the highest after day 4. To compare the early gene expression mediated by AAV serotype 1 and 2, we packaged into these capsids AAV-LacZ vectors that carried a CMV promoter driving LacZ gene expression and injected them to normal and ischemic hearts. Gene expression was detected by LacZ staining. The LacZ expression was detected in the normal and ischemic myocardium one day after AAV1-LacZ injection, while no LacZ expression could be detected in the hearts injected with AAV2-LacZ at this time. At day 14, 100% of the cardiomyocytes around needle injection sites were infected by AAV1-LacZ vector, whereas only sporadic cardiomyocytes were infected by AAV2-LacZ. These results indicate that AAV1 can mediate gene expression within one day. HRE regulated VEGF gene packaged in this capsid will induce very early gene expression and therefore enhance the therapeutic effect.

Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1α accumulation—Implications for prolyl hydroxylase activity and iron

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)–HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.

Novel function of neuronal PAS domain protein 1 in erythropoietin expression in neuronal cells

The basic helix loop helix-PAS (bHLH-PAS) transcription factors have diverse roles in physiologic responses to the environment and in early development. One bHLH-PAS protein, neuronal PAS domain protein 1 (NPAS1), is reported to be expressed only in the central nervous system beginning at the late embryonic stage, but its function is unknown. Using an anti-NPAS1 antibody, we have shown that NPAS1 expression in cerebral cortex was observed first around embryonic Day 16.5 (E16.5) and was then dispersed throughout the region as cortical development progressed. From the similarity with hypoxia-inducible factor-1alpha (HIF-1alpha), we investigated whether NPAS1 regulates transcription of erythropoietin (EPO), which is the target of HIF-1alpha and is expressed in the brain during the early developmental stage. In the present study, we have shown that NPAS1 binds to the enhancer region of Epo in vivo. A luciferase reporter assay revealed a repressive effect of NPAS1 on hypoxia-responsive element-regulated gene expression. These results raise the possibility that NPAS1 plays a role in late central nervous system development by modulating EPO expression in response to cellular oxygen level.

Molecular indicators of hypoxia in the blue crab Callinectes sapidus

Occurrence of hypoxia in estuarine waters is increasing, and recovery of impacted estu- aries is slow. Detection of early effects of hypoxia is needed for timely remedial action to be taken. Here we examine the use of hypoxia-responsive gene and protein expression profiles in the blue crab Callinectes sapidus as early indicators of impacts of hypoxia. We cloned 23 potential hypoxia- responsive genes, which were used to construct gene macroarrays. Crabs exposed to chronic hypoxia (2.5 ppm dissolved oxygen (DO)) for 15 d showed significant (p < 0.05) changes in gene expression of heat shock protein 70 (Hsp70), copper metallothionein (CuMt3), cytosolic MnSOD (cyt-MnSOD) and ribosomal proteins S15 and L23. In all cases except for CuMt3, gene expression decreased after 5 d exposure to hypoxia. Expression of Hsp70, CuMt3 and cyt-MnSOD also increased (p < 0.05) in nor- moxic crabs held for 15 d, suggesting confounding effects from confinement stress. Hemocyanin pro- tein concentrations changed significantly (p = 0.005) across the 15 d chronic hypoxia exposure. Hemocyanin in crabs exposed to 10 d intermittent hypoxia (2.5 to 8 ppm DO over a 24 h cycle) did not change, but cyt-MnSOD gene expression increased significantly (p = 0.037), whereas cytochrome c oxidase subunit 1 (ccox1) showed a 2.2-fold downregulation. Blue crabs collected from Pensacola Bay, Florida, showed significant (p < 0.006) downregulation of ccox1 and cyt-MnSOD gene ex- pression as well as hemocyanin protein levels at a diurnally hypoxic marsh site. Several hypoxia- responsive genes (elongation factor 2, cryptocyanin, hemocyanin) were also significantly elevated (p < 0.006) in intermolt versus premolt normoxic crabs. The identification of hypoxia-responsive genes and proteins in the blue crab is a promising first step towards the development of sensitive molecular tools for the detection of sublethal effects of hypoxia in estuarine-resident species.

245 HIF-1, hypoxia inducible factor-1 as a therapeutic target

This chapter focuses on oxygen-dependent regulation of erythropoiesis. Hypoxia inducible factor 1, subunit α (HIF-1α) is a central molecule in oxygen sensing and the response to hypoxia. HIF-1α is unstable in normoxic conditions: specific proline hydroxylases convert its proline residues 402 and 564 to hydroxyprolines, the hydroxylated HIF-1α is captured by the von Hippel Lindau protein (pVHL), ubiqutinated, and degraded in proteasomes. This chapter describes the methods for the quantitative detection of mRNA for hypoxia-regulated genes in the blood cells and for assessing the response of red blood cell progenitors to erythropoietin—a hormone playing an essential role in the adaptation to hypoxia. Real data of studies of the congenital defect of the germ-line mutation of VHL gene leading to the constitutive upregulation of hypoxia sensing, Chuvash polycythemia, are used as an example. Concepts related to the separation of plasma, platelets, mononuclear cells, and granulocytes from human peripheral blood, the preparation of EBV-transformed B lymphoblastoid cell lines (B-LCL), quantification of hypoxia-responsive gene expression by real-time RT-PCR is discussed along with the response of erythroid progenitors to erythropoietin in vitro.

The hypoxic tumor microenvironment and gene expression

Solid tumors are not static entities but are constantly responding to environmental signals as they grow and develop. One mechanism by which they respond to the adverse conditions of the tumor microenvironment is through coordinated changes in gene expression. The synchronized turning of genes on and off leads to biologic adaption to the adverse oxygen-poor environment. Because tumor hypoxia can be found in almost every solid tumor, it represents one of the most pervasive microenvironmental stresses that can impact malignant progression and therapeutic response. Interestingly, tumors that exhibit robust induction of hypoxia-responsive gene expression networks show a clinically more aggressive natural history. The contribution of hypoxia-responsive gene networks to malignant response is currently under investigation. An understanding of the coordinated functions of hypoxia induced and repressed genes can lead to a better understanding of the clinical significance of the hypoxic tumor phenotype.

192. Cardiac-Specific and Hypoxia-Responsive Vascular Endothelial Growth Factor Expression in Ischemic Hearts

A problem associated with angiogenic gene therapy is that uncontrolled gene expression of angiogenic factors may cause several side effects, including: hemangioma formation, retinopathy, occult tumor growth and atherosclerotic plaque progression. Previously, we used the hypoxia response element (HRE) isolated from the erythropoietin (Epo) gene enhancer to render vascular endothelial growth factor (VEGF) expression responsive to hypoxia induction in vitro and in ischemic myocardium in vivo. We found in our experiment that adeno-associate viral vector (AAV) can be detected in other organs in addition to the heart after intra-myocardial injection. Thus, unwanted effects may still occur even with HRE controlled VEGF expression, since hypoxia in other tissues in patients with heart failure may results in extra-cardiac VEGF gene expression. To ensure the safety of angiogenic therapy for ischemic heart disease, it is necessary that the gene expression is cardiac specific and hypoxia responsive. In this study, we explored the feasibility of using the cardiac myosin light-chain-2v (MLC-2v) promoter and Epo HRE to mediate cardiac-specific and hypoxia-responsive VEGF gene expression. We constructed an AAV vector with MLC-2v promoter and 9 copies of Epo HRE driving VEGF (MLCVEGF) expression. Gene expression mediated by this vector was studied in vitro by transfection of this vector into a rat cardiac muscle cell line (H9c2), a rat skeletal muscle cell line (L6) and a mouse fibroblast cell line (NIH3T3), and in vivo by direct injection of the vector to normal and ischemic mouse hearts. AAV vectors with CMV driving VEGF (CMVVEGF), and 9HRE plus minimum SV40 promoter driving VEGF (H9VEGF) were used as controls. RESULTS: With MLCVEGF infection, VEGF expression is higher in H9c2 than the other two non-cardiac cell lines, and the expression is hypoxia inducible. In vivo studies indicated that MLCVEGF increased VEGF expression by 74% in ischemic heart compared to normal heart. VEGF expression was detected in liver, kidney, spleen and gonad of CMVVEGF inoculated mice by RT-PCR, but no VEGF expression was detected in organs other than heart and liver of MLCVEGF inoculated mice. CONCLUSION: Our result indicates that MLC-2v promoter and 9 copies of HRE can mediate cardiac-specific and hypoxia-inducible gene expression in vitro and in vivo. Induction of angiogenesis and improvement of cardiac function by this vector, studies currently in progress, may provide a safe approach for the treatment of ischemic heart disease.

Analysis of the effect of aging on the response to hypoxia by cDNA microarray

Little is known about the detail of hypoxia-responsive gene expression patterns in advanced age, even though aging is thought to be partially associated with a decreased response to hypoxia. In the present study, we identified several hypoxia-inducible genes and investigated the effect of aging on hypoxic gene expression profiles using cDNA microarray analysis of young/old human diploid fibroblasts. Of 7458 genes in the microarray, we found that genes involved in angiogenesis, defense against oxidative stress, and transcription regulation are severely impaired in senescent cell, which is consistent with the fact that aged cells have attenuated responses to various stimuli.