Levels Of Hypoxia-inducible Factor Research Articles

Neocytolysis Is Mediated by down Regulation of Catalase By Mir-21 Resulting in defective Clearance of reticulocyte mitochondrial ROS

Oxygen homeostasis is tightly controlled by the number of red blood cells (RBCs). Hypoxia increases RBCs by enhanced erythropoiesis mediated by hypoxia-inducible factors (HIFs). Upon return to normoxia, the increase in RBCs is overcorrected by preferential destruction of young RBCs, a process termed neocytolysis. Neocytolysis was first described in astronauts and later in people descending from high altitude. The molecular mechanism of neocytolysis is obscure. We hypothesized that neocytolysis occurs because of rapid transient changes of HIF levels, resulting in increased reactive oxygen species (ROS) from mitochondria in reticulocytes and defective antioxidant protection of young RBCs generated in hypoxia.We developed a neocytolysis model by exposing mice to 12% oxygen (equivalent to 4500m altitude) for 10 days. This model recapitulates the RBC changes observed in humans exposed to hypoxia for ~30 days. Upon return to normoxia, ROS were markedly increased in reticulocytes and mature RBCs, but not in neutrophils, B- or T-cells, or monocytes. Reduction of ROS by antioxidant (N-acetyl-L cysteine) treatment attenuated hemolysis and decreased hematocrit.To test whether HIFs (transcription factors regulating hypoxic response) contribute to neocytolysis, we repeated these experiments using Chuvash mice (which bear a VHLR200Wmutation, resulting in constitutively high HIF). These mice also showed attenuated hemolysis and decreased hematocrit; in addition, their reticulocyte half-life was higher (36.6 vs.17.8 hours in wild type). Similar findings were also observed with treatment of mice with dimethyloxallyl glycine (DMOG), an inhibitor of prolyl hydroxylase (another negative regulator of HIF). These experiments indicate the essential role of HIF pathways in neocytolysis.Mitochondria are a major source of ROS in cells. During terminal differentiation of RBCs, mitochondria are removed from reticulocytes by mitophagy. After hypoxia treatment, mitochondria mass increased in reticulocytes concomitant with the reduction of HIF-regulated Bnip3L (a mediator of mitophagy) transcripts. These increased ROS were of mitochondrial origin, as detected by Mito-Sox staining.To pursue the mechanism behind the preferential destruction of young RBCs, we investigated antioxidant enzymes. After hypoxia treatment, catalase decreased by 30%, but not glutathione peroxidase, superoxide dismutase or NADH oxidase. The decreased catalase in RBCs produced during hypoxia was unexpected, as it was shown previously that catalase is regulated by HIF2 (as we also show, to a lower degree by HIF1 in our Hif1a-/-embryo) suggesting alternate negative regulator(s) of catalase in hypoxia. Several hypoxia-regulated microRNAs (miRs) are reported to control oxidative stress; we found that miR-451, miR-205 and miR-21 were expressed in erythroid progenitors and reticulocytes and induced after 10 day-hypoxia exposure. To verify whether these miRs regulate catalase expression, we overexpressed and downregulated these miRs in K562 and HEL erythroid cell lines, and found that only miR-21 regulated catalase. Further, we found increased miR-21 after 10 day-hypoxia exposure, with a concomitant decrease of catalase transcripts and activity resulting in impaired ROS scavenging.We conclude that neocytolysis is mediated by excessive generation of ROS from increased mitochondrial mass due to reduced Bnip3L in reticulocytes upon return to normoxia. The reticulocyte ROS then interact with hypoxia-produced young RBCs having miR-21-downregulated catalase, resulting in their preferential destruction. We show that increased mitochondrial ROS and miR-21-downregulated catalase provide the molecular basis of neocytolysis. DisclosuresNo relevant conflicts of interest to declare.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Restoring physiological levels of ascorbate slows tumor growth and moderates HIF-1 pathway activity in Gulo(-/-) mice.

Hypoxia-inducible factor-1 (HIF-1) governs cellular adaption to the hypoxic microenvironment and is associated with a proliferative, metastatic, and treatment-resistant tumor phenotype. HIF-1 levels and transcriptional activity are regulated by proline and asparagine hydroxylases, which require ascorbate as cofactor. Ascorbate supplementation reduced HIF-1 activation in vitro, but only limited data are available in relevant animal models. There is no information of the effect of physiological levels of ascorbate on HIF activity and tumor growth, which was measured in this study. C57BL/6 Gulo−/− mice (a model of the human ascorbate dependency condition) were supplemented with 3300 mg/L, 330 mg/L, or 33 mg/L of ascorbate in their drinking water before and during subcutaneous tumor growth of B16-F10 melanoma or Lewis lung carcinoma (LL/2). Ascorbate levels in tumors increased significantly with elevated ascorbate intake and restoration of wild-type ascorbate levels led to a reduction in growth of B16-F10 (log phase P < 0.001) and LL/2 tumors (lag growth P < 0.001, log phase P < 0.05). Levels of HIF-1α protein in tumors decreased as dietary ascorbate supplementation increased for both tumor models (P < 0.001). Similarly, tumor ascorbate was inversely correlated with levels of the HIF-1 target proteins CA-IX, GLUT-1, and VEGF in both B16-F10 and LL/2 tumors (P < 0.05). The extent of necrosis was similar between ascorbate groups but varied between models (30% for B16-F10 and 21% for LL/2), indicating that ascorbate did not affect tumor hypoxia. Our data support the hypothesis that restoration of optimal intracellular ascorbate levels reduces tumor growth via moderation of HIF-1 pathway activity.

Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen.

The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases; their catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role. PHD2 forms a stable complex with Fe(II) and 2OG; crystallographic and kinetic analyses indicate that an Fe(II)-co-ordinated water molecule, which must be displaced before O2 binding, is relatively stable in the active site of PHD2. We used active site substitutions to investigate whether these properties are related to the slow reaction of PHD2 with O2. While disruption of 2OG binding in a R383K variant did not accelerate O2 activation, we found that substitution of the Fe(II)-binding aspartate for a glutamate residue (D315E) manifested significantly reduced Fe(II) binding, yet maintained catalytic activity with a 5-fold faster reaction with O2. The results inform on how the precise active site environment of oxygenases can affect rates of O2 activation and provide insights into limiting steps in PHD catalysis.

Abstract 229: Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice

Abstract Background: Hypoxia-inducible factor-1 (HIF-1) upregulates the expression of hundreds of genes involved in adaption of tumours to the hypoxic microenvironment. HIF-1 levels and transcriptional activity are regulated by hydroxylase enzymes, which require ascorbate as a cofactor. Adequate supplementation of cells with ascorbate has been shown to reduce HIF-1 activation in vitro, and we hypothesise that a similar activity could affect tumour growth in vivo. In this study we investigated the effect of dietary ascorbate on tumour growth in Gulo-/- mice, a model of the human ascorbate deficiency condition. Methods: C57/Bl6 Gulo-/- mice were supplemented with 3300 mg/L, 330 mg/L or 33mg/L of ascorbate in their drinking water for one month before subcutaneous, syngeneic tumour implantation with either B16-F10 melanoma, or Lewis lung carcinoma (LL/2), and then for duration of the experiment. Levels of HIF-1α, and its targets, carbonic anhydrase-IX (CA-IX), and glucose transporter-1 (GLUT-1) were analysed by western blotting, and vascular endothelial growth factor (VEGF) levels by ELISA. Ascorbate concentrations of tissue, plasma and tumours were measured using high pressure liquid chromatography with electrochemical detection (HPLC-EC). Results: Across all organs and plasma samples, ascorbate content was in the physiological range (up to levels seen in wild-type mice) and was correlated with dietary ascorbate intake. Tumour ascorbate also reflected ascorbate intake and optimal ascorbate levels (3300 mg/L) significantly increased the time for LL/2 tumours to reach 200 mm3 (lag growth) (13.3±2.6 days), compared to these same tumours in mice supplemented with either 330 mg/L (9.1±2.6 days) or 33 mg/L (9.8±1.39 days) of ascorbate (p&amp;lt;0.001). Similarly, tumours took significantly longer to grow from 200-800 mm3 (log phase) when mice were supplemented with 3300 mg/L compared to lower supplementation (p&amp;lt;0.05). B16-F10 tumours showed no difference in the lag growth period between ascorbate treatments (p=0.49). However, supplementing mice with 3300 mg/mL of ascorbate lead to a significant reduction in the tumour growth rate during log phase (3.9±1.1 days), compared to mice supplemented with either 330 mg/L (2.0±0.90 days) or 33mg/L (2.1±0.71 days) (p&amp;lt;0.001). Levels of HIF-1α protein in tumours decreased as dietary ascorbate supplementation increased for both LL/2 (p&amp;lt;0.001) and B16F10 (p&amp;lt;0.001). Tumour ascorbate was significantly correlated with low levels of CA-IX, GLUT-1 and VEGF levels in both LL/2 and B16 tumours (all p&amp;lt;0.05). Conclusions: The data from the current study support the hypothesis that restoration of optimal intracellular ascorbate levels is associated with reduced HIF-1 levels and slower tumour growth. This data may have implications for the management of cancer, although clinical trials are required to assess whether human tumour levels of ascorbate can similarly be manipulated. Citation Format: Elizabeth J. Campbell, Stephanie M. Bozonet, Bridget A. Robinson, Margreet CM Vissers, Gabi U. Dachs. Restoring physiological levels of ascorbate alleviates HIF-1 activation and reduces tumour growth in Gulo-/- mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 229. doi:10.1158/1538-7445.AM2014-229

Abstract 727: Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis

Abstract Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727

Abstract 222: Disulfiram targets glioblsatoma stem like cells by modulating aldehyde dehydrogenase and hypoxia-NF-κB pathway

Abstract Introduction: Glioblastoma Multiforme (GBM) is the most common lethal brain tumor associated with dismal survival rate. It is widely accepted that GBM contains a small population of cancer cells (∼1%) expressing CSC markers that promotes therapeutic resistance. High levels of aldehyde dehydrogenase (ALDH) activity, a characteristic feature of CSCs, play a significant role in chemoresistance. Many recent studies have elucidated that intra-tumoral hypoxia induces CSC phenotypes in tumour via epithelial-to-mesenchymal transition (EMT). Hypoxia inducible factors (HIFs) are master transcriptional regulators under hypoxic conditions. However NF-κB, another key transcription factor, is also highly up-regulated in hypoxia induced CSCs. There are no clear insights on how HIFs and NF-κB together orchestrates the anti-apoptotic signalling, chemo-radiation resistance and maintenance of stemness in CSC phenotypes. Disulfiram (DS), an anti-alcoholism drug, in combination with copper (Cu) effectively reverses chemoresistance in many cancers. DS/Cu is a strong inhibitor of NF-κB and ALDH and induces apoptosis. In this study we used an in vitro GBM CSC model and NF-κB-p65 and HIFs transfected GBM cell lines to investigate the relationship between hypoxia induced HIFs, NF-κB activation and ALDH activity and their role in chemoresistance. We also examined the cytotoxicity of DS/Cu on GBM CSCs and its effect on the above pathways. Results In comparison with adhered cells, the GBM cellsgrown as spheres in specialized neurosphere medium (NS) or normal DMEM (SUS) showed high proportion of hypoxic cells and elevated levels of CSC and EMT markers (ALDH, CD133, CD44, OCT4, SOX2, NANOG, E-cadherin to N-cadherin switch) suggesting hypoxia induced EMT phenotypes inCSCs. Increased levels of HIFs, ALDH (1A3 and 3A1) and NF-κB activity were detected in CSCs. CSCs are highly resistant to conventional anticancer drugs like temozolomide, paclitaxel, vincristine and doxorubicin. GBM cells transfected with NF-κB-p65 displayed CSC markers, chemoresistance and nuclear translocation of HIFs. Although transfection of HIFs induced expression of CSC markers, the cells did not show NF-κB activation and are not resistant to anticancer drugs. These results indicate the pivotal role of NF-κB in maintaining CSC characteristics and chemoresistance. DS is cytotoxic to both GBM cell lines and the CSCs derived from them in a Cu-dependent manner.DS/Cu inhibits NF-κB and ALDH activity and triggers intrinsic apoptotic pathway. After short exposure (2 hours) to low concentration of DS/Cu, the CSCs are completely eradicated in the cell culture. Conclusions: NF-κB plays pivotal role in chemoresistance. DS/Cu inhibits NF-κB, eradicates CSCs and reverses resistance in GBM.DS is an FDA approved drug with low/no toxicity to normal tissues and can freely pass through the BBB. Further study may lead to quick translation of DS into clinical trials. Citation Format: Vinodh Kannappan, Peng Liu, Sarah Brown, Xiuwu Bian, Tawari Patricia Erebi, Angel L. Armesilla, John L. Darling, Weiguang Wang. Disulfiram targets glioblsatoma stem like cells by modulating aldehyde dehydrogenase and hypoxia-NF-κB pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 222. doi:10.1158/1538-7445.AM2014-222

O2.04 * TREATMENT WITH OMX-4.80, A TUMOR-PENETRATING TUNABLE OXYGEN CARRIER, REDUCES TUMOR HYPOXIA AND DRAMATICALLY ENHANCES RADIATION THERAPY IN INTRACRANIAL MODELS OF GLIOBLASTOMA

We have developed OMX-4.80, a tunable oxygen carrier from the H-NOX protein family, that preferentially accumulates in tumor tissue, reduces hypoxia and enhances radiotherapy (RT). Tumor hypoxia is associated with poor patient outcomes in multiple solid tumors including glioblastoma (GB) and is thought to be a major contributor to poor cancer responses to chemo and radiation therapy. Here, we show that OMX-4.80 administered i.v. into the tail vein of mice bearing orthotopic GB passes the blood-tumor barrier, penetrates deep into intracranial tumors, and significantly reduces tumor hypoxia in a dose-dependent manner. Hypoxia reduction is demonstrated by a significant decrease in levels of hypoxia inducible factor, HIF1α, and the external hypoxia marker pimonidazole as assessed by IHC and image analysis. Furthermore, hypoxia before and after treatment is compared in the individual tumors by concurrent immunostaining of tumors with a long-term hypoxia marker, Glut1, and short-lived HIF-1α. These data reveal >50% reduction in hypoxic tumor area after OMX-4.80 treatment confirming the effectiveness of OMX-4.80 in reducing hypoxia in individual tumors. When OMX-4.80 treatment was coupled with a single high dose of RT, we observed a 2.7-fold increase in tumor growth delay and 2.9-fold longer survival (p < 0.05, by logrank). Toxicology studies in rodents and dogs with single and multiple supratherapeutic doses of OMX-4.80 revealed a good safety profile affirming its suitability for clinical development. In conclusion, our results indicate that OMX-4.80 is an effective and promising RT enhancer that warrants clinical development in GB patients.

Preclinical evidence that SSR128129E – A novel small-molecule multi-fibroblast growth factor receptor blocker – Radiosensitises human glioblastoma

Resistance of glioblastoma to radiotherapy is mainly due to tumour cell radioresistance, which is partially controlled by growth factors such as fibroblast growth factor (FGF). Because we have previously demonstrated the role of FGF-2 in tumour cell radioresistance, we investigate here whether inhibiting FGF-2 pathways by targeting fibroblast growth factor receptor (FGFR) may represent a new strategy to optimise the efficiency of radiotherapy in glioblastoma.Treating radioresistant U87 and SF763 glioblastoma cells with the FGFR inhibitor, SSR12819E, radiosensitises these cells while the survival after irradiation of the more radiosensitive U251 and SF767 cells was not affected. SSR128129E administration to U87 cells increases the radiation-induced mitotic cell death. It also decreased cell membrane availability of the FGFR-1 mainly expressed in these cells, increased this receptor’s ubiquitylation, inhibited radiation-induced RhoB activation and modulated the level of hypoxia inducible factor, HIF-1α, a master regulator of hypoxia, thus suggesting a role of FGFR in the regulation of hypoxia pathways. Moreover, treating orthotopically U87 xenografted mice with SSR128129E before two subsequent local 2.5Gy irradiations significantly increased the animals neurological sign free survival (NSFS) compared to the other groups of treatment. These results strongly suggest that targeting FGFR with the FGFR blocker SSR128129E might represent an interesting strategy to improve the efficiency of radiotherapy in glioblastoma.

Ginsenoside Rg3 enhances radiosensitization of hypoxic oesophageal cancer cell lines through vascular endothelial growth factor and hypoxia inducible factor 1α

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.

Vasculogenesis: a crucial player in the resistance of solid tumours to radiotherapy

Tumours have two main ways to develop a vasculature: by angiogenesis, the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis, the formation of blood vessels from circulating cells. Because tumour irradiation abrogates local angiogenesis, the tumour must rely on the vasculogenesis pathway for regrowth after irradiation. Tumour irradiation produces a marked influx of CD11b(+) myeloid cells (macrophages) into the tumours, and these are crucial to the formation of blood vessels in the tumours after irradiation and for the recurrence of the tumours. This process is driven by increased tumour hypoxia, which increases levels of HIF-1 (hypoxia-inducible factor 1), which in turn upregulates SDF-1 (stromal cell-derived factor 1 or CXCL12), the main driver of the vasculogenesis pathway. Inhibition of HIF-1 or of its downstream target SDF-1 prevents the radiation-induced influx of the CD11b(+) myeloid cells and delays or prevents the tumours from recurring following irradiation. Others and we have shown that with a variety of tumours in both mice and rats, the inhibition of the SDF-1/CXCR4 pathway delays or prevents the recurrence of implanted or autochthonous tumours following irradiation or following treatment with vascular disrupting agents or some chemotherapeutic drugs such as paclitaxel. In addition to the recruited macrophages, endothelial progenitor cells (EPCs) are also recruited to the irradiated tumours, a process also driven by SDF-1. Together, the recruited proangiogenic macrophages and the EPCs reform the tumour vasculature and allow the tumour to regrow following irradiation. This is a new paradigm with major implications for the treatment of solid tumours by radiotherapy.

Chronodisruption increases cardiovascular risk in zebrafish via reduced clearance of senescent erythrocytes

The circadian clock and the hypoxic signaling pathway play critical roles in physiological homeostasis as well as in pathogenesis. The bi-directionality of the interaction between both pathways has been shown on physiological and only recently also on molecular level. But the consequences of a disturbed circadian rhythm for the hypoxic response and the cardiovascular system have never been addressed in any organism. Here we show that the hypoxic response of animals subjected to chronodisruption is reduced by approximately 30%, as reflected by decreased expression levels of hypoxia inducible factor 1 and its down-stream target genes erythropoietin, responsible for the generation of red blood cells (RBC) and vascular endothelial growth factor, which is essential for proper vascularization. Beside malformations of their vascular beds, chronodisrupted animals surprisingly revealed elevated numbers of senescent erythrocytes under normoxic conditions, due to a reduced clearance rate via apoptosis. Over-aged erythrocytes in turn are characterized by decreased oxygen transport capacities and an increased tendency for aggregation, explaining the higher mortality of chronodisrupted animals observed in our study. The present study shows for the first time that chronodisruption strongly interferes with the hypoxic signalling cascade, increasing the cardiovascular risk in zebrafish due to elevated proportions of senescent erythrocytes. The results might shed new light on the etiology of the increased cardiovascular risk observed among shiftworkers.

Expressions of Hypoxia-Inducible Factor-1 and Epithelial Cell Adhesion Molecule are Linked with Aggressive Local Recurrence of Hepatocellular Carcinoma After Radiofrequency Ablation Therapy

Radiofrequency ablation (RFA) is a widely used therapy for hepatocellular carcinoma (HCC). Several reports have demonstrated the aggressive local recurrence of HCC after RFA, suggesting that induction of further malignant transformation of HCC has occurred. Eighty-eight (88) patients with HCC who underwent hepatic resection were included in this study. Hepatectomy was indicated for local recurrence of HCC after RFA (n = 10, RFA group) and for HCC without prior RFA (n = 78, non-RFA group). Clinicopathological data and the patient's prognosis after hepatectomy were compared between the two groups. Expression levels of hypoxia-inducible factor-1 (HIF-1), epithelial cell adhesion molecule (EpCAM), CD44, and vascular endothelial growth factor messenger RNA (mRNA) in the tumor tissues were also examined. The RFA group showed higher frequency of portal vein invasion and less tumor differentiation compared with the non-RFA group (p < 0.05). Overall and disease-free survival rates in the RFA group were significantly worse than those in the non-RFA group (p < 0.05). HIF-1 and EpCAM mRNA expression levels in the RFA group were significantly higher than those in the non-RFA group (p < 0.05). These results suggest that local HCC recurrence after RFA shows an aggressive tumor phenotype and poor prognosis through the enhanced expressions of HIF-1 and EpCAM in the residual HCC tumors after insufficient or sub-lethal treatment by RFA.

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Hypoxia-Inducible Factor-1α in Pulmonary Arterial Smooth Muscle Cells and Hypoxia-induced Pulmonary Hypertension

Expression of IDH1 Mutant Protein R132H and SDHB in Adult and Pediatric Gliomas

Isocitrate dehydrogenase 1 (IDH1) and Succinate dehydrogenase (SDH) mutant tumors may have increased levels of hypoxia inducible factor (HIF), which leads to a tumoral pseudohypoxic profile. Increased HIF levels may promote tumor progression by the activation of numerous cellular processes including resistance to apoptosis, vascular remodeling and angiogenesis. A high frequency of IDH1 mutations has been detected in adult but not in pediatric gliomas. However, loss of SDH expression has not been investigated in gliomas. To further explore the profile and possible contribution of IDH1 and SDHB to glioma tumorigenesis, we investigated the expression of IDH1-R132 mutant and SDHB in a series of 94 histologically confirmed pediatric and adult gliomas. There were 13 diffuse intrinsic pontine gliomas and 81 adult gliomas: 10 lowgrade astrocytomas 8 oligodendrogliomas, 4 ependymomas and 59 glioblastomas (GBMs). Pediatric brain stem gliomas were negative for mutant IDH1 (0/13) and showed preserved SDHB expression (13/13). IDH1 mutant tumors included 6/10 astrocytomas, 5/8 oligodendrogliomas, 0/4 ependymomas and 13/59 GBMs. No adult or pediatric glioma exhibited loss of SDHB expression. IDH1 mutations are frequently present in low- and high-grade gliomas, but not in pediatric brain stem gliomas. It appears that IDH1, but not SDHB, contributes to HIF stabilization in adult gliomas. However, IDH1 or SDHB alterations do not appear to play a role in pediatric gliomagenesis.

The role of hypoxia inducible factor-1 in hepatocellular carcinoma.

Hypoxia is a common feature of many solid tumors, including hepatocellular carcinoma (HCC). Hypoxia can promote tumor progression and induce radiation and chemotherapy resistance. As one of the major mediators of hypoxic response, hypoxia inducible factor-1 (HIF-1) has been shown to activate hypoxia-responsive genes, which are involved in multiple aspects of tumorigenesis and cancer progression, including proliferation, metabolism, angiogenesis, invasion, metastasis and therapy resistance. It has been demonstrated that a high level of HIF-1 in the HCC microenvironment leads to enhanced proliferation and survival of HCC cells. Accordingly, overexpression, of HIF-1 is associated with poor prognosis in HCC. In this review, we described the mechanism by which HIF-1 is regulated and how HIF-1 mediates the biological effects of hypoxia in tissues. We also summarized the latest findings concerning the role of HIF-1 in the development of HCC, which could shed light on new therapeutic approaches for the treatment of HCC.

Glucagon-induced angiogenesis and tumor growth through the HIF-1-VEGF-dependent pathway in hyperglycemic nude mice.

In this study, we examined the effect glucagon-induced hyperglycemia on tumor growth as well as the role of the hypoxia-inducible factor 1 (HIF-1)-vascular endothelial growth factor (VEGF) pathway in this condition. A high concentration of glucose (HG) was utilized to treat HeLa cells under hypoxic or normoxic conditions, and transcriptional levels of HIF-1, VEGF, and basic fibroblast growth factor (bFGF) were evaluated. Moreover, the ability of an HIF-1 inhibitor to block the effect induced by HG was examined. By contrast, hyperglycemia was induced in nude mice by glucagon released from an osmotic pump, and microvessel density was determined with CD31 staining. Thus, the relationship among hyperglycemia, microvessel density, tumor growth, and the HIF-1 inhibitor were analyzed. We found that HG increased transcription of the VEGF gene, which is downstream of HIF-1. Moreover, HG impaired the function of HIF-1 inhibitors [HIF-1 small interfering RNA (siRNA) and berberine] to affect the VEGF transcription level in tumor cells. By contrast, hyperglycemia increased tumor microvessel density and promoted tumor growth, which was inhibited by the HIF-1 inhibitor. However, hyperglycemia attenuated the effect of the HIF-1 inhibitor. Glucagon-induced hyperglycemia influenced tumor microenvironments through the HIF-1-VEGF-dependent pathway and promoted tumor growth and resistance to HIF-1 inhibition treatments.

Interleukin-1β promotes hypoxia-induced apoptosis of glioblastoma cells by inhibiting hypoxia-inducible factor-1 mediated adrenomedullin production

Glioblastoma is the most common brain tumor in adults. Advanced glioblastomas normally contain hypoxic areas. The primary cellular responses to hypoxia are generally mediated by the transcription factor hypoxia-inducible factor 1 (HIF-1). Interleukin-1β (IL-1β) is a cytokine that is often present in the glioblastoma microenvironment and is known to be a modulator of glioblastoma progression. However, the role of IL-1β in regulating glioblastoma progression is still controversial. In this study, we found that in the human glioblastoma cell lines U87MG and U138MG, IL-1β inhibits the transactivation activity of HIF-1 by promoting the ubiquitin-independent proteasomal degradation of the oxygen-labile α-subunit of HIF-1 and downregulates the expression of the HIF-1 target gene adrenomedullin (AM). Apoptosis and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays showed that AM protects glioblastoma cells against hypoxia-induced apoptosis in a dose-dependent manner. Thus, in the presence of IL-1β more glioblastoma cells undergo hypoxia-induced cell death. Our findings suggest that when estimating the influence of IL-1β on the prognosis of glioblastoma patients, factors such as the degree of hypoxia, the expression levels of HIF-1 and AM should be taken into consideration. For the AM-producing glioblastoma cells, IL-1β represents a potent apoptosis inducer.

Competitive HIF Prolyl Hydroxylase Inhibitors Show Protection against Oxidative Stress by a Mechanism Partially Dependent on Glycolysis

The hypoxia inducible factor 1 (HIF-1) is a central transcription factor involved in the cellular and molecular adaptation to hypoxia and low glucose supply. The level of HIF-1 is to a large degree regulated by the HIF prolyl hydroxylase enzymes (HPHs) belonging to the Fe(II) and 2-oxoglutarate-dependent dioxygenase superfamily. In the present study, we compared competitive and noncompetitive HPH-inhibitor compounds in two different cell types (SH-SY5Y and PC12). Although the competitive HPH-inhibitor compounds were found to be pharmacologically more potent than the non-competitive compounds at inhibiting HPH2 and HPH1, this was not translated into the cellular effects of the compounds, where the non-competitive inhibitors were actually more potent than the competitive in stabilizing and translocatingHIF1αto the nucleus (quantified with Cellomics ArrayScan technology). This could be explained by the high cellular concentrations of the cofactor 2-oxoglutarate (2-OG) as the competitive inhibitors act by binding to the 2-OG site of the HPH enzymes. Both competitive and non-competitive HPH inhibitors protected the cells against 6-OHDA induced oxidative stress. In addition, the protective effect of a specific HPH inhibitor was partially preserved when the cells were serum starved and exposed to 2-deoxyglucose, an inhibitor of glycolysis, indicating that other processes than restoring energy supply could be important for the HIF-mediated cytoprotection.

Antagonism of PDGF-BB suppresses subretinal neovascularization and enhances the effects of blocking VEGF-A

Hypoxia-inducible factor-1 (HIF-1) plays an important role in retinal and subretinal neovascularization (NV). Increased levels of HIF-1 cause increased expression of vascular endothelial growth factor (VEGF-A) and current therapies for ocular NV focus on neutralizing VEGF-A, but there is mounting evidence that other HIF-1-responsive gene products may also participate. In this study, we tested the effect of a designed ankyrin repeat protein (DARPin) that selectively binds and antagonizes the hypoxia-regulated gene product PDGF-BB in three models of subretinal NV (relevant to neovascular age-related macular degeneration) and compared its effects to a DARPin that selectively antagonizes VEGF-A. Daily intraperitoneal injections of 10 mg/kg of the anti-PDGF-BB DARPin or 1 mg/kg of the anti-VEGF DARPin significantly suppressed subretinal NV from laser-induced rupture of Bruch's membrane. Injections of 1 mg/kg/day of the anti-PDGF-BB DARPin had no significant effect, but when combined with 1 mg/kg/day of the anti-VEGF-A DARPin there was greater suppression than injection of the anti-VEGF-A DARPin alone. In Vldlr (-/-) mice which spontaneously develop subretinal NV, intraocular injection of 1.85 μg of anti-PDGF-BB or anti-VEGF-A DARPin caused significant suppression of NV and when combined there was greater suppression than with either alone. The two DARPins also showed an additive effect in Tet/opsin/VEGF double transgenic mice, a particularly severe model of subretinal NV and exudative retinal detachment. In addition, intraocular injection of 1.85 μg of anti-PDGF-BB DARPin strongly suppressed ischemia-induced retinal NV, which is relevant to proliferative diabetic retinopathy and retinopathy of prematurity. These data demonstrate that PDGF-BB is another hypoxia-regulated gene product that along with VEGF-A contributes to ocular NV and suppression of both provides an additive effect.