oxalylglycine and cobaltous-chloride

Thixotropic clays have favorable properties for tissue regeneration. Hypoxia mimetic agents showed promising results in pre-clinical models for hard and soft tissue regeneration. It is unclear if clays can be used as carrier for hypoxia mimetic agent in a periodontal regenerative setting. Here, we tested the response of human fibroblasts of the periodontal soft tissue to synthetic clay hydrogels and assessed hypoxia mimetic agent release. Cells were cultured on synthetic clay hydrogels (5.00%-0.15%). We assessed viability and differentiation capacity with resazurin-based toxicity assays, MTT staining, Live-Dead staining, and alkaline phosphatase staining. To reveal the response of fibroblasts to hypoxia mimetic agent-loaded clay hydrogels, cells were exposed to clay supplemented with dimethyloxalylglycine, deferoxamine, l-mimosine, and CoCl

Topics: Amino Acids, Dicarboxylic; Biocompatible Materials; Cell Hypoxia; Cells, Cultured; Clay; Cobalt; Deferoxamine; Drug Delivery Systems; Fibroblasts; Humans; Hydrogels; Mimosine; Periodontium; Tissue Scaffolds

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the hypoxia inducible factor (HIF) complex. However, various compounds can also stabilize HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF-stabilizing compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in transforming growth factor-β3-containing media in the presence of HIF-stabilizing compounds. HIF-1α stabilization was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by quantitative polymerase chain reaction, and cartilage-like extracellular matrix production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localization. However, while the 2-oxoglutarate analog dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, desferrioxamine (DFX) and cobalt chloride (CoCl

Topics: Amino Acids, Dicarboxylic; Bone Marrow Cells; Cell Differentiation; Cell Hypoxia; Child; Chondrogenesis; Cobalt; Deferoxamine; Enzyme Inhibitors; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Mesenchymal Stem Cells

Hypoxia mimetic agents (HMAs) have been shown to have a positive influence on cellular functions in a multitude of tissue regenerative strategies. Novel experimental approaches use biomaterials as carriers for controlled delivery of these HMAs. Here, the cytotoxic aspects of biocompatibility are of key relevance. The MTT assay is widely used to evaluate cytotoxicity and proliferation. Based on the implications from the proceeding research we hypothesized that specific HMAs such as deferoxamine at high concentrations can interfere with the MTT assay. Thus, the aim of this study was to test the repercussions of the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl

Topics: 3T3 Cells; Amino Acids, Dicarboxylic; Animals; Biocompatible Materials; Cell Line; Cell Survival; Cobalt; Deferoxamine; Dimethyl Sulfoxide; Mice; Mimosine; Tetrazolium Salts; Thiazoles

Understanding the endogenous survival pathways induced by ischemic tolerance may yield targets for neuroprotection from stroke. One well-studied pathway reported to be evoked by preconditioning stimuli is the transcription factor HIF (hypoxia-inducible factor). However, whether HIF induction by ischemic insults is neuroprotective or toxic is still unclear. We examined the ability of three prolyl-hydroxylase inhibitors, which induce HIF, to protect hippocampal cultures from oxygen-glucose deprivation. Hippocampal cultures were exposed to ischemic preconditioning or various concentrations of cobalt chloride, deferoxamine (DFO) or dimethyloxylalyglycine (DMOG), prior to lethal oxygen-glucose deprivation (OGD). Cell survival of neurons and astrocytes was determined with dual-label immunocytochemistry. The induction of HIF targets was assessed in mixed as well as astrocyte-enriched cultures. Ischemic preconditioning, as well as low concentrations of cobalt and DFO, enhanced the survival of neurons following OGD. However, DMOG exacerbates OGD-induced neuronal death. At low concentrations, all three prolyl-hydroxylase (PHD) inhibitors increased the survival of astrocytes. Neuroprotective concentrations of cobalt induced the transcription of the cytokine erythropoietin (EPO) in astrocyte cultures. In addition, pretreatment with recombinant human erythropoietin (rH-EPO) also protected neurons from OGD. Our data suggest that HIF-induced EPO, released from astrocytes, protects neurons from OGD. However, the three PHD inhibitors each exhibited different neuroprotective profiles at low concentrations, suggesting that not all PHD inhibitors are created equal. The protective effects at low doses is reminiscent of HIF involvement in ischemic tolerance, in which sub-lethal insults induce HIF pathways resulting in neuroprotection, whereas the high-dose toxicity suggests that over-activation of HIF is not always protective. Therefore, the choice of inhibitor and dose may determine the clinical utility of these compounds. Deferoxamine exhibited little toxicity even at higher doses, and therefore appears a promising candidate for clinical use.

Topics: Amino Acids, Dicarboxylic; Animals; Astrocytes; Brain Ischemia; Cell Survival; Cells, Cultured; Cobalt; Deferoxamine; Erythropoietin; Hippocampus; Hypoxia-Inducible Factor 1; Immunohistochemistry; Ischemic Preconditioning; Mice; Mice, Inbred C57BL; Neurons; Neuroprotective Agents; Polymerase Chain Reaction; Prolyl-Hydroxylase Inhibitors

Hypoxia-inducible factors (HIFs) are transcriptional regulators that mediate the cellular response to low oxygen. Although HIF-1 is usually considered as the principal mediator of hypoxic adaptation, several tissues and different cell types express both HIF-1 and HIF-2 isoforms under hypoxia or when treated with hypoxia mimetic chemicals such as cobalt. However, the similarities or differences between HIF-1 and HIF-2, in terms of their tissue- and inducer-specific activation and function, are not adequately characterized. To address this issue, we investigated the effects of true hypoxia and hypoxia mimetics on HIF-1 and HIF-2 induction and specific gene transcriptional activity in two hepatic cancer cell lines, Huh7 and HepG2. Both hypoxia and cobalt caused rapid induction of both HIF-1α and HIF-2α proteins. Hypoxia induced erythropoietin (EPO) expression and secretion in a HIF-2-dependent way. Surprisingly, however, EPO expression was not induced when cells were treated with cobalt. In agreement, both HIF-1- and HIF-2-dependent promoters (of PGK and SOD2 genes, respectively) were activated by hypoxia while cobalt only activated the HIF-1-dependent PGK promoter. Unlike cobalt, other hypoxia mimetics such as DFO and DMOG activated both types of promoters. Furthermore, cobalt impaired the hypoxic stimulation of HIF-2, but not HIF-1, activity and cobalt-induced HIF-2α interacted poorly with USF-2, a HIF-2-specific co-activator. These data show that, despite similar induction of HIF-1α and HIF-2α protein expression, HIF-1 and HIF-2 specific gene activating functions respond differently to different stimuli and suggest the operation of oxygen-independent and gene- or tissue-specific regulatory mechanisms involving additional transcription factors or co-activators.

Topics: Amino Acids, Dicarboxylic; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Line, Tumor; Cell Nucleus; Cobalt; Deferoxamine; Erythropoietin; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Liver Neoplasms; Phosphoglycerate Kinase; Promoter Regions, Genetic; Propanolamines; Protein Binding; Pyrrolidines; RNA, Messenger; Superoxide Dismutase; Transcription, Genetic; Upstream Stimulatory Factors

The Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT, HIF-1β) is a member of the basic-Helix-Loop-Helix PER/ARNT/SIM (bHLH/PAS) protein family and a vital transcriptional regulator regarding development and physiological adaptation processes. ARNT is discussed to be linked with cancer, and other diseases. ARNT is known to be translocated into the cell nucleus, where accumulation of the protein takes place. ARNT is a heterodimerisation partner of the xenobiotic ligand activated Aryl Hydrocarbon Receptor (AhR), the Single Minded proteins (SIM), the cardiovascular helix-loop-helix factor 1 and the Hypoxia Inducible Factor proteins (HIF-α). ARNT is obligatory for HIF-1, HIF-2 and HIF-3 binding to DNA. Whereas degradation of the HIF-α subunits is suppressed by hypoxia, ARNT is generally regarded as constitutively expressed in excess within the cell, and stabilisation is commonly thought to be oxygen-independent. However, we provide evidence that the regulation of ARNT is far more complex. The aim of our study was to reevaluate the regulation of ARNT expression.. We examined cell lines of different origin like MCF-7 and T47D (human breast cancer), HeLa (human cervix carcinoma), Hep3B and HepG2 (human hepatoma), Kelly (human neuroblastoma), REPC (human kidney) and Cos7 (primary primate kidney) cells. We used immunoblot analysis, densitometry, RT-PCR and transient transfection.. Our results show that ARNT protein levels are influenced by hypoxia and hypoxia mimetics such as cobalt(II)-chloride (CoCl2) and dimethyloxalylglycine (DMOG) in a cell line specific manner. We demonstrate that this effect might be triggered by HIF-1α which plays an important role in the process of stabilizing ARNT in hypoxia.

Topics: Amino Acids, Dicarboxylic; Antimutagenic Agents; Aryl Hydrocarbon Receptor Nuclear Translocator; Cell Hypoxia; Cell Line, Tumor; Cobalt; HeLa Cells; Humans; Immunoblotting

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl(2) does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser(293), Ser(300), and Ser(232), respectively) in HepG2 cells. This same effect is also observed for site 1 in mouse NIH/3T3 fibroblasts and J774 macrophages. CoCl(2) unexpectedly decreases the mRNA expression for PDH kinase-2 in HepG2 cells, which likely explains the dephosphorylation of PDH observed. And nor does desferrioxamine promote the expected increase in PDH phosphorylation. Dimethyloxaloylglycine (a prolyl hydroxylase inhibitor) performs better in this regard, but failed to promote the stronger effects seen with hypoxia. Consequently, CoCl(2) and desferrioxamine are unreliable mimics of hypoxia for physiological events downstream of HIF-1α stabilization. Our study demonstrates that mimetic chemicals must be chosen with caution and evaluated thoroughly if bona fide cellular outcomes are to be promoted with fidelity.

Topics: Amino Acids, Dicarboxylic; Animals; Blotting, Western; Cell Hypoxia; Cell Line; Cobalt; Deferoxamine; Gene Expression Regulation, Enzymologic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mammals; Mice; Phosphorylation; Pyruvate Dehydrogenase Complex; RNA, Messenger

Pharmacological inhibitors of prolyl hydroxylases (PHDs) can induce a proangiogenic response that favors wound healing and bone regeneration. However, the response of periodontal cells to PHD inhibitors is unknown.. To determine the effects of PHD inhibitors on periodontal cells, we exposed human fibroblasts from the gingiva and the periodontal ligament to dimethyloxallyl glycine, desferrioxamine, l-mimosine and CoCl(2). Viability, proliferation, and protein synthesis were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), [(3)H]thymidine, and [(3)H]leucine incorporation, respectively. The levels of Ki67, hypoxia-inducible factor 1α (HIF-1α), p27, phosphorylated c-Jun N-terminal kinase (JNK) and phosphorylated p38 were determined by immunohistochemistry and western blotting. Vascular endothelial growth factor (VEGF) mRNA levels were measured by quantitative PCR. Protein levels of VEGF and interleukin (IL)-6 were evaluated by immunoassays.. We found that PHD inhibitors, while leaving cell viability unchanged, reduced proliferation and protein synthesis. This was paralleled by decreased Ki67 levels and increased p27 levels, suggesting that PHD inhibitors provoke growth arrest. Independently from this response, PHD inhibitors stabilized HIF-1α and increased the production of VEGF. This increase of VEGF was observed in the presence of proinflammatory IL-1 and pharmacological inhibitors of JNK and p38 signaling. Moreover, PHD inhibitors did not modulate expression of IL-6 and the phosphorylation of JNK and p38.. These results suggest that PHD inhibitors enhance the production of VEGF in periodontal fibroblasts, even in the presence of proinflammatory IL-1. The data further suggest that PHD inhibitors do not provoke a significant proinflammatory or anti-inflammatory response in this in vitro setting.

Topics: Amino Acids, Dicarboxylic; Antimutagenic Agents; Cell Proliferation; Cell Survival; Cells, Cultured; Cobalt; Cyclin-Dependent Kinase Inhibitor p27; Deferoxamine; Enzyme Inhibitors; Fibroblasts; Gingiva; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Inflammation Mediators; Interleukin-1; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Ki-67 Antigen; Mimosine; p38 Mitogen-Activated Protein Kinases; Periodontal Ligament; Procollagen-Proline Dioxygenase; Protein Kinase Inhibitors; Proteins; Siderophores; Vascular Endothelial Growth Factor A

The accumulation of hypoxia-inducible factor-1α (HIF-1α) is under the influence of hydrogen sulfide (H(2) S), which regulates hypoxia responses. The regulation of HIF-1α accumulation by H(2) S has been shown, but the mechanisms for this effect are largely elusive and controversial. This study aimed at addressing the controversial mechanisms for and the functional importance of the interaction of H(2) S and HIF-1α protein.. HIF-1α protein levels and HIF-1α transcriptional activity were detected by Western blotting and luciferase assay. The mechanisms for H(2) S-regulated HIF-1α protein levels were determined using short interfering RNA transfection, co-immunoprecipitation and 7-methyl-GTP sepharose 4B pull-down assay. Angiogenic activity was evaluated using tube formation assay in EA.hy926 cells.. The accumulation of HIF-1α protein under hypoxia (1% O(2) ) or hypoxia-mimetic conditions was reversed by sodium hydrosulfide (NaHS). This effect of NaHS was not altered after blocking the ubiquitin-proteasomal pathway for HIF-1α degradation; however, blockade of protein translation with cycloheximide abolished the effect of NaHS on the half-life of HIF-1α protein. Knockdown of eukaryotic translation initiation factor 2α (eIF2α) suppressed the effect of NaHS on HIF-1α protein accumulation under hypoxia. NaHS inhibited the expression of VEGF under hypoxia. It also decreased in vitro capillary tube formation and cell proliferation of EA.hy926 cells under hypoxia, but stimulated the tube formation under normoxia.. H(2) S suppresses HIF-1α translation by enhancing eIF2α phosphorylation under hypoxia. The interaction of H(2) S and HIF-1α inhibits the angiogenic activity of vascular endothelial cells under hypoxia through the down-regulation of VEGF.

Topics: Amino Acids, Dicarboxylic; Cell Line; Cobalt; Deferoxamine; Down-Regulation; Eukaryotic Initiation Factor-2; HEK293 Cells; Humans; Hydrogen Sulfide; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit

Prolyl hydroxylase (PHD) inhibitors can induce a proangiogenic response that stimulates regeneration in soft and hard tissues. However, the effect of PHD inhibitors on the dental pulp is unclear. The purpose of this study was to evaluate the effects of PHD inhibitors on the proangiogenic capacity of human dental pulp-derived cells.. To test the response of dental pulp-derived cells to PHD inhibitors, the cells were exposed to dimethyloxalylglycine, desferrioxamine, L-mimosine, and cobalt chloride. To assess the response of dental pulp cells to a capping material supplemented with PHD inhibitors, the cells were treated with supernatants from calcium hydroxide. Viability, proliferation, and protein synthesis were assessed by formazan formation, (3)[H]thymidine, and (3)[H]leucine incorporation assays. The effect on the proangiogenic capacity was measured by immunoassays for vascular endothelial growth factor (VEGF).. We found that all 4 PHD inhibitors can reduce viability, proliferation, and protein synthesis at high concentrations. At nontoxic concentrations and in the presence of supernatants from calcium hydroxide, PHD inhibitors stimulated the production of VEGF in dental pulp-derived cells. When calcium hydroxide was supplemented with the PHD inhibitors, the supernatants from these preparations did not significantly elevate VEGF levels.. These results show that PHD inhibitors can stimulate VEGF production of dental pulp-derived cells, suggesting a corresponding increase in their proangiogenic capacity. Further studies will be required to understand the impact that this might have on pulp regeneration.

Topics: Amino Acids, Dicarboxylic; Cells, Cultured; Cobalt; Deferoxamine; Dental Pulp; Dental Pulp Capping; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mimosine; Neovascularization, Physiologic; Procollagen-Proline Dioxygenase; Regeneration; Siderophores; Vascular Endothelial Growth Factor A

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

Topics: Amino Acids, Dicarboxylic; Angiotensin II; Animals; Aorta; Blood Pressure; Blotting, Northern; Blotting, Western; Cell Hypoxia; Cells, Cultured; Cobalt; Down-Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Hypoxia-Inducible Factor-Proline Dioxygenases; Male; Mice; Mice, Inbred C57BL; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Procollagen-Proline Dioxygenase; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Renin; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference

The respiratory epithelium forms an important barrier against inhaled pollutants and microorganisms, and its barrier function is often compromised during inflammatory airway diseases. Epithelial activation of hypoxia-inducible factor-1 (HIF-1) represents one feature of airway inflammation, but the functional importance of HIF-1 within the respiratory epithelium is largely unknown. Using primary mouse tracheal epithelial (MTE) cells or immortalized human bronchial epithelial cells (16HBE14o-), we evaluated the impact of HIF-1 activation on loss of epithelial barrier function during oxidative stress. Exposure of either 16HBE14o- or MTE cells to H(2)O(2) resulted in significant loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-dextran (4 kDa), and this was attenuated significantly after prior activation of HIF-1 by preexposure to hypoxia (2% O(2); 6 h) or the hypoxia mimics CoCl(2) or dimethyloxalylglycine (DMOG). Oxidative barrier loss was associated with reduced levels of the tight junction protein occludin and with hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO(2)H), events that were also attenuated by prior activation of HIF-1. Involvement of HIF-1 in these protective effects was confirmed using the pharmacological inhibitor YC-1 and by short-hairpin RNA knockdown of HIF-1α. The protective effects of HIF-1 were associated with induction of sestrin-2, a hypoxia-inducible enzyme known to reduce oxidative stress and minimize Prx hyperoxidation. Together, our results suggest that loss of epithelial barrier integrity by oxidative stress is minimized by activation of HIF-1, in part by induction of sestrin-2.

Topics: Amino Acids, Dicarboxylic; Animals; Cell Hypoxia; Cell Line; Cobalt; Dextrans; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Gene Knockdown Techniques; Humans; Hydrogen Peroxide; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Proteins; Mice; Mice, Inbred C57BL; Nuclear Proteins; Occludin; Oxidants; Oxidation-Reduction; Oxidative Stress; Permeability; Peroxiredoxins; Primary Cell Culture; Procollagen-Proline Dioxygenase; Respiratory Mucosa; RNA Interference; Trachea

The 5/6(th) nephrectomy or ablation/infarction (A/I) preparation has been used as a classic model of chronic kidney disease (CKD). We observed increased kidney oxygen consumption (Q(O2)) and altered renal hemodynamics in the A/I kidney that were normalized after combined angiotensin II (ANG II) blockade. Studies suggest hypoxia inducible factor as a protective influence in A/I. We induced hypoxia-inducible factor (HIF) and HIF target proteins by two different methods, cobalt chloride (CoCl(2)) and dimethyloxalyglycine (DMOG), for the first week after creation of A/I and compared the metabolic and renal hemodynamic outcomes to combined ANG II blockade. We also examined the HIF target proteins expressed by using Western blots and real-time PCR. Treatment with DMOG, CoCl(2), and ANG II blockade normalized kidney oxygen consumption factored by Na reabsorption and increased both renal blood flow and glomerular filtration rate. At 1 wk, CoCl(2) and DMOG increased kidney expression of HIF by Western blot. In the untreated A/I kidney, VEGF, heme oxygenase-1, and GLUT1 were all modestly increased. Both ANG II blockade and CoCl(2) therapy increased VEGF and GLUT1 but the cobalt markedly so. ANG II blockade decreased heme oxygenase-1 expression while CoCl(2) increased it. By real-time PCR, erythropoietin and GLUT1 were only increased by CoCl(2) therapy. Cell proliferation was modestly increased by ANG II blockade but markedly after cobalt therapy. Metabolic and hemodynamic abnormalities were corrected equally by ANG II blockade and HIF therapies. However, the molecular patterns differed significantly between ANG II blockade and cobalt therapy. HIF induction may prove to be protective in this model of CKD.

Topics: Amino Acids, Dicarboxylic; Angiotensin II; Animals; Carbonic Anhydrase IX; Carbonic Anhydrases; Cobalt; Enzyme Induction; Glucose Transporter Type 1; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Kidney Failure, Chronic; Male; Proliferating Cell Nuclear Antigen; Rats; Rats, Wistar

Previous work showed that dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary artery hypertension in rat via targeting smooth muscle cells. In our study, DHEA was tested on human pulmonary arterial smooth muscle cells (HPASMC) to identify its mechanism of action under hypoxia in vitro. We show that DHEA decreased HIF-1alpha accumulation under both "chemical hypoxia" with treatment by the iron chelator deferroxamin and gas hypoxia (1% O2). The mRNA levels of HIF-1alpha were unchanged whether or not DHEA was applied under chemical and gas hypoxia, as compared to controls in normoxia, suggesting a post-transcriptional effect of the steroid. Protein levels of prolyl hydroxylases responsible for HIF-1alpha degradation were not modified by DHEA treatment. In addition, a synthetic derivative of DHEA, 3beta-methyl-Delta5-androsten-17-one (which cannot be metabolized), was as active as DHEA on HIF-1alpha accumulation, as well as testosterone and 17beta-estradiol (E2). In HPASMC cultures under normoxia and both types of hypoxia, DHEA gave rise to Delta5-androstene-3beta,17beta-diol (ADIOL) and DHEA-sulfate (DHEA-S). Neither testosterone, nor E2 were found. In addition, ADIOL, DHEA-S, 7alpha-hydroxy-DHEA and Delta4-androstene-3,17-dione were ineffective on HIF-1alpha accumulation. The effect of DHEA per se reducing HIF-1alpha accumulation may be relevant to reduced hypoxia effects in pulmonary arterial hypertension.

Topics: Amino Acids, Dicarboxylic; Base Sequence; Cells, Cultured; Cobalt; Deferoxamine; Dehydroepiandrosterone; DNA Primers; Enzyme Inhibitors; Estradiol; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Iron Chelating Agents; Models, Cardiovascular; Myocytes, Smooth Muscle; Procollagen-Proline Dioxygenase; Pulmonary Artery; Testosterone

Cobalt is suspected to cause memory deficit in humans and was reported to induce neurotoxicity in animal models. We have studied the effects of cobalt in primary cultures of mouse astrocytes. CoCl(2) (0.2-0.8mM) caused dose-dependent ATP depletion, apoptosis (cell shrinkage, phosphatidylserine externalization and chromatin rearrangements) and secondary necrosis. The mitochondria appeared to be a main target of cobalt toxicity, as shown by the loss of mitochondrial membrane potential (DeltaPsi(m)) and release from the mitochondria of apoptogenic factors, e.g. apoptosis inducing factor (AIF). Pre-treatment with bongkrekic acid reduced ATP depletion, implicating the involvement of the mitochondrial permeability transition (MPT) pore. Cobalt increased the generation of oxygen radicals, but antioxidants did not prevent toxicity. There was also an impaired response to ATP stimulation, evaluated as a lower raise in intracellular calcium. Similarly to hypoxia and dymethyloxallyl glycine (DMOG), cobalt triggered stabilization of the alpha-subunit of hypoxia-inducible factor HIF-1 (HIF-1alpha). This early event was followed by an increased expression of HIF-1 regulated genes, e.g. stress protein HO-1, pro-apoptotic factor Nip3 and iNOS. Although all of the three stimuli activated the HIF-1alpha pathway and decreased ATP levels, the downstream effects were different. DMOG only inhibited cell proliferation, whereas the other two conditions caused cell death by apoptosis and necrosis. This points to cobalt and hypoxia not only inducing HIF-1alpha regulated genes but also affecting similarly other cellular functions, including metabolism.

Topics: Amino Acids, Dicarboxylic; Animals; Astrocytes; Calcium Signaling; Cell Hypoxia; Cells, Cultured; Cobalt; Dose-Response Relationship, Drug; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Oxygen; Reactive Oxygen Species

Low oxygen stimulates pulmonary vascular development and airway branching and involves hypoxia-inducible factor (HIF). HIF is stable and initiates expression of angiogenic factors under hypoxia, whereas normoxia triggers hydroxylation of the HIF-1alpha subunit by prolyl hydroxylases (PHDs) and subsequent degradation. Herein, we investigated whether chemical stabilization of HIF-1alpha under normoxic (20% O(2)) conditions would stimulate vascular growth and branching morphogenesis in early lung explants. Tie2-LacZ (endothelial LacZ marker) mice were used for visualization of the vasculature. Embryonic day 11.5 (E11.5) lung buds were dissected and cultured in 20% O(2) in the absence or presence of cobalt chloride (CoCl(2), a hypoxia mimetic), dimethyloxalylglycine (DMOG; a nonspecific inhibitor of PHDs), or desferrioxamine (DFO; an iron chelator). Vascularization was assessed by X-gal staining, and terminal buds were counted. The fine vascular network surrounding the developing lung buds seen in control explants disappeared in CoCl(2)- and DFO-treated explants. Also, epithelial branching was reduced in the explants treated with CoCl(2) and DFO. In contrast, DMOG inhibited branching but stimulated vascularization. Both DFO and DMOG increased nuclear HIF-1alpha protein levels, whereas CoCl(2) had no effect. Since HIF-1alpha induces VEGF expression, the effect of SU-5416, a potent VEGF receptor (VEGFR) blocker, on early lung development was also investigated. Inhibition of VEGFR2 signaling in explants maintained under hypoxic (2% O(2)) conditions completely abolished vascularization and slightly decreased epithelial branching. Taken together, the data suggest that DMOG stabilization of HIF-1alpha during early development leads to a hypervascular lung and that airway branching proceeds without the vasculature, albeit at a slower rate.

Topics: Amino Acids, Dicarboxylic; Animals; Chlorides; Cobalt; Deferoxamine; Dose-Response Relationship, Drug; Embryo, Mammalian; Epithelial Cells; Ferric Compounds; Hypoxia-Inducible Factor 1; In Vitro Techniques; Indoles; Lung; Mice; Morphogenesis; Neovascularization, Physiologic; Pyrroles; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2

Topics: Amino Acids, Dicarboxylic; Animals; Cobalt; Deferoxamine; Female; Humans; Hypoxia-Inducible Factor 1; Lung; Mice; Neovascularization, Physiologic; Pregnancy; Premature Birth; Receptors, Vascular Endothelial Growth Factor

Apelin, a novel peptide with significant cardioactive properties, is upregulated by insulin in adipocytes. However, the mechanism by which insulin promotes apelin production is unknown. Hypoxia-inducible factor-1 (HIF-1), a heterodimeric transcription factor involved in the angiogenic and metabolic responses to tissue hypoxia, has been shown to be activated by insulin in various settings. We therefore hypothesized that HIF-1 regulates insulin-mediated apelin expression in adipocytes. 3T3-L1 cells were differentiated into adipocytes in culture. For experiments, serum-starved 3T3-L1 cells were exposed to insulin and/or a 1% O(2) environment. Apelin expression was assessed using quantitative real-time PCR and ELISA. To directly assess the role of HIF-1 in apelin production, we differentiated mouse embryonic fibroblasts (MEFs) containing a targeted deletion of the HIF-1alpha gene into adipocytes and measured their response to insulin and hypoxia. Apelin expression in mature 3T3-L1 adipocytes was increased significantly by insulin and was attenuated by pharmacological inhibition of insulin signaling. Exposure of cells to either hypoxia or the chemical HIF activators cobalt chloride (CoCl(2)) and dimethyloxaloylglycine (DMOG) resulted in significant upregulation of apelin, consistent with a role for HIF in apelin induction. Moreover, hypoxia-, CoCl(2)-, DMOG-, and insulin-induced apelin expression were all attenuated in differentiated HIF-1alpha-deficient MEFs. In summary, in cultured 3T3-L1 adipocytes and differentiated MEFs, HIF-1 appears to be involved in hypoxia- and insulin-induced apelin expression.

Topics: 3T3-L1 Cells; Active Transport, Cell Nucleus; Adipocytes; Adipokines; Amino Acids, Dicarboxylic; Androstadienes; Animals; Apelin; Carrier Proteins; Cell Hypoxia; Cell Line, Transformed; Cell Nucleus; Cobalt; Enzyme Inhibitors; Gene Deletion; Gene Expression Regulation; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Intercellular Signaling Peptides and Proteins; Mice; Proto-Oncogene Proteins c-akt; Rotenone; Wortmannin

Stabilization of the hypoxia-inducible factor-1 (HIF-1) protein is essential for its role as a regulator of gene expression under low oxygen conditions. Here, employing a novel hydroxylation-specific antibody, we directly show that proline 564 of HIF-1alpha and proline 531 of HIF-2alpha are hydroxylated under normoxia. Importantly, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 hydroxylation is diminished with the treatment of hypoxia, cobalt chloride, desferrioxamine, or dimethyloxalyglycine, regardless of the E3 ubiquitin ligase activity of the von Hippel-Lindau (VHL) tumor suppressor gene. Furthermore, in VHL-deficient cells, HIF-1alpha Pro-564 and HIF-2alpha Pro-531 had detectable amounts of hydroxylation following transition to hypoxia, indicating that the post-translational modification is not reversible. The introduction of v-Src or RasV12 oncogenes resulted in the stabilization of normoxic HIF-1alpha and the loss of hydroxylated Pro-564, demonstrating that oncogene-induced stabilization of HIF-1alpha is signaled through the inhibition of prolyl hydroxylation. Conversely, a constitutively active Akt oncogene stabilized HIF-1alpha under normoxia independently of prolyl hydroxylation, suggesting an alternative mechanism for HIF-1alpha stabilization. Thus, these results indicate distinct pathways for HIF-1alpha stabilization by different oncogenes. More importantly, these findings link oncogenesis with normoxic HIF-1alpha expression through prolyl hydroxylation.

Topics: Amino Acids, Dicarboxylic; Animals; Chelating Agents; Cobalt; Deferoxamine; Fibroblasts; Hydroxylation; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunoblotting; Iron; Luciferases; Mice; Mice, Knockout; Proline; Protein Binding; Transcription Factors; Transfection