Hypoxia-inducible Lipid Droplet-associated (HILPDA) Is a Novel Peroxisome Proliferator-activated Receptor (PPAR) Target Involved in Hepatic Triglyceride Secretion

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Mattijsen, F.; Georgiadi, A.; Andasarie, T.; Szalowska, E.; Zota, A.; Krones-Herzig, A.; Kersten, A.H.

2014-01-01

Peroxisome proliferator-activated receptors (PPARs) play major roles in the regulation of hepatic lipid metabolism through the control of numerous genes involved in processes such as lipid uptake and fatty acid oxidation. Here we identify hypoxia-inducible lipid droplet-associated (Hilpda/Hig2) as a

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

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Huai-Ren Chang

2018-04-01

Full Text Available Background/Aims: Intermittent hypoxia (IH has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA group. When we treated cardiomyocytes with Na+-H+ exchange (NHE inhibitors (Amiloride and HOE642 or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline, intracellular calcium chelator or Na+-Ca2+ exchange (NCX inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

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Xu Yan

2014-01-01

Full Text Available Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1 on hypoxia/ischemia (H/I injury in cardiomyocytes in vitro and the mitochondrial apoptotic pathway mediated mechanism. Methods. Neonatal rat cardiomyocytes (NRCMs for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit. Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm. Its administration also inhibited activities of caspase-9 and caspase-3. Conclusion. Administration of GS-Rb1 during H/I in vitro is involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

Down-regulation of hypoxia-inducible factor-1 alpha and vascular endothelial growth factor by HEXIM1 attenuates myocardial angiogenesis in hypoxic mice.

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Yoshikawa, Noritada; Shimizu, Noriaki; Ojima, Hidenori; Kobayashi, Hiroshi; Hosono, Osamu; Tanaka, Hirotoshi

2014-10-24

Pulmonary hypertension (PH) sustains elevation of pulmonary vascular resistance and ultimately leads to right ventricular (RV) hypertrophy and failure and death. Recently, proangiogenic factors hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) have been known to promote left ventricular myocardial angiogenesis and lead to cardiac hypertrophy, and this would be involved in RV hypertrophy of PH patients. Previously, we revealed that overexpression of HEXIM1 prevents endothelin-1-induced cardiomyocyte hypertrophy and hypertrophic genes expression, and that cardiomyocyte-specific HEXIM1 transgenic mice ameliorates RV hypertrophy in hypoxia-induced PH model. Given these results, here we analyzed the effect of HEXIM1 on the expression of HIF-1α and VEGF and on myocardial angiogenesis of RV in PH. We revealed that overexpression of HEXIM1 prevented hypoxia-induced expression of HIF-1α protein and its target genes including VEGF in the cultured cardiac myocytes and fibroblasts, and that cardiomyocyte-specific HEXIM1 transgenic mice repressed RV myocardial angiogenesis in hypoxia-induced PH model. Thus, we conclude that HEXIM1 could prevent RV hypertrophy, at least in part, via suppression of myocardial angiogenesis through down-regulation of HIF-1α and VEGF in the myocardium under hypoxic condition. Copyright © 2014 Elsevier Inc. All rights reserved.

Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

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Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Yokokawa, Takumi; Endo, Yuriko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Iwanaka, Nobumasa [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Higashida, Kazuhiko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 (Japan); Taguchi, Sadayoshi [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)

2013-10-11

Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

International Nuclear Information System (INIS)

Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Taguchi, Sadayoshi

2013-01-01

Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O 2 for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

Protective effect of pomegranate seed oil against H2O2 -induced oxidative stress in cardiomyocytes

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Mehdi Bihamta

2017-01-01

Full Text Available Objective: It has been well documented that oxidative stress is involved in the pathogenesis of cardiac diseases. Previous studies have shown that pomegranate seed oil (PSO has antioxidant properties. This study was designed to investigate probable protective effects of PSO against hydrogen peroxide (H2O2-induced damage in H9c2 cardiomyocytes.Materials and Methods: The cells were pretreated 24 hr with PSO 1 hr before exposure to 200 µM H2O2. Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT assay. The level of reactive oxygen species (ROS and lipid peroxidation were measured by fluorimetric methods.Results: H2O2 significantly decreased cell viability which was accompanied by an increase in ROS production and lipid peroxidation and a decline in superoxide dismutase activity. Pretreatment with PSO increased viability of cardiomyocytes and decrease the elevated ROS production and lipid peroxidation. Also, PSO was able to restore superoxide dismutase activity.Conclusion: PSO has protective effect against oxidative stress-induced damage in cardiomyocytes and can be considered as a natural cardioprotective agent to prevent cardiovascular diseases.

The protective effect of lycopene on hypoxia/reoxygenation-induced endoplasmic reticulum stress in H9C2 cardiomyocytes.

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Gao, Yang; Jia, Pengyu; Shu, WenQi; Jia, Dalin

2016-03-05

Nowadays, drugs protecting ischemia/reperfusion (I/R) myocardium become more suitable for clinic. It has been confirmed lycopene has various protections, but lacking the observation of its effect on endoplasmic reticulum stress (ERS)-mediated apoptosis caused by hypoxia/reoxygenation (H/R). This study aims to clarify the protective effect of lycopene on ERS induced by H/R in H9C2 cardiomyocytes. Detect the survival rate, lactic dehydrogenase (LDH) activity, apoptosis ratio, glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP), c-Jun-N-terminal protein Kinase (JNK) and Caspase-12 mRNA and protein expression and phosphorylation of JNK (p-JNK) protein expression. LDH activity, apoptosis ratio and GRP78 protein expression increase in the H/R group, reduced by lycopene. The survival rate reduces in the H/R and thapsigargin (TG) groups; lycopene and 4-phenyl butyric acid (4-PBA) can improve it caused by H/R, lycopene also can improve it caused by TG. The apoptosis ratio, the expression of GRP78, CHOP and Caspase-12 mRNA and protein and p-JNK protein increase in the H/R and TG groups, weaken in the lycopene+H/R, 4-PBA+H/R and lycopene+TG groups. There is no obvious change in the expression of JNK mRNA or protein. Hence, our results provide the evidence that 10 μM lycopene plays an obviously protective effect on H/R H9C2 cardiomyocytes, realized through reducing ERS and apoptosis. The possible mechanism may be related to CHOP, p-JNK and Caspase-12 pathways. Copyright © 2016. Published by Elsevier B.V.

Antihypertrophic Effects of Small Molecules that Maintain Mitochondrial ATP Levels Under Hypoxia

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Hiroaki Nagai

2017-10-01

Full Text Available Since impaired mitochondrial ATP production in cardiomyocytes is thought to lead to heart failure, a drug that protects mitochondria and improves ATP production under disease conditions would be an attractive treatment option. In this study, we identified small-molecule drugs, including the anti-parasitic agent, ivermectin, that maintain mitochondrial ATP levels under hypoxia in cardiomyocytes. Mechanistically, transcriptomic analysis and gene silencing experiments revealed that ivermectin increased mitochondrial ATP production by inducing Cox6a2, a subunit of the mitochondrial respiratory chain. Furthermore, ivermectin inhibited the hypertrophic response of human induced pluripotent stem cell-derived cardiomyocytes. Pharmacological inhibition of importin β, one of the targets of ivermectin, exhibited protection against mitochondrial ATP decline and cardiomyocyte hypertrophy. These findings indicate that maintaining mitochondrial ATP under hypoxia may prevent hypertrophy and improve cardiac function, providing therapeutic options for mitochondrial dysfunction.

NanoSIMS Analysis of Intravascular Lipolysis and Lipid Movement across Capillaries and into Cardiomyocytes

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He, Cuiwen; Weston, Thomas A; Jung, Rachel S

2018-01-01

, mice were given an injection of [2H]triglyceride-enriched TRLs, and the movement of 2H-labeled lipids across capillaries and into cardiomyocytes was examined by NanoSIMS. TRL processing and lipid movement in tissues were extremely rapid. Within 30 s, TRL-derived lipids appeared in the subendothelial...

Lipid peroxidation in neonatal mouse brain subjected to two different types of hypoxia.

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Hasegawa, K; Yoshioka, H; Sawada, T; Nishikawa, H

1991-01-01

To elucidate the role of free radicals in the pathogenesis of neonatal hypoxic encephalopathy, we determined the content of thiobarbituric acid reactants (TBARs), as an index of lipid peroxidation related with a free radical reaction, in the brains of newborn mice during hypoxia and recovery from hypoxia. Hypoxic stress was induced by 100% nitrogen gas breathing (N2 group) or 100% carbon dioxide gas breathing (CO2 group). TBARs increased with 20 minutes of hypoxia and returned to the control level during the recovery period in both groups. The increase in TBARs in the CO2 group was greater than that in the N2 group. These results may suggest that free radical reaction occurs during the hypoxic period and that CO2 hypoxia is more effective on free radical production in the newborn brain than N2 hypoxia.

The protective effect of lipid emulsion in preventing bupivacaine-induced mitochondrial injury and apoptosis of H9C2 cardiomyocytes.

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Chen, Zhe; Jin, Zhousheng; Xia, Yun; Zhao, Shishi; Xu, Xuzhong; Papadimos, Thomas J; Wang, Quanguang

2017-11-01

Lipid emulsion (LE) has been shown to be effective in the resuscitation of bupivacaine-induced cardiac arrest, but the precise mechanism of this action has not been fully elucidated. Pursuant to this lack of information on the mechanism in which LE protects the myocardium during bupivacaine-induced toxicity, we explored mitochondrial function and cell apoptosis. H9C2 cardiomyocytes were used in study. Cells were randomly divided in different groups and were cultivated 6 h, 12 h, and 24 h. The mitochondria were extracted and mitochondrial ATP content was measured, as was mitochondrial membrane potential, the concentration of calcium ion (Ca2+), and the activity of Ca2+-ATP enzyme (Ca2+-ATPase). Cells from groups Bup1000, LE group, and Bup1000LE were collected to determine cell viability, cell apoptosis, and electron microscopy scanning of mitochondrial ultrastructure (after 24 h). We found that LE can reverse the inhibition of the mitochondrial function induced by bupivacaine, regulate the concentration of calcium ion in mitochondria, resulting in the protection of myocardial cells from toxicity induced by bupivacaine.

Attenuation of ischemia-reperfusion-induced alterations in intracellular Ca2+ in cardiomyocytes from hearts treated with N-acetylcysteine and N-mercaptopropionylglycine.

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Saini-Chohan, Harjot K; Dhalla, Naranjan S

2009-12-01

This study was undertaken to test whether Ca(2+)-handling abnormalities in cardiomyocytes after ischemia-reperfusion (I/R) are prevented by antioxidants such as N-acetyl L-cysteine (NAC), which is known to reduce oxidative stress by increasing the glutathione redox status, and N-(2-mercaptopropionyl)-glycine (MPG), which scavenges both peroxynitrite and hydroxyl radicals. For this purpose, isolated rat hearts were subjected to 30 min of global ischemia followed by 30 min of reperfusion, and cardiomyocytes were prepared to monitor changes in the intracellular concentration of free Ca(2+) ([Ca(2+)](i)). Marked depression in the left ventricular developed pressure and elevation in the left ventricular end-diastolic pressure in I/R hearts were attenuated by treatment with NAC or MPG. Cardiomyocytes obtained from I/R hearts showed an increase in the basal level of [Ca(2+)](i) as well as augmentation of the low Na(+)-induced increase in [Ca(2+)](i), with no change in the KCl-induced increase in [Ca(2+)](i). These I/R-induced alterations in Ca(2+) handling by cardiomyocytes were attenuated by treatment of hearts with NAC or MPG. Furthermore, reduction in the isoproterenol-, ATP-, ouabain-, and caffeine-induced increases in [Ca(2+)](i) in cardiomyocytes from I/R hearts were limited by treatment with NAC or MPG. The increases in the basal [Ca(2+)](i), unlike the KCl-induced increase in [Ca(2+)](i), were fully or partially prevented by both NAC and MPG upon exposing cardiomyocytes to hypoxia-reoxygenation, H(2)O(2), or a mixture of xanthine and xanthine oxidase. These results suggest that improvement in cardiac function of I/R hearts treated with NAC or MPG was associated with attenuation of changes in Ca(2+) handling by cardiomyocytes, and the results support the view that oxidative stress due to oxyradical generation and peroxynitrite formation plays an important role in the development of intracellular Ca(2+) overload in cardiomyocytes as a consequence of I/R injury.

LncRNA TUG1 serves an important role in hypoxia-induced myocardial cell injury by regulating the miR-145-5p-Binp3 axis

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Wu, Zhongwei; Zhao, Shengji; Li, Chunfu; Liu, Chaoquan

2018-01-01

The aim of the present study was to investigate the function of long non-coding RNA TUG1 in hypoxia-induced myocardial cell injury and to explore the potential molecular mechanisms. The cardiomyocyte cell line H9c2 was cultured under hypoxic and normoxic conditions. TUG1 expression under hypoxic conditions was then detected. The effects of TUG1 overexpression on viability, apoptosis, migration and invasion were assayed. In addition, the microRNA (miR)-145-5p expression was detected. Following H9c2 cell transfection with miR-145-5p mimics, the H9c2 cell viability, apoptosis, migration and invasion were also detected. Additionally, the target gene of miR-145-5p was assayed by Luciferase reporter assay. The protein expressions of Wnt-3a, Wnt5a, and β-catenin in H9c2 cells under hypoxic conditions were also determined. The results revealed that hypoxia induced injury in H9c2 cells, including inhibiting cell viability, migration and invasion, and promoting cell apoptosis. Overexpression of TUG1 aggravated hypoxia-induced injury in H9c2 cells. In addition, miR-145-5p was negatively regulated by TUG1, and TUG1 overexpression aggravated hypoxia-induced injury via the downregulation of miR-145-5p. Furthermore, B-cell lymphoma 2 interacting protein 3 (Bnip3) was a target of miR-145-5p, and overexpression of Bnip3 aggravated hypoxia-induced cell injury by activating Wnt/β-catenin signaling pathways in H9c2 cells. In conclusion, overexpression of TUG1 aggravated hypoxia-induced injury in cardiomyocytes by regulating the miR-145-5p-Binp3 axis. Activation of the Wnt/β-catenin signaling pathway may be a key mechanism to mediate the role of TUG1 in regulating hypoxia-induced myocardial injury. TUG1 may be an effective diagnostic marker and therapeutic target for myocardial ischemia. PMID:29207102

Cardiomyocyte Overexpression of FABP4 Aggravates Pressure Overload-Induced Heart Hypertrophy.

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Ji Zhang

Full Text Available Fatty acid binding protein 4 (FABP4 is a member of the intracellular lipid-binding protein family, responsible for the transportation of fatty acids. It is considered to express mainly in adipose tissues, and be strongly associated with inflammation, obesity, diabetes and cardiovasculardiseases. Here we report that FABP4 is also expressed in cardiomyocytes and plays an important role in regulating heart function under pressure overload. We generated heart-specific transgenic FABP4 (FABP4-TG mice using α myosin-heavy chain (α-MHC promoter and human FABP4 sequence, resulting in over-expression of FABP4 in cardiomyocytes. The FABP4-TG mice displayed normal cardiac morphology and contractile function. When they were subjected to the transverse aorta constriction (TAC procedure, the FABP4-TG mice developed more cardiac hypertrophy correlated with significantly increased ERK phosphorylation, compared with wild type controls. FABP4 over-expression in cardiomyocytes activated phosphor-ERK signal and up-regulate the expression of cardiac hypertrophic marker genes. Conversely, FABP4 induced phosphor-ERK signal and hypertrophic gene expressions can be markedly inhibited by an ERK inhibitor PD098059 as well as the FABP4 inhibitor BMS309403. These results suggest that FABP4 over-expression in cardiomyocytes can aggravate the development of cardiac hypertrophy through the activation of ERK signal pathway.

Hypoxia-induced lipid peroxidation in rat brain and protective effect of carnitine and phosphocreatine

Czech Academy of Sciences Publication Activity Database

Rauchová, Hana; Koudelová, J.; Drahota, Zdeněk; Mourek, J.

2002-01-01

Roč. 27, č. 9 (2002), s. 899-904 ISSN 0364-3190 R&D Projects: GA MŠk LN00A069 Grant - others:GA UK(XC) 22/2001 Institutional research plan: CEZ:AV0Z5011922 Keywords : hypobaric hypoxia-lipid peroxidation * carnitine * lactate/pyruvate ratio Subject RIV: ED - Physiology Impact factor: 1.672, year: 2002

Glucocorticoid Induced Leucine Zipper inhibits apoptosis of cardiomyocytes by doxorubicin

International Nuclear Information System (INIS)

Aguilar, David; Strom, Joshua; Chen, Qin M.

2014-01-01

Doxorubicin (Dox) is an indispensable chemotherapeutic agent for the treatment of various forms of neoplasia such as lung, breast, ovarian, and bladder cancers. Cardiotoxicity is a major concern for patients receiving Dox therapy. Previous work from our laboratory indicated that glucocorticoids (GCs) alleviate Dox-induced apoptosis in cardiomyocytes. Here we have found Glucocorticoid-Induced Leucine Zipper (GILZ) to be a mediator of GC-induced cytoprotection. GILZ was found to be induced in cardiomyocytes by GC treatment. Knocking down of GILZ using siRNA resulted in cancelation of GC-induced cytoprotection against apoptosis by Dox treatment. Overexpressing GILZ by transfection was able to protect cells from apoptosis induced by Dox as measured by caspase activation, Annexin V binding and morphologic changes. Western blot analyses indicate that GILZ overexpression prevented cytochrome c release from mitochondria and cleavage of caspase-3. When bcl-2 family proteins were examined, we found that GILZ overexpression causes induction of the pro-survival protein Bcl-xL. Since siRNA against Bcl-xL reverses GC induced cytoprotection, Bcl-xL induction represents an important event in GILZ-induced cytoprotection. Our data suggest that GILZ functions as a cytoprotective gene in cardiomyocytes. - Highlights: • Corticosteroids act as a cytoprotective agent in cardiomyocytes • Corticosteroids induce GILZ expression in cardiomyocytes • Elevated GILZ results in resistance against apoptosis induced by doxorubicin • GILZ induces Bcl-xL protein without inducing Bcl-xL mRNA

Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

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Wu Yihe

2012-04-01

Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

Overexpression of Hypoxia-Inducible Factor-1α Exacerbates Endothelial Barrier Dysfunction Induced by Hypoxia

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Pei Wang

2013-09-01

Full Text Available Background/Aims: The mechanisms involved in endothelial barrier dysfunction induced by hypoxia are incompletely understood. There is debate about the role of hypoxia-inducible factor-1α (HIF-1α in endothelial barrier disruption. The aim of this study was to investigate the effect of genetic overexpression of HIF-1α on barrier function and the underlying mechanisms in hypoxic endothelial cells. Methods: The plasmid pcDNA3.1/V5-His-HIF-1α was stably transfected into human endothelial cells. The cells were exposed to normoxia or hypoxia. The mRNA and protein expressions of HIF-1α were detected by RT-PCR and Western blot respectively. The barrier function was assessed by measuring the transendothelial electrical resistance (TER. The Western blot analysis was used to determine the protein expression of glucose transporter-1 (GLUT-1, zonular occludens-1 (ZO-1, occludin, and myosin light chain kinase (MLCK in endothelial cells. The mRNA expression of proinflammatory cytokines was detected by qRT-PCR. Results: Genetic overexpression of HIF-1α significantly increased the mRNA and protein expression of HIF-1α in endothelial cells. The overexpression of HIF-1α enhanced the hypoxia-induced increase of HIF-1α and GLUT-1 protein expression. HIF-1α overexpression not only exacerbated hypoxia-induced endothelial barrier dysfunction but also augmented hypoxia-induced up-regulation of MLCK protein expression. HIF-1α overexpression also enhanced IL-1β, IL-6 and TNF-α mRNA expression. Conclusion: We provide evidence that genetic overexpression of HIF-1α aggravates the hypoxia-induced endothelial barrier dysfunction via enhancing the up-regulation of MLCK protein expression caused by hypoxia, suggesting a potential role for HIF-1α in the pathogenesis of endothelial barrier dysfunction in hypoxia.

Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

Science.gov (United States)

Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

2013-08-01

Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

Migraine induced by hypoxia

DEFF Research Database (Denmark)

Arngrim, Nanna; Schytz, Henrik Winther; Britze, Josefine

2016-01-01

in the visual cortex were measured by proton magnetic resonance spectroscopy. The circumference of cranial arteries was measured by 3 T high-resolution magnetic resonance angiography. Hypoxia induced migraine-like attacks in eight patients compared to one patient after sham (P = 0.039), aura in three...... and possible aura in 4 of 15 patients. Hypoxia did not change glutamate concentration in the visual cortex compared to sham, but increased lactate concentration (P = 0.028) and circumference of the cranial arteries (P ... suggests that hypoxia may provoke migraine headache and aura symptoms in some patients. The mechanisms behind the migraine-inducing effect of hypoxia should be further investigated....

The role of hypoxia inducible factor 1 (HIF-1) in hypoxia induced apoptosis

NARCIS (Netherlands)

Greijer, A.E.; Wall, E. van der

2004-01-01

Apoptosis can be induced in response to hypoxia. The severity of hypoxia determines whether cells become apoptotic or adapt to hypoxia and survive. A hypoxic environment devoid of nutrients prevents the cell undergoing energy dependent apoptosis and cells become necrotic. Apoptosis regulatory

Hypoxia and oxidation levels of DNA and lipids in humans and animal experimental models

DEFF Research Database (Denmark)

Møller, Peter; Risom, Lotte; Lundby, Carsten

2008-01-01

The objective of this review was to evaluate the association between hypoxia and oxidative damage to DNA and lipids. Evaluation criteria encompassed specificity and validation status of the biomarkers, study design, strength of the association, dose-response relationship, biological plausibility......, analogous exposures, and effect modification by intervention. The collective interpretation indicates persuasive evidence from the studies in humans for an association between hypoxia and elevated levels of oxidative damage to DNA and lipids. The levels of oxidatively generated DNA lesions and lipid...... in subjects at high altitude. Most of the animal experimental models should be interpreted with caution because the assays for assessment of lipid peroxidation products have suboptimal validity....

Protection of Pentoxifylline against Testis Injury Induced by Intermittent Hypobaric Hypoxia

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Chen Yao

2016-01-01

Full Text Available To investigate the effect of pentoxifylline (PTX on spermatogenesis dysfunction induced by intermittent hypobaric hypoxia (IHH and unveil the underlying mechanism, experimental animals were assigned to Control, IHH+Vehicle, and IHH+PTX groups and exposed to 4 cycles of 96 h of hypobaric hypoxia followed by 96 h of normobaric normoxia for 32 days. PTX was administered for 32 days. Blood and tissue samples were collected 7 days thereafter. Serum malondialdehyde levels were used to assess lipid peroxidation; ferric-reducing antioxidant power (FRAP, superoxide dismutase, and catalase and glutathione peroxidase enzyme activities were assessed to determine antioxidant capacity in various samples. Testis histopathology was assessed after hematoxylin-eosin staining by Johnsen’s testicular scoring system. Meanwhile, testosterone synthase and vimentin amounts were assessed by immunohistochemistry. Sperm count, motility, and density were assessed to determine epididymal sperm quality. IHH treatment induced significant pathological changes in testicular tissue and enhanced serum lipid peroxide levels, while reducing serum FRAP, antioxidant enzyme activities, and testosterone synthase expression. Moreover, IHH impaired epididymal sperm quality and vimentin structure in Sertoli cells. Oral administration of PTX improved the pathological changes in the testis. IHH may impair spermatogenesis function of testicular tissues by inducing oxidative stress, but this impairment could be attenuated by administration of PTX.

ALDH2 Inhibition Potentiates High Glucose Stress-Induced Injury in Cultured Cardiomyocytes

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Guodong Pan

2016-01-01

Full Text Available Aldehyde dehydrogenase (ALDH gene superfamily consists of 19 isozymes. They are present in various organs and involved in metabolizing aldehydes that are biologically generated. For instance, ALDH2, a cardiac mitochondrial ALDH isozyme, is known to detoxify 4-hydroxy-2-nonenal, a reactive aldehyde produced upon lipid peroxidation in diabetic conditions. We hypothesized that inhibition of ALDH leads to the accumulation of unmetabolized 4HNE and consequently exacerbates injury in cells subjected to high glucose stress. H9C2 cardiomyocyte cell lines were pretreated with 10 μM disulfiram (DSF, an inhibitor of ALDH2 or vehicle (DMSO for 2 hours, and then subjected to high glucose stress {33 mM D-glucose (HG or 33 mM D-mannitol as an osmotic control (Ctrl} for 24 hrs. The decrease in ALDH2 activity with DSF pretreatment was higher in HG group when compared to Ctrl group. Increased 4HNE adduct formation with DSF pretreatment was higher in HG group compared to Ctrl group. Pretreatment with DSF leads to potentiated HG-induced cell death in cultured H9C2 cardiomyocytes by lowering mitochondrial membrane potential. Our results indicate that ALDH2 activity is important in preventing high glucose induced cellular dysfunction.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

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Chen, Rui; Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping; Liu, Chao; Lin, Zhoumeng; Xie, Wei-Bing; Wang, Huijun

2016-01-01

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

DNA damage-inducible transcript 4 (DDIT4) mediates methamphetamine-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes

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Chen, Rui [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Department of Forensic Medicine, Guangdong Medical University, Dongguan 523808 (China); Wang, Bin; Chen, Ling; Cai, Dunpeng; Li, Bing; Chen, Chuanxiang; Huang, Enping [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Liu, Chao [Guangzhou Forensic Science Institute, Guangzhou 510030 (China); Lin, Zhoumeng [Institute of Computational Comparative Medicine and Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506 (United States); Xie, Wei-Bing, E-mail: xieweib@126.com [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Wang, Huijun, E-mail: hjwang711@yahoo.cn [Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China)

2016-03-15

Methamphetamine (METH) is an amphetamine-like psychostimulant that is commonly abused. Previous studies have shown that METH can induce damages to the nervous system and recent studies suggest that METH can also cause adverse and potentially lethal effects on the cardiovascular system. Recently, we demonstrated that DNA damage-inducible transcript 4 (DDIT4) regulates METH-induced neurotoxicity. However, the role of DDIT4 in METH-induced cardiotoxicity remains unknown. We hypothesized that DDIT4 may mediate METH-induced autophagy and apoptosis in cardiomyocytes. To test the hypothesis, we examined DDIT4 protein expression in cardiomyocytes and in heart tissues of rats exposed to METH with Western blotting. We also determined the effects on METH-induced autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes using Western blot analysis, fluorescence microscopy and TUNEL staining. Our results showed that METH exposure increased DDIT4 expression and decreased phosphorylation of mTOR that was accompanied with increased autophagy and apoptosis both in vitro and in vivo. These effects were normalized after silencing DDIT4. On the other hand, rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes. These results suggest that DDIT4 mediates METH-induced autophagy and apoptosis through mTOR signaling pathway in cardiomyocytes. - Highlights: • METH exposure increases DDIT4 expression in cardiomyocytes. • DDIT4 mediates METH-induced autophagy and apoptosis in cardiomyocytes. • DDIT4 silencing protects cardiomyocytes against METH-caused autophagy and apoptosis.

The Adipokine Chemerin Induces Apoptosis in Cardiomyocytes

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Diego Rodríguez-Penas

2015-08-01

Full Text Available Background: The adipokine chemerin has been associated with cardiovascular disease. We investigated the effects of chemerin on viability and intracellular signalling in murine cardiomyocytes, and the effects of insulin and TNF-α on cardiomyocyte chemerin production. Methods: Hoechst dye vital staining and cell cycle analysis were used to analyse the viability of murine cardiac cells in culture. Western blot was used to explore the phosphorylation of AKT and caspase-9 activity in neonatal rat cardiomyocytes and HL-1 cells. Finally, RT-qPCR, ELISA and western blot were performed to examine chemerin and CMKLR1 expression after insulin and TNF-α treatment in cardiac cells. Results: Chemerin treatment increased apoptosis, reduced phosphorylation of AKT at Thr308 and increased caspase-9 activity in murine cardiomyocytes. Insulin treatment lowered chemerin and CMKLR1 mRNA and protein levels, and the amount of chemerin in the cell media, while TNF-α treatment increased chemerin mRNA and protein levels but decreased expression of the CMKLR1 gene. Conclusion: Chemerin induces apoptosis, reduces AKT phosphorylation and increases the cleavage of caspase-9 in murine cardiomyocytes. The expression of chemerin is regulated by important metabolic (insulin and inflammatory (TNF-α mediators at cardiac level. Our results suggest that chemerin could play a role in the physiopathology of cardiac diseases.

Cardiomyocyte Regeneration

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Toshio Nakanishi

2013-01-01

Full Text Available The heart was initially believed to be a terminally differentiated organ; once the cardiomyocytes died, no recovery could be made to replace the dead cells. However, around a decade ago, the concept of cardiac stem cells (CSCs in adult hearts was proposed. CSCs differentiate into cardiomyocytes, keeping the heart functioning. Studies have proved the existence of stem cells in the heart. These somatic stem cells have been studied for use in cardiac regeneration. Moreover, recently, induced pluripotent stem cells (iPSCs were invented, and methodologies have now been developed to induce stable cardiomyocyte differentiation and purification of mature cardiomyocytes. A reprogramming method has also been applied to direct reprogramming using cardiac fibroblasts into cardiomyocytes. Here, we address cardiomyocyte differentiation of CSCs and iPSCs. Furthermore, we describe the potential of CSCs in regenerative biology and regenerative medicine.

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells.

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Jeon, Daun; Park, Heon Joo; Kim, Hong Seok

2018-01-01

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial-mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

Exercise attenuates intermittent hypoxia-induced cardiac fibrosis associated with sodium-hydrogen exchanger-1 in rats

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Tsung-I Chen

2016-10-01

Full Text Available Purpose: To investigate the role of sodium–hydrogen exchanger-1 (NHE-1 and exercise training on intermittent hypoxia-induced cardiac fibrosis in obstructive sleep apnea (OSA, using an animal model mimicking the intermittent hypoxia of OSA. Methods: Eight-week-old male Sprague–Dawley rats were randomly assigned to control (CON, intermittent hypoxia (IH, exercise (EXE or IH combined with exercise (IHEXE groups. These groups were randomly assigned to subgroups receiving either a vehicle or the NHE-1 inhibitor cariporide. The EXE and IHEXE rats underwent exercise training on an animal treadmill for 10 weeks (5 days/week, 60 minutes/day, 24–30 m/minute, 2–10% grade. The IH and IHEXE rats were exposed to 14 days of IH (30 seconds of hypoxia - nadir of 2-6% O2 - followed by 45 seconds of normoxia for 8 hours/day. At the end of 10 weeks, rats were sacrificed and then hearts were removed to determine the myocardial levels of fibrosis index, oxidative stress, antioxidant capacity and NHE-1 activation. Results: Compared to the CON rats, IH induced higher cardiac fibrosis, lower myocardial catalase and superoxidative dismutase activities, higher myocardial lipid and protein peroxidation and higher NHE-1 activation (p < 0.05 for each, which were all abolished by cariporide. Compared to the IH rats, lower cardiac fibrosis, higher myocardial antioxidant capacity, lower myocardial lipid and protein peroxidation and lower NHE-1 activation were found in the IHEXE rats (p < 0.05 for each. Conclusion: IH-induced cardiac fibrosis was associated with NHE-1 hyperactivity. However, exercise training and cariporide exerted an inhibitory effect to prevent myocardial NHE-1 hyperactivity, which contributed to reduced IH-induced cardiac fibrosis. Therefore, NHE-1 plays a critical role in the effect of exercise on IH-induced increased cardiac fibrosis.

Impact of miR-208 and its Target Gene Nemo-Like Kinase on the Protective Effect of Ginsenoside Rb1 in Hypoxia/Ischemia Injuried Cardiomyocytes

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Xu Yan

2016-09-01

Full Text Available Background/Aims: Ginsenoside Rb1 (GS-Rb1 is one of the most important active pharmacological extracts of the Traditional Chinese Medicine ginseng, with extensive evidence of its cardioprotective properties. Mir-208 has been shown to act as a biomarker of acute myocardial infarction in vivo studies including man. However the impact of miR-208 on the protective effect of GS-Rb1 in hypoxia/ischemia injured cardiomyocytes remains unclear. The current study aims to investigate the target gene of miR-208 and the impact on the protective effect of GS-Rb1 in hypoxia/ischemia (H/I injuried cardiomyocytes. Materials and Methods: Primary cultures of neonatal rat cardiomyocytes (NRCMs was subjected to the H/I conditions with or without GS-Rb1. Cell viability was calculated by MTT assay and confirmed by flow cytometry analysis. Mir-208 was then detected by qRT-PCR. Luciferase reporter assay was carried out to detect the target gene of Mir-208. Then the NRCMs were transfected with miR-208 mimics and inhibitors to evaluate the impact on cardioprotective properties of Rb1. Results: The miR-208 expression level was clearly upregulated in the H/I treated NRCMs accompanied by the percentage of the apoptotic cells which could be reversed by GS-Rb1 pretreatment. The nemo-like kinase (NLK mRNA and protein expression levels were decreased in H/I group measured by RT-PCR and western blotting. Luciferase activity assay was then carried out to identify that NLK may be a direct target of mir-208. MTT assay showed that miR-208 inhibitor slightly decreased the protective effect of Rb1 on the H/I impaired NRCMs. However, results showed no statistical difference. Conclusions: These findings proved that NLK was a direct target of mir-208 and miR-208 act indirectly during Rb1 protecting H/I impaired NRCMs and further researches were needed to explore the relationship that microRNAs and other signal pathways in the protective effect of GS-Rb1 on the hypoxia/ischemia injuries in

Suppression of the expression of hypoxia-inducible factor-1α by RNA interference alleviates hypoxia-induced pulmonary hypertension in adult rats.

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Li, Ying; Shi, Bo; Huang, Liping; Wang, Xin; Yu, Xiaona; Guo, Baosheng; Ren, Weidong

2016-12-01

Hypoxia-inducible factor-1α (HIF-1α) has been implicated in the pathogenesis of hypoxic pulmonary hypertension (PH). However, the potential clinical value of HIF-1α as a therapeutic target in the treatment of PH has not yet been evaluated. In this study, an animal model of hypoxia-induced PH was established by exposing adult rats to 10% O2 for 3 weeks, and the effects of the lentivirus-mediated delivery of HIF-1α short hairpin RNA (shRNA) by intratracheal instillation prior to exposure to hypoxia on the manifestations of hypoxia-induced PH were assessed. The successful delivery of HIF-1α shRNA into the pulmonary arteries effectively suppressed the hypoxia-induced upregulation of HIF-1α, accompanied by the prominent attenuation the symptoms associated with hypoxia-induced PH, including the elevation of pulmonary arterial pressure, hypertrophy and hyperplasia of pulmonary artery smooth muscle cells (PASMCs), as well as the muscularization of pulmonary arterioles. In addition, the knockdown of HIF-1α in cultured rat primary PASMCs significantly inhibited the hypoxia-induced acceleration of the cell cycle and the proliferation of the PASMCs, suggesting that HIF-1α may be a direct mediator of PASMC hyperplasia in hypoxia-induced PH. In conclusion, this study demonstrates the potent suppressive effects of HIF-1α shRNA on hypoxia-induced PH and PASMC hyperplasia, providing evidence for the potential application of HIF-1α shRNA in the treatment of hypoxic PH.

Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

LENUS (Irish Health Repository)

Biniecka, Monika

2012-02-01

OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

Microtubular stability affects pVHL-mediated regulation of HIF-1alpha via the p38/MAPK pathway in hypoxic cardiomyocytes.

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Miao Teng

Full Text Available BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL, as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4 overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia

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Minamino, Tohru; Christou, Helen; Hsieh, Chung-Ming; Liu, Yuxiang; Dhawan, Vijender; Abraham, Nader G.; Perrella, Mark A.; Mitsialis, S. Alex; Kourembanas, Stella

2001-07-01

Chronic hypoxia causes pulmonary hypertension with smooth muscle cell proliferation and matrix deposition in the wall of the pulmonary arterioles. We demonstrate here that hypoxia also induces a pronounced inflammation in the lung before the structural changes of the vessel wall. The proinflammatory action of hypoxia is mediated by the induction of distinct cytokines and chemokines and is independent of tumor necrosis factor- signaling. We have previously proposed a crucial role for heme oxygenase-1 (HO-1) in protecting cardiomyocytes from hypoxic stress, and potent anti-inflammatory properties of HO-1 have been reported in models of tissue injury. We thus established transgenic mice that constitutively express HO-1 in the lung and exposed them to chronic hypoxia. HO-1 transgenic mice were protected from the development of both pulmonary inflammation as well as hypertension and vessel wall hypertrophy induced by hypoxia. Significantly, the hypoxic induction of proinflammatory cytokines and chemokines was suppressed in HO-1 transgenic mice. Our findings suggest an important protective function of enzymatic products of HO-1 activity as inhibitors of hypoxia-induced vasoconstrictive and proinflammatory pathways.

Mesenchymal Stem Cells Respond to Hypoxia by Increasing Diacylglycerols.

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Lakatos, Kinga; Kalomoiris, Stefanos; Merkely, Béla; Nolta, Jan A; Fierro, Fernando A

2016-02-01

Mesenchymal stem cells (MSC) are currently being tested clinically for a plethora of conditions, with most approaches relying on the secretion of paracrine signals by MSC to modulate the immune system, promote wound healing, and induce angiogenesis. Hypoxia has been shown to affect MSC proliferation, differentiation, survival and secretory profile. Here, we investigate changes in the lipid composition of human bone marrow-derived MSC after exposure to hypoxia. Using mass spectrometry, we compared the lipid profiles of MSC derived from five different donors, cultured for two days in either normoxia (control) or hypoxia (1% oxygen). Hypoxia induced a significant increase of total triglycerides, fatty acids and diacylglycerols (DG). Remarkably, reduction of DG levels using the phosphatidylcholine-specific phospholipase C inhibitor D609 inhibited the secretion of VEGF and Angiopoietin-2, but increased the secretion of interleukin-8, without affecting significantly their respective mRNA levels. Functionally, incubation of MSC in hypoxia with D609 inhibited the potential of the cells to promote migration of human endothelial cells in a wound/scratch assay. Hence, we show that hypoxia induces in MSC an increase of DG that may affect the angiogenic potential of these cells. © 2015 Wiley Periodicals, Inc.

Glycogen synthesis is induced in hypoxia by the hypoxia-inducible factor and promotes cancer cell survival

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Joffrey ePelletier

2012-02-01

Full Text Available The hypoxia-inducible factor 1 (HIF-1, in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1, were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these hypoxia-preconditioned cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO2 acts as an alarm that prepares the cells to face subsequent nutrient depletion and to survive.

Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

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Pelletier, Joffrey; Bellot, Grégory [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France); Gounon, Pierre; Lacas-Gervais, Sandra [Centre Commun de Microscopie Appliquée, University of Nice-Sophia Antipolis, Nice (France); Pouysségur, Jacques; Mazure, Nathalie M., E-mail: mazure@unice.fr [Institute of Developmental Biology and Cancer Research, CNRS-UMR 6543, Centre Antoine Lacassagne, University of Nice-Sophia Antipolis, Nice (France)

2012-02-28

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO{sub 2} acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

Glycogen Synthesis is Induced in Hypoxia by the Hypoxia-Inducible Factor and Promotes Cancer Cell Survival

International Nuclear Information System (INIS)

Pelletier, Joffrey; Bellot, Grégory; Gounon, Pierre; Lacas-Gervais, Sandra; Pouysségur, Jacques; Mazure, Nathalie M.

2012-01-01

The hypoxia-inducible factor 1 (HIF-1), in addition to genetic and epigenetic changes, is largely responsible for alterations in cell metabolism in hypoxic tumor cells. This transcription factor not only favors cell proliferation through the metabolic shift from oxidative phosphorylation to glycolysis and lactic acid production but also stimulates nutrient supply by mediating adaptive survival mechanisms. In this study we showed that glycogen synthesis is enhanced in non-cancer and cancer cells when exposed to hypoxia, resulting in a large increase in glycogen stores. Furthermore, we demonstrated that the mRNA and protein levels of the first enzyme of glycogenesis, phosphoglucomutase1 (PGM1), were increased in hypoxia. We showed that induction of glycogen storage as well as PGM1 expression were dependent on HIF-1 and HIF-2. We established that hypoxia-induced glycogen stores are rapidly mobilized in cells that are starved of glucose. Glycogenolysis allows these “hypoxia-preconditioned” cells to confront and survive glucose deprivation. In contrast normoxic control cells exhibit a high rate of cell death following glucose removal. These findings point to the important role of hypoxia and HIF in inducing mechanisms of rapid adaptation and survival in response to a decrease in oxygen tension. We propose that a decrease in pO 2 acts as an “alarm” that prepares the cells to face subsequent nutrient depletion and to survive.

Dedifferentiation, Proliferation, and Redifferentiation of Adult Mammalian Cardiomyocytes After Ischemic Injury.

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Wang, Wei Eric; Li, Liangpeng; Xia, Xuewei; Fu, Wenbin; Liao, Qiao; Lan, Cong; Yang, Dezhong; Chen, Hongmei; Yue, Rongchuan; Zeng, Cindy; Zhou, Lin; Zhou, Bin; Duan, Dayue Darrel; Chen, Xiongwen; Houser, Steven R; Zeng, Chunyu

2017-08-29

Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. β-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca 2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca 2+ -dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca 2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca 2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia

Axin1 up-regulated 1 accelerates stress-induced cardiomyocytes apoptosis through activating Wnt/β-catenin signaling.

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Ye, Xing; Lin, Junyi; Lin, Zebin; Xue, Aimin; Li, Liliang; Zhao, Ziqin; Liu, Li; Shen, Yiwen; Cong, Bin

2017-10-15

Stress-induced cardiomyocyte apoptosis contributes to the pathogenesis of a variety of cardiovascular diseases, but how stress induces cardiomyocyte apoptosis remains largely unclear. The present study aims to investigate the effects of Axin1 up-regulated 1 (Axud1), a novel pro-apoptotic protein, on the cardiomyocyte survival and the underlying mechanisms. To this end, a rat model under restraint stress (RS) was established and in vitro stress-induced cardiomyocytes culture was achieved. Our data showed that Axud1 was upregulated in the rat myocardia after exposure to RS. Anti-apoptotic Bcl-2 was decreased, whereas pro-apoptotic Bax and Cleaved caspase-3 (Cc3) were increased in a time-dependent manner. The Wnt/β-catenin signaling was observed to be interestingly activated in heart undergoing RS. In addition, the treatment of norepinephrine (NE) to in vitro cardiomyocytes increased Axud1 level and induced cell apoptosis. Wnt/β-catenin signaling was consistently activated. Knockdown of Axud1 using specific siRNA blunted NE-induced cardiomyocytes apoptosis and also inactivated the Wnt/β-catenin signaling. XAV-939, an inhibitor of Wnt/β-catenin signaling, partially reversed the pro-apoptotic effect of NE. In conclusion, Axud1 accelerated stress-induced cardiomyocytes apoptosis through activation of Wnt/β-catenin signaling pathway. Our data provided novel evidence that therapeutic strategies against Axud1 or Wnt/β-catenin signaling might be promising in relation to RS-induced myocardial injury. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Silibinin inhibits hypoxia-induced HIF-1α-mediated signaling, angiogenesis and lipogenesis in prostate cancer cells: In vitro evidence and in vivo functional imaging and metabolomics.

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Deep, Gagan; Kumar, Rahul; Nambiar, Dhanya K; Jain, Anil K; Ramteke, Anand M; Serkova, Natalie J; Agarwal, Chapla; Agarwal, Rajesh

2017-03-01

Hypoxia is associated with aggressive phenotype and poor prognosis in prostate cancer (PCa) patients suggesting that PCa growth and progression could be controlled via targeting hypoxia-induced signaling and biological effects. Here, we analyzed silibinin (a natural flavonoid) efficacy to target cell growth, angiogenesis, and metabolic changes in human PCa, LNCaP, and 22Rv1 cells under hypoxic condition. Silibinin treatment inhibited the proliferation, clonogenicity, and endothelial cells tube formation by hypoxic (1% O 2 ) PCa cells. Interestingly, hypoxia promoted a lipogenic phenotype in PCa cells via activating acetyl-Co A carboxylase (ACC) and fatty acid synthase (FASN) that was inhibited by silibinin treatment. Importantly, silibinin treatment strongly decreased hypoxia-induced HIF-1α expression in PCa cells together with a strong reduction in hypoxia-induced NADPH oxidase (NOX) activity. HIF-1α overexpression in LNCaP cells significantly increased the lipid accumulation and NOX activity; however, silibinin treatment reduced HIF-1α expression, lipid levels, clonogenicity, and NOX activity even in HIF-1α overexpressing LNCaP cells. In vivo, silibinin feeding (200 mg/kg body weight) to male nude mice with 22Rv1 tumors, specifically inhibited tumor vascularity (measured by dynamic contrast-enhanced MRI) resulting in tumor growth inhibition without directly inducing necrosis (as revealed by diffusion-weighted MRI). Silibinin feeding did not significantly affect tumor glucose uptake measured by FDG-PET; however, reduced the lipid synthesis measured by quantitative 1 H-NMR metabolomics. IHC analyses of tumor tissues confirmed that silibinin feeding decreased proliferation and angiogenesis as well as reduced HIF-1α, FASN, and ACC levels. Together, these findings further support silibinin usefulness against PCa through inhibiting hypoxia-induced signaling. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

High Uric Acid Induces Insulin Resistance in Cardiomyocytes In Vitro and In Vivo.

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Li Zhi

Full Text Available Clinical studies have shown hyperuricemia strongly associated with insulin resistance as well as cardiovascular disease. Direct evidence of how high uric acid (HUA affects insulin resistance in cardiomyocytes, but the pathological mechanism of HUA associated with cardiovascular disease remains to be clarified. We aimed to examine the effect of HUA on insulin sensitivity in cardiomyocytes and on insulin resistance in hyperuricemic mouse model. We exposed primary cardiomyocytes and a rat cardiomyocyte cell line, H9c2 cardiomyocytes, to HUA, then quantified glucose uptake with a fluorescent glucose analog, 2-NBDG, after insulin challenge and detected reactive oxygen species (ROS production. Western blot analysis was used to examine the levels of insulin receptor (IR, phosphorylated insulin receptor substrate 1 (IRS1, Ser307 and phospho-Akt (Ser473. We monitored the impact of HUA on insulin resistance, insulin signaling and IR, phospho-IRS1 (Ser307 and phospho-Akt levels in myocardial tissue of an acute hyperuricemia mouse model established by potassium oxonate treatment. HUA inhibited insulin-induced glucose uptake in H9c2 and primary cardiomyocytes. It increased ROS production; pretreatment with N-acetyl-L-cysteine (NAC, a ROS scavenger, reversed HUA-inhibited glucose uptake induced by insulin. HUA exposure directly increased the phospho-IRS1 (Ser307 response to insulin and inhibited that of phospho-Akt in H9C2 cardiomyocytes, which was blocked by NAC. Furthermore, the acute hyperuricemic mice model showed impaired glucose tolerance and insulin tolerance accompanied by increased phospho-IRS1 (Ser307 and inhibited phospho-Akt response to insulin in myocardial tissues. HUA inhibited insulin signaling and induced insulin resistance in cardiomyocytes in vitro and in vivo, which is a novel potential mechanism of hyperuricemic-related cardiovascular disease.

SOX6 and PDCD4 enhance cardiomyocyte apoptosis through LPS-induced miR-499 inhibition.

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Jia, Zhuqing; Wang, Jiaji; Shi, Qiong; Liu, Siyu; Wang, Weiping; Tian, Yuyao; Lu, Qin; Chen, Ping; Ma, Kangtao; Zhou, Chunyan

2016-02-01

Sepsis-induced cardiac apoptosis is one of the major pathogenic factors in myocardial dysfunction. As it enhances numerous proinflammatory factors, lipopolysaccharide (LPS) is considered the principal mediator in this pathological process. However, the detailed mechanisms involved are unclear. In this study, we attempted to explore the mechanisms involved in LPS-induced cardiomyocyte apoptosis. We found that LPS stimulation inhibited microRNA (miR)-499 expression and thereby upregulated the expression of SOX6 and PDCD4 in neonatal rat cardiomyocytes. We demonstrate that SOX6 and PDCD4 are target genes of miR-499, and they enhance LPS-induced cardiomyocyte apoptosis by activating the BCL-2 family pathway. The apoptosis process enhanced by overexpression of SOX6 or PDCD4, was rescued by the cardiac-abundant miR-499. Overexpression of miR-499 protected the cardiomyocytes against LPS-induced apoptosis. In brief, our results demonstrate the existence of a miR-499-SOX6/PDCD4-BCL-2 family pathway in cardiomyocytes in response to LPS stimulation.

Human-induced pluripotent stem cell-derived cardiomyocytes from cardiac progenitor cells: effects of selective ion channel blockade.

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Altomare, Claudia; Pianezzi, Enea; Cervio, Elisabetta; Bolis, Sara; Biemmi, Vanessa; Benzoni, Patrizia; Camici, Giovanni G; Moccetti, Tiziano; Barile, Lucio; Vassalli, Giuseppe

2016-12-01

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are likely to revolutionize electrophysiological approaches to arrhythmias. Recent evidence suggests the somatic cell origin of hiPSCs may influence their differentiation potential. Owing to their cardiomyogenic potential, cardiac-stromal progenitor cells (CPCs) are an interesting cellular source for generation of hiPSC-derived cardiomyocytes. The effect of ionic current blockade in hiPSC-derived cardiomyocytes generated from CPCs has not been characterized yet. Human-induced pluripotent stem cell-derived cardiomyocytes were generated from adult CPCs and skin fibroblasts from the same individuals. The effect of selective ionic current blockade on spontaneously beating hiPSC-derived cardiomyocytes was assessed using multi-electrode arrays. Cardiac-stromal progenitor cells could be reprogrammed into hiPSCs, then differentiated into hiPSC-derived cardiomyocytes. Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin showed higher upregulation of cardiac-specific genes compared with those of fibroblastic origin. Human-induced pluripotent stem cell-derived cardiomyocytes of both somatic cell origins exhibited sensitivity to tetrodotoxin, a blocker of Na +  current (I Na ), nifedipine, a blocker of L-type Ca 2+  current (I CaL ), and E4031, a blocker of the rapid component of delayed rectifier K +  current (I Kr ). Human-induced pluripotent stem cell-derived cardiomyocytes of cardiac origin exhibited sensitivity to JNJ303, a blocker of the slow component of delayed rectifier K +  current (I Ks ). In hiPSC-derived cardiomyocytes of cardiac origin, I Na , I CaL , I Kr , and I Ks were present as tetrodotoxin-, nifedipine-, E4031-, and JNJ303-sensitive currents, respectively. Although cardiac differentiation efficiency was improved in hiPSCs of cardiac vs. non-cardiac origin, no major functional differences were observed between hiPSC-derived cardiomyocytes of different somatic

Taurine ameliorated homocysteine-induced H9C2 cardiomyocyte apoptosis by modulating endoplasmic reticulum stress.

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Zhang, Zhimin; Zhao, Lianyou; Zhou, Yanfen; Lu, Xuanhao; Wang, Zhengqiang; Wang, Jipeng; Li, Wei

2017-05-01

Homocysteine (Hcy)-triggered endoplasmic reticulum (ER) stress-mediated endothelial cell apoptosis has been suggested as a cause of Hcy-dependent vascular injury. However, whether ER stress is the molecular mechanism linking Hcy and cardiomyocytes death is unclear. Taurine has been reported to exert cardioprotective effects via various mechanisms. However, whether taurine protects against Hcy-induced cardiomyocyte death by attenuating ER stress is unknown. This study aimed to evaluate the opposite effects of taurine on Hcy-induced cardiomyocyte apoptosis and their underlying mechanisms. Our results demonstrated that low-dose or short-term Hcy treatment increased the expression of glucose-regulated protein 78 (GRP78) and activated protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), which in turn prevented apoptotic cell death. High-dose Hcy or prolonged Hcy treatment duration significantly up-regulated levels of C/EBP homologous protein (CHOP), cleaved caspase-12, p-c-Jun N-terminal kinase (JNK), and then triggered apoptotic events. High-dose Hcy also resulted in a decrease in mitochondrial membrane potential (Δψm) and an increase in cytoplasmic cytochrome C and the expression of cleaved caspase-9. Pretreatment of cardiomyocytes with sodium 4-phenylbutyric acid (an ER stress inhibitor) significantly inhibited Hcy-induced apoptosis. Furthermore, blocking the PERK pathway partly alleviated Hcy-induced ER stress-modulated cardiomyocyte apoptosis, and down-regulated the levels of Bax and cleaved caspase-3. Experimental taurine pretreatment inhibited the expression of ER stress-related proteins, and protected against apoptotic events triggered by Hcy-induced ER stress. Taken together, our results suggest that Hcy triggered ER stress in cardiomyocytes, which was the crucial molecular mechanism mediating Hcy-induced cardiomyocyte apoptosis, and the adverse effect of Hcy could be prevented by taurine.

Sequestration of fatty acids in triglycerides prevents endoplasmic reticulum stress in an in vitro model of cardiomyocyte lipotoxicity

NARCIS (Netherlands)

Bosma, M.; Dapito, D.H.; Drosatos-Tampakaki, Z.; Huiping-Son, N.; Huang, L.S.; Kersten, A.H.; Drosatos, K.; Goldberg, I.J.

2014-01-01

We used human cardiomyocyte-derived cells to create an in vitro model to study lipid metabolism and explored the effects of PPAR gamma, ACSL1 and ATGL on fatty acid-induced ER stress. Compared to oleate, palmitate treatment resulted in less intracellular accumulation of lipid droplets and more ER

Hypoxia-inducible factor signalling mechanisms in the central nervous system.

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Corcoran, A; O'Connor, J J

2013-08-01

In the CNS, neurones are highly sensitive to the availability of oxygen. In conditions where oxygen availability is decreased, neuronal function can be altered, leading to injury and cell death. Hypoxia has been implicated in a number of central nervous system pathologies including stroke, head trauma and neurodegenerative diseases. Cellular responses to oxygen deprivation are complex and result in activation of short- and long-term mechanisms to conserve energy and protect cells. Failure of synaptic transmission can be observed within minutes following this hypoxia. The acute effects of hypoxia on synaptic transmission are primarily mediated by altering ion fluxes across membranes, pre-synaptic effects of adenosine and other actions at glutamatergic receptors. A more long-term feature of the response of neurones to hypoxia is the activation of transcription factors such as hypoxia-inducible factor. The activation of hypoxia-inducible factor is governed by a family of dioxygenases called hypoxia-inducible factor prolyl 4 hydroxylases (PHDs). Under hypoxic conditions, PHD activity is inhibited, thereby allowing hypoxia-inducible factor to accumulate and translocate to the nucleus, where it binds to the hypoxia-responsive element sequences of target gene promoters. Inhibition of PHD activity stabilizes hypoxia-inducible factor and other proteins thus acting as a neuroprotective agent. This review will focus on the response of neuronal cells to hypoxia-inducible factor and its targets, including the prolyl hydroxylases. We also present evidence for acute effects of PHD inhibition on synaptic transmission and plasticity in the hippocampus. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

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Liu, Yanlong [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Chunhong [Second Hospital, Jilin University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Wang, Yuhua [College of Food Science and Engineering, Jilin Agricultural University, Changchun (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Ma, Zhenhua [First Hospital, Xi' an Jiaotong University, Xi' an (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); McClain, Craig [Department of Medicine, University of Louisville, Louisville, KY (United States); Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States); Robley Rex Veterans Affairs Medical Center, Louisville, KY (United States); Li, Xiaokun [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Feng, Wenke, E-mail: wenke.feng@louisville.edu [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Department of Medicine, University of Louisville, Louisville, KY (United States); Alcohol Research Center, University of Louisville, Louisville, KY (United States)

2012-10-15

Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl{sub 2}), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl{sub 2} treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl{sub 2} administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl{sub 2}-induced reactive oxygen species (ROS) formation and completely negated CoCl{sub 2}-induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl{sub 2} administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl{sub 2} increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl{sub 2}-induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and

Cobalt chloride decreases fibroblast growth factor-21 expression dependent on oxidative stress but not hypoxia-inducible factor in Caco-2 cells

International Nuclear Information System (INIS)

Liu, Yanlong; Wang, Chunhong; Wang, Yuhua; Ma, Zhenhua; Xiao, Jian; McClain, Craig; Li, Xiaokun; Feng, Wenke

2012-01-01

Fibroblast growth factor-21 (FGF21) is a potential metabolic regulator with multiple beneficial effects on metabolic diseases. FGF21 is mainly expressed in the liver, but is also found in other tissues including the intestine, which expresses β-klotho abundantly. The intestine is a unique organ that operates in a physiologically hypoxic environment, and is responsible for the fat absorption processes including triglyceride breakdown, re-synthesis and absorption into the portal circulation. In the present study, we investigated the effects of hypoxia and the chemical hypoxia inducer, cobalt chloride (CoCl 2 ), on FGF21 expression in Caco-2 cells and the consequence of fat accumulation. Physical hypoxia (1% oxygen) and CoCl 2 treatment decreased both FGF21 mRNA and secreted protein levels. Gene silence and inhibition of hypoxia-inducible factor-α (HIFα) did not affect the reduction of FGF21 mRNA and protein levels by hypoxia. However, CoCl 2 administration caused a significant increase in oxidative stress. The addition of n-acetylcysteine (NAC) suppressed CoCl 2 -induced reactive oxygen species (ROS) formation and completely negated CoCl 2 -induced FGF21 loss. mRNA stability analysis demonstrated that the CoCl 2 administration caused a remarkable reduction in FGF21 mRNA stability. Furthermore, CoCl 2 increased intracellular triglyceride (TG) accumulation, along with a reduction in mRNA levels of lipid lipase, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), and an increase of sterol regulatory element-binding protein-1c (SREBP1c) and stearoyl-coenzyme A (SCD1). Addition of both NAC and recombinant FGF21 significantly attenuated the CoCl 2 -induced TG accumulation. In conclusion, the decrease of FGF21 in Caco-2 cells by chemical hypoxia is independent of HIFα, but dependent on an oxidative stress-mediated mechanism. The regulation of FGF21 by hypoxia may contribute to intestinal lipid metabolism and absorption. -- Graphical abstract: Physical

Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1α.

Science.gov (United States)

Yang, Yeon Ju; Na, Hwi Jung; Suh, Michelle J; Ban, Myung Jin; Byeon, Hyung Kwon; Kim, Won Shik; Kim, Jae Wook; Choi, Eun Chang; Kwon, Hyeong Ju; Chang, Jae Won; Koh, Yoon Woo

2015-11-01

Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poor prognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroid cancer. We showed that hypoxia inducible factor-1α (HIF-1α) induced aggressiveness in FTC cells and identified the underlying mechanism of the HIF-1α-induced invasive characteristics. Cells were cultured under controlled hypoxic environments (1% O₂) or normoxic conditions. The effect of hypoxia on HIF-1α, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-time PCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functional character of EMT. The involvement of HIF-1α and Twist in EMT were studied using gene overexpression or silencing. After orthotopic nude mouse model was established using the cells transfected with lentiviral shHIF-1α, tissue analysis was done. Hypoxia induces HIF-1α expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentin expression. We showed that overexpression of HIF-1α via transfection resulted in the aforementioned changes without hypoxia, and repression of HIF-1α with RNA interference suppressed hypoxia-induced HIF-1α and EMT. Furthermore, we also observed that Twist expression was regulated by HIF-1α. These were confirmed in the orthotopic FTC model. Hypoxia induced HIF-1α, which in turn induced EMT, resulting in the increased capacity for invasion and migration of cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategy to prevent invasive and metastatic FTC.

Hypoxia induces epithelial-mesenchymal transition via activation of SNAI1 by hypoxia-inducible factor -1α in hepatocellular carcinoma

International Nuclear Information System (INIS)

Zhang, Lin; Feng, Xiaobin; Dong, Jiahong; Qian, Cheng; Huang, Gang; Li, Xiaowu; Zhang, Yujun; Jiang, Yan; Shen, Junjie; Liu, Jia; Wang, Qingliang; Zhu, Jin

2013-01-01

High invasion and metastasis are the primary factors causing poor prognosis of patients with hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying these biological behaviors have not been completely elucidated. In this study, we investigate the molecular mechanism by which hypoxia promotes HCC invasion and metastasis through inducing epithelial-mesenchymal transition (EMT). The expression of EMT markers was analyzed by immunohistochemistry. Effect of hypoxia on induction of EMT and ability of cell migration and invasion were performed. Luciferase reporter system was used for evaluation of Snail regulation by hypoxia-inducible factor -1α (HIF-1α). We found that overexpression of HIF-1α was observed in HCC liver tissues and was related to poor prognosis of HCC patients. HIF-1α expression profile was correlated with the expression levels of SNAI1, E-cadherin, N-cadherin and Vimentin. Hypoxia was able to induce EMT and enhance ability of invasion and migration in HCC cells. The same phenomena were also observed in CoCl2-treated cells. The shRNA-mediated HIF-1α suppression abrogated CoCl2-induced EMT and reduced ability of migration and invasion in HCC cells. Luciferase assay showed that HIF-1α transcriptional regulated the expression of SNAI1 based on two hypoxia response elements (HREs) in SNAI1 promoter. We demonstrated that hypoxia-stabilized HIF1α promoted EMT through increasing SNAI1 transcription in HCC cells. This data provided a potential therapeutic target for HCC treatment

Hypoxia-induced retinopathy model in adult zebrafish

DEFF Research Database (Denmark)

Cao, Ziquan; Jensen, Lasse D.; Rouhi, Pegah

2010-01-01

Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available....... In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia......-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs....

Role of hypoxia and hypoxia inducible factor in physiological and pathological conditions

Directory of Open Access Journals (Sweden)

Mozhgan Jahani

2017-11-01

Full Text Available Introduction: Organisms are exposed to oxygen deprivation (Hypoxia in various physiological and pathological conditions. There are different conserve evolutionary responses to counterview with this stress that primary transcriptional response to stress related to hypoxia is interceded by hypoxia-inducible factor (HIF-1 in mammals. This factor can regulate different genes that have essential roles in adaptation to this condition. In this review, the role of this factor in physiological and pathological conditions under hypoxic condition has been evaluated after examining structural features and regulation characteristics of HIF-1. Methods: First, articles related to the keywords of hypoxia and HIF-1 (from 1991-2016 were searched from valid databases such as Springer Link, Google Scholar, PubMed and Science direct. Then, the articles correlated with hypoxia, HIF-1 and their roles in physiological and pathological conditions (120 articles were searched and just 64 articles were selected for this study. Result: According to studies, there are different genes in cells and organs that can be regulated by HIF-1. Activation of genes expression by this protein occurs through its linkage to cis-acting of 50 base pair hypoxia response element (HRE region located in their promotor and enhancer. Depending on circumstances, activation of these genes can be beneficial or harmful. Conclusion: Activation of different genes in hypoxia by HIF-1 has different effects on physiological and pathological conditions. Therefore, HIF-1, as a hypoxia-inducible factor in hypoxic conditions, plays an essential role in the adaptation of cells and organs to changes related to the presence of oxygen.

Atomic force microscopy observation of lipopolysaccharide-induced cardiomyocyte cytoskeleton reorganization.

Science.gov (United States)

Wang, Liqun; Chen, Tangting; Zhou, Xiang; Huang, Qiaobing; Jin, Chunhua

2013-08-01

We applied atomic force microscopy (AFM) to observe lipopolysaccharide (LPS)-induced intracellular cytoskeleton reorganization in primary cardiomyocytes from neonatal mouse. The nonionic detergent Triton X-100 was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized by AFM. Using three-dimensional technique of AFM, we were able to quantify the changes of cytoskeleton by the "density" and total "volume" of the cytoskeleton fibers. Compared to the control group, the density of cytoskeleton was remarkably decreased and the volume of cytoskeleton was significantly increased after LPS treatment, which suggests that LPS may induce the cytoskeleton reorganization and change the cardiomyocyte morphology. Copyright © 2013 Elsevier Ltd. All rights reserved.

Similar to spironolactone, oxymatrine is protective in aldosterone-induced cardiomyocyte injury via inhibition of calpain and apoptosis-inducing factor signaling.

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Ting-Ting Xiao

Full Text Available Accumulating evidence indicates that oxymatrine (OMT possesses variously pharmacological properties, especially on the cardiovascular system. We previously demonstrated that activated calpain/apoptosis-inducing factor (AIF-mediated pathway was the key molecular mechanism in aldosterone (ALD induces cardiomyocytes apoptosis. In the present study, we extended the experimentation by investigating the effect of OMT on cardiomyocytes exposed to ALD, as compared to spironolactone (Spiro, a classical ALD receptor antagonist. Cardiomyocytes were pre-incubated with OMT, Spiro or vehicle for 1 h, and then, cardiomyocytes were exposed to ALD 24 h. The cell injury was evaluated by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and lactate dehydrogenase (LDH leakage ratio. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL assay, annexin V/PI staining, and relative caspase-3 activity assay. Furthermore, expression of pro-apoptotic proteins including truncated Bid (tBid, calpain and AIF were evaluated by western blot analysis. ALD stimulation increased cardiomyocytes apoptosis, caspase-3 activity and protein expression of calpain, tBid and AIF in the cytosol (p<0.05. Pre-incubated with cardiomyocytes injury and increased caspase-3 activity were significantly attenuated (p<0.05. Furthermore, OMT suppressed ALD-induced high expression of calpain and AIF. And these effects of OMT could be comparable to Spiro. These findings indicated that OMT might be a potential cardioprotective-agent against excessive ALD-induced cardiotoxicity, at least in part, mediated through inhibition of calpain/AIF signaling.

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis.

Science.gov (United States)

Lee, Dae-Hee; Lee, Yong J

2008-10-01

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis. (c) 2008 Wiley-Liss, Inc.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

Science.gov (United States)

Su, Hongyan; Li, Jingyuan; Chen, Tongshuai; Li, Na; Xiao, Jie; Wang, Shujian; Guo, Xiaobin; Yang, Yi; Bu, Peili

2016-11-01

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

Effects of copper sulfate-oxidized or myeloperoxidase- modified LDL on lipid loading and programmed cell death in macrophages under hypoxia

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Vlaminck B

2014-09-01

Full Text Available Benoit Vlaminck,1 Damien Calay,1 Marie Genin,1 Aude Sauvage,1 Noelle Ninane,1 Karim Zouaoui Boudjeltia,2 Martine Raes,1 Carine Michiels1 1Laboratory of Biochemistry and Cellular Biology (URBC, Namur Research Institute for Life Sciences (NARILIS, University of Namur, Namur, Belgium; 2Laboratory of Experimental Medicine (ULB 222 Unit, Universite Libre de Bruxelles, CHU de Charleroi, Charleroi, Belgium Abstract: Atheromatous plaques contain heavily lipid-loaded macrophages that die, hence generating the necrotic core of these plaques. Since plaque instability and rupture is often correlated with a large necrotic core, it is important to understand the mechanisms underlying foam cell death. Furthermore, macrophages within the plaque are associated with hypoxic areas but little is known about the effect of low oxygen partial pressure on macrophage death. The aim of this work was to unravel macrophage death mechanisms induced by oxidized low-density lipoproteins (LDL both under normoxia and hypoxia. Differentiated macrophages were incubated in the presence of native, copper sulfate-oxidized, or myeloperoxidase-modified LDL. The unfolded protein response, apoptosis, and autophagy were then investigated. The unfolded protein response and autophagy were triggered by myeloperoxidase-modified LDL and, to a larger extent, by copper sulfate-oxidized LDL. Electron microscopy observations showed that oxidized LDL induced excessive autophagy and apoptosis under normoxia, which were less marked under hypoxia. Myeloperoxidase-modified LDL were more toxic and induced a higher level of apoptosis. Hypoxia markedly decreased apoptosis and cell death, as marked by caspase activation. In conclusion, the cell death pathways induced by copper sulfate-oxidized and myeloperoxidase-modified LDL are different and are differentially modulated by hypoxia. Keywords: Ox-LDL, myeloperoxidase, hypoxia, UPR, apoptosis, autophagy, macrophages

Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

NARCIS (Netherlands)

Leszczynska, K.B.; Foskolou, I.P.; Abraham, A.G.; Anbalagan, S.; Tellier, C.; Haider, S.; Span, P.N.; O'Neill, E.E.; Buffa, F.M.; Hammond, E.M.

2015-01-01

Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent

Hypoxia-induced p53 modulates both apoptosis and radiosensitivity via AKT

Science.gov (United States)

Leszczynska, Katarzyna B.; Foskolou, Iosifina P.; Abraham, Aswin G.; Anbalagan, Selvakumar; Tellier, Céline; Haider, Syed; Span, Paul N.; O’Neill, Eric E.; Buffa, Francesca M.; Hammond, Ester M.

2015-01-01

Restoration of hypoxia-induced apoptosis in tumors harboring p53 mutations has been proposed as a potential therapeutic strategy; however, the transcriptional targets that mediate hypoxia-induced p53-dependent apoptosis remain elusive. Here, we demonstrated that hypoxia-induced p53-dependent apoptosis is reliant on the DNA-binding and transactivation domains of p53 but not on the acetylation sites K120 and K164, which, in contrast, are essential for DNA damage–induced, p53-dependent apoptosis. Evaluation of hypoxia-induced transcripts in multiple cell lines identified a group of genes that are hypoxia-inducible proapoptotic targets of p53, including inositol polyphosphate-5-phosphatase (INPP5D), pleckstrin domain–containing A3 (PHLDA3), sulfatase 2 (SULF2), B cell translocation gene 2 (BTG2), cytoplasmic FMR1-interacting protein 2 (CYFIP2), and KN motif and ankyrin repeat domains 3 (KANK3). These targets were also regulated by p53 in human cancers, including breast, brain, colorectal, kidney, bladder, and melanoma cancers. Downregulation of these hypoxia-inducible targets associated with poor prognosis, suggesting that hypoxia-induced apoptosis contributes to p53-mediated tumor suppression and treatment response. Induction of p53 targets, PHLDA3, and a specific INPP5D transcript mediated apoptosis in response to hypoxia through AKT inhibition. Moreover, pharmacological inhibition of AKT led to apoptosis in the hypoxic regions of p53-deficient tumors and consequently increased radiosensitivity. Together, these results identify mediators of hypoxia-induced p53-dependent apoptosis and suggest AKT inhibition may improve radiotherapy response in p53-deficient tumors. PMID:25961455

Intermittent hypoxia induces hyperlipidemia in lean mice.

Science.gov (United States)

Li, Jianguo; Thorne, Laura N; Punjabi, Naresh M; Sun, Cheuk-Kwan; Schwartz, Alan R; Smith, Philip L; Marino, Rafael L; Rodriguez, Annabelle; Hubbard, Walter C; O'Donnell, Christopher P; Polotsky, Vsevolod Y

2005-09-30

Obstructive sleep apnea, a syndrome leading to recurrent intermittent hypoxia (IH), has been associated previously with hypercholesterolemia, independent of underlying obesity. We examined the effects of experimentally induced IH on serum lipid levels and pathways of lipid metabolism in the absence and presence of obesity. Lean C57BL/6J mice and leptin-deficient obese C57BL/6J-Lep(ob) mice were exposed to IH for five days to determine changes in serum lipid profile, liver lipid content, and expression of key hepatic genes of lipid metabolism. In lean mice, exposure to IH increased fasting serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, phospholipids (PLs), and triglycerides (TGs), as well as liver TG content. These changes were not observed in obese mice, which had hyperlipidemia and fatty liver at baseline. In lean mice, IH increased sterol regulatory element binding protein 1 (SREBP-1) levels in the liver, increased mRNA and protein levels of stearoyl-coenzyme A desaturase 1 (SCD-1), an important gene of TG and PL biosynthesis controlled by SREBP-1, and increased monounsaturated fatty acid content in serum, which indicated augmented SCD-1 activity. In addition, in lean mice, IH decreased protein levels of scavenger receptor B1, regulating uptake of cholesterol esters and HDL by the liver. We conclude that exposure to IH for five days increases serum cholesterol and PL levels, upregulates pathways of TG and PL biosynthesis, and inhibits pathways of cholesterol uptake in the liver in the lean state but does not exacerbate the pre-existing hyperlipidemia and metabolic disturbances in leptin-deficient obesity.

Hypoxia-Induced Autophagy Is Mediated through Hypoxia-Inducible Factor Induction of BNIP3 and BNIP3L via Their BH3 Domains▿ †

OpenAIRE

Bellot, Grégory; Garcia-Medina, Raquel; Gounon, Pierre; Chiche, Johanna; Roux, Danièle; Pouysségur, Jacques; Mazure, Nathalie M.

2009-01-01

While hypoxia-inducible factor (HIF) is a major actor in the cell survival response to hypoxia, HIF also is associated with cell death. Several studies implicate the HIF-induced putative BH3-only proapoptotic genes bnip3 and bnip3l in hypoxia-mediated cell death. We, like others, do not support this assertion. Here, we clearly demonstrate that the hypoxic microenvironment contributes to survival rather than cell death by inducing autophagy. The ablation of Beclin1, a major actor of autophagy,...

Signaling hypoxia by hypoxia-inducible factor protein hydroxylases: a historical overview and future perspectives

Science.gov (United States)

Bishop, Tammie; Ratcliffe, Peter J

2014-01-01

By the early 1900s, the close matching of oxygen supply with demand was recognized to be a fundamental requirement for physiological function, and multiple adaptive responses to environment hypoxia had been described. Nevertheless, the widespread operation of mechanisms that directly sense and respond to levels of oxygen in animal cells was not appreciated for most of the twentieth century with investigators generally stressing the regulatory importance of metabolic products. Work over the last 25 years has overturned that paradigm. It has revealed the existence of a set of “oxygen-sensing” 2-oxoglutarate dependent dioxygenases that catalyze the hydroxylation of specific amino acid residues and thereby control the stability and activity of hypoxia-inducible factor. The hypoxia-inducible factor hydroxylase pathway regulates a massive transcriptional cascade that is operative in essentially all animal cells. It transduces a wide range of responses to hypoxia, extending well beyond the classical boundaries of hypoxia physiology. Here we review the discovery and elucidation of these pathways, and consider the opportunities and challenges that have been brought into focus by the findings, including new implications for the integrated physiology of hypoxia and therapeutic approaches to ischemic/hypoxic disease. PMID:27774477

Cuticle ultrastructure, cuticular lipid composition, and gene expression in hypoxia-stressed Arabidopsis stems and leaves.

Science.gov (United States)

Kim, Hyojin; Choi, Dongsu; Suh, Mi Chung

2017-06-01

An increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis and may allow plants to cope with oxygen deficiency. The hydrophobic cuticle layer consisting of cutin polyester and cuticular wax is the first barrier to protect the aerial parts of land plants from environmental stresses. In the present study, we investigated the role of cuticle membrane in Arabidopsis responses to oxygen deficiency. TEM analysis showed that the epidermal cells of hypoxia-treated Arabidopsis stems and leaves possessed a thinner electron-translucent cuticle proper and a more electron-dense cuticular layer. A reduction in epicuticular wax crystal deposition was observed in SEM images of hypoxia-treated Arabidopsis stem compared with normoxic control. Cuticular transpiration was more rapid in hypoxia-stressed leaves than in normoxic control. Total wax and cutin loads decreased by approximately 6-12 and 12-22%, respectively, and the levels of C29 alkanes, secondary alcohols, and ketones, C16:0 ω-hydroxy fatty acids, and C18:2 dicarboxylic acids were also prominently reduced in hypoxia-stressed Arabidopsis leaves and/or stems relative to normoxic control. Genome-wide transcriptome and quantitative RT-PCR analyses revealed that the expression of several genes involved in the biosynthesis and transport of cuticular waxes and cutin monomers were downregulated more than fourfold, but no significant alterations were detected in the transcript levels of fatty acid biosynthetic genes, BCCP2, PDH-E1α, and ENR1 in hypoxia-treated Arabidopsis stems and leaves compared with normoxic control. Taken together, an increased permeability of the cuticle is closely associated with downregulation of genes involved in cuticular lipid synthesis in hypoxia-stressed Arabidopsis. The present study elucidates one of the cuticle-related adaptive responses that may allow plants to cope with low oxygen levels.

Selenium deficiency aggravates T-2 toxin-induced injury of primary neonatal rat cardiomyocytes through ER stress.

Science.gov (United States)

Xu, Jing; Pan, Shengchi; Gan, Fang; Hao, Shu; Liu, Dandan; Xu, Haibin; Huang, Kehe

2018-04-01

Keshan disease is a potentially fatal cardiomyopathy in humans. Selenium deficiency, T-2 toxin, and myocarditis virus are thought to be the major factors contributing to Keshan disease. But the relationship among these three factors is poorly described. This study aims to explore whether selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury and its underlying mechanism. Cardiomyocytes were isolated from neonatal rat and cultured at the physiological (2.0 μM) or lower concentrations of selenium with different concentrations of T-2 toxin. Our results showed that selenium deficiencies aggravated T-2 toxin-induced cardiomyocyte injury in a concentration-dependent manner as demonstrated by MTT bioassay, LDH activity, reactive oxygen species levels and caspase 3 protein expressions. T-2 toxin treatment significantly increased mRNA expressions for stress proteins GRP78 and CHOP in cardiomyocytes compared with the control. Selenium deficiencies further promoted GRP78, CHOP and p-eIF2α expressions. Knockdown of CHOP by the specific small interfering RNA eliminated the effect of selenium deficiencies on T-2 toxin-induced injury. It could be concluded that selenium deficiency aggravates T-2 toxin-induced cardiomyocyte injury through initiating more aggressive endoplasmic reticulum stress. Copyright © 2018 Elsevier B.V. All rights reserved.

Glycine reduces tissue lipid peroxidation in hypoxia-reoxygenation-induced necrotizing enterocolitis in rats

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Meyer Karine Furtado

2006-01-01

Full Text Available PURPOSE: To assess the protective effect of glycine in an experimental model of Neonatal Necrotizing Enterocolitis (NEC. METHODS: Fifty (50 neonatal Wistar rats, from a litter of six female rats and weighing 4 to 6 grams, were used. Five animals were cannibalized and the 45 remaining were distributed into three groups: the G1 normal control group (n=12; the G2 Group (n=16, of animals that underwent hypoxia-reoxygenation (HR; the G3 Group of animals (n=17 that underwent HR following a 5% intraperitoneal glycine infusion. The animals underwent hypoxia in a CO2 chamber receiving an air flow of 100% CO2 for 5 minutes and reoxygenation receiving an O2 flow at 100% for 5 minutes. One centimeter long small bowel and colon segments were prepared for histological analysis. The rest of the bowel was removed in a block and frozen at minus 80degreesC for homogenization and determination of tissue malondialdehyde (MDA. Tissue lesions were classified as Grade 0 to Grade 5, according to the level of damaged mucosa. RESULTS: The animals in Group G1 had levels of small bowel and colon lesion significantly smaller as compared to the animals in Groups G2 and G3. The G2 group had mean MDA values significantly higher than the animals in the G1 (p = .015 and G3 (p=0.021 groups. MDA values did not differ significantly (p = 0.992 for the animals in groups G1 and G3. CONCLUSION: Glycine reduces tissue MDA levels (a measurement of lipid peroxidation following HR in neonatal rats.

Hypoxia-induced dysfunction of rat diaphragm: role of peroxynitrite.

NARCIS (Netherlands)

Zhu, X.; Heunks, L.M.A.; Versteeg, E.M.M.; Heijden, E. van der; Ennen, L.; Kuppevelt, A.H.M.S.M. van; Vina, J.; Dekhuijzen, P.N.R.

2005-01-01

Oxidants may play a role in hypoxia-induced respiratory muscle dysfunction. In the present study we hypothesized that hypoxia-induced impairment in diaphragm contractility is associated with elevated peroxynitrite generation. In addition, we hypothesized that strenuous contractility of the diaphragm

Progress toward overcoming hypoxia-induced resistance to solid tumor therapy

International Nuclear Information System (INIS)

Karakashev, Sergey V; Reginato, Mauricio J

2015-01-01

Hypoxic tumors are associated with poor clinical outcome for multiple types of human cancer. This may be due, in part, to hypoxic cancer cells being resistant to anticancer therapy, including radiation therapy, chemotherapy, and targeted therapy. Hypoxia inducible factor 1, a major regulator of cellular response to hypoxia, regulates the expression of genes that are involved in multiple aspects of cancer biology, including cell survival, proliferation, metabolism, invasion, and angiogenesis. Here, we review multiple pathways regulated by hypoxia/hypoxia inducible factor 1 in cancer cells and discuss the latest advancements in overcoming hypoxia-mediated tumor resistance

Hypoxia-induced cytotoxic drug resistance in osteosarcoma is independent of HIF-1Alpha.

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Jennifer Adamski

Full Text Available Survival rates from childhood cancer have improved dramatically in the last 40 years, such that over 80% of children are now cured. However in certain subgroups, including metastatic osteosarcoma, survival has remained stubbornly poor, despite dose intensive multi-agent chemotherapy regimens, and new therapeutic approaches are needed. Hypoxia is common in adult solid tumours and is associated with treatment resistance and poorer outcome. Hypoxia induces chemotherapy resistance in paediatric tumours including neuroblastoma, rhabdomyosarcoma and Ewing's sarcoma, in vitro, and this drug resistance is dependent on the oxygen-regulated transcription factor hypoxia inducible factor-1 (HIF-1. In this study the effects of hypoxia on the response of the osteosarcoma cell lines 791T, HOS and U2OS to the clinically relevant cytotoxics cisplatin, doxorubicin and etoposide were evaluated. Significant hypoxia-induced resistance to all three agents was seen in all three cell lines and hypoxia significantly reduced drug-induced apoptosis. Hypoxia also attenuated drug-induced activation of p53 in the p53 wild-type U2OS osteosarcoma cells. Drug resistance was not induced by HIF-1α stabilisation in normoxia by cobalt chloride nor reversed by the suppression of HIF-1α in hypoxia by shRNAi, siRNA, dominant negative HIF or inhibition with the small molecule NSC-134754, strongly suggesting that hypoxia-induced drug resistance in osteosarcoma cells is independent of HIF-1α. Inhibition of the phosphoinositide 3-kinase (PI3K pathway using the inhibitor PI-103 did not reverse hypoxia-induced drug resistance, suggesting the hypoxic activation of Akt in osteosarcoma cells does not play a significant role in hypoxia-induced drug resistance. Targeting hypoxia is an exciting prospect to improve current anti-cancer therapy and combat drug resistance. Significant hypoxia-induced drug resistance in osteosarcoma cells highlights the potential importance of hypoxia as a target

Hypoxia-inducible factor-1α upregulation in microglia following hypoxia protects against ischemia-induced cerebral infarction.

Science.gov (United States)

Huang, Tao; Huang, Weiyi; Zhang, Zhiqiang; Yu, Lei; Xie, Caijun; Zhu, Dongan; Peng, Zizhuang; Chen, Jiehan

2014-10-01

Activated microglia were considered to be the toxic inflammatory mediators that induce neuron degeneration after brain ischemia. Hypoxia can enhance the expression of hypoxia-inducible factor-1α (HIF-1α) in microglia and cause microglial activation. However, intermittent hypoxia has been reported recently to be capable of protecting the body from myocardial ischemia. We established a high-altitude environment as the hypoxic condition in this study. The hypoxic condition displayed a neuroprotective effect after brain ischemia, and mice exposed to this condition presented better neurological performance and smaller infarct size. At the same time, a high level of HIF-1α, low level of isoform of nitric oxide synthase, and a reduction in microglial activation were also seen in ischemic focus of hypoxic mice. However, this neuroprotective effect could be blocked by 2-methoxyestradiol, the HIF-1α inhibitor. Our finding suggested that HIF-1α expression was involved in microglial activation in vitro and was regulated by oxygen supply. The microglia were inactivated by re-exposure to hypoxia, which might be due to overexpression of HIF-1α. These results indicated that hypoxic conditions can be exploited to achieve maximum neuroprotection after brain ischemia. This mechanism possibly lies in microglial inactivation through regulation of the expression of HIF-1α.

Hypoxia-inducible factor-1α induces multidrug resistance protein in colon cancer

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Lv Y

2015-07-01

Full Text Available Yingqian Lv, Shan Zhao, Jinzhu Han, Likang Zheng, Zixin Yang, Li Zhao Department of Oncology, The Second Hospital, Hebei Medical University, Shijiazhuang, Hebei Province, People’s Republic of China Abstract: Multidrug resistance is the major cause of chemotherapy failure in many solid tumors, including colon cancer. Hypoxic environment is a feature for all solid tumors and is important for the development of tumor resistance to chemotherapy. Hypoxia-inducible factor (HIF-1α is the key transcription factor that mediates cellular response to hypoxia. HIF-1α has been shown to play an important role in tumor resistance; however, the mechanism is still not fully understood. Here, we found that HIF-1α and the drug resistance-associated gene multidrug resistance associated protein 1 (MRP1 were induced by treatment of colon cancer cells with the hypoxia-mimetic agent cobalt chloride. Inhibition of HIF-1α by RNA interference and dominant-negative protein can significantly reduce the induction of MRP1 by hypoxia. Bioinformatics analysis showed that a hypoxia response element is located at -378 to -373 bp upstream of the transcription start site of MRP1 gene. Luciferase reporter assay combined with mutation analysis confirmed that this element is essential for hypoxia-mediated activation of MRP gene. Furthermore, RNA interference revealed that HIF-1α is necessary for this hypoxia-driven activation of MRP1 promoter. Importantly, chromatin immunoprecipitation analysis demonstrated that HIF-1α could directly bind to this HRE site in vivo. Together, these data suggest that MRP1 is a downstream target gene of HIF-1α, which provides a potential novel mechanism for HIF-1α-mediated drug resistance in colon cancer and maybe other solid tumors as well. Keywords: hypoxia, hypoxia-inducible factor-1α, multidrug resistance associated protein, transcriptional regulation, chemotherapy tolerance

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor.

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Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

2010-07-16

Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1alpha and HIF-2alpha immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1alpha-positive, 15 HIF-2alpha-positive and 10 positive for HIF-1alpha and HIF-2alpha. Expression of HIF-1alpha and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1alpha and HIF-2alpha in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2alpha in Ewing's. Downstream transcription was HIF-1alpha-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by >or= 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1alpha and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Upregulated copper transporters in hypoxia-induced pulmonary hypertension.

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Adriana M Zimnicka

Full Text Available Pulmonary vascular remodeling and increased arterial wall stiffness are two major causes for the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients and animals with pulmonary hypertension. Cellular copper (Cu plays an important role in angiogenesis and extracellular matrix remodeling; increased Cu in vascular smooth muscle cells has been demonstrated to be associated with atherosclerosis and hypertension in animal experiments. In this study, we show that the Cu-uptake transporter 1, CTR1, and the Cu-efflux pump, ATP7A, were both upregulated in the lung tissues and pulmonary arteries of mice with hypoxia-induced pulmonary hypertension. Hypoxia also significantly increased expression and activity of lysyl oxidase (LOX, a Cu-dependent enzyme that causes crosslinks of collagen and elastin in the extracellular matrix. In vitro experiments show that exposure to hypoxia or treatment with cobalt (CoCl2 also increased protein expression of CTR1, ATP7A, and LOX in pulmonary arterial smooth muscle cells (PASMC. In PASMC exposed to hypoxia or treated with CoCl2, we also confirmed that the Cu transport is increased using 64Cu uptake assays. Furthermore, hypoxia increased both cell migration and proliferation in a Cu-dependent manner. Downregulation of hypoxia-inducible factor 1α (HIF-1α with siRNA significantly attenuated hypoxia-mediated upregulation of CTR1 mRNA. In summary, the data from this study indicate that increased Cu transportation due to upregulated CTR1 and ATP7A in pulmonary arteries and PASMC contributes to the development of hypoxia-induced pulmonary hypertension. The increased Cu uptake and elevated ATP7A also facilitate the increase in LOX activity and thus the increase in crosslink of extracellular matrix, and eventually leading to the increase in pulmonary arterial stiffness.

Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

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Peng Zhang

2014-03-01

Full Text Available Hypoxia-inducible factors (HIFs play key roles in the cellular response to hypoxia. It is widely accepted that whereas HIF-1 and HIF-2 function as transcriptional activators, HIF-3 inhibits HIF-1/2α action. Contrary to this idea, we show that zebrafish Hif-3α has strong transactivation activity. Hif-3α is degraded under normoxia. Mutation of P393, P493, and L503 inhibits this oxygen-dependent degradation. Transcriptomics and chromatin immunoprecipitation analyses identify genes that are regulated by Hif-3α, Hif-1α, or both. Under hypoxia or when overexpressed, Hif-3α binds to its target gene promoters and upregulates their expression. Dominant-negative inhibition and knockdown of Hif-3α abolish hypoxia-induced Hif-3α-promoter binding and gene expression. Hif-3α not only mediates hypoxia-induced growth and developmental retardation but also possesses hypoxia-independent activities. Importantly, transactivation activity is conserved and human HIF-3α upregulates similar genes in human cells. These findings suggest that Hif-3 is an oxygen-dependent transcription factor and activates a distinct transcriptional response to hypoxia.

Graphene Sheet-Induced Global Maturation of Cardiomyocytes Derived from Human Induced Pluripotent Stem Cells.

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Wang, Jiaxian; Cui, Chang; Nan, Haiyan; Yu, Yuanfang; Xiao, Yini; Poon, Ellen; Yang, Gang; Wang, Xijie; Wang, Chenchen; Li, Lingsong; Boheler, Kenneth Richard; Ma, Xu; Cheng, Xin; Ni, Zhenhua; Chen, Minglong

2017-08-09

Human induced pluripotent stem cells (hiPSCs) can proliferate infinitely. Their ability to differentiate into cardiomyocytes provides abundant sources for disease modeling, drug screening and regenerative medicine. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) display a low degree of maturation and fetal-like properties. Current in vitro differentiation methods do not mimic the structural, mechanical, or physiological properties of the cardiogenesis niche. Recently, we present an efficient cardiac maturation platform that combines hiPSCs monolayer cardiac differentiation with graphene substrate, which is a biocompatible and superconductive material. The hiPSCs lines were successfully maintained on the graphene sheets and were able to differentiate into functional cardiomyocytes. This strategy markedly increased the myofibril ultrastructural organization, elevated the conduction velocity, and enhanced both the Ca 2+ handling and electrophysiological properties in the absence of electrical stimulation. On the graphene substrate, the expression of connexin 43 increased along with the conduction velocity. Interestingly, the bone morphogenetic proteins signaling was also significantly activated during early cardiogenesis, confirmed by RNA sequencing analysis. Here, we reasoned that graphene substrate as a conductive biomimetic surface could facilitate the intrinsic electrical propagation, mimicking the microenvironment of the native heart, to further promote the global maturation of hiPSC-CMs. Our findings highlight the capability of electrically active substrates to influence cardiomyocyte development. We believe that application of graphene sheets will be useful for simple, fast, and scalable maturation of regenerated cardiomyocytes.

Hypoxia compounds exercise-induced free radical formation in humans; partitioning contributions from the cerebral and femoral circulation

DEFF Research Database (Denmark)

Bailey, Damian M; Rasmussen, Peter; Evans, Kevin A

2018-01-01

This study examined to what extent the human cerebral and femoral circulation contribute to free radical formation during basal and exercise-induced responses to hypoxia. Healthy participants (5♂, 5♀) were randomly assigned single-blinded to normoxic (21% O2) and hypoxic (10% O2) trials...... hypoxia (P free radical-mediated lipid peroxidation subsequent to inadequate antioxidant defense. This was pronounced during exercise across the femoral circulation in proportion to the increase in local O2 uptake (r = -0.397 to -0.459, P = 0.037 to 0...... with measurements taken at rest and 30min after cycling at 70% of maximal power output in hypoxia and equivalent relative and absolute intensities in normoxia. Blood was sampled from the brachial artery (a), internal jugular and femoral veins (v) for non-enzymatic antioxidants (HPLC), ascorbate radical (A...

Hypoxia-inducible transcription factor-1α promotes hypoxia-induced A549 apoptosis via a mechanism that involves the glycolysis pathway

International Nuclear Information System (INIS)

Luo, FengMing; Liu, XiaoJing; Yan, NaiHong; Li, ShuangQing; Cao, GuiQun; Cheng, QingYing; Xia, QingJie; Wang, HongJing

2006-01-01

Hypoxia-inducible transcription factor-1α (HIF-1α), which plays an important role in controlling the hypoxia-induced glycolysis pathway, is a 'master' gene in the tissue hypoxia response during tumor development. However, its role in the apoptosis of non-small cell lung cancer remains unknown. Here, we have studied the effects of HIF-1α on apoptosis by modulating HIF-1α gene expression in A549 cells through both siRNA knock-down and over-expression. A549 cells were transfected with a HIF-1α siRNA plasmid or a HIF-1α expression vector. Transfected cells were exposed to a normoxic or hypoxic environment in the presence or absence of 25 mM HEPES and 2-deoxyglucose (2-DG) (5 mM). The expression of three key genes of the glycolysis pathway, glucose transporter type 1(GLUT1), phosphoglycerate kinase 1(PGK1), and hexokinase 1(HK1), were measured using real-time RT-PCR. Glycolysis was monitored by measuring changes of pH and lactate concentration in the culture medium. Apoptosis was detected by TUNEL assay and flow cytometry. Knocking down expression of HIF-1α inhibited the glycolysis pathway, increased the pH of the culture medium, and protected the cells from hypoxia-induced apoptosis. In contrast, over-expression of HIF-1α accelerated glycolysis in A549 cells, decreased the pH of the culture medium, and enhanced hypoxia-induced apoptosis. These effects of HIF-1α on glycolysis, pH of the medium, and apoptosis were reversed by treatment with the glycolytic inhibitor, 2-DG. Apoptosis induced by HIF-1α over-expression was partially inhibited by increasing the buffering capacity of the culture medium by adding HEPES. During hypoxia in A549 cells, HIF-1α promotes activity of the glycolysis pathway and decreases the pH of the culture medium, resulting in increased cellular apoptosis

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

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Dirksen Uta

2010-07-01

Full Text Available Abstract Background Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor. Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. Methods HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. Results 17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Conclusions Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

Science.gov (United States)

Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

2017-02-01

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

Energy Technology Data Exchange (ETDEWEB)

Xu, Aibin; Liu, Jingyi [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Institute of Cardiovascular Disease, General Hospital of Beijing Command, PLA, Beijing (China); Liu, Peilin; Jia, Min; Wang, Han [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Tao, Ling, E-mail: lingtao2006@gmail.com [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China)

2014-04-18

Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H{sub 2}O{sub 2} led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H{sub 2}O{sub 2} and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the

Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

International Nuclear Information System (INIS)

Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

2014-01-01

Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H 2 O 2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H 2 O 2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process

High Glucose-Induced Cardiomyocyte Death May Be Linked to Unbalanced Branched-Chain Amino Acids and Energy Metabolism

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Xi Zhang

2018-04-01

Full Text Available High glucose-induced cardiomyocyte death is a common symptom in advanced-stage diabetic patients, while its metabolic mechanism is still poorly understood. The aim of this study was to explore metabolic changes in high glucose-induced cardiomyocytes and the heart of streptozotocin-induced diabetic rats by 1H-NMR-based metabolomics. We found that high glucose can promote cardiomyocyte death both in vitro and in vivo studies. Metabolomic results show that several metabolites exhibited inconsistent variations in vitro and in vivo. However, we also identified a series of common metabolic changes, including increases in branched-chain amino acids (BCAAs: leucine, isoleucine and valine as well as decreases in aspartate and creatine under high glucose condition. Moreover, a reduced energy metabolism could also be a common metabolic characteristic, as indicated by decreases in ATP in vitro as well as AMP, fumarate and succinate in vivo. Therefore, this study reveals that a decrease in energy metabolism and an increase in BCAAs metabolism could be implicated in high glucose-induced cardiomyocyte death.

Role of hypoxia-inducible factor in diabetic myocardial hypertrophy ...

African Journals Online (AJOL)

Purpose: This study was carried out to investigate the role of hypoxia-inducible factor (HIF) in diabetic cardiomyopathy in vitro. Methods: Hypoxia was induced chemically in H9C2 cells (cardiac hypertrophy model), and the cells were treated with phenylephrine (PE), deferoxamine (DFO), PE + DFO, and HIF-1α siRNA under ...

Screening of hypoxia-inducible genes in sporadic ALS.

LENUS (Irish Health Repository)

Cronin, Simon

2008-10-01

Genetic variations in two hypoxia-inducible angiogenic genes, VEGF and ANG, have been linked with sporadic amyotrophic lateral sclerosis (SALS). Common variations in these genes may reduce the levels or functioning of their products. VEGF and ANG belong to a larger group of angiogenic genes that are up-regulated under hypoxic conditions. We hypothesized that common genetic variation across other members of this group may also predispose to sporadic ALS. To screen other hypoxia-inducible angiogenic genes for association with SALS, we selected 112 tagging single nucleotide polymorphisms (tgSNPs) that captured the common genetic variation across 16 VEGF-like and eight ANG-like hypoxia-inducible genes. Screening for association was performed in 270 Irish individuals with typical SALS and 272 ethnically matched unrelated controls. SNPs showing association in the Irish phase were genotyped in a replication sample of 281 Swedish sporadic ALS patients and 286 Swedish controls. Seven markers showed association in the Irish. The one modest replication signal observed in the Swedish replication sample, at rs3801158 in the gene inhibin beta A, was for the opposite allele vs. the Irish cohort. We failed to detect association of common variation across 24 candidate hypoxia-inducible angiogenic genes with SALS.

SIRT1 Functions as an Important Regulator of Estrogen-Mediated Cardiomyocyte Protection in Angiotensin II-Induced Heart Hypertrophy

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Tao Shen

2014-01-01

Full Text Available Background. Sirtuin 1 (SIRT1 is a member of the sirtuin family, which could activate cell survival machinery and has been shown to be protective in regulation of heart function. Here, we determined the mechanism by which SIRT1 regulates Angiotensin II- (AngII- induced cardiac hypertrophy and injury in vivo and in vitro. Methods. We analyzed SIRT1 expression in the hearts of control and AngII-induced mouse hypertrophy. Female C57BL/6 mice were ovariectomized and pretreated with 17β-estradiol to measure SIRT1 expression. Protein synthesis, cardiomyocyte surface area analysis, qRT-PCR, TUNEL staining, and Western blot were performed on AngII-induced mouse heart hypertrophy samples and cultured neonatal rat ventricular myocytes (NRVMs to investigate the function of SIRT1. Results. SIRT1 expression was slightly upregulated in AngII-induced mouse heart hypertrophy in vivo and in vitro, accompanied by elevated cardiomyocyte apoptosis. SIRT1 overexpression relieves AngII-induced cardiomyocyte hypertrophy and apoptosis. 17β-Estradiol was able to protect cardiomyocytes from AngII-induced injury with a profound upregulation of SIRT1 and activation of AMPK. Moreover, estrogen receptor inhibitor ICI 182,780 and SIRT1 inhibitor niacinamide could block SIRT1’s protective effect. Conclusions. These results indicate that SIRT1 functions as an important regulator of estrogen-mediated cardiomyocyte protection during AngII-induced heart hypertrophy and injury.

Hypoxia promotes apoptosis of neuronal cells through hypoxia-inducible factor-1α-microRNA-204-B-cell lymphoma-2 pathway.

Science.gov (United States)

Wang, Xiuwen; Li, Ji; Wu, Dongjin; Bu, Xiangpeng; Qiao, Yong

2016-01-01

Neuronal cells are highly sensitive to hypoxia and may be subjected to apoptosis when exposed to hypoxia. Several apoptosis-related genes and miRNAs involve in hypoxia-induced apoptosis. This study aimed to examine the role of HIF1α-miR-204-BCL-2 pathway in hypoxia-induced apoptosis in neuronal cells. Annexin V/propidium iodide assay was performed to analyze cell apoptosis in AGE1.HN and PC12 cells under hypoxic or normoxic conditions. The expression of BCL-2 and miR-204 were determined by Western blot and qRT-PCR. The effects of miR-204 overexpression or knockdown on the expression of BCL-2 were evaluated by luciferase assay and Western blot under hypoxic or normoxic conditions. HIF-1α inhibitor YC-1 and siHIF-1α were employed to determine the effect of HIF-1α on the up-regulation of miR-204 and down-regulation of BCL-2 induced by hypoxia. Apoptosis assay showed the presence of apoptosis induced by hypoxia in neuronal cells. Moreover, we found that hypoxia significantly down-regulated the expression of BCL-2, and increased the mRNA level of miR-204 in neuronal cells than that in control. Bioinformatic analysis and luciferase reporter assay demonstrated that miR-204 directly targeted and regulated the expression of BCL-2. Specifically, the expression of BCL-2 was inhibited by miR-204 mimic and enhanced by miR-204 inhibitor. Furthermore, we detected that hypoxia induced cell apoptosis via HIF-1α/miR-204/BCL-2 in neuronal cells. This study demonstrated that HIF-1α-miR-204-BCL-2 pathway contributed to apoptosis of neuronal cells induced by hypoxia, which could potentially be exploited to prevent spinal cord ischemia-reperfusion injury. © 2015 by the Society for Experimental Biology and Medicine.

A preclinical model for noninvasive imaging of hypoxia-induced gene expression; comparison with an exogenous marker of tumor hypoxia

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Wen Bixiu; Burgman, Paul; Zanzonico, Pat; O' Donoghue, Joseph; Li, Gloria C.; Ling, C. Clifton [Memorial Sloan-Kettering Cancer Center, Department of Medical Physics, New York (United States); Cai Shangde; Finn, Ron [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Serganova, Inna [Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States); Blasberg, Ronald; Gelovani, Juri [Memorial Sloan-Kettering Cancer Center, Department of Radiology, New York (United States); Memorial Sloan-Kettering Cancer Center, Department of Neurology, New York (United States)

2004-11-01

Hypoxia is associated with tumor aggressiveness and is an important cause of resistance to radiation therapy and chemotherapy. Assays of tumor hypoxia could provide selection tools for hypoxia-modifying treatments. The purpose of this study was to develop and characterize a rodent tumor model with a reporter gene construct that would be transactivated by the hypoxia-inducible molecular switch, i.e., the upregulation of HIF-1. The reporter gene construct is the herpes simplex virus 1-thymidine kinase (HSV1-tk) fused with the enhanced green fluorescent protein (eGFP) under the regulation of an artificial hypoxia-responsive enhancer/promoter. In this model, tumor hypoxia would up-regulate HIF-1, and through the hypoxia-responsive promoter transactivate the HSV1-tkeGFPfusion gene. The expression of this reporter gene can be assessed with the {sup 124}I-labeled reporter substrate 2'-fluoro-2'-deoxy-1-{beta}-d-arabinofuranosyl-5-iodouracil ({sup 124}I-FIAU), which is phosphorylated by the HSV1-tk enzyme and trapped in the hypoxic cells. Animal positron emission tomography (microPET) and phosphor plate imaging (PPI) were used in this study to visualize the trapped {sup 124}I-FIAU, providing a distribution of the hypoxia-induced molecular events. The distribution of {sup 124}I-FIAU was also compared with that of an exogenous hypoxic cell marker, {sup 18}F-fluoromisonidazole (FMISO). Our results showed that {sup 124}I-FIAU microPET imaging of the hypoxia-induced reporter gene expression is feasible, and that the intratumoral distributions of {sup 124}I-FIAU and {sup 18}F-FMISO are similar. In tumor sections, detailed radioactivity distributions were obtained with PPI which also showed similarity between {sup 124}I-FIAU and {sup 18}F-FMISO. This reporter system is sufficiently sensitive to detect hypoxia-induced transcriptional activation by noninvasive imaging and might provide a valuable tool in studying tumor hypoxia and in validating existing and future

[Over-expression of BDNF inhibits angiotensin II-induced apoptosis of cardiomyocytes in SD rats].

Science.gov (United States)

Cao, Jingli; Wu, Yingfeng; Liu, Geming; Li, Zhenlong

2018-03-01

Objective To investigate the role and molecular mechanism of brain-derived neurotrophic factor (BDNF) against the process of cardiomyocyte hypertrophy and apoptosis. Methods Cardiomyocyte hypertrophy were estabolished by angiotensin II (Ang II) in neonatal cardiomyocytes in vitro and incomplete ligature of abdominal aorta of SD rats in vivo. BDNF over-expressing recombinant vector pcDNA5-BDNF was transfected into cardiomyocytes by liposomes. Immunofluorescence staining was used to detect the effect of BDNF transfection on the surface area of myocardial cells. The effect of BDNF transfection on the apoptosis of cardiomyocytes was assayed by flow cytometry. Real-time fluorescent quantitative PCR was performed to detect the effect of over-expression of BDNF on the expressions of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) mRNAs in cardiomyocytes. Western blot assay was used to observe the changes of BDNF, ANP and BNP, calmodulin kinase 2 (CaMK2) and phosphorylated calmodulin kinase 2 (p-CaMK2), calcineurin (CaN), p-CaN, nuclear factor of activated T cells 3 (NFATC3) and p-NFATC3 protein expressions in the myocardial tissues and cardiomyocytes. Results The expression of BDNF protein increased significantly in cardiac hypertrophy animal and cell models in a time-dependent manner. Compared with the untransfected control cardiomyocytes, the surface area of cardiomyocytes, the rate of apoptosis, the levels of ANP and BNP mRNA and protein expression, the levels of p-CaMK2 and CaN protein in the BDNF over-expressed cardiomyocytes were remarkably reduced, while the level of p-NFATC3 protein rose significantly. Conclusion BDNF inhibits the apoptosis of cardiomyocytes induced by Ang II, and it plays the role by inhibiting CaMK2 and CaN signaling pathways.

Antioxidant mechanism of Rutin on hypoxia-induced pulmonary arterial cell proliferation.

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Li, Qian; Qiu, Yanli; Mao, Min; Lv, Jinying; Zhang, Lixin; Li, Shuzhen; Li, Xia; Zheng, Xiaodong

2014-11-18

Reactive oxygen species (ROS) are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC) proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4) in pulmonary artery endothelial cells (PAECs). Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α). Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC), a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

Antioxidant Mechanism of Rutin on Hypoxia-Induced Pulmonary Arterial Cell Proliferation

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Qian Li

2014-11-01

Full Text Available Reactive oxygen species (ROS are involved in the pathologic process of pulmonary arterial hypertension as either mediators or inducers. Rutin is a type of flavonoid which exhibits significant scavenging properties on oxygen radicals both in vitro and in vivo. In this study, we proposed that rutin attenuated hypoxia-induced pulmonary artery smooth muscle cell (PASMC proliferation by scavenging ROS. Immunofluorescence data showed that rutin decreased the production of ROS, which was mainly generated through mitochondria and NADPH oxidase 4 (Nox4 in pulmonary artery endothelial cells (PAECs. Western blot results provided further evidence on rutin increasing expression of Nox4 and hypoxia-inducible factor-1α (HIF-1α. Moreover, cell cycle analysis by flow cytometry indicated that proliferation of PASMCs triggered by hypoxia was also repressed by rutin. However, N-acetyl-L-cysteine (NAC, a scavenger of ROS, abolished or diminished the capability of rutin in repressing hypoxia-induced cell proliferation. These data suggest that rutin shows a potential benefit against the development of hypoxic pulmonary arterial hypertension by inhibiting ROS, subsequently preventing hypoxia-induced PASMC proliferation.

Andrographolide inhibits hypoxia-induced hypoxia-inducible factor 1α and endothelin 1 expression through the heme oxygenase 1/CO/cGMP/MKP-5 pathways in EA.hy926 cells.

Science.gov (United States)

Lin, Hung-Chih; Su, Shih-Li; Lin, Wan-Chun; Lin, Ai-Hsuan; Yang, Ya-Chen; Lii, Chong-Kuei; Chen, Haw-Wen

2018-03-01

Andrographolide is a potent anti-inflammatory agent found in Andrographis paniculata. Endothelin 1 (ET-1) is an endothelium-derived vasoconstrictor with pro-inflammatory properties secreted in response to hypoxia. Mitogen-activated protein kinase phosphatase 5 (MKP-5) is a dual-specificity phosphatase that dephosphorylates threonine and tyrosine residues of MAPKs. We showed previously that hypoxia-induced HIF-1α expression and ET-1 secretion are dependent on p38 MAPK in EA.hy926 cells. Here, we investigate what role MKP-5 plays in andrographolide's inhibition of hypoxia-induced expression of HIF-1α and ET-1. Hypoxic conditions were created using the hypoxia-mimetic agent CoCl 2 . Andrographolide enhanced HO-1 and MKP-5 expression and cellular cGMP content in addition to inhibiting hypoxia-induced ROS generation. Concomitantly, the HO-1 byproduct CO and the cGMP analogue 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) increased MKP-5 expression, and pretreatment with CO and 8-Br-cGMP inhibited hypoxia-induced HIF-1α and ET-1 expression. Transfection of HO-1 siRNA or pretreatment with the HO-1 inhibitor ZnPP-9 or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a specific inhibitor of soluble guanylate cyclase, reduced andrographolide-induced MKP-5 expression. Moreover, silencing MKP-5 or treatment with the phosphatase inhibitor vanadate abrogated andrographolide's suppressing hypoxia-induced p38 MAPK activation and HIF-1α expression. The inhibition of hypoxia-induced HIF-1α and ET-1 expression by andrographolide is likely associated with HO-1/CO/cGMP/MKP-5 pathways, which is involved in inhibiting hypoxia-induced p38 MAPK activation. © 2017 Wiley Periodicals, Inc.

Alamandine acts via MrgD to induce AMPK/NO activation against Ang II hypertrophy in cardiomyocytes.

Science.gov (United States)

de Jesus, Itamar Couto Guedes; Scalzo, Sergio; Alves, Fabiana; Marques, Kariny; Rocha-Resende, Cibele; Bader, Michael; Santos, Robson A Souza; Guatimosim, Silvia

2018-02-14

The renin-angiotensin system (RAS) plays a pivotal role in the pathogenesis of cardiovascular diseases. New members of this system have been characterized and shown to have biologically relevant actions. Alamandine and its receptor MrgD are recently identified components of RAS. In the cardiovascular system alamandine actions included vasodilation, antihypertensive and anti-fibrosis effects. Currently, the actions of alamandine on cardiomyocytes are unknown. Here our goal was twofold: (1) to unravel the signaling molecules activated by the alamandine/MrgD axis in cardiomyocytes; (2) to evaluate the ability of this axis to prevent against Angiotensin II (Ang II)-induced hypertrophy. In cardiomyocytes from C57BL/6 mice, alamandine treatment induced an increase in nitric oxide (NO) production, which was blocked by D-Pro 7 -Ang-(1-7), a MrgD antagonist. This NO rise correlated with increased phosphorylation of AMPK. Alamandine induced NO production was preserved in Mas -/- myocytes, and lost in MrgD -/- cells. Binding of fluorescent-labeled alamandine was observed in wild-type cells, but it was dramatically reduced in MrgD -/- myocytes. We also assessed the consequences of prolonged alamandine exposure to cultured neonatal rat cardiomyocytes (NRCMs) treated with Ang II. Treatment of NRCMs with alamandine prevented Ang II-induced hypertrophy. Moreover, antihypertrophic actions of alamandine were mediated via MrgD and NO, since they could be prevented by D-Pro 7 -Ang-(1-7) or inhibitors of NO synthase or AMPK. β-alanine, a MrgD agonist, recapitulated alamandine's cardioprotective effects in cardiomyocytes. Our data show that alamandine via MrgD induces AMPK/NO signaling to counterregulate Ang II induced hypertrophy. These findings highlight the therapeutic potential of the alamandine/MrgD axis in the heart.

Zinc promotes the death of hypoxic astrocytes by upregulating hypoxia-induced hypoxia-inducible factor-1alpha expression via poly(ADP-ribose) polymerase-1.

Science.gov (United States)

Pan, Rong; Chen, Chen; Liu, Wen-Lan; Liu, Ke-Jian

2013-07-01

Pathological release of excess zinc ions has been implicated in ischemic brain cell death. However, the underlying mechanisms remain to be elucidated. In stroke, ischemia-induced zinc release and hypoxia-inducible factor-1 (HIF-1) accumulation concurrently occur in the ischemic tissue. The present study tests the hypothesis that the presence of high intracellular zinc concentration is a major cause of modifications to PARP-1 and HIF-1α during hypoxia, which significantly contributes to cell death during ischemia. Primary cortical astrocytes and C8-D1A cells were exposed to different concentrations of zinc chloride. Cell death rate and protein expression of HIF-1 and Poly(ADP-ribose) polymerase (PARP)-1 were examined after 3-h hypoxic treatment. Although 3-h hypoxia or 100 μM of zinc alone did not induce noticeable cytotoxicity, their combination led to a dramatic increase in astrocytic cell death in a zinc-concentration-dependent manner. Exposure of astrocytes to hypoxia for 3 h remarkably increased the levels of intracellular zinc and HIF-1α protein, which was further augmented by added exogenous zinc. Notably, HIF-1α knockdown blocked zinc-induced astrocyte death. Moreover, knockdown of PARP-1, another important protein in the response of hypoxia, attenuated the overexpression of HIF-1α and reduced the cell death rate. Our studies show that zinc promotes hypoxic cell death through overexpression of the hypoxia response factor HIF-1α via the cell fate determine factor PARP-1 modification, which provides a novel mechanism for zinc-mediated ischemic brain injury. © 2013 John Wiley & Sons Ltd.

Hypoxia, hypoxia-inducible transcription factor, and macrophages in human atherosclerotic plaques are correlated with intraplaque angiogenesis

NARCIS (Netherlands)

Sluimer, Judith C.; Gasc, Jean-Marie; van Wanroij, Job L.; Kisters, Natasja; Groeneweg, Mathijs; Sollewijn Gelpke, Maarten D.; Cleutjens, Jack P.; van den Akker, Luc H.; Corvol, Pierre; Wouters, Bradly G.; Daemen, Mat J.; Bijnens, Ann-Pascale J.

2008-01-01

We sought to examine the presence of hypoxia in human carotid atherosclerosis and its association with hypoxia-inducible transcription factor (HIF) and intraplaque angiogenesis. Atherosclerotic plaques develop intraplaque angiogenesis, which is a typical feature of hypoxic tissue and expression of

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism, intervention, and long-term consequences.

Directory of Open Access Journals (Sweden)

Andrei Tintu

Full Text Available Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos.Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20 and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF and its scavenger soluble VEGF receptor-1 (sFlt-1 to investigate the potential role of this hypoxia-regulated cytokine.Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF(165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype.Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Protective effects of novel single compound, Hirsutine on hypoxic neonatal rat cardiomyocytes.

Science.gov (United States)

Wu, Li Xin; Gu, Xian Feng; Zhu, Yi Chun; Zhu, Yi Zhun

2011-01-10

Uncaria rhynchophylla is a traditional Chinese herb that has been applied in China for treatment of ailments of the cardiovascular system, but little is known about its active constituents and effect in cardiomyocytes. In present study, we investigated the cardioprotective effect of 0.1μΜ, 1μΜ and 10μΜ Hirsutine isolated from the methanolic extracts of Uncaria rhynchophylla by high performance liquid chromatography (HPLC) on neonatal rat cardiomyocytes treated with hypoxia to determine the mechanism underlying the protective effect with regard to cardiac anti-oxidant enzymes and apoptosis genes. Hirsutine significantly increased the viability of cardiomyocytes injured by hypoxia. Gene expression levels of proapoptotic genes (Bax, Fas and caspase-3) were significantly downregulated compared with the hypoxic control group (P<0.05), whereas the expression level of Bcl-2 was upregulated following Hirsutine treatment (P<0.05). Correspondingly, Hirsutine treatment increased Bcl-2 protein level and decreased Bax protein level. Assay investigating cardiac anti-oxidant enzymes provided further evidence for the protective effect of Hirsutine, as indicated by the induction of the anti-oxidant enzymes superoxide dismutase. The results of present study suggest that the mechanism of action of Hirsutine in hypoxic neonatal rat cardiomyocytes may be related to its anti-oxidant and anti-apoptotic properties. This may open an avenue for developing novel candidate compounds with cardioprotectiveeffect from unique Chinese plant. Copyright © 2010 Elsevier B.V. All rights reserved.

Influence of contrast agent dose and ultrasound exposure on cardiomyocyte injury induced by myocardial contrast echocardiography in rats.

Science.gov (United States)

Miller, Douglas L; Li, Peng; Dou, Chunyan; Gordon, David; Edwards, Chris A; Armstrong, William F

2005-10-01

To detect specific cardiomyocyte injury induced by myocardial contrast material-enhanced echocardiography (ie, myocardial contrast echocardiography) in rats and to ascertain the influences of contrast material dose and ultrasound exposure on this injury. All animal procedures were approved by the university committee for the use and care of animals. Myocardial contrast echocardiography with 1:4 electrocardiographic (ECG) triggering was performed at 1.5 MHz in 61 anesthetized rats. Evans blue (EB) dye was injected as the vital stain for cardiomyocyte injury. At the start of myocardial contrast echocardiography, which lasted 10 minutes, perflutren lipid microsphere-based contrast material was infused through the tail vein for 5 minutes. Premature heartbeats were counted from the ECG record. The numbers of EB-stained cells counted on sections of heart specimens obtained 24 hours after myocardial contrast echocardiography and then either fresh frozen or embedded in paraffin were determined by using fluorescence microscopy. Results were compared statistically by using t tests and Mann-Whitney rank sum tests. EB-stained cells were concentrated in the anterior region of the myocardium. In the paraffin-embedded specimens, EB-stained cells were often accompanied by but largely separate from areas of inflammatory cell infiltration. At end-systolic triggering with a 50 microL/kg dose of microsphere contrast material, the EB-stained cell count increased with increasing peak rarefactional pressure amplitude, with significantly increased cell counts at 1.6 MPa (P .1). EB-stained cell counts increased with increasing contrast material dose, from 10 to 50 microL/kg, at 2.0 MPa. Cardiomyocyte injury was induced by the interaction of ultrasound pulses with contrast agent microbubbles during myocardial contrast echocardiography in rats, and the numbers of injured cells increased with increasing contrast agent dose and ultrasound exposure. RSNA, 2005

The role of hypoxia inducible factor-1 alpha in bypassing oncogene-induced senescence.

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Mehtap Kilic Eren

Full Text Available Oncogene induced senescence (OIS is a sustained anti-proliferative response acutely induced in primary cells via activation of mitogenic oncogenes such as Ras/BRAF. This mechanism acts as an initial barrier preventing normal cells transformation into malignant cell. Besides oncogenic activation and DNA damage response (DDR, senescence is modulated by a plethora of other factors, and one of the most important one is oxygen tension of the tissue. The aim of this study was to determine the impact of hypoxia on RasV12-induced senescence in human diploid fibroblasts (HDFs. We showed here that hypoxia prevents execution of oncogene induced senescence (OIS, through a strong down-regulation of senescence hallmarks, such as SA- β-galactosidase, H3K9me3, HP1γ, p53, p21CIP1 and p16INK4a in association with induction of hypoxia inducible factor-1α (HIF-1α. In addition, hypoxia also decreased marks of H-RasV12-induced DDR in both cell lines through down-regulation of ATM/ATR, Chk1 and Chk2 phosphorylation as well as decreased γ-H2AX positivity. Utilizing shRNA system targeting HIF-1α we show that HIF-1α is directly involved in down regulation of p53 and its target p21CIP1 but not p16INK4a. In line with this finding we found that knock down of HIF-1α leads to a strong induction of apoptotic response, but not restoration of senescence in Ras expressing HDFs in hypoxia. This indicates that HIF-1α is an important player in early steps of tumorigenesis, leading to suppression of senescence through its negative regulation of p53 and p21CIP1. In our work we describe a mechanism through which hypoxia and specifically HIF-1α preclude cells from maintaining senescence-driven anti proliferative response. These findings indicate the possible mechanism through which hypoxic environment helps premalignant cells to evade impingement of cellular failsafe pathways.

Cancer cell-associated cytoplasmic B7–H4 is induced by hypoxia through hypoxia-inducible factor-1α and promotes cancer cell proliferation

Energy Technology Data Exchange (ETDEWEB)

Jeon, You-Kyoung [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Park, Sae-Gwang; Choi, Il-Whan [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Soo-Woong [Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Lee, Sang Min [Department of Internal Medicine, Division of Hematology/Oncology, Busan Paik Hospital, Inje University, Busan 614-735 (Korea, Republic of); Choi, Inhak, E-mail: miccih@inje.ac.kr [Department of Microbiology and Immunology, Inje University College of Medicine, Busan 614-735 (Korea, Republic of); Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan 614-735 (Korea, Republic of)

2015-04-03

Aberrant B7–H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7–H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7–H4 transcription in primary CD138{sup +} multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7–H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7–H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7–H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7–H4 expression. Furthermore, knockdown of cytoplasmic B7–H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7–H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7–H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment. - Highlights: • Hypoxia upregulates B7–H4 transcription and protein expression. • Hypoxia-induced B7–H4 is detected in the cytoplasm, but not on membrane. • ChIP assay reveals a binding of HIF-1α to B7–H4 promoter at HRE site. • Knockdown and pharmacological inhibition of HIF-1α reduce B7–H4 expression. • B7–H4 knockdown decrease the number of cells in S-phase of cell cycle.

DPP4 deficiency exerts protective effect against H2O2 induced oxidative stress in isolated cardiomyocytes.

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Hui-Chun Ku

Full Text Available Apart from the antihyperglycemic effects, DPP4 inhibitors and GLP-1 molecules are involved in the preservation of cardiac functions. We have demonstrated that DPP4-deficient rats possess resistance to endotoxemia and ischemia/reperfusion stress. However, whether the decrease of DPP4 activity simply augmented the GLP-1 signaling or that such decrease resulted in a change of cellular function remain unclear. Accordingly, we investigated the responses of H(2O(2-induced oxidative stress in adult wild-type and DPP4-deficient rats isolated cardiomyocytes. The coadministration of GLP-1 or DPP4 inhibitor was also performed to define the mechanisms. Cell viability, ROS concentration, catalase activity, glucose uptake, prosurvival, proapoptotic signaling, and contractile function were examined after cells exposed to H(2O(2. DPP4-deficient cardiomyocytes were found to be resistant to H(2O(2-induced cell death via activating AKT signaling, enhancing glucose uptake, preserving catalase activity, diminishing ROS level and proapoptotic signaling. GLP-1 concentration-dependently improved cell viability in wild-type cardiomyocyte against ROS stress, and the ceiling response concentration (200 nM was chosen for studies. GLP-1 was shown to decrease H(2O(2-induced cell death by its receptor-dependent AKT pathway in wild-type cardiomyocytes, but failed to cause further activation of AKT in DPP4-deficient cardiomyocytes. Acute treatment of DPP4 inhibitor only augmented the protective effect of low dose GLP-1, but failed to alter fuel utilization or ameliorate cell viability in wild-type cardiomyocytes after H(2O(2 exposure. The improvement of cell viability after H(2O(2 exposure was correlated with the alleviation of cellular contractile dysfunction in both DPP4-deficient and GLP-1 treated wild-type cardiomyocytes. These findings demonstrated that GLP-1 receptor-dependent pathway is important and exert protective effect in wild-type cardiomyocyte. Long term loss of

HIV-1 subtype C unproductively infects human cardiomyocytes in vitro and induces apoptosis mitigated by an anti-Gp120 aptamer.

Science.gov (United States)

Lopes de Campos, Walter R; Chirwa, Nthato; London, Grace; Rotherham, Lia S; Morris, Lynn; Mayosi, Bongani M; Khati, Makobetsa

2014-01-01

HIV-associated cardiomyopathy (HIVCM) is of clinical concern in developing countries because of a high HIV-1 prevalence, especially subtype C, and limited access to highly active antiretroviral therapy (HAART). For these reasons, we investigated the direct and indirect effects of HIV-1 subtype C infection of cultured human cardiomyocytes and the mechanisms leading to cardiomyocytes damage; as well as a way to mitigate the damage. We evaluated a novel approach to mitigate HIVCM using a previously reported gp120 binding and HIV-1 neutralizing aptamer called UCLA1. We established a cell-based model of HIVCM by infecting human cardiomyocytes with cell-free HIV-1 or co-culturing human cardiomyocytes with HIV-infected monocyte derived macrophages (MDM). We discovered that HIV-1 subtype C unproductively (i.e. its life cycle is arrested after reverse transcription) infects cardiomyocytes. Furthermore, we found that HIV-1 initiates apoptosis of cardiomyocytes through caspase-9 activation, preferentially via the intrinsic or mitochondrial initiated pathway. CXCR4 receptor-using viruses were stronger inducers of apoptosis than CCR5 utilizing variants. Importantly, we discovered that HIV-1 induced apoptosis of cardiomyocytes was mitigated by UCLA1. However, UCLA1 had no protective effective on cardiomyocytes when apoptosis was triggered by HIV-infected MDM. When HIV-1 was treated with UCLA1 prior to infection of MDM, it failed to induce apoptosis of cardiomyocytes. These data suggest that HIV-1 causes a mitochondrial initiated apoptotic cascade, which signal through caspase-9, whereas HIV-1 infected MDM causes apoptosis predominantly via the death-receptor pathway, mediated by caspase-8. Furthermore the data suggest that UCLA1 protects cardiomyocytes from caspase-mediated apoptosis, directly by binding to HIV-1 and indirectly by preventing infection of MDM.

[Role and related mechanism of S1P/S1P1 signal pathway during post conditioning of hypertrophic cardiomyocytes].

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Bao, X H; Li, H X; Tao, J; Li, X M; Yang, Y N; Ma, Y T; Chen, B D

2016-05-24

To study the role and mechanism of sphingosine-1-phosphate (S1P)/ sphingosine-1-phosphate receptor 1(S1P1) signal pathway during post conditioning of hypertrophic cardiomyocytes. Neonatal rat cardiomyocytes were isolated and cultured, then stimulated by norepinephrine (NE) to induce cardiomyocytes hypertrophy. Using tri-gas incubator to create hypoxia and reoxygenation enviroment to mimic ischemia-reperfusion and postconditioning. Hypertrophic cardiomyoctyes were divided into five groups according to the presence or absence of various drugs and postconditiong and relevant signal pathways changes were detected: (1) IPost group (hypoxia+ postconditioning); (2) IPost+ S1P group (cells were pretreated with S1P (1 μmol/L) for 2 h before IPost); (3) IPost+ W-146+ S1P group (cells in IPost+ W-146+ S1P group were pretreated with S1P1 inhibitor W-146 (0.4 μmol/L) for 20 min); (4) IPost+ PD98059+ S1P group (cells in IPost+ S1P group were pretreated with MAPK antagonist PD98059 (125 μmol/L) for 20 min); (5) IPost+ LY-294002+ S1P group (cells in IPost+ S1P group were pretreated with PI3K antagonist LY294002 (0.1 μmol/L) for 20 min). Apoptosis was detected by flow cytometry and protein expression of relevant signal pathways were detected by Western blot. (1)Apoptosis rate was significantly increased in hypoxia/reoxygenation (27.90±4.49)% group compared with normal control group (7.97±2.18)%, which could be significantly reduced in IPost group (15.90±1.77)% (all PS1P and IPost+ S1P+ LY-294002 groups than in IPost and IPost+ S1P+ W-146 and IPost+ S1P+ PD98059 group (all PS1P and IPost+ S1P+ LY-294002 group than in IPost and IPost+ S1P+ W-146 group and IPost+ S1P+ PD98059 group (all PS1P+ W-146 and IPost+ S1P+ PD98059 groups. p-ERK1/2 and p-Akt levels in IPost+ S1P+ W-146 group and IPost+ S1P+ PD98059 were similar as in IPost group. S1P can play protective role on NE induced cardiomyocytes hypertrophy during post conditioning through downregulating caspase-3 expression and

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

International Nuclear Information System (INIS)

Knowles, Helen J; Schaefer, Karl-Ludwig; Dirksen, Uta; Athanasou, Nicholas A

2010-01-01

Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma. HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration. 17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration. Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in in vivo induction of HIF. In vitro data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas

Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

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Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

2008-01-15

In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

Notch-1 mediates hypoxia-induced angiogenesis in rheumatoid arthritis.

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Gao, Wei; Sweeney, Catherine; Connolly, Mary; Kennedy, Aisling; Ng, Chin Teck; McCormick, Jennifer; Veale, Douglas J; Fearon, Ursula

2012-07-01

To examine the effect of hypoxia on Notch-1 signaling pathway components and angiogenesis in inflammatory arthritis. The expression and regulation of Notch-1, its ligand delta-like protein 4 (DLL-4) and downstream signaling components (hairy-related transcription factor 1 [HRT-1], HRT-2), and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions (1-3%) were assessed in synovial tissue specimens from patients with inflammatory arthritis and controls and in human dermal microvascular endothelial cells (HDMECs) by immunohistology, dual immunofluorescence staining (Notch-1/factor VIII), Western blotting, and real-time polymerase chain reaction. In vivo synovial tissue oxygen levels (tissue PO2) were measured under direct visualization at arthroscopy. HDMEC activation under hypoxic conditions in the presence of Notch-1 small interfering RNA (siRNA), the γ-secretase inhibitor DAPT, or dimethyloxalylglycine (DMOG) was assessed by Matrigel tube formation assay, migration assay, invasion assay, and matrix metalloproteinase 2 (MMP-2)/MMP-9 zymography. Expression of Notch-1, its ligand DLL-4, and HRT-1 was demonstrated in synovial tissue, with the strongest expression localized to perivascular/vascular regions. Localization of Notch-1 to synovial endothelium was confirmed by dual immunofluorescence staining. Notch-1 intracellular domain (NICD) expression was significantly higher in synovial tissue from patients with tissue PO2 of PO2 of >20 mm Hg (>3% O2). Exposure of HDMECs to 3% hypoxia induced HIF-1α and NICD protein expression and DLL-4, HRT-1, and HRT-2 messenger RNA expression. DMOG directly induced NICD expression, while Notch-1 siRNA inhibited hypoxia-induced HIF-1α expression, suggesting that Notch-1/HIF-1α signaling is bidirectional. Finally, 3% hypoxia-induced angiogenesis, endothelial cell migration, endothelial cell invasion, and proMMP-2 and proMMP-9 activities were inhibited by Notch-1 siRNA and/or the γ-secretase inhibitor DAPT. Our

[Rat cardiomyocyte remodeling after neonatal cryptosporidiosis. II. Elongation, excessive polyploidization and HIF-1alpha overexpression].

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Anatskaia, O V; Sidorenko, N V; Matveev, I V; Kropotov, A V; Vinogradov, A E

2012-01-01

Retrospective epidemyological studies evidence that infant diseases leave survivors with an increased susceptibility to cardiovascular diseases in later life. At the same time, the mechanisms of this link remain poorly understood. Based on medical statistics reporting that infectious gastroenteritis is the most common cause of maladies in babies, infants and children, we analysed the effects of moderate cryptosporidial gastroenteritis on the heart and ventricular cardiomyocyte remodelling in rats of the first month of life. The disease was challenged by a worldwide human protozoic pathogen Cryptosporidium parvum (Apicomplexa, Sporozoa). The main symptoms manifested in the growth retardation moderate diarrhea. Using real-time PCR, cytophotometry, confocal microscopy and image analysis, we indicated that cryptosporidiosis was associated, with the atrophy heart and the elongation, narrowing, protein content decrease and hyperpolyploidization of cardiomyocytes and the moderate overexpression of hypoxia inducible factor 1alpha (HIF-1alpha) mRNA. Cardiomyocyte shape remodeling and heart atrophy presented in all age groups. The severity of these changes, hovewer, declined gradually from younger to older groups. In contrast, hyperpolyploidization and HIF-1alpha mRNA overexpression were registered mainly among animals aged between 6 and 13 days, and were barely detected and non-significant in older age groups. In the rat the time period covering 6-13 days after birth is known to coincide with the intensive cardiomyocyte polyploidization and the switch from proliferation to hypertrophy. Thus, our data indicate that neonatal cryptosporidiosis may be potential cardiovascular diseases risk factor and that one of the critical time windows for the growing heart covers the time period when cardiomyocyte undergo polyploidization.

Expression of DDX3 is directly modulated by hypoxia inducible factor-1 alpha in breast epithelial cells.

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Mahendran Botlagunta

2011-03-01

Full Text Available DEAD box protein, DDX3, is aberrantly expressed in breast cancer cells ranging from weakly invasive to aggressive phenotypes and functions as an important regulator of cancer cell growth and survival. Here, we demonstrate that hypoxia inducible factor-1α is a transcriptional activator of DDX3 in breast cancer cells. Within the promoter region of the human DDX3 gene, we identified three putative hypoxia inducible factor-1 responsive elements. By luciferase reporter assays in combination with mutated hypoxia inducible factor-1 responsive elements, we determined that the hypoxia inducible factor-1 responsive element at position -153 relative to the translation start site is essential for transcriptional activation of DDX3 under hypoxic conditions. We also demonstrated that hypoxia inducible factor-1 binds to the DDX3 promoter and that the binding is specific, as revealed by siRNA against hypoxia inducible factor-1 and chromatin immunoprecipitation assays. Thus, the activation of DDX3 expression during hypoxia is due to the direct binding of hypoxia inducible factor-1 to hypoxia responsive elements in the DDX3 promoter. In addition, we observed a significant overlap in the protein expression pattern of hypoxia inducible factor-1α and DDX3 in MDA-MB-231 xenograft tumors. Taken together, our results demonstrate, for the first time, the role of DDX3 as a hypoxia-inducible gene that exhibits enhanced expression through the interaction of hypoxia inducible factor-1 with hypoxia inducible factor-1 responsive elements in its promoter region.

Dexamethasone impairs hypoxia-inducible factor-1 function

International Nuclear Information System (INIS)

Wagner, A.E.; Huck, G.; Stiehl, D.P.; Jelkmann, W.; Hellwig-Buergel, T.

2008-01-01

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription-factor composed of α- and β-subunits. HIF-1 is not only necessary for the cellular adaptation to hypoxia, but it is also involved in inflammatory processes and wound healing. Glucocorticoids (GC) are therapeutically used to suppress inflammatory responses. Herein, we investigated whether GC modulate HIF-1 function using GC receptor (GR) possessing (HepG2) and GR deficient (Hep3B) human hepatoma cell cultures as model systems. Dexamethasone (DEX) treatment increased HIF-1α levels in the cytosol of HepG2 cells, while nuclear HIF-1α levels and HIF-1 DNA-binding was reduced. In addition, DEX dose-dependently lowered the hypoxia-induced luciferase activity in a reporter gene system. DEX suppressed the hypoxic stimulation of the expression of the HIF-1 target gene VEGF (vascular endothelial growth factor) in HepG2 cultures. DEX did not reduce hypoxically induced luciferase activity in HRB5 cells, a Hep3B derivative lacking GR. Transient expression of the GR in HRB5 cells restored the susceptibility to DEX. Our study discloses the inhibitory action of GC on HIF-1 dependent gene expression, which may be important with respect to the impaired wound healing in DEX-treated patients

Both cardiomyocyte and endothelial cell Nox4 mediate protection against hemodynamic overload-induced remodelling.

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Zhang, Min; Mongue-Din, Heloise; Martin, Daniel; Catibog, Norman; Smyrnias, Ioannis; Zhang, Xiaohong; Yu, Bin; Wang, Minshu; Brandes, Ralf P; Schröder, Katrin; Shah, Ajay M

2018-03-01

NADPH oxidase-4 (Nox4) is an important reactive oxygen species (ROS) source that is upregulated in the haemodynamically overloaded heart. Our previous studies using global Nox4 knockout (Nox4KO) mice demonstrated a protective role of Nox4 during chronic abdominal aortic banding, involving a paracrine enhancement of myocardial capillary density. However, other authors who studied cardiac-specific Nox4KO mice reported detrimental effects of Nox4 in response to transverse aortic constriction (TAC). It has been speculated that these divergent results are due to cell-specific actions of Nox4 (i.e. cardiomyocyte Nox4 detrimental but endothelial Nox4 beneficial) and/or differences in the model of pressure overload (i.e. abdominal banding vs. TAC). This study aimed to (i) investigate whether the effects of Nox4 on pressure overload-induced cardiac remodelling vary according to the pressure overload model and (ii) compare the roles of cardiomyocyte vs. endothelial cell Nox4. Global Nox4KO mice subjected to TAC developed worse cardiac remodelling and contractile dysfunction than wild-type littermates, consistent with our previous results with abdominal aortic banding. Next, we generated inducible cardiomyocyte-specific Nox4 KO mice (Cardio-Nox4KO) and endothelial-specific Nox4 KO mice (Endo-Nox4KO) and studied their responses to pressure overload. Both Cardio-Nox4KO and Endo-Nox4KO developed worse pressure overload-induced cardiac remodelling and dysfunction than wild-type littermates, associated with significant decrease in protein levels of HIF1α and VEGF and impairment of myocardial capillarization. Cardiomyocyte as well as endothelial cell Nox4 contributes to protection against chronic hemodynamic overload-induced cardiac remodelling, at least in part through common effects on myocardial capillary density. © The Author 2017 Published by Oxford University Press on behalf of the European Society of Cardiology.

Effect of Berberine on PPARα/NO Activation in High Glucose- and Insulin-Induced Cardiomyocyte Hypertrophy

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Mingfeng Wang

2013-01-01

Full Text Available Rhizoma coptidis, the root of Coptis chinensis Franch, has been used in China as a folk medicine in the treatment of diabetes for thousands of years. Berberine, one of the active ingredients of Rhizoma coptidis, has been reported to improve symptoms of diabetes and to treat experimental cardiac hypertrophy, respectively. The objective of this study was to evaluate the potential effect of berberine on cardiomyocyte hypertrophy in diabetes and its possible influence on peroxisome proliferator-activated receptor-α (PPARα/nitric oxide (NO signaling pathway. The cardiomyocyte hypertrophy induced by high glucose (25.5 mmol/L and insulin (0.1 μmol/L (HGI was characterized in rat primary cardiomyocyte by measuring the cell surface area, protein content, and atrial natriuretic factor mRNA expression level. Protein and mRNA expression were measured by western blot and real-time RT-PCR, respectively. The enzymatic activity of NO synthase (NOS was measured using a spectrophotometric assay, and NO concentration was measured using the Griess assay. HGI significantly induced cardiomyocyte hypertrophy and decreased the expression of PPARα and endothelial NOS at the mRNA and protein levels, which occurred in parallel with declining NOS activity and NO concentration. The effect of HGI was inhibited by berberine (0.1 to 100 μmol/L, fenofibrate (0.3 μmol/L, or L-arginine (100 μmol/L. MK886 (0.3 μmol/L, a selective PPARα antagonist, could abolish the effects of berberine and fenofibrate. NG-nitro-L-arginine-methyl ester (100 μmol/L, a NOS inhibitor, could block the effects of L-arginine, but only partially blocked the effects of berberine. These results suggest that berberine can blunt HGI-induced cardiomyocyte hypertrophy in vitro, through the activation of the PPARα/NO signaling pathway.

Hypoxia and hypoxia inducible factor-1α are required for normal endometrial repair during menstruation.

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Maybin, Jacqueline A; Murray, Alison A; Saunders, Philippa T K; Hirani, Nikhil; Carmeliet, Peter; Critchley, Hilary O D

2018-01-23

Heavy menstrual bleeding (HMB) is common and debilitating, and often requires surgery due to hormonal side effects from medical therapies. Here we show that transient, physiological hypoxia occurs in the menstrual endometrium to stabilise hypoxia inducible factor 1 (HIF-1) and drive repair of the denuded surface. We report that women with HMB have decreased endometrial HIF-1α during menstruation and prolonged menstrual bleeding. In a mouse model of simulated menses, physiological endometrial hypoxia occurs during bleeding. Maintenance of mice under hyperoxia during menses decreases HIF-1α induction and delays endometrial repair. The same effects are observed upon genetic or pharmacological reduction of endometrial HIF-1α. Conversely, artificial induction of hypoxia by pharmacological stabilisation of HIF-1α rescues the delayed endometrial repair in hypoxia-deficient mice. These data reveal a role for HIF-1 in the endometrium and suggest its pharmacological stabilisation during menses offers an effective, non-hormonal treatment for women with HMB.

Mitochondrial Reactive Oxygen Species Trigger Hypoxia-Induced Transcription

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Chandel, N. S.; Maltepe, E.; Goldwasser, E.; Mathieu, C. E.; Simon, M. C.; Schumacker, P. T.

1998-09-01

Transcriptional activation of erythropoietin, glycolytic enzymes, and vascular endothelial growth factor occurs during hypoxia or in response to cobalt chloride (CoCl2) in Hep3B cells. However, neither the mechanism of cellular O2 sensing nor that of cobalt is fully understood. We tested whether mitochondria act as O2 sensors during hypoxia and whether hypoxia and cobalt activate transcription by increasing generation of reactive oxygen species (ROS). Results show (i) wild-type Hep3B cells increase ROS generation during hypoxia (1.5% O2) or CoCl2 incubation, (ii) Hep3B cells depleted of mitochondrial DNA (ρ 0 cells) fail to respire, fail to activate mRNA for erythropoietin, glycolytic enzymes, or vascular endothelial growth factor during hypoxia, and fail to increase ROS generation during hypoxia; (iii) ρ 0 cells increase ROS generation in response to CoCl2 and retain the ability to induce expression of these genes; and (iv) the antioxidants pyrrolidine dithiocarbamate and ebselen abolish transcriptional activation of these genes during hypoxia or CoCl2 in wild-type cells, and abolish the response to CoCl2 in ρ 0 cells. Thus, hypoxia activates transcription via a mitochondria-dependent signaling process involving increased ROS, whereas CoCl2 activates transcription by stimulating ROS generation via a mitochondria-independent mechanism.

Hypoxia-induced lipid peroxidation in the brain during postnatal ontogenesis

Czech Academy of Sciences Publication Activity Database

Rauchová, Hana; Vokurková, Martina; Koudelová, J.

2012-01-01

Roč. 61, Suppl.1 (2012), S89-S101 ISSN 0862-8408 R&D Projects: GA MŠk(CZ) 1M0510; GA ČR(CZ) GAP304/12/0259 Institutional research plan: CEZ:AV0Z50110509 Keywords : hypobaric hypoxia * reactive oxygen species (ROS) * polyunsaturated fatty acids * Na * K-ATPase * catalase * L-carnitine Subject RIV: FA - Cardiovascular Diseases incl. Cardiotharic Surgery Impact factor: 1.531, year: 2012

Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5 expression through both hypoxia inducible factor-1α and proteasome-mediated pathways.

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Jitesh D Kawedia

Full Text Available The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5, we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70% decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels.

Hypoxia and Hypoxia Mimetics Decrease Aquaporin 5 (AQP5) Expression through Both Hypoxia Inducible Factor-1α and Proteasome-Mediated Pathways

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Kawedia, Jitesh D.; Yang, Fan; Sartor, Maureen A.; Gozal, David; Czyzyk-Krzeska, Maria; Menon, Anil G.

2013-01-01

The alveolar epithelium plays a central role in gas exchange and fluid transport, and is therefore critical for normal lung function. Since the bulk of water flux across this epithelium depends on the membrane water channel Aquaporin 5 (AQP5), we asked whether hypoxia had any effect on AQP5 expression. We show that hypoxia causes a significant (70%) decrease in AQP5 expression in the lungs of mice exposed to hypoxia. Hypoxia and the hypoxia mimetic, cobalt, also caused similar decreases in AQP5 mRNA and protein expression in the mouse lung epithelial cell line MLE-12. The action of hypoxia and cobalt on AQP5 transcription was demonstrated by directly quantifying heternonuclear RNA by real-time PCR. Dominant negative mutants of Hypoxia Inducible Factor (HIF-1α) and HIF-1α siRNA blocked the action of cobalt, showing that HIF-1α is a key component in this mechanism. The proteasome inhibitors, lactacystin or proteasome inhibitor-III completely abolished the effect of hypoxia and cobalt both at the protein and mRNA level indicating that the proteasome pathway is probably involved not only for the stability of HIF-1α protein, but for the stability of unidentified transcription factors that regulate AQP5 transcription. These studies reveal a potentially important physiological mechanism linking hypoxic stress and membrane water channels. PMID:23469202

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

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Ying Huang

2013-11-01

Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

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Sada, Kiminori; Nishikawa, Takeshi; Kukidome, Daisuke; Yoshinaga, Tomoaki; Kajihara, Nobuhiro; Sonoda, Kazuhiro; Senokuchi, Takafumi; Motoshima, Hiroyuki; Matsumura, Takeshi; Araki, Eiichi

2016-01-01

We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS) generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs), cellular hypoxia increased after incubation with high glucose (HG). A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD) overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1), a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner.

Hyperglycemia Induces Cellular Hypoxia through Production of Mitochondrial ROS Followed by Suppression of Aquaporin-1.

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Kiminori Sada

Full Text Available We previously proposed that hyperglycemia-induced mitochondrial reactive oxygen species (mtROS generation is a key event in the development of diabetic complications. Interestingly, some common aspects exist between hyperglycemia and hypoxia-induced phenomena. Thus, hyperglycemia may induce cellular hypoxia, and this phenomenon may also be involved in the pathogenesis of diabetic complications. In endothelial cells (ECs, cellular hypoxia increased after incubation with high glucose (HG. A similar phenomenon was observed in glomeruli of diabetic mice. HG-induced cellular hypoxia was suppressed by mitochondria blockades or manganese superoxide dismutase (MnSOD overexpression, which is a specific SOD for mtROS. Overexpression of MnSOD also increased the expression of aquaporin-1 (AQP1, a water and oxygen channel. AQP1 overexpression in ECs suppressed hyperglycemia-induced cellular hypoxia, endothelin-1 and fibronectin overproduction, and apoptosis. Therefore, hyperglycemia-induced cellular hypoxia and mtROS generation may promote hyperglycemic damage in a coordinated manner.

PKA activity exacerbates hypoxia-induced ROS formation and hypoxic injury in PC-12 cells.

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Gozal, Evelyne; Metz, Cynthia J; Dematteis, Maurice; Sachleben, Leroy R; Schurr, Avital; Rane, Madhavi J

2017-09-05

Hypoxia is a primary factor in many pathological conditions. Hypoxic cell death is commonly attributed to metabolic failure and oxidative injury. cAMP-dependent protein kinase A (PKA) is activated in hypoxia and regulates multiple enzymes of the mitochondrial electron transport chain, thus may be implicated in cellular energy depletion and hypoxia-induced cell death. Wild type (WT) PC-12 cells and PKA activity-deficient 123.7 PC-12 cells were exposed to 3, 6, 12 and 24h hypoxia (0.1% or 5% O 2 ). Hypoxia, at 24h 0.1% O 2 , induced cell death and increased reactive oxygen species (ROS) in WT PC-12 cells. Despite lower ATP levels in normoxic 123.7 cells than in WT cells, hypoxia only decreased ATP levels in WT cells. However, menadione-induced oxidative stress similarly affected both cell types. While mitochondrial COX IV expression remained consistently higher in 123.7 cells, hypoxia decreased COX IV expression in both cell types. N-acetyl cysteine antioxidant treatment blocked hypoxia-induced WT cell death without preventing ATP depletion. Transient PKA catα expression in 123.7 cells partially restored hypoxia-induced ROS but did not alter ATP levels or COX IV expression. We conclude that PKA signaling contributes to hypoxic injury, by regulating oxidative stress rather than by depleting ATP levels. Therapeutic strategies targeting PKA signaling may improve cellular adaptation and recovery in hypoxic pathologies. Copyright © 2017 Elsevier B.V. All rights reserved.

Disparate roles of zinc in chemical hypoxia-induced neuronal death

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Sujeong eKim

2015-01-01

Full Text Available Accumulating evidence has provided a causative role of zinc (Zn2+ in neuronal death following ischemic brain injury. Using a hypoxia model of primary cultured cortical neurons with hypoxia-inducing chemicals, cobalt chloride (1 mM CoCl2, deferoxamine (3 mM DFX, and sodium azide (2 mM NaN3, we evaluated whether Zn2+ is involved in hypoxic neuronal death. The hypoxic chemicals rapidly elicited intracellular Zn2+ release/accumulation in viable neurons. The immediate addition of the Zn2+ chelator, CaEDTA or N,N,N’N’-tetrakis-(2-pyridylmethyl ethylenediamine (TPEN, prevented the intracellular Zn2+ load and CoCl2-induced neuronal death, but neither 3-hour-later Zn2+ chelation nor a non-Zn2+ chelator ZnEDTA (1 mM demonstrated any effects. However, neither CaEDTA nor TPEN rescued neurons from cell death following DFX- or NaN3-induced hypoxia, whereas ZnEDTA rendered them resistant to the hypoxic injury. Instead, the immediate supplementation of Zn2+ rescued DFX- and NaN3-induced neuronal death. The iron supplementation also afforded neuroprotection against DFX-induced hypoxic injury. Thus, although intracellular Zn2+ release/accumulation is common during chemical hypoxia, Zn2+ might differently influence the subsequent fate of neurons; it appears to play a neurotoxic or neuroprotective role depending on the hypoxic chemical used. These results also suggest that different hypoxic chemicals may induce neuronal death via distinct mechanisms.

Disparate roles of zinc in chemical hypoxia-induced neuronal death.

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Kim, Sujeong; Seo, Jung-Woo; Oh, Shin Bi; Kim, So Hee; Kim, Inki; Suh, Nayoung; Lee, Joo-Yong

2015-01-01

Accumulating evidence has provided a causative role of zinc (Zn(2+)) in neuronal death following ischemic brain injury. Using a hypoxia model of primary cultured cortical neurons with hypoxia-inducing chemicals, cobalt chloride (1 mM CoCl2), deferoxamine (3 mM DFX), and sodium azide (2 mM NaN3), we evaluated whether Zn(2+) is involved in hypoxic neuronal death. The hypoxic chemicals rapidly elicited intracellular Zn(2+) release/accumulation in viable neurons. The immediate addition of the Zn(2+) chelator, CaEDTA or N,N,N'N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), prevented the intracellular Zn(2+) load and CoCl2-induced neuronal death, but neither 3 hour later Zn(2+) chelation nor a non-Zn(2+) chelator ZnEDTA (1 mM) demonstrated any effects. However, neither CaEDTA nor TPEN rescued neurons from cell death following DFX- or NaN3-induced hypoxia, whereas ZnEDTA rendered them resistant to the hypoxic injury. Instead, the immediate supplementation of Zn(2+) rescued DFX- and NaN3-induced neuronal death. The iron supplementation also afforded neuroprotection against DFX-induced hypoxic injury. Thus, although intracellular Zn(2+) release/accumulation is common during chemical hypoxia, Zn(2+) might differently influence the subsequent fate of neurons; it appears to play a neurotoxic or neuroprotective role depending on the hypoxic chemical used. These results also suggest that different hypoxic chemicals may induce neuronal death via distinct mechanisms.

Paeoniflorin prevents hypoxia-induced epithelial–mesenchymal transition in human breast cancer cells

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Zhou Z

2016-04-01

Full Text Available Zhenyu Zhou,1,* Shunchang Wang,1,* Caijuan Song,2 Zhuang Hu11Department of Thyroid and Breast, Huaihe Hospital, Henan University, Kaifeng, 2Department of Immunization Program, Zhengzhou Center for Disease Control and Prevention, Zhengzhou, People’s Republic of China*These authors contributed equally to this workAbstract: Paeoniflorin (PF is a monoterpene glycoside extracted from the root of Paeonia lactiflora Pall. Previous studies have demonstrated that PF inhibits the growth, invasion, and metastasis of tumors in vivo and in vitro. However, the effect of PF on hypoxia-induced epithelial–mesenchymal transition (EMT in breast cancer cells remains unknown. Therefore, the objective of this study was to investigate the effect of PF on hypoxia-induced EMT in breast cancer cells, as well as characterize the underlying mechanism. The results presented in this study demonstrate that PF blocks the migration and invasion of breast cancer cells by repressing EMT under hypoxic conditions. PF also significantly attenuated the hypoxia-induced increase in HIF-1α level. Furthermore, PF prevented hypoxia-induced expression of phosphorylated PI3K and Akt in MDA-MB-231 cells. In conclusion, PF prevented hypoxia-induced EMT in breast cancer cells by inhibiting HIF-1α expression via modulation of PI3K/Akt signaling pathway. This finding provides evidence that PF can serve as a therapeutic agent for the treatment of breast cancer.Keywords: paeoniflorin, breast cancer, hypoxia, epithelial–mesenchymal transition, PI3K/Akt signaling pathway

Does Acute Normobaric Hypoxia Induce Anapyrexia in Adult Humans?

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Seo, Yongsuk; Gerhart, Hayden D; Vaughan, Jeremiah; Kim, Jung-Hyun; Glickman, Ellen L

2017-06-01

Seo, Yongsuk, Hayden D. Gerhart, Jeremiah Vaughan, Jung-Hyun Kim, and Ellen L. Glickman. Does acute normobaric hypoxia induce anapyrexia in adult humans? High Alt Med Biol. 18:185-190, 2017.-Exposure to hypoxia is known to induce a reduction in core body temperature as a protective mechanism, which has been shown in both animals and humans. The purpose of this study was to test if acute exposure to normobaric hypoxia (NH) induces anapyrexia in adult humans in association with decreased peripheral oxygen saturation (SpO 2 ). Ten healthy male subjects were seated in atmospheres of normobaric normoxia 21% (NN21), NH 17% (NH17), and 13% (NH13) O 2 for 60 minutes in a counterbalanced manner. Rectal temperature (Tre) was continuously monitored together with the quantification of metabolic heat production (MHP) and body heat storage (S). Baseline physiological measurements showed no differences between the three conditions. SpO 2 was significantly decreased in NH17 and NH13 compared with NN21 (p ≤ 0.001). Tre decreased following 60 minutes of resting in all conditions, but, independent of the conditions, showed no association between Tre and levels of hypoxic SpO 2 . There was also no significant difference in either MHP or S between conditions. The present results showed no evidence of hypoxia-induced anapyrexia in adult humans during 1 hour of resting after exposure to NH either at 13% or 17% O 2 .

Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

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Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

2014-04-01

In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.

Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

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Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

2014-11-01

Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ

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Cha, Min-Ji; Jang, Jin-Kyung; Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon; Lee, Chang Yeon; Park, Jun-Hee; Lee, Jiyun; Seo, Hyang-Hee; Choi, Eunhyun; Jeon, Woo-min; Hwang, Hye Jin; Shin, Hyun-Taek

2013-01-01

Highlights: •CaMKIIδ mediates H 2 O 2 -induced Ca 2+ overload in cardiomyocytes. •miR-145 can inhibit Ca 2+ overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca 2+ ) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca 2+ signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca 2+ -mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H 2 O 2 -mediated Ca 2+ overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca 2+ overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca 2+ -related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca 2+ overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses

Hypoxia-inducible factor-1 alpha has a key role in hypoxic preconditioning.

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Taie, Satoshi; Ono, Junichiro; Iwanaga, Yasuyuki; Tomita, Shuhei; Asaga, Takehiko; Chujo, Kosuke; Ueki, Masaaki

2009-08-01

Sublethal hypoxia induces tolerance to subsequent hypoxic insults in a process known as hypoxic preconditioning (HP). Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key transcription protein involved in the mechanism of HP. In this study, we investigated the effects of HP on tissue oxygenation and expression of HIF-1 alpha gene targets in the brain using neural cell-specific HIF-1 alpha-deficient mice. The animals were exposed to 8% oxygen for 3 hours. Twenty-four hours later, the oxygen partial pressure (pO(2)) of brain tissue and gene expression were measured during hypoxia. HP improved the pO(2) of brain tissue during subsequent hypoxia with upregulated inducible nitric oxide synthase in wild-type mice, whereas HP had no detectable effect in the mutant mice. Our results indicate that the protective effects of HP may be partially mediated by improving tissue oxygenation via HIF-1 alpha and inducible nitric oxide synthase.

Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

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Yi-Wen Chen

2013-01-01

Full Text Available Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3, caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP, Ena/VASP-like protein (Evl, and isopentenyl-diphosphate delta-isomerase 1 (Idi-1 were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

HIF-1 and NDRG2 contribute to hypoxia-induced radioresistance of cervical cancer Hela cells

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Liu, Junye; Zhang, Jing; Wang, Xiaowu; Li, Yan; Chen, Yongbin; Li, Kangchu; Zhang, Jian; Yao, Libo; Guo, Guozhen

2010-01-01

Hypoxia inducible factor 1 (HIF-1), the key mediator of hypoxia signaling pathways, has been shown involved in hypoxia-induced radioresistance. However, the underlying mechanisms are unclear. The present study demonstrated that both hypoxia and hypoxia mimetic cobalt chloride could increase the radioresistance of human cervical cancer Hela cells. Meanwhile, ectopic expression of HIF-1 could enhance the resistance of Hela cells to radiation, whereas knocking-down of HIF-1 could increase the sensitivity of Hela cells to radiation in the presence of hypoxia. N-Myc downstream-regulated gene 2 (NDRG2), a new HIF-1 target gene identified in our lab, was found to be upregulated by hypoxia and radiation in a HIF-1-dependent manner. Overexpression of NDRG2 resulted in decreased sensitivity of Hela cells to radiation while silencing NDRG2 led to radiosensitization. Moreover, NDRG2 was proved to protect Hela cells from radiation-induced apoptosis and abolish radiation-induced upregulation of Bax. Taken together, these data suggest that both HIF-1 and NDRG2 contribute to hypoxia-induced tumor radioresistance and that NDRG2 acts downstream of HIF-1 to promote radioresistance through suppressing radiation-induced Bax expression. It would be meaningful to further explore the clinical application potential of HIF-1 and NDRG2 blockade as radiosensitizer for tumor therapy.

Role of JAK-STAT pathway in reducing cardiomyocytes hypoxia/reoxygenation injury induced by S1P postconditioning.

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Wang, Yuqing; Wang, Dongfei; Zhang, Lizhi; Ye, Fangyu; Li, Mengmeng; Wen, Ke

2016-08-05

This experiment was designed to explore the protection of sphingosine1-phosphate (S1P) postconditioning on rat myocardial cells injured by hypoxia/reoxygenation acting via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal pathway. The data showed that S1P could significantly increase cell viability, lower the rate of apoptosis, decrease the content of lactate dehydrogenase (LDH) and caspase3 activity in the culture medium, increase the activity of total superoxide dismutase (T-SOD) and manganese superoxide dismutase (Mn-SOD), reduce the loss of mitochondrial membrane potential and the fluorescence intensity of intracellular calcium, as well as increase the phosphorylation of JAK2 and STAT3 in comparison with the H/R group. When the JAK inhibitor AG490 or the STAT inhibitor stattic were added, the effects of S1P were inhibited. Our date shows that S1P protects H9c2 cells from hypoxia/reoxygenation injury and that the protection by S1P was inhibited by AG490 and stattic. Therefore S1P protects H9c2 cells against hypoxia/reoxygenation injury via the JAK-STAT pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

Cardiotoxicity evaluation using human embryonic stem cells and induced pluripotent stem cell-derived cardiomyocytes.

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Zhao, Qi; Wang, Xijie; Wang, Shuyan; Song, Zheng; Wang, Jiaxian; Ma, Jing

2017-03-09

Cardiotoxicity remains an important concern in drug discovery. Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have become an attractive platform to evaluate cardiotoxicity. However, the consistency between human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in prediction of cardiotoxicity has yet to be elucidated. Here we screened the toxicities of four representative drugs (E-4031, isoprenaline, quinidine, and haloperidol) using both hESC-CMs and hiPSC-CMs, combined with an impedance-based bioanalytical method. It showed that both hESC-CMs and hiPSC-CMs can recapitulate cardiotoxicity and identify the effects of well-characterized compounds. The combined platform of hPSC-CMs and an impedance-based bioanalytical method could improve preclinical cardiotoxicity screening, holding great potential for increasing drug development accuracy.

Qidantongmai Protects Endothelial Cells Against Hypoxia-Induced ...

African Journals Online (AJOL)

induced damage. The ability of QDTM to modulate the serum VEGF-A level may play an important role in its effects on endothelial cells. Key words: Traditional Chinese Medicine, human umbilical vein endothelial cells, hypoxia, VEGF ...

Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.

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Nicolas Christoforou

Full Text Available The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS cells, which once differentiated allow for the enrichment of Nkx2-5(+ cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+ cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors' ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological

Induction of gastrin expression in gastrointestinal cells by hypoxia or cobalt is independent of hypoxia-inducible factor (HIF).

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Xiao, Lin; Kovac, Suzana; Chang, Mike; Shulkes, Arthur; Baldwin, Graham S; Patel, Oneel

2012-07-01

Gastrin and its precursors have been shown to promote mitogenesis and angiogenesis in gastrointestinal tumors. Hypoxia stimulates tumor growth, but its effect on gastrin gene regulation has not been examined in detail. Here we have investigated the effect of hypoxia on the transcription of the gastrin gene in human gastric cancer (AGS) cells. Gastrin mRNA was measured by real-time PCR, gastrin peptides were measured by RIA, and gastrin promoter activity was measured by dual-luciferase reporter assay. Exposure to a low oxygen concentration (1%) increased gastrin mRNA concentrations in wild-type AGS cells (AGS) and in AGS cells overexpressing the gastrin receptor (AGS-cholecystokinin receptor 2) by 2.1 ± 0.4- and 4.1 ± 0.3-fold (P factor hypoxia-inducible factor 1 (HIF-1) or knockdown of either the HIF-1α or HIF-1β subunit did not affect gastrin promoter inducibility under hypoxia indicated that the hypoxic activation of the gastrin gene is likely HIF independent. Mutational analysis of previously identified Sp1 regulatory elements in the gastrin promoter also failed to abrogate the induction of promoter activity by hypoxia. The observations that hypoxia up-regulates the gastrin gene in AGS cells by HIF-independent mechanisms, and that this effect is enhanced by the presence of gastrin receptors, provide potential targets for gastrointestinal cancer therapy.

Regulation of HIF prolyl hydroxylases by hypoxia-inducible factors.

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Aprelikova, Olga; Chandramouli, Gadisetti V R; Wood, Matthew; Vasselli, James R; Riss, Joseph; Maranchie, Jodi K; Linehan, W Marston; Barrett, J Carl

2004-06-01

Hypoxia and induction of hypoxia-inducible factors (HIF-1alpha and HIF-2alpha) is a hallmark of many tumors. Under normal oxygen tension HIF-alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel-Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re-introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF-1 and HIF-2 and found that siRNA targeted degradation of HIF-1alpha and HIF-2alpha results in decreased hypoxia-induced PHD3 expression. Ectopic overexpression of HIF-2alpha in two different cell lines provided a much better induction of PHD3 gene than HIF-1alpha. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF-2alpha. However, induction of PHD2 by hypoxia has HIF-1-independent and -dependent components. Short-term hypoxia (4 h) results in induction of PHD2 independent of HIF-1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA-mediated degradation of HIF-1alpha subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Copyright 2004 Wiley-Liss, Inc.

Hypoxia and hypoxia-inducible factors as regulators of T cell development, differentiation, and function

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McNamee, Eóin N.; Johnson, Darlynn Korns; Homann, Dirk

2014-01-01

Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies. PMID:22961658

A polysaccharide of Dendrobium officinale ameliorates H2O2-induced apoptosis in H9c2 cardiomyocytes via PI3K/AKT and MAPK pathways.

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Zhang, Jing-Yi; Guo, Ying; Si, Jin-Ping; Sun, Xiao-Bo; Sun, Gui-Bo; Liu, Jing-Jing

2017-11-01

Dendrobium officinale is one valuable traditional Chinese medicine, which has skyscraping medicinal value. Polysaccharide is the main active ingredient in D. officinale; its antioxidant activity is a hot research topic nowadays. Oxidative stress plays an important role in the pathological progress of a variety of cardiovascular disease, as one of key factors of cardiomyocyte apoptosis. This research adopts a model of H 2 O 2 induction-H9c2 cardiomyocytes apoptosis, aiming to study the effect of Dendrobium officinale Polysaccharide (DOP-GY) for cardiomyocyte apoptosis caused by oxidative stress and its possible mechanism. Our results showed that pretreatment of DOP-GY (low dose: 6.25μg/mL, medium dose: 12.5μg/mL, high dose: 25μg/mL) followed by a 2h incubation with 200μM H 2 O 2 elevated the survival rate, cutted the LDH leakage, reduced lipid peroxidation damage, improved the activity of the endogenous antioxidant enzymes. In addition, the pretreatment of DOP-GY significantly inhibited the production of ROS, declined of the mitochondrial membrane potential, down-regulated pro-apoptosis protein and up-regulated anti-apoptosis protein. The protective effect was correlated with the PI3K/Akt and MAPK signal pathway. Collectively, these observations suggest that DOY-GY has the potential to exert cardioprotective effects against H 2 O 2 -induced H9c2 cardiomyocyte apoptosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

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Ribeiro, Maisa; Teixeira, Sarah R; Azevedo, Monarko N; Fraga, Ailton C; Gontijo, Antônio Pm; Vêncio, Eneida F

2017-04-01

To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

Electrophysiological, haemodynamic, and mitochondrial alterations induced by levobupivacaine during myocardial ischemia in a pig model: protection by lipid emulsions?

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Mamou, Zahida; Descotes, Jacques; Chevalier, Philippe; Bui-Xuan, Bernard; Romestaing, Caroline; Timour, Quadiri

2015-10-01

Accidental intravascular or high-dose injection of local anesthetics (LA) can result in serious, potentially life-threatening complications. Indeed, adequate supportive measures and the administration of lipid emulsions are required in such complications. The study's objectives were threefold: (i) evaluate the myocardial toxicity of levobupivacaine when administered intravenously; (ii) investigate levobupivacaine toxicity on cardiomyocytes mitochondrial functions and cellular structure; (iii) assess the protective effects of a lipid emulsion in the presence or absence of myocardial ischemia. Domestic pigs randomized into two groups of 24 animals each, with either preserved coronary circulation or experimental myocardial ischemia. Six animals from each group received either: (i) single IV injection of saline, (ii) lipid emulsion (Intralipid(®) ), (iii) levobupivacaine, (iv) combination levobupivacaine-Intralipid(®) . Serially measured endpoints included: heart rate, duration of the monophasic action potentials (dMAP), mean arterial pressure, and peak of the time derivative of left ventricular pressure (LV dP/dtmax ). In addition, the following cardiomyocytes mitochondrial functions were measured: reactive oxygen species (ROS) production, oxidative phosphorylation, and calcium retention capacity (CRC) as well as the consequences of ROS production on lipids, proteins, and DNA. IV injection of levobupivacaine induced sinus bradycardia and reduced dMAP and LV dP/dtmax . At the mitochondrial level, oxygen consumption and CRC were decreased. In contrast, ROS production was increased leading to enhanced lipid peroxidation and structural alterations of proteins and DNA. Myocardial ischemia was associated with global worsening of all changes. Intralipid(®) quickly improved haemodynamics. However, beneficial effects of Intralipid(®) were less clear after myocardial ischemia. © 2015 Société Française de Pharmacologie et de Thérapeutique.

Arrhythmia causes lipid accumulation and reduced glucose uptake.

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Lenski, Matthias; Schleider, Gregor; Kohlhaas, Michael; Adrian, Lucas; Adam, Oliver; Tian, Qinghai; Kaestner, Lars; Lipp, Peter; Lehrke, Michael; Maack, Christoph; Böhm, Michael; Laufs, Ulrich

2015-01-01

Atrial fibrillation (AF) is characterized by irregular contractions of atrial cardiomyocytes and increased energy demand. The aim of this study was to characterize the influence of arrhythmia on glucose and fatty acid (FA) metabolism in cardiomyocytes, mice and human left atrial myocardium. Compared to regular pacing, irregular (pseudo-random variation at the same number of contractions/min) pacing of neonatal rat cardiomyocytes induced shorter action potential durations and effective refractory periods and increased diastolic [Ca(2+)]c. This was associated with the activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and AMP-activated protein kinase (AMPK). Membrane expression of fatty acid translocase (FAT/CD36) and (14)C-palmitic acid uptake were augmented while membrane expression of glucose transporter subtype 4 (GLUT-4) as well as (3)H-glucose uptake were reduced. Inhibition of AMPK and CaMKII prevented these arrhythmia-induced metabolic changes. Similar alterations of FA metabolism were observed in a transgenic mouse model (RacET) for spontaneous AF. Consistent with these findings samples of left atrial myocardium of patients with AF compared to matched samples of patients with sinus rhythm showed up-regulation of CaMKII and AMPK and increased membrane expression of FAT/CD36, resulting in lipid accumulation. These changes of FA metabolism were accompanied by decreased membrane expression of GLUT-4, increased glycogen content and increased expression of the pro-apoptotic protein bax. Irregular pacing of cardiomyocytes increases diastolic [Ca(2+)]c and activation of CaMKII and AMPK resulting in lipid accumulation, reduced glucose uptake and increased glycogen synthesis. These metabolic changes are accompanied by an activation of pro-apoptotic signalling pathways.

Experimental research on recombinant human endostatin-induced cardiomyocyte apoptosis in rats

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Jing QIN

2014-03-01

Full Text Available Objective　To explore the recombinant human endostatin (rh-ES-induced cardiotoxicity in rats and its mechanism. Methods　Twenty four female Wistar rats were randomly divided into four groups (6 each. Rats in low, moderate and high dose group received rh-ES with a dosage of 3, 6 and 12mg/(kg·d, respectively, by intraperitoneal injection, and rats in control group received the same amount of normal saline alone. Half of rats in each group were sacrificed by spinal dislocation after 4 weeks and 8 weeks of the treatment. Pathomorphologic and ultrastructural changes in rat's myocardial tissue were evaluated by light microscopy and transmission electron microscopy. Cardiomyocyte apoptosis was detected with TdT-mediated dUTP nick end labeling (TUNEL assay. Microvessel density (MVD in myocardial tissue was measured by immunohistochemically marking endothelial cell with CD34. Results　No pathomorphologic and ultrastrucural changes were found under light microscope and transmission electron microscope in the low dose and moderate dose groups, but cardiomyocyte damage were found in the high dose group. TUNEL assay revealed more apoptotic cells in high and moderate (only 8 weeks dose groups than in control group (P=0.033, P=0.000, and the apoptosis index was highest in the high dose group at 8 weeks. In addition, compared with the control group, MVD significantly increased in high dose groups at 4 weeks and 8 weeks (P<0.05. Conclusions　rh-ES induces the cardiotoxicity in rats, and cardiomyocyte apoptosis is involved in the pathological course of cardiac toxicity. DOI: 10.11855/j.issn.0577-7402.2014.01.02

Zeaxanthin Inhibits Hypoxia-Induced VEGF Secretion by RPE Cells through Decreased Protein Levels of Hypoxia-Inducible Factors-1α

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Richard Rosen

2015-01-01

Full Text Available Hypoxia is the most important stimulus leading to upregulation of VEGF in the retina and this is caused by accumulation of hypoxia-inducible factors-1α (HIF-1α protein. The effects of zeaxanthin, a natural phytochemical, on the VEGF and HIF-1α expression in the primary culture of human retinal pigment epithelial (RPE cells were studied. An in vitro RPE cell hypoxia model was established by placing cells under 1% oxygen pressure or by adding cobalt chloride (CoCl2 to the culture medium. RPE cells and conditioned media were collected from cultures treated with and without zeaxanthin under normoxic and hypoxic conditions. VEGF and HIF-1α protein and RNA levels were measured by ELISA kits and RT-PCR, respectively. Hypoxia caused a significant increase of VEGF expression and accumulation of HIF-1α in RPE cells. Zeaxanthin at 50–150 μM significantly inhibited the expression of VEGF and accumulation of HIF-1α protein caused by hypoxia but did not affect expression of VEGF and HIF-1α under normoxic conditions. This is the first report on the effect of zeaxanthin on VEGF and HIF-1α levels in cultured RPE cells and suggests that zeaxanthin may have potential value in the prevention and treatment of various retinal diseases associated with vascular leakage and neovascularization.

Hypoxia-Inducible Regulation of a Prodrug-Activating Enzyme for Tumor-Specific Gene Therapy

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Toru Shibata

2002-01-01

Full Text Available Previous studies have suggested that tumor hypoxia could be exploited for cancer gene therapy. Using hypoxia-responsive elements derived from the human vascular endothelial growth factor gene, we have generated vectors expressing a bacterial nitroreductase. (20NTR gene that can activate the anticancer prodrug CB1954. Stable transfectants of human HT1080 tumor cells with hypoxia-inducible vectors were established with G418 selection. Hypoxic induction of NTR protein correlated with increased sensitivity to in vitro exposure of HT 1080 cells to the prodrug. Growth delay assays were performed with established tumor xenografts derived from the same cells to detect the in vivo efficacy of CB1954 conversion to its cytotoxic form. Significant antitumor effects were achieved with intraperitoneal injections of CB1954 both in tumors that express NTR constitutively or with a hypoxia-inducible promoter. In addition, respiration of 10% O2 increased tumor hypoxia in vivo and enhanced the antitumor effects. Taken together, these results demonstrate that hypoxia-inducible vectors may be useful for tumor-selective gene therapy, although the problem of delivery of the vector to the tumors, particularly to the hypoxic cells in the tumors, is not addressed by these studies.

Radiation-induced hypoxia may perpetuate late normal tissue injury

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Vujaskovic, Zeljko; Anscher, Mitchell S.; Feng, Q.-F.; Rabbani, Zahid N.; Amin, Khalid; Samulski, Thaddeus S.; Dewhirst, Mark W.; Haroon, Zishan A.

2001-01-01

Purpose: The purpose of this study was to determine whether or not hypoxia develops in rat lung tissue after radiation. Methods and Materials: Fisher-344 rats were irradiated to the right hemithorax using a single dose of 28 Gy. Pulmonary function was assessed by measuring the changes in respiratory rate every 2 weeks, for 6 months after irradiation. The hypoxia marker was administered 3 h before euthanasia. The tissues were harvested at 6 weeks and 6 months after irradiation and processed for immunohistochemistry. Results: A moderate hypoxia was detected in the rat lungs at 6 weeks after irradiation, before the onset of functional or histopathologic changes. The more severe hypoxia, that developed at the later time points (6 months) after irradiation, was associated with a significant increase in macrophage activity, collagen deposition, lung fibrosis, and elevation in the respiratory rate. Immunohistochemistry studies revealed an increase in TGF-β, VEGF, and CD-31 endothelial cell marker, suggesting a hypoxia-mediated activation of the profibrinogenic and proangiogenic pathways. Conclusion: A new paradigm of radiation-induced lung injury should consider postradiation hypoxia to be an important contributing factor mediating a continuous production of a number of inflammatory and fibrogenic cytokines

Efficient and scalable purification of cardiomyocytes from human embryonic and induced pluripotent stem cells by VCAM1 surface expression.

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Hideki Uosaki

Full Text Available RATIONALE: Human embryonic and induced pluripotent stem cells (hESCs/hiPSCs are promising cell sources for cardiac regenerative medicine. To realize hESC/hiPSC-based cardiac cell therapy, efficient induction, purification, and transplantation methods for cardiomyocytes are required. Though marker gene transduction or fluorescent-based purification methods have been reported, fast, efficient and scalable purification methods with no genetic modification are essential for clinical purpose but have not yet been established. In this study, we attempted to identify cell surface markers for cardiomyocytes derived from hESC/hiPSCs. METHOD AND RESULT: We adopted a previously reported differentiation protocol for hESCs based on high density monolayer culture to hiPSCs with some modification. Cardiac troponin-T (TNNT2-positive cardiomyocytes appeared robustly with 30-70% efficiency. Using this differentiation method, we screened 242 antibodies for human cell surface molecules to isolate cardiomyocytes derived from hiPSCs and identified anti-VCAM1 (Vascular cell adhesion molecule 1 antibody specifically marked cardiomyocytes. TNNT2-positive cells were detected at day 7-8 after induction and 80% of them became VCAM1-positive by day 11. Approximately 95-98% of VCAM1-positive cells at day 11 were positive for TNNT2. VCAM1 was exclusive with CD144 (endothelium, CD140b (pericytes and TRA-1-60 (undifferentiated hESCs/hiPSCs. 95% of MACS-purified cells were positive for TNNT2. MACS purification yielded 5-10×10(5 VCAM1-positive cells from a single well of a six-well culture plate. Purified VCAM1-positive cells displayed molecular and functional features of cardiomyocytes. VCAM1 also specifically marked cardiomyocytes derived from other hESC or hiPSC lines. CONCLUSION: We succeeded in efficiently inducing cardiomyocytes from hESCs/hiPSCs and identifying VCAM1 as a potent cell surface marker for robust, efficient and scalable purification of cardiomyocytes from h

Hypoxia-inducible factor-1 plays a role in phosphate-induced vascular smooth muscle cell calcification.

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Mokas, Sophie; Larivière, Richard; Lamalice, Laurent; Gobeil, Stéphane; Cornfield, David N; Agharazii, Mohsen; Richard, Darren E

2016-09-01

Medial vascular calcification is a common complication of chronic kidney disease (CKD). Although elevated inorganic phosphate stimulates vascular smooth muscle cell (VSMC) osteogenic transdifferentiation and calcification, the mechanisms involved in their calcification during CKD are not fully defined. Because hypoxic gene activation is linked to CKD and stimulates bone cell osteogenic differentiation, we used in vivo and in vitro rodent models to define the role of hypoxic signaling during elevated inorganic phosphate-induced VSMC calcification. Cell mineralization studies showed that elevated inorganic phosphate rapidly induced VSMC calcification. Hypoxia strongly enhanced elevated inorganic phosphate-induced VSMC calcification and osteogenic transdifferentiation, as seen by osteogenic marker expression. Hypoxia-inducible factor-1 (HIF-1), the key hypoxic transcription factor, was essential for enhanced VSMC calcification. Targeting HIF-1 expression in murine VSMC blocked calcification in hypoxia with elevated inorganic phosphate while HIF-1 activators, including clinically used FG-4592/Roxadustat, recreated a procalcifying environment. Elevated inorganic phosphate rapidly activated HIF-1, even in normal oxygenation; an effect mediated by HIF-1α subunit stabilization. Thus, hypoxia synergizes with elevated inorganic phosphate to enhance VSMC osteogenic transdifferentiation. Our work identifies HIF-1 as an early CKD-related pathological event, prospective marker, and potential target against vascular calcification in CKD-relevant conditions. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

[Octanol preconditioning alleviates mouse cardiomyocyte swelling induced by simulated ischemia/reperfusion challenge in vitro].

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Luo, Yukun; Fang, Jun; Fan, Lin; Lin, Chaogui; Chen, Zhaoyang; Chen, Lianglong

2012-10-01

To investigate the role of connexin 43-formed hemichannels in cell volume regulation induced by simulated ischemia/reperfusion (SI/R). Mouse cardiomyocytes isolated on a Langendorff apparatus with enzyme solution were aliquoted into control, SI/R and SI/R +octanol groups. Calcein-AM was used to stain the cells and the cell volume was measured with confocal microscope by stack scanning. Trypan blue was used to measure the cell viability after the treatments. Calcein-AM staining and cofocal microscopy yielded stable and reproducible results for cell volume measurement. Mouse cardiomyocytes subjected to simulated SI/R showed obvious cell swelling as compared with the control cells [(126∓6)% vs 100%, Poctanol preconditioning significantly attenuated the cell swelling [(113∓6)%, Poctanol preconditioning obviously reduced the viability of the cells with SI/R challenge [(31∓2)%, Poctanol can alleviate the cell swelling to enhance the viability of the cardiomyocytes following SI/R.

Hypoxic pretreatment protects against neuronal damage of the rat hippocampus induced by severe hypoxia.

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Gorgias, N; Maidatsi, P; Tsolaki, M; Alvanou, A; Kiriazis, G; Kaidoglou, K; Giala, M

1996-04-01

The present study investigates whether under conditions of successive hypoxic exposures pretreatment with mild (15% O(2)) or moderate (10% O(2)) hypoxia, protects hippocampal neurones against damage induced by severe (3% O(2)) hypoxia. The ultrastructural findings were also correlated with regional superoxide dismutase (SOD) activity changes. In unpretreated rats severe hypoxia induced ultrastructural changes consistent with the aspects of delayed neuronal death (DND). However, in preexposed animals hippocampal damage was attenuated in an inversely proportional way with the severity of the hypoxic pretreatment. The ultrastructural hypoxic tolerance findings were also closely related to increased regional SOD activity levels. Thus the activation of the endogenous antioxidant defense by hypoxic preconditioning, protects against hippocampal damage induced by severe hypoxia. The eventual contribution of increased endogenous adenosine and/or reduced excitotoxicity to induce hypoxic tolerance is discussed.

HIF1 Contributes to Hypoxia-Induced Pancreatic Cancer Cells Invasion via Promoting QSOX1 Expression

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Chen-Ye Shi

2013-08-01

Full Text Available Background: Quiescin sulfhydryl oxidase 1 (QSOX1, which oxidizes sulfhydryl groups to form disulfide bonds in proteins, is found to be over-expressed in various pancreatic cancer cell lines and patients. QSOX1 promotes invasion of pancreatic cancer cells by activating MMP-2 and MMP-9. However, its regulatory mechanism remains largely undefined. Methods: Real-time PCR and Western blot were employed to detect the expression of QSOX1 in human pancreatic cancer cell lines under hypoxic condition. Luciferase reporter and ChIP assays were used to assess the regulation of QSOX1 by hypoxia-inducible factor 1 (HIF-1. Small interfering RNA (siRNA was applied to knock down endogenous expression of QSOX1. Matrigel-coated invasion chamber essays were conducted to detect the invasion capacity of QSOX1-depleted cells. Results: Both hypoxia and hypoxia mimicking reagent up-regulated the expression of QSOX1 in human pancreatic cancer cell lines. Knockdown of HIF-1α eliminated hypoxia induced QSOX1 expression. HIF-1α was found directly bound to two hypoxia-response elements (HRE of QSOX1 gene, both of which were required for HIF-1 induced QSOX1 expression. Moreover, QSOX1 silencing blocked hypoxia-induced pancreatic cancer cells invasion. Conclusion: QSOX1 is a direct target of HIF-1 and may contribute to hypoxia-induced pancreatic cancer cells invasion.

Cross-talk between lipid and protein carbonylation in a dynamic cardiomyocyte model of mild nitroxidative stress

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Eva Griesser

2017-04-01

Full Text Available Reactive oxygen and nitrogen species (ROS/RNS play an important role in the regulation of cardiac function. Increase in ROS/RNS concentration results in lipid and protein oxidation and is often associated with onset and/or progression of many cardiovascular disorders. However, interplay between lipid and protein modifications has not been simultaneously studied in detail so far. Biomolecule carbonylation is one of the most common biomarkers of oxidative stress. Using a dynamic model of nitroxidative stress we demonstrated rapid changes in biomolecule carbonylation in rat cardiomyocytes. Levels of carbonylated species increased as early as 15 min upon treatment with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1, and decreased to values close to control after 16 h. Total (lipids+proteins vs. protein-specific carbonylation showed different dynamics, with a significant increase in protein-bound carbonyls at later time points. Treatment with SIN-1 in combination with inhibitors of proteasomal and autophagy/lysosomal degradation pathways allowed confirmation of a significant role of the proteasome in the degradation of carbonylated proteins, whereas lipid carbonylation increased in the presence of autophagy/lysosomal inhibitors. Electrophilic aldehydes and ketones formed by lipid peroxidation were identified and relatively quantified using LC-MS/MS. Molecular identity of reactive species was used for data-driven analysis of their protein targets. Combination of different enrichment strategies with LC-MS/MS analysis allowed identification of more than 167 unique proteins with 332 sites modified by electrophilic lipid peroxidation products. Gene ontology analysis of modified proteins demonstrated enrichment of several functional categories including proteins involved in cytoskeleton, extracellular matrix, ion channels and their regulation. Using calcium mobilization assays, the effect of nitroxidative stress on the activity of several ion

Apigenin ameliorates hypertension-induced cardiac hypertrophy and down-regulates cardiac hypoxia inducible factor-lα in rats.

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Zhu, Zeng-Yan; Gao, Tian; Huang, Yan; Xue, Jie; Xie, Mei-Lin

2016-04-01

Apigenin is a natural flavonoid compound that can inhibit hypoxia-inducible factor (HIF)-1α expression in cultured tumor cells under hypoxic conditions. Hypertension-induced cardiac hypertrophy is always accompanied by abnormal myocardial glucolipid metabolism due to an increase of HIF-1α. However, whether or not apigenin may ameliorate the cardiac hypertrophy and abnormal myocardial glucolipid metabolism remains unknown. This study aimed to examine the effects of apigenin. Rats with cardiac hypertrophy induced by renovascular hypertension were treated with apigenin 50-100 mg kg(-1) (the doses can be achieved by pharmacological or dietary supplementation for an adult person) by gavage for 4 weeks. The results showed that after treatment with apigenin, the blood pressure, heart weight, heart weight index, cardiomyocyte cross-sectional area, serum angiotensin II, and serum and myocardial free fatty acids were reduced. It is important to note that apigenin decreased the expression level of myocardial HIF-1α protein. Moreover, apigenin simultaneously increased the expression levels of myocardial peroxisome proliferator-activated receptor (PPAR) α, carnitine palmitoyltransferase (CPT)-1, and pyruvate dehydrogenase kinase (PDK)-4 proteins and decreased the expression levels of myocardial PPARγ, glycerol-3-phosphate acyltransferase genes (GPAT), and glucose transporter (GLUT)-4 proteins. These findings demonstrated that apigenin could improve hypertensive cardiac hypertrophy and abnormal myocardial glucolipid metabolism in rats, and its mechanisms might be associated with the down-regulation of myocardial HIF-1α expression and, subsequently increasing the expressions of myocardial PPARα and its target genes CPT-1 and PDK-4, and decreasing the expressions of myocardial PPARγ and its target genes GPAT and GLUT-4.

Apelin Protects Primary Rat Retinal Pericytes from Chemical Hypoxia-Induced Apoptosis

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Li Chen

2015-01-01

Full Text Available Pericytes are a population of cells that participate in normal vessel architecture and regulate permeability. Apelin, as the endogenous ligand of G protein-coupled receptor APJ, participates in a number of physiological and pathological processes. To date, the effect of apelin on pericyte is not clear. Our study aimed to investigate the potential protection mechanisms of apelin, with regard to primary rat retinal pericytes under hypoxia. Immunofluorescence staining revealed that pericytes colocalized with APJ in the fibrovascular membranes dissected from proliferative diabetic retinopathy patients. In the in vitro studies, we first demonstrated that the expression of apelin/APJ was upregulated in pericytes under hypoxia, and apelin increased pericytes proliferation and migration. Moreover, knockdown of apelin in pericyte was achieved via lentivirus-mediated RNA interference. After the inhibition of apelin, pericytes proliferation was inhibited significantly in hypoxia culture condition. Furthermore, exogenous recombinant apelin effectively prevented hypoxia-induced apoptosis through downregulating active-caspase 3 expression and increasing the ratio of B cell lymphoma-2 (Bcl-2/Bcl-2 associated X protein (Bax in pericytes. These results suggest that apelin suppressed hypoxia-induced pericytes injury, which indicated that apelin could be a potential therapeutic target for retinal angiogenic diseases.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

2010-01-01

Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

2010-10-15

Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

Chronic hypoxia promotes pulmonary artery endothelial cell proliferation through H2O2-induced 5-lipoxygenase.

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Kristi M Porter

Full Text Available Pulmonary Hypertension (PH is a progressive disorder characterized by endothelial dysfunction and proliferation. Hypoxia induces PH by increasing vascular remodeling. A potential mediator in hypoxia-induced PH development is arachidonate 5-Lipoxygenase (ALOX5. While ALOX5 metabolites have been shown to promote pulmonary vasoconstriction and endothelial cell proliferation, the contribution of ALOX5 to hypoxia-induced proliferation remains unknown. We hypothesize that hypoxia exposure stimulates HPAEC proliferation by increasing ALOX5 expression and activity. To test this, human pulmonary artery endothelial cells (HPAEC were cultured under normoxic (21% O2 or hypoxic (1% O2 conditions for 24-, 48-, or 72 hours. In a subset of cells, the ALOX5 inhibitor, zileuton, or the 5-lipoxygenase activating protein inhibitor, MK-886, was administered during hypoxia exposure. ALOX5 expression was measured by qRT-PCR and western blot and HPAEC proliferation was assessed. Our results demonstrate that 24 and 48 hours of hypoxia exposure have no effect on HPAEC proliferation or ALOX5 expression. Seventy two hours of hypoxia significantly increases HPAEC ALOX5 expression, hydrogen peroxide (H2O2 release, and HPAEC proliferation. We also demonstrate that targeted ALOX5 gene silencing or inhibition of the ALOX5 pathway by pharmacological blockade attenuates hypoxia-induced HPAEC proliferation. Furthermore, our findings indicate that hypoxia-induced increases in cell proliferation and ALOX5 expression are dependent on H2O2 production, as administration of the antioxidant PEG-catalase blocks these effects and addition of H2O2 to HPAEC promotes proliferation. Overall, these studies indicate that hypoxia exposure induces HPAEC proliferation by activating the ALOX5 pathway via the generation of H2O2.

Expression of manganese superoxide dismutase in rat blood, heart and brain during induced systemic hypoxia

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Septelia I. Wanandi

2011-02-01

Full Text Available Background: Hypoxia results in an increased generation of ROS. Until now, little is known about the role of MnSOD - a major endogenous antioxidant enzyme - on the cell adaptation response against hypoxia. The aim of this study was to  determine the MnSOD mRNA expression and levels of specific activity in blood, heart and brain of rats during induced systemic hypoxia.Methods: Twenty-five male Sprague Dawley rats were subjected to systemic hypoxia in an hypoxic chamber (at 8-10% O2 for 0, 1, 7, 14 and 21 days, respectively. The mRNA relative expression of MnSOD was analyzed using Real Time RT-PCR. MnSOD specific activity was determined using xanthine oxidase inhibition assay.Results: The MnSOD mRNA relative expression in rat blood and heart was decreased during early induced systemic hypoxia (day 1 and increased as hypoxia continued, whereas the mRNA expression in brain was increased since day 1 and reached its maximum level at day 7. The result of MnSOD specific activity during early systemic hypoxia was similar to the mRNA expression. Under very late hypoxic condition (day 21, MnSOD specific activity in blood, heart and brain was significantly decreased. We demonstrate a positive correlation between MnSOD mRNA expression and specific activity in these 3 tissues during day 0-14 of induced systemic hypoxia. Furthermore, mRNA expression and specific activity levels in heart strongly correlate with those in blood.Conclusion: The MnSOD expression at early and late phases of induced systemic hypoxia is distinctly regulated. The MnSOD expression in brain differs from that in blood and heart revealing that brain tissue can  possibly survive better from induced systemic hypoxia than heart and blood. The determination of MnSOD expression in blood can be used to describe its expression in heart under systemic hypoxic condition. (Med J Indones 2011; 20:27-33Keywords: MnSOD, mRNA expression, ROS, specific activity, systemic hypoxia

Hypoxia-induced oxidative base modifications in the VEGF hypoxia-response element are associated with transcriptionally active nucleosomes.

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Ruchko, Mykhaylo V; Gorodnya, Olena M; Pastukh, Viktor M; Swiger, Brad M; Middleton, Natavia S; Wilson, Glenn L; Gillespie, Mark N

2009-02-01

Reactive oxygen species (ROS) generated in hypoxic pulmonary artery endothelial cells cause transient oxidative base modifications in the hypoxia-response element (HRE) of the VEGF gene that bear a conspicuous relationship to induction of VEGF mRNA expression (K.A. Ziel et al., FASEB J. 19, 387-394, 2005). If such base modifications are indeed linked to transcriptional regulation, then they should be detected in HRE sequences associated with transcriptionally active nucleosomes. Southern blot analysis of the VEGF HRE associated with nucleosome fractions prepared by micrococcal nuclease digestion indicated that hypoxia redistributed some HRE sequences from multinucleosomes to transcriptionally active mono- and dinucleosome fractions. A simple PCR method revealed that VEGF HRE sequences harboring oxidative base modifications were found exclusively in mononucleosomes. Inhibition of hypoxia-induced ROS generation with myxathiozol prevented formation of oxidative base modifications but not the redistribution of HRE sequences into mono- and dinucleosome fractions. The histone deacetylase inhibitor trichostatin A caused retention of HRE sequences in compacted nucleosome fractions and prevented formation of oxidative base modifications. These findings suggest that the hypoxia-induced oxidant stress directed at the VEGF HRE requires the sequence to be repositioned into mononucleosomes and support the prospect that oxidative modifications in this sequence are an important step in transcriptional activation.

MicroRNA-145 suppresses ROS-induced Ca{sup 2+} overload of cardiomyocytes by targeting CaMKIIδ

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Cha, Min-Ji [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jang, Jin-Kyung [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); Ham, Onju; Song, Byeong-Wook; Lee, Se-Yeon [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Lee, Chang Yeon; Park, Jun-Hee [Department of Integrated Omics for Biomedical Sciences, Graduate School, Yonsei University, 50 Yonsei-ro, Seodamun-gu, Seoul 120-759 (Korea, Republic of); Lee, Jiyun; Seo, Hyang-Hee [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Choi, Eunhyun [Severance Integrative Research Institute for Cerebral and Cardiovascular Disease, Yonsei University Health System, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Jeon, Woo-min [Department of Animal Resource, Sahmyook University, Seoul 139-742 (Korea, Republic of); Hwang, Hye Jin [Cardiovascular Research Institute, Yonsei University College of Medicine, 250 Seongsanno, Seodamun-gu, Seoul 120-752 (Korea, Republic of); Shin, Hyun-Taek [College of Pharmacy, Sookmyung Women’s University, 52 HyoChangWon-Gil, Yongsan-ku, Seoul 140-742 (Korea, Republic of); and others

2013-06-14

Highlights: •CaMKIIδ mediates H{sub 2}O{sub 2}-induced Ca{sup 2+} overload in cardiomyocytes. •miR-145 can inhibit Ca{sup 2+} overload. •A luciferase assay confirms that miR-145 functions as a CaMKIIδ-targeting miRNA. •Overexpression of miR-145 regulates CaMKIIδ-related genes and ameliorates apoptosis. -- Abstract: A change in intracellular free calcium (Ca{sup 2+}) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca{sup 2+} signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca{sup 2+}-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H{sub 2}O{sub 2}-mediated Ca{sup 2+} overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca{sup 2+} overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca{sup 2+}-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca{sup 2+} overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

Hypoxia regulates the expression of the neuromedin B receptor through a mechanism dependent on hypoxia-inducible factor-1α.

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Hyun-Joo Park

Full Text Available The neuromedin B receptor (NMB-R, a member of the mammalian bombesin receptor family, is frequently overexpressed in various tumors. In the present study, we found that exposure to hypoxic conditions increases the levels of NMBR mRNA and protein in breast cancer cells, which are tightly regulated by hypoxia-inducible factor-1α (HIF-1α. We confirmed the effect of HIF-1α on NMBR transcription by performing an NMBR promoter-driven reporter assay and then identified a functional hypoxia-responsive element (HRE in the human NMBR promoter region. Further, the binding of HIF-1α to the NMBR promoter was corroborated by electrophoretic mobility shift and chromatin immunoprecipitation assays, which showed that HIF-1α specifically and directly bound to the NMBR promoter in response to hypoxia. Immunohistochemical analysis of a xenograft and a human breast cancer tissue array revealed a significant correlation between NMB-R and HIF-1α expression. Taken together, our findings indicate that hypoxia induces NMB-R expression through a novel mechanism to regulate HIF-1α expression in breast cancer cells.

Hydrogen sulfide enhances nitric oxide-induced tolerance of hypoxia in maize (Zea mays L.).

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Peng, Renyi; Bian, Zhiyuan; Zhou, Lina; Cheng, Wei; Hai, Na; Yang, Changquan; Yang, Tao; Wang, Xinyu; Wang, Chongying

2016-11-01

Our data present H 2 S in a new role, serving as a multi-faceted transducer to different response mechanisms during NO-induced acquisition of tolerance to flooding-induced hypoxia in maize seedling roots. Nitric oxide (NO), serving as a secondary messenger, modulates physiological processes in plants. Recently, hydrogen sulfide (H 2 S) has been demonstrated to have similar signaling functions. This study focused on the effects of treatment with H 2 S on NO-induced hypoxia tolerance in maize seedlings. The results showed that treatment with the NO donor sodium nitroprusside (SNP) enhanced survival rate of submerged maize roots through induced accumulation of endogenous H 2 S. The induced H 2 S then enhanced endogenous Ca 2+ levels as well as the Ca 2+ -dependent activity of alcohol dehydrogenase (ADH), improving the capacity for antioxidant defense and, ultimately, the hypoxia tolerance in maize seedlings. In addition, NO induced the activities of key enzymes in H 2 S biosynthesis, such as L-cysteine desulfhydrases (L-CDs), O-acetyl-L-serine (thiol)lyase (OAS-TL), and β-Cyanoalanine Synthase (CAS). SNP-induced hypoxia tolerance was enhanced by the application of NaHS, but was eliminated by the H 2 S-synthesis inhibitor hydroxylamine (HA) and the H 2 S-scavenger hypotaurine (HT). H 2 S concurrently enhanced the transcriptional levels of relative hypoxia-induced genes. Together, our findings indicated that H 2 S serves as a multi-faceted transducer that enhances the nitric oxide-induced hypoxia tolerance in maize (Zea mays L.).

cGMP-Phosphodiesterase Inhibition Prevents Hypoxia-Induced Cell Death Activation in Porcine Retinal Explants.

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Lorena Olivares-González

Full Text Available Retinal hypoxia and oxidative stress are involved in several retinal degenerations including diabetic retinopathy, glaucoma, central retinal artery occlusion, or retinopathy of prematurity. The second messenger cyclic guanosine monophosphate (cGMP has been reported to be protective for neuronal cells under several pathological conditions including ischemia/hypoxia. The purpose of this study was to evaluate whether the accumulation of cGMP through the pharmacological inhibition of phosphodiesterase (PDE with Zaprinast prevented retinal degeneration induced by mild hypoxia in cultures of porcine retina. Exposure to mild hypoxia (5% O2 for 24h reduced cGMP content and induced retinal degeneration by caspase dependent and independent (PARP activation mechanisms. Hypoxia also produced a redox imbalance reducing antioxidant response (superoxide dismutase and catalase activities and increasing superoxide free radical release. Zaprinast reduced mild hypoxia-induced cell death through inhibition of caspase-3 or PARP activation depending on the cell layer. PDE inhibition also ameliorated the effects of mild hypoxia on antioxidant response and the release of superoxide radical in the photoreceptor layer. The use of a PKG inhibitor, KT5823, suggested that cGMP-PKG pathway is involved in cell survival and antioxidant response. The inhibition of PDE, therefore, could be useful for reducing retinal degeneration under hypoxic/ischemic conditions.

In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

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Uddin, Md Imam; Evans, Stephanie M; Craft, Jason R; Capozzi, Megan E; McCollum, Gary W; Yang, Rong; Marnett, Lawrence J; Uddin, Md Jashim; Jayagopal, Ashwath; Penn, John S

2016-08-05

Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

Overexpression of KCNJ2 in induced pluripotent stem cell-derived cardiomyocytes for the assessment of QT-prolonging drugs

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Min Li

2017-06-01

Full Text Available Human induced pluripotent stem cell (hiPSC-derived cardiomyocytes hold great potentials to predict pro-arrhythmic risks in preclinical cardiac safety screening, although the hiPSC cardiomyocytes exhibit rather immature functional and structural characteristics, including spontaneous activity. Our physiological characterization and mathematical simulation showed that low expression of the inward-rectifier potassium (IK1 channel is a determinant of spontaneous activity. To understand impact of the low IK1 expression on the pharmacological properties, we tested if transduction of hiPSC-derived cardiomyocytes with KCNJ2, which encodes the IK1 channel, alters pharmacological response to cardiac repolarization processes. The transduction of KCNJ2 resulted in quiescent hiPSC-derived cardiomyocytes, which need pacing to elicit action potentials. Significant prolongation of paced action potential duration in KCNJ2-transduced hiPSC-derived cardiomyocytes was stably measured at 0.1 μM E-4031, although the same concentration of E-4031 ablated firing of non-treated hiPSC-derived cardiomyocytes. These results in single cells were confirmed by mathematical simulations. Using the hiPSC-derived cardiac sheets with KCNJ2-transduction, we also investigated effects of a range of drugs on field potential duration recorded at 1 Hz. The KCNJ2 overexpression in hiPSC-derived cardiomyocytes may contribute to evaluate a part of QT-prolonging drugs at toxicological concentrations with high accuracy.

Cardiomyocyte hypertrophy induced by Endonuclease G deficiency requires reactive oxygen radicals accumulation and is inhibitable by the micropeptide humanin.

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Blasco, Natividad; Cámara, Yolanda; Núñez, Estefanía; Beà, Aida; Barés, Gisel; Forné, Carles; Ruíz-Meana, Marisol; Girón, Cristina; Barba, Ignasi; García-Arumí, Elena; García-Dorado, David; Vázquez, Jesús; Martí, Ramon; Llovera, Marta; Sanchis, Daniel

2018-06-01

The endonuclease G gene (Endog), which codes for a mitochondrial nuclease, was identified as a determinant of cardiac hypertrophy. How ENDOG controls cardiomyocyte growth is still unknown. Thus, we aimed at finding the link between ENDOG activity and cardiomyocyte growth. Endog deficiency induced reactive oxygen species (ROS) accumulation and abnormal growth in neonatal rodent cardiomyocytes, altering the AKT-GSK3β and Class-II histone deacethylases (HDAC) signal transduction pathways. These effects were blocked by ROS scavengers. Lack of ENDOG reduced mitochondrial DNA (mtDNA) replication independently of ROS accumulation. Because mtDNA encodes several subunits of the mitochondrial electron transport chain, whose activity is an important source of cellular ROS, we investigated whether Endog deficiency compromised the expression and activity of the respiratory chain complexes but found no changes in these parameters nor in ATP content. MtDNA also codes for humanin, a micropeptide with possible metabolic functions. Nanomolar concentrations of synthetic humanin restored normal ROS levels and cell size in Endog-deficient cardiomyocytes. These results support the involvement of redox signaling in the control of cardiomyocyte growth by ENDOG and suggest a pathway relating mtDNA content to the regulation of cell growth probably involving humanin, which prevents reactive oxygen radicals accumulation and hypertrophy induced by Endog deficiency. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

MicroRNA-195 induced apoptosis in hypoxic chondrocytes by targeting hypoxia-inducible factor 1 alpha.

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Bai, R; Zhao, A-Q; Zhao, Z-Q; Liu, W-L; Jian, D-M

2015-02-01

The chondrocytes, the resident cells of cartilage, are maintained and take effects in the whole life upon chronic hypoxic exposure, which hypoxia-inducible factor 1 alpha (HIF-1α) play pivotal roles in response to. Dysregulation of some microRNA (miRNAs) have also been identified to be involved in hypoxia-related physiologic and pathophysiologic responses in some tissues or cell lines. However, the mechanism of miRNAs reponse to hypoxia remain largely unknown in chondrocytes, including the microRNA-195 (miR-195). AIM To investigate the effects of microRNAs (miRNAs) and hypoxia-inducible factor 1 alpha (HIF-1α) on chondrocytes in physiologic environment. We compared the expression of miR-195 and HIF-1α mRNA on hypoxia with that on normoxia in ATDC 5 cells by qRT-PCR. Further experiments was performed to confirmed the relationships of miR-195 and HIF-1α by bioinformatics analysis and dual reporter gene assay. we also assessed the effect of miR-195 on apoptosis in hypoxic ATDC 5 cells by transfect with miR-195 mimics. It was found the downregulated miR-195 and upregulated HIF-1α were present in hypoxic ATDC 5 cells. miR-195 negatively regulated HIF-1α by targeting its 3'-untranslated region. Moreover, the founding indicated miR-195 greatly increased apoptosis and downregulated HIF-1α mRNA occurred simultaneously in hypoxic chondrocytes. We concluded that miR-195 induced apoptosis in hypoxic chondrocytes by directly targeting HIF-1α.

Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage

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Kumar Anil

2010-10-01

Full Text Available Abstract Background The ability to respond to changes in the extra-intracellular environment is prerequisite for cell survival. Cellular responses to the environment include elevating defense systems, such as the antioxidant defense system. Hypoxia-evoked reactive oxygen species (ROS-driven oxidative stress is an underlying mechanism of retinal ganglion cell (RGC death that leads to blinding disorders. The protein peroxiredoxin 6 (PRDX6 plays a pleiotropic role in negatively regulating death signaling in response to stressors, and thereby stabilizes cellular homeostasis. Results We have shown that RGCs exposed to hypoxia (1% or hypoxia mimetic cobalt chloride display reduced expression of PRDX6 with higher ROS expression and activation of NF-κB. These cells undergo apoptosis, while cells with over-expression of PRDX6 demonstrate resistance against hypoxia-driven RGC death. The RGCs exposed to hypoxia either with 1% oxygen or cobalt chloride (0-400 μM, revealed ~30%-70% apoptotic cell death after 48 and 72 h of exposure. Western analysis and real-time PCR showed elevated expression of PRDX6 during hypoxia at 24 h, while PRDX6 protein and mRNA expression declined from 48 h onwards following hypoxia exposure. Concomitant with this, RGCs showed increased ROS expression and activation of NF-κB with IkB phosphorylation/degradation, as examined with H2DCF-DA and transactivation assays. These hypoxia-induced adverse reactions could be reversed by over-expression of PRDX6. Conclusion Because an abundance of PRDX6 in cells was able to attenuate hypoxia-induced RGC death, the protein could possibly be developed as a novel therapeutic agent acting to postpone RGC injury and delay the progression of glaucoma and other disorders caused by the increased-ROS-generated death signaling related to hypoxia.

AICAR Protects against High Palmitate/High Insulin-Induced Intramyocellular Lipid Accumulation and Insulin Resistance in HL-1 Cardiac Cells by Inducing PPAR-Target Gene Expression

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Ricardo Rodríguez-Calvo

2015-01-01

Full Text Available Here we studied the impact of 5-aminoimidazole-4-carboxamide riboside (AICAR, a well-known AMPK activator, on cardiac metabolic adaptation. AMPK activation by AICAR was confirmed by increased phospho-Thr172-AMPK and phospho-Ser79-ACC protein levels in HL-1 cardiomyocytes. Then, cells were exposed to AICAR stimulation for 24 h in the presence or absence of the AMPK inhibitor Compound C, and the mRNA levels of the three PPARs were analyzed by real-time RT-PCR. Treatment with AICAR induced gene expression of all three PPARs, but only the Ppara and Pparg regulation were dependent on AMPK. Next, we exposed HL-1 cells to high palmitate/high insulin (HP/HI conditions either in presence or in absence of AICAR, and we evaluated the expression of selected PPAR-targets genes. HP/HI induced insulin resistance and lipid storage was accompanied by increased Cd36, Acot1, and Ucp3 mRNA levels. AICAR treatment induced the expression of Acadvl and Glut4, which correlated to prevention of the HP/HI-induced intramyocellular lipid build-up, and attenuation of the HP/HI-induced impairment of glucose uptake. These data support the hypothesis that AICAR contributes to cardiac metabolic adaptation via regulation of transcriptional mechanisms.

Hypoxia-Induced Signaling Promotes Prostate Cancer Progression: Exosomes Role as Messenger of Hypoxic Response in Tumor Microenvironment

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Deep, Gagan; Panigrahi, Gati K.

2017-01-01

Prostate cancer (PCA) is the leading malignancy in men and the second leading cause of cancer-related deaths. Hypoxia (low O2 condition) is considered an early event in prostate carcinogenesis associated with an aggressive phenotype. In fact, clinically, hypoxia and hypoxia-related biomarkers are associated with treatment failure and disease progression. Hypoxia-inducible factor 1 (HIF-1) is the key factor that is activated under hypoxia, and mediates adaptation of cells to hypoxic conditions through regulating the expression of genes associated with angiogenesis, epithelial-to-mesenchymal transition (EMT), metastasis, survival, proliferation, metabolism, stemness, hormone-refractory progression, and therapeutic resistance. Besides HIF-1, several other signaling pathways including PI3K/Akt/mTOR, NADPH oxidase (NOX), Wnt/β-catenin, and Hedgehog are activated in cancer cells under hypoxic conditions, and also contribute in hypoxia-induced biological effects in HIF-1-dependent and -independent manners. Hypoxic cancer cells cause extensive changes in the tumor microenvironment both local and distant, and recent studies have provided ample evidence supporting the crucial role of nanosized vesicles “exosomes” in mediating hypoxia-induced tumor microenvironment remodeling. Exosomes’ role has been reported in hypoxia-induced angiogenesis, stemness, activation of cancer-associated fibroblasts (CAFs), and EMT. Together, existing literature suggests that hypoxia plays a predominant role in PCA growth and progression, and PCA could be effectively prevented and treated via targeting hypoxia/hypoxia-related signaling pathways. PMID:27279239

Hypoxia-inducible factor-1 signalling promotes goblet cell hyperplasia in airway epithelium

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Polosukhin, Vasiliy V; Cates, Justin M; Lawson, William E; Milstone, Aaron P; Matafonov, Anton G; Massion, Pierre P; Lee, Jae Woo; Randell, Scott H; Blackwell, Timothy S

2018-01-01

Goblet cell hyperplasia is a common feature of chronic obstructive pulmonary disease (COPD) airways, but the mechanisms that underlie this epithelial remodelling in COPD are not understood. Based on our previous finding of hypoxia-inducible factor-1α (HIF-1α) nuclear localization in large airways from patients with COPD, we investigated whether hypoxia-inducible signalling could influence the development of goblet cell hyperplasia. We evaluated large airway samples obtained from 18 lifelong non-smokers and 13 former smokers without COPD, and 45 former smokers with COPD. In these specimens, HIF-1α nuclear staining occurred almost exclusively in COPD patients in areas of airway remodelling. In COPD patients, 93.2 ± 3.9% (range 65 – 100%) of goblet cells were HIF-1α positive in areas of goblet cell hyperplasia, whereas nuclear HIF-1α was not detected in individuals without COPD or in normal-appearing pseudostratified epithelium from COPD patients. To determine the direct effects of hypoxia-inducible signalling on epithelial cell differentiation in vitro, human bronchial epithelial cells (HBECs) were grown in air-liquid interface cultures under hypoxia (1% O2) or following treatment with a selective HIF-1α stabilizer, (2R)-[(4-biphenylylsulphonyl)amino]-N-hydroxy-3-phenyl-propionamide (BiPS). HBECs grown in hypoxia or with BiPS treatment were characterized by HIF-1α activation, carbonic anhydrase IX expression, mucus-producing cell hyperplasia and increased expression of MUC5AC. Analysis of signal transduction pathways in cells with HIF-1α activation showed increased ERK1/2 phosphorylation without activation of epidermal growth factor receptor, Ras, PI3K-Akt or STAT6. These data indicate an important effect of hypoxia-inducible signalling on airway epithelial cell differentiation and identify a new potential target to limit mucus production in COPD. PMID:21557221

Translational control is a major contributor to hypoxia induced gene expression

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Beucken, Twan van den; Magagnin, Michael G.; Jutten, Barry; Seigneuric, Renaud; Lambin, Philippe; Koritzinsky, Marianne; Wouters, Bradly G.

2011-01-01

Background and purpose: Hypoxia is a common feature of solid tumors that is associated with an aggressive phenotype, resistance to therapy and poor prognosis. Major contributors to these adverse effects are the transcriptional program activated by the HIF family of transcription factors as well as the translational response mediated by PERK-dependent phosphorylation of eIF2α and inhibition of mTORC1 activity. In this study we determined the relative contribution of both transcriptional and translational responses to changes in hypoxia induced gene expression. Material and methods: Total and efficiently translated (polysomal) mRNA was isolated from DU145 prostate carcinoma cells that were exposed for up to 24 h of hypoxia ( 2 ). Changes in transcription and translation were assessed using affymetrix microarray technology. Results: Our data reveal an unexpectedly large contribution of translation control on both induced and repressed gene expression at all hypoxic time points, particularly during acute hypoxia (2-4 h). Gene ontology analysis revealed that gene classes like transcription and signal transduction are stimulated by translational control whereas expression of genes involved in cell growth and protein metabolism are repressed during hypoxic conditions by translational control. Conclusions: Our data indicate that translation influences gene expression during hypoxia on a scale comparable to that of transcription.

Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor.

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Kwong, Raymond W M; Kumai, Yusuke; Tzaneva, Velislava; Azzi, Estelle; Hochhold, Nina; Robertson, Cayleih; Pelster, Bernd; Perry, Steve F

2016-12-15

The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P O 2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca 2+ levels and Ca 2+ uptake. Ca 2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca 2+ channel (ecac), but not plasma membrane Ca 2+ -ATPase (pmca2) or Na + /Ca 2+ -exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca 2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca 2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca 2+ balance during hypoxia, the results demonstrated that the reduction of Ca 2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca 2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression. © 2016. Published by The Company of Biologists Ltd.

Hypoxia activated EGFR signaling induces epithelial to mesenchymal transition (EMT.

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Ashish Misra

Full Text Available Metastasis is a multi-step process which requires the conversion of polarized epithelial cells to mesenchymal cells, Epithelial-Mesenchymal Transition (EMT. EMT is essential during embryonic morphogenesis and has been implicated in the progression of primary tumors towards metastasis. Hypoxia is known to induce EMT; however the molecular mechanism is still poorly understood. Using the A431 epithelial cancer cell line, we show that cells grown under hypoxic conditions migrated faster than cells grown under normal oxygen environment. Cells grown under hypoxia showed reduced adhesion to the extracellular matrix (ECM probably due to reduced number of Vinculin patches. Growth under hypoxic conditions also led to down regulation of E-cadherin and up regulation of vimentin expression. The increased motility of cells grown under hypoxia could be due to redistribution of Rac1 to the plasma membrane as opposed to increased expression of Rac1. EGF (Epidermal Growth Factor is a known inducer of EMT and growth of A431 cells in the absence of oxygen led to increased expression of EGFR (EGF Receptor. Treatment of A431 cells with EGF led to reduced cell adhesion to ECM, increased cell motility and other EMT characteristics. Furthermore, this transition was blocked by the monoclonal antibody Cetuximab. Cetuximab also blocked the hypoxia-induced EMT suggesting that cell growth under hypoxic conditions led to activation of EGFR signaling and induction of EMT phenotype.

Chronic intermittent hypoxia induces atherosclerosis via activation of adipose angiopoietin-like 4.

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Drager, Luciano F; Yao, Qiaoling; Hernandez, Karen L; Shin, Mi-Kyung; Bevans-Fonti, Shannon; Gay, Jason; Sussan, Thomas E; Jun, Jonathan C; Myers, Allen C; Olivecrona, Gunilla; Schwartz, Alan R; Halberg, Nils; Scherer, Philipp E; Semenza, Gregg L; Powell, David R; Polotsky, Vsevolod Y

2013-07-15

Obstructive sleep apnea is a risk factor for dyslipidemia and atherosclerosis, which have been attributed to chronic intermittent hypoxia (CIH). Intermittent hypoxia inhibits a key enzyme of lipoprotein clearance, lipoprotein lipase, and up-regulates a lipoprotein lipase inhibitor, angiopoietin-like 4 (Angptl4), in adipose tissue. The effects and mechanisms of Angptl4 up-regulation in sleep apnea are unknown. To examine whether CIH induces dyslipidemia and atherosclerosis by increasing adipose Angptl4 via hypoxia-inducible factor-1 (HIF-1). ApoE(-/-) mice were exposed to intermittent hypoxia or air for 4 weeks while being treated with Angptl4-neutralizing antibody or vehicle. In vehicle-treated mice, hypoxia increased adipose Angptl4 levels, inhibited adipose lipoprotein lipase, increased fasting levels of plasma triglycerides and very low density lipoprotein cholesterol, and increased the size of atherosclerotic plaques. The effects of CIH were abolished by the antibody. Hypoxia-induced increases in plasma fasting triglycerides and adipose Angptl4 were not observed in mice with germline heterozygosity for a HIF-1α knockout allele. Transgenic overexpression of HIF-1α in adipose tissue led to dyslipidemia and increased levels of adipose Angptl4. In cultured adipocytes, constitutive expression of HIF-1α increased Angptl4 levels, which was abolished by siRNA. Finally, in obese patients undergoing bariatric surgery, the severity of nocturnal hypoxemia predicted Angptl4 levels in subcutaneous adipose tissue. HIF-1-mediated increase in adipose Angptl4 and the ensuing lipoprotein lipase inactivation may contribute to atherosclerosis in patients with sleep apnea.

Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression.

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Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

2009-08-06

Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed.

Severe hypoxia induces chemo-resistance in clinical cervical tumors through MVP over-expression

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Lara, Pedro C; Lloret, Marta; Clavo, Bernardino; Apolinario, Rosa M; Henríquez-Hernández, Luis Alberto; Bordón, Elisa; Fontes, Fausto; Rey, Agustín

2009-01-01

Oxygen molecule modulates tumour response to radiotherapy. Higher radiation doses are required under hypoxic conditions to induce cell death. Hypoxia may inhibit the non-homologous end-joining DNA repair through down regulating Ku70/80 expression. Hypoxia induces drug resistance in clinical tumours, although the mechanism is not clearly elucidated. Vaults are ribonucleoprotein particles with a hollow barrel-like structure composed of three proteins: major vault protein (MVP), vault poly(ADP-ribose) polymerase, and telomerase associated protein-1 and small untranslated RNA. Over-expression of MVP has been associated with chemotherapy resistance. Also, it has been related to poor outcome in patients treated with radiotherapy alone. The aim of the present study was to assess the relation of Major Vault Protein expression and tumor hypoxia in clinical cervical tumors. MVP, p53 and angiogenesis, together with tumor oxygenation, were determined in forty-three consecutive patients suffering from localized cervix carcinoma. High MVP expression was related to severe hypoxia compared to low MVP expressing tumors (p = 0.022). Tumors over-expressing MVP also showed increased angiogenesis (p = 0.003). Besides it, in this study we show for the first time that severe tumor hypoxia is associated with high MVP expression in clinical cervical tumors. Up-regulation of MVP by hypoxia is of critical relevance as chemotherapy is currently a standard treatment for those patients. From our results it could be suggested that hypoxia not only induces increased genetic instability, oncogenic properties and metastatization, but through the correlation observed with MVP expression, another pathway of chemo and radiation resistance could be developed

Multi-parameter in vitro toxicity testing of crizotinib, sunitinib, erlotinib, and nilotinib in human cardiomyocytes

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Doherty, Kimberly R.; Wappel, Robert L.; Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Kramer, James W.; Brown, Arthur M.; Shell, Scott A.; Bacus, Sarah

2013-01-01

Tyrosine kinase inhibitors (TKi) have greatly improved the treatment and prognosis of multiple cancer types. However, unexpected cardiotoxicity has arisen in a subset of patients treated with these agents that was not wholly predicted by pre-clinical testing, which centers around animal toxicity studies and inhibition of the human Ether-à-go-go-Related Gene (hERG) channel. Therefore, we sought to determine whether a multi-parameter test panel assessing the effect of drug treatment on cellular, molecular, and electrophysiological endpoints could accurately predict cardiotoxicity. We examined how 4 FDA-approved TKi agents impacted cell viability, apoptosis, reactive oxygen species (ROS) generation, metabolic status, impedance, and ion channel function in human cardiomyocytes. The 3 drugs clinically associated with severe cardiac adverse events (crizotinib, sunitinib, nilotinib) all proved to be cardiotoxic in our in vitro tests while the relatively cardiac-safe drug erlotinib showed only minor changes in cardiac cell health. Crizotinib, an ALK/MET inhibitor, led to increased ROS production, caspase activation, cholesterol accumulation, disruption in cardiac cell beat rate, and blockage of ion channels. The multi-targeted TKi sunitinib showed decreased cardiomyocyte viability, AMPK inhibition, increased lipid accumulation, disrupted beat pattern, and hERG block. Nilotinib, a second generation Bcr-Abl inhibitor, led to increased ROS generation, caspase activation, hERG block, and an arrhythmic beat pattern. Thus, each drug showed a unique toxicity profile that may reflect the multiple mechanisms leading to cardiotoxicity. This study demonstrates that a multi-parameter approach can provide a robust characterization of drug-induced cardiomyocyte damage that can be leveraged to improve drug safety during early phase development. - Highlights: • TKi with known adverse effects show unique cardiotoxicity profiles in this panel. • Crizotinib increases ROS, apoptosis, and

Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

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Hongran Wang

2016-03-01

Full Text Available Induced pluripotent stem (iPS cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc, without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs, iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin, cardiac alpha myosin heavy chain (α-MHC, cardiac troponin T (cTnT, and connexin 43 (CX43, as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5 and gata binding protein 4 (gata4. The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI in the future.

Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer

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Havelund, Birgitte Mayland; Spindler, Karen-Lise Garm; Sørensen, Flemming Brandt

2010-01-01

Clinical Aspects of Hypoxia-inducible Factors in Colorectal Cancer  Birgitte Mayland Havelund1,4 MD, Karen-Lise Garm Spindler1,4 MD, PhD, Flemming Brandt Sørensen2,4 MD, DMSc, Ivan Brandslund3 MD, DMSc, Anders Jakobsen1,4 MD, DMSc.1Department of Oncology, 2Pathology and 3Biochemistry, Vejle...... Hospital, Vejle, Denmark4Institute of Regional Health Services Research, University of Southern Denmark, Odense DenmarkBackgroundPrognostic and predictive markers are needed for individualizing the treatment of colorectal cancer. Hypoxia-inducible factor 1α (HIF-1α) is a transcription-inducing factor which...... the predictive and prognostic value of HIF-1α in colorectal cancer.Materials and MethodsThe project is divided into 3 substudies:1. Biological and methodological aspects. The expression of HIF-1α measured by immunohistochemistry in paraffin embedded tissue is related to single nucleotide polymorphism (SNP...

Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy.

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Huang, Kun; Gao, Lu; Yang, Ming; Wang, Jiliang; Wang, Zheng; Wang, Lin; Wang, Guobin; Li, Huili

2017-08-01

Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hypoxia-independent upregulation of placental hypoxia inducible factor-1α gene expression contributes to the pathogenesis of preeclampsia.

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Iriyama, Takayuki; Wang, Wei; Parchim, Nicholas F; Song, Anren; Blackwell, Sean C; Sibai, Baha M; Kellems, Rodney E; Xia, Yang

2015-06-01

Accumulation of hypoxia inducible factor-1α (HIF-1α) is commonly an acute and beneficial response to hypoxia, whereas chronically elevated HIF-1α is associated with multiple disease conditions, including preeclampsia, a serious hypertensive disease of pregnancy. However, the molecular basis underlying the persistent elevation of placental HIF-1α in preeclampsia and its role in the pathogenesis of preeclampsia are poorly understood. Here we report that Hif-1α mRNA and HIF-1α protein were elevated in the placentas of pregnant mice infused with angiotensin II type I receptor agonistic autoantibody, a pathogenic factor in preeclampsia. Knockdown of placental Hif-1α mRNA by specific siRNA significantly attenuated hallmark features of preeclampsia induced by angiotensin II type I receptor agonistic autoantibody in pregnant mice, including hypertension, proteinuria, kidney damage, impaired placental vasculature, and elevated maternal circulating soluble fms-like tyrosine kinase-1 levels. Next, we discovered that Hif-1α mRNA levels and HIF-1α protein levels were induced in an independent preeclampsia model with infusion of the inflammatory cytokine tumor necrosis factor superfamily member 14 (LIGHT). SiRNA knockdown experiments also demonstrated that elevated HIF-1α contributed to LIGHT-induced preeclampsia features. Translational studies with human placentas showed that angiotensin II type I receptor agonistic autoantibody or LIGHT is capable of inducing HIF-1α in a hypoxia-independent manner. Moreover, increased HIF-1α was found to be responsible for angiotensin II type I receptor agonistic autoantibody or LIGHT-induced elevation of Flt-1 gene expression and production of soluble fms-like tyrosine kinase-1 in human villous explants. Overall, we demonstrated that hypoxia-independent stimulation of HIF-1α gene expression in the placenta is a common pathogenic mechanism promoting disease progression. Our findings reveal new insight to preeclampsia and highlight

Hypoxia attenuates inflammatory mediators production induced by Acanthamoeba via Toll-like receptor 4 signaling in human corneal epithelial cells

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Pan, Hong; Wu, Xinyi

2012-01-01

Highlights: ► Hypoxia attenuates Acanthamoeba-induced the production of IL-8 and IFN-β. ► Hypoxia inhibits TLR4 expression in a time-dependent manner in HCECs. ► Hypoxia inhibits Acanthamoeba-induced the activation of NF-κB and ERK1/2 in HCECs. ► Hypoxia decreases Acanthamoeba-induced inflammatory response via TLR4 signaling. ► LPS-induced the secretion of IL-6 and IL-8 is abated by hypoxia via TLR4 signaling. -- Abstract: Acanthamoeba keratitis (AK) is a vision-threatening corneal infection that is intimately associated with contact lens use which leads to hypoxic conditions on the corneal surface. However, the effect of hypoxia on the Acanthamoeba-induced host inflammatory response of corneal epithelial cells has not been studied. In the present study, we investigated the effect of hypoxia on the Acanthamoeba-induced production of inflammatory mediators interleukin-8 (IL-8) and interferon-β (IFN-β) in human corneal epithelial cells and then evaluated its effects on the Toll-like receptor 4 (TLR4) signaling, including TLR4 and myeloid differentiation primary response gene (88) (MyD88) expression as well as the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinases 1/2 (ERK1/2). We then studied the effect of hypoxia on a TLR4-specific inflammatory response triggered by the TLR4 ligand lipopolysaccharide (LPS). Our data showed that hypoxia significantly decreased the production of IL-8 and IFN-β. Furthermore, hypoxia attenuated Acanthamoeba-triggered TLR4 expression as well as the activation of NF-κB and ERK1/2, indicating that hypoxia abated Acanthamoeba-induced inflammatory responses by affecting TLR4 signaling. Hypoxia also inhibited LPS-induced IL-6 and IL-8 secretion, myeloid differentiation primary response gene (88) MyD88 expression and NF-κB activation, confirming that hypoxia suppressed the LPS-induced inflammatory response by affecting TLR4 signaling. In conclusion

Saururus cernuus lignans-Potent small molecule inhibitors of hypoxia-inducible factor-1

International Nuclear Information System (INIS)

Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou Yudong

2005-01-01

Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B 1 , manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC 50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors

Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes

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Desy S. Lee

2015-09-01

Full Text Available Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs. We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo, to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

Differentiation and characterization of rhesus monkey atrial and ventricular cardiomyocytes from induced pluripotent stem cells.

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Zhang, Xiaoqian; Cao, Henghua; Bai, Shuyun; Huo, Weibang; Ma, Yue

2017-04-01

The combination of non-human primate animals and their induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) provides not only transplantation models for cell-based therapy of heart diseases, but also opportunities for heart-related drug research on both cellular and animal levels. However, the subtypes and electrophysiology properties of non-human primate iPSC-CMs hadn't been detailed characterized. In this study, we generated rhesus monkey induced pluripotent stem cells (riPSCs), and efficiently differentiated them into ventricular or atrial cardiomyocytes by modulating retinoic acid (RA) pathways. Our results revealed that the electrophysiological characteristics and response to canonical drugs of riPSC-CMs were similar with those of human pluripotent stem cell derived CMs. Therefore, rhesus monkeys and their iPSC-CMs provide a powerful and practicable system for heart related biomedical research. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

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Louis, Xavier Lieben; Thandapilly, Sijo Joseph; Kalt, Wilhelmina; Vinqvist-Tymchuk, Melinda; Aloud, Basma Milad; Raj, Pema; Yu, Liping; Le, Hoa; Netticadan, Thomas

2014-08-01

The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

17β-Estradiol-induced interaction of ERα with NPPA regulates gene expression in cardiomyocytes.

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Mahmoodzadeh, Shokoufeh; Pham, Thi Hang; Kuehne, Arne; Fielitz, Britta; Dworatzek, Elke; Kararigas, Georgios; Petrov, George; Davidson, Mercy M; Regitz-Zagrosek, Vera

2012-12-01

17β-Oestradiol (E2) and its receptors (ERα and ERβ) are important regulators of physiological and pathological processes in the cardiovascular system. ER act in concert with other regulatory factors mediating oestrogenic effects. However, the underlying mechanisms modulating ER transcriptional activity are not fully elucidated. To gain better understanding of E2-induced ERα action in the human heart, we aimed to identify and functionally analyse interaction partners of ERα. Using yeast two-hybrid assays with a human heart cDNA library, we identified atrial natriuretic peptide precursor A (NPPA), a well-known cardiac hypertrophy marker, as a novel ERα interaction partner interacting in an E2-dependent manner. Mutation analyses and immunofluorescence data indicated that the LXXLL motif within NPPA is necessary for its E2-induced interaction with ERα, its action as a co-repressor of ERα, and its translocation into the nucleus of human and rat cardiomyocytes. Expression analysis and chromatin immunoprecipitation assays in a human left ventricular cardiomyocyte cell line, AC16, showed that NPPA interacts with E2/ERα, suppressing the transcriptional activity of ERα on E2-target genes, such as NPPA, connexin43, αactinin-2, nuclear factor of activated T-cells, and collagens I and III. We characterize for the first time an E2-regulated interaction of NPPA with ERα in cardiomyocytes, that may be crucial in physiological and/or pathological cardiac processes, thereby representing a potential therapeutic target.

A prognostic profile of hypoxia-induced genes for localised high-grade soft tissue sarcoma

DEFF Research Database (Denmark)

Aggerholm-Pedersen, Ninna; Sørensen, Brita Singers; Overgaard, Jens

2016-01-01

sarcoma (STS). METHODS: The hypoxia-induced gene quantification was performed by real-time quantitative PCR (RT-qPCR) of formalin-fixed, paraffin-embedded tissue samples. The gene expression cut-points were determined in a test cohort of 55 STS patients and used to allocate each patient into a more......BACKGROUND: For decades, tumour hypoxia has been pursued as a cancer treatment target. However, prognostic and predictive biomarkers are essential for the use of this target in the clinic. This study investigates the prognostic value of a hypoxia-induced gene profile in localised soft tissue...

Intermittent Hypoxia-Induced Cardiovascular Remodeling Is Reversed by Normoxia in a Mouse Model of Sleep Apnea.

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Castro-Grattoni, Anabel L; Alvarez-Buvé, Roger; Torres, Marta; Farré, Ramon; Montserrat, Josep M; Dalmases, Mireia; Almendros, Isaac; Barbé, Ferran; Sánchez-de-la-Torre, Manuel

2016-06-01

Intermittent hypoxia (IH) is the principal injurious factor involved in the cardiovascular morbidity and mortality associated with OSA. The gold standard for treatment is CPAP, which eliminates IH and appears to reduce cardiovascular risk. There is no experimental evidence on the reversibility of cardiovascular remodeling after IH withdrawal. The objective of the present study is to assess the reversibility of early cardiovascular structural remodeling induced by IH after resumption of normoxic breathing in a novel recovery animal model mimicking OSA treatment. We investigated cardiovascular remodeling in C57BL/6 mice exposed to IH for 6 weeks vs the normoxia group and its spontaneous recovery after 6 subsequent weeks under normoxia. Aortic expansive remodeling was induced by IH, with intima-media thickening and without lumen perimeter changes. Elastic fiber network disorganization, fragmentation, and estrangement between the end points of disrupted fibers were increased by IH. Extracellular matrix turnover was altered, as visualized by collagen and mucoid interlaminar accumulation. Furthermore, left ventricular perivascular fibrosis was increased by IH, whereas cardiomyocytes size was unaffected. These cardiovascular remodeling events induced by IH were normalized after recovery in normoxia, mimicking CPAP treatment. The early structural cardiovascular remodeling induced by IH was normalized after IH removal, revealing a novel recovery model for studying the effects of OSA treatment. Our findings suggest the clinical relevance of early detection and effective treatment of OSA in patients to prevent the natural course of cardiovascular diseases. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

Propofol ameliorates doxorubicin-induced oxidative stress and cellular apoptosis in rat cardiomyocytes

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Lai, H.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Yeh, Y.C. [Graduate Institute of Natural Healing Sciences, Nanhua University, Chiayi, Taiwan (China); Wang, L.C. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Ting, C.T.; Lee, W.L. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Lee, H.W. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Wang, K.Y. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine, Chung-Shan Medical University, Taichung, Taiwan (China); Wu, A. [College of Biological Science, University of California, Davis (United States); Su, C.S. [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Liu, T.J., E-mail: trliu@vghtc.gov.tw [Cardiovascular Center and Department of Anesthesiology, Taichung Veterans General Hospital, Taichung, Taiwan (China); Department of Medicine and Cardiovascular Research Center, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

2011-12-15

Background: Propofol is an anesthetic with pluripotent cytoprotective properties against various extrinsic insults. This study was designed to examine whether this agent could also ameliorate the infamous toxicity of doxorubicin, a widely-used chemotherapeutic agent against a variety of cancer diseases, on myocardial cells. Methods: Cultured neonatal rat cardiomyocytes were administrated with vehicle, doxorubicin (1 {mu}M), propofol (1 {mu}M), or propofol plus doxorubicin (given 1 h post propofol). After 24 h, cells were harvested and specific analyses regarding oxidative/nitrative stress and cellular apoptosis were conducted. Results: Trypan blue exclusion and MTT assays disclosed that viability of cardiomyocytes was significantly reduced by doxorubicin. Contents of reactive oxygen and nitrogen species were increased and antioxidant enzymes SOD1, SOD2, and GPx were decreased in these doxorubicin-treated cells. Mitochondrial dehydrogenase activity and membrane potential were also depressed, along with activation of key effectors downstream of mitochondrion-dependent apoptotic signaling. Besides, abundance of p53 was elevated and cleavage of PKC-{delta} was induced in these myocardial cells. In contrast, all of the above oxidative, nitrative and pro-apoptotic events could be suppressed by propofol pretreatment. Conclusions: Propofol could extensively counteract oxidative/nitrative and multiple apoptotic effects of doxorubicin in the heart; hence, this anesthetic may serve as an adjuvant agent to assuage the untoward cardiac effects of doxorubicin in clinical application. -- Highlights: Black-Right-Pointing-Pointer We evaluate how propofol prevents doxorubicin-induced toxicity in cardiomyocytes. Black-Right-Pointing-Pointer Propofol reduces doxorubicin-imposed nitrative and oxidative stress. Black-Right-Pointing-Pointer Propofol suppresses mitochondrion-, p53- and PKC-related apoptotic signaling. Black-Right-Pointing-Pointer Propofol ameliorates apoptosis and

Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms.

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Stenmark, Kurt R; Fagan, Karen A; Frid, Maria G

2006-09-29

Chronic hypoxic exposure induces changes in the structure of pulmonary arteries, as well as in the biochemical and functional phenotypes of each of the vascular cell types, from the hilum of the lung to the most peripheral vessels in the alveolar wall. The magnitude and the specific profile of the changes depend on the species, sex, and the developmental stage at which the exposure to hypoxia occurred. Further, hypoxia-induced changes are site specific, such that the remodeling process in the large vessels differs from that in the smallest vessels. The cellular and molecular mechanisms vary and depend on the cellular composition of vessels at particular sites along the longitudinal axis of the pulmonary vasculature, as well as on local environmental factors. Each of the resident vascular cell types (ie, endothelial, smooth muscle, adventitial fibroblast) undergo site- and time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors, and each resident cell type plays a specific role in the overall remodeling response. In addition, hypoxic exposure induces an inflammatory response within the vessel wall, and the recruited circulating progenitor cells contribute significantly to the structural remodeling and persistent vasoconstriction of the pulmonary circulation. The possibility exists that the lung or lung vessels also contain resident progenitor cells that participate in the remodeling process. Thus the hypoxia-induced remodeling of the pulmonary circulation is a highly complex process where numerous interactive events must be taken into account as we search for newer, more effective therapeutic interventions. This review provides perspectives on each of the aforementioned areas.

β3-adrenergic receptor activation induces TGFβ1 expression in cardiomyocytes via the PKG/JNK/c-Jun pathway.

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Xu, Zhongcheng; Wu, Jimin; Xin, Junzhou; Feng, Yenan; Hu, Guomin; Shen, Jing; Li, Mingzhe; Zhang, Youyi; Xiao, Han; Wang, Li

2018-06-05

In heart failure, the expression of cardiac β 3 -adrenergic receptors (β 3 -ARs) increases. However, the precise role of β 3 -AR signaling within cardiomyocytes remains unclear. Transforming growth factor β1 (TGFβ1) is a crucial cytokine mediating the cardiac remodeling that plays a causal role in the progression of heart failure. Here, we set out to determine the effect of β 3 -AR activation on TGFβ1 expression in rat cardiomyocytes and examine the underlying mechanism. The selective β 3 -AR agonist BRL37344 induced an increase in TGFβ1 expression and the phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun in β 3 -AR-overexpressing cardiomyocytes. Those effects of BRL37344 were suppressed by a β 3 -AR antagonist. Moreover, the inhibition of JNK and c-Jun activity by a JNK inhibitor and c-Jun siRNA blocked the increase in TGFβ1 expression upon β 3 -AR activation. A protein kinase G (PKG) inhibitor also attenuated β 3 -AR-agonist-induced TGFβ1 expression and the phosphorylation of JNK and c-Jun. In conclusion, the β 3 -AR activation in cardiomyocytes increases the expression of TGFβ1 via the PKG/JNK/c-Jun pathway. These results help us further understand the role of β 3 -AR signaling in heart failure. Copyright © 2018 Elsevier Inc. All rights reserved.

A novel adjustable automated system for inducing chronic intermittent hypoxia in mice.

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Polšek, Dora; Bago, Marcel; Živaljić, Marija; Rosenzweig, Ivana; Lacza, Zsombor; Gajović, Srećko

2017-01-01

Sleep apnea is a chronic, widely underdiagnosed condition characterized by disruption of sleep architecture and intermittent hypoxia due to short cessations of breathing. It is a major independent risk factor for myocardial infarction, congestive heart failure and stroke as well as one of the rare modifiable risk factors for Alzheimer's Dementia. Reliable animal disease models are needed to understand the link between sleep apnea and the various clinically linked disorders. An automated system for inducing hypoxia was developed, in which the major improvement was the possibility to efficiently adjust the length and intensity of hypoxia in two different periods. The chamber used a small volume of gas allowing for fast exchanges of different oxygen levels. The mice were kept in their cages adapted with the system on the cage lid. As a proof of principle, they were exposed to a three week period of intermittent hypoxia for 8 hours a day, with 90 s intervals of 5, 7% and 21% oxygen to validate the model. Treated (n = 8) and control mice (no hypoxia, n = 7) were handled in the same manner and their hippocampal brain regions compared by histology. The chamber provided a fast, reliable and precise intermittent hypoxia, without inducing noticeable side effects to the animals. The validation experiment showed that apoptotic neurons in the hippocampus were more numerous in the mice exposed to intermittent hypoxia than in the control group, in all tested hippocampal regions (cornu ammonis 1 (CA1) P apnea, which was validated by apoptosis of hippocampal neurons.

Limited effects of exogenous glucose during severe hypoxia and a lack of hypoxia-stimulated glucose uptake in isolated rainbow trout cardiac muscle

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Becker, Tracy A.; DellaValle, Brian; Gesser, Hans; Rodnick, Kenneth J.

2013-01-01

SUMMARY We examined whether exogenous glucose affects contractile performance of electrically paced ventricle strips from rainbow trout under conditions known to alter cardiomyocyte performance, ion regulation and energy demands. Physiological levels of d-glucose did not influence twitch force development for aerobic preparations (1) paced at 0.5 or 1.1 Hz, (2) at 15 or 23°C, (3) receiving adrenergic stimulation or (4) during reoxygenation with or without adrenaline after severe hypoxia. Contractile responses to ryanodine, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, were also not affected by exogenous glucose. However, glucose did attenuate the fall in twitch force during severe hypoxia. Glucose uptake was assayed in non-contracting ventricle strips using 2-[3H] deoxy-d-glucose (2-DG) under aerobic and hypoxic conditions, at different incubation temperatures and with different inhibitors. Based upon a lack of saturation of 2-DG uptake and incomplete inhibition of uptake by cytochalasin B and d-glucose, 2-DG uptake was mediated by a combination of facilitated transport and simple diffusion. Hypoxia stimulated lactate efflux sixfold to sevenfold with glucose present, but did not increase 2-DG uptake or reduce lactate efflux in the presence of cytochalasin B. Increasing temperature (14 to 24°C) also did not increase 2-DG uptake, but decreasing temperature (14 to 4°C) reduced 2-DG uptake by 45%. In conclusion, exogenous glucose improves mechanical performance under hypoxia but not under any of the aerobic conditions applied. The extracellular concentration of glucose and cold temperature appear to determine and limit cardiomyocyte glucose uptake, respectively, and together may help define a metabolic strategy that relies predominantly on intracellular energy stores. PMID:23685969

Hypoxia-induced metastasis model in embryonic zebrafish

DEFF Research Database (Denmark)

Rouhi, Pegah; Jensen, Lasse D.; Cao, Ziquan

2010-01-01

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring...... of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent Di......I-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average...

Palmitate diet-induced loss of cardiac caveolin-3: a novel mechanism for lipid-induced contractile dysfunction.

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Catherine J Knowles

Full Text Available Obesity is associated with an increased risk of cardiomyopathy, and mechanisms linking the underlying risk and dietary factors are not well understood. We tested the hypothesis that dietary intake of saturated fat increases the levels of sphingolipids, namely ceramide and sphingomyelin in cardiac cell membranes that disrupt caveolae, specialized membrane micro-domains and important for cellular signaling. C57BL/6 mice were fed two high-fat diets: palmitate diet (21% total fat, 47% is palmitate, and MCT diet (21% medium-chain triglycerides, no palmitate. We established that high-palmitate feeding for 12 weeks leads to 40% and 50% increases in ceramide and sphingomyelin, respectively, in cellular membranes. Concomitant with sphingolipid accumulation, we observed a 40% reduction in systolic contractile performance. To explore the relationship of increased sphingolipids with caveolins, we analyzed caveolin protein levels and intracellular localization in isolated cardiomyocytes. In normal cardiomyocytes, caveolin-1 and caveolin-3 co-localize at the plasma membrane and the T-tubule system. However, mice maintained on palmitate lost 80% of caveolin-3, mainly from the T-tubule system. Mice maintained on MCT diet had a 90% reduction in caveolin-1. These data show that caveolin isoforms are sensitive to the lipid environment. These data are further supported by similar findings in human cardiac tissue samples from non-obese, obese, non-obese cardiomyopathic, and obese cardiomyopathic patients. To further elucidate the contractile dysfunction associated with the loss of caveolin-3, we determined the localization of the ryanodine receptor and found lower expression and loss of the striated appearance of this protein. We suggest that palmitate-induced loss of caveolin-3 results in cardiac contractile dysfunction via a defect in calcium-induced calcium release.

Hypoxia-Induced Collagen Synthesis of Human Lung Fibroblasts by Activating the Angiotensin System

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Shan-Shan Liu

2013-12-01

Full Text Available The exact molecular mechanism that mediates hypoxia-induced pulmonary fibrosis needs to be further clarified. The aim of this study was to explore the effect and underlying mechanism of angiotensin II (Ang II on collagen synthesis in hypoxic human lung fibroblast (HLF cells. The HLF-1 cell line was used for in vitro studies. Angiotensinogen (AGT, angiotensin converting enzyme (ACE, angiotensin II type 1 receptor (AT1R and angiotensin II type 2 receptor (AT2R expression levels in human lung fibroblasts were analysed using real-time polymerase chain reaction (RT-PCR after hypoxic treatment. Additionally, the collagen type I (Col-I, AT1R and nuclear factor κappaB (NF-κB protein expression levels were detected using Western blot analysis, and NF-κB nuclear translocation was measured using immunofluorescence localization analysis. Ang II levels in HLF-1 cells were measured with an enzyme-linked immunosorbent assay (ELISA. We found that hypoxia increased Col-I mRNA and protein expression in HLF-1 cells, and this effect could be inhibited by an AT1R or AT2R inhibitor. The levels of NF-κB, RAS components and Ang II production in HLF-1 cells were significantly increased after the hypoxia exposure. Hypoxia or Ang II increased NF-κB-p50 protein expression in HLF-1 cells, and the special effect could be inhibited by telmisartan (TST, an AT1R inhibitor, and partially inhibited by PD123319, an AT2R inhibitor. Importantly, hypoxia-induced NF-κB nuclear translocation could be nearly completely inhibited by an AT1R or AT2R inhibitor. Furthermore pyrrolidine dithiocarbamate (PDTC, a NF-κB blocker, abolished the expression of hypoxia-induced AT1R and Col-I in HLF-1 cells. Our results indicate that Ang II-mediated NF-κB signalling via ATR is involved in hypoxia-induced collagen synthesis in human lung fibroblasts.

TCDD Induces the Hypoxia-Inducible Factor (HIF-1α Regulatory Pathway in Human Trophoblastic JAR Cells

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Tien-Ling Liao

2014-09-01

Full Text Available The exposure to dioxin can compromise pregnancy outcomes and increase the risk of preterm births. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD has been demonstrated to induce placental hypoxia at the end of pregnancy in a rat model, and hypoxia has been suggested to be the cause of abnormal trophoblast differentiation and placental insufficiency syndromes. In this study, we demonstrate that the non-hypoxic stimulation of human trophoblastic cells by TCDD strongly increased hypoxia inducible factor-1 alpha (HIF-1α stabilization. TCDD exposure induced the generation of reactive oxygen species (ROS and nitric oxide. TCDD-induced HIF-1α stabilization and Akt phosphorylation was inhibited by pretreatment with wortmannin (a phosphatidylinositol 3-kinase (PI3K inhibitor or N-acetylcysteine (a ROS scavenger. The augmented HIF-1α stabilization by TCDD occurred via the ROS-dependent activation of the PI3K/Akt pathway. Additionally, a significant increase in invasion and metallomatrix protease-9 activity was found in TCDD-treated cells. The gene expression of vascular endothelial growth factor and placental growth factor was induced upon TCDD stimulation, whereas the protein levels of peroxisome proliferator-activated receptor γ (PPARγ, PPARγ coactivator-1α, mitochondrial transcription factor, and uncoupling protein 2 were decreased. Our results indicate that an activated HIF-1α pathway, elicited oxidative stress, and induced metabolic stress contribute to TCDD-induced trophoblastic toxicity. These findings may provide molecular insight into the TCDD-induced impairment of trophoblast function and placental development.

MicroRNA-1 overexpression blunts cardiomyocyte hypertrophy elicited by thyroid hormone.

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Diniz, Gabriela Placoná; Lino, Caroline Antunes; Moreno, Camila Rodrigues; Senger, Nathalia; Barreto-Chaves, Maria Luiza Morais

2017-12-01

It is well-known that increased thyroid hormone (TH) levels induce cardiomyocyte growth. MicroRNAs (miRNAs) have been identified as key players in cardiomyocyte hypertrophy, which is associated with increased risk of heart failure. In this study, we evaluated the miR-1 expression in TH-induced cardiac hypertrophy, as well as the potential involvement of miR-1 in cardiomyocyte hypertrophy elicited by TH in vitro. The possible role of type 1 angiotensin II receptor (AT1R) in the effect promoted by TH in miR-1 expression was also evaluated. Neonatal rat cardiac myocytes (NRCMs) were treated with T 3 for 24 hr and Wistar rats were subjected to hyperthyroidism for 14 days combined or not with AT1R blocker. Real Time RT-PCR analysis indicated that miR-1 expression was decreased in cardiac hypertrophy in response to TH in vitro and in vivo, and this effect was unchanged by AT1R blocker. In addition, HDAC4, which is target of miR-1, was increased in NRCMs after T 3 treatment. A gain-of-function study revealed that overexpression of miR-1 prevented T 3 -induced cardiomyocyte hypertrophy and reduced HADC4 mRNA levels in NRCMs. In vivo experiments confirmed the downregulation of miR-1 in cardiac tissue from hyperthyroid animals, which was accompanied by increased HDAC4 mRNA levels. In addition, HDAC inhibitor prevented T 3 -induced cardiomyocyte hypertrophy. Our data reveal a new mechanistic insight into cardiomyocyte growth in response to TH, suggesting that miR-1 plays a role in cardiomyocyte hypertrophy induced by TH potentially via targeting HADC4. © 2017 Wiley Periodicals, Inc.

Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells

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Asghar, Muhammad Yasir; Bergelin, Nina; Jaakkola, Panu; Törnquist, Kid

2013-01-01

Sphingosine-1-phosphate (S1P) is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1) is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3), Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1. PMID:23824493

Sphingosine-1-Phosphate as a Regulator of Hypoxia-Induced Factor-1α in Thyroid Follicular Carcinoma Cells.

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Veronica Kalhori

Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid, which regulates several cancer-related processes including migration and angiogenesis. We have previously shown S1P to induce migration of follicular ML-1 thyroid cancer cells. Hypoxia-induced factor-1 (HIF-1 is an oxygen-sensitive transcription factor, which adapts cells to hypoxic conditions through increased survival, motility and angiogenesis. Due to these properties and its increased expression in response to intratumoral hypoxia, HIF-1 is considered a significant regulator of tumor biology. We found S1P to increase expression of the regulatory HIF-1α subunit in normoxic ML-1 cells. S1P also increased HIF-1 activity and expression of HIF-1 target genes. Importantly, inhibition or knockdown of HIF-1α attenuated the S1P-induced migration of ML-1 cells. S1P-induced HIF-1α expression was mediated by S1P receptor 3 (S1P3, Gi proteins and their downstream effectors MEK, PI3K, mTOR and PKCβI. Half-life measurements with cycloheximide indicated that S1P treatment stabilized the HIF-1α protein. On the other hand, S1P activated translational regulators eIF-4E and p70S6K, which are known to control HIF-1α synthesis. In conclusion, we have identified S1P as a non-hypoxic regulator of HIF-1 activity in thyroid cancer cells, studied the signaling involved in S1P-induced HIF-1α expression and shown S1P-induced migration to be mediated by HIF-1.

Impaired ALDH2 activity decreases the mitochondrial respiration in H9C2 cardiomyocytes.

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Mali, Vishal R; Deshpande, Mandar; Pan, Guodong; Thandavarayan, Rajarajan A; Palaniyandi, Suresh S

2016-02-01

Reactive oxygen species (ROS)-mediated reactive aldehydes induce cellular stress. In cardiovascular diseases such as ischemia-reperfusion injury, lipid-peroxidation derived reactive aldehydes such as 4-hydroxy-2-nonenal (4HNE) are known to contribute to the pathogenesis. 4HNE is involved in ROS formation, abnormal calcium handling and more importantly defective mitochondrial respiration. Aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)(+)-dependent isozymes which can detoxify endogenous and exogenous aldehydes into non-toxic carboxylic acids. Therefore we hypothesize that 4HNE afflicts mitochondrial respiration and leads to cell death by impairing ALDH2 activity in cultured H9C2 cardiomyocyte cell lines. H9C2 cardiomyocytes were treated with 25, 50 and 75 μM 4HNE and its vehicle, ethanol as well as 25, 50 and 75 μM disulfiram (DSF), an inhibitor of ALDH2 and its vehicle (DMSO) for 4 h. 4HNE significantly decreased ALDH2 activity, ALDH2 protein levels, mitochondrial respiration and mitochondrial respiratory reserve capacity, and increased 4HNE adduct formation and cell death in cultured H9C2 cardiomyocytes. ALDH2 inhibition by DSF and ALDH2 siRNA attenuated ALDH2 activity besides reducing ALDH2 levels, mitochondrial respiration and mitochondrial respiratory reserve capacity and increased cell death. Our results indicate that ALDH2 impairment can lead to poor mitochondrial respiration and increased cell death in cultured H9C2 cardiomyocytes. Copyright © 2015 Elsevier Inc. All rights reserved.

Cardioprotective effect of breviscapine: inhibition of apoptosis in H9c2 cardiomyocytes via the PI3K/Akt/eNOS pathway following simulated ischemia/reperfusion injury.

Science.gov (United States)

Wang, Jun; Ji, Shu-Yun; Liu, Si-Zhu; Jing, Rui; Lou, Wei-Juan

2015-09-01

Breviscapine (BE) is a standardized Chinese herbal medicine extracted from Erigeron breviscapus (Vant.) Hand.-Mazz. It has been widely used to treat cardiovascular and cerebrovascular diseases. However, there are no reports on the protective effects and underlying molecular mechanisms of BE action on myocardial ischemia/reperfusion (MI/R)-induced cardiomyocyte apoptosis. In the present study, we aimed to confirm the cardioprotective effect of BE from MI/R injury in vivo, and investigate the potential molecular mechanisms against simulated ischemia/reperfusion (SI/R)-induced cardiomyocyte apoptosis in vitro. The rat model of MI/R injury was induced by 30 min of transient vessel occlusion followed by 3 h of reperfusion. BE significantly reduced the myocardium infarct size and production of cardiac troponin (cTnl) in serum. In an in vitro experiment, H9c2 cardiomyocytes were incubated with vehicle or ischemic buffer during hypoxia; then, they were reoxygenated with or without BE. BE markedly improved the cell viability and decreased lactate dehydrogenase (LDH) release. We confirmed the anti-apoptotic effect of BE with the Hoechst 33258 staining assay, and this effect was associated with an increase in Bcl-2 and a decrease in active caspase-3 expression. Western blot analysis also showed that BE increased the phosphorylation of Akt and eNOS in H9c2 cells, and the protective effects of BE were partially inhibited by the phosphatidylinositol 3'-kinase (PI3K) specific inhibitor LY294002. Our results suggested that BE could provide significant cardioprotection against MI/R injury, and the potential mechanisms might involve suppression of cardiomyocyte apoptosis through activating the PI3K/Akt/eNOS signaling pathway.

Postnatal ablation of Foxm1 from cardiomyocytes causes late onset cardiac hypertrophy and fibrosis without exacerbating pressure overload-induced cardiac remodeling.

Directory of Open Access Journals (Sweden)

Craig Bolte

Full Text Available Heart disease remains a leading cause of morbidity and mortality in the industrialized world. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disorder and the most common cause of sudden cardiac death. Foxm1 transcription factor (also known as HFH-11B, Trident, Win or MPP2 plays an important role in the pathogenesis of various cancers and is a critical mediator of post-injury repair in multiple organs. Foxm1 has been previously shown to be essential for heart development and proliferation of embryonic cardiomyocytes. However, the role of Foxm1 in postnatal heart development and in cardiac injury has not been evaluated. To delete Foxm1 in postnatal cardiomyocytes, αMHC-Cre/Foxm1(fl/fl mice were generated. Surprisingly, αMHC-Cre/Foxm1(fl/fl mice exhibited normal cardiomyocyte proliferation at postnatal day seven and had no defects in cardiac structure or function but developed cardiac hypertrophy and fibrosis late in life. The development of cardiomyocyte hypertrophy and cardiac fibrosis in aged Foxm1-deficient mice was associated with reduced expression of Hey2, an important regulator of cardiac homeostasis, and increased expression of genes critical for cardiac remodeling, including MMP9, αSMA, fibronectin and vimentin. We also found that following aortic constriction Foxm1 mRNA and protein were induced in cardiomyocytes. However, Foxm1 deletion did not exacerbate cardiac hypertrophy or fibrosis following chronic pressure overload. Our results demonstrate that Foxm1 regulates genes critical for age-induced cardiomyocyte hypertrophy and cardiac fibrosis.

The protective effect of hypoxia and dithiothreitol on X-ray-induced genetic damage in Arabidopsis

International Nuclear Information System (INIS)

Sree Ramulu, K.; Veen, J.H. van der

1987-01-01

A study was made on the protective effect of hypoxia and dithiothreitol (DTT) on X-ray-induced ovule sterility and embryonic lethality in Arabidopsis. Both hypoxia and DTT gave a pronounced and additive reduction of radiation-induced genetic damage. The reduction was significantly higher for ovule sterility than for embryonic lethals. It is suggested that non-fertilized ovules contain a higher ratio of strand breaks/other damage than embryonic lethals do, for hypoxia and DTT are known specifically to give a reduction of strand breaks. (Auth.)

Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

Energy Technology Data Exchange (ETDEWEB)

Ragnum, Harald Bull [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Røe, Kathrine [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Division of Medicine, Department of Oncology, Akershus University Hospital, Lørenskog (Norway); Holm, Ruth; Vlatkovic, Ljiljana [Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Nesland, Jahn Marthin [Department of Pathology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Medical Faculty, University of Oslo, Oslo (Norway); Aarnes, Eva-Katrine [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Ree, Anne Hansen [Division of Medicine, Department of Oncology, Akershus University Hospital, Lørenskog (Norway); Medical Faculty, University of Oslo, Oslo (Norway); Flatmark, Kjersti [Department of Tumor Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Department of Gastrointestinal Surgery, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Seierstad, Therese [Department of Radiology and Nuclear Medicine, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Faculty of Health Sciences, Buskerud University College, Drammen (Norway); Lilleby, Wolfgang [Department of Oncology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway); Lyng, Heidi, E-mail: heidi.lyng@rr-research.no [Department of Radiation Biology, The Norwegian Radium Hospital, Oslo University Hospital, Oslo (Norway)

2013-11-15

Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to

Hypoxia-Independent Downregulation of Hypoxia-Inducible Factor 1 Targets by Androgen Deprivation Therapy in Prostate Cancer

International Nuclear Information System (INIS)

Ragnum, Harald Bull; Røe, Kathrine; Holm, Ruth; Vlatkovic, Ljiljana; Nesland, Jahn Marthin; Aarnes, Eva-Katrine; Ree, Anne Hansen; Flatmark, Kjersti; Seierstad, Therese; Lilleby, Wolfgang; Lyng, Heidi

2013-01-01

Purpose: We explored changes in hypoxia-inducible factor 1 (HIF1) signaling during androgen deprivation therapy (ADT) of androgen-sensitive prostate cancer xenografts under conditions in which no significant change in immunostaining of the hypoxia marker pimonidazole had occurred. Methods and Materials: Gene expression profiles of volume-matched androgen-exposed and androgen-deprived CWR22 xenografts, with similar pimonidazole-positive fractions, were compared. Direct targets of androgen receptor (AR) and HIF1 transcription factors were identified among the differentially expressed genes by using published lists. Biological processes affected by ADT were determined by gene ontology analysis. HIF1α protein expression in xenografts and biopsy samples from 35 patients receiving neoadjuvant ADT was assessed by immunohistochemistry. Results: A total of 1344 genes showed more than 2-fold change in expression by ADT, including 35 downregulated and 5 upregulated HIF1 targets. Six genes were shared HIF1 and AR targets, and their downregulation was confirmed with quantitative RT-PCR. Significant suppression of the biological processes proliferation, metabolism, and stress response in androgen-deprived xenografts was found, consistent with tumor regression. Nineteen downregulated HIF1 targets were involved in those significant biological processes, most of them in metabolism. Four of these were shared AR and HIF1 targets, including genes encoding the regulatory glycolytic proteins HK2, PFKFB3, and SLC2A1. Most of the downregulated HIF1 targets were induced by hypoxia in androgen-responsive prostate cancer cell lines, confirming their role as hypoxia-responsive HIF1 targets in prostate cancer. Downregulation of HIF1 targets was consistent with the absence of HIF1α protein in xenografts and downregulation in patients by ADT (P<.001). Conclusions: AR repression by ADT may lead to downregulation of HIF1 signaling independently of hypoxic fraction, and this may contribute to

Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes

International Nuclear Information System (INIS)

Chien, Peter Tzu-Yu; Lin, Chih-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

2015-01-01

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE 2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. - Graphical abstract: In summary, CORM-2 treatment induces Pyk2 transactivated PDGFR, which induces PI3K/Akt/MAPK activation, and then recruits Sp1/Foxo1 transcriptional factors to regulate HO-1 gene expression in primary human cardiomyocytes. - Highlights: • CORM-2 induces HO-1 expression. • Pyk2-dependent PDGFR activates PI3K/Akt/MAPK pathway in CORM-2-induced HO-1

Induction of HO-1 by carbon monoxide releasing molecule-2 attenuates thrombin-induced COX-2 expression and hypertrophy in primary human cardiomyocytes

Energy Technology Data Exchange (ETDEWEB)

Chien, Peter Tzu-Yu [Department of Physiology and Pharmacology and Health Ageing Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Lin, Chih-Chung; Hsiao, Li-Der [Department of Anesthetics, Chang Gung Memorial Hospital at Lin-Kou and College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan (China); Yang, Chuen-Mao, E-mail: chuenmao@mail.cgu.edu.tw [Department of Physiology and Pharmacology and Health Ageing Research Center, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan, Taiwan (China); Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Tao-Yuan, Taiwan (China)

2015-12-01

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE{sub 2} release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. - Graphical abstract: In summary, CORM-2 treatment induces Pyk2 transactivated PDGFR, which induces PI3K/Akt/MAPK activation, and then recruits Sp1/Foxo1 transcriptional factors to regulate HO-1 gene expression in primary human cardiomyocytes. - Highlights: • CORM-2 induces HO-1 expression. • Pyk2-dependent PDGFR activates PI3K/Akt/MAPK pathway in CORM-2-induced HO

Oolong tea prevents cardiomyocyte loss against hypoxia by attenuating p-JNK mediated hypertrophy and enhancing P-IGF1R, p-akt, and p-Badser136 activity and by fortifying NRF2 antioxidation system.

Science.gov (United States)

Shibu, Marthandam Asokan; Kuo, Chia-Hua; Chen, Bih-Cheng; Ju, Da-Tong; Chen, Ray-Jade; Lai, Chao-Hung; Huang, Pei-Jane; Viswanadha, Vijaya Padma; Kuo, Wei-Wen; Huang, Chih-Yang

2018-02-01

Tea, the most widely consumed natural beverage has been associated with reduced mortality risk from cardiovascular disease. Oolong tea is a partially fermented tea containing high levels of catechins, their degree of oxidation varies between 20%-80% causing differences in their active metabolites. In this study we examined the effect of oolong tea extract (OTE) obtained by oxidation at low-temperature for short-time against hypoxic injury and found that oolong tea provides cyto-protective effects by suppressing the JNK mediated hypertrophic effects and by enhancing the innate antioxidant mechanisms in neonatal cardiomyocytes and in H9c2 cells. OTE effectively attenuates 24 h hypoxia-triggered cardiomyocyte loss by suppressing caspase-3-cleavage and apoptosis in a dose-dependent manner. OTE also enhances the IGFIR/p-Akt associated survival-mechanism involving the elevation of p-Bad ser136 in a dose-dependent manner to aid cellular adaptations against hypoxic challenge. The results show the effects and mechanism of Oolong tea to provide cardio-protective benefits during hypoxic conditions. © 2017 Wiley Periodicals, Inc.

Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

NARCIS (Netherlands)

Weijer, Ruud; Broekgaarden, Mans; Krekorian, Massis; Alles, Lindy K.; van Wijk, Albert C.; Mackaaij, Claire; Verheij, Joanne; van der Wal, Allard C.; van Gulik, Thomas M.; Storm, Gert; Heger, Michal

2016-01-01

Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression levels of

Inhibition of hypoxia inducible factor 1 and topoisomerase with acriflavine sensitizes perihilar cholangiocarcinomas to photodynamic therapy

NARCIS (Netherlands)

Weijer, R.; Broekgaarden, M.; Krekorian, M.; Alles, L.K.; van Wijk, A.C; Mackaaij, C.; Verheij, J.; van der Wal, A.C.; van Gullik, T.M.; Storm, Gerrit; Heger, M.

2016-01-01

Background: Photodynamic therapy (PDT) induces tumor cell death by oxidative stress and hypoxia but also survival signaling through activation of hypoxia-inducible factor 1 (HIF-1). Since perihilar cholangiocarcinomas are relatively recalcitrant to PDT, the aims were to (1) determine the expression

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro

Energy Technology Data Exchange (ETDEWEB)

Zhou, Xiangjun [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Yao, Qisheng, E-mail: yymcyqs@126.com [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Sun, Xinbo; Gong, Xiaoxin; Yang, Yong; Chen, Congbo [Department of Urology, Taihe Hospital, Hubei University of Medicine, Hubei (China); Shan, Guang [Department of Urology, Renmin Hospital of Wuhan University, Hubei (China)

2017-03-01

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treated with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway. - Highlights: • Slit2 ameliorates inflammation after hypoxia-and LPS-induced epithelial cells injury

Intermittent hypercapnic hypoxia during sleep does not induce ventilatory long-term facilitation in healthy males.

Science.gov (United States)

Deacon, Naomi L; McEvoy, R Doug; Stadler, Daniel L; Catcheside, Peter G

2017-09-01

Intermittent hypoxia-induced ventilatory neuroplasticity is likely important in obstructive sleep apnea pathophysiology. Although concomitant CO 2 levels and arousal state critically influence neuroplastic effects of intermittent hypoxia, no studies have investigated intermittent hypercapnic hypoxia effects during sleep in humans. Thus the purpose of this study was to investigate if intermittent hypercapnic hypoxia during sleep induces neuroplasticity (ventilatory long-term facilitation and increased chemoreflex responsiveness) in humans. Twelve healthy males were exposed to intermittent hypercapnic hypoxia (24 × 30 s episodes of 3% CO 2 and 3.0 ± 0.2% O 2 ) and intermittent medical air during sleep after 2 wk washout period in a randomized crossover study design. Minute ventilation, end-tidal CO 2 , O 2 saturation, breath timing, upper airway resistance, and genioglossal and diaphragm electromyograms were examined during 10 min of stable stage 2 sleep preceding gas exposure, during gas and intervening room air periods, and throughout 1 h of room air recovery. There were no significant differences between conditions across time to indicate long-term facilitation of ventilation, genioglossal or diaphragm electromyogram activity, and no change in ventilatory response from the first to last gas exposure to suggest any change in chemoreflex responsiveness. These findings contrast with previous intermittent hypoxia studies without intermittent hypercapnia and suggest that the more relevant gas disturbance stimulus of concomitant intermittent hypercapnia frequently occurring in sleep apnea influences acute neuroplastic effects of intermittent hypoxia. These findings highlight the need for further studies of intermittent hypercapnic hypoxia during sleep to clarify the role of ventilatory neuroplasticity in the pathophysiology of sleep apnea. NEW & NOTEWORTHY Both arousal state and concomitant CO 2 levels are known modulators of the effects of intermittent hypoxia on

Cardiac injury of the newborn mammalian heart accelerates cardiomyocyte terminal differentiation

DEFF Research Database (Denmark)

Zebrowski, David C.; Jensen, Charlotte H.; Becker, Robert

2017-01-01

exhibited midbody formation consistent with successful abscission, whereas those from 3 day-old cardiomyocytes after apical resection exhibited midbody formation consistent with abscission failure. Lastly, injured hearts failed to fully regenerate as evidenced by persistent scarring and reduced wall motion......After birth cardiomyocytes undergo terminal differentiation, characterized by binucleation and centrosome disassembly, rendering the heart unable to regenerate. Yet, it has been suggested that newborn mammals regenerate their hearts after apical resection by cardiomyocyte proliferation. Thus, we...... increased rate of binucleation there was a nearly 2-fold increase in the number of cardiomyocytes in mitosis indicating that the majority of injury-induced cardiomyocyte cell cycle activity results in binucleation, not proliferation. Concurrently, cardiomyocytes undergoing cytokinesis from embryonic hearts...

Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

DEFF Research Database (Denmark)

Møller, P; Loft, S; Lundby, C

2001-01-01

; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage...... oxygen species, generated by leakage of the mitochondrial respiration or during a hypoxia-induced inflammation. Furthermore, the presence of DNA strand breaks may play an important role in maintaining hypoxia-induced inflammation processes. Hypoxia seems to deplete the antioxidant system of its capacity...

Cerium oxide nanoparticles protect rodent lungs from hypobaric hypoxia-induced oxidative stress and inflammation

Directory of Open Access Journals (Sweden)

Arya A

2013-11-01

Full Text Available Aditya Arya,1 Niroj Kumar Sethy,1 Sushil Kumar Singh,2 Mainak Das,3 Kalpana Bhargava1 1Peptide and Proteomics Division, Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organization, Delhi, 2Functional Materials Division, Solid State Physics Laboratory, Defence Research and Development Organization, Delhi, 3Biological Science and Bioengineering, Indian Institute of Technology, Kanpur, Uttar Pradesh, India Background: Cerium oxide nanoparticles (nanoceria are effective at quenching reactive oxygen species (ROS in cell culture and animal models. Although nanoceria reportedly deposit in lungs, their efficacy in conferring lung protection during oxidative stress remains unexplored. Thus, the study evaluated the protective efficacy of nanoceria in rat lung tissue during hypobaric hypoxia. Methods: A total of 48 animals were randomly divided into four equal groups (control [C], nanoceria treated [T], hypoxia [H], and nanoceria treated plus hypoxia [T+H]. Animals were injected intraperitoneally with either a dose of 0.5 µg/kg body weight/week of nanoceria (T and T+H groups or vehicle (C and H groups for 5 weeks. After the final dose, H and T+H animals were challenged with hypobaric hypoxia, while C and T animals were maintained at normoxia. Lungs were isolated and homogenate was obtained for analysis of ROS, lipid peroxidation, glutathione, protein carbonylation, and 4-hydroxynonenal-adduct formation. Plasma was used for estimating major inflammatory cytokines using enzyme-linked immunosorbent assay. Intact lung tissues were fixed and both transmission electron microscopy and histopathological examinations were carried out separately for detecting internalization of nanoparticles as well as altered lung morphology. Results: Spherical nanoceria of 7–10 nm diameter were synthesized using a microemulsion method, and the lung protective efficacy of the nanoceria evaluated during hypobaric hypoxia. With repeated

Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

Directory of Open Access Journals (Sweden)

Yi Miao

2013-02-01

Full Text Available Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS by measuring mitochondrial ROS (mtROS, mitochondrial DNA (mtDNA copy number, xanthine oxidase (XO and NADPH oxidase (NOX activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

Science.gov (United States)

Ghosh, Rajeshwary; Goswami, Sumanta K; Feitoza, Luis Felipe B B; Hammock, Bruce; Gomes, Aldrin V

2016-11-15

One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Hypoxia-induced autophagy is inhibited by PADI4 knockdown, which promotes apoptosis of fibroblast-like synoviocytes in rheumatoid arthritis

Science.gov (United States)

Fan, Tingting; Zhang, Changsong; Zong, Ming; Fan, Lieying

2018-01-01

Impaired apoptosis of rheumatoid arthritis (RA)-fibroblast-like synoviocytes (FLS) is pivotal in the process of RA. Peptidyl arginine deiminase type IV (PADI4) is associated with autoantibody regulation via histone citrullination in RA. The present study aimed to investigate the role of PADI4 in the apoptosis of RA-FLS. FLS were isolated from patients with RA and a rat model. The effects of PADI4 on RA-FLS were investigated in vitro and in vivo. Hypoxia-induced autophagy was induced by 1% O2 and was detected by immunohistochemical and immunofluorescence analysis; in addition, apoptosis was detected by flow cytometry. RA-FLS obtained from RA rat model exhibited significant proliferation under severe hypoxia conditions. Hypoxia also significantly induced autophagy and elevated the expression of PADI4. Subsequently, short hairpin RNA-mediated PADI4 knockdown was demonstrated to significantly inhibit hypoxia-induced autophagy and promote apoptosis in RA-FLS. The results of these in vitro and in vivo studies suggested that PADI4 may be closely associated with hypoxia-induced autophagy, and the inhibition of hypoxia-induced autophagy by PADI4 knockdown may contribute to an increase in the apoptosis of RA-FLS. PMID:29393388

Multiple roles of hypoxia in ovarian function: roles of hypoxia-inducible factor-related and -unrelated signals during the luteal phase

OpenAIRE

Nishimura, Ryo; Okuda, Kiyoshi

2015-01-01

There is increasing interest in the role of oxygen conditions in the microenvironment of organs because of the discovery of a hypoxia-specific transcription factor, namely hypoxia-inducible factor (HIF) 1. Ovarian function has several phases that change day by day, including ovulation, follicular growth and corpus luteum formation and regression. These phases are regulated by many factors, including pituitary hormones and local hormones, such as steroids, peptides and cytokines, as well as ox...

Complex Evaluation Oxygen Status and Lipid Metabolism Indexes in Newborns with Perinatal Hypoxia and Hypovolemic Shock

Directory of Open Access Journals (Sweden)

Svetlana A. Perepelitsa

2017-01-01

Full Text Available Aim. To asses of metabolism, lipid metabolism and oxygen status parameters in newborns with perinatal hypoxia.Materials and Methods. 53 newborn babies born with signs of severe hypoxia and low Apgar scoring equal to 2 at the 1st minute of life were enrolled in the study. Newborns were divided into 2 groups depending on the presence of the clinical presentation of shock: Group 1 «Shock» and Group 2 «Acute intranatal hypoxia» (AIH. All newborns underwent testing for blood gas and acid-base balance, lactate level. Cholesterol and triglyceride levels in the central venous blood were also tested immediately after the birth and on the 5th day of life. Mechanical ventilation mode and parameters were registered. The mean airway pressure (MAP and the oxygen saturation index (OSI were calculated.Results. Severe decompensated metabolic lactic acidosis was diagnosed in a «Shock» group newborns at birth, thus indicating severe perinatal hypoxia which had triggered the development of shock. As for the «AIH» group newborns, they had hyperlactatemia alone. The most severe hypoxemia at birth was diagnosed in newborns of the «Shock» group; the OSI value in these infants was significantly higher than that in «AIH» infants (P<0.01. Despite the treatment and mechanical ventilation, during the posthypoxic period, newborns from the «Shock» group were characterized by increased OSI values over 12 hours after birth. Significantly high levels of OSI persisted for 48 hours after the delivery. Severe hypotriglyceridemia and hypocholesterolemia were found in both group newborns.Conclusion. The study demonstrated that there was intranatal complex metabolism impairment in the case of perinatal hypoxia; at birth, it manifested by metabolic acidosis of various degrees of severity and imbalance of triglycerides and cholesterol levels. The longer and more severe hypoxia is, the more severe acid-base balance and blood lactate level impairment at birth become

Moderate ethanol administration accentuates cardiomyocyte contractile dysfunction and mitochondrial injury in high fat diet-induced obesity.

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Yuan, Fang; Lei, Yonghong; Wang, Qiurong; Esberg, Lucy B; Huang, Zaixing; Scott, Glenda I; Li, Xue; Ren, Jun

2015-03-18

Light to moderate drinking confers cardioprotection although it remains unclear with regards to the role of moderate drinking on cardiac function in obesity. This study was designed to examine the impact of moderate ethanol intake on myocardial function in high fat diet intake-induced obesity and the mechanism(s) involved with a focus on mitochondrial integrity. C57BL/6 mice were fed low or high fat diet for 16 weeks prior to ethanol challenge (1g/kg/d for 3 days). Cardiac contractile function, intracellular Ca(2+) homeostasis, myocardial histology, and mitochondrial integrity [aconitase activity and the mitochondrial proteins SOD1, UCP-2 and PPARγ coactivator 1α (PGC-1α)] were assessed 24h after the final ethanol challenge. Fat diet intake compromised cardiomyocyte contractile and intracellular Ca(2+) properties (depressed peak shortening and maximal velocities of shortening/relengthening, prolonged duration of relengthening, dampened intracellular Ca(2+) rise and clearance without affecting duration of shortening). Although moderate ethanol challenge failed to alter cardiomyocyte mechanical property under low fat diet intake, it accentuated high fat diet intake-induced changes in cardiomyocyte contractile function and intracellular Ca(2+) handling. Moderate ethanol challenge failed to affect fat diet intake-induced cardiac hypertrophy as evidenced by H&E staining. High fat diet intake reduced myocardial aconitase activity, downregulated levels of mitochondrial protein UCP-2, PGC-1α, SOD1 and interrupted intracellular Ca(2+) regulatory proteins, the effect of which was augmented by moderate ethanol challenge. Neither high fat diet intake nor moderate ethanol challenge affected protein or mRNA levels as well as phosphorylation of Akt and GSK3β in mouse hearts. Taken together, our data revealed that moderate ethanol challenge accentuated high fat diet-induced cardiac contractile and intracellular Ca(2+) anomalies as well as mitochondrial injury. Copyright �

Structural and functional analysis of coral Hypoxia Inducible Factor.

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Didier Zoccola

Full Text Available Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O2 concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1 is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O2 concentrations of the coral model Stylophora pistillata. In silico analysis showed that homologs of HIF-1 α (SpiHIF-1α and HIF-1β (SpiHIF-1β exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O2 concentration between Cnidarians and Mammals whose ancestors diverged about 1,200-1,500 million years ago.

Structural and functional analysis of coral Hypoxia Inducible Factor.

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Zoccola, Didier; Morain, Jonas; Pagès, Gilles; Caminiti-Segonds, Natacha; Giuliano, Sandy; Tambutté, Sylvie; Allemand, Denis

2017-01-01

Tissues of symbiotic Cnidarians are exposed to wide, rapid and daily variations of oxygen concentration. Indeed, during daytime, intracellular O2 concentration increases due to symbiont photosynthesis, while during night, respiration of both host cells and symbionts leads to intra-tissue hypoxia. The Hypoxia Inducible Factor 1 (HIF-1) is a heterodimeric transcription factor used for maintenance of oxygen homeostasis and adaptation to hypoxia. Here, we carried out a mechanistic study of the response to variations of O2 concentrations of the coral model Stylophora pistillata. In silico analysis showed that homologs of HIF-1 α (SpiHIF-1α) and HIF-1β (SpiHIF-1β) exist in coral. A specific SpiHIF-1 DNA binding on mammalian Hypoxia Response Element (HRE) sequences was shown in extracts from coral exposed to dark conditions. Then, we cloned the coral HIF-1α and β genes and determined their expression and transcriptional activity. Although HIF-1α has an incomplete Oxygen-dependent Degradation Domain (ODD) relative to its human homolog, its protein level is increased under hypoxia when tested in mammalian cells. Moreover, co-transfection of SpiHIF-1α and β in mammalian cells stimulated an artificial promoter containing HRE only in hypoxic conditions. This study shows the strong conservation of molecular mechanisms involved in adaptation to O2 concentration between Cnidarians and Mammals whose ancestors diverged about 1,200-1,500 million years ago.

Hypoxia Inducible Factors and Hypertension: Lessons from Sleep Apnea Syndrome

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Nanduri, Jayasri; Peng, Ying-Jie; Yuan, Guoxiang; Kumar, Ganesh K.; Prabhakar, Nanduri R.

2015-01-01

Systemic hypertension is one of the most prevalent cardiovascular diseases. Sleep disordered breathing (SDB) with recurrent apnea is a major risk factor for developing essential hypertension. Chronic intermittent hypoxia (CIH) is a hallmark manifestation of recurrent apnea. Rodent models patterned after the O2 profiles seen with SDB patients showed that CIH is the major stimulus for causing systemic hypertension. This article reviews the physiological and molecular basis of CIH-induced hypertension. Physiological studies have identified that augmented carotid body chemosensory reflex and the resulting increase in sympathetic nerve activity is a major contributor to CIH-induced hypertension. Analysis of molecular mechanisms revealed that CIH activates hypoxia-inducible factor (HIF)-1 and suppresses HIF-2- mediated transcription. Dysregulation of HIF-1- and HIF-2- mediated transcription leads to imbalance of pro-oxidant and anti-oxidant enzyme gene expression resulting in increased reactive species (ROS) generation in the chemosensory reflex which is central for developing hypertension. PMID:25772710

Distinct responses of protein turnover regulatory pathways in hypoxia- and semistarvation-induced muscle atrophy

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de Theije, Chiel C.; Langen, Ramon C. J.; Lamers, Wouter H.; Schols, Annemie M. W. J.; Köhler, S. Eleonore

2013-01-01

The balance of muscle protein synthesis and degradation determines skeletal muscle mass. We hypothesized that hypoxia-induced muscle atrophy and alterations in the regulation of muscle protein turnover include a hypoxia-specific component, in addition to the observed effects of reduction in food

Hypoxia inducible factor-1α-dependent epithelial to mesenchymal transition under hypoxic conditions in prostate cancer cells.

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Li, Mingchuan; Wang, Yong Xing; Luo, Yong; Zhao, Jiahui; Li, Qing; Zhang, Jiao; Jiang, Yongguang

2016-07-01

Prostate cancer is the most commonly diagnosed cancer in men and the second leading cause of cancer death. Hypoxia is an environmental stimulus that plays an important role in the development and cancer progression especially for solid tumors. The key regulator under hypoxic conditions is stabilized hypoxia-inducible factor (HIF)-1α. In the present study, immune-fluorescent staining, siRNAs, qRT-PC, immunoblotting, cell migration and invasion assays were carried out to test typical epithelial to mesenchymal transition under hypoxia and the key regulators of this process in PC3, a human prostate cancer cell line. Our data demonstrated that hypoxia induces diverse molecular, phenotypic and functional changes in prostate cancer cells that are consistent with EMT. We also showed that a cell signal factor such as HIF-1α, which might be stabilized under hypoxic environment, is involved in EMT and cancer cell invasive potency. The induced hypoxia could be blocked by HIF-1α gene silencing and reoxygenation of EMT in prostate cancer cells, hypoxia partially reversed accompanied by a process of mesenchymal-epithelial reverting transition (MErT). EMT might be induced by activation of HIF-1α-dependent cell signaling in hypoxic prostate cancer cells.

Hepcidin: A Critical Regulator Of Iron Metabolism During Hypoxia

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2011-01-01

inducible factor (HIF)/hypoxia response element ( HRE ) system, as well as recent evidence indicating that localized adipose hypoxia due to obesity may...mechanisms by which hypoxia affects hepcidin expression, to include a review of the hypoxia inducible factor (HIF)/hypoxia response element ( HRE ) system, as...a battery of genes are induced by the hypoxia inducible factor (HIF)/hypoxia response element ( HRE ) system. The HIF system senses O2 levels through

LncRNA TUG1 serves an important role in hypoxia-induced myocardial cell injury by regulating the miR‑145‑5p‑Binp3 axis.

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Wu, Zhongwei; Zhao, Shengji; Li, Chunfu; Liu, Chaoquan

2018-02-01

The aim of the present study was to investigate the function of long non‑coding RNA TUG1 in hypoxia‑induced myocardial cell injury and to explore the potential molecular mechanisms. The cardiomyocyte cell line H9c2 was cultured under hypoxic and normoxic conditions. TUG1 expression under hypoxic conditions was then detected. The effects of TUG1 overexpression on viability, apoptosis, migration and invasion were assayed. In addition, the microRNA (miR)‑145‑5p expression was detected. Following H9c2 cell transfection with miR‑145‑5p mimics, the H9c2 cell viability, apoptosis, migration and invasion were also detected. Additionally, the target gene of miR‑145‑5p was assayed by Luciferase reporter assay. The protein expressions of Wnt‑3a, Wnt5a, and β‑catenin in H9c2 cells under hypoxic conditions were also determined. The results revealed that hypoxia induced injury in H9c2 cells, including inhibiting cell viability, migration and invasion, and promoting cell apoptosis. Overexpression of TUG1 aggravated hypoxia‑induced injury in H9c2 cells. In addition, miR‑145‑5p was negatively regulated by TUG1, and TUG1 overexpression aggravated hypoxia‑induced injury via the downregulation of miR‑145‑5p. Furthermore, B‑cell lymphoma 2 interacting protein 3 (Bnip3) was a target of miR‑145‑5p, and overexpression of Bnip3 aggravated hypoxia‑induced cell injury by activating Wnt/β‑catenin signaling pathways in H9c2 cells. In conclusion, overexpression of TUG1 aggravated hypoxia‑induced injury in cardiomyocytes by regulating the miR‑145‑5p‑Binp3 axis. Activation of the Wnt/β‑catenin signaling pathway may be a key mechanism to mediate the role of TUG1 in regulating hypoxia‑induced myocardial injury. TUG1 may be an effective diagnostic marker and therapeutic target for myocardial ischemia.

High-fat feeding in cardiomyocyte-restricted PPARdelta knockout mice leads to cardiac overexpression of lipid metabolic genes but fails to rescue cardiac phenotypes.

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Li, Yuquan; Cheng, Lihong; Qin, Qianhong; Liu, Jian; Lo, Woo-kuen; Brako, Lowrence A; Yang, Qinglin

2009-10-01

Peroxisome proliferator-activated receptor delta (PPARdelta) is an essential determinant of basal myocardial fatty acid oxidation (FAO) and bioenergetics. We wished to determine whether increased lipid loading affects the PPARdelta deficient heart in transcriptional regulation of FAO and in the development of cardiac pathology. Cardiomyocyte-restricted PPARdelta knockout (CR-PPARdelta(-/-)) and control (alpha-MyHC-Cre) mice were subjected to 48 h of fasting and to a long-term maintenance on a (28 weeks) high-fat diet (HFD). The expression of key FAO proteins in heart was examined. Serum lipid profiles, cardiac pathology, and changes of various transduction signaling pathways were also examined. Mice subjected to fasting exhibited upregulated transcript expression of FAO genes in the CR-PPARdelta(-/-) hearts. Moreover, long-term HFD in CR-PPARdelta(-/-) mice induced a strikingly greater transcriptional response. After HFD, genes encoding key FAO enzymes were expressed remarkably more in CR-PPARdelta(-/-) hearts than in those of control mice. Despite the marked rise of FAO gene expression, corresponding protein expression remained low in the CR-PPARdelta(-/-) heart, accompanied by abnormalities in sarcomere structures and mitochondria that were similar to those of CR-PPARdelta(-/-) hearts with regular chow feeding. The CR-PPARdelta(-/-) mice displayed increased expression of PPARgamma co-activator-1alpha (PGC-1alpha) and PPARalpha in the heart with deactivated Akt and p42/44 MAPK signaling in response to HFD. We conclude that PPARdelta is an essential determinant of myocardial FAO. Increased lipid intake activates cardiac expression of FAO genes via PPARalpha/PGC-1alpha pathway, albeit it is not sufficient to improve cardiac pathology due to PPARdelta deficiency.

Effect of hypoxia on the incorporation of [2-3H] glycerol and [1-14C[-palmitate into lipids of various brain regions

International Nuclear Information System (INIS)

Alberghina, M.; Giuffrida, A.M.

1981-01-01

The lipid metabolism in guinea pig brain after intermittent hypoxia, prolonged for 80 hrs, was markedly impaired. The in vivo incorporation of [2-3H] glycerol and [1-14C] palmitate into lipids of microsomes, mitochondria, myelin, and synaptosomes, purified form cerebral hemispheres, was significantly lower in the hypoxic animals than in the controls. The same effect was observed on the incorporation of labeled precursors into lipids of mitochondria purified from cerebellum and brainstem. In particular, the labeling of th major phospholipids present - ie, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) - in the mitochondria of the three brain regions examined decreased after hypoxic treatment

Gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 signaling in pregnant rats.

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Zhou, Jianjun; Xiao, Daliao; Hu, Yali; Wang, Zhiqun; Paradis, Alexandra; Mata-Greenwood, Eugenia; Zhang, Lubo

2013-09-01

Preeclampsia is a life-threatening pregnancy disorder. However, its pathogenesis remains unclear. We tested the hypothesis that gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 (ET-1) signaling. Time-dated pregnant and nonpregnant rats were divided into normoxic and hypoxic (10.5% O2 from the gestational day 6-21) groups. Chronic hypoxia had no significant effect on blood pressure or proteinuria in nonpregnant rats but significantly increased blood pressure on day 12 (systolic blood pressure, 111.7 ± 6.1 versus 138.5 ± 3.5 mm Hg; P=0.004) and day 20 (systolic blood pressure, 103.4 ± 4.6 versus 125.1 ± 6.1 mm Hg; P=0.02) in pregnant rats and urine protein (μg/μL)/creatinine (nmol/μL) ratio on day 20 (0.10 ± 0.01 versus 0.20 ± 0.04; P=0.04), as compared with the normoxic control group. This was accompanied with asymmetrical fetal growth restriction. Hypoxia resulted in impaired trophoblast invasion and uteroplacental vascular remodeling. In addition, plasma ET-1 levels, as well as the abundance of prepro-ET-1 mRNA, ET-1 type A receptor and angiotensin II type 1 receptor protein in the kidney and placenta were significantly increased in the chronic hypoxic group, as compared with the control animals. Treatment with the ET-1 type A receptor antagonist, BQ123, during the course of hypoxia exposure significantly attenuated the hypoxia-induced hypertension and other preeclampsia-like features. The results demonstrate that chronic hypoxia during gestation induces preeclamptic symptoms in pregnant rats via heightened ET-1 and ET-1 type A receptor-mediated signaling, providing a molecular mechanism linking gestational hypoxia and increased risk of preeclampsia.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

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Yoichi Takakusagi

Full Text Available BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2, with minimal effect under aerobic conditions (21% O2. Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3, significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

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Takakusagi, Yoichi; Matsumoto, Shingo; Saito, Keita; Matsuo, Masayuki; Kishimoto, Shun; Wojtkowiak, Jonathan W; DeGraff, William; Kesarwala, Aparna H; Choudhuri, Rajani; Devasahayam, Nallathamby; Subramanian, Sankaran; Munasinghe, Jeeva P; Gillies, Robert J; Mitchell, James B; Hart, Charles P; Krishna, Murali C

2014-01-01

TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

Structural phenotyping of stem cell-derived cardiomyocytes.

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Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

2015-03-10

Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Induced pluripotent stem cell-derived cardiomyocytes for cardiovascular disease modeling and drug screening

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Sharma, Arun; Wu, Joseph C; Wu, Sean M

2013-01-01

Human induced pluripotent stem cells (hiPSCs) have emerged as a novel tool for drug discovery and therapy in cardiovascular medicine. hiPSCs are functionally similar to human embryonic stem cells (hESCs) and can be derived autologously without the ethical challenges associated with hESCs. Given the limited regenerative capacity of the human heart following myocardial injury, cardiomyocytes derived from hiPSCs (hiPSC-CMs) have garnered significant attention from basic and translational scienti...

Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension.

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Maston, Levi D; Jones, David T; Giermakowska, Wieslawa; Howard, Tamara A; Cannon, Judy L; Wang, Wei; Wei, Yongyi; Xuan, Weimin; Resta, Thomas C; Gonzalez Bosc, Laura V

2017-05-01

Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4 + T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4 + T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1 -/- , lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4 + , CD8 + , or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1 -/- mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4 + but not CD8 + T cells restored the hypertensive phenotype in RAG1 -/- mice. Interestingly, RAG1 -/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4 + cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension. Copyright © 2017 the American Physiological Society.

Flaxseed Oil Alleviates Chronic HFD-Induced Insulin Resistance through Remodeling Lipid Homeostasis in Obese Adipose Tissue.

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Yu, Xiao; Tang, Yuhan; Liu, Peiyi; Xiao, Lin; Liu, Liegang; Shen, Ruiling; Deng, Qianchun; Yao, Ping

2017-11-08

Emerging evidence suggests that higher circulating long-chain n-3 polyunsaturated fatty acids (n-3PUFA) levels were intimately associated with lower prevalence of obesity and insulin resistance. However, the understanding of bioactivity and potential mechanism of α-linolenic acid-rich flaxseed oil (ALA-FO) against insulin resistance was still limited. This study evaluated the effect of FO on high-fat diet (HFD)-induced insulin resistance in C57BL/6J mice focused on adipose tissue lipolysis. Mice after HFD feeding for 16 weeks (60% fat-derived calories) exhibited systemic insulin resistance, which was greatly attenuated by medium dose of FO (M-FO), paralleling with differential accumulation of ALA and its n-3 derivatives across serum lipid fractions. Moreover, M-FO was sufficient to effectively block the metabolic activation of adipose tissue macrophages (ATMs), thereby improving adipose tissue insulin signaling. Importantly, suppression of hypoxia-inducible factors HIF-1α and HIF-2α were involved in FO-mediated modulation of adipose tissue lipolysis, accompanied by specific reconstitution of n-3PUFA within adipose tissue lipid fractions.

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

International Nuclear Information System (INIS)

Zaja, Ivan; Bai, Xiaowen; Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G.; Bosnjak, Zeljko J.

2014-01-01

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

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Zaja, Ivan [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bai, Xiaowen, E-mail: xibai@mcw.edu [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bosnjak, Zeljko J. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States)

2014-10-31

Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

Aromatic Hydrocarbon Receptor Suppresses Prostate Cancer Bone Metastasis Cells-Induced Vasculogenesis of Endothelial Progenitor Cells under Hypoxia

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Shuai Huang

2016-07-01

Full Text Available Background/Aims: Hypoxia leads to the development of neovascularization in solid tumor by regulating VEGF expression. Aromatic hydrocarbon receptor (AHR, a receptor for dioxin-like compounds, functions as a transcription factor through dimerization with hypoxia-inducible factors 1β (HIF-1β and inhibits the secretion of vascular endothelial growth factor (VEGF. The purpose of this study was to explore whether AHR can suppress hypoxia-induced VEGF production in prostate bone metastasis cells and repress neovascularization in endothelial progenitor cells (EPCs, and, if so, through what mechanisms. Methods: PC-3 or LNCaP cells induced angiogenesis was detected by Matrigel-based tube formation assay, mRNA expression levels was measured by qRT-PCR, VEGF secretion level was determined by ELISA assay, respectively. Results: AHR activation inhibits hypoxia-induced adhesiveness and vasculogenesis of EPCs induced by PC-3 or LNCaP cells under hypoxia. Moreover, AHR activation suppressed hypoxia-induced VEGF production in PC-3 and LNCaP cells (48 ± 14% in PC-3, p = 0.000; 41 ± 14% in LNCaP, p = 0.000 by attenuating HIF-1α and HIF-1β level that in turn diminished the angiogenic ability of EPCs in vitro. Furthermore, we found the mRNA level of hypoxia-inducible factors 1α (HIF-1α (1.54 ± 0.13 fold in PC-3, p = 0.002, 1.62 ± 0.12 fold in LNCaP, p = 0.001 and HIF-1β (1.67 ± 0.23 fold in PC-3, p = 0.007; 1.75 ± 0.26 fold in LNCaP, p=0.008 were upregulated in prostate cancer bone metastasis PC-3 and LNCaP cell lines in response to hypoxia, and revealed that the regulation of VEGF by HIF-1α and HIF-1β was possibly mediated by the activation of phosphatidylinositol 3-kinase pathway. Conclusion: By providing a mechanistic insight into the modulation of neovascularization by AHR ligand, we suggest that AHR ligand has a strong potential of being a new therapeutic agent with applications in the field of bone metastatic prostate cancer.

Cytoprotective effects of fisetin against hypoxia-induced cell death in PC12 cells.

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Chen, Pei-Yi; Ho, Yi-Ru; Wu, Ming-Jiuan; Huang, Shun-Ping; Chen, Po-Kong; Tai, Mi-Hsueh; Ho, Chi-Tang; Yen, Jui-Hung

2015-01-01

Fisetin (3,7,3',4'-tetrahydroxyflavone), a flavonol compound of flavonoids, exhibits a broad spectrum of biological activities including anti-oxidant, anti-inflammatory, anti-cancer and neuroprotective effects. The aim of this study is to investigate the cytoprotective effect of fisetin and the underlying molecular mechanism against hypoxia-induced cell death in PC12 cells. The results of this study showed that fisetin significantly restored the cell viability of PC12 cells under both cobalt chloride (CoCl₂)- and low oxygen-induced hypoxic conditions. Treatment with fisetin successfully reduced the CoCl₂-mediated reactive oxygen species (ROS) production, which was accompanied by an increase in the cell viability of PC12 cells. Furthermore, we found that treatment of PC12 cells with fisetin markedly upregulated hypoxia-inducible factor 1α (HIF-1α), its nuclear accumulation and the hypoxia-response element (HRE)-driven transcriptional activation. The fisetin-mediated cytoprotection during CoCl₂ exposure was significantly attenuated through the administration of HIF-1α siRNA. Moreover, we demonstrated that MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK and phosphatidylinositol 3-kinase (PI3 K) inhibitors significantly blocked the increase in cell survival that was induced by fisetin treatment under hypoxic conditions. Consistently, increased phosphorylation of ERK, p38 and Akt proteins was observed in PC12 cells treated with fisetin. However, the fisetin-induced HRE-driven transcription was not affected by inhibition of these kinase signaling pathways. Current results reveal for the first time that fisetin promotes cell survival and protects against hypoxia-induced cell death through ROS scavenging and the activation of HIF1α-, MAPK/ERK-, p38 MAPK- and PI3 K/Akt-dependent signaling pathways in PC12 cells.

Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes

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Yao Xiao

2016-09-01

Full Text Available A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

Epigenetic control of hypoxia inducible factor-1α-dependent expression of placental growth factor in hypoxic conditions.

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Tudisco, Laura; Della Ragione, Floriana; Tarallo, Valeria; Apicella, Ivana; D'Esposito, Maurizio; Matarazzo, Maria Rosaria; De Falco, Sandro

2014-04-01

Hypoxia plays a crucial role in the angiogenic switch, modulating a large set of genes mainly through the activation of hypoxia-inducible factor (HIF) transcriptional complex. Endothelial cells play a central role in new vessels formation and express placental growth factor (PlGF), a member of vascular endothelial growth factor (VEGF) family, mainly involved in pathological angiogenesis. Despite several observations suggest a hypoxia-mediated positive modulation of PlGF, the molecular mechanism governing this regulation has not been fully elucidated. We decided to investigate if epigenetic modifications are involved in hypoxia-induced PlGF expression. We report that PlGF expression was induced in cultured human and mouse endothelial cells exposed to hypoxia (1% O 2), although DNA methylation at the Plgf CpG-island remains unchanged. Remarkably, robust hyperacetylation of histones H3 and H4 was observed in the second intron of Plgf, where hypoxia responsive elements (HREs), never described before, are located. HIF-1α, but not HIF-2α, binds to identified HREs. Noteworthy, only HIF-1α silencing fully inhibited PlGF upregulation. These results formally demonstrate a direct involvement of HIF-1α in the upregulation of PlGF expression in hypoxia through chromatin remodeling of HREs sites. Therefore, PlGF may be considered one of the putative targets of anti-HIF therapeutic applications.

The hypobaric hypoxia affects the oxidant balance in skeletal muscle regeneration of women

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Rosa Mancinelli

2016-07-01

Full Text Available Aim: The aim of this study was to determine whether a 14-day trekking expeditions, in high altitude hypoxic environment, triggers redox disturbance at the level of satellite cells (adult stem cells in young women.Methods: We collected muscle biopsies from Vastus Lateralis muscle for both single fiber analysis and satellite cells isolation. The samples collected before (PRE-Hypoxia and after (POST-Hypoxia the trekking in the Himalayas were compared. Satellite cells were investigated for oxidative stress (oxidant production, antioxidant enzyme activity and lipid damage, mitochondrial potential variation, gene profile of HIF and myogenic transcription factors (Pax7, MyoD, myogenin and miRNA expression (miR-1, miR-133, miR-206.Results: The nuclear domain analysis showed a significant fusion and consequent reduction of the Pax7+ satellite cells in the single mature fibers. The POST-Hypoxia myoblasts obtained by two out of six volunteers showed high superoxide anion production and lipid peroxidation along with impaired dismutase and catalase and mitochondrial potential. The transcription profile and miRNA expression were different for oxidized and non oxidized cells.Conclusions: The present study supports the phenomenon of hypobaric-hypoxia-induced oxidative stress and its role in the impairment of the regenerative capacity of satellite cells derived from the Vastus Lateralis muscle of young adult female subjects.

TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

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Hyou-Ju Jin

Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

The radiosensitivity of glioblastoma cell lines after hypoxia-induced Bax expression

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Chen, J.K.; Hu, L.J.; Kong, E.L.; Lamborn, K.R.; Deen, D.F.

2003-01-01

Full text: Radiation therapy is the most effective treatment after surgery for patients with malignant gliomas. However, the hypoxic cells exclusive to tumor tissue have proven resistant to both radiotherapy and many forms of chemotherapy. In order to specifically target these hypoxic cells, U-251 MG and U-87 MG human glioblastoma cells were stably transfected with constructs containing the suicide gene Bax under the regulation of nine copies of hypoxia-responsive elements (HREs). During hypoxia, the transcriptional complex hypoxia-inducible-factor 1 (HIF-1) binds to HRE and facilitates the transcription of downstream genes. Previously, hypoxia-induced Bax expression in transfected U-251 and U-87 clone cells has been shown to increase cell killing. The benefits of the gene therapy could be further expanded if Bax also acted to increase the sensitivity of these clone cells to radiation. To determine whether this was the case, parent and clone cells were irradiated with graded doses of X-rays under hypoxic conditions. These cells were then left hypoxic for varying durations of time, after which they were incubated for two weeks under aerated conditions to assay for clonogenic cell survival. After less than an hour under hypoxia, both U-251 and U-87 clone cells appeared significantly more sensitive to radiation than their respective parent cells. However, after longer amounts of time under anoxia, higher surviving fractions were found in each clone that were consistent with those of their respective parent cell line, showing that potentially lethal damage repair (PLDR) had occurred in the clone cells. Parent cells did not exhibit PLDR. Results are inconclusive at this point in time. Western blot analyses detailing the amount of Bax expression at each time point as well as further research exploring different durations of hypoxia will be necessary to reveal the nature of the correlation between Bax expression and radiosensitivity. Supported by NS-42927 and CA-85356

Group B streptococcal beta-hemolysin/cytolysin directly impairs cardiomyocyte viability and function.

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Mary E Hensler

Full Text Available BACKGROUND: Group B Streptococcus (GBS is a leading cause of neonatal sepsis where myocardial dysfunction is an important contributor to poor outcome. Here we study the effects of the GBS pore-forming beta-hemolysin/cytolysin (Bh/c exotoxin on cardiomyocyte viability, contractility, and calcium transients. METHODOLOGY/PRINCIPAL FINDINGS: HL-1 cardiomyocytes exposed to intact wild-type (WT or isogenic Deltabeta h/c mutant GBS, or to cell-free extracts from either strain, were assessed for viability by trypan blue exclusion and for apoptosis by TUNEL staining. Functionality of exposed cardiomyocytes was analyzed by visual quantitation of the rate and extent of contractility. Mitochondrial membrane polarization was measured in TMRE-loaded cells exposed to GBS beta h/c. Effects of GBS beta h/c on calcium transients were studied in fura-2AM-loaded primary rat ventricular cardiomyocytes. Exposure of HL-1 cardiomyocytes to either WT GBS or beta h/c extracts significantly reduced both rate and extent of contractility and later induced necrotic and apoptotic cell death. No effects on cardiomyocyte viability or function were observed after treatment with Deltabeta h/c mutant bacteria or extracts. The beta h/c toxin was associated with complete and rapid loss of detectable calcium transients in primary neonatal rat ventricular cardiomyocytes and induced a loss of mitochondrial membrane polarization. These effects on viability and function were abrogated by the beta h/c inhibitor, dipalmitoyl phosphatidylcholine (DPPC. CONCLUSIONS/SIGNIFICANCE: Our data show a rapid loss of cardiomyocyte viability and function induced by GBS beta h/c, and these deleterious effects are inhibited by DPPC, a normal constituent of human pulmonary surfactant.. These findings have clinical implications for the cardiac dysfunction observed in neonatal GBS infections.

Whole brain radiation-induced impairments in learning and memory are time-sensitive and reversible by systemic hypoxia.

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Junie P Warrington

Full Text Available Whole brain radiation therapy (WBRT is commonly used for treatment of primary and metastatic brain tumors; however, cognitive impairment occurs in 40-50% of brain tumor survivors. The etiology of the cognitive impairment following WBRT remains elusive. We recently reported that radiation-induced cerebrovascular rarefaction within hippocampal subregions could be completely reversed by systemic hypoxia. However, the effects of this intervention on learning and memory have not been reported. In this study, we assessed the time-course for WBRT-induced impairments in contextual and spatial learning and the capacity of systemic hypoxia to reverse WBRT-induced deficits in spatial memory. A clinical fractionated series of 4.5Gy WBRT was administered to mice twice weekly for 4 weeks, and after various periods of recovery, behavioral analyses were performed. To study the effects of systemic hypoxia, mice were subjected to 11% (hypoxia or 21% oxygen (normoxia for 28 days, initiated 1 month after the completion of WBRT. Our results indicate that WBRT induces a transient deficit in contextual learning, disruption of working memory, and progressive impairment of spatial learning. Additionally, systemic hypoxia completely reversed WBRT-induced impairments in learning and these behavioral effects as well as increased vessel density persisted for at least 2 months following hypoxia treatment. Our results provide critical support for the hypothesis that cerebrovascular rarefaction is a key component of cognitive impairment post-WBRT and indicate that processes of learning and memory, once thought to be permanently impaired after WBRT, can be restored.

A novel adjustable automated system for inducing chronic intermittent hypoxia in mice.

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Dora Polšek

Full Text Available Sleep apnea is a chronic, widely underdiagnosed condition characterized by disruption of sleep architecture and intermittent hypoxia due to short cessations of breathing. It is a major independent risk factor for myocardial infarction, congestive heart failure and stroke as well as one of the rare modifiable risk factors for Alzheimer's Dementia. Reliable animal disease models are needed to understand the link between sleep apnea and the various clinically linked disorders.An automated system for inducing hypoxia was developed, in which the major improvement was the possibility to efficiently adjust the length and intensity of hypoxia in two different periods. The chamber used a small volume of gas allowing for fast exchanges of different oxygen levels. The mice were kept in their cages adapted with the system on the cage lid. As a proof of principle, they were exposed to a three week period of intermittent hypoxia for 8 hours a day, with 90 s intervals of 5, 7% and 21% oxygen to validate the model. Treated (n = 8 and control mice (no hypoxia, n = 7 were handled in the same manner and their hippocampal brain regions compared by histology.The chamber provided a fast, reliable and precise intermittent hypoxia, without inducing noticeable side effects to the animals. The validation experiment showed that apoptotic neurons in the hippocampus were more numerous in the mice exposed to intermittent hypoxia than in the control group, in all tested hippocampal regions (cornu ammonis 1 (CA1 P <0.001; cornu ammonis 3 (CA3 P <0.001; and dentate gyrus (DG P = 0.023. In both, control and hypoxic conditions, there was a significantly higher number of apoptotic neurons in the DG compared to the CA1 and CA3 subfields (P <0.001.The new design of a hypoxic chamber provides a fast, adjustable and reliable model of obstructive sleep apnea, which was validated by apoptosis of hippocampal neurons.

Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

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Inadomi, Chiaki; Murata, Hiroaki; Ihara, Yoshito; Goto, Shinji; Urata, Yoshishige; Yodoi, Junji; Kondo, Takahito; Sumikawa, Koji

2012-01-01

Highlights: ► GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. ► NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. ► Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: comparing reoxygenation in cultured cardiomyocytes.

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Meynier, Alexandra; Razik, Hafida; Cordelet, Catherine; Grégoire, Stéphane; Demaison, Luc

2003-01-01

during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. 14C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.

Ageing and cardiorespiratory response to hypoxia.

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Lhuissier, François J; Canouï-Poitrine, Florence; Richalet, Jean-Paul

2012-11-01

The risk of severe altitude-induced diseases is related to ventilatory and cardiac responses to hypoxia and is dependent on sex, age and exercise training status. However, it remains unclear how ageing modifies these physiological adaptations to hypoxia. We assessed the physiological responses to hypoxia with ageing through a cross-sectional 20 year study including 4675 subjects (2789 men, 1886 women; 14-85 years old) and a longitudinal study including 30 subjects explored at a mean 10.4 year interval. The influence of sex, training status and menopause was evaluated. The hypoxia-induced desaturation and the ventilatory and cardiac responses to hypoxia at rest and exercise were measured. In men, ventilatory response to hypoxia increased (P ageing. Cardiac response to hypoxia was blunted with ageing in both sexes (P ageing. These adaptive responses were less pronounced or absent in post-menopausal women (P ageing in men while cardiac response is blunted with ageing in both sexes. Training aggravates desaturation at exercise in hypoxia, improves the ventilatory response and limits the ageing-induced blunting of cardiac response to hypoxia. Training limits the negative effects of menopause in cardiorespiratory adaptations to hypoxia.

Compound C prevents Hypoxia-Inducible Factor-1α protein stabilization by regulating the cellular oxygen availability via interaction with Mitochondrial Complex I

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Hagen Thilo

2011-04-01

Full Text Available Abstract The transcription factor Hypoxia-Inducible Factor-1α is a master regulator of the cellular response to low oxygen concentration. Compound C, an inhibitor of AMP-activated kinase, has been reported to inhibit hypoxia dependent Hypoxia-Inducible Factor-1α activation via a mechanism that is independent of AMP-activated kinase but dependent on its interaction with the mitochondrial electron transport chain. The objective of this study is to characterize the interaction of Compound C with the mitochondrial electron transport chain and to determine the mechanism through which the drug influences the stability of the Hypoxia-Inducible Factor-1α protein. We found that Compound C functions as an inhibitor of complex I of the mitochondrial electron transport chain as demonstrated by its effect on mitochondrial respiration. It also prevents hypoxia-induced Hypoxia-Inducible Factor-1α stabilization in a dose dependent manner. In addition, Compound C does not have significant effects on reactive oxygen species production from complex I via both forward and reverse electron flux. This study provides evidence that similar to other mitochondrial electron transport chain inhibitors, Compound C regulates Hypoxia-Inducible Factor-1α stability by controlling the cellular oxygen concentration.

Response of skeletal muscle mitochondria to hypoxia.

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Hoppeler, Hans; Vogt, Michael; Weibel, Ewald R; Flück, Martin

2003-01-01

This review explores the current concepts relating the structural and functional modifications of skeletal muscle mitochondria to the molecular mechanisms activated when organisms are exposed to a hypoxic environment. In contrast to earlier assumptions it is now established that permanent or long-term exposure to severe environmental hypoxia decreases the mitochondrial content of muscle fibres. Oxidative muscle metabolism is shifted towards a higher reliance on carbohydrates as a fuel, and intramyocellular lipid substrate stores are reduced. Moreover, in muscle cells of mountaineers returning from the Himalayas, we find accumulations of lipofuscin, believed to be a mitochondrial degradation product. Low mitochondrial contents are also observed in high-altitude natives such as Sherpas. In these subjects high-altitude performance seems to be improved by better coupling between ATP demand and supply pathways as well as better metabolite homeostasis. The hypoxia-inducible factor 1 (HIF-1) has been identified as a master regulator for the expression of genes involved in the hypoxia response, such as genes coding for glucose transporters, glycolytic enzymes and vascular endothelial growth factor (VEGF). HIF-1 achieves this by binding to hypoxia response elements in the promoter regions of these genes, whereby the increase of HIF-1 in hypoxia is the consequence of a reduced degradation of its dominant subunit HIF-1a. A further mechanism that seems implicated in the hypoxia response of muscle mitochondria is related to the formation of reactive oxygen species (ROS) in mitochondria during oxidative phosphorylation. How exactly ROS interfere with HIF-1a as well as MAP kinase and other signalling pathways is debated. The current evidence suggests that mitochondria themselves could be important players in oxygen sensing.

Chronic intermittent hypoxia induces cardiac inflammation and dysfunction in a rat obstructive sleep apnea model.

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Wei, Qin; Bian, Yeping; Yu, Fuchao; Zhang, Qiang; Zhang, Guanghao; Li, Yang; Song, Songsong; Ren, Xiaomei; Tong, Jiayi

2016-11-01

Chronic intermittent hypoxia is considered to play an important role in cardiovascular pathogenesis during the development of obstructive sleep apnea (OSA). We used a well-described OSA rat model induced with simultaneous intermittent hypoxia. Male Sprague Dawley rats were individually placed into plexiglass chambers with air pressure and components were electronically controlled. The rats were exposed to intermittent hypoxia 8 hours daily for 5 weeks. The changes of cardiac structure and function were examined by ultrasound. The cardiac pathology, apoptosis, and fibrosis were analyzed by H&E staining, TUNNEL assay, and picosirius staining, respectively. The expression of inflammation and fibrosis marker genes was analyzed by quantitative real-time PCR and Western blot. Chronic intermittent hypoxia/low pressure resulted in significant increase of left ventricular internal diameters (LVIDs), end-systolic volume (ESV), end-diastolic volume (EDV), and blood lactate level and marked reduction in ejection fraction and fractional shortening. Chronic intermittent hypoxia increased TUNNEL-positive myocytes, disrupted normal arrangement of cardiac fibers, and increased Sirius stained collagen fibers. The expression levels of hypoxia induced factor (HIF)-1α, NF-kB, IL-6, and matrix metallopeptidase 2 (MMP-2) were significantly increased in the heart of rats exposed to chronic intermittent hypoxia. In conclusion, the left ventricular function was adversely affected by chronic intermittent hypoxia, which is associated with increased expression of HIF-1α and NF-kB signaling molecules and development of cardiac inflammation, apoptosis and fibrosis. © 2016 by the Journal of Biomedical Research. All rights reserved.

Effects of gamma-ray radiation on activity and apoptosis of rat cardiomyocytes in vitro

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Hu Shunying; Jiang Changsheng; Chen Guowei; Duan Haifeng; Wang Rongliang; Wu Bin; Guo Zikuan; Wang Lisheng

2007-01-01

Objective: It is reported that radiation-induced myocardial degeneration in the rat is preceded by changes in capillary structure and function. The aim of the present study is to investigate direct effect of gamma ray radiation on activity and apoptosis of cultured rat cardiomyocytes in vitro. Methods: The study was performed using primary cell cultures of cardiomyocytes isolated from hearts of now-born rats. After being cultured for 72h in vitro, cardiomyocytes were irradiated with single dose of 5 Gy, 10 Gy, 20 Gy of gamma ray respectively. At 48h post-irradiation, the concentration of LDH in the supernatant of cell culture was tested using methods introduced by International Federation of clinical chemistry (IFCC), and apoptosis was determined with flow cytometry. The viability of myocytes was determined with crystal violet test and MTT test at 48h and 120h post-irradiation respectively. Results: LDH concentration in the supernatant of cell culture of cardiomyocytes were increased significantly with the irradiation dose augment. Flow cytometry confirmed the induction of apoptosis in response to different gamma ray doses irradiation at 48h after irradiation. The viable cardiomyocytes irradiated by gamma ray were significantly declined at 120h after irradiation compared to un-irradiated cells, however there were no significant difference between two groups at 48h post-irradiation. Dose-effect relationship was demonstrated between cardiomyocyte apoptosis, viability and irradiation dose in the study. Conclusion: The study demonstrates gamma ray radiation can cause direct damage to cultured cardiomyocytes, including inhibiting activity and inducing apoptosis of cardiomyocytes in vitro, which shows dose effect relationship. The mechanism of gamma ray irradiation induced injury to cardiomyocytes should be investigated further. (authors)

Hypoxia signaling pathways: modulators of oxygen-related organelles

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Schönenberger, Miriam J.; Kovacs, Werner J.

2015-01-01

Oxygen (O2) is an essential substrate in cellular metabolism, bioenergetics, and signaling and as such linked to the survival and normal function of all metazoans. Low O2 tension (hypoxia) is a fundamental feature of physiological processes as well as pathophysiological conditions such as cancer and ischemic diseases. Central to the molecular mechanisms underlying O2 homeostasis are the hypoxia-inducible factors-1 and -2 alpha (HIF-1α and EPAS1/HIF-2α) that function as master regulators of the adaptive response to hypoxia. HIF-induced genes promote characteristic tumor behaviors, including angiogenesis and metabolic reprogramming. The aim of this review is to critically explore current knowledge of how HIF-α signaling regulates the abundance and function of major O2-consuming organelles. Abundant evidence suggests key roles for HIF-1α in the regulation of mitochondrial homeostasis. An essential adaptation to sustained hypoxia is repression of mitochondrial respiration and induction of glycolysis. HIF-1α activates several genes that trigger mitophagy and represses regulators of mitochondrial biogenesis. Several lines of evidence point to a strong relationship between hypoxia, the accumulation of misfolded proteins in the endoplasmic reticulum, and activation of the unfolded protein response. Surprisingly, although peroxisomes depend highly on molecular O2 for their function, there has been no evidence linking HIF signaling to peroxisomes. We discuss our recent findings that establish HIF-2α as a negative regulator of peroxisome abundance and suggest a mechanism by which cells attune peroxisomal function with O2 availability. HIF-2α activation augments peroxisome turnover by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We discuss potential mechanisms by which HIF-2α might trigger pexophagy and place special emphasis on the potential pathological implications of HIF-2α-mediated pexophagy for human health. PMID:26258123

Cytoprotective effects of atmospheric-pressure plasmas against hypoxia-induced neuronal injuries

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Yan, Xu; Meng, Zhaozhong; Ouyang, Jiting; Qiao, Yajun; Li, Jiaxin; Jia, Mei; Yuan, Fang; (Ken Ostrikov, Kostya

2018-02-01

Atmospheric pressure plasma jet (APPJ) has recently been the focus of cytoprotective research due to the physiological roles of ROS and RNS. In the current study, we investigated the effect of APPJ treatment on the hypoxia (1% oxygen) induced cell injuries. SH-SY5Y cells were treated by APPJ for different duration and incubated in normoxic condition (20% oxygen) for 5 h followed by 24 h hypoxia treatment. Cell viability was evaluated by lactate dehydrogenase (LDH) release and further monitored using the electric cell-substrate impedance sensing (ECIS) system after APPJ treatment. Results showed that APPJ could reduce cell injuries after 24 h hypoxia, which was consistent with the ECIS results. Furthermore, extracellular NO and H2O2 production was significantly increased with the APPJ treatment. It was also interesting to find that APPJ treatment reduced SH-SY5Y cells proliferation in the hypoxic microenvironment during the first 20 h of hypoxia. Although more work was still need to clarify whether the cell viability maintenance was related to the cell proliferation during hypoxia, our results provide the first evidence of real-time cell viability changes after APPJ treatment under both normoxic and hypoxic conditions, which could provide evidence for the neuroprotective applications of APPJ.

An Automated Platform for Assessment of Congenital and Drug-Induced Arrhythmia with hiPSC-Derived Cardiomyocytes

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Wesley L. McKeithan

2017-10-01

Full Text Available The ability to produce unlimited numbers of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs harboring disease and patient-specific gene variants creates a new paradigm for modeling congenital heart diseases (CHDs and predicting proarrhythmic liabilities of drug candidates. However, a major roadblock to implementing hiPSC-CM technology in drug discovery is that conventional methods for monitoring action potential (AP kinetics and arrhythmia phenotypes in vitro have been too costly or technically challenging to execute in high throughput. Herein, we describe the first large-scale, fully automated and statistically robust analysis of AP kinetics and drug-induced proarrhythmia in hiPSC-CMs. The platform combines the optical recording of a small molecule fluorescent voltage sensing probe (VoltageFluor2.1.Cl, an automated high throughput microscope and automated image analysis to rapidly generate physiological measurements of cardiomyocytes (CMs. The technique can be readily adapted on any high content imager to study hiPSC-CM physiology and predict the proarrhythmic effects of drug candidates.

Pathophysiological response to hypoxia - from the molecular mechanisms of malady to drug discovery: epigenetic regulation of the hypoxic response via hypoxia-inducible factor and histone modifying enzymes.

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Mimura, Imari; Tanaka, Tetsuhiro; Wada, Youichiro; Kodama, Tatsuhiko; Nangaku, Masaomi

2011-01-01

The hypoxia response regulated primarily by hypoxia-inducible factor (HIF) influences metabolism, cell survival, and angiogenesis to maintain biological homeostasis. In addition to the traditional transcriptional regulation by HIF, recent studies have shown that epigenetic modulation such as histone methylation, acetylation, and DNA methylation could change the regulation of the response to hypoxia. Eukaryotic chromatin is known to be modified by multiple post-translational histone methylation and demethylation, which result in the chromatin conformation change to adapt to hypoxic stimuli. Interestingly, some of the histone demethylase enzymes, which have the Jumonji domain-containing family, require oxygen to function and are induced by hypoxia in an HIF-1-dependent manner. Recent studies have demonstrated that histone modifiers play important roles in the hypoxic environment such as that in cancer cells and that they may become new therapeutic targets for cancer patients. It may lead to finding a new therapy for cancer to clarify a new epigenetic mechanism by HIF and histone demethylase such as JMJD1A (KDM3A) under hypoxia.

IBMX protects human proximal tubular epithelial cells from hypoxic stress through suppressing hypoxia-inducible factor-1α expression.

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Hasan, Arif Ul; Kittikulsuth, Wararat; Yamaguchi, Fuminori; Musarrat Ansary, Tuba; Rahman, Asadur; Shibayama, Yuki; Nakano, Daisuke; Hitomi, Hirofumi; Tokuda, Masaaki; Nishiyama, Akira

2017-09-15

Hypoxia predisposes renal fibrosis. This study was conducted to identify novel approaches to ameliorate the pathogenic effect of hypoxia. Using human proximal tubular epithelial cells we showed that a pan-phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX) dose and time dependently downregulated hypoxia-inducible factor 1α (HIF-1α) mRNA expression, which was further augmented by addition of a transcriptional inhibitor, actinomycin D. IBMX also increased the cellular cyclic adenosine monophosphate (cAMP) level. Luciferase assay showed that blocking of protein kinase A (PKA) using H89 reduced, while 8-Br-cAMP agonized the repression of HIF-1α promoter activity in hypoxic condition. Deletion of cAMP response element binding sites from the HIF-1α promoter abrogated the effect of IBMX. Western blot and immunofluorescent study confirmed that the CoCl 2 induced increased HIF-1α protein in whole cell lysate and in nucleus was reduced by the IBMX. Through this process, IBMX attenuated both CoCl 2 and hypoxia induced mRNA expressions of two pro-fibrogenic factors, platelet-derived growth factor B and lysyl oxidase. Moreover, IBMX reduced production of a mesenchymal transformation factor, β-catenin; as well as protected against hypoxia induced cell-death. Taken together, our study showed novel evidence that the PDE inhibitor IBMX can downregulate the transcription of HIF-1α, and thus may attenuate hypoxia induced renal fibrosis. Copyright © 2017 Elsevier Inc. All rights reserved.

Hypoxia-induced resistance to doxorubicin and methotrexate in human melanoma cell lines in vitro.

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Sanna, K; Rofstad, E K

1994-07-15

Rodent cell lines can develop resistance to doxorubicin and methotrexate during hypoxic stress. This has so far not been observed in human tumor cell lines. The purpose of our communication is to show that doxorubicin and methotrexate resistance can also develop in human melanoma cells during exposure to hypoxia. Four cell lines (BEX-c, COX-c, SAX-c, WIX-c) have been studied. Cells were exposed to hypoxia (O2 concentration WIX-c. BEX-c and SAX-c were sensitive to methotrexate without hypoxia pre-treatment, whereas COX-c and WIX-c were resistant initially. Hypoxia-induced drug resistance was present immediately after reoxygenation and tended to decrease with time but remained statistically significant even 42 hr after reoxygenation.

Effects of cadmium on hypoxia-induced expression of hemoglobin and erythropoietin in larval sheepshead minnow, Cyprinodon variegatus

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Dangre, A.J.; Manning, S. [Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564 (United States); Brouwer, M., E-mail: marius.brouwer@usm.edu [Department of Coastal Sciences, University of Southern Mississippi, 703 East Beach Drive, Ocean Springs, MS 39564 (United States)

2010-08-15

Hypoxia and toxic metals are two common stressors found in the estuarine environment. To date little information is available on the combined effects of these stressors on early larval development in fish. We investigated the effect of cadmium and hypoxia exposure alone as well in combination on larval Cyprinodon variegatus. The LC{sub 10} for cadmium was determined to be 0.3 ppm in a 96 h acute exposure. This concentration was used in all studies. Cadmium in larvae increased significantly with exposure time (1, 3, 5 and 7 days post-hatch). The increase was proportional to body weight and not affected by hypoxia. Cadmium responsive genes were identified by suppression subtractive hybridization (SSH) in Cyprinodonvariegatus larvae after exposure to cadmium for 1, 3, 5 and 7 days. We obtained over 700 sequences from the cadmium cDNA library. Blast search of ESTs suggested that cadmium modulates multiple physiological processes. Pertinent to this study, cadmium was found to down-regulate both embryonic {alpha} and {beta} globin, which are expressed in erythrocytes generated during the first, or primitive, wave of erythropoiesis in teleosts. Hemoglobin (Hb) and erythropoietin (Epo) (the hormone that promotes red blood cell production) are known hypoxia-inducible genes. To explore the possibility that cadmium might offset the hypoxia-induced expression of Hb and Epo, we investigated the expression of both genes following hypoxia, cadmium and combined exposures for 1, 3, 5 and 7 days post-hatch. Since Epo had not yet been identified in C. variegatus we first successfully cloned a partial coding sequence of the C. variegatus hormone. Subsequent studies revealed that expression levels of Hb and Epo remained unchanged in the normoxic controls during the time course of the study. Hypoxia increased Epo expression relative to normoxic controls, on days 3, 5 and 7, while cadmium in hypoxia inhibited the increase. Only the changes on days 5 and 7 were statistically significant

Autoantibodies in dilated cardiomyopathy induce vascular endothelial growth factor expression in cardiomyocytes

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Saygili, Erol, E-mail: erol.saygili@med.uni-duesseldorf.de [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Noor-Ebad, Fawad; Schröder, Jörg W.; Mischke, Karl [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Saygili, Esra [Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, D-40225 Düsseldorf (Germany); Rackauskas, Gediminas [Department of Cardiovascular Medicine, Vilnius University Hospital Santariskiu Klinikos, Vilnius University (Lithuania); Marx, Nikolaus [Department of Cardiology, University RWTH Aachen, Pauwelsstr. 30, D-52074 Aachen (Germany); Kelm, Malte; Rana, Obaida R. [Division of Cardiology, Pulmonology, and Vascular Medicine, University Hospital Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf (Germany)

2015-09-11

Background: Autoantibodies have been identified as major predisposing factors for dilated cardiomyopathy (DCM). Patients with DCM show elevated serum levels of vascular endothelial growth factor (VEGF) whose source is unknown. Besides its well-investigated effects on angiogenesis, evidence is present that VEGF signaling is additionally involved in fibroblast proliferation and cardiomyocyte hypertrophy, hence in cardiac remodeling. Whether autoimmune effects in DCM impact cardiac VEGF signaling needs to be elucidated. Methods: Five DCM patients were treated by the immunoadsorption (IA) therapy on five consecutive days. The eluents from the IA columns were collected and prepared for cell culture. Cardiomyocytes from neonatal rats (NRCM) were incubated with increasing DCM-immunoglobulin-G (IgG) concentrations for 48 h. Polyclonal IgG (Venimmun N), which was used to restore IgG plasma levels in DCM patients after the IA therapy was additionally used for control cell culture purposes. Results: Elevated serum levels of VEGF decreased significantly after IA (Serum VEGF (ng/ml); DCM pre-IA: 45 ± 9.1 vs. DCM post–IA: 29 ± 6.7; P < 0.05). In cell culture, pretreatment of NRCM by DCM-IgG induced VEGF expression in a time and dose dependent manner. Biologically active VEGF that was secreted by NRCM significantly increased BNP mRNA levels in control cardiomyocytes and induced cell-proliferation of cultured cardiac fibroblast (Fibroblast proliferation; NRCM medium/HC-IgG: 1 ± 0.0 vs. NRCM medium/DCM-IgG 100 ng/ml: 5.6 ± 0.9; P < 0.05). Conclusion: The present study extends the knowledge about the possible link between autoimmune signaling in DCM and VEGF induction. Whether this observation plays a considerable role in cardiac remodeling during DCM development needs to be further elucidated. - Highlights: • Mechanisms of remodeling in dilated cardiomyopathy (DCM) are not fully understood. • Autoantibodies have been identified as major predisposing factors

Hypoxia-Inducible Factor-1α in carcinogenesis and progression of breast cancer

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Bos, R.

2004-01-01

This thesis is primarily focused on the previously hardly explored role of HIF-1 in breast cancer. HIF-1 is a transcription factor induced by hypoxia, but also by some oncogenes, tumor suppressor genes and growth factors. Activated HIF-1 can induce angiogenesis, glycolysis, erythropoiesis, and other

[Effects of interleukin-18 and hypoxia-inducible factor-1α in serum and gingival tissues of rat model with periodontitis exposed to chronic intermittent hypoxia].

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Wang, Bin; Wang, Xiaoqin

2015-08-01

This study evaluates the expression of interleukin-18 (IL-18) and hypoxia-inducible factor (HIF)-lα in rat periodontitis model exposed to normoxia and chronic intermittent hypoxia (CIH) environments. The possible correlation between periodontitis and obstructive sleep apnea-hypopnea syndrome (OSAHS) was also investigated. Methods: Thirty-two Sprague-Dawley (SD) rats were randomly assigned into four groups: normoxia control, normoxia periodontitis, hypoxia control, and hypoxia periodontitis groups. The periodontitis models were established by ligating the bilateral maxillary second molars and employing high-carbohydrate diets. Rats in hypoxia control and hypoxia periodontitis groups were exposed to CIH treatment mimicking a moderately severe OSAHS condition. All animals were sacrificed after eight weeks, and the clinical periodontal indexes were detected. The levels of IL-18 and HIF-1α in serum and gingival tissues were determined using enzyme-linked immunosorbent assay (ELISA). The correlation between attachment loss (AL) and the levels of IL-18 and HIF-lα in hypoxia periodontitis group was evaluated. The levels of IL-18 and HIF-lα in hypoxia periodontitis group were significantly higher than that in normoxia periodontitis and hypoxia control groups (Pperiodontal tissues, which is correlated with IL-18 and HIF-lα levels.

Efficient generation of transgene- and feeder-free induced pluripotent stem cells from human dental mesenchymal stem cells and their chemically defined differentiation into cardiomyocytes.

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Tan, Xiaobing; Dai, Qingli; Guo, Tao; Xu, Jingshu; Dai, Qingyuan

2018-01-22

Advance in stem cell research resulted in several processes to generate induced pluripotent stem cells (iPSCs) from adult somatic cells. In our previous study, the reprogramming of iPSCs from human dental mesenchymal stem cells (MSCs) including SCAP and DPSCs, has been reported. Herein, safe iPSCs were reprogrammed from SCAP and DPSCs using non-integrating RNA virus vector, which is an RNA virus carrying no risk of altering host genome. DPSCs- and SCAP-derived iPSCs exhibited the characteristics of the classical morphology with human embryonic stem cells (hESCs) without integration of foreign genes, indicating the potential of their clinical application. Moreover, induced PSCs showed the capacity of self-renewal and differentiation into cardiac myocytes. We have achieved the differentiation of hiPSCs to cardiomyocytes lineage under serum and feeder-free conditions, using a chemically defined medium CDM3. In CDM3, hiPSCs differentiation is highly generating cardiomyocytes. The results showed this protocol produced contractile sheets of up to 97.2% TNNT2 cardiomyocytes after purification. Furthermore, derived hiPSCs differentiated to mature cells of the three embryonic germ layers in vivo and in vitro of beating cardiomyocytes. The above whole protocol enables the generation of large scale of highly pure cardiomyocytes as needed for cellular therapy. Copyright © 2017. Published by Elsevier Inc.

A RNA antagonist of hypoxia-inducible factor-1alpha, EZN-2968, inhibits tumor cell growth

DEFF Research Database (Denmark)

Greenberger, Lee M; Horak, Ivan D; Filpula, David

2008-01-01

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism. Elevated expression of the alpha-subunit of HIF-1 (HIF-1alpha), which occurs in response to hypoxia or activation of growth facto...

Mitochondrial p38β and manganese superoxide dismutase interaction mediated by estrogen in cardiomyocytes.

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Han Liu

Full Text Available While etiology behind the observed acceleration of ischemic heart disease in postmenopausal women is poorly understood, collective scientific data suggest cardioprotective effects of the endogenous female sex hormone, estrogen. We have previously shown that 17β-estradiol (E2 protects cardiomyocytes exposed to hypoxia-reoxygenation (H/R by inhibiting p38α - p53 signaling in apoptosis and activating pro-survival p38β mitogen activated protein kinase (p38β MAPK, leading to suppression of reactive oxygen species (ROS post H/R. However, little is known about the mechanism behind the antioxidant actions of E2-dependent p38β. The aim of this study is to determine whether the cytoprotection by estrogen involves regulation of manganese superoxide dismutase (MnSOD, a major mitochondrial ROS scavenging enzyme, via cardiac p38β.We identified mitochondrial p38β by immunocytochemistry and by immunoblotting in mitochondria isolated from neonatal cardiomyocytes of Sprague-Dawley rats. E2 facilitated the mitochondrial localization of the active form of the kinase, phosphorylated p38β (p-p38β. E2 also reduced the H/R-induced mitochondrial membrane potential decline, augmented the MnSOD activity and suppressed anion superoxide generation, while the dismutase protein expression remained unaltered. Co-immunoprecipitation studies showed physical association between MnSOD and p38β. p38β phosphorylated MnSOD in an E2-dependent manner in in-vitro kinase assays.This work demonstrates for the first time a mitochondrial pool of active p38β and E2-mediated phosphorylation of MnSOD by the kinase. The results shed light on the mechanism behind the cytoprotective actions of E2 in cardiomyocytes under oxidative stress.

Absence of the inflammasome adaptor ASC reduces hypoxia-induced pulmonary hypertension in mice.

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Cero, Fadila Telarevic; Hillestad, Vigdis; Sjaastad, Ivar; Yndestad, Arne; Aukrust, Pål; Ranheim, Trine; Lunde, Ida Gjervold; Olsen, Maria Belland; Lien, Egil; Zhang, Lili; Haugstad, Solveig Bjærum; Løberg, Else Marit; Christensen, Geir; Larsen, Karl-Otto; Skjønsberg, Ole Henning

2015-08-15

Pulmonary hypertension is a serious condition that can lead to premature death. The mechanisms involved are incompletely understood although a role for the immune system has been suggested. Inflammasomes are part of the innate immune system and consist of the effector caspase-1 and a receptor, where nucleotide-binding oligomerization domain-like receptor pyrin domain-containing 3 (NLRP3) is the best characterized and interacts with the adaptor protein apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). To investigate whether ASC and NLRP3 inflammasome components are involved in hypoxia-induced pulmonary hypertension, we utilized mice deficient in ASC and NLRP3. Active caspase-1, IL-18, and IL-1β, which are regulated by inflammasomes, were measured in lung homogenates in wild-type (WT), ASC(-/-), and NLRP3(-/-) mice, and phenotypical changes related to pulmonary hypertension and right ventricular remodeling were characterized after hypoxic exposure. Right ventricular systolic pressure (RVSP) of ASC(-/-) mice was significantly lower than in WT exposed to hypoxia (40.8 ± 1.5 mmHg vs. 55.8 ± 2.4 mmHg, P right ventricular remodeling. RVSP of NLRP3(-/-) mice exposed to hypoxia was not significantly altered compared with WT hypoxia. Whereas hypoxia increased protein levels of caspase-1, IL-18, and IL-1β in WT and NLRP3(-/-) mice, this response was absent in ASC(-/-) mice. Moreover, ASC(-/-) mice displayed reduced muscularization and collagen deposition around arteries. In conclusion, hypoxia-induced elevated right ventricular pressure and remodeling were attenuated in mice lacking the inflammasome adaptor protein ASC, suggesting that inflammasomes play an important role in the pathogenesis of pulmonary hypertension. Copyright © 2015 the American Physiological Society.

Zinc-induced cardiomyocyte relaxation in a rat model of hyperglycemia is independent of myosin isoform

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Yi Ting

2012-11-01

Full Text Available Abstract It has been reported previously that diabetic cardiomyopathy can be inhibited or reverted with chronic zinc supplementation. In the current study, we hypothesized that total cardiac calcium and zinc content is altered in early onset diabetes mellitus characterized in part as hyperglycemia (HG and that exposure of zinc ion (Zn2+ to isolated cardiomyocytes would enhance contraction-relaxation function in HG more so than in nonHG controls. To better control for differential cardiac myosin isoform expression as occurs in rodents after β-islet cell necrosis, hypothyroidism was induced in 16 rats resulting in 100% β-myosin heavy chain expression in the heart. β-Islet cell necrosis was induced in half of the rats by streptozocin administration. After 6 wks of HG, both HG and nonHG controls rats demonstrated similar myofilament performance measured as thin filament calcium sensitivity, native thin filament velocity in the myosin motility assay and contractile velocity and power. Extracellular Zn2+ reduced cardiomyocyte contractile function in both groups, but enhanced relaxation function significantly in the HG group compared to controls. Most notably, a reduction in diastolic sarcomere length with increasing pacing frequencies, i.e., incomplete relaxation, was more pronounced in the HG compared to controls, but was normalized with extracellular Zn2+ application. This is a novel finding implicating that the detrimental effect of HG on cardiomyocyte Ca2+ regulation can be amelioration by Zn2+. Among the many post-translational modifications examined, only phosphorylation of ryanodine receptor (RyR at S-2808 was significantly higher in HG compared to nonHG. We did not find in our hypothyroid rats any differentiating effects of HG on myofibrillar protein phosphorylation, lysine acetylation, O-linked N-acetylglucosamine and advanced glycated end-products, which are often implicated as complicating factors in cardiac performance due to HG. Our

Hypoxia-Inducible Factor-1 as a Therapeutic Target in Endometrial Cancer Management

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Laura M. S. Seeber

2010-01-01

Full Text Available In the Western world, endometrial cancer (EC is the most common malignant tumor of the female genital tract. Solid tumors like EC outgrow their vasculature resulting in hypoxia. Tumor hypoxia is important because it renders an aggressive phenotype and leads to radio- and chemo-therapy resistance. Hypoxia-inducible factor-1 (HIF-1 plays an essential role in the adaptive cellular response to hypoxia and is associated with poor clinical outcome in EC. Therefore, HIF-1 could be an attractive therapeutic target. Selective HIF-1 inhibitors have not been identified. A number of nonselective inhibitors which target signaling pathways upstream or downstream HIF-1 are known to decrease HIF-1 protein levels. In clinical trials for the treatment of advanced and/or recurrent EC are the topoisomerase I inhibitor Topotecan, mTOR-inhibitor Rapamycin, and angiogenesis inhibitor Bevacizumab. Preliminary data shows encouraging results for these agents. Further work is needed to identify selective HIF-1 inhibitors and to translate these into clinical trials.

Regenerative responses after mild heart injuries for cardiomyocyte proliferation in zebrafish

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Itou, Junji; Akiyama, Ryutaro; Pehoski, Steve; Yu, Xiaodan; Kawakami, Hiroko; Kawakami, Yasuhiko

2014-01-01

Background The zebrafish heart regenerates after various severe injuries. Common processes of heart regeneration are cardiomyocyte proliferation, activation of epicardial tissue and neovascularization. In order to further characterize heart regeneration processes, we introduced milder injuries and compared responses to those induced by ventricular apex resection, a widely used injury method. We used scratching of the ventricular surface and puncturing of the ventricle with a fine tungsten needle as injury inducing techniques. Results Scratching the ventricular surface induced subtle cardiomyocyte proliferation and responses of the epicardium. Endothelial cell accumulation was limited to the surface of the heart. Ventricular puncture induced cardiomyocyte proliferation, endocardial and epicardial activation and neo-vascularization, similar to the resection method. However, the degree of the responses was milder, correlating with milder injury. Sham operation induced epicardial aldh1a2 expression but not tbx18 and WT1. Conclusions Puncturing the ventricle induces responses equivalent to resection at milder degrees in a shorter time frame and would be used as simple injury model. Scratching the ventricle did not induce heart regeneration and would be used for studying wound responses to epicardium. PMID:25074230

Lack of bcr and abr promotes hypoxia-induced pulmonary hypertension in mice.

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Min Yu

Full Text Available Bcr and Abr are GTPase activating proteins that specifically downregulate activity of the small GTPase Rac in restricted cell types in vivo. Rac1 is expressed in smooth muscle cells, a critical cell type involved in the pathogenesis of pulmonary hypertension. The molecular mechanisms that underlie hypoxia-associated pulmonary hypertension are not well-defined.Bcr and abr null mutant mice were compared to wild type controls for the development of pulmonary hypertension after exposure to hypoxia. Also, pulmonary arterial smooth muscle cells from those mice were cultured in hypoxia and examined for proliferation, p38 activation and IL-6 production. Mice lacking Bcr or Abr exposed to hypoxia developed increased right ventricular pressure, hypertrophy and pulmonary vascular remodeling. Perivascular leukocyte infiltration in the lungs was increased, and under hypoxia bcr-/- and abr-/- macrophages generated more reactive oxygen species. Consistent with a contribution of inflammation and oxidative stress in pulmonary hypertension-associated vascular damage, Bcr and Abr-deficient animals showed elevated endothelial leakage after hypoxia exposure. Hypoxia-treated pulmonary arterial smooth muscle cells from Bcr- or Abr-deficient mice also proliferated faster than those of wild type mice. Moreover, activated Rac1, phosphorylated p38 and interleukin 6 were increased in these cells in the absence of Bcr or Abr. Inhibition of Rac1 activation with Z62954982, a novel Rac inhibitor, decreased proliferation, p38 phosphorylation and IL-6 levels in pulmonary arterial smooth muscle cells exposed to hypoxia.Bcr and Abr play a critical role in down-regulating hypoxia-induced pulmonary hypertension by deactivating Rac1 and, through this, reducing both oxidative stress generated by leukocytes as well as p38 phosphorylation, IL-6 production and proliferation of pulmonary arterial smooth muscle cells.

Notch1 is required for hypoxia-induced proliferation, invasion and chemoresistance of T-cell acute lymphoblastic leukemia cells

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Zou Jie

2013-01-01

Full Text Available Abstract Background Notch1 is a potent regulator known to play an oncogenic role in many malignancies including T-cell acute lymphoblastic leukemia (T-ALL. Tumor hypoxia and increased hypoxia-inducible factor-1α (HIF-1α activity can act as major stimuli for tumor aggressiveness and progression. Although hypoxia-mediated activation of the Notch1 pathway plays an important role in tumor cell survival and invasiveness, the interaction between HIF-1α and Notch1 has not yet been identified in T-ALL. This study was designed to investigate whether hypoxia activates Notch1 signalling through HIF-1α stabilization and to determine the contribution of hypoxia and HIF-1α to proliferation, invasion and chemoresistance in T-ALL. Methods T-ALL cell lines (Jurkat, Sup-T1 transfected with HIF-1α or Notch1 small interference RNA (siRNA were incubated in normoxic or hypoxic conditions. Their potential for proliferation and invasion was measured by WST-8 and transwell assays. Flow cytometry was used to detect apoptosis and assess cell cycle regulation. Expression and regulation of components of the HIF-1α and Notch1 pathways and of genes related to proliferation, invasion and apoptosis were assessed by quantitative real-time PCR or Western blot. Results Hypoxia potentiated Notch1 signalling via stabilization and activation of the transcription factor HIF-1α. Hypoxia/HIF-1α-activated Notch1 signalling altered expression of cell cycle regulatory proteins and accelerated cell proliferation. Hypoxia-induced Notch1 activation increased the expression of matrix metalloproteinase-2 (MMP2 and MMP9, which increased invasiveness. Of greater clinical significance, knockdown of Notch1 prevented the protective effect of hypoxia/HIF-1α against dexamethasone-induced apoptosis. This sensitization correlated with losing the effect of hypoxia/HIF-1α on Bcl-2 and Bcl-xL expression. Conclusions Notch1 signalling is required for hypoxia/HIF-1α-induced proliferation

Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

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Shahrooz Ghaderi

2018-03-01

Full Text Available Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette. Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE, cardiac specific promoter, internal ribosome entry site (IRES, and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak. Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1% transgene expression. Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

Hypoxia-inducible factor 1-mediated human GATA1 induction promotes erythroid differentiation under hypoxic conditions.

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Zhang, Feng-Lin; Shen, Guo-Min; Liu, Xiao-Ling; Wang, Fang; Zhao, Ying-Ze; Zhang, Jun-Wu

2012-08-01

Hypoxia-inducible factor promotes erythropoiesis through coordinated cell type-specific hypoxia responses. GATA1 is essential to normal erythropoiesis and plays a crucial role in erythroid differentiation. In this study, we show that hypoxia-induced GATA1 expression is mediated by HIF1 in erythroid cells. Under hypoxic conditions, significantly increased GATA1 mRNA and protein levels were detected in K562 cells and erythroid induction cultures of CD34(+) haematopoietic stem/progenitor cells. Enforced HIF1α expression increased GATA1 expression, while HIF1α knockdown by RNA interference decreased GATA1 expression. In silico analysis revealed one potential hypoxia response element (HRE). The results from reporter gene and mutation analysis suggested that this element is necessary for hypoxic response. Chromatin immunoprecipitation (ChIP)-PCR showed that the putative HRE was recognized and bound by HIF1 in vivo. These results demonstrate that the up-regulation of GATA1 during hypoxia is directly mediated by HIF1.The mRNA expression of some erythroid differentiation markers was increased under hypoxic conditions, but decreased with RNA interference of HIF1α or GATA1. Flow cytometry analysis also indicated that hypoxia, desferrioxamine or CoCl(2) induced expression of erythroid surface markers CD71 and CD235a, while expression repression of HIF1α or GATA1 by RNA interference led to a decreased expression of CD235a. These results suggested that HIF1-mediated GATA1 up-regulation promotes erythropoiesis in order to satisfy the needs of an organism under hypoxic conditions. © 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Overexpression of glutaredoxin protects cardiomyocytes against nitric oxide-induced apoptosis with suppressing the S-nitrosylation of proteins and nuclear translocation of GAPDH

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Inadomi, Chiaki, E-mail: inadomic@nagasaki-u.ac.jp [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Murata, Hiroaki [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan); Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Ihara, Yoshito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Biochemistry, Wakayama Medical University, Wakayama 641-8509 (Japan); Goto, Shinji; Urata, Yoshishige [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Department of Stem Cell Biology, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Yodoi, Junji [Department of Biological Responses, Institute for Virus Research, Kyoto University, Kyoto 606-8507 (Japan); Kondo, Takahito [Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523 (Japan); Sumikawa, Koji [Department of Anesthesiology, Nagasaki University School of Medicine, Nagasaki 852-8501 (Japan)

2012-08-31

Highlights: Black-Right-Pointing-Pointer GRX1 overexpression protects myocardiac H9c2 cells against NO-induced apoptosis. Black-Right-Pointing-Pointer NO-induced nuclear translocation of GAPDH is suppressed in GRX overexpressors. Black-Right-Pointing-Pointer Oxidation of GAPDH by NO is less in GRX overexpressors than in controls. -- Abstract: There is increasing evidence demonstrating that glutaredoxin 1 (GRX1), a cytosolic enzyme responsible for the catalysis of protein deglutathionylation, plays distinct roles in inflammation and apoptosis by inducing changes in the cellular redox system. In this study, we investigated whether and how the overexpression of GRX1 protects cardiomyocytes against nitric oxide (NO)-induced apoptosis. Cardiomyocytes (H9c2 cells) were transfected with the expression vector for mouse GRX1 cDNA, and mock-transfected cells were used as a control. Compared with the mock-transfected cells, the GRX1-transfected cells were more resistant to NO-induced apoptosis. Stimulation with NO significantly increased the nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a pro-apoptotic protein, in the mock-transfected cells, but did not change GAPDH localization in the GRX1-transfected cells. Furthermore, we found that NO stimulation clearly induced the oxidative modification of GAPDH in the mock-transfected cells, whereas less modification of GAPDH was observed in the GRX1-transfected cells. These data suggest that the overexpression of GRX1 could protect cardiomyocytes against NO-induced apoptosis, likely through the inhibition of the oxidative modification and the nuclear translocation of GAPDH.

Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.

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Yang, Xiulan; Rodriguez, Marita; Pabon, Lil; Fischer, Karin A; Reinecke, Hans; Regnier, Michael; Sniadecki, Nathan J; Ruohola-Baker, Hannele; Murry, Charles E

2014-07-01

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) have great potential as a cell source for therapeutic applications such as regenerative medicine, disease modeling, drug screening, and toxicity testing. This potential is limited, however, by the immature state of the cardiomyocytes acquired using current protocols. Tri-iodo-l-thyronine (T3) is a growth hormone that is essential for optimal heart growth. In this study, we investigated the effect of T3 on hiPSC-CM maturation. A one-week treatment with T3 increased cardiomyocyte size, anisotropy, and sarcomere length. T3 treatment was associated with reduced cell cycle activity, manifest as reduced DNA synthesis and increased expression of the cyclin-dependent kinase inhibitor p21. Contractile force analyses were performed on individual cardiomyocytes using arrays of microposts, revealing an almost two-fold higher force per-beat after T3 treatment and also an enhancement in contractile kinetics. This improvement in force generation was accompanied by an increase in rates of calcium release and reuptake, along with a significant increase in sarcoendoplasmic reticulum ATPase expression. Finally, although mitochondrial genomes were not numerically increased, extracellular flux analysis showed a significant increase in maximal mitochondrial respiratory capacity and respiratory reserve capability after T3 treatment. Using a broad spectrum of morphological, molecular, and functional parameters, we conclude that T3 is a driver for hiPSC-CM maturation. T3 treatment may enhance the utility of hiPSC-CMs for therapy, disease modeling, or drug/toxicity screens. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development.

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Tsuji, Kenji; Kitamura, Shinji; Makino, Hirofumi

2014-04-25

The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly. In this study, we examined whether HIF regulates kidney development. We harvested kidneys from day 13 rat embryos (E13Ks) and cultured the organs under normoxic (20% O2/5% CO2) or hypoxic (5% O2/5% CO2) conditions. We evaluated the kidneys based on morphology and gene expression. E13Ks cultured under hypoxic conditions had significantly more ureteric bud (UB) branching than the E13Ks cultured under normoxic conditions. In addition, the mRNA levels of GDNF and GDNF receptor (GFR-α1), increased under hypoxic conditions in E13Ks. When we cultured E13Ks with the HIF-1α inhibitor digoxin or with siRNA targeting HIF-1α under hypoxic conditions, we did not observe increased UB branching. In addition, the expression of GDNF and GFR-α1 was inhibited under hypoxic conditions when the kidneys were treated with siRNA targeting HIF-1α. We also elucidated that hypoxia inhibited UB cell apoptosis and promoted the expression of FGF7 mRNA levels in metanephric mesenchymal (MM) cells in vitro. These findings suggest that hypoxic condition has important roles in inducing branching morphogenesis during kidney development. Hypoxia might mediate branching morphogenesis via not only GDNF/Ret but also FGF signaling pathway. Copyright © 2014 Elsevier Inc. All rights reserved.

Histone deacetylase inhibitors augment doxorubicin-induced DNA damage in cardiomyocytes.

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Ververis, Katherine; Rodd, Annabelle L; Tang, Michelle M; El-Osta, Assam; Karagiannis, Tom C

2011-12-01

Histone deacetylase inhibitors have emerged as a new class of anticancer therapeutics with suberoylanilide hydroxamic acid (Vorinostat) and depsipeptide (Romidepsin) already being approved for clinical use. Numerous studies have identified that histone deacetylase inhibitors will be most effective in the clinic when used in combination with conventional cancer therapies such as ionizing radiation and chemotherapeutic agents. One promising combination, particularly for hematologic malignancies, involves the use of histone deacetylase inhibitors with the anthracycline, doxorubicin. However, we previously identified that trichostatin A can potentiate doxorubicin-induced hypertrophy, the dose-limiting side-effect of the anthracycline, in cardiac myocytes. Here we have the extended the earlier studies and evaluated the effects of combinations of the histone deacetylase inhibitors, trichostatin A, valproic acid and sodium butyrate on doxorubicin-induced DNA double-strand breaks in cardiomyocytes. Using γH2AX as a molecular marker for the DNA lesions, we identified that all of the broad-spectrum histone deacetylase inhibitors tested augment doxorubicin-induced DNA damage. Furthermore, it is evident from the fluorescence photomicrographs of stained nuclei that the histone deacetylase inhibitors also augment doxorubicin-induced hypertrophy. These observations highlight the importance of investigating potential side-effects, in relevant model systems, which may be associated with emerging combination therapies for cancer.

Silymarin Component 2,3-dehydrosilybin Attenuates Cardiomyocyte Damage Following Hypoxia/Reoxygenation by Limiting Oxidative Stress

Czech Academy of Sciences Publication Activity Database

Gabrielová, E.; Křen, Vladimír; Jabůrek, Martin; Modriansky, M.

2015-01-01

Roč. 64, č. 1 (2015), s. 79-91 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GAP301/11/0662 Institutional support: RVO:61388971 ; RVO:67985823 Keywords : Silymarin * Dehydrosilybin * Neonatal rat cardiomyocytes Subject RIV: ED - Physiology Impact factor: 1.643, year: 2015

Hypoxia induces adipogenic differentitation of myoblastic cell lines

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Itoigawa, Yoshiaki [Tohoku University School of Medicine, Sendai (Japan); Juntendo University School of Medicine, Tokyo (Japan); Kishimoto, Koshi N., E-mail: kishimoto@med.tohoku.ac.jp [Tohoku University School of Medicine, Sendai (Japan); Okuno, Hiroshi; Sano, Hirotaka [Tohoku University School of Medicine, Sendai (Japan); Kaneko, Kazuo [Juntendo University School of Medicine, Tokyo (Japan); Itoi, Eiji [Tohoku University School of Medicine, Sendai (Japan)

2010-09-03

Research highlights: {yields} C2C12 and G8 myogenic cell lines treated by hypoxia differentiate into adipocytes. {yields} The expression of C/EBP{beta}, {alpha} and PPAR{gamma} were increased under hypoxia. {yields} Myogenic differentiation of C2C12 was inhibited under hypoxia. -- Abstract: Muscle atrophy usually accompanies fat accumulation in the muscle. In such atrophic conditions as back muscles of kyphotic spine and the rotator cuff muscles with torn tendons, blood flow might be diminished. It is known that hypoxia causes trans-differentiation of mesenchymal stem cells derived from bone marrow into adipocytes. However, it has not been elucidated yet if hypoxia turned myoblasts into adipocytes. We investigated adipogenesis in C2C12 and G8 murine myogenic cell line treated by hypoxia. Cells were also treated with the cocktail of insulin, dexamethasone and IBMX (MDI), which has been known to inhibit Wnt signaling and promote adipogenesis. Adipogenic differentiation was seen in both hypoxia and MDI. Adipogenic marker gene expression was assessed in C2C12. CCAAT/enhancer-binding protein (C/EBP) {beta}, {alpha} and peroxisome proliferator activating receptor (PPAR) {gamma} were increased by both hypoxia and MDI. The expression profile of Wnt10b was different between hypoxia and MDI. The mechanism for adipogenesis of myoblasts in hypoxia might be regulated by different mechanism than the modification of Wnt signaling.

Influence of chronic prenatal hypoxia on the specialized contact apparatus of rat heart ventricles during ontogeny

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N. S. Petruk

2014-08-01

Full Text Available Background. During prenatal development embryonic mammalian heart undergoes deep dynamic changes due to size, structure and functions. Pathological intrauterine hypoxia influences fetal development generally and cardiogeny particularly, it affects the structure and function of the heart muscle and can lead to a variety of cardiovascular abnormalities and congenital heart defects. It is known that as a result of chronic intrauterine hypoxia during the stages of prenatal ontogeny specialized intercellular apparatus of cardiomyocytes is damaged, which plays a role not only in the mechanical connections of the cardiomyocytes, but also in the electric cooperatives, metabolic conversions, the homeostasis of the ionic balance and transport morphogenetic signaling molecules which are involved into the mechanisms of cardiogeny. It contributes to the development of diseases that may be associated with impaired local distribution of specialized intercellular junctions, and manifests as arrhythmias and cardiac conduction. Objective. To determine the effects of chronic prenatal hypoxia on the structure and distribution of specialized intercellular junctions of typical cardiomyocytes in the rat ventricular myocardium at the stages of prenatal ontogeny and to evaluate morphometric parameters of intercalated disks in the postnatal period. Materials and methods. White rats were used as a material. Intrauterine hypoxia was modelled by intraperitoneal injection of sodium nitrite from 10th to 21st day of pregnancy. Hearts were investigated by the transmission electron microscopy during the stages of prenatal and postnatal ontogeny and in adult animals. Morphometric and statistical methods were applied. Pairwise comparisons between means of different groups were performed using Student’s t-test where, for each couple of normally distributed populations, the null hypothesis that the means are equal was verified. Results. The average length of desmosomes on the 20th

Inflammatory cytokine tumor necrosis factor α suppresses neuroprotective endogenous erythropoietin from astrocytes mediated by hypoxia-inducible factor-2α.

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Nagaya, Yoshiaki; Aoyama, Mineyoshi; Tamura, Tetsuya; Kakita, Hiroki; Kato, Shin; Hida, Hideki; Saitoh, Shinji; Asai, Kiyofumi

2014-12-01

Interest in erythropoietin (EPO) as a neuroprotective mediator has grown since it was found that systemically administered EPO is protective in several animal models of disease. However, given that the blood-brain barrier limits EPO entry into the brain, alternative approaches that induce endogenous EPO production in the brain may be more effective clinically and associated with fewer untoward side-effects. Astrocytes are the main source of EPO in the central nervous system. In the present study we investigated the effect of the inflammatory cytokine tumor necrosis factor α (TNFα) on hypoxia-induced upregulation of EPO in rat brain. Hypoxia significantly increased EPO mRNA expression in the brain and kidney, and this increase was suppressed by TNFα in vivo. In cultured astrocytes exposed to hypoxic conditions for 6 and 12 h, TNFα suppressed the hypoxia-induced increase in EPO mRNA expression in a concentration-dependent manner. TNFα inhibition of hypoxia-induced EPO expression was mediated primarily by hypoxia-inducible factor (HIF)-2α rather than HIF-1α. The effects of TNFα in reducing hypoxia-induced upregulation of EPO mRNA expression probably involve destabilization of HIF-2α, which is regulated by the nuclear factor (NF)-κB signaling pathway. TNFα treatment attenuated the protective effects of astrocytes on neurons under hypoxic conditions via EPO signaling. The effective blockade of TNFα signaling may contribute to the maintenance of the neuroprotective effects of EPO even under hypoxic conditions with an inflammatory response. © 2014 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Cinnamic aldehyde suppresses hypoxia-induced angiogenesis via inhibition of hypoxia-inducible factor-1α expression during tumor progression.

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Bae, Woom-Yee; Choi, Jae-Sun; Kim, Ja-Eun; Jeong, Joo-Won

2015-11-01

During tumor progression, hypoxia-inducible factor 1 (HIF-1) plays a critical role in tumor angiogenesis and tumor growth by regulating the transcription of several genes in response to a hypoxic environment and changes in growth factors. This study was designed to investigate the effects of cinnamic aldehyde (CA) on tumor growth and angiogenesis and the mechanisms underlying CA's anti-angiogenic activities. We found that CA administration inhibits tumor growth and blocks tumor angiogenesis in BALB/c mice. In addition, CA treatment decreased HIF-1α protein expression and vascular endothelial growth factor (VEGF) expression in mouse tumors and Renca cells exposed to hypoxia in vitro. Interestingly, CA treatment did not affect the stability of von Hippel-Lindau protein (pVHL)-associated HIF-1α and CA attenuated the activation of mammalian target of rapamycin (mTOR) pathway. Collectively, these findings strongly indicate that the anti-angiogenic activity of CA is, at least in part, regulated by the mTOR pathway-mediated suppression of HIF-1α protein expression and these findings suggest that CA may be a potential drug for human cancer therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Radiation-induced lipid peroxidation: influence of oxygen concentration and membrane lipid composition

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Wolters, H.; Tilburg, C.A.M. van; Konings, A.W.T.

1987-01-01

Radiation -induced lipid peroxidation phospholipid liposomes was investigated in terms of its dependence on lipid composition and oxygen concentration. Non-peroxidizable lipid incorporated in the liposomes reduced the rate of peroxidation of the peroxidizable phospholipid acyl chains, possibly by restricting the length of chain reactions. The latter effect is believed to be caused by interference of the non-peroxidizable lipids in the bilayer. At low oxygen concentration lipid peroxidation was reduced. The cause of this limited peroxidation may be a reduced number of radical initiation reactions possibly involving oxygen-derived superoxide radicals. Killing of proliferating mammalian cells, irradiated at oxygen concentrations ranging from 0 to 100%, appeared to be independent of the concentration of peroxidizable phospholipids in the cell membranes. This indicates that lipid peroxidation is not the determining process in radiation-induced reproductive cell death. (author)

The role of hypoxia-inducible factor-2 in digestive system cancers.

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Zhao, J; Du, F; Shen, G; Zheng, F; Xu, B

2015-01-15

Hypoxia is an all but ubiquitous phenomenon in cancers. Two known hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, primarily mediate the transcriptional response to hypoxia. Despite the high homology between HIF-1α and HIF-2α, emerging evidence suggests differences between both molecules in terms of transcriptional targets as well as impact on multiple physiological pathways and tumorigenesis. To date, much progress has been made toward understanding the roles of HIF-2α in digestive system cancers. Indeed, HIF-2α has been shown to regulate multiple aspects of digestive system cancers, including cell proliferation, angiogenesis and apoptosis, metabolism, metastasis and resistance to chemotherapy. These findings make HIF-2α a critical regulator of this malignant phenotype. Here we summarize the function of HIF-2 during cancer development as well as its contribution to tumorigenesis in digestive system malignancies.

Polycystin-2-dependent control of cardiomyocyte autophagy.

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Criollo, Alfredo; Altamirano, Francisco; Pedrozo, Zully; Schiattarella, Gabriele G; Li, Dan L; Rivera-Mejías, Pablo; Sotomayor-Flores, Cristian; Parra, Valentina; Villalobos, Elisa; Battiprolu, Pavan K; Jiang, Nan; May, Herman I; Morselli, Eugenia; Somlo, Stefan; de Smedt, Humbert; Gillette, Thomas G; Lavandero, Sergio; Hill, Joseph A

2018-05-01

Considerable evidence points to critical roles of intracellular Ca 2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca 2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca 2+ homeostasis and autophagy. Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2 F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca 2+ chelation using BAPTA-AM, whereas removal of extracellular Ca 2+ had no effect, pointing to a role of intracellular Ca 2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca 2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca 2+ -channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca 2+ . Furthermore, PC2 ablation was associated with impaired Ca 2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca 2+ stores. Finally, we provide evidence that Ca 2+ -mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. Together, this study unveils PC2 as a novel regulator of autophagy acting

Enzyme 15-lipoxygenase 1 promotes hypoxia-inducible factor 1α turnover and reduces vascular endothelial growth factor expression: implications for angiogenesis

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Zhong, Hua; Wang, Ruoxiang; Kelavkar, Uddhav; Wang, Christopher Y; Simons, Jonathan

2014-01-01

Hypoxia-inducible factor 1α (HIF-1α) is the regulatory subunit of the heterodimeric HIF-1 that plays a critical role in transcriptional regulation of genes in angiogenesis and hypoxic adaptation, while fatty acid metabolism mediated by lipoxygenases has been implicated in a variety of pathogeneses, including cancers. In this study, we report that 15-lipoxygenase 1 (15-LO1), a key member of the lipoxygenase family, promotes HIF-1α ubiquitination and degradation. Altering the level of 15-LO1 yields inverse changes in HIF-1α and HIF-1 transcriptional activity, under both normoxia and hypoxia, and even in CoCl 2 -treated cells where HIF-1α has been artificially elevated. The antagonistic effect of 15-LO1 is mediated by the Pro 564 /hydroxylation/26S proteasome system, while both the enzymatic activity and the intracellular membrane-binding function of 15-LO1 appear to contribute to HIF-1α suppression. Our findings provide a novel mechanism for HIF-1α regulation, in which oxygen-dependent HIF-1 activity is modulated by an oxygen-insensitive lipid metabolic enzyme

Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.

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Eldridge, Sandy; Guo, Liang; Mussio, Jodie; Furniss, Mike; Hamre, John; Davis, Myrtle

2014-10-01

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy. Published by Oxford University Press on behalf of Toxicological Sciences 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

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Asai, Yoichiro; Yamada, Tetsuya; Tsukita, Sohei; Takahashi, Kei; Maekawa, Masamitsu; Honma, Midori; Ikeda, Masanori; Murakami, Keigo; Munakata, Yuichiro; Shirai, Yuta; Kodama, Shinjiro; Sugisawa, Takashi; Chiba, Yumiko; Kondo, Yasuteru; Kaneko, Keizo; Uno, Kenji; Sawada, Shojiro; Imai, Junta; Nakamura, Yasuhiro; Yamaguchi, Hiroaki; Tanaka, Kozo; Sasano, Hironobu; Mano, Nariyasu; Ueno, Yoshiyuki; Shimosegawa, Tooru; Katagiri, Hideki

2017-05-01

Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic

KNK437, abrogates hypoxia-induced radioresistance by dual targeting of the AKT and HIF-1α survival pathways

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Oommen, Deepu; Prise, Kevin M.

2012-01-01

Highlights: ► KNK437, a benzylidene lactam compound, is a novel radiosensitizer. ► KNK437 inhibits AKT signaling and abrogates the accumulation of HIF-1α under hypoxia. ► KNK437 abrogates hypoxia induced resistance to radiation. -- Abstract: KNK437 is a benzylidene lactam compound known to inhibit stress-induced synthesis of heat shock proteins (HSPs). HSPs promote radioresistance and play a major role in stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α is widely responsible for tumor resistance to radiation under hypoxic conditions. We hypothesized that KNK437 sensitizes cancer cells to radiation and overrides hypoxia-induced radioresistance via destabilizing HIF-1α. Treatment of human cancer cells MDA-MB-231 and T98G with KNK437 sensitized them to ionizing radiation (IR). Surprisingly, IR did not induce HSPs in these cell lines. As hypothesized, KNK437 abrogated the accumulation of HIF-1α in hypoxic cells. However, there was no induction of HSPs under hypoxic conditions. Moreover, the proteosome inhibitor MG132 did not restore HIF-1α levels in KNK437-treated cells. This suggested that the absence of HIF-1α in hypoxic cells was not due to the enhanced protein degradation. HIF-1α is mainly regulated at the level of post-transcription and AKT is known to modulate the translation of HIF-1α mRNA. Interestingly, pre-treatment of cells with KNK437 inhibited AKT signaling. Furthermore, down regulation of AKT by siRNA abrogated HIF-1α levels under hypoxia. Interestingly, KNK437 reduced cell survival in hypoxic conditions and inhibited hypoxia-induced resistance to radiation. Taken together, these data suggest that KNK437 is an effective radiosensitizer that targets multiple pro-survival stress response pathways.

[Role of ATP-sensitive potassium channel activators in liver mitochondrial function in rats with different resistance to hypoxia].

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Tkachenko, H M; Kurhaliuk, N M; Vovkanych, L S

2003-01-01

Effects of ATP-sensitive potassium (KATP) channels opener pinacidil (0.06 mg/kg) and inhibitor glibenclamide (1 mg/kg) in rats with different resistance to hypoxia on indices of ADP-stimulation of mitochondrial respiration by Chance, calcium capacity and processes of lipid peroxidation in liver has been investigated. We used next substrates of oxidation: 0.35 mM succinate, 1 mM alpha-ketoglutarate. Additional analyses contain the next inhibitors: mitochondrial fermentative complex I-10 mkM rotenone, succinate dehydrogenase 2 mM malonic acid. It was shown that effects of pinacidil induced the increasing of oxidative phosporylation efficacy and ATP synthesis together with lowering of calcium capacity in rats with low resistance to hypoxia. Effects of pinacidil were leveled by glibenclamide. These changes are connected with the increasing of respiratory rate, calcium overload and intensification of lipid peroxidation processes. A conclusion was made about protective effect of pinacidil on mitochondrial functioning by economization of oxygen-dependent processes, adaptive potentialities of organisms with low resistance to hypoxia being increased.

Paradoxically, iron overload does not potentiate doxorubicin-induced cardiotoxicity in vitro in cardiomyocytes and in vivo in mice

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Guenancia, Charles [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Li, Na [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Hachet, Olivier [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Rigal, Eve [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cottin, Yves [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Cardiology Department, University Hospital, Dijon (France); Dutartre, Patrick; Rochette, Luc [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France); Vergely, Catherine, E-mail: cvergely@u-bourgogne.fr [INSERM UMR866, University of Burgundy, LPPCM, Faculties of Medicine and Pharmacy, Dijon (France)

2015-04-15

Doxorubicin (DOX) is known to induce serious cardiotoxicity, which is believed to be mediated by oxidative stress and complex interactions with iron. However, the relationship between iron and DOX-induced cardiotoxicity remains controversial and the role of iron chelation therapy to prevent cardiotoxicity is called into question. Firstly, we evaluated in vitro the effects of DOX in combination with dextran–iron on cell viability in cultured H9c2 cardiomyocytes and EMT-6 cancer cells. Secondly, we used an in vivo murine model of iron overloading (IO) in which male C57BL/6 mice received a daily intra-peritoneal injection of dextran–iron (15 mg/kg) for 3 weeks (D0–D20) and then (D21) a single sub-lethal intra-peritoneal injection of 6 mg/kg of DOX. While DOX significantly decreased cell viability in EMT-6 and H9c2, pretreatment with dextran–iron (125–1000 μg/mL) in combination with DOX, paradoxically limited cytotoxicity in H9c2 and increased it in EMT-6. In mice, IO alone resulted in cardiac hypertrophy (+ 22%) and up-regulation of brain natriuretic peptide and β-myosin heavy-chain (β-MHC) expression, as well as an increase in cardiac nitro-oxidative stress revealed by electron spin resonance spectroscopy. In DOX-treated mice, there was a significant decrease in left-ventricular ejection fraction (LVEF) and an up-regulation of cardiac β-MHC and atrial natriuretic peptide (ANP) expression. However, prior IO did not exacerbate the DOX-induced fall in LVEF and there was no increase in ANP expression. IO did not impair the capacity of DOX to decrease cancer cell viability and could even prevent some aspects of DOX cardiotoxicity in cardiomyocytes and in mice. - Highlights: • The effects of iron on cardiomyocytes were opposite to those on cancer cell lines. • In our model, iron overload did not potentiate anthracycline cardiotoxicity. • Chronic oxidative stress induced by iron could mitigate doxorubicin cardiotoxicity. • The role of iron in

The zebrafish miR-462/miR-731 cluster is induced under hypoxic stress via hypoxia-inducible factor 1α and functions in cellular adaptations.

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Huang, Chun-Xiao; Chen, Nan; Wu, Xin-Jie; Huang, Cui-Hong; He, Yan; Tang, Rong; Wang, Wei-Min; Wang, Huan-Ling

2015-12-01

Hypoxia, a unique and essential environmental stress, evokes highly coordinated cellular responses, and hypoxia-inducible factor (HIF) 1 in the hypoxia signaling pathway, an evolutionarily conserved cellular signaling pathway, acts as a master regulator of the transcriptional response to hypoxic stress. MicroRNAs (miRNAs), a major class of posttranscriptional gene expression regulators, also play pivotal roles in orchestrating hypoxia-mediated cellular adaptations. Here, global miRNA expression profiling and quantitative real-time PCR indicated that the up-regulation of the miR-462/miR-731 cluster in zebrafish larvae is induced by hypoxia. It was further validated that miR-462 and miR-731 are up-regulated in a Hif-1α-mediated manner under hypoxia and specifically target ddx5 and ppm1da, respectively. Overexpression of miR-462 and miR-731 represses cell proliferation through blocking cell cycle progress of DNA replication, and induces apoptosis. In situ detection revealed that the miR-462/miR-731 cluster is highly expressed in a consistent and ubiquitous manner throughout the early developmental stages. Additionally, the transcripts become restricted to the notochord, pharyngeal arch, liver, and gut regions from postfertilization d 3 to 5. These data highlight a previously unidentified role of the miR-462/miR-731 cluster as a crucial signaling mediator for hypoxia-mediated cellular adaptations and provide some insights into the potential function of the cluster during embryonic development. © FASEB.

The Effects of Portulaca oleracea on Hypoxia-Induced Pulmonary Edema in Mice.

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Yue, Tan; Xiaosa, Wen; Ruirui, Qi; Wencai, Shi; Hailiang, Xin; Min, Li

2015-03-01

Portulaca oleracea L. (PO) is known as "a vegetable for long life" due to its antioxidant, anti-inflammatory, and other pharmacological activities. However, the protective activity of the ethanol extract of PO (EEPO) against hypoxia-induced pulmonary edema has not been fully investigated. In this study, we exposed mice to a simulated altitude of 7000 meters for 0, 3, 6, 9, and 12 h to observe changes in the water content and transvascular leakage of the mouse lung. It was found that transvascular leakage increased to the maximum in the mouse lung after 6 h exposure to hypobaric hypoxia. Prophylactic administration of EEPO before hypoxic exposure markedly reduced the transvascular leakage and oxidative stress, and inhibited the upregulation of NF-kB in the mouse lung, as compared with the control group. In addition, EEPO significantly reduced the levels of proinflammatory cytokines and cell adhesion molecules in the lungs of mice, as compared with the hypoxia group. Our results show that EEPO can reduce initial transvascular leakage and pulmonary edema under hypobaric hypoxia conditions.

Effect of hepatocyte growth factor and angiotensin II on rat cardiomyocyte hypertrophy

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Chen, Ai-Lan [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Ou, Cai-Wen [The Fourth Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); He, Zhao-Chu [Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Liu, Qi-Cai [Experimental Medical Research Center, Guangzhou Medical University, Guangzhou (China); Dong, Qi [Department of Physiology, Guangzhou Medical University, Guangzhou (China); Chen, Min-Sheng [Guangzhou Key Laboratory of Cardiovascular Disease, Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China)

2012-10-15

Angiotensin II (Ang II) plays an important role in cardiomyocyte hypertrophy. The combined effect of hepatocyte growth factor (HGF) and Ang II on cardiomyocytes is unknown. The present study was designed to determine the effect of HGF on cardiomyocyte hypertrophy and to explore the combined effect of HGF and Ang II on cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from neonatal rat hearts and cultured in vitro. Cells were treated with Ang II (1 µM) alone, HGF (10 ng/mL) alone, and Ang II (1 µM) plus HGF (10 ng/mL) for 24, 48, and 72 h. The amount of [{sup 3}H]-leucine incorporation was then measured to evaluate protein synthesis. The mRNA levels of β-myosin heavy chain and atrial natriuretic factor were determined by real-time PCR to evaluate the presence of fetal phenotypes of gene expression. The cell size of cardiomyocytes was also studied. Ang II (1 µM) increased cardiomyocyte hypertrophy. Similar to Ang II, treatment with 1 µM HGF promoted cardiomyocyte hypertrophy. Moreover, the combination of 1 µM Ang II and 10 ng/mL HGF clearly induced a combined pro-hypertrophy effect on cardiomyocytes. The present study demonstrates for the first time a novel, combined effect of HGF and Ang II in promoting cardiomyocyte hypertrophy.

Sickle Mice Are Sensitive to Hypoxia/Ischemia-Induced Stroke but Respond to Tissue-Type Plasminogen Activator Treatment.

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Sun, Yu-Yo; Lee, Jolly; Huang, Henry; Wagner, Mary B; Joiner, Clinton H; Archer, David R; Kuan, Chia-Yi

2017-12-01

The effects of lytic stroke therapy in patients with sickle cell anemia are unknown, although a recent study suggested that coexistent sickle cell anemia does not increase the risk of cerebral hemorrhage. This finding calls for systemic analysis of the effects of thrombolytic stroke therapy, first in humanized sickle mice, and then in patients. There is also a need for additional predictive markers of sickle cell anemia-associated vasculopathy. We used Doppler ultrasound to examine the carotid artery of Townes sickle mice tested their responses to repetitive mild hypoxia-ischemia- and transient hypoxia-ischemia-induced stroke at 3 or 6 months of age, respectively. We also examined the effects of tPA (tissue-type plasminogen activator) treatment in transient hypoxia-ischemia-injured sickle mice. Three-month-old sickle cell (SS) mice showed elevated resistive index in the carotid artery and higher sensitivity to repetitive mild hypoxia-ischemia-induced cerebral infarct. Six-month-old SS mice showed greater resistive index and increased flow velocity without obstructive vasculopathy in the carotid artery. Instead, the cerebral vascular wall in SS mice showed ectopic expression of PAI-1 (plasminogen activator inhibitor-1) and P-selectin, suggesting a proadhesive and prothrombotic propensity. Indeed, SS mice showed enhanced leukocyte and platelet adherence to the cerebral vascular wall, broader fibrin deposition, and higher mortality after transient hypoxia-ischemia. Yet, post-transient hypoxia-ischemia treatment with tPA reduced thrombosis and mortality in SS mice. Sickle mice are sensitive to hypoxia/ischemia-induced cerebral infarct but benefit from thrombolytic treatment. An increased resistive index in carotid arteries may be an early marker of sickle cell vasculopathy. © 2017 American Heart Association, Inc.

Hypoxia-induced increases in serotonin-immunoreactive nerve fibers in the medulla oblongata of the rat.

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Morinaga, Ryosuke; Nakamuta, Nobuaki; Yamamoto, Yoshio

2016-10-01

Hypoxia induces respiratory responses in mammals and serotonergic neurons in the medulla oblongata participate in respiratory control. However, the morphological changes in serotonergic neurons induced by hypoxia have not yet been examined and respiratory controls of serotonergic neurons have not been clarified. We herein investigated the distribution of immunoreactivity for serotonin (5-hydroxytryptamine; 5-HT) in the medulla oblongata of control rats and rats exposed to 1-6h of hypoxia (10% O 2 ). We also examined the medulla oblongata by multiple immunofluorescence labeling for 5-HT, neurokinin 1 receptors (NK1R), a marker for some respiratory neurons in the pre-Bötzinger complex (PBC), and dopamine β-hydroxylase (DBH), a marker for catecholaminergic neurons. The number of 5-HT-immunoreactive nerve cell bodies in the raphe nuclei was higher in rats exposed to hypoxia than in control rats. The number of 5-HT-immunoreactive nerve fibers significantly increased in the rostral ventrolateral medulla of rats exposed to 1-6h of hypoxia, caudal ventrolateral medulla of rats exposed to 2-6h of hypoxia, and lateral part of the nucleus of the solitary tract and dorsal motor nucleus of the vagus nerve of rats exposed to 1-2h of hypoxia. Multiple immunofluorescence labeling showed that 5-HT-immunoreactive nerve fibers were close to NK1R-immunoreactive neurons in ventrolateral medulla and to DBH-immunoreactive neurons in the medulla. These results suggest that serotonergic neurons partly regulate respiratory control under hypoxic conditions by modulating the activity of NK1R-expressing and catecholaminergic neurons. Copyright © 2016 Elsevier GmbH. All rights reserved.

Enhanced carotid body chemosensory activity and the cardiovascular alterations induced by intermittent hypoxia

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Rodrigo eIturriaga

2014-12-01

Full Text Available The carotid body (CB plays a main role in the maintenance of the oxygen homeostasis. The hypoxic stimulation of the CB increases the chemosensory discharge, which in turn elicits reflex sympathetic, cardiovascular and ventilatory adjustments. An exacerbate carotid chemosensory activity has been associated with human sympathetic-mediated diseases such as hypertension, insulin resistance, heart failure and obstructive sleep apnea (OSA. Indeed, the CB chemosensory discharge becomes tonically hypereactive in experimental models of OSA and heart failure. Chronic intermittent hypoxia (CIH, a main feature of OSA, enhances CB chemosensory baseline discharges in normoxia and in response to hypoxia, inducing sympathetic overactivity and hypertension. Oxidative stress, increased levels of ET-1, Angiotensin II and pro-inflammatory cytokines, along with a reduced production of NO in the CB, have been associated with the enhanced carotid chemosensory activity. In this review, we will discuss new evidence supporting a main role for the CB chemoreceptor in the autonomic and cardiorespiratory alterations induced by intermittent hypoxia, as well as the molecular mechanisms involved in the CB chemosensory potentiation.

Enhanced carotid body chemosensory activity and the cardiovascular alterations induced by intermittent hypoxia

Science.gov (United States)

Iturriaga, Rodrigo; Andrade, David C.; Del Rio, Rodrigo

2014-01-01

The carotid body (CB) plays a main role in the maintenance of the oxygen homeostasis. The hypoxic stimulation of the CB increases the chemosensory discharge, which in turn elicits reflex sympathetic, cardiovascular, and ventilatory adjustments. An exacerbate carotid chemosensory activity has been associated with human sympathetic-mediated diseases such as hypertension, insulin resistance, heart failure, and obstructive sleep apnea (OSA). Indeed, the CB chemosensory discharge becomes tonically hypereactive in experimental models of OSA and heart failure. Chronic intermittent hypoxia (CIH), a main feature of OSA, enhances CB chemosensory baseline discharges in normoxia and in response to hypoxia, inducing sympathetic overactivity and hypertension. Oxidative stress, increased levels of ET-1, Angiotensin II and pro-inflammatory cytokines, along with a reduced production of NO in the CB, have been associated with the enhanced carotid chemosensory activity. In this review, we will discuss new evidence supporting a main role for the CB chemoreceptor in the autonomic and cardiorespiratory alterations induced by intermittent hypoxia, as well as the molecular mechanisms involved in the CB chemosensory potentiation. PMID:25520668

High-dose benfotiamine rescues cardiomyocyte contractile dysfunction in streptozotocin-induced diabetes mellitus.

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Ceylan-Isik, Asli F; Wu, Shan; Li, Qun; Li, Shi-Yan; Ren, Jun

2006-01-01

Diabetic cardiomyopathy is characterized by cardiac dysfunction. This study was designed to examine the effect of benfotiamine, a lipophilic derivative of thiamine, on streptozotocin (STZ)-induced cardiac contractile dysfunction in mouse cardiomyocytes. Adult male FVB mice were made diabetic with a single injection of STZ (200 mg/kg ip). Fourteen days later, control and diabetic (fasting plasma glucose > 13.9 mM) mice were put on benfotiamine therapy (100 mg.kg(-1).day(-1) ip) for another 14 days. Mechanical and intracellular Ca2+ properties were evaluated in left ventricular myocytes using an IonOptix MyoCam system. The following indexes were evaluated: peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening, resting and rise of intracellular Ca2+ in response to electrical stimulus, sarcoplasmic reticulum (SR) Ca2+ load, and intracellular Ca2+ decay rate (tau). Two- or four-week STZ treatment led to hyperglycemia, prolonged TPS and TR90, reduced SR Ca2+ load, elevated resting intracellular Ca2+ level and prolonged tau associated with normal PS, maximal velocity of shortening/relengthening, and intracellular Ca2+ rise in response to electrical stimulus. Benfotiamine treatment abolished prolongation in TPS, TR90, and tau, as well as reduction in SR Ca2+ load without affecting hyperglycemia and elevated resting intracellular Ca2+. Diabetes triggered oxidative stress, measured by GSH-to-GSSG ratio and formation of advanced glycation end product (AGE) in the hearts. Benfotiamine treatment alleviated oxidative stress without affecting AGE or protein carbonyl formation. Collectively, our results indicated that benfotiamine may rescue STZ-induced cardiomyocyte dysfunction but not AGE formation in short-term diabetes.

Emerging roles of hypoxia-inducible factors and reactive oxygen species in cancer and pluripotent stem cells

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Shigeo Saito

2015-06-01

Full Text Available Eukaryotic organisms require oxygen homeostasis to maintain proper cellular function for survival. During conditions of low oxygen tension (hypoxia, cells activate the transcription of genes that induce an adaptive response, which supplies oxygen to tissues. Hypoxia and hypoxia-inducible factors (HIFs may contribute to the maintenance of putative cancer stem cells, which can continue self-renewal indefinitely and express stemness genes in hypoxic stress environments (stem cell niches. Reactive oxygen species (ROS have long been recognized as toxic by-products of aerobic metabolism that are harmful to living cells, leading to DNA damage, senescence, or cell death. HIFs may promote a cancer stem cell state, whereas the loss of HIFs induces the production of cellular ROS and activation of proteins p53 and p16Ink4a, which lead to tumor cell death and senescence. ROS seem to inhibit HIF regulation in cancer cells. By contrast, controversial data have suggested that hypoxia increases the generation of ROS, which prevents hydroxylation of HIF proteins by inducing their transcription as negative feedback. Moreover, hypoxic conditions enhance the generation of induced pluripotent stem cells (iPSCs. During reprogramming of somatic cells into a PSC state, cells attain a metabolic state typically observed in embryonic stem cells (ESCs. ESCs and iPSCs share similar bioenergetic metabolisms, including decreased mitochondrial number and activity, and induced anaerobic glycolysis. This review discusses the current knowledge regarding the emerging roles of ROS homeostasis in cellular reprogramming and the implications of hypoxic regulation in cancer development.

Effects of natural and human-induced hypoxia on coastal benthos

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L. A. Levin

2009-10-01

Full Text Available Coastal hypoxia (defined here as <1.42 ml L⁻¹; 62.5 μM; 2 mg L⁻¹, approx. 30% oxygen saturation develops seasonally in many estuaries, fjords, and along open coasts as a result of natural upwelling or from anthropogenic eutrophication induced by riverine nutrient inputs. Permanent hypoxia occurs naturally in some isolated seas and marine basins as well as in open slope oxygen minimum zones. Responses of benthos to hypoxia depend on the duration, predictability, and intensity of oxygen depletion and on whether H₂S is formed. Under suboxic conditions, large mats of filamentous sulfide oxidizing bacteria cover the seabed and consume sulfide. They are hypothesized to provide a detoxified microhabitat for eukaryotic benthic communities. Calcareous foraminiferans and nematodes are particularly tolerant of low oxygen concentrations and may attain high densities and dominance, often in association with microbial mats. When oxygen is sufficient to support metazoans, small, soft-bodied invertebrates (typically annelids, often with short generation times and elaborate branchial structures, predominate. Large taxa are more sensitive than small taxa to hypoxia. Crustaceans and echinoderms are typically more sensitive to hypoxia, with lower oxygen thresholds, than annelids, sipunculans, molluscs and cnidarians. Mobile fish and shellfish will migrate away from low-oxygen areas. Within a species, early life stages may be more subject to oxygen stress than older life stages.

Hypoxia alters both the structure and function of benthic communities, but effects may differ with regional hypoxia history. Human-caused hypoxia is generally linked to eutrophication, and occurs adjacent to watersheds with large populations or agricultural activities. Many occurrences are seasonal, within estuaries, fjords or enclosed seas of the North Atlantic and the NW Pacific Oceans. Benthic faunal responses, elicited at oxygen levels below

Hypoxia-induced secretion of TGF-β1 in mesenchymal stem cell promotes breast cancer cell progression.

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Hung, Shun-Pei; Yang, Muh-Hwa; Tseng, Kuo-Fung; Lee, Oscar K

2013-01-01

In solid tumors, a decreased oxygen and nutrient supply creates a hypoxic microenvironment in the central region. This hypoxic condition induces molecular responses of normal and cancer cells in the local area, including angiogenesis, metabolic changes, and metastasis. In addition, other cells including mesenchymal stem cells (MSCs) have been reported to be recruited into the hypoxic area of solid tumors. In our previous study, we found that hypoxic condition induces the secretion of growth factors and cytokines in MSCs, and here we demonstrate that elevated secretion of transforming growth factor-β1 (TGF-β1) by MSCs under hypoxia promotes the growth, motility, and invasive ability of breast cancer cells. It was found that TGF-β1 promoter activity was regulated by hypoxia, and the major hypoxia-regulated element was located between bp -1030 to -666 in front of the TGF-β1 promoter region. In ChIP assay, the results revealed that HIF-1 was bound to the hypoxia response element (HRE) of TGF-β1 promoter. Collectively, the results indicate that hypoxia microenvironment can enhance cancer cell growth through the paracrine effects of the MSCs by driving their TGF-β1 gene expression and secretion. Therefore, extra caution has to be exercised when considering hypoxia pretreatment of MSCs before cell transplantation into patients for therapeutic purposes, particularly in patients susceptible to tumor growth.

Hypoxia-Inducible Histone Lysine Demethylases: Impact on the Aging Process and Age-Related Diseases

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Salminen, Antero; Kaarniranta, Kai; Kauppinen, Anu

2016-01-01

Hypoxia is an environmental stress at high altitude and underground conditions but it is also present in many chronic age-related diseases, where blood flow into tissues is impaired. The oxygen-sensing system stimulates gene expression protecting tissues against hypoxic insults. Hypoxia stabilizes the expression of hypoxia-inducible transcription factor-1α (HIF-1α), which controls the expression of hundreds of survival genes related to e.g. enhanced energy metabolism and autophagy. Moreover, many stress-related signaling mechanisms, such as oxidative stress and energy metabolic disturbances, as well as the signaling cascades via ceramide, mTOR, NF-κB, and TGF-β pathways, can also induce the expression of HIF-1α protein to facilitate cell survival in normoxia. Hypoxia is linked to prominent epigenetic changes in chromatin landscape. Screening studies have indicated that the stabilization of HIF-1α increases the expression of distinct histone lysine demethylases (KDM). HIF-1α stimulates the expression of KDM3A, KDM4B, KDM4C, and KDM6B, which enhance gene transcription by demethylating H3K9 and H3K27 sites (repressive epigenetic marks). In addition, HIF-1α induces the expression of KDM2B and KDM5B, which repress transcription by demethylating H3K4me2,3 sites (activating marks). Hypoxia-inducible KDMs support locally the gene transcription induced by HIF-1α, although they can also control genome-wide chromatin landscape, especially KDMs which demethylate H3K9 and H3K27 sites. These epigenetic marks have important role in the control of heterochromatin segments and 3D folding of chromosomes, as well as the genetic loci regulating cell type commitment, proliferation, and cellular senescence, e.g. the INK4 box. A chronic stimulation of HIF-1α can provoke tissue fibrosis and cellular senescence, which both are increasingly present with aging and age-related diseases. We will review the regulation of HIF-1α-dependent induction of KDMs and clarify their role in

2-Iminobiotin Superimposed on Hypothermia Protects Human Neuronal Cells from Hypoxia-Induced Cell Damage: An in Vitro Study

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Karina Zitta

2018-01-01

Full Text Available Perinatal asphyxia represents one of the major causes of neonatal morbidity and mortality. Hypothermia is currently the only established treatment for hypoxic-ischemic encephalopathy (HIE, but additional pharmacological strategies are being explored to further reduce the damage after perinatal asphyxia. The aim of this study was to evaluate whether 2-iminobiotin (2-IB superimposed on hypothermia has the potential to attenuate hypoxia-induced injury of neuronal cells. In vitro hypoxia was induced for 7 h in neuronal IMR-32 cell cultures. Afterwards, all cultures were subjected to 25 h of hypothermia (33.5°C, and incubated with vehicle or 2-IB (10, 30, 50, 100, and 300 ng/ml. Cell morphology was evaluated by brightfield microscopy. Cell damage was analyzed by LDH assays. Production of reactive oxygen species (ROS was measured using fluorometric assays. Western blotting for PARP, Caspase-3, and the phosphorylated forms of akt and erk1/2 was conducted. To evaluate early apoptotic events and signaling, cell protein was isolated 4 h post-hypoxia and human apoptosis proteome profiler arrays were performed. Twenty-five hour after the hypoxic insult, clear morphological signs of cell damage were visible and significant LDH release as well as ROS production were observed even under hypothermic conditions. Post-hypoxic application of 2-IB (10 and 30 ng/ml reduced the hypoxia-induced LDH release but not ROS production. Phosphorylation of erk1/2 was significantly increased after hypoxia, while phosphorylation of akt, protein expression of Caspase-3 and cleavage of PARP were only slightly increased. Addition of 2-IB did not affect any of the investigated proteins. Apoptosis proteome profiler arrays performed with cellular protein obtained 4 h after hypoxia revealed that post-hypoxic application of 2-IB resulted in a ≥ 25% down regulation of 10/35 apoptosis-related proteins: Bad, Bax, Bcl-2, cleaved Caspase-3, TRAILR1, TRAILR2, PON2, p21, p27, and phospho

Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome.

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Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

2014-04-18

Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H2O2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H2O2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process. Copyright © 2014 Elsevier Inc. All rights reserved.

Preventive effect of piracetam and vinpocetine on hypoxia-reoxygenation induced injury in primary hippocampal culture.

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Solanki, P; Prasad, D; Muthuraju, S; Sharma, A K; Singh, S B; Ilavzhagan, G

2011-04-01

The present study investigates the potential of Piracetam and Vinpocetine (nootropic drugs, known to possess neuroprotective properties) in preventing hypoxia-reoxygenation induced oxidative stress in primary hippocampal cell culture. The hippocampal culture was exposed to hypoxia (95% N(2), 5% CO(2)) for 3h and followed by 1h of reoxygenation (21% O(2) and 5% CO(2)) at 37 °C. The primary hippocampal cultures were supplemented with the optimum dose of Piracetam and Vinpocetine, independently, and the cultures were divided into six groups, viz. Control/Normoxia, Hypoxia, Hypoxia+Piracetam, Hypoxia+Vinpocetine, Normoxia + Piracetam and Normoxia+Vinpocetine. The cell-viability assays and biochemical oxidative stress parameters were evaluated for each of the six groups. Administration of 1mM Piracetam or 500 nM Vinpocetine significantly prevents the culture from hypoxia-reoxygenation injury when determined by Neutral Red assay, LDH release and Acetylcholine esterase activity. Results showed that Piracetam and Vinpocetine supplementation significantly prevented the fall of mitochondrial membrane potential, rise in ROS generation and reduction in antioxidant levels associated with the hypoxia-reoxygenation injury. In conclusion, the present study establishes that both Piracetam and Vinpocetine give neuroprotection against hypoxia-reoxygenation injury in primary hippocampal cell culture. Copyright © 2010 Elsevier Ltd. All rights reserved.

Autophagy-associated atrophy and metabolic remodeling of the mouse diaphragm after short-term intermittent hypoxia.

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Christian Giordano

Full Text Available Short-term intermittent hypoxia (IH is common in patients with acute respiratory disorders. Although prolonged exposure to hypoxia induces atrophy and increased fatigability of skeletal muscle, the response to short-term IH is less well known. We hypothesized that the diaphragm and limb muscles would adapt differently to short-term IH given that hypoxia stimulates ventilation and triggers a superimposed exercise stimulus in the diaphragm.We determined the structural, metabolic, and contractile properties of the mouse diaphragm after 4 days of IH (8 hours per day, 30 episodes per hour to a FiO2 nadir=6%, and compared responses in the diaphragm to a commonly studied reference limb muscle, the tibialis anterior. Outcome measures included muscle fiber size, assays of muscle proteolysis (calpain, ubiquitin-proteasome, and autophagy pathways, markers of oxidative stress and mitochondrial function, quantification of intramyocellular lipid and lipid metabolism genes, type I myosin heavy chain (MyHC expression, and in vitro contractile properties.After 4 days of IH, the diaphragm alone demonstrated significant atrophy (30% decrease of myofiber size together with increased LC3B-II protein (2.4-fold and mRNA markers of the autophagy pathway (LC3B, Gabarapl1, Bnip3, whereas active calpain and E3 ubiquitin ligases (MuRF1, atrogin-1 were unaffected in both muscles. Succinate dehydrogenase activity was significantly reduced by IH in both muscles. However, only the diaphragm exhibited increased intramyocellular lipid droplets (2.5-fold after IH, along with upregulation of genes linked to activated lipid metabolism. In addition, although the diaphragm showed evidence for acute fatigue immediately following IH, it underwent an adaptive fiber type switch toward slow type I MyHC-expressing fibers, associated with greater intrinsic endurance of the muscle during repetitive stimulation in vitro.Short-term IH induces preferential atrophy in the mouse diaphragm

SirT1 confers hypoxia-induced radioresistance via the modulation of c-Myc stabilization on hepatoma cells

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Xie Yuexia; Zhang Jianghong; Shao Chunlin; Xu Yanwu

2012-01-01

Intratumoral hypoxia is an important contributory factor to tumor cell resistance to radiotherapy. SirT1, a nicotinamide adenine dinucleotide (NAD + )-dependent histone/protein deacetylase, has been linked to the decrease of radiation-induced DNA damage and seems to be critical for cancer therapy. The purpose of this study was to investigate the role of SirT1 in hypoxia-induced radiation response on hepatoma cells. It was found that the administration with resveratrol, a putative SirT1 activator, enhanced the resistance of HepG2 cells against radiation-induced DNA damage of MN formation under hypoxia condition; while nicotinamide, a well-known SirT1 inhibitor, sensitized this radiation damage. Nevertheless, pretreatment of cells with 10058-F4, a specific inhibitor of c-Myc, almost eliminated the nicotinamide-induced radiosensitive effect. Further studies revealed that resveratrol inhibited c-Myc protein accumulation via up-regulation of SirT1 expression and deacetylase activity, and this loss of c-Myc protein was abolished by inhibiting its degradation in the presence of MG132, a potent inhibitor of proteasome. In contrast, nicotinamide attenuated c-Myc protein degradation induced by radiation under hypoxia through inhibition of SirT1 deacetylase activity. Our findings suggest that SirT1 could serve as a novel potent target of radiation-induced DNA damage and thus as a potential strategy to advance the efficiency of radiation therapy in hepatoma entities. (author)

Molecular basis for the regulation of hypoxia-inducible factor-1α levels by 2-deoxy-D-ribose.

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Ikeda, Ryuji; Tabata, Sho; Tajitsu, Yusuke; Nishizawa, Yukihiko; Minami, Kentaro; Furukawa, Tatsuhiko; Yamamoto, Masatatsu; Shinsato, Yoshinari; Akiyama, Shin-Ichi; Yamada, Katsushi; Takeda, Yasuo

2013-09-01

The angiogenic factor, platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP), stimulates the chemotaxis of endothelial cells and confers resistance to apoptosis induced by hypoxia. 2-Deoxy-D-ribose, a degradation product of thymidine generated by TP enzymatic activity, inhibits the upregulation of hypoxia-inducible factor (HIF) 1α, BNIP3 and caspase-3 induced by hypoxia. In the present study, we investigated the molecular basis for the suppressive effect of 2-deoxy-D-ribose on the upregulation of HIF-1α. 2-Deoxy-D-ribose enhanced the interaction of HIF-1α and the von Hippel-Lindau (VHL) protein under hypoxic conditions. It did not affect the expression of HIF-1α, prolyl hydroxylase (PHD)1/2/3 and VHL mRNA under normoxic or hypoxic conditions, but enhanced the interaction of HIF-1α and PHD2 under hypoxic conditions. 2-Deoxy-D-ribose also increased the amount of hydroxy-HIF-1α in the presence of the proteasome inhibitor MG-132. The expression levels of TP are elevated in many types of malignant solid tumors and, thus, 2-deoxy-D-ribose generated by TP in these tumors may play an important role in tumor progression by preventing hypoxia-induced apoptosis.

Analysis of the effects of exposure to acute hypoxia on oxidative lesions and tumour progression in a transgenic mouse breast cancer model

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Lunt Sarah

2008-05-01

Full Text Available Abstract Background Tumour hypoxia is known to be a poor prognostic indicator, predictive of increased risk of metastatic disease and reduced survival. Genomic instability has been proposed as one of the potential mechanisms for hypoxic tumour progression. Both of these features are commonly found in many cancer types, but their relationship and association with tumour progression has not been examined in the same model. Methods To address this issue, we determined the effects of 6 week in vivo acute hypoxic exposure on the levels of mutagenic lipid peroxidation product, malondialdehyde, and 8-oxo-7,8-dihydro-2'-deoxyguanosine DNA (8-oxo-dG lesions in the transgenic polyomavirus middle T (PyMT breast cancer mouse model. Results We observed significantly increased plasma lipid peroxidation and 8-oxo-dG lesion levels in the hypoxia-exposed mice. Consumption of malondialdehyde also induced a significant increase in the PyMT tumour DNA lesion levels, however, these increases did not translate into enhanced tumour progression. We further showed that the in vivo exposure to acute hypoxia induced accumulation of F4/80 positive tumour-associated macrophages (TAMs, demonstrating a relationship between hypoxia and macrophages in an experimental model. Conclusion These data suggest that although exposure to acute hypoxia causes an increase in 8-oxo-dG lesions and TAMs in the PyMT tumours, these increases do not translate into significant changes in tumour progression at the primary or metastatic levels in this strong viral oncogene-driven breast cancer model.

(Re-)programming of subtype specific cardiomyocytes.

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Hausburg, Frauke; Jung, Julia Jeannine; Hoch, Matti; Wolfien, Markus; Yavari, Arash; Rimmbach, Christian; David, Robert

2017-10-01

Adult cardiomyocytes (CMs) possess a highly restricted intrinsic regenerative potential - a major barrier to the effective treatment of a range of chronic degenerative cardiac disorders characterized by cellular loss and/or irreversible dysfunction and which underlies the majority of deaths in developed countries. Both stem cell programming and direct cell reprogramming hold promise as novel, potentially curative approaches to address this therapeutic challenge. The advent of induced pluripotent stem cells (iPSCs) has introduced a second pluripotent stem cell source besides embryonic stem cells (ESCs), enabling even autologous cardiomyocyte production. In addition, the recent achievement of directly reprogramming somatic cells into cardiomyocytes is likely to become of great importance. In either case, different clinical scenarios will require the generation of highly pure, specific cardiac cellular-subtypes. In this review, we discuss these themes as related to the cardiovascular stem cell and programming field, including a focus on the emergent topic of pacemaker cell generation for the development of biological pacemakers and in vitro drug testing. Copyright © 2017 Elsevier B.V. All rights reserved.

Protective effect of Edaravone against hypoxia-induced cytotoxicity in osteoblasts MC3T3-E1 cells.

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Cao, Bo; Chai, Chunxiang; Zhao, Sishun

2015-12-01

Edaravone is a newly developed clinical medicine for the treatment of acute cerebral infarction. Reduced blood supply to bones (hypoxia) has been involved in the pathological development of osteoporosis. In this study, we investigated the effect of Edaravone and its latent mechanism on hypoxia-induced cell toxicity in MC3T3-E1 cells. Cell viability was determined by the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) were determined by the fluorescence dyes 2',7'-dichlorofluorescein diacetate (DCFH-DA) and 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA), respectively. mRNA and proteins were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Edaravone significantly restored the hypoxia-induced reduction of MC3T3-E1 cell viability and inhibited lactate dehydrogenase release. In addition, we found that Edaravone inhibits the generation of ROS and NO. Hoechst staining results indicated that the nuclear condensation characteristic of apoptosis was increased in MC3T3-E1 cells after hypoxia exposure, which was significantly suppressed by Edaravone treatment. Mechanistically, we found that Edaravone markedly reduced the expression of cleaved caspase-3 and blunted the release of cytochrome c. These findings strongly suggested that Edaravone suppresses hypoxia-induced cytotoxicity in MC3T3-E1 cells. The pleiotropic effects of Edaravone on hypoxia exposure in osteoblasts suggest potential antiosteoporosis mechanisms of Edaravone. © 2015 International Union of Biochemistry and Molecular Biology.

Metallothionein as a compensatory component prevents intermittent hypoxia-induced cardiomyopathy in mice

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Yin, Xia; Zhou, Shanshan [The First Hospital of Jilin University, Changchun, 130021 (China); KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Zheng, Yang, E-mail: zhengyang@jlu.edu.cn [The First Hospital of Jilin University, Changchun, 130021 (China); Tan, Yi [KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Chinese–American Research Institute for Diabetic Complications, Wenzhou Medical College School of Pharmacy, Wenzhou, 325035 (China); Kong, Maiying [Department of Bioinformatics and Biostatistics, School of Public Health and Information Sciences, University of Louisville, Louisville, KY 40202 (United States); Wang, Bo [KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Department of Pathology, Inner Mongolia Forestry General Hospital, Yakeshi, 022150 (China); Feng, Wenke [Department of Medicine, School of Medicine, University of Louisville, Louisville, 40202 (United States); Epstein, Paul N. [KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Cai, Jun, E-mail: j0cai002@louisville.edu [KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Cai, Lu [KCHRI at the Department of Pediatrics, School of Medicine, University of Louisville, Louisville, 40202 (United States); Chinese–American Research Institute for Diabetic Complications, Wenzhou Medical College School of Pharmacy, Wenzhou, 325035 (China); Department of Medicine, School of Medicine, University of Louisville, Louisville, 40202 (United States)

2014-05-15

Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (IH) to induce cardiovascular disease, which may be related to oxidative damage. Metallothionein (MT) has been extensively proved to be an endogenous and highly inducible antioxidant protein expressed in the heart. Therefore, we tested the hypotheses that oxidative stress plays a critical role in OSA induced cardiac damage and MT protects the heart from OSA-induced cardiomyopathy. To mimic hypoxia/reoxygenation events that occur in adult OSA patients, mice were exposed to IH for 3 days to 8 weeks. The IH paradigm consisted of alternating cycles of 20.9% O{sub 2}/8% O{sub 2} F{sub I}O{sub 2} (30 episodes per hour) with 20 s at the nadir F{sub I}O{sub 2} for 12 h a day during daylight. IH significantly increased the ratio of heart weight to tibia length at 4 weeks with a decrease in cardiac function from 4 to 8 weeks. Cardiac oxidative damage and fibrosis were observed after 4 and 8 weeks of IH exposures. Endogenous MT expression was up-regulated in response to 3-day IH, but significantly decreased at 4 and 8 weeks of IH. In support of MT as a major compensatory component, mice with cardiac overexpression of MT gene and mice with global MT gene deletion were completely resistant, and highly sensitive, respectively, to chronic IH induced cardiac effects. These findings suggest that chronic IH induces cardiomyopathy characterized by oxidative stress-mediated cardiac damage and the antioxidant MT protects the heart from such pathological and functional changes. - Highlights: • The effect of intermittent hypoxia (IH) on cardiac metallothionein (MT) • Cardiac MT expression was up-regulated in response to 3-day IH. • Exposure to 4- or 8-week IH downregulated cardiac MT expression. • Overexpression of cardiac MT protects from IH-induced cardiac damage. • Global deletion of MT gene made the heart more sensitive to IH damage.

Metallothionein as a compensatory component prevents intermittent hypoxia-induced cardiomyopathy in mice

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Yin, Xia; Zhou, Shanshan; Zheng, Yang; Tan, Yi; Kong, Maiying; Wang, Bo; Feng, Wenke; Epstein, Paul N.; Cai, Jun; Cai, Lu

2014-01-01

Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (IH) to induce cardiovascular disease, which may be related to oxidative damage. Metallothionein (MT) has been extensively proved to be an endogenous and highly inducible antioxidant protein expressed in the heart. Therefore, we tested the hypotheses that oxidative stress plays a critical role in OSA induced cardiac damage and MT protects the heart from OSA-induced cardiomyopathy. To mimic hypoxia/reoxygenation events that occur in adult OSA patients, mice were exposed to IH for 3 days to 8 weeks. The IH paradigm consisted of alternating cycles of 20.9% O 2 /8% O 2 F I O 2 (30 episodes per hour) with 20 s at the nadir F I O 2 for 12 h a day during daylight. IH significantly increased the ratio of heart weight to tibia length at 4 weeks with a decrease in cardiac function from 4 to 8 weeks. Cardiac oxidative damage and fibrosis were observed after 4 and 8 weeks of IH exposures. Endogenous MT expression was up-regulated in response to 3-day IH, but significantly decreased at 4 and 8 weeks of IH. In support of MT as a major compensatory component, mice with cardiac overexpression of MT gene and mice with global MT gene deletion were completely resistant, and highly sensitive, respectively, to chronic IH induced cardiac effects. These findings suggest that chronic IH induces cardiomyopathy characterized by oxidative stress-mediated cardiac damage and the antioxidant MT protects the heart from such pathological and functional changes. - Highlights: • The effect of intermittent hypoxia (IH) on cardiac metallothionein (MT) • Cardiac MT expression was up-regulated in response to 3-day IH. • Exposure to 4- or 8-week IH downregulated cardiac MT expression. • Overexpression of cardiac MT protects from IH-induced cardiac damage. • Global deletion of MT gene made the heart more sensitive to IH damage

Activation of β-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptors δ (PPARδ in Neonatal Rat Cardiomyocytes

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Ming-Ting Chou

2012-01-01

Full Text Available Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells.

Unraveling the role of hypoxia-inducible factor (HIF)-1α and HIF-2α in the adaption process of human microvascular endothelial cells (HMEC-1) to hypoxia: Redundant HIF-dependent regulation of macrophage migration inhibitory factor.

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Hahne, Martin; Schumann, Peggy; Mursell, Mathias; Strehl, Cindy; Hoff, Paula; Buttgereit, Frank; Gaber, Timo

2018-03-01

Hypoxia driven angiogenesis is a prominent feature of tissue regeneration, inflammation and tumor growth and is regulated by hypoxia-inducible factor (HIF)-1 and -2. The distinct functions of HIFs in the hypoxia-induced angiogenesis and metabolic switch of endothelial cells are still unknown and therefore aim of this study. We investigated the role of HIF-1 and -2 in the adaptation of immortalized human microvascular endothelial cells (HMEC-1) to hypoxic conditions (1% O 2 ) in terms of angiogenesis, cytokine secretion, gene expression and ATP/ADP-ratio using shRNA-mediated reduction of the oxygen sensitive α-subunits of either HIF-1 or HIF-2 or the combination of both. Reduction of HIF-1α diminished cellular energy, hypoxia-induced glycolytic gene expression, and angiogenesis not altering pro-angiogenic factors. Reduction of HIF-2α diminished hypoxia-induced pro-angiogenic factors, enhanced anti-angiogenic factors and attenuated angiogenesis not altering glycolytic gene expression. Reduction of both HIFs reduced cell survival, gene expression of glycolytic enzymes and pro-angiogenic factors as compared to the corresponding control. Finally, we identified the macrophage migration inhibitory factor (MIF) to be redundantly regulated by HIF-1 and HIF-2 and to be essential in the process of hypoxia-driven angiogenesis. Our results demonstrate a major impact of HIF-1 and HIF-2 on hypoxia-induced angiogenesis indicating distinct but also overlapping functions of HIF-1 and HIF-2. These findings open new possibilities for therapeutic approaches by specifically targeting the HIF-1 and HIF-2 or their target MIF. Copyright © 2017 Elsevier Inc. All rights reserved.

Glutamatergic neurotransmission modulates hypoxia-induced hyperventilation but not anapyrexia

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Paula P.M. de

2004-01-01

Full Text Available The interaction between pulmonary ventilation (V E and body temperature (Tb is essential for O2 delivery to match metabolic rate under varying states of metabolic demand. Hypoxia causes hyperventilation and anapyrexia (a regulated drop in Tb, but the neurotransmitters responsible for this interaction are not well known. Since L-glutamate is released centrally in response to peripheral chemoreceptor stimulation and glutamatergic receptors are spread in the central nervous system we tested the hypothesis that central L-glutamate mediates the ventilatory and thermal responses to hypoxia. We measured V E and Tb in 40 adult male Wistar rats (270 to 300 g before and after intracerebroventricular injection of kynurenic acid (KYN, an ionotropic glutamatergic receptor antagonist, alpha-methyl-4-carboxyphenylglycine (MCPG, a metabotropic glutamatergic receptor antagonist or vehicle (saline, followed by a 1-h period of hypoxia (7% inspired O2 or normoxia (humidified room air. Under normoxia, KYN (N = 5 or MCPG (N = 8 treatment did not affect V E or Tb compared to saline (N = 6. KYN and MCPG injection caused a decrease in hypoxia-induced hyperventilation (595 ± 49 for KYN, N = 7 and 525 ± 84 ml kg-1 min-1 for MCPG, N = 6; P < 0.05 but did not affect anapyrexia (35.3 ± 0.2 for KYN and 34.7 ± 0.4ºC for MCPG compared to saline (912 ± 110 ml kg-1 min-1 and 34.8 ± 0.2ºC, N = 8. We conclude that glutamatergic receptors are involved in hypoxic hyperventilation but do not affect anapyrexia, indicating that L-glutamate is not a common mediator of this interaction.

Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

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Daniel R. Bayzigitov

2016-01-01

Full Text Available Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

Diacetoxyscirpenol as a new anticancer agent to target hypoxia-inducible factor 1

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Choi, Yong-Joon; Shin, Hyun-Woo; Chun, Yang-Sook; Leutou, Alain Simplice; Son, Byeng Wha; Park, Jong-Wan

2016-01-01

Hypoxia activates hypoxia-inducible factor 1, which promotes the progression of malignancy by stimulating angiogenesis and by augmenting the ability of tumors to survive. Thus, HIF-1 is one of the most compelling targets for treating cancers. The aim of this study was to find a small molecule that inhibits HIF-1 under hypoxia in cancer cells. 7,280 compounds in a chemical library were tested in a cancer cell line expressing luciferase HIF-dependently. Through three rounds of screening, we finally picked up a compound that originates from a marine bacterium parasitizing red alga. The antibiotic potently inhibited HIF-1 expression and its transcriptional activity in cancer cells exposed to hypoxia. Through two-step fractionation, diacetoxyscirpenol was purified and identified as a HIF-inhibiting ingredient. Mechanistically, diacetoxyscirpenol inhibits the synthesis of HIF-1α protein and also interferes with the dimerization of HIF-1α and ARNT. It attenuates HIF-mediated gene expression in cancer cells exposed to hypoxia, and by doing so reduces tumorigenic and angiogenic potentials of cancer cells. More importantly, diacetoxyscirpenol retarded tumor growth in mice, and reduced HIF-1α expression and vascular formation in the tumors. Overall, diacetoxyscirpenol is considered a potential drug deregulating the HIF-1 signaling pathway, and it could be beneficially employed for treating malignant tumors with hypoxic microenvironment. PMID:27613833

MicroRNA-145 Aggravates Hypoxia-Induced Injury by Targeting Rac1 in H9c2 Cells.

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Wang, Ximing; Zhang, Yanxia; Wang, Hongshan; Zhao, Genshang; Fa, Xianen

2017-01-01

Myocardial infarction (MI) is a leading cause of morbidity and mortality. Here, we sought to explore the potential role and underlying mechanism of miR-145 in MI. H9c2 cells were cultured under persistent hypoxia to simulate MI. The hypoxia-induced injury was assessed on the basis of cell viability, migration, invasion and apoptosis. The expression of miR-145 was evaluated by qRT-PCR and the influence of aberrantly expressed miR-145 on H9c2 cells under hypoxia was also estimated. Utilizing bioinformatics methods, the target genes of miR-145 were verified by luciferase reporter assay. Then, effects of abnormally expressed target gene on miR-145 silenced H9c2 cells were assessed. Finally, the phosphorylation levels of key kinases in the phosphatidylinositol-3-kinase (PI3K)/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways were detected by Western blot analysis. Hypoxia remarkably lowered viability, migration and invasion but promoted cell apoptosis. Meantime, the miR-145 level was up-regulated in H9c2 cells under hypoxia. Following experiments suggested that hypoxia-induced injury was exacerbated by miR-145 overexpression while was alleviated by miR-145 silence. Rac1 was predicted and further validated to be a target gene of miR-145. The influence of miR-145 silencing on H9c2 cells under hypoxia could be reversed by down-regulation of Rac1. Additionally, the phosphorylation levels of PI3K, AKT, MAPK and ERK were all elevated in miR-145 silenced cells and these alterations were reversed by down-regulation of Rac1. miR-145 silencing could protect H9c2 cells against hypoxia-induced injury by targeting Rac1, in which PI3K/AKT and MAPK/ERK pathways might be involved. © 2017 The Author(s). Published by S. Karger AG, Basel.

The endoplasmic reticulum stress-autophagy pathway is involved in apelin-13-induced cardiomyocyte hypertrophy in vitro

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Feng XIE; Di WU; Shi-fang HUANG; Jian-gang CAO; He-ning LI; Lu HE; Mei-qing LIU; Lan-fang LI; Lin-xi CHEN

2017-01-01

Apelin is the endogenous ligand for the G protein-coupled receptor APJ,and plays important roles in the cardiovascular system.Our previous studies showed that apelin-13 promotes the hypertrophy of H9c2 rat cardiomyocytes through the PI3K-autophagy pathway.The aim of this study was to explore what roles ER stress and autophagy played in apelin-13-induced hypertrophy of cardiomyocytes in vitro.Treatment of H9c2 cells with apelin-13 (0.001-2 μJmol/L) dose-dependently increased the production of ROS and the expression levels of NADPH oxidase 4 (NOX4).Knockdown of Nox4 with siRNAs effectively prevented the reduction of GSH/GSSG ratio in apelin-13-treated cells.Furthermore,apelin-13 treatment dose-dependently increased the expression of Bip and CHOP,two ER stress markers,in the cells.Knockdown of APJ or Nox4 with the corresponding siRNAs,or application of NADPH inhibitor DPI blocked apelin-13-induced increases in Bip and CHOP expression.Moreover,apelin-13 treatment increased the formation of autophagosome and ER fragments and the LC3 puncta in the ER of the cells.Knockdown of APJ,Nox4,Bip or CHOP with the corresponding siRNAs,or application of DPI or salubrinal attenuated apelin-13-induced overexpression of LC3-Ⅱ/Ⅰ and beclin 1.Finally,knockdown of Nox4,Bip or CHOP with the corresponding siRNAs,or application of salubrinal significantly suppressed apelin-13-induced increases in the cell diameter,volume and protein contents.Our results demonstrate that ER stress-autophagy is involved in apelin-13-induced H9c2 cell hypertrophy.

Late gestational intermittent hypoxia induces metabolic and epigenetic changes in male adult offspring mice.

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Khalyfa, Abdelnaby; Cortese, Rene; Qiao, Zhuanhong; Ye, Honggang; Bao, Riyue; Andrade, Jorge; Gozal, David

2017-04-15

Late gestation during pregnancy has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia, a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis and metabolic function in offspring. Here we show that late gestation intermittent hypoxia induces metabolic dysfunction as reflected by increased body weight and adiposity index in adult male offspring that is paralleled by epigenomic alterations and inflammation in visceral white adipose tissue. Fetal perturbations by OSA during pregnancy impose long-term detrimental effects manifesting as metabolic dysfunction in adult male offspring. Pregnancy, particularly late gestation (LG), has been associated with a relatively high prevalence of obstructive sleep apnoea (OSA). Intermittent hypoxia (IH), a hallmark of OSA, could impose significant long-term effects on somatic growth, energy homeostasis, and metabolic function in offspring. We hypothesized that IH during late pregnancy (LG-IH) may increase the propensity for metabolic dysregulation and obesity in adult offspring via epigenetic modifications. Time-pregnant female C57BL/6 mice were exposed to LG-IH or room air (LG-RA) during days 13-18 of gestation. At 24 weeks, blood samples were collected from offspring mice for lipid profiles and insulin resistance, indirect calorimetry was performed and visceral white adipose tissues (VWAT) were assessed for inflammatory cells as well as for differentially methylated gene regions (DMRs) using a methylated DNA immunoprecipitation on chip (MeDIP-chip). Body weight, food intake, adiposity index, fasting insulin, triglycerides and cholesterol levels were all significantly higher in LG-IH male but not female offspring. LG-IH also altered metabolic expenditure and locomotor activities in male offspring, and increased number of pro-inflammatory macrophages emerged in VWAT along with 1520 DMRs (P < 0.0001), associated with 693�

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression.

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Carlin, Sean; Pugachev, Andrei; Sun, Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C Clifton; Humm, John L

2009-10-01

To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer (18)F-fluoromisonidazole ((18)F-FMISO). Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe (124)I-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-iodouracil ((124)I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between (124)I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe (18)F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with (124)I-FIAU (3 h before sacrifice) and (18)F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between (18)F-FMISO and (124)I-FIAU on a pixel-by-pixel basis was performed. Correlation coefficients between (124)I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between (18)F-FMISO and (124)I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of this model for the

In vivo characterization of a reporter gene system for imaging hypoxia-induced gene expression

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Carlin, Sean; Pugachev, Andrei; Sun Xiaorong; Burke, Sean; Claus, Filip; O'Donoghue, Joseph; Ling, C. Clifton; Humm, John L.

2009-01-01

Purpose: To characterize a tumor model containing a hypoxia-inducible reporter gene and to demonstrate utility by comparison of reporter gene expression to the uptake and distribution of the hypoxia tracer 18 F-fluoromisonidazole ( 18 F-FMISO). Methods: Three tumors derived from the rat prostate cancer cell line R3327-AT were grown in each of two rats as follows: (1) parental R3327-AT, (2) positive control R3327-AT/PC in which the HSV1-tkeGFP fusion reporter gene was expressed constitutively, (3) R3327-AT/HRE in which the reporter gene was placed under the control of a hypoxia-inducible factor-responsive promoter sequence (HRE). Animals were coadministered a hypoxia-specific marker (pimonidazole) and the reporter gene probe 124 I-2'-fluoro-2'-deoxy-1-β-D-arabinofuranosyl-5-iodouracil ( 124 I-FIAU) 3 h prior to sacrifice. Statistical analysis of the spatial association between 124 I-FIAU uptake and pimonidazole fluorescent staining intensity was then performed on a pixel-by-pixel basis. Utility of this system was demonstrated by assessment of reporter gene expression versus the exogenous hypoxia probe 18 F-FMISO. Two rats, each bearing a single R3327-AT/HRE tumor, were injected with 124 I-FIAU (3 h before sacrifice) and 18 F-FMISO (2 h before sacrifice). Statistical analysis of the spatial association between 18 F-FMISO and 124 I-FIAU on a pixel-by-pixel basis was performed. Results: Correlation coefficients between 124 I-FIAU uptake and pimonidazole staining intensity were: 0.11 in R3327-AT tumors, -0.66 in R3327-AT/PC and 0.76 in R3327-AT/HRE, confirming that only in the R3327-AT/HRE tumor was HSV1-tkeGFP gene expression associated with hypoxia. Correlation coefficients between 18 F-FMISO and 124 I-FIAU uptakes in R3327-AT/HRE tumors were r=0.56, demonstrating good spatial correspondence between the two tracers. Conclusions: We have confirmed hypoxia-specific expression of the HSV1-tkeGFP fusion gene in the R3327-AT/HRE tumor model and demonstrated the utility of

Dystrophin-deficient cardiomyocytes derived from human urine: New biologic reagents for drug discovery

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Xuan Guan

2014-03-01

Full Text Available The ability to extract somatic cells from a patient and reprogram them to pluripotency opens up new possibilities for personalized medicine. Induced pluripotent stem cells (iPSCs have been employed to generate beating cardiomyocytes from a patient's skin or blood cells. Here, iPSC methods were used to generate cardiomyocytes starting from the urine of a patient with Duchenne muscular dystrophy (DMD. Urine was chosen as a starting material because it contains adult stem cells called urine-derived stem cells (USCs. USCs express the canonical reprogramming factors c-myc and klf4, and possess high telomerase activity. Pluripotency of urine-derived iPSC clones was confirmed by immunocytochemistry, RT-PCR and teratoma formation. Urine-derived iPSC clones generated from healthy volunteers and a DMD patient were differentiated into beating cardiomyocytes using a series of small molecules in monolayer culture. Results indicate that cardiomyocytes retain the DMD patient's dystrophin mutation. Physiological assays suggest that dystrophin-deficient cardiomyocytes possess phenotypic differences from normal cardiomyocytes. These results demonstrate the feasibility of generating cardiomyocytes from a urine sample and that urine-derived cardiomyocytes retain characteristic features that might be further exploited for mechanistic studies and drug discovery.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes.

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van Deel, Elza D; Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão-Pires, Inês; van der Velden, Jolanda

2017-07-15

This paper describes a novel model that allows exploration of matrix-induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function. Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca 2+ handling and myofilament function. Cell shortening and Ca 2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix. Matrix stiffness-impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness. Matrix stiffness-induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte-matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix-induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca 2+ handling but does not alter myofilament-generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness-induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness-induced changes in cardiomyocyte

Evaluation of Optogenetic Electrophysiology Tools in Human Stem Cell-Derived Cardiomyocytes

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Susann Björk

2017-11-01

Full Text Available Current cardiac drug safety assessments focus on hERG channel block and QT prolongation for evaluating arrhythmic risks, whereas the optogenetic approach focuses on the action potential (AP waveform generated by a monolayer of human cardiomyocytes beating synchronously, thus assessing the contribution of several ion channels on the overall drug effect. This novel tool provides arrhythmogenic sensitizing by light-induced pacing in combination with non-invasive, all-optical measurements of cardiomyocyte APs and will improve assessment of drug-induced electrophysiological aberrancies. With the help of patch clamp electrophysiology measurements, we aimed to investigate whether the optogenetic modifications alter human cardiomyocytes' electrophysiology and how well the optogenetic analyses perform against this gold standard. Patch clamp electrophysiology measurements of non-transduced stem cell-derived cardiomyocytes compared to cells expressing the commercially available optogenetic constructs Optopatch and CaViar revealed no significant changes in action potential duration (APD parameters. Thus, inserting the optogenetic constructs into cardiomyocytes does not significantly affect the cardiomyocyte's electrophysiological properties. When comparing the two methods against each other (patch clamp vs. optogenetic imaging we found no significant differences in APD parameters for the Optopatch transduced cells, whereas the CaViar transduced cells exhibited modest increases in APD-values measured with optogenetic imaging. Thus, to broaden the screen, we combined optogenetic measurements of membrane potential and calcium transients with contractile motion measured by video motion tracking. Furthermore, to assess how optogenetic measurements can predict changes in membrane potential, or early afterdepolarizations (EADs, cells were exposed to cumulating doses of E-4031, a hERG potassium channel blocker, and drug effects were measured at both spontaneous and

Hypoxia-inducible factor-2α-dependent hypoxic induction of Wnt10b expression in adipogenic cells.

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Park, Young-Kwon; Park, Bongju; Lee, Seongyeol; Choi, Kang; Moon, Yunwon; Park, Hyunsung

2013-09-06

Adipocyte hyperplasia and hypertrophy in obesity can lead to many changes in adipose tissue, such as hypoxia, metabolic dysregulation, and enhanced secretion of cytokines. In this study, hypoxia increased the expression of Wnt10b in both human and mouse adipogenic cells, but not in hypoxia-inducible factor (HIF)-2α-deficient adipogenic cells. Chromatin immunoprecipitation analysis revealed that HIF-2α, but not HIF-1α, bound to the Wnt10b enhancer region as well as upstream of the Wnt1 gene, which is encoded by an antisense strand of the Wnt10b gene. Hypoxia-conditioned medium (H-CM) induced phosphorylation of lipoprotein-receptor-related protein 6 as well as β-catenin-dependent gene expression in normoxic cells, which suggests that H-CM contains canonical Wnt signals. Furthermore, adipogenesis of both human mesenchymal stem cells and mouse preadipocytes was inhibited by H-CM even under normoxic conditions. These results suggest that O2 concentration gradients influence the formation of Wnt ligand gradients, which are involved in the regulation of pluripotency, cell proliferation, and cell differentiation.

Anti-addiction Drug Ibogaine Prolongs the Action Potential in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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Rubi, Lena; Eckert, Daniel; Boehm, Stefan; Hilber, Karlheinz; Koenig, Xaver

2017-04-01

Ibogaine is a plant alkaloid used as anti-addiction drug in dozens of alternative medicine clinics worldwide. Recently, alarming reports of life-threatening cardiac arrhythmias and cases of sudden death associated with the ingestion of ibogaine have accumulated. Using whole-cell patch clamp recordings, we assessed the effects of ibogaine and its main metabolite noribogaine on action potentials in human ventricular-like cardiomyocytes derived from induced pluripotent stem cells. Therapeutic concentrations of ibogaine and its long-lived active metabolite noribogaine significantly retarded action potential repolarization in human cardiomyocytes. These findings represent the first experimental proof that ibogaine application entails a cardiac arrhythmia risk for humans. In addition, they explain the clinically observed delayed incidence of cardiac adverse events several days after ibogaine intake. We conclude that therapeutic concentrations of ibogaine retard action potential repolarization in the human heart. This may give rise to a prolongation of the QT interval in the electrocardiogram and cardiac arrhythmias.

Intermittent hypoxia in obese Zucker rats: cardiometabolic and inflammatory effects.

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Briançon-Marjollet, Anne; Monneret, Denis; Henri, Marion; Joyeux-Faure, Marie; Totoson, Perle; Cachot, Sandrine; Faure, Patrice; Godin-Ribuot, Diane

2016-11-01

What is the central question of this study? This study addresses the relative impact of obesity and intermittent hypoxia in the pathophysiological process of obstructive sleep apnoea by investigating the metabolic, inflammatory and cardiovascular consequences of intermittent hypoxia in lean and obese Zucker rats. What is the main finding and its importance? We found that obesity and intermittent hypoxia have mainly distinct consequences on the investigated inflammatory and cardiometabolic parameters in Zucker rats. This suggests that, for a given severity of sleep apnea, the association of obesity and obstructive sleep apnoea may not necessarily be deleterious. Obstructive sleep apnoea is associated with obesity with a high prevalence, and both co-morbidities are independent cardiovascular risk factors. Intermittent hypoxia (IH) is thought to be the main factor responsible for the obstructive sleep apnoea-related cardiometabolic alterations. The aim of this study was to assess the respective impact of obesity and IH on the inflammatory and cardiometabolic state in rats. Lean and obese Zucker rats were exposed to normoxia or chronic IH, and we assessed metabolic and inflammatory parameters, such as plasma lipids and glucose, serum leptin and adiponectin, liver cytokines, nuclear factor-κB activity and cardiac endothelin-1 levels. Myocardial infarct size was also evaluated following in vitro ischaemia-reperfusion. Circulating lipids, insulin, homeostasis model assessment of insulin resistance (HOMA-IR), leptin and adiponectin levels were higher in obese versus lean rats. Chronic IH did not have a significant impact on metabolic parameters in lean rats. In obese rats, IH increased glycaemia and HOMA-IR. Liver interleukin-6 and tumour necrosis factor-α levels were elevated in lean rats exposed to IH; obesity prevented the increase in interleukin-6 but not in tumour necrosis factor-α. Finally, IH exposure enhanced myocardial sensitivity to infarction in both lean and

Multi-Vitamin B Supplementation Reverses Hypoxia-Induced Tau Hyperphosphorylation and Improves Memory Function in Adult Mice.

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Yu, Lixia; Chen, Yuan; Wang, Weiguang; Xiao, Zhonghai; Hong, Yan

2016-08-04

Hypobaric hypoxia (HH) leads to reduced oxygen delivery to brain. It could trigger cognitive dysfunction and increase the risk of dementia including Alzheimer's disease (AD). The present study was undertaken in order to examine whether B vitamins (B6, B12, folate, and choline) could exert protective effects on hypoxia-induced memory deficit and AD related molecular events in mice. Adult male Kunming mice were assigned to five groups: normoxic control, hypoxic model (HH), hypoxia+vitamin B6/B12/folate (HB), hypoxia+choline (HC), hypoxia+vitamin B6/B12/folate+choline (HBC). Mice in the hypoxia, HB, HC, and HBC groups were exposed to hypobaric hypoxia for 8 h/day for 28 days in a decompression chamber mimicking 5500 meters of high altitude. Spatial and passive memories were assessed by radial arm and step-through passive test, respectively. Levels of tau and glycogen synthase kinase (GSK)-3β phosphorylation were detected by western blot. Homocysteine (Hcy) concentrations were determined using enzymatic cycling assay. Mice in the HH group exhibited significant spatial working and passive memory impairment, increased tau phosphorylation at Thr181, Ser262, Ser202/Thr205, and Ser396 in the cortex and hippocampus, and elevated Hcy levels compared with controls. Concomitantly, the levels of Ser9-phosphorylated GSK-3β were significantly decreased in brain after hypoxic treatment. Supplementations of vitamin B6/B12/folate+choline could significantly ameliorate the hypoxia-induced memory deficits, observably decreased Hcy concentrations in serum, and markedly attenuated tau hyperphosphorylation at multiple AD-related sites through upregulating inhibitory Ser9-phosphorylated GSK-3β. Our finding give further insight into combined neuroprotective effects of vitamin B6, B12, folate, and choline on brain against hypoxia.

Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression

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Makino, Yuichi; Cao, Renhai; Svensson, Kristian; Bertilsson, Göran; Asman, Mikael; Tanaka, Hirotoshi; Cao, Yihai; Berkenstam, Anders; Poellinger, Lorenz

2001-11-01

Alteration of gene expression is a crucial component of adaptive responses to hypoxia. These responses are mediated by hypoxia-inducible transcription factors (HIFs). Here we describe an inhibitory PAS (Per/Arnt/Sim) domain protein, IPAS, which is a basic helix-loop-helix (bHLH)/PAS protein structurally related to HIFs. IPAS contains no endogenous transactivation function but demonstrates dominant negative regulation of HIF-mediated control of gene expression. Ectopic expression of IPAS in hepatoma cells selectively impairs induction of genes involved in adaptation to a hypoxic environment, notably the vascular endothelial growth factor (VEGF) gene, and results in retarded tumour growth and tumour vascular density in vivo. In mice, IPAS was predominantly expressed in Purkinje cells of the cerebellum and in corneal epithelium of the eye. Expression of IPAS in the cornea correlates with low levels of expression of the VEGF gene under hypoxic conditions. Application of an IPAS antisense oligonucleotide to the mouse cornea induced angiogenesis under normal oxygen conditions, and demonstrated hypoxia-dependent induction of VEGF gene expression in hypoxic corneal cells. These results indicate a previously unknown mechanism for negative regulation of angiogenesis and maintenance of an avascular phenotype.

Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

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Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling

2015-01-01

Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H 2 O 2 production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling

Rcan1-1L overexpression induces mitochondrial autophagy and improves cell survival in angiotensin II-exposed cardiomyocytes

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Duan, Hongyan; Li, Yongqiang; Yan, Lijie; Yang, Haitao; Wu, Jintao; Qian, Peng; Li, Bing; Wang, Shanling, E-mail: shanglingwang@126.com

2015-07-01

Mitochondrial autophagy is an important adaptive stress response and can be modulated by various key molecules. A previous study found that the regulator of calcineurin 1-1L (Rcan1-1L) may regulate mitochondrial autophagy and cause mitochondria degradation in neurocytes. However, the effect of Rcan1-1L on cardiomyocytes has not been determined. In the present study, we aimed to investigate the role of Rcan1-1L in angiotensin II (Ang II)-exposed human cardiomyocytes. Above all, Human adult cardiac myocytes (HACMs) were exposed to 200 nmol/L Ang II for 4 days. Enhanced H{sub 2}O{sub 2} production, cytochrome C release and mitochondrial permeability were observed in these cells, which were blocked by valsartan. Consistently, Ang II exposure significantly reduced cardiomyocyte viability. However, transfection of Rcan1-1L vector promoted cell viability and ameliorated the apoptosis caused by Ang II. Rcan1-1L clearly promoted mitochondrial autophagy in HACMs, with elevated autophagy protein (ATG) 5 and light chain 3 (LC3) expression. Transient mitochondrial biogenesis and reduced cytochrome C release was also induced by Rcan1-1L. Additionally, Rcan1-1L significantly inhibited calcineurin/nuclear factor of activated T cells (NFAT) signaling. We thus conclude that Rcan1-1L may play a protective role in Ang II-treated cardiomyocytes through the induction of mitochondrial autophagy, and may be an alternative method of cardiac protection. - Highlights: • Transfection of Rcan1-1L into HACMs promoted cell viability and reduced apoptosis. • Transfection of Rcan1-1L promoted mitochondrial autophagy in HACMs. • Rcan1-1L inhibited the calcineurin/nuclear factor of activated T cells signaling.

Neuroprotective Role of Intermittent Hypobaric Hypoxia in Unpredictable Chronic Mild Stress Induced Depression in Rats

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Deep, Satayanarayan; Prasad, Dipti; Singh, Shashi Bala; Khan, Nilofar

2016-01-01

Hypoxic exposure results in several pathophysiological conditions associated with nervous system, these include acute and chronic mountain sickness, loss of memory, and high altitude cerebral edema. Previous reports have also suggested the role of hypoxia in pathogenesis of depression and related psychological conditions. On the other hand, sub lethal intermittent hypoxic exposure induces protection against future lethal hypoxia and may have beneficial effect. Therefore, the present study was designed to explore the neuroprotective role of intermittent hypobaric hypoxia (IHH) in Unpredictable Chronic Mild Stress (UCMS) induced depression like behaviour in rats. The IHH refers to the periodic exposures to hypoxic conditions interrupted by the normoxic or lesser hypoxic conditions. The current study examines the effect of IHH against UCMS induced depression, using elevated plus maze (EPM), open field test (OFT), force swim test (FST), as behavioural paradigm and related histological and molecular approaches. The data indicated the UCMS induced depression like behaviour as evident from decreased exploration activity in OFT with increased anxiety levels in EPM, and increased immobility time in the FST; whereas on providing the IHH (5000m altitude, 4hrs/day for two weeks) these behavioural changes were ameliorated. The morphological and molecular studies also validated the neuroprotective effect of IHH against UCMS induced neuronal loss and decreased neurogenesis. Here, we also explored the role of Brain-Derived Neurotrophic Factor (BDNF) in anticipatory action of IHH against detrimental effect of UCMS as upon blocking of BDNF-TrkB signalling the beneficial effect of IHH was nullified. Taken together, the findings of our study demonstrate that the intermittent hypoxia has a therapeutic potential similar to an antidepressant in animal model of depression and could be developed as a preventive therapeutic option against this pathophysiological state. PMID:26901349

Neuroprotective Role of Intermittent Hypobaric Hypoxia in Unpredictable Chronic Mild Stress Induced Depression in Rats.

Directory of Open Access Journals (Sweden)

Neetu Kushwah

Full Text Available Hypoxic exposure results in several pathophysiological conditions associated with nervous system, these include acute and chronic mountain sickness, loss of memory, and high altitude cerebral edema. Previous reports have also suggested the role of hypoxia in pathogenesis of depression and related psychological conditions. On the other hand, sub lethal intermittent hypoxic exposure induces protection against future lethal hypoxia and may have beneficial effect. Therefore, the present study was designed to explore the neuroprotective role of intermittent hypobaric hypoxia (IHH in Unpredictable Chronic Mild Stress (UCMS induced depression like behaviour in rats. The IHH refers to the periodic exposures to hypoxic conditions interrupted by the normoxic or lesser hypoxic conditions. The current study examines the effect of IHH against UCMS induced depression, using elevated plus maze (EPM, open field test (OFT, force swim test (FST, as behavioural paradigm and related histological and molecular approaches. The data indicated the UCMS induced depression like behaviour as evident from decreased exploration activity in OFT with increased anxiety levels in EPM, and increased immobility time in the FST; whereas on providing the IHH (5000m altitude, 4hrs/day for two weeks these behavioural changes were ameliorated. The morphological and molecular studies also validated the neuroprotective effect of IHH against UCMS induced neuronal loss and decreased neurogenesis. Here, we also explored the role of Brain-Derived Neurotrophic Factor (BDNF in anticipatory action of IHH against detrimental effect of UCMS as upon blocking of BDNF-TrkB signalling the beneficial effect of IHH was nullified. Taken together, the findings of our study demonstrate that the intermittent hypoxia has a therapeutic potential similar to an antidepressant in animal model of depression and could be developed as a preventive therapeutic option against this pathophysiological state.

p53 dependent apoptotic cell death induces embryonic malformation in Carassius auratus under chronic hypoxia.

Directory of Open Access Journals (Sweden)

Paramita Banerjee Sawant

Full Text Available Hypoxia is a global phenomenon affecting recruitment as well as the embryonic development of aquatic fauna. The present study depicts hypoxia induced disruption of the intrinsic pathway of programmed cell death (PCD, leading to embryonic malformation in the goldfish, Carrasius auratus. Constant hypoxia induced the early expression of pro-apoptotic/tumor suppressor p53 and concomitant expression of the cell death molecule, caspase-3, leading to high level of DNA damage and cell death in hypoxic embryos, as compared to normoxic ones. As a result, the former showed delayed 4 and 64 celled stages and a delay in appearance of epiboly stage. Expression of p53 efficiently switched off expression of the anti-apoptotic Bcl-2 during the initial 12 hours post fertilization (hpf and caused embryonic cell death. However, after 12 hours, simultaneous downregulation of p53 and Caspase-3 and exponential increase of Bcl-2, caused uncontrolled cell proliferation and prevented essential programmed cell death (PCD, ultimately resulting in significant (p<0.05 embryonic malformation up to 144 hpf. Evidences suggest that uncontrolled cell proliferation after 12 hpf may have been due to downregulation of p53 abundance, which in turn has an influence on upregulation of anti-apoptotic Bcl-2. Therefore, we have been able to show for the first time and propose that hypoxia induced downregulation of p53 beyond 12 hpf, disrupts PCD and leads to failure in normal differentiation, causing malformation in gold fish embryos.

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

Hif1a inactivation rescues photoreceptor degeneration induced by a chronic hypoxia-like stress.

Science.gov (United States)

Barben, Maya; Ail, Divya; Storti, Federica; Klee, Katrin; Schori, Christian; Samardzija, Marijana; Michalakis, Stylianos; Biel, Martin; Meneau, Isabelle; Blaser, Frank; Barthelmes, Daniel; Grimm, Christian

2018-04-17

Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients.

Hypoxia-inducible factor 1–mediated characteristic features of cancer cells for tumor radioresistance

International Nuclear Information System (INIS)

Harada, Hiroshi

2016-01-01

Tumor hypoxia has been attracting increasing attention in the fields of radiation biology and oncology since Thomlinson and Gray detected hypoxic cells in malignant solid tumors and showed that they exert a negative impact on the outcome of radiation therapy. This unfavorable influence has, at least partly, been attributed to cancer cells acquiring a radioresistant phenotype through the activation of the transcription factor, hypoxia-inducible factor 1 (HIF-1). On the other hand, accumulating evidence has recently revealed that, even though HIF-1 is recognized as an important regulator of cellular adaptive responses to hypoxia, it may not become active and induce tumor radioresistance under hypoxic conditions only. The mechanisms by which HIF-1 is activated in cancer cells not only under hypoxic conditions, but also under normoxic conditions, through cancer-specific genetic alterations and the resultant imbalance in intermediate metabolites have been summarized herein. The relevance of the HIF-1–mediated characteristic features of cancer cells, such as the production of antioxidants through reprogramming of the glucose metabolic pathway and cell cycle regulation, for tumor radioresistance has also been reviewed

Ebselen by modulating oxidative stress improves hypoxia-induced macroglial Müller cell and vascular injury in the retina.

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Tan, Sih Min; Deliyanti, Devy; Figgett, William A; Talia, Dean M; de Haan, Judy B; Wilkinson-Berka, Jennifer L

2015-07-01

Oxidative stress is an important contributor to glial and vascular cell damage in ischemic retinopathies. We hypothesized that ebselen via its ability to reduce reactive oxygen species (ROS) and augment nuclear factor-like 2 (Nrf2) anti-oxidants would attenuate hypoxia-induced damage to macroglial Müller cells and also lessen retinal vasculopathy. Primary cultures of rat Müller cells were exposed to normoxia (21% O2), hypoxia (0.5% O2) and ebselen (2.5 μM) for up to 72 h. Oxygen-induced retinopathy (OIR) was induced in C57BL/6J mice while control mice were housed in room air. Mice received vehicle (saline, 5% dimethyl sulfoxide) or ebselen (10 mg/kg) each day between postnatal days 6-18. In cultured Müller cells, flow cytometry for dihydroethidium revealed that ebselen reduced the hypoxia-induced increase in ROS levels, whilst increasing the expression of Nrf2-regulated anti-oxidant genes, heme oxygenase 1, glutathione peroxidase-1, NAD(P)H dehydrogenase quinone oxidoreductase 1 and glutamate-cysteine ligase. Moreover, in Müller cells, ebselen reduced the hypoxia-induced increase in protein levels of pro-angiogenic and pro-inflammatory factors including vascular endothelial growth factor, interleukin-6, monocyte chemoattractant-protein 1 and intercellular adhesion molecule-1, and the mRNA levels of glial fibrillary acidic protein (GFAP), a marker of Müller cell injury. Ebselen improved OIR by attenuating capillary vaso-obliteration and neovascularization and a concomitant reduction in Müller cell gliosis and GFAP. We conclude that ebselen protects against hypoxia-induced injury of retinal Müller cells and the microvasculature, which is linked to its ability to reduce oxidative stress, vascular damaging factors and inflammation. Agents such as ebselen may be potential treatments for retinopathies that feature oxidative stress-mediated damage to glia and the microvasculature. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hypoxia induced expression of endogenous markers in vitro is highly influenced by pH

International Nuclear Information System (INIS)

Sorensen, Brita Singers; Alsner, Jan; Overgaard, Jens; Horsman, Michael R.

2007-01-01

Background: Genes such as carbonic anhydrase IX (Ca9), glucose transporter 1 (Glut1), lactate dehydrogenase A (LDH-A), osteopontin (OPN) and lysyl oxidase (LOX) have been suggested as hypoxic markers, but inconsistent results suggest that factors other than oxygen influence their expression. The current study is a detailed investigation using a range of pH values from 6.3 to 7.5 in two human cell lines to establish the pH dependency of hypoxia induced gene expression. Methods: Human tumour cell lines (uterine cervix squamous cell carcinoma (SiHa) and pharyngeal squamous cell carcinoma [FaDu DD ]) were used. Hypoxia was induced by gassing cells in airtight chambers with various oxygen concentrations (21%, 1%, 0.1%, 0.01% and 0%) for up to 24 h. The media were titrated to a range of pH values (7.5, 7.0, 6.7, 6.5 and 6.3). Gene expression was determined by real-time PCR. Results: In both SiHa and FaDu DD cells Ca9 and LOX reached the highest level of expression at 1% oxygen. In FaDu DD cells, a pH of 6.5 had a medium suppression effect on the hypoxia induced expression of Ca9. pH 6.3 resulted in severe suppression of expression for Ca9 and LOX in both SiHa and FaDu DD . Glut1 and LDH-A had a similar expression pattern to each other, with a maximum expression at 0.01% oxygen, in both cell lines. For these genes pH 6.5 and 6.3 changed the expression pattern in SiHa cells. OPN was up regulated at low oxygen in SiHa cells, but was not induced by hypoxia in FaDu DD cells. Conclusion: As tumour hypoxia occurs in a deprived microenvironment, other environmental factors, for example low pH, might interact with the effect of low oxygen concentration on gene expression. This study shows that pH in two cell lines has a profound influence on the oxygen dependent induction of certain endogenous hypoxic markers

17-AAG, a Hsp90 inhibitor, attenuates the hypoxia-induced expression of SDF-1alpha and ILK in mouse RPE cells.

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Wang, Ye Qing; Zhang, Xiao Mei; Wang, Xiao Dan; Wang, Bin Jie; Wang, Wei

2010-03-01

The aim of this study was to investigate the changes of SDF-1alpha and ILK expression in mouse retinal pigment epithelium (RPE) cells in response to hypoxia, and the effect of 17-Allylamino-17-demethoxygeldanamycin (17-AAG), a heat shock protein 90 (Hsp90) inhibitor, on the hypoxia-induced expression of SDF-1alpha and ILK. RPE cells were cultured with 200 micromol/L cobalt chloride (CoCl(2)) for different times (1, 3, 6, 12, 24, 72 h) to imitate chemical hypoxia. Pretreatment of 17-AAG was 1 h prior to hypoxic insult. Cellular viability after 17-AAG treatment was assessed by MTT assay, and the changes of SDF-1alpha and ILK expression were examined by RT-PCR and Western blot. Up-regulation of SDF-1alpha and ILK expression in response to hypoxia was observed. One hour pretreatment of 17-AAG could remarkably decreased the hypoxia-induced SDF-1alpha and ILK expression in vitro. Our results indicated that SDF-1alpha and ILK involved in the hypoxic response of RPE cells, and 1 h pretreatment of 17-AAG had an inhibitive effect on the hypoxia-induced SDF-1alpha and ILK expression.

Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

International Nuclear Information System (INIS)

Tse, Anna Chung-Kwan; Li, Jing-Woei; Chan, Ting-Fung; Wu, Rudolf Shiu-Sun; Lai, Keng-Po

2015-01-01

Highlights: • We demonstrate hypoxia induced miR-210 in ovarian follicular cells. • We show anti-apoptotic roles of miR-210 in ovarian follicular cells under hypoxia. • Apoptotic genes (DLC1, SLK, TNFRSF10B, RBM25, and USP7) are target of miR-210. • MiR-210 is vital for ovarian follicular cells proliferation in response to hypoxia. - Abstract: Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of mi

Hypoxia induces miR-210, leading to anti-apoptosis in ovarian follicular cells of marine medaka Oryzias melastigma

Energy Technology Data Exchange (ETDEWEB)

Tse, Anna Chung-Kwan [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Li, Jing-Woei; Chan, Ting-Fung [School of Life Sciences, Hong Kong Bioinformatics Centre, The Chinese University of Hong Kong, Hong Kong SAR (China); Wu, Rudolf Shiu-Sun [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China); Lai, Keng-Po, E-mail: balllai@hku.hk [School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR (China); State Key Laboratory in Marine Pollution, Hong Kong SAR (China)

2015-08-15

Highlights: • We demonstrate hypoxia induced miR-210 in ovarian follicular cells. • We show anti-apoptotic roles of miR-210 in ovarian follicular cells under hypoxia. • Apoptotic genes (DLC1, SLK, TNFRSF10B, RBM25, and USP7) are target of miR-210. • MiR-210 is vital for ovarian follicular cells proliferation in response to hypoxia. - Abstract: Hypoxia is a major global problem that impairs reproductive functions and reduces the quality and quantity of gametes and the fertilization success of marine fish. Nevertheless, the detailed molecular mechanism underlying hypoxia-induced female reproductive impairment remains largely unknown. There is increasing evidence that miRNA is vital in regulating ovarian functions and is closely associated with female fertility in humans. Certain miRNAs that regulate apoptotic genes can be induced by hypoxia, resulting in cell apoptosis. Using primary ovarian follicular cells of the marine medaka, Oryzias melastigma, as a model, we investigated the response of miR-210 to hypoxic stress in ovarian tissues to see if it would interrupt reproductive functions. A significant induction of miR-210 was found in primary ovarian follicular cells exposed to hypoxia, and gene ontology analysis further highlighted the potential roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis. A number of miR-210 target apoptotic genes, including Deleted in liver cancer 1 protein (DLC1), STE20-like serine/threonine-protein kinase (SLK), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), RNA binding motif protein 25 (RBM25), and Ubiquitin-specific-processing protease 7 (USP7), were identified. We further showed that ectopic expression of miR-210 would result in down-regulation of these apoptotic genes. On the other hand, the inhibition of miR-210 promoted apoptotic cell death and the expression of apoptotic marker – caspase 3 in follicular cells under hypoxic treatment, supporting the regulatory role of mi

TMEM45A is essential for hypoxia-induced chemoresistance in breast and liver cancer cells

International Nuclear Information System (INIS)

Flamant, Lionel; Roegiers, Edith; Pierre, Michael; Hayez, Aurélie; Sterpin, Christiane; De Backer, Olivier; Arnould, Thierry; Poumay, Yves; Michiels, Carine

2012-01-01

Hypoxia is a common characteristic of solid tumors associated with reduced response to radio- and chemotherapy, therefore increasing the probability of tumor recurrence. The aim of this study was to identify new mechanisms responsible for hypoxia-induced resistance in breast cancer cells. MDA-MB-231 and HepG2 cells were incubated in the presence of taxol or etoposide respectively under normoxia and hypoxia and apoptosis was analysed. A whole transcriptome analysis was performed in order to identify genes whose expression profile was correlated with apoptosis. The effect of gene invalidation using siRNA was studied on drug-induced apoptosis. MDA-MB-231 cells incubated in the presence of taxol were protected from apoptosis and cell death by hypoxia. We demonstrated that TMEM45A expression was associated with taxol resistance. TMEM45A expression was increased both in MDA-MB-231 human breast cancer cells and in HepG2 human hepatoma cells in conditions where protection of cells against apoptosis induced by chemotherapeutic agents was observed, i.e. under hypoxia in the presence of taxol or etoposide. Moreover, this resistance was suppressed by siRNA-mediated silencing of TMEM45A. Kaplan Meier curve showed an association between high TMEM45A expression and poor prognostic in breast cancer patients. Finally, TMEM45 is highly expressed in normal differentiated keratinocytes both in vitro and in vivo, suggesting that this protein is involved in epithelial functions. Altogether, our results unravel a new mechanism for taxol and etoposide resistance mediated by TMEM45A. High levels of TMEM45A expression in tumors may be indicative of potential resistance to cancer therapy, making TMEM45A an interesting biomarker for resistance

Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

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Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Sloan, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth W; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

2018-01-05

Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a

Long-term divergent tidal flat benthic community recovery following hypoxia-induced mortality

NARCIS (Netherlands)

Colen, van C.; Montserrat, F.; Vincx, M.; Herman, P.M.J.; Ysebaert, T.; Degraer, S.

2010-01-01

Macrobenthos recovery after hypoxia-induced mass mortality was assessed in an estuarine tidal mudflat during 3 years. During the first 2 years, a Pearson-Rosenberg type of community recovery took place along with the improving bottom water oxygen conditions. After 3 months, spionid polychaetes

Effect of hypoxia-inducible factor 1-alpha (HIF-1α) on proliferation ...

African Journals Online (AJOL)

Jane

2011-07-25

Jul 25, 2011 ... Full Length Research Paper. Effect of hypoxia-inducible factor 1-alpha ... 1Department of Neurosurgery, Ruijin Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200025,. China. 2Department of Neurosurgery, Chaoyang Hospital, Huainan, Anhui, China. 3Department of Neurosurgery ...

Baicalin inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via the AKT/HIF-1α/p27-associated pathway.

Science.gov (United States)

Zhang, Lin; Pu, Zhichen; Wang, Junsong; Zhang, Zhifeng; Hu, Dongmei; Wang, Junjie

2014-05-09

Baicalin, a flavonoid compound purified from the dry roots of Scutellaria baicalensis Georgi, has been shown to possess various pharmacological actions. Previous studies have revealed that baicalin inhibits the growth of cancer cells through the induction of apoptosis. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by enhanced pulmonary artery smooth muscle cell (PASMCs) proliferation and suppressed apoptosis. However, the potential mechanism of baicalin in the regulation of PASMC proliferation and the prevention of cardiovascular diseases remains unexplored. To test the effects of baicalin on hypoxia, we used rats treated with or without baicalin (100 mg·kg⁻¹ each rat) at the beginning of the third week after hypoxia. Hemodynamic and pulmonary pathomorphology data showed that right ventricular systolic pressures (RVSP), the weight of the right ventricle/left ventricle plus septum (RV/LV + S) ratio and the medial width of pulmonary arterioles were much higher in chronic hypoxia. However, baicalin treatment repressed the elevation of RVSP, RV/LV + S and attenuated the pulmonary vascular structure remodeling (PVSR) of pulmonary arterioles induced by chronic hypoxia. Additionally, baicalin (10 and 20 μmol·L⁻¹) treatment suppressed the proliferation of PASMCs and attenuated the expression of hypoxia-inducible factor-α (HIF-α) under hypoxia exposure. Meanwhile, baicalin reversed the hypoxia-induced reduction of p27 and increased AKT/protein kinase B phosphorylation p-AKT both in vivo and in vitro. These results suggested that baicalin could effectively attenuate PVSR and hypoxic pulmonary hypertension.

Cardiomyocytes undergo apoptosis in human immunodeficiency virus cardiomyopathy through mitochondrion- and death receptor-controlled pathways.

Science.gov (United States)

Twu, Cheryl; Liu, Nancy Q; Popik, Waldemar; Bukrinsky, Michael; Sayre, James; Roberts, Jaclyn; Rania, Shammas; Bramhandam, Vishnu; Roos, Kenneth P; MacLellan, W Robb; Fiala, Milan

2002-10-29

We investigated 18 AIDS hearts (5 with and 13 without cardiomyopathy) by using immunocytochemistry and computerized image analysis regarding the roles of HIV-1 proteins and tumor necrosis factor ligands in HIV cardiomyopathy (HIVCM). HIVCM and cardiomyocyte apoptosis were significantly related to each other and to the expression by inflammatory cells of gp120 and tumor necrosis factor-alpha. In HIVCM heart, active caspase 9, a component of the mitochondrion-controlled apoptotic pathway, and the elements of the death receptor-mediated pathway, tumor necrosis factor-alpha and Fas ligand, were expressed strongly on macrophages and weakly on cardiomyocytes. HIVCM showed significantly greater macrophage infiltration and cardiomyocyte apoptosis rate compared with non-HIVCM. HIV-1 entered cultured neonatal rat ventricular myocytes by macropinocytosis but did not replicate. HIV-1- or gp120-induced apoptosis of rat myocytes through a mitochondrion-controlled pathway, which was inhibited by heparin, AOP-RANTES, or pertussis toxin, suggesting that cardiomyocyte apoptosis is induced by signaling through chemokine receptors. In conclusion, in patients with HIVCM, cardiomyocytes die through both mitochondrion- and death receptor-controlled apoptotic pathways.

Enhanceosomes as integrators of hypoxia inducible factor (HIF) and other transcription factors in the hypoxic transcriptional response.

Science.gov (United States)

Pawlus, Matthew R; Hu, Cheng-Jun

2013-09-01

Hypoxia is a prevalent attribute of the solid tumor microenvironment that promotes the expression of genes through posttranslational modifications and stabilization of alpha subunits (HIF1α and HIF2α) of hypoxia-inducible factors (HIFs). Despite significant similarities, HIF1 (HIF1α/ARNT) and HIF2 (HIF2α/ARNT) activate common as well as unique target genes and exhibit different functions in cancer biology. More surprisingly, accumulating data indicates that the HIF1- and/or HIF2-mediated hypoxia responses can be oncogenic as well as tumor suppressive. While the role of HIF in the hypoxia response is well established, recent data support the concept that HIF is necessary, but not sufficient for the hypoxic response. Other transcription factors that are activated by hypoxia are also required for the HIF-mediated hypoxia response. HIFs, other transcription factors, co-factors and RNA poll II recruited by HIF and other transcription factors form multifactorial enhanceosome complexes on the promoters of HIF target genes to activate hypoxia inducible genes. Importantly, HIF1 or HIF2 requires distinct partners in activating HIF1 or HIF2 target genes. Because HIF enhanceosome formation is required for the gene activation and distinct functions of HIF1 and HIF2 in tumor biology, disruption of the HIF1 or HIF2 specific enhanceosome complex may prove to be a beneficial strategy in tumor treatment in which tumor growth is specifically dependent upon HIF1 or HIF2 activity. Copyright © 2013 Elsevier Inc. All rights reserved.

Subchronic inhalation of soluble manganese induces expression of hypoxia-associated angiogenic genes in adult mouse lungs

International Nuclear Information System (INIS)

Bredow, Sebastian; Falgout, Melanie M.; March, Thomas H.; Yingling, Christin M.; Malkoski, Stephen P.; Aden, James; Bedrick, Edward J.; Lewis, Johnnye L.; Divine, Kevin K.

2007-01-01

Although the lung constitutes the major exposure route for airborne manganese (Mn), little is known about the potential pulmonary effects and the underlying molecular mechanisms. Transition metals can mimic a hypoxia-like response, activating the hypoxia inducible factor-1 (HIF-1) transcription factor family. Through binding to the hypoxia-response element (HRE), these factors regulate expression of many genes, including vascular endothelial growth factor (VEGF). Increases in VEGF, an important biomarker of angiogenesis, have been linked to respiratory diseases, including pulmonary hypertension. The objective of this study was to evaluate pulmonary hypoxia-associated angiogenic gene expression in response to exposure of soluble Mn(II) and to assess the genes' role as intermediaries of potential pulmonary Mn toxicity. In vitro, 0.25 mM Mn(II) altered morphology and slowed the growth of human pulmonary epithelial cell lines. Acute doses between 0.05 and 1 mM stimulated VEGF promoter activity up to 3.7-fold in transient transfection assays. Deletion of the HRE within the promoter had no effect on Mn(II)-induced VEGF expression but decreased cobalt [Co(II)]-induced activity 2-fold, suggesting that HIF-1 may not be involved in Mn(II)-induced VEGF gene transcription. Nose-only inhalation to 2 mg Mn(II)/m 3 for 5 days at 6 h/day produced no significant pulmonary inflammation but induced a 2-fold increase in pulmonary VEGF mRNA levels in adult mice and significantly altered expression of genes associated with murine angiogenesis. These findings suggest that even short-term exposures to soluble, occupationally relevant Mn(II) concentrations may alter pulmonary gene expression in pathways that ultimately could affect the lungs' susceptibility to respiratory disease

Update on hypoxia-inducible factors and hydroxylases in oxygen regulatory pathways: from physiology to therapeutics

Directory of Open Access Journals (Sweden)

Ratcliffe P

2017-03-01

Full Text Available Peter Ratcliffe,1,2 Peppi Koivunen,3 Johanna Myllyharju,3 Jiannis Ragoussis,4 Judith VMG Bovée,5 Ines Batinic-Haberle,6 Claire Vinatier,7 Valérie Trichet,8 Florence Robriquet,9 Lisa Oliver,9 Betty Gardie9,10 1Target Discovery Institute, University of Oxford, 2The Francis Crick Institute, London, UK; 3Oulu Center for Cell-Matrix Research, Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland; 4McGill University and Genome Quebec Innovation Centre, McGill University, Montreal, Canada; 5Department of Pathology, Leiden University Medical Center, Leiden, the Netherlands; 6Department of Radiation Oncology, Duke University School of Medicine, Durham, NC, USA; 7INSERM UMR 1229, Regenerative Medicine and Skeleton-RMeS, Team STEP, University of Nantes, UFR Odontology, 8UMR 1238 INSERM, Université de Nantes, Faculté de Médecine, 9CRCINA, INSERM, Université de Nantes, Nantes, 10Ecole Pratique des Hautes Etudes, PSL Research University, Paris, France Abstract: The “Hypoxia Nantes 2016” organized its second conference dedicated to the field of hypoxia research. This conference focused on “the role of hypoxia under physiological conditions as well as in cancer” and took place in Nantes, France, in October 6–7, 2016. The main objective of this conference was to bring together a large group of scientists from different spheres of hypoxia. Recent advances were presented and discussed around different topics: genomics, physiology, musculoskeletal, stem cells, microenvironment and cancer, and oxidative stress. This review summarizes the major highlights of the meeting. Keywords: hypoxia, genomics, lipid metabolism, musculoskeletal, cancer, oxidative stress 

Hypoxia-inducible factor 1-alpha up-regulates the expression of phospholipase D2 in colon cancer cells under hypoxic conditions.

Science.gov (United States)

Liu, Maoxi; Du, Kunli; Fu, Zhongxue; Zhang, Shouru; Wu, Xingye

2015-01-01

Hypoxia is a common characteristic of solid tumors. Recent studies confirmed that phospholipase D2 (PLD2) plays significant roles in cancer progression. In this study, correlation between the expression of PLD2 and the change in the protein level of hypoxia-inducible factor 1-alpha (HIF1-α) was studied. Thirty human colon cancer tissues were examined for the expression of HIF1-α and PLD2 protein, and mRNA levels. SW480 and SW620 cells were exposed to normoxia (20 %) or hypoxia (Hypoxic stress induced PLD2 mRNA and protein expression in SW480 and SW620 cells. Cells transfected with HIF1-α siRNA showed attenuation of hypoxia stress-induced PLD2 expression. In vivo growth decreased in response to HIF1-α and PLD2 inhibition. These results suggest that PLD2 expression in colon cancer cells is up-regulated via HIF1-α in response to hypoxic stress and underscores the crucial role of HIF1-α-induced PLD2 in tumor growth.

Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

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Hiroyasu Kamei

2008-08-01

Full Text Available Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

Antenatal hypoxia induces programming of reduced arterial blood pressure response in female rat offspring: role of ovarian function.

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DaLiao Xiao

Full Text Available In utero exposure to adverse environmental factors increases the risk of cardiovascular disease in adulthood. The present study tested the hypothesis that antenatal hypoxia causes a gender-dependent programming of altered arterial blood pressure response (BP in adult offspring. Time-dated pregnant rats were divided into normoxic and hypoxic (10.5% O2 from days 15 to 21 of gestation groups. The experiments were conducted in adult offspring. Antenatal hypoxia caused intrauterine growth restriction, and resulted in a gender-dependent increase Angiotensin II (Ang II-induced BP response in male offspring, but significant decrease in BP response in female offspring. The baroreflex sensitivity was not significantly altered. Consistent with the reduced blood pressure response, antenatal hypoxia significantly decreased Ang II-induced arterial vasoconstriction in female offspring. Ovariectomy had no significant effect in control animals, but significantly increased Ang II-induced maximal BP response in prenatally hypoxic animals and eliminated the difference of BP response between the two groups. Estrogen replacement in ovariectomized animals significantly decreased the BP response to angiotensin II I only in control, but not in hypoxic animals. The result suggests complex programming mechanisms of antenatal hypoxia in regulation of ovary function. Hypoxia-mediated ovary dysfunction results in the phenotype of reduced vascular contractility and BP response in female adult offspring.

Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism

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Voss, Melanie J; Möller, Mischa F; Powe, Desmond G; Niggemann, Bernd; Zänker, Kurt S; Entschladen, Frank

2011-01-01

Some breast cancer patients receiving anti-angiogenic treatment show increased metastases, possibly as a result of induced hypoxia. The effect of hypoxia on tumor cell migration was assessed in selected luminal, post-EMT and basal-like breast carcinoma cell lines. Migration was assessed in luminal (MCF-7), post-EMT (MDA-MB-231, MDA-MB-435S), and basal-like (MDA-MB-468) human breast carcinoma cell lines under normal and oxygen-deprived conditions, using a collagen-based assay. Cell proliferation was determined, secreted cytokine and chemokine levels were measured using flow-cytometry and a bead-based immunoassay, and the hypoxic genes HIF-1α and CA IX were assessed using PCR. The functional effect of tumor-cell conditioned medium on the migration of neutrophil granulocytes (NG) was tested. Hypoxia caused increased migratory activity but not proliferation in all tumor cell lines, involving the release and autocrine action of soluble mediators. Conditioned medium (CM) from hypoxic cells induced migration in normoxic cells. Hypoxia changed the profile of released inflammatory mediators according to cell type. Interleukin-8 was produced only by post-EMT and basal-like cell lines, regardless of hypoxia. MCP-1 was produced by MDA-MB-435 and -468 cells, whereas IL-6 was present only in MDA-MB-231. IL-2, TNF-α, and NGF production was stimulated by hypoxia in MCF-7 cells. CM from normoxic and hypoxic MDA-MB-231 and MDA-MB-435S cells and hypoxic MCF-7 cells, but not MDA-MB-468, induced NG migration. Hypoxia increases migration by the autocrine action of released signal substances in selected luminal and basal-like breast carcinoma cell lines which might explain why anti-angiogenic treatment can worsen clinical outcome in some patients

Functional cardiotoxicity assessment of cosmetic compounds using human-induced pluripotent stem cell-derived cardiomyocytes.

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Chaudhari, Umesh; Nemade, Harshal; Sureshkumar, Poornima; Vinken, Mathieu; Ates, Gamze; Rogiers, Vera; Hescheler, Jürgen; Hengstler, Jan Georg; Sachinidis, Agapios

2018-01-01

There is a large demand of a human relevant in vitro test system suitable for assessing the cardiotoxic potential of cosmetic ingredients and other chemicals. Using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we have already established an in vitro cardiotoxicity assay and identified genomic biomarkers of anthracycline-induced cardiotoxicity in our previous work. Here, five cosmetic ingredients were studied by the new hiPSC-CMs test; kojic acid (KJA), triclosan (TS), triclocarban (TCC), 2,7-naphthalenediol (NPT), and basic red 51 (BR51) based on cytotoxicity as well as ATP assays, beating rate, and genomic biomarkers to determine the lowest observed effect concentration (LOEC) and no observed effect concentration (NOEC). The LOEC for beating rate were 400, 10, 3, >400, and 3 µM for KJA, TS, TCC, NPT, and BR51, respectively. The corresponding concentrations for cytotoxicity or ATP depletion were similar, with the exception of TS and TCC, where the cardiomyocyte-beating assay showed positive results at non-cytotoxic concentrations. Functional analysis also showed that the individual compounds caused different effects on hiPSC-CMs. While exposure to KJA, TS, TCC, and BR51 induced significant arrhythmic beating, NPT slightly decreased cell viability, but did not influence beating. Gene expression studies showed that TS and NPT caused down-regulation of cytoskeletal and cardiac ion homeostasis genes. Moreover, TS and NPT deregulated genomic biomarkers known to be affected also by anthracyclines. The present study demonstrates that hiPSC-CMs can be used to determine LOECs and NOECs in vitro, which can be compared to human blood concentrations to determine margins of exposure. Our in vitro assay, which so far has been tested with several anthracyclines and cosmetics, still requires validation by larger numbers of positive and negative controls, before it can be recommended for routine analysis.

Simulating sleep apnea by exposure to intermittent hypoxia induces inflammation in the lung and liver.

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da Rosa, Darlan Pase; Forgiarini, Luiz Felipe; Baronio, Diego; Feijó, Cristiano Andrade; Martinez, Dênis; Marroni, Norma Possa

2012-01-01

Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH). IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n = 6) or a simulated IH (SIH) (n = 6) for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α), nuclear factor kappa B (NF-κB), and tumor necrosis factor (TNF-α), inducible NO synthase (iNOS), vascular endothelial growth factor (VEGF), and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

Simulating Sleep Apnea by Exposure to Intermittent Hypoxia Induces Inflammation in the Lung and Liver

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Darlan Pase da Rosa

2012-01-01

Full Text Available Sleep apnea is a breathing disorder that results from momentary and cyclic collapse of the upper airway, leading to intermittent hypoxia (IH. IH can lead to the formation of free radicals that increase oxidative stress, and this mechanism may explain the association between central sleep apnea and nonalcoholic steatohepatitis. We assessed the level of inflammation in the lung and liver tissue from animals subjected to intermittent hypoxia and simulated sleep apnea. A total of 12 C57BL/6 mice were divided into two groups and then exposed to IH (n=6 or a simulated IH (SIH (n=6 for 35 days. We observed an increase in oxidative damage and other changes to endogenous antioxidant enzymes in mice exposed to IH. Specifically, the expression of multiple transcription factors, including hypoxia inducible factor (HIF-1α, nuclear factor kappa B (NF-κB, and tumor necrosis factor (TNF-α, inducible NO synthase (iNOS, vascular endothelial growth factor (VEGF, and cleaved caspase 3 were shown to be increased in the IH group. Overall, we found that exposure to intermittent hypoxia for 35 days by simulating sleep apnea leads to oxidative stress, inflammation, and increased activity of caspase 3 in the liver and lung.

Cardiomyocyte-Restricted Deletion of PPARβ/δ in PPARα-Null Mice Causes Impaired Mitochondrial Biogenesis and Defense, but No Further Depression of Myocardial Fatty Acid Oxidation

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Jian Liu

2011-01-01

Full Text Available It is well documented that PPARα and PPARβ/δ share overlapping functions in regulating myocardial lipid metabolism. However, previous studies demonstrated that cardiomyocyte-restricted PPARβ/δ deficiency in mice leads to severe cardiac pathological development, whereas global PPARα knockout shows a benign cardiac phenotype. It is unknown whether a PPARα-null background would alter the pathological development in mice with cardiomyocyte-restricted PPARβ/δ deficiency. In the present study, a mouse model with long-term PPARβ/δ deficiency in PPARα-null background showed a comparably reduced cardiac expression of lipid metabolism to those of single PPAR-deficient mouse models. The PPARα-null background did not rescue or aggravate the cardiac pathological development linked to cardiomyocyte-restricted PPARβ/δ deficiency. Moreover, PPARα-null did not alter the phenotypic development in adult mice with the short-term deletion of PPARβ/δ in their hearts, which showed mitochondrial abnormalities, depressed cardiac performance, and cardiac hypertrophy with attenuated expression of key factors in mitochondrial biogenesis and defense. The present study demonstrates that cardiomyocyte-restricted deletion of PPARβ/δ in PPARα-null mice causes impaired mitochondrial biogenesis and defense, but no further depression of fatty acid oxidation. Therefore, PPARβ/δ is essential for maintaining mitochondrial biogenesis and defense in cardiomyocytes independent of PPARα.

Vagal modulation of high mobility group box-1 protein mediates electroacupuncture-induced cardioprotection in ischemia-reperfusion injury.

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Zhang, Juan; Yong, Yue; Li, Xing; Hu, Yu; Wang, Jian; Wang, Yong-qiang; Song, Wei; Chen, Wen-ting; Xie, Jian; Chen, Xue-mei; Lv, Xin; Hou, Li-li; Wang, Ke; Zhou, Jia; Wang, Xiang-rui; Song, Jian-gang

2015-10-26

Excessive release of high mobility group box-1 (HMGB1) protein from ischemic cardiomyocytes activates inflammatory cascades and enhances myocardial injury after reperfusion. Here we report evidence that electroacupuncture of mice at Neiguan acupoints can inhibit the up-regulation of cardiac HMGB1 following myocardial ischemia and attenuate the associated inflammatory responses and myocardial injury during reperfusion. These benefits of electroacupuncture were partially reversed by administering recombinant HMGB1 to the mice, and further potentiated by administering anti-HMGB1 antibody. Electroacupuncture-induced inhibition of HMGB1 release was markedly reduced by unilateral vagotomy or administration of nicotinic receptor antagonist, but not by chemical sympathectomy. The cholinesterase inhibitor neostigmine mimicked the effects of electroacupuncture on HMGB1 release and myocardial ischemia reperfusion injury. Culture experiments with isolated neonatal cardiomyocytes showed that acetylcholine, but not noradrenaline, inhibited hypoxia-induced release of HMGB1 via a α7nAchR-dependent pathway. These results suggest that electroacupuncture acts via the vagal nerve and its nicotinic receptor-mediated signaling to inhibit HMGB1 release from ischemic cardiomyocytes. This helps attenuate pro-inflammatory responses and myocardial injury during reperfusion.

Simultaneous Assessment of Cardiomyocyte DNA Synthesis and Ploidy: A Method to Assist Quantification of Cardiomyocyte Regeneration and Turnover.

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Richardson, Gavin D

2016-05-23

Although it is accepted that the heart has a limited potential to regenerate cardiomyocytes following injury and that low levels of cardiomyocyte turnover occur during normal ageing, quantification of these events remains challenging. This is in part due to the rarity of the process and the fact that multiple cellular sources contribute to myocardial maintenance. Furthermore, DNA duplication within cardiomyocytes often leads to a polyploid cardiomyocyte and only rarely leads to new cardiomyocytes by cellular division. In order to accurately quantify cardiomyocyte turnover discrimination between these processes is essential. The protocol described here employs long term nucleoside labeling in order to label all nuclei which have arisen as a result of DNA replication and cardiomyocyte nuclei identified by utilizing nuclei isolation and subsequent PCM1 immunolabeling. Together this allows the accurate and sensitive identification of the nucleoside labeling of the cardiomyocyte nuclei population. Furthermore, 4',6-diamidino-2-phenylindole labeling and analysis of nuclei ploidy, enables the discrimination of neo-cardiomyocyte nuclei from nuclei which have incorporated nucleoside during polyploidization. Although this method cannot control for cardiomyocyte binucleation, it allows a rapid and robust quantification of neo-cardiomyocyte nuclei while accounting for polyploidization. This method has a number of downstream applications including assessing the potential therapeutics to enhance cardiomyocyte regeneration or investigating the effects of cardiac disease on cardiomyocyte turnover and ploidy. This technique is also compatible with additional downstream immunohistological techniques, allowing quantification of nucleoside incorporation in all cardiac cell types.

Hypoxia-Inducible Factor Pathway Inhibition Resolves Tumor Hypoxia and Improves Local Tumor Control After Single-Dose Irradiation

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Helbig, Linda; Koi, Lydia; Brüchner, Kerstin; Gurtner, Kristin; Hess-Stumpp, Holger; Unterschemmann, Kerstin; Pruschy, Martin

2014-01-01

Purpose: To study the effects of BAY-84-7296, a novel orally bioavailable inhibitor of mitochondrial complex I and hypoxia-inducible factor 1 (HIF-1) activity, on hypoxia, microenvironment, and radiation response of tumors. Methods and Materials: UT-SCC-5 and UT-SCC-14 human squamous cell carcinomas were transplanted subcutaneously in nude mice. When tumors reached 4 mm in diameter BAY-84-7296 (Bayer Pharma AG) or carrier was daily administered to the animals. At 7 mm tumors were either excised for Western blot and immunohistologic investigations or were irradiated with single doses. After irradiation animals were randomized to receive BAY-84-7296 maintenance or carrier. Local tumor control was evaluated 150 days after irradiation, and the dose to control 50% of tumors (TCD 50 ) was calculated. Results: BAY-84-7296 decreased nuclear HIF-1α expression. Daily administration of inhibitor for approximately 2 weeks resulted in a marked decrease of pimonidazole hypoxic fraction in UT-SCC-5 (0.5% vs 21%, P 50 , with an enhancement ratio of 1.37 (95% confidence interval [CI] 1.13-1.72) in UT-SCC-5 and of 1.55 (95% CI 1.26-1.94) in UT-SCC-14. BAY-84-7296 maintenance after irradiation did not further decrease TCD 50 . Conclusions: BAY-84-7296 resulted in a marked decrease in tumor hypoxia and substantially reduced radioresistance of tumor cells with the capacity to cause a local recurrence after irradiation. The data suggest that reduction of cellular hypoxia tolerance by BAY-84-7296 may represent the primary biological mechanism underlying the observed enhancement of radiation response. Whether this mechanism contributes to the improved outcome of fractionated chemoradiation therapy warrants further investigation

Low sodium intake does not impair renal compensation of hypoxia-induced respiratory alkalosis.

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Höhne, Claudia; Boemke, Willehad; Schleyer, Nora; Francis, Roland C; Krebs, Martin O; Kaczmarczyk, Gabriele

2002-05-01

Acute hypoxia causes hyperventilation and respiratory alkalosis, often combined with increased diuresis and sodium, potassium, and bicarbonate excretion. With a low sodium intake, the excretion of the anion bicarbonate may be limited by the lower excretion rate of the cation sodium through activated sodium-retaining mechanisms. This study investigates whether the short-term renal compensation of hypoxia-induced respiratory alkalosis is impaired by a low sodium intake. Nine conscious, tracheotomized dogs were studied twice either on a low-sodium (LS = 0.5 mmol sodium x kg body wt-1 x day-1) or high-sodium (HS = 7.5 mmol sodium x kg body wt-1 x day-1) diet. The dogs breathed spontaneously via a ventilator circuit during the experiments: first hour, normoxia (inspiratory oxygen fraction = 0.21); second to fourth hour, hypoxia (inspiratory oxygen fraction = 0.1). During hypoxia (arterial PO2 34.4 +/- 2.1 Torr), plasma pH increased from 7.37 +/- 0.01 to 7.48 +/- 0.01 (P respiratory alkalosis was not impaired by a low sodium intake. The increased sodium excretion during hypoxia seems to be combined with a decrease in plasma aldosterone and angiotensin II in LS as well as in HS dogs. Other factors, e.g., increased mean arterial blood pressure, minute ventilation, and renal blood flow, may have contributed.

Hypoxia Inducible Factor-1α (HIF-1 α and its Role in Tumour Progression to Malignancy