Category: Moms

Autophagy and hypoxia

Autophagy and hypoxia

Autophagy and hypoxia 54, — The above Immunity-boosting exercise suggested that the autophagy activation in IH htpoxia be hjpoxia double-edged sword: pro-survival Autophqgy the proliferative stage, while pro-death Autophgy the involuting stage. Based Autophagy and hypoxia the severity and duration of hypoxia, each HIF and non-HIFmediated response can involve multiple alternative pathways such as apoptosis and autophagy among others, to promote hypoxia resistance. Clinical Medicine. Oncotarget 7, — At early steps during autophagy induction, active ULK1 regulates the recruitment of Atg14L-containing vacuolar sorting protein 34 VPS34 complex 13 Reciprocal influence of the p53 and the hypoxic pathways. Autophagy and hypoxia

Autophagy and hypoxia -

PCR cycling conditions were as follows: 42°C for 10 min and 95°C for 20 sec for the reverse transcription, and 95°C for 10 sec and 60°C for 30 sec for the PCR reaction, repeated for 35 cycles. For the quantitative analysis of the mRNA expression of Beclin 1, autophagy-related gene 5 Atg5 and Atg7, qPCR was conducted using a LightCycler ® system Roche Diagnostics GmbH, Mannheim, Germany ; 1 µg RNA per sample was converted to cDNA and used for qPCR.

The Beclin 1 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. sc; , Santa Cruz, CA, USA , and the Atg5 cat. Danvers, MA USA. Cell extracts were prepared by a standard protocol using Cell Lysis Buffer Cell Signaling Technology, Inc.

Goat anti-mouse IgG or goat anti-rabbit IgG Pierce Biotechnology, Inc. Cell viability was determined using an MTT assay Thermo Fisher Scientific, Inc. At 24 h after the treatment, the incubation medium in the test wells was replaced with 50 µl 1X MTT solution, and the cells were incubated for 2 h at 37°C.

Following incubation, the MTT solution was discarded, and µl dimethylsulfoxide DMSO was added to dissolve the precipitate completely at room temperature.

The optical density was then measured at nm using a spectrophotometer. All experiments were performed in three separate experiments. For GFP-LC3 dot number analysis, relative mRNA expression of Beclin 1, Atg5 and Atg7 to β-actin, and MTT measurements, data are presented as the mean ± standard error.

Data were analyzed by Student's t-test using GraphPad Prism 5 GraphPad Software, Inc. H9c2 cells were transfected with GFP-LC3, a biomarker for autophagy, and exposed to hypoxic conditions for 24 h.

LC3, which comprises the two isoforms LC3-I and LC3-II, typically exhibits diffuse cytosolic distribution. Representative fluorescence images, shown in Fig. The ultrastructures of the H9c2 cells with hypoxia or with rapamycin treatment were observed using electron microscopy, and prominent features of cells with hypoxia or rapamycin treatment were found in the form of autophagic vacuoles and autolysosomes in the cytoplasm data not shown.

Hypoxia induces autophagy in cardiomyocytes. A-C The cardiomyocytes were transfected with a plasmid that expressed a GFP-LC3 fusion protein. Following fixation, the cells were immediately visualized using fluorescence microscopy. The data were normally distributed and were statistically analyzed using the Student's t-test.

Experiments were independently performed in triplicate. GFP, green fluorescent protein; LC3, microtubule-associated protein 1 light chain 3; rapa, rapamycin. When autophagy is activated, the LC3-I protein localized in the cytoplasm is cleaved, converted into LC3-II and inserted into autophagosome membranes As part of a type III phosphoinositide-3 kinase complex, the autophagy gene Beclin 1 is required for the formation of the autophagic vesicles In addition, ATG products, such as Atg5 and Atg7, play essential roles in autophagy.

As shown in Fig. Notably, these results indicate that hypoxia and rapamycin treatment induce autophagy in H9c2 cells.

Hypoxia promotes the conversion of LC3-I to LC3-II and upregulates the expression of Atg5, Atg7 and Beclin 1 in cardiocmyocytes.

The total cell mRNA was analyzed using a reverse transcription quantitative polymerase chain reaction. All results were independently repeated three times.

LC3, microtubule-associated protein 1 light chain 3; Atg, autophagy-related gene. To reveal the precise mechanisms underlying the activation of autophagy in H9c2 cells, the change in HIF-1α expression was further investigated with a pcDNA3.

HIF-1, as a key transcription factor, plays a pivotal role in hypoxia. The HIF-1 overexpression with eukaryotic plasmid or HIF-1 knockdown with siRNA was induced in the H9c2 cells. Furthermore, HIF-1 overexpression markedly aggravated the transformation of LC3-I to LC3-II Fig.

By contrast, transfection with HIF-1 siRNA, which blocked HIF-1 expression, significantly prevented LC3-II production, as well as the expression of Atg5, Atg7 and Beclin 1 Fig.

The results therefore showed that the hypoxia-induced autophagy in H9c2 cells was dependent on HIF-1α. In combination, the results suggest that the HIF-1 pathway regulates the activation of autophagy in H9c2 cells under the hypoxic condition.

Hypoxia induces autophagy in cardiomyocytes through the HIF-1α pathway. A-D The cardiomyocytes were transfected with a plasmid that expressed a GFP-LC3 fusion protein and A pcDNA3. Following fixation, cells were immediately visualized using fluorescence microscopy.

E and F Following transfection with pcDNA-HIF-1α or HIF-1α siRNA for 24 h, the cells were lysed and subjected to western blotting with the antibodies indicated. All results are averaged for three independent experiments.

HIF-1α, hypoxia-inducible factor 1α; GFP, green fluorescent protein; LC3, microtubule-associated protein 1 light chain 3; siRNA, small interfering RNA; Atg, autophagy-related gene. The initial experiments demonstrated that HIF-1 induced significant autophagy and high expression levels of autophagy-associated molecules in H9c2 cells.

To further determine the effect of HIF-1α-mediated autophagy under hypoxia on cell viability, the MTT assay was conducted for H9c2 cells under several conditions: Hypoxia, normoxia, post-rapamycin treatment, HIF-1α overexpression and HIF-1α knockdown.

To further investigate the role of HIF-1α in the hypoxia-induced cell viability reduction, the viability of H9c2 cells was re-examined with HIF-1α overexpression or knockdown.

In combination, these results suggest that HIF-1α-induced autophagy attenuates the hypoxia-reduced viability of H9c2 cells. Viability of H9c2 cells under hypoxia or following HIF-1α expression regulation.

A Viability of H9c2 cells under normoxia, hypoxia or under hypoxia and following nM rapamycin treatment. B and C Viability of H9c2 cells following HIF-1α overexpression or knockdown, B under normoxia or C hypoxia. Normal H9c2 cells were taken as a control.

All results are representative of four independent experiments. Furthermore, Macrophage autophagy has been indicated to play a protective role in advanced atherosclerosis in a mouse model 45 ; however, the protective role of autophagy in cardiomyocytes under hypoxia has not yet been elucidated.

In the present study, increased autophagic activity was detected in cardiomyocytes under hypoxia. To the best of our knowledge, this is the first study to report the effects of hypoxia on autophagy induction in cardiomyocytes.

Several approaches were adopted to determine the levels of autophagy, including the analysis of fluorescent LC3-GFP dots and the mRNA and protein expression levels of autophagy-associated molecules.

Cardiomyocytes were exposed to hypoxia, and an evident increase was found in the number of autophagic vacuoles and autolysosomes in the cytoplasm, suggesting enhanced autophagy formation. The occurrence of autophagy was also confirmed with rapamycin treatment in the H9c2 cell model. The analysis of autophagy-associated molecule levels confirmed the induction of autophagy by hypoxia or rapamycin: The expression of LC3-II increased in cells post-hypoxia or -rapamycin treatment, and the Beclin 1, Atg5 and Atg7 expression increased in cardiomyocytes post-hypoxia or -rapamycin treatment at both the mRNA and protein levels.

In summary, autophagy could be induced by hypoxia in cardiomyocytes in vitro. HIF-1 is a transcription factor that is essential in the regulation of gene expression to maintain oxygen homeostasis HIF-1 has been demonstrated to coordinate adaptive responses to hypoxia at both the cellular and systemic levels 18 , 47 , Furthermore, HIF-1 has been shown to regulate the expression of hundreds of target genes involved in angiogenesis, erythropoiesis, metabolism, autophagy and other adaptive responses to hypoxia To elucidate the mechanisms by which cardiomyocytes respond to hypoxia, an HIF-1α overexpression plasmid and HIF-1α siRNA were used to manipulate the HIF-1α expression and to investigate the regulatory role of HIF-1α in hypoxia-induced autophagy.

The results demonstrated that the hypoxia-induced autophagy in cardiomyocytes involved the HIF-1 pathway. A significant increase in autophagy-specific autolysosomes was observed via the LC3-GFP reporter vector under a fluorescence microscope in HIF-1α-treated cells.

By contrast, HIF-1α knockdown with siRNA treatment led to considerably less autolysosome formation in the cardiomyocytes. Furthermore, the cleavage and recruitment of LC3 to autophagosomes, as well as the expression of autophagy-associated molecules, were enhanced by HIF-1α overexpression and attenuated by HIF-1α knockdown.

In combination, these results indicated that the HIF-1 pathway was involved in hypoxia-induced autophagy in cardiomyocytes. Hypoxia treatment and HIF-1α overexpression were both found to induce autophagy; however, their effects on cell viability differed.

Hypoxia appeared to reduce the cell viability, though accompanied by HIF-1α stimulating, whereas HIF-1α could significantly inhibit the reduction of cardiomyocyte viability.

However, the overexpression of HIF-1α ameliorated the reduction in cell viability induced by hypoxia, while the downregulation of HIF-1α enhanced the hypoxia-induced cell viability reduction.

The autophagy induced by HIF-1 may, therefore, facilitate cardiomyocytes to overcome the hypoxic injury and increase survival. In conclusion, the results of the present study suggest that hypoxia can induce autophagy in cardiomyocytes through activation of the HIF-1 pathway.

HIF-1α upregulation can increase autophagy and ameliorate the hypoxia-induced cell viability reduction. The present study presents a promising foundation for the development of methods to prevent hypoxic-ischemic injury to cardiomyocytes.

The present study was supported by a grant from the Inner Mongolia Department of Education Project no.

NJZY Gautier-Veyret E, Arnaud C, Bäck M, Pépin JL, Petri MH, Baguet JP, Tamisier R, Lévy P and Stanke-Labesque F: Intermittent hypoxia-activated cyclooxygenase pathway: Role in atherosclerosis.

Eur Respir J. Bovill EG and van der Vliet A: Venous valvular stasis-associated hypoxia and thrombosis: What is the link? Annu Rev Physiol. Circ Res. Talukder MA, Elnakish MT, Yang F, Nishijima Y, Alhaj MA, Velayutham M, Hassanain HH and Zweier JL: Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury.

Am J Physiol Heart Circ Physiol. Goldhaber JI and Weiss JN: Oxygen free radicals and cardiac reperfusion abnormalities. Cheng TO: Coronary heart disease in China. Hosp Med. Barth J, Schneider S and von Känel R: Lack of social support in the etiology and the prognosis of coronary heart disease: A systematic review and meta-analysis.

Psychosom Med. Ostádal B: Myocardial ischemic injury and protection. Exp Clin Cardiol. Ost'ádal B, Ost'ádalova I, Skárka L, Kolár F and Kopecký J: Ischemic injury of the developing heart.

Tong W, Xiong F, Li Y and Zhang L: Hypoxia inhibits cardiomyocyte proliferation in fetal rat hearts via upregulating TIMP Am J Physiol Regul Integr Comp Physiol. Botting KJ, McMillen IC, Forbes H, Nyengaard JR and Morrison JL: Chronic hypoxemia in late gestation decreases cardiomyocyte number but does not change expression of hypoxia-responsive genes.

J Am Heart Assoc. Ramjiawan A, Bagchi RA, Blant A, Albak L, Cavasin MA, Horn TR, McKinsey TA and Czubryt MP: Roles of histone deacetylation and AMP kinase in regulation of cardiomyocyte PGC-1α gene expression in hypoxia. Am J Physiol Cell Physiol. Muraguchi T, Kawawa A and Kubota S: Prohibitin protects against hypoxia-induced H9c2 cardiomyocyte cell death.

Biomed Res. Clin Exp Pharmacol Physiol. Shohet RV and Garcia JA: Keeping the engine primed: HIF factors as key regulators of cardiac metabolism and angiogenesis during ischemia.

J Mol Med Berl. Taylor CT: Mitochondria and cellular oxygen sensing in the HIF pathway. Biochem J. Semenza GL: Hypoxia-inducible factor 1: Regulator of mitochondrial metabolism and mediator of ischemic preconditioning.

Biochim Biophys Acta. Mol Neurobiol 37 1 :7— Bras M, Queenan B, Susin SA Programmed cell death via mitochondria: different modes of dying. Biochem Mosc — Article CAS Google Scholar. Bellot G, Garcia-Medina R, Gounon P, Chiche J, Roux D, Pouysségur J, Mazure NM Hypoxia-induced autophagy is mediated through hypoxia-inducible factor induction of BNIP3 and BNIP3L via their BH3 domains.

Mol Cell Biol — Article PubMed Central CAS PubMed Google Scholar. Mazure MN, Pouyssegur J Hypoxia-induced autophagy: cell death or cell survival? Curr Opin Cell Biol — Mariño G, Niso-Santano M, Baehrecke EH, Kroemer G Self-consumption: the interplay of autophagy and apoptosis.

Nat Rev Mol Cell Biol — Article PubMed Central PubMed Google Scholar. Xu M, Zhang HL Death and survival of neuronal and astrocytic cells in ischemic brain injury: a role of autophagy. Acta Pharmacol Sin — Xia DY, Li W, Qian HR, Yao S, Liu JG, Qi XK Ischemia preconditioning is neuroprotective in a rat cerebral ischemic injury model through autophagy activation and apoptosis inhibition.

Braz J Med Biol Res — Kuma A, Hatano M, Matsui M, Yamamoto A, Nakaya H, Yoshimori T, Ohsumi Y, Tokuhisa T, Mizushima N The role of autophagy during the early neonatal starvation period. Nature — Antioxid Redox Signal — Gao L, Jiang T, Guo J, Liu Y, Cui G, Gu L, Su L, Zhang Y Inhibition of autophagy contributes to ischemic postconditioning-induced neuroprotection against focal cerebral ischemia in rats.

PLoS One 7:e Schaaf MB, Cojocari D, Keulers TG, Jutten B, Starmans MH, de Jong MC, Begg AC, Savelkouls KG, Bussink J, Vooijs M, Wouters BG, Rouschop KM The autophagy associated gene, ULK1, promotes tolerance to chronic and acute hypoxia. Radiother Oncol — Zhang H, Bosch-Marce M, Shimoda LA, Tan YS, Baek JH, Wesley JB, Gonzalez FJ, Semenza GL Mitochondrial autophagy is an HIFdependent adaptive metabolic response to hypoxia.

J Biol Chem 16 — Chen JL, Lin HH, Kim KJ, Lin A, Ou JH, Ann DK PKC delta signaling: a dual role in regulating hypoxic stress-induced autophagy and apoptosis. Autophagy 5 2 — Kim J, Guan KL Regulation of the autophagy initiating kinase ULK1 by nutrients: roles of mTORC1 and AMPK.

Cell Cycle 10 9 — Cam H, Easton JB, High A, Houghton PJ mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF-1α. Mol Cell 40 4 — Papandreou I, Lim AL, Laderoute K, Denko NC Hypoxia signals autophagy in tumor cells via AMPK activity, independent of HIF-1, BNIP3, and BNIP3L.

Cell Death Differ 15 10 — Mattson MP Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1 2 — Algeciras-Schimnich A, Shen L, Barnhart BC, Murmann AE, Burkhardt JK, Peter ME Molecular ordering of the initial signaling events of CD Adams JM Ways of dying: multiple pathways to apoptosis.

Genes Dev — Ashkenazi A Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer — Li H, Zhu H, Xu CJ, Yuan J Cleavage of BID by caspase8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell — Kroemer G, Galluzzi L, Brenner C Mitochondrial membrane permeabilization in cell death.

Physiol Rev — Tokar T, Ulicny J The mathematical model of the Bcl-2 family mediated MOMP regulation can perform a non-trivial pattern recognition. PLoS One 8 12 :e Phesse TJ, Myant KB, Cole AM, Ridgway RA, Pearson H, Muncan V, van den Brink GR, Vousden KH, Sears R, Vassilev LT, Clarke AR, Sansom OJ Endogenous c-Myc is essential for pinduced apoptosis in response to DNA damage in vivo.

Cell Death Differ 21 6 — Gallagher PJ, Blue EK Post-translational regulation of the cellular levels of DAPK.

Apoptosis 19 2 — Toxicol Lett 3 — Li Q, Zhang T, Wang J, Zhang Z, Zhai Y, Yang GY, Sun X Rapamycin attenuates mitochondrial dysfunction via activation of mitophagy in experimental ischemic stroke.

Biochem Biophys Res Commun 2 — Zhang X, Yan H, Yuan Y, Gao J, Shen Z, Cheng Y, Shen Y, Wang RR, Wang X, Hu WW, Wang G, Chen Z Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance. Autophagy — Cancer Biother Radiopharm 26 3 — Article PubMed Google Scholar.

Grishchuk Y, Ginet V, Truttmann AC, Clarke PG, Puyal J Beclin 1-independent autophagy contributes to apoptosis in cortical neurons. Autophagy 7 10 — Grohm J, Plesnila N, Culmsee C Bid mediates fission, membrane permeabilization and peri-nuclear accumulation of mitochondria as a prerequisite for oxidative neuronal cell death.

Brain Behav Immun 24 5 — J Biol Chem 40 — Transplantation 84 9 — Puyal J, Vaslin A, Mottier V, Clarke PG Postischemic treatment of neonatal cerebral ischemia should target autophagy. Ann Neurol 66 3 — Young MM, Takahashi Y, Khan O, Park S, Hori T, Yun J, Sharma AK, Amin S, Hu CD, Zhang J, Kester M, Wang HG Autophagosomal membrane serves as platform for intracellular death-inducing signaling complex iDISC -mediated caspase-8 activation and apoptosis.

J Biol Chem — Russell JC, Whiting H, Szuflita N, Hossain MA Nuclear translocation of X-linked inhibitor of apoptosis XIAP determines cell fate after hypoxia ischemia in neonatal brain. J Neurochem 3 — Nachmias B, Ashhab Y, Ben-Yehuda D The inhibitor of apoptosis protein family IAPs : an emerging therapeutic target in cancer.

Semin Cancer Biol — Nezis IP, Shravage BV, Sagona AP, Lamark T, Bjørkøy G, Johansen T, Rusten TE, Brech A, Baehrecke EH, Stenmark H Autophagic degradation of dBruce controls DNA fragmentation in nurse cells during late Drosophila melanogaster oogenesis.

J Cell Biol — Mizushima N, Yoshimori T, Ohsumi Y The role of atg proteins in autophagosome formation. Annu Rev Cell Dev Biol — Oral O, Oz-Arslan D, Itah Z, Naghavi A, Deveci R, Karacali S, Gozuacik D Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death.

Apoptosis — Kim J, Huang WP, Klionsky DJ Membrane recruitment of Aut7p in the autophagy and cytoplasm to vacuole targeting pathways requires Aut1p, Aut2p, and the autophagy conjugation complex.

Betin VM, Lane JD Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. J Cell Sci — Luo S, Rubinsztein DC Apoptosis blocks Beclin 1-dependent autophagosome synthesis: an effect rescued by Bcl-xL.

Cell Death Differ — Li H, Wang P, Sun Q, Ding WX, Yin XM, Sobol RW, Stolz DB, Yu J, Zhang L Following cytochrome crelease, autophagy is inhibited during chemotherapy-induced apoptosis by caspasemediated cleavage of Beclin Cancer Res Cancer Res — Lamy L, Ngo VN, Emre NC, Shaffer AL 3rd, Yang Y, Tian E, Nair V, Kruhlak MJ, Zingone A, Landgren O, Staudt LM Control of autophagic cell death by caspase in multiple myeloma.

Cancer Cell — Download references. This work was supported by National Natural Science Foundation of China Grant No.

Autophagy and hypoxia Chen, Wei Zhang, Uypoxia Li, Autoophagy Ren, Ning Xu, Xnd Liu, Feng-Qin Promoting optimal digestion, Zhi-Jun Sun, Jun Autphagy, Yi-Fang Zhao, Yhpoxia autophagy in endothelial cells: a double-edged sword in the progression Autophagy and hypoxia infantile haemangioma? The aim of this study was to investigate the precise role of hypoxia-induced autophagy in endothelial cells, and whether it contributes to the distinctive progression of infantile haemangioma IH. The endothelial cells EOMA and HUVECs were cultured under hypoxic conditions for indicated times 0—72 h. The results showed that short exposure of the endothelial cells to hypoxia resulted in increased cell survival and proliferation, accompanied by occurrence of autophagy. Prolonged hypoxia-induced autophagy, correlating with increased cell death, was also detected afterwards. Copyright: Autophagyy Gui Autophagy and hypoxia al. Autopbagy is an open access article distributed Antioxidant drinks for hydration the terms of Wnd Commons Autophagy and hypoxia License. As the most prevalent disease afflicting humans 67coronary artery disease is mainly caused by the hypoxic-ischemic injury to cardiomyocytes 89. Myocardial ischemia and infarction develop when the blood supply to the myocardium decreases or is discontinued. The mechanism underlying myocardial ischemia and infarction has yet to be fully elucidated; however, the oxygen deprivation, i.

Video

The 7 Powerful Ways to Increase Autophagy

Author: Zololmaran

0 thoughts on “Autophagy and hypoxia

Leave a comment

Yours email will be published. Important fields a marked *

Design by ThemesDNA.com