Loss of Autophagy Leads to Megakaryocytes Differentiation Failure and Defective Platelets Function

Abstract Background Megakaryocytes (MKs), large progenitor cells residing in the bone marrow, are the source of platelets. In 2009, Colosetti P et al. reported that PMA and SB could induce megakaryocytic differentiation of the chronic myelogenous leukemia cell line K562 by triggering autophagy. It gives us the first insight of autophagy induction in in vitro megakaryocytic differentiation. Although Feng W et al. reported autophagy also exists in human platelets, the role of autophagy in megakaryocyte-platelet commitment axis remains poorly understood. In this study, we elucidated the biological effects of autophagy deficiency on megakaryopoiesis and thrombosis using hematopoietic system conditionally atg7 knockout mice. Methods and Materials To evaluate the biological effects of autophagy deficiency on platelets, the following experiments were performed: (1) complete blood count of wild type and atg7-/- mice, (2) the tail bleeding time assay of wild type and Atg7-/- mice, (3) the effect of atg7 knockout on platelet aggregation and activation of CD62P and JON/A (αIIbβ3) were analyzed by flow cytometry. To assess whether the observed changes in platelets of atg7-/- mice result in aberrations of megakaryopoiesis, the following experiments were performed: (1) the percentage of BM CD41+CD61+ cells was analyzed by flow cytometry, (2) the AchE activity assay of platelets and murine BM Lin- cells cultured with murine TPO and SCF, (3) Morphology of megakaryocytes derived from BM Lin- cells was evaluated by Wright-Giemsa staining, (4) megakaryocytic differentiation from BM Lin- cells was evaluated by CD41/forward-scatter (FSC) dot plot. To evaluate the role of reactive oxygen species in MK differentiation, the Lin- cells were stained with MitoTracker Green and MitoSox Red and then analyzed by flow cytometry. Results (1) The number of platelets in the peripheral blood of atg7-/- mice was significantly decreased (WT: 904.2±75.5, Atg7+/-: 942.8±136.3, Atg7-/-: 330.5±282.2, p<0.01), while the size of platelets (MPV) was increased compared with WT mice (WT: 5.6±0.1, Atg7+/-: 5.7±0.1, atg7-/-: 6.8±0.5, p<0.01). (2) The bleeding time was significantly longer (WT: 51.5±14.8s, Atg7-/-: 915.2±282.9s, p<0.01) and thrombin-induced platelet aggregation was decreased (WT: 97.5±2.5, Atg7+/-: 62.5±7.5, Atg7-/-: 7.75±7.25, p<0.05) in Atg7-/- mice than in wild-type mice. (3) The activation of CD62P (WT: 14.5±0.09, atg7+/-: 11.17±0.06, atg7-/-: 3.2±0.03, p<0.01) and JON/A (αIIbβ3) (WT: 48.1±0.1, atg7+/-: 13.5±0.1, atg7-/-: 5.9±0.2, p<0.01) was decreased in atg7-/- platelets. These results indicated that atg7-dependent autophagy is important for thrombosis and platelet function. (4) The percentage of CD41+CD61+ cells was decreased in bone marrow of atg7-/- mice (WT: 30.4±0.6, atg7+/-: 27.9±1.3, atg7-/-: 18.9±0.3, p<0.01). (5) In mice lacking autophagy, both the Lin- cells stimulated by TPO (WT: 0.35±0.03, atg7-/-: 0.22±0.05, p<0.01) and the platelets collected through the inferior vena cava (WT: 0.099±0.005, atg7-/-: 0.05±0.009, p<0.01) had significantly lower AChE activity compared with WT mice. (6) Low level of CD41+/FSChigh cells were seen in the in vitro culture of atg7-/- BM Lin- cells with TPO and SCF ( WT:5.6±2.2, Atg7-/-: 0.2±0.03, p<0.01). These results reflected a significant reduction in MK differentiation from autophagy defective hematopoietic progenitors. An accumulation of mitochondria (WT: 547.3±7.0, atg7-/-: 737.8±126.6, p<0.01) and mitochondrial superoxide (WT: 280.2±4.8, atg7-/-: 343.8±42.4, p<0.05) was found in atg7-/- BM Lin- cells, which may severely disturb the progress of MK differentiation. Conclusion Autophagy is essential for the megakaryopoiesis and thrombosis by maintaining mitochondrial homeostasis. Elevated reactive oxygen species might be the cause of megakaryocytic differentiation blockade. Disclosures No relevant conflicts of interest to declare.

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A Novel Tetrapeptide Derivative Exhibits In Vitro Inhibition of Neutrophil-Derived Reactive Oxygen Species and Lysosomal Enzymes Release

Oxidative Medicine and Cellular Longevity ◽

10.1155/2013/853210 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Sumitra Miriyala ◽

Manikandan Panchatcharam ◽

Meera Ramanujam ◽

Rengarajulu Puvanakrishnan

Keyword(s):

Reactive Oxygen Species ◽

Superoxide Anion ◽

Lysosomal Enzymes ◽

Reactive Oxygen ◽

Peptide Sequence ◽

Ros Generation ◽

Oxygen Species ◽

Complement Factors

Neutrophil infiltration plays a major role in the pathogenesis of myocardial injury. Oxidative injury is suggested to be a central mechanism of the cellular damage after acute myocardial infarction. This study is pertained to the prognostic role of a tetrapeptide derivative PEP1261 (BOC-Lys(BOC)-Arg-Asp-Ser(tBu)-OtBU), a peptide sequence (39–42) of lactoferrin, studied in the modulation of neutrophil functions in vitro by measuring the reactive oxygen species (ROS) generation, lysosomal enzymes release, and enhanced expression of C proteins. The groundwork experimentation was concerned with the isolation of neutrophils from the normal and acute myocardial infarct rats to find out the efficacy of PEP1261 in the presence of a powerful neutrophil stimulant, phorbol 12-myristate 13 acetate (PMA). Stimulation of neutrophils with PMA resulted in an oxidative burst of superoxide anion and enhanced release of lysosomal enzymes and expression of complement proteins. The present study further demonstrated that the free radicals increase the complement factors in the neutrophils confirming the role of ROS. PEP1261 treatment significantly reduced the levels of superoxide anion and inhibited the release of lysosomal enzymes in the stimulated control and infarct rat neutrophils. This study demonstrated that PEP1261 significantly inhibited the effect on the ROS generation as well as the mRNA synthesis and expression of the complement factors in neutrophils isolated from infarct heart.

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Cell-biological effects of zinc oxide spheres and rods from the nano- to the microscale at sub-toxic levels

Cell Biology and Toxicology ◽

10.1007/s10565-020-09571-z ◽

2020 ◽

Author(s):

M. Olejnik ◽

M. Kersting ◽

N. Rosenkranz ◽

K. Loza ◽

M. Breisch ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Zinc Oxide ◽

Zinc Acetate ◽

Culture Medium ◽

Biological Effects ◽

Oxygen Species ◽

Cell Culture Medium ◽

Oxide Particles ◽

Zinc Oxide Particles

AbstractZinc oxide particles were synthesized in various sizes and shapes, i.e., spheres of 40-nm, 200-nm, and 500-nm diameter and rods of 40∙100 nm2 and 100∙400 nm2 (all PVP-stabilized and well dispersed in water and cell culture medium). Crystallographically, the particles consisted of the hexagonal wurtzite phase with a primary crystallite size of 20 to 100 nm. The particles showed a slow dissolution in water and cell culture medium (both neutral; about 10% after 5 days) but dissolved within about 1 h in two different simulated lysosomal media (pH 4.5 to 4.8). Cells relevant for respiratory exposure (NR8383 rat alveolar macrophages) were exposed to these particles in vitro. Viability, apoptosis, and cell activation (generation of reactive oxygen species, ROS, release of cytokines) were investigated in an in vitro lung cell model with respect to the migration of inflammatory cells. All particle types were rapidly taken up by the cells, leading to an increased intracellular zinc ion concentration. The nanoparticles were more cytotoxic than the microparticles and comparable with dissolved zinc acetate. All particles induced cell apoptosis, unlike dissolved zinc acetate, indicating a particle-related mechanism. Microparticles induced a stronger formation of reactive oxygen species than smaller particles probably due to higher sedimentation (cell-to-particle contact) of microparticles in contrast to nanoparticles. The effect of particle types on the cytokine release was weak and mainly resulted in a decrease as shown by a protein microarray. In the particle-induced cell migration assay (PICMA), all particles had a lower effect than dissolved zinc acetate. In conclusion, the biological effects of zinc oxide particles in the sub-toxic range are caused by zinc ions after intracellular dissolution, by cell-to-particle contacts, and by the uptake of zinc oxide particles into cells.

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Superoxide Dismutase Influences the Virulence of Cryptococcus neoformans by Affecting Growth within Macrophages

Infection and Immunity ◽

10.1128/iai.71.1.173-180.2003 ◽

2003 ◽

Vol 71 (1) ◽

pp. 173-180 ◽

Cited By ~ 179

Author(s):

Gary M. Cox ◽

Thomas S. Harrison ◽

Henry C. McDade ◽

Carlos P. Taborda ◽

Garrett Heinrich ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Superoxide Dismutase ◽

Cryptococcus Neoformans ◽

Fungal Infections ◽

Reactive Oxygen ◽

Oxygen Species ◽

Wild Type ◽

Pathogenic Yeast ◽

Antioxidant Defense Systems

ABSTRACT Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and molecular oxygen and has been shown to contribute to the virulence of many human-pathogenic bacteria through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. To examine the contribution of SOD to the pathogenesis of fungal infections, we cloned the Cu,Zn SOD-encoding gene (SOD1) from the human-pathogenic yeast Cryptococcus neoformans and made mutants via targeted disruption. The sod1 mutant strains had marked decreases in SOD activity and were strikingly more susceptible to reactive oxygen species in vitro. A sod1 mutant was significantly less virulent than the wild-type strain and two independent reconstituted strains, as measured by cumulative survival in the mouse inhalational model. In vitro studies established that the sod1 strain had attenuated growth compared to the growth of the wild type and a reconstituted strain inside macrophages producing reduced amounts of nitric oxide. These findings demonstrate that (i) the Cu,Zn SOD contributes to virulence but is not required for pathogenicity in C. neoformans; (ii) the decreased virulence of the sod1 strain may be due to increased susceptibility to oxygen radicals within macrophages; and (iii) other antioxidant defense systems in C. neoformans can compensate for the loss of the Cu,Zn SOD in vivo.

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Role of reactive oxygen species in organophosphate insecticide phosalone toxicity in erythrocytes in vitro

Toxicology in Vitro ◽

10.1016/s0887-2333(02)00133-9 ◽

2003 ◽

Vol 17 (2) ◽

pp. 153-157 ◽

Cited By ~ 49

Author(s):

I Altuntas ◽

N Delibas ◽

D.K Doguc ◽

S Ozmen ◽

F Gultekin

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Oxygen Species ◽

Organophosphate Insecticide

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Melatonin, an Endogenous Hormone, Modulates Th17 Cells via the Reactive Oxygen Species/TXNIP/HIF-1α Axis to Alleviate Autoimmune Uveitis

10.21203/rs.3.rs-1192974/v1 ◽

2022 ◽

Author(s):

Dan Liang ◽

Jun Huang ◽

Zhuang Li ◽

Yunwei Hu ◽

Zuoyi Li ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Flow Cytometry ◽

T Cells ◽

Th17 Cells ◽

Reactive Oxygen ◽

Treg Cells ◽

Oxygen Species ◽

T Helper ◽

Autoimmune Uveitis

Abstract Background Melatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis. Methods EAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1-20 (IRBP1−20). melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were employed to evaluate the disease progression. T lymphocytes accumulation, the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR. In vivo and in vitro experiments, T helper 1 (Th1), T helper 17 (Th17) and regulatory T (Treg) cells were detected via flow cytometry, the level reactive oxygen species(ROS) from CD4+ cells were tested via flow cytometry, and the expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α)proteins were also quantified via western blot analysis, to elucidate the mechanism of melatonin inhibiting EAU. Results Melatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells and potentiated Treg cells by suppressing their transcription factors. In vitro studies corroborated that melatonin restrains the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU. Conclusions Melatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

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Endotoxin Engages Mitochondrial Quality Control via an iNOS-Reactive Oxygen Species Signaling Pathway in Hepatocytes

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/4745067 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Anthony Cyr ◽

Lauran Chambers ◽

Paul K. Waltz ◽

Sean P. Whelan ◽

Lauryn Kohut ◽

...

Keyword(s):

Nitric Oxide ◽

Reactive Oxygen Species ◽

Quality Control ◽

Mitochondrial Biogenesis ◽

Organ Injury ◽

Oxygen Species ◽

Wild Type ◽

Mitochondrial Quality Control

Background. Organ injury and dysfunction in sepsis accounts for significant morbidity and mortality. Adaptive cellular responses in the setting of sepsis prevent injury and allow for organ recovery. We and others have shown that part of the adaptive response includes regulation of cellular respiration and maintenance of a healthy mitochondrial population. Herein, we hypothesized that endotoxin-induced changes in hepatocyte mitochondrial respiration and homeostasis are regulated by an inducible nitric oxide synthase/nitric oxide (iNOS/NO)-mitochondrial reactive oxygen species (mtROS) signaling axis, involving activation of the NRF2 signaling pathway. Methods. Wild-type (C57Bl/6) or iNos-/- male mice were subjected to intraperitoneal lipopolysaccharide (LPS) injections to simulate endotoxemia. Individual mice were randomized to treatment with NO-releasing agent DPTA-NONOate, mtROS scavenger MitoTEMPO, or vehicle controls. Other mice were treated with scramble or Nrf2-specific siRNA via tail vein injection. Primary murine hepatocytes were utilized for in vitro studies with or without LPS stimulation. Oxygen consumption rates were measured to establish mitochondrial respiratory parameters. Western blotting, confocal microscopy with immunocytochemistry, and rtPCR were performed for analysis of iNOS as well as markers of both autophagy and mitochondrial biogenesis. Results. LPS treatment inhibited aerobic respiration in vitro in wild-type but not iNos-/- cells. Experimental endotoxemia in vivo or in vitro induced iNOS protein and mtROS production. However, induction of mtROS was dependent on iNOS expression. Furthermore, LPS-induced hepatic autophagy/mitophagy and mitochondrial biogenesis were significantly attenuated in iNos-/- mice or cells with NO or mtROS scavenging. These responses were rescued in iNos-/- mice via delivery of NO both in vivo and in vitro. Conclusions. These data suggest that regulation of mitochondrial quality control following hepatocyte LPS exposure is dependent at least in part on a NO-mtROS signaling network. Further investigation to identify specific agents that modulate this process may facilitate the prevention of organ injury in sepsis.

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Root Extracts of Two Cultivars of Paeonia Species: Lipid Composition and Biological Effects on Different Cell Lines: Preliminary Results

Molecules ◽

10.3390/molecules26030655 ◽

2021 ◽

Vol 26 (3) ◽

pp. 655

Author(s):

Natalia Calonghi ◽

Giovanna Farruggia ◽

Carla Boga ◽

Gabriele Micheletti ◽

Elena Fini ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Flow Cytometry ◽

Biological Activity ◽

Cell Lines ◽

Doubling Time ◽

Biological Effects ◽

Lipid Fraction ◽

Phase Imaging ◽

Oxygen Species ◽

Root Extracts

The roots of two cultivars of Paeonia, namely Paeonia officinalis “Rubra Plena” and Paeonia “Pink Hawaiian Coral”, have been extracted with chloroform. The composition of the lipid fraction, analyzed by GC–MS technique, revealed the absence of paeonol and the presence of phenol, benzoic acid, fatty acid—and some sterol—derivatives. The chloroformic extracts have been tested on normal and several cancer cell lines but showed antiproliferative activity only on the ovarian carcinoma and the osteosarcoma. The biological activity of extracts was investigated mainly by confocal microscopy, flow cytometry and quantum phase imaging. The results indicated that the root extracts induced a hyperpolarization of mitochondria and an increase in reactive oxygen species levels, without inducing cell death. These effects are associated to an increased doubling time and a retarded confluence.

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Acetylation of H4K12 in porcine oocytes during in vitro aging: potential role of ooplasmic reactive oxygen species

Theriogenology ◽

10.1016/j.theriogenology.2010.09.031 ◽

2011 ◽

Vol 75 (4) ◽

pp. 638-646 ◽

Cited By ~ 24

Author(s):

Mao-Sheng Cui ◽

Xian-Long Wang ◽

Da-Wei Tang ◽

Jing Zhang ◽

Yan Liu ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Potential Role ◽

Reactive Oxygen ◽

Oxygen Species ◽

In Vitro Aging

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The Small GTPase RacA Mediates Intracellular Reactive Oxygen Species Production, Polarized Growth, and Virulence in the Human Fungal Pathogen Aspergillus fumigatus

Eukaryotic Cell ◽

10.1128/ec.00288-10 ◽

2010 ◽

Vol 10 (2) ◽

pp. 174-186 ◽

Cited By ~ 31

Author(s):

Haiyan Li ◽

Bridget M. Barker ◽

Nora Grahl ◽

Srisombat Puttikamonkul ◽

Jeremey D. Bell ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Aspergillus Fumigatus ◽

Invasive Pulmonary Aspergillosis ◽

Reactive Oxygen ◽

Small Gtpase ◽

Oxygen Species ◽

Wild Type ◽

Content Type ◽

Diphenylene Iodonium

ABSTRACTAspergillus fumigatusis the predominant mold pathogen in immunocompromised patients. In this study, we present the first characterization of the small GTPase RacA inA. fumigatus. To gain insight into the function ofracAin the growth and pathogenesis ofA. fumigatus, we constructed a strain that lacks a functionalracAgene. The ΔracAstrain showed significant morphological defects, including a reduced growth rate and abnormal conidiogenesis on glucose minimal medium. In the ΔracAstrain, apical dominance in the leading hyphae is lost and, instead, multiple axes of polarity emerge. Intriguingly, superoxide production at the hyphal tips was reduced by 25% in the ΔracAstrain. Treatment of wild-type hyphae with diphenylene iodonium, an inhibitor of NADPH oxidase, resulted in phenotypes similar to that of the ΔracAstrain. These data suggest that ΔracAstrain phenotypes may be due to a reduction or alteration in the production of reactive oxygen species. Most surprisingly, despite these developmental and growth abnormalities, the ΔracAstrain retained at least wild-type virulence in both an insect model and two immunologically distinct murine models of invasive pulmonary aspergillosis. These results demonstrate thatin vitrogrowth phenotypes do not always correlate within vivovirulence and raise intriguing questions about the role of RacA inAspergillusvirulence.

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The Role of Reactive Oxygen Species in the Light-Induced Deformation of DNA

Zeitschrift für Naturforschung B ◽

10.1515/znb-1984-0922 ◽

1984 ◽

Vol 39 (9) ◽

pp. 1276-1280 ◽

Cited By ~ 1

Author(s):

R. Baumann ◽

M. Herrmann ◽

H. Parlar

Keyword(s):

Reactive Oxygen Species ◽

Excited States ◽

Reactive Oxygen ◽

Oxygen Species ◽

In Vivo Experiments ◽

Pyrimidine Bases ◽

Effect Of Oxygen

Dimerizations and reactions with water of pyrimidine bases are the primary steps held responsible for the deformation of DNA at short wavelengths in vitro and in vivo experiments. However the influence of oxygen in combination with water on the UV deformation at wavelengths representative for troposphere is evident from the observed data and both together are needed to change the DNA structure. The only plausible explanation for the effect of oxygen is the formation of reactive oxygen species during the UV irradiation of DNA. In the present work the deformation of DNA by different oxygen species like singlet oxygen (1O2), superoxideanion (O2-), hydroxyradical (·OH), ozone (O3) and hydrogenperoxide (H2O2) is excluded with the help of chemical-trapping experiments. The photo-induced transformation proceeds via excited states of DNA. which react with groundstate oxygen to afford peroxide.

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