scholarly journals Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress

2021 ◽  
Vol 8 ◽  
Author(s):  
Ayan Rajgarhia ◽  
Kameshwar R. Ayasolla ◽  
Nahla Zaghloul ◽  
Jorge M. Lopez Da Re ◽  
Edmund J. Miller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
Superoxide Dismutase ◽  
Cardiac Fibrosis ◽  
Extracellular Superoxide Dismutase ◽  
Hypoxic Stress ◽  
Fibroblast Proliferation ◽  
Gene Methylation ◽  
Hypoxic Group

Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A) and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia, but its effects on these epigenetic changes have not been fully explored. To test our hypothesis, we used an in-vitro model: wild-type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG). The studied animals were housed in hypoxia (10% O2) for 21 days. The right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western blot analyses. Primary C57BL6 mouse cardiac fibroblast tissue culture was used to study the in-vitro model, the downstream effects of RASSF-1 expression and methylation, and its relation to ERK1/2. Our findings showed a significant increase in cardiac fibrosis markers: Collagen 1, alpha smooth muscle actin (ASMA), and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p < 0.05). The expression of DNA methylation enzymes (DNMT 1&3b) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p < 0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p < 0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p < 0.05). Methylation of the RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs. 0.75, respectively). Based on our findings, we can speculate that EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

scholarly journals Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress

2020 ◽  
Author(s):  
Ayan Rajgarhia ◽  
Kameshwar Ayyasola ◽  
Nahla Zaghloul ◽  
Jorge M. Lopez Da Re ◽  
Edmund J. Miller ◽  
...  
Keyword(s):  
Dna Methylation ◽  
In Vitro Model ◽  
Cardiac Fibrosis ◽  
Extracellular Superoxide Dismutase ◽  
Hypoxic Stress ◽  
Fibroblast Proliferation ◽  
Gene Methylation ◽  
Vitro Model ◽  
Hypoxic Group

Abstract Background: Chronic hypoxic stress induces epigenetic modifications mainly DNA methylation in cardiac fibroblasts, inactivating tumor suppressor genes (RASSF1A), and activating kinases (ERK1/2) leading to fibroblast proliferation and cardiac fibrosis. The Ras/ERK signaling pathway is an intracellular signal transduction critically involved in fibroblast proliferation. RASSF1A functions through its effect on downstream ERK1/2. The antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), decreases oxidative stress from chronic hypoxia but it’s effects on these epigenetic changes has not been fully explored. Objectives: In-vitro model: Wild type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG) were housed in hypoxia (10% O2) for 21 days. Right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western Blot analyses. in-vitro model, downstream effects of RASSF-1 expression and methylation and its relation to ERK1/2, were studied using primary C57BL6 mouse cardiac fibroblast tissue culture.Results: There were significant increases in markers of cardiac fibrosis : Collagen 1, Alpha Smooth Muscle Actin (ASMA) and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p< 0.05). Expression of DNA methylation enzymes (DNMT 1,2) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p<0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p<0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p<0.05). Methylation of RASSF1A promoter region was significantly reduced in the TG hypoxic group compared to the WT hypoxic group (0.59 vs 0.75 respectively). Conclusions: EC-SOD significantly attenuates RASSF1A gene methylation and can alleviate cardiac fibrosis induced by hypoxia.

scholarly journals Extracellular Superoxide Dismutase (EC-SOD) Regulates Gene Methylation and Cardiac Fibrosis During Chronic Hypoxic Stress

2020 ◽  
Author(s):  
Ayan Rajgarhia ◽  
Kameshwar Ayyasola ◽  
Nahla Zaghloul ◽  
Jorge M. Lopez Da Re ◽  
Edmund J. Miller ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Cardiac Fibrosis ◽  
Smooth Muscle Actin ◽  
Extracellular Superoxide Dismutase ◽  
Hypoxic Stress ◽  
Gene Methylation ◽  
Alpha Smooth Muscle Actin ◽  
Extra Copy ◽  
Rassf1a Gene ◽  
Hypoxic Group

AbstractBackgroundChronic hypoxic stress induces epigenetic modifications in cardiac fibroblasts, such as inactivation of tumor suppressor genes (RASSF1A), and activation of kinases (ERK1/2). The effects of the antioxidant enzyme, extracellular superoxide dismutase (EC-SOD), on these epigenetic changes has not been fully explored.ObjectivesTo define the effect of EC-SOD overexpression on cardiac fibrosis induced by chronic hypoxia.MethodsWild type C57B6 male mice (WT) and transgenic males with an extra copy of human hEC-SOD (TG) were housed in hypoxia (10% O2) for 21 days. Right ventricular tissue was studied for cardiac fibrosis markers using RT-PCR and Western Blot analyses. Downstream effects were studied, for both RASSF-1 expression and methylation and its relation to ERK1/2, using in-vivo & in-vitro modelsResultsThere were significant increases in markers of cardiac fibrosis : Collagen 1, Alpha Smooth Muscle Actin (ASMA) and SNAIL, in the WT hypoxic animals as compared to the TG hypoxic group (p< 0.05). Expression of DNA methylation enzymes (DNMT 1,2) was significantly increased in the WT hypoxic mice as compared to the hypoxic TG mice (p<0.001). RASSF1A expression was significantly lower and ERK1/2 was significantly higher in hypoxia WT compared to the hypoxic TG group (p<0.05). Use of SiRNA to block RASSF1A gene expression in murine cardiac fibroblast tissue culture led to increased fibroblast proliferation (p<0.05). Methylation of RASSF1A promoter region showed a significant reduction in the TG hypoxic group compared to the WT hypoxic group (0.59 vs 0.75 respectively).ConclusionsEC-SOD significantly attenuates RASSF1A gene methylation, and plays a pivotal role cardiac fibrosis induced by hypoxia.

scholarly journals RasOncogene-Mediated Progressive Silencing of Extracellular Superoxide Dismutase in Tumorigenesis

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Francesca Cammarota ◽  
Gabriella de Vita ◽  
Marco Salvatore ◽  
Mikko O. Laukkanen
Keyword(s):  
Superoxide Dismutase ◽  
Cancer Cells ◽  
Self Regulation ◽  
Thyroid Cell ◽  
Extracellular Superoxide Dismutase ◽  
Mrna Synthesis ◽  
Ras Activation ◽  
Degree Of Differentiation ◽  
In Cells

Extracellular superoxide dismutase (SOD3) is a secreted enzyme that uses superoxide anion as a substrate in a dismutase reaction that results in the formation of hydrogen peroxide. Both of these reactive oxygen species affect growth signaling in cells. Although SOD3 has growth-supporting characteristics, the expression ofSOD3is downregulated in epithelial cancer cells. In the current work, we studied the mechanisms regulatingSOD3expressionin vitrousing thyroid cell models representing different stages of thyroid cancer. We demonstrate that a low level of RAS activation increasesSOD3mRNA synthesis that then gradually decreases with increasing levels of RAS activation and the decreasing degree of differentiation of the cancer cells. Our data indicate thatSOD3regulation can be divided into two classes. The first class involves RAS–driven reversible regulation ofSOD3expression that can be mediated by the following mechanisms: RAS GTPase regulatory genes that are responsible forSOD3self-regulation; RAS-stimulated p38 MAPK activation; and RAS-activated increased expression of themir21microRNA, which inversely correlates withsod3mRNA expression. The second class involves permanent silencing ofSOD3mediated by epigenetic DNA methylation in cells that represent more advanced cancers. Therefore, the work suggests thatSOD3belongs to the group ofrasoncogene-silenced genes.

DNA methylation in lowbush blueberry (Vaccinium angustifolium Ait.) propagated by softwood cutting and tissue culture

2018 ◽  
Vol 98 (5) ◽  
pp. 1035-1044 ◽  
Author(s):  
Juran C. Goyali ◽  
Abir U. Igamberdiev ◽  
Samir C. Debnath
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
Growth Conditions ◽  
Vaccinium Angustifolium ◽  
Lowbush Blueberry ◽  
Restriction Sites ◽  
Methylation Sensitive Amplification Polymorphism ◽  
Softwood Cutting

Plant DNA methylation is one of the frequent epigenetic variations induced by tissue culture. Global DNA methylation was evaluated in lowbush blueberry (Vaccinium angustifolium Ait.) wild clone QB9C and cultivar Fundy propagated by conventional softwood cutting (SC) and tissue culture (TC) using the methylation-sensitive amplification polymorphism (MSAP) technique. In all, 106 and 107 DNA fragments were amplified using 16 selective primer combinations in SC plants of QB9C and Fundy, respectively. In micropropagated QB9C and Fundy plants, there were 105 and 109 amplified fragments, respectively. Overall, 25% of restriction sites were methylated at the cytosine nucleotide in QB9C plants propagated by SC compared with 19% in Fundy. In contrast, a total of 29% and 20% of restriction sites were methylated at cytosine in micropropagated QB9C and Fundy plants, respectively. Tissue culture plants demonstrated higher methylation events than SC plants in both genotypes. Previously, methylation polymorphism has been detected in TC plants but not in SC counterparts. Different patterns of DNA methylation and polymorphism in the plants propagated in in vitro and in vivo conditions suggest the possibility of involvement of these fragments in the processes of regulating plant growth and development under prevailing growth conditions.

Plasma extracellular superoxide dismutase and erythrocyte Cu, Zn-containing superoxide dismutase in alcoholics treated with disulfiram

1986 ◽  
Vol 70 (4) ◽  
pp. 365-369 ◽  
Author(s):  
Michael Öhman ◽  
Stefan L. Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Chronic Alcoholism ◽  
Term Treatment ◽  
Extracellular Superoxide Dismutase ◽  
Healthy Controls ◽  
Efficient Inhibitor ◽  
Long Term Treatment

1. Disulfiram has long been used in the treatment of chronic alcoholism. It is in vivo partially reduced to diethyldithiocarbamate, which is an efficient inhibitor of Cu, Zn-containing superoxide dismutase both in vitro and in vivo. The recently described extracellular superoxide dismutase is even more sensitive to diethyldithiocarbamate than Cu, Zn-superoxide dismutase. 2. To test for the possibility that long term treatment with disulfiram leads to inhibition of the superoxide dismutases, plasma extracellular superoxide dismutase and erythrocyte Cu, Zn-superoxide dismutase were determined in 12 disulfiram-treated alcoholics, and compared with 11 non-treated alcoholics and 19 healthy controls. 3. Plasma extracellular superoxide dismutase was moderately reduced (about 20%) in the disulfiram-treated alcoholics as compared with the non-treated alcoholics and the healthy controls. No effect of disulfiram treatment on erythrocyte Cu, Zn-superoxide dismutase activity was demonstrated.

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

2020 ◽  
Author(s):  
Piotr Tomasz Bednarek ◽  
Renata Orłowska
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
De Novo ◽  
Culture Conditions ◽  
Ion Concentration ◽  
Tissue Cultures ◽  
Regeneration Medium ◽  
Green Plants ◽  
Anther Cultures

Abstract BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

scholarly journals Repurposing mesalazine against cardiac fibrosis in vitro

2020 ◽  
Author(s):  
Maximilian Hoffmann ◽  
Theresa A. Kant ◽  
Ramona Emig ◽  
Johanna S. E. Rausch ◽  
Manja Newe ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
Drug Repurposing ◽  
Fibroblast Cell ◽  
Cardiac Fibroblast ◽  
Cell Stiffness ◽  
Fibroblast Proliferation ◽  
Time Saving ◽  
Solvent Control ◽  
Vitro Model

Abstract Cardiovascular diseases are exacerbated and driven by cardiac fibrosis. TGFβ induces fibroblast activation and differentiation into myofibroblasts that secrete excessive extracellular matrix proteins leading to stiffening of the heart, concomitant cardiac dysfunction, and arrhythmias. However, effective pharmacotherapy for preventing or reversing cardiac fibrosis is presently unavailable. Therefore, drug repurposing could be a cost- and time-saving approach to discover antifibrotic interventions. The aim of this study was to investigate the antifibrotic potential of mesalazine in a cardiac fibroblast stress model. TGFβ was used to induce a profibrotic phenotype in a human cardiac fibroblast cell line. After induction, cells were treated with mesalazine or solvent control. Fibroblast proliferation, key fibrosis protein expression, extracellular collagen deposition, and mechanical properties were subsequently determined. In response to TGFβ treatment, fibroblasts underwent a profound phenoconversion towards myofibroblasts, determined by the expression of fibrillary αSMA. Mesalazine reduced differentiation nearly by half and diminished fibroblast proliferation by a third. Additionally, TGFβ led to increased cell stiffness and adhesion, which were reversed by mesalazine treatment. Collagen 1 expression and deposition—key drivers of fibrosis—were significantly increased upon TGFβ stimulation and reduced to control levels by mesalazine. SMAD2/3 and ERK1/2 phosphorylation, along with reduced nuclear NFκB translocation, were identified as potential modes of action. The current study provides experimental pre-clinical evidence for antifibrotic effects of mesalazine in an in vitro model of cardiac fibrosis. Furthermore, it sheds light on possible mechanisms of action and suggests further investigation in experimental and clinical settings.

scholarly journals Superoxide-dependent consumption of nitric oxide in biological media may confound in vitro experiments

2003 ◽  
Vol 369 (2) ◽  
pp. 399-406 ◽  
Author(s):  
Robert G. KEYNES ◽  
Charmaine GRIFFITHS ◽  
John GARTHWAITE
Keyword(s):  
Tissue Culture ◽  
Superoxide Dismutase ◽  
Culture Medium ◽  
Superoxide Radical ◽  
Cultured Cells ◽  
Soluble Guanylate Cyclase ◽  
Subsequent Reaction ◽  
Biological Media ◽  
State Concentration

NO functions ubiquitously as a biological messenger but has also been implicated in various pathologies, a role supported by many reports that exogenous or endogenous NO can kill cells in tissue culture. In the course of experiments aimed at examining the toxicity of exogenous NO towards cultured cells, we found that most of the NO delivered using a NONOate (diazeniumdiolate) donor was removed by reaction with the tissue-culture medium. Two NO-consuming ingredients were identified: Hepes buffer and, under laboratory lighting, the vitamin riboflavin. In each case, the loss of NO was reversed by the addition of superoxide dismutase. The effect of Hepes was observed over a range of NONOate concentrations (producing up to 1μM NO). Furthermore, from measurements of soluble guanylate cyclase activity, Hepes-dependent NO consumption remained significant at the low nanomolar NO concentrations relevant to physiological NO signalling. The combination of Hepes and riboflavin (in the light) acted synergistically to the extent that, instead of a steady-state concentration of about 1μM being generated, NO was undetectable (<10nM). Again, the consumption could be inhibited by superoxide dismutase. A scheme is proposed whereby a ‘vicious cycle’ of superoxide radical (O2•-) formation occurs as a result of oxidation of Hepes to its radical species, fuelled by the subsequent reaction of O2•- with NO to form peroxynitrite (ONOO-). The inadvertent production of ONOO- and other reactive species in biological media, or the associated loss of NO, may contribute to the adverse effects, or otherwise, of NO in vitro.

scholarly journals Non-enzymic glycation of human extracellular superoxide dismutase

1991 ◽  
Vol 279 (1) ◽  
pp. 263-267 ◽  
Author(s):  
T Adachi ◽  
H Ohta ◽  
K Hirano ◽  
K Hayashi ◽  
S L Marklund
Keyword(s):  
Superoxide Dismutase ◽  
Heparan Sulphate ◽  
Normal Subjects ◽  
Enzymic Activity ◽  
Diabetic Patients ◽  
Extracellular Superoxide Dismutase ◽  
Heparin Binding ◽  
A Minor ◽  
Heparin Affinity

The secretory enzyme extracellular superoxide dismutase (EC-SOD) is in plasma heterogenous with regard to heparin-affinity and can be divided into three fractions, A that lacks affinity, B with intermediate affinity and C with high affinity. The C fraction forms an equilibrium between the plasma phase and heparan sulphate proteoglycan on the surface of the endothelium. In vitro EC-SOD C could be time-dependently glycated. The enzymic activity was not affected in glycated EC-SOD, but the high heparin-affinity was lost in about half of the studied glycated fraction. Addition of heparin decreased the glycation in vitro, and EC-SOD C modified with the lysine-specific reagent trinitrobenzenesulphonic acid could not be glycated in vitro. The findings suggest that the glycation sites are localized rather far away from the active site and may occur on lysine residues in the heparin-binding domain in the C-terminal end of the enzyme. The proportion of glycated EC-SOD in serum of diabetic patients was considerably higher than in normal subjects. Of the subfractions, EC-SOD B was by far the most highly glycated, followed by EC-SOD A. EC-SOD C was glycated only to be a minor extent. The findings suggest that glycation is one of the factors that contribute to the heterogeneity in heparin-affinity of plasma EC-SOD. Since this phenomenon is increased in diabetes, the cell-surface-associated EC-SOD may be decreased in this disease, increasing the susceptibility of cells to superoxide radicals produced in the extracellular space.

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