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

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.

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

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2021.669975 ◽

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.

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

10.1101/2020.06.16.154302 ◽

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.

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Injection of mesenchymal stromal cells into a mechanically stimulated in vitro model of cardiac fibrosis has paracrine effects on resident fibroblasts

Cytotherapy ◽

10.1016/j.jcyt.2014.01.416 ◽

2014 ◽

Vol 16 (7) ◽

pp. 906-914 ◽

Cited By ~ 12

Author(s):

Peter A. Galie ◽

Jan P. Stegemann

Keyword(s):

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

In Vitro Model ◽

Cardiac Fibrosis ◽

Paracrine Effects ◽

Vitro Model

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PACAP Modulates the Autophagy Process in an In Vitro Model of Amyotrophic Lateral Sclerosis

International Journal of Molecular Sciences ◽

10.3390/ijms21082943 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2943 ◽

Cited By ~ 4

Author(s):

Agata Grazia D’Amico ◽

Grazia Maugeri ◽

Salvatore Saccone ◽

Concetta Federico ◽

Sebastiano Cavallaro ◽

...

Keyword(s):

Cell Death ◽

Amyotrophic Lateral Sclerosis ◽

Motor Neuron ◽

Motor Neurons ◽

In Vitro Model ◽

Erk Signaling ◽

Hypoxic Stress ◽

Vitro Model ◽

Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of complex etiology leading to motor neuron degeneration. Many gene alterations cause this pathology, including mutation in Cu, Zn superoxide dismutase (SOD1), which leads to its gain of function. Mutant SOD1 proteins are prone to aberrant misfolding and create aggregates that impair autophagy. The hypoxic stress is strictly linked to the disease progression since it induces uncontrolled autophagy activation and the consequent high rates of cell death. Previously, we showed that pituitary adenylate cyclase-activating polypeptide (PACAP) exerts neurotrophic activity in cultured mSOD1 motor neurons exposed to serum deprivation. To date, no studies have examined whether the protective effect of PACAP on mSOD1 cells exposed to hypoxic insult is mediated through the regulation of the autophagy process. In the present study, we used the neuroblastoma-spinal cord-34 (NSC-34) cell line, stably expressing human wild type or mutant SOD1 G93A, to represent a well characterized in vitro model of a familial form of ALS. These cells were exposed to 100-µM desferrioxamine mesylate salt for 24h, to mimic the hypoxic stress affecting motor neurons during the disease progression. Our results showed that PACAP treatment significantly reduced cell death and hypoxia-induced mSOD1 accumulation by modulating the autophagy process in G93A motor neurons, as revealed by the decreased LC3II and the increased p62 levels, two autophagy indicators. These results were also confirmed by evaluating the vacuole formation detected through light chain 3 (LC3) immunofluorescence. Furthermore, the PACAP effects on autophagy seem to be mediated through the activation of the MAPK/ERK signaling pathway. Overall, our data demonstrated that PACAP exerts an ameliorative effect on the mSOD1 motor neuron viability by modulating a hypoxia-induced autophagy process through activation of MAPK/ERK signaling cascade.

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Polychlorinated biphenyls (PCBs) alter DNA methylation and genomic integrity of sheep fetal cells in a simplified in vitro model of pregnancy exposure

Toxicology in Vitro ◽

10.1016/j.tiv.2017.09.017 ◽

2018 ◽

Vol 46 ◽

pp. 39-46 ◽

Cited By ~ 6

Author(s):

Debora A. Anzalone ◽

Silvestre Sampino ◽

Marta Czernik ◽

Domenico Iuso ◽

Grazyna E. Ptak

Keyword(s):

Dna Methylation ◽

Polychlorinated Biphenyls ◽

In Vitro Model ◽

Genomic Integrity ◽

Fetal Cells ◽

Vitro Model

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Repurposing mesalazine against cardiac fibrosis in vitro

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/s00210-020-01998-9 ◽

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.

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