Crosstalk between redox regulatory pathways and epigenetic processes in tissue and cell culture models of cardiovascular complications

Oxidative stress is caused by an imbalance between the production of reactive oxygen species (ROS) and the neutralization of these species by antioxidant systems, leading to a disruption of redox signaling, oxidative damage and potential pathological consequences, including cardiovascular complications. There is a growing body of evidence that ROS influence epigenetic pathways by affecting the function or levels of epigenetic modulators, such as histone modifying enzymes. Since epigenetic modifications are increasingly recognized as major players in cardiovascular disease development and progression, investigation of the interplay between redox signaling and epigenetic regulation is of particular interest in this context. A readout assay for the detection of protein S-nitros(yl)ation, a redox modification, was established using an approach consisting of light-induced homolysis of nitros(yl)ated proteins and subsequent immuno-spin trapping of generated protein radicals via 5,5 dimethyl-1 pyrroline N-oxide (DMPO) and a respective antibody. In order to investigate ROS-induced epigenetic changes, a suitable model system with elevated ROS formation had to be established. Culturing endothelial cells under hyperglycemic conditions is known to generate increased oxidative stress. However, no substantial effects on ROS formation and histone methylation and acetylation patterns could be observed in the endothelial cell line EA.hy926 upon hyperglycemia, possibly due to a systematical technical error that, however, could not be identified despite multiple methodological variations. In mice deficient in the antioxidant protein glutathione peroxidase-1 (GPx-1) endothelial dysfunction and enhanced ROS levels have been reported previously, an effect that was further potentiated by aging. Epigenetic analysis of this model led to the hypothesis of potential dityrosine cross-linking between histone 3 and histone 4 accompanied by enhanced histone 3 lysine 9 dimethylation upon increased oxidative stress. However, upon further investigation by mass spectrometry and exclusion of antibody cross-reactivity to IgGs in the animal samples this assumption was revealed to be false. In a published study by our group it was demonstrated that empagliflozin, a selective sodium-glucose co transporter 2 inhibitor (SGLT2i), reduced glucotoxicity and thereby prevented the development of endothelial dysfunction, reduced oxidative stress and exhibited anti-inflammatory effects in ZDF rats, an animal model for type 2 diabetes mellitus (T2DM). Investigation of involved epigenetic mechanisms by chromatin immunoprecipitation (ChIP) analysis revealed an effect of empagliflozin treatment on expression of glucotoxicity- and inflammation-markers in diabetic animals via altered histone methylation patterns. Finally, the interplay of increased ROS formation and epigenetic alterations was studied in H9c2 cardiomyocytes with a doxorubicin-induced cardiotoxic phenotype. It was discovered that doxorubicin treatment affected the expression of certain epigenetic modulators in correlation with increased oxidative stress markers. Given that epigenetic changes are reversible, they represent potential intervention targets as well as biomarkers that can be addressed for drug discovery. Thus, establishing a comprehensive understanding of the interplay of ROS and epigenetic mechanisms in cardiovascular related diseases may lead to the development of novel and precisely targeted treatment options.Der Ausdruck „oxidativer Stress“ bezeichnet ein Ungleichgewicht zwischen der Entstehung freier reaktiver Sauerstoffradikale (ROS) und deren Neutralisierung durch anti-oxidative Mechanismen. Oxidativer Stress führt zur Beeinträchtigung der zellulären Redox-Regulation, Entstehung oxidativer Schädigungen und letztendlich zu möglichen pathologischen Konsequenzen, wie z.B. kardiovaskulären Erkrankungen. Immer mehr Forschungsresultate deuten darauf hin, dass ROS epigenetische Prozesse beeinflussen, indem sie die Funktion oder Konzentration epigenetischer Modulatoren, wie z.B. Histon-Modifikatoren, verändern. Da epigenetische Modifikationen zunehmend mit der Entwicklung und dem Fortschreiten kardiovaskulärer Erkrankungen in Verbindung gebracht werden, ist es von großem Interesse das Zusammenspiel von Redox-Signalwegen und epigenetischer Regulierung vor diesem Hintergrund zu untersuchen. In dieser Arbeit wurde zunächst ein Testverfahren zur Detektion von Protein S-Nitros(yl)ierung, einer Redox-Modifikation, erstellt. Dieses beinhaltete die Licht-induzierte Homolyse der nitros(yl)ierten Proteine, Immuno-Spin Trapping der entstehenden Radikale mithilfe von 5,5 Dimethyl-1 pyrroline N-oxid (DMPO) und Detektion durch einen entsprechenden Antikörper. Um ROS-induzierte epigenetische Veränderungen zu untersuchen, musste ein geeignetes Modellsystem etabliert werden, welches erhöhten oxidativen Stress aufweist. Es ist bekannt, dass Endothelzellen bei Kultivierung mit erhöhter Glukose-Konzentration vermehrt ROS bilden. Dennoch konnten gegenwärtig in diesem Modell keine erheblichen Effekte auf die ROS-Bildung und Histon-Modifikationen festgestellt werden, was möglicherweise auf ein systematisches methodologisches Problem zurückzuführen ist, das trotz intensiver Variation der Versuchsbedingungen nicht identifiziert werden konnte. In einer vorherigen Studie wurde in Mäusen mit Knock out des anti-oxidativen Enzyms Glutathionperoxidase-1 (GPx-1) eine endotheliale Dysfunktion und erhöhter oxidativer Stress verzeichnet, wobei dieser Effekt bei höherem Alter verstärkt war. Epigenetische Analysen dieses Modells führten zu der Hypothese, dass bei hohem oxidativen Stress Dityrosin-Brücken (=kovalente Bindungen) zwischen Histon 3 und Histon 4 gebildet werden, zusammen mit verstärkter Dimethylierung von Lysin 9 an Histon 3 (H3K9me2). Allerdings stellte sich bei weiteren Untersuchungen mittels Massenspektrometrie und Verwendung von Sekundärantikörpern mit garantierter Abwesenheit von Kreuzreaktivitäten gegenüber den IgGs der Tierproben heraus, dass diese Annahme falsch war. In einer von uns publizierten Studie wurde gezeigt, dass Empagliflozin, ein selektiver Inhibitor des Natrium-Glukose Cotransporters 2, die Glukotoxizität in ZDF Ratten, einem Modell für Typ 2 Diabetes Mellitus, verminderte und dadurch sowohl die Entwicklung einer endothelialen Dysfunktion verhinderte, als auch oxidativen Stress verringerte und anti-entzündlich wirkte. Die Charakterisierung involvierter epigenetischer Mechanismen mittels Chromatin-Immunopräzipitation (ChIP)-basierter Analyse zeigte einen Effekt von Empagliflozin auf die Expression bestimmter Glukotoxizitäts- und Entzündungsmarker in diabetischen Tieren durch veränderte Histon-Methylierungsmuster. Abschließend wurde das Zusammenspiel von ROS-Entstehung und epigenetischen Veränderungen in H9c2 Kardiomyozyten unter Bedingungen von Doxorubicin-induziertem Kardiotoxizitätsphänotyp analysiert. Dabei wurde festgestellt, dass die Doxorubicin-Behandlung in Korrelation mit erhöhtem oxidativen Stress die Expression bestimmter epigenetischer Modulatoren beeinflusste. Da epigenetische Veränderungen reversibel sind, stellen sie potenzielle Angriffspunkte zur therapeutischen Intervention dar und könnten auch als Biomarker pathologischer Prozesse fungieren. Daher ist ein umfassendes Verständnis des Zusammenwirkens von ROS und epigenetischer Mechanismen in kardiovaskulären Erkrankungen von großem Wert für die Entwicklung neuer und gezielter Behandlungsmöglichkeiten

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS).

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed.The EU-ROS consortium (COST Action BM1203) was supported by the European Cooperation in Science and Technology (COST). The present overview represents the final Action dissemination summarizing the major achievements of COST Action BM1203 (EU-ROS) as well as research news and personal views of its members. Some authors were also supported by COST Actions BM1005 (ENOG) and BM1307 (PROTEOSTASIS), as well as funding from the European Commission FP7 and H2020 programmes, and several national funding agencies

New Therapeutic Implications of Endothelial Nitric Oxide Synthase (eNOS) Function/Dysfunction in Cardiovascular Disease

The Global Burden of Disease Study identified cardiovascular risk factors as leading causes of global deaths and life years lost. Endothelial dysfunction represents a pathomechanism that is associated with most of these risk factors and stressors, and represents an early (subclinical) marker/predictor of atherosclerosis. Oxidative stress is a trigger of endothelial dysfunction and it is a hall-mark of cardiovascular diseases and of the risk factors/stressors that are responsible for their initiation. Endothelial function is largely based on endothelial nitric oxide synthase (eNOS) function and activity. Likewise, oxidative stress can lead to the loss of eNOS activity or even “uncoupling” of the enzyme by adverse regulation of well-defined “redox switches” in eNOS itself or up-/down-stream signaling molecules. Of note, not only eNOS function and activity in the endothelium are essential for vascular integrity and homeostasis, but also eNOS in perivascular adipose tissue plays an important role for these processes. Accordingly, eNOS protein represents an attractive therapeutic target that, so far, was not pharmacologically exploited. With our present work, we want to provide an overview on recent advances and future therapeutic strategies that could be used to target eNOS activity and function in cardiovascular (and other) diseases, including life style changes and epigenetic modulations. We highlight the redox-regulatory mechanisms in eNOS function and up- and down-stream signaling pathways (e.g., tetrahydrobiopterin metabolism and soluble guanylyl cyclase/cGMP pathway) and their potential pharmacological exploitation

CD40L controls obesity-associated vascular inflammation, oxidative stress, and endothelial dysfunction in high fat diet-treated and db/db mice

CD40 ligand (CD40L) signaling controls vascular oxidative stress and related dysfunction in angiotensin-II-induced arterial hypertension by regulating vascular immune cell recruitment and platelet activation. Here we investigated the role of CD40L in experimental hyperlipidemia. Male wild type and CD40L-/- mice (C57BL/6 background) were subjected to high fat diet for sixteen weeks. Weight, cholesterol, HDL, and LDL levels, endothelial function (isometric tension recording), oxidative stress (NADPH oxidase expression, dihydroethidium fluorescence) and inflammatory parameters (inducible nitric oxide synthase, interleukin-6 expression) were assessed. CD40L expression, weight, leptin and lipids were increased, and endothelial dysfunction, oxidative stress and inflammation were more pronounced in wild type mice on a high fat diet, all of which was almost normalized by CD40L deficiency. Similar results were obtained in diabetic db/db mice with CD40/TRAF6 inhibitor (6877002) therapy. In a small human study higher serum sCD40L levels and an inflammatory phenotype were detected in the blood and Aorta ascendens of obese patients (body mass index > 35) that underwent by-pass surgery. CD40L controls obesity-associated vascular inflammation, oxidative stress and endothelial dysfunction in mice and potentially humans. Thus, CD40L represents a therapeutic target in lipid metabolic disorders which is a leading cause in cardiovascular diseas

The SGLT2 inhibitor empagliflozin improves the primary diabetic complications in ZDF rats

Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, β-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF‐Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial)

European contribution to the study of ROS:a summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)

Abstract The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

Low incidence of SARS-CoV-2, risk factors of mortality and the course of illness in the French national cohort of dialysis patients

International audienceThe aim of this study was to estimate the incidence of COVID-19 disease in the French national population of dialysis patients, their course of illness and to identify the risk factors associated with mortality. Our study included all patients on dialysis recorded in the French REIN Registry in April 2020. Clinical characteristics at last follow-up and the evolution of COVID-19 illness severity over time were recorded for diagnosed cases (either suspicious clinical symptoms, characteristic signs on the chest scan or a positive reverse transcription polymerase chain reaction) for SARS-CoV-2. A total of 1,621 infected patients were reported on the REIN registry from March 16th, 2020 to May 4th, 2020. Of these, 344 died. The prevalence of COVID-19 patients varied from less than 1% to 10% between regions. The probability of being a case was higher in males, patients with diabetes, those in need of assistance for transfer or treated at a self-care unit. Dialysis at home was associated with a lower probability of being infected as was being a smoker, a former smoker, having an active malignancy, or peripheral vascular disease. Mortality in diagnosed cases (21%) was associated with the same causes as in the general population. Higher age, hypoalbuminemia and the presence of an ischemic heart disease were statistically independently associated with a higher risk of death. Being treated at a selfcare unit was associated with a lower risk. Thus, our study showed a relatively low frequency of COVID-19 among dialysis patients contrary to what might have been assumed