Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

BACKGROUND/AIMS: Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. METHODS: To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. RESULTS: All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. CONCLUSION: Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Effects of Rheum rhaponticum and Rheum rhabarbarum extracts on haemostatic activity of blood plasma components and endothelial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional medicine recommends the use of Rheum rhaponticum L. and R. rhabarbarum L. to treat over thirty complaints, including disorders related to the cardiovascular system such as heartache, pains in the pericardium, epistaxis and other types of haemorrhage, blood purification as well as disorders of venous circulation. AIM OF THE STUDY: This work was dedicated to examining for the first time the effects of extracts from petioles and roots of R. rhaponticum and R. rhabarbarum, as well as two stilbene compounds (rhapontigenin and rhaponticin) on the haemostatic activity of endothelial cells and functionality of blood plasma components of the haemostatic system. MATERIALS AND METHODS: The study was based on three main experimental modules, including the activity of proteins of the human blood plasma coagulation cascade and the fibrinolytic system as well as analyses of the haemostatic activity of human vascular endothelial cells. Additionally, interactions of the main components of the rhubarb extracts with crucial serine proteases of the coagulation cascade and fibrinolysis (i.e. thrombin, the coagulation factor Xa and plasmin) were analyzed in silico. RESULTS: The examined extracts displayed anticoagulant properties and significantly reduced the tissue factor-induced clotting of human blood plasma (by about 40%). Inhibitory effects of the tested extracts on thrombin and the coagulation factor Xa (FXa) were found as well. For the extracts, the IC50 was ranging from 20.26 to 48.11 µg/ml. Modulatory effects on the haemostatic response of endothelial cells, including the release of von Willebrand factor, tissue-type plasminogen activator and the plasminogen activator inhibitor-1, have been also found. CONCLUSIONS: Our results indicated for the first time that the examined Rheum extracts influenced the haemostatic properties of blood plasma proteins and endothelial cells, with the prevalence of the anticoagulant action. The anticoagulant effect of the investigated extracts may be partly attributed to the inhibition of the FXa and thrombin activities, the key serine proteases of the blood coagulation cascade.

How Warburg-Associated Lactic Acidosis Rewires Cancer Cell Energy Metabolism to Resist Glucose Deprivation.

Lactic acidosis, a hallmark of solid tumour microenvironment, originates from lactate hyperproduction and its co-secretion with protons by cancer cells displaying the Warburg effect. Long considered a side effect of cancer metabolism, lactic acidosis is now known to play a major role in tumour physiology, aggressiveness and treatment efficiency. Growing evidence shows that it promotes cancer cell resistance to glucose deprivation, a common feature of tumours. Here we review the current understanding of how extracellular lactate and acidosis, acting as a combination of enzymatic inhibitors, signal, and nutrient, switch cancer cell metabolism from the Warburg effect to an oxidative metabolic phenotype, which allows cancer cells to withstand glucose deprivation, and makes lactic acidosis a promising anticancer target. We also discuss how the evidence about lactic acidosis' effect could be integrated in the understanding of the whole-tumour metabolism and what perspectives it opens up for future research.

Rheum rhaponticum and Rheum rhabarbarum Extracts as Modulators of Endothelial Cell Inflammatory Response.

BACKGROUND: Inflammation, endothelial dysfunction, and alterations in blood physiology are key factors contributing to atherosclerosis and other cardiovascular disorders. Hence, modulation of endothelial function and reducing its pro-inflammatory and pro-thrombotic activity is considered one of the most important cardioprotective strategies. This study aimed to evaluate the anti-inflammatory potential of rhubarb extracts isolated from petioles and underground organs of Rheum rhabarbarum L. (garden rhubarb) and R. rhaponticum L. (rhapontic rhubarb) as well as two stilbenoids, typically found in these plants, i.e., rhapontigenin (RHPG) and its glycoside, rhaponticin (RHPT). METHODS: Analysis of the anti-inflammatory effects of the indicated rhubarb-derived substances involved different aspects of the endothelial cells' (HUVECs) response: release of the inflammatory mediators; cyclooxygenase (COX-2) and 5-lipoxygenase (5-LOX) expression as well as the recruitment of leukocytes to the activated HUVECs. The ability of the rhubarb-derived extracts to inhibit COX-2 and 5-LOX activities was examined as well. The study was supplemented with the in silico analysis of major components of the analyzed extracts' interactions with COX-2 and 5-LOX. RESULTS: The obtained results indicated that the examined plant extracts and stilbenes possess anti-inflammatory properties and influence the inflammatory response of endothelial cells. Biochemical and in silico tests revealed significant inhibition of COX-2, with special importance of rhaponticin, as a compound abundant in both plant species. In addition to the reduction in COX-2 gene expression and enzyme activity, a decrease in the cytokine level and leukocyte influx was observed. Biochemical tests and computational analyses indicate that some components of rhubarb extracts may act as COX-2 inhibitors, with marginal inhibitory effect on 5-LOX.

Ketone Bodies as Metabolites and Signalling Molecules at the Crossroad between Inflammation and Epigenetic Control of Cardiometabolic Disorders.

For many years, it has been clear that a Western diet rich in saturated fats and sugars promotes an inflammatory environment predisposing a person to chronic cardiometabolic diseases. In parallel, the emergence of ketogenic diets, deprived of carbohydrates and promoting the synthesis of ketone bodies imitating the metabolic effects of fasting, has been shown to provide a possible nutritional solution to alleviating diseases triggered by an inflammatory environment. The main ketone body, ß-hydroxybutyrate (BHB), acts as an alternative fuel, and also as a substrate for a novel histone post-translational modification, ß-hydroxybutyrylation. ß-hydroxybutyrylation influences the state of chromatin architecture and promotes the transcription of multiple genes. BHB has also been shown to modulate inflammation in chronic diseases. In this review, we discuss, in the pathological context of cardiovascular risks, the current understanding of how ketone bodies, or a ketogenic diet, are able to modulate, trigger, or inhibit inflammation and how the epigenome and chromatin remodeling may be a key contributor.

Fat not so bad? The role of ketone bodies and ketogenic diet in the treatment of endothelial dysfunction and hypertension.

The ketogenic diet (KD), a high-fat, low-carbohydrate dietary approach that is based on the induction of extensive ketone bodies (KB) metabolism, is recently receiving a lot of attention due to its application as effective intervention for multiple metabolic disorders including cardiovascular diseases. Despite its already established clinical use, especially in the treatment of drug-resistant epilepsy, GLUT1 deficiency syndromes and, in selected cases, obesity; the systemic impact of is not yet fully understood. Here, we discuss the evidence for and against the application of ketogenic diets, or ketone bodies precursors, in the etiology of hypertension and endothelial cells dysfunction. We attempt to identify the benefits and potential risks of chronic use of the ketogenic diet, also considering the molecular effects that KB exerts at multiple levels.

Ketogenic diet administration to mice after a high-fat-diet regimen promotes weight loss, glycemic normalization and induces adaptations of ketogenic pathways in liver and kidney.

OBJECTIVE: The ketogenic diet (KD), characterized by very limited dietary carbohydrate intake and used as nutritional treatment for GLUT1-deficiency syndromes and pharmacologically refractory epilepsy, may promote weight loss and improve metabolic fitness, potentially alleviating the symptoms of osteoarthritis. Here, we have studied the effects of administration of a ketogenic diet in mice previously rendered obese by feeding a high fat diet (HFD) and submitted to surgical destabilization of the medial meniscus to mimic osteoarthritis. METHODS: 6-weeks old mice were fed an HFD for 10 weeks and then switched to a chow diet (CD), KD or maintained on a HFD for 8 weeks. Glycemia, ß-hydroxybutyrate (BHB), body weight and fat mass were compared among groups. In liver and kidney, protein expression and histone post-translational modifications were assessed by Western blot, and gene expression by quantitative Real-Time PCR. RESULTS: After a 10 weeks HDF feeding, administration for 8 weeks of a KD or CD induced a comparable weight loss and decrease in fat mass, with better glycemic normalization in the KD group. Histone ß-hydroxybutyrylation, but not histone acetylation, was increased in the liver and kidney of mice fed the KD and the rate-limiting ketogenic enzyme HMGCS2 was upregulated - at the gene and protein level - in liver and, to an even greater extent, in kidney. KD-induced HMGCS2 overexpression may be dependent on FGF21, whose gene expression was increased by KD in liver. CONCLUSIONS: Over a period of 8 weeks, KD is more effective than a chow diet to induce metabolic normalization. Besides acting as a fuel molecule, BHB may exert its metabolic effects through modulation of the epigenome - via histone ß-hydroxybutyrylation - and extensive transcriptional modulation in liver and kidney.

The selected epigenetic effects of phthalates: DBP, BBP and their metabolites: MBP, MBzP on human peripheral blood mononuclear cells (In Vitro).

Phthalates are classified as non-genotoxic carcinogens. These compounds do not cause direct DNA damage but may induce indirect DNA lesions leading to cancer development. In the presented paper we have studied the effect of di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), and their metabolites, such as mono-n-butyl phthalate (MBP) and monobenzyl phthalate (MBzP) on selected epigenetic parameters in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with tested phthalates at 0.001, 0.01 and 0.1 µg/mL for 24 h. Next, global DNA methylation, methylation in the promoter regions of tumor suppressor genes (P16, TP53) and proto-oncogenes (BCL2, CCND1) were assessed as well as the expression profile of the indicated genes was analysed. The obtained results have revealed significant reduction of global DNA methylation level in PBMCs exposed to BBP, MBP and MBzP. Phthalates changed methylation pattern of the tested genes, decreased expression of P16 and TP53 genes and increased the expression of BCL2 and CCND1. In conclusion, our results have shown that the examined phthalates disturbed the processes of methylation and expression of tumor suppressor genes (P16, TP53) and protooncogenes (BCL2, CCND1) in human PBMCs.

The Action of Vitamin D in Adipose Tissue: Is There the Link between Vitamin D Deficiency and Adipose Tissue-Related Metabolic Disorders?

Adipose tissue plays an important role in systemic metabolism via the secretion of adipocytokines and storing and releasing energy. In obesity, adipose tissue becomes dysfunctional and characterized by hypertrophied adipocytes, increased inflammation, hypoxia, and decreased angiogenesis. Although adipose tissue is one of the major stores of vitamin D, its deficiency is detective in obese subjects. In the presented review, we show how vitamin D regulates numerous processes in adipose tissue and how their dysregulation leads to metabolic disorders. The molecular response to vitamin D in adipose tissue affects not only energy metabolism and adipokine and anti-inflammatory cytokine production via the regulation of gene expression but also genes participating in antioxidant defense, adipocytes differentiation, and apoptosis. Thus, its deficiency disturbs adipocytokines secretion, metabolism, lipid storage, adipogenesis, thermogenesis, the regulation of inflammation, and oxidative stress balance. Restoring the proper functionality of adipose tissue in overweight or obese subjects is of particular importance in order to reduce the risk of developing obesity-related complications, such as cardiovascular diseases and diabetes. Taking into account the results of experimental studies, it seemed that vitamin D may be a remedy for adipose tissue dysfunction, but the results of the clinical trials are not consistent, as some of them show improvement and others no effect of this vitamin on metabolic and insulin resistance parameters. Therefore, further studies are required to evaluate the beneficial effects of vitamin D, especially in overweight and obese subjects, due to the presence of a volumetric dilution of this vitamin among them.

Citrullination in the pathology of inflammatory and autoimmune disorders: recent advances and future perspectives.

Numerous post-translational modifications (PTMs) govern the collective metabolism of a cell through altering the structure and functions of proteins. The action of the most prevalent PTMs, encompassing phosphorylation, methylation, acylations, ubiquitination and glycosylation is well documented. A less explored protein PTM, conversion of peptidylarginine to citrulline, is the subject of this review. The process of citrullination is catalysed by peptidylarginine deiminases (PADs), a family of conserved enzymes expressed in a variety of human tissues. Accumulating evidence suggest that citrullination plays a significant role in regulating cellular metabolism and gene expression by affecting a multitude of pathways and modulating the chromatin status. Here, we will discuss the biochemical nature of arginine citrullination, the enzymatic machinery behind it and also provide information on the pathological consequences of citrullination in the development of inflammatory diseases (rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, periodontitis and COVID-19), cancer and thromboembolism. Finally, developments on inhibitors against protein citrullination and recent clinical trials providing a promising therapeutic approach to inflammatory disease by targeting citrullination are discussed.

The Role of Lysine-Specific Demethylase 1 (LSD1) in Shaping the Endothelial Inflammatory Response.

BACKGROUND/AIMS: Inflammation is the body's natural response to stress in the broadest sense. The regulatory mechanisms that control this process, some of which are still unclear, are needed to balance the immune response, but also when insufficient, can cause immunodeficiency resulting in infection, cancer, neurodegeneration or other serious disorders. In this study, we focused on defining the role of lysine-specific demethylase 1 (LSD1), an enzyme involved in modulating the methylation state of lysine, including histone and non-histone proteins, in shaping the inflammatory profile of endothelial cells. METHODS: To determine the role of LSD1 in the inflammatory response of ECs, cells were stimulated with lipopolysaccharide (100 ng/ml LPS) in the presence and absence of an LSD1 inhibitor (2-PCPA). A transcription model of LSD1 deficient cells (HMEC-1 LSD1 KD) obtained by lentiviral shRNA transduction was also used. The indicated cellular models were analyzed by gene profiling, monitoring of p65 shuttling by Western blotting and immunofluorescence staining. Also chromatin immunoprecipitation (ChIP) was performed to identify the interactions between selected: IL-6/p65 and LSD1. RESULTS: Analysis of both experimental models revealed an altered inflammatory response following both LSD1 inhibition and LSD1 silencing. We observed decreased U-937 monocytes recruitment to LPS-activated endothelial cells and decreased extracellular secretion of many proinflammatory cytokines, also confirmed at the transcript level by RT-qPCR. Monitoring of the LPS-induced p65 translocation revealed inhibition of the NF-kB subunit in LSD1 KD vs nonT as well as due to pretreatment of 2-PCPA cells. Gene profiling performed with RNA microarrays confirmed the obtained biochemical data at the transcript level. CONCLUSION: In conclusion, the conducted studies showed a proinflammatory profile of LSD1 activity in endothelial cells, revealed by the inhibition of the enzyme activity and confirmed at the transcriptional level by the inhibition of its expression. Although we found significant changes in the modification of interactions between monocytes and endothelial cells as well as in cytokine/chemokine release and expression that were consistent with the altered NF-κB-p65 translocation into the nucleus, we did not identify a direct interaction between LSD1 and the transcription factor. Our finding may have important implications for prevention of cardiovascular diseases at their first stage - activation of the endothelium as well as for tumor cell biology, providing evidence for the use of LSD1 inhibitors to reduce the inflammatory response, which enhances tumor tissue remodeling, angiogenesis and metastasis.

Chronic and Transient Hyperglycemia Induces Changes in the Expression Patterns of IL6 and ADIPOQ Genes and Their Associated Epigenetic Modifications in Differentiating Human Visceral Adipocytes.

Adipokines secreted by hypertrophic visceral adipose tissue (VAT) instigate low-grade inflammation, followed by hyperglycemia (HG)-related metabolic disorders. The latter may develop with the participation of epigenetic modifications. Our aim was to assess how HG influences selected epigenetic modifications and the expression of interleukin 6 (IL-6) and adiponectin (APN; gene symbol ADIPOQ) during the adipogenesis of human visceral preadipocytes (HPA-v). Adipocytes (Ads) were chronically or transiently HG-treated during three stages of adipogenesis (proliferation, differentiation, maturation). We measured adipokine mRNA, protein, proven or predicted microRNA expression (RT-qPCR and ELISA), and enrichment of H3K9/14ac, H3K4me3, and H3K9me3 at gene promoter regions (chromatin immunoprecipitation). In chronic HG, we detected different expression patterns of the studied adipokines at the mRNA and protein levels. Chronic and transient HG-induced changes in miRNA (miR-26a-5p, miR-26b-5p, let-7d-5p, let-7e-5p, miR-365a-3p, miR-146a-5p) were mostly convergent to altered IL-6 transcription. Alterations in histone marks at the IL6 promoter were also in agreement with IL-6 mRNA. The open chromatin marks at the ADIPOQ promoter mostly reflected the APN transcription during NG adipogenesis, while, in the differentiation stage, HG-induced changes in all studied marks were in line with APN mRNA levels. In summary, HG dysregulated adipokine expression, promoting inflammation. Epigenetic changes coexisted with altered expression of adipokines, especially for IL-6; therefore, epigenetic marks induced by transient HG may act as epi-memory in Ads. Such changes in the epigenome and expression of adipokines could be instrumental in the development of inflammation and metabolic deregulation of VAT.

Metabolomic Approaches to Study Chemical Exposure-Related Metabolism Alterations in Mammalian Cell Cultures.

Biological organisms are constantly exposed to an immense repertoire of molecules that cover environmental or food-derived molecules and drugs, triggering a continuous flow of stimuli-dependent adaptations. The diversity of these chemicals as well as their concentrations contribute to the multiplicity of induced effects, including activation, stimulation, or inhibition of physiological processes and toxicity. Metabolism, as the foremost phenotype and manifestation of life, has proven to be immensely sensitive and highly adaptive to chemical stimuli. Therefore, studying the effect of endo- or xenobiotics over cellular metabolism delivers valuable knowledge to apprehend potential cellular activity of individual molecules and evaluate their acute or chronic benefits and toxicity. The development of modern metabolomics technologies such as mass spectrometry or nuclear magnetic resonance spectroscopy now offers unprecedented solutions for the rapid and efficient determination of metabolic profiles of cells and more complex biological systems. Combined with the availability of well-established cell culture techniques, these analytical methods appear perfectly suited to determine the biological activity and estimate the positive and negative effects of chemicals in a variety of cell types and models, even at hardly detectable concentrations. Metabolic phenotypes can be estimated from studying intracellular metabolites at homeostasis in vivo, while in vitro cell cultures provide additional access to metabolites exchanged with growth media. This article discusses analytical solutions available for metabolic phenotyping of cell culture metabolism as well as the general metabolomics workflow suitable for testing the biological activity of molecular compounds. We emphasize how metabolic profiling of cell supernatants and intracellular extracts can deliver valuable and complementary insights for evaluating the effects of xenobiotics on cellular metabolism. We note that the concepts and methods discussed primarily for xenobiotics exposure are widely applicable to drug testing in general, including endobiotics that cover active metabolites, nutrients, peptides and proteins, cytokines, hormones, vitamins, etc.

The selected epigenetic effects of aminomethylphosphonic acid, a primary metabolite of glyphosate on human peripheral blood mononuclear cells (in vitro).

Aminomethylphosphonic acid (AMPA) is a primary metabolite of glyphosate and amino-polyphosphonate. We have determined the effect of AMPA on selected epigenetic parameters and major cell cycle drivers in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with AMPA at 0.5, 10 and 250 µM for 24 h. The performed analysis included: global DNA methylation by colorimetric measurement of 5-methylcytosine in DNA, methylation in the promoter regions of selected tumor suppressor genes (P16, P21, TP53) and proto-oncogenes (BCL2, CCND1) as well as the expression profile of the indicated genes by Real-Time PCR assays. The obtained results have revealed significant reduction of global DNA methylation level in PBMCs exposed to AMPA. Investigated xenobiotic changed methylation pattern of the P21 and TP53 suppressor gene promoters, but in case of other analyzed genes: P16, BCL2 and CCND1 no statistically significant changes have been noted. Gene profiling have shown that AMPA only changed the expression of CCND1. Summing up, our results have revealed a small potential disturbance in methylation processes and the absence of changes in expression of tested tumor suppressor genes (P16, P21, TP53) and protooncogenes (BCL2) in human PBMCs exposed to AMPA.

Epigallocatechin-3-gallate (EGCG) Alters Histone Acetylation and Methylation and Impacts Chromatin Architecture Profile in Human Endothelial Cells.

Epigallocatechin gallate (EGCG), the main green tea polyphenol, exerts a wide variety of biological actions. Epigenetically, the catechin has been classified as a DNMTs inhibitor, however, its impact on histone modifications and chromatin structure is still poorly understood. The purpose of this study was to find the impact of EGCG on the histone posttranslational modifications machinery and chromatin remodeling in human endothelial cells of both microvascular (HMEC-1) and vein (HUVECs) origin. We analyzed the methylation and acetylation status of histones (Western blotting), as well as assessed the activity (fluorometric assay kit) and gene expression (qPCR) of the enzymes playing a prominent role in shaping the human epigenome. The performed analyses showed that EGCG increases histone acetylation (H3K9/14ac, H3ac), and methylation of both active (H3K4me3) and repressive (H3K9me3) chromatin marks. We also found that the catechin acts as an HDAC inhibitor in cellular and cell-free models. Additionally, we observed that EGCG affects chromatin architecture by reducing the expression of heterochromatin binding proteins: HP1α, HP1γ. Our results indicate that EGCG promotes chromatin relaxation in human endothelial cells and presents a broad epigenetic potential affecting expression and activity of epigenome modulators including HDAC5 and 7, p300, CREBP, LSD1 or KMT2A.

The Epigenetic Profile of Tumor Endothelial Cells. Effects of Combined Therapy with Antiangiogenic and Epigenetic Drugs on Cancer Progression.

Tumors require a constant supply of nutrients to grow which are provided through tumor blood vessels. To metastasize, tumors need a route to enter circulation, that route is also provided by tumor blood vessels. Thus, angiogenesis is necessary for both tumor progression and metastasis. Angiogenesis is tightly regulated by a balance of angiogenic and antiangiogenic factors. Angiogenic factors of the vascular endothelial growth factor (VEGF) family lead to the activation of endothelial cells, proliferation, and neovascularization. Significant VEGF-A upregulation is commonly observed in cancer cells, also due to hypoxic conditions, and activates endothelial cells (ECs) by paracrine signaling stimulating cell migration and proliferation, resulting in tumor-dependent angiogenesis. Conversely, antiangiogenic factors inhibit angiogenesis by suppressing ECs activation. One of the best-known anti-angiogenic factors is thrombospondin-1 (TSP-1). In pathological angiogenesis, the balance shifts towards the proangiogenic factors and an angiogenic switch that promotes tumor angiogenesis. Here, we review the current literature supporting the notion of the existence of two different endothelial lineages: normal endothelial cells (NECs), representing the physiological form of vascular endothelium, and tumor endothelial cells (TECs), which are strongly promoted by the tumor microenvironment and are biologically different from NECs. The angiogenic switch would be also important for the explanation of the differences between NECs and TECs, as angiogenic factors, cytokines and growth factors secreted into the tumor microenvironment may cause genetic instability. In this review, we focus on the epigenetic differences between the two endothelial lineages, which provide a possible window for pharmacological targeting of TECs.

Modulation of Cellular Biochemistry, Epigenetics and Metabolomics by Ketone Bodies. Implications of the Ketogenic Diet in the Physiology of the Organism and Pathological States.

Ketone bodies (KBs), comprising ß-hydroxybutyrate, acetoacetate and acetone, are a set of fuel molecules serving as an alternative energy source to glucose. KBs are mainly produced by the liver from fatty acids during periods of fasting, and prolonged or intense physical activity. In diabetes, mainly type-1, ketoacidosis is the pathological response to glucose malabsorption. Endogenous production of ketone bodies is promoted by consumption of a ketogenic diet (KD), a diet virtually devoid of carbohydrates. Despite its recently widespread use, the systemic impact of KD is only partially understood, and ranges from physiologically beneficial outcomes in particular circumstances to potentially harmful effects. Here, we firstly review ketone body metabolism and molecular signaling, to then link the understanding of ketone bodies' biochemistry to controversies regarding their putative or proven medical benefits. We overview the physiological consequences of ketone bodies' consumption, focusing on (i) KB-induced histone post-translational modifications, particularly ß-hydroxybutyrylation and acetylation, which appears to be the core epigenetic mechanisms of activity of ß-hydroxybutyrate to modulate inflammation; (ii) inflammatory responses to a KD; (iii) proven benefits of the KD in the context of neuronal disease and cancer; and (iv) consequences of the KD's application on cardiovascular health and on physical performance.

Effects of ketogenic diet and ketone bodies on the cardiovascular system: Concentration matters.

Ketone bodies have emerged as central mediators of metabolic health, and multiple beneficial effects of a ketogenic diet, impacting metabolism, neuronal pathologies and, to a certain extent, tumorigenesis, have been reported both in animal models and clinical research. Ketone bodies, endogenously produced by the liver, act pleiotropically as metabolic intermediates, signaling molecules, and epigenetic modifiers. The endothelium and the vascular system are central regulators of the organism's metabolic state and become dysfunctional in cardiovascular disease, atherosclerosis, and diabetic micro- and macrovascular complications. As physiological circulating ketone bodies can attain millimolar concentrations, the endothelium is the first-line cell lineage exposed to them. While in diabetic ketoacidosis high ketone body concentrations are detrimental to the vasculature, recent research revealed that ketone bodies in the low millimolar range may exert beneficial effects on endothelial cell (EC) functioning by modulating the EC inflammatory status, senescence, and metabolism. Here, we review the long-held evidence of detrimental cardiovascular effects of ketoacidosis as well as the more recent evidence for a positive impact of ketone bodies-at lower concentrations-on the ECs metabolism and vascular physiology and the subjacent cellular and molecular mechanisms. We also explore arising controversies in the field and discuss the importance of ketone body concentrations in relation to their effects. At low concentration, endogenously produced ketone bodies upon uptake of a ketogenic diet or supplemented ketone bodies (or their precursors) may prove beneficial to ameliorate endothelial function and, consequently, pathologies in which endothelial damage occurs.

Glyphosate affects methylation in the promoter regions of selected tumor suppressors as well as expression of major cell cycle and apoptosis drivers in PBMCs (in vitro study).

We have determined the effect of glyphosate on selected epigenetic parameters and major cell cycle drivers in human peripheral blood mononuclear cells (PBMCs). The cells were incubated with glyphosate at 0.5, 10 and 100 µM. The analysis included: global DNA methylation, methylation in the promoter regions of tumor suppressor genes (P16, P21, TP53) and proto-oncogenes (BCL2, CCND1) by the Real-Time PCR and the expression profile of the indicated genes by Real-Time PCR. The obtained results have revealed significant reduction of global DNA methylation level in PBMCs exposed to glyphosate. Tested compound changed methylation pattern of the P21 and TP53 suppressor gene promoters, but in case of other analyzed genes: P16, BCL2 and CCND1 we did not identify any statistically significant changes. Gene profiling showed that glyphosate changed the expression of genes involved in the regulation of cell cycle and apoptosis. Glyphosate decreased expression of P16 and TP53 as well as an increase in the expression of BCl2, CCND1 and P21. Summing up, our results have shown a potential disturbance in methylation processes and gene expression in human PBMCs exposed to glyphosate, but the observed changes do not prejudge about the final metabolic effects, which are depended on many other factors.

Silencing Lysine-Specific Histone Demethylase 1 (LSD1) Causes Increased HP1-Positive Chromatin, Stimulation of DNA Repair Processes, and Dysregulation of Proliferation by Chk1 Phosphorylation in Human Endothelial Cells.

: The methylation of histone lysine residues modifies chromatin conformation and regulates the expression of genes implicated in cell metabolism. Lysine-specific demethylase 1 (LSD1) is a flavin-dependent monoamine oxidase that can demethylate mono- and dimethylated histone lysines 4 and 9 (H3K4 and H3K9). The removal of methyl groups from the lysine residues of histone and non-histone proteins was found to be an important regulatory factor of cell proliferation. However, its role has not been fully elucidated. In this study, we assessed LSD1-mediated cell cycle progression using a human endothelial cell model. The short hairpin RNA knockdown of LSD1 inhibits the G2/M phase of cell cycle progression by checkpoint kinase 1 (Chk1) phosphorylation (S137). We observed elevated DNA damage, which was consistent with the increased detection of double-strand breaks as well as purines and pyrimidines oxidation, which accompanied the activation of ATR/ATRIP signaling by H2AXS139 phosphorylation. The irreversible pharmacological inhibition of LSD1 by 2-phenylcyclopropylamine (2-PCPA) inactivated its enzymatic activity, causing significant changes in heterochromatin and euchromatin conformation assessed by chromatin assembly factor 1 subunit A (CAF1A) and heterochromatin protein 1 isoform α and γ (HP1α/γ) immunofluorescence analysis. We conclude that the knockdown of LSD1 in endothelial cells leads to increased HP1-positive chromatin, the stimulation of DNA repair processes, and the dysregulation of proliferation machinery.