Sirtuin 1 and Sirtuin 3 in Granulosa Cell Tumors

Sirtuins (SIRTs) are NAD⁺-dependent deacetylases that regulate proliferation and cell death. In the human ovary, granulosa cells express sirtuin 1 (SIRT1), which has also been detected in human tumors derived from granulosa cells, i.e., granulosa cell tumors (GCTs), and in KGN cells. KGN cells are an established cellular model for the majority of GCTs and were used to explore the role of SIRT1. The SIRT1 activator SRT2104 increased cell proliferation. By contrast, the inhibitor EX527 reduced cell numbers, without inducing apoptosis. These results were supported by the outcome of siRNA-mediated silencing studies. A tissue microarray containing 92 GCTs revealed nuclear and/or cytoplasmic SIRT1 staining in the majority of the samples, and also, SIRT2-7 were detected in most samples. The expression of SIRT1–7 was not correlated with the survival of the patients; however, SIRT3 and SIRT7 expression was significantly correlated with the proliferation marker Ki-67, implying roles in tumor cell proliferation. SIRT3 was identified by a proteomic analysis as the most abundant SIRT in KGN. The results of the siRNA-silencing experiments indicate involvement of SIRT3 in proliferation. Thus, several SIRTs are expressed by GCTs, and SIRT1 and SIRT3 are involved in the growth regulation of KGN. If transferable to GCTs, these SIRTs may represent novel drug targets.     

Increased Histone Acetylation and Decreased Expression of Specific Histone Deacetylases in Ultraviolet-Irradiated and Intrinsically Aged Human Skin In Vivo

Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.     

Five Days Periodic Fasting Elevates Levels of Longevity Related Christensenella and Sirtuin Expression in Humans

Periodic fasting (PF) is an increasingly popular approach that assists in the management of metabolic and inflammatory diseases as well as in preventing mechanisms involved in aging. However, little is known about the effects of fasting on gut microbiota and its impact on the epigenetic regulation of metabolically relevant enzymes, especially sirtuins (SIRTs). We analyzed the effect of periodic fasting on the human gut microbiota, SIRTs expression, and mitochondrial content in 51 males and females. The participants fasted under supervision for five consecutive days following the Buchinger fasting guidelines. Ketogenesis, selected mRNAs, miRNAs, mitochondrial (mt) DNA, and gut composition were analyzed before and after PF. PF triggered a significant switch in metabolism, as indicated by the increase in ß-hydroxybutyrate (BHB) and pyruvate dehydrogenase kinase isoform 4 (PDK4) expression in the capillary blood. MtDNA, SIRT1, SIRT3, and miRlet7b-5p expression in blood cells were elevated, whereas SIRT6 and miR125b-5p were not affected. Following fasting, gut microbiota diversity increased, and a statistically significant correlation between SIRT1 gene expression and the abundance of Prevotella and Lactobacillus was detected. The abundance of longevity related Christensenella species increased after fasting and inversely correlated with age as well as body mass index (BMI). Thus, this represents the first study that showing that fasting not only changes the composition of the gut microbiota, making it more diverse, but also affects SIRT expression in humans.     

A Fluorescent Probe for Imaging Sirtuin Activity in Living Cells,Based on One‐Step Cleavage of the Dabcyl Quencher

Sirtuins (SIRTs) are a family of NAD⁺‐dependent histone deacetylases. In mammals, dysfunction of SIRTs is associated with age‐related metabolic diseases and cancers, so SIRT modulators are considered attractive therapeutic targets. However, current screening methodologies are problematic, and no tools for imaging endogenous SIRT activity in living cells have been available until now. In this work we present a series of simple and highly sensitive new SIRT activity probes. Fluorescence of these probes is activated by SIRT‐mediated hydrolytic release of a 4‐(4‐dimethylaminophenylazo)benzoyl (Dabcyl)‐based FRET quencher moiety from the ?‐amino group of lysine in a nonapeptide derived from histone H3K9 and bearing a C‐terminal fluorophore. The probe SFP3 detected activities of SIRT1, ‐2, ‐3, and ‐6, which exhibit deacylase activities towards long‐chain fatty acyl groups. We then truncated the molecular structure of SFP3 in order to improve both its stability to peptidases and its membrane permeability, and developed probe KST‐F, which showed specificity for SIRT1 over SIRT2 and SIRT3. We show that KST‐F can visualize endogenous SIRT1 activity in living cells.     

Grenzflächen in der Medizin

Interfaces in medicine. Some 6 × 10¹³ individual cells and a variety of differential structures make human organism a highly complex interfacial system. Interfaces play a predominant role in almost all areas of medicine. A number of topical developments which have led to important advances in medical research and practice are selected from the wealth of known phenomena.     

Biomarkers and Mechanisms of Oxidative Stress—Last 20 Years of Research with an Emphasis on Kidney Damage and Renal Transplantation

Oxidative stress is an imbalance between pro- and antioxidants that adversely influences the organism in various mechanisms and on many levels. Oxidative damage occurring concomitantly in many cellular structures may cause a deterioration of function, including apoptosis and necrosis. The damage leaves a molecular “footprint”, which can be detected by specific methodology, using certain oxidative stress biomarkers. There is an intimate relationship between oxidative stress, inflammation, and functional impairment, resulting in various diseases affecting the entire human body. In the current narrative review, we strengthen the connection between oxidative stress mechanisms and their active compounds, emphasizing kidney damage and renal transplantation. An analysis of reactive oxygen species (ROS), antioxidants, products of peroxidation, and finally signaling pathways gives a lot of promising data that potentially will modify cell responses on many levels, including gene expression. Oxidative damage, stress, and ROS are still intensively exploited research subjects. We discuss compounds mentioned earlier as biomarkers of oxidative stress and present their role documented during the last 20 years of research. The following keywords and MeSH terms were used in the search: oxidative stress, kidney, transplantation, ischemia-reperfusion injury, IRI, biomarkers, peroxidation, and treatment.     

A Molecular Perspective on Sirtuin Activity

The protein acetylation of either the α-amino groups of amino-terminal residues or of internal lysine or cysteine residues is one of the major posttranslational protein modifications that occur in the cell with repercussions at the protein as well as at the metabolome level. The lysine acetylation status is determined by the opposing activities of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), which add and remove acetyl groups from proteins, respectively. A special group of KDACs, named sirtuins, that require NAD⁺ as a substrate have received particular attention in recent years. They play critical roles in metabolism, and their abnormal activity has been implicated in several diseases. Conversely, the modulation of their activity has been associated with protection from age-related cardiovascular and metabolic diseases and with increased longevity. The benefits of either activating or inhibiting these enzymes have turned sirtuins into attractive therapeutic targets, and considerable effort has been directed toward developing specific sirtuin modulators. This review summarizes the protein acylation/deacylation processes with a special focus on the current developments in the sirtuin research field.     

Protective action of CLA against oxidative inactivation of paraoxonase 1, an antioxidant enzyme

The effect of CLA on paraoxonase 1 (PON1), one of the antioxidant proteins associated with HDL, was investigated for its protective action against oxidative inactivation as well as its stabilization activity. When cis-9 (c9),trans-11 (t11)-CLA and t10,c12-CLA were examined for their protective activity against ascorbate/Cu²⁻-induced inactivation of PON1 in the presence of Ca²⁺, two CLA isomers exhibited a remarkable protection (E _max, 71–74%) in a concentration-dependent manner (50% effective concentration, 3–4 μM), characterized by a saturation pattern. Such a protective action was also reproduced with oleic acid, but not linoleic acid. Rather, linoleic acid antagonized the protective action of CLA isomers in a noncompetitive fashion. Additionally, the two CLA isomers also protected PON1 from oxidative inactivation by H₂O₂ or cumene hydroperoxide. The concentration-dependent protective action of CLA against various oxidative inactivation systems suggests that the protective action of CLA isomers may be mediated through their selective binding to a specific binding site in a PON1 molecule. Separately, the inactivation of PON1 by p-hydroxymercuribenzoate (PHMB), a modifier of the cysteine residue, was also prevented by CLA isomers, suggesting the possible existence of the cysteine residue in the binding site of CLA. The c9,t11-CLA isomer seems to be somewhat more effective than t10,c12-CLA in protecting against the inactivation of PON1 by either peroxides or PHMB, in contrast to the similar efficacy of these two CLA isomers in preventing ascorbate/Cu²⁺-induced inactivation of PON1. Separately, CLA isomers successfully stabilized PON1, but not linoleic acid. These data suggest that the two CLA isomers may play a beneficial role in protecting PON1 from oxidative inactivation as well as in its stabilization.     

Oxidative Stress in the Pathogenesis of Keratoconus and Fuchs Endothelial Corneal Dystrophy

Due to its localization and function, the cornea is regularly exposed to sunlight and atmospheric oxygen, mainly dioxygen, which produce reactive oxygen species (ROS). Therefore, corneal cells are particularly susceptible to oxidative stress. The accumulation of ROS in the cornea may affect signal transduction, proliferation and may also promote cell death. The cornea has several enzymatic and non-enzymatic antioxidants involved in ROS scavenging, but in certain conditions they may not cope with oxidative stress, leading to diseases of the eye. Keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD) are multifactorial diseases of the cornea, in which pathogenesis is not fully understood. However, increased levels of oxidative stress markers detected in these disorders indicate that oxidative stress may play an important role in their development and progression. These markers are: (i) decreased levels of non-enzymatic antioxidants, and (ii) decreased expression of genes encoding antioxidative enzymes, including thioredoxin reductase, peroxiredoxins, superoxide dismutase, glutathione S-transferase, and aldehyde dehydrogenase. Moreover, the FECD endothelium displays higher levels of oxidative DNA damage, especially in mitochondrial DNA (mtDNA), whereas KC cornea shows abnormal levels of some components of oxidative phosphorylation encoded by mtDNA. In this review we present some considerations and results of experiments supporting the thesis on the important role of oxidative stress in KC and FECD pathology.     

15-Lipoxygenase-Mediated Modification of HDL3 Impairs eNOS Activation in Human Endothelial Cells

Caveolae are cholesterol and glycosphingolipids-enriched microdomains of plasma membranes. Caveolin-1 represents the major structural protein of caveolae, that also contain receptors and molecules involved in signal transduction pathways. Caveolae are particularly abundant in endothelial cells, where they play important physiological and pathological roles in regulating endothelial cell functions. Several molecules with relevant functions in endothelial cells are localized in caveolae, including endothelial nitric oxide synthase (eNOS), which regulates the production of nitric oxide, and scavenger receptor class B type I (SR-BI), which plays a key role in the induction of eNOS activity mediated by high density lipoproteins (HDL). HDL have several atheroprotective functions, including a positive effect on endothelial cells, as it is a potent agonist of eNOS through the interaction with SR-BI. However, the oxidative modification of HDL may impair their protective role. In the present study we evaluated the effect of 15-lipoxygenase-mediated modification of HDL₃ on the expression and/or activity of some proteins localized in endothelial caveolae and involved in the nitric oxide generation pathway. We found that after modification, HDL₃ failed to activate eNOS and to induce NO production, due to both a reduced ability to interact with its own receptor SR-BI and to a reduced expression of SR-BI in cells exposed to modified HDL. These findings suggest that modification of HDL may reduce its endothelial-protective role also by interfering with vasodilatory function of HDL.     

Brief Review of Endometriosis and the Role of Trace Elements

Endometriosis is a chronic, estrogen-dependent, inflammatory condition that is defined as the presence of endometrial glands and stroma outside the uterine cavity. Despite the progress in research into the mechanisms leading to the development of endometriosis, its cause has not yet been established. It seems to be possible that the formation of oxidative stress may be one of the main causes of the development of endometriosis. There is much research that studies the potential role of trace elements in the appearance of endometrial-like lesions. Most studies focus on assessing the content of selected trace elements in the blood, urine, or peritoneal fluid in women with endometriosis. Meanwhile, little is known about the content of these elements in endometrial-like implants, which may be helpful in developing the theory of endometriosis. Investigations that are more comprehensive are needed to confirm a hypothesis that some trace elements play a role in the pathomechanism of endometriosis.     

Insulin and α-Tocopherol Enhance the Protective Effect of Each Other on Brain Cortical Neurons under Oxidative Stress Conditions and in Rat Two-Vessel Forebrain Ischemia/Reperfusion Injury

Clinical trials show that insulin administered intranasally is a promising drug to treat neurodegenerative diseases, but at high doses its use may result in cerebral insulin resistance. Identifying compounds which could enhance the protective effects of insulin, may be helpful to reduce its effective dose. Our aim was thus to study the efficiency of combined use of insulin and α-tocopherol (α-T) to increase the viability of cultured cortical neurons under oxidative stress conditions and to normalize the metabolic disturbances caused by free radical reaction activation in brain cortex of rats with two-vessel forebrain ischemia/reperfusion injury. Immunoblotting, flow cytometry, colorimetric, and fluorometric techniques were used. α-T enhanced the protective and antioxidative effects of insulin on neurons in oxidative stress, their effects were additive. At the late stages of oxidative stress, the combined action of insulin and α-T increased Akt-kinase activity, inactivated GSK-3beta and normalized ERK1/2 activity in cortical neurons, it was more effective than either drug action. In the brain cortex, ischemia/reperfusion increased the lipid peroxidation product content and caused Na⁺,K⁺-ATPase oxidative inactivation. Co-administration of insulin (intranasally, 0.25 IU/rat) and α-T (orally, 50 mg/kg) led to a more pronounced normalization of the levels of Schiff bases, conjugated dienes and trienes and Na⁺,K⁺-ATPase activity than administration of each drug alone. Thus, α-T enhances the protective effects of insulin on cultured cortical neurons in oxidative stress and in the brain cortex of rats with cerebral ischemia/reperfusion injury.     

Modulatory Effect of Myokines on Reactive Oxygen Species in Ischemia/Reperfusion

There is a growing body of evidence showing the importance of physical activity against acute ischemic events in various organs. Ischemia/reperfusion injury (I/R) is characterized by tissue damage as a result of restriction and subsequent restoration of blood supply to an organ. Oxidative stress due to increased reactive oxygen species formation and/or insufficient antioxidant defense is considered to play an important role in I/R. Physical activity not only decreases the general risk factors for ischemia but also confers direct anti-ischemic protection via myokine production. Myokines are skeletal muscle-derived cytokines, representing multifunctional communication channels between the contracting skeletal muscle and other organs through an endocrine manner. In this review, we discuss the most prominent members of the myokines (i.e., brain-derived neurotrophic factor (BDNF), cathepsin B, decorin, fibroblast growth factors-2 and -21, follistatin, follistatin-like, insulin-like growth factor-1; interleukin-6, interleukin-7, interleukin-15, irisin, leukemia inhibitory factor, meteorin-like, myonectin, musclin, myostatin, and osteoglycin) with a particular interest in their potential influence on reactive oxygen and nitrogen species formation or antioxidant capacity. A better understanding of the mechanism of action of myokines and particularly their participation in the regulation of oxidative stress may widen their possible therapeutic use and, thereby, may support the fight against I/R.     

Intense Pulsed Light Attenuates UV-Induced Hyperimmune Response and Pigmentation in Human Skin Cells

The skin of an organism is affected by various environmental factors and fights against aging stress via mechanical and biochemical responses. Photoaging induced by ultraviolet B (UVB) irradiation is common and is the most vital factor in the senescence phenotype of skin, and so, suppression of UVB stress-induced damage is critical. To lessen the UVB-induced hyperimmune response and hyperpigmentation, we investigated the ameliorative effects of intense pulsed light (IPL) treatment on the photoaged phenotype of skin cells. Normal human epidermal keratinocytes and human epidermal melanocytes were exposed to 20 mJ/cm² of UVB. After UVB irradiation, the cells were treated with green (525–530 nm) and yellow (585–592 nm) IPL at various time points prior to the harvest step. Subsequently, various signs of excessive immune response, including expression of proinflammatory and melanogenic genes and proteins, cellular oxidative stress level, and antioxidative enzyme activity, were examined. We found that IPL treatment reduced excessive cutaneous immune reactions by suppressing UVB-induced proinflammatory cytokine expression. IPL treatment prevented hyperpigmentation, and combined treatment with green and yellow IPL synergistically attenuated both processes. IPL treatment may exert protective effects against UVB injury in skin cells by attenuating inflammatory cytokine and melanogenic gene overexpression, possibly by reducing intracellular oxidative stress. IPL treatment also preserves antioxidative enzyme activity under UVB irradiation. This study suggests that IPL treatment is a useful strategy against photoaging, and provides evidence supporting clinical approaches with non-invasive light therapy.     

Erythrocyte Deformability and Na,K-ATPase Activity in Various Pathophysiological Situations and Their Protection by Selected Nutritional Antioxidants in Humans

The physicochemical and functional properties of erythrocytes are worsened in a variety of diseases. Erythrocyte deformability refers to their ability to adjust their shape according to external forces exerted against them in the circulation. It is influenced by the functionality of the Na,K-ATPase enzyme, which is localized in their membranes. The proposed review is focused on knowledge regarding changes in erythrocyte Na,K-ATPase activity, and their impact on erythrocyte deformability in various pathophysiological situations observed exclusively in human studies, as well as on the potential erytroprotective effects of selected natural nutritional antioxidants. A clear link between the erythrocyte properties and the parameters of oxidative stress was observed. The undesirable consequences of oxidative stress on erythrocyte quality and hemorheology could be at least partially prevented by intake of diverse antioxidants occurring naturally in foodstuffs. Despite intensive research concerning the effect of antioxidants, only a small number of investigations on erythrocyte properties in humans is available in databases. It is worth shifting attention from animal and in vitro experiments and focusing more on antioxidant administration in human studies in order to establish what type of antioxidant, in what concentration, and in which individuals it may provide a beneficial effect on the human organism, by protecting erythrocyte properties.     

Impaired mitochondrial respiratory functions and oxidative stress in streptozotocin-induced diabetic rats

We have previously shown a tissue-specific increase in oxidative stress in the early stages of streptozotocin (STZ)-induced diabetic rats. In this study, we investigated oxidative stress-related long-term complications and mitochondrial dysfunctions in the different tissues of STZ-induced diabetic rats (>15 mM blood glucose for 8 weeks). These animals showed a persistent increase in reactive oxygen and nitrogen species (ROS and RNS, respectively) production. Oxidative protein carbonylation was also increased with the maximum effect observed in the pancreas of diabetic rats. The activities of mitochondrial respiratory enzymes ubiquinol: cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV) were significantly decreased while that of NADH:ubiquinone oxidoreductase (Complex I) and succinate:ubiquinone oxidoreductase (Complex II) were moderately increased in diabetic rats, which was confirmed by the increased expression of the 70 kDa Complex II sub-unit. Mitochondrial matrix aconitase, a ROS sensitive enzyme, was markedly inhibited in the diabetic rat tissues. Increased expression of oxidative stress marker proteins Hsp-70 and HO-1 was also observed along with increased expression of nitric oxide synthase. These results suggest that mitochondrial respiratory complexes may play a critical role in ROS/RNS homeostasis and oxidative stress related changes in type 1 diabetes and may have implications in the etiology of diabetes and its complications.     

Crosstalk between MicroRNA and Oxidative Stress in Primary Open-Angle Glaucoma

Reactive oxygen species (ROS) plays a key role in the pathogenesis of primary open-angle glaucoma (POAG), a chronic neurodegenerative disease that damages the trabecular meshwork (TM) cells, inducing apoptosis of the retinal ganglion cells (RGC), deteriorating the optic nerve head, and leading to blindness. Aqueous humor (AH) outflow resistance and intraocular pressure (IOP) elevation contribute to disease progression. Nevertheless, despite the existence of pharmacological and surgical treatments, there is room for the development of additional treatment approaches. The following review is aimed at investigating the role of different microRNAs (miRNAs) in the expression of genes and proteins involved in the regulation of inflammatory and degenerative processes, focusing on the delicate balance of synthesis and deposition of extracellular matrix (ECM) regulated by chronic oxidative stress in POAG related tissues. The neutralizing activity of a couple of miRNAs was described, suggesting effective downregulation of pro-inflammatory and pro-fibrotic signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), transforming growth factor-beta 2 (TGF-β2), Wnt/β-Catenin, and PI3K/AKT. In addition, with regards to the elevated IOP in many POAG patients due to increased outflow resistance, Collagen type I degradation was stimulated by some miRNAs and prevented ECM deposition in TM cells. Mitochondrial dysfunction as a consequence of oxidative stress was suppressed following exposure to different miRNAs. In contrast, increased oxidative damage by inhibiting the mTOR signaling pathway was described as part of the action of selected miRNAs. Summarizing, specific miRNAs may be promising therapeutic targets for lowering or preventing oxidative stress injury in POAG patients.     

Radical Roles for RAGE in the Pathogenesis of Oxidative Stress in Cardiovascular Diseases and Beyond

Oxidative stress is a central mechanism by which the receptor for advanced glycation endproducts (RAGE) mediates its pathological effects. Multiple experimental inquiries in RAGE-expressing cultured cells have demonstrated that ligand-RAGE interaction mediates generation of reactive oxygen species (ROS) and consequent downstream signal transduction and regulation of gene expression. The primary mechanism by which RAGE generates oxidative stress is via activation of NADPH oxidase; amplification mechanisms in the mitochondria may further drive ROS production. Recent studies indicating that the cytoplasmic domain of RAGE binds to the formin mDia1 provide further support for the critical roles of this pathway in oxidative stress; mDia1 was required for activation of rac1 and NADPH oxidase in primary murine aortic smooth muscle cells treated with RAGE ligand S100B. In vivo, in multiple distinct disease models in animals, RAGE action generates oxidative stress and modulates cellular/tissue fate in range of disorders, such as in myocardial ischemia, atherosclerosis, and aneurysm formation. Blockade or genetic deletion of RAGE was shown to be protective in these settings. Indeed, beyond cardiovascular disease, evidence is accruing in human subjects linking levels of RAGE ligands and soluble RAGE to oxidative stress in disorders such as doxorubicin toxicity, acetaminophen toxicity, neurodegeneration, hyperlipidemia, diabetes, preeclampsia, rheumatoid arthritis and pulmonary fibrosis. Blockade of RAGE signal transduction may be a key strategy for the prevention of the deleterious consequences of oxidative stress, particularly in chronic disease.