The role of glutathione peroxidase-1 in health and disease.

Glutathione peroxidase 1 (GPx1) is an important cellular antioxidant enzyme that is found in the cytoplasm and mitochondria of mammalian cells. Like most selenoenzymes, it has a single redox-sensitive selenocysteine amino acid that is important for the enzymatic reduction of hydrogen peroxide and soluble lipid hydroperoxides. Glutathione provides the source of reducing equivalents for its function. As an antioxidant enzyme, GPx1 modulates the balance between necessary and harmful levels of reactive oxygen species. In this review, we discuss how selenium availability and modifiers of selenocysteine incorporation alter GPx1 expression to promote disease states. We review the role of GPx1 in cardiovascular and metabolic health, provide examples of how GPx1 modulates stroke and provides neuroprotection, and consider how GPx1 may contribute to cancer risk. Overall, GPx1 is protective against the development and progression of many chronic diseases; however, there are some situations in which increased expression of GPx1 may promote cellular dysfunction and disease owing to its removal of essential reactive oxygen species.

Selenium, a Micronutrient That Modulates Cardiovascular Health via Redox Enzymology.

Selenium (Se) is a trace nutrient that promotes human health through its incorporation into selenoproteins in the form of the redox-active amino acid selenocysteine (Sec). There are 25 selenoproteins in humans, and many of them play essential roles in the protection against oxidative stress. Selenoproteins, such as glutathione peroxidase and thioredoxin reductase, play an important role in the reduction of hydrogen and lipid hydroperoxides, and regulate the redox status of Cys in proteins. Emerging evidence suggests a role for endoplasmic reticulum selenoproteins, such as selenoproteins K, S, and T, in mediating redox homeostasis, protein modifications, and endoplasmic reticulum stress. Selenoprotein P, which functions as a carrier of Se to tissues, also participates in regulating cellular reactive oxygen species. Cellular reactive oxygen species are essential for regulating cell growth and proliferation, protein folding, and normal mitochondrial function, but their excess causes cell damage and mitochondrial dysfunction, and promotes inflammatory responses. Experimental evidence indicates a role for individual selenoproteins in cardiovascular diseases, primarily by modulating the damaging effects of reactive oxygen species. This review examines the roles that selenoproteins play in regulating vascular and cardiac function in health and disease, highlighting their antioxidant and redox actions in these processes.

A genome-wide positioning systems network algorithm for in silico drug repurposing.

Recent advances in DNA/RNA sequencing have made it possible to identify new targets rapidly and to repurpose approved drugs for treating heterogeneous diseases by the 'precise' targeting of individualized disease modules. In this study, we develop a Genome-wide Positioning Systems network (GPSnet) algorithm for drug repurposing by specifically targeting disease modules derived from individual patient's DNA and RNA sequencing profiles mapped to the human protein-protein interactome network. We investigate whole-exome sequencing and transcriptome profiles from ~5,000 patients across 15 cancer types from The Cancer Genome Atlas. We show that GPSnet-predicted disease modules can predict drug responses and prioritize new indications for 140 approved drugs. Importantly, we experimentally validate that an approved cardiac arrhythmia and heart failure drug, ouabain, shows potential antitumor activities in lung adenocarcinoma by uniquely targeting a HIF1α/LEO1-mediated cell metabolism pathway. In summary, GPSnet offers a network-based, in silico drug repurposing framework for more efficacious therapeutic selections.

Early pregnancy vitamin D status and risk of preeclampsia.

BACKGROUND: Low vitamin D status in pregnancy was proposed as a risk factor of preeclampsia. METHODS: We assessed the effect of vitamin D supplementation (4,400 vs. 400 IU/day), initiated early in pregnancy (10-18 weeks), on the development of preeclampsia. The effects of serum vitamin D (25-hydroxyvitamin D [25OHD]) levels on preeclampsia incidence at trial entry and in the third trimester (32-38 weeks) were studied. We also conducted a nested case-control study of 157 women to investigate peripheral blood vitamin D-associated gene expression profiles at 10 to 18 weeks in 47 participants who developed preeclampsia. RESULTS: Of 881 women randomized, outcome data were available for 816, with 67 (8.2%) developing preeclampsia. There was no significant difference between treatment (N = 408) or control (N = 408) groups in the incidence of preeclampsia (8.08% vs. 8.33%, respectively; relative risk: 0.97; 95% CI, 0.61-1.53). However, in a cohort analysis and after adjustment for confounders, a significant effect of sufficient vitamin D status (25OHD ≥30 ng/ml) was observed in both early and late pregnancy compared with insufficient levels (25OHD <30 ng/ml) (adjusted odds ratio, 0.28; 95% CI, 0.10-0.96). Differential expression of 348 vitamin D-associated genes (158 upregulated) was found in peripheral blood of women who developed preeclampsia (FDR <0.05 in the Vitamin D Antenatal Asthma Reduction Trial [VDAART]; P < 0.05 in a replication cohort). Functional enrichment and network analyses of this vitamin D-associated gene set suggests several highly functional modules related to systematic inflammatory and immune responses, including some nodes with a high degree of connectivity. CONCLUSIONS: Vitamin D supplementation initiated in weeks 10-18 of pregnancy did not reduce preeclampsia incidence in the intention-to-treat paradigm. However, vitamin D levels of 30 ng/ml or higher at trial entry and in late pregnancy were associated with a lower risk of preeclampsia. Differentially expressed vitamin D-associated transcriptomes implicated the emergence of an early pregnancy, distinctive immune response in women who went on to develop preeclampsia. TRIAL REGISTRATION: ClinicalTrials.gov NCT00920621. FUNDING: Quebec Breast Cancer Foundation and Genome Canada Innovation Network. This trial was funded by the National Heart, Lung, and Blood Institute. For details see Acknowledgments.

Systems Pharmacology and Rational Polypharmacy: Nitric Oxide-Cyclic GMP Signaling Pathway as an Illustrative Example and Derivation of the General Case.

Impaired nitric oxide (NO˙)-cyclic guanosine 3', 5'-monophosphate (cGMP) signaling has been observed in many cardiovascular disorders, including heart failure and pulmonary arterial hypertension. There are several enzymatic determinants of cGMP levels in this pathway, including soluble guanylyl cyclase (sGC) itself, the NO˙-activated form of sGC, and phosphodiesterase(s) (PDE). Therapies for some of these disorders with PDE inhibitors have been successful at increasing cGMP levels in both cardiac and vascular tissues. However, at the systems level, it is not clear whether perturbation of PDE alone, under oxidative stress, is the best approach for increasing cGMP levels as compared with perturbation of other potential pathway targets, either alone or in combination. Here, we develop a model-based approach to perturbing this pathway, focusing on single reactions, pairs of reactions, or trios of reactions as targets, then monitoring the theoretical effects of these interventions on cGMP levels. Single perturbations of all reaction steps within this pathway demonstrated that three reaction steps, including the oxidation of sGC, NO˙ dissociation from sGC, and cGMP degradation by PDE, exerted a dominant influence on cGMP accumulation relative to other reaction steps. Furthermore, among all possible single, paired, and triple perturbations of this pathway, the combined perturbations of these three reaction steps had the greatest impact on cGMP accumulation. These computational findings were confirmed in cell-based experiments. We conclude that a combined perturbation of the oxidatively-impaired NO˙-cGMP signaling pathway is a better approach to the restoration of cGMP levels as compared with corresponding individual perturbations. This approach may also yield improved therapeutic responses in other complex pharmacologically amenable pathways.

Inhibition of cellular methyltransferases promotes endothelial cell activation by suppressing glutathione peroxidase 1 protein expression.

S-adenosylhomocysteine (SAH) is a negative regulator of most methyltransferases and the precursor for the cardiovascular risk factor homocysteine. We have previously identified a link between the homocysteine-induced suppression of the selenoprotein glutathione peroxidase 1 (GPx-1) and endothelial dysfunction. Here we demonstrate a specific mechanism by which hypomethylation, promoted by the accumulation of the homocysteine precursor SAH, suppresses GPx-1 expression and leads to inflammatory activation of endothelial cells. The expression of GPx-1 and a subset of other selenoproteins is dependent on the methylation of the tRNA(Sec) to the Um34 form. The formation of methylated tRNA(Sec) facilitates translational incorporation of selenocysteine at a UGA codon. Our findings demonstrate that SAH accumulation in endothelial cells suppresses the expression of GPx-1 to promote oxidative stress. Hypomethylation stress, caused by SAH accumulation, inhibits the formation of the methylated isoform of the tRNA(Sec) and reduces GPx-1 expression. In contrast, under these conditions, the expression and activity of thioredoxin reductase 1, another selenoprotein, is increased. Furthermore, SAH-induced oxidative stress creates a proinflammatory activation of endothelial cells characterized by up-regulation of adhesion molecules and an augmented capacity to bind leukocytes. Taken together, these data suggest that SAH accumulation in endothelial cells can induce tRNA(Sec) hypomethylation, which alters the expression of selenoproteins such as GPx-1 to contribute to a proatherogenic endothelial phenotype.

Both selenium deficiency and modest selenium supplementation lead to myocardial fibrosis in mice via effects on redox-methylation balance.

SCOPE: Selenium has complex effects in vivo on multiple homeostatic mechanisms such as redox balance, methylation balance, and epigenesis, via its interaction with the methionine-homocysteine cycle. In this study, we examined the hypothesis that selenium status would modulate both redox and methylation balance and thereby modulate myocardial structure and function. METHODS AND RESULTS: We examined the effects of selenium-deficient (<0.025 mg/kg), control (0.15 mg/kg), and selenium-supplemented (0.5 mg/kg) diets on myocardial histology, biochemistry and function in adult C57/BL6 mice. Selenium deficiency led to reactive myocardial fibrosis and systolic dysfunction accompanied by increased myocardial oxidant stress. Selenium supplementation significantly reduced methylation potential, DNA methyltransferase activity and DNA methylation. In mice fed the supplemented diet, inspite of lower oxidant stress, myocardial matrix gene expression was significantly altered resulting in reactive myocardial fibrosis and diastolic dysfunction in the absence of myocardial hypertrophy. CONCLUSION: Our results indicate that both selenium deficiency and modest selenium supplementation leads to a similar phenotype of abnormal myocardial matrix remodeling and dysfunction in the normal heart. The crucial role selenium plays in maintaining the balance between redox and methylation pathways needs to be taken into account while optimizing selenium status for prevention and treatment of heart failure.

High-resolution imaging of selenium in kidneys: a localized selenium pool associated with glutathione peroxidase 3.

AIM: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.

Both maximal expression of selenoproteins and selenoprotein deficiency can promote development of type 2 diabetes-like phenotype in mice.

Selenium (Se) is an essential trace element in mammals that has been shown to exert its function through selenoproteins. Whereas optimal levels of Se in the diet have important health benefits, a recent clinical trial has suggested that supplemental intake of Se above the adequate level potentially may raise the risk of type 2 diabetes mellitus. However, the molecular mechanisms for the effect of dietary Se on the development of this disease are not understood. In the present study, we examined the contribution of selenoproteins to increased risk of developing diabetes using animal models. C57BL/6J mice (n=6-7 per group) were fed either Se-deficient Torula yeast-based diet or diets supplemented with 0.1 and 0.4 parts per million Se. Our data show that mice maintained on an Se-supplemented diet develop hyperinsulinemia and have decreased insulin sensitivity. These effects are accompanied by elevated expression of a selective group of selenoproteins. We also observed that reduced synthesis of these selenoproteins caused by overexpression of an i(6)A(-) mutant selenocysteine tRNA promotes glucose intolerance and leads to a diabetes-like phenotype. These findings indicate that both high expression of selenoproteins and selenoprotein deficiency may dysregulate glucose homeostasis and suggest a role for selenoproteins in development of diabetes.

Regulation of the extracellular antioxidant selenoprotein plasma glutathione peroxidase (GPx-3) in mammalian cells.

Plasma glutathione peroxidase (GPx-3) is a selenocysteine-containing extracellular antioxidant protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides. Selenoprotein expression involves the alternate recognition of a UGA codon as a selenocysteine codon and requires signals in the 3'-untranslated region (UTR), including a selenocysteine insertion sequence (SECIS), as well as specific translational cofactors. To ascertain regulatory determinants of GPx-3 expression and function, we generated recombinant GPx-3 (rGPX-3) constructs with various 3'-UTR, as well as a Sec73Cys mutant. In transfected Cos7 cells, the Sec73Cys mutant was expressed at higher levels than the wild type rGPx-3, although the wild type rGPx-3 had higher specific activity, similar to plasma purified GPx-3. A 3'-UTR with only the SECIS was insufficient for wild type rGPx-3 protein expression. Selenocompound supplementation and co-transfection with SECIS binding protein 2 increased wild type rGPx-3 expression. These results demonstrate the importance of translational mechanisms in GPx-3 expression.

Selenium supplementation improves antioxidant capacity in vitro and in vivo in patients with coronary artery disease The SElenium Therapy in Coronary Artery disease Patients (SETCAP) Study.

BACKGROUND: Selenium is a central determinant of antioxidative glutathione peroxidase 1 (GPx-1) expression and activity. The relevance of selenium supplementation on GPx-1 in coronary artery disease (CAD) needs to be established. We assessed the effect of selenium supplementation on GPx-1 in cell culture and on endothelial function in a prospective clinical trial. METHODS: Human coronary artery endothelial cells were incubated with 5.78 to 578 nmol/L sodium selenite, Se-methyl-selenocysteine hydrochloride, or seleno-l-methionine. Glutathione peroxidase 1 mRNA and protein expression and activity were measured. Coronary artery disease patients (n = 465) with impaired endothelial function (flow-mediated dilation [FMD] <8%) were randomly assigned to receive 200 or 500 microg sodium selenite daily or matching placebo during a 12-week period. We tested the effect on red blood cell GPx-1 activity and brachial artery FMD. Furthermore, differences in biomarkers of oxidative stress and inflammation were measured. RESULTS: Sodium selenite and Se-methyl-selenocysteine hydrochloride increased GPx-1 protein and activity in a dose-dependent manner (P < .0001). The intention-to-treat groups comprised 433 CAD patients. Glutathione peroxidase 1 activity increased from 37.0 U/gHb (31.3-41.7) to 41.1 U/gHb (35.2-48.4) (P < .0001) in the 200 microg and from 38.1 U/gHb (33.2-43.8) to 42.6 U/gHb (35.0-49.1) (P < .0001) in the 500 microg sodium selenite group treated for 12-weeks. No relevant changes were observed for FMD or biomarkers of oxidative stress and inflammation. CONCLUSIONS: Sodium selenite supplementation increases GPx-1 activity in endothelial cells and in CAD patients. Future studies have to demonstrate whether long-term CAD outcome can be improved.

Aminoglycosides decrease glutathione peroxidase-1 activity by interfering with selenocysteine incorporation.

Cellular glutathione peroxidase is a key intracellular antioxidant enzyme that contains a selenocysteine residue at its active site. Selenium, a selenocysteine incorporation sequence in the 3'-untranslated region of the glutathione peroxidase mRNA, and other translational cofactors are necessary for "read-through" of a UGA stop codon that specifies selenocysteine incorporation. Aminoglycoside antibiotics facilitate read-through of premature stop codons in prokayotes and eukaryotes. We studied the effects of G418, an aminoglycoside, on cellular glutathione peroxidase expression and function in mammalian cells. Insertion of a selenocysteine incorporation element along with a UGA codon into a reporter construct allows for read-through only in the presence of selenium. G418 increased read-through in selenium-replete cells as well as in the absence of selenium. G418 treatment increased immunodetectable endogenous or recombinant glutathione peroxidase but reduced the specific activity of the enzyme. Tandem mass spectrometry experiments indicated that G418 caused a substitution of l-arginine for selenocysteine. These data show that G418 can affect the biosynthesis of this key antioxidant enzyme by promoting substitution at the UGA codon.

Homocysteine down-regulates cellular glutathione peroxidase (GPx1) by decreasing translation.

Hyperhomocysteinemia contributes to vascular dysfunction and an increase in the risk of cardiovascular disease. An elevated level of homocysteine in vivo and in cell culture systems results in a decrease in the activity of cellular glutathione peroxidase (GPx1), an intracellular antioxidant enzyme that reduces hydrogen peroxide and lipid peroxides. In this study, we show that homocysteine interferes with GPx1 protein expression without affecting transcript levels. Expression of the selenocysteine (SEC)-containing GPx1 protein requires special translational cofactors to "read-through" a UGA-stop codon that specifies SEC incorporation at the active site of the enzyme. These factors include a selenocysteine incorporation sequence (SECIS) in the 3'-untranslated region of the GPx1 mRNA and cofactors involved in the biosynthesis and translational insertion of SEC. To monitor SEC incorporation, we used a reporter gene system that has a UGA codon within the protein-coding region of the luciferase mRNA. Addition of either the GPx1 or GPx3 SECIS element in the 3'-untranslated region of the luciferase gene stimulated read-through by 6-11-fold in selenium-replete cells; absence of selenium prevented translation. To alter cellular homocysteine production, we used methionine in the presence of aminopterin, a folate antagonist, co-administered with hypoxanthine and thymidine (HAT/Met). This treatment increased homocysteine levels in the media by 30% (p < 0.01) and decreased GPx1 enzyme activity by 45% (p = 0.0028). HAT/Met treatment decreased selenium-mediated read-through significantly (p < 0.001) in luciferase constructs containing the GPx1 or GPx3 SECIS element; most importantly, the suppression of selenium-dependent read-through was similar whether an SV40 promoter or the GPx1 promoter was used to drive transcription of the SECIS-containing constructs. Furthermore, HAT/Met had no effect on steady-state GPx1 mRNA levels but decreased GPx1 protein levels, suggesting that this effect is not transcriptionally mediated. These data support the conclusion that homocysteine decreases GPx1 activity by altering the translational mechanism essential for the synthesis of this selenocysteine-containing protein.

Increased myocardial dysfunction after ischemia-reperfusion in mice lacking glucose-6-phosphate dehydrogenase.

BACKGROUND: Free radical injury contributes to cardiac dysfunction during ischemia-reperfusion. Detoxification of free radicals requires maintenance of reduced glutathione (GSH) by NADPH. The principal mechanism responsible for generating NADPH and maintaining GSH during periods of myocardial ischemia-reperfusion remains unknown. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, generates NADPH in a reaction linked to the de novo production of ribose. We therefore hypothesized that G6PD is essential for maintaining GSH levels and protecting the heart during ischemia-reperfusion injury. METHODS AND RESULTS: Susceptibility to myocardial ischemia-reperfusion injury was determined in Langendorff-perfused hearts isolated from wild-type mice (WT) and mice lacking G6PD (G6PD(def)) (20% of WT myocardial G6PD activity). During global zero-flow ischemia, cardiac function was similar between WT and G6PD(def) hearts. On reperfusion, however, cardiac relaxation and contractile performance were greatly impaired in G6PD(def) myocardium, as demonstrated by elevated end-diastolic pressures and decreased percent recovery of developed pressure relative to WT hearts. Contractile dysfunction in G6PD(def) hearts was associated with depletion of total glutathione stores and impaired generation of GSH from its oxidized form. Increased ischemia-reperfusion injury in G6PD(def) hearts was reversed by treatment with the antioxidant MnTMPyP but unaffected by supplementation of ribose stores. CONCLUSIONS: These results demonstrate that G6PD is an essential myocardial antioxidant enzyme, required for maintaining cellular glutathione levels and protecting against oxidative stress-induced cardiac dysfunction during ischemia-reperfusion.

Homocysteine and atherothrombosis: diagnosis and treatment.

Hyperhomocysteinemia has long been recognized as a risk factor for cardiovascular disease. Many cross-sectional and retrospective case-control studies have shown an association between elevated total homocysteine levels and coronary, peripheral, and cerebral vascular disease; prospective studies, however, have been inconsistent. Overall, there is evidence to suggest a modest association between elevated homocysteine levels and cardiovascular disease risk. Folate supplementation has been shown to reduce plasma homocysteine even when levels are in the normal range. Clinical studies suggest that lowering plasma homocysteine may improve endothelial dysfunction, a marker of atherothrombotic risk. The long-term effects of folate supplementation on homocysteine levels and cardiovascular disease risk await the results of ongoing clinical trials. However, several recent studies suggest a benefit for reduction of plasma homocysteine levels, as individuals with lower homocysteine have reduced cardiovascular event rates.