Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

BACKGROUND: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. METHODS: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. RESULTS: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1ß sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. CONCLUSIONS: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

High glucose-induced ubiquitination of G6PD leads to the injury of podocytes.

Oxidative stress contributes substantially to podocyte injury, which plays an important role in the development of diabetic kidney disease. The mechanism of hyperglycemia-induced oxidative stress in podocytes is not fully understood. Glucose-6-phosphate dehydrogenase (G6PD) is critical in maintaining NADPH, which is an important cofactor for the antioxidant system. Here, we hypothesized that high glucose induced ubiquitination and degradation of G6PD, which injured podocytes by reactive oxygen species (ROS) accumulation. We found that G6PD protein expression was decreased in kidneys of both diabetic patients and diabetic rodents. G6PD activity was also reduced in diabetic mice. Overexpressing G6PD reversed redox imbalance and podocyte apoptosis induced by high glucose and palmitate. Inhibition of G6PD with small interfering RNA induced podocyte apoptosis. In kidneys of G6PD-deficient mice, podocyte apoptosis was significantly increased. Interestingly, high glucose had no effect on G6PD mRNA expression. Decreased G6PD protein expression was mediated by the ubiquitin proteasome pathway. We found that the von Hippel-Lindau (VHL) protein, an E3 ubiquitin ligase subunit, directly bound to G6PD and degraded G6PD through ubiquitylating G6PD on K366 and K403. In summary, our data suggest that high glucose induces ubiquitination of G6PD by VHL E3 ubiquitin ligase, which leads to ROS accumulation and podocyte injury.-Wang, M., Hu, J., Yan, L., Yang, Y., He, M., Wu, M., Li, Q., Gong, W., Yang, Y., Wang, Y., Handy, D. E., Lu, B., Hao, C., Wang, Q., Li, Y., Hu, R., Stanton, R. C., Zhang, Z. High glucose-induced ubiquitination of G6PD leads to the injury of podocytes.

Pre-clinical model of severe glutathione peroxidase-3 deficiency and chronic kidney disease results in coronary artery thrombosis and depressed left ventricular function.

Background: Chronic kidney disease (CKD) patients have deficient levels of glutathione peroxidase-3 (GPx3). We hypothesized that GPx3 deficiency may lead to cardiovascular disease in the presence of chronic kidney disease due to an accumulation of reactive oxygen species and decreased microvascular perfusion of the myocardium. Methods. To isolate the exclusive effect of GPx3 deficiency in kidney disease-induced cardiac disease, we studied the GPx3 knockout mouse strain (GPx3-/-) in the setting of surgery-induced CKD. Results. Ribonucleic acid (RNA) microarray screening of non-stimulated GPx3-/- heart tissue show increased expression of genes associated with cardiomyopathy including myh7, plac9, serpine1 and cd74 compared with wild-type (WT) controls. GPx3-/- mice underwent surgically induced renal mass reduction to generate a model of CKD. GPx3-/- + CKD mice underwent echocardiography 4 weeks after injury. Fractional shortening (FS) was decreased to 32.9 ± 5.8% in GPx3-/- + CKD compared to 62.0% ± 10.3 in WT + CKD (P < 0.001). Platelet aggregates were increased in the myocardium of GPx3-/- + CKD. Asymmetric dimethylarginine (ADMA) levels were increased in both GPx3-/- + CKD and WT+ CKD. ADMA stimulated spontaneous platelet aggregation more quickly in washed platelets from GPx3-/-. In vitro platelet aggregation was enhanced in samples from GPx3-/- + CKD. Platelet aggregation in GPx3-/- + CKD samples was mitigated after in vivo administration of ebselen, a glutathione peroxidase mimetic. FS improved in GPx3-/- + CKD mice after ebselen treatment. Conclusion: These results suggest GPx3 deficiency is a substantive contributing factor to the development of kidney disease-induced cardiac disease.

Selenoprotein Gene Nomenclature.

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

S-adenosylhomocysteine induces inflammation through NFkB: A possible role for EZH2 in endothelial cell activation.

S-adenosylhomocysteine (SAH) can induce endothelial dysfunction and activation, contributing to atherogenesis; however, its role in the activation of the inflammatory mediator NFkB has not been explored. Our aim was to determine the role of NFkB in SAH-induced activation of endothelial cells. Furthermore, we examined whether SAH, as a potent inhibitor of S-adenosylmethionine-dependent methyltransferases, suppresses the function of EZH2 methyltransferase to contribute to SAH-induced endothelial cell activation. We found that excess SAH increases the expression of adhesion molecules and cytokines in human coronary artery endothelial cells. Importantly, this up-regulation was suppressed in cells expressing a dominant negative form of the NFkB inhibitor, IkB. Moreover, SAH accumulation triggers the activation of both the canonical and non-canonical NFkB pathways, decreases EZH2, and reduces histone 3 lysine 27 trimethylation. EZH2 knockdown recapitulated the effects of excess SAH on endothelial activation, i.e., it induced NFkB activation and the subsequent up-regulation of adhesion molecules and cytokines. Our findings suggest that suppression of the epigenetic regulator EZH2 by excess SAH may contribute to NFkB activation and the consequent vascular inflammatory response. These studies unveil new targets of SAH regulation, demonstrating that EZH2 suppression and NFkB activation mediated by SAH accumulation may contribute to its adverse effects in the vasculature.

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.

Comparison of Protein N-Homocysteinylation in Rat Plasma under Elevated Homocysteine Using a Specific Chemical Labeling Method.

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated "total homocysteine" concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between "total homocysteine" and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine ß-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation.

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.

Aldosterone impairs vascular reactivity by decreasing glucose-6-phosphate dehydrogenase activity.

Hyperaldosteronism is associated with impaired vascular reactivity; however, the mechanisms by which aldosterone promotes endothelial dysfunction remain unknown. Glucose-6-phosphate dehydrogenase (G6PD) modulates vascular function by limiting oxidant stress to preserve bioavailable nitric oxide (NO(*)). Here we show that aldosterone (10(-9)-;10(-7) mol/l) decreased endothelial G6PD expression and activity in vitro, resulting in increased oxidant stress and decreased NO(*) levels-similar to what is observed in G6PD-deficient endothelial cells. Aldosterone decreased G6PD expression by increasing expression of the cyclic AMP-response element modulator (CREM) to inhibit cyclic AMP-response element binding protein (CREB)-mediated G6PD transcription. In vivo, infusion of aldosterone decreased vascular G6PD expression and impaired vascular reactivity. These effects were abrogated by spironolactone or vascular gene transfer of G6pd. These findings demonstrate that aldosterone induces a G6PD-deficient phenotype to impair endothelial function; aldosterone antagonism or gene transfer of G6pd improves vascular reactivity by restoring G6PD activity.

Aldosterone inactivates the endothelin-B receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized, in part, by decreased endothelial nitric oxide (NO(·)) production and elevated levels of endothelin-1. Endothelin-1 is known to stimulate endothelial nitric oxide synthase (eNOS) via the endothelin-B receptor (ET(B)), suggesting that this signaling pathway is perturbed in PAH. Endothelin-1 also stimulates adrenal aldosterone synthesis; in systemic blood vessels, hyperaldosteronism induces vascular dysfunction by increasing endothelial reactive oxygen species generation and decreasing NO(·) levels. We hypothesized that aldosterone modulates PAH by disrupting ET(B)-eNOS signaling through a mechanism involving increased pulmonary endothelial oxidant stress. METHODS AND RESULTS: In rats with PAH, elevated endothelin-1 levels were associated with elevated aldosterone levels in plasma and lung tissue and decreased lung NO(·) metabolites in the absence of left-sided heart failure. In human pulmonary artery endothelial cells, endothelin-1 increased aldosterone levels via peroxisome proliferator-activated receptor gamma coactivator-1α/steroidogenesis factor-1-dependent upregulation of aldosterone synthase. Aldosterone also increased reactive oxygen species production, which oxidatively modified cysteinyl thiols in the eNOS-activating region of ET(B) to decrease endothelin-1-stimulated eNOS activity. Substitution of ET(B)-Cys405 with alanine improved ET(B)-dependent NO(·) synthesis under conditions of oxidant stress, confirming that Cys405 is a redox-sensitive thiol that is necessary for ET(B)-eNOS signaling. In human pulmonary artery endothelial cells, mineralocorticoid receptor antagonism with spironolactone decreased aldosterone-mediated reactive oxygen species generation and restored ET(B)-dependent NO(·) production. Spironolactone or eplerenone prevented or reversed pulmonary vascular remodeling and improved cardiopulmonary hemodynamics in 2 animal models of PAH in vivo. CONCLUSIONS: Our findings demonstrate that aldosterone modulates an ET(B) cysteinyl thiol redox switch to decrease pulmonary endothelium-derived NO(·) and promote PAH.

Systems analysis of oxidant stress in the vasculature.

Systems biology and network analysis are emerging as valuable tools for the discovery of novel relationships, the identification of key regulatory factors, and the prediction of phenotypic changes in complex biological systems. Redox homeostasis in the vasculature is maintained by an intricate balance between oxidant-generating and antioxidant systems. When these systems are perturbed, conditions are permissive for oxidant stress, which, in turn, promotes vascular dysfunction and structural remodeling. Owing to the number of elements involved in redox regulation and the different vascular pathophenotypes associated with oxidant stress, vascular oxidant stress represents an ideal system to study by network analysis. Networks offer a method to organize experimentally derived factors, including proteins, metabolites, and DNA, that are represented as nodes into an unbiased comprehensive platform for study. Through analysis of the network, it is possible to determine essential or regulatory nodes, identify previously unknown connections between nodes, and locate modules, which are groups of nodes located within the same neighborhood that function together and have implications for phenotype. Investigators have only recently begun to construct oxidant stress-related networks to examine vascular structure and function; however, these early studies have provided mechanistic insight to further our understanding of this complicated biological system.

Tumor necrosis factor-α-mediated suppression of dual-specificity phosphatase 4: crosstalk between NFκB and MAPK regulates endothelial cell survival.

We investigated the effects of tumor necrosis factor-α (TNF-α) exposure on mitogen-activated protein kinase signaling in human microvascular endothelial cells. TNF-α caused a significant suppression of a dual specificity phosphatase, DUSP4, that regulates ERK1/2 activation. Thus, we hypothesized that suppression of DUSP4 enhances cell survival by increasing ERK1/2 signaling in response to growth factor stimulation. In support of this concept, TNF-α pre-exposure increased growth factor-mediated ERK1/2 activation, whereas overexpression of DUSP4 with an adenovirus decreased ERK1/2 compared to an empty adenovirus control. Overexpression of DUSP4 also significantly decreased cell viability, lessened recovery in an in vitro wound healing assay, and decreased DNA synthesis. Pharmacological inhibition of NFκB activation or a dominant negative construct of the inhibitor of κB significantly lessened TNF-α-mediated suppression of DUSP4 expression by 70-84% and attenuated ERK activation, implicating NFκB-dependent pathways in the TNF-α-mediated suppression of DUSP4 that contributes to ERK1/2 signaling. Taken together, our findings show that DUSP4 attenuates ERK signaling and reduces cell viability, suggesting that the novel crosstalk between NFκB and MAPK pathways contributes to cell survival.

SDF-1α in glycan nanoparticles exhibits full activity and reduces pulmonary hypertension in rats.

To establish a homing signal in the lung to recruit circulating stem cells for tissue repair, we formulated a nanoparticle, SDF-1α NP, by complexing SDF-1α with dextran sulfate and chitosan. The data show that SDF-1α was barely released from the nanoparticles over an extended period of time in vitro (3% in 7 days at 37 °C); however, incorporated SDF-1α exhibited full chemotactic activity and receptor activation compared to its free form. The nanoparticles were not endocytosed after incubation with Jurkat cells. When aerosolized into the lungs of rats, SDF-1α NP displayed a greater retention time compared to free SDF-1α (64 vs 2% remaining at 16 h). In a rat model of monocrotaline-induced lung injury, SDF-1α NP, but not free form SDF-1α, was found to reduce pulmonary hypertension. These data suggest that the nanoparticle formulation protected SDF-1α from rapid clearance in the lung and sustained its biological function in vivo.

Integration of circulating microRNAs and transcriptome signatures identifies early-pregnancy biomarkers of preeclampsia.

BACKGROUND: MicroRNAs (miRNAs) have been implicated in the pathobiology of preeclampsia, a common hypertensive disorder of pregnancy. In a nested matched case-control cohort within the Vitamin D Antenatal Asthma Reduction Trial (VDAART), we previously identified peripheral blood mRNA signatures related to preeclampsia and vitamin D status (≤30 ng/mL) during gestation from 10 to 18 weeks, using differential expression analysis. METHODS: Using quantitative PCR arrays, we conducted profiling of circulating miRNAs at 10-18 weeks of gestation in the same VDAART cohort to identify differentially expressed (DE) miRNAs associated with preeclampsia and vitamin D status. For the validation of the expression of circulating miRNA signatures in the placenta, the HTR-8/SVneo trophoblast cell line was used. Targets of circulating miRNA signatures in the preeclampsia mRNA signatures were identified by consensus ranking of miRNA-target prediction scores from four sources. The connected component of target signatures was identified by mapping to the protein-protein interaction (PPI) network and hub targets were determined. As experimental validation, we examined the gene and protein expression of IGF1R, one of the key hub genes, as a target of the DE miRNA, miR-182-5p, in response to a miR-182-5p mimic in HTR-8/SVneo cells. RESULTS: Pregnant women with preeclampsia had 16 circulating DE miRNAs relative to normal pregnancy controls that were also DE under vitamin D insufficiency (9/16 = 56% upregulated, FDR < .05). Thirteen miRNAs (13/16 = 81.3%) were detected in HTR-8/SVneo cells. Overall, 16 DE miRNAs had 122 targets, of which 87 were unique. Network analysis demonstrated that the 32 targets of DE miRNA signatures created a connected subnetwork in the preeclampsia module with CXCL8, CXCL10, CD274, MMP9 and IGF1R having the highest connectivity and centrality degree. In an in vitro validation experiment, the introduction of an hsa-miR-182-5p mimic resulted in significant reduction of its target IGF1R gene and protein expression within HTR-8/SVneo cells. CONCLUSIONS: The integration of the circulating DE miRNA and mRNA signatures associated preeclampsia added additional insights into the subclinical molecular signature of preeclampsia. Our systems and network biology approach revealed several biological pathways, including IGF-1, that may play a role in the early pathophysiology of preeclampsia. These pathways and signatures also denote potential biomarkers for the early stages of preeclampsia and suggest possible preventive measures.

Homocysteine induces cardiomyocyte dysfunction and apoptosis through p38 MAPK-mediated increase in oxidant stress.

Elevated plasma homocysteine (Hcy) is a risk factor for cardiovascular disease. While Hcy has been shown to promote endothelial dysfunction by decreasing the bioavailability of nitric oxide and increasing oxidative stress in the vasculature, the effects of Hcy on cardiomyocytes remain less understood. In this study we explored the effects of hyperhomocysteinemia (HHcy) on myocardial function ex vivo and examined the direct effects of Hcy on cardiomyocyte function and survival in vitro. Studies with isolated hearts from wild type and HHcy mice (heterozygous cystathionine-beta synthase deficient mice) demonstrated that HHcy mouse hearts had more severely impaired cardiac relaxation and contractile function and increased cell death following ischemia reperfusion (I/R). In isolated cultured adult rat ventricular myocytes, exposure to Hcy for 24 h impaired cardiomyocyte contractility in a concentration-dependent manner, and promoted apoptosis as revealed by terminal dUTP nick-end labeling and cleaved caspase-3 immunoblotting. These effects were associated with activation of p38 MAPK, decreased expression of thioredoxin (TRX) protein, and increased production of reactive oxygen species (ROS). Inhibition of p38 MAPK by the selective inhibitor SB203580 (5 µM) prevented all of these Hcy-induced changes. Furthermore, adenovirus-mediated overexpression of TRX in cardiomyocytes significantly attenuated Hcy-induced ROS generation, apoptosis, and impairment of myocyte contractility. Thus, Hcy may increase the risk for CVD not only by causing endothelial dysfunction, but also by directly exerting detrimental effects on cardiomyocytes.

Glutathione peroxidase-1 deficiency augments proinflammatory cytokine-induced redox signaling and human endothelial cell activation.

Glutathione peroxidase-1 (GPx-1) is a crucial antioxidant enzyme, the deficiency of which promotes atherogenesis. Accordingly, we examined the mechanisms by which GPx-1 deficiency enhances endothelial cell activation and inflammation. In human microvascular endothelial cells, we found that GPx-1 deficiency augments intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by redox-dependent mechanisms that involve NFκB. Suppression of GPx-1 enhanced TNF-α-induced ROS production and ICAM-1 expression, whereas overexpression of GPx-1 attenuated these TNF-α-mediated responses. GPx-1 deficiency prolonged TNF-α-induced IκBα degradation and activation of ERK1/2 and JNK. JNK or NFκB inhibition attenuated TNF-α induction of ICAM-1 and VCAM-1 expression in GPx-1-deficient and control cells, whereas ERK1/2 inhibition attenuated only VCAM-1 expression. To analyze further signaling pathways involved in GPx-1-mediated protection from TNF-α-induced ROS, we performed microarray analysis of human microvascular endothelial cells treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), encoding an antagonist of MAPK signaling, was down-regulated by GPx-1 suppression. Targeted DUSP4 knockdown enhanced TNF-α-mediated ERK1/2 pathway activation and resulted in increased adhesion molecule expression, indicating that GPx-1 deficiency may augment TNF-α-mediated events, in part, by regulating DUSP4.

Glutathione peroxidase-3 deficiency promotes platelet-dependent thrombosis in vivo.

BACKGROUND: Glutathione peroxidase-3 (GPx-3) is a selenocysteine-containing plasma protein that scavenges reactive oxygen species in the extracellular compartment. A deficiency of this enzyme has been associated with platelet-dependent thrombosis, and a promoter haplotype with reduced function has been associated with stroke risk. METHODS AND RESULTS: We recently developed a genetic mouse model to assess platelet function and thrombosis in the setting of GPx-3 deficiency. The GPx-3((-/-)) mice showed an attenuated bleeding time and an enhanced aggregation response to the agonist ADP compared with wild-type mice. GPx-3((-/-)) mice displayed increased plasma levels of soluble P-selectin and decreased plasma cyclic cGMP compared with wild-type mice. ADP infusion-induced platelet aggregation in the pulmonary vasculature produced a more robust platelet activation response in the GPx-3((-/-)) than wild-type mice; histological sections from the pulmonary vasculature of GPx-3((-/-)) compared with wild-type mice showed increased platelet-rich thrombi and a higher percentage of occluded vessels. Cremaster muscle preparations revealed endothelial dysfunction in the GPx-3((-/-)) compared with wild-type mice. With a no-flow ischemia-reperfusion stroke model, GPx-3((-/-)) mice had significantly larger cerebral infarctions compared with wild-type mice and platelet-dependent strokes. To assess the neuroprotective role of antioxidants in this model, we found that manganese(III) meso-tetrakis(4-benzoic acid)porphyrin treatment reduced stroke size in GPx-3((-/-)) mice compared with vehicle-treated controls. CONCLUSIONS: These findings demonstrate that GPx-3 deficiency results in a prothrombotic state and vascular dysfunction that promotes platelet-dependent arterial thrombosis. These data illustrate the importance of this plasma antioxidant enzyme in regulating platelet activity, endothelial function, platelet-dependent thrombosis, and vascular thrombotic propensity.

Hyperhomocysteinemia from trimethylation of hepatic phosphatidylethanolamine during cholesterol cholelithogenesis in inbred mice.

UNLABELLED: Because hyperhomocysteinemia can occur in cholesterol gallstone disease, we hypothesized that this may result from trimethylation of phosphatidylethanolamine (PE), which partakes in biliary phosphatidylcholine (PC) hypersecretion during cholesterol cholelithogenesis. We fed murine strains C57L/J, C57BL/6J, SWR/J, AKR/J, PE N-methyltransferase (PEMT) knockout (KO), PEMT heterozygous (HET), and wildtype (WT) mice a cholesterol/cholic acid lithogenic diet (LD) for up to 56 days and documented biliary lipid phase transitions and secretion rates. We quantified plasma total homocysteine (tHcy), folate, and vitamin B12 in plasma and liver, as well as biliary tHcy and cysteine secretion rates. Rate-limiting enzyme activities of PC synthesis, PEMT and cytidine triphosphate: phosphocholine cytidylyltransferase (PCT), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) were measured in liver homogenates. Other potential sources of plasma tHcy, glycine N-methyltransferase (GNMT) and guanidinoacetate N-methyltransferase (GAMT), were assayed by gene expression. Plasma tHcy and PEMT activities became elevated during cholelithogenesis in gallstone-susceptible C57L, C57BL/6, and SWR mice but not in the gallstone-resistant AKR mice. Persisting in C57L mice, which exhibit the greatest Lith gene burden, these increases were accompanied by elevated hepatic SAM/SAH ratios and augmented biliary tHcy secretion rates. Counter-regulation included remethylation of Hcy to methionine concurrent with decreased folate and vitamin B12 levels and Hcy transsulfuration to cysteine. Concomitantly, methylenetetrahydrofolate reductase (Mthfr), betaine-homocysteine methyltransferase (Bhmt), and cystathionine-ß-synthase (Cbs) were up-regulated, but Gnmt and Gamt genes were down-regulated. PEMT KO and HET mice displayed biliary lipid secretion rates and high gallstone prevalence rates similar to WT mice without any elevation in plasma tHcy levels. CONCLUSION: This work implicates up-regulation of PC synthesis by the PEMT pathway as a source of elevated plasma and bile tHcy during cholesterol cholelithogenesis.

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.

Comprehensive network medicine-based drug repositioning via integration of therapeutic efficacy and side effects.

Despite advances in modern medicine that led to improvements in cardiovascular outcomes, cardiovascular disease (CVD) remains the leading cause of mortality and morbidity globally. Thus, there is an urgent need for new approaches to improve CVD drug treatments. As the development time and cost of drug discovery to clinical application are excessive, alternate strategies for drug development are warranted. Among these are included computational approaches based on omics data for drug repositioning, which have attracted increasing attention. In this work, we developed an adjusted similarity measure implemented by the algorithm SAveRUNNER to reposition drugs for cardiovascular diseases while, at the same time, considering the side effects of drug candidates. We analyzed nine cardiovascular disorders and two side effects. We formulated both disease disorders and side effects as network modules in the human interactome, and considered those drug candidates that are proximal to disease modules but far from side-effects modules as ideal. Our method provides a list of drug candidates for cardiovascular diseases that are unlikely to produce common, adverse side-effects. This approach incorporating side effects is applicable to other diseases, as well.