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High glucose sensitizes adult cardiomyocytes to ischaemia/reperfusion injury through nitrative thioredoxin inactivation
Abstract
Ischaemic cardiac injury is significantly increased in diabetic patients, but its underlying mechanisms remain incompletely understood. The current study attempted to identify new molecular mechanisms potentially contributive to hyperglycaemic-exaggeration of myocardial ischaemic injury.
Adult mouse cardiomyocytes were cultured in normal-glucose (NG, 5.5 mM) or high-glucose (HG, 25 mM) medium. Twelve hours after NG or HG pre-culture, cardiomyocytes were subjected to 3 h of simulated ischaemia (SI), followed by 3 h of reperfusion (R) in NG medium. Prior to and after SI/R, the following were determined: cardiomyocyte death and apoptosis, sustained oxidative/nitrative stress and thioredoxin (Trx) activity, expression, and nitration. Compared with NG-cultured cardiomyocytes, 12 h HG culture significantly increased superoxide and peroxynitrite production, increased Trx-1 nitration, and reduced Trx activity (P < 0.01). Despite being subject to identical SI/R procedures and conditions, cells pre-cultured in HG sustained greater injury, evidenced by elevated lactate dehydrogenase release and caspase-3 activation (P < 0.01). Moreover, SI/R induced greater superoxide/peroxynitrite overproduction and greater Trx-1 nitration and inactivation in HG pre-cultured cardiomyocytes than in NG pre-cultured cardiomyocytes. Finally, the supplementation of human Trx-1, superoxide scavenger, or peroxynitrite decomposition catalyst in HG pre-cultured cells reduced Trx-1 nitration, preserved Trx-1 activity, and normalized SI/R injury to levels observed in NG pre-cultured cardiomyocytes.
High glucose sensitized cardiomyocytes to ischaemia/reperfusion injury through nitrative Trx-1 inactivation. Interventions restoring Trx-1 activity in the diabetic heart may represent novel therapies attenuating cardiac injury in diabetic patients.
... In contrast to our results, Subramani et al. [38] showed a reduction in infarct size in aged mice (20-26 months) with Trx1 overexpression, but in their model, the overexpression was not cardiac-specific as in our study. However, these authors demonstrated an increase in Trx-reductase activity even in aged mice, which is contrary to numerous experimental studies that show that Trx-reductase activity is reduced in the presence of comorbidities such as age [18,34,39,40]. Furthermore, these results are difficult to interpret since they do not compare Trx1 activity and expression in aged and young mice. ...
... Furthermore, these results are difficult to interpret since they do not compare Trx1 activity and expression in aged and young mice. Finally, the results of Subramani et al. [38] were opposite to those of several authors showing that Trx1 overexpression might not be enough to exert a protective effect in aging hearts because there is a Trx1 inactivation by nitration [18,34,37,[39][40][41]. For this reason, further studies are needed to elucidate the role of Trx1 in I/R injury and aging, particularly in the middle age of life. ...
... In contrast to our results, Subramani et al. [38] showed a reduction in infarct size in aged mice (20)(21)(22)(23)(24)(25)(26) months) with Trx1 overexpression, but in their model, the overexpression was not cardiac-speci c as in our study. However, these authors demonstrated an increase in Trx-reductase activity even in aged mice, which is contrary to numerous experimental studies that show that Trx-reductase activity is reduced in the presence of comorbidities such as age [18,34,39,40]. Furthermore, these results are di cult to interpret since they do not compare Trx1 activity and expression in aged and young mice. ...
... Subramani et al. [38] were opposite to those of several authors showing that Trx1 overexpression might not be enough to exert a protective effect in aging hearts because there is a Trx1 inactivation by nitration[18,34,37,[39][40][41]. For this reason, further studies are needed to elucidate the role of Trx1 in I/R injury and aging, particularly in the middle age of life.A limitation of our study is the isolated isovolumic heart technique because it is an acute and shortduration experimental model and for this reason, the full postischemic function recovery cannot be detected. ...
Thioredoxin-1 (Trx1) has cardioprotective effects on ischemia/reperfusion (I/R) injury, although its role in ischemic postconditioning (PostC) in middle-aged mice is not understood. This study aimed to evaluate if combining two cardioprotective strategies, such as Trx1 overexpression and PostC, could exert a synergistic effect in reducing infarct size in middle-aged mice. Young or middle-aged wild-type mice (Wt), transgenic mice overexpressing Trx1, and dominant negative (DN-Trx1) mutant of Trx1 mice were used. Mice hearts were subjected to I/R or PostC protocol. Infarct size, hydrogen peroxide (H 2 O 2 ) production, protein nitration, Trx1 activity, mitochondrial function, and Trx1, pAkt and pGSK3β expression were measured. PostC could not reduce infarct size even in the presence of Trx1 overexpression in middle-aged mice. This finding was accompanied by a lack of Akt and GSK3β phosphorylation, and Trx1 expression (in Wt group). Trx1 activity was diminished and H 2 O 2 production and protein nitration were increased in middle age. The respiratory control rate dropped after I/R in Wt-Young and PostC restored this value, but not in middle-aged groups. Our results showed that Trx1 plays a key role in the PostC protection mechanism in young but not middle-aged mice, even in the presence of Trx1 overexpression.
... Biologically Trx system has several roles such as Trx1 attenuates cardiac hypertrophy by scavenging ROS (Ye et al., 2020;Yoshioka et al., 2004;Luan et al., 2009), plays anti-apoptotic role by interacting with ASK-1 (Liu and Min, 2002), acts as proton donor for ribonucleotide reductase in DNA synthesis (Avval and Holmgren, 2009), performs immunomodulation and stimulation of eosinophils (Mendoza et al., 2020;Zhou et al., 2020;Zhou et al., 2019;Torii et al., 2010,) and redox regulation of transcription factors such as NFκB that reduces inflammation (Heilman and Watson, 2008;Matthews et al., 1992). Trx has also shown to increase the processivity of T7 DNA polymerase in T7 and M13 phages (Tran et al., 2012;Bedford et al., 1997). ...
... Likewise, several other promising results have been documented by Trx mediated gene therapy for therapeutic implementation in disease conditions. It was reported that intramyocardial administration of adenoviral vectors containing Trx1 gene reduced diabetes mediated angiogenesis impairment, oxidative stress, and fibrosis of cardiomyocytes in myocardial infarcted type 1 diabetes rat model (Samuel et al., 2010;Luan et al., 2009). Trx1 mediated gene therapy using mesenchymal stem cells also induced pro-angiogenic factors and reduced fibrosis in the infracted rat myocardium (Suresh et al., 2015). ...
Nitric Oxide is a very well known gaseous second messenger molecule and vasorelaxant agent involved in a variety of signaling in the body such as neurotransmission, ion channel modulation, and inflammation modulation. However, it’s reversible covalent attachment to thiol groups of cysteine residues under nitrosative stress leading to aberrant protein S-nitrosylation (PSNO) has been reported in several pathological conditions in the body stemming from neurodegenerative diseases, cancer, cardiovascular system, and immune system disorders. In the cell, PSNOs are partly unstable and transit to a more stable disulfide state serving as an intermediate step towards disulfide formation thus eliciting the biological response. Scientists have identified several cellular thiol-dependent disulfide reductases that have the intrinsic capability to reverse the modification by reducing the stable disulfides formed in PSNOs and thereby rescue S-nitrosylation-induced altered proteins. The physiological roles of these major cellular ubiquitous S-denitrosylases and their probable implementations have not been fully explored. Gaining knowledge from current research and development this review provides a deeper insight into understanding the interplay and role of the major ubiquitous S-denitrosylases in maintaining cellular redox homeostasis. This review umbrellas the mechanism of Thioredoxin, TRP14, and Glutaredoxin systems and highlights their substrates specificities at different cellular conditions, physiological roles, and importance in diseased conditions that would allow researchers to investigate effective therapeutic interventions for nitrosative stress-related diseases and disorders.
... Similar with redox, metabolic and pathophysiological changes are evoked by a high level of glucose that may interact via common metabolic pathways and biomolecular and electrical changes to exacerbate complications of diabetes. Ischemic cardiac injury is significantly increased in diabetic hearts [8]. In a study, mouse cardiomyocytes were cultured in normal-glucose (5.5 mM) or high-glucose (25 mM) medium, high-glucose-sensitized cardiomyocytes to ischemia/ reperfusion injury through nitrative thioredoxin (a small protein ubiquitously expressed in living cells, and its activity in the diabetic heart may attenuate cardiac injury) inactivation [8]. ...
... Ischemic cardiac injury is significantly increased in diabetic hearts [8]. In a study, mouse cardiomyocytes were cultured in normal-glucose (5.5 mM) or high-glucose (25 mM) medium, high-glucose-sensitized cardiomyocytes to ischemia/ reperfusion injury through nitrative thioredoxin (a small protein ubiquitously expressed in living cells, and its activity in the diabetic heart may attenuate cardiac injury) inactivation [8]. Hyperglycemia is also an important risk factor for long QT syndromes by inhibiting the cardiac rapid component delayed rectifier K + current (Iks), and perfusing the isolated hearts with high-glucose-containing Krebs solution (33 mM) demonstrated an increase in QT interval in non-diabetic rats [9]. ...
PurposeCellular changes occurring in diabetic cardiomyopathy include disturbances of calcium and sodium homeostasis. Voltage-gated sodium channels are responsible for the initiation of cardiac action potentials, and the excitability would create relevance. The effect of ranolazine as a sodium channel blocker on atrium electromechanical parameters is investigated and compared with lidocaine in streptozocin-treated diabetic rats.Methods
After an 8-week induction of diabetes type I, the effect of cumulative concentrations of ranolazine and lidocaine on the electrophysiology of isolated atrium was studied. Ranolazine’s effects were evaluated on cardiac sodium current in normal- and high-glucose medium, with whole-cell patch-clamp technique.ResultsRanolazine at therapeutic concentrations had no significant statistical effect on refractory period in normal and diabetic isolated heart. Ranolazine (10 μM) caused a hyperpolarizing shift of V1/2 for steady-state inactivation in normal media, while it significantly elicited a depolarizing shift in high-glucose media (p < 0.05).Conclusion
It is concluded that in the isolated rat atrium preparation, ranolazine and lidocaine have no beneficial on diabetic cardiomyopathy. Although refractoriness and contractility were not much different in normal and diabetic atria, there was a definite effect of ranolazine and lidocaine on sodium current in varying concentrations. This may have significance in future therapeutics.
... Trx1 is mainly located in cytosol but also translocates to the nucleus, whereas Trx2 is located only in mitochondria. Our previous study has demonstrated that inactivation of Trx1 increases vulnerability of diabetic hearts to ischemia/reperfusion (I/R) injury [5,6]. Increasing evidence suggests that Trx1 modulates the process of angiogenesis [7]. ...
... Accumulating evidence supports the notion that Trx might represent a promising therapeutic target for the prevention and treatment of cardiovascular disease [3,4]. We have previously reported that Trx1 inactivation increased vulnerability of diabetic hearts to I/R injury [5,6]. Trx system was also implicated to play a role in endothelial cell homeostasis and key angiogenic process, including endothelial cell migration, proliferation, and survival. ...
Diabetes mellitus (DM)-induced impairment of collateral formation has been demonstrated in subjects with coronary artery disease, which contributes to unfavorable prognosis among diabetic individuals. In our previous studies, thioredoxin1 (Trx1) activity was shown to be decreased in diabetic cardiac tissues, but the reason of Trx1 inactivation and whether it mediates the impaired angiogenesis in ischemic myocardium is still to be identified. As thioredoxin-interacting protein (TXNIP), an endogenous inhibitor of Trx, is overexpressed in DM due to carbohydrate response element within its promoter, we hypothesized that inhibition of Trx1 by enhanced TXNIP expression in endothelial cells may play a role in hyperglycemia-induced impairment of angiogenesis. In the present study, we found that high glucose-mediated increase of TXNIP expression and TXNIP-Trx1 interaction induced the impairment in endothelial cell function and survival, since these detrimental effects are rescued by silencing TXNIP with small interfering RNA. In diabetic mice, TXNIP knockdown or recombinant human Trx1 treatment counteracted the impairment of angiogenesis, alleviated myocardial ischemic injury, and improved survival rate. All these data implicate that TXNIP upregulation and subsequently the increased formation of TXNIP-Trx1 complex is a novel pathologic pathway by which DM induces insufficient angiogenesis and thereby exacerbates myocardial ischemia injury.
... In the clinical condition, the rapid reintroduction of blood flow post-reperfusion can lead to an immediate supply of oxygen and nutrients, triggering heightened inflammation and oxidative stress, thereby potentially causing tissue damage. A study suggested that high glucose sensitizes cardiomyocytes to ischemia-reperfusion (IR) injury [20]. Another study proposed that intracellular and mitochondrial calcium overload may contribute to reperfusion injury by exacerbating oxidative stress [21]. ...
Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.
... Previous studies revealed the impact of high glucose on the expression of TRX1, PGC-1a, NRF1 and TFAM. In the study by Luan et al. (2009), the activity of TRX1 was decreased in diabetic cardiac tissues. As suggested by Hou et al. (2020), the mechanism may involve TRX-interacting protein (TXNIP), an endogenous inhibitor of TRX. ...
Context
Oxidative injury in a high-glucose (HG) environment may be a mechanism of diabetic retinopathy (DR) and edaravone can protect retinal ganglion cells by scavenging ROS.
Objective
To explore the effect of edaravone on HG-induced injury.
Materials and methods
First, Müller cells were cultured by different concentrations of glucose for different durations to obtain a suitable culture concentrations and duration. Müller cells were then divided into Control, HG + Vehicle, HG + Eda-5 μM, HG + Eda-10 μM, HG + Eda-20 μM, and HG + Eda-40 μM groups. Cells were cultured by 20 mM glucose and different concentrations of edaravone for 72 h.
Results
The IC50 of glucose at 12–72 h is 489.3, 103.5, 27.92 and 20.71 mM, respectively. When Müller cells were cultured in 20 mM glucose for 72 h, the cell viability was 52.3%. Edaravone significantly increased cell viability compared to Vehicle (68.4% vs 53.3%; 78.6% vs 53.3%). The EC50 of edaravone is 34.38 μM. HG induced high apoptosis rate (25.5%), while edaravone (20 and 40 μM) reduced it to 12.5% and 6.89%. HG increased the DCF fluorescence signal (189% of Control) and decreased the mitochondrial membrane potential by 57%. Edaravone significantly decreased the DCF fluorescence signal (144% and 132% of Control) and recovered the mitochondrial membrane potential to 68% and 89% of Control. Furthermore, HG decreased the expression of TRX1, PGC-1α, NRF1 and TFAM, which were restored by edaravone.
Discussion and conclusion
These findings provide a new potential approach for the treatment of DR and indicated new molecular targets in the prevention of DR.
... Several Michael acceptors compounds have been designed to be able to inhibit TrxR by covalent interaction with the catalytic selenocysteine residue in the enzyme's active site (see for instance [5]). Trx can also be inactivated by nitration caused by peroxynitrite [104,105,176], which is formed in a reaction between superoxide anion radical and NO [177]. ...
Thioredoxin reductases (TrxRs) belong to the pyridine nucleotide disulfide oxidoreductase family enzymes that reduce thioredoxin (Trx). The couple TrxR and Trx is one of the major antioxidant systems that control the redox homeostasis in cells. The thioredoxin system, comprised of TrxR Trx and NADPH, exerts its activities via a disulfide-dithiol exchange reaction. Inhibition of TrxR is an important clinical goal in all conditions in which the redox state is perturbed. The present review focuses on the most critical aspects of the cellular functions of TrxRs and their inhibition mechanisms by metal ions or chemicals, through direct targeting of TrxRs or their substrates or protein interactors. To update the involvement of overactivation/dysfunction of TrxRs in various pathological conditions, human diseases associated with TrxRs genes were critically summarized by publicly available genome-wide association study (GWAS) catalogs and literature. The pieces of evidence presented here justify why TrxR is recognized as one of the most critical clinical targets and the growing current interest in developing molecules capable of interfering with the functions of TrxR enzymes.
... HG treatment sensitized adult cardiomyocytes to ischemia/reperfusion (I/R) injury [34]. Myocardial hypoxia results in metabolic changes and irreversible damage leading to cardiomyocyte death [35]. ...
Lesions caused by high glucose (HG), hypoxia/reperfusion (H/R), and the coexistence of both conditions in cardiomyocytes are linked to an overproduction of reactive oxygen species (ROS), causing irreversible damage to macromolecules in the cardiomyocyte as well as its ultrastructure. Fenofibrate, a peroxisome proliferator-activated receptor alpha (PPARα) agonist, promotes beneficial activities counteracting cardiac injury. Therefore, the objective of this work was to determine the potential protective effect of fenofibrate in cardiomyocytes exposed to HG, H/R, and HG+H/R. Cardiomyocyte cultures were divided into four main groups: (1) control (CT), (2) HG (25 mM), (3) H/R, and (4) HG+H/R. Our results indicate that cell viability decreases in cardiomyocytes undergoing HG, H/R, and both conditions, while fenofibrate improves cell viability in every case. Fenofibrate also decreases ROS production as well as nicotinamide adenine dinucleotide phosphate oxidase (NADPH) subunit expression. Regarding the antioxidant defense, superoxide dismutase (SOD Cu2+/Zn2+ and SOD Mn2+), catalase, and the antioxidant capacity were decreased in HG, H/R, and HG+H/R-exposed cardiomyocytes, while fenofibrate increased those parameters. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) increased significantly in treated cells, while pathologies increased the expression of its inhibitor Keap1. Oxidative stress-induced mitochondrial damage was lower in fenofibrate-exposed cardiomyocytes. Endothelial nitric oxide synthase was also favored in cardiomyocytes treated with fenofibrate. Our results suggest that fenofibrate preserves the antioxidant status and the ultrastructure in cardiomyocytes undergoing HG, H/R, and HG+H/R preventing damage to essential macromolecules involved in the proper functioning of the cardiomyocyte.
... Trx1 belongs to the thioredoxin protein group, which localizes to both the cytosol and nucleus of heart cells [15][16][17][18]. Overexpression of Trx-1 in mice reduced cardiac hypertrophy induced by aortic banding, while Trx-1 deficiency resulted in cardiac hypertrophy and increased oxidative stress under baseline conditions [19]. ...
Recent data show that cardiac hypertrophy contributes substantially to the overall heart failure burden. Mitochondrial dysfunction is a common feature of cardiac hypertrophy. Recent studies have reported that isosteviol inhibits myocardial ischemia-reperfusion injury in guinea pigs and H9c2 cells. This work investigated the protective mechanisms of isosteviol sodium (STVNa) against isoproterenol (Iso)-induced cardiac hypertrophy. We found that STVNa significantly inhibited H9c2 cell and rat primary cardiomyocyte cell surface, restored mitochondrial membrane potential (MMP) and morphological integrity, and decreased the expression of mitochondrial function-related proteins Fis1 and Drp1. Furthermore, STVNa decreased reactive oxygen species (ROS) levels and upregulated the expression of antioxidant factors, Thioredoxin 1 (Trx1) and Peroxiredoxin 2 (Prdx2). Moreover, STVNa restored the activity of histone deacetylase 4 (HDAC4) in the nucleus. Together, our data show that STVNa confers protection against Iso-induced myocardial hypertrophy primarily through the Prdx2/ROS/Trx1 signaling pathway. Thus, STVNA is a potentially effective treatment for cardiac hypertrophy in humans.
... Evidences show that the thioredoxin activity, which is regulated by thioredoxin-interacting protein, controls cardiac hypertrophy [86]. Inactivation of nitrative thioredoxin promotes cardiomyocyte injury induced by high glucose and cardiac ischemia/reperfusion [87]. Thioredoxin activity can also be inhibited by methylglyoxal, aggravating cardiomyocyte ischemia/reperfusion injury [88]. ...
ROS functions as a second messenger and modulates multiple signaling pathways under the physiological conditions. However, excessive intracellular ROS causes damage to the molecular components of the cell, which promotes the pathogenesis of various human diseases. Cardiovascular diseases are serious threats to human health with extremely high rates of morbidity and mortality. Dysregulation of cell death promotes the pathogenesis of cardiovascular diseases and is the clinical target during the disease treatment. Numerous studies show that ROS production is closely linked to the cell death process and promotes the occurrence and development of the cardiovascular diseases. In this review, we summarize the regulation of intracellular ROS, the roles of ROS played in the development of cardiovascular diseases, and the programmed cell death induced by intracellular ROS. We also focus on anti-ROS system and the potential application of anti-ROS strategy in the treatment of cardiovascular diseases.
... As maternal glucose freely crosses the placenta, offspring of diabetic mothers are exposed to high blood glucose levels during gestation [3]. Studies in cultured adult cardiomyocytes have shown that high glucose increases the expression of NOX2 [36], the predominant isoform of NADPH oxidase in heart. However, it is not known if maternal diabetes up-regulates myocardial NOX2 expression in normal glycemic and glucose-tolerant offspring. ...
Offspring of diabetic mothers are at risk of cardiovascular diseases in adulthood. However, the underlying molecular mechanisms are not clear. We hypothesize that prenatal exposure to maternal diabetes up-regulates myocardial NOX2 expression and enhances ischaemia/reperfusion (I/R) injury in the adult offspring. Maternal diabetes was induced in C57BL/6 mice by streptozotocin. Glucose-tolerant adult offspring of diabetic mothers and normal controls were subjected to myocardial I/R injury. Vascular endothelial growth factor (VEGF) expression, ROS generation, myocardial apoptosis and infarct size were assessed. The VEGF-Akt (protein kinase B)-mammalian target of rapamycin (mTOR)-NOX2 signalling pathway was also studied in cultured cardiomyocytes in response to high glucose level. In the hearts of adult offspring from diabetic mothers, increases were observed in VEGF expression, NOX2 protein levels and both Akt and mTOR phosphorylation levels as compared to the offspring of control mothers. After I/R, ROS generation, myocardial apoptosis and infarct size were all significantly higher in the offspring of diabetic mothers relative to offspring of control mothers, and these differences were diminished by in vivo treatment with the NADPH oxidase inhibitor apocynin. In cultured cardiomyocytes, high glucose increased mTOR phosphorylation, which was inhibited by the PI3 kinase inhibitor LY294002. Notably, high glucose-induced NOX2 protein expression and ROS production were inhibited by rapamycin. In conclusion, maternal diabetes promotes VEGF-Akt-mTOR-NOX2 signalling and enhances myocardial I/R injury in the adult offspring. Increased ROS production from NOX2 is a possible molecular mechanism responsible for developmental origins of cardiovascular disease in offspring of diabetic mothers.
... Cultivation of murine cardiomyocytes in high-glucose conditions was accompanied by increased O 2 -and ONOO-production, Trx1 nitration and a subsequent reduction of Trx1 activity. Simulated ischemia/reperfusion in high-glucose medium aggravated the observed effects, whereas supplementation with human Trx1 or antioxidants prevented ROS overproduction, Trx1 inhibition and cellular injury [182] Similarly, diabetic animals were characterized by increased vascular TXNIP expression and decreased Trx activity, all being prevented by insulin treatment [183]. Myocardial TrxR activity was also significantly decreased in diabetic (streptozotocin-induced) rats but not in Rac1 KO animals, being indicative of the role Rac1/NADPH oxidase signaling in the regulation of TrxR activity [184]. ...
Mammalian thioredoxin reductase (TrxR) is a selenoprotein with three existing isoenzymes (TrxR1, TrxR2, and TrxR3), which is found primarily intracellularly but also in extracellular fluids. The main substrate thioredoxin (Trx) is similarly found (as Trx1 and Trx2) in various intracellular compartments, in blood plasma, and is the cell’s major disulfide reductase. Thioredoxin reductase is necessary as a NADPH-dependent reducing agent in biochemical reactions involving Trx. Genetic and environmental factors like selenium status influence the activity of TrxR. Research shows that the Trx/TrxR system plays a significant role in the physiology of the adipose tissue, in carbohydrate metabolism, insulin production and sensitivity, blood pressure regulation, inflammation, chemotactic activity of macrophages, and atherogenesis. Based on recent research, it has been reported that the modulation of the Trx/TrxR system may be considered as a new target in the management of the metabolic syndrome, insulin resistance, and type 2 diabetes, as well as in the treatment of hypertension and atherosclerosis. In this review evidence about a possible role of this system as a marker of the metabolic syndrome is reported.
... Cardiac expression of GPx levels is reduced in the diabetic apolipoprotein Edeficient mice [37]. Meanwhile, attempts to attenuate I/R injury using enzymatic and nonenzymatic antioxidants have not been universally successful in DM [38,39]. ...
Diabetes mellitus (DM) displays a high morbidity. The diabetic heart is susceptible to myocardial ischemia/reperfusion (MI/R) injury. Impaired activation of prosurvival pathways, endoplasmic reticulum (ER) stress, increased basal oxidative state, and decreased antioxidant defense and autophagy may render diabetic hearts more vulnerable to MI/R injury. Oxidative stress and mTOR signaling crucially regulate cardiometabolism, affecting MI/R injury under diabetes. Producing reactive oxygen species (ROS) and reactive nitrogen species (RNS), uncoupling nitric oxide synthase (NOS), and disturbing the mitochondrial quality control may be three major mechanisms of oxidative stress. mTOR signaling presents both cardioprotective and cardiotoxic effects on the diabetic heart, which interplays with oxidative stress directly or indirectly. Antihyperglycemic agent metformin and newly found free radicals scavengers, Sirt1 and CTRP9, may serve as promising pharmacological therapeutic targets. In this review, we will focus on the role of oxidative stress and mTOR signaling in the pathophysiology of MI/R injury in diabetes and discuss potential mechanisms and their interactions in an effort to provide some evidence for cardiometabolic targeted therapies for ischemic heart disease (IHD).
... Apolipoprotein A (ApoA-1) is a major high density lipoprotein and studies have shown that nitrated ApoA-I levels are increased in T2D patients [137] and LC-MS/MS analysis indicated MPO mediated nitration of Tyr18 in atherosclerotic tissues [138]. Increased thioredoxin nitration is also associated with ischemia/reperfusion injury in cardiomyocytes under elevated glucose levels [139]. ...
Diabetes Mellitus is a chronic metabolic disorder that is often associated with various complications including micro- and macro- angiopathies. The increase in mortality rates associated with these complications is one among the major factors that compels to define proper therapeutic strategies. Protein signatures including their post-translational modifications that are modulated under various disease conditions enabled the biomarker discovery processes and thereby facilitate to predict and diagnose the disease onset at an earlier stage. The identification of the post-translationally modified proteins not only enabled the biomarker prediction associated with various disease conditions but also has a great impact in target based drug discovery process. Mass spectrometry based proteome profiling aided the identification and characterization of these site specific modifications that had a huge impact on various functional aspects in a biological context. In this review, we tried to compile and discuss the information available regarding some of the modified proteins with regard to deregulated glucose metabolism. Several commonly observed modifications including glycosylation, hydroxylation, phosphorylation, nitration, nitrosylation and carbonylation that seem to have a major role in the management of diabetes disorders are summarized. Moreover, the importance and impact of less explored post-translational modifications such as palmitoylation, carbamylation, deamidation and SUMOylation that are associated to diabetes related complications are described.
... High glucose exposure has also been shown to directly suppress Trx activity in vitro and leads to an excessive injury response in an ischemia-reperfusion injury model. This suggests that Trx may have a cardioprotective role in DM [64]. This is consistent with suggested function of Trx as a regulator of cardiac hypertrophy. ...
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality among patients with diabetes mellitus (DM). DM can lead to multiple cardiovascular complications, including coronary artery disease (CAD), cardiac hypertrophy, and heart failure (HF). HF represents one of the most common causes of death in patients with DM and results from DM-induced CAD and diabetic cardiomyopathy. Oxidative stress is closely associated with the pathogenesis of DM and results from overproduction of reactive oxygen species (ROS). ROS overproduction is associated with hyperglycemia and metabolic disorders, such as impaired antioxidant function in conjunction with impaired antioxidant activity. Long-term exposure to oxidative stress in DM induces chronic inflammation and fibrosis in a range of tissues, leading to formation and progression of disease states in these tissues. Indeed, markers for oxidative stress are overexpressed in patients with DM, suggesting that increased ROS may be primarily responsible for the development of diabetic complications. Therefore, an understanding of the pathophysiological mechanisms mediated by oxidative stress is crucial to the prevention and treatment of diabetes-induced CVD. The current review focuses on the relationship between diabetes-induced CVD and oxidative stress, while highlighting the latest insights into this relationship from findings on diabetic heart and vascular disease.
... Irrespective of nitration or nitrosylation, nitrosative stress induced complications have a significant role in sepsis, neurodegeneration, vascular complications, myocardial infaction, cardiac ischemic disease, insulin resistance, diabetes, and diabetic cardiomyopathy [180][181][182][183] . ...
... Sepsis is known to induce acute hyperglycemia [3,4], and its progression is usually accompanied with the change of glycemia levels. Especially, the concentration of glucose has shown to accelerate the aggravation of sepsis [3,5]. ...
Sepsis is defined as a systemic inflammatory response syndrome that disorders the functions of host immune system, including the imbalance between pro- and anti-inflammatory responses mediated by immune macrophages. Sepsis could also induce acute hyperglycemia. Studies have shown that the silent mating type information regulation 2 homolog 1 (SIRT1), an NAD+-dependent deacetylase, mediates NF-κb deacetylation and inhibits its function. Therefore, SIRT1 is likely to play an important role in high glucose-mediated inflammatory signalings. Here we demonstrate that high glucose significantly downregulates both the mRNA and protein levels of SIRT1 and upregulates the mRNA level and the release of two pro-inflammatory cytokines, IL-1β and TNF-α, in RAW264.7 macrophages. Interestingly, the reduced level of SIRT1 by high glucose is remarkably upregulated by SIRT1 activator SRT1720, while the level and the release of IL-1β and TNF-α significantly decrease with the use of SRT1720. However, when the function of SIRT1 is inhibited by EX527 or its expression is suppressed by RNAi, the upregulated level and release of IL-1β and TNF-α by high glucose are further increased. Taken together, these findings collectively suggest that SIRT1 is an important regulator in many high glucose-related inflammatory diseases such as sepsis.
... This suggests that hyperglycemia sensitizes cardiomyocytes to a hypertrophic response, and that addition of a further strain such as osmotic pressure or oxidative stress is needed to activate the hypertrophic response. Consistent with this, cardiomyocytes cultured in high glucose are more sensitive to acute stress such as I/R injury (99). This is likely due to a combination of factors, including mitochondrial sensitization, osmotic stress, oxidation of cellular components by ROS, and decreased autophagy. ...
Diabetes is strongly associated with increased incidence of heart disease and mortality due to development of diabetic cardiomyopathy. Even in the absence of cardiovascular disease, cardiomyopathy frequently arises in diabetic patients. Current treatment options for cardiomyopathy in diabetic patients are the same as for non-diabetic patients and do not address the causes underlying the loss of contractility. Recent Advances: Although there are numerous distinctions between Type 1 and Type 2 diabetes, recent evidence suggests that the two disease states converge on mitochondria as an epicenter for cardiomyocyte damage.
Accumulation of dysfunctional mitochondria contributes to cardiac tissue injury in both acute and chronic conditions. Removal of damaged mitochondria by macroautophagy, termed "mitophagy", is critical for maintaining cardiomyocyte health and contractility under both normal conditions and during stress. However, very little is known about the involvement of mitophagy in the pathogenesis of diabetic cardiomyopathy. A growing interest in this topic has given rise to a wave of publications that aim to decipher the status of autophagy and mitophagy in Type 1 and Type 2 diabetes.
This review summarizes these recent studies with the goal of drawing conclusions about the activation or suppression of autophagy and mitophagy in the diabetic heart. A better understanding of how autophagy and mitophagy are affected in the diabetic myocardium is still needed, as well as whether they can be targeted therapeutically.
... Rekhraj at al. recently described, that in patient with ischemic heart disease, high dose off allopurinol was capable to reduce left ventricular muscle mass and improve the symptoms of the disease [65]. [152,153] A substantial amount of experimental evidence suggests that there is reduced nitric oxide (NO) availability in diabetic tissues. Different mechanisms have been proposed to be responsible for the diabetesinduced dysfunction of NO production, bioavailability and/or signaling. ...
Diabetes is a recognized risk factor for cardiovascular diseases and heart failure. Diabetic cardiovascular dysfunction also underscores the development of diabetic retinopathy, nephropathy and neuropathy. Despite the broad availability of antidiabetic therapy, glycaemic control still remains a major challenge in the management of diabetic patients. Hyperglycaemia triggers formation of advanced glycosylation end products(AGEs), activates protein kinase C, enhances polyol pathway, glucose autoxidation, which coupled with elevated levels of free fatty acids, and leptin have been implicated in increased generation of superoxide anion by mitochondria, NADPH oxidases and xanthine oxidoreductase in diabetic vasculature and myocardium. Superoxide anion interacts with nitric oxide forming the potent toxin peroxynitrite via diffusion limited reaction, which in concert with other oxidants triggers activation of stress kinases, endoplasmic reticulum stress, mitochondrial and poly(ADP-ribose) polymerase 1-dependent cell death, dysregulates autophagy/mitophagy, inactivates key proteins involved in myocardial calcium handling/contractility and antioxidant defense, activates matrix metalloproteinases and redox-dependent pro-inflammatory transcription factors (e.g. nuclear factor kappaB) promoting inflammation, AGEs formation, eventually culminating in myocardial dysfunction, remodeling and heart failure. Understanding the complex interplay of oxidative/nitrosative stress with pro-inflammatory, metabolic and cell death pathways is critical to devise novel targeted therapies for diabetic cardiomyopathy, which will be overviewed in this brief synopsis. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.
... We chose this high glucose concentration since it is able to mimic a comparable cardiomyocyte injury as seen in in vivo myocardium exposed to hyperglycaemia, and it has been well accepted and widely used in previous in vitro studies. 35,36 In summary, our study in a preclinical large animal model demonstrates that hyperglycaemia blunts insulin protection against MI/R injury and reveals the role of O-GlcNAc modification in hyperglycaemia-induced impairment of insulin cardioprotection. These findings highlight the significance of hyperglycaemia prevention in GIK cardioprotection against MI/R injury and suggest inhibition of O-GlcNAc modification of insulin-signalling proteins as a potential cardioprotective strategy for hyperglycaemia-associated myocardial injury. ...
Aims:
Experimental evidence has shown significant cardioprotective effects of insulin, whereas clinical trials produced mixed results without valid explanations. This study was designed to examine the effect of hyperglycaemia on insulin cardioprotective action in a preclinical large animal model of myocardial ischaemia/reperfusion (MI/R).
Methods and results:
Anaesthetized dogs were subjected to MI/R (30 min/4 h) and randomized to normal plasma insulin/euglycaemia (NI/NG), normal-insulin/hyperglycaemia (NI/HG), high-insulin/euglycaemia (HI/NG), and high-insulin/hyperglycaemia (HI/HG) achieved by controlled glucose/insulin infusion. Endogenous insulin production was abolished by peripancreatic vessel ligation. Compared with the control animals (NI/NG), hyperglycaemia (NI/HG) significantly aggravated MI/R injury. Insulin elevation at clamped euglycaemia (HI/NG) protected against MI/R injury as evidenced by reduced infarct size, decreased necrosis and apoptosis, and alleviated inflammatory and oxidative stress (leucocyte infiltration, myeloperoxidase, and malondialdehyde levels). However, these cardioprotective effects of insulin were markedly blunted in hyperglycaemic animals (HI/HG). In vitro mechanistic study in neonatal rat cardiomyocytes revealed that insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and Akt was significantly attenuated by high glucose, accompanied by markedly increased IRS-1 O-GlcNAc glycosylation following hypoxia/reoxygenation. Inhibition of hexosamine biosynthesis with 6-diazo-5-oxonorleucine abrogated high glucose-induced O-GlcNAc modification and inactivation of IRS-1/Akt as well as cell injury.
Conclusions:
Our results, derived from a canine model of MI/R, demonstrate that hyperglycaemia blunts insulin protection against MI/R injury via hyperglycaemia-induced glycosylation and subsequent inactivation of insulin-signalling proteins. Our findings suggest that prevention of hyperglycaemia is critical for achieving maximal insulin cardioprotection for the ischaemic/reperfused hearts.
... The interaction of Trx and ASK1 was significantly decreased in the diabetic group, while p38 kinase activity was increased. The interactions among Trx, ASK1, p38 kinase, and apoptosis have been shown in many studies of ischemia/reperfusion injury [37,38]. Besides, Trx inhibition may also elicit aggravated free radical damage which could be another reason for cardiomyocyte apoptosis found in diabetes, reperfusion injury, and other diseases. ...
Cardiac complications are the leading cause of death in diabetes. However, the mechanism of diabetes in inducing myocardial injury and apoptosis, and whether the thioredoxin (Trx) system is involved remain unclear. In this study, male Sprague-Dawley rats were randomly divided into two groups: the control and the diabetes groups, and then were randomly divided into five different timepoints (the 1st, 2nd, 4th, 12th, and 24th week). The results showed that diabetes-induced cardiac injury was enhanced in the type 2 diabetes rats, as evidenced by aggravated cardiac dysfunction, biochemical indicators, and increased myocardial apoptosis (TUNEL and caspase-3 activity). The activity of myocardial Trx and Trx reductase (TR) in diabetic rats was significantly decreased from the second week and continually aggravated with the disease progression. In diabetic rats, the mRNA expression of Trx1, Trx2, TR1, and TR2 was decreased first and then increased after the fourth week. Meanwhile, the protein expression of these Trx system members was significantly increased at the 12th week. Trx nitration was cleared, the Trx/ASK1 interaction was significantly decreased, and the activity of p38 was significantly enhanced in cardiac tissues at the 12th week. These results demonstrated that diabetes may cause myocardial injury and apoptosis, and the extent of which was accompanied with the development of the disease. The mechanism is associated with the development of diabetes and the decreased activity of Trx and TR. The reasons for decreased Trx activity may include: decrease of Trx and TR protein expression; nitration modification of Trx; and up-regulation of TXNIP expression.
... Trx has thus been suggested to be a negative regulator of cardiac hypertrophy (Ago & Sadoshima, 2007). High glucose conditions directly impair cardiomyocyte Trx activity in vitro, exaggerating a subsequent ischemia-reperfusion (I-R) injury response, suggesting that endogenous Trx activity is cardioprotective, and that this protection may be impaired in the diabetic heart (Luan et al., 2009). In recent years, the role of thioredoxin interacting protein (TxNIP), the endogenous Trx inhibitor, in the cardiovascular complications of diabetes and other cardiovascular disorders has attracted significant interest. ...
... For the long term continuous exposure to high-glucose, CMECs were cultured in 6 well plates. When CMECs became confluent, media were replaced to high glucose medium (25 mmol/L) for 6 h, 12 h and 24 h respectively as described previously [21]. In the control group, CMECs were maintained in normal glucose medium (5.5 mmol/L). ...
Cardiac microvascular endothelial cells (CMECs) dysfunction contributes to cardiovascular complications in diabetes, whereas, the underlying mechanism is not fully clarified. FoxO transcription factors are involved in apoptosis and reactive oxygen species (ROS) production. Therefore, the present study was designed to elucidate the potential role of FoxO3a on the CMECs injury induced by high glucose.
CMECs were isolated from hearts of adult rats and cultured in normal or high glucose medium for 6 h, 12 h and 24 h respectively. To down-regulate FoxO3a expression, CMECs were transfected with FoxO3a siRNA. ROS accumulation and apoptosis in CMECs were assessed by dihydroethidine (DHE) staining and TUNEL assay respectively. Moreover, the expressions of Akt, FoxO3a, Bim and BclxL in CMECs were assessed by Western blotting assay.
ROS accumulation in CMECs was significantly increased after high glucose incubation for 6 to 24 h. Meanwhile, high glucose also increased apoptosis in CMECs, correlated with decreased the phosphorylation expressions of Akt and FoxO3a. Moreover, high glucose incubation increased the expression of Bim, whereas increased anti-apoptotic protein BclxL. Furthermore, siRNA target FoxO3a silencing enhanced the ROS accumulation, whereas suppressed apoptosis in CMECs. FoxO3a silencing also abolished the disturbance of Bcl-2 proteins induced by high glucose in CMECs.
Our data provide evidence that high glucose induced FoxO3a activation which suppressed ROS accumulation, and in parallel, resulted in apoptosis of CMECs.
... Furthermore, it has been also demonstrated that the activities of other enzymes from the antioxidant system family are down-regulated in STZ-induced diabetic rat heart [14,15,17,18]. In support of these findings, it has been demonstrated that high glucose sensitized cardiomyocytes to ischemia/reperfusion injury through Trx-1 inactivation [20]. In addition, it has been also reported that an antioxidant, resveratrol, exerts its cardioprotective effect by upregulating Trx-1 mRNA levels [13]. ...
Increased oxidative stress contributes to heart dysfunction via impaired Ca(2+) homeostasis in diabetes. Abnormal RyR2 function related with altered cellular redox state is an important factor in the pathogenesis of diabetic cardiomyopathy, while its underlying mechanisms remain poorly understood. In the present study, we used a streptozotocin-induced rat model of diabetic cardiomyopathy and tested a hypothesis that diabetes-related alteration in RyR2 function is related with ROS-induced posttranslational modifications. For this, we used heart preparations from either a diabetic rat or a sodium selenate (NaSe)-treated (0.3 mg/kg for 4 weeks) diabetic rat as well as either NaSe- (100 nmol/L) or thioredoxin (Trx; 5 μmol/L)-incubated (30 min) diabetic cardiomyocytes. Experimental approaches included imaging of intracellular free-Ca(2+) ([Ca(2+)]i) under both electrically stimulated and resting Fluo-3-loaded cardiomyocytes. RyR2-mediated SR-Ca(2+) leak was significantly enhanced in diabetic cardiomyocytes, resulting in reduced amplitude and prolonged time courses of [Ca(2+)]i transients compared to those of controls. Both SR-Ca(2+) leak and [Ca(2+)]i transients were normalized by treating diabetic rats with NaSe or by incubating diabetic myocytes with NaSe or Trx. Moreover, exposure of diabetic cardiomyocytes to antioxidants significantly improved [Ca(2+)]i handling factors such as phosphorylation/protein levels of RyR2, amount of RyR2-bound FKBP12.6 and activities of both protein kinase A and CaMKII. NaSe treatment also normalized the oxidative stress/antioxidant defense biomarkers in plasma as well as Trx activity and nuclear factor-κB phosphorylation in the diabetic rat heart. Collectively, these findings suggest that redox modification through Trx-system besides the glutathione system contributes to abnormal function of RyR2s in hyperglycemic cardiomyocytes, presenting a potential therapeutic target for treating diabetics to preserve cardiac function.
... ROS production, which serves as an index for oxidative stress in viable cardiomyocytes, was measured as described previously. 28 For details, see Supplementary material online. ...
Aims:
Apelin, an endogenous cytokine, has a number of biological effects on the cardiovascular system, including a cardioprotective effect and calcium modulation. Because the intracellular calcium abnormality is considered to play an important role in cardiac dysfunction induced by ischaemia-reperfusion (I/R), the aim of this study was to examine the effects of apelin-13 on I/R-induced changes in cardiac performance and sarcoplasmic reticulum (SR) function.
Methods and results:
Isolated rat hearts were subjected to global ischaemia followed by reperfusion in the absence or presence of apelin-13 and inhibitors of some survival kinases. We found that depressed cardiac performance induced by I/R was attenuated by apelin-13. Furthermore, apelin-13 depressed oxidative stress during I/R. SR function depressed during I/R was partly reversed by apelin-13. SR oxidative modification levels were increased in I/R and reversed by apelin. Inhibitors of phosphatidylinositol-3-kinase and protein kinase C abolished the effects of apelin. Apelin-13 maintained the Ca(2+) transient against I/R in cardiomyocytes.
Conclusion:
Apelin protects SR function and cardiac performance during I/R by attenuating oxidation of sarco(endo)plasmic reticulum Ca(2+)-ATPase and RyR.
... The relationship between diabetic complications and oxidative and/or nitrosative stress is believed to be related to increased superoxide generation and 3-NT accumulation (2,16). In this study, we confirmed that high levels of superoxide and 3-NT accompany development of cardiac remodeling in the late stage of diabetes. ...
We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8 and 16 weeks after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA: 3-ketoacid-coenzyme A transferase 1 (SCOT), was identified by mass spectrometry. While total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp374 showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).
... Moreover, the SAG is increased by elevated levels of glucose-6phosphate dehydrogenase-derived NADPH in obese and hyperglycemic rats [32]. Furthermore, hyperglycemia reduces the levels of intracellular antioxidant protein thioredoxin, thereby increasing SAG [33]. ROS are involved in the induction of cardiac hypertrophy and fibrosis by activating tyrosine kinase, protein kinase C, MAPK, and TGFβ [34][35][36][37]. ...
This study investigated the possible synergistic role of obesity in hypertension-induced cardiac remodeling and its modulation by gemfibrozil treatment in rats. Male Wistar rats were fed a high-fat diet (HFD) for 90 days. Normal rats were subjected to hypertension by partial abdominal aortic constriction (PAAC) for 28 days. In the HFD+PAAC control group, rats on HFD were subjected to PAAC on the 62nd day and were sacrificed on the 90th day. HFD and PAAC individually resulted in significant cardiac hypertrophy and fibrosis along with increased oxidative stress and mean arterial blood pressure (MABP) in rats as evidenced by various morphological, biochemical, and histological parameters. Moreover, the HFD + PAAC control group showed marked cardiac remodeling compared to rats subjected to HFD or PAAC alone. The HFD+gemfibrozil and HFD+PAAC+gemfibrozil groups showed significant reduction in cardiac remodeling along with reduction in oxidative stress and MABP. Hence, it may be concluded that oxidative stress plays a key role in obesity-mediated synergistic effects on induction and progression of PAAC-induced cardiac remodeling, and its deleterious effects could be reversed by gemfibrozil treatment in rats through its antioxidant activity.
Here we aimed to establish an in vitro engineered heart tissue (EHT) co-morbidity mimicking model of ischemia-reperfusion injury and diabetes. EHTs were generated from primary neonatal rat cardiomyocytes. Hyperglycemic conditions or hyperosmolar controls were applied for one day to model acute hyperglycemia and for seven days to model chronic hyperglycemia. 120 min' simulated ischemia (SI) was followed by 120 min' reperfusion (R) and 1-day follow-up reperfusion (FR). Normoxic controls (N) were not subjected to SI/R. Half of the EHTs was paced, the other half was left unpaced. To assess cell injury, lactate-dehydrogenase (LDH) concentration was measured. Beating force and activity (frequency) were monitored as cardiomyocyte functional parameters. LDH-release indicated relevant cell injury after SI/N in each experimental condition, with much higher effects in the chronically hyperglycemic/hyperosmolar groups. SI stopped beating of EHTs in each condition, which returned during reperfusion, with weaker recovery in chronic conditions than in acute conditions. Acutely treated EHTs showed small LDH-release and ~ 80% recovery of force during reperfusion and follow-up, while chronically treated EHTs showed a marked LDH-release, only ~30% recovery with reperfusion and complete loss of beating activity during 24 h follow-up reperfusion. We conclude that EHTs respond differently to SI/R injury in acute and chronic hyperglycemia/hyperosmolarity, and that our EHT model is a novel in vitro combination of diabetes and ischemia-reperfusion.
Type 2 Diabetes (T2D) is a leading cause for major macrovascular complications like Coronary Heart disease (CHD). The number of CHD deaths attributable to diabetes has been increasing alarmingly. Oxidative stress induced by several metabolic derangements/pathways including hyperglycemia, hyperinsulinemia, dyslipidemia, insulin resistance and diminished antioxidant capacity, is one of the proposed pathogenic mechanisms for progression of CHD in type 2 diabetic subjects. Risk of CHD in type 2 diabetics is also influenced by several genetic factors apart from confounding risk factors. Several single nucleotide polymorphisms (SNPs) in oxidative stress related genes like CYBA, TXNIP, TRXR2, MPO and PARP-1 are known to be associated with diabetes induced CHD. Hence, understanding the molecular and genetic pathophysiological mechanisms contributed by oxidative stress is vital to the prevention and management of diabetes-induced CHD.
Accumulating evidence indicates the occurrence and development of diabetic complications relates to not only constant high plasma glucose, but also glucose fluctuations which affect various kinds of molecular mechanisms in various target cells and tissues. In this review, we detail reactive oxygen species and their potentially damaging effects upon glucose fluctuations and resultant downstream regulation of protein signaling pathways, including protein kinase C, protein kinase B, nuclear factor-κB, and the mitogen-activated protein kinase signaling pathway. A deeper understanding of glucose-fluctuation-related molecular mechanisms in the development of diabetic complications may enable more potential target therapies in future.
Significance:
Redox signaling is one of the key elements involved in cardiovascular diseases. Two important molecules are the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and the oxidoreductase thioredoxin-1 (Trx-1). Recent Advances: During the previous years, a lot of studies investigated Nrf2 and Trx-1 as protective proteins in cardiovascular disorders. Moreover, post-translational modifications of those molecules were identified that play an important role in the cardiovascular system. This review will summarize changes in the vasculature in atherosclerosis and ischemia reperfusion injury of the heart and the newest findings achieved with Nrf2 and Trx-1 therein. Interestingly, Nrf2 and Trx-1 can act together as well as independently of each other in protection against atherosclerosis and ischemia and reperfusion injury.
Critical issues:
In principle, pharmacological activation of a transcription factor-like Nrf2 can be dangerous, since a transcription regulator has multiple targets and the pleiotropic effects of such activation should not be ignored. Moreover, overactivation of Nrf2 as well as long-term treatment with Trx-1 could be deleterious for the cardiovascular system.
Future directions:
Therefore, the length of treatment with Nrf2 activators and/or Trx-1 has first to be studied in more detail in cardiovascular disorders. Moreover, a combination of Nrf2 activators and Trx-1 should be investigated and taken into consideration. Antioxid. Redox Signal. 26, 630-644.
A precise control of oxidation/reduction of protein thiols is essential for intact cardiac physiology. Irreversible oxidative modifications have been proposed to play a role in the pathogenesis of cardiovascular diseases. An imbalance of redox homeostasis with diminution of antioxidant capacities predisposes the heart to oxidant injury. There is growing interest in endoplasmic reticulum (ER) stress in the cardiovascular field, since perturbation of redox homeostasis in the ER is sufficient to cause ER stress. Because a number of human diseases are related to altered redox homeostasis and defects in protein folding, many research efforts have been devoted in recent years to understanding the structure and enzymatic properties of the thioredoxin superfamily. The thioredoxin superfamily has been well documented as thiol oxidoreductases to exert a role in various cell signaling pathways. The redox properties of the thioredoxin motif account for the different functions of several members of the thioredoxin superfamily. While thioredoxin and glutaredoxin primarily act as antioxidants by reducing protein disulfides and mixed disulfide, another member of the superfamily, protein disulfide isomerase (PDI), can act as an oxidant by forming intrachain disulfide bonds that contribute to proper protein folding. Increasing evidence suggests a pivotal role of PDI in the survival pathway that promotes cardiomyocyte survival and leads to more favorable cardiac remodeling. Thus, the thiol redox state is important for cellular redox signaling and survival pathway in the heart. This review summarizes the key features of major members of the thioredoxin superfamily directly involved in cardiac physiology and pathology. © 2015 American Physiological Society. Compr Physiol 5: 513-530, 2015.
Abstract The interaction between antioxidant glutathione and the free thiol in susceptible cysteine residues of proteins leads to reversible protein S-glutathionylation. This reaction ensures cellular homeostasis control (as a common redox-dependent post-translational modification associated with signal transduction) and intervenes in oxidative stress-related cardiovascular pathology (as initiated by redox imbalance). The purpose of this review is to evaluate the recent knowledge on protein S-glutathionylation in terms of chemistry, broad cellular intervention, specific quantification, and potential for therapeutic exploitation. The data bases searched were Medline and PubMed, from 2009 to 2014 (term: glutathionylation). Protein S-glutathionylation ensures protection of protein thiols against irreversible over-oxidation, operates as a biological redox switch in both cell survival (influencing kinases and protein phosphatases pathways) and cell death (by potentiation of apoptosis), and cross-talks with phosphorylation and with S-nitrosylation. Collectively, protein S-glutathionylation appears as a valuable biomarker for oxidative stress, with potential for translation into novel therapeutic strategies.
Aims:
Cardiac structural genes have been implicated as causative factors for congenital heart diseases (CHDs). NEXN is an F-actin binding protein and previously identified as a disease gene causing cardiomyopathies. Whether NEXN contributes to CHDs aetiologically remains unknown. Here, we explored the function of NEXN in cardiac development.
Methods and results:
First, we determine the role of NEXN in cardiac differentiation using mouse P19cl6 in vitro model; we demonstrated that NEXN inhibited cardiac contractile markers, serving as a negative regulator. Interestingly, we found this effect was mediated by GATA4, a crucial transcription factor that controls cardiac development by knockdown, overexpression, and rescue experiment, respectively. We then generated transgenic mouse models and surprisingly, we discovered cardiac-selective expression of the NEXN gene caused atrial septal defects (ASDs). Next, to search for the mutations in NEXN gene in patients suffering from ASDs, we sequenced the exon and exon-intron joint regions of the NEXN gene in 150 probands with isolated ASDs and identified three mutations in the conserved region of NEXN (c.-52-78C>A, K199E, and L227S), which were not found in 500 healthy controls. Finally, we characterize the related mechanisms and found all mutations inhibited GATA4 expression.
Conclusion:
We identify NEXN as a novel gene for ASD and its function to inhibit GATA4 established a critical regulation of an F-actin binding protein on a transcription factor in cardiac development.
Oxidative stress has been implicated in the pathogenesis of various cardiovascular diseases, including ischemic heart disease and heart failure. The peroxisome proliferator-activated receptor gamma (PPARγ) agonist improves insulin sensitivity and limits tissue inflammation and cellular apoptosis, but there are few data on the relationship between the PPARγ agonist, rosiglitazone (RSG), and the thioredoxin (TRx) system in oxidatively stressed cardiomyocytes (CMCs). Here we provide evidence that the PPARγ agonist RSG protects rat CMCs from hydrogen peroxide (H(2)O(2))-induced apoptosis by TRx overexpression. The expression levels of pAkt/Akt, pErk/Erk, survivin, Bcl-2/Bax-α, and manganese-superoxide dismutase were increased by RSG pretreatment in H(2)O(2)-injured rat CMCs. On the contrary, the expression levels of caspase-3 and p53 were decreased by RSG pretreatment. These effects of RSG were reversed by chemical inhibitors of TRx and the PPARγ antagonist. This suggests that RSG protects rCMCs from H(2)O(2)-induced oxidative stress through TRx overexpression and a PPARγ-dependent mechanism.
Diabetes and its complications are a major public health burden in the developed world. The major cause of diabetic complications is abnormal growth of new blood vessels. This dysfunctional neovascularization results in significant morbidity and mortality in patients with diabetes and, as such, is a major focus of basic and clinical investigation. It has become clear that hyperglycemia disrupts tissue-level signaling in response to hypoxia and ischemia, impairs the vasculogenic potential of circulating stem cells and fundamentally alters the structure and function of key neovascularization proteins, including hypoxia-inducible factor-1. These mechanistic and pathophysiologic studies have revealed new therapeutic targets to restore normal neovascularization and to ameliorate and prevent diabetic vascular complications.
Reactive oxygen or nitrogen species play an integral role in both myocardial injury and repair. This dichotomy is differentiated at the level of species type, amount, duration of free radical generated. Homeostatic mechanisms designed to prevent free radical generation in the first instance, scavenge, or enzymatically convert them to less toxic forms and water, play crucial roles in maintenance of cellular structure and function. The outcome between functional recovery and dysfunction is dependent upon the inherent ability of these homeostatic antioxidant defenses to withstand acute free radical generation, in the order of seconds to minutes. Alternatively, pre-existent antioxidant capacity (from intracellular and extracellular sources) may regulate the degree of free radical generation. This converts reactive oxygen and nitrogen species to the role of second messenger involved in cell signalling. The adaptive capacity of the cell is altered by the balance between death or survival signal converging at the level of the mitochondria, with distinct pathophysiologic consequences that extends the period of injury from hours to days and weeks. Hyperglycemia, hyperlipidemia, and insulin resistance enhance oxidative stress in diabetic myocardium that cannot adapt to ischemia reperfusion. Altered glucose flux, mitochondrial derangements and nitric oxide synthase uncoupling in the presence of decreased antioxidant defense and impaired prosurvival cell signalling may render the diabetic myocardium more vulnerable to injury, remodelling and heart failure. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Significance:
Nitric oxide (NO) regulates a diverse range of cellular processes, including vasodilation, neurotransmission, and antimicrobial and anti-tumor activities. S-nitrosylation with the formation of S-nitrosothiols (RSNOs) is an important feature of NO signaling regulating protein function. In mammalian cells, glutathione (GSH), S-nitrosoglutathione reductase (GSNOR), and thioredoxin (Trx) have been identified as the major protein denitrosylases.
Recent advances:
Human cytosolic/nuclear Trx1 in the disulfide form can be nitrosylated at Cys73 and transnitrosylate target proteins, including caspase 3. Thus, similar to GSH, which by forming S-nitrosoglutathione (GSNO) can transnitrosylate proteins, Trx can either denitrosylate or nitrosylate proteins depending on its oxidation state.
Critical issues:
In this review, we discuss the regulation of cellular processes by reversible S-nitrosylation and Trx-mediated cellular homeostasis of RSNOs and S-nitrosoproteins.
Future directions:
Functions of RSNOs in vivo and their pharmacological uses have not yet been fully studied. Further investigations on the role of Trx systems in relation to biologically relevant RSNOs, their functions, and the mechanisms of denitrosylation will facilitate the development of drugs and therapies. Antioxid. Redox Signal. 18, 259-269.
S-nitrosylation (or S-nitrosation) by Nitric Oxide (NO), i.e., the covalent attachment of a NO group to a cysteine thiol and formation of S-nitrosothiols (R-S-N=O or RSNO), has emerged as an important feature of NO biology and pathobiology. Many NO-related biological functions have been directly associated with the S-nitrosothiols and a considerable number of S-nitrosylated proteins have been identified which can positively or negatively regulate various cellular processes including signaling and metabolic pathways. SCOPE OF THE REVIEW: Taking account of the recent progress in the field of research, this review focuses on the regulation of cellular processes by S-nitrosylation and Trx-mediated cellular homeostasis of S-nitrosothiols.
Thioredoxin (Trx) system in mammalian cells utilizes thiol and selenol groups to maintain a reducing intracellular environment to combat oxidative/nitrosative stress. Reduced glutathione (GSH) and Trx system perform the major role in denitrosylation of S-nitrosylated proteins. However, under certain conditions, oxidized form of mammalian Trx can be S-nitrosylated and then it can trans-S-nitrosylate target proteins, such as caspase 3.
Investigations on the role of thioredoxin system in relation to biologically relevant RSNOs, their functions, and the mechanisms of S-denitrosylation facilitate the development of drugs and therapies. This article is part of a Special Issue entitled Regulation of Cellular Processes.
It is well-known that insulin acts as an important hormone, controlling energy metabolism, cellular proliferation and biosynthesis of functional molecules to maintain a biological homeostasis. Over the past few years, intensive insulin therapy has been believed to be benefit for the outcome of diabetic patients, in which the suppression of oxidative stress plays a role. Moreover, insulin is accepted as a key component of glucose-insulin-potassium, a treatment which has been believed to exert significant cardiovascular protective effect via the reduction of oxidative stress. Furthermore, accumulating evidence has suggested that insulin exerts important redox-regulating actions in various insulin-sensitive target organs, implying the systematic antioxidative role of insulin as a hormone. It is time for us to revisit insulin effects, through summarizing and evaluating the novel functions of insulin and their mechanisms. This review focuses on the antioxidative effect of insulin and highlights insulin-induced regulation of various antioxidant enzymes via insulin signaling pathways and the cross talk between key transcription factors, including nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NF-κB) which are responsible for the transcription of antioxidant enzymes, leading to reduced generation of reactive oxygen species (ROS) and the enhancement of the elimination of ROS.
Oxidative stress may cause a loss of tetrahydrobiopterin (BH4), a co-factor of nitric oxide synthase (NOS), decrease the bioavailability of NO and aggravate ischemia/reperfusion (I/R) injury in diabetic heart. We hypothesized that ascorbic acid (AA) and N-acetyl cysteine (NAC) protect the diabetic heart from I/R injury by increasing BH4/dihydrobiopterin (BH2) ratio and inhibiting uncoupling of NOS. Diabetes mellitus was induced in rats by streptozotocin treatment, and the hearts were isolated and perfused. BH4 and BH4/BH2 ratio decreased in the diabetic heart associated with increased production of superoxide and nitrotyrosine (NT). Treatment with AA or NAC significantly increased BH4/BH2 ratio in the diabetic heart associated with decreased production of superoxide and NT and increased generation of nitrate plus nitrite (NOx). Pre-treatment with AA or NAC before 30 min ischemia followed by 120 min reperfusion improved left ventricular (LV) function and reduced infarct size in the diabetic but not non-diabetic hearts. The NOS inhibitor, L-NAME, inhibited the increase in the generation of superoxide, NT and NOx, but aggravated LV function and increased infarct size in the diabetic heart. L-NAME also abrogated the increase in NOx and improvement of LV function and the infarct size-limiting effect induced by AA or NAC in the diabetic heart. These results suggest that AA and NAC increase BH4/BH2 ratio and prevent NOS uncoupling in the diabetic heart. Resultant increase in the bioavailability of NO renders the diabetic heart toleratant to I/R injury.
The diabetic heart is known to be susceptible to ischemia/reperfusion (I/R) injury by increased oxidative stress. Although oxidative stress upregulates inducible nitric oxide (iNOS), the role of iNOS in I/R injury in the diabetic heart has been poorly understood. Because iNOS-derived nitric oxide (NO) plays a crucial role in cardioprotection against I/R injury, we hypothesized that inhibition of iNOS uncoupling would restore tolerance to I/R injury in the diabetic heart. The present study demonstrated that iNOS-derived superoxide generation was reduced, and that the NO bioavailability was increased, by treatment with the NOS-cofactor, tetrahydrobiopterin (BH4), before I/R in the hearts isolated from diabetic rats. This was associated with a reduction of infarct size and improvement of left ventricular (LV) function after I/R. The cardioprotective effect of BH4 was abrogated by treatment with a thiol reducing agent dithiothreitol (DTT), but not a NO-sensitive guanylyl cyclase inhibitor ODQ, suggesting that iNOS-derived NO-mediated cardioprotection occurs through protein S-nitrosylation but not cGMP-dependent signaling in the diabetic heart. Indeed, protein S-nitrosylation was increased by treatment with BH4 in the diabetic heart and was inhibited by DTT. These results suggest that the inhibition of iNOS uncoupling unmasks tolerance to I/R injury through enhanced protein S-nitrosylation in the diabetic rat heart.
This study is to investigate changes of tolerance to ischemia-reperfusion (IR) injury and protection of ischemic preconditioning (IPC) of type1 diabetes rat heart. Type1 diabetes rat model was made by single injection of streptozotocin and divided into 4wk (D-4w) group and 8wk (D-8w) group randomly. The two groups were further assigned to six teams: D-4w-con, D-4w-IR, D-4w-IPC, D-8w-con, D-8w-IR and D-8w-IPC team. Normal rats were correspondingly divided into N-4w-con, N-4w-IR, N-4w-IPC, N-8w-con, N-8w-IR and N-8w-IPC team. Heart function, myocardial infarct size, release of lactate dehydrogenase (LDH) and creatine kinase (CK) were detected. And injury increment rate, injury rate and protection rate were calculated. Ultrastructure of cardiomyocyte was observed by electron microscope. The myocardial injury increment rate, infarct sizes in D-con group were all significantly higher than those in N-con rat group, and the degree of injury in D-8w rat hearts was exacerbated compared to D-4w rat hearts. The myocardial injury rate and the injury of cardiomyocyte ultrastructure in D-4w-IR team were lower compared with N-4w-IR group. Compared with D-4w-IR team, IPC decreased infarct sizes in D-4w-IPC group, but its protection rate was lower than that in N-4w-IPC team. Infarct sizes in D-8w-IPC team had no significance with those in D-8w-IR team. The fundament cardiac function of type1 diabetes rat heart was weakened. Diabetes self could induce heart injury, which could aggravate along with time. The tolerance of 4w type1 diabetes rat to IR injury is stronger but the protection of IPC was weaker compared to normal rat. The protection of IPC disappears in D-8w group.
Diabetes increases myocardial ischemia/reperfusion (I/R) injury. However, the underlying mechanisms remain incompletely understood. This study investigated the role of Rac1 signaling and calpain in exacerbated I/R injury in diabetic hearts. Mice with cardiac-specific deletion of Rac1 (Rac1-ko) and transgenic mice with cardiac-specific superoxide dismutase-2 (SOD2) or calpastatin overexpression were rendered diabetic with streptozotocin. Isolated perfused hearts were subjected to global I/R. After I/R, Rac1 activity was significantly enhanced in diabetic compared with nondiabetic hearts. Diabetic hearts displayed more severe I/R injury than nondiabetic hearts, as evidenced by more lactate dehydrogenase release and apoptosis and decreased cardiac function. These adverse impacts of diabetes were abrogated in Rac1-ko hearts or by perfusion with the Rac1 inhibitor NSC23766. In an in vivo I/R mouse model, infarct size was much smaller in diabetic Rac1-ko compared with wild-type mice. Inhibition of Rac1 signaling prevented NADPH oxidase activation, reactive oxygen species production, and protein carbonyl accumulation, leading to inhibition of calpain activation. Furthermore, SOD2 or calpastatin overexpression significantly reduced I/R injury in diabetic hearts and improved cardiac function after I/R. In summary, Rac1 activation increases I/R injury in diabetic hearts and the role of Rac1 signaling is mediated, at least in part, through calpain activation.
Type 2 diabetes is an age-related disease associated with vascular pathologies, including severe blindness, renal failure, atherosclerosis, and stroke. Reactive oxygen species (ROS), especially mitochondrial ROS, play a key role in regulating the cellular redox status, and an overproduction of ROS may in part underlie the pathogenesis of diabetes and other age-related diseases. Cells have evolved endogenous defense mechanisms against sustained oxidative stress such as the redox-sensitive transcription factor nuclear factor E2-related factor 2 (Nrf2), which regulates antioxidant response element (ARE/electrophile response element)-mediated expression of detoxifying and antioxidant enzymes and the cystine/glutamate transporter involved in glutathione biosynthesis. We hypothesize that diminished Nrf2/ARE activity contributes to increased oxidative stress and mitochondrial dysfunction in the vasculature leading to endothelial dysfunction, insulin resistance, and abnormal angiogenesis observed in diabetes. Sustained hyperglycemia further exacerbates redox dysregulation, thereby providing a positive feedback loop for severe diabetic complications. This review focuses on the role that Nrf2/ARE-linked gene expression plays in regulating endothelial redox homeostasis in health and type 2 diabetes, highlighting recent evidence that Nrf2 may provide a therapeutic target for countering oxidative stress associated with vascular disease and aging.
During the United Kingdom Prospective Diabetes Study (UKPDS), patients with type 2 diabetes mellitus who received intensive glucose therapy had a lower risk of microvascular complications than did those receiving conventional dietary therapy. We conducted post-trial monitoring to determine whether this improved glucose control persisted and whether such therapy had a long-term effect on macrovascular outcomes.
Of 5102 patients with newly diagnosed type 2 diabetes, 4209 were randomly assigned to receive either conventional therapy (dietary restriction) or intensive therapy (either sulfonylurea or insulin or, in overweight patients, metformin) for glucose control. In post-trial monitoring, 3277 patients were asked to attend annual UKPDS clinics for 5 years, but no attempts were made to maintain their previously assigned therapies. Annual questionnaires were used to follow patients who were unable to attend the clinics, and all patients in years 6 to 10 were assessed through questionnaires. We examined seven prespecified aggregate clinical outcomes from the UKPDS on an intention-to-treat basis, according to previous randomization categories.
Between-group differences in glycated hemoglobin levels were lost after the first year. In the sulfonylurea-insulin group, relative reductions in risk persisted at 10 years for any diabetes-related end point (9%, P=0.04) and microvascular disease (24%, P=0.001), and risk reductions for myocardial infarction (15%, P=0.01) and death from any cause (13%, P=0.007) emerged over time, as more events occurred. In the metformin group, significant risk reductions persisted for any diabetes-related end point (21%, P=0.01), myocardial infarction (33%, P=0.005), and death from any cause (27%, P=0.002).
Despite an early loss of glycemic differences, a continued reduction in microvascular risk and emergent risk reductions for myocardial infarction and death from any cause were observed during 10 years of post-trial follow-up. A continued benefit after metformin therapy was evident among overweight patients. (UKPDS 80; Current Controlled Trials number, ISRCTN75451837.)
Smooth muscle cells, macrophages, glial cells, keratinocytes, and transformed cells have been established as synthesis sites for vascular endothelial growth factor (VEGF). The modulating effects of VEGF are essentially limited to endothelial cells (ECs), the only cell type consistently shown to express VEGF receptors. VEGF has thus been considered to act exclusively via a paracrine pathway. We sought to determine whether the role of human ECs might, under selected conditions, extend beyond that of a target to involve contingency synthesis of VEGF. In both unstimulated human umbilical vein ECs (HUVECs) and human derma-derived microvascular ECs (HMECs), Northern analysis detected no VEGF transcripts. Phorbol-12-myristate 13-acetate (10(-7) M) treatment, however, induced VEGF mRNA expression in both HUVECs and HMECs, peaking at 3 and 6 h, respectively, and returning to undetectable levels by 12 h. In vitro exposure of HUVECs to a hypoxic environment (pO2 = 35 mm of mercury) for 12, 24, and 48 h and exposure of HMECs for 6, 12, 24, and 48 h induced VEGF mRNA in a time-dependent fashion. Re-exposure to normoxia (pO2 = 150 mm of mercury) for 24 h after 24 h of hypoxia returned VEGF mRNA transcripts to undetectable levels in HUVECs. Cobalt chloride and nickel chloride treatment each induced VEGF mRNA in ECs. Cycloheximide treatment further augmented expression of VEGF mRNA induced by cobalt chloride, nickel chloride, and hypoxia in HUVECs. VEGF protein production in hypoxia HUVECs was demonstrated immunohistochemically. Conditioned media from hypoxic HUVECs caused a 2-fold increase in the incorporation of tritiated thymidine. Finally, immune precipitates of anti-KDR probed with anti-Tyr(P) antibodies demonstrated evidence of receptor autophosphorylation in hypoxic but not normoxic HUVECs. These findings thus establish the potential for an autocrine pathway that may augment and/or amplify the paracrine effects of VEGF in stimulating angiogenesis.
Apoptosis signal-regulating kinase (ASK) 1 was recently identified as a mitogen-activated protein (MAP) kinase kinase kinase which activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways and is required for tumor necrosis factor (TNF)-alpha-induced apoptosis; however, the mechanism regulating ASK1 activity is unknown. Through genetic screening for ASK1-binding proteins, thioredoxin (Trx), a reduction/oxidation (redox)-regulatory protein thought to have anti-apoptotic effects, was identified as an interacting partner of ASK1. Trx associated with the N-terminal portion of ASK1 in vitro and in vivo. Expression of Trx inhibited ASK1 kinase activity and the subsequent ASK1-dependent apoptosis. Treatment of cells with N-acetyl-L-cysteine also inhibited serum withdrawal-, TNF-alpha- and hydrogen peroxide-induced activation of ASK1 as well as apoptosis. The interaction between Trx and ASK1 was found to be highly dependent on the redox status of Trx. Moreover, inhibition of Trx resulted in activation of endogenous ASK1 activity, suggesting that Trx is a physiological inhibitor of ASK1. The evidence that Trx is a negative regulator of ASK1 suggests possible mechanisms for redox regulation of the apoptosis signal transduction pathway as well as the effects of antioxidants against cytokine- and stress-induced apoptosis.
The production of reactive oxygen species (e.g., superoxide) by endothelial cells is relevant to tissue injury during ischemia-reperfusion, and may also play a role in intracellular signaling pathways. However, the molecular identities of the enzymes responsible for endothelial superoxide production are poorly defined, although xanthine oxidase, NADH/NADPH oxidoreductases and nitric oxide synthase are among proteins suggested to contribute. Recent studies suggest that an NADH/NADPH oxidase similar to that found in neutrophils is an important source of superoxide in vascular smooth muscle.
We investigated whether a phagocyte-type NADH/NADPH oxidase complex is present in rat cultured coronary microvascular endothelial cells. The expression of NADPH oxidase components was studied by RT-PCR and Western blot analyses, while functional activity was assessed by measurement of superoxide production by lucigenin-enhanced chemiluminescence.
The major component of the phagocyte-type NADH/NADPH oxidase complex, a cytochrome b558 heterodimer, was shown to be present both at mRNA and protein levels, using oligonucleotide primers designed from published neutrophil and vascular smooth muscle sequences and anti-neutrophil antibodies respectively. Functional activity of the enzyme was also confirmed by NADPH-evoked superoxide production in cell homogenates, which was inhibited either by the superoxide chelator Tiron or by diphenyleneiodonium, an inhibitor of the oxidase.
A functional phagocyte-type NADPH oxidase is expressed in coronary microvascular endothelial cells, where it may contribute to the physiological and/or pathophysiological effects of reactive oxygen species. These data, together with reports of the presence of a similar oxidase in other non-phagocytic cell types, suggest that this enzyme complex is widely expressed in many tissues where it may subserve signaling and other functions.
Because high D-glucose significantly stimulates endothelial cell death, we examined the molecular mechanisms of high D-glucose-induced endothelial apoptosis. Treatment of human aortic endothelial cells with high D-glucose (25 mmol/l), but not mannitol and L-glucose, resulted in a significant decrease in cell number and a significant increase in apoptotic cells as compared with a physiological concentration (5 mmol/l). Interestingly, high D-glucose treatment significantly increased bax protein, accompanied by translocation of bax protein from cytosol to mitochondria-enriched heavy membrane fraction. In contrast, the expression and distribution of bcl-2 protein were not altered by high D-glucose. In addition, the activity of caspase-3 proteases was increased after exposure to high glucose, whereas caspase inhibitors prevented endothelial cell death induced by high D-glucose. On the other hand, p38 mitogen-activated protein kinase (MAPK) was markedly phosphorylated and showed sustained phosphorylation after stimulation. A specific inhibitor of p38 MAPK, SB 203580, and the overexpression of kinase-inactive p38 MAPK significantly attenuated cell death induced by high D-glucose in human aortic endothelial cells, whereas at 6 h after high D-glucose treatment, SB 203580 and overexpression of kinase-inactive p38 MAPK did not attenuate caspase-3 activation induced by high D-glucose. Importantly, caspase inhibitors significantly attenuated the sustained phosphorylation of p38 MAPK induced by high D-glucose. Thus, we finally focused the MAPK kinase (MEK) kinase 1 (MEKK1) to further examine the cross-talk between p38 MAPK and the bax-caspase proteases pathway. High D-glucose treatment induced MEKK1 cleavage, whereas caspase inhibitors significantly attenuated the cleavage. Importantly, kinase-inactive MEKK1 also blocked the phosphorylation of p38 MAPK induced by high D-glucose. Here, we demonstrated that high D-glucose induced apoptosis in human endothelial cells through activation of the bax-caspase proteases pathway and through phosphorylation of p38 MAPK mediated by MEKK1. Phosphorylation of p38 MAPK downstream of the bax-caspase pathway may play a pivotal role in endothelial apoptosis mediated by high D-glucose.
Recent evidence from cultured endothelial cell studies suggests that phosphorylation of endothelial nitric oxide synthase (eNOS) through the PI3-kinase-Akt pathway increases NO production. This study was designed to elucidate the signaling pathway involved in the antiapoptotic effect of insulin in vivo and to test the hypothesis that phosphorylation of eNOS by insulin may participate in the cardioprotective effect of insulin after myocardial ischemia and reperfusion.
Male Sprague-Dawley rats were subjected to 30 minutes of myocardial ischemia and 4 hours of reperfusion. Rats were randomized to receive vehicle, insulin, insulin plus wortmannin, or insulin plus L-NAME. Treatment with insulin resulted in 2.6-fold and 4.3-fold increases in Akt and eNOS phosphorylation and a significant increase in NO production in ischemic/reperfused myocardial tissue. Phosphorylation of Akt and eNOS and increase of NO production by insulin were completely blocked by wortmannin, a PI3-kinase inhibitor. Pretreatment with L-NAME, a nonselective NOS inhibitor, had no effect on Akt and eNOS phosphorylation but significantly reduced NO production. Moreover, treatment with insulin markedly reduced myocardial apoptotic death (P<0.01 versus vehicle). Pretreatment with wortmannin abolished the antiapoptotic effect of insulin. Most importantly, pretreatment with L-NAME also significantly reduced the antiapoptotic effect of insulin (P<0.01 versus insulin).
These results demonstrated that in vivo administration of insulin activated Akt through the PI3-kinase-dependent mechanism and reduced postischemic myocardial apoptotic death. Phosphorylation of eNOS and the concurrent increase of NO production contribute significantly to the antiapoptotic effect of insulin.
Mammalian thioredoxin (TRX) with redox-active dithiol in the active site plays multiple roles in intracellular signaling and resistance against oxidative stress. TRX is induced by a variety of stresses including infectious agents as well as hormones and chemicals. TRX is secreted from activated cells such as HTLV-I-transformed T-cells as a redox-sensitive molecule with cytokine-like and chemokine-like activities. The promoter of the TRX gene contains a series of stress-responsive elements. In turn, TRX promotes activation of transcription factors such as NF-kappa B, AP-1, and p53. We have reported that natural substances including estrogen, prostaglandins, and cAMP induce mRNA, protein, and secretion of TRX. These agents seemed to exert their physiological functions including cytoprotective actions partly through the induction of TRX without massive oxidative stress, which induces TRX strongly as well as other stress proteins. We report here a new TRX inducer substance, geranylgeranylacetone (GGA), which is originally derived from a natural plant constituent and has been used in the clinical field as an anti-ulcer drug. We have demonstrated that GGA induces the messenger RNA and protein of TRX and affects the activation of transcription factors, AP-1 and NF-kappa B, and that GGA blunted ethanol-induced cytotoxicity of cultured hepatocytes and gastrointestine mucosal cells. We will discuss a possible novel molecular mechanism of GGA, which is to protect cells via the induction of TRX and activation of transcription factors such as NF-kappa B and AP-1. Identification of the particular TRX-inducing components may contribute to the elucidation of the molecular basis of the "French Paradox," in which good red wines are beneficial for the cardiovascular system.
To identify proteins undergoing glutathionylation (formation of protein-glutathione mixed disulfides) in human T cell blasts, we radiolabeled the glutathione pool with (35)S, exposed cells to the oxidant diamide, and analyzed cellular proteins by two-dimensional electrophoresis. One of the proteins undergoing glutathionylation was identified by molecular weight, isoelectric point, and immunoblotting as thioredoxin (Trx). Incubation of recombinant human Trx with glutathione disulfide or S-nitrosoglutathione led to the formation of glutathionylated Trx, identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. The glutathionylation site was identified as Cys-72. Glutathionylation of rhTrx abolished its enzymatic activity as insulin disulfide reductase in the presence of NADPH and Trx reductase. Activity was, however, regained with sigmoidal kinetics, indicating a process of autoactivation due to the ability of Trx to de-glutathionylate itself. These data suggest that the intracellular glutathione/glutathione disulfide ratio, an indicator of the redox state of the cell, can regulate Trx functions reversibly through thiol-disulfide exchange reactions.
Peroxynitrite is a cytotoxic oxidant formed from nitric oxide (NO) and superoxide. Tyrosine nitration, a footprint of peroxynitrite, has been demonstrated in the pancreatic islets as well as in the cardiovascular system of diabetic subjects. Delineation of the pathogenetic role of peroxynitrite in disease conditions requires the use of potent, in vivo active peroxynitrite decomposition catalysts. The aim of the current work was to produce a potent peroxynitrite decomposition catalyst and to test its effects in rodent models of diabetes and its complications.
FP15 was synthesized and analyzed using standard chemical methods. Diabetes was triggered by the administration of streptozotocin. Tyrosine nitration was measured immunohistochemically. Cardiovascular and vascular measurements were conducted according to standard physiologic methods.
FP15, a potent porphyrinic peroxynitrite decomposition catalyst, potently inhibited tyrosine nitration and peroxynitrite-induced cytotoxicity in vitro and in vivo. FP15 treatment (3-10 mg/kg/d) dose dependently and reduced the incidence and severity of diabetes mellitus in rats subjected to multiple low doses of streptozotocin, as well as in nonobese mice developing spontaneous autoimmune diabetes. Furthermore, treatment with FP15 protected against the development of vascular dysfunction (loss of endothelium-dependent relaxations) and the cardiac dysfunction (loss of myocardial contractility) in diabetic mice. FP15 treatment reduced tyrosine nitration in the diabetic pancreatic islets.
The current results demonstrate the importance of endogenous peroxynitrite generation in the pathogenesis of autoimmune diabetes and diabetic cardiovascular complications. Peroxynitrite decomposition catalysts may be of therapeutic utility in diabetes and other pathophysiologic conditions.
Thioredoxin (Trx1) is a redox-active protein containing two active site cysteines (Cys-32 and Cys-35) that cycle between the dithiol and disulfide forms as Trx1 reduces target proteins. Examination of the redox characteristics of this active site dithiol/disulfide couple is complicated by the presence of three additional non-active site cysteines. Using the redox Western blot technique and matrix assisted laser desorption ionization time-of-flight mass spectrometry mass spectrometry, we determined the midpoint potential (E0) of the Trx1 active site (-230 mV) and identified a second redox-active dithiol/disulfide (Cys-62 and Cys-69) in an alpha helix proximal to the active site, which formed under oxidizing conditions. This non-active site disulfide was not a substrate for reduction by thioredoxin reductase and delayed the reduction of the active site disulfide by thioredoxin reductase. Within actively growing THP1 cells, most of the active site of Trx1 was in the dithiol form, whereas the non-active site was totally in the dithiol form. The addition of increasing concentrations of diamide to these cells resulted in oxidation of the active site at fairly low concentrations and oxidation of the non-active site at higher concentrations. Taken together these results suggest that the Cys-62-Cys-69 disulfide could provide a means to transiently inhibit Trx1 activity under conditions of redox signaling or oxidative stress, allowing more time for the sensing and transmission of oxidative signals.
Apoptosis contributes to myocardial ischemia/reperfusion (MI/R) injury, and both thioredoxin (Trx) and nitric oxide have been shown to exert antiapoptotic effects in vitro. Recent evidence suggests that this particular action of Trx requires S-nitrosation at Cys-69. The present study sought to investigate whether or not exogenously applied Trx reduces MI/R injury in vivo and to which extent this effect depends on S-nitrosation. Adult mice were subjected to 30 min of MI and treated with either vehicle or human Trx (hTrx, 2 mg/kg, i.p.) 10 min before reperfusion. Native hTrx was incorporated into myocardial tissue as shown by immunostaining, and reduced MI/R injury as evidenced by decreased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining, DNA fragmentation, caspase-3 activity, and infarct size. When hTrx was partially S-nitrosated by preincubation with S-nitrosoglutathione, its cardioprotective effect was markedly enhanced. Treatment with hTrx significantly reduced p38 mitogen-activated protein kinase (MAPK) activity, and this effect was also potentiated by S-nitrosation. To further address the role of S-nitrosation for the overall antiapoptotic effect to Trx, the action of Escherichia coli Trx (eTrx) was investigated in the same model. Whereas eTrx inhibited MI/R-induced apoptosis to a degree similar to hTrx, S-nitrosation of this protein, which lacks Cys-69, failed to further enhance its antiapoptotic action. Collectively, our results demonstrate that systemically applied Trx is taken up by the myocardium to exert potent cardioprotective effects in vivo, offering interesting therapeutic avenues. In the case of hTrx, these effects are further potentiated by S-nitrosation, but this posttranslational modification is not essential for protection.
• reperfusion injury
• apoptosis
• antioxidant
• posttranslational regulation
Oxidative stress plays an important role in atherosclerotic vascular disease, and several recent studies were focused on thioredoxin-1 (Trx-1) and its potential protective role against oxidative stress. Since human monocyte-derived macrophages (HMDM) are important cells in several inflammatory diseases including atherosclerosis, we conducted this study to evaluate the impact of extracellular recombinant human Trx-1 (rhTrx-1) on gene expression in lipopolysaccharide-activated HMDM. Our results showed that rhTrx-1 was capable of reducing interleukin (IL)-1beta mRNA and protein synthesis in a dose-dependent manner. This effect was partly mediated through a reduction of NF-kappaB activation as analyzed by transient transfection and gel shift assays. In addition, we showed that the attenuation of NF-kappaB activity was the result of the reduction of both p50 and p65 subunit mRNA and protein synthesis on one hand and of the induction of I-kappaBalpha mRNA and protein expression on the other hand. Moreover, inhibition of endogenous Trx-1 mRNA was also observed, suggesting a contribution to the diminution of NF-kappaB activity since endogenous Trx-1, in contrast to the exogenous Trx-1, activates the NF-kappaB system. Finally, H2O2-oxidized rhTrx-1 reduced IL-1beta mRNA synthesis in lipopolysaccharide-activated HMDM. This result highly suggested that the rhTrx-1 used in this study could be oxidized in the culture medium and, in turn, reduced IL-1beta mRNA and protein synthesis. Taken together, these data indicated a potential new mechanism through which extracellular rhTrx-1 exerts an anti-inflammatory function in HMDM.
Thioredoxin (Trx) is an oxidoreductase that prevents free radical-induced cell death in cultured cells. Here we assessed the mechanism(s) underlying the cardioprotective effects of Trx in vivo.
The effects of myocardial ischemia (30 min) and reperfusion were measured in mice, with assays of myocardial apoptosis, superoxide production, NOx and nitrotyrosine content, and myocardial infarct size. Recombinant human Trx (rhTrx, 0.7-20 mg kg(-1), i.p.) was given 10 min before reperfusion.
Treatment with 2 mg kg(-1) rhTrx significantly decreased myocardial apoptosis and reduced infarct size (P<0.01). Nitrotyrosine content of cardiomyocytes was markedly reduced in rhTrx-treated animals (P<0.01). To further identify the mechanisms by which rhTrx may exert its anti-nitrative effect, iNOS expression and production of NOx and superoxide were determined. Treatment with rhTrx had no significant effect on iNOS expression or NOx content in the ischemic/reperfused heart. However, it markedly upregulated mSOD and reduced tissue superoxide content. To further establish a causative link between the anti- peroxynitrite effect and the cardioprotective effect of rhTrx, cultured adult cardiomyocytes were incubated with SIN-1, a peroxynitrite donor, (50 microM for 3 h) resulting in a nitrotyrosine content comparable to that seen in the ischemic/reperfused heart and causing significant cardiomyocyte apoptosis (P<0.01). Treatment with rhTrx markedly decreased SIN-1 induced apoptosis (P<0.01).
These results demonstrate that Trx is a novel anti-apoptotic and cardioprotective molecule that exerts its cardioprotective effects by reducing ischemia/reperfusion-induced oxidative/nitrative stress.
Several clinical studies have demonstrated that levels of adiponectin are significantly reduced in patients with type 2 diabetes and that adiponectin levels are inversely related to the risk of myocardial ischemia. The present study was designed to determine the mechanism by which adiponectin exerts its protective effects against myocardial ischemia/reperfusion.
Adiponectin-/- or wild-type mice were subjected to 30 minutes of myocardial ischemia followed by 3 hours or 24 hours (infarct size and cardiac function) of reperfusion. Myocardial infarct size and apoptosis, production of peroxynitrite, nitric oxide (NO) and superoxide, and inducible NO synthase (iNOS) and gp91(phox) protein expression were compared. Myocardial apoptosis and infarct size were markedly enhanced in adiponectin-/- mice (P<0.01). Formation of NO, superoxide, and their cytotoxic reaction product, peroxynitrite, were all significantly higher in cardiac tissue obtained from adiponectin-/- than from wild-type mice (P<0.01). Moreover, myocardial ischemia/reperfusion-induced iNOS and gp91(phox) protein expression was further enhanced, but endothelial NOS phosphorylation was reduced in cardiac tissue from adiponectin-/- mice. Administration of the globular domain of adiponectin 10 minutes before reperfusion reduced myocardial ischemia/reperfusion-induced iNOS/gp91(phox) protein expression, decreased NO/superoxide production, blocked peroxynitrite formation, and reversed proapoptotic and infarct-enlargement effects observed in adiponectin-/- mice.
The present study demonstrates that adiponectin is a natural molecule that protects hearts from ischemia/reperfusion injury by inhibition of iNOS and nicotinamide adenine dinucleotide phosphate-oxidase protein expression and resultant oxidative/nitrative stress.
Hyperglycemia is an independent risk factor for diabetic heart failure. However, the mechanisms that mediate hyperglycemia-induced cardiac damage remain poorly understood. The transcription factor GATA4 is essential for cardiac homeostasis, and its protein levels are dramatically reduced in the heart in response to diverse pathologic stresses. In this study, we investigated if hyperglycemia affects GATA4 expression in cardiomyocytes and if enhancing GATA4 signaling could attenuate hyperglycemia-induced cardiomyocyte injury. In cultured rat cardiomyocytes, high glucose (HG, 25 or 40 mm) markedly reduced GATA4 protein levels as compared with normal glucose (NG, 5.5 mm). Equal amount of mannitol did not affect GATA4 protein expression (NG, 100 +/- 12%; mannitol, 97 +/- 8%, versus HG, 43 +/- 16%, p < 0.05). The GATA4 mRNA content, either steady-state or polysome-associated, remained unchanged. HG-induced GATA4 reduction was reversed by MG262, a specific proteasome inhibitor. HG did not activate the ubiquitin proteasome system (UPS) in cardiomyocytes as indicated by a UPS reporter, nor did it increase the peptidase activities or protein expression of the proteasomal subunits. However, the mRNA levels of ubiquitin-protein isopeptide ligase (E3) carboxyl terminus of Hsp70-interacting protein (CHIP) were markedly increased in HG-treated cardiomyocytes. CHIP overexpression promoted GATA4 protein degradation, whereas small interfering RNA-mediated CHIP knockdown prevented HG-induced GATA4 depletion. Moreover, overexpression of GATA4 blocked HG-induced cardiomyocyte death. Also, GATA4 protein levels were diminished in the hearts of streptozotocin and db/db diabetic mice (44 +/- 7% and 67 +/- 13% of control, p < 0.05), which correlated with increased CHIP mRNA abundance. In summary, increased GATA4 protein degradation may be an important mechanism that contributes to hyperglycemic cardiotoxicity.
Peroxynitrite--the product of the diffusion-controlled reaction of nitric oxide with superoxide radical--is a short-lived oxidant species that is a potent inducer of cell death. Conditions in which the reaction products of peroxynitrite have been detected and in which pharmacological inhibition of its formation or its decomposition have been shown to be of benefit include vascular diseases, ischaemia-reperfusion injury, circulatory shock, inflammation, pain and neurodegeneration. In this Review, we first discuss the biochemistry and pathophysiology of peroxynitrite and then focus on pharmacological strategies to attenuate the toxic effects of peroxynitrite. These include its catalytic reduction to nitrite and its isomerization to nitrate by metalloporphyrins, which have led to potential candidates for drug development for cardiovascular, inflammatory and neurodegenerative diseases.
Reactive oxygen species are produced by various stressors derived from internal and external sources, including endogenous metabolic activities. Glucose metabolism is one of the most primitive sources for energy production for most cells; however, it may at the same time yield hazardous oxidative stress via simultaneous oxidant production. The protective mechanism against oxidative stress is thus an indispensable biological function. Recently, genetic mutation loci affecting life span were isolated from experimental model organisms, and several locus products were found to be closely linked with machinery either producing or defending oxidative stress. Thioredoxin (TRX) is a small protein having strong antioxiradical quenching capabilities and other multiple functions depending on the cellular redox state. In this review, we focus on the role of TRX in the aging process (senescence) as a redox-regulating molecule against oxidative stress. We also discuss the possibility of the TRX system serving as an index marker for cellular proliferation and senescence.
Thioredoxin 2 (Trx-2) is a small redox protein containing the thioredoxin active site Trp-Cys-Gly-Pro-Cys that is localized
to the mitochondria by a mitochondrial leader sequence and encoded by a nuclear gene (Trx-2). Trx-2 plays an important role in cell viability and the regulation of apoptosis in vitro. To investigate the role of Trx-2
in mouse development, we studied the phenotype of mice that have the Trx-2 gene silenced by mutational insertion. Homozygous mutant embryos do not survive to birth and die after implantation at Theiler
stage 15/16. The homozygous mutant embryos display an open anterior neural tube and show massively increased apoptosis at
10.5 days postcoitus and are not present by 12.5 days postcoitus. The timing of the embryonic lethality coincides with the
maturation of the mitochondria, since they begin oxidative phosphorylation during this stage of embryogenesis. In addition,
embryonic fibroblasts cultured from homozygous Trx-2-null embryos were not viable. Heterozygous mice are fertile and have no discernible phenotype visible by external observation,
despite having decreased Trx-2 mRNA and protein. These results show that the mitochondrial redox protein Trx-2 is required for normal development of the
mouse embryo and for actively respiring cells.
There is a wealth of clinical data showing the relationship between diabetes mellitus and atherosclerosis and its clinical complications. To dissect this relationship, investigators have attempted, usually unsuccessfully, to create a small-animal model in which diabetes accelerates vascular lesion development. This effort has often been complicated by development of hyperlipidemia leading to difficulty in differentiating the effects of hyperglycemia from those of lipid abnormalities. A study in the current issue of the JCI provides data on a new mouse model in which atherosclerosis initiation is accelerated in diabetic mice and is reduced by insulin therapy. Moreover, these animals have greater intra-arterial hemorrhage, which might be due to less stable plaques (see the related article beginning on page 659).
Background— Recent evidence from cultured endothelial cell studies suggests that phosphorylation of endothelial nitric oxide synthase (eNOS) through the PI3-kinase– Akt pathway increases NO production. This study was designed to elucidate the signaling pathway involved in the antiapoptotic effect of insulin in vivo and to test the hypothesis that phosphorylation of eNOS by insulin may participate in the cardioprotective effect of insulin after myocardial ischemia and reperfusion.
Methods and Results— Male Sprague-Dawley rats were subjected to 30 minutes of myocardial ischemia and 4 hours of reperfusion. Rats were randomized to receive vehicle, insulin, insulin plus wortmannin, or insulin plus L-NAME. Treatment with insulin resulted in 2.6-fold and 4.3-fold increases in Akt and eNOS phosphorylation and a significant increase in NO production in ischemic/reperfused myocardial tissue. Phosphorylation of Akt and eNOS and increase of NO production by insulin were completely blocked by wortmannin, a PI3-kinase inhibitor. Pretreatment with L-NAME, a nonselective NOS inhibitor, had no effect on Akt and eNOS phosphorylation but significantly reduced NO production. Moreover, treatment with insulin markedly reduced myocardial apoptotic death ( P <0.01 versus vehicle). Pretreatment with wortmannin abolished the antiapoptotic effect of insulin. Most importantly, pretreatment with L-NAME also significantly reduced the antiapoptotic effect of insulin ( P <0.01 versus insulin).
Conclusions— These results demonstrated that in vivo administration of insulin activated Akt through the PI3-kinase–dependent mechanism and reduced postischemic myocardial apoptotic death. Phosphorylation of eNOS and the concurrent increase of NO production contribute significantly to the antiapoptotic effect of insulin.
The ability of mismatch binding proteins, in particular the Escherichia coli MutS protein, to bind DNA containing mismatched base pairs or one to four unpaired bases makes them ideally suited to use in mutation/polymorphism detection assays. Several methods, including nuclease protection, band shift, and filter binding assays, have been employed with varying degrees of success. New assays, including genemic mismatch scanning, immobilized mismatch binding protein, and direct detection of mismatch binding protein, are being developed and appear promising. Mismatch binding protein-based assays have the potential for widespread use in both basic research and clinical applications.
Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.
While a damaged endothelium is recognised to be a key accessory to diabetic macroangiopathy, awareness is developing that impairments concerning endothelium- and nitric oxide (NO)-dependent microvascular function, may contribute to several other corollaries of diabetes, such as hypertension, dyslipidaemia and in vivo insulin resistance. There are now several reports describing elevations in specific oxidant stress markers in both insulin resistance syndrome (IRS) and diabetes, together with determinations of reduced total antioxidant defence and depletions in individual antioxidants. Such a pro-oxidant environment in diabetes may disrupt endothelial function through the inactivation of NO, resulting in the attenuation of a fundamental anti-atherogenic and euglycaemic vascular influence. Indeed, experimental and clinical data suggest that the supplementation of insulin resistant or diabetic states with antioxidants such as vitamin E, normalises oxidant stress and improves both endothelium-dependent vasodilation and insulin sensitivity. However, the promising potential efficacy of antioxidant therapy in cardiovascular disease and diabetes, in either a primary or secondary preventative role, awaits definitive clinical demonstration.
Thioredoxin 1 (Trx) is a known redox regulator that is implicated in the redox control of cell growth and apoptosis inhibition. Here we show that Trx is essential for maintaining the content of S-nitrosylated molecules in endothelial cells. Trx itself is S-nitrosylated at cysteine 69 under basal conditions, and this S-nitrosylation is required for scavenging reactive oxygen species and for preserving the redox regulatory activity of Trx. S-nitrosylation of Trx also contributes to the anti-apoptotic function of Trx. Thus, Trx can exert its complete redox regulatory and anti-apoptotic functions in endothelial cells only when cysteine 69 is S-nitrosylated.
There is growing evidence that cardiovascular disease is associated with progressive changes in the production of free radicals and radical-derived reactive species. These intermediates react with all major cellular constituents and may serve several physiological and pathophysiological functions. The nitration of protein tyrosine residues has been used as a footprint for in vivo production of radical and nonradical reactive species. Tyrosine nitration may alter protein function and metabolism and therefore, provides for further dysfunctional changes. This review focuses on an appearance of tyrosine nitrated proteins in cardiovascular tissues under different settings of cardiovascular disease. Sources of reactive species, putative mechanisms of protein nitration in vivo, as well as protein nitration under normal physiological conditions, are also described. The goal of this review is to attract more attention to identification of specific proteins, which undergo tyrosine nitration and to study a correlation between their altered function and pathology. Understanding how protein nitration affects disease progression may offer a unique option for design of antioxidant therapy for the treatment of cardiovascular complications. At the same time, protein nitration can be a biological marker of efficiency of antioxidant therapy.
Reactive oxygen species (ROS) have been implicated in the pathogenesis of vascular dysfunction in diabetes mellitus, and NAD(P)H oxidase is known as the most important source of ROS in the vasculatures. To determine whether NAD(P)H oxidase is a major participant in the critical intermediary signaling events in high glucose (HG, 25 mM)-induced proliferation of vascular smooth muscle cells (VSMC), we investigated in explanted aortic VSMC from rats the role of NAD(P)H oxidase on the HG-related cellular proliferation and superoxide production. VSMC under HG condition had increased proliferative capacity that was inhibited by tiron (1 mM), a cell membrane permeable superoxide scavenger, but not by SOD, which is not permeable to cell membrane. The nitroblue tetrazolium staining in the HG-exposed VSMC was more prominent than that of VSMC under normal glucose (5.5 mM) condition, which was significantly inhibited by DPI (10 microM), an NAD(P)H oxidase inhibitor, but not by inhibitors for other oxidases such as NADH dehydrogenase, xanthine oxidase, and nitric oxide synthase. In the VSMC under HG condition, the enhanced NAD(P)H oxidase activity with increased membrane translocation of Rac1 was observed, but the protein expression of p22phox and gp91phox was not increased. These data suggest that HG-induced changes in VSMC proliferation are related to the intracellular production of superoxide through enhanced activity of NAD(P)H oxidase.
Redox control has emerged as a fundamental biological control mechanism. One of the major redox control systems is the thioredoxin system comprised of thioredoxin (TRX) and thioredoxin reductase (TR). Together they form a powerful system involved in many central intracellular and extracellular processes including cell proliferation, the redox regulation of gene expression and signal transduction, protection against oxidative stress, anti-apoptotic functions, growth factor and co-cytokine effects, and regulation of the redox state of the extracellular environment. Over recent years this system has increasingly been linked to the development and expression of cancer phenotypes. In this report immunocytochemical approaches have been used to simultaneously determine the expression and localisation of both TRX and TR in primary human cancers, including breast cancer, thyroid, prostate and colorectal carcinoma, and malignant melanoma. In aggressive invasive mammary carcinomas and advanced malignant melanomas, thioredoxin was highly over-expressed compared to tumours of lesser aggressive nature. TRX expression was found in both nuclear and cytoplasmic location in the neoplastic cells. Furthermore, increased levels of TRX positively correlate with thioredoxin reductase (TR) expression and localisation. These results, which are the first immunocytochemical studies on the in vivo expression and localisation of TRX and TR in melanomas, thyroid, prostate and colorectal carcinomas and the first reports of TR expression in breast carcinomas, significantly extend the range of human cancers for which such data is available. Overall the results support the conclusion that aggressive tumours greatly over-express both TRX and TR. Such tumours have a high proliferation capacity, a low apoptosis rate and an elevated metastatic potential strongly implicating the involvement of the TRX system in the processes of oncogenesis and tumourogenesis and confirming its potential as a target for anticancer therapy for a wide range of human tumours.
Local overproduction of nitric oxide is seen in early stages of diabetes, which can react with superoxide (O(2)(-)) to form peroxynitrite (ONOO(-)). The aim of this study was to examine the effect of scavengers for nitric oxide, O(2)(-), ONOO(-) and NOS cofactor tetrahydrobiopterin (BH(4)) on high glucose-induced cardiac contractile dysfunction.
Ventricular myocytes were cultured for 24 h with either normal (N, 5.5 mmol/l) or high (25.5 mmol/l) glucose, with or without the nitric oxide scavengers haemoglobin (100 nmol/l), PTIO (100 micromol/l), the NOS inhibitor L-NMMA (100 micromol/l), superoxide dismutase (SOD, 500 U/ml), the ONOO(-) scavengers urate (100 micromol/l), MnTABP (100 micromol/l), BH(4) (10 micromol/l) and its inactive analogue NH(4) (10 micromol/l), and the GTP cyclohydrolase I inhibitor DAHP (1 mmol/l). Myocyte mechanics, NOS protein expression and activity were evaluated.
High glucose myocytes showed reduced peak shortening, decreased maximal velocity of shortening/relengthening (+/- dL/dt), prolonged relengthening (TR(90)) and normal shortening duration (TPS) associated with reduced cytosolic Ca(2+) rise compared to normal myocytes. The high glucose-induced abnormalities were abrogated or attenuated by urate, MnTBAP, L-NMMA, BH(4), and SOD, whereas unaffected by haemoglobin, PTIO and NH(4). L-NMMA reduced peak shortening while PTIO and DAHP depressed +/- dL/dt and prolonged TPS or TR(90) in normal myocytes. High glucose increased NOS activity, protein expression of eNOS but not iNOS, which were attenuated by L-NMMA and BH(4), respectively.
These results suggested that NOS cofactor, NO and ONOO(-) play a role in glucose-induced cardiomyocyte contractile dysfunction and in the pathogenesis of diabetic cardiomyopathy.
There is a wealth of clinical data showing the relationship between diabetes mellitus and atherosclerosis and its clinical complications. To dissect this relationship, investigators have attempted, usually unsuccessfully, to create a small-animal model in which diabetes accelerates vascular lesion development. This effort has often been complicated by development of hyperlipidemia leading to difficulty in differentiating the effects of hyperglycemia from those of lipid abnormalities. A study in the current issue of the JCI provides data on a new mouse model in which atherosclerosis initiation is accelerated in diabetic mice and is reduced by insulin therapy. Moreover, these animals have greater intra-arterial hemorrhage, which might be due to less stable plaques.
Intracellular proteins involved in oxidative stress and apoptosis are nitrated in diseased tissues but not in normal tissues; definitive evidence to support a causative link between a specific protein that is nitratively modified with tissue injury in a specific disease is limited, however. The aims of the present study were to determine whether thioredoxin (Trx), a novel antioxidant and antiapoptotic molecule, is susceptible to nitrative inactivation and to establish a causative link between Trx nitration and postischemic myocardial apoptosis.
In vitro exposure of human Trx-1 to 3-morpholinosydnonimine resulted in significant Trx-1 nitration and almost abolished Trx-1 activity. 3-morpholinosydnonimine-induced nitrative Trx-1 inactivation was completely blocked by MnTE-2-PyP(5+) (a superoxide dismutase mimetic) and markedly attenuated by PTIO (a nitric oxide scavenger). Administration of either reduced or oxidized Trx-1 in vivo attenuated myocardial ischemia/reperfusion injury (>50% reduction in apoptosis and infarct size, P<0.01). However, administration of nitrated Trx-1 failed to exert a cardioprotective effect. In cardiac tissues obtained from ischemic/reperfused heart, significant Trx-1 nitration was detected, Trx activity was markedly inhibited, Trx-1/ASK1 (apoptosis signal-regulating kinase-1) complex formation was abolished, and apoptosis signal-regulating kinase-1 activity was increased. Treatment with either FP15 (a peroxynitrite decomposition catalyst) or MnTE-2-PyP(5+) 10 minutes before reperfusion blocked nitrative Trx inactivation, attenuated apoptosis signal-regulating kinase-1 activation, and reduced postischemic myocardial apoptosis.
These results strongly suggest that nitrative inactivation of Trx plays a proapoptotic role under those pathological conditions in which production of reactive nitrogen species is increased and that antinitrating treatment may have therapeutic value in those diseases, such as myocardial ischemia/reperfusion, in which pathological apoptosis is increased.
Endothelial dysfunction is one manifestation of the many changes induced in the arterial wall by the metabolic abnormalities accompanying diabetes and insulin resistance. In type 1 diabetes, endothelial dysfunction is most consistently found in advanced stages of the disease. In other patients, it is associated with nondiabetic insulin resistance and probably precedes type 2 diabetes. In obesity and insulin resistance, increased secretion of proinflammatory cytokines and decreased secretion of adiponectin from adipose tissue, increased circulating levels of free fatty acids, and postprandial hyperglycemia can all alter gene expression and cell signaling in vascular endothelium, cause vascular insulin resistance, and change the release of endothelium-derived factors. In diabetes, sustained hyperglycemia causes increased intracellular concentrations of glucose metabolites in endothelial cells. These changes cause mitochondrial dysfunction, increased oxidative stress, and activation of protein kinase C. Dysfunctional endothelium displays activation of vascular NADPH oxidase, uncoupling of endothelial nitric oxide synthase, increased expression of endothelin 1, a changed balance between the production of vasodilator and vasoconstrictor prostanoids, and induction of adhesion molecules. This review describes how these and other changes influence endothelium-dependent vasodilation in patients with insulin resistance and diabetes. The clinical utility of endothelial function testing and future therapeutic targets is also discussed.
Reduced thioredoxin denitrosylates caspase-3, a key protein involved in cell death.
Hyperglycemia is common and associated with markedly increased mortality rates in patients hospitalized with acute coronary syndromes (ACS). Despite the fact that several studies have documented this association, hyperglycemia remains underappreciated as a risk factor, and it is frequently untreated in ACS patients. This is in large part due to limitations of prior studies, and the remaining critical gaps in our understanding of the relationship between hyperglycemia and poor outcomes. The main objective of the present statement is to summarize the current state of knowledge regarding the association between elevated glucose and patient outcomes in ACS and to outline the most important knowledge gaps in this field. These gaps include the need to specifically define hyperglycemia, develop optimal ways of measuring and tracking glucose values during ACS hospitalization, and better understand the physiological mechanisms responsible for poor outcomes associated with hyperglycemia. The most important issue, however, is whether elevated glucose is a direct mediator of adverse outcomes in ACS patients or just a marker of greater disease severity. Given the marked increase in short- and long-term mortality associated with hyperglycemia, there is an urgent need for definitive large randomized trials to determine whether treatment strategies aimed at glucose control will improve patient outcomes and to define specific glucose treatment targets. Although firm guidelines will need to await completion of these clinical trials, the present statement also provides consensus recommendations for hyperglycemia management in patients with ACS on the basis of the available data.
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