RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

Background

Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.

Methods/Findings

Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.

Conclusion/Significance

This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.     

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells

Aim

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.

Method

RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.

Results

The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine¹¹⁷⁷ phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N⁵-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.

Conclusion

This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.     

Inhibition of PI3K-Akt Signaling Blocks Exercise-Mediated Enhancement of Adult Neurogenesis and Synaptic Plasticity in the Dentate Gyrus

Background

Physical exercise has been shown to increase adult neurogenesis in the dentate gyrus and enhances synaptic plasticity. The antiapoptotic kinase, Akt has also been shown to be phosphorylated following voluntary exercise; however, it remains unknown whether the PI3K-Akt signaling pathway is involved in exercise-induced neurogenesis and the associated facilitation of synaptic plasticity in the dentate gyrus.

Methodology/Principal Findings

To gain insight into the potential role of this signaling pathway in exercise-induced neurogenesis and LTP in the dentate gyrus rats were infused with the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or vehicle control solution (icv) via osmotic minipumps and exercised in a running wheel for 10 days. Newborn cells in the dentate gyrus were date-labelled with BrdU on the last 3 days of exercise. Then, they were either returned to the home cage for 2 weeks to assess exercise-induced LTP and neurogenesis in the dentate gyrus, or were killed on the last day of exercise to assess proliferation and activation of the PI3K-Akt cascade using western blotting.

Conclusions/Significance

Exercise increases cell proliferation and promotes survival of adult-born neurons in the dentate gyrus. Immediately after exercise, we found that Akt and three downstream targets, BAD, GSK3β and FOXO1 were activated. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 blocked exercise-induced phosphorylation of Akt and downstream target proteins. This had no effect on exercise-induced cell proliferation, but it abolished most of the beneficial effect of exercise on the survival of newly generated dentate gyrus neurons and prevented exercise-induced increase in dentate gyrus LTP. These results suggest that activation of the PI3 kinase-Akt signaling pathway plays a significant role via an antiapoptotic function in promoting survival of newly formed granule cells generated during exercise and the associated increase in synaptic plasticity in the dentate gyrus.     

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes

Introduction

Synovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.

Methods

Human chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm²). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.

Results

Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.

Conclusions

These data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Exercise-Induced Blood Lactate Increase Does Not Change Red Blood Cell Deformability in Cyclists

Background

The effect of exercise-induced lactate production on red blood cell deformability and other blood rheological changes is controversial, given heavy-exercise induces biochemical processes (e.g., oxidative stress) known to perturb haemorheology. The aim of the present study was to examine the haemorheological response to a short-duration cycling protocol designed to increase blood lactate concentration, but of duration insufficient to induce significant oxidative stress.

Methods

Male cyclists and triathletes (n = 6; 27±7 yr; body mass index: 23.7±3.0 kg/m²; peak oxygen uptake 4.02±0.51 L/min) performed unloaded (0 W), moderate-intensity, and heavy-intensity cycling. Blood was sampled at rest and during the final minute of each cycling bout. Blood chemistry, blood viscosity, red blood cell aggregation and red blood cell deformability were measured.

Results

Blood lactate concentration increased significantly during heavy-intensity cycling, when compared with all other conditions. Methaemoglobin fraction did not change during any exercise bout when compared with rest. Blood viscosity at native haematocrit increased during heavy-intensity cycling at higher-shear rates when compared with rest, unloaded and moderate-intensity cycling. Heavy-intensity exercise increased the amplitude of red blood cell aggregation in native haematocrit samples when compared with all other conditions. Red blood cell deformability was not changed by exercise.

Conclusion

Acute exercise perturbs haemorheology in an intensity dose-response fashion; however, many of the haemorheological effects appear to be secondary to haemoconcentration, rather than increased lactate concentration.     

Rapid Rather than Gradual Weight Reduction Impairs Hemorheological Parameters of Taekwondo Athletes through Reduction in RBC-NOS Activation

Purpose

Rapid weight reduction is part of the pre-competition routine and has been shown to negatively affect psychological and physiological performance of Taekwondo (TKD) athletes. This is caused by a reduction of the body water and an electrolyte imbalance. So far, it is unknown whether weight reduction also affects hemorheological properties and hemorheology-influencing nitric oxide (NO) signaling, important for oxygen supply to the muscles and organs.

Methods

For this purpose, ten male TKD athletes reduced their body weight by 5% within four days (rapid weight reduction, RWR). After a recovery phase, athletes reduced body weight by 5% within four weeks (gradual weight reduction, GWR). Each intervention was preceded by two baseline measurements and followed by a simulated competition. Basal blood parameters (red blood cell (RBC) count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean cellular hemoglobin and mean cellular hemoglobin concentration), RBC-NO synthase activation, RBC nitrite as marker for NO synthesis, RBC deformability and aggregation parameters were determined on a total of eight investigation days.

Results

Basal blood parameters were not affected by the two interventions. In contrast to GWR, RWR decreased activation of RBC-NO synthase, RBC nitrite, respective NO concentration and RBC deformability. Additionally, RWR increased RBC aggregation and disaggregation threshold.

Conclusion

The results point out that a rapid weight reduction negatively affects hemorheological parameters and NO signaling in RBC which might limit performance capacity. Thus, GWR should be preferred to achieve the desired weight prior to a competition to avoid these negative effects.     

Effects of Moderate Aerobic Exercise Training on Hemorheological and Laboratory Parameters in Ischemic Heart Disease Patients

Background and Design

In this study we set out to determine the effects of long-term physical training on hemorheological, laboratory parameters, exercise tolerability, psychological factors in cardiac patients participating in an ambulatory rehabilitation program.

Methods

Before physical training, patients were examined by echocardiography, tested on treadmill by the Bruce protocol, and blood was drawn for laboratory tests. The enrolled 79 ischemic heart disease patients joined a 24-week cardiac rehabilitation training program. Blood was drawn to measure hematocrit (Hct), plasma and whole blood viscosity (PV, WBV), red blood cell (RBC) aggregation and deformability. Hemorheological, clinical chemistry and psychological measurements were repeated 12 and 24 weeks later, and a treadmill test was performed at the end of the program.

Results

After 12 weeks Hct, PV, WBV and RBC aggregation were significantly decreased, RBC deformability exhibited a significant increase (p<0.05). Laboratory parameters (triglyceride, uric acid, hsCRP and fibrinogen) were significantly decreased (p<0.05). After 24 weeks the significant results were still observed. By the end of the study, IL-6 and TNF-α levels displayed decreasing trends (p<0.06). There was a significant improvement in MET (p<0.001), and the BMI decrease was also significant (p<0.05). The vital exhaustion parameters measured on the fatigue impact scale indicated a significant improvement in two areas of the daily activities (p<0.05).

Conclusions

Regular physical training improved the exercise tolerability of patients with ischemic heart disease. Previous publications have demonstrated that decreases in Hct and PV may reduce cardiovascular risk, while a decrease in RBC aggregation and an increase in deformability improve the capillary flow. Positive changes in laboratory parameters and body weight may indicate better oxidative and inflammatory circumstances and an improved metabolic state. The psychological findings point to an improvement in the quality of life.     

Nitric oxide-induced activation of the AMP-activated protein kinase α2 subunit attenuates IκB kinase activity and inflammatory responses in endothelial cells

Background

In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO).

Methodology/Principal Findings

Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2^-/- mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2^-/- mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2^+/+ versus AMPKα2^-/- mice.

Conclusions

These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK.     

Vascular endothelial growth factor receptor-2 couples cyclo-oxygenase-2 with pro-angiogenic actions of leptin on human endothelial cells

Background

The adipocyte-derived hormone leptin influences the behaviour of a wide range of cell types and is now recognised as a pro-angiogenic and pro-inflammatory factor. In the vasculature, these effects are mediated in part through its direct leptin receptor (ObRb)-driven actions on endothelial cells (ECs) but the mechanisms responsible for these activities have not been established. In this study we sought to more fully define the molecular links between inflammatory and angiogenic responses of leptin-stimulated human ECs.

Methodology/Principal Findings

Immunoblotting studies showed that leptin increased cyclo-oxygenase-2 (COX-2) expression (but not COX-1) in cultured human umbilical vein ECs (HUVEC) through pathways that depend upon activation of both p38 mitogen-activated protein kinase (p38^MAPK) and Akt, and stimulated rapid phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) on Tyr¹¹⁷⁵. Phosphorylation of VEGFR2, p38^MAPK and Akt, and COX-2 induction in cells challenged with leptin were blocked by a specific leptin peptide receptor antagonist. Pharmacological inhibitors of COX-2, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and p38^MAPK abrogated leptin-induced EC proliferation (assessed by quantifying 5-bromo-2′-deoxyuridine incorporation, calcein fluorescence and propidium iodide staining), slowed the increased migration rate of leptin-stimulated cells (in vitro wound healing assay) and inhibited leptin-induced capillary-like tube formation by HUVEC on Matrigel. Inhibition of VEGFR2 tyrosine kinase activity reduced leptin-stimulated p38^MAPK and Akt activation, COX-2 induction, and pro-angiogenic EC responses, and blockade of VEGFR2 or COX-2 activities abolished leptin-driven neo-angiogenesis in a chick chorioallantoic membrane vascularisation assay in vivo.

Conclusions/Significance

We conclude that a functional endothelial p38^MAPK/Akt/COX-2 signalling axis is required for leptin''s pro-angiogenic actions and that this is regulated upstream by ObRb-dependent activation of VEGFR2. These studies identify a new function for VEGFR2 as a mediator of leptin-stimulated COX-2 expression and angiogenesis and have implications for understanding leptin''s regulation of the vasculature in both non-obese and obese individuals.     

Opposing Functions of Akt Isoforms in Lung Tumor Initiation and Progression

Background

The phosphatidylinositol 3-kinase–regulated protein kinase, Akt, plays an important role in the initiation and progression of human cancer. Mammalian cells express three Akt isoforms (Akt1–3), which are encoded by distinct genes. Despite sharing a high degree of amino acid identity, phenotypes observed in knockout mice suggest that Akt isoforms are not functionally redundant. The relative contributions of the different Akt isoforms to oncogenesis, and the effect of their deficiencies on tumor development, are not well understood.

Methods

Here we demonstrate that Akt isoforms have non-overlapping and sometimes opposing functions in tumor initiation and progression using a viral oncogene-induced mouse model of lung cancer and Akt isoform-specific knockout mice.

Results

Akt1 ablation significantly delays initiation of lung tumor growth, whereas Akt2 deficiency dramatically accelerates tumorigenesis in this mouse model. Ablation of Akt3 had a small, not statistically significant, stimulatory effect on tumor induction and growth by the viral oncogene. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling and Ki67 immunostaining of lung tissue sections revealed that the delayed tumor induction in Akt1^−/− mice was due to the inhibitory effects of Akt1 ablation on cell growth and survival. Conversely, the accelerated growth rate of lung tumors in Akt2^−/− and Akt3^−/− mice was due to increased cell proliferation and reduced tumor cell apoptosis. Investigation of Akt signaling in tumors from Akt knockout mice revealed that the lack of Akt1 interrupted the propagation of signaling in tumors to the critical downstream targets, GSK-3α/β and mTOR.

Conclusions

These results demonstrate that the degree of functional redundancy between Akt isoforms in the context of lung tumor initiation is minimal. Given that this mouse model exhibits considerable similarities to human lung cancer, these findings have important implications for the design and use of Akt inhibitors for the treatment of lung cancer.     

Regulation of Proapoptotic Mammalian ste20–Like Kinase MST2 by the IGF1-Akt Pathway

Background

Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown.

Methodology/Principal Findings

Using immunoblotting, in vitro kinase and in vivo labeling assays, we show that IGF1 inhibits MST2 cleavage and activation induced by DNA damage through the phosphatidylinosotol 3-kinase (PI3K)/Akt pathway. Akt phosphorylates a highly conserved threonine-117 residue of MST2 in vitro and in vivo, which leads to inhibition of MST2 cleavage, nuclear translocation, autophosphorylation-Thr180 and kinase activity. As a result, MST2 proapoptotic and growth arrest function was significantly reduced. Further, inverse correlation between pMST2-T117/pAkt and pMST2-T180 was observed in human breast tumors.

Conclusions/Significance

Our findings demonstrate for the first time that extracellular cell survival signal IGF1 regulates MST2 and that Akt is a key upstream regulator of MST2.     

Metformin Protects Rat Hepatocytes against Bile Acid-Induced Apoptosis

Background

Metformin is used in the treatment of Diabetes Mellitus type II and improves liver function in patients with non-alcoholic fatty liver disease (NAFLD). Metformin activates AMP-activated protein kinase (AMPK), the cellular energy sensor that is sensitive to changes in the AMP/ATP-ratio. AMPK is an inhibitor of mammalian target of rapamycin (mTOR). Both AMPK and mTOR are able to modulate cell death.

Aim

To evaluate the effects of metformin on hepatocyte cell death.

Methods

Apoptotic cell death was induced in primary rat hepatocytes using either the bile acid glycochenodeoxycholic acid (GCDCA) or TNFα in combination with actinomycin D (actD). AMPK, mTOR and phosphoinositide-3 kinase (PI3K)/Akt were inhibited using pharmacological inhibitors. Apoptosis and necrosis were quantified by caspase activation, acridine orange staining and Sytox green staining respectively.

Results

Metformin dose-dependently reduces GCDCA-induced apoptosis, even when added 2 hours after GCDCA, without increasing necrotic cell death. Metformin does not protect against TNFα/ActD-induced apoptosis. The protective effect of metformin is dependent on an intact PI3-kinase/Akt pathway, but does not require AMPK/mTOR-signaling. Metformin does not inhibit NF-κB activation.

Conclusion

Metformin protects against bile acid-induced apoptosis and could be considered in the treatment of chronic liver diseases accompanied by inflammation.     

Aurin Tricarboxylic Acid Protects against Red Blood Cell Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinemia

Objectives

Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage.

Design and Methods

We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera.

Results

We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes.

Conclusions

We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.     

Diazeniumdiolate mediated nitrosative stress alters nitric oxide homeostasis through intracellular calcium and S-glutathionylation of nitric oxide synthetase

Background

PABA/NO is a diazeniumdiolate that acts as a direct nitrogen monoxide (NO) donor and is in development as an anticancer drug. Its mechanism of action and effect on cells is not yet fully understood.

Methodology/Principal Findings

We used HPLC and mass spectrometry to identify a primary nitroaromatic glutathione metabolite of PABA/NO and used fluorescent assays to characterize drug effects on calcium and NO homeostasis, relating these to endothelial nitric oxide synthase (eNOS) activity. Unexpectedly, the glutathione conjugate was found to be a competitive inhibitor of sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA) presumably at the same site as thapsigargin, increasing intracellular Ca²⁺ release and causing auto-regulation of eNOS through S-glutathionylation.

Conclusions/Significance

The initial direct release of NO after PABA/NO was followed by an eNOS-mediated generation of NO as a consequence of drug-induced increase in Ca²⁺ flux and calmodulin (CaM) activation. PABA/NO has a unique dual mechanism of action with direct intracellular NO generation combined with metabolite driven regulation of eNOS activation.     

Effect of Antioxidant Supplementation on Exercise-Induced Cardiac Troponin Release in Cyclists: A Randomized Trial

Background

Cardiac troponin is the biochemical gold standard to diagnose acute myocardial infarction. Interestingly however, elevated cardiac troponin concentrations are also frequently observed during and after endurance-type exercise. Oxidative stress associated with prolonged exercise has been proposed to contribute to cardiac troponin release. Therefore, the aim of this study was to assess the effect of 4 week astaxanthin supplementation (a potent cartenoid antioxidant) on antioxidant capacity and exercise-induced cardiac troponin release in cyclists.

Methods

Thirty-two well-trained male cyclists (age 25±5, weight 73±7 kg, maximum O₂ uptake 60±5 mL·kg⁻¹·min⁻¹, W_max 5.4±0.5 W·kg⁻¹; mean ± SD) were repeatedly subjected to a laboratory based standardized exercise protocol before and after 4 weeks of astaxanthin (20 mg/day), or placebo supplementation in a double-blind randomized manner. Blood samples were obtained at baseline, at 60 min of cycling and immediately post-exercise (≈ 120 min).

Results

The pre-supplementation cycling trial induced a significant rise of median cardiac troponin T concentrations from 3.2 (IQR 3.0–4.2) to 4.7 ng/L (IQR 3.7–6.7), immediately post-exercise (p<0.001). Four weeks of astaxanthin supplementation significantly increased mean basal plasma astaxanthin concentrations from non-detectable values to 175±86 µg·kg⁻¹. However, daily astaxanthin supplementation had no effect on exercise-induced cardiac troponin T release (p = 0.24), as measured by the incremental area under the curve. Furthermore, the elevation in basal plasma astaxanthin concentrations was not reflected in changes in antioxidant capacity markers (trolox equivalent antioxidant capacity, uric acid, and malondialdehyde). Markers of inflammation (high-sensitivity C-reactive protein) and exercise-induced skeletal muscle damage (creatine kinase) were equally unaffected by astaxanthin supplementation.

Conclusion

Despite substantial increases in plasma astaxanthin concentrations, astaxanthin supplementation did not improve antioxidant capacity in well-trained cyclists. Accordingly, exercise-induced cardiac troponin T concentrations were not affected by astaxanthin supplementation.

Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01241877","term_id":"NCT01241877"}}NCT01241877     

Erythrocytes do not activate purified and platelet soluble guanylate cyclases even in conditions favourable for NO synthesis

Background

Direct interaction between Red blood cells (RBCs) and platelets is known for a long time. The bleeding time is prolonged in anemic patients independent of their platelet count and could be corrected by transfusion of RBCs, which indicates that RBCs play an important role in hemostasis and platelet activation. However, in the last few years, opposing mechanisms of platelet inhibition by RBCs derived nitric oxide (NO) were proposed. The aim of our study was to identify whether RBCs could produce NO and activate soluble guanylate cyclase (sGC) in platelets.

Methods

To test whether RBCs could activate sGC under different conditions (whole blood, under hypoxia, or even loaded with NO), we used our well-established and highly sensitive models of NO-dependent sGC activation in platelets and activation of purified sGC. The activation of sGC was monitored by detecting the phosphorylation of Vasodilator Stimulated Phosphoprotein (VASP^S239) by flow cytometry and Western blot. ANOVA followed by Bonferroni’s test and Student’s t-test were used as appropriate.

Results

We show that in the whole blood, RBCs prevent NO-mediated inhibition of ADP and TRAP6-induced platelet activation. Likewise, coincubation of RBCs with platelets results in strong inhibition of NO-induced sGC activation. Under hypoxic conditions, incubation of RBCs with NO donor leads to Hb-NO formation which inhibits sGC activation in platelets. Similarly, RBCs inhibit activation of purified sGC, even under conditions optimal for RBC-mediated generation of NO from nitrite.

Conclusions

All our experiments demonstrate that RBCs act as strong NO scavengers and prevent NO-mediated inhibition of activated platelets. In all tested conditions, RBCs were not able to activate platelet or purified sGC.

     

Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

Background

Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E₂ (PGE₂) are probably also released and could explain the refractory period observed in patients with EIB.

Objective

This study aimed to evaluate the protective effect of PGE₂ on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction.

Methods

We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE₂ and prostanoid receptor (EP) antagonists for EP_1–4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed.

Results

Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK) pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE₂ significantly reduced mannitol-induced degranulation through EP₂ and EP₄ receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone.

Conclusions

Our data show a protective role for the PGE₂ receptors EP₂ and EP₄ following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.     

Dual Role of Endothelial Nitric Oxide Synthase in Oxidized LDL-Induced,p66Shc-Mediated Oxidative Stress in Cultured Human Endothelial Cells

Background

The aging gene p66^Shc, is an important mediator of oxidative stress-induced vascular dysfunction and disease. In cultured human aortic endothelial cells (HAEC), p66^Shc deletion increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B. However, the putative role of the NO pathway on p66^Shc activation remains unclear. This study was designed to elucidate the regulatory role of the eNOS/NO pathway on p66^Shc activation.

Methods and Results

Incubation of HAEC with oxidized low density lipoprotein (oxLDL) led to phosphorylation of p66^Shc at Ser-36, resulting in an enhanced production of superoxide anion (O₂ ^-). In the absence of oxLDL, inhibition of eNOS by small interfering RNA or L-NAME, induced p66^Shc phosphorylation, suggesting that basal NO production inhibits O₂ ^- production. oxLDL-induced, p66^Shc-mediated O₂- was prevented by eNOS inhibition, suggesting that when cells are stimulated with oxLDL eNOS is a source of reactive oxygen species. Endogenous or exogenous NO donors, prevented p66^Shc activation and reduced O_2- production. Treatment with tetrahydrobiopterin, an eNOS cofactor, restored eNOS uncoupling, prevented p66^Shc activation, and reduced O₂- generation. However, late treatment with tetrahydropterin did not yield the same result suggesting that eNOS uncoupling is the primary source of reactive oxygen species.

Conclusions

The present study reports that in primary cultured HAEC treated with oxLDL, p66^Shc-mediated oxidative stress is derived from eNOS uncoupling. This finding contributes novel information on the mechanisms of p66^Shc activation and its dual interaction with eNOS underscoring the importance eNOS uncoupling as a putative antioxidant therapeutical target in endothelial dysfunction as observed in cardiovascular disease.     

Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin,a Major Red Wine Polyphenol

Background

Red wine polyphenols can prevent cardiovascular and inflammatory diseases. Resveratrol, the most extensively studied constituent, is unlikely to solely account for these beneficial effects because of its rather low abundance and bioavailability. Malvidin is far the most abundant polyphenol in red wine; however, very limited data are available about its effect on inflammatory processes and kinase signaling pathways.

Methods & Findings

The present study was carried out by using RAW 264.7 macrophages stimulated by bacterial lipopolysaccharide in the presence and absence of malvidin. From the cells, activation of nuclear factor-kappaB, mitogen-activated protein kinase, protein kinase B/Akt and poly ADP-ribose polymerase, reactive oxygen species production, mitogen-activated protein kinase phosphatase-1 expression and mitochondrial depolarization were determined. We found that malvidin attenuated lipopolysaccharide-induced nuclear factor-kappaB, poly ADP-ribose polymerase and mitogen-activated protein kinase activation, reactive oxygen species production and mitochondrial depolarization, while upregulated the compensatory processes; mitogen-activated protein kinase phosphatase-1 expression and Akt activation.

Conclusions

These effects of malvidin may explain the previous findings and at least partially account for the positive effects of moderate red wine consumption on inflammation-mediated chronic maladies such as obesity, diabetes, hypertension and cardiovascular disease.