Redox signaling responses to laminar shear stress in vascular endothelial cells

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-06-201

Protocol for a randomized controlled dismantling study of an internet-based intervention for depressive symptoms: exploring the contribution of behavioral activation and positive psychotherapy strategies

Background: there are evidence-based interventions for depression that include different components. However, the efficacy of their therapeutic components is unknown. Another important issue related to depression interventions is that, up to now, their therapeutic components have only focused on reducing negative symptoms rather than on improving positive affect and well-being. Because the low levels of positive affect are more strongly linked to depression than to other emotional disorders, it is important to include this variable as an important treatment target. Positive psychotherapeutic strategies (PPs) could help in this issue. The results obtained so far are consistent and promising, showing that Internet-based interventions are effective in treating depression. However, most of them are also multi-component, and it is important to make progress in investigating what each component contributes to the intervention. Methods: the current study will be a three-armed, simple-blinded, randomized controlled clinical trial with a dismantling design. 192 participants will be randomly assigned to: a) an Internet-based Global Protocol condition, which includes traditional therapeutic components of evidence-based treatments for depression (Motivation for change, Psychoeducation, Cognitive Therapy, Behavioral Activation (BA), Relapse Prevention) and PPs component, offering strategies to enhance positive mood and promote psychological strengths; b) an Internet-based BA Protocol condition (without the PPs component), and c) an Internet-based PPs Protocol condition (without the BA component). Primary outcome measures will be the BDI-II and PANAS. Secondary outcomes will include other variables such as depression, anxiety and stress, quality of life, resilience, and wellbeing related measures. Treatment acceptance and usability will also be measured. Participants will be assessed at pre-, post-treatment, 3-, 6- and 12- month follow- ups. The data will be analyzed based on the Intention-to-treat principle. Per protocol analyses will also be performed. Discussion: to the best of our knowledge, this is the first randomized dismantling intervention study for depression with the aim of exploring the contribution of a PPs component and the BA component in an Internet-based intervention. The three protocols are online interventions, helping to reach many people who need psychological treatments and otherwise would not have access to them

NOX4-dependent Hydrogen peroxide promotes shear stress-induced SHP2 sulfenylation and eNOS activation

© 2015 Elsevier Inc.All rights reserved. Laminar shear stress (LSS) triggers signals that ultimately result in atheroprotection and vasodilatation. Early responses are related to the activation of specific signaling cascades. We investigated the participation of redox-mediated modifications and in particular the role of hydrogen peroxide (H2O2) in the sulfenylation of redox-sensitive phosphatases. Exposure of vascular endothelial cells to short periods of LSS (12 dyn/cm2) resulted in the generation of superoxide radical anion as detected by the formation of 2-hydroxyethidium by HPLC and its subsequent conversion to H2O2, which was corroborated by the increase in the fluorescence of the specific peroxide sensor HyPer. By using biotinylated dimedone we detected increased total protein sulfenylation in the bovine proteome, which was dependent on NADPH oxidase 4 (NOX4)-mediated generation of peroxide. Mass spectrometry analysis allowed us to identify the phosphatase SHP2 as a protein susceptible to sulfenylation under LSS. Given the dependence of FAK activity on SHP2 function, we explored the role of FAK under LSS conditions. FAK activation and subsequent endothelial NO synthase (eNOS) phosphorylation were promoted by LSS and both processes were dependent on NOX4, as demonstrated in lung endothelial cells isolated from NOX4-null mice. These results support the idea that LSS elicits redox-sensitive signal transduction responses involving NOX4-dependent generation of hydrogen peroxide, SHP2 sulfenylation, and ulterior FAK-mediated eNOS activation.Ministerio deEconomía y Competitividad, SAF2012-31338(S.L.),CSD2007-00020(S. L.), SAF2010-37926(J.V.); Instituto de Salud CarlosIII, REDinREN RD12/0021/0009(S.L.), ProteoRed-PT13/0001/0017(J.V.), RETIC-RD12/0042/0056(J.V.); Deutsche Forschungsgemeinschaft (SFB815/TP1toK.S.andR.P.B.andSCHR1241/1-1toK.S.); German Center for Cardiovascular Research; ComunidaddeMadrid “Fi-broteam” S2010/BMD-2321(S.L.);and Fundación Renal “Iñigo Alvarez deToledo” (S.L.). This work was also supported by European Cooperationin Science and Technology actionsBM-1203(EU-ROS) and BM-1005(ENOGAS) (S.L.).A.M.S.is supported by the British Heart Foundation.TheCBMSO receives institutional support from Fundación Ramón ArecesPeer Reviewe

Hydrogen peroxide signaling in vascular endothelial cells

Redox signaling is implicated in different physiological and pathological events in the vasculature. Among the different reactive oxygen species, hydrogen peroxide (H2O2) is a very good candidate to perform functions as an intracellular messenger in the regulation of several biological events.In this review, we summarize the main physiological sources of H2O2 in the endothelium and the molecular mechanisms by which it is able to act as a signaling mediator in the vasculature. © 2014 The Authors.Ministerio de Economía y Competitividad SAF 2012-31338 (S.L.), CSD 2007-00020 (S.L.),Fundación Renal “Iñigo Alvarez de Toledo”Peer Reviewe

Hydrogen peroxide signaling in vascular endothelial cells

Redox signaling is implicated in different physiological and pathological events in the vasculature. Among the different reactive oxygen species, hydrogen peroxide (H2O2) is a very good candidate to perform functions as an intracellular messenger in the regulation of several biological events. In this review, we summarize the main physiological sources of H2O2 in the endothelium and the molecular mechanisms by which it is able to act as a signaling mediator in the vasculature

Role of PTEN in modulation of ADP-dependent signaling pathways in vascular endothelial cells

ADP plays critical signaling roles in the vascular endothelium. ADP receptors are targeted by several cardiovascular drugs, yet the intracellular pathways modulated by ADP are incompletely understood. These studies have identified important roles for the phosphatase PTEN in ADP-dependent modulation of the endothelial isoform of nitric oxide synthase (eNOS) as well as of lipid and protein kinase pathways in endothelial cells. We find that ADP-promoted eNOS activation as well as phosphorylation of p38 MAPK are enhanced by siRNA-mediated PTEN knockdown. However, the increase in ADP-dependent eNOS activation promoted by PTEN knockdown is abrogated by siRNA-mediated knockdown of p38 MAPK. These findings indicate that PTEN tonically suppresses both p38 phosphorylation as well as ADP-stimulated eNOS activity. A key enzymatic activity of PTEN is its role as a lipid phosphatase, catalyzing the dephosphorylation of phosphoinositol-3,4,5-trisphosphate (PIP3) to phosphoinositol-4,5-bisphosphate (PIP2). We performed biochemical analyses of cellular phospholipids in endothelial cells to show that siRNA-mediated PTEN knockdown leads to a marked increase in PIP3. Because these complex lipids activate the small GTPase Rac1, we explored the role of PTEN in ADP-modulated Rac1 activation. We used a FRET biosensor for Rac1 to show that ADP-dependent Rac1 activation is blocked by siRNA-mediated PTEN knockdown. We then exploited a FRET biosensor for PIP3 to show that the striking ADP-dependent increase in intracellular PIP3 is entirely blocked by PTEN knockdown. These studies identify a key role for PTEN in the modulation of lipid mediators involved in ADP receptor-regulated endothelial signaling pathways involving eNOS activation in vascular endothelial cells. © 2013 Elsevier B.V.National Institutes of Health (HL46457, HL48743, GM36259); Ministerio de Economía y Competitividad; Fundación Renal “Iñigo Álvarez deToledo”; Fundación “Ramón Areces”Peer Reviewe

A “fluorescence switch” technique increases the sensitivity of proteomic detection and identification of S‐nitrosylated proteins

Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The ‘‘biotin switch’’ technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a ‘‘fluorescence switch’’ technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This ‘‘fluorescence switch’’ approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.Gobierno de EspañaDepto. de Bioquímica y Biología MolecularTRUEpu

Diurnal and seasonal variation of BTEX in the air of Monterrey, Mexico: preliminary study of sources and photochemical ozone pollution

Atmospheric levels of benzene, toluene, ethylbenzene, and xylenes (BTEX) were measured in an urban site located in Nuevo Leon, Mexico, during summer and autumn 2013. A total of 60 samples were collected using carbon-packed cartridges at 0900, 1200, and 1500 h and then analyzed using gas chromatography with flame ionization detector. Meterological parameters and criteria air pollutants were measured and correlated with BTEX by a principal component analysis (PCA). The relative abundance of BTEX followed the order: benzene > toluene > ethylbenzene > p-xylene with mean concentrations of 55.24 μg m−3, 22.24 μg m−3, 6.94 μg m−3, and 4.17 μg m−3, respectively, during summer. Mean concentrations during autumn were 21.079 μg m−3 for benzene, 3.648 μg m−3 for toluene, 2.521 μg m−3 for ethylbenzene, and 2.115 μg m−3 for p-xylene. All measured BTEX showed clear diurnal and seasonal patterns. The highest mean levels for benzene were obtained during the midday. Toluene, ethylbenzene, and p-xylene showed the highest levels during afternoon period. BTEX levels were higher when wind blew from NE and ESE during summer and from ESE during autumn. The municipalities of Apodaca and Guadalupe are located in these directions where important industries, high traffic volume, many oil and gas service stations, and the biggest airport in this region are found. These sources could contribute to the BTEX concentrations measured during the sampling period

Critical role of hydrogen peroxide signaling in the sequential activation of p38 MAPK and eNOS in laminar shear stress

Laminar shear stress (LSS) is a protective hemodynamic regulator of endothelial function and limits the development of atherosclerosis and other vascular wall diseases related to pathophysiological generation of reactive oxygen species. LSS activates several endothelial signaling responses, including the activation of MAPKs and eNOS. Here, we explored the mechanisms of activation of these key endothelial signaling pathways. Using the cone/plate model we found that LSS (12 dyn/cm 2) rapidly promotes endothelial intracellular generation of superoxide and hydrogen peroxide (H 2O 2). Physiological concentrations of H 2O 2 (flux of 0.1 nM/min and 15 οM added extracellularly) significantly activated both eNOS and p38 MAPK. Pharmacological inhibition of NADPH oxidases (NOXs) and specific knockdown of NOX4 decreased LSS-induced p38 MAPK activation. Whereas the absence of eNOS did not alter LSS-induced p38 MAPK activation, pharmacological inhibition and knockdown of p38α MAPK blocked H 2O 2- and LSS-induced eNOS phosphorylation and reduced •NO levels. We propose a model in which LSS promotes the formation of signaling levels of H 2O 2, which in turn activate p38α MAPK and then stimulate eNOS, leading to increased •NO generation and protection of endothelial function. © 2012 Elsevier Inc. All rights reserved.Ministerio de Ciencia e Innovación; Ayuda CSIC–Universidad de la República (Uruguay) de Cooperación BilateralPeer Reviewe