Virtual issue by COST Action BM1203 (EU-ROS) “Emerging concepts in redox biology and oxidative stress”

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Protective role for miR-9-5p in the fibrogenic transformation of human dermal fibroblasts

[Background] Excessive accumulation of extracellular matrix (ECM) proteins is the hallmark of fibrotic diseases, including skin fibrosis. This response relies on the activation of dermal fibroblasts that evolve into a pro-fibrogenic phenotype. One of the major players in this process is the cytokine transforming growth factor-β (TGF-β). MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression affecting a wide range of pathophysiological events including fibrogenesis. MicroRNA-9-5p (miR-9-5p) has been shown to exert a protective role in lung and peritoneal fibrosis. This study aimed to evaluate the role of miR-9-5p in skin fibrosis.[Results] miR-9-5p is up-regulated in TGF-β1-treated human dermal fibroblasts (HDFs). In silico identification of miR-9-5p targets spotted the type II TGF-β receptor (TGFBR2) as a potential TGF-β signaling-related effector for this miRNA. Consistently, over-expression of miR-9-5p in HDFs down-regulated TGFBR2 at both the mRNA and protein levels and reduced the phosphorylation of Smad2 and the translocation of Smad2/3 to the nucleus. In keeping, over-expression of miR-9-5p significantly delayed TGF-β1-dependent transformation of dermal fibroblasts, decreasing the expression of ECM protein collagen, type I, alpha 1 (Col1α1), and fibronectin (FN), the amount of secreted collagen proteins, and the expression of the archetypal myofibroblast marker alpha-smooth muscle actin (α-SMA). By contrast, specific inhibition of miR-9-5p resulted in enhanced presence of fibrosis markers. The expression of miR-9-5p was also detected in the skin and plasma in the mouse model of bleomycin-induced dermal fibrosis. Using lentiviral constructs, we demonstrated that miR-9-5p over-expression was also capable of deterring fibrogenesis in this same model.[Conclusions] miR-9-5p significantly prevents fibrogenesis in skin fibrosis. This is mediated by an abrogation of TGF-β-mediated signaling through the down-regulation of TGFBR2 expression in HDFs. These results may pave the way for future diagnostic or therapeutic developments for skin fibrosis based on miR-9-5p.This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) SAF 2012-31338 and CSD 2007-00020 (SL), Instituto de Salud Carlos III REDinREN RD12/0021/0009, Comunidad de Madrid “Fibroteam” S2010/BMD-2321), and Fundación Renal “Iñigo Alvarez de Toledo,” all from Spain. This study was supported by the European Cooperation in Science and Research COST actions BM-1203 (EU-ROS) and BM-1005 (ENOGAS) (SL). The CBMSO receives institutional support from Fundación “Ramón Areces”. Verónica Miguel is currently supported by the MINECO program of Formación de Personal Investigador (FPI BES-2013-065986). Marta Fierro-Fernández was supported by a postdoctoral grant of the MINECO “Juan de la Cierva” Program. Oscar Busnadiego was a fellow of the FPI program.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

L-Plastin S-glutathionylation promotes reduced binding to β-actin and affects neutrophil functions

© 2015 Elsevier Inc. All rights reserved. Posttranslational modifications (PTMs) of cytoskeleton proteins due to oxidative stress associated with several pathological conditions often lead to alterations in cell function. The current study evaluates the effect of nitric oxide (DETA-NO)-induced oxidative stress-related S-glutathionylation of cytoskeleton proteins in human PMNs. By using in vitro and genetic approaches, we showed that S-glutathionylation of L-plastin (LPL) and β-actin promotes reduced chemotaxis, polarization, bactericidal activity, and phagocytosis. We identified Cys-206, Cys-283, and Cys-460 as S-thiolated residues in the β-actin-binding domain of LPL, where cys-460 had the maximum score. Site-directed mutagenesis of LPL Cys-460 further confirmed the role in the redox regulation of LPL. S-Thiolation diminished binding as well as the bundling activity of LPL. The presence of S-thiolated LPL was detected in neutrophils from both diabetic patients and db/db mice with impaired PMN functions. Thus, enhanced nitroxidative stress may results in LPL S-glutathionylation leading to impaired chemotaxis, polarization, and bactericidal activity of human PMNs, providing a mechanistic basis for their impaired functions in diabetes mellitus.Ministerio de Economía y Competitividad (MINECO),SAF 2012–388.Award of research fellowships to M.D.,A.K.S.,D.A.,and S.N. from the Council of Scientific and Industrial Research, India.Peer Reviewe

MiR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2

© 2015 The Authors. Uncontrolled extracellular matrix (ECM) production by fibroblasts in response to injury contributes to fibrotic diseases, including idiopathic pulmonary fibrosis (IPF). Reactive oxygen species (ROS) generation is involved in the pathogenesis of IPF. Transforming growth factor-β1 (TGF-β1) stimulates the production of NADPH oxidase 4 (NOX4)-dependent ROS, promoting lung fibrosis (LF). Dysregulation of microRNAs (miRNAs) has been shown to contribute to LF. To identify miRNAs involved in redox regulation relevant for IPF, we performed arrays in human lung fibroblasts exposed to ROS. miR-9-5p was selected as the best candidate and we demonstrate its inhibitory effect on TGF-β receptor type II (TGFBR2) and NOX4 expression. Increased expression of miR-9-5p abrogates TGF-β1-dependent myofibroblast phenotypic transformation. In the mouse model of bleomycin-induced LF, miR-9-5p dramatically reduces fibrogenesis and inhibition of miR-9-5p and prevents its anti-fibrotic effect both in vitro and in vivo. In lung specimens from patients with IPF, high levels of miR-9-5p are found. In omentum-derived mesothelial cells (MCs) from patients subjected to peritoneal dialysis (PD), miR-9-5p also inhibits mesothelial to myofibroblast transformation. We propose that TGF-β1 induces miR-9-5p expression as a self-limiting homeostatic response.Ministerio de Economía y Competitividad (MINECO) SAF 2012-31338 (SL), SAF 2013-47611 (MLC) and CSD 2007-00020 (SL), Instituto de Salud Carlos III REDinREN RD12/0021/0009 (SL and LGB) and FIS PS12/00094 (LGB), Comunidad de Madrid “Fibroteam” S2010/BMD-2321 (SL and MLC) and Fundación Renal “Iñigo Alvarez de Toledo” (SL), all from Spain. Supported by European Cooperation in Science and Research COST actions BM-1203 (EU-ROS) and BM-1005 (ENOGAS) (SL). The CBMSO receives institutional support from Fundación “Ramón Areces”.Peer Reviewe

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

Los pasos debidos en el proceso investigador

4 p.La metodología de algo tan amplio como el proceso investigador puede estudiarse desde diferentes perspectivas. Entre ellas se sitúan la filosofía de la ciencia, la psicología y la pedagogía. Como es lógico existe una información acumulada tan abundante sobre este tema en cualquiera de las áreas citadas que sobrepasa cualquier intento de resumirla en pocas páginas. Sin embargo, a la luz de las nuevas corrientes de pensamiento sobre el quehacer clínico diario-medicina basada en el hecho probadoparece necesario revisar el proceso que nos permite progresar en el conocimiento de un problema de una manera ordenada. Para ello este artículo pretende revisar brevemente 3 aspectos: a) Elementos necesarios para llevar a cabo una investigación en el campo de la medicina clínica o experimental. b) Características diferenciales de la investigación experimental y clínica. c) Requisitos y limitaciones a la hora de establecer una relación causa-efecto.Peer reviewe

La ciclosporina A produce anión superóxido intracelular en el endotelio

5 p.-4 fig.Muchas enfermedades están asociadas con una sobreproducción de radicales libres del oxígeno generadores de daño celular. Entre estos radicales, el anión superóxido ha recibido especial atención por su participación en patologías con alta prevalencia, como por ejemplo, el daño por reperfusión (tras infarto miocárdico o accidente cerebrovascular) o los procesos inflamatorios como la artritis. La importancia del anión superóxido en términos de utilidad terapéutica queda reflejado en el esfuerzo, culminado ya al menos en animales1, por encontrar agentes mimetizadores de la actividad superóxido dismutasa (eliminación de anión superóxido) con estabilidad química y biológica in vivo. Hasta ahora se ha considerado que los efectos tóxicos de los fármacos inmunosupresores inhibidores de la calcineurina, como es la CsA, podían depender también de la inhibición de este enzima. Entre estos efectos tóxicos se encuentra el daño sobre el endotelio vascular, manifestado en ocasiones como hipertensión o como síndromes que recuerdan a la afectación vascular de la anemia hemolítica microangiopática (microangiopatía trombótica). Hasta la fecha no se ha demostrado relación entre la inhibición de la actividad fosfatasa de la calcineurina y estos efectos secundarios, por lo que se hace necesario estudiar otros mecanismos alternativos que permitan explicarlos.Javier Navarro Antolín es becario del Fondo de Investigación Sanitaria (FIS) (BEFI 98/9070). Los estudios comentados en este trabajo han sido financiados en parte por la Sociedad Española de Nefrología (AYUDA 2/98), el Plan Nacional de I+D (CICYT SAF 97-0035) y la Comunidad Autónoma de Madrid (CAM 08.4/0032/1998).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. © 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

Disfunción endotelial en la aterosclerosis: papel protector de las estatinas

11 p.-6 fig.[EN]Atherosclerosis is a chronic disease of the vascular wall characterized by macrophage accumulation, vascular smooth muscle cell proliferation and extracellular matrix intimal formation. A deficient function in the L-arginine-nitric oxide (NO)-cGMP pathway or an excess of endothelin-1 (ET-1) have been related with the endothelial dysfunction associated to arteriosclerosis. This derangement is one of the first steps in the atherogenetic process. Inhibitors of 3-hydroxi-3-methylglutaryl CoA reductase (statins) have been succesfully employed in the treatment of dyslipidemia and atherosclerosis. These drugs reduce cholesterol levels by inhibiting mevalonate synthesis, a precursor step in the sterol biosynthetic pathway. Recent clinical studies have reported that, aside from their cholesterol lowering action, statins may ameliorate endothelial function by additional mechanisms. Based on these observations, we have studied the potential effect of statins on the expression of the endothelial vasoactive factors, ET-1 and NO in bovine aortic endothelial cells (BAEC). Our results show that Atorvastatin and Simvastatin significantly reduce the synthesis of ET-1 and the expression of the pre-proET-1 mRNA. This effect is also evident in the presence of proatherogenic concentrations of oxidized low density lipoproteins (oxLDLs). Although no significant modification of endothelial NO synthase levels was observed in the presence of these drugs alone, they clearly reduced oxLDL-mediated inhibition in BAEC. Thus, statins may contribute to the regulation of vascular tone by modifying the expression of endothelial vasoactive factors.[ES]La aterosclerosis es un trastorno crónico de la pared del vaso caracterizado por la acumulación de macrófagos, la proliferación de células musculares lisas y la elaboración de matriz extracelular en la íntima. Se ha observado que una función deficiente de la vía L-arginina-óxido nítrico (NO)-GMPc o bien un exceso de endotelina-1 (ET-1) es responsable, al menos en parte, de la disfunción endotelial asociada a la arteriosclerosis. Esta disfunción del endotelio constituye una de las primeras etapas del desarrollo de la aterosclerosis. En los últimos años se han empleado con éxito los inhibidores del enzima 3-hidroxi-3-metilglutaril coenzima A (HMG-CoA) reductasa (estatinas) en el tratamiento de las dislipemias y la aterosclerosis. Estos fármacos reducen los niveles de colesterol a través de la inhibición de la síntesis de mevalonato, el precursor de los esteroles en la célula. Estudios clínicos recientes han sugerido la idea de que, además de su papel como agentes hipocolesterolemiantes, las estatinas pueden mejorar la función endotelial por mecanismos adicionales. Basándonos en estas observaciones, hemos estudiado el efecto potencial de las estatinas sobre la expresión de los factores vasoactivos ET-1 y NO en células endoteliales de aorta bovina (CEAB). Nuestros resultados demuestran que la Atorvastatina y la Simvastatina reducen significativamente la síntesis de ET-1 y la expresión de su precursor, la pre-proET-1. Asimismo, el efecto inhibidor de la expresión del RNAm de la pre-proET-1 persiste en la presencia de concentraciones proaterogénicas de lipoproteínas de baja densidad oxidadas (LDLox). Aunque estos fármacos por si mismos no modifican significativamente la expresión de la NO sintasa endotelial, son capaces de reducir claramente la inhibición de la expresión provocada por las LDLox en CEAB. Por lo tanto, las estatinas podrían modular el tono vascular a través de la modificación de la expresión de factores endoteliales vasoactivos.Este trabajo ha sido financiado por Parke-Davis España, y en parte por la CICYT (SAF 97-0035) y la Unión Europea (BIOMED 2, BMH1-CT92-1893).Peer reviewe

Efecto de la adenosina sobre la contracción glomerular y de las células mesangiales en cultivo

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina. Fecha de lectura: 4 de Julio de 198