TRAF3IP2 mediates TWEAK/TWEAKR-induced pro-fibrotic responses in cultured cardiac fibroblasts and the heart

Persistent inflammation promotes development and progression of heart failure (HF). TWEAK (TNF-Related WEAK Inducer Of Apoptosis), a NF-κB- and/or AP-1-responsive proinflammatory cytokine that signals via TWEAK receptor (TWEAKR), is expressed at high levels in human and preclinical models of HF. Since the adapter molecule TRAF3IP2 (TRAF3 Interacting Protein 2) is an upstream regulator of various proinflammatory pathways, including those activated by NF-κB and AP-1, we hypothesized that targeting TRAF3IP2 inhibits TWEAK-induced proinflammatory and pro-fibrotic responses in vitro and in vivo. Consistent with the hypothesis, forced expression of TRAF3IP2 upregulated TWEAK and its receptor expression in cultured adult mouse cardiac fibroblasts (CF). Further, exogenous TWEAK upregulated TRAF3IP2 expression in a time- and dose-dependent manner, suggesting a positive-feedback regulation of TRAF3IP2 and TWEAK. TWEAK also promoted TRAF3IP2 nuclear translocation. Confirming its critical role in TWEAK signaling, silencing TRAF3IP2 inhibited TWEAK autoregulation, TWEAKR upregulation, p38 MAPK, NF-κB and AP-1 activation, inflammatory cytokine expression, MMP and TIMP1 activation, collagen expression and secretion, and importantly, proliferation and migration. Recapitulating these in vitro results, continuous infusion of TWEAK for 7 days increased systolic blood pressure (SBP), upregulated TRAF3IP2 expression, activated p38 MAPK, NF-κB and AP-1, induced the expression of multiple proinflammatory and pro-fibrotic mediators, and interstitial fibrosis in hearts of wild type mice. These proinflammatory and pro-fibrotic changes occurred in conjunction with myocardial hypertrophy and contractile dysfunction. Importantly, genetic ablation of TRAF3IP2 inhibited these TWEAK-induced adverse cardiac changes independent of increases in SBP, indicating that TRAF3IP2 plays a causal role, and thus a therapeutic target, in chronic inflammatory and fibro-proliferative diseases

RECK suppresses interleukin-17/TRAF3IP2-mediated MMP-13 activation and human aortic smooth muscle cell migration and proliferation

Sustained inflammation and matrix metalloproteinase (MMP) activation contribute to vascular occlusive/proliferative disorders. Interleukin-17 (IL-17) is a proinflammatory cytokine that signals mainly via TRAF3 Interacting Protein 2 (TRAF3IP2), an upstream regulator of various critical transcription factors, including AP-1 and NF-κB. Reversion inducing cysteine rich protein with kazal motifs (RECK) is a membrane-anchored MMP inhibitor. Here we investigated whether IL-17A/TRAF3IP2 signaling promotes MMP-13-dependent human aortic smooth muscle cell (SMC) proliferation and migration, and determined whether RECK overexpression blunts these responses. Indeed, IL-17A treatment induced (a) JNK, p38 MAPK, AP-1, NF-κB, and CREB activation, (b) miR-21 induction, (c) miR-27b and miR-320 inhibition, (d) MMP-13 expression and activation, (e) RECK suppression, and (f) SMC migration and proliferation, all in a TRAF3IP2-dependent manner. In fact, gain of TRAG3IP2 function, by itself, induced MMP-13 expression and activation, and RECK suppression. Furthermore, treatment with recombinant MMP-13 stimulated SMC migration in part via ERK activation. Importantly, RECK gain-of-function attenuated MMP-13 activity without affecting its mRNA or protein levels, and inhibited IL-17A- and MMP-13-induced SMC migration. These results indicate that increased MMP-13 and decreased RECK contribute to IL-17A-induced TRAF3IP2-dependent SMC migration and proliferation, and suggest that TRAF3IP2 inhibitors or RECK inducers have the potential to block the progression of neointimal thickening in hyperplastic vascular diseases

Three-dimensional nucleation with diffusion controlled growth: a comparative study of electrochemical phase formation from aqueous and deep eutectic solvents

The nucleation of Ag onto vitreous carbon from aqueous 3 M NaCl or 0.6 M NaClO4 and deep eutectic solvent (DES) 1:2 M mixture of choline chloride:urea solutions containing Ag+, has been studied analyzing the chronoamperometric response to single potential steps. From the coordinates of the maxima observed in the current responses, the nucleation frequencies A (s(-1)) and number densities of nucleation sites N-0 (cm(-2)) were obtained from the standard model of nucleation with diffusion-controlled three-dimensional growth. Analysis of the overpotential dependence of nucleation frequencies using the classical electrochemical nucleation theory allowed to calculate the Gibbs free energy of nucleation Delta(G) over tilde (n(c)) and critical nucleus size n(c) as well as the exchange current density j(o), transfer coefficient alpha and surface tension sigma of silver nuclei. The kinetics of Ag+ reduction is two orders of magnitude slower in DES compared to both aqueous systems studied, and values of alpha << 0.5 where found in both aqueous and DES media, indicating either that the intermediate state for metal ion reduction is located close to the initial state, i.e., the solvated or complexed metal ion in solution, or that the metal ion is specifically adsorbed on the surface and the symmetry factor involved requires an alternative electron transfer formalism. The low Delta(G) over tilde (n(c)) and n(c) values observed indicate that the discharge of a single Ag ion on the surface already becomes a supercritical nucleus, involving a very low Gibbs energy barrier, characteristic of a non-activated process. (C) 2016 Elsevier B.V. All rights reserved

Pseudo-single crystal electrochemistry on polycrystalline electrodes : visualizing activity at grains and grain boundaries on platinum for the Fe2+/Fe3+ redox reaction

The influence of electrode surface structure on electrochemical reaction rates and mechanisms is a major theme in electrochemical research, especially as electrodes with inherent structural heterogeneities are used ubiquitously. Yet, probing local electrochemistry and surface structure at complex surfaces is challenging. In this paper, high spatial resolution scanning electrochemical cell microscopy (SECCM) complemented with electron backscatter diffraction (EBSD) is demonstrated as a means of performing ‘pseudo-single-crystal’ electrochemical measurements at individual grains of a polycrystalline platinum electrode, while also allowing grain boundaries to be probed. Using the Fe2+/3+ couple as an illustrative case, a strong correlation is found between local surface structure and electrochemical activity. Variations in electrochemical activity for individual high index grains, visualized in a weakly adsorbing perchlorate medium, show that there is higher activity on grains with a significant (101) orientation contribution, compared to those with (001) and (111) contribution, consistent with findings on single-crystal electrodes. Interestingly, for Fe2+ oxidation in a sulfate medium a different pattern of activity emerges. Here, SECCM reveals only minor variations in activity between individual grains, again consistent with single-crystal studies, with a greatly enhanced activity at grain boundaries. This suggests that these sites may contribute significantly to the overall electrochemical behavior measured on the macroscale

Insulin regulates neurovascular coupling through astrocytes

Mice with insulin receptor (IR)-deficient astrocytes (GFAP-IR knockout [KO] mice) show blunted responses to insulin and reduced brain glucose uptake, whereas IRdeficient astrocytes show disturbed mitochondrial responses to glucose. While exploring the functional impact of disturbed mitochondrial function in astrocytes, we observed that GFAP-IR KO mice show uncoupling of brain blood flow with glucose uptake. Since IR-deficient astrocytes show higher levels of reactive oxidant species (ROS), this leads to stimulation of hypoxia-inducible factor-1¿ and, consequently, of the vascular endothelial growth factor angiogenic pathway. Indeed, GFAP-IR KO mice show disturbed brain vascularity and blood flow that is normalized by treatment with the antioxidant N-acetylcysteine (NAC). NAC ameliorated high ROS levels, normalized angiogenic signaling and mitochondrial function in IR-deficient astrocytes, and normalized neurovascular coupling in GFAP-IR KO mice. Our results indicate that by modulating glucose uptake and angiogenesis, insulin receptors in astrocytes participate in neurovascular coupling.We are thankful to M.Garcia and R. Cañadas for technical support. This work was funded by Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) (Instituto de Salud CarlosIII, Spain) to I.T.A., A.G., and T.I.; an Inter-CIBER project (PIE14/00061) to I.T.A.that forms part of the projects PID2019-104376RB-I00 (I.T.A.) and RTI2018-094887-B-I00 (M.N.) funded by MCIN/AEI/10.13039/501100011033; a grant from Junta de Andalucia Consejería de Economía y Conocimiento (P18-RT-2233 to A.G.) cofinanced by Programa Operativo FEDER 2014–2020; a grant from Instituto de Salud Carlos III Spain (cofinanced by FEDER funds from the European Union; PI21/00915 to A.G.); Grant PID2020-115218RB-I00 to T.I. funded by Ministerio de Ciencia e Innovación/Agencia Española de Investigación (MCIN/AEI/10.13039/501100011033); and a grant from Comunidad de Madrid through the European Social Fund (ESF)–financed programme Neurometabolismo-Comunidad de Madrid (NEUROMETAB-CM) (B2017/BMD-3700 to I.T.A.and T.I.). M.N. was also supported by the Spanish Ministry of Science and Innovation (Ramón y Cajal RYC-2016-20414). J.P.-U. was contracted by CIBERNED

Brain Effective Connectivity During Motor-Imagery and Execution Following Stroke and Rehabilitation

Brain areas within the motor system interact directly or indirectly during motor-imagery and motor-execution tasks. These interactions and their functionality can change following stroke and recovery. How brain network interactions reorganize and recover their functionality during recovery and treatment following stroke are not well understood. To contribute to answering these questions, we recorded blood oxygenation-level dependent (BOLD) functional magnetic resonance imaging (fMRI) signals from10 stroke survivors and evaluated dynamical causal modeling (DCM)-based effective connectivity among three motor areas: primary motor cortex (M1), premotor cortex (PMC) and supplementary motor area (SMA), during motor-imagery and motor-execution tasks. We compared the connectivity between affected and unaffected hemispheres before and after mental practice and combined mental practice and physical therapy as treatments. The treatment (intervention) period varied in length between 14 to 51 days but all patients received the same dose of 60 h of treatment. Using Bayesian model selection (BMS) approach in the DCMapproach, wefound that, after intervention, the same network dominated during motor-imagery and motor-execution tasks butmodulatory parameters suggested a suppressive influence of SM A on M1 during the motor-imagery task whereas the influence of SM A on M1 was unrestricted during themotor-execution task.We found that the intervention caused a reorganization of the network during both tasks for unaffected as well as for the affected hemisphere. Using Bayesian model averaging (BMA) approach, we found that the intervention improved the regional connectivity among the motor areas during both the tasks. The connectivity between PMCandM1was stronger inmotor-imagery taskswhereas the connectivity from PMC to M1, SM A to M1 dominated in motor-execution tasks. There was significant behavioral improvement (p = 0.001) in sensation and motor movements because of the intervention as reflected by behavioral Fugl-Meyer (FMA)measures,whichwere significantly correlated (p=0.05)with a subset of connectivity. These findings suggest that PMC andM1 play a crucial role duringmotor-imagery aswell as during motorexecution task. In addition,M1 causesmore exchange of causal information amongmotor areas during a motorexecution task than during a motor-imagery task due to its interaction with SM A. This study expands our understanding of motor network involved during two different tasks, which are commonly used during rehabilitation following stroke. A clear understanding of the effective connectivity networks leads to a better treatment in helping stroke survivors regain motor ability

Direct In Vivo Evidence for Tumor Propagation by Glioblastoma Cancer Stem Cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC

Transparent nanocrystalline yttria-stabilized-zirconia calvarium prosthesis

UnlabelledLaser-based diagnostics and therapeutics show promise for many neurological disorders. However, the poor transparency of cranial bone (calvaria) limits the spatial resolution and interaction depth that can be achieved, thus constraining opportunity in this regard. Herein, we report preliminary results from efforts seeking to address this limitation through use of novel transparent cranial implants made from nanocrystalline yttria-stabilized zirconia (nc-YSZ). Using optical coherence tomography (OCT) imaging of underlying brain in an acute murine model, we show that signal strength is improved when imaging through nc-YSZ implants relative to native cranium. As such, this provides initial evidence supporting the feasibility of nc-YSZ as a transparent cranial implant material. Furthermore, it represents a crucial first step towards realization of an innovative new concept we are developing, which seeks to eventually provide a clinically-viable means for optically accessing the brain, on-demand, over large areas, and on a chronically-recurring basis, without need for repeated craniectomies.From the clinical editorIn this study, transparent nanocrystalline yttria-stabilized-zirconia is used as an experimental "cranium prosthesis" material, enabling the replacement of segments of cranial bone with a material that allows for optical access to the brain on a recurrent basis using optical imaging methods such as OCT

Determination of Specific Electrocatalytic Sites in the Oxidation of Small Molecules on Crystalline Metal Surfaces

The identification of active sites in electrocatalytic reactions is part of the elucidation of mechanisms of catalyzed reactions on solid surfaces. However, this is not an easy task, even for apparently simple reactions, as we sometimes think the oxidation of adsorbed CO is. For surfaces consisting of non-equivalent sites, the recognition of specific active sites must consider the influence that facets, as is the steps/defect on the surface of the catalyst, cause in its neighbors; one has to consider the electrochemical environment under which the “active sites” lie on the surface, meaning that defects/steps on the surface do not partake in chemistry by themselves. In this paper, we outline the recent efforts in understanding the close relationships between site-specific and the overall rate and/or selectivity of electrocatalytic reactions. We analyze hydrogen adsorption/desorption, and electro-oxidation of CO, methanol, and ammonia. The classical topic of asymmetric electrocatalysis on kinked surfaces is also addressed for glucose electro-oxidation. The article takes into account selected existing data combined with our original works.M.J.S.F. is grateful to PNPD/CAPES (Brazil). J.M.F. thanks the MCINN (FEDER, Spain) project-CTQ-2016-76221-P