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Free Radical Biology and Medicine (FRBM)

NEIL1 stimulates neurogenesis and suppresses neuroinflammation after stress

Publication date: September 2019 Source: Free Radical Biology and Medicine, Volume 141 Author(s): Beimeng Yang, David M. Figueroa, Yujun Hou, Mansi Babbar, Stephanie L. Baringer, Deborah L. Croteau, Vilhelm A. Bohr AbstractCellular exposure to ionizing radiation leads to oxidatively generated DNA damage, which has been implicated in neurodegenerative diseases. DNA damage is repaired by the evolutionarily conserved base excision repair (BER) system. Exposure of mice to ionizing radiation affects neurogenesis and neuroinflammation. However, the consequences of deficient DNA repair on adult neurogenesis and neuroinflammation are poorly understood despite their potential relevance for homeostasis. We previously reported that loss of NEIL1, an important DNA glycosylase involved in BER, is associated with deficiencies in spatial memory, olfaction, and protection against ischemic stroke in mice. Here, we show that Neil1−/− mice display an anxiety-mediated behavior in the open field test, a deficient recognitive memory in novel object recognition and increased neuroinflammatory response under basal conditions. Further, mice lacking NEIL1 have decreased neurogenesis and deficient resolution of neuroinflammation following gamma irradiation (IR)-induced stress compared to WT mice. Neil1−/− IR-exposed mice also exhibit increased DNA damage and apoptosis in the hippocampus. Interestingly, behavioral tests two weeks after IR showed impaired stress response in the Neil1−/− mice. Our data indicate that NEIL1 plays an important role in adult neurogenesis and in the resolution of neuroinflammation. Graphical abstract

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The myeloperoxidase product, hypochlorous acid, reduces thrombus formation under flow and attenuates clot retraction and fibrinolysis in human blood

Publication date: September 2019 Source: Free Radical Biology and Medicine, Volume 141 Author(s): Tomasz Misztal, Agata Golaszewska, Maria Magdalena Tomasiak-Lozowska, Marta Iwanicka, Natalia Marcinczyk, Agnieszka Leszczynska, Ewa Chabielska, Tomasz Rusak AbstractHypochlorite (HOCl), a strong oxidant and antimicrobial agent, has been proposed to be associated with hemostatic abnormalities during inflammatory response. However, its complex impact on hemostasis is not completely understood. In this report we studied the effect of clinically relevant (micromolar) HOCl concentrations on thrombus formation under flow, kinetics of platelet-fibrin clot formation, its architecture, retraction, and lysis. We found that HOCl (up to 500 µM) did not affect kinetics of coagulation measured in whole blood. HOCl (500-1000 µM) markedly diminished thrombus formation under flow. Clot retraction rate was reduced by HOCl dose-dependently (50-500 µM). HOCl (125-500 µM) inhibited fibrinolysis in whole blood and in platelet-depleted plasma, dose-dependently. Activity of plasmin was reduced by HOCl at concentrations started from 500 µM. HOCl (up to 500 µM) did not reduce plasminogen binding to fibrin under flow. HOCl (125-500 µM) modulated architecture of fibrin- and platelet-fibrin clots towards structures made of thin and densely packed fibers. Exposure of pure fibrinogen to HOCl (10-1000 µM) resulted in formation of dityrosine and was associated with altered fibrin structure derived from such modified fibrinogen. HOCl-altered fibrin net structure was not related with modulation of platelet procoagulant response, thrombin generation, and factor XIII activity. We conclude that, in human blood, clinically relevant HOCl concentrations may inhibit thrombus formation under flow, clot retraction and fibrinolysis. Fibrinolysis and clot retraction seem to be the most sensitive to HOCl-evoked inhibition. HOCl-modified fibrinogen and altered clot structure associated with it are likely to be primary sources of attenuated fibrinolysis. Graphical

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Hydroxytyrosol prevents PM2.5-induced adiposity and insulin resistance by restraining oxidative stress related NF-κB pathway and modulation of gut microbiota in a murine model

Publication date: September 2019 Source: Free Radical Biology and Medicine, Volume 141 Author(s): Ningning Wang, Yanan Ma, Zhuoqun Liu, Lei Liu, Keming Yang, Yaguang Wei, Yang Liu, Xin Chen, Xiance Sun, Deliang Wen AbstractExposure to fine particular matter (≤2.5 μM, PM2.5) contributes to increased risk of obesity and type 2 diabetes. Hydroxytyrosol (HT), a simple polyphenol found in virgin olive oil, is considered to be beneficial for cardiovascular and metabolic disorders. The current study determined whether HT could improve PM2.5-induced adiposity and insulin resistance (IR), and explored the underlying mechanisms. Fifteen adult female C57BL/6j mice on a chow diet were randomly divided into three groups receiving (1) sterile PBS, (2) PM2.5 suspended in sterile PBS (1 mg/mL) and (3) PM2.5+HT (50 mg/kg/day). PM2.5/PBS exposure was administered by oropharynx instillation every other day and HT supplementation was achieved by gavage every day. Four-week PM2.5 exposure did not affect body weight, but significantly increased visceral fat mass. The abdominal adiposity coincided with adipocyte hypertrophy and proliferation in visceral white adipose tissue (WAT), as well as decreased metabolic activity in brown adipose tissue and subcutaneous WAT. PM2.5 enhanced the oxidative stress by diminishing antioxidant enzyme activities in liver and serum, whereas contents of 4-hydroxynonenal (4-HNE), malondialdehyde (MDA) levels in liver and serum were elevated. These changes were accompanied by macrophage infiltration and activation of NF-κB pathway in the liver. Moreover, PM2.5 exposure led to glucose intolerance and insulin insensitivity, impaired hepatic glycogenesis, and decreased insulin-stimulated Akt phosphorylation in peripheral tissues. Importantly, HT treatment prevented PM2.5-induced visceral adipogenesis, oxidative stress, hepatic inflammation and NF-κB activation, systemic and peripheral IR. In vitro, after HepG2 cells were incubated with PM2.5 (0, 5, 25, 50, 100 and 200 μg/mL), reduced glutathione depletion and

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miR-15b induces premature ovarian failure in mice via inhibition of α-Klotho expression in ovarian granulosa cells

Publication date: September 2019 Source: Free Radical Biology and Medicine, Volume 141 Author(s): Te Liu, Yan Liu, Yongyi Huang, Jiulin Chen, Zhihua Yu, Chuan Chen, Lingyun Lai AbstractA thorough understanding of epigenetics regulatory mechanisms of premature ovarian failure (POF) is still lacking. Here, we found that cyclophosphamide induced significantly decrease in α-Klotho (Kl) expression in mouse ovarian granulosa cells (mOGCs), suggesting that cyclophosphamide inhibited Kl expression. Cyclophosphamide also significantly accelerated ageing and led to a decline in the pregnancy rate of C. elegans. We subsequently noted that the pathological condition exhibited by Kl−/- mice was similar to that observed in cyclophosphamide-induced POF mice. Furthermore, the mOGCs in both types of mice showed significant signs of oxidative stress damage, including decreased SOD and ATP, increased ROS levels. Detailed analyses revealed that the decreased Kl expression led to the reduced expression of autophagy-related proteins in mOGCs, which resulted in decreased autophagy activity. Finally, we found that cyclophosphamide attenuated the autophagy function of mOGCs via upregulating microRNA-15b expression, which silenced the endogenous Kl mRNA expression and stimulated the activity of the downstream TGFβ1/Smad pathway. Therefore, we demonstrated that Kl was one of the key inhibitory factors in the development of POF. It elucidated the underlying epigenetic regulatory mechanism, whereby cyclophosphamide-dependent microRNA-15b inhibited Kl expression, leading to the reduced ability of mOGCs to induce autophagy and ROS scavenging, ultimately causing POF. Graphical abstractA thorough understanding of epigenetics regulatory mechanisms of premature ovarian failure (POF) is still lacking. Here, we found that cyclophosphamide induced significantly decrease in α-Klotho (Kl) expression in mouse ovarian granulosa cells (mOGCs), suggesting that cyclophosphamide inhibited Kl expression. Cyclophosphamide also significantly accelerated ageing

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Plin5 deficiency exacerbates pressure overload-induced cardiac hypertrophy and heart failure by enhancing myocardial fatty acid oxidation and oxidative stress

Publication date: September 2019 Source: Free Radical Biology and Medicine, Volume 141 Author(s): Chao Wang, Yuan Yuan, Jie Wu, Yuanlin Zhao, Xing Gao, Yihua Chen, Chao Sun, Liming Xiao, Pengfei Zheng, Peizhen Hu, Zengshan Li, Zhe Wang, Jing Ye, Lijun Zhang AbstractWhile cardiac hypertrophy and heart failure are accompanied by significant alterations in energy metabolism, more than 50–70% of energy is obtained from fatty acid β-oxidation (FAO) in adult hearts under physiological conditions. Plin5 is involved in the metabolism of lipid droplets (LDs) and is highly abundant in oxidative tissues including heart, liver and skeletal muscle. Plin5 protects the storage of triglyceride (TG) in LDs by inhibiting lipolysis, thereby suppressing excess FAO and preventing excessive oxidative stress in the heart. In this study, we investigated the roles of Plin5 in cardiac hypertrophy and heart failure in mice treated with transverse aortic constriction (TAC). The results indicated that Plin5 deficiency aggravated myocardial hypertrophy in the TAC-treated mice and exacerbated the TAC-induced heart failure. We also found that Plin5 deficiency reduced the cardiac lipid accumulation and upregulated the levels of PPARα and PGC-1α, which stimulate mitochondrial proliferation. Moreover, Plin5 deficiency aggravated the TAC-induced oxidative stress. We consistently found that Plin5 knockdown disrupted TG storage and elevated FAO and lipolysis in H9C2 rat cardiomyocytes. In addition, Plin5 knockdown also provoked mitochondrial proliferation and lipotoxic injury in H9C2 cells. In conclusion, Plin5 deficiency increases myocardial lipolysis, elevates FAO and oxidative burden, and thereby exacerbates cardiac hypertrophy and heart failure in TAC-treated mice. Graphical abstract

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Redox Biology

Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli

Publication date: September 2019 Source: Redox Biology, Volume 26 Author(s): Hanne R. Gerding, Christiaan Karreman, Andreas Daiber, Johannes Delp, Daniel Hammler, Martin Mex, Stefan Schildknecht, Marcel Leist AbstractTyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t1/2 = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein. Graphical abstract

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ReactELISA method for quantifying methylglyoxal levels in plasma and cell cultures

Publication date: September 2019 Source: Redox Biology, Volume 26 Author(s): Rasmus Kold-Christensen, Karina Kragh Jensen, Emil Smedegård-Holmquist, Lambert Kristiansen Sørensen, Jakob Hansen, Karl Anker Jørgensen, Peter Kristensen, Mogens Johannsen AbstractMethylglyoxal (MG) is a toxic glycolytic by-product associated with increased levels of inflammation and oxidative stress and has been linked to ageing-related diseases, such as diabetes and Alzheimer's disease. As MG is a highly reactive dicarbonyl compound, forming both reversible and irreversible adducts with a range of endogenous nucleophiles, measuring endogenous levels of MG are quite troublesome. Furthermore, as MG is a small metabolite it is not very immunogenic, excluding conventional ELISA for detection purposes, thus only more instrumentally demanding LC-MS/MS-based methods have demonstrated convincing quantitative data. In the present work we develop a novel bifunctional MG capture probe as well as a high specificity monoclonal antibody to finally setup a robust reaction-based ELISA (ReactELISA) method for detecting the highly reactive and low-level (nM) metabolite MG in human biological specimens. The assay is tested and validated against the current golden standard LC-MS/MS method in human blood plasma and cell-culture media. Furthermore, we demonstrate the assays ability to measure small perturbations of MG levels in growth media caused by a small molecule drug buthionine sulfoximine (BSO) of current clinical relevance. Finally, the assay is converted into a homogenous (no-wash) AlphaLISA version (ReactAlphaLISA), which offers the potential for operationally simple screening of further small molecules capable of perturbing cellular MG. Such compounds could be of relevance as probes to gain insight into MG metabolism as well as drug-leads to alleviate ageing-related diseases. Graphical abstract

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Anthocyanins protect the gastrointestinal tract from high fat diet-induced alterations in redox signaling, barrier integrity and dysbiosis

Publication date: September 2019 Source: Redox Biology, Volume 26 Author(s): Eleonora Cremonini, Elena Daveri, Angela Mastaloudis, Ana M. Adamo, David Mills, Karen Kalanetra, Shelly N. Hester, Steve M. Wood, Cesar G. Fraga, Patricia I. Oteiza AbstractThe gastrointestinal (GI) tract can play a critical role in the development of pathologies associated with overeating, overweight and obesity. We previously observed that supplementation with anthocyanins (AC) (particularly glycosides of cyanidin and delphinidin) mitigated high fat diet (HFD)-induced development of obesity, dyslipidemia, insulin resistance and steatosis in C57BL/6J mice. This paper investigated whether these beneficial effects could be related to AC capacity to sustain intestinal monolayer integrity, prevent endotoxemia, and HFD-associated dysbiosis. The involvement of redox-related mechanisms were further investigated in Caco-2 cell monolayers. Consumption of a HFD for 14 weeks caused intestinal permeabilization and endotoxemia, which were associated with a decreased ileum expression of tight junction (TJ) proteins (occludin, ZO-1 and claudin-1), increased expression of NADPH oxidase (NOX1 and NOX4) and NOS2 and oxidative stress, and activation of redox sensitive signals (NF-κB and ERK1/2) that regulate TJ dynamics. AC supplementation mitigated all these events and increased GLP-2 levels, the intestinal hormone that upregulates TJ protein expression. AC also prevented, in vitro, tumor necrosis factor alpha-induced Caco-2 monolayer permeabilization, NOX1/4 upregulation, oxidative stress, and NF-κB and ERK activation. HFD-induced obesity in mice caused dysbiosis and affected the levels and secretion of MUC2, a mucin that participates in intestinal cell barrier protection and immune response. AC supplementation restored microbiota composition and MUC2 levels and distribution in HFD-fed mice. Thus, AC, particularly delphinidin and cyanidin, can preserve GI physiology in HFD-induced obesity in part through redox-regulated mechanisms. This can in part exp

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The dynamic uptake and release of SOD3 from intracellular stores in macrophages modulates the inflammatory response

Publication date: September 2019 Source: Redox Biology, Volume 26 Author(s): Lili Hu, Elias D. Zachariae, Ulrike G. Larsen, Frederik Vilhardt, Steen V. Petersen AbstractSuperoxide dismutase 3 (SOD3) is an extracellular enzyme with the capacity to modulate extracellular redox conditions by catalyzing the dismutation of superoxide to hydrogen peroxide. In addition to synthesis and release of this extracellular protein via the secretory pathway, several studies have shown that the protein also localizes to intracellular compartments in neutrophils and macrophages. Here we show that human macrophages release SOD3 from an intracellular compartment within 30 min following LPS stimulation. This release acutely increases the level of SOD3 on the cell surface as well as in the extracellular environment. Generation of the intracellular compartment in macrophages is supported by endocytosis of extracellular SOD3 via the LDL receptor-related protein 1 (LRP1). Using bone marrow-derived macrophages established from wild-type and SOD3−/− mice, we further show that the pro-inflammatory profile established in LPS-stimulated cells is altered in the absence of SOD3, suggesting that the active release of this protein affects the inflammatory response. The internalization and acute release from stimulated macrophages indicates that SOD3 not only functions as a passive antioxidant in the extracellular environment, but also plays an active role in modulating redox signaling to support biological responses. Graphical abstract

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A novel NOX2 inhibitor attenuates human neutrophil oxidative stress and ameliorates inflammatory arthritis in mice

Publication date: September 2019 Source: Redox Biology, Volume 26 Author(s): Fu-Chao Liu, Huang-Ping Yu, Po-Jen Chen, Hsuan-Wu Yang, Shih-Hsin Chang, Cherng-Chyi Tzeng, Wei-Jen Cheng, You-Ren Chen, Yeh-Long Chen, Tsong-Long Hwang AbstractNeutrophil infiltration plays a significant pathological role in inflammatory diseases. NADPH oxidase type 2 (NOX2) is a respiratory burst oxidase that generates large amounts of superoxide anion (O2•−) and subsequent other reactive oxygen species (ROS). NOX2 is an emerging therapeutic target for treating neutrophilic inflammatory diseases. Herein, we show that 4-[(4-(dimethylamino)butoxy)imino]-1-methyl-1H-benzo[f]indol-9(4H)-one (CYR5099) acts as a NOX2 inhibitor and exerts a protective effect against complete Freund's adjuvant (CFA)-induced inflammatory arthritis in mice. CYR5099 restricted the production of O2•− and ROS, but not the elastase release, in human neutrophils activated with various stimulators. The upstream signaling pathways of NOX2 were not inhibited by CYR5099. Significantly, CYR5099 inhibited NOX2 activity in activated human neutrophils and in reconstituted subcellular assays. In addition, CYR5099 reduced ROS production, neutrophil infiltration, and edema in CFA-induced arthritis in mice. Our findings suggest that CYR5099 is a NOX2 inhibitor and has therapeutic potential for treating neutrophil-dominant oxidative inflammatory disorders. Graphical abstract

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