bims-noxint 2021-03-07 papers

bims-noxint

Biomed News

on NADPH oxidases in tumorigenesis

Issue of 2021‒03‒07
six papers selected by
Laia Caja Puigsubira
Uppsala University

Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice.
Profibrotic epithelial TGF-β1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.
Human Placental NADPH Oxidase Mediates sFlt-1 and PlGF Secretion in Early Pregnancy: Exploration of the TGF-β1/p38 MAPK Pathways.
NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway.
ROS Live Cell Imaging During Neuronal Development.
DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk.

Theranostics. 2021 ;11(8): 3796-3812

Impairment of type H vessels by NOX2-mediated endothelial oxidative stress: critical mechanisms and therapeutic targets for bone fragility in streptozotocin-induced type 1 diabetic mice.

Xiao-Fan Hu, Geng Xiang, Tian-Ji Wang, Yu-Bo Ma, Yang Zhang, Ya-Bo Yan, Xiong Zhao, Zi-Xiang Wu, Ya-Fei Feng, Wei Lei.

  Rationale: Mechanisms underlying the compromised bone formation in type 1 diabetes mellitus (T1DM), which causes bone fragility and frequent fractures, remain poorly understood. Recent advances in organ-specific vascular endothelial cells (ECs) identify type H blood vessel injury in the bone, which actively direct osteogenesis, as a possible player. Methods: T1DM was induced in mice by streptozotocin (STZ) injection in two severity degrees. Bony endothelium, the coupling of angiogenesis and osteogenesis, and bone mass quality were evaluated. Insulin, antioxidants, and NADPH oxidase (NOX) inhibitors were administered to diabetic animals to investigate possible mechanisms and design therapeutic strategies. Results: T1DM in mice led to the holistic abnormality of the vascular system in the bone, especially type H vessels, resulting in the uncoupling of angiogenesis and osteogenesis and inhibition of bone formation. The severity of osteopathy was positively related to glycemic levels. These pathological changes were attenuated by early-started, but not late-started, insulin therapy. ECs in diabetic bones showed significantly higher levels of reactive oxygen species (ROS) and NOX 1 and 2. Impairments of bone vessels and bone mass were effectively ameliorated by treatment with anti-oxidants or NOX2 inhibitors, but not by a NOX1/4 inhibitor. GSK2795039 (GSK), a NOX2 inhibitor, significantly supplemented the insulin effect on the diabetic bone. Conclusions: Diabetic osteopathy could be a chronic microvascular complication of T1DM. The impairment of type H vessels by NOX2-mediated endothelial oxidative stress might be an important contributor that can serve as a therapeutic target for T1DM-induced osteopathy.

Keywords:  Diabetic bone fragility; Endothelial damage; NADPH oxidase 2.; Oxidative stress; Type H vessels

DOI:  https://doi.org/10.7150/thno.50907
Am J Physiol Lung Cell Mol Physiol. 2021 Mar 01. 320(3): L356-L367

Profibrotic epithelial TGF-β1 signaling involves NOX4-mitochondria cross talk and redox-mediated activation of the tyrosine kinase FYN.

Carmen Veith, Milena Hristova, Karamatullah Danyal, Aida Habibovic, Christopher M Dustin, John E McDonough, Bart M Vanaudenaerde, Michael Kreuter, Marc A Schneider, Nicolas Kahn, Frederik J van Schooten, Agnes W Boots, Albert van der Vliet.

  Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-β (TGF-β) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-β signaling is not known. Here, we demonstrate that TGF-β1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-β1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-β-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-β1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.

Keywords:  NOX4; SRC; idiopathic pulmonary fibrosis; mitochondria; redox signaling

DOI:  https://doi.org/10.1152/ajplung.00444.2019
Antioxidants (Basel). 2021 Feb 12. pii: 281. [Epub ahead of print]10(2):

Human Placental NADPH Oxidase Mediates sFlt-1 and PlGF Secretion in Early Pregnancy: Exploration of the TGF-β1/p38 MAPK Pathways.

Isabelle Hernandez, Audrey Chissey, Jean Guibourdenche, Roger Atasoy, Xavier Coumoul, Thierry Fournier, Jean-Louis Beaudeux, Amal Zerrad-Saadi.

  Preeclampsia, a hypertensive disorder occurring during pregnancy, is characterized by excessive oxidative stress and trophoblast dysfunction with dysregulation of soluble Fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) production. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (Nox) is the major source of placental superoxide in early pregnancy and its activation with the subsequent formation of superoxide has been demonstrated for various agents including Transforming Growth Factor beta-1 (TGF-β1), a well-known p38 MAPK pathway activator. However, the bridge between Nox and sFlt-1 remains unknown. The purpose of this study was to explore the possible signaling pathway of TGF-β1/Nox/p38 induced sFlt-1 production in human chorionic villi (CV).METHODS: Human chorionic villi from first trimester placenta (7-9 Gestational Weeks (GW)) were treated with TGF-β1 or preincubated with p38 inhibitor, SB203580. For NADPH oxidase inhibition, CV were treated with diphenyleneiodonium (DPI). The protein levels of phospho-p38, p38, phospho-Mothers Against Decapentaplegic homolog 2 (SMAD2), and SMAD2 were detected by Western blot. The secretion of sFlt-1 and PlGF by chorionic villi were measured with Electrochemiluminescence Immunologic Assays, and NADPH oxidase activity was monitored by lucigenin method.
RESULTS: We demonstrate for the first time that NADPH oxidase is involved in sFlt-1 and PlGF secretion in first trimester chorionic villi. Indeed, the inhibition of Nox by DPI decreases sFlt-1, and increases PlGF secretions. We also demonstrate the involvement of p38 MAPK in sFlt-1 secretion and Nox activation as blocking the p38 MAPK phosphorylation decreases both sFlt-1 secretion and superoxide production. Nevertheless, TGF-β1-mediated p38 activation do not seem to be involved in regulation of the first trimester placental angiogenic balance and no crosstalk was found between SMAD2 and p38 MAPK pathways.
CONCLUSIONS: Thus, the placental NADPH oxidase play a major role in mediating the signal transduction cascade of sFlt-1 production. Furthermore, we highlight for the first time the involvement of p38 activation in first trimester placental Nox activity.

Keywords:  NADPH oxidase; SMAD2; first trimester; p38 MAPK; preeclampsia; pregnancy; sFlt-1

DOI:  https://doi.org/10.3390/antiox10020281
Front Cell Dev Biol. 2021 ;9 612554

NADPH and Mito-Apocynin Treatment Protects Against KA-Induced Excitotoxic Injury Through Autophagy Pathway.

Na Liu, Miao-Miao Lin, Si-Si Huang, Zi-Qi Liu, Jun-Chao Wu, Zhong-Qin Liang, Zheng-Hong Qin, Yan Wang.

  Aim: Previous research recognizes that NADPH can produce reduced glutathione (GSH) as a coenzyme and produce ROS as a substrate of NADPH oxidase (NOX). Besides, excessive activation of glutamate receptors results in mitochondrial impairment. The study aims at spelling out the effects of NADPH and Mito-apocynin, a NOX inhibitor which specifically targets the mitochondria, on the excitotoxicity induced by Kainic acid (KA) and its mechanism.Methods: The in vivo neuronal excitotoxicity model was constructed by stereotypically injecting KA into the unilateral striatum of mice. Administrated NADPH (i.v, intravenous) 30 min prior and Mito-apocynin (i.g, intragastric) 1 day prior, respectively, then kept administrating daily until mice were sacrificed 14 days later. Nissl staining measured the lesion of striatum and survival status of neurons. Cylinder test of forelimb asymmetry and the adhesive removal test reflected the behavioral deficit caused by neural dysfunction. Determined Total superoxide dismutase (T-SOD), malondialdehyde (MDA), and GSH indicated oxidative stress. Western blot presented the expression levels of LC3-II/LC3-I, SQSTM1/p62, TIGAR, and NOX4. Assessed oxygen consumption rate using High-Resolution Respirometry. In vitro, the MitoSOX Indicator reflected superoxide released by neuron mitochondria. JC-1 and ATP assay Kit were used to detect mitochondrial membrane potential (MMP) and energy metabolism, respectively.
Results: In this study, we have successfully established excitotoxic model by KA in vivo and in vitro. KA induced decreased SOD activity and increased MDA concentration. KA cause the change of LC3-II/LC3-I, SQSTM1/p62, and TIGAR expression, indicating the autophagy activation. NADPH plays a protective role in vivo and in vitro. It reversed the KA-mediated changes in LC3, SQSTM1/p62, TIGAR, and NOX4 protein expression. Mito-apocynin inhibited KA-induced increases in mitochondrial NOX4 expression and activity. Compared with NADPH, the combination showed more significant neuroprotective effects, presenting more neurons survive and better motor function recovery. The combination also better inhibited the over-activated autophagy. In vitro, combination of NADPH and Mito-apocynin performed better in restoring mitochondria membrane potential.
Conclusion: In summary, combined administration of NADPH and NOX inhibitors offers better neuroprotection by reducing NADPH as a NOX substrate to generate ROS. The combined use of NADPH and Mito-apocynin can better restore neurons and mitochondrial function through autophagy pathway.

Keywords:  Mito-apocynin; NADPH; NOX; ROS; autophagy; excitotoxicity; mitochondria

DOI:  https://doi.org/10.3389/fcell.2021.612554
J Vis Exp. 2021 02 09.

ROS Live Cell Imaging During Neuronal Development.

Aslihan Terzi, S M Sabbir Alam, Daniel M Suter.

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

DOI: https://doi.org/10.3791/62165
J Clin Invest. 2021 Mar 02. pii: 141676. [Epub ahead of print]

DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk.

Helmut Grasberger, Andrew T Magis, Elisa Sheng, Matthew P Conomos, Min Zhang, Lea S Garzotto, Guoqing Hou, Shrinivas Bishu, Hiroko Nagao-Kitamoto, Mohamad El-Zataari, Sho Kitamoto, Nobuhiko Kamada, Ryan Stidham, Yasutada Akiba, Jonathan Kaunitz, Yael Haberman, Subra Kugathasan, Lee A Denson, Gilbert S Omenn, John Y Kao.

  A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multi-omic analyses using 2,872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance (155 unique variants with allele frequency < 1%; 12.9% carrier rate). We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C (FDR=2.6e-5), which is induced also in mucosal biopsies of IBD patients. DUOX2 deficient mice replicated increased IL17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in IBD patients corroborated IL17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL17C induction provided a rationale for variant stratification in case-control studies that substantiated DUOX2 as an IBD risk gene (pooled OR = 1.54 [95% CI 1.09-2.18]; P = 7.1e-4). Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.

Keywords:  Gastroenterology; Genetic variation; Inflammatory bowel disease; Innate immunity

DOI:  https://doi.org/10.1172/JCI141676