title: The imbalanced redox status in senescent endothelial cells is due to dysregulated Thioredoxin-1 and NADPH oxidase 4.
abstract: Environmental stressors as well as genetic modifications are known to enhance oxidative stress and aging processes. Mitochondrial and nuclear dysfunctions contribute to the onset of aging. One of the most important redox regulators in primary human endothelial cells is Thioredoxin-1 (Trx-1), a 12 kD protein with additional anti-apoptotic properties. Cellular generators of reactive oxygen species are NADPH oxidases (NOXs), of which NOX4 shows highest expression levels in endothelial cells. Therefore, the aim of the study was to investigate how Trx-1 and NOX4 are regulated during stress-induced premature senescence in endothelial cells. We treated primary human endothelial cells for two weeks with H2O2 to generate stress-induced premature senescence in these cells. In this model senescence-associated β-Galactosidase and nuclear p21 as senescence markers are increased. Moreover, total and mitochondrial reactive oxygen species formation is enhanced. An imbalanced redox homeostasis is detected by elevated NOX4 and decreased Trx-1 levels. This can be rescued by lentiviral expression of Trx-1. Moreover, the lysosomal protease Cathepsin D is over-activated, which results in reduced Trx-1 protein levels. Inhibition of "over-active" Cathepsin D by the specific, cell-permeable inhibitor pepstatin A abolishes the increase in nuclear p21 protein, ROS formation and degradation of Trx-1 protein, thus leading to blockade of stress-induced premature senescence by stabilizing the cellular redox homeostasis. Aortic Trx-1 levels are decreased and Cathepsin D activity is increased in NOX4 transgenic mice exclusively expressing NOX4 in the endothelium when compared to their wildtype littermates. Thus, loss of Trx-1 and upregulation of NOX4 importantly contribute to the imbalance in the redox-status of senescent endothelial cells ex vivo and in vivo.
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: christinegoy100@gmail.com.
authors.forename: Christine
authors.initials: C
authors.lastname: Goy
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: czypiorski@gmail.com.
authors.forename: Philip
authors.initials: P
authors.lastname: Czypiorski
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: Joachim.Altschmied@uni-duesseldorf.de.
authors.forename: Joachim
authors.initials: J
authors.lastname: Altschmied
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: Sascha.Jakob@uni-duesseldorf.de.
authors.forename: Sascha
authors.initials: S
authors.lastname: Jakob
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: Lothar.Rabanter@uni-duesseldorf.de.
authors.forename: Lothar L
authors.initials: LL
authors.lastname: Rabanter
authors.affiliation: King's College London BHF Centre of Excellence, The James Black Centre, London SE5 9NU, UK. Electronic address: alison.brewer@kcl.ac.uk.
authors.forename: Alison C
authors.initials: AC
authors.lastname: Brewer
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: Niloofar.Ale-Agha@uni-duesseldorf.de.
authors.forename: Niloofar
authors.initials: N
authors.lastname: Ale-Agha
authors.affiliation: IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: Nadine.Dyballa@uni-duesseldorf.de.
authors.forename: Nadine
authors.initials: N
authors.lastname: Dyballa-Rukes
authors.affiliation: King's College London BHF Centre of Excellence, The James Black Centre, London SE5 9NU, UK. Electronic address: ajay.shah@kcl.ac.uk.
authors.forename: Ajay M
authors.initials: AM
authors.lastname: Shah
authors.affiliation: Central Institute of Clinical Chemistry and Laboratory Medicine, University of Duesseldorf, 40225 Duesseldorf, Germany; IUF - Leibniz Research Institute for Environmental Medicine at the University of Duesseldorf gGmbH, 40225 Duesseldorf, Germany. Electronic address: juhae001@uni-duesseldorf.de.
authors.forename: Judith
authors.initials: J
authors.lastname: Haendeler
mesh_terms: D000818:Animals; D002402:Cathepsin D / Q000378:metabolism; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D005786:Gene Expression Regulation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D015398:Signal Transduction; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D016877:Oxidants; C491203:TXN protein, human; D013879:Thioredoxins; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C533922:CTSD protein, human; D002402:Cathepsin D
keywords: Cathepsin D; Endothelial cells; Imbalanced redox homeostasis; NADPH oxidase 4; Senescence; Thioredoxin-1