Calcium-dependent blood-brain barrier breakdown by NOX5 limits postreperfusion benefit in stroke
Ana I. Casas, … , Christoph Kleinschnitz, Harald H.H.W. Schmidt
Ana I. Casas, … , Christoph Kleinschnitz, Harald H.H.W. Schmidt
Published March 18, 2019
Citation Information: J Clin Invest. 2019;129(4):1772-1778. https://doi.org/10.1172/JCI124283.
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Concise Communication Neuroscience

Calcium-dependent blood-brain barrier breakdown by NOX5 limits postreperfusion benefit in stroke

Abstract

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calcium-activated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenation-induced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Authors

Ana I. Casas, Pamela W.M. Kleikers, Eva Geuss, Friederike Langhauser, Thure Adler, Dirk H. Busch, Valerie Gailus-Durner, Martin Hrabê de Angelis, Javier Egea, Manuela G. Lopez, Christoph Kleinschnitz, Harald H.H.W. Schmidt

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Figure 4

NOX5 inhibition before reoxygenation reduced cell permeability to basal levels using a human in vitro ischemia model.

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NOX5 inhibition before reoxygenation reduced cell permeability to basal ...
(A) HBMECs were seeded on Transwell inserts, where cell permeability was assessed using Evans blue dye. (B) After incubation and seeding under normoxic conditions, HBMECs were subjected to 6 hours of hypoxia and 24 hours of reoxygenation. Cells were treated with both NOX4 (M13) and NOX5 inhibitors (ML090) at early (20 minutes before reoxygenation) and late (20 minutes after reoxygenation) time points. (C) Cell permeability was assessed by measuring Evans blue fluorescence after hypoxia. Evans blue diffusion was significantly reduced in cells subjected to early treatment of ML090 (0.01 μM), which preferably inhibits NOX5, and M13 (0.2 μM), which is mainly a NOX4 inhibitor (*P < 0.05, 2-tailed unpaired t test). In contrast, late treatment was only effective after M13 (0.2 μM) treatment compared with that of nontreated cells (##P < 0.01). Reox, reoxygenation. (D) NOX5 activation takes place within the first 30 minutes to 1 hour after hypoxia (see Figure 3), while NOX4 activation peak appears at around 5 hours after ischemia (20). Data are represented as mean ± SEM in all experiments. NOX4i, NOX4 inhibitor.