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 April 1, 2019; First 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 Therapeutics

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 1

Generation and validation of the NOX5-KI mouse.

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Generation and validation of the NOX5-KI mouse. (A) Representative schem...
(A) Representative schematic of the humanized NOX5 mice. NOX5 gene located on the X chromosome results in females with a double copy of the gene (NOX5-KI/KI), while gene load is half in male mice (NOX5-KI/Y) (B) Construction of the humanized NOX5-KI mice. First, WT and deleted Hprt constructs are shown. The targeting vector to insert NOX5 (human NOX5 cDNA, blue box) was coupled to the human Hprt promoter (red box) and exons 1 and 2. The human Hprt promoter is under control of the Tie2 gene (purple box) to create NOX5 expression. Hatched black and red boxes represent murine and human Hprt exons, respectively. Solid line represents intronic sequences. (C) NOX5 gene expression was measured by qPCR (Ct) in different organs (lung, brain, kidney, aorta, and mouse brain capillary endothelial cells [MBCEC]); macrophages from bone marrow (undifferentiated [M0], inflammatory [M1], and active [M2]); and hematopoietic cells isolated from spleen (monocytes [Mono], neutrophils [Neu], B cells, and T cells). Tissues from WT mice did not show NOX5 gene expression, while significant expression was detected in NOX5-KI mice. Ct values of n = 4 are shown.