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New rationale for NOX4/5 inhibitors for neuroprotective strategy after stroke

 

Ischemic stroke is a predominant cause of disability worldwide. The thrombolytic or mechanical occlusion has to be removed immediately to supply the brain with blood again. However, the reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier (BBB). The reason for the increase of calcium in the blood was unknown so far.

In a collaborative effort, scientists from the University of Maastricht, Duisburg-Essen, Würzburg, Munich and Madrid found out, that the enzyme 5 NADPH oxidase (NOX5), a calcium activated, reactive-oxygen-species (ROS)-forming enzyme, is responsible for the Calcium elevation in the blood after reperfusion.

The team of scientists generated a humanized knock-in (KI) mouse model to analyze the impact of the NOX5 enzyme after stroke induction and reperfusion in mice. To mimic the physiological human expression pattern of NOX5 in mice, the mouse line express the human NOX5 gene under control of the Tie2 promoter, which physiologically regulates endothelial and hematopoietic gene expression in human cells. The leukocyte proportions in NOX5-KI mice were indistinguishable from those in WT mice, indicating no major phenotype change in the immune cell origin. In addition, no phenotype was observed with respect to life span, cognition, or neuromotor function.

As a first result, NOX5 caused acute, post-reoxygenation ROS formation in the in vitro organotypic hippocampal cultures from NOX5-KI mice. The increased in vitro ROS production was related to Ca2+ overload. In vivo, post ischemic ROS formation, infarct volume and functional outcomes were worsened in NOX5-KI mice. The post stroke calcium seems to be a key mechanism for blood-brain barrier disruption. To monitor the pharmacologically inhibition of NOX5, a primary culture of human brain microvascular endothelial cells (HBMECs) was set up. The acute post-reoxygenation leakiness of human microvascular brain endothelial cells was prevented by pharmacological NOX4 and NOX5 inhibition, while NOX5 inhibitor was only protective when added early.

The results of the study support further evaluation of post stroke reperfusion in the presence of NOX inhibition for limiting the stroke-induced damage. The findings provide a clear rationale for further development of pharmacological NOX4/5 inhibitors as a first-in-class neuroprotective strategy after stroke.

Casas AI, Kleikers PW, Geuss E, Langhauser F, Adler T, Busch DH, Gailus-Durner V, Hrabe de Angelis M, Egea J, Lopez MG, Kleinschnitz C, Schmidt HH.                                                                               Calcium-dependent blood-brain barrier breakdown by NOX5 limits postreperfusion benefit in stroke.  J Clin Invest. 2019 Mar 18;130:1772-1778. doi: 10.1172/JCI124283. eCollection 2019 Mar 18.