Pre-ischaemic empagliflozin treatment attenuates blood-brain barrier disruption via β-catenin mediated protection of cerebral endothelial cells

Abstract

Aims: Microvascular endothelial cells dysfunction can significantly worsen ischaemic stroke outcomes by disrupting tight junctions and increasing the acquisition of adhesion molecules, accelerating blood-brain barrier (BBB) disruption and pro-inflammatory response. The identification of drugs that improve endothelial cell function may be crucial for ischaemic stroke. It has been validated that empagliflozin (EMPA), a novel antidiabetic drug, protects endothelial cells regardless of the diabetic status of the patient. However, the impact of EMPA on stroke outcomes is unclear. We hypothesized that EMPA would exert a beneficial effect on ischaemic stroke outcome by protecting microvascular endothelial cells against tight junction disruption and the increase of adhesion molecules.

Methods and results: Young adult male mice were administered with EMPA or vehicle (dimethyl sulfoxide) daily for 7 days before being subjected to transient middle cerebral artery occlusion (tMCAO). Neurological deficits were evaluated for up to 28 days post-tMCAO. Infarct volume, BBB disruption, and inflammatory status were assessed 1 day after tMCAO.bEnd.3 cells and primary brain microvascular endothelial cells were treated with EMPA or vehicle under oxygen and glucose deprivation/reperfusion (OGD/R), and the lactate dehydrogenase release, transendothelial electrical resistance, leakage of fluorescein isothiocyanate-dextran, and tight junction and adhesion molecules proteins were examined. Mechanistic studies probing the effect of EMPA on endothelial cells were conducted by RNA-seq. EMPA treatment before ischaemia markedly improved infarct volume, BBB disruption, and inflammation 1-day post-tMCAO, and further enhanced neurobehavioral function up to 28 days. Pre-treatment of EMPA attenuated endothelial cell dysfunction under OGD/R conditions. In mechanistic terms, RNA-seq data from isolated cerebral microvessels revealed that the Wnt/β-catenin signalling pathway was preserved in the EMPA group, in contrast to the vehicle group. Pre-treatment with EMPA inhibited β-catenin ubiquitination and promoted β-catenin translocation from the cytoplasm to the nucleus to improve endothelial cell function. Importantly, the β-catenin inhibitor XAV-939 eliminated this protective function of EMPA.

Conclusion: EMPA administration before tMCAO attenuated ischaemia/reperfusion-induced BBB disruption and inflammation via β-catenin-mediated protection of cerebral microvascular endothelial cells. Therefore, EMPA shows potential for improving stroke outcomes as an adjunctive preventive strategy.

Keywords: BBB; Cerebral endothelial cell; Empagliflozin; Inflammation; Ischaemic stroke; β-catenin.