LRRC8A in endothelial cells contributes to the aberrant blood-brain barrier integrity in ischaemic stroke

BACKGROUND: The increased permeability of the blood-brain barrier (BBB) is a critical contributor to the high mortality following ischaemic stroke. However, the mechanisms regulating BBB integrity remain poorly understood. Leucine-rich repeat-containing 8A (LRRC8A) is a chloride channel critical for cellular volume homeostasis and plays a key role in regulating neuronal injury during ischaemia. However, its impact on BBB function is currently unclear. METHODS: A transient middle cerebral artery occlusion model was established to investigate the impact of LRRC8A on BBB integrity. Laser speckle contrast imaging was used to monitor cortical blood flow. Primary mouse and human brain microvascular endothelial cells (m/hBMVECs) were subjected to oxygen-glucose deprivation (OGD) and re-oxygenation for varying durations. Patch-clamp recordings were performed to measure volume-regulated chloride currents. Immunostaining was conducted to evaluate protein expression. Cell permeability was evaluated with transwell assay. RESULTS: LRRC8A deletion in endothelial cells ameliorates the infarct area and mitigates BBB leakage. Ischaemia dramatically upregulates the expression of LRRC8A in endothelial cells, concurrently downregulating tight junction proteins. OGD exposure augments the VRCC current mediated by LRRC8A in BMVECs. In contrast, inhibiting LRRC8A promotes the expression of ZO-1 and VE-cadherin, thereby preserving the integrity of endothelial cells. With-no-lysine kinase 1 (WNK1) inhibition contributes to LRRC8A-induced BBB damage post-ischaemic stroke. Eupatorin, a newly identified LRRC8A inhibitor, exerts neuroprotective effects against ischaemic stroke. CONCLUSIONS: LRRC8A in BMVECs plays a pivotal role in modulating BBB integrity, a process regulated by WNK1. As an LRRC8A inhibitor, Eupatorin holds the potential for ischaemic stroke therapy.