Cathepsin‐B is a Mediator of Barrier Dysfunction and Hyperpermeability in Blood‐Brain Barrier Endothelial Cells

Abstract

Cathepsin B belongs to the papain‐like family of cysteine proteases and is an upstream mediator of the NLRP3 inflammasome pathway that leads to interleukin 1β (IL‐1β) secretion. Interleukin 1β is a potent inducer of tight junction disruption and hyperpermeability in blood‐brain barrier (BBB) endothelial cells. However, the involvement of cathepsin B in BBB dysfunction and hyperpermeability is not clearly known. Understanding this pathway is critical to preserving BBB integrity and protection against brain edema. In order to study this relationship, rat brain microvascular endothelial cells were transfected with active cathepsin B. Tight junction organization and changes in permeability were evaluated based on immunofluorescence, immunoblot analysis of zonula occudens‐1 (ZO‐1) and Transwell monolayer permeability assay using FITC‐dextran (10‐kDa). Cathepsin B activity and cell viability were evaluated fluorometrically. F‐actin stress fiber formation was studied using rhodamine phalloidin staining. Active cathepsin B induced ZO‐1 dislocation/tight junction disorganization, F‐actin stress fiber formation and monolayer hyperpermeability significantly. These effects were inhibited by pretreatment with NLRP3 inhibitors isoliquiritigenin and MCC950. Active cathepsin B had no significant effect on ZO‐1 protein expression or cell viability. Furthermore, cathepsin B inhibitor E‐64d attenuated hydrogen peroxide‐induced cathepsin B activity and monolayer hyperpermeability significantly. These results suggest that cathepsin B is a mediator of BBB dysfunction/hyperpermeability. Cathepsin B inhibition has protective effects against barrier dysfunction and hyperpermeability in BBB endothelial cells. One of the mechanisms by which cathepsin B induces barrier dysfunction is via the NLRP3 inflammasome pathway.