Endocytic Pathways and Actin Remodeling Mediate Everolimus-Induced VE-Cadherin Disorganization and Barrier...

VE-cadherin is a key component of endothelial adherens junctions, and its disorganization contributes to vascular dysfunction. While rapamycin analogs like everolimus (EVL) are clinically linked to endothelial barrier dysfunction (EBD), the underlying molecular mechanisms remain poorly defined. This study investigates how EVL alters VE-cadherin organization, trafficking, cytoskeletal architecture, and barrier function in endothelial cells.

Human umbilical vein endothelial cells (HUVECs) were treated with 500 nM EVL for 4 or 24 h. Junctional VE-cadherin organization was quantified using confocal microscopy and the Junction Analyzer Program. Cytoskeletal changes were assessed via F-actin anisotropy, and pharmacologic inhibitors (chlorpromazine, chloroquine, and brefeldin A) were used to block clathrin-mediated endocytosis, lysosomal degradation, and Golgi trafficking, respectively. Barrier function was evaluated using TEER and 4 kDa FITC-dextran transwell assays.

EVL reduced continuous VE-cadherin and increased punctate junctions in a time-dependent but partially reversible manner. Inhibiting endocytosis or lysosomal degradation preserved VE-cadherin continuity, while Golgi disruption blocked recovery. EVL also increased F-actin anisotropy, reflecting enhanced stress-fiber alignment within individual cells, but transiently uncoupled intracellular actin organization from coordinated cytoskeletal alignment across the monolayer. Functionally, EVL decreased TEER and increased dextran permeability by 2.24-2.63-fold, indicating significant barrier disruption.

EVL compromises endothelial barrier integrity by promoting VE-cadherin internalization and lysosomal degradation, accompanied by cytoskeletal remodeling and a Golgi-dependent, partial restoration of junctional VE-cadherin. These findings highlight endocytic, degradative, and Golgi-mediated trafficking pathways as key modulators of EVL-induced endothelial barrier dysfunction and provide mechanistic insight into the vascular effects of rapalog-based mTOR inhibition.