BackgroundTumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α–mediated endothelial cell barrier dysfunction and microvascular hyperpermeability.MethodsRat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monola... More
BackgroundTumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α–mediated endothelial cell barrier dysfunction and microvascular hyperpermeability.MethodsRat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using β-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α–induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically.ResultsZ-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α–induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α–induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α–induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability.ConclusionsOur results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α–induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL–based drug development against microvascular hyperpermeability and tissue edema.
