Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large

Background: Tumor necrosis factor-alpha (TNF-alpha), 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-alpha-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. Methods: Rat 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 beta-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 mu M) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 mu M) for 48 hours were performed to study their respective effects on TNF-alpha-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 mu g/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. Results: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-alpha-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-alpha-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-alpha-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. Conclusions: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-alpha-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. (C) 2013 Elsevier Inc. All rights reserved.