title: Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.
abstract: BACKGROUND
  Tumor 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.
  
  
  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 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.
  
  
  RESULTS
  Z-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.
  
  
  CONCLUSIONS
  Our 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.
authors.affiliation: Department of Surgery, Morehouse School of Medicine, Atlanta, Georgia 30310, USA.
authors.forename: Devendra A
authors.initials: DA
authors.lastname: Sawant
authors.forename: Binu
authors.initials: B
authors.lastname: Tharakan
authors.forename: Rickesha L
authors.initials: RL
authors.lastname: Wilson
authors.forename: Hayden W
authors.initials: HW
authors.lastname: Stagg
authors.forename: Felicia A
authors.initials: FA
authors.lastname: Hunter
authors.forename: Ed W
authors.initials: EW
authors.lastname: Childs
mesh_terms: D022005:Adherens Junctions / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000235:genetics / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004487:Edema / Q000378:metabolism* / Q000473:pathology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008168:Lung / Q000166:cytology; D009842:Oligopeptides / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D051020:bcl-X Protein / Q000378:metabolism* / Q000494:pharmacology; D051176:beta Catenin / Q000378:metabolism
chemical_list: D051023:BH3 Interacting Domain Death Agonist Protein; C494813:Bcl2l1 protein, rat; C494816:Bid protein, rat; C495268:Ctnnb1 protein, rat; D015853:Cysteine Proteinase Inhibitors; D009842:Oligopeptides; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; D051176:beta Catenin; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; D053148:Caspase 3; D053181:Caspase 8
keywords: Adherens junction; Apoptotic signaling; BID; Bcl-2-homology domain 3-interacting domain death agonist; Vascular permeability; β-Catenin