Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.
Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 μM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using β-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 μM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.
Department of Surgery, Morehouse School of Medicine, 720 Westview Drive SW, Atlanta, GA, 30310, USA.
Devendra A
DA
Sawant
Rickesha L
RL
Wilson
Binu
B
Tharakan
Hayden W
HW
Stagg
Felicia A
FA
Hunter
Ed W
EW
Childs
D022005:Adherens Junctions / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002199:Capillary Permeability; D053148:Caspase 3 / Q000378:metabolism; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology*; D053078:Membrane Potential, Mitochondrial; D055806:Microvessels / Q000166:cytology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C505410:Casp3 protein, rat; D053148:Caspase 3