title: Glycogen synthase kinase 3 inhibitor protects against microvascular hyperpermeability following hemorrhagic shock.
abstract: BACKGROUND
  Hemorrhagic shock (HS)-induced microvascular hyperpermeability involves disruption of endothelial cell adherens junctions leading to increase in paracellular permeability. β-Catenin, an integral component of the adherens junctional complex and Wnt pathway, and caspase 3 via its apoptotic signaling regulate endothelial cell barrier integrity. We have hypothesized that inhibiting phosphorylation of β-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 would attenuate microvascular hyperpermeability following HS.
  
  
  METHODS
  In Sprague-Dawley rats, HS was induced by withdrawing blood to reduce mean arterial pressure to 40 mm Hg for 60 minutes followed by resuscitation. Rats were given SB216763 (600 μg/kg) intravenously 10 minutes before shock. To study microvascular permeability, the rats were intravenously injected with fluorescein isothiocyanate (FITC)-albumin (50 mg/kg), and its flux across the mesenteric postcapillary venules was determined using intravital microscopy. In cell culture studies, rat lung microvascular endothelial cell monolayers grown on Transwell plates were pretreated with SB216763 (5 μM) followed by BAK (5 μg/mL) and caspase 3 (5 μg/mL) protein transfection. FITC-albumin (5 mg/mL) flux across cell monolayers indicates change in monolayer permeability. Activity of canonical Wnt pathway was determined by luciferase assay. Caspase 3 enzyme activity was assayed fluorometrically.
  
  
  RESULTS
  The HS group showed significant increase in FITC-albumin extravasation (p < 0.05) compared with sham. SB216763 significantly decrease HS-induced FITC-albumin extravasation (p < 0.05). Pretreatment with SB216763 protected against a BAK-induced increase in rat lung microvascular endothelial cell monolayer permeability and caspase 3 activity but failed to show similar results with a caspase 3-induced increase in monolayer permeability. Wnt3a treatment showed an increase in β-catenin-dependent T-cell factor-mediated transcription.
  
  
  CONCLUSIONS
  Inhibiting phosphorylation of β-catenin and caspase 3 activity using glycogen synthase kinase 3-specific inhibitor SB216763 help regulates HS-induced microvascular hyperpermeability.
authors.affiliation: From the Department of Surgery (D.A.S., F.A.H., E.W.C.), Morehouse School of Medicine, Atlanta, Georgia; Texas A&M Health Science Center College of Medicine and Scott & White Health Care (B.T.), Temple, Texas.
authors.forename: Devendra A
authors.initials: DA
authors.lastname: Sawant
authors.forename: Binu
authors.initials: B
authors.lastname: Tharakan
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 / Q000502:physiology; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors*; D007211:Indoles / Q000494:pharmacology*; D008297:Male; D008301:Maleimides / Q000494:pharmacology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012771:Shock, Hemorrhagic / Q000503:physiopathology*; D015398:Signal Transduction
chemical_list: D007211:Indoles; D008301:Maleimides; C417521:SB 216763; D038362:Glycogen Synthase Kinase 3; D053148:Caspase 3