title: Reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell hyperpermeability following oxygen-glucose deprivation and reoxygenation.
abstract: OBJECTIVE
  Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this.
  
  
  METHODS
  RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively.
  
  
  RESULTS
  OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly.
  
  
  CONCLUSIONS
  These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.
authors.affiliation: Departments of Surgery and Pediatrics, Texas A&M University Health Science Center College of Medicine and Scott & White Healthcare, Temple, Texas, USA.
authors.forename: Himakarnika
authors.initials: H
authors.lastname: Alluri
authors.forename: Hayden W
authors.initials: HW
authors.lastname: Stagg
authors.forename: Rickesha L
authors.initials: RL
authors.lastname: Wilson
authors.forename: Robert P
authors.initials: RP
authors.lastname: Clayton
authors.forename: Devendra A
authors.initials: DA
authors.lastname: Sawant
authors.forename: Madhavi
authors.initials: M
authors.lastname: Koneru
authors.forename: Madhava R
authors.initials: MR
authors.lastname: Beeram
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Davis
authors.forename: Binu
authors.initials: B
authors.lastname: Tharakan
mesh_terms: D000818:Animals; D001812:Blood-Brain Barrier / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D001929:Brain Edema / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D002199:Capillary Permeability; D053148:Caspase 3 / Q000378:metabolism*; D015687:Cell Hypoxia; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; C505410:Casp3 protein, rat; D053148:Caspase 3; D005947:Glucose
keywords: blood-brain barrier; endothelial cells; ischemia reperfusion; vascular hyperpermeability