title: Cerebral hypoxia/ischemia selectively disrupts tight junctions complexes in stem cell-derived human brain microvascular endothelial cells.
abstract: BACKGROUND
  Cerebral hypoxia/ischemia (H/I) is an important stress factor involved in the disruption of the blood-brain barrier (BBB) following stroke injury, yet the cellular and molecular mechanisms on how the human BBB responds to such injury remains unclear. In this study, we investigated the cellular response of the human BBB to chemical and environmental H/I in vitro.
  
  
  METHODS
  In this study, we used immortalized hCMEC/D3 and IMR90 stem-cell derived human brain microvascular endothelial cell lines (IMR90-derived BMECs). Hypoxic stress was achieved by exposure to cobalt chloride (CoCl2) or by exposure to 1 % hypoxia and oxygen/glucose deprivation (OGD) was used to model ischemic injury. We assessed barrier function using both transendothelial electrical resistance (TEER) and sodium fluorescein permeability. Changes in cell junction integrity were assessed by immunocytochemistry and cell viability was assessed by trypan-blue exclusion and by MTS assays. Statistical analysis was performed using one-way analysis of variance (ANOVA).
  
  
  RESULTS
  CoCl2selectively disrupted the barrier function in IMR90-derived BMECs but not in hCMEC/D3 monolayers and cytotoxic effects did not drive such disruption. In addition, hypoxia/OGD stress significantly disrupted the barrier function by selectively disrupting tight junctions (TJs) complexes. In addition, we noted an uncoupling between cell metabolic activity and barrier integrity.
  
  
  CONCLUSIONS
  In this study, we demonstrated the ability of IMR90-derived BMECs to respond to hypoxic/ischemic injury triggered by both chemical and environmental stress by showing a disruption of the barrier function. Such disruption was selectively targeting TJ complexes and was not driven by cellular apoptosis. In conclusion, this study suggests the suitability of stem cell-derived human BMECs monolayers as a model of cerebral hypoxia/ischemia in vitro.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 South Coulter Street, Amarillo, TX, USA.
authors.forename: Shyanne
authors.initials: S
authors.lastname: Page
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 South Coulter Street, Amarillo, TX, USA.
authors.forename: Alli
authors.initials: A
authors.lastname: Munsell
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 South Coulter Street, Amarillo, TX, USA. abraham.al-ahmad@ttuhsc.edu.
authors.forename: Abraham J
authors.identifier: http://orcid.org/0000-0001-7463-3204
authors.initials: AJ
authors.lastname: Al-Ahmad
mesh_terms: D001812:Blood-Brain Barrier / Q000378:metabolism*; D002199:Capillary Permeability / Q000502:physiology; D018929:Cell Culture Techniques; D015687:Cell Hypoxia / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D003035:Cobalt; D017097:Electric Impedance; D042783:Endothelial Cells / Q000378:metabolism*; D019793:Fluorescein; D005456:Fluorescent Dyes; D005947:Glucose / Q000172:deficiency*; D006801:Humans; D020925:Hypoxia-Ischemia, Brain; D007150:Immunohistochemistry; D055806:Microvessels / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism; D019108:Tight Junctions / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D003035:Cobalt; C018021:cobaltous chloride; D005947:Glucose; D019793:Fluorescein