title: Loss of focal adhesion kinase enhances endothelial barrier function and increases focal adhesions.
abstract: OBJECTIVE
  To determine the role of FAK in the regulation of endothelial barrier function.
  
  
  METHODS
  Stable FAK knockdown HLEC were generated by lentiviral infection of FAK shRNA. Measurements of isometric tension and transendothelial electrical resistance were performed.
  
  
  RESULTS
  A FAK knockdown human pulmonary endothelial cell line was generated by lentiviral infection with FAK shRNA and resulted in greater than 90% reduction in FAK protein with no change in Pyk2 protein. Loss of FAK altered cell morphology and actin distribution in both pre- and post-confluent endothelial cells. Large, polygonal shaped endothelial cells with randomly organized stress fibers were identified in pre-confluent cultures, while in confluent monolayers, endothelial cells were irregularly shaped with actin bundles present at cell margins. An increase in the number and size of vinculin plaques was detected in FAK-depleted cells. FAK knockdown monolayers generated a greater transendothelial electrical resistance than controls. Thrombin treatment induced similar changes in TER in both FAK knockdown and control cell lines. FAK-depleted endothelial cells developed a higher stable basal isometric tension compared to control monolayers, but the increase in tension stimulated by thrombin does not differ between the cell lines. Basal myosin II regulatory light chain phosphorylation was unaltered in FAK-depleted cells. In addition, loss of FAK enhanced VE-cadherin localization to the cell membrane without altering VE-cadherin protein levels.
  
  
  CONCLUSIONS
  The loss of FAK in endothelial cells enhanced cell attachment and strengthened cell-cell contacts resulting in greater basal tension leading to formation of a tighter endothelial monolayer.
authors.affiliation: Department of Physiology and Pharmacology, West Virginia University School of Medicine, Morgantown, West Virginia; Center for Cardiovascular and Respiratory Sciences, West Virginia University School of Medicine, Morgantown, West Virginia.
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Arnold
authors.forename: Zoe M
authors.initials: ZM
authors.lastname: Goeckeler
authors.forename: Robert B
authors.initials: RB
authors.lastname: Wysolmerski
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D017097:Electric Impedance; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D051417:Focal Adhesion Kinase 1 / Q000235:genetics / Q000378:metabolism*; D022001:Focal Adhesions / Q000201:enzymology* / Q000235:genetics; D055785:Gene Knockdown Techniques; D006801:Humans; D008168:Lung / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D018345:Mice, Knockout
chemical_list: D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human; C496051:Ptk2 protein, mouse
keywords: endothelial cells; focal adhesions; isometric tension; permeability