title: ZO-1 redistribution and F-actin stress fiber formation in pulmonary endothelial cells after thermal injury.
abstract: BACKGROUND
  In response to isolated inflammatory stimuli, changes in endothelial cell morphology that enhance paracellular flow of solutes result from F-actin stress fiber formation, myosin phosphorylation, and actin anchoring protein (ZO-1) modifications. We hypothesized that myosin light chain kinase inhibition would diminish burn-enhanced endothelial monolayer permeability by secondarily preventing F-actin and actin anchoring protein rearrangements.
  
  
  METHODS
  Human pulmonary microvascular endothelial cells were treated for 4 hours with 20% human burn serum (isolated from patients with > 45% total body surface area thermal injury or healthy volunteers). Select cultures were pretreated with myosin light chain kinase inhibitors (ML-9). Permeability was assessed by migration of bovine serum albumin across cell monolayers. Cells were stained with rhodamine-phalloidin and anti-ZO-1 antisera and examined by means of confocal microscopy.
  
  
  RESULTS
  Burn serum significantly enhanced monolayer permeability to albumin, whereas pretreatment with ML-9 limited this effect. Control cells maintained cortical F-actin and peripheral ZO-1 distributions (1a, b), whereas burn serum induced transcellular F-actin stress fiber formation and a diffuse ZO-1 staining (2a, b). ML-9 prevented burn-induced actin rearrangements, but not the diffuse redistribution of ZO-1.
  
  
  CONCLUSIONS
  These data demonstrate that endothelial F-actin stress fiber formation and ZO-1 redistribution contribute to postburn loss of pulmonary endothelial monolayer integrity. Although myosin phosphorylation appears to be required for endothelial F-actin stress fiber formation, redistribution of actin-membrane anchoring proteins appears to be regulated independently after thermal injury.
authors.affiliation: Department of Surgery, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., MC 9158, Dallas, TX 75325-9158, USA. joseph.murphy@utsouthwestern.edu
authors.forename: Joseph T
authors.initials: JT
authors.lastname: Murphy
authors.forename: Steve
authors.initials: S
authors.lastname: Duffy
mesh_terms: D000199:Actins / Q000648:ultrastructure*; D000328:Adult; D002056:Burns / Q000097:blood / Q000150:complications* / Q000276:immunology; D019559:Capillary Leak Syndrome / Q000209:etiology* / Q000378:metabolism / Q000473:pathology*; D002199:Capillary Permeability; D016022:Case-Control Studies; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000473:pathology*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007249:Inflammation; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis / Q000648:ultrastructure*; D018994:Myosin Light Chains / Q000037:antagonists & inhibitors; D009218:Myosins / Q000378:metabolism / Q000648:ultrastructure; D062793:Occludin; D010750:Phosphoproteins / Q000032:analysis / Q000648:ultrastructure*; D010766:Phosphorylation; D020545:Respiratory Mucosa / Q000473:pathology*; D022502:Stress Fibers / Q000473:pathology*; D062826:Zonula Occludens-1 Protein
chemical_list: D000199:Actins; D008565:Membrane Proteins; D018994:Myosin Light Chains; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D018799:Intercellular Adhesion Molecule-1; D009218:Myosins