ZO-1 redistribution and F-actin stress fiber formation in pulmonary endothelial cells after thermal injury.

Abstract

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. 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. 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. 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.