Quantification of adherens junction disruption and contiguous paracellular protein leak in human lung endothelial cells under septic conditions.

Abstract

Sepsis is associated with dysfunction of MVEC resulting in organ edema and inflammation. VE-cadherin, a component of MVEC adherens junctions, may be disrupted in sepsis. However, the direct connection between individual MVEC VE-cadherin disruption and increased paracellular permeability is uncertain. Human pulmonary MVEC were cultured on a biotin matrix and treated with cytomix, as a model of sepsis, vs PBS. MVEC permeability was assessed by trans-MVEC monolayer leak of Oregon green 488-conjugated avidin, which bound subcellular biotin to localize sites of paracellular leak. Leak was correlated with individual cell-specific MVEC surface VE-cadherin continuity by fluorescence microscopy. Cytomix treatment reduced total MVEC VE-cadherin density, disrupted surface VE-cadherin continuity, was associated with intercellular gap formation, and enhanced paracellular avidin leak. Cytomix-induced MVEC paracellular avidin leak was strongly correlated temporally and was highly contiguous with focal MVEC surface VE-cadherin disruption. Total cellular VE-cadherin density was less strongly correlated with MVEC paracellular avidin leak and individual cell-specific focal surface VE-cadherin discontinuity. These data support a mechanistic link between septic human lung MVEC VE-cadherin disruption and contiguous paracellular protein leak, and will permit more detailed assessment of individual cell-specific mechanisms of septic MVEC barrier dysfunction.