PKC-dependent, burn-induced adherens junction reorganization and barrier dysfunction in pulmonary microvascular endothelial cells

Abstract

-Rat lung microvascular endothelial cell monolayers were exposed to donor plasma from burned rats (25% total body surface area) at 1:3 dilution for 30 min. Immunofluorescence analysis revealed that concomitant with gap formation alterations were seen in the adherens junction (AJ) proteins ␤-catenin and vascular endothelial-cadherin. Both of these components were shown to exist in a smooth, uniform arrangement at the cell periphery in untreated cells. However, upon exposure to burn plasma, this uniformity was lost, and the AJ proteins showed a disrupted, zipper-like pattern at the cells' edge. In addition, these proteins were absent from areas of gap formation between the cells, and an increase in punctate staining throughout the cells suggests they were internalized in response to burn plasma. Measurements of both transendothelial electrical resistance and FITC-albumin flux across the cell monolayer were used to assess barrier integrity. Our study found that exposure to burn plasma rapidly caused the electrical resistance across confluent monolayers to decrease and albumin flux to increase, phenomena associated with barrier dysfunction. Furthermore, all the above responses to burn plasma were attenuated when cells were pretreated with the PKC inhibitor bisindolylmaleimide, suggesting that PKC is required for burn-induced pulmonary endothelial dysfunction.protein kinase C; cadherin; ␤-catenin; serine phosphorylation THE WALL OF MICROVASCULAR exchange vessels is composed of endothelial cells that connect to each other via closely opposed intercellular junctions. These junctions maintain the semipermeable property of the endothelial barrier and control the transvascular passage of solutes, fluid, and blood cells. The adherens junctions (AJ; formed by transmembrane adhesive proteins called cadherins) are important complexes involved in the regulation of paracellular permeability in microvascular endothelium. Vascular endothelial (VE)-cadherin is associated with the actin cytoskeleton through a family of proteins called catenins, including ␣-catenin, ␤-catenin, and plakoglobin (11,13). Whereas the AJ guard against macromolecular leakage by holding the cells together, the contractile forces generated by actomyosin interaction tend to pull the tightly connected endothelial cells apart. The balance between these two forces maintains the endothelial monolayer in a semipermeable status, and disruption of this equilibrium can lead to barrier dysfunction.Endothelial permeability is affected by many agonists including thrombin, histamine, phorbol esters, growth factors, and polymorphonuclear leukocytes (1, 3-5, 24, 25). Characteristic phenomena resulting from endothelial cell exposure to various agonists are phosphorylation of myosin light chain (MLC), actomyosin contractile forces, and modification/reorganization of the AJ (23-25, 27). We have shown that activated neutrophils induce tyrosine phosphorylation of VE-cadherin and ␤-catenin followed by gap formation and hyperpermeability (25). Furthermore, we found that applicat...

Counter Regulatory Effects of PKC βII and PKC δ on Coronary Endothelial Permeability

“…The endothelial barrier property was determined by measuring TER as described previously 15,16 (see supplemental materials, available online at http://atvb.ahajournals.org, for details).…”

“…4,19,20 Phosphorylation of intercellular junctional structures is a key molecular event underlying vascular leakage in inflammation. For certain molecules known to participate in cell contraction or adhesion and thus affect permeability (eg, myosin light chain, catenins, nitric oxide synthase, and MAP kinases 16,[21][22][23] ), serine or threonine phosphorylation are required for their activation and downstream targeting. As potent serine/threonine kinases, PKCs are capable of signal transduction in the endothelium that ultimately affects the barrier property.…”

“…In support of this hypothesis, we have previously reported that phosphorylation of junctional molecules contributes to paracellular hyperpermeability. 16,21 Other studies also show PKC-dependent disruption of junctional integrity coupled with altered VE-cadherin structure 33,34 or VE-cadherin/␤-catenin association. 35 In contrast to the ␤isoform, PKC␦ was concentrated in the cytosol and did not change its location on activation.…”

A role for endothelial-derived matrix metalloproteinase-2 in breast cancer cell transmigration across the endothelial-basement membrane barrier

“…Co-culture and transendothelial migration assay HLMVEC (10 5 ) were seeded on the top chamber of the aforementioned transwell apparatus and grown to confluence as previously described [17,18]. The medium was replaced on a daily basis.…”

Role of src-suppressed C kinase substrate in rat pulmonary microvascular endothelial hyperpermeability stimulated by inflammatory cytokines

“…Samples collected from sides of the filter were analyzed by a fluorospectrophotometer (WFY-28, Tuopu; Tianjin, China) to reckon FITC-labeled dextran concentrations of both chambers. The permeability coefficient (P d, cm/s), based on the flux of FITC-labeled dextran across the monolayer, was calculated according to the following equation: [22]. Values of P d were all reported as the percentage of the basal P d obtained by dividing the P d values of each treated monolayer by the P d values of the control monolayer at 48 h post-seeding.…”