The stabilization of endothelial cell (EC) barrier function within newly formed capillaries is a critical feature of angiogenesis. We examined human lung EC barrier regulation elicited by hepatocyte growth factor (HGF), a recognized angiogenic factor and EC chemoattractant. HGF rapidly and dose-dependently elevated transendothelial electrical resistance (TER) of EC monolayers (>50% increase at 100 ng/ml), with immunofluorescence microscopic evidence of both cytoplasmic actin stress fiber dissolution and strong augmentation of the cortical actin ring. HGF rapidly stimulated phosphatidylinositol 3'-kinase, ERK, p38 mitogen-activated protein kinase, and protein kinase C activities. Pharmacological inhibitor studies demonstrated each pathway to be intimately involved in HGF-induced increases in TER, cortical actin thickening, and phosphorylation of the Ser/Thr glycogen synthase kinase-3beta (GSK-3beta), a potential target for the HGF barrier-promoting response. GSK-3beta phosphorylation was strongly correlated with reductions in both HGF-induced TER and enhanced beta-catenin immunoreactivity observed at cell-cell junctions. Our data suggest a model in which HGF-mediated EC cytoskeletal rearrangement and barrier enhancement depend critically on the activation of a complex kinase cascade that converges at GSK-3beta to increase the availability of beta-catenin, thereby enhancing endothelial junctional integrity and vascular barrier function.

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets (ϳ40% maximum, 2.5 ϫ 10 6 platelets/ml). Platelet supernatants, derived from 2.5 ϫ 10 6 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a G i␣-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: [689][690][691][692][693][694][695][696][697][698][699][700][701] 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P. endothelium; lung; vasculature; injury; G protein; differentiation; cell differentiating gene THE VASCULAR ENDOTHELIUM IS a biologically complex tissue that forms a semipermeable barrier between the intravascular fluid compartment and the interstitium of various organs. Integrity of the endothelial cell (EC) monolayer is essential for homeostasis, and perturbations of the barrier function of the endothelium are now recognized as a cardinal feature of diverse and important pathobiological processes including acute lung injury and atherogenesis. The lung vasculature contains an enormous surface area and is particularly sensitive to the dynamic features of endothelial barrier dysregulation, where increased vascular permeability leads to exudation of fluid and solutes from the intravascular space into the pulmonary interstitium. Extensive increases in lung vascular permeability result in flooding of the alveolar air spaces (pulmonary edema), which is the hallmark pathophysiological derangement of the adult respiratory distress syndrome. Circulating blood platelets have been noted for many decades to be essential to the maintenance of the endothelium as a semipermeable barrier. In vitro and in vivo models have described profound defects in EC barrier function after perfusion wit...

NaF, a potent G protein activator and Ser/Thr phosphatase inhibitor, significantly increased albumin permeability and decreased transcellular electrical resistance (TER), indicating endothelial cell (EC) barrier impairment. EC barrier dysfunction induced by NaF was accompanied by the development of actin stress fibers, intercellular gap formation, and significant time-dependent increases in myosin light chain (MLC) phosphorylation. However, despite rapid, albeit transient, activation of Ca(2+)/calmodulin-dependent MLC kinase (MLCK), the specific MLCK inhibitor ML-7 failed to affect NaF-induced MLC phosphorylation, actin cytoskeletal rearrangement, and reductions in TER, suggesting a limited role of MLCK in NaF-induced EC activation. In contrast, strategies to reduce Rho (C3 exoenzyme or toxin B) or to inhibit Rho-associated kinase (Y-27632 or dominant/negative RhoK) dramatically reduced MLC phosphorylation and actin stress fiber formation and significantly attenuated NaF-induced EC barrier dysfunction. Consistent with this role for RhoK activity, NaF selectively inhibited myosin-specific phosphatase activity, whereas the total Ser/Thr phosphatase activity remained unchanged. These data strongly suggest that MLC phosphorylation, mediated primarily by RhoK, and not MLCK, participates in NaF-induced EC actin cytoskeletal changes and barrier dysfunction.