Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.Endothelial cells lining the inner surface of microvessels form a semipermeable barrier that actively participates in blood-tissue exchange of plasma fluid, proteins and cells. The precise regulation of endothelial permeability is essential for maintaining circulatory homeostasis and the physiological function of different organs. As a result, microvascular barrier dysfunction and endothelial hyperpermeability represent crucial events in the development of a variety of disease processes, such as adult respiratory distress syndrome (ARDS), ischemia-reperfusion (I-R) injury, diabetic vascular complications, and tumour metastasis. Better insight into the molecular mechanisms underlying pathogenic conditions related to microvascular hyperpermeability is required for developing effective therapeutic strategies. Following intensive studies over the past few decades, it is now understood that endothelial permeability is mediated through a transcellular pathway (across cells) and a paracellular pathway (between cells), both of which are highly regulated by mechanical forces and biochemical signals. Transcellular versus paracellular permeabilityAn important molecular mechanism underlying transcellular permeability is macromolecule transcytosis via caveoli -specialised plasmalemmal vesicles containing caveolin-1. The involvement of caveolin-1 in regulating cardiovascular functions associated with endothelial barrier properties has been demonstrated through studies using transgenic and knockout NIH Public Access Author ManuscriptExpert Rev Mol Med. Author manuscript; available in PMC 2010 February 24. Published in final edited form as:Expert Rev Mol Med. ; 11: e19. doi:10.1017/S1462399409001112. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript animals (Refs 1,2,3,4). Upon binding to...

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Microvascular barrier dysfunction is implicated in the initiation and progression of inflammation, posttraumatic complications, sepsis, ischaemia-reperfusion injury, atherosclerosis, and diabetes. Under physiological conditions, a precise equilibrium between endothelial cell-cell adhesion and actin-myosin-based centripetal tension tightly controls the semi-permeability of microvascular barriers. Myosin light chain kinase (MLCK) plays an important role in maintaining the equilibrium by phosphorylating myosin light chain (MLC), thereby inducing actomyosin contractility and weakening endothelial cell-cell adhesion. MLCK is activated by numerous physiological factors and inflammatory or angiogenic mediators, causing vascular hyperpermeability. In this review, we discuss experimental evidence supporting the crucial role of MLCK in the hyperpermeability response to key cell signalling events during inflammation. At the cellular level, in vitro studies of cultured endothelial monolayers treated with MLCK inhibitors or transfected with specific inhibiting peptides have demonstrated that induction of endothelial MLCK activity is necessary for hyperpermeability. Ex vivo studies of live microvessels, enabled by development of the isolated, perfused venule method, support the importance of MLCK in endothelial permeability regulation in an environment that more closely resembles in vivo tissues. Finally, the role of MLCK in vascular hyperpermeability has been confirmed with in vivo studies of animal disease models and the use of transgenic MLCK210 knockout mice. These approaches provide a more complete view of the role of MLCK in vascular barrier dysfunction.

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The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which ␤-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junctioncytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on ␤-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that ␤-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for ␤-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, ␤-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to ␤-catenin and VE-cadherin.The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ), 1 tight junctions, gap junctions, and complexus adherentes (1, 2). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including ␣-catenin, ␤-catenin, and plakoglobin (3, 4). The endothelial permeability is affected by many agonists including ␣-thrombin, histamine, and phorbol esters (5-9) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10 -14). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10, 11). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11-14), little is k...