Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.

We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension. ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. (

SUMMARY. Increased permeability of the pulmonary microvasculature is felt to cause acute noncardiogenic lung edema, and histological studies of edematous lungs show gaps between apparently healthy endothelial cells. To determine whether alterations in endothelial cell cytoskeletons would alter endothelial permeability, we exposed monolayers of pulmonary artery endothelial cells grown on micropore filters to cytochalasin B or D. Cytochalasin exposed monolayers demonstrated a 2-to 3-fold increase in endothelial permeability that was readily reversible by washing the monolayers free of cytochalasins. Parallel phase contrast and fluorescence microscopy demonstrated retraction of cell cytoplasm and disruption of bundles of microfilaments in cytochalasin exposed cells. These changes also were readily reversed after washing the cells free of cytochalasins. To test the relevance of these findings to an in situ microvasculature, we added cytochalasin B to the perfusate of isolated rabbit lungs and observed that cytochalasin B caused a high permeability lung edema. These studies suggest that endothelial cell cytoskeletons may be important determinants of endothelial permeability. (Circ Res 51: 657-661, 1982) ACUTE noncardiogenic lung edema is believed to result from increased permeability of the pulmonary microvasculature (Staub, 1978). Although many animal and ex vivo models of noncardiogenic lung edema have been studied, the mechanism of increased endothelial permeability remains unknown. Several studies of the systemic circulation have suggested that increased endothelial permeability induced by histamine is associated with endothelial cells pulling apart and gaps forming between cells (Majno and Palade, 1961;Majno et al., 1967). Hurley (1982) has provided evidence that similar gaps in the continuity of the endothelium may be important in high permeability lung edema.The permeability of epithelial surfaces has been studied more thoroughly than that of endothelial surfaces, and several studies have suggested a role of microfilaments of the cytoskeleton in regulating epithelial permeability (Meza et al., 1980;Bentzel et al., 1980;Duffey et al., 1981). We hypothesized that the cytoskeleton might also contribute to regulation of endothelial permeability. To test this, we added the microfilament disrupting agents, cytochalasin B and D, to monolayers of endothelial cells grown on micropore filters, and we observed that cytochalasins caused a readily reversible increase in endothelial permeability. The increased permeability was associated with disruption of the microfilament apparatus and formation of gaps between adjacent endothelial cells, whereas the recovery was associated initially with the localization of actin to the junction of adjacent cells and later reorganization into the normal pattern of microfilaments for endothelial cells. To support the relevance of these in vitro findings to an intact microvasculature, we added cytochalasin B to the perfusate of isolated lungs, and we found that cytochalasin B increased ...

To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

IntroductionPyocyanin, a secretory product of Pseudomonas aeruginosa, has the capacity to undergo redox cycling under aerobic conditions with resulting generation of superoxide and hydrogen peroxide. By using spin trapping techniques in conjunction with electron paramagnetic resonance spectrometry (EPR), superoxide was detected during the aerobic reduction of pyocyanin by NADH or porcine endothelial cells. No evidence of hydroxyl radical formation was detected. Chromium oxalate eliminated the EPR spectrum of the superoxide-derived spin adduct resulting from endothelial cell exposure to pyocyanin, suggesting superoxide formation close to the endothelial cell plasma membrane. We have previously reported that iron bound to the P. aeruginosa siderophore pyochelin (ferripyochelin) catalyzes the formation of hydroxyl free radical from superoxide and hydrogen peroxide via the Haber-Weiss reaction. In the present study, spin trap evidence of hydroxyl radical formation was detected when NADH and pyocyanin were allowed to react in the presence of ferripyochelin. Similarly, endothelial cell exposure to pyocyanin and ferripyochelin also resulted in hydroxyl radical production which appeared to occur in close proximity to the cell surface. As assessed by 51Cr release, endothelial cells which were treated with pyocyanin or ferripyochelin alone demonstrated minimal injury. However, endothelial cell exposure to the combination of pyochelin and pyocyanin resulted in 55% specific 51Cr release. Injury was not observed with the substitution of iron-free pyochelin and was diminished by the presence of catalase or dimethyl thiourea. These data suggest the possibility that the P. aeruginosa secretory products pyocyanin and pyochelin may act synergistically via the generation of hydroxyl radical to damage local tissues at sites of pseudomonas infection. (J. Clin. Invest. 1992. 90:2187-2196