Oxidants increase paracellular permeability in a cultured epithelial cell line.

Inflammation of epithelia is an important step in the pathophysiology of a wide variety of diseases. Because reactive oxygen metabolites are important effector molecules of acute inflammation, we examined the effect of oxidants on the barrier function of a cultured epithelium, Madin Darby Canine Kidney cells, by measuring the transepithelial electrical conductance, Gt, of monolayers grown on permeable supports. We found that H2O2, added directly or generated with glucose oxidase, increased Gt. Similar effects were observed with addition of xanthine and xanthine oxidase, a system which enzymatically generates superoxide radical O2-. The oxidant-induced increase in Gt was reversible if the exposure to oxidants was not prolonged (less than 20 min), and if the concentration of H2O2 was less than 5 X 10(-3) M. The increase in Gt suggested that oxidants increase the permeability of the paracellular pathway, a suggestion supported by an oxidant-induced increase in the permeability to 14C-mannitol, which primarily crosses epithelia via the extracellular route. In addition to functional changes in the epithelial monolayer, oxidants changed the cell morphology; after H2O2 exposure, the cells tended to pull apart, most prominently at their basolateral surfaces. These changes were heterogeneous with most areas showing no changes. Some of the morphologic changes could be reversed if the exposure to H2O2 was limited. We also observed a disruption of the normal pattern of the actin-cytoskeleton, particularly in the area of cell to cell junctions, as demonstrated by fluorescent staining of f-actin with rhodamine phallicidin. These functional and structural findings indicate that oxidants increase the permeability of the paracellular pathway in a cultured epithelium. The changes can be reversible, and are accompanied by alterations in organization of the cell cytoskeleton. These studies demonstrate the dynamic nature of the interaction between epithelial cells and oxygen metabolites.

The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells.

Histamine causes adjacent endothelial cells to retract from each another. We examined phosphorylation of the 20-kD myosin light chain (MLC20) in human umbilical vein endothelial cells (HUVECs) exposed to histamine to determine if we could find evidence to support the hypothesis that retraction of these cells in response to histamine represents an actomyosin-initiated contraction of the endothelial cytoskeleton. We found that MLC20 in HUVECs was constitutively phosphorylated with approximately 0.2 mol phosphate/mol MLC20. Histamine increased MLC20 phosphorylation by 0.18 +/- 0.05 mol phosphate/mol MLC20. This peak increase in phosphorylation occurred 30 s after initiating histamine exposure, persisted through 90s, and returned to control levels by 5 min. Agents that increase HUVEC cAMP prevent cell retraction in response to histamine. An increase in HUVEC cAMP decreased MLC20 phosphorylation by 0.18 +/- 0.02 mol phosphate/mol MLC20 and prevented the increase in MLC20 phosphorylation after exposure to histamine. Tryptic peptide maps of phosphorylated myosin light chain indicated that myosin light chain kinase phosphorylated MLC20 in HUVECs under basal, cAMP-, and histamine-stimulated conditions. Phosphoaminoacid analysis of the monophosphorylated peptide indicated that, in contrast to smooth muscle cells, ser19 and thr18 monophosphorylation occurs in HUVECs. On the basis of our results, modulation of myosin light chain kinase activity may be an important regulatory step in the control of endothelial barrier function.

Interaction of the Pseudomonas aeruginosa secretory products pyocyanin and pyochelin generates hydroxyl radical and causes synergistic damage to endothelial cells. Implications for Pseudomonas-associated tissue injury.

Pyocyanin, 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.

Histamine and thrombin modulate endothelial focal adhesion through centripetal and centrifugal forces.

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.

Differential opening of the brain endothelial barrier following neutralization of the endothelial luminal anionic charge in vitro.

This study was undertaken to determine the effect of neutralization of brain endothelial cell luminal membrane anionic charge on endothelial permeability properties. Mouse brain microvessel endothelial cells were grown to confluence on a nitrocellulose filter. The permeability of the endothelium to Evan's blue dye (EBD) (molecular weight 960) and fluoresceinated dextran (FITC-D) (molecular weight 20,000), both polar molecules, was assessed before and after the exposure of the endothelium to cationic ferritin (CF) or native ferritin (NF). The use of CF resulted in a significant increase in permeability of the endothelium to EBD compared to NF. This result indicates that ablation of endothelial surface anionic charge enhances endothelial transport of a small, polar-charged molecule. Cationic ferritin did not increase the permeability of FITC-D compared to NF. This negative result is not surprising because FITC-D differs from EBD in terms of charge and solubility as well as in size. The electrical resistance of the endothelial cell layer after the application of CF was unchanged from baseline values suggesting a transcellular rather than a paracellular route of the EBD leakage.

Activated endothelial cells in culture suppress baroreceptors in the carotid sinus of dog.

Vascular stretch increases the activity of arterial baroreceptors along with the production and release of substances from the endothelium. We hypothesized that endothelial factors modulate the sensitivity of baroreceptors during increases in arterial pressure. Baroreceptor activity was recorded from single fibers innervating the isolated carotid sinus of dogs anesthetized with chloralose after removal of the endothelium (balloon denudation) and after replacing into the denuded sinus bovine aortic endothelial cells cultured on microcarrier beads. The endothelial cells were activated with either the calcium ionophore A23187 (2 microM) or bradykinin (10 microM). The threshold pressure (n = 7) determined with a slow ramp increase in static pressure averaged 73 +/- 7 (SEM) mm Hg during exposure to naked beads and was increased significantly (96 +/- 18 mm Hg; p less than 0.05) during exposure to endothelial cell cultures. During stepwise increases in pressure, activity (n = 6) averaged 14 +/- 5, 40 +/- 8, and 54 +/- 8 spikes/sec at 75, 125, and 175 mm Hg during exposure to naked beads and decreased significantly to 2 +/- 2, 30 +/- 11, and 35 +/- 12 spikes/sec at equivalent pressures during exposure to the cell cultures. The activity was restored after replacement of the cell cultures with naked beads. The suppressed activity was not caused by changes in carotid sinus diameter or strain (sonomicrometers) or by the chemical activators that were also added to the naked beads. The results indicate that chemically activated endothelial cells release an inhibitory factor that suppresses baroreceptor activity.

Purification of dystrophin-related protein (utrophin) from lung and its identification in pulmonary artery endothelial cells.

Dystrophin-related protein (DRP or utrophin) is an autosomal homologue of dystrophin, a membrane cytoskeletal protein encoded by the Duchenne muscular dystrophy gene. In contrast to dystrophin which is predominantly expressed in muscle, DRP is expressed in various tissues. Here we report the purification and biochemical characterization of DRP from lung which shows the highest levels of DRP expression among adult tissues. DRP was purified from the high alkaline extract of lung membranes using heparin-agarose column chromatography followed by anti-DRP immunoaffinity column chromatography. DRP was expressed in the cultured pulmonary artery endothelial cells. Expression of DRP in endothelial cells could explain its abundant expression in lung. In analogy to dystrophin of muscle cells, DRP could be playing an important role in the mechanical stress mechanisms of endothelial cells.

Pulmonary immune complex deposition in Wegener's granulomatosis.

Two male patients with pulmonary manifestations of Wegener's granulomatosis are presented. One had an elevated rheumatoid factor, and both had elevated levels of immunoglobulin E. Both demonstrated characteristic necrotizing granulomatous lesions on light microscopy of lung tissue. Immunohistologic analysis of lung tissue demonstrated a granular deposition of immunoglobulin G and complement. Raji cell assay of sera demonstrated elevated levels of circulating immune complexes in the sera of the one patient tested prior to any therapy. These findings support the hypothesis that immune complex deposition contributes to the pathogenesis of Wegener's granulomatosis.

Swan-Ganz catheter location and left atrial pressure determine the accuracy of the wedge pressure when positive end-expiratory pressure is used.

In 43 percent of 30 consecutive open heart surgery patients, Swan-Ganz catheter tips lodged within 1 cm of or above the left atrium. When in this position the wedge pressure measured by the catheter was not an accurate estimate of left atrial pressure when positive end-expiratory pressure (PEEP) was used, especially when left atrial pressure was low. Catheters located below the left atrium were accurate at all levels of PEEP tested. The position of Swan-Ganz catheters should be confirmed by a lateral chest roentgenogram when PEEP is used, and catheter tips not below the left atrium should be repositioned.

Effects of calcium on transendothelial albumin transfer and electrical resistance.

Nicolaysen, and more recently Kern and Malik, reported that chelation of calcium increased microvascular hydraulic conductivity and albumin permeability in isolated perfused lungs. To begin to understand how calcium affects endothelial function we examined the effect of calcium chelation on an in vitro endothelium. Chelation of calcium with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid increased the rate of transendothelial albumin transfer by 125%. Reincubation of the endothelium in calcium-repleted medium restored the rate of transfer to its original value. Chelation of extracellular calcium abolished transendothelial electrical resistance. The transendothelial electrical resistance was also restored to normal by reincubation of the endothelium in calcium-repleted medium. Chelation of extracellular calcium caused adjacent endothelial cells to retract from one another, and normal apposition of adjacent cells was restored after reincubation in calcium-repleted medium. Chelation of extracellular calcium produced a centripetal retraction of the peripheral band of actin in individual endothelial cells, and the actin band resumed its normal location after reincubation in calcium-repleted medium. Calcium is an important determinant of endothelial integrity and alterations in calcium produce dynamic changes in endothelial barrier properties and in endothelial-cell shape.

Cationic neutrophil proteins increase transendothelial albumin movement.

Neutrophils play a role in the development of pulmonary edema in many models of the adult respiratory distress syndrome, but the mechanism of their action is not completely understood. We asked whether two neutrophil secretory products, human neutrophil cationic protein (NCP) and human neutrophil elastase (HNE), would nonenzymatically alter the movement of albumin across a cultured endothelial monolayer. Both enzymes were inactivated by heating before use. HNE was additionally enzymatically inactivated with a chloromethylketone oligopeptide (CMK) inhibitor and with alpha 1-proteinase inhibitor (alpha 1-PI). Heated NCP, heated HNE, and CMK-complexed HNE all increased transendothelial albumin transfer. The cation protamine also increased albumin transfer across the endothelium and this increase was blocked by heparin. Alpha 1-PI and fetal bovine serum also prevented the cationic proteins from increasing albumin transfer. Using the release of lactate dehydrogenase as a marker of cytotoxicity, heated HNE was toxic to endothelial cells, heated NCP had only minimal toxicity, and protamine had no toxicity. Changes in endothelial cell shape with gap formation was seen after exposure to both heated HNE and heated NCP. Both the cytotoxicity associated with heated HNE and the cell shape changes associated with heated NCP and heated HNE could be blocked by heparin. These results suggest that in addition to neutrophil proteases and reactive O2 molecules, neutrophil-derived cationic proteins can directly and nonenzymatically contribute to edema formation during acute inflammation.

Polymorphonuclear leukocyte: arachidonate edema.

Polymorphonuclear leukocytes (PMN) are important participants in many models of acute lung edema. Enhanced metabolism of arachidonate is also characteristic of many of these models. We found that PMN and arachidonate, but neither alone, increased alveolar capillary permeability of isolated perfused lungs and increased transfer of albumin across monolayers of endothelial cells cultured on micropore filters. Inhibition of PMN, but not endothelial cyclooxygenase, blunted the edematous process. Neither PMN proteases nor PMN-derived oxidants were involved. The edemagenic activity was not found in supernatants of PMN and arachidonate, and unstable prostaglandins did not alter endothelial albumin transfer. The edemagenic process was not inhibited by blocking leukotriene synthesis, and endothelial albumin transfer was not increased by direct addition of leukotrienes to endothelium. These data demonstrate that PMN and arachidonate can interact to increase endothelial permeability and that PMN cyclooxygenase activity is important for this process. This interaction is of potential significance to the acute inflammatory process in the lung vasculature.

Antioxidant activity of some antiproteases.

Proteolytic enzymes and reactive oxygen molecules participate in the pathogenesis of models of acute inflammation, and antiproteases have been used to determine proteolytic participation in these models. However, antiproteases have been demonstrated to directly inhibit inflammatory cells. In this report, significant antioxidant effects of antiproteases are described. These effects involve both scavenging of oxygen radicals and direct inhibition of enzymes capable of producing oxygen radicals. These results suggest using caution in implicating proteolytic mechanisms solely on the basis of antiprotease inhibition of expected phenomena.