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D M Shasby

Publications and source records attributed to D M Shasby.

At least 55 records · Page 3Linked to original sources
PubMed1987

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.

Antimicrobial Cationic Peptides↗
PubMed1987

Transendothelial transfer of macromolecules in vitro.

The transendothelial transfer of macromolecules has been difficult to study because of the complexities of the in vivo models. We have developed a model of an endothelium cultured on a permeable support and used it to characterize the transendothelial transfer of albumin. Porcine pulmonary artery endothelial cells form a single layer of cells lining the gelatin-impregnated polycarbonate micropore filters, and the cells develop junctional structures similar to endothelial tight junctions observed in vivo. The monolayer resists the flow of electrical current, and the resistance is sensitive to extracellular calcium concentrations. Albumin transfer across the cultured monolayers was found to be asymmetric, and the rate of transfer from interstitium to lumen was greater than that from lumen to interstitium. The asymmetric transfer occurred against a concentration gradient and was abolished by treating the monolayer with NaCN. Increasing albumin concentrations increased the rate of interstitial to luminal transfer, and the process demonstrated saturation at an interstitial albumin concentration of 725 microM. These data point out the usefulness of the in vitro preparation to identify potentially important aspects of transendothelial transport that would be difficult to detect in vivo.

Albumins↗
PubMed1986

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.

Albumins↗
PubMed1985

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.

Animals↗
PubMed1985

Exposure to fatty acid increases human low density lipoprotein transfer across cultured endothelial monolayers.

Human low density lipoproteins radiolabeled with 125I transfer across confluent monolayers of cultured porcine pulmonary artery endothelial cells. The amount transferred was dependent on the low density lipoprotein concentration and was not saturable at concentrations up to 300 micrograms protein per 0.5 ml medium. Gel filtration showed that more than 90% of the 125I which crossed the endothelial monolayer remained associated with low density lipoproteins, indicating that appreciable amounts of lipoprotein were not degraded during the transfer process. When the endothelial monolayer was exposed for 24 hours to culture media supplemented with 100-300 microM fatty acid complexed with 100 microM albumin, the amount of low density lipoprotein subsequently transferred increased by 65% to 150%. The extent of the increase was dependent on the type of fatty acid added and its concentration. At 200 microM, albumin-bound oleic and linoleic acids increased low density lipoprotein transfer, whereas palmitic, linolenic, arachidonic, and eicosapentaenoic acids did not. These results are consistent with the hypothesis that exposure of the endothelium to elevated concentrations of fatty acid may allow excessive amounts of cholesterol-rich lipoproteins to enter the arterial intima.

Animals↗
PubMed1985

Active transendothelial transport of albumin. Interstitium to lumen.

Cultured porcine pulmonary artery endothelial monolayers actively transport albumin from interstitium to lumen. The active process favors interstitial to luminal transport by a factor of 10 even in the face of a 2:1 luminal:interstitial albumin gradient. The active process is abolished by treatment with 1 mM sodium cyanide. Dextran is not actively transported. This process could be important in determining transvascular fluid balance and transfer of macromolecules across the endothelium.

Animals↗
PubMed1985

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.

Antioxidants↗
PubMed1985

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.

Animals↗
PubMed1985

Reversible oxidant-induced increases in albumin transfer across cultured endothelium: alterations in cell shape and calcium homeostasis.

To determine whether reactive oxygen molecules could directly and reversibly increase the transfer of albumin across an endothelial barrier, we measured albumin transfer across monolayers of endothelium cultured on micropore filters before and after exposure to xanthine and xanthine oxidase. Xanthine and xanthine oxidase increased endothelial albumin transfer in a dose-dependent fashion. Parallel phase contrast and fluorescence microscopy demonstrated retraction of adjacent cells from one another and disruption of the actin filaments. The oxidant-induced increases in albumin transfer and changes in cell shape were reversed by removing xanthine oxidase and then incubating the monolayers for 3 1/2 hours in tissue culture media enriched with fetal bovine serum. However, incubation in tissue culture media without serum resulted in progressive injury and cell death. Hence, the brief exposure to oxidants initiated a progressive injury process that was reversed by incubation in serum. Because intracellular and extracellular calcium are important determinants of cell shape, and because some oxidized membrane lipids act as calcium ionophores, we asked whether oxidants altered endothelial calcium homeostasis. Xanthine-xanthine oxidase increased release of 45Ca++ from preloaded cells. The calcium antagonist lanthanum chloride prevented xanthine-xanthine oxidase increases in endothelial albumin transfer and prevented the changes in cell shape; chelation of extracellular calcium inhibited lysis of endothelium by xanthine-xanthine oxidase; and the calcium ionophore A23187 increased endothelial albumin transfer and mimicked the oxidant-induced changes in cell shape. Lanthanum chloride inhibited these effects of A23187. These data suggest that oxygen radicals can reversibly increase endothelial permeability to macromolecules, that this is associated with reversible changes in endothelial cell shape and actin filaments, and that the changes in cell shape are related to oxidant-induced changes in endothelial calcium homeostasis.

Animals↗
PubMed1984

Lung parenchymal injury induced by bleomycin.

Bleomycin, a chemotherapeutic agent used to treat various malignancies, is thought to directly injure lung tissue. Granulocytes, which can also injure lung tissue, are found in large numbers in the initial stages of bleomycin-induced lung injury. An in vitro method, using 3H-uridine-labeled rat lung explants as the target tissue, was used to determine whether bleomycin could directly injure lung tissue. Bleomycin itself injured the explants in a dose-dependent manner. Supernatants of explants exposed to bleomycin exhibited chemotactic activity for granulocytes. The bleomycin-exposed tissues, but not their supernatants, induced granulocytes to produce superoxide anion. These studies demonstrated that bleomycin can directly injure lung tissue at doses approaching peak serum levels in humans, and that granulocytes may be attracted to the lung by a chemotactic factor released by lung tissue injured by bleomycin. These studies suggested that granulocytes may play a significant role in bleomycin-induced lung injury.

Animals↗
PubMed1983

Granulocytes and phorbol myristate acetate increase permeability to albumin of cultured endothelial monolayers and isolated perfused lungs. Role of oxygen radicals and granulocyte adherence.

Human granulocytes and phorbol myristate acetate (PMA) increased permeability to albumin of monolayers of cultured endothelial cells grown on micropore filters. Granulocytes from a patient with chronic granulomatous disease and PMA did not increase endothelial permeability to albumin, demonstrating that the increase in permeability is dependent on granulocyte-derived oxygen radicals. When granulocytes were separated from the endothelial cells by a micropore filter, granulocytes and PMA no longer increased endothelial permeability to albumin, demonstrating that PMA-stimulated granulocytes must be closely approximated to endothelial cells to increase endothelial permeability. The relevance of these in vitro findings to an intact microvasculature was confirmed by demonstrating that agents that reduce granulocyte adherence to endothelium reduce edema formed in isolated lungs by granulocytes and PMA, an oxygen radical dependent process. Pretreatment of granulocytes with cytochalasin B or addition of 2% dextran to isolated lung perfusates reduced granulocyte adherence and markedly reduced edema formation in isolated lungs. These studies demonstrate that PMA-stimulated granulocytes must be closely apposed to endothelial cells to increase endothelial permeability through an oxygen-radical-dependent mechanism, and they suggest that reduction of granulocyte adherence may protect against granulocyte-dependent edema.

Animals↗
PubMed1983

The complement-mediated binding of soluble antibody/dsDNA immune complexes to human neutrophils.

The complement-mediated binding of soluble antibody/3H-dsDNA immune complexes (prepared in vitro) to human polymorphonuclear leukocytes (PMN) has been investigated quantitatively. Studies with isolated complement components in conjunction with experiments on the binding of these complexes to human red blood cells suggest that the binding to both cell types is mediated predominantly by CR1 (C4b-C3b) receptors but that CR3 (iC3b or C3d-g) receptors may play a role in binding to PMN but probably not to RBC. Our results also indicate that under the standard conditions of these assays (37 degrees C, 20 to 40 min incubations) there is no significant internalization of the soluble antibody/dsDNA immune complexes after they are bound by the PMN.

Antigen-Antibody Complex↗
PubMed1982

Reduction of the edema of acute hyperoxic lung injury by granulocyte depletion.

Increased numbers of granulocytes are found in lungs acutely injured by hyperoxia, but their contribution to lung injury remains unknown. We found that circulating granulocytes markedly increased (P less than 0.01) in rabbits exposed to hyperoxia for 72 h and that the numbers of granulocytes in lung lavages also increased and were correlated (r = 0.72, P less than 0.01) with the degree of edematous lung injury. Furthermore, when rabbits were treated with nitrogen mustard (1.75 mg/kg) and developed sustained granulocytopenia, exposure to hyperoxia for 72 h resulted in fewer granulocytes in lung lavages and less edematous lung injury. In contrast, when rabbits were similarly treated with nitrogen mustard but did not maintain sustained granulocytopenia throughout the exposure to hyperoxia, increased numbers of granulocytes were found in lung lavages and the degree of edematous lung injury increased to levels not different from those observed in oxygen-exposed rabbits that had not been treated with nitrogen mustard. These findings suggest that granulocytes may contribute to production of edema in acute oxygen toxicity.

Animals↗
PubMed1982

Role of endothelial cell cytoskeleton in control of endothelial permeability.

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 from 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.

Animals↗
PubMed1982

Granulocytes mediate acute edematous lung injury in rabbits and in isolated rabbit lungs perfused with phorbol myristate acetate: role of oxygen radicals.

Acute edematous lung injury is associated with a marked increase in the number of granulocytes in the alveoli and microvasculature of the lung. Phorbol myristate acetate (PMA) causes granulocytes to adhere, aggregate, and release oxygen radicals and granular enzymes. We found that intravenously injected PMA caused a protein-rich edema in lungs of control rabbits but not in granulocytopenic rabbits pretreated with nitrogen mustard. Specifically, control rabbits treated with PMA had higher lung weight to body weight ratios (6.4 +/- 1.0 X 10(-3)) and lung lavage albumin concentrations (190 +/- 44 mg/dl) than granulocytopenic rabbits pretreated with nitrogen mustard and then given PMA (4.74 +/- 0.23 X 10(-3) and 9.9 +/- 3.8 mg/dl, respectively). To further clarify the role of granulocytes in the production of edema, additional experiments were conducted in an isolated perfused rabbit lung. Addition of purified granulocytes and PMA to the balanced salt perfusate caused lung edema, whereas neither granulocytes nor PMA alone caused edema. Specifically, increases in lung weights (42 +/- 9.2 g) and albumin concentrations (1,182 +/- mg/dl) in lung lavages from isolated lungs exposed to granulocytes and PMA were greater than increases in lung weights or albumin concentrations in lung lavages from isolated lungs exposed to granulocytes alone (2.0 +/- 0.4 g and 15 +/- 0.6 mg/dl), or to PMA alone (6.0 +/- 0.6 g and 81 +/- 34 mg/dl). To determine the contribution of oxygen radicals to the pathogenesis of the edema, chronic granulomatous disease granulocytes, which are deficient in oxygen radical production, were added to the isolated lung perfusate. Chronic granulomatous disease granulocytes and PMA did not cause edema in isolated lungs (delta lung weight 1.0 +/- 0.2 g and lavage albumin 12 +/- 5.0 mg/dl) whereas granulocytes from normal human subjects and PMA did (delta lung weight 43 +/- 5.2 g and lavage albumin 1,120 +/- 54 mg/dl). These data suggest that oxygen radicals released from stimulated granulocytes contribute to the pathogenesis of acute edematous lung injury.

Agranulocytosis↗
PubMed1982

Oxygen-radical-mediated permeability edema and vasoconstriction in isolated perfused rabbit lungs.

Oxygen radicals have been implicated in the pathogenesis of permeability pulmonary edema. To determine directly if O2 radicals can cause increased alveolar-capillary membrane (ACM) permeability and low-pressure permeability edema, we chemically produced O2 radicals in the sale perfusates of isolated rabbit lungs. The O2 radicals generated by xanthine oxidase caused protein-rich edema and increases in lung perfusion pressures that were inhibitable by catalase (hydrogen peroxide scavenger) or dimethylthiourea (hydroxyl radical scavenger) but not by superoxide dismutase. To determine the effect of O2 radicals on ACM permeability without interference from increased perfusion pressures, we used papaverine to maintain baseline perfusion pressures during O2 radical exposure and then assessed ACM integrity by evaluating the response of isolated lungs to elevated outflow pressures (10 mmHg for 10 min). Under these conditions, increased ACM permeability manifested by weight gains and lavage albumin accumulations occurred in lungs treated with xanthine oxidase but not in control lungs. We conclude that O2 radicals can cause increased ACM permeability and vasoconstriction in isolated lungs.

Animals↗