Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability.

We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.

Evidence for cellular heterogeneity in primary cultures of human orbital fibroblasts.

Orbital fibroblasts in culture display phenotypic attributes that distinguish them from fibroblasts derived from other anatomical regions. The current studies were conducted to define potential cellular heterogeneity among orbital fibroblasts with regard to 1) differential expression of Thy-1, a 25-kilodalton glycoprotein associated with cell signaling; 2) cells undergoing a change in shape in response to prostaglandin E2 (PGE2); and 3) differences in morphology and Thy-1 expression between single cell-derived clonal fibroblast strains. On the basis of flow cytometric analysis using an anti-Thy-1 monoclonal antibody, 65% of intact orbital fibroblasts expressed surface Thy-1 (n = 5; range, 54-71%). In contrast, greater than 95% of the fibroblasts present in the five dermal strains tested were Thy-1 positive. A total of six strains of orbital fibroblasts were assessed for their shape change response to a 4-h treatment with PGE2 (100 nmol/L). A mean of 37% of the fibroblasts present in each culture responded to PGE2 (range, 22-50%). In contrast, only 1% of dermal fibroblasts exhibited any change in morphology. Three separate clones were generated from a single parent strain of Graves' orbital fibroblasts. These clones consisted of homogeneous appearing cells; however, substantial clone to clone differences in morphology were stably expressed for several population doublings. Thy-1 was expressed uniformly in cells of two clones, whereas the third was Thy-1 negative. Factor VIII and smooth muscle-specific alpha-actin were undetectable in any of the orbital or dermal cultures examined. Thus, Thy-1 expression is uniform in fibroblasts from certain anatomical regions such as the skin and heterogeneous in cells derived from human lung and orbit. These findings suggest that human orbital connective tissue may have a complexity not previously appreciated.

Extracellular matrix hyaluronan is a determinant of the endothelial barrier.

We measured the hydraulic conductivity (Lp) of the extracellular matrix (ECM) obtained after detaching bovine pulmonary microvascular endothelial (BPMVEC) and bovine pulmonary arterial endothelial cell (BPAEC) monolayers from the ECM at different days postseeding. From day 1 to day 5 in culture, the total Lp (i.e., of cell monolayer + ECM) decreased from basal values of 17.1 +/- 4.0 to 8.5 +/- 1.6 x 10(-6) cm.s-1.cmH2O-1 in BPAEC (P < 0.05) and 7.6 +/- 1.1 to 3.7 +/- 0.8 in BPMVEC (P < 0.05), respectively, and on day 5 the total Lp values were lower in BPMVEC than in BPAEC (P < 0.05). On the 5th day, ECM Lp was 55.0 +/- 8.3 in BPAEC and 10.7 +/- 0.9 cm.s-1.cmH2O-1 in BPMVEC (P < 0.05), indicating that the contribution of ECM to the total Lp was greater in BPMVEC than in BPAEC. Treatment of [3H]acetate-labeled ECM with Streptomyces hyaluronidase (HAse; 6 U/ml for 10 min) released sixfold greater radioactivity in BPMVEC compared with untreated BPMVEC controls; a similar treatment of BPAEC did not release detectable radioactivity indicative of a higher hyaluronan content in the BPMVEC ECM. HAse treatment reduced the differences in total Lp between BPMVEC and BPAEC at different days postseeding. Moreover, on the 5th day after seeding, the ECM Lp of BPMVEC increased to a greater extent after HAse treatment than the ECM of BPAEC. These data indicate that the hyaluronan component of the ECM is an important determinant of the endothelial liquid-exchange barrier.

Growth characteristics of pulmonary artery smooth muscle cells from fawn-hooded rats.

We compared the proliferative rates of vascular smooth muscle cells (VSMC) from pulmonary arteries of pulmonary hypertensive fawn-hooded rats (FHR) with VSMC from normotensive Sprague-Dawley rats (SDR). VSMC from FHR grew at increased rates and reached higher densities at all serum concentrations studied (5-20%) than the VSMC from SDR. The VSMC from FHR also responded to epidermal growth factor (EGF) at low serum concentrations, as evidenced by significantly greater DNA synthetic rates, than the control VSMC. The increased growth in these cells could be due to increased number and/or affinity of EGF receptors because of the higher specific binding of 125I-EGF to the VSMC from FHR. The VSMC from FHR and SDR were equally sensitive to the antiproliferative effects of heparin, suggesting that the heparin-sensitive pathways are not altered in the VSMC from FHR. These results suggest that the development of pulmonary hypertension in FHR may be related to the higher proliferative capacity of the pulmonary VSMC, which may be coupled to increased activity of the EGF receptors on these cells.

Overexpression of manganous superoxide dismutase (MnSOD) in pulmonary endothelial cells confers resistance to hyperoxia.

Treatment of cells or organisms with agents that increase the expression of MnSOD confers resistance to certain types of oxidative damage. However, since these treatments also affect other cellular systems with antioxidant capabilities, the role of MnSOD remains uncertain. To better determine whether increased MnSOD expression confers increased resistance to oxidant stress, a eukaryotic expression vector harboring a mouse MnSOD cDNA was constructed. Bovine lung microvessel endothelial cells were co-transfected with the MnSOD expression vector and pSV2-neo, which contains the neor gene and provides a dominant selectable marker. Control clones were generated by transfecting the cells with psV2-neo alone. Stably transfected cell lines were selected and cell lines overexpressing MnSOD were confirmed by Northern blotting, immunoblot analysis, and activity gels. The activities of copper/zinc superoxide dismutase, catalase, and glutathione peroxidase were examined, and no increase in activity of any of these enzymes was detected. Cells were exposed to hyperoxic challenge by treatment with 95% O2 and 5% CO2 for 24 h. Viability was assessed by a clonogenic assay. The cell lines that overexpressed MnSOD showed a twofold increase in survival compared to control cells. These results demonstrate a significant resistance to hyperoxia induced oxidative stress in endothelial cells overexpressing MnSOD.

Segmental differentiation of permeability, protein glycosylation, and morphology of cultured bovine lung vascular endothelium.

The barrier function, surface biochemistry, and morphology of confluent monolayers of endothelial cells isolated from different segments of the bovine lung vasculature [microvessels (BLMVEC), vein (BPVEC) and artery (BPAEC)] were grown in culture and compared. A number of common cell surface proteins were identified along with two proteins of 46 and 48 kDa found exclusively on BPVEC. Lectin affinity chromatography revealed multiple glycosylation differences. The lectins, Arachis hypogaea (AHA) and Lycopersicum esculentum (LEA) agglutinins, interacted with several glycoproteins of BLMVEC but not of BPAEC. Bandeiraea simplicifolia (BS-1) and Caragana arborescens (CAA) agglutinins recognized several glycoproteins of BPVEC and BPAEC but not BLMVEC. Permeabilities were much lower for BLMVEC than BPAEC or BPVEC monolayers, with a range of about 16-fold less for sucrose to 2-fold less for albumin. Electron microscopy revealed that BLMVEC have a greater surface density of plasmalemmal vesicles (approximately 4-fold) and more extensively developed intercellular junctions with more focal membrane adhesion sites per junction (approximately 9-fold) than the other cells. We conclude that: i) BLMVEC monolayers form a much more restrictive barrier to molecular transport as a result of the tighter junctional formation; and ii) endothelial surface glycoproteins may be differentially glycosylated depending on their segmental location within the vasculature.

Lipopolysaccharide binding protein and CD14 interaction induces tumor necrosis factor-alpha generation and neutrophil sequestration in lungs after intratracheal endotoxin.

It has been proposed that lipopolysaccharide (LPS) bound to the 60-kD LPS binding protein (LBP) forms an LPS/LBP complex that, in turn, binds to the CD14 receptor on monocytes/macrophages and stimulates the release of cytokines. We examined the role of LBP and CD14 in tumor necrosis factor-alpha (TNF-alpha) production and neutrophil (polymorphonuclear leukocyte [PMN]) sequestration in lungs induced by intratracheal instillation of LPS using rabbit lungs perfused at constant flow with lactated Ringer-albumin solution. LPS alone (Salmonella minnesota, wild type; 20 ng) or in the presence of LBP (500 ng) was injected intratracheally. In some experiments, human PMNs (5 x 10(7)) were added to the perfusate after a 2-hour period of perfusion. Samples of lung perfusate were collected every 30 minutes for 180 minutes when bronchoalveolar lavage was also performed. TNF-alpha concentrations in the perfusate and bronchoalveolar lavage fluid were determined by use of a bioassay with L-929 fibroblasts, and PMN accumulation in lungs was determined by myeloperoxidase assay of lung homogenates. LPS alone did not significantly increase TNF-alpha production or lung PMN accumulation, whereas the LPS/LBP complex increased TNF-alpha concentration in perfusate twofold and PMN accumulation twofold compared with the effect of LPS alone. Intratracheal instillation of anti-CD14 monoclonal antibody MY4 (40 micrograms) with the LPS/LBP complex prevented TNF-alpha release and PMN sequestration, whereas an isotype-matched control monoclonal antibody was ineffective. Therefore, LBP in the airspace enhances the LPS effect on TNF-alpha production via a CD14-dependent pathway, and as a result, CD14 activation can contribute to lung PMN sequestration.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of tumor necrosis factor after pulmonary artery occlusion and reperfusion.

Cytokines may function as mediators of reperfusion tissue injury in lungs. Because the lung contains resident macrophages that can serve as potential sources of cytokines, we examined the possibility that pulmonary artery occlusion by reperfusion is a factor in mediating the release of cytokines. After left lung ischemia induced by a 24-h period of left pulmonary artery occlusion, we observed a transient increase in TNF-alpha concentration in lung effluent in rabbits during the period reperfusion. The peak TNF-alpha levels ranged from 55 to 335 pg/ml, and a mean peak time was at 45 to 60 min after the initiation of reperfusion. The TNF-alpha concentrations then decreased towards baseline. TNF-alpha was detected in control plasma or in plasma from sham-operated animals. Less than 10 pg/ml of endotoxin was detected in any samples. Lung tissue myeloperoxidase content, a measure of neutrophil infiltration, increased progressively during the 2-h reperfusion period. The time course of generation of TNF-alpha preceded the maximal rise in lung tissue myeloperoxidase activity. The data show that lung ischemia/reperfusion results in transient generation of TNF-alpha, which is known to mediate neutrophil sequestration. Neutrophil sequestration and resulting lung injury after reperfusion may be dependent on generation of TNF-alpha at the onset of reperfusion.

Endothelin-1 stimulates DNA synthesis and proliferation of pulmonary artery smooth muscle cells.

Endothelin-1 (ET-1), a 21-amino acid peptide released from the endothelium, elicits a variety of biological effects that include vascular smooth muscle cell (VSMC) contraction, release of secondary mediators, and cell proliferation. The present study was undertaken to examine the proliferative potential of ET-1 toward pulmonary artery VSMC in culture. In the presence of low serum and epidermal growth factor (EGF), ET-1 stimulated marked DNA synthesis and proliferation of VSMC. The contributing factor from serum appeared to be platelet-derived growth factor (PDGF) because the antibody to PDGF eliminated the stimulatory activity. The antibody to EGF also prevented the stimulation, suggesting that both PDGF and EGF are required for the full expression of the VSMC growth-promoting activity of ET-1. A paradoxical aspect of ET-1 effect on VSMC was the ability of ET-1 to inhibit the EGF-stimulated DNA synthesis when the two factors were added together to a high baseline DNA synthetic activity. The inhibition was prevented if ET-1 was added 12-18 h after the addition of EGF or if ET-1 and EGF were added to a protein kinase C-depleted VSMC. The inhibition by ET-1 may be mediated by protein kinase C activation followed by inhibition of EGF binding to its receptor. The results indicate that ET-1 under appropriate conditions can modulate the growth of pulmonary artery VSMC in both positive and negative directions.

Influence of extracellular matrix in tumor necrosis factor-induced increase in endothelial permeability.

We examined the possibility that alterations of the extracellular matrix (ECM) contribute to the tumor necrosis factor-alpha (TNF-alpha)-induced increase in endothelial monolayer permeability. Endothelial permeability to 125I-labeled albumin was determined using bovine pulmonary microvessel endothelial cell (BPMVE) monolayers grown to confluence on microporous (0.8 microns diam) gelatin- and fibronectin-coated polycarbonate filters. Treatment of BPMVE with TNF-alpha (10(2) to 10(4) U/ml for 4-24 h) produced concentration- and time-dependent increases in endothelial permeability that paralleled the changes in morphology from cobblestone to elongated cells and the formation of prominent intercellular gaps and actin stress fibers. We examined the role of ECM in these changes using filters coated with ECM made by the BPMVE. Fresh BPMVE seeded onto filters coated with ECM produced by TNF-alpha-treated BPMVE had two- to threefold higher 125I-albumin permeability values than BPMVE monolayers seeded onto filters coated with ECM from control cells (P < 0.05). BPMVE seeded onto ECM from TNF-alpha-treated BPMVE also developed intercellular gaps and centralized actin filaments characteristic of the TNF-alpha-treated BPMVE. This effect was not attributable to TNF-alpha adsorbed to ECM. Polyacrylamide gel electrophoresis of ECM extracted from BPMVE treated with TNF-alpha showed decreased fibronectin. These findings suggest that the TNF-alpha-induced increase in endothelial permeability involves the loss of fibronectin and remodeling of the ECM. The increase in endothelial permeability may be secondary to decreased endothelial cell-ECM contacts resulting in elongation of cells and formation of intercellular gaps.

Culture and characterization of pulmonary microvascular endothelial cells.

Surface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein, and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins [molecular weight (M(r)) 160-220 and 40 kDa] binding to the Ricinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (M(r) = 342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive to the flux of larger tracer molecules, ovalbumin (M(r) 43 kDa) and albumin (M(r) = 69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic difference in the cultured pulmonary microvessel EC barrier function.

Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function.

We have developed an electrical method to study endothelial cell shape changes in real time in order to examine the mechanisms of alterations in the endothelial barrier function. Endothelial shape changes were quantified by using a monolayer of endothelial cells grown on a small (10(-3) cm2) evaporated gold electrode and measuring the changes in electrical impedance. Bovine pulmonary microvessel endothelial cells and bovine pulmonary artery endothelial cells were used to study the effects of alpha-thrombin on cell-shape dynamics by the impedance measurement. alpha-Thrombin produced a dose-dependent decrease in impedance that occurred within 0.5 min in both cell types, indicative of retraction of endothelial cells and widening of interendothelial junctions because of "rounding up" of the cells. The alpha-thrombin-induced decrease in impedance persisted for approximately 2 hr, after which the value recovered to basal levels. Pretreatment of endothelial cells with the protein kinase C inhibitor, calphostin C, or with 8-bromoadenosine 3',5'-cyclic monophosphate prevented the decreased impedance, suggesting that the endothelial cell change is modulated by activation of second-messenger pathways. The alpha-thrombin-induced decrease in impedance was in agreement with the previously observed increases in transendothelial albumin permeability and evidence of formation of intercellular gaps after alpha-thrombin challenge. The impedance measurement may be a valuable in vitro method for the assessment of mechanisms of decreased endothelial barrier function occurring with inflammatory mediators. Since the rapidly occurring changes in endothelial cell shape in response to mediators such as thrombin are mediated activation of second-messenger pathways, the ability to monitor endothelial cell dynamics in real time may provide insights into the signal-transduction events mediating the increased endothelial permeability.

IL-6 enhances TNF-alpha- and IL-1-induced increase of Mn superoxide dismutase mRNA and O2 tolerance.

Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha (IL-1), and interleukin-6 (IL-6) are multifunctional cytokines produced by a number of cells in response to endotoxin. We have previously demonstrated (M.-F. Tsan, J. E. White, T. A. Santana, and C. Y. Lee. J. Appl. Physiol. 68: 1211-1219, 1990, and M.-F. Tsan, C. Y. Lee, and J. E. White. J. Appl. Physiol. 71: 688-697, 1991) that tracheal insufflation of 5 micrograms of TNF-alpha or 1 microgram of IL-1 markedly protects rats against O2 toxicity and enhances pulmonary Mn superoxide dismutase (Mn SOD) activity. We now report that TNF-alpha and IL-1 at subprotective doses, e.g., 1 and 0.2 micrograms, respectively, act synergistically in protecting rats against O2 toxicity. Likewise, TNF-alpha and IL-1 at 0.005 microgram/ml each act synergistically in enhancing endothelial cell Mn SOD, but not Cu,Zn SOD mRNA levels. IL-6 at 5 or 10 micrograms provides no protective effect in rats against O2 toxicity and at up to 0.5 microgram/ml has no apparent effect on endothelial cell Mn or Cu,Zn SOD mRNA levels. However, IL-6 markedly enhances TNF-alpha- and IL-1-induced increases in Mn SOD mRNA levels and O2 tolerance. These results support an important role of Mn SOD in the protection against O2 toxicity.

Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability.

BACKGROUND: We examined the effects of hydrogen peroxide (H2O2) on endothelial permeability and the possible role of protein kinase C (PKC) activation in mediating the response. EXPERIMENTAL DESIGN: Pulmonary microvessel endothelial cell monolayers were grown to confluency on gelatin- and fibronectin-coated microporous filters. Endothelial permeability was measured by determining the transendothelial clearance rate of [125I]albumin. The monolayers in all cases were challenged for 1 hour with H2O2. In some experiments, the monolayers were preincubated with PKC inhibitors H7 (an isoquinolinylsulphonamide derivative) (0.05 mM) or calphostin C (5 x 10(-6) mM) or with the inactive isoquinolinylsulphonamide analog, HA1004 (0.05 mM), before the H2O2 challenge. RESULTS: Addition of H2O2 (0 to 0.5 mM) to endothelial monolayers in the absence of PKC inhibitors resulted in a concentration-dependent increases in endothelial permeability and the response occurred without LDH release and morphologic evidence of cytolysis. The increase in permeability was significantly reduced by H7 and calphostin C, but not by HA1007. Immunocytochemical localization of PKC indicated that PKC isotype II was abundant in these cells and that it was distributed uniformly in the cytosol. H2O2 induced translocation of PKC to the cell membrane indicating enzyme activation. H7 and calphostin C prevented the H2O2-induced PKC translocation, whereas HA1004 had no effect. Both PKC inhibitors also prevented cell "rounding" and formation of interendothelial gaps, whereas HA1004 was ineffective. CONCLUSIONS: The results indicate that PKC activation is an important determinant of the H2O2-induced increase in endothelial permeability.

Fibrin contact increases endothelial permeability to albumin.

We studied the effects of contact of bovine pulmonary artery endothelial cell monolayers with fibrin on the endothelial barrier function. Fibrin formed by clotting purified fibrinogen (0.5 to 3.0 mg/ml) with alpha-thrombin (1 U/ml) was added to endothelial monolayers and permeability measurements were made after fibrin removal. Fibrin incubation for 3 hours resulted in 2- to 5-fold increases in transendothelial 125I-albumin permeability. Permeability returned to baseline value within 3 hours after fibrin removal. Direct contact with fibrin was necessary for the response, since fibrin separated from the endothelium did not increase permeability. Contact with agarose (2 mg/ml) or fibrinogen (0.5 to 3.0 mg/ml) also did not increase endothelial permeability. Transmission electron microscopic examination indicated normal appearance of interendothelial junctions at a time when albumin permeability was increased and no overt evidence of endothelial injury. Incubation of fibrin with endothelial monolayers at 4 degrees C prevented the increase in albumin permeability. We examined the possibility that increased albumin transcytosis was responsible for fibrin's effect using 14C-sucrose (Mr = 342D), a lipid insoluble tracer. Fibrin increased sucrose flux by 1.5-fold compared to 2- to 5-fold increases in albumin flux. The results indicate that fibrin contact with the endothelial cell increases endothelial permeability. The effect of fibrin may involve activation of temperature-sensitive bulk phase transcytosis of albumin.

Tumor necrosis factor-alpha-mediated decrease in glutathione increases the sensitivity of pulmonary vascular endothelial cells to H2O2.

We examined the effects of tumor necrosis factor-alpha (TNF alpha) stimulation of endothelial cells on the increase in endothelial permeability induced by H2O2. Bovine pulmonary microvascular endothelial cells (BPMVEC) were grown to confluence on a microporous filter and the 125I-albumin clearance rate across the monolayer was determined. Pretreatment with TNF alpha (100 U/ml) for 6 h had no direct effect on transendothelial 125I-albumin permeability. However, TNF alpha pretreatment enhanced the susceptibility of BPMVEC to H2O2; that is, H2O2 (10 microM) alone had no direct effect, whereas H2O2 increased 125I-albumin permeability more than threefold when added to monolayers pretreated for 6 h with TNF alpha. Determination of lactate dehydrogenase release indicated that increased permeability was not due to cytolysis. We measured the intracellular contents of GSH and catalase to determine their possible role in mediating the increased susceptibility to H2O2. TNF alpha treatment (100 U/ml for 6 h) decreased total GSH content and concomitantly increased the oxidized GSH content, but did not alter the cellular catalase activity. The role of GSH was examined by pretreating endothelial cells with 2 mM GSH for 3 h, which produced an 80% increase in intracellular GSH content. GSH repletion inhibited the increased sensitivity of the TNF alpha-treated endothelial cells to H2O2. We tested the effects of xanthine oxidase (XO) inhibition since XO activation may be a source of oxidants responsible for the decrease in cellular GSH content. Pretreatment with 0.5 mM oxypurinol attenuated the synergistic effect of TNF alpha and H2O2 on endothelial permeability. The results indicate that decreased oxidant buffering capacity secondary to TNF alpha-induced reduction in intracellular GSH content mediates the increased susceptibility of endothelial cells to H2O2. This mechanism may contribute to oxidant-dependent vascular endothelial injury in septicemia associated with TNF alpha release.

Mn and Cu/Zn SOD expression in cells from LPS-sensitive and LPS-resistant mice.

We examined the effect of lipopolysaccharide (LPS) treatment on the expression of manganese and copper/zinc superoxide dismutase (MnSOD and Cu/ZnSOD) mRNA and protein in resident peritoneal macrophages and lung endothelial cells derived from LPS-sensitive (LPS-s) and LPS-resistant (LPS-r) mice. Macrophages from both LPS-s and LPS-r mice treated with LPS for 24 h produced increased levels of MnSOD mRNA and protein. In contrast, levels of lung endothelial cell MnSOD mRNA and protein from LPS-s mice were increased by LPS treatment, while no increases in these parameters were observed in endothelial cells from LPS-r mice. Tumor necrosis factor-alpha (TNF alpha) treatment, however, did increase levels of MnSOD mRNA in both LPS-s and LPS-r endothelial cells to an equal extent. Both macrophage and endothelial cell Cu/ZnSOD mRNA and protein levels were not significantly affected by LPS treatment. These results demonstrate that the mutation that affects susceptibility to LPS in LPS-r mice exerts a differential influence on MnSOD inducibility in a cell specific manner.

Lectin binding to gp60 decreases specific albumin binding and transport in pulmonary artery endothelial monolayers.

The effect of albumin binding to cultured bovine pulmonary artery endothelial cell (BPAEC) monolayers on the transendothelial flux of 125I-labelled bovine serum albumin (BSA) was examined to determine its possible role on albumin transcytosis. The transport of 125I-BSA tracer across BPAEC grown on gelatin- and fibronectin-coated filters (0.8 microns pore diam.) was affected by the presence of unlabelled BSA in the medium in that transendothelial 125I-BSA permeability decreased, reaching a 40% reduction at BSA concentrations equal to or greater than 5 mg/ml. BSA binding to BPAEC monolayers was saturated at concentration of 10 mg/ml with an apparent binding affinity of 6 x 10(-7) M. In contrast, gelatin added to the medium altered neither 125I-BSA binding nor transport. Several lectins were tested for their ability to inhibit 125I-BSA binding and transport. One lectin, Ricinus communis (RCA), reduced 125I-BSA binding by 70% and transport by 40%. Other lectins, Ulex europaeus, Triticum vulgare, and Glycine max decreased neither 125I-BSA binding nor transport. The reduction of 125I-BSA transport by RCA was not observed in the presence of saturating levels of BSA, indicating that RCA influenced only the albumin-dependent component of transport. RCA, but not other lectins, precipitated a 60 kDa plasmalemmal glycoprotein from cell lysates of surface radioiodinated BPAEC monolayers. This 60 kDa glycoprotein appears to be the equivalent of gp60 identified previously as an albumin binding glycoprotein in rat microvascular endothelium. In summary, approximately 40% of albumin transport across BPAEC monolayers is dependent on albumin binding. This component of albumin transport is inhibited by 80% by the binding of RCA to gp60. These results suggest that binding of albumin to gp60 on pulmonary artery endothelial cell membrane is a critical determinant of transendothelial albumin flux involving mechanisms such as plasmalemmal vesicular transcytosis.

Regulation of antioxidant enzyme expression in LPS-treated bovine retinal pigment epithelial and corneal endothelial cells.

Retinal pigment epithelial (RPE) and corneal endothelial (CE) cells, because of their locations and functions, are continuously exposed to toxic oxidants. Protection from these toxic materials may be due, in part, to the action of endogenous antioxidant enzymes. We have established the presence of mRNAs that encode antioxidant enzymes in bovine RPE and CE cells and have determined the effect of bacterial lipopolysaccharide (LPS) on their expression. The most striking change in antioxidant enzyme expression is an increase in the level of mitochondrial manganous superoxide dismutase (MnSOD) mRNA in the LPS-treated RPE and CE cells. This increase in mRNA expression is accompanied by a slight increase in MnSOD activity as determined by SOD activity gels.

Thrombin-mediated release of lipids from pulmonary artery endothelial cells promotes neutrophil adherence.

We previously have described the ability of alpha-thrombin (the native procoagulant enzyme) to stimulate adherence of neutrophils to pulmonary artery endothelial cells. In the present study, we observed that conditioned medium factors released by alpha-thrombin (10(-8) M) treatment of cultured ovine pulmonary artery endothelial cells increased neutrophil adherence to naive pulmonary artery endothelial monolayers. This effect was independent of any residual alpha-thrombin present in the medium. In contrast to thrombin-induced neutrophil adherence, adherence of neutrophils mediated by the conditioned medium was not inhibited by the anti-CD18 monoclonal antibody 60.3, indicating a CD18-independent mechanism. The factors generated by the action of alpha-thrombin on endothelial cells also resulted in concentration-dependent neutrophil migration. The neutrophil adherence- and migration-promoting activities were isolated in the ether portion after extraction of the conditioned medium. Chromatographic analysis showed that the active components (which resolved into two peaks by reversed-phase high-performance liquid chromatography) were relatively hydrophilic low molecular weight lipids without phosphorus or amino acids. Reconstitution of these peaks indicated that they mediated neutrophil adhesion and migration responses. The results indicate that lipid factors promoting neutrophil adhesion and migration are generated by the action of thrombin on pulmonary artery endothelial cells. The generation of these factors may contribute to the amplification of the lung inflammatory response after pulmonary intravascular coagulation induced by thrombin.