Endothelial cells of bovine pulmonary artery lack receptors for C3b and for the Fc portion of immunoglobulin G.

Bovine pulmonary endothelial cells do not possess receptors for the 3b component of complement (C3b) or for the Fc portion of immunoglobulin G. The lack of these receptors may help explain the nonthrombogenic function of endothelial cells. Our findings rule out the possibility that endothelial cells participate in pulmonary immune complex disease through the binding of C3b or Fc fragments.

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.

Analysis of thermal adaptation in the HSL enzyme family.

The recently solved three-dimensional (3D) structures of two thermostable members of the carboxylesterase/lipase HSL family, namely the Alicyclobacillus (formerly Bacillus) acidocaldarius and Archaeoglobus fulgidus carboxylesterases (EST2 and AFEST, respectively) were compared with that of the mesophilic homologous counterpart Brefeldine A esterase from Bacillus subtilis. Since the 3D homology models of other members of the HSL family were also available, we performed a structural alignment with all these sequences. The resulting alignment was used to assess the amino acid "traffic rule" in the HSL family. Quite surprisingly, the data were in very good agreement with those recently reported from two independent groups and based on the comparison of a huge number of homologous sequences from the genus Bacillus, Methanococcus and Deinococcus/Thermus. Taken as a whole, the data point to the statistical meaning of defined amino acid conversions going from psychrophilic to hyperthermophilic sequences. We identified and mapped several such changes onto the EST2 structure and observed that such mutations were localized mostly in loops regions or alpha-helices and were mostly excluded from the active site. A site-directed mutagenesis of two of the identified residues confirmed they were involved in thermal stability.

Generation of neutrophil chemotactic activity by phorbol ester-stimulated calf pulmonary artery endothelial cells.

Chemotactic activity for human polymorphonuclear leukocytes (PMNL) was detected in serum-free conditioned media 1 to 4 hr after monolayers of calf pulmonary artery endothelial cells were pretreated with phorbol myristate acetate (PMA). Chemotactic activity was increased in conditioned media following pretreatment with either PMA or the less lipophilic active phorbol ester, 4-beta-phorbol-12,13-dibutyrate (P(Bu)2) in a dose-dependent manner. Chemotactic activity of conditioned media from PMA-treated endothelial cells was confirmed by checkerboard analysis. The chemotactic activity in conditioned media from PMA-pretreated endothelial cells was completely inhibited by pretreating endothelial cells with either cycloheximide, actinomycin D, or the lipooxygenase inhibitor, diethylcarbamazine. Furthermore, the chemotactic activity was heat-stable, inhibited by trypsin treatment, and present in both aqueous and lipid phases after ether extraction. The data demonstrate that pulmonary artery endothelial cells exposed to active phorbol esters release potent chemotactic factor(s) for PMNL. These findings suggest a role for activators of protein kinase C in mediating endothelial cell release of chemotactic factor(s) that may be important in the directed migration of circulating leukocytes to sites of vascular injury.

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.

Expression of bovine and mouse endothelial cell antioxidant enzymes following TNF-alpha exposure.

Endothelial cells are primary targets for injury by reactive oxygen species. Endothelial catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganous superoxide dismutase (MnSOD) provide potential antioxidant enzymatic defenses against oxidant-induced cellular damage. Previous studies in vivo and in vitro have demonstrated that in certain cell types exposure to oxidants may increase the expression of one or more of these antioxidant enzymes, thus providing greater intracellular potential to withstand oxidant-induced cell stress. To test whether endothelial antioxidant enzyme expression is influenced by similar oxidant-induced stresses in vitro, we have exposed endothelial cells to tumor necrosis factor-alpha (TNF-alpha) and have measured levels of catalase, CuZnSOD and MnSOD mRNA, and protein. Our results demonstrate a selective increase of MnSOD mRNA, with coordinate increases of both MnSOD protein and enzyme activity in endothelial cells treated for 24/h with TNF-alpha. In contrast, levels of catalase and CuZnSOD mRNA and protein remained unchanged in these cells after TNF-alpha treatment. These observations were made in microvessel endothelial cells derived from murine and bovine sources. Our results indicate that TNF-alpha can act specifically to increase enzymatic antioxidant potential in endothelial cells by induction of a particular antioxidant enzyme encoding mRNA species. These data demonstrate the capacity of endothelial cells to mount an antioxidant defense in response to exposure to an inducer of oxidative damage.

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.

Assessment of the efficacy of iodine-131 for thyroid ablation.

It is customary to ablate residual tissue after near-total thyroidectomy for thyroid carcinoma by administering 131I. A recent trend has been to use lower 131I doses. This study was designed to assess the efficacy of thyroid ablation by 1110 MBq of 131I (30 mCi) in patients who had near-total thyroidectomy for papillary, mixed or follicular thyroid carcinoma. Four months after surgery, a whole-body scan was done using 185 MBq (5 mCi) of 131I after withdrawal of L-thyroxine for 5-6 wk. Residual thyroid area was then measured by planimetry of the thyroid scan. Patients received ablation therapy within 5 days after scanning and one or more subsequent scans were performed 6 mo later. Forty-four patients were treated to ablate residual functional thyroid tissue. Of these, 12 (27%) had successful ablation. Total body areas (1.63 +/- 0.16 versus 1.83 +/- 0.30, p < 0.03) and residual thyroid tissue (1.4 +/- 1.4 versus 2.0 +/- 1.2 cm2, p < 0.05) were less in patients with total thyroid ablation while there was a trend for a smaller incidence of associated goiter in those patients (1/12 versus 13/32, p < 0.07). Nine of the 17 (53%) patients with a total body area less than 1.9 m2 and/or with a residual thyroid tissue less than 2.1 cm2 and/or without associated previous associated diffuse or multinodular goiter had a total thyroid ablation, while 3 of the 27 (11%) patients who did not have these characteristics had a successful therapy (p < 0.005). Our data suggest that 1110 MBq (30 mCi) of 131I can achieve total ablation of residual thyroid tissue after near-total thyroidectomy particularly in patients with lower total body area and smaller residual thyroid tissue without associated previous diffuse or multinodular goiter.

Inhibition of human scleral fibroblast proliferation with heparin.

Proliferation of fibroblasts is a serious problem in ocular trauma and surgical wound healing. Depending on the location of the injury, the growth of fibroblasts can lead to different problems. In glaucoma filtering surgery, fibroblast proliferation may contribute to scar tissue formation and premature wound closure. Fibroblastic growth in proliferative vitreoretinopathy may lead to the formation of preretinal membranes, which can contract, causing retinal detachment. In an effort to find a more effective method of inhibiting ocular fibroblast proliferation, we have investigated the effect of heparin, a sulfated polysaccharide, on the proliferation of fibroblasts obtained from the sclera of donor eyes. Heparin inhibits the incorporation of 3H-thymidine in a dose-dependent manner in the presence of fetal bovine serum (FBS). This inhibition is partially reversed by endothelial cell growth factor (ECGF). The heparin antagonist protamine sulfate causes a reversal of heparin inhibition and, in some instances, a significant increase in 3H-thymidine incorporation compared to serum controls. Heparin was equally effective in inhibiting cell proliferation in control and heparin-protamine sulfate-pretreated medium. These results were apparently unrelated to a direct toxic effect on cells, as a Trypan Blue exclusion assay showed no significant difference in viability when heparin treated cells were compared to control cells. Direct cell counts showed that heparin was effective in inhibiting cell proliferation over a long time period, but only if it was reinstilled every 2 days. Heparin treatment shows promise as a method for controlling fibroblast proliferation in the eye.

Temperature-induced denaturation of beta-glycosidase from the archaeon Sulfolobus solfataricus.

The beta-glycosidase isolated from the extreme thermophilic archaeon Sulfolobus solfataricus, grown at 87 degrees C, is a tetrameric protein with a molecular mass of 240 kDa. This enzyme is barely active at 30 degrees C and has optimal activity, over 95 degrees C, at pH 6.5. Its thermal stability was investigated at pH 10.1 and 10.6 by means of functional studies, circular dichroism and differential scanning calorimetry. There was no evidence of thermal activation of the enzyme and the temperature-induced denaturation was irreversible and not well represented by the two-state transition model. A more complex process occurred, involving the dissociation and unfolding of subunits, and subsequent nonspecific association and/or aggregation. Denaturation temperature was around 85 degrees C, depending on protein concentration. The denaturation enthalpy change was between 7,500 and 9,800 kJ.mol-1, depending on the pH. The collapse of the native structure around 85 degrees C was confirmed by circular dichroism measurements and time-dependent activity studies. Finally, preliminary investigations were performed on the recombinant enzyme expressed in Escherichia coli.

Transendothelial albumin flux: evidence against asymmetric transport.

We examined the recent proposition (Circ. Res. 57: 903-905, 1985) that the interstitium-to-luminal transport of albumin is an active phenomenon. Studies were made using cultured bovine and sheep pulmonary-artery endothelial cells. The transendothelial 125I-albumin flux from the luminal-to-abluminal side was compared with the flux from the abluminal-to-luminal side. The endothelial cells were grown to confluence on gelatinized-polycarbonated filters separating the abluminal from the luminal compartments. The albumin concentration in each compartment was 1 g/100 ml to equalize the oncotic pressure gradients. The effect of hydrostatic pressure was eliminated by maintaining equal fluid levels in both compartments. The transendothelial albumin flux across the monolayer was measured by adding the 125I-albumin tracer either on the luminal or the abluminal side. A double-isotope method was also used to study bidirectional transendothelial flux of albumin at the same time for the same cultured endothelium. The results indicated that albumin flux from the luminal-to-abluminal side was equal to the flux from the abluminal-to-luminal side. Both bovine and sheep pulmonary artery endothelial cells in culture behave symmetrically for albumin, suggesting that albumin is not actively transported from the interstitium to the lumen.

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability.

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.

Measurement of albumin permeability across endothelial monolayers in vitro.

We have developed an experimental system to measure the permeability of the cultured endothelial monolayer. The luminal-to-abluminal flux of 125I-albumin across cultured pulmonary endothelium was expressed as a clearance rate equal to the permeability-surface area product. After clearance rate measurement for a 30-min base-line period, a test agent was added to the luminal side, and the clearance rate was remeasured during a 30-min experimental period. In control studies the base-line clearance rate was 0.343 +/- 0.017 microliter/min. After correction for the diffusional resistances of the filter and unstirred layers, the calculated permeability of the endothelial monolayer was 1.2 X 10(-5) cm/s. When culture medium was the test agent, the experimental clearance rate was unchanged from the base-line value. After addition of 4 mM oleic acid to the luminal chamber, the clearance rate was 0.528 +/- 0.017 microliter/min compared with a base-line value of 0.330 +/- 0.008 microliter/min (P less than 0.005). This method allows the calculation of endothelial permeability with correction for unstirred layers and the use of each monolayer as its own control.

Evidence for regulation of endothelial plasminogen-activating system by polymorphonuclear leukocyte elastase.

Incubation of unstimulated polymorphonuclear leukocytes with cultured bovine pulmonary artery endothelial cells increased the activity of endothelial plasminogen activator. On the other hand, polymorphonuclear leukocytes stimulated by serum-opsonized zymosan decreased the plasminogen activator activity. A specific elastase inhibitor increased the enhancing effect of the unstimulated polymorphonuclear leukocytes and reversed the suppressing effect of the stimulated polymorphonuclear leukocytes. Catalytically active elastase suppressed the plasminogen activator activity and increased the activity of plasminogen activator inhibitor. In contrast, inactivated elastase enhanced the activity of plasminogen activator. Both, active and inactive forms of elastase bound to the endothelial cells. These findings suggest that elastase modulates the endothelial plasminogen-activating system, apparently by binding to the endothelial cells.

Hydration enthalpy of model peptides: N-acetyl amino acid amides.

Determination of hydration parameters for the solute-solvent interactions of model peptide molecules can provide quantitative information on the factors affecting the folding and stability of proteins in aqueous solutions. Standard hydration enthalpies are calculated by combination of the standard sublimation and solution enthalpy data, experimentally determined. The results for some N-acetyl amino acid amides, assumed as model for peptides, are reported and the trend of hydration enthalpies with increasing complexity of the model molecules is discussed on the basis of the group additivity method. Further the direct proportionality between hydration enthalpy and non-polar accessible surface area (ASA) of each amino acid residue is emphasized. Finally it is pointed out that there exists a convergence temperature for the enthalpy associated with the hydration process of these model compounds and its value TH* = 93 +/- 7 degrees C is close to that found for small globular proteins (i.e. TH* = 100 +/- 6 degrees C). This finding can give some insights to clarify the emergence of convergence behaviour in the unfolding process.

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.

Association of alpha-crystallin with actin in cultured lens cells.

The nature of the beaded filaments in the lens fiber cell has been debated for some time. One explanation is that beaded filaments represent an association of alpha-crystallin with actin filaments. By using a double labelling technique that allowed us to view actin filaments and alpha-crystallin in the same cell we have demonstrated that some of the alpha-crystallin in lens cells is indeed associated with actin.

The liquid amide transfer model and the unfolding thermodynamics of small globular proteins.

In this paper, the solid cyclic dipeptide model developed by Murphy and Gill is analysed in order to point out that, apart from general thermodynamic features shown by well-characterized small globular proteins, only the polar and apolar contributions to the net denaturation heat capacity change are necessary to calculate the so-called protein stability curve, delta dGzero versus temperature. We propose that these specific heat capacity contributions can be determined in a reliable manner by a group additivity analysis of the transfer process of liquid amides from pure liquid phase into water. This suggests that the unfolding process, thought of as the transfer of amino acid residues from the protein 'core' to contact with water molecules, can be modelled based on the transfer process of organic amides. The reliability of the model is tested in comparison with literature data.

The effects of polyols on the thermal stability of calf thymus DNA.

The effects on thermal denaturation of calf thymus DNA (ct-DNA) and its conformational changes induced by the presence in solution of different polyols, namely glycerol, i-erytritol, L( -- ) and D( + ) arabitol, D-mannitol, D-sorbitol and myo-inositol, have been investigated by means of differential scanning calorimetry (DSC) and circular dichroism (CD). By increasing the concentration of these additives a decrease in both the denaturation enthalpy (deltadH) and temperature of the maximum of the denaturation peak (Tmax) of DNA is observed. The values of these thermodynamic parameters depend on both the nature and concentration of the solute. The overall destabilization of DNA molecule has been related to the different capability of polyhydric alcohols to interact with the polynucleotide solvation sites replacing water and to the modification of the electrostatic interactions between the polynucleotide and its surrounding atmosphere of counterions. The particular behaviour of L( -- ) arabitol, which showed a much greater destabilizing ability compared to the other polyols, was further investigated and attributed to a direct more effective interaction with the double helix of DNA. CD spectra showed only a slight alteration of DNA-B structure in the presence of all the molecules here studied, except for L( -- ) arabitol where the DNA molecule seems to undergo a meaningful conformational change. The salt concentration dependence of DNA thermal stability in the presence of L( -- ) arabitol indicates a conformational change of polynucleotide towards a more extended conformation.

Influence of charged groups on the conformational stability of succinoglycan in dilute aqueous solution.

Viscosity, optical activity, and differential scanning calorimetry data clearly point out that partial and/or total removal of charged substituent groups, i.e. succinate and pyruvyl residues, from succinoglycan lead to water soluble derivatives exhibiting a higher stability order----disorder conformational changes with respect to the native polysaccharide. The new succinoglycan derivatives also exhibit very little, if any, hysteresis upon 'renaturation' (cooling) as opposed to the case of the parent polymer. The absence of ionized groups is thus beneficial, thermodynamically and kinetically, to the attainment in dilute aqueous solution of an ordered conformation by the uncharged succinoglycan backbone, as allowed by the regular enchainment of its constituent sugar residues.