Sir James Black and propranolol. The role of the basic sciences in the history of cardiovascular pharmacology

Human endothelial cells are injured by the action of leukocytes. We investigated the role of nitric oxide (NO) in the induction of injury to human pulmonary artery endothelial cells. NO has been a putative source of cytotoxic reactive oxygen species in some settings. Incubation of endothelial cells with neutrophils increased the release of lactate dehydrogenase activity and preloaded fura-2 from endothelial cells, indicating that neutrophils induce endothelial cell injury. This effect was augmented by treatment with carboxy-PTIO, which traps NO in the medium, or with L-NAME, an inhibitor of NO synthase. When endothelial cells were incubated with neutrophils stimulated by phorbol myristate acetate, an activator of protein kinase C, endothelial cell damage was further enhanced and the amount of NO in the medium was decreased. Dibutyryl cyclic AMP, a cell-permeable analogue of cyclic AMP, protected against neutrophil-induced endothelial cell injury and increased NO release into the medium. The effects of dibutyryl cyclic AMP were abrogated by treatment with H-89, a potent inhibitor of cyclic AMP-dependent protein kinase. The protective effect on neutrophil-induced endothelial cell injury by dibutyryl cyclic AMP was abolished by addition of carboxy-PTIO or L-NAME. Thus, our studies suggest that NO, presumably released from endothelial cells, protects against endothelial injury by activated neutrophils and the protective effect by cyclic AMP during coculture with activated neutrophils is mediated through the action of NO. However, when monocytes activated by lipopolysaccharide and IFN-gamma were used instead of neutrophils, endothelial cells were likewise injured, but a much higher level of NO was detected and injury was diminished by addition of carboxy-PTIO to the medium. These observations suggest that the high levels of NO released by activated monocytes contribute to endothelial injury, whereas low levels of NO protect endothelial cells against injury by neutrophils.     

Lecithinized superoxide dismutase attenuates phorbol myristate acetate-induced injury in isolated dog lung

Lecithinized superoxide dismutase, a lecithin derivative bound to recombinant human CuZn superoxide dismutase, has a higher affinity for cells such as polymorphonuclear leukocytes and endothelial cells than recombinant human CuZn superoxide dismutase has. We determined the protective effects of lecithinized superoxide dismutase on the increased microvascular permeability induced by phorbol myristate acetate (PMA) in isolated dog lungs. Microvascular permeability was assessed by the capillary filtration coefficient (Kf,c) and solvent drag reflection coefficient (sigma(f)). PMA (13.3 microg) increased microvascular permeability, as evidenced by an increase in Kf,c and the small sigma(f) value. Lecithinized superoxide dismutase at both low (4800 U) and high doses (48,000 U) inhibited the PMA-induced increase in Kf,c, but only the high dose of lecithinized superoxide dismutase attenuated the decrease in sigma(f). Recombinant human CuZn superoxide dismutase did not affect the PMA-induced increase in vascular permeability at either a low (4800 U) or a high dose (48,000 U). These findings suggest that lecithinized superoxide dismutase has a protective effect against oxygen radical-induced lung injury in isolated dog lungs.     

Effect of antithyroid drugs on hydroxyl radical formation and alpha-1-proteinase inhibitor inactivation by neutrophils: therapeutic implications

The release of proteolytic enzymes and generation of strong oxidants such as the hydroxyl radical by activated neutrophils has been proposed to play an important role in mediating toxin-induced liver injury. The antithyroid drug propylthiouracil protects against liver injury induced by many hepatotoxic agents and markedly reduces mortality in patients with alcoholic liver disease. However, the mechanism(s) by which propylthiouracil protects against liver injury is not well understood. The present studies investigate the effect of antithyroid drugs on proteolytic enzyme activity and on hydroxyl radical generation from activated neutrophils. In the presence of hydrogen peroxide and chloride, neutrophil myeloperoxidase, an enzyme from the same gene superfamily as thyroid peroxidase, generates hypochlorous acid which inactivates alpha-1-proteinase inhibitor (A1PI) present in serum. This inactivation allows neutrophil-released proteolytic enzymes to attack cells. In the present study myeloperoxidase activity was inhibited fully at therapeutic concentrations by antithyroid drugs (propylthiouracil and methimazole). Antithyroid drugs fully prevented hypochlorous acid formation, and prevented neutrophil-mediated inactivation of A1PI, with concomitant blockage of proteolytic activity. Conversely, generation of both superoxide and hydroxyl radicals by activated neutrophils was unaffected by propylthiouracil. The production of these oxygen radicals was fully inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride, however. These studies indicate that antithyroid drugs are unlikely to prevent cell injury by inhibiting hydroxyl radical generation or by scavenging hydroxyl radicals, but are likely to exert their hepatoprotective anti-inflammatory action by inhibiting neutrophil myeloperoxidase, an enzyme akin to thyroid peroxidase.     

Bystander tumoricidal effect and gap junctional communication in lung cancer cell lines

Calcium dobesilate, a vascular protective agent, was tested in vitro for its scavenging action against oxygen free radicals. Calcium dobesilate was as potent as rutin to scavenge hydroxyl radicals (IC50 = 1.1 vs 0.7 microM, respectively). It was also able to scavenge superoxide radicals, but with 23 times less potency than rutin (IC50 = 682 vs 30 microM, respectively). Calcium dobesilate significantly reduced platelet activating factor (PAF)-induced chemiluminescence in human PMN cells and lipid peroxidation by oxygen free radicals in human erythrocyte membranes, although these actions required calcium dobesilate concentrations > or = 50 microM. Finally, in cultured bovine aortic endothelial cells, magnesium dobesilate reduced the increase in cytosolic free calcium induced by hydrogen peroxide and inhibited phenazine methosulfate-induced cell potassium loss. In conclusion, calcium dobesilate was effective in scavenging hydroxyl radicals in vitro, at therapeutically relevant concentrations. Conversely, higher concentrations of the compound were required to scavenge superoxide radicals or to protect the cells against the deleterious effects of intracellular reactive oxygen species. Further studies in vivo are required to determine if these antioxidant properties of calcium dobesilate can play a role in its vascular protective mechanisms.     

Overexpression of human superoxide dismutase inhibits oxidation of low-density lipoprotein by endothelial cells

Oxidation of LDL in the subendothelial space has been proposed to play a key role in atherosclerosis. Endothelial cells produce superoxide anions (O2.-) and oxidize LDL in vitro; however, the role of O2.- in endothelial cell-induced LDL oxidation is unclear. Incubation of human LDL (200 microg/mL) with bovine aortic endothelial cells (BAECs) for 18 hours resulted in a 4-fold increase in LDL oxidation compared with cell-free incubation (22.5+/-1.1 versus 6.3+/-0.2 [mean+/-SEM] nmol malondialdehyde/mg LDL protein, respectively; P<0.05). Under similar conditions, incubation of LDL with porcine aortic endothelial cells resulted in a 5-fold increase in LDL oxidation. Inclusion of exogenous copper/zinc superoxide dismutase (Cu/ZnSOD, 100 microg/mL) in the medium reduced BAEC-induced LDL oxidation by 79%. To determine whether the intracellular SOD content can have a similar protective effect, BAECs were infected with adenoviral vectors containing cDNA for human Cu/ZnSOD (AdCu/ZnSOD) or manganese SOD (AdMnSOD). Adenoviral infection increased the content and activity of either Cu/ZnSOD or MnSOD in the cells and reduced cellular O2.- release by two thirds. When cells infected with AdCu/ZnSOD or AdMnSOD were incubated with LDL, formation of malondialdehyde was decreased by 77% and 32%, respectively. Two other indices of LDL oxidation, formation of conjugated dienes and increased LDL electrophoretic mobility, were similarly reduced by SOD transduction. These data suggest that production of O2.- contributes to endothelial cell-induced oxidation of LDL in vitro. Furthermore, adenovirus-mediated transfer of cDNA for human SOD, particularly Cu/ZnSOD, effectively reduces oxidation of LDL by endothelial cells.     

Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane

BACKGROUND: Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. METHODS: Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. RESULTS: These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma membranes was demonstrated by the subsequent growth and replication of these cells in culture. CONCLUSIONS: These findings indicate that XO is present on the outside surface of the endothelial cell plasma membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.     

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Previous reports have shown that exposure of vascular endothelial and smooth muscle cells to exogenous amyloid beta (Abeta) peptide results in cell damage and toxicity via oxidative injury. In this study we demonstrate that overexpression of the amyloid precursor protein (APP) is toxic to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells. Intracellular coexpression of the free radical scavenger proteins metallothionein or MnSOD abolished the toxic effect of APP overexpression in endothelial cells. Our results demonstrate that endothelial cells are specifically susceptible to intracellular overexpression of APP and free radical generation is the likely mechanism of cell damage due to APP overexpression.     

Use of the Comet assay to investigate the role of superoxide in glutathione-induced DNA damage

Although glutathione is an important scavenging molecule within the cell, it can also act as a pro-oxidant and at biological concentrations (1 mM) can induce DNA damage. We have used a sensitive cell-free Comet assay for DNA strand breakage to investigate this damage and to try to determine the active species involved. We show a substantial protection against glutathione-mediated DNA damage by superoxide dismutase (200 U/ml) and complete protection by combined superoxide dismutase and catalase. Damage is also prevented by EDTA but only at 100 mM and is not prevented by the chelating agent diethylenetriamine-pentaacetic acid (100 microM). Although superoxide is known to potentiate DNA damage by other reactive species, none of these indirect mechanisms seem to account for our results and it is possible that superoxide may damage DNA directly. Under the same experimental conditions, S-nitrosoglutathione requires ultraviolet A photolysis to cause DNA strand breakage and superoxide dismutase increases the level of this damage. When intact human lymphocytes are incubated with glutathione (1 mM) in phosphate buffer, DNA damage is also observed, but in this case it is completely preventable by catalase, with no protective effect of superoxide dismutase. Since cellular scavenging systems are not completely protective against reactive species formed from autooxidation of extracellular glutathione and since glutathione and oxygen are ubiquitously present within cells, our results imply that cells may have a mechanism of preventing autooxidation, rather than simply relying on scavenging the reactive species formula.     

Prevention of mitochondrial injury by manganese superoxide dismutase reveals a primary mechanism for alkaline-induced cell death

Alkalosis is a clinical complication resulting from various pathological and physiological conditions. Although it is well established that reducing the cellular proton concentration is lethal, the mechanism leading to cell death is unknown. Mitochondrial respiration generates a proton gradient and superoxide radicals, suggesting a possible link between oxidative stress, mitochondrial integrity, and alkaline-induced cell death. Manganese superoxide dismutase removes superoxide radicals in mitochondria, and thus protects mitochondria from oxidative injury. Cells cultured under alkaline conditions were found to exhibit elevated levels of mitochondrial membrane potential, reactive oxygen species, and calcium which was accompanied by mitochondrial damage, DNA fragmentation, and cell death. Overexpression of manganese superoxide dismutase reduced the levels of intracellular reactive oxygen species and calcium, restored mitochondrial transmembrane potential, and prevented cell death. The results suggest that mitochondria are the primary target for alkaline-induced cell death and that free radical generation is an important and early event conveying cell death signals under alkaline conditions.     

The role of ferredoxin-NADP+ reductase in the concerted cell defense against oxidative damage -- studies using Escherichia coli mutants and cloned plant genes

Ferredoxin-NADP+ reductases (FNR) participate in cellular defense against oxidative damage. Escherichia coli mutants deficient in FNR are abnormally sensitive to methyl viologen and hydrogen peroxide. Tolerance to these oxidants was regained by expression of plant FNR, superoxide dismutase, or catalase genes in the mutant cells. FNR contribution to the concerted defense against viologen toxicity under redox-cycling conditions was similar to that of the two major E. coli superoxide dismutases together, as judged by the phenotypes displayed by relevant mutant strains. However, FNR expression in sodA sodB strains failed to increase their tolerance to viologens, indicating that the FNR target is not the superoxide radical. Sensitivity of FNR-deficient cells to oxidants is related to extensive DNA damage. Incubation of the mutant bacteria with iron chelators or hydroxyl radical scavengers provided significant protection against viologens or peroxide, suggesting that oxidative injury in FNR-deficient cells was mediated by intracellular iron through the formation of hydroxyl radicals in situ. The NADP(H)-dependent activities of the reductase were necessary and sufficient for detoxification, without participation of either ferredoxin or flavodoxin in the process. Possible mechanisms by which FNR may exert its protective role are discussed.     

Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils

Nitric oxide (.NO) plays a central role in the pathogenesis of diverse inflammatory and infectious disorders. The toxicity of .NO is thought to be engendered, in part, by its reaction with superoxide (O2.-), yielding the potent oxidant peroxynitrite (ONOO-). However, evidence for a role of ONOO- in vivo is based largely upon detection of 3-nitrotyrosine in injured tissues. We have recently demonstrated that nitrite (NO2-), a major end-product of .NO metabolism, readily promotes tyrosine nitration through formation of nitryl chloride (NO2Cl) and nitrogen dioxide (.NO2) by reaction with the inflammatory mediators hypochlorous acid (HOCl) or myeloperoxidase. We now show that activated human polymorphonuclear neutrophils convert NO2- into NO2Cl and .NO2 through myeloperoxidase-dependent pathways. Polymorphonuclear neutrophil-mediated nitration and chlorination of tyrosine residues or 4-hydroxyphenylacetic acid is enhanced by addition of NO2- or by fluxes of .NO. Addition of 15NO2- led to 15N enrichment of nitrated phenolic substrates, confirming its role in polymorphonuclear neutrophil-mediated nitration reactions. Polymorphonuclear neutrophil-mediated inactivation of endothelial cell angiotensin-converting enzyme was exacerbated by NO2-, illustrating the physiological significance of these reaction pathways to cellular dysfunction. Our data reveal that NO2- may regulate inflammatory processes through oxidative mechanisms, perhaps by contributing to the tyrosine nitration and chlorination observed in vivo.     

Purification and partial characterization of an acid proteinase from Dirofilaria immitis

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.     

Inhibitory effect of bifemelane on superoxide generation by activated neutrophils measured using a simple chemiluminescence method

We evaluated the effect of 4-(2-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane hydrochloride) on superoxide production by human neutrophils using an MCLA-dependent chemiluminescence assay. Bifemelane hydrochloride dose-dependently inhibited superoxide production by neutrophils stimulated with phorbol myristate acetate, opsonized zymosan, or N-formyl-methionyl-leucyl-phenylalanine, while it had no effect on superoxide production by a hypoxanthine-xanthine oxidase system. These results indicate that bifemelane hydrochloride does not have a scavenging effect, but has an inhibitory effect on superoxide generation by neutrophils. Although this drug is commonly used for treating chronic cerebral infarction, it may also have a protective effect on acute ischemia/reperfusion injury.     

Reactive oxygen species in reoxygenation injury of rat brain capillary endothelial cells

OBJECTIVE: To clarify the mechanism of anoxia/reoxygenation (A/R) injury of rat brain capillary endothelial cells (BCEC). METHODS: BCEC isolated from Sprague-Dawley rats by enzymatic treatment and centrifugation were subjected to anoxia (95% N2, 5% CO2) for 20 minutes and then to reoxygenation (95% air, 5% CO2) for 3 hours. Enzyme inhibitors, including oxypurinol, indomethacin, and N(G)-nitro-L-arginine methyl ester, or specific free-radical scavengers, such as superoxide dismutase, catalase, and the ferric iron chelator deferoxamine, were added before A/R injury. The BCEC were incubated in a range of Ca2+ concentrations from 1 to 0.01 mmol/L during A/R injury. Cytotoxicity was assayed by release of intracellular lactate dehydrogenase (LDH). RESULTS: With A/R injury, LDH release from the control group (no protective agents) significantly increased (44.8 +/- 3.3%), compared with a small increase in a normoxic group. BCEC treated with oxypurinol, indomethacin, or N(G)-nitro-L-arginine methyl ester showed suppression of LDH release. LDH release was almost totally suppressed by superoxide dismutase and partially by catalase or deferoxamine. The LDH release was partly dependent on calcium concentration. CONCLUSION: BCEC subjected to A/R become potent generators of free radicals, especially superoxide anion. Free radical production depends on both xanthine oxidase and cyclooxygenase pathways. Peroxynitrite and extracellular Ca2+ both contribute importantly to reoxygenation injury of BCEC.     

Dextran sulphates protect porcine arterial endothelial cells from free radical injury

1. The ability of dextran sulphate to protect cultured porcine arterial endothelial cells injured by addition of xanthine and xanthine oxidase (X/XO) or hydrogen peroxide to cell medium was examined using a variety of drug preparations. Cell damage was assessed by determining cell viability (by trypan blue exclusion) and release of lactate dehydrogenase into the medium. 2. Dextran sulphates of average molecular weight (M(r)) 5000, 8000 (hydrogenated or unhydrogenated) at 0.05, 0.5, 5 and 50 micrograms ml-1 medium, added 24 h prior to X/XO, protected cells, whereas dextran sulphate M(r) 500,000 was protective only at 0.5 microgram ml-1. 3. None of the dextran sulphates used showed any toxic effect on cells in concentrations up to 500 micrograms ml-1 medium. 4. When the duration of pretreatment with dextran sulphate M(r) 8000 was varied, 6 h was required for a protective effect on cells damaged by X/XO, which was enhanced with durations of 16 and 24 h. 5. Dextran sulphates had a similar protective effect on cells damaged by hydrogen peroxide. 6. This study suggest that dextran sulphates may prevent conditions resulting from free radical injury.     

Artemia salina as a test organism for measuring superoxide-mediated toxicity

The purpose of this study was to examine the possibility of using Artemia salina as a test organism in the search for compounds having the ability to protect against superoxide-mediated toxicity. The basic procedure for the assay using Artemia salina was performed as described in previous literature, with minor modifications. We found that Artemia salina are extremely sensitive to menadione bisulfite, a compound whose toxicity is probably mediated by intracellular superoxide generation. Desferrioxamine (desferal), a compound with known protective effects, was shown to display dramatic protective activity in our system. We also observed that an inhibitor of endogenous superoxide dismutase (SOD) activity increased the toxicity of menadione toward Artemia salina. In conclusion, this simple, inexpensive, and convenient assay could be a valuable addition to a screening effort in the search for compounds that will be protective against damage by superoxide or other active oxygen species.     

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.     

Thyrotropin receptor expression in adrenal, kidney, and thymus

Inflammatory conditions characterized by neutrophil activation are associated with a variety of chronic diseases. Reactive oxygen species are produced by activated neutrophils and produce DNA damage which may lead to tissue damage. Previous studies have shown that activated murine neutrophils induce DNA strand breaks in a target plasmacytoma cell, RIMPC 2394. We studied the effect of a water soluble nitroxide anti-oxidant, Tempol, on murine neutrophil induction of DNA strand breaks in this system. Murine neutrophils were isolated from the peritoneal cavity of BALB/cAn mice after an i.p. injection of pristane oil. Neutrophils were activated by the phorbol ester PMA and co-incubated with RIMPC 2394 cells. Control alkaline elution studies revealed progressive DNA strand breaks in RIMPC cells with time. The addition of Tempol to the incubation mixture prevented DNA damage in a dose dependent fashion. Five mM Tempol provided complete protection. Tempol protection against DNA strand breaks was similar for both stimulated neutrophils and exogenously added hydrogen peroxide. Measurement of hydrogen peroxide produced by stimulated neutrophils demonstrated that Tempol did not decrease hydrogen peroxide concentration. Oxidation of reduced metals, thereby interfering with the production of hydroxyl radical, is the most likely mechanism of nitroxide protection, although superoxide dismutase (SOD) like activity and scavenging of carbon-based free radicals may also account for a portion of the observed protection. The anti-oxidant activity of Tempol inhibited DNA damage by activated neutrophils. The nitroxides as a class of compounds may have a role in the investigation and modification of inflammatory conditions.