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Biomedical subjects

M A Shatos

Publications and source records attributed to M A Shatos.

At least 19 recordsLinked to original sources
PubMed2001

Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro.

PURPOSE: To isolate, culture, and characterize goblet cells from the conjunctiva of rats. METHODS: Conjunctival tissue was surgically removed from Sprague-Dawley rats. Goblet cells were then isolated from the nictitating membrane and fornix using explant cultures. Cells derived from the explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. They were characterized using an enzyme-linked lectin assay (ELLA) with the lectin Ulex europaeus agglutinin-1 (UEA-1), Western blot analysis, PCR, light and electron microscopy, specialized histochemistry and indirect immunofluorescence microscopy. RESULTS: Goblet cells were successfully isolated from conjunctival explants by scraping nongoblet cells from the culture vessel. To date, cultures have been passaged a minimum of three times without the loss of their specific cellular markers. Cells identified as goblet cells fulfilled the following criteria: positive staining for alcian blue/periodic acid Schiff reagent, cytokeratin (CK)-7, the lectins UEA-I and Helix pomatia agglutinin (HPA), MUC5AC, and M(3) muscarinic receptor; detection of MUC5AC mRNA using RT-PCR; and negative staining for CK-4, M(1) muscarinic receptor, and Banderia simplicifolia lectin. The authors also measured, using the ELLA, substantial amounts of UEA-I-detectable high-molecular-weight glycoproteins and MUC5AC released into the medium. CONCLUSIONS: Cultured goblet cells retain many characteristics of goblet cells in vivo and thus may serve as a useful tool in delineating the pathobiology of the ocular surface.

Animals↗
PubMed1999

Effects of alpha-thrombin on superoxide dismutase levels in human cerebral microvascular endothelial cells.

BACKGROUND: Sequelae of traumatic brain injury include generation of oxygen-free radicals and fibrin deposition, which worsen the initial injury. Superoxide dismutases (SODs) scavenge and bind to the free-radical superoxide anion (O2-), potentially defending against oxidative stress. In the present study, we investigated the production of SOD within human cerebral microvascular endothelial (HCME) cells after exposure to alpha-thrombin, hypothesizing that manganese SOD (MnSOD) expression is increased. Our aims were to determine whether alterations in SOD are observed at the mRNA level, to examine whether a particular species is preferentially expressed, and to determine the requirement of the active site of alpha-thrombin. METHODS: HCME cells were characterized and grown to confluence. Control cells and cells exposed to 10 nmol/L alpha-thrombin were harvested for mRNA isolation using reverse transcriptase-polymerase chain reaction. Quantitation of mRNA production determined the levels of copper-zinc SOD and MnSOD. Active site blocked alpha-thrombin was used as a negative control and determined the specificity of the response. RESULTS: The cells in culture were identified as endothelial after fulfilling criteria, such as positive immunocytochemical staining for factor VIII/von Willebrand factor antigen and binding of Ulex europaeus agglutinin-1 lectin. Levels of MnSOD mRNA were elevated at all time points in response to alpha-thrombin, whereas the cytosolic form was undetectable. HCME cells that were exposed to active site-blocked alpha-thrombin produced mRNA levels of MnSOD that were increased above those of controls, but this increase was half that of mRNA levels of MnSOD produced by HCME cells that were exposed to alpha-thrombin. CONCLUSION: Our study showed for the first time that alpha-thrombin partially modulates SOD in HCME cells, causing a preferential increase in MnSOD. Further investigation into secondary brain injury will provide insights into the role of alpha-thrombin in the mechanism of free radical-induced alterations, potentially improving the outcome of patients with head injury.

Cells, Cultured↗
PubMed1999

Biochemical properties potentially rendering human brain microvasculature vulnerable to proteolytic injury associated with the use of fibrinolytic drugs.

BACKGROUND: Determinants of predisposition to intracranial bleeding in response to the administration of thrombolytic drugs have not yet been well characterized. OBJECTIVE: To delineate factors involved, by characterizing susceptibility of human cerebral microvascular endothelium (HCME) to injury associated with inflammatory cytokines, levels of which are typically elevated in blood in patients who have suffered a myocardial infarction or stroke and been treated with thrombolytic drugs. METHODS: Elaboration of fibrinolytic system proteins by HCME exposed either to interleukin-1 beta or to tumor necrosis factor-alpha (TNF) in serum-free medium for 24 h was characterized. Cell-conditioned medium was assayed for tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and plasminogen activator inhibitor type 1--(PAI-1) by enzyme-linked immunosorbent assay. To determine whether the induction of u-PA was mediated by oxygen-centered radicals, the following were added to media: superoxide dismutase (a scavenger of O2-.), catalase (a scavenger of O2-. and H2O2) and dimethylthiourea (a scavenger of OH.). RESULTS: Interleukin-1 beta had no effect upon elaboration of fibrinolytic system proteins by HCME. By contrast, TNF selectively increased elaboration of u-PA. Accumulation of t-PA and PAI-1 remained unchanged. Accumulation of u-PA was inhibited by cycloheximide, implying that there was a requirement for protein synthesis. Dimethylthiourea abolished the increase elaboration of u-PA induced by TNF completely, catalase did so partially, and SOD did not do so at all. CONCLUSION: The propensity of HCME to elaborate u-PA rather than PAI-1 appears to render cerebral microvasculature particularly vulnerable to proteolytic attack in settings in which inflammatory cytokines are elaborated locally or in which their concentrations in blood are elevated.

Brain↗
PubMed1997

Human brain pericytes differentially regulate expression of procoagulant enzyme complexes comprising the extrinsic pathway of blood coagulation.

After vascular injury, pericytes may function in blood coagulation events that lead to thrombin formation due to their subendothelial location in the microvasculature. Pericytes from human cerebral cortex microvessels were isolated and characterized, and their ability to express and regulate procoagulant enzyme complexes was determined. Tissue factor was detected on the cell surface of cultured human brain pericytes by immunocytochemistry and was shown to form a functional complex with factor (F) VIIa to effect both FIX and FX activation. Treatment of pericytes with the calcium ionophore A23187 increased the observed tissue factor activity twofold to fivefold, which was shown to be due to an enhancement of cofactor activity and not the release of endogenous antigen stores. Pericytes also provided the appropriate membrane surface required for the assembly of a functional prothrombinase complex, so that in the presence of FVa and FXa, they effected thrombin formation 50 to 100 times faster than any other cell examined to date. In marked contrast to observations in other cell systems, pericyte expression of prothrombinase activity remained unaltered after treatment with A23187. As has been shown for platelets, the membrane receptor on pericytes for FXa assembly into the prothrombinase complex appears to at least partially consist of the FXa receptor effector cell protease receptor-1. These combined data indicate that pericytes can activate and propagate the coagulant response through the extrinsic pathway and that the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation. These observations support the concept that pericytes may play an important role in regulating coagulation events after cerebrovascular injury.

Blood Coagulation↗
PubMed1997

Human vascular endothelium heterogeneity. A comparative study of cerebral and peripheral cultured vascular endothelial cells.

BACKGROUND AND PURPOSE: Hormones, neurotransmitters, and autacoids play a key role in the regulation of vascular tone as a result of their interaction with the endothelium. The aim of this study was to compare selected properties of three human endothelial cell lines isolated from cerebral pial arteries (PEC) and two peripheral vessels, the superficial temporal (SEC) and omental (OEC) arteries. METHODS: Intracellular free calcium concentration ([Ca2+]i) and receptor protein expression were measured in characterized primary cultures of human endothelial cells. RESULTS: All cell lines labeled positively for factor VIII/von Willebrand factor. Growth rate and constitutive release of endothelin-1, expressed as a function of protein, were both significantly lower in cerebral cells (PEC) than in endothelial cells derived from peripheral vessels. Basal [Ca2+]i measured with the fluorescent calcium indicator fura 2-AM (2 mumol/L) did not differ in either of the three cell lines. Although PEC responded to endothelin-1 (0.1 mumol/L) and vasoactive intestinal peptide (1 mumol/L) by a twofold to threefold increase in [Ca2+]i, OEC were unresponsive to these peptides. Moreover, the calcium response to alpha-thrombin (10 nmol/L) was greater in cerebral (PEC) than in peripheral (SEC, OEC) endothelial cells, while bradykinin (100 nmol/L) increased [Ca2+]i to a similar level in all three cell types. CONCLUSIONS: This study demonstrates that endothelial cells from different sites of the vasculature exhibit different growth rates and vary in their response to agonists.

Adolescent↗
PubMed1996

Suppression of plasminogen activator inhibitor-1 release from human cerebral endothelium by plasminogen activators. A factor potentially predisposing to intracranial bleeding.

BACKGROUND: Intracranial bleeding is the most catastrophic potential complication of treatment with thrombolytic agents. To identify potential factors that may contribute to this problem, we characterized elaboration by human brain endothelial cells of plasminogen activator inhibitor-1 (PAI-1) and measured PAI-1 mRNA levels. METHODS AND RESULTS: When human cerebral microvascular endothelial cells (HCMEC), pial arterial endothelial cells, and middle meningeal arterial endothelial cells were exposed to 10 to 1000 ng/mL recombinant tissue-type plasminogen activator (RTPA), urokinase-type plasminogen activator (UPA), or streptokinase/ plasminogen (37 U streptokinase plus 2 mumol/L plasminogen) for 24 hours, they exhibited concentration-dependent decreases in elaboration of PAI-1 of 65 +/- 3%, 48 +/- 3%, and 59 +/- 8%. UPA and streptokinase/plasminogen elicited decreases of 33 +/- 8% and 35 +/- 4%, respectively, that were specific with respect to the protease agonists as to total protein synthesis and cell type; ie, neither human umbilical vein endothelial cells nor cerebral pericytes exhibited inhibition of PAI-1 elaboration. No decrease in HCMEC PAI-1 elaboration was induced by coagulation factor XB (10 nmol/L). A 2.7 +/- 0.5-fold increase was induced by alpha-thrombin (10 nmol/L). PAI-1 secretion from HCMEC decreased within 4 hours of exposure to 100 ng/mL RTPA. In HCMEC exposed to RTPA for 8 hours, PAI-1 mRNA decreased from 176 +/- 20 to 43 +/- 2.2 pg/microgram RNA. CONCLUSIONS: These results indicate that brain endothelial cells exposed to RTPA exhibit paradoxically diminished elaboration of PAI-1. This property may render brain vasculature vulnerable to attack by serine proteases, thereby predisposing to injury and initiating an underlying subsequent intracerebral hemorrhage in patients given plasminogen activators for treatment of coronary thrombosis.

Aprotinin↗
PubMed1996

The effects of hyperosmolarity on the viability and function of endothelial cells.

Hypertonic solutions have been demonstrated to be efficacious in the treatment of hypovolemic shock. Their continued use when serum osmolarity is elevated may be harmful because they induce cellular dehydration. Because the hyperosmotic tolerance of cells is largely unknown, we determined the effects of increased media osmolarity on in vitro endothelial cell viability and function following periods of normoxia, anoxia, and anoxia with reoxygenation. Bovine aortic endothelial cells were exposed to hypertonic media of 330-570 mOsm/liter for 6-30 hr. Cell viability and function were ascertained utilizing trypan blue exclusion, lactate dehydrogenase (LDH) enzyme release, and cell replating assays. Endothelial cells exposed to media of 460 mOsm/liter demonstrated no significant decrease in the percentage of viable cells (69.81 +/- 6.03 vs 70.64 +/- 4.62% for controls), LDH activity (334.67 +/- 7.91 vs 228.03 +/- 191.28 Berger-Broida U/ml), and replating efficiency (58.27 +/- 42.07 vs 59.10 +/- 5.79%) after 30 hr of normoxic incubation. Hypertonic media up to 570 mOsm/liter did not adversely affect cell viability following a 6-hr anoxic insult. A 6-hr anoxic insult followed by 24 hr of reoxygenation in media of 530 and 570 mOsm/liter resulted in significantly increased viability and replating efficiency compared to 30 hr of normoxia. Our data demonstrate that in vitro endothelial cells tolerate media osmolarity of up to 460 mOsm/liter without apparent decrement in viability or replating efficiency even in adverse conditions of anoxia and reoxygenation. Our data also suggest that exposure to anoxia may induce tolerance of endothelial cells to hyperosmotic media.

Animals↗
PubMed1996

alpha-Thrombin upregulates G alpha i3 in human vascular endothelial cells.

BACKGROUND AND PURPOSE: During thrombosis, alpha-thrombin becomes sequestered by fibrin and the subendothelial basement membrane, and it is available to interact with the vasculature over prolonged periods. In this study, we investigated the long-term effect of alpha-thrombin on G alpha i3 and G alpha s levels in human vascular endothelial cells (EC). Because obesity is associated with changes in receptor signaling in many animal models, we also explored the influence of this clinical risk factor. METHODS: Primary cultures of human EC were exposed to alpha-thrombin for 16 hours, and immunologically detectable G alpha i3 and G alpha s levels were measured. RESULTS: alpha-Thrombin (100 nmol/L) increased G alpha i3 levels in EC derived from the cerebral microvasculature and superficial temporal artery (4.2 +/- 1.2-fold and 2.8 +/- 0.32-fold, respectively) but had no significant effect on EC derived from omental artery (P > .6) or from the superficial temporal artery of obese (body mass index > or = 28 kg/m2) patients (P > .4). The expression of G alpha s was unchanged in all cell types (P > or = .1). Two other circulating peptides, vasoactive intestinal peptide and endothelin-1, failed to alter the expression of either G protein in EC from the cerebral microvasculature, further demonstrating the specificity of the alpha-thrombin effect. However, thrombin receptor activating protein-14 mimicked the alpha-thrombin response and increased G alpha i3 in EC derived from the cerebral microvasculature and superficial temporal artery. CONCLUSIONS: We conclude that alpha-thrombin increases G alpha i3 expression in some EC through activation of its tethered liganded receptor. Obesity appears to suppress this action of alpha-thrombin.

Adult↗
PubMed1995

Alpha-thrombin stimulates urokinase production and DNA synthesis in cultured human cerebral microvascular endothelial cells.

alpha-Thrombin regulation of endothelial cell (EC) fibrinolysis has been documented by using endothelia derived from a number of anatomic locations but not with those derived from the human cerebral vasculature. In the present study, the fibrinolytic properties of human cerebral microvascular ECs and their regulation by alpha-thrombin are delineated and contrasted with those of human umbilical vein and foreskin microvascular ECs. In cerebral ECs, alpha-thrombin elicited a unique dose-dependent increase in urokinase production and DNA synthesis. Maximal stimulation, observed with 10 nmol/L alpha-thrombin, resulted in a 30- to 50-fold increase in urokinase production and a concomitant fourfold increase in DNA synthesis; the increase in urokinase was reflected in higher steady-state levels of urokinase mRNA. The major urokinase product secreted is the single-chain form of the enzyme. No effect was observed with the addition of other proteases or catalytically inactive variants of alpha-thrombin. A thrombin receptor agonist peptide upregulated urokinase production but had no effect on DNA synthesis, suggesting that fibrinolysis is mediated by the thrombin receptor but that proliferation is regulated by a different pathway. These findings suggest the possibility that the cerebral microvasculature may be a specialized region of the vascular system in which urokinase-type plasminogen activator, not tissue-type plasminogen activator, is the key catalyst of fibrin lysis when the brain responds to thrombotic events and that alpha-thrombin may regulate repair of the cerebral microvascular system.

Blotting, Northern↗
PubMed1994

Sublethal percussion trauma in vitro causes a persisting derangement in the nonthrombogenic properties of brain endothelial cells.

The delivery of a blow to the head represents a transfer of energy, part of which manifests itself as a short-lived pressure change within the skull. An in vitro model was developed to test whether cerebral endothelial cell hemostatic function is altered with exposure to this type of pressure event. Human cerebral microvascular endothelium (HCME) cells were subjected to rapid (2-5 msec) changes in pressure (delta atmosphere = 1.2-10), the sublethal range defined (delta atmosphere < or = 6.5), and the nonthrombogenic status of sublethally percussed HCME cells assessed using the adherence of alpha-thrombin activated platelets as an indicator. The HCME cells had lost their normal capacity to suppress adherence of activated platelets when evaluated 1 hour or 24 hours after percussion. Adherence of activated platelets to percussed HCME cells was blocked by the addition of PGI2, an inhibitor of platelet adherence, when evaluated at 1 hour but not 24 hours after percussion, indicating that percussed HCME cells were undergoing further derangement of their nonthrombogenic mechanisms. Percussed HCME cells cultured for 24 hours in medium containing scavengers of oxygen free radicals recovered their capacity to block platelet adherence. We conclude that sublethal percussion immediately compromises the nonthrombogenic character of HCME cells and initiates the development of a persisting prothrombotic state in HCME cells. This derangement appears linked to increased production of reactive oxygen species by percussed HCME cells.

Brain↗
PubMed1993

Herpes simplex virus decreases endothelial cell plasminogen activator inhibitor.

Herpes simplex virus (HSV) infection is histopathologically associated with vascular injury, fibrinoid necrosis and inflammatory cell infiltrates. We have previously shown in vitro that HSV infection of human umbilical vein endothelial cells (HUVEC) promotes a procoagulant phenotype manifest by the induction of tissue factor, the loss of thrombomodulin, and an increase in platelet adhesion. In these studies we examined the effects of HSV infection on HUVEC plasminogen activator inhibitor type 1 (PAI-1) and tissue plasminogen activator (t-PA). HSV infection caused the loss of PAI-1 in the extracellular matrix (ECM) and that released into the supernatant of HUVEC. Both activity and antigen levels of the Serpin inhibitor are diminished as a result of HSV infection. The loss of inhibitor is not secondary to diminished vitronectin (Vn), the primary binding protein of PAI-1 in the ECM, but appears to be secondary to decreased synthesis at the RNA level. Tissue plasminogen activator (t-PA) synthesis is also decreased in endothelial HSV infection. PAI-1 loss may further promote a procoagulant phenotype in HSV infection in vivo.

Cells, Cultured↗
PubMed1992

Modulation of the fibrinolytic response of cultured human vascular endothelium by extracellularly generated oxygen radicals.

Clinical and experimental data indicate that activated oxygen species interfere with vascular endothelial cell function. Here, the impact of extracellular oxidant injury on the fibrinolytic response of cultured human umbilical vein endothelial (HUVE) cells was investigated at the protein and mRNA levels. Xanthine (50 microM) and xanthine oxidase (100 milliunits), which produces the superoxide anion radical (O2-) and hydrogen peroxide (H2O2), was used to sublethally injure HUVE cells. Following a 15-min exposure, washed cells were incubated for up to 24 h in serum-free culture medium. Tissue-type plasminogen activator (t-PA) antigen, plasminogen activator inhibitor-1 (PAI-1) antigen, and PAI-1 activity were determined in 1.25 ml of conditioned medium and t-PA and PAI-1 mRNA in the cell extracts of 2 x 10(6) HUVE cells. Control cells secreted 3.9 +/- 1.3 ng/ml (mean +/- S.D., n = 12) within 24 h. Treatment with xanthine/xanthine oxidase for 15 min induced a 2.8 +/- 0.4-fold increase (n = 12, p less than 0.05) of t-PA antigen secretion after 24 h. The t-PA antigen was recovered predominantly in complex with PAI-1. The oxidant injury caused a 3.0 +/- 0.8-fold increase (n = 9, p less than 0.05) in t-PA mRNA within 2 h. Total protein synthesis was unaltered by xanthine/xanthine oxidase. The oxidant scavengers superoxide dismutase and catalase, in combination, abolished the effect of xanthine/xanthine oxidase on t-PA secretion and t-PA mRNA synthesis. Xanthine/xanthine oxidase treatment of HUVE cells did not affect the PAI-1 secretion in conditioned medium nor the PAI-1 mRNA levels in cell extracts. Thus extracellular oxidant injury induces t-PA but not PAI-1 synthesis in HUVE cells.

Antigens↗
PubMed1991

Development and characterization of a rapid-onset rodent inhalation model of asbestosis for disease prevention.

A short-term inhalation model of asbestosis was developed in rodents to examine possible preventive approaches to lung disease. Fischer 344 (F344) rats were exposed for 10 and 20 days to National Institute of Environmental Health Sciences (NIEHS) crocidolite asbestos while sham controls were exposed to air only. To determine quantitative biochemical indicators of asbestos-induced lung disease, bronchoalveolar lavage (BAL) fluids were analyzed for lactic dehydrogenase (LDH), alkaline phosphatase, angiotensin-converting enzyme (ACE), and protein. Total and differential cell counts were performed on cell pellets from BAL. Lungs from additional rats were processed for histopathology, measurement of hydroxyproline, and autoradiography after injection of rats with 3H-thymidine. Exposure to asbestos for 10 and 20 days caused increases in LDH, alkaline phosphatase, and protein in BAL. In contrast, ACE was undetectable in BAL fluids from sham or asbestos-exposed rats. At both time periods, the percentages of polymorphonuclear leukocytes (PMNs) and lymphocytes in BAL were increased in asbestos-exposed rats. Total cell numbers in BAL were increased significantly at 20 days in animals inhaling asbestos. Exposure to asbestos for 10 and 20 days caused elevated amounts of hydroxyproline in lung and the development of fibrotic lesions. Asbestos-exposed rats exhibited increased numbers of interstitial cells and airspace epithelial cells incorporating 3H-thymidine, whereas labeled bronchiolar epithelial cells were not elevated significantly. The quantitative changes in asbestos-associated enzyme levels, cell types and protein in BAL, as well as increases in hydroxyproline and morphologic evidence of fibrosis, are useful indices of asbestos-related lung injury which enable preventive and therapeutic approaches to disease.

Administration, Inhalation↗
PubMed1990

Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture.

The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.

Aerobiosis↗
PubMed1990

Cross-tolerance to hyperoxia following cadmium aerosol pretreatment.

Preexposure of male Lewis rats to Cd aerosols (1.6 mg Cd/m3, 3 hr/day, 5 days/week, for 4 weeks) has been found to produce a marked degree of tolerance to hyperoxia (greater than 96% O2). Cd-pretreated animals were still alive after 8 days of continuous exposure to oxygen. In contrast, hyperoxia was fatal to all air-preexposed animals within 54-62 hr. Lungs of Cd-pretreated animals were characterized by hyperplasia and/or hypertrophy of the type II alveolar cell compartment which may have enabled them to more rapidly repair oxidant damage resulting from hyperoxia. Cd pretreatment augmented enzymatic antioxidant enzyme activities, including total lung Se-dependent glutathione peroxidase, catalase, glutathione reductase, and Mn-superoxide dismutase, and caused elevations in pulmonary nonprotein thiols and metallothionein (MT). MT, a thiol-rich, low-molecular-weight protein, was 400-fold higher in Cd-pretreated animals and bound more than 80% of the total Cd in the lung. We have hypothesized that MT serves as an expendable yet renewable cellular target for free radical damage during oxygen exposure. A systemic acute-phase response, characterized by alterations in plasma Zn and Cu concentrations and increased ceruloplasmin oxidase activity, was initiated in Cd-pretreated animals by the fourth day of hyperoxia. This response was accompanied by improvement in pulmonary status and extensive pulmonary repair.

Aerosols↗
PubMed1990

Inhibition of lung injury, inflammation, and interstitial pulmonary fibrosis by polyethylene glycol-conjugated catalase in a rapid inhalation model of asbestosis.

Several in vitro studies suggest the involvement of active oxygen metabolites in cell damage caused by asbestos. To determine if lung injury, inflammation, and asbestosis could be inhibited in vivo in a rapid-onset, inhalation model of disease, a novel method of chronic administration of antioxidant enzymes was developed. In brief, Fischer 344 rats were treated with polyethylene glycol-conjugated (PEG-) superoxide dismutase or catalase in osmotic pumps over a 10-day (5 days/wk for 2 wk) or 20-day (5 days/wk for 2 wk) period of exposure to crocidolite asbestos. Control rats included sham-exposed animals and those exposed to asbestos but receiving chemically inactivated enzymes. After 10 days of exposure to asbestos, lactic dehydrogenase (LDH), alkaline phosphatase, and total protein in bronchoalveolar lavage (BAL) were measured in one group of rats. Total and differnetial cell counts in BAL also were assessed. After 20 days of exposure, lungs of an additional group of rats were evaluated by histopathology and by measurement of hydroxyproline. Asbestos-associated elevations in LDH, protein, and total cell numbers in BAL were reduced in rats receiving PEG-catalase. Decreases in numbers of alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes occurred in these animals. Exposure to asbestos for 20 days caused significant increases in both the amount of hydroxyproline in lung and the severity and extent of fibrotic lesions as determined by histopathology. These indicators of asbestosis were inhibited in a dosage-dependent fashion in rats receiving PEG-catalase. Use of inactivated PEG-catalase failed to boost serum levels of catalase and did not inhibit asbestos-induced elevation of hydroxyproline in lung.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Prevention of asbestos-induced cell death in rat lung fibroblasts and alveolar macrophages by scavengers of active oxygen species.

Cell injury and inflammation caused by asbestos are critical to the pathogenesis of pulmonary fibrosis (asbestosis). Our goal in studies here was to investigate the possible modulation of asbestos-related cell death using antioxidants in both target and effector cells of asbestosis. After exposure to crocidolite asbestos at a range of concentrations (2.5-25 micrograms/cm2 dish), the viability of a normal rat lung fibroblast line (RL-82) and freshly isolated alveolar macrophages (AM) was determined by exclusion of trypan blue and nigrosin, respectively. In comparison to fibroblasts, AM were more resistant to the cytotoxic effects of asbestos. Cytotoxic concentrations of asbestos then were added to both cell types in combination with the antioxidants, superoxide dismutase (SOD), a scavenger of superoxide (O2-.), and catalase, an enzyme scavenging H2O2. Dimethylthiourea (DMTU), a scavenger of the hydroxyl radical (OH.) and deferoxamine, an iron chelator, also were evaluated in similar studies. Results showed significant dosage-dependent reduction (P less than 0.001) of asbestos-associated cell death with all agents. In contrast, asbestos-induced toxicity was not ameliorated after addition of chemically inactivated SOD and catalase or bovine serum albumin. Results above suggest asbestos-induced cell damage is mediated by active oxygen species. In this regard, the iron associated with the fiber and/or its interaction with cell membranes might be critical in driving a modified Haber-Weiss (Fenton-type) reaction resulting in production of OH(.).

Animals↗