Air pollution upregulates endothelial cell procoagulant activity via ultrafine particle-induced oxidant signaling and tissue factor expression.

Air pollution exposure is associated with cardiovascular events triggered by clot formation. Endothelial activation and initiation of coagulation are pathophysiological mechanisms that could link inhaled air pollutants to vascular events. Here we investigated the underlying mechanisms of increased endothelial cell procoagulant activity following exposure to soluble components of ultrafine particles (soluble UF). Human coronary artery endothelial cells (HCAEC) were exposed to soluble UF and assessed for their ability to trigger procoagulant activity in platelet-free plasma. Exposed HCAEC triggered earlier thrombin generation and faster fibrin clot formation, which was abolished by an anti-tissue factor (TF) antibody, indicating TF-dependent effects. Soluble UF exposure increased TF mRNA expression without compensatory increases in key anticoagulant proteins. To identify early events that regulate TF expression, we measured endothelial H2O2 production following soluble UF exposure and identified the enzymatic source. Soluble UF exposure increased endothelial H2O2 production, and antioxidants attenuated UF-induced upregulation of TF, linking the procoagulant responses to reactive oxygen species (ROS) formation. Chemical inhibitors and RNA silencing showed that NOX-4, an important endothelial source of H2O2, was involved in UF-induced upregulation of TF mRNA. These data indicate that soluble UF exposure induces endothelial cell procoagulant activity, which involves de novo TF synthesis, ROS production, and the NOX-4 enzyme. These findings provide mechanistic insight into the adverse cardiovascular effects associated with air pollution exposure.

Protecting the permeability pore and mitochondrial biogenesis.

Recent evidence links the pathogenesis of multiple organ dysfunction syndrome (MODS) in sepsis to mitochondrial damage. Our hypothesis is that cellular mechanisms maintaining mitochondrial function must be protected in order to prevent MODS. Recent animal experiments indicate that host defences which target and kill microbes, in part via reactive oxygen and nitrogen production, also injure mitochondria, thus activating mitochondrial cell death pathways. To limit such collateral damage, the cell up-regulates and imports into mitochondria several nuclear-encoded proteins for antioxidant defence and mitochondrial DNA (mtDNA) replication. Fully integrated responses lead to mitochondrial biogenesis, which may alter cellular phenotype to avoid mitochondrial permeability transition, apoptosis, or energy failure. Key to the cell's vulnerability to oxidant generation by the innate immune response is the mtDNA content. MtDNA depletion is opposed by oxidation reduction (redox) signals that communicate the extent of mitochondrial damage to the nucleus. Molecular studies suggest that redox mechanisms activate two biogenic transcription factors, nuclear respiratory factors 1 and 2, which forestall a deterioration of oxidative phosphorylation during infection. Biogenic failure or an intrinsic biogenic arrest could hasten degradation of mitochondrial function and drive the cell to apoptosis or necrosis. By implication, novel protective strategies for biogenesis hold promise for the prevention of MODS.

Metabolic capacity regulates iron homeostasis in endothelial cells.

The sensitivity of endothelial cells to oxidative stress and the high concentrations of iron in mitochondria led us to test the hypotheses that (1) changes in respiratory capacity alter iron homeostasis, and (2) lack of aerobic metabolism decreases labile iron stores and attenuates oxidative stress. Two respiration-deficient (rho(o)) endothelial cell lines with selective deletion of mitochondrial DNA (mtDNA) were created by exposing a parent endothelial cell line (EA) to ethidium bromide. Surviving cells were cloned and mtDNA-deficient cell lines were demonstrated to have diminished oxygen consumption. Total cellular and mitochondrial iron levels were measured, and iron uptake and compartmentalization were measured by inductively coupled plasma atomic emission spectroscopy. Iron transport and storage protein expression were analyzed by real-time polymerase chain reaction and Western blot or ELISA, and total and mitochondrial reactive oxygen species (ROS) generation was measured. Mitochondrial iron content was the same in all three cell lines, but both rho(o) lines had lower iron uptake and total cellular iron. Protein and mRNA expressions of major cytosolic iron transport constituents were down-regulated in rho(o) cells, including transferrin receptor, divalent metal transporter-1 (-IRE isoform), and ferritin. The mitochondrial iron-handling protein, frataxin, was also decreased in respiration-deficient cells. The rho(o) cell lines generated less mitochondrial ROS but released more extracellular H(2)O(2), and demonstrated significantly lower levels of lipid aldehyde formation than control cells. In summary, rho(o) cells with a minimal aerobic capacity had decreased iron uptake and storage. This work demonstrates that mitochondria regulate iron homeostasis in endothelial cells.

Oxygen-induced mitochondrial biogenesis in the rat hippocampus.

The hypothesis that damage to mitochondrial DNA by reactive oxygen species increases the activity of nuclear and mitochondrial transcription factors for mitochondrial DNA replication was tested in the in vivo rat brain. Mitochondrial reactive oxygen species generation was stimulated using pre-convulsive doses of hyperbaric oxygen and hippocampal mitochondrial DNA content and neuronal and mitochondrial morphology and cell proliferation were evaluated at 1, 5 and 10 days. Gene expression was subsequently evaluated to assess nuclear and mitochondrial-encoded respiratory genes, mitochondrial transcription factor A, and nuclear respiratory transcription factors-1 and -2. After 1 day, a mitochondrial DNA deletion emerged involving Complex I and IV subunit-encoding regions that was independent of overt neurological or cytological O(2) toxicity, and resolved before the onset of cell proliferation. This damage was attenuated by blockade of neuronal nitric oxide synthase. Compensatory responses were found in nuclear gene expression for manganese superoxide dismutase, mitochondrial transcription factor A, and nuclear respiratory transcription factor-2. Enhanced nuclear respiratory transcription factor-2 binding activity in hippocampus was accompanied by a nearly three-fold boost in mitochondrial DNA content over 5 days. The finding that O(2) activates regional mitochondrial DNA transcription, replication, and mitochondrial biogenesis in the hippocampus may have important implications for maintaining neuronal viability after brain injury.

Tissue factor in experimental acute lung injury.

Acute lung injury (ALI) is characterized by fibrin deposition in the tissue and vascular spaces. Coagulation is activated after exposure to endotoxin or bacteria, and a procoagulant environment rapidly develops in the vascular, interstitial, and alveolar spaces of the lung. These changes are tissue factor (TF)-dependent and associated with increases in inflammatory cytokines. Procoagulant changes also occur in the lungs of patients with the acute respiratory distress syndrome (ARDS), suggesting that epithelial inflammation activates the extrinsic pathway. Many inflammatory mediators have specific effects on coagulation; however, the role of TF in regulation of pulmonary inflammatory responses is less clear. Here we report initial data on blockade of TF-initiated coagulation in baboons with Escherichia coli sepsis-induced ALI, using active site-inactivated FVIIa (FVIIai ASIS). Treatment with FVIIai prevented plasma fibrinogen depletion and attenuated fibrin deposition in the tissues. The drug also decreased systemic cytokine responses and inflammatory changes in the lung, including neutrophil infiltration, and decreased edema. Coagulation blockade with FVIIai improved lung function by preserving gas exchange and compliance, decreased pulmonary hypertension, and enhanced renal function. These results show that TF-FVIIa complex is an important regulatory site for the pathologic response of the lung to sepsis.

Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons.

Sepsis-induced tissue factor (TF) expression activates coagulation in the lung and leads to a procoagulant environment, which results in fibrin deposition and potentiates inflammation. We hypothesized that preventing initiation of coagulation at TF-Factor VIIa (FVIIa) complex would block fibrin deposition and control inflammation in sepsis, thereby limiting acute lung injury (ALI) and other organ damage in baboons. A model of ALI was used in which adult baboons were primed with killed Escherichia coli (1 x 10(9) CFU/kg), and bacteremic sepsis was induced 12 h later by infusion of live E. coli at 1 x 10(10) CFU/kg. Animals in the treatment group were given a competitive inhibitor of TF, site-inactivated FVIIa (FVIIai), intravenously at the time of the infusion of live bacteria and monitored physiologically for another 36 h. FVIIai dramatically protected gas exchange and lung compliance, prevented lung edema and pulmonary hypertension, and preserved renal function relative to vehicle (all p < 0.05). Treatment attenuated sepsis-induced fibrinogen depletion (p < 0.01) and decreased systemic proinflammatory cytokine responses, for example, interleukin 6 (p < 0.01). The protective effects of TF blockade in sepsis-induced ALI were confirmed by using tissue factor pathway inhibitor. The results show that TF-FVIIa complex contributes to organ injury in septic primates in part through selective stimulation of proinflammatory cytokine release and fibrin deposition.

Inhaled carbon monoxide and hyperoxic lung injury in rats.

Because carbon monoxide (CO) has been proposed to have anti-inflammatory properties, we sought protective effects of CO in pulmonary O(2) toxicity, which leads rapidly to lung inflammation and respiratory failure. Based on published studies, we hypothesized that CO protects the lung against O(2) by selectively increasing expression of antioxidant enzymes, thereby decreasing oxidative injury and inflammation. Rats exposed to O(2) with or without CO [50-500 parts/million (ppm)] for 60 h were compared for lung wet-to-dry weight ratio (W/D), pleural fluid volume, myeloperoxidase (MPO) activity, histology, expression of heme oxygenase-1 (HO-1), and manganese superoxide dismutase (Mn SOD) proteins. The brains were evaluated for histological evidence of damage from CO. In O(2)-exposed animals, lung W/D increased from 4.8 in normal rats to 6.3; however, only CO at 200 and 500 ppm decreased W/D significantly (to 5.9) during O(2) exposure. Large volumes of pleural fluid accumulated in all rats, with no significant CO treatment effect. Lung MPO values increased after O(2) and were not attenuated by CO treatment. CO did not enhance lung expression of oxidant-responsive proteins Mn SOD and HO-1. Animals receiving O(2) and CO at 200 or 500 ppm showed significant apoptotic cell death in the cortex and hippocampus by immunochemical staining. Thus significant protection by CO against O(2)-induced lung injury could not be confirmed in rats, even at CO concentrations associated with apoptosis in the brain.

Antibody to intercellular adhesion molecule 1 (CD54) decreases survival and not lung injury in baboons with sepsis.

Neutrophil influx into the lung is an important event in the pathogenesis of acute lung injury in gram-negative sepsis. We hypothesized that administration of a monoclonal antibody to intercellular adhesion molecule 1 (ICAM-1, CD54), a molecule mediating neutrophil adhesion to endothelial cells, would decrease neutrophil sequestration and transmigration in the lung and attenuate lung injury in Escherichia coli sepsis. Sepsis was induced in 12 baboons primed with heat-killed E. coli (1 x 10(9) CFU/kg) 12 h before infusion of live bacteria (1 x 10(10) CFU/kg). Six animals received monoclonal antibody to CD54 (1 mg/kg) intravenously at the time of live E. coli infusion. After 48 h or when blood pressure could not be maintained, tissues were harvested and bronchoalveolar lavage (BAL) samples were obtained. Median survival time was decreased in anti-CD54-treated animals. This group also had decreased mean arterial pressure, increased metabolic acidosis, and decreased urine output. Measures of lung injury including gas exchange, lung lavage protein and lactate dehydrogenase (LDH), lung thiobarbituric acid-reactive species, and lung histology, including alveolar neutrophil volumes, were unaffected by treatment. The effect of anti-CD54 on neutrophil influx into tissues as measured by myeloperoxidase was organ specific. These data show that monoclonal antibody to CD54 does not ameliorate acute lung injury in E. coli sepsis, and septic primates given anti-CD54 have worsened metabolic parameters and decreased survival.

Proinflammatory cytokines increase in sepsis after anti-adhesion molecule therapy.

Cytokine mediators and leukocyte-endothelial cell adhesion molecules are critical and interdependent components of the acute inflammatory response in sepsis. We hypothesized that the administration of monoclonal antibodies to intercellular adhesion molecule-1 (CD54) or E- and L-selectin (CD62E/L) would decrease serum levels of the proinflammatory cytokines interleukin-1beta (IL-1), IL-6, and IL-8 and tumor necrosis factor receptor (TNFR-1) in baboons during sepsis. Adult male baboons received infusions of 1 x 10(9) colony forming units (CFU)/kg heat-killed Escherichia coli (E. coli) followed 12 h later by live E. coli (1 x 10(10) CFU/kg). At the time of live bacterial infusion, six septic animals were treated with a monoclonal antibody to CD54 and six with an antibody to CD62E and L (1 mg/kg). Eight untreated septic animals served as controls. Sequentially drawn serum samples were assayed for IL-1, IL-6, IL-8, and TNFR-1 using enzyme-linked immunoassay (ELISA). Data were compared using Mann-Whitney U tests and Chi-square analyses. Median survival was decreased in both treatment groups compared to controls (P < 0.05). Peak IL-1 level was higher than controls in septic animals treated with anti-CD54 but not anti-CD62E/L (P < 0.05, P = NS, respectively). Elevations in IL-6, IL-8, and TNFR-1 were increased and prolonged in both antibody treated groups compared to controls (P < 0.05). These results provide the first in vivo evidence that leukocyte-endothelial adhesion molecules CD54 and CD62E/L regulate cytokine production in sepsis.

Detection of granulocyte-macrophage colony-stimulating factor in patients with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is an idiopathic lung disease in which the alveolar spaces are filled with surfactant. Recently, it has been proposed that PAP is caused by deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) because GM-CSF-knockout mice develop the disease. To examine this possibility, we tested the two hypotheses that lung GM-CSF levels are low and that alveolar macrophages (AM) do not respond to GM-CSF in patients with PAP. Data from 10 adult patients with PAP who underwent therapeutic whole-lung lavage were compared with those of 10 healthy volunteers who underwent bronchoalveolar lavage (BAL) by fiberoptic bronchoscopy. Bronchoalveolar lavage fluid (BALF) and plasma were collected and analyzed for total protein and levels of GM-CSF, interleukin-3, and tumor necrosis factor (TNF)-alpha. Isolated AM were cultured with or without lipopolysaccharide (LPS) or GM-CSF, and production of GM-CSF and TNF-alpha was measured after 24 h. GM-CSF in BALF and plasma was higher in PAP than in control subjects (p

Expression of heme oxygenase-1 in the lung in chronic hypoxia.

Heme oxygenase (HO)-1 is an oxygen-dependent enzyme that may regulate vascular tone and cell proliferation through the production of carbon monoxide (CO). We tested the hypothesis that HO-1 is upregulated in the lung in chronic hypoxia by exposing male Sprague-Dawley rats to 17,000 feet (395 Torr) for 0, 1, 3, 7, 14, or 21 days. After exposure, blood gases, carboxyhemoglobin (COHb) levels, and hematocrit were measured, and the lungs were either inflation fixed for immunohistochemistry or frozen for later measurement of HO enzyme activity, Western blot for HO-1 protein, and RT-PCR for HO-1 mRNA. The heart was excised and weighed, and the right-to-left heart weight ratio was determined. During hypoxia, the hematocrit increased progressively, reaching significantly higher values than the control value after 3 days. COHb levels increased above the control value after 1 day of hypoxia and increased progressively between 14 and 21 days, whereas arterial PO(2) and arterial PCO(2) did not vary significantly. HO-1 protein determined by Western blot increased for the first 7 days and declined thereafter; however, enzyme activity was elevated only after 1 day. Changes in HO-1 during hypoxia were localized by immunohistochemistry to inflammatory cells (early) and newly muscularized arterioles (later). Lung HO-1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was increased after 1 and 21 days. The data indicate that lung HO-1 protein and activity are upregulated only during early chronic hypoxia, whereas persistent COHb elevations indicate high endogenous CO production rates at nonpulmonary sites. If CO has antiproliferative properties, the lack of HO enzyme activity in the lung may be permissive for pulmonary vascular proliferation in hypoxia.

Oxygen toxicity.

That hyperoxia induces "a profound modification in the metabolism of tissues" is an old observation. The mechanisms by which the physiologic, pathologic, and biomechanical perturbations caused by oxygen relate to the in vivo manifestations of oxygen toxicity, however, have not been explained fully. This article reviews cellular mechanisms of toxicity, outlines the clinical manifestations of oxygen toxicity, and discusses methods of monitoring and modifying oxygen tolerance.

Induction of ferritin and heme oxygenase-1 by endotoxin in the lung.

Heme oxygenase (HO)-1 expression is increased by forms of oxidative stress that also induce ferritin. Even though this could result from release of iron by heme degradation, we hypothesized that ferritin expression in the lung after endotoxin [lipopolysaccharide (LPS)] would occur independently of HO-1 because iron sequestration is an important response to infection. We tested this hypothesis by instilling saline or LPS (1 mg) into lungs of rats and measuring ferritin expression, HO-1 expression and activity, and HO-1 and ferritin mRNAs at different times. Lungs were also inflation fixed for immunohistochemistry for HO-1 and ferritin. Studies were performed with and without the HO inhibitor tin protoporphyrin. Ferritin and HO-1 labeling were minimal (macrophages only) in control lungs. By 4 h after LPS instillation, ferritin staining was present in bronchial epithelium and macrophages, became diffuse at 16 h, and was nearly gone by 48-72 h. HO-1 was detectable in macrophages 4 and 16 h after LPS instillation, increased in macrophages and bronchial epithelium at 24 h, and diffusely increased in bronchial epithelium and the alveolar region at 48-72 h. Lung ferritin content increased significantly by 4 h and peaked at 16 h before declining. HO-1 protein was present by Western blot in control lung, stable at 4 h, and increased by 24 h after LPS instillation, whereas HO enzyme activity had increased by 4 h after LPS instillation. After complete inhibition of HO enzyme activity with tin protoporphyrin, ferritin increased threefold at 4 h and sixfold at 24 h after LPS instillation. HO-1 mRNA increased by 4 h and was sustained at 24 h, whereas ferritin mRNA did not change after LPS instillation. These results indicate that intratracheal LPS rapidly induces ferritin protein in the lung independently of its mRNA synthesis or HO enzyme activity. LPS induces HO-1 mRNA, which is followed by increased expression of protein.

Bacterial priming increases lung injury in gram-negative sepsis.

Sepsis syndrome is a leading cause of acute respiratory distress syndrome (ARDS), but the development of acute lung injury is highly variable for reasons that are poorly understood. We hypothesized that nonlethal systemic exposure to gram-negative bacteria, with its consequent activation of inflammatory processes, would increase functional and structural lung injury on a second exposure to live organisms, as compared with exposure of naive animals. Sixteen adult baboons received 1 to 2 x 10(10) colony-forming-units (cfu)/kg Escherichia coli by intravenous infusion. Eight animals received live bacteria as a single infusion, whereas the other eight received 10% of the total dose as heat-killed organisms (priming dose) 12 h before the live infusion. Pulmonary gas exchange and hemodynamics were monitored for 48 h or until blood pressure could not be maintained. The animals were killed and one lung was processed for electron microscopy and morphometry. Group data were compared through analysis of variance (ANOVA). The systemic circulatory responses to the bacterial challenge were similar, although less severe shock occurred in primed animals. In contrast, primed animals had increased structural damage involving lung epithelium and endothelium, and showed increased cellularity of the interstitium. The morphologic evidence of increased lung injury in septic animals with prior exposure to heat-killed bacteria suggests that prior activation of systemic inflammatory responses is a contributing factor in the pathogenesis of ARDS.

Differential expression of arginase and iNOS in the lung in sepsis.

The primary metabolic fates of L-arginine are conversion to L-citrulline by nitric oxide synthase (NOS) and to L-ornithine by arginase. In the lung, arginine utilization is increased after the inducible form of NOS (iNOS) is expressed during inflammation. The expression of arginase in normal lung and after sepsis, and its potential relationships with iNOS, however, are not known. Since arginase and iNOS share the substrate L-arginine, we tested the hypothesis that lung arginase would be co-induced with iNOS in sepsis and its cellular distribution would be related to that of iNOS in the lung. Lungs from cecal ligation and puncture (CLP) and sham-operated (S) rats were harvested 6 or 16 hours after the procedures. Lung wet-to-dry weight ratio, myeloperoxidase content, and lipid peroxidation products were measured as indices of lung injury. Western blot analyses were performed with polyclonal antibodies against two isoforms of rat arginase (I and II) and iNOS. Additional lungs from CLP and S animals were inflation-fixed for immunohistochemistry using the same antibodies. We found by Western blot that arginase II at 39 kDa was the main isoform present in normal rat lung. The enzyme was distributed diffusely in alveolar and bronchial epithelial cells, endothelial cells, and alveolar macrophages. After CLP, arginase II was almost undetectable in rat lungs at 16 hours. In contrast, in normal lung, the iNOS was not detectable by Western blot or immunohistochemistry. After CLP, strong expression of iNOS was found in similar cell types to arginase II. These data demonstrate loss of constitutive expression of arginase II in rat lung as iNOS is upregulated by the response to sepsis.

Lung-specific induction of heme oxygenase-1 and hyperoxic lung injury.

Heme oxygenase (HO)-1, which catalyzes heme breakdown, is induced by oxidative stress and may protect against oxidative injury. We hypothesized that induction of HO-1 by hemoglobin (Hb) in the lung would protect the rat from pulmonary O2 toxicity. Rats given intratracheal (i.t.) Hb showed lung-specific induction of HO-1 by 8 h by Western analysis. Rats were then pretreated for 8 h before 60 h of exposure to 100% O2 with either IT normal saline, HB, or Hb plus the HO-1 inhibitor tinprotoporphyrin (SnPP). Both the Hb + O2 and Hb + O2 + SnPP animals had less lung injury than normal saline controls as indicated by lower pleural fluid volumes and wet-to-dry weight ratios (P < 0.01). The improvement in injury in the two Hb-treated groups was the same despite a 61% decrease in HO enzyme activity in the Hb + SnPP group after 60 h of O2. In addition, inhibition of HO activity with SnPP alone before O2 exposure did not augment the extent of hyperoxic lung injury. These results demonstrate that IT Hb induces lung HO-1 in the rat and protects against hyperoxia; however, the protection is not mediated by increased HO enzyme activity.

Antibody to E- and L-selectin does not prevent lung injury or mortality in septic baboons.

Recruitment of polymorphonuclear leukocytes (PMN) through upregulation of cellular adhesion molecules is a proposed mechanism of injury in sepsis and acute respiratory distress syndrome (ARDS). We hypothesized that pretreatment of baboons with a monoclonal antibody to human E- and L-selectin (EL-246) during sepsis would decrease PMN influx into tissues and result in less organ injury during gram-negative sepsis. We studied 14 anesthetized, ventilated adult baboons; six animals received 1 mg/kg of EL-246 before infusion of an LD100 of live Escherichia coli and six received the E. coli infusion without antibody therapy. Two other animals received 1 mg/kg of EL-246 intravenously without an infusion of bacteria. Intermittent measurements were made of circulatory pressures, cardiac output, urine output, arterial blood gases, ventilation:perfusion ratio (VA/Q), and hematologic status. The experiments were ended at 48 h or at the time of death. Tissues were harvested for pathology and biochemical measurements. The E. coli infusions were associated with a hyperdynamic state, pulmonary hypertension, systemic hypotension, decreased urine output (UOP), and metabolic acidosis. The antibody partly blocked PMN migration, but there were few significant physiologic or biochemical differences between the EL-246-treated and untreated animals. In the antibody-treated animals, UOP was decreased, metabolic acidosis was worsened, and median survival time was decreased significantly. We conclude that treatment with an antibody to E- and L-selectin in gram-negative sepsis does not improve gas exchange or protect against lung injury, and is associated with decreased survival time in primates.

Oxidative metabolism in rat hepatocytes and mitochondria during sepsis.

We hypothesized that cellular oxygen consumption is abnormal during sepsis as a result of increased oxidative stress and selective mitochondrial damage. In a rat model of sepsis (cecal ligation and puncture), we studied the respiratory characteristics of isolated hepatocytes and liver mitochondria 16 h after onset of septic injury. Endogenous respiration by isolated cells was decreased during sepsis, while cyanide-resistant (nonmitochondrial) respiration was unaffected. Maximal oxygen consumption in ADP-supplemented, permeabilized hepatocytes was decreased with succinate as the substrate, but not with malate + glutamate or TMPD + ascorbate. In contrast, maximum oxygen consumption (State 3) by isolated liver mitochondria increased up to 35% during sepsis using either succinate or malate + glutamate as substrate. The electrophoretic features and mobility of nondenatured mitochondrial respiratory complexes were similar in control and septic hepatocytes, with the exception of decreased Complex V protein in sepsis. Structural evaluation of mitochondria in fixed liver slices by electron microscopy showed mitochondrial swelling in most of the septic animals. Measurements of oxidative stress during sepsis suggested an increase in hydroxylation of salicylate by isolated hepatocytes, and mitochondrial protein carbonyl content was increased significantly. Induction of iNOS in hepatocytes after 16 h of sepsis was variable, and little release of the oxidation products of NO. was detected. These findings are interpreted to mean that hepatocytes contain a mixed population of injured and hyperfunctional mitochondria during sepsis.

Aerosolized manganese SOD decreases hyperoxic pulmonary injury in primates. I. Physiology and biochemistry.

Prolonged hyperoxia causes lung injury and respiratory failure secondary to oxidative tissue damage mediated, in part, by the superoxide anion. We hypothesized that aerosol treatment with recombinant human manganese superoxide dismutase (rhMnSOD) would attenuate hyperoxic lung damage in primates. Adult baboons were anesthetized and ventilated with 100% oxygen for 96 h or until death. Six animals were treated with aerosolized rhMnSOD (3 mg . kg-1 . day-1 in divided doses), and six control animals did not receive enzyme therapy. Physiological variables were recorded every 12 h, and ventilation-perfusion ratio relationships were evaluated by using the multiple inert-gas elimination technique. After the experiments, surfactant composition and lung edema were measured. We found that rhMnSOD significantly decreased pulmonary shunt fraction (P < 0.01) and preserved arterial oxygenation (P < 0.01) during hyperoxia. The rhMnSOD increased lung phospholipids, phosphatidylcholine and disaturated phosphatidylcholine, and decreased lung edema in this model. Testing of higher and lower doses of MnSOD (1 and 10 mg . kg-1 . day-1) in two other groups of baboons produced variable physiological protection, suggesting a "window" of effective dosage. We conclude that aerosolized MnSOD (3 mg . kg-1 . day-1) affords significant preservation of pulmonary gas exchange during hyperoxic lung injury.