Carolyn G. Robbins

Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells

Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO + hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia + NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO + hyperoxia. These results indicate that combined NO + hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO+hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia+ NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4-5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO+hyperoxia. These results indicate that combined NO+hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.

Improved pulmonary distribution of recombinant human Cu/Zn superoxide dismutase, using a modified ultrasonic nebulizer

Prophylactic, intratracheal instillation of recombinant human Cu/Zn superoxide dismutase (rhSOD) has been shown to lessen lung injury produced by 48 h of hyperoxia and mechanical ventilation in neonatal piglets. However, instillation of small volumes of rhSOD intratracheally would not be expected to result in uniform pulmonary distribution. Aerosolization is a technique that may improve pulmonary distribution of drugs, but is limited by the poor efficiency of most nebulizers. A newly modified ultrasonic nebulizer was tested to assess pulmonary distribution of rhSOD compared to that achieved by intratracheal instillation. rhSOD was dual‐labeled with technetium‐99m (99mTc) and a fluorescent analog (permitting quantitative and qualitative assessments of pulmonary distribution), and administered to neonatal piglets by intratracheal instillation or by aerosolization.

Intracellular uptake of recombinant superoxide dismutase after intratracheal administration

We have previously demonstrated that recombinant human copper-zinc superoxide dismutase (rhCu,ZnSOD) is rapidly incorporated into cells of airways, respiratory bronchioles, and alveoli after intratracheal administration. The present study examines whether this cellular uptake is specific for rhCu,ZnSOD or whether other proteins are similarly incorporated into lung cells. Twenty-two newborn piglets (2-3 days old, 1.2-2.0 kg) were intubated and mechanically ventilated. Eight piglets received fluorescently labeled recombinant human manganese superoxide dismutase (rhMnSOD), six received fluorescently labeled albumin, two received free (unbound) fluorescent label intratracheally, and two piglets served as untreated controls. To determine whether endogenous surfactant was important in the process of intracellular uptake, four additional piglets were made surfactant deficient by repeated bronchoalveolar lavage and then given rhCu,ZnSOD intratracheally. All animals were killed after 30-60 min. Lung sections were examined blindly by laser confocal microscopy. Similar to our previous observations with rhCu,ZnSOD, intracellular uptake of rhMnSOD and albumin was noted throughout the lung. The free label did not localize intracellularly. The uptake of proteins did not appear to be affected by surfactant deficiency. rhMnSOD administration was associated with a greater than twofold increase in lung MnSOD activity. Data suggest that the cellular uptake of antioxidants and other proteins in the lung may reflect a nonspecific host defense system for clearing proteins from the lumen of airways and alveoli.

Recombinant Human CuZn Superoxide Dismutase (rhSOD) as an Adjunctive Therapy for Inhaled Nitric Oxide (NO)

Recombinant Human CuZn Superoxide Dismutase (rhSOD) as an Adjunctive Therapy for Inhaled Nitric Oxide (NO)

The Effects of Human Recombinant Granulocyte Colony Stimulating Factor (G-CSF) on Long Injury from Hyperoxia and Mechanical Ventilation in the Neonatal Piglet

The Effects of Human Recombinant Granulocyte Colony Stimulating Factor (G-CSF) on Long Injury from Hyperoxia and Mechanical Ventilation in the Neonatal Piglet

Lung Apoptosis In Response To Therapy With Nitric Oxide (No) And Hyperoxia(HYP) • 1733

Previous in vivo studies indicate that prolonged exposure to NO+HYP causes significant lung injury in newborn piglets. Peroxynitrite-mediated oxidant damage has been implicated in the pathogenesis of this injury. However, little is known about the in vivo mechanisms of cell injury or the mode of subsequent cell death. Our previous in vitro studies in human alveolar epithelial (A549) cells demonstrated that NO+HYP directly induced acute and synergistic cytotoxicity, but the mode of cell death was found to be nonapoptotic. To determine if NO+HYP results in apoptosis in vivo, we studied 1-3 day old piglets, mechanically ventilated for 48h with 100% O2, 100 PPM NO + 21% O2, 5 PPM NO + 100% O2, 100 PPM NO + 90% O2 or unventilated control animals(n=2/group). Animals were sacrificed, lungs removed and instillation-fixed in buffered formalin and embedded in paraffin. TUNEL (TdT dUTP Nick End Labeling) assays were performed on sections of lung tissue (right lower lobe) and the number of apoptotic (TUNEL+) nuclei/field were quantified. Unventilated animals had 3.5±0.5 (mean ± SE) apoptotic nuclei/field while animals ventilated with 100 PPM NO + 21% O2 had 7.0±2, 100% O2 had 27±3, 5 PPM NO + 100% O2 had 31±4, and 100 PPM NO+ 90% O2 had 68±9 (p< 0.05, ANOVA). Preliminary data suggest that combined therapy with NO+HYP results in increased apoptosis in the lung which may correlate with previously documented pulmonary injury.

PROTEIN NITRATION IN THE LUNG IN RESPONSE TO NITRIC OXIDE (NO) AND HYPEROXIA(HYP). † 1579

Previous in vivo studies have indicated that prolonged exposure to NO + HYP(but not NO alone) causes significant lung injury in neonatal piglets. Peroxynitrate (ONOO), the product of NO and superoxide, can cause protein nitration and oxidant tissue injury and is thought to be a causative agent in the injury process. Nitrotyrosine (NT), a stable byproduct of ONOO, has been used as a specific indicator of ONOO formation. To determine whether NT formation occurs in vitro, cultured human alveolar epithelial cells (A549) were grown in room air (RA), 95% oxygen (HYP), 2mM SNAP (an NO donor) or HYP + SNAP. Western blot analysis, using a polyclonal antibody against NT, revealed NT-containing proteins at 25 and 35 kD from cells exposed to SNAP or SNAP + HYP. In vivo studies were then performed with 1-3 d old piglets, mechanically ventilated with RA (n=2), 100% oxygen (n=3), 100 PPM NO (n=4) or 100 PPM NO and 90% oxygen (n=4) for 48 h. Animals were sacrificed, the lungs removed and Western blot analysis performed for NT. Results demonstrated one major 35 kD protein in lung tissue from animals exposed to NO or NO + 90% oxygen, but not in animals exposed to RA or 100% oxygen. Control blots with preabsorbed antibody showed no specific staining. These results indicate that while NT may be a specific marker of ONOO formation, the presence of NT alone may not be a specific indicator of lung tissue injury. In addition, these data suggest that sufficient amounts of superoxide may be present both in tissue culture and in vivo to yield ONOO in the absence of an additional superoxide donor. Further studies are necessary to more clearly elucidate the role of protein nitration in lung injury.

PULMONARY ENDOTHELIAL CELLS ARE MORE RESISTANT THAN PULMONARY EPITHELIAL CELLS TO SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE (NO) AND HYPEROXIA (HYP).† 1553

PULMONARY ENDOTHELIAL CELLS ARE MORE RESISTANT THAN PULMONARY EPITHELIAL CELLS TO SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE (NO) AND HYPEROXIA (HYP).† 1553

SYNERGISTIC CYTOTOXICITY FROM NITRIC OXIDE AND HYPEROXIA. |[dagger]| 2042

Nitric oxide (NO) has been discovered to be a potent vasodilator. Endogenous NO is produced by many cell types and is important in regulating basal vasomotor tone. Exogenous NO has recently been used very successfully to treat pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen therapy. Unfortunately, there are few reports on the combined toxicity of NO and hyperoxia, either of which can be toxic alone. Together, the potential for the formation of peroxynitrite(ONOO˙) - an extremely reactive and toxic free radical - is increased. To directly test this potential toxicity, cultured human alveolar epithelial(A549) cells were grown in room air (control), hyperoxia (95% O2), 2 mM SNAP (S-nitroso-N-acetyl penicillamine - an NO donor) and hyperoxia + SNAP. Live cells were counted in each of the groups above daily for the next six days, and media and gasses were refreshed each day. Cells began to die in hyperoxia after 4 to 5 d, but cell counts were relatively unchanged in NO. However, cells exposed to both NO and hyperoxia began to die on day 2, and died rapidly thereafter. This cytotoxic effect was clearly synergistic, the death rate far exceeding any additive effect. Some cells were grown on cover slips for later immunocytochemistry for nitrotyrosine, which is a stable byproduct of peroxynitrite (which is too reactive to detect directly). Immunoreactive nitrotyrosine was most abundant only in cells exposed to NO + hyperoxia. Western blots indicate that a variety of proteins become nitrated under various conditions. However, a unique band appeared on blots only after culture in NO + hyperoxia. These experiments indicate that NO and hyperoxia have a synergistic cytotoxic effect on alveolar cells in culture, which is probably mediated by production of peroxynitrite.(Funded by grants from the NIH-NHLBI, ALA and Winthrop-University Hospital)

INTRACELLULAR UPTAKE OF HUMAN SUPEROXIDE DISMUTASE FOLLOWING INTRATRACHEAL ADMINISTRATION. † 1964

INTRACELLULAR UPTAKE OF HUMAN SUPEROXIDE DISMUTASE FOLLOWING INTRATRACHEAL ADMINISTRATION. † 1964