Pulmonary Diseases

Metabolic Shift in Lung Mitochondria After Toxic Exposure to Cigarette Smoke Amit Agarwal1 and Enrique Cadenas1 1University of Southern California, United States Cigarette smoking (CS) leads to alteration in cellular redox status, a hallmark in the pathogenesis of chronic obstructive pulmonary disease (COPD). the current study was undertaken to determine the role of CS in the development of mitochondrial dysfunction due to oxidative stress as a consequence of altered redox status. Male A/J mice were exposed to CS generated by a smoking machine for 4 or 8 wk. a recovery group was exposed to CS for 8 wk and allowed to recover for 2 wk. Our data indicates that short-term cigarette smoke exposure leads to altered metabolism of glucose due to oxidative modification of GAPDH, a central glycolytic enzyme and a concurrent increase in the pentose phosphate pathway of glucose metabolism. on the other hand, the activity and expression of mitochondrial respiratory chain complexes II, IV, and V were found to increase after 8 weeks of CS exposure. Microarray analysis of gene expression in mouse lungs after exposure to CS for 8 wk revealed upregulation of a group of genes involved in metabolism, electron transfer chain, oxidative phosphorylation, mitochondrial transport and dynamics, and redox regulation. to follow up on the source of substrates for mitochondrial respiratory chain mediated oxidative phosphorylation, we studied the effect of cigarette smoke, on primary alveolar Type II cells from mice exposed to cigarette smoke. the Type II alveolar cells showed a decrease in mitochondrial respiration on glucose and increased respiration on fatty acids (palmitate). the cells also showed an increase in expression of FAT/CD36 and CPT1 after CS exposure. Phosphatidylcholine was found to decrease after CS exposure along-with an increase in the cytosolic PLA2 activity. Thus, palmitate present in alveolar cells for surfactant synthesis could serve as an energy substrate in the event of altered glucose metabolism in alveolar cells.

Haptoglobin Therapy in a Rodent Model of Severe Pulmonary Vascular Disease Mediated by Hemoglobin Reduces Disease Progression David Irwin1, Paul Buehler2, Paul Eigenberger1, Christina Lisk1, Joanne Maltzhan3, Kathryn Hassel1, Eva Nozik-Grayck1, and Zoe Loomis1 1University of Colorado Denver, United States, 2FDA, United States, 3University of Colorado Denver, United States Introduction: Cell-free hemoglobin exposure is a pathogenic modifier of vascular and peripheral organ dysfunction and when combined with tissue hypoxia, as typifies many disease and injury states, its effects are more pronounced. Haptaglobin is an acute phase protein responsible for binding and clearing Hb through the macrophage CD163 receptor mechanism. Haptoglobin infusion may be a viable therapy to attenuate vascular diseases associated with chronic exposure to Hb. We hypothesized that in a rodent model of severe pulmonary vascular disease mediated by Hb in the presence of tissue hypoxia, haptoglobin therapy

would attenuate Hbs vasculotoxic effects and slow the progression towards pulmonary arterial hypertension. Methods: Using programmable micro-pumps, male Sprague Dawley rats were exposed for 5 weeks to either the combination of Hb (35 mg/day) and chronic hypoxia or hypoxia alone in the presence or absence of haptolglobin therapy (300 mg per week). Blood pressures, cardiac output, right ventricular hypertrophy, indices of pulmonary vascular remodeling and markers of inflammation were evaluated. Results: Rats exposed to the combination of Hb and hypoxia had lower cardiac outputs, increased systemic and pulmonary vascular resistance, right ventricular hypertrophy, and pulmonary vascular remodeling compared to rats exposed to hypoxia. Further, when compared to hypoxia alone, the combination of Hb and hypoxia had increased lung perivascular leukocytes, CD163 positive macrophages, and ICAM-1 expression. Haptoglobin treatment attenuated all these responses in the rats exposed to Hb and hypoxia, but did not completely restore these indices to those values observed in animals exposed to hypoxia alone. Conclusion: This study demonstrated the proof of concept that haptoglobin therapy reduces the vascular toxicity and disease modifying effects of free Hb by slowing the progression of pulmonary vascular disease to pulmonary hypertension.

Loss of Vascular EC-SOD Increases the Pro-Inflammatory Cytokine IL-18 in Chronic Hypoxia-Induced Pulmonary Hypertension Laura Alejandra Guerra1, Keyla Tumas2, Timothy Stidham2, Richard Johnson2, Crystal Woods2, John Hartney3, Russell Bowler3, Rashmin Savani4, Eva Nozik-Grayck2, and Leah Villegas2 1University of Texas at El Paso, United States, 2University of Colorado Anschutz Medical Campus, United States, 3National Jewish Hospital, United States, 4University of Texas Southwestern Medical Center, United States The antioxidant enzyme extracellular superoxide dismutase (EC-SOD or SOD3) contains a positively charged C-terminal heparin-binding domain (HBD) that enables it to bind to the extracellular matrix, contributing to its strong vascular localization. a single nucleotide polymorphism (SNP) in the HBD (R213G) in humans decreases the matrix binding affinity of EC-SOD and increases risk of cardiovascular disease. Loss of EC-SOD contributes to injury in animal models of lung and vascular disease characterized by inflammation and fibrosis, including pulmonary hypertension (PH). We recently reported that chronic hypoxia increases lung expression of the proinflammatory cytokine IL-18 and that the increase in IL-18 is blocked by treatment with an EC-SOD mimetic. We thus hypothesized that a loss of vascular EC-SOD due to decreased production or redistribution will modulate IL-18 expression. We tested the following mouse strains exposed to normoxia (Nx), 14 days (14dHX), or 21 days (21dHX) of hypobaric hypoxia: wild type (WT), total body knockout of EC-SOD (KO), and a new strain with knock-in of the human EC-SOD with the R213G SNP (R213G). in R213G mice, there was an allele dependent decrease in PA EC-SOD expression and concomitant increase in plasma EC-SOD. Lung IL-18 mRNA and protein expression at baseline, analyzed by QPCR and western blot, were higher in KO, and hypoxia lead to an earier rise and higher levels of IL-18 compared to WT. While lung IL-18 protein content was higher in R213G at baseline compared to WT, it did

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not increase further at 14d or 21dHx. We conclude that loss of vascular EC-SOD modulates the regulation of IL-18 in the lung at baseline and in response to chronic hypoxia. However, the impact of low vascular EC-SOD due to loss of expression versus altered tissue distribution may have different effects on IL-18 expression or activation.

Glutathione Redox Regulation of Lung Epithelial Sodium Channel (ENaC) Activity Nicholle M Johnson1, Lisa Kreiner1, Charles a Downs2, Lou Ann Brown1,3, Jason M Hansen1,4, and My N. Helms1,3 1Emory University School of Medicine, United States, 2Emory University Nell Hodgson Woodruff School of Nursing, United States, 3Center for Developmental Lung Biology, United States, 4Center for Cystic Fibrosis Research, United States Bronchopulmonary dysplasia (BPD) is a respiratory distress syndrome that occurs in 1-out-of-4 pre-term and low birth weight infants. Oxidative stress (depleted stores of GSH), inflammation, arrested alveolar development, and excess fluid accumulation are the risk factors presented at birth. in the present study, we utilized single channel patch clamp recordings to test the hypothesis that changes in the glutathione redox potential (Eh) alters normal epithelial sodium channel activity (ENaC; the rate limiting factor for salt and water clearance out of the lung). One major finding from our study is that there was a linear decrease in ENaC activity as Eh became less negative (n=19; P<0.05). Specifically, applying 400 M GSSG to the cell bath significantly decreased ENaC open probability (Po) from 0.39 + 0.06 to 0.13 + 0.05, thus implicating GSSG as an important pro-injury regulatory factor in edematous lung disorders such as BPD. Real-time fluoroscopic detection of lung fluid volumes confirm that tracheal instillation of 100 L GSSG (400 M) significantly attenuates the rate of alveolar fluid clearance, and biochemical assays indicate that S-glutathionylation promotes proteolytic degradation of -ENaC subunit. Post-natal (PN) pups intranasally exposed to 400 M GSSG (on PN days 1-15), as well as 1mM amiloride treated pups (on PN days 1-15), presented with significant attenuation of lung maturation (as measured by H&E labeling and radial alveolar counts) compared to control. Together, the data indicate that S-glutathionylation of ENaC decreases the rate of lung fluid clearance and could alter alveolar development; suggesting that antioxidant therapies targeted at sustaining lung ENaC activity could improve health outcomes for BPD patients.

Oxidation of Glutathione by Hypochlorous Acid in the Airways of Children with Cystic Fibrosis Anthony Kettle1, Rufus Turner1, Catherine L Gangell2, Irada Khalilova1, D Timothy Harwood1, Anna Chapman1, Christine C Winterbourn1, and Peter Sly2 1University of Otago, Christchurch, New Zealand, 2University of Queensland, Australia Glutathione (GSH) is an important antioxidant in the lungs. Low concentrations of GSH in the airways of patients with cystic

fibrosis (CF) may result from poor transport by a defective cystic fibrosis transmembrane conductance regulator. Alternatively, enhanced oxidation of GSH may occur during inflammation. We measured GSH, oxidized glutathione (GSSG), and glutathionylation of proteins in bronchoalveolar lavage (BAL) from young children with CF (n=167) and non-CF disease controls (n=21). Myeloperoxidase and biomarkers of its product hypochlorous acid, including glutathione sulfonamide (GSA) and 3-chlorotyrosine, were also measured. the concentration of GSH was lower (2.2 vs 3.9 μM, p =0.030) in BAL from children with CF whereas GSA was higher (6·4 vs 2·3 nM, p=0·051). GSA was correlated with myeloperoxidase (r=0·57; p<0·0001) and 3-chlorotyrosine (r=0·72; p<0·0001). GSSG was correlated with these biomarkers as well as GSA (r=0.46; p<0.0001). the percentage of glutathione attached to proteins was higher (11.3 vs 1.1%, p < 0·001) in CF (n=25) than controls (n=12). Neutrophilic inflammation during pulmonary infections in CF resulted in higher levels of myeloperoxidase (p<0·0001), GSA (p=0·0001) and 3-chlorotyrosine (p=0·002) in BAL. the concentration of glutathione is lower in the airways of young children with CF, especially those with pulmonary infections. Increased oxidation of GSH by hypochlorous acid and its attachment to proteins contribute to this deficiency. Therapies targeted against production of hypochlorous acid have potential to boost antioxidant defence and slow the onset and progression of CF lung disease.

Oxidation of Methionine in Calprotectin by Neutrophils in the Airways of Children with Cystic Fibrosis Nicholas J Magon1, Rufus Turner1, Nina Dickerhof1, Richard Gearry1, Mark Hampton1, Peter Sly2, and Anthony Kettle1 1University of Otago, Christchurch, New Zealand, 2University of Queensland, Australia Calprotectin is a prime target for oxidants generated by neutrophils at sites of inflammation. This abundant protein is a heterodimer of S100A8 and S100A9, and is secreted from activated neutrophils in inflamed tissues. Recent work has suggested that calprotectin is modified during the neutrophil oxidative burst. Dehydromethionine is a stable cyclic product formed on free and N-terminal methionines by hypohalous acids. We identified dehydromethionine as a novel modification on the N-terminus of S100A8. It was formed along with methionine sulfoxide, a non-specific marker of oxidation, when calprotectin was treated with reagent hypochlorous acid or that generated by myeloperoxidase and activated neutrophils. We developed a sensitive LC/MS assay for detecting the S100A8 N-terminal tryptic peptide, MLTELEK, and its oxidation products, the diastereoisomers of dehydro-MLTELEK, and MLTELEK sulfoxide in clinical samples. We observed both oxidation products in the bronchoalveolar lavage fluid (BAL) of children with cystic fibrosis and together these accounted for 94% of the total MLTELEK species detected. Furthermore, dehydro-MLTELEK was higher in children with cystic fibrosis than disease controls (p = 0.036), raised in cystic fibrosis children with a respiratory infection (p = 0.003), and correlated with bacterial density in BAL (r = 0.54, p = 0.021). the detection of dehydromethionine on S100A8 in these samples demonstrates that it is a relatively stable and biologically relevant modification that may be useful as a specific biomarker of neutrophil activation and subsequent generation of hypochlorous acid. the high level of oxidation observed in vivo may affect the normal function of calprotectin and contribute to the pathology of

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