Method for depletion of mitochondria DNA in human bronchial epithelial cells.

Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2',3'-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA. The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis.•BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA).•Cells' mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR).•Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer.

Mitochondrial DNA Copy Number Variation in Asthma Risk, Severity, and Exacerbations.

Rationale: Although airway oxidative stress and inflammation are central to asthma pathogenesis, there is limited knowledge of the relationship of asthma risk, severity, or exacerbations to mitochondrial dysfunction, which is pivotal to oxidant generation and inflammation. Objectives: We investigated whether mitochondrial DNA copy number (mtDNA-CN) as a measure of mitochondrial function is associated with asthma diagnosis, severity, oxidative stress, and exacerbations. Methods: We measured mtDNA-CN in blood in two cohorts. In the UK Biobank (UKB), we compared mtDNA-CN in mild and moderate-severe asthmatics to non-asthmatics. In the Severe Asthma Research Program (SARP), we evaluated mtDNA-CN in relation to asthma severity, biomarkers of oxidative stress and inflammation, and exacerbations. Measures and Main Results: In UK Biobank, asthmatics (n = 29,768) have lower mtDNA-CN compared to non-asthmatics (n = 239,158) (beta, -0.026 [95% CI, -0.038 to -0.014], P = 2.46×10-5). While lower mtDNA-CN is associated with asthma, mtDNA-CN did not differ by asthma severity in either UKB or SARP. Biomarkers of inflammation show that asthmatics have higher white blood cells (WBC), neutrophils, eosinophils, fraction exhaled nitric oxide (FENO), and lower superoxide dismutase (SOD) than non-asthmatics, confirming greater oxidative stress in asthma. In one year follow-up in SARP, higher mtDNA-CN is associated with reduced risk of three or more exacerbations in the subsequent year (OR 0.352 [95% CI, 0.164 to 0.753], P = 0.007). Conclusions: Asthma is characterized by mitochondrial dysfunction. Higher mtDNA-CN identifies an exacerbation-resistant asthma phenotype, suggesting mitochondrial function is important in exacerbation risk.

Method for Depletion of Mitochondria DNA in Human Bronchial Epithelial Cells.

Introduction: Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines derived from primary airway epithelium that are depleted of mitochondrial DNA. Methods: We treated BET-1A and BEAS-2B cells with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA). Cells' mtDNA copy number were verified by quantitative polymerase chain reaction (qPCR) in comparison to nuclear DNA (nDNA). Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer. Results: One week of EtBr treatment led to ~95% reduction of mtDNA copy number (mtDNA-CN) in cells (mtDNA-CN, mean±SE, baseline vs. treatment: BEAS-2B, 820 ± 62 vs. 56 ± 9; BET-1A, 957 ± 52 vs. 73 ± 2), which was further reduced by addition of 25 µM ddC (mtDNA-CN: BEAS-2B, 2.8; BET-1A, 47.9). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA (mtDNA-CN: BEAS-2B, 0.1; BET-1A, 0.3). The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ~two-fold in cells when mtDNA was eliminated [ECAR (mpH/min/103 cells), baseline vs. treatment: BEAS-2B, 0.50 ± 0.03 vs. 0.94 ± 0.10 P=0.005; BET-1A, 0.80 ± 0.04 vs. 1.14 ± 0.06 P=0.001]. Conclusion: Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis. This cell model system can be used to further test mitochondrial mechanisms of inflammation in bronchial epithelial cells.

Metabolism in Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by impaired regulation of pulmonary hemodynamics and vascular growth. Alterations of metabolism and bioenergetics are increasingly recognized as universal hallmarks of PAH, as metabolic abnormalities are identified in lungs and hearts of patients, animal models of the disease, and cells derived from lungs of patients. Mitochondria are the primary organelle critically mediating the complex and integrative metabolic pathways in bioenergetics, biosynthetic pathways, and cell signaling. Here, we review the alterations in metabolic pathways that are linked to the pathologic vascular phenotype of PAH, including abnormalities in glycolysis and glucose oxidation, fatty acid oxidation, glutaminolysis, arginine metabolism, one-carbon metabolism, the reducing and oxidizing cell environment, and the tricarboxylic acid cycle, as well as the effects of PAH-associated nuclear and mitochondrial mutations on metabolism. Understanding of the metabolic mechanisms underlying PAH provides important knowledge for the design of new therapeutics for treatment of patients.

Cisplatin-Mediated Upregulation of APE2 Binding to MYH9 Provokes Mitochondrial Fragmentation and Acute Kidney Injury.

Cisplatin chemotherapy is standard care for many cancers but is toxic to the kidneys. How this toxicity occurs is uncertain. In this study, we identified apurinic/apyrimidinic endonuclease 2 (APE2) as a critical molecule upregulated in the proximal tubule cells (PTC) following cisplatin-induced nuclear DNA and mitochondrial DNA damage in cisplatin-treated C57B6J mice. The APE2 transgenic mouse phenotype recapitulated the pathophysiological features of C-AKI (acute kidney injury, AKI) in the absence of cisplatin treatment. APE2 pulldown-MS analysis revealed that APE2 binds myosin heavy-Chain 9 (MYH9) protein in mitochondria after cisplatin treatment. Human MYH9-related disorder is caused by mutations in MYH9 that eventually lead to nephritis, macrothrombocytopenia, and deafness, a constellation of symptoms similar to the toxicity profile of cisplatin. Moreover, cisplatin-induced C-AKI was attenuated in APE2-knockout mice. Taken together, these findings suggest that cisplatin promotes AKI development by upregulating APE2, which leads to subsequent MYH9 dysfunction in PTC mitochondria due to an unrelated role of APE2 in DNA damage repair. This postulated mechanism and the availability of an engineered transgenic mouse model based on the mechanism of C-AKI provides an opportunity to identify novel targets for prophylactic treatment of this serious disease. SIGNIFICANCE: These results reveal and highlight an unexpected role of APE2 via its interaction with MYH9 and suggest that APE2 has the potential to prevent acute kidney injury in patients with cisplatin-treated cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/713/F1.large.jpg.

Anti-inflammatory Roles of Glucocorticoids Are Mediated by Foxp3+ Regulatory T Cells via a miR-342-Dependent Mechanism.

Glucocorticoids (GC) are the mainstay treatment option for inflammatory conditions. Despite the broad usage of GC, the mechanisms by which GC exerts its effects remain elusive. Here, utilizing murine autoimmune and allergic inflammation models, we report that Foxp3+ regulatory T (Treg) cells are irreplaceable GC target cells in vivo. Dexamethasone (Dex) administered in the absence of Treg cells completely lost its ability to control inflammation, and the lack of glucocorticoid receptor in Treg cells alone resulted in the loss of therapeutic ability of Dex. Mechanistically, Dex induced miR-342-3p specifically in Treg cells and miR-342-3p directly targeted the mTORC2 component, Rictor. Altering miRNA-342-3p or Rictor expression in Treg cells dysregulated metabolic programming in Treg cells, controlling their regulatory functions in vivo. Our results uncover a previously unknown contribution of Treg cells during glucocorticoid-mediated treatment of inflammation and the underlying mechanisms operated via the Dex-miR-342-Rictor axis.

Integrative proteomics and phosphoproteomics in pulmonary arterial hypertension.

Pulmonary arterial endothelial cells (PAEC) are mechanistically linked to origins of pulmonary arterial hypertension (PAH). Here, global proteomics and phosphoproteomics of PAEC from PAH (n = 4) and healthy lungs (n = 5) were performed using LC-MS/MS to confirm known pathways and identify new areas of investigation in PAH. Among PAH and control cells, 170 proteins and 240 phosphopeptides were differentially expressed; of these, 45 proteins and 18 phosphopeptides were located in the mitochondria. Pathologic pathways were identified with integrative bioinformatics and human protein-protein interactome network analyses, then confirmed with targeted proteomics in PAH PAEC and non-targeted metabolomics and targeted high-performance liquid chromatography of metabolites in plasma from PAH patients (n = 30) and healthy controls (n = 12). Dysregulated pathways in PAH include accelerated one carbon metabolism, abnormal tricarboxylic acid (TCA) cycle flux and glutamate metabolism, dysfunctional arginine and nitric oxide pathways, and increased oxidative stress. Functional studies in cells confirmed abnormalities in glucose metabolism, mitochondrial oxygen consumption, and production of reactive oxygen species in PAH. Altogether, the findings indicate that PAH is typified by changes in metabolic pathways that are primarily found in mitochondria.

Nitric oxide alters hyaluronan deposition by airway smooth muscle cells.

Asthma is a chronic inflammatory disease that is known to cause changes in the extracellular matrix, including changes in hyaluronan (HA) deposition. However, little is known about the factors that modulate its deposition or the potential consequences. Asthmatics with high levels of exhaled nitric oxide (NO) are characterized by greater airway reactivity and greater evidence of airway inflammation. Based on these data and our previous work we hypothesized that excessive NO promotes the pathologic production of HA by airway smooth muscle cells (SMCs). Exposure of cultured SMCs to various NO donors results in the accumulation of HA in the form of unique, cable-like structures. HA accumulates rapidly after exposure to NO and can be seen as early as one hour after NO treatment. The cable-like HA in NO-treated SMC cultures supports the binding of leukocytes. In addition, NO produced by murine macrophages (RAW cells) and airway epithelial cells also induces SMCs to produce HA cables when grown in co-culture. The modulation of HA by NO appears to be independent of soluble guanylate cyclase. Taken together, NO-induced production of leukocyte-binding HA by SMCs provides a new potential mechanism for the non-resolving airway inflammation in asthma and suggests a key role of non-immune cells in driving the chronic inflammation of the submucosa. Modulation of NO, HA and the consequent immune cell interactions may serve as potential therapeutic targets in asthma.

Arginine metabolic endotypes related to asthma severity.

AIMS: Arginine metabolism via inducible nitric oxide synthase (iNOS) and arginase 2 (ARG2) is higher in asthmatics than in healthy individuals. We hypothesized that a sub-phenotype of asthma might be defined by the magnitude of arginine metabolism categorized on the basis of high and low fraction of exhaled nitric oxide (FENO). METHODS: To test this hypothesis, asthmatics (n = 52) were compared to healthy controls (n = 51) for levels of FENO, serum arginase activity, and airway epithelial expression of iNOS and ARG2 proteins, in relation to clinical parameters of asthma inflammation and airway reactivity. In parallel, bronchial epithelial cells were evaluated for metabolic effects of iNOS and ARG2 expression in vitro. RESULTS: Asthmatics with high FENO (≥ 35 ppb; 44% of asthmatics) had higher expression of iNOS (P = 0.04) and ARG2 (P = 0.05) in the airway, indicating FENO is a marker of the high arginine metabolic endotype. High FENO asthmatics had the lowest FEV1% (P < 0.001), FEV1/FVC (P = 0.0002) and PC20 (P < 0.001) as compared to low FENO asthmatics or healthy controls. Low FENO asthmatics had near normal iNOS and ARG2 expression (both P > 0.05), and significantly higher PC20 (P < 0.001) as compared to high FENO asthmatics. In vitro studies to evaluate metabolic effects showed that iNOS overexpression and iNOS+ARG2 co-expression in a human bronchial epithelial cell line led to greater reliance on glycolysis with higher rate of pyruvate going to lactate. CONCLUSIONS: The high FENO phenotype represents a large portion of the asthma population, and is typified by greater arginine metabolism and more severe and reactive asthma.

Antioxidant defense and oxidative damage vary widely among high-altitude residents.

OBJECTIVES: People living at high altitude experience unavoidable low oxygen levels (hypoxia). While acute hypoxia causes an increase in oxidative stress and damage despite higher antioxidant activity, the consequences of chronic hypoxia are poorly understood. The aim of the present study is to assess antioxidant activity and oxidative damage in high-altitude natives and upward migrants. METHODS: Individuals from two indigenous high-altitude populations (Amhara, n = 39), (Sherpa, n = 34), one multigenerational high-altitude population (Oromo, n = 42), one upward migrant population (Nepali, n = 12), and two low-altitude reference populations (Amhara, n = 29; Oromo, n = 18) provided plasma for measurement of superoxide dismutase (SOD) activity as a marker of antioxidant capacity, and urine for measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) as a marker of DNA oxidative damage. RESULTS: High-altitude Amhara and Sherpa had the highest SOD activity, while highland Oromo and Nepalis had the lowest among high-altitude populations. High-altitude Amhara had the lowest DNA damage, Sherpa intermediate levels, and high-altitude Oromo had the highest. CONCLUSIONS: High-altitude residence alone does not associate with high antioxidant defenses; residence length appears to be influential. The single-generation upward migrant sample had the lowest defense and nearly the highest DNA damage. The two high-altitude resident samples with millennia of residence had higher defenses than the two with multiple or single generations of residence.

Alternative hematological and vascular adaptive responses to high-altitude hypoxia in East African highlanders.

Elevation of hemoglobin concentration, a common adaptive response to high-altitude hypoxia, occurs among Oromo but is dampened among Amhara highlanders of East Africa. We hypothesized that Amhara highlanders offset their smaller hemoglobin response with a vascular response. We tested this by comparing Amhara and Oromo highlanders at 3,700 and 4,000 m to their lowland counterparts at 1,200 and 1,700 m. To evaluate vascular responses, we assessed urinary levels of nitrate (NO3-) as a readout of production of the vasodilator nitric oxide and its downstream signal transducer cyclic guanosine monophosphate (cGMP), along with diastolic blood pressure as an indicator of vasomotor tone. To evaluate hematological responses, we measured hemoglobin and percent oxygen saturation of hemoglobin. Amhara highlanders, but not Oromo, had higher NO3- and cGMP compared with their lowland counterparts. NO3- directly correlated with cGMP (Amhara R2 = 0.25, P < 0.0001; Oromo R2 = 0.30, P < 0.0001). Consistent with higher levels of NO3- and cGMP, diastolic blood pressure was lower in Amhara highlanders. Both highland samples had apparent left shift in oxyhemoglobin saturation characteristics and maintained total oxyhemoglobin content similar to their lowland counterparts. However, deoxyhemoglobin levels were significantly higher, much more so among Oromo than Amhara. In conclusion, the Amhara balance minimally elevated hemoglobin with vasodilatory response to environmental hypoxia, whereas Oromo rely mainly on elevated hemoglobin response. These results point to different combinations of adaptive responses in genetically similar East African highlanders.

Phosphorylation inactivation of endothelial nitric oxide synthesis in pulmonary arterial hypertension.

The impairment of vasodilator nitric oxide (NO) production is well accepted as a typical marker of endothelial dysfunction in vascular diseases, including in the pathophysiology of pulmonary arterial hypertension (PAH), but the molecular mechanisms accounting for loss of NO production are unknown. We hypothesized that low NO production by pulmonary arterial endothelial cells in PAH is due to inactivation of NO synthase (eNOS) by aberrant phosphorylation of the protein. To test the hypothesis, we evaluated eNOS levels, dimerization, and phosphorylation in the vascular endothelial cells and lungs of patients with PAH compared with controls. In mechanistic studies, eNOS activity in endothelial cells in PAH lungs was found to be inhibited due to phosphorylation at T495. Evidence pointed to greater phosphorylation/activation of protein kinase C (PKC) α and its greater association with eNOS as the source of greater phosphorylation at T495. The presence of greater amounts of pT495-eNOS in plexiform lesions in lungs of patients with PAH confirmed the pathobiological mechanism in vivo. Transfection of the activating mutation of eNOS (T495A/S1177D) restored NO production in PAH cells. Pharmacological blockade of PKC activity by ß-blocker also restored NO formation by PAH cells, identifying one mechanism by which ß-blockers may benefit PAH and cardiovascular diseases through recovery of endothelial functions.

Increased mitochondrial arginine metabolism supports bioenergetics in asthma.

High levels of arginine metabolizing enzymes, including inducible nitric oxide synthase (iNOS) and arginase (ARG), are typical in asthmatic airway epithelium; however, little is known about the metabolic effects of enhanced arginine flux in asthma. Here, we demonstrated that increased metabolism sustains arginine availability in asthmatic airway epithelium with consequences for bioenergetics and inflammation. Expression of iNOS, ARG2, arginine synthetic enzymes, and mitochondrial respiratory complexes III and IV was elevated in asthmatic lung samples compared with healthy controls. ARG2 overexpression in a human bronchial epithelial cell line accelerated oxidative bioenergetic pathways and suppressed hypoxia-inducible factors (HIFs) and phosphorylation of the signal transducer for atopic Th2 inflammation STAT6 (pSTAT6), both of which are implicated in asthma etiology. Arg2-deficient mice had lower mitochondrial membrane potential and greater HIF-2α than WT animals. In an allergen-induced asthma model, mice lacking Arg2 had greater Th2 inflammation than WT mice, as indicated by higher levels of pSTAT6, IL-13, IL-17, eotaxin, and eosinophils and more mucus metaplasia. Bone marrow transplants from Arg2-deficient mice did not affect airway inflammation in recipient mice, supporting resident lung cells as the drivers of elevated Th2 inflammation. These data demonstrate that arginine flux preserves cellular respiration and suppresses pathological signaling events that promote inflammation in asthma.

Eotaxin-Rich Proangiogenic Hematopoietic Progenitor Cells and CCR3+ Endothelium in the Atopic Asthmatic Response.

Angiogenesis is closely linked to and precedes eosinophilic infiltration in asthma. Eosinophils are recruited into the airway by chemoattractant eotaxins, which are expressed by endothelial cells, smooth muscles cells, epithelial cells, and hematopoietic cells. We hypothesized that bone marrow-derived proangiogenic progenitor cells that contain eotaxins contribute to the initiation of angiogenesis and inflammation in asthma. Whole-lung allergen challenge of atopic asthma patients revealed vascular activation occurs within hours of challenge and before airway inflammation. The eotaxin receptor CCR3 was expressed at high levels on submucosal endothelial cells in patients and a murine model of asthma. Ex vivo exposure of murine endothelial cells to eotaxins induced migration and angiogenesis. In mechanistic studies, wild-type mice transplanted with eotaxin-1/2-deficient bone marrow had markedly less angiogenesis and inflammation in an atopic asthma model, whereas adoptive transfer of proangiogenic progenitor cells from wild-type mice in an atopic asthma model into the eotaxin-1/2-deficient mice led to angiogenesis and airway inflammation. The findings indicate that Th2-promoting hematopoietic progenitor cells are rapidly recruited to the lung upon allergen exposure and release eotaxins that coordinately activate endothelial cells, angiogenesis, and airway inflammation.

Reciprocal Changes in Phosphoenolpyruvate Carboxykinase and Pyruvate Kinase with Age Are a Determinant of Aging in Caenorhabditis elegans.

Aging involves progressive loss of cellular function and integrity, presumably caused by accumulated stochastic damage to cells. Alterations in energy metabolism contribute to aging, but how energy metabolism changes with age, how these changes affect aging, and whether they can be modified to modulate aging remain unclear. In locomotory muscle of post-fertile Caenorhabditis elegans, we identified a progressive decrease in cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), a longevity-associated metabolic enzyme, and a reciprocal increase in glycolytic pyruvate kinase (PK) that were necessary and sufficient to limit lifespan. Decline in PEPCK-C with age also led to loss of cellular function and integrity including muscle activity, and cellular senescence. Genetic and pharmacologic interventions of PEPCK-C, muscle activity, and AMPK signaling demonstrate that declines in PEPCK-C and muscle function with age interacted to limit reproductive life and lifespan via disrupted energy homeostasis. Quantifications of metabolic flux show that reciprocal changes in PEPCK-C and PK with age shunted energy metabolism toward glycolysis, reducing mitochondrial bioenergetics. Last, calorie restriction countered changes in PEPCK-C and PK with age to elicit anti-aging effects via TOR inhibition. Thus, a programmed metabolic event involving PEPCK-C and PK is a determinant of aging that can be modified to modulate aging.

Targeting mTORC1-mediated metabolic addiction overcomes fludarabine resistance in malignant B cells.

UNLABELLED: MTOR complex-1(mTORC1) activation occurs frequently in cancers, yet clinical efficacy of rapalogs is limited because of the associated activation of upstream survival pathways. An alternative approach is to inhibit downstream of mTORC1; therefore, acquired resistance to fludarabine (Flu), a purine analogue and antimetabolite chemotherapy, active agent for chronic lymphocytic leukemia (CLL) was investigated. Elevated phospho-p70S6K, also known as RPS6KB1 (ribosomal protein S6 kinase, 70kDa, polypeptide 1) (T389), an mTORC1 activation marker, predicted Flu resistance in a panel of B-cell lines, isogenic Flu-resistant (FluR) derivatives, and primary human CLL cells. Consistent with the anabolic role of mTORC1, FluR cells had higher rates of glycolysis and oxidative phosphorylation than Flu-sensitive (FluS) cells. Rapalogs (everolimus and rapamycin) induced moderate cell death in FluR and primary CLL cells, and everolimus significantly inhibited glycolysis and oxidative phosphorylation in FluR cells. Strikingly, the higher oxidative phosphorylation in FluR cells was not coupled to higher ATP synthesis. Instead, it contributed primarily to an essential, dihydroorotate dehydrogenase catalyzed, step in de novo pyrimidine biosynthesis. mTORC1 promotes pyrimidine biosynthesis by p70S6 kinase-mediated phosphorylation of CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; Ser1859) and favors S-phase cell-cycle progression. We found increased phospho-CAD (S1859) and higher S-phase population in FluR cells. Pharmacological inhibition of de novo pyrimidine biosynthesis using N-phosphonacetyl-l-aspartate and leflunomide, RNAi-mediated knockdown of p70S6K, and inhibition of mitochondrial respiration were selectively cytotoxic to FluR, but not FluS, cells. These results reveal a novel link between mTORC1-mediated metabolic reprogramming and Flu resistance identifying mitochondrial respiration and de novo pyrimidine biosynthesis as potential therapeutic targets. IMPLICATIONS: This study provides the first evidence for mTORC1/p70S6K-dependent regulation of pyrimidine biosynthesis in a relevant disease setting.

Early effect of tidal volume on lung injury biomarkers in surgical patients with healthy lungs.

BACKGROUND: The early biological impact of short-term mechanical ventilation on healthy lungs is unknown. The authors aimed to characterize the immediate tidal volume (VT)-related changes on lung injury biomarkers in patients with healthy lungs and low risk of pulmonary complications. METHODS: Twenty-eight healthy patients for knee replacement surgery were prospectively randomized to volume-controlled ventilation with VT 6 (VT6) or 10 (VT10) ml/kg predicted body weight. General anesthesia and other ventilatory parameters (positive end-expiratory pressure, 5 cm H2O, FIO2, 0.5, respiratory rate titrated for normocapnia) were managed similarly in the two groups. Exhaled breath condensate and blood samples were collected for nitrite, nitrate, tumor necrosis factor-α, interleukins-1ß, -6, -8, -10, -11, neutrophil elastase, and Clara Cell protein 16 measurements, at the onset of ventilation and 60 min later. RESULTS: No significant differences in biomarkers were detected between the VT groups at any time. The coefficient of variation of exhaled breath condensate nitrite and nitrate decreased in the VT6 but increased in the VT10 group after 60-min ventilation. Sixty-minute ventilation significantly increased plasma neutrophil elastase levels in the VT6 (35.2 ± 30.4 vs. 56.4 ± 51.7 ng/ml, P = 0.008) and Clara Cell protein 16 levels in the VT10 group (16.4 ± 8.8 vs. 18.7 ± 9.5 ng/ml, P = 0.015). Exhaled breath condensate nitrite correlated with plateau pressure (r = 0.27, P = 0.042) and plasma neutrophil elastase (r = 0.44, P = 0.001). Plasma Clara Cell protein 16 correlated with compliance (r = 0.34, P = 0.014). CONCLUSIONS: No tidal volume-related changes were observed in the selected lung injury biomarkers of patients with healthy lungs after 60-min ventilation. Plasma neutrophil elastase and plasma Clara Cell protein 16 might indicate atelectrauma and lung distention, respectively.

A novel method for pulmonary research: assessment of bioenergetic function at the air-liquid interface.

Air-liquid interface cell culture is an organotypic model for study of differentiated functional airway epithelium in vitro. Dysregulation of cellular energy metabolism and mitochondrial function have been suggested to contribute to airway diseases. However, there is currently no established method to determine oxygen consumption and glycolysis in airway epithelium in air-liquid interface. In order to study metabolism in differentiated airway epithelial cells, we engineered an insert for the Seahorse XF24 Analyzer that enabled the measure of respiration by oxygen consumption rate (OCR) and glycolysis by extracellular acidification rate (ECAR). Oxidative metabolism and glycolysis in airway epithelial cells cultured on the inserts were successfully measured. The inserts did not affect the measures of OCR or ECAR. Cells under media with apical and basolateral feeding had less oxidative metabolism as compared to cells on the inserts at air-interface with basolateral feeding. The design of inserts that can be used in the measure of bioenergetics in small numbers of cells in an organotypic state may be useful for evaluation of new drugs and metabolic mechanisms that underlie airway diseases.

Nebulized nitrite protects rat lung grafts from ischemia reperfusion injury.

OBJECTIVES: Nebulization is a potential method for delivering therapeutic agents to lung grafts. Recent evidence suggests that nitrite may mitigate ischemia-reperfusion injury via a nitric oxide-dependent pathway. METHODS: Syngeneic orthotopic left lung transplantation was performed in rats after 7 hours of cold ischemia. Sodium nitrite (3 mg) or phosphate-buffered saline (controls) was delivered before procurement via nebulization. RESULTS: Nitrite treatment was associated with better oxygenation, lower peak airway pressure, lower wet/dry ratio, reduced myeloperoxidase level and macrophage infiltration, increased cyclic guanosine monophosphate (cGMP) levels, and decreased levels of interleukin 6, interleukin 1-ß, inducible nitric oxide synthase, and intercellular adhesion molecule-1 at 2 hours after reperfusion. Treatment with 2-(4-carboxypheny)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger, reversed the beneficial effects of nitrite and decreased cGMP concentration in grafts. A dose-response curve of nitrite was performed at the following doses: 0.3 mg (N0.1), 3.0 mg (N1.0), 5.25 mg (N1.75), 7.5 mg (N2.5), and 15.0 mg (N5.0). All treatments, excluding N1.0, resulted in poorer oxygenation, higher peak airway pressures, and higher wet/dry ratio. Higher dosage groups (N1.75, N2.5, and N5.0) exhibited positive immunostaining of nitrotyrosine and increased the intensity of nitrotyrosine in immunoblotting. CONCLUSIONS: These data suggest that nebulized nitrite limits lung ischemia-reperfusion injury and may prove a clinically useful strategy but requires appropriate dosing to limit oxidative injury at high doses.