Inhibiting lung lining fluid glutathione metabolism with GGsTop as a novel treatment for asthma.

Asthma is characterized by airway inflammation. Inflammation is associated with oxidant stress. Airway epithelial cells are shielded from this stress by a thin layer of lung lining fluid (LLF) which contains an abundance of the antioxidant glutathione. LLF glutathione metabolism is regulated by γ-glutamyl transferase (GGT). Loss of LLF GGT activity in the mutant GGT(enu1) mouse causes an increase in baseline LLF glutathione content which is magnified in an IL-13 model of allergic airway inflammation and protective against asthma. Normal mice are susceptible to asthma in this model but can be protected with acivicin, a GGT inhibitor. GGT is a target to treat asthma but acivicin toxicity limits clinical use. GGsTop is a novel GGT inhibitor. GGsTop inhibits LLF GGT activity only when delivered through the airway. In the IL-13 model, mice treated with IL-13 and GGsTop exhibit a lung inflammatory response similar to that of mice treated with IL-13 alone. But mice treated with IL-13 and GGsTop show attenuation of methacholine-stimulated airway hyper-reactivity, inhibition of Muc5ac and Muc5b gene induction, decreased airway epithelial cell mucous accumulation and a fourfold increase in LLF glutathione content compared to mice treated with IL-13 alone. Mice treated with GGsTop alone are no different from that of mice treated with saline alone, and show no signs of toxicity. GGsTop could represent a valuable pharmacological tool to inhibit LLF GGT activity in pulmonary disease models. The associated increase in LLF glutathione can protect lung airway epithelial cells against oxidant injury associated with inflammation in asthma.

Outcomes of a ventilator-associated pneumonia bundle on rates of ventilator-associated pneumonia and other health care-associated infections in a long-term acute care hospital setting.

Long-term trends in ventilator-associated pneumonia (VAP) rates, and other health care-associated infections, were examined prior to, during, and after introduction of a VAP bundle in a long-term acute care hospital setting. VAP incidence rate declined in a step-wise fashion and reached a null value. Incidence rates of bacteremia from any cause declined in a similar fashion. The incidence rates of vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus colonization or infection rates also decreased, but that of Clostridium difficile infection did not. VAP in the long-term acute care hospital setting can be controlled over time with implementation of Centers for Disease Control and Prevention-based VAP bundle. This outcome also may decrease certain other health care-associated infections.

Transcription factor Klf4, induced in the lung by oxygen at birth, regulates perinatal fibroblast and myofibroblast differentiation.

The fluid-filled lung exists in relative hypoxia in utero (∼25 mm Hg), but at birth fills with ambient air where the partial pressure of oxygen is ∼150 mm Hg. The impact of this change was studied in mouse lung with microarrays to analyze gene expression one day before, and 2, 6, 12 and 24 hours after birth into room air or 10% O(2). The expression levels of >150 genes, representing transcriptional regulation, structure, apoptosis and antioxidants were altered 2 hrs after birth in room air but blunted or absent with birth in 10% O(2). Kruppel-like factor 4 (Klf4), a regulator of cell growth arrest and differentiation, was the most significantly altered lung gene at birth. Its protein product was expressed in fibroblasts and airway epithelial cells. Klf4 mRNA was induced in lung fibroblasts exposed to hyperoxia and constitutive expression of Klf4 mRNA in Klf4-null fibroblasts induced mRNAs for p21(cip1/Waf1), smooth muscle actin, type 1 collagen, fibronectin and tenascin C. In Klf4 perinatal null lung, p21(cip1/Waf1)mRNA expression was deficient prior to birth and associated with ongoing cell proliferation after birth; connective tissue gene expression was deficient around birth and smooth muscle actin protein expression was absent from myofibroblasts at tips of developing alveoli; p53, p21(cip1/Waf1) and caspase-3 protein expression were widespread at birth suggesting excess apoptosis compared to normal lung. We propose that the changing oxygen environment at birth acts as a physiologic signal to induce lung Klf4 mRNA expression, which then regulates proliferation and apoptosis in fibroblasts and airway epithelial cells, and connective tissue gene expression and myofibroblast differentiation at the tips of developing alveoli.

Inhibiting Glutathione Metabolism in Lung Lining Fluid as a Strategy to Augment Antioxidant Defense.

Glutathione is abundant in the lining fluid that bathes the gas exchange surface of the lung. On the one hand glutathione in this extracellular pool functions in antioxidant defense to protect cells and proteins in the alveolar space from oxidant injury; on the other hand, it functions as a source of cysteine to maintain cellular glutathione and protein synthesis. These seemingly opposing functions are regulated through metabolism by gamma-glutamyl transferase (GGT, EC 2.3.2.2). Even under normal physiologic conditions, lung lining fluid (LLF) contains a concentrated pool of GGT activity exceeding that of whole lung by about 7-fold and indicating increased turnover of glutathione at the epithelial surface of the lung. With oxidant stress LLF GGT activity is amplified even further as glutathione turnover is accelerated to meet the increased demands of cells for cysteine. Mouse models of GGT deficiency confirmed this biological role of LLF GGT activity and revealed the robust expansiveness and antioxidant capacity of the LLF glutathione pool in the absence of metabolism. Acivicin, an irreversible inhibitor of GGT, can be utilized to augment LLF fluid glutathione content in normal mice and novel GGT inhibitors have now been defined that provide advantages over acivicin. Inhibiting LLF GGT activity is a novel strategy to selectively augment the extracellular LLF glutathione pool. The enhanced antioxidant capacity can maintain lung epithelial cell integrity and barrier function under oxidant stress.

Hyperoxia-induced lung injury in gamma-glutamyl transferase deficiency is associated with alterations in nitrosative and nitrative stress.

gamma-Glutamyl transferase (GGT) regulates glutathione metabolism and cysteine supply. GGT inactivation in GGT(enu1) mice limits cysteine availability causing cellular glutathione deficiency. In lung, the resultant oxidant burden is associated with increased nitric oxide (NO) production, yet GGT(enu1) mice still exhibit higher mortality in hyperoxia. We hypothesized that NO metabolism is altered under severe oxidant stress and contributes to lung cellular injury and death. We compared lung injury, NO synthase (NOS) expression, nitrate/nitrite production, nitroso product formation, peroxynitrite accumulation, and cell death in wild-type and GGT(enu1) mice in normoxia and hyperoxia. The role of NOS activity in cell death was determined by NOS inhibition. Exposure of wild-type mice to hyperoxia caused increased lung injury, altered NO metabolism, and induction of cell death compared with normoxia, which was attenuated by NOS inhibition. Each of these lung injury indices were magnified in hyperoxia-exposed GGT(enu1) mice except nitrosation, which showed a diminished decrease compared with wild-type mice. NOS inhibition attenuated cell death only slightly, likely due to further exacerbation of oxidant stress. Taken together, these data suggest that apoptosis in hyperoxia is partially NO-dependent and reiterate the importance of cellular glutathione in lung antioxidant defense. Therefore, reduced denitrosylation of proteins, possibly resulting in impaired cellular repair, and excessive apoptotic cell death likely contribute to increased lung injury and mortality of GGT(enu1) mice in hyperoxia.

Epidemiology of ventilator-associated pneumonia in a long-term acute care hospital.

OBJECTIVE: To characterize the epidemiology and microbiology of ventilator-associated pneumonia (VAP) in a long-term acute care hospital (LTACH). DESIGN: Retrospective study of prospectively identified cases of VAP. SETTING: Single-center, 207-bed LTACH with the capacity to house 42 patients requiring mechanical ventilation, evaluated from April 1, 2006, through January 31, 2008. METHODS: Data on the occurrence of VAP were collected prospectively as part of routine infection surveillance at Radius Specialty Hospital. After March 2006, Radius Specialty Hospital implemented a bundle of interventions for the prevention of VAP (hereafter referred to as the VAP-bundle approach). A case of VAP was defined as a patient who required mechanical ventilation at Radius Specialty Hospital for at least 48 hours before any symptoms of pneumonia appeared and who met the Centers for Disease Control and Prevention criteria for VAP. Sputum samples were collected from a tracheal aspirate if there was clinical suspicion of VAP, and these samples were semiquantitatively cultured. RESULTS: During the 22-month study period, 23 cases of VAP involving 19 patients were associated with 157 LTACH admissions (infection rate, 14.6%), corresponding to a rate of 1.67 cases per 1,000 ventilator-days, which is a 56% reduction from the VAP rate of 3.8 cases per 1,000 ventilator-days reported before the implementation of the VAP-bundle approach (P< .001). Microbiological data were available for 21 (91%) of 23 cases of VAP. Cases of VAP in the LTACH were frequently polymicrobial (mean number +/- SD, 1.78+/-1.0 pathogens per case of VAP), and 20 (95%) of 21 cases of VAP had at least 1 pathogen (Pseudomonas species, Acinetobacter species, gram-negative bacilli resistant to more than 3 antibiotics, or methicillin-resistant Staphylococcus aureus) cultured from a sputum sample. LTACH patients with VAP were more likely to have a neurological reason for ventilator dependence, compared with LTACH patients without VAP (69.6% of cases of VAP vs 39% of cases of respiratory failure; P= .014). In addition, patients with VAP had a longer length of LTACH stay, compared with patients without VAP (median length of stay, 131 days vs 39 days; P= .002). In 6 (26%) of 23 cases of VAP, the patient was eventually weaned from use of mechanical ventilation. Of the 19 patients with VAP, 1 (5%) did not survive the LTACH stay. CONCLUSIONS: The VAP rate in the LTACH is lower than the VAP rate reported in acute care hospitals. Cases of VAP in the LTACH were frequently polymicrobial and were associated with multidrug-resistant pathogens and increased length of stay. The guidelines from the Centers for Disease Control and Prevention that are aimed at reducing cases of VAP appear to be effective if applied in the LTACH setting.

Regulation of Fgf10 gene expression in murine mesenchymal cells.

Fgf10 has a prominent role in organogenesis. In the developing lung, Fgf10 is dynamically expressed in the distal mesenchyme from where it diffuses and activates its epithelial receptor, Fgfr2b, to trigger budding. Little is known about how Fgf10 expression is regulated. Here we have identified a mouse lung-specific mesenchymal cell line, MLg, which expresses endogenous Fgf10 and responds to known regulators of Fgf10 in a way that is reminiscent of the early lung. To gain insights into the mechanisms involved in the transcriptional regulation of Fgf10 in these cells, we have cloned and analyzed approximately a 4.5 kb region of the mouse Fgf10 promoter. Promoter deletion analysis and Luciferase reporter assays revealed an upstream region of the Fgf10 promoter with selective enhancer activity in the MLg, but not in the non-lung-derived cell line NIH3T3. Our data suggest that a potential lung mesenchyme-specific enhancer may exist within this region of the Fgf10 promoter.

Lung lining fluid glutathione attenuates IL-13-induced asthma.

GGT(enu1) mice, deficient in gamma-glutamyl transferase and unable to metabolize extracellular glutathione, develop intracellular glutathione deficiency and oxidant stress. We used intratracheal IL-13 to induce airway inflammation and asthma in wild-type (WT) and GGT(enu1) mice to determine the effect of altered glutathione metabolism on bronchial asthma. WT and GGT(enu1) mice developed similar degrees of lung inflammation. In contrast, IL-13 induced airway epithelial cell mucous cell hyperplasia, mucin and mucin-related gene expression, epidermal growth factor receptor mRNA, and epidermal growth factor receptor activation along with airway hyperreactivity in WT mice but not in GGT(enu1) mice. Lung lining fluid (extracellular) glutathione was 10-fold greater in GGT(enu1) than in WT lungs, providing increased buffering of inflammation-associated reactive oxygen species. Pharmacologic inhibition of GGT in WT mice produced similar effects, suggesting that the lung lining fluid glutathione protects against epithelial cell induction of asthma. Inhibiting GGT activity in lung lining fluid may represent a novel therapeutic approach for preventing and treating asthma.

Fibulin-5 gene expression in human lung fibroblasts is regulated by TGF-beta and phosphatidylinositol 3-kinase activity.

Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor-beta (TGF-beta) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steady-state level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-beta markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF-beta induction. Electrophoretic gel mobility shift assay revealed that TGF-beta strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3-dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF-beta was blocked by pretreatment with TGF-beta receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002, and actinomycin D. Basal and TGF-beta-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-beta-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-beta-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-beta/Smad signaling and requires the PI3-kinase/Akt pathway.

Hypoxia results in an HIF-1-dependent induction of brain-specific aldolase C in lung epithelial cells.

Aldolase C (EC 4.1.2.13) is a brain-specific aldolase isoform and a putative target of the transcription factor hypoxia-inducible factor (HIF)-1. We identified aldolase C as a candidate hypoxia-regulated gene in mouse lung epithelial (MLE) cells using differential display. We show that the message accumulates in a robust fashion when MLE cells are exposed to 1% oxygen and is inversely related to oxygen content. Induction in hypoxia is dependent on protein synthesis. We localized a hypoxia-responsive element (HRE) in the aldolase C promoter using a series of deletion and heterologous expression studies. The HRE overlaps with a region of the proximal aldolase C promoter that is also related to its brain-specific expression. The HRE contains an Arnt (HIF-1beta) and an HIF-1alpha site. We show that induction in hypoxia is dependent on the HIF-1 site and that HIF-1alpha protein is present, by gel-shift assay, within nuclear complexes of MLE cells in hypoxia. Aldolase C mRNA expression is developmentally regulated in the fetal lung, rapidly downregulated in the newborn lung at birth, and inducible in the adult lung when exposed to hypoxia. This pattern of regulation is not seen in the brain. This preservation of this HRE in the promoters of four other species suggests that aldolase C may function as a stress-response gene.

Development and prospective validation of a model for predicting weaning in chronic ventilator dependent patients.

BACKGROUND: Approximately ten percent of patients placed on mechanical ventilation during acute illness will require long-term ventilator support. Unfortunately, despite rehabilitation, some will never be liberated from the ventilator. A method of predicting weaning outcomes for these patients could help conserve resources and minimize frustrating failed weaning attempts for this population. The objective of this investigation was to identify predictors of weaning outcome for patients admitted to a chronic ventilator unit (CVU). METHODS: This was a retrospective analysis with prospective validation. The study setting was a 25 bed CVU within a rehabilitation hospital. The training group consisted of 43 patients referred to our facility for weaning after > 3 weeks of mechanical ventilation. A multivariate model to predict weaning outcome was constructed in this group and applied to a prospective group of 31 patients followed during an 18-month period. RESULTS: A modified Glasgow Coma Scale (GCS) and the presence of sustained spontaneous respirations (SSR), defined as the presence of 2 breaths recorded above the ventilator settings on four occasions, were highly predictive of weaning success within six months of CVU admission. Patients with a modified GCS > or = 8 were 6.5 times more likely to wean than those with a modified GCS < 8 (95% confidence interval 1.6-26.3) and those with SSR were 25.5 times more likely to wean than those without SSR (95% confidence interval 4.3-51.9). CONCLUSIONS: In our population of CVU patients, simple parameters that were available on admission and did not directly reflect cardiopulmonary function were useful predictors of weaning outcome.

Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair.

Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins alphavbeta3, alphavbeta5, and alpha9beta1. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1beta abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair.

Gamma-glutamyl transferase deficiency results in lung oxidant stress in normoxia.

gamma-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGT(enu1) mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGT(enu1) lung. Glutathione content was barely decreased in GGT(enu1) lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGT(enu1) alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGT(enu1) lung. When GGT(enu1) mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.

DANCE in developing and injured lung.

We identified rat developing arteries and neural crest derivatives with multiple epidermal growth factor-like domains (DANCE) as a developmentally regulated gene using suppression-subtractive hybridization. Northern analysis confirmed a fivefold induction of this mRNA transcript between fetal day 18 and 20 that persisted through postnatal day 17. The level was declining at postnatal day 21 and was similar in adult lung to that at fetal day 18. In adults DANCE mRNA abundance was highest in lung, kidney, and spleen, lower in heart, skeletal muscle, and brain, but absent from liver and thymus. It was abundant in pulmonary artery endothelium and a lung epithelial type 2 cell line, barely detectable in vascular smooth muscle, and absent in fibroblasts. In situ hybridization revealed a regulated pattern of expression in endothelial cells of fetal, postnatal, and adult lung. Because DANCE mRNA was inducible in systemic arteries during recovery from injury, we searched for induction in lung injured by hyperoxia. Mouse DANCE mRNA abundance was unchanged during an acute 3-day exposure period, induced threefold 5 days into the recovery phase, and returned to baseline at days 8, 11, and 14. In situ hybridization at day 5 suggested a diffuse pattern of induction. DANCE may play a role in lung endothelial cell biology during development repair after injury.