AJP: Lung Cellular and Molecular Physiology recent issues

Physiological determinants of the pulmonary filtration coefficient

Parker, J. C., Townsley, M. I. — 2008-07-28

Current emphasis on translational application of genetic models of lung disease has renewed interest in the measurement of the gravimetric filtration coefficient (K_f) as a means to assess vascular permeability changes in isolated perfused lungs. The K_f is the product of the hydraulic conductivity and the filtration surface area, and is a sensitive measure of vascular fluid permeability when the pulmonary vessels are fully recruited and perfused. We have observed a remarkable consistency of the normalized baseline K_f values between species with widely varying body weights from mice to sheep. Uniformity of K_f values can be attributed to the thin alveolar capillary barrier required for gas exchange and the conserved matching of lung vascular surface area to the oxygen requirements of the body mass. An allometric correlation between the total lung filtration coefficient (K_f,t) and body weight in several species (r² = 1.00) had a slope that was similar to those reported for alveolar and pulmonary capillary surface areas and pulmonary diffusion coefficients determined by morphometric methods in these species. A consistent K_f is dependent on accurately separating the filtration and vascular volume components of lung weight gain, then K_f is a consistent and repeatable index of lung vascular permeability.

From the farm to the lab: the pig as a new model of cystic fibrosis lung disease

Verkman, A. S. — 2008-07-28

The porcine lung as a potential model for cystic fibrosis

2008-07-28

Airway disease currently causes most of the morbidity and mortality in patients with cystic fibrosis (CF). However, understanding the pathogenesis of CF lung disease and developing novel therapeutic strategies have been hampered by the limitations of current models. Although the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) has been targeted in mice, CF mice fail to develop lung or pancreatic disease like that in humans. In many respects, the anatomy, biochemistry, physiology, size, and genetics of pigs resemble those of humans. Thus pigs with a targeted CFTR gene might provide a good model for CF. Here, we review aspects of porcine airways and lung that are relevant to CF.

TTF-1 response element is critical for temporal and spatial regulation and necessary for hormonal regulation of human surfactant protein-A2 promoter activity

Liu, D., Yi, M., Smith, M., Mendelson, C. R. — 2008-07-28

Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara cells, and is developmentally and hormonally regulated in fetal lung. Using transfected human fetal type II cells, we previously observed that ~300 bp of 5'-flanking DNA mediated cAMP and interleukin-1 (IL-1) stimulation and dexamethasone (Dex) inhibition of hSP-A2 promoter activity. This region contains response elements for estrogen-related receptor element (ERRE, –241 bp), thyroid transcription factor (TTF)-1/Nkx2.1 (TTF-binding protein, –171 bp), upstream stimulatory factor 1/2 (E-box, –80 bp), and stimulatory protein (Sp) 1 (G/T-box, –62 bp), which are essential for basal and cAMP induction of hSP-A2 expression. To define genomic regions necessary for developmental, hormonal, and tissue-specific regulation of hSP-A2 expression in vivo, we analyzed transgenic mice carrying hGH reporter genes comprised of 313 bp of hSP-A2 gene 5'-flanking DNA ± mutation in the TBE or 175 bp of 5'-flanking DNA, containing TBE, E-box and G/T-box, but lacking ERRE. Transgenes containing 313 or 175 bp of hSP-A2 5'-flanking DNA were expressed in a lung cell-specific manner and developmentally regulated in concert with the endogenous mouse SP-A gene. In cultured lung explants from hSP-A_–313:hGH transgenic fetal mice, cAMP and IL-1 induced and Dex inhibited transgene expression. However, the 175-bp hSP-A2 genomic region was insufficient to mediate hormonal regulation of hSP-A2 promoter activity. The finding that expression of the hSP-A_–313TBEmut:hGH transgene was essentially undetectable in fetal lung and was not hormonally regulated in transgenic fetal lung explants underscores the critical importance of the TBE in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression.

Lung-selective gene responses to alveolar hypoxia: potential role for the bone morphogenetic antagonist gremlin in pulmonary hypertension

2008-07-28

Pulmonary hypoxia is a common complication of chronic lung diseases leading to the development of pulmonary hypertension. The underlying sustained increase in vascular resistance in hypoxia is a response unique to the lung. Thus we hypothesized that there are genes for which expression is altered selectively in the lung in response to alveolar hypoxia. Using a novel subtractive array strategy, we compared gene responses to hypoxia in primary human pulmonary microvascular endothelial cells (HMVEC-L) with those in cardiac microvascular endothelium and identified 90 genes (forming 9 clusters) differentially regulated in the lung endothelium. From one cluster, we confirmed that the bone morphogenetic protein (BMP) antagonist, gremlin 1, was upregulated in the hypoxic murine lung in vivo but was unchanged in five systemic organs. We also demonstrated that gremlin protein was significantly increased by hypoxia in vivo and inhibited HMVEC-L responses to BMP stimulation in vitro. Furthermore, significant upregulation of gremlin was measured in lungs of patients with pulmonary hypertensive disease. From a second cluster, we showed that CXC receptor 7, a receptor for the proangiogenic chemokine CXCL12, was selectively upregulated in the hypoxic lung in vivo, confirming that our subtractive strategy had successfully identified a second lung-selective hypoxia-responsive gene. We conclude that hypoxia, typical of that encountered in pulmonary disease, causes lung-specific alterations in gene expression. This gives new insights into the mechanisms of pulmonary hypertension and vascular loss in chronic lung disease and identifies gremlin 1 as a potentially important mediator of vascular changes in hypoxic pulmonary hypertension.

Retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury

2008-07-28

We studied the capacity of adult human bone marrow-derived cells (BMDC) to incorporate into distal lung of immunodeficient mice following lung injury. Immunodeficient NOD/SCID and NOD/SCID/β₂ microglobulin (β₂M)^null mice were administered bleomycin (bleo) or saline intranasally. One, 2, 3 and 4 days after bleo or saline, human BMDC labeled with CellTracker Green CMFDA (5-chloromethylfluorescein diacetate) were infused intravenously. Retention of CMFDA⁺ cells was maximal when delivered 4 days after bleo treatment. Seven days after bleo, <0.005% of enzymatically dispersed lung cells from NOD/SCID mice were CMFDA⁺, which increased 10- to 100-fold in NOD/SCID/β₂M^null mice. Preincubation of BMDC with Diprotin A, a reversible inhibitor of CD26 peptidase activity that enhances the stromal-derived factor-1 (SDF-1/CXCL12)/CXCR4 axis, resulted in a 30% increase in the percentage of CMFDA⁺ cells retained in the lung. These data indicate that human BMDC can be identified in lungs of mice following injury, albeit at low levels, and this may be modestly enhanced by manipulation of the SDF-1/CXCR4 axis. Given the overall low number of human cells detected, methods to increase homing and retention of adult BMDC, and consideration of other stem cell populations, will likely be required to facilitate engraftment in the treatment of lung injury.

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

2008-07-28

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10–50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15–290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.

Peroxisome proliferator-activated receptor-{gamma} in cystic fibrosis lung epithelium

2008-07-28

The pathophysiology of cystic fibrosis (CF) inflammatory lung disease is not well understood. CF airway epithelial cells respond to inflammatory stimuli with increased production of proinflammatory cytokines as a result of increased NF-B activation. Peroxisome proliferator-activated receptor- (PPAR) inhibits NF-B activity and is reported to be reduced in CF. If PPAR participates in regulatory dysfunction in the CF lung, perhaps PPAR ligands might be useful therapeutically. Cell models of CF airway epithelium were used to evaluate PPAR expression and binding to NF-B at basal and under conditions of inflammatory stimulation by Pseudomonas aeruginosa or TNF/IL-1β. An animal model of CF was used to evaluate the potential of PPAR agonists as therapeutic agents in vivo. In vitro, PPAR agonists reduced IL-8 and MMP-9 release from airway epithelial cells in response to PAO1 or TNF/IL-1β stimulation. Less NF-B bound to PPAR in CF than normal cells, in two different assays; PPAR agonists abrogated this reduction. PPAR bound less to its target DNA sequence in CF cells. To test the importance of the reported PPAR inactivation by phosphorylation, we observed that inhibitors of ERK, but not JNK, were synergistic with PPAR agonists in reducing IL-8 secretion. In vivo, administration of PPAR agonists reduced airway inflammation in response to acute infection with P. aeruginosa in CF, but not wild-type, mice. In summary, PPAR inhibits the inflammatory response in CF, at least in part by interaction with NF-B in airway epithelial cells. PPAR agonists may be therapeutic in CF.

Depletion of resident alveolar macrophages does not prevent Fas-mediated lung injury in mice

2008-07-28

Activation of the Fas/Fas ligand (FasL) system in the lungs results in a form of injury characterized by alveolar epithelial apoptosis and neutrophilic inflammation. Studies in vitro show that Fas activation induces apoptosis in alveolar epithelial cells and cytokine production in alveolar macrophages. The main goal of this study was to determine the contribution of alveolar macrophages to Fas-induced lung inflammation in mice, by depleting alveolar macrophages using clodronate-containing liposomes. Liposomes containing clodronate or PBS were instilled by intratracheal instillation. After 24 h, the mice received intratracheal instillations of the Fas-activating monoclonal antibody Jo2 or an isotype control antibody and were studied 18 h later. The Jo2 MAb induced increases in bronchoalveolar lavage fluid (BALF) total neutrophils, lung caspase-3 activity, and BALF total protein and worsened histological lung injury in the macrophage-depleted mice. Studies in vitro showed that Fas activation induced the release of the cytokine KC in a mouse lung epithelial cell line, MLE-12. These results suggest that the lung inflammatory response to Fas activation is not primarily dependent on resident alveolar macrophages and may instead depend on cytokine release by alveolar epithelial cells.

Cytosolic phospholipase A2{alpha} activation induced by S1P is mediated by the S1P3 receptor in lung epithelial cells

Chen, L.-Y., Woszczek, G., Nagineni, S., Logun, C., Shelhamer, J. H. — 2008-07-28

Cytosolic phospholipase A₂ (cPLA₂) activation is a regulatory step in the control of arachidonic acid (AA) liberation for eicosanoid formation. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator involved in the regulation of many important proinflammatory processes and has been found in the airways of asthmatic subjects. We investigated the mechanism of S1P-induced AA release and determined the involvement of cPLA₂ in these events in A549 human lung epithelial cells. S1P induced AA release rapidly within 5 min in a dose- and time-dependent manner. S1P-induced AA release was inhibited by the cPLA₂ inhibitors methyl arachidonyl fluorophosphonate (MAFP) and pyrrolidine derivative, by small interfering RNA-mediated downregulation of cPLA₂, and by inhibition of S1P-induced calcium flux, suggesting a significant role of cPLA₂ in S1P-mediated AA release. Knockdown of the S1P3 receptor, the major S1P receptor expressed on A549 cells, inhibited S1P-induced calcium flux and AA release. The S1P-induced calcium flux and AA release was associated with sphingosine kinase 1 (Sphk1) expression and activity. Furthermore, Rho-associated kinase, downstream of S1P3, was crucial for S1P-induced cPLA₂ activation. Our data suggest that S1P acting through S1P3, calcium flux, and Rho kinase activates cPLA₂ and releases AA in lung epithelial cells. An understanding of S1P-induced cPLA₂ activation mechanisms in epithelial cells may provide potential targets to control inflammatory processes in the lung.

Chemical conjugation of {Delta}F508-CFTR corrector deoxyspergualin to transporter human serum albumin enhances its ability to rescue Cl- channel functions

2008-07-28

The most common mutation of the cystic fibrosis (CF) gene, the deletion of Phe508, encodes a protein (F508-CFTR) that fails to fold properly, thus mutated F508-cystic fibrosis transmembrane conductance regulator (CFTR) is recognized and degraded via the ubiquitin-proteasome endoplasmic reticulum-associated degradation pathway. Chemical and pharmacological chaperones and ligand-induced transport open options for designing specific drugs to control protein (mis)folding or transport. A class of compounds that has been proposed as having potential utility in F508-CFTR is that which targets the molecular chaperone and proteasome systems. In this study, we have selected deoxyspergualin (DSG) as a reference molecule for this class of compounds and for ease of cross-linking to human serum albumin (HSA) as a protein transporter. Chemical cross-linking of DSG to HSA via a disulfide-based cross-linker and its administration to cells carrying F508-CFTR resulted in a greater enhancement of F508-CFTR function than when free DSG was used. Function of the selenium-dependent oxidoreductase system was required to allow intracellular activation of HSA-DSG conjugates. The principle that carrier proteins can deliver pharmacological chaperones to cells leading to correction of defective CFTR functions is therefore proven and warrants further investigations.

Role of brain-derived neurotrophic factor in hyperoxia-induced enhancement of contractility and impairment of relaxation in lung parenchyma

2008-07-28

Prolonged hyperoxic exposure contributes to neonatal lung injury, and airway hyperreactivity is characterized by enhanced contraction and impaired relaxation of airway smooth muscle. Our previous data demonstrate that hyperoxia in rat pups upregulates expression of brain-derived neurotrophic factor (BDNF) mRNA and protein, disrupts NO-cGMP signaling, and impairs cAMP production in airway smooth muscle. We hypothesized that BDNF-tyrosine kinase B (TrkB) signaling plays a functional role in airway hyperreactivity via upregulation of cholinergic mechanisms in hyperoxia-exposed lungs. Five-day-old rat pups were exposed to ≥95% oxygen or room air for 7 days and administered daily tyrosine kinase inhibitor K-252a (50 µg·kg^–1·day^–1 ip) to block BDNF-TrkB signaling or vehicle. Lungs were removed for HPLC measurement of ACh or for in vitro force measurement of lung parenchymal strips. ACh content doubled in hyperoxic compared with room air-exposed lungs. K-252a treatment of hyperoxic pups restored ACh content to room air levels. Hyperoxia increased contraction and impaired relaxation of lung strips in response to incremental electrical field stimulation. K-252a administration to hyperoxic pups reversed this increase in contraction and decrease in relaxation. K-252a or TrkB-Fc was used to block the effect of exogenous BDNF in vitro. Both K-252a and TrkB-Fc blocked the effects of exogenous BDNF. Hyperoxia decreased cAMP and cGMP levels in lung strips, and blockade of BDNF-TrkB signaling restored cAMP but not cGMP to control levels. Therefore, hyperoxia-induced increase in activity of BDNF-TrkB receptor signaling appears to play a critical role in enhancing cholinergically mediated contractile responses of lung parenchyma.

Effect of severe calorie restriction on the lung in two strains of mice

Bishai, J. M., Mitzner, W. — 2008-07-28

There is a body of literature in animal models that has suggested the development of emphysema following severe calorie restriction. This has led to the notion of "nutritional emphysema" that might have relevance in COPD patients. There have been few studies, however, that have looked closely at both the mechanics and lung structure in the same animals. In the present work, we examined lung mechanics and histological changes in two strains of mice that have substantial differences in alveolar size, the C57BL/6 and C3H/HeJ strains. We quantified the dynamic elastance and resistance at 2.5 Hz, the quasistatic pressure volume curve, and the alveolar chord lengths in lungs inflated to a lung capacity at 25–30 cmH₂O. We found that after 2 or 3 wk of calorie restriction to 1/3 their normal diet, the lungs became stiffer with increased resistance. In addition, the lung capacity was also decreased. These mechanical changes were reversed after 2 wk on a normal ad libitum diet. Histology of the postmortem fixed lungs showed no changes in the mean alveolar chord lengths with calorie restriction. Although the baseline mechanics and alveolar size were quantitatively different in the two strains, both strains showed similar qualitative changes during the starvation and refeeding periods. Thus, in two strains of mice with genetically determined differences in alveolar size, neither the mechanics nor the histology show any evidence of emphysema-like changes with this severe caloric insult.

Critical role of Cdc42 in mediating endothelial barrier protection in vivo

2008-07-28

Activation of the Rho GTPase Cdc42 has been shown in endothelial cell monolayers to prevent disassembly of interendothelial junctions and the increase in endothelial permeability. Here, we addressed the in vivo role of Cdc42 activity in mediating endothelial barrier protection in lungs by generating mice expressing the dominant active mutant V12Cdc42 protein in vascular endothelial cells targeted via the VE-cadherin promoter. These mice developed normally and exhibited constitutively active GTP-bound Cdc42. The increase in lung vascular permeability and gain in tissue water content in response to intraperitoneal lipopolysaccharide challenge (7 mg/kg) were markedly attenuated in the transgenic mice. To address the basis of the protective effect, we observed that expression of V12Cdc42 mutant in endothelial monolayers reduced the decrease in transendothelial electrical resistance, a measure of opening of interendothelial junctions, thus indicating that Cdc42 activity preserved junctional integrity. RhoA activity in V12Cdc42-expressing endothelial monolayers was reduced compared with untransfected cells, suggesting that activated Cdc42 functions by counteracting the canonical RhoA-mediated mechanism of endothelial hyperpermeability. Therefore, Cdc42 activity of microvessel endothelial cells is a critical determinant of junctional barrier restrictiveness and may represent a means of therapeutically modulating increased lung vascular permeability and edema formation.

Hydrogen peroxide decreases endothelial nitric oxide synthase promoter activity through the inhibition of AP-1 activity

Kumar, S., Sun, X., Wedgwood, S., Black, S. M. — 2008-07-28

Previously, we have reported that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in pulmonary arterial endothelial cells (PAECs) in response to hydrogen peroxide (H₂O₂). Thus the objective of this study was to identify the cis-element(s) and transcription factor(s) responsible for oxidant-mediated downregulation of the eNOS gene. Initial promoter experiments in PAECs treated with H₂O₂ revealed a significant decrease in activity of a promoter fragment containing 840 bp of upstream sequence of the human eNOS gene fused to a luciferase reporter. However, a promoter construct containing only 640 bp of upstream sequence had a significantly attenuated response to H₂O₂ challenge. As the 840-bp promoter construct had a putative binding site for the transcription factor activator protein-1 (AP-1) that was lacking in the 640-bp construct, we evaluated the effect of H₂O₂ on promoter activity after mutation of the AP-1 binding sequence (TGAGTCA at –661 to TGAGTtg in the 840-bp construct). Similar to the results seen with the 640 bp, the AP-1 mutant promoter had a significantly attenuated response to H₂O₂. EMSA revealed decreased binding of AP-1 during H₂O₂ treatment. Supershift analysis indicated that the AP-1 complex consisted of a c-Jun and FosB heterodimer. Furthermore, in vitro EMSA analysis indicated the c-Jun binding was significantly decreased after H₂O₂ exposure. Using chromatin immunoprecipitation analysis, we demonstrated decreased binding of AP-1 to the eNOS promoter in vivo in response to H₂O₂. These data suggest a role of decreased AP-1 binding likely through c-Jun in the H₂O₂-mediated decrease in eNOS promoter activity.

Reduction in systemic epithelial ion transport in septicemia-related pulmonary edema due to changes in amiloride-insensitive sodium transport?

Eisenhut, M. — 2008-07-28

Animal models of chronic obstructive pulmonary disease

Wright, J. L., Cosio, M., Churg, A. — 2008-07-03

The mechanisms involved in the genesis of chronic obstructive pulmonary disease (COPD) are poorly defined. This area is complicated and difficult to model because COPD consists of four separate anatomic lesions (emphysema, small airway remodeling, pulmonary hypertension, and chronic bronchitis) and a functional lesion, acute exacerbation; moreover, the disease in humans develops over decades. This review discusses the various animal models that have been used to attempt to recreate human COPD and the advantages and disadvantages of each. None of the models reproduces the exact changes seen in humans, but cigarette smoke-induced disease appears to come the closest, and genetically modified animals also, in some instances, shed light on processes that appear to play a role.

Challenges in translating plasma proteomics from bench to bedside: update from the NHLBI Clinical Proteomics Programs

2008-07-03

The emerging scientific field of proteomics encompasses the identification, characterization, and quantification of the protein content or proteome of whole cells, tissues, or body fluids. The potential for proteomic technologies to identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of clinical disease states holds great promise for clinical use. However, there are many challenges in translating plasma proteomics from bench to bedside, and relatively few plasma biomarkers have successfully transitioned from proteomic discovery to routine clinical use. Key barriers to this translation include the need for "orthogonal" biomarkers (i.e., uncorrelated with existing markers), the complexity of the proteome in biological samples, the presence of high abundance proteins such as albumin in biological samples that hinder detection of low abundance proteins, false positive associations that occur with analysis of high dimensional datasets, and the limited understanding of the effects of growth, development, and age on the normal plasma proteome. Strategies to overcome these challenges are discussed.

Cystic fibrosis: ironing out the problem of infection?

Reid, D. W., Anderson, G. J., Lamont, I. L. — 2008-07-03

The {Delta}F508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

2008-07-03

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing F508-CFTR significantly enhanced P. aeruginosa biofilm formation, and F508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on F508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing F508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The F508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Pulmonary alveolar epithelial uptake of S-nitrosothiols is regulated by L-type amino acid transporter

2008-07-03

Nitric oxide (NO) effects are often mediated via S-nitrosothiol (SNO) formation; SNO uptake has recently been shown to be mediated in some cell types via system L-type amino acid transporters (LAT-1, 2). Inhaled NO therapy may exert some biological effects via SNO formation. We therefore sought to determine if pulmonary epithelial SNO uptake depended on LAT or peptide transporter 2 (PEPT2). Both LAT-1 and PEPT2 proteins were detected by immunoblot and immunocytochemistry in L2 cells and rat lung. We tested SNO uptake through the transporters by exposing rat alveolar epithelial cells (L2 and type II) to RSNOs: S-nitrosoglutathione, S-nitrosocysteinylglycine (SNO-Cys-Gly), S-nitrosocysteine (CSNO), and to NO donor diethylamine NONOate (DEA-NONOate). SNO was detected in cell lysates by ozone chemiluminescence. NO uptake was detected by fluorescence in alveolar epithelial cells loaded with 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) diacetate cultured in submersion and exposed to RSNOs and DEA NONOate. Addition of l-Cys but not d-Cys to RSNOs or DEA NONOate increased SNO and DAF-FM signal that was inhibited by coincubation with LAT competitors. Incubation of cells with PEPT2 substrate SNO-Cys-Gly showed no increase in SNO or DAF-FM signal unless incubated with l-Cys. This was unaffected by PEPT2 inhibition. We conclude that RSNOs (thionitrites, S-nitrosothiols) and NO enter alveolar epithelial cells predominantly by S-nitrosation of l-Cys, which is then imported through LAT.

Superoxide dismutase protects against apoptosis and alveolar enlargement induced by ceramide

2008-07-03

The molecular events leading to emphysema development include generation of oxidative stress and alveolar cell apoptosis. Oxidative stress upregulates ceramides, proapoptotic signaling sphingolipids that trigger further oxidative stress and alveolar space enlargement, as shown in an experimental model of emphysema due to VEGF blockade. As alveolar cell apoptosis and oxidative stress mutually interact to mediate alveolar destruction, we hypothesized that the oxidative stress generated by ceramide is required for its pathogenic effect on lung alveoli. To model the direct lung effects of ceramide, mice received ceramide intratracheally (Cer_12:0 or Cer_8:0; 1 mg/kg) or vehicle. Apoptosis was inhibited with a general caspase inhibitor. Ceramide augmentation shown to mimic levels found in human emphysema lungs increased oxidative stress, and decreased, independently of caspase activation, the lung superoxide dismutase activity at 48 h. In contrast to their wild-type littermates, transgenic mice overexpressing human Cu/Zn SOD were significantly protected from ceramide-induced superoxide production, apoptosis, and air space enlargement. Activation of lung acid sphingomyelinase in response to ceramide treatment was abolished in the Cu/Zn SOD transgenic mice. Since cigarette smoke-induced emphysema in mice is similarly ameliorated by the Cu/Zn SOD overexpression, we hypothesized that cigarette smoke may induce ceramides in the mouse lung. Utilizing tandem mass spectrometry, we documented increased lung ceramides in adult mice exposed to cigarette smoke for 4 wk. In conclusion, ceramide-induced superoxide accumulation in the lung may be a critical step in ceramide's proapoptotic effect in the lung. This work implicates excessive lung ceramides as amplifiers of lung injury through redox-dependent mechanisms.

Localized elasticity measured in epithelial cells migrating at a wound edge using atomic force microscopy

2008-07-03

Restoration of lung homeostasis following injury requires efficient wound healing by the epithelium. The mechanisms of lung epithelial wound healing include cell spreading and migration into the wounded area and later cell proliferation. We hypothesized that mechanical properties of cells vary near the wound edge, and this may provide cues to direct cell migration. To investigate this hypothesis, we measured variations in the stiffness of migrating human bronchial epithelial cells (16HBE cells) ~2 h after applying a scratch wound. We used atomic force microscopy (AFM) in contact mode to measure the cell stiffness in 1.5-µm square regions at different locations relative to the wound edge. In regions far from the wound edge (>2.75 mm), there was substantial variation in the elastic modulus in specific cellular regions, but the median values measured from multiple fields were consistently lower than 5 kPa. At the wound edge, cell stiffness was significantly lower within the first 5 µm but increased significantly between 10 and 15 µm before decreasing again below the median values away from the wound edge. When cells were infected with an adenovirus expressing a dominant negative form of RhoA, cell stiffness was significantly decreased compared with cells infected with a control adenovirus. In addition, expression of dominant negative RhoA abrogated the peak increase in stiffness near the wound edge. These results suggest that cells near the wound edge undergo localized changes in cellular stiffness that may provide signals for cell spreading and migration.

Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Firth, A. L., Yuill, K. H., Smirnov, S. V. — 2008-07-03

Voltage-gated K⁺ (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (I_Kv) revealed the presence of a mitochondria-mediated Mg²⁺ and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.

Involvement of RhoA/Rho kinase signaling in protection against monocrotaline-induced pulmonary hypertension in pneumonectomized rats by dehydroepiandrosterone

2008-07-03

RhoA/Rho kinase (ROCK) signaling plays a key role in the pathogenesis of experimental pulmonary hypertension (PH). Dehydroepiandrosterone (DHEA), a naturally occurring steroid hormone, effectively inhibits chronic hypoxic PH, but the responsible mechanisms are unclear. This study tested whether DHEA was also effective in treating monocrotaline (MCT)-induced PH in left pneumonectomized rats and whether inhibition of RhoA/ROCK signaling was involved in the protective effect of DHEA. Three weeks after MCT injection, pneumonectomized rats developed PH with severe vascular remodeling, including occlusive neointimal lesions in pulmonary arterioles. In lungs from these animals, we detected cleaved (constitutively active) ROCK I as well as increases in activities of RhoA and ROCK and increases in ROCK II protein expression. Chronic DHEA treatment (1%, by food for 3 wk) markedly inhibited the MCT-induced PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 33 ± 5 and 16 ± 1 mmHg, respectively) and severe pulmonary vascular remodeling in pneumonectomized rats. The MCT-induced changes in RhoA/ROCK-related protein expression were nearly normalized by DHEA. A 3-wk DHEA treatment (1%) started 3 wk after MCT injection completely inhibited the progression of PH (mean pulmonary artery pressures after treatment with 0% and 1% DHEA were 47 ± 3 and 30 ± 3 mmHg, respectively), and this treatment also resulted in 100% survival in contrast to 30% in DHEA-untreated rats. These results suggest that inhibition of RhoA/ROCK signaling, including the cleavage and constitutive activation of ROCK I, is an important component of the impressive protection of DHEA against MCT-induced PH in pneumonectomized rats.

Particulate matter exposure induces persistent lung inflammation and endothelial dysfunction

2008-07-03

Epidemiologic and animal studies have shown that exposure to particulate matter air pollution (PM) is a risk factor for the development of atherosclerosis. Whether PM-induced lung and systemic inflammation is involved in this process is not clear. We hypothesized that PM exposure causes lung and systemic inflammation, which in turn leads to vascular endothelial dysfunction, a key step in the initiation and progression of atherosclerosis. New Zealand White rabbits were exposed for 5 days (acute, total dose 8 mg) and 4 wk (chronic, total dose 16 mg) to either PM smaller than 10 µm (PM₁₀) or saline intratracheally. Lung inflammation was quantified by morphometry; systemic inflammation was assessed by white blood cell and platelet counts and serum interleukin (IL)-6, nitric oxide, and endothelin levels. Endothelial dysfunction was assessed by vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP). PM₁₀ exposure increased lung macrophages (P < 0.02), macrophages containing particles (P < 0.001), and activated macrophages (P < 0.006). PM₁₀ increased serum IL-6 levels in the first 2 wk of exposure (P < 0.05) but not in weeks 3 or 4. PM₁₀ exposure reduced ACh-related relaxation of the carotid artery with both acute and chronic exposure, with no effect on SNP-induced vasodilatation. Serum IL-6 levels correlated with macrophages containing particles (P = 0.043) and ACh-induced vasodilatation (P = 0.014 at week 1, P = 0.021 at week 2). Exposure to PM₁₀ caused lung and systemic inflammation that were both associated with vascular endothelial dysfunction. This suggests that PM-induced lung and systemic inflammatory responses contribute to the adverse vascular events associated with exposure to air pollution.

Transforming growth factor-{beta} signaling mediates hypoxia-induced pulmonary arterial remodeling and inhibition of alveolar development in newborn mouse lung

2008-07-03

Hypoxia causes abnormal neonatal pulmonary artery remodeling (PAR) and inhibition of alveolar development (IAD). Transforming growth factor (TGF)-β is an important regulator of lung development and repair from injury. We tested the hypothesis that inhibition of TGF-β signaling attenuates hypoxia-induced PAR and IAD. Mice with an inducible dominant-negative mutation of the TGF-β type II receptor (DNTGFβRII) and nontransgenic wild-type (WT) mice were exposed to hypoxia (12% O₂) or air from birth to 14 days of age. Expression of DNTGFβRII was induced by 20 µg/g ZnSO₄ given intraperitoneally daily from birth. PAR, IAD, cell proliferation, and expression of extracellular matrix (ECM) proteins were assessed. In WT mice, hypoxia led to thicker, more muscularized resistance pulmonary arteries and impaired alveolarization, accompanied by increases in active TGF-β and phosphorylated Smad2. Hypoxia-induced PAR and IAD were greatly attenuated in DNTGFβRII mice given ZnSO₄ compared with WT control mice and DNTGFβRII mice not given ZnSO₄. The stimulatory effects of hypoxic exposure on pulmonary arterial cell proliferation and lung ECM proteins were abrogated in DNTGFβRII mice given ZnSO₄. These data support the conclusion that TGF-β plays an important role in hypoxia-induced pulmonary vascular adaptation and IAD in the newborn animal model.

Augmented inducible nitric oxide synthase expression and increased NO production reduce sepsis-induced lung injury and mortality in myeloperoxidase-null mice

2008-07-03

The myeloperoxidase (MPO)-hydrogen peroxide-halide system is an efficient oxygen-dependent antimicrobial component of polymorphonuclear leukocyte (PMN)-mediated host defense. However, MPO deficiency results in few clinical consequences indicating the activation of compensatory mechanisms. Here, we determined possible mechanisms protecting the host using MPO^–/– mice challenged with live gram-negative bacterium Escherichia coli. We observed that MPO^–/– mice unexpectedly had improved survival compared with wild-type (WT) mice within 5–12 h after intraperitoneal E. coli challenge. Lungs of MPO^–/– mice also demonstrated lower bacterial colonization and markedly attenuated increases in microvascular permeability and edema formation after E. coli challenge compared with WT. However, PMN sequestration in lungs of both groups was similar. Basal inducible nitric oxide synthase (iNOS) expression was significantly elevated in lungs and PMNs of MPO^–/– mice, and NO production was increased two- to sixfold compared with WT. Nitrotyrosine levels doubled in lungs of WT mice within 1 h after E. coli challenge but did not change in MPO^–/– mice. Inhibition of iNOS in MPO^–/– mice significantly increased lung edema and reduced their survival after E. coli challenge, but iNOS inhibitor had the opposite effect in WT mice. Thus augmented iNOS expression and NO production in MPO^–/– mice compensate for the lack of HOCl-mediated bacterial killing, and the absence of MPO-derived oxidants mitigates E. coli sepsis-induced lung inflammation and injury.

Differences in STIM1 and TRPC expression in proximal and distal pulmonary arterial smooth muscle are associated with differences in Ca2+ responses to hypoxia

Lu, W., Wang, J., Shimoda, L. A., Sylvester, J. T. — 2008-07-03

Hypoxic pulmonary vasoconstriction (HPV) requires Ca²⁺ influx through store-operated Ca²⁺ channels (SOCC) in pulmonary arterial smooth muscle cells (PASMC) and is greater in distal than proximal pulmonary arteries (PA). SOCC may be composed of canonical transient receptor potential (TRPC) proteins and activated by stromal interacting molecule 1 (STIM1). To assess the possibility that HPV is greater in distal PA because store-operated Ca²⁺ entry (SOCE) is greater in distal PASMC, we measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) and SOCE in primary cultures of PASMC using fluorescent microscopy and the Ca²⁺-sensitive dye fura 2. Both hypoxia (4% O₂) and KCl (60 mM) increased [Ca²⁺]_i. Responses to hypoxia, but not KCl, were greater in distal cells. We measured SOCE in PASMC perfused with Ca²⁺-free solutions containing cyclopiazonic acid to deplete Ca²⁺ stores in sarcoplasmic reticulum and nifedipine to prevent Ca²⁺ entry through L-type voltage-operated Ca²⁺ channels. Under these conditions, the increase in [Ca²⁺]_i caused by restoration of extracellular Ca²⁺ and the decrease in fura 2 fluorescence caused by Mn²⁺ were greater in distal PASMC, indicating greater SOCE. Moreover, the increase in SOCE caused by hypoxia was also greater in distal cells. Real-time quantitative polymerase chain reaction analysis of PASMC and freshly isolated deendothelialized PA tissue demonstrated expression of STIM1 and five of seven known TRPC isoforms (TRPC1 > TRPC6 > TRPC4 >> TRPC3 TRPC5). For both protein, as measured by Western blotting, and mRNA, expression of STIM1, TRPC1, TRPC6, and TRPC4 was greater in distal than proximal PASMC and PA. These results provide further support for the importance of SOCE in HPV and suggest that HPV is greater in distal than proximal PA because greater numbers and activation of SOCC in distal PASMC generate bigger increases in [Ca²⁺]_i.

Paradoxical role of alveolar macrophage-derived granulocyte-macrophage colony-stimulating factor in pulmonary host defense post-bone marrow transplantation

2008-07-03

Impaired host defense post-bone marrow transplant (BMT) is related to overproduction of prostaglandin E₂ (PGE₂) by alveolar macrophages (AMs). We show AMs post-BMT overproduce granulocyte-macrophage colony-stimulating factor (GM-CSF), whereas GM-CSF in lung homogenates is impaired both at baseline and in response to infection post-BMT. Homeostatic regulation of GM-CSF may occur by hematopoietic/structural cell cross talk. To determine whether AM overproduction of GM-CSF influenced immunosuppression post-BMT, we compared mice that received BMT from wild-type donors (control BMT) or mice that received BMT from GM-CSF–/– donors (GM-CSF–/– BMT) with untransplanted mice. GM-CSF–/– BMT mice were less susceptible to pneumonia with Pseudomonas aeruginosa compared with control BMT mice and showed antibacterial responses equal to or better than untransplanted mice. GM-CSF–/– BMT AMs displayed normal phagocytosis and a trend toward enhanced bacterial killing. Surprisingly, AMs from GM-CSF–/– BMT mice overproduced PGE₂, but expression of the inhibitory EP₂ receptor was diminished. As a consequence of decreased EP₂ receptor expression, we found diminished accumulation of cAMP in response to PGE₂ stimulation in GM-CSF–/– BMT AMs compared with control BMT AMs. In addition, GM-CSF–/– BMT AMs retained cysteinyl leukotriene production and normal TNF- response compared with AMs from control BMT mice. GM-CSF–/– BMT neutrophils also showed improved bacterial killing. Although genetic ablation of GM-CSF in hematopoietic cells post-BMT improved host defense, transplantation of wild-type bone marrow into GM-CSF–/– recipients demonstrated that parenchymal cell-derived GM-CSF is necessary for effective innate immune responses post-BMT. These results highlight the complex regulation of GM-CSF and innate immunity post-BMT.

Transforming growth factor-{beta}1 protects against pulmonary artery endothelial cell apoptosis via ALK5

Lu, Q. — 2008-07-03

Transforming growth factor (TGF)-β1 has been reported to cause endothelial cell apoptosis. However, conflicting data have also demonstrated that TGF-β1 promotes endothelial cell survival. In this study, the effect of TGF-β1 on apoptosis of cultured bovine pulmonary artery endothelial cells (PAEC) induced by multiple stimuli was investigated. TGF-β1 protected against apoptosis of bovine PAEC induced by serum deprivation or the VEGF receptor inhibitor SU-5416, but not by UV light exposure or TNF. Neither caspase-8 nor caspase-12 was activated by serum deprivation or the VEGF receptor blocker. However, blockade of VEGF receptors activated caspase-9, an effect that was abolished by TGF-β1. Furthermore, serum deprivation and inhibition of VEGF receptors significantly decreased the protein level of Bcl-2, an effect that was also abrogated by TGF-β1. In addition, the baseline level of Bcl-2 was enhanced by TGF-β1 and reduced by inhibition of activin receptor-like kinase 5 (ALK5), a TGF-β1 type I receptor. Furthermore, inhibition of ALK5 caused apoptosis of bovine PAEC. These results suggest that TGF-β1 signaling is critical for maintenance of bovine PAEC survival. Finally, the protective effects of TGF-β1 on bovine PAEC apoptosis and Bcl-2 reduction were abolished by ALK5 inhibition, but not by inhibition of non-SMAD signaling pathways. Also, TGF-β1 activated SMAD2 and SMAD1/5, an effect that was abolished by ALK5 inhibition. The results of this study suggest that TGF-β1 protects against bovine PAEC apoptosis, possibly through ALK5-mediated Bcl-2 induction and subsequent inhibition of the mitochondria-mediated intrinsic pathway of apoptosis. Understanding the mechanism by which TGF-β1 promotes endothelial cell survival may provide a better treatment for apoptosis-dependent vascular diseases, such as emphysema.

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

Haufe, D., Koenigshausen, E., Knels, L., Wendel, M., Stehr, S. N., Koch, T. — 2008-07-03

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.

EGF antagonizes TGF-{beta}-induced tropoelastin expression in lung fibroblasts via stabilization of Smad corepressor TGIF

Yang, S., Nugent, M. A., Panchenko, M. P. — 2008-07-03

We previously reported that neutrophil elastase (NE) downregulates transforming growth factor-β (TGF-β)-maintained tropoelastin mRNA levels in lung fibroblasts through transactivation of the epidermal growth factor (EGF) receptor (EGFR)/Mek/Erk pathway, which is dependent on the NE-initiated release of soluble EGFR ligands. In the present study, we investigated the mechanism by which EGF downregulates tropoelastin expression. We found that EGF downregulates tropoelastin expression through inhibition of TGF-β signaling. We show that EGF does not prevent the TGF-β-induced nuclear accumulation of Smad2/3; rather, EGF stabilizes the short-lived Smad transcriptional corepressor TG-interacting factor (TGIF) via EGFR/Mek/Erk-mediated phosphorylation of TGIF. Elevation of TGIF levels, either by TGIF overexpression or prevention of TGIF degradation, is sufficient to inhibit TGF-β-induced tropoelastin expression. Moreover, TGIF is essential for EGF-mediated downregulation of tropoelastin expression, inasmuch as small interfering RNA knockdown of TGIF blocked EGF-induced downregulation of tropoelastin. Finally, we demonstrated that NE treatment, which releases EGF-like growth factors, causes stabilization of TGIF through the EGFR/Mek/Erk pathway. These results suggest that EGFR/Mek/Erk signaling specifically antagonizes the proelastogenic action of TGF-β in lung fibroblasts by stabilizing the Smad transcriptional corepressor TGIF.

Utility of magnetic resonance imaging and nuclear magnetic resonance-based metabolomics for quantification of inflammatory lung injury

2008-07-03

Magnetic resonance imaging (MRI) and metabolic nuclear magnetic resonance (NMR) spectroscopy are clinically available but have had little application in the quantification of experimental lung injury. There is a growing and unfulfilled need for predictive animal models that can improve our understanding of disease pathogenesis and therapeutic intervention. Integration of MRI and NMR could extend the application of experimental data into the clinical setting. This study investigated the ability of MRI and metabolic NMR to detect and quantify inflammation-mediated lung injury. Pulmonary inflammation was induced in male B6C3F1 mice by intratracheal administration of IL-1β and TNF- under isoflurane anesthesia. Mice underwent MRI at 2, 4, 6, and 24 h after dosing. At 6 and 24 h lungs were harvested for metabolic NMR analysis. Data acquired from IL-1β+TNF--treated animals were compared with saline-treated control mice. The hyperintense-to-total lung volume (HTLV) ratio derived from MRI was higher in IL-1β+TNF--treated mice compared with control at 2, 4, and 6 h but returned to control levels by 24 h. The ability of MRI to detect pulmonary inflammation was confirmed by the association between HTLV ratio and histological and pathological end points. Principal component analysis of NMR-detectable metabolites also showed a temporal pattern for which energy metabolism-based biomarkers were identified. These data demonstrate that both MRI and metabolic NMR have utility in the detection and quantification of inflammation-mediated lung injury. Integration of these clinically available techniques into experimental models of lung injury could improve the translation of basic science knowledge and information to the clinic.

Metalloelastase in lungs and alveolar macrophages is modulated by extracellular substance P in mice

Xu, J., Xu, F., Barrett, E. — 2008-07-03

Metalloelastase (MMP-12), mainly produced by macrophages, has been shown to play a key role in the pathogenesis of emphysema in animal models. Chronic cigarette smoke increases pulmonary MMP-12, which is closely correlated with an elevation of pulmonary substance P (SP). Because alveolar macrophages (AMs) contain the neurokinin-1 receptor (NK1R), we tested whether SP was able to trigger the upregulation of MMP-12 synthesis in AMs by acting on the NK1R. AMs isolated from bronchoalveolar lavage cells in C3H/HeN mice were cultured with control medium or SP that was coupled without or with NK1R antagonists (CP-99,994 or aprepitant) for 24 h. We found that SP significantly increased the mRNA of MMP-12 and NK1R by 11-fold and 82%, respectively, in AMs (P < 0.05), and these responses were abolished by NK1R antagonists with little change in the cells' viability. Because pulmonary SP is primarily released by bronchopulmonary C-fibers (PCFs), we further asked whether destruction of PCFs would reduce SP and MMP-12. Two groups of mice were pretreated with vehicle and neonatal capsaicin (NCAP) to degenerate PCFs, respectively. Our results show that NCAP treatment significantly decreased mRNA and protein levels of SP associated with a reduction NK1R and MMP-12 in the lungs and AMs. These findings suggest that SP has a modulatory effect on pulmonary MMP-12 by acting on NK1R to trigger MMP-12 syntheses in the AMs.

MAP kinases mediate interleukin-13 effects on calcium signaling in human airway smooth muscle cells

2008-07-03

Interleukin-13 (IL-13) has been strongly implicated in the pathogenesis of allergic asthma through animal models that have shown that IL-13 is both necessary and sufficient to cause airway hyperresponsiveness (AHR). Airway smooth muscle (ASM) is a primary effector of AHR, and IL-13 increases the responsiveness of ASM, by increasing Ca²⁺ release intracellularly, to bronchoconstrictors such as histamine. The mechanisms and signaling pathways mediating this effect are incompletely understood. We have investigated the pathways through which IL-13 regulates the Ca²⁺ response to histamine in primary human ASM cell cultures. Functional IL-13 receptors were demonstrated by IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and mitogen-activated protein kinases (MAPKs). IL-13 increased Ca²⁺ responses to histamine. The augmentation of Ca²⁺ signaling was not affected by inhibition of STAT6 or p38 MAPK signaling but was prevented by concurrent inhibition of c-jun N-terminal kinase (JNK) and extracellular signal-related kinase (ERK) MAPKs. This inhibition did not affect the IL-13-induced increase in histamine receptors. We conclude that IL-13 induces potentiation of Ca²⁺ responses to contractile agonists by affecting mechanisms downstream of receptors. JNK and ERK MAPKs modulate these mechanisms.

Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis

Li, X., Molina-Molina, M., Abdul-Hafez, A., Uhal, V., Xaubet, A., Uhal, B. D. — 2008-07-03

Earlier work from this laboratory showed that local generation of angiotensin (ANG) II is required for the pathogenesis of experimental pulmonary fibrosis and that ANG peptides are expressed robustly in the lungs of patients with idiopathic pulmonary fibrosis (IPF). Angiotensin converting enzyme-2 (ACE-2) degrades the octapeptide ANG II to form the heptapeptide ANG1-7 and thereby limits ANG II accumulation. On this basis, we hypothesized that ACE-2 would be protective against experimental lung fibrogenesis and might be downregulated in human and experimental lung fibrosis. In lung biopsy specimens from patients with IPF, ACE-2 mRNA and enzyme activity were decreased by 92% (P < 0.01) and 74% (P < 0.05), respectively. ACE-2 mRNA and activity were also decreased similarly in the lungs of bleomycin-treated rats and C57-BL6 mice. In mice exposed to low doses of bleomycin, lung collagen accumulation was enhanced by intratracheal administration of either ACE-2-specific small interfering RNAs (siRNAs) or the peptide DX₆₀₀, a competitive inhibitor of ACE-2 (P < 0.05). Administration of either ACE-2 siRNA or DX₆₀₀ significantly increased the ANG II content of mouse lung tissue above the level induced by bleomycin alone. Coadministration of the ANG II receptor antagonist saralasin blocked the DX₆₀₀-induced increase in lung collagen. Moreover, purified recombinant human ACE-2, delivered to mice systemically by osmotic minipump, attenuated bleomycin-induced lung collagen accumulation. Together, these data show that ACE-2 mRNA and activity are severely downregulated in both human and experimental lung fibrosis and suggest that ACE-2 protects against lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II.

Glucocorticoid regulation of CD38 expression in human airway smooth muscle cells: role of dual specificity phosphatase 1

Kang, B. N., Jude, J. A., Panettieri, R. A., Walseth, T. F., Kannan, M. S. — 2008-07-03

The enzymatic activity of CD38, ADP-ribosyl cyclase, synthesizes the calcium mobilizing molecule cyclic ADP-ribose from β-NAD. In human airway smooth muscle (HASM) cells, CD38 expression is augmented by the inflammatory cytokine, TNF-, causing increased intracellular calcium response to agonists. The transcriptional and posttranscriptional regulation of CD38 expression involves signaling through MAPKs and requires activation of NF-B and activator protein-1 (AP-1). The cytokine-augmented CD38 expression is decreased by anti-inflammatory glucocorticoids due to inhibition of NF-B activation and other mechanisms. In this study, we investigated glucocorticoid regulation of CD38 expression in HASM cells through the MKP-1. In HASM cells, dexamethasone and TNF- induced MKP-1 expression (both mRNA and protein) rapidly. Dexamethasone decreased TNF--induced phosphorylation of the major MAPKs, i.e., ERK, p38, and JNK, and decreased the activation of NF-B and AP-1. Dexamethasone also decreased CD38 expression induced by TNF-, and part of this effect was attributable to decreased transcript stability. In cells transfected with MKP-1-specific small interfering RNAs (siRNAs), there was significant attenuation of MKP-1 expression and partial, but nonsignificant, reversal of dexamethasone inhibition of CD38 expression. These results indicate that regulation of CD38 expression in HASM cells by glucocorticoids involves decreased signaling through MAPKs and activation of transcription factors. The glucocorticoid effects on decreased CD38 expression and function result from regulation through transcription and transcript stability.

Increased transcription of cytokine genes in human lung epithelial cells through activation of a TRPM8 variant by cold temperatures

Sabnis, A. S., Reilly, C. A., Veranth, J. M., Yost, G. S. — 2008-07-03

Recognition of temperature is a critical element of sensory perception and allows mammals to evaluate both their external environment and internal status. The respiratory epithelium is constantly exposed to the external environment, and prolonged inhalation of cold air is detrimental to human airways. However, the mechanisms responsible for adverse effects elicited by cold air on the human airways are poorly understood. Transient receptor potential melastatin family member 8 (TRPM8) is a well-established cold- and menthol-sensing cation channel. We recently discovered a functional cold- and menthol-sensing variant of the TRPM8 ion channel in human lung epithelial cells. The present study explores the hypothesis that this TRPM8 variant mediates airway cell inflammatory responses elicited by cold air/temperatures. Here, we show that activation of the TRPM8 variant in human lung epithelial cells leads to increased expression of several cytokine and chemokine genes, including IL-1, -1β, -4, -6, -8, and -13, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-. Our results provide new insights into mechanisms that potentially control airway inflammation due to inhalation of cold air and suggest a possible role for the TRPM8 variant in the pathophysiology of asthma.

Muscarinic receptor M1 and phosphodiesterase 1 are key determinants in pulmonary vascular dysfunction following perinatal hypoxia in mice

2008-07-03

Perinatal adverse events such as limitation of nutrients or oxygen supply are associated with the occurrence of diseases in adulthood, like cardiovascular diseases and diabetes. We investigated the long-term effects of perinatal hypoxia on the lung circulation, with particular attention to the nitric oxide (NO)/cGMP pathway. Mice were placed under hypoxia in utero 5 days before delivery and for 5 days after birth. Pups were then bred in normoxia until adulthood. Adults born in hypoxia displayed an altered regulation of pulmonary vascular tone with higher right ventricular pressure in normoxia and increased sensitivity to acute hypoxia compared with controls. Perinatal hypoxia dramatically decreased endothelium-dependent relaxation induced by ACh in adult pulmonary arteries (PAs) but did not influence NO-mediated endothelium-independent relaxation. The M₃ muscarinic receptor was implicated in the relaxing action of ACh and M₁ muscarinic receptor (M₁AChR) in its vasoconstrictive effects. Pirenzepine or telenzepine, two preferential inhibitors of M₁AChR, abolished the adverse effects of perinatal hypoxia on ACh-induced relaxation. M₁AChR mRNA expression was increased in lungs and PAs of mice born in hypoxia. The phosphodiesterase 1 (PDE1) inhibitor vinpocetine also reversed the decrease in ACh-induced relaxation following perinatal hypoxia, suggesting that M₁AChR-mediated alteration of ACh-induced relaxation is due to the activation of calcium-dependent PDE1. Therefore, perinatal hypoxia leads to an altered pulmonary circulation in adulthood with vascular dysfunction characterized by impaired endothelium-dependent relaxation and M₁AChR plays a predominant role. This raises the possibility that muscarinic receptors could be key determinants in pulmonary vascular diseases in relation to "perinatal imprinting."

The inhaled Rho kinase inhibitor Y-27632 protects against allergen-induced acute bronchoconstriction, airway hyperresponsiveness, and inflammation

Schaafsma, D., Bos, I. S. T., Zuidhof, A. B., Zaagsma, J., Meurs, H. — 2008-07-03

Recently, we have shown that allergen-induced airway hyperresponsiveness (AHR) after the early (EAR) and late (LAR) asthmatic reaction in guinea pigs could be reversed acutely by inhalation of the Rho kinase inhibitor Y-27632. The present study addresses the effects of pretreatment with inhaled Y-27632 on the severity of the allergen-induced EAR and LAR, the development of AHR after these reactions, and airway inflammation. Using permanently instrumented and unrestrained ovalbumin (OA)-sensitized guinea pigs, single OA challenge-induced EAR and LAR, expressed as area under the lung function (pleural pressure, P_pl) time-response curve, were measured, and histamine PC₁₀₀ (provocation concentration causing a 100% increase of P_pl) values were assessed 24 h before, and at 6 and 24 h after, the OA challenge (after the EAR and LAR, respectively). Thirty minutes before and 8 h after OA challenge, saline or Y-27632 (5 mM) was nebulized. After the last PC₁₀₀ value, bronchoalveolar lavage (BAL) was performed, and the inflammatory cell profile was determined. It was demonstrated that inhalation of Y-27632 before allergen challenge markedly reduced the immediate allergen-induced peak rise in P_pl, without significantly reducing the overall EAR and LAR. Also, pretreatment with Y-27632 considerably protected against the development of AHR after the EAR and fully prevented AHR after the LAR. These effects could not be explained by a direct effect of Y-27632 on the histamine responsiveness, because of the short duration of the acute bronchoprotection of Y-27632 (<90 min). In addition, Y-27632 reduced the number of total inflammatory cells, eosinophils, macrophages, and neutrophils recovered from the BAL. Altogether, inhaled Y-27632 protects against acute allergen-induced bronchoconstriction, development of AHR after the EAR and LAR, and airway inflammation in an established guinea pig model of allergic asthma.

Induction of IL-8 by Mycoplasma pneumoniae membrane in BEAS-2B cells

2008-07-03

Mycoplasma pne umoniae is an extracellular pathogen, residing on mucosal surfaces of the respiratory and genital tracts. The lack of cell walls in mycoplasmas facilitates the direct contact of the bacterial membrane with the host cell. The cell membrane of mycoplasma is the major inducer of the host pathogenic response. Airway diseases caused by M. pneumoniae include bronchiolitis, bronchitis, and rarely bronchiectasis. In such disorders, neutrophil infiltration of the airways predominates. More recently, M. pneumoniae has been implicated in the pathogenesis of asthma. Epithelial cells play an important role in recruiting inflammatory cells into the airways. Since M. pneumoniae infection of human epithelial cells induces expression of IL-8—a potent activator of neutrophils—we investigated the signaling and transcriptional mechanisms by which mycoplasma membrane induces expression of this chemokine. In BEAS-2B human bronchial epithelial cells, mycoplasma membrane fraction (MMF) increased IL-8 mRNA and protein production. Activation of the transcriptional elements activating protein-1, nuclear factor-interleukin-6, and particularly NF-B are essential for optimal IL-8 production by MMF. The mitogen-activated protein kinases individually played a modest role in MMF-induced IL-8 production. Toll-like receptor-2 did not play a significant role in MMF-induction of IL-8. Antibiotics with microbicidal activity against M. pneumoniae are also known to have anti-inflammatory effects. Whereas clarithromycin, azithromycin, and moxifloxacin individually were able to inhibit TNF--induction of IL-8, each failed to inhibit MMF-induction of IL-8.

Ciliated epithelial cell lifespan in the mouse trachea and lung

Rawlins, E. L., Hogan, B. L. M. — 2008-07-03

The steady-state turnover of epithelial cells in the lung and trachea is highly relevant to investigators who are studying endogenous stem cells, manipulating gene expression in vivo, or using viral vectors for gene therapy. However, the average lifetime of different airway epithelial cell types has not previously been assessed using currently available genetic techniques. Here, we use Cre/loxP genetic technology to indelibly label a random fraction of ciliated cells throughout the airways of a cohort of mice and follow them in vivo for up to 18 mo. We demonstrate that ciliated airway epithelial cells are a terminally differentiated population. Moreover, their average half-life of 6 mo in the trachea and 17 mo in the lung is much longer than previously available estimates, with significant numbers of labeled cells still present after 18 mo.