AJP: Lung Cellular and Molecular Physiology recent issues

Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure

Stenmark, K. R., Meyrick, B., Galie, N., Mooi, W. J., McMurtry, I. F. — Fri, 20 Nov 2009 16:08:59 PST

At present, six groups of chronic pulmonary hypertension (PH) are described. Among these, group 1 (and 1') comprises a group of diverse diseases termed pulmonary arterial hypertension (PAH) that have several pathophysiological, histological, and prognostic features in common. PAH is a particularly severe and progressive form of PH that frequently leads to right heart failure and premature death. The diagnosis of PAH must include a series of defined clinical parameters, which extend beyond mere elevations in pulmonary arterial pressures and include precapillary PH, pulmonary hypertensive arteriopathy (usually with plexiform lesions), slow clinical onset (months or years), and a chronic time course (years) characterized by progressive deterioration. What appears to distinguish PAH from other forms of PH is the severity of the arteriopathy observed, the defining characteristic of which is "plexogenic arteriopathy." The pathogenesis of this arteriopathy remains unclear despite intense investigation in a variety of animal model systems. The most commonly used animal models ("classic" models) are rodents exposed to either hypoxia or monocrotaline. Newer models, which involve modification of classic approaches, have been developed that exhibit more severe PH and vascular lesions, which include neointimal proliferation and occlusion of small vessels. In addition, genetically manipulated mice have been generated that have provided insight into the role of specific molecules in the pulmonary hypertensive process. Unfortunately, at present, there is no perfect preclinical model that completely recapitulates human PAH. All models, however, have provided and will continue to provide invaluable insight into the numerous pathways that contribute to the development and maintenance of PH. Use of both classic and newly developed animal models will allow continued rigorous testing of new hypotheses regarding pathogenesis and treatment. This review highlights progress that has been made in animal modeling of this important human condition.

Thinking small, but with big league consequences: procoagulant microparticles in the alveolar space

Zimmerman, G. A. — Fri, 20 Nov 2009 16:08:59 PST

Procoagulant alveolar microparticles in the lungs of patients with acute respiratory distress syndrome

Bastarache, J. A., Fremont, R. D., Kropski, J. A., Bossert, F. R., Ware, L. B. — Fri, 20 Nov 2009 16:08:59 PST

Coagulation and fibrinolysis abnormalities are observed in acute lung injury (ALI) in both human disease and animal models and may contribute to ongoing inflammation in the lung. Tissue factor (TF), the main initiator of the coagulation cascade, is upregulated in the lungs of patients with ALI/acute respiratory distress syndrome (ARDS) and likely contributes to fibrin deposition in the air space. The mechanisms that govern TF upregulation and activation in the lung are not well understood. In the vascular space, TF-bearing microparticles (MPs) are central to clot formation and propagation. We hypothesized that TF-bearing MPs in the lungs of patients with ARDS contribute to the procoagulant phenotype in the air space during acute injury and that the alveolar epithelium is one potential source of TF MPs. We studied pulmonary edema fluid collected from patients with ARDS compared with a control group of patients with hydrostatic pulmonary edema. Patients with ARDS have higher concentrations of MPs in the lung compared with patients with hydrostatic edema (25.5 IQR 21.3–46.9 vs. 7.8 IQR 2.3–27.5 µmol/l, P = 0.009 by Mann-Whitney U-test). These MPs are enriched for TF, have procoagulant activity, and likely originate from the alveolar epithelium [as measured by elevated levels of RAGE (receptor for advanced glycation end products) in ARDS MPs compared with hydrostatic MPs]. Furthermore, alveolar epithelial cells in culture release procoagulant TF MPs in response to a proinflammatory stimulus. These findings suggest that alveolar epithelial-derived MPs are one potential source of TF procoagulant activity in the air space in ARDS and that epithelial MP formation and release may represent a unique therapeutic target in ARDS.

More life for a "terminal" cell

Borok, Z., Crandall, E. D. — Fri, 20 Nov 2009 16:08:59 PST

Rat alveolar type I cells proliferate, express OCT-4, and exhibit phenotypic plasticity in vitro

Gonzalez, R. F., Allen, L., Dobbs, L. G. — Fri, 20 Nov 2009 16:08:59 PST

Alveolar type I (TI) cells are large, squamous cells that cover 95–99% of the internal surface area of the lung. Although TI cells are believed to be terminally differentiated, incapable of either proliferation or phenotypic plasticity, TI cells in vitro both proliferate and express phenotypic markers of other differentiated cell types. Rat TI cells isolated in purities of >99% proliferate in culture, with a sixfold increase in cell number before the cells reach confluence; >50% of the cultured TI cells are Ki67+. At cell densities of 1–2 cells/well, ~50% of the cells had the capacity to form colonies. Under the same conditions, type II cells do not proliferate. Cultured TI cells express RTI40 and aquaporin 5, phenotypic markers of the TI cell phenotype. By immunofluorescence, Western blotting, and Q-PCR, TI cells express OCT-4A (POU5F1), a transcription factor associated with maintenance of the pluripotent state in stem cells. Based on the expression patterns of various marker proteins, TI cells are distinct from either of two recently described putative pulmonary multipotent cell populations, the bronchoalveolar stem cell or the OCT-4+ stem/progenitor cell. Although TI cells in adult rat lung tissue do not express either surfactant protein C (SP-C) or CC10, respective markers of the TII and Clara cell phenotypes, in culture TI cells can be induced to express both SP-C and CC10. Together, the findings that TI cells proliferate and exhibit phenotypic plasticity in vitro raise the possibility that TI cells may have similar properties in vivo.

Unveiling cell phenotypes in lung vascular remodeling

Johns, R. A., Yamaji-Kegan, K. — Fri, 20 Nov 2009 16:08:59 PST

Sustained hypoxia leads to the emergence of cells with enhanced growth, migratory, and promitogenic potentials within the distal pulmonary artery wall

Fri, 20 Nov 2009 16:08:59 PST

All forms of chronic pulmonary hypertension (PH) are characterized by structural remodeling of the pulmonary artery (PA) media, a process previously attributed solely to changes in the phenotype of resident smooth muscle cells (SMC). However, recent experimental evidence in both systemic and pulmonary circulations suggests that other cell types, including circulating and local progenitors, contribute significantly to this process. The goal of this study was to determine if hypoxia-induced remodeling of distal PA (dPA) media involves the emergence of cells with phenotypic and functional characteristics distinct from those of resident dPA SMC and fibroblasts. In vivo, in contrast to the phenotypically uniform SMC composition of dPA media in control calves, the remodeled dPA media of neonatal calves with severe hypoxia-induced PH comprised cells exhibiting a distinct phenotype, including the expression of hematopoetic (CD45), leukocytic/monocytic (CD11b, CD14), progenitor (cKit), and motility-associated (S100A4) cell markers. Consistent with these in vivo observations, primary cell cultures isolated from dPA media of hypertensive calves yielded not only differentiated SMC, but also smaller, morphologically rhomboidal (thus termed here "R") cells that transiently expressed CD11b, constitutively expressed the mesenchymal cell marker type I procollagen, expressed high mRNA levels of progenitor cell markers cKit, CD34, CD73, as well as for inflammatory mediators, IL-6 and MCP-1, and, with time in culture, gained expression of a myofibroblast marker, -SM-actin. R cells exhibited highly augmented proliferative, migratory, invasive, and potent promitogenic capabilities, which were due, at least in part, to the production of PDGFs, SDF-1/CXCL12, and S100A4. These data suggest that the cellular mechanisms of dPA remodeling include the emergence of cells with phenotypic and functional characteristics markedly distinct from those of resident dPA cells.

The olfactory system is affected by steroid aerosol treatment in mice

Mucignat-Caretta, C., Bondi, M., Rubini, A., Calabrese, F., Barbato, A. — Fri, 20 Nov 2009 16:09:00 PST

Asthma needs continuous treatment often for years. In humans, some drugs are administered via aerosol, therefore they come in contact with both respiratory and olfactory mucosa. We explored the possibility that antiasthma corticosteroid treatment could influence the olfactory function by passage through the nose. A group of mice was exposed twice daily for 42 days to fluticasone propionate aerosol and was compared with a control group. Olfactory behavior, respiratory mechanics, histology, and immunoreactivity in the olfactory system were assessed. Fluticasone-treated mice were slower in retrieving a piece of hidden food, but both groups were similarly fast when the food was visible. When a clearly detectable odor was present in the environment, all mice behaved in a similar way. Respiratory mechanics indices were similar in all mice except for the viscose resistance, which was reduced in fluticasone-treated mice. Olfactory mucosa of fluticasone-treated mice was thicker than that of controls. Slight but consistent differences in staining were present for Olfactory Marker Protein but not for other proteins. A mild impairment of olfactory function is present in mice chronically treated with fluticasone aerosol, apparently accompanied by slight modifications of the olfactory receptor cells, and suggests monitoring of olfactory function modifications in long-term steroid users.

Tie2-mediated loss of peroxisome proliferator-activated receptor-{gamma} in mice causes PDGF receptor-{beta}-dependent pulmonary arterial muscularization

Fri, 20 Nov 2009 16:09:00 PST

Peroxisome proliferator-activated receptor (PPAR)- is reduced in pulmonary arteries (PAs) of patients with PA hypertension (PAH), and we reported that deletion of PPAR in smooth muscle cells (SMCs) of transgenic mice results in PAH. However, the sequelae of loss of PPAR in PA endothelial cells (ECs) are unknown. Therefore, we bred Tie2-Cre mice with PPAR^flox/flox mice to induce EC loss of PPAR (Tie2 PPAR^–/–), and we assessed PAH by right ventricular systolic pressure (RVSP), RV hypertrophy (RVH), and muscularized distal PAs in room air (RA), after chronic hypoxia (CH), and after 4 wk of recovery in RA (Rec-RA). The Tie2 PPAR^–/– mice developed spontaneous PAH in RA with increased RVSP, RVH, and muscularized PAs vs. wild type (WT); both genotypes exhibited a similar degree of PAH following chronic hypoxia, but Tie2 PPAR^–/– mice had more residual PAH compared with WT mice after Rec-RA. The Tie2 PPAR^–/– vs. WT mice in RA had increased platelet-derived growth factor receptor-β (PDGF-Rβ) expression and signaling, despite an elevation in the PPAR target apolipoprotein E, an inhibitor of PDGF signaling. Inhibition of PDGF-Rβ signaling with imatinib, however, was sufficient to reverse the PAH observed in the Tie2 PPAR^–/– mice. Thus the disruption of PPAR signaling in EC is sufficient to cause mild PAH and to impair recovery from CH-induced PAH. Inhibition of heightened PDGF-Rβ signaling is sufficient to reverse PAH in this genetic model.

Regulation of inflammation by Rac2 in immune complex-mediated acute lung injury

Fri, 20 Nov 2009 16:09:00 PST

Acute lung injury (ALI) is an inflammatory disorder associated with recruitment and activation of neutrophils in lungs. Rac2, a member of the Rho GTPase subfamily, is an essential regulator of neutrophil degranulation, superoxide release, and chemotaxis. Here, we hypothesized that Rac2 is important in mediating lung injury. Using a model of IgG immune complex-mediated ALI, we showed that injury was attenuated in rac2^–/– mice compared with wild-type (WT) mice undergoing ALI, with significant decreases in alveolar leukocyte numbers, vascular leakage, and the inflammatory mediators, myeloperoxidase (MPO) and matrix metalloproteinases (MMPs). Reduced injury in rac2^–/– mice was not associated with diminished cytokine and chemokine production, since bronchoalveolar lavage (BAL) levels of IL-17, TNF, CCL3, CXCL1, and CXCL2 were similarly increased in WT and rac2^–/– mice with ALI compared with sham-treated mice (no ALI). BAL levels of MMP-2 and MMP-9 were significantly decreased in the airways of rac2^–/– mice with ALI. Immunohistochemical analysis revealed that MMP-2 and MMP-9 expression was evident in alveolar macrophages and interstitial neutrophils in WT ALI. In contrast, MMP-positive cells were less prominent in rac2^–/– mice with ALI. Chimeric mice showed that Rac2-mediated lung injury was dependent on hematopoietic cells derived from bone marrow. We propose that lung injury in response to immune complex deposition is dependent on Rac2 in alveolar macrophages and neutrophils.

Inhaled nitric oxide improves lung structure and pulmonary hypertension in a model of bleomycin-induced bronchopulmonary dysplasia in neonatal rats

Tourneux, P., Markham, N., Seedorf, G., Balasubramaniam, V., Abman, S. H. — Fri, 20 Nov 2009 16:09:00 PST

Whether inhaled nitric oxide (iNO) prevents the development of bronchopulmonary dysplasia (BPD) in premature infants is controversial. In adult rats, bleomycin (Bleo) induces lung fibrosis and pulmonary hypertension, but the effects of Bleo on the developing lung and iNO treatment on Bleo-induced neonatal lung injury are uncertain. Therefore, we sought to determine whether early and prolonged iNO therapy attenuates changes of pulmonary vascular and alveolar structure in a model of BPD induced by Bleo treatment of neonatal rats. Sprague-Dawley rat pups were treated with Bleo (1 mg/kg ip daily) or vehicle (controls) from day 2 to 10, followed by recovery from day 11 to 19. Treatment groups received early (days 2–10), late (days 11–19), or prolonged iNO therapy (10 ppm; days 2–19). We found that compared with controls, Bleo increased right ventricular hypertrophy (RVH), and pulmonary arterial wall thickness, and reduced vessel density alveolarization. In each iNO treatment group, iNO decreased RVH (P < 0.01) and wall thickness (P < 0.01) and restored vessel density after Bleo (P < 0.05). iNO therapy improved alveolarization for each treatment group after Bleo; however, the values remained abnormal compared with controls. Prolonged iNO treatment had greater effects on lung structure after bleomycin than late treatment alone. We conclude that Bleo induces lung structural changes that mimic BPD in neonatal rats, and that early and prolonged iNO therapy prevents right ventricle hypertrophy and pulmonary vascular remodeling and partially improves lung structure.

Role of Toll-like receptor 5 in the innate immune response to acute P. aeruginosa pneumonia

Morris, A. E., Liggitt, H. D., Hawn, T. R., Skerrett, S. J. — Fri, 20 Nov 2009 16:09:00 PST

Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and an important pathogen in patients with chronic lung disease, such as cystic fibrosis and bronchiectasis. The contribution of Toll-like receptor 5 (TLR5) to the innate immune response to this organism is incompletely understood. We exposed wild-type and TLR5-deficient (Tlr5^–/–) mice to aerosolized P. aeruginosa at low and high inocula and assessed bacterial clearance, lung inflammation, and cytokine production 4 and 24 h after infection. Bacterial clearance was impaired in Tlr5^–/– mice after low-inoculum, but not high-inoculum, infection. Early bronchoalveolar accumulation of neutrophils was reduced in Tlr5^–/– mice after low- and high-dose infection. Cytokine responses, including markedly impaired monocyte chemoattractant protein-1 production 4 h after low- and high-inoculum challenge, were selectively altered in Tlr5^–/– mice. In contrast, there was no impairment of bacterial clearance, neutrophil recruitment, or monocyte chemoattractant protein-1 production in Tlr5^–/– mice after infection with a nonflagellated isotypic strain of P. aeruginosa. Thus TLR5-mediated recognition of flagellin is involved in activating pulmonary defenses against P. aeruginosa and contributes to antibacterial resistance in a manner that is partially inoculum dependent. These data are the first to demonstrate a unique role for TLR5 in the innate immune response to P. aeruginosa lung infection.

Hypoxia-induced alveolar epithelial-mesenchymal transition requires mitochondrial ROS and hypoxia-inducible factor 1

Fri, 20 Nov 2009 16:09:00 PST

Patients with acute lung injury develop hypoxia, which may lead to lung dysfunction and aberrant tissue repair. Recent studies have suggested that epithelial-mesenchymal transition (EMT) contributes to pulmonary fibrosis. We sought to determine whether hypoxia induces EMT in alveolar epithelial cells (AEC). We found that hypoxia induced the expression of -smooth muscle actin (-SMA) and vimentin and decreased the expression of E-cadherin in transformed and primary human, rat, and mouse AEC, suggesting that hypoxia induces EMT in AEC. Both severe hypoxia and moderate hypoxia induced EMT. The reactive oxygen species (ROS) scavenger Euk-134 prevented hypoxia-induced EMT. Moreover, hypoxia-induced expression of -SMA and vimentin was prevented in mitochondria-deficient ⁰ cells, which are incapable of ROS production during hypoxia. CoCl₂ and dimethyloxaloylglycine, two compounds that stabilize hypoxia-inducible factor (HIF)- under normoxia, failed to induce -SMA expression in AEC. Furthermore, overexpression of constitutively active HIF-1 did not induce -SMA. However, loss of HIF-1 or HIF-2 abolished induction of -SMA mRNA during hypoxia. Hypoxia increased the levels of transforming growth factor (TGF)-β1, and preincubation of AEC with SB431542, an inhibitor of the TGF-β1 type I receptor kinase, prevented the hypoxia-induced EMT, suggesting that the process was TGF-β1 dependent. Furthermore, both ROS and HIF- were necessary for hypoxia-induced TGF-β1 upregulation. Accordingly, we have provided evidence that hypoxia induces EMT of AEC through mitochondrial ROS, HIF, and endogenous TGF-β1 signaling.

Airway surface liquid depth measured in ex vivo fragments of pig and human trachea: dependence on Na+ and Cl- channel function

Fri, 20 Nov 2009 16:09:00 PST

The airway surface liquid (ASL) is the thin fluid layer lining the airways whose depth may be reduced in cystic fibrosis. Prior measurements of ASL depth have been made in airway epithelial cell cultures. Here, we established methodology to measure ASL depth to ~1-µm accuracy in ex vivo fragments of freshly obtained human and pig tracheas. Airway fragments were mounted in chambers designed for perfusion of the basal surface and observation of the apical, fluorescently stained ASL by scanning confocal microscopy using a high numerical aperture lens immersed in perfluorocarbon. Measurement accuracy was verified using standards of specified fluid thickness. ASL depth in well-differentiated primary cultures of human nasal respiratory epithelium was 8.0 ± 0.5 µm (SE 10 cultures) under basal conditions, 8.4 ± 0.4 µm following ENaC inhibition by amiloride, and 14.5 ± 1.2 µm following CFTR stimulation by cAMP agonists. ASL depth in human trachea was 7.0 ± 0.7 µm under basal conditions, 11.0 ± 1.7 µm following amiloride, 17.0 ± 3.4 µm following cAMP agonists, and 7.1 ± 0.5 µm after CFTR inhibition. Similar results were found in pig trachea. This study provides the first direct measurements of ASL depth in intact human airways and indicates the involvement of ENaC sodium channels and CFTR chloride channels in determining ASL depth. We suggest that CF lung disease may be caused by the inability of CFTR-deficient airways to increase their ASL depth transiently following secretory stimuli that in non-CF airways produce transient increases in ASL depth.

Differential expression of platelet-activating factor acetylhydrolase in lung macrophages

Howard, K. M. — Fri, 20 Nov 2009 16:09:00 PST

Platelet-activating factor (PAF) acetylhydrolase plays a crucial role inactivating the potent inflammatory mediator, PAF. PAF is implicated in the initiation and propagation of acute lung injury. Although PAF acetylhydrolase is a constitutively active plasma protein, increased PAF production during inflammatory events may necessitate an increase in PAF acetylhydrolase activity in the local environment. A series of experiments were conducted to determine whether the systemic administration of LPS to Sprague-Dawley rats resulted in enhanced expression of PAF acetylhydrolase in lung tissue. Ribonuclease protection assays revealed a dramatic increase in PAF acetylhydrolase mRNA, which peaked at 24 h following in vivo LPS administration. The increase in PAF acetylhydrolase mRNA was dose dependent and was detected when as little as 10 µg/kg of LPS was administered. Western blot analyses of lung tissue homogenates confirmed an increased production of PAF acetylhydrolase protein in response to LPS. In addition, Western blot analyses revealed the rat PAF acetylhydrolase protein exhibited heterogeneous molecular weights with predominant species migrating at 63 and 67 kDa. Some of the molecular weight heterogeneity likely resulted from extensive glycosylation of the secreted protein. Immunohistochemical analyses of lung tissue sections and colocalization experiments revealed a heterogenous population of cells that express the plasma-type PAF acetylhydrolase. Lung interstitial macrophages were PAF acetylhydrolase positive, but surprisingly, alveolar macrophages did not increase expression of PAF acetylhydrolase in response to systemic LPS administration. In addition, rat granulocytes consisting primarily of neutrophils were strongly positive for PAF acetylhydrolase in the LPS-exposed lung tissue. The absence of immunoreactive PAF acetylhydrolase in alveolar macrophages obtained from bronchial alveolar lavage confirmed that systemic LPS administration resulted in enhanced PAF acetylhydrolase expression only in a subset of lung macrophages.

Hypoxia promotes human pulmonary artery smooth muscle cell proliferation through induction of arginase

Chen, B., Calvert, A. E., Cui, H., Nelin, L. D. — Fri, 20 Nov 2009 16:09:00 PST

Vascular remodeling and smooth muscle cell proliferation are hallmark pathogenic features of pulmonary artery hypertension (PAH). Alterations in the metabolism of l-arginine via arginase and nitric oxide synthase play a critical role in the endothelial dysfunction seen in PAH. l-arginine metabolism by arginase produces l-ornithine and urea. l-ornithine is a precursor for polyamine and proline synthesis, ultimately leading to an increase in cellular proliferation. Given the integral role of the smooth muscle layer in the pathogenesis of hypoxia-induced PAH, we hypothesized that hypoxia would increase cellular proliferation via arginase induction in human pulmonary artery smooth muscle cells (hPASMC). We found that arginase II mRNA and protein expression were significantly increased in cultured hPASMC exposed to 1% O₂ for 24 and 48 h, which coincided with an increase in arginase activity at 48 h. There were no hypoxia-induced changes in levels of arginase I mRNA or protein in cultured hPASMC. Exposure to hypoxia resulted in more than one and a half times as many viable cells after 120 h than normoxic exposure. The addition of the arginase inhibitor, S-(2-boronoethyl)-l-cysteine, completely prevented both the hypoxia-induced increase in arginase activity and proliferation in hPASMC. Furthermore, transfection of small interfering RNA (siRNA) targeting arginase II in hPASMC resulted in knockdown of arginase II protein levels and complete prevention of the hypoxia-induced cellular proliferation. These data support our hypothesis that hypoxia increases proliferation of hPASMC through the induction of arginase II.

Hyperoxia disrupts vascular endothelial growth factor-nitric oxide signaling and decreases growth of endothelial colony-forming cells from preterm infants

Fri, 20 Nov 2009 16:09:00 PST

Exposure of preterm infants to hyperoxia impairs vascular growth, contributing to the development of bronchopulmonary dysplasia and retinopathy of prematurity. Disruption of vascular endothelial growth factor (VEGF)-nitric oxide (NO) signaling impairs vascular growth. Endothelial progenitor cells (EPCs) may play an important role in vascular growth. Endothelial colony-forming cells (ECFCs), a type of EPC, from human preterm cord blood are more susceptible to hyperoxia-induced growth impairment than term ECFCs. Therefore, we hypothesized that hyperoxia disrupts VEGF-NO signaling and impairs growth in preterm ECFCs and that exogenous VEGF or NO preserves growth in hyperoxia. Growth kinetics of preterm cord blood-derived ECFCs (gestational ages, 27–34 wk) were assessed in room air (RA) and hyperoxia (40–50% oxygen) with or without VEGF, NO, or N-nitro-l-arginine. VEGF, VEGF receptor-2 (VEGFR-2), and endothelial NO synthase (eNOS) protein expression and NO production were compared. Compared with RA controls, hyperoxia significantly decreased growth, VEGFR-2 and eNOS expression, and NO production. VEGF treatment restored growth in hyperoxia to values measured in RA controls and significantly increased eNOS expression in hyperoxia. NO treatment also increased growth in hyperoxia. N-nitro-l-arginine treatment inhibited VEGF-augmented growth in RA and hyperoxia. We conclude that hyperoxia decreases growth and disrupts VEGF-NO signaling in human preterm ECFCs. VEGF treatment restores growth in hyperoxia by increasing NO production. NO treatment also increases growth during hyperoxia. Exogenous VEGF or NO may protect preterm ECFCs from the adverse effects of hyperoxia and preservation of ECFC function may improve outcomes of preterm infants.

Age-dependent endothelial nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice

Fri, 20 Nov 2009 16:09:00 PST

Endoglin is a TGF-β superfamily receptor critical for endothelial cell function. Mutations in this gene are associated with hereditary hemorrhagic telangiectasia type I (HHT1), and clinical signs of disease are generally more evident later in life. We previously showed that systemic vessels of adult Eng heterozygous (Eng^+/–) mice exhibit increased vasorelaxation due to uncoupling of endothelial nitric oxide synthase (eNOS). We postulated that these changes may develop with age and evaluated pulmonary arteries from newborn and adult Eng^+/– mice for eNOS-dependent, acetylcholine (ACh-induced) vasorelaxation, compared with that of age-matched littermate controls. While ACh-induced vasorelaxation was similar in all newborn mice, it was significantly increased in the adult Eng^+/– vs. control vessels. The vasodilatory responses were inhibited by l-NAME suggesting eNOS dependence. eNOS uncoupling was observed in lung tissues of adult, but not newborn, heterozygous mice and was associated with increased production of reactive O₂ species (ROS) in adult Eng^+/– vs. control lungs. Interestingly, ROS generation was higher in adult than newborn mice and so were the levels of NADPH oxidase 4 and SOD 1, 2, 3 isoforms. However, enzyme protein levels and NADPH activity were normal in adult Eng^+/– lungs indicating that the developmental maturation of ROS generation and scavenging cannot account for the increased vasodilatation observed in adult Eng^+/– mice. Our data suggest that eNOS-dependent H₂O₂ generation in Eng^+/– lungs accounts for the heightened pulmonary vasorelaxation. To the extent that these mice mimic human HHT1, age-associated pulmonary vascular eNOS uncoupling may explain the late childhood and adult onset of clinical lung manifestations.

Role of lung iron in determining the bacterial and host struggle in cystic fibrosis

Reid, D. W., Anderson, G. J., Lamont, I. L. — Mon, 26 Oct 2009 14:24:03 PDT

Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It is a multiorgan system disease that affects the lungs, gastrointestinal tract, liver, and pancreas. The majority of morbidity and mortality in CF relates to chronic airway infection with a variety of bacterial species, commencing in very early infancy, which results in lung destruction and ultimately organ failure (41, 43). This review focuses on iron homeostasis in the CF lung and its role in determining the success and chronicity of Pseudomonas aeruginosa infection. There have been previous excellent reviews regarding iron metabolism in the lower respiratory tract and mechanisms of P. aeruginosa iron acquisition, and we direct readers to these articles for further background reading (31, 53, 58, 77, 96). In this review, we have brought the "two sides of the coin" together to provide a holistic overview of the relationship between host and bacterial iron homeostasis and put this information into the context of current understanding on infection in the CF lung.

Could N-acetylcysteine slow progression of idiopathic pulmonary fibrosis by inhibiting EMT?

Wolters, P. J. — Mon, 26 Oct 2009 14:24:03 PDT

N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition

Felton, V. M., Borok, Z., Willis, B. C. — Mon, 26 Oct 2009 14:24:03 PDT

The ability of transforming growth factor-β1 (TGF-β1) to induce epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AEC) in vitro and in vivo, together with the demonstration of EMT in biopsies of idiopathic pulmonary fibrosis (IPF) patients, suggests a role for TGF-β1-induced EMT in disease pathogenesis. We investigated the effects of N-acetylcysteine (NAC) on TGF-β1-induced EMT in a rat epithelial cell line (RLE-6TN) and in primary rat alveolar epithelial cells (AEC). RLE-6TN cells exposed to TGF-β1 for 5 days underwent EMT as evidenced by acquisition of a fibroblast-like morphology, downregulation of the epithelial-specific protein zonula occludens-1, and induction of the mesenchymal-specific proteins -smooth muscle actin (-SMA) and vimentin. These changes were inhibited by NAC, which also prevented Smad3 phosphorylation. Similarly, primary alveolar epithelial type II cells exposed to TGF-β1 also underwent EMT that was prevented by NAC. TGF-β1 decreased cellular GSH levels by 50–80%, whereas NAC restored them to ~150% of those found in TGF-β1-treated cells. Treatment with glutathione monoethyl ester similarly prevented an increase in mesenchymal marker expression. Consistent with its role as an antioxidant and cellular redox stabilizer, NAC dramatically reduced intracellular reactive oxygen species production in the presence of TGF-β1. Finally, inhibition of intracellular ROS generation during TGF-β1 treatment prevented alveolar EMT, but treatment with H₂O₂ alone did not induce EMT. We conclude that NAC prevents EMT in AEC in vitro, at least in part through replenishment of intracellular GSH stores and limitation of TGF-β1-induced intracellular ROS generation. We speculate that beneficial effects of NAC on pulmonary function in IPF may be mediated by inhibitory effects on alveolar EMT.

Mechanisms of alveolar epithelial chloride absorption

Ingbar, D. H., Bhargava, M., O'Grady, S. M. — Mon, 26 Oct 2009 14:24:03 PDT

Characteristics of Cl- uptake in rat alveolar type I cells

Johnson, M., Allen, L., Dobbs, L. — Mon, 26 Oct 2009 14:24:03 PDT

Although Cl^– transport in fetal lung is important for fluid secretion and normal lung development, the role of Cl^– transport in adult lung is not well understood. In physiological studies, the cystic fibrosis transmembrane regulator (CFTR) plays a role in fluid absorption in the distal air spaces of adult lung, and alveolar type II cells cultured for 5 days have the capacity to transport Cl^–. Although both alveolar type I and type II cells express CFTR, it has previously not been known whether type I cells transport Cl^–. We studied Cl^– uptake in isolated type I cells directly, using either radioisotopic tracers or halide-sensitive fluorescent indicators. By both methods, type I cells take up Cl^–. In the presence of β-adrenergic agonist stimulation, Cl^– uptake can be inhibited by CFTR antagonists. Type I cells express both the Cl^–/HCO₃^– anion exchanger AE2 and the voltage-gated Cl^– channels CLC5 and CLC2. Inhibitors of AE2 also block Cl^– uptake in type I cells. Together, these results demonstrate that type I cells are capable of Cl^– uptake and suggest that the effects seen in whole lung studies establishing the importance of Cl^– movement in alveolar fluid clearance may be, in part, the result of Cl^– transport across type I cells.

The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease

Nichols, D. P., Ziady, A. G., Shank, S. L., Eastman, J. F., Davis, P. B. — Mon, 26 Oct 2009 14:24:03 PDT

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-B activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

Mon, 26 Oct 2009 14:24:03 PDT

Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1 on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1 enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.

Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

Mon, 26 Oct 2009 14:24:04 PDT

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF- and ROS in a Rac1-dependent manner. TNF- was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia

Cercone, M. A., Schroeder, W., Schomberg, S., Carpenter, T. C. — Mon, 26 Oct 2009 14:24:04 PDT

Ephrin family receptor tyrosine kinases are mediators of angiogenesis that may also regulate endothelial barrier function in the lung. Previous work has demonstrated that stimulation of EphA ephrin receptors causes increased vascular leak in the intact lung and increased permeability in cultured endothelial cells. Whether EphA receptors are involved in the permeability changes associated with lung injury is unknown. We studied this question in young rats exposed to viral respiratory infection combined with exposure to moderate hypoxia, a previously described lung injury model. We found that the EphA2 receptor is expressed in normal lung and that EphA2 expression is markedly upregulated in the lungs of hypoxic infected (HV) rats compared with normal control animals. Immunohistochemistry showed increased EphA2 expression principally in areas of edematous alveolar septae. In HV rats, EphA2 antagonism with either the soluble decoy receptor EphA2/Fc or with monoclonal anti-EphA2 antibody reduced albumin extravasation and histological evidence of edema formation (P < 0.01). Vascular leak in HV rats is mediated in large part by increased lung endothelin (ET) levels. In HV rats, ET receptor antagonism with bosentan resulted in reduced EphA2 mRNA and protein expression (P < 0.01). Experiments with cultured rat lung microvascular endothelial cells demonstrated that ET increases endothelial EphA2 expression. These results suggest that EphA2 expression is increased in lung injury, contributes to vascular leak in the injured lung, and is regulated in endothelial cells by ET. EphA2 may be a previously unrecognized contributor to the pathophysiology of lung injury.

Abrogation of TGF-{beta}1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition

Guo, W., Shan, B., Klingsberg, R. C., Qin, X., Lasky, J. A. — Mon, 26 Oct 2009 14:24:04 PDT

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with no known effective pharmacological therapy. The fibroblastic foci of IPF contain activated myofibroblasts that are the major synthesizers of type I collagen. Transforming growth factor (TGF)-β1 promotes differentiation of fibroblasts into myofibroblasts in vitro and in vivo. In the current study, we investigated the molecular link between TGF-β1-mediated myofibroblast differentiation and histone deacetylase (HDAC) activity. Treatment of normal human lung fibroblasts (NHLFs) with the pan-HDAC inhibitor trichostatin A (TSA) inhibited TGF-β1-mediated -smooth muscle actin (-SMA) and ₁ type I collagen mRNA induction. TSA also blocked the TGF-β1-driven contractile response in NHLFs. The inhibition of -SMA expression by TSA was associated with reduced phosphorylation of Akt, and a pharmacological inhibitor of Akt blocked TGF-β1-mediated -SMA induction in a dose-dependent manner. HDAC4 knockdown was effective in inhibiting TGF-β1-stimulated -SMA expression as well as the phosphorylation of Akt. Moreover, the inhibitors of protein phosphatase 2A and 1 (PP2A and PP1) rescued the TGF-β1-mediated -SMA induction from the inhibitory effect of TSA. Together, these data demonstrate that the differentiation of NHLFs to myofibroblasts is HDAC4 dependent and requires phosphorylation of Akt.

Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells

Sorokina, E. M., Feinstein, S. I., Milovanova, T. N., Fisher, A. B. — Mon, 26 Oct 2009 14:24:04 PDT

Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA₂ (aiPLA₂) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31–40 from the Prdx6 NH₂-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31–40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA₂ activity depend on a novel 10-aa peptide located at positions 31–40 of the protein.

Influence of cytoskeletal structure and mechanics on epithelial cell injury during cyclic airway reopening

Mon, 26 Oct 2009 14:24:04 PDT

Although patients with acute respiratory distress syndrome require mechanical ventilation, these ventilators often exacerbate the existing lung injury. For example, the cyclic closure and reopening of fluid-filled airways during ventilation can cause epithelial cell (EpC) necrosis and barrier disruption. Although much work has focused on minimizing the injurious mechanical forces generated during ventilation, an alternative approach is to make the EpC less susceptible to injury by altering the cell's intrinsic biomechanical/biostructural properties. In this study, we hypothesized that alterations in cytoskeletal structure and mechanics can be used to reduce the cell's susceptibility to injury during airway reopening. EpC were treated with jasplakinolide to stabilize actin filaments or latrunculin A to depolymerize actin and then exposed to cyclic airway reopening conditions at room temperature using a previously developed in vitro cell culture model. Actin stabilization did not affect cell viability but significantly improved cell adhesion primarily due to the development of more numerous focal adhesions. Surprisingly, actin depolymerization significantly improved both cell viability and cell adhesion but weakened focal adhesions. Optical tweezer based measurements of the EpC's micromechanical properties indicate that although latrunculin-treated cells are softer, they also have increased viscous damping properties. To further investigate the effect of "fluidization" on cell injury, experiments were also conducted at 37°C. Although cells held at 37°C exhibited no changes in cytoskeletal structure, they did exhibit increased viscous damping properties and improved cell viability. We conclude that fluidization of the actin cytoskeleton makes the EpC less susceptible to the injurious mechanical forces generated during cyclic airway reopening.

Cigarette smoke extract induces COX-2 expression via a PKC{alpha}/c-Src/EGFR, PDGFR/PI3K/Akt/NF-{kappa}B pathway and p300 in tracheal smooth muscle cells

Mon, 26 Oct 2009 14:24:04 PDT

Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E₂ (PGE₂) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKC/c-Src/EGFR, PDGFR/PI3K/Akt/NF-B signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKC (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKC, Src, or Akt. Furthermore, CSE-stimulated NF-B p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-B in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-B (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE₂ generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE₂ generation was mediated through PKC/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-B complex.

In vitro translocation of quantum dots and influence of oxidative stress

Geys, J., De Vos, R., Nemery, B., Hoet, P. H. M. — Mon, 26 Oct 2009 14:24:04 PDT

In vivo, translocation of inhaled nanoparticles to the circulation has been demonstrated. However, the interaction of nanoparticles with the lung epithelium is not understood. In this study, we investigated, in vitro, the translocation of nano-sized quantum dots (QDs; 25 pmol/ml) through a tight monolayer of primary isolated rat alveolar epithelial cells. The influence of surface charge on translocation was examined using nonfunctionalized QDs, amine-QDs, and carboxyl-QDs. The interaction between nanoparticles and the lung epithelium was monitored by repeatedly measuring the transepithelial electrical resistance (TEER) and by examining the cell layer with confocal microscopy. The effect of oxidative stress was tested by incubating the cells with tert-butyl hydroperoxide (t-BOOH; 75 µM or 1 or 10 mM); the antioxidant N-acetyl-l-cysteine was also used to assess the role of particle-mediated oxidative stress. No translocation through a tight monolayer of primary rat alveolar epithelial cells was observed for any of the different types of QDs. In general, an increase in TEER was found after incubation with QDs. A condition of low oxidative stress did not enhance translocation. In contrast, conditions of high stress (1 or 10 mM t-BOOH or due to QDs toxicity) with disruption of the cell layer, as shown in a decreased TEER, resulted in substantial translocation. In conclusion, no translocation of QDs was found through a tight monolayer of primary rat alveolar epithelial cells, regardless of the QDs surface charge. QDs did not impair the barrier function of the epithelial cells. In conditions with disruption of the cell-cell barrier, translocation was demonstrated.

Peroxisome proliferator-activated receptor-{gamma} ligands induce heme oxygenase-1 in lung fibroblasts by a PPAR{gamma}-independent, glutathione-dependent mechanism

Mon, 26 Oct 2009 14:24:04 PDT

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor- (PPAR) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPAR ligands 15d-PGJ₂ and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPAR independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ₂ contain an /β unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPAR ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ₂ and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ₂ or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ₂ and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPAR ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.

Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats

Mon, 26 Oct 2009 14:24:04 PDT

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O₂ for 14 days with or without 5.5% CO₂. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO₂ had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO₂ provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

Elastase- and LPS-exposed mice display altered responses to rhinovirus infection

Mon, 26 Oct 2009 14:24:04 PDT

Viral infection is associated with approximately one-half of acute exacerbations of chronic obstructive pulmonary disease (COPD), which in turn, accelerate disease progression. In this study, we infected mice exposed to a combination of elastase and LPS, a constituent of cigarette smoke and a risk factor for development of COPD, with rhinovirus serotype 1B, and examined animals for viral persistence, airway resistance, lung volume, and cytokine responses. Mice exposed to elastase and LPS once a week for 4 wk showed features of COPD such as airway inflammation and obstruction, goblet cell metaplasia, reduced lung elastance, increased total lung volume, and increased alveolar chord length. In general, mice exposed to elastase or LPS alone showed intermediate effects. Compared with rhinovirus (RV)-infected PBS-exposed mice, RV-infected elastase/LPS-exposed mice showed persistence of viral RNA, airway hyperresponsiveness, increased lung volume, and sustained increases in expression of TNF, IL-5, IL-13, and muc5AC (up to 14 days postinfection). Furthermore, virus-induced IFNs, interferon response factor-7, and IL-10 were deficient in elastase/LPS-treated mice. Mice exposed to LPS or elastase alone cleared virus similar to PBS-treated control mice. We conclude that limited exposure of mice to elastase/LPS produces a COPD-like condition including increased persistence of RV, likely due to skewing of the immune response towards a Th2 phenotype. Similar mechanisms may be operative in COPD.

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways

Zhang, M., Lin, L., Lee, S.-J., Mo, L., Cao, J., Ifedigbo, E., Jin, Y. — Mon, 26 Oct 2009 14:24:04 PDT

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1^–/–) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using "gain" and "loss" of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression.

PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells

Mon, 26 Oct 2009 14:24:04 PDT

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1–3% O₂) resulted in time- and O₂-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.

Pulmonary effects of keratinocyte growth factor in newborn rats exposed to hyperoxia

Mon, 26 Oct 2009 14:24:04 PDT

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of keratinocyte growth factor (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O₂ from birth, increased viability induced by intraperitoneal injection of KGF (2 µg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

Effects of hypercapnia with and without acidosis on hypoxic pulmonary vasoconstriction

Mon, 26 Oct 2009 14:24:04 PDT

Acute respiratory disorders and permissive hypercapnic strategy may lead to alveolar hypoxia and hypercapnic acidosis. However, the effects of hypercapnia with or without acidosis on hypoxic pulmonary vasoconstriction (HPV) and oxygen diffusion capacity of the lung are controversial. We investigated the effects of hypercapnic acidosis and hypercapnia with normal pH (pH corrected with sodium bicarbonate) on HPV, capillary permeability, gas exchange, and ventilation-perfusion matching in the isolated ventilated-perfused rabbit lung. No alteration in vascular tone was noted during normoxic hypercapnia with or without acidosis compared with normoxic normocapnia. Hypercapnia with normal pH resulted in a transient increase in HPV during the course of consecutive ventilation maneuvers, whereas hypercapnic acidosis increased HPV over time. Hypercapnic acidosis decreased exhaled NO during hypoxia more than hypercapnia with normal pH and normocapnia, whereas intravascular NO release was unchanged. However, inhibition of NO synthesis by nitro-l-arginine (l-NNA) resulted in a loss of the increased HPV caused by hypercapnic acidosis but not that caused by hypercapnia with normal pH. Furthermore, capillary permeability increased during hypoxic hypercapnia with normal pH but not hypoxic hypercapnic acidosis. This effect was NO-dependent because it disappeared during l-NNA administration. Ventilation-perfusion matching and arterial Po₂ were improved according to the strength of HPV in hypercapnia compared with normocapnia during Tween nebulization-induced lung injury. In conclusion, the increased HPV during hypercapnic acidosis is beneficial to lung gas exchange by improving ventilation-perfusion matching and preserving the capillary barrier function. These effects seem to be linked to NO-mediated pathways.

Inhibition of geranylgeranyltransferase inhibits bronchial smooth muscle hyperresponsiveness in mice

Chiba, Y., Sato, S., Hanazaki, M., Sakai, H., Misawa, M. — Mon, 26 Oct 2009 14:24:04 PDT

Recent studies revealed an involvement of RhoA/Rho-kinase in the contraction of bronchial smooth muscle (BSM), and this pathway has now been proposed as a new target for asthma therapy. A posttranslational geranylgeranylation of RhoA is required for its activation. Thus selective inhibition of geranylgeranyltransferase may be a novel strategy for treatment of the BSM hyperresponsiveness in asthmatics. To test this hypothesis, we investigated the effect of a geranylgeranyltransferase inhibitor, GGTI-2133, on antigen-induced BSM hyperresponsiveness by using mice with experimental asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also were treated with GGTI-2133 (5 mg/kg ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a BSM hyperresponsiveness to acetylcholine with the increased expressions of RhoA and the anti-farnesyl-positive 21-kDa proteins, probably geranylgeranylated RhoA. The in vivo GGTI-2133 treatments significantly inhibited BSM hyperresponsiveness induced by antigen exposure. In another series of experiments, BSM tissues isolated from the repeatedly antigen-challenged mice were cultured for 48 h in the absence or presence of GGTI-2133. Under these conditions, the putative geranylgeranylated RhoA was decreased in a GGTI-2133 concentration-dependent manner. The in vitro incubation with GGTI-2133 also inhibited BSM hyperresponsiveness induced by antigen exposure. These findings suggest that GGTI-2133 inhibits antigen-induced BSM hyperresponsiveness, probably by reducing downstream signal transduction of RhoA. Selective geranylgeranyltransferase inhibitors may be beneficial for the treatment of airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

Persistent bronchiolar remodeling following brief ventilation of the very immature ovine lung

Mon, 26 Oct 2009 14:24:04 PDT

Children and adults who were mechanically ventilated following preterm birth are at increased risk of reduced lung function, suggesting small airway dysfunction. We hypothesized that short periods of mechanical ventilation of very immature lungs can induce persistent bronchiolar remodeling that may adversely affect later lung function. Our objectives were to characterize the effects of brief, positive-pressure ventilation per se on the small airways in very immature, surfactant-deficient lungs and to determine whether the effects persist after the cessation of ventilation. Fetal sheep (0.75 of term) were mechanically ventilated in utero with room air (peak inspiratory pressure 40 cmH₂O, positive end-expiratory pressure 4 cmH₂O, 65 breaths/min) for 6 or 12 h, after which tissues were collected; another group was studied 7 days after 12-h ventilation. Age-matched unventilated fetuses were controls. The mean basement membrane perimeter of airways analyzed was 548.6 ± 8.5 µm and was not different between groups. Immediately after ventilation, 21% of airways had epithelial injury; in airways with intact epithelium, there was more airway smooth muscle (ASM) and less collagen, and the epithelium contained more mucin-containing and apoptotic cells and fewer proliferating cells. Seven days after ventilation, epithelial injury was absent but the epithelium was thicker, with greater cell turnover; there were increased amounts of bronchiolar collagen and ASM and fewer alveolar attachments. The increase in ASM was likely due to cellular hypertrophy rather than hyperplasia. We conclude that brief mechanical ventilation of the very immature lung induces remodeling of the bronchiolar epithelium and walls that lasts for at least 7 days; such changes could contribute to later airway dysfunction.

Mesenchymal stem cells produce Wnt isoforms and TGF-{beta}1 that mediate proliferation and procollagen expression by lung fibroblasts

Salazar, K. D., Lankford, S. M., Brody, A. R. — Mon, 26 Oct 2009 14:24:04 PDT

Studies have been carried out previously to determine whether mesenchymal stem cells (MSC) influence the progression of pulmonary fibrosis. Here, we asked whether MSC (derived from mouse bone marrow and human umbilical cord blood) produce factors that mediate lung fibroblast (LF) growth and matrix production. MSC-conditioned media (CM) were found by ELISA to contain significant amounts of PDGF-AA and transforming growth factor-β₁ (TGF-β₁). Proliferation was increased in a concentration-dependent manner in LF cell lines and primary cells cultured in MSC-CM, but neither anti-PDGF antibodies nor PDGF receptor-specific antibodies affected proliferation, nor did a number of other antibodies to well-known mitogenic factors. However, proliferation was significantly inhibited by the Wnt signaling antagonist, secreted frizzled related protein-1 (sFRP-1). In addition, anti-Wnt1 and anti-Wnt2 antibodies attenuated MSC-CM-induced proliferation, and increased expression of Wnt7b was identified. As would be expected in cells activated by Wnt, nuclear β-catenin was increased. The amount of TGF-β₁ in MSC-CM and its biological activity were revealed by activation at acidic pH. The stem cells synthesized and released TGF-β₁ that increased ₁-procollagen gene expression by LF target cells. Addition of anti-TGF-β to the MSC-CM blocked upregulation of collagen gene expression. These data demonstrate that MSC from mice and humans produce Wnt proteins and TGF-β₁ that respectively stimulate LF proliferation and matrix production, two hallmarks of fibroproliferative lung disease. It will be essential to determine whether these factors can play a role in attempts to use MSC for therapeutic approaches.