Lung Injury Induces Alveolar Type 2 Cell Hypertrophy and Polyploidy with Implications for Repair and Regeneration.

Epithelial polyploidization after injury is a conserved phenomenon recently shown to improve barrier restoration during wound healing. Whether lung injury can induce alveolar epithelial polyploidy is not known. We show that bleomycin injury induces alveolar type 2 cell (AT2) hypertrophy and polyploidy. AT2 polyploidization is also seen in short term ex vivo cultures, where AT2-to-AT1 transdifferentiation is associated with substantial binucleation due to failed cytokinesis. Both hypertrophic and polyploid features of AT2 cells can be attenuated by inhibiting the integrated stress response using the small molecule ISRIB. These data suggest that AT2 hypertrophic growth and polyploidization may be a feature of alveolar epithelial injury. Because AT2 cells serve as facultative progenitors for the distal lung epithelium, a propensity for injury-induced binucleation has implications for AT2 self-renewal and regenerative potential upon reinjury, which may benefit from targeting the integrated stress response.

Impaired phagocytic function in CX3CR1+ tissue-resident skeletal muscle macrophages prevents muscle recovery after influenza A virus-induced pneumonia in old mice.

Skeletal muscle dysfunction in survivors of pneumonia disproportionately affects older individuals in whom it causes substantial morbidity. We found that skeletal muscle recovery was impaired in old compared with young mice after influenza A virus-induced pneumonia. In young mice, recovery of muscle loss was associated with expansion of tissue-resident skeletal muscle macrophages and downregulation of MHC II expression, followed by a proliferation of muscle satellite cells. These findings were absent in old mice and in mice deficient in Cx3cr1. Transcriptomic profiling of tissue-resident skeletal muscle macrophages from old compared with young mice showed downregulation of pathways associated with phagocytosis and proteostasis, and persistent upregulation of inflammatory pathways. Consistently, skeletal muscle macrophages from old mice failed to downregulate MHCII expression during recovery from influenza A virus-induced pneumonia and showed impaired phagocytic function in vitro. Like old animals, mice deficient in the phagocytic receptor Mertk showed no macrophage expansion, MHCII downregulation, or satellite cell proliferation and failed to recover skeletal muscle function after influenza A pneumonia. Our data suggest that a loss of phagocytic function in a CX3CR1+ tissue-resident skeletal muscle macrophage population in old mice precludes satellite cell proliferation and recovery of skeletal muscle function after influenza A pneumonia.

High CO2 Levels Impair Lung Wound Healing.

Delayed lung repair leads to alveolopleural fistulae, which are a major cause of morbidity after lung resections. We have reported that intrapleural hypercapnia is associated with delayed lung repair after lung resection. Here, we provide new evidence that hypercapnia delays wound closure of both large airway and alveolar epithelial cell monolayers because of inhibition of epithelial cell migration. Cell migration and airway epithelial wound closure were dependent on Rac1-GTPase activation, which was suppressed by hypercapnia directly through the upregulation of AMP kinase and indirectly through inhibition of injury-induced NF-κB-mediated CXCL12 (pleural CXC motif chemokine 12) release, respectively. Both these pathways were independently suppressed, because dominant negative AMP kinase rescued the effects of hypercapnia on Rac1-GTPase in uninjured resting cells, whereas proteasomal inhibition reversed the NF-κB-mediated CXCL12 release during injury. Constitutive overexpression of Rac1-GTPase rescued the effects of hypercapnia on both pathways as well as on wound healing. Similarly, exogenous recombinant CXCL12 reversed the effects of hypercapnia through Rac1-GTPase activation by its receptor, CXCR4. Moreover, CXCL12 transgenic murine recipients of orthotopic tracheal transplantation were protected from hypercapnia-induced inhibition of tracheal epithelial cell migration and wound repair. In patients undergoing lobectomy, we found inverse correlation between intrapleural carbon dioxide and pleural CXCL12 levels as well as between CXCL12 levels and alveolopleural leak. Accordingly, we provide first evidence that high carbon dioxide levels impair lung repair by inhibiting epithelial cell migration through two distinct pathways, which can be restored by recombinant CXCL12.

Cardiac glycosides decrease influenza virus replication by inhibiting cell protein translational machinery.

Cardiac glycosides (CGs) are used primarily for cardiac failure and have been reported to have other effects, including inhibition of viral replication. Here we set out to study mechanisms by which CGs as inhibitors of the Na-K-ATPase decrease influenza A virus (IAV) replication in the lungs. We found that CGs inhibit influenza virus replication in alveolar epithelial cells by decreasing intracellular potassium, which in turn inhibits protein translation, independently of viral entry, mRNA transcription, and protein degradation. These effects were independent of the Src signaling pathway and intracellular calcium concentration changes. We found that short-term treatment with ouabain prevented IAV replication without cytotoxicity. Rodents express a Na-K-ATPase-α1 resistant to CGs. Thus we utilized Na-K-ATPase-α1-sensitive mice, infected them with high doses of influenza virus, and observed a modest survival benefit when treated with ouabain. In summary, we provide evidence that the inhibition of the Na-K-ATPase by CGs decreases influenza A viral replication by modulating the cell protein translational machinery and results in a modest survival benefit in mice.

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α1-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells (CreSPC/HOIL-1Lfl/fl ) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1-Na,K-ATPase construct bearing an S18A (α1-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and lung injury.

High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and ß-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

High CO2 levels cause skeletal muscle atrophy via AMP-activated kinase (AMPK), FoxO3a protein, and muscle-specific Ring finger protein 1 (MuRF1).

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO2 had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO2 had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO2 induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1(-/-) mice exposed to high CO2 did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO2, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO2 induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO2 activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.

Hypercapnia impairs lung neutrophil function and increases mortality in murine pseudomonas pneumonia.

Hypercapnia, an elevation of the level of carbon dioxide (CO2) in blood and tissues, is a marker of poor prognosis in chronic obstructive pulmonary disease and other pulmonary disorders. We previously reported that hypercapnia inhibits the expression of TNF and IL-6 and phagocytosis in macrophages in vitro. In the present study, we determined the effects of normoxic hypercapnia (10% CO2, 21% O2, and 69% N2) on outcomes of Pseudomonas aeruginosa pneumonia in BALB/c mice and on pulmonary neutrophil function. We found that the mortality of P. aeruginosa pneumonia was increased in 10% CO2-exposed compared with air-exposed mice. Hypercapnia increased pneumonia mortality similarly in mice with acute and chronic respiratory acidosis, indicating an effect unrelated to the degree of acidosis. Exposure to 10% CO2 increased the burden of P. aeruginosa in the lungs, spleen, and liver, but did not alter lung injury attributable to pneumonia. Hypercapnia did not reduce pulmonary neutrophil recruitment during infection, but alveolar neutrophils from 10% CO2-exposed mice phagocytosed fewer bacteria and produced less H2O2 than neutrophils from air-exposed mice. Secretion of IL-6 and TNF in the lungs of 10% CO2-exposed mice was decreased 7 hours, but not 15 hours, after the onset of pneumonia, indicating that hypercapnia inhibited the early cytokine response to infection. The increase in pneumonia mortality caused by elevated CO2 was reversible when hypercapnic mice were returned to breathing air before or immediately after infection. These results suggest that hypercapnia may increase the susceptibility to and/or worsen the outcome of lung infections in patients with severe lung disease.

Evolutionary conserved role of c-Jun-N-terminal kinase in CO2-induced epithelial dysfunction.

Elevated CO(2) levels (hypercapnia) occur in patients with respiratory diseases and impair alveolar epithelial integrity, in part, by inhibiting Na,K-ATPase function. Here, we examined the role of c-Jun N-terminal kinase (JNK) in CO(2) signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO(2) levels rapidly induced activation of JNK leading to downregulation of Na,K-ATPase and alveolar epithelial dysfunction. Hypercapnia-induced activation of JNK required AMP-activated protein kinase (AMPK) and protein kinase C-ζ leading to subsequent phosphorylation of JNK at Ser-129. Importantly, elevated CO(2) levels also caused a rapid and prominent activation of JNK in Drosophila S2 cells and in C. elegans. Paralleling the results with mammalian epithelial cells, RNAi against Drosophila JNK fully prevented CO(2)-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO(2) signaling was additionally demonstrated by the ability of mutations in the C. elegans JNK homologs, jnk-1 and kgb-2 to partially rescue the hypercapnia-induced fertility defects but not the pharyngeal pumping defects. Together, these data provide evidence that deleterious effects of hypercapnia are mediated by JNK which plays an evolutionary conserved, specific role in CO(2) signaling in mammals, diptera and nematodes.

Hypoxia leads to Na,K-ATPase downregulation via Ca(2+) release-activated Ca(2+) channels and AMPK activation.

To maintain cellular ATP levels, hypoxia leads to Na,K-ATPase inhibition in a process dependent on reactive oxygen species (ROS) and the activation of AMP-activated kinase α1 (AMPK-α1). We report here that during hypoxia AMPK activation does not require the liver kinase B1 (LKB1) but requires the release of Ca(2+) from the endoplasmic reticulum (ER) and redistribution of STIM1 to ER-plasma membrane junctions, leading to calcium entry via Ca(2+) release-activated Ca(2+) (CRAC) channels. This increase in intracellular Ca(2+) induces Ca(2+)/calmodulin-dependent kinase kinase ß (CaMKKß)-mediated AMPK activation and Na,K-ATPase downregulation. Also, in cells unable to generate mitochondrial ROS, hypoxia failed to increase intracellular Ca(2+) concentration while a STIM1 mutant rescued the AMPK activation, suggesting that ROS act upstream of Ca(2+) signaling. Furthermore, inhibition of CRAC channel function in rat lungs prevented the impairment of alveolar fluid reabsorption caused by hypoxia. These data suggest that during hypoxia, calcium entry via CRAC channels leads to AMPK activation, Na,K-ATPase downregulation, and alveolar epithelial dysfunction.

Extracellular signal-regulated kinase (ERK) participates in the hypercapnia-induced Na,K-ATPase downregulation.

Hypercapnia has been shown to impair alveolar fluid reabsorption (AFR) by decreasing Na,K-ATPase activity. Extracellular signal-regulated kinase pathway (ERK) is activated under conditions of cellular stress and has been known to regulate the Na,K-ATPase. Here, we show that hypercapnia leads to ERK activation in a time-dependent manner in alveolar epithelial cells (AEC). Inhibition of ERK by U0126 or siRNA prevented both the hypercapnia-induced Na,K-ATPase endocytosis and impairment of AFR. Moreover, ERK inhibition prevented AMPK activation, a known modulator of hypercapnia-induced Na,K-ATPase endocytosis. Accordingly, these data suggest that hypercapnia-induced Na,K-ATPase endocytosis is dependent on ERK activation in AEC and that ERK plays an important role in hypercapnia-induced impairment of AFR in rat lungs.

Myosin-Va restrains the trafficking of Na+/K+-ATPase-containing vesicles in alveolar epithelial cells.

Stimulation of Na(+)/K(+)-ATPase activity in alveolar epithelial cells by cAMP involves its recruitment from intracellular compartments to the plasma membrane. Here, we studied the role of the actin molecular motor myosin-V in this process. We provide evidence that, in alveolar epithelial cells, cAMP promotes Na(+)/K(+)-ATPase recruitment to the plasma membrane by increasing the average speed of Na(+)/K(+)-ATPase-containing vesicles moving to the cell periphery. We found that three isoforms of myosin-V are expressed in alveolar epithelial cells; however, only myosin-Va and Vc colocalized with the Na(+)/K(+)-ATPase in intracellular membrane fractions. Overexpression of dominant-negative myosin-Va or knockdown with specific shRNA increased the average speed and distance traveled by the Na(+)/K(+)-ATPase-containing vesicles, as well as the Na(+)/K(+)-ATPase activity and protein abundance at the plasma membrane to similar levels as those observed with cAMP stimulation. These data show that myosin-Va has a role in restraining Na(+)/K(+)-ATPase-containing vesicles within intracellular pools and that this restrain is released after stimulation by cAMP allowing the recruitment of the Na(+)/K(+)-ATPase to the plasma membrane and thus increased activity.

Overexpression of the Na-K-ATPase alpha2-subunit improves lung liquid clearance during ventilation-induced lung injury.

Mechanical ventilation with high tidal volumes (HV(T)) impairs lung liquid clearance (LLC) and downregulates alveolar epithelial Na-K-ATPase. We have previously reported that the Na-K-ATPase alpha(2)-subunit contributes to LLC in normal rat lungs. Here we tested whether overexpression of Na-K-ATPase alpha(2)-subunit in the alveolar epithelium would increase clearance in a HV(T) model of lung injury. We infected rat lungs with a replication-incompetent adenovirus that expresses Na-K-ATPase alpha(2)-subunit gene (Adalpha(2)) 7 days before HV(T) mechanical ventilation. HV(T) ventilation decreased LLC by approximately 50% in untreated, sham, and Adnull-infected rats. Overexpression of Na-K-ATPase alpha(2)-subunit prevented the decrease in clearance caused by HV(T) and was associated with significant increases in Na-K-ATPase alpha(2) protein abundance and activity in peripheral lung basolateral membrane fractions. Ouabain at 10(-5) M, a concentration that inhibits the alpha(2) but not the Na-K-ATPase alpha(1), decreased LLC in Adalpha(2)-infected rats to the same level as sham and Adnull-infected lungs, suggesting that the increased clearance in Adalpha(2) lungs was due to Na-K-ATPase alpha(2) expression and activity. In summary, we provide evidence that augmentation of the Na-K-ATPase alpha(2)-subunit, via gene transfer, may accelerate LLC in the injured lung.

AMP-activated protein kinase regulates CO2-induced alveolar epithelial dysfunction in rats and human cells by promoting Na,K-ATPase endocytosis.

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

Carbonic anhydrase II and alveolar fluid reabsorption during hypercapnia.

Carbonic anhydrase II (CAII) plays an important role in carbon dioxide metabolism and intracellular pH regulation. In this study, we provide evidence that CAII is expressed in both type I (AECI) and type II (AECII) alveolar epithelial cells by RT-PCR and Western blotting in freshly isolated rat cells. These results were further confirmed by double immunostaining with CAII antibodies and AECI- or AECII-specific markers in freshly isolated alveolar epithelial cells and rat lung tissues. Inhibition of CAII by acetazolamide or methazolamide delayed the decrease in the intracellular pH observed during hypercapnia in cultured AECI, AECII, and AECI-like cells. In an isolated-perfused rat lung model, alveolar fluid reabsorption significantly decreased during high CO(2) exposure, which was not prevented by carbonic anhydrase inhibition. Thus, we provide evidence that CAII is expressed in rat alveolar epithelial cells and does not regulate lung alveolar fluid reabsorption during hypercapnia.

High CO2 levels impair alveolar epithelial function independently of pH.

BACKGROUND: In patients with acute respiratory failure, gas exchange is impaired due to the accumulation of fluid in the lung airspaces. This life-threatening syndrome is treated with mechanical ventilation, which is adjusted to maintain gas exchange, but can be associated with the accumulation of carbon dioxide in the lung. Carbon dioxide (CO2) is a by-product of cellular energy utilization and its elimination is affected via alveolar epithelial cells. Signaling pathways sensitive to changes in CO2 levels were described in plants and neuronal mammalian cells. However, it has not been fully elucidated whether non-neuronal cells sense and respond to CO2. The Na,K-ATPase consumes approximately 40% of the cellular metabolism to maintain cell homeostasis. Our study examines the effects of increased pCO2 on the epithelial Na,K-ATPase a major contributor to alveolar fluid reabsorption which is a marker of alveolar epithelial function. PRINCIPAL FINDINGS: We found that short-term increases in pCO2 impaired alveolar fluid reabsorption in rats. Also, we provide evidence that non-excitable, alveolar epithelial cells sense and respond to high levels of CO2, independently of extracellular and intracellular pH, by inhibiting Na,K-ATPase function, via activation of PKCzeta which phosphorylates the Na,K-ATPase, causing it to endocytose from the plasma membrane into intracellular pools. CONCLUSIONS: Our data suggest that alveolar epithelial cells, through which CO2 is eliminated in mammals, are highly sensitive to hypercapnia. Elevated CO2 levels impair alveolar epithelial function, independently of pH, which is relevant in patients with lung diseases and altered alveolar gas exchange.

Phosphorylation and ubiquitination are necessary for Na,K-ATPase endocytosis during hypoxia.

As a cellular adaptative response, hypoxia decreases Na,K-ATPase activity by triggering the endocytosis of its alpha(1) subunit in alveolar epithelial cells. Here, we present evidence that the ubiquitin conjugating system is important in the Na,K-ATPase endocytosis during hypoxia and that ubiquitination of Na,K-ATPase alpha(1) subunit occurs at the basolateral membrane. Endocytosis and ubiquitination were prevented when the Ser 18 in the PKC phosphorylation motif of the Na,K-ATPase alpha(1) subunit was mutated to an alanine, suggesting that phosphorylation at Ser-18 is required for ubiquitination. Mutation of the four lysines surrounding Ser 18 to arginine prevented Na,K-ATPase ubiquitination and endocytosis during hypoxia; however, only one of them was sufficient to restore hypoxia-induced endocytosis. We provide evidence that ubiquitination plays an important role in cellular adaptation to hypoxia by regulating Na,K-ATPase alpha(1)-subunit endocytosis.

Airborne particulate matter inhibits alveolar fluid reabsorption in mice via oxidant generation.

Ambient particulate matter is increasingly recognized as a significant contributor to human cardiopulmonary morbidity and mortality in the United States and worldwide. We sought to determine whether exposure to ambient particulate matter would alter alveolar fluid clearance in mice. Mice were exposed to a range of doses of a well-characterized particulate matter collected from the ambient air in Düsseldorf, Germany through a single intratracheal instillation, and alveolar fluid clearance and measurements of lung injury were made. Exposure to even very low doses of particulate matter (10 microg) resulted in a significant reduction in alveolar fluid clearance that was maximal 24 h after the exposure, with complete resolution after 7 d. This was paralleled by a decrease in lung Na,K-ATPase activity. To investigate the mechanism of this effect, we measured plasma membrane Na,K-ATPase abundance in A549 cells and Na,K-ATPase activity in primary rat alveolar type II cells after exposure to particulate matter in the presence or absence of the combined superoxide dismutase and catalase mimetic EUK-134 (5 microM). Membrane but not total protein abundance of the Na,K-ATPase was decreased after exposure to particulate matter, as was Na,K-ATPase activity. This decrease was prevented by the combined superoxide dismutase/catalase mimetic EUK-134. The intratracheal instillation of particulate matter results in alveolar epithelial injury and decreased alveolar fluid clearance, conceivably due to downregulation of the Na,K-ATPase.

In vivo timing of onset of transgene expression following adenoviral-mediated gene transfer.

Recombinant adenoviruses are efficient gene transfer vehicles that could be used for treatment of acute diseases. However, the time required for adenoviruses to produce physiologically relevant levels of transgene in vivo is unknown. To address this question rat lungs were infected with an E1a(-)/E3a(-) adenovirus that contains an hCMV-driven human beta(2)-adrenergic receptor (beta(2)AR) cDNA. Human beta(2)AR message and protein expression were noted 2-4 h postinfection without evidence of pseudotransduction. beta(2)AR function (cAMP production) was increased at 6 h postinfection. To determine when beta(2)AR gene transfer affects downstream catecholamine-sensitive pathways, we measured lung Na,K-ATPase expression and alveolar fluid clearance (AFC). beta(2)AR gene transfer increased Na,K-ATPase number by 80% at 6 h, and AFC by 20% at 8 h postinfection. These data indicate that recombinant adenoviruses can produce physiologically significant levels of transgene within hours of infection and that they may be suitable for gene therapies for acute, rapidly progressive diseases.