The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.
pulmonary macrophages; alveolar macrophages; interstitial macrophages; macrophage polarization; lung fibrosis
Acute exposure to particulate matter (PM) air pollution causes thrombotic cardiovascular events, leading to increased mortality rates; however, the link between PM and cardiovascular dysfunction is not completely understood. We have previously shown that the release of IL-6 from alveolar macrophages is required for a prothrombotic state and acceleration of thrombosis following exposure to PM. Here, we determined that PM exposure results in the systemic release of catecholamines, which engage the β2-adrenergic receptor (β2AR) on murine alveolar macrophages and augment the release of IL-6. In mice, β2AR signaling promoted the development of a prothrombotic state that was sufficient to accelerate arterial thrombosis. In primary human alveolar macrophages, administration of a β2AR agonist augmented IL-6 release, while the addition of a beta blocker inhibited PM-induced IL-6 release. Genetic loss or pharmacologic inhibition of the β2AR on murine alveolar macrophages attenuated PM-induced IL-6 release and prothrombotic state. Furthermore, exogenous β2AR agonist therapy further augmented these responses in alveolar macrophages through generation of mitochondrial ROS and subsequent increase of adenylyl cyclase activity. Together, these results link the activation of the sympathetic nervous system by β2AR signaling with metabolism, lung inflammation, and an enhanced susceptibility to thrombotic cardiovascular events.
The development of organ fibrosis after injury requires activation of transforming growth factor β1 which regulates the transcription of profibrotic genes. The systemic administration of a proteasomal inhibitor has been reported to prevent the development of fibrosis in the liver, kidney and bone marrow. It is hypothesised that proteasomal inhibition would prevent lung and skin fibrosis after injury by inhibiting TGF-β1-mediated transcription.
Bortezomib, a small molecule proteasome inhibitor in widespread clinical use, was administered to mice beginning 7 days after the intratracheal or intradermal administration of bleomycin and lung and skin fibrosis was measured after 21 or 40 days, respectively. To examine the mechanism of this protection, bortezomib was administered to primary normal lung fibroblasts and primary lung and skin fibroblasts obtained from patients with idiopathic pulmonary fibrosis and scleroderma, respectively.
Bortezomib promoted normal repair and prevented lung and skin fibrosis when administered beginning 7 days after the initiation of bleomycin. In primary human lung fibroblasts from normal individuals and patients with idiopathic pulmonary fibrosis and in skin fibroblasts from a patient with scleroderma, bortezomib inhibited TGF-β1-mediated target gene expression by inhibiting transcription induced by activated Smads. An increase in the abundance and activity of the nuclear hormone receptor PPARγ, a repressor of Smad-mediated transcription, contributed to this response.
Proteasomal inhibition prevents lung and skin fibrosis after injury in part by increasing the abundance and activity of PPARγ. Proteasomal inhibition may offer a novel therapeutic alternative in patients with dysregulated tissue repair and fibrosis.
The repair of the bronchiolar epithelium damaged by cell-mediated, physical, or chemical insult requires epithelial cell migration over a provisional matrix composed of complexes of extracellular matrix molecules, including fibronectin and laminin. These matrix molecules support migration and enhance cell adhesion. When cells adhere too tightly to their matrix they fail to move; but if they adhere too little, they are unable to develop the traction force necessary for motility. Thus, we investigated the relative contributions of laminin and fibronectin to bronchiolar cell adhesion and migration using the immortalized bronchial lung epithelial cell line (BEP2D) and normal human bronchial epithelial (NHBE) cells, both of which assemble a laminin α3β3γ2 (LM332)/fibronectin-rich matrix. Intriguingly, BEP2D and NHBE cells migrate significantly faster on an LM332-rich matrix than on fibronectin. Moreover, addition of fibronectin to LM332 matrix suppresses motility of both cell types. Finally, fibronectin enhances the adhesion of both BEP2D and NHBE cells to LM332-coated surfaces. These results suggest that fibronectin fine tunes LM332-mediated migration by boosting bronchiolar cell adhesion to substrate. We suggest that, during epithelial wound healing of the injured airway, fibronectin plays an important adhesive role for laminin-driven epithelial cell motility by promoting a stable cellular interaction with the provisional matrix.
motility; adhesion; extracellular matrix
During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.
We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepRfl/fl) or macrophages and alveolar type II cells (LysM-Cre/Leprfl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.
The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre+/+/LepRfl/fl and LysM-Cre+/+/LepRfl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre+/+/LepRfl/fl and LysM-Cre+/+/LepRfl/fl mice exhibited improved survival.
Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.
The development of non-viral methods for efficient gene transfer to the lung is highly desired for the treatment of a number of pulmonary diseases. We have developed a non-invasive procedure using electroporation to transfer genes to the lungs of rats. Purified plasmid (100 to 600 μg) was delivered to the lungs of anesthetized rats through an endotracheal tube and a series of square wave pulses were delivered via electrodes placed on the chest. Relatively uniform gene expression was observed in multiple cell types and layers throughout the lung, including airway and alveolar epithelial cells, airway smooth muscle cells, and vascular endothelial cells and was dose- and pulse length-dependent. Most importantly, no inflammatory response was detected. To demonstrate efficacy of this approach, the β1 subunit of the Na+,K+-ATPase was transferred to the lungs of rats with or without electroporation, and three days later, alveolar fluid clearance was measured. Animals electroporated with the β1 subunit plasmid showed a two-fold increase in alveolar fluid clearance and Na+,K+-ATPase activity as compared to animals receiving all other plasmids, with or without electroporation. These results demonstrate that electroporation is an effective method to increase clearance by introducing therapeutic genes (Na+,K+-ATPase) into the rat lung.
plasmid; electroporation; acute lung injury; edema
Lung cells are exposed to cyclic stretch during normal respiration and during positive pressure mechanical ventilation administered to support gas exchange. Dystroglycan is a ubiquitously expressed matrix receptor that is required for normal basement membrane formation during embryogenesis and for maintaining the function of skeletal muscle myocytes and neurons where it links cells to matrix. We previously reported that equibiaxial stretch of primary alveolar epithelial cells activated the MAP kinase pathway ERK1/2 through a mechanism that required an interaction between dystroglycan and matrix. We determined whether this mechanism of mechanotransduction activates other signaling cascades in lung epithelium. Exposure of rat epithelial alveolar type II cells (AEC) to cyclic mechanical stretch resulted in activation of 5′ AMP-activated protein kinase (AMPK). This response was not affected by pretreatment of AEC with the ERK inhibitor PD98059 but was inhibited by knockdown in dystroglycan expression. Moreover, production of reactive oxygen species was enhanced in mechanically stimulated AEC in which dystroglycan was knocked down. This enhancement was reversed by treatment of AEC with an AMPK activator. Activation of AMPK was also observed in lung homogenates from mice after 15 minutes of noninjurious mechanical ventilation. Furthermore, knockdown of dystroglycan in the lungs of mice using an adenovirus encoding a dystroglycan shRNA prevented the stretch-induced activation of AMPK. These results suggest that exposure to cyclic stretch activates the metabolic sensing pathway AMPK in the lung epithelium and supports a novel role for dystroglycan in this mechanotransduction.
stretch; lung injury; mechanical ventilation; acute respiratory distress syndrome
Bactericidal antibiotics with diverse mechanisms of action induce generation of mitochondrial reactive oxygen species in mammalian cells (Kalghatgi et al., this issue).
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 μg) 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 abscence of the combined superoxide dismutase and catalase mimetic EUK-134 (5 μM). 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.
antioxidant; lung injury; Na,K-ATPase; pollution; ROS
Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for its transactivation or repression activity, resulted in hyper-inflammation, lung injury and increased mortality in LPS-treated mice while reduced bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged pro-inflammatory cytokine expression. Upon stimulation, Miz1 was phosphorylated at Ser178, which is required for recruiting histone deacetylase 1 to repress transcription of C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying resolution of LPS-induced inflammation.
Pulmonary fibrosis is a disease that results in loss of normal lung architecture, but the signaling events that drive tissue destruction are incompletely understood. Wnt/β-catenin signaling is important in normal lung development, but whether abnormal signaling occurs in lung fibrosis due to systemic sclerosis and the consequences of β-catenin signaling toward the fibrogenic phenotype remain poorly defined. In this study, we show nuclear β-catenin accumulation in fibroblastic foci from lungs of patients with systemic sclerosis–associated advanced pulmonary fibrosis. Forced activation of β-catenin signaling in three independently derived sources of normal human lung fibroblasts promotes proliferation and migratory activities but is not sufficient to activate classic markers of fibroblast activation, such as TGF-β, type 1 collagen, α-smooth muscle actin, and connective tissue growth factor. These findings indicate that activation of β-catenin signaling in pulmonary fibroblasts may be a common feature of lung fibrosis, contributing to fibroproliferative and migratory activities associated with the disease.
Wnt/β-catenin signaling; scleroderma; fibrosis
Elevated CO2 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 CO2 signaling in mammalian alveolar epithelial cells as well as in diptera, nematodes and rodent lungs. In alveolar epithelial cells, elevated CO2 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 CO2 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 CO2-induced downregulation of Na,K-ATPase in Drosophila S2 cells. The importance and specificity of JNK CO2 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 CO2 signaling in mammals, diptera and nematodes.
Rationale: Diabetic patients have a lower incidence of acute respiratory distress syndrome (ARDS), and those who develop ARDS are less likely to die. The mechanisms that underlie this protection are unknown.
Objectives: To determine whether leptin resistance, a feature of diabetes, prevents fibroproliferation after lung injury.
Methods: We examined lung injury and fibroproliferation after the intratracheal instillation of bleomycin in wild-type and leptin-resistant (db/db) diabetic mice. We examined the effect of leptin on transforming growth factor (TGF)-β1–mediated transcription in primary normal human lung fibroblasts. Bronchoalveolar lavage fluid (BAL) samples from patients with ARDS and ventilated control subjects were obtained for measurement of leptin and active TGF-β1 levels.
Measurements and Main Results: Diabetic mice (db/db) were resistant to lung fibrosis. The db/db mice had higher levels of peroxisome proliferator–activated receptor-γ (PPARγ), an inhibitor of the transcriptional response to TGF-β1, a cytokine critical in the pathogenesis of fibroproliferative ARDS. In normal human lung fibroblasts, leptin augmented the transcription of profibrotic genes in response to TGF-β1 through a mechanism that required PPARγ. In patients with ARDS, BAL leptin levels were elevated and correlated with TGF-β1 levels. Overall, there was no significant relationship between BAL leptin levels and clinical outcomes; however, in nonobese patients, higher BAL leptin levels were associated with fewer intensive care unit– and ventilator-free days and higher mortality.
Conclusions: Leptin signaling is required for bleomycin-induced lung fibrosis. Leptin augments TGF-β1 signaling in lung fibroblasts by inhibiting PPARγ. These findings provide a mechanism for the observed protection against ARDS observed in diabetic patients.
acute lung injury; fibrosis; lung; diabetes mellitus
HMG-CoA reductase inhibitors such as rosuvastatin may have immunomodulatory and anti-inflammatory effects that may reduce the severity of influenza A infection. We hypothesized that rosuvastatin would decrease viral replication, attenuate lung injury, and improve mortality following influenza A infection in mice.
C57Bl/6 mice were treated daily with rosuvastatin (10 mg/kg/day) supplemented in chow (or control chow) beginning three days prior to infection with either A//Udorn/72 [H3N2] or A/WSN/33 [H1N1] influenza A virus (1×105 pfu/mouse). Plaque assays were used to examine the effect of rosuvastatin on viral replication in vitro and in the lungs of infected mice. We measured cell count with differential, protein and cytokines in the bronchoalveolar lavage (BAL) fluid, histologic evidence of lung injury, and wet-to-dry ratio on Day 1, 2, 4, and 6. We also recorded daily weights and mortality.
The administration of rosuvastatin had no effect on viral clearance of influenza A after infection. Weight loss, lung inflammation and lung injury severity were similar in the rosuvastatin and control treated mice. In the mice infected with influenza A (A/WSN/33), mortality was unaffected by treatment with rosuvastatin.
Statins did not alter the replication of influenza A in vitro or enhance its clearance from the lung in vivo. Statins neither attenuated the severity of influenza A-induced lung injury nor had an effect on influenza A-related mortality. Our data suggest that the association between HMG CoA reductase inhibitors and improved outcomes in patients with sepsis and pneumonia are not attributable to their effects on influenza A infection.
Rationale: Acute lung injury and the acute respiratory distress syndrome are characterized by increased lung oxidant stress and apoptotic cell death. The contribution of epithelial cell apoptosis to the development of lung injury is unknown.
Objectives: To determine whether oxidant-mediated activation of the intrinsic or extrinsic apoptotic pathway contributes to the development of acute lung injury.
Methods: Exposure of tissue-specific or global knockout mice or cells lacking critical components of the apoptotic pathway to hyperoxia, a well-established mouse model of oxidant-induced lung injury, for measurement of cell death, lung injury, and survival.
Measurements and Main Results: We found that the overexpression of SOD2 prevents hyperoxia-induced BAX activation and cell death in primary alveolar epithelial cells and prolongs the survival of mice exposed to hyperoxia. The conditional loss of BAX and BAK in the lung epithelium prevented hyperoxia-induced cell death in alveolar epithelial cells, ameliorated hyperoxia-induced lung injury, and prolonged survival in mice. By contrast, Cyclophilin D–deficient mice were not protected from hyperoxia, indicating that opening of the mitochondrial permeability transition pore is dispensable for hyperoxia-induced lung injury. Mice globally deficient in the BH3-only proteins BIM, BID, PUMA, or NOXA, which are proximal upstream regulators of BAX and BAK, were not protected against hyperoxia-induced lung injury suggesting redundancy of these proteins in the activation of BAX or BAK.
Conclusions: Mitochondrial oxidant generation initiates BAX- or BAK-dependent alveolar epithelial cell death, which contributes to hyperoxia-induced lung injury.
cell death; epithelium; Bcl-2 proteins; acute respiratory distress syndrome
AMP-activated protein kinase (AMPK) is a sensor of cellular energy status found in metazoans that is known to be activated by stimuli that increase the cellular AMP/ATP ratio. Full activation of AMPK requires specific phosphorylation within the activation loop of the catalytic domain of the α-subunit by upstream kinases such as the serine/threonine protein kinase LKB1. Here we show that hypoxia activates AMPK through LKB1 without an increase in the AMP/ATP ratio. Hypoxia increased reactive oxygen species (ROS) levels and the antioxidant EUK-134 abolished the hypoxic activation of AMPK. Cells deficient in mitochondrial DNA (ρ0 cells) failed to activate AMPK during hypoxia but are able to in the presence of exogenous H2O2. Furthermore, we provide genetic evidence that ROS generated within the mitochondrial electron transport chain and not oxidative phosphorylation is required for hypoxic activation of AMPK. Collectively, these data indicate that oxidative stress and not an increase in the AMP/ATP ratio is required for hypoxic activation of AMPK.
AMP-activated kinase; Hypoxia; LKB1; Mitochondria; Reactive oxygen species; Free radicals
Exposure of human populations to chronically elevated levels of ambient particulate matter air pollution < 2.5 μm in diameter (PM2.5) has been associated with an increase in lung cancer incidence. Over 70% of lung cancer cell lines exhibit promoter methylation of the tumor suppressor p16, an epigenetic modification that reduces its expression. We exposed mice to concentrated ambient PM2.5 via inhalation, 8 hours daily for 3 weeks and exposed primary murine alveolar epithelial cells to daily doses of fine urban PM (5 µg/cm2). In both mice and alveolar epithelial cells, PM exposure increased ROS production, expression of the DNA methyltransferase 1 (DNMT1), and methylation of the p16 promoter. In alveolar epithelial cells, increased transcription of DNMT1 and methylation of the p16 promoter were inhibited by a mitochondrially targeted antioxidant and a JNK inhibitor. These findings provide a potential mechanism by which PM exposure increases the risk of lung cancer.
Alcohol intake increases the risk of acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) and is associated with poor outcomes in patients who develop these syndromes. No specific therapies are currently available to treat or decrease the risk of ARDS in patients with alcoholism. We have recently shown increased levels of lung adenosine inhibit alveolar fluid clearance, an important predictor of outcome in patients with ARDS. We hypothesized that alcohol might worsen lung injury by increasing lung adenosine levels, resulting in impaired active Na+ transport in the lung.
We treated wild-type mice with alcohol administered i.p. to achieve blood alcohol levels associated with moderate to severe intoxication and measured the rate of alveolar fluid clearance and Na,K-ATPase expression in peripheral lung tissue and assessed the effect of alcohol on survival during exposure to hyperoxia. We used primary rat alveolar type II cells to investigate the mechanisms by which alcohol regulates alveolar Na+ transport.
Exposure to alcohol reduced alveolar fluid clearance, downregulated Na,K-ATPase in the lung tissue and worsened hyperoxia-induced lung injury. Alcohol caused an increase in BAL fluid adenosine levels. A similar increase in lung adenosine levels was observed after exposure to hyperoxia. In primary rat alveolar type II cells alcohol and adenosine decreased the abundance of the Na,K-ATPase at the basolateral membrane via a mechanism that required activation of the AMPK.
Alcohol decreases alveolar fluid clearance and impairs survival from acute lung injury. Alcohol induced increases in lung adenosine levels may be responsible for reduction in alveolar fluid clearance and associated worsening of lung injury.
Excitement surrounding the attractive physical and chemical characteristics of single walled carbon nanotubes (SWCNTs) has been tempered by concerns regarding their potential health risks. Here we consider the lung toxicity of nanoscale dispersed SWCNTs (mean diameter ~ 1 nm). Because dispersion of the SWCNTs increases their aspect ratio relative to as-produced aggregates, we directly test the prevailing hypothesis that lung toxicity associated with SWCNTs compared with other carbon structures is attributable to the large aspect ratio of the individual particles. Thirty days after their intratracheal administration to mice, the granuloma-like structures with mild fibrosis in the large airways observed in mice treated with aggregated SWCNTs were absent in mice treated with nanoscale dispersed SWCNTs. Examination of lung sections from mice treated with nanoscale dispersed SWCNTs revealed uptake of the SWCNTs by macrophages and gradual clearance over time. We conclude that the toxicity of SWCNTs in vivo is attributable to aggregation of the nanomaterial rather than the large aspect ratio of the individual nanotubes. Biocompatible nanoscale dispersion provides a scalable method to generate purified preparations of SWCNTs with minimal toxicity, thus allowing them to be used safely in commercial and biomedical applications.
Exposure of human populations to ambient particulate matter (PM) air pollution significantly contributes to the mortality attributable to ischemic cardiovascular events. We reported that mice treated with intratracheally instilled PM develop a prothrombotic state that requires the release of IL-6 by alveolar macrophages. We sought to determine whether exposure of mice to PM increases the levels of PAI-1, a major regulator of thrombolysis, via a similar or distinct mechanism.
Methods and Principal Findings
Adult, male C57BL/6 and IL-6 knock out (IL-6−/−) mice were exposed to either concentrated ambient PM less than 2.5 µm (CAPs) or filtered air 8 hours daily for 3 days or were exposed to either urban particulate matter or PBS via intratracheal instillation and examined 24 hours later. Exposure to CAPs or urban PM resulted in the IL-6 dependent activation of coagulation in the lung and systemically. PAI-1 mRNA and protein levels were higher in the lung and adipose tissue of mice treated with CAPs or PM compared with filtered air or PBS controls. The increase in PAI-1 was similar in wild-type and IL-6−/− mice but was absent in mice treated with etanercept, a TNF-α inhibitor. Treatment with etanercept did not prevent the PM-induced tendency toward thrombus formation.
Mice exposed to inhaled PM exhibited a TNF-α-dependent increase in PAI-1 and an IL-6-dependent activation of coagulation. These results suggest that multiple mechanisms link PM-induced lung inflammation with the development of a prothrombotic state.
It is estimated that, combined, 400,000 people are diagnosed with idiopathic pulmonary fibrosis (IPF) or acute lung injury/acute respiratory distress syndrome annually in the United States, and both diseases are associated with an unacceptably high mortality rate. Although these disorders are distinct clinical entities, they share pathogenic mechanisms that may provide overlapping therapeutic targets. One example is fibroblast activation, which occurs concomitant with acute lung injury as well as in the progressive fibrosis of IPF. Both clinical entities are characterized by elevations of the profibrotic cytokine, transforming growth factor (TGF)-β1. Protein degradation by the ubiquitin–proteasomal system modulates TGF-β1 expression and signaling. In this review, we highlight the effects of proteasomal inhibition in various animal models of tissue fibrosis and mechanisms by which it may regulate TGF-β1 expression and signaling. At present, there are no effective therapies for fibroproliferative acute respiratory distress syndrome or IPF, and proteasomal inhibition may provide a novel, attractive target in these devastating diseases.
acute respiratory distress syndrome; transforming growth factor-β1; Smad; ubiquitination
Previous studies from our lab have demonstrated that upon exposure to physiologic levels of cyclic stretch, alveolar epithelial cells demonstrate a significant decrease in the amount of polymerized tubulin (Geiger et al., Gene Therapy 2006;13:725–731). However, not all microtubules are disassembled, although the mechanisms or implications of this were unknown. Using immunofluorescence microscopy, Western blotting, and immunohistochemistry approaches, we have compared the levels of acetylated tubulin in stretched and unstretched A549 cells and in murine lungs. In cultured cells exposed to cyclic stretch (10% change in basement membrane surface area at 0.25 Hz), nearly all of the remaining microtubules were acetylated, as demonstrated using immunofluorescence microscopy. In murine lungs ventilated for 20 minutes at 12 to 20 ml/kg followed by 48 hours of spontaneous breathing or for 3 hours at 16 to 40 ml/kg, levels of acetylated tubulin were increased in the peripheral lung. In both our in vitro and in vivo studies, we have found that mild to moderate levels of cyclic stretch significantly increases tubulin acetylation in a magnitude- and duration-dependent manner. This appears to be due to a decrease in histone deacetylase 6 activity (HDAC6), the major tubulin deacetylase. Since it has been previously shown that acetylated microtubules are positively correlated to a more stable population of microtubules, this result suggests that microtubule stability may be increased by cyclic stretch, and that tubulin acetylation is one way in which cells respond to changes in exogenous mechanical forces.
microtubule; histone deacetylase 6; acetylation; alveolar epithelium