Alveolar Space Enlargement Research Articles

Type 1 diabetes contributes to combined pulmonary fibrosis and emphysema in male alpha 1 antitrypsin deficient mice.

Type 1 diabetes (T1D) is a metabolic disease characterized by hyperglycemia and can affect multiple organs, leading to life-threatening complications. Increased prevalence of pulmonary disease is observed in T1D patients, and diabetes is a leading cause of comorbidity in several lung pathologies. A deficiency of alpha-1 antitrypsin (AAT) can lead to the development of emphysema. Decreased AAT plasma concentrations and anti-protease activity are documented in T1D patients. The objective of this study was to determine whether T1D exacerbates the progression of lung damage in AAT deficiency. First, pulmonary function testing (PFT) and histopathological changes in the lungs of C57BL/6J streptozotocin (STZ)-induced T1D mice were investigated 3 and 6 months after the onset of hyperglycemia. PFT demonstrated a restrictive pulmonary pattern in the lungs of STZ-injected mice, along with upregulation of mRNA expression of pro-fibrotic markers Acta2, Ccn2, and Fn1. Increased collagen deposition was observed 6 months after the onset of hyperglycemia. To study the effect of T1D on the progression of lung damage in AAT deficiency background, C57BL/6J AAT knockout (KO) mice were used. Control and STZ-challenged AAT KO mice did not show significant changes in lung function 3 months after the onset of hyperglycemia. However, histological examination of the lung demonstrated increased collagen accumulation and alveolar space enlargement in STZ-induced AAT KO mice. AAT pretreatment on TGF-β-stimulated primary lung fibroblasts reduced mRNA expression of pro-fibrotic markers ACTA2, CCN2, and FN1. Induction of T1D in AAT deficiency leads to a combined pulmonary fibrosis and emphysema (CPFE) phenotype in male mice.

USP13 deficiency aggravates cigarette smoke‐induced alveolar space enlargement through stabilization of TXNIP

Deubiquitinating enzymes regulate cellular function through deubiquitination and stabilization of substrate proteins. We have shown that USP13 deficiency promotes lung inflammation. Cigarette smoke (CS) causes lung connective tissue disruption and oxidative stress in lung cells, resulting in the development of emphysema; however, the role of USP13 in the CS exposure‐induced emphysema has not been well studied. USP13‐deficient mice and wild type mice were exposed to 4 unfiltered cigarettes for 5 days each week, over the course of 6 months. While emphysema was not observed in wild type mice, CS exposure increased γ‐H2AX levels in lung tissues, suggesting a model of mild smoke exposure was established. Under the mild CS exposure condition, USP13‐deficient mice show significant increases in alveolar CL, indicating that USP13‐deficient mice are susceptible to CS‐induced alveolar enlargement. We have shown that USP13 knockout reduced fibronectin expression in lungs. Further, we found that collagen levels were reduced in USP13‐siRNA‐transfected lung fibroblast cells. It is possible that a loss of connective tissue function contributes to alveolar enlargement in USP13‐deficient mice. Oxidative stress is a hallmark of CS‐induced emphysema. We investigated the role of USP13 in the expression of an oxidative stress markers, named thioredoxin interacting protein (TXNIP), which modulates cellular redox states and NLRP3 inflammasome activity. TXNIP expression levels have been shown to be decreased in smokers compared with non‐smokers. TXNIP is unstable in the presence of protein synthesis inhibitor cycloheximide (CHX). Pretreatment with proteasome inhibitor, MG‐132, attenuated TXNIP degradation, suggesting that TXNIP degradation is mediated by the proteasome. Knockout or knockdown of USP13 in lungs or lung fibroblast cells significantly reduced TXNIP levels. TXNIP degradation is increased in USP13 knockdown cells. Further, the reduction of TXNIP was observed in USP13‐inhibitor‐treated mouse lung epithelial cells. This study provides evidence showing that USP13 deficiency plays a role in alveolar space enlargement.

Postnatal Ozone Exposure Disrupts Alveolar Development, Exaggerates Mucoinflammatory Responses, and Suppresses Bacterial Clearance in Developing Scnn1b-Tg+ Mice Lungs

Increased levels of ambient ozone, one of the six criteria air pollutants, result in respiratory tract injury and worsening of ongoing lung diseases. However, the effect of ozone exposure on the respiratory tract undergoing active lung development and simultaneously experiencing mucoinflammatory lung diseases, such as cystic fibrosis, remains unclear. To address these questions, we exposed Scnn1b transgenic (Scnn1b-Tg+) mice, a mouse model of cystic fibrosis-like lung disease, and littermate wild-type (WT) mice to ozone from postnatal days (PND) 3-20 and examined the lung phenotypes at PND21. As compared with filtered air (FA)-exposed WT mice, the ozone-exposed WT mice exhibited marked alveolar space enlargement, in addition to significant eosinophilic infiltration, type 2 inflammation, and mucous cell metaplasia. Ozone-exposed Scnn1b-Tg+ mice also exhibited significantly increased alveolar space enlargement, which was also accompanied by exaggerated granulocytic infiltration, type 2 inflammation, and a greater degree of mucus obstruction. The alveolar space enlargement in ozone-exposed WT, FA-exposed Scnn1b-Tg+, and ozone-exposed Scnn1b-Tg+ mice was accompanied by elevated levels of MMP12 protein in macrophages and Mmp12 mRNA in the lung homogenates. Finally, although bacterial burden was largely resolved by PND21 in FA-exposed Scnn1b-Tg+ mice, ozone-exposed Scnn1b-Tg+ mice exhibited compromised bacterial clearance, which was also associated with increased levels of IL-10, an immunosuppressive cytokine, and marked mucus obstruction. Taken together, our data show that ozone exposure results in alveolar space remodeling during active phases of lung development and markedly exaggerates the mucoinflammatory outcomes of pediatric-onset lung disease, including bacterial infections, granulocytic inflammation, mucus obstruction, and alveolar space enlargement.

USP13 Deficiency Aggravates Cigarette-smoke-induced Alveolar Space Enlargement.

Alveolar enlargement is a pathological feature of emphysema. Long-term exposure to cigarette smoke (CS) is a high-risk factor for the development of emphysema. Abnormal protein ubiquitination has been implicated to regulate the development of human disorders, however, the role of protein ubiquitination in emphysema has not been well-studied. In this study, we attempted to investigate if a deubiquitinase, USP13, regulates the development of emphysema. Under a mild CS exposure condition, USP13-deficient mice show significant increases in alveolar chord length, indicating that USP13-deficient mice are susceptible to CS-induced alveolar enlargement. It has been shown that USP13 knockout reduced fibronectin expression in lungs. Here, we found that collagen levels were reduced in USP13 siRNA-transfected lung fibroblast cells. This suggests that a loss of extracellular matrix in connective tissues contributes to alveolar enlargement in USP13-deficient mice in response to CS exposure. Further, we investigated the role of USP13 in the expression of oxidative stress markers TXNIP and HMOX1. An increase in HMOX1 abundance was observed in USP13 knockdown lung fibroblast and epithelial cells. Overexpression of USP13 reduced HMOX1 protein levels in lung fibroblast cells, suggesting that modulation of USP13 levels may affect oxidative stress. Knockdown of USP13 significantly reduced TXNIP levels in lungs or lung fibroblast cells. A protein stability pulse-chase assay showed that TXNIP is instable within USP13 knockdown lung fibroblast cells. Further, the reduction of TXNIP was observed in USP13 inhibitor-treated lung epithelial cells. USP13-deficient mice also show higher levels of IgG in bronchoalveolar lavage fluid. This study provides evidence showing that USP13 deficiency plays a role in alveolar space enlargement.

Postnatal ozone exposure disrupts alveolar development, exaggerates mucoinflammatory responses, and suppresses bacterial clearance in developing Scnn1b-Tg+ mice lungs.

Abstract Increased levels of ambient ozone result in the respiratory tract injury and worsening of ongoing lung diseases. However, the effect of ozone exposure on the respiratory tract undergoing active lung development and simultaneously experiencing mucoinflammatory lung diseases such as cystic fibrosis (CF) remains unclear. To address these questions, we exposed sodium channel non-voltage gated 1, beta subunit overexpressing transgenic mice (Scnn1b-Tg+), a mouse model of CF-like lung disease, and littermate wild type (WT) mice to ozone from postnatal day (PND) 3–20 and examined the lung phenotypes at PND21. As compared to filtered air (FA)-exposed WT mice, the ozone-exposed WT mice exhibited marked alveolar space enlargement, significant eosinophilic infiltration, type-2 inflammation, and mucous cell metaplasia. Ozone-exposed Scnn1b-Tg+ mice also exhibited significantly increased alveolar space enlargement, exaggerated granulocytic infiltration, type-2 inflammation, and greater degree of mucoobstruction. The alveolar space enlargement in ozone-exposed WT, and ozone-exposed Scnn1b-Tg+ mice was accompanied by elevated levels of MMP12 protein in macrophages and Mmp12 mRNA in the lung homogenates. Finally, while bacterial burden was largely resolved by PND21 in FA-exposed Scnn1b-Tg+ mice, ozone-exposed Scnn1b-Tg+ mice exhibited compromised bacterial clearance which was also associated with increased levels of IL-10, an immunosuppressive cytokine, and marked mucoobstruction. Taken together, our data show that ozone exposure results in alveolar space remodeling during active phases of lung development and markedly exaggerates the developmental history of an ongoing mucoinflammatory pediatric onset lung disease.

Sirtuin 3 Inhibits Airway Epithelial Mitochondrial Oxidative Stress in Cigarette Smoke-Induced COPD.

Mitochondrial damage in airway epithelial cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Sirtuin 3 (Sirt3) is a mitochondrial deacetylase regulating mitochondrial function, but its role in the pathogenesis of COPD is still unknown. The aim of the present study was to investigate the effect of Sirt3 on airway epithelial mitochondria in cigarette smoke-induced COPD. Our present study has shown serious airway inflammation, alveolar space enlargement, and mitochondrial damage of the airway epithelium in COPD rats. Compared to the control rats, Sirt3 protein expression was significantly decreased in the airway epithelium and lung tissue homogenate from COPD rats. In airway epithelial cells (BEAS-2B), cigarette smoke extract (CSE) treatment significantly decreased mRNA and protein expression of Sirt3 and manganese superoxide dismutase (MnSOD), as well as MnSOD activity in a concentration and time-dependent manner. Sirt3 siRNA further significantly intensified the decreases in MnSOD expression and activity and aggravated mitochondrial oxidative stress and cell injury when airway epithelial cells were treated with 7.5% CSE. In contrast, Sirt3 overexpression significantly prevented the decrease of MnSOD expression and activity and improved mitochondrial oxidative stress and cell injury in CSE-treated airway epithelial cells. These data suggest that Sirt3 inhibits airway epithelial mitochondrial oxidative stress possibly through the regulation of MnSOD, thereby contributing to the pathogenesis of COPD.

Ablation of IL-33 Suppresses Th2 Responses but Is Accompanied by Sustained Mucus Obstruction in the Scnn1b Transgenic Mouse Model.

Cystic fibrosis is characterized by dehydration of the airway surface liquid layer with persistent mucus obstruction. Th2 immune responses are often manifested as increased mucous cell density (mucous cell metaplasia) associated with mucus obstruction. IL-33 is a known inducer of Th2 immune responses, but its roles in mucus obstruction and related phenotypes in a cystic fibrosis-like lung disease model (i.e., Scnn1b-Tg-positive [Tg+]) mouse, remain unclear. Accordingly, IL-33 knockout (IL-33KO) Tg+ mice were examined and compared with IL-33 heterozygous (IL-33HET) Tg+ mice. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had complete absence of bronchoalveolar lavage fluid eosinophilia, accompanied with significant reduction in bronchoalveolar lavage fluid concentration of IL-5, a cytokine associated with eosinophil differentiation and recruitment, and IL-4, a major Th2 cytokine. As compared with IL-33HET/Tg+ mice, IL-33KO/Tg+ mice had significantly reduced levels of Th2-associated gene signatures (Slc26a4, Clca1, Retnla, and Chi3l4), along with complete loss of intracellular mucopolysaccharide staining in the airway epithelium. As compared with IL-33HET/Tg+ mice, although the IL-33KO/Tg+ mice had significantly reduced levels of MUC5AC protein expression, they showed no reduction in the degree of mucus obstruction, MUC5B protein expression, bacterial burden, and neonatal mortality. Interestingly, the histological features, including subepithelial airway inflammation and alveolar space enlargement, were somewhat exaggerated in IL-33KO/Tg+ mice compared with IL-33HET/Tg+ mice. Taken together, our data indicate that although IL-33 modulates Th2 inflammatory responses and MUC5AC protein production, mucus obstruction is not dependent on IL-33.

Reduced transient receptor potential vanilloid 2 expression in alveolar macrophages causes COPD in mice through impaired phagocytic activity

BackgroundDefective phagocytosis in alveolar macrophages is associated with chronic obstructive pulmonary disease (COPD). Transient receptor potential cation channel subfamily V member 2 (TRPV2), a type of nonselective cation channel pertinent to diverse physiological functions, regulates macrophage phagocytosis. However, the role of TRPV2 in COPD remains poorly understood. Here, we explored the role of TRPV2 in the development of COPD.MethodsMacrophage TRPV2 expression and phagocytosis function were measured in MH-S cells (a murine alveolar macrophage cell line) and a cigarette smoke exposure mouse model.ResultsTRPV2 expression and phagocytosis function were reduced when MH-S cells were exposed to cigarette smoke extract (CSE). TRPV2 knockdown by siRNA decreased phagocytosis in MH-S cells. Consistently, TRPV2 expression was reduced in alveolar macrophages prepared from bronchoalveolar lavage samples of mice which were exposed to cigarette smoke for 2 months. In addition, the alveolar space was progressively enlarged during development in TRPV2 knockout (TRPV2KO) mice. Moreover, exposure to cigarette smoke for 2 months significantly induced alveolar space enlargement in TRPV2KO mice, but not in wild-type (WT) mice. The phagocytic function of alveolar macrophages from TRPV2KO mice was reduced, compared with macrophages from WT mice.ConclusionsTRPV2 expression is profoundly downregulated in alveolar macrophages at early time points of cigarette smoke exposure. Reduced TRPV2-mediated phagocytic function renders the lung susceptible to cigarette smoke-induced alveolar space enlargement. TRPV2 may provide a therapeutic target for COPD induced by cigarette smoke.

Low-Level Laser Therapy Reduces Lung Inflammation in an Experimental Model of Chronic Obstructive Pulmonary Disease Involving P2X7 Receptor.

Chronic obstructive pulmonary disease (COPD) is a progressive disease characterized by irreversible airflow limitation, airway inflammation and remodeling, and enlargement of alveolar spaces. COPD is in the top five leading causes of deaths worldwide and presents a high economic cost. However, there are some preventive measures to lower the risk of developing COPD. Low-level laser therapy (LLLT) is a new effective therapy, with very low cost and no side effects. So, our objective was to investigate if LLLT reduces pulmonary alterations in an experimental model of COPD. C57BL/6 mice were submitted to cigarette smoke for 75 days (2x/day). After 60 days to smoke exposure, the treated group was submitted to LLLT (diode laser, 660 nm, 30 mW, and 3 J/cm2) for 15 days and euthanized for morphologic and functional analysis of the lungs. Our results showed that LLLT significantly reduced the number of inflammatory cells and the proinflammatory cytokine secretion such as IL-1β, IL-6, and TNF-α in bronchoalveolar lavage fluid (BALF). We also observed that LLLT decreased collagen deposition as well as the expression of purinergic P2X7 receptor. On the other hand, LLLT increased the IL-10 release. Thus, LLLT can be pointed as a promising therapeutic approach for lung inflammatory diseases as COPD.

Assessment of pulmonary microstructural changes by hyperpolarized 129Xe diffusion-weighted imaging in an elastase-instilled rat model of emphysema.

The purpose of this study was to explore the feasibility of hyperpolarized 129Xe diffusion-weighted imaging (DWI) for the evaluation of pulmonary microstructural changes in the presence of pancreatic porcine elastase (PPE)-induced pulmonary emphysema rat model. Sixteen male Sprague-Dawley (SD) rats were randomly divided into two groups, the emphysema model group and control group. Experimental emphysematous models were made by instilling elastase into rat lungs of model group, the control group were instilled with isodose saline. Hyperpolarized 129Xe magnetic resonance imaging (MRI) and histology were performed in all 16 rats after 30 days. DWIs were performed on a Bruker 7.0 T micro MRI, and the apparent diffusion coefficients (ADCs) were measured in all rats. Mean linear intercepts (MLIs) of pulmonary alveoli were measured on histology. The statistical analyses were performed about the correlation between the mean ADC of hyperpolarized 129Xe in the whole lung and MLI of pulmonary histology metric. The pulmonary emphysematous model was successfully confirmed by the histology and all scans were also successful. The ADC value of 129Xe in the model group (0.0313±0.0005 cm2/s) was significantly increased compared with that of the control group (0.0288±0.0007 cm2/s, P<0.0001). Morphological differences such as MLI of pulmonary alveoli were observed between the two groups, the MLI of pulmonary alveoli in model group significantly increased (91±5 µm) than that of control group (50±3 µm, P<0.0001). Furthermore, the ADCs was moderately correlated with MLIs (r=0.724, P<0.01). These results indicate that 129Xe ADC value can quantitatively reflect the alveolar space enlargement and it is a promising biomarker for the detection of pulmonary emphysema.

Early Impairment of Lung Mechanics in a Murine Model of Marfan Syndrome.

Early morbidity and mortality in patients with Marfan syndrome (MFS) -a connective tissue disease caused by mutations in fibrillin-1 gene- are mainly caused by aorta aneurysm and rupture. However, the increase in the life expectancy of MFS patients recently achieved by reparatory surgery promotes clinical manifestations in other organs. Although some studies have reported respiratory alterations in MFS, our knowledge of how this connective tissue disease modifies lung mechanics is scarce. Hence, we assessed whether the stiffness of the whole lung and of its extracellular matrix (ECM) is affected in a well-characterized MFS mouse model (FBN1C1039G/+). The stiffness of the whole lung and of its ECM were measured by conventional mechanical ventilation and atomic force microscopy, respectively. We studied 5-week and 9-month old mice, whose ages are representative of early and late stages of the disease. At both ages, the lungs of MFS mice were significantly more compliant than in wild type (WT) mice. By contrast, no significant differences were found in local lung ECM stiffness. Moreover, histopathological lung evaluation showed a clear emphysematous-like pattern in MFS mice since alveolar space enlargement was significantly increased compared with WT mice. These data suggest that the mechanism explaining the increased lung compliance in MFS is not a direct consequence of reduced ECM stiffness, but an emphysema-like alteration in the 3D structural organization of the lung. Since lung alterations in MFS are almost fully manifested at an early age, it is suggested that respiratory monitoring could provide early biomarkers for diagnosis and/or follow-up of patients with the Marfan syndrome.

Chronic Exposure to Water-Pipe Smoke Induces Alveolar Enlargement, DNA Damage and Impairment of Lung Function

Background/Aim: Epidemiological evidence indicates that water-pipe smoking (WPS) adversely affects the respiratory system. However, the mechanisms underlying its effects are not well understood. Recent experimental studies reported the occurrence of lung inflammation and oxidative stress following acute and subacute exposure to WPS. Here, we wanted to verify the extent of inflammation and oxidative stress in mice chronically-exposed to WPS and to evaluate, for the first time, its effect on alveolar injury and DNA damage and their association with impairment of lung function. Methods: Mice were nose-only exposed to mainstream WPS (30 min/day; 5 days/week for 6 consecutive months). Control mice were exposed using the same protocol to atmospheric air only. At the end of the exposure period, several respiratory parameters were assessed. Results: In bronchoalveolar lavage fluid, WPS increased neutrophil and lymphocyte numbers, lactate dehydrogenase, myeloperoxidase and matrix metallopeptidase 9 activities, as well as several proinflammatory cytokines. In lung tissue, lipid peroxidation, reactive oxygen species, superoxide dismutase activity and reduced glutathione were all increased by WPS exposure. Along with oxidative stress, WPS exposure significantly increased lung DNA damage index. Histologically the lungs of WPS-exposed mice had foci of mixed inflammatory cells infiltration in the interalveolar interstitium which consisted of neutrophils, lymphocytes and macrophages. Interestingly, we found dilated alveolar spaces and alveolar ducts with damaged interalveolar septae, and impairment of lung function following WPS exposure. Conclusion: We show the persistence of lung inflammation and oxidative stress in mice chronically-exposed to WPS and demonstrate, for the first time, the occurrence of DNA damage and enlargement of alveolar spaces and ducts associated with impairment of lung function. Our findings provide novel mechanistic elucidation for the long-term effects of WPS on the respiratory system.

Role of Cigarette Smoke-Induced Aggresome Formation in Chronic Obstructive Pulmonary Disease-Emphysema Pathogenesis.

Cigarette smoke (CS) exposure is known to induce proteostasis imbalance that can initiate accumulation of ubiquitinated proteins. Therefore, the primary goal of this study was to determine if first- and secondhand CS induces localization of ubiquitinated proteins in perinuclear spaces as aggresome bodies. Furthermore, we sought to determine the mechanism by which smoke-induced aggresome formation contributes to chronic obstructive pulmonary disease (COPD)-emphysema pathogenesis. Hence, Beas2b cells were treated with CS extract (CSE) for in vitro experimental analysis of CS-induced aggresome formation by immunoblotting, microscopy, and reporter assays, whereas chronic CS-exposed murine model and human COPD-emphysema lung tissues were used for validation. In preliminary analysis, we observed a significant (P < 0.01) increase in ubiquitinated protein aggregation in the insoluble protein fraction of CSE-treated Beas2b cells. We verified that CS-induced ubiquitin aggregrates are localized in the perinuclear spaces as aggresome bodies. These CS-induced aggresomes (P < 0.001) colocalize with autophagy protein microtubule-associated protein 1 light chain-3B(+) autophagy bodies, whereas U.S. Food and Drug Administration-approved autophagy-inducing drug (carbamazepine) significantly (P < 0.01) decreases their colocalization and expression, suggesting CS-impaired autophagy. Moreover, CSE treatment significantly increases valosin-containing protein-p62 protein-protein interaction (P < 0.0005) and p62 expression (aberrant autophagy marker; P < 0.0001), verifying CS-impaired autophagy as an aggresome formation mechanism. We also found that inhibiting protein synthesis by cycloheximide does not deplete CS-induced ubiquitinated protein aggregates, suggesting the role of CS-induced protein synthesis in aggresome formation. Next, we used an emphysema murine model to verify that chronic CS significantly (P < 0.0005) induces aggresome formation. Moreover, we observed that autophagy induction by carbamazepine inhibits CS-induced aggresome formation and alveolar space enlargement (P < 0.001), confirming involvement of aggresome bodies in COPD-emphysema pathogenesis. Finally, significantly higher p62 accumulation in smokers and severe COPD-emphysema lungs (Global Initiative for Chronic Obstructive Lung Disease Stage III/IV) as compared with normal nonsmokers (Global Initiative for Chronic Obstructive Lung Disease Stage 0) substantiates the pathogenic role of autophagy impairment in aggresome formation and COPD-emphysema progression. In conclusion, CS-induced aggresome formation is a novel mechanism involved in COPD-emphysema pathogenesis.

Proliferative activity of liver growth factor is associated with an improvement of cigarette smoke-induced emphysema in mice.

Cigarette smoke (CS)-induced emphysema is a major component of chronic obstructive pulmonary disease (COPD). COPD treatment is based on the administration of bronchodilators and corticosteroids to control symptoms and exacerbations, however, to date, there are no effective therapies to reverse disease progression. Liver growth factor (LGF) is an albumin-bilirubin complex with mitogenic properties, whose therapeutic effects have previously been reported in a model of emphysema and several rodent models of human disease. To approach the therapeutic effect of LGF in a model of previously established emphysema, morphometric and lung function parameters, matrix metalloproteinase (MMP) activity and the expression of several markers, such as VEGF, PCNA, 3NT and Nrf2, were assessed in air-exposed and CS-exposed C57BL/6J male mice with and without intraperitoneal (i.p.) injection of LGF. CS-exposed mice presented a significant enlargement of alveolar spaces, higher alveolar internal area and loss of lung function that correlated with higher MMP activity, higher expression of 3NT and lower expression of VEGF. CS-exposed mice injected with LGF, showed an amelioration of emphysema and improved lung function, which correlated with lower MMP activity and 3NT expression and higher levels of VEGF, PCNA and Nrf2. Taken together, this study suggests that LGF administration ameliorates CS-induced emphysema, highlights the ability of LGF to promote alveolar cell proliferation and may be a promising strategy to revert COPD progression.

Prevention of Alveolar Destruction and Airspace Enlargement in a Mouse Model of Pulmonary Lymphangioleiomyomatosis (LAM)

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic disease characterized by neoplastic growth of atypical smooth muscle-like LAM cells, destruction of lung parenchyma, obstruction of lymphatics, and formation of lung cysts, leading to spontaneous pneumothoraces (lung rupture and collapse) and progressive loss of pulmonary function. The disease is caused by mutational inactivation of the tumor suppressor gene tuberous sclerosis complex 1 (TSC1) or TSC2. By injecting TSC2-null cells into nude mice, we have developed a mouse model of LAM that is characterized by multiple random TSC2-null lung lesions, vascular endothelial growth factor-D expression, lymphangiogenesis, destruction of lung parenchyma, and decreased survival, similar to human LAM. The mice show enlargement of alveolar airspaces that is associated with progressive growth of TSC2-null lesions in the lung, up-regulation of proinflammatory cytokines and matrix metalloproteinases (MMPs) that degrade extracellular matrix, and destruction of elastic fibers. TSC2-null lesions and alveolar destruction were differentially inhibited by the macrolide antibiotic rapamycin (which inhibits TSC2-null lesion growth by a cytostatic mechanism) and a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, simvastatin (which inhibits growth of TSC2-null lesions by a predominantly proapoptotic mechanism). Treatment with simvastatin markedly inhibited MMP-2, MMP-3, and MMP-9 levels in lung and prevented alveolar destruction. The combination of rapamycin and simvastatin prevented both growth of TSC2-null lesions and lung destruction by inhibiting MMP-2, MMP-3, and MMP-9. Our findings demonstrate a mechanistic link between loss of TSC2 and alveolar destruction and suggest that treatment with rapamycin and simvastatin together could benefit patients with LAM by targeting cells with TSC2 dysfunction and preventing airspace enlargement.

Chronic Inflammation in the Pathogenesis of COPD and Lung Cancer

Chronic obstructive pulmonary disease (COPD) and lung cancer are leading causes of morbidity and mortality worldwide. At least 85 to 90% of all COPD and human lung cancers are related to cigarette smoke (CS), but genetic susceptibility and environmental factors also contribute to the pathogenesis. Chronic airway inflammation has been indicated as a major risk factor in COPD and has been suggested to be associated with an increased risk of human cancer, including lung cancer. Therefore, we hypothesize that COPD and lung cancer may share chronic inflammation as one of the common pathogenic mechanisms. We developed two animal exposure paradigms to elucidate roles of chronic inflammation in COPD and lung cancer. In the COPD study, mice were separated into four treatment groups: filtered-air control, lipopolysaccharides (LPS), CS, and LPS1CS. For the lung cancer study, mice were assigned into four different treatment groups: saline control, LPS, NNK (a major cigarette smoke carcinogen), and LPS1NNK. Mice were killed after 4 to 6 months of treatment and lung tissues were collected to assess alveolar space enlargement, lung tumors, and the inflammatory response. The LPS group showed increased inflammation but no alveolar space enlargement or tumor formation. LPS1CS displayed enhanced alveolar space enlargement when compared with CS alone, but neither group developed lung tumor. On the lung cancer study, we observed a more than sixfold increase in tumor numbers in the LPS1NNK group compared with the NNK alone group, but no alveolar space enlargement was observed. There were significant increases of inflammatory cells, particularly macrophages, in all treatment groups except NNK and controls. The increased alveolar macrophage population displayed an alternatively activated M2 phenotype in both COPD and lung cancer models. Our results suggest that chronic inflammation enhances emphysemalike alveolar space enlargement and also synergistically promotes cigarette smoke carcinogen–induced lung tumorigenesis.

Measurement of 129 Xe gas apparent diffusion coefficient anisotropy in an elastase-instilled rat model of emphysema

Hyperpolarized noble gas ((3)He and (129)Xe) apparent diffusion coefficient (ADC) measurements have shown remarkable sensitivity to microstructural (i.e., alveolar) changes in the lung, particularly emphysema. The ADC of hyperpolarized noble gases depends strongly on the diffusion time (Δ), and (3)He ADC has been shown to be anisotropic for Δ ranging from a few milliseconds down to a few hundred microseconds. In this study, the anisotropic nature of (129)Xe diffusion and its dependence on Δ were investigated both numerically, in a budded cylinder model, and in vivo, in an elastase-instilled rat model of emphysema. Whole lung longitudinal ADC (D(L)) and transverse ADC (D(T)) were measured for Δ = 6, 50, and 100 ms at 73.5 mT, and correlated with measurements of the mean linear intercept (L(m)) obtained from lung histology. A significant increase (P = 0.0021) in D(T) was measured for Δ = 6 ms between the sham (0.0021 ± 0.0005 cm(2)/s) and elastase-instilled (0.005 ± 0.001 cm(2)/s) cohorts, and a strong correlation was measured between D(T) (Δ = 6 ms) and L(m), with a Pearson's correlation coefficient of 0.90. This study confirms that (129)Xe D(T) increases correlate with alveolar space enlargement due to elastase instillation in rats.

Targeted Deletion of Jun/AP-1 in Alveolar Epithelial Cells Causes Progressive Emphysema and Worsens Cigarette Smoke–Induced Lung Inflammation

Chronic obstructive pulmonary disease appears to occur slowly and progressively over many years, with both genetic factors and environmental modifiers contributing to its pathogenesis. Although the c-Jun/activator protein 1 transcriptional factor regulates cell proliferation, apoptosis, and inflammatory responses, its role in lung pathogenesis is largely unknown. In this study, we report decreased expression levels of c-Jun mRNA and protein in the lung tissues of patients with advanced chronic obstructive pulmonary disease, and the genetic deletion of c-Jun specifically in alveolar epithelial cells causes progressive emphysema with lung inflammation and alveolar air space enlargement, which are cardinal features of emphysema. Although mice lacking c-Jun specifically in lung alveolar epithelial cells appear normal at the age of 6 weeks, when exposed to long-term cigarette smoke, c-Jun-mutant mice display more lung inflammation with perivascular and peribronchiolar infiltrates compared with controls. These results demonstrate that the c-Jun/activator protein 1 pathway is critical for maintaining lung alveolar cell homeostasis and that loss of its expression can contribute to lung inflammation and progressive emphysema.

Sepsis by using Cecal Ligation and Single Puncture Causes Alveolar Space Enlargement in LPA2 Knockout Mice

Lysophosphatidic acid (LPA) plays a dual-function in lung inflammatory diseases. LPA receptors contribute to the pathogenesis of asthma, acute lung injury, and fibrosis. Here, we investigate the role of LPA receptor type 2 (LPA2) in sepsis-induced lung inflammation and injury. Sepsis was induced using cecal ligation and single puncture (CLP) with 27 gauge needle. Plasma interleukin-6 (IL-6) and KC levels were elevated in septic wild type and LPA2-/- mice, while septic LPA2-/- mice reduces plasma KC, not IL-6 levels, compared to septic wild type mice. Bronchoalveolar lavage (BAL) KC levels increased in septic wild type and LPA2-/- mice, while the sepsis had no effect on BAL IL-6 levels, protein leak, and inflammatory cell infiltration in the lungs in wild type and LPA2-/- mice. Hematoxylin and eosin (H&E) staining revealed that septic LPA2-/- mice aggravated alveolar space enlargement. Western blotting analysis of lung tissues demonstrate that the level of cortactin, an F-actin binding protein, was decreased in septic LPA2-/- mice, when compared to wild type mice. The level of immunoglobulin G (IgG) in BAL fluids significantly increased in septic LPA2-/- mice, when compared to septic wild type mice and sham mice. Furthermore, we found that sham and septic LPA2-/- mice increased surfactant proteins B, C, and D (SP-B, SP-C, and SP-D) expression in lungs, while SP-A levels in lungs was decreased in sham and septic LPA2-/- mice. These results suggest LPA2 may regulate cortactin and surfactant protein expression in the lung. LPA2 and its downstream signaling may play a protective role against sepsis induced emphysema like disease.

Stimulation of sphingosine 1-phosphate signaling as an alveolar cell survival strategy in emphysema.

Vascular endothelial growth factor receptor (VEGFR) inhibition increases ceramides in lung structural cells of the alveolus, initiating apoptosis and alveolar destruction morphologically resembling emphysema. The effects of increased endogenous ceramides could be offset by sphingosine 1-phosphate (S1P), a prosurvival by-product of ceramide metabolism. The aims of our work were to investigate the sphingosine-S1P-S1P receptor axis in the VEGFR inhibition model of emphysema and to determine whether stimulation of S1P signaling is sufficient to functionally antagonize alveolar space enlargement. Concurrent to VEGFR blockade in mice, S1P signaling augmentation was achieved via treatment with the S1P precursor sphingosine, S1P agonist FTY720, or S1P receptor-1 (S1PR1) agonist SEW2871. Outcomes included sphingosine kinase-1 RNA expression and activity, sphingolipid measurements by combined liquid chromatography-tandem mass spectrometry, immunoblotting for prosurvival signaling pathways, caspase-3 activity and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assays, and airspace morphometry. Consistent with previously reported de novo activation of ceramide synthesis, VEGFR inhibition triggered increases in lung ceramides, dihydroceramides, and dihydrosphingosine, but did not alter sphingosine kinase activity or S1P levels. Administration of sphingosine decreased the ceramide-to-S1P ratio in the lung and inhibited alveolar space enlargement, along with activation of prosurvival signaling pathways and decreased lung parenchyma cell apoptosis. Sphingosine significantly opposed ceramide-induced apoptosis in cultured lung endothelial cells, but not epithelial cells. FTY720 or SEW2871 recapitulated the protective effects of sphingosine on airspace enlargement concomitant with attenuation of VEGFR inhibitor-induced lung apoptosis. Strategies aimed at augmenting the S1P-S1PR1 signaling may be effective in ameliorating the apoptotic mechanisms of emphysema development.