Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

Suppression of cigarette smoke induced MMP1 expression by selective serotonin re-uptake inhibitors.

Globally, COPD remains a major cause of disability and death. In the United States alone, it is estimated that approximately 14 million people suffer from the disease. Given the high disease burden and requirement for chronic, long-term medical care associated with COPD, it is essential that new disease modifying agents are developed to complement the symptomatic therapeutics currently available. In the present report, we have identified a potentially novel therapeutic agent through the use of a high throughput screen based on the knowledge that cigarette smoke induces the proteolytic enzyme MMP1 leading to destruction of the lung in COPD. A construct utilizing the cigarette responsive promoter element of MMP-1 was conjugated to a luciferase reporter and utilized in an in vitro assay to screen the NIH Molecular Libraries Small Molecule Repository to identify putative targets that suppressed luciferase expression in response to cigarette smoke extract (CSE). Selective serotonin reuptake inhibitors potently inhibited luciferase expression and were further validated. SSRI treatment suppressed MMP-1 production in small airway epithelial cells exposed to (CSE) in vitro as well as in smoke exposed rabbits. In addition, SSRI treatment inhibited inflammatory cytokine production while rescuing cigarette smoke induced downregulation in vivo of the anti-inflammatory lipid transporter ABCA1, previously shown by our laboratory to be lung protective. Importantly, SSRI treatment prevented lung destruction in smoke exposed rabbits as measured by morphometry. These studies support further investigation into SSRIs as a novel therapeutic for COPD may be warranted.

Cigarette smoke extract induces airway epithelial cell death via repressing PRMT6/AKT signaling.

Chronic obstructive pulmonary disease (COPD) is a severe public health threat world-wide. Cigarette smoke (CS)-induced airway epithelial cell death is a major pathway of pathogenesis in emphysema, a subtype of COPD. Protein arginine methyltransferase 6 (PRMT6) is a type I PRMT that catalyzes mono- and di-methylation on arginine residues within histone and non-histone proteins to modulate a variety of life processes, such as apoptosis. However, its role in CS-induced lung epithelial death has not been fully elucidated. Here we report that PRMT6 was decreased in mouse lung tissues from a cigarette smoke extract (CSE)-mediated experimental emphysematous model and in CSE treated or cigarette smoke exposed lung epithelial cells. Depletion of PRMT6 increased the protein levels of phosphatase PTEN and PI3K regulatory subunit p85 but decreased a downstream kinase PDK1, resulting in AKT dephosphorylation and thereafter, lung epithelial cell death. Knockout of PRMT6 inhibited epithelial survival and promoted CSE-mediated epithelial cell death, while ectopic expression of PRMT6 protein partially reversed epithelial cell death via PI3K/AKT-mediated cell survival signaling in CSE cellular models. These findings demonstrate that PRMT6 plays a crucial role in CS-induced bronchial epithelial cell death that may be a potential therapeutic target against the airway cell death in CS-induced COPD.

The Protective Effect of HBO1 on Cigarette Smoke Extract-Induced Apoptosis in Airway Epithelial Cells.

Purpose: Epigenetic modification is one of most important mechanisms underlying the pathogenesis of chronic obstructive pulmonary disease (COPD). The purpose of this study was to determine whether histone acetyltransferase binding to ORC1 (HBO1) can protect against cigarette smoke (CS)-induced cell apoptosis and sustain normal histone acetylation in COPD. Methods: Human lung tissue samples were obtained from patients who underwent lung resection. The emphysema mouse model and HBO1 overexpressing mice were each established by intraperitoneal injection with cigarette smoke extract (CSE) or intratracheal lentiviral vectors instillation. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays were used to assess apoptotic ratio in mice. The apoptosis of human bronchial epithelial cells (HBECs) was assayed by flow cytometry. HBO1, B-cell lymphoma-2 (BCL-2), and H3K14ac protein expression were detected by Western blotting. HBO1 mRNA expression was measured by quantitative real-time polymerase chain reaction. Results: Protein expression of HBO1 was decreased significantly in lung tissue from COPD patients and CSE-treated emphysema mouse models. Overexpression of HBO1 attenuated CSE-induced emphysematous changes, as well as apoptosis in the lungs of COPD mice. In vitro, the HBO1 protein degraded in a time- and dose-dependent course with CSE treatment. With flow cytometry, we proved that HBO1 could reverse the apoptosis of HBECs induced by CSE. Furthermore, HBO1 overexpression promoted the expression of anti-apoptotic BCL-2 protein and enhanced H3K14 acetylation in airway epithelial cells. Conclusion: These findings demonstrate that the key histone modulator HBO1 plays a protective role in COPD pathogenesis that may shed light on potential therapeutic targets to inhibit the progress of COPD.

Mice overexpressing latent matrix metalloproteinase-2 develop lung emphysema after short-term exposure to cigarette smoke extract.

Chronic obstructive pulmonary disease is the major growing cause of mortality and morbidity worldwide, and it is going to become the third most common cause of death by 2020. Chronic obstructive pulmonary disease is pathologically characterized by lung emphysema and small airway inflammation. Animal models are very important to get insights into the disease pathogenesis but current models of chronic obstructive pulmonary disease take a long time to develop. The need of a new model is compelling. In the present study we focus on the role of matrix metalloproteinases in the pathogenesis of chronic obstructive pulmonary disease and hypothesized that lung overexpression of latent matrix metalloproteinases-2 would allow the development of emphysema after short-term exposure to cigarette smoke extract inhalation. Human latent matrix metalloproteinases-2 transgenic mouse expressing high level of the protein in the lungs and wild type mouse were exposed to aerosolized cigarette smoke extract for two weeks. Transgenic mice showed significant lung emphysematous changes, increased infiltration of inflammatory cells and enhanced lung concentrations of inflammatory cytokines in the lungs compared to their wild type counterparts after inhalation of cigarette smoke extract. This novel mouse model will be a very useful tool for evaluating the mechanistic pathways and for development of novel therapies in cigarette smoke-associated lung emphysema.

Suppression of PTPN6 exacerbates aluminum oxide nanoparticle-induced COPD-like lesions in mice through activation of STAT pathway.

BACKGROUND: Inhaled nanoparticles can deposit in the deep lung where they interact with pulmonary cells. Despite numerous studies on pulmonary nanotoxicity, detailed molecular mechanisms of specific nanomaterial-induced lung injury have yet to be identified. RESULTS: Using whole-body dynamic inhalation model, we studied the interactions between aluminum oxide nanoparticles (Al2O3 NPs) and the pulmonary system in vivo. We found that seven-day-exposure to Al2O3 NPs resulted in emphysema and small airway remodeling in murine lungs, accompanied by enhanced inflammation and apoptosis. Al2O3 NPs exposure led to suppression of PTPN6 and phosphorylation of STAT3, culminating in increased expression of the apoptotic marker PDCD4. Rescue of PTPN6 expression or application of a STAT3 inhibitor, effectively protected murine lungs from inflammation and apoptosis, as well as, in part, from the induction of chronic obstructive pulmonary disease (COPD)-like effects. CONCLUSION: In summary, our studies show that inhibition of PTPN6 plays a critical role in Al2O3 NPs-induced COPD-like lesions.

The protective effect of PRMT6 overexpression on cigarette smoke extract-induced murine emphysema model.

BACKGROUND: Cigarette smoke exposure is the most common risk factor for emphysema, which is one of the major pathologies of COPD. Protein arginine methyltransferase 6 (PRMT6) is a nuclear enzyme that specially catalyzes dimethylation of R2 in histone H3 (H3R2me2a). H3R2me2a prevents trimethylation of H3K4 (H3K4me3), which is located in the transcription start sites of genes in mammalian genomes. We attempted to determine the expression of PRMT6 in human samples, and investigate whether the upregulation of PRMT6 expression can attenuate the development of cigarette smoke extract (CSE)-induced emphysema. Further experiments were performed to elucidate the molecular mechanisms involved. MATERIALS AND METHODS: Human lung tissues were obtained from patients undergoing pneumonectomy for benign pulmonary lesions. BALB/c mice were treated with lentiviral vectors intratracheally and injected with CSE three times. The protein expression of PRMT6, H3R2me2a, and H3K4me3 in human and mouse samples, as well as B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), and endothelial nitric oxide synthase (eNOS) in mice were detected in lung homogenates by Western blotting. The mRNA expression of cyclooxygenase-2, interleukin-6, Bcl-2, Bax, and eNOS in mice was measured by quantitative real-time polymerase chain reaction. RESULTS: The expression of PRMT6 was significantly downregulated in the pulmonary parenchyma in smokers with COPD as well as in mice treated with CSE. Overexpression of PRMT6 was detected in the CSE + Lenti-PRMT6 group of mice, which reversed the expression of H3R2me2a and H3K4me3. Inflammation, apoptosis, and oxidative stress levels were severe in the CSE-treated emphysema mice compared with the control group, which was inhibited by the overexpression of PRMT6. CONCLUSION: The overexpression of PRMT6 might inhibit inflammation, apoptosis, and oxidative stress in CSE-induced emphysema mediated by H3R2me2a.

COPD in individuals with the PiMZ alpha-1 antitrypsin genotype.

Since the discovery of severe alpha-1 antitrypsin deficiency as a genetic risk factor for emphysema, there has been ongoing debate over whether individuals with intermediate deficiency with one protease inhibitor Z allele (PiMZ, or MZ) are at some risk for emphysema. This is important, because MZ individuals comprise 2-5% of the general population. In this review we summarise the evidence about the risks of the MZ population to develop emphysema or asthma. We discuss the different study designs that have tried to answer this question. The risk of emphysema is more pronounced in case-control than in population-based studies, perhaps due to inadequate power. Carefully designed family studies show an increased risk of emphysema in MZ smokers. This is supported by the rapid decline in lung function of MZ individuals when compared to the general population after massive environmental exposures. The risk of asthma in MZ subjects is less studied, and more literature is needed before firm conclusions can be made. Augmentation therapy in MZ individuals is not supported by any objective studies. MZ smokers are at increased risk for emphysema that is more pronounced when other environmental challenges are present.

The copper dependent-lysyl oxidases contribute to the pathogenesis of pulmonary emphysema in chronic obstructive pulmonary disease patients.

Abnormalities in the elastic fiber biology are seen in pulmonary emphysema (PE). The copper-dependent lysyl oxidases regulate the production and accumulation of elastic fibers in the connective tissue. This study focused on the relationship between lysyl oxidase (LOX), LOX-like protein 1 (LOXL1), and LOXL2 and PE pathogenesis. Lung samples with or without PE from patients with chronic obstructive lung disease (n=35) were used. Protein levels of elastin, LOX, LOXL1, LOXL2, hypoxia inducible factor 1-alpha (HIF-1α), copper metabolism domain containing-1 (COMMD1), and phosphatase and tensin homolog (PTEN) were assayed using microscopic and biochemical methods The emphysematous areas were characterized by enlargement of the alveoli, destruction of the alveolar structure, accumulation of macrophages in the alveolar lumens, and showed increased HIF-1α immunoreactivity. Additionally, the emphysematous areas had significantly lower elastin, LOX, LOXL1, LOXL2, HIF-1α, COMMD1, and PTEN protein levels than the non-emphysematous areas. We suppose that the reductions in the HIF-1α levels led to decreases in the protein levels of active LOX, LOXL1, and LOXL2. These decreases might cause abnormalities in the elastic fiber biology. HIF-1α activation induced by decreased COMMD1 and protease activation induced by decreased PTEN might contribute to the development of PE. Finally, methods aimed at increasing the protein levels of LOXs, COMMD1 and PTEN might be effective for treating PE.

Alpha-1 Antitrypsin-Deficient Macrophages Have Increased Matriptase-Mediated Proteolytic Activity.

Alpha-1 antitrypsin (AAT) deficiency-associated emphysema is largely attributed to insufficient inhibition of neutrophil elastase released from neutrophils. Correcting AAT levels using augmentation therapy only slows disease progression, and that suggests a more complex process of lung destruction. Because alveolar macrophages (Mɸ) express AAT, we propose that the expression and intracellular accumulation of mutated Z-AAT (the most common mutation) compromises Mɸ function and contributes to emphysema development. Extracellular matrix (ECM) degradation is a hallmark of emphysema pathology. In this study, Mɸ from individuals with Z-AAT (Z-Mɸ) have greater proteolytic activity on ECM than do normal Mɸ. This abnormal Z-Mɸ activity is not abrogated by supplementation with exogenous AAT and is likely the result of cellular dysfunction induced by intracellular accumulation of Z-AAT. Using pharmacologic inhibitors, we show that several classes of proteases are involved in matrix degradation by Z-Mɸ. Importantly, compared with normal Mɸ, the membrane-bound serine protease, matriptase, is present in Z-Mɸ at higher levels and contributes to their proteolytic activity on ECM. In addition, we identified matrix metalloproteinase (MMP)-14, a membrane-anchored metalloproteinase, as a novel substrate for matriptase, and showed that matriptase regulates the levels of MMP-14 on the cell surface. Thus, high levels of matriptase may contribute to increased ECM degradation by Z-Mɸ, both directly and through MMP-14 activation. In summary, the expression of Z-AAT in Mɸ confers increased proteolytic activity on ECM. This proteolytic activity is not rescued by exogenous AAT supplementation and could thus contribute to augmentation resistance in AAT deficiency-associated emphysema.

Matrix Metalloproteinase-28 Is a Key Contributor to Emphysema Pathogenesis.

Chronic obstructive pulmonary disease (COPD) comprises chronic bronchitis and emphysema, and is a leading cause of morbidity and mortality. Because tissue destruction is the prominent characteristic of emphysema, extracellular proteinases, particularly those with elastolytic ability, are often considered to be key drivers in this disease. Several human and mouse studies have implicated roles for matrix metalloproteinases (MMPs), particularly macrophage-derived proteinases, in COPD pathogenesis. MMP-28 is expressed by the pulmonary epithelium and macrophage, and we have found that it regulates macrophage recruitment and polarization. We hypothesized that MMP-28 has contributory roles in emphysema via alteration of macrophage numbers and activation. Because of the established association of emphysema pathogenesis to macrophage influx, we evaluated the inflammatory changes and lung histology of Mmp28-/- mice exposed to 3 and 6 months of cigarette smoke. At earlier time points, we found altered macrophage polarization in the smoke-exposed Mmp28-/- lung consistent with other published findings that MMP-28 regulates macrophage activation. At both 3 and 6 months, Mmp28-/- mice had blunted inflammatory responses more closely resembling nonsmoked mice, with a reduction in neutrophil recruitment and CXCL1 chemokine expression. By 6 months, Mmp28-/- mice were protected from emphysema. These results highlight a previously unrecognized role for MMP-28 in promoting chronic lung inflammation and tissue remodeling induced by cigarette smoke and highlight another potential target to modulate COPD.

Distinct emphysema subtypes defined by quantitative CT analysis are associated with specific pulmonary matrix metalloproteinases.

BACKGROUND: Emphysema is characterised by distinct pathological sub-types, but little is known about the divergent underlying aetiology. Matrix-metalloproteinases (MMPs) are proteolytic enzymes that can degrade the extracellular matrix and have been identified as potentially important in the development of emphysema. However, the relationship between MMPs and emphysema sub-type is unknown. We investigated the role of MMPs and their inhibitors in the development of emphysema sub-types by quantifying levels and determining relationships with these sub-types in mild-moderate COPD patients and ex/current smokers with preserved lung function. METHODS: Twenty-four mild-moderate COPD and 8 ex/current smokers with preserved lung function underwent high resolution CT and distinct emphysema sub-types were quantified using novel local histogram-based assessment of lung density. We analysed levels of MMPs and tissue inhibitors of MMPs (TIMPs) in bronchoalveolar lavage (BAL) and assessed their relationship with these emphysema sub-types. RESULTS: The most prevalent emphysema subtypes in COPD subjects were mild and moderate centrilobular (CLE) emphysema, while only small amounts of severe centrilobular emphysema, paraseptal emphysema (PSE) and panlobular emphysema (PLE) were present. MMP-3, and -10 associated with all emphysema sub-types other than mild CLE, while MMP-7 and -8 had associations with moderate and severe CLE and PSE. MMP-9 also had associations with moderate CLE and paraseptal emphysema. Mild CLE occurred in substantial quantities irrespective of whether airflow obstruction was present and did not show any associations with MMPs. CONCLUSION: Multiple MMPs are directly associated with emphysema sub-types identified by CT imaging, apart from mild CLE. This suggests that MMPs play a significant role in the tissue destruction seen in the more severe sub-types of emphysema, whereas early emphysematous change may be driven by a different mechanism. TRIAL REGISTRATION: Trial registration number NCT01701869 .

Alpha-1 Antitrypsin Deficiency Today: New Insights in the Immunological Pathways.

More than 50 years ago, the observation that absence of the α1 band from protein electrophoresis is associated with severe emphysema established the link between α1-antitrypsin deficiency (AATD) and lung damage. From this discovery, the classic paradigm of protease/antiprotease imbalance was derived, linking lung destruction in patients with AATD to the unopposed effect of proteases. By extension, this paradigm was also applied to patients with 'common' chronic obstructive pulmonary disease, in whom large increases in smoke-induced proteases could overwhelm the antiprotease capability of AAT. However, it has become increasingly evident that AAT has important anti-inflammatory and immunoregulatory activities which, beside its antiprotease function, may be critically involved in lung destruction. From this perspective, we will consider recent evidence, based on epidemiological, clinical and immunopathological studies, suggesting that it is time to move on from the original protease/antiprotease paradigm toward a more complex view of the condition, which embraces its immunomodulating functions. Of importance, the potent immunoregulatory, tolerogenic role of AAT may support its therapeutic use in a number of diseases other than AATD, particularly in immune-related disorders.

Mitochondrial E3 Ubiquitin Protein Ligase 1 Mediates Cigarette Smoke-Induced Endothelial Cell Death and Dysfunction.

By virtue of the critical roles of Akt in vascular endothelial cell (EC) survival and function, cigarette smoke-induced Akt reduction may contribute to EC death and dysfunction in smokers' lungs. One of the negative Akt regulatory mechanisms is K48-linked Akt ubiquitination and subsequent proteasomal degradation. Here, we assessed the involvement of mitochondrial E3 ubiquitin protein ligase 1 (MUL1), recently revealed as a novel Akt ubiquitin E3 ligase, in cigarette smoke-induced Akt ubiquitination and its contribution to pulmonary EC death and dysfunction. In human lung microvascular ECs (HLMVECs), cigarette smoke extract (CSE) noticeably elevated MUL1 expression and K48-linked Akt ubiquitination, whereas Akt, p-Akt, eNOS, and p-eNOS levels were decreased. MUL1 knockdown suppressed CSE-induced Akt ubiquitination/degradation and cytoplasmic reductions of Akt and p-Akt. Furthermore, MUL1 knockdown attenuated reductions of eNOS and p-eNOS and alleviated EC survival, migration, and tube formation in the presence of CSE exposure. In addition, overexpression of K284R Akt, a mutant for a MUL1-ubiquitination site, produced similar effects. In HLMVECs exposed to CSE, Akt-MUL1 interaction was increased in coimmunoprecipitation and in situ proximity ligation assays. Similarly, the proximity ligation assay signals were elevated in rat lungs exposed to cigarette smoke for 3 months, during which Mul1 levels were noticeably increased. Finally, we found that CSE-mediated MUL1 induction in HLMVECs is mediated by retinoic acid receptor-related orphan receptor α. Taken together, these data suggest that cigarette smoke-induced MUL1 elevation mediates Akt ubiquitination/degradation, potentially leading to pulmonary EC death and functional impairment.

Heme oxygenase-1-mediated autophagy protects against pulmonary endothelial cell death and development of emphysema in cadmium-treated mice.

Pulmonary exposure to cadmium, a major component of cigarette smoke, has a dramatic impact on lung function and the development of emphysema. Cigarette smoke exposure induces heme oxygenase-1 (HO-1), a cytoprotective enzyme. In this study, we employed a truncated mouse model of emphysema by intratracheal instillation of cadmium (CdCl2) solution (0.025% per 1 mg/kg body wt) in HO-1(+/+), HO-1(-/-), and overexpressing humanized HO-1 bacterial artificial chromosome (hHO-1BAC) mice. We evaluated the role of HO-1 in cadmium-induced emphysema in mice by analyzing histopathology, micro-computed tomography scans, and lung function tests. CdCl2-exposed HO-1(-/-) mice exhibited more severe emphysema compared with HO-1(+/+) or hHO-1BAC mice. Loss of pulmonary endothelial cells (PECs) from the alveolar capillary membrane is recognized to be a target in emphysema. PECs from HO-1(+/+), HO-1(-/-), and hHO-1BAC were employed to define the underlying molecular mechanism for the protection from emphysema by HO-1. Electron microscopy, expression of autophagic markers (microtubule-associated protein 1B-light chain 3 II, autophagy protein 5, and Beclin1) and apoptotic marker (cleaved caspase 3) suggested induction of autophagy and apoptosis in PECs after CdCl2 treatment. CdCl2-treated HO-1(-/-) PECs exhibited downregulation of autophagic markers and significantly increased cleaved caspase 3 expression and activity (∼4-fold higher). Moreover, hHO-1BAC PECs demonstrated upregulated autophagy and absence of cleaved caspase 3 expression or activity. Pretreatment of HO-1(+/+) PECs with rapamycin induced autophagy and resulted in reduced cell death upon cadmium treatment. Induction of autophagy following CdCl2 treatment was found to be protective from apoptotic cell death. HO-1 induced protective autophagy in PECs and mitigated cadmium-induced emphysema.

Rosiglitazone attenuates the metalloprotease/anti-metalloprotease imbalance in emphysema induced by cigarette smoke: involvement of extracellular signal-regulated kinase and NFκB signaling.

OBJECTIVE: We investigated how rosiglitazone attenuated cigarette smoke (CS)-induced emphysema in a rat model. In particular, we focused on its possible effects on the imbalance between metalloprotease (MMP) and anti-MMP activity, mitogen-activated protein kinase (MAPK) phosphorylation, and nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) signaling pathway over-activation. METHODS: A total of 36 Wistar rats were divided into three groups (n=12 each): animals were exposed to CS for 12 weeks in the absence (the CS group) or presence of 30 mg/kg rosiglitazone (the rosiglitazone-CS [RCS] group); a control group was treated with the rosiglitazone vehicle only, without any CS exposure. Histopathology of lung tissue in all groups was evaluated to grade severity of the disease. Expression levels of peroxisome proliferator-activated receptor γ (PPARγ), MMP2, and MMP9 in lung tissue were determined and compared using Western blotting and immunohistochemistry. Activation of MAPKs, NFκB, and the nuclear factor of kappa light polypeptide gene enhancer in B-cell inhibitor, alpha (IκBα) phosphorylation in lung tissue was examined by Western blotting. RESULTS: Emphysema-related pathology, based on inter-alveolar wall distance and alveolar density, was less severe in the RCS group than in the CS group. Compared with the CS group, levels of PPARγ were higher in the RCS group, and levels of MMP2 and MMP9 proteins were lower in the RCS rats. Levels of activated MAPKs and NFκB were also lower, while the IκBα phosphorylation was increased in the lung tissue of RCS rats. CONCLUSION: Our findings suggest that oral administration of rosiglitazone attenuates the metalloprotease activity induced by CS, and the underlying mechanism might involve the activation of signaling pathways dependent on MAPKs or NFκB. Our results further suggest that PPARγ contributes to the pathogenesis of emphysema as well as airway inflammation induced by CS.

Autoprocessing of neutrophil elastase near its active site reduces the efficiency of natural and synthetic elastase inhibitors.

An imbalance between neutrophil-derived proteases and extracellular inhibitors is widely regarded as an important pathogenic mechanism for lung injury. Despite intense efforts over the last three decades, attempts to develop small-molecule inhibitors for neutrophil elastase have failed in the clinic. Here we discover an intrinsic self-cleaving property of mouse neutrophil elastase that interferes with the action of elastase inhibitors. We show that conversion of the single-chain (sc) into a two-chain (tc) neutrophil elastase by self-cleavage near its S1 pocket altered substrate activity and impaired both inhibition by endogenous α-1-antitrypsin and synthetic small molecules. Our data indicate that autoconversion of neutrophil elastase decreases the inhibitory efficacy of natural α-1-antitrypsin and small-molecule inhibitors, while retaining its pathological potential in an experimental mouse model. The so-far overlooked occurrence and properties of a naturally occurring tc-form of neutrophil elastase necessitates the redesign of small-molecule inhibitors that target the sc-form as well as the tc-form of neutrophil elastase.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis.

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-ß-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Matrix metalloproteinases in destructive lung disease.

Matrix metalloproteinases (MMPs) play essential physiologic roles in numerous processes ranging from development to wound repair. Unfortunately, given the broad substrate specificity of the MMP family as a whole, aberrant degradation of extracellular matrix proteins can result in destructive disease. Emphysema, the result of destroyed lung elastin and collagen matrix, is the prototypical example of such a destructive process. More recent data has highlighted that MMPs play much more elaborate physiologic and pathophysiologic roles than simple matrix protein cleavage. Key pathophysiological roles for MMPs in emphysema will be discussed herein.

Telomerase mutations in smokers with severe emphysema.

Mutations in the essential telomerase genes TERT and TR cause familial pulmonary fibrosis; however, in telomerase-null mice, short telomeres predispose to emphysema after chronic cigarette smoke exposure. Here, we tested whether telomerase mutations are a risk factor for human emphysema by examining their frequency in smokers with chronic obstructive pulmonary disease (COPD). Across two independent cohorts, we found 3 of 292 severe COPD cases carried deleterious mutations in TERT (1%). This prevalence is comparable to the frequency of alpha-1 antitrypsin deficiency documented in this population. The TERT mutations compromised telomerase catalytic activity, and mutation carriers had short telomeres. Telomerase mutation carriers with emphysema were predominantly female and had an increased incidence of pneumothorax. In families, emphysema showed an autosomal dominant inheritance pattern, along with pulmonary fibrosis and other telomere syndrome features, but manifested only in smokers. Our findings identify germline mutations in telomerase as a Mendelian risk factor for COPD susceptibility that clusters in autosomal dominant families with telomere-mediated disease including pulmonary fibrosis.