Loss of ADAM15 in female mice does not worsen pressure overload cardiomyopathy, independent of ovarian hormones.

Cardiac hypertrophy is a common feature in several cardiomyopathies. We previously reported that loss of ADAM15 (disintegrin and metalloproteinase 15) worsened cardiac hypertrophy and dilated cardiomyopathy following cardiac pressure overload. Here, we investigated the impact of ADAM15 loss in female mice following cardiac pressure overload induced by transverse aortic constriction (TAC). Female Adam15-/-mice developed the same degree of cardiac hypertrophy, dilation and dysfunction as the parallel female wildtype (WT) mice at 6 weeks post-TAC. To determine if this is due to the protective effects of estrogen which could mask the negative impact of Adam15 loss, WT and Adam15-/- mice underwent ovariectomy (OVx) 2 weeks prior to TAC. Cardiac structure and function analyses were performed at 6 weeks post-TAC. OVx similarly impacted females of both genotypes post-TAC. Calcineurin (Cn) activity was increased post-OVx-TAC, and more in Adam15-/- mice, however this increase was not reflected in the total-to-phospho NFAT levels. Integrin α7 expression, which was upstream of Cn activation in male Adam15-/--TAC mice, remained unchanged in female mice. However, activation of the Mitogen Activated Protein Kinases (ERK, JNK, P38) were greater in Adam15-/--OVx-TAC compared to WT-OVx-TAC mice. In addition, ADAM15 protein levels were significantly increased post-TAC in male but not in female WT mice. Myocardial fibrosis was comparable in non-OVx WT-TAC and Adam15-/--TAC mice. OVx increased the perivascular fibrosis more in Adam15-/- compared to WT mice post-TAC. Our data demonstrate that loss of ovarian hormones did not fully replicate the male phenotype in the female Adam15-/- mice post-TAC. Since ADAM15 levels were increased in males but not in females post-TAC, it is plausible that ADAM15 does not play a prominent role in post-TAC events in female mice. Our findings highlight the significance of factors other than sex hormones in mediating cardiomyopathies in females, which require a more thorough understanding.

Association of clonal hematopoiesis with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, -5.7%; adjusted 95% CI, -8.8% to -2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.

Acute lung injury (ALI) is still associated with high mortality. Growing evidence suggests that Club Cell Protein 16 (CC16) plays a protective role against ALI. However, the doses of recombinant CC16 (rCC16) used in preclinical studies are supraphysiological for clinical applications. Extracellular vesicles (EVs) are nanovesicles endogenously generated by mammalian cells. Our study demonstrated that CC16 is released via small EVs and EV-encapsulated CC16 (sEV-CC16) and has anti-inflammatory activities, which protect mice from lipopolysaccharide (LPS) or bacteria-induced ALI. Additionally, sEV-CC16 can activate the DNA damage repair signaling pathways. Consistent with this activity, we observed more severe DNA damage in lungs from Cc16 knockout (KO) than wild-type (WT) mice. Mechanistically, we elucidated that CC16 suppresses nuclear factor κB (NF-κB) signaling activation by binding to heat shock protein 60 (HSP60). We concluded that sEV-CC16 could be a potential therapeutic agent for ALI by inhibiting the inflammatory and DNA damage responses by reducing NF-κB signaling.

ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-ß1.

The a disintegrin and metalloproteinase (ADAM) family of proteinases alter the extracellular environment and are involved in the development of T cells and autoimmunity. The role of ADAM family members in Th17 cell differentiation is unknown. We identified ADAM9 to be specifically expressed and to promote Th17 differentiation. Mechanistically, we found that ADAM9 cleaved the latency-associated peptide to produce bioactive transforming growth factor ß1, which promoted SMAD2/3 phosphorylation and activation. A transcription factor inducible cAMP early repressor was found to bind directly to the ADAM9 promoter and to promote its transcription. Adam9-deficient mice displayed mitigated experimental autoimmune encephalomyelitis, and transfer of Adam9-deficient myelin oligodendrocyte globulin-specific T cells into Rag1-/- mice failed to induce disease. At the translational level, an increased abundance of ADAM9 levels was observed in CD4+ T cells from patients with systemic lupus erythematosus, and ADAM9 gene deletion in lupus primary CD4+ T cells clearly attenuated their ability to differentiate into Th17 cells. These findings revealed that ADAM9 as a proteinase provides Th17 cells with an ability to activate transforming growth factor ß1 and accelerates its differentiation, resulting in aberrant autoimmunity.

Dysregulation of miR-103a Mediates Cigarette Smoking-induced Lipid-laden Macrophage Formation.

Cigarette smoke (CS) is considered a major risk factor for chronic obstructive pulmonary disease (COPD) that is currently the third leading cause of death in the United States. Studies have indicated that patients with COPD have elevated blood low-density lipoprotein levels, which may contribute to the dysregulation of lipid metabolism. Accumulating data show that microRNAs (miRNAs) are involved in various human diseases. However, the role of microRNAs in the pathogenesis of COPD remains poorly defined. In this study, we found that miR-103a expression was significantly reduced in alveolar macrophages from smokers and patients with COPD versus that in alveolar macrophages from nonsmokers. Our data indicated that reactive oxygen species negatively regulate miR-103a in macrophages. Functionally, miR-103a modulates the expressions of genes involved in lipid metabolism and directly targets low-density lipoprotein receptors in macrophages. Furthermore, overexpression of miR-103a suppressed the accumulation of lipid droplets and reduced the reactive oxygen species, both in vitro and in vivo. Taken together, our findings indicate that downregulation of miR-103a contributes to cigarette smoke-induced lipid-laden macrophage formation and plays a critical role in lipid homeostasis in lung macrophages in the pathogenesis of COPD.

A Novel Protective Role for Matrix Metalloproteinase-8 in the Pulmonary Vasculature.

Rationale: Mechanical signaling through cell-matrix interactions plays a major role in progressive vascular remodeling in pulmonary arterial hypertension (PAH). MMP-8 (matrix metalloproteinase-8) is an interstitial collagenase involved in regulating inflammation and fibrosis of the lung and systemic vasculature, but its role in PAH pathogenesis remains unexplored. Objectives: To evaluate MMP-8 as a modulator of pathogenic mechanical signaling in PAH. Methods: MMP-8 levels were measured in plasma from patients with pulmonary hypertension (PH) and controls by ELISA. MMP-8 vascular expression was examined in lung tissue from patients with PAH and rodent models of PH. MMP-8-/- and MMP-8+/+ mice were exposed to normobaric hypoxia or normoxia for 4-8 weeks. PH severity was evaluated by right ventricular systolic pressure, echocardiography, pulmonary artery morphometry, and immunostaining. Proliferation, migration, matrix component expression, and mechanical signaling were assessed in MMP-8-/- and MMP-8+/+ pulmonary artery smooth muscle cells (PASMCs). Measurements and Main Results: MMP-8 expression was significantly increased in plasma and pulmonary arteries of patients with PH compared with controls and induced in the pulmonary vasculature in rodent PH models. Hypoxia-exposed MMP-8-/- mice had significant mortality, increased right ventricular systolic pressure, severe right ventricular dysfunction, and exaggerated vascular remodeling compared with MMP-8+/+ mice. MMP-8-/- PASMCs demonstrated exaggerated proliferation and migration mediated by altered matrix protein expression, elevated integrin-ß3 levels, and induction of FAK (focal adhesion kinase) and downstream YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) activity. Conclusions: MMP-8 is a novel protective factor upregulated in the pulmonary vasculature during PAH pathogenesis. MMP-8 opposes pathologic mechanobiological feedback by altering matrix composition and disrupting integrin-ß3/FAK and YAP/TAZ-dependent mechanical signaling in PASMCs.

Metformin: Experimental and Clinical Evidence for a Potential Role in Emphysema Treatment.

Rationale: Cigarette smoke (CS) inhalation triggers oxidative stress and inflammation, leading to accelerated lung aging, apoptosis, and emphysema, as well as systemic pathologies. Metformin is beneficial for protecting against aging-related diseases. Objectives: We sought to investigate whether metformin may ameliorate CS-induced pathologies of emphysematous chronic obstructive pulmonary disease (COPD). Methods: Mice were exposed chronically to CS and fed metformin-enriched chow for the second half of exposure. Lung, kidney, and muscle pathologies, lung proteostasis, endoplasmic reticulum (ER) stress, mitochondrial function, and mediators of metformin effects in vivo and/or in vitro were studied. We evaluated the association of metformin use with indices of emphysema progression over 5 years of follow-up among the COPDGene (Genetic Epidemiology of COPD) study participants. The association of metformin use with the percentage of emphysema and adjusted lung density was estimated by using a linear mixed model. Measurements and Main Results: Metformin protected against CS-induced pulmonary inflammation and airspace enlargement; small airway remodeling, glomerular shrinkage, oxidative stress, apoptosis, telomere damage, aging, dysmetabolism in vivo and in vitro; and ER stress. The AMPK (AMP-activated protein kinase) pathway was central to metformin's protective action. Within COPDGene, participants receiving metformin compared with those not receiving it had a slower progression of emphysema (-0.92%; 95% confidence interval [CI], -1.7% to -0.14%; P = 0.02) and a slower adjusted lung density decrease (2.2 g/L; 95% CI, 0.43 to 4.0 g/L; P = 0.01). Conclusions: Metformin protected against CS-induced lung, renal, and muscle injury; mitochondrial dysfunction; and unfolded protein responses and ER stress in mice. In humans, metformin use was associated with lesser emphysema progression over time. Our results provide a rationale for clinical trials testing the efficacy of metformin in limiting emphysema progression and its systemic consequences.

ADAM (a Disintegrin and Metalloproteinase) 15 Deficiency Exacerbates Ang II (Angiotensin II)-Induced Aortic Remodeling Leading to Abdominal Aortic Aneurysm.

OBJECTIVE: ADAM (a disintegrin and metalloproteinase) 15-a membrane-bound metalloprotease from the ADAM (disintegrin and metalloproteinase) family-has been linked to endothelial permeability, inflammation, and metastasis. However, its function in aortic aneurysm has not been explored. We aimed to determine the function of ADAM15 in the pathogenesis of aortic remodeling and aneurysm formation. Approach and Results: Male Adam15-deficient and WT (wild type) mice (10 weeks old), on standard laboratory diet, received Ang II (angiotensin II; 1.5 mg/kg per day) or saline (Alzet pump) for 2 or 4 weeks. Ang II increased ADAM15 in WT aorta, while Adam15-deficiency resulted in abdominal aortic aneurysm characterized by loss of medial smooth muscle cells (SMCs), elastin fragmentation, inflammation, but unaltered Ang II-mediated hypertension. In the abdominal aortic tissue and primary aortic SMCs culture, Adam15 deficiency decreased SMC proliferation, increased apoptosis, and reduced contractile properties along with F-actin depolymerization to G-actin. Ang II triggered a markedly greater increase in THBS (thrombospondin) 1 in Adam15-deficient aorta, primarily the medial layer in vivo, and in aortic SMC in vitro; increased SSH1 (slingshot homolog 1) phosphatase activity and cofilin dephosphorylation that promoted F-actin depolymerization and G-actin accumulation. rhTHBS1 (recombinant THBS1) alone was sufficient to activate the cofilin pathway, increase G-actin, and induce apoptosis of aortic SMCs, confirming the key role of THBS1 in this process. Further, in human abdominal aortic aneurysm specimens, decreased ADAM15 was associated with increased THBS1 levels and loss of medial SMCs. CONCLUSIONS: This study is the first to demonstrate a key role for ADAM15 in abdominal aortic aneurysm through regulating the SMC function, thereby placing ADAM15 in a critical position as a potential therapeutic target for abdominal aortic aneurysm.

Tissue Inhibitor of Metalloproteinase-1 Promotes Polymorphonuclear Neutrophil (PMN) Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces.

Matrix metalloproteinase (MMP)-8 and -9 released by degranulating polymorphonuclear cells (PMNs) promote pericellular proteolysis by binding to PMN surfaces in a catalytically active tissue inhibitor of metalloproteinases (TIMP)-resistant forms. The PMN receptor(s) to which MMP-8 and MMP-9 bind(s) is not known. Competitive binding experiments showed that Mmp-8 and Mmp-9 share binding sites on murine PMN surfaces. A novel form of TIMP-1 (an inhibitor of soluble MMPs) is rapidly expressed on PMN surfaces when human PMNs are activated. Membrane-bound TIMP-1 is the PMN receptor for pro- and active MMP-8 and -9 as shown by the following: 1) TIMP-1 is strikingly colocalized with MMP-8 and -9 on activated human PMN surfaces and in PMN extracellular traps; 2) minimal immunoreactive and active Mmp-8 or Mmp-9 are detected on the surface of activated Timp-1-/- murine PMNs; and 3) binding of exogenous Timp-1 (but not Timp-2) to Timp-1-/- murine PMNs reconstitutes the binding of exogenous pro-Mmp-8 and pro-Mmp-9 to the surface of Timp-1-/- PMNs. Unlike full-length pro-Mmp-8 and pro-Mmp-9, mutant pro-Mmp proteins lacking the COOH-terminal hemopexin domain fail to bind to Mmp-8-/-x Mmp-9-/- murine PMNs. Soluble hemopexin inhibits the binding of pro-Mmp-8 and pro-Mmp-9 to Mmp-8-/-x Mmp-9-/- murine PMNs. Thus, the COOH-terminal hemopexin domains of pro-Mmp-8 and pro-Mmp-9 are required for their binding to membrane-bound Timp-1 on murine PMNs. Exposing nonhuman primates to cigarette smoke upregulates colocalized expression of TIMP-1 with MMP-8 and MMP-9 on peripheral blood PMN surfaces. By anchoring MMP-8 and MMP-9 to PMN surfaces, membrane-bound TIMP-1 plays a counterintuitive role in promoting PMN pericellular proteolysis occurring in chronic obstructive pulmonary disease and other diseases.

ADAM15 expression is increased in lung CD8+ T cells, macrophages, and bronchial epithelial cells in patients with COPD and is inversely related to airflow obstruction.

BACKGROUND: A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by activated leukocytes, and fibroblasts in vitro. Whether ADAM15 expression is increased in the lungs of COPD patients is not known. METHODS: ADAM15 gene expression and/or protein levels were measured in whole lung and bronchoalveolar lavage (BAL) macrophage samples obtained from COPD patients, smokers, and non-smokers. Soluble ADAM15 protein levels were measured in BAL fluid (BALF) and plasma samples from COPD patients and controls. Cells expressing ADAM15 in the lungs were identified using immunostaining. Staining for ADAM15 in different cells in the lungs was related to forced expiratory volume in 1 s (FEV1), ratio of FEV1 to forced vital capacity (FEV1/FVC), and pack-years of smoking history. RESULTS: ADAM15 gene expression and/or protein levels were increased in alveolar macrophages and whole lung samples from COPD patients versus smokers and non-smokers. Soluble ADAM15 protein levels were similar in BALF and plasma samples from COPD patients and controls. ADAM15 immunostaining was increased in macrophages, CD8+ T cells, epithelial cells, and airway α-smooth muscle (α-SMA)-positive cells in the lungs of COPD patients. ADAM15 immunostaining in macrophages, CD8+ T cells and bronchial (but not alveolar) epithelial cells was related inversely to FEV1 and FEV1/FVC, but not to pack-years of smoking history. ADAM15 staining levels in airway α-SMA-positive cells was directly related to FEV1/FVC. Over-expressing ADAM15 in THP-1 cells reduced their release of matrix metalloproteinases and CCL2. CONCLUSIONS: These results link increased ADAM15 expression especially in lung leukocytes and bronchial epithelial cells to the pathogenesis of COPD.

Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Fatty acid binding protein 4 (FABP4), an intracellular lipid chaperone and adipokine, is expressed by lung macrophages, but the function of macrophage-FABP4 remains elusive. We investigated the role of FABP4 in host defense in a murine model of Pseudomonas aeruginosa pneumonia. Compared with wild-type (WT) mice, FABP4-deficient (FABP4-/-) mice exhibited decreased bacterial clearance and increased mortality when challenged intranasally with P. aeruginosa. These findings in FABP4-/- mice were associated with a delayed neutrophil recruitment into the lungs and were followed by greater acute lung injury and inflammation. Among leukocytes, only macrophages expressed FABP4 in WT mice with P. aeruginosa pneumonia. Chimeric FABP4-/- mice with WT bone marrow were protected from increased mortality seen in chimeric WT mice with FABP4-/- bone marrow during P. aeruginosa pneumonia, thus confirming the role of macrophages as the main source of protective FABP4 against that infection. There was less production of C-X-C motif chemokine ligand 1 (CXCL1) in FABP4-/- alveolar macrophages and lower airway CXCL1 levels in FABP4-/- mice. Delivering recombinant CXCL1 to the airways protected FABP4-/- mice from increased susceptibility to P. aeruginosa pneumonia. Thus, macrophage-FABP4 has a novel role in pulmonary host defense against P. aeruginosa infection by facilitating crosstalk between macrophages and neutrophils via regulation of macrophage CXCL1 production.-Liang, X., Gupta, K., Rojas Quintero, J., Cernadas, M., Kobzik, L., Christou, H., Pier, G. B., Owen, C. A., Çataltepe, S. Macrophage FABP4 is required for neutrophil recruitment and bacterial clearance in Pseudomonas aeruginosa pneumonia.

Club Cell Secretory Protein Deficiency Leads to Altered Lung Function.

RATIONALE: CC16 (club cell secretory protein-16), a member of the secretoglobin family, is one of the most abundant proteins in normal airway secretions and has been described as a serum biomarker for obstructive lung diseases. OBJECTIVES: To determine whether low CC16 is a marker for airway pathology or is implicated in the pathophysiology of progressive airway damage in these conditions. METHODS: Using human data from the birth cohort of the Tucson Children's Respiratory Study, we examined the relation of circulating CC16 levels with pulmonary function and responses to bronchial methacholine challenge from childhood up to age 32 years. In wild-type and CC16-/- mice, we set out to comprehensively examine pulmonary physiology, inflammation, and remodeling in the naive airway. MEASUREMENTS AND MAIN RESULTS: We observed that Tucson Children's Respiratory Study participants in the lowest tertile of serum CC16 had significant deficits in their lung function and enhanced airway hyperresponsiveness to methacholine challenge from 11 years throughout young adult life. Similarly, CC16-/- mice had significant deficits in lung function and enhanced airway hyperresponsiveness to methacholine as compared with wild-type mice, which were independent of inflammation and mucin production. As compared with wild-type mice, CC16-/- mice had significantly elevated gene expression of procollagen type I, procollagen type III, and α-smooth muscle actin, areas of pronounced collagen deposition and significantly enhanced smooth muscle thickness. CONCLUSIONS: Our findings support clinical observations by providing evidence that lack of CC16 in the lung results in dramatically altered pulmonary function and structural alterations consistent with enhanced remodeling.

RNA-sequencing across three matched tissues reveals shared and tissue-specific gene expression and pathway signatures of COPD.

BACKGROUND: Multiple gene expression studies have been performed separately in peripheral blood, lung, and airway tissues to study COPD. We performed RNA-sequencing gene expression profiling of large-airway epithelium, alveolar macrophage and peripheral blood samples from the same subset of COPD cases and controls from the COPDGene study who underwent bronchoscopy at a single center. Using statistical and gene set enrichment approaches, we sought to improve the understanding of COPD by studying gene sets and pathways across these tissues, beyond the individual genomic determinants. METHODS: We performed differential expression analysis using RNA-seq data obtained from 63 samples from 21 COPD cases and controls (includes four non-smokers) via the R package DESeq2. We tested associations between gene expression and variables related to lung function, smoking history, and CT scan measures of emphysema and airway disease. We examined the correlation of differential gene expression across the tissues and phenotypes, hypothesizing that this would reveal preserved and private gene expression signatures. We performed gene set enrichment analyses using curated databases and findings from prior COPD studies to provide biological and disease relevance. RESULTS: The known smoking-related genes CYP1B1 and AHRR were among the top differential expression results for smoking status in the large-airway epithelium data. We observed a significant overlap of genes primarily across large-airway and macrophage results for smoking and airway disease phenotypes. We did not observe specific genes differentially expressed in all three tissues for any of the phenotypes. However, we did observe hemostasis and immune signaling pathways in the overlaps across all three tissues for emphysema, and amyloid and telomere-related pathways for smoking. In peripheral blood, the emphysema results were enriched for B cell related genes previously identified in lung tissue studies. CONCLUSIONS: Our integrative analyses across COPD-relevant tissues and prior studies revealed shared and tissue-specific disease biology. These replicated and novel findings in the airway and peripheral blood have highlighted candidate genes and pathways for COPD pathogenesis.

CFTR regulates B cell activation and lymphoid follicle development.

BACKGROUND: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. METHODS: The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr -/-) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr -/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr -/- mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr -/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr -/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls. CONCLUSIONS: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.

Plasma metabolomics and clinical predictors of survival differences in COPD patients.

BACKGROUND: Plasma metabolomics profile (PMP) in COPD has been associated with clinical characteristics, but PMP's relationship to survival has not been reported. We determined PMP differences between patients with COPD who died an average of 2 years after enrollment (Non-survivors, NS) compared to those who survived (S) and also with age matched controls (C). METHODS: We studied prospectively 90 patients with severe COPD and 30 controls. NS were divided in discovery and validation cohorts (30 patients each) and the results compared to the PMP of 30 S and C. All participants completed lung function tests, dyspnea scores, quality of life, exercise capacity, BODE index, and plasma metabolomics by liquid and gas chromatography / mass spectometry (LC/MS, LC/MS2, GC/MS). Statistically, we used Random Forest Analysis (RFA) and Support Vector Machine (SVM) to determine metabolites that differentiated the 3 groups and compared the ability of metabolites vs. clinical characteristics to classify patients into survivors and non-survivors. RESULTS: There were 79 metabolites statistically different between S and NS [p < 0.05 and false discovery rate (q value) < 0.1]. RFA and SVM classification of COPD survivors and non-survivors had a predicted accuracy of 74 and 85% respectively. Elevation of tricyclic acid cycle intermediates branched amino acids depletion and increase in lactate, fructose and xylonate showed the most relevant differences between S vs. NS suggesting alteration in mitochondrial oxidative energy generation. PMP had similar predictive power for risk of death as information provided by clinical characteristics. CONCLUSIONS: A plasma metabolomic profile characterized by an oxidative energy production difference between survivors and non-survivors was observed in COPD patients 2 years before death.

A Disintegrin and A Metalloproteinase-9 (ADAM9): A Novel Proteinase Culprit with Multifarious Contributions to COPD.

INTRODUCTION: Proteinases with a disintegrin and a metalloproteinase domain (ADAMs) have not been well studied in COPD. We investigated whether ADAM9 is linked to COPD in humans and mice. METHODS: ADAM9 blood and lung levels were measured in COPD patients versus controls, and air- versus cigarette smoke (CS)-exposed wild-type (WT) mice. WT and Adam9-/- mice were exposed to air or CS for 1-6 months, and COPD-like lung pathologies were measured. RESULTS: ADAM9 staining was increased in lung epithelial cells and macrophages in smokers and even more so in COPD patients and correlated directly with pack-year smoking history and inversely with airflow obstruction and/or FEV1 % predicted. Bronchial epithelial cell ADAM9 mRNA levels were higher in COPD patients than controls and correlated directly with pack-year smoking history. Plasma, BALF and sputum ADAM9 levels were similar in COPD patients and controls. CS exposure increased Adam9 levels in WT murine lungs. Adam9-/- mice were protected from emphysema development, small airway fibrosis, and airway mucus metaplasia. CS-exposed Adam9-/- mice had reduced lung macrophage counts, alveolar septal cell apoptosis, lung elastin degradation, and shedding of VEGFR2 and EGFR in BALF samples. Recombinant ADAM9 sheds EGF and VEGF receptors from epithelial cells to reduce activation of the Akt pro-survival pathway and increase cellular apoptosis. CONCLUSIONS: ADAM9 levels are increased in COPD lungs and linked to key clinical variables. Adam9 promotes emphysema development, and large and small airway disease in mice. Inhibition of ADAM9 could be a therapeutic approach for multiple COPD phenotypes.

A Disintegrin and Metalloproteinase Domain-8: A Novel Protective Proteinase in Chronic Obstructive Pulmonary Disease.

RATIONALE: ADAM8 (a disintegrin and metalloproteinase domain-8) is expressed by leukocytes and epithelial cells in health, but its contribution to the pathogenesis of chronic obstructive pulmonary disease (COPD) is unknown. OBJECTIVES: To determine whether the expression of ADAM8 is increased in the lungs of patients with COPD and cigarette smoke (CS)-exposed mice, and whether ADAM8 promotes the development of COPD. METHODS: ADAM8 levels were measured in lung, sputum, plasma, and/or BAL fluid samples from patients with COPD, smokers, and nonsmokers, and wild-type (WT) mice exposed to CS versus air. COPD-like lung pathologies were compared in CS-exposed WT versus Adam8-/- mice. MEASUREMENTS AND MAIN RESULTS: ADAM8 immunostaining was reduced in macrophages, and alveolar and bronchial epithelial cells in the lungs of patients with COPD versus control subjects, and CS- versus air-exposed WT mice. ADAM8 levels were similar in plasma, sputum, and BAL fluid samples from patients with COPD and control subjects. CS-exposed Adam8-/- mice had greater airspace enlargement and airway mucus cell metaplasia than WT mice, but similar small airway fibrosis. CS-exposed Adam8-/- mice had higher lung macrophage counts, oxidative stress levels, and alveolar septal cell death rates, but lower alveolar septal cell proliferation rates and soluble epidermal growth factor receptor BAL fluid levels than WT mice. Adam8 deficiency increased lung inflammation by reducing CS-induced activation of the intrinsic apoptosis pathway in macrophages. Human ADAM8 proteolytically shed the epidermal growth factor receptor from bronchial epithelial cells to reduce mucin expression in vitro. Adam8 bone marrow chimera studies revealed that Adam8 deficiency in leukocytes and lung parenchymal cells contributed to the exaggerated COPD-like disease in Adam8-/- mice. CONCLUSIONS: Adam8 deficiency increases CS-induced lung inflammation, emphysema, and airway mucus cell metaplasia. Strategies that increase or prolong ADAM8's expression in the lung may have therapeutic efficacy in COPD.

Hhip haploinsufficiency sensitizes mice to age-related emphysema.

Genetic variants in Hedgehog interacting protein (HHIP) have consistently been associated with the susceptibility to develop chronic obstructive pulmonary disease and pulmonary function levels, including the forced expiratory volume in 1 s (FEV1), in general population samples by genome-wide association studies. However, in vivo evidence connecting Hhip to age-related FEV1 decline and emphysema development is lacking. Herein, using Hhip heterozygous mice (Hhip(+/-)), we observed increased lung compliance and spontaneous emphysema in Hhip(+/-) mice starting at 10 mo of age. This increase was preceded by increases in oxidative stress levels in the lungs of Hhip(+/-) vs. Hhip(+/+) mice. To our knowledge, these results provide the first line of evidence that HHIP is involved in maintaining normal lung function and alveolar structures. Interestingly, antioxidant N-acetyl cysteine treatment in mice starting at age of 5 mo improved lung function and prevented emphysema development in Hhip(+/-) mice, suggesting that N-acetyl cysteine treatment limits the progression of age-related emphysema in Hhip(+/-) mice. Therefore, reduced lung function and age-related spontaneous emphysema development in Hhip(+/-) mice may be caused by increased oxidative stress levels in murine lungs as a result of haploinsufficiency of Hhip.

Lung-Function Trajectories Leading to Chronic Obstructive Pulmonary Disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is thought to result from an accelerated decline in forced expiratory volume in 1 second (FEV1) over time. Yet it is possible that a normal decline in FEV1 could also lead to COPD in persons whose maximally attained FEV1 is less than population norms. METHODS: We stratified participants in three independent cohorts (the Framingham Offspring Cohort, the Copenhagen City Heart Study, and the Lovelace Smokers Cohort) according to lung function (FEV1 ≥80% or <80% of the predicted value) at cohort inception (mean age of patients, approximately 40 years) and the presence or absence of COPD at the last study visit. We then determined the rate of decline in FEV1 over time among the participants according to their FEV1 at cohort inception and COPD status at study end. RESULTS: Among 657 persons who had an FEV1 of less than 80% of the predicted value before 40 years of age, 174 (26%) had COPD after 22 years of observation, whereas among 2207 persons who had a baseline FEV1 of at least 80% of the predicted value before 40 years of age, 158 (7%) had COPD after 22 years of observation (P<0.001). Approximately half the 332 persons with COPD at the end of the observation period had had a normal FEV1 before 40 years of age and had a rapid decline in FEV1 thereafter, with a mean (±SD) decline of 53±21 ml per year. The remaining half had had a low FEV1 in early adulthood and a subsequent mean decline in FEV1 of 27±18 ml per year (P<0.001), despite similar smoking exposure. CONCLUSIONS: Our study suggests that low FEV1 in early adulthood is important in the genesis of COPD and that accelerated decline in FEV1 is not an obligate feature of COPD. (Funded by an unrestricted grant from GlaxoSmithKline and others.).

Emphysema and extrapulmonary tissue loss in COPD: a multi-organ loss of tissue phenotype.

We tested whether emphysema progression accompanies enhanced tissue loss in other body compartments in 1817 patients from the ECLIPSE chronic obstructive pulmonary disease (COPD) cohort.Clinical and selected systemic biomarker measurements were compared in subjects grouped by quantitative tomography scan emphysema quartiles using the percentage of low attenuation area (LAA%). Lowest and highest quartile patients had amino-acid metabolomic profiles. We related LAA% to 3 years decline in lung function (forced expiratory volume in 1 s (FEV1)), body mass index (BMI), fat-free mass index (FFMI) and exacerbations, hospitalisations and mortality rates.Participants with more baseline emphysema had lower FEV1, BMI and FFMI, worse functional capacity, and less cardiovascular disease but more osteoporosis. Systemic C-reactive protein and interleukin-6 levels were similar among groups, but club cell protein 16 was higher and interleukin-8, surfactant protein D and soluble receptor for advanced glycation end product were lower with more emphysema. Metabolomics differed between extreme emphysema quartiles. Patients with more emphysema had accelerated FEV1, BMI and FFMI decline and more exacerbations, hospitalisations and mortality.COPD patients with more emphysema undergo excessive loss of pulmonary and extrapulmonary tissue, which is probably related to abnormal tissue maintenance. Because of worse clinical outcomes, we propose this subgroup be named the multi-organ loss of tissue (MOLT) COPD phenotype.