Susan H. Conradi

The Role of Matrix Metalloproteinase-9 in Cigarette Smoke–induced Emphysema

RATIONALE Matrix metalloprotease (MMP)-9 is an elastolytic endopeptidase produced by activated macrophages that may be involved in the development of human pulmonary emphysema and could be inhibited with existing compounds. Mouse models have demonstrated that excess MMP-9 production can result in permanent alveolar destruction. OBJECTIVES To determine if MMP-9 causes cigarette smoke-induced emphysema using MMP-9 knockout mice and human samples. METHODS Mouse lungs were analyzed for inflammation and airspace enlargement using a mainstream smoke-exposure model. Human macrophage mRNA was isolated from subjects with emphysema by laser capture microdissection. Human blood monocyte mRNA was isolated from subjects with greater than 30 pack-year smoking history. Human gene expression was determined by quantitative polymerase chain reaction and compared with emphysema severity determined by automated computed tomography analysis. Plasma Clara cell secretory protein and surfactant protein-D were quantified to measure ongoing lung injury. MEASUREMENTS AND MAIN RESULTS Mice deficient in MMP-9 develop the same degree of cigarette smoke-induced inflammation and airspace enlargement as strain-matched controls. Macrophages are the predominant source of MMP-9 production in human emphysema specimens and similar quantities of macrophage MMP-9 mRNA is present in areas of lung with and without emphysema. Circulating monocytes produce more MMP-9 in individuals with advanced emphysema severity despite no correlation of MMP-9 with markers of ongoing lung damage. CONCLUSIONS These results suggest that MMP-9 in humans who smoke is similar to smoke-exposed mice, where MMP-9 is present in emphysematous lung but not correlated with the emphysema. To the degree that the mechanisms of emphysema in humans who smoke resemble the mouse model, these data suggest specific inhibition of MMP-9 is unlikely to be an effective therapy for cigarette smoke-induced emphysema. Clinical trial registered with www.clinicaltrials.gov (NCT 00757120).

Oxidative Damage to Nucleic Acids in Severe Emphysema

BACKGROUND Oxidative stress is a key element in the pathogenesis of emphysema, but oxidation of nucleic acids has been largely overlooked. The aim of this study was to investigate oxidative damage to nucleic acids in severe emphysematous lungs. METHODS Thirteen human severe emphysematous lungs, including five with alpha(1)-antitrypsin deficiency (AATD), were obtained from patients receiving lung transplantation. Control lung tissue was obtained from non-COPD lungs (n = 8) and donor lungs (n = 8). DNA and RNA oxidation were investigated by immunochemistry. Morphometry (mean linear intercept [Lm] and CT scan) and immunostaining for CD68 and neutrophil elastase also were performed. RESULTS Nucleic acid oxidation was increased in alveolar wall cells in emphysematous lungs compared to non-COPD and donor lungs (p < 0.01). In emphysematous lungs, oxidative damage to nucleic acids in alveolar wall cells was increased in the more severe emphysematous areas assessed by histology (Lm, > 0.5 mm; p < 0.05) and CT scan (< -950 Hounsfield units; p < 0.05). Compared to classic emphysema, AATD lungs exhibited higher levels of nucleic acid oxidation in macrophages (p < 0.05) and airway epithelial cells (p < 0.01). Pretreatments with DNase and RNase demonstrated that RNA oxidation was more prevalent than DNA oxidation in alveolar wall cells. CONCLUSIONS We demonstrated for the first time that nucleic acids, especially RNA, are oxidized in human emphysematous lungs. The correlation between the levels of oxidative damage to nucleic acids in alveolar wall cells and the severity of emphysema suggest a potential role in the pathogenesis of emphysema.

Elastin expression in very severe human COPD.

Alveolar elastic fibres are key targets of proteases during the pathogenesis of chronic obstructive pulmonary disease (COPD). In the current study, we hypothesised that a response to injury leads to enhanced alveolar elastin gene expression in very severe COPD. Lung samples obtained from 43 patients, including 11 with very severe COPD (stage 4), 10 donors, 10 with moderate/severe COPD (stage 2–3) and 12 non-COPD subjects, were analysed for elastin mRNA expression by real-time RT-PCR and in situ hybridisation. Alveolar elastic fibres were visualised using Harts staining of sections of frozen inflated lungs obtained from 11 COPD stage 4 patients and three donor lungs. Compared with donors, non-COPD and stage 2–3 COPD, elastin mRNA expression was significantly increased in very severe COPD lungs (12-fold change), and localised in situ hybridisation induced elastin expression to alveolar walls. Compared with donors, alveolar elastic fibres also comprised a greater volume fraction of total lung tissue in very severe COPD lungs (p<0.01), but elastic fibre content was not increased per lung volume, and desmosine content was not increased. The present study demonstrates enhanced alveolar elastin expression in very severe COPD. The efficiency of this potential repair mechanism and its regulation remain to be demonstrated.

Cigarette Smoke Induces Nucleic-Acid Oxidation in Lung Fibroblasts

Oxidative stress is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD), but the molecular pathway connecting oxidative damage to tissue destruction remains to be fully defined. We suggest that reactive oxygen species (ROS) oxidatively damage nucleic acids, and this effect requires multiple repair mechanisms, particularly base excision pathway components 8-oxoguanine-DNA glycosylase (OGG1), endonuclease III homologue 1 (NTH1), and single-strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), as well as the nucleic acid-binding protein, Y-box binding protein 1 (YB1). This study was therefore designed to define the levels of nucleic-acid oxidation and expression of genes involved in the repair of COPD and in corresponding models of this disease. We found significant oxidation of nucleic acids localized to alveolar lung fibroblasts, increased levels of OGG1 mRNA expression, and decreased concentrations of NTH1, SMUG1, and YB1 mRNA in lung samples from subjects with very severe COPD compared with little or no COPD. Mice exposed to cigarette smoke exhibited a time-dependent accumulation of nucleic-acid oxidation in alveolar fibroblasts, which was associated with an increase in OGG1 and YB1 mRNA concentrations. Similarly, human lung fibroblasts exposed to cigarette smoke extract exhibited ROS-dependent nucleic-acid oxidation. The short interfering RNA (siRNA)-dependent knockdown of OGG1 and YB1 expression increased nucleic-acid oxidation at the basal state and after exposure to cigarette smoke. Together, our results demonstrate ROS-dependent, cigarette smoke-induced nucleic-acid oxidation in alveolar fibroblasts, which may play a role in the pathogenesis of emphysema.

Increased Iron Sequestration in Alveolar Macrophages in Chronic Obtructive Pulmonary Disease

Free iron in lung can cause the generation of reactive oxygen species, an important factor in chronic obstructive pulmonary disease (COPD) pathogenesis. Iron accumulation has been implicated in oxidative stress in other diseases, such as Alzheimer’s and Parkinson’s diseases, but little is known about iron accumulation in COPD. We sought to determine if iron content and the expression of iron transport and/or storage genes in lung differ between controls and COPD subjects, and whether changes in these correlate with airway obstruction. Explanted lung tissue was obtained from transplant donors, GOLD 2–3 COPD subjects, and GOLD 4 lung transplant recipients, and bronchoalveolar lavage (BAL) cells were obtained from non-smokers, healthy smokers, and GOLD 1–3 COPD subjects. Iron-positive cells were quantified histologically, and the expression of iron uptake (transferrin and transferrin receptor), storage (ferritin) and export (ferroportin) genes was examined by real-time RT-PCR assay. Percentage of iron-positive cells and expression levels of iron metabolism genes were examined for correlations with airflow limitation indices (forced expiratory volume in the first second (FEV1) and the ratio between FEV1 and forced vital capacity (FEV1/FVC)). The alveolar macrophage was identified as the predominant iron-positive cell type in lung tissues. Futhermore, the quantity of iron deposit and the percentage of iron positive macrophages were increased with COPD and emphysema severity. The mRNA expression of iron uptake and storage genes transferrin and ferritin were significantly increased in GOLD 4 COPD lungs compared to donors (6.9 and 3.22 fold increase, respectively). In BAL cells, the mRNA expression of transferrin, transferrin receptor and ferritin correlated with airway obstruction. These results support activation of an iron sequestration mechanism by alveolar macrophages in COPD, which we postulate is a protective mechanism against iron induced oxidative stress.

Accurate Measurement of Small Airways on Low-Dose Thoracic CT Scans in Smokers

BACKGROUND Partial volume averaging and tilt relative to the scan plane on transverse images limit the accuracy of airway wall thickness measurements on CT scan, confounding assessment of the relationship between airway remodeling and clinical status in COPD. The purpose of this study was to assess the effect of partial volume averaging and tilt corrections on airway wall thickness measurement accuracy and on relationships between airway wall thickening and clinical status in COPD. METHODS Airway wall thickness measurements in 80 heavy smokers were obtained on transverse images from low-dose CT scan using the open-source program Airway Inspector. Measurements were corrected for partial volume averaging and tilt effects using an attenuation- and geometry-based algorithm and compared with functional status. RESULTS The algorithm reduced wall thickness measurements of smaller airways to a greater degree than larger airways, increasing the overall range. When restricted to analyses of airways with an inner diameter < 3.0 mm, for a theoretical airway of 2.0 mm inner diameter, the wall thickness decreased from 1.07 ± 0.07 to 0.29 ± 0.10 mm, and the square root of the wall area decreased from 3.34 ± 0.15 to 1.58 ± 0.29 mm, comparable to histologic measurement studies. Corrected measurements had higher correlation with FEV₁, differed more between BMI, airflow obstruction, dyspnea, and exercise capacity (BODE) index scores, and explained a greater proportion of FEV1 variability in multivariate models. CONCLUSIONS Correcting for partial volume averaging improves accuracy of airway wall thickness estimation, allowing direct measurement of the small airways to better define their role in COPD.

Equating quantitative emphysema measurements on different CT image reconstructions.

PURPOSE To mathematically model the relationship between CT measurements of emphysema obtained from images reconstructed using different section thicknesses and kernels and to evaluate the accuracy of the models for converting measurements to those of a reference reconstruction. METHODS CT raw data from the lung cancer screening examinations of 138 heavy smokers were reconstructed at 15 different combinations of section thickness and kernel. An emphysema index was quantified as the percentage of the lung with attenuation below -950 HU (EI950). Linear, quadratic, and power functions were used to model the relationship between EI950 values obtained with a reference 1 mm, medium smooth kernel reconstruction and values from each of the other 14 reconstructions. Preferred models were selected using the corrected Akaike information criterion (AICc), coefficients of determination (R2), and residuals (conversion errors), and cross-validated by a jackknife approach using the leave-one-out method. RESULTS The preferred models were power functions, with model R2 values ranging from 0.949 to 0.998. The errors in converting EI950 measurements from other reconstructions to the 1 mm, medium smooth kernel reconstruction in leave-one-out testing were less than 3.0 index percentage points for all reconstructions, and less than 1.0 index percentage point for five reconstructions. Conversion errors were related in part to image noise, emphysema distribution, and attenuation histogram parameters. Conversion inaccuracy related to increased kernel sharpness tended to be reduced by increased section thickness. CONCLUSIONS Image reconstruction-related differences in quantitative emphysema measurements were successfully modeled using power functions.

Measuring small airways in transverse CT images correction for partial volume averaging and airway tilt.

RATIONALE AND OBJECTIVES Airway wall dimensions can be determined in vivo using transverse computed tomographic (CT) images, but the measurement of airway phantoms shows that the wall thickness is consistently overestimated for small airways. This phantom study was performed to derive and test corrections to the measurements on the basis of consideration of partial volume averaging and tilt effects. MATERIALS AND METHODS A lung phantom with six polycarbonate tubes embedded in foam was scanned, and the cross-sectional dimensions of the tubes were determined using the full width at half maximum, zero crossing, and phase congruency edge detection methods. Equations were derived using the reported wall intensity to correct for partial volume averaging. Corrections for the overestimation of the wall thickness due to the tilt of the tube with respect to the CT z-axis were also derived. RESULTS All three methods (full width at half maximum, zero crossing, and phase congruency) overestimated the wall thickness of the small polycarbonate tubes. It was verified that two sources of error were partial volume averaging and tilt that was introduced when the phantom was positioned with tube axes at an angle to the CT z-axis. The corrections were applied to the measured tube wall dimensions and substantially reduced the deviation of the CT measurements from the true values. CONCLUSIONS Correcting for partial volume effects and airway tilt greatly increases the accuracy of simulated airway wall measurements in transverse CT images.

Original investigationMeasuring Small Airways in Transverse CT Images: Correction for Partial Volume Averaging and Airway Tilt

RATIONALE AND OBJECTIVES Airway wall dimensions can be determined in vivo using transverse computed tomographic (CT) images, but the measurement of airway phantoms shows that the wall thickness is consistently overestimated for small airways. This phantom study was performed to derive and test corrections to the measurements on the basis of consideration of partial volume averaging and tilt effects. MATERIALS AND METHODS A lung phantom with six polycarbonate tubes embedded in foam was scanned, and the cross-sectional dimensions of the tubes were determined using the full width at half maximum, zero crossing, and phase congruency edge detection methods. Equations were derived using the reported wall intensity to correct for partial volume averaging. Corrections for the overestimation of the wall thickness due to the tilt of the tube with respect to the CT z-axis were also derived. RESULTS All three methods (full width at half maximum, zero crossing, and phase congruency) overestimated the wall thickness of the small polycarbonate tubes. It was verified that two sources of error were partial volume averaging and tilt that was introduced when the phantom was positioned with tube axes at an angle to the CT z-axis. The corrections were applied to the measured tube wall dimensions and substantially reduced the deviation of the CT measurements from the true values. CONCLUSIONS Correcting for partial volume effects and airway tilt greatly increases the accuracy of simulated airway wall measurements in transverse CT images.