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Chronic obstructive pulmonary disease (COPD) is a progressive disease with both environmental and genetic risk factors. Genome-wide association studies (GWAS) have identified multiple genomic regions influencing risk of COPD. To thoroughly investigate the genetic etiology of COPD, however, it is also important to explore the role of copy number variants (CNVs) because the presence of structural variants can alter gene expression and can be causal for some diseases. Here, we investigated effects of polymorphic CNVs on quantitative measures of pulmonary function and chest computed tomography (CT) phenotypes among subjects enrolled in COPDGene, a multisite study. COPDGene subjects consist of roughly one-third African American (AA) and two-thirds non-Hispanic white adult smokers (with or without COPD). We estimated CNVs using PennCNV on 9,076 COPDGene subjects using Illumina's Omni-Express genome-wide marker array. We tested for association between polymorphic CNV components (defined as disjoint intervals of copy number regions) for several quantitative phenotypes associated with COPD within each racial group. Among the AAs, we identified a polymorphic CNV on chromosome 5q35.2 located between two genes (FAM153B and SIMK1, but also harboring several pseudo-genes) giving genome-wide significance in tests of association with total lung capacity (TLCCT ) as measured by chest CT scans. This is the first study of genome-wide association tests of polymorphic CNVs and TLCCT . Although the ARIC cohort did not have the phenotype of TLCCT , we found similar counts of CNV deletions and amplifications among AA and European subjects in this second cohort.

African Americans are admixed with genetic contributions from European and African ancestral populations. Admixture mapping leverages this information to map genes influencing differential disease risk across populations. We performed admixture and association mapping in 3,300 African American current or former smokers from the COPDGene Study. We analyzed estimated local ancestry and SNP genotype information to identify regions associated with FEV1 /FVC, the ratio of forced expiratory volume in one second to forced vital capacity, measured by spirometry performed after bronchodilator administration. Global African ancestry inversely associated with FEV1 /FVC (P = 0.035). Genome-wide admixture analysis, controlling for age, gender, body mass index, current smoking status, pack-years smoked, and four principal components summarizing the genetic background of African Americans in the COPDGene Study, identified a region on chromosome 12q14.1 associated with FEV1 /FVC (P = 2.1 × 10(-6) ) when regressed on local ancestry. Allelic association in this region of chromosome 12 identified an intronic variant in FAM19A2 (rs348644) as associated with FEV1 /FVC (P = 1.76 × 10(-6) ). By combining admixture and association mapping, a marker on chromosome 12q14.1 was identified as being associated with reduced FEV1 /FVC ratio among African Americans in the COPDGene Study.

Bronchopulmonary dysplasia (BPD), a chronic lung disease affecting preterm neonates, is associated with significant childhood and adult health problems. Histopathologic features of BPD include impaired vascular and distal airway development. We previously showed that activation of hypoxia-inducible factors (HIFs) by inhibition of prolyl hydroxylase domain-containing proteins (PHDs) is feasible and that it stimulates vascular endothelial growth factor (VEGF)-dependent angiogenesis in vitro. We tested the hypothesis that enhancement of angiogenesis by activation of HIFs improves lung growth and function in prematurely born neonates in vivo. Preterm baboons (125 day+14 day pro re nata O2 model, corresponding to 27 human gestational weeks) were treated for 14 days with intravenous (i.v.) FG-4095, a PHD inhibitor. Notably, 77% of diminished total alveolar surface area in untreated controls was recovered by FG-4095 treatment. Functional significance of the structural changes was indicated by improved oxygenation and lung compliance in FG-4095-treated newborns. Surfactant proteins B and C and saturated phosphatidylcholine were unchanged. Incidence of spontaneous ductus arteriosus closure was increased, likely contributing to lower ratio of pulmonary to systemic blood flow in FG-4095 group. These findings indicate that HIF stimulation by PHD inhibition ameliorates pathological and physiological consequences of BPD.

Peroxynitrite is a toxic oxidant species produced from nitric oxide (NO) and superoxide. We have recently observed that the cell-permeable superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) inhibits the suppression of mitochondrial respiration elicited by authentic peroxynitrite in vitro. Here we have investigated the relative potency of MnTBAP and a range of related compounds in terms of inhibition of peroxynitrite-induced oxidation and cytotoxicity. In addition, we tested the effects of MnTBAP on the vascular and the cellular energetic failure in rodent models of endotoxic shock. We observed a dose-related inhibition of the peroxynitrite-induced oxidation of dihydrorhodamine 123 to rhodamine by MnTBAP, ZnTBAP and FeTBAP, but not by MnTMPyP [(5,10,15,20-tetrakis(N-methyl-4′-pirydyl)porphinato)-manganese (III)]. In addition, MnTBAP, ZnTBAP and FeTBAP, but not MnTMPyP prevented the suppression of mitochondrial respiration by authentic peroxynitrite in cultured J774 macrophages. In rat cultured aortic smooth muscle cells, MnTBAP protected against the suppression of mitochondrial respiration in response to authentic peroxynitrite, immunostimulation and nitric oxide (NO) donor compounds. MnTBAP slightly reduced the amount of nitrite/nitrate produced in response to immunostimulation in these cells. Administration of MnTBAP, 15 mg kg−1 i.v., before the administration of endotoxin (15 mg kg−1, i.v.) to rats ameliorated the development of vascular hyporeactivity and the development of endothelial dysfunction in the thoracic aorta ex vivo. MnTBAP also prevented the endotoxin-induced decrease in mitochondrial respiration, the development of DNA single strand breaks, and the depletion of intracellular NAD+ in peritoneal macrophages ex vivo. MnTBAP did not inhibit the expression by endotoxin of the inducible NO synthase in lung samples. MnTBAP did not alter survival rate in mice challenged with high dose endotoxin. Our findings, taken together with previous data demonstrating protective effects of NO synthase inhibitors against the endotoxin-induced contractile and energetic failure in the models of shock used in the current study, and with the known ability of peroxynitrite to cause cellular energy depletion, suggest a role for peroxynitrite in the pathogenesis of cellular energetic failure and contractile dysfunction in endotoxin shock. British Journal of Pharmacology (1997) 120, 259–267; doi:10.1038/sj.bjp.0700872

SUMMARY In morphometric studies of lung tissue, accurate determination of the total number of any specific type of cell, such as the endothelial cell, requires knowledge of the shape of the cell nucleus. This knowledge of shape is necessary to calculate the mean caliper diameter, which, in turn, is an indispensable element of the equation for the number of nuclei (therefore cells) per unit volume (Nv). Nine human lung endothelial cell nuclei were therefore reconstructed in dental wax from serial sections and were found to be pleomorphic triaxial ellipsoids. Five of these approached an oblate shape. The axes of these nine nuclear models were directly measured and a computer program using numerical integration was written to determine the mean caliper diameters of these ellipsoids. The estimated mean semi-axes of the nine nuclei were (±SD) 5·98 ± 1·61, 3·61 ± 0·70, 1·36 ± 0·34; and the estimated overall mean caliper diameter for the total population of human lung endothelial nuclei was 7·97 ± 1·27 μm.

Pulmonary structure and function were quantitatively investigated over the lifespan of the Fischer 344 rat by morphometric and physiologic techniques. Male animals 1 week, 6 weeks, 5 months, 14 months, and 26 months of age and female animals 5 months, 14 months, and 26 months of age were studied. All alveolar tissue compartments demonstrated significant increases in volume, surface area, and cell number during the first 5 months of life. From 5 to 26 months of age, remodelling in the epithelial and interstitial compartments continued to take place while the endothelial compartment remained relatively unchanged. In the epithelial compartment the ratio of type II cells to type I cells lining the alveolar surface decreased as age increased. In the interstitial compartment the volume of the noncellular components of the interstitium increased by 39% in males and by 89% in females from 5 to 26 months of age. Physiologic measurements of lung volumes in males at 6 weeks, 14 months, and 26 months demonstrated progressive increases in vital capacity (VC) and total lung capacity (TLC). Morphometric pulmonary-diffusion capacity (DLO2) increased in males from 1 week to 5 months of age and remained relatively unchanged from 5 to 26 months of age in both sexes.

SUMMARY In the morphometry laboratory a practical, accurate, and computationally simple procedure is needed when the estimation of the number of spherical particles per unit volume (Nv) is pursued. In addition, this procedure should be able to deal with the very real problem of profiles too small to be counted. Two computationally simple methods, the size class analysis method and the mean profile diameter method, were examined in detail. Computer-generated random profiles from various size-distributions of spheres were analysed by both methods. The percents error to be expected with various sphere distributions with a relatively large number of missed small profiles were determined for both methods. The accuracy of the size class analysis method was poor when a significant number of small profiles were missed. In this situation the accuracy of this method could be greatly improved by applying a simple modification to the procedure. A size class was identified which was larger than the largest missed profile but smaller than the diameter of the smallest sphere in the population. All contributions of this size class and all smaller size classes were deleted from the computation of Nv. The mean profile diameter method was found to be difficult to apply to distributions containing missed small profiles. Missed small profiles were handled more predictably by the modified size class analysis method.

We tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in the protein's structural conformation occur that would affect its immunoreactivity. In spite of a higher sensitivity observed for Ig-gold, we preferred to use protein A-gold in our system because of the low nonspecific labeling and the more precise antigen detection by the latter immunomarker.