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Published ahead of print on July 29, 2004, doi:10.1165/rcmb.2004-0008OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 595-600, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0008OC

Emphysema Lung Tissue Gene Expression Profiling

Heiko A. Golpon, Christopher D. Coldren, Martin R. Zamora, Gregory P. Cosgrove, Mark D. Moore, Rubin M. Tuder, Mark W. Geraci and Norbert F. Voelkel

Pulmonary Hypertension Center, COPD Center, Division of Pulmonary Sciences and Critical Care Medicine, Department of Pathology, University of Colorado Health Sciences Center, Denver; National Jewish Medical and Research Center, Denver, Colorado; and Pulmonary Division, University of Magdeburg Medical Center, Magdeburg, Germany

Address correspondence to: Norbert F. Voelkel, M.D., Division of Pulmonary Sciences and Critical Care Medicine, 4200 East Ninth Avenue, C272, Denver, CO 80262. E-mail: norbert.voelkel{at}uchsc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Emphysema occurs in a subgroup of patients with chronic obstructive pulmonary disease and patients with the genetic defect of alpha1-antitrypsin deficiency who have a smoking history of many years' duration. Emphysema is generally the result of a chronic and progressive destruction of the alveolar structures, which is believed to be driven by chronic inflammation, infections, oxidative stress, and an imbalance of protease and antiprotease activity. Here, we use microarray technology to characterize the gene expression profile of lung tissue samples obtained from patients with advanced emphysema and that obtained from healthy subjects. We hypothesized that the gene expression profile of emphysema lung tissue is distinct when compared with the expression profile of normal lungs. We report that severely emphysematous tissue is characterized by a global decrease in gene expression and by an increased abundance of transcripts encoding proteins involved in inflammation, immune responses, and proteolysis. Whereas the gene expression profile is to some degree shared between "usual" emphysema and alpha1-antitrypsin deficiency–related emphysema, there are statistically significant differences in the modulation of groups of genes associated with protein and energy metabolism, and immune function, which allow distinction between these two emphysema types on the lung tissue level.

Abbreviations: alpha1-antitrypsin, AAT • "usual" emphysematous lung tissue, EML • normal lung tissue, NML • polymerase chain reaction, PCR


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic obstructive lung diseases are a group of chronic and often progressive and finally debilitating diseases, which include alveolar airspace enlargement, as seen in the emphysema of smokers and patients with the genetic disorder of alpha1-antitrypsin (AAT) deficiency (1). Patients with emphysema demonstrate lung hyperinflation and quite frequently their emphysema is ubiquitously distributed over the five lung lobes. The etiology of lung emphysema is most frequently related to cigarette smoking; however, not every smoker develops emphysema (2). The protease/antiprotease concept of emphysema pathobiology originally evolved from the description of pulmonary emphysema in patients with AAT deficiency (3), and a number of recent animal experiments also support the concept of a component of proteolytic tissue and cell damage (4–6). In this context, we have recently reported that lung tissue obtained from patients with severe emphysema demonstrates frequent apoptotic events in the alveolar septal cell structures (7). Based on our recent experience with whole lung tissue extract gene profiling in primary pulmonary hypertension (8), we now apply the microarray gene profiling technology to lung tissue resected from patients with emphysema and AAT deficiency–related emphysema. We report here that lungs from patients with severe emphysema show a global reduction in gene expression, but a higher abundance of the transcripts of genes encoding proteins involved in inflammatory responses, immune responses, and proteolysis when compared with normal lung tissue (NML). The tissue gene expression profile is, to some extent, shared by emphysema and AAT-deficiency emphysema, although a number of genes define a distinction between these two forms of emphysema.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patient Lung Tissue Samples
We obtained lungs from 11 patients with severe emphysema who were either undergoing lung volume reduction surgery (Patient 2) or lung transplant surgery (all other patients) (Table 1). Pulmonary obstructive changes were documented by lung function studies. Six of the emphysema patients had been previously diagnosed as AAT-deficient. The NML samples were obtained from Tissue Transformation Technology Inc. (Edison, NJ). These samples were from organs donated for transplant, but unused due to age or size mismatch (Table 1); none of these individuals had clinical evidence of airflow limitation. All the lung tissue samples were obtained in accordance with Institutional Review Board (IRB) guidelines, carefully inspected by one of us (R.M.T. or G.P.C), and declared histologically normal (n = 5) or emphysematous (n = 11). In each case, nucleic acid samples were prepared from a small (~ 3 mm3), grossly homogeneous piece of peripheral tissue.


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  		TABLE 1. Patient data

 
Gene Expression Analysis
Standard methods were used for RNA preparation, reverse transcription, array target labeling and hybridization, and real-time polymerase chain reaction (PCR) analysis. Details on these methods can be found in the online data supplement. Tabular gene expression data are published in Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/), submission GSE1122.

Data Analysis
Expression values were quantified and array quality control was performed using the statistical algorithms implemented in Affymetrix Microarray Suite 5.0 (Affymetrix, Foster City, CA), and the details of these algorithms are documented at the manufacturer's website. Signal values for all arrays were scaled to a uniform target value of 500, and the resulting tabular data were further analyzed with BRB ArrayTools v2.1 d1 (National Cancer Institute Biometric Research Branch, Bethesda, MD), developed by Dr. Richard Simon and Amy Peng Lam. Gene ontology analysis was conducted using GenMAPP and MAPPFinder (9). The z-score assigned to each category by MAPPFinder reflects the degree to which the expression of genes in that category was greater than that expected by chance. A high, positive z-score indicates that a large number of genes in that category are differentially expressed between the compared conditions. Tile plots were generated using GeneCluster 2.0 (10). The array generated using tissue from AAT-deficient Patient 8 was excluded from the analysis due to low global signal intensity.

Statistical Analysis
The scaled expression data set was filtered to include only genes that were assessed to be present (detection, P < 0.05) in at least two of the samples. Of the 6,086 potential genes on the Affymetrix HuGeneFL chip (Affymetrix), 4,100 passed this filtering criterion and were subject to further analysis. Genes comprising the gene expression signature differentiating the three classes were tabulated using the "class comparison" function in ArrayTools; genes with a univariate F-test P-value < 0.001 were found, and permutation and false discovery rate analyses were performed. Additional class comparison analysis was performed between pairs of classes using t tests and permutation tests. Functional assignment of changes in expression was conducted using MAPPFinder, and the superabundance of changes in any category was assessed using a 10% false discovery rate cutoff for differentially expressed genes. Expression values for genes mentioned in the text or displayed in figures were subject to one-way analysis of variance with Newman-Keuls correction for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Table 1 displays the clinical data of the patients, including the patients' pulmonary function data. Clearly, all the patients with either "usual" emphysema or AAT deficiency–related lung disease had severe lung function impairment; however, none of the patients was a current smoker at the time of surgery or tissue collection. To evaluate the regional variability of gene expression in the lung, NML was sampled from upper and lower lobes, and from central and peripheral regions, and subjected to gene chip analysis. Regional variability in gene expression was found to be small relative to variation between individuals (data not shown).

Statistically validated modulation of gene expression was found between normal and emphysematous lungs. Figure 1 shows the expression pattern of 150 genes, which had been found to have the most significant differences in their expression between normal and emphysematous lung tissue samples. An annotated list of these genes is available in the online supplement (Table E1). The dendrogram shows the relatedness of the expression patterns of all of the patients' lung tissue samples based on this 150-gene signature. Binary comparisons to NML of "usual" emphysema and AAT deficiency–related emphysema individually shows that each disease is characterized by a different pattern of gene expression, but that each manifests as a decreased transcript abundance for roughly 80% of the differentially expressed genes (Tables E2 and E3 in the online supplement). The multidimensional scaling plot (Figure 2) also shows the segregation of the normal, AAT deficiency-associated emphysema, and "usual" emphysematous lung tissue (EML) based on the gene expression data sets.



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  		Figure 1. Dendrogram illustrating relatedness of the 15 lung samples based on the 150-gene signature, and the title plot showing relative expression levels for each gene in each sample. Genes in the signature were selected based on F-test significance (P = 0.001). Dendrogram represents average linkage-centered correlation. Tiles show relative expression range from –3{sigma} (dark blue) to +3{sigma} (dark red). ADL, AAT deficiency–related EML.

 


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  		Figure 2. Multidimensional scaling plot illustrating the relatedness of the 15 lung samples based on the 150 gene signature. A sphere represents each sample, and the position of each sphere in space is determined by the three largest principal components of variance for that sample within the gene expression signature. Good separation exists between normal lung samples (green circles), AAT deficiency–related emphysema lung samples (red circles), and "usual" emphysema lung samples (blue circles) based on this gene expression signature.

 
In addition to the supervised analysis, unsupervised clustering was performed on the entire data set, including all of 4,100 genes that were present in 2 or more of the 15 samples. This global analysis segregates NML from emphysemic tissue reproducibly (11) (robustness index R = 0.912 for two clusters) with the exception that one "usual" emphysema sample, Patient 1, is grouped with the normal lung samples. No segregation of "usual" and AAT deficiency–associated emphysema is seen on the global level.

Real-time PCR analysis of the genes encoding transforming growth factor ß receptor III, ras homolog gene family, member A, heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A), glutathione peroxidase 3 (plasma), tryptase ß1, secretory leukocyte protease inhibitor, FBJ murine osteosarcoma viral oncogene homolog B, and matrix metalloproteinase 7 was used to confirm the relative expression of these genes, and the results from these experiments agree with the microarray results. Figure 3 shows examples of differently expressed genes and data regarding expression verification by quantitative PCR.



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  		Figure 3. Gene expression for 3 exemplary genes (glutathione peroxidase 3 [plasma], transforming growth factor ß receptor III, matrix metalloproteinase 7) was assessed by microarray analysis (A, C, E) and quantitative real-time PCR (B, D, F). Examples demonstrating the different patterns of expression found when NML (light gray bars) and tissue from patients with "usual" emphysema (dark gray bars) and with AAT deficiency–associated emphysema (black bars) were compared. Quantitative real-time PCR expression levels were normalized to 18S ribosomal RNA and are expressed in arbitrary units. Change significance was estimated using one-way analysis of variance with Newman-Keuls multiple test correction. The relative expression levels of the genes encoding transforming growth factor ß receptor III, ras homolog gene family, member A, heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor A), glutathione peroxidase 3 [plasma], tryptase ß1, secretory leukocyte protease inhibitor, FBJ murine osteosarcoma viral oncogene homolog B, and matrix metalloproteinase 7 were verified by quantitative PCR.

 
The functional categorization of the statistically significant differences in gene expression between NML samples and tissue from patients with AAT deficiency associated emphysema shows a markedly different pattern than that developed from the comparison of normal tissue with tissue from patients with "usual" emphysema (Figure 4). Tables 2 and 3 list categories of differentially expressed between NML samples and samples from patients with "usual" and AAT deficiency associated emphysema. Table 4 lists gene transcripts, which are increased in their abundance in "usual" emphysema but not in AAT deficiency–related emphysema, and Table 5 displays 2 genes, which are decreased in the lung tissues from patients with "usual" emphysema, but not AAT deficiency–related emphysema.



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  		Figure 4. Gene ontology (GO) categories that have a superabundance of differentially expressed genes in either EML (maroon bars) or AAT deficiency–related emphysematous lung tissue (blue bars) when compared with NML. The z-score assigned to each category reflects the degree to which the expression of genes in that category was greater than that expected by chance. A high, positive z-score indicates that a large number of genes in that category are differentially expressed between the compared conditions. The four-digit GO category ID is listed for each category.

 

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  		TABLE 2. Gene transcripts increased in abundance in emphysema and alpha1-antitrypsin deficiency–related emphysema

 

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  		TABLE 3. Gene transcripts decreased in abundance in emphysema and alpha1-antitrypsin deficiency–related emphysema

 

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  		TABLE 4. Gene transcripts increased in their abundance in emphysema but not alpha1-antitrypsin deficiency–related emphysema

 

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  		TABLE 5. Gene transcripts decreased in their expression in emphysema but not alpha1-antitrypsin deficiency–related emphysema

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our analysis and stringent statistical evaluation of the gene expression pattern in lung tissue shows surprisingly modest differences between individuals with normal lung function and patients with end-stage emphysema, given the dramatic structural and histologic changes characteristic of emphysema. We found that the level of gene expression for individual genes was frequently similar in the lung tissues from patients with "usual" emphysema or AAT deficiency–related emphysema, (Table 2, Figure 1, and Table E1 in the online supplement), yet a reduction in protein biosynthesis capacity is a clear characteristic of the lung gene expression pattern of patients with AAT deficiency–related emphysema (Figure 4 and Table E4 in the online supplement), but this is a less pronounced theme of the "usual" emphysema. It is also of interest that multiple genes encoding functionally related proteins, which often are localized on different chromosomes, are concordantly altered in their expression. As suggested by Figure 1, significant heterogeneity exists within the members of each group. That such heterogeneity is present on the level of gene expression is to be expected in the human population, and our focus on statistical significance (rather than mean fold-changes in expression) reflects the challenges of working with this type of data. It is perhaps remarkable that the level of gene expression heterogeneity in the lung is roughly equal in all three groups.

The analysis of individual genes appears to indicate that the lung emphysema gene expression profile can be broadly described as one in which genes encoding enzymes involved in protein degradation and processing are altered in their expression; one example of altered expression is the decreased expression of the genes encoding the proteasome 26S, {alpha} type 1, and ß 7 subunits (Table E1 in the online supplement). A relatively small number of genes encoding proteins involved in inflammation are differentially affected when normal and "usual" EMLs are compared, and this difference is not detected in the comparison of normal and AAT deficiency–related emphysema (Figures 2 and 4). This is consistent with recent reports describing an increased number of inflammatory cells present in the lungs of patients with severe emphysema (12), and raises the question as to whether an influx of inflammatory cells is a characteristic shared by AAT deficiency–related emphysema.

We found an upregulation in both forms of emphysema in the expression of the MEL transforming oncogene (13) and the vav-1 (14, 15) and RAB 1A oncogenes. Also increased in expression in the emphysema tissues was the gene encoding the deoxyhypusine synthase, which catalyses the oxidative cleavage of spermidine, the expression of the gene encoding glucose phosphate isomerase, phosphogluconate dehydrogenase, phospholipase C ß2, protein tyrosine phosphatase, and mitogen-activated protein kinase kinase kinase 3 (see Table E1 in the online supplement). There was no significant difference in the expression of genes encoding proteases, with the exception of cathepsin B, which was increased in expression in the lungs of patients with "usual" emphysema, caspase 9, which was increased in the lung tissue from patients with AAT deficiency–related emphysema, and matrix metalloproteinase 7, which was increased in both conditions. The genes encoding claudin, a component of tight junctions, a Ca++ transporting ATPase, activin A receptor II, and ALCAM, a protein expressed in pulmonary microvascular endothelial cells (16), demonstrated significant decreases in expression in emphysematous lungs (Table E1 in the online supplement).

Our data provide only a snapshot of tissue—but not cell-specific—information, and the gene expression signature almost certainly reflects severe, end-stage lung disease. It is possible, and perhaps likely, that lung tissue from patients with mild or early disease is characterized by a different gene expression profile than described here. We were surprised to find, on the gene expression level, altered expression of a paucity of genes coding for proteins involved in inflammation and proteolysis, and also that there are sets of genes that discriminate between tissues from patients with "usual" and AAT deficiency–related emphysema (Figure 4). Our earlier finding of enhanced apoptosis of cells in emphysematous lungs may be supported by the overexpression of the gene, ‘requiem apoptosis response’, and decreased expression of the Bcl-2–interacting protein. Calreticulin, a multifunctional protein located on the cell surface and in the endoplasmic reticulum of many cells, has been shown to be involved in the phagocytic removal of apoptosed cells by macrophages (17) by binding to the {alpha}2 macroglobulin receptor CD91 (18). Perhaps the calreticulin gene is upregulated in its expression in an "attempt" to facilitate the removal of apoptosed cells. On the other hand, overexpression of calreticulin has also been shown to promote apoptosis of cardiomyoblasts (19), possibly by decreasing mitochondrial Ca++ and mitochondrial membrane potential, thus damaging the mitochondria (20). Aquaporin 1 is expressed in the lung in endothelial cells (21, 22), and its decreased gene expression in emphysematous lungs may represent a loss of lung capillaries and endothelial cells (Figure 3); a similar explanation may apply to the decreased expression of the gene encoding ephrin B1.The ephrin B family of proteins are receptor tyrosine kinases that regulate endothelial cell attachment and promote neovascularization (23–25).

Moesin belongs to the highly related ezrin, radixin, and moesin (ERM) proteins, which have been proposed to link transmembrane proteins to the actin cytoskeleton and to play a role in signaling pathways in epithelial cells (26), and also in the early phase of apoptosis (27). Whether the increased expression of the gene encoding moesin in the emphysema lungs relates to the maintenance of epithelial cell integrity and control of apoptosis is presently unclear. We propose that lung cell apoptosis in emphysema may not only be caused by decreased vascular endothelial growth factor and vascular endothelial growth factor receptor expression and signaling (28), but also by impaired mitochondrial energy metabolism (29, 30), as illustrated by decreased expression of genes encoding mitochondrial ribosomal proteins and several cytochrome c oxidase subunits (Table E2 in the online supplement). Mitochondria have been widely implicated in the chain of events leading to apoptosis (29–31), mediated by oxidative stress and redox imbalance. Cleary, lung tissue enzyme activity studies will be necessary to support this concept.

As stated, the signature gene expression pattern of end-stage emphysematous lungs—regardless of whether the emphysema is related to AAT deficiency or not—is one of overall decreased expression (Figure 1, and Tables E2 and E3 in the online supplement). Against this general backdrop, a very small number of genes show a significant enhancement of expression when compared with those from NML. Here we highlight the genes which are upregulated in their expression in "usual" emphysema: hematopoietic protein 1, 1.6-fold; interleukin-2R{gamma}, 1.46-fold; CD 72 antigen, 3.2-fold; major histocompatibility complex class II, DM {alpha}, 2.7-fold; stromal cell–derived factor 1 (32), 2.4-fold (Table E3 in the online supplement). Likewise there is robust enhancement of expression of a remarkably small number of genes in AAT deficiency–related EML: M-phase phosphoprotein 1, 4.2-fold; nuclear factor 1, A Type, 1.7-fold; myosin IC, 2-fold; thymopoietin, 2.1-fold; Krueppel-related zinc finger protein, 2.1-fold; chemokine receptor GTP-binding protein, 2.8-fold; transforming growth factor ß2, 1.7-fold; cyclooxygenase 2, 3-fold; protein tyrosine phosphatase J, 4.8-fold (Table E2 in the online supplement).

The causes of, or "reasons" for, this change in gene expression in emphysematous lungs when compared with normal lungs are not clear, yet the emphysema expression pattern in these end-stage lungs is unlikely explained entirely by inflammation; instead, expression differences may be the consequence of tissue ischemia, oxidative stress (33), impaired energy metabolism, and presence or absence of biologically active AAT.


   Acknowledgments
 
The authors thank Sylk Sotto-Santiago for her contributions to this research. This work was supported by the Bixler-COPD Foundation, a grant from the Deutsche Forschungsgemeinschaft, Bonn–Bad Godesberg, Germany (to H.A.G.), National Institutes of Health R01 HL 66554-01 (to N.F.V.), and National Heart, Lung, and Blood Institute 1 R01 HL 72340-01 (to M.W.G.). N.F.V. is the Hart Family Professor of Emphysema Research.


   Footnotes
 
H.A.G. and C.D.C. contributed equally to this work.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Conflict of Interest Statement: H.A.G. has no declared conflicts of interest; C.D.C. has no declared conflicts of interest; M.R.Z. has no declared conflicts of interest; G.P.C. has no declared conflicts of interest; M.D.M. has no declared conflicts of interest; R.M.T. has no declared conflicts of interest; M.W.G. has no declared conflicts of interest; and N.F.V. has no declared conflicts of interest.

Received in original form January 8, 2004

Received in final form June 28, 2004


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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