- Email: [email protected]
Uses of Expression Microarrays in Studies of Pulmonary Fibrosis, Asthma, Acute Lung Injury, and Emphysema
identical to a familial mutation in infants with the same phenotype strongly supports the hypothesis that the mutations were causally related to their lung disease. Missense SP-C mutations that resulted in amino-acid substitutions in residues highly conserved across species (P30 L, I73T, G100V, Y104H, P115 L, I126R, T187N, and L188R), as well as a frameshift mutation (140delA) associated with expression of a stable transcript, were identified in 10 other infants, 6 of whom had a family history of lung disease. None of the identified mutations were found on 100 control chromosomes, indicating that they are not common polymorphisms. We conclude that mutations in the SP-C gene are a cause of both familial and sporadic ILD. While the pathophysiologic mechanisms remain to be elucidated, the finding of mutations on one allele suggests a dominant negative effect on SP-C or pro-SP-C function or metabolism.
Reference 1 Nogee LM, Dunbar AE III, Wert SE, et al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 2001; 344:573–579
Mapping Susceptibility Genes for the Induction of Pulmonary Fibrosis in Mice* Richard K. Barth, PhD; LeRoy A. Hanchett, PhD; and Clare M. Baecher-Allan, PhD
(CHEST 2002; 121:21S) Abbreviations: FGF ⫽ fibroblast growth factor; QTL ⫽ quantitative trait locus; TNF ⫽ tumor necrosis factor
fibrosis is a potentially fatal disease that can P ulmonary result from radiation or chemotherapeutic treatment
of malignancy, exposure to certain irritants, and idiopathic events. Our studies have focused on the genetic mechanisms underlying this disease through the analysis of inbred mouse strain variation in susceptibility to fibrosis induction. We have identified an inbred mouse strain (DBA/2) that is highly susceptible to bleomycin-induced pulmonary fibrosis and is genetically very dissimilar to the standard fibrosis-sensitive strain, C57BL/6, but similar to the standard fibrosis-resistant strain, BALB/c. Analysis of a set of backcross progeny generated between DBA/2 and BALB/c strains indicates that susceptibility to development of pulmonary fibrosis is controlled primarily by a few (two to three) independent genetic loci. Genetic linkage
*From the University of Rochester Cancer Center and Department of Microbiology and Immunology, University of Rochester, Rochester, NY. Supported by National Heart, Lung, and Blood Institute grant R01-HL58128. Correspondence to: Richard K. Barth, PhD, Associate Professor of Oncology in Microbiology and Immunology, University of Rochester Cancer Center, 601 Elmwood Ave, Box 704, Rochester, NY 14642; e-mail: [email protected].
using quantitative trait locus (QTL) analysis has led to the chromosomal assignment of two of these susceptibility loci. One susceptibility gene is located within a subregion of chromosome 6 that contains a cluster of genes that are members of the tumor necrosis factor (TNF)-receptor family, including the 55-kd TNF-␣1 receptor. The second susceptibility gene has been mapped to the telomeric end of chromosome 13, within an interval encompassing fibroblast growth factor (FGF)-10, a member of the FGF gene family that is expressed predominantly in the developing lung. Analysis of allelic variation in these candidate genes is underway in order to evaluate their utility as genetic markers for fibrosis susceptibility and to elucidate their possible role in influencing the disease process.
Uses of Expression Microarrays in Studies of Pulmonary Fibrosis, Asthma, Acute Lung Injury, and Emphysema* Roger S. Mitchell Lecture Dean Sheppard, MD
Expression microarrays are a powerful tool that could provide new information about the molecular pathways regulating common lung diseases. To exemplify how this tool can be useful, selected examples of informative experiments are reviewed. In studies relevant to asthma, the cytokine interleukin-13 has been shown to produce many of the phenotypic features of this disease, but the cellular targets in the airways and the molecular pathways activated are largely unknown. We have used microarrays to begin to dissect the different transcriptional responses of primary lung cells to this cytokine. In experiments designed to identify global transcriptional programs responsible for regulating lung inflammation and pulmonary fibrosis, we performed microarray experiments on lung tissue from wild-type mice and mice lacking a member of the integrin family know to be involved in activation of latent transforming growth factor (TGF)-. In addition to identifying distinct cluster of genes involved in each of these processes, these studies led to the identification of novel pathways by which TGF- can regulate acute lung injury and emphysema. Together, these examples demonstrate how careful *From the Lung Biology Center, Center for Occupational and Environmental Health, Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco, San Francisco, CA. Correspondence to: Dean Sheppard, MD, Lung Biology Center, University of California, San Francisco, Box 0854, San Francisco, CA 94143; e-mail: [email protected] CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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application and thorough analysis of expression microarrays can facilitate the discovery of novel molecular targets for intervening in common lung diseases. (CHEST 2002; 121:21S–25) Key words: acute lung injury; integrins; microarrays; pulmonary fibrosis Abbreviations: IL ⫽ interleukin; MMP ⫽ matrix metalloproteinase; STAT ⫽ signal transducer and activator of transcription; TGF ⫽ transforming growth factor; Th2 ⫽ T-helper 2
E
xpression microarrays are a recently developed method that should soon allow pulmonary investigators to simultaneously evaluate messenger RNA concentrations for every transcript from a given genome simultaneously in a single experiment.1 Although this approach has all of the limitations of classical methods for measuring RNA abundance (eg, reverse transcriptase-polymerase chain reaction, Northern blotting, or ribonuclease protection assays), it has the major advantage of eliminating the need to design specific reagents and conditions for each target of interest, one at a time. Because expression arrays are currently expensive, and because the results of a single experiment can generate enormous amounts of data, thoughtful experimental design and improved methods for data analysis are likely to be critical determinants of how successful investigators are in applying this new tool. In this review are discussed a few examples of how we have utilized expression microarrays to develop a few new insights into the molecular pathways underlying common lung diseases.
Effects of Interleukin-13 on Primary Lung Cells T lymphocytes of the T-helper 2 (Th2) subtype have been shown to be prominent in the airways of patients with asthma.2 In murine models, T-cell transfer studies3 have demonstrated that cells differentiated into Th2 cells in vitro are sufficient to induce most of the phenotypic features of allergic asthma (ie, airway responsiveness, mucus metaplasia, and eosinophilic inflammation). The principal product of Th2 cells responsible for inducing all of these features during the effector phase of the immune response appears to be interleukin (IL)-13.4,5 IL-13 modifies cell behavior by activating the signal transducer and activator of transcription (STAT)-6, which translocates to the nucleus and regulates transcription of target genes.6 However, the cellular targets of IL-13 responsible for each of the phenotypic features of asthma and the specific genes whose expression is regulated in these target cells remain largely unknown. Since IL-13 works in large measure by regulating gene expression, these questions seem ideally suited to expression array experiments. As a first step to address these questions, Lee et al7 utilized Affymetrix hu6500 GeneChips (Affymetrix; Santa Clara, CA) to evaluate expression of approximately 6,500 genes in primary cultures of airway epithelial cells, airway smooth-muscle cells, and lung fibroblasts 6 h after addition of either IL-13 or an equivalent volume of phosphate22S
Figure 1. Venn diagram of overlap among 50 genes most highly induced in primary lung cells by IL-13. No genes were induced in all three cell types.
buffered saline solution to subconfluent cultures. First, to determine whether each cell type was capable of activating the same STAT-6 signaling pathway, they performed STAT-6 immunoprecipitations followed by antiphosphotyrosine Western blot tests and demonstrated that STAT-6 was phosphorylated in response to IL-13 in each case. Surprisingly, however, despite activation of the same canonical signaling pathway, the genes induced and inhibited by IL-13 were virtually nonoverlapping in these cells. In fact, although several hundred genes were induced in at least one cell type, there was not a single gene that met our criteria for induction in all three cell types (Fig 1). One clue to why the pattern of gene expression was so different came from evaluation of the transcription factors induced in each cell type. Although transcription factors were among the most prominently induced genes, completely distinct groups of transcription factors were induced in each cell type.7 The most prominent genes induced in airway smooth-muscle cells were signaling effectors and receptors and contractile proteins. Different signaling receptors and effectors were prominently induced in fibroblasts, suggesting that IL-13 might be priming each of these cell types to respond to other signals. In epithelial cells, the most prominent genes induced were components of the extracellular matrix. In summary, these studies demonstrated a striking effect of cellular differentiation in determining the transcriptional response to IL-13 in primary lung cells. The patterns of gene expression of each cell type provide some clues to the likely in vivo cellular and molecular targets of IL-13 that could contribute to allergic asthma. Of course, an important caveat is that these responses may not fully reflect the in vivo transcriptional responses because of the well-known effects of in vitro culture conditions on cellular differentiation.
Combined Use of Gene Knockouts and Expression Arrays to Identify IN VIVO Pathways Involved in Acute Lung Injury, Pulmonary Fibrosis, and Emphysema The development of lines of mice expressing homozygous null mutations of individual genes provides an opportunity to utilize expression microarrays to identify
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy
molecular pathways downstream of these inactivated genes. This approach is especially attractive when applied to knockout lines with disease-related phenotypes because it has the potential to identify previously unexpected features of molecular pathogenesis. For example, several years ago we generated a line of mice lacking expression of the epithelial integrin, ␣v6.8 These mice are dramatically protected from pulmonary fibrosis despite developing enhanced pulmonary inflammation.8,9 Based on in vitro observations, we suspected that both the protection from fibrosis and enhancement of inflammation were a consequence of loss of a normal pathway for activation of latent transforming growth factor (TGF)-.9 Expression array experiments on lung tissue from these mice, at baseline and after treatment with the fibrosis-inducing drug, bleomycin, have provided both expected and surprising insights into the molecular mechanisms underlying pulmonary fibrosis, emphysema, and acute lung injury.10 With regard to pulmonary fibrosis, cluster analysis based on pairwise comparisons of gene expression after saline solution or at two time points after bleomycin in wild-type and 6 knockout mice identified two distinct clusters of genes whose expression was increased in response to bleomycin.10 One cluster consisted of 63 genes that were expressed at higher levels at baseline in the knockout mice but were expressed at similar levels in both strains after bleomycin. We reasoned that these genes might be regulators of lung inflammation, since the knockout mice had substantial inflammation, but there was a robust inflammatory response to bleomycin in both strains. This hypothesis turned out to be substantially correct, since 38 of these genes were known regulators of inflammation. The other 25 genes in this cluster are thus candidate regulators of inflammation. The second cluster consisted of 66 genes that were expressed at the same level at baseline, but were preferentially induced by bleomycin in wild-type mice. We reasoned that these genes might encode regulators of the fibrotic response, since wild-type mice were substantially more susceptible to pulmonary fibrosis. Again, the genes in this group encoded 42 proteins that were known matrix components, matrix response genes, or known mediators of tissue remodeling, suggesting that our hypothesis was substantially correct. Interestingly, most of the known TGF-–inducible genes on the arrays we used were included in this cluster, providing support for the hypothesis that 6 knockout mice are protected from pulmonary fibrosis as a consequence of failure to activate TGF-. Further insights into a previously unexpected in vivo pathway came from examination of the differences in baseline gene expression between 6 knockout and wildtype mice. From scattergram analysis, a small group of only four genes stood out as most dramatically induced in the lungs of 6 knockout mice (Fig 2). Of these, the most highly induced was the gene encoding matrix metalloelastase (matrix metalloproteinase [MMP]-12). MMP-12 was of interest since it is a macrophage-restricted protease that has been strongly implicated in the induction of emphysema in mice.11 Quantitative polymerase chain reaction of RNA from alveolar macrophages obtained from wild-type or 6 knockout mice confirmed that MMP-12 messenger
Figure 2. Mean values for expression of approximately 6,000 murine genes (arbitrary units) in lungs from 6 knockout (-/-) or wild-type (⫹/⫹) mice. Genes whose values were within twofold of each other in lungs from each strain of mice are omitted.
RNA concentration was increased ⬎ 200 fold in macrophages from 6 knockout animals. We therefore wondered whether loss of ␣v6 might contribute to the development of emphysema through induction of this metalloprotease. Indeed, when we performed quantitative morphometry of aging 6 knockout mice, it became clear that these animals had a progressive increase in alveolar diameter characteristic of emphysema. This effect was clearly a consequence of MMP-12 induction, because when we crossed 6 knockout mice onto an MMP-12 knockout background the induction of emphysema was completely abolished. This effect, like the role of this integrin in the development of pulmonary fibrosis, is likely to depend on ␣v6-mediated activation of TGF-, since TGF- is an extremely potent inhibitor of MMP-12 induction in vitro.12,13 Additional insights into the in vivo functions of the integrin ␣v6 and TGF- itself came from analysis of the time course of induction of these TGF-–inducible genes. Utilizing a method called self-organizing maps, we identified a small subcluster of 11 genes that were all preferentially induced by bleomycin in wild-type mice and were all induced with a similar time course.10 All 11 of these genes were known to be TGF- inducible. Interestingly, all of these genes were already substantially induced by 2 to 5 days after treatment with bleomycin, a time point well in advance of the first detectable fibrosis. In addition to causing fibrosis, bleomycin is a potent cause of acute lung injury in mice (and humans), and this effect is maximal approximately 5 days after treatment.14 We therefore wondered whether TGF- itself might participate in induction or modulation of the pulmonary edema that characterizes acute lung injury. To examine this possibility, we first compared bleomycin-induced pulmonary edema in wild-type and 6 knockout mice and found that 6 knockout mice were completely protected from bleomycin-induced pulmonary edema, despite the expected enhancement of the acute inflammatory response to bleomycin.14 CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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Although the finding that 6 knockout mice were protected from pulmonary edema suggested a possible role for TGF- as an effector in this process, we could not exclude an unrelated protective effect of loss of this integrin, for example as a compensatory response to chronic inflammation. To more directly examine the role of TGF- itself, we examined the effects of a potent TGF- inhibitor (a chimeric molecule composed of the extracellular domain of the high-affinity TGF-II receptor fused to the Ig Fc domain15,16) on wild-type mice treated with bleomycin. This chimeric inhibitor also completely prevented bleomycin-induced pulmonary edema, thus directly implicating TGF- in this process.14 Furthermore, this effect was not limited to pulmonary edema induced by bleomycin, since the TGF-–receptor chimera also prevented endotoxin-induced pulmonary edema in wild-type mice. Thus, based on an unexpected pattern of gene expression in response to bleomycin, we were able to identify a novel effector of pulmonary edema in acute lung injury and have identified the integrin ␣v6 and TGF- itself as potential therapeutic targets for improving the treatment of this largely untreatable group of disorders.
Pilot Study of Human Pulmonary Fibrosis In contrast to the lung injury and pulmonary fibrosis induced by bleomycin in genetically identical mice, human pulmonary fibrosis is etiologically and temporally heterogeneous and obviously occurs in people who are genetically heterogeneous. Interpretation of microarray analysis of tissue samples obtained from such patients is thus clearly more challenging and likely to pose greater problems in distinguishing true signals from noise. Nonetheless, we have begun pilot studies to assess the utility of this approach. In our first effort, lung biopsy samples from five patients with pathologic features consistent with usual interstitial pneumonitis were analyzed and compared to samples from three resected lungs with normal histologic findings and RNA from a pool of five normal lungs. Because of the heterogeneity of these samples, a critical first step was deciding which genes were differentially expressed in a meaningful fashion. For this determination, we utilized methods specially designed for this purpose to generate an “information score” for each of the 8,400 genes being analyzed. The first method consisted of first computationally determining, for each gene, the optimal value that would separate control from experimental (patient) values, and then counting the number of values that were “misclassified” into the wrong group. A second method involved mathematically determining a Gaussian distribution curve for patient and control values for each gene and then calculating the overlap between these distributions. With these approaches we identified 164 genes that were likely to be informative in this data set. Encouragingly, many of these overlapped with the genes present in the “fibrosis cluster” from our studies of murine pulmonary fibrosis. Of these, the individual gene with the highest information score was the metalloprotease martilysin (MMP-7). MMP-7 is of interest because it has been reported to be involved in a number of processes that go beyond its role in degrading components of the extracellular matrix, including activation of tumor necrosis factor-␣17 and generation of 24S
soluble Fas ligand,18 two effects that could be predicted to enhance pulmonary fibrosis. We therefore examined the role this protein played in fibrosis in more detail. Immunostaining demonstrated dramatic induction of MMP-7 in epithelial cells overlying fibroblastic foci in other patients with usual interstitial pneumonitis. Furthermore, MMP-7 knockout mice, on two different genetic backgrounds, were substantially protected from bleomycin-induced pulmonary fibrosis. Thus, microarray analysis, even on a small number of samples from patients with pulmonary fibrosis, successfully identified at least one unexpected protein that appears to contribute to pathogenesis of this disease.
Future Directions Despite the encouraging results of the initial studies described above, it is clear that optimal application of microarray technology to the study of diseases of complex organs like the lung will be limited by spatial and temporal heterogeneity of disease, and by dramatic differences in cellular composition of affected and unaffected tissue. It will therefore be critical to develop improved methods for unbiased amplification of small RNA samples so that meaningful information can be obtained by utilizing microarrays on small tissue samples and pure cell populations (eg, samples obtained by microdissection of tissue sections). The combination of these approaches with proteomic analysis and follow-up functional evaluation of identified candidates is likely to greatly accelerate our understanding of molecular pathogenesis over the next several years.
References 1 Albelda SM, Sheppard D. Functional genomics and expression profiling: be there or be square. Am J Respir Cell Mol Biol 2000; 23:265–269 2 Robinson DS, Hamid QW, Ying S, et al. Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992; 326:298 –304 3 Cohn L, Tepper JS, Bottomly K. IL-4-independent induction of airway hyperresponsiveness by Th2, but not Th1, cells. J Immunol 1998; 161:3813–3816 4 Grunig G, Warnock M, Wakil AE, et al. Interleukin 13 and interleukin 4 mediate the asthmatic phenotype through interleukin 4 receptor ␣. Science 1998; 282:226 –228 5 Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258–2261 6 Lin J-X, Migone T-S, Friedman M, et al. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotrophy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 1995; 2:331–339 7 Lee JH, Kaminski N, Dolganov G, et al. Interleukin-13 induces dramatically different transcriptional programs in three human airway cell types. Am J Respir Cell Mol Biol 2001; 25:474 – 485 8 Huang XZ, Wu JF, Cass D, et al. Inactivation of the integrin -6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J Cell Biol 1996; 133:921–928 9 Munger JS, Huang XZ, Kawakatsu H, et al. The integrin ␣v6 binds and activates latent TGF1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999; 96:319 –328 10 Kaminski N, Allard J, Pittet J-F, et al. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and remodeling. Proc Natl Acad Sci U S A 2000; 97:1778 –1783
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy
11 Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997; 277:2002–2004 12 Werner F, Jain MK, Feinberg MW, et al. Transforming growth factor-1 inhibition of macrophage activation is mediated via Smad3. J Biol Chem 2000; 275:36653–36658 13 Feinberg MW, Jain MK, Werner F, et al. Transforming growth factor- 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages. J Biol Chem 2000; 275:25766 –25773 14 Pittet J-F, Griffiths MJD, Geiser T, et al. TGF is a critical mediator of acute lung injury. J Clin Invest 2001; 107:1529 –1536 15 Wang Q, Wang Y, Hyde DM, et al. Reduction of bleomycin induced lung fibrosis by transforming growth factor beta soluble receptor in hamsters. Thorax 1999; 54:805– 812 16 Smith JD, Bryant SR, Couper LL, et al. Soluble transforming growth factor- type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999; 84:1212–1222 17 Haro H, Crawford HC, Fingleton B, et al. Matrix metalloproteinase-7– dependent release of tumor necrosis factor-␣ in a model of herniated disc resorption. J Clin Invest 2000; 105:143–150 18 Mitsiades N, Yu WH, Poulaki V, et al. Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity. Cancer Res 2001; 61:577–581
Allelic Imbalance Demonstrated by Microsatellite Analysis of Lung Samples From Patients With Familial Pulmonary Fibrosis* Alan Q. Thomas, MD; Jennifer Carneal, BS; Cheryl Markin, BS; Kirk B. Lane, PhD; John A. Phillips III, MD; and James E. Loyd, MD
(CHEST 2002; 121:25S) Abbreviations: FIPF ⫽ familial idiopathic pulmonary fibrosis; IPF ⫽ idiopathic pulmonary fibrosis; LOH ⫽ loss of heterozygosity; MSI ⫽ microsatellite instability
pulmonary fibrosis (IPF) is a fatal, proliferaI diopathic tive lung disease of unknown etiology. The relative risk of lung cancer in patients with IPF is seven times that of smoking control subjects, suggesting chronic DNA damage and/or repair dysfunction. Vassilakis et al1 demonstrated loss of heterozygosity (LOH) and microsatellite
*From the Division of Allergy, Pulmonary, and Critical Care Medicine (Drs. Thomas, Lane, Loyd, and Ms. Markin), and Division of Medical Genetics (Ms. Carneal and Dr. Phillips), Vanderbilt University School of Medicine, Nashville, TN. This work was supported by HL-07123 and by the Vanderbilt University Discovery Program. Correspondence to: Alan Q. Thomas, MD, Research Fellow, Center for Lung Research, Department of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, 1211 22nd Ave, Nashville, TN 37232
instability (MSI) in the DNA from sputum of patients with IPF. We hypothesized that LOH and MSI might be involved in the pathogenesis of familial IPF (FIPF) and could possibly identify candidate loci. Thus, we analyzed the incidence of LOH and MSI at six highly polymorphic microsatellite marker loci from 11 patients with FIPF and 4 patients with familial primary pulmonary hypertension. Microsatellite markers were analyzed on an ABI 310. Applied Biosystems, Foster City, CA). The allele ratio of each patient was calculated using the following previously described formula: (T1/T2)/(N1/N2), where N1 and N2 are areas under the allele peaks in blood, and T1 and T2 are areas under the allele peaks in lung tissue. Four of 11 patients with FIPF (36%) exhibited LOH by at least one marker, including D17S579, D9S59, and D8S133, all adjacent to putative tumor suppressor genes. No patients with familial primary pulmonary hypertension showed LOH, and MSI was not detected in either group. These findings suggest that allelic imbalance occurs in patients with FIPF patients, which may play a role in the pathogenesis of the disease and identify candidate loci for relevant genes.
Reference 1 Vassilakis DA, Sourvinos G, Pantelidis P, et al. Extended genetic alterations in a patient with pulmonary sarcoidosis, a benign disease. Sarcoidosis Vasc Diffuse Lung Dis 2001; 18:307–310
Overexpression of Matrix Metalloproteinase-7 in Pulmonary Fibrosis* Gregory P. Cosgrove, MD; Marvin I. Schwarz, MD, FCCP; Mark W. Geraci, MD; Kevin K. Brown, MD, FCCP; and G. Scott Worthen, MD
(CHEST 2002; 121:25S–26S) Key words: genomics; idiopathic pulmonary fibrosis; matrix metalloproteinase; matrix metalloproteinase-7; nonspecific interstitial pneumonitis; oligonucleotide microarray; pulmonary fibrosis Abbreviations: IPF ⫽ idiopathic pulmonary fibrosis; MMP ⫽ matrix metalloproteinase; NSIP ⫽ nonspecific interstitial pneumonitis; TIMP ⫽ tissue inhibitor of metalloproteinase
metalloproteinases (MMPs) are inducible zincM atrix dependent proteinases involved in a variety of nor-
mal biological processes, including connective tissue deg-
*From the Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and the Department of Medicine, National Jewish Medical and Research Center, Denver, CO. This study was supported by the National Heart, Lung, and Blood Institute/National Institutes of Health grant 5 T32 HL07085–26. Correspondence to: Gregory P. Cosgrove, MD, 1400 Jackson St, D403, Denver, CO 80206; e-mail: [email protected] CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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Reference 1 Nogee LM, Dunbar AE III, Wert SE, et al. A mutation in the surfactant protein C gene associated with familial interstitial lung disease. N Engl J Med 2001; 344:573–579
Mapping Susceptibility Genes for the Induction of Pulmonary Fibrosis in Mice* Richard K. Barth, PhD; LeRoy A. Hanchett, PhD; and Clare M. Baecher-Allan, PhD
(CHEST 2002; 121:21S) Abbreviations: FGF ⫽ fibroblast growth factor; QTL ⫽ quantitative trait locus; TNF ⫽ tumor necrosis factor
fibrosis is a potentially fatal disease that can P ulmonary result from radiation or chemotherapeutic treatment
of malignancy, exposure to certain irritants, and idiopathic events. Our studies have focused on the genetic mechanisms underlying this disease through the analysis of inbred mouse strain variation in susceptibility to fibrosis induction. We have identified an inbred mouse strain (DBA/2) that is highly susceptible to bleomycin-induced pulmonary fibrosis and is genetically very dissimilar to the standard fibrosis-sensitive strain, C57BL/6, but similar to the standard fibrosis-resistant strain, BALB/c. Analysis of a set of backcross progeny generated between DBA/2 and BALB/c strains indicates that susceptibility to development of pulmonary fibrosis is controlled primarily by a few (two to three) independent genetic loci. Genetic linkage
*From the University of Rochester Cancer Center and Department of Microbiology and Immunology, University of Rochester, Rochester, NY. Supported by National Heart, Lung, and Blood Institute grant R01-HL58128. Correspondence to: Richard K. Barth, PhD, Associate Professor of Oncology in Microbiology and Immunology, University of Rochester Cancer Center, 601 Elmwood Ave, Box 704, Rochester, NY 14642; e-mail: [email protected].
using quantitative trait locus (QTL) analysis has led to the chromosomal assignment of two of these susceptibility loci. One susceptibility gene is located within a subregion of chromosome 6 that contains a cluster of genes that are members of the tumor necrosis factor (TNF)-receptor family, including the 55-kd TNF-␣1 receptor. The second susceptibility gene has been mapped to the telomeric end of chromosome 13, within an interval encompassing fibroblast growth factor (FGF)-10, a member of the FGF gene family that is expressed predominantly in the developing lung. Analysis of allelic variation in these candidate genes is underway in order to evaluate their utility as genetic markers for fibrosis susceptibility and to elucidate their possible role in influencing the disease process.
Uses of Expression Microarrays in Studies of Pulmonary Fibrosis, Asthma, Acute Lung Injury, and Emphysema* Roger S. Mitchell Lecture Dean Sheppard, MD
Expression microarrays are a powerful tool that could provide new information about the molecular pathways regulating common lung diseases. To exemplify how this tool can be useful, selected examples of informative experiments are reviewed. In studies relevant to asthma, the cytokine interleukin-13 has been shown to produce many of the phenotypic features of this disease, but the cellular targets in the airways and the molecular pathways activated are largely unknown. We have used microarrays to begin to dissect the different transcriptional responses of primary lung cells to this cytokine. In experiments designed to identify global transcriptional programs responsible for regulating lung inflammation and pulmonary fibrosis, we performed microarray experiments on lung tissue from wild-type mice and mice lacking a member of the integrin family know to be involved in activation of latent transforming growth factor (TGF)-. In addition to identifying distinct cluster of genes involved in each of these processes, these studies led to the identification of novel pathways by which TGF- can regulate acute lung injury and emphysema. Together, these examples demonstrate how careful *From the Lung Biology Center, Center for Occupational and Environmental Health, Cardiovascular Research Institute, Department of Medicine, University of California, San Francisco, San Francisco, CA. Correspondence to: Dean Sheppard, MD, Lung Biology Center, University of California, San Francisco, Box 0854, San Francisco, CA 94143; e-mail: [email protected] CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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application and thorough analysis of expression microarrays can facilitate the discovery of novel molecular targets for intervening in common lung diseases. (CHEST 2002; 121:21S–25) Key words: acute lung injury; integrins; microarrays; pulmonary fibrosis Abbreviations: IL ⫽ interleukin; MMP ⫽ matrix metalloproteinase; STAT ⫽ signal transducer and activator of transcription; TGF ⫽ transforming growth factor; Th2 ⫽ T-helper 2
E
xpression microarrays are a recently developed method that should soon allow pulmonary investigators to simultaneously evaluate messenger RNA concentrations for every transcript from a given genome simultaneously in a single experiment.1 Although this approach has all of the limitations of classical methods for measuring RNA abundance (eg, reverse transcriptase-polymerase chain reaction, Northern blotting, or ribonuclease protection assays), it has the major advantage of eliminating the need to design specific reagents and conditions for each target of interest, one at a time. Because expression arrays are currently expensive, and because the results of a single experiment can generate enormous amounts of data, thoughtful experimental design and improved methods for data analysis are likely to be critical determinants of how successful investigators are in applying this new tool. In this review are discussed a few examples of how we have utilized expression microarrays to develop a few new insights into the molecular pathways underlying common lung diseases.
Effects of Interleukin-13 on Primary Lung Cells T lymphocytes of the T-helper 2 (Th2) subtype have been shown to be prominent in the airways of patients with asthma.2 In murine models, T-cell transfer studies3 have demonstrated that cells differentiated into Th2 cells in vitro are sufficient to induce most of the phenotypic features of allergic asthma (ie, airway responsiveness, mucus metaplasia, and eosinophilic inflammation). The principal product of Th2 cells responsible for inducing all of these features during the effector phase of the immune response appears to be interleukin (IL)-13.4,5 IL-13 modifies cell behavior by activating the signal transducer and activator of transcription (STAT)-6, which translocates to the nucleus and regulates transcription of target genes.6 However, the cellular targets of IL-13 responsible for each of the phenotypic features of asthma and the specific genes whose expression is regulated in these target cells remain largely unknown. Since IL-13 works in large measure by regulating gene expression, these questions seem ideally suited to expression array experiments. As a first step to address these questions, Lee et al7 utilized Affymetrix hu6500 GeneChips (Affymetrix; Santa Clara, CA) to evaluate expression of approximately 6,500 genes in primary cultures of airway epithelial cells, airway smooth-muscle cells, and lung fibroblasts 6 h after addition of either IL-13 or an equivalent volume of phosphate22S
Figure 1. Venn diagram of overlap among 50 genes most highly induced in primary lung cells by IL-13. No genes were induced in all three cell types.
buffered saline solution to subconfluent cultures. First, to determine whether each cell type was capable of activating the same STAT-6 signaling pathway, they performed STAT-6 immunoprecipitations followed by antiphosphotyrosine Western blot tests and demonstrated that STAT-6 was phosphorylated in response to IL-13 in each case. Surprisingly, however, despite activation of the same canonical signaling pathway, the genes induced and inhibited by IL-13 were virtually nonoverlapping in these cells. In fact, although several hundred genes were induced in at least one cell type, there was not a single gene that met our criteria for induction in all three cell types (Fig 1). One clue to why the pattern of gene expression was so different came from evaluation of the transcription factors induced in each cell type. Although transcription factors were among the most prominently induced genes, completely distinct groups of transcription factors were induced in each cell type.7 The most prominent genes induced in airway smooth-muscle cells were signaling effectors and receptors and contractile proteins. Different signaling receptors and effectors were prominently induced in fibroblasts, suggesting that IL-13 might be priming each of these cell types to respond to other signals. In epithelial cells, the most prominent genes induced were components of the extracellular matrix. In summary, these studies demonstrated a striking effect of cellular differentiation in determining the transcriptional response to IL-13 in primary lung cells. The patterns of gene expression of each cell type provide some clues to the likely in vivo cellular and molecular targets of IL-13 that could contribute to allergic asthma. Of course, an important caveat is that these responses may not fully reflect the in vivo transcriptional responses because of the well-known effects of in vitro culture conditions on cellular differentiation.
Combined Use of Gene Knockouts and Expression Arrays to Identify IN VIVO Pathways Involved in Acute Lung Injury, Pulmonary Fibrosis, and Emphysema The development of lines of mice expressing homozygous null mutations of individual genes provides an opportunity to utilize expression microarrays to identify
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy
molecular pathways downstream of these inactivated genes. This approach is especially attractive when applied to knockout lines with disease-related phenotypes because it has the potential to identify previously unexpected features of molecular pathogenesis. For example, several years ago we generated a line of mice lacking expression of the epithelial integrin, ␣v6.8 These mice are dramatically protected from pulmonary fibrosis despite developing enhanced pulmonary inflammation.8,9 Based on in vitro observations, we suspected that both the protection from fibrosis and enhancement of inflammation were a consequence of loss of a normal pathway for activation of latent transforming growth factor (TGF)-.9 Expression array experiments on lung tissue from these mice, at baseline and after treatment with the fibrosis-inducing drug, bleomycin, have provided both expected and surprising insights into the molecular mechanisms underlying pulmonary fibrosis, emphysema, and acute lung injury.10 With regard to pulmonary fibrosis, cluster analysis based on pairwise comparisons of gene expression after saline solution or at two time points after bleomycin in wild-type and 6 knockout mice identified two distinct clusters of genes whose expression was increased in response to bleomycin.10 One cluster consisted of 63 genes that were expressed at higher levels at baseline in the knockout mice but were expressed at similar levels in both strains after bleomycin. We reasoned that these genes might be regulators of lung inflammation, since the knockout mice had substantial inflammation, but there was a robust inflammatory response to bleomycin in both strains. This hypothesis turned out to be substantially correct, since 38 of these genes were known regulators of inflammation. The other 25 genes in this cluster are thus candidate regulators of inflammation. The second cluster consisted of 66 genes that were expressed at the same level at baseline, but were preferentially induced by bleomycin in wild-type mice. We reasoned that these genes might encode regulators of the fibrotic response, since wild-type mice were substantially more susceptible to pulmonary fibrosis. Again, the genes in this group encoded 42 proteins that were known matrix components, matrix response genes, or known mediators of tissue remodeling, suggesting that our hypothesis was substantially correct. Interestingly, most of the known TGF-–inducible genes on the arrays we used were included in this cluster, providing support for the hypothesis that 6 knockout mice are protected from pulmonary fibrosis as a consequence of failure to activate TGF-. Further insights into a previously unexpected in vivo pathway came from examination of the differences in baseline gene expression between 6 knockout and wildtype mice. From scattergram analysis, a small group of only four genes stood out as most dramatically induced in the lungs of 6 knockout mice (Fig 2). Of these, the most highly induced was the gene encoding matrix metalloelastase (matrix metalloproteinase [MMP]-12). MMP-12 was of interest since it is a macrophage-restricted protease that has been strongly implicated in the induction of emphysema in mice.11 Quantitative polymerase chain reaction of RNA from alveolar macrophages obtained from wild-type or 6 knockout mice confirmed that MMP-12 messenger
Figure 2. Mean values for expression of approximately 6,000 murine genes (arbitrary units) in lungs from 6 knockout (-/-) or wild-type (⫹/⫹) mice. Genes whose values were within twofold of each other in lungs from each strain of mice are omitted.
RNA concentration was increased ⬎ 200 fold in macrophages from 6 knockout animals. We therefore wondered whether loss of ␣v6 might contribute to the development of emphysema through induction of this metalloprotease. Indeed, when we performed quantitative morphometry of aging 6 knockout mice, it became clear that these animals had a progressive increase in alveolar diameter characteristic of emphysema. This effect was clearly a consequence of MMP-12 induction, because when we crossed 6 knockout mice onto an MMP-12 knockout background the induction of emphysema was completely abolished. This effect, like the role of this integrin in the development of pulmonary fibrosis, is likely to depend on ␣v6-mediated activation of TGF-, since TGF- is an extremely potent inhibitor of MMP-12 induction in vitro.12,13 Additional insights into the in vivo functions of the integrin ␣v6 and TGF- itself came from analysis of the time course of induction of these TGF-–inducible genes. Utilizing a method called self-organizing maps, we identified a small subcluster of 11 genes that were all preferentially induced by bleomycin in wild-type mice and were all induced with a similar time course.10 All 11 of these genes were known to be TGF- inducible. Interestingly, all of these genes were already substantially induced by 2 to 5 days after treatment with bleomycin, a time point well in advance of the first detectable fibrosis. In addition to causing fibrosis, bleomycin is a potent cause of acute lung injury in mice (and humans), and this effect is maximal approximately 5 days after treatment.14 We therefore wondered whether TGF- itself might participate in induction or modulation of the pulmonary edema that characterizes acute lung injury. To examine this possibility, we first compared bleomycin-induced pulmonary edema in wild-type and 6 knockout mice and found that 6 knockout mice were completely protected from bleomycin-induced pulmonary edema, despite the expected enhancement of the acute inflammatory response to bleomycin.14 CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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Although the finding that 6 knockout mice were protected from pulmonary edema suggested a possible role for TGF- as an effector in this process, we could not exclude an unrelated protective effect of loss of this integrin, for example as a compensatory response to chronic inflammation. To more directly examine the role of TGF- itself, we examined the effects of a potent TGF- inhibitor (a chimeric molecule composed of the extracellular domain of the high-affinity TGF-II receptor fused to the Ig Fc domain15,16) on wild-type mice treated with bleomycin. This chimeric inhibitor also completely prevented bleomycin-induced pulmonary edema, thus directly implicating TGF- in this process.14 Furthermore, this effect was not limited to pulmonary edema induced by bleomycin, since the TGF-–receptor chimera also prevented endotoxin-induced pulmonary edema in wild-type mice. Thus, based on an unexpected pattern of gene expression in response to bleomycin, we were able to identify a novel effector of pulmonary edema in acute lung injury and have identified the integrin ␣v6 and TGF- itself as potential therapeutic targets for improving the treatment of this largely untreatable group of disorders.
Pilot Study of Human Pulmonary Fibrosis In contrast to the lung injury and pulmonary fibrosis induced by bleomycin in genetically identical mice, human pulmonary fibrosis is etiologically and temporally heterogeneous and obviously occurs in people who are genetically heterogeneous. Interpretation of microarray analysis of tissue samples obtained from such patients is thus clearly more challenging and likely to pose greater problems in distinguishing true signals from noise. Nonetheless, we have begun pilot studies to assess the utility of this approach. In our first effort, lung biopsy samples from five patients with pathologic features consistent with usual interstitial pneumonitis were analyzed and compared to samples from three resected lungs with normal histologic findings and RNA from a pool of five normal lungs. Because of the heterogeneity of these samples, a critical first step was deciding which genes were differentially expressed in a meaningful fashion. For this determination, we utilized methods specially designed for this purpose to generate an “information score” for each of the 8,400 genes being analyzed. The first method consisted of first computationally determining, for each gene, the optimal value that would separate control from experimental (patient) values, and then counting the number of values that were “misclassified” into the wrong group. A second method involved mathematically determining a Gaussian distribution curve for patient and control values for each gene and then calculating the overlap between these distributions. With these approaches we identified 164 genes that were likely to be informative in this data set. Encouragingly, many of these overlapped with the genes present in the “fibrosis cluster” from our studies of murine pulmonary fibrosis. Of these, the individual gene with the highest information score was the metalloprotease martilysin (MMP-7). MMP-7 is of interest because it has been reported to be involved in a number of processes that go beyond its role in degrading components of the extracellular matrix, including activation of tumor necrosis factor-␣17 and generation of 24S
soluble Fas ligand,18 two effects that could be predicted to enhance pulmonary fibrosis. We therefore examined the role this protein played in fibrosis in more detail. Immunostaining demonstrated dramatic induction of MMP-7 in epithelial cells overlying fibroblastic foci in other patients with usual interstitial pneumonitis. Furthermore, MMP-7 knockout mice, on two different genetic backgrounds, were substantially protected from bleomycin-induced pulmonary fibrosis. Thus, microarray analysis, even on a small number of samples from patients with pulmonary fibrosis, successfully identified at least one unexpected protein that appears to contribute to pathogenesis of this disease.
Future Directions Despite the encouraging results of the initial studies described above, it is clear that optimal application of microarray technology to the study of diseases of complex organs like the lung will be limited by spatial and temporal heterogeneity of disease, and by dramatic differences in cellular composition of affected and unaffected tissue. It will therefore be critical to develop improved methods for unbiased amplification of small RNA samples so that meaningful information can be obtained by utilizing microarrays on small tissue samples and pure cell populations (eg, samples obtained by microdissection of tissue sections). The combination of these approaches with proteomic analysis and follow-up functional evaluation of identified candidates is likely to greatly accelerate our understanding of molecular pathogenesis over the next several years.
References 1 Albelda SM, Sheppard D. Functional genomics and expression profiling: be there or be square. Am J Respir Cell Mol Biol 2000; 23:265–269 2 Robinson DS, Hamid QW, Ying S, et al. Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N Engl J Med 1992; 326:298 –304 3 Cohn L, Tepper JS, Bottomly K. IL-4-independent induction of airway hyperresponsiveness by Th2, but not Th1, cells. J Immunol 1998; 161:3813–3816 4 Grunig G, Warnock M, Wakil AE, et al. Interleukin 13 and interleukin 4 mediate the asthmatic phenotype through interleukin 4 receptor ␣. Science 1998; 282:226 –228 5 Wills-Karp M, Luyimbazi J, Xu X, et al. Interleukin-13: central mediator of allergic asthma. Science 1998; 282:2258–2261 6 Lin J-X, Migone T-S, Friedman M, et al. The role of shared receptor motifs and common Stat proteins in the generation of cytokine pleiotrophy and redundancy by IL-2, IL-4, IL-7, IL-13, and IL-15. Immunity 1995; 2:331–339 7 Lee JH, Kaminski N, Dolganov G, et al. Interleukin-13 induces dramatically different transcriptional programs in three human airway cell types. Am J Respir Cell Mol Biol 2001; 25:474 – 485 8 Huang XZ, Wu JF, Cass D, et al. Inactivation of the integrin -6 subunit gene reveals a role of epithelial integrins in regulating inflammation in the lung and skin. J Cell Biol 1996; 133:921–928 9 Munger JS, Huang XZ, Kawakatsu H, et al. The integrin ␣v6 binds and activates latent TGF1: a mechanism for regulating pulmonary inflammation and fibrosis. Cell 1999; 96:319 –328 10 Kaminski N, Allard J, Pittet J-F, et al. Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and remodeling. Proc Natl Acad Sci U S A 2000; 97:1778 –1783
Thomas L. Petty 44th Annual Aspen Lung Conference: Pulmonary Genetics, Genomics, Gene Therapy
11 Hautamaki RD, Kobayashi DK, Senior RM, et al. Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science 1997; 277:2002–2004 12 Werner F, Jain MK, Feinberg MW, et al. Transforming growth factor-1 inhibition of macrophage activation is mediated via Smad3. J Biol Chem 2000; 275:36653–36658 13 Feinberg MW, Jain MK, Werner F, et al. Transforming growth factor- 1 inhibits cytokine-mediated induction of human metalloelastase in macrophages. J Biol Chem 2000; 275:25766 –25773 14 Pittet J-F, Griffiths MJD, Geiser T, et al. TGF is a critical mediator of acute lung injury. J Clin Invest 2001; 107:1529 –1536 15 Wang Q, Wang Y, Hyde DM, et al. Reduction of bleomycin induced lung fibrosis by transforming growth factor beta soluble receptor in hamsters. Thorax 1999; 54:805– 812 16 Smith JD, Bryant SR, Couper LL, et al. Soluble transforming growth factor- type II receptor inhibits negative remodeling, fibroblast transdifferentiation, and intimal lesion formation but not endothelial growth. Circ Res 1999; 84:1212–1222 17 Haro H, Crawford HC, Fingleton B, et al. Matrix metalloproteinase-7– dependent release of tumor necrosis factor-␣ in a model of herniated disc resorption. J Clin Invest 2000; 105:143–150 18 Mitsiades N, Yu WH, Poulaki V, et al. Matrix metalloproteinase-7-mediated cleavage of Fas ligand protects tumor cells from chemotherapeutic drug cytotoxicity. Cancer Res 2001; 61:577–581
Allelic Imbalance Demonstrated by Microsatellite Analysis of Lung Samples From Patients With Familial Pulmonary Fibrosis* Alan Q. Thomas, MD; Jennifer Carneal, BS; Cheryl Markin, BS; Kirk B. Lane, PhD; John A. Phillips III, MD; and James E. Loyd, MD
(CHEST 2002; 121:25S) Abbreviations: FIPF ⫽ familial idiopathic pulmonary fibrosis; IPF ⫽ idiopathic pulmonary fibrosis; LOH ⫽ loss of heterozygosity; MSI ⫽ microsatellite instability
pulmonary fibrosis (IPF) is a fatal, proliferaI diopathic tive lung disease of unknown etiology. The relative risk of lung cancer in patients with IPF is seven times that of smoking control subjects, suggesting chronic DNA damage and/or repair dysfunction. Vassilakis et al1 demonstrated loss of heterozygosity (LOH) and microsatellite
*From the Division of Allergy, Pulmonary, and Critical Care Medicine (Drs. Thomas, Lane, Loyd, and Ms. Markin), and Division of Medical Genetics (Ms. Carneal and Dr. Phillips), Vanderbilt University School of Medicine, Nashville, TN. This work was supported by HL-07123 and by the Vanderbilt University Discovery Program. Correspondence to: Alan Q. Thomas, MD, Research Fellow, Center for Lung Research, Department of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University School of Medicine, 1211 22nd Ave, Nashville, TN 37232
instability (MSI) in the DNA from sputum of patients with IPF. We hypothesized that LOH and MSI might be involved in the pathogenesis of familial IPF (FIPF) and could possibly identify candidate loci. Thus, we analyzed the incidence of LOH and MSI at six highly polymorphic microsatellite marker loci from 11 patients with FIPF and 4 patients with familial primary pulmonary hypertension. Microsatellite markers were analyzed on an ABI 310. Applied Biosystems, Foster City, CA). The allele ratio of each patient was calculated using the following previously described formula: (T1/T2)/(N1/N2), where N1 and N2 are areas under the allele peaks in blood, and T1 and T2 are areas under the allele peaks in lung tissue. Four of 11 patients with FIPF (36%) exhibited LOH by at least one marker, including D17S579, D9S59, and D8S133, all adjacent to putative tumor suppressor genes. No patients with familial primary pulmonary hypertension showed LOH, and MSI was not detected in either group. These findings suggest that allelic imbalance occurs in patients with FIPF patients, which may play a role in the pathogenesis of the disease and identify candidate loci for relevant genes.
Reference 1 Vassilakis DA, Sourvinos G, Pantelidis P, et al. Extended genetic alterations in a patient with pulmonary sarcoidosis, a benign disease. Sarcoidosis Vasc Diffuse Lung Dis 2001; 18:307–310
Overexpression of Matrix Metalloproteinase-7 in Pulmonary Fibrosis* Gregory P. Cosgrove, MD; Marvin I. Schwarz, MD, FCCP; Mark W. Geraci, MD; Kevin K. Brown, MD, FCCP; and G. Scott Worthen, MD
(CHEST 2002; 121:25S–26S) Key words: genomics; idiopathic pulmonary fibrosis; matrix metalloproteinase; matrix metalloproteinase-7; nonspecific interstitial pneumonitis; oligonucleotide microarray; pulmonary fibrosis Abbreviations: IPF ⫽ idiopathic pulmonary fibrosis; MMP ⫽ matrix metalloproteinase; NSIP ⫽ nonspecific interstitial pneumonitis; TIMP ⫽ tissue inhibitor of metalloproteinase
metalloproteinases (MMPs) are inducible zincM atrix dependent proteinases involved in a variety of nor-
mal biological processes, including connective tissue deg-
*From the Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, and the Department of Medicine, National Jewish Medical and Research Center, Denver, CO. This study was supported by the National Heart, Lung, and Blood Institute/National Institutes of Health grant 5 T32 HL07085–26. Correspondence to: Gregory P. Cosgrove, MD, 1400 Jackson St, D403, Denver, CO 80206; e-mail: [email protected] CHEST / 121 / 3 / MARCH, 2002 SUPPLEMENT
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