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• W2003332851 abstract "Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis. Increased production of tumor necrosis factor (TNF)-α and matrix metalloproteinases (MMPs) is a feature of inflammatory lung diseases, including emphysema and fibrosis, but the divergent pathological characteristics that result indicate involvement of other processes in disease pathogenesis. Transgenic mice overexpressing TNF-α in type II alveolar epithelial cells under the control of the surfactant protein (SP)-C promoter develop pulmonary inflammation and emphysema but are resistant to induction of fibrosis by administration of bleomycin or transforming growth factor-β. To study the molecular mechanisms underlying the development of this phenotype, we used a microarray approach to characterize the pulmonary transcriptome of SP-C/TNF-α mice and wild-type littermates. Four-month-old SP-C/TNF-α mice displayed pronounced pulmonary inflammation, airspace enlargement, increased MMP-2 and MMP-9 levels, and altered expression of 2332 probes. The functional assessment of genes with increased expression revealed enrichment of inflammatory/immune responses and proteases, whereas genes involved in protease inhibition, angiogenesis, cross-linking of basement membrane proteins, and myofibroblast differentiation were predominantly decreased. Comparison with multiple lung disease models identified a set of genes unique to the SP-C/TNF-α model and revealed that lack of extracellular matrix production distinguished SP-C/TNF-α mice from fibrosis models. Activation of inflammatory and proteolytic pathways and disruption of maintenance and repair processes are central features of emphysema in this TNF-overexpression model. Impairment of myofibroblast differentiation and extracellular matrix production may underlie resistance to induction of fibrosis. The pulmonary immune system protects the lungs from the harmful effects of inhaled xenobiotics and pathogens, in part through the release of pro-inflammatory mediators. These mediators activate professional phagocytes that work to clear the lungs of contaminating material. Although transient activation of the inflammatory system can be effective in responding to immune challenges in the lungs without causing lasting injury, chronic inflammatory processes can contribute to destruction of lung architecture, even after elimination of the noxious agent, ultimately leading to pathological conditions, such as emphysema. For example, increased levels of macrophages and neutrophils persist in the lungs of former smokers with chronic obstructive pulmonary disorder, even after smoking cessation, resulting in sustained increases in the production of proteolytic enzymes.1Owen C.A. Roles for proteinases in the pathogenesis of chronic obstructive pulmonary disease.Int J Chron Obstruct Pulmon Dis. 2008; 3: 253-268PubMed Google Scholar Although an imbalance of proteases and protease inhibitors is thought to be a primary cause of the substantial remodeling and destruction of alveolar walls that occur in emphysema,2Churg A. Wright J.L. Proteases and emphysema.Curr Opin Pulm Med. 2005; 11: 153-159Crossref PubMed Scopus (114) Google Scholar other processes, including apoptosis,3Henson P.M. Vandivier R.W. Douglas I.S. Cell death, remodeling, and repair in chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2006; 3: 713-717Crossref PubMed Scopus (106) Google Scholar cellular senescence,4Tsuji T. Aoshiba K. Nagai A. Alveolar cell senescence in patients with pulmonary emphysema.Am J Respir Crit Care Med. 2006; 174: 886-893Crossref PubMed Scopus (350) Google Scholar and dysfunctional repair systems,3Henson P.M. Vandivier R.W. Douglas I.S. Cell death, remodeling, and repair in chronic obstructive pulmonary disease.Proc Am Thorac Soc. 2006; 3: 713-717Crossref PubMed Scopus (106) Google Scholar, 5Kasahara Y. Tuder R.M. Taraseviciene-Stewart L. Le Cras T.D. Abman S. Hirth P.K. Waltenberger J. Voelkel N.F. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema.J Clin Invest. 2000; 106: 1311-1319Crossref PubMed Scopus (954) Google Scholar have also been postulated to play a role. It is clear that several interrelated cellular and molecular events contribute to pathological conditions resulting from a chronic inflammatory state in the lungs.6Wright J.L. Churg A. Current concepts in mechanisms of emphysema.Toxicol Pathol. 2007; 35: 111-115Crossref PubMed Scopus (21) Google Scholar Understanding how diverse factors contribute collectively to the development of lung pathological conditions remains an active area of research. Models that allow disease phenotypes to be examined and compared are useful to gain a better understanding of processes underlying pulmonary diseases and to characterize the role of specific agents in disease pathogenesis.7Lewis C.C. Yang J.Y. Huang X. Banerjee S.K. Blackburn M.R. Baluk P. McDonald D.M. Blackwell T.S. Nagabhushanam V. Peters W. Voehringer D. Erle D.J. Disease-specific gene expression profiling in multiple models of lung disease.Am J Respir Crit Care Med. 2008; 177: 376-387Crossref PubMed Scopus (90) Google Scholar Tumor necrosis factor (TNF)-α is a pleiotropic cytokine implicated in several lung pathological conditions, including fibrosis, emphysema, asthma, and connective tissue breakdown associated with cigarette smoking.8Churg A. Wang R.D. Tai H. Wang X. Xie C. Wright J.L. Tumor necrosis factor-alpha drives 70% of cigarette smoke-induced emphysema in the mouse.Am J Respir Crit Care Med. 2004; 170: 492-498Crossref PubMed Scopus (308) Google Scholar, 9Mukhopadhyay S. Hoidal J.R. Mukherjee T.K. Role of TNFalpha in pulmonary pathophysiology.Respir Res. 2006; 7: 125Crossref PubMed Scopus (395) Google Scholar, 10Piguet P.F. Collart M.A. Grau G.E. Sappino A.P. Vassalli P. Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis.Nature. 1990; 344: 245-247Crossref PubMed Scopus (507) Google Scholar To evaluate the involvement of TNF-α in inflammatory lung disease, transgenic mice that constitutively express TNF-α in alveolar type II epithelial cells under the control of the surfactant protein (SP)-C promoter were generated.11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar These mice develop chronic inflammation and airspace enlargement.11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar, 12Fujita M. Shannon J.M. Irvin C.G. Fagan K.A. Cool C. Augustin A. Mason R.J. Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol. 2001; 280: L39-L49PubMed Google Scholar On the basis of the interstitial inflammation and increased collagen deposition, SP-C/TNF-α mice were initially characterized as a model of idiopathic pulmonary fibrosis.11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar However, subsequent work by Fujita et al12Fujita M. Shannon J.M. Irvin C.G. Fagan K.A. Cool C. Augustin A. Mason R.J. Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol. 2001; 280: L39-L49PubMed Google Scholar demonstrated that the transgenic mice were characterized by large lung volumes and decreased elastic recoil, a phenotype more similar to emphysema. Moreover, although hydroxyproline content was increased per lung, there was no increase when values were normalized to lung weight, suggesting that the effect was related to an increase in the size of the lungs, rather than to fibrosis.12Fujita M. Shannon J.M. Irvin C.G. Fagan K.A. Cool C. Augustin A. Mason R.J. Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol. 2001; 280: L39-L49PubMed Google Scholar Physiological and morphometric imaging analyses confirmed that this model displayed features of emphysema, also revealing some consolidation of airspaces consistent with fibrosis.13Lundblad L.K. Thompson-Figueroa J. Leclair T. Sullivan M.J. Poynter M.E. Irvin C.G. Bates J.H. Tumor necrosis factor-alpha overexpression in lung disease: a single cause behind a complex phenotype.Am J Respir Crit Care Med. 2005; 171: 1363-1370Crossref PubMed Scopus (200) Google Scholar Remarkably, although TNF-α has been implicated in the pathogenesis of fibrosis in humans,14Piguet P.F. Ribaux C. Karpuz V. Grau G.E. Kapanci Y. Expression and localization of tumor necrosis factor-alpha and its mRNA in idiopathic pulmonary fibrosis.Am J Pathol. 1993; 143: 651-655PubMed Google Scholar, 15Grutters J.C. du Bois R.M. Genetics of fibrosing lung diseases.Eur Respir J. 2005; 25: 915-927Crossref PubMed Scopus (90) Google Scholar and anti–TNF-α antibodies attenuate bleomycin- or silica-induced fibrosis in animals,10Piguet P.F. Collart M.A. Grau G.E. Sappino A.P. Vassalli P. Requirement of tumour necrosis factor for development of silica-induced pulmonary fibrosis.Nature. 1990; 344: 245-247Crossref PubMed Scopus (507) Google Scholar, 16Piguet P.F. Collart M.A. Grau G.E. Kapanci Y. Vassalli P. Tumor necrosis factor/cachectin plays a key role in bleomycin-induced pneumopathy and fibrosis.J Exp Med. 1989; 170: 655-663Crossref PubMed Scopus (494) Google Scholar the lungs of SP-C/TNF-α mice are resistant to initiation of fibrosis by instillation of transforming growth factor (TGF)-β or bleomycin.17Fujita M. Shannon J.M. Morikawa O. Gauldie J. Hara N. Mason R.J. Overexpression of TNF-alpha diminishes pulmonary fibrosis induced by bleomycin or TGF-beta.Am J Respir Cell Mol Biol. 2003; 29: 669-676Crossref PubMed Scopus (116) Google Scholar Together, the data suggest that factors other than TNF-α alone are important in determining the fate of injured lungs. Given the pleiotropic nature of TNF-α signaling and its association with several lung pathological conditions with differing phenotypes, an understanding of the molecular pathways modulated by TNF-α expression in the lungs should be of use in characterizing disease pathogenesis and identifying potential therapeutic targets for further investigation. Global screening approaches offer powerful tools to assess biological pathways that drive disease phenotypes.7Lewis C.C. Yang J.Y. Huang X. Banerjee S.K. Blackburn M.R. Baluk P. McDonald D.M. Blackwell T.S. Nagabhushanam V. Peters W. Voehringer D. Erle D.J. Disease-specific gene expression profiling in multiple models of lung disease.Am J Respir Crit Care Med. 2008; 177: 376-387Crossref PubMed Scopus (90) Google Scholar To gain a better understanding of the molecular processes underlying the chronic inflammatory state and resistance to induction of fibrosis of the SP-C/TNF-α mice, in the present study, we used a microarray approach to compare the level of expression of approximately 21,000 genes in the lungs of TNF-overexpressing mice and wild-type littermates. We then contrasted the SP-C/TNF-α lung transcriptome with 12 other models of lung disease to identify key molecular pathways that distinguish disease phenotypes. We report that constitutive expression of TNF-α in the lungs results in several effects on critical pathways implicated in the pathogenesis of emphysema, including increased expression of genes involved in inflammation and matrix remodeling and reduced expression of genes involved in lung repair, maintenance pathways, and myofibroblast differentiation. SP-C/TNF-α male mice (C57BL/6 background)11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar were originally provided by Dr. Robert Mason (National Jewish Medical and Research Center, Denver, CO). The SP-C/TNF-α male mice were crossed with C57BL/6 female mice (Charles River Laboratories, St. Constant, QC, Canada) and maintained as a heterozygous line by repeated backcrossing. Male transgenic mice and their wild-type littermates were genotyped by PCR analysis of genomic DNA isolated from ear punches, as previously described.11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar Animals were aged 132 ± 5 days at the time of the experiment. Mice were housed in individual Plexiglas cages on wood-chip bedding under high-efficiency particulate filtered air and held to a 12-hour dark-light cycle. Food and water were provided ad libitum. All experimental protocols were reviewed and approved by the Animal Care Committee of Health Canada. Mice were anesthetized by administration of sodium pentobarbital (60 mg/kg, i.p.). For histological examination, lungs (n = 3 per genotype) were inflated at 25 cm H2O static pressure by intratracheal instillation of 4% paraformaldehyde in PBS. The lungs were excised, immersed in fixative, and stored at 4°C. Tissue blocks were dehydrated in ethanol and embedded in paraffin. Sections (0.75 μm thick) were stained with H&E. For microarray analyses, five mice per genotype were used, whereas for real-time PCR analyses, lavage cytological examination, and gelatin zymography, an independent set of four mice per genotype were used. Lungs were washed by bronchoalveolar lavage with saline (37°C) at 30 mL/kg body weight, flash frozen in liquid nitrogen, and stored at −80°C. Lavage fluid was centrifuged (400 × g for 10 minutes at 4°C) to remove cells and frozen at −80°C. Cells were counted using a Coulter Multisizer II (Beckman Coulter Canada Inc., Mississauga, ON, Canada), and differential cell counts were obtained from cytospin preparations. Bronchoalveolar lavage fluid from SP-C/TNF-α and wild-type mice was evaluated for matrix metalloproteinase (MMP) activity by gelatin zymography. Equal quantities of bronchoalveolar lavage fluid (20 μL) were loaded on 10% SDS-acrylamide gels containing 1 mg/mL gelatin (Sigma-Aldrich Canada Ltd., Oakville, ON, Canada) and run for 1 hour at 200 mV. Each gel contained prestained mol. wt. markers (Bio-Rad Laboratories Canada Ltd., Mississauga, ON, Canada) and MMP-2 standards (Calbiochem, La Jolla, CA). Gels were incubated in Zymogram Renaturation Buffer (Bio-Rad Laboratories Canada Ltd.) for 30 minutes, followed by overnight incubation at 37°C in Zymogram Development Buffer (Bio-Rad Laboratories Canada Ltd.). After incubation, gels were stained in 0.5% Coomassie Blue R-250 (Sigma-Adrich Canada Ltd.) staining solution (in 40% methanol/10% acetic acid) for 1 hour and then destained in a solution of 40% methanol/10% acetic acid. Clear bands were assessed by densitometric analysis using NIH shareware. To verify MMP activity, control gels were incubated under the same conditions in buffer containing 25 mmol/L EDTA. Frozen lung samples were homogenized in TRIzol reagent (Invitrogen Canada Inc., Burlington, ON, Canada), and total RNA was isolated according to the manufacturer's instructions. The isolated RNA was further purified by spin-column cleanup using RNeasy Mini Kits (Qiagen Inc., Mississauga, ON, Canada). RNA was quantified using the RiboGreen RNA Quantitation Reagent and Kit (Molecular Probes, Eugene, OR), and quality was confirmed using the RNA 6000 NanoLab Chip Kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada). Archived microarray data for age-matched transgenic animals and wild-type littermates (n = 5 per genotype) from our inhalation facility18Thomson E.M. Williams A. Yauk C.L. Vincent R. Toxicogenomic analysis of susceptibility to inhaled urban particulate matter in mice with chronic lung inflammation.Part Fibre Toxicol. 2009; 6: 6Crossref PubMed Scopus (24) Google Scholar were used for assessment of the lung transcriptome. Microarray data are available at the National Center for Biotechnology Information Gene Expression Omnibus (accession no. GSE11037; http://www.ncbi.nlm.nih.gov/projects/geo). Briefly, 2.5 μg of RNA was amplified and labeled using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Canada Inc.), according to the manufacturer's instructions. Agilent Mouse G4121A Microarrays were hybridized with 5 μg of Cy5-labeled lung RNA (one lung per array). Cy3-labeled Universal Mouse Reference RNA (Agilent Technologies Canada Inc.; 5 μg) was used as a common reference on all arrays. A randomized block design was used for the order of sample hybridizations. Arrays were scanned using a ScanArray Express scanner (Perkin-Elmer Life Sciences, Woodbridge, ON, Canada), and data were acquired with ImaGene 5.5 (BioDiscovery, El Segundo, CA). Lowess normalization19Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.Nucleic Acids Res. 2002; 30: e15Crossref PubMed Scopus (2812) Google Scholar was performed using SAS/STAT software, version 8.2 of the SAS System for Windows (1999–2001; SAS Institute Inc., Cary, NC). The logarithm base 2 relative intensities were used for subsequent analyses, and the MAANOVA library20Wu H. Kerr M.K. Cui X. Churchill G.A. MAANOVA: a software package for the analysis of spotted cDNA microarray experiments.in: Parmigiani G. Garrett E.S. Irizarry R.A. Zeger S. The Analysis of Gene Expression Data: Methods and Software. Springer-Verlag, New York2003: 313-431Crossref Google Scholar in R was used to identify differentially expressed genes. The Fs statistic21Cui X.G. Hwang J.T.G. Qiu J. Blades N.J. Churchill G.A. Improved statistical tests for differential gene expression by shrinking variance components.Biostatistics. 2005; 6: 59-75Crossref PubMed Scopus (382) Google Scholar was used as a shrinkage estimator for the gene-specific variance components, and P values for all statistical tests were estimated using the permutation method (1000 permutations with residual shuffling). These P values were then adjusted for multiple comparisons using the false-discovery rate (FDR) approach,22Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc. 1995; 57: 289-300Google Scholar and genes were considered differentially expressed if they had an FDR-adjusted P < 0.05. The group means for the fold-change calculation (transgenic versus wild type) were based on the adjusted relative intensity for each gene after subtraction of nuisance factors (eg, day of hybridization) from the normalized ratio. Gene ontology enrichment analysis and functional annotation clustering were performed using the Database for Annotation, Visualization and Integrated Discovery23Dennis Jr, G. Sherman B.T. Hosack D.A. Yang J. Gao W. Lane H.C. Lempicki R.A. DAVID: Database for Annotation, Visualization, and Integrated Discovery.Genome Biol. 2003; 4: P3Crossref PubMed Google Scholar, 24Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat Protoc. 2009; 4: 44-57Crossref PubMed Scopus (24210) Google Scholar (http://david.abcc.ncifcrf.gov/home.jsp) using the array probe list as the background population. Analysis of KEGG pathways (Kyoto Encyclopedia of Genes & Genomes online resource, http://www.genome.ad.jp/kegg/pathway.html) was also performed. Microarray data for 12 murine models of lung disease were obtained from the National Center for Biotechnology Information Gene Expression Omnibus site (accession no. GSE4231; http://www.ncbi.nlm.nih.gov/projects/geo). Because five different platforms were used in the analyses, GenBank accession identification numbers were used to identify probes common to all platforms. On arrays that contained multiple probes for a given GenBank accession identification number, the lowess normalized19Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation.Nucleic Acids Res. 2002; 30: e15Crossref PubMed Scopus (2812) Google Scholar log2 of the ratios was averaged. Data were then merged across the five platforms, yielding 7148 unique GenBank accession identification numbers. Differentially expressed genes were identified as described for the SP-C/TNF-α arrays. Because of low power to detect significant effects for several of the models, a less stringent statistical filter of a 1.5-fold cutoff and an unadjusted P < 0.05 was used. Hierarchical clustering was conducted using the top 50 genes with an unadjusted P < 0.05 ranked by fold change for each disease model, excluding the SP-C/TNF-α model. The distance metric was one minus the Pearson correlation, and average linkage was used. Real-time PCR was performed on samples used for microarray experiments (n = 5 per genotype) or on independent biological samples from age-matched animals (n = 4 per genotype). cDNA, 20 ng, was combined with Quantitect (Qiagen Inc.) primer assays and real-time iQ SYBR Green supermix (Bio-Rad Laboratories Canada Ltd.), according to the product protocol, and run at an annealing temperature of 55°C on the Lightcycler 480 (Roche Diagnostics, Laval, QC, Canada). Fluorescence was monitored at every cycle during the 72°C elongation step. Postrun melt curves were routinely inspected to verify product purity. Expression was calculated relative to the TATA-box binding protein, which exhibited unchanged expression between genotypes (data not shown), using the ΔΔCT method. Statistical significance was assessed by t-test (Sigma-Stat version 3.0; SPSS Inc., Chicago, IL). Histological assessment of the inflated lungs confirmed that SP-C/TNF-α mice bred in our laboratory had enlarged alveolar spaces and thickened septa compared with wild-type littermates (Figure 1, A–D), as expected.11Miyazaki Y. Araki K. Vesin C. Garcia I. Kapanci Y. Whitsett J.A. Piguet P.F. Vassalli P. Expression of a tumor necrosis factor-alpha transgene in murine lung causes lymphocytic and fibrosing alveolitis: a mouse model of progressive pulmonary fibrosis.J Clin Invest. 1995; 96: 250-259Crossref PubMed Scopus (326) Google Scholar, 12Fujita M. Shannon J.M. Irvin C.G. Fagan K.A. Cool C. Augustin A. Mason R.J. Overexpression of tumor necrosis factor-alpha produces an increase in lung volumes and pulmonary hypertension.Am J Physiol Lung Cell Mol Physiol. 2001; 280: L39-L49PubMed Google Scholar Increased cellularity was apparent in both the alveoli and the interstitium. Bronchoalveolar lavage recovered 10-fold higher numbers of cells in the transgenic mice compared with wild-type animals (Figure 1E), consisting of an eightfold increase in macrophages and increased numbers of neutrophils and lymphocytes (Figure 1F). Because MMPs are implicated in basement membrane remodeling and airspace enlargement, we examined MMP levels in bronchoalveolar lavage fluid by gelatin zymography. Clear bands representing areas of enzymatic activity were detected at 72 and 105 kDa in SP-C/TNF-α mice (Figure 2), corresponding to the expected mol. wt. of pro-MMP-2 and pro-MMP-9, respectively, whereas only a faint band at 72 kDa was observed in wild-type animals. The enzyme activity was inhibited by incubation of gels in buffer containing EDTA (data not shown). Comparison of the lung transcriptome of wild-type and transgenic animals resulted in the identification of 2332 differentially expressed probes (FDR-adjusted P < 0.05; see Supplemental Table S1 at http://ajp.amjpathol.org). Of the differentially expressed probes, 1283 (55%) were increased and 1049 (45%) were decreased. Fold changes ranged from 34-fold increased to sevenfold decreased expression relative to wild-type animals, with most genes (67%) exhibiting a fold change of less than two and 95% exhibiting a fold change of less than four (summarized in Table 1). Cluster analysis performed on unfiltered transcriptional profiles revealed a clear genotype effect, with samples from transgenic and wild-type animals separated on two main branches (Figure 3A). To generate a more conservative list of significant genes, a twofold cutoff filter was applied to the list of significant genes (Figure 3B). This filtering resulted in a list of 760 probes, of which 472 (62%) were increased and 288 (38%) were decreased. Probes with the greatest increase in expression in the TNF lungs included Ig genes [eg, Ig-joining chain, 34-fold; Ig κ chain variable 38, 17-fold; Ig heavy chain 1a (serum IgG2a), 16-fold; Ig λ chain, variable 1, 12-fold], consistent with the lymphocytic infiltration; the acute-phase response proteins serum amyloid A3 (30-fold) and serum amyloid A1 (10-fold); C-type lectin domain family 4, member d (17-fold); and TNF-α (17-fold). Probes with the greatest decrease in expression included procollagen C-terminal enhancer protein 2a (sevenfold), a glycoprotein that potentiates collagen cleavage; genes involved in biotransformation of endogenous and exogenous compounds, including cytochrome P450, family 1, subfamily a, polypeptide 1 (sixfold), sulfotransferase family 1D, member 1 (fivefold), and flavin-containing monooxygenase 3 (fourfold); cytochrome P450, family 2, subfamily s, polypeptide 1 (fourfold); the scaffolding protein tetraspanin 7 (fivefold); insulin-like growth factor binding proteins 2 (threefold) and 6 (threefold); and the tissue inhibitor of metalloproteinase-3 (TIMP-3; threefold). Differential expression of a panel of seven probes chosen to represent a range of fold changes (threefold decrease to fivefold increase relative to wild type) was confirmed by real-time PCR (t-test, P < 0.05 for all genes; see Supplemental Figure S1 at http://ajp.amjpathol.org). In general, the difference in gene expression between genotypes was greater according to the PCR analysis than was observed in the microarray data, as is common with microarray analyses, but the direction of change was consistent.Table 1Fold-Change Distribution of Differentially Expressed Probes (SP-C/TNF-α versus Wild-Type) with FDR-Adjusted P < 0.05Fold-change rangeTotal probesIncreased expressionDecreased expression1–1.57754253501.5–27973864112–46463692774–6685996–8262428–10440>1016160 Open table in a new tab Functional analysis was performed on the list of 760 probes with FDR-adjusted P < 0.05 and a greater than twofold difference in gene expression. Functional analysis of terms related to cellular localization revealed that the products of differentially expressed genes were predominantly associated with the extracellular space, plasma membrane, or lysosome (Table 2). The most significant enrichment of terms relating to molecular function was for chemokines, cytokines, and antigen binding (Table 3), consistent with the pronounced inflammatory/immune response. Functional analysis of biological processes showed that the 472 genes with increased expression in the lungs of transgenic mice were strongly enriched for terms related to immune system processes and inflammation (Table 4). In contrast, the 288 genes with decreased expression were enriched for terms relating to blood vessel development and cytoskeletal organization and biogenesis (T" @default.
• W2003332851 created "2016-06-24" @default.
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• W2003332851 date "2012-04-01" @default.
• W2003332851 modified "2023-09-27" @default.
• W2003332851 title "Overexpression of Tumor Necrosis Factor-α in the Lungs Alters Immune Response, Matrix Remodeling, and Repair and Maintenance Pathways" @default.
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