Rationale: Damage to airway epithelium is followed by deposition of extracellular matrix (ECM) and migration of adjacent epithelial cells. We have shown that epithelial cells from children with asthma fail to heal a wound in vitro.
Objectives: To determine whether dysregulated ECM production by the epithelium plays a role in aberrant repair in asthma.
Methods: Airway epithelial cells (AEC) from children with asthma (n = 36), healthy atopic control subjects (n = 23), and healthy nonatopic control subjects (n = 53) were investigated by microarray, gene expression and silencing, transcript regulation analysis, and ability to close mechanical wounds.
Measurements and Main Results: Time to repair a mechanical wound in vitro by AEC from healthy and atopic children was not significantly different and both were faster than AEC from children with asthma. Microarray analysis revealed differential expression of multiple gene sets associated with repair and remodeling in asthmatic AEC. Fibronectin (FN) was the only ECM component whose expression was significantly lower in asthmatic AEC. Expression differences were verified by quantitative polymerase chain reaction and ELISA, and reduced FN expression persisted in asthmatic cells over passage. Silencing of FN expression in nonasthmatic AEC inhibited wound repair, whereas addition of FN to asthmatic AEC restored reparative capacity. Asthmatic AEC failed to synthesize FN in response to wounding or cytokine/growth factor stimulation. Exposure to 5′, 2′deoxyazacytidine had no effect on FN expression and subsequent analysis of the FN promoter did not show evidence of DNA methylation.
Conclusions: These data show that the reduced capacity of asthmatic epithelial cells to secrete FN is an important contributor to the dysregulated AEC repair observed in these cells.
Injury of the airway epithelium is normally repaired by a process involving the deposition of extracellular matrix proteins by airway epithelial cells, promoting adhesion and migration to the site of injury. Dysregulated repair processes leading to epithelial remodeling may be important in asthma pathogenesis.
This study found that fibronectin (FN) is necessary for normal wound repair, and that asthmatic epithelial cells have persistently reduced FN production and deficient wound repair in vitro that can be restored with exogenous FN.
Under normal circumstances damage and loss of epithelial cells is followed by repair of the denuded luminal surface by the adjacent epithelium. The AEC surrounding the injury are triggered to synthesize and deposit extracellular matrix (ECM) on the exposed basement membrane to promote adhesion and migration of adjacent epithelial cells into the injury site (16). Fibronectin (FN) is one of the primary ECM proteins produced by AEC (17) and profoundly influences the survival, proliferation, and differentiation of these cells, suggesting it is an important contributor to epithelial wound repair (18–20).
Given our previous observations of incomplete epithelial repair by asthmatic epithelium (14), we hypothesized that intrinsic differences specifically relating to proliferation, differentiation, and migration contribute to the dysregulated epithelial response to injury. We investigated the global gene expression profile of pediatric asthmatic epithelium and identified FN as a target for further investigation. We show that knockdown of FN gene in normal cells impairs wound closure, whereas addition of FN to asthmatic cells largely restores reparative capacity. Fibronectin gene expression in asthmatic cells could not be induced through growth factor/cytokine treatment or mechanical wounding. Finally, we provide evidence to suggest that epigenetic regulation is not responsible for silencing FN expression in asthmatic epithelial cells. These observations suggest that functional alterations in epithelial wound repair are present early in the course of childhood asthma and that reduced production of FN is a key component of this process.
Detailed descriptions of methodologies are provided in the online supplement.
Children (2.4–16.9 yr) with and without mild asthma were recruited before elective surgery. Asthma was defined by a physician diagnosis and positive responses on American Thoracic Society/European Respiratory Society respiratory questionnaires (21, 22). Children with asthma had not taken inhaled/oral glucocorticosteroids for at least 1 month before surgery. Atopy was determined by a positive radioallergosorbent result to a panel of common allergens, including house dust mite, grass pollens, milk, mold, peanut, egg white, and animal dander. Epithelial cells were collected by bronchial brushing and cultured as previously described (8, 23, 24) and classified as healthy nonatopic (pAECHNA), healthy atopic (pAECHA), or atopic asthmatic (pAECAA).
Two wound models were used. To assess wound repair kinetics, circular wounds were created (25) and wound closure calculated as outlined in the online supplement. To assess the effects of small interfering RNA (siRNA), smaller wounds were made using a plastic pipette tip as described (14). Details are in the online supplement. In these experiments, supernatants, RNA, and cell pellets were collected at 24, 48, and 72 hours post wounding. For studies involving exogenous FN addition, confluent cultures were wounded. Purified human fibronectin (0–50 μg; Roche Diagnostics, Penzberg, Germany) was added and wound closure monitored.
Total RNA was extracted using an RNeasy mini kit (QIAGEN, Hilden, Germany). Fibronectin gene expression was determined via two-step reverse transcriptase polymerase chain reaction (RT-PCR) using the TaqMan Universal PCR Master mix and Taqman Gene Expression Assay (ID:Hs00365058_ml; Applied Biosystems, Melbourne, Victoria, Australia). Expression was normalized to 18S rRNA (Applied Biosystems).
Soluble and cell lysate FN was measured by ELISA (Biomedical Technologies Inc, Stoughton, MA). Protein levels were normalized to cell number and expressed as pg/ml/106 cells.
RNA from 16 hybridizations (pAECHNA = 9; pAECAA = 7) was quantified, assessed for quality using an Agilent Bioanalyser, and processed as previously described (23) for hybridization to Affymetrix Human Genome U133A Arrays. Data were normalized by GC Robust Multiarray Algorithm and differential gene expression between the groups was assessed.
siRNAs directed against human FN were commercially manufactured using previously published sequence information (https://www1.qiagen.com/GeneGlobe; QIAGEN). Gene silencing was performed as detailed in the online supplement.
Quiesced cells were treated with either transforming growth factor-β1 (10 ng/ml), dexamethasone (10 μM), phorbol-12-myristate-13-acetate (10 ng/ml), or cycloheximide (10 μM) for 24 hours or homocysteine (0.1 mM) for 6 hours. Cells were collected 3, 6, and 24 hours post treatment and FN gene expression examined.
Cultures of pAECAA at 50% confluence were placed in basal medium for 24 hours and then incubated with either the DNA methyltransferase inhibitor 5′-aza-2′deoxycytidine (AZA) (5 μM) for 96 hours or the histone deacetylase inhibitor (HDAC) trichostatin A (TSA) (15 nM). At 3, 6, and 24 hours post treatment, FN gene expression was analyzed by quantitative (qPCR).
Cultures of pAECAA at 50% confluence were washed and incubated with media AZA (5 μM) for 96 hours. DNA was extracted and bisulfite treatment of 0.5 to 1 μg genomic DNA per sample performed using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA). Modified DNA was amplified by PCR using primer pairs specific for regions spanning from −770 to −640 and +380 to +583 bp from the FN transcription start site, purified using the DNA Clean and Concentrator kit (Zymo Research), and directly sequenced using an ABI 3130xl 16-capillary genetic analyzer.
Statistical significance was assessed using Mann-Whitney nonparametric analysis. Experiments were performed three to six times using matched samples and all values presented are means ± SD. All P values less than 0.05 were considered to be significant. We have previously shown little subject variation within each phenotype and validated this experimental approach (8).
The clinical characteristics and demographics for all subjects recruited to the study are provided in Table 1. Of the 112 subjects recruited for this study, 53 were nonasthmatic, showed no response to any of the allergens tested, and had a group mean total IgE of 57 IU/L. A further 23 subjects without asthma tested positive to one or more allergens and had a total group mean IgE of 256 IU/L. Thirty-six children were classified as atopic asthmatic with the majority also suffering from hay fever, eczema, or both. All subjects with asthma had mild disease and a total group mean IgE of 572 IU/L.
Average Age (yr)
Age Range (yr)
Average IgE (kU/L)
Hay Fever & Eczema
|Healthy nonatopic||53||M||25||8.9 ± 4.6||2.5–12.7||49.7||3||2||1|
|F||28||6.6 ± 3.0||2.9–16.1||63.7||4||5||0|
|Healthy atopic||23||M||14||7.3 ± 3.7||2.4–15.6||256||4||3||2|
|F||9||10.1 ± 4.4||2.4–13.9||257||3||2||2|
|Atopic asthmatic||36||M||23||8.6 ± 3.5||2.4–16.9||729||4||2||12|
|F||13||8.5 ± 3.0||3.6–13.2||414||4||3||4|
We have recently shown that in the absence of exogenous stimuli, pAECAA cells proliferate faster than pAECHNA cells (8, 14). To examine whether this correlated with differences in wound closure capacity, pAECHNA, pAECHA, and pAECAA cells were grown to confluence and mechanically wounded. Although both pAECHNA and pAECHA cells were able to effectively close the wound within a similar time frame (7–9 d), pAECAA showed a dramatic impairment in wound healing ability, failing to completely close wounds even after 10 days (Figure 1). We followed repair in a subset of these cells over 30 days and found maximum wound closure to be less than 70% (data not shown).
To examine dysregulated repair of asthmatic epithelium, we undertook a global analysis of gene expression using Affymetrix microarrays using freshly harvested epithelial cells from children with and without asthma (Figure 2). Based on a log2 ratio greater than or equal to 1.5 and false discovery rate (FDR) of 0.25, 764 genes were identified with increased gene expression (see Table E1 in the online supplement), including genes associated with apoptosis, metabolism, signal pathways and transduction, transcription, and transport. In addition, a further 848 genes showed decreased expression (Table E2), including those involved in cell adhesion, cell cycle, chemotaxis, immune response, protein transport, and cell motility. The complete data set is publicly available in the Gene Expression Omnibus public repository (http://www.ncbi.nlm.nih.gov/geo/) (accession no. GSE18965) in a format that complies with the Minimal Information About a Microarray Experiment guidelines. Gene ontology analysis of these differentially expressed genes identified a number of different processes, including biological processes (Table 2, Table E3), cellular components (Tables E4A and E4B), and molecular function (Tables E5A and E5B), that were significantly different. Each table illustrates the top 15 gene ontology terms with the most significantly increased (Tables E3, E4A, and E5A) or decreased (Table 2, Tables E4B and E5B) expression.
Gene Ontology Term
Genes in Ontology
|GO:0009607||Response to biotic stimulus||829||125||8.11E-17|
|GO:0009613||Response to pest, pathogen, or parasite||493||81||4.74E-13|
|GO:0051707||Response to other organism||498||81||8.26E-13|
|GO:0009605||Response to external stimulus||457||71||2.10E-10|
|GO:0009611||Response to wounding||356||60||2.25E-10|
|GO:0006950||Response to stress||942||112||2.01E-08|
|GO:0019884||Antigen presentation, exogenous antigen||14||9||6.63E-08|
|GO:0050896||Response to stimulus||1737||177||1.10E-07|
|GO:0019886||Antigen processing, exogenous antigen via MHC class II||15||9||1.55E-07|
When examined more closely, 60 probes in the gene set involved in response to wounding showed significantly decreased expression in the asthmatic epithelium. These included genes involved in chemokine binding (CCL3,5,18), toll-like receptor (TLR) signaling (TLR2, 8, CD14), and interleukin production and signaling (IL1A, IL1B, ILR1, IL8RA) (Table E6). FN-1 was the only extra cellular matrix component to be significantly decreased out of this probe list. Gene Set Enrichment Analysis (GSEA) was then performed to identify coordinated changes in genes with common biological function. Using GSEA of gene sets that included FN, we found six gene sets demonstrating core enrichment in pAECHNA compared with pAECAA (Table E7), including the “response to wounding” gene set (Figure E1). Strikingly, of the 60 probes in this gene set, only one ECM component, FN-1, was significantly decreased in asthmatic cells (2.1-fold; Table E6), validating our initial observations. This finding was further confirmed by qPCR, which showed a fivefold reduction in FN gene expression in pAECAA (Figure 3A; P = 0.003). The decrease in FN gene expression was mirrored by a substantially reduced production of FN protein by pAECAA (Figure 3B; P < 0.001) measured in both culture supernatants and cell lysates. The diminished capacity to synthesize FN was maintained over repeated passage (Figure 3C; P < 0.05). To address whether this may be an effect of atopy, we also assessed FN expression in pAECHA and found gene expression to be similar to pAECHNA (P = 0.675; Figure 3A). Although the amount of FN protein released by pAECHA was significantly lower than pAECHNA (P = 0.004 and P = 0.01 for lysate and soluble FN production, respectively), the amounts were still significantly higher than that from pAECAA (P = 0.003 and P = 0.001 for lysate and soluble FN production, respectively; Figure E2A). Because atopy may precede the development of asthma, we assessed the effect of age on the rate of wound closure and FN gene and protein production and found that these parameters were not related to age (data not shown).
We then used siRNA to determine the contribution of FN to epithelial wound repair. Using this approach FN gene expression was reduced by 89% in pAECHNA and 85% in pAECAA (Figure E2B). In pAECHNA, FN gene expression increased more than fivefold during the first 48 hours of wound repair (Figure 4A), returning to baseline levels by 72 hours, coinciding with complete wound closure (Figure 4B). As expected, FN knockdown dramatically inhibited wound repair (Figure 4B). To determine whether FN was necessary for wound repair we added FN back to cultures in which FN gene expression was silenced. As shown in Figure 4C, the addition of FN promoted complete wound closure by 72 hours, suggesting that FN is necessary and sufficient for epithelial restoration under normal conditions.
In contrast to pAECHNA, FN gene expression in pAECAA was not altered by wounding (Figure 5A) and coincident with this, wound closure over the same period was minimal. Furthermore, when FN was silenced in pAECAA, wound repair was virtually abolished (Figure 5B). Addition of FN to pAECAA enhanced wound repair in a concentration-dependent manner. Although full wound closure was not achieved by 72 hours, approximately 70% of the wound had closed in the presence of FN compared with approximately 20% in the untreated cultures (Figure 5B; P < 0.001).
Having confirmed a functional role for FN in epithelial wound repair, we next sought to identify possible mechanisms that could be responsible for suppressing its expression in pAECAA. In silico analysis of the FN promoter revealed several putative transcription factor consensus sequences, including glucocorticoid response element (GRE), cAMP response element (CRE), iron response transcription activator (ATF-2), and Smad binding sites. We then exposed pAECHNA and pAECAA to selective agonists known to activate these transcription factor consensus sequences. However, as shown in Figures 6A−6C, stimulation of GRE, CRE, ATF-2, or Smad (Figures 6A−6D) had little effect on inducing FN expression.
The down-regulation of FN gene expression through the hypermethylation of 5′CpG islands in its promoter region has been suggested to play a functional role in the development and progression of certain malignancies (26). We evaluated the extent of CpG methylation in the FN promoter as well as 100 bp upstream of the transcriptional start site in pAECAA using direct sequencing of bisulfite-converted genomic DNA. However, we found no evidence of DNA methylation in the regions tested (Figure 7A). Similarly, treatment with the DNA methyltransferase inhibitor AZA had little effect in activating FN gene expression (Figure 7B). We also investigated the potential regulatory roles of HDAC on FN gene expression. Exposure to the HDAC inhibitor TSA, which would normally result in increased levels of histone acetylation, also failed to stimulate transcription and resulting FN gene expression even over extended exposure periods (Figure 7B). Combined exposure to TSA and AZA had no effect (Figure 7B).
We have previously shown biochemical and functional differences between the epithelium of children with and without asthma (8), and that asthmatic epithelial cells exhibit dysregulated responses to injury (14). In the current study, we extend these earlier findings by undertaking a global gene expression analysis on freshly isolated epithelial cells obtained from children with and without asthma focusing on expression of ECM genes and others associated with wound repair. Gene ontology analysis revealed that the expression of the gene set “response to wounding” was reduced in pAECAA. Within this gene set, FN was the only ECM protein significantly down-regulated in pAECAA. This finding was confirmed by qPCR and analysis of protein expression. Furthermore, knocking down FN expression in pAECHNA resulted in a significant impairment in wound healing and addition of FN to these cells fully rescued wound healing capacity. In pAECAA addition of FN significantly improved, but did not completely restore, wound healing ability. Addition of growth factors and cytokines known to induce FN expression did not impact the reduced FN expression in pAECAA. Similarly, wounding itself had no effect on FN gene expression in pAECAA. Finally, we found no evidence of this in pAECAA. Collectively our data demonstrate that the response to wounding is dysregulated in AEC from children with asthma, and that a reduced ability to synthesize FN is a key factor in this response.
The epithelium is constantly faced with inflammatory and potentially injurious stimuli. To maintain the integrity of the barrier, rapid repair mechanisms are necessary (27, 28). Under normal conditions, repair is believed to follow a coordinated series of processes involving inflammation, proliferation, and reepithelialization, which is accompanied by remodeling of the underlying ECM (18, 20). We show that pAECHNA were able to fully repair wounds 1 mm wide within 7 days. This time is similar to that seen in animal studies in vivo (18, 29). In contrast, pAECAA exhibit a significantly impaired ability to complete wound repair, failing to fully close wounds even after 30 days. Although dysregulated wound repair in asthmatic epithelium has been previously suggested (30, 31), to our knowledge these data are the first to functionally assess the wound-healing capacity of pediatric asthmatic airway epithelium. Importantly, our findings provide further mechanistic support for the concept that compromised repair processes are an innate feature of asthmatic epithelium.
Microarray technology has been used to identify gene profiles associated with a number of lung diseases, including asthma, pulmonary fibrosis, and chronic obstructive pulmonary disease in the context of understanding diseases in adults (32–39). We used microarray to identify gene expression differences in children with asthma, focusing on the epithelium. Globally, we identified 1,612 genes as differentially expressed (764 increased, 848 decreased) in pAECAA and that expression of a large numbers of genes involved in the “response to wounding” gene ontology were reduced in these cells. This included a number of genes previously implicated in the pathophysiology of asthma, as well as a number of genes not previously known to be involved in this condition. These findings strongly support a fundamental alteration in the epithelium that contributes to onset and progression of asthma. In addition to the deficient wound repair response, other signals in the asthmatic epithelium point to deficient immune responses, consistent with two recent investigations demonstrating that asthmatic AEC have reduced capacity to control rhinovirus infection related to deficient innate immune response (40, 41).
Fibronectin is a large multifunctional glycoprotein that promotes cell migration, attachment, and spreading and has been shown to play an important role in epithelial wound repair (14, 42–46). In the “response to wounding” ontology, FN expression was significantly decreased in asthmatic cells. This was confirmed by qPCR analysis and at the protein level. These results differ from those of others (47) who have demonstrated that epithelial cells from adult patients with asthma release spontaneously or after stimulation more FN than their healthy counterparts. We think it is unlikely that the differences in FN production are the result of the sampling technique per se, but that they are likely to be related to cohort differences, namely, pediatric mild asthma and adult moderate to severe asthma. Similarly, the relative contribution of epithelial cell–specific FN as measured in this study versus the contribution of plasma leakage, epithelium, and other tissue sources measured in BAL in vivo studies (48) is unknown. We show that silencing FN in pAECHNA profoundly disrupted wound repair to a rate comparable to pAECAA. Moreover, addition of FN dose-dependently restored the reparative ability of these cells, confirming that FN is necessary and sufficient for full epithelial wound closure. In contrast, although addition of FN significantly improved the rate and extent of wound repair in pAECAA, full wound closure was not achieved, suggesting the involvement of other as-yet unknown interacting factors. These may include specific FN integrins and/or growth factor receptors, both of which are under investigation.
Because FN synthesis in pAECAA cells is constitutively low compared with pAECHNA, we next examined whether pAECAA could be stimulated to synthesize and secrete FN. Mechanical wounding, a nonspecific but powerful stimulus, did not induce FN synthesis in pAECAA cells. Analysis of the FN promoter revealed a number of transcription factor consensus sequences, such as Sp1, GRE, CRE, and ATF-2 sequences (27, 49–61). However, activation of these binding sites after treatment with transforming growth factor-β1, dexamethasone, phorbol-12-myristate-13-acetate, or homocysteine also failed to increase FN expression.
The reduction in FN expression seen in pAECAA persisted over serial passage suggesting that it is not dependent on an in vivo environment. Epigenetic control of gene expression by either methylation of CpG motifs in the promoter region or histone modifications can inhibit gene transcription by restricting the binding of specific transcription factors or recruiting repressive chromatin-modifying complexes such as HDACs. Methylation of CpG islands within the promoter region of several ECM genes, including FN, have been described in several cancers and are highly correlated with reduced FN expression (25, 61). In silico analysis of the FN gene revealed three CpG islands in the promoter and a total of 24 throughout the entire gene. We used two approaches to investigate whether the FN promoter was methylated. First, we treated proliferating cells with the DNA methyl transferase inhibitor, AZA. However, this had no effect on FN expression. To confirm this finding, we used bisulfite sequencing and again found no evidence that the FN promoter was methylated. Furthermore, treatment with the potent HDAC inhibitor TSA also had little effect on FN gene expression in pAECAA, suggesting that histone deacetylation was not involved in FN suppression. Current studies are focusing on microRNAs and in particular miR-17, which has been shown to regulate FN gene expression in other disease settings (62–64).
In summary, we reveal that the global gene expression profile of epithelium from children with mild asthma is intrinsically different from that of nonatopic children. Using this approach, qPCR, and ELISA, we show that expression of the ECM protein is reduced in asthmatic cells. Our data suggest that this defect was not related to control of FN expression by DNA methylation or histone acetylation. Given that FN is an essential component of the provisional ECM, providing a surface for epithelial migration and proliferation, our data highlight a potential mechanism for the abnormal epithelial repair seen in asthmatic airways.
The authors thank Drs. Scott Burgess, Amanda Griffiths, Rus Awang, Paul McNamara, and David Mullane for performing the bronchial brushings. They also thank Ms. Kak-Ming Ling, Ms. Andrea Mladinovic, Dr. Angela Fonceca, and Dr. Catherine Lane for technical assistance.
|1.||The Childhood Asthma Management Program Research Group. Long-term effects of budesonide or nedocromil in children with asthma. N Engl J Med 2000;343:1054–1063.|
|2.||Bisgaard H, Hermansen MN, Loland L, Halkjaer LB, Buchvald F. Intermittent inhaled corticosteroids in infants with episodic wheezing. N Engl J Med 2006;354:1998–2005.|
|3.||Guilbert TW, Morgan WJ, Zeiger RS, Mauger DT, Boehmer SJ, Szefler SJ, Bacharier LB, Lemanske RF Jr, Strunk RC, Allen DB, et al. Long-term inhaled corticosteroids in preschool children at high risk for asthma. N Engl J Med 2006;354:1985–1997.|
|4.||Martinez FD. Inhaled corticosteroids and asthma prevention. Lancet 2006;368:708–710.|
|5.||Murray CS, Woodcock A, Langley SJ, Morris J, Custovic A. Secondary prevention of asthma by the use of inhaled fluticasone propionate in wheezy infants (IFWIN): double-blind, randomised, controlled study. Lancet 2006;368:754–762.|
|6.||Pauwels RA, Pedersen S, Busse WW, Tan WC, Chen YZ, Ohlsson SV, Ullman A, Lamm CJ, O'Byrne PM. Early intervention with budesonide in mild persistent asthma: a randomised, double-blind trial. Lancet 2003;361:1071–1076.|
|7.||Knight DA, Holgate ST. The airway epithelium: structural and functional properties in health and disease. Respirology 2003;8:432–446.|
|8.||Kicic A, Sutanto EN, Stevens PT, Knight DA, Stick SM. Intrinsic biochemical and functional differences in bronchial epithelial cells of children with asthma. Am J Respir Crit Care Med 2006;174:1110–1118.|
|9.||Jeffery PK, Wardlaw AJ, Nelson FC, Collins JV, Kay AB. Bronchial biopsies in asthma. An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989;140:1745–1753.|
|10.||Montefort S, Roberts JA, Beasley R, Holgate ST, Roche WR. The site of disruption of the bronchial epithelium in asthmatic and non-asthmatic subjects. Thorax 1992;47:499–503.|
|11.||Montefort S, Roche WR, Holgate ST. Bronchial epithelial shedding in asthmatics and non-asthmatics. Respir Med 1993;87(Suppl B):9–11.|
|12.||Shebani E, Shahana S, Janson C, Roomans GM. Attachment of columnar airway epithelial cells in asthma. Tissue Cell 2005;37:145–152.|
|13.||Yoshihara S, Yamada Y, Abe T, Linden A, Arisaka O. Association of epithelial damage and signs of neutrophil mobilization in the airways during acute exacerbations of paediatric asthma. Clin Exp Immunol 2006;144:212–216.|
|14.||Stevens PT, Kicic A, Sutanto EN, Knight DA, Stick SM. Dysregulated repair in asthmatic paediatric airway epithelial cells: the role of plasminogen activator inhibitor-1. Clin Exp Allergy 2008;38:1901–1910.|
|15.||Holgate ST. The airway epithelium is central to the pathogenesis of asthma. Allergol Int 2008;57:1–10.|
|16.||Sottile J, Hocking DC, Swiatek PJ. Fibronectin matrix assembly enhances adhesion-dependent cell growth. J Cell Sci 1998;111:2933–2943.|
|17.||Sacco O, Silvestri M, Sabatini F, Sale R, Defilippi AC, Rossi GA. Epithelial cells and fibroblasts: structural repair and remodelling in the airways. Paediatr Respir Rev 2004;5(Suppl A):S35–40.|
|18.||Erjefalt JS, Erjefalt I, Sundler F, Persson CG. In vivo restitution of airway epithelium. Cell Tissue Res 1995;281:305–316.|
|19.||Hastie AT, Kraft WK, Nyce KB, Zangrilli JG, Musani AI, Fish JE, Peters SP. Asthmatic epithelial cell proliferation and stimulation of collagen production: human asthmatic epithelial cells stimulate collagen type III production by human lung myofibroblasts after segmental allergen challenge. Am J Respir Crit Care Med 2002;165:266–272.|
|20.||Zahm JM, Chevillard M, Puchelle E. Wound repair of human surface respiratory epithelium. Am J Respir Cell Mol Biol 1991;5:242–248.|
|21.||Asher MI, Keil U, Anderson HR, Beasley R, Crane J, Martinez F, Mitchell EA, Pearce N, Sibbald B, Stewart AW, et al. International study of asthma and allergies in childhood (ISAAC): rationale and methods. Eur Respir J 1995;8:483–491.|
|22.||Ferris BG. Epidemiology standardization project (American Thoracic Society). Am Rev Respir Dis 1978;118:1–120.|
|23.||Lane C, Burgess S, Kicic A, Knight D, Stick S. The use of non-bronchoscopic brushings to study the paediatric airway. Respir Res 2005;6:53.|
|24.||Lane C, Knight D, Burgess S, Franklin P, Horak F, Legg J, Moeller A, Stick S. Epithelial inducible nitric oxide synthase activity is the major determinant of nitric oxide concentration in exhaled breath. Thorax 2004;59:757–760.|
|25.||Vermeer PD, Einwalter LA, Moninger TO, Rokhlina T, Kern JA, Zabner J, Welsh MJ. Segregation of receptor and ligand regulates activation of epithelial growth factor receptor. Nature 2003;422:322–326.|
|26.||Chiba T, Yokosuka O, Fukai K, Hirasawa Y, Tada M, Mikata R, Imazeki F, Taniguchi H, Iwama A, Miyazaki M, et al. Identification and investigation of methylated genes in hepatomas. Eur J Cancer 2005;41:1185–1194.|
|27.||Erjefalt JS, Erjefalt I, Sundler F, Persson CG. Microcirculation-derived factors in airway epithelial repair in vivo. Microvasc Res 1994;48:161–178.|
|28.||Kotton DN, Summer R, Fine A. Lung stem cells: new paradigms. Exp Hematol 2004;32:340–343.|
|29.||Erjefalt JS, Sundler F, Persson CG. Epithelial barrier formation by airway basal cells. Thorax 1997;52:213–217.|
|30.||Puddicombe SM, Polosa R, Richter A, Krishna MT, Howarth PH, Holgate ST, Davies DE. Involvement of the epithelial growth factor receptor in epithelial repair in asthma. FASEB J 2000;14:1362–1374.|
|31.||Puddicombe SM, Torres-Lozano C, Richter A, Bucchieri F, Lordan JL, Howarth PH, Vrugt B, Albers R, Djukanovic R, Holgate ST, et al. Increased expression of p21waf cyclin-dependent kinase inhibitor in asthmatic bronchial epithelium. Am J Respir Cell Mol Biol 2003;28:61–68.|
|32.||Lee BH, Kim MS, Rhew JH, Park RW, de Crombrugghe B, Kim IS. Transcriptional regulation of fibronectin gene by phorbol myristate acetate in hepatoma cells: a negative role for NF-κB. J Cell Biochem 2000;76:437–451.|
|33.||Kaminski N, Zuo F, Cojocaro G, Yakhini Z, Ben-Dor A, Morris D, Sheppard D, Pardo A, Selman M, Heller RA. Use of oligonucleotide microarrays to analyze gene expression patterns in pulmonary fibrosis reveals distinct patterns of gene expression in mice and humans. Chest 2002;121:31S–32S.|
|34.||Laprise C, Sladek R, Ponton A, Bernier MC, Hudson TJ, Laviolette M. Functional classes of bronchial mucosa genes that are differentially expressed in asthma. BMC Genomics 2004;5:21.|
|35.||Ning W, Li CJ, Kaminski N, Feghali-Bostwick CA, Alber SM, Di YP, Otterbein SL, Song R, Hayashi S, Zhou Z, et al. Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease. Proc Natl Acad Sci USA 2004;101:14895–14900.|
|36.||Lilly CM, Tateno H, Oguma T, Israel E, Sonna LA. Effects of allergen challenge on airway epithelial cell gene expression. Am J Respir Crit Care Med 2005;171:579–586.|
|37.||Bhattacharya S, Srisuma S, Demeo DL, Reilly JJ, Bueno R, Silverman EK, Mariani TJ. Microarray data-based prioritization of chronic obstructive pulmonary disease susceptibility genes. Proc Am Thorac Soc 2006;3:472.|
|38.||Chambellan A, Cruickshank PJ, McKenzie P, Cannady SB, Szabo K, Comhair SA, Erzurum SC. Gene expression profile of human airway epithelium induced by hyperoxia in vivo. Am J Respir Cell Mol Biol 2006;35:424–435.|
|39.||Woodruff PG, Boushey HA, Dolganov GM, Barker CS, Yang YH, Donnelly S, Ellwanger A, Sidhu SS, Tao-Pick TP, Pantoja C, et al. Genome-wide profiling identifies epithelial cell genes associated with asthma and with treatment response to corticosteroids. Proc Natl Acad Sci USA 2007;104:15858–15863.|
|40.||Wark PA, Johnston SL, Bucchieri F, Powell R, Puddicombe S, Laza-Stanca V, Holgate ST, Davies DE. Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. J Exp Med 2005;201:937–947.|
|41.||Contoli M, Message SD, Laza-Stanca V, Edwards MR, Wark PA, Bartlett NW, Kebadze T, Mallia P, Stanciu LA, Parker HL, et al. Role of deficient type III interferon-λ production in asthma exacerbations. Nat Med 2006;12:1023–1026.|
|42.||Horiba K, Fukuda Y. Synchronous appearance of fibronectin, integrin α5 β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing. Virchows Arch 1994;425:425–434.|
|43.||Kanno S, Fukuda Y. Fibronectin and tenascin in rat tracheal wound healing and their relation to cell proliferation. Pathol Int 1994;44:96–106.|
|44.||Herard AL, Pierrot D, Hinnrasky J, Kaplan H, Sheppard D, Puchelle E, Zahm JM. Fibronectin and its α5 β1-integrin receptor are involved in the wound-repair process of airway epithelium. Am J Physiol 1996;271:L726–L733.|
|45.||Sottile J, Hocking DC, Langenbach KJ. Fibronectin polymerization stimulates cell growth by RGD-dependent and -independent mechanisms. J Cell Sci 2000;113:4287–4299.|
|46.||Hocking DC, Chang CH. Fibronectin matrix polymerization regulates small airway epithelial cell migration. Am J Physiol Lung Cell Mol Physiol 2003;285:L169–L179.|
|47.||Campbell AM, Chanez P, Vignola AM, Bousquet J, Couret I, Michel FB, Godard P. Functional characteristics of bronchial epithelium obtained by brushing from asthmatic and normal subjects. Am Rev Respir Dis 1993;147:529–534.|
|48.||Meerschaert J, Kelly EA, Mosher DF, Busse WW, Jarjour NN. Segmental antigen challenge increase fibronectin in bronchoalveolar lavage fluid. Am J Respir Crit Care Med 1999;159:619–625.|
|49.||Dean DC, Newby RF, Bourgeois S. Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor β, and cAMP in human cell lines. J Cell Biol 1988;106:2159–2170.|
|50.||Bernath VA, Muro AF, Vitullo AD, Bley MA, Baranao JL, Kornblihtt AR. Cyclic AMP inhibits fibronectin gene expression in a newly developed granulosa cell line by a mechanism that suppresses cAMP-responsive element-dependent transcriptional activation. J Biol Chem 1990;265:18219–18226.|
|51.||Bowlus CL, McQuillan JJ, Dean DC. Characterization of three different elements in the 5′-flanking region of the fibronectin gene which mediate a transcriptional response to cAMP. J Biol Chem 1991;266:1122–1127.|
|52.||Nakajima T, Nakamura T, Tsunoda S, Nakada S, Oda K. E1A-responsive elements for repression of rat fibronectin gene transcription. Mol Cell Biol 1992;12:2837–2846.|
|53.||Guller S, Wozniak R, Krikun G, Burnham JM, Kaplan P, Lockwood CJ. Glucocorticoid suppression of human placental fibronectin expression: implications in uterine-placental adherence. Endocrinology 1993;133:1139–1146.|
|54.||Miao S, Suri PK, Shu-Ling L, Abraham A, Cook N, Milos P, Zern MA. Role of the cyclic AMP response element in rat fibronectin gene expression. Hepatology 1993;17:882–890.|
|55.||Srebrow A, Muro AF, Werbajh S, Sharp PA, Kornblihtt AR. The CRE-binding factor ATF-2 facilitates the occupation of the CCAAT box in the fibronectin gene promoter. FEBS Lett 1993;327:25–28.|
|56.||Alonso CR, Pesce CG, Kornblihtt AR. The CCAAT-binding proteins CP1 and NF-I cooperate with ATF-2 in the transcription of the fibronectin gene. J Biol Chem 1996;271:22271–22279.|
|57.||Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE. Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1998;1:611–617.|
|58.||Dennler S, Itoh S, Vivien D, ten Dijke P, Huet S, Gauthier JM. Direct binding of Smad3 and Smad4 to critical TGF β-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J 1998;17:3091–3100.|
|59.||Albrecht M, Janssen M, Konrad L, Renneberg H, Aumuller G. Effects of dexamethasone on proliferation of and fibronectin synthesis by human primary prostatic stromal cells in vitro. Andrologia 2002;34:11–21.|
|60.||Zaniolo K, Gingras ME, Audette M, Guerin SL. Expression of the gene encoding poly(ADP-ribose) polymerase-1 is modulated by fibronectin during corneal wound healing. Invest Ophthalmol Vis Sci 2006;47:4199–4210.|
|61.||He Z, Liu S, Guo M, Mao J, Hughson MD. Expression of fibronectin and HIF-1α in renal cell carcinomas: relationship to von Hippel-Lindau gene inactivation. Cancer Genet Cytogenet 2004;152:89–94.|
|62.||Wang Q, Wang Y, Minto AW, Wang J, Shi Q, Quigg RJ. MicroRNA-377 is up-regulate and can lead to increased fibronectin production in diabetic nephropathy. FASEB J 2008;22:4126–4135.|
|63.||Zhang X, Liu S, Hu T, Liu S, He Y, Sun S. Up-regulated microRNA-143 transcribed by nuclear factor κB enhances hepatocarcinoma metastasis by repressing fibronectin production. Hepatology 2009;50:490–499.|
|64.||Shan SW, Lee DY, Deng Z, Shatseva T, Jeyapalan Z, Du WW, Zhang Y, Xuan JW, Yee S-P, Siragam V, et al. MicroRNA MiR-17 retards tissue growth and represses fibronectin expression. Nat Cell Biol 2009;11:1031–1038.|