Quantification of collagen and proteoglycan deposition in a murine model of airway remodelling
© Reinhardt et al. 2005
Received: 27 September 2004
Accepted: 08 April 2005
Published: 08 April 2005
Sub-epithelial extracellular matrix deposition is a feature of asthmatic airway remodelling associated with severity of disease, decline in lung function and airway hyperresponsiveness. The composition of, and mechanisms leading to, this increase in subepithelial matrix, and its importance in the pathogenesis of asthma are unclear. This is partly due to limitations of the current models and techniques to assess airway remodelling.
In this study we used a modified murine model of ovalbumin sensitisation and challenge to reproduce features of airway remodelling, including a sustained increase in sub-epithelial matrix deposition. In addition, we have established techniques to accurately and specifically measure changes in sub-epithelial matrix deposition, using histochemical and immunohistochemical staining in conjunction with digital image analysis, and applied these to the measurement of collagen and proteoglycans.
24 hours after final ovalbumin challenge, changes similar to those associated with acute asthma were observed, including inflammatory cell infiltration, epithelial cell shedding and goblet cell hyperplasia. Effects were restricted to the bronchial and peribronchial regions with parenchymal lung of ovalbumin sensitised and challenged mice appearing histologically normal. By 12 days, the acute inflammatory changes had largely resolved and increased sub-epithelial staining for collagen and proteoglycans was observed. Quantitative digital image analysis confirmed the increased deposition of sub-epithelial collagen (33%, p < 0.01) and proteoglycans (32%, p < 0.05), including decorin (66%, p < 0.01). In addition, the increase in sub-epithelial collagen deposition was maintained for at least 28 days (48%, p < 0.001).
This animal model reproduces many of the features of airway remodelling found in asthma and allows accurate and reproducible measurement of sub-epithelial extra-cellular matrix deposition. As far as we are aware, this is the first demonstration of increased sub-epithelial proteoglycan deposition in an animal model of airway remodelling. This model will be useful for measurement of other matrix components, as well as for assessment of the molecular mechanisms contributing to, and agents to modulate airway remodelling.
Asthma is a chronic disease with increasing prevalence characterised by persistent bronchial hyperreactivity . There is evidence of epithelial cell damage, inflammatory cell infiltration, together with hypertrophy and hyperplasia of goblet cells, submucosal glands and airway smooth muscle. Additionally, there is increased deposition of extracellular matrix molecules including collagen types I , III [3–5] and V [3, 5–7], fibronectin , tenascin [5, 8] and the proteoglycans, lumican, biglycan, versican and decorin [9, 10] in the sub-epithelial lamina reticularis. This leads to a 2-3-fold increase in the thickness of the lamina reticularis [11, 12] that has been correlated with increased fibroblast/myofibroblast number .
The importance of increased airway extracellular matrix deposition in the pathogenesis of asthma is uncertain. However, increasing evidence suggests it may play a significant role. Severity of asthma and decline in FEV1 correlate with sub-epithelial fibrosis [5, 13, 14]. Airway hyperresponsiveness correlates with bronchial wall thickening , sub-epithelial fibrosis [5, 7, 13], airway wall proteoglycan immunoreactivity  and airway fibroblast proteoglycan production . In addition, mathematical modelling suggests that thickening of the sub-epithelial layer will result in an altered folding pattern of the airway with fewer circumferential folds and an increased tendency to airway obstruction . The mechanisms responsible for increased sub-epithelial extracellular matrix deposition in asthma are unknown, although they are thought to involve a complex interaction between cells and mediators in the airway wall [18, 19]. Furthermore, the effects of current treatments on this aspect of the pathology are equivocal [6, 20–24].
Further studies are therefore required to determine the precise mechanisms involved in the sub-epithelial deposition of extracellular matrix proteins, its importance in relation to airway function and the effects of existing and new therapeutic agents on the process. Mechanistic studies of airway remodelling are difficult in man. Consequently, there is a need for good, reproducible animal models of airway remodelling and techniques to assess these parameters, preferably in mice, which would allow study of genetically-modified strains. Increased sub-epithelial deposition of collagen has been demonstrated in mouse models [25–28], but there is little or no information on other extra-cellular matrix proteins. The sensitivity of mice to airway remodelling is variable [29, 30] and protocols to achieve sustained airway remodelling can take several months . In addition, the methods used to assess sub-epithelial matrix deposition are either qualitative  or, where quantitative methods have been used, they are often subjective, laborious [25, 32], or not specific to measurement of airway changes .
In this study we have used C57Bl6/SV129 mice, a strain commonly used in the generation of genetically-modified animals, to develop a model which exhibits many characteristics of asthmatic airways and produces a rapid, reproducible and persistent increase in sub-epithelial extracellular matrix. In conjunction with this we developed accurate and sensitive histological methods using computer-assisted image analysis to specifically quantitate changes in sub-epithelial collagen and proteoglycan deposition.
Ovalbumin sensitisation and challenge
All animal studies were undertaken with appropriate local ethical and government regulatory approval. SV129/C57BL/6 mice were bred at University College London from breeding pairs obtained from the Jackson Laboratory, Bar Harbor, ME. Two to three month old mice were sensitised by intra-peritoneal injection of 10 μg chicken ovalbumin (grade V, Sigma, Poole, U.K.) in 0.1 ml normal saline or saline alone on two occasions 10 days apart. Twenty-one days after the second sensitisation, mice were challenged with six daily intratracheal instillations of 400 μg ovalbumin in 50 μl normal saline or saline alone. Briefly, mice were anaesthetised using an intra-peritoneal injection of 0.1 ml Saffan™ anaesthetic (9 mg alfaxalone and 3 mg alfadolone acetate/ml, Schering-Plough Animal Health, Welwyn Garden City, U.K.). An intra-venous cannula (22G/25 mm, BOC Ohmeda AB, Helsingborg, Sweden) was introduced through the mouth into the trachea and ovalbumin was instilled using a Hamilton syringe passed through the lumen of the cannula. Sham challenged mice received normal saline. Mice were killed in groups either one or twelve days after the final challenge to assess acute airway changes and airway remodelling respectively. Animals were injected intra-peritoneally with 0.1 ml Euthatal (200 mg pentobarbitone sodium/ml, Rhône Mérieux Ltd., Harlow, U.K.). A longitudinal ventral incision was made in the abdomen and the major vessels were sectioned. Lungs were removed and either wax embedded or frozen as described below.
Lung tissue preparation for histochemical and immunohistochemical staining of paraffin wax sections
Insufflation of the bronchial tree was performed using tracheal instillation of 4% paraformaldehyde at 20 cm 4% paraformaldehyde. The trachea was then ligated and the thoracic contents were removed. Lungs were fixed in 4% paraformaldehyde at 4°C overnight and then transferred to 15% sucrose in PBS at 4°C overnight. They were rinsed in 50% ethanol/H2O and stored in 70% ethanol/H2O prior to further dehydration and processing using an automated vacuum tissue processor (Leica TP1050, Leica Microsystems (U.K.) Ltd., Milton Keynes, U.K.). Lungs were embedded in paraffin wax using an embedding unit (Blockmaster III, Raymond A. Lamb, London, U.K.). Transverse 3 μm sections were cut for histochemical staining and 5 μm sections for immunohistochemistry. After sacrifice, care was taken to perform instillation at a constant pressure of paraformaldehyde. Lungs from each experiment were processed together in precisely the same way and sections were cut and stained at the same time. Automated staining was also pivotal to the uniform production of stained sections for image analysis.
Lung tissue preparation for proteoglycan staining of frozen sections
Insufflation of the bronchial tree was performed using tracheal instillation of a 1 : 3 mixture of OCT embedding matrix (Cell Path plc, Newtown, U.K.) : PBS at a pressure of 25 cm of this mixture. Due to the increased viscosity of this mixture, compared with paraformaldehyde, a higher pressure was required to attain the same degree of inflation. The trachea was ligated and thoracic contents were removed. Lungs were transferred to 50 ml polypropylene tubes containing OCT embedding matrix and the tubes were placed into a container of iso-pentane, chilled using liquid nitrogen. Frozen sections were cut at a thickness of 7 μm, placed on polylysine-coated slides and stored at -20°C before staining.
Paraffin wax embedded sections (3 μm) were stained with haematoxylin and eosin to demonstrate tissue architecture and inflammatory cell infiltration, and para-amino salicylic acid/Alcian blue to demonstrate mucous cells. A modified Martius Scarlet Blue (MSB) trichrome stain was employed for the localisation and quantification of collagen using computerised image analysis.
Immunohistochemistry for type III collagen and decorin was performed on paraffin embedded sections (5 μm). Sections were dewaxed, rehydrated and antigen retrieval achieved by either proteinase K digestion (10 μg/ml) for 10 minutes at room temperature for type III collagen or by microwaving sections in 10 mM citrate buffer, pH6, for 10 min for decorin. Sections were washed in PBS and endogenous peroxidase activity was blocked with 3% hydrogen peroxide (Sigma, Poole, U.K.) for 30 min at RT. Sections were then washed in PBS and incubated with 4% (v/v) goat serum (DakoCytomation, Ely, U.K.) for 20 min at RT to block non-specific binding sites. Excess serum was removed by blotting and the sections were incubated with either a 1 : 200 dilution of rabbit anti-human type III collagen antibody (Chemicon, Chandlers Ford, UK) or a 1 : 80 000 dilution of Decorin antibody, LF-113  (kindly provided by Dr L.W. Fisher, National Institutes of Health, Bethesda, MA. U.S.A.), overnight at 4°C. The sections were washed and incubated with 0.5% (v/v) goat anti-rabbit biotin-labelled secondary antibody (DakoCytomation, Ely, U.K.) for 60 min at RT. Further washes in PBS were performed before the sections were incubated with 0.5% (v/v) streptavidin (DakoCytomation, Ely, U.K.) for 30 min at RT. The streptavidin was removed by three washes in TBS and the sections were treated with DAB for 10 min. They were then washed in tap water, counterstained in Mayer's haematoxylin, differentiated in acid alcohol, dehydrated and mounted.
Frozen sections (7 μm) were stained with Cupromeronic Blue™ (Seikagaku Corporation, Tokyo, Japan) using critical electrolyte concentration (CEC) methodology . Cupromeronic Blue is an intensely coloured and electron-dense cationic dye used for the detection of sulphated polyanions including proteoglycans . Sections were rinsed for 5 min in distilled water and placed into the staining solution overnight. This consisted of 0.05% Cupromeronic Blue in 25 mM sodium acetate buffer (pH 5.8) containing 2.5% glutaraldehyde and 250 mM magnesium chloride. Different CEC were trialled before 250 mM of magnesium chloride was selected because it provided optimal staining. This CEC is appropriate for detection of small leucine-rich proteoglycans such as decorin (J.E. Scott, personal communication). The next day slides were washed in 0.025 M sodium acetate buffer (pH 5.8) containing 250 mM magnesium chloride. They were dehydrated to xylene and coverslips were applied.
Measurement of airway sub-epithelial matrix components using image analysis
Sections were examined by light microscopy using a x10 objective. Airways were selected using the following pre-defined criteria. Suitable airways were: complete, of an appropriate size to be contained within a high power field, not attached to other airways and cut in a plane perpendicular to their length (the minimum internal diameter : maximum internal diameter ratio was more than 0.5 in all cases). All suitable airways in each section were analysed. Images were digitised using a digital video camera (JVC KY-F55B, Imaging Associates, Thame, U.K.) with a resolution of 768 × 576 (vertical × horizontal) pixels. Pixel size was converted into micrometers and image analysis was performed using image analysis software (Zeiss KS300 Release 3.0, Imaging Associates, Thame, U.K.). Thresholding using pre-defined RGB criteria for airspace was performed. This allowed lumen airspace to be differentiated from airway wall. Airway lumen perimeter was then measured for each suitable airway. Thresholding using predefined RGB criteria for extra-cellular matrix constituents was performed. This produces a superimposed digitised image of both airway and parenchymal matrix. Next, a 'scrapping' procedure was completed in which pixels not adjoining at least ten other pixels were deleted. This provided better definition of airway matrix as links between airway and parenchymal matrix were removed. In this way, lumen perimeter and matrix values were obtained for each airway. Results were expressed as area of sub-epithelial matrix/unit airway perimeter (μm2/μm).
Data are presented as means ± SEM. Statistical evaluations were performed using ANOVA or unpaired t-tests for single group comparisons. A p-value of less than 0.05 was considered significant.
Many of the histological changes associated with airway remodelling in asthma are reproduced in this murine model following ovalbumin sensitisation and intra-tracheal challenge. Characteristic differences were seen one and twelve days after final challenge.
Histological changes seen 24 hours after the final challenge
Histological changes seen 12 days after the final challenge
Increased sub-epithelial collagen deposition
In these studies the area of sub-epithelial collagen staining was expressed with respect to length of airway lumen perimeter. Therefore differences could reflect changes in lumen perimeter as well as collagen deposition. However, we found no significant difference in lumen perimeter between control groups (991 ± 40 μm) and the ovalbumin sensitised/ovalbumin challenged group (1066 ± 55 μm) indicating that the changes observed were directly attributable to an increase in sub-epithelial collagen deposition.
To determine whether ovalbumin sensitisation and challenge had a differential effect on sub-epithelial collagen depending on airway size, airway data were sub-divided into lumen perimeters of greater or less than 1000 μm (figure 3B). In airways with lumen perimeters greater than 1000 μm, values for control groups were 5.84 ± 0.42 μm2/μm for saline sensitised/challenged mice, 7.00 ± 0.61 μm2/μm for saline sensitised/ovalbumin challenged mice and 6.36 ± 0.48 μm2/μm for ovalbumin sensitised/saline challenged mice. There were no significant differences between these groups. Sub-epithelial collagen/unit lumen perimeter was 9.73 ± 0.73 μm2/μm in ovalbumin sensitised/challenged mice, representing a 53% increase in collagen compared with the mean of the controls (p < 0.01). Ovalbumin sensitisation and challenge did not appear to increase the area of sub-epithelial collagen compared with saline sensitisation and challenge in airways with a lumen perimeter less than 1000 μm. Values for control groups were 6.63 ± 0.55 μm2/μm for saline sensitised/challenged mice (number of airways analysed, n = 43), 5.03 ± 0.25 μm2/μm for saline sensitised/ovalbumin challenged mice (n = 66) and 5.19 ± 0.31 μm2/μm for ovalbumin sensitised/saline challenged mice (n = 50). Sub-epithelial collagen/unit lumen perimeter was 6.58 ± 0.40 μm2/μm in ovalbumin sensitised/challenged mice (n = 88).
In a further experiment sub-epithelial collagen was measured 28 days after final challenge in order to assess whether the increase in collagen persisted over time. Saline sensitised/saline challenged mice were compared with ovalbumin sensitised/ovalbumin challenged mice. Only one control group was used as no significant differences had been shown between control groups in the previous experiment. Sub-epithelial collagen/unit lumen perimeter was 3.69 ± 0.22 μm2/μm in saline sensitised/saline challenged mice and 5.46 ± 0.42 μm2/μm in ovalbumin sensitised/challenged mice, a 48% increase in collagen (p < 0.001). In airways with lumen perimeters greater than 1000 μm, values for saline sensitised/challenged mice were 4.43 ± 0.35 μm2/μm (n = 34) and 6.92 ± 0.79 μm2/μm (n = 21) in ovalbumin sensitised/challenged mice, corresponding to a 56% increase in collagen (p < 0.002) and similar to that observed at 12 days. In contrast to the 12-day data, in airways with lumen perimeters less than 1000 μm, values for saline sensitised/challenged mice were 2.88 ± 0.16 μm2/μm (n = 31) and 4.58 ± 0.43 μm2/μm (n = 35) in ovalbuminsensitised/challenged mice, corresponding to a 59% increase in collagen (p < 0.001). The increase in sub-epithelial collagen is therefore maintained for at least four weeks.
Sections from two groups at 12 days were compared using the same techniques by an independent observer. Sub-epithelial collagen/unit lumen perimeter was 4.16 ± 0.21 μm2/μm in saline sensitised/saline challenged mice and 5.65 ± 0.24 μm2/μm in ovalbumin sensitised/challenged mice. This represents an increase of 36% (p < 0.001) and is of a similar magnitude to that obtained by the first observer.
Increased sub-epithelial proteoglycan deposition
Increased sub-epithelial decorin deposition
In control airways staining for decorin was weak with stronger staining associated with blood vessels (figure 5C). In contrast, in the airways of ovalbumin sensitised/challenged mice decorin immunohistochemistry demonstrated a well-localised, intense band of staining beneath the epithelium (figure 5D). Immunostaining was not seen elsewhere in lung sections. Sub-epithelial decorin/unit lumen perimeter was 1.66 ± 0.27 μm2/μm in control mice and 2.76 ± 0.28 μm2/μm in ovalbumin sensitised/challenged mice. This equates with an increase of 66% in decorin in ovalbumin sensitised/challenged mice (p < 0.01) (figure 6).
Using a modified ovalbumin sensitisation/challenge protocol in a mouse strain often used for the generation of transgenics and knockouts, many of the features of airway remodelling have been reproduced. At 24 hours after final challenge, epithelial changes similar to those found in acute asthma are seen. There is epithelial cell loss, mucous cell metaplasia and inflammatory cell infiltration of the airway wall. This model, in contrast to several others published in the literature, has the advantage of not producing any obvious parenchymal changes. 12 days after final challenge, the acute inflammatory changes have largely resolved and increased deposition of sub-epithelial collagen and proteoglycans, including decorin, are demonstrated.
The current protocol is based on the widely used ovalbumin sensitisation and challenge method [25, 26, 37, 38] using two intra-peritoneal sensitising injections and six daily intra-tracheal challenges of ovalbumin. The doses of ovalbumin for sensitisation and challenge fall within the ranges used in previous studies but the timings have been optimised to produce reproducible airway remodelling with increased and persistent deposition of extra-cellular matrix proteins including collagens and proteoglycans. Challenges were given by direct intra-tracheal instillation rather than by aerosol as this may more accurately control the dose of ovalbumin received by each mouse. Intra-tracheal instillation rather than aerosol inhalation may also have contributed to the reduction in parenchymal effects seen in this study.
We found similar increases in collagen deposition 12 and 28 days after final challenge when we analysed all airways together. However, when airways were sub-divided into groups with lumen perimeters of greater or less than 1000 μm the difference remained significant for larger airways at both time points but was only significant in smaller airways at the later time. A similar study in rats also found significant changes in smaller airways only at later times . The reason for this is unclear but could be due to remodelling occurring more slowly in smaller airways, although further studies would be required to confirm this. The studies of Palmans and co-workers in rats categorised airway size according to basement membrane length and divided airways into groups with basement membrane length ≤1000 μm, >1000 μm and ≤2000 μm, and >2000 μm. In the present study with mice, airway size was based on the lumen perimeter as this could be measured more accurately with the stains used. Both basement membrane length and lumen perimeter measurements have previously been validated as markers of airway size that are independent of lung volume . In our study very few airways had a perimeter of greater than 2000 μm. Therefore, airways were grouped as ≤1000 μm or >1000 μm.
The number of challenges varies dramatically between different published protocols used to induce airway remodelling. Two opposing theoretical considerations exist. In favour of a greater number of challenges is the, essentially intuitive, belief that remodelling only results from chronic, repetitive injury and repair [26, 39]. In favour of fewer challenges is the risk of immunological tolerance developing as more challenges are given. This is not just a hypothetical risk as it has been demonstrated that prolonged exposure of mice to intra-nasal or aerosolised ovalbumin results in peripheral CD4(+) T-cell unresponsiveness, reduced ovalbumin-specific IgE production and suppression of airway inflammation [41, 42]. We therefore used the minimum number of challenges that produced a significant and reproducible increase in sub-epithelial matrix.
Allergic airways disease is generally thought of as a Th2 driven response and most previous models of ovalbumin induced airway remodelling in mice have been developed in the BALB/c strain which has a strong Th2 response. The C57BL/6 and SV129 background strains do not generally exhibit strong Th2 responses  and may therefore not be ideal in some respects. However, the C57BL/6 strain has previously been used in studies of ovalbumin-induced airway remodelling  and the SV129 strain exhibits similar fibroproliferative responses to those of C57BL/6 in other models of fibrogenesis. Whilst we have not assessed the Th1/Th2 balance in the current studies, the fact that significantly increased and sustained deposition of sub-epithelial extracellular matrix proteins was observed, suggests that a strong Th2 response may not be critical to the development of airway remodelling. Further studies would be required to confirm this.
The sensitivity of different mouse strains is a considerable problem when modelling the various pathologic features of asthma including airway remodelling [29, 30]. This has lead to the development of a multitude of ovalbumin sensitisation and challenge protocols in order to optimise the modelling of particular features of asthma in different mouse strains. The protocol developed in this study has used mixed strain mice, which are commonly used in the generation of genetically engineered mice and is commercially available. Ideally, genetically engineered mice should be backcrossed to a pure strain. However, due to constraints of time and cost, this is often not achieved. We therefore believe the model described here is readily reproducible and will be useful for studies utilising genetically engineered mice to investigate mechanisms involved in airway remodelling.
An important feature of this study is the use of digital image analysis for the quantification of sub-epithelial extra-cellular matrix deposition. Previous studies in mice have used a variety of histological methods including projection of airway images onto grids and point-counting , measurement of airway wall thickness with a microscope eyepiece reticle and measurement of immunostaining intensity from black and white photomicrographs . Other studies have attempted to quantitate airway extra-cellular matrix deposition using different techniques. These include measurement of hydroxyproline in whole lung [30, 33] or in micro-dissected airways , and measurement of bronchoalveolar lavage fluid (BALF) cellular fibronectin content . Significant disadvantages exist with each of these methods. Measurements derived from whole lung or BALF cannot be airway specific and this problem will be exacerbated in models that induce significant parenchymal changes. Accurate micro-dissection of airways as small as those found in the mouse is extremely difficult, particularly when we are concerned with a layer as thin and variable as the lamina reticularis. Computerised image analysis of stained sections has a number of advantages. It is reproducible between observers and allows accurate determination of extra-cellular matrix in the sub-epithelial layer of each airway. Anatomically, murine airways are frequently associated with other airways and blood vessels. Use of computerised image analysis allows individual airways to be isolated from surrounding structures. This facilitates selection of airway from non-airway matrix. The technique is also highly adaptable, allowing use of different stains to quantify different extra-cellular matrices.
The increase in sub-epithelial collagen in ovalbumin sensitised/challenged mice that we found using six consecutive challenges over six days is similar to that observed in other acute  and more chronic (3–12 weeks) [26, 28, 30, 33] ovalbumin challenge studies. For example Blyth and co-workers observed a 55% increase in sub-epithelial reticulin staining after 6 ovalbumin challenges, three days apart, which was maintained for 50 days . Similarly, Temelkovski and co-workers described a 55% increase in the thickness of the sub-epithelial layer after three ovalbumin challenges/week for 4 weeks, with further increases if challenges were continued for up to 8 weeks . Furthermore, these changes are of a similar order of magnitude to those observed in the airways of asthmatics.
To further confirm that the sub-epithelial area of blue staining measured from MSB stained sections truly reflected an increase in collagen deposition we also immunostained sections for type III collagen. The percentage increases in the area of staining obtained for type III collagen and MSB staining following ovalbumin sensitisation/challenge were very similar. However, the values for type III collagen were about one third of those obtained from the MSB sections. This most likely reflects the difference between staining specifically for type III collagen and MSB, which is thought to stain all fibrillar collagens. Types I and III collagens represent greater than 95% of total lung collagen with type III collagen contributing approximately 30% of this total . The proportional area of staining for type III collagen compared with the total blue MSB stained area is therefore consistent with the distribution of collagen types in the lung.
Previous studies examining increased sub-epithelial extra-cellular matrix deposition in murine models of airway remodelling have measured changes in collagen [28, 32, 45, 48] and BAL cellular fibronectin [37, 49]. Similarly, airway collagen and fibronectin have been measured in a rat model . A unique feature of this study, therefore, is its application to measurement of increased sub-epithelial proteoglycans, including decorin.
The proteoglycans lumican, biglycan, versican, hyaluronan and decorin have been identified in biopsy studies of normal and asthmatic human airways. Lumican, biglycan, and versican immunostaining is predominantly found in the sub-epithelial layer of control airways . Versican and hyaluronan are also localised around and internal to airway smooth muscle bundles . Data on decorin immunostaining in controls are conflicting. Minimal airway decorin was demonstrated in one study , whereas extensive sub-epithelial staining was apparent in another . In this study we show a similar sub-epithelial localisation of proteoglycan staining in mouse airways. We also show that decorin is localised to airway sub-epithelial extra-cellular matrix in mice as described previously for human , bovine  and rat  airways, and is increased following ovalbumin sensitisation and challenge. Since this part of the study used only saline sensitised/saline challenged animals as a control we cannot exclude the possibility that increased proteoglycan deposition is due to non-specific effects of instilling large amounts of ovalbumin into the airways. However, this seems unlikely given the demonstrated lack of effect of ovalbumin on collagen deposition in the saline sensitised/ovalbumin challenged mice (figures 2 and 3) and the evidence for increased sub-epithelial proteoglycan deposition in asthmatic airways.
Lumican, biglycan and versican are increased in the sub-epithelial layer of asthmatic airways compared with controls [10, 24]. In our study, Cupromeronic Blue staining of proteoglycans was increased in ovalbumin sensitised/challenged mice with a similar sub-epithelial localisation to that observed in asthmatic airways, suggesting this is a good model of the changes observed in humans .
The finding of increased proteoglycans in the sub-epithelial layer of the airways of asthmatics and ovalbumin sensitised/challenged mice is of particular interest. Proteoglycans have an important role in determining organisation of collagen . Decorin, particularly, is an important regulator of collagen fibril assembly [55, 56]. Proteoglycans bind growth factors relevant to extra-cellular matrix deposition in the sub-epithelial layer. Perlecan binds specifically to fibroblast growth factor-7 . A number of proteoglycans bind transforming growth factor-β, including decorin, biglycan and fibromodulin . The increased immunostaining for decorin observed in ovalbumin sensitised/challenged mice is consistent with its sub-epithelial co-localisation with increased extra-cellular matrix, and TGF-β which has previously been shown to be increased . The increase in sub-epithelial proteoglycans may be an important factor in the development of sub-epithelial airway remodelling.
This animal model reproduces many of the features of airway remodelling found in asthma and allows accurate and reproducible measurement of sub-epithelial extra-cellular matrix deposition. As far as we are aware this is the first demonstration of increased sub-epithelial proteoglycans in an animal model of airway remodelling. Proteoglycans and collagen are increased by a similar magnitude to that demonstrated in asthmatics, providing a useful model of this aspect of the pathology. The properties of these matrix components suggest that they will have direct and indirect effects on airway function and the regulation of airway remodelling. The model will be invaluable in the assessment of the molecular mechanisms contributing to sub-epithelial airway remodelling and of agents to modulate such remodelling.
This work was supported by Grants from the Wellcome Trust (grant No. 060109) and Asthma UK.
- ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee: Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema. Lancet 1998, 351:1225–1232.View ArticleGoogle Scholar
- Roche WR, Beasley R, Williams JH, Holgate ST: Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989, 1:520–524.View ArticlePubMedGoogle Scholar
- Wilson JW, Li X: The measurement of reticular basement membrane and submucosal collagen in the asthmatic airway. Clin Exp Allergy 1997, 27:363–371.View ArticlePubMedGoogle Scholar
- Chu HW, Halliday JL, Martin RJ, Leung DY, Szefler SJ, Wenzel SE: Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease. Am J Respir Crit Care Med 1998, 158:1936–1944.View ArticlePubMedGoogle Scholar
- Hoshino M, Nakamura Y, Sim J, Shimojo J, Isogai S: Bronchial subepithelial fibrosis and expression of matrix metalloproteinase-9 in asthmatic airway inflammation. J Allergy Clin Immunol 1998, 102:783–788.View ArticlePubMedGoogle Scholar
- Jeffery PK, Godfrey RW, Adelroth E, Nelson F, Rogers A, Johansson SA: Effects of treatment on airway inflammation and thickening of basement membrane reticular collagen in asthma. A quantitative light and electron microscopic study. Am Rev Respir Dis 1992, 145:890–899.View ArticlePubMedGoogle Scholar
- Cho SH, Seo JY, Choi DC, Yoon HJ, Cho YJ, Min KU, Lee GK, Seo JW, Kim YY: Pathological changes according to the severity of asthma. Clin Exp Allergy 1996, 26:1210–1219.View ArticlePubMedGoogle Scholar
- Laitinen A, Altraja A, Kampe M, Linden M, Virtanen I, Laitinen LA: Tenascin is increased in airway basement membrane of asthmatics and decreased by an inhaled steroid. Am J Respir Crit Care Med 1997, 156:951–958.View ArticlePubMedGoogle Scholar
- Huang J, Olivenstein R, Taha R, Hamid Q, Ludwig M: Enhanced proteoglycan deposition in the airway wall of atopic asthmatics. Am J Respir Crit Care Med 1999, 160:725–729.View ArticlePubMedGoogle Scholar
- Roberts CR: Is asthma a fibrotic disease? Chest 1995, 107:111S-117S.View ArticlePubMedGoogle Scholar
- Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR: Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1990, 3:507–511.View ArticlePubMedGoogle Scholar
- Jeffery PK, Wardlaw AJ, Nelson FC, Collin JV, Kay AB: Bronchial biopsies in asthma: An ultrastructural, quantitative study and correlation with hyperreactivity. Am Rev Respir Dis 1989, 140:1745–1753.View ArticlePubMedGoogle Scholar
- Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A, Olivieri D: Airways remodeling is a distinctive feature of asthma and is related to severity of disease. Chest 1997, 111:852–857.View ArticlePubMedGoogle Scholar
- Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P, Hamid Q: Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 1997, 17:326–333.View ArticlePubMedGoogle Scholar
- Boulet L, Belanger M, Carrier G: Airway responsiveness and bronchial-wall thickness in asthma with or without fixed airflow obstruction. Am J Respir Crit Care Med 1995, 152:865–871.View ArticlePubMedGoogle Scholar
- Westergren-Thorsson G, Chakir J, Lafreniere-Allard MJ, Boulet LP, Tremblay GM: Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects. Int J Biochem Cell Biol 2002, 34:1256–1267.View ArticlePubMedGoogle Scholar
- Wiggs BR, Hrousis CA, Drazen JM, Kamm RD: On the mechanism of mucosal folding in normal and asthmatic airways. J Appl Physiol 1997, 83:1814–1821.PubMedGoogle Scholar
- Akers IA, McAnulty RJ, Laurent GJ: Fibroblasts and myofibroblasts. In Cellular mechanisms in airways inflammation. Edited by: Page CP, Banner KH Spina D. Basel: Birkhauser Verlag; 2000:159–198.View ArticleGoogle Scholar
- McAnulty RJ, Laurent GJ: Fibroblasts. In Asthma and COPD: Basic mechanisms and clinical management. Edited by: Barnes PB, Drazen JD Rennard SR Thomson NT. London: Academic Press; 2002:139–144.View ArticleGoogle Scholar
- Olivieri D, Chetta A, Del Donno M, Bertorelli G, Casalini A, Pesci A, Testi R, Foresi A: Effect of short-term treatment with low-dose inhaled fluticasone propionate on airway inflammation and remodeling in mild asthma: a placebo-controlled study. Am J Respir Crit Care Med 1997, 155:1864–1871.View ArticlePubMedGoogle Scholar
- Hoshino M, Nakamura Y, Sim JJ, Yamashiro Y, Uchida K, Hosaka K, Isogai S: Inhaled corticosteroid reduced lamina reticularis of the basement membrane by modulation of insulin-like growth factor (IGF)-I expression in bronchial asthma. Clin Exp Allergy 1998, 28:568–577.View ArticlePubMedGoogle Scholar
- Hoshino M, Takahashi M, Takai Y, Sim J: Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol 1999, 104:356–363.View ArticlePubMedGoogle Scholar
- Boulet LP, Turcotte H, Laviolette M, Naud F, Bernier MC, Martel S, Chakir J: Airway hyperresponsiveness, inflammation, and subepithelial collagen deposition in recently diagnosed versus long-standing mild asthma. Influence of inhaled corticosteroids. Am J Respir Crit Care Med 2000, 162:1308–1313.View ArticlePubMedGoogle Scholar
- Flood-Page P, Menzies-Gow A, Phipps S, Ying S, Wangoo A, Ludwig MS, Barnes N, Robinson D, Kay AB: Anti-IL-5 treatment reduces deposition of ECM proteins in the bronchial subepithelial basement membrane of mild atopic asthmatics. J Clin Invest 2003, 112:1029–1036.View ArticlePubMedPubMed CentralGoogle Scholar
- Blyth DI, Pedrick MS, Savage TJ, Hessel EM, Fattah D: Lung inflammation and epithelial changes in a murine model of atopic asthma. Am J Respir Cell Mol Biol 1996, 14:425–438.View ArticlePubMedGoogle Scholar
- Temelkovski J, Hogan SP, Shepherd DP, Foster PS, Kumar RK: An improved murine model of asthma: selective airway inflammation, epithelial lesions and increased methacholine responsiveness following chronic exposure to aerosolised allergen. Thorax 1998, 53:849–856.View ArticlePubMedPubMed CentralGoogle Scholar
- Kumar RK, Foster PS: Murine model of chronic human asthma. Immunol Cell Biol 2001, 79:141–144.View ArticlePubMedGoogle Scholar
- Cho JY, Miller M, Baek KJ, Han JW, Nayar J, Lee SY, McElwain K, McElwain S, Friedman S, Broide DH: Inhibition of airway remodeling in IL-5-deficient mice. J Clin Invest 2004, 113:551–560.View ArticlePubMedPubMed CentralGoogle Scholar
- Kumar RK, Foster PS: Modeling allergic asthma in mice: pitfalls and opportunities. Am J Respir Cell Mol Biol 2002, 27:267–272.View ArticlePubMedGoogle Scholar
- Shinagawa K, Kojima M: Mouse model of airway remodeling: strain differences. Am J Respir Crit Care Med 2003, 168:959–967.View ArticlePubMedGoogle Scholar
- Kumagai K, Ohno I, Imai K, Nawata J, Hayashi K, Okada S, Senoo H, Hattori T, Shirato K: The involvement of matrix metalloproteinases in basement membrane injury in a murine model of acute allergic airway inflammation. Clin Exp Allergy 2002, 32:1527–1534.View ArticlePubMedGoogle Scholar
- Tang W, Geba GP, Zheng T, Ray P, Homer RJ, Kuhn C III, Flavell RA, Elias JA: Targeted expression of IL-11 in the murine airway causes lymphocytic inflammation, bronchial remodeling, and airways obstruction. J Clin Invest 1996, 98:2845–2853.View ArticlePubMedPubMed CentralGoogle Scholar
- Tanaka H, Masuda T, Tokuoka S, Komai M, Nagao K, Takahashi Y, Nagai H: The effect of allergen-induced airway inflammation on airway remodeling in a murine model of allergic asthma. Inflamm Res 2001, 50:616–624.View ArticlePubMedGoogle Scholar
- Schaefer L, Hausser H, Altenburger M, Ugorcakova J, August C, Fisher LW, Schaefer RM, Kresse H: Decorin, biglycan and their endocytosis receptor in rat renal cortex. Kidney Int 1998, 54:1529–1541.View ArticlePubMedGoogle Scholar
- Scott JE: Histochemistry of Alcian blue. 3. The molecular biological basis of staining by Alcian blue 8GX and analogous phthalocyanins. Histochemie 1972, 32:191–212.View ArticlePubMedGoogle Scholar
- Scott JE: Proteoglycan histochemistry-a valuable tool for connective tissue biochemists. Coll Relat Res 1985, 5:541–575.View ArticlePubMedGoogle Scholar
- Trifilieff A, Fujitani Y, Coyle AJ, Kopf M, Bertrand C: IL-5 deficiency abolishes aspects of airway remodelling in a murine model of lung inflammation. Clin Exp Allergy 2001, 31:934–942.View ArticlePubMedGoogle Scholar
- Henderson WR Jr, Tang LO, Chu SJ, Tsao SM, Chiang GK, Jones F, Jonas M, Pae C, Wang H, Chi EY: A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model. Am J Respir Crit Care Med 2002, 165:108–116.View ArticlePubMedGoogle Scholar
- Palmans E, Kips JC, Pauwels RA: Prolonged allergen exposure induces structural airway changes in sensitized rats. Am J Respir Crit Care Med 2000, 161:627–635.View ArticlePubMedGoogle Scholar
- James AL, Pare PD, Hogg JC: Effects of lung volume, bronchoconstriction, and cigarette smoke on morphometric airway dimensions. J Appl Physiol 1988, 64:913–919.View ArticlePubMedGoogle Scholar
- Tsitoura DC, Blumenthal RL, Berry G, Dekruyff RH, Umetsu DT: Mechanisms preventing allergen-induced airways hyperreactivity: role of tolerance and immune deviation. J Allergy Clin Immunol 2000, 106:239–246.View ArticlePubMedGoogle Scholar
- Sakai K, Yokoyama A, Kohno N, Hamada H, Hiwada K: Prolonged antigen exposure ameliorates airway inflammation but not remodeling in a mouse model of bronchial asthma. Int Arch Allergy Immunol 2001, 126:126–134.View ArticlePubMedGoogle Scholar
- Charles PC, Weber KS, Cipriani B, Brosnan CF: Cytokine, chemokine and chemokine receptor mRNA expression in different strains of normal mice: implications for establishment of a Th1/Th2 bias. J Neuroimmunol 1999, 100:64–73.View ArticlePubMedGoogle Scholar
- Tsuchiya T, Nishimura Y, Nishiuma T, Kotani Y, Funada Y, Yoshimura S, Yokoyama M: Airway remodelling of murine chronic antigen exposure model. J Asthma 2003, 40:935–944.View ArticlePubMedGoogle Scholar
- Kenyon NJ, Ward RW, McGrew G, Last JA: TGF-beta1 causes airway fibrosis and increased collagen I and III mRNA in mice. Thorax 2003, 58:772–777.View ArticlePubMedPubMed CentralGoogle Scholar
- Laurent GJ, McAnulty RJ, Corrin B, Cockerill P: Biochemical and histological changes in pulmonary fibrosis induced in rabbits with intratracheal bleomycin. Eur J Clin Invest 1981, 11:441–448.View ArticlePubMedGoogle Scholar
- Kumar RK, Herbert C, Yang M, Koskinen AM, Mckenzie AN, Foster PS: Role of interleukin-13 in eosinophil accumulation and airway remodelling in a mouse model of chronic asthma. Clin Exp Allergy 2002, 32:1104–1111.View ArticlePubMedGoogle Scholar
- Masuda T, Tanaka H, Komai M, Nagao K, Ishizaki M, Kajiwara D, Nagai H: Mast cells play a partial role in allergen-induced subepithelial fibrosis in a murine model of allergic asthma. Clin Exp Allergy 2003, 33:705–713.View ArticlePubMedGoogle Scholar
- Trifilieff A, El Hashim A, Bertrand C: Time course of inflammatory and remodeling events in a murine model of asthma: effect of steroid treatment. Am J Physiol Lung Cell Mol Physiol 2000, 279:L1120-L1128.PubMedGoogle Scholar
- Pare PD, Roberts CR, Bai TR, Wiggs BJ: The functional consequences of airway remodeling in asthma. Monaldi Arch Chest Dis 1997, 52:589–596.PubMedGoogle Scholar
- Redington AE, Roche WR, Holgate ST, Howarth PH: Co-localization of immunoreactive transforming growth factor-beta 1 and decorin in bronchial biopsies from asthmatic and normal subjects. J Pathol 1998, 186:410–415.View ArticlePubMedGoogle Scholar
- Brahimi-Horn MC, Deudon E, Paul A, Mergey M, Mailleau C, Basbaum C, Dohrman A, Capeau J: Identification of decorin proteoglycan in bovine tracheal serous cells in culture and localization of decorin mRNA in situ. Eur J Cell Biol 1994, 64:271–280.PubMedGoogle Scholar
- Wang Y, Sakamoto K, Khosla J, Sannes PL: Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung. Am J Physiol Lung Cell Mol Physiol 2002, 282:L484-L490.View ArticlePubMedGoogle Scholar
- Scott JE: Structure and function in extracellular matrices depend on interactions between anionic glycosaminoglycans. Pathol Biol (Paris) 2001, 49:284–289.View ArticleGoogle Scholar
- Vogel KG, Paulsson M, Heinegard D: Specific inhibition of type I and type II collagen fibrillogenesis by the small proteoglycan of tendon. Biochem J 1984, 223:587–597.View ArticlePubMedPubMed CentralGoogle Scholar
- Danielson KG, Baribault H, Holmes DF, Graham H, Kadler KE, Iozzo RV: Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility. J Cell Biol 1997, 136:729–743.View ArticlePubMedPubMed CentralGoogle Scholar
- Mongiat M, Taylor K, Otto J, Aho S, Uitto J, Whitelock JM, Iozzo RV: The protein core of the proteoglycan perlecan binds specifically to fibroblast growth factor-7. J Biol Chem 2000, 275:7095–7100.View ArticlePubMedGoogle Scholar
- Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E: Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem J 1994, 302:527–534.View ArticlePubMedPubMed CentralGoogle Scholar
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