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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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In bleomycin (BM)-induced lung fibrosis, alterations have been shown in the expression and deposition of small proteoglycans (PGs). Less, however, is known about changes in large PGs. We investigated changes in large aggregating (versican [VS]), basement membrane (heparan sulfate proteoglycan [HSPG]), and small (biglycan and fibromodulin) PGs during the development of BM-induced pulmonary fibrosis. BM (1.5 U) was instilled intratracheally into male Sprague-Dawley rats. Control rats received saline. At 7, 14, and 28 d after administration of BM, lungs were excised; one lung was fixed in formalin and 5-µm sections were cut and stained with hematoxylin-eosin. The other lung was used for PG extraction. PGs were extracted with guanidine and were separated through composite gel polyacrylamide gel electrophoresis (PAGE) (large PGs) and sodium dodecylsulfate-PAGE (small PGs). Gels were either stained or electrophoretically transferred and probed with antibodies to VS, HSPG, biglycan, and fibromodulin. Histologic samples showed prominent inflammation, with abundant proteinaceous material, most evident in the samples obtained at 7 and 14 d after administration of BM. By 28 d after BM, much of the inflammatory response had resolved, and heterogeneous distribution of fibrosis was observed. Immunoblots showed a relative abundance of VS at 7 and 14 d. Control lungs stained minimally for VS. Levels of HSPG, biglycan, and fibromodulin were increased maximally at 14 d after administration of BM. Immunocytochemistry showed intense immunostaining of biglycan and fibromodulin in the areas of injured lung tissue from rats 14 and 28 d after BM administration. Control lungs revealed minimal staining for small PGs. Our findings indicate that changes in all subclasses of PGs occur during the development of BM-induced pulmonary fibrosis.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Pulmonary fibrosis is a chronic inflammatory interstitial lung disease characterized by massive synthesis, deposition, and rearrangement of extracellular matrix (ECM) macromolecules such as collagens, elastic fibers, and noncollagenous glycoproteins (1). The last-named components include proteoglycans (PGs), which are a heterogeneous family of genetically unrelated core proteins with covalently attached glycosaminoglycan (GAG) side chains (2). PGs affect tissue mechanical properties as a result of their large hydrodynamic volumes, and play an important role in ECM assembly in situations involving tissue development and repair (2). The peripheral lung has been shown to contain different classes of PGs (2). Versican (VS), a large chondroitin sulfate-containing PG, binds specifically with hyaluronic acid forming macromolecular aggregates in the interstitial matrix. In addition to VS several nonaggregating small, leucine-rich repeat proteoglycans (SLRPs) with one (decorin) or two (biglycan) GAG chains have been described in the lung interstitium (3). Recently, lumican has been identified as the predominant SLRP in the human peripheral lung (4). Additionally, heparan sulfate-containing proteoglycans, including perlecan of the epithelial basement membrane and syndecan of the cell surface, have been detected in the lung (2, 5).
Although increased synthesis and accumulation of collagen and elastin components have been extensively reported in fibrotic lung disease (1), the nature of changes in PGs has not been well characterized. Studies with rats indicate alterations in the expression and deposition of SLRPs during the development of bleomycin (BM)-induced pulmonary fibrosis: an increase in the messenger RNA (mRNA) and protein expression of biglycan, and a simultaneous decrease in decorin, have been reported. Conversely, no change in fibromodulin message was detected (3). Evidence from human studies suggests that VS may play an important role in a variety of fibroproliferative disorders, including pulmonary fibrosis (6). However, little information is available about temporal changes in the expression of VS during repair and remodeling in pulmonary fibrosis. There is essentially no information about changes in basement membrane PGs, nor about the site of deposition of small PGs. Therefore, we undertook the present study to (1) determine whether alterations in the expression of all major subclasses of PGs occur in BM-induced pulmonary fibrosis in the rat; (2) to identify the distribution of these molecules within the lung; and (3) to obtain information about the temporal schema of these changes.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Materials
Reagents were purchased from the following sources: BM (Blenoxane) from Bristol-Myers Squibb (Princeton, NJ); agarose from Park Scientific Limited (Northampton, UK); acrylamide, 6-aminohexanoic acid, benzamidine hydrochloride, chondroitinase avidin-biotin complex (ABC), dimethylformamide (DMF), ethylenediamine tetraacetic acid (EDTA), N-ethylmaleimide, Fast Red salt, glucuronolactone, guanidine hydrochloride, levamisole, N,N'-methylene-bisacrylamide, N,N,N',N'-tetramethylethylenediamine, papain, phenylmethylsulfonyl fluoride, sodium acetate, Trizma base, and Tween 20 from Sigma (Oakville, ON, Canada); antibody diluent, rabbit antimouse and swine antirabbit antibodies and universal blocking solution from DAKO (Mississauga, ON, Canada); monoclonal antibodies (mAbs) 12C5 and C17 from Developmental Studies Hybridoma Bank (Iowa City, IA); and enhanced chemiluminescence (ECL) detection reagents for Western blotting, hyper film, hybond ECL nitrocellulose membrane, and streptavidin-biotinylated horseradish peroxidase (HRP) complex from Amersham Pharmacia Biotech Inc. (Canada). All other reagents were of analytical grade.
Methods
Pulmonary fibrosis was induced in male Sprague-Dawley rats (weight: 275 to 350 g) by a single intratracheal instillation of 1.5 U of BM in 0.3 ml of saline. Control rats received an equal volume of saline only. At 7, 14, and 28 d after BM administration, animals were killed in accordance with standard ethical procedures, and the lungs were excised and processed for histopathology, immunocytochemistry, and proteoglycan analysis. Six animals were studied in each of the six groups.
Histology
The lungs and trachea were exposed by thoracotomy, and one lung was inflation-fixed overnight through the trachea with 10% neutral buffered formalin (at 25 cm H2O transpulmonary pressure). After fixation, lung tissues were paraffin embedded and cut into 5-µm sections. Representative sections were stained with hematoxylin-eosin (H&E) to visualize the overall tissue architecture.
Extraction of PGs
The second lung of each animal was frozen in 10 mM acetate buffer,
pH 6.0, and cut into 20-µm sections with a cryostat (4). The PGs were
extracted for 48 h at 4° C with 10 volumes of 4 M guanidine hydrochloride and 100 mM sodium acetate, pH 6.0, containing protease inhibitors (4). The PG extracts were then centrifuged at 15,000 rpm for
30 min, and the supernatants were dialyzed exhaustively against 10 mM
sodium acetate/10 mM Tris-HCl, pH 7.3, and distilled water. After dialysis, PGs were precipitated from the extracts with 9 volumes of ethanol (7). Briefly, 100 µl of the extract was mixed with 900 µl of 95%
ethanol in a 1.5 ml Eppendorf centrifuge tube. After incubation overnight at 
20° C, the sample was centrifuged at 12,000 rpm for 20 min
in a microfuge. The pellet was again suspended in 100 µl of 0.5 M sodium acetate and mixed with 900 µl of 95% ethanol, and precipitation
was repeated as described earlier. The precipitated PGs were dried
and stored at 20° C until further analysis.
Extraction of GAGs
Weighed portions (100 mg wet tissue) of the lung tissues were finely minced with scissors and then digested with papain (20:1, tissue: enzyme) in 0.5 ml of digestion buffer (0.5 M Tris-HCl/20 mM EDTA/ 20 mM cysteine HCl) at 60° C for 24 h. After digestion, proteins were precipitated with trichloroacetic acid (final concentration: 10%). The supernatants were dialyzed exhaustively against distilled water, and were precipitated with 5 volumes of 95% ethanol containing 0.5 M sodium acetate. The precipitated GAGs were dried and resuspended in distilled water. The total GAG content of lung tissues and PG extracts was estimated by measuring uronic acid.
Uronic Acid Assay
Uronic acid content was determined in guanidine HCl extracts and papain digests by the method of Bitter and Muir (8). Briefly, 750 µl of sulfuric acid/borate reagent was added to an Eppendorf tube and cooled on ice. Next, 125 µl of the standard or test solution was carefully added onto the surface of the borate-sulfuric acid mixture and allowed to diffuse for 10 min, with continued cooling on ice. The tubes were gently mixed and briefly vortexed to ensure homogeneity. Following this, the tubes were heated to 100° C for 10 min and cooled in an ice bath, and 25 µl of carbazole was added to the mixture. The tubes were then once again vortex mixed, heated to 100° C for 15 min, and cooled, and were read at 525 nm. Glucuronolactone was used as a standard.
Composite Agarose-PAGE and Western Blotting
Electrophoretic separation of large PGs was performed in a composite gel (0.6% agarose:1.2% polyacrylamide) according to the method of Heinegard and associates (7), with slight modification. Samples, usually ethanol precipitates, were run into the gel at 60 V and then separated at 160 V until the bromophenol blue marker migrated to 3 cm. Separated PGs were either stained with toluidine blue or electrophoretically transferred and probed with antibodies to VS or large basement membrane heparan sulfate proteoglycans (HSPGs).
After electrophoresis, separated PGs were transferred to nitrocellulose membranes, using a BIORAD (Mississauga, ON, Canada) blotter apparatus (20 V overnight at 4° C). After blocking, membranes were probed with mAbs 12C5 (1:2,000) or C17 (1:1,000), to detect VS (9) or large basement membrane HSPGs (10), respectively, in Tris-buffered saline with Tween (TBST) (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 1 h at room temperature (RT). After washing with TBST, membranes were incubated with a biotinylated rabbit antimouse secondary antibody (1:2,500) for 1 h at RT, washed again with TBST, and then incubated in streptavidin-biotinylated HRP complex (1:3,000) for 45 min at RT. After washing of membranes, antibody binding was visualized through ECL. PGs extracted from normal rat aorta and kidney were used as positive controls for VS and HSPG, respectively.
SDS-PAGE and Immunoblotting of Biglycan and Fibromodulin
Specific polyclonal antipeptide IgG and antipeptide sera preparations that have been shown to interact with biglycan and fibromodulin, respectively, in human cartilage samples (11, 12) were used to identify the expression of these small PGs during the development of BM- induced lung fibrosis. Preliminary experiments with these antibodies revealed immunoreactivity for biglycan and fibromodulin in rat lungs. PG extracts were digested with chondroitinase ABC (0.1 U/ml at 37° C for 4 h), run in a 10% SDS-PAGE gel, and then analyzed by immunoblotting. After electrophoresis, the separated proteins were electrophoretically transferred to nitrocellulose membranes and blocked for 1 h at RT. After blocking, membranes were washed with TBST and then incubated with primary antibodies for 1 h at RT to detect biglycan (1:500) or fibromodulin (1:500) core proteins. After washing with TBST, membranes were incubated with a 1:1,000 dilution of biotin-labeled swine antirabbit secondary antibody for 1 h at RT. After further washing with TBST, membranes were incubated in streptavidin-biotinylated HRP complex (1:3,000) for 45 min at RT, washed once again, and then visualized by ECL.
Quantitation of PGs in Western Blots
Densitometric analysis of both the large and small PGs was accomplished with Sigma gel software version 1.0 (Jandel Scientific, Chicago, IL). This method produces a graph of the peaks in each lane corresponding to the electrophoresis gel, and then integrates to calculate the areas under the peaks. The mean values of six observations are reported.
Immunocytochemistry
Paraffin-embedded sections were deparaffinized, hydrated, and incubated in a universal blocking solution for 1 h at RT. Sections were then rinsed with TBS and incubated with antibodies to biglycan or fibromodulin (1:500 in antibody diluent) for 1 h at RT. After washing with TBS, sections were incubated with a biotin-labeled swine antirabbit IgG for 1 h at RT, washed again, and incubated with alkaline phosphatase-conjugated avidin for 1 h. After further washing, tissue sections were developed with Fast Red salt (1 mg/ml in alkaline phosphatase substrate) for 10 min at RT and were counterstained with Mayer's hematoxylin.
Statistical Analysis
All data are given as the mean ± SD of results for six rats. One-way analysis of variance (ANOVA) with post hoc Bonferroni's correction was used to determine the significance of changes in PGs on Western blots. Two-way ANOVA with a post hoc t test was used to determine the statistical significance of changes in uronic acid.
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RESULTS |
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INTRODUCTION
METHODS
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DISCUSSION
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Histopathology
Evidence of BM-induced lung injury was apparent in H&E-stained lung sections (Figures 1a through 1d). Histologic analysis showed a prominent infiltration of inflammatory cells in the alveolar space and the interstitium at 7 and 14 d after BM administration. Heterogeneous distribution of fibrosis with a decrease in inflammation was evident at 28 d after BM instillation. Lung sections from control rats (at 7 d) showed normal alveolar architecture.
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Uronic Acid Content
The concentration of uronic acid was higher in BM-treated lungs than in controls at all time points studied (Figure 2). The time-course studies indicated that BM-treated lungs contained greater amounts of uronic acid at 14 d than at either 7 or 28 d after BM administration. BM administration caused a 1.6-fold increase in lung uronic acid at 7 d as compared with control lungs. Similarly, BM-treated lungs showed a 2.7-fold increase in uronic acid at 14 d, whereas a 2.0-fold increase was observed at 28 d as compared with control rats. There was no significant difference in the uronic acid content of BM-treated lungs at 14 and 28 d.
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PG Extraction Efficiency
To determine the percentage of extracted PGs, we estimated the uronic acid content in PG extracts (4 M guanidine hydrochloride extraction) and papain-digested lung tissues. From 82 to 88% of PGs were extracted from the lung tissue (Table 1). There was no significant difference between the control and BM groups in the percentage of extracted PGs.
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Composite Gel Analysis of Large PGs
Electrophoresis of guanidine HCl extracts in composite agarose-polyacrylamide gel revealed differences in the intensity of toluidine blue staining of large PGs (Figure 3). The intensity of toluidine blue staining differed in samples from the control and BM groups: an increase in toluidine blue staining was observed at 7 and 14 d in BM treated as compared with control groups. An increase in toluidine blue staining was noticed at 28 d after administration of BM, but the magnitude of the increase was less than at Day 7 and Day 14 after BM administration. Control lungs showed relatively weaker staining of large PGs.
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VS
Large PGs separated on composite gel were probed with the mAb 12C5 to investigate the expression of VS during the development of BM-induced pulmonary fibrosis (Figure 4). Immunoblot analysis revealed two major bands staining for VS. The data showed marked differences in the expression of VS in the control and BM groups. The intensity of the bands in samples from fibrotic lung were significantly greater than in samples from control lungs, although equal amounts of tissue extracts were applied to each lane of the electrophoresis gel. Quantitative analysis of immunoblots showed a significant increase (i.e., 2.8-fold for control versus Day 7 after BM; 5.5-fold for control versus Day 14 after BM; and 1.9-fold for control versus Day 28 after BM) in the amounts of VS in BM-treated lungs as compared with normal lungs. Results of quantitative analysis also showed that the greatest increase in VS occurred at 14 d after BM. Control lungs had the least staining for VS. PGs extracted from normal rat aorta were used as a positive control.
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HSPGs
Immunochemical analysis of large basement membrane HSPGs was done through Western blot analysis (Figure 5). In the Western blot analysis, strong staining was observed for HSPGs at Days 14 and 28 as compared with Day 7 after BM instillation. Analysis of HSPGs by immunoblotting revealed two closely migrating bands. Quantitative analysis of HSPG expression revealed greater amounts at Day 14 (a 2.9-fold increase) and Day 28 (a 1.9-fold increase) than in controls. In contrast, there was no difference in HSPG expression in controls and BM-treated rats at Day 7. PGs extracted from normal rat kidney served as a positive control.
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SLRPs
SDS-PAGE and subsequent Western blot analysis of lung tissue samples after chondroitinase ABC digestion showed a molecular weight distribution of 43,000 to 46,000 daltons calculated from the electrophoretic mobility of the migrating PGs and compared with molecular weight markers. Our results are in agreement with the findings of others, who have identified the 43-kD band as biglycan (Figure 6) (3) and the 46-kD band as fibromodulin core protein (Figure 7) (13). Expression of these small PGs was also quantitated through densitometric scanning, which demonstrated that the relative content of biglycan and fibromodulin changed markedly with the development of pulmonary fibrosis. An increase in the intensity of these bands was observed at Day 14 after BM administration as compared with Day 7 or Day 28. The expression of both biglycan and fibromodulin was also enhanced at Day 28 as compared with that of controls. However, there was no significant difference between the control and Day 7 BM-treated rats in the content of these SLRPs.
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Immunolocalization of Biglycan and Fibromodulin
Biglycan (Figures 1e and 1f) and fibromodulin (Figures 1g and 1h) were also examined immunocytochemically, to address the spatial distribution of these PGs during the development of BM-induced lung fibrosis. The distribution of biglycan and fibromodulin was more diffuse and extensive in BM-treated rats. A marked amount of immunostaining of biglycan and fibromodulin was observed in areas of lung injury and developing fibrosis of rats at 14 and 28 d after BM administration. Control lungs showed only minimal staining of these PGs around airways and blood vessels.
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DISCUSSION |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Abnormal accumulation of ECM macromolecules is believed to be the underlying mechanism in the development of pulmonary fibrosis in both animals and humans (1). Numerous studies, including those done in our own laboratory, have indicated increased synthesis and accumulation of collagens, elastin, and fibronectin in the lungs of animals with BM-induced pulmonary fibrosis (1, 14). Recent studies have shown alterations in the expression of small PGs in the rat model of lung fibrosis induced by BM (3). However, it is not known whether the pulmonary fibrosis seen in this model is associated with changes in deposition of other PG subclasses. The present study demonstrates that the lung content of the three different PG subclasses changes markedly during the development of BM- induced pulmonary fibrosis in the rat (see Table 2 for summary of results).
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Although earlier studies with humans have reported VS deposition during the repair phase of both granulomatous and nongranulomatous inflammatory processes (6), the temporal expression and changes in the relative levels of VS have not been defined. The results of time-course studies of the expression of VS in the present study indicate that the relative levels of VS were increased at 7 and 14 d in the fibrotic lung as compared with the control lung. These observations confirm that an increase in the synthesis of VS is a characteristic, early feature in fibrotic lungs. Qualitative analysis of large PGs through the use of composite gel electrophoresis (7) and subsequent immunoblotting revealed two major bands (Figures 3 and 4). The striking similarity between the electrophoretic mobility of VS in our studies and the electrophoretic profiles of VS in human fetal skin (15) further suggests the existence of two populations of aggregating PGs in the lung.
VS, the largest member of the PGs encoded by the hyalectan gene family (16), is a large, hyaluronic acid (HA)-binding
chondroitin sulfate PG that is expressed in a variety of tissues
including embryonic lung, aorta, cornea, and skeletal muscle
(17). VS is also present in adult tissues of various organs, including most smooth-muscle cells, the central and peripheral
nervous system (18), and blood vessels (19). VS has been implicated in the development of atherosclerotic lesions (20),
and abnormal deposits of VS have been identified in the
stroma of various tumors, particularly in HA-rich regions (21).
These observations are of particular importance in light of the
putative function of VS vis-a-vis cell proliferation, cell migration, and ECM synthesis by connective-tissue cells. In addition, the unique temporal upregulation of VS revealed in the
present study, and the spatial expression of VS in myofibroblast-rich regions in various fibrotic lung diseases (6), indicate
a mechanistic role for VS in the remodelling observed in pulmonary fibrosis. The high concentration of chondroitin sulfate
side chains in VS, and its formation of large supramolecular
aggregates with HA, would increase the water content of the
ECM during cell remodelling after injury, and might contribute to the formation of the open edematous matrix required
for cell migration and proliferation (2). Enhanced deposition
of VS in the early stages of fibrosis may reflect the activity of
transforming growth factor (TGF)-
and other growth factors in
the wound-healing environment. VS has been shown to be upregulated in human skin fibroblasts treated with TGF- (22). Indeed, TGF- enhances the synthesis of many ECM molecules
and is associated with the development of tissue fibrosis (3,
23). Our results clearly demonstrate that VS was induced during the early inflammatory and remodelling phase of lung injury, and highlight VS as an immediate early-response connective tissue macromolecule in BM-induced lung injury. To our
knowledge, this is the first report characterizing the temporal and quantitative expression of VS during the development of
BM-induced pulmonary fibrosis.
As compared with VS, relatively little is known about basement membrane HSPG in pulmonary fibrosis. The present study shows that basement membrane HSPGS were altered during the development of BM-induced pulmonary fibrosis. The levels of HSPG were increased at Day 14 and Day 28, whereas no significant differences were observed in the earlier stage of BM-induced lung injury. Immunoblot results identified two closely migrating bands, which is not surprising, since HSPGs are a heterogeneous family of macromolecules that occur in multiple forms (2, 24). The biologic significance of the increased deposition of HSPGs is not clear; however, alterations in the basement membrane are likely to influence the processes of both lung injury and repair. HSPGs can bind growth factors, cytokines, cell adhesion molecules, matrix proteins, proteases, and antiproteases (25). HSPGs also have many postulated functions, including effects on cell attachment and spreading, maintenance of cell shape, growth control, anticoagulation, ultrafiltration, and matrix assembly (25). In addition, HSPGs may sequester heparin-binding growth factors, such as fibroblast growth factor (FGF)-2, and potentiate their interaction with high-affinity receptors (26). It has been postulated that FGFs may regulate cell migration, proliferation, and gene expression, and may have a role in many developmental and pathologic processes, including wound healing and angiogenesis (27). Binding of HSPGs to FGFs may protect FGFs from proteolytic degradation (28) and promote endothelial and epithelial regrowth on denuded basal lamina (29). In this respect, it is interesting to note that alveolar type II cells in culture are known to synthesize HSPG (30). Lung HSPGs have been localized to basal laminae, including the alveolar and capillary basal laminae (31). The rat model of pulmonary fibrosis used in our study is a consequence of acute and chronic inflammation caused by BM. Following injury with type I cell loss, type II cells proliferate and spread over the denuded basal lamina (32). Increased production of basement membrane HSPG may therefore be important in the remodelling of basement membrane seen in pulmonary fibrosis (32).
The present study also identified changes in the expression
of the small PGs biglycan and fibromodulin. We observed that
the expression of biglycan was increased maximally at 14 d after administration of BM. The marked upregulation of biglycan in our study is compatible with observations in a previous
study of the expression of biglycan in BM-induced pulmonary
fibrosis (3). However, the previous study did not examine the
spatial expression of biglycan in BM-induced pulmonary fibrosis. In the present study, lung tissues from rats at 14 d after
BM administration revealed intense immunostaining for biglycan in areas of developing fibrosis. Changes in biglycan immunoreactivity were consistent with changes seen in the amount of biglycan in extracted tissue during the same period. Biglycan has also been shown to undergo changes during chronic
hyperoxia in rats, another model of fibrotic injury (33). A member of the family of SLRPs, biglycan is predominantly associated with the pericellular matrix in developing tissues and organs (34). The core protein of biglycan may interact with
several different matrix proteins and influence matrix assembly (35). Biglycan may also interact with fibronectin (36) and
types I (37) and VI collagen (38). In addition, biglycan binds
to TGF- (35), a cytokine implicated in pulmonary fibrosis.
Another observation in our study was the expression of fibromodulin in fibrotic lungs. Quantitation of banding patterns indicated that the expression of fibromodulin was highest at 14 d after BM administration. Immunocytochemical examination showed strong expression of fibromodulin in areas of injured tissue at 14 d after administration of BM, which was consistent with the increased expression of fibromodulin evidenced by Western blot analysis. Fibromodulin mRNA expression has previously been reported to show no difference in control and fibrotic lungs (3); however, actual protein expression was not examined. The discrepancy between report of this finding and our results suggest that regulation of fibromodulin expression may occur through posttranscriptional mechanisms. Fibromodulin belongs to the family of SLRPs whose core protein has attachment sites for keratan sulfate chains (16). Fibromodulin, like decorin, binds to type I and type II collagens and influences both the rate of fibrillogenesis and the structure of resulting fibrils (39). Thus, altered fibromodulin expression would be expected to have consequences for the organization of collagen in pathologic processes such as pulmonary fibrosis.
In conclusion, our results indicate that changes in all subclasses of PGs occur during the development of BM-induced pulmonary fibrosis in rats. The early increase in PGs observed in the present study, which preceded reported changes in collagen levels (40), strongly supports a mechanistic role for PGs in the processes that ultimately lead to pulmonary fibrosis. We can speculate about how the temporal pattern of changes in PGs may result in lung fibrosis. Increased deposition of VS during the early inflammatory response may play a key role in increasing the water content of the ECM, thereby enhancing the migration of cells at sites of inflammation. Because of its ability to bind growth factors and other ECM macromolecules, the increase in HSPG may be relevant to the structural organization of basement membranes in pulmonary fibrosis. Additionally, the later increases in biglycan and fibromodulin would affect collagen fibrillogenesis and the enhanced collagen matrix seen in pulmonary fibrosis.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. M. S. Ludwig, Meakins-Christie Laboratories, McGill University, 3626 St. Urbain Street, Montreal, QC, H2X 2P2 Canada. E-mail: mara{at}meakins.lan.mcgill.ca
(Received in original form September 24, 1999 and in revised form December 7, 1999).
Dr. Ludwig is a research scholar of the Fonds de la Recherche en Santé du Québec.Acknowledgments: The monoclonal antibodies 12C5, developed by Dr. R. Asher, and C17 developed by Dr. J. R. Sanes, were obtained from the Developmental Studies Hybridoma Bank of the University of Iowa Department of Biological Sciences, Iowa City, IA.
Supported by the J. T. Costello Memorial Research Fund and the Medical Research Council of Canada.
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References |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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