SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and Plasticity
Detection of chondroitin sulfates and decorin in developing fetal and neonatal rat lung

Yiqiong Wang, Kaori Sakamoto, Jody Khosla, and Philip L. Sannes

Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chondroitin sulfates and their related proteoglycans are components of extracellular matrix that act as key determinants of growth and differentiation characteristics of developing lungs. Changes in their immunohistochemical distribution during progressive organ maturation were examined with monospecific antibodies to chondroitin sulfate, a nonbasement membrane chondroitin sulfate proteoglycan, and the specific chondroitin sulfate-containing proteoglycan decorin in whole fetuses and lungs from newborn and adult rats. Alveolar and airway extracellular matrix immunostained heavily in the prenatal rat for both chondroitin sulfate and chondroitin sulfate proteoglycan, whereas decorin was confined to developing airways and vessels. These sites retained their respective levels of reactivity with all antibodies through 1-10 days postnatal but thereafter became progressively more diminished and focal in alveolar regions. The heavy staining seen early in development was interpreted to reflect a significant and wide distribution of chondroitin sulfates, chondroitin sulfate proteoglycans, and decorin in rapidly growing tissues, whereas the reduced and more focal reactivity observed at later time points coincided with known focal patterns of localization of fibrillar elements of the extracellular matrix and a more differentiated state.

extracellular matrix; basal laminae; growth factors; mesenchyme


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MAMMALIAN LUNG DEVELOPMENT involves complex relationships between cells and extracellular matrices that result in proliferation and/or differentiation (3, 11, 12). A number of studies have indicated that a variety of growth factors and extracellular matrix (ECM) components contribute to pulmonary tissue growth and morphogenesis (7, 20, 21, 24). The importance of growth factors, such as epidermal growth factor, insulin-like growth factor, acidic fibroblast growth factor (FGF-1), basic fibroblast growth factor (FGF-2), transforming growth factor-alpha (TGF-alpha ), and keratinocyte growth factor (FGF-7), are well established (7, 12-15, 20, 25, 29,), whereas much remains to be learned of the role(s) that individual and collective components of the ECM play in these complex processes (19, 21, 24, 27, 31). The interplay between these multiple influences is predictably both temporal and structural, so, to gain an understanding of their potential importance, it is particularly crucial to accurately define the precise microanatomic distribution of key molecules and the point in development when they appear.

Previous work has demonstrated the localization of laminin, heparan sulfate proteoglycan (HSPG), chondroitin sulfate (CS), basement membrane CSPG (BM-CSPG), and entactin in developing mammalian lung (21). These components of ECMs, especially those of the specialized BM regions, generally appear to promote growth and/or differentiation in concert with a select group of growth factors. CS alone or as part of the macromolecular structure of CSPGs has been shown to have biological properties that appear to influence cell proliferation and differentiation in a number of in vitro settings (30). The CS-containing PG decorin has been shown to bind to TGF-beta (32) and to alter its antiproliferative and fibrogenic effects in the lung (28). Recent studies on isolated type II alveolar epithelial cells suggest that, in conjunction with heparin-binding growth factors, in particular FGF-2, desulfated forms of CS promote thymidine incorporation into DNA, whereas its sulfated forms are inhibitory (22). Similarly, when combined with laminin substrata, desulfated CS significantly enhances DNA incorporation in type II cells treated with FGF-7 compared with laminin alone (23). CS is known to be a structural glycosaminoglycan (GAG) associated with specific PGs of BM and interstitial ECMs (4, 11), which have been immunolocalized in the developing postnatal and adult rat lung (21). CS and related molecules, therefore, have an established presence in postnatal developing (17) and adult lungs (5, 21, 31), but little is known about their microanatomic distributions in prenatal development. We hypothesized that the expression of CS-containing molecules in ECM would change at specific time points during the course of pre- and postnatal lung development. Accordingly, we extended and compared previous studies on postnatal and adult lung development to prenatal lung development using the same antibody probe to CS and antibodies to a non-BM-CSPG and the CS-containing PG decorin. Such information could be critical for determining the potential role(s) of these components in regional and site-specific maturation and differentiation of the normal and/or the pathogenesis states associated with altered growth and/or differentiation of the lung.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparations. All animal protocols were approved by the institutional animal care and use committee. Pregnant female adult, virus antibody-free rats (timed pregnancies; Fisher 344, Charles River Breeding Laboratories, Wilmington, MA) were killed by intraperitoneal injection with pentobarbital sodium, and fetuses (14, 16, 18, and 20 days of gestation) were surgically removed and immediately killed by decapitation. Newborn pups (days 0, 1, 5, 7, 10, and 25 postnatal) and nonpregnant adult rats were killed by intraperitoneal injection of pentobarbital sodium, and lungs were removed and fixed by insufflation with 4% paraformaldehyde overnight. The fetuses were sequentially cross-sectioned from shoulder to abdomen in 2- to 3-mm-thick segments. These thick segments or isolated lungs from postnatal and adult rats were dehydrated and processed routinely for paraffin embedding. Tissue blocks were embedded in paraffin, and 5-µm sections were cut and mounted on chrome alum-coated glass slides and stored at room temperature.

Immunostaining preparations. Monoclonal anti-CS (CS-56) in the form of mouse ascites fluid was purchased from Miles Immunobiologicals (Elkhardt, IN) or Sigma (St. Louis, MO). Its specificity has been established as CS portion of CSPG in previous studies by Avnur and Geiger (1); it reacts with CS A and C but not B (dermatan sulfate). Optimal dilutions were established for our purposes at 1:50. Polyclonal rabbit antiserum to a non-BM-CSPG, which recognizes the antigenic determinants present on the sulfate GlcU-GalNAc disaccharide unmasked by chondroitinase ABC digestion and which does not react with any core protein, was purchased from Chemicon (Temecula, CA). Optimal dilutions for our preparations were determined to be 1:3,000. A polyclonal rabbit antiserum to synthetic mouse decorin (LF-113) was obtained from Dr. Larry W. Fisher (6) and used at a dilution of 1:500.

Digestion protocols entailed incubating deparaffinized tissue sections with chondroitinase ABC (Proteus vulgaris, Sigma) at 0.5 µl/ml in 0.05 M Tris · HCl, pH 8.0, with 0.05 M NaCl for 30 (decorin) or 90 (CSPG) min at 37°C.

Biotinylated secondary antibodies and peroxidase-labeled avidin-biotin complexes were purchased from Vector Laboratories (Burlingame, CA) and used in accordance with the specifications of the manufacturer.

Immunostaining procedures. Sections were deparaffinized to alcohol and endogenous peroxidase quenched with 0.1% hydrogen peroxide in methanol (30 min). Some sections were digested with 50 mg/ml testicular hyaluronidase (type I-S, 340 U/mg, Sigma) in 0.05 M Tris buffer, pH 7.6, for 30 min at 37°C or chondroitinase ABC as described above. Sections were then covered with nonimmune serum from either goat (for polyclonal procedures) or horse (for monoclonal procedures) for 30 min at room temperature, followed by the primary antiserum or ascites fluid without intervening wash. The diluent used was a commercially available phosphate-buffered saline (PBS), pH 7.4 (Sigma), containing 1% bovine serum albumin (Sigma). After dilution, incubations in primary antisera or ascites fluid were for 2 h at room temperature in a humidified chamber. After being washed with PBS buffer, sections were incubated for 30 min in biotinylated secondary antisera directed against the host species of the primary antibodies or antisera according to the directions of the supplier (Vector Laboratories). These preparations were adsorbed against rat serum to reduce nonspecific background reactivity. After being washed with buffer, sections were incubated with a commercially obtained (Vector Laboratories) avidin-biotin peroxidase complex (ABC-P) for 30 min to 45 min according to the manufacturer's specifications. To visualize the ABC-P, sections were treated for 5-10 min with a solution containing 0.3 mg/ml diaminobenzidine HCl and 0.1% hydrogen peroxide in 0.05 M Tris · HCl buffer, pH 7.6, at room temperature. Wet sections were evaluated to determine sensitivity of the detection and then in some cases counterstained with hematoxylin and mounted in Permount (after appropriate dehydration).

Histochemical controls consisted of substitution of normal rabbit serum for the primary antisera in polyclonal procedures and nonimmune mouse ascites fluid in monoclonal procedures. All other steps in the procedures were identical to those detailed above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The immunoperoxidase reaction product indicative of the localization of CS, CSPG, and decorin was discrete, dense, and almost exclusively extracellular in fixed sections of rat fetal, postnatal, and adult rat lungs. Reactive sites were fibrillar in nature and appeared interconnected, forming a continuous network that interlaced all the extracellular volume between the cells of the epithelium and interstitium. In spite of staining vastly different epitopes, the reactive sites for CS and CSPG were nearly identical (except where indicated). The major difference was the intensity of staining, which tended to be greater with CSPG. Accordingly, to maximize the detail of the overall developmental sequence, we do not present duplicates of individual time points for CS/CSPG localization, but they are used for comparison with decorin localization.

In prenatal day 14 and 16 rats, immunoreactivity for both CS and CSPG filled the ECM regions of what are known to be rapidly developing airways (Fig. 1A), whereas decorin was not detected. This changed at prenatal day 16 when the latter was detected around developing airways and vessels (Fig. 1B). At prenatal day 20, CSPG and CS immunoreaction product was well defined in all regions containing ECM (Fig. 1, C and D). Anti-CSPG was somewhat more reactive than anti-CS in identical sections at this time point. Airways and vessels typically had multiple layers of dense reactivity. At this same time point, decorin remained confined to areas of ECM around major developing airways and vessels (Fig. 1E). Of note was the lack of cartilage reactivity for CS, although it was highly reactive with CSPG (data not shown). All other reactive sites were essentially identical for CS and CSPG at all time points assessed, with the latter being distinctly stronger. Epithelial cells were consistently nonreactive at all prenatal time points (Fig. 1, A-E), whereas mesenchymal/interstial cells were not readily distinguishable.


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Fig. 1.   A: 14-day prenatal rat lung immunostained with mouse anti-chondroitin sulfate proteoglycan (CSPG). Rudiments of bronchi (b) are surrounded by heavily stained extracellular matrix (ECM). No counterstain. Bar = 150 µm. B: 16-day prenatal rat lung immunostained with rabbit anti-decorin. Distinct but focal staining surround rudiments of central bronchi (b) and vessels; remainder of lung parenchyma is unstained. No counterstain. C: 20-day prenatal rat lung immunostained with rabbit anti-CSPG. Developing airway (b) and vessels and numerous alveolar structures are surrounded by distinctly stained ECM. No counterstain. D: 20-day prenatal rat lung immunostained with mouse anti-CS. Developing airways (b), vessels, and numerous alveolar structures are surrounded by heavily stained ECM. No counterstain. E: 20-day prenatal rat lung immunostained with rabbit anti-decorin. Developing airways (b) and vessels are all well-stained ECM, whereas the surrounding parenchyma lacks reactivity. v, Vessels. Bar in B-E = 300 µm.

At postnatal day 1, the ECM of bronchiolar structures, their associated vessels, and alveolar regions were highly reactive for CS (Fig. 2A) and CSPG, whereas decorin was unchanged (Fig. 2B). At postnatal day 5, the dense immunoreactivity for CS (Fig. 2C) and CSPG was evident in all ECM regions of airways and alveolar structures. In alveolar regions, the reactivity of the ECM appeared more punctate and discontinuous in the CS-stained preparations compared with the CSPG-stained. Decorin staining remained confined to larger airway and vessel walls, although alveolar regions demonstrated faint reactivity in developing septae (Fig. 2D). The discontinuous nature of the reaction product for CS and CSPG in alveolar ECM was increasingly evident at 7 days postnatal (Fig. 2E). These punctate deposits of immunoreactivity were especially apparent in developing septae (Fig. 2E), which were beginning to be distinguishable in the decorin-stained lungs. Postnatal day 10 lungs were essentially identical to day 7 for all three procedures (data not shown).


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Fig. 2.   A: 1-day postnatal rat lung immunostained with mouse anti-CS. A small airway and vessel and surrounding alveolar parenchyma show a regular, dense pattern of staining of the ECM. Bar = 150 µm. B: 1-day postnatal rat lung immunostained with rabbit anti-decorin. Positively stained ECM surrounds a bronchiole (b) and its accompanying arterial vessel but not the adjacent parenchyma. Bar = 300 µm. C: 5-day postnatal rat lung immunostained with mouse anti-CS. The ECM of small vessels developing alveolar structures near an alveolar duct strain heavily. Bar = 150 µm. D: 5-day postnatal rat lung immunostained with rabbit anti-decorin. Small airway and vessel have distinct staining, whereas the surrounding parenchyma is only lightly stained. Bar = 300 µm. E: 7-day postnatal rat lung immunostained for the localization of CS showing distinct, punctate staining of parenchymal ECM. a, Small airway; ad, alveolar duct. Bar = 150 µm.

Day 25 and adult lungs, however, were noticeably different in their immunoreactivity for CS, CSPG, and decorin, compared with the early stages detailed above. Interestingly, although overall reactivity for alveolar regions was dramatically reduced for CS and CSPG (Fig. 3, A and B), strong decorin staining (Fig. 3C) was now evident in sites that also stained for CS and CSPG. The other common reactive sites associated with the ECM of bronchi, bronchioles, and vessels remained unchanged. The reduction in alveolar staining was especially true for CS, which was detectable only in small fibrillar wisps of reactivity embedded in the thin subepithelial ECM (Fig. 3A). Airway reactivity by comparison was much greater than alveolar regions but diminished compared with earlier stages of development. Vessels in the adult were nearly uniformly unreactive for CS, with the exception of some smaller arteries in close association with bronchioles (Fig. 3A). Veins were unreactive. This was less true of CSPG reactivity, which was associated with all vessels and was comparatively heavier. This difference, not obvious in specimens from earlier stages of development, was not a result of incubation times or dilutions, which were kept constant throughout the study. Notably, the BM of the alveolar regions in the 25-day-old and adult rats in particular was devoid of reaction product. This was not true of the airways where the BM was easily distinguishable with anti-CS, whereas with anti-CSPG, it essentially blended into the underlying ECM. Decorin staining was very discrete and was isolated to the ECM of alveolar septae (Fig. 3C) and small vessels associated with airways.


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Fig. 3.   A: adult rat lung immunostained for the localization of CS. Reactive ECM (arrows) surrounds an alveolar duct. Bar = 150 µm. B: adult rat lung immunostained for the localization of CSPG. Reactivity is strongest around alveolar ducts, small vessels, whereas the remaining parenchymal ECM is only lightly reactive. Bar = 300 µm. C: adult rat lung immunostained for the localization of decorin. ECMs surrounding alveolar ducts are distinctly stained, whereas the remaining parenchyma is not reactive. Bar = 150 µm. D: 7-day postnatal rat lung treated with normal rabbit serum followed by biotinylated secondary antibody and standard ABC sequences as detailed in MATERIALS AND METHODS. No visible contrast is evident. Bar = 300 µm. E: adult rat lung digested with hyaluronidase then immunostained for the localization of CSPG shows no contrast density. Bar = 300 µm. F: adult rat lung digested with chondroitinase AC and then immunostained for the localization of CS also shows minimal contrast density. Bar = 300 µm.

All control sections in which normal ascites fluid (1:400) or normal rabbit serum (1:2,000) was substituted for the primary antiserum had no immunoreactivity (Fig. 3D). Hyaluronidase digestion before immunostaining removed all reactive sites for CS and CSPG (Fig. 3E), whereas chondroitinase AC digestion before immunostaining removed most reactive sites for CS (Fig. 3F). Chondroitinase AC digestion before immunostaining prevented most specific immunostaining for CSPG (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented demonstrate minor differences in immunolocalization patterns between the monoclonal anti-CS and the polyclonal anti-CSPG preparations in pre- and postnatal developing rat lungs, which in turn differ significantly from that seen with the specific CS-containing PG decorin. Although the antibodies to the former recognize totally different epitopes of CS (see MATERIALS AND METHODS), they appeared to localize the same molecule or molecular complex. The fact that anti-CSPG was much stronger in adult animals than anti-CS may relate to the relative abundance of the staining epitope present on mature CS molecules in reactive sites. It is significant, however, that neither reacts with the core protein of any PG, such as BM-CSPG, which is also found in the lung (21). This contrasted dramatically with the non-BM CS-containing PG decorin, which was consistently found associated with developing airways and vessels but was entirely lacking in developing alveolar regions. Although the anti-CS and -CSPG observed reacted with ECMs in the pre- and postnatal periods, it was not restricted to BM regions as described for the BM-CSPG (21). Reactive sites for CS and CSPG progressively diminished from postnatal day 7 on to adulthood, where they were principally confined to ECMs of large and small airways, arteries, and alveolar septae. The notable exceptions were pulmonary vessels, which were intensely reactive with CSPG and not reactive for CS (except for arterioles closely associated with bronchioles). Decorin shared localization sites in developing large airways and vessels with CS and CSPG, but unlike the latter, it was not observed in alveolar regions until day 5, when it was observed in focal, discrete sites of presumed septal development. In the mature, adult lungs, it was more widely distributed. The consistently strong reactivity in larger vessels and airways remained the same as in earlier times in development, whereas distinct reactivity was clearly evident around distal small airways, their accompanying vessels, and the alveolar septae of alveolar ducts. Nearly all CS-, CSPG-, and decorin-reactive sites in the adult seemed to coincide with regions known to contain elastic fibers and collagen (10), which are known to contain and be associated with sulfated GAGs and their respective PGs (18, 28). The immunolocalization of CS/CSPG(s) and decorin in neonatal, postnatal, and adult rat lungs significantly extends the knowledge established by previous reports on CSPGs in mammalian lungs. For example, the dense immunoreactivity observed here gives the impression of a presence of CS/CSPG that vastly exceeds the cuprolinic blue-reactive sites that correlate with a large CSPG described by Rutten et al. (17) in the developing mouse lung and van Kuppevelt et al. (31) in adult human lung parenchyma. This may in part reflect the differences in technical approaches utilized: immunostaining of specific antigenic determinants on CS molecules as opposed to charged anionic end groups on the same molecules. Nevertheless, the sparse, irregular, and punctate pattern of immunoreactivity seen in adult rat with anti-CS/CSPG and decorin in the centriacinar and alveolar regions was somewhat similar to the "striking irregular presence" of CSPGs described with cuprolinic blue in human lung pulmonary parenchyma (31). It is possible, if not likely, that immunoreactive sites represent a variety of different CS/CSPGs and that only a portion of these sites is accounted for by the cuprolinic blue staining, which van Kuppevelt et al. (31) indicated was at its highest level at day 2 of postnatal development. It is worthy of note that similar to the immunoreactive sites for CS/CSPG, a progressive reduction in cuprolinic blue reactivity was reported with advancing age (17). Radhakrishnamurthy et al. (15) showed that although the total GAG content of developing rabbit lungs decreased with advancing age, the percentage of CS remained high (40-50%) compared with other GAGs. This differs from temporal changes in GAG biosynthesis by isolated fibroblasts from developing fetal rat lungs, which were at their highest levels at 18 days gestation and declined to day 21 (3). They found HSPG to predominate, with CSPG levels highest at day 18 in cells peripheral to epithelial surfaces (those adjacent expressed a different GAG profile). These collective observations portend several conclusions: 1) CS/CSPG levels and distribution may differ significantly between species; 2) CS/CSPG levels are higher in prenatal development and rapidly diminish from postnatal development to adulthood.

The functional significance of specific or "group" CS/CSPGs in the developing lung is intriguing, given the amount present and where they are found. Their high levels relative to other GAGs and their microanatomic location suggest potential roles in a variety of developmental processes. For example, a CSPG has been found to be concentrated in regions where second-order branching is known to occur (2). Interestingly, inhibition of GAG synthesis has been shown to impair epithelial differentiation and branching morphogenesis in embryonic lung rudiments in vitro (27). A variety of ECMs have been shown to be involved in this important process (13, 25, 29, 34), so it may be unlikely that any one of these molecules has exclusive control. The wide distribution and degree of immunoreactivity of CS/CSPGs in the present report are likely to reflect a variety of PG forms. The number of functions ascribed to these CS/CSPGs are also considerable, from inhibiting proliferation and promoting differentiation (30) to interacting with and/or binding to specific soluble growth factors (16, 22). Decorin has been shown to bind to TGF-beta extracellularly and to reduce its capacity to influence cell proliferation and ECM biosynthesis (28), and recently its transient transgene expression has been used to effectively block bleomycin-induced fibrosis (8). Alveolar epithelial cells are know to biosynthesize CS-containing PGs (9, 26), where they have been linked with inhibition of heparin-binding growth factor-stimulated DNA (22). These complex relationships with growth factors, both promotional and inhibitory, may constitute important mechanisms operative in fetal/postnatal lung development. Their variety, quantity, and location clearly raise some compelling possibilities about how ECMs and growth factors modulate and modify the maturation in the mammalian lung.


    ACKNOWLEDGEMENTS

The authors gratefully acknowledge Dr. Larry W. Fisher, the National Institute of Dental Research, for the gift of rabbit anti-decorin and Wendy Savage and Doug Wagner of Biomedical Communications, North Carolina State University for photographic support.


    FOOTNOTES

This study was supported by National Heart, Lung, and Blood Institute Public Health Service Grant HL-44497 and a grant from the state of North Carolina.

Address for reprint requests and other correspondence: P. L. Sannes, Dept. of Anatomy, Physiol. Sciences, & Radiology, Coll. of Veterinary Medicine, North Carolina St. Univ., 4700 Hillsborough St., Raleigh, NC 27606 (E-mail: philip_sannes{at}ncsu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00160.2001

Received 8 May 2001; accepted in final form 25 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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