ARTICLE |
Correspondence to: Riitta KaarteenahoWiik, Dept. of Internal Medicine, PO Box 5000 (Kajaanintie 50), FIN-90014 Oulun Yliopisto, Finland.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tenascin-C is an extracellular matrix (ECM) glycoprotein expressed in human tissues during organogenesis and in fibrotic and neoplastic processes. We hypothesized that its expression would increase in human lung in neonatal disorders such as infant respiratory distress syndrome (RDS) and bronchopulmonary dysplasia (BPD). Tenascin-C expression was studied by immunohistochemistry (IHC) and mRNA in situ hybridization (ISH). The extent of tenascin-C immunoreactivity was scored as absent (0), low (+), moderate (++), strong (+++), or very strong (++++) separately in different types of pulmonary cells in controls (seven cases), RDS (19 cases), and BPD (12 cases). In controls, tenascin-C expression was low (+) underneath alveolar and bronchiolar epithelium, moderate (++) in intima of veins, and strong (+++) around chondrocytes. In RDS, tenascin-C expression was moderate (++) or strong (+++) underneath both bronchiolar and often detached alveolar epithelium underlying hyaline membranes in the walls of dilated alveoli. In particular, the patients with RDS who survived for 1 day or more had strong expression of tenascin-C within alveolar walls. In patients with BPD, tenascin-C was very strongly (++++) expressed in the remodeled fibrotic alveolar walls underneath regenerative epithelium. Increased expression of tenascin-C mRNA was seen below the alveolar and bronchiolar epithelia in RDS and BPD. The cells in these locations showed -smooth muscle actin immunoreactivity, suggesting a myofibroblast phenotype. In conclusion, tenascin-C is highly expressed in the walls of alveoli and bronchioli in RDS and BPD, suggesting an association between the expression of this protein and the presence of these disorders. (J Histochem Cytochem 50:423431, 2002)
Key Words: extracellular matrix, hyaline membrane disease
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN 1967, Northway and co-workers described bronchopulmonary dysplasia (BPD) in pre-term infants who developed a chronic lung disease (CLD) after having received assisted ventilation for hyaline membrane disease, i.e., respiratory distress syndrome (RDS) (
Interactions between epithelial and mesenchymal cells play an important role both in lung development and in fibrotic disorders of the lung. It is now evident that cytokines control these processes. Transforming growth factor-ß (TGF-ß) is a cytokine that controls the expression of several extracellular matrix (ECM) glycoproteins. It is increased at the site of lung injury in patients with pulmonary fibrosis and BPD (
The aim of the present study was to investigate the distribution of tenascin-C in RDS and BPD and to compare the expression of tenascin-C in these disorders to control cases of the same gestational ages. Most clinical definitions consider 28 days of age as the beginning of BPD. However, early pathological changes of BPD can be recognized within a few days after birth (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients and Handling of Specimens
Histopathologically typical cases of RDS and BPD were retrieved from the files of the Department of Pathology, Oulu University Hospital. The study protocol was approved by the Ethical Committee of the Medical Faculty of the University of Oulu. The patients were 38 autopsied babies who had died at Oulu University Hospital between 1987 and 1999. Autopsies had been performed within 3 days. A total of 19 patients developed RDS requiring mechanical ventilation after birth, and 12 patients developed BPD after having RDS. The controls (seven cases) included in the study were newborn infants (2328 weeks of gestational age) who had died within 1 hr after delivery for different reasons without lung disorders. The study group consisted of 19 cases with RDS, i.e., hyaline membrane disease, and 12 cases of BPD with typical morphological findings. BPD was classified on morphological grounds into four stages according to Rosan: stage I, acute (24 days); stage II, regenerative (48 days); stage III, transitional (86 days); and stage IV, chronic (after 16 days) (
|
|
Lung tissues from either right or left lung removed at autopsy were fixed in 10% formalin, then dehydrated and embedded in paraffin. Sections of 4 µm were stained with hematoxylineosin. The entire material was re-evaluated and one representative tissue block from each case was selected for immunohistochemical (IHC) studies in the entire study material. In 12 cases (four cases with RDS, four cases with BPD, and four control cases) one tissue block was selected for tenascin-C mRNA in situ hybridization (ISH). In these cases the autopsy had been performed within 1 day after death. Some mRNAs have been shown to be stable within a 24-hr period under postmortem conditions enabling application of ISH techniques to postmortem material (
Anti-tenascin-C Antibody and IHC Staining
A monoclonal antibody (MAb) 143DB7, known to react with the two major isoforms of tenascin-C, was used. The Mab 143DB7 was developed to detect tenascin-C in formaldehyde-fixed tissue and has been characterized in detail elsewhere (
Sections 4 µm thick were deparaffinized in xylene and rehydrated in graded ethanol. Endogenous peroxidase was consumed by incubating the sections in 0.1% hydrogen peroxide in absolute methanol for 20 min. Before immunostaining, the sections were treated with 0.4% pepsin (Merck; Darmstadt, Germany) at 37C for 30 min. For the immunostaining, the avidinbiotinperoxidase complex method was used as previously described (
MAb 143DB7 at a dilution of 1:1000 of the hybridoma supernatant was used as the primary antibody. The sections were incubated with the primary antibody at 4C overnight, followed by a biotinylated rabbit anti-mouse secondary antibody (at a dilution of 1:300 for 30 min) and the avidinbiotinperoxidase complex (both from Dakopatts; Glostrup, Denmark). The color was developed with diaminobenzidine. Sections were counterstained with a light hematoxylin stain and mounted with Eukitt (Kindler; Freiburg, Germany). The negative control consisted of substituting the primary antibody with PBS (pH 7.2) or serum isotype control (Zymed Laboratories; San Francisco, CA).
To identify the phenotype of the tenascin-C-expressing cells, the sections were stained with a commercially available antibody against -smooth muscle actin (Clone 1A4 from Sigma BioSciences; St Louis, MO). The extent of
-smooth muscle actin immunoreactivity was scored as absent (0), low (+), moderate (++), strong (+++), or very strong (++++) in different types of lung cells.
Preparation of Tissue Sections for ISH
Four-µm-thick sections from paraffin-embedded lung biopsies were collected on clean Superfrost Plus glass slides (Erie; Portsmouth, NH), paraffin was removed by xylene, and tissues rehydrated through a graded ethanol series. After three immersions in PBS, pH 7.2, the sections were treated in 0.2 M HCl for 20 min, twice in PBS for 3 min each, followed by proteinase K (100 µg/ml in PBS) treatment for 15 min at 37C. Then tissue sections were transferred into 0.025 M glycine for 30 sec. Tissues were postfixed in 4% paraformaldehyde (Fluka; Buchs, Switzerland) in PBS, pH 7.2 (all solutions made with 0.1% diethyl pyrocarbonate-treated water). After postfixation, tissue sections were transferred into 0.025 M glycine in PBS for 3 min and then acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) for 10 min (
Preparation of RNA Probes
A cDNA fragment (bases 8141316) of the full-length human tenascin-C cDNA (
The 503-bp PCR product containing a specific sequence for tenascin-C was subcloned into a TA vector (TA Cloning Kit; Invitrogen, Carlsbad, CA). Sense and antisense RNA probes were generated from a linearized template by using a riboprobe transcription kit (Promega; Madison, WI) and the probes were labeled with [35S]-UTP (Amersham; Little Chalfont, UK). The radioactively labeled RNA probes were purified by centrifugation through Bio-Gel P-30 columns (Bio-Spin 30; Bio-Rad, Richmond, CA). Each in vitro transcription reaction yielded RNA probes of high specific activity (typically 4.56 x 108 dpm/1 µg DNA template and 5070% incorporation).
In Situ Hybridization
The hybridization mixture contained the [35S]-labeled RNA probe (1.2 x 105 dpm/µl), 50% deionized formamide (Gibco BRL; Rockville, MD), 5 mM dithiothreitol, 500 µg/ml yeast tRNA (Gibco), 2 mg/ml bovine serum albumin (Gibco), and 4 x SSC. The samples were hybridized overnight at 50C while covered with Parafilm (
To evaluate the specificity of the 35S-labeled antisense tenascin-C probes to the tissue sections of lung specimens, control experiments were performed using 35S-labeled sense tenascin-C probes separately for each sample.
The hybridized tissue sections of lung samples were examined by light microscopy and the number of grains over the cells evaluated in general and especially at the locations where tenascin-C immunoreactivity was seen. Cells or cell groups hybridized with the 35S-labeled antisense tenascin probe were considered positive if they contained more grains than corresponding cells and tissue areas that had been hybridized with the 35S-labeled sense tenascin-C probe.
Scoring of Tenascin-C Immunoreactivity and Statistical Analysis
The extent of tenascin-C immunoreactivity was scored as absent (0), low (+), moderate (++), strong (+++), or very strong (++++) separately in and around different types of pulmonary cells in controls and study groups. In each case, the entire section of lung tissue (roughly 12 cm2) was evaluated. Two investigators (RKW and PP) performed the analysis of tenascin-C immunoreactivity independently. The inter-observer repeatability was excellent (=0.60) as measured using the Cohen's kappa statistics as described earlier (
The significance of the associations was determined using Fisher's exact probability test designed for small sample groups.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Controls
Tenascin-C immunoreactivity, scored as low (+) in all seven cases, was observed as thin and linear fibers underneath the basement membranes of the cuboidal alveolar epithelium consisting evidently of Type II prepneumocytes (Fig 1A). Low immunoreactivity (+) for tenascin-C was also detected underneath the bronchiolar epithelium and among smooth muscle cells in the walls of bronchi in 7/7 cases. Low (+) immunoreactivity was shown in the interstitium in all cases. In veins, the immunoreactivity for tenascin-C was moderate (++) in the intima, i.e., beneath the endothelial cells, in 7/7 cases. In contrast, no immunoreactivity for tenascin-C (0) was observed in the endothelial cells and intima of arteries. Media of veins was negative in all cases. Occasionally, in some small arteries a faint immunoreactivity for tenascin-C was shown in media. Strong immunoreactivity for tenascin-C (+++) was detected in and around chondrocytes in the walls of the bronchi in every control case. No immunoreactivity (0) was observed in mesothelial cells or submesothelial connective tissue of the pleura (Table 3).
|
|
RDS
In alveolar walls, the immunoreactivity for tenascin-C was usually strong (+++ in 14/19 cases) underneath the detached alveolar epithelium underlying the hyaline membranes, which were mainly negative for tenascin-C. The expression within alveolar walls was scored as moderate in five cases that had survived less than 1 day (++; case numbers 1, 3, 6, 9, and 10 in Table 2). The tenascin-C-immunopositive fibers were both linear and reticular (Fig 1C and Fig 1D). Low (+, 12/19 cases) or moderate (++, 7/19 cases) tenascin-C immunoreactivity was seen in the cells of interstitium. Also underneath the bronchiolar epithelium, the expression of tenascin-C was moderate (++, 5/19 cases) or strong (+++, 14/19 cases), while the expression among the smooth muscle cells of the bronchi was moderate (++) in all cases. Strong immunoreactivity for tenascin-C (+++) was detected in and around chondrocytes in 19/19 cases. In the intima and the endothelial cells of the veins, the expression of tenascin-C varied from low to moderate (+, 3/19 cases; ++, 16/19 cases, respectively), whereas the endothelial cells of the arteries were all negative (0). No immunoreactivity (0) was observed in the mesothelial cells or submesothelial connective tissue of the pleura (see Table 3).
BPD
A very strong and wide IHC expression for tenascin-C (++++) was observed in the fibrotic interstitium of the remodeled alveolar walls underneath the regenerative epithelium in every case (Fig 1E and Fig 1F), including the patients who had survived only 5 days (see Table 2). Also under the bronchiolar epithelium, the immunoreactivity for tenascin-C varied from strong (+++, 2/12 cases) to very strong (++++, 10/12 cases), while the expression among the smooth muscle cells of the bronchi was moderate (++, in 6/12 cases) or strong (+++, in 6/12 cases). As in controls and the RDS group, tenascin-C expression in and around chondrocytes was strong (+++) in every case. In the endothelial cells and intima of the veins, the tenascin-C immunoreactivity was low (+, 11/12 cases) or moderate (++, 1/12 cases), whereas in the endothelial cells of the arteries it was absent (0). No immunoreactivity for tenascin-C was observed in the pleura or mesothelium. For a summary of the results see Table 3.
mRNA ISH Findings
The number of mRNA grains was diffusely increased in all 12 cases in tissue hybridized with 35S-labeled antisense tenascin-C RNA probe compared to those hybridized with 35S-labeled sense RNA probe. In controls, the number of cells expressing tenascin-C mRNA varied from 20 to 200 underneath the alveolar epithelium and from 30 to 150 underneath the bronchiolar epithelium in one section of lung tissue (roughly 1 cm2). The corresponding numbers of tenascin-C mRNA-expressing cells below the alveolar and the bronchiolar epithelium were over 500 in both RDS and BPD (Fig 2A2D) (see Table 4). IHC staining demonstrated that the cells in these locations were positive for -smooth muscle actin, suggesting a myofibroblast phenotype. The number of tenascin mRNA grains was increased in the endothelial cells of veins and chondrocytes, but the number of positive cells in these locations did not vary in controls and diseased lung. In general, the expression of tenascin-C mRNA by ISH studies corresponded well with the findings of the IHC studies.
|
|
IHC Findings for -Smooth Muscle Actin
In controls, IHC staining for -smooth muscle actin showed intracellular positivity in the single row of cells below both the alveolar (Fig 1B) and the bronchiolar epithelium scored as low (+). The positive cells within the alveolar walls were localized beneath the basement membranes of the alveolar epithelium, and these cells corresponded to obviously myofibroblast-type cells.
-Smooth actin positivity is a typical phenotype for lung myofibroblasts (
-smooth muscle actin was observed also in the media of arteries, scored as very strong (++++), and in the cells underneath the endothelia of veins (++). Very strong expression for
-smooth muscle actin in the media of arteries is caused by the abundant smooth muscle cells. A few interstitial cells were faintly positive for
-smooth muscle actin, whereas the submesothelial connective tissue cells of the pleura were negative.
The IHC reactivity for -smooth muscle actin was stronger in the spindle-shaped cells underlying the alveolar epithelium in patients with RDS than in controls, and also in cells below the bronchiolar epithelium (scored as moderate ++ or strong +++) and in the interstitium. In patients with BPD, the cells expressing
-smooth muscle actin were highly increased in number underneath both the alveolar (Fig 1G) and the bronchiolar epithelium (scored as strong +++ or very strong ++++) (Table 5). Bundles of
-smooth muscle actin-positive cells, which were obviously myofibroblasts, accumulated especially in the areas of newly formed interstitial fibrosis (Fig 1G). Some positively stained cells were also detected in pleural submesothelial connective tissue. The findings in the arteries and veins were similar in control and disease groups, i.e., patients with RDS and BPD.
|
Statistical Analysis
In comparing the control group (n=7) to the group of the patients with RDS and BPD (n=31), the latter group showed significantly more strong or very strong tenascin-C immunoreactivity below alveolar epithelium (p<0.00001 by Fishers' exact probability test; Table 6). In comparing the cases with RDS and BPD that had survived less than 1 day (n=11) to the cases that survived 1 day or longer (n=20), the latter group showed significantly more often strong or very strong IHC expression for tenascin-C underneath alveolar epithelium than the previous group (p=0.002 by Fishers' exact probability test; Table 7).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The role of various ECM glycoproteins, such as fibronectin and collagen, has been widely studied in both RDS and BPD (
In control cases with a gestational age of 2328 weeks, tenascin-C was localized in alveolar walls, whereas in adult normal alveolar interstitium its expression is absent (-smooth muscle-positive cells, which were apparently myofibroblasts, greatly increases in the newly formed fibrosis of BPD, corresponding to the same localization as the increased expression of tenascin-C protein and mRNA. Moreover, myofibroblasts have been shown to increase in number and to form bundles of cells encircling terminal air spaces some days after lung injury in neonates (
The IHC expression for tenascin-C within alveolar and bronchiolar walls was stronger in patients with RDS and BPD than in controls. In comparing the cases with RDS and BPD, the scores of tenascin-C were higher in BPD. This is understandable because the fibrotic process is more advanced in BPD than in RDS. By IHC, however, the scores of RDS and BPD showed no difference, which may indicate that the increased expression of tenascin-C mRNA begins rapidly after the injury. In adult pulmonary fibrotic disorders, tenascin-C has been shown to increase, especially in active, newly formed fibrosis (
Tenascin-C has been shown to be elevated in epithelial lining fluid (ELF) and in serum of adult patients with pulmonary fibrotic disorders (
In conclusion, we observed that tenascin-C is highly expressed within alveolar walls underneath detached alveolar epithelium underlying hyaline membranes in patients with RDS who survive for 1 day or more. It was very strongly expressed in remodeled fibrotic alveolar walls underneath regenerative alveolar epithelium in every patient with BPD, of whom some had survived only for 5 days. This suggests that there is an association between the expression of tenascin-C and the presence of these disorders and, moreover, that its expression increases rapidly after injury.
![]() |
Acknowledgments |
---|
Supported by the Finnish Anti-Tuberculosis Association Foundation and the Paulo Foundation.
The technical assistance of Ms Mirja Vahera, Ms Erja Tomperi, Ms Annikki Huhtela, Ms Heli Auno, Ms Riitta Vuento, Mr Hannu Wäänänen, and Mr Tapio Leinonen is gratefully acknowledged.
Received for publication July 3, 2001; accepted October 11, 2001.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson RW, Stricland MB, Tsai SH, Haglin JJ (1973) Light microscopic and ultrastructural study of the adverse effects of oxygen therapy on the neonate lung. Am J Pathol 73:327-331[Medline]
Boudreau N, Werb Z, Bissell MJ (1996) Supression of apoptosis by basement membrane requires three-dimensional tissue organization and withdrawal from the cell cycle. Proc Natl Acad Sci USA 93:3509-3513
Broekelmann TJ, Limper AH, Colby TV, McDonald JA (1991) Transforming growth factor ß1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc Natl Acad Sci USA 88:6642-6646[Abstract]
Cherukupalli K, Larson JE, Rotschild A, Thurlbeck WM (1996) Biochemical, clinical, and morphologic studies on lungs of infants with bronchopulmonary dysplasia. Pediatr Pulmonol 22:215-229[Medline]
ChiquetEhrismann R (1991) Anti-adhesive molecules of extracellular matrix. Curr Opin Cell Biol 3:800-804[Medline]
ChiquetEhrismann R (1995) Tenascins, a growing family of extracellular matrix proteins. Experientia 51:853-862[Medline]
Delemos RA, Coalson JJ (1992) The contribution of experimental models to our understanding of the pathogenesis and treatment of bronchopulmonary dysplasia. Clin Perinatol 19:521-539[Medline]
Erickson HP (1993) Tenascin-C, tenascin-R and tenascin-X: a family of talented proteins in search of functions. Curr Opin Cell Biol 5:869-876[Medline]
Gerdes JS, Harris MC, Polin RA (1988) Effects of dexamethasone and indomethacin on elastase, alpha 1-proteinase inhibitor, and fibronectin in bronchoalveolar lavage fluid from neonates. J Pediatr 113:727-731[Medline]
Hack M, Horbar JD, Malloy MH, Tyson JE, Wright E, Wright L (1991) Very low birth weight outcomes of the National Institute of Child Health and Human Developmental Neonatal Network. Pediatrics 87:587-597[Abstract]
KaarteenahoWiik R, Mertaniemi P, Sajanti E, Soini Y, Pääkkö P (1998) Tenascin is increased in epithelial lining fluid in fibrotic lung disorders. Lung 176:371-380[Medline]
KaarteenahoWiik R, Tani T, Sormunen R, Soini Y, Virtanen I, Pääkkö P (1996) Tenascin immunoreactivity as a prognostic marker in usual interstitial pneumonia. Am J Respir Crit Care Med 154:511-518[Abstract]
Kaminski N, Allard JD, Pittet JF, Zuo F, Griffiths MJD, Morris D, Huang X, Sheppard D, Heller RA (2000) Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proc Natl Acad Sci USA 95:15623-15628
Kanno S, Fukuda Y (1994) Fibronectin and tenascin in rat tracheal wound healing and their relation to cell proliferation. Pathol Int 44:96-106[Medline]
Koch M, WehrleHaller B, Baumgartner S, Spring J, Brubacher D, Chiquet M (1991) Epithelial synthesis of tenascin at tips growing bronchi and graded accumulation in basement mambrane and mesenchyme. Exp Cell Res 194:297-300[Medline]
Kuhn C, McDonald JA (1991) The roles of the myofibroblasts in the idiopathic pulmonary fibrosis. Am J Pathol 138:1257-1265[Abstract]
Meiners S, Nur-e-Kamal MSA, Mercado MLT (2001) Identification of a neurite outgrowth-promoting motif within the alternatively spliced region of human tenascin-C. J Neurosci 21:7215-7225
Northway WH, Rosan RC, Porter DY (1967) Pulmonary disease following respiratory therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N Engl J Med 27:357-368
Northway WH, Jr (1992) Bronchopulmonary dysplasia: twenty-five years later. Pediatrics 89:969-973[Medline]
Pääkkö P, KaarteenahoWiik R, Pöllänen R, Soini Y (2000) Tenascin mRNA expression at the foci of recent injury in usual interstitial pneumonia. Am J Respir Crit Care Med 161:967-972
Pääkkö P, Nuorva K, Kamel D, Soini Y (1992) Evidence by in situ hybridization that c-erbB-2 proto-oncogene expression is a marker of malignancy and is expressed in lung adenocarcinomas. Am J Respir Cell Mol Biol 7:325-334[Medline]
Rennard SI, Crystal RG (1981) Fibronectin in human bronchopulmonary lavage fluid. J Clin Invest 69:113-122
Rhodes PG, Hall RT, Leonidas JC (1975) Chronic pulmonary disease in neonates with assisted ventilation. Pediatrics 55:788-796[Abstract]
Rosan CR (1975) Hyaline membrane disease and a related spectrum of neonatal pneumopathies. In Rosenberg HS, Bolande RP, eds. Perspectives in Pediatric Pathology. Chicago; Yearbook Medical Publishers, 1, 6-60
Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fuakayama M, Stier LE, Pääkkö PK, Gilardi P, StratfordPerricaudet LD, Perricaudet LD, Jallat S, Pavirani A, Lecocq JP, Crystal RG (1991) Adenovirus-mediated transfer of a recombinant 1-antitrypsin gene to the lung epithelium in vivo. Science 252:431-434[Medline]
Silcocks PBS (1983) Measuring repeatability and validity of histological diagnosisa brief review with some practical examples. J Clin Pathol 36:1269-1275[Abstract]
Sinkin RA, Roberts M, LoMonaco MB, Sanders RJ, Metlay LA (1998) Fibronectin expression in bronchopulmonary dysplasia. Pediatr Dev Pathol 1:494-502[Medline]
Siri A, Carnemolla B, Saginati M, Leprini A, Casari G, Baralle F, Zardi L (1991) Human tenascin: primary structure, pre-mRNA splicing patterns and localization of the epitopes recognized by two monoclonal antibodies. Nucleic Acids Res 19:525-531[Abstract]
Soini Y, Pääkkö P, Nuorva K, Kamel D, Linnala A, Virtanen I, Lehto V-P (1993) Tenascin immunoreactivity in lung tumors. Am J Clin Pathol 100:145-150[Medline]
Stocker JT (1986) Pathologic features of long-standing "healed" bronchopulmonary dysplasia: a study of 28 3- to 40-month-old infants. Hum Pathol 17:943-961[Medline]
Torikata C, Villiger B, Kuhn C, III, McDonald JA (1985) Ultrastructural distribution of fibronectin in normal and fibrotic lung. Lab Invest 52:399-408[Medline]
Toti P, Buonocore G, Tanganelli P, Catella AM, Palmeri MLD, Vatti R, Seemayer TA (1997) Bronchopulmonary dysplasia of the premature baby: an immunohistochemical study. Pediatr Pulmonol 24:22-28[Medline]
Vartio T, Laitinen L, Narvanen O (1987) Differential expession of the ED sequence-containing form of cellular fibronectin in embryonic and adult human tissues. J Cell Sci 88:419-430[Abstract]
Walker E, McNicol AM (1992) In situ hybridization demonstrates the stability of mRNA in post-mortem rat tissues. J Pathol 168:67-73[Medline]
Zhao Y (1999) Tenascin is expressed in the mesenchyme of the embryonic lung and down-regulated by dexamethasone in early organogenesis. Biochem Biophys Res Commun 263:597-602[Medline]