Temporal Expression of AlphaSmooth Muscle Actin and Drebrin in Septal Interstitial Cells during Alveolar Maturation
Department of Anatomy (MY,HK,TS) and Department of Obstetrics and Gynecology (MY,KK), Juntendo University School of Medicine, Tokyo, Japan
Correspondence to: Dr. Hidetake Kurihara, Department of Anatomy, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: hidetake{at}med.juntendo.ac.jp
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Summary |
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(J Histochem Cytochem 53:735744, 2005)
Key Words: collagen desmin elastin intermediate filament lung microfilament myofibroblast
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Introduction |
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In the stages of lung maturation, specific temporalspatial interactions between mesenchymal and epithelial cells are required to establish an effective airblood barrier (Chuang and McMahon 2003). In lung, alveolar epithelial cells secrete platelet-derived growth factor (PDGF), the receptor of which is expressed in interstitial fibroblasts (Bostrom et al. 1996
; Lindahl et al. 1997
). A mouse PDGF-A null allele has been shown to be homozygous lethal. Postnatally surviving PDGF-Adeficient mice develop lung emphysema because of the failure of alveolar septation. This is apparently caused by the loss of specialized interstitial fibroblasts in alveoli and associated elastin fiber deposits (Bostrom et al. 1996
). Although the mechanism that governs the outgrowth of secondary septa is not known, interstitial matrix proteins, especially elastin, seem to be required for the initiation and progression of alveolization (James et al. 1998
; Noguchi et al. 1989
). It is suggested that the precursor of elastin, tropoelastin, is synthesized and secreted by a specialized interstitial cells (fibroblasts) in the alveoli (Myers et al. 1983
); however, the interstitial fibroblasts that play a role in alveolar maturation are not fully characterized.
Generally, fibroblasts are responsible for the production and secretion of extracellular matrix components, proteolytic enzymes, and various cytokines (Sappino et al. 1990). A line of evidence suggests that fibroblasts are likely to have specialized functions in each of these tissues. It has been accepted that the fibroblasts are heterogeneous (Komuro 1990
). Several kinds of fibroblasts exist in the alveolar interstitial space, but it is difficult to distinguish the fibroblasts in the tissue by their morphological characteristics.
Alphasmooth muscle actin (alpha-SMA) is a differentiation marker of smooth muscle cells and is also present in a special type of fibroblast called myofibroblast. Previous reports demonstrated that cells containing alpha-SMA are found in maturing alveolar interstitium (Vaccaro and Brody 1978; Mitchell et al. 1990
; Wagner et al. 2003
). Myofibroblasts expressing alpha-SMA are instrumental in wound contraction during normal wound healing (Powell et al. 1999
; Tomasek et al. 2002
). Alpha-SMA within myofibroblasts becomes organized in filamentous bundles, called stress fibers, that allow the retractile movement producing wound contraction (Grinnell 1994
).
Recently, an actin-binding protein, developmentally regulated brain protein (drebrin), has been localized in the specialized interstitial cells of renal glomerulus (i.e., mesangial cells) (Peitsch et al. 2003). Drebrins are a family of actin-binding proteins originally identified in neuronal cells (Peitsch et al. 2001
; Shirao et al. 1992
). Drebrin has been involved in the regulation of actin filament organization, especially in the formation of neuronal cell processes and cell protrusions of motile cells, especially lamellipodia and filopodia (Peitsch et al. 2001
). Electron microscopy (EM) observation indicates that both mesangial cells and alveolar interstitial cells are tightly associated with the neighboring vasculature by their elongated cell protrusions. This finding led us to investigate the expression of drebrin in alveolar interstitial cells.
Skalli et al. (1989) have distinguished four different types of myofibroblasts according to their expression of alpha-SMA and the intermediate filaments, desmin and/or vimentin. Thus, we tried to examine which kinds of intermediate filament proteins are expressed in alveolar interstitial cells during maturation. The expression of intermediate filaments depends on cell type at the phase of cellular differentiation. In general, epithelial cells express cytokeratin-type intermediate filaments, whereas mesenchymal cells express vimentin-type ones. Desmin-type intermediate filaments, which are expressed predominantly in skeletal muscle cells, cardiac myocytes, and certain smooth muscle cells, are also found in some non-muscle cells. A major function shared by several types of cytoplasmic intermediate filaments is to stabilize cellular architecture against the mechanical forces to which it is subjected (Fuchs and Weber, 1994
). The alveoli need steady and tidy skeletal elements to maintain their unique structure in such a severe environment; however, the function of intermediate filaments in lung has not been clarified.
In the present study, we characterized the alveolar interstitial cells expressing unique cytoskeletal proteins during postnatal alveolar maturation using immunocytochemistry and EM.
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Materials and Methods |
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Animals
All the procedures performed on laboratory animals were approved by the Institutional Animal Care and Use Committee of Juntendo University School of Medicine, and all the animal experiments were carried out in compliance with the guidelines for animal experimentation of Juntendo University School of Medicine. Wistar rats (embryonic day 20 to 6 weeks old) were obtained from Charles River Japan (Kanagawa, Japan). They were kept in an air-conditioned room and maintained on a commercial stock diet and tap water ad libitum. Gestational age was calculated from the first day when a vaginal plug was seen.
Confocal Laser-scanning Microscopy
Rat lungs were perfused with 4% paraformaldehyde fixative buffered with 0.1 M phosphate buffer (PB, pH 7.4) under anesthesia with Nembutal (Dainippon Pharmaceutical; Osaka, Japan) and then were cut into small pieces. These samples were immersed in the same fixative for approximately 30 min. After washing with PBS, the tissue was immersed successively in PBS solutions containing 10%, 15%, and 20% sucrose for 4 hr, 12 hr, and 4 hr, respectively. Cryosections (thickness, 510 µm) were cut using a Jung Frigocut 2800E (Leica; Wetzlar, Germany) and then mounted on silane-coated glass slides (Dako; Carpinteria, CA). The cryosections were rinsed with PBS and blocked in blocking solution (0.1% BSA in PBS). The sections were incubated for 2 hr at RT with mouse monoclonal anti-alpha-SMA antibody (1:100), mouse monoclonal anti-desmin antibody (1:100), rabbit anti-laminin antibody (1:100), rabbit anti-rat elastin antibody (1:50), or goat polyclonal anti-drebrin antibody (1:100). Subsequently, the sections were incubated for 1 hr at RT with TRITC-conjugated anti-mouse IgG (1:200), TRITC-conjugated anti-rabbit IgG (1:200), or FITC-conjugated anti-rabbit IgG (1:100). Fluorescence specimens were viewed with confocal laser scanning microscope LSM510 (Carl Zeiss; Oberkochen, Germany).
Transmission Electron Microscopy
Rat lungs were perfused with 2.5% glutaraldehyde fixative buffered with 0.1 M PB (pH 7.4) under anesthesia with Nembutal and immersed in the same fixative for approximately 12 hr. The specimens were sectioned at 200-µm thickness with a Microslicer (Dosaka; Kyoto, Japan) and processed further by modified cold dehydration technique. The samples were successively immersed in 0.1% OsO4 in 0.1 M PB for 30 min, in 5% extracts of oolong tea in 0.05 M maleate buffer for 3 hr, and in 1% uranyl acetate in the same maleate buffer solution. Subsequently, the specimens were dehydrated with a graded series of acetone at 0C to 30C before being embedded in Epon 812 (Oken; Tokyo, Japan). These procedures enabled detailed morphological observations of the extracellular matrices and intracellular fibrils. Ultrathin sections (80 nm) were processed with a diamond knife, transferred to copper grids (50 mesh) that had been coated with Formvar membrane (Oken), stained with uranyl acetate and lead citrate, and observed in a Hitachi 7100 transmission electron microscope (Hitachi; Tokyo, Japan).
Immunoperoxidase Labeling on Cryostat Sections
Paraformaldehyde-fixed lung tissues from embryonic day 20 and postnatal day 2, 5, 12, and 21 animals were cut on a cryostat into 50-µm thick sections. Cryostat sections were incubated overnight at RT with mouse monoclonal anti-alpha-SMA antibody (1:50) followed by incubation for 2 hr in F(ab')2 fragments of goat anti-mouse IgG conjugated to horseradish peroxidase (1:100). The sections were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), reacted with diaminobenzidine and were processed for EM.
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Results |
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Elastic fibers are abundant in septal interstitium, and the relationship between elastin and alpha-SMApositive cells was examined by double immunostaining with anti-elastin antibody and anti-alpha-SMA antibody (Figure 2). Until postnatal day 12 (Figures 2A, 2B, and 2C), a large amount of elastic fibers surrounded the primitive alveolar cavities, and alpha-SMApositive slender cells were always associated with the elastic fibers. At the period of alveolar maturation (Figures 2C and 2D), the elastic fibers were concentrated in the tips of the elongated secondary septa with alpha-SMApositive round cells. In adult rat lung, the tips of the secondary septa contained a concentrated population of elastic fibers and exhibited no signals for alpha-SMA. In the region of alveolar ducts, the third type of alpha-SMApositive cells were found together with considerable amounts of elastic fibers (Figure 2E).
Electron Microscopy of Septal Interstitial Cells
Alpha-SMApositive round cells at the tips of secondary alveolar septa were found on postnatal days 12 and 21. Electron microscopy showed that the alpha-SMApositive cells had well-developed rough endoplasmic reticulum and Golgi apparatus in the cytoplasm and that their elongated processes were surrounded by elastic fibers and collagen fibers on postnatal days 12 and 21 (Figures 3A and 3B). In the adult rat lung, alpha-SMApositive cells were not seen in the alveolar septa, but interstitial cells surrounded by elastin and collagen fibers were still located in the interstitium (Figures 3C and 3D).
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Discussion |
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Our data on the cellular localization of alpha-SMA during lung maturation indicate that alpha-SMA is located exclusively at the protrusions of the septal myofibroblasts. Myofibroblasts are recognized to play a central role in closing wound tissue through their capacity to produce a strong contractile force (Serini and Gabbiani 1999). Exogenous expression of alpha-SMA was induced to enhance fibroblast contractile activity (Hinz et al. 2001
). Alpha-SMA expressed in the interstitial cells located within the alveolar walls might play a role in making the tensile forces of the alveolar parenchyma (Adler et al. 1989
). EM observations also suggest that the filopodia-like protrusions of the cells contain numerous actin bundles. Cell process formation is closely related to cellular adhesion to other cells or to the extracellular matrix and to cell movements. These events are achieved by the assembly of actin and actin-binding proteins (Tomasek et al. 2002
). We could detect that drebrin, a family of actin-binding proteins, is expressed in septal alpha-SMApositive cells. We found that drebrin is located in the cell processes of septal myofibroblast-like cells during alveolar maturation. The transient expression of drebrin in the myofibroblasts suggests that the formation of cellular projection is related to the elongation of the secondary septum. Drebrin is also found in the mesangial cells, which provide the mechanical support for glomerular capillaries by generating an inwardly directed counterforce and regulating the glomerular capillary wall tension by contraction and relaxation (Kriz et al. 1990
). Like mesangial cells, septal myofibroblasts might play a role in regulating the capillary wall tension via the interaction between the cell processes and the elastic fibers located beneath the capillary. Interestingly, drebrin is not expressed in the interstitial cells in the alveolar ducts expressing alpha-SMA during maturation, suggesting that the alpha-SMApositive interstitial cells in the alveolar ducts are distinct from the septal myofibroblast-like cells.
The septal myofibroblast-like cells are always surrounded by elastic fibers. The cells elongate their cell processes containing alpha-SMA to the elastic fibers during the septal formation. Because it appears that alpha-SMApositive fibroblasts have much larger focal adhesions with the substratum than those of alpha-SMAnegative fibroblasts in vitro (Dugina et al. 1998), it is thought that the interaction between the cellular protrusions and elastic fibers is important to make a contractile force. Elastic properties of the lung result from the presence of elastic fibers in the extracellular space. The 72-kDa biosynthetic precursor, tropoelastin, is secreted into the extracellular space where it becomes highly cross-linked into a rubberlike network through the activity of the copper-requiring enzyme lysyl oxidase (Rosenbloom et al. 1993
). Myers et al. (1983)
have demonstrated that the maximal rates of elastin synthesis are observed in lung explants from 7- to 12-day-old rats, and the rate of elastin synthesis during this period was five to eight times greater than the rate observed in adult rat lung. Furthermore, Noguchi and Samaha (1991)
reported that with alveolar septal formation the message for elastin in the interstitium increased progressively from day 17 of gestation, reaching a peak at postnatal days 7 to 11. In general, the signal declined significantly by postnatal day 21, and elastogenesis was virtually absent in the adult. These findings are consistent with our immunocytochemical data for alpha-SMA, suggesting that the expression of alpha-SMA in interstitial cells is closely associated with elastogenesis in the interstitium during septal formation.
It would be interesting to know the fate of alpha-SMAcontaining cells (e.g., septal myofibroblast-like cells) at the septal interstitium after lung maturation. We checked the cellular loss of alpha-SMAcontaining cells during maturation using the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, which is useful for detecting apoptotic cells. As a result, we were unable to obtain any evidence on the apoptosis of alpha-SMAcontaining cells throughout the whole period. On the contrary, we found cells showing similar ultrastructural characteristics surrounded by elastic fibers located in the septal interstitium in adult rat lung. These results indicate that phenotypical changes in alpha-SMApositive cells occur after alveolar maturation.
PDGF-Adeficient mice develop lung emphysema secondary to the failure of alveolar septation. Bostrom et al. (1996) reported that these mice lack lung alveolar alpha-SMApositive cells and exhibit reduced deposition of elastin fibers in the lung parenchyma. The PDGFs appear to regulate the generation of specific populations of myofibroblasts during mammalian development. PDGF-A is crucial for alveolar myofibroblast ontogeny. They also proposed that lung PDGF-R alpha+ cells are progenitors of the tropoelastin-positive alveolar SMC, and that postnatal alveogenesis failure in PDGF-A/ mice is due to a prenatal block in the distal spreading of PDGF-R alpha+ cells along the tubular lung epithelium during the canalicular stage of lung development. Our findings also indicate that the alpha-SMAcontaining cells are positive against anti-PDGF-A receptor antibody, and that PDGF signaling might be necessary to induce the expression of alpha-SMA and specialized actin filament organization for cell protrusion in which alpha-SMA and drebrin are involved.
Taken together, the unique localization and transient expression of alpha-SMA suggests that the septal myofibroblasts play an important role in septal formation.
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Footnotes |
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Literature Cited |
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Adler KB, Low RB, Leslie KO, Mitchell J, Evans JN (1989) Contractile cells in normal and fibrotic lung. Lab Invest 60:473485[Medline]
Bostrom H, Willetts K, Pekny M, Leveen P, Lindahl P, Hedstrand H, Pekna M, et al. (1996) PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell 85:863873[CrossRef][Medline]
Burri PH (1974) The postnatal growth of the rat lung. 3. Morphology. Anat Rec 180:7798[CrossRef][Medline]
Chuang PT, McMahon AP (2003) Branching morphogenesis of the lung: new molecular insights into an old problem. Trends Cell Biol 13:8691[CrossRef][Medline]
Dugina V, Alexandrova A, Chaponnier C, Vasiliev J, Gabbiani G (1998) Rat fibroblasts cultured from various organs exhibit differences in alpha-smooth muscle actin expression, cytoskeletal pattern, and adhesive structure organization. Exp Cell Res 238:481490[CrossRef][Medline]
Fuchs E, Weber K (1994) Intermediate filaments: structure, dynamics, function, and disease. Annu Rev Biochem 63:345382[CrossRef][Medline]
Gabbiani G, Schmid E, Winter S, Chaponnier C, de Ckhastonay C, Vandekerckhove J, Weber K, et al. (1981) Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific alpha-type actin. Proc Natl Acad Sci USA 78:298302
Grinnell F (1994) Fibroblasts, myofibroblasts, and wound contraction. J Cell Biol 124:401404[CrossRef][Medline]
Hinz B, Celetta G, Tomasek JJ, Gabbiani G, Chaponnier C (2001) Alpha-smooth muscle actin expression upregulates fibroblast contractile activity. Mol Biol Cell 12:27302741
James MF, Rich CB, Trinkaus-Randall V, Rosenbloom J, Foster JA (1998) Elastogenesis in the developing chick lung is transcriptionally regulated. Dev Dyn 213:170181[CrossRef][Medline]
Kapanci Y, Ribaux C, Chaponnier C, Gabbiani G (1992) Cytoskeletal features of alveolar myofibroblasts and pericytes in normal human and rat lung. J Histochem Cytochem 40:19551963
Kaplan NB, Grant MM, Brody JS (1985) The lipid interstitial cell of the pulmonary alveolus: age and species differences. Am Rev Respir Dis 132:13071312[Medline]
Komuro T (1990) Re-evaluation of fibroblasts and fibroblast-like cells. Anat Embryol (Berl) 182:103112[Medline]
Kriz W, Elger M, Lemley K, Sakai T (1990) Structure of the glomerular mesangium: a biomechanical interpretation. Kidney Int Suppl 30:S29[Medline]
Leslie KO, Mitchell JJ, Woodcock-Mitchell JL, Low RB (1990) Alpha smooth muscle actin expression in developing and adult human lung. Differentiation 44:143149[Medline]
Lindahl P, Karlsson L, Hellstrom M, Gebre-Medhin S, Willetts K, Heath JK, Betsholtz C (1997) Alveogenesis failure in PDGF-A-deficient mice is coupled to lack of distal spreading of alveolar smooth muscle cell progenitors during lung development. Development 124:39433953
Massaro D, Teich N, Maxwell S, Massaro GD, Whitney P (1985) Postnatal development of alveoli. J Clin Invest 76:12971305[Medline]
Massaro GD, Massaro D (1996) Formation of pulmonary alveoli and gas-exchange surface area: quantitation and regulation. Annu Rev Physiol 58:7392[CrossRef][Medline]
Mitchell JJ, Reynolds SE, Leslie KO, Low RB, Woodcock-Mitchell J (1990) Smooth muscle cell markers in developing rat lung. Am J Respir Cell Mol Biol 3:515523[Medline]
Myers B, Dubick M, Last JA, Rucker RB (1983) Elastin synthesis during perinatal lung development in the rat. Biochim Biophys Acta 761:1722[Medline]
Noguchi A, Reddy R, Kursar JD, Parks WC, Mecham RP (1989) Smooth muscle isoactin and elastin in fetal bovine lung. Exp Lung Res 15:537552[Medline]
Noguchi A, Samaha H (1991) Developmental changes in tropoelastin gene expression in the rat lung studied by in situ hybridization. Am J Respir Cell Mol Biol 5:571578[Medline]
Peitsch WK, Hofmann I, Pratzel S, Grund C, Kuhn C, Moll I, Langbein L, et al. (2001) Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia. Eur J Cell Biol 80:567579[Medline]
Peitsch WK, Hofmann I, Endlich N, Pratzel S, Kuhn C, Spring H, Grone HJ, et al. (2003) Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli. J Am Soc Nephrol 14:14521463
Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB (1999) Myofibroblasts. I. Paracrine cells important in health and disease. Am J Physiol 277:C19[Medline]
Rosenbloom J, Abrams WR, Mecham R (1993) Extracellular matrix 4: the elastic fiber. FASEB J 7:12081218
Sanchez-Esteban J, Wang Y, Cicchiello LA, Rubin LP (2002) Cyclic mechanical stretch inhibits cell proliferation and induces apoptosis in fetal rat lung fibroblasts. Am J Physiol Lung Cell Mol Physiol 282:L448456
Sappino AP, Schurch W, Gabbiani G (1990) Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest 63:144161[Medline]
Serini G, Gabbiani G (1999) Mechanisms of myofibroblast activity and phenotypic modulation. Exp Cell Res 250:273283[CrossRef][Medline]
Shirao T, Kojima N, Obata K (1992) Cloning of drebrin A and induction of neurite-like processes in drebrin-transfected cells. Neuroreport 3:109112[Medline]
Skalli O, Schurch W, Seemayer T, Lagace R, Montandon D, Pittet B, Gabbiani G (1989) Myofibroblasts from diverse pathologic settings are heterogeneous in their content of actin isoforms and intermediate filament proteins. Lab Invest 60:275285[Medline]
Thurlbeck WM (1975) Postnatal growth and development of the lung. Am Rev Respir Dis 111:803844[Medline]
Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3:349363[CrossRef][Medline]
Vaccaro C, Brody JS (1978) Ultrastructure of developing alveoli. I. The role of the interstitial fibroblast. Anat Rec 192:467479[CrossRef][Medline]
Vidic B, Ujevic N, Shabahang MM, van de Zande F (1989) Differentiation of interstitial cells and stromal proteins in the secondary septum of early postnatal rat: effect of maternal chronic exposure to whole cigarette smoke. Anat Rec 223:165173[CrossRef][Medline]
Wagner TE, Frevert CW, Herzog EL, Schnapp LM (2003) Expression of the integrin subunit a8 in murine lung development. J Histochem Cytochem 51:13071315