Conceived and designed the experiments: PDB APP JKB MBH BBM. Performed the experiments: PDB APP JKB ACA MDL AMG. Analyzed the data: PDB APP JKB MBH. Contributed reagents/materials/analysis tools: GSP. Wrote the paper: PDB MBH. Manuscript review: GSP BBM MBH.
The authors have declared that no competing interests exist.
In bronchopulmonary dysplasia (BPD), alveolar septae are thickened with collagen and α-smooth muscle actin, transforming growth factor (TGF)-β-positive myofibroblasts. Periostin, a secreted extracellular matrix protein, is involved in TGF-β-mediated fibrosis and myofibroblast differentiation. We hypothesized that periostin expression is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice, an animal model for this disease. We also examined periostin expression in neonatal lung mesenchymal stromal cells and lung tissue of hyperoxia-exposed neonatal mice and human infants with BPD. Two-to-three day-old wild-type and periostin null mice were exposed to air or 75% oxygen for 14 days. Mesenchymal stromal cells were isolated from tracheal aspirates of premature infants. Hyperoxic exposure of neonatal mice increased alveolar wall periostin expression, particularly in areas of interstitial thickening. Periostin co-localized with α-smooth muscle actin, suggesting synthesis by myofibroblasts. A similar pattern was found in lung sections of infants dying of BPD. Unlike wild-type mice, hyperoxia-exposed periostin null mice did not show larger air spaces or α-smooth muscle-positive myofibroblasts. Compared to hyperoxia-exposed wild-type mice, hyperoxia-exposed periostin null mice also showed reduced lung mRNA expression of α-smooth muscle actin, elastin, CXCL1, CXCL2 and CCL4. TGF-β treatment increased mesenchymal stromal cell periostin expression, and periostin treatment increased TGF-β-mediated DNA synthesis and myofibroblast differentiation. We conclude that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis.
Increased survival of very premature infants has been accompanied by an increased incidence of bronchopulmonary dysplasia (BPD)
We have isolated mesenchymal stromal cells from the tracheal aspirates of premature infants
Based on the above evidence that TGF-β plays an important role in the pathogenesis of BPD, we hypothesized that periostin expression is required for the myofibroblastic differentiation and alveolar simplication in hyperoxia-exposed neonatal mice, a commonly used animal model for this disease. We also examined the effects of periostin on mesenchymal stromal cell myofibroblastic differentiation
Two-to-three day-old wild-type C57BL/6J mice were exposed to air or 75% oxygen for 14 days, as described
Two-to-three day-old wild-type C57BL/6J mice were exposed to air or 75% oxygen for 14 days. Compared to air-exposed mice (panels A–D). hyperoxic exposure caused the development of fewer and larger airspaces (E). Fluorescence microscopy showed increased deposition of α-actin (red), elastin (green) and collagen-I (blue, F). Colocalization of α-actin, elastin and collagen-I appears white (arrow, inset). G. Immunohistochemical stains showed periostin expression in the alveolar walls, particularly in areas of interstitial thickening. H. Periostin expression (green) colocalized with α-smooth muscle actin (red) and collagen (blue), suggesting synthesis by myofibroblasts Colocalization of periostin and collagen appears light blue; colocalization of α-actin, periostin and collagen-I appears white (arrow, inset). Colocalization was also present at the tips of secondary crests (arrowhead). Original magnification, 200×. These results are typical of three individual experiments.
We also analyzed whole lung extracts for periostin protein expression by immunoblotting (
Whole lung lysates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with anti-periostin (A). Results from two normoxic wild-type mice and two hyperoxic wild-type mice are shown here; lysates from normoxic and hyperoxic periostin null mice are added for comparison. Immunoblots showed a full-length 90 kD isoform as well as two smaller bands. (B). Group mean data showing a significant increase in periostin expression with hyperoxic exposure (n = 4 for each group, one way ANOVA).
Periostin staining of lung sections from infants dying of BPD were compared with those from full-term infants. Immunohistochemical staining in the lungs of full-term infants was localized to the airway subepithelium (
The lung of a full-term infants dying of a non-pulmonary cause is shown in (A). There is significant staining in the airway subepithelium, with miminal staining of the airway epithelium or alveolar walls. B–D. Staining of lung sections from three individual infants dying of BPD showed increased periostin expression, particularly in the subepithelium and fibroblastic foci. E. We also examined periostin (green) and α-smooth muscle actin (red) expression by fluorescence microscopy. Lungs of full-term infants showed periostin expression in the airway subepithelium which was distinct from the adjacent smooth muscle. F–H. Lungs of three individual infants with BPD were also examined for periostin expression. Lungs showed colocalization of periostin and α-actin in interstitial alveolar myofibroblasts (F and G, arrows, insets). Colocalization of periostin and α-actin (yellow-orange) was also found at the tips of secondary crests (H, arrow, inset). Original magnification, 200×.
Two-to-three day-old B6;129-
Two-day-old periostin null mice were exposed to air or 75% oxygen for 14 days. Compared to air-exposed wild-type mice (A), hyperoxia-exposed wild-type mice showed alveolar simplication (panel B). In contrast, air- (C) and hyperoxia-exposed periostin null mice (D) showed normal alveolar architecture. (E). Hypoalveolarization in wild-type hyperoxia-exposed mice was associated with a statistically significant increase in mean alveolar chord length (n = 4, one-way ANOVA).
Air-exposed wild-type mice showed α-smooth muscle actin staining (red) in the airway and arterial walls, but no expression in the alveolar interstitia (
Lung sections were stained for α-actin (red), periostin (green) and collagen I (blue); colocalization appears white. Unlike air-exposed wild-type mice (panel A), hyperoxia-exposed wild-type mice showed thickening of the interstitial space with α-smooth muscle-, periostin- and collagen type I-positive myofibroblasts (B). Air- (C) and hyperoxia-exposed periostin null mice (D) did not show alveolar myofibroblasts. These results are typical of three individual experiments.
We examined the effects of periostin knockout on lung mRNA expression, as measured by quantitative PCR. Consistent with the observed increase in periostin-positive interstitial myofibroblasts, hyperoxic exposure significantly increased the expression of α-smooth muscle actin, elastin and periostin (
mRNA was measured by quantitative PCR. We examined mRNA expression of
We also examined right ventricular thickness in air- and hyperoxia-exposed wild-type and periostin null mice (
TGF-β treatment has been shown to increase periostin expression in cardiac myocytes, aortic smooth muscle, and bronchial epithelium. We treated neonatal lung mesenchymal cells with 10 ng/ml TGF-β for 72 h and assessed periostin mRNA and protein expression by qRT-PCR and ELISA, respectively. TGF-β treatment tended to increase periostin mRNA levels, but the changes were not statistically significant (
Neonatal lung mesenchymal stromal cells were treated with 10 ng/ml TGF-β for 72 h and periostin mRNA and protein expression assessed by qPCR and ELISA, respectively. A. mRNA expression tended to increase with TGF-β treatment (n = 4). B. TGF-β treatment significantly increased periostin protein abundance (n = 7, one-way ANOVA).
We examined the interactive effects of periostin (0–500 ng/ml) and TGF-β (0–10 ng/ml) on cellular [3H]-thymidine incorporation by two-way ANOVA (
Mesenchymal stromal cells (n = 4) were incubated with [3H]-thymidine and treated with either TGF-β or periostin. [3H]-thymidine incorporation was assessed by scintillation counting. We used two-way ANOVA with Fisher's least significant difference multiple comparison test to assess the individual and combined effects of TGF-β and periostin on neonatal lung mesenchymal stromal cell DNA synthesis. In the presence of 500 ng/ml periostin, 10 ng/ml TGF-β significantly increased DNA synthesis compared to periostin alone. Also, in the presence of 10 ng/ml TGF-β, 500 ng/ml periostin significantly increased DNA synthesis compared to other periostin concentrations.
The effects of periostin (50 ng/ml) on TGF-β-induced α-smooth muscle actin and elastin mRNA expression were examined in mesenchymal stromal cells, using two-way ANOVA for statistical analysis (
A. Periostin (50 ng/ml) was incubated with mesenchymal stromal cells (n = 6) in the presence or absence of TGF-β (2–10 ng/ml). α-smooth muscle actin and elastin mRNA expression were measured by qPCR. We used two-way ANOVA with Fisher's least significant difference multiple comparison test to assess the individual and combined effects of TGF-β and periostin on neonatal lung mesenchymal stromal cell α-actin and elastin gene expression. TGF-β significantly increased α-actin and elastin expression in both the presence and absence of periostin. However, periostin significantly increased α-actin and elastin expression only in the presence of TGF-β. In other experiments, cells were stained with anti-α-smooth muscle actin (red), anti-collagen I (green) and anti-elastin (blue) (B, no treatment; C, periostin, 50 ng/ml; D, TGF-β, 2 ng/ml; E, periostin, 50 ng/ml and TGF-β, 2 ng/ml). Merged and individual images are shown (original magnification, 200×, results are representative of three individual experiments).
We previously characterized the gene expression profile of neonatal lung mesenchymal stromal cells
The isolation of mesenchymal stromal cells from neonatal tracheal aspirates confers a nearly 23 fold increase in the odds of developing BPD
To further examine the possible contribution of periostin to BPD pathogenesis, we compared the responses to hyperoxia of wild-type mice to periostin null mice. As expected. wild-type mice show hypoalveolarization and interstitial fibrosis when exposed to hyperoxia during the alveolar period of lung development. Lung mRNA expression of α-actin, elastin and periostin was also increased. In contrast, periostin null mice showed normal alveolar development. Further, in contrast to control mice, the alveolar walls of periostin null mice did not show thickening with α-smooth muscle-positive myofibroblasts or deposition of collagen or elastin. Lung mRNA expression of α-actin and elastin was not increased in hyperoxia-exposed periostin null mice. These results suggest that periostin is required for hypoalveolarization and interstitial fibrosis in hyperoxia-exposed neonatal mice.
TGF-β overexpression in neonatal mouse lungs induces proliferation of α-smooth muscle actin-positive cells within the septal walls
Matricellular proteins are non-structural proteins that are secreted and sequestered in the extracellular matrix, where they interact with integrins, growth factors, proteases, cytokines and other extracellular matrix proteins. Among various functions, they regulate TGF-β activation, adhesion, migration, fiber deposition and angiogenesis, as well as cell proliferation, differentiation and survival. All are involved in fibrosis and increased matrix deposition, and most are expressed at low levels in normal adult tissue but upregulated during development, wound healing and tissue remodeling. Thus, many of the effects of TGF-β may be mediated by matricellular proteins. Few studies have examined the role of matricellular proteins in neonatal lung injury. Overexpression of connective tissue growth factor (CTGF) during development results in a lung phenotype analogous to BPD (33). CTGF expression is also increased after high tidal volume ventilation in newborn rat lungs (34). Lysyl oxidase, which crosslinks collagen and elastin, is proteolytically activated by CTGF and periostin
Increasing evidence suggests that periostin plays a role in lung inflammation. Subepithelial periostin deposition and fibrosis are present in the bronchial tissue of both ovalbumin-sensitized and ovalbumin-inhaled mice and patients with asthma
There are important limitations to our study. First, the periostin null mice we used were developed in the 129 strain and backcrossed to C57BL/6. Since neither the 129 or C57BL/6 strain are perfect controls, we employed C57BL/6 mice for this purpose. Nevertheless, strain differences in the response to hyperoxia could have contributed to the protective effect of the periostin knockout
In conclusion, we have shown that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice and infants with BPD, and is required for hyperoxia-induced hypoalveolarization and interstitial fibrosis. Our data also demonstrate that neonatal lung mesenchymal stromal cells are not only potent sources of periostin, but also respond to periostin treatment by differentiation into myofibroblasts. These data significantly extend previous results showing that an excess of TGF-β and CTGF each induce the BPD phenotype in naïve animals
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of the University of Michigan Medical School (protocol ID#10397). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Two-to-three day-old wild-type C57BL/6J and B6;129-
Lungs were perfused with 5 mM EDTA and inflated to 30 cm H2O pressure with 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Sections were stained with hematoxylin and eosin and mean alveolar chord length determined
Mouse lungs were washed in PBS and homogenized in a buffer containing 50 mM Tris (pH 7.5), 100 mM NaCl, 50 mM NaF, 40 mM β-glycerophosphate, 2 mM EDTA, 200 µM Na3VO4, 1% Triton X-100, 1% SDS and 1% sodium deoxycholate containing complete protease inhibitors (Roche Diagnostics, Indianapolis, IN). After centrifugation for 30 min at 4°C at 10,000×
Human lung tissue was obtained from the University of Rochester Lung Biorepository under a protocol approved by the Institutional Review Board of Strong Memorial Hospital (Rochester, NY). Specimens were obtained from 3 infants who died in the intensive care nursery (
Patient | Gender | Race | Gestational Age (wks) | Birth Weight (g) | Lived | BPD |
52 | F | Mixed | 32 3/7 | 1630 | yes | no |
56 | F | Caucasian | 26 6/7 | 1425 | yes | no |
58 | M | Caucasian | 27 6/7 | 1615 | yes | yes |
68 | M | Caucasian | 27 | 1265 | yes | yes |
83 | F | Caucasian | 24 4/7 | 735 | yes | yes |
84 | M | Caucasian | 27 5/7 | 845 | yes | yes |
85 | F | Caucasian | 28 5/7 | 1180 | yes | yes |
87 | F | African-American | 29 2/7 | 1080 | yes | no |
88 | F | Caucasian | 28 5/7 | 1210 | yes | yes |
90 | M | Caucasian | 28 | 1160 | yes | yes |
92 | M | Caucasian | 28 | 800 | no | yes |
93 | M | Caucasian | 28 5/7 | 1395 | yes | no |
97 | M | Caucasian | 31 3/7 | 1620 | yes | no |
Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Valencia, CA), then transcibed to first-strand cDNA using Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). First-strand cDNA was then used to quantify the expression of periostin (POSTN), α-smooth muscle actin (ACTA2), elastin (ELN), collagen type Iα1 (COLIA1), CXCL1, CXCL2, CCL4, VEGF-A, KDR/VEGFR2/Flk1, PECAM1/CD31 and the housekeeping gene glyceraldehyde dehydrogenase (GAPDH). The primers used are shown in
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Isolation of neonatal lung mesenchymal stromal cells was approved by the University of Michigan Institutional Review Board. Cells were isolated from tracheal aspirates of premature infants undergoing mechanical ventilation (
Hearts from normoxic or hyperoxic mice were removed, fixed in formalin, and processed for hematoxylin and eosin staining. Images of the right ventricular outer wall were made at 10× magnification and measured using NIH ImageJ. A hemacytometer was used to standardize the number of pixels/micrometer.
Mesenchymal stromal cells were grown on fibronectin-coated glass slides (BD Biosciences), fixed in 1% paraformaldehyde and probed with antibodies against α-smooth muscle actin, collagen I and elastin.
Mouse lung mesenchymal cells were plated into 12-well dishes at 100,000 cells/well, allowed to attach overnight, and incubated in serum-free medium supplemented with 500,000 cpm [3H]-thymidine. After 8 h, cells were treated with TGF-β1 or mouse periostin. Cells were maintained in the medium for 48 h. At the end of this time, the radioactive medium was removed, wells were washed three times with PBS, and the DNA precipitated with 10% trichloroacetic acid at 4°C. After 30 min, the acid was removed and the precipitate was washed three times with 70% ethanol at 4°C and solubilized at room temperature for 30 min with 1 mL 0.5 N NaOH and 1% Triton X-100. Solubilized DNA was counted in a liquid scintillation counter.
Cell supernatants were analyzed for soluble periostin by ELISA (Adipo Bioscience, Santa Clara, CA).
All data were described as mean±SEM. For simple comparisons of two or more groups, we used one-way analysis of variance (ANOVA). When examining the influence of two factors, we used two-way ANOVA. Significant differences between groups were pinpointed by Fisher's least significant difference multiple comparisons test.