We previously characterized the gene expression profile of neonatal lung mesenchymal stromal cells 
. Among other genes, we found that, compared to lung fibroblasts, mesenchymal stromal cells showed increased expression of POSTN
encodes periostin, a secreted non-structural extracellular matrix protein which regulates TGF-β-mediated fibrosis 
and myofibroblast differentiation 
. Previous studies have shown that periostin is also expressed by human bone marrow-derived mesenchymal stem cells 
The isolation of mesenchymal stromal cells from neonatal tracheal aspirates confers a nearly 23 fold increase in the odds of developing BPD 
. Overexpression of TGF-β in neonatal mouse lungs induces proliferation of α-actin-positive cells within the alveolar septal walls and hypoalveolarization 
, a phenotype analogous to BPD. On this basis, we hypothesized that periostin may play a prominent role in BPD pathogenesis. To test this, we measured periostin expression in mice exposed to hyperoxia during the first two weeks of life, a period which corresponds to the alveolar stage of human lung development 
. We also examined periostin expression in the lungs of human infants with BPD. We found that periostin expression is increased in the lungs of hyperoxia-exposed neonatal mice. Periostin was present in the alveolar walls, particularly in areas of interstitial thickening associated with α-smooth muscle actin-positive myofibroblasts. In human BPD lungs, the distribution of staining was similar, with prominent staining in the subepithelium of thickened alveolar walls and fibroblastic foci. A similar distribution was recently described in patients with usual interstitial pneumonia and fibrotic-type nonspecific interstitial pneumonia 
. The colocalization of periostin and α-smooth muscle actin suggests that periostin was expressed in part by alveolar myofibroblasts.
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 
. On this basis, we examined the effects of periostin on neonatal lung mesenchymal stromal cell proliferation and myofibroblastic differentiation. TGF-β and periostin had synergistic effects on DNA synthesis. Further, treatment with periostin enhanced TGF-β-induced α-smooth muscle actin, elastin and collagen type I expression, outcomes indicative of myofibroblast differentiation. These data are consistent with the notion that the observed effect of TGF-β on myofibroblast proliferation and differentiation, which contributes to interstitial thickening and fibrosis in vivo
, is mediated in part by periostin expression. Since periostin null mice were protected from hyperoxia-induced hypoalveolarization, we speculate that abnormal myofibroblast proliferation and differentiation, mediated by aberrant periostin expression, is incompatible with secondary crest development.
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 
, and increased in the lungs of infants with BPD and hyperoxia-exposed mice (35). Since overstabilization of the extracellular matrix by excessive lysyl oxidase activity might impede the normal matrix remodeling that is required for pulmonary alveolarization, it is conceivable that the periostin knockout mice are protected from hypoalveolarization due to reduced lysyl oxidase activation.
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 
. Periostin mRNA expression is upregulated in bronchial epithelial cells of asthmatic subjects 
. Periostin null mice show defect in allergen-induced eosinophil recruitment to the lungs 
. In the present study, the lungs of periostin null mice showed attenuated expression of the neutrophil and monocyte chemoattractants CXCL1, CXCL2 and CCL4. CXCR2, the receptor for CXCL1 and CXCL2, is required for double-stranded RNA-induced neutrophil sequestration and hypoalveolarization in newborn mice 
, and NF-κB signaling in fetal lung macrophages disrupts airway morphogenesis 
. Together, these data are consistent with the notion that periostin promotes hyperoxia-induced lung inflammation in neonatal mice.
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 
. However, a detailed comparison of C57BL/6 and 129/Sv strains showed similar susceptibility to hyperoxia, including changes in lung neutrophils, IL-6, and expression of collagen type Iα2 and fibronectin. Further, expression of collagens type IIIα1 and IVα3 was increased after hyperoxic exposure in the 129/S strain, suggesting that these mice are capable of a fibrotic response when exposed to hyperoxia. Second, we have not completely characterized the periostin knockout mice, which appear to have a subtle pulmonary phenotype. At post-natal age 16–17, air-exposed animals showed increased alveolar macrophages and cellularity, as well as decreased expression of angiogenesis-related genes. Nevertheless, periostin null mice did not show pulmonary hypertension, as evidenced by a normal right ventricular wall thickness. This uncoupling of alveolar development and expression of angiogenesis-related genes remains unexplained, but could relate to an acceleration of alveolarization in periostin null mice.
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 
. Since periostin is a downstream effector of TGF-β, periostin may represent a promising therapeutic target for BPD.