Tracheal Aspirate Levels of the Matricellular Protein SPARC Predict Development of Bronchopulmonary Dysplasia

<div><p>Background</p><p>Isolation of tracheal aspirate mesenchymal stromal cells (MSCs) from premature infants has been associated with increased risk of bronchopulmonary dysplasia (BPD). MSCs show high levels of mRNAs encoding matricellular proteins, non-structural extracellular proteins that regulate cell-matrix interactions and participate in tissue remodeling. We hypothesized that lung matricellular protein expression predicts BPD development.</p><p>Methods</p><p>We collected tracheal aspirates and MSCs from mechanically-ventilated premature infants during the first week of life. Tracheal aspirate and MSC-conditioned media were analyzed for seven matricellular proteins including SPARC (for Secreted Protein, Acidic, Rich in Cysteine, also called osteonectin) and normalized to secretory component of IgA. A multiple logistic regression model was used to determine whether tracheal aspirate matricellular protein levels were independent predictors of BPD or death, controlling for gestational age (GA) and birth weight (BW).</p><p>Results</p><p>We collected aspirates from 89 babies (38 developed BPD, 16 died before 36 wks post-conceptual age). MSC-conditioned media showed no differences in matricellular protein abundance between cells from patients developing BPD and cells from patients who did not. However, SPARC levels were higher in tracheal aspirates from babies with an outcome of BPD or death (<i>p</i><0.01). Further, our logistic model showed that tracheal aspirate SPARC (p<0.02) was an independent predictor of BPD/death. SPARC deposition was increased in the lungs of patients with BPD.</p><p>Conclusions</p><p>In mechanically-ventilated premature infants, tracheal aspirate SPARC levels predicted development of BPD or death. Further study is needed to determine the value of SPARC as a biomarker or therapeutic target in BPD.</p></div

Increased SPARC expression in human lungs of infants with BPD.

<p>Lung sections were immunostained with rabbit anti-human SPARC, labeled with biotinylated anti-rabbit IgG and developed using the Vectastain Elite ABC kit and diaminobenzidine. A-C. Lungs of three full term infants dying of non-pulmonary causes are shown. There is little or no SPARC staining (brown) in the alveolar walls. D. The lung of an infant born at 26 weeks gestational age who died on postnatal day 13. Panel E is the IgG control for panel A. F-I. Lung sections from four infants dying of BPD. Three infants (F-H) show increased SPARC expression in the thickened alveolar interstitium. Panel J is the IgG control for panel F. (Original magnification, 200X).</p

Normalized matricellular proteins and risk of developing BPD or death.

<p>Normalized SPARC levels were significantly higher in babies developing BPD or death prior to 36 weeks postmenstrual age (*p = 0.0185, Mann Whitney U test). There were no other significant differences in normalized matricellular protein concentration between infants who did not develop BPD and infants who developed BPD or died prior to 36 weeks postmenstrual age. Tracheal aspirate matricellular protein and secretory component of IgA (scIgA) concentrations were measured by ELISA or multiplex immune assay. The data show individual matricellular protein levels normalized to scIgA concentration. Medians and interquartile ranges are shown.</p

Hyperoxic exposure is associated with α-actin and periostin-double positive myofibroblasts in wild-type but not periostin null mice.

<p>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.</p

Hyperoxic exposure on neonatal mice increases lung periostin content.

<p>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).</p

Increased periostin expression in the lungs of infants with BPD.

<p>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×.</p

Periostin knockout prevents hypoventilation and myofibroblast differentiation in hyperoxia-exposed neonatal mice.

<p>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).</p

Effects of periostin and TGF-β on mesenchymal stromal cell DNA synthesis.

<p>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.</p

Effects of periostin and TGF-β on mesenchymal stromal cell myofibroblastic differentiation.

<p>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).</p

Hyperoxia induces a BPD phenotype in neonatal mice.

<p>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.</p