Changes in the Expression of Genes Known to be Involved in the Canonical Wnt Pathway

<p>The figure was generated using Ingenuity Pathways Analysis (Ingenuity Systems, <a href="http://www.ingenuity.com/" target="_blank">http://www.ingenuity.com/</a>). Increased genes are in increasing shades of red, decreased are in increasing shades of green. Combined red and green is when different members are changed in different directions.</p

Schematic Overview of the Epithelial–Mesenchymal Transition as Modulated by TGF-β and BMPs

<p>In IPF lungs, TGF-β3, gremlin, and LEF-1 are up-regulated, while BMP-2 is down-regulated. Dysregulation of TGF-β/BMP pathway induces a fibrotic phenotype. EMT, so important in development, is likely to represent one possible response of the alveolar epithelial cell facing sustained injury. Increased expression of BMPs may not only antagonize TGF-β-induced EMT, enhancing re-epithelialization, but also may cause the opposite process, mesenchymal-to-epithelial transition (MET).</p

Localization of Osteopontin in IPF Lungs

<p>Immunoreactive protein was revealed with AEC, and samples were counterstained with hematoxylin. Two representative IPF lung samples exhibited strong epithelial staining (original magnification, 40×) (A,B). Control lung showed no staining (C). Negative control section from IPF lung in which the primary antibody was replaced with nonimmune serum also showed no staining (40×) (D).</p

Increased Expression of CC16 in Patients with Idiopathic Pulmonary Fibrosis

<div><p>Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology. The pathogenic mechanisms are unclear, but evidence indicates that aberrantly activated alveolar epithelial cells secrete a variety of mediators which induce the migration, proliferation and activation of fibroblasts and finally the excessive accumulation of extracellular matrix with the consequent destruction of the lung parenchyma. CC16 (approved symbol SCGB1A1), a putative anti-inflammatory protein produced by “club” cells in the distal airways, has not been evaluated in IPF lungs. In this study, we determined the serum and bronchoalveolar lavage (BAL) levels as well as the lung cell localization of this protein. Also, we explored the usefulness of serum levels of CC16 for the differential diagnosis of IPF (n = 85), compared with non-IPF interstitial lung diseases [chronic hypersensitivity pneumonitis (cHP; n = 85) and connective tissue diseases (CTD-ILD; n = 85)]. CC16 was significantly increased in serum and BAL fluids of IPF patients and was found not only in club cells but also in alveolar epithelial cells. When compared with non-IPF patients and controls, serum levels were significantly increased (p<0.0001). Sensitivity and specificity for CC16 (cut-off 41ng/mL) were 24% and 90%, positive predictive value 56% and negative predictive value 69%. These findings demonstrate that CC16 is upregulated in IPF patients suggesting that may participate in its pathogenesis. Although higher than the serum levels of non-IPF patients it shows modest sensitivity to be useful as a potential biomarker for the differential diagnosis.</p></div

Effect of Osteopontin on MMP-7 Gene and Protein Expression and Activity in A549 Epithelial Cells

<div><p>(A) Northern blot of 20 μg total cellular RNA per lane extracted from control and A549 cells stimulated with 0.4, 1, and 2 μg/ml osteopontin.</p> <p>(B) Densitometry of Northern blot and normalization of MMP-7 to 18S demonstrates a 3- to 4-fold increase in MMP-7 over control.</p> <p>(C) Real-time PCR showing up-regulation of MMP-7 expression by osteopontin (*<i>p</i> < 0.01) and inhibition by anti-α_vβ₃, anti-CD44, anti-EGFR, and GRGDS.</p> <p>(D) Western blot demonstrating an increase of immunoreactive MMP-7 in conditioned medium from A549 epithelial cells stimulated with osteopontin. Activation of pro-MMP-7 by APMA is shown in leftmost lane.</p> <p>(E) Zymography of conditioned media in 12.5% SDS gels containing bovine CM-transferrin (0.3 mg/ml) and heparin as substrate.</p> <p>C, control; OPN, osteopontin.</p></div

Osteopontin Expression Levels by Microarray Analysis from Controls and IPF Lungs

<div><p>Total RNA was used to generate double-stranded cDNA and biotin-labeled cRNA. Fragmented cRNA was hybridized to Codelink Uniset I slides and stained and scanned as described in Materials and Methods.</p> <p>(A) A log scale scatter plot of the average of intensity of all the genes on the arrays in controls (x-axis) and IPF (y-axis). Colored points indicate 178 genes that were significantly changed (<i>p</i> < 0.01 in TNoM and Student's t-test). Points are colored by their fold ratios; progressive shades of blue indicate increase, and progressive shades of red indicate decrease. Points colored in gray did not reach significance. Oblique solid line indicates the line of equality. Two green dashed lines are lines that depict 10-fold change.</p> <p>(B) Osteopontin levels in individual samples are shown. The y-axis is expression level in arbitrary fluorescence levels (log scale). The blue quadrangles are osteopontin levels in individual samples. Heat map shows sample osteopontin levels normalized to the geometric mean of osteopontin in controls and log base 2-transformed.</p></div

Weakest Link Models of Osteopontin and MMP-7

<p>IPF samples are depicted in solid dots and controls in open dots; the y-axis is MMP-7 expression and x-axis is osteopontin expression. Black solid line is the curve of optimal use showing that the expression levels for MMP-7 and osteopontin jointly interact to determine the IPF phenotype. The probability contour plot is shown in terms of the observed expression data (scale is log base 2 for gene expression data) (A); and probability contour plot is shown in terms of percentiles of the data (scale is percentiles) (B).</p

Osteopontin Levels in BAL Fluid

<p>Quantification of osteopontin by ELISA was performed in BAL fluid samples from 18 IPF patients and 10 healthy individual controls. An increased concentration of soluble osteopontin was found in the BAL fluid obtained from IPF patients compared with healthy controls. The data represent the mean ± standard deviation (SD). * <i>p</i> < 0.01.</p

Effect of Osteopontin on Fibroblasts and Epithelial Cell Migration

<div><p>Fibroblasts (A) and A549 epithelial cells (B) were placed in the upper compartment of a Boyden-type chamber, and Ham's F-12 medium containing 5% BSA alone or with 10 μg/ml of osteopontin was added to the lower compartment. After 8 h of incubation, the migrating cells were stained, and the absorbance of the stained solution was measured by ELISA. In parallel experiments, osteopontin-stimulated cells were treated with anti-α_vβ₃, anti-CD44, and GRGDS. Each bar represents the mean ± SD of three experiments; *<i>p</i> < 0.01; **<i>p</i> < 0.05.</p> <p>OPN, osteopontin</p></div

Osteopontin and MMP-7 Colocalization in IPF Lungs

<div><p>(A−C) MMP-7 staining is shown in green (A), osteopontin is shown as red staining (B), and overlap of staining is shown in yellow (C), suggest colocalization of MMP-7 and osteopontin in alveolar epithelial cells in IPF lungs (60×).</p> <p>(D) A lower-magnification image (20×) of the same region (A–C) with the same color coding. The white rectangle depicts area shown in (A−C).</p></div