The ALK-5 inhibitor, SB525334, attenuates bleomycin-induced lung fibrosis.

<p>SB525334 was administered prophylactically (<b>A</b>) or therapeutically (<b>B</b>) at a dose of 60 mg/kg in chow beginning 1 day prior or 5 days after bleomycin instillation, respectively. SB525334 inhibited the lung fibrosis under both dosing conditions. Data are expressed as a percentage of the total collagen I stained area in the bleomycin vehicle control group ± SEM of n = 10–12 bleomycin treated mice in the prophylactic study and n = 6–9 in the therapeutic study. Saline treated control groups consisted of n = 5–7 mice. Significance (relative to the bleomycin vehicle control) was determined using a 2-tailed t-testand is denoted as **p<0.01 or ***p<0.001.</p

Bleomycin induces inflammatory and fibrotic changes in the lungs of mice.

<p>The numbers of BALF inflammatory cells increased after either a single (<b>A</b>) or repetitive (<b>D</b> bleomycin (black bars) instillation compared to the saline treated controls (white bars). Both a single (<b>B</b>) and repetitive (<b>E</b>) instillation of bleomycin led to increased fibrotic tissue in the lung. Changes in work of inflation (WoI), a measure of lung mechanics, were minimal in both the single (<b>C</b>) and repetitive (<b>F</b>) systems. Data are expressed as mean ± SEM of n = 7–8 mice. Significance (relative to the time-matched control at each time point) was determined using a Student’s t-test and is denoted as follows: *p<0.05; **p<0.01; and ***p<0.001.</p

Heatmap of gene set enrichment of mouse bleomycin-induced signatures in clinical IPF datasets.

<p>GSEA was performed for gene sets comprised of up-regulated genes in response to bleomycin at each time point from the mouse model. Enrichment of each gene set (denoted in rows) was determined against ranked lists of genes from clinical datasets comparing IPF vs. non-IPF conditions as well as IPF with acute exacerbation vs. stable IPF from two datasets (GSE2052, GSE10667, denoted in columns). Enrichment scores were plotted in a heatmap where gene sets enriched in IPF samples (nominal p<0.05, FDR <0.25) were denoted in red with intensity based on enrichment score (calculated in GSEA).</p

Unsupervised hierarchical clustering of genes differentially expressed between bleomycin and saline treated mice across time points.

<p>A union of 730 genes differentially expressed between bleomycin and saline treatments (fold-difference >2, false discovery rate (FDR) <0.05) were clustered across all mouse samples. Three distinct clusters of genes were revealed corresponding to phases of the bleomycin response, with most up-regulated genes being specific to the 7–14 days post-bleomycin treatment clusters. Inflammation phase (1–2 days) samples also clustered together and showed up-regulation of a subset of genes, while late fibrosis phase (21–35 days) samples also showed moderate up-regulation of a subset of genes.</p

Supervised hierarchical clustering of custom panels of genes across all mouse samples.

<p>Clustering of genes was performed for a panel of matrix metalloproteases (MMPs) and lysyl oxidase-like (LOXL) enzymes (<b>A</b>), of regulators of TGFβ signaling (B), and genes encoding matrix proteins (<b>C</b>) where samples were ordered based on treatment and time point.</p