The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of high unmet medical need. Although bromodomain (Brd) and extra terminal domain isoforms have recently been implicated in mediating inflammatory and oncologic indications, their roles in lung fibrosis have not been comprehensively assessed. We investigated the role of Brd on the profibrotic responses of lung fibroblasts (LFs) in patients with rapidly progressing IPF and a mouse bleomycin model of lung fibrosis. The enhanced migration, proliferation, and IL-6 release observed in LFs from patients with rapidly progressing IPF are attenuated by pharmacologic inhibition of Brd4. These changes are accompanied by enhanced histone H4 lysine5 acetylation and association of Brd4 with genes involved in the profibrotic responses in IPF LFs as demonstrated using chromatin immunoprecipitation and quantitative PCR. Oral administration of 200 mg/kg per day Brd4 inhibitor JQ1 in a therapeutic dosing regimen substantially attenuated lung fibrosis induced by bleomycin in C57BL/6 mice. In conclusion, this study shows that the Brd4 inhibitor JQ1, administered in a therapeutic dosage, is capable of inhibiting the profibrotic effects of IPF LFs and attenuates bleomycin-induced lung fibrosis in mice. These results suggest that Brd4 inhibitors may represent a novel therapy for the treatment of rapidly progressing IPF.

Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extraterminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Lysophosphatidic acid is a class of bioactive phospholipid that mediates most of its biological effects through LPA receptors, of which six isoforms have been identified. The recent results from LPA1 knockout mice suggested that blocking LPA1 signaling could provide a potential novel approach for the treatment of idiopathic pulmonary fibrosis. Here, we report the design and synthesis of pyrazole- and triazole-derived carbamates as LPA1-selective and LPA1/3 dual antagonists. In particular, compound 2, the most selective LPA1 antagonist reported, inhibited proliferation and contraction of normal human lung fibroblasts (NHLF) following LPA stimulation. Oral dosing of compound 2 to mice resulted in a dose-dependent reduction of plasma histamine levels in a murine LPA challenge model. Furthermore, we applied our novel antagonists as chemistry probes and investigated the contribution of LPA1/2/3 in mediating the pro-fibrotic responses. Our results suggest LPA1 as the major receptor subtype mediating LPA-induced proliferation and contraction of NHLF.

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