ABSTRACT: BACKGROUND:Idiopathic pulmonary fibrosis (IPF) is a rapidly progressing disease with challenging management. To find novel effective therapies, better preclinical models are needed for the screening of anti-fibrotic compounds. Activated fibroblasts drive fibrogenesis and are the main cells responsible for the accumulation of extracellular matrix (ECM). Here, a prolonged Scar-in-a-Jar assay was combined with clinically validated biochemical markers of ECM synthesis to evaluate ECM synthesis over time. To validate the model as a drug screening tool for novel anti-fibrotic compounds, two approved compounds for IPF, nintedanib and pirfenidone, and a compound in development, omipalisib, were tested. METHODS:Primary human lung fibroblasts from healthy donors were cultured for 12?days in the presence of ficoll and were stimulated with TGF-?1 with or without treatment with an ALK5/TGF-?1 receptor kinase inhibitor (ALK5i), nintedanib, pirfenidone or the mTOR/PI3K inhibitor omipalisib (GSK2126458). Biomarkers of ECM synthesis were evaluated over time in cell supernatants using ELISAs to assess type I, III, IV, V and VI collagen formation (PRO-C1, PRO-C3, PRO-C4, PRO-C5, PRO-C6), fibronectin (FBN-C) deposition and ?-smooth muscle actin (?-SMA) expression. RESULTS:TGF-?1 induced synthesis of PRO-C1, PRO-C6 and FBN-C as compared with unstimulated fibroblasts at all timepoints, while PRO-C3 and ?-SMA levels were not elevated until day 8. Elevated biomarkers were reduced by suppressing TGF-?1 signalling with ALK5i. Nintedanib and omipalisib were able to reduce all biomarkers induced by TGF-?1 in a concentration dependent manner, while pirfenidone had no effect on ?-SMA. CONCLUSIONS:TGF-?1 stimulated synthesis of type I, III and VI collagen, fibronectin and ?-SMA but not type IV or V collagen. Synthesis was increased over time, although temporal profiles differed, and was modulated pharmacologically by ALK5i, nintedanib, pirfenidone and omipalisib. This prolonged 12-day Scar-in-a-Jar assay utilising biochemical markers of ECM synthesis provides a useful screening tool for novel anti-fibrotic compounds.
