In Idiopathic pulmonary fibrosis (IPF), inflammation and widespread fibrosis result from repeated injury of the alveolar epithelium. Myofibroblasts are considered to play a major role in fibrosis through excessive deposition of extracellular matrix (ECM) during wound healing processes. Resident fibroblasts differentiate into more contractile myofibroblasts (Fibroblast-to-Myofibroblast Transition; FMT), secreting many ECM proteins. Epithelial-to-Mesenchymal Transition (EMT) of bronchial epithelial cells (HBEC) may account for increased numbers of fibroblasts, which can subsequently transform into myofibroblasts. We have developed two validated assays, mimicking both FMT and EMT processes in vitro using lung cells derived from IPF patients or healthy donors, to evaluate clinical candidates.
Primary human fibroblasts and HBEC derived from IPF patients, were isolated in-house from commercially sourced tissue. Cells from healthy donors were commercially sourced. Transdifferentiation of cells, cultured in 384-well plates, was induced with TGF-β1 and assessed by high content imaging (INCell 2200) following immunostaining for αSMA (FMT) and fibronectin (FN1, EMT). Concentration response curves were evaluated for the clinically approved drug nintedanib for its effect on both processes relevant to IPF.
TGF-β1 consistently induced αSMA expression in fibroblasts and FN1 expression in HBEC in a concentration-dependent manner, indicative of FMT and EMT, respectively. TGF-β1 mediated αSMA and FN1 expression could be fully restrained by treatment with the ALK-5 inhibitor; SB525334. Nintedanib consistently induced a concentration-dependent reduction of TGF-β1-mediated αSMA and FN1 expression, consistent with its anti-fibrotic indication in vivo. Nintedanib showed signs of potential cytotoxicity in FMT, evaluated by the effect on the number of nuclei, therewith leading to a likely overestimated inhibitory effect on αSMA expression. In HBEC, TGF-β1 induces a G1 cellcycle arrest by increased expression and/or stabilization of cyclin-dependent kinase inhibitors. Conversely, nintedanib induced an increase in number of nuclei in HBEC, indicative of neutralizing the TGF-β1 induced G1 arrest.
In conclusion, both FMT and EMT assays are compatible with high throughput screening of pharmacological agents, like small molecules, RNAi vectors and biologics and can therefore be a useful translatable tool for evaluating the potency and efficacy of prospective anti-fibrotic drugs.