Novel high-throughput myofibroblast assays identify agonists with therapeutic potential in pulmonary fibrosis that act via EP.sub.2 and EP.sub.4 receptors

Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile [alpha]-smooth muscle actin ([alpha]-SMA)-expressing myofibroblasts that produce and secrete ECM are key effector cells of the disease and therefore represent a viable target for potential novel anti-fibrotic treatments. We used primary normal human lung fibroblasts (NHLF) in two novel high-throughput screening assays to discover molecules that inhibit or revert fibroblast-to-myofibroblast differentiation. A phenotypic high-content assay (HCA) quantified the degree of myofibroblast differentiation, whereas an impedance-based assay, multiplexed with MS / MS quantification of [alpha]-SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E.sub.1 (PGE.sub.1) alprostadil, which very effectively and potently attenuated and even reversed TGF-[beta]1-induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF-[beta]1-induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP.sub.2 and EP.sub.4, respectively). However, selective activation of the EP.sub.2 or the EP.sub.4 receptor was already sufficient to prevent or reverse TGF-[beta]1-induced NHLF myofibroblast transition. Our high-throughput assays identified chemical structures with potent anti-fibrotic properties acting through potentially novel mechanisms.