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Pathological features of pulmonary fibrosis include accumulation of myofibroblasts and increased extracellular matrix (ECM) deposition in lung tissue. Contractile α–smooth muscle actin (α–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 α–SMA and collagen 1 alpha 1 (COL1) protein, provided a measure of contractility and ECM formation. The synthetic prostaglandin E1 (PGE1) alprostadil, which very effectively and potently attenuated and even reversed TGF–β1–induced myofibroblast differentiation, was identified by screening a library of approved drugs. In TGF–β1–induced myofibroblasts the effect of alprostadil was attributed to activation of prostanoid receptor 2 and 4 (EP2 and EP4, respectively). However, selective activation of the EP2 or the EP4 receptor was already sufficient to prevent or reverse TGF–β1–induced NHLF myofibroblast transition. Our high–throughput assays identified chemical structures with potent anti–fibrotic properties acting through potentially novel mechanisms. [ABSTRACT FROM AUTHOR]

Intratracheal administration of bleomycin induces fibrosis in the lung, which is mainly assessed by histopathological grading that is subjective. Current literature highlights the need of reproducible and quantitative pulmonary fibrosis analysis. If some quantitative studies looked at fibrosis parameters separately, none of them quantitatively assessed both aspects: lung tissue remodeling and collagenization. To ensure reliable quantification, support vector machine learning was used on digitalized images to design a fully automated method that analyzes two important aspects of lung fibrosis: (i) areas having substantial tissue remodeling with appearance of dense fibrotic masses and (ii) collagen deposition. Fibrotic masses were identified on low magnification images and collagen detection was performed at high magnification. To insure a fully automated application the tissue classifier was trained on several independent studies that were performed over a period of four years. The detection method generates two different values that can be used to quantify lung fibrosis development: (i) percent area of fibrotic masses and (ii) percent of alveolar collagen. These two parameters were validated using independent studies from bleomycin- and saline-treated animals. A significant change of these lung fibrosis quantification parameters- increased amount of fibrotic masses and increased collagen deposition- were observed upon intratracheal administration of bleomycin and subsequent significant beneficial treatments effects were observed with BIBF-1120 and pirfenidone. [ABSTRACT FROM AUTHOR]