Novel high–throughput myofibroblast assays identify agonists with therapeutic potential in pulmonary fibrosis that act via EP2 and EP4 receptors

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.

肺纤维化（pulmonary fibrosis）的病理特征表现为肺组织内肌成纤维细胞（myofibroblasts）聚集以及细胞外基质（extracellular matrix, ECM）沉积增加。能够产生并分泌细胞外基质、表达收缩型α-平滑肌肌动蛋白（α–smooth muscle actin, α–SMA）的肌成纤维细胞，是该疾病的关键效应细胞，因此可作为新型抗纤维化治疗手段的潜在靶点。本研究采用原代正常人肺成纤维细胞（primary normal human lung fibroblasts, NHLF），依托两种新型高通量筛选实验，旨在筛选可抑制或逆转成纤维细胞向肌成纤维细胞分化的活性分子。其中，表型高内涵筛选实验（phenotypic high–content assay, HCA）可量化肌成纤维细胞的分化程度；而基于阻抗的检测实验结合α-SMA与胶原蛋白1α1（collagen 1 alpha 1, COL1）蛋白的MS/MS定量分析，能够同时评估细胞收缩能力与细胞外基质生成情况。通过对获批药物库进行筛选，本研究发现合成前列腺素E1（prostaglandin E1, PGE1）类药物前列地尔（alprostadil）可高效且强力地减弱甚至逆转转化生长因子β1（transforming growth factor β1, TGF-β1）诱导的肌成纤维细胞分化。在TGF-β1诱导的肌成纤维细胞中，前列地尔的作用机制与其激活前列腺素受体2（prostanoid receptor 2, EP2）和前列腺素受体4（prostanoid receptor 4, EP4）相关。不过，仅选择性激活EP2或EP4受体，就足以阻止或逆转TGF-β1诱导的NHLF向肌成纤维细胞的转化。本研究的高通量筛选实验成功发现了一批具有强力抗纤维化活性、且可能通过全新机制发挥作用的化学结构分子。