Institute of Pharmacology and Toxicology, University Medical Center, Goettingen, Germany.
Randall Centre for Cell and Molecular Biophysics, Kings College London, London, UK.
Naunyn Schmiedebergs Arch Pharmacol. 2023 Aug;396(8):1687-1699. doi: 10.1007/s00210-023-02421-9. Epub 2023 Feb 17.
The anti-fibrotic drug pirfenidone (PFD) is currently in clinical testing for the treatment of heart failure with preserved ejection fraction; however, its effects on human cardiac cells have not been fully investigated. Therefore, we aimed to characterize the impact of PFD on human cardiac fibroblasts (CF) in 2D culture as well as in 3D-engineered connective tissues (ECT). We analyzed proliferation by automated cell counting and changes in signaling by immunoblotting. We generated ECT with different geometries to modify the cellular phenotype and investigated the effects of PFD on cell number and viability as well as on cell cycle activity. We further studied its effect on ECT compaction, contraction, stiffening, and strain resistance by ECT imaging, pole deflection analysis, and ultimate tensile testing. Our data demonstrate that PFD inhibits human CF proliferation in a concentration-dependent manner with an IC of 0.43 mg/ml and its anti-mitogenic effect was further corroborated by an inhibition of MEK1/2, ERK1/2, and riboprotein S6 (rpS6) phosphorylation. In ECT, a lower cell cycle activity was found in PFD-treated ECT and fewer cells resided in these ECT after 5 days of culture compared to the control. Moreover, ECT compaction as well as ECT contraction was impaired. Consequently, biomechanical analyses demonstrated that PFD reduced the stiffness of ECT. Taken together, our data demonstrate that the anti-fibrotic action of PFD on human CF is based on its anti-mitogenic effect in 2D cultures and ECT.
抗纤维化药物吡非尼酮（PFD）目前正在进行临床试验，以治疗射血分数保留的心力衰竭；然而，其对人体心脏细胞的影响尚未得到充分研究。因此，我们旨在研究 PFD 在二维培养的人类心脏成纤维细胞（CF）以及三维工程化结缔组织（ECT）中的作用。我们通过自动细胞计数分析增殖情况，并通过免疫印迹分析信号变化。我们生成了具有不同几何形状的 ECT，以改变细胞表型，并研究了 PFD 对细胞数量和活力以及细胞周期活性的影响。我们还通过 ECT 成像、极点偏转分析和极限拉伸测试进一步研究了其对 ECT 压实、收缩、变硬和应变阻力的影响。我们的数据表明，PFD 以浓度依赖的方式抑制人 CF 的增殖，其 IC 为 0.43mg/ml，其抗有丝分裂作用进一步得到了 MEK1/2、ERK1/2 和核糖体蛋白 S6（rpS6）磷酸化抑制的证实。在 ECT 中，PFD 处理的 ECT 中的细胞周期活性较低，与对照组相比，培养 5 天后，这些 ECT 中的细胞数量较少。此外，ECT 压实和 ECT 收缩受损。因此，生物力学分析表明，PFD 降低了 ECT 的刚度。总之，我们的数据表明，PFD 对人 CF 的抗纤维化作用基于其在 2D 培养物和 ECT 中的抗有丝分裂作用。
