避免肌成纤维细胞凋亡是纤维化疾病的标志,最终导致持续的肌成纤维细胞活化,细胞外基质(ECM)沉积,和重塑。靶向肌成纤维细胞凋亡正在成为逆转已建立的纤维化的新型治疗策略。我们最近发现,在基质硬度驱动的成纤维细胞向肌成纤维细胞转分化过程中,在僵硬激活的肌成纤维细胞中,“线粒体启动”(准备进行凋亡)显着增加。因此,肌成纤维细胞,传统上被视为抗凋亡细胞,当生存途径被阻断时,似乎准备死亡,我们称之为“准备死亡”的细胞状态。“这种易于凋亡的表型是由负载在成肌纤维细胞线粒体中的高水平促凋亡蛋白驱动的,这需要同时上调促存活BCL-2蛋白以抑制线粒体凋亡并确保存活。这里,我们描述了一种称为BH3谱分析的方法,该方法测量了myo/成纤维细胞凋亡引发以及它们对存活的抗凋亡依赖性。此外,我们描述了如何使用BH3谱分析来预测肌成纤维细胞对靶向促存活BCL-2蛋白的治疗剂的反应,也称为BH3模拟药物。最后,我们描述了通过膜联蛋白V染色评估肌成纤维细胞对外来细胞凋亡敏感性的方法。
Evasion of apoptosis by myofibroblasts is a hallmark of fibrotic diseases, ultimately leading to persistent myofibroblast activation, extracellular matrix (ECM) deposition, and remodeling. Targeting myofibroblast apoptosis is emerging as a novel therapeutic strategy to reverse established fibrosis. We have recently discovered that in the process of fibroblast-to-myofibroblast transdifferentiation driven by matrix stiffness, the \"mitochondrial priming\" (readiness to undergo apoptosis) is dramatically increased in stiffness-activated myofibroblasts. Thus, myofibroblasts, traditionally viewed as apoptosis-resistant cells, appear poised to die when survival pathways are blocked, a cellular state we call \"primed for death.\" This apoptosis-prone phenotype is driven by high levels of pro-apoptotic proteins loaded in myofibroblast\'s mitochondria, which require concomitant upregulation of pro-survival BCL-2 proteins to suppress mitochondrial apoptosis and ensure survival. Here, we describe a method called BH3 profiling which measures myo/fibroblast apoptotic priming as well as their antiapoptotic dependencies for survival. In addition, we describe how BH3 profiling can be used to predict myofibroblast responses to therapeutic agents targeting pro-survival BCL-2 proteins, also known as BH3 mimetic drugs. Finally, we describe methods to assess myofibroblast sensitivity to extrinsic apoptosis via Annexin V staining.