Methods for Studying Myofibroblast Apoptotic Pathways.

Abstract

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.