Panel Discussion

Aortic valve stenosis (AVS) is a chronic, deadly disease that occurs in at least two percent of patients over 60 years of age in western countries. In AVS, the aortic valve leaflets thicken and stiffen as they become calcified. This results in disrupted blood flow as the valve can no longer open and close normally; over time, the chronic stiffening of the valve leaflet leads to enlargement of the left ventricle and ultimately contributes to heart failure. From a cellular perspective, AVS occurs as quiescent valvular interstitial cells (VICS) activate to persistent myofibroblasts. These myofibroblasts are characterized by an increase in pro-calcific factors and especially in alpha smooth muscle actin (⍺SMA). Mechanical and secretory changes that accompany this phenotypic change have been documented, but the epigenetic regulation of this transition has not been thoroughly investigated. Chromatin remodeling is an epigenetic change where histones can regulate the accessibility of chromatin. Additionally, chromatin remodeling has been strongly linked to cell fate determination in VICs, indicating that epigenetic mechanisms are involved. The in vitro culture stiffness has also been shown to play an extensive role in the activation of quiescent VICs to myofibroblasts. Hydrogels pose a unique opportunity to study this phenotypic transition through mimicking diseased tissue versus healthy tissue extracellular matrix (ECM). These covalently crosslinked polymer networks can be tailored to specific stiffnesses, and networks can be controllably softened to replicate these tissues. By utilizing photo-degradable hydrogels to mimic a stiff, fibrotic ECM versus a healthy, soft ECM, the epigenetic changes regulating AVS can be investigated. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) was used to investigate changes in the accessibility of chromatin in combination with RNA sequencing (RNA-seq) to assess the downstream effects of changes in chromatin accessibility for quiescent VICs versus myofibroblasts. Differentially expressed chromatin regions were then compared to RNA-seq results from stiff and soft hydrogel VIC cultures to identify possible regulatory genes. Four genes were found to be differentially expressed that directly relate to changes in chromatin accessibility: ACTA2, SERPINE1, ID3, and PAMR1. Additionally, 18 transcription factors (TFs) were implicated in this cellular transition. We hypothesize that these genes may be involved in myofibroblast regulation, and therefore regulating these will lead to significant changes in myofibroblast activation when cultured on the appropriate stiffness. These epigenetic changes provide new insight into potential mechanisms that regulate the stiffness-induced transition from quiescent VICs to myofibroblasts and that therefore may regulate AVS.