PROJECT SUMMARY Aortic valve stenosis (AVS) is a progressive disease where fibroblast-like valvular interstitial cells (VICs) become persistently activated myofibroblasts, which contribute to pathologic aortic valve leaflet stiffening. AVS is treated with valve replacement surgeries, which may be avoided if small molecule drug combinations could be identified to inhibit persistent myofibroblast activation. However, the molecular mechanisms regulating persistent myofibroblast activation are unknown and may vary from patient-to-patient and/or by sex. My proposed R00 research seeks to continue characterizing the sex- and patient-specific differences that lead to persistent myofibroblast activation during AVS and optimize drug combinations to inhibit myofibroblast activation as a function of patient-specific cues. We hypothesize (i) sex-linked differences in how male and female VICs respond to mechanical cues and (ii) patient-specific biochemical cues found in AVS patient sera contribute to persistent activation and subsequent myofibroblast response to small molecule drugs. In Aim 1 (completed), we have characterized genes that escape X-chromosome inactivation that regulate myofibroblast activation uniquely in female VICs seeded on PEG hydrogels. In Aim 2 (in progress), we will generate persistently activated myofibroblasts in human AVS patient sera and determine alterations (e.g. open chromatin regions) in the VIC epigenome due to patient-specific serum factors using Assay for Transposase-Accessible Chromatin with sequencing (ATAC-seq). In Aim 3 (proposed), we will identify optimal combinations of small molecule drugs to inhibit persistent myofibroblast activation in the presence of AVS patient serum using a differential evolution algorithm. In sum, the proposed research will address an urgent, unmet need for sex-specific and precision medicine approaches for identifying molecular mechanisms of myofibroblast persistence, which may provide a bridge toward non-surgical AVS therapies.