Understanding valvular fibroblast mechanical memory using photo-tunable PEG hydrogels

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PROJECT SUMMARY Aortic valve stenosis (AVS) is a progressive disease characterized by excessive deposition of the extracellular matrix (ECM) components in the aortic valve, leading to increased valve stiffness and eventual heart failure. Unfortunately, the only current treatment is invasive surgical valve replacement or repair. A non-surgical alternative for treating AVS would reduce complications related to surgery, however, developing a pharmacological treatment has been limited by an incomplete understanding of disease progression. The clinical consensus is that early stages of AVS are characterized by persistent activation of resident fibroblasts (VICs). In healthy tissue, VICs transiently activate to myofibroblasts to repair injured tissue. In disease, chronic exposure to increased tissue stiffness prevents reversal of myofibroblast to quiescent VICs, resulting in persistently activated myofibroblasts. This time-dependent myofibroblast persistence implies VICs possess a mechanical memory of their past environments. Mesenchymal stem cells also possess a mechanical memory which is maintained through chromatin remodeling. In this proposal, we seek to understand the regulatory mechanisms responsible for myofibroblast persistence that will provide insights into AVS progression and identify potential therapy targets. We hypothesize that chromatin remodeling plays a role in myofibroblast persistence. In Aim I, we will determine the role of epigenetics in myofibroblast persistence. First, we will identify the mechanical cues that lead to transiently or persistently activated myofibroblasts. We will use photo-tunable PEG hydrogels where the hydrogel modulus may be tuned via UV light exposure to achieve moduli that mimic the stiffness of healthy and fibrotic tissues. We will initially culture VICs for varying times on stiff hydrogels, followed by an in situ modulus reduction to a softer hydrogel stiffness to mimic native tissue. After recovery at specified time points, VICs will be analyzed for persistence using established myofibroblast markers. To identify if mechanical cues play a role in chromatin remodeling, we will identify chromatin architecture differences between transient and persistent myofibroblasts by 1) immunofluorescence of methylation and acetylation, 2) RT-qPCR to measure the gene expression of common chromatin modifiers, 3) MATLAB algorithm to measure chromatin condensation. Finally, we will use chromatin remodeling inhibitors to determine if epigenetics plays a role in persistence. We will culture VICs under conditions to induce myofibroblast persistence, with and without the inhibitor, to determine if persistence is altered measured by the established myofibroblast markers. In Aim II, we will characterize molecular differences between the transient and persistently activated myofibroblasts by examining the RNA transcriptome of each myofibroblast population and identifying differentially expressed genes and signaling pathways between the two. We will validate regulatory pathways by blocking candidates identified from the transcriptome analysis with siRNAs.