Understanding valvular fibroblast mechanical memory using photo-tunable PEG hydrogels

项目总结 主动脉瓣狭窄(AVS)是一种进展性疾病，其特征是细胞外过度沉积。 主动脉瓣中含有基质(ECM)成分，导致瓣膜僵硬增加，最终导致心力衰竭。 不幸的是，目前唯一的治疗方法是侵入性外科瓣膜置换或修复。一种非手术的 治疗动静脉曲张的替代方案将减少与手术相关的并发症，然而，开发一种 药物治疗由于对疾病进展的不完全了解而受到限制。临床部 一致认为，AVS的早期特征是常驻成纤维细胞(VIC)的持续激活。 在健康组织中，VICS瞬间激活成肌成纤维细胞以修复受损组织。在疾病中，慢性暴露 增加组织硬度可防止肌成纤维细胞逆转为静止期VIC，导致持续性 激活的肌成纤维细胞。这种时间依赖性的肌成纤维细胞持久性表明VICS具有机械 对他们过去环境的记忆。间充质干细胞也拥有机械记忆，这是 通过染色质重塑来维持。在这项建议中，我们试图了解监管机制 负责肌成纤维细胞的持久性，这将为AVS的进展提供洞察力并确定潜在的 治疗目标。我们假设染色质重塑在肌成纤维细胞持久性中起作用。在AIM I中， 我们将确定表观遗传学在肌成纤维细胞持久性中的作用。首先，我们将识别机械线索 这会导致瞬时或持续激活的肌成纤维细胞。我们将在以下情况下使用可光调的聚乙二醇水凝胶 水凝胶的模数可以通过紫外线照射来调节，以达到模拟健康人的僵硬的模数 和纤维性组织。我们首先会在硬性水凝胶上培养不同时间的VICs，然后是原位模数。 减少到更柔软的水凝胶硬度，以模仿天然组织。在指定时间点恢复后，受害者将 使用已建立的肌成纤维细胞标记物进行持续性分析。以确定机械提示是否起作用 在染色质重塑中，我们将识别暂时性和持续性染色质结构的差异。 肌成纤维细胞通过1)甲基化和乙酰化的免疫荧光，2)RT-qPCR检测基因 常用的染色质修饰物的表达，3)用MatLab算法测量染色质缩合。最后， 我们将使用染色质重塑抑制剂来确定表观遗传学是否在持久性中起作用。我们将培养 在VICS条件下诱导肌成纤维细胞持续存在，使用和不使用抑制剂，以确定是否 持续性通过已建立的肌成纤维细胞标记物来衡量。在AIM II中，我们将描述 通过检测RNA检测瞬时和持久激活的肌成纤维细胞的分子差异 不同肌成纤维细胞群的转录组及其差异表达基因和信号转导 两者之间的路径。我们将通过阻止从 用siRNA进行转录组分析。

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.