Understanding valvular fibroblast mechanical memory using photo-tunable PEG hydrogels

使用光可调 PEG 水凝胶了解瓣膜成纤维细胞机械记忆

• 批准号: 9541843
• 负责人: Cierra Walker
• 金额: $ 3.75万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-15 至 2021-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9541843
• 关键词: Acetylation; Adult; Algorithms; Aortic Valve Stenosis; Architecture; Back; Biophysics; Cell Culture Techniques; Cells; Chromatin; Chromatin Remodeling Factor; Chronic; Clinical; Collagen; Consensus; Cues; Deposition; Development; Disease; Disease Progression; Dose; Environment; Epigenetic Process; Exhibits; Exposure to; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression; Genes; Genetic; Goals; Heart Valves; Heart failure; Histone Acetylation; Hydrogels; Immunofluorescence Immunologic; In Situ; Injury; Interstitial Collagenase; Lead; Measures; Mechanics; Memory; Mesenchymal Stem Cells; Methylation; Modulus; Molecular; Myofibroblast; Operative Surgical Procedures; Pathologic; Pathway interactions; Pharmacological Treatment; Physical condensation; Play; Population; Process; Progressive Disease; RNA; Recovery; Regulatory Pathway; Research; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Smooth Muscle Actin Staining Method; Specific qualifier value; Stress Fibers; Surgical Valves; Time; Tissue Therapy; Tissues; Ultraviolet Rays; aortic valve; aortic valve disorder; chromatin remodeling; connective tissue growth factor; differential expression; ethylene glycol; histone methylation; inhibitor/antagonist; injured; insight; interstitial cell; mRNA Expression; prevent; repaired; response; small molecule; targeted treatment; transcriptome; valve replacement

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.

项目摘要 主动脉瓣狭窄（AVS）是一种进行性疾病，其特征是细胞外沉积过多 主动脉瓣中的矩阵（ECM）组件，导致瓣膜刚度增加和最终心力衰竭。 不幸的是，目前唯一的治疗方法是侵入性手术瓣膜更换或修复。非手术 但是，治疗AV的替代方法可以减少与手术有关的并发症，但是， 对疾病进展的不完全了解，药理学治疗受到限制。临床 共识是，AV的早期阶段的特征是持续成纤维细胞（VIC）的持续激活。 在健康的组织中，VIC会瞬时激活肌纤维细胞以修复受伤的组织。在疾病中，慢性暴露 增加组织刚度可防止肌成纤维细胞逆转静止的vics，导致持续 激活的肌纤维细胞。这种时间依赖的肌纤维细胞持久性意味着VIC具有机械性 记忆他们过去的环境。间充质干细胞还具有机械记忆 通过染色质重塑保持。在此提案中，我们试图了解监管机制 负责肌纤维细胞持久性，这将为AVS的进展提供见解并确定潜力 治疗靶标。我们假设染色质重塑在肌纤维细胞持久性中起作用。在目标一世中 我们将确定表观遗传学在肌纤维细胞持久性中的作用。首先，我们将确定机械提示 这导致瞬时或持续激活的肌纤维细胞。我们将使用光粘的PEG水凝胶 水凝胶模量可以通过紫外线暴露来调节以实现模拟健康刚度的模量 和纤维化组织。我们最初将在僵硬的水凝胶上培养不同的时间，然后是原位模量 还原至模拟天然组织的较软的水凝胶刚度。在指定时间点恢复后，VICS将 使用已建立的肌纤维细胞标记来分析持久性。确定机械提示是否发挥作用 在染色质重塑中，我们将识别瞬态和持久性之间的染色质结构差异 肌纤维细胞1）甲基化和乙酰化的免疫荧光，2）RT-QPCR测量基因 常见染色质修饰剂的表达，3）MATLAB算法以测量染色质缩合。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。我们将文化 条件下的VIC诱导有或没有抑制剂的肌纤维细胞持久性，以确定是否是否 持久性通过已建立的肌纤维细胞标记来改变。在AIM II中，我们将描述 瞬时和持续激活的肌纤维细胞之间的分子差异通过检查RNA 每个肌纤维细胞种群的转录组，并识别差异表达的基因和信号传导 两者之间的途径。我们将通过阻止从该候选人确定的候选人来验证调节途径 用siRNAS进行转录组分析。