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: 9766817
• 负责人: Cierra Walker
• 金额: $ 3.8万
• 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/9766817
• 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.

项目概要 主动脉瓣狭窄（AVS）是一种进行性疾病，其特征是细胞外物质过度沉积 主动脉瓣中的基质（ECM）成分，导致瓣膜硬度增加并最终导致心力衰竭。 不幸的是，目前唯一的治疗方法是侵入性手术瓣膜置换或修复。一种非手术 治疗 AVS 的替代方案将减少与手术相关的并发症，但是，开发一种 由于对疾病进展的不完全了解，药物治疗受到限制。临床上 人们一致认为，AVS 早期阶段的特点是常驻成纤维细胞 (VIC) 持续激活。 在健康组织中，VIC 会短暂激活肌成纤维细胞以修复受损组织。在疾病中，长期暴露 增加组织硬度可防止肌成纤维细胞逆转为静止 VIC，从而导致持续 激活的肌成纤维细胞。这种时间依赖性肌成纤维细胞持久性意味着 VIC 具有机械作用 对过去环境的记忆。间充质干细胞还具有机械记忆 通过染色质重塑来维持。在本提案中，我们寻求了解监管机制 负责肌成纤维细胞的持久性，这将提供对 AVS 进展的见解并识别潜在的 治疗目标。我们假设染色质重塑在肌成纤维细胞的持久性中发挥作用。在《目标一》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 导致肌成纤维细胞短暂或持续激活。我们将使用光可调 PEG 水凝胶，其中 水凝胶模量可以通过紫外光照射来调节，以达到模拟健康硬度的模量 和纤维化组织。我们首先将在硬水凝胶上培养 VIC 不同时间，然后进行原位模量 降低至较软的水凝胶硬度以模仿天然组织。在指定时间点恢复后，VIC 将 使用已建立的肌成纤维细胞标记物分析其持久性。确定机械线索是否发挥作用 在染色质重塑中，我们将识别短暂性和持久性之间的染色质结构差异 肌成纤维细胞通过 1) 甲基化和乙酰化的免疫荧光，2) RT-qPCR 来测量基因 常见染色质修饰剂的表达，3）MATLAB算法测量染色质凝聚。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发挥作用。我们将文化 在有或没有抑制剂的情况下，在诱导肌成纤维细胞持久性的条件下进行 VIC，以确定是否 通过已建立的肌成纤维细胞标记来测量持久性的改变。在目标 II 中，我们将描述 通过检查 RNA 来区分瞬时和持续激活的肌成纤维细胞之间的分子差异 每个肌成纤维细胞群体的转录组并鉴定差异表达的基因和信号传导 两者之间的路径。我们将通过阻止从 使用 siRNA 进行转录组分析。