Engineered Hydrogels with Reversible Moduli to Probe Myofibroblast Activation

具有可逆模量的工程水凝胶可探测肌成纤维细胞的激活

• 批准号: 8783970
• 负责人: Adrianne Rosales
• 金额: $ 5.15万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8783970
• 关键词: Address; Back; Cell Culture Techniques; Cells; Chemicals; Crosslinker; Cues; Development; Disease; Disease Progression; Elasticity; Engineering; Environment; Ethylene Glycols; Excision; Exposure to; Extracellular Matrix; Family suidae; Fibroblasts; Fibrosis; Gel; Goals; Growth Factor; Heart Valve Diseases; Heart Valves; Hour; Hydrogels; In Situ; Injury; Investigation; Lead; Length; Ligands; Light; Measures; Mechanics; Mediating; Molecular; Molecular Conformation; Monitor; Myofibroblast; PI3K/AKT; Pathway interactions; Peptides; Phenotype; Physiological; Play; Polymers; Polystyrenes; Process; Property; Proto-Oncogene Proteins c-akt; Reaction; Regulation; Research; Role; Signal Pathway; Signal Transduction; Smooth Muscle Actin Staining Method; Stenosis; Stress Fibers; Structure; Surface; Testing; Time; Tissues; aortic valve; base; cell type; crosslink; density; ethylene glycol; extracellular; insight; interstitial; interstitial cell; irradiation; monomer; novel therapeutics; public health relevance; research study; response; response to injury; synthetic peptide; tissue culture; treatment strategy; wound healing; wound injury

DESCRIPTION (provided by applicant): A significant cause of heart valve disease is valvular fibrosis and stenosis, or the stiffening and thickening of valvular tissue. Valvular interstitial clls (VICs) are the most predominant cell type in the heart valve and are thought to play a key role in disease progression. VICs become activated to form myofibroblasts, which control the formation of the extracellular matrix in response to valvular injury; however, abnormal or prolonged VIC activation can lead to excess tissue formation. In addition to soluble factors, there is evidence that environmental stiffness is a critical factor in VIC activation. When VICs are cultured on stif substrates versus soft substrates, markers of activation are increased, such as ¿-smooth muscle actin (¿-SMA) stress fibers. In addition AKT activity is upregulated, indicating that the PI3K/AKT signaling pathway is important to mediating the VIC response to mechanical cues. Unfortunately, many traditional cell culture substrates are unnaturally stiff and inherently lead t VIC activation. In addition, it historically has been difficult to probe dynamic changes in stiffnes without also changing the chemical composition of the substrate (e.g., by degrading gel structure), leading to confounding changes in network connectivity or ligand density. To address these issues, the proposed research aims to develop a cell culture substrate with reversible mechanical properties to probe the effects of dynamic stiffening and softening on myofibroblast activation. This will be accomplished in two aims. In Aim 1, the goal is to develop a peptide-crosslinked poly(ethylene glycol) hydrogel substrate that can reversibly stiffen upon exposure to controlled wavelengths of light. Specifically, a photoisomerization reaction will occur upon light exposure, causing a change in the peptide crosslinker conformation that will yield a corresponding change in hydrogel stiffness. Upon irradiation with an orthogonal wavelength of light, the peptide chains will relax and reverse the hydrogel to its original state. Light exposure will therefore serve as a noninvasive switch between stiff, activating conditions and soft, de-activating conditions of VIC cell culture. It is hypothesized that VICs will sense stiffening cues via changes in the PI3K/AKT signaling pathway and that activation will depend upon the length of the culture time on the stiffened substrates. Furthermore, it is anticipated that the time required to deactivate the VICs will also depend on the duration of culture on the stiffened substrates. This hypothesis will be carefully investigated in Aim 2, in which the reversibly tunable substrates from Aim 1 will be used to measure the dynamic response of AKT activity to in situ stiffening and softening of substrate modulus at various cell culture times. These changes in AKT activity will be correlated with markers of VIC activation, such as ¿-SMA expression, to determine the relationship between matrix stiffness, the PI3K/AKT signaling pathway, and cellular phenotype. The proposed research will lend insight to the molecular level basis of VIC activation/deactivation in response to environmental cues, which may help the field identify new strategies for the treatment of valvular disease and the reversal of diseased phenotypes.

描述（由申请人提供）：心脏瓣膜疾病的一个重要原因是瓣膜纤维化和狭窄，或瓣膜组织硬化和增厚。瓣膜间质细胞（VIC）是心脏瓣膜中最主要的细胞类型，被认为在疾病进展中起关键作用。VIC被激活以形成肌成纤维细胞，肌成纤维细胞控制细胞外基质的形成以响应瓣膜损伤;然而，异常或延长的维克激活可导致过量的组织形成。除了可溶性因素外，有证据表明环境刚性是维克激活的关键因素。当VIC在硬基质与软基质上培养时，激活标记物增加，例如<$-平滑肌肌动蛋白（<$-SMA）应力纤维。此外，AKT活性上调，表明PI 3 K/AKT信号通路对于介导维克对机械刺激的反应是重要的。不幸的是，许多传统的细胞培养基质是不自然的坚硬的，并且固有地导致t维克活化。此外，历史上难以在不改变基底的化学组成的情况下探测刚度的动态变化（例如，通过降解凝胶结构），导致网络连接性或配体密度的混杂变化。为了解决这些问题，拟议的研究旨在开发一种具有可逆机械性能的细胞培养基质，以探索动态硬化和软化对肌成纤维细胞活化的影响。这将通过两个目标来实现。在目的1中，目标是开发肽交联的聚（乙二醇）水凝胶基质，其可以在暴露于受控波长的光时可逆地交联。具体地，光异构化反应将在光暴露时发生，引起肽交联剂构象的变化，这将产生水凝胶刚度的相应变化。在用正交波长的光照射时，肽链将松弛并将水凝胶逆转到其原始状态。光暴露 因此将作为维克细胞培养的刚性活化条件和柔性失活条件之间的非侵入性转换。假设VIC将通过PI 3 K/AKT信号传导途径的变化感知硬化线索，并且激活将取决于在硬化基质上培养时间的长度。此外，预计灭活VIC所需的时间也将取决于在硬化基质上培养的持续时间。这一假设将在目标2中仔细研究，其中来自目标1的可逆可调底物将用于测量AKT活性对不同细胞培养时间下底物模量的原位硬化和软化的动态响应。AKT活性的这些变化将与维克活化的标志物（例如Δ-SMA表达）相关，以确定基质硬度、PI 3 K/AKT信号传导途径和细胞表型之间的关系。拟议的研究将深入了解维克激活/失活的分子水平基础，以响应环境线索，这可能有助于该领域确定治疗瓣膜病和逆转疾病表型的新策略。