Engineered Hydrogels with Reversible Moduli to Probe Myofibroblast Activation

描述(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(VICs)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，该细胞控制细胞外基质的形成，以响应瓣膜损伤；然而，VIC异常或长时间激活可导致过量的组织形成。除了可溶性因素，有证据表明环境僵硬是VIC激活的关键因素。当VIC被培养在STIF基质上而不是软基质上时，激活的标志物增加，例如-平滑肌肌动蛋白(SMA)应激纤维。此外，AKT活性上调，表明PI3K/AKT信号通路在VIC对机械刺激的反应中起重要作用。不幸的是，许多传统的细胞培养底物不自然地僵硬，固有地导致VIC激活。此外，历史上很难在不改变底物化学成分的情况下探测刚性的动态变化(例如，通过降解凝胶结构)，从而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，本研究旨在开发一种具有可逆力学性能的细胞培养基质，以探讨动态僵化和软化对肌成纤维细胞激活的影响。这将通过两个目标实现。在目标1中，目标是开发一种多肽交联型聚乙二醇水凝胶基质，该基质可在受控波长的光照射下可逆地变硬。具体地说，光暴露时会发生光异构化反应，导致多肽交联剂构象的变化，从而导致水凝胶硬度的相应变化。当用正交光波长照射时，多肽链将松弛并将水凝胶反转到其原始状态。光曝光 因此，将作为VIC细胞培养的僵硬、激活条件和软性、失活条件之间的非侵入性切换。假设VICS将通过PI3K/AKT信号通路的变化来感知硬化信号，并且激活将取决于在硬化的底物上培养的时间的长度。此外，预计停用VICS所需的时间也将取决于在硬化的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活性在不同细胞培养时间对底物模数的原位硬化和软化的动态响应。AKT活性的这些变化将与VIC激活的标志，如？-SMA的表达相关联，以确定基质僵硬、PI3K/AKT信号通路和细胞表型之间的关系。这项拟议的研究将深入了解VIC对环境提示的激活/去激活的分子水平基础，这可能有助于该领域确定治疗瓣膜疾病和逆转疾病表型的新策略。

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.