Engineered Hydrogels with Reversible Moduli to Probe Myofibroblast Activation

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（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被激活形成肌成纤维细胞，在瓣膜损伤时控制细胞外基质的形成；然而，异常或延长的VIC激活可导致多余的组织形成。除了可溶性因素外，有证据表明环境刚度是VIC激活的关键因素。当vic在硬基质上与软基质上培养时，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PI3K/AKT信号通路在介导VIC对机械信号的反应中起重要作用。不幸的是，许多传统的细胞培养基质不自然地坚硬，固有地导致VIC激活。此外，在不改变底物的化学成分（例如，通过降解凝胶结构）的情况下，很难探测刚度的动态变化，从而导致网络连通性或配体密度的混淆变化。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。在目标1中，目标是开发一种肽交联聚（乙二醇）水凝胶底物，该底物在暴露于受控波长的光下可以可逆地变硬。具体来说，在光照射下会发生光异构化反应，引起肽交联剂构象的变化，从而导致水凝胶硬度的相应变化。当用正交波长的光照射时，肽链会松弛并使水凝胶逆转到原来的状态。曝光

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.