Engineered Hydrogels with Reversible Moduli to Probe Myofibroblast Activation

Rosales, Adrianne

Abstract

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.

Public Health Relevance

Valvular interstitial cells (VICs) are the prevalent cell type in heart valves and can contribute to valvular fibrosis upon prolonged activation. Because a stiff extracellular environment plays a key role in VIC activation, the proposed research will probe the role of dynamic changes in matrix stiffness on VIC activation via a new cell culture substrate with reversible mechanical properties. It is anticipated that the insight gained from this research will inform the development of strategies to reverse VIC activation, leading to new therapeutic treatments for heart valve disease.