Regulation of Valve Interstitial Cell Phenotype and Function Using Biomimetic Hydrogels

The aortic valve regulates the unidirectional flow of oxygenated blood from the left ventricle to the systemic circulation. When severe congenital defects occur in aortic valves, valve replacement is inevitable in children. However, current options including mechanical valves and bioprosthetic valves, lack the ability to grow and remodel, which necessitates multiple valve replacements as children grow. Tissue engineering provides a possible avenue to generate a living valve substitute that can grow and remodel via combining cells, scaffolds and environmental cues. The cells used in this work were valvular interstitial cells (VICs), the predominant cell population in the valves, and responsible for extracellular matrix (ECM) synthesis in the valve tissue. VICs are highly heterogeneous and dynamic in phenotype, with the majority assuming a quiescent, fibroblast phenotype in healthy adult valves1,2. During valve injury or disease conditions, VICs may undergo myofibroblast activation or osteogenic differentiation3,4. Myofibroblast activation is characterized by the expression of smooth muscle -actin (SMA), and may cause valve fibrosis3,4; osteogenic differentiation is characterized by the upregulation of alkaline phosphatase (ALP), followed by tissue calcification, which is the leading cause of valve disease in the elderly (>60 years of age) and the failure of bioprosthetic valves5. However, the most common method of in vitro VIC culture on two-dimensional (2D) stiff substrates leads to myofibroblast activation of VICs. For better physiological relevance and future application in valve substitutes, there is a need to understand and regulate VIC behaviors within three-dimensional (3D) scaffolds that are more reminiscent to their native environments. This dissertation describes the development of a poly(ethylene glycol) (PEG)-based hydrogel platform to support VIC growth in 3D, and the exploration of free and immobilized bioactive cues to dictate VIC phenotype and behaviors toward the development of a living valve substitute.

Otherwise bioinert, PEG hydrogels were functionalized with cell-adhesive ligands RGDS and proteolytically degradable sequences (GGGPQGIWGQGK). The functionalized hydrogels supported VIC growth, proliferation and ECM remodeling (secretion of matrix metalloproteinase-2 and deposition of collagens) in 3D during the culture period of 4 weeks. The soft hydrogels with compressive moduli of ~4.3 kPa quickly reverted VICs from myofibroblast activation to a quiescent phenotype upon encapsulation, evidenced by the loss of αSMA expression. The functionalized PEG hydrogels are preferable to 2D stiff substrates for preservation of the native phenotype of VICs and resistance to calcification.

In an effort to potentially promote deposition of ECM components by encapsulated VICs, ascorbic acid (AA), which is a cofactor in the post-translational modification of collagen molecules6 and has been reported to increase collagen section by several other cell types7–9, was added to the culture media of cell-laden hydrogels. AA treatment promoted VIC-mediated ECM remodeling without negatively influencing their quiescent phenotype in hydrogels. AA also enhanced VIC spreading and proliferation while inhibiting apoptosis.

ECM-mimicking adhesive peptides with specific affinity to different receptors were immobilized on PEG hydrogels in order to regulate VIC adhesion, phenotype and ECM production. Expression of adhesion receptors by VICs was assessed via flow cytometry and used to guide the choice of peptides studied. The peptide RGDS with affinity to multiple integrin receptors, and specific receptor-targeting peptides DGEA (integrin 21), YIGSR (67 kDa laminin/elastin receptor; 67LR), and VAPG (67LR) were chosen based on the receptor expression profiles as well as the potential outcomes of each receptor binding. DGEA, YIGSR, and VAPG alone were insufficient to induce stable VIC adhesion. As a result, these peptides were studied in combination with 1 mM RGDS. For VICs cultured on 2D hydrogel surfaces, YIGSR and VAPG down-regulated the expression of αSMA (myofibroblast activation marker) whereas DGEA promoted VIC adhesion and VIC-mediated ECM deposition while inhibiting the activity of ALP (osteogenic differentiation marker). Further, YIGSR and DGEA in combination promoted ECM deposition while inhibiting both myofibroblastic and osteogenic differentiation. However, VICs behaved differently when cultured within 3D hydrogels, with VICs assuming a quiescent, fibroblastic phenotype without any calcification under all peptide conditions tested. DGEA promoted ECM deposition by VICs within hydrogels without causing VIC activation.

The results of this research provide a clearer understanding of VIC biology and pathology under biomimetic conditions and lay the groundwork for constructing living valve substitutes using the tissue engineering approach. The hydrogel platform developed in this work may also be applied to study the initiation and progression of valvular diseases.