Modulating Glycosaminoglycan Fine Structure in Valvular Interstitial Cells
Wong, Fergus Forbes
Grande-Allen, K. Jane
Doctor of Philosophy
The effect of glycosaminoglycan (GAG) fine structure on valvular interstitial cell (VIC) interactions with matrix is poorly understood. In heart valves, proteoglycans (PGs) and GAGs are differentially expressed across valve types, and become abundant with age and during disease development. The consequence of maladaptive PG/GAG expression is unclear and the implications of PG/GAG changes on valvular interstitial cell behavior need to be explored. However, the complexity of GAG structure has made it difficult to study the role of fine structural moieties. GAGs may vary in polymer length and each monomer may undergo modifications, resulting in a highly variable and difficult to analyze structure. The present work describes the development of a GAG modulation toolkit and an exploration of how GAG fine structure influences VIC behavior. Adeno-associated virus (AAV) transduction efficiency is characterized and optimized for VICs, resulting in the most effective method for VIC gene delivery to date. AAV gene delivery is leveraged to build a library of vectors to regulate gene expression of GAG biosynthesis enzymes in order to modulate GAG fine structure. The effect of GAG fine structure on VIC-matrix interactions is explored using quantitative functional assays to reveal a role for GAG chain length and sulfation in VIC adhesion, migration, and contraction. Overall, these studies advance the repertoire of tools used to study and manipulate VICs, and expand the understanding of how GAG fine structure influences VIC functions.