Effects of Cell Seeding Density, Extracellular Matrix Composition, and Geometry on Yes-Associated Protein...

Corneal fibroblasts are central to normal and abnormal wound healing in the cornea. During the wound healing process, several biochemical and biophysical signals that are present in the extracellular matrix (ECM) play critical roles in regulating corneal fibroblast behavior. The translocation and activation of Yes-associated protein (YAP)-a main transcriptional factor in the Hippo signaling pathway-is one example of mechanotransduction involving these signals. However, how corneal fibroblasts integrate these simultaneous cues is unknown. In this study, we utilized well-defined micropatterns of aligned collagen fibrils and other ECM proteins to explore the effects of cell density, topography, geometric confinement, and ECM composition on the translocation of YAP in corneal fibroblasts. We observed that when human corneal fibroblasts (HTKs) were confined to narrow micropatterns (50 μm and 100 μm) of proteins, there was a high degree of cell alignment irrespective of cell seeding density. However, the location of YAP was dependent upon the cell seeding density, ECM composition, and topography. YAP was more nuclear-localized on substrates coated with aligned collagen fibrils or fibronectin as compared to substrates coated with monomeric collagen, random collagen fibrils, or poly-L-Lysine. In addition, we also observed that YAP nuclear localization was significantly reduced when HTKs were cultured on aligned collagen fibrils, monomeric collagen, or fibronectin in the presence of monoclonal blocking antibodies against α₅ or β₁ integrin subunits. Finally, we observed that HTK cells formed fibrillar fibronectin on both monomeric collagen and aligned collagen fibrils. These findings provide new insights into how simultaneous biochemical and biophysical cues affect YAP localization in corneal fibroblasts.