Modulation of Hsp90 signaling to limit corneal fibrosis and improve ocular drug penetration

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ABSTRACT Keratoablative surgeries, including photorefractive keratectomy (PRK), phototherapeutic keratectomy (PTK), and laser assisted in situ keratomileusis (LASIK), are commonly performed procedures to correct refractive error and treat anterior stromal corneal diseases. The success of these surgeries requires a well-coordinated corneal wound healing response to limit post-operative corneal fibrosis. Heat shock protein 90 (Hsp90) is a key molecular chaperone responsible for the correct folding of many cellular proteins. In addition, Hsp90 has been shown to regulate two signaling pathways important to wound healing, transforming growth factor β (TGF-β) and the Hippo (mainly YAP and TAZ) pathways, by (1) stabilizing the activated TGF-β receptor complex and (2) targeting YAP and TAZ for degradation by the proteasome. Our lab and others have demonstrated the importance of both cytoactive factors and biophysical cues on determining the responses of corneal stromal cells. For example, corneal fibroblasts stimulated with TGF-β and grown on stiff substrates, mimicking a corneal wound bed, will upregulate αSMA expression and transdifferentiate to myofibroblasts. Clinically, excessive numbers or sustained persistence of myofibroblasts can be associated with development of corneal fibrosis and haze. YAP and TAZ, two important mechanotransducers, “sense” the matrix stiffness surrounding the cell. When grown on stiffer substrates, YAP and TAZ will localize the nucleus, resulting in the expression of multiple downstream molecules, including TGF-β. We propose to inhibit Hsp90 to modulate both the TGF-β and TAZ signaling pathways to limit αSMA expression and corneal fibrosis/haze. Experiments with knockout mice and with chemical inhibitors of these pathways are designed to help dissect the interaction between TGF-β and TAZ in the context of corneal wound healing. In addition, we have investigated the role Hsp90 inhibition in corneal epithelial cells. We demonstrate that treatment of stratified corneal epithelial cells with an Hsp90 inhibitor can result in the disruption of paracellular tight junctions, characterized by reduced trans- epithelial electrical resistance and loss of ZO-1 localization at the epithelial cell borders. We propose to define the toxicity, time course and effect on permeability of an Hsp90 inhibitor on corneal epithelial cells, both in vitro and in vivo. Results from these experiments could lead to the development of a novel method for increasing drug permeability, helping to overcome one the of the largest barrier to topical drug delivery, the corneal epithelial tight junction. This proposal is focused on two independent outcomes, (1) limiting corneal fibrosis/haze during wound healing and (2) increase corneal permeability to promote topical drug penetration, that are harmonized through the inhibition of Hsp90. Overall, findings from this proposal could prove to be clinically significant and lead to the development of novel therapeutic approaches to the benefit of patients.