Corneal Epithelial-Stromal Interactions During Regeneration and Fibrosis

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SUMMARY Corneal wound healing is a complex process involving corneal epithelial cell proliferation, myofibroblast generation, and extracellular matrix (ECM) deposition. In corneal wound models, the disruption of the Bowman's layer that separates the epithelium from stroma is often observed, and this disruption may lead to a different pathological (fibrotic) state, which we propose can be mediated by the intercellular signaling between epithelial and stromal cells in part via paracrine factors. TGF-β is a pleiotropic cytokine that exists in three isoforms (TGF-β1, -β2, and -β3), which exert biological effects through signaling pathways to maintain corneal integrity and wound healing. Previously, we found that TGF-β1 is involved in corneal fibrotic wound healing by stimulating myofibroblast differentiation; whereas, TGF-β3 application after corneal wounding reversed and diminished the fibrotic response in vitro and in vivo, respectively. Despite the functional differences in TGF-β isoforms, the molecular mechanisms in dampening fibrosis remain poorly understood. Amid other paracrine factors, extracellular vesicles (EVs) are recognized as mediators for cell-cell communication. EVs can selectively engulf a part of their parental cell and become enriched in a repertoire of bioactive cargo (e.g., proteins or lipids), and offset their cargo into recipient cells by ensuing physiological changes. We have shown that corneal epithelial cell-derived EVs can trigger myofibroblast differentiation and generate an ECM microenvironment that promotes myofibroblast persistence, which is key for corneal scarring; however, the bioactive cargo driving this remains unclear. Of disease relevance, keratoconus (KCN) is of interest because it leads to corneal stromal scarring even without any acute trauma or known underlying etiology. Pathologically, KCN is similar to our wound-healing models in that the KCN corneas: 1) develop gaps in Bowman's layer that allow direct contact between epithelium and stroma; 2) have EVs present between epithelial cells and stroma; and 3) develop myofibroblast differentiation that leads to scarring under the breaks in Bowman's layer. TGF-β's role in myofibroblast differentiation and ECM remodeling suggests involvement in KCN's pathogenesis, either in a causative or secondary repair role, leading to structural changes in KCN. In this proposal, we hypothesize that loading TGF-β3 onto EVs and applying to corneal wounds will drive healing without scarring by dampening the fibrotic response and preventing the onset of scar formation; however, TGF-β1-EVs will enhance the scarring in corneal wounds. Through these experiments we will also determine bioactive cargo from KCN-epithelial cell derived EVs that is contributing to KCN corneal scarring and disease progression. Relevance to Public Health—Collectively, this proposal will provide key mechanistic insights into corneal EV biology and their corresponding cargo, as well as identify the bioactive EV cargo that could be used to predict corneal scarring in KCN patients in the future.