Mechanisms of corneal stromal activation during regeneration and fibrosis by exosomes

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SUMMARY Corneal scarring (or fibrosis) is known to cause visual impairment in moderate to severe wounds, and with no single effective therapy for either the prevention or treatment, the need for new tools to treat and reduce the risk of scarring is urgent. Cell-cell communication is a vital component for the cornea that encompasses homeostasis, regeneration, and fibrosis. We have shown that the wound-healing mechanisms of corneal epithelial cells are comprised in their secretome, with extracellular vesicles (EVs) being a key vector. During EV production, the EVs selectively engulf a part of their parental cell and become enriched in a repertoire of bioactive cargo (e.g., proteins, lipids, and RNAs). We recently reported that human corneal fibroblasts (hCF) treated by TGF-β1 increased expression of fibrotic markers that established a fibrotic matrix, unlike TGF-β3 (anti-fibrotic). Also, our data suggests that EVs derived from human corneal epithelial cells (hCE-EV) can trigger hCF differentiation, and the proteome of hCE-EV containing TGF-β1 or -β3 could be a driving mechanism that could contribute to the mechanistic action of corneal scar formation. Although, despite the wound-healing and corneal stromal activation capacity of hCE-EVs, our understanding of their bioactive properties and molecular mechanisms for such effects remains unclear. In this proposal, we hypothesize that the EV subsets, hCE (TGF-β1KD/-β3KD)-EVs, will trigger corneal myofibroblast differentiation to generate an activated stromal microenvironment that supports fibrotic healing. We propose the following Aims to test this hypothesis: Aim 1) Modulate and characterize hCE-EV subsets; Aim 2) Define stromal activation capacity of hCE-EV subsets in 2D and 3D models in vitro; and Aim 3) Determine how hCE-EV subsets effect corneal scarring in vivo. Relevance to Public Health—Collectively, this proposal will provide important insight for hCE- EV biology and corneal fibroblast-target cell interaction, which can be leveraged to develop novel EV-based therapies for the prevention and treatment of corneal scarring.