Impact of Exosomes Released by Different Corneal Cell Types on the Wound Healing Properties of Human Corneal Epithelial Cells

Abstract

Corneal wound healing involves communication between the different cell types that constitute the three cellular layers of the cornea (epithelium, stroma and endothelium), a process ensured in part by a category of extracellular vesicles called exosomes. In the present study, we isolated exosomes released by primary cultured human corneal epithelial cells (hCECs), corneal fibroblasts (hCFs) and corneal endothelial cells (hCEnCs) and determined whether they have wound healing characteristics of their own and to which point they modify the genetic and proteomic pattern of these cell types. Exosomes released by all three cell types significantly accelerated wound closure of scratch-wounded hCECs in vitro compared to controls (without exosomes). Profiling of activated kinases revealed that exosomes from human corneal cells caused the activation of signal transduction mediators that belong to the HSP27, STAT, β-catenin, GSK-3β and p38 pathways. Most of all, data from gene profiling analyses indicated that exosomes, irrespective of their cellular origin, alter a restricted subset of genes that are completely different between each targeted cell type (hCECs, hCFS, hCEnCs). Analysis of the genes specifically differentially regulated for a given cell-type in the microarray data using the Ingenuity Pathway Analysis (IPA) software revealed that the mean gene expression profile of hCECs cultured in the presence of exosomes would likely promote cell proliferation and migration whereas it would reduce differentiation when compared to control cells. Collectively, our findings represent a conceptual advance in understanding the mechanisms of corneal wound repair that may ultimately open new avenues for the development of novel therapeutic approaches to improve closure of corneal wounds.

Isolation and Characterization of Human Conjunctival Mesenchymal Stromal Cells and Their Extracellular Vesicles

“…There are some interesting studies analyzing this in the cornea. 13 , 14 , 31 , 32 Those studies also highlight that the basement membrane appears to limit EV diffusion, what could mean that the impact of EVs released by stromal cells may depend on the basement membrane integrity. However, in different diseases, epithelial function and structure is damaged, and basement membrane integrity can be affected.…”

“…The conjunctiva is composed of a stratified epithelium containing squamous epithelial cells and goblet cells and by a loose stroma containing, among other cells, conjunctival MSCs (Conj-MSCs). 12 There are several studies investigating ocular surface EVs, most of them focused on EVs derived from corneal epithelium, 13 – 15 with just one study analyzing conjunctival epithelial EVs. 16 Regarding ocular MSC-derived EVs, Shojaati et al showed that EVs isolated from MSCs from corneal stromal stem cells (CSSCs) had an essential role in the regeneration of the cornea after wounding.…”

Current Advances in Corneal Stromal Stem Cell Biology and Therapeutic Applications

“…Experiments in transgenic mice with reduced Klf4 expression resulted in increased epithelial cell layers and a disrupted barrier function, highlighting Klf4's importance in maintaining the equilibrium between the stroma and epithelium [99]. This change is linked to the process of epithelial-to-mesenchymal transition [100]. The loss of Klf4 expression can engender worsening of stromal wounds and atypical transformation of keratocytes into myofibroblasts [101][102][103].…”

“…The searching in this catalog of exosome donors is first refined by markers for the desirable molecules that will determine the vesicles performance in vivo [152,153]. The exosome source affects the exosome markers, which then guide the exosomes functionality [100]. Each of these steps will undoubtedly be the subject of future studies.…”