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Transparency of the human cornea is responsible for clear vision, which is maintained by a monolayer of non-proliferative human corneal endothelial cells (HCEnCs). Dysfunction of these cells can result in irreversible corneal blindness. It is important to identify key factors that limit the proliferation of HCEnCs and thus attempt to reverse them. Extracellular vesicles contain cargo which includes microRNAs (miRNAs) that can modulate a cellular function. In non small cell lung cancer, expression of miR-195-5p has been shown to inhibit proliferation; therefore, we aimed to investigate the inhibitory effect of miR-195-5p in inducing the proliferation of HCEnCs. Human corneal endothelial cell line (HCEC-12) and primary HCEnCs were cultured with miR-195-5p scramble, mimic or inhibitor. Corneal tissues from human cadaveric and FECD donors, and from pigs, mice and rabbits, were used for RT-PCR. miR-195-5p showed an abundance value of 11,363.31 a.u. When normalized against HCEnCs from cadaveric donors, FECD tissues showed a significant upregulation of miR-195-5p (p < 0.05) but was significantly downregulated in pig (p < 0.001), mouse (p < 0.01) and rabbit (p < 0.001) CEnCs, which have known proliferative capacity. Proliferation, cell doubling, and wound healing rates were significantly higher when miR-195-5p was inhibited. Inhibiting miR-195-5p showed a significant improvement in viability (HEC staining), decreased cell apoptosis (TdT-dNTP staining) and expression of ZO-1, NA+/K+-ATPase and Ki-67 markers. Expression of miR-195-5p is found in HCEnCs and FECD cells, which restricts the proliferation of these cells. However, inhibiting miR-195-5p can induce the proliferation of HCEnCs, which opens exciting directions for future research in prolonging FECD pathogenesis by increasing the proliferative capacity of HCEnCs using anti-miR therapy in vivo. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs), specifically exosomes, carry a cell-type dependent cargo that is transported to the recipient cell and translated in the presence of a required machinery. Differences in the cargo carried by the corneal and conjunctival-derived EVs could be the agent that triggers the transdifferentiation of these two cell populations. Therefore, this study investigates the role of EVs in triggering the plasticity of corneal and conjunctival epithelial cells and identifies prospective miRNA and genes responsible for maintaining ocular surface homeostasis. The EVs were extracted from the conditioned media (after starving) of corneal epithelial (hTCEpi) and conjunctival (HCjE-Gi) cell lines using ultracentrifugation. HCjE-Gi cells were cultured with hTCEpi-derived EVs and vice-versa. The EVs were characterized as exosomes using Nanosight and Flow cytometry. KRT3 and KRT12 were used as associated corneal markers, whereas KRT7 and KRT13 were used as associated conjunctival markers with ΔNp63 as a differentiation marker. Shift of these markers was an indication of transdifferentiation. The cargo of the extracted exosomes from both the cell types was explored using next-generation sequencing. The hTCEpi-derived EVs induced conjunctival epithelial cells to express the corneal-associated markers KRT3 and KRT12, losing their conjunctival phenotype at both the mRNA and protein level. Simultaneously, HCjE-Gi-derived EVs induced corneal epithelial cells to express the conjunctival associated markers KRT7 and KRT13, losing their corneal phenotype. This process of differentiation was accompanied by an intermediate step of cell de-differentiation showed by up-regulation in the expression of epithelial stem cell marker ΔNp63, also shown on the ex vivo human cadaveric donor corneas. miRNA molecules (total of 11 including precursor and mature) with significant differences in their relative abundance between the two populations (p < 0.05) were found and investigated. miR-9-5p expression was higher in HCjE-Gi cells and HCjE-Gi-derived EVs when compared to hTCEpi cells and hTCEPi-derived EVs (p < 0.001). The results suggest that EVs released by the two cell types have the ability to influence the transdifferentiation of human conjunctival and corneal epithelial cells. miR-9-5p could have a role in stem cell homeostasis and cell differentiation via HES-1 gene. [ABSTRACT FROM AUTHOR]