Non-pigmented ciliary epithelium derived extracellular vesicles uptake mechanism by the trabecular meshwork.

Abstract

Consistent with increasing findings, extracellular vesicles (EVs), consider as a major constituents of the aqueous humor, have a role as signaling mediators in glaucoma. Following secretion, EVs hold immense promise for utilization as bio-therapeutics and drug delivery vehicles due to their nature as biological nanoparticles that facilitate intercellular molecular transport. Yet, the specific pathway utilizing for transferring signals by EVs in the ocular drainage system is not fully understood. Hence, the objective of this study was to examine internalization mechanisms by which Non-Pigmented Ciliary Epithelium (NPCE)-derived EVs deliver their signals to the Trabecular Meshwork (TM) cells. EVs were isolated and size and concentration were determined. Internalization study of treated EVs with Proteinase-K to achieve removal of surface membrane proteins on EVs was conducted. Energy dependent uptake mechanism was examined under various temperatures. Using uptake inhibitors endocytosis, phagocytosis, and Wnt-TGFβ2 signaling were investigated. TM cells exposed to NPCE EVs demonstrate a significant decrease in the levels of two proteins in two Wnt-TGFb2 signaling proteins levels: p-GSK3β and β-catenin. A significant decrease in the uptake by TM cells of Proteinase-K-treated EVs was found, followed by attenuation of the Wnt-TGFβ2 proteins expression. Energy dependent uptake revealed a significant decrease in EVs internalization. The exposure of TM cells to endocytosis uptake inhibitors abolished the decrease of the Wnt-TGFβ2 proteins levels. Exposure to phagocytosis uptake inhibitor resulted in a partial inhibition of NPCE EVs effect in TM cells. The attenuation of proteins expression levels following uptake inhibitors treatment or EVs membrane proteins removal indicates that Wnt-TGFβ2 signaling in TM cells is mediated through NPCE EVs surface proteins in an active manner that involves endocytosis-dependent routes.