The role of extracellular vesicles (EVs) as signal mediators has been described in many biological fields. How many EVs are needed to deliver the desired physiological signal is yet unclear. Using a normal trabecular meshwork (NTM) cell culture exposed to non‐pigmented ciliary epithelium (NPCE)–derived EVs, a relevant model for studying the human ocular drainage system, we addressed the EVs dose–response effects on the Wnt signaling. The objective of the study was to investigate the dosing effects of NPCE‐derived EVs on TM Wnt signaling. EVs were isolated by PEG 8000 method from NPCE and RPE cells (used as controls) conditioned media. Concentrations were determined by Tunable Resistive Pulse Sensing method. Various exosomes concentration were incubated with TM cells, for the determination of mRNA (β‐Catenin, Axin2 and LEF1) and protein (β‐Catenin, GSK‐3β) expression using real‐time quantitative PCR and Western blot, respectively. Exposure of NTM cells for 8 hrs to low EVs concentrations was associated with a significant decreased expression of β‐Catenin, GSK‐3β, as opposed to exposure to high exosomal concentrations. Pro‐MMP9 and MMP9 activities were significantly enhanced in NTM cells treated with high EV concentrations of (X10) as compared to low EV concentrations of either NPCE‐ or RPE‐derived EVs and to untreated control. Our data support the concept that EVs biological effects are concentration‐dependent at their target site. Specifically in the present study, we described a general dose–response at the gene and MMPs activity and a different dose–response regarding key canonical Wnt proteins expression.

Primary open-angle glaucoma is a leading cause of irreversible blindness, often associated with increased intraocular pressure. Extracellular vesicles (EVs) carry a specific composition of proteins, lipids and nucleotides have been considered as essential mediators of cell-cell communication. Their potential impact for crosstalk between tissues responsible for aqueous humour production and out-flow is largely unknown.The study objective was to investigate the effects of EVs derived from non-pigmented ciliary epithelium (NPCE) primary cells on the expression of Wnt proteins in a human primary trabecular meshwork (TM) cells and define the mechanism underlying exosome-mediated regulation that signalling pathway. Consistent with the results in TM cell line, EVs released by both primary NPCE cells and NPCE cell line showed diminished pGSK3β phosphorylation and decreased cytosolic levels of β-catenin in primary TM cells. At the molecular level, we showed that NPCE exosome treatment downregulated the expression of positive GSKβ regulator-AKT protein but increased the levels of GSKβ negative regulator-PP2A protein in TM cells. NPCE exosome protein analysis revealed 584 miRNAs and 182 proteins involved in the regulation of TM cellular processes, including WNT/β-catenin signalling pathway, cell adhesion and extracellular matrix deposition. We found that negative modulator of Wnt signalling miR-29b was abundant in NPCE exosomal samples and treatment of TM cells with NPCE EVs significantly decreased COL3A1 expression. Suggesting that miR-29b can be responsible for decreased levels of WNT/β-catenin pathway. Overall, this study highlights a potential role of EVs derived from NPCE cells in modulating ECM proteins and TM canonical Wnt signalling. K E Y W O R D S exosomes, extracellular matrix, extracellular vesicles, non-pigmented ciliary epithelium, primary open-angle glaucoma, trabecular meshwork, Wnt | 4647 LERNER Et aL.

The importance of extracellular vesicles (EVs) as signaling mediators has been emphasized for several pathways with only limited data regarding their role as protective messages during oxidative stress (OS). The ocular drainage system is unique by being continuously exposed to OS and having a one-way flow of the aqueous humor carrying EVs taking role in glaucoma disease. Here, we aimed to examine the ability of EVs derived from the non-pigmented ciliary epithelium (NPCE)—the aqueous humor producing cells exposed to OS—to deliver protecting messages to the trabecular meshwork (TM)—the aqueous humor draining cells—a process with significance to the pathophysiology of glaucoma disease. EVs extracted from media of NPCE cells exposed to non-lethal OS and their unstressed control were incubated with TM cells. The effects of EVs derived from oxidative stressed cells on the activation of the nuclear factor erythroid 2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1), a major OS pathway, and of the Wnt pathway, known for its role in primary open-angle glaucoma, were evaluated. EVs derived from oxidized NPCE cells significantly protected TM cells from direct OS. The TM cells uptake of EVs from oxidized NPCE and their cytosolic Nrf2 levels were significantly higher at 8 h post-exposure. EVs derived from oxidized NPCE cells significantly attenuated Wnt protein expression in TM cells and activated major antioxidant genes as measured by qRT-PCR. TM cells exposed to EVs derived from oxidized NPCE cells exhibited significantly lower OS and higher super oxide dismutase and catalase activity. Finally, we were able to show that carbonylated proteins and products of oxidized protein are presented in significantly higher levels in EVs derived from oxidized NPCE cells, supporting their suggested role in the signaling process. We hypothesize that these findings may have implications beyond understanding the pathophysiology of glaucoma disease and that transmitting signals that activate the antioxidant system in target cells represent a broad response common to many tissues communication.

Reactive oxygen species (ROS) plays a key role in the pathogenesis of primary open-angle glaucoma (POAG), a chronic neurodegenerative disease that damages the trabecular meshwork (TM) cells, inducing apoptosis of the retinal ganglion cells (RGC), deteriorating the optic nerve head, and leading to blindness. Aqueous humor (AH) outflow resistance and intraocular pressure (IOP) elevation contribute to disease progression. Nevertheless, despite the existence of pharmacological and surgical treatments, there is room for the development of additional treatment approaches. The following review is aimed at investigating the role of different microRNAs (miRNAs) in the expression of genes and proteins involved in the regulation of inflammatory and degenerative processes, focusing on the delicate balance of synthesis and deposition of extracellular matrix (ECM) regulated by chronic oxidative stress in POAG related tissues. The neutralizing activity of a couple of miRNAs was described, suggesting effective downregulation of pro-inflammatory and pro-fibrotic signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), transforming growth factor-beta 2 (TGF-β2), Wnt/β-Catenin, and PI3K/AKT. In addition, with regards to the elevated IOP in many POAG patients due to increased outflow resistance, Collagen type I degradation was stimulated by some miRNAs and prevented ECM deposition in TM cells. Mitochondrial dysfunction as a consequence of oxidative stress was suppressed following exposure to different miRNAs. In contrast, increased oxidative damage by inhibiting the mTOR signaling pathway was described as part of the action of selected miRNAs. Summarizing, specific miRNAs may be promising therapeutic targets for lowering or preventing oxidative stress injury in POAG patients.

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.

Primary open-angle glaucoma is established by the disruption of trabecular meshwork (TM) function. The disruption leads to increased resistance to the aqueous humor (AH), generated by the non-pigmented ciliary epithelium (NPCE). Extracellular vesicles (EVs) participate in the communication between the NPCE and the TM tissue in the ocular drainage system. The potential use of NPCE-derived EVs to deliver siRNA to TM cells has scarcely been explored. NPCE-derived EVs were isolated and loaded with anti-fibrotic (SMAD7) siRNA. EV’s structural integrity and siRNA loading efficiency were estimated via electron microscopy and fluorescence. Engineered EVs were added to pre-cultured TM cells and qRT-PCR was used to verify the transfer of selected siRNA to the cells. Western blot analysis was used to evaluate the qualitative effects on Wnt-TGFβ2 proteins’ expression. EVs loaded with exogenous siRNA achieved a 53% mRNA knockdown of SMAD7 in TM cells, resulting in a significant elevation in the levels of β-Catenin, pGSK3β, N-Cadherin, K-Cadherin, and TGFβ2 proteins in TM cells. NPCE-derived EVs can be used for efficient siRNA molecule delivery into TM cells, which may prove to be beneficial as a therapeutic target to lower intraocular pressure (IOP).

The present research aims to determine whether the application of non‐pigmented ciliary epithelium cells derived extracellular vesicles to human trabecular meshwork cells affects the formation and secretion of collagen type I to the extracellular matrix formation. Following the extraction of non‐pigmented ciliary epithelium derived extracellular vesicles by a precipitation method, their size and concentration were determined using tunable resistive pulse sensing technology. Extracellular vesicles were incubated with trabecular meshwork cells for 3 days. Morphological changes of collagen type I in the extracellular matrix of trabecular meshwork cells were visualized using confocal microscopy and scanning electron microscopy. A Sirius Red assay was used to determine the total amount of collagen. Finally, collagen type I expression levels in the extracellular matrix of trabecular meshwork cells were quantified by cell western analysis. We found that non‐pigmented ciliary epithelium extracellular vesicles were very effective at preventing collagen fibres formation by the trabecular meshwork cells, and their secretion to the extracellular matrix was significantly reduced (P < .001). Morphological changes in the extracellular matrix of trabecular meshwork cells were observed. Our study indicates that non‐pigmented ciliary epithelium extracellular vesicles can be used to control collagen type I fibrillogenesis in trabecular meshwork cells. These fibrils net‐like structure is responsible for remodelling the extracellular matrix. Moreover, we suggest that targeting collagen type I fibril assembly may be a viable treatment for primary open‐angle glaucoma abnormal matrix deposition of the extracellular matrix.

Background Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. Purpose To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. Methods TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. Results EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including β-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. Conclusion Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.