Non-Pigmented Ciliary Epithelium-Derived Extracellular Vesicles Loaded with SMAD7 siRNA Attenuate Wnt Signaling in Trabecular Meshwork Cells In Vitro.

Saray Tabak,Valeria Feinshtein,Elie Beit-Yannai,Sofia Schreiber-Avissar

doi:10.3390/ph14090858

Saray Tabak, Valeria Feinshtein + Show 2 more

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https://doi.org/10.3390/ph14090858

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Journal: Pharmaceuticals	Publication Date: Aug 27, 2021
Citations: 11	License type: CC BY 4.0

Affiliation: Ben-Gurion University of the Negev

Abstract

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).

Highlights

Primary open-angle glaucoma (POAG), the predominant form of glaucoma [1,2], is a chronic, degenerative optic neuropathy characterized by progressive visual field loss [3]that leads to irreversible blindness [2]
Since TGFβ2 enhances the adhesion of cells through Cadherins and βCatenin expression in human trabecular meshwork (TM) cells [14], we further investigated the crosstalk of TGFβ2 and Wnt signaling in TM cells
To ensure that the tested parameters for achieving maximal siRNA loading into the Extracellular vesicles (EVs) did not lead to the structural instability of the non-pigmented ciliary epithelium (NPCE)-derived vesicles, we used Tunable Resistive Pulse Sensing (TRPS) technology and Cryo-transmission electron microscopy (TEM) analysis

Summary

Introduction

Primary open-angle glaucoma (POAG), the predominant form of glaucoma [1,2], is a chronic, degenerative optic neuropathy characterized by progressive visual field loss [3]that leads to irreversible blindness [2]. From the anterior chamber of the eye the non-pigmented ciliary epithelium-NPCE serves as the site of AH production, while the trabecular meshwork (TM), and Schlemm’s canal are the principal locations of AH outflow. Contraction of the ciliary muscle causes expansion of the TM and opening of Schlemm’s canal, which subsequently increases the conductivity of AH through the TM [6]. The canonical Wnt signaling pathway in TM cells could provide insights leading to new glaucoma therapies [7]. The canonical Wnt signaling pathway is a critical regulator of IOP, and plays a role in extracellular matrix (ECM) expression in TM cells through regulation of matrix metalloproteinases’ (MMPs) activity [8,9,10]. Elevation of cytosolic β-Catenin levels in TM cells increases the expression of N-Cadherin [14]

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