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

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).

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Extracellular Vesicles Mediate Anti-Oxidative Response—In Vitro Study in the Ocular Drainage System

International Journal of Molecular Sciences ◽

10.3390/ijms21176105 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6105 ◽

Cited By ~ 1

Author(s):

Natalie Lerner ◽

Itay Chen ◽

Sofia Schreiber-Avissar ◽

Elie Beit-Yannai

Keyword(s):

Extracellular Vesicles ◽

Aqueous Humor ◽

Open Angle Glaucoma ◽

Drainage System ◽

In Vitro Study ◽

Ciliary Epithelium ◽

Target Cells ◽

Related Factor ◽

Antioxidant Genes

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.

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Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells

Scientific Reports ◽

10.1038/s41598-021-98766-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Megumi Watanabe ◽

Yosuke Ida ◽

Hiroshi Ohguro ◽

Chiaki Ota ◽

Fumihito Hikage

Keyword(s):

Trabecular Meshwork ◽

Ex Vivo ◽

Open Angle Glaucoma ◽

Smooth Muscle Actin ◽

Three Dimensional ◽

Tissue Inhibitor Of Metalloproteinase ◽

Three Dimension ◽

Trabecular Meshwork Cells ◽

2D And 3D

AbstractTo establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

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Cross-Talk between Ciliary Epithelium and Trabecular Meshwork Cells In-Vitro: A New Insight into Glaucoma

PLoS ONE ◽

10.1371/journal.pone.0112259 ◽

2014 ◽

Vol 9 (11) ◽

pp. e112259 ◽

Cited By ~ 2

Author(s):

Natalie Lerner ◽

Elie Beit-Yannai

Keyword(s):

Cross Talk ◽

Trabecular Meshwork ◽

Ciliary Epithelium ◽

Trabecular Meshwork Cells ◽

Insight Into

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Functional Implications of Cross-Linked Actin Networks in Trabecular Meshwork Cells

Cellular Physiology and Biochemistry ◽

10.1159/000487170 ◽

2018 ◽

Vol 45 (2) ◽

pp. 783-794 ◽

Cited By ~ 6

Author(s):

Laura Duffy ◽

Steven O’Reilly

Keyword(s):

Trabecular Meshwork ◽

Open Angle Glaucoma ◽

Collagen Gel ◽

Mechanical Stretch ◽

Actin Networks ◽

Trabecular Meshwork Cells ◽

Microscopy Technique ◽

Collagen Gel Contraction

Background/Aims: The Trabecular meshwork (TM) is the tissue responsible for outflow resistance and therefore intraocular pressure. TM cells contain a contractile apparatus that is composed of actin stress fibres which run parallel to the axis of the cell and are responsible for facilitating contraction. Cross-Linked Actin Networks (CLANs) are polygonal arrangements of actin that form a geodesic network found predominantly in TM cells both in situ and in vitro. The aim of this work is to determine the functional significance of CLANs in TM cells and to assess the effect of mechanical stretch stimulation on the induction (or not) of CLANs. Methods: We used collagen gel contraction models to demonstrate functional impairment of cells when induced to express CLANs in situ. Cyclic mechanical stretch was used to stimulate cells and measure CLANs Results: CLANs inhibited contraction and cyclic mechanical stretch induced CLANs. Furthermore, we also demonstrated that using shape alone we could predict the appearance of CLANs using a simple light microscopy technique. Conclusion: Taken together we have now shown, for the first time, a functional deficit In TM cells with CLANs Furthermore that shape alone can predict the appearance of CLAN containing cells. CLANs can now be linked to a functional effect and may underlie the appearance of CLANs with the pathology of primary open angle glaucoma (POAG).

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Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma

Journal of Clinical Medicine ◽

10.3390/jcm10010078 ◽

2020 ◽

Vol 10 (1) ◽

pp. 78

Author(s):

April Nettesheim ◽

Myoung Sup Shim ◽

Angela Dixon ◽

Urmimala Raychaudhuri ◽

Haiyan Gong ◽

...

Keyword(s):

Trabecular Meshwork ◽

Cathepsin B ◽

Molecular Mechanisms ◽

Culture Media ◽

Ecm Remodeling ◽

Trabecular Meshwork Cells ◽

Proteolytic Cascade

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFβ/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

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