Eye / Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21683
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Browsing Eye / Vision by Author "Mao, Weiming"
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Item Application of the CRISPR interference method in regulating TGFβ2 in the trabecular meshwork(2017-03-14) Nguyen, Nghia; Webber, Hannah; Bermudez, Jaclyn; Clark, Abbot; Mao, Weiming; Goan, DanielIntroduction: Glaucoma is an eye disease that damages the optic nerve and leads to gradual loss of vision. Glaucoma is the second leading cause of blindness globally. The trabecular meshwork (TM) is an ocular tissue responsible for controlling and drainage the aqueous humor which is a fluid that fills the eye. In primary open angle glaucoma (POAG), the most frequent type of glaucoma, there is a dysfunction within the TM that decreases the outflow of aqueous humor and elevates intraocular pressure (IOP). Transforming growth factor beta 2 (TGFβ2) is a protein that controls cell growth, differentiation, proliferation, and apoptosis. Many studies have shown that elevated TGFβ2 induces glaucoma phenotypes in the eye, including elevated IOP. Therefore, lowering TGFβ2 levels in the TM is a potential therapeutic strategy for treating glaucomatous changes in the TM as well as lowering IOP. Since our published study showed that elevated TGFβ2 is likely due to histone hyperacetylation, the purpose of this study was to determine whether the novel CRISPR interference technology, which is able to deacetylate histones in a gene-specific manner, is suitable for the manipulation of TGFβ2 levels in the TM. Methods: Four sets of oligos were designed close to the transcriptional start site of the TGFβ2 gene using the online CRISPR sgRNA design tool (http://crispr.mit.edu/) for the construction of sgRNAs. These oligos were sub-cloned into the target sgRNA expression vector (Addgene). The dCas9-KRAB expression vector was purchased from Addgene. The sgRNA expression vector and dCas9-KRAB vector were co-transfected in transformed human TM cells (GTM3). Four days after transfection, we isolated mRNA and protein for quantitative PCR (qPCR) and Western immunoblotting analyses. Results: The expression of dCas9-KRAB and/or sgRNA did not show toxicity to GTM3 cells. qPCR analysis showed that the 2 two-vector system dramatically repressed the level of TGFβ2 in GTM3 cells. Conclusions: The CRISPR/dCAS9 interference method is effective in lowering the level of TGFβ2 in the HTM. Further studies are required to determine the specificity and suitability of this technology in other genes and primary human TM cells.Item Effects of Wnt/beta-catenin and SMAD/TGF-beta crosstalk on cadherins in the trabecular meshwork(2017-03-14) Mao, Weiming; Clark, Abbot; Webber, HannahPurpose: We have shown cross-inhibition of the primary open angle glaucoma (POAG)-associated Wnt/beta-catenin and SMAD/TGFb signaling pathways in trabecular meshwork (TM) but the downstream effects of this crosstalk are unknown. Wnt transcription factor and cadherin accessory protein, beta-catenin, plays a role in cadherins junction stability. We studied the role of Wnt/beta-catenin and SMAD/TGFb signaling on cadherins junctions in the TM and TM cell adhesion. Methods: Confluent primary TM (NTM) cells (donated from Alcon) were treated for 2 or 24 hours with or without 100ng/mL Wnt3a or 5ng/mL TGFb2 and their membrane-bound protein, conditioned media, and total protein were isolated for western immunoblotting. Samples were probed for fibronectin (FN), PAI-1, p-Smad3, beta-catenin, GAPDH, Pan-, K-, or OB-cadherin. Some NTM cells were grown to 80% confluence then transfected with 0.3nM or 0.5nM K-cadherin, OB-cadherin, or non-targeting siRNA. Forty-eight or 72 hours after transfection, cells were harvested for western immunoblotting or immunofluorescence. NTM cells were plated for the Acea iCelligence system to determine cellular impedance with data collected every 30 minutes to establish baseline. After 24 hours, culture medium was replaced with transfection mixes as described. Cell impedance was continuously measured for an additional 96 hours. Bright field images of the cells were taken. Results: Wnt3a treatment resulted in increased K-cadherin expression. Co-treatment of Wnt3a and TGFb2 decreased the expression of K-cadherin. Three days after transfection with 0.5nM K-cadherin siRNA, decreased K-cadherin expression was observed in both whole cell lysate and membrane-bound fractions. Transfection with K-cadherin siRNA decreased NTM cellular impedance compared to non-targeting siRNA control. Conclusions: Crosstalk between Wnt/beta-catenin and SMAD/TGFb signaling pathways in the TM may regulate the expression of cadherins as well as NTM cell adhesion.