Eye/Vision
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21759
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Browsing Eye/Vision by Author "Mao, Weiming"
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Item EFFECT OF LYSYL OXIDASE (LOX) AND TISSUE TRANSGLUTAMINASE (TGM2) GENES ON HUMAN TRABECULAR MESHWORK CELLS(2014-03) Kirkland, Kyle A.; Bermudez, Jaclyn Y.; Mao, Weiming; Clark, Abbot F.Purpose: Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM). Methods: LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells. Results: LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells. Conclusions: TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-β2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.Item EPIGENETIC REGULATION OF GLAUCOMA-ASSOCIATED GROWTH FACTORS IN THE TRABECULAR MESHWORK(2014-03) Bermudez, Jaclyn Y.; Webber, Hannah; Cheng, Yi-Qiang; Clark, Abbot F.; Mao, WeimingGlaucoma is a leading cause of blindness in the U.S. and worldwide. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function. Therefore, it is important to study how glaucoma-associated growth factors in the TM are regulated. We investigated how heritable changes in gene activity regulate the TM without altering the DNA sequence. Purpose (a): Glaucoma is a leading cause of blindness in the U.S. and worldwide. This disease leads to progressive, irreversible damage to the optic nerve and visual function. The primary risk factor of primary open angle glaucoma (POAG), the major type of glaucoma, is elevated intraocular pressure (IOP). IOP elevation in glaucoma patients is due to glaucomatous insults to the trabecular meshwork (TM) and compromised TM function, which increase aqueous humor outflow resistance. In the glaucomatous TM (GTM), there is excessive extracellular matrix (ECM) protein deposition. Many studies have suggested that cell signaling pathways, such as the transforming growth factor beta (TGF-β) and Wnt signaling pathways, play key roles in TM homeostasis.The growth factors that are associated with these pathways, including TGFβ2, Gremlin and sFRP1, are found to be at higher levels in the GTM cells compared to normal TM cells. Little is known about the role of epigenetics in regulating glaucoma-associated growth factors in the TM. One of the major epigenetic regulatory mechanisms is histone acetylation.We hypothesize that histone acetylation is responsible for the increased expression of glaucoma associated factors in the TM. Methods (b): Primary human TM cell cultures were treated with 10nM Thailandepsin (TDP-A), a histone deacetylase inhibitor (HDACi), or 1% DMSO as vehicle control for 4 days. Cells were harvested for qPCR to compare gene expression levels or for ChIP assays to compare promoter associated histone acetylation status. We also treated paired perfusion cultured bovine anterior segments with DMSO or TDP-A for 7 to 10 days. The IOP change of the treated bovine eyes was monitored and recorded. Data were analyzed by using Student’s t-test or one-way ANOVA. P values less than 0.05 were considered significant. Results (c): TDP-A significantly elevated the expression of sFRP-1 and TGFβ2 (n=3, p2 as well as elevated IOP. Conclusions (d): Histone acetylation may play an important role in the dysregulation of growth factors in the TM. This mechanism provides a unique opportunity to elucidate the etiology of POAG. Also, TDP-A is a potent HDACi that can be used as a powerful tool in glaucoma research.