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Find video protocols related to scientific articles indexed in Pubmed.
Biology of the extracellular matrix: an overview.
J. Glaucoma
PUBLISHED: 10-03-2014
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The extracellular matrix (ECM) is an intricate network composed of an array of multidomain macromolecules organized in a cell/tissue-specific manner. Components of the ECM link together to form a structurally stable composite, contributing to the mechanical properties of tissues. The ECM is also a reservoir of growth factors and bioactive molecules. It is a highly dynamic entity that is of vital importance, determining and controlling the most fundamental behaviors and characteristics of cells such as proliferation, adhesion, migration, polarity, differentiation, and apoptosis.
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Differential expression of genes in cells cultured from juxtacanalicular trabecular meshwork and Schlemm's canal.
J Ocul Pharmacol Ther
PUBLISHED: 03-10-2014
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The purpose of this study was to distinguish differences in gene expression between cells cultured from the juxtacanalicular trabecular meshwork (JCTM) and those from Schlemm's canal (SC), to gain clues to differences between those cell types, and to add to our baseline knowledge of gene expression differences in these cell types for later comparison between cells from nonprimary open-angle glaucoma (POAG) and POAG outflow tissues.
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Cellular processing of myocilin.
PLoS ONE
PUBLISHED: 01-01-2014
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Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines.
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Effects of mutations and deletions in the human optineurin gene.
Springerplus
PUBLISHED: 01-01-2014
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Optineurin is a gene associated with normal tension glaucoma (NTG) and amyotrophic lateral sclerosis (ALS). Foci formation and functional consequences including Golgi fragmentation, impairment of vesicle trafficking and apoptosis were observed previously upon overexpression and/or mutation of optineurin. In the current study, a total of 15 GFP tagged constructs that included NTG (E50K and 2 bp-AG insertion), ALS (exon 5 deletion, R96L, Q398X, and E478G) and non-disease (L157A and D474N) associated mutants and a series of deletion fragments were cloned into mammalian expression vectors and transfected into RGC5 and/or Neuro2A cells to evaluate whether their expression confer the optineurin phenotypes. The cells were monitored for foci formation and stained by immunofluorescence with anti-GM130 to analyze the Golgi integrity. Transferrin uptake experiments were performed to evaluate the protein trafficking process and apoptosis was assessed with the active caspase 3/7 detection kit. We demonstrated that cells expressing E50K and R96L optineurin exhibited all of the optineurin phenotypes. Q398X mutant did not induce foci formation, but triggered Golgi fragmentation, impairment of transferrin uptake and increase in apoptosis. The 2 bp-AG insertion mutant had a nuclear localization, compromised the transferrin uptake and strongly induced apoptosis. The foci formation, which might not predict the rest of the phenotypes, appeared to require both the leucine zipper and ubiquitin binding domains of the optineurin sequence. Interactions of optineurin with proteins including Rab8, myosin VI, huntingtin and transferrin receptor might directly determine whether the Golgi and protein trafficking phenotypes would be manifested. Examination of mutants and deletion fragments located at various sites of optineurin gene provide clues as to what regions of the gene may play a critical role in the development of pathologic consequences.
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Sustained release of matrix metalloproteinase-3 to trabecular meshwork cells using biodegradable PLGA microparticles.
Mol. Pharm.
PUBLISHED: 07-18-2013
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Accumulation of extracellular matrix (ECM) materials in the trabecular meshwork (TM) is believed to be a contributing factor to intraocular pressure (IOP) elevation, a risk factor/cause of primary open angle glaucoma, a major blinding disease. Matrix metalloproteinase-3 (MMP-3) is one of the proteinases that can effectively degrade ECM elements such as fibronectin, and MMP-3 delivery to the TM represents a promising approach for IOP reduction and treatment of glaucoma. In this study, we tested the feasibility of using polymeric microparticles to achieve a slow and sustained release of active MMP-3 to cultured human TM cells. ?-Casein, with molecular weight (24 kDa) and hydrophobicity similar to those of the active MMP-3 fragment (19.2 kDa), was first employed as a model for initial testing. ?-casein was encapsulated into poly(lactic-co-glycolic acid) (PLGA) microparticles using a double emulsion procedure at an encapsulation efficiency of approximately 45%. The PLGA microparticles were chosen given their biocompatibility and the proven capacity of sustained release of encapsulated molecules. The release test conducted in the culture medium showed a slow and sustained release of the protein over 20 days without a significant initial burst release. Active MMP-3 was subsequently encapsulated into PLGA microparticles with an encapsulation efficiency of approximately 50%. A biofunctional assay utilizing human TM cells was set up in which the reduction of fibronectin was used as an indicator of enzyme activity. It was observed that fibronectin staining was markedly reduced by the medium collected from MMP-3-microparticle-treated cultures compared to that from blank- and ?-casein-microparticle controls, which was validated using a direct MMP-3 activity assay. The controlled release of MMP-3 from the microparticles resulted in sustained degradation of fibronectin up to 10 days. This proof-of-concept undertaking represents the first study on the controlled and sustained release of active MMP-3 to TM cells via encapsulation into PLGA microparticles as a potential treatment of glaucoma.
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sCD44 overexpression increases intraocular pressure and aqueous outflow resistance.
Mol. Vis.
PUBLISHED: 01-01-2013
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CD44 plays major roles in multiple physiologic processes. The ectodomain concentration of the CD44 receptor, soluble CD44 (sCD44), is significantly increased in the aqueous humor of primary open-angle glaucoma (POAG). The purpose of this study was to determine if adenoviral constructs of CD44 and isolated 32-kDa sCD44 change intraocular pressure (IOP) in vivo and aqueous outflow resistance in vitro.
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Decellularized human cornea for reconstructing the corneal epithelium and anterior stroma.
Tissue Eng Part C Methods
PUBLISHED: 12-22-2011
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In this project, we strived to develop a decellularized human cornea to use as a scaffold for reconstructing the corneal epithelium and anterior stroma. Human cadaver corneas were decellularized by five different methods, including detergent- and nondetergent-based approaches. The success of each method on the removal of cells from the cornea was investigated. The structural integrity of decellularized corneas was compared with the native cornea by electron microscopy. The integrity of the basement membrane of the epithelium was analyzed by histology and by the expression of collagen type IV, laminin, and fibronectin. Finally, the ability of the decellularized corneas to support the growth of human corneal epithelial cells and fibroblasts was assessed in vitro. Corneas processed using Triton X-100, liquid nitrogen, and poly(ethylene glycol) resulted in incomplete removal of cellular material. Corneas processed with the use of sodium dodecyl sulfate (SDS) or with sodium chloride (NaCl) plus nucleases successfully removed all cellular material; however, only the NaCl plus nuclease treatment kept the epithelial basement membrane completely intact. Corneas processed with NaCl plus nuclease supported both fibroblast and epithelial cell growth in vitro, while corneas treated with SDS supported the growth of only fibroblasts and not epithelial cells. Decellularized human corneas provide a scaffold that can support the growth of corneal epithelial cells and stromal fibroblasts. This approach may be useful for reconstructing the anterior cornea and limbus using autologous cells.
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Novel mutation and three other sequence variants segregating with phenotype at keratoconus 13q32 susceptibility locus.
Eur. J. Hum. Genet.
PUBLISHED: 11-02-2011
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Keratoconus (KTCN), a non-inflammatory corneal disorder characterized by stromal thinning, represents a major cause of corneal transplantations. Genetic and environmental factors have a role in the etiology of this complex disease. Previously reported linkage analysis revealed that chromosomal region 13q32 is likely to contain causative gene(s) for familial KTCN. Consequently, we have chosen eight positional candidate genes in this region: MBNL1, IPO5, FARP1, RNF113B, STK24, DOCK9, ZIC5 and ZIC2, and sequenced all of them in 51 individuals from Ecuadorian KTCN families and 105 matching controls. The mutation screening identified one mutation and three sequence variants showing 100% segregation under a dominant model with KTCN phenotype in one large Ecuadorian family. These substitutions were found in three different genes: c.2262A>C (p.Gln754His) and c.720+43A>G in DOCK9; c.2377-132A>C in IPO5 and c.1053+29G>C in STK24. PolyPhen analyses predicted that c.2262A>C (Gln754His) is possibly damaging for the protein function and structure. Our results suggest that c.2262A>C (p.Gln754His) mutation in DOCK9 may contribute to the KTCN phenotype in the large KTCN-014 family.
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Myocilin and Optineurin: Differential Characteristics and Functional Consequences.
Taiwan J Ophthalmol
PUBLISHED: 11-01-2011
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Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells and their axons. This review describes the characteristics of myocilin and optineurin protein products and summarizes the consequences of ectopically expressed wild type and mutant myocilin and optineurin in trabecular meshwork and/or neuronal cells. Myocilin and optineurin exhibit differential characteristics and have divergent functional consequences. They contribute to the development of glaucoma likely via distinct mechanisms.
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In vivo serial imaging of regenerating corneal nerves after surgical transection in transgenic thy1-YFP mice.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-01-2011
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To determine the effect of lamellar transection surgery on the nerve fiber density (NFD) and pattern of nerve regeneration in the cornea of thy1-YFP transgenic mice.
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Processing of optineurin in neuronal cells.
J. Biol. Chem.
PUBLISHED: 11-08-2010
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Optineurin is a gene linked to amyotrophic lateral sclerosis, Paget disease of bone, and glaucoma, a major blinding disease. Mutations such as E50K were identified in glaucoma patients. We investigated herein the involvement of ubiquitin-proteasome pathway (UPP) and autophagy, two major routes for protein clearance, in processing of optineurin in a retinal ganglion cell model line RGC5 and neuronal PC12 cells. It was found that the endogenous optineurin level in neuronal cells was increased by treatment of proteasomal inhibitor but not by autophagic and lysosomal inhibitors. Multiple bands immunoreactive to anti-ubiquitin were seen in the optineurin pulldown, indicating that optineurin was ubiquitinated. In cells overexpressing wild type and E50K optineurin, the level of the proteasome regulatory ?5 subunit (PSMB5, indicative of proteasome activity) was reduced, whereas that for autophagy marker microtubule-associated protein 1 light chain 3 was enhanced compared with controls. Autophagosome formation was detected by electron microscopy. The foci formed after optineurin transfection were increased upon treatment of an autophagic inhibitor but were decreased by treatment of an inducer, rapamycin. Moreover, the level of optineurin-triggered apoptosis was reduced by rapamycin. This study thus provides compelling evidence that in a normal homeostatic situation, the turnover of endogenous optineurin involves mainly UPP. When optineurin is up-regulated or mutated, the UPP function is compromised, and autophagy comes into play. A decreased PSMB5 level and an induced autophagy were also demonstrated in vivo in retinal ganglion cells of E50K transgenic mice, validating and making relevant the in vitro findings.
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Suppression of transforming growth factor-? effects in rabbit subconjunctival fibroblasts by activin receptor-like kinase 5 inhibitor.
Mol. Vis.
PUBLISHED: 09-09-2010
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Transforming growth factor-? (TGF-?) activity has been implicated in subconjunctival scarring in eyes following glaucoma filtration surgery (GFS). The purpose of this study is to determine whether an inhibitor for activin receptor-like kinase (ALK) 5 (also known as TGF-? receptor type I) could suppress TGF-? activity and thereby promote filtering bleb survival after GFS in a rabbit model.
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The role of crumbs genes in the vertebrate cornea.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 09-01-2010
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To evaluate the role of crumbs genes and related epithelial polarity loci in the vertebrate cornea.
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Impairment of protein trafficking upon overexpression and mutation of optineurin.
PLoS ONE
PUBLISHED: 04-15-2010
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Glaucoma is a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and axons. Optineurin is one of the candidate genes identified so far. A mutation of Glu(50) to Lys (E50K) has been reported to be associated with a more progressive and severe disease. Optineurin, known to interact with Rab8, myosin VI and transferrin receptor (TfR), was speculated to have a role in protein trafficking. Here we determined whether, and how optineurin overexpression and E50K mutation affect the internalization of transferrin (Tf), widely used as a marker for receptor-mediated endocytosis.
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Wnt gene expression in human trabecular meshwork cells.
Mol. Vis.
PUBLISHED: 01-22-2010
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The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as beta-catenin (CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated.
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Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.
PLoS ONE
PUBLISHED: 01-12-2010
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Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.
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Differential effects of myocilin and optineurin, two glaucoma genes, on neurite outgrowth.
Am. J. Pathol.
PUBLISHED: 12-03-2009
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Myocilin and optineurin are two genes linked to glaucoma, a major blinding disease characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. To investigate the effects of force-expressed wild-type and mutant myocilin and optineurin on neurite outgrowth in neuronal cells, we transiently transfected cells with pEGFP-N1 (mock control) as well as myocilin and optineurin plasmids including pMYOC(WT)-EGFP, pMYOC(P370L)-EGFP, pMYOC(1-367)-EGFP, pOPTN(WT)-EGFP, and pOPTN(E50K)-EGFP. PC12 cells transfected with pEGFP-N1 produced, as anticipated, long and extensive neuritis on nerve growth factor induction. The neurite length in those cells transfected with myocilin constructs was shortened and the number of neurites was also reduced. A similar inhibitory effect on neurite outgrowth was also elicited by myocilin transfection in RGC5 cells. In contrast, neither transfection of the optineurin constructs pOPTN(WT)-EGFP and pOPTN(E50K)-EGFP nor the myocilin and optineurin small-interfering RNA treatments induced significant alterations in neurite outgrowth. Transfection with the wild-type optineurin construct, but not with that of the wild-type myocilin, increased the apoptotic activity in cells. These results demonstrated that the two glaucoma genes, myocilin and optineurin, exhibited differential effects on neurite outgrowth. They may contribute to the development of neurodegenerative glaucoma via distinct mechanisms.
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Effects of Sp1 overexpression on cultured human corneal stromal cells.
Genes Cells
PUBLISHED: 09-16-2009
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Sp1, a transcription factor, is upregulated in keratoconus, a cornea-thinning disease. Keratoconus corneas have also been shown to contain increased levels of degradative enzymes such as cathepsin B and decreased proteinase inhibitors such as alpha1-proteinase inhibitor (alpha1-PI). We transfected cultured human corneal stromal cells to overexpress Sp1. The resulting effects on cathepsin B and alpha1-PI levels as well as the cellular proliferative and apoptotic activities were examined by Western blotting and cytochemical staining. It was found that the Sp1 transfected cells contained a greater amount of cathepsin B than did mock transfected controls. The activity of cathepsin B was also increased. By contrast, the protein level of alpha1-PI was lowered in corneal stromal cells upon Sp1 overexpression. The Sp1-induced alterations thus mimicked closely those observed in keratoconus, supporting the notion that Sp1 upregulation may be a key factor contributing directly to the disease development. Furthermore, the apoptotic activity was unaffected in Sp1 transfectants but the proliferation was inhibited, consistent with the idea that Sp1 may play a role in differentiation of corneal cells.
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Histopathologic and immunohistochemical studies of keratoglobus.
Arch. Ophthalmol.
PUBLISHED: 08-12-2009
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To examine histopathologic and immunohistochemical features of human corneal buttons from patients who developed keratoglobus.
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Establishment of inducible wild type and mutant myocilin-GFP-expressing RGC5 cell lines.
PLoS ONE
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Myocilin is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Gln368stop (Q368X) and Pro370Leu (P370L) have been identified in patients. The exact role of myocilin and its functional association with glaucoma are still unclear. In the present study, we established tetracycline-inducible (Tet-on) wild type and mutant myocilin-green fluorescence protein (GFP) expressing RGC5 stable cell lines and studied the changes in cell migration and barrier function upon induction.
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Wnt activation by wild type and mutant myocilin in cultured human trabecular meshwork cells.
PLoS ONE
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Myocilin is a gene linked to the most prevalent form of glaucoma, a major blinding disease. The trabecular meshwork (TM), a specialized eye tissue, is believed to be involved, at least in part, in the development of glaucoma. The Pro³?? to Leu (P370L) mutation of myocilin is associated with severe glaucoma phenotypes and Gln³?? stop (Q368X) is the most common myocilin mutation reported. Myocilin, upon overexpression, has been shown to induce phenotypes that include a loss of actin stress fibers, an increase in the cAMP level and protein kinase A (PKA) activity, as well as a reduction in the RhoA activity. We examined herein whether Wnt signaling pathway is involved in the myocilin phenotypes and whether P370L and Q368X mutants also display biological effects similar to those of the wild type myocilin.
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Cellular and molecular biology of optineurin.
Int Rev Cell Mol Biol
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Optineurin is a gene linked to glaucoma, amyotrophic lateral sclerosis, other neurodegenerative diseases, and Pagets disease of bone. This review describes the characteristics of optineurin and summarizes the cellular and molecular biology investigations conducted so far on optineurin. Data from a number of laboratories indicate that optineurin is a cytosolic protein containing 577 amino acid residues. Interacting with proteins such as myosin VI, Rab8, huntingtin, transferrin receptor, and TANK-binding kinase 1, optineurin is involved in basic cellular functions including protein trafficking, maintenance of the Golgi apparatus, as well as NF-?B pathway, antiviral, and antibacteria signaling. Mutation or alteration of homeostasis of optineurin (such as overexpression or knockdown) results in adverse consequences in the cells, leading to the development of neurodegenerative diseases including glaucoma.
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