D Margaret Hunt

University of South Carolina, Columbia, SC, United States

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Publications (7)23.51 Total impact

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    ABSTRACT: In proliferative vitreoretinopathy retinal pigment epithelial (RPE) cells undergo epithelial-mesenchymal transformation (EMT). Vitreous and transforming growth factor-beta (TGFbeta) have been implicated in this EMT. The role of TGFbeta in the vitreous-mediated transformation of low-passage human RPE cells was investigated. Cells were treated with vitreous or TGFbeta2. SB431542 was used to inhibit TGFbeta signaling. Morphology was investigated using phase-contrast or confocal microscopy. Motility was measured using a monolayer-wounding assay. Invasion was determined using basement membrane matrix-based assays. Gene expression was measured by quantitative PCR, immunohistochemistry, or immunoblotting. Changes in phosphorylation or cellular localization of Smad -2, -3, or -4 indicated a TGFbeta-like activity in vitreous. Cortical actin filaments in untreated cells were replaced by stress fibers after TGFbeta treatment, but peripheral actin aggregates were seen in vitreous-treated cells. SB431542 did not block the morphologic change induced by vitreous. Vitreous-treated cells exhibited increased motility and invasion, whereas TGFbeta-treated cells did not. However, SB431542 decreased vitreous-meditated changes in motility and invasion. The levels of mRNA for genes indicative of myofibroblast differentiation (alpha-SMA and CTGF) were increased by treatment with TGFbeta but suppressed by vitreous. TGFbeta or vitreous caused increased expression of Snail1. Vitreous or TGFbeta caused a fibroblast-like morphology and induced Snail1, a marker of EMT. TGFbeta activity in vitreous was necessary but not sufficient for the vitreous-induced motile, invasive phenotype. However, differences in the cytoskeletal organization and in the expression of CTGF and alpha-SMA suggested that TGFbeta-treatment caused differentiation along a myofibroblast pathway, whereas vitreous treatment suppressed myofibroblast formation.
    Investigative ophthalmology & visual science 08/2009; 50(12):5965-74. · 3.43 Impact Factor
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    ABSTRACT: In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast-like and participate in the formation of contractile membranes, which can lead to retinal detachment. Vitreous treatment of RPE cells in vitro results in similar morphologic changes. This study was conducted to examine vitreous-induced modulation of gene expression in RPE cells. Low-passage human RPE cell lines derived from three donors were each treated for 6, 12, 24, or 48 hours with complete medium or complete medium containing 25% vitreous. Changes in mRNA levels were examined by using microarrays. Real-time quantitative PCR (qPCR) was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblot analysis were used to examine protein expression. Vitreous treatment caused a progressive reprogramming of gene expression. qPCR confirmed vitreous modulation of mRNA levels of 10 of 10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Downregulated genes included genes associated with differentiated RPE cells. Upregulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/bone morphogenetic protein (BMP) pathway and the focal adhesion pathway may play a role in this process. BMP-2 protein and mRNA were increased. Despite the biological variation in vitreous and RPE donors, vitreous reproducibly modulated a limited number of mRNAs. Many of these changes were consistent with the more fibroblast-like appearance of vitreous-treated cells and with the pathobiology of PVR. TGF-beta and BMP-2 may be important modulators of vitreous-induced changes in gene expression.
    Investigative Ophthalmology &amp Visual Science 04/2007; 48(4):1853-63. · 3.44 Impact Factor
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    ABSTRACT: When human retinal pigment epithelial (RPE) cells come in contact with vitreous, they undergo changes in gene expression that include inflammatory and anti-oxidant responses. The effects of vitreous on expression of heme oxygenase-1 (HO-1), metallothionein (MT) -1a and -2a, and c-fos were investigated. Activator protein-1 (AP-1) binding sites are located in the promoter region of HO-1 and MT genes and the effects of vitreous on c-fos activity were investigated. Low passage cultures of human RPE cells were grown in the presence or absence of vitreous or transforming growth factor-beta (TGF-beta). The expression of HO-1 and MTs was measured by real time PCR and, in the case of HO-1, by immunoblotting and immunofluorescence microscopy. Specific inhibitors were used to investigate possible signaling pathways. The effect of vitreous on activation of AP-1 transcription factor was determined by immunoblotting, electrophoretic mobility shift assays, or immunofluorescence microscopy. Incubation of RPE cells with vitreous resulted in increased expression of HO-1, MT-1a and MT-2a. TGF-beta caused an increase in HO-1 expression, although not to the extent mediated by vitreous, but had little effect on MT expression. Addition of inhibitors of TGF-beta signaling (SB431542 or TGF-beta-neutralizing antibodies) decreased the vitreous induction of HO-1. Several reactive oxygen species (ROS) quenchers inhibited the TGF-beta-induced or vitreous-induced elevation of HO-1 mRNA but had no effect on vitreous-mediated induction of MT expression. Inhibitors of the mitogen-activated protein kinase (p38MAPK; SB203580) and Jun N-terminal kinase (JNK; SP600125) pathways inhibited vitreous-induction of HO-1. C-fos, a component of AP-1 transcription factor complexes, exhibited increased expression and activation in the presence of vitreous. TGF-beta, a known component of vitreous, can account for some but not all of the regulation of the anti-oxidant, anti-inflammatory HO-1 gene in human RPE cells, but it does not participate in the vitreous-mediated upregulation of MTs. Both vitreous and TGF-beta signals increased HO-1 expression via ROS but the latter were not involved in vitreous-mediated MT expression. Increased p38, JNK, and c-fos activation may be implicated in vitreous modulation of HO-1.
    Molecular vision 02/2007; 13:66-78. · 1.99 Impact Factor
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    ABSTRACT: Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G(1)/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis.
    International Journal of Cancer 02/2004; 108(1):71-7. · 6.20 Impact Factor
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    ABSTRACT: To investigate the alterations in gene expression when human retinal pigment epithelial (RPE) cells in culture are treated with vitreous as a model for the changes that occur in proliferative vitreoretinopathy. Human RPE cells were cultured with or without human vitreous or collagen. RNA was extracted and reverse transcribed. The RNAs expressed were compared by using DNA macroarrays. Messenger RNA levels were also measured using real-time reverse transcription polymerase chain reaction. Protein expression was examined by immunoblot analysis. Immunoassays were used to determine levels of prostaglandin E(2). Vitreous treatment of RPE cells resulted in increased expression of two critical enzymes in the synthesis of prostaglandin E(2): membrane-associated prostaglandin E-synthase (mPGES) and cyclooxygenase (COX)-2. Increased levels of mPGES RNA and protein were still present at 48 hours of treatment, but the increase in COX-2 mRNA and protein was transient. The increase in the expression of mPGES was associated with an increase in the production of prostaglandin E(2) that was observed at 12 and 24 hours of treatment but not at 48 hours. Treatment with 100 microg collagen I per ml medium did not cause increased expression of mPGES and COX-2, even though both collagen- and vitreous-treatment caused a morphologic change in the RPE cells to a more fibroblast-like phenotype. Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response.
    Investigative Ophthalmology &amp Visual Science 05/2003; 44(4):1767-74. · 3.44 Impact Factor
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    ABSTRACT: Hepatocyte growth factor (HGF) has been implicated in retinal pigment epithelial (RPE) cell proliferation and migration that occurs in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). The aim of this study is to investigate HGF induced signaling pathways that lead to RPE cell migration. Localization of beta-catenin was determined by immunofluorescence. HGF induced migration of ARPE-19 cells was studied using a quantitative migration assay after wounding in the presence of a DNA polymerase inhibitor, and in the presence or absence of a mitogen activated protein kinase (MAP kinase) kinase inhibitor. C-jun expression was determined by semi-quantitative RT-PCR and by Northern blot analysis. P42/p44 MAP kinase activity was determined by western blot and by an immunoprecipitation kinase assay. Tyrosine phosphorylation of the HGF receptor (HGFR or c-met) and beta-catenin was determined by immunoprecipitation and western blot analysis. Transactivation activity of beta-catenin was determined by luciferase reporter gene analysis. Beta-catenin and E-cadherin were co-localized on the basal surface of the RPE in vivo. Diffusion of the cell surface-localized beta-catenin occurs in migratory cells in vitro in the presence of HGF. HGF induced a MAP kinase dependent ARPE-19 cell migration, which is accompanied with a transient increase of c-jun expression and concomitant increases of MAP kinase activity, tyrosine phosphorylation of HGFR and beta-catenin, increased cytosolic levels of beta-catenin, and transactivation activity of beta-catenin. Tyrosine phosphorylation of HGFR and beta-catenin occurs in the primary or passaged RPE cultures or proliferative ARPE-19 cells, but not freshly isolated RPE or differentiated ARPE-19 cells. This study defines the signal transduction pathways activated by HGF in RPE cells, leading to an increase in the MAP kinase activity and free pool of beta-catenin, and changes in gene expression. These findings are consistent with the hypothesis that both beta-catenin and MAP kinases are components of the HGF induced RPE migration that occurs in proliferative retinal diseases.
    Molecular vision 01/2003; 8:483-93. · 1.99 Impact Factor
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    ABSTRACT: The retina expresses metallothionein (MT) which has been reported to protect cells against oxidative stress and apoptosis. The types of MT expressed by human retinal cells were identified by laser capture microdissection and RT--PCR and it was found that MT-2a is expressed by retinal pigment epithelial (RPE) cells, photoreceptor cells, inner nuclear layer cells and ganglion cells while MT-1a is expressed by RPE cells and MT-3 by cells of the neural retina. MT is induced in cultured human RPE cells under stress conditions such as the presence of glucocorticoids, interleukin-1/TNF alpha, oxygen and TGF beta 1. Cultured human D407 RPE cells were transfected with plasmids that allowed the expression of MT to be controlled via the tet operator protein by the level of tetracycline in the medium. These experiments showed that elevation of MT levels by transfection of RPE cells protects them against toxic levels of cadmium, heme- and iron-induced oxidation and UV light-induced apoptosis.
    Experimental Eye Research 02/2002; 74(1):83-92. · 3.03 Impact Factor