Vitreous Modulation of Gene Expression in Low Passage Human Retinal Pigment Epithelial Cells

Investigative Ophthalmology and Visual Science (2007) In press

PURPOSE. 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 morphological changes. This study examined vitreous-induced modulation of gene expression in RPE cells.
METHODS. Low passage human RPE cell lines derived 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 using microarrays. Real-time quantitative PCR was used to measure mRNA expression of a subset of genes in cells from three additional donors. Immunohistochemistry and immunoblotting were used to examine expression of bone morphogenetic protein-2 (BMP-2).
RESULTS. Vitreous-treatment caused a progressive re-programming of gene expression. QPCR confirmed vitreous modulation of mRNA levels for 10/10 genes. Changes consistent with a transition from an epithelial to a mesenchymal phenotype were observed. Down-regulated genes included genes associated with differentiated RPE cells. Up-regulated genes included genes associated with stress and inflammation. Pathway analysis indicated that the transforming growth factor-beta/BMP pathway and the focal adhesion pathway may play a role in this process.
CONCLUSIONS. 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.

Hartung, R., Parapuram, SK., Ganti, R., Hunt, DM, Chalam, KV. and Hunt, RC

VITREOUS INDUCES AN ANTI-OXIDANT RESPONSE MEDIATED IN PART BY TGF-ΒETA AND REACTIVE OXYGEN SPECIES GENERATION IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS

Molecular Vision (2007) In press

PURPOSE: When human retinal pigment epithelial cells come in contact with vitreous, they undergo changes in gene expression including inflammatory and anti-oxidant responses. The effect of vitreous on expression of heme oxygenase-1 (HO-1) and metallothionein (MT) -1a and -2a was investigated. 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.
METHODS: Low passage cultures of human retinal pigment epithelial cells were grown in the presence or absence of vitreous or transforming growth factor β (TGFβ). 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.
RESULTS: Incubation of RPE cells with vitreous resulted in increased expression of HO-1, MT-1a and MT-2a. TGFβ 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 TGFβ signaling (SB431542 or TGFβ-neutralizing antibodies) decreased the vitreous induction of HO-1. Several reactive oxygen species (ROS) quenchers inhibited the TGFβ-induced or vitreous-induced elevation of HO-1 mRNA but had no effect on vitreous-mediated induction of MT expression. Inhibitors of the p38MAPK (SB203580) and Jun N-terminal kinase (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.
CONCLUSIONS: TGFβ, 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 does not participate in the vitreous-mediated up-regulation of MTs. Both vitreous and TGFβ signal 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.
 

Parapuram SK, Ganti R, Hunt RC, Hunt DM.

Vitreous induces components of the prostaglandin E2 pathway in human retinal pigment epithelial cells.
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1767-74

PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: Treatment of human RPE cells with vitreous induces changes in gene expression that are indicative of an inflammatory response

Liou GI, Matragoon S, Samuel S, Behzadian MA, Tsai NT, Gu X, Roon P, Hunt DM, Hunt RC, Caldwell RB, Marcus DM.

MAP kinase and beta-catenin signaling in HGF induced RPE migration
Mol Vis. 2002 Dec 20;8:483-93

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

HGF regulation of RPE proliferation in an IL-1beta/retinal hole-induced rabbit model of PVR.
Mol Vis. 2002 Dec 20;8:494-501

PURPOSE: To understand molecular events that lead to retinal pigment epithelial (RPE) cell proliferation and migration during the early phases of proliferative vitreoretinopathy (PVR) in a rabbit model. METHODS: Retinal holes were created and interleukin-1beta(IL-1beta) was injected intravitreally. Eyes were examined by indirect ophthalmoscopy and eyecup pieces containing retinal holes were analyzed at different times after the surgery up to 4 weeks. RPE proliferation and migration were examined by immunohistochemistry. Tyrosine phosphorylation of extracellular signal regulated kinase (ERK) and hepatocyte growth factor receptor (HGFR or c-met) was determined by immunoprecipitation and western blot analysis. Tyrosine phosphorylation of c-met and morphological studies was performed on vitreous treated ARPE-19 cells. Expression of c-jun was determined by Northern blot analysis. Matrix metalloproteinase (MMP) content in vitreous was assessed by zymography. RESULTS: Indirect ophthalmoscopy identified formation of epiretinal membrane and immunohistochemistry identified proliferative and migratory RPE and other cells in the posterior segment containing retinal holes at 4 weeks post-surgery. Tyrosine phosphorylation of ERK and c-met occurred in this segment within 30 min of surgery. ARPE-19 cells treated with vitreous from the 24 h post-surgical eyes, but not with control vitreous or IL-1beta, showed morphological changes and tyrosine phosphorylation of c-met. Northern blot analysis in this segment identified upregulation of c-jun within 30 min of surgery and the expression peaked at 72 h. Zymographic analysis of vitreous identified MMP-9 in 12-72 h post-surgery. CONCLUSIONS: These data suggest that the presence of retinal holes and IL-1beta may lead to activation of HGF, mitogen activated protein kinases (MAPK), c-jun and extracellular matrix remodeling, resulting in proliferative and migratory cells in the wounded retina.

Lu H, Hunt DM, Ganti R, Davis A, Dutt K, Alam J, Hunt RC. 

Metallothionein protects retinal pigment epithelial cells against apoptosis and oxidative stress.  Exp Eye Res 2002 Jan;74(1):83-92

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. 

Meitinger D, Hunt DM, Shih DT, Fox JC, Hunt RC. 

Vitreous-induced modulation of integrins in retinal pigment epithelial cells: effects of fibroblast growth factor-2.  Exp Eye Res 2001 Nov;73(5):681-92

Growth in the presence of vitreous results in transformation of human RPE cells from an epithelioid to a fibroblast-like appearance and leads to an elevation of the expression of alpha(5) and alpha(2) integrins, while the level of alpha(3) integrin is reduced. These changes are inhibited by the presence of FGF-2. Vitreous treatment increases mobility, as does antibody neutralization of FGF-2 or antibody blockade of FGF receptors. The vitreous-induced rise in mobility depends on an increase in alpha(5) integrin expression since it is inhibited by anti-alpha(5) integrin antibodies. Expression of alpha(5) integrin as a result of infection of RPE cells with an alpha(5) integrin-encoding adenovirus induced morphological transformation and an increase in mobility similar to that seen with vitreous. It is concluded that a decrease in FGF-2 plays an important role in vitreous-induced alterations of RPE cell morphology, integrin expression and mobility. High FGF-2 levels prevent at least some of the increased mobility of RPE cells induced by vitreous. This is mediated via extracellular FGF-2 binding to FGF receptor(s) since antibodies to FGF-2 or to its receptor(s) mimic the effects of vitreous. Changes in mobility and morphology involve altered alpha(5) integrin expression since mobility is blocked by antibodies against these proteins while elevated alpha(5) integrin expression increases mobility and leads to morphological changes