Search PubMed for publications by Dr Margaret Hunt
Ganti, R., Hunt, RC., Parapuram, SK and
Hunt, DM
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.
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
Chen, W-H., Hunt, D.M. and Hunt R.C. Changes in the expression of anti-oxidant
protective proteins in the rat retina during pre- and post-natal development. Investigative
Ophthalmology and Visual Science 40: 744-751, 1999
Purpose:
In retinopathy of prematurity, capillary growth in the retina is
attenuated. Subsequent cyclic elevation of oxygen levels leads to renewed capillary growth
that may eventually result in retinal detachment. It is hypothesized that the sensitivity
of the premature retina to oxidative shock results from its lack of anti-oxidant
protective proteins. Methods: The expression of heme oxygenase-1,
metallothionein,
superoxide dismutase and catalase mRNAs was measured in retinae of rats from 6 days before
birth to 4 days after birth using in situ hybridization and semi-quantitative
reverse transcriptase polymerase chain reaction with Southern blotting. Results: Superoxide
dismutase mRNA was expressed to a similar extent at all time points. Metallothionein mRNA
expression, which was high at embryonic day 16 (E16) and E18, fell to low levels
by the time of birth and remained low at least to 4 days after birth. Catalase
mRNA expression was low until birth and rose until at least post-natal day 4. Heme
oxygenase-1 mRNA showed low expression at E16 and E18, rose before birth and then
diminished. Conclusions: Four anti-oxidant protein mRNAs expressed by the rat
retina show very different patterns of expression. Two of these proteins, heme oxygenase-1
and catalase are expressed at relatively low levels until around the time of birth. The
former is important in protection against heme-mediated generation of reactive oxygen
species while the latter protects against hydrogen peroxide-generated damage. As a result
of the low expression of these mRNAs, and presumably the proteins encoded by them, the
premature rat (and probably the premature human) is likely to be born without a full
complement of anti-oxidant defenses. |
