The 2000 Meeting of the International Congress of Cell Biology, September, 2000 click here
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The 1998 Meeting of the Association for Research in Vision and Ophthalmology (ARVO) May 1998 click here
The 1997 Meeting of the American Society for Cell Biology (ASCB) December 1997 click here
The 1997 Meeting of the Association for Research in Vision and Ophthalmology (ARVO) May 1997 click here
International Cell Biology Abstract. 2000
Human retinal pigment epithelial cells can express MT1-MMP and collagenase-3 (MMP13). D. M. Hunt, O. Olasimbo, D. Meitinger, Fairey, P.W., R. C. Hunt. Department of Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29212, USA.
Retinal pigment epithelial (RPE) cells form a barrier epithelium at the back of the retina and normally do not divide. However, in certain pathological circumstances, RPE cells can gain access to the vitreous humor, proliferate and become more fibroblast-like in morphology. The RPE cells plus other cells form epiretinal membranes containing extracellular matrix and embedded cells. Contraction of these membranes can result in retinal detachment and loss of sight. Vitreous treatment of RPE cells in culture causes an epithelioid-fibroblastic transformation similar to that seen in vivo. Using gene arrays, we observed that the in vitro transformation is accompanied by an increase in mRNA for MT1-MMP, a transmembrane metalloproteinase which can be activated in the trans-Golgi network. Vitreous treatment also results in increased expression of total MT1-MMP protein measured by semi-quantitative immunofluorescence or by immunoblotting techniques. Surface biotinylation with a membrane-impermeable reagent indicated that vitreous caused increased surface expression of MT1-MMP. MT1-MMP cleaves a variety of substrates, including extracellular matrix components, MMP-2 (gelatinase A) and collagenase-3 (MMP-13). MMP-2 has been reported to be present in the vitreous and other parts of the eye in a latent form, but we can find no reports investigating expression of collagenase-3 in the eye. Since collagenase-3 cleaves fibrillar collagens, particularly collagen II, the major collagenous component of vitreous, we have commenced studies to see if RPE cells can express this metalloproteinase. Using RT-PCR techniques, we have found that they can do so in the presence of certain inducing factors, at least at the mRNA level.
OVER-EXPRESSION OF METALLOTHIONEIN-1 BUT NOT HEME OXYGENASE-1 PROTECTS RPE CELLS AGAINST OXIDATIVE DAMAGE ((R.C.Hunt, D.M. Hunt and H.Lu)) Department of Microbiology and Immunology, University of South Carolina, Columbia, South Carolina
Purpose. Retinal pigment epithelial (RPE) cells synthesize a number of proteins that may protect the retina against oxidative shock. Among these are metallothionein-1 (MT-1) and heme oxygenase-1 (HO-1). The function of MT-1 is obscure. To determine whether these proteins can indeed protect RPE cells, they were expressed under the control of a switchable promotor. Methods. A line of human RPE cells (D407) was stably transfected with a plasmid that encodes a tetracycline-responsive transcription factor. These cells were then stably transfected with HO-1 or MT-1-encoding plasmids in which mRNA expression is controlled by the level of tetracycline in the medium. Levels of mRNA in the presence and absence of tetracycline were measured by Northern blotting while protein was measured by immunofluorescence and Western blotting. Cells were exposed to a free radical generator (xanthine oxidase/hypoxanthine) in the presence of various catalysts including heme and iron and oxidative damage was assessed in chromium-release assays. Results. D407 RPE cells normally express undetectable levels of HO-1 and MT-1 but both proteins are induced by a variety of agents of oxidative stress. Tetracycline-induced expression of either HO-1 or MT-1 in the transfected cells was dependent on the level of tetracycline in the medium. Elevation of MT-1 protected against both heme- and iron-mediated oxidative assault but over expression of HO-1 had no protective effect against ether heme or iron. However, elevation of HO-1, unlike elevation of MT-1, increased RPE cell proliferation. Conclusions. These data suggest that MT-1 is an anti-oxidant protein but over-expression of HO-1 does not confer resistance to oxygen toxicity. This may result from the elevation of redox-active iron as a result of heme metabolism. The effect of HO-1 expression in cell proliferation suggests that heme metabolites, such as iron, may act as nutrients.
MODULATION OF FGF-2 EXPRESSION BY VITREOUS HUMOR OR ANTI-SENSE MRNA IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS LEADS TO MORPHOLOGICAL ALTERATIONS AND CHANGES IN CELL MOTILITY. (( D. Meitinger, D.M. Hunt, W. Chen, P. Fairey, R.C. Hunt)) Department of Microbiology, University of South Carolina School of Medicine, Columbia, SC))
Purpose. When vitreous humor comes in contact with human RPE cells, the latter undergo an epithelial to mesenchymal transition similar to that which occurs in proliferative vitreoretinopathy. The basis of this alteration was investigated. Methods. RPE cells were incubated with vitreous humor and adenovirus constructs that encode either -galactosidase (lac z) or anti-sense FGF-2 mRNAs. Alterations in surface molecule levels were measured by fluorescence immuno-cytometry. Alterations in mRNA expression were measured by ribonuclease protection assays. Mobility was measured by a Boyden chamber assay. Results. The vitreous-induced alteration in morphology was accompanied by an increase in cell motility, a decrease in 3 integrin and an increase in 5 integrin. Ribonuclease protection assays showed that this was accompanied by a reduction in the amount of FGF-2 mRNA. Addition of recombinant FGF-2 to vitreous-treated cells reversed the changes in integrin expression and motility. RPE cells were infected with an adenovirus construct (ad-asbFGF) that led to the synthesis of an antisense FGF-2 mRNA. This resulted in a reduction of FGF-2 synthesis by the cells as shown by immuno-blotting, compared to cells infected by a control adenovirus construct containing the lac z gene. Reduction of FGF-2 in the ad-asbFGF-infected cells led to a morphological change similar to that observed in vitreous humor-treated cells and to an increase in cell motility. Conclusions. FGF-2 levels are important in maintaining RPE cell epithelial morphology. This may depend on the expression of certain cell surface integrin molecules.
NIH grant EY10516 and DFG Me1532/1-1 None
CHANGES IN INTEGRIN EXPRESSION BY FGF-2 IN
RETINAL PIGMENT EPITHELIAL CELLS EXPOSED TO VITREOUS HUMOR ((D. Meitinger, S. Riddell,
D.M. Hunt and R.C. Hunt)) University of South Carolina, Columbia
Purpose. When RPE cells come into contact with vitreous humor, as
occurs in proliferative vitreoretinopathy, they undergo a morphological transition from a
epithelial to a more mesenchymal appearance and secrete an altered extracellular matrix to
form epiretinal membranes. The changes in gene expression that accompany the change in
morphology have been investigated. Methods. Messenger RNA
differential display has been used to identify major changes in gene expression.
Quantitative fluorescence cytometry and immunoprecipitation have been used to identify
associated changes. Results. When RPE cells are exposed to
vitreous humor, a major reduction in FGF-2 (bFGF) mRNA expression occurs accompanied by a
loss of epithelial morphology. This change can be partially reversed by addition of FGF-2.
Accompanying the change in morphology is an increase in the rate of accumulation and
steady-state levels of alpha5 integrin (the fibronectin receptor) and in alphav
/ beta3 integrins (the vitronectin receptor). The increase in synthesis is not
a result of increased cell proliferation in the presence of vitreous humor. The level of
beta1 integrins does not change while the level of alpha3 integrin
falls. The increase in the level of alpha5 integrin is reversible by addition
of FGF-2. Conclusions. The morphological change shown by RPE cells
in the presence of vitreous humor may result from lower FGF-2 synthesis since this protein
often acts as a differentiation-stabilizing factor. Morphological changes may reflect
cell-extracellular matrix interactions since vitreous humor and FGF-2 alter integrin
metabolism.
Supported by NIH grant EY10515
CHANGES IN ANTI-OXIDANT PROTEINS DURING
RAT RETINAL DEVELOPMENT ((W. Chen1, D.M. Hunt1, A. Smith2
and R.C. Hunt1)) University of South Carolina, Columbia1; University
of Missouri-Kansas City2.
Purpose. The retina is especially prone to damage by reactive
oxygen species as a result of oxygenation, irradiation and its high content of
polyunsaturated fatty acids. The formation of reactive oxygen species is catalyzed by iron
and heme. Since oxidative assault is likely to occur at birth, the production of mRNAs for
two anti-oxidant proteins, metallothionein-1 and heme oxygenase-1 was studied during the
development of the rat retina. Methods. Rat eyes were fixed in
paraformaldehyde and embedded in paraffin. Retinal sections were subjected to in situ
hybridization with digoxygenin-labeled probes for the two anti-oxidant proteins and for
actin. Sense probes for each mRNA were also used. Results. At 16
and 21 days after fertilization, rat embryonic retina expressed metallothionein-1 mRNA. At
birth, however, metallothionein-1 mRNA was decreased. No expression of heme oxygenase-1
mRNA was observed at 16 days post-fertilization but by the time of birth, this mRNA
increased and was expressed for at least several days thereafter. Actin mRNA was
synthesized throughout the period investigated. No signal was seen in the neural retina
from the sense probes. Conclusions. Heme oxygenase-1 mRNA, which
encodes the first enzyme in the catabolism of heme and a major protective agent against
oxidative damage is increased at a time when the fetus is likely to experience oxidative
assault, that is when it leaves the hypoxic environment of the uterus. Thus it is possible
that premature birth may result in a neonate with an inappropriately developed
anti-oxidant defense system and this may be a factor in retinopathy of prematurity.
Supported by NIH grant EY10516
SYNTHESIS OF HAPTOGLOBIN BY THE NEURAL
RETINA ((H. Lu1, W. Chen1, A. Smith2, D.M. Hunt1,
K. Dutt3 and R.C. Hunt1)) University of South Carolina, Columbia1;
University of Missouri-Kansas City2; Morehouse University, Atlanta3
Purpose. Anti-oxidant proteins are of great importance in
the retina during oxidative assault and inflammation. Extracellularly, protection against
reactive oxygen species (RO2S) is effected by the production of proteins that
bind iron, the major catalyst of (RO2S) production. Free iron is bound by
transferrin while heme iron is bound by hemopexin. A third protective protein, haptoglobin
binds hemoglobin that is released by erythrocytes but is also important in immunity since
it is an acute phase protein that responds to changes in the levels of several
inflammatory cytokines, particularly interleukin-1 and interleukin-6, altering neutrophil
metabolism, the proliferation of B-lymphocytes and antibody production. It has already
been shown that transferrin and hemopexin are made in the retina. The production of
haptoglobin was therefore investigated. Methods. Reverse
transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization were
used to detect haptoglobin mRNA while immunofluorescence was used to detect the protein. Results.
Haptoglobin can be detected in human retina sections by immunofluorescence. RT-PCR showed
that haptoglobin mRNA is made by neural retina but not by RPE cells dissected from human
donor eyes. Cultured photoreceptor cells expressed haptoglobin mRNA but a line of human
RPE cells (D407) did not. In situ hybridization, using sections of human retina
and digoxygenin probes that detect haptoglobin mRNA, showed that the site of synthesis of
the protein is cells of the inner and outer nuclear layers. Conclusions.
Haptoglobin joins hemopexin and transferrin as an anti-oxidant protein made by cells of
the neural retina. Retinal synthesis of these proteins probably occurs because
liver-synthesized proteins cannot gain access to the retina because of the blood retinal
barrier.
Supported by NIH grant EY 10516
Abstracts for the Annual Meeting of the American Society for Cell Biology Meeting, Washington, D.C. December 1997
REDUCTION IN THE LEVELS OF FGF-2 IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS LEADS TO LOSS OF EPITHELIAL CHARACTERISTICS AND CHANGES IN CELL MOBILITY. ((D.M. Hunt, D. Meitinger, W. Chen, P. Fairey and R.C. Hunt)) Department of Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208
When vitreous humor comes in contact with human RPE cells, the latter undergo an epithelial to mesenchymal transition in a manner similar to that which occurs in vivo in proliferative vitreoretinopathy. The cells exhibit an altered actin cytoskeleton structure and become more mobile. Differential mRNA display showed that the morphological transition on vitreous humor treatment is accompanied by a reduction in the amount of FGF-2 mRNA which was confirmed by in situ hybridization and ribonuclease protection assays. Western blotting also showed that the level of FGF-2 protein was reduced. Addition of recombinant FGF-2 to vitreous-treated cells reversed their increased mobility while mobility of untreated cells was increased when they were incubated with anti-FGF-2 antibodies to neutralize FGF-2 synthesized by the cells. Mobility of untreated cells was also increased by incubation with anti-FGF receptor (anti-flg) antibodies that block binding of endogenous FGFs. These data suggest that FGF-2 plays an important role in the morphological and mobility changes that occur in the presence of vitreous humor. To investigate these findings further, human RPE cells were infected with an adenovirus construct (ad-asbFGF) that led to the synthesis of an mRNA that was antisense to that encoding FGF-2 mRNA. This resulted in a reduction of FGF-2 synthesis by the cells over a period of 72 hours, as shown by Western blotting, while cells infected by a control adenovirus construct containing the lac z gene did not show a change in FGF-2. Reduction of FGF-2 levels in the ad-asbFGF- infected cells led to a morphological change similar to that observed in vitreous humor-treated cells and to an increase in cell mobility.
Supported by NIH grant EY10516 and a Fellowship from DGF (Me1532/1-1)
DECREASED FGF-2 EXPRESSION IN HUMAN RETINAL PIGMENT EPITHELIAL CELLS LEADS TO AN EPITHELIAL TO MESENCHYMAL MORPHOLOGY CHANGE AND TO CHANGES IN CELL SURFACE MOLECULES. ((D. Meitinger, D.M. Hunt and R.C. Hunt)) Department of Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208
Human RPE cells treated with vitreous humor lose their epithelial shape, overgrow one another and become more mobile. It has previously been shown that a reduction in FGF-2 accompanies these changes in vitro. The changes in mobility and shape suggest that cell surface adhesion molecules may be altered as a result of the contact with vitreous humor and the synthesis of these proteins was therefore investigated. It was found that the level of alpha5 integrin rises as the cells change from an epithelial to a mesenchymal appearance while the level of alpha3 integrin falls. N-cadherin and beta1 and alpha2 integrin levels are little changed. The rise in alpha5 and the fall in alpha 3 integrins are reversed by addition of recombinant FGF-2 to vitreous-treated cells suggesting that cell surface molecule synthesis may be controlled by FGF-2. To determine whether this is the case, RPE cells were treated with an adenovirus construct that expresses antisense FGF-2 mRNA. This led to a change from epithelial to mesenchymal morphology and to alterations in the surface levels of alpha3 and alpha5 integrins. This suggests that the morphological change induced by vitreous and controlled by FGF-2 may result from altered integrin expression. To determine whether this is the case, RPE cells were also infected with an adenovirus construct that expresses alpha5 integrin mRNA. This led to an increase in the surface expression of this integrin and to a change from an epithelial to a mesenchymal morphology. The increase in alpha5 integrin expression also led to an increase in cell mobility suggesting that the expression of this surface adhesion molecule may contribute to the observed changes in mobility.
Supported by NIH grant EY10516 and a Fellowship from DGF (Me1532/1-1)
CHANGES IN ANTI-OXIDANT PROTEINS DURING RAT RETINAL DEVELOPMENT ((W. Chen, D.M. Hunt, A. Smith and R.C. Hunt)) Department of Microbiology, University of South Carolina Medical School, Columbia SC 29208 and Division of Molecular Biology and Biochemistry, University of Missouri, Kansas City., MO 64110
The retina is especially prone to damage by reactive oxygen species as a result of its high level of oxygenation, its exposure to irradiation and its high content of polyunsaturated fatty acids. Oxidative assault is especially damaging to the retina of the premature infant and can lead to retinopathy of prematurity. Why such sensitivity to reactive oxygen species occurs is unknown but it may be the result of the premature infant being born without a normal anti-oxidant protective system since the latter is unnecessary in the low oxygen tension of the womb. Oxidative assault is likely to be exacerbated in the premature infant by the use of an oxygen incubator. To determine whether anti-oxidant proteins develop late in gestation of the rat, in situ hybridization and semi-quantitative reverse transcriptase-polymerase chain reaction and Southern blotting were used to measure the activity of heme oxygenase-1, metallothionein-1, superoxide dismutase and catalase in the developing rat eye. Little expression of heme oxygenase-1 mRNA was observed at 16 days of embryonic development (E16) but by the time of birth (embryonic day 22), this mRNA was greatly increased but fell in amount thereafter. At E16 and E18, metallothionein-1 mRNA was expressed but fell to undetectable levels by birth. Catalase mRNA levels were low prior to birth and rose in amount in the first few days of post-natal life. Superoxide dismutase and actin mRNAs were synthesized at similar levels throughout the period investigated. These data imply that heme oxygenase-1 mRNA, which encodes the first enzyme in the catabolism of heme and is major protective agent against oxidative damage is increased at a time when the fetus is likely to experience oxidative assault. Thus it is possible that premature birth may result in a neonate with an inappropriately developed anti-oxidant defense system and this may be a factor in retinopathy of prematurity.
ISOLATION OF GENES WHOSE EXPRESSION IS MODULATED DURING EPITHELIAL-MESENCHYMAL TRANSDIFFERENTIATION OF HUMAN RETINAL PIGMENT EPITHELIAL CELLS. ((P.W.Fairey IV, D.M.Hunt and R.C.Hunt)) Department of Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208.
Proliferative vitreoretinopathy (PVR) is a disease in which retinal cells which are normally quiescent gain access to, and proliferate in the vitreous humor. Retinal pigment epithelial (RPE) cells have been implicated in playing an important role in PVR. RPE cells, along with other cells, proliferate, de-differentiate, and elaborate an extracellular matrix forming epiretinal membranes which may contract, resulting in retinal detachment and loss of sight. Upon exposure to human vitreous, low passage human RPE cells and several RPE cell lines exhibit marked morphological changes consistent with the epithelial-mesenchymal transformation seen in PVR. The differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to detect changes in mRNA expression in the presence of vitreous. Total RNA samples were obtained from vitreous-treated or control cultures of RPE cells at 24 hours of treatment and used in DDRT-PCR. We encountered several problems with the methods including lack of reproducibility, a low number of bands, detection of short PCR fragments only (less that 500 bp) and failure to amplify excised differentially expressed bands for further study. Modifications were made to the procedure including altered primer concentrations, nucleotide concentrations, enzyme concentrations and buffer compositions. We have detected several genes which are differentially expressed, including a cell cycle regulator, a translation factor, and a regulator of cytoskeletal organization. Altered mRNA levels for genes of interest were confirmed by ribonuclease protection assays. This increases the number of genes whose expression is known to be modulated by exposure to vitreous. It is anticipated that this approach should help to elucidate some of the events in PVR and perhaps other epithelial-mesenchymal transformations. Supported by NIH grant EY10516, an RPS award from the University of South Carolina and an NAS grant through Sigma Xi.
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