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From the Department of Ophthalmology,*
Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands; The
Gaubius Laboratory,
Leiden, The Netherlands;
the Department of Pathology,§
Haartman
Institute, University of Helsinki, Helsinki, Finland; and Department of
Ophthalmo-Immunology,
Netherlands Ophthalmic
Research Institute, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On the other hand, in the course of CNV, RPE cells are observed to encapsulate the new vessels, with their basal side toward the endothelial cells, which coincides with the arrest of further vascular growth.4 These morphological observations in vivo suggest that the RPE can have a function in controlling CNV.
Based on these observations, it seems that the RPE has a dual role in its interactions with the CC, but how the integrity of the RPE/CC microunit is regulated in healthy conditions, how the photoreceptor layer remains avascular, and why pathological changes in the RPE layer lead to choroidal atrophy or to an angiogenic response from the choroid are essentially unknown. It is likely, however, that mediator molecules play an essential role in these intercellular interactions.
Such interactions have previously been investigated in cell cultures of the constituent cells, but from these studies conflicting results have emerged. Whereas some authors were able to demonstrate that RPE cells in vitro produce an inhibitor of endothelial proliferation,5 others found mitogenic activity for endothelium in RPE conditioned culture medium.6
We hypothesize that a polarized secretion of growth/inhibitory factors may explain these apparently conflicting in vivo and in vitro observations. Growth factors produced by RPE in vitro include platelet-derived growth factor-BB, transforming growth factor-ß, and vascular endothelial growth factor-A (VEGF).7 VEGF, a vascular permeability and angiogenesis factor, is a good candidate for a role in maintaining the CC. VEGF has been shown to increase vascular permeability in skin and muscle partly by inducing a fenestrated endothelial phenotype.8 Furthermore, it can act as a vascular survival factor in vivo.9 In a coculture experiment of RPE and CC endothelium in vitro, RPE-derived VEGF was shown to stimulate tube formation of the underlying endothelial cells.10
The aim of the present study was to evaluate the polarity of VEGF secretion by cultured human RPE cells and to investigate the localization of VEGF receptors in situ in human and monkey eye tissue sections.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four human donor eyes (age of the donors: 9, 15, 17, and 24 years), obtained from the Corneabank, Amsterdam, were used as a source of primary RPE cells. The RPE cells (further designated as RPE cell lines) were isolated within 24 hours post mortem.11 RPE cells were isolated as described by Holtkamp et al11 In short, the cornea, the anterior segment, the optic nerve, vitreous, and neural retina were removed from the eye, and the RPE cells were dissociated from the eye with trypsin by two subsequent incubations at 37°C. Cells obtained from the second incubation were plated in 24-well plates (Costar, Cambridge, MA) at 105 cells/well in Iscove's modified Dulbecco's medium (IMDM) (Gibco BRL) supplemented with 20% fetal calf serum (FCS) (Gibco BRL), penicillin (100 U/ml; Gibco BRL), and streptomycin (100 mg/ml; Gibco BRL). Nonadherent cells were removed after 2 days by washing and refreshing the culture medium. At confluence, cells were detached by trypsin treatment and passed to culture flasks at approximately 4 x 104 cells/cm2. For the present experiments, RPE cells were used between the sixth and 11th passages. To investigate whether the RPE cell lines were not contaminated during the isolation procedure, the cells were analyzed morphologically and by immunohistochemistry.
For immunohistochemistry, cells were cultured on tissue chamber slides (Lab-tek; Nunc, Naperville, IL) and stained with antibodies specific to cytokeratin 8/18 (CAM 5:2; Beckton-Dickinson, San Jose, CA) or specific to glucose transporter-1 protein (a kind gift from L. Andersson, University of Uppsala, Uppsala, Sweden). As a negative control an antibody against a nonhuman bacterial protein was used (mouse negative control immunoglobulins; DAKO).
All cells were cultured at 37°C and at 5% CO2. Medium was changed twice a week.
RPE Cell Monolayers on Transwell Filters
RPE cells were cultured on transwell filters (12-mm diameter, 0.4-mm pore size; Costar) according to the method of Holtkamp et al.11 Briefly, filters were coated with 160 ml of a 1:40 dilution of Matrigel (Collaborative Biomedical Products, Bedford, MA) in medium and air dried overnight. The RPE cell lines were seeded as 1.6 x 105 cells/cm2 in a volume of 200 ml/filter, in IMDM supplemented with 1% normal human serum (NHS) (CLB, Amsterdam, the Netherlands). In the lower compartment, 1000 ml medium was added, thereby leveling the height of the liquid levels to prevent hydrostatic pressure. After 2 days IMDM/1%NHS was added to a final volume of 750 ml in the upper compartment and 1500 ml in the lower compartment.
Transepithelial resistance (TER) was measured once a week with an Endohm chamber and an ohmmeter (World Precision Instruments, Sarasota, FL). TER measurements were corrected for background by subtracting the TER value of a matrigel-coated filter without cells. Filters with RPE monolayers were used for experiments approximately 3 weeks after plating.
Microscopy of RPE Cell Monolayers on Transwell Filters
The possible formation of multilayers of RPE cells was checked by light microscopy on sections of the filters. The filters were washed with phosphate-buffered saline (PBS), frozen in liquid N2, embedded in Tissue-Tek (Miles, Elkhart, IN), cut into 10-µm-thick sections, fixed in acetone for 5 minutes, and air dried. The sections were stained with hematoxylin for 10 minutes, followed by staining with eosin for 1 minute.
The presence of tight junctions was determined by immunofluorescence microscopy. Filters were washed with PBS (pH 7.4), fixed with 4% paraformaldehyde, washed again with PBS, permeabilized by incubation with 0.2% Triton X-100 for 15 minutes, blocked with 5% normal goat serum (Jackson Lab), and incubated with polyclonal antibodies against the tight junction-associated protein ZO-1 (Zymed Laboratories, San Francisco, CA; 1:200) for 1 hour. After washing with PBS, filters were incubated for 1 hour with fluorescein isothiocyanate-conjugated swine anti-rabbit immunoglobulins (Nordic, El Toro, CA; 1:100), washed again in PBS, and sealed with vectashield before being observed in a fluorescence microscope (Leica).
As described previously,11 the integrity of the RPE cell monolayers was analyzed by electron microscopy (EM). For scanning EM, filters were washed with Hanks' buffered salt solution, after which the cells were fixed in a 80 mmol/L cacodylate-buffered solution containing 1% glutaraldehyde and 1.25% paraformaldehyde (pH 7.3). After fixation for several days, the filters were dehydrated with ethanol, critical-point dried with CO2 as the intermediate, and coated with a few nanometers of platinum. The filters were inspected and micrographed with a Philips SEM 505 scanning electron microscope (Philips Industries, Eindhoven, the Netherlands) with a secondary emission detector.
For transmission EM, filters were postfixed in OsO4, dehydrated in a graded series of ethanol, and embedded in Epon. Ultrathin sections were cut, stained with uranyl acetate and lead citrate, and inspected and photographed in a Philips EM 400 electron microscope (Philips Industries) equipped with an EDAX PV9800 system and an ultrathin window detector (EDAX PV9760/53).
VEGF Secretion of RPE Cell Monolayers on Transwell Filters
Experiments were performed with 12 transwell filters with confluent RPE cells from each donor. Medium was removed from the 48 filters and replaced with fresh medium containing 1% NHS just before the experiments. The filters were incubated in normal growth conditions (24 filters, 37°C, 20% O2, 5% CO2, moist environment) or in hypoxic conditions (24 filters, 37°C, 1% O2, 5% CO2, moist environment) for 24 hours (24 filters) or 48 hours (24 filters). Thus each of the four conditions investigated was tested in triplicate. After 24 hours or 48 hours medium from the upper and lower compartments was collected, snap-frozen, and stored at -20°C until further analysis.
Levels of VEGF production of the RPE cells toward the basal or the apical side were assayed by enzyme-linked immunoabsorbent assay (R&D Systems, Abingdon, England), according to the manufacturer's description. Each sample of medium was tested in triplicate.
Statistical Analysis
For statistical data analysis, the Independent Samples t-test was used after investigation of equality of variances by Levene's test for equality of variances (SPSS Software, SPSS Gorinchem, The Netherlands).
Human and Primate Tissue Samples
Eyes from 30 persons with no known eye disease were obtained from the Corneabank, Amsterdam, within 5–15 hours after death. Furthermore, fresh normal eyes from two Cynomolgus monkeys (Macaca fascicularis) were kindly provided by the University of Nijmegen, the Netherlands. All animal experiments in this study were performed in accordance with the ARVO Resolution on the Use of Animals in Ophthalmic and Vision Research. Samples of the posterior segment were dissected. All tissues were snap-frozen and stored at -70°C.
Immunohistochemistry
Air-dried cryosections (10 µm) were fixed in cold acetone for 10 minutes and postfixed for 2 minutes with Zamboni fixative (2% paraformaldehyde in a saturated picric acid solution) and stained by an indirect immunoperoxidase procedure. To reduce nonspecific staining, sections were preincubated in 10% normal goat serum (Jacksons Immuno Research Laboratories, West Grove, PA; catalog no. 005-000-121) in PBS for 15 minutes and then incubated overnight at 4°C with the following antibodies: anti-endothelial monoclonal antibody (mAb) PAL-E (1:1000), anti-VEGF receptor KDR, anti-VEGF receptor flt-1 (both a kind gift of Dr. H. A. Weich, Department of Gene Expression, GBF, 38124 Braunschweig, Germany; 1:200), and anti-VEGF receptor flt-4 (raised at the Haartman Institute, University of Helsinki, Helsinki, Finland; 1:400). Sections were washed and incubated with subsequent compounds of a sensitive avidin-biotin-based Histostain plus kit (Zymed 85–9043) according to the manufacturer's instructions. Sections were washed again, and staining was performed using 3-amino-9-ethyl carbazole (AEC) containing 0.01% H2O2.12 The reaction was terminated by rinsing the sections with H2O. Counterstaining was performed with hematoxylin. Sections were washed and mounted in glycerol/glycerine (Sigma; GG-1). In control sections the first antibodies were omitted.
All sections were examined by two independent observers in a masked fashion. Microvascular staining was graded as follows: no staining (-), weak staining (±), or evident staining (+).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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After seeding on transwell filters (12 filters for each donor),
the human retinal pigment epithelial (RPE) cell monolayers (passage
between 6 and 11) showed a gradual increase in TER. The TER of the
matrigel-coated filters without cells was 15 Ohm·cm2.
Maximum net TER was reached after approximately 3 weeks. The TER of the
four RPE cell lines ranged from 30 ± 1.2 to 100 ± 4.3
Ohm·cm2
but showed little variation within each cell line
(Figure 1)
.
|
. Transmission electron microscopic
studies revealed a monolayer of cells with microvilli and tight
junctions at the side of the upper compartment of the transwell system
(Figure 2B)
, and deposition of basement membrane material between the
lower side of the cells and the filter, indicating that the upper
compartment represents the apical ("retinal") side of the RPE
monolayer and the lower compartment the basal ("choroidal") side.
The presence of tight junctional complexes was suggested by
immunofluorescence staining of the cell layers for the tight
junction-associated protein ZO-1. ZO-1 was present at one level around
every cell, in both the cell lines with low (35 Ohm·cm2,
Figure 2C
) and high (100 Ohm·cm2, Figure 2D
) TER.
Staining for ZO-1 also showed that the density of cells on the filters
was similar for all four RPE cell lines (approximately 106
cells/well). Taken together, these results indicated that the cultured
RPE cells are very similar to differentiated resting RPE in
vivo.
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The RPE cell lines produced vascular endothelial growth factor-A
(VEGF) under normal culture circumstances (20% O2, 5%
CO2). The total production of VEGF varied between the
different RPE cell lines, ranging from 1056 ± 64 to 7410 ±
456 pg/filter (106
cells) (24-hour incubation) (Figure 3)
. Interestingly, the TER values of the
four RPE cell lines correlated with the total VEGF production (compare
Figures 1 and 3
).
|
. When RPE monolayers were kept in
hypoxic conditions (1% O2, 5% CO2), VEGF
production increased between 1.3- and 1.5-fold to the basal side but
much less to the apical side. At 24 hours, this basal increase (and
consequently higher polarization) was statistically significant for
donors B, C, and D (P < 0.05, Figure 4
). As a
consequence, in hypoxic conditions the amount of VEGF secreted toward
the basal side was 3–10 times higher than the amount secreted to the
apical side.
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Retina and choroid of 30 human donor eyes and two monkey eyes were
stained with antibodies against VEGF receptors flt-1 (VEGFR-1), KDR
(VEGFR-2), and flt-4 (VEGFR-3) and with a marker for fenestrated
endothelium, PAL-E. Staining with the antibody against PAL-E strongly
outlined the endothelial cells of the choriocapillaris as well as the
endothelial cells of the other choroidal vessels in both the human
donor eyes and the monkey eyes (Figure 5A)
. Staining of the sections with
antibodies against the VEGF receptors KDR and flt-4 showed a particular
staining pattern of the choriocapillaris in both human and monkey eyes
(Figure 5, C and D)
. Only on the side of the choriocapillaris
endothelium that faces the RPE was a granular staining for KDR and
flt-4 observed. On the scleral side of the choriocapillaris and in the
larger choroidal vessels, only sporadic staining for KDR and flt-4 was
seen (Figure 5, C and D)
. The microvasculature of adjacent retina was
negative. The staining for flt-4 in the choriocapillaris was even more
pronounced than that for KDR. In contrast, staining with anti-flt-1
antibodies showed, in addition to marked staining of the inner CC, weak
staining in the other choroidal vessels (Figure 5B)
and staining of the
entire retinal microvasculature in a pattern suggestive of expression
of this receptor by both endothelial cells and pericytes (results not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several authors have previously demonstrated that cultured human RPE cells produce VEGF, among other growth factors.7,13-18 In fact, VEGF was found to be responsible for most, if not all, mitogenic activity for endothelial cells in RPE conditioned medium.6 In vivo, RPE cells can dedifferentiate and act as multipotent cells in ocular wound healing and proliferative vitreoretinopathy. Therefore, one may argue that human RPE cells in vitro produce VEGF as a result of the specific culture conditions, which may induce an "injury-type" phenotype of cells, comparable to those observed in vivo in wound healing and other pathological conditions. However, several in vivo studies indicate that also in the resting normal eye,1,6,17,18 VEGF is produced constitutively by RPE cells, suggesting a physiological role of the growth factor in the eye. Furthermore, of our four different RPE cell lines, those with a higher TER produced higher amounts of VEGF. This may indicate that a higher degree of differentiation of RPE cells in vitro, as reflected by a higher TER,19 leads to a higher production of VEGF. In these tight RPE monolayers with the highest TER, VEGF secretion was also most polarized, with up to 10 times more VEGF produced to the basal side as compared to the apical side. Previous in vitro studies have found that RPE cells secrete between 0.6 and 70 ng VEGF/106 cells.6,20 This correlates well with our results, as the concentrations of VEGF found in the RPE conditioned medium ranged from 0.2 ng/ml on the apical side to 11 ng/ml on the basal side in hypoxic conditions. These concentrations of VEGF are in the biologically active range, as observed in experiments with cultured endothelium.21-23 From such experiments, VEGF has emerged as an important permeability and angiogenesis factor.21-23 In vitro, VEGF is mitogenic for endothelial cells and stimulates their migration. When injected in vivo into muscle or skin, it rapidly induces endothelial fenestrations in the continuous endothelium of these tissues.8 In other conditions, as in the developing retina, VEGF has been shown to act as a survival factor for endothelium.9
The relation between the choriocapillaris and the RPE has long been a focus of attention from both clinicians and basal scientists. Anatomically, the choroidal vascular plexus is modeled toward the RPE, as if drawn to it by some mysterious force. Furthermore, the choriocapillaris endothelial cells have fenestrations only on their side facing the RPE. It is also clear from clinical observations that the choriocapillaris will disappear after loss of the RPE in geographic age-related macular degeneration (AMD) and in other conditions.24,25 Animal models also demonstrate that RPE loss leads to choriocapillaris atrophy, preceded by loss of the endothelial fenestrations.2,26 All of these data suggest a strong modulating influence of the RPE on the choriocapillaris.
It has previously been suggested that VEGF may be involved in this respect, based on the observed production of VEGF by cultured RPE. Furthermore, in a coculture experiment, VEGF derived from RPE monolayers induced capillary tube formation of underlying endothelial cells in a type I collagen gel.3 These studies did not specify the degree of differentiation of the RPE cells or the direction of secretion of VEGF, however. In fact, these results were confusing, because, when VEGF is secreted by RPE toward the retina in vivo, this would be expected to induce retinal neovascularization. Such neovascularization was demonstrated recently in a transgenic mouse model with constitutive VEGF production by photoreceptor cells.27 However, in the normal eye, the outer retina is, of course, completely avascular. The data presented in this study suggest that the constitutive secretion of VEGF by RPE in vivo may well be highly polarized and be restricted to the basal choroidal side.
Our observations suggest that VEGF is involved in a physiological paracrine relation between the RPE and the choriocapillaris. The observed specific localization of the VEGFR-2, KDR, at the inner choriocapillaris, which is facing the basal side of the RPE cells, further supports this notion, as expression of VEGFR-2 by these cells would allow paracrine signaling by VEGF-A. In contrast, immunohistochemical staining for the VEGFR-1, flt-1, was also found in other vessels of the choroid and retina, in a pattern suggestive of both endothelial and pericyte staining. It has been proposed that these two VEGF receptors (KDR and flt-1) mediate different functions of VEGF-A.28 Therefore, differential expression of the receptors in tissues may allow the regulation of VEGF-A activity. A third member of the VEGF receptor family, flt-4, uses other gene products, VEGF-C and VEGF-D, as ligands, whereas its affinity for VEGF-A is negligible.29,30 Our finding that flt-4 is present in the vessel segments of the choriocapillaris that contain KDR suggests that cooperativity may occur between different types of VEGF molecules. It is unknown, however, whether RPE cells indeed produce VEGF-C or VEGF-D.
In light of the known effects of VEGF on endothelial cells in vivo and in vitro, VEGF may well be very important in both the trophic influence of RPE on the choriocapillaris, as well as in inducing its highly permeable phenotype, reflected by the abundant fenestrations located on the RPE side of the choriocapillaris.31 Recently, Roberts and Palade8 and Esser et al32 found that VEGF is able to induce endothelial fenestrations, both in vivo and in vitro. Likewise, secretion of VEGF by RPE cells to the basal side could induce the fenestrations of the choriocapillaris. It has to be resolved whether the secretion of VEGF-A is sufficient or VEGF-A and VEGF-C or other factors cooperate in this effect. A recent study indicates that such a cooperation exists in endothelial proliferation and tube formation in vitro.33 Moreover, the VEGF receptors KDR and flt-4 are mainly localized on the RPE side of the choriocapillaris, providing a selectively responsive area of the existing blood vessels. By such a paracrine mechanism VEGF secreted by the RPE cells may also act as a trophic factor on the choriocapillaris.
These findings may also have important implications for the understanding of the pathogenesis of choroidal neovascularization as occurs in AMD. Choriocapillaris atrophy and an increased thickness of Bruch's membrane and relative impermeability to water-soluble compounds, like VEGF, have been reported in aging and AMD.34,35 One may speculate that the latter changes in Bruch's membrane may not only lead to CC atrophy resulting from insufficient amounts of VEGF reaching the choroid, but may lead, on the other hand, to accumulation of VEGF on the basal side of the RPE, as has indeed been observed in AMD in immunohistochemical studies.36 Here, VEGF may reach a critical concentration and induce CNV under the RPE. This mechanism may be further enhanced by local hypoxia, as we observed up-regulated basolateral production of VEGF by RPE cells in hypoxic conditions. In fact, a relative outer retinal hypoxia has been suggested to occur in AMD as a result of CC atrophy and thickening of Bruch's membrane.34,36 In other situations with CNV, defects in the RPE monolayer could lead to misdirection of secreted VEGF and subsequent classical subretinal neovascularization.
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Acknowledgements |
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Footnotes |
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Supported by the Stichting Blindenhulp, the Diabetes Fonds Nederland (grant 95.103), the Praeventiefonds (grant 26-1822-3), the Edward en Marianne Blaauwfonds, and the Stichting "De Drie Lichten."
Accepted for publication April 30, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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